Nghiên cứu thành phần hóa học và hoạt tính sinh học của cây bù dẻ trơn (uvaria boniana), bù dẻ hoa vàng (uvaria hamiltonii), lãnh công màu hung (fissistigma cupreonitens) và lãnh công xám (fissistigma glaucesens) t

1 MINISTRY OF EDUCATION AND TRAINING VINH UNIVERSITY NGUYEN THANH TAM STUDY ON THE CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITY OF UVARIA BONIANA, UVARIA HAMILTONII, FISSISTIGMA CUPREONITENS AND FISSISTIGMA GLAUCESENS SPECIALIZATION: ORGANIC CHEMISTRY Code: 9.44.01.14 PhD THESIS SUMMARY NGHE AN – 2019 The project was completed at The School of Natural Sciences Education – Vinh University and Taynguyen Institute for Scientific Research Supervisors: Assoc Prof Dr Hoang Van Luu Dr Nguyen Huu Toan Phan Reviewer 1: Assoc Prof Dr Reviewer 2: Assoc Prof Dr Reviewer 3: Assoc Prof Dr The thesis will be defended at the High-tech buildings at Vinh University, 182 Le Duan, Vinh City, Nghe An Province, 2020 At h 00, The thesis can be found at: The National Library of Vietnam The Library Nguyen Thuc Hao, Vinh University INTRODUCTION Preamble Vietnam is located in Southeast Asia, with a humid tropical climate, diverse and abundant in plants According to recent statistics, the Vietnamese plants have over 10,000 species, of which about 3,200 species of plants have been used in traditional medicine and 600 species produce essential oils This is a very valuable natural resource and has great impact on people life and health In Vietnam, the Annonaceae family has 201 species (29 genera) including many endemic species such as Alphonsea sonlaensis Ban, Artabotrys hienianus Ban, Artabotrys vietnamensis Ban, Artabotrys vinhensis Ast., Fissistigma taynghuyenense Ban, Fissistigma tonkinense (Fin & Gagnep ) Merr., Goniothalamus donnaiensis Fin & Gagnep., Goniothalamus vietnamensis Ban., Melodorum kontumense Ban, Fissistigma Griff is an important genus of the family Annonaceae with about 80 species and widely distributed in Asia and Australia, especially in Southeast Asia such as Malaysia, Indonesia, Thailand and China, Cambodia, Laos and Vietnam Some species of the genus Fissistigma are medicinal plants for treatment of muscular atrophy, liver, hepatosplenomegaly, sciatica, arthritis, inflammatory and tumor Uvaria L genus is a large genus of the Annonaceae family, with about 175 species worldwide and about 17 species in Viet Nam The trees of this genus are mainly climbing or bushy, rarely woody Although the Annonaceae family tree has a very high economic value as well as valuable biological activity as popular folk medicine, the studies on its chemical composition have not been conducted much in Vietnam For that reason, we have chosen the research project: "Research on chemical composition and biological activity of Fissistigma and Uvaria genus of Annonaceae " in order to contribute to determine the chemical composition of the plants and find the source of raw materials for pharmaceutical industry Research objects The research objects of thesis are extracts of Uvaria boniana Fin & Gagnep., Uvaria hamiltonii Hook.f et Thoms., Fissistigma cupreonitens Merr & Chun and Fissistigma glaucesens (Hance) Merr in Việt Nam Research tasks The research tasks include: - Use suitable solvents to extract to get corresponding extracts from Uvaria boniana Fin & Gagnep., Uvaria hamiltonii Hook.f et Thoms., Fissistigma cupreonitens Merr & Chun and Fissistigma glaucesens (Hance) Merr - Separate and identify structures of the isolated compounds - Test for biological activities of the concerned compounds Research methods - Methods for collection samples: collection, identification, storage and preservation dried samples at the laboratory temperature (20-300C) - Methods for separating, purifying bioactive compounds: extraction of the materials; extraction with solvents of different polarity at room temperature e.g hexane, ethyl acetate, cloroform, butanol… Isolation of compounds by a combination of chromatographic techniques; column chromatography (CC), flash chromatography (FC), and thin layer chromatography (TLC), high performance liquid chromatography (HPLC) - Methods for structure elucidation by UV, IR, mass spectrometry (MS), as well as one- and two dimensional NMR techniques (1H-NMR, 13C-NMR, 2D- 1H-1H-COSY, HMBC, HSQC) - Methods for bioactivity testing: Methods used to test antibacterial activity and antioxidant activity of isolated compounds New contributions of the thesis In this research, we have new results: - From the extracts of Uvaria boniana Fin & Gagnep eight compounds were isolated and structurally determined: uvaridacol G ; 4-methyl-4-[(2Z)-3’phenylprop-2’-en-1’-yl]cyclohex-2-en-1-one; 3,7- dimethoxy quercetin 4’- O[α-L- rhamnopyranosyl-(1  2) -β-D- glucopyranoside; -sitosterol and stigmasterol, 6-methoxyzeylenol; aristolactam AII; stigmasta-4,22-dien-3-on It was first time these compounds were isolated from Uvaria boniana - From the extracts of Uvaria hamiltonii Hook.f et Thoms eight compounds were been isolated and structurally determined: quercetin, luteolin7-O-glucoside, luteolin-4’-O-glucoside, rutin, rhoifolin, glutinol, zeylenol, lupeol It was first time these compounds were isolated from Uvaria hamiltonii - From the extracts of Fissistigma glaucescens (Hance) Merr.) six compounds were isolated and structurally determined: four alkaloids: aristolactam BII, velutinam, aristolactam BI, pukatein and 02 flavonoids: apigenin-8-C-β-D-galactopyranoside and rutin This is the first time, Velutinam, pukatein and apigenin-8-C-β-D-galactopyranoside were isolated from Fissistigma glaucescens - From the extracts of Fissistigma cupreonitens Merr & Chun six compounds were isolated and structurally determined: 04 flavonoids: 6hydroxy-5,7,8-trimetoxy flavanon, 2’,5’-dihydroxy-3’,4’,6’-trimetoxy chalcon, quercetin, rutin and 02 steroids: -sitosterol; -sitosterol-3-O--Dglucopyranoside had been isolated and the structures determined This is the first time, 6-hydroxy-5,7,8-trimetoxy flavanon, 2’,5’-dihydroxy-3’,4’,6’trimetoxy chalcon were isolated from Fissistigma cupreonitens Merr & Chun - Testing for antibacterial activity and antioxidant activity of isolated compounds Structure of the thesis The thesis is displayed in a total of 135 pages with 26 tables, 39 figures, diagrams and 150 references Its major sections include: Introduction (4 pages), overview (25 pages), methods and experiment (30 pages), results and discussion (69 pages), conclusion (1 pages), published works (1 page) and references (14 pages) In addition, there is an appendix with 60 spectra of the isolated compounds CHAPTER 1: OVERVIEW The thesis has conducted a literature review on: Uvaria - The diversity and distribution of Uvaria - The chemical composition of Uvaria - Biological activity of Uvaria 1.1 Uvaria boniana -The diversity and distribution of Uvaria boniana - The chemical composition of Uvaria boniana - Biological activity of Uvaria boniana 1.2 Uvaria hamiltonii - The diversity and distribution of Uvaria hamiltonii - The chemical composition of Uvaria hamiltonii - Biological activity of Uvaria hamiltonii Fissistigma - The diversity and distribution of Fissistigma - The chemical composition of Fissistigma - Biological activity of Fissistigma 2.1 Fissistigma glaucesens - The diversity and distribution of Fissistigma glaucesens - The chemical composition of Fissistigma glaucesens - Biological activity of Fissistigma glaucesens 2.2 Fissistigma cupreonitens - The diversity and distribution of Fissistigma cupreonitens - The chemical composition of Fissistigma cupreonitens - Biological activity of Fissistigma cupreonitens CHAPTER 2: METHODS AND EXPERIMENT 2.1 Methods 2.1.1 Method for collecting samples The plant materials include Uvaria boniana Fin & Gagnep., Uvaria hamiltonii Hook.f et Thoms., Fissistigma cupreonitens Merr & Chun, and Fissistigma glaucesens (Hance) Merr were collected Pù Huống, Pu Mát- Nghệ An, Viet Nam in August 2015 Samples were identified by associate professor Tran Huy Thai (working at Institute of Ecology and Biological