CN111233950B - Method for extracting caffeic acid derivatives from echinacea purpurea - Google Patents
Method for extracting caffeic acid derivatives from echinacea purpurea Download PDFInfo
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Abstract
A method for extracting caffeic acid derivatives from Echinacea purpurea comprises the following steps: 1) Carrying out enzyme deactivation treatment on the raw materials, and carrying out high-alcohol reflux extraction; 2) Concentrating until no alcohol smell exists, and centrifuging; 3) Passing through macroporous resin, gradient eluting, and collecting target section eluate; 4) Decolorizing the target section eluate, concentrating, filtering, adjusting pH to 2-5, passing through polyamide resin, and gradient eluting to obtain three sections of target section eluate; 5) Concentrating the target eluate, heating, cooling to separate out crystal, and vacuum drying to obtain chlorogenic acid, caffeic acid, chicoric acid, and echinacoside. The method can be used for preparing the caffeic acid derivatives with high purity, the content can reach 80 percent, chlorogenic acid, caffeic acid, echinacoside and chicoric acid can be simultaneously prepared, the yield can reach more than 88 percent, and the industrial production can be realized; the process is safe and reliable and is environment-friendly.
Description
Technical Field
The invention belongs to the technical field of separation and preparation of traditional Chinese medicines, and mainly relates to a method for extracting caffeic acid derivatives from echinacea purpurea.
Technical Field
The main components of Echinacea purpurea comprise caffeic acid derivatives (chlorogenic acid, caffeic acid, echinacoside and chicoric acid), alkylamides and polysaccharides, and have good pharmacological activity. At present, echinacea purpurea extracts are available, but the content of chicoric acid and the content of caffeic acid derivatives (chlorogenic acid, caffeic acid, echinacoside and chicoric acid) are lower and are only about 4 percent generally, and the further development and utilization of the echinacea purpurea extracts are limited by the low-content extracts. Nigel B.Perry et al [ J.Agric.food Chem,2001, 49 (4): 1702-1706] prepared a chicoric acid control with a purity of 95% using preparative chromatography. However, the preparation chromatographic equipment has high requirements, difficult amplification production, high cost and toxic and harmful reagents, so the preparation chromatographic equipment can only be used for producing standard products, but cannot be used for industrial production. In the prior art, a process for simultaneously extracting and purifying chlorogenic acid, caffeic acid, echinacoside and chicoric acid from echinacea purpurea does not exist.
The echinacea purpurea caffeic acid derivatives are relatively complex and mainly comprise chlorogenic acid, caffeic acid, echinacoside and chicoric acid. The cichoric acid crystal is in thread shape and needle shape, and its molecular formula is C 22 H 18 O 12 The molecular weight is 474.37, chicoric acid is easily soluble in ethanol, methanol and acetone, slightly soluble in ethyl acetate and diethyl ether, insoluble in petroleum ether, benzene and chloroform, and easily soluble in hot water, but only 0.5% soluble at 25 ℃; the molecular formula of echinacoside is C 35 H 46 O 20 The molecular weight is 786.73, the white amorphous powder is easily soluble in polar solvents such as methanol and ethanol, insoluble in non-polar solvents such as petroleum ether, soluble in alkali, glacial acetic acid and pyridine, slightly soluble in common organic solvents and insoluble in water; caffeic acid has a molecular formula of C 9 H 8 O 4 The crystal is yellow crystal, the crystal precipitated from the concentrated solution does not contain crystal water, the crystal precipitated from the dilute solution contains molecular crystal water, is slightly soluble in cold water and is easily soluble in hot water, cold ethanol and ethyl acetate; chlorogenic acid molecular formula C 16 H 18 O 9 The hemihydrate is white or yellowish needle-shaped crystal, the solubility of chlorogenic acid in water at 25 deg.C is about 4%, and the chlorogenic acid is easily dissolved in polar solvents such as ethanol, acetone, methanol, etc.,slightly soluble in ethyl acetate and insoluble in lipophilic organic solvents such as chloroform, diethyl ether and benzene.
