Study on chemical constituents and biological activities of tacca Vietnamensis and tacca chantrieri growing in Vietnam

mulated BV2 cells at concentration of 80 μM Comp. Inhibition (%) Comp. Inhibition (%) Comp. Inhibition (%) TV1 45.1 ± 2.2 TV5 72.0 ± 2.5 TV8 42.2 ± 1.8 TV2 43.2 ± 1.8 TV6 40.0 ± 2.0 TV9 40.1 ± 3.0 TV3 63.2 ± 1.5 TV7 46.9 ± 2.2 Butein* (10 µM) 90.0 ± 5.0 TV4 67.5 ± 2.1 Table 2.2. Inhibitory NO effects of compounds TV3-TV5 in the LPS-stimulated BV2 cells Comp. IC50 (µM) Comp. IC50 (µM) TV3 52.1 ± 3.6 TV5 43.7 ± 4.2 TV4 47.3 ± 6.0 Butein* 4.3 ± 0.5 - 13 compounds (TC1-TC13) were evaluated for the inhibitory activities of nitric oxide production in LPS-stimulated BV2 cells. Table 2.3. Inhibition activities of TC1-TC13 on NO production in the LPS-stimulated BV2 cells at concentration of 80 μM Comp. Inhibition (%) Comp. Inhibition (%) Comp. Inhibition (%) TC1 85.1 ± 4.5 TC6 47.4 ± 2.5 TC11 40.8 ± 2.0 TC2 63.8 ± 3.6 TC7 42.0 ± 2.1 TC12 36.8 ± 2.8 TC3 43.2 ± 2.4 TC8 42.0 ± 3.0 TC13 28.7 ± 1.9 TC4 47.1 ± 2.5 TC9 45.7 ± 2.2 Butein (10 µM) 78.0 ± 4.2 TC5 46.5 ± 3.3 TC10 44.3 ± 2.1 6 Table 2.4. Inhibitory NO effects of compounds TC1-TC2 in the LPS-stimulated BV2 cells Comp. IC50 (µM) Comp. IC50 (µM) TC1 12.4 ± 2.4 Butein 4.3 ± 0.8 TC2 59.0 ± 3.5 2.5.2. Results on cytotoxic activity of compounds from Tacca vietnamensis and Tacca chantrieri - 13 compounds (TC1-TC13) were evaluated for cytotoxic activity on four human cancer cell lines, including PC-3, LNCaP, MDA-MB-231 and HepG2. Table 2.6. The effects of compounds on the growth of PC3, LNCaP, MDA-MB-231 cell lines Comp. IC50 (µM) PC-3 LNCaP MDA-MB-231 TC2 24.5 ± 1.2 19.0 ± 1.5 20.9 ± 1.6 TC7 30.7 ± 1.5 19.1 ± 1.4 24.2 ± 1.5 TC9 30.8 ± 2.0 20.2 ± 1.2 49.3 ± 3.2 TC13 17.9 ± 1.8 18.8 ± 1.3 22.0 ± 2.0 Ellipticine 1.1 ± 0.1 0.7 ± 0.1 0.8 ± 0.1 CHAPTER 3: DISCUSSIONS 3.1. Chemical structure of isolated compounds This section presents the detailed results of spectral analysis and structure determination of 22 isolated compounds from Tacca vietnamensis and Tacca chantrieri. * 9 compounds from Tacca vietnamensis ( Figure 3.2): Taccavietnamoside A (TV1), taccavietnamoside B (TV2), taccavietnamoside C (TV3), taccavietnamoside D (TV4), taccavietnamoside E (TV5), (24S,25R)-spirost-5-en-3β,24-diol 3-O-α-L-rhamnopyranosyl- (1→2)-[α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranoside (TV6); (24S,25R)-spirost-5-en-3β,24-diol 3-O-α-L-rhamnopyranosyl-(1→2)-[β-D- glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranoside (TV7); chantrieroside A (TV8) and plantagineoside A (TV9). * 13 compounds from Tacca chantrieri (Figure 3.1): Chantriolide D (TC1), chantriolide E (TC2), chantriolide A (TC3), chantriolide B (TC4), chantriolide C (TC5), (3R,5R)-3,5-dihydroxy-1,7-bis (3,4- dihydroxyphenyl)heptane (TC6), (3R,5R)-3,5-dihydroxy-1,7-bis(3,4- 7 dihydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC7), (3R,5R)-3,5- dihydroxy-1,7-bis(4-hydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC8), (3R,5R)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4- hydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC9), (6S,9R) roseoside (TC10), 2-hydroxyphenol-1-O-β-D-glucopyranoside (TC11), 1-O-syringoyl-β-D-glucopyranoside (TC12) and benzyl-β-D- glucopyranosyl (1→6)-β-D-glucopyranoside (TC13). Figure 3.2. Chemical structure of compounds from Tacca vietnamensis Figure 3.3. Chemical structure of compounds from Tacca chantrieri 3.1.1. Spectral characteristics of taccalonolide and withanolide compounds 3.1.2. Spectral characteristics of spirostanol saponin 3.1.3. Chemical structure of isolated compounds from Tacca vietnamensis: 3.1.3.1 Compound TV1: Taccavietnamoside A (new compound) 8 Figure 3.4. Chemical structure of TV1 and taccasuboside C (65) Compound TV1 was obtained as a white amorphous powder and its molecular formula was determined as C45H72O18 on the basic of HR-ESI-MS pseudo-ion at m/z 923.4607 [M+Na]+ (Calcd for [C45H72O18Na]+, 923.4611). The 1H-NMR spectra of TV1 appeared signals including an olefinic protons at δH 5.28 (br s), four methyl groups at δH 0.95 (s), 0.99 (s), 1.20 (d, J = 6.5 Hz) and 1.59 (s), which suggested the structure of steroid skeleton. In addition to these, three anomeric protons at δH 4.85 (d, J = 7.5 Hz), 5.71 (br s) and 5.81 (br s), indicated the presence of three sugar moieties.  13C-NMR and DEPT data of TV1 showed the presence of 45 carbons, including 5 non-protonated carbons at δC 37.0, 40.9, 68.5, 111.5 and 140.7; 24 methine carbons at δC 31.5, 35.8, 50.2, 56.5, 62.3, 66.0, 69.8, 69.9, 70.5, 72.3, 72.4, 72.5, 72.7, 73.5, 73.7, 77.8, 77.9, 78.3, 81.8, 87.2, 99.8, 102.5, 103.7 and 121.7; 10 methylen carbons at δC 21.0, 30.0, 31.9, 32.2, 37.4, 38.6, 40.0, 45.1, 62.2 and 69.1 and 6 methyl groups at δC 14.5, 16.4, 18.3,18.6, 19.3 and 26.1. The HMBC correlations between H-4 (δH 2.64 and 2.70) and C-5 (δC 140.7)/C-6 (δC 121.7); between H-19 (δH 0.95) and C-5 (δC 140,7) confirmed the position of double bond at C-5/C-6. Moreover, the acetal group at C-22 was confirmed by 13C-NMR chemical shift of C-22 (δC 111.5) as well as the HMBC correlations between H-20 (δH 3.00)/H-21 (δH 1.20)/H-26 (δH 3.60 and 4.13) and C-22 (δC 111.5). Analysis the data of 1H-, 13C-NMR and DEPT spectra, chemical shift of C-22 (δC111.5- spiro ring) and the published documents [19, 62], which suggest the compound of TV1 is a spirostanol saponin. The NMR data of TV1 (Table 3.1) were similar to those of taccasuboside C [19] except for signals at C-23, C-24 and C-25 of aglycone: Chemical shift of C-23, C-24, C-25 of TV1 are δC 66.0, 45.1 and 68.5, respectively 9 (Taccasuboside C: δC 64.6, 43.6, and 70.0 [19], recorded in pyridine-d5), which suggested the different configuration at C-25. The configurations of hydroxyl groups at C-23 and C-25 were defined as equatorial orientation by ROESY observation between H-21 (δH 1.20) and H-23 (δH 3.99); and between H-23 (δH 3.99) and H-27 (δH 1.59). Sugars obtained by acid hydrolysis of TV1 were identified