阅读说明：本技术 一种从迷迭香中提取咖啡酸的方法 (Method for extracting caffeic acid from rosemary ) 是由 罗华 张有发 黄六仔 卢小刚 于 2020-06-28 设计创作，主要内容包括：本发明公开了一种从迷迭香中提取咖啡酸的方法,该方法包括以下步骤：(1)将迷迭香破碎,用乙醇在80～83℃提取,得提取液,将提取液固液分离,得澄清的提取液；(2)将澄清的提取液浓缩至Brix40～60%,冷却,加入碳源；(3)将以乳酸菌和酵母菌构成的混菌液接种至步骤(2)中,调整pH值,发酵得到液体；(4)将液体加水至Brix11～12%,并将pH值调至4~6,固液分离,得澄清液；(5)将澄清液加入大孔吸附树脂中,洗涤大孔吸附树脂、减压浓缩至Brix30～35%；(6)对浓缩液脱色过滤,再次浓缩,干燥得咖啡酸提取物。该方法有效成分含量高、对环境友好、提取效率高,成本较低、适合工业化生产。(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 clear extracting solution to Brix 40-60%, cooling, and adding a carbon source; (3) inoculating the mixed bacteria liquid consisting of 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 the Brix 11-12% is reached, 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 30-35%; (6) decolorizing and filtering the concentrated solution, concentrating again, and drying to obtain caffeic acid extract. The method has high effective component content, environment friendliness, high extraction efficiency, low cost, and suitability for industrial production.)

1. A method for extracting caffeic acid from rosemary is characterized by comprising the following steps:

s1, crushing rosemary, extracting with ethanol at 80-83 ℃, and carrying out solid-liquid separation on the extracting solution to obtain a clear extracting solution;

s2, concentrating the clear extracting solution obtained in the step S1 to Brix 40-60% to obtain a concentrated solution, and adding a carbon source;

s3, inoculating zymophyte into the concentrated solution added with the carbon source in the step S2, adjusting 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 11-12%, adjusting the pH value to 4-6, and performing 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 unadsorbed impurities; washing the macroporous adsorption resin with desorption solution with the pH value of 4-6, collecting the washed desorption solution, and concentrating under reduced pressure to Brix 30-35%;

s6, decolorizing and filtering the concentrated solution, concentrating the filtrate again, and drying to obtain the caffeic acid extract.

2. The method according to claim 1, wherein in step S1 rosemary is crushed to a diameter of 0.5cm or less and the extraction is a percolation extraction.

3. The method according to claim 1, wherein the ethanol concentration in the step S1 is 50% -60%.

4. 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.

5. The method according to claim 1, wherein the fermentation tubes comprise 10-90% by mass of lactic acid bacteria and 10-90% by mass of yeast.

6. The method according to claim 1, wherein the fermentation tubes comprise 60-80% of lactic acid bacteria and 20-40% of yeast by mass.

7. The method according to claim 1, wherein the fermentation tubes comprise 70% of lactic acid bacteria and 30% of yeast by mass.

8. The method of claim 1, wherein the macroporous adsorbent resin is NKA-9 macroporous resin, D1O1 macroporous resin, D301 macroporous basic resin, D201 macroporous basic resin, 001 x 7Na 732.

9. The method of claim 1, wherein the macroporous adsorbent resin is a medium polarity macroporous adsorbent resin; preferably, the macroporous adsorption resin is NKA-9 macroporous adsorption resin.

10. The method according to claim 1, wherein the desorption solution is an ethanol solution with a concentration of 50-60% by volume; the active carbon filter cloth for decolorization and filtration has 325 meshes; the drying is vacuum drying or spray drying until the water content of the caffeic acid extract is not more than 5%.

Technical Field

The invention relates to a method for extracting caffeic acid from rosemary, and belongs to the technical field of biological medicines.

Background

Rosmarinus officinalis is a plant of Rosmarinus of Labiatae. The rosemary is a plant with abundant use value, is originally produced in mediterranean regions, is widely cultivated in countries in the world at present, and is used for enhancing memory, treating language disorder, conditioning menstruation, growing hair, promoting digestion and protecting liver in European countries more than 2000 years ago. Rosemary, recorded in Bencao Shi Yi, is pungent, warm and non-toxic. Mainly for foul breath. At present, China has large-area planting in Yunnan, Guizhou, Guangxi, Hainan, Xinjiang and other places.

