Summary of Chemical doctoral thesis: Study on chemical composition and develop extraction technology process to make valuable products from Docynia indica (Wall.) Decne) fruits in Vietnam

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Research to isolate, determine chemical structure and probe the biological activity of chemical components in apple and Docynia indica fruits; Research to optimize the extraction technology process to create products from Docynia indica fruits.

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Nội dung Text: Summary of Chemical doctoral thesis: Study on chemical composition and develop extraction technology process to make valuable products from Docynia indica (Wall.) Decne) fruits in Vietnam

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY …………***………… LE XUAN DUY STUDY ON CHEMICAL COMPOSITION AND DEVELOP EXTRACTION TECHNOLOGY PROCESS TO MAKE VALUABLE PRODUCTS FROM DOCYNIA INDICA (WALL.) DECNE) FRUITS IN VIETNAM Major: Chemical engineering Code: 9.52.03.01 SUMMARY OF CHEMICAL DOCTORAL THESIS HA NOI – 2020
This thesis was completed at Graduate University of Science and Technology - Vietnam Academy of Science and Technology. Supervisors: 1. Assoc. Prof. PhD. Vu Dinh Hoang School of Chemical Engineering - Hanoi University of Science and Technology 2. Assoc. Prof. PhD. Nguyen Manh Cuong Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology Examiner 1: … Examiner 2: … Examiner 3: …. The thesis defense was monitored by the Graduate University level Board of Examiners, held at: Graduate University of Science and Technology - 18 Hoang Quoc Viet - Cau Giay - Ha Noi. At ……….. , ….………………….. 2020 The thesis is available in Vietnam National Library and Library of Graduate University of Science and Technology
PUBLISHED SCIENTIFIC WORKS 1. Xuan Duy Le, Manh Cuong Nguyen, Dinh Hoang Vu, Minh Quan Pham, Quoc Long Pham, Quang Tung Nguyen, Tuan Anh Nguyen, Van Thinh Pham, Long Giang Bach, Tuong Van Nguyen. “Optimization of Microwave-Assisted Extraction of Total Phenolic and Total Flavonoid from Fruits of Docynia indica (Wall.) Decne. Using Response Surface Methodology”. Processes 7.8 (2019): 485. (SCI-E, Q2), Doi:10.3390/pr7080485. 2. Le Xuan Duy, Le Ba Vinh, Gao Dan, Vu Dinh Hoang, Tran Quoc Toan, Young Ho Kim, Nguyen Manh Cuong “Soluble epoxide hydrolase inhibitors from Docynia indica (Wall.) Decne.”. Nat. Prod. Res. (2020): 1-6 (SCI-E, Q2), DOI: 10.1080/14786419.2020.1774759. 3. Le Xuan Duy, Tran Quoc Toan, Le Tat Thanh, Cam Thi Inh, Do Huu Nghi, Ha Viet Hai, Vu Dinh Hoang, Do Thi Nguyet, Pham Quoc Long. “ Primary study on the chemical composition from the fruits of Docynia indica in Viet nam”. Vietnam J. Technol 53 (4C) (2015): 81-87. 4. Le Xuan Duy, Tran Quoc Toan, Do Huu Nghi, Le Tat Thanh, Vu Dinh Hoang, Young Ho Kim, Nguyen Manh Cuong. “Triterpene acids from Docynia indica fruits and their cytotoxic activity”. Vietnam J. Technol 56 (2018): 199-204 5. Le Xuan Duy, Nguyen Manh Cuong, Vu Dinh Hoang, Pham Minh Quan, Lai Phuong Phuong Thao, Pham Quoc Long, Tran Quoc Toan. “Process optimization for the extraction of phenolics from the fruits of Vietnam Docynia indica (Wall.) Decne.” Proceeding of international workshop (2019), pp. 506-513 (ISBN:978-604-965-263-9).
