Summary of Chemistry doctoral thesis: Study on chemical constituents and biological activities of several Antidesma species growing in Vietnam

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The objectives of the thesis: Study on chemical constituents of three Antidesma species including A. acidum, A. ghaesembilla, and A. hainanensis growing in Vietnam; evaluation of biological activities of isolated metabolites to find potential compounds.

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Nội dung Text: Summary of Chemistry doctoral thesis: Study on chemical constituents and biological activities of several Antidesma species growing in Vietnam

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ----------------------------- LE CANH VIET CUONG STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF SEVERAL ANTIDESMA SPECIES GROWING IN VIETNAM Major: Organic chemistry Code: 62.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi - 2017
This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Adviser 1: Assoc. Prof. Dr. Phan Van Kiem Adviser 2: Assoc. Prof. Dr. Le Mai Huong 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 2017 Thesis can be found in The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology.
1 INTRODUCTION 1. The urgency of the thesis The World Health Organization (WHO) had estimated that as much as 80 percent population relies on traditional medicines in deverloping countries, especially medicinal plants. In the process of drugs discovery and development, traditional knowledge and experiential databases were considered to be one of the important factors which could be increased success rate of lead compounds by reducing the consumed time and money spent as well as toxic occurrence. Therefore, investigations on medicinal plants still remain attractive and stimulate attention of scientists worldwide. According to Dictionary of Vietnamese medicinal plants (Vo Van Chi, 2003), in Vietnam, there were 11 species of the Antidesma genus which were used to treat stomachache, syphilis, measles, chickenpox, menstrual diseases, ... Phytochemical investigation of Antidesma genus led to the isolation of numerous alkaloids, terpenoids, steroids, megastigmanes, flavonoids, lignans and other phenolic compounds. In addition, a lot of extractions and isolated compounds exhibited potent anticancer, anti-oxidant, antidiabetic, antimicrobial activity, .... Therefore, thesis title was chosen to be "Study on chemical constituents and biological activities of several Antidesma species growing in Vietnam". 2. The objectives of the thesis Study on chemical constituents of three Antidesma species including A. acidum, A. ghaesembilla, and A. hainanensis growing in Vietnam. Evaluation of biological activities of isolated metabolites to find potential compounds. 3. The main contents of the thesis 1. Isolation of compounds from the leaves of A. acidum, A. ghaesembilla, and A. hainanensis; 2. Determination of chemical structures of the isolated compounds;
2 3. Evaluation on the cytotoxic activity of the isolated compounds; 4. Evaluation on the anti-inflammatory activity of isolated compounds. CHAPTER 1: OVERVIEW Overview of internal and international researches related to our study CHAPTER 2: EXPERIMENT AND RESULTS 2.1. Plant materials - The leaves of A. acidum were collectted at Tam Dao, Vinh Phuc, Vietnam in March, 2013. - The leaves of A. ghaesembilla were collectted at Buon Don, Dak Lak, Vietnam in March, 2013. - The leaves of A. hainanensis were collectted at Tam Dao, Vinh Phuc, Vietnam in December, 2014. 2.2. Methods 2.2.1. Methods for isolation of secondary metabolites Chromatographic methods such as thin layer chromatography (TLC), column chromatography (CC). 2.2.2. Methods for determination of chemical struture of compounds Physical parameters and modern spectroscopic methods such as optical rotation ([]D), electrospray ionization mass spectrometry (ESI-MS) and high-resolution ESI-MS (HR-ESI-MS), one/two-dimention nuclear magnetic resonance (NMR) spectra, circular dichroism spectrum (CD). 2.2.3. Methods for evaluation of biological activities - Cytotoxic activity was determined by the MTT assay. - Anti-inflammatory activity of the compounds was assessed on the basis of inhibiting NO production in lipopolysaccharide (LPS) activated BV2 cells. 2.3. Isolation of compounds 2.3.1. Isolation of compounds from A. hainanensis
