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MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF THESIS THESIS Specialization: Biotechnology Industry code: 7420201 NGUYEN THI THU HAU ANTIOXIDANT AND ENZYME TYROSINASE INHIBITOR OF PINEAPPLE (Ananas comosus) YEAR 2022 WORK COMPLETED AT CAN THO UNIVERSITY Main instructor: Assoc.Prof.Dr Tran Nhan Dung Sub-instructor: Assoc Prof.Dr Huynh Van Ba The thesis is defended before the university doctoral thesis evaluation board Meeting at: …………… , Can Tho University) At hour day month year Review 1: Review 2: Review 3: Confirmation of review by the Chairman of the Council Thesis can be found at the library: - Learning Resource Center, Can Tho University; - Vietnam National Library i LIST OF PUBLICATIONS Domestic magazine Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Tong Van Hai., 2020 A survey on antioxidant activation from ethanol extraction from the leaves, stems and peels of pineapple (Ananas comosus) at Tac Cau, Kien Giang region Journal of Agriculture and Rural Development, No (2020), 74-80 Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Trinh Chi Bao, 2020 Survey activity antioxidant from extracts ethanol the stems and fruit pineapple (Ananas comosus) at Tac Cau, Kien Giang region Journal of Medicine, No (1), 45-49 Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Nguyen Minh Chon, Nguyen Duc Do, Vo Thi Yen Linh, Le Thi Thu Doan, Nguyen Thi Truc Anh, 2020 Investigation of antioxidant activity and tyrosinase inhibition of methanol extract from pineapple leaves at Tac Cau, Kien Giang province Vietnam Journal of Agricultural Science and Technology, No 10 (2020), 109-114 Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Vo Thi Yen Linh, Le Thi Thu Doan, Thi Ngoc Quyen, Bui Tran Hoang An, 2020 A survey on antioxidant activation from ethanol extraction from the peels and fruit of pineapple (Ananas comosus) of green and ripe stages at Tac Cau, Kien Giang region Journal of Tra Vinh University, 12 (2020), 1-11 Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Nguyen Minh Chon, Nguyen Duc Do, Pham Thi Be Tu, Tran Thi Ngoc Ly, Lam Huynh Duc, Pham Thi Ai Vi, Ly Le Quyen, Le Thi Ngoc Nga, 2021 Antioxidant and inhibition of tyrosinase form methanol extraction of stems and fruits pineapple (Ananas comosus) at Tac Cau, Kien Giang province Journal of Agriculture and Rural Development, No 3+4 (2020), 68-74 Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Nguyen Minh Chon, Nguyen Duc Do, Pham Thi Be Tu, 2021 Investigation of antioxidant and tyrosinase inhibitory activities from the methanol extract of pineapple peel (Ananas comosus (L.) Merr.) collected in Tac Cau area, Kien Giang Journal of Medicine and Pharmacy, March 17 (2021), 21-28 Proceedings of the domestic conference ii Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Ninh Khac Huyen Tran, Lam Thi Kim Ngan, Vuong Tu Ky, Tran Ngoc Trong, Le Thi Thu Doan, 2020 A survey on antioxidant activation from ethanol extraction from the peels and fruit of pineapple (Ananas comosus) of green and ripe stages at Tac Cau, Kien Giang region Proceedings of the National Biotechnology Conference 2020, 194 – 199 Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Vo Thi Yen Linh, Le Thi Thu Doan, Thi Ngoc Quyen, Bui Tran Hoang An, 2020 A survey on antioxidant activation from ethanol extraction from the peels and fruit of pineapple (Ananas comosus) of green and ripe stages at Tac Cau, Kien Giang region Proceedings of the Conference on Application of Biotechnology in Agriculture and Fisheries Production (2nd time), 2020, iii CHƯƠNG 1: CHAPTER 1: INTRODUCTION 1.1 Pose the problem Pineapple (Ananas comosus (L.) Merr.) of the bromeliaceae family, native to Paraguay, is the third most valuable fruit in the world after bananas and citrus (Vrianty et al al., 2019) In the body of organisms (including humans), free radicals are always produced, which leads to the destruction of organelles and internal structures of cells due to mutations that lead to dangerous and degenerative diseases cells (Ghasemzadeh, 2012; Vrianty et al., 2019) Antioxidant compounds are compounds that slow down or prevent the growth of free radicals that protect cells and the body The disadvantages of cell degeneration can be prevented by supplementing with antioxidants from food and medicine (Ghasemzadeh, 2012; Ha & Thu, 2009; Vrianty et al., 2019) Tyrosinase is the main enzyme in the biosynthetic pathway of melanin, a complex of pigment granules in the skin, hair, retinal mucosa, and melanin surrounding the nerve axons Melasma is the phenomenon of increasing the content of melanin to protect the skin, helping the skin against ultraviolet rays (Bonesi et al., 2019; F Kong et al., 2000; Pires et al., 2021) Chemically synthesized tyrosinase inhibitors are not really safe and effective Therefore, the problem of finding compounds that inhibit tyrosinase from nature is studied for application in the fields of medicine, food industry, environmental treatment and cosmetics (Lai et al., 2018; Yuan et al., 2020) In the world and in Vietnam, there have been