Tóm tắt Luận án Chemical constituents and biological activities of two starfish anthenea sibogae and anthenea aspera collected from Vietnamese coast

es MeOH (t = 45 0C) F6.3.3 (3,2 mg), F6.3.4 (5,8 mg), F6.4.1 (13,5 mg), CH2Cl2 (3x500 ml) Dissolved in H2O (1.0 L). Elution AgNO3; EtOH F6.3 (87 mg) F6.4 (25 mg) F6.3.2 (2,3 mg) HPLC (Diasfer-110-C18) 80%MeOH; 0,5 ml/min ASB5 (2,4 mg; tR = 35 min) ASB6 (2,1 mg; tR = 39,8 min)ASB8 (1,2mg; tR = 30,4 min) ASB9 (0,6 mg; tR = 27,6 min) ASB10 (1,0 mg; tR = 27,5 min) ASB11 (1,9 mg; tR = 58,2 min) ASB7 (0,3 mg; tR = 31,7 min) 83%MeOH; 0,5 ml/min 75% EtOH,; 25 ml/min 85% MeOH: 0,5 ml/min HPLC (Diasorb-130-C16T) HPLC (Diasfer-110-C18) HPLC (Diasfer-110-C18) Figure 3.1. Diagram for the isolation procedure of starfish A. sibogae 3.1.2. Physical properties and spectral data of isolated compounds 3.2. Extraction and isolation procedure for starfish A. aspera This section details how to isolate compounds from A. aspera. The separation of compounds was summarized in the diagram in Figure 3.2. 3.2.1. Sample treatment of starfish A. aspera 5 Fresh Anthenea aspera (10 Kg) Crude EtOH 213 g 1. Chopped 2. Extracted by EtOH (3x8L), filtered 3. Evaporated Crude EtOAc (SDE) 68 g Crude hexane (SDH) 45 g MeOH (1Lx3) n-hexane (1Lx3) EtOAc (1Lx3) Crude MeOH (SDM) 96 g SDH4 SDH6 SDE1 SDE3 AA7 17 mg SDM1 ... SDH9 SDH1 hexane/CH2Cl2 100:0-0:100 .. Hexane/EtOAc gradien AA1 0,17 g AA2 0,53 g AA3 1,02 g AA4 15 mg CH2Cl2/ MeOH 05/1 SDM4 RP-C18 MeOH/H2O/NH4OH 70/29/1 AA5 48 mg CHCl3/MeOH gradien SDE7 SDE9 AA6 30 mg CHCl3/MeOH/H2O 4/1/0,1 AA8 33 mg CHCl3/MeOH/H2O 5/1/0,1 CH2Cl2/ MeOH 10:0-8:2 . SDM7 CH2Cl2/ MeOH 3/1 AA10 10 mg AA11 8 mg AA9 9 mg CHCl3/MeOH/H2O 2/1/0,1 CH2Cl2/ MeOH 100:0-0:100 . SDM11 Figure 3.2. Diagram for the isolation procedure of starfish A. aspera 3.2.2. Physical properties and spectral data of isolated compounds. CHAPTER 4 - RESULTS AND DISCUSSION This chapter presents the results of isolation and structural elucidation of isolated compounds from 2 starfish A. aspera and A. sibogae, cytotoxic activity, tumor suppression activity on soft agar and anti-proliferative activity of some isolated compounds. 4.1. Structural elucidation of isolated compounds from the starfish A. sibogae This section details the results of the structural determination of 11 compounds isolating from A. sibogae, including 6 new compounds and 5 known compounds. ASB1. Cholesterol ASB2. Thymine 6 ASB3. L-tyrosine ASB4. Tryptophan ASB5. Anthenoside S1 (new compound) ASB6. Anthenoside S2 (new compound) ASB7. Anthenoside S3 (new compound) ASB8. Anthenoside S4 (new compound) ASB9. Anthenoside S5 (new compound) ASB10. Anthenoside S6 (newcompound) ASB11. Mixture of Anthenoside J và Anthenoside K 7 4.1.5. ASB5 compound: (20R, 22E)-7-O- (6-O-methyl-β-D-galactofuranosyl) - 16-O- (3-O-methyl- β -D-glucopyranosyl) -24-nor- 