Nghiên cứu thành phần hóa học, hoạt tính sinh học của loài trichosanthes baviensis gagnep (qua lâu ba vì), trichossanthes anguina l (dưa núi) và trichosanthes kirilowii maxim (qua lâu nhân) tt a

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY - Hoang Thi Yen Hoàng Thị Yến STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF Trichosanthes baviensis, Trichosanthes anguina AND Trichosanthes kirilowii Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOTORAL THESIS Hanoi - 2019 This thesis was completed at: Graduate University of Science and Technology, Vietnam Academy of Science and Technology Advisor 1: Prof Chau Van Minh Advisor 2: PhD Nguyen Xuan Nhiem Reviewer 1: Reviewer 2: Reviewer 3: This thesis will be defended at Graduate University of Science and Technology, Vietnam Academy of Science and Technology at hour date month 2019 The thesis can be found in: - The Library of Graduate University of Science and Technology, Vietnam Academy of Science and Technology - Vietnam National Library 27 LIST OF PUBLISHED ARTICLES Nguyen Xuan Nhiem, Hoang Thi Yen, Bui Thi Thuy Luyen, Bui Huu Tai, Pham Van Hoan, Nguyen Phuong Thao, Hoang Le Tuan Anh, Ninh Khac Ban, Phan Van Kiem, Chau Van Minh, Jang Hoon Kim, Jeon Mi Ni and Young Ho Kim Chemical components from the leaves of Trichosanthes baviensis and their tyrosinase inhibitory activity Bulletin of the Korean chemical society, 2015, 36 (2), 703-706 Nguyen Xuan Nhiem, Hoang Thi Yen, Phan Van Kiem, Chau Van Minh, Bui Huu Tai, Hoang Le Tuan Anh, SeonJu Park, Nanyoung Kim, and Seung Hyun Kim 1H and 13C NMR assignments of tricanguinas A -B, coumarin monoterpenes from Trichosanthes anguina L Magnetic Resonance in Chemistry, 2014, 53(2), 178-180 Chau Van Minh, Nguyen Xuan Nhiem, Hoang Thi Yen, Phan Van Kiem, Bui Huu Tai, Hoang Le Tuan Anh, Truong Thi Thu Hien, SeonJu Park, Nanyoung Kim, and Seung Hyun Kim Chemical constituents of Trichosanthes kirilowii and their cytotoxic activities Archives of Pharmacal Research, 2015, 38(8), 14431448 Hoang Thi Yen, Hoang Le Tuan Anh, Duong Thi Hai Yen, Pham Hai Yen, Duong Thi Dung, Do Thi Ha, Phan Van Kiem, Chau Van Minh, Nguyen Xuan Nhiem Phenolic glycosides from Trichosanthes baviensis Journal of Medicinal Materials, 2014, 19(5), 283-287 Hoang Thi Yen, Hoang Le Tuan Anh, Dan Thi Thuy Hang, Pham Hai Yen, Pham Thi Trang Tho, Do Thi Ha, Phan Van Kiem, Chau Van Minh, Nguyen Xuan Nhiem Megastigmane glycosides from Trichosanthes anguina L Journal of Medicinal Materials, 2014, 19(5), 299-303 26 cytotoxic activity on A-549 cell line with IC50 of 2.7 µM, stronger than those of mitoxantrone (IC50 of 7.2 µM) Compounds TK1, TK6 and TK8 exhibited moderate cytotoxic activity with IC50 values ranging from 14.7 to 49.4 µM Compounds TK2 and TK3 exhibited weak cytotoxic activity on the cell lines A-549 and HT-29 RECOMMENDATIONS From the results of chemical constituents and biological activities of T baviensis, T anguina and T kirilowii, we demonstrate that: TB6 from T baviensis exhibited strong inhibitory tyrosinase activity Therefore, futher studies are required on the whitening effect of this compound TK7 from T kirilowii exhibited significant cytototic activity on two human cancer cell lines, HT-29 and OVCAR Therefore, futher studies (in vivo model) of this compounds are required NEW FINDINGS OF THE THESIS This is the first study on the chemical constituents and biological activities of T baviensis, T anguina, and T kirilowii grown Vietnam new compounds and 26 known compounds isolated from T baviensis, T anguina and T kirilowii The new compounds were named as 9.26-epoxymultiflorenol (TB1), tricanguina (TA1), tricanguina B (TA2), and trichobenzolignan (TK1) Compounds from T baviensis were evaluated for tyrosinase inhibitory activity for the first time INTRODUCTION Vietnam is located in a tropical monsoon climate, mountainous and hilly