CN110156601B - Method for preparing high-purity chlorogenic acid from stevia rebaudiana - Google Patents
Method for preparing high-purity chlorogenic acid from stevia rebaudiana Download PDFInfo
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Abstract
The invention discloses a method for preparing high-purity chlorogenic acid from stevia rebaudiana, belonging to the technical field of natural product chemistry. The invention takes stevia rebaudiana leaves as raw materials, water as an extraction medium, the leaching liquor is purified by polyamide resin or polyamide resin after being filtered and flocculated, water is used as an eluent, and the eluent is concentrated and crystallized to prepare a product with the chlorogenic acid mass content of more than 98%. Meanwhile, in the process of purifying chlorogenic acid by using resin, other collected components can be used for separating and extracting other stevia polyphenols and stevioside. In general, the method realizes the preparation of high-purity chlorogenic acid by taking stevia rebaudiana leaves as raw materials, the mass content of the chlorogenic acid in the product can reach more than 98 percent, the process operation is simple, organic solvents are hardly used, and the method is environment-friendly and safe.
Description
Technical Field
The invention relates to a method for preparing high-purity chlorogenic acid from stevia rebaudiana, in particular to a method for extracting the high-purity chlorogenic acid from the stevia rebaudiana by a water medium method, belonging to the technical field of natural product chemistry.
Background
Chlorogenic acid, also known as caffeoylquinic acid, belongs to one of chlorogenic acids, is depside generated from caffeic acid and quinic acid (1-hydroxyhexahydro gallic acid), and is a phenylpropanoid compound generated by plants in the aerobic respiration process through the shikimic acid pathway.
Chlorogenic acid is an important bioactive substance and has great application value in the fields of medicine, health and the like. Chlorogenic acid has certain pharmacological effects on digestive system, blood system and reproductive system, and has wide antibacterial, antiinflammatory, cholagogic and hemostatic effects; chlorogenic acid has strong antibacterial and antiviral effects, and can effectively inhibit Staphylococcus aureus, hemolytic streptococcus, Bacillus dysenteriae, and Salmonella typhi; chlorogenic acid is an effective phenolic antioxidant, and can effectively remove hydroxyl free radicals and superoxide anions, thereby protecting organism from damage of oxidation; in addition, chlorogenic acid also has a certain protective effect on cardiovascular and has an obvious blood pressure reducing effect. Due to various efficacies and physiological activities of chlorogenic acid, the extraction and purification of chlorogenic acid are attracting more and more attention.
Chlorogenic acid is widely found in plants and mainly comes from honeysuckle, eucommia, coffee beans, acer truncatum leaves, burdock leaves, sweet potato leaves and the like. Stevia rebaudiana Bertoni is perennial herb of Compositae, contains stevioside 8% -12% of dry leaves of stevia rebaudiana Bertoni, and is mainly used for extracting natural sweetener stevioside. Besides steviol glycosides, the alcohol, water or alcohol-water extracts of stevia contain steviol, flavonoids, quinic and caffeic acids and derivatives, amino acids and fatty acids and derivatives, and other impurities such as tannins, pigments and pectins that need to be removed. The phenolic compounds in stevia include simple phenolic acids, chlorogenic acids, flavonoids, flavonols, flavanols, isoflavones, etc. For stevia rebaudiana, research at home and abroad mainly focuses on the preparation and separation of stevioside, and few reports are reported on the extraction and separation of polyphenol and chlorogenic acid in the stevia rebaudiana. The polyphenol in the stevia rebaudiana is discarded along with the extraction waste residue; or the extraction from the waste residue has low purity and poor extraction efficiency.
