Research on chemical constituents and biological activities of callicarpa candicans and callicarpa macrophylla growing in Vietnam

flower. The constituents with the main content determined in the Nang essential oil of her plant are E- caryophyllene (5.07%), α- selinene (5.66%), δ-cadinene (5.44%). The chemical composition of the fresh leaves of Eucalyptus tree has 50 components, accounting for 90.59% of total content. The main component identified in essential oil is Phytol (11.03%). 4.2. The compounds were isolated from the leaves of the Big Leaf and the Taurus From the residue of n-hexane and ethyl acetate extracts from the leaves of the Tzu tree leaves, up to 10 compounds were isolated and chemically structured, including 7 terpenoid compounds: callimacrophylla B (M8), 3β- hydroxyolean-12-ene. (M2), β-amyrin (M3), ursolic acid (M6), oleanolic acid (M10), callimacrophylla A (M1) and ent-1β-acetoxy-7β, 14α-dihydroxy-16- kauren-15-on (M7) ) and 3 steroid compounds: spinasterol (M5), β-sitosterol (M4) and daucosterol (M9). In particular, callimacrophylla A (M1) and callimacrophylla B (M8) are two new compounds. From the residue of n-hexane and ethyl acetate extracts of Nang Nang leaves (C. candicans) 11 compounds were isolated and identified chemical structure, including 4 flavonoid compounds: 5-hydroxy-7.4'-dimethoxyflavon (C1), 5-hydroxy-3 ', 4', 7-trimethoxyflavon (C3), genkwanin compound (C9) and cynaroside (C10); 5 terpenoid compounds: ursolic acid (M6), 2α-hydroxy- ursolic acid (C7), 2α, 3β, 23-trihydroxyurs-12-en-28-oic acid (C8), seco- hinokiol (C5) and methyl seco -hinokiol (C6) and 2 steroid compounds: β- sitosterol (M4) and daucosterol (M9). In it, the compound methyl seco-hinokiol was isolated for the first time from nature. The main chemical composition of the two genera Callicarpa species studied are mainly flavonoid, diterpennoid and triterpenoid compounds. Several compounds were isolated in two species: ursolic acid, β-sitosterol and daucosterol. 7 Triterpenoid compounds isolated from 2 research species show that the main chemical components are ursane and oleane frame compounds, suitable for the main chemical components of triterpenoid compounds in Callicarpa genus in the literature. Father. The diterpene compounds isolated from two research species show that the main chemical components are ent-kaurane and abietane frame compounds, which are suitable for the main chemical components of diterpenoid compounds in Callicarpa in the literature. announced. The compounds isolated from leaves of two studied species Table 4.26. The compounds were isolated from 2 studied species Tên hợp chất Lớp chất Loài phân lập KL (mg) Tính mới callimacrophylla A (M1) Diterpneoid C. macrophylla 10,8 M ent-1α-acetoxy-7β,14 α-dihydroxy-kaur- 16-en-15-on (M7) Diterpenoid C. macrophylla 12,5 H seco-hinokiol (C5) Diterpenoid C. candicans 22,8 H methyl seco-hinokiol (C6) Diterpenoid C. candicans 31,0 H 3β-hydroxyolean-12-ene (M2) Triterpenoid