The objectives of the thesis: Study on the chemical compositions of three species: Allophylus livescens, Chirita halongesis and Oldenlandia pinifolia growing in Viet Nam to find bioactive compounds as the basis for further research.
Trang 1MINISTRY OF EDUCATION
AND TRAINING
VIETNAM ACADEMY
OF SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
-
KHIEU THI TAM
STUDY ON CHEMICAL CONSTITUENTS AND CYTOTOXIC
ACTIVITY OF 3 SPECIES ALLOPHYLUS LIVESCENS GAGNEP,
CHIRITA HALONGENSIS KIEW& T.H.NGUYEN AND
OLDENLANDIA PINIFOLIA (WALL EX G.DON) KUNTZE] IN
Trang 2This thesis is completed at: Vietnam Academy of Science and
Technology
Scientific instructors:
Assoc Dr NGUYEN THI HOANG ANH
Assoc Dr TRAN VAN LOC
Trang 3INTRODUCTION
1 The urgency of the thesis
Today, the economic development, the need for care and protection of the health of the community becomes more urgent Thus, the demand for medicine
in preventing and treatment of incurable diseases especially cancer is increasing
The plant resources in Viet Nam are rich and multiple with over 12000 species of 309 plant families Among them, 3900 species have been used for medicine However, the relationship between the chemical structure and pharmacological activity in many cases has been not clarified
Allophylus livescens and Chirita halongesis, two endemic species of Ha
Long Bay, have not yet studied on chemical compositions and biological
activities Oldenlandia pinifolia is used in traditional medicine for treatment of
inflammation, pain and other diseases and there has been only one report on the phytochemistry of this species
To contribute to understanding, research and systematic evaluation of biologically active compounds from coastal plants and mangroves, and to propose directions for the exploitation and conservation of genetic resources of the rare plant species in Vietnam, we conducted research on the chemical
constituents and cytotoxic activity of 3 species: Allophylus livescens Gagnep.,
Chirita halongesis Kiew & T H Nguyen and Oldenlandia pinifolia (Wall ex
G Don) Kuntze growing in Viet Nam
2 The objectives of the thesis
Study on the chemical compositions of three species: Allophylus
livescens, Chirita halongesis and Oldenlandia pinifolia growing in Viet Nam to
find bioactive compounds as the basis for further research
3 The main contents of the thesis
- Isolation of compounds from three species Allophylus livescens, Chirita
halongesis and Oldenlandia pinifolia collected in Viet Nam by column
chromatography
- Determination of chemical structure of isolated compounds based on
IR, MS, 1D-NMR, 2D-NMR spectroscopic analysis
- Evaluation of cytotoxic activity of extracts and some isolated compounds