At present, raw materials for industrially extracting and purifying chlorogenic acid mainly come from honeysuckle, folium cortex eucommiae, coffee beans and the like. Chinese patent CN103288644A describes a method for extracting chlorogenic acid from flos Lonicerae, cleaning flos Lonicerae, drying, and pulverizing to obtain powder; extracting with water for multiple times, adjusting pH, adsorbing with macroporous resin, eluting with alcohol to obtain crude extract, extracting, separating, concentrating, and drying to obtain chlorogenic acid. Chinese patent CN1974527A discloses a method for preparing high-purity chlorogenic acid and total flavonoids from folium Eucommiae, which comprises pulverizing dried folium Eucommiae, and extracting with ethanol; then adsorbing by a macroporous resin column, passing through a silica gel column, concentrating under reduced pressure, and recrystallizing by acetone or water to obtain a refined chlorogenic acid product with the purity of 96-99%. Patent CN109096110A describes a process for separating and purifying 99% chlorogenic acid from coffee bean, which comprises pulverizing coffee bean, sieving to obtain coarse powder, extracting with ethanol, stirring, centrifuging, filtering, adsorbing the filtrate with macroporous adsorbent resin, treating the eluate with nanofiltration membrane, decolorizing, filtering, concentrating under reduced pressure, crystallizing to obtain crude product, and recrystallizing with diluted ethanol to obtain 99% chlorogenic acid. Patent CN03134575.1A discloses a process for extracting chlorogenic acid from Acer truncatum Bunge leaves, which comprises extracting chlorogenic acid from Acer truncatum Bunge leaves at low temperature, controlling time, pH value and material-liquid ratio, and has high solid solution extraction rate, good effect, and simple operation, and the obtained chlorogenic acid has a purity of about 40%. Patent CN1651398A discloses a process for extracting chlorogenic acid from burdock leaves, which comprises washing fresh burdock leaves, removing impurities, degreasing with chloroform, decolorizing, extracting with acid water under reflux, extracting with ethyl acetate, concentrating under reduced pressure, adsorbing with AB-8 resin column, eluting with ethanol, concentrating the eluate, and recrystallizing to obtain pure chlorogenic acid. Patent CN200510094347.8 describes a method for extracting and purifying chlorogenic acid from sunflower meal, which comprises extracting sunflower meal with ethanol aqueous solution, concentrating the extractive solution to remove alcohol, precipitating with acid to separate protein, extracting with ethyl acetate, separating and concentrating to obtain crude chlorogenic acid with purity of 70%, and further purifying with D101 macroporous resin to obtain chlorogenic acid with content of 92%.
In summary, most of the raw materials for extracting chlorogenic acid at present are Chinese herbal medicines with relatively high content of chlorogenic acid and materials with abundant resources. Stevia rebaudiana leaves are used as raw materials, related reports of chlorogenic acid obtained through extraction and separation are few, in patent CN102924544A, stevia rebaudiana leaves are used as raw materials, stevioside and chlorogenic acid are prepared step by step, the stevia rebaudiana leaves are soaked in cold water to be extracted, ferrous salt is added, the pH value is adjusted to be flocculated, filtrate and filter cake are obtained through filtration, and the filtrate is subjected to adsorption, desorption and refining treatment through macroporous adsorption resin to obtain stevioside; dissolving the filter cake with water, adjusting acid, adsorbing with macroporous adsorbent resin, resolving, concentrating, drying, and recrystallizing with alcohol to obtain chlorogenic acid. Patent CN105001281A discloses a method for synchronously producing stevioside, flavone and chlorogenic acid, stevia leaves are subjected to water extraction and flocculation, then are filtered to obtain filter mud and filtrate, and the filtrate is subjected to resin purification to obtain a stevioside product; dissolving the filter mud with alcohol water solution, adsorbing with macroporous adsorption resin, performing gradient analysis, preparing chlorogenic acid with low-concentration alcohol analysis solution, concentrating the high-concentration alcohol analysis solution, and extracting with organic solvent to obtain flavone. The method realizes simultaneous extraction of stevioside, flavone and chlorogenic acid, and improves the comprehensive utilization value of stevia, but the obtained chlorogenic acid has low purity.
It can be seen that the current process for extracting chlorogenic acid is relatively mature, but some problems also exist. Firstly, the source of the raw materials is the problem, the source of the raw materials of the chlorogenic acid is single, most of the chlorogenic acid is Chinese herbal medicines and materials with scarce resources, and the chlorogenic acid is concentrated on the honeysuckle, the coffee beans and the eucommia leaves. Secondly, the process has too many extraction steps, uses more organic reagents, increases the cost and has potential safety hazard. More importantly, in the prior art for extracting stevia rebaudiana polyphenol from stevia rebaudiana leaves, a stevia rebaudiana polyphenol mixture is mostly obtained, and is extracted from filter residues obtained by filtering stevia rebaudiana leaves after water extraction and flocculation, so that the extraction efficiency and the product purity are low. In addition, in the prior art, most of polyamide resin used in other plant extraction processes is particles with 30-60 meshes (namely 0.25-0.5mm), which is not beneficial to large-scale extraction.