C. macrophylla 10,3 L β-amyrin (M3) Triterpenoid C. macrophylla 12,7 L ursolic acid (M6) Triterpenoid C. macrophylla C. candicans 15,5 15,7 L L 8 callimacrophylla B (M8) Triterpenoid C. macrophylla 10,1 M 2α-hydroxy-ursolic acid (C7) Triterpenoid C. candicans 12,5 L 2α,3β,23-trihydroxyurs-12-en-28-oic acid (C8) Triterpenoid C. candicans 11,5 L oleanolic acid (M10) Triterpenoid C. macrophylla 8,5 spinasterol (M5) Steroid C. macrophylla 11,2 L β–sitosterol (M4) Steroid C. macrophylla C. candicans 20,0 19,0 daucosterol (M9) Steroid C. macrophylla C. candicans 16,5 5-hydroxy-7,4’-dimethoxyflavon (C1) Flavonoid C. candicans 8,5 L 5-hydroxy-3’,4’,7-trimethoxyflavon (C3) Flavonoid C. candicans 11,2 L genkwanin (C9) Flavonoid C. candicans 13,0 L cynaroside (C10) Flavonoid C. candicans 10,8 L M: New compound; L: For the first time isolated from species; H: First isolated from them The two newly obtained compounds are 1 substance belonging to the class of diterpenoid and triterpenoid. They are structured based on the following spectral data 4.2.1.1. Callimacrophylla A (M1) - New compound Compound 1 was obtained as a white crystal and its molecular formula was deduced as C20H28O3 by HR-ESI mass spectrum (found m/z 387.1940 [Mþ35Cl]-, calcd. For C20H32O535Cl: 387.1938; m/z 389.1918 [Mþ37Cl]-, calcd. for C20H32O537Cl: 389.1909).). Hình 4.7. Phổ ESI-MS của M1 1H-NMR spectrum of 1 exhibited two methyl singlet signals at dH 0.65 and 1.02, one oxygenated methine at dH 3.66 (dd, J ¼ 10.5, 5.5 Hz), the doublet of doublet signals oftwo oxygenated methylene at dH 2.85/3.18 and 3.45/3.54, and other signals from dH 0.93 to 4.78 ppm. (Bảng 4.3). 9 Hình 4.8. Phổ 1H-NMR của M1 Hình 4.9. Phổ 13C-NMR của M1 The 13C-NMR spectrum of 1 showed signals of 20 carbon atoms, which were sorted by DEPT spectra into one ketone group at dC 219.3; one oxygenated methine at dC 69.5; two oxygenated methylenes at dC 61.4 and 69.9; two methylgroups at dC 17.4 and 17.7, four quaternary carbons at dC 36.9, 38.5, 58.4, and 78.4; six methylene and three methine groups. The above results suggested that compound 1 to be a diterpene with the ent-kaurane skeleton similar to ent-7a,16b,17-trihydroxy-18- acetoxykaur-15-one except for the disappearance of acetoxy group at C-18 instead of the addition of hydroxy group at this position in 1 (Giang et al. 2013). Assignments of 1H- and 13C-NMR data of 1 were completed by HSQC and HMBC spectra in comparison with the corresponding data of ent-7a,16b,17-trihydroxy-18- acetoxykaur-15-one (Giang et al. 2013) 101]. 10 Hình 4.10. Phổ DEPT của M1 In the HSQC spectra, protons at dH 0.65, 1.02, and 3.62 had cross peaks with carbons at dC 17.4, 17.7, and 69.5, respectively; while protons at dH 2.85/3.18 and 3.45/3.54 had cross peaks with carbons at dC 69.9 and 64.1, respectively. Furthermore, four proton signals at dH 4.30, 4.43, 4.49, and 4.78 did not have HSQC cross peaks with any carbons suggesting that compound 1 had four hydroxy groups (hình 4.6 và bảng 