CN108440293A - A method of preparing high-purity chlorogenic acid by raw material of woodbine - Google Patents
A method of preparing high-purity chlorogenic acid by raw material of woodbine Download PDFInfo
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Abstract
The invention discloses a kind of methods preparing high-purity chlorogenic acid as raw material using woodbine.Its technical characterstic be using the aerial part of woodbine honeysuckle, Honeysuckle flower include bud, flower, leaf and stem be raw material, using supercritical extract or ultrasonic wave extraction extraction woodbine in chlorogenic acid.By ethyl acetate extraction, the means removal of impurities of alkalization precipitation, carrys out the chlorogenic acid in extract-enriched using acidizing extraction, its purity is made to can reach 50% or more.High-purity chlorogenic acid crystal can be directly further precipitated by concentrated crystallization.This method raw material sources are extensive, easy to operate, can save traditional chlorogenic acid extraction and resin adsorption in subtractive process and the operating process such as prepared by column chromatography, can both obtain high-purity chlorogenic acid product, the value with extensive industrial applications.
Description
Technical field
The invention belongs to pharmaceutical chemistry technical fields, are related to a kind of green using woodbine as feed purification preparation high-purity
The method of ortho acid.
Background technology
Honeysuckle (Lonicerajaponica Thunb.) is the dry flower of Caprifoliaceae woodbine honeysuckle.Chinese medicine
Theory thinks that honeysuckle is cold in nature sweet in flavor, enters the warps such as lung, the heart, stomach, and there is clearing heat and detoxicating, dispelling wind and heat from the body, anti-inflammatory, qi-restoratives to treat the work(of wind
Effect, cures mainly disease under turgor, warm disease heating, the diseases such as heat toxin large carbuncle and tumour.Honeysuckle flower is caprifoliaceae plant largeflower-like honeysuckle flower
(Lonicera macranthoides Hand.-Mazz.), fulvoushair honeysuckle flower (Lonicerafulvotomentosa Hsu et
S.C.Cheng), lonicera hypoglauca miq (Lonicera hypoglauca Miq.), Lonicera confusa (Lonicera confuse DC.)
Dry flower.Two kinds of honeysuckle flowers, which belong to, belongs to easy mixed Chinese medicine not of the same race, just by the same mountain of honeysuckle since 2005 editions pharmacopeia
Honeysuckle flower is separately treated, but completely the same in the description to two kinds of medicinal material meridian distribution of property and flavor and major function.Honeysuckle, mountain silver
Flower is common large Chinese medicine, there is the effect of clearing heat and detoxicating, dispelling wind and heat from the body, 1/5~1/3 prescriptions of traditional Chinese medicine, more than 200 kinds
It is widely used in Chinese patent drug, 500 numerous foods and health food and daily chemical products.Modern pharmacology research shows honeysuckle
With Honeysuckle flower antibacterial, it is antiviral, anti-inflammatory, anti-oxidant, bring down a fever, liver protection, immunological regulation, have in terms of antitumor activity it is similar
Pharmacological action.In addition to Heilungkiang, the Inner Mongol, Ningxia, Qinghai, Xinjiang, Hainan and Tibet, all parts of the country are distributed honeysuckle.
But the main place of production of current honeysuckle concentrates on the ground such as Henan, Shandong, Hebei, wherein with " the close honeysuckle flower " in the Henan, " Ji in Shandong
Honeysuckle flower " and the honeysuckle genuineness of Hebei Julu are stronger.Mountain and hill area, main product are generally planted or be grown on to Honeysuckle flower
The distribution in area is more extensive than honeysuckle, is concentrated mainly on Anhui, Zhejiang, Jiangxi, Fujian, Hubei, Hunan, Guangdong, Guangxi, four
The southern areas such as river, Yunnan and Guizhou.Chemical constitution study shows that honeysuckle and Honeysuckle flower contain volatile oil, flavones, organic
The Multiple components such as acid, iridoid glycoside, Content of Chlorogenic Acid, flavones and total iridoid be considered as honeysuckle main pharmacodynamics at
Divide [the Agriculture of Anhui science such as Xia Wei, 2017,45 (33):126-127；The such as Wang Lingna food and biotechnology journal, 2017,
36(11):1673-1689；The China medicine companies such as text celebrating, 2018,27 (2):1-5].Newest content analysis finds that chlorogenic acid is in flower
Content highest in flower bud is 27.22mg/g, is stem (19.68mg/g) and leaf (12.80mg/g) successively；Galuteolin contains in stem
Highest is measured, about 2.31mg/g is leaf 1.66mg/g and flower 1.07mg/g (such as Xu little Bo food industry science and technology, 2018) successively.
