Method for extracting limonin from pomelo kernels by response surface experiment method

Abstract

A method for extracting limonin from grapefruit kernels by adopting a response surface experimental method comprises the following steps: separating and drying: separately separating and collecting grapefruit kernels of grapefruits, weighing fresh grapefruit kernels, drying, and weighing dry grapefruit kernels; squeezing and crushing: squeezing the dried pomelo kernels to obtain kernel oil and kernel residues, respectively collecting the kernel oil and the kernel residues, crushing the kernel residues to obtain pomelo kernel powder, and weighing the weight of the pomelo kernels; extracting, namely adding a solvent into the powder, dissolving the powder in the solvent, extracting in a set extraction time in an ultrasonic manner to obtain an extracting solution, and evaporating and spin-drying the extracting solution through a rotary evaporator to obtain a limonin extract; and detection: dissolving, filtering and centrifuging the limonin extract by using acetonitrile, taking supernate as a sample solution for later use, and detecting the sample solution to obtain the extraction rate of limonin. The influence of each factor is analyzed by using a response method, and the extraction efficiency and stability are improved.

Description

Technical Field The invention relates to the technical field of grapefruit processing, in particular to a method for extracting limonin from grapefruit seeds by using a response surface experimental method. Background Art Pomelo is rich in limonoid compounds, with limonin playing the main role. The content is higher in the pomelo pit (seed), and less in the peel (about 1 / 10,000 to 5 / 100,000). Pomelo is not only of high nutritional value, but also has the effects of strengthening the stomach, moistening the lungs, replenishing blood, clearing the intestines, facilitating bowel movements, digesting food, and detoxifying alcohol. my country is rich in pomelo resources. Pomelo pits are by-products of pomelo processing and are often discarded as waste. Using extraction methods to develop and utilize waste can obtain considerable economic benefits and has broad industrial prospects. Limonin compounds are mainly found in the fruits of Rutaceae plants and have multiple biological activities such as anti-tumor, insect repellent, antiviral, analgesic, anti-inflammatory, and hypnotic. In addition to the above biological effects, limonin analogs also have antioxidant activity, antibacterial properties, HIV inhibition, cholesterol reduction, obvious diuretic effect, improvement of cardiovascular and cerebrovascular circulation and sleep, antiviral, cytochrome regulation, etc. They have good health functions and can be used in functional food additives, anti-cancer foods, pesticides, feed additives, etc. At present, although there are many reports on the extraction of limonin, most of the extractants are single extractants. However, due to the influence of temperature and ultrasonic power, the best solvents at different temperatures are different. For example, for the extraction of limonin and nomilin, Shao Jinliang et al. used a constant temperature of 35°C and ultrasonic extraction, and ethyl acetate was the best extractant, while Luo Xirong et al. used 37°C and ultrasonic extraction, and dichloromethane was the best extractant. However, these methods have the disadvantages of low extraction rate and long time consumption. In addition, there are few research reports on the process of extracting limonin using mixed solvents as extractants, and there are also few research reports on the use of response surface methodology to optimize the yield of limonin. Response Surface Methodology (RSM) is a simulation analysis method based on mathematical statistics. RSM design test methods include central composite design, full factor design, Box-Behnken design, etc. Compared with traditional statistical methods, RSM method can more intuitively judge the optimal area through graphics; it uses the results of previous tests to reasonably design a limited number of tests, establish a mathematical model, fit the global