Method for extracting jujuboside by ultrasonic-assisted deep-eutectic solvent

Abstract

The invention belongs to the technical field of food processing, and particularly relates to a method for extracting jujuboside through an ultrasonic-assisted deep-eutectic solvent. According to the method, semen ziziphi spinosae powder is subjected to ultrasonic extraction by adopting a DES solvent, and the jujuboside extract is obtained. The jujuboside is extracted based on ultrasonic-assisted DES, the extraction time is shorter, and the extraction rate is higher. The DES solution prepared by the method has the characteristics of recoverability, greenness, no pollution and capability of overcoming the defects that a traditional organic reagent is easy to volatilize and has toxic properties, and the safety and higher nutritional value of the product are ensured while the extraction efficiency is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food processing, and particularly relates to a method for extracting jujube seed saponins by ultrasonic-assisted deep eutectic solvent. BACKGROUND

[0002] Jujube seed (Ziziphi Semen), also known as jujube kernel and jujube kernel, is the seed of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chiu, and is a homologous product of medicine and food. It is recorded in the Chinese medical classic Shennong Bencao Jing that jujube seed has the effects of "stabilizing five viscera, lightening the body and prolonging life", and is a medicinal material with the functions of nourishing heart and calming mind, tonifying liver, promoting saliva secretion and astringing sweat. Further modern pharmacological studies show that the main chemical components of jujube seed are triterpenoid saponins, fatty acids, alkaloids, flavonoids and amino acids, and jujube seed has multiple medicinal values such as reducing cholesterol and blood lipids, antioxidant, anti-convulsion, anti-inflammatory, anti-depression and enhancing immunity. Triterpenoid saponins are the main source of the medicinal effect of jujube seed, and have superior functions of sedation, calming and hypnotic. The extraction of triterpenoid saponins from jujube seed has been widely studied in the food processing industry.

[0003] At present, the main method for extracting jujube seed saponins is mainly dependent on organic solvents (such as methanol and ethanol) and high-temperature reflux method, and there are problems such as high solvent toxicity, high energy consumption, long extraction time and the like. The yield of saponins in the traditional process is only 1.2-1.8%, and subsequent complex purification is required to remove solvent residues. Therefore, it is urgent to explore a safe, efficient and green extraction method.

[0004] Deep eutectic solvent (DES) is a stable solvent formed by the combination of hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) through intermolecular hydrogen bond interaction. As a new type of green solvent, DES has many advantages such as non-volatility, simple preparation, low toxicity, environmental protection and recyclability, and has been widely used in the fields of organic synthesis, nanomaterial preparation and natural product extraction. However, the components in jujube seed are complex, containing interfering substances such as polysaccharides and flavonoids, which may lead to low purity of the extract, poor mass transfer efficiency and increased burden of subsequent purification. At present, there is no deep eutectic solvent system that can effectively extract jujube seed saponins. SUMMARY

[0005] In view of the research blank in the prior art, the application provides a method for extracting jujube seed saponins by ultrasonic-assisted deep eutectic solvent. The application uses ultraviolet spectroscopy (UV) to qualitatively analyze the extract, and optimizes the extraction process.

[0006] To achieve the above object, the technical scheme adopted by the present application is: The present application provides a method for extracting jujube kernel saponins by ultrasonic-assisted deep eutectic solvent, comprising the following steps: S1, dry the cleaned jujube kernels to constant weight, grind into powder, and obtain jujube kernel powder for standby; S2, DES solvent preparation: select choline chloride as a hydrogen bond donor, mix it with urea, heat and stir until it becomes a uniform clear transparent liquid state, adjust the water content, and obtain the DES solvent; S3, jujube kernel saponin extraction: weigh the jujube kernel powder and put it into the DES solvent, stir uniformly, and ultrasonic extract to obtain jujube kernel saponin extract.

[0007] Preferably, in step S2, the temperature of heating and stirring is 80-100℃; the molar ratio of choline chloride to urea is 2:1-1:4; and the water content of the DES solvent is 0-40%.

[0008] Preferably, in step S2, the molar ratio of choline chloride to urea is 1:1, and the water content of the DES solvent is 25%.

[0009] Preferably, in step S3, the solid-liquid ratio of the jujube kernel powder to the DES solvent is 1g:20-60mL.

[0010] Preferably, in step S3, the ultrasonic extraction is carried out at 30-70℃, 240-480W power for 30-150min.

[0011] Preferably, in step S3, the solid-liquid ratio of the jujube kernel powder to the DES solvent is 1:50, and the ultrasonic extraction is carried out at 60℃, 312W power for 120min.

