Summary of Chemistry dotoral thesis: Study on chemical constituents and biological activities of Trichosanthes baviensis, Trichosanthes anguina, and Trichosanthes kirilowii

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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.

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Nội dung Text: Summary of Chemistry dotoral thesis: Study on chemical constituents and biological activities of Trichosanthes baviensis, Trichosanthes anguina, and Trichosanthes kirilowii

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING 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
2 27 This thesis was completed at: Graduate University of Science and LIST OF PUBLISHED ARTICLES Technology, Vietnam Academy of Science and Technology 1. 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, Advisor 1: Prof. Chau Van Minh Jang Hoon Kim, Jeon Mi Ni and Young Ho Kim. Chemical Advisor 2: PhD. Nguyen Xuan Nhiem components from the leaves of Trichosanthes baviensis and their tyrosinase inhibitory activity. Bulletin of the Korean chemical society, 2015, 36 (2), 703-706. 2. 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 Reviewer 1: assignments of tricanguinas A -B, coumarin monoterpenes from Trichosanthes anguina L. Magnetic Resonance in Chemistry, Reviewer 2: 2014, 53(2), 178-180. 3. Chau Van Minh, Nguyen Xuan Nhiem, Hoang Thi Yen, Phan Van Reviewer 3: 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), 1443- 1448. This thesis will be defended at Graduate University of Science and 4. Hoang Thi Yen, Hoang Le Tuan Anh, Duong Thi Hai Yen, Pham Technology, Vietnam Academy of Science and Technology at hour Hai Yen, Duong Thi Dung, Do Thi Ha, Phan Van Kiem, Chau date month 2019. Van Minh, Nguyen Xuan Nhiem. Phenolic glycosides from Trichosanthes baviensis. Journal of Medicinal Materials, 2014, 19(5), 283-287. The thesis can be found in: 5. Hoang Thi Yen, Hoang Le Tuan Anh, Dan Thi Thuy Hang, Pham - The Library of Graduate University of Science and Technology, Hai Yen, Pham Thi Trang Tho, Do Thi Ha, Phan Van Kiem, Vietnam Academy of Science and Technology. Chau Van Minh, Nguyen Xuan Nhiem. Megastigmane glycosides - Vietnam National Library. from Trichosanthes anguina L.. Journal of Medicinal Materials, 2014, 19(5), 299-303.
26 3 cytotoxic activity on A-549 cell line with IC50 of 2.7 µM, stronger INTRODUCTION than those of mitoxantrone (IC50 of 7.2 µM). Compounds TK1, TK6 Vietnam is located in a tropical monsoon climate, and TK8 exhibited moderate cytotoxic activity with IC50 values mountainous and hilly terrain, so the climate conditions are also very ranging from 14.7 to 49.4 µM. Compounds TK2 and TK3 exhibited diverse, with many typical and sub-climatic regions. These factors weak cytotoxic activity on the cell lines A-549 and HT-29. effect on ecological conditions, dense, moist, evergreen, or sparse, semi-deciduous tropical vegetation and subtropical vegetation in the RECOMMENDATIONS high mountain areas. From the results of chemical constituents and biological According to estimates of Vietnamese botanists, there are activities of T. baviensis, T. anguina and T. kirilowii, we demonstrate about 12,000 species in Vietnam, of which, about one third is used as that: traditional oriental medicine. Many of these species have been used TB6 from T. baviensis exhibited strong inhibitory tyrosinase in traditional medicine for other purposes for human life such as activity. Therefore, futher studies are required on the whitening effect of Eurycoma longifolia, Mentha arvensis, Momordica charantia, this compound. Angelica sinensis, Panax vietnamensis, and Gymnema sylvestre,... TK7 from T. kirilowii exhibited significant cytototic activity on In addition to the abundance of species, Vietnamese two human cancer cell lines, HT-29 and OVCAR. Therefore, futher medicinal resources have value in the treatment of diseases in folk. studies (in vivo model) of this compounds are required. Medicinal plants are used in a single form or in combination with NEW FINDINGS OF THE THESIS each other to create ancient remedies, which exist up to today. In 1. This is the first study on the chemical constituents and biological addition, hundreds of medicinal plants have been proved by modern activities of T. baviensis, T. anguina, and T. kirilowii grown Vietnam. medicine - pharmaceutical science to prove their therapeutic value. 