4 The contribution of the thesis
This is the first time the chemical constituents and cytotoxic activities of
Allophylus livescens and Chirita halongenis species have been studied in
Vietnam as well as in the world Two compounds were first isolated from
Allophylus genus: 1,6,10,14-phytatetraen-3-ol and catechin Oleanolic acid,
decaffeoylacetoside and β-hydroxy acteoside were found for the first time in
Chirita genus
This is the first study on the cytotoxic activity of Oldenlandia pinofolia
Trang 4species in Vietnam and in the world From this species, 14 compounds have
been isolated and structurally elucidated, consisting of a novel compound (1,4,6-trihydroxy-2-methyl-anthraquinone) and two compounds (lutein và
afzelin) isolated for first time from Oldenlandia genus The n-butanol extract
and four isolated compounds inhibited the proliferation of chronic myelogenous leukemia cells, induced apoptosis, and activated caspase 3
(p<0.05)
5 The structure of the thesis
The thesis includes preface (2), contents (131 pages, divided into 4 charptes), conclusion and recommendation (2 pages) There are also list of abbreviations, list of tables, list of figures, published scientific works related to this thesis and references and appendices
THE CONTENT OF THE THESIS
CHAPTER 1: OVERVIEW
1.1 Overview of Allophylus genus
1.1.1 Botanical introduction
1.1.2 Use in traditional medicine
1.1.3 Studied results on the chemical composition and biological activity 1.1.4 Allophylus livescens
Until now, there have been no phytochemical and biological activity work about this species
1.2 Overview of Chirita genus
1.2.1 Botanical introduction
1.2.2 Use in traditional medicine
1.2.3 Study results on the chemical composition and biological activity
1.2.4 Chirita halongensis
Chirita halongensis, an endemic species of Ha Long Bay, detected in the
year 2000 and until now has not been chemically and biologically investigated
1.3 Overview of Oldenlandia genus
1.3.1 Botanical introduction
1.3.2 Use in traditional medicine
1.3.3 Studied results on the chemical composition and biological activity 1.3.4 Oldenlandia pinifolia
Oldenlandia pinifolia (Wall Ex G Don) Kuntze (synonym Hedyotis pinifolia Wall Ex G Don) is a small herb growing in sandy areas from Hue to
the south of Vietnam To the best of our knowledge, there has been only one report on the phytochemistry of this species
1.4 Iridoid compounds
1.4.1 Introduction
1.4.2 Classification
Trang 51.4.3 Biological activity
CHAPTER 2: PLANT METERIALS AND STUDY METHODS
2.1 Plant material and determination of scientific name
- Allophylus livescens Gagnep was collected in Ha Long bay in April 2013 and
