Study on chemical composition and develop extraction technology process to make valuable products from docynia indica (wall.) decne) fruits in Vietnam

evaporator to obtain 652 g of a methanol extraction residue. Dissolve the methanol residue with distilled water and extract it in turn with solvents increasing in polarities n-hexane, dichloromethane, and ethyl acetate. The distribution extracts are filtered and concentrated to obtain the corresponding extract residues, n-hexane (36,2 g); dichloromethane residue (65 g); ethyl acetate residue (258.8 g), and water residue (289.4 g). 3.1.1. Diagram of isolation of compounds from EtOAc residues of Docynia indica fruits. Take 114 g of ethyl acetate extraction residue (EtOAc), and perform the chromatography of the regular phase silica gel column with the CH2Cl2/MeOH gradient elution solvent (100: 0  0/100) divided into 8 symbol segments from E1 to E8. Examine the fractions obtained by thin- layer chromatography (TLC), color current with 5% H2SO4 reagent, or check with UV scanners at 254 nm and 365 nm. Gather the same fractions, then evaporate to chase away the solvent to obtain the extract of the fractions. From segments, E1 to E8 continues to conduct chromatographic methods for isolation and collection of clean compounds. The isolation diagram is shown in figure 3.1.1. 6 Figure 3.1.1. Diagram of isolation of chemical components from a Docynia indica fruits 3.1.2. Physical parameters and spectral data of compounds (see thesis) 3.2. Phenolic extraction process and research model establishment 3.2.1. Extraction by soxhlet method Prepare the soxhlet extraction kit. Accurately weigh 10g of raw Docynia indica fruits powder into a filter paper bag, tie it tightly. Put the filter bag containing the ingredients into the extraction compartment. Fill a 1000 mL flask with 500 mL of 96% food ethanol. Install a reflux condenser to connect the soxhlet extraction device. Turn on the electric stove to heat to 650C, the extraction time is 10 hours. At the end of the extraction process, the extract is evaporated to evaporate the solvent to obtain the total extract. Quantification and analysis of total phenolic, total flavonoid content will then be conducted. Total extract high content Y (%) is calculated according to the weight of the extracted sample. Study results are used as a standard for comparison with the following phenolic extraction methods 3.2.2. Extraction using microwave 3.2.2.1. Conduct experiments 100g of Docynia indica fruits powder is placed in a 1000 ml flask, adding 500 ml of solvent (mixed with the concentration of ethanol and pH to be studied) to the flask. Put the flask in the microwave and install the reflux condenser. Turn on the condenser, turn on the microwave at the 7 capacity levels to be studied. The extraction time is calculated starting with the microwave on. At the end of the extraction process, the extract is filtered through a buchner filter and concentrated to obtain a total extract. Determination of total extract high content (%); total phenolic content (mg GAE / g extract) and total flavonoid (mg QE / g extract) in extract. 