GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY VU THI QUYNH CHI

Transcription

1 MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY VU THI QUYNH CHI STUDY ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF Tacca vietnamensis AND Tacca chantrieri GROWING IN VIETNAM Major: Organic chemistry Code: SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi

2 This thesis was completed at: Graduate University Science and Technology - Vietnam Academy of Science and Technology Supervisor 1: Dr. Nguyen Xuan Nhiem Institute of Marine Biochemistry - Vietnam Academy of Science and Technology Supervisor 2: Dr. Pham Hai Yen Institute of Marine Biochemistry - Vietnam Academy of Science and Technology 1 st Reviewer: 2 nd Reviewer: 3 rd Reviewer: The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at date month 2018 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 Vietnam.

3 1 INTRODUCTION 1. The urgency of the thesis Vietnam has a long tradition of traditional medicine using a variety of herbs for treating diseases and enhancing health. Vietnam has about 12,000 species of higher plants. Of these, nearly 5,000 species are used as medicinal plants [1, 2]. The medicinal plant resources have played important role due to the great potential in research and development of drugs in the treatment of diseases. Many compounds from medicinal plants and animal were discovered and used as drugs for treating diseases and enhancing health. However, many of medicinal plants still need to be studied chemical constituents as well as biological activities to find out bioactive compounds. The Tacca species, the well-known medicinal plants were used for the treatment of diseases such as gastric ulcer, enteritis, hepatitis, etc., get the attention of scientists around the world. The studies have showed that the extract and compounds from Tacca species exhibited various biological activities such as cytotoxic, microtubules, anti-inflammatory, anti-fungal, antimicrobial, and anti-bacterial activities, etc. In Vietnam, there are some species of Tacca such as Tacca chantrieri, a traditional medicine was used for the treatment of rheumatism. Tacca vietnamensis roots and tubers are used as medicines such as Tacca chantrieri. Their leaves were used as vegetable. There are few researches on the chemical components and biological activities of Tacca species grown in Vietnam. Until so far, there are only 3 publications on Tacca plantaginea and Tacca chantrieri [1, 4-6]. Therefore, to identify bioactive compounds from Tacca species, I chosen thesis topic "Study on chemical constituents and biological activities of Tacca vietnmensis and Tacca chantrieri species growing in Vietnam". 2. The aim of the thesis Study on chemical constituents of two Tacca species including Tacca vietnamensis and Tacca chantrierri growing in Vietnam.

4 2 Evaluate cytotoxic and inflammatory activities of isolates to find out bioactive compounds. 3. The main contents of the thesis 1. Isolate compounds from the rhizomes of T. vietnamensis and T. chantrierri; 2. Elucidate chemical structures of the isolated compounds; 3. Evaluate the cytotoxic activity of the isolated compounds; 4. Evaluate the anti-inflammatory activity of isolated compounds. CHAPTER 1: OVERVIEW Overview of national and international researches related to my study of the chemical constituents and biological activity of Tacca and about cancer and inflammation Introduction to Tacca genus The genus Tacca (Taccaceae) includes 17 species in the world. In Vietnam, Tacca genus includes 6 species. They are all herbal plants and distributed predominately in Southeast Asia, Pacific islands, and Africa,... Their rhizomes have been used in traditional medicine to treat gastric ulcer, enteritis, and hepatitis, etc. The chemical constituents of Tacca include steroidal, diarylheptanoids and their glucosides, and some other compounds. The phytochemical investigations of this genus confirmed the presence of diarylheptanoids and steroidal saponins. In addition, these compounds showed cytotoxic and anti-inflammatory activity [1, 3-6] Introduction to Tacca vietnamensis and Tacca chantrieri Tacca vietnamensis Thin et Hoat is an endemic plant in Vietnam. However, there has not been studied about phytochemical investigation of this plant. Tacca chantrieri André is perennial plant growing in Vietnam and some tropical countries. The phytochemical investigations of this plant confirmed the presence of diarylheptanoids, steroidal saponins,

5 Introduction to cancer Introduction to cancer and some treatments; Some types of cancer drugs are naturally derived Introduction to inflammation Introduction of inflammation, anti-inflammatory drugs and some products from nature have anti-inflammatory activity. CHAPTER 2: EXPERIMENTAL AND RESULTS 2.1. Plant materials The rhizomes of Tacca vietnamensis Thin et Hoat were collected in Bachma National park, Thua Thien Hue, Vietnam. The rhizomes of Tacca chantrieri André were collected in Tamdao, Vinhphuc, Vietnam Methods Methods for isolation Chromatographic methods such as thin layer chromatography (TLC), column chromatography (CC) Methods for structural elucidation Physical parameters and modern spectroscopic methods such as optical rotation ([ ] D), electrospray ionization mass spectrometry (ESI-MS) and high-resolution ESI-MS (HR-ESI-MS), one/two-dimension nuclear magnetic resonance (NMR) spectra, circular dichroism spectrum (CD) Biological assays - Cytotoxic activity was determined by the MTT assay. - Anti-inflammatory activity of the compounds was assessed on the basis of inhibiting NO production in lipopolysaccharide (LPS) activated BV2 cells Isolation of compounds This section presents outlines of the general methods to isolate pure substances from the plants samples Isolation of compounds from Tacca vietnanensis:

6 4 This section presents the process of isolating the compounds from Tacca vietnamensis. Figure 2.1. Isolation of compounds from Tacca vietnamensis Isolation of compounds from Tacca chantrieri: This section presents the process of isolating the compounds from Tacca chantrieri. Figure 2.2. Isolation of compounds from Tacca chantrieri

7 Physical properties and spectroscopic data of the isolated compounds Physical properties and spectroscopic data of the isolated compounds from Tacca vietnamensis This section presents physical properties and spectroscopic data of 9 compounds from Taccca vietnamensis Physical properties and spectroscopic data of the isolated compounds from Tacca chantrieri This section presents physical properties and spectroscopic data of 13 compounds from Tacca chantrieri Results on biological activities of isolated compounds Results on anti-inflammatory activity of compounds from Tacca vietnamensis and Tacca chantrieri - 9 compounds (TV1-TV9) were evaluated for the inhibitory activities of nitric oxide production in LPS-stimulated BV2 cells. Table 2.1. Inhibition activities of TV1-TV9 on NO production in the LPS-stimulated BV2 cells at concentration of 80 μm Comp. Inhibition (%) Comp. Inhibition (%) Comp. Inhibition (%) TV ± 2.2 TV ± 2.5 TV ± 1.8 TV ± 1.8 TV ± 2.0 TV ± 3.0 TV ± 1.5 TV ± 2.2 Butein * TV ± 2.1 (10 µm) 90.0 ± 5.0 Table 2.2. Inhibitory NO effects of compounds TV3-TV5 in the LPS-stimulated BV2 cells Comp. IC50 (µm) Comp. IC50 (µm) TV ± 3.6 TV ± 4.2 TV ± 6.0 Butein * 4.3 ± compounds (TC1-TC13) were evaluated for the inhibitory activities of nitric oxide production in LPS-stimulated BV2 cells. Table 2.3. Inhibition activities of TC1-TC13 on NO production in the LPS-stimulated BV2 cells at concentration of 80 μm Comp. Inhibition (%) Comp. Inhibition (%) Comp. Inhibition (%) TC ± 4.5 TC ± 2.5 TC ± 2.0 TC ± 3.6 TC ± 2.1 TC ± 2.8 TC ± 2.4 TC ± 3.0 TC ± 1.9 TC ± 2.5 TC ± 2.2 Butein (10 TC ± 3.3 TC ± 2.1 µm) 78.0 ± 4.2

8 6 Table 2.4. Inhibitory NO effects of compounds TC1-TC2 in the LPS-stimulated BV2 cells Comp. IC50 (µm) Comp. IC50 (µm) TC ± 2.4 Butein 4.3 ± 0.8 TC ± Results on cytotoxic activity of compounds from Tacca vietnamensis and Tacca chantrieri - 13 compounds (TC1-TC13) were evaluated for cytotoxic activity on four human cancer cell lines, including PC-3, LNCaP, MDA-MB-231 and HepG2. Table 2.6. The effects of compounds on the growth of PC3, LNCaP, MDA-MB-231 cell lines Comp. IC50 (µm) PC-3 LNCaP MDA-MB-231 TC ± ± ± 1.6 TC ± ± ± 1.5 TC ± ± ± 3.2 TC ± ± ± 2.0 Ellipticine 1.1 ± ± ± 0.1 CHAPTER 3: DISCUSSIONS 3.1. Chemical structure of isolated compounds This section presents the detailed results of spectral analysis and structure determination of 22 isolated compounds from Tacca vietnamensis and Tacca chantrieri. * 9 compounds from Tacca vietnamensis ( Figure 3.2): Taccavietnamoside A (TV1), taccavietnamoside B (TV2), taccavietnamoside C (TV3), taccavietnamoside D (TV4), taccavietnamoside (1 2)-[α-L-rhamnopyranosyl-(1 3)]-β-D-glucopyranoside (24S,25R)-spirost-5-en-3β,24-diol (TV6); E (TV5), (24S,25R)-spirost-5-en-3β,24-diol 3-O-α-L-rhamnopyranosyl- 3-O-α-L-rhamnopyranosyl-(1 2)-[β-Dglucopyranosyl-(1 4)-α-L-rhamnopyranosyl-(1 3)]-β-D-glucopyranoside (TV7); chantrieroside A (TV8) and plantagineoside A (TV9). * 13 compounds from Tacca chantrieri (Figure 3.1): Chantriolide D (TC1), chantriolide E (TC2), chantriolide A (TC3), chantriolide B (TC4), chantriolide C (TC5), (3R,5R)-3,5-dihydroxy-1,7-bis (3,4- dihydroxyphenyl)heptane (TC6), (3R,5R)-3,5-dihydroxy-1,7-bis(3,4-