[0012] The present application has the following advantages: (1) The present application is based on ultrasonic-assisted DES extraction of jujube kernel saponins, which has shorter extraction time and higher extraction rate. (2) The DES solution configured in the present application is recyclable, green and non-polluting, which overcomes the characteristics of traditional organic reagents that are easy to volatilize and have toxic properties, improves the extraction efficiency, and ensures the safety and high nutritional value of the product. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 is the absorbance of jujube kernel saponins; Figure 2 is the standard curve of jujube kernel saponins; Figure 3 is the extraction rate graph of the water content of the deep eutectic solvent; Figure 4Extraction yield graph of eutectic solvent molar ratio influencing factor Figure 5 Extraction yield graph of material liquid ratio influencing factor Figure 6 Extraction yield graph of extraction temperature influencing factor Figure 7 Extraction yield graph of ultrasonic time influencing factor Figure 8 Extraction yield graph of ultrasonic power influencing factor Figure 9 Three-dimensional response surface graph. DETAILED DESCRIPTION

[0014] Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0015] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by one of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0016] Embodiment 1 (1) Dry raw jujube seeds are purchased, washed, and dried in a constant temperature oven at 60°C until constant weight, then ground by a sealed grinder and passed through an 80-mesh sieve to obtain jujube seed powder; (2) Preparation of DES solvent: a. Accurately weigh a certain amount of HBD and HBA and mix them.

[0017] b. Heat and stir at 80-100°C until a uniform transparent liquid is formed to obtain a basic DES solution; c. Take a certain volume of the basic DES solution prepared in step 2 and add a certain volume ratio of water to obtain a DES solvent with a certain water content; (3) Put 2g of jujube seed powder into a conical flask containing the DES solvent with the corresponding water content for ultrasonic combination reaction, set the extraction temperature, time, and ultrasonic power according to the operation instructions, and perform extraction.

[0018] (4) Centrifuge the extraction solvent (10min, 4000r / min) and take the supernatant.

[0019] Effect embodiment 1 (1) Determination of absorbance of jujube seed saponins: Dilute jujube seed saponin A powder with ethanol, accurately pipette 0.5 ml of jujube seed saponin A standard and 0.2 ml of ethanol blank into 10 ml stoppered test tubes, evaporate the solvent, add 0.2 ml of 5% vanillin glacial acetic acid solution and 0.8 ml of perchloroacetic acid to each test tube, shake well, heat in a 60°C water bath for 15 min, immediately cool in an ice water bath for 5 min, accurately add 5.0 ml of glacial acetic acid and shake well, measure the absorbance using a UV spectrophotometer at 200-800, the optimal absorption peak was found to be 609, the results are shown in ( Figure 1 ).

[0020] (2) Determination of the yield of jujube seed saponins: Jujube seed saponin A powder was diluted with ethanol to accurately prepare a 1 mg / ml jujube seed saponin A standard solution. 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3 ml of the standard solution and 0.2 ml of ethanol (control group) were pipetted and the absorbance was measured at a wavelength of 609 nm. A standard curve was plotted with the mass concentration of jujube seed saponins as the abscissa and the absorbance as the ordinate: y = 2.2977*x - 0.0343 ( R 2 =0.9925), where: y is the mass of jujube seed saponins (mg); x is the absorbance value, and the results are shown in ( Figure 2 ).

[0021] (3) Single-factor experiment (a) Effect of moisture content on the extraction yield of jujube seed saponins DES solvent was prepared according to the method provided in Example 1. The extraction ultrasonic power was fixed at 240 W, the material-to-liquid ratio at 1:40, the extraction temperature at 60°C, the molar ratio at 1:1, and the extraction time at 120 min. Different water contents (0%, 10%, 20%, 30%, and 40%) were set for extraction of jujube seed saponins, and the extraction yield was calculated. The results are shown in [Figure 1]. Figure 3 .

[0022] pass Figure 3 It can be seen that the extraction rate of jujube seed saponins is the highest when the water content of the DES solvent is 20%.

[0023] (b) Effect of molar ratio on the extraction yield of jujube seed saponins DES solvent was prepared according to the above method. The extraction ultrasonic power was fixed at 240 W, the solid-liquid ratio at 1:40, the extraction temperature at 60℃, the water content at 30%, and the extraction time at 120 min. Different molar ratios of 2:1, 1:1, 1:2, 1:3, and 1:4 were set for the extraction of jujube seed saponins, and the extraction yield was calculated. The results are shown in […]. Figure 4 .

[0024] pass Figure 4It can be seen that the extraction rate of jujube seed saponins is the highest when the molar ratio is 1:1.