5α-cholesta-8(14), 22(23) - diene-3α, 6β, 7β, 16α-tetraol (anthenoside S1, new compound) The molecular formula of ASB5 was determined to be of C40H66O14 from the [M + Na] + sodium adduct ion peak at m/z 793,4346 in the (+)-HR-ESI-MS spectrum. The 1 H- and 13 C-NMR spectroscopic data reffered to the steroidal nucleus of ASB5 revealed the chemical shifts of H- and C-atoms of two angular Me groups H3–C(18) [δ(H) 0,91 (s), δ(C) 20,3] and H3–C(19) [δ(H) 0,84 (s); δ(C) 15,5], an 8(14) double bond (δ(C) 126,6; 147,6), two HC–O groups, including H‒ C(3) [δ(H) 4,07 ‒ 4,08 (m, W = 11,8); δ(C) 67,5] and H‒C(6) [δ(H) 3,64 (t, J = 2,8); δ(C) 75,2], as well as two HC–O groups bearing Omonosaccharide residues, including H‒C(7) [δ(H) 4,23 (d, J = 2,8); δ(C) 78,5] and H‒C(16) [δ(H) 4,63 (td, J = 9,0; 5,0); δ(C) 79,2]. The width of the multiplet of H‒C(3) about 12 Hz corresponded well to the 3α-OH configuration while the width of the multiplet of H‒C(3) at the 3β-OH configuration is more than 30 Hz. The H- and C-atom resonances of the H3‒C(18), H3‒C(19), H‒C(3), H‒C(6), H‒C(7), and H‒C(16) were similar to the same signals in the NMR spectra of anthenoside Q and testified about a Δ8(14)-3α,6β,7β,16α-tetrahydroxysteroidal nucleus glycosylated at the C(7) and C(16) positions in ASB5. The NMR spectra of the aglycon side chain showed the presence of three secondary Me groups H3–C(21) [δ(H) 1,10 (d, J = 7,0); δ(C) 23,8], H3–C(26) [δ(H) 0,98 (d, J = 6,7); δ(C) 23,2], and H3–C(27) [δ(H) 0,97 (d, J = 6,7); δ(C) 23,1], and a 22(23) double bond [δ(H) 5,74 (ddd, J = 15,3; 8,8; 1,2), 5,30 (dd, J = 15,3; 6,8); δ(C) 133,9; 137,2]. Based on these data, a (22E)-Δ22-24-nor-cholestane side chain has been assumed in ASB5. A thorough analysis of the COSY, HSQC, HMBC, and ROESY spectra led to the assignment of all the H- and C-atom resonances of the steroidal moiety in ASB5 (Tables 4.6 and 4.7, Fig. 4.1.27). The H- and C-atom sequences at H-C(1) to H-C(7), H-C(9) 8 to H-C(12) through H-C(11), H-C(15) to H-C(17), H-C(17) to H-C(20), H-C(20) to H-C(22), H-C(23) to H3-C(27) were ascertained using the COSY and HSQC experiments. The total structure of the steroidal aglycon of ASB5 was supported by the key HMBC correlations H-C(6)/C(8), C(10); H-C(15)/C(8), C(14), C(17); H3-C(18)/C(12), C(13), C(14), C(17); H3-C(19)/С(1), С(9), С(10); H3- C(21)/C(17), C(20), C(22); H-C(22)/C(25); and H-C(23)/C(20), C(25), C(26), C(27). The key ROESY cross-peaks, such as H3-C(19)/Hβ-C(2), Hβ-C(4), Hβ- C(11); H3-C(18)/Hβ-C(12), Hβ-C(15), H-C(16); H-C(5)/Hα-C(1), Hα-C(9); Hα- C(4)/Hα-C(6); Hβ-C(4)/H-C(19); and Hα‒C(7)/Hα-C(15), along with the values of the coupling constants of H-C(6), H-C(7), and H-C(16), confirmed the 3α,6β,7β,16α relative configurations of O-bearing substituents and 5α/9α/10β/13β steroidal nucleus in ASB5. The resonance of H3-C(21) at δ(H) 