CN1587251A discloses a process for extracting high-purity chicoric acid from echinacea, which is characterized in that chloroform is adopted to treat echinacea, methanol is used for extraction, ethyl acetate is used for three times of extraction after extraction, and finally acetonitrile reverse phase chromatographic column is used for purification to obtain a high-purity chicoric acid product. The process uses chloroform and methanol which are harmful to the environment and human body, and is easy to remain in chicoric acid products, and the acetonitrile reverse phase chromatographic column is expensive, the production efficiency is low, and the cost is high. CN1473602A disclosesExtraction and preparation process of echinacea purpureaThe method comprises the steps of ethanol reflux extraction, impurity removal, concentration, vacuum drying, column loading and the like. It has the following disadvantages: (1) the extract is directly subjected to microfiltration treatment, so that the microfiltration membrane is easy to block, and the production efficiency is low, so that the method is not suitable for large-scale production. (2) Concentrating the obtained extract, and drying under reduced pressure to obtain final extract with low content of chicoric acid due to poor heat resistance of polyphenols such as chicoric acid and large loss of chicoric acid caused by long-term heating. CN1957961A discloses a method for preparing Echinacea purpurea extract, wherein fresh Echinacea purpurea whole plant is adopted as raw material, and the Echinacea purpurea extract is obtained by extracting and concentrating. The method has the following defects: (1) the preparation process takes fresh echinacea medicinal materials as research objects, although the content of chicoric acid in fresh echinacea medicinal materials is higher, the production of the echinacea extract is limited by seasons, and the industrial production of the echinacea extract is limited by the medicine source problem of the echinacea extract. (2) The process production mainly takes the chicoric acid content as an index, and the caffeic acid, echinacoside and chlorogenic acid in the medicinal materials are not considered, so that the waste of medicinal material resources is caused to a certain extent. (3) The preparation method comprises extracting the above Chinese medicinal materials with high concentration ethanol for 2 times, adsorbing large amount of ethanol in the residue, and producing Echinacea purpurea extract with high cost. CN101367728B adopts commercially available 3-5% Echinacea purpurea extract as raw material, and is dissolved in waterFiltering, adding acid to adjust pH to 1-4, purifying with macroporous adsorbent resin, sequentially eluting with three eluents with different gradients, collecting 6-45 column volumes of eluate as caftaric acid, and collecting 60-100 column volumes of eluate as chicoric acid; after decompression and evaporation to dryness, the monocaffeyltartaric acid and the chicoric acid with the purity of more than 95 percent are obtained by recrystallization. The method has the following defects: (1) the preparation process takes the echinacea purpurea extract as a research object, and increases the cost of industrial production. (2) The volume of the elution solvent is too large, and the mass production is inconvenient. (3) The effective components only separate chicoric acid and cafe mono tartaric acid, and chlorogenic acid and echinacoside, etc. are not considered.
The method has the defects that the effective components in the medicinal materials are not fully utilized, so that the resource waste is caused, and the equipment used for preparing the high-content product is expensive, so that the industrial production cannot be realized.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide the method for extracting the caffeic acid derivatives from the echinacea purpurea, the obtained caffeic acid derivatives have high content, chlorogenic acid, caffeic acid, echinacoside and chicoric acid can be simultaneously separated and purified, the preparation cost is low, and the method is safe and environment-friendly.
The invention is realized by the following technical scheme:
a method for extracting caffeic acid derivatives from Echinacea purpurea comprises the following steps:
1) Preparing echinacea purpurea raw material, adding acid water with weight 3-5 times of that of echinacea purpurea raw material into an extraction tank, heating to 60-100 ℃, adding echinacea purpurea raw material, stirring and keeping the temperature for 5-30min (performing enzyme deactivation treatment); adding 80-95% ethanol water solution (added with ethanol water solution of high concentration, and mixed with acid water to obtain ethanol water solution of low concentration), maintaining the temperature for 0.5-2 hr, and discharging the material solution; adding 40-60% ethanol water solution 8-12 times the weight of Echinacea purpurea raw material into the residue, reflux extracting or extracting at 75-85 deg.C for 0.5-2 hr for 1-2 times, and filtering; collecting all the feed liquid to obtain an extracting solution;
the extraction times are too few, which is not favorable for fully extracting the effective components in the raw materials; too many extraction times, limited extraction of the active ingredients, high cost of the extraction solvent and increased impurity content in the extract.