many studies on antioxidant capacity from pineapple fruit pulp, leaves, and skin, but there has not been an overall comparative assessment of antioxidant capacity of leaves, stem, fruit pulp and pineapple peel At the same time, there are few studies on the ability of pineapple extracts to inhibit tyrosinase activity The purpose of the thesis is to study on “Antioxidant capacity and tyrosinase inhibition of pineapple plant” (Ananas comosus (L.) Merr.)” in order to improve the application value of pineapple in other fields such as pharmaceuticals and cosmetics 1.2 Dissertation Objectives Evaluation of antioxidant capacity and tyrosinase inhibition of pineapple leaf, stem, fruit pulp and peel extracts (Ananas comosus (L.) Merr.) Qualitative and quantitative composition of secondary plant compounds (of which the most important are polyphenols and flavonoids) of crude extracts and compare the antioxidant capacity and tyrosinase inhibition of the extracts of the high sample has the ability to effectively inhibit tyrosinase 1.3 Research content Content 1: Preliminary survey to select high extraction solvent and raw material areas and identification of pineapple species in Tac Cau area Content 2: Investigate the antioxidant capacity (DPPH reduction activity, Fe3+ ion reduction activity to Fe2+ ions, Cu2+ ion reduction activity to Cu+ ions) and tyrosinase inhibition of Tac Cau pineapple high methanol Content 3: Fractional separation chromatography of pineapple peel methanol extract and investigation of antioxidant and tyrosinase inhibition of highly active fractions Content 4: Investigate the ability of inhibiting melanin production of highly active fraction from methanol extract F1 of pineapple peel on B16F10 cell line Content 5: Investigate compounds in pineapple peel F1 methanol extract that have antioxidant and tyrosinase inhibitory properties through GC-MS spectrum analysis 1.4 Subjects and scope of research 1.4.1 Research subjects The study was carried out on pineapple plants (leaves, stems, flesh and pods) collected in Hon Dat (Long Xuyen Quadrangle) and Tac Cau (Western Hau River ecoregion) of Kien Giang province 1.4.2 Research scope The study focused on the antioxidant capacity (conducted by methods of DPPH reduction, Fe3+ ion reduction, Cu2+ ion reduction) and the ability to inhibit tyrosinase enzyme activity of extracts from leaves, stems, and flesh pineapple fruit and bark The studies were carried out from May 2018 to January 2021 at the Biotechnology Research and Development Institute, Can Tho University and the Center for Experimental Practice Management, Kien Giang University 1.5 Meaning of research 1.5.1 Scientific significance The thesis provides useful scientific information on the antioxidant and tyrosinase inhibitory ability of pineapple extracts for application in fields such as medicine, pharmacy and cosmetic chemistry 1.5.2 Practical significance Research results show the potential of using by-products in the pineapple processing industry, preparing extracts from pineapple peels using raw materials in medicine and pharmacy to help reduce environmental pollution and increase value of pineapple products in pineapple farming industry 1.6 The novelty of the thesis The thesis has determined that pineapple leaves, stems, fruit pulp and peel have antioxidant capacity and inhibit tyrosinase activity The pineapple peel methanol extract has higher antioxidant capacity (Cu2+ deionization) and inhibits tyrosinase activity than the methanol extract from pineapple leaves, stems, and flesh The content of total polyphenols and total flavonoids was related to the antioxidant capacity of pineapple extract and to the ability to inhibit tyrosinase activity of pineapple extract The results of inhibiting melanin production on the B16F10 melanoma cell line of the F1 fraction extracted from pineapple peel methanol extract open a new direction of application of pineapple by-products in medicine and pharmacy (prevention and treatment of related diseases) related to the formation of melanin such as Alzheimer's, Parkinson's, freckles, melasma and diseases related to melanoma cell lines) CHƯƠNG 2: CHAPTER 2: RESEARCH METHODS 2.1 Research media 2.1.1 Time and place Time: From May 2018 to January 2021 Location: Biotechnology Research and Development Institute, Can Tho University and Center for Experimental Practice Management, Kien Giang University 2.1.2 Materials Leaves, stem, fruit pulp and peel of pineapple (Ananas comosus (L.) Merr.) were collected in two different ecological regions of Kien Giang province, Vietnam (Western Song Hau ecoregion - Tac Cau pineapple and natural habitat) Long Xuyen Quadrangle - Hon Dat pineapple) The pineapple samples studied were identified based on morphological characteristics according to the Vietnamese plant classification system according to Pham Hoang Ho, (1993) Criteria for selecting experimental samples: leaves at the top, fruit and pineapple skin are aged from 24 to 26 weeks after flowering, pineapple stems are aged from to years old Cell B16F10 was provided by the ATCC cell bank (USA).Phương