Tóm tắt Luận án Study on chemical constituents and biological activities of several antidesma species growing in Vietnam

ide (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 Comp. IC50 (µM) Comp. IC50 (µM) 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 Comp. Inhibition (%) Comp. Inhibition (%) Comp. Inhibition (%) 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 Comp. Inhibition (%) Comp. Inhibition (%) Comp. Inhibition (%) 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- 7,7'-epoxylignan-4,4',9-triol 4-O-β-D- glucopyranoside (new compound) AH2: 9-O-formylaviculin (new compound) AH3: (+)-Isolariciresinol 9-O-β-D- glucopyranoside AH4: (–)-Lyoniresinol AH5: (+)-Lyoniresinol-9-O-β-D- glucopyranoside AH6: 1-O-(2,4-dihydroxy-6- methoxyphenyl)-6-O-(4-hydroxy-3,5- dimethoxybenzoyl)-β-D-glucopyranoside AH7: 4-O-[6-O-(4-hydroxy-3,5- dimethoxybenzoyl)-β-D-glucopyranosyl]- 3-hydroxyphenethyl alcohol AH8: 4-Hydroxymethyl-2-methoxyphenyl- 6-O-syringoyl-β-D-glucopyranoside 9 AH9: Phenethyl α-L-arabinofuranosyl- (1→6)-O-β-D-glucopyranoside AH10: Syringoyl-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- glucopyranoside AH16: N–trans-feruloyloctopamide AH17: trans-Linalool-3,6-oxide 7-O-β-D- glucopyranoside AH18: Lotusanine B 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 (- 0,36) AH1 λmax (Δε): 236 (+1,60) và 219 (−0,23) 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 δCa 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- Methoxycinnamyl alcohol AC12: Vanilin 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 1H- and 13C-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# δCa 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 (new compound) AG2: Antidesoic acid B (new compound) AG3: Vitexin AG4: Orientin AG5: Isovitexin AG6: Homoorientin AG7: Luteolin-4′-O-β-D-glucopyranoside AG8: Amentoflavone AG9: Vanillyl alcohol 4-O- β-D-glucopyranoside AG10: 4-Hydroxy-3,5- dimethoxybenzyl-O-β-D- glucopyranoside AG11: 3-Hydroxy-4,5- dimethoxyphenyl-O-β-D- glucopyranoside AG12: 3,4,5- Trimethoxyphenyl-O-β-D- glucopyranoside AG13: Sinapyl alcohol 4- O-β-D-glucopyranoside AG14: (–)-Syringaresinol 19 3.4. Biological activities of isolated compounds 3.4.1. Cytotoxic activity of compounds from A. acidum Twelve compounds (AC1-AC12) from A. acidum were evaluated for cytotoxic activitiy against HL-60 leukemia cells (Table 2.1., and 2.2.). As the results, compound AC1 showed significant cytotoxic activity on HL- 60 with a IC50 value of 4.8 µM, compared with mitoxantrone an anticancer agent, IC50 of 6.8 µM. Compounds AC3-AC8 and AC10 exhibited moderate activity with the IC50 values ranging of 8.0-44.7 µM. Furthermore, the isolated compounds were evaluated on HEL-299 normal cell line. They did not inhibit the growth of HEL-299 cell line (IC50 >100 µM). Compound AC1 had the strongest cytotoxic activity and chose for investigation whether its inhibitory effect might arise from the induction of apoptosis. Apoptotic cell death has typical characteristics, such as chromatin condensation, membrane blebbing, cell shrinkage, and increased population of sub-G1 hypodiploid cells. When treated with AC1 for 24 and 48 h, the apoptotic characteristics were observed such as increasing apoptotic bodies. The Bcl-2 family is separated into antiapoptotic proteins, such as Bcl-2, and pro-apoptotic proteins, such as Bax. The Bax induces apoptosis by the releasing of cytochrome c from mitochondria. In contrast, Bcl-2 inhibits the releasing of cytochrome c. During apoptosis, released cytochrome c induces the cleavage of caspase-9, which is followed by the cleavage of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP). Therefore, to investigate the possible mechanism underlying the induction of apoptosis, we examined the levels of apoptosis-related proteins. When treated with AC1, we could observe the alteration of expression of apoptosis-related proteins such as up- regulation of Bax, down-regulation of Bcl-2, cleaves of caspase-3 and cleaves of PARP. The above evidence indicated that AC1 induced apoptosis via alteration of expression of apoptosis-related proteins in HL- 60 cells. 