CN111676264B - Method for extracting caffeic acid from rosemary - Google Patents
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Abstract
The invention discloses a method for extracting caffeic acid from rosemary, which comprises the following steps: (1) Crushing rosemary, extracting with ethanol at 80-83 ℃ to obtain an extracting solution, and carrying out solid-liquid separation on the extracting solution to obtain a clear extracting solution; (2) Concentrating the clarified extract to Brix of 40-60%, cooling, and adding carbon source; (3) Inoculating mixed bacteria liquid formed by lactic acid bacteria and saccharomycetes into the step (2), adjusting the pH value, and fermenting to obtain liquid; (4) Adding water into the liquid until Brix is 11-12%, adjusting the pH value to 4-6, and carrying out solid-liquid separation to obtain a clarified liquid; (5) Adding the clarified liquid into macroporous adsorption resin, washing the macroporous adsorption resin, and concentrating under reduced pressure until Brix is 30-35%; (6) Decolorizing and filtering the concentrated solution, concentrating again, and drying to obtain caffeic acid extract. The method has the advantages of high content of effective components, environmental friendliness, high extraction efficiency and low cost, and is suitable for industrial production.
Description
Technical Field
The invention relates to a method for extracting caffeic acid from rosemary, and belongs to the technical field of biological medicine.
Background
Rosemary is a plant of the genus rosemary of the family Labiatae. The rosemary is a plant with abundant use value, is native to Mediterranean regions, is widely cultivated in countries around the world, and is used for enhancing memory, treating language disorder, conditioning menstruation, promoting hair growth, promoting digestion and protecting liver in some European countries before 2000. In Ben Cao Shi Yi (materia Medica pickup), rosemary is pungent, warm and nontoxic. And (3) the main bad smell. At present, the regions of Yunnan, guizhou, guangxi, hainan, xinjiang and the like in China are planted in a large area.
For the plant research, there are a great deal of reports and patents abroad, and the components of the plant research mainly comprise diterpenoid phenols, flavonoids, triterpenes, essential oil and the like, and the plant research is widely applied to the aspects of medicines, cosmetics, foods and the like. Rosemary is widely used as a food additive nowadays, and the antibacterial and antioxidant effects of rosemary can improve the food quality and prolong the preservation time. In recent years, attention of rosemary has been raised, and many reports have been made about chemical components and pharmacological actions of rosemary. The rosmarinic acid contained in rosemary is absorbed and metabolized in vivo in the form of conjugate or methylation, and the absorbed rosmarinic acid is mostly degraded into the conjugate and/or methylation of caffeic acid, ferulic acid and cinnamic acid, and then gradually secreted in urine. The composition has various pharmacological effects of resisting bacteria, inflammation, viruses, oxidization and apoptosis, scavenging free radicals, inhibiting immunity and the like, particularly has the protective effect on myocardial cell injury, resisting nerve cell apoptosis, inducing T cell apoptosis, resisting cancer, having strong anti-amyloid generation effect on Alzheimer's beta-amyloid fibril and the like, and has very wide application prospect and market value.
Caffeic acid, also known as hydrolyzed caffeic tannic acid, 3, 4-dihydroxycinnamic acid, 3- (3, 4-dihydroxyphenyl) -2-acrylic acid, is a cinnamic acid compound; is yellow crystal, slightly soluble in cold water, and easily soluble in hot water and cold ethanol. Is commonly found in common herbal medicines such as radix Angelicae sinensis and rhizoma Ligustici Chuanxiong, and is commonly found in flowers, leaves or fruits of plants of Compositae, rosaceae, sapindaceae, ranunculaceae, nymphaeaceae, rutaceae, polygonaceae, patriniaceae, labiatae, and Eucommiaceae. Has antibacterial, antiviral, antivenin, antidiarrheal, antifertility, antitumor and other pharmacological activities, and also has blood cooling and hemostatic effects, and is used for treating hypertension, coronary heart disease, arrhythmia, etc. As a representative compound of hydroxycinnamic acid, caffeic acid has a strong antioxidant activity.