For the plant research, a large number of reports and patents are provided abroad, and the components of the plant mainly comprise diterpene phenols, flavonoids, triterpenes, essential oil and the like, and the plant is widely applied to the aspects of medicines, cosmetics, foods and the like. Rosemary is also widely used as food additive, and its antibacterial and antioxidant effects can improve food quality and prolong fresh-keeping time. In recent years, the interest of rosemary has been increasing, and many reports have been made about the chemical components and pharmacological actions of rosemary. Rosemary contains rosmarinic acid which is absorbed and metabolized in vivo in a conjugate or methylated form, and most of the absorbed rosmarinic acid is degraded into a conjugate of caffeic acid, ferulic acid and cinnamic acid, or methylated form, and then gradually secreted into urine. Has various pharmacological actions such as antibacterial action, anti-inflammatory action, antivirus action, antioxidation action, anti-apoptosis action, free radical removal action, immunosuppression action and the like, particularly has the protection action on myocardial cell injury, nerve cell apoptosis resistance, T cell apoptosis induction and anticancer action, has strong amyloid generation resistance on Alzheimer beta-amyloid fibrils, and the like, so that the application prospect and market value of the novel anti-tumor medicine are very wide.

Caffeic acid is also called hydrolytic caffeotannic acid, 3, 4-dihydroxy cinnamic acid, 3- (3, 4-dihydroxy phenyl) -2-acrylic acid, and is cinnamic acid compound; it is yellow crystal, slightly soluble in cold water, and easily soluble in hot water and cold ethanol. Is commonly found in common plant herbs 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, Polypodiaceae, Rutaceae, Labiatae and Eucommiaceae. Has antibacterial, antiviral, anti-snake venom, anti-diarrhea, anti-fertility, and anti-tumor pharmacological activities, and also has blood cooling and hemostatic effects, and can be used for treating hypertension, coronary heart disease, arrhythmia, etc. Caffeic acid, a representative compound of hydroxycinnamic acids, has a strong antioxidant activity.

At present, no report of extracting and preparing caffeic acid from rosemary is found. Mainly by extracting caffeic acid from plants rich in caffeic acid, which is usually not high in content, the direct extraction of caffeic acid from rosemary is more rarely harvested, so that a new way for obtaining caffeic acid is needed.

The research finds that rosemary contains more rosmarinic acid and a small amount of caffeic acid, the invention provides a preparation method of caffeic acid, rosmarinic acid is extracted and then is converted into caffeic acid through fermentation, so that the yield of caffeic acid obtained from rosemary is greatly improved, and the efficient utilization of rosemary resources is realized.

Disclosure of Invention

The invention aims to provide an efficient and economic method for extracting caffeic acid from rosemary, and simultaneously improve the content of the caffeic acid in the extract.

A method for extracting caffeic acid from rosemary is characterized by comprising the following steps:

s1, crushing rosemary, extracting with ethanol at 80-83 ℃, and carrying out solid-liquid separation on the extracting solution to obtain a clear extracting solution;

s2, concentrating the clear extracting solution obtained in the step S1 to Brix 40-60% to obtain a concentrated solution, and adding a carbon source;

s3, inoculating zymophyte into the concentrated solution added with the carbon source in the step S2, adjusting 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 11-12%, adjusting the pH value to 4-6, and performing 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 unadsorbed impurities; washing the macroporous adsorption resin with desorption solution with the pH value of 4-6, collecting the washed desorption solution, and concentrating under reduced pressure to Brix 30-35%;

s6, decolorizing and filtering the concentrated solution, concentrating the filtrate again, and drying to obtain the caffeic acid extract.

Preferably, in the step S1, rosemary is crushed to a diameter of 0.5cm or less.

Preferably, the ethanol concentration in the step S1 is 50% to 60%.

The ethanol concentration is 50% -60% because the solubility of the rosemary caffeic acid is relatively proper at the concentration, so that the alcohol-soluble impurities brought into the rosemary caffeic acid as little as possible can be avoided, the burden of the post-treatment process is increased, and the production efficiency is improved.

Preferably, the extraction of step (1) is a percolation extraction.

Preferably, the carbon source is glucose.

Preferably, the solid-liquid separation is filtration or centrifugation.

Preferably, the fermentation bacteria comprise, by mass, 10-90% of lactic acid bacteria and 10-90% of yeast.

Preferably, the fermentation bacteria comprise 60-80% of lactic acid bacteria and 20-40% of yeast by mass percentage.

Preferably, the fermentation bacteria comprise 70% of lactic acid bacteria and 30% of yeast in percentage by mass.

Caffeine is a xanthine alkaloid compound, caffeine is prepared through biotransformation, lactic acid bacteria and saccharomycetes are the most effective biological bacteria for plant biological fermentation, the lactic acid bacteria and the saccharomycetes are probiotic bacteria, a fermentation process of generating lactic acid is theoretically realized through fermentation of a large number of lactic acid bacteria and anaerobic glycolysis, and ethanol fermentation is two main fermentation forms in an organism, so that smooth fermentation can be promoted. And a small amount of yeast is added for fermentation, so that the sugar is decomposed into carbon dioxide and water under the aerobic condition, the growth of the yeast is fast, and the sugar is decomposed into alcohol and carbon dioxide under the anoxic condition, so that the fermentation process is expanded, and the bioconversion process of caffeine is facilitated. Therefore, it is preferable that the ratio of lactic acid bacteria is larger.

