Extraction process, process optimization and content determination method of fructus cinnamoii oil

Abstract

The invention discloses an extraction process, a process optimization method and a content determination method of fructus cinnamoii oil. According to the method, a supercritical CO2 extraction method (SFE-CO2) is adopted for extracting the fructus cinnamoii oil, on the basis of a single factor experiment, the extraction process of the fructus cinnamoii oil is optimized through a response surface method in combination with an entropy weight method, the obtained optimal extraction process is as follows: the temperature is 34 DEG C, the pressure is 21 Mpa, the time is 41 min, the flow is 12 L / h, and the average yield of the fructus cinnamoii oil is 25.73% under the conditions of the temperature being 34 DEG C, the pressure being 21 Mpa, the time being 41 min and the flow being 12 L / h. The extraction process disclosed by the invention is stable and feasible in quality and high in extraction rate, and the extracted fructus cinnamoii oil is high in effective component content.

Description

Technical Field

[0001] The invention belongs to the technical field of vegetable oil processing, and particularly relates to an extraction process, process optimization and content determination method of Litsea cubeba oil. Background Art

[0002] Supercritical CO2 extraction (SFE-CO2) is an advanced technology that uses CO2 in a supercritical state under high pressure to extract and separate various organic substances. In recent years, it has been widely used in the separation and application of volatile oil substances as well as in the fields of medicine, food, cosmetics, etc. Compared with other methods, SFE-CO2 has obvious advantages such as high extraction rate and protection of effective ingredients. It uses CO2 as the extraction solvent, which is non-toxic, pollution-free, cheap and abundant in source, making it suitable for the extraction and separation of natural medicines.

[0003] Litsea cubeba (Litsea cubeba) is the dried, mature fruit of the Lauraceae plant Cinnamomum migao HWLi. Also known as Maodanmu, Qingtumu, Bajiaodai, Qingtu Bajiao, and False Lemon Fruit, it is primarily distributed in Guizhou, Yunnan, and Guangxi. It warms the middle and dispels cold, regulates qi, and relieves pain. It is commonly used to treat stomachache, abdominal pain, chest pain, vomiting, and chest tightness. It is a specialty folk medicine in southwestern China for treating heart and stomach pain. Litsea cubeba oil, extracted from Litsea cubeba, has been used to protect against myocardial ischemia, combat arrhythmia, atrial fibrillation, hypoxia, relax visceral smooth muscle, and has antiviral, anti-inflammatory, and analgesic properties.

[0004] Because there are many highly volatile and heat-sensitive substances in the oil of Litsea cubeba, and SFE-CO2 has the advantages of high extraction rate, short production cycle, and protection of the original chemical components during extraction, the team of this invention conducted research based on the establishment and verification of the GC detection method, and optimized the SFE-CO2 extraction method of Litsea cubeba oil by combining the Box-Behnken response surface methodology with the entropy weight method. Summary of the Invention

[0005] The invention aims to provide an extraction process for Litsea cubeba oil.

[0006] Another object of the present invention is to provide a method for optimizing the extraction process of Litsea cubeba oil.

[0007] Another object of the present invention is to provide a content determination method used in the method for optimizing the extraction process of Litsea cubeba oil.

[0008] The extraction process of the macrocarpon oil of the present invention comprises the following steps: using an electronic balance to weigh the macrocarpon crushed to 20 mesh, and loading the crushed macrocarpon into an SFE-CO2 extraction kettle; introducing CO2, setting the operating table parameters as temperature 15-55°C, pressure 15-35 MPa, time 20-80 min, and CO2 flow rate 6-16 L / h, and performing SFE-CO2 extraction.

[0009] Preferably, the extraction process of the macrocarpon oil of the present invention is: using an electronic balance to weigh the macrocarpon crushed to 20 mesh, and loading it into an SFE-CO2 extraction kettle; introducing CO2, setting the operating table parameters to temperature 25-45°C, pressure 20-30 MPa, time 40-80 min, and CO2 flow rate 10-14 L / h, and performing SFE-CO2 extraction.

[0010] Further preferably, the extraction process of the macrocarpon oil of the present invention is:

[0011] The method for optimizing the extraction process of Litsea cubeba oil of the present invention comprises the following steps:

[0012] Step 1: Weigh the Litsea cubeba fruit crushed into 20 mesh, put it into the SFE-CO2 extraction kettle, introduce CO2, set the operating table parameters as temperature, pressure, time and CO2 flow rate, and perform SFE-CO2 extraction;

[0013] Step 2: Taking the contents of methyl caprate, methyl undecanoate, methyl laurate, and methyl tridecanoate in Litsea cubeba oil and the comprehensive score of Litsea cubeba oil yield as the evaluation indicators, the extraction temperature, extraction pressure, extraction time, and CO2 flow rate of Litsea cubeba oil were investigated;

[0014] Step 3: Based on the results obtained in step 2, perform factor level experimental design based on the Box-Behnken response surface;

[0015] Step 4: Obtain the entropy weight of each indicator through the entropy weight method and calculate the comprehensive score;

[0016] Step 5: Use Design-Expert 11 to perform variance analysis and quadratic multiple regression fitting on the experimental data to obtain the model equation between extraction temperature, extraction pressure, extraction time, CO2 and comprehensive score;

[0017] Step 6: Obtain the optimal extraction conditions of the SFE-CO2 extraction process of Litsea cubeba oil based on the model equation.

[0018] The calculation method of the comprehensive score in step 4 of the present invention is: comprehensive score = methyl decanoate × 0.2465 + methyl undecanoate × 0.1483 + methyl laurate × 0.1583 + methyl tridecanoate × 0.1379 + yield × 0.3090.

[0019] The model equation of step 5 of the present invention is Y=0.6502-0.0536*A+0.1406*B+0.1615*C+0.2631*D-0.0738*AB+0.0408*AC-0.0975*AD+0.0510*BC+0.1549*BD-0.0870*CD-0.2298*A 2 -0.0784*B 2 -0.2241*C 2 -0.1148*D 2 , where Y is the comprehensive score, A is the extraction temperature, B is the extraction pressure, C is the extraction time, and D is the CO2 flow rate.

[0020] The optimal extraction conditions in step 6 of the present invention are: temperature 34° C., pressure 21 MPa, time 41 min, and flow rate 12 L / h.

[0021] The content determination method used in the method for optimizing the extraction process of Litsea cubeba oil of the present invention is as follows:

[0022] (1) Preparation of test solution:

[0023] Accurately weigh 0.1g of Litsea cubeba oil, add 4mL of 0.5mol / L KOH-MeOH solution, saponify in a 60℃ water bath for 15min, remove and cool; add 8mL of 25% hydrochloric acid, place in a 60℃ water bath for 15min, remove and cool; then add 4mL of n-hexane and shake for 5min; finally, add 4mL of saturated sodium chloride solution and let it stand for 10min. The supernatant is collected, evaporated under reduced pressure, and redissolved in 2mL of n-hexane to pass through a microporous filter membrane (0.22μm) before testing.

[0024] (2) Preparation of mixed reference solution:

[0025] Accurately pipette 62.9565 mg of methyl decanoate, 63.4750 mg of methyl undecanoate, 62.575 mg of methyl laurate, and 64.4810 mg of methyl tridecanoate, and accurately weigh them. Dissolve them in chromatographically pure n-hexane and make up to the volume in a 5 mL volumetric flask to prepare a mixed reference substance stock solution containing 12.5913 mg / mL of methyl decanoate, 12.6950 mg / mL of methyl undecanoate, 12.5150 mg / mL of methyl laurate, and 12.8962 mg / mL. Place in a -20°C refrigerator until use.

