Method for improving quality of Ajowan fruit essential oil

Abstract

The invention discloses a method for improving the quality of Ajowan fruit essential oil, which comprises the following steps of: obtaining the Ajowan fruit essential oil containing thymol, p-cymene and gamma-terpinene by adopting a steam distillation-water extraction method, a steam distillation-water separation extraction method, a supercritical CO2 extraction method, a solvent extraction method, a Soxhlet extraction method and an ultrasonic-assisted extraction method; a GC-FID (Gas Chromatography-Flame Ionization Detector) technology and a high-performance thin-layer chromatography system are adopted to carry out component analysis on the obtained essential oil, and the essential oil yield and three active components and main components of thymol, p-cymene and gamma-terpinene are taken as comprehensive score indexes. The quality of the essential oil and the extraction effect of active ingredients are improved.

Description

Technical Field

[0001] The invention relates to a method for improving the quality of asocarp essential oil. Background Art

[0002] The Uyghur medicine Ayurveda fruit is the dried mature fruit of the plant Ayurveda (Trachyspermumammi (L.) Scpague) of the genus Trachyspermum of the family Umbelliferae. It is a plant that is unique to Xinjiang, my country and can be used as both medicine and food. According to the medical literature in the Uyghur Medicine Records and the Xinjiang Chinese Herbal Medicine Handbook, Ayurveda fruit has the effects of treating dryness, coldness and dampness, dispersing cold air, eliminating food stagnation, expelling stones and treating skin diseases. It is often used for cold paralysis, weak tendons and veins, stomach pain due to cold, frequent hiccups, nausea and loss of appetite, difficulty urinating, stones in the body, itchy skin, vitiligo and eczema.

[0003] The main characteristic components and active ingredients of Trachyspermumammi (L.) Sprague essential oil are terpenoid compounds thymol, p-cymene and γ-terpinene. The total content of these three components in the essential oil is as high as 94%, and they all have significant biological activity. In particular, thymol has a wide spectrum of biological activities, such as antibacterial, antioxidant, anti-inflammatory and immune regulation activities, and is highly safe. It is often used in making spices, drugs and indicators.

[0004] In recent years, there have been reports on the extraction technology of essential oil of Ayurveda fruit, such as Alimijiang Aobulitalipu using steam distillation to extract its volatile oil, with an extraction rate of 2.12% (w / w); Tohati Aji Maimaiti Aili used microwave extraction and Soxhlet extraction to extract essential oil of Ayurveda fruit; in addition, foreign studies reported supercritical CO2 extraction and solvent extraction. However, after applying these methods to the extraction of essential oil of Ayurveda fruit, it was found that there were problems such as low extraction rate, low purity of essential oil, large proportion of pigments and fatty substances, and waste of resources. Therefore, the existing technology for extracting essential oil of Ayurveda fruit needs to be improved urgently, so as to achieve the requirements of high extraction rate of essential oil, good quality, high content of effective ingredients and easy crystallization, laying a foundation for the future purification of natural active ingredient thymol. Summary of the invention

[0005] The object of the present invention is to provide a method for improving the quality of euphorbia pulegosa essential oil, the method adopts steam distillation-co-water extraction method, steam distillation-water-insulated extraction method, supercritical CO2 extraction method, solvent extraction method, Soxhlet extraction method or ultrasound-assisted extraction method, namely, obtaining euphorbia pulegosa essential oil containing thymol, p-cymene and γ-terpinene, and then, using GC-FID instrument and high performance thin layer chromatography system to analyze the components of the obtained essential oil, and taking the essential oil yield and three active components and main components thymol, p-cymene and γ-terpinene as comprehensive score indicators. The quality of the essential oil and the effect of extracting the active components thereof are improved.

[0006] The method for improving the quality of the essential oil of Ashoka fruit of the present invention adopts steam distillation-co-water extraction method, steam distillation-water-insulated extraction method, supercritical CO2 extraction method, solvent extraction method, Soxhlet extraction method or ultrasound-assisted extraction method for extraction, and the specific operation is carried out according to the following steps:

[0007] Steam distillation-water extraction method:

[0008] a. After the Asoko fruit is crushed by a grinder, it is passed through a 40-mesh sieve and placed in a 2000 mL hard round-bottom flask at a liquid-to-solid ratio of 14 mL / g. The flask is soaked for 49 min and heated with an electric heating mantle. The timing starts after boiling and the water is adjusted to a slightly boiling state and maintained for 6 h.

[0009] b. Transfer the oil and water in the oil-water separator to a test tube with a piston, and quickly seal it with a sealing film. After standing for 10 hours, separate the oil and water, and weigh it to obtain the ayurvedic essential oil containing thymol, p-cymene and γ-terpinene;

[0010] Steam distillation-water extraction method:

[0011] a. Add purified water to the bottom of the extraction tank, put a layer net, spread pure cotton gauze, sprinkle 100.0g of Ashoka fruit powder, install the tank cover, connect the oil-water separator and condenser, and then turn on the SHT high-power precision digital display electric heating jacket to heat it. After boiling, immediately reduce the heating and keep it in a slightly boiling state for 6 hours;

[0012] b. Transfer the oil and water in the oil-water separator to a test tube with a piston, and quickly seal it with a sealing film. After standing for 10 hours, separate the oil and water, and weigh it to obtain the ayurvedic essential oil containing thymol, p-cymene and γ-terpinene;

[0013] Supercritical CO2 extraction method:

[0014] Accurately weigh 26.0 g of Asobosu fruit powder and put it into the sample kettle, then weigh the weight of the sample collection bottle and record the data, then connect it to the sample outlet, finally turn on the instrument and set the critical pressure to 196 MPa, the critical temperature to 50 ° C, the collection valve temperature to 100 ° C, the extraction time to 2 hours, after the extraction is completed, remove the sample collection bottle and weigh it, so as to obtain Asobosu fruit essential oil containing thymol, p-cymene and γ-terpinene;

[0015] Solvent extraction method:

[0016] Accurately weigh 10.0 g of Asobosu fruit powder and put it into a 250 mL conical flask with a piston, add 100 mL of n-hexane solvent and mix well, soak and extract for 24 hours and then filter, concentrate the filtrate by rotary evaporator to obtain an oily extract, weigh the mass of 5 mL of the empty bottle and record the data, then transfer the essential oil in the round-bottom flask to the weighed bottle, finally weigh the weight of the bottle and the essential oil, and obtain Asobosu fruit essential oil containing thymol, p-cymene and γ-terpinene;

[0017] Soxhlet extraction method:

[0018] Accurately weigh 12.0 g of Asobosu fruit powder and wrap it with filter paper, put the Asobosu fruit powder wrapped with filter paper into an extractor, put in an electric heating mantle, add 120 mL of n-hexane solvent, connect a condenser, start heating, and start timing when the solvent liquid surface is observed to boil. After 5 hours of extraction time, filter with filter paper, and concentrate the filtrate with a rotary evaporator to obtain an oily extract containing thymol, p-cymene and γ-terpinene. Asobosu fruit essential oil;

[0019] Ultrasonic assisted extraction method:

[0020] Accurately weigh 10.0 g of Ashoka fruit powder and put it into a 250 mL conical flask with a piston, add 100 mL of n-hexane solvent, and then put it into an ultrasonic instrument with an operating frequency of 53 KHz, a power of 100%, and a set temperature of 40°C. After ultrasonication for 20 minutes, let it cool and filter with filter paper. The filtrate is concentrated by rotary evaporator to obtain an oily extract of Ashoka fruit essential oil containing thymol, p-cymene and γ-terpinene.

[0021] The method for improving the quality of the essential oil of Ashoka fruit of the present invention, the Ashoka fruit essential oil containing thymol, p-cymene and γ-terpinene obtained by the method, and the yield thereof are calculated, and the calculation formula of the essential oil yield (Yield%) is:

[0022]

[0023] Wherein, M1 is the mass of essential oil, and M2 is the mass of Ayurveda fruit after pretreatment.

[0024] The method for improving the quality of the essential oil of Ayurveda fruit is to obtain the essential oil of Ayurveda fruit containing thymol, p-cymene and γ-terpinene by the method of the present invention, and to evaluate its active ingredients: the essential oil is subjected to GC-FID main component and active ingredient thymol, p-cymene and γ-terpinene and thin layer chromatography system HPTLC fingerprint and 3D chromatogram purity analysis, and the capillary used for GC-FID analysis is HP-INNOWAX capillary column (60m×0.25mm, 0.2 5μm), carrier gas was high-purity N2, flow rate was 1.3mL / min, average linear velocity was 25.432cm / sec, injection volume was 1.0μL, split ratio was 30:1 and injection port temperature was 230℃; program heating: temperature was 50℃ for 3min, then increased to 70℃ for 3min at 2℃ / min, and finally increased to 200℃ for 8min at 5℃ / min; sample treatment: 10μL essential oil was added to 1mL solvent, and the essential oil was subjected to thin layer chromatography system (model TLC Scanner4 System) analysis, the silica gel plate size was 10mm×10mm, the sample volume was 2.0μL, the developing solvent was n-hexane:ethyl acetate with a volume ratio of 8:1, and the 254nm and 366nm HPTLC fingerprints and 190nm, 254nm, 300nm and 366nm 3D chromatograms were observed. The silica gel plate containing essential oil components was developed with vanillin developer and then scanned with white light.

[0025] The method for improving the quality of the essential oil of Ashoka fruit of the present invention is to optimize the extraction process of the steam distillation-co-water extraction (HD) extraction method, and design single factor and response surface experiments:

[0026] Step (1) single factor experiment: several groups of single factor experiments were designed with extraction time, liquid-to-solid ratio, soaking time and crushing particle size as four single factors, and the essential oils obtained by extraction were analyzed by GC-FID one by one;

[0027] Step (2) According to the peak area of ​​(1), the standard curves of thymol, p-cymene and γ-terpinene are drawn by external standard method to ensure that the three main components in the essential oil of Ashoka are within the linear range. In addition, the GC-FID analysis methodology is investigated to determine the precision of the instrument, the repeatability of the method, the stability of the essential oil of Ashoka and the recovery rate of spike addition. The results show that the RSD and the recovery rate are within the limit. Therefore, it meets the requirements for establishing the content analysis of the essential oil of Ashoka;

[0028] Based on steps (1) and (2), the comprehensive score formula is established as follows:

[0029]

[0030] Where y is the comprehensive score%, C1 is the concentration of thymol, C2 is the concentration of p-cymene and C3 is the concentration of γ-terpinene;

[0031] Response surface method optimization: Taking the comprehensive score as the response value, according to the Box-Benhnken experimental design principle, a response surface experiment was conducted on the basis of the single factor experiment in step (1), and the multiple regression equation was simulated as follows: y = 157.85193-17.08068A-5.17038B+0.90696C-0.24609D+2.00744AB-0.73060AC

[0032] +0.032974AD+0.16571BC+0.047360BD+0.038953CD-0.32634A 2 -0.29058B 2 -0.31981C 2 -0.00751972D 2

[0033] Where y is the comprehensive score of the response value (%), A is the extraction time (h), B is the liquid-to-solid ratio (mL / g), C is the soaking time (h) and D is the crushing particle size (mesh);