3.2.2.2. Design the experimental plan matrix The research technological factors include 4 factors: Z1: Extraction time (minutes); Z2: Extracted ethanol concentration (%); Z3: Microwave power (W) and Z4: pH of extraction solvent. The target functions are Y1 (total phenolic content), Y2 (total flavonoid content), and Y3 (total extractable high content). The encoding variables of Z1, Z2, Z3, and Z4 are denoted A, B, C, and D respectively. Select the survey model according to Box-Willson with k = 4, choose lever arm α = 1.414 and number solution at the center is 3. The total number of experiments of the matrix is 27 experiments. 3.2.3. Extraction using ultrasound 3.2.3.1. Conduct experiments 100g of material powder was placed in a 1000 mL or 2000 mL 3 neck flask, extraction solvent using 65% ethanol, and 5.4 solvent pH was added to the flask at study rates (according to experimental arrangement. test). Install a condenser, thermometer, and transducer ultrasonic device, then heat with an electric stove, ultrasonic extraction at the conditions of temperature, ultrasonic power, and time to study. At the end of the extraction process, the extract is filtered through a Buchner filter and concentrated to obtain a total extract. Determination of total extract high content (%); total phenolic content (mg GAE / g extract) and total flavonoid (mg QE / g extract) in extract. 3.2.3.2. Design the experimental plan matrix The research technological factors include 4 factors: Z1: Solvent/material ratio (v / w), Z2: Ultrasonic extraction temperature (0C), Z3: Ultrasonic power (W), and Z4: Extraction time (minutes). The target functions are Y1 (total phenolic content), and Y2 (total extract high content). The encoded variables of Z1, Z2, Z3, and Z4 are denoted A, B, C, and D respectively. Select the survey model according to Box-Behnken with k = 4, the number of experiments at the center is 3. Total the experiments of the matrix are 27 experiments. 3.2.4. Extraction by reflux method 8 3.2.4.1. Conduct experiments 100g of Docynia indica fruits powder was added to a 2000 mL flask, extraction solvent using 65% ethanol, and 5.4 solvent pH were added to the flask at study rates. Install condenser and conduct heating by the electric stove, reflux extraction at the temperature and time conditions needing research. At the end of the extraction process, the extract is filtered through a buchner filter and concentrated to obtain a total extract. Determination of total extract high content (%); total phenolic content (mg GAE / g extract) and total flavonoid (mg QE / g extract) in extract. 3.2.4.2. Design the experimental plan matrix The research technological factors include 3 factors: Z1: extraction time (minutes), Z2: ratio of solvent / material (v / w) and Z3: extraction temperature (0C). The target functions are Y1 (total phenolic content), and Y2 (total extract high content). The encoding variables of Z1, Z2, and Z3 are denoted A, B, and C. For k = 3, choose the lever arm α = 1.215 and the number of experiments at the center is 1. The match is 15 experiments 3.3. Spray-drying process and model design 3.3.1. Conduct experiments The extract is filtered and concentrated to obtain 2.1 liters of the solution with a dry matter content of 15-20%. Add the drying aid maltodextrin at the research rate to the concentrated extract. Turn on the extractor agitator, turn on the flow pump, adjust the supply flow rate according to the research conditions and conduct the spray-drying process, the drying agent (hot air) is blown into the injection chamber. The drying agent temperature is changed according to the research conditions. Equipment used for research is Buchi B290 mini spray dryer, fluid injection speed (15-35 mL/min), inlet hot air temperature (120 - 2200C), maximum hot air flow 35 m3/hour, evaporation capacity 1 L H2O / hour (Institute of Biotechnology - Food, Hanoi University of science andTechnology). The powder after spray drying was analyzed for total phenolic content and moisture content. 