Chlorogenic acid (Chlorogenic acid, CGA) is 3 hydroxyl condensations by caffeinic 1 carboxyl and chinic acid
At the natural phenolic acid class compound of ester, also known as 3-O-caffeoylquinic acid (3-O-caffeoylquinic acid), average molecular
Quality 354.31,208 DEG C of fusing point, semihydrate are acicular crystal, [α]20 D=-37 (C=2.8) dissolve in methanol, ethyl alcohol, micro-
It is dissolved in water, ether, ethyl acetate, solubility is poor in chloroform, benzene.In woodbine honeysuckle and Honeysuckle flower Content of Chlorogenic Acid class
Substance is mainly collected in cauline leaf and bud, and the amount in root is seldom.In the growth course of cauline leaf, the chlorogenic acid amount of full-bloom stage is most
The amount of height, setting phase is declined, and amount when fruit maturation is begun to ramp up again, and in withered early period, the content of chlorogenic acid is again extensive
Again to a relatively high level.
1, the extracting method of chlorogenic acid
Due to having ester bond, the unstable parts such as unsaturated double-bond and polyphenol, extraction process in chlorogenic acid molecular structure
In hydrolysis and the migration of intramolecular ester group can occur cause its structure that isomerization occurs, therefore how efficiently to extract honeysuckle and mountain
Chlorogenic acid in honeysuckle flower is always the emphasis studied.It is usually extracted from plant using ethyl alcohol, acetone, methanol isopolarity solvent green
Ortho acid.The extracting method of chlorogenic acid substance includes water extraction, organic solvent extraction, enzymatic isolation method, supercritical fluid extraction at present
Follow the example of, a variety of methods such as microwave assisting method, ultrasonic wave auxiliary law [the food security quality testing journals such as Pan Wanlian, 2017,8
(11):4269-4276]。
It is solvent that water is mostly used in pharmaceutical industry of Chinese medicine, and heating boils extraction to improve the leaching rate of chlorogenic acid, but
The corresponding dissolution for also increasing water-solubility impurity, the yield of chlorogenic acid be only 3.6% [the food research and developments such as Li Jie,
2018,39(2):62-67].Wu Junwei etc. optimizes the flocculated extraction process of honeysuckle decocting using uniform design, passes through
The yield of pH5.0 sour water chlorogenic acid extractings is up to 4.8% [such as Wu Junwei China veterinary drug magazine, 2006,40 (8):23-25].
Wei Jun etc. has investigated the extraction process of ethyl alcohol using ethanol reflux extraction using Yongzhou Honeysuckle flower as raw material by orthogonal test,
It is 2.86% [such as Zhang Weijun Hu'nan University of Science and Engineering journal, 2017,38 (10) that chlorogenic acid, which extracts yield,:51-56].Xiao Wen equalitys
Chlorogenic acid in honeysuckle is extracted using 75% ethanol refluxing process, the yield of soaking reflux chlorogenic acid is 3.81% [Xiao Wen
Equality, Agriculture of Anhui science, 2011,39 (35):675-676].It is that raw material passes through 70% that Shu Xiaoshun etc., which once utilized lonicera hypoglauca miq flower,
80 DEG C of refluxing extraction chlorogenic acids of ethyl alcohol, yield up to 4.85% [the research and development of natural products such as Shu Xiaoshun, 2013,25
(10):1448-1451]。
Li Wanlin etc. has investigated Microwave Extraction condition, 300W, 60 DEG C of microwave 3min, and Chlorogenic Acid of Flos Lonicerae yield is reachable
5.16% [Chinese herb feed additive recent studies on, 2013,5 (35):35-38].Equally use 700W, 60 DEG C of microwave 5min In Flower Buds of Lonicera Japonica Thunb
The yield of Content of Chlorogenic Acid is up to 6.41% [.Sep.Purif.Technol. such as Zhang Bin, 2008,62 (2):480-483].