9 7 dihydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC7), (3R,5R)-3,5- dihydroxy-1,7-bis(4-hydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC8), (3R,5R)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4- hydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC9), (6S,9R) roseoside (TC10), 2-hydroxyphenol-1-O-β-D-glucopyranoside (TC11), 1-O-syringoyl-β-D-glucopyranoside (TC12) and benzyl-β-dglucopyranosyl (1 6)-β-D-glucopyranoside (TC13). Figure 3.2. Chemical structure of compounds from Tacca vietnamensis Figure 3.3. Chemical structure of compounds from Tacca chantrieri Spectral characteristics of taccalonolide and withanolide compounds Spectral characteristics of spirostanol saponin Chemical structure of isolated compounds from Tacca vietnamensis: Compound TV1: Taccavietnamoside A (new compound)

10 8 Figure 3.4. Chemical structure of TV1 and taccasuboside C (65) Compound TV1 was obtained as a white amorphous powder and its molecular formula was determined as C 45H 72O 18 on the basic of HR-ESI-MS pseudo-ion at m/z [M+Na] + (Calcd for [C 45H 72O 18Na] +, ). The 1 H-NMR spectra of TV1 appeared signals including an olefinic protons at δ H 5.28 (br s), four methyl groups at δ H 0.95 (s), 0.99 (s), 1.20 (d, J = 6.5 Hz) and 1.59 (s), which suggested the structure of steroid skeleton. In addition to these, three anomeric protons at δ H 4.85 (d, J = 7.5 Hz), 5.71 (br s) and 5.81 (br s), indicated the presence of three sugar moieties. 13 C-NMR and DEPT data of TV1 showed the presence of 45 carbons, including 5 non-protonated carbons at δ C 37.0, 40.9, 68.5, and 140.7; 24 methine carbons at δ C 31.5, 35.8, 50.2, 56.5, 62.3, 66.0, 69.8, 69.9, 70.5, 72.3, 72.4, 72.5, 72.7, 73.5, 73.7, 77.8, 77.9, 78.3, 81.8, 87.2, 99.8, 102.5, and 121.7; 10 methylen carbons at δ C 21.0, 30.0, 31.9, 32.2, 37.4, 38.6, 40.0, 45.1, 62.2 and 69.1 and 6 methyl groups at δ C 14.5, 16.4, 18.3,18.6, 19.3 and The HMBC correlations between H-4 (δ H 2.64 and 2.70) and C-5 (δ C 140.7)/C-6 (δ C 121.7); between H-19 (δ H 0.95) and C-5 (δ C 140,7) confirmed the position of double bond at C-5/C-6. Moreover, the acetal group at C-22 was confirmed by 13 C-NMR chemical shift of C-22 (δ C 111.5) as well as the HMBC correlations between H-20 (δ H 3.00)/H-21 (δ H 1.20)/H-26 (δ H 3.60 and 4.13) and C-22 (δ C 111.5). Analysis the data of 1 H-, 13 C-NMR and DEPT spectra, chemical shift of C-22 (δ C spiro ring) and the published documents [19, 62], which suggest the compound of TV1 is a spirostanol saponin. The NMR data of TV1 (Table 3.1) were similar to those of taccasuboside C [19] except for signals at C-23, C-24 and C-25 of aglycone: Chemical shift of C-23, C-24, C-25 of TV1 are δ C 66.0, 45.1 and 68.5, respectively