2. 4 new compounds and 26 known compounds isolated from T. Over the past few decades, traditional medicine has provided baviensis, T. anguina and T. kirilowii. The new compounds were modern medicine with more than 40% of all drugs or drugs-derived. named as 9.26-epoxymultiflorenol (TB1), tricanguina (TA1), Therefore, studies have focused on the scientific assessment of tricanguina B (TA2), and trichobenzolignan (TK1). traditional plant-derived drugs. Among the above-mentioned plants, 3. Compounds from T. baviensis were evaluated for tyrosinase Some Trichosanthes species are widely cultivated and many species inhibitory activity for the first time. 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
4 25 activities of Trichosanthes baviensis, Trichosanthes anguina, and and (3R,9S) 3,9-dihydroxymegastigman-5-ene 9-O-β-D- Trichosanthes kirilowii". glucopyranoside (TA5), isopentyl 1-O-β-D-glucopyranoside (TA8); The objectives of the thesis 1 known compound: icariside B1 (TA6). Study on chemical constituents of three Trichosanthes species 3. 8 compounds were isolated and elucidated from T. kirilowii, (T. baviensis, T. anguina, and T. kirilowii) grown in Vietnam. including: Evaluation of cytotoxic and tyrosinase enzyme inhibitory  1 new compound: trichobenzolignan (TK1) activities of isolated compounds from the above mentioned  3 known compounds were reported from Trichosanthes genus for Trichosanthes species. the first time: arvenin I (TK8), ligballinol (TK2) and ehletianol C The main contents of the thesis (TK4); 4 known compounds: (-)-pinoresinol (TK3), luteolin-7-O- 1. Isolation of compounds from T. baviensis, T. anguina, and T. glucoside (TK5), chrysoeriol-7-O-β-D-glucoside (TK6) and 10α- kirilowii. cucurbita-5,24-dien-3β-ol (TK7). 4. TB1-TB10 from T. baviensis were evaluated for their tyrosinase 2. Determination of chemical structures of isolated compounds. inhibitory activity. Compound TB6 exhibited potent tyrosinase 3. Evaluation of cytotoxic and tyrosinase enzyme inhibitory inhibitory activity with IC50 of 3.9 ± 1.5 µM. Compounds TB3, TB4, activities of isolated compounds. 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, CHAPTER 1. OVERVIEW TB8, and TB9 exhibited moderate activity with IC50 values of 14.5 ± This chapter provides an overview on the Trichosanthes genus 3.4, 11.4 ± 2.8, 10.3 ± 2.5, 11.5 ± 4.1, 16.7 ± 2.1, 10.1 ± 3.1 µM about chemical components and biological activities in all over the respectively. Compounds TB1-TB10 were evaluated for tyrosinase world and in Vietnam. inhibitory activity for the first time. 1. 1. Introduction to Trichosanthes genus 5. TA1-TA8 from T. anguina were evaluated for their tyrosinase 1.1.1. Plant characteristics of Trichosanthes genus inhibitory activity. Trichosanthes genus is a large genus in Cucurbitaceae, Compound TA7 exhibited significant tyrosinase inhibitory activity, consisting of about 100 species and received great interest of compared to those of the positive control, kojic acid (IC50 of 14.6 ± scientists all over the world. According to statistics and preliminary 3.3 µM) with IC50 is 8.5±3.3 µM. Compounds TA1-TA6 exhibited description of Prof. Pham Hoang Ho, Vietnam has about 12 species moderate activity with IC50 values ranging from 21.3 to 46.7 µM. of Trichosanthes, including 2 endemic species of Vietnam (T. Compound TA8 did not show activity. baviensis and T. pierrei [1]). 6. The cytotoxic activity of TK1-TK8 against four human cancer cell 1.1.2. The review of Trichosanthes in traditional medicine. lines: A-549, HT-29, OVCAR and MCF-7. As the results, compound Some species in the Trichosanthes genus (T. ovigera Blume, TK7 exhibited the strongest cytotoxic activity on two human cancer T. cucumerina L.) have been widely cultivated and used as cell lines, HT-29 and OVCAR with IC50 values are 4.1 and 6.5 µM vegetables. Many species have been used in folk medicine for the respectively, compared to those of mitoxantrone (IC50 values of 3.1 and 8.4 µM respectively). Compound TK5 exhibited the significant
