CN1687008A - Method for extracting compound of phenolic acid from leaves of woodbind - Google Patents
Method for extracting compound of phenolic acid from leaves of woodbind Download PDFInfo
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- CN1687008A CN1687008A CN 200510038498 CN200510038498A CN1687008A CN 1687008 A CN1687008 A CN 1687008A CN 200510038498 CN200510038498 CN 200510038498 CN 200510038498 A CN200510038498 A CN 200510038498A CN 1687008 A CN1687008 A CN 1687008A
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Abstract
The preparation method of chlorogenic acid and isochlorogenic acid compound uses leaf of lonicera macranthoides as raw material and adopts the following steps: defatting, using ethyl alcohol with a certain concentration to make extraction, concentrating ethyl alcohol extract, feeding the concentrated ethyl alcohol extract into macroporous resin column, eluting by using 0-10% ethyl alcohol to obtain chlorogenic acid and its isomer, then eluting with 20-35% ethyl alcohol to obtain isochlorogenic acid and its isomer; mixing then according to different ratio and making treatment so as to obtain total phenelic acid, its content can be up to 80%.
Description
Technical field
The invention belongs to from leaves of woodbind the method for extracting compound of phenolic acid, be specially that extraction separation has the chlorogenic acid of strong Green Tea Extract, resistance of oxidation effect and the method for isochlorogenic acid from leaves of woodbind.
Background technology
Chlorogenic acid in the Japanese Honeysuckle and isochlorogenic acid (DCQ or two caffeoyl quinic acids) and derivative thereof all have antivirus action (King PJ, Ma G, Miao W, et al.Structure-activity relationships:analogues of thedicaffeoylquinic and dicaffeoyltartaric acids as potent inhibitors of humanimmunodeficiency virus type 1 intergerase and repiciation[J] J.Med Chem, 1999,42 (3): 497-509), has stronger anti-radical action in addition, Heilmann and Weiss etc. utilize the human neutrophil to stimulate with zymosan or FMLP and produce respiratory burst, detect the generation situation of oxyradical, found that DCQ has the very strong ability of catching superoxide anion and hydroxy radical qiao (Heilmann J, Merfort T, Weiss M.Radical scavenger activityof different 3,4-dihydroxyflavonols and 1,5-dicaffeyolquinic acid studied byInhibition of chemiluninescence[J] .Planta Med, 1995,61 (5): 435-438; Fraga CG, Martino VS, Ferraro GE, et al.Flavonoids as antioxidants evaluated by in vitro andin situ liver chemiluninescence[J] .Biochem Pharmacol, 1987,36 (5): 717-720.).
The research document of the chlorogenic acid of extraction separation or total phenolic acid is a lot of from Japanese Honeysuckle.As: CN1439410A extracts method antibiotic, the antiviral activity position from Japanese Honeysuckle, by water alcohol method, macroporous adsorptive resins wash-out, polyamide column wash-out obtain honey suckle active partly.For another example: the technology of CN1199728 chlorogenic acid extracting from Japanese Honeysuckle, Japanese Honeysuckle is added acidic ethanol soak, heating and refluxing extraction, the dregs of a decoction add the acidic ethanol refluxing extraction again.Because chlorogenic acid is more stable under acidic conditions.
Woodbine has largeflower-like honeysuckle flower, lonicera hypoglauca miq (L.hypoglauca), Lonicera confusa DC. (L.confusa), L. similis Hemsl (L.similes), hair style honeysuckle (L.dasystyla), flower bud of Coralline Honeysuckle (L.chrysantha) etc.Largeflower-like honeysuckle flower is the dry flower of woodbine L.macranthoieds Hand.-Mazz..The resource of leaves of woodbind is horn of plenty and cheap more, though also contain chlorogenic acid and isochlorogenic acid in the leaves of woodbind, but, influence the extraction separation of chlorogenic acid and isochlorogenic acid owing to contain plurality of impurities such as a large amount of greases, tannin, polysaccharide, protein, chlorophyll.
Summary of the invention
The technical problem to be solved in the present invention is how to study from the resource rational utilization angle, utilizes the more leaf of horn of plenty and cheap woodbine of resource, but the method that the industrialization of extraction separation chlorogenic acid and isochlorogenic acid is used.And research is through once extracting, separate simultaneously the method that obtains chlorogenic acid and isochlorogenic acid.This extraction and separation method can make chlorogenic acid and isochlorogenic acid structure, drug effect keep stable, improves content and yield.
