Summary of Chemical doctoral thesis: Study on chemical constituents and biological activities of two species Markhamia stipulata var. canaenses V.S. Dang and Stereospermum binhchaunesis V.S. Dang of Bignoniaceae

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The objectives of the thesis: Study on chemical constituents of two species Markhamia stipulata var. canaense V.S. Dang and Stereospermum binhchaunesis V.S. Dang; evaluation of biological activities of isolated compounds to find potential compounds.

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Nội dung Text: Summary of Chemical doctoral thesis: Study on chemical constituents and biological activities of two species Markhamia stipulata var. canaenses V.S. Dang and Stereospermum binhchaunesis V.S. Dang of Bignoniaceae

1 MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ----------------------------- NG NGO TRONG NGHIA O STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF TWO SPECIES MARKHAMIA STIPULATA VAR. CANAENSE V.S. DANG AND STEREOSPERMUM BINHCHAUENSIS V.S. DANG OF BIGNONIACEAE Major: Chemistry of natural compounds Code: 9440117 SUMMARY OF CHEMICAL DOCTORAL THESIS HO CHI MINH, 2020
This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Adviser 1: Assoc. Prof. Dr. Mai Dinh Tri Adviser 2: Assoc. Prof. Dr. Tran Cong Luan 1st Reviewer: 2nd Reviewer: 3rd Reviewer: The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology At ……hour…….day……. month …….2020 Thesis can be found in - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology. - The National library of Viet Nam
1 INTRODUCTION 1. The urgency of the thesis Vietnam is a country located in the tropical monsoon region with an extremely rich and diverse plant source, including many valuable medicinal plants that our ancestors have used for a long time to treat a variety of diseases that show the importance of medicinal plant resources. In recent years, according to the socio-economic development, the demand for health care has been constantly increasing, scientists are going on looking for active substances with curative properties of natural origin. Many natural compounds have been isolated and widely applied. They are used to produce medicines, plant protection drugs, as raw materials for food, pharmaceutical and cosmetic industries by safety, few side effects, environmentally friendly. Therefore, the search and study of the chemical composition and biological activity of medicinal plants in order to detect attractive active compounds from herbs is a very necessary issue. The Markhamia and the Stereospermum genus of Bignoniaceae famila are distributed in tropical and subtropical regions of Africa, China and Southeast Asian countries such as Vietnam, Laos, Cambodia, and Thailand. The research results of some species in these 2 genus have discovered many interesting biological properties such as hypoglycemia, antibacterial, anti-inflammatory, antioxidant, and analgesic ... Markhamia stipulata var. canaense V.S. Dang and Stereospermum binhchauensis V. S. Dang are two newly discovered species in 2015. Up to now, there have not been any studies on the chemical composition and biological activity of the two species. So based on that science, we chose the topic "Study on chemical constituents and biological activities of two species Markhamia stipulata var. canaenses V.S. Dang and Stereospermum binhchaunesis V.S. Dang of Bignoniaceae” contribute to the scientific basis for the study and application of these two species in the future.
2 2. The objectives of the thesis Study on chemical constituents of two species Markhamia stipulata var. canaense V.S. Dang and Stereospermum binhchaunesis V.S. Dang. Evaluation of biological activities of isolated compounds to find potential compounds. 3. The main contents of the thesis 1. Isolation of compounds from the leaves of Markhamia stipulata var. canaense V.S. Dang and Stereospermum binhchaunesis V.S. Dang. 2. Determination of chemical structures of the isolated compounds. 3. Evaluation on the cytotoxic activity and -glucosidase inhibiting of the isolated compounds. CHƯƠNG 1: OVERVIEW The overview of internal and international researches related to our study. CHƯƠNG 2: METHODS 2.1. Plant materials The leaves of M. stipulata var. canaense V.S. Dang were collected in Ca Na, Thuan Nam District, Ninh Thuan Province, Vietnam, in June 2016, and identified by Dr. Van Son Dang, Institute of Tropical Biology. The leaves of S. binhchauensis V.S. Dang were collected in Binh Chau, Phuoc Buu District, Ba Ria Province, Vietnam, in June 2016, and identified by Dr. Van Son Dang, Institute of Tropical Biology. 2.2. Chemicals This section presents solvents and chemicals used in experiments 2.3. Isolation Methods This section presents methods for isolating pure compounds: thin-layer chromatography (TLC) and column chromatography
3 2.4. Methods for determination of chemical struture of compounds General methods to determine the chemical structure of the compound are a combination of physical parameters and modern spectroscopic methods including Mass spectrometry and high-resolution mass spectrometry (HR-ESI- MS), magnetic resonance spectrum (1D, 2D-NMR), ultraviolet-visible (UV), infrared (IR) and optical rotation ([]D). 2.5. Methods for evaluation of biological activities This section presents chemicals and equipment for the biological activity test method. CHƯƠNG 3: EXPERIMENTALS 3.1. Extraction This section presents the process of making methanol extracts and partitioned extract from M. stipulata var. canaense V.S. Dang and S. binhchauensis V.S. Dang. 3.2. Isolation This session presents in detail the isolated procedure of 28 compounds from M. stipulata var. canaense V.S. Dang and S. binhchauensis V.S. Dang as shown in schemes 4.1, 4.2, 4.3, 4.4.
