Summary of Chemical doctoral thesis: Studying the chemical composition and biological activity of the Sheic species (Callicarpa candicans) and the large-leafed Tuzhou (Callicarpa macrophylla) in Vietnam

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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.

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Nội dung Text: Summary of Chemical doctoral thesis: Studying the chemical composition and biological activity of the Sheic species (Callicarpa candicans) and the large-leafed Tuzhou (Callicarpa macrophylla) in Vietnam

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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: 1. Assoc.Prof. Dr. Pham Thi Hong Minh Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology 2. 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.
1 PREAMBLE 1. 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: anti- inflammatory, 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. . 2. 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. 3. 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 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. 4. 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 1. 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 2. 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
3 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- [4,5- dimethylthiazol-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 8 strains: Vi Gram, mycelium and yeast. The tests were conducted at the Department of Biologically active, Institute of Natural Products Chemistry, VAST Chapter 3. 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 2 studied species.
4 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
5 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 4. RESULTS AND DISCUSSION Chapter 4 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
6 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 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 KL Tính Tên hợp chất Lớp chất Loài phân lập (mg) mới callimacrophylla A (M1) Diterpneoid C. macrophylla 10,8 M ent-1α-acetoxy-7β,14 α-dihydroxy-kaur- Diterpenoid C. macrophylla 12,5 H 16-en-15-on (M7) 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 C. macrophylla 15,5 L ursolic acid (M6) Triterpenoid C. candicans 15,7 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 Triterpenoid C. candicans 11,5 L acid (C8) oleanolic acid (M10) Triterpenoid C. macrophylla 8,5 spinasterol (M5) Steroid C. macrophylla 11,2 L Steroid C. macrophylla 20,0 β–sitosterol (M4) C. candicans 19,0 C. macrophylla daucosterol (M9) Steroid 16,5 C. candicans 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 1 H-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) 20,3 2,03 (m) 2 17,3 1,38 (1H, m)/ 1,57 (1H, m) 1,85 (overlap) 39,2 2,01 (m) 3 34,7 1,11 (1H, m)/ 1,40 (1H, m) 1,42 (m) 4 36,9 - 44,2 5 44,9 - 48,1 1,63 (dd, 11,9, 1,7) 30,5 1,75 (m) 6 26,9 1,75 (1H, m)/ 1,66 (1H, m) 1,40 (m) 78,1 2,28 (dt, 13,3, 4,1) 7 69,5 3,62 (1H, dd, 10,5; 5,5) 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 - 19,1 3,66 (dd, 10,7, 6,0) 11 17,9 1,20 (1H, m)/ 1,53 (1H, m) 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 - 3,45 (1H, dd, 6,5; 12,0) 66,7 6,02 (br s) 17 61,4 5,16 (br s) 3,54 (1H, dd, 4,5; 12,0) 2,85 (1H, dd, 5,0; 10,5) 71,0 3,64 (d, 10,5) 18 69,9 3,32 (d, 10,5) 3,18 (1H, dd, 5,0; 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 1 H-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 13 C-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 M8 [94] Vị trí δC δH (mult., J Hz) #δC #δH (mult., J Hz) 2,75 (1H, dt, 6,5; 3,5) 38,9 2,75(lH,ddd, 3,5;3,5;13,5) 1 38,9 1,13 (1H, m) 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) IC50 (µg/mL) Form name 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 14,65 23,87 56,21 microwave