4.3). Hình 4.11. Phổ HSQC của M1 11 Hình 4.12. Phổ HMBC của M1 In the HMBC spectra of 1, the correlations from protons at dH 0.65 and 2.85/3.18 to carbons C-3 (dC 34.7), C-4 (dC 36.9), C-5 (dC 44.9), from H-20 (dH 1.02) to C-1 (dC 38.6), 2 D. T. LAM ET AL. C-5 (dC 44.9), C-9 (dC 52.6), C-10 (dC 38.5), and from H-7 (dH 3.45/33.54) to C-13 (dC 38.7), C-15 (dC 219.3), C-16 (dC 78.4) were observed, confirming that three hydroxy groups were at C-18, C-6 and C-17, and the assignments of NMR data of the concerned positions. The last hydroxy group was at C-7 confirming by the HMBC correlations from hydroxy proton at dH 4.30 to C-6 (dC 26.9), C-7 (dC 65.9), C-8 (dC 58.4). In addition, HMBC correlations from 18-OH (dH 4.49) to C-18 and C-4, from 16- OH (dH 4.78) to C-13, C-15, C-16, and from 17-OH (dH 4.43) to C-17 and C- 16 were observed. On the other hand, the carbon chemical shifts of C-18 (dC 69.9) and C-19 (dC 17.4) of 1 were similar to the corresponding values of siderone (18-hydroxy derivative, dC-18 ¼ 71.0 and dC-19 ¼ 16.9) (Venturella et al. 1983), and difference from the corresponding values of diterpene SL-II (19-hydroxy derivative, dC-19 64.3 and dC-18 22.8) (Piozzi et al.1980). Above evidence further confirmed that the hydroxy group was at C-18. The large coupling constant of H-7 (J ¼ 10.5 Hz) of 1 comparing to the broad singlet of H-7 of 7b,16a,17-trihydroxy-ent-kauran-19-oic acid (Nhiem et al. 2015) confirmed equatorial orientation (a-configuration) of 7-OH group in 1. The carbon chemical shifts of C-16 (dC 78.4) and C-17 (dC 61.4) of 1 were consistent with the corresponding values of ent- 7a,16b,17-trihydroxykaur-18- acetoxy-15-one (dC-16 77.4 and dC-17 63.1) (Giang et al.2013) and difference from 16a,17-dihydroxy-15-oxo-ent-kaur-19-oic acid (dC-16 83.0 and dC-17 12 65.3) (Braca et al. 2004) suggesting the b-configuration of 16-OH group. From the above evidence, compound 1 was determined to be ent-7a,16b,17,18- tetrahydroxykaur-15-one, a new compound and named as callimacrophylla A. Figure 4.6. Chemical structure, the main interaction HMBCHC) of M1 Table 4.3. Spectrum data of 1H- and 13C-NMR of M1 and reference substance TT Hợp chất M1 [103] δC δH (mult., J Hz) #δC #δH (mult., J Hz) 1 38,6 0,57 (1H, m)/ 1,66 (1H, m) 41,7 3,57 (m) 2 17,3 1,38 (1H, m)/ 1,57 (1H, m) 20,3 2,03 (m) 1,85 (overlap) 3 34,7 1,11 (1H, m)/ 1,40 (1H, m) 39,2 2,01 (m) 1,42 (m) 4 36,9 - 44,2 5 44,9 - 48,1 1,63 (dd, 11,9, 1,7) 6 26,9 1,75 (1H, m)/ 1,66 (1H, m) 30,5 1,75 (m) 1,40 (m) 7 69,5 3,62 (1H, dd, 10,5; 5,5) 78,1 2,28 (dt, 13,3, 4,1) 1,33 (overlap) 8 58,4 - 49,0 - 9 52,6 0,94 (1H, d, 9,0) 51,1 1,85 (overlap) 10 38,5 1,20 (1H, m)/ 1,53 (1H, m) 40,4 - 11 17,9 1,20 (1H, m)/ 1,53 (1H, m) 19,1 3,66 (dd, 10,7, 6,0) 1,68 (m) 12 28,0 1,18 (1H, m)/ 1,60 (1H, m) 27,6 1,98 (m) 1,57 (m) 13 38,7 2,21 (1H, br d, 3,5) 46,1 2,95 (br s) 14 25,3 1,66 (1H, m)/ 2,38 (1H, m) 37,5 2,45 (d, 11,8) 1,33 (overlap) 15 219,3 - 219,3 - 16 78,4 - 82,9 - 17 61,4 3,45 (1H, dd, 6,5; 12,0) 