Summary of chemistry doctoral thesis: Study on chemical constituents and biological activities from the leaves of Excoecaria agallocha L. and Excoecaria cochinchinensis Lour.

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The objectives of the thesis: Isolation and determination of chemical structures of the isolated compounds from the leaves of Excoecaria agallocha L. and Excoecaria cochinchinensis Lour.; studied the cytotoxic, anti-inflammatory, and antimicrobial activities of the isolated compounds to find the bioactive compounds.

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Nội dung Text: Summary of chemistry doctoral thesis: Study on chemical constituents and biological activities from the leaves of Excoecaria agallocha L. and Excoecaria cochinchinensis Lour.

1 MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND ECHNOLOGY ------------------------ Lai Hop Hieu STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES FROM THE LEAVES OF EXCOECARIA AGALLOCHA L. AND EXCOECARIA COCHINCHINENSIS Lour. Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi – 2021
2 This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Adviser 1: Prof. Dr. Ngo Dai Quang Adviser 2: Dr. Nguyen Van Thanh 1st Reviewer: …………………………………………………….... 2nd Reviewer: : ……………………………………………………. 3rd Reviewer: : …………………………………………………….. The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at hour date month 2021. Thesis can be found in: - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology - National Library
1 INTRODUCTION 1. The urgency of the thesis Throughout human history, marine microorganisms and natural plants have become potential sources in the discovery of novel drugs for the treatment of human diseases. Nowadays, more than 70% of anti- cancer drugs in the market are derived from natural products or synthesized based on the structure of natural compounds. Besides cancer, which is a major issue of concern for scientists, the emergence of antibiotic drug resistance is also a big threat to human health worldwide. Antibiotic drug resistance occurs when microorganisms such as viruses, fungi or parasites change their mechanism of action in response to the existing antimicrobial treatments. Several factors contribute to antibiotic resistance such as the overuse/misuse of antibiotics and the self-medication with antibiotics. The important role of natural bioactive compounds has been investigated from traditional medicine to modern medicine. Their value is not only for direct use as a medicine but also as a structure lead compound for the discovery and development of new drugs. In an attempt to investigate and research medicinal materials for public health care programs, the study on natural compounds which exhibit several biological activities such as cytotoxicity, anti-cancer, anti- microorganisms... for treatment of cancer and antibiotic multidrug- resistance is one of the main goals of scientists around the world. Marine organisms and mangrove plants raise much attention to the scientists in the field of biomedicine and pharmacology. Several studies have been carried out to investigate new bioactive compounds derived from mangrove plants. Therefore, the thesis namely “Study on chemical constituents and biological activities from the leaves of Excoecaria agallocha L. and Excoecaria cochinchinensis Lour.” was conducted to investigate potential bioactive compounds from E. agallocha and E. cochinchinensis in order to demonstrate more clearly the therapeutic uses in traditional medicine and increase the scientific value of these plants in Vietnam. 2. The objectives of the thesis  Isolation and determination of chemical structures of the isolated compounds from the leaves of Excoecaria agallocha L. and Excoecaria cochinchinensis Lour.
2  Studied the cytotoxic, anti-inflammatory, and antimicrobial activities of the isolated compounds to find the bioactive compounds. 3. The main contents of the thesis  Isolation of compounds from the leaves of Excoecaria agallocha and E. cochinchinensis using various chromatographic separations. Determination of chemical structures of the isolated compounds.  Evaluation of the cytotoxic, anti-inflammatory, and antimicrobial activities of the isolated metabolites to find out potential compounds. CHAPTER I. OVERVIEW This chapter presents the overview of domestic and international studies related to the chemical compositions and biological activities of E. agallocha and E. cochinchinensis. CHAPTER II. RESEARCH OBJECTIVE AND RESEARCH METHODOLOGY II.1. Research objective Figure II.1. E. agallocha Figure II.2. E. cochinchinensis The leaves of E. agallocha were collected in Xuan Thuy, Nam Dinh, Vietnam in July 2013. The leaves of E. cochinchinensis were collected in Van Giang, Hung Yen, Vietnam in April 2016. Two samples were identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. The voucher specimens were deposited at the Institute of Ecology and Biological Resources and Institute of Marine Biochemistry, VAST, Vietnam.