24 5 The results showed that TK7 exhibited the strongest treatments of antidote, diuretic, blood sugar, skin diseases, inhibitory activity of human cancer cell lines, HT-29 and OVCAR, headaches, ... with IC50 values of 4.1 and 6.5 µM, respectively, compared to those 1.1.3. The review of Trichosanthes chemical constituents of mitoxantrone, IC50 values of 3.1 and 8.4 µM, respectively). TK5 In recent years, there have been many studies on chemical exhibited strong cytotoxic activity on A-549 cancer cell line with constituents and biological activities of Trichosanthes species. IC50 value of 2.7 µM, compared to those of mitoxantrone (IC50 of 7.2 According to published papers in the liturature, the chemical µM). Compounds TK1, TK6 and TK8 exhibited moderate cytotoxic constituents of the Trichosanthes genus include main classes: activity with IC50 ranging from 14.7 to 49.4 µM. The compounds triterpenoids, steroids, flavonoids, lignans, alkaloids, and some other TK2 and TK3 exhibited moderate cytototix activity on human cancer minor compounds. Especially, cucurbitanes-the class of triterpenoids cell lines, A-549 and HT-29. Compound TK4 did not show cytotoxic are quite common compounds in the species of Trichosanthes. The activity [86]. chemical constituents studies mainly focused on 8 species: T. anguina, T. cucumerina, T. cucumeroides, T. dioica, T. fructus, T. CONCLUSIONS kirilowii, T. pericarpium and T. tricuspidata. Details overview of the From the T. baviensis, T. anguina, and T. kirilowii grown in chemical constituents, see the thesis. Vietnam, 30 compounds have been isolated and evaluated the 1.1.3.1. Triterpenoids biological activities. There are 64 triterpenoids, 1-64 from four species T. 1. 14 compounds were isolated and elucidated from T. baviensis, cucumerina, T. kirilowii, T. tricuspidata, and T. anguina. including: Steroid compounds  1 new compound: 9,26-epoxymultiflorenol (TB1). There are 26 steroids, 65-90 from four species T. dioica, T.  8 known compounds were reported from Trichosanthes genus kirilowii, T. rosthornii and T. tricuspidata. for the first time: β-amyrin acetate (TB2), lup-20(29)-en-3β-ol 1.1.3.2. Flavonoids (TB6), ergosta-6,22-dien-3β,5α,8α-triol (TB3), nicotiflorin (TB12), There are 20 flavonoids 91-110 from two species T. kirilowii (+)-lyoniresinol 9'-O-β-D-glucopyranoside (TB7), (-)-lyoniresinol 9'- and T. pericarpium. O-β-D-glucopyranoside (TB8), demethoxybergenin (TB9), icariside 1.1.3.3. Lignans F2 (TB11); 5 known compounds: spinasterol (TB4), 4α,14α- There are 5 lignans 110-114 from T. kirilowii. dimethyl-9,19-cyclo-5α,9β-ergost-24(28)-en-3β-ol (TB5), bergenin 1.1.3.4. Alkaloids (TB10), (6S,9S)-roseoside (TB13), and thymidine (TB14). There are 17 alkaloids 115-131 from T. kirilowii. 2. 8 compounds were isolated and elucidated from T. anguina, 1.1.4. The review of Trichosanthes biological activities including: The studies of scientists in the world mainly focus on 8  2 new compounds: tricanguina A (TA1) and tricanguina B (TA2). Trichosanthes species compounds from Trichosanthes species  5 known compounds were reported from Trichosanthes genus for exhibited significant bioligical activities. A number of interesting the first time: kaemferol 3-O-β-D-glucopyranoside (1→3) O-β-D- activities such as cytotoxic, anti-inflammatory, antibacterial, glucopyranoside (TA7), corchoionoside B (TA3), icariside B5 (TA4)