11 9 (Taccasuboside C: δ C 64.6, 43.6, and 70.0 [19], recorded in pyridine-d 5), which suggested the different configuration at C-25. The configurations of hydroxyl groups at C-23 and C-25 were defined as equatorial orientation by ROESY observation between H-21 (δ H 1.20) and H-23 (δ H 3.99); and between H-23 (δ H 3.99) and H-27 (δ H 1.59). Sugars obtained by acid hydrolysis of TV1 were identified as D-glucose and L-rhamnose based on GC analysis (identified as TMS derivatives). In addition, the HMBC cross peaks from rha H-1 ( H 5.81) to glc C-2 ( C 78.3); from rha H-1 ( H 5.71) to glc C-3 ( C 87.2) and from glc H-1 ( H 4.85) to C-3 ( C 77.8) confirmed the sugar linkages as α-l-rhamnopyranosyl- (1 2)-O-[α-L-rhamnopyranosyl-(1 3)]-β-D-glucopyranoside, with the location of sugar moiety at C-3 of aglycone. This was also in good agreement with the 13 C NMR data of trisaccharide reported for taccasuboside C from Tacca subflabellata [19]. Thus, the structure of TV1 was elucidated to be (23S,25R)-spirost-5-en-3β,23,25-triol 3-O-α-L-rhamnopyranosyl-(1 2)-[α-Lrhamnopyranosyl-(1 3)]-β-D-glucopyranoside and named taccavietnamoside A. Figure 3.5. The important HMBC and ROESY correlations of TV1 Figure 3.6. HR-ESI-MS of TV1 Table 3.1. NMR spectral data of TV1 and reference compound C C # C a,b H a,c (mult., J, Hz) Aglycone (m)/1.66 (m) (m)/2.06 (m) (m) (dd. 12.0, 12.0) 2.70 (br d, 12.0) (br s) (m)/1.81 (m) (m) (m)

12 Glc Rha Rha 10 C C # C a,b H a,c (mult., J, Hz) (m) (m)/1.71 (m) (m) (m)/1.97 (m) (m) (t,. 8.5) (s) (s) (q, 7.0) (d, 6.5) (br d, 8.5) (br d, 12.0) 2.57 (m) (d, 10.5) 4.13 (d, 10.5) (s) 3-O (d, 7.5) (dd, 7.5, 8.5) (dd, 8.5, 9.0) (dd, 8.5, 9.0) (m) (br d, 11.5) 4.41 (br d, 11.5) 2 -O (br s) (br s) (dd, 2.5, 9.0) (m) (m) (d, 6.0) 3 -O (br s) (br s) (dd, 2.5, 9.0) (m) (m) (d, 6.0) a Recorded in C 5D 5N, b 125 MHz, c 500 MHz, # δ C of taccasuboside C [19]

13 11 Figure H-NMR spectrum of TV1 Figure C-NMR spectrum of TV1 Figure 3.9. DEPT spectrum TV1 Figure HSQC spectrum of TV1 Figure HMBC spectrum của TV1 Figure ROESY spectrum of TV Compound TV2: Taccavietnamoside B (new compound) Figure Chemical structure of TV2 and reference compound TV1 Compound TV2 was obtained as a white amorphous powder and its molecular formula was determined as C 51H 82O 23 on the basic of HR-ESI-MS pseudo-ion at m/z [M+Na] + (Calcd for [C 51H 82O 23Na] +, ). The 1 H-NMR spectra of TV2 appeared signals including an olefinic protons at δ H 5.27 (br s), four methyl groups at δ H 0.96 (s), 0.99 (s), 1.21 (d, J = 7.0 Hz) and 1.59 (s), which suggested the structure of steroid skeleton. In addition, four

14 12 anomeric protons at δ H 4.85 (d, J = 8.0 Hz), 5.21 (d, J = 8.0 Hz), 5.71 (br s), and 5.76 (br s), indicated the presence of four sugar units. 13 C-NMR and DEPT spectra of TV2 showed the presence of 51 carbons: including 5 non-protonated carbons at δ C 37.0, 41.0, 68.5, and 140.7; 29 methine carbons at δ C 31.5, 35.8, 50.2, 56.6, 62.3, 66.0, 68.7, 69.7, 69.8, 71.4, 72.0, 72.3, 72.4, 72.7, 73.7, 76.3, 77.8, 78.0, 78.3, 78.5, 78.6, 81.8, 84.3, 86.2, 99.8, 102.5, 103.1, and 121.7; 11 methylen carbons at δ C 21.0, 30.0, 32.0, 32.3, 37.4, 38.8, 40.1, 45.2, 62.1, 62.5, and 69.2; and 6 methyl carbons at δ C 14.5, 16.5, 18.2, 18.6, 19.3, and The NMR data and chemical shift at C-22 (δ C spiro ring) on 13 C-NMR spectrum, which suggested TV2 is a spirostanol saponin. The 1 H- and 13 C-NMR data of TV2 were similar to those of taccavietnamoside A (TV1), except for the addition of a sugar unit at C-4 : signals of anomeric proton at δ H 5.21 (d, J = 8.0) and 6 carbons at δ C 62.5, 71.4, 76.3, 78.3, 78.6 and Sugars obtained by acid hydrolysis of TV2 were identified as D-glucose and L-rhamnose based on GC analysis (identified as TMS derivatives). In addition, the HMBC cross peaks from rha H-1 (δ H 5.76) to glc C-2 (δ C 78.5), from glc H-1 (δ H 5.21) tới rha C-4 (δ C 84.3), from rha H-1 (δ H 5.71) to glc C-3 (δ C 86.2), and from glc H-1 (δ H 4.85) to C- 3 (δ C 77.8) confirmed the sugar linkages as O-α-L-rhamnopyranosyl-(1 2)- O-[β-D-glucopyranosyl-(1 4)-O-α-L-rhamnopyranosyl-(1 3)]-β-Dglucopyranoside and the location of sugar at C-3 of aglycone. This sugar moiety was also reported from Tacca chantrieri [29]. Consequently, the structure of TV2 was determined to be (23S,25R)-spirost-5-en-3β,23,25-triol 3-O-α-L-rhamnopyranosyl-(1 2)-[β-D-glucopyranosyl-(1 4)-α-Lrhamnopyranosyl-(1 3)]-β-D-glucopyranoside and named taccavietnamoside B. Figure The important HMBC and COSY correlations of TV2 Figure HR-ESI-MS of TV2