There is no report of extracting caffeic acid from rosemary. The caffeic acid is mainly extracted from plants rich in caffeic acid, but the content of caffeic acid in plants is not high, and the caffeic acid is obtained from rosemary directly and is not very well harvested, so that a new way needs to be searched for to obtain the caffeic acid.
The research shows that the rosemary contains more rosmarinic acid and less caffeic acid, and the invention provides a preparation method of caffeic acid, which is used for extracting the rosmarinic acid and then converting the rosmarinic acid into the caffeic acid through fermentation, thereby greatly improving the yield of the caffeic acid obtained from the rosemary and realizing the efficient utilization of rosemary resources.
Disclosure of Invention
The invention aims to provide a method for efficiently and economically extracting caffeic acid from rosemary, and simultaneously, the content of caffeic acid in the extract is increased.
A method for extracting caffeic acid from rosemary, comprising the steps of:
s1, crushing rosemary, extracting with ethanol at 80-83 ℃, and carrying out solid-liquid separation on the extract to obtain clear extract;
s2, concentrating the clarified extract obtained in the step S1 to 40-60% Brix to obtain a concentrated solution, and adding a carbon source;
s3, inoculating fermentation bacteria into the concentrated solution added with the carbon source in the step S2, regulating the pH value to 4-6, and fermenting to obtain caffeic acid liquid;
s4, adding water into the caffeic acid liquid obtained in the step S3 to Brix of 11-12%, adjusting the pH value to 4-6, and carrying out solid-liquid separation to obtain clear supernatant;
s5, adding the supernatant obtained in the step S4 into macroporous adsorption resin, and washing the macroporous adsorption resin with water to remove impurities which are not adsorbed; washing macroporous adsorption resin with desorption solution with pH of 4-6, collecting the washed desorption solution, and concentrating under reduced pressure to Brix 30-35%;
s6, decoloring and filtering the concentrated solution, concentrating the filtrate again, and drying to obtain the caffeic acid extract.
Preferably, in the step S1, the rosemary is crushed to a diameter of less than 0.5 cm.
Preferably, the concentration of ethanol in the step S1 is 50% -60%.
The concentration of the ethanol is 50% -60%, and because the solubility of the rosmarinic acid in the concentration is proper, the impurity bringing the alcohol solubility is avoided as few as possible, the burden of the post treatment process is increased, and the production efficiency is improved.
Preferably, the extraction in step (1) is a leakage extraction.
Preferably, the carbon source is glucose.
Preferably, the solid-liquid separation is filtration or centrifugation.
Preferably, the fermentation bacteria comprise 10-90% of lactic acid bacteria and 10-90% of saccharomycetes by mass percent.
Preferably, the fermentation bacteria comprise 60-80% of lactic acid bacteria and 20-40% of saccharomycetes by mass percent.
Preferably, the fermentation bacteria comprise 70% of lactic acid bacteria and 30% of saccharomycetes in percentage by mass.
Caffeine is a xanthine alkaloid compound, caffeine is prepared through biotransformation, lactobacillus and saccharomycetes are the most effective biological bacteria for plant biological fermentation, and are probiotic bacteria, and in theory, the fermentation process of generating lactic acid through anaerobic glycolysis by a large number of lactobacillus fermentation, and ethanol fermentation is the same as two main fermentation forms in organisms, so that the fermentation can be promoted to be smoothly carried out. And a small amount of saccharomycetes are matched for fermentation, so that sugar is decomposed into carbon dioxide and water under the condition of oxygen, the saccharomycetes grow faster, and under the condition of oxygen deficiency, the saccharomycetes decompose the sugar into alcohol and carbon dioxide, so that the fermentation process is expanded, and the bioconversion process of caffeine is facilitated. Therefore, a larger proportion of lactic acid bacteria is preferred.
Preferably, the fermentation temperature is 35-37 ℃ and the period is 7-10 days.
Preferably, the pH-adjusted solution is dilute hydrochloric acid.
Preferably, the macroporous adsorption resin is NKA-9 macroporous resin, D1O1 macroporous resin, D301 macroporous basic resin, D201 macroporous basic resin, 001×7Na732.