determined by Dr Tran Thi Phuong Anh, Vietnam National Museum of Nature,
Vietnam Academy of Science and Technology (VAST) A voucher specimen (No VHH.HL 4.2013.1) was deposited in Institute of Chemistry-Vietnam Academy of Science and Technology (VAST)
- Chirita halongensis Kiew & T.H.Nguyen was collected in Ha Long bay,
Quang Ninh province in October 2013 and determined by Dr Tran Thi Phuong
Anh, Vietnam National Museum of Nature, Vietnam Academy of Science and
Technology (VAST) A voucher specimen (No VHH.HL 10.2013.2) was deposited in Institute of Chemistry-Vietnam Academy of Science and Technology (VAST)
- Oldenlandia pinifolia Wall Ex G Don was collected in Phu Vang, Thua
Thien Hue province in October 2014 and determined by Dr Do Xuan Cam, Hue university A voucher specimen (No VHH.TTH 10.2014.1) was deposited
in Institute of Chemistry-Vietnam Academy of Science and Technology (VAST)
2.2 Methods for treatment and extraction
Leaves, twigs of Allophylus livescens and whole plants of Chirita
halongensis, Oldenlandia pinifolia were extracted by using a general procedure
2.3 Methods for isolation of secondary metabolites
Chromatographic methods: thin layer chromatography (TLC), column chromatography (CC)
2.4 Methods for determination of chemical structure
Spectroscopic methods: Electron Spray Ionisation Mass Spectroscopy (ESI-MS) and High Resolution Electron Spray Ionisation Mass Spectroscopy (HR-ESI-MS), one/two-dimention nuclear magnetic resonance spectra (NMR)
2.5 Methods for biological assays
2.5.1 Cytotoxic activity
The test determined the total cell protein content based on the optical density measured when the cellular protein component was stained with Sulforhodamine B (SRB)
Trang 6Allophylus livescens's leaves and twigs
H 2 O aqueous
Figure 3.1 Isolation of compounds from Allophylus livescens
3.1.2 Physical and spectroscopic data of isolated compounds from Allophylus livescens
3.1.2.1 1,6,10,14 Phytatetraen-3-ol (AL1)
Oil, Rf = 0.48 (n-hexane:CH2Cl2 = 95:5, v/v) ESI-MS, m/z = 291
[M+H]+, 1H-NMR (500 MHz, CD3OD): H (ppm) 5.21 (d, 17.5, H-1); 5.05 (d, 11.0, H-1); 5.91 (dd, 10.5 & 17.5; H-2); 1.55 (m, H-4); 1.95 – 2.09 (m, H-5); 5.14 (t, 7.0; H-6); 1.95 – 2.09 (m, H-8, H-9, H-12, H-13); 5.11 (t, 6.5; H-10); 5.11 (t, 6.5, H-14); 1.68 (s, H-16); 1.59 (s, H-17); 1.27 (s, H-18); 1.60 (s, H-19, H-20) 13C-NMR (125 MHz, CD3OD): C (ppm) 111.6 (C-1); 145.1 (C-2); 73.4 (C-3); 42.1 (C-4); 22.7 (C-5); 124.4 (C-6); 135.5 (C-7); 39.7 (C-8); 26.5 (C-9); 124.1 (C-10); 135.0 (C-11); 39.7 (C-12); 26.7 (C-13); 124.2 (C-14); 131.2 (C-15); 25.6 (C-16); 17.6 (C-17); 27.8 (C-18); 16.0 (C-19, C-20)
3.1.2.2 Catechin (AL2)