[0034] The regression equation was solved to obtain the optimal extraction process conditions with high comprehensive scores of Ayurveda fruit: extraction time 6.0h, liquid-to-solid ratio 14mL / g, soaking time 0.81h and crushing particle size 42.99 mesh. Based on the constraints of actual operation, the optimal extraction conditions optimized by the modified model were used to verify the Ayurveda fruit essential oil. The extraction conditions were extraction time 6.0h, liquid-to-solid ratio 14mL / g, soaking time 49min and crushing particle size 40.0 mesh;

[0035] Single factor experiment: extraction time, liquid-to-solid ratio, soaking time and crushing particle size were four single factors to design several groups of single factor experiments, and the extracted essential oils were analyzed for the content of active ingredients by GC-FID;

[0036] According to the peak area, the standard curves of thymol, p-cymene and γ-terpinene were drawn by external standard method; in addition, the methodological investigation showed that the precision RSD of the three main components was less than 1.0%, indicating that the instrument was good; the repeatability RSD was ≤0.02%, indicating that the method had good repeatability; the stability RSD was ≤0.01%, indicating that the essential oil had good stability within 24 hours; the spike recovery rate was within the recovery rate limit range of 85%-99% of the analytical method validation, indicating that the analytical results were very close to the theoretical values, and the sample determination obtained accurate data; therefore, it met the requirements for establishing the analysis of the content of essential oil components;

[0037] The comprehensive score formula is:

[0038]

[0039] In the formula, y is the comprehensive score%, C1 is the concentration of thymol, C2 is the concentration of p-cymene, and C3 is the concentration of γ-terpinene;

[0040] Response surface method optimization: Taking the comprehensive score as the response value, according to the response surface Box-Benhnken experimental design principle, a response surface experiment was conducted on the basis of the single factor experiment, and the multiple regression equation obtained by simulation was as follows:

[0041] y=157.85193-17.08068A-5.17038B+0.90696C-0.24609D+2.00744AB-0.73060AC

[0042] +0.032974AD+0.16571BC+0.047360BD+0.038953CD-0.32634A 2 -0.29058B 2 -0.31981C 2 -0.00751972D 2

[0043] Wherein, y is the comprehensive score (%), A is the extraction time (h), B is the liquid-to-solid ratio (mL / g), C is the soaking time (h), and D is the crushing particle size (mesh). BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a flow chart of the extraction method of the present invention;

[0045] Figure 2 The essential oils of Ayurveda fruit obtained by 6 extraction methods of the present invention, including HD: steam distillation-co-hydration essential oil, SD: steam distillation-water separation extraction essential oil, SFE: supercritical CO2 extraction essential oil, LLE: solvent extraction essential oil, SE: Soxhlet extraction essential oil, US: ultrasound-assisted extraction essential oil and GC-FID total ion chromatograms of three main component standards (thymol, p-cymene, γ-terpinene);

[0046] Figure 3 The yields of the essential oil of Ashoka fruit obtained by the six extraction methods of the present invention, the intuitive graph of the comprehensive scores and the peak area values ​​of the three main components;

[0047] Figure 4The essential oil of Ayurveda fruit obtained by 6 extraction methods of the present invention, wherein HD: steam distillation-co-hydration essential oil, SD: steam distillation-water separation extraction essential oil, SFE: supercritical CO2 extraction essential oil, LLE: solvent extraction essential oil, SE: Soxhlet extraction essential oil, US: ultrasound-assisted extraction essential oil and three main component standards (thymol, p-cymene, γ-terpinene) form a 3D chromatogram of 9 points at 190nm;

[0048] Figure 5 The essential oil of Ashoka fruit obtained by 6 extraction methods of the present invention, wherein HD: steam distillation-co-hydration essential oil, SD: steam distillation-water separation extraction essential oil, SFE: supercritical CO2 extraction essential oil, LLE: solvent extraction essential oil, SE: Soxhlet extraction essential oil, US: ultrasound-assisted extraction essential oil and three main component standards (thymol, p-cymene, γ-terpinene) form a 3D chromatogram of 9 points at 254nm;

[0049] Figure 6 The essential oil of Ashoka fruit obtained by 6 extraction methods of the present invention, wherein HD: steam distillation-co-hydration essential oil, SD: steam distillation-water separation extraction essential oil, SFE: supercritical CO2 extraction essential oil, LLE: solvent extraction essential oil, SE: Soxhlet extraction essential oil, US: ultrasound-assisted extraction essential oil and three main component standards (thymol, p-cymene, γ-terpinene) form a 3D chromatogram of 9 points at 300nm;

[0050] Figure 7 The essential oil of Ashoka fruit obtained by 6 extraction methods of the present invention, wherein HD: steam distillation-co-hydration essential oil, SD: steam distillation-water separation extraction essential oil, SFE: supercritical CO2 extraction essential oil, LLE: solvent extraction essential oil, SE: Soxhlet extraction essential oil, US: ultrasound-assisted extraction essential oil and three main component standards (thymol, p-cymene, γ-terpinene) form a 3D chromatogram of 9 points at 366nm;

[0051] Figure 8 The essential oils of Ayurveda fruit obtained by 6 extraction methods of the present invention, wherein HD: steam distillation-co-hydration essential oil, SD: steam distillation-water separation extraction essential oil, SFE: supercritical CO2 extraction essential oil, LLE: solvent extraction essential oil, SE: Soxhlet extraction essential oil, US: ultrasound-assisted extraction essential oil and three main component standards (thymol, p-cymene, γ-terpinene) at (A) 254nm, (B) 366nm and (C) white light scanning HPTLC fingerprints;