[0026] (3) Gas chromatography conditions:

[0027] The injection port temperature was 200°C; the detector temperature was 230°C; the split ratio was 20:1; the column flow rate was 1 mL / min; the injection volume was 5 μL; the heating program was as follows: initial temperature at 40°C, hold for 1 min, 40°C to 200°C, heating at 5°C per minute, hold for 2 min; or initial temperature at 60°C, hold for 1 min, 60°C to 160°C, heating at 15°C per minute, hold for 0 min, 160°C to 170°C, heating at 0.5°C per minute, hold for 0 min, 170°C to 230°C, heating at 3°C per minute, hold for 30 min.

[0028] (4) Determination:

[0029] The solutions of steps (1) and (2) are sampled and measured according to the gas chromatography conditions of step (3), and the peak areas are recorded and the contents of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate are calculated.

[0030] Preferably, the content determination method used in the method for optimizing the extraction process of Litsea cubeba oil of the present invention is as follows:

[0031] (1) Preparation of test solution:

[0032] Accurately weigh 0.1g of Litsea cubeba oil, add 4mL of 0.5mol / L KOH-MeOH solution, saponify in a 60℃ water bath for 15min, remove and cool; add 8mL of 25% hydrochloric acid, place in a 60℃ water bath for 15min, remove and cool; then add 4mL of n-hexane and shake for 5min; finally, add 4mL of saturated sodium chloride solution and let it stand for 10min. The supernatant is collected, evaporated under reduced pressure, and redissolved in 2mL of n-hexane to pass through a microporous filter membrane (0.22μm) before testing.

[0033] (2) Preparation of mixed reference solution:

[0034] Accurately pipette 62.9565 mg of methyl decanoate, 63.4750 mg of methyl undecanoate, 62.575 mg of methyl laurate, and 64.4810 mg of methyl tridecanoate, and accurately weigh them. Dissolve them in chromatographically pure n-hexane and make up to the volume in a 5 mL volumetric flask to prepare a mixed reference substance stock solution containing 12.5913 mg / mL of methyl decanoate, 12.6950 mg / mL of methyl undecanoate, 12.5150 mg / mL of methyl laurate, and 12.8962 mg / mL. Place in a -20°C refrigerator until use.

[0035] (3) Gas chromatography conditions:

[0036] The injection port temperature was 200°C; the detector temperature was 230°C; the split ratio was 20:1; the column flow rate was 1 mL / min; the injection volume was 5 μL; the heating program was as follows: initial temperature 60°C, hold for 1 min, 60°C to 160°C, heating at 15°C / min, hold for 0 min, 160°C to 170°C, heating at 0.5°C / min, hold for 0 min, 170°C to 230°C, heating at 3°C / min, hold for 30 min;

[0037] (4) Determination:

[0038] The solutions of steps (1) and (2) are sampled and measured according to the gas chromatography conditions of step (3), and the peak areas are recorded and the contents of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate are calculated.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] 1. The yield and comprehensive evaluation of the macrocarpon oil extracted by the extraction process of the present invention are high, and the process is stable and feasible. The optimal SFE-CO2 extraction process parameters of the macrocarpon oil obtained by the present invention are optimized by the response surface methodology combined with the entropy weight method: temperature 33.9344°C, pressure 21.0487Mpa, time 41.1534min, flow rate 12.0317L / h, and the predicted comprehensive score is 0.7611. Combined with actual conditions, the optimal parameter results are revised to: temperature 34°C, pressure 21Mpa, time 41min, flow rate 12L / h. At this time, the average yield of macrocarpon oil is 25.73%, and the average comprehensive evaluation is 0.7541, which is relatively close to the predicted value comprehensive score of 0.7611, indicating that the extraction process is stable and feasible.

[0041] 2. The extraction process of the present invention is simple, easy and stable. The results of the regression model variance analysis show that the Box-Behnken model F value is 3.3030, P = 0.0163 < 0.05, indicating that the regression model obtained in the experiment has extremely high significance and statistical significance; the P value of the lack of fit term is 0.1194 > 0.05, indicating that the lack of fit term is not significant, that is, the model fits the experimental data well and can be used to analyze the relationship between each factor and the response value. The variance analysis of each factor found that the linear term DD, the interaction term BD and the quadratic term A 2 、C 2 The above results show that the regression model can well reflect the changes in the response value, and this model can be used to analyze and predict the impact of each single factor on the comprehensive score of the extracted Litsea cubeba oil.

[0042] 3. The entropy weight method of the present invention calculates the weights of the yields of methyl decanoate, methyl undecanoate, methyl laurate, methyl tridecanoate and Litsea cubeba oil to be 0.2465, 0.1483, 0.1583, 0.1379 and 0.3090, respectively. Therefore, the calculation method for obtaining the comprehensive score is: comprehensive score = methyl decanoate × 0.2465 + methyl undecanoate × 0.1483 + methyl laurate × 0.1583 + methyl tridecanoate × 0.1379 + yield × 0.3090.

[0043] 4. The present invention investigates the gas chromatography programmed temperature conditions for determining the contents of methyl caprate, methyl undecanoate, methyl laurate, and methyl tridecanoate in Litsea cubeba oil, and determines that the temperature program conditions are as follows: an initial temperature of 60°C, maintained for 1 minute, a temperature increase of 15°C per minute from 60°C to 160°C, maintained for 0 minute, a temperature increase of 0.5°C per minute from 160°C to 170°C, maintained for 0 minute, and a temperature increase of 3°C per minute from 170°C to 230°C, maintained for 30 minutes.

[0044] 5. The present invention investigated the methyl esterification method of Litsea cubeba oil. The results showed that the acid-base combination method had a better esterification effect than the acid treatment method and the alkali treatment method. Therefore, the acid-base combination method was selected for subsequent experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Results of programmed temperature conditions screening (wherein, A is an initial temperature of 40°C, held for 1 min, 40°C to 200°C, heating at 5°C per minute, held for 2 min; B is an initial temperature of 60°C, held for 1 min, 60°C to 160°C, heating at 15°C per minute, held for 0 min, 160°C to 170°C, heating at 0.5°C per minute, held for 0 min, 170°C to 230°C, heating at 3°C per minute, held for 30 min; C is a spectrum of a n-hexane solution of methyl decanoate; D is a spectrum of a n-hexane solution of methyl undecanoate; E is a spectrum of a n-hexane solution of methyl laurate; F is a spectrum of a n-hexane solution of methyl tridecanoate);

[0046] Figure 2 Screening results of different methylation methods (A is acid treatment method, B is alkali treatment method, and C is acid-base combined method);

[0047] Figure 3 The results of the content determination of the standard solution at each concentration gradient (where A is the mixed reference solution of group ①; B is the mixed reference solution of group ②; C is the mixed reference solution of group ③; D is the mixed reference solution of group ④; E is the mixed reference solution of group ⑤);

[0048] Figure 4 The effect of extraction temperature on the overall score;

[0049] Figure 5 The effect of extraction pressure on the overall score;

[0050] Figure 6 The effect of extraction time on the overall score;

[0051] Figure 7 The impact of CO2 flow rate on the comprehensive score;

[0052] Figure 8 Effect of temperature and pressure interaction on comprehensive score (including response surface plot (left) and contour plot (right));

[0053] Figure 9 Effect of temperature and time interaction on comprehensive score (including response surface plot (left) and contour plot (right));

[0054] Figure 10 Effect of temperature and flow rate interaction on comprehensive score (including response surface plot (left) and contour plot (right));

[0055] Figure 11 Effect diagram of the interaction between pressure and time on the comprehensive score (including response surface plot (left) and contour plot (right));

[0056] Figure 12 Effect of pressure and flow rate interaction on comprehensive score (including response surface plot (left) and contour plot (right));

[0057] Figure 13 The impact of the interaction between time and flow on the comprehensive score (including response surface plot (left) and contour plot (right)). DETAILED DESCRIPTION

[0058] The technical solution of the present invention is further described in detail below through specific embodiments.