Resources) They were stored at School of Biochemical Technology-Environment 2.1.2 Methods for sample treatment and extraction Samples were collected in plant growing season Collected samples then were washed, dried at 40 °C Samples were extracted using suitable solvent by ultrasound-assisted device 2.1.3 Methods for analysis and isolation of compounds To analyse and isolate compounds, chromatography methods such as :Thin layer chromatography (TLC); column chromatography (CC); flash column chromatography (FC); high performance liquid chromatography (HPLC); and fractional crystallization have been used 2.1.4 Methods for structural determination Structure of isolated compounds were identified by combination of modern spectroscopic methods: - Ultraviolet (UV), infrared (IR), mass spectrometry (ESI-MS, HR-ESIMS), nuclear magnetic resonance spectroscopic 1H-NMR, 13C-NMR, DEPT and HSQC, HMBC, 1H-1H COSY 2.1.5 Methods of bioactivity assay Antimicorbial and antioxidant activity was conducted at the bioassay Lab, Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology 2.2 Chemicals and equipment 2.2.1 Chemicals: Solvents for extraction are industrial hexane, methanol, butanol, ethylacetate, acetone, and distilled The solvents for thin layer chromatography, fast column chromatography are purely analytical (PA) 2.2.2 Equipment: - Melting points were measured on Yanaco MP-S3 - Optical rotation were measured on Jasco DIP -370 - UV were run in Hitachi UV-3210 - Mass spectrometry were run in Bruker Dailtonics APEX II 30eV spectrometherr and micrOTOF-QII 10187 - Nuclear magnetic resonance spectroscopy (1H-NMR, 13C-NMR, DEPT and HSQC, HMBC, 1H-1H COSY) were run in Brucker AVANCE III-700 using TMS as reference compound - Column chromatography (CC); flash column chromatography (FC) used 60,70-230 Kieselgel and 230-240 (Merck) - Thin layer chromatography (TLC) was plated with kieselgel 60 and identified by H2SO4 10% solution at 1100 C in 10 mnutes, and UV lamp at 254 nm and 368 nm identified by I2 2.3 Study on compounds from leaf of Uvaria boniana 2.3.1 Extracting and isolating substances The leaf of Uvaria boniana (6.0 kg) were air-dried, powdered and soaked with methanol at room temperature, the combined extracts were concentrated under reduced pressure to give the residue (254.0 g) The residue was suspended into water and partitioned with ethyl axetate and butanol, successively to afford the ethyl acetate (UBE) (3172 g), butanol (UBW) (40 g), and water (10 g) residues respectively, after removal of the corresponding solvent The ethyl acetate residue was purified by silica gel column chromatography eluted with hexane: acetone gradients (100:0-1:1 vv) to afford many fractions, which were combined to 10 fractions (UBE1 to UBE10) Fraction UBE1 was purified by silica gel column chromatography eluted with hexane: acetone (15:1) to afford further seven subfractions UBE1.1 – E1.7 Subfraction UBE1.1 (2,6 g) was purified by silica gel column chromatography eluted with hexane: acetone (15:1) to afford compound UBM4 (38 mg) Subfraction UBE1.4 (2,5 g) was was purified by silica gel column chromatography eluted with hexane: acetone (9:1) to provide compounds UBM1 (12 mg) and UBM2 (27 mg) Fraction UBE2 (5,6 g) was purified by silica gel column chromatography eluted with CHCl3: MeOH (50:1 to 1:1) to afford further five subfractions UBE2.1 – E2.5 Subfraction UBE2.2 (1,2 g) was purified by silica gel column chromatography eluted with CHCl3: MeOH (20:1) to afford compound UBM7 (19 mg) Subfraction UBE2.4 (0,84 g) was purified by silica gel column chromatography eluted with CHCl3: MeOH (15:1) to afford compound UBM8 (14 mg) Fraction UBE3 (3,8 g) was purified by silica gel column chromatography eluted with CHCl3: MeOH (30:1 to 6:1) to afford further five subfractions UBE3.1 – E3.5 Subfraction UBE3.4 (0,96 g) was purified by silica gel column chromatography eluted with CHCl3: MeOH (10:1) to afford compound UBM6 (15 mg) Fraction UBE3.7 (1,9 g) was purified by silica gel column