terrain, so the climate conditions are also very diverse, with many typical and sub-climatic regions These factors effect on ecological conditions, dense, moist, evergreen, or sparse, semi-deciduous tropical vegetation and subtropical vegetation in the high mountain areas According to estimates of Vietnamese botanists, there are about 12,000 species in Vietnam, of which, about one third is used as traditional oriental medicine Many of these species have been used in traditional medicine for other purposes for human life such as Eurycoma longifolia, Mentha arvensis, Momordica charantia, Angelica sinensis, Panax vietnamensis, and Gymnema sylvestre, In addition to the abundance of species, Vietnamese medicinal resources have value in the treatment of diseases in folk Medicinal plants are used in a single form or in combination with each other to create ancient remedies, which exist up to today In addition, hundreds of medicinal plants have been proved by modern medicine - pharmaceutical science to prove their therapeutic value Over the past few decades, traditional medicine has provided modern medicine with more than 40% of all drugs or drugs-derived Therefore, studies have focused on the scientific assessment of traditional plant-derived drugs Among the above-mentioned plants, Some Trichosanthes species are widely cultivated and many species grow naturally in Vietnam and other countries Moreover, Trichosanthes species are used as vegetables, some of them are used as folk medicine for treatment of antidotes, diuretics, reducing blood sugar, skin diseases, cure headache, etc, For the purpose of researching to clarify the chemical compositions and biological activities of some Trichosanthes species, I have chosen the Doctor thesis entitled "Study on chemical constituents and biological activities of Trichosanthes baviensis, Trichosanthes anguina, and Trichosanthes kirilowii" The objectives of the thesis Study on chemical constituents of three Trichosanthes species (T baviensis, T anguina, and T kirilowii) grown in Vietnam Evaluation of cytotoxic and tyrosinase enzyme inhibitory activities of isolated compounds from the above mentioned Trichosanthes species The main contents of the thesis Isolation of compounds from T baviensis, T anguina, and T kirilowii Determination of chemical structures of isolated compounds Evaluation of cytotoxic and tyrosinase enzyme inhibitory activities of isolated compounds CHAPTER OVERVIEW This chapter provides an overview on the Trichosanthes genus about chemical components and biological activities in all over the world and in Vietnam 1 Introduction to Trichosanthes genus 1.1.1 Plant characteristics of Trichosanthes genus Trichosanthes genus is a large genus in Cucurbitaceae, consisting of about 100 species and received great interest of scientists all over the world According to statistics and preliminary description of Prof Pham Hoang Ho, Vietnam has about 12 species of Trichosanthes, including endemic species of Vietnam (T baviensis and T pierrei [1]) 1.1.2 The review of Trichosanthes in traditional medicine Some species in the Trichosanthes genus (T ovigera Blume, T cucumerina L.) have been widely cultivated and used as vegetables Many species have been used in folk medicine for the 25 and (3R,9S) 3,9-dihydroxymegastigman-5-ene 9-O-β-Dglucopyranoside (TA5), isopentyl 1-O-β-D-glucopyranoside (TA8); known compound: icariside B1 (TA6) compounds were isolated and elucidated from T kirilowii, including:  new compound: trichobenzolignan (TK1)  known compounds were reported from Trichosanthes genus for the first time: arvenin I (TK8), ligballinol (TK2) and ehletianol C (TK4); known compounds: (-)-pinoresinol (TK3), luteolin-7-Oglucoside (TK5), chrysoeriol-7-O-β-D-glucoside (TK6) and 10αcucurbita-5,24-dien-3β-ol (TK7) TB1-TB10 from T baviensis were evaluated for their tyrosinase inhibitory