Process for extracting ellagic acid from walnut endopleura

Abstract

The invention provides a process for extracting ellagic acid from walnut endopleura, and relates to the technical field of ellagic acid extraction. The process for extracting ellagic acid from walnut endopleura comprises the following steps: step 1, raw material pretreatment: crushing the walnut endopleura, and sieving with a 20-80-mesh sieve after crushing to obtain an intermediate material I; step 2, ultrasonic-assisted extraction: mixing the intermediate material I in the step 1 with ethanol, performing ultrasonic treatment on the mixture for 1-1.2 hours under the conditions that the frequency is 53 kHz, the power is 240 W and the temperature is 60 DEG C to destroy a cell structure, and then extracting to obtain an extracting solution; step 3, acid hydrolysis: mixing and stirring the extracting solution in the step 2 and a 1mo l / L hydrochloric acid solution according to a mass-volume ratio of 1: 2, then centrifuging, and extracting supernate; and 4, purifying a crude product. Through the processes of crushing of the walnut skin, ultrasonic extraction, acid hydrolysis and purification, the yield of ellagic acid extracted from the walnut skin is very high, the waste utilization cost is low, the extraction of ellagic acid from the walnut skin belongs to a creative technology, and the process is simple.

Description

Technical Field

[0001] The present invention relates to the technical field of ellagic acid extraction, and specifically to a process for extracting ellagic acid from the inner seed coat of walnuts. Background Art

[0002] Ellagic acid is a natural polyphenolic compound with various biological activities such as antioxidant, anti-inflammatory, and anti-cancer, and is widely used in the pharmaceutical, food, and cosmetic industries.

[0003] In the prior art, the extraction of ellagic acid mostly uses traditional solvent extraction or enzymatic hydrolysis methods, which have problems such as low extraction rate, complex process, and high cost. The inner seed coat of walnuts, as agricultural waste, is rich in ellagic acid, but there has been no report on its efficient extraction process. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a process for extracting ellagic acid from the inner seed coat of walnuts, which solves the problems of low extraction rate and high cost of ellagic acid.

[0005] To achieve the above objectives, the present invention is realized through the following technical solutions: A process for extracting ellagic acid from the inner seed coat of walnuts, comprising the following steps:

[0006] Step 1. Raw material pretreatment:

[0007] Crush the inner seed coat of walnuts, and after crushing, pass through a 20 - 80 mesh sieve to obtain intermediate material 1;

[0008] Step 2. Ultrasonic-assisted extraction:

[0009] Mix intermediate material 1 in Step 1 with ethanol, and the mass ratio of the intermediate material 1 to ethanol in the material-liquid is 1:12. Ultrasonically treat it at a frequency of 53 kHz, a power of 240 W, and a temperature of 60 °C for 1 - 1.2 h to destroy the cell structure and then carry out extraction to obtain an extract;

[0010] Step 3. Acid hydrolysis:

[0011] Mix the extract in Step 2 with 1 mol / L hydrochloric acid solution according to a mass-to-volume ratio of 1:2, stir, and then centrifuge to obtain the supernatant;

[0012] Step 4. Purification of crude product:

[0013] Concentrate the supernatant in Step 3. Then, add 1% sodium hydroxide solution to the supernatant according to the mass ratio of walnut inner pericarp to the volume of alkali of 1:1, add water and petroleum ether for extraction to remove fat-soluble impurities according to the mass ratio of walnut inner seed coat to the liquid volume of 1:10, and add water and petroleum ether for extraction to remove fat-soluble impurities according to the mass ratio of walnut inner seed coat to the liquid mass volume of 1:1. Centrifuge respectively and collect the aqueous phase, adjust the pH to 2-3, precipitate respectively, and vacuum dry to obtain ellagic acid.

[0014] Preferably, the concentration of the ethanol solution in Step 2 is 50%, the extraction temperature is 70 degrees Celsius, and the extraction time is 80-90 minutes.