[0059] Example 1 SFE-CO2 extraction process of Litsea cubeba oil

[0060] (1) Using an electronic balance, weigh the Litsea cubeba seeds crushed to 20 mesh and put them into a SFE-CO2 extraction kettle;

[0061] (2) CO2 was introduced, and the operating table parameters were set to temperature 34°C, pressure 21 MPa, time 41 min, and CO2 flow rate 12 L / h for SFE-CO2 extraction.

[0062] Example 2 SFE-CO2 extraction process of Litsea cubeba oil

[0063] (1) Using an electronic balance, weigh the Litsea cubeba seeds crushed to 20 mesh and put them into a SFE-CO2 extraction kettle;

[0064] (2) CO2 was introduced, and the operating table parameters were set to temperature 55°C, pressure 35 MPa, time 80 min, and CO2 flow rate 16 L / h for SFE-CO2 extraction.

[0065] Example 3 SFE-CO2 extraction process of Litsea cubeba oil

[0066] (1) Using an electronic balance, weigh the Litsea cubeba seeds crushed to 20 mesh and put them into a SFE-CO2 extraction kettle;

[0067] (2) CO2 was introduced, and the operating table parameters were set to temperature 15°C, pressure 15 MPa, time 20 min, and CO2 flow rate 6 L / h for SFE-CO2 extraction.

[0068] Example 4 SFE-CO2 extraction process of Litsea cubeba oil

[0069] (1) Using an electronic balance, weigh the Litsea cubeba seeds crushed to 20 mesh and put them into a SFE-CO2 extraction kettle;

[0070] (2) CO2 was introduced, and the operating table parameters were set to temperature 45°C, pressure 30 MPa, time 60 min, and CO2 flow rate 14 L / h for SFE-CO2 extraction.

[0071] Example 5 SFE-CO2 extraction process of Litsea cubeba oil

[0072] (1) Using an electronic balance, weigh the Litsea cubeba seeds crushed to 20 mesh and put them into a SFE-CO2 extraction kettle;

[0073] (2) CO2 was introduced, and the operating table parameters were set to temperature 25°C, pressure 20 MPa, time 30 min, and CO2 flow rate 8 L / h for SFE-CO2 extraction.

[0074] Example 6 SFE-CO2 Extraction Process of Litsea glutinosa Oil

[0075] (1) Using an electronic balance, weigh the Litsea cubeba seeds crushed to 20 mesh and put them into a SFE-CO2 extraction kettle;

[0076] (2) CO2 was introduced, and the operating table parameters were set to temperature 35°C, pressure 25 MPa, time 40 min, and CO2 flow rate 10 L / h for SFE-CO2 extraction.

[0077] Example 7 SFE-CO2 Extraction Process for Litsea glutinosa Oil

[0078] (1) Using an electronic balance, weigh the Litsea cubeba seeds crushed to 20 mesh and put them into a SFE-CO2 extraction kettle;

[0079] (2) CO2 was introduced, and the operating table parameters were set to temperature 35°C, pressure 30 MPa, time 40 min, and CO2 flow rate 12 L / h for SFE-CO2 extraction.

[0080] Example 8 Optimization of SFE-CO2 Extraction Process of Litsea glutinosa Oil by Response Surface Methodology Combined with Entropy Weight Method

[0081] (1) Using an electronic balance, weigh the Litsea cubeba seeds crushed to 20 mesh, load them into a SFE-CO2 extraction kettle, introduce CO2, set the operating table parameters to temperature, pressure, time and CO2 flow rate, and perform SFE-CO2 extraction;

[0082] (2) Taking the contents of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate in Litsea cubeba oil and the comprehensive score of Litsea cubeba oil yield as the investigation indicators, the extraction temperature, extraction pressure, extraction time, and CO2 flow rate of Litsea cubeba oil were investigated. The investigation range of extraction temperature was 15-55℃, the investigation range of extraction pressure was 15-35Mpa, the investigation range of extraction time was 20-80min, and the investigation range of CO2 flow rate was 6-16L / h;

[0083] (3) Based on the results obtained in step (2), a factor level experimental design was performed based on the Box-Behnken response surface;

[0084] (4) The entropy weight of each indicator was obtained by the entropy weight method, and the comprehensive score was calculated as follows: comprehensive score = methyl decanoate × 0.2465 + methyl undecanoate × 0.1483 + methyl laurate × 0.1583 + methyl tridecanoate × 0.1379 + yield × 0.3090;

[0085] (5) Using Design-Expert 11, variance analysis and quadratic multiple regression fitting were performed on the experimental data, and the model equation between extraction temperature, extraction pressure, extraction time, CO2 and comprehensive score was obtained as Y = 0.6502-0.0536*A+0.1406*B+0.1615*C+0.2631*D-0.0738*AB+0.0408*AC-0.0975*AD+0.0510*BC+0.1549*BD-0.0870*CD-0.2298*A 2 -0.0784*B 2 -0.2241*C 2 -0.1148*D 2 , where Y is the comprehensive score, A is the extraction temperature, B is the extraction pressure, C is the extraction time, and D is the CO2 flow rate;

[0086] (6) According to the model equation, the optimal extraction conditions for the SFE-CO2 extraction process of Litsea cubeba oil were obtained as follows: temperature 34℃, pressure 21MPa, time 41min, and CO2 flow rate 12L / h.

[0087] Example 9: Determination of the Content of Methyl Decanoate, Methyl Undecanoate, Methyl Laurate, and Methyl Tridecanoate

[0088] (1) Preparation of test solution:

[0089] Accurately weigh 0.1g of Litsea cubeba oil, add 4mL of 0.5mol / L KOH-MeOH solution, saponify in a 60℃ water bath for 15min, remove and cool; add 8mL of 25% hydrochloric acid, place in a 60℃ water bath for 15min, remove and cool; then add 4mL of n-hexane and shake for 5min; finally, add 4mL of saturated sodium chloride solution and let it stand for 10min. The supernatant is collected, evaporated under reduced pressure, and redissolved in 2mL of n-hexane to pass through a microporous filter membrane (0.22μm) before testing.