3 This section presents the process of isolating the compounds from A. hainanensis. Figure 2.4. Isolation of compounds from A. hainanensis 2.3.2. Isolation of compounds from A. acidum This section presents the process of isolating the compounds from A. acidum. Figure 2.5. Isolation of compounds from A. acidum
4 2.3.3. Isolation of compounds from A. ghaesembilla This section presents the process of isolating the compounds from A. ghaesembilla. Figure 2.6. Isolation of compounds from A. ghaesembilla 2.4. Physical properties and spectroscopic data of the isolated compounds 2.4.1. Physical properties and spectroscopic data of the isolated compounds from A. hainanensis This section presents physical properties and spectroscopic data of 18 compounds from A. hainanensis. 2.4.2. Physical properties and spectroscopic data of the isolated compounds from A. acidum This section presents physical properties and spectroscopic data of 12 compounds from A. acidum. 2.4.3. Physical properties and spectroscopic data of the isolated compounds from A. ghaesembilla
5 This section presents physical properties and spectroscopic data of 14 compounds from A. ghaesembilla. 2.5. Results on biological activities of isolated compounds 2.5.1. Results on cytotoxic activity of compounds from A. acidum - 12 compounds (AC1-AC12) were evaluated for their cytotoxic activities against HL-60 leukemia cells by MTT assay. Table 2.1. % inhibition on HL-60 cells of compounds AC1-AC12 at concentration of 100 μM Comp. Inhibition (%) Comp. Inhibition (%) AC1 95.18 ± 0.55 AC7 95.22 ± 0.20 AC2 42.10 ± 2.68 AC8 95.29 ± 0.53 AC3 93.95 ± 0.74 AC9 47.30 ± 3.20 AC4 95.00 ± 0.46 AC10 95.55 ± 0.12 AC5 94.99 ± 0.98 AC11 48.93 ± 4.44 AC6 95.51 ± 0.11 AC12 36.17 ± 3.96 Table 2.2. The effects of compounds AC1, AC3 – AC8, and AC10 on the growth of HL-60 leukemia and HEL-299 normal cells IC50 (µM) IC50 (µM) Comp. Comp. HL-60 HEL-299 HL-60 HEL-299 AC1 4.8 ± 0.2 >100 AC6 22.5 ± 0.9 >100 AC3 26.4 ± 0.6 >100 AC7 28.1 ± 0.2 >100 AC4 8.0 ± 0.9 >100 AC8 25.4 ± 0.8 >100 AC5 24.8 ± 0.7 >100 AC10 44.7 ± 3.3 >100 PC* 6.8 ± 0.9 >100 *) Mitoxantrone was used as a positive control (PC).
6 2.5.2. Results on anti-inflammatory activity of compounds from A. hainanensis - 18 compounds (AH1-AH18) were evaluated for their anti- inflammatory activity on the basis of inhibiting NO production in lipopolysaccharide (LPS) activated BV2 cells. Table 2.3. % inhibition on NO production in lipopolysaccharide (LPS) activated BV2 cells of compounds AH1-AH12 at concentration of 80 μM Inhibition Inhibition Inhibition Comp. Comp. Comp. (%) (%) (%) AH1 90.1 ± 5.0 AH7 92.0 ± 4.1 AH13 62.2 ± 3.7 AH2 79.8 ± 4.6 AH8 96.7 ± 3.7 AH14 89.6 ± 6.7 AH3 83.2 ± 6.3 AH9 26.2 ± 4.3 AH15 95.3 ± 3.8 AH4 73.1 ± 5.3 AH10 41.8 ± 6.6 AH16 35.4 ± 3.6 AH5 86.5 ± 5.2 AH11 33.7 ± 5.8 AH17 24.0 ± 4.9 AH6 47.4 ± 7.5 AH12 38.7 ± 6.2 AH18 87.5 ± 4.1 PC* 85.0 ± 5.1 * Butein (10 µM) was used as a positive control (PC). Table 2.4. Inhibitory effects on NO production in lipopolysaccharide (LPS) activated BV2 cells of compounds AH1-AH5, AH7, AH8, AH13- AH15, and AH18 Comp. IC50 (µM) Comp. IC50 (µM) AH1 10.8 ± 1.1 AH8 5.3 ± 0.4 AH2 15.1 ± 1.2 AH13 48.2 ± 6.8 AH3 21.2 ± 3.1 AH14 8.6 ± 1.1 AH4 67.9 ± 26.0 AH15 5.0 ± 0.2 AH5 19.0 ± 0.9 AH18 7.4 ± 1.8 AH7 26.3 ± 1.3 PC* 3.8 ± 0.6 * Butein was used as a positive control (PC).