1 INTRODUCTION 1. The urgency of the thesis Since ancient times, people have known how to use natural plant sources for health care, prevention, and treatment of diseases. In recent years, the trend of using herbs for prevention and treatment has increased. Today, herbs are not only used in raw form according to traditional medicine but have been applied modern technologies in the field of Chemistry, Biology combined with pharmaceutical technology to create valuable products. High nutritional value, strong pharmacological properties. Vietnam is located in a tropical monsoon climate, so it has a rich and diverse plant source, estimated to have over 13,000 species. According to ethnic medicine, there are more than 4000 species used as medicine or for human health care. With the orientation of developing medicinal plants to serve daily life, nowadays, many areas for cultivation and planting of medicinal plants have been formed on a large scale. Especially, in some Northwestern provinces such as Yen Bai, Son la, Dien Bien in recent years, Docynia indica fruits are strongly developing to serve the needs of consumers. Docynia indica (Wall.) Decne, belongs to the Rosaceae family. In our country, Docynia indica fruit are distributed at altitudes from 1500m to 2000m in the Northwest mountainous provinces. According to traditional medicine, Docynia indica fruit has a sour taste, a little sweetness, warmth, has a flavor effect, belongs to the digestive group of chemicals, mainly treats digestive disorders due to eating a lot of meat, grease, eat indigestion, help to eat delicious dried Docynia indica fruits is a medicine of oriental medicine that helps gastric juice increase secretion of bile acids and gastric juice pepsin, prevent lipid metabolism disorders and reduce blood fat ... Recent modern studies have shown fruit extracts Docynia indica fruits has antibacterial, anti-glucoside and lipid metabolism disorders; Several chemical ingredients isolated from Docynia indica fruits have anti-inflammatory and hypoglycemic effects. The research and processing of Docynia indica fruits to create health care products, support for the treatment of a number of diseases is very interested. However, up to now, studies on Docynia indica fruits both domestically and internationally have been very modest. Therefore, it is necessary to have in-depth studies on the chemical composition as well as the biological activity of the docynia indica, and to expand research into technologies for the extraction and enrichment of chemical components with high biological activity. The active ingredient group phenolic) in
2 Docynia indica fruits to meet the requirements for the formulation of high-quality products. From the above problems, we choose the topic “Study on chemical composition and develop extraction technology process to make valuable products from Docynia indica (Wall.) Decne) fruits in Vietnam”. 2. The research objectives of the thesis Research to isolate, determine chemical structure and probe the biological activity of chemical components in apple and Docynia indica fruits; Research to optimize the extraction technology process to create products from Docynia indica fruits 3. The main research contents of the thesis • Study on isolation and structure of some chemical components in ethyl acetate extract of Docynia indica (Wall.) Decne): Preparation of extract residue, isolation of compounds by chromatic method sign, determine chemical structure by modern physical and chemical methods. • Evaluation of biological activity: Cardioprotective, antioxidant and inhibitory effects of some cancer cell lines of total extracts, fractionated extracts and some compounds isolated. • Researching and optimizing the extraction and drying technology processes to create phenolic rich phenolic Docynia indica fruits extract powder at laboratory scale 4. The structure of the thesis The thesis consists of 147 pages with 76 tables and 63 pictures. The dissertation's layout is as follows: Introduction 2 pages, overview 26 pages, subjects and research methods 8 pages, experiment 11 pages, results and discussion 87 pages, conclusions and recommendations 03 pages, 01 page published list of published works, 9 pages for references CHAPTER 1. OVERVIEW The overview consists of four main parts: Part 1 introduces the genus of Docynia indicas (Docynia), the rose family (Rosaceae); information about Docynia indica fruits trees, Docynia indica fruits such as distribution and production of Docynia indica fruits in our country; Chemical composition and biological effects of Docynia indica fruits too. Part 2 introduces the exploitation, processing, and use of Docynia indica fruits in our country, the current technology of processing Docynia indica fruits. Part 3 introduces the group of phenolic active ingredients that occupy a large amount of Docynia indica fruits, general characteristics,
3 and modern phenolic extraction methods. Part 4 introduces the surface response method (RSM) and technology process optimization CHAPTER 2. SUBJECTS AND METHODOLOGY 2.1. Research subjects Docynia indica (Wall.) Decne apple, collected in Bac Yen District, Son La Province in September 2014 and October 2017. The sample is scientifically identified by Dr. Nguyen Quoc Binh, Museum of Nature Vietnam - VAST. 2.2. Research Methods 2.2.1. Quantitative method of some chemical components 2.2.1.1. Quantitative method of total phenolic The total phenolic content was determined according to the method of Singleton et al. (1999) using Folin-Ciocalteu reagent and gallic acid standard. Total phenolic content = C.V/m; With C: Phenolic concentration determined according to the standard curve (µg/ mL); V: Total extract volume (mL); m: High extractable mass corresponding to the amount of sample extracted (g) 2.2.1.2. Quantitative method for total flavonoids Total flavonoid content was determined by the method of Zhishen et al. (1999). The principle of the method is based on the yellow complexation between the flavonoids and the AlCl3 solution. The color intensity proportional to the flavonoid content was determined at 415 nm. Quercetin is used as the reference standard Total flavonoid content = C.V/m; With C: Flavonoid concentration determined according to the standard curve (µg / mL); V: Total extract volume (mL); m: High extractable mass corresponding to the amount of sample extracted (g) 2.2.2. Extraction, isolation, chemical structure determination 2.2.2.1. Extraction method + Microwave extraction method: Microwave-assisted extraction + Ultrasonic extraction method: Extraction with support of ultrasound + Reflux extraction method: Extraction with heating by the electric stove, the solvent is circulated by the condenser system Soxhlet extraction method: Using soxhlet extraction equipment to extract high quantitative extraction.