[0025] (c) Effect of solid-liquid ratio on the extraction yield of jujube seed saponins DES solvent was prepared according to the method provided in Example 1. The extraction ultrasonic power was fixed at 240 W, molar ratio at 1:1, extraction temperature at 60℃, water content at 30%, and extraction time at 120 min. Different material-to-liquid ratios of 1:20, 1:30, 1:40, 1:50, and 1:60 were set for the extraction of jujube seed saponins, and the extraction yield was calculated. The results are shown in […]. Figure 5 .

[0026] pass Figure 5 It can be seen that the extraction rate of jujube seed saponins is the highest when the material-to-liquid ratio is 1:50.

[0027] (d) Effect of extraction temperature on the extraction yield of jujube seed saponins DES solvent was prepared according to the method provided in Example 1. The extraction ultrasonic power was fixed at 240 W, molar ratio of 1:1, solid-liquid ratio of 1:40, water content of 30%, and extraction time of 120 min. Different extraction temperatures of 30, 40, 50, 60, and 70 °C were set for the extraction of jujube seed saponins, and the yield was calculated. The results are shown in [Figure 1]. Figure 6 .

[0028] pass Figure 6 It can be seen that the extraction rate of jujube seed saponins is the highest when the extraction temperature is 60℃.

[0029] (e) Effect of extraction time on the extraction yield of jujube seed saponins DES solvent was prepared according to the method provided in Example 1. The extraction ultrasonic power was fixed at 240 W, molar ratio 1:1, solid-liquid ratio 1:40, water content 30%, and extraction temperature 60℃. Extraction times of 30, 60, 90, 120, and 150 °C were set for the extraction of jujube seed saponins, and the yield was calculated. The results are shown in [Figure 1]. Figure 7 .

[0030] pass Figure 7 It can be seen that the extraction rate of jujube seed saponins is the highest when the extraction time is 120℃.

[0031] (f) Effect of ultrasonic power on the extraction yield of jujube seed saponins DES solvent was prepared according to the method provided in Example 1. The extraction time was fixed at 120 min, molar ratio 1:1, solid-liquid ratio 1:40, water content 30%, and extraction temperature 60℃. Different ultrasonic powers (240, 300, 360, 420, and 480) were used to extract jujube seed saponins, and the extraction yield was calculated. The results are shown in [Figure 1]. Figure 8 .

[0032] pass Figure 8 It can be seen that the extraction rate of jujube seed saponins is the highest when the ultrasonic power is 360w.

[0033] Example 2 (a) Plackett-Burman test Based on the single-factor experimental results of the above-mentioned effect examples, following the Plackett-Burman experimental principle, with the extraction yield of jujube seed saponins as the response value, and ultrasonic temperature (A), water content (B), ultrasonic time (C), molar ratio (D), material-liquid ratio (E), and ultrasonic power (F) as independent variables, the significance of the differences in the effects of each factor on the extraction yield of jujube seed saponins was investigated. An experiment with N=12 experiments was created, and each group of experiments was repeated 3 times. The specific experimental design is shown in Tables 1 and 2.

[0034] Table 1. Plackett-Burman Experimental Design Factors and Levels Table 2 Plackett-Burman Experimental Design and Results Analysis of variance was performed on the regression model, and the results are shown in Table 3. The table shows that temperature, moisture content, and ultrasonic power have a more significant impact on the extraction rate of jujube seed saponins. P <0.05); Experimental model P value( P A correlation coefficient <0.01 indicates that the model has reached a significant level; R² =0.9499, indicating a good correlation; adjusted coefficient of determination R2Adj =0.8897, indicating that the regression model's predicted value can achieve an accuracy of 88.97%; the lower the coefficient of variation (CV), the higher the reliability and accuracy of the experiment is generally. CV=6.20% indicates that the Plackett-Burman experiment has good reliability and accuracy; generally, an effective signal-to-noise ratio (Adeq Precisior) greater than 4.0 is considered reasonable. The precision of this experiment is 11.139.

[0035] Table 3. Factors, levels, and significance analysis of the Plackett-Burman experiment. Note: * indicates a significant effect (P < 0.05), — indicates a non-significant effect.

[0036] (ii) Response surface methodology Based on the Plackett-Burman experimental results and following the Box-Behnken experimental principle, a three-factor, three-level response surface optimization experiment was constructed with the amount of jujube seed saponins extracted as the response value and ultrasonic temperature (A), moisture content (B), and ultrasonic power (C) as independent variables. Each experiment was repeated three times. The specific experimental design is shown in Table 4.