Disclosure of Invention
[ problem ] to
Solves the problems of single extraction source, complex preparation process, large amount of organic solvent and low yield of chlorogenic acid.
[ solution ]
The invention takes stevia rebaudiana leaves as raw materials and water as extraction media to prepare high-purity chlorogenic acid. The technical scheme provided by the invention comprises the following steps:
(1) leaching and flocculating stevia rebaudiana leaves: soaking dry stevia leaves in water, performing countercurrent extraction, filtering, collecting filtrate to obtain stevia extract, adjusting pH of the stevia extract to acidity, adding chitosan into the stevia extract while stirring, standing for flocculation, and collecting supernatant after flocculation;
(2) and (3) chromatographic purification: filter-pressing the flocculated supernatant with plate-and-frame filter, adsorbing the filtrate with polyamide resin, and collecting the effluent liquid for other use; then, eluting the polyamide resin by using acidified water, and collecting water washing liquor for extracting chlorogenic acid; eluting the polyamide resin by using methanol or ethanol solution with the volume fraction of more than 80 percent, collecting alcohol analysis solution, concentrating, spraying and drying the alcohol analysis solution for other use;
(3) and (3) recrystallization: adsorbing the water washing liquid obtained in the step (2) by using polyamide resin, eluting the polyamide resin by using acidified water, collecting the water washing liquid to obtain enriched water washing liquid, concentrating, adjusting the pH and the temperature, crystallizing while stirring, and performing suction filtration to obtain a filter cake; and then using water as a recrystallization solvent, adjusting the pH, and recrystallizing until the mass content of chlorogenic acid is more than 98%.
In an embodiment of the invention, in the step (1), when stevia rebaudiana leaves are leached, the mass ratio of the dry stevia rebaudiana leaves to water is 1: (3-12), and the water temperature for leaching is 20-60 ℃; adjusting pH of the sweet stevia extractive solution to 3.5-5.0. Preferably, the water temperature for leaching is 35-60 ℃; adjusting pH of the sweet stevia extractive solution to 3.5-4.0.
In one embodiment of the invention, in the step (1), the deacetylation degree of the chitosan is 80% -90%, the mass of the added chitosan is 0.1% -0.2% of the mass of the dry stevia leaves, and the standing flocculation time is 20min-1 h.
In one embodiment of the present invention, the polyamide resin used for enrichment in the steps (2) and (3) is a polyamide resin or a polyamide-like resin; comprises one of polyamide 6(PA6), LX-16, LX-8, polyamide 66(PA66) or poly m-xylylene adipamide (MXD 6).
In one embodiment of the present invention, the acidified water in step (2) and step (3) is an acidic aqueous solution obtained by adjusting the pH to 3.5 to 5.0 with a dilute acid, and the amount of acidified water used is 3 to 5 column volumes.
In one embodiment of the present invention, in the step (3), the pH is adjusted to 3.5 to 5.0, the crystallization temperature is adjusted to 0 ℃ to 25 ℃, and the crystallization solvent is water.
In one embodiment of the present invention, in the step (3), the recrystallization solvent is water, the pH is adjusted to 3.5 to 5.0, the crystallization temperature is 0 ℃ to 25 ℃, the number of recrystallization times is 1 to 2, and the stirring speed is 10 to 30 rpm.
In an embodiment of the present invention, the effluent and alcohol eluent in step (2) contain other polyphenols and steviol glycosides, etc., and the other polyphenols and steviol glycosides can be obtained by further separation and extraction.
[ advantageous effects ]
The method provided by the invention is simple to operate, and the purity of the obtained chlorogenic acid can reach as high as 99%; the chlorogenic acid is prepared through a whole process of water medium, and is green and environment-friendly.
The method provided by the invention expands the utilization field of stevia rebaudiana, does not influence the extraction of stevioside and stevia polyphenol while preparing high-purity chlorogenic acid, and realizes the comprehensive utilization of the stevia rebaudiana.