[0052] Fig. 9 The standard curves of the three main components (thymol, p-cymene, and γ-terpinene) of the present invention;

[0053] Fig.10 The effect of the steam distillation-co-water extraction method single factor experiment on the comprehensive score of Ayurveda foeniculum essential oil of the present invention;

[0054] Fig.11 It is a response surface diagram of the influence of different factors on the comprehensive score of Ayurveda fruit essential oil in the present invention;

[0055] Fig.12 It is a contour map of the influence of different factors on the comprehensive score of Ayurveda fruit essential oil in the present invention. DETAILED DESCRIPTION

[0056] In order to make the innovative contents of the above invention easier to understand, the present invention is described in detail in combination with the accompanying drawings and embodiments of the present invention, but is not limited to the present invention.

[0057] Example 1

[0058] Steam distillation-water extraction method (HD):

[0059] a. After the Asoko fruit is crushed by a grinder, it is passed through a 40-mesh sieve and placed in a 2000 mL hard round-bottom flask at a liquid-to-solid ratio of 14 mL / g. The flask is soaked for 49 min and heated with an electric heating mantle. The timing starts after boiling and the water is adjusted to a slightly boiling state and maintained for 6 h.

[0060] b. Turn off the electric heating jacket, transfer the oil and water in the oil-water separator to a test tube with a piston, and quickly seal it with a sealing film. After standing for 10 hours, separate the oil and water, and weigh it to obtain the Ayurvedic essential oil containing thymol, p-cymene and γ-terpinene; weigh it, and the results are shown in Table 1. The color of the essential oil is colorless, clear and transparent, and the yield is 3.55%; after the obtained essential oil is placed in a refrigerator at a temperature of -20°C and stored for 3 days, stable white crystals appear, and the main component of the crystalline part is thymol.

[0061] Example 2

[0062] Steam distillation-water extraction method (SD):

[0063] a. Add purified water to the bottom of the extraction tank, put a layer net, spread pure cotton gauze, sprinkle 100.0g of Ashoka fruit powder, install the tank cover, connect the oil-water separator and condenser, and then turn on the SHT high-power precision digital display electric heating jacket to heat it. After boiling, immediately reduce the heating and keep it in a slightly boiling state for 6 hours;

[0064] b. The oil and water in the oil-water separator were transferred to a test tube with a piston, and the tube was quickly sealed with a sealing film. After standing for 10 hours, the oil and water were separated. The yield of essential oil is shown in Table 1, that is, the essential oil of Ashoka fruit containing thymol, p-cymene and γ-terpinene was obtained, and the essential oil was colorless, clear and transparent.

[0065] Example 3

[0066] Supercritical CO2 extraction (SFE):

[0067] Accurately weigh 26.0 g of Ayurveda fruit powder and put it into the sample kettle, then weigh the weight of the sample collection bottle and record the data, connect the sample collection bottle to the sample outlet, and wait for the Ayurveda fruit essential oil to be collected. Turn on the instrument and set the critical pressure to 196 MPa, the critical temperature to 50 ° C, the collection valve temperature to 100 ° C, and the extraction time to 2 h. After the extraction is completed, remove the sample collection bottle and weigh it, as shown in Table 1, to obtain Ayurveda fruit essential oil containing thymol, p-cymene and γ-terpinene. The essential oil is a yellow, clear and transparent liquid and contains fine medicinal powder.

[0068] Example 4

[0069] Solvent Extraction (LLE):

[0070] Accurately weigh 10.0 g of Ashoka fruit powder and put it into a 250 mL conical flask with a piston, add 100 mL of n-hexane solvent and mix well. Soak and extract for 24 hours and then filter. The filtrate is concentrated by rotary evaporator to obtain an oily extract. Weigh the mass of 5 mL of the empty bottle and record the data. Then transfer the essential oil in the round-bottom flask to the weighed bottle. Finally, weigh the weight of the bottle and the essential oil to obtain Ashoka fruit essential oil containing thymol, p-cymene and γ-terpinene. The essential oil is a dark yellow oil.

[0071] Example 5

[0072] Soxhlet Extraction (SE):

[0073] Accurately weigh 12.0 g of Ashoka fruit powder and wrap it with filter paper, put the Ashoka fruit powder wrapped with filter paper into an extractor, put in an electric heating mantle, add 120 mL of n-hexane solvent, connect a condenser, start heating, and start timing when the solvent liquid surface is observed to boil. After 5 hours of extraction, filter with filter paper, and concentrate the filtrate with a rotary evaporator to obtain an oily extract containing thymol, p-cymene and γ-terpinene. The essential oil is a dark yellow oil.

[0074] Example 6

[0075] Ultrasound-assisted extraction (US):

[0076] Accurately weigh 10.0 g of Ashoka fruit powder and put it into a 250 mL conical flask with a piston, add 100 mL of n-hexane solvent, and then put it into an ultrasonic instrument with an operating frequency of 53 KHz, a power of 100%, and a set temperature of 40 ° C. After ultrasonication for 20 minutes, let it cool, filter with filter paper, and concentrate the filtrate with a rotary evaporator to obtain an oily extract of Ashoka fruit essential oil containing thymol, p-cymene and γ-terpinene, and the essential oil is a dark yellow oil.

[0077] The calculation formula of the essential oil yield (Yield%) of Examples 1-6 is:

[0078]

[0079] Where M1 is the mass of essential oil, and M2 is the mass of sample after pretreatment.

