阅读说明：本技术 一种从丹凤牡丹籽粕中提取白藜芦醇的制备方法 (Preparation method for extracting resveratrol from paeonia ostii seed meal ) 是由 王冰 韩刚启 王娟 刘建丽 李华敏 刘小倩 王子涵 李眉欣 李家文 张澳元 于 2020-11-24 设计创作，主要内容包括：一种从丹凤牡丹籽粕中提取白藜芦醇的制备方法,属于天然产物技术领域,其特征在于：以丹凤牡丹籽粕为原料、经过酶法提取、超声提取、萃取、利用高压制备液相色谱分离纯化,进而实现其产品回收率高、纯度好的基本工艺流程。通过本发明的技术条件制备牡丹籽壳白藜芦醇,工艺操作简单,易实施,具有制备过程简单、产率高、纯度高和原料价廉的特点,可以广泛应用于保健品、食品和护肤品领域,具有很好的商业前景。(A preparation method for extracting resveratrol from peony seed meal belongs to the technical field of natural products, and is characterized in that: the method takes the peony seed meal as a raw material, and realizes the basic process flow of high product recovery rate and good purity by enzymatic extraction, ultrasonic extraction, extraction and high-pressure preparative liquid chromatography separation and purification. The peony seed shell resveratrol prepared by the technical conditions has the characteristics of simple process operation, easy implementation, simple preparation process, high yield, high purity and low cost of raw materials, can be widely applied to the fields of health-care products, foods and skin care products, and has good commercial prospect.)

1. A preparation method for extracting resveratrol from peony seed meal provides a basic process flow for realizing high product recovery rate and good purity by taking the peony seed meal as a raw material and carrying out enzymatic extraction, ultrasonic extraction and high-pressure preparative liquid chromatography separation and purification, and is characterized by comprising the following steps:

(1) pretreatment of raw materials: drying the paeonia ostii seed meal until the water content is less than 10%, crushing, and screening by using a sieve of 20-60 meshes to obtain fine powder of the paeonia ostii seed meal;

(2) enzyme extraction: adding water with the mass volume 2-10 times of that of the fine peony seed meal powder, adjusting the pH value to 4.0-6.0, adding 0.1-1% of one or more enzymes of cellulase, hemicellulase, beta-glucosidase and xylanase, and stirring at a low speed for 1-5 hours at the temperature of 40-60 ℃; carrying out enzymolysis;

(3) ultrasonic extraction: after enzymolysis, adding ethanol water with volume fraction of 60-90% into the feed liquid, wherein the volume ratio of the ethanol water to the feed liquid is 1: 5-1: 20 (g: mL), then performing ultrasonic treatment with the power of 80-150W for 1-2 h, the extraction temperature of 30-60 ℃, extracting for 1-3 times, filtering, combining filtrates, performing reduced pressure evaporation on the filtrate at the temperature of 40-60 ℃, recovering an organic solvent, and performing vacuum drying or freeze drying on a concentrate to obtain a crude extract of the polydatin;

(4) and (3) extraction: adding distilled water into the resveratrol crude extract according to the mass-volume ratio of 1: 10-1: 20 (g: mL), stirring and dissolving at 50-60 ℃, filtering, adding petroleum ether with the volume ratio of 1: 1 into the filtrate, extracting for 3-5 times, combining petroleum ether extract phases, and concentrating under reduced pressure at 40-60 ℃ to obtain a petroleum ether extract; continuously performing isovolumetric extraction on the water layer for 3-5 times by using ethyl acetate, combining ethyl acetate extract phases, adjusting the pH value of the ethyl acetate extract phases to be 8.0-10.0 by using alkali, standing, filtering, and concentrating under reduced pressure until the volume of extract liquid is 1/15-1/20, and crystallizing to obtain a veratrol glycoside crude extract;

(5) hydrolysis and enrichment: adding 2-10 times of 2-10% diluted acid into the obtained crude extract of the polydatin according to the volume ratio, hydrolyzing for 4-8 h, cooling and standing for 24h after hydrolysis and enrichment, and filtering and washing acid to obtain a crude product of the polydatin;

(6) separation and purification: dissolving the obtained resveratrol crude product in ethyl acetate, and separating and purifying by using high-pressure preparative liquid chromatography, wherein the separation conditions of the high-pressure preparative liquid chromatography are as follows: a chromatographic column: welch Ultimate XB-C18(10 μm, 21.2 mm. times.250 mm); flow rate: 25 mL/min; sample introduction volume: 6 mL; mobile phase A: an aqueous solution containing 0.5% (v/v) acetic acid; mobile phase B: methanol; isocratic elution: 40 percent; ultraviolet detection wavelength: 308 nm; collecting the eluent of the resveratrol section, and then carrying out reduced pressure concentration until the eluent is crystallized in small volume to obtain a crude resveratrol product with higher purity;

(7) the product is as follows: and repeatedly recrystallizing the obtained resveratrol crude product with higher purity by using ethanol to obtain a white crystalline solid resveratrol product with the purity of more than 99 percent.

