Method for preparing rosemary water-soluble and fat-soluble antioxidants simultaneously

Abstract

The present application discloses a method for preparing rosemary water-soluble and fat-soluble antioxidants at the same time, comprising the following steps: obtaining dried rosemary stems and leaves as raw materials; using ethanol water as an extraction solvent to soak the raw materials into a solid-liquid mixture; using a chromatography column to elute a mixed extract from the solid-liquid mixture; using a rotary evaporation method to separate the water-soluble components and fat-soluble components in the mixed extract; the water-soluble components contain rosemary water-soluble antioxidants; and the fat-soluble components contain rosemary fat-soluble antioxidants. The preparation method of the present application has a simple and efficient process, a high extraction rate, a large output, is easy to realize industrialization and mass production, has good product safety, the solvent used in the preparation process can be recycled, has low production cost, generates less hazardous waste, and has little environmental pollution.

Description

Technical Field

[0001] The present application relates to the technical field of separation and purification of natural compounds, and in particular to a method for simultaneously preparing rosemary water-soluble and fat-soluble antioxidants. Background Art

[0002] In recent years, with the continuous improvement of people's living standards, the safety of artificial preservatives in food, cosmetics and daily necessities has attracted increasing attention, and the research and development and application of pure natural antioxidants have become a research hotspot in the fields of food and chemical industry. Rosemary is a plant of the genus Rosmarinus in the Lamiaceae family. It is currently recognized as a plant with high antioxidant effect and is safe and non-toxic. It is known as the third generation of green food antioxidants and has broad application prospects and huge market potential. At present, the commercially available rosemary antioxidants include two types: fat-soluble antioxidants and water-soluble antioxidants. According to GB 1886.172-2016 National Food Safety Standard Food Additive Rosemary Extract, the content of rosmarinic acid, the main active ingredient in water-soluble antioxidants, should not be less than 5%. Rosemarinic acid has strong antioxidant and antibacterial capabilities and can withstand high temperatures above 100°C. It can be widely used in various beverages, aquatic products, and natural water-soluble pigments. The content of carnosic acid, the main active ingredient in fat-soluble antioxidants, should not be less than 10%. Because of its good thermal stability, it can withstand high temperatures of 190°C to 240°C. It is mainly used in oils, dairy products, oil-rich foods, candies, baked foods and other foods.

[0003] At present, the research on rosemary extract mainly focuses on the preparation and application of fat-soluble antioxidants, and the main preparation methods are ethanol extraction or supercritical CO2 extraction. The preparation of fat-soluble antioxidants by ethanol extraction usually requires the separation of rosemary essential oil by steam distillation. During the production process, carnosic acid is easily oxidized and degraded, which affects the yield and antioxidant effect of the extract; the preparation of fat-soluble antioxidants by supercritical CO2 extraction has high production costs, a single product and a darker color, and it is difficult to achieve industrial production. Rosemary water-soluble antioxidants are usually by-products of the production of rosemary essential oil and fat-soluble antioxidants. The content of rosmarinic acid in the extract is low, and the subsequent purification and refining steps are complicated, and the production cost is high. Summary of the invention

[0004] The purpose of the present application is to provide a method for simultaneously preparing rosemary water-soluble and fat-soluble antioxidants in view of the deficiencies of the prior art.

[0005] In order to achieve the above technical objectives, the technical solutions adopted in this application are as follows:

[0006] A method for simultaneously preparing rosemary water-soluble and fat-soluble antioxidants comprises the following steps:

[0007] Obtain dried rosemary stems and leaves as raw material;

[0008] Using ethanol water as an extraction solvent to soak the raw material into a solid-liquid mixture;

[0009] Using a chromatography column to elute the solid-liquid mixture into a mixed extract;

[0010] Separating the water-soluble components and the fat-soluble components in the mixed extract by rotary evaporation;

[0011] The water-soluble component contains rosemary water-soluble antioxidant;

[0012] The fat-soluble component contains rosemary fat-soluble antioxidant.

