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MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY …………***………… VU THI THU LE RESEARCH ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF CALLICARPA CANDICANS AND CALLICARPA MACROPHYLLA GROWING IN VIETNAM Major: Chemistry of natural compounds Code: 9.44.01.17 SUMMARY OF CHEMICAL DOCTORAL THESIS HA NOI - 2020 This thesis was completed at Graduate University of Science and Technology - Vietnam Academy of Science and Technology Supervisors: Assoc.Prof Dr Pham Thi Hong Minh Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology Prof Dr Pham Quoc Long Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology Examiner 1: Assoc.Prof Dr Phan Minh Giang University of Science – Vietnam National University Examiner 2: Dr Đỗ Hữu Nghị Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology The thesis defense was monitored by the Graduate University level Board of Examiners, held at: Graduate University of Science and Technology - 18 Hoang Quoc Viet - Cau Giay - Ha Noi At ……… , ….………………… 2020 The thesis is available in Vietnam National Library and Library of Graduate University of Science and Technology PREAMBLE The urgency of the thesis Vietnam is located in a tropical climate region with extremely rich natural resources and biodiversity with many valuable medicinal herbs The increasing use of traditional medicinal herbs or from naturally occurring compounds has taken an important position in medicine Herbal remedies are the subject for scientists to fully study the nature of the active ingredients in natural plants, the results of the study will contribute to a better explanation of the effects Treatment of traditional medicinal plants is still often used in folk In the world, studies of the genus Callicarpa mainly focus on: botanical, pharmacology, plant chemistry and clinical Pharmacological studies conducted on crude extracts or pure compounds provide the scientific basis for traditional use Biological activity studies mainly focused on activities: antiinflammatory, hemostasis, memory loss, oxidation, antibacterial Studies of chemical composition and biological activity in the world show that diterpenoid and triterpenoid compounds have very good anticancer activity, which is the most abundant chemical component in Callicarpa genus The research to find out the active ingredients of plants has been receiving the attention of scientists from many countries around the world, including Vietnam This is also the reason for the topic "Studying the chemical composition and biological activity of the Sheic species (Callicarpa candicans) and the large-leafed Tuzhou (Callicarpa macrophylla) in Vietnam." be selected for research The research objectives of the thesis Isolate and determine the chemical structure of compounds from C candicans and C macrophylla species collected in Vietnam Assessment of toxic activity on some cancer cell lines: liver (Hep-G2), lung (Lu-1) and breast (MCF-7) of isolated clean compounds Researching the chemical composition of C candicans and C macrophylla essential oils The main research content of the thesis 1.Determining the chemical structure of compounds isolated from leaves of C candicans and C macrophylla in Vietnam 2.Determining the essential chemical composition of leaves of C candicans and C macrophylla in Vietnam by conventional steam entraining methods and microwave-assisted distillation methods 3.Evaluation of toxic activity on three cancer cell lines (liver - HepG2, prostate - PC3, lung - A549) and in vitro antimicrobial testing of essential oils obtained from C candicans leaves and C macrophylla Evaluation of toxic activity on three strains of liver cancer (Hep-G2), lung (Lu-1) and breast (MCF-7) in vitro of clean compounds isolated from leaves of C candicans and C macrophylla Chapter OVERVIEW The literature review is a collection of national and international research on: 1.1 General characteristics of botany Callicarpa 1.2 Pharmacological effects of the genus Callicarpa 1.3 Chemical composition of plants genus Callicarpa 1.4 Biological activity of genus Callicarpa plants Chapter SUBJECTS AND METHODS OF THE STUDY This section describes in detail the sample handling processes, the methods of generating sediments, chromatography and the isolation of compounds; determine the chemical composition of essential oils and methods of bioactive test 2.1 Materials and research methods 2.1.1 Plant samples Leaves, stems, branches and fruits (Callicarpa candicans (Burm.f.) Hochr.) And Tu Chau big leaves (Callicarpa macrophylla Vahl) collected in Tam Dao district - Vinh Phuc in October 2015 and Dai Tu district, province Thai Nguyen in October 2017, Dr Nguyen Quoc Binh (Vietnam Museum of Nature - VAST) identifies the scientific name and denominator model stored at the Vietnam Museum of Nature 2.1.2 Method of isolation of compounds from tree samples The analysis and separation of plant extracts are made by different chromatographic methods such as thin layer chromatography (TLC), normal column chromatography (CC) with static phase is silica gel (Merck), Inverted phase chromatography with static phase is YMC RP 18 (Merck), molecular wire chromatography with static phase is sephadex LH-20 (Merck) and stationary phase chromatography is silica gel 2.1.3 Methods of determining chemical structure The structure of the compounds is determined by the combination of physical parameters with modern spectroscopic methods such as melting point (Mp), polarity ([α] D), mass gas chromatography spectroscopy (GC-MS), electronic atomization mass spectrometry (ESI-MS), high resolution mass spectrometry (HR-ESI-MS), nuclear magnetic resonance spectroscopy including 1-dimensional spectrum (1H-, 13C -NMR and DEPT) 2-dimensional spectrum (COZY, HSQC, HMBC and NOESY) 2.2 Method of extracting essential oil 2.2.1 The steam-distillation distillation method uses a micro-essential oil distillator 2.2.2 The method of steam distillation involves the use of microwaves 2.2.3 Method of analyzing essential oil chemical composition Determine the chemical composition of essential oil by combining GC – MS system with standard library and Mass Finder 4.0 retention time locking software 2.3 Biological activity test method The cytotoxic activity was tested by SRB method on three cancers: liver (Hep-G2), lung (LU-1) and breast (MCF-7) and MTT method [3- [4,5dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide] on three lines of liver cancer (Hep-G2), prostate (PC3) and lung (A549) The method of assessing antimicrobial activity was conducted to evaluate the antibiotic activity of extracted samples by methods of Vander Bergher and Vlietlinck (1991), and McKane & Kandel (1996) on strains: Vi Gram, mycelium and yeast The tests were conducted at the Department of Biologically active, Institute of Natural Products Chemistry, VAST Chapter EXPERIMENTAL The empirical section described in detail the processes: Sample processing and isolation of clean substances