A method for extracting limonene

Abstract

This invention discloses a method for extracting limonin, comprising the following steps: S1, preparing an extract containing limonin; S2, adding macroporous resin to the extract and stirring, the macroporous resin being used to adsorb limonin in the extract; S3, removing the macroporous resin from the extract and placing it in a chromatography column to collect the eluent; S4, evaporating and concentrating the eluent to obtain a limonin extract, using ethyl acetate as a washing solvent, ultrasonically dissolving the limonin extract, then adding pure water for washing, concentrating and evaporating the washing solution to obtain a limonin sample; S5, performing TLC directional analysis on the limonin sample to check whether the limonin sample is qualified. This invention improves the extraction rate of limonin and yields a high extraction content.

Description

Technical Field

[0001] This invention relates to the field of limonene treatment technology, specifically a method for extracting limonene. Background Technology

[0002] Limonene, also known as berberine lactone or evodiamine lactone, has a variety of biological activities such as anti-tumor, insect repellent, antiviral, analgesic, anti-inflammatory, and hypnotic effects. It is an important natural pharmaceutical raw material and can be used to treat pneumonia, as a functional food additive, anti-cancer food, insecticide, and feed additive.

[0003] Limonenes refer to mixtures composed of limonin and its analogues. These mixtures primarily consist of limonin and nomiline, both triterpenoids and products of plant secondary metabolism. They are mainly found in various Rutaceae and Meliaceae plants and are the main substances causing the bitterness of citrus juices. They possess a wide range of biological activities, such as insecticidal, antiviral, antitumor, and antibacterial effects. The effects of limonenes in anticancer, analgesic, cholesterol-regulating, atherosclerosis-preventing, and insecticidal applications are receiving increasing attention, but research in this area is still in its early stages in China. Because limonin and nomiline have similar structures and pharmacological effects, they are collectively referred to as limonenes and are usually used together without separation. Currently, limonenes on the market are mainly extracted from sweet orange or grapefruit seeds.

[0004] Extraction of limonene compounds typically involves using organic solvents, such as petroleum ether, from sweet orange or grapefruit seeds. This method is costly, hazardous, and causes significant environmental pollution. Furthermore, it yields low levels of limonene compounds and generally suffers from low extraction rates and time-consuming processes. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for extracting limonene.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: A method for extracting limonene, comprising the following steps: S1, Prepare an extract containing limonene; S2, Add macroporous resin to the extract and stir. The macroporous resin is used to adsorb limonene in the extract. The macroporous resin and extract are added in a ratio of 1:3 to 1:5. The macroporous resin is expressed in grams by mass and the extract is expressed in milliliters by volume. S3, Take out the macroporous resin from the extract and put it into the chromatography column. Select ethanol solution as the eluent to elute the macroporous resin and collect the eluent. S4. The eluent was concentrated by rotary evaporation to obtain limonin extract. Ethyl acetate was used as the washing solvent to dissolve the limonin extract by ultrasonication. Then, pure water was added for washing. The washing solution was concentrated and evaporated to obtain limonin sample. S5. Perform TLC directional analysis on the limonene sample to check whether the limonene sample is qualified.

[0007] As a further improvement, the macroporous resin is selected as AB-8 resin.

[0008] As a further improvement, in step S2, the stirring speed is set to 110 rad / min to 130 rad / min, and the duration is 14-16 minutes.

[0009] As a further improvement, in step S3, the concentration of the ethanol solution is 60% to 80%.

[0010] As a further improvement, during the elution process in step S3, the flow rate of the ethanol solution is controlled at 0.4 mL / min to 0.6 mL / min.

[0011] As a further improvement, in step S5, ethyl acetate and petroleum ether are selected as the developing agents for the limonene sample, and acetonitrile is selected as the spotting solvent. The limonene sample is placed on a silica gel plate and processed by the developing agent and the spotting solvent. Ehrlich's reagent was chosen as the colorimetric agent for the developed limonene on the silica gel plate.

[0012] As a further improvement, the ethyl acetate and petroleum ether are mixed in a volume ratio of 4-7:3-6.

[0013] As a further improvement, the Ehrlich reagent is composed of p-dimethylaminobenzaldehyde dissolved in a sulfuric acid-ethanol mixture, wherein p-dimethylaminobenzaldehyde and the sulfuric acid-ethanol mixture are added at a mass-volume ratio of 1-2:1, and the sulfuric acid-ethanol mixture is mixed in a volume ratio of 6-7:3-4.

[0014] As a further improvement, in step S5, after performing TLC directional analysis on limonene, the silica gel powder that has not been impregnated with the color developer on the silica gel plate is cut off, and the silica gel powder is stirred and extracted to recover the limonene from the silica gel powder.