Drawings
FIG. 1 HPLC chart of chlorogenic acid obtained in example 1 (chlorogenic acid content 99.1%)
FIG. 2 HPLC chart of chlorogenic acid obtained in comparative example 1 of example 1 (chlorogenic acid content 92.49%)
FIG. 3 HPLC chart of chlorogenic acid as an intermediate obtained in example 2 (chlorogenic acid content: 63.2%)
FIG. 4 HPLC chart of chlorogenic acid obtained in example 2 (chlorogenic acid content 98.5%)
FIG. 5 HPLC chart of chlorogenic acid obtained in example 3 (chlorogenic acid content 99%)
FIG. 6 HPLC chart of chlorogenic acid (chlorogenic acid content 80.68%) obtained in comparative example 2 of example 3
FIG. 7 HPLC chart of chlorogenic acid obtained in example 4 (chlorogenic acid content 98.0%)
FIG. 8 HPLC chart of chlorogenic acid prepared with macroporous resin XDA-8 of comparative example 3 (chlorogenic acid content 60.44%)
Detailed Description
Content detection method of chlorogenic acid (HPLC): the content measurement of chlorogenic acid is modified appropriately according to standard T/CCCHMHPIE 1.16-2016 of China medical and health product Committee for import and export. Chlorogenic acid is used as a standard substance. Chromatographic conditions are as follows: detection wavelength of the ultraviolet detector: 330 nm. A chromatographic column: c18(2) in the formula,
4.6X 250 mm; column temperature: at 25 ℃. Mobile phase: acetonitrile-water-formic acid gradient elution with a flow rate of 1.0 mL/min. The amount of sample was 10. mu.L. Mobile phase A: acetonitrile-water-formic acid (250:750:1, mL/mL), mobile phase B: acetonitrile-water-formic acid (100:900:1, mL/mL), gradient run conditions are as follows in Table 1:
TABLE 1
| Time/min | Mobile phase A% | Mobile phase A% |
| 0-20 | 0 | 100 |
| 20-35 | 100 | 0 |
| 35-37 | 100→0 | 0→100 |
| 37-45 | 0 | 100 |
Except for special indication, the deacetylation degree of the chitosan is 80-90%. Among the polyamides and polyamide-like resins used, polyamide 6(PA6), designation 1013B, density of 1.14g/cm3The particle diameter is about 1-3mm, and the product is produced by Nihon Shuyu Xingshi Kabushiki Kaisha; polyamide 66(PA66), designation EPR27, with a density of 1.14g/cm3The diameter of the particles is about 1-3mm, and the product is produced by engineering plastics Limited of Hippocampus; LX-16 and LX-8, selected to have a particle diameter of about 0.8 to 1.25mm, available from Sean blue, advanced science and technology materials, Inc.; poly (m-xylylene adipamide) (MXD6), trade name S6002, density 1.22g/cm3The particle diameter is 1-2.2mm, and is available from Mitsubishi gas chemical corporation.
The present invention will be further described with reference to specific examples.
Example 1:
placing 1kg of dried leaves of stevia rebaudiana Bertoni in a storage tank, extracting with 3X 4L of water at 35 deg.C, and adjusting pH of the obtained stevia rebaudiana Bertoni extract to 3.5 with 0.1mol/L hydrochloric acid. Adding 120mL of 1 wt% chitosan solution into the extractive solution under stirring, standing for 50min, and removing impurities by flocculation. Filtering the supernatant after flocculation and impurity removal by using a plate frame, and adsorbing the filtered supernatant by using PA6 resin, and reserving the effluent for other use; eluting the resin with 3 times of column volume of water, and collecting water eluate for extracting chlorogenic acid. Then, the PA6 resin was resolved with 80 vol% aqueous methanol, and the alcohol resolved solution was collected and further concentrated and dried for other use. Adsorbing chlorogenic acid in the enriched water eluate with PA6 resin, eluting with 3 times of column volume water, collecting and concentrating water eluate, adjusting pH of the eluate to 3.5 with 0.1mol/L hydrochloric acid, crystallizing at 20 deg.C and 10rpm for 12 hr, and vacuum filtering to obtain filter cake; further, water was used as a recrystallization solvent, and the mixture was crystallized again at 20 ℃ and pH 3.5 at 10rpm for 18 hours. Filtering, drying the residue to obtain chlorogenic acid with content of 99.1% (HPLC chromatogram is shown in figure 1).
Comparative example 1: extraction was carried out at pH 7.0, and the conditions were otherwise the same as in example 1.