chromatography eluted with CHCl3: MeOH (7:1) to afford compound UBM5 (12 mg) The butanol residue was subjected to 100:0, 40:1, 30:1; 10:1, 4:1, 2:1) by silica gel column chromatography eluted with CHCl3:CH3OH gradients (100:0, 40:1, 30:1; 10:1, 4:1, 2:1) to give many fractions The faction UBB4 was purified by silica gel column chromatography eluted with CHCl3:CH3OH gradients (10:1, 8:1) to furnish compound UBM3 (17 mg) 2.4 Study on chemical constituents of leaf of Uvaria hamiltonii 2.4.1 Extracting and isolating substances The leaf of Uvaria hamiltonii (1.5 kg) were air-dried and powdered and soaked with methanol at 40 °C for 10 days, and the combined extracts were concentrated under reduced pressure to give deep brown syrup (207.0 g) The crude extract was suspended into water and partitioned with n-hexane and cloroform, ethyl acetate successively to afford n-hexane (39.2 g), cloroform (41.0 g), ethyl acetate (35.7 g) respectively, after removal of the corresponding solvent The ethyl acetate residue was purified by silica gel column chromatography eluted with CHCl3:CH3OH gradients (100:0; 50:1; 39:1; 30:1; 20:1; 15:1; 9:1; 4:1; 2:1; 1:1) to afford six fractions (UHE1 to UHE6) Subfraction UHE6 was subjected to the sephadex LH-20 column chromatography (100 gam, 60 x cm) using CH3OH:H2O as eluent (1:1, 1:0) to afford further four subfractions UHE6-1 - E6-4 Fraction UHE6-3 (229 mg) was subjected to silica gel column chromatography (200 gam, 60 x cm) using CHCl3:CH3OH as eluent (5:1, 3:1, 1:1) to provide four subfractions UHE6.3-1 E63-4 Subfraction E6.3-1 was subjected to the reverse phase (RP -18 ) column chromatography (100 gam, 60 x cm) using CH3OH:H2O as eluent (1:1) to afford compound UHM1 The water residue was subjected to the sephadex HP-20 column chromatography eluted with H2O, MeOH-H2O (25:75, 50:50, 75:25, 100:0) to form five subfractions (UHW1-UHW5) Fraction UHW3 (11.3 g) was subjected to silica gel column chromatography (200 gam, 60 x cm) using CHCl3:CH3OH as eluent (6:1, 4:1, 2:1, 1:1) to afford five subfractions (UHW3.1-W3.5) Subfraction UHW3-5 was subjected to the sephadex LH-20 column chromatography eluted (100 g, 60 x cm) with CH3OH:H2O (1:1, 1:0) to prvide four subfractions UHW3.5-1 – W3.5-4 Subfraction W3.5-4 was subjected to the reverse phase (RP -18 ) column chromatography eluted (100 g, 60 x cm) with CH3OH:H2O (1:1) to afford UHM4 (23 mg) Subfraction UHW3.4 was subjected to the sephadex LH-20 column chromatography eluted (100 g, 60 x cm) with CH3OH:H2O (1:4, 1:0) to afford five subfractions (UHW3.4.1-UHW3.4.5) Subfraction UHW3.4-4 (54 mg) was subjected to the reverse phase (RP -18 ) column chromatography eluted (100 gam, 60 x cm) with CH3OH:H2O (1:4, 1:1) to afford six subfractions (UHW3.4.4.1-W3.4.4.6) Fraction UHW3.4.4-3 (36 mg) was subjected to silica gel column chromatography eluted (200 g, 60 x cm) with CH2Cl2:Acetone (2:1) to afford UHM2 (8 mg) and UHM5 (12 mg) Subfraction UHW3-2 was subjected to the sephadex LH-20 column chromatography eluted (100 g, 60 x cm) with CH3OH:H2O (1:4, 1:0) to afford five subfractions (UHW3.2.1-UHW3.2.5) Subfraction W3.2-4 (70mg) was subjected to the reverse phase (RP -18) column chromatography eluted (100 gam, 60 x cm) with CH3OH:H2O (1:1) to afford UHM3 (11 mg) 10 The chloroform residue was purified by silica gel column chromatography eluted with CHCl3:CH3OH gradients (100:0; to 0:100) to afford five fractions (UHC1 to UHC5) Subfraction UHC3 (10,83 g) was subjected to the sephadex LH-20 column chromatography (100 gam, 60 x cm) using CH3OH:H2O as eluent (1:1, 1:0) to afford further four subfractions UHC3.1 – C3.4 Fraction UHC3.2 (0,11 g) was subjected to silica gel column chromatography RP-18 using CH3OH:H2O as eluent (1:1) to provide compound UHM8 (15mg) The hexane residue was purified by silica gel column chromatography eluted with hexane:EtOAc gradients to afford five fractions (UHH1 to UHH9) Subfraction UHH1 (10,83 g) was purified by silica gel column chromatography eluted with using CH3OH:MeOH as