For addressing the above problem, it is as follows to the invention provides technical scheme.
The preparation method of a kind of chlorogenic acid and isochlorogenic acid is characterized in that comprising the steps:
A) getting leaves of woodbind is raw material, carries out degreasing with low polar solvent;
B) get leaf after the degreasing of step a), use extraction using alcohol, ethanol extract;
C) ethanol extract enrichment step b) gets concentrated solution;
D) get the concentrated solution of step c), last macroporous resin column, earlier with 0～10% ethanol elution, collection 0～10% ethanol eluate; Use 20～40% ethanol elutions again, collect 20～40% ethanol eluates;
E) get 0～10% ethanol eluate of step d), after concentrating, adding ethanol earlier is 45～60% to containing the alcohol amount, removes precipitation, supernatant concentration, adding ethanol again is 75～85% to containing the alcohol amount, removes precipitation, gets supernatant concentration, the chlorogenic acid crude product;
F) get 20～40% ethanol eluates of step d), be concentrated into do not have the alcohol flavor after, transfer pH to 2～3 with hydrochloric acid, use ethyl acetate extraction, concentrate acetic acid ethyl acetate extract, get the isochlorogenic acid crude product.
Raw materials used grayer-hair honeysuckle leaf, leaf of Lonicera hypoglauca Miq., Lonicera confusa DC. leaf, the L. similis Hemsl leaf of being selected from this preparation method's the step a); Low polar solvent is selected from sherwood oil, gasoline, benzene, and degreasing time is no less than 2 hours; In the step b) with 60～80% alcohol reflux; The concentrated solution of step c) does not have the alcohol flavor; The macroporous resin of step d) is selected from D101, AB-8, HPD100, HPD300, D1 or D2.
In the preferable methods, raw materials used grayer-hair honeysuckle leaf, the leaf of Lonicera hypoglauca Miq. of being selected from the step a); Be meant with the sherwood oil backflow after 2～10 hours filtering sherwood oil liquid with petroleum ether degreasing; In the step b) with 70～75% alcohol reflux.
Macroporous resin column on the step d) among the above-mentioned preparation method earlier with 5～10% ethanol elutions, is collected 5～10% ethanol eluates; Use 30～35% ethanol elutions again, collect 30～35% ethanol eluates.After the ethanol eluate of step e) concentrated, adding ethanol was removed precipitation to containing alcohol amount 50% earlier, and supernatant concentration is to relative density 1.06～1.18, and adding ethanol again is 80% to containing the alcohol amount, removes precipitation, gets supernatant concentration, gets the chlorogenic acid crude product.
Among the described preparation method, get the chlorogenic acid crude product of step e), use the 0-10% dissolve with ethanol, remove the water liquid behind the ethanol, transfer pH2～3 with concentrated hydrochloric acid after, use ethyl acetate extraction, concentrate acetic acid ethyl acetate extract, chlorogenic acid.
Among the aforementioned preparation method, get the chlorogenic acid crude product of step e), after merging with the isochlorogenic acid of step f), earlier with 50～80% dissolve with ethanol, after concentrating, with behind concentrated hydrochloric acid accent pH to 2～3, use ethyl acetate extraction again, with the extraction liquid activated carbon decolorizing, concentrate, obtain total phenolic acid.
More preferably among the preparation method, get the chlorogenic acid crude product of step e), use the 0-10% dissolve with ethanol, remove the water liquid behind the ethanol, transfer pH2～3 with concentrated hydrochloric acid after, use ethyl acetate extraction, concentrate acetic acid ethyl acetate extract, chlorogenic acid, after merging with the isochlorogenic acid of step f) again, earlier with 60～70% dissolve with ethanol, after concentrating, again with behind concentrated hydrochloric acid accent pH to 2～3, use ethyl acetate extraction, with the extraction liquid activated carbon decolorizing, concentrate, obtain total phenolic acid.