[0015] As a further improvement, in the stirring extraction, acetonitrile is used as the desorption solvent. After adding acetonitrile to silica gel powder, stirring is carried out for 9-11 minutes at a stirring speed of 300-350 rad / min.

[0016] The present invention has the following beneficial technical effects: By introducing steps such as macroporous resin adsorption, ethanol solution elution, and ethyl acetate washing, the high cost, hazards, and environmental pollution problems caused by the large-scale use of petroleum ether in traditional methods are effectively avoided. This method aims to provide a simple, efficient, and relatively pure route for the preparation of limonene, thereby overcoming the shortcomings of existing technologies such as low extraction rate and long processing time. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the effect of stirring speed on the adsorption of macroporous resin in this invention; Figure 2 This is a schematic diagram illustrating the effect of stirring time on macroporous resin adsorption in this invention; Figure 3 This is a schematic diagram showing the ratio of macroporous resin to limonene in this invention. Figure 4 This is a schematic diagram illustrating the effect of different flow rates of the eluent on macroporous resin in this invention. Figure 5 This is a schematic diagram showing the effect of different flow rates on the elution rate of macroporous resin in this invention. Figure 6 This is a schematic diagram illustrating the effect of different ethanol concentrations on the elution of limonene in this invention; Figure 7 This is a schematic diagram illustrating the effect of different ethanol concentrations on the elution rate of macroporous resin in this invention. Figure 8 This is a schematic diagram illustrating the effect of different washing solvents on limonene in this invention; Figure 9 This invention illustrates the effect of different proportions of developing solvent on the Rf value of spots. Figure 10 This is a schematic diagram illustrating the effect of using acetonitrile as the extraction solvent in this invention on limonene; Figure 11 This is a schematic diagram illustrating the effect of ethyl acetate as the stirring extraction solvent on limonene in this invention; Figure 12 This is a schematic diagram illustrating the effect of dichloromethane as the stirring extraction solvent on limonene in this invention; Figure 13 This is a schematic diagram illustrating the effect of stirring time during stirring extraction in this invention; Figure 14 This is a schematic diagram illustrating the effect of stirring speed during stirring extraction in this invention; Figure 15 This is a schematic diagram of the standard curve of limonene in this invention. Detailed Implementation

[0018] The embodiments of the present invention are described in detail below.

[0019] Example 1 A method for extracting limonene, comprising the following steps: S1. Prepare an extract containing limonin. The extract refers to the initial solution containing the target compound limonin. This solution is usually obtained after preliminary treatment of the plant material and serves as the starting material for subsequent separation and purification steps. For example, the plant material containing limonin can be pulverized and then extracted using conventional methods such as water extraction, alcohol extraction, or a combination of both. Alternatively, it can be obtained through enzymatic hydrolysis followed by filtration.

[0020] S2, add AB-8 macroporous resin to the extract and stir at 120 rad / min for 15 minutes. During stirring, the macroporous resin adsorbs limonene in the extract. The macroporous resin and extract are added in a 1:3 ratio, where the macroporous resin is expressed in grams by mass and the extract in milliliters by volume.

[0021] S3. Take out the macroporous resin from the extract and put it into the chromatography column. Select ethanol solution as the eluent to elute the macroporous resin. Collect the eluent, in which the ethanol concentration is 60%. Use a peristaltic pump to control the flow rate of the ethanol solution at 0.5 mL / min to elute the macroporous resin.

[0022] S4. The eluent is concentrated by rotary evaporation to obtain limonin extract. Ethyl acetate is used as the washing solvent to dissolve the limonin extract by ultrasonication, followed by washing with pure water. The washing solution is then concentrated and evaporated to dryness to obtain the limonin sample. The ratio of ethyl acetate to limonin eluent can be 1:5.

[0023] S5. Perform TLC directional analysis on the limonene sample to check if the limonene sample is up to standard. Specifically: Ethyl acetate and petroleum ether were mixed in a ratio of 4.5:5.5 as the developing solvent for limonene samples, and acetonitrile was selected as the spotting solvent. The limonene samples were placed on a silica gel plate and processed by the developing solvent and the spotting solvent. Ehrlich's reagent was selected as the colorimetric agent for limonene developed on silica gel plates. Ehrlich's reagent is composed of p-dimethylaminobenzaldehyde dissolved in a sulfuric acid-ethanol mixture, wherein p-dimethylaminobenzaldehyde and the sulfuric acid-ethanol mixture are added at a mass-volume ratio of 1.25:1, and the sulfuric acid-ethanol mixture is mixed at a volume ratio of 6.5:3.5. After TLC directional analysis of limonene, the silica gel powder that was not impregnated with the colorimetric reagent was cut off from the silica gel plate and stirred for extraction. Acetonitrile was used as the eluent. Acetonitrile was added to the silica gel powder and stirred for 10 minutes at a stirring speed of 300 rad / min to recover limonene from the silica gel powder.