eluent (1:1, 1:0) to afford further five subfractions UHH1.1 – H.4 Fraction UHH1.4 (0,194 g) was purified by silica gel column chromatography eluted with hexane:CH2Cl2 gradients to give compounds UHM6 (10 mg) and UHM7 (10 mg) 16 2.7 Biological activity test 2.7.1 Antimicrobial activity tested Antimicrobial activity was conducted to evaluate the antibiotic activity of extracted samples on 96-well microtiter plates according to modern methods discribed Vander Bergher and Vlietlinck (1991) ), and McKane & Kandel (1996) 2.7.2 Anti-oxidant activity It is an approved method for rapid determination of antioxidant activity based on the ability to trap free radicals generated by DPPH (1,1-diphenyl-2picrylhydrazyl) (Brand-Williams et al 1995, Shela et al 2003, Kumar et al 2013) The reagent is dissolved in dimethyl sulfoxide (DMSO 100%) and DPPH is diluted in 96% ethanol Absorption of DPPH at a wavelength of 515 nm was determined by an ELISA reader after adding DPPH to the sample solution on a 96-well microtitre plates The results of the tests are expressed as the average of at least replicate tests ± standard deviation (p ≤ 0.05) CHAPTER 3: RESULTS AND DICUSSION 3.1 Uvaria boniana 3.1.1 Isolated compounds The leaves of Uvaria boniana were extracted with methanol Solvent was evaporated under reduced pressure to produced the methanolic residue This residue was suspended in water and then partitioned with ethyl axetate and butanol, successively to afford the ethyl acetate, butanol, and water residues respectively, after removal of the corresponding solvent From the extracts of the leaves of Uvaria boniana, eight compounds were isolated by column chromatography and structurally determined by spectrscopic methods (table 3.1) The results of the study are shown in Table 3.1 Table 3.1 Isolated compounds from Uvaria boniana Symbol Name of compounds UBM1 Uvaridacol G 4-Methyl-4-[(2Z)-3’-phenylprop-2’-en-1’-yl]cyclohex-2en-1-one 3,7- Dimethoxy quercetin 4’- O- [α-Lrhamnopyranosyl- (1  2) -β-D- glucopyranoside UBM2 UBM3 Mass (mg) 12 mg 27 mg 17mg UBM4 -Sitosterol 38 mg UBM5 Stigmasterol 12 mg 17 UBM6 6-methoxyzeylenol 15 mg UBM7 Aristolactam AII 19 mg UBM8 Stigmasta-4,22-dien-3-on 14 mg 3.1.2 Determining the structure of compounds 3.1.2.1 Compound UBM1 The isolated UBM1 compound is amorphos solid The molecular formula of C21H20O7 was confirmed by the positive-mode HR-ESI-MS with m/z 385.1285 [M+H]+, calcd for C21H20O7 In the 1H-NMR and 13C-NMR spectra of 1, it shows the signal of benzoyl groups, oxymethine groups, oxymethylene groups and methine olefinic groups However, they are determined by the shift to the low field of H-2 at δ H 5.76 (1H, d, J = 8.0 Hz) and the shift to the high field of H-3 at δ H 4.29 (1H, t, J = 13.5, 7.0 Hz) 13C-NMR spectrum shows the signals of 21 carbon atoms, including carbon C-O atoms, olefinic carbon atoms [δC 131,5 (C-5); 130,3 (C-6)], benzoyl groups [δC 129,2 (C-1 ’); 130,4 (C-2 ', C-6'); 127.3 (C-3 ’, C5’); 131.1 (C-1 ’); 131,4 (C-2 '', C-6 ''); 129,2 (C-3 '', C-5 ''); 133.7 (C-4 ’); 133.8 (C-4 ''))] In the HMBC spectrum of UBM1, the long correlations of H-7 protons at δH 4.75 and 4.68 with C-2 (δC 76.3), C-6 (δC 130.3) and C-1 (δC) 75,5) suggest the connection of C-2, C-6 and C-7 to quaternary carbon C-1 Correlations of H-2 protons at δH 5,76 and aromatic protons at δH 8.07 (H-2, H6) with ester carbon at C 167,1 (C-7 '') indicate the position of the two benzoyl groups are at C-2 and C-7 positions These spectral data are completely consistent with the data reported for compound uvaridacol G in the literature Therefore, UBM1 was identified as uvaridacol G Table 3.2: 1H and 13C NMR chemical shifts, HMBC of UBM1 Vị trí DEPT δC (ppm) δH (ppm, multi., J in Hz) δC (ppm) [19] δH (ppm) [19] C 75,5 CH 76,3 5,76 (1H, d, J = 8,0 Hz) 79,6 5,33( 1H, d, J =10,5 Hz) CH 70,9 4,29 (1H, t, J = 13,5, 7,0 Hz) 73,9 4,16( dd, J = 10,5, 7,8 Hz) CH 69,7 4,43 (1H, t, J = 10,5, 4,5 Hz) 72,2 4,40 (m) 74,3 18 CH 131,5 CH 130,3 CH2 68,0 1’ C 129,2 2’ CH 130,4 3’ 