Preferably, the macroporous adsorption resin is a mesoporous macroporous adsorption resin.
Preferably, the macroporous adsorption resin is NKA-9 macroporous resin or D1O1 macroporous resin.
More preferably, the macroporous adsorbent resin is NKA-9 macroporous adsorbent resin.
The NKA-9 macroporous adsorption resin is an adsorbent taking divinylbenzene as a framework structure, and benzene rings connected to a main chain are planes with uniform electron distribution, have strong adsorption capacity for some molecules with similar properties and various cyclic aromatic contents, and increase along with the enhancement of the lipophilicity of adsorbed molecules. Widely used for separating ginkgo flavone, polyphenol, caffeic acid and the like. It has recently shown unique effects in the separation of natural products, especially in the separation and purification of water-soluble contents.
Preferably, the desorption solution is ethanol solution with the volume percentage concentration of 50-60%.
Preferably, the activated carbon filter cloth for decoloration and filtration has a mesh number of 325 mesh.
Preferably, the drying is vacuum drying or spray drying, until the water content of the caffeic acid extract is not more than 5%.
At present, similar substances are usually extracted by alcohol, and impurities are removed by long-time precipitation at low temperature after concentration, and the main defects are high temperature requirement (below 4 ℃) and incomplete impurity removal, so that the production cost is high, the content of active ingredients is low, and industrialization is difficult. The caffeic acid prepared by the traditional chemical synthesis method has the advantages of long reaction time, non-ideal purity, high residue of harmful substances in the product, non-ideal caffeic acid product and the like. According to the invention, rosmarinic acid and caffeic acid are extracted through 60% -65% ethanol, and after concentration, the rosmarinic acid is converted into caffeic acid through fermentation, so that the yield of the caffeic acid obtained from rosemary is greatly improved, and the efficient utilization of rosemary resources is realized. Before the separation of the resin column, the invention also adds water to the obtained liquid until Brix is 10-12%, and adjusts the pH value to 4-6 by acid, under the condition, the impurities in the solution are rapidly precipitated under the acidic condition, the low-temperature long-time standing is not needed, the separation is carried out immediately, and the production period is greatly shortened. Separating with macroporous adsorbent resin column to obtain product with high effective components. The concentrated solution is decolorized and filtered by using 325 mesh active carbon filter cloth, so that partial colored substances and water insoluble impurities can be removed. The content of target product in the extract is generally 10-20% by using the current common extraction method, and the content of caffeic acid in the extract can reach 50-60% by using the method of the invention. The product prepared by the invention is mainly used as a medicine raw material.
Compared with the prior art, the invention has the following beneficial effects:
1. the method has the advantages of good extraction consistency of caffeic acid, simple process and suitability for industrial popularization.
2. The invention adopts mixed bacteria composed of lactobacillus and saccharomycetes and rosmarinic acid to ferment at the pH value of 4-6 and Brix of 40-60 percent to obtain caffeic acid, and 6-P-beta glucosidase participating in the metabolism of phenolic compounds is contained in the fermentation process, and Gallate decarboxylase and P-coumaric acid carboxylase convert the rosmarinic acid into the caffeic acid, thereby realizing the improvement of the content of the caffeic acid.
3. The invention adopts the leakage method to extract the caffeic acid, so that the effective components in the rosemary are quickly and fully dissolved in a short time, the reaction condition is mild, the structure protection of active substances is facilitated, new organic impurities are not introduced, the safety of the final product is ensured, and the environmental pollution is also reduced.
4. The method has reasonable process, high content of effective components, high extraction efficiency and simple operation, and is suitable for industrial production.