3,54 (1H, dd, 4,5; 12,0) 66,7 6,02 (br s) 5,16 (br s) 18 69,9 2,85 (1H, dd, 5,0; 10,5) 3,18 (1H, dd, 5,0; 10,5) 71,0 3,64 (d, 10,5) 3,32 (d, 10,5) 13 19 17,4 0,65 (3H, s) 16,8 0,88 (s) 20 17,7 1,02 (3H, s) 16,1 1,44 (s) 7-OH 4,30 (1H, d, 5,0) 16-OH 4,78 (1H, s) 17-OH 4,43 (1H, dd, 4,5; 6,5) 18-OH 4,49 (1H, dd, 5,0; 10,0) #δH và #δC của scopariusol L (1H: 500 MHz, 13C: 125 MHz, pyridine-d5) [102] 4.2.2.1. Callimacrophylla B compound (M8) - New compound Compound 2 was obtained as a white crystal and its molecular formula was deduced as C32H50O4 by HR-ESI mass spectrum (found m/z 499.3786 [M þ H]þ, calcd. 499.3786 for C32H50O4 1H-NMR spectrum of compound M8 appeared 6 groups of methyl singlet at H 0.83 (3H, s, H3-28); 1.17 (3H, s, H3-26); 1,18 (3H, s, H3-25); 1,34 (3H, s, H3-27); 0.89 (6H, s, H3-23 and H3-24), 2 methyl groups as doublet at 0,H 0.80 (3H, J = 6.5 Hz, H3-29) and 0.93 (3H, J = 6.5 Hz, H3-30) and 1 methyl group at H 2.05 (3H, s, CH3CO) of acetoxy group. The 1H-NMR spectrum of M8 also showed the presence of 6 methane groups [H 4,53 (1H, dd, J = 11.5; 4,5, H-3); 0.82 (1H, m, H-5); 2.50 (1H, s, H-9), 2.43 (1H, dd, J = 11.5; 1,5, H- 18); 1.42 (1H, m, H-19) and 1.08 (1H, m, H-20)] and 8 methine groups have a chemical shift between 5H 0.95-2.75 (H- 1, H-2, H-6, H-7, H-15, H-16, H-21 and H-22), a proton singlet at H 6.27 (1H, s, 12-OH) ( Table 4.7) Hình 4.17. Phổ HR-ESI-MS của M8 Hình 4.18. Phổ 1H-NMR của M8 13C-NMR spectra combined with DEPT spectroscopy showed that compound M8 has 32 carbon atoms, including 8 quaternary carbon [C 38,0 (C-4); 45.5 (C-8); 37.0 (C-10); 195.2 (C-11); 144.5 (C-12); 134.4 (C-13); 41.7 14 (C-14) and 33.4 (C-17)], 6 methine groups [C 80.5 (C-3); 55.0 (C-5); 59.7 (C- 9); 48.9 (C-18); 39.3 (C-19) and 40.8 (C-20)], 8 methylene groups [C 38.9 (C-1); 23.5 (C-2); 17.4 (C-6); 32.9 (C-7); 27.3 (C-15); 27.5 (C-16); 31.2 (C- 21) and 41.2 (C-22)] and 8 methyl groups [C 28.0 (C-23); 16.6 (C-24); 16.7 (C-25); 18.6 (C-26); 21.0 (C-27); 28.8 (C-28); 16.6 (C-29) and 20.9 (C-30)]. In addition, there are signals of 01 acetoxy group at 170C 170.9 (CH3CO) and 21.3 (CH3CO) also obtained from 13C-NMR spectrum. Hình 4.19. Phổ 13C-NMR của M8 All of the above data suggests that compound M8 is a ursane triterpene containing an acetoxy group and has a structure similar to 3β-acetoxy-urs-12-ene- 11-one [94], except for differences. on the chemical displacement of carbon atoms at C-11, C-12 and C-13. When comparing NMR data of M8 and 3β-acetoxy-urs-12- ene-11-one compounds (TLTK), we can see in TLTK of proton signal of olefin group at H 5,54 (1H, s) directly linked to C-12 is a quaternary carbon (not linked to hydrogen) with a chemical shift C of 144.5 ppm (C-12). Also at the C-12 position associated with the hydroxy group is also shown by high resolution mass spectrometry. In addition, it was also determined by the proton nuclear interaction of protons at H 6.27 (1H, s, 12-OH) with carbon atoms at C 195.2 (C-11); 144.5 (C- 12) and 134.4 (C-13). In addition, links on the HMBC spectrum between H-18 (2,4H 2.43) and C-12 (144.5) / C-13 (134C 134.4) indicate the position of the double bond at C-12 / C-13 and ketones group at C-11 (Figure 4.16) 15 Hình 4.20. HSQC của M8 Hình 4.21. Phổ HMBC của M8 Besides, the link between methyl protons at H-23 / H-24 (H 0.89) and C- 3 (C 80.5) / C-4 (C 38.0) and C- 5 (C 55,0) as well as the link between H-3 (H 4,53) / CH3CO (H 2,05) with carbon atoms at C 170,9 (CH3CO), combined with constant the large coupling number of H-3 (J = 11.5 Hz) on the 1H-NMR spectrum confirms that the acetoxy group bound at C-3 has direction có. In addition, the H-3 proton has a α direction determined by the bonds from H-2α (H 1.65) and H3-23 (H 0.89) to H- 3 (H 4.53). as well as from H3-25 (H 1.18) to H-2β (H 1.72) on the ROESY spectrum. On the other hand, links between H-20 and H-29 / H- 19 and between H-2 protons and H-1 / H-3 were also found on the 1H-1H COZ spectrum. 16 Figure 4.22 Spectrum 1H-1H Cozy and NOESY of M8 Combine spectral data with HMBC, 1H-1H COZY and ROESY and compare with spectral data of 3β-acetoxy-urs-12-ene-11-one compounds [94] in the reference document that allows confirmation Compound M8 is 3β- acetoxy-urs-12-ene-11-one-12-ol. This is a new compound and is named callimacrophylla B Figure 4.16. Chemical structure, the main interaction HMBC (HC) of M8 Table 4.7. Spectrum data 1H- and 13C-NMR of M8 and reference substance Vị trí M8 [94] δC δH (mult., J Hz) #δC #δH (mult., J Hz) 1 38,9 2,75 (1H, dt, 6,5; 3,5) 1,13 (1H, m) 38,9 2,75(lH,ddd, 3,5;3,5;13,5) 2 23,5 1,65 (1H, m) / 1,72 (1H, m) 23,6 3 80,5 4,53 (1H, dd, 11,5; 4,5) 80,7 4,51 (lH, dd, 4,6; 11,7) 4 38,0 - 38,1 5 55,0 0,82 (1H, m) 55,1 6 17,4 1,43 (1H, m)/ 1,58 (1H, m) 17,5 7 32,9 1,43 (1H, m)/ 1,68 (1H, m) 32,8 8 45,5 - 45,2 9 59,7 2,50 (1H, s) 61,5 2,34 (1H, s) 10 37,0 - 36,7 17 11 195,2 - 199,5 12 144,5 - 130,5 5,54 (1H, s) 13 134,4 - 164,8 14 41,7 - 43,7 15 27,3 0,95 (1H, m)/ 2,10 (1H, m) 27,2 16 27,5 1,17 (1H, m)/ 1,90 (1H, m) 27,3 17 33,4 - 33,9 18 48,9 2,43 (1H, dd, 11,5; 1,5) 59,1 19 39,3 1,42 (1H, m) 39,2 20 40,8 1,08 (1H, m) 39,3 21 31,2 1,25 (1H, m)/ 1,43 (1H, m) 30,9 22 41,2 1,39 (1H, m)/ 1,47 (1H, m) 40,9 23 28,0 0,89 (3H, s) 28,1 0,87 (3H, s) 24 16,6 0,89 (3H, s) 16,7 0,88 (3H, s) 25 16,7 1,18 (3H, s) 16,5 1,16 (3H, s) 26 18,6 1,17 (3H, s) 18,6 1,18 (3H, s) 27 21,0 1,34 (3H, s) 20,5 1,29 (3H, s) 28 28,8 0,83 (3H, s) 28,9 0,81 (3H, s) 29 16,6 0,80 (3H, d, 6,5) 17,5 0,80 (3H,d, 6,0) 30 20,9 0,93 (3H, d, 6,5) 21,2 0,94 (3H, d, 6,0) CH3CO 170,9 - 170,9 - CH3CO 21,3 2,05 (3H, s) 21,1 2,04 (3H, s) 12-OH 6,27 (1H, s) #δH và #δC của 3β-acetoxy-urs-12-ene-11-one (1H: 500 MHz,13C:125MHz,CDCl3) [94] 4.3. Evaluate the biological activity of Nang nang and Chau Tu large leaves 4.3.1. Evaluate the biological activity of the essential oil of Nang nang leaves and Tu Chau leaves The results showed that dried her leaf essential oil showed weak activity on Hep-G2 liver cancer cell line (IC50 = 94.5 µg / mL). Fresh leaf oil extracted by conventional steam-distillation distillation method and Zenzi leaves were not shown to be active on test cancer cell lines Table 4.20. Results of in vitro cytotoxic activity of Lady and Tu Chau leaf oil on large cell lines: liver (Hep-G2), prostate cancer (PC3) and lung cancer ( A549) Form name IC50 (µg/mL) Hep-G2 PC3 A549 Dry leaf essential oil 94,53 >100 >100 Ordinary fresh leaves essential oil >100 >100 >100 Fresh leaves essential oil uses a microwave 14,65 23,87 56,21 18 Essential oil of fresh Tuzhou leaves >100 >100 >100 Control (paclitaxel) 4,03 3,48 3,69 The results also indicate that the microwave-assisted distillation method has obtained the constituents or mixture of constituents in the leaves essential oil. ordinary water. That has important implications for the next research direction of the essential oils of Her in particular and essential oils from other plants in general. Table 4.21. Test results of the antimicrobial activity of the essential oil of Nang nang leaves and Tu Chau leaves Tên mẫu MIC (g/mL) EC PA BS SA AN FO SC CA Dry leaf essential oil >200 >200 >200 >200 >200 >200 >200 >200 Ordinary fresh leaves essential oil >200 >200 >200 >200 >200 200 >200 200 Fresh leaves essential oil uses a microwave >200 >200 >200 >200 >200 100 >200 200 Zingzhou essential oil >200 >200 >200 >200 >200 >200 >200 >200 EC: Escherichia coli, PA: Pseudomonas aeruginosa, BS: Bacillus subtillis, SA: Staphylococcus aureus, AN: Aspergillus niger, FO: Fusarium oxysporum, SC: Saccharomyces cerevisiae, CA: Candida albicans 4.3.2 Evaluation of in vitro cytotoxic activity of magnetic extract from species tu chau la to and Nang nang The data from Table 4.22 shows that the ethanol residue from leaves, fruits, stems of large leaves of Chau Chau and She exhibited good inhibitory activity on 3 cancer cell lines tested with CS values (%) from 29.69  0.8 to 89.62  1.2%. In particular, the ethanol residue of the leaves of Big Chau and Tu Chau (L.CM and L.CC) has a good inhibitory effect with CS values (%) of 47.84  2.1 and 56, respectively. 28  2,6 (for Hep-G2 cancer cell line); 39.40  2.2 and 29.69  0.8 (for Lu-1 cancer cell line) and 30.23  1.5 and 35.18  1.0 (for cancer cell line) letter MCF-7). Preliminary results of the cytotoxic activity of the three cancer cell lines Hep-G2, Lu-1 and MCF-7 showed that the leaves of the Chau Chau leaves and the Lady were more effective than the fruits. and the trunk of their branches. Therefore, the next chemical composition research focused on the leaves of the Big tree and the tree Table 4.22. In vitro cytotoxic activity total ethanol residues from leaves, berries and stems of Big tree leaves 19 TT KH mẫu Nồng độ đầu (g/mL) Giá trị CS (%) Dòng tế bào Hep-G2 Lu-1 MCF-7 DMSO - 100 100 100 Chứng (+) 5 1,34  0,8 2,66  0,9 1,21  0,71 1 Q.CC 20 89,29  1,1 33,04  1,4 40,43  2,79 2 T.CC 20 89,62  1,2 33,53  1,6 91,29  0,32 3 L.CC 20 56,28  2,6 29,69  0,8 35,18  1,0 4 Q.CM 20 