[0013] Preferably, the raw materials are crushed before soaking and then sieved through a 20-40 mesh sieve.

[0014] Optionally, the ethanol water is an ethanol aqueous solution with a volume percentage of 50% to 95%.

[0015] More preferably, the soaking time is 2 to 4 hours.

[0016] Further optionally, the column diameter-to-height ratio of the solid-liquid mixture in the chromatography column is 1:2.5-10.

[0017] Furthermore, the eluent of the solid-liquid mixture is the extraction solvent, the elution flow rate is 1 to 3 BV / h, and the elution is performed at room temperature.

[0018] Preferably, the rotary evaporation method is carried out at 40-50° C.; the collected evaporated liquid is dissolved in the water phase to obtain the water-soluble component; the collected precipitate is dissolved in the ethanol phase to obtain the fat-soluble component.

[0019] Furthermore, after the water-soluble components and fat-soluble components are decolorized and dried respectively, a rosemary water-soluble antioxidant extract and a rosemary fat-soluble antioxidant extract are obtained.

[0020] Optionally, the decolorization uses a decolorizing agent to adsorb the pigment, and the decolorizing agent is selected from any one of activated carbon, activated clay and macroporous resin.

[0021] Compared with the prior art, this application has the following advantages:

[0022] The present application uses dried rosemary stems and leaves as materials, utilizes column chromatography extraction to prepare rosemary mixed extract, and performs rotary evaporation on the mixed extract to separate water-soluble components and fat-soluble components in the mixed extract, and respectively allows the water-soluble components and fat-soluble components to stand to obtain rosemary water-soluble and fat-soluble antioxidants. The preparation method of the present application has a simple and efficient process, a high extraction rate, a large output, is easy to realize industrialization and batch production, has good product safety, and the solvent used in the preparation process can be recycled, has low production cost, generates less hazardous waste, and has little environmental pollution.

[0023] The column chromatography extraction technology adopted in the present application does not use a stationary phase, and directly fills the raw materials fully soaked in the extracting solution into the chromatography column for elution. By controlling the elution conditions, rosemary water-soluble antioxidants (rosmarinic acid) and rosemary fat-soluble antioxidants (carnosic acid) can be efficiently extracted simultaneously. The extraction conditions are mild, the process is simple, the cost is low, the yield is high, the degradation of rosmarinic acid and carnosic acid can be effectively prevented, and the method is suitable for large-scale industrial production.

[0024] The whole process of this application is green and environmentally friendly, and the solvent is only ethanol aqueous solution, which is non-toxic and has low odor, ensuring the safety of the two antioxidants as food additives. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The present invention provides a process flow chart of the method for simultaneously preparing rosemary water-soluble and fat-soluble antioxidants.

[0026] Figure 2 The present invention provides the results of thin layer chromatography analysis of rosemary extract and antioxidants obtained by the method of the present application.

[0027] Figure 3 The present invention provides the results of high performance liquid chromatography analysis of the water-soluble components and fat-soluble components of rosemary obtained by the method of the present application. DETAILED DESCRIPTION

[0028] The present application is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0029] refer to Figure 1 In one possible embodiment of the method for simultaneously preparing rosemary water-soluble and fat-soluble antioxidants of the present application, the following steps are used to simultaneously extract the water-soluble antioxidants and fat-soluble antioxidants of rosemary:

[0030] S01: obtaining dried rosemary stems and leaves as raw materials;

[0031] After the stems and leaves of rosemary are harvested, they are dried at low temperature (about 50°C) to avoid rotting and mildew of the fresh materials, while preventing significant loss of volatile oil components, making it easier to extract various components in rosemary through different technical paths, such as extracting essential oils, antioxidant components or medicinal components of rosemary.