1,10 (δ(H) 1,10 for (20R)-Δ22- and δ(H) 1,00 for (20S)-Δ22-steroids) as well as the ROESY correlations of H3-C(18)/H-C(20), H3-C(21); H3-C(21)/Hβ-C(12); and H-C(22)/H- C(16) allowed us to assume the (20R)-configuration. As a result, we proposed the (20R,22E)-24-nor-5α-cholesta-8(14),22(23)-diene-3α,6β,7β,16α-tetraol structure as the aglycon moiety of ASB5. The 1 H-NMR spectrum of ASB5 showed two resonances of anomeric H- atoms at δ(H) 4,33 and 5,02, correlated in the HSQC experiment with corresponding C-atom signals at δ(C) 102,9 and 108,4, resp. The (+)-ESI-MS/MS spectrum of the [M + Na] + ion peak at m/z 793 exhibited fragment ion peaks at m/z 599 ([(M + Na) – С7H14O6] +) and 217 ([С7H14O6 + Na] +). The (‒)-ESI- MS/MS spectrum of the [M ‒H]- ion peak at m/z 769 displayed fragment ion peaks at m/z 575 ([(M – H) – С7H14O6] -) and 193 ([С7H13O6] - ). All peaks corresponded to the loss of O-methyl-hexose residue. The chemical shifts and coupling constants of H-C(1)-H-C(6) of two O-methyl-hexose units were determined by the irradiation of anomeric H-atoms in the 1D TOCSY 9 experiments. Moreover, the H- and C-atom signals of the monosaccharide residues of ASB5 were established using 2D-NMR experiments (Tables 4.6 and 4.7). These chemical shifts and the corresponding coupling constants coincided well with those of terminal 3-O-methyl-β-glucopyranosyl and 6-O-methyl-β- galactofuranosyl residues. Acid hydrolysis of glycoside 1 with 2M TFA was carried out to ascertain the stereochemical series of its monosaccharide units. Alcoholysis of the obtained monosaccharides by (R)-(-)-2-octanol followed by acetylation, GC analysis, and comparison of retention times of acetylated (-)-2- octyl glycoside derivatives with the corresponding derivatives of standard monosaccharides allowed us to establish the D-configuration of the 3-O-methyl- glucose and 6-O-methyl-galactose (Experimental Section). The attachment positions of the monosaccharide units to the steroidal aglycon were defined by the HMBC and ROESY spectra, where the cross-peaks between H-C(1’) of 3-OMe- Glcp and C(16), H-C(16) of the aglycon and H-C(1’’) of 6-OMe-Galf and C(7), H-C(7) of the aglycon were observed (Fig. 1.27). On the basis of all the above mentioned data, the structure of anthenoside S1 was elucidated to be (20R,22E)-7- O-(6-O-methyl-β-D-galactofuranosyl)-16-O-(3-O-methyl-β-D-glucopyranosyl)- 24-nor-5α-cholesta-8(14),22(23)-diene-3α,6β,7β,16α-tetraol (ASB5). Fig. 4.1.27. Chemical structure ASB5 10 Table 4.6. 