activity Compound TB6 exhibited potent tyrosinase inhibitory activity with IC50 of 3.9 ± 1.5 µM Compounds TB3, TB4, and TB10 exhibited significant activity with IC50 of 6.9 ± 2.2, 9.5 ± 3.1, 9.3 ± 2.1 µM respectively Compounds TB1, TB2, TB5, TB7, TB8, and TB9 exhibited moderate activity with IC50 values of 14.5 ± 3.4, 11.4 ± 2.8, 10.3 ± 2.5, 11.5 ± 4.1, 16.7 ± 2.1, 10.1 ± 3.1 µM respectively Compounds TB1-TB10 were evaluated for tyrosinase inhibitory activity for the first time TA1-TA8 from T anguina were evaluated for their tyrosinase inhibitory activity Compound TA7 exhibited significant tyrosinase inhibitory activity, compared to those of the positive control, kojic acid (IC50 of 14.6 ± 3.3 µM) with IC50 is 8.5±3.3 µM Compounds TA1-TA6 exhibited moderate activity with IC50 values ranging from 21.3 to 46.7 µM Compound TA8 did not show activity The cytotoxic activity of TK1-TK8 against four human cancer cell lines: A-549, HT-29, OVCAR and MCF-7 As the results, compound TK7 exhibited the strongest cytotoxic activity on two human cancer cell lines, HT-29 and OVCAR with IC50 values are 4.1 and 6.5 µM respectively, compared to those of mitoxantrone (IC50 values of 3.1 and 8.4 µM respectively) Compound TK5 exhibited the significant 24 The results showed that TK7 exhibited the strongest inhibitory activity of human cancer cell lines, HT-29 and OVCAR, with IC50 values of 4.1 and 6.5 µM, respectively, compared to those of mitoxantrone, IC50 values of 3.1 and 8.4 µM, respectively) TK5 exhibited strong cytotoxic activity on A-549 cancer cell line with IC50 value of 2.7 µM, compared to those of mitoxantrone (IC50 of 7.2 µM) Compounds TK1, TK6 and TK8 exhibited moderate cytotoxic activity with IC50 ranging from 14.7 to 49.4 µM The compounds TK2 and TK3 exhibited moderate cytototix activity on human cancer cell lines, A-549 and HT-29 Compound TK4 did not show cytotoxic activity [86] CONCLUSIONS From the T baviensis, T anguina, and T kirilowii grown in Vietnam, 30 compounds have been isolated and evaluated the biological activities 14 compounds were isolated and elucidated from T baviensis, including:  new compound: 9,26-epoxymultiflorenol (TB1)  known compounds were reported from Trichosanthes genus for the first time: β-amyrin acetate (TB2), lup-20(29)-en-3β-ol (TB6), ergosta-6,22-dien-3β,5α,8α-triol (TB3), nicotiflorin (TB12), (+)-lyoniresinol 9'-O-β-D-glucopyranoside (TB7), (-)-lyoniresinol 9'O-β-D-glucopyranoside (TB8), demethoxybergenin (TB9), icariside F2 (TB11); known compounds: spinasterol (TB4), 4α,14αdimethyl-9,19-cyclo-5α,9β-ergost-24(28)-en-3β-ol (TB5), bergenin (TB10), (6S,9S)-roseoside (TB13), and thymidine (TB14) compounds were isolated and elucidated from T anguina, including:  new compounds: tricanguina A (TA1) and tricanguina B (TA2)  known compounds were reported from Trichosanthes genus for the first time: kaemferol 3-O-β-D-glucopyranoside (1→3) O-β-Dglucopyranoside (TA7), corchoionoside B (TA3), icariside B5 (TA4) treatments of antidote, diuretic, blood sugar, skin diseases, headaches, 1.1.3 The review of Trichosanthes chemical constituents In recent years, there have been many studies on chemical constituents and biological activities of Trichosanthes species According to published papers in the liturature, the chemical constituents of the Trichosanthes genus include main classes: triterpenoids, steroids, flavonoids, lignans, alkaloids, and some other minor compounds Especially, cucurbitanes-the class of triterpenoids are quite common compounds in the species of Trichosanthes The chemical constituents studies mainly focused on species: T anguina, T cucumerina, T cucumeroides, T dioica, T fructus, T kirilowii, T pericarpium and T tricuspidata Details overview of the chemical constituents, see the thesis 1.1.3.1 Triterpenoids There are 64 triterpenoids, 1-64 from four species T cucumerina, T kirilowii, T