Leaves of woodbind is grayer-hair honeysuckle leaf for example, behind extraction using alcohol, concentrates, and through D101 macroporous resin and other model such as AB-8, HPD100, HPD300, D1 or D2, water and 10% ethanol elution are perhaps directly used 10% ethanol elution.Water intaking and 10% ethanol elution part or directly use 10% ethanol elution part, concentrate as near do not have the alcohol flavor after, with 50% and 80% ethanol secondary alcohol precipitation, get the chlorogenic acid crude product; Use 35% ethanol elution, collect 35% ethanol eluate, concentrate as near do not have the alcohol flavor after, transfer PH to 2 with concentrated hydrochloric acid, ethyl acetate extraction, with extraction liquid concentrate the isochlorogenic acid crude product.Chlorogenic acid crude product and isochlorogenic acid crude product can be distinguished purifying.After also this two parts merging can being concentrated, transfer PH to 2 with concentrated hydrochloric acid again, ethyl acetate extraction purifying and refining can get total phenolic acid part, and content reaches more than 80%.Chlorogenic acid isomer 3-caffeoyl quinic acid in the extract: 5-caffeoyl quinic acid: the content ratio between the 4-caffeoyl quinic acid is about 4.92～6.23: 0.85～1.23: 1; Isochlorogenic acid isomer 3, the two caffeoyl quinic acids of 5-, 3, the two caffeoyl quinic acids and 4 of 4-, the content ratio between the two caffeoyl quinic acids of 5-is about 0.19～0.48: 1.6～2.5: 1.
The present invention is from the resource rational utilization angle, utilize the more leaf of the woodbine of horn of plenty of resource, as leaf, the especially grayer-hair honeysuckle leaf of grayer-hair honeysuckle leaf and lonicera hypoglauca miq, Lonicera confusa DC., L. similis Hemsl, extraction separation chlorogenic acid and isochlorogenic acid.From economic benefit, the cost of material of leaf is well below the price of bud, and the present invention has significantly reduced production cost.
And the present invention once extracts, separates simultaneously and obtain chlorogenic acid and isochlorogenic acid, can be used for disposable extraction separation chlorogenic acid and isochlorogenic acid.The present invention is the preparation technology that the index composition is studied total phenolic acid with chlorogenic acid, isochlorogenic acid, and chlorogenic acid, isochlorogenic acid can be carried out quality control as intermediate.
The present invention adopts the good separating effect of macroporous adsorbent resin to carry out enrichment, and handle its purifying and refining in conjunction with alcohol precipitation and acidic conditions, HPLC detects monomer content, the total phenolic content of spectrophotometry obtains at last that the monomer chlorogenic acid content can reach more than 70% in the high-purity chlorogenic acid prepared product; The monomer chlorogenic acid content can reach more than 50% in total phenolic acid prepared product, and total phenolic content can reach more than 80%.And utilize angle from the content and the reasonable resources of target product, carried out preferably extracting medicinal material.This extraction and separation method significantly improves the content of chlorogenic acid and isochlorogenic acid and yield, makes chlorogenic acid and isochlorogenic acid keep stable.This method technological operation is simple, and convenient, cost is low, no hazardous solvent, and the production security height is fit to suitability for industrialized production.
Description of drawings
Fig. 1: A: the liquid chromatogram of water elution liquid
Fig. 2: B: the liquid chromatogram of macroporous resin 10% ethanol eluate
Fig. 3: C: the liquid chromatogram of macroporous resin 20% ethanol eluate
Fig. 4: D: the liquid chromatogram of macroporous resin 35% ethanol eluate
Fig. 5: E: the liquid chromatogram of total phenolic acid part
Respectively identify the implication at peak among the above-mentioned figure: 1-chlorogenic acid (3-caffeoyl quinic acid); 2-chlorogenic acid isomer; 3-chlorogenic acid isomer 4. isochlorogenic acid a; 5-isochlorogenic acid b, 6 isochlorogenic acid c; 7-feruloylquinic acid class; 8-feruloylquinic acid class
Embodiment
Embodiment one
1 content assaying method
Instrument and reagent high performance liquid chromatograph Aglientl100 series, binary pump G1312A, automatic sampler, diode-array detector, Chemstation is chem workstation A.06.01, chromatographic column: Aglient Zorbax 80A, Extend-C18 (4.6mm * 250mm, 5cm), experimental water (Robust, Guangzhou food-drink company limited), acetonitrile is a chromatographically pure, and phosphoric acid is analytical pure.