pháp nghiên cứu 2.1.3 Content 1: Preliminary survey to select raw materials and extraction solvents Objectives: to determine the raw material area and type of extraction solvent for further studies of the thesis Evaluation of antioxidant capacity of pineapple ethanol extract in Hon Dat and Tac Cau a Extraction from parts of pineapple plants collected at Hon Dat and Tac Cau in ethanol solvent Experimental layout: determine the moisture content of raw materials by drying method (Appendix 9.6, Vietnam Pharmacopoeia V) by infrared moisture drying balance MX50 AND (Japan) Experiments were arranged and conducted according to Phụng, 2007; Rodriguez., 2017; Vrianty et al., 2019 with correction (Table 3.1) Evaluation criteria: Efficiency (%) = (CT mass (g)/Sample weight (g)) x 100 (1) Table 3.1: Experimental arrangement for extracting pineapple leaf, stem, flesh and peel extracts in Hon Dat and Tac Cau in ethanol Experiment L_EHĐ T_EHĐ TQ_EHĐ V_EHĐ L_ETC T_ETC TQ_ETC V_ETC Parts of the Plant Leaves Trunk Fruit flesh Peel Leaves Trunk Fruit flesh Peel Ecological zone Solvent Sample/solvent ratio (w/v) Hon Đat Ethanol 1:4 Tac Cau Ethanol 1:4 b Investigation of antioxidant capacity of pineapple ethanol extract in Hon Dat and Tac Cau areas Experimental arrangement: The experiment was performed according to the description of (Jovanović et al., 2018; Li et al., 2014) with correction Dilute 0.1 Mm DPPH solution; Phase the extract to the appropriate concentration range (investigated, mixed with methanol); Dilute the standard vitamin C solution to the concentration range (12.5 - 150 µg/mL) (mix with methanol) Implementation process Place mL of the extracts at the prepared concentrations in turn into a test tube, add mL of 0.1 mM DPPH After 30 of incubation in the dark, the OD was measured using a spectrophotometer at 517 nm The control sample was carried out in the same way but replaced the extract with methanol Build vitamin C standard curve: build vitamin C standard curve with the same procedure as for extract Evaluation criteria: Based on the OD value of vitamin C and extracts at 517 nm, calculate the percentage of preparation at different concentrations according to formula (2) The DPPH-reducing activity was expressed as a percentage of inhibition: Percentage inhibition (%) = (A0 - A)/A0 x 100 (2) From the percentage inhibition value at the concentrations of each extract sample, construct a calibration curve y = ax + b From this, infer the IC50 value (the concentration of extract or vitamin C at which 50% inhibition) The lower the IC50 value, the higher the crude sample will have the higher antioxidant activity and vice versa Experiment 2: Investigation of antioxidant capacity of pineapple extract in Tac Cau area extracted in ethanol/methanol solvent a Extraction from parts of pineapple plant collected at Tac Cau in ethanol/methanol Table 3.2: Experimental arrangement for preparation of crude extracts of pineapple leaves, stems, flesh and skin in different ecological regions and solvents Experiment L_ETC T_ETC TQ_ETC V_ETC L_MTC T_MTC TQ_MTC V_MTC Parts of the Plant Leaves Trunk Fruit flesh Peel Leaves Trunk Fruit flesh Peel Ecological zone Solvent Ethanol Sample/solvent ratio (w/v) 1:4 Tac Cau Methanol 1:4 Experimental layout and monitoring criteria: similar to experiment Experiment 3: Sequencing genes to identify Tac Cau pineapple species Purpose: This study aims to redefine the species of pineapple in Tac Cau area (endemic to Kien Giang province a DNA Extraction ollow the CTAB (Cetyl trimethyl ammonnium bromide) procedure, described by Rogers and Bendich, (1988) Use a standard commercial kit for sample purification (PCR Clean up) The process follows the author Tran Nhan Dung et al , 2012 b Perform PCR reaction In good quality DNA samples, primers that amplify the ITS sequence region (Baldwin et al., 1995) are used to perform PCR reactions as follows: + ITS1: 5’-TCC GTA GGT GAA CCT GCG G-3’ + ITS4: 5’-TCC TCC GCT TAT TGA TAT GC-3’ Experiment 9: Investigation of antioxidant capacity and tyrosinase inhibition of pineapple peel extracts Objective:: identify the high fraction sample with the strongest tyrosinase inhibitory activity to test the inhibition of melanin production on the melanoma cell line B16F10 and analyze the GC-MS spectrum Experimental layout and monitoring criteria: similar to experiment 5a, 5b 2.1.6 Content 4: Investigation of the ability to inhibit melanin production on melanoma cell line B16F10 of high fractions F1 Experiment 10: Investigation of inhibition of melanin production by pineapple peel F1 fraction on B16F10 cell line Objective: Determination of the inhibitory activity of melanin production of pineapple peel F1 fraction on the melanocyte cell line B16F10 Experimental arrangement: Samples were sent to the Biological Laboratory, Institute of Biotechnology under the Vietnam Academy of Science and Technology Experiments were carried out according to (Kim et al., 2014; Lee and Cho, 2018; L Q Loan et al., 2018) with correction Conduct experiments: B16F10 cells were grown in a 6-well plate at a concentration