Liu Yamin etc. reports the technique of ultrasonic wave assisted extraction Honeysuckle flower Content of Chlorogenic Acid, upon extracting between be 50min, solid-liquid ratio (W/
V it is) 1:50, ultrasonic power 300W, under the conditions of ultrasonic frequency is 59KHz, chlorogenic acid yield has reached 8.9% [Liu Ya
Quick equal food industry science and technology, 2014,35 (1):186-190].Cao Yuan etc. compare steam distillation, ultrasonic extraction and
Alcohol extracting method extracts the quality of Chlorogenic Acid of Flos Lonicerae, and finally thinks that ultrasonic extraction is the best approach, and yield can reach
5.62% [treasure's traditional Chinese medical science traditional Chinese medicines, 2008,19 (12) when the such as Cao Yuan:2857-2858].In addition there are ultrahigh-pressure extraction method [Qin Xia
Equal hubei agricultural sciences, 2010,49 (9):215-217], a variety of extraction processes such as biological enzyme [the food industry such as military space virtue,
2012,33(4):70-71].Clock tinkling of pieces of jade etc. has once inquired into the supercritical CO of honeysuckle-leaf2Abstraction technique extracts in honeysuckle-leaf
The yield of volatile oil and chlorogenic acid, chlorogenic acid can reach 6.98% [Asia-Pacific the such as clock tinkling of pieces of jade traditional medicine, 2013,9 (2):43-46].
In conclusion in being to extract several extracting methods of raw material with honeysuckle plant, carried using microwave-ultrasonic wave added method
The yield highest (5.93%) of chlorogenic acid, ultrasonic extraction (5.62%) and microwave method (5.16%) is taken to take second place；And water extraction,
The chlorogenic acid yield of alcohol extracting method is only 4.8% and 3.81%.And using In Flower Buds of Lonicera Japonica Thunb as the yield of the chlorogenic acid of the microwave method of raw material
Highest (6.41%), hence it is evident that higher than alcohol extracting method (4.85%) and water extraction (4.8%) yield (the chemical reagent such as Sunyang,
2017,39(3):257-262)。
The method that Chinese patent also discloses that a variety of chlorogenic acid extractings from honeysuckle, including water extraction and alcohol precipitation method (Shen
Please number：CN201710713254.1；CN201010552254.6；CN97109109.9)；Microwave after alcohol impregnates, high pressure and super
Sound wave extraction method (application number：CN201510639356.4；CN201510158278.6；CN201410229487.0；
CN201310439267.6；CN201310550799.7；CN200510059798.8)；Extraction method (Shen again after cellulase processing
Please number：CN201410098541.2；CN201310057205.9；CN201010124495.0；CN201110284568.7)；From
Sub- liquid ultrasonic zymohydrolysis extracting method (application number：CN106631799A；CN201310232265.X)；Water carries-liquid-liquid extraction method (Shen
Please number：) and supercritical CO CN201010577116.32Extraction (application number：The extracting methods such as CN200510020265.9).
2, chlorogenic acid isolates and purifies
In the isolation and purification method of chlorogenic acid, Amberlyst process, membrane separation technique, cyclodextrin bonded gel media method,
High-speed countercurrent chromatography and Thin-layer chromatography have application.Gong Zhihua etc. compares NKA-2, D1400, and polyamide etc. is several
As a result kind macroporous absorbent resin shows that the adsorption effect of NKA-2 is best to the effect of Chlorogenic Acid of Flos Lonicerae adsorption and desorption,
After 80% ethyl alcohol is eluent, the purity of chlorogenic acid up to 45~53% [the analysis test journals such as Gong Zhihua, 2011,30
(1):85-90].Chen Yuan etc. optimizes the condition of D101 type macroreticular resins elution, and chlorogenic acid is obtained with 10% ethanol eluate
Purity highest, and the chlorogenic acid yield highest that 30% ethanol elution obtains.Further extracts and crystallize, green original by ethyl acetate
The purity of acid is up to 98.7% [the research and development of natural products such as Chen Yuan, 2014,26 (9):1503-1509].The ratios such as model distant view
Compared with different types of microfiltration membranes, ultrafiltration membrane and reverse osmosis membrane to the filtering feature of chlorogenic acid extracting solution and the influence of rejection,
The best rate of recovery of chlorogenic acid up to 67.8%, product purity up to 13.2% or more [the Food Sciences such as model distant view, 2010,31
(20):43-46].Wu is just very equal using water flux as index, compares two kinds of microfiltration membranes of MF1 and MF2, and two kinds of UF1 and UF2 are super
Two kinds of NF membranes of filter membrane and NF1 and NF2 finally make the purity of chlorogenic acid by 2.8% the filtering feature of chlorogenic acid extracting solution
27% [the just very equal Food Sciences of Wu, 2007,28 (11) are risen to:227-231].Zhang Yimin etc. uses oligomerization cyclodextrin key
The chlorogenic acid in gel column separating purification honeysuckle is closed, when with water:Ethyl alcohol:Acetic acid=85:10:5 mixed solutions, load capacity are
Isocratic elution purifying chlorogenic acid is carried out under 0.5mg/mL room temperature, recovery rate is up to 65%, and purity is up to 97% or more [Zhang Yimin
Equal process engineering journals, 2009,9 (5):970-974].