5. The extract prepared by the invention has stable purity and the caffeic acid content is between 50 and 60 percent.
6. The solvent of the invention can be recycled, and is economical and environment-friendly.
Drawings
FIG. 1 is a graphical representation of the effect of rosmarinic acid on ethanol extraction at various concentrations in example 1;
FIG. 2 is a graphical representation of the effect of rosmarinic acid on fermentation broth in example 1;
wherein the lactic acid bacteria are: the saccharomycete comprises the following components in percentage by weight: 5. d7: 3. e9:1, a step of;
FIG. 3 is a graphical representation of the selection of conditions for various macroporous adsorbent resins for rosmarinic acid in example 2;
wherein A is NKA-9 macroporous resin, B is D1O1 macroporous resin, C is D301 macroporous alkaline resin, D is D201 macroporous alkaline resin, E is 001×7Na732
The ordinate of fig. 1 to 3 is the caffeic acid content of the product.
FIG. 4 is a high performance liquid chromatography detection chart of caffeic acid in example 3.
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Example 1
Weighing 500g of dried rosemary pieces, adding 10L of ethanol (30%, 40%, 50%, 60%, 70% and 80%), extracting under reflux for 2 hours, filtering, concentrating the filtrate under reduced pressure to 40-60% of Brix, standing, cooling, and adding 50g of glucose for uniform mixing. Adding 5mL of mixed bacteria liquid of 70% of lactobacillus and 30% of saccharomycetes into the concentrated solution, uniformly mixing, controlling the temperature at 37 ℃, carrying out heat preservation and culture for 7 days, adding water into the obtained liquid to reach Brix of 10-12%, adjusting the pH value to 4-6 by using dilute hydrochloric acid, and centrifuging to obtain clarified centrifugate; passing the supernatant through NKA-9 macroporous adsorbent resin; after the column is finished, eluting impurities which are not adsorbed by the resin by using pure water, desorbing by using 50-60% ethanol solution with the pH of 4-6, starting to receive liquid when effluent is slightly acidic, flushing the effluent to be neutral by using deionized water after finishing the application of the caffeic acid solution, stopping the application of water and the receiving liquid, concentrating under reduced pressure to Brix 30-35%, decolorizing and filtering the concentrated solution by using 325-mesh active carbon filter cloth, concentrating again, drying in vacuum, and detecting the caffeic acid content.
As shown in the results of FIG. 1, when the ethanol concentration is 30% -40%, the caffeic acid content is 22.1% -42.7%, and the caffeic acid content is gradually increased along with the increase of the ethanol proportion. At an ethanol concentration of 60, the caffeic acid content was 57.1% to the highest value, which then decreased with increasing ethanol concentration.
Therefore, the extraction is preferably performed with an ethanol concentration of 50% -60%.
Example 2
Weighing 500g of dried rosemary pieces, adding 10L of 60% -65% ethanol, carrying out reflux extraction for 2 hours, filtering, concentrating the filtrate under reduced pressure to 40% -60% Brix, standing, cooling, and adding 50g of glucose for uniform mixing. Lactic acid bacteria: adding 5mL of mixed bacteria liquid with the saccharomycete proportion of (A1:9, B3:7, C5:5, D7:3 and E9:1) into the concentrated solution, uniformly mixing, controlling the temperature at 37 ℃, carrying out heat preservation and culture for 7 days, adding water into the obtained liquid until Brix is 10-12%, regulating the pH value by dilute hydrochloric acid to 4-6, and centrifuging to obtain clear centrifugate; passing the supernatant through NKA-9 macroporous adsorbent resin; after the column is finished, eluting impurities which are not adsorbed by the resin by using pure water, desorbing by using 50-60% ethanol solution with the pH of 4-6, starting to receive liquid when effluent is slightly acidic, flushing the effluent to be neutral by using deionized water after finishing the application of the caffeic acid solution, stopping the application of water and the receiving liquid, concentrating under reduced pressure to Brix 30-35%, decolorizing and filtering the concentrated solution by using 325-mesh active carbon filter cloth, concentrating again, drying in vacuum, and detecting the caffeic acid content.
As shown in the results of fig. 2, lactic acid bacteria: when the ratio of the saccharomycetes is 1:9, the content of the caffeic acid is only 14.8%, and the content of the caffeic acid is gradually increased along with the increase of the ratio of the lactic acid bacteria. In lactic acid bacteria: when the ratio of the saccharomycetes is 7:3, the content of the caffeic acid reaches 57.4 percent to reach the highest value, and then the caffeic acid is reduced along with the increase of the lactobacillus.