Placing 1kg of dried leaves of stevia rebaudiana Bertoni in a storage tank, extracting with 3X 4L of water at 35 deg.C, and adjusting pH of the obtained stevia rebaudiana Bertoni extract to 7.0 with 0.05mol/L of sodium bicarbonate. Adding 120mL of 1 wt% chitosan solution into the extractive solution under stirring, standing for 50min, and removing impurities by flocculation. Filtering the supernatant after flocculation and impurity removal by using a plate frame, and adsorbing the filtered supernatant by using PA6 resin, and reserving the effluent for other use; eluting the resin with 3 times of column volume of water, and collecting water eluate for extracting chlorogenic acid. Then, the PA6 resin was resolved with 80 vol% aqueous methanol, and the alcohol resolved solution was collected and further concentrated and dried for other use. Adsorbing chlorogenic acid in the enriched water eluate with PA6 resin, eluting with 3 times of column volume water, collecting and concentrating water eluate, adjusting pH of the eluate to 7.0 with 0.05mol/L sodium bicarbonate, and crystallizing at 20 deg.C and 10rpm for 12 hr; crystallization was again carried out at 20 ℃ and pH 7.0 at 10rpm for 18 h. Filtering, drying the residue to obtain chlorogenic acid with content of 92.49% (HPLC chromatogram is shown in figure 2). Comparing fig. 1 and fig. 2, it can be seen that the purity of the chlorogenic acid obtained is reduced to 92.4% after the pH is adjusted to neutral.
Example 2:
placing 1kg of dried leaves of stevia rebaudiana Bertoni in a storage tank, extracting with 3X 2.5L of water at 60 deg.C, and adjusting pH of the obtained stevia rebaudiana Bertoni extract to 4.5 with 0.1mol/L of acetic acid. Adding 100mL of 1% chitosan solution into the extractive solution under stirring, standing for 20min, and removing impurities by flocculation. Filtering the supernatant after deflocculation and impurity removal by using a plate frame, and adsorbing the filtered supernatant by using PA66 resin, and reserving the effluent for other use; eluting the resin with 4 times column volume of water, and using the water eluate as chlorogenic acid extraction. Then, the PA66 resin was resolved with 80% ethanol, and the alcohol-resolved solution was further concentrated and dried for other use. Adsorbing chlorogenic acid in the water eluate with PA66 resin, eluting with 4 times of column volume water, and collecting chlorogenic acid content of 63.2% (HPLC chromatogram is shown in FIG. 3); after concentration, adjusting the pH value to 4.5 by using 0.1mol/L hydrochloric acid, and then crystallizing for 12 hours at 0 ℃ and 10 rpm; crystallization was again carried out at 0 ℃ and pH 4.5 at 10rpm for 12 h. Filtering, drying the residue to obtain chlorogenic acid with content of 98.5% (HPLC chromatogram is shown in FIG. 4).
Example 3:
placing 1kg of dried leaves of stevia rebaudiana Bertoni in a storage tank, extracting with 3X 3L of water at 50 deg.C, and adjusting pH of the obtained stevia rebaudiana Bertoni extract to 4.0 with 0.1mol/L hydrochloric acid. Adding 120mL of 1% chitosan solution into the extractive solution under stirring, standing for 50min, and removing impurities by flocculation. Filtering the supernatant after deflocculation and impurity removal by using a plate frame, and adsorbing the filtered supernatant by LX-16 resin, and reserving the effluent for other purposes; eluting the resin with 5 times column volume of water, and using the eluate as chlorogenic acid extraction. Then, the LX-16 resin is resolved by 85% methanol, and the alcohol resolved solution is further concentrated and dried for other use. Adsorbing chlorogenic acid in the enriched water eluate with LX-16 resin, eluting with 5 times of column volume water, collecting and concentrating water eluate, adjusting pH of the eluate to 4.0 with 0.1mol/L hydrochloric acid, and crystallizing at 20 deg.C and 10rpm for 12 hr; crystallization was again carried out at 20 ℃ and pH 4.0 at 10rpm for 16 h. Filtering, drying the residue to obtain chlorogenic acid with content of 99.0% (HPLC chromatogram is shown in figure 5).
Comparative example 2: the degree of deacetylation of chitosan was reduced, and the other conditions were the same as in example 3.