About the refined of chlorogenic acid, Chinese patent is mainly based on macroporous resin adsorption, wherein Chinese patent (application
Number：CN107056615A；CN201410795415.2；CN201410080316.6；CN201310412781.0；
CN201310390214.X；CN201310390213.5；CN201310179170.6；CN201210453316.7；
CN201110292698.5；CN201110288112.8；CN201010196913.7；CN200910116384.2；
CN200810124130.0；CN02133448.X；CN02133448.X it) individually discloses after alcohol extracting in the plants such as honeysuckle
It is detached using macroporous absorbent resin and reverse phase absorption resin adsorption (application number：CN201410149292.5), Ultra filtration membrane
(application number：CN201110361835.6 purifying process).Then polyamide, silica gel column chromatography, mesolow, half are further utilized
It prepares liquid phase separation and purifies (application number：CN201310390213.5；CN201510498084.0；CN201310714715.9；
CN201310390214.X；CN1793105A；CN200410035758.5), extraction and continuous countercurrent extraction and recrystallization etc.
Method (application number：CN200910115669.4；CN200910047915.7；CN200910047915.7；
CN200910029959.7；CN01144170.4 the method for) obtaining high-purity chlorogenic acid monomer.A kind of aluminium is utilized in addition, also having
Salt precipitation prepares the report (application number of high-purity chlorogenic acid：CN201210157091.0).
Invention content
Purpose of the present invention is to solve during chlorogenic acid isolates and purifies using Amberlyst process, cyclodextrin bonded gel media
The post separations method such as method, high-speed countercurrent chromatography and Thin-layer chromatography is time-consuming and laborious, and purifying is of high cost, and organic solvent is used
Measure the problems such as big.It is raw material, including flower that the present invention, which provides a kind of aerial part using woodbine honeysuckle, Honeysuckle flower,
Flower bud, flower, leaf and stem, the method for extracting and preparing high-purity chlorogenic acid.This method utilizes supercritical technology or ultrasonic wave extraction skill
The extract of art is cleaned by the means of ethyl acetate extraction, alkalization precipitation, can be direct using acidification ethyl acetate extraction
So that the purity of chlorogenic acid is reached 50% or more, simplifies chlorogenic acid and isolate and purify process, it further can be with by concentrated crystallization
High-purity chlorogenic acid crystal is directly precipitated.
Technical scheme of the present invention
A method of high-purity chlorogenic acid being prepared by raw material of woodbine, this method is original using woodbine
Material, using ultrasonic wave extraction or supercritical CO2Extracting process extracts the chlorogenic acid in woodbine, is extracted by ethyl acetate
It takes, the means removal of impurities for the precipitation that alkalizes, carrys out the chlorogenic acid in extract-enriched using acidizing extraction, further pass through condensing crystallizing
Prepare high-purity chlorogenic acid crystal.
The woodbine is the aerial part of honeysuckle and Honeysuckle flower, specifically includes bud, flower, leaf and stem.
The supercritical CO2Extracting process is, using the broken powder of woodbine as raw material, can be connect on the technology
20%~30% ethyl alcohol of use (preferably 20%) received is used as entrainer, 100~150bar of pressure limit (150bar) best to extract
Take temperature be 35~55 DEG C (preferably 45.3 DEG C), flow velocity be 4~12mL/min (preferably 9.29mL/min), extraction time 0.5~
The supercritical CO of 1h (preferably 0.5h)2Extracting process extracts the chlorogenic acid in woodbine, organic molten through removal is concentrated under reduced pressure
Chlorogenic acid extracting solution is obtained after agent.
The supersonic extracting method is, using the broken powder of woodbine as raw material, is subjected on the technology
70% or more use supersonic extracting method of the ethanol solution as Extraction solvent, extract the green original in woodbine
Acid obtains chlorogenic acid extracting solution after being concentrated under reduced pressure and removing organic solvent.
The removal of impurities means of the chlorogenic acid extracting solution are, for above-mentioned supercritical CO2Extracting process or ultrasonic wave extraction side
The chlorogenic acid extracting solution that method obtains, first adopts and is extracted with ethyl acetate, and removes ethyl acetate soluble impurity, then by adding alkali to adjust
After pH, centrifugation or filtering removal precipitation obtain refined extracting solution.