1 H-NMR data of compounds ASB5-ASB10 Vị trí ASB5 ASB6 ASB7 ASB8 ASB9 ASB10 1 1,51-1,55 (m) 1,29-1,31 (m) 1,51- 1,55 (m) 1,28- 1,30 (m) 1,50-1,56 (m) 1,28-1,31 (m) 1,51-1,55 (m) 1,28-1,34 (m) 1,52- 1,55 (m) 1,29- 1,32 (m) 1,50- 1,54 (m) 1,28- 1,30 (m) 2 1,61-1,63 (m) 1,60- 1,64 (m) 1,60-1,64 (m) 1,60-1,62 (m) 1,60- 1,63 (m) 1,60- 1,63 (m) 3 4,07-4,08 (m) 4,06- 4,08 (m) 4,06-4,08 (m) 4,06-4,08 (m) 4,07- 4,09 (m) 4,07- 4,09 (m) 4 1,96 (td, J = 14,0; 2,8) 1,37 (br d, J = 14,0) 1,96 (td, J = 14,0; 2,8) 1,36 (td, J=14,0) 1,96 (td, J = 14,0, 2,8) 1,36 (br d, J = 14,0) 1,96 (td, J =14,0;2,8) 1,36 (br d, J = 14,0) 1,98 (td, J = 13,7, 2,8) 1,38 (br d, J = 13,7) 1,96 (td, J = 14,0; 2,8) 1,37 (brd, J = 14,0) 5 2,12 (dt, J = 14,0, 2,8) 2,12 (dt, J =14,0; 2,8) 2,12 (dt, J = 14,0; 2,8) 2,13 (dt, J =14,0, 2,8) 2,16 (td, J =13,7; 2,8) 2,13 (dt, J = 14,0, 2,8) 6 3,64 (t, J = 2,8) 3,63 (t, J = 2,8) 3,64(t, J = 2,8) 3,64 (t, J = 2,8) 3,53 (t, J = 2,8) 3,61- 3,63 (m) 7 4,23 (d, J = 2,8) 4,22 (d, J = 2,8) 4,26 (d, J = 2,8) 4,27 (d, J = 2,8) 4,25 (d, J = 2,8) 4,23 (d, J = 2,8) 8 - - - - - - 9 2,25-2,28 (m) 2,25- 2,28 (m) 2,26-2,28 (m) 2,25-2,29 (m) 2,24- 2,26 (m) 2,25- 2,28 (m) 10 - - - - - - 11 1,63-1,67 (m) 1,52-1,55 (m) 1,63- 1,66 (m) 1,51- 1,55 (m) 1,62-1,66 (m) 1,50-1,56 (m) 1,62-1,66 (m) 1,51-1,56 (m) 1,65- 1,68 (m) 1,50- 1,56 (m) 1,63- 1,67 (m) 1,51- 1,57 (m) 12 1,82 (dt, J 1,81 (dt, 1,85 (dt, J = 1,87-1,89 1,80 (dt, 1,82 (dt, 11 = 12,5;3,3) 1,23-1,27 (m) J = 12,4; 3,4) 1,23- 1,27 (m) 12,0; 3,3) 1,28-1,32 (m) (m) 1,30-1,34 (m) J = 12,0; 3,4) 1,19- 1,22 (m) J = 12,3; 3,5) 1,24 ‒ 1,28 (m) 13 - - - - - - 14 - - - - - - 15 2,86 (ddd, J = 17,0; 9,0, 2,8) 2,64(ddd, J = 17,0; 5,0, 2,1) 2,85 (ddd, J = 17,0; 9,0, 2,8) 2,64 (ddd, J = 17,0; 5,0, 2,1) 2,84 (ddd, J = 17,0; 8,7; 3,0) 2,68 (ddd, J = 17,0; 4,1; 1,8) 2,85 (ddd, J = 17,0; 8,6; 2,9) 2,62-2,69 (m) 2,93 (ddd, J = 16,8; 9,0; 3,0) 2,38- 2,41 (m) 2,87 (ddd, J = 17,0; 9,0, 3,1) 2,60 (ddd, J = 17,0; 5,2, 1,8) 16 4,63(td, J = 9,0; 5,0) 4,62 (td, J = 9,0; 5,0) 4,55 (td, J = 8,7; 4,1) 4,57 (td, J = 8,6, 4,2) 4,47 (ddd, J = 9,8, 9,0; 6,0) 4,46 (td, J = 9,0; 5,2) 17 1,53 (dd, J = 9,0; 4,0) 1,54 (dd, J = 9,0; 4,0) 1,49-1,51 (m) 1,51 (dd, J = 8,6, 6,7) 1,46 (dd, J = 9,8; 2,7) 1,48 (dd, J = 9,0; 4,6) 18 0,91 (s) 0,91 (s) 0,94 (s) 0,95 (s) 0,89 (s) 0,93 (s) 19 0,84 (s) 0,84 (s) 0,85 (s) 0,85 (s) 0,83 (s) 0,85 (s) 20 2,38-2,42 (m) 2,38- 2,42 (m) 1,67-1,74 (m) 1,69-1,75 (m) 2,37- 2,42 (m) 1,66- 1,71 (m) 21 1,10 (d, J = 7,0) 1,10 (d, J = 7,1) 1,03 (d, J = 6,8) 1,03 (d, J = 6,8) 1,09 (d, J = 7,3) 1,06 (d, J = 6,8) 22 5,74 (ddd, J = 15,3; 8,8, 1,2) 5,76 (d, J = 15,3; 8,8, 1,2) 1,71-1,76 (m) 1,43-1,47 (m) 1,85-1,89 (m) 1,52-1,56 (m) 5,65 (dd, J = 15,4, 7,3) 1,79- 1,86 (m) 1,43- 1,47 (m) 23 5,30 (dd, J = 15,3, 5,31(dd, J = 15,3; 2,07-2,12 (m) 1,88-1,93 (m) 2,19-2,23 (m) 5,37 (dd, J = 2,21- 2,24 (m) 12 6,8) 6,8) 1,87-1,91 (m) 15,4, 7,3) 1,92- 1,97 (m) 24 - - 5,14 (br t, J =7,5) - 1,92(br td, J =7,3, 3,3) - 25 2,22-2,30 (m) 2,26- 2,29 (m) - 2,24-2,31 (m) 1,56- 1,62 (m) 2,24- 2,30 (m) 26 0,98 (d, J = 6,7) 0,98 (d, J = 6,6) 1,67 (s) 1,03 (d, J = 6,9) 0,89 (d, J = 6,5) 1,04 (d, J = 6,9) 27 0,97 (d, J = 6,7) 0,97 (d, J = 6,6) 1,60 (s) 1,03 (d, J = 6,9) 0,89 (d, J = 6,5) 1,04 (d, J = 6,9) 28 4,72 (d, J = 1,3) 4,70 (br s) 4,75 (br s) 4,72 (br d, J =1,3) 3-OMe-β- D-Glcp 4-OMe- β-D- Glcp 3-OMe-β-D- Glcp 3-OMe-β- D-Glcp 3-OMe- β-D- Galf β-D- Galf 1’ 4,33 (d, J = 7,7) 4,29 (d, J =7,8) 4,32 (d, J = 7,7) 4,32 (d, J = 7,8) 4,97 (br s) 4,95 (d, J = 2,4) 2’ 3,23 (dd, J = 9,3; 7,7) 3,17 (dd, J =9,3; 7,8) 3,21(dd, J = 9,1, 7,7) 3,22 (dd, J =9,1, 7,8) 4,00 (dd, J = 2,5; 1,1) 3,96 (dd, J = 4,8; 2,4) 3’ 3,09 (t, J = 9,3) 3,46 (t, J = 9,3) 3,08 (t, J = 9,1) 3,08 (t, J = 9,1) 3,74 (dd, J = 5,6; 2,5) 4,04 (dd, J = 7,2; 4,8) 4’ 3,36 (t, J = 9,3) 3,10 (t, J = 9,3) 3,32 (t, J = 9,1) 3,27-3,29 (m) 4.08 (dd, J = 5.6, 3.5) 3,88 (dd, J = 7,2; 2,4) 5’ 3,27 (ddd, J = 9,3;5,5, 3,26 (ddd, J = 9,3; 5,1, 3,25 (ddd, J = 9,1; 5,7; 2,5) 3,26 (m) 3.73- 3.75 (m) 3,70- 3,73 (m) 13 2,5) 2,2) 6’ 3,88(dd, J=11,6; 2,5) 3,70(dd, J=11,6; 5,5) 3,86(dd, J=11,6; 2,2) 3,71(dd, J=11,6; 5,1) 3,86(dd, J=11,6, 2,5) 3,65(dd,J=11,6, 5,7) 3,86(dd, J=11,6, 2,5) 3,63(dd, J=11,6, 5,6) 3,65(br d, J=6,1) 3,62 (dd, J = 11,2; 7,2) 3,59 (dd, J = 11,2, 4,5) OMe 3,63 (s) 3,56 (s) 3,62 (s) 3,62 (s) 3,41 (s) 6-OMe-β- D-Galf 6-OMe- β-D-Galf 6-OMe-β-D- Galf 6-OMe-β- D-Galf 6-OMe- β-D- Galf 1’’ 5,02 (d, J = 2,0) 5,01 (d, J = 2,0) 5,05 (d, J = 2,0) 5.04 (d, J = 2.0) 4.99 (d, J = 2.3) 2’’ 3,90 (dd, J = 3,7; 2,0) 3,89- 3,91 (m) 3,90 (dd, J = 4,3, 2,0) 3.90 (dd, J =3.6, 2.0) 3.91 (dd, J = 3.8, 2.3) 3’’ 3,94 (dd, J = 6,2; 3,7) 3,94 (dd, J = 6,1; 3,6) 3,90-3,96 (m) 3.93 (dd, J = 6.8, 3.6) 3.95 (dd, J = 6.2, 3.8) 4’’ 3,90- 3,91(m) 3,89- 3,91 (m) 3,91(dd, J = 5,8, 3,4) 3.92 (dd, J = 6.8, 3.5) 3.88 (dd, J = 6.2, 3.9) 5’’ 3,82 (ddd, J = 7,0; 5.2; 3,4) 3,82 (ddd, J = 7,0; 5,2, 3,3) 3,82 (ddd, J = 7.2, 4.8, 3.4) 3.82 (ddd, J = 7.1, 4.8, 3.5) 3.82 ‒ 3.85 (m) 6’’ 3,53 (dd, J = 10,3; 5,2) 3,52 (dd, J = 10,3;7,0) 3,53 (dd, J = 10,1; 5,2) 3,52 (dd, J = 10,1; 7,0) 3,53(dd, J = 10,1, 4,8) 3,52 (dd, J = 10,1; 7,2) 3,54 (dd, J = 10.1, 4.8) 3,52 (dd, J = 10.1, 7,1) 3,53 (d, J = 6,0) OMe 3,38 (s) 3,38 (s) 3,38 (s) 3,38(s) 3,39(s) 14 Measured in CD3OD, 700 MHz Table 4.7. 13 C-NMR spectroscopic data of compounds ASB5-ASB10 Position ASB5 ASB6 ASB8 ASB9 ASB10 1 34.6 34.5 34.6 34.5 34.5 2 29.6 29.6 29.6 29.5 29.6 3 67.5 67.5 67.3 67.4 67.5 4 33.3 33.3 33.2 33.4 33.3 5 38.0 38.0 37.9 37.7 38.0 6 75.2 75.5 75.5 77.4 75.2 7 78.5 78.7 78.7 72.1 78.4 8 126.6 126.6 126.6 128.4 127.0 9 45.9 45.9 45.6 45.8 45.8 10 38.8 38.8 38.9 38.7 38.9 11 19.5 19.5 19.6 19.4 19.5 12 37.2 37.1 37.6 36.9 37.3 13 45.4 45.4 45.4 44.9 45.1 14 147.6 147.6 148.0 146.8 147.4 15 34.3 34.5 34.2 33.5 33.8 16 79.2 79.4 80.1 76.9 77.7 17 62.8 62.8 62.7 62.6 62.7 18 20.3 20.3 19.7 20.5 20.1 19 15.5 15.4 15.5 15.3 15.4 20 37.2 37.1 34.1 37.1 32.9 21 23.8 23.8 20.8 24.6 21.4 22 133.9 133.8 34.6 137.7 33.8 23 137.2 137.4 33.0 129.5 33.3 24 - - 158.4 43.2 157.7 25 32.3 32.3 34.9 29.9 