[0015] Preferably, the mixing and stirring in Step 3 is carried out at 60 degrees Celsius for 60 minutes.

[0016] Preferably, the extraction rate of ellagic acid is 2.784 ug / g, detected by HPLC and under the environment of 254 nm wavelength.

[0017] The present invention provides a process for extracting ellagic acid from walnut inner seed coat. It has the following beneficial effects:

[0018] Through the processes of crushing, ultrasonic extraction, acid hydrolysis and purification of walnut peel, the present invention has a very high yield of extracting ellagic acid from walnut peel, low waste utilization cost, and the extraction of ellagic acid from walnut peel belongs to a creative technology with a simple process. Brief Description of the Drawings

[0019] Figure 1 It is the influence of the solid-liquid ratio on the extraction rate under the single-factor conditions of the present invention;

[0020] Figure 2 It is the influence of the volume ratio of ethanol aqueous solution on the extraction rate under the single-factor conditions of the present invention;

[0021] Figure 3 It is the influence of the extraction time on the extraction rate under the single-factor conditions of the present invention;

[0022] Figure 4 It is the influence of the extraction temperature on the extraction rate under the single-factor conditions of the present invention. Detailed Description of the Invention

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0024] Example 1:

[0025] As Figures 1-4 shown, an embodiment of the present invention provides a process for extracting ellagic acid from the inner seed coat of walnuts, comprising the following steps:

[0026] Step 1. Raw material pretreatment:

[0027] Crush the inner seed coat of walnuts, and after crushing, pass through an 80-mesh sieve to obtain Intermediate Material 1. Crushing increases the specific surface area of the inner seed coat of walnuts and improves the efficiency of collecting ellagic acid;

[0028] Step 2. Ultrasonic-assisted extraction:

[0029] Mix Intermediate Material 1 in Step 1 with ethanol. The mass ratio of Intermediate Material 1 to ethanol in the material-liquid is 1:12. Ultrasonically treat it at a frequency of 53 kHz, a power of 240 W, and a temperature of 60 °C for 1 h to disrupt the cell structure and then perform extraction to obtain an extract. The concentration of the ethanol solution is 50%, the extraction temperature is 70 °C, and the extraction time is 80 minutes to obtain ellagic acid;

[0030] Step 3. Acid hydrolysis:

[0031] Mix the extract in Step 2 with a 1 mol / L hydrochloric acid solution according to a mass-to-volume ratio of 1:2, stir the mixture at 60 °C for 60 minutes, then perform centrifugation, and extract the supernatant. At this time, ellagic acid is converted from the bound state to the free state;

[0032] Step 4. Crude product purification:

[0033] Concentrate the supernatant in Step 3. Then, add a 1% sodium hydroxide solution to the supernatant according to a ratio of the mass of the inner middle skin of walnuts to the volume of the alkali of 1:1, add water and petroleum ether for extraction to remove fat-soluble impurities according to a ratio of the mass of the inner seed coat of walnuts to the liquid volume of 1:10, add water and petroleum ether for extraction to remove fat-soluble impurities according to a ratio of the mass of the inner seed coat of walnuts to the liquid mass volume of 1:1. After centrifugation respectively, collect the aqueous phase, adjust the pH to 2-3, precipitate respectively, and vacuum dry to obtain ellagic acid. Then, use acetonitrile-water gradient elution, the elution time is 0-30 minutes, acetonitrile 5% → 100%; the flow rate is 1.0 mL / min, the column temperature is 30 °C to obtain ellagic acid, the extraction rate of ellagic acid is 2.784 ug / g, and it is detected by HPLC under an environment of a wavelength of 254 nm.

