Extraction method of Brassica rapa seed oil

Abstract

The invention discloses an extraction method of Brassica rapa seed oil, and belongs to the technical field of compound extraction. According to the method, green solvent dimethyl ether (DME) is combined with a response surface method to optimize process parameters (pressure, temperature and time) for extracting the turnip seed oil, fatty acid composition of the obtained turnip seed oil is analyzed through gas chromatography, and saponified matter of the turnip seed oil is prepared and used for follow-up activity and functional evaluation. After optimization, under the conditions that the extraction pressure is 6 MPa, the extraction temperature is 47 DEG C and the extraction time is 83 min, the DME extraction rate (31.72%) of the method is higher than that (30.94%) of Soxhlet extraction of a traditional extraction method. More importantly, rare unsaturated fatty acids such as nervonic acid and the like are effectively retained by lower extraction temperature, and 21 fatty acids are identified by component analysis. The unique fatty acid composition guaranteed by an optimized process is directly associated with the remarkable biological activity of the fatty acid composition. Core advantages of the method in turnip seed oil extraction are proved, and scientific basis and technical support are provided for industrialization of turnip seed oil in the field of cosmetics.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for extracting chamae sac oil, and belongs to the technical field of compound extraction. BACKGROUND

[0002] Chamae is also known as turnip, which belongs to Brassica rapa L. subsp. rapa. Turnip seed is the dried seed of Brassica rapa L. subsp. rapa. It has the effects of removing damp-heat to resolve jaundice and resolving stagnation to promote urination. According to the Record of Materia Medica, “turnip seed can induce perspiration and induce vomiting, and can also improve eyesight and detoxify, and its effects are very significant.” Shin JS et al. found that turnip extract can significantly inhibit rat paw swelling and reduce arthritis index, serum rheumatoid factor and nuclear transcription factor receptor activator ligand / osteoprotegerin (RANKL / OPG) ratio. The active ingredients in turnip seed oil, such as glucosinolates, flavonoids and polysaccharides, have good potential medicinal activities in terms of anti-tumor, antioxidant, anti-inflammatory and immune enhancement.

[0003] Studies have shown that turnip seed oil has important value, but its extraction efficiency and activity retention are highly dependent on the extraction process. The current common extraction processes such as supercritical CO2, Soxhlet extraction and cold pressing have inherent defects such as high energy consumption, solvent pollution risk and degradation of heat-sensitive components, which seriously restrict the industrial application process. Dong Haiyan et al. used supercritical CO2 extraction technology to extract turnip seed oil, and optimized the process conditions (31.65 MPa, 46.10 ℃, 80.40 min) by response surface method, and the extraction rate was 31.55%, but this technology still faces challenges such as high energy consumption, complex operation process and high equipment cost. In the Soxhlet extraction method, there are too many residual organic solvents, and high temperature destroys heat-sensitive substances, resulting in a decrease in oil quality. Therefore, the development of environmentally friendly, efficient and energy-saving green extraction technology has become an urgent need for the industry. SUMMARY

[0004] To solve the problems in the prior art, the present application provides a method for extracting chamae seed oil, which optimizes the process parameters (pressure, temperature and time) for extracting turnip seed oil by using the green solvent dimethyl ether (DME). As a new type of green solvent, DME has high polarity characteristics due to the ether bond in its molecular structure, and its physical properties show a significantly low boiling point (-24.8 °C) and low density (0.66 g / cm 3), which enables it to efficiently dissolve a wide range of substances from non-polar lipids to moderately polar compounds, even achieving precise extraction of target components in aqueous substrates such as microalgae and plant tissues. Compared to traditional organic solvents, DME has low toxicity, high selectivity, and environmental friendliness, and has been certified as GRAS (Generally Recognized as Safe) by the FDA, the European Union EFSA (Document No. 2012 / 234), and the Australian and New Zealand Food Standards Agency (Standard 1.3.3), which lays a safety foundation for its application in food-grade natural product extraction. In the application of extracting turnip seed oil, DME can avoid the heat sensitivity defects and toxicity residue risks of traditional solvents due to its unique compatibility with water-lipid complex systems, thereby improving the quality of oil and expanding its market potential in high-end food and pharmaceutical fields.

[0005] With the significant growth in market demand for natural green ingredients, turnip seed oil, as an ideal substitute for synthetic additives, has shown great application potential in the development of high-end skincare products. To maximize the extraction rate of active ingredients, dimethyl ether (DME) extraction technology is introduced, which utilizes its low temperature, easy recovery, and low residue characteristics under subcritical conditions to gently preserve heat-sensitive active substances while improving mass transfer efficiency. This technology optimizes extraction parameters through response surface methodology and combines gas chromatography system analysis of fatty acid composition and comprehensive evaluation of activity and allergenicity in vitro experiments to provide theoretical support and practical basis for the scientific application of turnip seed oil in the cosmetics field.