20 The PI3K/AKT signaling pathways regulate cell survival, cell growth and apoptosis. Especially, activated AKT contribute to the survival and growth of cancer cell via c-myc. The c-myc is frequently over-expressed in various types of tumors. In order to investigate intracellular signaling AC1 induced, we analyzed the phosphorylation of AKT and the level of c-myc by Western blotting. As results, the treatment of AC1 decreased phosphorylation of AKT and down- regulation of c-myc in conditions that could induce apoptosis in HL-60 cells. These findings suggested that the apoptosis-inducing effects of AC1 are mediated by down-regulation of p-AKT and c-myc. 3.4.2. Anti-inflammatory activity of compounds from A. hainanensis Among isolated compounds from A. hainanensis, compounds AH8, AH14, AH15 and AH18 significantly displayed inhibitory effects on NO production in BV2 cells with IC50 of 5.3, 8.6, 5.0, and 7,4 µM, respectively (butein was used as a positive control, IC50 of 3,8 µM). Compounds AH1-AH3, AH5, and AH7 moderately decreased NO production with the IC50 values ranging of 10.8 - 26.3 µM. Although the number of compound analogous is not huge enough, however our results might primarily show that lignan glycosides (AH1–AH3, AH5) exhibited higher inhibitory effects than an aglycone (AH4). Futhermore, compounds  AH14 and AH15 with a carbonyl group at C-9 exhibited higher inhibitory effects than other megastigmane  derivatives (AH11-AH13). 3.4.3. Anti-inflammatory activity of compounds from A. ghaesembilla Among isolated compounds from A. ghaesembilla, compounds AG4 and AG8 significantly displayed inhibitory effects on NO production in BV2 cells with IC50 of 9.5 and 5.4 µM, respectively (butein was used as a positive control, IC50 of 3,8 µM). Compounds AG1-AG3, AG5-AG8, and AG9-AG14 moderately decreased NO production with with the IC50 values ranging of 21.4 - 72.7 µM. 21 CONCLUSIONS This is the first study on chemical constituents and biological activities of A. hainanensis, A. acidum, and A. ghaesembilla growing in Vietnam. 1. Chemical investigations Using various chromatographic methods, 44 compounds were isolated from A. hainanensis, A. acidum, and A. ghaesembilla. Their chemical structures were determined by NMR, electrospray ionization (ESI)-MS, circular dichroism (CD) spectroscopic methods, and as well as by comparison with those reported in the literature. - Eighteen compounds were isolated and identified from A. hainanensis leaves. Among them, two new compounds, (7S,7'R,8S,8'R)- 3,3'-dimethoxy-7,7'-epoxylignan-4,4',9-triol 4-O-β-D-glucopyranoside (AH1), and 9-O-formylaviculin (AH2) were obtained. Three compounds, β-D-glucopyranosyl phaseate (AH11), megastigm-7-ene-3-ol-9-one 3-O- α-L-arabinofuranosyl-(1→6)-O-β-D-glucopyranoside (AH15) and lotusanine B (AH18) were isolated from Euphorbiaceae family for the first time. Three compounds, (+)-isolariciresinol 9-O-β-D- glucopyranoside (AH3), (+)-lyoniresinol-9-O-β-D-glucopyranoside (AH5) and ampelopsisionoside (AH12) were isolated from Antidesma genus for the first time. Ten remaning compounds were previously reported from Antidesma genus, including (–)-lyoniresinol (AH4), 1-O- (2,4-dihydroxy-6-methoxyphenyl)-6-O-(4-hydroxy-3,5- dimethoxybenzoyl)-β-D-glucopyranoside (AH6), 4-O-[6-O-(4- hydroxy-3,5-dimethoxybenzoyl)-β-D-glucopyranosyl]-3-hydroxy phenethyl alcohol (AH7), 4-hydroxymethyl-2-methoxyphenyl-6-O- syringoyl-β-D-glucopyranoside (AH8), phenethyl α-L-arabinofuranosyl- (1→6)-O-β-D-glucopyranoside (AH9), syringoyl-O-β-D-glucopyranoside (AH10), alangioside A (AH13), alangionoside L (AH14), N–trans- feruloyloctopamide (AH16) and trans-linalool-3,6-oxide 7-O-β-D- glucopyranoside (AH17). 