6 23 antifungal, anti-oxidant activities ... of which, cytotoxic activity is Compounds from T. baviensis have shown strong tyrosinase prominent with strong inhibitory on different cancer cell lines. inhibitory activity, suggesting further researchs on mechanism of 1.1.4.1. Cytotoxic activities tyrosinase inhibitory activity of T. baviensis. Therefore, in vivo 1.1.4.2. Anti-inflammatory activities studies are needed to improve the potential of applications in the 1.1.4.3. Anti-oxidant activities cosmetic industry, to protect the skin from external factors. The 1.1.4.4. Antibacterial and antifungal activities research results will contribute to the application opportunities in the Detailed overview of biological activities, see the thesis. production of drugs to treat diseases related to skin pigmentation 1.1.5. The reviews of Trichosanthes in Vietnam disorders. According to published documents, there are two studies on 3.3.2. Cytotoxic activity of compounds from T. kirilowii chemical constituents and biological activities of two Trichosanthes According to previous studies, compounds isolated from T. species: T. kirilowii and T. tricuspidata in Vietnam. kirilowii exhibited many interesting activities, especially cytoxic 1.2. Introduction about T. baviensis, T. anguina and T. kirilowii activities, such as A-549, SK-OV-3, SK-MEL-2, XF-498, HCT-15, 1.2.1. T. baviensis UO-31, CCRF-CEM, SR, NCI-H460, NCI-H522, HCT-116, U251, 1.2.2. T. anguina OVCAR-3, OVCAR-6, SN12C, SK-Mel-2, B16F1, breast cancer 1.2.3. T. kirilowii (SR-BR-3, MCF-7, T47D and MDA-MB-435), colon cancer (Caco- 2) [4], [14], [37]. CHAPTER 2: EXPERIMENT AND RESULTS Among the compounds from T. kirilowii, karounidiol (45) 2.1. Plant materials exhibited strong anticancer activity on many cancer cell lines UO-31 The stems and leaves of T. baviensis were collected in Ba Vi, with IC50 of 1.98 μM, leukemia CCRF-CEM with IC50 of 1.63 μM; Hanoi, Vietnam in September 2011; the stems and leaves of T. CRL-2262 with IC50 of 1.75 μM, A-549 with IC50 of 1.76 μM; NCI- anguina were collected in Hoabinh in Agust 2013, Vietnam; the root H460 with IC50 of 1.91 μM, NCI-H522 with IC50 of 3.56 μM, colon of T. kirilowii was collected in Hoabinh, Vietnam, September 2012. cancer HCT-116 with IC50 of 2.01 μM, U251 with IC50 of 2.02 μM, The scientific names of those Trichosanthes were identified by Prof. OVCAR-3 with IC50 of 1.79 μM; OVCAR-6 with IC50 of 2.06 μM Ninh Khac Ban, Institute of Marine Biochemistry, Vietnam Academy and kidney cancer SN12C with IC50 of 2,36 μM [14]. The isoetin 5'- of Science and Technology. 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 7 In 2005, Khan and co-authors isolated eight cycloartane 2.2. Research methods triterpenoid compounds and tested tyrosinase inhibitory activity. 2.2.1. Isolation methods Results showed that seven of the eight substances had better 2.2.1.1. Thin layer chromatography (TLC) inhibitory activity than kojic acid (IC50 = 16.67±0.52); some have 2.2.1.2. Column chromatography (CC) strong activity, IC50 = 1.32 ± 0.37 [84]. 2.2.1.3. Purification of substances In 2014, Khan and co-authors isolated β-amyrin acetate 2.2.2. Structural elucidation methods (TB2) from Madhuca latifolia fruit and evaluated tyrosinase 2.2.2.1. High resolution electrospary mass spectrum (HR-ESI-MS) inhibitory activity. As the results, TB2 showed significant tyrosinase 2.2.2.2. Nuclear magnetic resonance spectroscopy (NMR) inhibitory with IC50 of 23.12±0.07 [85]. 2.2.2.3. Circular dichlorism (CD) In a publication by Park and co-authors in 2017, the 2.2.2.4. Optical rotation [α]) constituent bergenin in some cosmetics inhibited the growth of 2.2.3. Biological assays tyrosinase enzymes. 2.2.3.1. Cytotoxic assay From the results of the chemical constituents of T. baviensis - 2.2.3.2. Tyrosinase enzyme assay an endemic specie in Vietnam, all compounds exhibited strong 2.3. Isolation of compounds tyrosinase inhibitory activity. Compounds exhibited the stronger This section presents outlines of the general methods to tyrosinase inhibitory activity than those of positive control, kojic acid isolate pure substances from the plants samples. (IC50 is 14.6 ± 3.3 µM). Especially, TB6 exhibited the strongest 2.3.1. Isolation of compounds from T. baviensis 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 Figure 2.2. The isolation scheme of compounds from T. baviensis 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.