3 II.2. Research methodology II.2.1. Methods for extraction The samples were cut into pieces and extracted three times with MeOH at room temperature (for 3 days) or in an ultrasonic bath (three times, each time 45 min). Evaporation of the solvent in vacuo obtained a residue, which was suspended in distilled water and partitioned in turn with n-hexane, CH2Cl2, and EtOAc. 2.2.2. Methods for metabolites isolation Combining a number of chromatographic methods including thin- layer chromatography (TLC), column chromatography (CC), silica gel, RP-18, and Sephadex LH-20... II.2.2. Methods for determination of the chemical structure of compounds The general method used to determine the chemical structure of compounds is the combination between physical parameters and modern spectroscopic including optical rotation ([α]D), electrospray ionization mass spectrometry (ESI-MS), and high-resolution ESI-MS (HR-ESI-MS), one/two- dimension nuclear magnetic resonance (NMR) spectra. II.2.3. Methods for evaluation of biological activities  Cytotoxic activity was evaluated against three human cancer cell lines, MCF-7 (human breast cancer cells), LU-1 (human lung adenocarcinoma), and KB (human epidermoid carcinoma) by the MTT and SRB assays.  Anti-inflammatory activity of isolated compounds was assessed based on inhibiting NO production in lipopolysaccharide (LPS) activated RAW264.7 cells.  The antimicrobial activity of the isolated metabolites against a selected panel of the Gram-positive (Bacillus subtillis ATCC11774 and Staphylococcus aureus ATCC11632) and Gram-negative (Escherichia coli ATCC25922, and Pseudomonas aeruginosa ATCC27853) bacteria, as well as a set of yeast molds (Aspergillus niger 439, Fusarium oxysporum M42, Candida albicans ATCC7754, and Saccharomyces cerevisiae SH 20), were also determined.
4 CHAPTER III. EXPERIMENT AND EMPIRICAL RESULTS III.1. Isolation of compounds III.1.1. Isolation of compounds from E. agallocha This part showed the extraction and isolation experiments of the compounds isolated from the leaves of E. agallocha. The leaves of Excoecaria agallocha A: Acetone CC: Chromatography column (2.5 kg dried) D: Dichloromethane Extraction with ultrasonic bath MeOH, 3 times, 45min, 45-60oC. M: Methanol H: n-Hexane MeOH extract W: water (A, 200 g) Add water (1L) Add CHCl3 (1L×3 times) CHCl3/H2O 1:1 CHCl3 fraction H2O layer (94 g) n-Hexane/60% Aq. MeOH Add EtOAc (1L×3 times) n-Hexane fraction MeOH fraction EtOAc fraction H2O layer (H, 80 g) (C, 14 g) (E, 7 g) (W) n-Hexane, n-Hexane-acetone gradien 100:1, 70:1,...0:100 C-1 C-2 C-3 (2.6 g) ( 1.1 g) CC, Silica gel (10.1 g) HA 3:1, DA 4:1 C-3A C-3B C-3C RP-18, CC, RP-18, CC, MW 1:1 MW 1:2 EA-5 EA-3 (5,8 mg) (8 mg) Figure III.1. Isolation of compounds from the CHCl3 fraction of E. agallocha Cặn ethylfraction EtOAc acetate (E, 7 g) RP-C18, CC, H2O/MeOH :2/1 E-1 E-2 E-3 E-4 (0,45 g) (1,5 g) (2,7g) (2,24 mg) Sephadex H2O/MeOH:2/1 E-2B E-2B CC, CH2Cl2/MeOH: 2/1 v/v EA-9 E-1 E-2 E-3 (8,2 mg) (0,45 g) (1,5 g) (2,7g) Sephadex Sephadex H2O/MeOH:1/1 H2O/MeOH:1/1 EA-4 EA-6 EA-7 (10,2 mg) (12 mg) (7 mg) Figure III.2. Isolation of compounds from the EtOAc fraction of E. agallocha