Orange power, Rf = 0.56 (CHCl3:MeOH:H2O = 6.5:3.5:0.5, v/v) 1H-NMR (500 MHz, CD3OD): H (ppm) 4.60 (d, 7.5 Hz, H-2); 4.00 (m, H-3); 2.53 (dd, 16.0, 8.1 Hz, H-4a); 2.87 (dd, 16.0; 8.1 Hz, H-4b); 5.88 (d, 2.3 Hz, H-6); 5.96 (d, 2.3 Hz, H-8); 6.86 (d, 1.9 Hz, H-2’); 6.79 (d, 8.1 Hz, H-5’); 6.74 (dd, 8.1; 1.9 Hz, H-6’).13C-NMR (125 MHz, CD3OD): C (ppm) 82.8 (C-2); 68.8 (C-3); 28.5 (C-4); 156.9 (C-5); 95.5 (C-6); 157.5 (C-7); 96.3 (C-8); 157.8 (C-9); 100.8 (C-10); 132.2 (C-1’); 115.3 (C-2’); 146.2 (C-3’); 146.2 (C-4’); 116.1 (C-5’); 120.0 (C-6’)
3.1.2.3 Stigmasterol(AL3)
White needles, Rf = 0.55 (n-hexane:EtOAc = 3:1, v/v) 1H-NMR (500
Trang 7MHz, CDCl3): H (ppm) 3.51 (m, H-3); 5.34 (m, H-5); 0.92 (d, 6.5 Hz, CH319); 5.01 (dd, 8.5; 15.0 Hz, H-20); 5,15 (dd, 8.5; 15.0 Hz, H-21); 0.84 (t, 7.2
-Hz, CH3-24); 0.83 (d, 6.5 Hz, CH3-26); 0.80 (d, 6.6 Hz, CH3-27); 0.70 (s, CH328); 1.03 (s, CH3-29)
-3.1.2.4 -sitosterol(AL4)
White needles, Rf = 0.55 (n-hexane:EtOAc = 3:1, v/v) 1H-NMR (500 MHz, CDCl3): H (ppm) 3.51 (m, H-3); 5.34 (m, H-5); 0.92 (d, 6.5 Hz, CH3-19); 0.85 (t, 7.2 Hz, CH3-24); 0.83 (d, 6.5 Hz, CH3-26); 0.81 (d, 6.6 Hz, CH3-27); 0.68 (s, CH3-28); 1.02 (s, CH3-29)
3.1.2.5 -sitosterol glucoside (AL5)
White needles, Rf = 0.45 (CH2Cl2:MeOH = 9:1, v/v)
3.2 Isolation of compounds from Chirita halongensis
The whole palnt of Chirita halongensis
H 2 O aqueous CH2
Figure 3.2 Isolation of compounds from Chirita halongensis
3.2.2 Physical and spectroscopic data of isolated compounds from Chirita halongensis
3.2.2.1 7-Hydroxytectoquinone (CH1):
Yellow needles, Rf = 0.30 (CH2Cl2:MeOH = 4.8:0.2, v/v) 1H NMR (500 MHz, CD3OD): H (ppm) 8.07 (brs, H-1); 7.68 (dd, 1.0 & 8.0 Hz, H-3); 8.16 (d, 8.0 Hz; H-4); 8.17 (d, 8.5 Hz, H-5); 7.21 (dd, 2.5 & 8.5 Hz, H-6); 7.59 (d, 2.5
Hz, H-8); 2.54 (s, 2-CH3).13C NMR (125MHz, CD3OD): C (ppm) 128.2 (C-1); 146.3 (C-2); 136.1 (C-3); 128.2 (C-4); 132.8 (C-4a); 130.9 (C-5); 122.3 (C-6);
Trang 8164.7 7); 113.5 8); 137.1 8a); 184.6 9); 134.9 9a); 183.3 10); 127.2 (C-10a); 21.8 (2-Me)
Amorphous white power, Rf = 0.3 (n-hexane:EtOAc = 1:1, v/v)
(-)-ESI-MS m/z: 471.0 [M-H]-, molecular formula: C30H48O4 1H-NMR (500 MHz, CDCl3 & CD3OD): H (ppm) 3.83 (br s, H-3); 2.19 (d, 11.0 Hz, H-18); 3.70 (d, 11.0 Hz, Ha-24); 3.46 (d, 11.0 Hz, Hb-24) 13C-NMR (125 MHz, CD3OD): C
(ppm) 34.4 (C-1); 24.5 (C-2); 71.3 (C-3), 44.0 (C-4); 50.6 (C-5); 19.5 (C-6); 34.6 (C-7); 40.9 (C-8); 48.8 (C-9, C-17); 37.9 (C-10); 25.3 (C-11); 126.9 (C-12); 139.6 (C-13); 43.3 (C-14); 29.2 (C-15); 26.1 (C-16); 54.4 (C-18); 40.5 (C-19); 40.4 (C-20); 30.7 (C-21); 38.1 (C-22); 22.8 (C-23); 66.3 (C-24); 16.3 (C-25); 17.6 (C-26); 24.1 (C-27); 181.6 (C-28); 17.7 (C-29); 21.6 (C-30)