[0032] In the preparation method of the present application, the dried rosemary raw material needs to be crushed and passed through a 20-40 mesh sieve. Crushing is beneficial to increase the contact surface area during the subsequent contact with the extraction solvent, so as to facilitate the dissolution of the target extract into the extraction solvent. However, the crushed particles should not be too small, otherwise the extraction solvent will have difficulty passing through the gaps between the particles and sufficient elution cannot be achieved.

[0033] S02: using ethanol water as an extraction solvent to soak the raw material to form a solid-liquid mixture;

[0034] The water-soluble antioxidant in rosemary is mainly rosmarinic acid, which is an acid containing polyphenolic hydroxyl groups. It was first isolated and extracted from rosemary in 1958 and got its name. Rosmarinic acid is widely distributed, from lower mosses to higher dicotyledons, mainly distributed in Lamiaceae, Boraginaceae, Cucurbitaceae, Tiliaceae, and Umbelliferae. Rosmarinic acid is a water-soluble substance. Low-content rosemary sources are light yellow to brown powder, and high-content is white powder; it is easy to absorb moisture; it has a special herbal smell of rosemary; it turns blue-green when it encounters ferric chloride-potassium ferrocyanide solution.

[0035] The fat-soluble antioxidants in rosemary are mainly carnosic acid, which is also known as carnosic acid. It is easily soluble in oils and fats but insoluble in water. It is a colorless to light yellow powder crystal with the characteristics of high efficiency antioxidant, safety and non-toxicity, and high temperature resistance.

[0036] According to the characteristics of rosmarinic acid and carnosic acid, in this step, water-soluble rosmarinic acid and fat-soluble carnosic acid need to be leached from the raw materials at the same time, so ethanol water, an ethanol aqueous solution with a volume percentage of 50% to 95%, is used as the extraction solvent. Next, the soaking time of the raw materials and ethanol water should be at least 2 hours, so that the raw materials are fully swollen, and the soaking time should not be too long. The dissolution efficiency of the target extract has a peak value. Too long soaking time is not conducive to optimizing the input-output ratio of the entire preparation process. It has been verified by experiments that the soaking time should not exceed 4 hours. Further, in order to increase the concentration of the target extract in the subsequent eluent, the ethanol water used during soaking should not be too much, and the ethanol water just covers the raw materials when the raw materials are fully swollen, which meets the requirements.

[0037] S03: using a chromatography column to elute the solid-liquid mixture into a mixed extract;

[0038] For the convenience of observation, the embodiment of the present application preferably uses a glass chromatography column to elute the target extract from the soaked solid-liquid mixture. Specifically, referring to the column packing method of the stationary phase of the column chromatography method, the solid-liquid extract is loaded into the glass chromatography column so that the column diameter-to-height ratio of the solid-liquid mixture in the chromatography column is 1:2.5-10.

[0039] Alternatively, the aforementioned dried, pulverized raw materials and extraction solvent may be first loaded into a glass chromatography column and immersed in the glass chromatography column. If bubbles or voids appear in the solid-liquid mixture, they may be treated by vibration or vacuuming.

[0040] During elution, the ethanol water is used as the eluent, and the elution flow rate is controlled between 1 and 3 BV / h at room temperature. The eluted liquid is a mixed extract containing water-soluble components and fat-soluble components. The volume ratio of ethanol in the ethanol water can be appropriately adjusted during the elution process to control the harvest amount and elution time of the water-soluble components and the fat-soluble components, respectively.

[0041] refer to Figure 2 The eluates flowing out at different times were subjected to thin layer chromatography analysis. In the ethanol-water eluate with a fixed ethanol-water volume ratio, water-soluble rosmarinic acid was eluted first, and fat-soluble carnosic acid was still collected in the second half of the elution process. This result provides two insights: first, compared with rosmarinic acid, fat-soluble carnosic acid is more enriched in the stems and leaves of rosemary; second, the volume ratio of ethanol in ethanol-water can be adjusted during the elution process, so that the enrichment levels of rosmarinic acid and carnosic acid in the eluates collected at different times are higher, which is conducive to simplifying the subsequent purification steps.