5 Water layer Diaion HP-20 MeOH-H2O (gradient 0:100, 25:75, 50:50, v/v) W-1 W-2 W-3 W-4 Silica gel CC, Silica gel CC, CHCl3-MeOH (50:1, 25:1, v/v) CHCl3-MeOH (30:1, 20:1, v/v) W-2A W-2B W-3C W-3A W-3B W-3D RP-18 CC RP-18 CC MeOH-H2O (3:3, v/v) Acetone-H2O (1:3,5, v/v) W-3A1 W-3A2 W-3B1 W-3B2 W-3B3 Silica gel CC, Sephadex LH-20, Sephadex LH-20, n-Hexane-acetone (5:2) MeOH-H2O (1:2) MeOH-H2O (1:2) EA-8 EA-1 EA-2 (4 mg) (3.5 mg) (5 mg) Figure III.3. Isolation of compounds from the water layer of E. agallocha III.1.2. Isolation of compounds from E. cochinchinensis This section presents the process of isolating 13 compounds from the leaves of E. cochinchinensis. The leaves of Excoecaria cochinchinensis (3 kg dried) Extraction with ultrasonic bath MeOH, 3 time, 45min, 45-55oC MeOH extract (M, 450 g) Add water (1L) n-Hexane/H2O 1:1 Add n-hexane (1.5L × 3 time) n-Hexane fraction H2O layer (H, 120 g) add EtOAc (1.5L × 3 time) EtOAc/H2O 1:1 EtOAc fraction (E, 100 g) H2O layer (W) Figure III.4. The partitioned MeOH extract of E. cochinchinensis
6 Water layer Diaion HP-20 MeOH-H2O (gradient 0:100, 25:75, 50:50, v/v) W-1 W-2 W-3 W-4 (85 g) Silica gel CC, Silica gel CC, CHCl3-MeOH (50:1, 25:1, v/v) CHCl3-MeOH (30:1, 20:1, v/v) W-2A W-2B W-3A W-3B W-3C W-3D RP-C18 CC Acetone-H2O (1:3,5, v/v) W-3B1 W-3B2 W-3B3 Sephadex LH-20 (MeOH-H2O, 1:2) EC-2 EC-6 (8 mg) (3 mg) Silica gel CC, n-Hexane-acetone (5:2); Sephadex LH-20, MeOH-H2O (1:1) EC-11 EC-10 EC-9 EC-8 (2,7 mg) (6,6 mg) (3 mg) (3 mg) Silica gel CC, CH2Cl2-MeOH-H2O (5:1:0,1); RP-C18, MeOH-H2O (1:1) EC-5 EC-4 EC-3 EC-1 (5,5 mg) (2 mg) (2 mg) (2,2 mg) Silica gel CC, CH2Cl2-MeOH-H2O (6:1:0,05); Sephadex LH-20, MeOH-H2O (1:1) EC-13 EC-12 EC-7 (20 mg) (21 mg) (3 mg) Figure III.7. Isolation of compounds from the water layer of E. cochinchinensis III.1.3. Physical properties and spectroscopic data of the isolated compounds III.1.3.1. Physical properties and spectroscopic data of the isolated compounds from E. agallocha This section presents physical properties and spectroscopic data of 09 compounds from E. agallocha. III.1.3.2. Physical properties and spectroscopic data of the isolated compounds from E. cochinchinensis This section presents physical properties and spectroscopic data of 13 compounds from E. cochinchinensis.
7 III.2. Results on cytotoxic activities of isolated compounds III.2.1. Results on cytotoxic activity of extract from E. agallocha Table III.1. The effects of the MeOH extract from E. agallocha Cell line Sample KB LU-1 MCF7 % IC50 % IC50 % IC50 inhibition (µg/mL) inhibition (µg/mL) inhibition (µg/mL) MeOH 81.90 19.77 85.03 15.23 65.38 57.57 extract Ellipticine 97.18 0.39 96.35 0.50 95.73 0.48 10 µg/mL III.2.2. Results on antimicrobial activity of compounds from E. agallocha Table III.3. The effects of isolated compounds from E. agallocha Minimum inhibitory concentration (MIC, g/mL) Samples Gram (-) Gram (+) Fungus * * * * * * Ec Pa Bc Sa An Fo Sc* Ca* Streptomycin - - 7.188 14.375 - - - - (57,5 g/mL) Nystatin - - - - 23.125 11.563 5.781 11.563 (92,5 g/mL) Tetracyclin 5.5 11 - - - - - - (44 g/mL) EA-1 >50 - - - - - - >50 EA-2 - - >50 - >50 - - - EA-3 - - - - >50 - - - EA-4 - - >50 - - - - - EA-5 - >50 - - - 50 - - EA-6 - - - - - - - - EA-7 - - - - - - - - EA-8 - - - - - - - - EA-9 >50 - - - >50 - >50 - MeOH - - 200 - - - - - extract Streptomycin, nystatin, and tetracyclin were used as the positive control. Ec (Escherichia coli), Pa (Pseudomonas aeruginosa), Bc (Bacillus subtillis), Sa (Staphylococcus aureus), An (Aspergillus niger), Fo (Fusarium oxysporum), Sc (Saccharomyces cerevisiae), and Ca (Candida albicans). (-) No detection.