8 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 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 Figure 2.4. Isolation of compounds from T. kirilowii the key role of tyrosinase in both mammalian melanogenesis and fruit or fungi enzymatic browning. They are widely used in 2.4. Physical properties and spectroscopic data of the isolated dermatological treatments and also applied in cosmetics. Therefore, compounds the development of safe and effective tyrosinase inhibitors have This section provides physical properties and spectroscopic become important for improving food quality and preventing data of 30 compounds from T. baviensis, T. anguina and T. kirilowii. 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 9 MeO MeO 2.5. Biological activities results O O 2.5.1. Tyrosinase inhibitory activity of compounds from T. baviensis and Me MeO O MeO O Me O O O H O T. anguina. O O O Table 2.1. Tyrosinase inhibitory activity of TB1-TB10 Me Me Me Hợp chất IC50 (μM) 10' TA1 TA2 Me TB1 14.5 ± 3.4 O OH O HO OH OH TB2 11.4 ± 2.8 O OH O HO OH OH O TB3 6.9 ± 2.2 OH O Me TA3 HO Me TA5 Me TB4 9.5 ± 3.1 O TB5 10.3 ± 2.5 Me OH H TB6 3.9 ± 1.5 OH O HO O OH TB7 11.5 ± 4.1 OH O Me HO O Me TB8 16.7 ± 2.1 TA4 O OH HO HO TA6 OH TB9 10.1 ± 3.1 OH TB10 9.3 ± 2.1 HO O Kojic acid 14.6 ± 3.3 HO O O HO HO O Me Table 2.2. Tyrosinase enzyme inhibitory activity of TA1-TA8 OH O HO O OH Me Hợp chất IC50 (μM) HO TA8 HO O O OH TA1 21.3 ± 2.8 HO HO OH TA7 TA2 36.8±2.6 TA3 33.9±3.1 TA4 42.2±3.6 Figure 3.64. Chemical structures of compounds from T. anguina TA5 46.7±2.0 TA6 22.7±1.4 TA7 8.5±3.3 TA8 >100 Kojic acid 14.6 ± 3.3
10 19 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 MCF-7 TK1 36.4 16.2 21.6 26.5 TK2 >100 45.5 >100 >100 TK3 52.4 60.9 >100 >100 TK4 >100 >100 >100 >100 TK5 2.7 16.0 14.5 32.7 TK6 21.1 40.7 32.1 15.8 TK7 11.3 4.1 6.5 17.3 TK8 17.0 49.4 14.7 42.8 Mitoxantrone 7.2 3.1 8.4 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 11 Table 3.20. NMR spectral data of TK1 and reference compound Table 3.1. NMR spectral data of TB1 C δCa,# δCa,b δHa,c (mult., J = Hz) C δCa,b δHa,c (mult., J = Hz) 1 128.6 134.2 - 1 30.8 1.43 (m)/1.63 (m) 2, 6 127.9 128.2 7.16 (d, 8.0) 2 27.2 1.65 (m) 3, 5 117.8 116.3 6.73 (d, 8.0) 3 79.3 3.21 (dd, 5.4, 9.0) 4 158.7 158.5 - 4 39.0 - 7 87.9 88.7 5.46 (d, 6.0) 5 45.1 1.47 (m) 8 55.2 54.7 3.43 (m) 6 23.5 2.07 (ddd, 2.4, 10.8, 18.0)/2.16 (dt, 4.8, 18.0) 9 65.3 65.1 3.80 (m) 7 113.3 5.25 (dd, 2.4, 4.8) 1' 135.7 129.5 - 8 143.7 - 2' 125.9 123.7 7.34 (s) 9 85.8 - 3' 137.7 131.5 - 10 38.4 - 4' 159.4 161.0 - 11 30.3 1.55 (m)/1.79 (dd, 4.2, 6.0) 5' 109.8 110.0 6.72 (d, 8.0) 12 36.3 1.45 (m)/1.56 (m) 6' 129.7 128.7 7.20 (d, 8.0) 13 40.3 - 7' 132.0 6.54 (d, 16.0) 14 