7 2.5.3. Results on anti-inflammatory activity of compounds from A. ghaesembilla - 14 compounds (AG1-AG14) were evaluated for their anti- inflammatory activity on the basis of inhibiting NO production in lipopolysaccharide (LPS) activated BV2 cells. Table 2.5. % inhibition on NO production in lipopolysaccharide (LPS) activated BV2 cells of compounds AG1-AG14 at concentration of 80 μM Inhibition Inhibition Inhibition Comp. Comp. Comp. (%) (%) (%) AG1 79.0 ± 5.6 AG6 62.4 ± 5.5 AG11 56.0 ± 6.3 AG2 96.0 ± 5.0 AG7 74.3 ± 5.7 AG12 95.0 ± 5.1 AG3 83.3 ± 4.6 AG8 105.0 ± 2.6 AG13 64.7 ± 4.9 AG4 77.2 ± 5.1 AG9 66.3 ± 4.0 AG14 40.1 ± 4.3 AG5 81.2 ± 4.4 AG10 68.3 ± 4.0 PC* 85.0 ± 5.1 * Butein (10 µM) was used as a positive control (PC). Table 2.6. Inhibitory effects on NO production in lipopolysaccharide (LPS) activated BV2 cells of compounds AG1-AG13 Comp. IC50 (µM) Comp. IC50 (µM) AG1 37.3 ± 8.7 AG8 5.4 ± 0.6 AG2 23.8 ± 3.1 AG9 48.3 ± 7.3 AG3 36.3 ± 3.8 AG10 50.2 ± 5.4 AG4 9.5 ± 1.3 AG11 72.7 ± 20.9 AG5 32.4 ± 9.9 AG12 21.4 ± 4.4 AG6 62.4 ± 11.2 AG13 44.3 ± 8.9 AG7 56.6 ± 5.7 PC* 3.8 ± 0.6 * Butein was used as a positive control (PC).
8 CHAPTER 3: DISCUSSIONS 3.1. Chemical structure of compounds from A. hainanensis This section presents the detailed results of spectral analysis and structure determination of 18 isolated compounds from A. hainanensis. AH1: (7S,7'R,8S,8'R) -3,3'-Dimethoxy- AH2: 9-O-formylaviculin (new compound) 7,7'-epoxylignan-4,4',9-triol 4-O-β-D- glucopyranoside (new compound) AH3: (+)-Isolariciresinol 9-O-β-D- AH4: (–)-Lyoniresinol glucopyranoside AH5: (+)-Lyoniresinol-9-O-β-D- AH6: 1-O-(2,4-dihydroxy-6- glucopyranoside methoxyphenyl)-6-O-(4-hydroxy-3,5- dimethoxybenzoyl)-β-D-glucopyranoside AH7: 4-O-[6-O-(4-hydroxy-3,5- AH8: 4-Hydroxymethyl-2-methoxyphenyl- dimethoxybenzoyl)-β-D-glucopyranosyl]- 6-O-syringoyl-β-D-glucopyranoside 3-hydroxyphenethyl alcohol
9 AH9: Phenethyl α-L-arabinofuranosyl- AH10: Syringoyl-O-β-D-glucopyranoside (1→6)-O-β-D-glucopyranoside AH11: β-D-Glucopyranosyl phaseate AH12: Ampelopsisionoside AH13: Alangioside A AH14: Alangionoside L AH15: Megastigm-7-ene-3-ol-9-one 3-O- α-L-arabinofuranosyl-(1→6)-O-β-D- AH16: N–trans-feruloyloctopamide glucopyranoside AH17: trans-Linalool-3,6-oxide 7-O-β-D- AH18: Lotusanine B glucopyranoside
10 Detailed methods for determination of chemical structure of a new compound was introduced as bellowing. 3.1.1. Compound AH1: (7S,7'R,8S,8'R) -3,3'-Dimethoxy-7,7'- epoxylignan-4,4',9-triol 4-O-β-D-glucopyranoside (new compound) Compound AH1 was obtained as a pale yellow amorphous powder. Its molecular formula was determined to be C26H34O11 by high resolution electrospray ionization (HR-ESI)-MS (m/z 545.1995 [M+Na]+; Calcd for C26H34O11Na, 545.1999) and 13C-NMR analysis, indicating ten indices of hydrogen deficiency. The 1H-NMR spectra of AH1 appeared resonant signals including six olefinic protons of two set of ABX spin-coupled systems at δH 7.18 (1H, d, J=8.5 Hz), 7.10 (1H, d, J=1.5 Hz), 7.08 (1H, d, J=1.5 Hz), 6.99 (1H, dd, J=1.5, 8.5 Hz), 6.97 (1H, dd, J=1.5, 8.5 Hz), and 6.84 (1H, d, J=8.5 Hz); two oxygenated methines at δH 5.20 (1H, d, J=8.5 Hz), 4.40 (1H, d, J=9.0 Hz); an anomeric proton at δH 4.92 (1H, d, J=7.5 Hz), two methoxy groups at δH 3.91 and 3.88 (each 3H, s); and a secondary methyl group at δH 1.14 (d, J=6.5 Hz). Figure 3.1. HR-ESI-MS of AH1 Figure 3.2. 