4 Schemes 4.1. Isolation compounds from extract MSH
5 Schemes 4.2. Isolation compounds from extract MSE
6 Schemes 4.3. Isolation compounds from extract SBH
7 Schemes 4.4. Isolation compounds from extract SBE
8 3.3. Physical properties and spectroscopic data of the isolated compounds from M. stipulata var. canaense V.S. Dang This section presents physical properties and spectroscopic data of 17 compounds from M. stipulata var. canaense V.S. Dang 3.4. Physical properties and spectroscopic data of the isolated compounds from S. binhchauensis V.S. Dang This section presents physical properties and spectroscopic data of 11 compounds from S. binhchauensis V.S. Dang 3.5. Biological activities of isolated compounds This section presents methods for testing cytotoxic and α-glucosidase inhibiting activity. CHƯƠNG 4. RESULTS AND DISCUSSIONS 4.1. Isolated compounds From n-hexane extract (MSH) and EtOAc extract (MSE) of leave M. stipulata var. canaense V.S. Dang isolated 17 compounds. From n-hexane extract (SBH) and EtOAc extract (SBE) of leave S. binhchauensis V.S. Dang isolated 11 compounds. 4.2. Determination the chemical structure of isolated compounds This section presents results of spectral analysis and determines the structure of 28 compounds isolated from Markhamia stipulata V.S. Dang and Stereospermum binhchaunesis V.S. Dang species. MS01 : Mollic acid MS02: Markhacanasin A
9 MS03: Markhacanasin B MS04: Markhacanasin C MS05: Epi- Markhacanasin C MS06: Acid Oleanoic MS07: Acid Ursolic MS08: Acid 6β,19α-dihydroxyursolic
10 MS09: Apigenin MS10: Luteolin MS11: 4’,7-dimethylapigenin MS12: Naringenin MS13: Apigenin 7-O-β-D- glucopyranoside MS14: Luteolin 7-O-β-D- glucopyranoside MS15: Tectoquinone MS16: 1-hexadecanoyl-sn-glycerol
11 MS17: Markhasphingolipid A SB01: Chrysin 5,7-dimethyl ehter SB02: Kaempferol SB04: Luteolin 7-O-β-D- SB03: Luteolin glucopyranoside SB05: Naringenin 7-O-β-D- glucopyranoside SB06: Eriodictyol 7-O-β-D- glucopyranoside
12 SB07: Specioside SB08: Lupeol SB10: Bergapten SB09: Acid oleanoic SB11: ,7-dihyroxychromone Below details the method for determining the structure of the new compounds MS02. MS02 was obtained as a white amorphous powder. The molecular formula was established as C30H48O5 by HR-ESI-MS data ([M+NH4]+ m/z 506.3826, calcd. 506.3845).