[0024] TLC-directed analysis is used to preliminarily determine the presence and purity of limonene in the sample. Specifically, the procedure involves spotting the sample onto a silica gel plate and then developing it using a developing solvent. After development, a colorimetric reagent can be sprayed to develop the color, and the Rf value and color of the spots are observed to determine whether the limonene sample meets the requirements.

[0025] Example 2 A method for extracting limonene, comprising the following steps: S1. Prepare an extract containing limonin. The extract refers to the initial solution containing the target compound limonin. This solution is usually obtained after preliminary treatment of the plant material and serves as the starting material for subsequent separation and purification steps. For example, the plant material containing limonin can be pulverized and then extracted using conventional methods such as water extraction, alcohol extraction, or a combination of both. Alternatively, it can be obtained through enzymatic hydrolysis followed by filtration.

[0026] S2, add AB-8 macroporous resin to the extract and stir at 110 rad / min for 14 minutes. During stirring, the macroporous resin adsorbs limonene in the extract. The macroporous resin and extract are added in a ratio of 1:4, where the macroporous resin is expressed in grams by mass and the extract is expressed in milliliters by volume.

[0027] S3. Take out the macroporous resin from the extract and put it into the chromatography column. Select ethanol solution as the eluent to elute the macroporous resin. Collect the eluent, in which the ethanol concentration is 70%. Use a peristaltic pump to control the flow rate of the ethanol solution at 0.4 mL / min to elute the macroporous resin.

[0028] S4. The eluent is concentrated by rotary evaporation to obtain limonin extract. Ethyl acetate is used as the washing solvent to dissolve the limonin extract by ultrasonication, followed by washing with pure water. The washing solution is then concentrated and evaporated to dryness to obtain the limonin sample. The ratio of ethyl acetate to limonin eluent can be 1:5.

[0029] S5. Perform TLC directional analysis on the limonene sample to check if the limonene sample is up to standard. Specifically: Ethyl acetate and petroleum ether were mixed in a volume ratio of 5.5:4.5 as the developing solvent for limonene samples, and acetonitrile was selected as the spotting solvent. The limonene samples were placed on a silica gel plate and processed by the developing solvent and the spotting solvent. Ehrlich's reagent was selected as the colorimetric agent for limonene developed on silica gel plates. Ehrlich's reagent is composed of p-dimethylaminobenzaldehyde dissolved in a sulfuric acid-ethanol mixture, wherein p-dimethylaminobenzaldehyde and the sulfuric acid-ethanol mixture are added at a mass-volume ratio of 1:1, and the sulfuric acid and ethanol in the sulfuric acid-ethanol mixture are mixed at a volume ratio of 6:3. After TLC directional analysis of limonene, the silica gel powder that was not impregnated with the colorimetric reagent was cut off from the silica gel plate and stirred for extraction. Acetonitrile was used as the eluent. Acetonitrile was added to the silica gel powder and stirred for 9 minutes at a stirring speed of 330 rad / min to recover limonene from the silica gel powder.

[0030] TLC-directed analysis is used to preliminarily determine the presence and purity of limonene in the sample. Specifically, the procedure involves spotting the sample onto a silica gel plate and then developing it using a developing solvent. After development, a colorimetric reagent can be sprayed to develop the color, and the Rf value and color of the spots are observed to determine whether the limonene sample meets the requirements.

[0031] Example 3 A method for extracting limonene, comprising the following steps: S1. Prepare an extract containing limonin. The extract refers to the initial solution containing the target compound limonin. This solution is usually obtained after preliminary treatment of the plant material and serves as the starting material for subsequent separation and purification steps. For example, the plant material containing limonin can be pulverized and then extracted using conventional methods such as water extraction, alcohol extraction, or a combination of both. Alternatively, it can be obtained through enzymatic hydrolysis followed by filtration.

[0032] S2, add AB-8 macroporous resin to the extract and stir at 130 rad / min for 16 minutes. During stirring, the macroporous resin adsorbs limonene in the extract. The macroporous resin and extract are added in a ratio of 1:5, where the macroporous resin is expressed in grams by mass and the extract in milliliters by volume.