4’ 5’ CH CH CH 127,3 133,7 127,3 6’ CH 130,4 7’ 1’’ C=O C 166,8 131,1 2’’ CH 131,4 3’’ 4’’ 5’’ CH CH CH 129,2 133,8 129,2 6’’ CH 131,4 7’’ C=O 167,1 7a 7b 5,95 (1H, dd, J = 11,5, 2,5 Hz) 5,79 (1H, d, J = 11,0 Hz) 4,75 (1H, d, J = 11,0 Hz) 4,68 (1H, d, J = 11,0 Hz) 130,2 129,7 67,0 5,90 (dd, J = 10,5, 2,3 Hz) 5,74 (d, J = 10,5 Hz) 4,5 (d, J = 11,5 Hz) 129,3 8,03 (2H, d, J = 6,5 Hz) 7,49 (2H, m) 7,63 (1H, m) 7,49 (2H, m) 8,03 (2H, d, J = 6,5 Hz) 129,7 128,5 133,3 128,5 129,7 7,95 (d, J = 7,8 Hz) 7,39 (m) 7,55 (m) 7,39 (m) 7,95 (d, J = 7,8 Hz) 166,2 129,9 8,07 (2H, d, J = 6,5 Hz) 7,49 (2H, m) 7,63 (1H, m) 7,49 (2H, m) 8,07 (2H, d, J = 6,5 Hz) 130,0 128,7 133,9 128,7 130,0 8,03( d, J = 7,8 Hz) 7,39 (m) 7,55 (m) 7,39 (m) 8,03 (d, J = 7,8 Hz) 167,9 Uvaridacol G (UBM1) Today, scientists are interested in finding priority agents to slow down the survival of pancreatic cancer cells under low nutritional conditions as a new approach to treating this type of cancer According to Awale et al., PC compounds isolated from U dac bodies such as uvaridacol A − C are slightly toxic to pancreatic cancer cell lines PANC-1, PSN-1, MIA PaCa-2 and KLM- with PC50 values in the concentration range of 100-200 µM Meanwhile, the 19 dimer PC compound, grandifloracin, showed very good activity with these cell lines with PC50 values of 14.5, 32.6, 17.5 and 32.7, respectively μM The authors also isolated uvaridacol E, uvaridacol F, uvaridacol H which have a toxic effect on pancreatic cancer cell line PANC-1 Therefore, they are considered as potential agents to develop new drugs towards the treatment of pancreatic cancer in humans Fig 3.1: The 13C-NMR of UBM1 Fig 3.2: The 13C-NMR of UBM1 20 Fig 3.3: The DEPT of UBM1 Fig 3.4: The HMBC of UBM1 21 Fig 3.5: The HSQC of UBM1 3.2 Uvaria hamilton 3.2.1 Isolated compounds The leaves of Uvaria hamilton were extracted with methanol Solvent was evaporated under reduced pressure to produced the methanolic residue This residue was suspended in water and then partitioned with chloroform to afford the chloroform and water residues respectively, after removal of the corresponding solvent From the extracts of the leaves of Uvaria hamilton, eight compounds were isolated by column chromatography and structurally determined by spectrscopic methods (table 3.3) Table 3.3 Isolated compounds from Uvaria hamilton Symbol Name Mass Quercetin 15 mg UHM1 UHM2 Luteolin-7-O-β-glucoside, mg UHM3 Luteolin 4’-O-glucopyranoside 11 mg UHM4 Rutin 23 mg UHM5 Rhoifolin 12 mg UHM6 Glutinol 15 mg UHM7 Zeylenol 10 mg UHM8 Lupeol 17 mg 22 3.3 Fissistigma cupreonitens Merr & Chun 3.3.1 Isolated compounds The leaves of Fissistigma cupreonitens were extracted with methanol Solvent was evaporated under reduced pressure to produced the methanolic residue This residue was suspended in water and then partitioned with ethyl acetate to afford the corresponding ethyl acetate residue, after removal of the corresponding solvent From the ethyl acetate extract of the leaves of Fissistigma cupreonitens, six compounds were isolated by column chromatography and structurally determined by spectrscopic methods (table 3.4) Table 3.4 Isolated compounds from Fissistigma cupreonitens Symbol Name Mass FCM1 -Sitosterol 25 mg FCM2 6-Hydroxy-5,7,8-trimetoxy flavanon 15 mg FCM3 2’,5’-Dihydroxy-3’,4’,6’-trimetoxy chalcon 19 mg FCM4 Quercetin 13 mg FCM5 Rutin 14 mg FCM6 -Sitosterol-3-O--D-glucopyranoside 12 mg 3.4 Fissistigma glaucescens (Hance) Merr 3.4.1 Isolated compounds The leaves of Fissistigma glaucescens were extracted with methanol Solvent was evaporated under reduced pressure to produced the methanolic residue This residue was suspended in water and then partitioned with ethyl acetate to afford the corresponding ethyl acetate residue, after removal of the corresponding solvent From the ethyl acetate extract of the leaves of Fissistigma glaucescens, six compounds were isolated by column chromatography and structurally determined by spectrscopic methods (table 3.4) 23 Table 3.5 Isolated compounds