2) Concentrating the extracting solution obtained in the step 1) under reduced pressure until no alcohol smell exists, controlling the total mass concentration of solutes to be 10-15wt%, centrifuging, and obtaining clear liquid for later use;
3) Enabling the clear liquid obtained in the step 2) to pass through a non-polar macroporous resin or a weak-polar macroporous resin at the flow rate of 1-2BV/h, sequentially eluting by using 1-2BV of pure water, 1-3BV of ethanol water solution with the volume concentration of 10-30% and 2-4BV of ethanol water solution with the volume concentration of 50-80%, and collecting ethanol water solution eluent with the volume concentration of 50-80%, namely high-content caffeic acid derivative eluent;
4) Adding activated carbon which is 2-5wt% of the solute in the eluent into the eluent with high content of caffeic acid derivatives obtained in the step 3), then decompressing and concentrating at 60-70 ℃ until no alcohol smell exists, centrifuging while hot to obtain filtrate;
in the step, the solute is caffeic acid derivatives including chlorogenic acid, caffeic acid, echinacoside and chicoric acid;
5) Adjusting the pH of the filtrate obtained in the step 4) to 2-5 (preferably adjusting the pH by hydrochloric acid), passing through polyamide resin, and sequentially eluting by pure water and ethanol aqueous solutions with different concentrations in a first section, a second section and a third section, wherein the ethanol aqueous solution with the volume concentration of 25-35% in the first section, the ethanol aqueous solution with the volume concentration of 50-60% in the second section and the ethanol aqueous solution with the volume concentration of 70-80% in the third section;
the first section of eluent is chlorogenic acid eluent, is decompressed and concentrated until the mass content of the chlorogenic acid is 30-40%, is heated to 85-90 ℃, is cooled to separate out crystals, is subjected to solid-liquid separation, and is dried in vacuum to obtain the chlorogenic acid;
the second stage of eluent is the eluent of the mixture of caffeic acid and echinacoside, the mixture of caffeic acid and echinacoside is concentrated under reduced pressure until the mass content is 30% -40%, the temperature is raised to 85-90 ℃, then the mixture is cooled to separate out crystal, solid-liquid separation is carried out, and the solid state is dried in vacuum to obtain the caffeic acid; concentrating the liquid under reduced pressure, and vacuum drying to obtain echinacoside;
and the third section of eluent is chicory acid pickling and dehydrating solution, the third section of eluent is concentrated under reduced pressure until the mass content of the chicory acid is 30-40%, the temperature is raised to 85-90 ℃, crystals are separated out by cooling, solid-liquid separation is carried out, and the solid state is dried in vacuum, so that the chicory acid is obtained.
Further, in the step 1), the echinacea raw material is crushed before the enzyme deactivation treatment.
Further, in step 1), the acid water is an aqueous solution of a volatile acid. Preferably, the pH is 2 to 5. The volatile acid is preferably hydrochloric acid. The polyphenol oxidase has survival conditions of proper temperature (30-50 deg.C), proper pH (6-7), and sufficient substrate, and can be deactivated under peracid or alkalescence condition, but the effective component to be extracted is organic acid, and the polyphenol oxidase is easily hydrolyzed under heat under alkalescence condition. Resulting in loss of the active ingredient. Therefore, proper acidity and high temperature are selected to synergistically kill the enzyme, and the adsorption quantity of the resin can be improved.
Further, in the step 3), the nonpolar macroporous resin or the weak polar macroporous resin is at least one of LX-20 macroporous resin, LX-60 macroporous resin, XDA-6 macroporous resin and the like.
Research shows that if polar macroporous resin is adopted, the separation effect is poor, and the product yield and purity are reduced; by adopting the nonpolar macroporous resin or the weak-polarity macroporous resin, the product yield and the purity can be improved; and LX-20 macroporous resin, LX-60 macroporous resin or XDA-6 macroporous resin is adopted, the effect is best, and the product yield and purity can be greatly improved.
Further, in the step 5), the volume of the first section of ethanol aqueous solution is 2-3BV, the volume of the second section of ethanol aqueous solution is 1-2BV, and the volume of the third section of ethanol aqueous solution is 1.5-2.5BV.
Further, in the step 5), the volume of the pure water is 1-2BV.
Further, the ethanol can be recovered from the ethanol-containing solution in each step.
The method takes echinacea purpurea as a raw material, ethanol water solution is adopted for reflux extraction after enzyme deactivation treatment, and macroporous resin purification and polyamide resin separation technology are combined to prepare the high-content caffeic acid derivative.
