CN113200857A - Process for purifying origanum rosemarium acid antioxidant by two-phase solvent extraction method - Google Patents
Process for purifying origanum rosemarium acid antioxidant by two-phase solvent extraction method Download PDFInfo
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- CN113200857A CN113200857A CN202110301239.2A CN202110301239A CN113200857A CN 113200857 A CN113200857 A CN 113200857A CN 202110301239 A CN202110301239 A CN 202110301239A CN 113200857 A CN113200857 A CN 113200857A
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- rosmarinic acid
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- 238000000638 solvent extraction Methods 0.000 title claims abstract description 30
- 230000003078 antioxidant effect Effects 0.000 title claims abstract description 16
- 239000003963 antioxidant agent Substances 0.000 title claims abstract description 15
- 241001529744 Origanum Species 0.000 title claims description 12
- 235000011203 Origanum Nutrition 0.000 title claims description 12
- 239000002253 acid Substances 0.000 title abstract description 8
- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 claims abstract description 102
- 240000007673 Origanum vulgare Species 0.000 claims abstract description 34
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/04—Solvent extraction of solutions which are liquid
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/04—Solvent extraction of solutions which are liquid
- B01D11/0415—Solvent extraction of solutions which are liquid in combination with membranes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/04—Solvent extraction of solutions which are liquid
- B01D11/0488—Flow sheets
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D21/00—Separation of suspended solid particles from liquids by sedimentation
- B01D21/01—Separation of suspended solid particles from liquids by sedimentation using flocculating agents
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/58—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B9/00—Essential oils; Perfumes
- C11B9/02—Recovery or refining of essential oils from raw materials
- C11B9/027—Recovery of volatiles by distillation or stripping
Abstract
The invention discloses a technology for purifying oregano rosemaric acid antioxidant by a two-phase solvent extraction method, which takes oregano as a raw material to prepare oregano rosemaric acid, and then purifies the rosemaric acid by the two-phase solvent extraction method; extracting with 15% -30% ethanol water solution at a ratio of 1:8 for 2 times, adding flocculant and membrane filter, concentrating, adjusting pH with hydrochloric acid or other acid to 2-3, extracting with n-butanol at a ratio of 1:1.5, concentrating to obtain concentrate, dissolving pH =2-3 with water, removing insoluble substances, extracting the clear solution with methyl acetate at a ratio of 1:1.5, and drying to obtain oregano rosemaric acid antioxidant product (content about 70%).
Description
Technical Field
The invention relates to the field of preparation and purification of plant active ingredients, in particular to a process for purifying origanum rosemarium acid antioxidant by a two-phase solvent extraction method.
Background art:
origanum vulgare L, also called dysentery stopping grass, elsholtzia haichowensis and mentha microphylla are perennial herb plants in the family of Labiatae, are natural medicinal and edible plants, can be used as medicines for all the herbs, and can be used as food seasoning to increase the fragrance of food or remove the smell of meat. Oregano is considered a powerful natural antibiotic and studies have shown that it is even capable of fighting against a super staphylococcus aureus bacterium that is not currently available to any antibiotic. The antioxidant effect of oregano is superior to that of other medicinal herbs, and the antioxidant capacity of oregano is 4 times that of blueberry, 12 times that of orange and 42 times that of apple according to evaluation by the American Ministry of agriculture. The research on the chemical components of oregano mainly comprises reports of chemical components such as volatile oil, organic acid, flavone, triterpene and sterol (oregano essential oil, rosmarinic acid and ursolic acid) and the like serving as novel plant antibiotics, and researches show that the tea tree essential oil and the oregano essential oil have a good inhibition effect on trichophyton mentagrophytes, and the oregano essential oil has a better effect than the tea tree essential oil. An invention patent of 'a pesticide composition containing oregano and rosemary extracts and a preparation method and application thereof' (application publication number CN109497100A) applied by Chengdu Xingyang crop science Co., Ltd in 2019 belongs to the technical field of botanical