8 III.2.3. Results on anti-inflammatory activity of isolated compounds from E. cochinchinensis Table III.4. Effects of compounds on the LPS-induced NO production on RAW264.7 cells from E. cochinchinensis Concentration Compounds Inhibition (%) Growth of cell (%) (µM) EC-1 38.72 ± 0.56 76.12 ± 1.36 EC-2 46.78 ± 0.35 78.89 ± 1.32 EC-3 75.83 ± 0.77 86.65 ± 1.54 EC-4 31.09 ± 1.60 56.49 ± 0.97 EC-5 42.02 ± 1.01 75.51 ± 1.52 EC-6 68.18 ± 0.67 73.59 ± 0.67 EC-7 100 39.22 ± 1.07 78.60 ± 2.18 EC-8 94.96 ± 0.26 80.41 ± 1.66 EC-9 82.91 ± 1.03 83.19 ± 2.37 EC-10 27.87 ± 0.81 84.55 ± 0.98 EC-11 30.47 ± 0.69 82.91 ± 1.43 EC-12 38.38 ± 0.19 70.16 ± 1.77 EC-13 35.01 ± 0.76 55.50 ± 2.54 0.3 33.89 ± 0.51 95.35 ± 0.75 Cardamonin 3 88.80 ± 0.51 86.00 ± 1.55 Cardamonin was used as a positive control. Data are presented as the mean ± standard deviation (SD) of at least three independent experiments performed in triplicate. Table III.5. The IC50 values of selected compounds Compounds IC50 values (µM) EC-3 13.80 ± 1.23 EC-6 58.10 ± 2.04 EC-8 6.17 ± 0.25 EC-9 12.02 ± 0.73 Cardamonin 1.57 ± 0.24 Cardamonin was used as a positive control. Data are presented as the mean ± standard deviation (SD) of at least three independent experiments performed in triplicate.
9 CHAPTER IV. DISCUSSIONS IV.1. Determination of the chemical structure of compounds from E. agallocha This section presents the detailed results of spectral analysis and structure determination of 09 isolated compounds from E. agallocha. The detailed methods for the determination of the chemical structure of a new compound are introduced in the following section. IV.1.1. Excoecarin L (EA-1, new compound) Figure IV.1. Structure of EA-1 and keys COSY, HMBC correlations and reference compounnd Intens. +MS, 1.5min #90 x105 355.1761 3.0 343.1897 2.5 2.0 1.5 1.0 373.1984 0.5 397.2241 311.1806 325.1961 339.2025 382.1945 388.3920 0.0 300 310 320 330 340 350 360 370 380 390 m/z Figure IV.2. HR-ESI-MS spectrum of EA-1 Compound EA-1 was obtained as an amorphous white powder. Its molecular formula was determined by HR-ESI-MS as C19H28O4 on the basis of the [M + Na]+ sodiated-molecular ion peak observed at m/z 343.1897 (calcd. for C19H28O4Na+, 343.1880). The 13C NMR and HSQC spectra revealed the presence of 19 carbon atoms corresponding to four quaternary carbons, six methines, eight methylenes, and one methyl. Among them, two oleﬁnic methines (δC 135.2 and 135.5), four oxygenated carbons (two methylenes, one methine, and one quaternary carbon resonating at δC 68.66, 69.4, 71.1, and 98.6, respectively) were evident. With six degrees of unsaturation established from the molecular formula, compound EA-1 was suggested to contain ﬁve rings and one double-bond. The 1H NMR spectrum conﬁrmed the presence of one sec- methyl group [δH 1.12 (3H, d, J = 7.0 Hz, H-18)], one oxymethine group
10 Hình III.3. Phổ 1H NMR của hợp chất EA-1 Hình III.4. Phổ 13C NMR của hợp chất EA-1 Figure VI.5. HSQC spectrum of EA-1 [δH 3.75 (1H, ddd, J = 4.0, 11.0, 11.5 Hz, H-6)], two oxymethylene groups [δH 3.40 (1H, d, J = 11.0 Hz, Ha-17)/3.45 (1H, d, J = 11.0 Hz, Hb-17) and 3.80 (1H, d, J = 9.5 Hz, Ha-20)/3.89 (1H, dd, J = 3.5, 9.5 Hz, Hb-20)], and two oleﬁnic protons of a disubstituted double bond [δH 5.73 (1H, d, J = 6.0 Hz, H-15) and 5.66 (1H, d, J = 6.0 Hz, H-16)] (Table IV.1).