13 13 The C-NMR and DEPT spectrum, showed MS02 has thirty carbons including: one carbonyl carbon, two olefinic carbons, three oxygenated methine carbons, five quaternary carbons, four methine carbons, nine methylene carbons, six methyl carbons. The presence of six methyl groups, one methyl carbon at δC 9.0 (C-29), two olefinic carbons at δC 131.7 (C-25) and 126.2 (C-24) and one carbonyl carbon 180.5 (C-28) which indicated a cycloart-24-en-28-oic acid as a skeleton[58,59]. Thus MS02 was a cycloartane-type triterpenoid bearing three hydroxyl groups. The 1H-NMR data of MS02 revealed a pair of doublets of an AX system at δH 0.47 (d, J = 4.5 Hz, H-19a), 0.83 (d, J = 4.5 Hz, H-19b) which characteristic of typical resonances of a cyclopropyl methylene group in a cycloartane-type framework; one olefinic proton at δH 5.12 (t, J = 7.0 Hz, H-24); three oxygenated methine protons at δH 3.58 (brs, H-1), 4.54 (d, J = 5.0 & 12.0 Hz, H-3) and 3.59 (m, H-7); five tertiary methyl groups at δH 1.03-1.69 and one secondary methyl group δH 0.94 (d, J = 6.0 Hz, H-21). The HMBC spectrum (Fig. 4.2) designated correlations between oxygenated methine proton at δH 3.58 (H-1) and carbons at δC 71.2 (C-3) and 37.4 (C-5); between proton at δH 4.54 (H-3) and carbons at δC 55.5 (C-4) and 37.4 (C-5); between proton at δH 3.59 (H-7) and carbons at δC 37.4 (C-5); so, three hydroxyl group attached to C-1, C-3 and C-7, respectively. Moreover, these connectivities were also conﬁrmed by correlations observed in the COSY spectrum between H-1 (δH 3.58) and H-2a (δH 1.82), H-2b (δH 1.88) and H-3 (δH 4.54); between H-5 (δH 2.78) and H-6a (δH 1.17), H-6b (δH 1.45) and H-7 (δH 3.59) and H-8 (δH 1.63). In addition, the NOESY spectral data of MS02 (Fig. 4.2) evinced the cyclopropyl methylene protons (H-19) which β-type were close in space to proton (H-1); the
14 methine proton (H-5) which α-type was close in space to protons (H-3, H-7). Thus, three hydroxyl groups of carbons C-1, C-3 and C-7 were α, β and β-type, respectively. Based on data of IR, UV, HR-ESI-MS, 1D, 2D-NMR and compared with previous published data[58,59]; the structure of MS02 was identified as 1α,3β,7β-trihydroxycycloart-24-en-28-oic acid, and named Markhacanasin A. Fig 4.2: The selected HMBC, COSY and NOESY correlations of 1 Fig 4.3: Chemical structures of MS02 4.3. Biological activity of compounds 4.3.1. Cytotoxic activity Results of cytotoxic activity of fraction from leaves of M. stipulata var. canaense V.S. Dang are presented in table 4.2
15 Table 4.2. The cytotoxic percentage (%) of fractions from leaves of M.stipulata var. canaense V.S. Dang at concentration of 100 µg/mL. Cell line No. Sample MCF-7 HeLa Hep G2 1 MSH 72.11 ± 1.96 52.60 ± 1.20 49.64 ± 2.27 2 MSH.I 19.95 ± 4.26 0.78 ± 4.78 2.00 ± 0.49 3 MSH.II 55,81 ± 2.16 35.49 ± 1.86 39.34 ± 1.73 4 MSH.III 86.84 ± 2.78 95.03 ± 0.79 92.71 ± 0.99 5 MSH.IV 95.83 ± 0.64 95.59 ± 1.62 90.91 ± 1.13 6 MSH.V 79.00 ± 0.06 92.75 ± 1.83 61.89 ± 2.13 7 MSE 28.98±4.00 6.72 ± 2.99 13.57 ± 6.22 8 MSE.I 35.57 ± 1.79 13.61 ± 2.48 23.65 ± 4.96 9 MSE.II 60.29 ± 1.94 37.48 ± 3.52 46.13 ± 1.78 10 MSE.III 41.36 ± 4.16 6.08 ± 2.21 12.50 ± 3.24 11 MSE.IV 21.67 ± 4.74 1.92 ± 1.01 -9.57 ± 4.30 12 MSE.V 5.98 ± 6.27 -7.33 ± 1.26 -7.88 ± 5.41 13 Camptothecin 51.85 ± 1.32 65.08 ± 2.59 51.85 ± 0.32 Results showed that the fraction MSH.IV, which had toxic activity on 3 cancer cell lines MCF-7, HeLa, Hep G2, were at the highest concentration of 100 µg / mL with the cytotoxic percentage. 95.83 ± 0.64%, 95.59 ± 1.62%, 90.91 ± 1.13% Results of cytotoxic activity of fraction from leaves of S.binhchauesis V.S. Dang are presented in table 4.3