34.9 26 23.2 23.2 22.6 22.8 22.5 27 23.1 23.1 22.4 22.3 28 108.6 107.2 3-OMe-β-D- Glcp 4-OMe- β-D- Glcp 3-OMe-β- D-Glcp 3-OMe- β-D- Galf β-D- Galf 1’ 102.9 103.0 102.6 108.2 107.6 2’ 75.2 75.2 75.1 80.9 83.7 3’ 87.9 78.2 87.8 88.9 78.3 4’ 71.5 81.2 71.7 84.3 84.4 15 5’ 77.8 77.2 77.9 73.2 72.4 6’ 63.2 62.7 63.4 65.1 65.4 OMe 61.0 60.8 61.0 58.1 6-OMe -β- D-Galf 6-OMe- β-D- Galf 6-OMe-β- D-Galf 6- OMe- β-D- Galf 1’’ 108.4 108.4 108.5 108.4 2’’ 83.4 83.4 83.4 83.5 3’’ 78.7 78.7 78.7 78.7 4’’ 85.0 85.0 85.0 85.0 5’’ 70.8 70.8 70.8 70.8 6’’ 75.5 75.5 75.5 75.5 OMe 59.4 59.4 59.4 59.4 Measured in CD3OD, 176 MHz 4.2. Structure elucidation of isolated compounds from the starfish A. aspera 4 sterols and 7 other compounds were isolated for the first time from hexane, ethyl acetate and methanol extracts of starfish A. aspera collected in Northeast Vietnam. AA1. Cholesterol AA2. Lathosterol AA3. Cholest-4-ene-3β,6β-diol AA4.Cholestan-3β,5α,6β,15α,16β-26-hexol AA5. Cyclo(L-glycine-L-proline) AA6. L-glycine-L-propyl 16 AA7.Cyclo(L-alanine-4-hydroxyl-L-prolyl AA8. L-Phenylalanine AA9. Tyramine AA10. Thymine AA11. Uracil 4.2.1. AA1 compound: cholesterol Compound AA1 has melting point, Rf and NMR spectrum coincide with compound ASB1 4.2.2. AA2 compound: Lathosterol (Cholest-7,8-ene-3-ol) Fig. 4.2.8. Chemical structure of AA2 Table 4.14. NMR spectrum data of AA2 and reference substance Position #C C a,c H a,b (mult., J, Hz) 1 37.1 37.1 1.82 m/ 1.07 m 2 31.3 31.4 1.80 m/ 1.61 m 3 70.7 71.1 3.60 m 4 37.8 38.0 1.27 m/ 1.72 m 5 40.2 40.3 1.40 m 6 29.6 29.7 1.76 m 7 117.2 117.4 5.15 m 8 139.3 139.6 - 9 49.4 49.5 1.61 m 10 34.1 34.2 - 11 21.5 21.6 1.57 m, 1.45 m 12 39.5 39.5 1.20; 2.02 m 13 43.2 43.4 - 17 14 54.9 55.0 1.80 overlap 15 22.9 23.0 1.40 m; 1.52 m 16 27.9 27.9 1,88 m; 1.26 m 17 56.1 56.1 1.20 m 18 11.8 11.8 0.53 s 19 12.9 13.0 0.79 s 20 36.1 36.0 1.36 m 21 18.8 18.8 0.92 d (6.5) 22 36.1 36.2 0.99 m; 1.34 m 23 23.9 23.9 1.14 m, 1.34 m 24 39.4 39.6 1.13-1.10 m 25 27.9 28.0 1.52 m 26 22.5 22.6 0.86 d (7.0) 27 22.7 22.8 0.87 d (7.0) a CD3OD, b 500 MHz, c 125 MHz, #δC data of [58] 4.2.3. AA3 compound: cholest-4-ene-3,6-diol Fig. 4.2.13. Chemical structure of AA3 Table 4.15. NMR spectrum data of AA3 and reference substance Position C a,c H a,b (mult., J, Hz) C δC a,c H a,b (mult., J, Hz) 1 37.4 1.76 (m); 1.32 (m) 15 25.2 1.64 (m); 1.40 (m) 2 29.6 1.91 (m); 1.49 (m) 16 29.2 1.88 ( m), 1.32 (m) 3 69.2 4.11-4.16 (trùng H-6) 17 57.6 1.14 (m) 4 121.4 5.67 (d, J 1.5 Hz) 18 12.4 0.75 (s) 5 149.5 - 19 19.2 1.08 (s) 6 68.6 4.11-4.16 (trùng H-3) 20 37.1 1.43 (m) 7 43.7 0.87 (m) 21 19.2 0.95 (d, J 6.5) 8 35.8 1.58 (m) 22 37.3 1.39 (m); 1.05 (m) 9 55.9 0.75 (m) 23 24.9 1.14-1.22 (m) 10 38.9 - 24 40.7 1.10-1.21 (m) 11 22.1 1.39; 1.53 (m) 25 29.1 1.55 (m) 12 41.1 2.04-2.06 (m) 26 23.2 0.89 (d, 6.5) 18 13 43.2 - 27 22.9 0.86 (d, 6.5) 