15 13 Table 3.2. NMR spectral data of TV2 and reference compound C C # C a,b DEPT H a,c (mult., J, Hz) Aglycone CH (m)/1.66 (m) CH (m)/2.06 (m) CH 3.86 (m) CH (dd, 12.0, 12.0)/2.69 (dd, 4.5, 12.0) C CH 5.27 (d, 4.5) CH (m)/1.80 (m) CH 1.48 (m) CH 0.86 (m) C CH (m) CH (m)/1.71 (m) C CH 1.05 (m) CH (m)/1.97 (m) CH 4.60 (m) CH 1.88 (t, 7.5) CH (s) CH (s) CH 3.00 (q, 7.0) CH (d, 7.0) C CH 3.97 (br d, 8.5) CH (br d, 11.0)/2.54 (t, 11.0) C CH (d, 10.5)/4.12 (d, 10.5) CH (s) 3-O-Glc CH 4.85 (d, 8.0) CH 4.00 (t, 8.0) CH 4.12 (m) CH 4.05 (t, 8.5) CH 3.76 (m) CH (dd, 3.0, 12.0)/4.40 (dd, 5.0, 12.0) 2 -O-Rha CH 5.76 (br s) CH 4.69 (br s) CH 4.47 (dd, 3.0, 9.0) CH 4.25 (m) CH 4.80 (m) CH (d, 6.5) 3 -O-Rha CH 5.71 (br s) CH 4.82 (br s) CH 4.54 (dd, 2.5, 9.0)

16 14 C C # C a,b DEPT H a,c (mult., J, Hz) CH 4.39 (m) CH 4.76 (m) CH (d, 6.0) 4 -O-Glc CH 5.21 (d, 8.0) CH 4.05 (m) CH 4.02 (m) CH 4.23 (t, 9.0) CH 3.76 (m) CH (dd, 3.0, 12.0)/4.40 (dd, 5.0, 12.0) a Recorded in C 5D 5N, b 125 MHz, c 500 MHz, # δ C of taccavietnamoside A (TV1) Figure H-NMR spectrum of TV2 Figure C-NMR spectrum of TV2 Figure DEPT spectrum of TV2 Figure HSQC spectrum of TV2 Figure HMBC spectrum of TV2 Figure COSY of TV2 Figure ROESY of TV2

17 Chemical structure of isolated compounds from Tacca chantrieri Compound TC1: Chantriolide D (new compound) Figure Chemical structure of TC1 and taccanlonolide M (13) Compound TC1 was obtained as a white amorphous powder. Its molecular formula was assigned as C 35H 50O 15 on the basic of HR-ESI-MS pseudo-ion at m/z [M+H] + (Calcd for [C 35H 51O 15] +, ) and m/z [M+Na] + (Calcd for [C 35H 50O 15Na] +, ). The 1 H-NMR spectra of TC1 exhibited signals for four methyl groups at δ H 0.76 (3H, s), 1.13 (3H, s), 0.80 (3H, d, J = 6.0 Hz) and 1.17 (3H, d, J = 6.0 Hz), two methyl acetyl groups at δ H 1.91 (3H, s) and 2.06 (3H, s), which suggested the structure a steroid with two acetyl groups. In addition, anomeric protons at δ H 4.20 (d, J = 8.0 Hz), indicated the presence of a sugar unit. The 13 C-NMR and DEPT spectra of TC1 revealed the presence of 35 carbons, including: 2 ketone carbons at δ C and 211.7; 2 acetyl carbonyl carbons at δ C and 170.5, 3 non-protonated carbons at δ C 41.9, 42.6, and 81.0; 18 methine carbons at δ C 31.7, 37.1, 41.6, 41.8, 51.0, 51.8, 53.7, 54.3, 54.9, 70.8, 72.7, 74.3, 74.6, 77.1, 77.4, 78.2, 86.0 and 105.5; 4 methylene carbons at δ C 25.2, 29.6, 44.5, and 61.9 and 6 methyl carbons at δ C 13.3, 15.3, 15.3, 19.8, 20.2 and All these data coupled with a literature survey indicated that TC1 was a steroidal glucoside [15]. The HMBC correlations HMBC between H-6 (δ H 4.22) and C-5 (δ C 81.0)/C-7 (δ C 206.0)/C-10 (δ C 42.6); between H-14 (δ H 2.74)/H-16 (δ H 1.46)/H-17 (δ H 1.65) and C-15 (δ C 211.7) confirmed the positions of two hydroxy groups at C-5 and C-6, two ketone groups at C-7 and C-15. The 13 C-NMR chemical shift to a higher field at C-2 (δ C 51.0), C-3 (δ C 54.9) and correlation HMBC from H-4 (δ H 2.37) to C-2 (δ C 50.1)/C-3 (δ C 54.9) suggested the epoxy group at C-2/C-3. Two