The invention has the following advantages:
1. the raw materials are subjected to heat preservation treatment by high-temperature acid water, so that polyphenol oxidase can be damaged, the degradation phenomenon of active ingredients in the extraction process is reduced, and the used acid is volatile acid, so that the yield of the active ingredients is not influenced;
2. because the echinacea purpurea contains components such as alkylamides and chlorophyll, and the caffeic acid derivative is easy to dissolve in organic solvents such as ethanol, and the like, and is extracted by adopting a low-concentration ethanol water solution, impurities with small polarity can be effectively prevented from being extracted, a certain purification effect can be achieved, and a certain decoloration effect can be achieved;
3. impurities (glycoprotein, polysaccharide and the like) with larger polarity are removed through primary purification, high-purity chlorogenic acid, caffeic acid, echinacoside and chicoric acid can be simultaneously prepared through secondary purification and separation by adjusting the polarity of an elution solvent, and the active ingredients are concentrated to certain solid content concentration by utilizing the characteristics of insolubility in water, solubility in hot water and the like of the active ingredients, heated for assisting dissolution, cooled for crystallization, can be crystallized and separated out, and achieve the effect of further purification;
4. the process has strong operability and low cost, is suitable for industrial production, and has safe and reliable production and environmental protection.
Detailed Description
The present invention is further illustrated by the following examples.
In the specification, unless otherwise stated, the concentration of the ethanol aqueous solution is the volume concentration of ethanol therein, and the other percentages are mass percentages.
Example 1
The embodiment comprises the following steps:
1) Weighing 100kg of echinacea raw material, crushing for later use, adding acid water (the acid water is hydrochloric acid and the pH value is adjusted to be = 2) which is 4 times of the weight of the echinacea raw material into an extraction tank, heating to 60 ℃, adding the echinacea raw material, stirring and preserving heat for 30min, adding ethanol water solution with the volume concentration of 95% which is 5 times of the weight of the echinacea raw material, preserving heat and refluxing for 1h, and discharging the feed liquid; adding 50% ethanol water solution with volume concentration 10 times of the weight of Echinacea purpurea raw material into the residue, reflux-extracting for 1.5 hr under heat preservation, and filtering; collecting all the feed liquid to obtain an extracting solution;
2) Concentrating the extracting solution obtained in the step 1) under reduced pressure until no alcohol smell exists, controlling the total mass concentration of solutes to be 10wt%, centrifuging, and reserving clear liquid for later use;
3) Enabling the clear liquid obtained in the step 2) to pass through LX-60 macroporous resin at the flow rate of 1BV/h, sequentially eluting by adopting 2BV of pure water, 3BV of 20% ethanol water solution in volume concentration and 4BV of 80% ethanol water solution in volume concentration, and collecting 80% ethanol water solution eluent in volume concentration, namely high-content caffeic acid derivative eluent;
4) Adding activated carbon which is 5 percent of the mass of the solute in the eluent into the eluent with high content of caffeic acid derivatives obtained in the step 3), then decompressing and concentrating at 60 ℃ until no alcohol smell exists, and centrifuging while hot to obtain filtrate;
in the step, the solute is caffeic acid derivatives including chlorogenic acid, caffeic acid, echinacoside and chicoric acid;
5) Adjusting the pH of the filtrate obtained in the step 4) to 2 (adjusting the pH with hydrochloric acid), separating and purifying with polyamide resin, and sequentially eluting with 2BV of pure water, 2BV of 25% by volume of ethanol aqueous solution (first section), 1BV of 50% by volume of ethanol aqueous solution (second section) and 2.5BV of 70% by volume of ethanol aqueous solution (third section);
the first section of eluent is chlorogenic acid eluent, is concentrated under reduced pressure until the mass content of the chlorogenic acid is 40 percent, is heated to 85 ℃, is cooled to separate out crystals, is subjected to solid-liquid separation, and is dried in vacuum to obtain the chlorogenic acid;
the second stage of eluent is caffeic acid and echinacoside mixture eluent, and is concentrated under reduced pressure until the mass content of the caffeic acid and echinacoside mixture is 40%, the temperature is raised to 85 ℃, then the mixture is cooled to separate out crystal, solid-liquid separation is carried out, and the solid state is dried in vacuum to obtain caffeic acid; concentrating the liquid under reduced pressure, and vacuum drying to obtain echinacoside;
and the third section of eluent is chicory acid pickling and dehydrating solution, the third section of eluent is concentrated under reduced pressure until the mass content of the chicory acid is 40 percent, the temperature is raised to 85 ℃, crystals are separated out by cooling, solid-liquid separation is carried out, and the solid state is dried in vacuum, thus obtaining the chicory acid.