as D-glucose and L-rhamnose based on GC analysis (identified as TMS derivatives). In addition, the HMBC cross peaks from rha H-1′′ (H 5.81) to glc C-2′ (C 78.3); from rha H-1′′′ (H 5.71) to glc C-3′ (C 87.2) and from glc H-1′ (H 4.85) to C-3 (C 77.8) confirmed the sugar linkages as α-L-rhamnopyranosyl- (1→2)-O-[α-L-rhamnopyranosyl-(1→3)]-β-D-glucopyranoside, with the location of sugar moiety at C-3 of aglycone. This was also in good agreement with the 13C NMR data of trisaccharide reported for taccasuboside C from Tacca subflabellata [19]. Thus, the structure of TV1 was elucidated to be (23S,25R)-spirost-5-en-3β,23,25-triol 3-O-α-L-rhamnopyranosyl-(1→2)-[α-L- rhamnopyranosyl-(1→3)]-β-D-glucopyranoside and named taccavietnamoside A. Figure 3.5. The important HMBC and ROESY correlations of TV1 Figure 3.6. HR-ESI-MS of TV1 Table 3.1. NMR spectral data of TV1 and reference compound C C# Ca,b Ha,c(mult., J, Hz) Aglycone 1 37.5 37.4 0.91 (m)/1.66 (m) 2 30.1 30.0 1.80 (m)/2.06 (m) 3 77.9 77.8 3.88 (m) 4 38.7 38.6 2.64 (dd. 12.0, 12.0) 2.70 (br d, 12.0) 5 140.8 140.7 - 6 121.8 121.7 5.28 (br s) 7 32.4 32.2 1.45 (m)/1.81 (m) 8 31.6 31.5 1.48 (m) 9 50.3 50.2 0.85 (m) 10 C C# Ca,b Ha,c(mult., J, Hz) 10 37.2 37.0 - 11 21.1 21.0 1.38 (m) 12 40.2 40.0 1.11 (m)/1.71 (m) 13 41.1 40.9 - 14 56.7 56.5 1.05 (m) 15 32.3 31.9 1.45 (m)/1.97 (m) 16 81.9 81.8 4.60 (m) 17 62.6 62.3 1.88 (t,. 8.5) 18 16.6 16.4 0.99 (s) 19 19.4 19.3 0.95 (s) 20 35.8 35.8 3.00 (q, 7.0) 21 14.9 14.5 1.20 (d, 6.5) 22 112.2 111.5 - 23 64.6 66.0 3.99 (br d, 8.5) 24 43.6 45.1 2.47 (br d, 12.0) 2.57 (m) 25 70.0 68.5 - 26 69.3 69.1 3.60 (d, 10.5) 4.13 (d, 10.5) 27 26.9 26.1 1.59 (s) 3-O- Glc 1′ 99.9 99.8 4.85 (d, 7.5) 2′ 78.4 78.3 4.00 (dd, 7.5, 8.5) 3′ 87.5 87.2 4.12 (dd, 8.5, 9.0) 4′ 69.9 69.8 4.00 (dd, 8.5, 9.0) 5′ 78.1 77.9 3.77 (m) 6′ 62.3 62.2 4.29 (br d, 11.5) 4.41 (br d, 11.5) 2′-O- Rha 1′′ 102.7 102.5 5.81 (br s) 2′′ 72.5 72.3 4.72 (br s) 3′′ 72.9 72.7 4.46 (dd, 2.5, 9.0) 4′′ 73.9 73.7 4.29 (m) 5′′ 69.9 69.9 4.82 (m) 6′′ 18.7 18.6 1.72 (d, 6.0) 3′-O- Rha 1′′′ 103.9 103.7 5.71 (br s) 2′′′ 72.5 72.4 4.81 (br s) 3′′′ 72.7 72.5 4.48 (dd, 2.5, 9.0) 4′′′ 73.6 73.5 4.29 (m) 5′′′ 70.7 70.5 4.75 (m) 6′′′ 18.5 18.3 1.62 (d, 6.0) a Recorded in C5D5N, b125 MHz, c 500 MHz, # δC of taccasuboside C [19] 11 Figure 3.7. 1H-NMR spectrum of TV1 Figure 3.8. 13C-NMR spectrum of TV1 Figure 3.9. DEPT spectrum TV1 Figure 3.10. HSQC spectrum of TV1 Figure 3.11. HMBC spectrum của TV1 Figure 3.12. ROESY spectrum of TV1 3.1.3.2 Compound TV2: Taccavietnamoside B (new compound) Figure 3.13. Chemical structure of TV2 and reference compound TV1 Compound TV2 was obtained as a white amorphous powder and its molecular formula was determined as C51H82O23 on the basic of HR-ESI-MS pseudo-ion at m/z 1085.5133 [M+Na]+ (Calcd for [C51H82O23Na]+, 1085.5139). The 1H-NMR spectra of TV2 appeared signals including an olefinic protons at δH 5.27 (br s), four methyl groups at δH 0.96 (s), 0.99 (s), 1.21 (d, J = 7.0 Hz) and 1.59 (s), which suggested the structure of steroid skeleton. In addition, four 12 anomeric protons at δH 4.85 (d, J = 8.0 Hz), 5.21 (d, J = 8.0 Hz), 5.71 (br s), and 5.76 (br s), indicated the presence of four sugar units.  