11 Figure VI.6. HMBC spectrum of EA-1 Figure VI.7. COSY spectrum of EA-1 Detailed analysis of correlations provided by COSY and HMBC experiments (Fig. IV.1) revealed that the planar structure of EA-1 was similar to that of agallochin I, previously isolated from the same species, except for the presence of an additional hydroxy group at C-17. In fact, the HMBC cross-peaks from H-17 to C-12, C-13, C-14, and C-16 placed the hydroxy group at C-17, whereas the other hydroxy group and the methyl group were placed at C-6 and C-4, respectively, due to the COSY correlations of H-18/H-4/H-5/H6/H-7. The downﬁeld chemical shift of the quaternary carbon at δC 98.6 (C-3) in conjunction with the HMBC correlations from H-20 to C-1, C-3, C-5, and C-10 indicated that the ether bridge was positioned between C-20 and C-3, and the last hydroxy group was located at C-3.
12 Figure VI.8. Keys NOESY correlations of EA-1 Figure IV.9. NOESY spectrum of EA-1 The relative stereochemistry of EA-1 was obtained through analysis of 1H NMR coupling constants and NOESY experiment. Speciﬁcally, the large J-values (J = 11.0 - 12.5 Hz) of H-5, H-6, Ha-7, and H-9 indicated the axial orientation of these protons. The NOE correlations between H-5/H-9, Ha-1, Ha-7; Ha-7/Ha-14, H-9; Hb-20/H- 15; H-15/H-16 and Ha-20/Ha-11, Hb-1, Ha-2 conﬁrmed the structure of beyer-15-ene diterpenoid skeleton. Finally, the conﬁgurations at C-4 and C-6 were determined on the basis of the NOE correlations between H- 6/Hb-20, H-15, H-4 and between H3-18/Hb-2 (Fig. IV.8-IV.9). Therefore, compound EA-1 was elucidated as 3β,20-epoxy-3,6α,17- trihydroxy-19-nor-beyer-15-ene (excoecarin L).
13 Table IV.1. The NMR data of EA-1 and reference compound # No. δCa,b [35] δCc,d δCc,e (mult., J in Hz) 1 31.2 32.7 1.27 (1H, m) 2.09 (1H, ddd, 3.5, 12.5, 12.5) 2 26.9 28.2 1.71 (1H, m) 2.02 (1H, ddd, 3.5, 12.0, 13.5) 3 98.3 98.6 - 4 41.5 42.7 1.96 (1H, m) 5 56.9 57.9 1.03 (1H, dd, 5.0, 11.0) 6 69.5 71.1 3.75 (1H, ddd, 4.0, 11.0, 11.5) 7 44.7 46.2 1.43 (1H, dd, 11.5, 13.0) 1.85 (1H, dd, 4.0, 13.0) 8 49.4 50.2 - 9 44.5 46.4 1.20 (1H, dd, 4.5, 12.5) 10 36.3 37.5 - 11 20.7 21.4 1.08 (1H, m)/1.72 (1H, m) 12 31.9 28.0 1.28 (1H, m)/1.36 (1H, m) 13 43.7 51.1 - 14 60.4 56.6 1.09 (1H, m) 1.67 (1H, dd, 2.5, 9.5) 15 133.3 135.5 5.73 (1H, d, 6.0) 16 137.9 135.2 5.66 (1H, d, 6.0) 17 24.4 68.6 3.40 (1H, d, 11.0) 3.45 (1H, d, 11.0) 18 19.3 19.6 1.12 (3H, d, 7.0) 20 68.5 69.4 3.80 (1H, dd, 5.0, 9.5) 3.89 (1H, dd, 3.5, 9.5) a CDCl3, b75MHz; cCD3OD, d125MHz, e500MHz. #δC of agallochin I [35].