HPLC analysis and detection prove that the chlorogenic acid content is 88.57wt% (0.15 Kg), the caffeic acid content is 91.25wt% (0.96 Kg), the echinacoside content is 85.76wt% (0.019 KG), the chicoric acid content is 93.21wt% (1.94 Kg), and the total polyphenol extraction rate reaches 88.44wt%.
Example 2
The embodiment comprises the following steps:
1) Weighing 100kg of echinacea raw material, crushing for later use, adding acid water (the acid water is hydrochloric acid, and the pH value is adjusted to be = 5) which is 4 times of the weight of the echinacea raw material into an extraction tank, heating to 100 ℃, adding the echinacea raw material, stirring and preserving heat for 5min, adding ethanol water solution with the volume concentration of 95% which is 6 times of the weight of the echinacea raw material, preserving heat and refluxing for 1h, and discharging the feed liquid; adding 60% ethanol water solution with volume concentration 10 times of the weight of Echinacea purpurea raw material into the residue, reflux-extracting for 1.5 hr under heat preservation, and filtering; collecting all the feed liquid to obtain an extracting solution;
2) Concentrating the extracting solution obtained in the step 1) under reduced pressure until no alcohol smell exists, controlling the total mass concentration of solutes to be 15%, centrifuging, and obtaining clear liquid for later use;
3) Enabling the clear liquid obtained in the step 2) to pass through LX-20 macroporous resin at the flow rate of 2BV/h, sequentially eluting with 2BV of pure water, 1BV of ethanol water solution with the volume concentration of 10% and 4BV of ethanol water solution with the volume concentration of 50%, and collecting ethanol water solution eluent with the volume concentration of 50%, namely high-content caffeic acid derivative eluent;
4) Adding activated carbon which is 2 percent of the mass of the solute in the eluent into the eluent with high caffeic acid derivative content obtained in the step 3), then concentrating the eluent under reduced pressure at 70 ℃ until no alcohol smell exists, and centrifuging the eluent while the eluent is hot to obtain filtrate;
in the step, the solute is caffeic acid derivatives including chlorogenic acid, caffeic acid, echinacoside and chicoric acid;
5) Adjusting the pH of the filtrate obtained in the step 4) to 5 by using hydrochloric acid, separating and purifying by using polyamide resin, and eluting by using 1BV of pure water, 3BV of ethanol water solution with the volume concentration of 30% (first section), 2BV of ethanol water solution with the volume concentration of 60% (second section) and 1.5BV of ethanol water solution with the volume concentration of 80% (third section) in sequence;
the first section of eluent is chlorogenic acid eluent, is decompressed and concentrated until the mass content of the chlorogenic acid is 30 percent, is heated to 90 ℃, is cooled to separate out crystals, is subjected to solid-liquid separation, and is dried in vacuum to obtain the chlorogenic acid;
the second stage of eluent is caffeic acid and echinacoside mixture eluent, and is concentrated under reduced pressure until the mass content of the caffeic acid and echinacoside mixture is 30%, the temperature is raised to 90 ℃, then the mixture is cooled to separate out crystals, solid-liquid separation is carried out, and the solid is dried in vacuum, thus obtaining caffeic acid; concentrating the liquid under reduced pressure, and vacuum drying to obtain echinacoside;
and the third section of eluent is chicory acid pickling and dehydrating solution, the third section of eluent is concentrated under reduced pressure until the mass content of the chicory acid is 30 percent, the temperature is raised to 90 ℃, crystals are separated out by cooling, solid-liquid separation is carried out, and the solid state is dried in vacuum, thus obtaining the chicory acid.
HPLC analysis and detection shows that the chlorogenic acid content is 88.77wt% (0.15 KG), the caffeic acid content is 91.53wt% (0.98 KG), the echinacoside content is 87.31wt% (0.019 KG), the chicoric acid content is 92.21wt% (2.00 KG), and the total polyphenol extraction rate reaches 90.32%.