pesticides, and provides a pesticide composition containing oregano and rosemary extracts. The invention patent applied by the Yichun academy of sciences in 2019, which is a composite plant extract, and a preparation method and application thereof. The invention patent of Hunan green sprangletop biotechnology, Inc. in 2020, "a method for the comprehensive extraction of oregano" (application publication No. CN111187437A) can obtain essential oil, rosmarinic acid and ursolic acid, but the ursolic acid is a fat-soluble component and cannot be extracted under the alcoholic strength of 60%. The invention discloses a majoram oil preparation, a preparation method and application thereof in 2018 of a large pollution-free pesticide research service center in the Yanglingcuo (application publication number CN108719345A), and the invention discloses a majoram oil preparation, a preparation method and application thereof in 2018 of a large pollution-free pesticide research service center in the Yanglingcuo (application publication number CN108739876A), wherein the majoram oil is used for preparing the preparation. The invention discloses a method for extracting natural high-content oregano phenol (application publication No. CN103655688A) invented in 2014 by agriculture and animal husbandry biological pharmaceutical Co., Ltd in Fujian, and relates to a method for extracting natural high-content oregano phenol. The patent does not mention what the high content is. The patent adopts a two-phase solvent extraction method for purifying the oregano rosmarinic acid antioxidant for the first time, and can provide reference for the comprehensive development and utilization of oregano resources for enterprises.
Disclosure of Invention
The invention aims to provide a process for purifying origanum rosemarium acid antioxidant by a two-phase solvent extraction method, which is designed aiming at the problem that the solution contains more polysaccharide, pigment and macromolecular protein impurities during water extraction, so that the product is dark in color and easy to damp, and after essential oil is obtained by water distillation, ethanol is added to ensure that the concentration of the essential oil is 15-30%, so that the impurities are reduced. Filtering and decolorizing by flocculation membrane to obtain purified rosmarinic acid solution, and further purifying rosmarinic acid by two-phase distribution method.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a technology for purifying origanum vulgare rosmarinic acid antioxidant by a two-phase solvent extraction method is characterized by comprising the following steps:
(1) preparing origanum vulgaris essential oil: adding dried origanum vulgare into the aqueous solution according to the mass-volume ratio of 1:5, and extracting 0.2-0.3% of origanum vulgare essential oil by a steam distillation method;
(2) preparing a crude extract of origanum vulgare rosmarinic acid: adding alcohol into the extract obtained in the step (1) to enable the alcohol concentration to be 15-30%, performing reflux extraction, performing extraction once, and combining the extracting solutions;
(3) filtering and decoloring by a flocculation membrane: adding a polymeric flocculant into the rosmarinic acid aqueous solution obtained in the step (2) for dissolving, filtering through a 800nm tubular membrane and a roll-type membrane with the molecular weight cut-off of 1000, and filtering the permeate through a roll-type membrane with the molecular weight cut-off of 150 to obtain a concentrated solution;
(4) two-phase solvent extraction to prepare higher purity rosmarinic acid: adding n-butanol into the clear liquid of the concentrated solution obtained in the step (3) according to the proportion of 1:1.5 for extraction once, and concentrating to obtain a concentrate;
(5) two-phase solvent extraction to prepare higher high-concentration rosmarinic acid: and (4) extracting and purifying the crude rosmarinic acid obtained in the step (4) for the second time by using an organic solvent extraction method, adding methyl acetate into clear liquid according to the ratio of 1:1.5 for extracting once, concentrating to obtain a concentrate, and drying to obtain a product.
Preferably, the flocculation membrane filtration decoloration step is as follows: and (3) adding dicyandiamide formaldehyde-dicyandiamide formaldehyde ammonium salt flocculant into the rosmarinic acid aqueous solution obtained in the step (2) for dissolving, centrifuging to remove precipitates, filtering through a 800nm tubular membrane, filtering the permeate through a roll-type membrane with the molecular weight cutoff of 1000Da, and filtering the permeate through a roll-type membrane with the molecular weight cutoff of 150 to obtain a concentrated solution, wherein the content of a dry product is about 15%.