1H-NMR spectrum of AH1 The 13C-NMR spectra of AH1 revealed signals of 26 carbons which were divided into six non-protonated carbons, 15 methines, two methylenes, and three methyl carbons. Among them, an anomeric and five oxygenated aliphatic carbons (δC 102.8, 78.2, 77.8, 74.9, 71.4, 62.5) were assigned for a glucose unit. Carbon signals of two methoxy groups were observed at δC 56.7 and 56.5. Remaining 18 carbons belonged
11 to aglycone moiety. Aforementioned data, the deshielded oxygenated methines C-7 (δC 82.6), C-7′ (δC 89.3) and ten indices of hydrogen deficiency of AH1 suggested that AH1 was a tetrahydrofuran lignan glycoside [Phytochemistry, 55, 843 (2000)]. Figure 3.3. 13C-NMR spectrum of AH1 Figure 3.4. DEPT spectrum of AH1 Figure 3.5. HSQC spectrum of AH1 Figure 3.6. 1H-1H COSY spectrum of AH1 The correlation spectroscopy (COSY) cross peaks observed, including H- 7 (δH 5.20)/ H-8 (δH 2.40)/ H-8′ (δH 2.08)/ H-7′ (δH 4.40), H-8 (δH 2.40)/H- 9 (δH 3.29, 3.21), and H-8′/ H-9′ (δH 1.14) which was supported for a tetrahydrofuran lignan skeleton. The chemical shift of C-9 (δC 63.6) and heteronuclear multiple bond connectivity (HMBC) correlations of H-9 (δH 3.29, 3.21) and carbons C-7 (δC 82.6)/ C-8 (δC 54.4)/ C-8′ (δC 46.2)
12 indicated for a free hydroxyl group at C-9. The HMBC correlations from both protons H-2 (δH 7.08) and H-6 (δH 6.99) to carbons C-4 (δC 147.2) and C-7 (δC 82.6), from anomeric proton H-1″ (δH 4.92) to C-4 were demonstrated for the location of an O-glycosidic linkage at C-4. Similarly, a free hydroxyl group at C-4′ was also confirmed by its carbon chemical shift (δC 147.6), and HMBC correlations of protons H-2′ (δH 7.10), H-6′ (δH 6.97) with C-4′ and C-7′ (δC 89.3). Two methoxy groups located at C-3 and C-3′ which were proved by strong HMBC correlations of H-5 (δH 7.18), 3-OCH3 (δH 3.88)/C-3 (δC 150.4); and H-5′ (δH 6.84), 3′-OCH3 (δH 3.91)/C-3′ (δC 149.1), respectively. Figure 3.7. HMBC spectrum of AH1 Next, the absolute configuration of AH1 was established by nuclear overhauser effect spectroscopy (NOESY) and circular dichroism (CD) spectrum. The NOESY correlations of H-7 (δH 5.20)/H-8 (δH 2.40)/H-9′ (δH 1.14)/H-7′ (δH 4.40) suggested that they were in close proximity and assumed locating all β-orientations. Furthermore, the CD spectrum of AH1 displayed close similarity Cotton effects [λmax (mdeg) 238 (+0.28) and 223 (−0.36)] with those of schisphenlignan G [λmax (Δε): 236 (+1.60) and 219 (−0.23)] [Fitoterapia, 86, 171 (2013)], indicating that AH1 had the same 7S,7′R,8S,8′R-configurations. The 7S and 7′R configurations was further explained by a coupled CD curve possessing positive exciton chirality rule in the CD spectrum of AH1 [J. Nat. Prod. 74, 1444 (2011)].