Technical Field

The invention relates to a method for preparing resveratrol from paeonia ostii seed shells, and belongs to the technical field of natural products.

Background

Peony (Paeonia suffruticosa Andr.) belongs to Paeoniaceae (Paeonia aeeae), Paeonia (Paeonia) and perennial woody plants. Peony root bark has good medicinal value, and is recorded in Ben Cao Jing two thousand years ago. Until the period of sui Tang, peony becomes a valuable ornamental plant. The peony seeds are seeds of peony buds, are black, hard in peel, bitter in taste, irregular and round, and are slightly larger than soybeans. Peony seeds can treat waist and leg pain in folks, and have the functions of diminishing inflammation and resisting oxidation. The research of the chemistry and the pharmacology of the traditional Chinese medicine finds that the peony seeds have the effects of resisting bacteria, diminishing inflammation, calming, reducing blood sugar, resisting allergy, regulating immunity and the like. The peony seed meal is solid residue meal after peony seed oil pressing, and researches show that the peony seed meal also contains a large amount of polyphenol active substances. Including resveratrol and its derivatives. Resveratrol (Resveratrol) has the chemical name of 3, 5, 4 '-trihydroxystilbene (3, 5, 4' -trihydroxystilbene), is a non-flavonoid polyphenol compound, and is known to be derived from natural plants including grapes, giant knotweed, peony, mulberry, peanuts, and the like. Resveratrol is usually present in the form of a glycoside in combination with glucose, and in small amounts in the free form in plants. Resveratrol is a natural antioxidant, and has biological activities of reducing blood lipid, resisting virus and tumor, etc. In recent years, health care products containing resveratrol as an active ingredient have appeared on the market. The common methods currently used for extracting resveratrol include organic solvent extraction method and super-solvent extraction methodSonic extraction method, microwave-assisted extraction, and supercritical CO extraction₂Extraction technology, enzymolysis method and the like. Research shows that the organic solvent extraction method is simple and easy to operate and low in production cost, but has the disadvantages of large solvent consumption, long heating time and low extraction efficiency. The microwave-assisted extraction method has the advantages of high selectivity, short operation time and the like, but the microwave-assisted extraction method has obvious thermal effect and great harm to the body of workers due to microwave leakage. Supercritical CO₂The extraction method has the advantages of high efficiency, low energy consumption, environmental protection and the like, but the technical content requirement is high, the equipment operation cost is high, and the extraction method is difficult to be put into large-scale industrial production. At present, ultrasonic waves are used for extracting resveratrol in peony seed meal, but the yield and the industrialization degree of the yield are still not satisfactory. Meanwhile, the enzymatic method is used as a method for extracting the phenolic substances, and has the advantages of high product recovery rate, small solvent consumption, mild conditions of the enzymatic hydrolysis method, difficulty in causing limited resource waste and pollution and the like. If the two are combined, the ultrasonic wave can better promote the material to be crushed, the surface contact area between a solid phase and a liquid phase is increased, and meanwhile, the enzyme can damage cell walls and reduce the mass transfer resistance, so that the extraction of a target object is facilitated. In recent years, with the rise of the peony seed oil industry, a large amount of oil peony seed meal is generated. The oil peony seed meal is partially used as a fertilizer, but is more discarded and has extremely low utilization rate, so the peony seed meal becomes a main byproduct. At present, most researchers are researching how to extract resveratrol from grapes and other plants, but reports about how to extract resveratrol from paeonia ostii peony seeds are few, abundant and cheap oil peony seed meal resources are deeply developed, high-purity resveratrol with bioactivity is obtained, waste materials can be changed into things of value, and good social and economic benefits are achieved. Therefore, the invention provides a method for preparing resveratrol from peony seed shells, which is a method for preparing resveratrol from peony seed shells and aims to solve the problems that the recovery rate of resveratrol products in peony seed meal is improved and the purity of the resveratrol products is increased, and further realize the basic process flow of high product recovery rate and high purity by utilizing enzymatic extraction, ultrasonic extraction, extraction and high-pressure preparation liquid chromatography separation and purificationIs necessary.