60,02  2,3 47,31  1,5 38,86  2,26 5 T.CM 20 64,66  2,2 55,64  2,8 90,22  0,15 6 L.CM 20 47,84  2,1 39,40  2,2 30,23  1,5 Q: fruit, T: stems, L: leaves, CC: C. candicans, CM: C. macrophylla) Continue to test the activity of the n-hexane, EtOAc and methanol residues of big leaf and conifer tree leaves for cancer cell lines Hep-G2, Lu-1 and MCF- 7. With the exception of methanol fractional sludge, which hardly shows activity, both residual fractional residues of the leaves of these two species have different effects, in which the n-hexane segment (L. CM.H) of Big tree and EtOAc (L.CC.E) of Her leaves showed the strongest activity with low CS (%) and low CS (%) values from 12.49  1,4 - 30,17  0,1%. This result is explained by the presence of strong cytotoxic activity groups such as terpenoids and flavonoids in the segments of weak and moderate polarization Table 4.23. The cytotoxic activity of the residues extracted from the leaves of the Big tree and the leafy tree TT KH mẫu Nồng độ đầu (g/mL) Giá trị CS (%) Dòng tế bào Hep-G2 LU-1 MCF-7 DMSO - 100 100 100 Chứng (+) 5 1,34  0,8 2,66  0,9 1,21  0,71 1 L.CM.H 20 20,18  0,8 12,49  1,4 11,61  2,11 2 L.CM.E 20 98,03  0,9 18,20  1,3 40,43  2,79 3 L.CM.M 20 100 100 91,29  0,32 4 L.CC.H 20 98,84  0,9 73,04  1,5 38,86  2,26 5 L.CC.E 20 30,17  0,1 15,69  2,3 19,93  0,11 6 L.CC.M 20 100 100 90,22  0,15 (L: lá, H: n-hexane, E: ethyl acetate, M: methanol, CC: C. candicans, CM: C. macrophylla) 4.3.3. Evaluation of in vitro cytotoxic activity of clean compounds isolated from Big leaf and Tui Chau leaves 20 Table 4.24. In vitro cytotoxic activity test of substances on cancer cell lines (Hep-G2), lung (Lu-1) and breast (MCF-7) TT Hợp chất Giá trị IC50 (g/mL) Dòng tế bào HepG2 Lu1 MCF7 Ellipticine 0,29 0,51 0,48 1 methyl seco-hinokiol (C6) 8,65 8,53 2,20 2 seco-hinokiol (C5) 8,25 9,13 2,46 3 callimacrophylla A (M1) 2,72 2,68 1,57 4 ent-1β-acetoxy-7β,14α-dihydroxy-16-kauren-15-on (M7) 0,31 1,55 0,23 5 callimacrophylla B (M8) 0,32 1,87 0,28 6 ursolic acid (M6) 0,25 0,31 0,21 7 2α,3β,23-trihydroxyurs-12-en-28-oic acid (C8) - - - 8 β-amyrin (M3) - - - 9 3β-hydroxyolean-12-ene (M2) 5,85 - 2,36 10 spinasterol (M5) 8,22 8,29 1,82 11 5-hydroxy-7,4’-dimethoxyflavon (C1) - - - 12 5-hydroxy-3’,4’,7-trimethoxyflavon (C3) - - - 13 genkwanin (C9) - - - 14 cynaroside (C10) - - - Assessment of toxic activity of tested cancer cell lines: Table 4.24 shows that the anti-cancer activity of liver cancer (Hep-G2), lung (Lu-1) and breast (MCF-7) of terpenoid compounds is stronger than that of steroid and flavonoid compounds. The compounds showed a more cytotoxic activity against breast cancer cells (MCF-7) than the liver cancer cell lines (Hep-G2) and lungs (Lu-1). Anti-cell activity against liver cancer (Hep-G2), lung (Lu-1) and breast (MCF-7) among 14 active compounds showed 6 active inactive compounds with IC50> 100 ( g / mL) include: 5-hydroxy-7.4'- dimethoxyflavon (C1), 5-hydroxy-3 ', 4', 7-trimethoxyflavon (C3), 2α, 3β, 