relationship of the function, and evaluate and optimize the test by describing the relationship between the response value and each factor to obtain the best test condition model. Summary of the invention The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a method for extracting limonin from pomelo seeds. A method for extracting limonin from pomelo seed by using response surface methodology comprises the following steps: Separation and drying: After the grapefruit seeds are separated and collected, the fresh weight of the grapefruit seeds is weighed, and the seeds are dried at a constant temperature of 50-60° C. for 8-10 hours, and the dry weight is weighed after drying; Pressing and crushing: Pressing the dried pomelo seeds to obtain seed oil and seed residue, collecting the seed oil and the seed residue respectively, crushing the seed residue to less than 40 meshes to obtain pomelo seed powder, and weighing the pomelo seeds; Extraction, adding a solvent to the powder to dissolve the powder in the solvent, then extracting by ultrasonication for a set extraction time to obtain an extract, and evaporating the extract to dryness by a rotary evaporator to obtain a limonin extract; Detection: The limonin extract is dissolved in acetonitrile, filtered, and centrifuged, and the supernatant is taken as a sample solution for standby use. The sample solution is tested to obtain the extraction rate of limonin. The response surface experimental method was used to analyze different usage ratios of ethyl acetate and dichloromethane. Three different usage ratios were put into the test, namely, the usage ratio of ethyl acetate was greater than the usage ratio of dichloromethane, the usage ratio of ethyl acetate was equal to the usage ratio of dichloromethane, and the usage ratio of ethyl acetate was less than the usage ratio of dichloromethane. The result showed that the usage ratio of ethyl acetate was greater than the usage ratio of dichloromethane was the preferred solution. The solvent is a mixture of ethyl acetate and dichloromethane, wherein the volume ratio of ethyl acetate to dichloromethane is 1:1-1:10, and the preferred ratio of ethyl acetate to dichloromethane is 1:4. When ultrasonic extraction is performed, the temperature is maintained at 30-40°C, the extraction time is 30-60 minutes, and the ultrasonic power is 40-60 khz. When the dosage ratio of ethyl acetate and dichloromethane in the solvent is determined, several groups of solvents and powders with different dosage ratios are tested, and the amount of solvent is greater than that of powder, and the dosage ratio of powder to solvent is obtained as 1:8-1:20, and the unit is g / ml. The addition ratio of the powder to the solvent is 1:11, and the unit is g / ml. The ratio of ethyl acetate to dichloromethane in the solvent and the ratio of solvent to powder are determined to be 30-60 minutes, the temperature is maintained at 30-40° C., and the ultrasonic power is 40-60 khz. According to the obtained usage ratio of ethyl acetate and dichloromethane in the solvent, the addition ratio of solvent and powder, and the extraction time, multiple linear regression and binomial fitting were performed and the regression equation was established: Y=+1.80025+0.027719A-0.007493B+0.003499C-0.010132AB+0.031357AC+0.009298BC-0.050093A2-0.027756B2-0.117008C2, Wherein, Y represents the response value and the measured limonin content, A represents the ultrasonic extraction time, in min; B represents the addition ratio of solvent to powder, in g / mL; C represents the usage ratio of ethyl acetate to dichloromethane in the solvent. In the extraction of limonin, the extraction time is greater than the added amount ratio of the solvent to the powder, and the added amount ratio of the solvent to the powder is greater than the amount ratio of ethyl acetate to dichloromethane in the solvent. The pomelo includes Shatian pomelo, Wendan pomelo, and honey pomelo, and Shatian pomelo is preferred. Compared with the prior art, the present invention has the following beneficial technical effects: The mixed solvent consisting of ethyl acetate and dichloromethane is used as an extracting liquid to