[0080] Example 7

[0081] The essential oils obtained in Examples 1-6 were subjected to GC-FID analysis of the three main components, thymol, p-cymene and γ-terpinene:

[0082] Essential oil solution treatment: 10 μL essential oil was taken and added to 1 mL solvent; standard solution treatment: 50.0 mg thymol was weighed and added to a 10 mL volumetric flask, and then the volume was fixed with n-hexane; 20 μL of p-cymene and γ-terpinene were taken and added to 10 mL solvent, followed by single-standard GC-FID analysis; the GC-FID analysis capillary was an HP-INNOWAX capillary column (60 m×0.25 mm, 0.25 μm), the carrier gas was high-purity N2, the flow rate was 1.3 mL / min, the average linear velocity was 25.432 cm / sec, the injection volume was 1.0 μL, the split ratio was 30:1, the injection port temperature was 230°C, and the program temperature was: 50°C for 3 min, then increased to 70°C at 2°C / min for 3 min, and finally increased to 200°C at 5°C / min for 8 min. The results are as follows: Figure 2 As shown, the peak areas of thymol are HD>SD>SFE>SE>US>LLE, the peak areas of p-cymene are SD>HD>SE>SFE>US>LLE, and the peak areas of γ-terpinene are SD>HD>SFE>SE>US>LLE; p-cymene and γ-terpinene are extremely volatile components, and when extracted with SFE, they may flow out of the sample collection bottle with CO2, and the content of p-cymene and γ-terpinene collected is less than that of thymol, so the detected peak area is relatively small;

[0083] The yields of essential oils extracted by the above different extraction methods and the peak areas of their three main components were summarized, and the comprehensive scores were calculated by the formula (as shown in Table 1), which is:

[0084]

[0085] In the formula, y is the comprehensive score%, P1 is the peak area of ​​thymol, P2 is the peak area of ​​p-cymene and P3 is the peak area of ​​γ-terpinene; from Table 1 and intuitive comparison Figure 3 Available,

[0086] Table 1 Comprehensive scores of different extraction methods

[0087]

[0088]

[0089] It can be seen from Table 1 that the peak area of ​​thymol, a broad-spectrum active ingredient extracted by steam distillation-co-water extraction (HD), is the largest at 2012.34, and its comprehensive score is also the highest at 56.61%.

[0090] Example 8

[0091] The essential oils obtained in Examples 1-6 and the three main components of thymol, p-cymene and γ-terpinene were analyzed by thin layer chromatography (HPTLC fingerprint and 3D chromatogram in Table 2):

[0092] First, a 10 mm × 10 mm silica gel plate was selected and a straight line was drawn at 1 mm from the other end of the sample as the solvent front line. Samples were spotted using a thin layer chromatography spotter. For essential oil solution treatment, 10 μL of essential oil was drawn and added to 1 mL of solvent. For standard solution treatment, 50.0 mg of thymol was weighed and added to a 10 mL volumetric flask, and then the volume was fixed with n-hexane. 20 μL of p-cymene and γ-terpinene were drawn and added to 10 mL of solvent, respectively. The spotting volume was 2.0 μL. The order of the essential oils spotted was steam distillation-co-water extraction essential oil (HD), steam distillation-water extraction essential oil (SD), supercritical CO2 extraction essential oil (SFE), solvent extraction essential oil (LLE), Soxhlet extraction essential oil (SE), ultrasound-assisted extraction essential oil (US), thymol, p-cymene and γ-terpinene; secondly, n-hexane:ethyl acetate with a volume ratio of 8:1 was selected as the developing agent to develop the essential oil in the silica gel plate, and the developing agent was taken out after reaching the solvent front line and waited for the solvent to evaporate; finally, a 3D UV scanner was placed to scan at 190nm, 254nm, 300nm and 366nm to obtain a 3D chromatogram, and the corresponding results are shown in Figure 4 And the specific data are shown in Table 2;

[0093] Table 2 3D chromatogram peak area

[0094]

[0095]

[0096] From Table 2 and Figure 4 , Figure 5 , Figure 6 and Figure 7 It can be seen that the thymol extracted by steam distillation-co-water extraction (HD) showed a maximum peak area of ​​29444.4AU at 190nm. The essential oils extracted by supercritical CO2 extraction (SFE), solvent extraction (LLE), Soxhlet extraction (SE) and ultrasound-assisted extraction (US) showed an uncertain component peak at 0.58Rf with an area of ​​up to 11948.8AU, which did not match the main component standard, indicating that it was not the main component compound and was likely a fatty acid compound; the 3D chromatogram at 254nm showed that the main component was thymol , which is the same as the peak position of the standard product-thymol; the 3D chromatogram of supercritical CO2 extraction essential oil (SFE) at 300nm shows a peak area of ​​888.2AU at 0.31Rf, which is an uncertain component; the 3D chromatogram of supercritical CO2 extraction essential oil (SFE), solvent extraction essential oil (LLE), Soxhlet extraction essential oil (SE) and ultrasound-assisted extraction essential oil (US) at 366nm highlights the peak area of ​​unknown compounds at 0.03Rf, indicating that SFE essential oil, LLE essential oil, SE essential oil and US essential oil contain large polar compounds, which are not volatile components;

[0097] After the above experimental steps, the silica gel plate was transferred to a conventional UV instrument and the HPTLC fingerprint was obtained after observation at 254nm and 366nm. The corresponding results are shown in Figure 8 A and Figure 8 B; Figure 8 A shows that from left to right at 0.39Rf, the UV spots show from strong to weak, which is consistent with the 3D chromatogram results at 254nm; Figure 8 B shows that at low Rf values, supercritical CO2 extraction (SFE) of essential oils has yellow fluorescence, while solvent extraction (LLE), Soxhlet extraction (SE) and ultrasound-assisted extraction (US) of essential oils have blue fluorescence, indicating that essential oils contain a variety of highly polar components. The results are consistent with the 3D chromatogram at 366 nm. Figure 1 The results showed that the essential oil extracted by steam distillation-co-water extraction (HD) method had high purity.