49.0 - 8' 127.0 6.20 (dt, 6.0, 16.0) 15 22.0 1.25 (m)/1.87 (ddd, 4.2, 7.8, 14.4) 16 34.6 1.17 (m)/1.53 (m) 9' 63.9 4.18 (d, 6.0) a 17 32.2 - CD3OD, b100 MHz, c400 MHz, #δC of cupressoside B (TK1a)[75] 18 45.5 1.55 (m) 3.2. Chemical structure of isolated compounds 19 36.1 1.16 (dd, 10.2, 13.8)/1.32 (dd, 6.0, 13.8) 20 28.8 - This section presents the structure determination of 30 21 33.3 1.24 (m)/1.43 (m) compounds isolated from T. baviensis, T. anguina and T. kirilowii. 22 36.2 0.87 (m)/1.43 (m) 23 28.3 1.02 (s) 24 15.4 0.93 (s) 25 14.5 0.90 (s) 26 78.1 3.25 (d, 7.8)/4.52 (d, 7.8) 27 19.7 0.84 (s) 28 29.1 0.94 (s) 29 33.5 0.95 (s) 30 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 1H- NMR spectrum of TB1 showed the following signals: one olefinic
12 17 proton at δH 5.25, two oxymethylene protons at δH 3.25 and 4.52, and DEPT spectra of TK1 revealed signals for 18 carbons, including five seven tertiary methyl groups at δH 0.84 (3H, s), 0.90 (3H, s), 0.93 quaternary at δC 129.5, 131.5, 134.2, 158.5, and 161.0, eleven (3H, s), 0.94 (3H, s), 0.95 (6H, s), and 1.02 (3H, s), assigned to methine at δC 54.7, 88.7, 110.0, 116.3 (2xC), 123.8, 127.1, 128.2 oleane-type triterpene. The 13C-NMR and distortionless enhancement (2xC), 128.7, 132.0, and two oxymethylene carbons at δC 63.9 and by polarization transfer (DEPT) spectra of TB1 revealed 30 carbon 65.1. The 1H- and 13CNMR data of TK1 suggested the presence of signals including 8 quaternary, 4 methine, 11 methylene, and 7 dihydrobenzofuran skeleton and similar to those of cupressoside B methyl carbons. The 1H- and 13C-NMR data of TB1 were similar to (TK1a) except for the different from propyl moiety at C-1’ [75]. The those of multiflorenol except for the additional oxygen bridge of C- HMBC correlations between H-7 (δH 5.45) and C-1 (δC 134.2), C- 9/C-26. All the carbons were assigned to relevant protons by means 2/C-6 (δC 128.2), and C-3’ (δC 131.5), C-4’ (δC 161.0), between H- of an heteronuclear multiple quantum correlation (HMQC) 2/H-6 (δH 7.16) and C-1 (δC 134.2), C-4 (δC 158.5), and C-7 (δC 88.7) experiment. The heteronuclear multiple bond correlation (HMBC) suggested the position of p-hydroxylphenyl at C-7. The HMBC correlations between H-23 (δH 1.02)/H-24 (δH 0.93) and C-3 (δ 79.3), correlations from H-7’ (δH 6.54) to C-1’ (δC 129.5), C-2’ (δC 123.8), C-4 (δC 39.1), C-5 (δC 45.1), suggested the hydroxyl group at C-3 and C-6’ (δC 128.7), C-8’ (δC 127.1), and C-9’ (δC 63.9) confirmed the two methyl groups at C-4. The β-orientation configuration of the presence of double bond at C-7’/C-8’ and hydroxyl group at C-9’. hydroxyl group at C-3 was proved based on the coupling constant The E configuration of the double bond was based