[0090] (2) Preparation of mixed reference solution:

[0091] Accurately pipette 62.9565 mg of methyl decanoate, 63.4750 mg of methyl undecanoate, 62.575 mg of methyl laurate, and 64.4810 mg of methyl tridecanoate, and accurately weigh them. Dissolve them in chromatographically pure n-hexane and make up to the volume in a 5 mL volumetric flask to prepare a mixed reference substance stock solution containing 12.5913 mg / mL of methyl decanoate, 12.6950 mg / mL of methyl undecanoate, 12.5150 mg / mL of methyl laurate, and 12.8962 mg / mL. Place in a -20°C refrigerator until use.

[0092] (3) Gas chromatography conditions:

[0093] Inlet temperature: 200°C; detector temperature: 230°C; split ratio: 20:1; column flow rate: 1 mL / min; injection volume: 5 μL; heating program: initial temperature: 60°C, hold for 1 min, 60°C to 160°C, increase by 15°C per minute, hold for 0 min, 160°C to 170°C, increase by 0.5°C per minute, hold for 0 min, 170°C to 230°C, increase by 3°C per minute, hold for 30 min;

[0094] (4) Determination:

[0095] The solutions of steps (1) and (2) are sampled and measured according to the gas chromatography conditions of step (3), and the peak areas are recorded and the contents of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate are calculated.

[0096] Example 10 Determination of the Content of Methyl Caprate, Methyl Undecanoate, Methyl Laurate, and Methyl Tridecanoate

[0097] (1) Preparation of test solution:

[0098] Accurately weigh 0.1g of Litsea cubeba oil, add 4mL of 0.5mol / L KOH-MeOH solution, saponify in a 60℃ water bath for 15min, remove and cool; add 8mL of 25% hydrochloric acid, place in a 60℃ water bath for 15min, remove and cool; then add 4mL of n-hexane and shake for 5min; finally, add 4mL of saturated sodium chloride solution and let it stand for 10min. The supernatant is collected, evaporated under reduced pressure, and redissolved in 2mL of n-hexane to pass through a microporous filter membrane (0.22μm) before testing.

[0099] (2) Preparation of mixed reference solution:

[0100] Accurately pipette 62.9565 mg of methyl decanoate, 63.4750 mg of methyl undecanoate, 62.575 mg of methyl laurate, and 64.4810 mg of methyl tridecanoate, and accurately weigh them. Dissolve them in chromatographically pure n-hexane and make up to the volume in a 5 mL volumetric flask to prepare a mixed reference substance stock solution containing 12.5913 mg / mL of methyl decanoate, 12.6950 mg / mL of methyl undecanoate, 12.5150 mg / mL of methyl laurate, and 12.8962 mg / mL. Place in a -20°C refrigerator until use.

[0101] (3) Gas chromatography conditions:

[0102] Inlet temperature: 200°C; detector temperature: 230°C; split ratio: 20:1; column flow rate: 1 mL / min; injection volume: 5 μL; heating program: initial temperature: 40°C, hold for 1 min, then 40°C to 200°C, heating at 5°C / min, hold for 2 min;

[0103] (4) Determination:

[0104] The solutions of steps (1) and (2) are sampled and measured according to the gas chromatography conditions of step (3), and the peak areas are recorded and the contents of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate are calculated.

[0105] In order to verify the effectiveness of the present invention, the invention team conducted a series of experiments, as follows:

[0106] 1 Instruments and Materials

[0107] 1.1 Instruments and Equipment

[0108] The experimental instruments and equipment information are shown in Table 1.

[0109] Table 1 Experimental instruments and equipment information

[0110]

[0111] 1.2 Materials and Reagents

[0112] The source of sample information is shown in Table 2, and the experimental reagents are shown in Table 3.

[0113] Table 2 Source information of Litsea cubeba

[0114]

[0115] Table 3 Test reagents

[0116]

[0117] 2 Methods

[0118] 2.1 Screening of GC conditions

[0119] 2.1.1 Screening of programmed temperature conditions

[0120] After methyl esterification of the Litsea cubeba oil sample, individual fatty acid methyl ester standard solutions and mixed fatty acid methyl ester standard solutions were injected into a GC chromatograph for analysis and qualitative analysis of the chromatographic peaks. The GC temperature program conditions are shown in Tables 4 and 5.

[0121] Table 4 GC temperature program conditions 1

[0122]

[0123] Table 5 GC program temperature conditions 2

[0124]

[0125] 2.1.2 Screening of methyl esterification methods for Litsea cubeba oil

[0126] Methods for methyl esterification of fatty acids include acid treatment, alkali treatment, boron trifluoride (BF3), simplified alkaline methyl esterification, diazomethane, and tetramethylammonium hydroxide. This study investigated the effects of acid treatment, alkali treatment, and combined acid-base methyl esterification on the fatty acid content of Litsea cubeba oil and determined a method for methyl esterification of Litsea cubeba oil.

[0127] 2.1.2.1 Acid treatment method

[0128] The acid treatment method generally uses a sulfuric acid (H2SO4)-MeOH solution for methyl esterification. Accurately weigh 0.1g of oil sample into a 10.0mL stoppered test tube and add 2mL (0.5mol / L) H2SO4-MeOH solution as a catalyst. Place in a water bath shaker at 40°C for 15 minutes, then remove and allow to stand at room temperature for 15 minutes. Add 2mL of n-hexane and allow to stand at room temperature for 10 minutes. Aspirate 1.0mL of the clear supernatant and filter through an organic filter (0.22μm) before testing.

[0129] 2.1.2.2 Alkali treatment method

[0130] Weigh 0.1 g of oil sample, add 2 mL of 1% KOH-MeOH into a 10.0 mL stoppered test tube, place in a water bath shaker and heat at 70°C for 15 min, let stand at room temperature for 15 min, add 2 mL of n-hexane, let stand at room temperature for 10 min, aspirate 1.0 mL of the upper clear liquid, filter through an organic filter membrane (0.22 μm) and then test.

[0131] 2.1.2.3 Acid-base combination method

[0132] Weigh 0.1 g of Litsea cubeba oil, add 4 mL of 0.5 mol / L KOH-MeOH solution, saponify in a 60°C water bath for 15 min, remove and cool; add 8 mL of 25% hydrochloric acid, place in a 60°C water bath for 15 min, remove and cool; then add 4 mL of n-hexane and shake for 5 min; finally, add 4 mL of saturated sodium chloride solution and let it stand for 10 min. Take the supernatant, evaporate to dryness under reduced pressure, add 2 mL of n-hexane to dissolve it again, and pass it through a microporous filter membrane (0.22 μm) before testing.

[0133] 2.1.3 Methodological Investigation

[0134] 2.1.3.1 Preparation of test solution

[0135] Accurately weigh 0.1 g of Litsea cubeba oil and treat it using the preferred methyl esterification method under "2.1.2" to prepare the test solution.

[0136] 2.1.3.2 Preparation of mixed reference solution

[0137] Accurately pipette 62.9565 mg of methyl decanoate, 63.4750 mg of methyl undecanoate, 62.575 mg of methyl laurate, and 64.4810 mg of methyl tridecanoate as reference substances, accurately weigh them, dissolve them in n-hexane and make up to the volume in a 5 mL volumetric flask to prepare a mixed reference substance stock solution containing 12.5913 mg / mL of methyl decanoate, 12.6950 mg / mL of methyl undecanoate, 12.5150 mg / mL of methyl laurate, and 12.8962 mg / mL of methyl tridecanoate, and place it in a -20 °C refrigerator for use.