tricuspidata, and T anguina Steroid compounds There are 26 steroids, 65-90 from four species T dioica, T kirilowii, T rosthornii and T tricuspidata 1.1.3.2 Flavonoids There are 20 flavonoids 91-110 from two species T kirilowii and T pericarpium 1.1.3.3 Lignans There are lignans 110-114 from T kirilowii 1.1.3.4 Alkaloids There are 17 alkaloids 115-131 from T kirilowii 1.1.4 The review of Trichosanthes biological activities The studies of scientists in the world mainly focus on Trichosanthes species compounds from Trichosanthes species exhibited significant bioligical activities A number of interesting activities such as cytotoxic, anti-inflammatory, antibacterial, antifungal, anti-oxidant activities of which, cytotoxic activity is prominent with strong inhibitory on different cancer cell lines 1.1.4.1 Cytotoxic activities 1.1.4.2 Anti-inflammatory activities 1.1.4.3 Anti-oxidant activities 1.1.4.4 Antibacterial and antifungal activities Detailed overview of biological activities, see the thesis 1.1.5 The reviews of Trichosanthes in Vietnam According to published documents, there are two studies on chemical constituents and biological activities of two Trichosanthes species: T kirilowii and T tricuspidata in Vietnam 1.2 Introduction about T baviensis, T anguina and T kirilowii 1.2.1 T baviensis 1.2.2 T anguina 1.2.3 T kirilowii CHAPTER 2: EXPERIMENT AND RESULTS 2.1 Plant materials The stems and leaves of T baviensis were collected in Ba Vi, Hanoi, Vietnam in September 2011; the stems and leaves of T anguina were collected in Hoabinh in Agust 2013, Vietnam; the root of T kirilowii was collected in Hoabinh, Vietnam, September 2012 The scientific names of those Trichosanthes were identified by Prof Ninh Khac Ban, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology 23 Compounds from T baviensis have shown strong tyrosinase inhibitory activity, suggesting further researchs on mechanism of tyrosinase inhibitory activity of T baviensis Therefore, in vivo studies are needed to improve the potential of applications in the cosmetic industry, to protect the skin from external factors The research results will contribute to the application opportunities in the production of drugs to treat diseases related to skin pigmentation disorders 3.3.2 Cytotoxic activity of compounds from T kirilowii According to previous studies, compounds isolated from T kirilowii exhibited many interesting activities, especially cytoxic activities, such as A-549, SK-OV-3, SK-MEL-2, XF-498, HCT-15, UO-31, CCRF-CEM, SR, NCI-H460, NCI-H522, HCT-116, U251, OVCAR-3, OVCAR-6, SN12C, SK-Mel-2, B16F1, breast cancer (SR-BR-3, MCF-7, T47D and MDA-MB-435), colon cancer (Caco2) [4], [14], [37] Among the compounds from T kirilowii, karounidiol (45) exhibited strong anticancer activity on many cancer cell lines UO-31 with IC50 of 1.98 μM, leukemia CCRF-CEM with IC50 of 1.63 μM; CRL-2262 with IC50 of 1.75 μM, A-549 with IC50 of 1.76 μM; NCIH460 with IC50 of 1.91 μM, NCI-H522 with IC50 of 3.56 μM, colon cancer HCT-116 with IC50 of 2.01 μM, U251 with IC50 of 2.02 μM, OVCAR-3 with IC50 of 1.79 μM; OVCAR-6 with IC50 of 2.06 μM and kidney cancer SN12C with IC50 of 2,36 μM [14] The isoetin 5'methyl ether compound (93) significantly inhibited human lung cancer (A-549), malignant skin cancer (SK-Mel-2), and malignant mouse cancer (B16F1) with IC50 values of 0.92, 8.00, 7.23 µg/mL, respectively [29] Compounds (TK1-TK8) have been evaluated for their cytotoxic activityon four human cancer cell lines including: A-549, HT-29, OVCAR, and MCF-7 Mitoxantrone, an anti-cancer drug was used as positive control 22 In 2005, Khan and co-authors isolated eight cycloartane triterpenoid compounds and tested tyrosinase inhibitory activity Results showed that seven of the eight substances had better inhibitory activity than kojic acid (IC50 = 16.67±0.52); some have strong