Example 3
The embodiment comprises the following steps:
1) Weighing 100kg of echinacea raw material, crushing for later use, adding acid water (the acid water is hydrochloric acid, and the pH value is adjusted to be = 3) which is 4 times of the weight of the echinacea raw material into an extraction tank, heating to 90 ℃, adding the echinacea raw material, stirring and preserving heat for 15min, adding ethanol water solution with the volume concentration of 85% which is 6 times of the weight of the echinacea raw material, preserving heat and refluxing for 1h, and discharging the feed liquid; adding 50% ethanol water solution with volume concentration 10 times of Echinacea purpurea raw material weight into the residue, reflux extracting for 1.5 hr under heat preservation, and filtering; collecting all the feed liquid to obtain an extracting solution;
2) Concentrating the extracting solution obtained in the step 1) under reduced pressure until no alcohol smell exists, controlling the total mass concentration of solutes to be 10wt%, centrifuging, and obtaining clear liquid for later use;
3) Enabling the clear liquid obtained in the step 2) to pass through XDA-6 macroporous resin at the flow rate of 1BV/h, sequentially eluting with 2BV of pure water, 2BV of 20% ethanol water solution in volume concentration and 3BV of 70% ethanol water solution in volume concentration, and collecting 70% ethanol water solution eluent in volume concentration to obtain high-content caffeic acid derivative eluent;
4) Adding activated carbon which is 2 percent of the mass of the solute in the eluent into the eluent with high caffeic acid derivative content obtained in the step 3), concentrating under reduced pressure at 65 ℃ until no alcohol smell exists, and centrifuging while hot to obtain filtrate; in the step, the solute is caffeic acid derivatives including chlorogenic acid, caffeic acid, echinacoside and chicoric acid;
5) Adjusting the pH of the filtrate obtained in the step 4) to 5 by using hydrochloric acid, passing through polyamide resin, and eluting by using 1BV of pure water, 2.5BV of 35% ethanol water solution (first section), 1.5BV of 55% ethanol water solution (second section) and 2BV of 75% ethanol water solution (third section) in sequence.
The first section of eluent is chlorogenic acid eluent, is decompressed and concentrated until the mass content of the chlorogenic acid is 40 percent, is heated to 90 ℃, is cooled to separate out crystals, is subjected to solid-liquid separation, and is dried in vacuum to obtain the chlorogenic acid;
the second stage of eluent is caffeic acid and echinacoside mixture eluent, and is concentrated under reduced pressure until the mass content of the caffeic acid and echinacoside mixture is 40%, the temperature is raised to 90 ℃, then the mixture is cooled to separate out crystals, solid-liquid separation is carried out, and the solid is dried in vacuum, thus obtaining caffeic acid; concentrating the liquid under reduced pressure, and vacuum drying to obtain echinacoside;
and the third section of eluent is chicory acid pickling and dehydrating solution, the third section of eluent is concentrated under reduced pressure until the mass content of the chicory acid is 40 percent, the temperature is raised to 90 ℃, crystals are separated out by cooling, solid-liquid separation is carried out, and the solid state is dried in vacuum, thus obtaining the chicory acid.
HPLC analysis and detection shows that the chlorogenic acid content is 90.55wt% (0.15 KG), the caffeic acid content is 91.59wt% (0.99 KG), the echinacoside content is 89.21wt% (0.019 KG), the chicoric acid content is 93.50wt% (2.00 KG), and the total polyphenol extraction rate reaches 91.26wt%.
Comparative example 1
In this comparative example, except that the enzyme deactivation treatment was not performed, that is, there was no step (1) 'adding acid water (hydrochloric acid, pH = 2) in an extraction tank in an amount of 4 times the weight of the echinacea raw material, raising the temperature to 60 ℃, adding the echinacea raw material, stirring and maintaining the temperature for 30min,' and the operation of directly adding the echinacea raw material to the extraction tank, the other parameters were the same as those in example 1.
The product obtained in this comparative example was analyzed and determined by HPLC to have a chlorogenic acid content of 84.88wt% (0.14 Kg), a caffeic acid content of 87.69wt% (0.90 Kg), an echinacoside content of 80.22wt% (0.018 KG), a chicoric acid content of 88.53wt% (1.83 Kg), and a total polyphenol extraction rate of 79.14wt%.
Comparative example 2
This comparative example was carried out in the same manner as example 1 except that VC was used as an acid in the enzyme deactivation treatment.
HPLC analysis and detection of the product obtained in the comparative example show that the chlorogenic acid content is 85.02wt% (0.14 Kg), the caffeic acid content is 87.77wt% (0.92 Kg), the echinacoside content is 80.25wt% (0.019 KG), the chicoric acid content is 88.91wt% (1.87 Kg), and the total polyphenol extraction rate reaches 81.33wt%.