[0138] 2.1.3.3 Linear relationship investigation

[0139] Accurately pipette the mixed reference solution, dilute it with n-hexane to different multiples, and then inject it into the sample for measurement in sequence. Record the peak area and draw a standard curve with the concentration of the reference solution as the horizontal axis and the peak area as the vertical axis.

[0140] 2.1.3.5 Precision test

[0141] Accurately aspirate the same mixed reference solution, inject the sample continuously for 6 times, and record the retention time and RSD value of chromatographic peak area of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate.

[0142] 2.1.3.5 Repeatability test

[0143] Take 6 portions of the same methyl esterified test sample, record the retention time and chromatographic peak area of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate after sampling, and calculate the RSD values of the retention times of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate.

[0144] 2.1.3.6 Stability test

[0145] Take the same methyl esterified sample, place it at room temperature, and inject it for determination at 0, 2, 4, 8, 12, and 24 hours, respectively. Calculate the RSD values of the retention time and peak area of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate.

[0146] 2.1.3.7 Sample recovery rate investigation

[0147] Take the methyl esterified sample and add the reference solution containing methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate in a ratio of approximately 1:1 of the content of each component in the test sample. Prepare 6 parallel portions and detect according to the chromatographic conditions. Calculate the average sample recovery rate.

[0148] 2.2 Optimization of SFE-CO2 extraction process of Litsea cubeba oil by response surface methodology combined with entropy weight method

[0149] 2.2.1 Sample preparation

[0150] Same as under “2.1.3.1”.

[0151] 2.2.2 Single factor experiment on SFE-CO2 extraction of Litsea cubeba oil

[0152] Litsea cubeba → manual impurity removal → crushing and screening → loading into extraction kettle → introducing CO2 → adjusting parameters → increasing temperature and pressure (volatilizing CO2) → Litsea cubeba oil.

[0153] 50 g of Litsea cubeba crushed to 20 mesh size was weighed using an electronic balance and loaded into a SFE-CO2 extraction kettle. CO2 was introduced and the operating table parameters were set. The extraction temperature, extraction pressure, extraction time and CO2 flow rate were used as variables to perform SFE-CO2 extraction to extract Litsea cubeba oil. The Litsea cubeba oil extraction rate was calculated according to the following formula:

[0154]

[0155] The extraction rate of Litsea cubeba oil and the comprehensive score of the contents of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate were used as evaluation indicators, and the extraction temperature, extraction pressure, extraction time and CO2 flow rate were used as evaluation factors to optimize the SFE-CO2 working parameters. The optimization experiments were carried out in sequence based on the particle size of 20 mesh, extraction pressure of 25 MPa, extraction temperature of 35℃, extraction time of 40 min, CO2 flow rate of 6 L / h and separation kettle II pressure of 6 MPa.

[0156] 2.2.3 Calculation of comprehensive score using entropy weight method

[0157] The entropy weight method is an objective weighting method. In its specific application, the entropy weight method uses information entropy to calculate the entropy weight of each indicator based on the degree of variation of each indicator. The entropy weight is then used to modify the weight of each indicator, thereby obtaining a more objective indicator weight. Therefore, the entropy weight method is used to calculate the comprehensive score of the yield of Litsea cubeba oil and the content of the four components in the oil. The entropy weight method is calculated using the following steps:

[0158] 1) Data standardization

[0159]

[0160] Among them, formula 1 represents the normalized calculation process of positive indicators, while formula 2 is the normalized calculation process of negative indicators. j ) is the minimum value of the data in the jth column; max(x j ) is the maximum value of the data in column j. The normalized result is shifted by 0.00001 to ensure smooth operation of subsequent operations.

[0161] 2) Calculate the specific gravity p ij value

[0162]

[0163] Here, n is the number of experiments, i = 1, 2, 3, ···, n.

[0164] 3) Calculate the information entropy e of each indicator j

[0165]

[0166] Among them, 0≦e j ≦1.

[0167] 4) Calculate the differential coefficient g j

[0168] g j =1-e j (5).

[0169] 5) Determination of the weight of each indicator

[0170]

[0171] Among them, m is the evaluation index, j = 1, 2, 3, ···, m.

[0172] 6) Calculation of comprehensive evaluation indicators

[0173]

[0174] 2.2.3 Comprehensive score calculation

[0175] The entropy weight method was used to quantify the content of each component as a weight indicator. The weights of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate were calculated using this method. The overall score for each test number was calculated as follows: methyl decanoate × methyl decanoate weight + methyl undecanoate × methyl undecanoate weight + methyl laurate × methyl laurate weight + methyl tridecanoate × methyl tridecanoate weight + yield × yield weight.

[0176] 2.2.3.1 Effect of extraction temperature on comprehensive score

[0177] The powder of Litsea cubeba medicinal material was weighed, and the extraction temperature was used as the only variable under the conditions of CO2 flow rate 6L / h, extraction time 40min, and extraction pressure controlled at 25MPa. The effects of extraction temperature on the comprehensive score at 15, 25, 35, 45, and 55℃ were investigated.

[0178] 2.2.3.2 Effect of extraction pressure on comprehensive score

[0179] Weigh the powder of Litsea cubeba medicinal material. Under the conditions of extraction temperature, extraction time of 40 min and CO2 flow rate of 6 L / h under “2.2.3.1”, take extraction pressure as the only variable and investigate the effect of pressure of 15, 20, 25, 30 and 35 MPa on the comprehensive score.

[0180] 2.2.3.3 Effect of extraction time on comprehensive score

[0181] Weigh the powder of Litsea cubeba medicinal material. Under the conditions of extraction temperature under "2.2.3.1" and extraction pressure under "2.2.3.2", and CO2 flow rate controlled at 6 L / h, with extraction time as the only variable, investigate the effect of extraction time of 20, 30, 40, 60, and 80 min on the comprehensive score.

[0182] 2.2.3.4 Impact of CO2 flow rate on comprehensive score

[0183] Weigh the powder of Litsea cubeba medicinal material, and examine the effects of CO2 flow rate of 6, 8, 10, 12, 14 and 16 L / h on the comprehensive score under the extraction temperature under "2.2.3.1", the extraction pressure under "2.2.3.2", and the extraction time under "2.2.3.3". Use CO2 flow rate as the only variable.

[0184] 2.2.4 Response surface design for SFE-CO2 extraction of Litsea cubeba oil

[0185] The Box-Behnken response surface experimental design was used to screen the optimal combination of factors.

[0186] Based on a single-factor experiment, extraction temperature (A), extraction pressure (B), extraction time (C), and CO2 flow rate (D) were selected as response variables. The contents of four fatty acids (methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate) and the extraction yield of Litsea cubeba oil were used as evaluation indicators, with the overall score as the response value. A four-factor, three-level response surface analysis experiment was designed using the Box-Behnken principle. The design factors and level codes for the response surface experiment are shown in Table 6.

[0187] Table 6 Response surface factor level table for extracting Litsea cubeba oil

[0188]

[0189] 2.2.5 Determination of content of 11 batches of Litsea cubeba oil samples

[0190] Take each batch of Litsea cubeba medicinal materials and extract them using SFE-CO2 technology to obtain Litsea cubeba oil. Prepare the test sample according to "2.1.2" and determine it according to the chromatographic conditions under "2.1.1". Record the peak area and calculate its content.