of 1x105 cells/mL, and continued to incubate the cells with the study sample at different concentrations in the presence of αMSH (10 nM) for 48 h After days of incubation at 37oC and 5% CO2, cells were harvested and washed with PBS The cell residue was then dissolved in 1N NaOH solution containing 10% DMSO and incubated at 80oC for hour After measuring the OD value at 405/450 nm Rating Criteria: The ability to inhibit melanin production of the studied samples was compared with the negative control without sample incubation according to the formula (10): % Inhibits melanin production =100%- ((ODmẫu – OD– αMSH)/(OD+α MSH – OD-α MSH)) x 100 (10) 2.1.7 Content 5: GC-MS spectrum analysis of pineapple peel F1 fractions Experiment 11: GC-MS mass spectrometry analysis of pineapple peel F1 fractions Objective: The study was carried out to determine in the F1 fraction of pineapple peel there are compounds and groups of compounds that 11 have antioxidant and tyrosinase inhibitory properties through a series of previous studies Experimental arrangement: The experiment was performed according to the description of (Ikram et al., 2020) with correction Sample phase: The sample phase is carried out at the Institute of Biotechnology Research and Development, Can Tho University Dilute CPD F1 pineapple peel in acetone (France) or methanol (Germany) to get mL of a solution with a concentration of 100 ppm Method of preparation: Weigh 1mg + 1mL to get a concentration of 1000 ppm; Withdraw 100 microliters of stock solution 1000 ppm + 900 microliters of solvent to obtain a concentration of 100 ppm; 10 ppm and ppm phase Performing analysis: After being prepared, samples are sent for GCMS analysis at Can Tho Technical Center for Standards, Metrology and Quality The sample analysis temperature program is installed on the Shimadzu GCMS-QP2010 system Processing GC-MS data: Based on ion spectroscopy combined with data from NIST, WILEY libraries and previous de-spectral information to identify compounds present in F1 analytical samples 2.2 Data processing methods Raw data were calculated, processed and charted using Excel 2016 Statistical data were processed by Minitab 16 software such as: analysis of variance ANOVA, coefficient of variation (CV%), average comparison Average the difference using Tukey's test 12 CHƯƠNG 3: CHAPTER 3: KẾT QUẢ VÀ THẢO LUẬN 3.1 Preliminary survey results for raw materials 3.1.1 High antioxidant results of pineapple ethanol extract in two ecological zones Hon Dat and Tac Cau Results of sampling, processing and extraction of extracts from leaves, stems, flesh and skin of pineapple in Tac Cau and Hon Dat in ethanol solvent Table 4.1: Results of extracting pineapple extracts at Hon Dat and Tac Cau Parts of Experiment the Plant Solvent Weight (g) Eco zone Extraction Humidity (%) Fresh samples Leaves L_EHĐ 20,03±0,29 81,17±1,33 Trunk T_EHĐ 32,6±0,77 63,23±2,05 Hon Đat 112,68±2,72 84,6±1,05 Fruit flesh TQ_EHĐ Peel V_EHĐ 51,69±0,43 Ethanol 80,9±1,25 Leaves L_ETC 21,08±0,10 81,03±0,42 Trunk T_ETC 35,01±0,20 63,7±0,92 Tac Cau 129,95±6,68 84,3±0,62 Fruit flesh TQ_ETC Peel V_ETC 54,35±0,80 81,6± ,96 CT 5,1 5,5 7,3 5,6 5,2 5,1 7,6 5,9 Performance (%) ± 0,03e 3,3 ± 0,08d 11,27±0,27b 5,17±0,04c 2,11±0,01e 3,5±0,02d 12,99±0,67a 5,44±0,08c Preliminary survey to select the material area (Ecoregion of Long Xuyen Quadrangle - Hon Dat - and the West River Hau ecoregion - Tac Cau) and suitable solvents (ethanol/methanol) The moisture content of fresh materials showed that the moisture content in pineapple body was about 81.03 - 81.17%, that of pandan leaves was 63.23 - 63.7%, that of fruit flesh was 84.3 - 84.6% and that of peel the result is 80.9 – 81.6% The extract was dried at 45oC until the moisture content reached from 5.1 to 7.6% (standard of Vietnam Pharmacopoeia V) Statistical processing results show that, when using the same type of sample, there was no difference in high extraction efficiency High antioxidant results of pineapple ethanol extract in two ecological zones Hon Dat and Tac Cau The results of investigation of antioxidant activity of pineapple extracts in two ecological regions extracted in ethanol solvent are shown in Table 4.2 13 Table 4.2: Results of antioxidant activity of pineapple ethanol collected in Hon Dat and Tac Cau ecological areas by DPPH reduction method Experiment L_EHĐ T_EHĐ TQ_EHĐ V_EHĐ L_ETC T_ETC TQ_ETC V_ETC Ascorbic acid Ecological zone De-DPPH (IC50 µg/mL) 104,08 ± 1,89ef 251,92 ± 7,69b 292,93 ± 8,86a 143,63 ± 2,64d 76,74 ± 1,35g 196,52 ± 17,92c 220,41 ± 9,85c 112,85 ± 8,66e 84,07 ± 2,12fg Hon Đat Tac Cau control sample Thus, the Tac Cau ecoregion showed a higher antioxidant capacity than the extracts from the Hon Dat ecoregion The IC50 index of leaf samples from Tac Cau area was lowest (76.74 ± 1.35) µg/mL, which was lower than that of ascorbic acid control (84.07 ± 2.12) µg/mL When comparing the antioxidant results on each pair of similar parts of pineapple in two different ecological regions, the leaf sample of Tac Cau (IC50 = 76.74 µg/mL) showed a higher antioxidant capacity than 36 times