activity, IC50 = 1.32 ± 0.37 [84] In 2014, Khan and co-authors isolated β-amyrin acetate (TB2) from Madhuca latifolia fruit and evaluated tyrosinase inhibitory activity As the results, TB2 showed significant tyrosinase inhibitory with IC50 of 23.12±0.07 [85] In a publication by Park and co-authors in 2017, the constituent bergenin in some cosmetics inhibited the growth of tyrosinase enzymes From the results of the chemical constituents of T baviensis an endemic specie in Vietnam, all compounds exhibited strong tyrosinase inhibitory activity Compounds exhibited the stronger tyrosinase inhibitory activity than those of positive control, kojic acid (IC50 is 14.6 ± 3.3 µM) Especially, TB6 exhibited the strongest tyrosinase inhibitory activity (IC50: 3.9 ± 1.5 µM) Compounds TB3, TB4, and TB10 exhibited significant tyrosinase inhibitory activity (IC50 values are 6.9 ± 2.2, 9.5 ± 3.1, 9.3 ± 2.1 µM respectively) Compounds TB1, TB2, TB5, TB7, TB8, and TB9 exhibited moderate tyrosinase inhibitory activity with IC50 values of 14.5 ± 3.4, 11.4 ± 2.8, 10.3 ± 2.5, 11.5 ± 4.1, 16.7 ± 2.1, and 10.1 ± 3.1 µM, respectively) This is the first time the compounds from T baviensis have been evaluated for tyrosinase inhibitory activity The tyrosinase inhibitory activity of compounds from T anguina were also evaluated As the results, compound TA7 exhibited stronger inhibitory activity (IC50 of 8.5±3.3 µM) than those of the positive control, kojic acid (IC50 of 14.6 ± 3.3 µM) Compounds TA1-TA6 exhibited moderate tyrosinase inhibitory activity with IC50 from 21.3 to 46.7 µM Compound TA8 did not exhibit tyrosinase inhibitory activity at the tested concentration 2.2 Research methods 2.2.1 Isolation methods 2.2.1.1 Thin layer chromatography (TLC) 2.2.1.2 Column chromatography (CC) 2.2.1.3 Purification of substances 2.2.2 Structural elucidation methods 2.2.2.1 High resolution electrospary mass spectrum (HR-ESI-MS) 2.2.2.2 Nuclear magnetic resonance spectroscopy (NMR) 2.2.2.3 Circular dichlorism (CD) 2.2.2.4 Optical rotation [α]) 2.2.3 Biological assays 2.2.3.1 Cytotoxic assay 2.2.3.2 Tyrosinase enzyme assay 2.3 Isolation of compounds This section presents outlines of the general methods to isolate pure substances from the plants samples 2.3.1 Isolation of compounds from T baviensis Figure 2.2 The isolation scheme of compounds from T baviensis 21 2.3.2 Isolation of compounds from T anguina Figure 2.3 The isolation scheme of compounds from T anguina 2.3.3 Isolation of compounds from T kirilowii Figure 2.4 Isolation of compounds from T kirilowii 2.4 Physical properties and spectroscopic data of the isolated compounds This section provides physical properties and spectroscopic data of 30 compounds from T baviensis, T anguina and T kirilowii Figure 3.65 Chemical structures of compounds from T kirilowii 3.3 Biological activities of isolated compounds 3.3.1 Tyrosinase inhibitory activity Tyrosinase inhibitors have been a great concern solely due to the key role of tyrosinase in both mammalian melanogenesis and fruit or fungi enzymatic browning They are widely used in dermatological treatments and also applied in cosmetics Therefore, the development of safe and effective tyrosinase inhibitors have become important for improving food quality and preventing pigmentation disorders and other melaninrelated human health issues Plants are rich source of bioactive compounds that are mostly free side effects Thus, interest in finding natural tyrosinase inhibitors also increasing [82], [83] 20 MeO MeO MeO Me Me MeO O O O O O O Me TA2 Me OH OH O O HO OH OH Me T anguina O OH O HO O H 2.5.1 Tyrosinase inhibitory activity of compounds from T baviensis and O O Table 2.1 Tyrosinase inhibitory activity of TB1-TB10 Me TA1 10' O O O Me 2.5 Biological activities results OH O OH HO TA3 TA5 Me O Me Me OH O HO OH O Me H OH O OH TA4 OH HO O HO HO Me O OH TA6 OH O HO Hợp