[0042] S04: separating the water-soluble components and the fat-soluble components in the mixed extract by rotary evaporation;

[0043] The boiling point of rosmarinic acid is 694.7±55.0℃ (760mmHg) and the boiling point of carnosic acid is 490.0±45.0℃. The difference in boiling points can be used to separate the two by rotary evaporation. The temperature of the rotary evaporation is controlled to be maintained at 40-50℃. Under this condition, the water-soluble components become evaporating liquid and leave the mixed extract, and the fat-soluble components become precipitates and remain in the round-bottom flask.

[0044] S05-1&S05-2: Redissolve the evaporated liquid and the precipitate respectively;

[0045] After rotary evaporation, the collected evaporated liquid is dissolved in the aqueous phase to obtain the water-soluble component, and the collected precipitate is dissolved in the ethanol phase to obtain the fat-soluble component.

[0046] refer to Figure 3, the water-soluble components (water-soluble extracts) and fat-soluble components (fat-soluble extracts) were analyzed by high performance liquid chromatography, respectively, which confirmed that rosmarinic acid only existed in the water-soluble components, and that the water-soluble components contained other active substances in addition to rosmarinic acid, which corresponded to the experimental results of thin-layer chromatography; it was also confirmed that carnosic acid only existed in the fat-soluble components, and that there were fewer other active substances in the fat-soluble components, which also corresponded to the experimental results of thin-layer chromatography.

[0047] It can be concluded that the water-soluble component contains rosemary water-soluble antioxidants, and the fat-soluble component contains rosemary fat-soluble antioxidants.

[0048] S06-1&S06-2: Respectively purify the reconstituted water-soluble components and fat-soluble components.

[0049] Specifically, after decolorizing and drying the water-soluble components and fat-soluble components, a rosemary water-soluble antioxidant extract and a rosemary fat-soluble antioxidant extract are obtained. Since the previous steps have enriched and removed impurities to the target extract to the maximum extent, the so-called refining aims to make the product better in appearance and more stable in quality. The decolorization uses a decolorant to adsorb the pigment, and the decolorant is selected from any one of activated carbon, activated clay and macroporous resin.

[0050] The method for simultaneously preparing rosemary water-soluble and fat-soluble antioxidants of the present application is described below through specific examples.

[0051] Example 1

[0052] A method for simultaneously preparing a rosemary water-soluble and fat-soluble antioxidant, specifically comprising the following steps: (1) taking 10 g of dried rosemary stems and leaves, crushing them and passing them through a 20-mesh sieve; (2) soaking the crushed rosemary material in a 70% (V / V) ethanol aqueous solution, fully swelling it for 2 hours, and then loading it into a glass chromatography column, using a 70% (V / V) ethanol aqueous solution as an eluent, with a diameter-to-height ratio of 1:5 and an elution flow rate of 1 BV / h for column chromatography extraction; (3) collecting 10 BV of the eluent, rotary evaporating it to a water phase, adding 10% (m / V) activated white clay, decolorizing it at 40°C for 1 hour, concentrating and drying it to obtain a rosemary water-soluble antioxidant; (4) collecting the precipitate in (3), adding ethanol to dissolve it, adding 10% (m / V) activated white clay, decolorizing it at 40°C for 1 hour, concentrating and drying it to obtain a rosemary fat-soluble antioxidant.

[0053] Example 2

[0054] A method for simultaneously preparing a rosemary water-soluble and fat-soluble antioxidant, specifically comprising the following steps: (1) taking 5g of dried rosemary stems and leaves, crushing them and passing them through a 20-mesh sieve; (2) soaking the crushed rosemary material in a 50% (V / V) ethanol aqueous solution, fully swelling it for 2h, and then loading it into a glass chromatography column, using a 50% (V / V) ethanol aqueous solution as an eluent, with a diameter-to-height ratio of 1:10 and an elution flow rate of 2BV / h for column chromatography extraction; (3) collecting 8BV of the eluent, rotary evaporating it to a water phase, adding 10% (m / V) activated carbon, decolorizing it at 40°C for 1h, concentrating and drying it to obtain a rosemary water-soluble antioxidant; (4) collecting the precipitate in (3), adding ethanol to dissolve it, adding 10% (m / V) activated carbon, decolorizing it at 40°C for 1h, concentrating and drying it to obtain a rosemary fat-soluble antioxidant.