The extracting solution adds alkali to adjust pH, and sodium hydroxide, potassium hydroxide or calcium hydroxide solution adjustment pH value, pH can be used
Ranging from pH9~11, preferably pH are 10.
The method for the chlorogenic acid that the acidizing extraction comes in extract-enriched is, for refined extracting solution obtained above,
PH value, ranging from pH1~3 pH, wherein it is preferred that pH2 are adjusted by using hydrochloric acid or sulfuric acid solution；Volume ratio (V is used again:V)1:
1 ethyl acetate extracts repeatedly, and extraction times are 3~5, and preferably 4 times, combined ethyl acetate extract liquor obtains chlorogenic acid enrichment
Liquid.
The condensing crystallizing process is to be concentrated into above-described chlorogenic acid ethyl acetate pregnant solution by being concentrated under reduced pressure
3~5 times of amount (M of mass volume ratio are added in dried powder:V methanol) or ethyl alcohol dissolving add 8~10 times of amount (V of volume ratio:
V dichloromethane low temperature) is stood overnight, and high-purity chlorogenic acid crystal is directly precipitated.
High-purity chlorogenic acid crystal prepared by the method for the present invention, can be used for the intermediate and standard items of biological medicine.
The advantages of the present invention：
It using woodbine cauline leaf and bud is that raw material extracts and prepare high-purity chlorogenic acid that the present invention, which provides a kind of,
Method.This method passes through ethyl acetate extraction, alkali with supercritical technology or the chlorogenic acid extracting crude extract of ultrasonic wave extraction
The means removal of impurities for changing precipitation can directly obtain chlorogenic acid of the purity 50% or more using acidification ethyl acetate extraction, into
99.5% or more high-purity chlorogenic acid crystal can directly be precipitated by concentrated crystallization for one step.This method equipment requirement letter
Single, the column chromatography during simplifying and being isolated and purified instead of chlorogenic acid, the techniques such as membrane filtration are extracted by soda acid.This
It is extensive to invent the raw material sources used, it is easy to operate, prepared chlorogenic acid high-purity, the valence with extensive industrial applications
Value.
Description of the drawings
Fig. 1 is the flow chart that high-purity chlorogenic acid is prepared using woodbine as raw material.
Fig. 2 is the Pareto chart of Plackett-Burman designs.
Fig. 3 is steepest hill climbing test temperature, flow velocity and chlorogenic acid mass fraction relationship, and wherein A is temperature, and B is flow velocity.
Fig. 4 is supercritical extraction process response surface optimization.
Fig. 5 is the investigation of the best PH of alkali impurity removal process.
Fig. 6 is the investigation of the best PH of acid extraction technique, and wherein A is HPLC test maps, and B is chlorogenic acid content column figure.
Fig. 7 is the investigation of acid extraction technique extraction times, and wherein A is HPLC test maps, and B is chlorogenic acid content column figure.
Fig. 8 is the HPLC test maps of the chlorogenic acid content of ethyl acetate layer extract.
Fig. 9 is the microphoto of chlorogenic acid crystallization.
Figure 10 is the HPLC test maps of technique final products.
Specific implementation mode
The investigation and implementation of 1. supercritical CO 2 extraction technology of embodiment
Due to the unstability of chlorogenic acid, when extraction, is unable to high temperature, strong light and long-time heating, therefore supercritical fluid extracts
It is a kind of ideal method to follow the example of.The present invention utilizes orthogonal experiment and sound using the recovery rate of chlorogenic acid as index
Face design method is answered to optimize the process conditions of supercritical fluid extraction, with the woodbine gold of Hebei province Julu County plantation
As raw material, 50 DEG C dry 12 hours the branches and leaves of honeysuckle flower (Lonicerajaponica Thunb.), are crushed with high speed disintegrator,
And cross 20 mesh sieve.Chlorogenic acid content therein, instrument are surveyed using HPLC methods：Shimadzu high performance liquid chromatograph LC-15C is furnished with two
First pump, SPD-15C UV detector, CTO-15C column ovens, LC-Solution 15C work stations, Agilent Eclipse
Plus C18 chromatographic columns (4.6 × 100mm, 3.5 μm).Chromatographic condition：Mobile phase A:0.1% formic acid water, Mobile phase B:Acetonitrile.When
Between program:B phases are set as：0.01-10min, 2-8%；10-15min, 8%；15-20min, 8-10%；20-25min, 10-
15%；25-35min, 15-20%；35-50min, 20-100%；50-55min, 100-2%；55-60min, 2%.Flow velocity is
1mL/min, column temperature are 30 DEG C, Detection wavelength 327nm.