that of Hon Dat leaves (IC50 = 104.08 µg/mL) Similarly, the antioxidant capacity of Tac Cau pineapple peel (IC50 = 112.85 µg/mL) is 1.27 times higher than that of Hon Dat pineapple peel (IC50 = 143.63 µg/mL); sample of Tac Cau fruit (IC50 = 220.41 µg/mL) was 1.33 times higher than that of Hon Dat fruit (IC50 = 292.93 µg/mL); Tac Cau pineapple stem sample (IC50 = 196.52 µg/mL) was 1.28 times higher than that of Hon Dat pineapple stem sample (IC50 = 251.92 µg/mL) The research results are consistent with the previous study by Rodriguez., (2017), which studied TPC, TFC and antioxidant capacity of pineapple plants in different ecological regions in Bzazil Results in different ecological regions showed different results in terms of antioxidant capacity, total polyphenol content and total flavonoid content Tac Cau islet has rich alluvial soil due to the accretion of rivers and typical topography is brackish water, alkaline and salty soil, but the quality of juicy pineapple is grown on a large area of more than 2,700 hectares provide a large source of raw materials for production and research 14 At the same time, the research results are similar to the previous study by Yuris and Siow, 2014, which studied the antioxidant capacity of fruit pulp in pineapple varieties Joseplune, Morris and Sarawak in different ecological regions of the species Ananas comosus As a result, different ecological regions, different material varieties and different solvents, the ability to reduce DPPH is also different Therefore, the leaves, stem, fruit pulp and pineapple peel were selected in the Tac Cau ecoregion to conduct research for further experiments.Kết kháng oxy hóa cao chiết ethanol/methanol dứa vùng Tắc Cậu, Kiên Giang Results of sampling, processing and extraction of extracts from leaves, stems, flesh and skin of pineapple in Tac Cau in ethanol/methanol solvent Each type of solvent will have the ability to extract different groups of compounds Two types of solvents commonly used in the extraction of natural compounds are ethanol and methanol because of their low cost and high efficiency, large solvent recovery and low toxicity (Phụng, 2007; Trúc et al., 2020; Vrianty et al., 2019) The results of preparing extracts from leaves, stems, fruit pulp and pineapple peel in Tac Cau area, Kien Giang province in ethanol and methanol are shown in Table 4.3 Table 4.3: Results of sampling, processing and extraction of pineapple extract at Tac Cau in ethanol/methaol Weight (g) Part Experiment Solvent Extraction Leaves L_ETC 21,03±0,29 Trunk T_ETC 32,2±0,77 EtOH Fruit flesh TQ_ETC 119,34±3,74 Peel V_ETC 52,36±0,73 Leaves L_MTC 22,08±0,99 Trunk T_MTC 34,35±0,20 MeOH Fruit flesh TQ_MTC 127,95±8,13 Peel V_MTC 53,68±1,33 Humidity * (%) Fresh CT samples 80,83±0,99 5,0 62,57±2,1 5,4 84,6±1,0 7,1 81,27±1,3 5,4 81,97±0,76 5,1 64,37±0,77 5,3 83,97±1,19 7,2 81,97±1,0 5,7 Performance (%) 2,1±0,03d 3,2±0,15c 11,93±0,37a 5,24±0,07b 2,21±0,1d 3,44±0,19c 12,81±0,81a 5,37±0,13b Thus, when using materials from Tac Cau pineapple leaves, stems, fruit pulp and peel extracted in ethanol and methanol, the statistical processing results of extraction efficiency did not differ between samples of the same set parts but only the differences between the parts Extraction efficiency goes from high to low in the following order: pulp 15 → rind → stem → leaf Continue to study on the DPPH reduction activity of the extracts extracted in ethanol and methanol solvents to choose the most suitable solvent for further studies of the thesis Antioxidative results of pineapple ethanol/methanol extracts collected at Tac Cau The results of the investigation of the antioxidant activity of ethanol and methanol extracts of leaves, stems, fruit flesh and pineapple peels in the Tac Cau ecoregion are shown by the IC value of 50 (inhibition concentration of 50%) through the reduction activity DPPH (Table 4.4) The lower the IC 50 value, the stronger the antioxidant activity and vice versa Table 4.4: Antioxidant activity of pineapple ethanol and methanol extract in Tac Cau ecoregion, Kien Giang Experiment L_MTC T_MTC TQ_MTC V_MTC L_ETC T_ETC TQ_ETC V_ETC Ascorbic acid Solvent De-DPPH (IC50 µg/mL) 41,13 ± 0,46g 138,59 ± 11,10d 154,47 ± 4,13c 64,79 ± 4,19f 77,98 ± 1,48f 180,92 ± 6,27b 199,28 ± 3,41a 111,38 ± 7,14e 73,01 ± 2,97f Methanol Ethanol control sample All treatments showed antioxidant capacity through DPPH free radical scavenging test results Pineapple methanol extract showed higher antioxidant activity than pineapple ethanol extract IC 50 of Tac Cau leaf methanol extract was lowest ( 41.13 ± 0.46) µg/mL, which was 1.8 times lower than that of ascorbic acid control (73.01 ± 2.12) µg/Ml The lowest antioxidant capacity was high in fruit pulp ethanol with the highest IC 50 index of 199.28 ± 3.41 µg/mL When comparing the antioxidant activity on each of the extracts extracted in two different solvents, the highest antioxidant capacity was high in leaf methanol (IC 50 = 41.13 ± 0.46 µg/mL) the second is pod ethanol (IC 50 = 64.79 ± 4.19 µg/mL) and foliar ethanol (IC 50 = 77.98 ± 1.48 µg/mL) and the