3.3.2. Design the experimental plan matrix The research technological factors include 3 factors: Z1: Content of drying aid maltodextrin (%, w/w), Z2: Inlet hot air temperature (0C), and Z3: Spray rate (mL/min) ). The target function is Y1 (total phenolic content of the product after spray drying), Y2 (moisture content of the product after spray drying). The encoded variables of Z1, Z2, and Z3 are denoted A, B, and C. Select the survey model according to Box-Behnken with k = 3, the number of experiments at the center is 1. The total number of experiments of The matrix is 15 experiments. 9 CHAPTER 4. RESULTS AND DISCUSSION 4.1. Compounds isolated from EtOAc residue from Docynia indica From the Docynia indica ethyl acetate extract isolated and identified 25 compounds including: +19 phenolic compounds (TM1, TM2, TM3, TM5, TM7, TM8, TM9, TM10, TM12, TM13, TM15, TM16, TM17, TM18, TM30, TM33, TM35, TM36, TM37). In which there is a new compound called 3S-Thunberginol C 6-O-β- D-glucopyranoside (TM17). + 5 triterpenoid compounds (TM20, TM22, TM23, TM24, TM25) + 1 derivative of linear acids: (TM6) Table 4.1. Compounds isolated from EtOAC residue from Docynia indica TT code Name weight (mg) 1 TM1 Chlorogenic acid methyl ester 45,5 2 TM2 Quercetin 21,0 3 TM3 Protocatechuic acid 6,8 4 TM5 Hyperin 13,5 5 TM6 4-methyl malate 32,0 6 TM7 Naringenin-7-O- β-D- glucopyranoside 11,0 7 TM8 Phlorizin 10,8 8 TM9 3-methoxy, 4-hydroxy-benzoic acid 12,2 9 TM10 Astilbin 22,0 10 TM12 Gallic acid 19,0 11 TM13 Methyl gallate 6,5 12 TM15 Chrysin 9,5 13 TM16 Naringenin 5,6 14 TM17 (New) 3S-Thunberginol C 6-O-β- D- glucopyranoside 6,5 15 TM18 1-O-coumaroyl-β-D-glucopyranose 12,0 16 TM20 Pomolic acid 8,0 17 TM22 Euscaphic acid 7,6 18 TM23 23-Hydroxy ursolic acid 10,5 19 TM24 Ursolic acid 22,0 20 TM25 Maslinic acid 11,2 21 TM30 (2R/S)-5,7,3’,5’-tetrahydroxy- flavanone 7-O-β-D glucopyranosie 8,2 22 TM33 Phloretin-2’-O-(β-D-xylopyranosyl- 11,3 10 (16)-O-β-D glucopyranoside) 23 TM35 Cis-p-coumaric acid 4-O-β-D- glucopyranoside 7,6 24 TM36 Myricitrin 7,2 25 TM37 2’,6’-dihydroxy-3’,4’- dimethoxychalcone 4,4 4.1.1. Compound 3S-Thunberginol C 6-O-β- D-glucopyranoside (TM17) - New compound The compound TM17 is obtained as a white solid. On high resolution mass spectrometry HR-ESI-MS of TM17 appeared pseudo- molar ions are [M - H] ¯ at m / z 433.1119, [M + 35Cl] ¯ at m/z 469.0890 and [M + 37Cl] ¯ at m/z 471.0870. Theoretical calculation for ions [C21H21O10] ¯ has m/z 433.1129, ions [C21H22ClO10] ¯ have m/z 469,0896 corresponding to isotope 35Cl and 471,0876 corresponding to isotope 37Cl. From the data on high resolution mass spectrometry, the molecular formula of TM17 is determined to be C21H22O10. The polarity of the compound TM17 is [α] D = - 690 (c = 0.1, MeOH). Figure 4.1.1.1. HR-ESI-MS spectrum of compound TM17 On the 1H-NMR spectrum of compound TM17 appeared spin system AABB [δH 6.80 (d, J = 8.5 Hz, H-3 ′, 5 ′); 7.32 (d, J = 8.0 Hz, H-2 ′, 6 ′)] allows for the determination of a double potential phenyl ring in compound TM17. In addition, the aromatic ring of isocoumarin nucleus is characterized by a resonant signal with chemical