chất TB1 TB2 TB3 TB4 TB5 TB6 TB7 TB8 TB9 TB10 Kojic acid IC50 (μM) 14.5 ± 3.4 11.4 ± 2.8 6.9 ± 2.2 9.5 ± 3.1 10.3 ± 2.5 3.9 ± 1.5 11.5 ± 4.1 16.7 ± 2.1 10.1 ± 3.1 9.3 ± 2.1 14.6 ± 3.3 HO O HO HO HO HO HO OH O O O OH O TA8 O Me Me OH O OH HO HO TA7 Figure 3.64 Chemical structures of compounds from T anguina Table 2.2 Tyrosinase enzyme inhibitory activity of TA1-TA8 Hợp chất TA1 TA2 TA3 TA4 TA5 TA6 TA7 TA8 Kojic acid IC50 (μM) 21.3 ± 2.8 36.8±2.6 33.9±3.1 42.2±3.6 46.7±2.0 22.7±1.4 8.5±3.3 >100 14.6 ± 3.3 10 2.5.2 Cytotoxic activity of compounds from T kirilowii Table 2.3 Cytotoxic activity of TK1-TK8 IC50 (µM) Hợp chất A-549 HT-29 OVCAR 36.4 16.2 21.6 TK1 >100 45.5 >100 TK2 52.4 60.9 >100 TK3 >100 >100 >100 TK4 2.7 16.0 14.5 TK5 21.1 40.7 32.1 TK6 11.3 4.1 6.5 TK7 17.0 49.4 14.7 TK8 7.2 3.1 8.4 Mitoxantrone 19 MCF-7 26.5 >100 >100 >100 32.7 15.8 17.3 42.8 10.3 CHAPTER 3: DISCUSSIONS 3.1 Determination of chemical structures of isolated compounds This section presents the detailed results of spectral analysis and structure determination of 30 isolated compounds from from T baviensis, T anguina and T kirilowii 3.1.1 Determination of chemical structures of compounds from T baviensis 3.1.1.1 Compound TB1: 9,26-epoxymultiflorenol (new compound) Figure 3.63 Chemical structures of compounds from T baviensis Figure 3.1 Structures of compound TB1 and reference compound TB1a 18 Table 3.20 NMR spectral data of TK1 and reference compound C δCa,# δCa,b δHa,c (mult., J = Hz) 128.6 134.2 2, 127.9 128.2 7.16 (d, 8.0) 3, 117.8 116.3 6.73 (d, 8.0) 158.7 158.5 87.9 88.7 5.46 (d, 6.0) 55.2 54.7 3.43 (m) 65.3 65.1 3.80 (m) 1' 135.7 129.5 2' 125.9 123.7 7.34 (s) 3' 137.7 131.5 4' 159.4 161.0 5' 109.8 110.0 6.72 (d, 8.0) 6' 129.7 128.7 7.20 (d, 8.0) 7' 132.0 6.54 (d, 16.0) 8' 127.0 6.20 (dt, 6.0, 16.0) 9' 63.9 4.18 (d, 6.0) a CD3OD, b100 MHz, c400 MHz, #δC of cupressoside B (TK1a)[75] 3.2 Chemical structure of isolated compounds This section presents the structure determination of 30 compounds isolated from T baviensis, T anguina and T kirilowii 11 Table 3.1 NMR spectral data of TB1 δHa,c (mult., J = Hz) 30.8 1.43 (m)/1.63 (m) 27.2 1.65 (m) 79.3 3.21 (dd, 5.4, 9.0) 39.0 45.1 1.47 (m) 23.5 2.07 (ddd, 2.4, 10.8, 18.0)/2.16 (dt, 4.8, 18.0) 113.3 5.25 (dd, 2.4, 4.8) 143.7 85.8 38.4 30.3 1.55 (m)/1.79 (dd, 4.2, 6.0) 36.3 1.45 (m)/1.56 (m) 40.3 49.0 22.0 1.25 (m)/1.87 (ddd, 4.2, 7.8, 14.4) 34.6 1.17 (m)/1.53 (m) 32.2 45.5 1.55 (m) 36.1 1.16 (dd, 10.2, 13.8)/1.32 (dd, 6.0, 13.8) 28.8 33.3 1.24 (m)/1.43 (m) 36.2 0.87 (m)/1.43 (m) 28.3 1.02 (s) 15.4 0.93 (s) 14.5 0.90 (s) 78.1 3.25 (d, 7.8)/4.52 (d, 7.8) 19.7 0.84 (s) 29.1 0.94 (s) 33.5 0.95 (s) 31.7 0.95 (s) a CDCl3, b150 MHz, c600 MHz Compound TB1 was obtained as a colorless wax and the HRESI-MS- m/z 463.3536 [M+Na]+ of TB1 indicated the molecular formula to be C30H48O2 (calcd 463.3547 for [C30H48O2Na]+) The 1HNMR spectrum of TB1 showed the following signals: one olefinic C 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 δCa,b 12 17 proton at δH 5.25, two oxymethylene protons at δH 3.25 and 4.52, and seven tertiary methyl groups at δH 0.84 (3H, s), 0.90 (3H, s), 0.93 (3H, s), 0.94 (3H, s), 0.95 (6H, s), and 1.02 (3H, s), assigned to oleane-type triterpene The 13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectra of TB1 revealed 30 carbon signals including quaternary, methine, 11 methylene, and methyl carbons The 1H- and 13C-NMR data of TB1 were similar to those of multiflorenol except for the additional oxygen bridge of C9/C-26 All the carbons were assigned to relevant protons by means of an heteronuclear multiple quantum correlation (HMQC) experiment The heteronuclear multiple bond correlation (HMBC) correlations between H-23 (δH 1.02)/H-24 (δH 0.93) and C-3 (δ 79.3), C-4 (δC 39.1), C-5 (δC 45.1), suggested the hydroxyl group at C-3 and two methyl groups at C-4 The β-orientation