Preferably, the two-phase solvent extraction produces higher purity rosmarinic acid: and (4) adjusting the pH value of the concentrated solution obtained in the step (3) to 2-3, centrifuging to remove insoluble substances, and changing the water phase into water-insoluble impurities. Extracting the clear liquid with n-butanol at a ratio of 1:1.5, concentrating to obtain concentrate, removing water solution to obtain water-soluble impurities, and concentrating n-butanol phase under reduced pressure to obtain rosmarinic acid with content of about 35%.
Preferably, the two-phase solvent extraction produces higher concentrations of rosmarinic acid: and (4) extracting and purifying the 35% crude rosmarinic acid obtained in the step (4) for the second time by using an organic solvent extraction method, adjusting the pH value to be 2-3, centrifuging to remove insoluble substances, adding methyl acetate into clear liquid according to the proportion of 1:1.5, extracting for one time, concentrating to obtain a concentrate, and drying to obtain a product with the concentration of about 70%.
Preferably, the preparation method of the dicyandiamide formaldehyde-dicyandiamide formaldehyde ammonium salt flocculant comprises the following steps: 100mL of 37% formaldehyde was placed in a 500mL four-necked flask or a three-necked flask equipped with a stirrer, a thermometer, and a condenser. 50-52 g of dicyandiamide is added under stirring, and the temperature naturally rises to about 38 ℃. 10 g of aluminum chloride is added at 40 ℃, and the solution becomes clear after reaction for about 30 min. Adding 30-33 g of modifier ammonium chloride. Controlling the temperature to be about 40 ℃ (the temperature is raised in the reaction process), stirring until the solution is transparent (about 1h), adding 100mL of water, raising the temperature to 65-70 ℃, reacting for 2-2.5 h, and observing the viscosity. Before cooling, 100mL of water was added and stirred for 30 min.
As a process for purifying the antioxidant of the oregano rosemaric acid by adopting a two-phase solvent extraction method for the first time, relevant process parameters need to be creatively tested and selected according to the physicochemical characteristics of the oregano rosemaric acid, and the test results are as follows:
1. the effects of n-butanol extraction of water phase and alcohol-containing water phase
After 90% of samples are dissolved in 15% ethanol and pure water in the water phase (0.2mg/mL), the water phase is extracted by adding n-butanol with the same volume, and the content of rosmarinic acid in the upper and lower phases is determined, and the results are shown in the following table:
TABLE 1 proportion of rosmarinic acid in upper and lower phases
From the experimental data it can be seen that: dissolving the standard substance with 15% ethanol and water, extracting with n-butanol, and extracting 15% ethanol phase with rosmarinic acid 70% in ethanol water solution. The ph neutral aqueous phase was extracted and approximately 70% of the rosmarinic acid was partitioned in the organic phase. Therefore, the rosmarinic acid sample is dissolved by water.
2. N-butanol extraction of aqueous phases of different pH
Respectively dissolving 90% of rosmarinic acid samples by water phases with different pH values, extracting by using n-butanol with the same volume, after phase splitting, respectively detecting upper and lower phases by high performance liquid chromatography, wherein the peak area ratio is as follows:
TABLE 2 partitioning of n-butanol extraction of aqueous phase rosmarinic acid of different pH
From the experimental data it can be seen that: the mixture ratio of rosmarinic acid at upper and lower phase is 0.1-3, 98% is in organic phase, and when pH is 2-3, rosmarinic acid can be enriched to extract rosmarinic acid with high efficiency, and simultaneously remove impurity insoluble in upper phase under acidic condition.
3. Two-phase solvent extractant selection
Dissolving the raw materials in acid water, wherein the material-liquid ratio is 1:8, centrifuging to remove insoluble substances, extracting with diethyl ether, methyl acetate, ethyl acetate, butyl acetate, chloroform, n-butanol, petroleum ether and n-hexane respectively according to the volume ratio of 1:1 for two times, and detecting the content of the upper phase and the last lower phase of rosmarinic acid after the two times of extraction. The results are shown in table 3 below:
TABLE 3 different solvent extraction effectiveness
The experimental results show that: the top phase had the highest total rosmarinic acid, followed by methyl acetate, ethyl acetate and diethyl ether, when extracted with n-butanol. The extraction times are twice.