The present embodiment supercritical CO 2 extraction technology uses Waters Investigator Analytical/
Semi-Prep SFC repackings are furnished with main pump and the auxiliary pump of solvent, fraction collection system, interpolation pipe carbon dioxide steel cylinder, titanium dioxide
Carbon steel bottle pumps and the equipment of extraction kettle, carbon dioxide supercritical extraction chlorogenic acid.Five factors, two level is used first
Plackett-Burman contrived experiments select two levels (being shown in Table 1) of suitable height for each factor, have investigated dioxy
Change the major influence factors of carbon supercritical extract.Wherein, entrainer chooses 70% ethyl alcohol, using Minitab17 come contrived experiment
(being shown in Table 2).
The selection of 1. each factor level value of table
Table 2.Plackett-Burman experimental designs and chlorogenic acid content measurement result
By Minitab17 analysis factor design results, obtain the Pareto chart of factor contribution margin (see Fig. 2).As shown in Figure 2
It is temperature and flow velocity respectively to contribute maximum two factors to chlorogenic acid yield, and it is excellent next to carry out technique to the two factors
Change and investigates.
And by steepest hill climbing test come the central point (see Fig. 3) of this two factor of temperature and flow velocity, by other three
Factor selects level value appropriate：Pressure 150bar, entrainer ratio 20%, extraction time 0.5h.Different temperature is respectively set
Degree and flow velocity, the central point (being shown in Table 3) of each factor is determined in conjunction with Minitab response surface experimental designs, investigates its optimised process
Condition.
3 response surface design factor level value of table
The results are shown in Table 4, draws response surface figure (see Fig. 4) according to response surface design and experimental result, determines super face
It is 45.3 DEG C that boundary, which extracts best temperature, flow velocity 9.29mL/min.
4 response surface of table designs and experimental result
With reference to the optimum condition of above-mentioned response surface, the powder 10g of 20 mesh sieve, supercritical carbon dioxide extraction are taken after crushing and crossed
The temperature taken is set as 45 DEG C, and using 20% ethyl alcohol as entrainer, flow velocity 9mL/min is carried out by the above-mentioned optimised process that obtains
Chlorogenic acid extracts.Medicinal extract is concentrated and dried to obtain after being enriched with to chlorogenic acid, after combining extraction liquid, using in above-described embodiment 1
HPLC methods, detect chlorogenic acid content therein be 6.7%, it is almost the same with theoretical value (6.8%).
The extraction of 2. supercritical ultrasonics technology Chlorogenic Acid of Flos Lonicerae of embodiment
Using Guangxi Xincheng County produce Honeysuckle flower Lonicera hypoglauca Miq. cauline leaf as Honeysuckle flower raw material,
For raw material, its powder 10g is taken, is added 1:70% ethanol solution of 30 (W/V) volume ratios, ultrasonic power 300W, ultrasound
Wave frequency rate is 60KHz, and ultrasonic extraction 30 minutes is concentrated under reduced pressure to obtain chlorogenic acid extract 1.95g, and uses above-mentioned implementation after filtering
HPLC methods in example 1 detect chlorogenic acid content therein can be to 5.9%.
The investigation of 3. alkali edulcoration purification technique of embodiment
The chlorogenic acid extraction medicinal extract 0.76g that 2 supercritical ultrasonics technology of above-described embodiment obtains is taken, 30mL deionized waters, ultrasound is added
It is set fully to dissolve within 30 minutes.It is extracted repeatedly with monoploid product ethyl acetate respectively, until ethyl acetate layer achromaticity and clarification
(totally 5 times).Water intaking layer, is divided into 6 parts, adjusts PH6~11 with NaOH respectively, 3000rpm centrifugations 10 after staticly settling 3 hours
Minute, obtained insoluble matter precipitation, which is dried, weighs, and investigates alkali dust removal rate.The results are shown in Figure 5, confirms alkali impurity removal process
Best PH be 10.
The investigation of 4. acid extraction recovery process of embodiment
The chlorogenic acid extraction medicinal extract 0.76g that 2 supercritical ultrasonics technology of above-described embodiment obtains is taken again, repeats the alkali of above-described embodiment 3
Clean optimised process, takes the water layer supernatant solution after centrifugation, is equally divided into 6 parts, respectively with dense HCL tune PH1~6, is used in combination one times
The ethyl acetate of volume is extracted.It collects ethyl acetate extract layer to be spin-dried for, wherein 1mg powder is taken to be dissolved in 1mL70% first respectively
In alcohol, the measurement of chlorogenic acid content is carried out using HPLC methods.The results are shown in Figure 6, and the best PH of acid extraction recovery process should be big
In 2.