22 - Twelve compounds were isolated and identified from A. acidum leaves. Among them, one compound, 5-demethyltoddaculin (AC6) were obtained from natural sources for the first time. Seven compounds, clauszoline B (AC1), clauszoline H (AC2), mukonal (AC3), 7- methoxymukonal (AC4), heptaphyline (AC5), xanthoxyletin (AC7) and alloxanthoxyletin (AC8) were isolated from Antidesma genus for the first time. Ten remaning compounds were previously reported from Antidesma genus, including (E)-p-propenylphenol O-β-D-glucopyranoside (AC9), p- methoxycinnamaldehyde (AC10), trans-4-methoxycinnamyl alcohol (AC11), vanilin (AC12). - Forteen compounds were isolated and identified from A. ghaesembilla leaves. Among them, two new compounds, antidesoic acid A (AG1), and antidesoic acid B (AG2) were obtained. Eight compounds, vitexin (AG3), orientin (AG4), isovitexin (AG5), homoorientin (AG6), 4-hydroxy-3,5-dimethoxybenzyl-O-β-D-glucopyranoside (AG10), 3- hydroxy-4,5-dimethoxyphenyl-O-β-D-glucopyranoside (AG11), 3,4,5- trimethoxyphenyl-O-β-D-glucopyranoside (AG12), sinapyl alcohol 4-O- β-D-glucopyranoside (AG13) were isolated from Antidesma genus for the first time. Four remaning compounds were previously reported from Antidesma genus, including luteolin-4′-O-β-D-glucopyranoside (AG7), amentoflavone (AG8), vanillyl alcohol 4-O-β-D-glucopyranoside (AG9), (–)-syringaresinol (AG14). 2. Biological activity - Twelve isolated compounds (AC1-AC12) from A. acidum species were evaluated for cytotoxic activity on HL-60 cancer cell line. As the results, compounds AC1 and AC4 showed significant cytotoxic activity on HL-60 cells with the IC50 values of 4.8 and 8.0 µM, respectively, compared with mitoxantrone an anticancer agent, IC50 of 6.8 µM. Compounds AC3, AC5-AC8, AC10 exhibited moderate activity with IC50 values ranging of 22.5-44.7 µM. Compound AC1 was further chosen for investigation whether the inhibitory effect of AC1 might arise from the induction of apoptosis. Results indicated that AC1 induced 23 apoptosis via alteration of expression of apoptosis-related proteins in HL- 60 cells such as up-regulation of Bax, down-regulation of Bcl-2, cleaves of caspase-3 and cleaves of PARP. - Eighteen compounds (AH1-AH18) were tested for their inhibitory activity on NO production in activated BV2 cells. As the results, compounds AH8, AH14, AH15, and AH18 showed potent inhibitory activity on NO production in LPS-stimulated BV2 cells with IC50 values of 5.3, 8.6, 5.0, and 7.4 µM, respectively, compared with butein (positive control, IC50 of 3.8 µM). - Forteen compounds (AG1-AG14) were tested for their inhibitory activity on NO production in activated BV2 cells. As the results, compounds AG4 and AG8 showed potent inhibitory activity on NO production in LPS-stimulated BV2 cells with IC50 values of 9.5 and 5.4 µM, respectively, compared with butein agent (positive control, IC50 of 3.8 µM). RECOMMENDATIONS - Compound AC4 showed potent anti-proliferation on HL-60 cancer cell line. Further studies to clarify activity mechanism and pharmacoglogical study of this compound should be carried. - Compounds AH8, AH14, AH15, AH18, AG4, and AG8 showed potent inhibitory activity on NO production in LPS-stimulated BV2 with IC50 values ranging of 5.0-9.5 µM. Further experiments should be required to aprove anti-inflammatory activity of them before in vivo studies. NEW FINDINGS OF THE THESIS 1. This is the first study of chemical constituents and biological activities of A. acidum, A. ghaesembilla, and A. hainanensis growing in Vietnam. 2. 44 compounds were isolated and identified f