4. Preparation of dicyandiamide formaldehyde-dicyandiamide formaldehyde ammonium salt flocculant
Dicyandiamide formaldehyde-dicyandiamide formaldehyde ammonium salt flocculant: 100mL of 37% formaldehyde was placed in a 500mL four-necked flask or a three-necked flask equipped with a stirrer, a thermometer, and a condenser. 50-52 g of dicyandiamide is added under stirring, and the temperature naturally rises to about 38 ℃. 10 g of aluminum chloride is added at 40 ℃, and the solution becomes clear after reaction for about 30 min. Adding 30-33 g of modifier ammonium chloride. Controlling the temperature to be about 40 ℃ (the temperature is raised in the reaction process), stirring until the solution is transparent (about 1h), adding 100mL of water, raising the temperature to 65-70 ℃, reacting for 2-2.5 h, and observing the viscosity. Before cooling, 100mL of water was added and stirred for 30 min.
Compared with the prior art, the invention has the following advantages:
the invention provides a technology for purifying oregano rosemaric acid antioxidant by a two-phase solvent extraction method, which adopts a steam distillation method to obtain oregano essential oil, adjusts the concentration of ethanol to reduce impurities such as sugar, protein and the like, and extracts and filters a filter membrane to prepare higher content rosemaric acid (15%). Parameters such as a two-phase extracting agent, extraction pH and the like are optimized in more detail, and 70% rosmarinic acid is prepared by two-phase extraction and purification.
Drawings
Fig. 1 is a schematic view of the process for extracting origanum rosemarinic acid according to the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The process for purifying origanum rosemarium acid antioxidant by a two-phase solvent extraction method as shown in figure 1 is prepared by the following steps:
1) preparing origanum vulgaris essential oil: adding the dried origanum vulgare into the aqueous solution according to the mass-volume ratio of 1:5, and extracting the origanum vulgare essential oil by adopting an on-water distillation method.
2) Preparing a crude rosmarinic acid extract: adding alcohol into the material solution with essential oil removed to reach concentration of 15-30%, reflux extracting, extracting once, and mixing extractive solutions.
3) Filtering and decoloring by a flocculation membrane: adding high molecules into the rosmarinic acid aqueous solution obtained in the step 2), flocculating, centrifuging to remove precipitates, filtering through a 800nm tubular membrane, filtering the permeate through a roll-type membrane with the molecular weight cutoff of 1000, and filtering the permeate through a roll-type membrane with the molecular weight cutoff of 150 to obtain a concentrated solution. The content is about 15% (dry product).
4) Two-phase solvent extraction to prepare higher concentration rosmarinic acid: adjusting pH of the concentrated solution obtained in the step 3) to 2-3 with hydrochloric acid, centrifuging to remove insoluble substances, adding n-butanol into the clear solution according to the proportion of 1:1.5, extracting once, and concentrating to obtain a concentrate with the content of about 35%.
5) Two-phase solvent extraction to prepare higher high-concentration rosmarinic acid: dissolving the 35% crude rosmarinic acid obtained in the step 4) according to the material-liquid ratio of 1:6 (adjusting the pH value to 2-3), performing secondary extraction and purification by using an organic solvent extraction method, centrifuging to remove insoluble substances, adding methyl acetate into clear liquid according to the ratio of 1:1.5, extracting for one time, concentrating to obtain a concentrate, and drying to obtain a product with the concentration of about 70%.
Example 2
The process for purifying the origanum rosemarium acid antioxidant by the two-phase solvent extraction method comprises the following steps:
1) preparing origanum vulgaris essential oil: 1000 g of dried oregano grass is added into a self-made 10-liter extraction tank, aqueous solution is added according to the mass volume ratio of 1:6 of feed liquid, and oregano essential oil (about 0.2-0.3%) is extracted by a steam distillation method.
2) Preparing a crude rosmarinic acid extract: adding alcohol into the feed liquid with essential oil removed to make the concentration be 20-30%, reflux extracting, extracting once, mixing extractive solutions, and filtering with four layers of gauze.