from Fissistigma glaucescens Symbol Name Mass FGM1 Aristolactam BII 18 mg FGM2 Velutinam 18 mg FGM3 Aristolactam BI 16 mg FGM4 Pukatein 26 mg FGM5 Apigenin-8-C-β-D-galactopyranoside 20 mg FGM6 Rutin 24 mg 3.5 Antimicrobial and antioxidant activities of isolated compounds The isolated compounds were tested for biological activity: antimicrobial activity and antioxidant activity Due to the amount of compounds UHM2, UHM3, UHM5 is too0 small, we only tested biological activity of the five othe compounds 3.5.1 Antimicrobial activity The results, showed that, the compounds UBM2, UBM3, UHM7, FCM2, FGM1 did not show microbiological activity, while the rest had antimicrobial activity with the tested strains in experiment concentration (100 (g/ml)) The UHM8 compound isolated from CHCl3 extract is supposed to be the main compound causing antimicrobial activity according to the above results 3.5.1 Antioxidant activity The results, showed that, the compounds UBM6, UHM4, UHM7, FCM3, FGM2, and FGM4 were negative with antioxidant activity testing, while The analysis of biologically active compounds, that have antioxidant activity of 24 compounds The results, showed that when testing the antioxidant activity of the compounds UBM6, UHM4, UHM7, FCM3, FGM2, FGM4 showed negative results with antioxidant activity, the remaining substances showed antioxidant activity The results are also consistent with the above observation that two ethyl acetate and water extracts were active at the test concentrations (compounds UHM2, UHM5 isolated from water extract were also reported to be actively antioxidant) 24 THE CHEMICAL COMPOUND ISOLATED FROM Uvaria boniana (1) Uvaridacol G (2) 4-Methyl-4-[(2Z)-3’-phenylprop-2’ -en-1’-yl]cyclohex-2-en-1-one (3) 3,7- Dimethoxy quercetin 4’- O- [α-L- rhamnopyranosyl- (1  2) -β-Dglucopyranoside (4) -Sitosterol (6)6-Methoxyzeylenol (7) Aristolactam AII (5) Stigmasterol (8) Stigmasta-4,22-dien-3-on 25 THE CHEMICAL COMPOUND ISOLATED FROM Uvaria hamiltonii OH HO O HO OH O (1) quercetin HO OH 1'' 1' O O 5'' OH 5 OH O OH OH OH O 4' 1' O 3'' HO OH O 1'' 5'' OH 3' 4' 1' O OH OH OH 4' OH (2) luteolin-7-O-glucoside O (3) luteolin-4’-O-glucoside OH 4' HO OH 3' O 4' OH O HO O 1'' O 1' O OH OH O 6''' H3C O 5''' O OH 5'' O HO 1'' O 3'' 1''' 6'' 3''' O O OH 1''' 10 OH OH OH HO H3C OH OH OH OH (4) rutin (5) rhoifolin 30 H3C CH3 29 20 19 CH3 12 18 11 26 CH3 CH3 CH3 23 H3C (7) zeylenol 28 27 24 CH3 (6) glutinol 16 HO HO 15 10 22 13 25 (8) lupeol THE CHEMICAL COMPOUND ISOLATED FROM Fissistigma cupreonitens 29 28 21 18 HO 14 O 26 10 10 (1) -sitosterol O (2) 6-hydroxy-5,7,8-trimetoxy flavanon H3CO 6' 4' OCH 5' OH 5' 6, HO O CH3O 2' OH 1' 2' (3) 2’,5’-dihydroxy-3’,4’,6’-trimetoxy chalcon OH 3' OH 10 O 4' 1' 3' 6' CH3O 5' 1' HO 15 4' 2' CH3O 25 16 24 17 13 19 CH3O 27 23 12 11 3' 22 20 OH OH O (4) Quercetin 26 5' 4' 6, HO O 6''' 2' 3' 10 OH OH 1' O O O O OH 6" 5" 1'' HO 2" 3" 5''' CH3 4''' O 4" 1''' HO OH OH OH 2''' 3''' O O HO HO OH OH OH (6) -sitosterol-3-O--D-glucopyranosit (5) Rutin THE CHEMICAL COMPOUND ISOLATED FROMFissistigma glaucescens O H3CO O NH 10 a H3CO 5a H HO N O 10 4a 8a R (1) Aristolactam BII (2) Velutinam (3) Aristolactam BI OH OH '' HO '' 5'' O HO 6' OH OH 2' O 4' 6, 3' OH O 1'' HO (4) Pukatein 5' 4'' O H OH OCH3 5' O 6''' 2' 3' OH 10 O OH 1' OH O O O 6" 5" 1'' HO (5) Apigenin-8-C-β-D-galactopyranoside 2" 3" 5''' CH3 4''' O 4" 1''' HO 2''' 3''' OH OH (6) Rutin OH OH 27 CONCLUSION Studies on the chemical composition and bioactivity of the compounds isolated from (Uvaria boniana Fin, & Gagnep,), (Uvaria hamiltonii Hook,f, et Thoms,), (Fissistigma cupreonitens Merr, & Chun) and (Fissistigma glaucesens (Hance) Merr,) in Việt Nam, we have the following findings: From the extracts of Uvaria boniana Fin & Gagnep, eight compounds have been isolated and structurally determined including: uvaridacol G ; 4-methyl-4[(2Z)-3’-phenylprop-2’-en-1’-yl]cyclohex-2-en-1-one; 3,7- dimethoxy quercetin 4’- O- [α-L- rhamnopyranosyl-(1  2) -β-D- glucopyranoside; -sitosterol