Placing 1kg of dried leaves of stevia rebaudiana Bertoni in a storage tank, extracting with 3X 3L of water at 50 deg.C, and adjusting pH of the obtained stevia rebaudiana Bertoni extract to 4.0 with 0.1mol/L hydrochloric acid. Adding 120mL of 1% chitosan (degree of deacetylation is 70%) solution into the extractive solution under stirring, standing for 50min, and removing impurities by flocculation. Filtering the supernatant after deflocculation and impurity removal by using a plate frame, and adsorbing the filtered supernatant by LX-16 resin, and reserving the effluent for other purposes; eluting the resin with 5 times column volume of water, and using the eluate as chlorogenic acid extraction. Then, the LX-16 resin is resolved by 85% methanol, and the alcohol resolved solution is further concentrated and dried for other use. Adsorbing chlorogenic acid in the enriched water eluate with LX-16 resin, eluting with 5 times of column volume water, collecting and concentrating water eluate, adjusting pH of the eluate to 4.0 with 0.1mol/L hydrochloric acid, and crystallizing at 20 deg.C and 10rpm for 12 hr; crystallization was again carried out at 20 ℃ and pH 4.0 at 10rpm for 16 h. Filtering, drying the residue to obtain chlorogenic acid with content of 80.68% (HPLC chromatogram is shown in FIG. 6).
Example 4:
placing 1kg of dried leaves of stevia rebaudiana Bertoni in a storage tank, extracting with 3X 2L of water at 60 deg.C, and adjusting pH of the obtained stevia rebaudiana Bertoni extract to 4.0 with 0.1mol/L hydrochloric acid. Adding 100mL of 1% chitosan solution into the extractive solution under stirring, standing for 40min, and removing impurities by flocculation. Filtering the supernatant after deflocculation and impurity removal by using a plate frame, and adsorbing the filtered supernatant by LX-8 resin, and reserving the effluent for other use; the resin was then eluted with water and the eluate was used to extract chlorogenic acid. Then, the LX-8 resin is resolved by 90 percent ethanol, and the alcohol resolved solution is further concentrated and dried for other use. Enriching chlorogenic acid in water eluate with LX-8 resin, concentrating the eluate after resin enrichment, adjusting pH of the eluate to 4.0 with 0.1mol/L hydrochloric acid, and crystallizing at 10 deg.C and 30rpm for 12 hr; crystallization was again carried out at 10 ℃ and pH 4.0 at 30rpm for 16 h. Filtering, drying the residue to obtain chlorogenic acid with content of 98.0% (HPLC chromatogram is shown in FIG. 7).
Comparative example 3:
placing 1kg of dried leaves of stevia rebaudiana Bertoni in a storage tank, extracting with 3X 3L of water at 50 deg.C, and adjusting pH of the obtained stevia rebaudiana Bertoni extract to 3.5 with 0.1mol/L hydrochloric acid. Adding 100mL of 1% chitosan solution into the extractive solution under stirring, standing for 30min, and removing impurities by flocculation. Filtering the supernatant after deflocculation and impurity removal by using a plate frame, and adsorbing the filtered supernatant by XDA-8 resin, and reserving the effluent for other use; the resin was then eluted with water and the eluate was used to extract chlorogenic acid. Then, the XDA-8 resin was resolved with 85% methanol, and the alcohol-resolved solution was further concentrated and dried for other use. Enriching chlorogenic acid in water eluate with XDA-8 resin, concentrating the eluate after resin enrichment, adjusting pH of the eluate to 3.5 with 0.1mol/L hydrochloric acid, crystallizing at 5 deg.C and 10rpm for 12 hr; crystallization was again carried out at 5 ℃ and pH 3.5 at 10rpm for 12 h. Filtering, drying the residue to obtain chlorogenic acid with content of 60.44% (HPLC chromatogram is shown in figure 8).