13C-NMR and DEPT spectra of TV2 showed the presence of 51 carbons: including 5 non-protonated carbons at δC 37.0, 41.0, 68.5, 111.5 and 140.7; 29 methine carbons at δC 31.5, 35.8, 50.2, 56.6, 62.3, 66.0, 68.7, 69.7, 69.8, 71.4, 72.0, 72.3, 72.4, 72.7, 73.7, 76.3, 77.8, 78.0, 78.3, 78.5, 78.6, 81.8, 84.3, 86.2, 99.8, 102.5, 103.1, 106.4 and 121.7; 11 methylen carbons at δC 21.0, 30.0, 32.0, 32.3, 37.4, 38.8, 40.1, 45.2, 62.1, 62.5, and 69.2; and 6 methyl carbons at δC 14.5, 16.5, 18.2, 18.6, 19.3, and 26.2. The NMR data and chemical shift at C-22 (δC111.5- spiro ring) on 13C-NMR spectrum, which suggested TV2 is a spirostanol saponin. The 1H- and 13C-NMR data of TV2 were similar to those of taccavietnamoside A (TV1), except for the addition of a sugar unit at C-4″″: signals of anomeric proton at δH 5.21 (d, J = 8.0) and 6 carbons at δC 62.5, 71.4, 76.3, 78.3, 78.6 and 106.4. Sugars obtained by acid hydrolysis of TV2 were identified as D-glucose and L-rhamnose based on GC analysis (identified as TMS derivatives). In addition, the HMBC cross peaks from rha H-1″ (δH 5.76) to glc C-2′ (δC 78.5), from glc H-1″″ (δH 5.21) tới rha C-4‴ (δC 84.3), from rha H-1‴ (δH 5.71) to glc C-3′ (δC 86.2), and from glc H-1′ (δH 4.85) to C- 3 (δC 77.8) confirmed the sugar linkages as O-α-L-rhamnopyranosyl-(1→2)- O-[β-D-glucopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→3)]-β-D- glucopyranoside and the location of sugar at C-3 of aglycone. This sugar moiety was also reported from Tacca chantrieri [29]. Consequently, the structure of TV2 was determined to be (23S,25R)-spirost-5-en-3β,23,25-triol 3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)-α-L- rhamnopyranosyl-(1→3)]-β-D-glucopyranoside and named taccavietnamoside B. Figure 3.14. The important HMBC and COSY correlations of TV2 Figure 3.15. HR-ESI-MS of TV2 13 Table 3.2. NMR spectral data of TV2 and reference compound C C# Ca,b DEPT Ha,c (mult., J, Hz) Aglycone 1 37.4 37.4 CH2 0.92 (m)/1.66 (m) 2 30.0 30.0 CH2 1.80 (m)/2.06 (m) 3 77.8 77.8 CH 3.86 (m) 4 38.6 38.8 CH2 2.63 (dd, 12.0, 12.0)/2.69 (dd, 4.5, 12.0) 5 140.7 140.7 C - 6 121.7 121.7 CH 5.27 (d, 4.5) 7 32.2 32.3 CH2 1.42 (m)/1.80 (m) 8 31.5 31.5 CH 1.48 (m) 9 50.2 50.2 CH 0.86 (m) 10 37.0 37.0 C - 11 21.0 21.0 CH2 1.38 (m) 12 40.0 40.1 CH2 1.11 (m)/1.71 (m) 13 40.9 41.0 C - 14 56.5 56.6 CH 1.05 (m) 15 31.9 32.0 CH2 1.43 (m)/1.97 (m) 16 81.8 81.8 CH 4.60 (m) 17 62.3 62.3 CH 1.88 (t, 7.5) 18 16.4 16.5 CH3 0.99 (s) 19 19.3 19.3 CH3 0.96 (s) 20 35.8 35.8 CH 3.00 (q, 7.0) 21 14.5 14.5 CH3 1.21 (d, 7.0) 22 111.5 111.5 C - 23 66.0 66.0 CH 3.97 (br d, 8.5) 24 45.1 45.2 CH2 2.47 (br d, 11.0)/2.54 (t, 11.0) 25 68.5 68.5 C - 26 69.1 69.2 CH2 3.60 (d, 10.5)/4.12 (d, 10.5) 27 26.1 26.2 CH3 1.59 (s) 3-O-Glc 1′ 99.8 99.8 CH 4.85 (d, 8.0) 2′ 78.3 78.5 CH 