Comparative example 3
This comparative example was the same as example 1 except that an AB-8 resin (weakly polar resin) was used for the initial purification in step 3).
The product obtained in the comparative example was analyzed and tested by HPLC, the chlorogenic acid content was 84.98wt% (0.15 Kg), the caffeic acid content was 86.88wt% (0.94 Kg), the echinacoside content was 80.54wt% (0.019 KG), the chicoric acid content was 89.21wt% (1.88 Kg), and the total polyphenol extraction rate was 82.16wt%.
Comparative example 4
This comparative example was carried out in the same manner as in example 1 except that NKA-2 resin (polar resin) was used for the initial purification in step 3).
The product obtained in the comparative example was analyzed and detected by HPLC, and found to have a chlorogenic acid content of 83.25wt% (0.146 Kg), a caffeic acid content of 87.44wt% (0.90 Kg), an echinacoside content of 80.46wt% (0.018 KG), a chicoric acid content of 87.92wt% (1.85 Kg), and a total polyphenol extraction rate of 79.52wt%.
Claims (7)
1. A method for extracting caffeic acid derivatives from Echinacea purpurea is characterized by comprising the following steps:
1) Preparing echinacea purpurea raw material, adding acid water into an extraction tank, heating, adding the echinacea purpurea raw material, stirring, preserving heat and carrying out enzyme deactivation treatment; adding 80-95 vol% ethanol water solution, maintaining the temperature, and discharging the liquid; adding 40-60% ethanol water solution into the residue, reflux extracting or heat preserving extracting, and filtering; collecting all the feed liquid to obtain an extracting solution; the acid is hydrochloric acid; the pH of the acid water is 2-5;
2) Concentrating the extracting solution obtained in the step 1) under reduced pressure until no alcohol smell exists, centrifuging, and obtaining clear liquid for later use;
3) Passing the clear liquid obtained in the step 2) through a non-polar macroporous resin or a weak-polar macroporous resin, eluting by using pure water, an ethanol water solution with the volume concentration of 10-30% and an ethanol water solution with the volume concentration of 50-80% in sequence, and collecting the ethanol water solution eluent with the volume concentration of 50-80%, namely the high-content caffeic acid derivative eluent;
4) Adding activated carbon into the high-content caffeic acid derivative eluent obtained in the step 3), then concentrating under reduced pressure until no alcohol smell exists, and centrifuging to obtain filtrate;
5) Adjusting the pH of the filtrate obtained in the step 4) to 2-5, passing through polyamide resin, and sequentially eluting by using pure water and ethanol aqueous solutions with different concentrations in a first section, a second section and a third section, wherein the ethanol aqueous solution with the volume concentration of 25-35% is used in the first section, the ethanol aqueous solution with the volume concentration of 50-60% is used in the second section, and the ethanol aqueous solution with the volume concentration of 70-80% is used in the third section;
the first section of eluent is chlorogenic acid eluent, and is subjected to reduced pressure concentration, temperature rise to 85-90 ℃, cooling to separate out crystals, solid-liquid separation, and solid drying to obtain chlorogenic acid;
the second stage of eluent is caffeic acid and echinacoside mixture eluent, and is concentrated under reduced pressure, heated to 85-90 deg.C, cooled to separate out crystal, subjected to solid-liquid separation, and dried to obtain caffeic acid; concentrating the liquid under reduced pressure, and drying to obtain echinacoside;
and the eluent in the third section is chicory acid pickling and dehydrating solution, concentrating under reduced pressure, heating to 85-90 ℃, cooling to separate out crystals, carrying out solid-liquid separation, and drying the solid to obtain the chicoric acid.