shift [δH 6.54 (br s, H- 5); 6.52 (d, J = 2 Hz, H-7, TM17a); 6.51 (d, J = 2 Hz, H-7, TM17b)], while the lactone ring appears with resonant signals at δH 5.61 (t, J = 2.5 [M + 35Cl]¯ [M - H]¯ [M + 37Cl]¯ CTPT: C21H22O10 11 Hz, H-3, TM17a); 5.59 (t, J = 2.5 Hz, H-3, TM17b); 3.11 and 3.07 (H-4a); 3.34 (m, H-4b). Figure 4.1.1.2. The 1H-NMR spectrum of compound TM17 13C-NMR spectrum combined DEPT spectrum showed 21 signals of carbon atom including 2 carbon of methylene group (-CH2), 12 carbon of methine group (-CH), and 7 carbon not bound to hydrogen. In which 1 signal of ketone group linked to oxygen atom (O-C=O) characteristic of dihydroisocoumarin frame appears at 169.25/169.18 ppm, 2 pairs of symmetrical carbon signal of B ring at δC (ppm) 128.22/128.20 and 115.18 show that the ring B is substituted at 2 symmetrical positions on the ring (C-1 'and C4'), 2 typical carbon signals of the lactone ring at δC (ppm) 80.0 and 33.66; Besides, the appearance of 6 carbon signals at the chemical shift δC (ppm) 99.77/99.67; 73.06; 76.44; 69.51; 77.11 and 60.52 suggest these are six-carbon signals of a sugar congener. On 2-dimensional spectrum HMBC showed the interaction of proton anomeric δH (ppm) 4.99 (1H, d, J = 7.5 Hz) /4.97 (1H, d, J = 8.0 Hz) with carbon atom C-6 of aglycon. The interaction constant of proton anomer J = 7.5 - 8.0 Hz allows to confirm that this is a beta sugar. HMBC spectrum also shows interactions between H-7 (δH 5.51 / 5.52) with C-5 (δC 107.37 / 107.27), C-9 (δC 102.56 / 102.54); H-4 (δH 3.08 / 3.34) with C-5 (δC 107.37 / 107.27), C-9 (δC 102.56 / 102.54); H-5 (δH 6.55) with C-4 (δC 33.66); H-3 (δH 5.61 / 5.59) with C-10 (δC 142.15), C-6 '(δC 128.22 / 128.20), C-2' (δC 128.22 / 128.20); H-2 '(δH 7.32), H-6' (δH 7.32) with C- 12 4 '(δC 157.76); H3 '(δH 6.80), H-5' (δH 6.80) with C-1 '(δC 128.47 / 128,45). The above interactions allow locating two hydroxyl substituent groups at C-8 and C-4 '. Figure 4.1.1.3. 13C-NMR spectrum of compound TM17 Figure 4.1.1.5. HSQC spectrum and HMBC spectrum of compound TM17 The signals on the above 1H-NMR and 13C-NMR spectrum above can confirm that the aglycon part of TM17 is Tshirtbinol C [72] The TM17 hydrolysis and high-performance liquid chromatography with standard controls identified the sugar congeners in the TM17 molecule as β-D glucose 13 Figure 4.1.1.6. HPLC chromatogram for determination of sugar congeners In the chemical structure of compound TM17, the carbon C-3 position is the antagonistic carbon. Therefore, to determine the absolute configuration of compound TM17, we measure circular dichroism (CD spectrum). Results on CD spectrum show negative cotton effects occurring at wavelengths 227 nm (Δε -2.29), 255 nm (Δε -4.85) and 305 nm (Δε -1.21). In which, the negative cotton effect occurs most strongly at the wavelength of 255 nm (Δε -4.85) (Figure 4.1.1.9). Comparing the CD spectrum of compound TM17 with compounds with chemical structure and relative carbon position similar to compound TM17 such as 3S- hydrangenol 4'-O-glucoside, 3S-elasticberginol I 4 '-O-glucoside, and 3S- florahydroside. In the three compounds mentioned above, on their CD spectrum, the strongest negative cotton effects occurred at 260 nm (Δε - 3.76), 255 nm (Δε -8.30), and 255 nm (Δε -0.79) respectively [72- 74]. This indicates that the compound TM17 is suitable for the 3S configuration of 3-aryl dihydroisocoumarin. Therefore, compound TM17 is confirmed to be 