3.2.2.3 Ursolic acid (CH3)
Amorphous white power, Rf = 0.5 (CH2Cl2:MeOH = 94:6, v/v)
(-)-ESI-MS m/z: 455.2 [M-H]-, molecular formula: C30H48O3 1H-NMR (500 MHz, CDCl3): H (ppm) 2.98 (m, H-3), 5.11 (m, H-12); 2.09 (d, 11.3 Hz, H-18); 0.88
(s, Me-23); 0.66 (s, Me-24); 0.85 (s, Me-25); 0.73 (s, Me-26); 1.02 (s, Me-27);
0.79 (d, 6.4 Hz, Me-29); 0.89 (d, 8.7 Hz, Me-30) 13C-NMR (125 MHz, CDCl3):
C (ppm)38.2 (C-1); 26.9 (C-2); 76.9 (C-3); 38.4 (C-4); 54.8 (C-5); 18.0 (C-6); 30.2 (C-7); 39.1 (C-8); 47.0 (C-9); 36.5 (C-10); 23.8 (C-11); 124.6 (C-12); 138.2 (C-13); 41.6 (C-14); 32.7 (C-15); 22.8 (C-16); 46.83 (C-17); 52.4 (C-18); 38.4 (C-19); 38.5 (C-20); 27.5 (C-21); 36.3 (C-22); 28.3 (C-23); 16.91 (C-24); 16.1 (C-
25); 15.2 (C-26); 23.3 (C-27); 178.3 (C-28); 17.0 (C-29); 21.1 (C-30)
3.2.2.4 Oleanolic acid (CH4)
Amorphous white power, Rf = 0.48 (CH2Cl2:MeOH = 94:6, v/v)
(-)-ESI-MS m/z: 455.2 [M-H]-, molecular formula: C30H48O3 1H-NMR (500 MHz, CDCl3): H (ppm) 5.27 (1H, t, 3.5 Hz, H-12); 3.20 (1H, dd, 4.0; 11.0 Hz, H-3); 2.81 (1H, dd, 4.0; 13.5 Hz, H-18); 1.12; 0.97; 0.91; 0.90; 0.89; 0.76; 0.74 (each signal 3H, s, Me-23, 24, 25, 26, 27, 29, 30) 13C-NMR (125 MHz, CDCl3): C
(ppm)38.4 (C-1); 27.7 (C-2); 79.1 (C-3); 38.8 (C-4), 55.3 (C-5); 18.3 (C-6); 32.7 (C-7); 39.3 (C-8); 47.7 (C-9); 37.1 (C-10); 23.0 (C-11); 122.7 (C-12); 143.6 (C-13); 41.7 (C-14); 27.2 (C-15); 23.4 (C-16); 46.5 (C-17); 41.1 (C-18); 45.9 (C-19); 30.7 (C-20); 33.8 (C-21); 32.5 (C-22); 28.1 (C-23); 15.6 (C-24); 15.3 (C-25); 17.1 (C-26); 25.9 (C-27); 181.6 (C-28); 33.1 (C-29); 23.6 (C-30)
Amorphous pale yellow power, Rf = 0.3 (EtOAc:MeOH:H2O = 4:0.5:0.1,
v/v) (-)-ESI-MS m/z: 315.0 [M-H]-, molecular formula C14H20O8 1H NMR (500 MHz, CD3OD): H (ppm) 6.71 (d, 1.5 Hz, H-2); 6.69 (d, 8.5 Hz, H-5); 6.57 (dd, 1.5 & 8.5 Hz, H-6); 2.80 (m, H-7); 3.72 (m, H-8); 4.05 (m, H-8); 4.31 (d, 8.0 Hz, Glc-1); 3.20 (t, 8.0 Hz, Glc-2); 3.28 – 3.39 (m, Glc-3, 4, 5); 3.68 (dd,
Trang 95.0 & 12.0 Hz, Glc-6); 3.88 (dd, 1.5 & 12.0 Hz, Glc-6) 13C-NMR (125 MHz,
CD3OD): C (ppm) 131.6 (C-1); 117.1(C-2); 146.1 (C-3); 144.6 (C-4); 116.3 (C-5); 121.3 (C-6); 36.6 (C-7); 72.1 (C-8); 104.4 (Glc-1); 75.1(Glc-2); 77.9 (Glc-3); 71.6 (Glc-4); 78.1 (Glc-5); 62.7 (Glc-6)
3.2.2.6 Acteoside (CH6)
Amorphous power, Rf = 0.56 (CH2Cl2:MeOH:H2O = 3.75:1.0:0.1, v/v)
1
H-NMR (500 MHz, CD3OD): H (ppm) 6.73 (d, 2.0 Hz, H-2); 6.71 (d, 8.0 Hz, H-5); 6.58 (dd, 2.0; 8.5 Hz, H-6); 3.75 (m, H-8); 4.06 (m, H-8); 2.81 (m, H-7);
7.09 (d, J = 1.5 Hz, H-2’); 6.81 (d, 8.0 Hz, H-5’); 6.98 (dd, 1.5 & 8.0 Hz, H-6’);