14 Figure IV.26. The structures of 9 compounds isolated from E. agallocha IV.2. Determination of chemical structure of isolated compounds from E. cochinchinensis VI.2.1. 6α,7α-Epoxy-4β,5β,9α,13α-tetrahydroxy-rhamnofola-1,15-dien- 3-one 20-O-β-D-glucopyranoside (EC-1, new compound) Figure IV.27. Structure of EC-1 and reference compound Compound EC-1 was isolated as a white, amorphous powder. Its molecular formula was determined to be C26H38O12 by the negative HR-QTOF-MS ion peaks at m/z 541.2297 [M - H]– (calcd for C26H37O12–, 541.2291), 577.2063 [M + Cl]– (calcd for C26H38ClO–, 577.2057), and 587.2346 [M + HCOO]– (calcd for C27H39O–, 587.2345), indicating eight degrees of unsaturation (Fig. IV.28).
15 Figure IV.28. HR-ESI-MS spectrum of EC-1 Figure IV.28. 1H NMR spectrum of EC-1 Figure IV.29. 13C NMR spectrum of EC-1
16 Figure IV.30. HSQC spectrum of EC-1 13 The C NMR and HSQC spectra revealed the presence of 26 carbon atoms including 6 non-protonated carbons, 13 methines, 4 methylenes, and 3 methyls. Among them, a typical α,β-unsaturated carbonyl moiety [δC 209.9 (C-3), 134.8 (C-2), 163.1 (C-1)], two other olefinic carbons [δC 145.9 (C-15), and 117.3 (C-16)], three oxygenated tertiary carbons [δC 74.8 (C-4), 64.6 (C-6), and 77.3 (C-9)], three oxymethines [δC 71.4 (C-13), 68.1 (C-5), and 62.3 (C-7)], and an oxymethylene [δC 74.2 (C-20)], along with a glucopyranosyl unit [δC 104.8 (C-1′), 75.2 (C-2′), 78.0 (C-3′), 71.7 (C-4′), 78.0 (C-5′), and 62.8 (C-6′)] were observed (Table IV.9). Since one carbonyl group and two double bonds accounted for three degrees of unsaturation, EC-1 was determined to be a pentacyclic compound. Accordingly, the 1H NMR spectrum showed the existence of three methyls [δH 1.70 (3H, s, H-17), 0.96 (3H, d, J = 7.0 Hz, H-18), and 1.76 (3H, d, J = 2.0 Hz, H-19)], one terminal double bond [δH 4.98 (1H, d, J = 2.0 Hz, H-16a)/5.04 (1H, br s, H-16b)], and one trisubstituted double bond [δH 7.66 (1H, br s, H-1)] (Fig. IV.28-IV.29). The large coupling constant of the anomeric proton [δH 4.33 (1H, d, J = 7.5 Hz, H-1′) confirmed the β-glucosidic linkage (Fig. IV.26). Careful comparison of the 1H and 13C NMR spectroscopic data for diterpenoidal nucleus of 1 (Table IV.9) with those of venenatin, a daphnane-type diterpenoid, revealed that they were very similar and these compounds had the same structure of A and B rings.
17 Figure IV.30. Keys COSY, HMBC, and NOESY correlations of EC-1 Figure IV.32. HMBC spectrum of EC-1 This deduction was also confirmed by COSY and HMBC correlations as shown in Fig. IV.30. Besides, the COSY cross-peaks of H-7/H-8/H-14/H-13/H-12/H-11/H-18 in combination with the HMBC correlations from H3-18 to C-9, C-11, C-12, from H-7 to C-9 and C-14, from H-8 to C-9, C-11, C-13, C-14, C-15, from H3-17 to C-14, C-15, C- 16, and from H2-16 to C-14, C-17 established structure of C ring, which was fused to the B ring at the C-8 and C-9, and substituted with two hydroxy groups at C-9 and C-13, a methyl group at C-11, and an
18 Figure III.33. COSY spectrum of EC-1 Figure IV.34. NOESY spectrum of EC-1 isopropenyl moiety at C-14. The downfield chemical shift of the oxymethylene carbon at δC 74.2 (C-20) along with the HMBC correlation from H-1′ to C-20 indicated the position of glucosyl moiety. Thus, EC-1 was established as a rhamnofolane diterpene glucoside. The relative configuration of EC-1 was assigned by analysing proton-proton coupling constants and NOESY data. The H-14 signal (dd, J = 10.0, 12.5 Hz) exhibited two large coupling with the H-8 (br d, J =12.5 Hz) and H-13 (ddd, J = 4.5, 10.0, 10.5 Hz) revealed the trans- diaxial orientation of these protons. The equatorial orientation of Ha-12 (ddd, J = 4.5, 4.5, 12.5 Hz) was deduced by the small coupling constants with its vicinal protons. In the NOESY spectrum, the correlations from