third is rind ethanol (IC 50 =) 111.38 ± 7.14 µg/mL) fourth was high methanol body (IC 50 = 138.59 ± 11.10 µg/mL), fifth was high methanol fruit pulp (IC 50 = 154.47 ± 4, 13 µg/mL) and the lowest 16 antioxidant capacity was high ethanol stem and pineapple pulp Tac Cau, Kien Giang (IC 50 = 180.92 ± 6.27 µg/mL and IC 50 = 199.28 ± 3.41) µg/mL) The results of antioxidant activity of extracts in different solvents and organelles showed different results The study results are also consistent with the study by Jovanovic et al., 2018, which evaluated the antioxidant capacity of the peel, apical leaves, stem, and juice of pineapple (Ananas comosus [L.] Merr.) by DPPH free radical scavenging method The results showed that pineapple peel and stem extracts in absolute methanol had the highest antioxidant capacity (IC 50 = 1.745 ± 0.046 mg/mL), while the lowest oxidizing capacity was high in the pulp extract in aqueous solution The medium is water (IC 50 = 88 ± 2.09 mg/mL) In addition, the difference in DPPH free radical scavenging capacity is also influenced by soil conditions, climatic conditions as well as plant age and season (L Q Loan et al., 2018) Results of gene sequencing of Tac Cau pineapple plant Morphological characteristics The research object is pineapple in Kien Giang, the sample was identified by Nguyen Van Phuoc, Department of Crop Science, Kien Giang University based on morphological characteristics according to Pham Hoang Ho, (1993) and according to the classification system species of Vietnamese herb, identified the research object belongs to the pineapple genus Queen, belongs to the species Ananas Comosus Gene sequencing results The results of comparing the ITS sequences of pineapple samples used in this study showed the similarity from 99.07 to 100% with the ITS sequences of Ananas comosus with the genetic codes HM104185.1, XM_020251500, HM768296.1 The results also showed the similarity of the ITS sequence of pineapple samples with related plants The results also show the similarity of ITS sequences of pineapple samples with some other related plants Specifically, this sequence is similar to the Guzmania hybrid with 98.56% similarity, Elaeis guineensis with 98.20% similarity, Cocos nucifera with 85.20% similarity According to the morphological characteristics and distribution of species in the genus Ananas published on the NCBI data bank, pineapple has similar characteristics to the Tac Cau pineapple tree sample under 17 study When comparing the morphological characteristics of pineapple samples from the Tac Cau area with those of Ananas comosus (L.) Merr described by the author Pham Hoang Ho found a similarity The combination of ITS gene sequence and morphological characteristics described in the book "Trees of Vietnam" by Pham Hoang Ho, 2003, can confirm that the pineapple sample under study is anas comosus (L.) Merr 3.2 Results of antioxidant capacity and tyrosinase inhibition of Tac Cau pineapple high methanol 3.2.1 Quantitative results of total polyphenols and flavonoids in pineapple methanol extract The biological activity of antioxidant and tyrosinase inhibition is highly dependent on the polyphenol and flavonoid content of the extract Therefore, it is necessary to study to determine the content of total polyphenols and total flavonoids The quantitative results of total polyphenols and total flavonoids in the methanol extracts of leaves, stems, fruit pulp, and pineapple peel are shown in Table 4.5 Table 4.5: Contents of total polyphenols, total flavonoids of leaves, stems, fruit pulp, and skin of Tac Cau pineapple Experimen L_MTC T_MTC TQ_MTC V_MTC Total polyphenol content (mg GAE/g) 242,00 ± 2,69b 187,63 ± 2,79c 50,32 ± 1,56d 291,43 ± 0,87a Total flavonoid content (mg QE/g CPĐ) 44,70 ± 1,97a 3,78 ± 0,39c 4,89 ± 0,64c 35,16 ± 2,34b The results of Table 4.5 showed that all extracts samples had the presence of total polyphenols and total flavonoids and there were statistical differences The polyphenol content in Tac Cau pineapple peel methanol was the highest (291.43 mg GAE/g) and the lowest was highest in Tac Cau pineapple pulp methanol (50.32 mg GAE/g) and ranked from low to high as follows: fruit pulp stem leaf and the highest sample was the methanol extract of the fruit rind The flavonoid content was highest in leaf methanol extract (44.7 mg QE/g) and lowest in pineapple stem methanol (3.78 mg QE/g) 18 3.2.2 Antioxidant results of Tac Cau pineapple high methanol The antioxidant activity of the extract was shown by DPPH reduction activity, Fe 3+ reduction activity, Cu 2+ reduction activity, and IC value of 50 (50% free radical inhibitory concentration) (Table 4.6) Table 4.6: Antioxidant activity of pineapple methanol extract Experimen L_MTC T_MTC TQ_MTC V_MTC Acid ascorbic De-DPPH(*) 31,27 ± 3,91c 131,14 ± 11,37a 143,55 ± 14,59a 62,63 ± 4,66b 84,07 ± 2,12 Value IC50 (µg/mL) De-Fe3+ (*) 110,25 ± 2,76c 168,69 ± 4,44b 203,25 ± 3,99a 97,72 ± 0,42d 7,95±0,34 De-Cu2+ (*) 270,65 ± 8,31c 752,56 ± 14,37a 373,53 ± 16,30b 220,95 ± 8,21d 260,33 ± 10,58 In general, the crude extracts have antioxidant activity