6.30 (d, 16.0 Hz, H-8’); 7.62 (d, 16.0 Hz, H-7’); 4.40 (d, 8.0 Hz, H-1”); 3.3 – 4.1 (m, H-2”, 3”); 4.95 (t, 9.0 Hz, H-4”); 3.3 – 4.1 (m, H-5”); 5.22 (brs, H-1”’); 3.3 -4.1 (m, H-2”’, H-3”’, H-4”’, H-5”’); 1.12 (d, 6.5 Hz, H-6”’).13C-NMR (125 MHz, CD3OD): C (ppm) 131.5 (C-1); 116.5 (C-2); 144.6 (C-3); 146.0 (C-4); 117.1 (C-5); 121.3 (C-6); 72.3 (C-8); 36.5 (C-7); 127.6 (C-1’); 114.7 (C-2’); 149.7 (C-3’); 146.7 (C-4’); 116.3 (C-5’); 123.2 (C-6’); 168.3 (C-9’); 115.3 (C-8’); 148.0 (C-7’); 104.1 (C-1”); 76.1 (C-2”); 81.6 (C-3”); 70.4 (C-4”); 75.9 (C-5”); 62.3 (C-6”); 103.0 (C-1”’); 72.0 (C-2”’); 72.2 (C-3”’); 73.8 (C-4”’); 70.6 (C-5”’); 18.4 (C-6”’)
3.2.2.7 Isoacteoside (CH7)
Amorphous power, Rf = 0.44 (CH2Cl2:MeOH:H2O = 3.75:1.0:0.1, v/v)
1
H-NMR (500 MHz, CD3OD): H (ppm) 6.70 (d, 1.5 Hz, H-2); 6.67 (d, 8.0 Hz, H-5); 6.55 (dd, 1.5; 8.0 Hz, H-6); 3.73 (m, H-8); 4.05 (m, H-8); 2.80 (m, H-7); 7.06 (d, 2.0 Hz, H-2’); 6.79 (d, 8.0 Hz, H-5’); 6.90 (dd, 2.0 & 8.5 Hz, H-6’); 6.30 (d, 16.0 Hz, H-8’); 7.58 (d, 16.0 Hz, H-7’); 4.33 (d, 8.0 Hz, H-1”); 3.4 – 4.0 (m, H-2”, H-3”, H-4”, H-5”); 4.39 (brt, 5.5 Hz, H-6”); 4.52 (dd, 1.5; 11.5
Hz, H-6”); 5.21 (brs, H-1”’); 3.4 – 4.0 (m, H-2”’, H-3”’, H-4”’,H-5”’); 1.28 (d, 6.0 Hz, H-6”’) 13C-NMR (125 MHz, CD3OD): C (ppm) 131.4 (C-1); 117.1 (C-2); 146.0 (C-3); 144.6 (C-4); 116.4 (C-5); 121.3 (C-6); 72.4 (C-8); 36.6 (C-7); 127.7 (C-1’’); 115.1 (C-2’); 146.7 (C-3’); 149.5 (C-4’); 116.6 (C-5’); 123.2 (C-6’); 169.2 (C-9); 114.8 (C-8’); 147.2 (C-7’); 104.3 (C-1”); 75.3 (C-2”); 84.0 (C-3”); 70.0 (C-4”); 75.6 (C-5”); 64.6 (C-6”); 102.7 (C-1”’); 72.2 (C-2”’); 72.3 (C-3”’); 74.0 (C-4”’); 70.4 (C-5”’); 17.9 (C-6”’)
3.2.2.8 Decaffeoylacteoside (CH8)
Amorphous power, Rf = 0.56 (CH2Cl2:MeOH:H2O = 3.5:1.0:0.1, v/v)
(-)-ESI-MS m/z: 461.0 [M-H]-, molecular formula: C20H30O12 1H-NMR (500 MHz,
CD3OD): H (ppm) 6.71 (d, 1.5 Hz, H-2); 6.69 (d, 8.0 Hz, H-5); 6.57 (dd, 2.0; 8.0 Hz, H-6); 3.72 (m, H-8); 4.01 (m, H-8); 2.80 (m, H-7); 4.31 (d, 8.0 Hz, H-1”); 3.3 – 4.1 (m, H-2”, H-3”, H-4”, H-5”); 3.37 (brt, 9,5 Hz, H-6”); 3.42 (brt, 9.5 Hz, H-6”); 5.17 (d, 1.0 Hz, H-1”’); 3.3 – 4.1 (m, H-2”’, H-3”’, H-4”’, H-
Trang 105”’); 1.26 (d, 7,0 Hz, H-6”’) và 13
C-NMR (125 MHz, CD3OD): C (ppm) 131.5 (C-1); 116.3 (C-2); 144.6 (C-3); 146.1(C-4); 117.1 (C-5); 121.2 (C-6); 72.1 (C-8); 36.5 (C-7); 104.2 (C-1”); 75.6 (C-2”); 84.5 (C-3”); 70.1 (C-4”); 77.8 (C-5”); 62.7 (C-6”); 102.8 (C-1”’); 72.2 (C-2”’); 72.3 (C-3”’); 74.0 (C-4”’); 70.2 (C-5”’); 17.9 (C-6”’)
Amorphous power, Rf = 0.38 (CH2Cl2: MeOH: H2O = 3.5:1.0:0.5, v/v) 1NMR (500 MHz, CD3OD): H (ppm) 6.73 (d, 2.0 Hz, H-2); 6.76 (d, 8.0 Hz, H-5); 6.87 (dd, 2.0; 8.0 Hz, H-6); 3.57 (m, H-8); 4.00 (m, H-8); 4.77 (m, H-7); 7.07 (d, 2.0 Hz, H-2’); 6.80 (d, 8.0 Hz, H5’); 6.98 (dd, 2.0 & 8.0 Hz, H-6’); 6.29 (d, 16.0 Hz, H-8’); 7.62 (d, 16.0 Hz, H-7’); 4.40 (d, 8.0 Hz, H-1”); 3.5 – 4.0 (m, H-2”, H-3”); 4.96 (t, 9.0 Hz, H-4”); 3.5 – 4.0 (m, H-5”); 3.42 (brt, 8.5