16 Table 4.3. The cytotoxic percentage (%) of fractions from leaves of S. binhchauesis V.S. Dang at concentration of 100 µg/mL. Cell line No. Sample MCF-7 HeLa Hep G2 1 SBH.I 12.92 ± 4.62 10.77 ± 0.45 0.71 ± 4.98 2 SBH.II 34.48 ± 3.22 12.91 ± 2.46 17.63 ± 5.64 3 SBH.III 44.41 ± 0.85 19.64 ± 0.94 20.14 ± 4.48 4 SBH.IV 41.94 ± 3.19 24.30 ± 1.19 17.37 ± 4.05 5 SBH.V 29.12 ± 3.15 18.83 ± 2.87 -3.02 ± 3.88 6 SBE.I 68.51 ± 1.71 56.31 ± 3.52 33.37 ± 3.39 7 SBE.II 60.56 ± 1.07 83.26 ± 5.56 53.79 ± 1.20 8 SBE.III 44.09 ± 1.47 44.57 ± 3.83 44.07 ± 0.61 9 SBE.IV 32.55 ± 5.50 35.89 ± 1.89 14,72 ± 8.33 10 SBE.V 1.31 ± 6.09 -4.97 ± 6.57 -12.43 ± 2.03 13 Camptothecin* 51.85 ± 1.32 65.08 ± 2.59 51.85 ± 0.32 *concentration at 0.01 µg/mL with MCF-7, 0.07 µg/mL with HepG2, and 1.00 µg/mL with HeLa Results showed that the fraction SBE.II, which had toxic activity on 3 cancer cell lines MCF-7, HeLa, Hep G2, were at the highest concentration of 100 µg / mL with the cytotoxic percentage 60.56 ± 1.07%, 83.26 ± 5.56%, 53.79 ± 1.20%. Results of cytotoxic activity of compound are presented in table 4.4 and table 4.5.
17 Table 4.4: The cytotoxic percentage (%) of compounds at concentration of 100 µg / mL. Cell line No. Sample MCF-7 HeLa Hep G2 NCI-H460 Jurkat 1 MS02* 92.72 ± 0.11 85.92 ± 3.99 91.25 ± 1.91 94.52 ± 1.67 91.84 ± 1.21 2 MS03 29.58 ± 3.06 6.66 ± 2.08 10.31 ± 1.59 -1.10 ± 3.13 21.54±3.78 3 MS04 5.06 ± 2.03 - - - - 4 MS05 72.16±0.34 - - - - 5 MS07 27.67 ± 1.57 86.36±3.69 29.50 ± 1.84 - - 6 MS08 43.38 ± 2.11 26.19 ± 1.25 5.57 ± 1.02 - - 7 MS10 71.39 ± 0.09 - 71.20 ± 2.46 - - 8 MS13 77.41 ± 0.82 - - - - 9 MS14 5.06 ± 2.03 - - - - 10 MS16 75.08 ± 2.81 63.72 ± 1.74 66.76 ± 1.68 - - 11 MS17 19.69 ± 3.18 12.76 ± 2.36 -4.43 ± 3.21 - - - Not test * Concentration at 50 µg/mL The results showed that the compounds MS03, MS04, MS08, MS17 do not show cytotoxic activity at concentrations of 100 µg /ml. Compounds MS05, MS07 show only cytotoxic activity on 1 cancer cell line. Compounds MS02 and MS16 exhibit high cytotoxic activity on cancer cell lines MCF-7, HeLa, Hep G2. Especially, MS02 showed high activity on 5 cell lines at a concentration of 50 µg/mL. Table 4.5: IC50 of compounds Cell line (IC50, µg/mL) No. Sample MCF-7 HeLa Hep G2 NCI-H460 Jurkat 1 MS02 16.51 ± 29.55 ± 25.26 ± 24.21 ± 14.72 ±
18 0.22 1.64 1.06 0.38 0.38 53.38 ± 2 MS05 - - - - 0.56 61.88 ± 3 MS13 - - - - 0.90 48.51 ± 63.30 ± 57.94 ± 4 MS16 - - 1.78 0.27 4.82 0.005 ± 0.089 ± 0.079 ± 0.003 ± 0.005 ± 5 Camptothecin 0.001 0.088 0.023 0.000 0.001 The results showed that MS02 showed good inhibition of cancer cell lines with IC50 values from 14 -28 µg/mL. 4.3.2. Inhibiting activity of α-glucosidase enzyme Results of α-glucosidase inhibiting activity of fraction from leaves of M.stipulata var. canaense V. S. Dang are presented in table 4.6 Table 4.6: Results of α-glucosidase inhibiting activity of fraction from leaves of M.stipulata var. canaense V. S. Dang No. Sample IC50 (µg/mL) 1 MSHI 527.530 2 MSHII - 3 MSHIII 141.785 4 MSHIV 145.908 5 MSHV - 6 MSEI - 7 MSEII 71.436 8 MSEIII 51.339 9 MSEIV 93.333 10 MSEV 70.909