from the leaves of two species of Large-leafed Tuzhou (C macrophylla) and Nang nang (C candicans); Physical constants and spectral data of compounds isolated from studied species 3.1 Big tree leaves of Zizhu (C macrophylla) 3.1.1 Acquire extracts from the leaves of the Ziocuo tree The process of processing the leaf sample of Tu Chau tree is large Diagram 3.1 Extraction diagram of Tu Chau leaves (C macrophylla) 3.1.2 Isolation and purification of substances from the leaves of the Tu Chau leaves The process of isolating compounds from n-hexane and ethyl acetate residues of the leaves of Chau Chau tree is as big as Figure 3.2 Diagram 3.2 Isolation of n-hexane and ethyl acetate residues of broadleaf wattle tree 3.2 Her candlelight leaves (C candicans) 3.2.1 Obtain extracts from the leaves of Nang nàng The process of treating her leaves is as Figure 3.3 Diagram 3.3 Diagram of extracting leaves of Nang (C candicans) leaves 3.2.2 Isolate and refine substances from the extract of Nang leaves The process of isolation of compounds from n-hexane residues and ethyl acetate of Her leaves as Figure 3.4 Diagram 3.4 Diagram of isolation of n-hexane and ethyl acetate residues from Her leaves Chapter RESULTS AND DISCUSSION Chapter presents how to determine the structure of the isolated compounds, the chemical composition of essential oils, and the results of the bioactive test of the components 4.1 Chemical composition of the essential oil of Nang nang and Tu Chau leaves GC - MS analysis in the composition of dried leaves She has 39 components, accounting for 92.57% of the total content 25 sesquiterpene hydrocarbons (62.98%) and 11 sesquiterpenes contain oxygen (22.46%) The main component identified in essential oil is α-Gurjunene (21.97%) In the essential oil of fresh leaves, there are 47 components discovered, accounting for 93.17% of the total content of essential oils Among them, there are 28 sesquiterpene hydrocarbons (69.84%), 12 sesquiterpen (16.50%) In particular, the largest constituent content is α-gurjunene (21.31%) The chemical composition of essential oil from the Nang Nang flower said there are 47 components, accounting for 92.86% of the total content The high content constituents are determined in the essential oil of 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 terpenoid compounds: callimacrophylla B (M8), 3βhydroxyolean-12-ene (M2), β-amyrin (M3), ursolic acid (M6), oleanolic acid (M10), callimacrophylla A (M1) and ent-1β-acetoxy-7β, 14α-dihydroxy-16kauren-15-on (M7) ) and 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 flavonoid compounds: 5-hydroxy-7.4'-dimethoxyflavon (C1), 5-hydroxy-3 ', 4', 7-trimethoxyflavon (C3), genkwanin compound (C9) and cynaroside (C10); terpenoid compounds: ursolic acid (M6), 2α-hydroxyursolic acid (C7), 2α, 3β, 23-trihydroxyurs-12-en-28-oic acid (C8), secohinokiol (C5) and methyl seco -hinokiol (C6) and steroid compounds: βsitosterol (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 Triterpenoid compounds isolated from 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 studied species Tên hợp chất callimacrophylla A (M1) ent-1α-acetoxy-7β,14 α-dihydroxy-kaur16-en-15-on (M7) seco-hinokiol (C5) methyl seco-hinokiol (C6) 3β-hydroxyolean-12-ene (M2) β-amyrin (M3) ursolic acid (M6) Diterpneoid C macrophylla KL Tính (mg) 10,8 M Diterpenoid C macrophylla 12,5 H Diterpenoid Diterpenoid Triterpenoid Triterpenoid 22,8 31,0 10,3 12,7 15,5 15,7 H H L L L L Lớp chất Loài phân lập C candicans C candicans C