Chromatographic condition moving phase: 0.2% phosphoric acid solution-acetonitrile (88: 12), keep 0-12min, change to (83: 17) at 12-15min moving phase proportional linearity, 15-30min keeps ratio (83: 17); Change to (50: 50) in the 30-60min linearity.Flow velocity is 1.0mLmin
-1, the detection wavelength is 327nm, 30 ℃ of column temperatures.
The selection of 2 medicinal materials
The all available the inventive method extraction separation of the leaf compound of phenolic acid of largeflower-like honeysuckle flower, lonicera hypoglauca miq (L.hypoglauca), Lonicera confusa DC. (L.confusa), L. similis Hemsl (L.similes), hair style honeysuckle (L.dasystyla), flower bud of Coralline Honeysuckle (L.chrysantha) in the woodbine.Wherein most preferred honeysuckle, the leaf of plant was the leaf of largeflower-like honeysuckle flower, because of wherein phenolic acid compound content is higher.The results are shown in following table.
Table 1 Japanese Honeysuckle chlorogenic acid content not of the same race compares
Sequence number sample chlorogenic acid content
1 honeysuckle bud 2.24%
2 lonicera hypoglauca miq buds 2.93%
3 Lonicera confusa DC. buds 3.36%
4 largeflower-like honeysuckle flower buds 7.44%
5 grayer-hair honeysuckle leafs 4.94%
6 largeflower-like honeysuckle flower stems 0.95%
Show that from the chlorogenic acid contents measurement result amount that is contained in the largeflower-like honeysuckle flower bud is obviously than the honeysuckle height, and for the largeflower-like honeysuckle flower different sites, the amount that is contained in the leaf is 4.94%, apparently higher than the honeysuckle bud, content is low in its stem.
So preferably use the dry leave of the woodbine largeflower-like honeysuckle flower L.macranthoieds Hand.-Mazz. in the Xiushan Mountain, Chongqing City county, the properties and characteristics of medicinal material, powder characteristics, the physics and chemistry diagnostic characteristics all meets the content under Chinese Plants will " largeflower-like honeysuckle flower " item.
3, separation purifying technique
After extracting solution suitably concentrates, the macroporous resin segmentation, distinguish water successively, 10% alcohol, 20% alcohol, 35% alcohol, 40%, 50% pure wash-out is collected every part elutriant, is settled to the 100ml volumetric flask, high performance liquid phase check and analysis result shows that chlorogenic acid mainly concentrates in water and 10% alcohol (i.e. 0～10% ethanol) elutriant, and isochlorogenic acid mainly concentrates in ethanol (the not comprising 10% ethanol) elutriant more than 10%, and with the most of wash-out of isochlorogenic acid, wash-out is complete during to 40% ethanol until 35% ethanol eluate.Easy and simple to handle for suitability for industrialized production, can preferably directly use 10% ethanol elution chlorogenic acid, get the chlorogenic acid crude product behind the concentrate eluant; And then directly use 35% ethanol elution isochlorogenic acid, concentrate eluant gets the isochlorogenic acid crude product.
The purifying of chlorogenic acid: in 10% ethanol eluate of macroporous resin column enrichment chlorogenic acid, but impurity such as more polysaccharide, protein are arranged also simultaneously.So carry out the alcohol precipitation experiment, improve chlorogenic acid purity.Take the secondary alcohol deposition method, after elutriant is concentrated, add ethanol for the first time and make that to contain the alcohol amount be 45～60% to carry out alcohol precipitation, the alcohol amount of preferably containing reaches 50% concentration and carries out alcohol precipitation, leave standstill 24 hours, centrifugal, get supernatant liquor, after the supernatant concentration, adding for the second time ethanol is 75～85% to carry out alcohol precipitation to containing the alcohol amount, preferably contains the alcohol amount and reaches 80% concentration and carry out alcohol precipitation, centrifugal, get supernatant liquor, supernatant concentration.Concentrated solution is transferred PH2～3 with concentrated hydrochloric acid, and ethyl acetate extraction 3 times promptly.Chlorogenic acid content can reach more than 70%.
The purifying of isochlorogenic acid: after 35% ethanol eluate of macroporous resin column concentrates, transfer PH to 2,, extraction liquid is merged with the ethyl acetate extraction of 2 times of amounts 3 times with concentrated hydrochloric acid, concentrated.Mother liquor is used n-butanol extraction again, finds that the compound of phenolic acid content in the butanol extraction liquid is very low, illustrates that ethyl acetate extraction can compare for 3 times fully.After acetic acid ethyl acetate extract concentrates, detect total content with ultraviolet spectrophotometry and can reach more than 80%, this part extract that obtains mainly contains isochlorogenic acid, is the isochlorogenic acid part.