13 Figure 3.8. The important HMBC and NOESY correlations of AH1 max (mdeg): 238 (+ 0,28) và 223 (- λmax (Δε): 236 (+1,60) và 219 0,36) (−0,23) AH1 Schisphenlignan G (AH1a) Figure 3.9. The chemical structure and CD spectrum values of AH1 and the reference compound Finally, acid hydrolysis of AH1 obtained D-glucose which was confirmed by TLC and GC analysis, indicating the presence of D- glucose in the structure of AH1 [Chem. Pharm. Bull., 57, 986 (2009)]. Consequently, compound AH1 was determined to be (7S,7′R,8S,8′R)- 3,3′-dimethoxy-7,7′-epoxylignan-4,4′,9-triol 4-O-β-D-glucopyranoside. Figure 3.10. NOESY spectrum of AH1 Figure 3.11. CD spectrum of AH1
14 Table 3.1. NMR spectral data of AH1 and reference compound C δ Ca DEPT δHa (mult., J in Hz) 1 135.8 C - 2 112.7 CH 7.08 (d, 1.5) 3 150.4 C - 4 147.2 C - 5 117.4 CH 7.18 (d, 8.5) 6 120.7 CH 6.99 (dd, 1.5, 8.5) 7 82.6 CH 5.20 (d, 8.5) 8 54.4 CH 2.40 (m) 9 63.6 CH2 3.29 (dd, 7.5, 10.5) 3.21 (dd, 6.5, 10.5) 1 133.0 C - 2 111.7 CH 7.10 (d, 1.5) 3 149.1 C - 4 147.6 C - 5 116.2 CH 6.84 (d, 8.5) 6 120.7 CH 6.97 (dd, 1.5, 8.5) 7 89.3 CH 4.40 (d, 9.0) 8 46.2 CH 2.08 (m) 9 16.6 CH3 1.14 (d, 6.5) 3-OCH3 56.7 CH3 3.88 (s) 3-OCH3 56.5 CH3 3.91 (s) 4-OGlc 1 102.8 CH 4.92 (d, 7.5) 2 74.9 CH 3.53 (dd, 7.5, 9.0) 3 77.8 CH 3.49 (dd, 9.0, 9.0) 4 71.4 CH 3.42* 5 78.2 CH 3.42* 6 62.5 CH2 3.71 (dd, 5.0, 12.0) 3.90* a) Recorded in CD3OD; *)Overlapped signals.
15 3.2. Chemical structure of compounds from A. acidum This section presents the detailed results of spectral analysis and structure determination of 12 isolated compounds from A. acidum. AC1: Clauszoline B AC2: Clauszoline H AC3: Mukonal AC4: 7-Methoxymukonal AC5: Heptaphyline AC6: 5-Demethyltoddaculin AC7: Xanthoxyletin AC8: Alloxanthoxyletin AC9: (E)-p-Propenylphenol O-β-D-glucopyranoside AC10: p-Methoxycinnamaldehyde AC11: trans-4- AC12: Vanilin Methoxycinnamyl alcohol Below, detailed method for determination of chemical structure of clauszoline B (AC1), which had the strongest inhibitory activity against HL-60 cells.