configuration of the hydroxyl group at C-3 was proved based on the coupling constant (JH-2β/H-3 = 9.0 Hz; JH-2α/H-3 = 5.4 Hz) and comparing the 13C-NMR chemical shifts of C-3 (δC 79.3), C-4 (δC 39.1) in TB1 with those of 3β-hydroxyolean-12-en-28-oic acid (C-3 [δC 78.8], C-4 [δC 38.9]) and 3α-hydroxyolean-12-en-28-oic acid (C-3 [δC 76.2], C-4 [δC 37.2]) [56] The HMBC correlations between H-25 (δH 0.90) and C-1 (δC 30.8), C-5 (δC 45.1), C-9 (δC 85.8), C-10 (δC 38.4) suggested that the methyl group was at C-10 In addition, the HMBC correlations between H-26 (δH 3.25 and 4.52) and C-8 (δC 143.7), C-9 (δC 85.8), C-13 (δC 40.3), C-14 (δC 49.0), and C-15 (δC 22.0), suggested the double bond at C-7/C-8 and oxygen bridge of C-9/C-26 DEPT spectra of TK1 revealed signals for 18 carbons, including five quaternary at δC 129.5, 131.5, 134.2, 158.5, and 161.0, eleven methine at δC 54.7, 88.7, 110.0, 116.3 (2xC), 123.8, 127.1, 128.2 (2xC), 128.7, 132.0, and two oxymethylene carbons at δC 63.9 and 65.1 The 1H- and 13CNMR data of TK1 suggested the presence of dihydrobenzofuran skeleton and similar to those of cupressoside B (TK1a) except for the different from propyl moiety at C-1’ [75] The HMBC correlations between H-7 (δH 5.45) and C-1 (δC 134.2), C2/C-6 (δC 128.2), and C-3’ (δC 131.5), C-4’ (δC 161.0), between H2/H-6 (δH 7.16) and C-1 (δC 134.2), C-4 (δC 158.5), and C-7 (δC 88.7) suggested the position of p-hydroxylphenyl at C-7 The HMBC correlations from H-7’ (δH 6.54) to C-1’ (δC 129.5), C-2’ (δC 123.8), C-6’ (δC 128.7), C-8’ (δC 127.1), and C-9’ (δC 63.9) confirmed the presence of double bond at C-7’/C-8’ and hydroxyl group at C-9’ The E configuration of the double bond was based on the coupling constant between H-7’ and H-8’, JH-7’/H-8’ = 16.0 Hz The large coupling constant of H-7 and H-8, JH-7/H-8 = 6.0 Hz confirmed the configurations of two protons at C-7 and C-8 to be trans The CD spectrum of TK1 λ = 244 nm (Δε: -4,3) and λ = 222 nm (Δε: +2,6) proved the configurations at C-7 and C-8 to be 7R and 8S by comparing with those of cupressoside B (a negative peat at 238 nm and a positive peak at 221 nm) [75] Based on the above evidence, compound TK1 was elucidated to be 2-(4-hydroxyphenyl)-5-(3-hydroxyprop-1E-en-1-yl)-2R,3Sdihydrobenzofuran and named trichobenzolignan Figure 3.2 Chemical structure and important HMBC, COSY, and NOESY correlations of compound TB1 Figure 3.49 Chemical structure and important HMBC correlations of compound TK1 13 16 Hình 3.25 Phổ CD TA1 3.1.3 Determination of chemical structures of compounds from T kirilowii Relative stereochemistry of TB1 was also confirmed by nuclear overhauser effect spectroscopy (NOESY) spectra analysis NOESY correlations including Hβ-6 (δH 2.07) correlated with H3-24 (δH 0.93) and H3-25 (δH 0.90), H3-25 correlated with Ha-26 (δH 3.25), Hb-26 (δH 4.52) correlated with H-18 (δH 1.55) and H3-28 (δH 0.94) suggested β-orientations of H3-24, H3-25, H2-26, H-18, and H3-28 However, NOESY correlations includingH3-23 (δH 1.02) correlated with H-3 (δH 3.21) and H-5 (δH 1.47), Hα-15 (δH 1.87) correlated with H3- 27 (δH 0.84) indicated α-configurations of H-3, H3-23, H-5, and H3-27 Consequently, the structure of TB1 was determined to be 9,26-epoxymultiflorenol 3.1.2 Determination of chemical structures of compounds from T anguina 3.1.2.1 Compound TA1: tricanguina A (new Compound) 3.1.3.1 Compound TK1: trichobenzolignan (new Compound) HO GlcO O O 4' 1' 3' 7' 9' OH TK1 OH OH OH TK1a Figure 3.48 Chemical structure of compound TK1 and reference compound Compound TK1 was obtained as a white amorphous powder and its molecular formula was determined to be C18H18O4 by HR-EIMS at m/z 298.1203 (Calcd C18H18O4 for 298.1205) The 1H-NMR spectrum of TK1 showed signals for four protons of 1,4-disubstituted benzene at δH 6.73 (2H, d, J = 8.0 Hz) and 7.16 (2H, d, J = 8.0 Hz); three protons of 