The purifying of total phenolic acid: isochlorogenic acid part and high-purity chlorogenic acid are partly merged, and ratio is by dry product weight 1～5: 5～1; Use earlier 70% dissolve with ethanol, transfer PH to 2～3 with concentrated hydrochloric acid again after concentrating.Under acidic conditions, extract repeatedly 3 times with ethyl acetate, extraction liquid merges, and uses activated carbon decolorizing, concentrates, and promptly obtains total phenolic acid part.
Total phenolic content in the table 2 grayer-hair honeysuckle leaf different treatment process
| Sample | Chlorogenic acid amount (g) | Phenolic acid amount (g) | Medicinal extract amount (g) | Total phenolic content |
| | ????1.700 ????0.3242 ????2.024 ????1.922 ????1.894 ????/ ????/ ????1.785 | ????/ ????/ ????3.060 ????2.665 ????2.151 ????1.2975 ????1.210 ????2.818 | ? ? ????8.69 ????4.804 ????2.630 ????2.474 ????1.466 ????3.433 | ????/ ????/ ????35.26％ ????55.49％ ????81.74％ ????52.45％ ????82.53％ ????82.09％ |
Content in the total phenolic acid prepared product that obtains at last between the chlorogenic acid isomer is than being peak 1: peak 2: peak 3 (5.91: 1.01: 1); Content between the isochlorogenic acid isomery basis is than being peak 4: peak 5: peak 6 (0.43: 2: 1).The content ratio of chlorogenic acid and isochlorogenic acid is 1.8: 1.
The Study on antioxidation of the compound of phenolic acid that 5 the present invention extract
5.1 materials and methods
Medicine:, mainly be the composition of chlorogenic acid different isomerization body by the chlorogenic acid part (chlorogenic acid content reaches 80%) of the inventive method preparation; Isochlorogenic acid part (composition of the isomer of 3 kind of two caffeoyl quinic acid); Total phenolic acid part (total phenolic content reaches more than 80%, and the monomer chlorogenic acid content reaches 50%) (concentration is calculated with the medicinal extract amount); The Japanese Honeysuckle alcohol extract, Japanese Honeysuckle water extract (concentration is calculated with the crude drug amount).
Reagent: pyrogallol (pyrogallol, Zun Yi second laboratory, analytical pure), the amino Phthalocyclohydrazide (luminol,3-aminophthalic acid cyclic hydrazide, Luminol, Sigma company) of 3-.
Method: remove free radical O
2 -Ability detects, get each 10 μ l of testing sample and in measuring cup, (make blank) with methyl alcohol, add 1mmol/L pyrogallol solution 20 μ l, original position is injected 970 μ l luminol,3-aminophthalic acid cyclic hydrazides (1mmol/L)-carbonic acid buffer (pH10.2), reaction cumulative volume 1ml, start luminously, time-delay 10s measures the 240s inner glow intensity.Generally be 50% o'clock concentration IC with luminous inhibiting rate
50Weigh the removing ability of sample to free radical.IC
50Be worth more for a short time, it is strong more to show that sample is removed the ability of free radical, and promptly resistance of oxidation is strong more.
Experiment shows: extraction separation chlorogenic acid part, isochlorogenic acid part and total phenolic acid part all demonstrate very strong Green Tea Extract ability from grayer-hair honeysuckle leaf, and its IC50 is near monomeric compound.Its resistance of oxidation of prepared product that explanation obtains according to the inventive method is much higher than the water extract or the alcohol extract of grayer-hair honeysuckle leaf.The Green Tea Extract ability of alcohol extract obviously is better than the water extract, because the liposoluble ingredient content in the alcohol extract is apparently higher than the water extract, this is consistent with the Liquid Detection result, illustrates that phenolic acid is the activeconstituents of antioxygenation in the Japanese Honeysuckle.The intensity of alcohol extract is followed successively by grey felt hair bud＞grayer-hair honeysuckle leaf＞honeysuckle bud, may also be (seeing Table 3) that the total phenolic content difference owing to chlorogenic acid and so on causes.