[0191] 2.2.6 Data processing and analysis

[0192] The experimental results were recorded and statistically analyzed using EXCEL tables. Each experiment was repeated three times. Prism 8.0 software was used to draw single-factor diagrams, and Design Expert 11 software was used to perform response surface analysis.

[0193] 3 Results and Analysis

[0194] 3.1 Screening of GC conditions

[0195] 3.1.1 Screening of programmed temperature conditions

[0196] The results of the initial temperature program conditions (i.e. GC temperature program conditions 1) are as follows Figure 1 A. The poor peak separation may be due to the mismatch between the heating rate and the polarity of the target or the unreasonable setting of the critical temperature range. Therefore, the heating program was optimized, that is, the GC heating program condition 2: the initial temperature is 60℃ and maintained for 1 minute, then increased to 160℃ at 15℃ / min, then increased to 170℃ at 0.5℃ / min, and finally increased to 230℃ at 3℃ / min and maintained for 30 minutes. The results are as follows Figure 1 B. After optimization, the separation of each component was significantly improved and the analysis time was shortened. To confirm the peak order of the target, n-hexane solutions of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate were prepared and analyzed. The results showed that the components peaked in the order of increasing carbon chain length, as shown in Figure 2. Figure 1 As shown in CF.

[0197] 3.1.2 Results of screening methods for methyl esterification of Litsea cubeba oil

[0198] Depend on Figure 2 GC analysis of Litsea cubeba oil treated with the three methyl esterification methods (acid, alkaline, and combined acid-base) revealed that the combined acid-base method produced significantly higher total peak areas than either the single acid or base method, and also detected a greater number of components. This result demonstrates the comprehensive methyl esterification capabilities of the combined acid-base method, and therefore has been established as the standard pretreatment method for subsequent fatty acid composition analysis.

[0199] 3.1.3 Methodological Investigation Results

[0200] 3.1.3.1 Establishment of standard curve

[0201] The mixed reference substance was diluted into 5 groups of standard solutions with a concentration gradient from ① to ⑤. The mass concentrations of the components to be tested in the mixed reference solution were as follows from high to low: methyl decanoate: ①12.5913, ②6.2957, ③3.1479, ④1.5739, ⑤0.787 mg / mL; methyl undecanoate: ①12.695, ②6.3475, ③3.1738, ④1.5869, ⑤0.7934 mg / mL; methyl laurate: ①12.515, ②6.2575, ③3.1288, ④1.5644, ⑤0.7821 mg / mL; methyl tridecanoate: ①12.8962, ②6.4481, ③3.2241, ④1.612, ⑤0.806 mg / mL. The chromatographic results are shown in Table 1. Figure 3Standard curves were plotted using the concentration of each standard solution as the horizontal axis and the peak area as the vertical axis. As shown in Table 7, each component exhibited a good linear relationship with the peak area over the concentration range. The limit of quantification (LOQ) was calculated using a signal-to-noise ratio of 10:1, and the limit of detection (LOD) was calculated using a signal-to-noise ratio of 3:1.

[0202] Table 7 Linear relationship determination results

[0203]

[0204] 3.1.3.2 Precision test

[0205] As shown in Tables 8 and 9, the calculated RSDs of the retention times of the common peaks were 0.12%, 0.14%, 0.11%, and 0.07%, respectively, and the RSDs of the peak areas were 1.23%, 1.51%, 1.48%, and 2.00%, respectively, indicating good instrument precision.

[0206] Table 8 Precision retention time determination results

[0207]

[0208]

[0209] Table 9 Precision peak area determination results

[0210]

[0211] 3.1.3.3 Repeatability test

[0212] The results are shown in Tables 10 and 11. The RSD values of the retention times of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate were 0.09%, 0.19%, 0.05%, and 0.08%, respectively, and the RSD values of the peak areas were 2.78%, 2.50%, 2.55%, and 2.59%, respectively, indicating that the method has good reproducibility.

[0213] Table 10 Repeatability retention time measurement results

[0214]

[0215] Table 11 Repeatability peak area measurement results

[0216]

[0217]

[0218] 3.1.3.4 Stability test

[0219] As shown in Tables 12 and 13, the calculated RSDs of the retention times of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate were 0.16%, 0.40%, 0.10%, and 0.12%, respectively, and the RSDs of the peak areas were 2.51%, 2.67%, 1.76%, and 2.67%, respectively, indicating that the test solution had good stability within 24 h.

[0220] Table 12 Stability retention time determination results

[0221]

[0222] Table 13 Stability peak area determination results

[0223]

[0224] 3.1.3.5 Investigation of sample recovery

[0225] The results are shown in Table 14. The results showed that the average recoveries of methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate were 100.17%, 99.14%, 98.67%, and 97.65%, respectively, with RSDs of 2.14%, 2.30%, 1.33%, and 2.86%, respectively.

[0226] Table 14 Recovery test results of four components in Litsea cubeba oil (n=6)

[0227]

[0228]

[0229] 3.1.3.6 Assay of 11 batches of Litsea cubeba oil samples

[0230] The results of content determination of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate in 11 batches of Litsea cubeba medicinal materials are shown in Table 15. The content of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate in production area C1 (Luo* Town, Luo* County) is the highest.

[0231] Table 15 Contents of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate in Litsea cubeba from different origins

[0232]

[0233] 3.1.4 Summary

[0234] In this experiment, an optimized gas chromatography analysis method was established. Using n-hexane as the solvent, the chromatographic conditions were set as follows: inlet temperature 200°C, detector temperature 230°C, split ratio 20:1, column flow rate 1mL / min, and injection volume 5μL. The optimized program temperature conditions were: initial temperature 60°C maintained for 1 min, then increased to 160°C at 15°C / min, then increased to 170°C at 0.5°C / min, and finally increased to 230°C at 3°C / min and maintained for 30 min. This method achieved good separation of the target components methyl decanoate, methyl undecanoate, methyl laurate, and methyl tridecanoate. The linear regression equation for methyl decanoate is y=42.929x+24.678, R 2 =0.9995, and a good linear relationship was found in the range of 0.7870 to 12.5913 mg / mL; the linear regression equation of methyl undecanoate was y=46.541x+4.8953, R 2 =0.9994, and a good linear relationship was found in the range of 0.7934 to 12.6950 mg / mL; the linear regression equation of methyl laurate was y=45.775x+8.1006, R 2 =0.9993, and a good linear relationship was found in the range of 0.7821 to 12.515 mg / mL; the linear regression equation of methyl tridecanoate was y=48.724x+3.3843, R 2 =0.9995, and a good linear relationship was found in the range of 0.8060-12.8962 mg / mL (R 2 ≥0.9993). Analysis and comparison of 11 batches of Litsea cubeba oil samples revealed that the content of methyl esterification products in the herbal material produced in Luoqun Town, Luodian County, Guizhou Province was significantly higher than that in other batches, providing a high-quality raw material foundation for subsequent process optimization.

[0235] 3.2 Optimization of SFE-CO2 extraction process of Litsea cubeba oil by response surface methodology combined with entropy weight method

[0236] 3.2.1 Results of single-factor test on the extraction of Litsea cubeba oil by SFE-CO2

[0237] 3.2.1.1 Effect of extraction temperature on the extraction rate and content of Litsea cubeba oil

[0238] The effects of extraction temperature on the content and yield of four fatty acids are shown in Table 16. Figure 4As shown in the figure, the comprehensive score reaches its maximum at 35°C, likely due to the enhanced molecular diffusion and extraction efficiency achieved by moderately elevated temperatures. However, above 35°C, the density of the supercritical fluid decreases, weakening its solubility and subsequently decreasing the extraction efficiency of the target component. Based on this, 35°C was identified as the central temperature for the response surface optimization design in this study.