1,2,4-trisubstituted aromatic ring with ABX coupling patterns at δH 6.72 (1H, d, J = 8.0 Hz), 7.20 (1H, dd, J = 2.0, 8.0 Hz), and 7.33 (1H, d, J = 2.0 Hz), and two olefin protons at δH 6.19 (1H, dt, J = 6.0, 16.0 Hz) and 6.54 (1H, d, J = 16.0 Hz) The 13C-NMR and Figure 3.23 Chemical structure of compound TA1 and reference compound Compound TA1 was obtained as a colorless amorphous powder, and its molecular formula was determined to be C21H24O7 by HR-ESI-MS at m/z 389.1578 [M+H]+ (calcd C21H25O7 for 389.1595) The 1HNNR spectrum showed one olefinic proton at δH 6.15 (s), three aromatic protons at δH 6.36 (d, J = 9.6 Hz), 7.00 (s), and 7.89 (d, J = 9.6 Hz), two methoxy groups at δH 3.89 (s) and 3.99 (s), two quaternary methyl groups at δH 1.90 (s), 2.09 (s), and one secondary methyl group at δH 1.15 (d, J = 6.4 Hz) The 13C NMR and DEPT spectra of TA1 displayed three carbonyls at δC 162.82, 200.70, and 211.57, six quaternary at δC 116.45, 141.85, 144.09, 145.74, 151.42, and 157.41, five methine at δC 38.65, 105.92, 115.73, 124.28, and 14 146.04, two methylene at δC 48.52 and 77.67, and five methyl carbons at δC 16.84, 21.00, 27.79, 57.02, and 62.95 Analysis of the H and 13C NMR data of indicated the presence of a coumarin and monoterpene moieties The 1H and 13C NMR data of TA1 were similar to those of 8-(2,3-dihydroxy-3-methylbutoxy)-6,7dimethoxycoumarin except formonoterpene moiety at C-8 The HMBC correlations from two methoxy groups (δH 3.89 and 3.99) to C-6 (δC 151.42) and C-7 (δC 141.85), respectively, confirmed the presence of two methoxy groups at C-6 and C-7 In addition, COSY correlations were observed between H-3 (δH 6.36)/H-4 (δH 7.89) These suggested the coumarin moiety to be 6,7-dimethoxy-8hydroxycoumarin The HMBC correlations between H-1′ (δH 4.98) and C-2′ (δC 211.57)/C-3′ (δC 38.65); between H-10′ (δH 1.15) and C-2′ (δC 211.57)/C-3′ (δC 38.65)/C-4′ (δC 48.52) suggested the carbonyl and methyl groups at C-2′ and C-3′, respectively In addition, the HMBC correlations from H-8′ (δH 1.90)/H-9′ (δH 2.09) to C-6′ (δC 124.28)/C7′ (δC 157.41); from H-6′ (δH 6.15) to C-5′/C-7′/C-8′/C-9′ confirmed the carbonyl and double bond at C-5′ and C-6′/C-7′, respectively Moreover, the C-1′ of monoterpene moiety was linked to C-8 of coumarin via oxygen bridge, proved by the HMBC correlations between H-1′ (δH 4.98) and C-8 (δC 145.74) Figure 3.24 Chemical structure and important HMBC and COSY correlations of compound TA1 15 The positive Cotton effect λ = 278 nm (Δε: + 8.05) in the CD spectrum of TA1 proved the configuration at C-3 to be R by comparison with those of similar structure: (S)-3-methylhexane-2,5dione (a negative Cotton effect at λ = 280nm) Based on the above evidence, the structure of TA1 was elucidated as 8-((3R)-3,7dimethyloct-6-en-2,5-dione-1-oxy)-6,7-dimethoxycoumarin, a new compound named tricanguina A Table 3.12 NMR spectral data of compound TA1 C 10 6-OCH3 7-OCH3 1' 2' 3' 4' 5' 6' 7' 8' 9' 10' δCa,b 162.8 115.7 146.0 105.9 151.4 141.8 145.7 144.0 116.5 57.0 62.6 77.7 211.6 38.7 48.5 ΔHa,c (mult., J = Hz) 6.35 (d, 9.6) 7.89 (d, 9.6) 7.00 (s) 3.89 (s) 3.99 (s) 4.98 (s) 3.25 (m) 2.62 (dd, 4.4, 17.6) 3.00 (dd, 9.2, 17.6) 200.7 124.3 6.16 (s) 157.4 28.0 1.90 (s) 21.0 2.09 (s) 16.8 1.15 (d, 6.4) a CD3OD, b100 MHz, c400 MHz ... chemical constituents and biological activities of T baviensis, T anguina, and T kirilowii grown Vietnam new compounds and 26 known compounds isolated from T baviensis, T anguina and T kirilowii. .. AND RESULTS 2.1 Plant materials The stems and leaves of T baviensis were collected in Ba Vi, Hanoi, Vietnam in September 2011; the stems and leaves of T anguina were collected in Hoabinh in Agust... Thuy Hang, Pham Hai Yen, Pham Thi Trang Tho, Do Thi Ha, Phan Van Kiem, Chau Van Minh, Nguyen Xuan Nhiem Megastigmane glycosides from Trichosanthes anguina L Journal of Medicinal Materials, 2014,