extract the pomelo pits, thereby improving the extraction efficiency and realizing the recycling of the discarded pomelo pits. The operation is simple and has high economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Schematic diagram of the standard curve of limonin in the present invention; Figure 2 This is a schematic diagram of the single factor experimental results of the mixed solvent ratio in the present invention; Figure 3 This is a schematic diagram of the single factor experimental results of the liquid-to-solid ratio in the present invention; Figure 4 This is a schematic diagram of the single factor experimental results of ultrasonic extraction time in the present invention; Figure 5 Three-dimensional surface diagram of the interaction between the mixed solvent ratio and the liquid-to-solid ratio; Figure 6 Contour plot of the interaction between the mixed solvent ratio and the liquid-to-solid ratio; Figure 7 Three-dimensional surface plot of the interaction between mixed solvent ratio and extraction time; Figure 8 Contour plot of the interactive effect of mixed solvent ratio and extraction time; Fig. 9 Three-dimensional surface diagram of the interaction between extraction time and liquid-to-solid ratio; Fig.10 Contour plot of the interaction between extraction time and liquid-to-solid ratio. DETAILED DESCRIPTION In order to further understand the features, technical means, specific objectives and functions of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. The following is an explanation of the pomelo variety, Shatian pomelo. The following is a list of the instruments and reagents involved. Experimental samples: Drug Name Specification / Purity Manufacturer Limonin 98％ Stanford Chemicals Acetonitrile Analytical grade Sinopharm Chemical Reagent Co., Ltd. Acetonitrile Chromatographic pure Tedia USA, Inc. Ethyl acetate Analytical grade Sinopharm Chemical Reagent Co., Ltd. Dichloromethane Analytical grade Sinopharm Chemical Reagent Co., Ltd. Anhydrous ethanol Analytical grade Sinopharm Chemical Reagent Co., Ltd. Experimental instruments: First, a standard curve of limonin needs to be prepared. Weigh 15.06 mg of limonin into a 15 ml conical flask, add 3.765 ml of acetonitrile, use 40 khz ultrasound to fully dissolve the limonin, transfer to a 5 ml volumetric flask, dilute to volume with chromatographically pure acetonitrile, shake well, and seal and store at 3 ° C for later use. Use a pipette to draw an appropriate amount of limonin standard reference stock solution, and use chromatographically pure acetonitrile to prepare standard solution reference solutions with mass concentrations of 5.00, 50.00, 100.00, 500.00, 1000.00, and 2000 μg / ml of normilin, and seal and store at 3 ° C for later use. The experimental results show that the reference Figure 1 As shown, the standard curve of limonin showed a good linear relationship. The standard curve was y=5009.50053x-12956.74079, R 2 =0.99998; this standard curve is used to quantify the limonin in the samples. Embodiment 1 A method for extracting limonin from pomelo pomelo seeds comprises the following steps: Separation and drying: After the pomelo seeds of Shatian pomelo are separated and collected, the fresh weight is weighed according to the preset single weight, and the seeds are dried at a constant temperature of 55°C for 8 hours. After drying, the dry weight is weighed. For example, if the single weight is set to 50g, the 50g pomelo seeds are weighed again after drying to determine the weight after removing a certain amount of water. Pressing and crushing: the dried pomelo seeds are pressed to obtain seed oil and seed residue, the seed oil and seed residue are collected separately, and the seed residue is crushed into 40 meshes to obtain pomelo seed powder. Extraction, add solvent to the powder to dissolve the powder in the solvent, then extract by ultrasonic means, keep the temperature at 30°C, the extraction time is 30 minutes, the ultrasonic power is maintained at 40khz, to obtain an extract, and evaporate the extract using a rotary evaporator to obtain limonin, the solvent is mixed by ethyl acetate and dichloromethane in a volume ratio of 1:1, the