2. The method of extracting caffeic acid derivatives from echinacea purpurea according to claim 1, comprising the steps of:
1) Preparing echinacea purpurea raw material, adding acid water with weight 3-5 times of that of the echinacea purpurea raw material into an extraction tank, heating to 60-100 ℃, adding the echinacea purpurea raw material, stirring, keeping the temperature for 5-30min, and performing enzyme deactivation treatment; adding 80-95% high concentration ethanol water solution 5-8 times the weight of Echinacea purpurea material, maintaining for 0.5-2 hr, and discharging the material liquid; adding 40-60% ethanol water solution 8-12 times the weight of Echinacea purpurea raw material into the residue, reflux extracting or extracting at 75-85 deg.C for 0.5-2 hr for 1-2 times, and filtering; collecting all the feed liquid to obtain an extracting solution; the acid is hydrochloric acid; the pH of the acid water is 2-5;
2) Concentrating the extracting solution obtained in the step 1) under reduced pressure until no alcohol smell exists, controlling the total mass concentration of solutes to be 10-15wt%, centrifuging, and obtaining clear liquid for later use;
3) Enabling the clear liquid obtained in the step 2) to pass through a non-polar macroporous resin or a weak-polar macroporous resin at the flow rate of 1-2BV/h, sequentially eluting by using 1-2BV of pure water, 1-3BV of ethanol water solution with the volume concentration of 10-30% and 2-4BV of ethanol water solution with the volume concentration of 50-80%, and collecting ethanol water solution eluent with the volume concentration of 50-80%, namely high-content caffeic acid derivative eluent;
4) Adding activated carbon which is 2-5wt% of the solute in the eluent into the eluent with high content of caffeic acid derivatives obtained in the step 3), then decompressing and concentrating at 60-70 ℃ until no alcohol smell exists, centrifuging while hot to obtain filtrate;
5) Adjusting the pH of the filtrate obtained in the step 4) to 2-5, passing through polyamide resin, and sequentially eluting by using pure water and ethanol aqueous solutions with different concentrations in a first section, a second section and a third section, wherein the ethanol aqueous solution with the volume concentration of 25-35% is used in the first section, the ethanol aqueous solution with the volume concentration of 50-60% is used in the second section, and the ethanol aqueous solution with the volume concentration of 70-80% is used in the third section;
the first section of eluent is chlorogenic acid eluent, is decompressed and concentrated until the mass content of the chlorogenic acid is 30-40%, is heated to 85-90 ℃, is cooled to separate out crystals, is subjected to solid-liquid separation, and is dried in vacuum to obtain the chlorogenic acid;
the second stage of eluent is the eluent of the mixture of caffeic acid and echinacoside, the mixture of caffeic acid and echinacoside is concentrated under reduced pressure until the mass content is 30% -40%, the temperature is raised to 85-90 ℃, then the mixture is cooled to separate out crystal, solid-liquid separation is carried out, and the solid state is dried in vacuum to obtain the caffeic acid; concentrating the liquid under reduced pressure, and vacuum drying to obtain echinacoside;
and the eluent in the third section is chicory acid pickling and dehydrating solution, the solution is concentrated under reduced pressure until the mass content of the chicory acid is 30 to 40 percent, the temperature is raised to 85 to 90 ℃, crystals are precipitated by cooling, solid-liquid separation is carried out, and the solid state is dried in vacuum, thus obtaining the chicoric acid.
3. The method for extracting caffeic acid derivatives from echinacea purpurea according to claim 1 or 2, wherein in the step 1), the echinacea purpurea raw material is crushed before the enzyme inactivation treatment.
4. The method for extracting caffeic acid derivatives from echinacea purpurea according to claim 1 or 2, wherein in the step 3), the non-polar macroporous resin or the weak-polar macroporous resin is at least one of LX-20 macroporous resin, LX-60 macroporous resin and XDA-6 macroporous resin.
5. The method for extracting caffeic acid derivatives from echinacea purpurea according to claim 1 or 2, wherein in the step 5), the volume of the first ethanol aqueous solution is 2-3BV, the volume of the second ethanol aqueous solution is 1-2BV, and the volume of the third ethanol aqueous solution is 1.5-2.5BV.
6. The method for extracting caffeic acid derivatives from echinacea purpurea according to claim 1 or 2, wherein the volume of pure water in step 5) is 1-2BV.
7. The method for extracting caffeic acid derivatives from echinacea purpurea according to claim 1 or 2, wherein the pH of the filtrate is adjusted with hydrochloric acid in step 5).
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| CN104693034A (en) * | 2015-03-27 | 2015-06-10 | 中国药科大学 | Purifying method for chicoric acid in echinacea purpurea |
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| CN101367728A (en) * | 2008-10-09 | 2009-02-18 | 湖南师范大学 | Method for purifying chicoric acid and monocaffeyltartaric acid from echinacea purpurea extract |
| CN104693034A (en) * | 2015-03-27 | 2015-06-10 | 中国药科大学 | Purifying method for chicoric acid in echinacea purpurea |
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