3S-Thunberginol C 6-O-β-D-glucopyranoside. This is a new compound isolated from nature for the first time. However, on the spectrum, 1H-NMR and 13C-NMR appeared dual signals appear in pairs of signals very close together (as shown in spectrum data table 4.1.1). We hypothesize that the compound TM17 exists simultaneously in 2 different stable profiles, so when measuring the magnetic resonance spectrum of the nucleus, the above phenomenon will occur. To support this hypothesis, we proceed to calculate the stable energy theory of the possible configurations of compound TM17 through the calculation of the relative enthalpy (ΔH) of this compound, using the DFT method. (density functional theory method). Theoretical calculation results showed that 02 half-boat profiles exist in stable energy state with relative enthalpy, respectively Δ H = 0.0 kcal/mol and Δ H = 0.2 kcal/mol. 14 This clarifies our hypothesis that the compound 3S-Thunberginol C 6-O- β- D-glucopyranoside exists in two different stable profiles (a / b). Figure 4.1.1.7. Strucrute of TM17 Figure 4.1.1.8. CD spectrum of TM17 (a) (b) Figure 4.1.1.9. Two half-boat durable profiles of TM17 compound Table 4.1.1. NMR spectroscopy data of compound TM17 and T-shirt comparator C Ref. (DMSO-d6) [72] TM17 (DMSO-d6) δH (ppm) δC (ppm) δH (ppm) δC (ppm) 1 169.4 169.25 / 169.18 3 5.54 (1H, t, J= 3.0 Hz) 79.7 5.61 (1H, t, J = 2.5 Hz) / 5.59 (1H, t, J = 2.5 Hz) 80.0 4 3.03 (1H, dd, J= 3.0; 17.0 Hz, H-4a) 3.24 (1H, dd, J= 3.0; 17.0 Hz, H-4b) 33.6 3.11 (1H, t, J= 2.5 Hz, H- 4a)/3.07 (1H, t, J= 2.5 Hz, H-4a) 3.35 (overlap, H-4b) 33.66 15 5 6.3 (1H, d, J= 2.0 Hz) 106.8 6.55 (1H, br s) 107.37 / 107.27 6 164.4 162.99 7 6.22 (1H, d, J = 2.0 Hz) 100.9 6.52 (1H, d, J= 2.0 Hz) / 6.51 (1H, d, J = 2.0 Hz) 101.76 8 11.1 (-OH) 163.3 11.09 (-OH) 163.28 9 100.3 102.56 / 102.54 10 142.2 142.15 1’ 128.6 128.47 / 128.45 2’ 7.31 (1H, d, J=9.0 Hz) 128.0 7.32 (1H, d, J = 8.0 Hz) 128.22 / 128.20 3’ 6.8 (1H, d, J=9.0 Hz) 115.1 6.80 (1H, d, J = 8.5 Hz) 115.18 4’ 157.6 157.76 5’ 6.8 (1H, d, J=9.0 Hz) 115.1 6.80 (1H, d, J = 8.5 Hz) 115.18 6’ 7.31 (1H, d, J=9.0 Hz) 128.0 7.32 (1H, d, J = 8.0 Hz) 128.22 / 128.20 1’’ 4.99 (1H, d, J = 7.5 Hz) / 4.97 (1H, d, J = 8.0 Hz) 99.77 / 99.67 2’’ 3.24, m 73.06 3’’ 3.37, m 76.44 4’’ 3.18, m 69.51 5’’ 3.2, m 77.11 6’’ 3.46 (1H, m) 3.76 (1H, m) 60.52 4.2. Evaluate the biological activity of extracts and compounds isolated 4.2.1. Cardiovascular protective activity (sEH) 4.2.1.1. For extracts The n-hexane fractional extract did not show activity at 37.5 and 75 µM, exhibiting low activity at 150 µM. The remaining fractions exhibit decreasing activity: high ethyl acetate> high dichloromethane> high total methanol> high total methanol. Table 4.2.2.1. Results assess sEH activity with extract TT Extraction fraction Test concentration (µM) Percent inhibition (%) 1 Dichloromethane 37.5 28.0 ± 3.4 16 2 75 57.9 ± 0.1 3 150 83.8 ± 2.7 4 Ethyl acetate 37.5 36.7 ± 2.5 5 75 68.7 ± 3.5 6 150 92.7 ± 1.2 7 Water 37.5 25.4 ± 1.2 8 75 45.9 ± 0.2 9 150 68.4 ± 0.1 10 Total extract methanol 37.5 4.9 ± 0.5 11 75 12.8 ± 1.7 12 150 21.1 ± 2.2 4.2.2.2. For isolated compounds There are 8 out of 25 active compounds including TM1, TM5, TM8, TM10, TM16, TM24, TM33 and TM37. The IC50 (µM) values of these eight compounds ranged from 10.0 ± 0.6 to 88.4 ± 0.2. In which, two compounds TM9 and TM37 showed the strongest activity when IC50 values were 19.3 ± 2.2 and 10.0 ± 0.6 µM, respectively. Table 4.2.2.2. Results of sEH activity test with the compounds isolated TT code Percent inhibition at 100 µM concentration (%) IC50 (µM) Control (+) Auda 16.8 ± 0.5 nM 1 TM1 73.3 ± 1.1 41.9 ± 1.1 2 TM5 72.8 ± 0.2 30.5 ± 0.1 3 TM9 >100 19.3 ± 2.2 4 TM10 79.9 ± 0.4 22.9 ± 0.2 5 TM16 76.8 ± 0.06 24.7 ± 2.5 6 TM24 74.0 ± 2.8 36.1 ± 0.6 7 TM33 52.7 ± 0.2 88.4 ± 0.2 8 TM37 >100 10.0 ± 0.6 4.2.2. Evaluation of antioxidant activity Out of a total of 25 compounds tested, 14 were active with SC50 (µg / mL) values in the range (18.05 ± 0.69 to 49.34 ± 1.22). Especially, of the 14 active compounds, 1 compound exhibited greater activity than the positive control, TM12 compound with SC50 value of 18.05 (µg / mL). Table 4.2.1. Results of antioxidant activity of isolated compounds code SC50 (µg/mL) code SC50 (µg/mL) 17 control (+) Ascorbic acid 26.40 ± 0.44 TM12 18.05 ± 0.69 TM1 35.48 ± 2.02 TM15 30.12 ± 0,15 TM2 31.37 ± 0.12 TM16 33.47 ± 0,51 TM3 28.18 ± 1.18 TM17 40.51 ± 0.78 TM5 29.17 ± 1.67 TM30 41.81 ± 0.45 TM7 49.34 ± 1.22 TM36 42.76 ± 2.12 TM9 29.11 ± 0.17 TM37 29,12 ± 0.38 TM10 42.32 ± 0.42 4.2.3. Evaluation of the cytotoxic activity of extracts Only 2 extracts of n-hexane and dichloromethane (CH2Cl2) fractions showed inhibitory activity on cervical cancer cells with IC50 values of 99.24 µg / mL and 67.2 µg / mL, respectively. The remaining fractional extracts showed no activity. 4.3. Laboratory optimization study of phenolic extraction from Docynia indica fruits. 4.3.1. Soxhlet extraction results obtained total extracts Soxhlet extraction has very high extraction efficiency because it is the most exhaustive method. The high content of total extract achieved an average of 36.07%. The total phenolic content and total flavonoid content are also very high with average values of 28.9 (mg GAE/g extract) and 20.0 (mg QE/ g extract), respectively. 4.3.2. Research model and optimization of phenolic extraction process by microwave extraction 4.3.2.1. Effect of univariate factors on the objective function 4.3.2.2. Modeling and defining the regression equation of the objective function. From the experimental data on the influence of univariate technology parameters on the target function, we choose the research model according to the second-order model of Box-Willson. The basic levels (or basic levels) of the factors and the coefficient α = 1.414 (with k = 4) are shown in Table 4.3.2.2a Table 4.3.2.2a. Experimental levels of technological variables The variable name, variable interval Level Independent Variables Codes Avariable Range (Δ) -α -1 0 1 +α Z1: Extraction time (min) A 15 9 15 30 45 51 Z2: Ethanol concentration (%) B 20 32 40 60 80 88 18 Z3: Microwave power (W) C 160 175 240 400 560 625 Z4: Solvent pH D 2 1.2 2 4 6 6.8 Use design expert software to build an experimental plan matrix with 27 experiments and evaluate the convergence of the model through analysis of variance. The research model results are determined to be consistent with the experiment. After removing the unimportant factor. The target function is determined and represented by the quadratic regression equation as follows: + Y1 = 29.42 + 2.93A + 0.88B + 1.78C + 0.76D - 0.89AB + 1.09AD - 1.02BC - 1.3B2 - 1.1D2 (1) + Y2 = 21.22 + 2.32A + 0.68B + 0.99C + 0.41D - 0.59AB + 0.62AD - 1.11BC + 0.39BD - 0.92B2 - 0.57D2 (2) + Y3 = 29.65 + 2.55A + 1.06B + 1.05C + 0.61D - 0.46AC + 0.49AD - 0.97BC - 0.91B2 (3) 