on the coupling (JH-2β/H-3 = 9.0 Hz; JH-2α/H-3 = 5.4 Hz) and comparing the 13C-NMR constant between H-7’ and H-8’, JH-7’/H-8’ = 16.0 Hz. The large chemical shifts of C-3 (δC 79.3), C-4 (δC 39.1) in TB1 with those of coupling constant of H-7 and H-8, JH-7/H-8 = 6.0 Hz confirmed the 3β-hydroxyolean-12-en-28-oic acid (C-3 [δC 78.8], C-4 [δC 38.9]) configurations of two protons at C-7 and C-8 to be trans. The CD and 3α-hydroxyolean-12-en-28-oic acid (C-3 [δC 76.2], C-4 [δC spectrum of TK1 λ = 244 nm (Δε: -4,3) and λ = 222 nm (Δε: +2,6) 37.2]) [56]. The HMBC correlations between H-25 (δH 0.90) and C-1 proved the configurations at C-7 and C-8 to be 7R and 8S by (δC 30.8), C-5 (δC 45.1), C-9 (δC 85.8), C-10 (δC 38.4) suggested that comparing with those of cupressoside B (a negative peat at 238 nm the methyl group was at C-10. In addition, the HMBC correlations and a positive peak at 221 nm) [75]. between H-26 (δH 3.25 and 4.52) and C-8 (δC 143.7), C-9 (δC 85.8), Based on the above evidence, compound TK1 was elucidated C-13 (δC 40.3), C-14 (δC 49.0), and C-15 (δC 22.0), suggested the to be 2-(4-hydroxyphenyl)-5-(3-hydroxyprop-1E-en-1-yl)-2R,3S- double bond at C-7/C-8 and oxygen bridge of C-9/C-26. dihydrobenzofuran and named trichobenzolignan. Figure 3.2. Chemical structure and important HMBC, COSY, and Figure 3.49. Chemical structure and important HMBC correlations NOESY correlations of compound TB1 of compound TK1
16 13 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. Hình 3.25. Phổ CD của TA1 anguina 3.1.3. Determination of chemical structures of compounds from T. kirilowii 3.1.2.1. Compound TA1: tricanguina A (new Compound) 3.1.3.1. Compound TK1: trichobenzolignan (new Compound) HO 4 GlcO 1 O O 7 4' 8 1' 3' 7' 9 9' OH OH OH OH TK1 TK1a Figure 3.23. Chemical structure of compound TA1 and reference compound Figure 3.48. Chemical structure of compound TK1 and reference Compound TA1 was obtained as a colorless amorphous compound powder, and its molecular formula was determined to be C21H24O7 by Compound TK1 was obtained as a white amorphous powder HR-ESI-MS at m/z 389.1578 [M+H]+ (calcd C21H25O7 for 389.1595). and its molecular formula was determined to be C18H18O4 by HR-EI- The 1HNNR spectrum showed one olefinic proton at δH 6.15 (s), MS at m/z 298.1203 (Calcd C18H18O4 for 298.1205). The 1H-NMR three aromatic protons at δH 6.36 (d, J = 9.6 Hz), 7.00 (s), and 7.89 spectrum of TK1 showed signals for four protons of 1,4-disubstituted (d, J = 9.6 Hz), two methoxy groups at δH 3.89 (s) and 3.99 (s), two benzene at δH 6.73 (2H, d, J = 8.0 Hz) and 7.16 (2H, d, J = 8.0 Hz); quaternary methyl groups at δH 1.90 (s), 2.09 (s), and one secondary three protons of 1,2,4-trisubstituted