macrophylla C macrophylla C macrophylla Triterpenoid C candicans 12 65.3) (Braca et al 2004) suggesting the b-configuration of 16-OH group From the above evidence, compound was determined to be ent-7a,16b,17,18tetrahydroxykaur-15-one, a new compound and named as callimacrophylla A Figure 4.6 Chemical structure, the main interaction HMBCHC) of M1 Table 4.3 Spectrum data of 1H- and 13C-NMR of M1 and reference substance TT δC 38,6 Hợp chất M1 δH (mult., J Hz) 0,57 (1H, m)/ 1,66 (1H, m) 17,3 1,38 (1H, m)/ 1,57 (1H, m) #δC 41,7 20,3 34,7 1,11 (1H, m)/ 1,40 (1H, m) 39,2 36,9 44,9 - 26,9 1,75 (1H, m)/ 1,66 (1H, m) 44,2 48,1 30,5 69,5 3,62 (1H, dd, 10,5; 5,5) 78,1 10 58,4 52,6 38,5 0,94 (1H, d, 9,0) 1,20 (1H, m)/ 1,53 (1H, m) 11 17,9 1,20 (1H, m)/ 1,53 (1H, m) 49,0 51,1 40,4 19,1 12 28,0 1,18 (1H, m)/ 1,60 (1H, m) 27,6 13 38,7 2,21 (1H, br d, 3,5) 14 25,3 1,66 (1H, m)/ 2,38 (1H, m) 46,1 37,5 15 16 219,3 78,4 17 61,4 18 69,9 3,45 (1H, dd, 6,5; 12,0) 3,54 (1H, dd, 4,5; 12,0) 2,85 (1H, dd, 5,0; 10,5) 3,18 (1H, dd, 5,0; 10,5) 219,3 82,9 66,7 71,0 [103] #δH (mult., J Hz) 3,57 (m) 2,03 (m) 1,85 (overlap) 2,01 (m) 1,42 (m) 1,63 (dd, 11,9, 1,7) 1,75 (m) 1,40 (m) 2,28 (dt, 13,3, 4,1) 1,33 (overlap) 1,85 (overlap) 3,66 (dd, 10,7, 6,0) 1,68 (m) 1,98 (m) 1,57 (m) 2,95 (br s) 2,45 (d, 11,8) 1,33 (overlap) 6,02 (br s) 5,16 (br s) 3,64 (d, 10,5) 3,32 (d, 10,5) 13 0,88 (s) 19 17,4 0,65 (3H, s) 16,8 1,44 (s) 20 17,7 1,02 (3H, s) 16,1 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 #δC scopariusol L (1H: 500 MHz, 13C: 125 MHz, pyridine-d5) [102] 4.2.2.1 Callimacrophylla B compound (M8) - New compound Compound 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 ỵ H]ỵ, calcd 499.3786 for C32H50O4 H-NMR spectrum of compound M8 appeared 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), 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 methyl group at H 2.05 (3H, s, CH3CO) of acetoxy group The 1H-NMR spectrum of M8 also showed the presence of 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, H18); 1.42 (1H, m, H-19) and 1.08 (1H, m, H-20)] and 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 M8 Hình 4.18 Phổ 1H-NMR M8 13 C-NMR spectra combined with DEPT spectroscopy showed that compound M8 has 32 carbon atoms, including 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)], methine groups [C 80.5 (C-3); 55.0 (C-5); 59.7 (C9); 48.9 (C-18); 39.3 (C-19) and 40.8 (C-20)], 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 (C21) and 41.2 (C-22)] and 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 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-ene11-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-12ene-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 (C12) 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 M8 Hình 4.21 Phổ HMBC M8 Besides, the link between methyl protons at H-23 / H-24 (H 0.89) and C3 (C 80.5) / C-4 (C 38.0) and C- (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 H3 (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 / H19 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 3βacetoxy-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 (HC) of M8 Table 4.7 Spectrum data 1H- and 13C-NMR of M8 and reference substance Vị trí δC 38,9 10 23,5 80,5 38,0 55,0 17,4 32,9 45,5 59,7 37,0 M8 δH (mult., J Hz) 2,75 (1H, dt, 6,5; 3,5) 1,13 (1H, m) 1,65 (1H, m) / 1,72 (1H, m) 4,53 (1H, dd, 11,5; 4,5) 0,82 (1H, m) 1,43 (1H, m)/ 1,58 (1H, m) 1,43 (1H, m)/ 1,68 (1H, m) 2,50 (1H, s) - [94] #δH (mult., J Hz) #δC 38,9 