[0006] The technical scheme adopted by the present application is: a method for extracting turnip seed oil, comprising the following steps: S1, a single factor experiment design is used to evaluate the independent influence of extraction pressure, extraction temperature and extraction time on the yield of turnip seed oil; S2, the test process is: start the pre-cooler to stabilize the pump head temperature at 0 ℃, weigh the powdered turnip seed sample and place it in the extraction tank, and connect the supercritical system pipeline, complete the system assembly and immediately perform airtightness verification; after the pump head temperature reaches the set value, open the dimethyl ether gas cylinder main valve and access valve in turn, set the extraction pressure and flow rate parameters on the control interface, and then drive the high-pressure pump to gradually increase the pressure; when the system pressure and temperature reach the preset values and are stable, start the timer to record the extraction time; S3, based on the single factor experiment results, set the extraction pressure, extraction temperature and extraction time, with -1, 0 and 1 representing the low, medium and high levels of the independent variables, and the yield of turnip seed oil as the indicator, a three-factor three-level test scheme is constructed by using Box-Behnken design; S4, the test matrix is designed and generated by Design-Expert 13.0.1 software, and the influence of the interaction of each factor on the yield is analyzed; S5, extraction pressure, extraction temperature, extraction time are three main factors affecting the extraction rate of turnip seed oil; the experimental data are analyzed by Design-Expert 13.0.1 software to obtain a quadratic multiple regression model: Y = 0.3010 + 0.0000x1 + 0.0005x2 + 0.0007x3 + 0.0005x1x2 + 0.0007x1x3 +0.0001x2x3 - 0.0150x1 2 - 0.0109x2 2 - 0.0070x3 2 ; Wherein, X1 is the extraction pressure, X2 is the extraction temperature, X3 is the extraction time; S6, the optimal process conditions of DME extraction of turnip seed oil are obtained by solving the quadratic multiple regression model.

[0007] Further, in the step S1, the extraction pressure is 0.8-20 MPa, the extraction temperature is 25-65 DEG C and the extraction time is 30-180 min.

[0008] Further, in the step S3, the low, medium and high levels of extraction pressure are 2.5, 5.0, 7.5 MPa, the low, medium and high levels of extraction temperature are 40, 45, 50 DEG C, and the low, medium and high levels of extraction time are 30, 60, 90 min.

[0009] Further, in the step S6, the optimal process conditions of DME extraction of turnip seed oil are obtained by solving the quadratic multiple regression model, and the optimal process conditions are: the extraction pressure is 6 MPa, the extraction temperature is 47 DEG C, and the extraction time is 83 min.

[0010] The beneficial effects of the application are: the method aims to use green solvent dimethyl ether (DME) to combine response surface method to optimize the process parameters (pressure, temperature, time) of extracting turnip seed oil, and the fatty acid composition of the obtained turnip seed oil is analyzed by gas chromatography, and the saponified product is prepared for subsequent activity and functional evaluation. Based on the RAW264.7 cell inflammation model induced by lipopolysaccharide (Lipopolysaccharide, LPS), the inhibitory effect of turnip seed oil on cell viability and inflammatory factors (NO, IL-6, TNF-alpha) is evaluated, and its clearance And The study investigated the cosmetic properties of DME, including its in vitro antioxidant capacity, tyrosinase inhibitory activity, and sensitization. Results showed that under optimized conditions of 5 MPa, 45 ℃, and 60 min, the DME extraction rate (31.72%) was higher than that of the traditional Soxhlet extraction method (30.94%), and it maintained high extraction efficiency even under low pressure of 0.8 MPa (22.48%), highlighting its energy-saving and environmentally friendly advantages. More importantly, the lower extraction temperature effectively preserved rare unsaturated fatty acids such as nervonic acid, and component analysis identified a total of 21 fatty acids. This unique fatty acid composition, ensured by the optimized process, is directly related to its significant bioactivity. The saponification of turnip seed oil... and The clearance rates reached 90.32% and 86.69% respectively, while significantly improving cell viability. It also inhibits the release of inflammatory factors. This study systematically demonstrated the core advantages of DME extraction technology in turnip seed oil extraction, and for the first time constructed a "process-activity" chain based on multi-dimensional activity evaluation, providing scientific basis and technical support for the industrialization of turnip seed oil in the cosmetics field. Attached Figure Description

[0011] Figure 1 This is a line graph showing the optimal process for extracting fatty acids from DME.