liquid ratio of solvent to powder is 9:1, the unit is g / ml, that is, 9ml of solvent is added to 1g of powder. Embodiment 2 A method for extracting limonin from pomelo pomelo seeds comprises the following steps: Separation and drying: After the pomelo seeds of Shatian pomelo are separated and collected, the fresh weight is weighed according to the preset single weight, and the seeds are dried at a constant temperature of 50℃ for 10 hours. After drying, the dry weight is weighed. For example, if the single weight is set to 50g, the 50g pomelo seeds are weighed again after drying to obtain the weight after removing a certain amount of water. Pressing and crushing: the dried pomelo seeds are pressed to obtain seed oil and seed residue, the seed oil and seed residue are collected separately, and the seed residue is crushed to 35 mesh to obtain pomelo seed powder. Extraction, adding solvent to the powder to dissolve the powder in the solvent, and then extracting by ultrasonic means, keeping the temperature at 40°C, the extraction time for 40 minutes, and the ultrasonic power at 50 khz to obtain an extract, and evaporating the extract using a rotary evaporator to obtain limonin, the solvent is mixed by ethyl acetate and dichloromethane in a volume ratio of 1:3, the liquid ratio of the solvent to the powder is 10:1, the unit is g / ml, that is, 10 ml of solvent is added to 1 g of powder. Embodiment 3 A method for extracting limonin from pomelo pomelo seeds comprises the following steps: Separation and drying: After the pomelo seeds of Shatian pomelo are separated and collected, the fresh weight is weighed according to the preset single weight, and the seeds are dried at a constant temperature of 60℃ for 8 hours. After drying, the dry weight is weighed. For example, if the single weight is set to 50g, the 50g pomelo seeds are weighed again after drying to determine the weight after removing a certain amount of water. Pressing and crushing: the dried pomelo seeds are pressed to obtain seed oil and seed residue, the seed oil and seed residue are collected separately, and the seed residue is crushed into 40 meshes to obtain pomelo seed powder. Extraction, add solvent to the powder to dissolve the powder in the solvent, then extract by ultrasonic means, keep the temperature at 36°C, the extraction time is 41 minutes, the ultrasonic power is maintained at 40khz, to obtain an extract, and evaporate the extract using a rotary evaporator to obtain limonin, the solvent is mixed by ethyl acetate and dichloromethane in a volume ratio of 1:4, the liquid ratio of solvent to powder is 11:1, the unit is g / ml, that is, 11ml of solvent is added to 1g of powder. Embodiment 4 A method for extracting limonin from pomelo pomelo seeds comprises the following steps: Separation and drying: After the pomelo seeds of Shatian pomelo are separated and collected, the fresh weight is weighed according to the preset single weight, and the seeds are dried at a constant temperature of 60℃ for 8 hours. After drying, the dry weight is weighed. For example, if the single weight is set to 50g, the 50g pomelo seeds are weighed again after drying to determine the weight after removing a certain amount of water. Pressing and crushing: the dried pomelo seeds are pressed to obtain seed oil and seed residue, the seed oil and seed residue are collected separately, and the seed residue is crushed into 30 meshes to obtain pomelo seed powder. Extraction, adding solvent to the powder to dissolve the powder in the solvent, and then extracting by ultrasonic means, maintaining the temperature at 40°C, the extraction time for 60 minutes, and the ultrasonic power at 50 khz to obtain an extract, and evaporating the extract using a rotary evaporator to obtain limonin, the solvent is mixed by ethyl acetate and dichloromethane in a volume ratio of 1:5, the liquid ratio of the solvent to the powder is 15:1, the unit is g / ml, that is, 15 ml of solvent is added to 1 g of powder. After testing and analyzing the limonin obtained in Examples 1 to 4, it was found that the main factors affecting the extraction efficiency and stability of limonin from grapefruit seeds are the