4.3.2.3. Extraction process optimization The extraction process should be optimized so that all three target functions Y1, Y2, and Y3 are maximum. This is solved by solving the optimization problem by Design expert 7.0 software according to the aspiration function method with priority levels (from 1 to 5). In this problem, with the set targets, we choose the priority for the target functions as follows: Function Y1 (level 5); function Y2 (level 3); function Y3 (level 2). In terms of technological parameters as table 4.3.2.4, the predicted value of the target functions in turn is Y1 = 33.64 (mg GAE / g); Y2 = 25.1 (mg QE / g) and Y3 = 33.33 (%). Table 4.3.2.3a. Optimized results of technology variables codes Independent Variables A B C D Extraction time (min) Ethanol concentration (%) Microwave power (W) Solvent pH 1.34 0.23 0.26 0.7 50.1 64.6 441.6 5.4 At the optimal conditions, conducting experiments comparing experimental results with theoretical calculation results shows that the difference is very small. Demonstrate construction model has high accuracy. 4.3.3. Research model and optimization of phenolic extraction process by ultrasonic extraction method 4.3.3.1. Effect of univariate factors on the objective function 4.3.3.2. Modeling and defining the regression equation of the objective function. 19 This problem is based on experimental data on the influence of univariate technology parameters on the target function, they choose the research model according to the 2nd order model of Box-Behnken. The original levels (0), the low (-1), and the high level (+1), of the factors (with k = 4) and the range of variation are shown in Table 4.3.3.2a. Table 4.3.3.2a. Experimental levels of technological variables Independent Variables code Avariable Range (Δ) Level -1 0 1 Z1: solvent/material (v/w) A 2 5 7 9 Z2: Extraction temperature (0C) B 15 30 45 60 Z3: ultrasonic power (W) C 40 100 140 180 Z4: Extraction time (min) D 15 45 60 75 Use design expert software to build an experimental plan matrix with 29 experiments and evaluate the convergence of the model through analysis of variance. The research model results are determined to be consistent with the experiment. After removing the unimportant factor. The target function is determined and represented by the quadratic regression equation as follows: + Y1 = 33.60 + 1.35B – 0.94C + 1.1D – 1.52AC + 1.7BC + 2.49BD + 1.63CD – 2.45A2 – 4.8B2 – 4.58C2 – 3.30D2 (1) + Y2 = 28.59 + 2.44A + 1.75C + 5.97D + 2.39AB + 5.33AD – 2.27BC – 4.72A2 – 2.75B2 – 2.9C2 – 2.17D2 (2) 4.3.3.3. Extraction process optimization Similar to the previous problem, the raw material extraction of Docynia indica fruits needs to be optimized so that both the target functions Y1 and Y2 reach the maximum value. This is solved by solving the optimization problem with Design expert 7.0 software according to the aspirational function method with priority levels (from 1 to 5). In this problem, with the set objectives, the student chooses the priority for the target functions as follows: Function Y1 (level 5), function Y2 (level 3). At the conditions of technological parameters as shown in Table 4.3.3.4, the predicted values of the target functions are Y1 = 33.0 (mg GAE / g) and Y2 = 32.83 (%), respectively. Table 4.3.3.3. Optimized results of technology variables code Independent Variables A B C D Solvent/ material (v/w) Extracti on tempera ture (0C) ultraso nic power (W) Extraction