3) Filtering and decoloring by a flocculation membrane: adding 0.5-1% dicyandiamide formaldehyde-dicyandiamide formaldehyde ammonium salt flocculant polymeric flocculant into the rosmarinic acid aqueous solution obtained in the step 2), centrifuging to remove precipitates, passing through a 200nm tubular membrane, filtering the permeate through a roll-type membrane with the molecular weight cutoff of 1500, and filtering the permeate through a roll-type membrane with the molecular weight cutoff of 150 to obtain a concentrated solution. The content is about 15% (dry product).
4) Two-phase solvent extraction to prepare higher concentration rosmarinic acid: adjusting pH of the concentrated solution obtained in the step 3) to 2-3 with sulfuric acid, centrifuging to remove insoluble substances, adding n-butanol into the clear solution according to the ratio of 1:1.5, extracting once, and concentrating to obtain a concentrate with the content of about 35%.
5) Two-phase solvent extraction to prepare higher high-concentration rosmarinic acid: dissolving the 35% crude rosmarinic acid obtained in the step 4) according to the material-liquid ratio of 1:6 (adjusting the pH value to 2-3 with sulfuric acid), adding methyl acetate into the clear liquid according to the ratio of 1:1.5, extracting once, concentrating to obtain a concentrate, and drying to obtain a product with the concentration of about 70%.
Claims (2)
1. A technology for purifying origanum vulgare rosmarinic acid antioxidant by a two-phase solvent extraction method is characterized by comprising the following steps:
1) preparing a crude extract of origanum vulgare rosmarinic acid: adding water solution into dried origanum vulgaris at a material liquid mass volume ratio of 1:5, distilling to extract origanum vulgaris essential oil, adding alcohol to make the concentration be 15-30%, reflux extracting, performing extraction once, and mixing extractive solutions;
2) filtering and decoloring by a flocculation membrane: dissolving the rosmarinic acid aqueous solution obtained in the step 1) in dicyandiamide formaldehyde-dicyandiamide formaldehyde ammonium salt flocculant, centrifuging to remove precipitates, filtering through an 800nm tubular membrane, filtering the permeate through a roll membrane with the molecular weight cutoff of 1000, and filtering the permeate through a roll membrane with the molecular weight cutoff of 150 to obtain a concentrated solution, wherein the content of a dry product is about 15%;
3) two-phase solvent extraction to prepare higher concentration rosmarinic acid: adjusting the pH value of the concentrated solution obtained in the step 2) to be 2-3, centrifuging to remove insoluble substances, adding n-butanol into clear liquid according to the proportion of 1:1.5, extracting once, and concentrating to obtain a concentrate with the content of about 35%;
4) two-phase solvent extraction to prepare higher high-concentration rosmarinic acid: and 3) performing secondary extraction and purification on the 35% rosmarinic acid crude product obtained in the step 3) by using an organic solvent extraction method, adjusting the pH value to be 2-3, centrifuging to remove insoluble substances, adding methyl acetate into clear liquid according to the proportion of 1:1.5 for extraction once, concentrating to obtain a concentrate, and drying to obtain a rosmarinic acid product with the content of about 70%.
2. The two-phase solvent extraction process for purifying origanum vulgare rosmarinic acid antioxidant according to claim 1, wherein the dicyandiamide formaldehyde-dicyandiamide formaldehyde ammonium salt flocculant is prepared by the following steps: adding 100mL of 37% formaldehyde into a 500mL four-neck flask or a three-neck flask which is provided with a stirrer, a thermometer and a condenser, adding 50-52 g of dicyandiamide under stirring, naturally raising the temperature to about 38 ℃, adding 10 g of aluminum chloride at 40 ℃, and reacting for about 30min until the solution becomes clear; adding 30-33 g of modifier ammonium chloride, heating in the reaction process, controlling the temperature to be about 40 ℃, stirring until the solution is transparent, adding 100mL of water, heating to 65-70 ℃, reacting for 2-2.5 hours, observing the viscosity, adding 100mL of water before cooling, and stirring for 30 min.
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