[0033] S3. Take the macroporous resin out of the extract and put it into the chromatography column. Select ethanol solution as the eluent to elute the macroporous resin. Collect the eluent, in which the ethanol concentration is 80%. Use a peristaltic pump to control the flow rate of the ethanol solution at 0.6 mL / min to elute the macroporous resin.

[0034] S4. The eluent is concentrated by rotary evaporation to obtain limonin extract. Ethyl acetate is used as the washing solvent to dissolve the limonin extract by ultrasonication, followed by washing with pure water. The washing solution is then concentrated and evaporated to dryness to obtain the limonin sample. The ratio of ethyl acetate to limonin eluent can be 1:5.

[0035] S5. Perform TLC directional analysis on the limonene sample to check if the limonene sample is up to standard. Specifically: Ethyl acetate and petroleum ether were mixed in a volume ratio of 6.5:3.5 as the developing solvent for limonene samples, and acetonitrile was selected as the spotting solvent. The limonene samples were placed on a silica gel plate and processed by the developing solvent and the spotting solvent. Ehrlich's reagent was selected as the colorimetric agent for limonene developed on silica gel plates. Ehrlich's reagent is composed of p-dimethylaminobenzaldehyde dissolved in a sulfuric acid-ethanol mixture, wherein p-dimethylaminobenzaldehyde and the sulfuric acid-ethanol mixture are added at a mass-volume ratio of 1:1, and the sulfuric acid and ethanol in the sulfuric acid-ethanol mixture are mixed at a volume ratio of 7:4. After TLC directional analysis of limonene, silica gel powder that had not been impregnated with the colorimetric reagent was cut off from the silica gel plate and stirred for extraction. Acetonitrile was used as the eluent, and acetonitrile was added to the silica gel powder and stirred for 11 minutes at a stirring speed of 350 rad / min to recover limonene from the silica gel powder.

[0036] TLC-directed analysis is used to preliminarily determine the presence and purity of limonene in the sample. Specifically, the procedure involves spotting the sample onto a silica gel plate and then developing it using a developing solvent. After development, a colorimetric reagent can be sprayed to develop the color, and the Rf value and color of the spots are observed to determine whether the limonene sample meets the requirements.

[0037] The following comparative examples illustrate the necessity and advantages of selecting specific components and parameters in this application.

[0038] The following comparisons are made using two macroporous resins: AB-8 macroporous resin and D101 macroporous resin.

[0039] I. Selection of Macroporous Resins 1. Comparison of AB-8 macroporous resin and D101 macroporous resin Weigh appropriate amounts of each of the two macroporous resins and pour them into 250 mL beakers. Add 95% ethanol until it reaches about 1 cm above the resin. Soak for 24 h until fully swollen. Skim off any floating macroporous resin. After filtration, pour the macroporous resin into a chromatography column. Use the wet column loading method and pure water to elute impurities from the macroporous resin loaded onto the column. Use a peristaltic pump to control the flow rate at 0.2 mL / min until the eluent is colorless, clear, and free of alcohol odor.

[0040] 2. Screening and treatment of macroporous resins Take 5 g each of the two treated macroporous resins AB-8 and D101, stir for 15 min, stir at a speed of 100 Rad / min, and screen the macroporous resins under the condition that the liquid-to-solid ratio of macroporous resin to limonene extract is 1:4. After the experiment is completed, the supernatant is collected, and the content of residual limonene in the supernatant is detected by HPLC. The adsorption rate is calculated, and the resin with the best adsorption rate is selected.

[0041] As shown in Table 1

[0042] AB-8 macroporous resin showed the best adsorption effect on limonoids in the sample extract, with an adsorption rate of 80.49%. Therefore, AB-8 has a high adsorption rate and adsorption capacity for limonoids in the sample solution, and the adsorption rate of 80.49% for limonoids by AB-8 macroporous resin is significantly better than that of D101 resin (75.54%).

[0043] In addition, AB-8 resin is a commonly used nonpolar macroporous adsorption resin. Its framework structure is typically a styrene-divinylbenzene copolymer, possessing a large specific surface area and pore size distribution, providing abundant adsorption sites. The nonpolar surface characteristics of this resin enable it to exhibit good affinity for organic compounds with a certain degree of hydrophobicity (such as limonene). During adsorption, limonene molecules can bind to the surface of AB-8 resin through physical adsorption mechanisms such as hydrophobic interactions and van der Waals forces, thereby achieving separation from the extract.