[0012] Figure 2 The results show the response surface and contour plots of the effects of pressure and temperature on the extraction rate (time period: 60 min).

[0013] Figure 3 The results show the response surface and contour plot of the effects of pressure and time on the extraction rate (at a temperature of 45 °C).

[0014] Figure 4 The results show the response surface and contour plots of the effects of temperature and time on the extraction rate (pressure 5 MPa).

[0015] Figure 5 This is the total peak chromatogram of turnip seed oil obtained by gas chromatography.

[0016] Figure 6 The results show the scavenging ability of turnip seed oil saponified products and positive control vitamin C against DPPH and ABTS+ free radicals.

[0017] Figure 7 The effect of saponified substances and the positive control kojic acid on the inhibition rate of tyrosinase activity was investigated.

[0018] Figure 8 It is the toxicity of turnip seed oil saponification to RAW264.7 cells.

[0019] Figure 9Effects of the saponified turnip seed oil on the inflammatory model of RAW264.7 cells. Wherein, a is the effect of the saponified turnip seed oil on the cell survival rate in the LPS-induced inflammatory model of RAW264.7 cells; b is the cell morphology of the control group; c is the cell morphology of the model group; d, e, and f are the cell morphologies of the RAW264.7 cells after the LPS model is treated by the low, medium, and high dose of the saponified turnip seed oil, respectively.

[0020] Figure 10 Effects of the saponified turnip seed oil on inflammatory factors. In the figure, a, b, and c are the effects of the saponified turnip seed oil on the levels of NO, IL-6, and TNF-α in the LPS-induced inflammatory model of RAW264.7 cells. DETAILED DESCRIPTION

[0021] The present application will be described in detail below by examples. It is necessary to point out here that the following examples are only used to further illustrate the present application, but not limited thereto, and unless otherwise specified.

[0022] The specific embodiments of the present application will be described in detail below in combination with the technical solutions: The turnip seeds required in the laboratory were collected from Wushi County and Keping County, Aksu Prefecture, Xinjiang Uygur Autonomous Region, China. After harvesting, the turnip seeds were sequentially screened to remove stems, leaves, sand, and moldy seed impurities, and the seeds with uniform color and full maturity were manually sorted and reserved. Subsequently, the seeds were washed with tap water, rinsed twice with distilled water, and then crushed and sieved by a flow-type crusher (DYQ-188, YONGLIPHARMACEUTICAL MACHINERY CO., China) to form a powder (particle size of 180 μm). Finally, the powder was sealed, stored away from light, and stored at room temperature to ensure the purity, uniformity, and stability of the bioactive components of the seeds, thereby meeting the requirements of subsequent tests.

[0023] FBS fetal bovine serum and DMEM high-sugar medium were purchased from Gibco Company (USA). The penicillin-streptomycin mixture was purchased from Biyun Tian Company (China). PBS was purchased from Shanghai Cytiva Company (China). Lipopolysaccharide was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (China). Mouse IL-6 ELISA kit, mouse TNF-α ELISA kit, and mouse IL-1β ELISA kit were all purchased from Wuhan Sanyou Biotechnology Co., Ltd. (China). CCK8 kit was purchased from Wuhan Sanyou Biotechnology Co., Ltd. (China). Nitric oxide (NO) detection kit was purchased from Biyun Tian Biotechnology Co., Ltd. (China). Sulforaphane (purity ≥98%) was purchased from Chengdu Jianteng Biotechnology Co., Ltd. (China). Example 1

[0024] (1) Single-factor experiment A single-factor experimental design was used to evaluate the independent effects of extraction pressure (0.8–20 MPa), extraction temperature (25–65 °C), and extraction time (30–180 min) on the yield of turnip seed oil. A supercritical system (SF0.1, SEPAREX, France) was used.