Disclosure of Invention

In order to overcome the problems of improving the recovery rate of a resveratrol product and increasing the purity of the resveratrol product by taking paeonia ostii seed meal as a raw material, the invention provides a method for preparing resveratrol from paeonia ostii seed shells, and the method for preparing the resveratrol from the paeonia ostii seed shells provides a basic process flow for realizing high product recovery rate and high purity by taking the paeonia ostii seed meal as a raw material and performing enzymatic extraction, ultrasonic extraction, extraction and high-pressure preparation liquid chromatography separation and purification.

The technical scheme adopted by the invention for solving the technical problems is as follows:

the invention relates to a method for preparing resveratrol from peony seed shells, which is characterized by comprising the following steps:

1. pretreatment of raw materials: drying the paeonia ostii seed meal until the water content is less than 10%, crushing, and screening by using a sieve of 20-60 meshes to obtain the paeonia ostii seed meal fine powder.

2. Enzyme extraction: adding water with the mass volume 2-10 times of that of the fine peony seed meal powder, adjusting the pH value to 4.0-6.0, adding 0.1-1% of one or more enzymes of cellulase, hemicellulase, beta-glucosidase and xylanase, and stirring at a low speed for 1-5 hours at the temperature of 40-60 ℃; and (4) enzymolysis.

3. Ultrasonic extraction: after enzymolysis, adding ethanol water with volume fraction of 60-90% into the feed liquid, wherein the volume ratio of the ethanol water to the feed liquid is 1: 5-1: 20 (g: mL), then performing ultrasonic treatment with the power of 80-150W for 1-2 h, the extraction temperature of 30-60 ℃, extracting for 1-3 times, filtering, combining filtrates, performing reduced pressure evaporation on the filtrate at the temperature of 40-60 ℃, recovering an organic solvent, and performing vacuum drying or freeze drying on the concentrate to obtain a crude extract of the polydatin.

4. And (3) extraction: adding distilled water into the resveratrol crude extract according to the mass-volume ratio of 1: 10-1: 20 (g: mL), stirring and dissolving at 50-60 ℃, filtering, adding petroleum ether with the volume ratio of 1: 1 into the filtrate, extracting for 3-5 times, combining petroleum ether extract phases, and concentrating under reduced pressure at 40-60 ℃ to obtain a petroleum ether extract; continuously performing isovolumetric extraction on the water layer for 3-5 times by using ethyl acetate, combining ethyl acetate extract phases, adjusting the pH value of the ethyl acetate extract phases to be 8.0-10.0 by using alkali, standing, filtering, and concentrating under reduced pressure until the volume of extract liquid is 1/15-1/20, and crystallizing to obtain the veratryl glucoside crude extract.

5. Hydrolysis and enrichment: and adding 2-10 times of diluted acid into the obtained crude extract of the polydatin according to the volume ratio, hydrolyzing for 4-8 h, cooling, standing for 24h after hydrolysis and enrichment, and filtering and washing acid to obtain a crude product of the polydatin.

6. Separation and purification: dissolving the obtained resveratrol crude product in ethyl acetate, and separating and purifying by using high-pressure preparative liquid chromatography, wherein the separation conditions of the high-pressure preparative liquid chromatography are as follows: a chromatographic column: welch Ultimate XB-C18(10 μm, 21.2 mm. times.250 mm); flow rate: 25 mL/min; sample introduction volume: 6 mL; mobile phase A: an aqueous solution containing 0.5% (v/v) acetic acid; mobile phase B: methanol; isocratic elution: 40 percent; ultraviolet detection wavelength: 308 nm. Collecting the eluent of the resveratrol section, and then carrying out reduced pressure concentration until the eluent is crystallized in small volume to obtain a crude resveratrol product with higher purity.

7. The product is as follows: and repeatedly recrystallizing the obtained resveratrol crude product with higher purity by using ethanol to obtain a white crystalline solid resveratrol product with the purity of more than 99 percent.

The method for preparing the resveratrol from the peony seed shells has the beneficial effects that: repeatedly recrystallizing the obtained resveratrol crude product with higher purity by using ethanol to obtain a white crystalline solid resveratrol product with the purity of more than 99 percent; secondly, the invention adopts an enzyme extraction method, can effectively destroy cell walls, reduce mass transfer resistance and greatly improve the extraction yield; the invention adopts ultrasonic extraction and high-pressure preparation liquid chromatography for separation and purification, and can effectively improve the content and purity of resveratrol; the process is simple to operate, easy to implement and high in purity.

Drawings

FIG. 1 is a liquid chromatogram of a peony seed husk resveratrol product from a process for preparing resveratrol from peony seed husks.

FIG. 2 is an infrared spectrum of a peony seed meal resveratrol product from a method of preparing resveratrol from peony seed hulls.