combination ratio of the two components in the solvent, the liquid ratio of the solvent to the powder obtained after crushing, and the extraction time of the ultrasonic extraction process. These three factors are the key factors affecting the extraction efficiency and stability. When using the response surface methodology, multiple experiments are mainly conducted on three factors: solvent ratio, liquid-to-solid ratio, and extraction time to obtain better results. For the convenience of explanation, the solvent ratio refers to the ratio of ethyl acetate to dichloromethane in the solvent; the liquid-to-solid ratio refers to the ratio of solvent to powder; and the extraction time refers to the duration of ultrasonic extraction. Single-factor multi-level experiments were carried out on the three single factors of solvent ratio, liquid-to-solid ratio and extraction time to further determine the optimal dosage ratio. Each factor was experimented at low, medium and high levels, and a three-factor three-level experimental table was designed with limonin content as the response value. The following is an experimental description of the three factors separately. Effect of solvent ratio on limonin extraction. The dried and pressed nuclear residue was crushed into 40 meshes, and 2g of nuclear residue was weighed and placed in 7 beakers. The solvent composed of ethyl acetate and dichloromethane was used as the extractant at a liquid-to-solid ratio of 10:1. The ratios of ethyl acetate and dichloromethane were 1:0, 5:1, 2:1, 1:1, 1:2, 1:5, and 0:1, respectively. Three parallel experiments were designed for each point. The extraction time was 45min, the extraction temperature was 35℃, and the ultrasonic power was 40khz for ultrasonic extraction. The extract was collected by filtration, dried by rotary evaporator, and dissolved in 5mL of acetonitrile. After dissolution, it was transferred to a centrifuge tube and centrifuged at 4000r / min for 15min. The upper layer of liquid was aspirated and filtered through a 0.22μm filter membrane before injection. The detector wavelength was 210nm. The chromatographic column model was WONDASIL C18-WR (5UM 4.6X150MM), mobile phase: acetonitrile (A)-water (B) (A:B, 45%:55%), column temperature: 35℃, injection volume 10μL, flow rate 1.0min / ml. Use high performance liquid chromatography to determine the peak area of ​​limonin in the sample, calculate the yield of limonin at each point using the standard curve of limonin, draw the curve, and refer to Figure 2 shown. Effect of liquid-to-solid ratio on limonin extraction. The dried and squeezed nuclear residue was crushed into 40 meshes, and 2g of nuclear residue was weighed and placed in 4 beakers respectively. Three parallel experiments were designed for each point according to the liquid-to-solid ratio of 5:1, 10:1, 15:1, and 20:1. A mixed solvent of ethyl acetate and dichloromethane was used as the extractant in a ratio of 1:1. The extraction time was 45min and the extraction temperature was 35℃ for ultrasonic extraction. The extract was collected by filtration, dried by rotary evaporator, and dissolved in acetonitrile. After dissolution, the supernatant was centrifuged and taken as the sample solution. The peak area of ​​limonin in the sample was determined by high performance liquid chromatography, and the yield of limonin at each point was calculated using the standard curve of limonin. The curve was drawn for reference. Figure 3 shown. Effect of extraction time on limonin extraction The dried and squeezed nuclear residue was crushed into 40 mesh, and 2g of nuclear residue was weighed into 5 beakers respectively. The liquid-to-solid ratio was 10:1, and the mixed solvent of ethyl acetate and dichloromethane was 1:1 as the extractant. The extraction time was 30min, 35min, 40min, 45min, and 50min. Three parallel experiments were designed for each point. The extracts were collected by filtration according to different extraction times, and the extracts were dried by rotary evaporator and dissolved in acetonitrile. After dissolution, the supernatant was centrifuged and taken as the sample solution. The peak area of ​​limonin in the sample was determined