[0044] 3. Determine the adsorption conditions of macroporous resins. (1) Determine the stirring speed for macroporous resin adsorption. Three 5 g portions of AB-8 and D101 macroporous resins were weighed and placed in 50 mL beakers. 20 mL of limonene extract (approximately 0.6 mg / mL) was added to each resin. The mixtures were stirred at speeds of 80 rad / min, 100 rad / min, and 120 rad / min for 15 min. After the experiment, the supernatant was collected, and the remaining limonene content was determined by HPLC. The adsorption rate of the macroporous resins was calculated, and the adsorption rates of the two resins were compared to select the resin with the higher adsorption rate.

[0045] like Figure 1As shown, at stirring speeds of 80 rad / min, 100 rad / min, and 120 rad / min, the adsorption of limonene by macroporous resin AB-8 initially increases and then decreases with increasing stirrer speed, reaching a maximum of 80% at 120 rad / min. In contrast, the adsorption of limonene by macroporous resin D101 initially decreases and then increases with increasing stirrer speed, but overall, its adsorption performance is not as good as that of macroporous resin AB-8. Therefore, a stirring speed of 120 rad / min is selected as the optimal stirring speed.

[0046] (2) Determine the stirring time for macroporous resin adsorption. Two different macroporous resins were selected after treatment. Three 5 g portions of each resin were weighed and placed in 50 mL beakers. 20 mL of limonene extract at a concentration of approximately 0.6 mg / mL was added to each beaker. The beakers were stirred for different times at a speed of 100 rad / min for 5 min, 10 min, 15 min, 20 min, and 25 min. After the experiment, the supernatant was collected and the remaining limonene content was detected by HPLC. The adsorption rate of the macroporous resins was calculated, and the adsorption rates of the two resins were compared to select the resin with the higher adsorption rate.

[0047] Ensure thorough mixing of the macroporous resin and limonene in the extract to promote effective contact between them. The speed range is set to balance mixing efficiency with avoiding physical damage to the macroporous resin or generating adverse shear forces. The stirring duration refers to the total time the stirring process is maintained; its purpose is to provide sufficient time for limonene molecules to diffuse fully and be adsorbed by the macroporous resin until the adsorption process reaches or approaches equilibrium.

[0048] like Figure 2 As shown, under different stirring times of 5 min, 10 min, 15 min, 20 min, and 25 min, the adsorption rate of limonene by both macroporous resins increased with increasing stirring time. However, the adsorption rate of limonene by D101 resin was not as good as that by AB-8 resin. Therefore, macroporous resin AB-8 was selected for further investigation. For macroporous resin AB-8, the adsorption rate did not increase significantly after stirring for 15 min. Therefore, considering the time cost, 15 min was selected as the optimal stirring time.

[0049] (3) Determine the liquid-to-solid ratio for macroporous resin adsorption. Two different macroporous resins were selected after treatment. Three 5 g portions of each resin were weighed and placed in 50 mL beakers. 20 mL of limonene extract at a concentration of approximately 0.6 mg / mL was added to each beaker. The beakers were stirred at 100 rad / min for 15 min. Different liquid-to-solid ratios of 1:3, 1:4, and 1:5 were investigated. After the experiment, the supernatant was collected and the remaining limonene content was detected by HPLC. The adsorption rate of the macroporous resins was calculated, and the adsorption rates of the two resins were compared to select the resin with the higher adsorption rate.

[0050] like Figure 3 As shown, with the increase of the liquid-to-solid ratio (mass of macroporous resin: volume of limonene extract), the adsorption rate of macroporous resin AB-8 for limonene gradually decreases. Therefore, it can be seen that the increase of the liquid-to-solid ratio will lead to the decrease of the adsorption rate. Thus, a liquid-to-solid ratio of 1:3 is the optimal liquid-to-solid ratio.

[0051] 4. Determine the conditions for dynamic desorption of macroporous resins (1) Determine the elution flow rate for dynamically eluting limonene from macroporous resin. Three macroporous resins, each 5 g saturated with adsorption, were packed into three chromatography columns of the same size. A 60% ethanol-water solution was used as the eluent. Elution was performed using a peristaltic pump at flow rates of 0.5 mL / min, 1.0 mL / min, and 1.5 mL / min, respectively. Ten tubes of eluting were collected, each containing 15 mL of eluting solution, and numbered sequentially. The limonene content in all eluting solutions was determined using HPLC. The total resolution and the amount of eluted limonene were calculated. Trend lines for the elution rate and for the limonene content at different flow rates were plotted. Finally, the optimal flow rate was determined through comparison.

[0052] like Figure 4 As shown, the limonene content eluted by macroporous resin AB-8 decreased with increasing eluent flow rate, indicating that increasing flow rate reduces elution efficiency. Figure 5 It can be concluded that the elution rate of limonene by macroporous resin AB-8 is gradually decreasing. Therefore, it can be concluded that increasing the flow rate will lead to a decrease in the elution rate. Thus, a flow rate of 0.5 mL / min is the optimal elution rate.