[0098] Finally, a vanillin developer containing 10% concentrated sulfuric acid (developer configuration: 3.0 g vanillin + 50 mL anhydrous ethanol + 0.5 mL concentrated sulfuric acid) was used to develop the silica gel plate containing essential oil components by wet spraying, heated on a thin layer plate heater to stabilize the color of the spots, then cooled to room temperature, and then scanned with white light; the results are shown in the figure. Figure 8As shown in C, thymol was clearly observed. In addition, supercritical CO2 extraction essential oil (SFE), solvent extraction essential oil (LLE), Soxhlet extraction essential oil (SE) and ultrasound-assisted extraction essential oil (US) showed spots of different colors at 0.58Rf and low Rf, which is consistent with the above experimental conclusions.

[0099] The essential oil obtained by steam distillation-co-water extraction (HD) has high purity, is a colorless, clear and transparent liquid, has a high comprehensive score and no pigments or fatty substances are observed, achieving the effect of improving the quality of Ayurveda essential oil and its active ingredients.

[0100] Example 9

[0101] According to the steam distillation-co-water extraction method (HD) of Example 1, the peak areas of the three main components of the essential oil GC-FID analysis were analyzed. The GC-FID analysis capillary was an HP-INNOWAX capillary column (60m×0.25mm, 0.25μm), the carrier gas was high-purity N2, the flow rate was 1.3mL / min, the average linear velocity was 25.432cm / sec, the injection volume was 1.0μL, the split ratio was 30:1, and the injection port temperature was 230°C. The temperature was programmed as follows: 50°C for 3 min, then raised to 70°C at 2°C / min and held at that temperature for 3 min, and finally raised to 200°C at 5°C / min and held at that temperature for 8 min. The external standard method was used to determine the mixed concentrations of several groups of thymol, p-cymene and γ-terpinene and draw a standard curve. The standard curve equations were: y=386.61x+34.531 for thymol, y=467.44x+8.9094 for p-cymene and y=436.01x+10.581 for γ-terpinene. Fig. 9 ; The precision of the GC-FID instrument, the repeatability of the method, the stability of the samples and the recovery of the spiked samples were also determined:

[0102] Precision test: Prepare three main component mixed standard solutions close to the sample concentration, inject 7 times continuously, record the chromatogram and the peak areas of the three main components, and then convert the peak areas into concentrations according to the standard curve equation. The RSD (%) values ​​are shown in Table 3. The calculation formula of RSD (%) is: Where SD is the standard deviation, is the average concentration;

[0103] Repeatability test: Take 7 portions of Ayurveda fruit of the same batch number (AYWG-YP-230323), respectively use the same pretreatment and steam distillation-water extraction method (HD) to extract 7 portions of essential oil, continuously inject and analyze under the established determination conditions, record the TLC chromatogram and the peak areas of the three main components, and then convert the peak areas into concentrations according to the calibration curve equation, and obtain the RSD (%) value as shown in Table 3;

[0104] Stability test: Select essential oils obtained by steam distillation-co-water extraction method and prepare them into solutions. Samples were injected 9 times at time 0h, 2h, 4h, 6h, 8h, 10h, 12h, 18h and 24h, and the chromatogram and the peak areas of the three main components were recorded. The peak areas were then converted into concentrations according to the calibration curve equation, and the RSD (%) values ​​were obtained as shown in Table 3.

[0105] Spiked recovery rate: Set blank spike recovery and three groups of 0.5:1, 1:1 and 2:1 (standard product: sample) sample spike recovery. Each group of sample spike recovery experiments were repeated three times. The chromatogram and the peak areas of the three main components were recorded. The peak areas were then converted into concentrations according to the standard curve equation. The spike recovery rates are shown in Table 3. The spike recovery rate formula is: Where P is the spike recovery rate (%), Ca is the sample concentration, Cb is the spiked sample concentration, and Cc is the spiked concentration;

[0106] Table 3 RSD (%) average value and spike recovery %

[0107]

[0108]

[0109] As shown in Table 3, the RSD (%) values ​​of precision, repeatability and stability are all less than 1%, which meets the requirements for establishing the analysis; Table 3 shows that the spike recovery rate is 85%-99%, which is within the recovery rate limit of the analytical method verification, indicating that the analytical measurement results are close to the theoretical values, and the sample measurement obtains accurate data; Fig. 9 It can be seen that the R of the three principal component standard curves 2 ≥0.9992, which proves that the three standard curves meet the linearity requirements and can be used in subsequent experiments to convert peak area values ​​into concentration values;

[0110] Example 10

[0111] According to the conclusion of Example 8, a single factor experiment was carried out on the steam distillation-water extraction (HD) method essential oil, mainly comprising:

[0112] Determination of extraction time: Accurately weigh 100.0g of Ashoka fruit powder that has been crushed and passed through a 40-mesh sieve, put it into a 2000mL hard round-bottom flask, and add 1200mL of secondary purified water. Soak for 0h, heat with an electric heating mantle, and start timing after boiling. The extraction time is 3h, 4h, 5h, 6h, 7h and 8h. The effect of extraction time on the essential oil yield and the content of the three main components is studied. The results are as follows: Fig.10 As shown in A, the optimal extraction time is 6 h, and the comprehensive score of this single factor is 95.86%;