[0025] First, start the precooler to stabilize the pump head temperature at 0℃. Accurately weigh 50.00 g of powdered turnip seed sample and place it in the extraction tank, then connect it to the supercritical system pipeline. Immediately after system assembly, verify the airtightness. Once the pump head temperature reaches the set value, sequentially open the main valve of the dimethyl ether cylinder and the main valve of the access circuit. Set the extraction pressure and flow rate parameters on the control interface, then drive the high-pressure pump to gradually increase the pressure. When the system pressure and temperature both reach the preset values ​​and stabilize, start the timer to record the extraction time. During this process, the parameters must be kept constant to ensure experimental reproducibility. It is particularly important to note that during the pressurization stage, when the pressure approaches the set value (difference ≤ 1 MPa), the throttle valve must be adjusted in advance to achieve precise pressure control. Also, after the temperature reaches the set value, allow the system to stabilize for 10-15 minutes. Based on existing research, dimethyl ether extraction technology can precisely regulate the affinity between solvent and solute through the synergistic control of temperature and pressure, achieving highly efficient and selective separation. This characteristic is significantly superior to traditional liquid extraction methods. (See Table 1 and...) Figure 1 As shown in Figure a, the optimal extraction conditions indicate that within the pressure range of 0.8 MPa to 5 MPa, the extraction rate increases to a peak of 27.94% with increasing pressure. However, beyond the critical pressure, the yield decreases due to the fluid density saturation effect (the pressure's regulatory effect on density weakens). Excessive pressure not only reduces selectivity and increases the risk of impurity leaching, damaging oil quality, but also significantly increases costs due to the increased pressure resistance and sealing requirements of the equipment.

[0026] Temperature control, such as Figure 1 As shown in Figure b, the extraction rate increases to 27.00% in the range of 35°C to 45°C, but further heating will reduce the mass transfer efficiency due to the increased tendency of solvent vaporization. This is a nonlinear response formed by the initial positive effect of temperature increase on promoting molecular motion and increasing the diffusion coefficient to improve the mass transfer rate.

[0027] Under conditions of 5 MPa pressure and 45 °C temperature, the yield increased significantly with time when the extraction time was ≤ 60 min. Figure 1 As shown in Figure c, the rate of decrease subsequently slows or declines due to the establishment of mass transfer equilibrium and possible component degradation, reflecting the dynamic characteristics of mass transfer kinetics. In summary, dimethyl ether extraction requires strict balancing of temperature and pressure parameters to avoid overcritical operation, in order to achieve synergistic optimization of efficiency, quality, and economy.

[0028] Table 1 Optimal fatty acid extraction conditions

[0029] (2) Optimization scheme for response surface methodology Based on the results of single-factor experiments, extraction pressures were set at 2.5, 5.0, and 7.5 MPa; extraction temperatures at 40, 45, and 50℃; and extraction times at 30, 60, and 90 min. -1, 0, and 1 represented the low, medium, and high levels of the independent variable, respectively. The yield of turnip seed oil was calculated as (…). The Box-Behnken design was used to construct a three-factor, three-level experimental scheme for the indicators (see Table 2). During the analysis, a 95% confidence level was considered for all variables, and the influence of the independent variables on the response variable was assessed using pure error, with a significance level set at [value missing]. The experimental matrix was generated using Design-Expert 13.0.1 software. The system analyzed the impact of the interaction of various factors on the yield and finally determined the optimal process parameters.

[0030] Table 2 Response Surface Design Factors and Levels

[0031] Single-factor results showed that extraction pressure, temperature, and time were the three main factors affecting the extraction rate of turnip seed oil. Regression analysis was performed on the experimental data in Table 3 using Design-Expert 13.0.1 software, yielding a quadratic multiple regression model:

[0032] Where X1 is the extraction pressure, X2 is the extraction temperature, and X3 is the extraction time; Analysis of variance was performed on model (7), and the results are shown in Table 3. The response surface methodology results of the interaction of the three factors on the extraction rate are shown in Table 3. Figures 2-4 The analysis of variance in Table 4 shows that: , This indicates that model (7) is highly significant and the differences between different treatments are extremely significant; , This indicates that the model's lack of fit is not significant, and no systematic bias was observed; the adjusted coefficient of determination of the model... This indicates that the model can explain 99.37% of the changes in the response values. Therefore, the model has a good fit, small experimental error, and is suitable for analysis and prediction of turnip seed oil extraction rate.

[0033] from Figure 2 ~ Figure 4It can be seen intuitively that pressure is the most significant factor affecting the extraction of DME from turnip seed oil, followed by time and temperature. By solving the equation of the regression model (1), the maximum content of 31.2% was obtained by equation fitting, i.e. the optimal process conditions for DME extraction of turnip seed oil are: extraction pressure 6 MPa, extraction temperature 47 ℃, extraction time 83 min.

[0034] Based on the optimal process conditions determined by the above response surface analysis, 3 DME verification tests were carried out, and the average extraction rate of turnip seed oil was 31.72%. Although there is a slight difference from the predicted value, considering the optimization logic of the model, the extraction rate is still within the reasonable response range. It proves that the DME extraction process model optimized by response surface method is feasible.