Above-mentioned process conditions are repeated, after PH is adjusted to 2, is repeatedly extracted using monoploid product ethyl acetate, is taken respectively
Each ethyl acetate layer solution 1mL is added 70% methanol 1mL dissolvings, the content of its chlorogenic acid is detected with HPLC methods, is examined after being spin-dried for
Examine best extraction times.The results are shown in Figure 7, and after continuous extraction 4 times, the rate of recovery of chlorogenic acid can reach 90% or more (figure
7), it is thus determined that extraction times should be greater than 4 times.Merge above-mentioned ethyl acetate layer extract liquor, solid powder is obtained in concentrate drying
61.5mg measures its Content of Chlorogenic Acid and reaches 52.3%.
The implementation of 5. abstraction impurity removal purifying process of embodiment
The medicinal extract for taking 1 supercritical extract of above-described embodiment to obtain is added 1:30 times of (M:V deionized water), ultrasonic 30min
It is set fully to dissolve.It is extracted respectively 5 times with 1 times of volume of ethylacetate, removal ethyl acetate soluble part.Water intaking layer, respectively
PH6~11 is adjusted with KOH and adjusts PH 10, is stood overnight, is filtered to remove precipitation.Supernatant is taken, PH 2 is adjusted with dense HCL, with 1 times
The ethyl acetate of volume distinguishes 4 extractions.Combined ethyl acetate layer is concentrated under reduced pressure drying, obtains solid powder.It is quantitative through HPLC
It tests and analyzes (Fig. 8), it is 51.5% to calculate solid Content of Chlorogenic Acid mass fraction., compared with the chlorogenic acid content in medicinal material, yield
It is 60.2%, reaches purification effect.
6. crystal refining of embodiment purifies
According to chlorogenic acid in different solvents different solubility, using crystallization processes to above-described embodiment crude extract carry out essence
System.Above-described embodiment 2 is repeated, the medicinal extract 1.0g for taking ultrasonic wave extraction to obtain is added 1:30 times of (M:V deionized water), ultrasound
30min makes it fully dissolve.It is extracted respectively 5 times with 1 times of volume of ethylacetate, removal ethyl acetate soluble part.Water intaking layer,
PH to pH 10 is adjusted with CaOH respectively, stands overnight, is filtered to remove precipitation.Supernatant is taken, PH 2 is adjusted with the sulfuric acid of 2M, then with 1
The ethyl acetate of times volume distinguishes 4 extractions.Combined ethyl acetate layer is concentrated under reduced pressure drying, obtains solid powder 85.6mg.Essence
The close crude extract powder 60.0mg for weighing above-described embodiment 6 and obtaining is added a small amount of 200 μ L methanol and makes it dissolve, adds 8 times of bodies
Long-pending dichloromethane 2mL stands for 24 hours at 4 DEG C, high-purity chlorogenic acid crystal (Fig. 9) is precipitated.Through HPLC content detections, green original
Acid content is 99.7%.
The implementation of 7. extraction purification process for refining of embodiment
The bud dried powder 30g (crossing 20 mesh sieve) for taking the close honeysuckle flower (Lonicerajaponica Thunb.) in Henan, is pressed
Total technological process (Fig. 1), is extracted using supercritical carbon dioxide, and optimised process is obtained by above-mentioned, and temperature is set as 45 DEG C,
Using 20% ethyl alcohol as entrainer, flow velocity 9mL/min, pressure is set as 150bar.After being enriched with to chlorogenic acid, merge extraction
It is concentrated and dried to obtain medicinal extract 2.76g after taking liquid.Distilled water 50mL, ultrasound is added in medicinal extract 2.5g after taking supercritical extract to concentrate
30min dispersing and dissolvings.The ethyl acetate accumulated with 50mL monoploids extracts 5 times to ethyl acetate layer clarification respectively.Take remaining water
Layer adjusts PH10 with NaOH, and 3000rpm is centrifuged 15 minutes and removed precipitation after standing 5 hours.Supernatant water phase is gone, with dense HCL tune
PH1.5, then respectively with 1 times of volume of ethylacetate continuous extraction 4 times, combined ethyl acetate extract liquor, concentrate drying obtains solid
Powder 219.2mg.Above-mentioned solid powder 200mg is taken, the dissolving of 1mL absolute ethyl alcohols is added, adds dichloromethane 10mL, 4 DEG C quiet
It sets and precipitates crystal 36.7mg overnight.