23- trihydroxyurs-12-en- 28-oic acid (C8), Genkwanin (C9) and cynaroside (C10) and β-amyrin (M3). The remaining compounds showing strong activity are methyl seco-hinokiol (C6), seco-hinokiol (C5), ent-1 α-acetoxy-7β, 14α- dihydroxy-16-kauren-15-on (M7) , callimacrophylla B (M8), ursolic acid (M6), spinasterol (M5), 3β-hydroxyolean-12-ene (M2), callimacrophylla A (M1) with 0.2 g / mL <IC50 <9.2 g / mL. Especially, ursolic acid (M6) has anti-cancer activity against liver cancer (Hep-G2), lung (Lu-1) and breast (MCF-7) very strong with IC50 value = 0.25; 0.31 and 0.21 g / mL are smaller than the Ellipticine reference concentration with IC50 = 2.9; 0.51 and 0.48 g / mL. Compounds ent-1α -acetoxy-7β, 14α-dihydroxy-16-kauren-15-on (M7) and callimacrophylla B (M8) exhibit strong MCF-7 breast cancer cell activity 21 with IC50 = 0.23 and 0.21 g / mL, stronger than the Ellipticine control with IC50 = 0.48 g / mL Two compounds abietane (methyl seco-hinokiol (C6), seco-hinokiol (C5)) and a steroid compound (spinasterol (M5) exhibit potent cytotoxicity against breast cancer cell lines (MCF-7) with IC50 values in the range of 1.82 g / mL <IC50 <2.46 /g / mL but exhibits toxic activity for liver cancer cell line (Hep- G2), lung (Lu-1) with a value of 8.22 g / mL <IC50 <9.13 g / mL, about 5 times worse than breast cancer (MCF-7). The cytotoxicity of breast cancer cells (MCF-7) is not significantly stronger than that of liver cancer cells (Hep-G2), lung (Lu-1), except for 3β-hydroxyolean-12- Ene (M2) does not show cytotoxic activity against lung cancer (Lu-1), cytotoxic activity against breast cancer (MCF-7) is better for cancer cell line. Hepatic letter (Hep-G2) with an IC50 value of 2.36 g / mL and 5.85 g / mL, respectively. For liver cancer (Hep-G2), lung (Lu-1) ent-kaurane diterpenoid (callimacrophylla A (M1) and ent-1β-acetoxy-7β, 14α-dihydroxy-16-kauren-15 frame compounds -on (M7)) and ursane-triterpenoid (callimacrophylla B (M8) and ursolic acid (M6) with 0.25 g / mL <IC50 <2.72 g / mL exhibiting stronger cytotoxic activity 8 times more than abietane frame compounds (methyl seco-hinokiol (C6), seco- hinokiol (C5)) and steroids (spinasterol (M5)) with 8.22 g / mL <IC50 <9.13 g For breast cancer (MCF-7), these compounds exhibited similar cytotoxic activity with a value of 0.21 g / mL <IC50 <2.46 mLg / mL. Except for ursane- triterpenoid: 2α, 3β, 23-trihydroxyurs-12-en-28-oic acid (C8) did not show activity The hydroxyl group at C-3 in the seco-hinokiol compound (C5) is replaced by the CH3O- group at the C-3 position in methyl seco-hinokiol (C6) of the two abietane-diterpenoid frame compounds leading to activity. cytotoxic activity for 25 g / mL and 8.65 g / mL cancers but less than lung (Lu-1) and breast (MCF-7) cell lines (IC50 value respectively 9.13 g / ml and 8.53 g / ml for the Lu-1 cancer line; 2.46 g / mL and 2.20 g / mL for the MCF-7 cancer line) however the difference is not significan The disappearance of hydroxy group at C-18 position; appear