4 2.2.2.2. Methods of isolation of chemical components + Column chromatography: normal phase silica gel, reverse phase, Sephadex ... + Thin layer chromatography: performed on pre-coated thin plates, detecting material streaks by UV light at the wavelengths of 254 and 365 nm or color detection by reagents. 2.2.2.3. Methods of chemical structure determination Combining physical and chemical parameters (melting point, polarity) with modern spectroscopy methods such as mass spectrometry (ESI-MS), high-resolution mass spectrometry (HR-ESI-MS), resonance spectrum 2- dimensional 1-dimensional (1H-, 13C-NMR, and DEPT) nucleus (HSQC, HMBC, NOESY ...). 2.2.3. Biological activity assessment method + Cardioprotective activity (sEH) via soluble epoxide hydrolase inhibition mechanism was evaluated based on fluorescence analysis with positive control using Auda. + Determination of antioxidant activity by DPPH method (1,1-diphenyl-2- picrylhydrazyl). The absorption of DPPH at the wavelength λ = 515 nm is determined by the ELISA reader after applying DPPH to the test sample solution on a 96-well micro-session. + Cytotoxic activity was tested by MTT method [3- [4,5-dimethylthiazol- 2-yl] -2,5-diphenyltetrazolium bromide] on two liver cancer cell lines (Hep-G2), and cervical cancer (HeLa) The tests were conducted at Faculty of Pharmacy, Chungnam University, Korea and Department of Experimental Biology, Institute of Natural Products Chemistry – VAST 2.2.4. Experimental planning method and technology process optimization Experimental planning method and technological process optimization are applied according to the quadratic model of Box-Willson or Box-Behnken depending on each specific problem. After building the model, we need to rate the convergence of the model through the analysis of variance. If the model converges, we determine the regression equation and optimize the technological parameters by Harrington's aspirational function (1965). Use Design Expert 7.0.0 software to model and optimize process technology parameters.
5 CHAPTER 3. EXPERIMENTAL 3.1. Preparation of total and fractional extracts 15 kg of fresh Docynia indica fruits are graded, washed, and sliced. Dry the yeast at 1100C for 20 minutes and dry the sample to dry at 600C (moisture content ≤ 10%). Dried Docynia indica fruits are finely ground by a mill (diameter 0.2 mm sieve) to obtain 5 kg of powder. 2 kg of Docynia indica fruits powder is extracted ultrasound with methanol at room temperature, solvent/material ratio (3/1, v / w). Filter the extract and concentrate using a reduced pressure rotary 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 weight TT code Name (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- 11,0 glucopyranoside 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 3S-Thunberginol C 6-O-β- D- 6,5 (New) glucopyranoside 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- 8,2 flavanone 7-O-β-D glucopyranosie 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- 7,6 glucopyranoside 24 TM36 Myricitrin 7,2 25 TM37 2’,6’-dihydroxy-3’,4’- 4,4 dimethoxychalcone 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). [M + 35Cl]¯ CTPT: C21H22O10 [M + 37Cl]¯ [M - H]¯ 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
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 δH (ppm) δC (ppm) (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) / 80.0 5.59 (1H, t, J = 2.5 Hz) 4 3.03 (1H, dd, J= 3.0; 33.6 3.11 (1H, t, J= 2.5 Hz, H- 33.66 17.0 Hz, H-4a) 4a)/3.07 (1H, t, J= 2.5 Hz, 3.24 (1H, dd, J= 3.0; H-4a) 17.0 Hz, H-4b) 3.35 (overlap, H-4b)
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 = 2.0 100.9 6.52 (1H, d, J= 2.0 Hz) / 101.76 Hz) 6.51 (1H, d, J = 2.0 Hz) 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) / 99.77 / 4.97 (1H, d, J = 8.0 Hz) 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) 60.52 3.76 (1H, m) 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 Test Percent fraction concentration inhibition (%) (µM) 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 37.5 4.9 ± 0.5 11 methanol 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 IC50 (µM) 100 µM concentration (%) Control (+) 16.8 ± 0.5 nM Auda 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 (+) 26.40 ± 0.44 TM12 18.05 ± 0.69 Ascorbic acid 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 Avariable Variables Codes 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