Table 3 is removed the comparison of active oxygen radical ability
Sample IC
50(ug/ml)
Chlorogenic acid (3-caffeoyl quinic acid) 5.17
Coffic acid 3.43
Chlorogenic acid butyl ester 8.72
The isomer of chlorogenic acid (1-caffeoyl quinic acid) 5.90
Chlorogenic acid part 14.59
Isochlorogenic acid part 10.49
Total phenolic acid part 12.0
Honeysuckle bud alcohol extract 172.1
Honeysuckle bud aqueous extract 206.17
Largeflower-like honeysuckle flower bud alcohol extract 70.03
Largeflower-like honeysuckle flower bud aqueous extract 130.35
Grayer-hair honeysuckle leaf alcohol extract 106.8
Grayer-hair honeysuckle leaf water extract 151
Embodiment two
Get grayer-hair honeysuckle leaf medicinal material 20kg, be ground into 40 order powder, after 80 ℃ of Soxhlet methods of sherwood oil are extracted and were carried out degreasing in 6 hours, 60% ethanol of 8 times of volumes of medicinal material weight, secondary, each 1 hour are extracted in 85 ℃ of thermal backflows.Extracting solution merges, decompression and solvent recovery.The gained concentrated solution is through the segmentation of D101 macroporous resin, and first water wash-out is used 10%, 35% ethanol elution more successively.Water and 10% alcohol eluen are merged, after concentrating, adding ethanol earlier is 50% to containing the alcohol amount, alcohol precipitation, disgorging, filtrate is concentrated into (70 ℃) relative density 1.08, adding ethanol again is 80% to containing the alcohol amount, alcohol precipitation, centrifuging and taking supernatant liquor, concentrated vacuum-drying promptly gets chlorogenic acid crude product 1.30kg, and purity is 70.3%.
In addition with 35% pure wash-out part, be concentrated into do not have the alcohol flavor after, transfer PH to 2 with concentrated hydrochloric acid, with the ethyl acetate extraction of 2 times of amounts 3 times, extraction liquid is merged, concentrated, gained medicinal extract promptly gets isochlorogenic acid crude product 0.72kg through vacuum-drying, purity is 81.23%.
With above-mentioned chlorogenic acid crude product and isochlorogenic acid crude product with 70% dissolve with ethanol after, being concentrated into does not have the alcohol flavor, transfers PH to 2 with concentrated hydrochloric acid again, ethyl acetate extraction concentrates acetic acid ethyl acetate extract, can get total phenolic acid part 1.6kg, content reaches more than 80%.
Embodiment three
Get grayer-hair honeysuckle leaf medicinal material 30kg, be ground into 40 order powder, after 85 ℃ of Soxhlet methods of sherwood oil were extracted and carried out degreasing in 5 hours, 75% alcohol heat reflux of 10 times of volumes of medicinal material weight extracted secondary, each 1 hour.The extracting solution decompression and solvent recovery, concentrated solution separates through the D101 macroporous resin, directly uses 10% ethanol elution, collects 10% ethanol eluate; Use 40% ethanol elution again, collect 40% ethanol eluate.Get 10% ethanol eluate, after concentrating, adding ethanol earlier is 50% to containing the alcohol amount, alcohol precipitation, disgorging, filtrate is concentrated into (70 ℃) relative density about 1.10, adding ethanol again is 80% to containing the alcohol amount, alcohol precipitation, centrifuging and taking supernatant liquor, concentrated vacuum-drying promptly gets chlorogenic acid crude product 1.9kg, and purity is 72.5%.
In addition with 40% ethanol elution part, be concentrated into do not have the alcohol flavor after, transfer PH to 2 with concentrated hydrochloric acid, with the ethyl acetate extraction of 3 times of amounts 3 times, extraction liquid is merged, concentrate, gained medicinal extract gets isochlorogenic acid crude product 1.02kg through vacuum-drying, purity is 80.50%.
After above-mentioned chlorogenic acid crude product and isochlorogenic acid crude product merged with 70% ethanol, concentrate, transfer PH to 3 with concentrated hydrochloric acid again, the ethyl acetate extraction purifying can get total phenolic acid part 2.25kg, and content reaches more than 80%.