[0037] Table 4. Factors and levels in the Box-Behnken experimental design Table 5 Box-Behnken Experimental Design and Results An analysis of variance was performed on the regression model, and the results are shown in Table 6. The model has high variance. F value( F = 50.97) and lower P value( P A correlation coefficient <0.0001 indicates that the model has reached a significant level. R² =0.9850, indicating a good correlation; adjusted coefficient of determination R2Adj =0.9656, indicating that the regression model's predicted value can achieve an accuracy of 96.56%; the lower the coefficient of variation (CV), the higher the reliability and accuracy of the experiment is generally. CV=3.17% indicates that the Plackett-Burman experiment has good reliability and accuracy; generally, an effective signal-to-noise ratio (Adeq Precisior) greater than 4.0 is considered reasonable. The precision of this experiment is 21.372.

[0038] Table 6. Factors, levels, and significance analysis of the Box-Behnken experiment. Note: * indicates a significant effect (P < 0.05), — indicates a non-significant effect.

[0039] Figure 9The three-dimensional response surface plots reflect the influence of the interaction of three factors on the extraction yield of jujube seed saponins. All three-dimensional response surface plots open downwards and have a maximum point, indicating that as the ultrasonic temperature, ultrasonic power, and water content gradually increase pairwise, the extraction yield of jujube seed saponins gradually rises to a peak and then decreases, consistent with the trend observed in the single-factor experiments. Analysis of the fitted equations and variance analysis results, using Design Expert software, yielded the optimal extraction conditions as follows: ultrasonic temperature 59.5928℃, ultrasonic power 311.666W, and water content 24.9384%. Based on actual extraction conditions and processes, for ease of practical operation, the extraction conditions were adjusted to ultrasonic temperature 60℃, ultrasonic power 312W, and water content 25%. Three verification experiments were conducted under these conditions, and the extraction yield of jujube seed saponins was 25.753 ± 0.173 mg / g. This shows that the optimized process parameters obtained in the experiment can effectively optimize the extraction of jujube seed saponins and improve the extraction efficiency.

[0040] Comparative Example 1 2g of jujube seed powder prepared under the same conditions was used. 1,4-Butanediol was selected as the hydrogen bond donor and choline chloride as the hydrogen bond acceptor. DES solvent was prepared according to the method provided in Example 1. The extraction ultrasonic power was fixed at 240W, molar ratio at 1:1, extraction temperature at 60℃, water content at 30%, extraction time at 120min, and material-liquid ratio at 1:40. The extraction experiment was carried out. The extraction solvent was centrifuged (10min, 4000r / min), and the supernatant was collected. The extraction yield was calculated to be 14.299mg / g, which is much lower than the 21.233mg / g of the urea-choline chloride combination extraction yield under the same conditions.

[0041] Comparative Example 2 Take 2g of jujube seed powder prepared under the same conditions and extract it by reflux with 70% ethanol at a material-to-liquid ratio of 1:50. After stirring to mix evenly, heat to reflux and start timing at a slight boiling state. Reflux for 1 hour for each group. Take out the supernatant and add 70% ethanol according to the material-to-liquid ratio to repeat the reflux extraction. Reflux for a total of 3 groups. Centrifuge the extraction solvent (10 min, 4000 r / min). Take the supernatant and concentrate by rotary evaporation. The extraction yield is calculated to be 9.418 mg / g.

Claims

1. A method for extracting jujube seed saponins using an ultrasound-assisted eutectic solvent, characterized in that, Includes the following steps: S1. Dry the cleaned jujube kernels to constant weight, grind them into powder, and set aside the jujube kernel powder. S2, DES solvent preparation: Choline chloride is selected as the hydrogen bond donor, and it is mixed with urea and heated and stirred until it presents a uniform, clear and transparent liquid state. The water content is adjusted to obtain DES solvent. S3. Jujube seed saponin extraction: Weigh jujube seed powder and put it into DES solvent, stir evenly, and extract by ultrasonication to obtain jujube seed saponin extract.

2. The method according to claim 1, characterized in that, In step S2, the heating and stirring temperature is 80-100℃; the molar ratio of choline chloride and urea is 2:1-1:4; and the water content of the DES solvent is 0-40%.

3. The method according to claim 2, characterized in that, In step S2, the molar ratio of choline chloride to urea is 1:1, and the water content of the DES solvent is 25%.

4. The method according to any one of claims 1-3, characterized in that, In step S3, the ratio of the jujube seed powder to the DES solvent is 1g:20-60mL.

5. The method according to claim 1 or 4, characterized in that, In step S3, the ultrasonic extraction is performed at 30-70℃ and 240-480W power for 30-150 minutes.

6. The method according to claim 5, characterized in that, In step S3, the material-to-liquid ratio is 1:50, and the ultrasonic extraction is performed at 60°C and 312W power for 120 minutes.

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