4.00 (t, 8.0) 3′ 87.2 86.2 CH 4.12 (m) 4′ 69.8 69.7 CH 4.05 (t, 8.5) 5′ 77.9 78.0 CH 3.76 (m) 6′ 62.2 62.1 CH2 4.29 (dd, 3.0, 12.0)/4.40 (dd, 5.0, 12.0) 2′-O-Rha 1′′ 102.5 102.5 CH 5.76 (br s) 2′′ 72.3 72.4 CH 4.69 (br s) 3′′ 72.7 72.7 CH 4.47 (dd, 3.0, 9.0) 4′′ 73.7 73.7 CH 4.25 (m) 5′′ 69.9 69.8 CH 4.80 (m) 6′′ 18.6 18.6 CH3 1.72 (d, 6.5) 3′-O-Rha 1′′′ 103.7 103.1 CH 5.71 (br s) 2′′′ 72.4 72.0 CH 4.82 (br s) 3′′′ 72.5 72.3 CH 4.54 (dd, 2.5, 9.0) 14 C C# Ca,b DEPT Ha,c (mult., J, Hz) 4′′′ 73.5 84.3 CH 4.39 (m) 5′′′ 70.5 68.7 CH 4.76 (m) 6′′′ 18.3 18.2 CH3 1.66 (d, 6.0) 4′′′-O-Glc 1′′′′ 106.4 CH 5.21 (d, 8.0) 2′′′′ 76.3 CH 4.05 (m) 3′′′′ 78.6 CH 4.02 (m) 4′′′′ 71.4 CH 4.23 (t, 9.0) 5′′′′ 78.3 CH 3.76 (m) 6′′′′ 62.5 CH2 4.29 (dd, 3.0, 12.0)/4.40 (dd, 5.0, 12.0) a Recorded in C5D5N, b125 MHz, c 500 MHz, #δC of taccavietnamoside A (TV1) Figure 3.16. 1H-NMR spectrum of TV2 Figure 3.17. 13C-NMR spectrum of TV2 Figure 3.18. DEPT spectrum of TV2 Figure 3.19. HSQC spectrum of TV2 Figure 3.20. HMBC spectrum of TV2 Figure 3.21. COSY of TV2 Figure 3.22. ROESY of TV2 15 3.1.4. Chemical structure of isolated compounds from Tacca chantrieri 3.1.4.1 Compound TC1: Chantriolide D (new compound) Figure 3.23. Chemical structure of TC1 and taccanlonolide M (13) Compound TC1 was obtained as a white amorphous powder.  Its molecular formula was assigned as C35H50O15 on the basic of HR-ESI-MS pseudo-ion at m/z 711.3237 [M+H]+ (Calcd for [C35H51O15]+, 711.3222) and m/z 733.3055 [M+Na]+ (Calcd for [C35H50O15Na]+, 733.3042). The 1H-NMR spectra of TC1 exhibited signals for four methyl groups at δH 0.76 (3H, s), 1.13 (3H, s), 0.80 (3H, d, J = 6.0 Hz) and 1.17 (3H, d, J = 6.0 Hz), two methyl acetyl groups at δH 1.91 (3H, s) and 2.06 (3H, s), which suggested the structure a steroid with two acetyl groups. In addition, anomeric protons at δH 4.20 (d, J = 8.0 Hz), indicated the presence of a sugar unit.  The 13C-NMR and DEPT spectra of TC1 revealed  the presence of  35  carbons, including: 2 ketone carbons at δC 206.0 and 211.7; 2 acetyl carbonyl carbons at δC 170.2 and 170.5, 3 non-protonated  carbons at δC 41.9, 42.6, and 81.0; 18 methine carbons at δC 31.7, 37.1, 41.6, 41.8, 51.0, 51.8, 53.7, 54.3, 54.9, 70.8, 72.7, 74.3, 74.6, 77.1, 77.4, 78.2, 86.0 and 105.5; 4 methylene carbons at δC 25.2, 29.6, 44.5, and 61.9 and 6 methyl carbons at δC 13.3, 15.3, 15.3, 19.8, 20.2 and 21.0. All these data coupled with a literature survey indicated that TC1 was a steroidal glucoside [15]. The HMBC correlations HMBC between H-6 (δH 4.22) and C-5 (δC 81.0)/C-7 (δC 206.0)/C-10 (δC 42.6); between H-14 (δH 2.74)/H-16 (δH 1.46)/H-17 (δH 1.65) and C-15 (δC 211.7) confirmed the positions of two hydroxy groups at C-5 and C-6, two ketone groups at C-7 and C-15. The 13C-NMR chemical shift to a higher field at C-2 (δC 51.0), C-3 (δC 54.9) and correlation HMBC from H-4 (δH 2.37) to C-2 (δC 50.1)/C-3 (δC 54.9) suggested the epoxy group at C-2/C-3. Two 16 acetoxy groups at C-1 and C-12 were confirmed by