18 16 acetoxy groups at C-1 and C-12 were confirmed by HMBC correlations from HMBC to H-1 (δ H 4.67) and H-12 (δ H 4.93) to acetyl carbonyls (δ C and 170.5), respectively. The HMBC correlations between H-19 (δ H 1.13) and C- 1(δ C 72.7)/C-5 (δ C 81.0)/C-9 (δ C 37.1)/C-10 (δ C 42.6); between H-18 (δ H 0.76) and C-12 (δ C 74.3), C-13 (δ C 41.9), C-14 (δ C 54.3), C-17 (δ C 51.8); between H-21 (δ H 0.80) and C-17 (δ C 51.8), C-20 (δ C 31.7), C-22 (δ C 44.5); between H-25 (δ H 1.17) and C-16 (δ C 53.7), C-23 (δ C 86.0), C-24 (δ C 41.8) confirmed the positions of four methyl groups at C-10, C-13, C-20 and C-24, respectively. Acid hydrolysis of TC1 gave D-glucose (identified as TMS derivative by GC). The location of the sugar moiety at C-23 was confirmed by HMBC correlation from glc H-1 (δ H 4.20) to aglycone C-23 (δ C 86.0). The configuration of the oxygenated groups at C-1, C-2, C-6, C-12 was defined as α-orientations, based on the similarity of the 13 C-NMR spectral data from C-1 to C-19 of TC1 and taccanlonolide M [15]. In addition, the α-orientations of the oxygenated groups at C-1, C-2, C-6, C-12 were based on the observation of ROE correlations on ROESY spectrum between H-18 (δ H 0.76) and H-12 (δ H 4.93)/H-8 (δ H 2.59); H-19 (δ H 1.13) and H-1 (δ H 4.67)/H-2 (δ H 3.57)/H-6 (δ H 4.22)/H-8 (δ H 2.59). The α-orientation of oxygenated group at C-23 was based on the ROE correlations between H-23 (δ H 3.10) and H-16 (δ H 1.46)/H- 25 (δ H 1.17). Thus, the structure of TC1 was determined and named chantriolide D. Figure The important HMBC correlations of TC1 Table 3.9. NMR spectral data of TC1 and reference compound C # C a,b C DEPT a,c H (mult., J, Hz) Aglycone CH 4.67 (d, 5.0) CH 3.57 (t, 5.0) CH 3.51 (m) CH (d, 16.0)/2.07* C -

19 17 C # C a,b C DEPT a,c H (mult., J, Hz) CH 4.22* C CH 2.59 (dd, 11.5, 12.0) CH 2.21 (dd, 4.0, 12.0) C CH (dd, 4.0, 15.0)/1.79 (br d, 15.0) CH 4.93 (br s) C CH 2.74 (d, 11.5) C CH 1.46 (dd, 11.5, 11.5) CH 1.65 (dd, 11.5, 11.5) CH (s) CH (s) CH 1.52 (m) CH (d, 6.0) CH */2.13 (m) CH 3.10* CH 1.63 (m) CH (d, 6.0) 1-OAc CH (s) 12-OAc CH (s) 23-OGlc CH 4.20 (d, 8.0) CH 2.98 (t, 8.0) CH 3.17* CH 3.10* CH 3.10* CH (dd, 4.0, 11.5)/3.66 (br d, 11.5) a Recorded in CD3OD, b 125MHz, c 500MHz, # C of taccanlonolide M [15], * Overlapped signals Compound TC2: Chantriolide E (new) Compound TC2 molecular formula was assigned as C 36H 51O 15+Cl on the basic of HR-ESI-MS pseudo-ion at m/z [M+Na] + (Calcd for [C 36H 51O 15ClNa] +, ). The 1 H-NMR spectra of TC2 appeared signals of four methyl group protons: three tertiary methyl groups at δ H 0.94 (3H, s), 1.09 (3H, s) and 2.14 (3H, s), one second methyl group at δ H 1.01 (3H, d, J = 7.0 Hz); one methyl acetyl group δ H 2.13 (H, br s); one anomeric proton at δ H 4.36 (H, d, J = 8.0 Hz). The 13 C-NMR and DEPT spectra of TC2 showed the signals of 36 carbons including 3 carbonyl carbons at δ C 167.9, 172.3, 2 and 218.1; 5 non-protonated carbon at δ C 42.0, 47.9, 74.7, 123.8, and 159.6; 17 methine carbons at δ C 30.5, 35.4,