Therefore, the mixed bacteria liquid with the proportion of 70% of lactobacillus and 30% of saccharomycetes is preferable to ferment.
Example 2
Weighing 500g of dried rosemary pieces, adding 10L of 60% -65% ethanol, carrying out reflux extraction for 2 hours, filtering, concentrating the filtrate under reduced pressure to 40% -60% Brix, standing, cooling, and adding 50g of glucose for uniform mixing. Adding 5mL of mixed bacteria liquid of 70% lactic acid bacteria and 30% saccharomycetes into the concentrated solution, uniformly mixing, controlling the temperature at 37 ℃, carrying out heat preservation and culture for 7 days, adding water into the obtained liquid until Brix is 10-12%, adjusting the pH value by dilute hydrochloric acid to 4-6, and centrifuging to obtain clarified centrifugate; passing the supernatant through macroporous adsorbent resin (A: NKA-9 macroporous resin, B: D1O1 macroporous resin, C: D301 macroporous basic resin, D: D201 macroporous basic resin, E:001×7Na732); after the column is finished, eluting impurities which are not adsorbed by the resin by using pure water, desorbing by using 50-60% ethanol solution with the pH of 4-6, starting to receive liquid when effluent is slightly acidic, flushing the effluent to be neutral by using deionized water after finishing the application of the caffeic acid solution, stopping the application of water and the receiving liquid, concentrating under reduced pressure to Brix 30-35%, decolorizing and filtering the concentrated solution by using 325-mesh active carbon filter cloth, concentrating again, drying in vacuum, and detecting the caffeic acid content.
As shown in FIG. 3, the effect of different adsorption resins on the total coumarin content was very pronounced. The adsorption resin is D1O1, and the total coumarin content is only 12.6%. The adsorption resin is D301 and NKA-9, and the total coumarin content can reach 42.4% and 58.4%.
Therefore, a medium-polarity NKA-9 macroporous resin is preferred.
Example 3
Weighing 10kg of dried rosemary pieces, putting the dried rosemary pieces into a 200L extraction tank, adding 120L of 60% -65% ethanol, and stirring; and (3) when the jacket steam is opened to heat the jacket to 80-83 ℃, circulating for 2 hours, then starting normal percolation, collecting the leakage liquid until the Brix value is less than 1%, stopping collecting, filtering, concentrating the filtrate under reduced pressure to 40-60% of Brix, standing, cooling, and adding 1kg of glucose for uniform mixing. Adding 10mL of mixed bacteria liquid of 70% lactic acid bacteria and 30% saccharomycetes into the concentrated solution, uniformly mixing, controlling the temperature at 37 ℃, carrying out heat preservation and culture for 7 days, adding water into the obtained liquid to reach Brix of 10-12%, adjusting the pH value to 4-6 by using dilute hydrochloric acid, and centrifuging to obtain clarified centrifugate; passing the centrifuged supernatant through 25L of NKA-9 macroporous adsorbent resin at a flow rate of 25L/h; after the column is finished, eluting impurities which are not adsorbed by the resin by using 150L of pure water at a flow rate of 50L/h, desorbing by using 25L of 50-60% ethanol solution with pH of 4-6 at a flow rate of 10L/h, and starting to receive liquid when the effluent is slightly acidic. Washing with deionized water to neutrality after finishing the application of the caffeic acid solution, stopping the application of water and receiving liquid, concentrating under reduced pressure to Brix 30-35%, decolorizing and filtering the concentrated solution with 325 mesh active carbon filter cloth, concentrating the filtrate again to solid content above 50%, and vacuum drying to obtain 462g dry block with caffeic acid content of 57.3%. The liquid chromatography data of caffeic acid is shown in fig. 4A.