16 3.2.1. Compound AC1 : Clauszoline B The 1H-NMR spectrum of AC1 showed signals for two tertiary methyl groups at δH 1.51 (6H, s), four aromatic protons at δH 6.85 (1H, s), 6.92 (1H, d, J = 7.5 Hz), 7.45 (1H, d, J = 7.5 Hz), and 8.09 (1H, s), two olefinic protons at δH 5.63 (d, J = 10.0 Hz) and 6.48 (d, J = 10.0 Hz), and one aldehyde proton at δH 9.91 (s). The 13C-NMR and DEPT spectra of AC1 showed the signals of 18 carbons including nine non-protonated carbons at δC 77.2, 115.6, 118.1, 118.4, 124.9, 129.3, 138.2, 145.8, and 161.3, six methines at δC 97.1, 111.7, 119.6, 122.7, 127.4, and 129.4, one aldehyde at δC 195.2, and two methyl carbons at δC 28.2. The analytical 1 13 H- and C-NMR data of AC1 indicated the structure of AC1 to be pyranocarbazole alkaloid and identical to clauszoline B [Chem. Eur. J., 20, 8536 (2014)]. Figure 3.41. The chemical structure and important HMBC correlations of AC1 The HMBC cross peaks from aldehyde proton (δH 9.91) to C-2 (δC 161.3), C-3 (δC 115.6), C-4 (δC 127.4) confirmed the position of aldehyde group at C-3. The HMBC correlations between H-1′ (δC 6.48) and C-6 (δC 119.6)/C-7 (δC 118.1)/C-8 (δC 138.2)/C-2′ (δC 129.4)/C-3′ (δC 77.2); between H-4′/H-5′ (δH 1.51) and C-2′ (δC 129.4)/C-3′ (δC 77.2) suggested the presence of the isoprene unit with the double bond at C-1′/C-2′ and this unit was located at C-7. Thus, the structure of AC1 was elucidated as
17 clauszoline B. This compound was reported from Antidesma genus for the first time. Table 3.20. NMR spectral data of AC1 and reference compound C δC # δ Ca DEPT δHa (mult., J in Hz) 1 96.72 97.1 CH 6.85 (s) 1a 146.58 145.8 C - 2 160.73 161.3 C - 3 115.47 115.6 C - 4 127.68 127.4 CH 8.09 (s) 4a 118.04 118.4 C - 5 111.81 111.7 CH 7.45 (d, 7.5) 5a 125.13 124.9 C - 6 119.21 119.6 CH 6.92 (d, 7.5) 7 118.04 118.1 C - 8 138.23 138.2 C - 8a 129.77 129.3 C - 1′ 122.63 122.7 CH 6.48 (d, 10.0) 2′ 129.35 129.4 CH 5.63 (d, 10.0) 3′ 76.69 77.2* C - 4′ 27.28 28.2 CH3 1.51 (s) 5′ 27.28 28.2 CH3 1.51 (s) 3-CHO 195.76 195.2 CH 9.91 (s) a) Recorded in CDCl3; #)C of clauszoline B recorded in acetone-d6 [ Chem. Eur. J., 20, 8536 (2014)]. *) Signal was obscured. 3.3. Chemical structure of compounds from A. ghaesembilla This section presents the detailed results of spectral analysis and structure determination of 14 isolated compounds from A. ghaesembilla.
18 AG1: Antidesoic acid A AG2: Antidesoic acid B AG3: Vitexin (new compound) (new compound) AG4: Orientin AG5: Isovitexin AG6: Homoorientin AG7: Luteolin-4′-O-β-D-glucopyranoside AG8: Amentoflavone AG9: Vanillyl alcohol 4-O- AG10: 4-Hydroxy-3,5- AG11: 3-Hydroxy-4,5- β-D-glucopyranoside dimethoxybenzyl-O-β-D- dimethoxyphenyl-O-β-D- glucopyranoside glucopyranoside AG12: 3,4,5- AG13: Sinapyl alcohol 4- AG14: Trimethoxyphenyl-O-β-D- O-β-D-glucopyranoside (–)-Syringaresinol glucopyranoside