14 57.4 1.07 (m) a CD3OD, b 500 MHz, c 125 MHz, #δC data of [77] 4.2.4. AA4 compound: cholestane 3,5,6,15,16,26-hexol Fig. 4.2.21. Chemical structure of AA4 Table 4.16. NMR spectrum data of AA4 and reference substance Position #C C ac H ab , mult (J = Hz) HMBC (HC) NOESY 1 31.7 31.7 1.79 m; 1.51 m C-5, C-19 2 33.5 33.5 1.62 m; 1.35 m C-10 3 68.4 68.3 4.03 m (5.5) 4 41.6 41.5 2.08 dd (11.5; 13.0) C-3, C-5 5 76.6 76.6 - 6 76.6 76.4 3.49 dd (2.5; 3.0) C-4, C-5, C-8, C-10 H-4, H-7 7 35.4 35.2 1.89 m C-6, C-8, C-9, C-14 8 32.2 31.1 2.01 m C-7, C-9, C-14 9 46.7 46.6 1.41 m 10 39.5 39.3 - 11 22.0 21.9 1.38 m C-13 12 42.1 42.0 1.98 m; 1.20 m C-9, C-14 13 44.9 44.7 - 14 61.2 60.9 0.98 m C-13, C-16, C-18 15 85.0 85.1 3.76 dd (2.5; 10.0) C-8, C-14, C-16 H-18 16 83.2 83.0 3.99 dd (2.5; 7.5) C-13, C-15 H-17 17 60.1 59.9 1.27 m C-13, C-18, C-20 18 15.2 15.1 0.93 s C-12, C-13, C-14, C-17 19 19 17.2 17.3 1.20 s C-1, C-5, C-9, C-10 20 31.0 31.0 1.89 m C-17 21 18.6 18.6 0.98 d (5.5) C-17, C-20, C-22 22 37.5 37.4 1.08 m C-21 23 24.8 24.8 1.46 m; 1.23 m C-24 24 35.0 34.9 1.43 m; 1.06 m C-27 25 37.0 37.0 1.58 m 26 68.6 68.4 3.45 dd (6.0; 10.5); 3.34 overlapped C-24, C-25, C-27 27 17.3 17.4 0,93 d (6,5) C-24, C-25, C-26 a CD3OD, b 500 MHz, c 125 MHz, #δC data of [78] 4.2.5. AA5 compound: cyclo(L-glycine-L-proline) Fig. 4.2.27. Chemical structure of AA5 Table 4.17. NMR spectrum data of AA5 and reference substance Position , H a cδ (mult, J, Hz) cyclo(L- Gly-L- Pro) [79] , H a cδ (mult, J, Hz) (AA5) ,b C aδ (AA5) HMBC (H→C) (AA5) ,d e C δ L-Gly-L- Pro [80] Gly 1 - 163.6 175.6 2 4.10 3.87 (dd) 4.10 * 3.90 (dd, 4.5; 16.5) 46.5 C-1, C-1′ 46.2 NH 7.15 7.35 (brs) C-1, C-2′ Pro 1′ 170.1 169.3 2′ 4.11 4.10* 58.5 C-3’, C-4’ 59.6 20 3′ 1.75-2.55 2.38 (m)/2.06 * (2.34-2.41) 28.4 C-1′, C-2′, C-4′, C-5′ 29.2 4′ 1.75-2.55 1.92 (m)/2.06 * (1.86-2.11) 22.3 C-2′, C-3′, C-5′ 23.6 5′ 3.58 (m) 3.64 (m)/3.56 (m) (3.58, m) 45.2 C-1, C-2′, C-3′, C-4′ 43.0 a CDCl3, , b 125 MHz, c 500 MHz, * signal overlap, d D2O, e CD3OD. 4.2.6. AA6 compound: L-glycine-L-prolin Fig. 4.2.31. Chemical structure of AA6 Table 4.18. NMR spectrum data of AA6 and reference substance Position #δC [80] L-Gly-L-Pro ,a b C δ (AA6) , a c H δ (mult, J.,Hz) (AA6) HMBC (H→C) (AA6) Gly 1 175.6 172.0 2 46.2 47.0 4.12 (ddd, 17,0; 2,0; 1,0) 3.76 (d, 17.0) C-1, C-1’ NH Pro 1′ 169.3 166.5 2′ 59.6 59.9 4.25 (m) C-3’ 3′ 29.2 29.3 2.35 (m) 1.99 (m) 4′ 23.6 23.3 2.04 (m) 1.96 (m) C-5’ 5′ 46.3 46.3 3.52-3.60 (m) C-4’ a MeOD–d4, # D2O, b 125 MHz, c 500 MHz. 21 4.2.7. AA7 compound: cyclo(L-alanyl-4-hydroxyl-L-prolyl) Fig. 4.2.37. Chemical structure of AA7 Table 4.19. NMR spectrum data of AA7 and reference substance Position #C C ac H ab , mult (J = Hz) HMBC (HC) 1 163.6 169.1 - 2 46.5 52.1 4.26 C-1 3 15.7 - C-1 1’ 170.1 172.8 - 2’ 58.5 58.9 4.54 C-1’ 3’ 28.4 38.2 2.30 (dd, dd, 6.5; 13.5) C-1’ 4’ 22.3 69.1 4.49 2.11 (ddd, 4.0 ; 11.0; 13.5) - 5’ 45.2 55.2 3.69 (dd,4.5; 13.0) 3.45 (d,13.0) C-1 N-H - - 4.63 - a CD3OD, b 500 MHz, c 125 MHz, #δC data of [81]. 