[0053] (2) Determine the eluent concentration for dynamically eluting limonene from macroporous resin. Five g of each of four macroporous resins that had been saturated with adsorption were selected and packed into four chromatography columns of the same size. Eluents of 20%, 40%, 60%, and 80% ethanol were used, respectively. Elution was performed using a peristaltic pump at a flow rate of 1.0 mL / min. 15 mL of eluent was collected per tube. The limonene content in all eluents was determined by HPLC. The total resolution and the amount of eluted limonene were calculated. Trend lines for the elution rate and for the limonene content at different flow rates were plotted. Finally, the optimal eluent concentration was determined by comparison.

[0054] Based on the experimental data results, such as Figure 6 and Figure 7 As shown.

[0055] according to Figure 6 The experimental results showed that the elution rate of limonene increased significantly with the increase of ethanol eluent concentration, reaching a peak at 80% ethanol concentration, indicating that increasing ethanol concentration increases the elution efficiency of limonene in macroporous resin AB-8; according to Figure 7 The experimental results showed that the elution rate of limonene by macroporous resin AB-8 was increasing, but the elution rate was relatively flat after reaching 60%-80% ethanol. Therefore, 60% ethanol was selected as the optimal loading concentration.

[0056] 5. Determine the optimal solvent for washing the limonene eluent.

[0057] (1) Ethyl acetate and dichloromethane were selected as solvents for washing the limonene eluent. The 75 ml crude product solution, eluted with 60% ethanol, was completely evaporated to dryness. It was then dissolved in 15 ml of ethyl acetate and 15 ml of dichloromethane, respectively, and sonicated for 15 min. Afterward, it was washed with 5 times its volume of water. The aqueous phase was washed three times with the organic phase, and the organic phase retained from the first three washes was washed once with saturated sodium chloride brine and dehydrated with anhydrous magnesium sulfate. After the organic phase washing solution was completely evaporated to dryness, 2 ml of the evaporated product was dissolved in acetonitrile and added to a 2 ml sample for HPLC analysis.

[0058] Comparing the peak area ratios of ethyl acetate and dichloromethane at the elution time of limonene, it was found that ethyl acetate had a higher peak area ratio at the elution time of limonene.

[0059] according to Figure 8 The experimental results express the desorption rate of limonene in the eluent under two different washing solvents, ethyl acetate and dichloromethane, based on... Figure 8 When the washing solvent is ethyl acetate, the amount of limonene washed in the eluent is greater than that in dichloromethane. Therefore, ethyl acetate is chosen as the optimal washing solvent.

[0060] 6. Determine the optimal conditions for adsorbing limonene using TLC directional analysis, i.e., thin-layer chromatography.

[0061] (1) Ethyl acetate and petroleum ether were selected as the developing solvents for thin-layer chromatography and the ratio of developing solvents was ethyl acetate: petroleum ether = 4.5: 5.5, 5.5: 4.5, 6.5: 3.5. The developing solvents and ratios with appropriate resolution were adopted.

[0062] Figure 9 In this study, as the petroleum ether content in the developing solvent increases, the distance between the two spots on the silica gel plate becomes farther and farther, and the Rf value continuously increases. This indicates that an increase in petroleum ether content leads to an increase in Rf. Considering that in most TLC qualitative analyses, an Rf value between 0.2 and 0.8 is considered the optimal range, the volume ratio of ethyl acetate to petroleum ether is 6.5:3.5, which is the optimal developing solvent ratio.

[0063] (2) Since dichloromethane and ethyl acetate are volatile, acetonitrile was chosen as the solvent for TLC.

[0064] From the perspective of solvent volatility, dichloromethane and ethyl acetate have boiling points of 39.6℃ and 77.1℃, respectively, both belonging to low-boiling-point organic solvents (boiling point <100℃). They have relatively high saturated vapor pressures at room temperature (46.5 kPa for dichloromethane and 10.3 kPa for ethyl acetate at 20℃), making them prone to rapid volatilization due to ambient temperature fluctuations or open conditions during operation. When used as sample dissolution solvents, rapid solvent evaporation during spotting can lead to premature precipitation of the sample on the silica gel plate, forming locally concentrated spots, or even tailing or spot splitting, interfering with the qualitative identification of the target compound.