[0034] Example 2:

[0035] As Figures 1-4As shown in the figure, an embodiment of the present invention provides a process for extracting ellagic acid from the inner seed coat of walnuts. Take 5 g of crushed inner seed coat of walnuts, add 60 mL of 50% ethanol aqueous solution, and perform ultrasonic extraction for 80 minutes at 53 kHz, 240 W, and 70 °C. After centrifugation, take the supernatant, add 10 mL of 1 mol / L hydrochloric acid, and stir and react at 60 °C for 60 minutes. After concentrating the reaction solution, dissolve it with 5 mL of 1% NaOH, add 50 mL of water and 10 mL of petroleum ether for extraction, and centrifuge to discard the organic phase. Adjust the pH of the aqueous phase to 2.5 with concentrated hydrochloric acid to precipitate, and vacuum dry to obtain ellagic acid. The content and purity of ellagic acid are detected by HPLC (mobile phase: acetonitrile-water gradient elution), and finally the extraction rate of ellagic acid is 2.784 μg / g.

[0036] The experiments are as follows:

[0037] Reagents:

[0038] Ellagic acid, sodium hydroxide, 36% concentrated HCl, ethanol, formic acid, petroleum ether, acetonitrile, distilled water.

[0039] Instruments:

[0040] Electronic balance (FA2004N), power-adjustable bench-top ultrasonic cleaner (KQ3200SM), magnetic stirrer (MS-H280-Pro / MS-H-ProA), rotary evaporation condenser (R-100), circulating water multi-purpose vacuum pump (SHB-Ⅲ), centrifuge (H1850), vacuum drying oven (DZF-6020), Shimadzu high performance liquid chromatograph (LC-20A).

[0041] Plot the curve:

[0042] Prepare a standard stock solution of ellagic acid (100 μg / mL)

[0043] Accurately weigh 10.0 mg of ellagic acid into a 100 mL brown volumetric flask, dissolve it with 5 mL of 1% NaOH solution (heating can be used for dissolution), then add 40 mL of 50% ethanol, and finally make up the volume to the scale with 50% ethanol. Prepare it freshly for use.

[0044] Prepare a standard working solution of ellagic acid:

[0045] Take 1.25 mL, 2.50 mL, 5.00 mL, 7.5 mL, and 10 mL of the standard stock solution (2.1) into 25 mL brown volumetric flasks, and dilute to the scale with 50% ethanol; form standard series solutions with concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL together with the stock solution. Prepare it freshly for use.

[0046] Experimental process:

[0047] Single-factor experiment:

[0048] Fix the ultrasonic conditions (ultrasonic for 1 h at a frequency of 53 kHz, a power of 240 W, and a temperature of 60 °C), and conduct single-factor experiments on the feeding amount, solvent ratio, extraction time, and extraction temperature. See Table 1

[0049] Table 1 Single-factor extraction process of ellagic acid

[0050]

[0051] Preparation of crude product:

[0052] Take 5 g of crushed walnut seed coat in a 250 mL beaker, add 50 mL of 40% ethanol, ultrasonic for 60 min at 53 Khz - 40% - 60 °C, centrifuge at 10000 r / min for 3 min, take the supernatant, add 10 mL of 1 mol HCl, and stir and react at 60 °C for 60 min. After the reaction, filter by suction, and concentrate the filtrate at 80 °C to obtain the crude product to be measured.

[0053] Purification of crude product:

[0054] Take the above crude product, dissolve it in 5 mL of 1% NaOH, add 50 mL of distilled water and 10 mL of petroleum ether for extraction. After stratification, discard the upper organic phase, collect the lower aqueous phase, centrifuge at 10000 r / min for 10 min, discard the supernatant impurities, vacuum the precipitate to obtain the sample to be detected.

[0055] Sample determination:

[0056] Take 10 samples to be measured in a 25 mL volumetric flask, dissolve them with 2 mL of 1% NaOH, make up the volume to the mark with 40% ethanol, filter, and determine with a high performance liquid chromatograph.

[0057] Single-factor experiment on solid-liquid ratio:

[0058] As Figure 1 shown, although the extraction rate increases with the increase of the solid-liquid ratio, but after the solid-liquid ratio exceeds 1:12, the increase amplitude is not large. In order to save solvent and post-treatment costs, therefore, 1:12 is selected as the final solid-liquid ratio in this process. At this time, the extraction content is: 1.785 ug / g.