Example 5:
placing 1kg of dried leaves of stevia rebaudiana Bertoni in a storage tank, extracting with 3X 3L of water at 40 deg.C, and adjusting pH of the obtained stevia rebaudiana Bertoni extract to 4.0 with 0.1mol/L hydrochloric acid. Adding 120mL of 1% chitosan solution into the extractive solution under stirring, standing for 50min, and removing impurities by flocculation. Filtering the supernatant after deflocculation and impurity removal by using a plate frame, and adsorbing the filtered supernatant by MXD6 resin to obtain an effluent liquid for other purposes; eluting the resin with 3 times of column volume of water, and using the eluate as chlorogenic acid extraction. The MXD6 resin was then digested with 85% methanol, and the alcohol-resolved solution was further concentrated and dried for further use. Adsorbing chlorogenic acid in the enriched water eluate with MXD6 resin, eluting with 3 times of column volume water, collecting and concentrating the effluent water eluate, adjusting pH of the eluate to 4.0 with 0.1mol/L hydrochloric acid, and crystallizing at 20 deg.C and 10rpm for 12 hr; crystallization was again carried out at 20 ℃ and pH 4.0 at 10rpm for 16 h. Filtering, and oven drying to obtain chlorogenic acid with content of 98.9%.
Although the present invention has been described with respect to the preferred embodiments, it should be understood that the invention is not limited thereto, and various changes and modifications can be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. A method for preparing chlorogenic acid from stevia rebaudiana is characterized by taking water as an extraction medium and comprising the following steps:
(1) leaching and flocculating stevia rebaudiana leaves: soaking dry stevia leaves in water, performing countercurrent extraction, filtering, collecting filtrate to obtain stevia extract, adjusting pH of the obtained stevia extract to 3.5-5.0, adding chitosan with deacetylation degree of 80-90% into the above stevia extract under stirring, standing for flocculation, and collecting flocculated supernatant;
(2) and (3) chromatographic purification: filter-pressing the flocculated supernatant with plate-and-frame filter, adsorbing the filtrate with polyamide resin, and collecting the effluent liquid for other use; then, eluting the polyamide resin by using acidified water, and collecting water washing liquor for extracting chlorogenic acid; eluting the polyamide resin by using methanol or ethanol solution with the volume fraction of more than 80 percent, collecting alcohol analysis solution, concentrating, spraying and drying the alcohol analysis solution for other use;
(3) and (3) recrystallization: adsorbing the water washing liquid obtained in the step (2) by using polyamide resin, eluting the polyamide resin by using acidified water, collecting the water washing liquid to obtain enriched water washing liquid, concentrating, adjusting the pH to 3.5-5.0, adjusting the temperature to 0-25 ℃, stirring while crystallizing, and performing suction filtration to obtain a filter cake; then using water as a recrystallization solvent, adjusting the pH value to 3.5-5.0, and recrystallizing until the mass content of chlorogenic acid is more than 98%;
the polyamide resin used for enrichment in the step (2) and the step (3) is one of polyamide 6, LX-16, LX-8, polyamide 66 or poly m-xylylene adipamide.
2. The method for preparing chlorogenic acid from stevia rebaudiana bertoni according to claim 1, wherein in the step (1), when stevia rebaudiana bertoni leaves are leached, the mass ratio of dry stevia rebaudiana bertoni leaves to water is 1: (3-12), and the water temperature for leaching is 20-60 ℃; adjusting pH of the sweet stevia extractive solution to 3.5-5.0.
3. The method for preparing chlorogenic acid from stevia rebaudiana bertoni according to claim 1 or 2, wherein in step (1), the deacetylation degree of chitosan is 80% -90%, the mass of chitosan added is 0.1% -0.2% of the feeding mass of dry stevia rebaudiana bertoni leaves, and the standing flocculation time is 20min-1 h.
4. The method of claim 1, wherein the acidified water used in steps (2) and (3) is an acidic aqueous solution obtained by adjusting pH to 3.5-5.0 with a dilute acid, and the acidified water is used in an amount of 3-5 column volumes.
5. The method of claim 1, wherein in step (3), the pH is adjusted to 3.5-5.0, the crystallization temperature is adjusted to 0-25 ℃, and the crystallization solvent is water.
6. The method of claim 1, wherein in the step (3), the recrystallization solvent is water, the pH is adjusted to 3.5 to 5.0, the crystallization temperature is 0 ℃ to 25 ℃, the number of recrystallization times is 1 to 2, and the stirring speed is 10 to 30 rpm.
7. The method for preparing chlorogenic acid from stevia rebaudiana as claimed in claim 1, wherein the effluent and alcohol desorption solution in step (2) contain other polyphenols and steviol glycosides, and the other polyphenols and steviol glycosides can be obtained by further separation and extraction.
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| CN104151378A (en) * | 2014-08-12 | 2014-11-19 | 济南汉定生物工程有限公司 | Rebaudioside M purification method |
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