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 and 001 × 7Na 732.

Preferably, the macroporous adsorbent resin is a medium-polarity macroporous adsorbent resin.

Preferably, the macroporous adsorption resin is NKA-9 macroporous resin or D1O1 macroporous resin.

More preferably, the macroporous adsorption resin is NKA-9 macroporous adsorption resin.

The NKA-9 macroporous adsorption resin is an adsorbent with divinylbenzene as a framework structure, a benzene ring connected to a main chain is a plane with uniformly distributed electrons, and the NKA-9 macroporous adsorption resin has strong adsorption capacity for molecules with similar properties and various cyclic aromatic compounds and is increased along with the enhancement of lipophilicity of adsorbed molecules. Is widely used for separating ginkgo flavone, polyphenol, caffeic acid and the like. In recent years, it has been shown to have a unique effect on the separation and purification of natural products, particularly water-soluble compounds.

Preferably, the desorption solution is an ethanol solution with the volume percentage concentration of 50-60%.

Preferably, the active carbon filter cloth for decolorization and filtration has a 325-mesh screen.

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, the similar substances are usually extracted by alcohol, and are precipitated at low temperature for a long time after being concentrated to remove impurities, 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 the industrialization is difficult. The caffeic acid prepared by the traditional chemical synthesis method has the defects of long reaction time, unsatisfactory purity, high residue of harmful substances in the product, unsatisfactory caffeic acid product and the like. The method extracts the rosmarinic acid and the caffeic acid by 60-65% ethanol, and converts the rosmarinic acid into the caffeic acid by fermentation after concentration, thereby greatly improving the yield of obtaining the caffeic acid from the rosemary and realizing the efficient utilization of rosemary resources. Before resin column separation, the invention also adds water into the obtained liquid to Brix 10-12%, and adjusts the pH value to 4-6 with acid, under the condition, the impurities in the solution are rapidly precipitated under the acidic condition, and do not need to be stood for a long time at low temperature, and are separated immediately, thereby greatly shortening the production period. Separating with macroporous adsorbent resin column to obtain the product with high effective component content. The concentrated solution is decolorized and filtered by 325-mesh active carbon filter cloth, and partial colored substances and water-insoluble impurities can be removed. In the conventional extraction method, the content of a target product in the extract is generally 10-20%, but the content of caffeic acid in the extract can reach 50-60% by using the method provided by the invention. The product prepared by the invention is mainly used as a raw material of medicines.

Compared with the prior art, the invention has the following beneficial effects:

1. the method has the advantages of good extraction continuity of the caffeic acid, simple process and suitability for industrial popularization.

2. The method comprises the step of fermenting rosemarinic acid and mixed bacteria consisting of lactic acid bacteria and yeast at the pH value of 4-6 and the Brix 40-60% to obtain the caffeic acid, wherein the fermentation process contains 6-P-beta-glucosidase, Gallate decarboxylase and P-coumaric acid carboxylase which participate in phenolic compound metabolism to convert the rosemarinic acid into the caffeic acid, so that the content of the caffeic acid is improved.

3. The invention adopts a leakage method to extract caffeic acid, so that the effective components in the rosemary can be quickly and fully dissolved out in a short time, the reaction conditions are mild, the structure protection of active substances is facilitated, new organic impurities cannot be introduced, the safety of final products is ensured, and the environmental pollution is reduced.

4. The method has the advantages of reasonable process, high content of effective components, high extraction efficiency, simple operation, and suitability for industrial production.

5. The extract prepared by the invention has stable purity, and the caffeic acid content is 50-60%.

6. The solvent of the invention can be recycled, and is economic and environment-friendly.

Drawings

FIG. 1 is a graph of the effect of caffeic acid on ethanol extraction of various concentrations selected in example 1;

FIG. 2 is a graphical analysis of the conditions for influencing fermentation broths of different ratios with caffeic acid rosemary in example 1;

wherein the ratio of lactic acid bacteria: the yeast mixture ratio is A1:9, B3:7 and C5: 5. d7: 3. e9: 1;

FIG. 3 is a graph of the selective elution of caffeic acid from rosemary in example 2 under different conditions on macroporous adsorbent resins;

wherein A is NKA-9 macroporous resin, B is D1O1 macroporous resin, C is D301 macroporous basic resin, D201 macroporous basic resin, E is 001 × 7Na732

The ordinate of fig. 1-3 is the caffeic acid content of the product.

FIG. 4 is a high performance liquid chromatography assay of caffeic acid of example 3.

Detailed Description

The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.