Embodiment four
Get Lonicera confusa DC. leaf medicinal material 20kg, be ground into 40 order powder, after 85 ℃ of Soxhlet methods of sherwood oil were extracted and carried out degreasing in 6 hours, 90 ℃ of thermal backflows of 60% ethanol of 12 times of volumes of medicinal material weight were extracted each 1 hour three times.Extracting solution merges, decompression and solvent recovery.The gained concentrated solution is used 10%, 35% ethanol elution successively through the segmentation of D101 macroporous resin.After 10% ethanol eluate being concentrated into (70 ℃) relative density and being 1.13, adding ethanol earlier is 60% to containing the alcohol amount, alcohol precipitation, disgorging, filtrate concentrates, and adding ethanol again is 85% to containing the alcohol amount, alcohol precipitation, centrifuging and taking supernatant liquor, concentrated vacuum-drying promptly get chlorogenic acid crude product 1.28kg, and purity is 71.8%.
In addition with 35% ethanol elution part, be concentrated into do not have the alcohol flavor after, transfer PH to 3 with concentrated hydrochloric acid, with the ethyl acetate extraction of 2 times of amounts 3 times, extraction liquid is merged, concentrated, gained medicinal extract promptly gets isochlorogenic acid crude product 0.70kg through vacuum-drying, purity is 82.35%.
After above-mentioned chlorogenic acid crude product and isochlorogenic acid crude product merged with 70% ethanol, being concentrated into did not have the alcohol flavor, transfers PH to 2 with concentrated hydrochloric acid again, and the ethyl acetate extraction purifying can get total phenolic acid part 1.46kg, and content reaches more than 80%.
Embodiment five
Get L. similis Hemsl leaf medicinal material 20kg, be ground into 40 order powder, after 6 hours, secondary, each 1 hour are extracted in 90 ℃ of thermal backflows of 70% ethanol of 8 times of volumes of the material weight of getting it filled with the gasoline degreasing.Extracting solution merges, decompression and solvent recovery.The gained concentrated solution is used 5%, 30% ethanol elution successively through the segmentation of AB-8 macroporous resin.After 5% alcohol eluen concentrated, adding ethanol earlier is 45% to containing the alcohol amount, alcohol precipitation, it is 1.18 that disgorging, filtrate are concentrated into (70 ℃) relative density, and adding ethanol again is 75% to containing the alcohol amount, alcohol precipitation, centrifuging and taking supernatant liquor, concentrated vacuum-drying promptly get chlorogenic acid crude product 1.28kg, and purity is 70.9%.
In addition with 30% pure wash-out part, be concentrated into do not have the alcohol flavor after, transfer PH to 2 with concentrated hydrochloric acid, with the ethyl acetate extraction of 2 times of amounts 3 times, extraction liquid is merged, concentrated, gained medicinal extract promptly gets isochlorogenic acid crude product 0.74kg through vacuum-drying, purity is 81.65%.
After above-mentioned chlorogenic acid crude product and isochlorogenic acid crude product merged with 50% ethanol, concentrate, transfer PH to 2 with concentrated hydrochloric acid again, the ethyl acetate extraction purifying can get total phenolic acid part 1.50kg, and content reaches more than 80%.
Embodiment six
Get leaf of Lonicera hypoglauca Miq. medicinal material 25kg, be ground into 40 order powder, after 85 ℃ of Soxhlet methods of sherwood oil are extracted and were carried out degreasing in 4 hours, 80% alcohol reflux secondary of 12 times of volumes of medicinal material weight, each 1 hour.Extracting solution merges, decompression and solvent recovery.The gained concentrated solution is used 10%, 40% ethanol elution successively through the segmentation of HPD100 macroporous resin.After 10% alcohol eluen concentrated, adding ethanol earlier was 50% to containing the alcohol amount, alcohol precipitation, disgorging, filtrate is concentrated into does not have the alcohol flavor, and adding ethanol again is 80% to containing the alcohol amount, alcohol precipitation, centrifuging and taking supernatant liquor, concentrated vacuum-drying promptly get chlorogenic acid crude product 1.61kg, and purity is 73.1%.
In addition with 40% pure wash-out part, be concentrated into do not have the alcohol flavor after, transfer PH to 2 with concentrated hydrochloric acid, with the ethyl acetate extraction of 2 times of amounts 3 times, extraction liquid is merged, concentrated, gained medicinal extract promptly gets isochlorogenic acid crude product 0.91kg through vacuum-drying, purity is 81.0%.