through reducing DPPH, reducing Fe 3+, reducing Cu 2+ Tac Cau (L_MTC) pineapple leaf extract has antioxidant activity with DPPH reduction activity (IC 50 = 31.27 ± 3.91 µg/mL), times stronger than that of methanol_shell sample (V_MTC) with value (IC 50) = 62.63 ± 4.66 µg/mL) and 4.6 times higher than that of pineapple pulp methanol extract (IC 50 = 143.55 ± 14.59 µg/mL), and 4.2 times higher than that of pineapple pulp pineapple stem methanol sample (IC50 = 131,14 ± 11,37 µg/mL) In contrast, investigating the antioxidant activity of the extract by reducing Fe 3+ , reducing Cu 2+ , the sample V_MTC had the strongest antioxidant activity through the lowest IC 50 value Methanol_pineapple peel has antioxidant activity with Fe 2+ and Cu 2+ ion reduction activitie (IC50 = 97,72 ± 0,42 µg/mL, IC50 = 220,95 ± 8,21 µg/mL) In the structure of tyrosinase enzyme, the active site has the participation of two Cu 2+ ions Therefore, the method of testing the antioxidant capacity by reducing Cu 2+ ion method shows the potential of the method in studying the relationship between antioxidant and inhibiting tyrosinase enzyme activity 3.2.3 The relationship between TPC and TFC oxidant capacity of Tac Cau pineapple plant The results also showed a positive relationship between total polyphenol content and total flavonoid content with antioxidant capacity of extracts from leaves, stems, fruit pulp and pineapple peel through 19 DPPH and deionization ability Cu 2+ Thus, samples of pineapple leaves and skin with high polyphenol and flavonoid content showed high antioxidant activity Similarly, the polyphenol and total flavonoid content of the stem and fruit pulp were low, so the antioxidant activity was also low 3.2.4 Tyrosinase inhibition results of pineapple high methanol Tac Cau area The inhibitory activity of tyrosinase enzyme of Tac Cau pineapple methanol extracts is shown in Table 4.7 Table 4.7: Tyrosinase inhibition of Tac Cau pineapple methanol extracts Experiment L_MTC T_MTC TQ_MTC V_MTC Acid Kojic TYR inhibitory (IC50 µg/mL) 150,16 ± 17,34c 777,68 ± 12,00a 414,94 ± 17,76b 94,12 ± 2,79d 23,35 ± 1,62 Survey results of tyrosinase inhibitory activity showed that methanol_pineapple peel had the strongest activity with the lowest IC 50 value (IC 50 = 94.12 ± 2.79 µg/mL) Tac Cau pineapple peel methanolic extract (V_MTC) has a tyrosinase inhibitory activity 1.6 times higher than that of methanol extract with the value (IC 50 = 150.16 ± 17.34 µg/mL), 8.3 times higher than that of the leaf methanol extract (IC 50 = 150.16 ± 17.34 µg/mL) with the high methanol_body sample with the value (IC 50 = 777.68 ± 12 µg/mL) and 4.4 times higher than that of the pineapple pulp methanol with the value (IC 50 = 414.94 ± 17.76 µg/mL) ) At the same time, all four samples of pineapple extract had lower tyrosinase inhibitory activity than the control Kojic acid (IC 50 = 23.35 ± 1.62 µg/mL) 3.3 Antioxidant and tyrosinase inhibitory results of pineapple peel extracts 3.3.1 Fractional separation chromatography of pineapple peel methanol extract From 58 grams of pineapple peel methanol extract, main segments were obtained: F 1, F 2, F 3, F 4, F5 with the results shown (Table 4.8) Table 4.8: Fractional high modulation results Extract segment F1 Weight (g) 1,99 20 Performance (%) 3,43 Extract segment F2 F3 F4 F5 Weight (g) 3,57 11,76 19,98 12,69 Performance (%) 6,16 20,28 34,45 21,87 3.3.2 Quantitative results of total polyphenols and total flavonoids in pineapple peel fractions Quantitative results Table 4.9 shows that the total polyphenol content, flavonoid highest total in F value was 430.89 ± 6.44 respectively (mg GAE / g CPD) and 220.05 ± 6.03 ( mg QE/g CPD) The content of total polyphenols and total flavonoids was lowest in F with values of 96.17 ± 4.76 (mg GAE/g CPD) and 42.17 ± 0.91 (mg QE/g CPD) respectively Table 4.9: Quantitative results of TPC and TFC of pineapple peel fractions Experimen F1 F2 F3 F4 F5 Total polyphenol content (mg GAE/g) 430,89 ± 6,44a 167,36 ± 1,43c 124,50 ± 2,58d 96,17 ± 4,76e 243,39 ± 3,09b Total flavonoid content (mg QE/g CPĐ) 220,05 ± 6,03a 166,22 ± 4,86b 64,64 ± 1,09d 42,17 ± 0,91e 82,73 ± 1,98c 3.3.3 Antioxidant and tyrosinase inhibitory results of pineapple peel extracts Results of in vitro antioxidant investigation of pineapple peel extracts Table 4.10: Antioxidant activity of pineapple peel extracts Experimen F1 F2 F3 F4 F5 CT Value IC50 (µg/mL) De-DPPH(*) De-Cu2+ (*) f 14,52 ± 0,44 18,78 ± 2,55f 55,75 ± 0,74e 138,39 ± 1,71d 141,51 ± 7,56c 371,46 ± 3,16c 288,33 ± 13,38a 550,74 ± 17,63a b 186,42 ± 9,45 394,20 ± 6,12b d 98,82 ± 1,05 102,50 ± 0,29e In vitro tyrosinase inhibition results of pineapple peel extracts Table 4.11: Tyrosinase inhibitory ability of pineapple peel extracts CPĐ F1 F2 F3 F4 F5 TYR inhibitory (IC50 µg/mL) 84,98 ± 5,06cd 111,84 ± 4,03c 174,50 ± 2,46b 232,79 ± 29,34a 185,97 ± 0,50b 21 CPĐ Cao thơ Acid kojic TYR inhibitory (IC50 µg/mL) 62,85 ± 1,45d 19,78 ± 1,29 Among the higher segment, the pineapple shell tyrosinase inhibitory activity was strongest in the segment F value (IC 50 = 84.98 ± 5.06 microg / mL) higher