by high performance liquid chromatography, and the yield of limonin at each point was calculated using the standard curve of limonin. The curve was drawn as follows: Figure 4 shown. Response surface design. Using Design-Expert software and the Box-Behnken experimental design method, the extraction time (min) (A), liquid-to-solid ratio (mL·g-1) (B), and mixed solvent ratio (mL) (C) were used as factors. The optimal experimental areas of each factor determined in the single factor investigation were set, and the limonin content (mg) was used as the response value to design a three-factor three-level experimental table. The factors and levels of the response surface experiment are shown in Table 1. There were a total of 17 test points, including 12 factorial points to investigate the influence of each factor on the response value; and 5 center points to investigate the experimental error. Through the data obtained from the above experiments, multiple linear regression and binomial fitting were performed and the regression equation was established to obtain the response surface diagram with limonin content as the response value. Figure 5-10 As shown in the figure, through surface plot analysis and verification, it was concluded that the optimal extraction process parameters for limonin in Shatian pomelo seeds were liquid-to-solid ratio of 1:11 (g / mL), mixed solvent ratio of ethyl acetate: dichloromethane of 1:4, and extraction time of 41 min. The regression equation is as follows: Y = +1.80025 + 0.027719A-0.007493B + 0.003499C-0.010132AB + 0.031357AC + 0.009298BC-0.050093A2-0.027756B2-0.117008C2. In the formula, Y represents the response value and the measured content of limonin, A represents the ultrasonic extraction time, in min; B represents the addition ratio of solvent to powder, in g / mL; C represents the dosage ratio of ethyl acetate to dichloromethane in the solvent. As shown in Table 2, Table 1 Table 2. Model fitting and analysis of variance The overall regression equation of the analysis model was F = 39.35, P < 0.01, indicating that the model was statistically significant. The lack of fit P was 0.9482 > 0.05, and there was no lack of fit factor, indicating that the established regression equation could fit the true level of limonin in the experimental results; the F value reflects the influence of each factor on the content, and the results show that the influence of each factor is A-extraction time > B-liquid-to-solid ratio > the proportion of mixed solvents; the quadratic terms AC, A2, B2, and C2 have significant effects on the response value (P < 0.05), which are C2 > A2 > AC > B2, indicating that there is a significant interaction between A and C. Verification experiment: The optimal extraction process conditions of limonin predicted by the regression model obtained by response surface analysis software are liquid-to-solid ratio of 11:1, mixed solvent ratio of ethyl acetate: dichloromethane of 1:4, and extraction time of 41min. In order to verify the reliability of the experiment, the above optimal extraction process conditions were adopted to extract limonin. Three portions of nuclear residue were weighed, each 2g, and the verification experiment was carried out according to the predicted optimal process conditions. The average extraction rate of limonin was calculated to be 0.3167%, and the RSD with the predicted value of 0.3175% was 0.08%, which verified that the established regression model was reliable and the optimized method was practical. Repeatability experiment: Take the same batch of samples, dry, crush, and sieve, take 2.00g of each in parallel, weigh accurately, and prepare the test solution according to the test solution preparation method. The RSD value is 0.91%, indicating that the method has good repeatability. Recovery experiment: Take three samples of known content, each weighing 2.00g, and prepare the test solution according to the test solution preparation method. Add 1mL of three reference solutions of low, medium and high concentrations respectively, and measure. The calculated recovery rate is 100.6% and the RSD is 1.8%, indicating that the recovery rate of this method is good.