[0065] In comparison, acetonitrile has a boiling point of 81.6℃ and a saturated vapor pressure of 11.5 kPa at 20℃. Its volatility is significantly lower than that of dichloromethane (approximately one-quarter of dichloromethane) and slightly higher than that of ethyl acetate, but the difference is small. When used as a sample dissolution solvent, it ensures slow evaporation during the spotting process, allowing the sample to be evenly distributed on the silica gel plate, forming round, clear spots, and avoiding abnormal chromatographic behavior caused by excessively high local concentrations.

[0066] Furthermore, considering the relationship between solvent polarity and target compound solubility, acetonitrile's polarity parameter (P'=5.8) falls between that of dichloromethane (P'=3.1) and ethyl acetate (P'=4.4), providing moderate solubility for limonin (a weakly polar triterpenoid compound) and nomiline. Acetonitrile's moderate volatility, good solubility, and reliable experimental repeatability make it a preferred alternative to dichloromethane and ethyl acetate for TLC, providing a stable and reliable experimental basis for the qualitative analysis of limonin and nomiline.

[0067] (3) Ehrlich's reagent was selected as the colorimetric reagent for developing limonene on silica gel plates. Dissolve 125 mg of p-dimethylaminobenzaldehyde in 100 ml of a sulfuric acid-ethanol mixture, where the sulfuric acid to ethanol ratio is 6.5:3.5.

[0068] 7. Single-factor screening for stirred extraction (1) Determine the optimal solvent for the extraction of limonene by stirring silica gel powder.

[0069] The silica powder that was not impregnated with the colorimetric reagent at the control point of the silica gel plate was collected and extracted with 15 ml of ethyl acetate, dichloromethane, and acetonitrile respectively. The mixture was then stirred at 200 rad / min for 30 min. The solution and silica powder were filtered, and the solution was completely evaporated to dryness. Then, 2 ml of acetonitrile was added to dissolve the solution, and the solution was placed in a sample vial for HPLC detection.

[0070] Experimental results are as follows Figure 10 , 11 Figure 12 shows the desorption rates of limonene in silica gel powder under three different solvents: ethyl acetate, dichloromethane, and acetonitrile. The desorption rate peaks in acetonitrile, indicating that acetonitrile increases the desorption efficiency of limonene in silica gel powder. Ethyl acetate and dichloromethane show much lower desorption rates for limonene in silica gel powder than acetonitrile. Therefore, acetonitrile is the preferred solvent. Figure 10 Acetonitrile was selected as the optimal solvent for stirring and extraction.

[0071] (2) Determine the optimal stirring speed for the extraction of limonene by stirring silica powder.

[0072] Cut off the silica gel powder that was not impregnated with the colorimetric reagent corresponding to the control point on the silica gel plate, add 15 ml of acetonitrile, and stir at speeds of 100 rad / min, 200 rad / min, 300 rad / min, and 400 rad / min for 30 min respectively. Filter the solution and silica gel powder, evaporate the solution completely to dryness, add 2 ml of acetonitrile to dissolve it, put it into a sample vial, and perform HPLC detection.

[0073] Comparing the peak area ratio of limonene elution time at four stirring speeds of 100 rad / min, 200 rad / min, 300 rad / min, and 400 rad / min, the results are as follows: Figure 14 As shown, with the increase of the stirring speed of the silica gel powder, the desorption of limonene in the silica gel powder by the solvent first increases and then decreases, and reaches a maximum value at 300 rad / min, with a maximum adsorption rate. Therefore, a stirring speed of 300 rad / min is selected as the optimal stirring speed.

[0074] (3) Determine the optimal stirring time for the extraction of limonene by stirring silica gel powder.

[0075] Cut off the silica gel powder that was not impregnated with the colorimetric reagent corresponding to the control point on the silica gel plate, add 15 ml of acetonitrile, and stir at 200 rad / min for 10 min, 20 min, and 30 min. Filter the solution and silica gel powder, take the solution and evaporate it completely to dryness, add 2 ml of acetonitrile to dissolve it, put it into a sample vial, and perform HPLC detection.

[0076] Comparing the peak area percentages of limonene elution at stirring times of 10 min, 20 min, and 30 min, the results are as follows: Figure 13 As shown, with the increase of stirring time, the desorption rate of limonene in silica gel powder by the solvent shows a decreasing trend, and reaches a maximum value in 10 min, with a maximum adsorption rate of 10 min. Therefore, a stirring time of 10 min is selected as the optimal stirring time.

[0077] refer to Figure 15 As shown in the test results, the peak area related parameter for the elution time of limonene is y = 5E+06x - 35369 (y = 5,000,000x). 35369) R 2 = 0.9995.

