MINISTRY OF EDUCATION AND TRAINING VINH UNIVERSITY NGUYEN THANH TAM STUDY ON THE CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITY OF UVARIA BONIANA, UVARIA HAMILTONII, FISSISTIGMA CUPREO
Trang 1MINISTRY 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
Trang 2The project was completed at The School of Natural Sciences Education – Vinh University and
Taynguyen Institute for Scientific Research
Supervisors: 1 Assoc Prof Dr Hoang Van Luu
2 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 8 h 00,
The thesis can be found at:
1 The National Library of Vietnam
2 The Library Nguyen Thuc Hao, Vinh University
Trang 3INTRODUCTION
1 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
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
Trang 4- Test for biological activities of the concerned compounds
- 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
5 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 ; phenylprop-2’-en-1’-yl]cyclohex-2-en-1-one; 3,7- dimethoxy quercetin 4’- O- [α-L- rhamnopyranosyl-(12) -β-D- glucopyranoside; -sitosterol and stigmasterol, 6-methoxyzeylenol; aristolactam AII; stigmasta-4,22-dien-3-on
4-methyl-4-[(2Z)-3’-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, 7-O-glucoside, luteolin-4’-O-glucoside, rutin, rhoifolin, glutinol, zeylenol,
luteolin-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: hydroxy-5,7,8-trimetoxy flavanon, 2’,5’-dihydroxy-3’,4’,6’-trimetoxy chalcon, quercetin, rutin and 02 steroids: -sitosterol; -sitosterol-3-O--D-glucopyranoside 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’-
6-trimetoxy chalcon were isolated from Fissistigma cupreonitens Merr & Chun
Trang 5- Testing for antibacterial activity and antioxidant activity of isolated
compounds
6 Structure of the thesis
The thesis is displayed in a total of 135 pages with 26 tables, 39 figures,
3 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:
1 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
2 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
Trang 6The 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, MS), nuclear magnetic resonance spectroscopic 1H-NMR, 13C-NMR, DEPT and HSQC, HMBC, 1H-1H COSY
HR-ESI-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
Trang 72.3 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)
Trang 92.4 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 3 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 3 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 3 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 3 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 3 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 3 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 3 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 3 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 3 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 3 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 3 cm) with CH3OH:H2O (1:1) to afford UHM3 (11 mg)
Trang 10The 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 3 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
Trang 122.5 Study on chemical constituents of leaf of Fissistigma cupreonitens
2.5.1 Extracting and isolating substances
The leaf of Fissistigma cupreonitens (8,6 kg) were dried at 40 °C,
powdered and soaked with methanol at room temperature for 1 week The combined extracts were concentrated under reduced pressure to give a residue (1024.0 g) The residue was suspended into water and extracted with n-hexane, ethyl acetate and n-butanol, successively to afford n-hexane (122 g), ethyl acetate (271 g), n-butanol (145.0 g) residues, respectively, after removal of the corresponding solvents
The ethyl acetate residue was fractionated by silica gel column chromatography eluted with hexane and axetone gradients (100:0; 50:1; 39:1; 30:1; 20:1; 15:1; 9:1; 4:1; 2:1; 1:1 ) to afford 10 fractions (FCE1 to FCE10) Fraction FCE1 (11.3 g) was subjected to silica gel column chromatography eluted (200 gam, 60 x 3 cm) with hexane and axetone gradients (15:1, 9:1) to to afford seven subfractions (FCE1.1-FCE1.7) Fraction F1-1 (11.3 g) was subjected to silica gel column chromatography (200 g, 60 x 3 cm) eluted with hexane and
acetone (15:1) to provide compound FCM1 (-sitosterol: 172 mg) Fraction F1-3 (15.3 g) was subjected to silica gel column chromatography (200 g, 60 x 3 cm)
eluted with hexane and axetone gradients (9:1, 4:1) to give compounds FCM 2 (6-hydroxy-5,7,8-trimetoxy flavanon: 35 mg) and FCM3 (2’,5’-dihydroxy-
3’,4’,6’-trimetoxy chalcon: 29 mg) Fraction F1-4 (9.3 g) was subjected to silica gel column chromatography (200 g, 60 x 3 cm) eluted with CHCl3:CH3OH (20:1)
to furnish compound FCM 4 (quercetin: 83 mg)
The butanol residue was fractionated by silica gel column chromatography eluted with CHCl3:CH3OH gradients (30:1; 20:1; 10:1; 5:1) to afford 10 fractions (FBM1 to FBM10) Fraction FBM5 (4.3 g) was subjected to silica gel column chromatography (200 g, 60 x 3 cm) eluted with CHCl3: CH3OH (10:1; 6:1) to
afford compound FCM 5 (rutin: 34 mg) Fraction FBM6 (4.3 g) was subjected to
silica gel column chromatography (200 g, 60 x 3 cm) eluted with CHCl3: CH3OH
(10:1; 6:1) to give compound FCM 6 (β-sitosterol-3-O-β-D-glucopyranosit: 112
mg)
Trang 142.6 Study on chemical constituents of leaf of Fissistigma glaucescens
2.6.1 Extracting and isolating substances
The leaf of Fissistigma glaucescens (10.0 kg) were air-dried, powdered and
soaked with methanol at room temperature for 7 days The combined extracts were concentrated under reduced pressure to give a methanolic residue (1212.0 g) The residue was suspended into water and extracted with n-hexane, clororform and n-butanol, successively to afford n-hexane (142 g), chlororform (291 g), n-butanol (155.0 g) residues, respectively, after removal of the corresponding solvent
The chlororform residue was fractionated by silica gel column chromatography eluted with chloroform-methanol gradients (100:0, 98:1: 95:5; 90:10; 80:2) to afford 12 fractions (FGC1 to FGC12) Fraction FGC2 (9.3 g) was subjected to silica gel column chromatography (200 g, 60 x 3 cm) eluted with
hexane and acetone (15:1) to afford compound FGC5 (91 mg) Fraction FGC3
(9.3 g) was subjected to silica gel column chromatography (200 g, 60 x 3 cm)
eluted with hexane and acetone (9:1, 6:1) to give compounds FGC4 (26 mg) and
FGC1 (18 mg) Fraction FGC4 (9.3 g) was subjected to silica gel column
chromatography (200 g, 60 x 3 cm) eluted with chloroform and methanol (95:5)
to provide compounds FGC3 (16 mg) and FGC2 (18 mg)
The butanol residue was fractionated by silica gel column chromatography eluted with CHCl3:CH3OH (30:1; 20:1; 10:1; 5:1) to afford 10 fractions (FGB1 to FGB10) Fraction FGB5 (9.3 g) was subjected to silica gel column chromatography (200 g, 60 x 3 cm) eluted with CHCl3: CH3OH (10:1; 6:1) to
afford compound FGC6 (24 mg)