[0035] Table 3 Box-Behnken experimental design and data analysis

[0036] Table 4 Analysis of variance of the model

[0037] Note: ; .

[0038] Example 2 Fatty acid composition in turnip seed oil In view of the highest yield and good stability of the seed oil under the optimal process (6 MPa, 47 ℃, 83 min), we further analyzed the fatty acid composition of the seed oil extracted under this condition to clarify its material basis. The gas chromatography analysis spectrum of turnip seed oil is shown in Figure 5 , and the main fatty acid components of turnip seed oil are shown in Table 5. It can be seen that, through gas chromatography analysis, 21 kinds of fatty acid components are identified in turnip seed oil, of which saturated fatty acids only account for 4.61%, mainly palmitic acid (2.64%) and stearic acid (1.08%); the content of unsaturated fatty acids is 92.98%, mainly with erucic acid (47.43%), oleic acid (13.70%) and linoleic acid (13.15%), and retains the rare heat-sensitive component nervonic acid (1.10%) which is easily damaged by traditional high-temperature process. Liu et al. extracted fatty acids from Xanthoceras sorbifolia Bunge seeds from different regions by various traditional extraction methods such as cold pressing, hot pressing and leaching, and did not detect nervonic acid. This is due to the low-temperature extraction characteristics of DME, which avoids the degradation of nervonic acid due to high temperature. Yan et al. experiments confirmed that erucic acid can be efficiently converted to nervonic acid in rat models, which is a key substance for the synthesis of brain myelin sheath. This conversion significantly enhances the activity of oligodendrocytes, promotes the myelination of nerve fibers, and improves nerve signal transmission. Therefore, plant oils with high erucic acid content have important disease application potential.

[0039] Table 5 Main fatty acid components of turnip seed oil

[0040] Example 3 Comparison of different extraction processes Soxhlet extraction method: reference to Valasi et al. method with slight modification. Soxhlet extraction (B-811, BUCHI Labortechnik AG., Swiss Confederation) used petroleum ether (PE, boiling range 30 ~ 60 ℃) as extraction solvent, 6.00 g raw material was accurately weighed and wrapped in filter paper tube placed in the extraction tube, the receiving bottle was preloaded with 80 mL petroleum ether and zeolite, the condensation device was assembled and heated in 70 ~ 80 ℃ water bath, so that the solvent continuously immersed the sample and circulated backflow by siphon action; the whole extraction process lasted for 180 min to ensure that the fatty acids were fully dissolved, after the end of the experiment, the device was disassembled, the receiving bottle was placed in water bath evaporation to about 1 ~ 2 mL of solvent left, then transferred to 110 ℃ oven drying for 1 hour, after cooling, the mass of the receiving bottle was weighed, the extraction amount of fatty acids was calculated by the mass difference and the extraction rate was obtained. This method is based on the principle of organic solvent circulation extraction, petroleum ether with boiling range of 30 ~ 60 ℃ can dissolve lipid components, 180 min of continuous backflow ensures complete extraction, and the water bath temperature is strictly higher than the boiling point of the solvent by 10 ~ 20 ℃ to avoid the risk of violent boiling.

[0041]

[0042] In particular, A1 is the final mass of the receiving bottle; B is the empty bottle mass; C is the raw material mass Based on the data in Table 6, the DME process exhibits the comprehensive advantages of green, efficient and low pressure adaptability. At 5 MPa and 45 ℃, an extraction rate of 31.72% is achieved in only 60 minutes, which is higher than that of Soxhlet extraction of 30.94%, which confirms the high efficiency of DME extraction.

[0043] Table 6 Comparison of different extraction processes

[0044] Example 4 Preparation and application of seakale seed oil saponified product Fatty acids in natural oils exist in the form of triglycerides, and the interference of fat-soluble impurities and the effect of hydrophobicity on the droplets may affect the accuracy and repeatability of activity evaluation. Through saponification treatment, alkaline hydrolysis converts triglycerides into water-soluble free fatty acid sodium salt, and then PBS is added to reconstitute a homogeneous system, which significantly improves the bioavailability of active ingredients, providing a standardized pretreatment basis for cell experiments such as antioxidant and anti-inflammatory.