20 , 41.3, 56.4, 57.3, 57.4, 60.4, 71.6, 74.6, , 76.7, 77.9, 78.0, 78.7 and 103.9; 6 methylene carbons at δ C 25.4, 33.1, 38.1, 43.8, 62.8 and 63.5; 5 methyl carbons at δ C 13.4, 14.8, 15.5, 20.7 and Figure Chemical structure of TC2 and plantagiolide I (46) The NMR spectra data of TC2 were similar to those of plantagiolide I [5], the main difference was the absence of the acetoxy group at C-2. The HMBC correlation between H-19 (δh 0.94) and C-1 (δ C 76.7)/C-5 (δ C 74.7)/C-9 (δ C 30.5)/C-10 (δ C 42.0); H-18 (δ H 1.09) and C-12 (δ C 75.1)/C-13 (δ C 47.9)/C-14 (δ C 41.3)/C-17 (δ C 57.4); H-21 (δ H 1.01) and C-17 (δ C 57.4)/C-20 (δ C 36.5)/C-22 (δ C 78.7); H-28 (δ H 2.14) and C-23 (δ C 33.1)/C-24 (δ C 159.6)/C-25 (δ C 123.8) showed position of 4 methyl groups at C-10, C-13, C-20 and C-24. The HMBC correlation from methyl proton (δ H 2.13), aglycone H-12 (δ H 5.18) to acetoxy carbonyl groups (δ C 172.3) confirmed position of this acetoxy group at C-12. The 13 C-NMR chemical shift of C-6, C-7 was shifted to a higher field [C-6 (δ C 57.3), C-7 (δ C 56.4)] and the HMBC correlation from H-6 (δ H 2.99) to C- 5 (δ C 74.7), suggesting the presence of a epoxy ring at C-6/C-7 and OH group at C-5. The HMBC correlation from H-27 (δ H 4.65) to C-24 (δ C 159.6)/C-25 (δ C 123.8)/C-26 (δ C 167.9) showed position of carbonyl group at C-26 and double bond at C-24/C-25. The HMBC correlation from H-15 (δ H 2.49)/H-17 (δ H 2.72) to C-16 (δ C 218.1), suggesting the presence of oxo group at C-16. Acid hydrolysis of TC2 gave D-glucose

21 19 (identified as TMS derivative by GC). The sugar at C-27 was proved by HMBC correlation between glc H-1 (δ H 4.36) to C-27 (δ C 63.5). The 13 C NMR chemical shift of C-3 (δ C 60.4) was shifted to higher field compared with that of the oxymethine carbon C-2 (δ C 74.6), suggesting the presence of a chlorine atom at C-3. The HR-ESI-MS of TC2 showed pseudo-molecular ion peaks at m/z [C 36H 51O 15Cl 35 +Na] + and m/z [C 36H 51O 15Cl 37 +Na] + (Calcd for [C 36H 51O 15Cl 35 +Na] + : and [C 36H 51O 15Cl 37 +Na] + : ), confirming the presence of chlorine atom in TC2. The configuration of chlorine at C-3 was determined as β (equatorial) by the large coupling constant, J = 10.0 Hz, between H-2 and H-3. The constitution of TC2 was confirmed by a detailed interpretation of 2D-NMR spectra, including HSQC, HMBC, COSY, and ROESY. Thus, the structure of 2 was established and named chantriolide E. Figure The important HMBC correlations of TC2 Table NMR spectral data of TC2 and reference compound C C # C a,b DEPT H a,c (mult., J, Hz) Aglycone CH 3.57 (d, 4.0) CH 3.94 (dd, 4.0, 10.0) CH 4.36 (m) CH2 2.19*/2.33 (dd. 6.6, 13.5) C CH 2.99 (d, 3.0) CH 3.36 (dd, 2.0, 3.0) CH 2.19 (m) CH 2.27 (m) C CH (t, 12.0)/2.01*