HPLC methods are used to measure its chlorogenic acid content for 99.9%, such as Figure 10.A small amount of above-mentioned chlorogenic acid product is taken, with X5 types
Micro melting point apparatus surveys fusing point, and product fusing point is 204.8 DEG C, essentially identical with 205.4 DEG C of chlorogenic acid standard items fusing point.
Claims (10)
1. a kind of method preparing high-purity chlorogenic acid as raw material using woodbine, it is characterised in that：Utilize woodbine
For raw material, using ultrasonic wave extraction or supercritical CO2Extracting process extracts the chlorogenic acid in woodbine, passes through ethyl acetate
Extraction, the means removal of impurities of alkalization precipitation, carry out the chlorogenic acid in extract-enriched using acidizing extraction, are further tied by concentrating
Crystalline substance prepares high-purity chlorogenic acid crystal.
2. the method according to claim 1 for preparing high-purity chlorogenic acid as raw material using woodbine, it is characterised in that：
The woodbine is the aerial part of honeysuckle and Honeysuckle flower, specifically includes bud, flower, leaf and stem.
3. the method according to claim 1 for preparing high-purity chlorogenic acid as raw material using woodbine, it is characterised in that：
The supercritical CO2Extracting process is, using the broken powder of woodbine as raw material, acceptable use on the technology
20%~30% ethyl alcohol is as entrainer, 100~150bar of pressure limit, and best extraction temperature is 35~55 DEG C, and flow velocity is 4~
12mL/min, the supercritical CO of 0.5~1h of extraction time2Extracting process extracts the chlorogenic acid in woodbine, dense through depressurizing
Chlorogenic acid extracting solution is obtained after contracting removal organic solvent.
4. the method according to claim 1 for preparing high-purity chlorogenic acid as raw material using woodbine, it is characterised in that：
The supersonic extracting method is, using the broken powder of woodbine as raw material, acceptable use on the technology
Supersonic extracting method of 70% or more the ethanol solution as Extraction solvent extracts the chlorogenic acid in woodbine, through subtracting
Chlorogenic acid extracting solution is obtained after pressure concentration removal organic solvent.
5. being existed according to claim 3 or 4 methods for preparing high-purity chlorogenic acid as raw material using woodbine, feature
In：The removal of impurities means of the chlorogenic acid extracting solution are, for the chlorogenic acid extracting solution of the claims 3 or 4, first use acetic acid
Ethyl ester extract, remove ethyl acetate soluble impurity, then by add alkali adjust pH after, centrifugation or filtering removal precipitation, obtain essence
Extracting solution processed.
6. the method according to claim 5 for preparing high-purity chlorogenic acid as raw material using woodbine, it is characterised in that：
The extracting solution adds alkali to adjust pH, sodium hydroxide, potassium hydroxide or calcium hydroxide solution adjustment pH value can be used, pH is ranging from
PH9~11.
7. the method for preparing high-purity chlorogenic acid as raw material using woodbine according to claim 6, it is characterised in that：Institute
It is 10 to state pH.
8. the method for preparing high-purity chlorogenic acid as raw material using woodbine according to claim 5, it is characterised in that：Institute
The method for stating the chlorogenic acid that acidizing extraction comes in extract-enriched is, for the refined extracting solution that the claims 5 obtain, to lead to
It crosses using hydrochloric acid or sulfuric acid solution adjustment pH value, ranging from pH1~3 pH, wherein it is preferred that pH2；Volume ratio (V is used again:V)1:1
Ethyl acetate extract repeatedly, extraction times be 3~5, preferably 4 times, combined ethyl acetate extract liquor obtains chlorogenic acid pregnant solution.
9. the method according to claim 1 for preparing high-purity chlorogenic acid as raw material using woodbine, it is characterised in that：
The condensing crystallizing process is to be concentrated to dryness chlorogenic acid ethyl acetate pregnant solution according to any one of claims 8 by being concentrated under reduced pressure
3~5 times of amount (M of mass volume ratio are added in dry powder:V methanol) or ethyl alcohol dissolving add 8~10 times of amount (V of volume ratio:V)
Dichloromethane low temperature stand overnight, directly be precipitated high-purity chlorogenic acid crystal.
10. the application of high-purity chlorogenic acid crystal prepared by claim 1 the method, it is characterised in that：Prepared is high-purity
Degree chlorogenic acid crystal can be used for the intermediate and standard items of biological medicine.
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