[0045] The method was modified slightly from that of Cheng et al. The water bath (HH.S11-1, Shanghai Boxun MedicalBiological Instrument Corp., China) was preheated to 75 ℃. 1.00 g of edible oil was weighed into the reaction flask, and 2.5 mL of deionized water, 0.2 g of sodium hydroxide, and a rotor were added sequentially. After placing a condenser above the water bath, the reaction flask was submerged in the water bath, and the temperature was adjusted to 95 ℃ and stirred for 1 h. After the reaction was complete, the reaction system was observed; there should be no solid sodium hydroxide residue and no floating edible oil residue on the surface of the solution. The flask was dried, the cooling water was removed, and approximately 2.5 mL of PBS was added. The mixture was shaken until the saponified product was completely dissolved. If it could not be completely dissolved, 1-2 mL of PBS was added. After dissolving, the mixture was cooled to room temperature with cold water. The mass m (mg) of the saponified product was calculated according to formula (2). Then weigh m / 10 g of the saponified product into a 50 mL centrifuge tube. The remaining sample can be temporarily stored in the centrifuge tube for later use. Add 20 mL of 10% BSA-PBS solution, adjust the pH to between 7.2 and 7.4, and make up to 25 mL with PBS. Store at 4 °C for later use.

[0046]

[0047] Where A2 is the final mass of the bottle after saponification; A1 is the mass of the empty bottle; B is the mass of the turnip seed oil sample; and C is the mass of the rotor. (1) Antioxidant activity like Figure 6 As shown, when the saponified concentration is 1.00 mg / mL, it has a significant effect on DPPH and ABTS. + The free radical scavenging rates reached 90.32% and 86.69%, respectively, and their IC50 values ​​were... 50 The values ​​were 0.33 mg / mL and 0.42 mg / mL, respectively. A dose-response relationship was observed between saponification and antioxidant capacity, and this relationship was statistically significant. This may be related to its high content of unsaturated fatty acids, which can scavenge free radicals by providing hydrogen atoms. This superior performance is significantly lower than that of subcritical butane-extracted sea buckthorn fruit oil (ABTS). + IC 50 The concentration of turnip seed oil saponified (0.89 mg / mL) confirmed its broad-spectrum inhibitory ability against different free radical systems, which is superior to most plant oils and essential oils, highlighting its excellent antioxidant efficacy in natural oil systems. Therefore, turnip seed oil saponified products, with their strong natural antioxidant capacity, provide a highly promising direction for the development of novel natural antioxidants.

[0048] Figure 6 The half-maximum inhibitory concentration (IC50) was used in the study.50 As a core pharmacodynamic indicator, calculations were performed using SPSS Statistics 27.0 (IBM Corp.). A Probit regression model was employed to analyze the experimentally obtained gradient concentration data using logarithmic regression. 10 After transformation, the concentrations are used as covariates, and the inhibition rate is used as the response variable. The total number of observations is uniformly assigned a value of 100. After model fitting, the concentration values ​​corresponding to a probability of 0.50 are extracted from the "Confidence Limitation" table of the output results as the IC50. 50 Point estimates and record their 95% confidence intervals.

[0049] (2) Inhibits tyrosinase activity like Figure 7 As shown, the inhibition of tyrosinase activity by turnip seed oil saponification exhibits a significant dose-dependent characteristic. In the low concentration range (0.005–0.10 mg / mL), the inhibition rate is weak, indicating a weak binding force between the sample molecules and tyrosinase within this concentration range. When the concentration is increased to 0.20–0.40 mg / mL, the inhibition rate sharply increases to 24.80%–43.21%, revealing its effective blocking of the enzymatic reaction process above this threshold concentration. When the concentration is further increased to the high concentration range (0.80–1.0 mg / mL), the inhibition rate reaches 58.38%–70.50%, confirming its medium-to-strong inhibitory potential. Based on the dose-response curve, its IC50 value is calculated to be... 50 The value was 0.54 mg / mL. Although the efficacy was significant, it was weaker than that of kojic acid control (IC50). 50 (The concentration was 0.06 mg / mL). Notably, 0.40 mg / mL was the critical efficacy inflection point, with an inhibition rate of 43.21%.

[0050] (3) Anti-inflammatory activity: (3.1) Effect of saponification on the viability of RAW264.7 cells The cytotoxic effects of turnip seed oil saponins on RAW264.7 cells were determined using the CCK-8 assay. The results are as follows: Figure 8 As shown in the figure. Compared with the control group, the cell survival rate of turnip seed oil saponified products showed an increasing trend in the concentration range of 0.01~1.00 mg / mL, without any toxic effects. It can be seen that the cell growth rate increased sharply in the concentration range of 0.01~2.00 mg / mL. Therefore, 0.025, 0.05, and 0.10 mg / mL were selected as low, medium, and high dosages for subsequent experiments.

[0051] Figure 8 In Figure A, one-way ANOVA showed highly significant differences among the treatment groups. ). Differences between groups were annotated by letter notation (e.g. a, b, c) based on Waller-Duncan post hoc test (k-ratio = [k value]). Groups sharing the same letter were not statistically different .