aromatic ring with ABX coupling methyl group at δH 1.15 (d, J = 6.4 Hz). The 13C NMR and DEPT patterns at δH 6.72 (1H, d, J = 8.0 Hz), 7.20 (1H, dd, J = 2.0, 8.0 Hz), spectra of TA1 displayed three carbonyls at δC 162.82, 200.70, and and 7.33 (1H, d, J = 2.0 Hz), and two olefin protons at δH 6.19 (1H, 211.57, six quaternary at δC 116.45, 141.85, 144.09, 145.74, 151.42, dt, J = 6.0, 16.0 Hz) and 6.54 (1H, d, J = 16.0 Hz). The 13C-NMR and and 157.41, five methine at δC 38.65, 105.92, 115.73, 124.28, and
14 15 146.04, two methylene at δC 48.52 and 77.67, and five methyl The positive Cotton effect λ = 278 nm (Δε: + 8.05) in the CD carbons at δC 16.84, 21.00, 27.79, 57.02, and 62.95. Analysis of the spectrum of TA1 proved the configuration at C-3 to be R by 1 H and 13C NMR data of 1 indicated the presence of a coumarin and comparison with those of similar structure: (S)-3-methylhexane-2,5- monoterpene moieties. The 1H and 13C NMR data of TA1 were similar to those of 8-(2,3-dihydroxy-3-methylbutoxy)-6,7- dione (a negative Cotton effect at λ = 280nm). Based on the above dimethoxycoumarin except formonoterpene moiety at C-8. The evidence, the structure of TA1 was elucidated as 8-((3R)-3,7- HMBC correlations from two methoxy groups (δH 3.89 and 3.99) to dimethyloct-6-en-2,5-dione-1-oxy)-6,7-dimethoxycoumarin, a new 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 compound named tricanguina A. correlations were observed between H-3 (δH 6.36)/H-4 (δH 7.89). Table 3.12. NMR spectral data of compound TA1 These suggested the coumarin moiety to be 6,7-dimethoxy-8- C δCa,b ΔHa,c (mult., J = Hz) hydroxycoumarin. 2 162.8 - The HMBC correlations between H-1′ (δH 4.98) and C-2′ (δC 3 115.7 6.35 (d, 9.6) 211.57)/C-3′ (δC 38.65); between H-10′ (δH 1.15) and C-2′ (δC 4 146.0 7.89 (d, 9.6) 211.57)/C-3′ (δC 38.65)/C-4′ (δC 48.52) suggested the carbonyl and 5 105.9 7.00 (s) methyl groups at C-2′ and C-3′, respectively. In addition, the HMBC 6 151.4 - correlations from H-8′ (δH 1.90)/H-9′ (δH 2.09) to C-6′ (δC 124.28)/C- 7 141.8 - 7′ (δC 157.41); from H-6′ (δH 6.15) to C-5′/C-7′/C-8′/C-9′ confirmed 8 145.7 - the carbonyl and double bond at C-5′ and C-6′/C-7′, respectively. 9 144.0 - Moreover, the C-1′ of monoterpene moiety was linked to C-8 10 116.5 - of coumarin via oxygen bridge, proved by the HMBC correlations 6-OCH3 57.0 3.89 (s) between H-1′ (δH 4.98) and C-8 (δC 145.74). 7-OCH3 62.6 3.99 (s) 1' 77.7 4.98 (s) 2' 211.6 - 3' 38.7 3.25 (m) 4' 48.5 2.62 (dd, 4.4, 17.6) 3.00 (dd, 9.2, 17.6) 5' 200.7 - 6' 124.3 6.16 (s) 7' 157.4 - 8' 28.0 1.90 (s) Figure 3.24. Chemical structure and important HMBC and 9' 21.0 2.09 (s) COSY correlations of compound TA1 10' 16.8 1.15 (d, 6.4) a CD3OD, b100 MHz, c400 MHz