HMBC correlations from HMBC to H-1 (δH 4.67) and H-12 (δH 4.93) to acetyl carbonyls (δC 170.2 and 170.5), respectively. The HMBC correlations between H-19 (δH 1.13) and C- 1(δC 72.7)/C-5 (δC 81.0)/C-9 (δC 37.1)/C-10 (δC 42.6); between H-18 (δH 0.76) and C-12 (δC 74.3), C-13 (δC 41.9), C-14 (δC 54.3), C-17 (δC 51.8); between H-21 (δH 0.80) and C-17 (δC 51.8), C-20 (δC 31.7), C-22 (δC 44.5); between H-25 (δH 1.17) and C-16 (δC 53.7), C-23 (δC 86.0), C-24 (δC 41.8) confirmed the positions of four methyl groups at C-10, C-13, C-20 and C-24, respectively. Acid hydrolysis of TC1 gave D-glucose (identified as TMS derivative by GC). The location of the sugar moiety at C-23 was confirmed by HMBC correlation from glc H-1′ (δH 4.20) to aglycone C-23 (δC 86.0). The configuration of the oxygenated groups at C-1, C-2, C-6, C-12 was defined as α-orientations, based on the similarity of the 13C-NMR spectral data from C-1 to C-19 of TC1 and taccanlonolide M [15]. In addition, the α-orientations of the oxygenated groups at C-1, C-2, C-6, C-12 were based on the observation of ROE correlations on ROESY spectrum between H-18 (δH 0.76) and H-12 (δH 4.93)/H-8 (δH 2.59); H-19 (δH 1.13) and H-1 (δH 4.67)/H-2 (δH 3.57)/H-6 (δH 4.22)/H-8 (δH 2.59). The α-orientation of oxygenated group at C-23 was based on the ROE correlations between H-23 (δH 3.10) and H-16 (δH 1.46)/H- 25 (δH 1.17). Thus, the structure of TC1 was determined and named chantriolide D. Figure 3.24. The important HMBC correlations of TC1 Table 3.9. NMR spectral data of TC1 and reference compound C C# Ca,b DEPT Ha,c (mult., J, Hz) Aglycone 1 73.0 72.7 CH 4.67 (d, 5.0) 2 49.1 51.0 CH 3.57 (t, 5.0) 3 55.3 54.9 CH 3.51 (m) 4 29.7 29.6 CH2 2.37 (d, 16.0)/2.07* 5 81.3 81.0 C - 17 C C# Ca,b DEPT Ha,c (mult., J, Hz) 6 78.4 78.2 CH 4.22* 7 205.8 206.0 C - 8 42.0 41.6 CH 2.59 (dd, 11.5, 12.0) 9 37.5 37.1 CH 2.21 (dd, 4.0, 12.0) 10 42.2 42.6 C - 11 25.7 25.2 CH2 1.41 (dd, 4.0, 15.0)/1.79 (br d, 15.0) 12 73.9 74.3 CH 4.93 (br s) 13 42.1 41.9 C - 14 55.4 54.3 CH 2.74 (d, 11.5) 15 210.8 211.7 C - 16 53.2 53.7 CH 1.46 (dd, 11.5, 11.5) 17 51.4 51.8 CH 1.65 (dd, 11.5, 11.5) 18 13.4 13.3 CH3 0.76 (s) 19 15.5 15.3 CH3 1.13 (s) 20 31.0 31.7 CH 1.52 (m) 21 19.4 19.8 CH3 0.80 (d, 6.0) 22 43.8 44.5 CH2 1.13*/2.13 (m) 23 86.4 86.0 CH 3.10* 24 42.0 41.8 CH 1.63 (m) 25 15.3 CH3 1.17 (d, 6.0) 1-OAc 170.3 170.2 - 20.7 20.2 CH3 1.91 (s) 12-OAc 170.6 170.5 - 21.0 21.0 CH3 2.06 (s) 23-OGlc 1′ 105.5 CH 4.20 (d, 8.0) 2′ 74.6 CH 2.98 (t, 8.0) 3′ 77.4 CH 3.17* 4′ 70.8 CH 3.10* 5′ 77.1 CH 3.10* 6′ 61.9 CH2 3.47 (dd, 4.0, 11.5)/3.66 (br d, 11.5) a Recorded in CD3OD, b125MHz, c 500MHz, # C of taccanlonolide M [15], * Overlapped signals 3.1.4.1 Compound TC2: Chantriolide E (new) Compound TC2 molecular formula was assigned as C36H51O15+Cl on the basic of HR-ESI-MS pseudo-ion at m/z 781.2854 [M+Na]+ (Calcd for [C36H51O15ClNa]+, 781.2809). The 1H-NMR spectra of TC2 appeared signals of four methyl group protons: three tertiary methyl groups at δH 0.94 (3H, s), 1.09 (3H, s) and 2.14 (3H, s), one second methyl group at δH 1.01 (3H, d, J = 7.0 Hz); one methyl acetyl group δH 2.13 (H, br s); one anomeric proton at δH 4.36 (H, d, J = 8.0 Hz). The 13C-NMR and DEPT spectra of TC2 showed the signals of 36 carbons including 3 carbonyl carbons at δC 167.9, 172.3, 2 and 218.1; 5 non-protonated carbon at δC 42.0, 47.9, 74.7, 123.8, and 159.6; 17 methine carbons at δC 30.5, 35.4, 18 36.5, 41.3, 56.4, 57.3, 57.4, 60.4, 71.6, 74.6, 75.1×2, 76.7, 77.9, 78.0, 78.7 and 103.9; 6 methylene carbons at δC 25.4, 33.1, 38.1, 43.8, 62.8 and 63.5; 5 methyl carbons at δC 13.4, 14.8, 15.5, 20.7 and 21.4. Figure 3.25. Chemical structure of TC2 and plantagiolide I (46) The NMR spectra data of TC2 were similar to those of plantagiolide I [5], the main difference was the absence of the acetoxy group at C-2. The HMBC correlation between H-19 (δH 0.94) and C-1 (δC 76.7)/C-5 (δC 74.7)/C-9 (δC 30.5)/C-10 (δC 42.0); H-18 (δH 1.09) and C-12 (δC 75.1)/C-13 (δC 47.9)/C-14 (δC 41.3)/C-17 (δC 57.4); H-21 (δH 1.01) and C-17 (δC 57.4)/C-20 (δC 36.5)/C-22 (δC 78.7); H-28 (δH 2.14) and C-23 (δC 33.1)/C-24 (δC 159.6)/C-25 (δC 123.8) showed position of 4 methyl groups at C-10, C-13, C-20 and C-24. The HMBC correlation from methyl proton (δH 2.13), aglycone H-12 (δH 5.18) to acetoxy carbonyl groups (δC 172.3) confirmed position of this acetoxy group at C-12. The 13C-NMR chemical shift of C-6, C-7 was shifted to a higher field [C-6 (δC 57.3), C-7 (δC 56.4)] and the HMBC correlation from H-6 (δH 2.99) to C- 5 (δC 74.7), suggesting the presence of a epoxy ring at C-6/C-7 and OH group at C-5. The HMBC correlation from H-27 (δH 4.65) to C-24 (δC 159.6)/C-25 (δC 123.8)/C-26 (δC 167.9) showed position of carbonyl group at C-26 and double bond at C-24/C-25. The HMBC correlation from H-15 (δH 2.49)/H-17 (δH 2.72) to C-16 (δC 218.1), suggesting the presence of oxo group at C-16. Acid hydrolysis of TC2 gave D-glucose 19 (identified as TMS derivative by GC). The sugar at C-27 was proved by HMBC correlation between glc H-1′ (δH 4.36) to C-27 (δC 63.5). The 13C NMR chemical shift of C-3 (δC 60.4) was shifted to higher field compared with that of the oxymethine carbon C-2 (δC 74.6), suggesting the presence of a chlorine atom at C-3. The HR-ESI-MS of TC2 showed pseudo-molecular ion peaks at m/z 781.2854 [C36H51O15Cl35+Na]+ and m/z 783.2891 [C36H51O15Cl37+Na]+ (Calcd for [C36H51O15Cl35+Na]+: 781.2809 and [C36H51O15Cl37+Na]+: 783.2802), confirming the presence of chlorine atom in TC2. The configuration of chlorine at C-3 was determined as β (equatorial) by the large coupling constant, J = 10.0 Hz, between H-2 and H-3. The constitution of TC2 was confirmed by a detailed interpretation of 2D-NMR spectra, including HSQC, HMBC, COSY, and ROESY. Thus, the structure of 2 was established and named chantriolide E. Figure 3.26. The important HMBC correlations of TC2 Table 3.10. NMR spectral data of TC2 and reference compound C C# Ca,b DEPT Ha,c (mult., J, Hz) Aglycone 