[0055] In summary, the present invention discloses a method for preparing rosemary water-soluble and fat-soluble antioxidants at the same time, wherein dried rosemary stems and leaves are used as materials, a rosemary extract is prepared by column chromatography extraction, the extract is rotary evaporated to an aqueous phase, the precipitate is redissolved with ethanol, and the aqueous phase and the ethanol phase are decolorized and dried to obtain rosemary water-soluble and fat-soluble antioxidants. The preparation method of the present invention has a simple and efficient process, a high extraction rate, a large output, is easy to realize industrialization and batch production, has good product safety, the solvent used in the preparation process can be recycled, has low production cost, generates less hazardous waste, and has little environmental pollution.

[0056] The above embodiments are preferred implementation modes of the present application, but are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present application should be equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A method for simultaneously preparing rosemary water-soluble and fat-soluble antioxidants, characterized in that: The following steps are involved: Dried rosemary stems and leaves are obtained as raw materials; the raw materials are crushed before soaking and then sieved through a 20-40 mesh sieve; Using ethanol water as an extraction solvent to soak the dried pulverized raw material to form a solid-liquid mixture, the state of the solid-liquid mixture is that when the dried pulverized raw material is fully swollen, the extraction solvent just covers the swollen raw material; the extraction solvent is used to simultaneously leach the water-soluble components and fat-soluble components of the dried pulverized raw material; Without using a stationary phase, the solid-liquid mixture is loaded into a chromatography column with reference to a stationary phase loading method of column chromatography, so that the column diameter-to-height ratio of the solid-liquid mixture in the chromatography column is 1:2.5-10; or, the dried and crushed raw materials and the extraction solvent are first loaded into the chromatography column, and the column is soaked, so that the column diameter-to-height ratio of the solid-liquid extract formed by the soaking is 1:2.5-10; The solid-liquid mixture is eluted into a mixed extract by using the extraction solvent as an eluent; the solid-liquid mixture is eluted at a flow rate of 1 to 3 BV / h at room temperature; the mixed extract comprises a portion enriched in water-soluble components, a portion enriched in fat-soluble components, and a portion containing the water-soluble components and fat-soluble components, which are harvested at different elution times using the extraction solvent with different ethanol volume ratios; Separating the water-soluble component and the fat-soluble component in the portion containing the water-soluble component and the fat-soluble component by rotary evaporation; The water-soluble components in the part enriched with water-soluble components and the part containing the water-soluble components and fat-soluble components contain rosemary water-soluble antioxidants; The fat-soluble components in the part enriched with fat-soluble components and the part containing the water-soluble components and fat-soluble components contain rosemary fat-soluble antioxidants.

2. The method according to claim 1, characterized in that The ethanol water is an ethanol water solution with a volume percentage of 50% to 95%.

3. The method according to claim 1, characterized in that The soaking time is 2 to 4 hours.

4. The method according to claim 1, characterized in that The rotary evaporation method is carried out at 40-50° C.; the collected evaporated liquid is dissolved in the water phase to obtain the water-soluble component; the collected precipitate is dissolved in the ethanol phase to obtain the fat-soluble component.

5. The method according to claim 4, characterized in that After the water-soluble components and fat-soluble components are decolorized and dried respectively, a rosemary water-soluble antioxidant extract and a rosemary fat-soluble antioxidant extract are obtained.

6. The method according to claim 5, characterized in that The decolorization adopts a decolorizing agent to adsorb the pigment, and the decolorizing agent is selected from any one of activated carbon, activated clay and macroporous resin.

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