[0239] Table 16 Effect of extraction temperature on the content of four fatty acids in Litsea cubeba oil (mg / g crude drug)

[0240]

[0241] 3.2.1.2 Effect of extraction pressure on comprehensive score

[0242] The effects of extraction pressure on the content and yield of four fatty acids are shown in Table 17. Figure 5 As shown, it shows a trend of first increasing and then decreasing. Under constant temperature conditions, as the pressure increases, the density of the supercritical fluid increases, the solvent solubility is enhanced, and the comprehensive score increases accordingly and reaches a peak at 25MPa. However, too high a pressure (>25MPa) may cause the material to be compacted, hindering the dissolution of the effective ingredients, resulting in a decrease in the score. When the pressure exceeds 30MPa, the yield does not increase, but the component content increases. Analysis shows that this is because the excessive pressure causes local cell wall breakage and component outflow. In this study, 25MPa was selected as the pressure optimization center point.

[0243] Table 17 Effect of extraction pressure on the content of four fatty acids in Litsea cubeba oil (mg / g crude drug)

[0244]

[0245] 3.2.1.3 Effect of extraction time on comprehensive score

[0246] The effects of extraction time on the content and yield of the four fatty acids are shown in Table 18. Figure 6 As shown in the figure, under fixed temperature and pressure conditions, the effect of extraction time on the overall score shows an initial upward and then downward trend. As time increases, the material comes into more complete contact with CO2, and the score continues to rise, reaching a peak at 40 minutes. After reaching the peak, the score decreases, likely due to the near-complete extraction of the target component, while some volatile components are lost in the continued flow of CO2. Based on this, 40 minutes was determined as the optimal extraction time.

[0247] Table 18 Effect of extraction time on the content of four fatty acids in Litsea cubeba oil (mg / g crude drug)

[0248]

[0249] 3.2.1.4 Impact of CO2 flow rate on comprehensive score

[0250] The effects of CO2 flow rate on the content and yield of four fatty acids are shown in Table 19. The effects of flow rate on the comprehensive score are shown in Table 19. Figure 7 As shown, the impact of flow rate on the comprehensive score shows an initial upward and then downward trend. As the CO2 flow rate increases, the contact efficiency between the fluid and the material improves, promoting an increase in the extraction rate. The comprehensive score rises accordingly, reaching a peak at 12 L / h. However, when the flow rate exceeds this threshold, the excessively high flow rate results in insufficient CO2 residence time in the extraction kettle, reducing the solvent's efficiency in dissolving the target components and causing a decrease in the score. Based on this, this study identified 12 L / h as the optimal CO2 flow rate.

[0251] Table 19 Effect of extraction flow rate on the content of four fatty acids in Litsea cubeba oil (mg / g crude drug)

[0252]

[0253] 3.2.2 Response surface analysis of SFE-CO2 extraction of Litsea cubeba oil

[0254] 3.2.2.1 Response surface experiment results

[0255] Twenty-nine response surface experiments were designed using the software Design-Expert 11, with three replicates for each experiment. The entropy weight method was used to calculate the weights for the yields of methyl decanoate, methyl undecanoate, methyl laurate, methyl tridecanoate, and Litsea cubeba oil, respectively: 0.2465, 0.1483, 0.1583, 0.1379, and 0.3090. The overall score was calculated as follows: methyl decanoate × 0.2465 + methyl undecanoate × 0.1483 + methyl laurate × 0.1583 + methyl tridecanoate × 0.1379 + yield × 0.3090. The overall score for each experiment was calculated by taking the average of the extraction yield and the four fatty acid contents. The experimental design and results are shown in Table 20.

[0256] Table 20 Box-Behnken test design and results

[0257]

[0258]

[0259] 3.2.2.2 Variance results and analysis

[0260] Design-Expert 11 was used to perform variance analysis and quadratic multiple regression fitting on the experimental data. The variance analysis results are shown in Table 21. The significance test probability of the model is P = 0.0163 < 0.05, which means that the model is statistically significant. The lack-of-fit term P = 0.1194 > 0.05, indicating that the lack-of-fit term is not significant, that is, the model fits the experimental data well and can be used to analyze the relationship between each factor and the response value. Corrected determination coefficient R 2 Adj The F value can be used to test the influence of each variable on the response value. The F value shows that the order of the influence of the extraction process conditions on the comprehensive score is CO2 flow rate (D) > extraction time (C) > extraction pressure (B) > extraction temperature (A). The linear term DD, the interaction term BD and the quadratic term A 2 、C 2 The comprehensive score was significantly affected, and the final multivariate quadratic regression equation was Y = 0.6502-0.0536*A+0.1406*B+0.1615*C+0.2631*D-0.0738*AB+0.0408*AC-0.0975*AD+0.0510*BC+0.1549*BD-0.0870*CD-0.2298*A 2 -0.0784*B 2 -0.2241*C 2 -0.1148*D 2 .

[0261] Table 21 Analysis of variance

[0262]

[0263]

[0264] Note: * indicates P<0.05, significant difference.

[0265] 3.2.2.3 Response surface analysis

[0266] Design-Expert 11 software was used to draw the response surface diagram and contour diagram of the interaction effect between factors on the comprehensive score. Figure 8-13 The steeper the response surface slope, the greater the impact of the experimental factor on the response value; the more the contour lines tend toward an ellipse, the stronger the interaction between the factors, while the more they tend toward a circle, the weaker the interaction. The figure shows that the response surfaces for flow and pressure are slightly steeper, while the interactions between the other factors are relatively flat, which is generally consistent with the results of the variance analysis of the regression model.

[0267] 3.2.2.4 Process Validation of Extraction Process

[0268] The experimental results are shown in Table 22. The optimal extraction process conditions fitted by the software were: temperature 33.9344°C, pressure 21.0487 MPa, time 41.1534 min, and flow rate 12.0317 L / h. The predicted comprehensive score was 0.7611. Based on actual conditions, the results were revised to: temperature 34°C, pressure 21 MPa, time 41 min, and flow rate 12 L / h. A verification experiment was conducted, and the actual comprehensive score was 0.7541, which is close to the predicted comprehensive score, indicating that the process is stable and feasible.

[0269] Table 22 Process verification test results

[0270]

[0271] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A process for extracting Litsea cubeba oil, characterized in that: The extraction process comprises the following steps: using an electronic balance to weigh Litsea cubeba crushed into 20 mesh, and loading the crushed Litsea cubeba into an SFE-CO2 extraction kettle; introducing CO2, setting the operating table parameters as temperature 15-55°C, pressure 15-35 MPa, time 20-80 min, and CO2 flow rate 6-16 L / h, and performing SFE-CO2 extraction.

2. The extraction process of Litsea cubeba oil according to claim 1, wherein: The extraction process comprises the following steps: using an electronic balance to weigh Litsea cubeba crushed into 20 mesh, and loading the crushed Litsea cubeba into an SFE-CO2 extraction kettle; introducing CO2, setting the operating table parameters as temperature 25-45° C., pressure 20-30 MPa, time 40-80 min, and CO2 flow rate 10-14 L / h, and performing SFE-CO2 extraction.