[0052] (3.2) Effect of saponification on the viability of LPS-induced RAW264.7 cell inflammation model As shown in Figure 9 A, compared with the LPS group, the cell viability of the positive control group (dexamethasone) was significantly increased. When the cells were co-treated with LPS and different concentrations of saponification of turnip seed oil (0.025, 0.05, 0.10 mg / mL), the cell viability was also significantly increased (P < 0.05) (Fig. 3B), but it was not dose-dependent. Compared with the control group, the cell survival rate in the LPS group showed a weak upward trend, and the statistical difference was significant (P < 0.05) (Fig. 3C). To further confirm whether the modeling was successful, we observed the morphology of the cells in the control and LPS groups under an inverted microscope. As shown in Figure 9 B and Figure 9 C, compared with the control group, the cell morphology in the LPS group changed significantly. As shown in Figure 9 D, compared with the LPS group, the cell morphology was significantly improved after treatment with saponification of turnip seed oil, and the number of round, full, and transparent live cells increased significantly.

[0053] Figure 9 Fig. 3D, a is the effect of saponification of turnip seed oil on the cell survival rate in the LPS-induced RAW264.7 cell inflammation model; b is the morphology of the cells in the control group; c is the morphology of the cells in the model group; d, e, and f are the morphologies of the RAW264.7 cells treated with low, medium, and high doses of saponification of turnip seed oil, respectively; (3.3) Anti-inflammatory effect of saponification on the LPS-induced RAW264.7 cell inflammation model IL-6, TNF-a, and NO content are classic inflammatory markers. By evaluating the inhibitory effect of saponification of turnip seed oil on the secretion level of inflammatory factors in cells, the anti-inflammatory activity can be determined. As shown in Figure 10 Fig. 4A, compared with the control group, the levels of NO, IL-6, and TNF-a in the LPS group increased significantly (P < 0.05) (Fig. 4B). ​). Compared with the LPS group, the dexamethasone positive control group and the 0.025, 0.05, 0.10 mg / mL different concentrations of saponins of Seipium torvum seeds could significantly reduce the levels of inflammatory factors such as NO, IL-6 and TNF-α ), but the effect of inhibiting inflammatory factors was not dose-dependent. The experimental results showed that when the administration group was compared with the positive control group (dexamethasone) and LPS was co-treated, the secretion levels of NO, IL-6 and TNF-α were slightly higher than those of the positive control group, but compared with the model group induced by LPS, the concentrations of these inflammatory factors showed a significant downward trend, and the statistical analysis showed that the difference was significant ). This finding showed that although the anti-inflammatory efficacy of saponins was not as good as that of the positive control, it could still effectively inhibit key inflammatory mediators, highlighting the significant anti-inflammatory activity of saponins of Seipium torvum seed oil, which was mainly due to the blocking of nuclear translocation and inhibition phosphorylation of subunit P65, thereby suppressing activation of the signaling pathway, and the inflammation regulation mechanism achieved by down-regulating the expression of pro-inflammatory cytokines and oxidative stress pathway, which suggested that saponins of Seipium torvum seed oil had market value for treating inflammation.

[0054] (4) Sensitization If there is no overlap between the peaks in the test substance and the polypeptide peaks, i.e. the test substance does not co-elute with the polypeptide chain, then the consumption rate of cysteine and lysine polypeptides can be calculated according to the change in peak area, and the average value of the elimination rate of the two polypeptide chains can be calculated. The percentage of polypeptide consumption is combined to judge the sensitization grade, and the result is predicted. The average value of the percentage of polypeptide consumption is between 0% and 6.38%, which is negative, and the result greater than this range indicates positive with different reaction grades.

[0055] As shown in Table 7, the polypeptide consumption rate (2.32%) of saponins of Seipium torvum seed oil in the DPRA test was significantly lower than the sensitization determination threshold, and the reaction grade was "none", and the DPRA prediction result determined that it was a non-sensitizing substance. This result conforms to the classification standard of non-sensitizing chemicals in the OECD guidelines, indicating that it lacks skin sensitization risk, and provides a reliable in vitro experimental basis for the safety evaluation of cosmetic raw materials, supporting its application safety in the formula.