[0059] As Figure 2 shown, the extraction rate is the highest when the ethanol content is 50%. Therefore, 50% ethanol aqueous solution is selected as the final extraction solution in this process. At this time, the extraction content is: 1.734 ug / g.

[0060] As Figure 3As shown, although the extraction rate increases with the increase of extraction time, the growth trend is not obvious after the extraction time reaches 50 minutes. Considering the actual production requirements, the extraction time is selected as 60 minutes in this process. At this time, the extraction content is: 1.535 ug / g.

[0061] As Figure 4 shown, although the extraction rate increases with the increase of extraction temperature, the higher the extraction temperature, the more ethanol volatilizes, which affects the extraction efficiency. Considering the potential risks in the actual production process, the extraction temperature is selected as 70 °C in this process. At this time, the extraction content is: 1.728 ug / g.

[0062] Orthogonal experiment:

[0063] Through the processing of the single-factor experiment data, it can be known that under the condition of 254 nm, the extraction efficiency is the highest when the solvent ratio is 50%. Therefore, the solvent ratio of 50% is fixed, and the orthogonal experiment is carried out on the feed amount, extraction time, and extraction temperature to explore the optimal conditions. See Table 2.

[0064] Table 2 Orthogonal experiment scheme for ellagic acid extraction process

[0065]

[0066] Therefore, it is inferred that the optimal process for extracting ellagic acid from the inner seed coat of walnuts is: the feed ratio is 1:12, the extraction time is 80 minutes, and the extraction temperature is 70 °C. At this time, the extraction rate reaches 2.784 ug / g.

[0067] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A process for extracting ellagic acid from the inner seed coat of walnuts, characterized in that: It includes the following steps: Step 1, raw material pretreatment: Crush the inner seed coat of walnuts, and after crushing, pass through a 20 - 80 mesh sieve to obtain Intermediate Material 1; Step 2, ultrasonic - assisted extraction: Mix Intermediate Material 1 from Step 1 with ethanol. The mass - to - liquid ratio of Intermediate Material 1 to ethanol is 1:

12. Ultrasonicate it at a frequency of 53 kHz, a power of 240 W, and a temperature of 60 °C for 1 - 1.2 h to disrupt the cell structure and then carry out extraction to obtain an extract; Step 3, acid hydrolysis: Mix the extract from Step 2 and 1 mol / L hydrochloric acid solution according to a mass - to - volume ratio of 1:2, stir, and then centrifuge to extract the supernatant; Step 4, crude product purification: Concentrate the supernatant from Step 3. Then, add 1% sodium hydroxide solution to the supernatant according to a ratio of the mass of the inner middle skin of walnuts to the volume of the alkali of 1:1, add water and petroleum ether for extraction to remove fat - soluble impurities according to a ratio of the mass of the inner seed coat of walnuts to the liquid volume of 1:10, add water and petroleum ether for extraction to remove fat - soluble impurities according to a ratio of the mass of the inner seed coat of walnuts to the liquid mass - volume of 1:

1. Centrifuge respectively and collect the aqueous phase, adjust the pH to 2 - 3, precipitate respectively, and vacuum - dry to obtain ellagic acid.

2. The process for extracting ellagic acid from the inner seed coat of walnuts according to claim 1, characterized in that: The concentration of the ethanol solution in Step 2 is 50%, the extraction temperature is 70 °C, and the extraction time is 80 - 90 minutes.

3. A process for extracting ellagic acid from the inner seed coat of walnuts according to claim 1, characterized in that: The mixing and stirring in Step 3 is carried out at 60 °C for 60 minutes.

4. A process for extracting ellagic acid from the inner seed coat of walnuts according to claim 1, characterized in that: The extraction rate of ellagic acid is 2.784 μg / g, detected by HPLC under a wavelength of 254 nm.

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