3. The extraction process of Litsea cubeba oil according to claim 2, characterized in that: The extraction process is as follows: using an electronic balance to weigh the Litsea cubeba crushed into 20 mesh, and loading it into an SFE-CO2 extraction kettle; introducing CO2, setting the operating table parameters as temperature 34°C, pressure 21 MPa, time 41 min, and CO2 flow rate 12 L / h to perform SFE-CO2 extraction.

4. A method for optimizing the extraction process of Litsea cubeba oil according to any one of claims 1 to 3, characterized in that: The optimization method comprises the following steps: Step 1: Weigh the Litsea cubeba fruit crushed into 20 mesh, put it into the SFE-CO2 extraction kettle, introduce CO2, set the operating table parameters as temperature, pressure, time and CO2 flow rate, and perform SFE-CO2 extraction; Step 2: Using the contents of methyl caprate, methyl undecanoate, methyl laurate, and methyl tridecanoate in Litsea cubeba oil and the comprehensive score of Litsea cubeba oil yield as evaluation indicators, a single factor investigation was conducted on the extraction temperature, extraction pressure, extraction time, and CO2 flow rate of Litsea cubeba oil; Step 3: Based on the results obtained in step 2, a four-factor three-level response surface analysis experiment was designed using the Box-Behnken principle; Step 4: Calculate the weight of each indicator by the entropy weight method to obtain the comprehensive score of the yield of Litsea cubeba oil and the content of the four components in the oil, and calculate the comprehensive score of different test numbers; Step 5: Use Design-Expert 11 to perform variance analysis and quadratic multiple regression fitting on the experimental data to obtain the model equation between extraction temperature, extraction pressure, extraction time, CO2 and comprehensive score; Step 6: Obtain the optimal extraction conditions of the SFE-CO2 extraction process of Litsea cubeba oil based on the model equation.

5. The method for optimizing the extraction process of Litsea cubeba oil according to claim 4, characterized in that: The comprehensive score in step 4 is calculated as follows: comprehensive score = methyl decanoate × 0.2465 + methyl undecanoate × 0.1483 + methyl laurate × 0.1583 + methyl tridecanoate × 0.1379 + yield × 0.3090.

6. The method for optimizing the extraction process of Litsea cubeba oil according to claim 4, characterized in that: The model equation in step 5 is Y=0.6502-0.0536*A+0.1406*B+0.1615*C+0.2631*D-0.0738*AB+0.0408*AC-0.0975*AD+0.0510*BC+0.1549*BD-0.0870*CD-0.2298*A 2 -0.0784*B 2 -0.2241*C 2 -0.1148*D 2 , where Y is the comprehensive score, A is the extraction temperature, B is the extraction pressure, C is the extraction time, and D is the CO2 flow rate.

7. The method for optimizing the extraction process of Litsea cubeba oil according to claim 4, characterized in that: The optimal extraction conditions in step 6 are: temperature 34°C, pressure 21 MPa, time 41 min, and CO2 flow rate 12 L / h.

8. A content determination method used in the method for optimizing the extraction process of Litsea cubeba oil according to any one of claims 4 to 6, characterized in that: The content determination method is as follows: (1) Preparation of test solution: Accurately weigh 0.1g of Litsea cubeba oil, add 4mL of 0.5mol / L KOH-MeOH solution, saponify in a 60℃ water bath for 15min, remove and cool; add 8mL of 25% hydrochloric acid, place in a 60℃ water bath for 15min, remove and cool; then add 4mL of n-hexane and shake for 5min; finally, add 4mL of saturated sodium chloride solution and let it stand for 10min. Take the supernatant, evaporate to dryness under reduced pressure, add 2mL of n-hexane to redissolve, filter through a 0.22μm microporous membrane, and then test; (2) Preparation of mixed reference solution: Accurately pipette 62.9565 mg of methyl decanoate, 63.4750 mg of methyl undecanoate, 62.575 mg of methyl laurate, and 64.4810 mg of methyl tridecanoate, and accurately weigh them. Dissolve them in chromatographically pure n-hexane and make up to the volume in a 5 mL volumetric flask to prepare a mixed reference substance stock solution containing 12.5913 mg / mL of methyl decanoate, 12.6950 mg / mL of methyl undecanoate, 12.5150 mg / mL of methyl laurate, and 12.8962 mg / mL. Place in a -20°C refrigerator until use. (3) Gas chromatography conditions: The injection port temperature was 200°C; the detector temperature was 230°C; the split ratio was 20:1; the column flow rate was 1 mL / min; the injection volume was 5 μL; the heating program was as follows: initial temperature at 40°C, hold for 1 min, 40°C to 200°C, heating at 5°C per minute, hold for 2 min; or initial temperature at 60°C, hold for 1 min, 60°C to 160°C, heating at 15°C per minute, hold for 0 min, 160°C to 170°C, heating at 0.5°C per minute, hold for 0 min, 170°C to 230°C, heating at 3°C per minute, hold for 30 min. (4) Determination: The solutions of steps (1) and (2) are sampled and measured according to the gas chromatography conditions of step (3), and the peak areas are recorded and the contents of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate are calculated.

9. The content determination method used in the method for optimizing the extraction process of Litsea cubeba oil according to claim 8, characterized in that: The content determination method is as follows: (1) Preparation of test solution: Accurately weigh 0.1g of Litsea cubeba oil, add 4mL of 0.5mol / L KOH-MeOH solution, saponify in a 60℃ water bath for 15min, remove and cool; add 8mL of 25% hydrochloric acid, place in a 60℃ water bath for 15min, remove and cool; then add 4mL of n-hexane and shake for 5min; finally, add 4mL of saturated sodium chloride solution and let it stand for 10min. Take the supernatant, evaporate to dryness under reduced pressure, add 2mL of n-hexane to redissolve, filter through a 0.22μm microporous membrane, and then test; (2) Preparation of mixed reference solution: Accurately pipette 62.9565 mg of methyl decanoate, 63.4750 mg of methyl undecanoate, 62.575 mg of methyl laurate, and 64.4810 mg of methyl tridecanoate, and accurately weigh them. Dissolve them in chromatographically pure n-hexane and make up to the volume in a 5 mL volumetric flask to prepare a mixed reference substance stock solution containing 12.5913 mg / mL of methyl decanoate, 12.6950 mg / mL of methyl undecanoate, 12.5150 mg / mL of methyl laurate, and 12.8962 mg / mL. Place in a -20°C refrigerator until use. (3) Gas chromatography conditions: The injection port temperature was 200°C; the detector temperature was 230°C; the split ratio was 20:1; the column flow rate was 1 mL / min; the injection volume was 5 μL; the heating program was as follows: initial temperature 60°C, hold for 1 min, 60°C to 160°C, heating at 15°C / min, hold for 0 min, 160°C to 170°C, heating at 0.5°C / min, hold for 0 min, 170°C to 230°C, heating at 3°C / min, hold for 30 min; (4) Determination: The solutions of steps (1) and (2) are sampled and measured according to the gas chromatography conditions of step (3), and the peak areas are recorded and the contents of methyl decanoate, methyl undecanoate, methyl laurate and methyl tridecanoate are calculated.

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