[0056] Table 7 Comparison of DPRA prediction model standard and test results of saponins of Seipium torvum seed oil

[0057] The advantages of subcritical dimethyl ether extraction technology in the extraction of turnip seed oil were demonstrated, and a "process-activity" correlation system was established, providing a scientific basis for its industrialization application in the cosmetics field. First, subcritical dimethyl ether extraction technology showed significant advantages compared to traditional methods. Under the optimized conditions of about 6 MPa, its extraction rate was significantly higher than that of Soxhlet extraction, ultrasonic extraction, and supercritical CO2 extraction. Notably, even at ultra-low pressure (0.8 MPa), this technology still maintained high extraction efficiency, reducing energy consumption and equipment costs, which meets the development needs of green and sustainable extraction technology. Second, the fatty acid composition of turnip seed oil extracted by subcritical dimethyl ether was characterized by high unsaturated fatty acids (92.98%), mainly including erucic acid, oleic acid, and linoleic acid, and containing nervonic acid. This composition not only gives it nutritional value but also lays the material foundation for its biological activity. Third, multidimensional activity evaluation confirmed that turnip seed oil has the potential to be used as a cosmetic raw material: its significant antioxidant capacity can resist skin aging related to oxidative stress; negative results of direct peptide binding test prove its non-sensitizing property, ensuring skin safety; tyrosinase inhibition activity suggests its application prospects in whitening preparations; and its ability to down-regulate pro-inflammatory factors (NO, IL-6, TNF-α) in lipopolysaccharide-induced macrophage models verifies its anti-inflammatory efficacy, which is crucial for soothing sensitive skin. In summary, the innovative application of subcritical dimethyl ether technology in turnip seed oil extraction optimizes the process parameters and clarifies its composition and activity characteristics. The established "process-activity" chain connects extraction technology and functional application, providing strong support for the development of turnip seed oil as a natural, safe, and multifunctional cosmetic raw material. Future research can focus on in vivo efficacy evaluation and formula development to further promote its industrialization.

[0058] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for extracting Chamaegu seed oil, characterized in that, Includes the following steps: S1. A single-factor experimental design was used to evaluate the independent effects of extraction pressure, extraction temperature and extraction time on the yield of turnip seed oil. S2. The experimental procedure is as follows: The precooler is started to stabilize the pump head temperature at 0 ℃. Powdered turnip seed samples are weighed and placed in the extraction tank, and connected to the supercritical system pipeline. After the system is assembled, the airtightness is verified immediately. After the pump head temperature reaches the set value, the main valve of the dimethyl ether cylinder and the main valve of the passage are opened in sequence. The extraction pressure and flow rate parameters are set on the control interface, and then the high-pressure pump is driven to gradually increase the pressure. When the system pressure and temperature reach the preset values ​​and stabilize, the timer is started to record the extraction time. S3. Based on the results of the single-factor experiment, extraction pressure, extraction temperature, and extraction time were set, with -1, 0, and 1 representing the low, medium, and high levels of the independent variable, respectively. The yield of turnip seed oil was used as the criterion. Y Indicators, using Box-Behnken design to construct a three-factor, three-level experimental scheme; S4. Using Design-Expert 13.0.1 software, design and generate an experimental matrix, and systematically analyze the impact of the interaction of various factors on the yield; S5, extraction pressure, extraction temperature, and extraction time are the three main factors affecting the extraction rate of turnip seed oil. Using DesignExpert 13.0.1 software, regression analysis was performed on the experimental data to obtain a quadratic multiple regression model: Y = 0.3010 + 0.0000x1 + 0.0005x2 + 0.0007x3 + 0.0005x1x2 + 0.0007x1x3 +0.0001x2x3 - 0.0150x1 2 - 0.0109x2 2 - 0.0070x3 2 ; Where X1 is the extraction pressure, X2 is the extraction temperature, and X3 is the extraction time; S6. Solve the quadratic multiple regression model to obtain the optimal process conditions for DME extraction of turnip seed oil.

2. The method for extracting Chamaegu seed oil according to claim 1, characterized in that: In step S1, the extraction pressure is 0.8 ~ 20 MPa, the extraction temperature is 25 ~ 65 ℃, and the extraction time is 30 ~ 180 min during the evaluation.

3. The method for extracting Chamaegu seed oil according to claim 1, characterized in that: In step S3, the extraction pressure is set to low, medium, and high levels of 2.5, 5.0, and 7.5 MPa, the extraction temperature is set to low, medium, and high levels of 40, 45, and 50 ℃, and the extraction time is set to low, medium, and high levels of 30, 60, and 90 min.

4. The method for extracting Chamaegu seed oil according to claim 1, characterized in that: In step S6, by solving the quadratic multiple regression model, the optimal process conditions for DME extraction of turnip seed oil are obtained as follows: extraction pressure 6 MPa, extraction temperature 47 ℃, and extraction time 83 min.

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