and stigmasterol , 6-methoxyzeylenol; aristolactam AII; stigmasta-4,22-dien-3-on This was first time these compounds were isolated from Uvaria boniana From the extracts of Uvaria hamiltonii Hook.f et Thoms 08 compounds have been isolated and structurally determined including: quercetin, luteolin-7-Oglucoside, luteolin-4’-O-glucoside, rutin, rhoifolin, glutinol, zeylenol, lupeol This was first time these compounds were isolated from Uvaria hamiltonii From the extracts of Fissistigma glaucescens (Hance) Merr.) 06 compounds have been isolated and structurally determined including: 04 compounds ancaloit aristolactam BII, velutinam, aristolactam BI, pukatein and 02 compounds flavonoid apigenin-8-C-β-D-galactopyranoside and rutin Velutinam, pukatein and apigenin-8-C-β-D-galactopyranoside were first time isolated from Fissistigma glaucescens From the extracts of Fissistigma cupreonitens Merr & Chun 06 compounds have been isolated and structurally determined including: 04 compounds flavonoid: 6-hydroxy-5,7,8-trimetoxy flavanon, 2’,5’-dihydroxy3’,4’,6’-trimetoxy chalcon, quercetin, rutin and 02 compounds steroid: sitosterol; -sitosterol-3-O--D-glucopyranoside 6-hydroxy-5,7,8-trimetoxy flavanon, 2’,5’-dihydroxy-3’,4’,6’-trimetoxy chalcon were first time isolated from Fissistigma cupreonitens Merr & Chun We have tested bioactivity of the extracs and isolated compounds including microbiological activity and antioxidant activity The antioxidant activity testing result showed that compounds UBM6, UHM4, UHM7, FCM3, FGM2, FGM4 exhibited negative effect, while the remaining substances exhibited antioxidant activity The results are also consistent with the above observation of biological activity of ethyl acetate and water extracts at testing concentration (compounds UHM2, UHM5 isolated from water extract was also reported with antioxidant activity) The antimicrobial activity testing result showed that compounds UBM2, UBM3, UHM7, FCM2, FGM1 did not show microbiological activity, while the other compounds had antimicrobial activity the tested strains in the running concentration (100 (g/ml)) The UHM8 compound isolated from CHCl3 extract 28 is supposed to be the main compound causing antimicrobial activity according to the above results 29 PUBLISHED WORKS Nguyen Thanh Tam, Đoan Manh Dung, Vo Cong Dung, Bui Van Nguyen, Nguyen Thi Thu, Hoang Van Luu, Nguyen Huy Hung, Tran Đinh Thang (2014), Alkaloids and flavonoids from Fissistigma glaucescens (Hance) Merr, in Vietnam Nam, Vietnam Journal of Science and Technology, 52(5A) 55-61 Hoang Van Luu, Nguyen Thanh Tam, Đoàn Mạnh Dũng, Tran Loi Loi, Nguyen Huy Hung, Tran Đinh Thang (2014), Flavonoids and steroids from Fissitigma cupreonitens Merr, & Chun in Vietnam, Vietnam Journal of Science and Technology, 52(5B) 662-667 Nguyen Thanh Tam, Nguyen Ngoc Tuan, Hoang Van Trung, Le Thi My Chau, Dinh Thi Trung Anh, Hoang Van Luu (2018), Chemical constituents from the leaves of Uvaria boniana in Vietnam, Vietnam Journal of Sciences and Technology, 56(5) Nguyen Thanh Tam, Nguyen Thi Dieu Thuan, Nguyen Thi Thu Hien, Pham Van Huyen, Nguyen Huu Toan Phan (2019), Flavonoids from the leaves of Uvaria hamiltonii (Annonaceae), Vietnam Journal of Chemistry, 57(4e3,4) 287290 Nguyen Thanh Tam, Nguyen Thi Ngan, Hoang Van Trung, Hoang Van Luu (2018), Chemical composition of leaves of Uvaria boniana in Việt Nam, Vinh Journal of Science, 47 (4) 50-54 30 ... antioxidant activity, the remaining substances showed antioxidant activity The results are also consistent with the above observation that two ethyl acetate and water extracts were active at the test... antioxidant activity The results are also consistent with the above observation of biological activity of ethyl acetate and water extracts at testing concentration (compounds UHM2, UHM5 isolated from... This residue was suspended in water and then partitioned with ethyl acetate to afford the corresponding ethyl acetate residue, after removal of the corresponding solvent From the ethyl acetate