Hz, 6”); 3.84 (brt, 9.0 Hz, 6”); 5.22 (brs, 1”’); 3.5 – 4.0 (m, 2”’, 3”’, H-4”’, H-5”’); 1.12 (d, 6.0 Hz, H-6”’) 13
H-C-NMR (125 MHz, CD3OD): C
(ppm) 133.7 (C-1); 114.6 (C-2); 146.3 (C-3); 146.1 (C-4); 116.5 (C-5); 119.0 (C-6); 76.7 (C-8); 74.2 (C-7); 127.7 (C-1’’); 115.3 (C-2’); 146.9 (C-3’); 149.8 (C-4’); 116.2 (C-5’); 123.2 (C-6’); 168.3 (C-9’); 114.7 (C-8’); 148.0 (C-7’); 104.6 (C-1”); 76.4 (C-2”); 81.3 (C-3”); 70.5 (C-4”); 76.1 (C-5”); 62.3 (C-6”); 102.9 (C-1”’); 72.1 (C-2”’); 72.4 (C-3”’); 73.8 (C-4”’); 70.4 (C-5”’); 18.4 (C-6”’)
3.3 Isolation of compounds from Oldenlandia pinifolia
The whole palnt of Oldenlandia pinifolia
H 2 O aqueous HP5
Figure 3.3 Isolation of compounds from Oldenlandia pinifolia
3.3.2 Physical and spectroscopic data of isolated compounds from Oldenlandia pinifolia
3.3.2.1 1,4,6-Trihydroxy-2-methyl-anthraquinone (HP1)
Trang 11Orange powder, Rf = 0.45 (n-hexane:CH2Cl2 = 4:1, v/v) HR-ESI-MS: m/z =
3.3.2.3 1,6-Dihydroxy-2-methylanthraquinone (HP3)
Orange powder, Rf = 0.47 (n-hexane:CHCl3:EtOAc = 1.0:1.5:1.0,
v/v).(-)-ESI-MS m/z: 253.0 [M-H]-, molecular formula: C15H10O4 1H-NMR (500 MHz, DMSO-d6): H (ppm) 7.61 (d, 7.5 Hz, H-3); 7.55 (d, 7.5 Hz, H-4); 7.44 (d, 2.5
Hz, H-5); 7.21 (dd, 2.5; 8.5 Hz, H-7); 8.08 (d, 8.5 Hz, H-8); 13.08 ( s, 1-OH); 2.27 (s, 2-CH3) 13C-NMR (125 MHz, DMSO-d6): C (ppm) 159.9 (C-1); 114.6 (C-2); 136.8 (C-3); 118.6 (C-4); 112.5 (C-5); 163.9 (C-6); 121.4 (C-7); 129.8 (C-8); 187.6 (C-9); 181.7 (C-10); 131.1 (C-4a); 124.4 (C-8a); 134.2 (C-9a); 135.6 (C-10a); 15.7 (2-CH3)
3.3.2.4 Digiferruginol (HP4)
Orange-yellow needles, Rf = 0.5 (CH2Cl2: MeOH = 9:1, v/v) (-)-ESI-MS
DMSO-d6), δH (ppm) 7.77 (d, 7.5 Hz, H-3); 7.92 (d, 8.0 Hz, H-4); 8.20 (m, H-5); 7.95
(m, H-6, H-7); 7.92 (m, H-7); 8.25 (m, H-8); 4.66 (d, 5.0 Hz, 2 -CH2OH); 5.46 (t, 5.5 Hz, 2-CH2OH); 12.77 (s, 1-OH) 13C NMR (125 MHz, DMSO- d6), C
(ppm) 158.4 (C-1); 138.2 (C-2); 131.3 (C-3); 118.8 (C-4); 126.8 (C-5); 134.5 (C-6); 135.1 (C-7); 126.6 (C-8); 188.7 (C-9); 181.8 (C-10); 133.6 (C-5a); 133.2 (C-8a); 114.9 (C-9a); 132.8 (C-10a); 57.4 (CH2OH)
3.3.2.5 Lutein(HP5)
Orange-red powder, Rf = 0.44 (n-hexane: EtOAc = 3.75:1.25, v/v) ESI-MS m/z: 569.3 [M+H]+, molecular formula: C40H56O2 1H NMR (500 MHz, CDCl3): H (ppm) 1.48 (t, 12.0 Hz, H2-2); 4.00 (m, H-3); 2.04 (dd, 17.0, 10.0