1 73.7 76.7 CH 3.57 (d, 4.0) 2 76.6 74.6 CH 3.94 (dd, 4.0, 10.0) 3 56.9 60.4 CH 4.36 (m) 4 43.7 43.8 CH2 2.19*/2.33 (dd. 6.6, 13.5) 5 74.2 74.7 C - 6 56.6 57.3 CH 2.99 (d, 3.0) 7 55.4 56.4 CH 3.36 (dd, 2.0, 3.0) 8 34.7 35.4 CH 2.19 (m) 9 30.0 30.5 CH 2.27 (m) 10 41.6 42.0 C - 11 24.9 25.4 CH2 1.73 (t, 12.0)/2.01* 20 C C# Ca,b DEPT Ha,c (mult., J, Hz) 12 74.0 75.1 CH 5.18 (br s) 13 47.0 47.9 C - 14 40.7 41.3 CH 2.50 (m) 15 37.6 38.1 CH2 2.22 (m)/2.49 (m) 16 215.9 218.1 C - 17 56.4 57.4 CH 2.72 (d, 7.5) 18 14.7 14.8 CH3 1.09 (s) 19 15.6 15.5 CH3 0.94 (s) 20 35.6 36.5 CH 2.38 (m) 21 13.2 13.4 CH3 1.01 (d, 7.0) 22 77.3 78.7 CH 4.92 (m) 23 32.4 33.1 CH2 2.40 (m)/2.50 (m) 24 156.8 159.6 C - 25 123.7 123.8 C - 26 165.6 167.9 C - 27 63.5 63.5 CH2 4.48 (d, 11.5)/4.65 (d, 11.5) 28 20.6 20.7 CH3 2.14 (s) 12-OAc 170.6 172.3 C - 21.2 21.4 CH3 2.13 (s) 27-OGlc 1′ 104.9 103.9 CH 4.36 (d, 8.0) 2′ 75.4 75.1 CH 3.20 (t, 8.0) 3′ 78.6 78.0 CH 3.37 (m) 4′ 71.8 71.6 CH 3.32 (m) 5′ 78.8 77.9 CH 3.30 (m) 6′ 62.9 62.8 CH2 3.70 (dd, 2.0, 12.0)/3.89 (dd, 5.4, 12.0) a Recorded in CD3OD, b125MHz, c500MHz, #C of plantagiolide I [5], * Overlapped signals Figure 3.27. HR-ESI-MS spectrum of TC2 Figure 3.28. 1H-NMR spectrum of TC2 Figure 3.29. 13C-NMR spectrum of TC2 Figure 3.30. DEPT spectrum of TC2 21 Figure 3.31.HSQC spectrum of TC2 Figure 3.32.HMBC spectrum of TC2 Figure 3.33.COSY spectrum of TC2 Figure 3.34.ROESY spectrum of TC2 3.2. Biological activities of isolated compounds 3.2.1. Anti-inflammatory activity of isolated compounds 22 compounds from Tacca vietnamensis and Tacca chantrieri were evaluated for their inhibitory activity on NO production in BV2 cells, LPS-stimulated. As results, compounds TV3-TV5 inhibited NO production in BV2 cells, LPS-stimulated, with IC50 values of 52.1 ± 3.6 µM, 47.3 ± 6.0 µM, 43.7 ± 4.2 µM, respectively. Butein was used as a positive control, IC50 of 4.3 ± 0.5 µM. Chantriolide D (TC1) and chantriolide E (TC2) inhibited NO production in BV2 cells, LPS- stimulated, with IC50 of 12.4 ± 2.4 µM and 59.0 ± 3.5 μM. Butein was used as a positive control, IC50 of 4.3 ± 0.8 µM. 3.2.2. Cytotoxic activities of isolated compound from Tacca chantrieri 13 compounds from Tacca chantrieri were evaluated for cytotoxic activities toward four human cancer lines, including PC-3, LNCaP, MDA-MB-231 and HepG2 cells. The results showed that the new withanolide glucoside (chantriolide E) exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP, and MDA-MB-231 with IC50 of 24.5 ± 1.2 µM, 19.0 ± 1.5 µM, 20.9 ± 1.6 µM, respectively. TC7 exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP and MDA-MB-231 with IC50 of 30.7 ± 1.5, 19.1 ± 1.4 and 22 24.2 ± 1.5 µM, respectively. TC9 exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP and MDA-MB-231 with IC50 of 30.8 ± 2.0, 20.2 ± 1.2 and 49.3 ± 3.2 µM. TC13 exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP and MDA- MB-231 with IC50 of 17.9 ± 1.8, 18.8 ± 1.3 and 22.0 ± 2.0 µM, respectively. Ellipticine was us