4.2.8. AA8 compound: L-phenylalanine Fig. 4.2.43. Chemical structure of AA8 Table 4.20. NMR spectrum data of AA8 and reference substance Position #C C ac H ab , mult (J = Hz) HMBC (HC) 1 135.3 137.3 - 2 128.7 130.0 7.28-7.38 (m, 5H, H-Ar); 3 129.7 130.4 7.28-7.38 (m, 5H, H-Ar); 4 130.7 128.4 7.28-7.38 (m, 5H, H-Ar); 22 5 129.7 130.4 7.28-7.38 (m, 5H, H-Ar); 6 128.7 130.0 7.28-7.38 (m, 5H, H-Ar); 7 37.8 38.3 3.33 (dd, 1H, J = 4.5; 14.5 Hz, H-7), 3.02 (dd, 1H, J = 9.0; 14.5 Hz, H-7). C-8, C-2, C-6, C-1, C-9 8 55.5 57.6 3.79 (dd, 1H, J = 4.5; 9.0 Hz, H-8); C-7, C-1, C-9 9 174.2 173.8 - a CD3OD, b 500 MHz, c 125 MHz, #δH data of [82] in CD3OD. 4.2.9. AA9 compound: tyramine Fig. 4.2.46. Chemical structure of AA9 Table 4.21. NMR spectrum data of AA9 and reference substance Position #C C ac H ab , mult (J = Hz) 1 128.3 128.5 - 2 130.5 130.8 7.11 d (8.5) 3 116.2 116.7 6.79 d (8.5) 4 156.6 157.8 - 5 116.2 116.7 6.79 d (8.5) 6 130.5 130.8 7.11 d (8.5) 7 35.1 34.0 2.88 dd (8.0; 7.0) 8 42.3 42.3 3.13 dd (8.0; 7.0) C=O 177.0 - CH3 10.5 - a CD3OD, b 500 MHz, c 125 MHz, #δC data of [83] in CD3OD. 4.2.10. AA10 compound: thymine Phổ 1H-NMR cũng như Rf và điểm nóng chảy của AA10 hoàn toàn đồng nhất với các dữ liệu ASB2. 4.2.11. AA11 compound: uracil 23 Fig. 4.2.51. Chemical structure of AA11 Table 4.22. NMR spectrum data of AA11 and reference substance Position #C C ac H ab , mult (J = Hz) 1 167.5 164.3 - 2 110.4 100.2 5.44 ppm (J =7,5Hz, H-2) 3 139.2 142.1 7.38 ppm (J =7,5Hz, H-3) 4 150.3 151.5 - 5 12.1 - - N-H 11.8 11.0 a CD3OD, b 500 MHz, c 125 MHz, #δc data of TLTK [84,85] 4.3. Anticancer activity of steroid glycosides from starfish species A. sibogae 4.3.1. Cytotoxic activity ASB5-ASB11 compounds were tested for cytotoxicity on human breast cancer cell lines T-47D using MTS method. Cisplatin was used as positive control. The results showed that compounds ASB5, ASB6, ASB8 and the mixture ASB11 as well as cisplatin were not cytotoxic to T-47D cell line at concentrations up to 150 μM after 24 hours and 48 hours. 4.3.2 Anti-proliferative activity Compounds ASB5, ASB6 and ASB8 did not show significant proliferative inhibitory activity against T-47D cell lines at a concentration of 50 μM, while the mixture ASB11 inhibited T-47D cell proliferation after 24 h, 48 hours and 72 hours at the same concentration cisplatin. After 24 hours of ASB11 mixture decreased T-47D cell proliferation by 10% while control cisplatin decreased T-47D cell proliferation by 20%. After 48 hours of ASB11 mixture decreased T-47D cell proliferation by 20% while cisplatin decreased T-47D cell proliferation by 50%. The ASB11 mixture (50 μM) reduced T-47D cell 24 proliferation after 72 hours by 47%,