With above-mentioned chlorogenic acid crude product and isochlorogenic acid crude product with 60% dissolve with ethanol after, being concentrated into does not have the alcohol flavor, transfers PH to 2 with concentrated hydrochloric acid again, the ethyl acetate extraction purifying can get total phenolic acid part 1.8kg, content reaches more than 80%.
Claims (8)
1, the preparation method of a kind of chlorogenic acid and isochlorogenic acid is characterized in that comprising the steps:
A) getting leaves of woodbind is raw material, carries out degreasing with low polar solvent;
B) get leaf after the degreasing of step a), use extraction using alcohol, ethanol extract;
C) ethanol extract enrichment step b) gets concentrated solution;
D) get the concentrated solution of step c), last macroporous resin column, earlier with 0～10% ethanol elution, collection 0～10% ethanol eluate; Use 20～40% ethanol elutions again, collect 20～40% ethanol eluates;
E) get 0～10% ethanol eluate of step d), after concentrating, adding ethanol earlier is 45～60% to containing the alcohol amount, removes precipitation, supernatant concentration, adding ethanol again is 75～85% to containing the alcohol amount, removes precipitation, gets supernatant concentration, the chlorogenic acid crude product;
F) get 20～40% ethanol eluates of step d), be concentrated into do not have the alcohol flavor after, transfer pH to 2～3 with hydrochloric acid, use ethyl acetate extraction, concentrate acetic acid ethyl acetate extract, get the isochlorogenic acid crude product.
2, the preparation method of claim 1 is characterized in that raw materials used grayer-hair honeysuckle leaf, leaf of Lonicera hypoglauca Miq., Lonicera confusa DC. leaf, the L. similis Hemsl leaf of being selected from the step a); Low polar solvent is selected from sherwood oil, gasoline, benzene, and degreasing time is no less than 2 hours; In the step b) with 60～80% alcohol reflux; The concentrated solution of step c) does not have the alcohol flavor; The macroporous resin of step d) is selected from D101, AB-8, HPD100, HPD300, D1 or D2.
3, the preparation method of claim 2 is characterized in that raw materials used grayer-hair honeysuckle leaf, the leaf of Lonicera hypoglauca Miq. of being selected from the step a); Be meant with the sherwood oil backflow after 2～10 hours filtering sherwood oil liquid with petroleum ether degreasing; In the step b) with 70～75% alcohol reflux.
4, the preparation method of claim 1 is characterized in that macroporous resin column on the step d), earlier with 5～10% ethanol elutions, collects 5～10% ethanol eluates; Use 30～35% ethanol elutions again, collect 30～35% ethanol eluates.
5, the preparation method of claim 1, after the ethanol eluate that it is characterized in that step e) concentrates, add ethanol earlier to containing alcohol amount 50%, remove precipitation, supernatant concentration is to relative density 1.06～1.18, and adding ethanol again is 80% to containing the alcohol amount, removes precipitation, get supernatant concentration, get the chlorogenic acid crude product.
6, the preparation method of claim 1 is characterized in that getting the chlorogenic acid crude product of step e), uses the 0-10% dissolve with ethanol, removes the water liquid behind the ethanol, transfer pH2～3 with concentrated hydrochloric acid after, use ethyl acetate extraction, concentrate acetic acid ethyl acetate extract, chlorogenic acid.
7, the preparation method of claim 1, it is characterized in that, get the chlorogenic acid crude product of step e), after merging with the isochlorogenic acid of step f), earlier with 50～80% dissolve with ethanol, after concentrating, after transferring pH to 2～3 with concentrated hydrochloric acid again, use ethyl acetate extraction, with the extraction liquid activated carbon decolorizing, concentrate, obtain total phenolic acid.
8, the preparation method of claim 7 is characterized in that, gets the chlorogenic acid crude product of step e), use the 0-10% dissolve with ethanol, remove the water liquid behind the ethanol, transfer pH2～3 with concentrated hydrochloric acid after, use ethyl acetate extraction, concentrate acetic acid ethyl acetate extract, get chlorogenic acid, after merging with the isochlorogenic acid of step f) again, earlier with 60～70% dissolve with ethanol, concentrated after, after transferring pH to 2～3 with concentrated hydrochloric acid again, use ethyl acetate extraction, with the extraction liquid activated carbon decolorizing, concentrate, obtain total phenolic acid.
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