(+)-Hz, H-4ax); 2.33 – 2.42 ( m, H-4eq); 6.12 (s, H-7, H-8); 6.15 (m, H-10); 6.67 (m, H-11, H-15, H-11’, H-15’); 6.36 (d, 15.0 Hz, H-12); 6.25 (brd, 9.0 Hz, H-14); 1.07 (s, 1-(CH3)2); 1.97 (s, 9-CH3, 13-CH3); 1.37 (dd, 13.0; 7.0 Hz, H-2’ax); 1.84 (dd, 13.0; 6.0 Hz, H-2’eq); 4.25 (m, H-3’); 5.54 (brs, H-4’); 2.33 – 2.42 (m, H-6’); 5.43 (dd, 10.0; 15.5 Hz, H-7’); 6.15 (m, H-8’, H-10’); 6.36 (d, 15.0 Hz, H-12’); 6.25 (brd, 9.0 Hz, H-14’); 0.85 (s, 1’-CH3); 1.00 ( s, 1’-CH3);
Trang 126.58-1.63 (s, 5’-CH3); 1.91 (s, 9’-CH3); 1.97 (s, 13’-CH3) 13C NMR (125 MHz, CDCl3): C (ppm) 37.1 (C-1); 48.5 (C-2); 65.1 (C-3); 42.6 (C-4); 126.2 (C-5); 137.6 (C-6); 125.6 (C-7); 138.5 (C-8); 135.7 (C-9); 131.3 (C-10); 124.8 (C-11); 137.7 (C-12); 136.5 C-13); 132.6 (C-14); 130.1 (C-15); 28.7 (1-Me); 30.3 (1-Me); 21.6 (5 –Me); 12.8 (9-Me, 13-Me); 34.0 (C-1’); 44.6 (C-2’); 65.9 (C-3’); 125.6 (C-4’); 137.8 (C-5’); 55.0 (C-6’); 128.7 (C-7’); 138.0 (C-8’); 135.1 (C-9’); 130.8 (C-10’); 124.5 (11’); 137.6 (C-12’); 136.4 (C-13’); 132.6 (C-14’); 130.0 (C-15’); 24.3 (1’-Me); 29.5 (1’-Me); 22.9 (5’-Me); 14.1 (9’-Me); 13.1 (13’-Me)
White power, Rf = 0.54 (CH2Cl2: MeOH: H2O = 4.0:1.0:0.1, v/v)
(+)-ESI-MS m/z: 437.0 [M+Na]+, molecular formula: C18H22O11 1H NMR (500 MHz, CD3OD): H (ppm) 5.97 (d, 1.0 Hz, H-1); 7.32 (d, 2.0 Hz, H-3); 3.70 (m, H-5); 5.59 (brd, 6.5 Hz, H-6); 5.75 (brs, H-7); 3.32 (m, H-9); 4.69 (dd, 14.0 Hz, 1.0 Hz, H-10a); 4.80 (dd, 14.0, 1.0 Hz, H-10b); 2.10 (s, CH3CO); 4.71 (d, 8.0
Hz, Glc-1); 3.22 (dd, 9.0, 8.0 Hz, Glc-2); 3.31 – 3.40 (m, Glc-3, 4, 5); 3.94 (dd, 12.0, 2.0 Hz, Glc-6); 3.70 (dd, 12.0; 6.0 Hz, Glc-6) 13C NMR (125 MHz,
CD3OD): C (ppm) 93.3 1); 150.3 3); 106.1 4); 37.4 5); 86.3 6); 128.9 (C-7); 144.2 (C-8); 45.2 (C-9); 61.9 (C-10); 172.3 (C-11); 172.6 (CH3CO); 20.6 (CH3CO); 100.0 (Glc-1); 74.6 (Glc-2); 78.3 (Glc-3); 71.6 (Glc-4); 77.8 (Glc-5); 62.8 (Glc-6)
(C-3.3.2.9 Deacetyl asperuloside (HP9)
White power, Rf = 0.67 (CH2Cl2:MeOH = 4:1, v/v) (-)-ESI-MS m/z:
370.9 [M-H]-, molecular formula: C16H20O10 1H NMR (500 MHz, CD3OD): H
(ppm) 5.97 (d, 1.5 Hz, H-1); 7.31 (d, 2.0 Hz, H-3); 3.70 (m, H-5); 5.58 (dd, 1.5; 6.5 Hz, H-6); 5.66 (brs, H-7); 3.32 (m, H-9); 4.21 (brs, H-10); 4.70 (d, 8.0 Hz, Glc-1); 3.21 (dd, 9.0; 8.0 Hz, Glc-2); 3.30 – 3.60 (m, Glc-3, Glc-4, Glc-5); 3.94 (dd, 12.0; 2.0 Hz, Glc-6); 3.84 (dd, 12.0; 6.0 Hz, Glc-6) 13C NMR (125 MHz,
CD3OD): C (ppm) 93.3 1); 150.2 3); 106.5 4); 37.5 5); 86.6 6); 125.7 (C-7); 149.8 (C-8); 45.0 (C-9); 60.1 (C-10); 172.8 (C-11); 99.9 (Glc-1); 74.7 (Glc-2); 78.4 (Glc-3); 71.6 (Glc-4); 77.9 (Glc-5); 62.8 (Glc-6)
(C-3.3.2.10 Asperulosidic acid (HP10)
White power, Rf = 0.56 (CH2Cl2:MeOH = 4:1, v/v) 1H NMR (500 MHz,
CD3OD): H (ppm) 5.07 (d, 9.0 Hz, H-1); 7.63 (s, H-3); 3.05 (m, H-5); 4.85 (m, H-6); 6.04 (brs, H-7); 2.65 (t, 8.0 Hz, H-9); 4.97 (brd, 14.5 Hz, H-10a); 4.83