[0059] 1.3.2.5.3 Stability test: Take the same sample solution and measure it at 0, 2, 4, 6, 12, and 24 hours respectively, record the peak area, and the RSD is 1.3%. The results show that the sample solution is stable for at least 24 hours. The results show that the extraction time, liquid-to-solid ratio and the ratio of mixed solvent have significant effects on the extraction rate of limonin. The variance analysis and verification test of the response equation of the extraction rate of limonin show that this method is reasonable and feasible. Through a systematic method investigation, while meeting the requirements of improving the extraction rate and stability of limonin, the optimal extraction conditions of limonin in grapefruit seeds are obtained: using dried and squeezed kernel residue as raw material, extraction temperature of 36℃, liquid-to-solid ratio of 1:11 (g / ml), extraction time of 41min, and the ratio of mixed solvent ethyl acetate: dichloromethane of 1:4. Under these conditions, the extraction rate of limonin is 0.3167%, which is very close to the predicted value of 0.3175%. It should be noted that the above description is not a limitation of the present invention. Without departing from the creative concept of the present invention, any obvious replacement is within the protection scope of the present invention.

Claims

1. A method for extracting limonin from pomelo seed using response surface methodology, characterized in that: The following steps are involved: Separation and drying: After the grapefruit seeds are separated and collected, the fresh weight of the grapefruit seeds is weighed, and the seeds are dried at a constant temperature of 50-60° C. for 8-10 hours, and the dry weight is weighed after drying; Pressing and crushing: Pressing the dried pomelo seeds to obtain seed oil and seed residue, collecting the seed oil and the seed residue respectively, crushing the seed residue to less than 40 meshes to obtain pomelo seed powder, and weighing the pomelo seeds; Extraction, adding a solvent to the powder to dissolve the powder in the solvent, then extracting by ultrasonication for a set extraction time to obtain an extract, and evaporating the extract to dryness by a rotary evaporator to obtain a limonin extract; Detection: The limonin extract is dissolved in acetonitrile, filtered, and centrifuged, and the supernatant is taken as a sample solution for standby use. The sample solution is tested to obtain the extraction rate of limonin.

2. The method for extracting limonin from pomelo seed according to claim 1, characterized in that: The response surface experimental method was used to analyze different usage ratios of ethyl acetate and dichloromethane. Three different usage ratios were put into the test, namely, the usage ratio of ethyl acetate was greater than the usage ratio of dichloromethane, the usage ratio of ethyl acetate was equal to the usage ratio of dichloromethane, and the usage ratio of ethyl acetate was less than the usage ratio of dichloromethane. The result showed that the usage ratio of ethyl acetate was greater than the usage ratio of dichloromethane was the preferred solution.

3. The method for extracting limonin from pomelo seed according to claim 2, characterized in that: The solvent is a mixture of ethyl acetate and dichloromethane, wherein the volume ratio of ethyl acetate to dichloromethane is 1:1-1:10, and the preferred ratio of ethyl acetate to dichloromethane is 1:

4.

4. The method for extracting limonin from pomelo seed according to claim 3, characterized in that: When ultrasonic extraction is performed, the temperature is maintained at 30-40°C, the extraction time is 30-60 minutes, and the ultrasonic power is 40-60 khz.

5. The method for extracting limonin from pomelo seed according to claim 4, characterized in that: When the dosage ratio of ethyl acetate and dichloromethane in the solvent is determined, several groups of solvents and powders with different dosage ratios are tested, and the amount of solvent is greater than that of powder, and the dosage ratio of powder to solvent is obtained as 1:8-1:20, and the unit is g / ml.

6. The method for extracting limonin from pomelo seed according to claim 5, characterized in that: The addition ratio of the powder to the solvent is 1:11, and the unit is g / ml.

7. The method for extracting limonin from pomelo seed according to claim 6, characterized in that: The ratio of ethyl acetate to dichloromethane in the solvent and the ratio of solvent to powder are determined to be 30-60 minutes, the temperature is maintained at 30-40° C., and the ultrasonic power is 40-60 khz.

8. The method for extracting limonin from pomelo seed according to claim 7, characterized in that: According to the obtained usage ratio of ethyl acetate and dichloromethane in the solvent, the addition ratio of solvent and powder, and the extraction time, multiple linear regression and binomial fitting were performed and the regression equation was established: Y=+1.80025+0.027719A-0.007493B+0.003499C-0.010132AB+0.031357AC+0.009298BC-0.050093A2-0.027756B2-0.117008C2, Wherein, Y represents the response value and the measured limonin content, A represents the ultrasonic extraction time, in min; B represents the addition ratio of solvent to powder, in g / mL; C represents the usage ratio of ethyl acetate to dichloromethane in the solvent.

9. The method for extracting limonin from pomelo seed according to claim 8, characterized in that: In the extraction of limonin, the extraction time is greater than the added amount ratio of the solvent to the powder, and the added amount ratio of the solvent to the powder is greater than the amount ratio of ethyl acetate to dichloromethane in the solvent.

10. The method for extracting limonin from pomelo seed according to claim 1, characterized in that: The pomelo includes Shatian pomelo, Wendan pomelo, and honey pomelo, and Shatian pomelo is preferred.

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