[0078] In addition, for the TLC directional analysis procedure, the limonene sample is first dissolved in a spotting solvent (acetonitrile) and then precisely coated onto the starting line of a silica gel plate. The spotted silica gel plate is then placed vertically in a developing tank containing a developing solvent (a mixture of ethyl acetate and petroleum ether). The developing solvent rises along the silica gel plate through capillary action. During this process, the developing solvent acts as the mobile phase, while the silica gel acts as the stationary phase, resulting in different adsorption and desorption reactions for the various components in the sample. Because limonene and its impurities have different affinities for the stationary and mobile phases, their migration rates on the silica gel plate also differ, thus achieving the separation of the components. This process ensures the effective separation of limonene from other components in the sample.

[0079] For limonene, Ehrlich's reagent reacts with specific groups in its structure, forming clearly visible spots of characteristic color on a silica gel plate. This specific color development avoids background interference or the development of non-target compounds that may occur when using general colorimetric reagents, greatly improving the accuracy and reliability of limonene identification. By observing the color, shape, and Rf value of the colored spots, the presence and purity of limonene can be directly determined.

[0080] Specifically, p-Dimethylaminobenzaldehyde, the core active ingredient of Ehrlich's reagent, functions by reacting with the specific structure of limonene to produce a colorimetric reaction, thereby forming visible spots of the target compound on a silica gel plate. A sulfuric acid-ethanol mixture serves as both a solvent and a reaction medium. Sulfuric acid provides the necessary acidic environment to promote the colorimetric reaction and may also participate in the formation of the colorimetric product. Ethanol acts as an organic solvent, dissolving p-dimethylaminobenzaldehyde and mixing with sulfuric acid to form a homogeneous solvent system. Simultaneously, it adjusts the polarity of the mixture to ensure the stability of the colorimetric reagent and the uniformity of spraying.

[0081] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. However, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for extracting limonene, characterized in that, Includes the following steps: S1, Prepare an extract containing limonene; S2, Add macroporous resin to the extract and stir. The macroporous resin is used to adsorb limonene in the extract. The macroporous resin and extract are added in a ratio of 1:3 to 1:

5. The macroporous resin is expressed in grams by mass and the extract is expressed in milliliters by volume. S3, Take out the macroporous resin from the extract and put it into the chromatography column. Select ethanol solution as the eluent to elute the macroporous resin and collect the eluent. S4. The eluent was concentrated by rotary evaporation to obtain limonin extract. Ethyl acetate was used as the extraction solvent to dissolve the limonin extract by ultrasonication. Then, pure water was added for extraction. The extract was concentrated and evaporated to obtain limonin sample. S5. Perform TLC directional analysis on the limonene sample to check whether the limonene sample is qualified.

2. The method for extracting limonin according to claim 1, characterized in that, The macroporous resin selected is AB-8 resin.

3. The method for extracting limonin according to claim 1, characterized in that, In step S2, the stirring speed is set to 110 rad / min to 130 rad / min, and the duration is 14-16 minutes.

4. The method for extracting limonin according to claim 1, characterized in that, In step S3, the concentration of the ethanol solution is 60% to 80%.

5. The method for extracting limonin according to claim 1 or 4, characterized in that, During the elution process in step S3, the flow rate of the ethanol solution is controlled at 0.4 mL / min to 0.6 mL / min.

6. The method for extracting limonin according to claim 1, characterized in that, In step S5, ethyl acetate and petroleum ether are selected as the developing solvents for limonene samples, and acetonitrile is selected as the spotting solvent. The limonene sample is placed on a silica gel plate and processed by the developing solvent and the spotting solvent. Ehrlich's reagent was chosen as the colorimetric agent for the developed limonene on the silica gel plate.

7. The method for extracting limonin according to claim 6, characterized in that, The ethyl acetate and petroleum ether were mixed in a volume ratio of 4-7:3-6.

8. The method for extracting limonin according to claim 6, characterized in that, The Ehrlich reagent is composed of p-dimethylaminobenzaldehyde dissolved in a sulfuric acid-ethanol mixture, wherein p-dimethylaminobenzaldehyde and the sulfuric acid-ethanol mixture are added at a mass-volume ratio of 1-2:1, and the sulfuric acid-ethanol mixture is mixed at a volume ratio of 6-7:3-4.

9. The method for extracting limonin according to claim 6, characterized in that, In step S5, after performing TLC directional analysis on limonene, the silica gel powder that has not been impregnated with the color developer is cut off from the silica gel plate, and the silica gel powder is stirred and extracted to recover the limonene from the silica gel powder.

10. The method for extracting limonin according to claim 9, characterized in that, In the stirring extraction process, acetonitrile is used as the desorption solvent. Acetonitrile is added to silica gel powder and stirred for 9-11 minutes at a stirring speed of 300-350 rad / min.

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