[0113] Determination of liquid-to-solid ratio: Accurately weigh 100.0g of Ashoka fruit powder that has been crushed and passed through a 40-mesh sieve, put it into a 2000mL hard round-bottom flask, add 800mL, 1000mL, 1200mL, 1400mL, and 1600mL of secondary purified water, soak for 0h, and the extraction time is 6h. The effect of liquid-to-solid ratio on the essential oil yield and the content of the three main components was studied. The results are as follows: Fig.10 As shown in B, the best liquid-to-solid ratio is 12 mL / g, and the comprehensive score of this single factor is 95.81%;

[0114] Determination of soaking time: Accurately weigh 100.0g of Ashoka fruit powder that has been crushed and passed through a 40-mesh sieve, put it into a 2000mL hard round-bottom flask, and add 1200mL of secondary purified water. Soak for 0h, 2h, 4h, 6h, 8h, and 10h. The extraction time is 6h. The effect of soaking time on the essential oil yield and the content of the three main components is studied. The results are as follows: Fig.10 As shown in C, the best immersion time is 0h, and the comprehensive score of this single factor is 97.88%;

[0115] Determination of grinding particle size: After the Ayuwei fruit was crushed by a grinder for 5 seconds, it was sieved. The medicinal materials that failed to pass through the sieve were continued to be crushed for 5 seconds. The crushing and sieving were repeated. 18 mesh, 24 mesh, 30 mesh, 40 mesh, 50 mesh, and 60 mesh sieves were selected as the grinding particle size for investigation. Then 100.0g of powder was accurately weighed and put into a 2000mL hard round-bottom flask, and 1200mL of purified water was added to soak for 0h. The extraction time was 6h. The effect of grinding particle size on the essential oil yield and the content of the three main components was studied. The results are as follows Fig.10 As shown in D, the best crushing particle size is 30 mesh, and the comprehensive score of this single factor is 94.97%;

[0116] The essential oil obtained above was analyzed by GC-FID. The essential oil solution was treated by taking 10 μL of essential oil and adding it to 1 mL of solvent. The GC-FID analysis capillary was HP-INNOWAX capillary column (60 m×0.25 mm, 0.25 μm), the carrier gas was high-purity N2, the flow rate was 1.3 mL / min, the average linear velocity was 25.432 cm / sec, the injection volume was 1.0 μL, the split ratio was 30:1, the injection port temperature was 230°C, and the program temperature was: 50°C for 3 min, then raised to 70°C for 3 min at 2°C / min, and finally raised to 200°C for 8 min at 5°C / min. The peak area values ​​of the three main components were substituted into the corresponding standard curve equation (such as Fig. 9 ), calculate the concentration value and obtain the comprehensive score. The comprehensive score formula is:

[0117]

[0118] Where y is the comprehensive score%, C1 is the concentration of thymol, C2 is the concentration of p-cymene and C3 is the concentration of γ-terpinene;

[0119] Finally, use Excel worksheet to draw the single factor influence curve, such as Fig. 9 Display the corresponding optimal single factor influence value.

[0120] Embodiment 11

[0121] This example uses a response surface experiment to optimize the steam distillation-water extraction method (HD) experiment, and the specific contents are as follows:

[0122] Based on the single factor experimental data, the single factor influence range was selected to design a three-level response surface scheme experiment, as shown in Table 4:

[0123] Table 4 Response surface design scheme

[0124]

[0125] Design-Expert V8.0.6 software was used to import the response surface design scheme to carry out the Box-Behnken response surface experimental design. The four factors of extraction time (h), liquid-to-solid ratio (mL / g), soaking time (h) and crushing particle size (mesh) were taken as the research objects. The Box-Benhnken four-factor three-level experiment was designed with the comprehensive score % as the response value. A total of 29 groups of experimental analysis were conducted. The results are shown in Table 5. The comprehensive score of Ayurveda fruit essential oil is in the range of 78.89%-93.19%; the comprehensive score of Ayurveda fruit essential oil corresponding to the experimental conditions of extraction time 6.0h, liquid-to-solid ratio 12mL / g, soaking time 0.0h and crushing particle size 30 mesh is 93.19%;

[0126] Table 5 Response surface experimental design and results

[0127]

[0128]

[0129] The P value of the variance analysis table of the regression model can represent the strength of each independent variable and the interaction between independent variables. As shown in Table 6, the P values ​​of model, A and AB are less than 0.0001, indicating that these three sources of variation are highly significant; the P value of the source of variation C is less than 0.001, indicating that the soaking time of C is moderately significant; the source of variation D 2 P<0.01, indicating that D 2 The particle size is significant; the model's lack of fit term P = 0.7551, indicating that the lack of fit is not significant and can fit the actual experimental results. 2 =0.9451 and adjusted coefficient of determination R 2=0.8702, indicating that the experimental value and the predicted value of the model have a high correlation and a good degree of fit; therefore, this model can be used to analyze and predict the steam distillation-co-water extraction (HD) conditions of Ayurvedic essential oil;

[0130] In addition, the larger the factor F value of the regression model coefficient, the greater the impact on the response value. As shown in Table 6, the influence of the four single factors on the comprehensive score is in the order of A extraction time > C soaking time > D crushing particle size > B liquid-to-solid ratio. The experimental results were subjected to regression analysis to obtain the multiple regression equation:

[0131] y=157.85193-17.08068A-5.17038B+0.90696C-0.24609D+2.00744AB-0.73060AC

[0132] +0.032974AD+0.16571BC+0.047360BD+0.038953CD-0.32634A 2 -0.29058B 2 -0.31981C 2 -0.00751972D 2 ;