2,75(lH,ddd, 3,5;3,5;13,5) 23,6 80,7 38,1 55,1 17,5 32,8 45,2 61,5 36,7 4,51 (lH, dd, 4,6; 11,7) 2,34 (1H, s) 17 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 CH3CO CH3CO 12-OH 195,2 144,5 134,4 41,7 27,3 27,5 33,4 48,9 39,3 40,8 31,2 41,2 28,0 16,6 16,7 18,6 21,0 28,8 16,6 20,9 170,9 21,3 0,95 (1H, m)/ 2,10 (1H, m) 1,17 (1H, m)/ 1,90 (1H, m) 2,43 (1H, dd, 11,5; 1,5) 1,42 (1H, m) 1,08 (1H, m) 1,25 (1H, m)/ 1,43 (1H, m) 1,39 (1H, m)/ 1,47 (1H, m) 0,89 (3H, s) 0,89 (3H, s) 1,18 (3H, s) 1,17 (3H, s) 1,34 (3H, s) 0,83 (3H, s) 0,80 (3H, d, 6,5) 0,93 (3H, d, 6,5) 2,05 (3H, s) 6,27 (1H, s) 199,5 130,5 164,8 43,7 27,2 27,3 33,9 59,1 39,2 39,3 30,9 40,9 28,1 16,7 16,5 18,6 20,5 28,9 17,5 21,2 170,9 21,1 5,54 (1H, s) 0,87 (3H, s) 0,88 (3H, s) 1,16 (3H, s) 1,18 (3H, s) 1,29 (3H, s) 0,81 (3H, s) 0,80 (3H,d, 6,0) 0,94 (3H, d, 6,0) 2,04 (3H, s) #δH #δC 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 Dry leaf essential oil Ordinary fresh leaves essential oil Fresh leaves essential oil uses a microwave IC50 (µg/mL) PC3 >100 A549 >100 >100 >100 >100 14,65 23,87 56,21 Hep-G2 94,53 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 MIC (g/mL) Tên mẫu EC PA BS SA AN FO SC CA Dry leaf essential >200 >200 >200 >200 >200 >200 >200 >200 oil Ordinary fresh >200 >200 >200 >200 >200 200 >200 200 leaves essential oil Fresh leaves essential oil uses a >200 >200 >200 >200 >200 100 >200 200 microwave Zingzhou essential >200 >200 >200 >200 >200 >200 >200 >200 oil 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 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 DMSO Chứng (+) Q.CC T.CC L.CC Q.CM T.CM L.CM Nồng độ đầu (g/mL) 20 20 20 20 20 20 Hep-G2 100 1,34 0,8 89,29 1,1 89,62 1,2 56,28 2,6 60,02 2,3 64,66 2,2 47,84 2,1 Giá trị CS (%) Dòng tế bào Lu-1 100 2,66 0,9 33,04 1,4 33,53 1,6 29,69 0,8 47,31 1,5 55,64 2,8 39,40 2,2 MCF-7 100 1,21 0,71 40,43 2,79 91,29 0,32 35,18 1,0 38,86 2,26 90,22 0,15 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 MCF7 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 (L: lá, Giá trị CS (%) Dòng tế bào Hep-G2 LU-1 MCF-7 DMSO 100 100 100 Chứng (+) 1,34 0,8 2,66 0,9 1,21 0,71 20 L.CM.H 20,18 0,8 12,49 1,4 11,61 2,11 20 L.CM.E 98,03 0,9 18,20 1,3 40,43 2,79 20 100 100 L.CM.M 91,29 0,32 20 L.CC.H 98,84 0,9 73,04 1,5 38,86 2,26 20 L.CC.E 30,17 0,1 15,69 2,3 19,93 0,11 20 100 100 L.CC.M 90,22 0,15 H: n-hexane, E: ethyl acetate, M: methanol, CC: C candicans, CM: C macrophylla) KH mẫu Nồng độ đầu (g/mL) 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 10 11 12 13 14 Hợp chất Ellipticine methyl seco-hinokiol (C6) seco-hinokiol (C5) callimacrophylla A (M1) ent-1β-acetoxy-7β,14α-dihydroxy-16-kauren-15-on (M7) callimacrophylla B (M8) ursolic acid (M6) 2α,3β,23-trihydroxyurs-12-en-28-oic acid (C8) β-amyrin (M3) 3β-hydroxyolean-12-ene (M2) spinasterol (M5) 5-hydroxy-7,4’-dimethoxyflavon (C1) 5-hydroxy-3’,4’,7-trimethoxyflavon (C3) genkwanin (C9) cynaroside (C10) Giá trị IC50 (g/mL) Dòng tế bào HepG2 Lu1 MCF7 0,29 0,51 0,48 8,65 8,53 2,20 8,25 9,13 2,46 2,72 2,68 1,57 0,31 1,55 0,23 0,32 1,87 0,28 0,25 0,31 0,21 5,85 2,36 8,22 8,29 1,82 - 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 active inactive compounds with IC50> 100 ( g / mL) include: 5-hydroxy-7.4'dimethoxyflavon (C1), 5-hydroxy-3 ', 4', 7-trimethoxyflavon (C3), 2α, 3β, 23trihydroxyurs-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, 14αdihydroxy-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
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