than 1.3 times the segment F (IC 50 = 111.84 ± 4.03 µg/mL) is 2.05 times higher than that of the F segment (IC 50 = 174.50 ± 2.46 µg/mL), 2.7 times higher times that of the F segment (IC 50 = 232.79 ± 29.34 g/mL) and 2.2 times higher than that of the F segment (IC 50 = 185.97 ± 0.50 µg/mL) Simultaneously, both the pineapple peel extract and the extracts had lower tyrosinase inhibitory activity compared to the control Kojic acid (IC 50 = 19.78 ± 1.29 µg/mL) 3.3.4 Relationship between TPC, TFC with antioxidant capacity and tyrosinase inhibition of pineapple peel extracts Table 4.12: Relationship between DPPH reduction, Cu 2+ reduction and tyrosinase inhibition Gía trị IC50 (µg/mL) Extract TPC TFC De-DPPH(*) F1 F2 F3 F4 F5 control sample 430,89±6,44a 167,36±1,43c 124,50±2,58d 96,17±4,76e 243,39±3,09b 220,05±6,03a 166,22±4,86b 64,64±1,09d 42,17±0,91e 82,73±1,98c 14,52±0,44f 55,75±0,74e 141,51±7,56c 288,33±13,38a 186,42±9,45b 89,48±0,26 De-Cu2+ (*) 18,78±2,55f 138,39±1,71d 371,46±3,16c 550,74±17,63a 394,20±6,12b 277,43±11,93 TYR inhibitory (**) 84,98±5,06cd 111,84±4,03c 174,50±2,46b 232,79±29,34a 185,97±0,50b 19,78±1,29 3.4 Results inhibit melanin production on B16F10 pigment cells of higher segments F1 pineapple shell Table 4:13: The percentage of samples melanin inhibiting F compared to control Percentage of inhibiting melanin production of the sample (%) Concentration (µg/mL) Ratio SD 200 14,92 1,35 25 50,79 4,49 10 26,35 0,45 0.5 9,52 0,90 Experimental cell residue images 22 (control sample) 0,69 Shell form F at a concentration of 200 micrograms / mL has begun to show a strong ability to inhibit melanin production on B16F10 cell lines compared to the concentration of 25 and 10 µg/mL Shell form F at concentrations 25 µg/mL and 10 µg/mL showed clear inhibition of melanin ability compared with negative controls 50.79% and 26.35 at%, respectively 3.5 Results of GC-MS mass spectrometry analysis of high-segment F1 pineapple peel samples According to the results of common GC-MS, then of the 34 substances/compounds in the high segment F peel pineapple 9, substances/compounds have been shown to have inhibitory activity of tyrosinase such as 3-Methylcyclopentanone, succinic acid, ferulic acid, p-cumaric acid, cumaric acid, 2,4'-Dihydroxy-3'-methoxyacetophenone, 3,4-Pyridinedimethanol, 6-methyl, cyclohexylideneacetic acid, benzoic acid (Table 4.18) (An et al., 2010; Mai et al., 2012; Matos et al., 2011; Taofiq et al., 2017; Xu et al., 2020) 23 CHƯƠNG 4: CONCLUSION AND PROPOSAL 4.1 Conclude Pineapple in Lao Tac Cau (Kien Giang) has superior advantages over pineapples in Hon Dat (Kien Giang) in terms of antioxidant properties as well as tyrosinase enzyme inhibitory activity in melanin biosynthesis There are differences in the distribution of secondary plant compounds in the leaves and skins of pineapples Polyphenol compounds are high in the pods, while the flavonoid content is high in the leaves Investigation of antioxidant activity of methanol extract from pineapple parts showed that the leaves at the tip of the fruit had the strongest DPPH reducing activity compared to the stem, flesh, and skin of the pineapple Meanwhile, the reducing activity of Fe 3+ and Cu 2+ was highest in pineapple peel Besides, pineapple peel also showed strong tyrosinase inhibitory activity compared to the rest of the pineapple plant Chromatography for high fractionation of methanol pods on silica gel with organic solvents with increasing polarity n-hexan > n-hexane: ethyl acetate > ethyl acetate > ethyl acetate: methanol > methanol for fractions F1 (n-hexan), F2 (n-hexane: ethyl acetate (3:7, v/v), F3 (ethyl acetate (100%, v/v), F4 (ethyl acetate: methanol (3:7, v/v), F5 (methanol (100%, v/v) In which segments F shown activity DPPH reduction, reduction of Cu 2+ and inhibition of tyrosinase in vitro strongest At the same time segment F is also capable of inhibiting melanin production on high B16F10 cell lines Analysis GS-MS shows in high segments F in the presence of compounds such as succinic acid, Ferulic acid, p-coumaric acid, cinnamic acid, Benzoic acid, 2-Ethylhexyl, these are compounds capable of strong antioxidant and tyrosinase inhibitory properties 4.2 Suggestion Surveying more cytotoxic activity of methanol extracts of bark and high segments F peel pineapple to applications in the medical, pharmaceutical, and cosmetic products Completing the production process of pineapple peel extract 24 i ... 3.3.2 Quantitative results of total polyphenols and total flavonoids in pineapple peel fractions Quantitative results Table 4.9 shows that the total polyphenol content, flavonoid highest total in... Tac Cau area The inhibitory activity of tyrosinase enzyme of Tac Cau pineapple methanol extracts is shown in Table 4.7 Table 4.7: Tyrosinase inhibition of Tac Cau pineapple methanol extracts Experiment... quantitative results of total polyphenols and total flavonoids in the methanol extracts of leaves, stems, fruit pulp, and pineapple peel are shown in Table 4.5 Table 4.5: Contents of total polyphenols,
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