[0133] Where y is the comprehensive score (%), A is the extraction time (h), B is the liquid-to-solid ratio (mL / g), C is the soaking time (h) and D is the crushing particle size (mesh);

[0134] Table 6 Analysis of variance table of response surface regression model

[0135]

[0136]

[0137] Note: *p<0.01; **p<0.001; ***p<0.0001

[0138] Both the response surface and contour lines can reflect the impact of each variable on the response value, and can predict the optimal conditions and optimal values ​​of the response; Fig.11 It can be seen that factors AB, AC, AD, BC, BD, and CD affect the comprehensive score of Ayurveda oleracea essential oil to varying degrees, affecting both the yield of Ayurveda oleracea essential oil and the contents of thymol, p-cymene, and γ-terpinene. Fig.12 It can be seen that the contour lines of AB, AC, and AD are elliptical, indicating that there is an obvious interaction between these factors.

[0139] Example 12

[0140] This example verifies the response surface optimization conditions:

[0141] The model was optimized using Design-Expert 8.0.6 software, and the optimal steam distillation-co-water extraction conditions were obtained as follows: extraction time 6.0h, liquid-to-solid ratio 14mL / g, soaking time 0.81h and crushing particle size 42.99 mesh, with a predicted comprehensive score of 95.65%. After correction according to actual conditions, the response surface optimization and verification extraction conditions were as follows: extraction time 6.0h, liquid-to-solid ratio 14mL / g, soaking time 49min and crushing particle size 40 mesh. The experiments were conducted in parallel three times, and the final actual comprehensive score was 96.07%, which was very close to the predicted value. Therefore, this response surface model and related parameters are accurate and reliable, and can predict the optimal conditions of the experiment, and can be used to improve the quality of Ayurveda essential oil and the extraction process of its active ingredients.

Claims

1. A method for improving the quality of Ashoka fruit essential oil, characterized in that, The method adopts steam distillation-co-water extraction method, steam distillation-water-insulated extraction method, supercritical CO2 extraction method, solvent extraction method, Soxhlet extraction method or ultrasound-assisted extraction method, and the specific operation is carried out according to the following steps: Steam distillation-water extraction method: a. Grind the Asoko fruit with a grinder, pass it through a 40-mesh sieve, and put it into a 2000 mL hard round-bottom flask at a liquid-to-solid ratio of 14 g / mL. Soak it for 49 min and heat it with an electric heating mantle. Start timing after boiling, and keep it at a slightly boiling state on the water surface for 6 h. b. Transfer the oil and water in the oil-water separator to a test tube with a piston, and quickly seal it with a sealing film. After standing for 10 hours, separate the oil and water, and weigh them to obtain the mixture containing thymol, p-cymene and γ - Ayurvedic essential oil containing terpinene; Steam distillation-water extraction method: a. Add secondary purified water to the bottom of the extraction tank, put a layer net on it, spread pure cotton gauze, sprinkle 100.0 g of Ashoka fruit powder on it, install the tank cover, connect the oil-water separator and condenser, and then turn on the SHT high-power precision digital display electric heating jacket to heat it. After boiling, immediately reduce the heating and keep it in a slightly boiling state for 6 hours; b. Transfer the oil and water in the oil-water separator to a test tube with a piston, and quickly seal it with a sealing film. After standing for 10 hours, separate the oil and water, and weigh them to obtain the mixture containing thymol, p-cymene and γ - Ayurvedic essential oil containing terpinene; Supercritical CO2 extraction method: Accurately weigh 26.0 g of Asoko fruit powder and put it into the sample kettle, then weigh the weight of the sample collection bottle and record the data, then connect it to the sample outlet, finally turn on the instrument and set the critical pressure to 196 MPa, the critical temperature to 50 °C, the collection valve temperature to 100 °C, the extraction time to 2 h, and after the extraction is completed, remove the sample collection bottle and weigh it to obtain the extract containing thymol, p-cymene and γ - Ayurvedic essential oil containing terpinene; Solvent extraction method: Accurately weigh 10.0 g of Asobosu fruit powder into a 250 mL conical flask with a piston, add 100 mL of n-hexane solvent and mix well. After soaking and extracting for 24 h, filter the filtrate and concentrate it on a rotary evaporator to obtain an oily extract. Weigh the mass of the 5 mL empty bottle and record the data. Then transfer the essential oil in the round-bottom flask to the weighed bottle. Finally, weigh the weight of the bottle and the essential oil to obtain the extract containing thymol, p-cymene and γ - Ayurvedic essential oil containing terpinene; Soxhlet extraction method: Accurately weigh 12.0 g of Asobosu fruit powder and wrap it with filter paper. Put the Asobosu fruit powder wrapped with filter paper into the extractor, put in the electric heating mantle, add 120 mL of n-hexane solvent, connect the condenser, start heating, and start timing when the solvent liquid surface boils. After 5 h of extraction, filter with filter paper, and concentrate the filtrate with a rotary evaporator to obtain an oily extract containing thymol, p-cymene and γ - Ayurvedic essential oil containing terpinene; Ultrasonic assisted extraction method: Accurately weigh 10.0 g of Asobosu fruit powder and put it into a 250 mL conical flask with a piston, add 100 mL of n-hexane solvent, and then put it into an ultrasonicator with an operating frequency of 53 KHz, a power of 100%, and a set temperature of 40 °C. After ultrasonication for 20 min, let it cool and filter it with filter paper. The filtrate was concentrated by rotary evaporator to obtain an oily extract containing thymol, p-cymene and γ -Ayurvedic essential oil containing terpinene.

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