FIG. 3 is a thermal analysis curve of a peony seed meal resveratrol product of a method for preparing resveratrol from peony seed hulls.

Detailed Description

The following examples are illustrative of the present invention and should not be construed as limiting thereof.

Example 1:

crushing the paeonia ostii seed meal, and sieving the crushed paeonia ostii seed meal by using a sieve with 20-60 meshes to obtain fine powder of the paeonia ostii seed meal; adding water with the mass volume of 2-10 times, adjusting the pH value to 4.0-6.0, adding 0.1-1% of one or more enzymes of cellulase, hemicellulase, beta-glucosidase and xylanase, and stirring at a low speed for 1-5 hours at the temperature of 40-60 ℃; and (4) enzymolysis. After enzymolysis, adding ethanol water with volume fraction of 60-90% into the feed liquid, wherein the volume ratio of the ethanol water to the feed liquid is 1: 5-1: 20 (g: mL), then performing ultrasonic treatment with the power of 80-150W for 1-2 h, the extraction temperature of 30-60 ℃, extracting for 1-3 times, filtering, combining filtrates, performing reduced pressure evaporation on the filtrate at the temperature of 40-60 ℃, recovering an organic solvent, and performing vacuum drying or freeze drying on a concentrate to obtain a crude extract of the polydatin; adding distilled water into the crude extract of the polydatin according to the mass-volume ratio of 1: 10-1: 20 (g: mL), stirring and dissolving at 50-60 ℃, filtering, adding petroleum ether with the volume ratio of 1: 1 into the filtrate, extracting for 3-5 times, combining the petroleum ether extract phases, and concentrating under reduced pressure at 40-60 ℃ to obtain a petroleum ether extract; continuously performing isovolumetric extraction on the water layer for 3-5 times by using ethyl acetate, combining ethyl acetate extract phases, adjusting the pH value of the ethyl acetate extract phases to be 8.0-10.0 by using alkali, standing, filtering, and concentrating under reduced pressure until the volume of extract liquid is 1/15-1/20, and crystallizing to obtain a veratrol glycoside crude extract; adding 2-10 times of 2-10% diluted acid according to the volume ratio, hydrolyzing for 4-8 h, cooling, standing for 24h after hydrolysis and enrichment, filtering, and washing with acid to obtain a resveratrol crude product; dissolving the crude resveratrol product in ethyl acetate, and separating and purifying by using high-pressure preparative liquid chromatography, wherein the separation conditions of the high-pressure preparative liquid chromatography are as follows: a chromatographic column: welch Ultimate XB-C18(10 μm, 21.2 mm. times.250 mm); flow rate: 25 mL/min; sample introduction volume: 6 mL; mobile phase A: an aqueous solution containing 0.5% (v/v) acetic acid; mobile phase B: methanol; isocratic elution: 40 percent; ultraviolet detection wavelength: 308 nm. Collecting the eluent of the resveratrol section, and then carrying out reduced pressure concentration until the eluent is crystallized in small volume to obtain a crude resveratrol product with higher purity; and finally, repeatedly recrystallizing by using ethanol to obtain a white crystalline solid resveratrol product with the purity of more than 99 percent.

Example 2:

the chromatographic conditions of the resveratrol are as follows: a chromatographic column: sinochrom C18 ODS column (250 mm. times.4.6 mm, 5 μm); mobile phase: methanol and water are 50: 50; detection wavelength: 308 nm; flow rate: 0.8 mL/min; sample introduction volume: 20 mu L of the solution; column temperature: at a temperature of 27 ℃. The HPLC chromatogram of the peony seed meal resveratrol product only has one identifiable chromatographic peak, and the retention time is 7.893min, as shown in figure 1. Scanning in the wavelength range 200-800 nm, wherein the maximum absorption wavelength is only 219 and 308 nm. The compound is resveratrol through structural identification. The spectral characteristics are as follows: infrared spectrum: 3250cm^-1Has phenolic hydroxyl absorption peak at 965cm^-1The characteristic absorption of trans-C ═ C-, all of which are characteristic absorption peaks of resveratrol chemical structure and have 1620cm^-1，1540cm^-1，1500cm^-1And characteristic absorption peaks of benzene rings. And the infrared spectrum of the resveratrol is matched with the related literature. The infrared spectrogram of the resveratrol product from peony seed meal is shown in figure 2.

Example 3:

the thermal stability of a white crystalline solid resveratrol product is researched, and the result shows that the resveratrol has good thermal stability below 240 ℃, and the resveratrol has an obvious endothermic peak near 254.39 ℃ and corresponds to the melting point of the medicine. The thermal analysis curve of the peony seed meal resveratrol product is shown in figure 3.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined by the appended claims and their equivalents.