Example 4
Weighing 300kg of dried rosemary pieces, putting the dried rosemary pieces into a 5000L extraction tank, adding 3000L of 60% -65% ethanol, and stirring; when the jacket steam is opened and heated to 80-83 ℃, circulation is carried out for 2 hours, normal percolation is started, the leakage liquid is collected until the Brix value is less than 1%, collection is stopped, filtration is carried out, the filtrate is concentrated to 40-60% of Brix under reduced pressure, standing is carried out, cooling is carried out, and 30kg of glucose is added and mixed uniformly. Adding 300mL of mixed bacteria solution of 70% lactic acid bacteria and 40% saccharomycetes into the concentrated solution, uniformly mixing, controlling the temperature at 37 ℃, carrying out heat preservation and culture for 7 days, adding water into the obtained solution to reach Brix of 10-12%, adjusting the pH value to 4-6 by using dilute hydrochloric acid, and centrifuging to obtain clarified centrifugate; passing the centrifuged supernatant through 1000L of NKA-9 macroporous adsorbent resin at a flow rate of 1000L/h; after the column is finished, 3000L pure water is used for eluting impurities which are not adsorbed by the resin at a flow rate of 2000L/h, then 1500L of 50-60% ethanol solution with pH of 4-6 is used for desorbing at a flow rate of 500L/h, and when the effluent is slightly acidic, the liquid starts to be received. Washing with deionized water to neutrality after finishing the application of the caffeic acid solution, stopping the application of water and receiving liquid, concentrating under reduced pressure to Brix 30-35%, decolorizing and filtering the concentrated solution with 325 mesh active carbon filter cloth, concentrating the filtrate again to solid content above 50%, and vacuum drying to obtain 13.27kg dry block with caffeic acid content of 58.1%. The liquid chromatography data of caffeic acid is shown in fig. 4B. The extraction rate of total coumarin is 58.1% after amplified extraction, and the total coumarin is consistent with the pilot scale level, and the chromatogram is to show that the product composition and content are highly consistent, so that the caffeic acid content in industrial production is greatly improved, and the industrial application prospect is excellent.
The foregoing examples are set forth in order to provide a more thorough description of the present invention, and are not intended to limit the scope of the invention, since modifications of the invention in various equivalent forms will occur to those skilled in the art upon reading the present invention, and are within the scope of the invention as defined in the appended claims.
Claims (4)
1. A method for extracting caffeic acid from rosemary, comprising the steps of:
s1, crushing rosemary, extracting with ethanol at 80-83 ℃, and carrying out solid-liquid separation on the extracting solution to obtain clear extracting solution;
s2, concentrating the clarified extract obtained in the step S1 to 40-60% Brix to obtain a concentrated solution, and adding a carbon source;
s3, inoculating fermentation bacteria into the concentrated solution added with the carbon source in the step S2, regulating the pH value to 4-6, and fermenting to obtain caffeic acid liquid;
s4, adding water into the caffeic acid liquid obtained in the step S3 to Brix of 11-12%, adjusting the pH value to 4-6, and carrying out solid-liquid separation to obtain clear supernatant;
s5, adding the supernatant obtained in the step S4 into macroporous adsorption resin, and washing the macroporous adsorption resin with water to remove impurities which are not adsorbed; washing macroporous adsorption resin with desorption solution with pH of 4-6, collecting the washed desorption solution, and concentrating under reduced pressure to Brix 30-35%;
s6, decoloring and filtering the concentrated solution, concentrating the filtrate again, and drying to obtain a caffeic acid extract;
the concentration of ethanol in the step S1 is 50% -60%;
the fermentation bacteria comprise 70% of lactic acid bacteria and 30% of saccharomycetes by mass percent;
the macroporous adsorption resin is NKA-9 macroporous adsorption resin.
2. The method according to claim 1, wherein in step S1, rosemary is crushed to a diameter of less than 0.5cm, and the extraction is a leakage extraction.
3. The method of claim 1, wherein the carbon source is glucose; the fermentation temperature is 35-37 ℃ and the period is 7-10 days; the solid-liquid separation is filtration or centrifugation.
4. The method of claim 1, wherein the desorption solution is an ethanol solution with a volume percentage concentration of 50-60%; the mesh number of the activated carbon filter cloth for decoloring and filtering is 325 meshes; the drying is vacuum drying or spray drying, and the water content of the caffeic acid extract is not more than 5%.
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