22 20 C C # C a,b DEPT H a,c (mult., J, Hz) CH 5.18 (br s) C CH 2.50 (m) CH (m)/2.49 (m) C CH 2.72 (d, 7.5) CH (s) CH (s) CH 2.38 (m) CH (d, 7.0) CH 4.92 (m) CH (m)/2.50 (m) C C C CH (d, 11.5)/4.65 (d, 11.5) CH (s) 12-OAc C CH (s) 27-OGlc CH 4.36 (d, 8.0) CH 3.20 (t, 8.0) CH 3.37 (m) CH 3.32 (m) CH 3.30 (m) CH (dd, 2.0, 12.0)/3.89 (dd, 5.4, 12.0) a Recorded in CD3OD, b 125MHz, c 500MHz, # C of plantagiolide I [5], * Overlapped signals Figure HR-ESI-MS spectrum of TC2 Figure H-NMR spectrum of TC2 Figure C-NMR spectrum of TC2 Figure DEPT spectrum of TC2

23 21 Figure 3.31.HSQC spectrum of TC2 Figure 3.32.HMBC spectrum of TC2 Figure 3.33.COSY spectrum of TC2 Figure 3.34.ROESY spectrum of TC Biological activities of isolated compounds Anti-inflammatory activity of isolated compounds 22 compounds from Tacca vietnamensis and Tacca chantrieri were evaluated for their inhibitory activity on NO production in BV2 cells, LPS-stimulated. As results, compounds TV3-TV5 inhibited NO production in BV2 cells, LPS-stimulated, with IC 50 values of 52.1 ± 3.6 µm, 47.3 ± 6.0 µm, 43.7 ± 4.2 µm, respectively. Butein was used as a positive control, IC 50 of 4.3 ± 0.5 µm. Chantriolide D (TC1) and chantriolide E (TC2) inhibited NO production in BV2 cells, LPSstimulated, with IC 50 of 12.4 ± 2.4 µm and 59.0 ± 3.5 μm. Butein was used as a positive control, IC 50 of 4.3 ± 0.8 µm Cytotoxic activities of isolated compound from Tacca chantrieri 13 compounds from Tacca chantrieri were evaluated for cytotoxic activities toward four human cancer lines, including PC-3, LNCaP, MDA-MB-231 and HepG2 cells. The results showed that the new withanolide glucoside (chantriolide E) exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP, and MDA-MB-231 with IC 50 of 24.5 ± 1.2 µm, 19.0 ± 1.5 µm, 20.9 ± 1.6 µm, respectively. TC7 exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP and MDA-MB-231 with IC 50 of 30.7 ± 1.5, 19.1 ± 1.4 and

24 ± 1.5 µm, respectively. TC9 exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP and MDA-MB-231 with IC 50 of 30.8 ± 2.0, 20.2 ± 1.2 and 49.3 ± 3.2 µm. TC13 exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP and MDA- MB-231 with IC 50 of 17.9 ± 1.8, 18.8 ± 1.3 and 22.0 ± 2.0 µm, respectively. Ellipticine was used as a positive control (IC 50 of 1.1 ± 0.1, 0.7 ± 0.1, 0.8 ± 0.1µM, respectively). CONCLUSIONS This is the first study on chemical constituents and biological activities of Tacca vietnamensis and biological activities of Tacca chantrieri growing in Vietnam. 1. Nine compounds were isolated and identified from Tacca vietnamensis. Among them, five new compounds: 5 spirostanol saponin: taccavietnamosides A-E (TV1-TV5). Four known compounds: 3 spirostanol glycoside: (24S,25R)-spirost-5-en-3β,24-diol 3-O-α-L- rhamnopyranosyl-(1 2)-[α-L-rhamnopyranosyl-(1 3)]-β-D- glucopyranoside (TV6), (24S,25R)-spirost-5-en-3β,24-diol 3-O-α-L- rhamnopyranosyl-(1 2)-[β-D-glucopyranosyl-(1 4)-α-L- rhamnopyranosyl-(1 3)]-β-D-glucopyranoside (TV7), chantrieroside A (TV8); one diaryl heptanoid glycoside: plantagineoside A (TV9). 2. Thirteen compounds were isolated and identified from Tacca chantrieri, including: Two new compounds: one taccalonolide: Chantriolide D (TC1) and one withanolide glucoside: Chantriolide E (TC2). Eleven known compounds: Three withanolide glycoside: Chantriolide A (TC3), chantriolide B (TC4) and chantriolide C (TC5); four knowed diaryl heptanoid glycoside: (3R,5R)-3,5-dihydroxy-1,7-bis (3,4-dihydroxyphenyl)heptane (TC6), (3R,5R)-3,5-dihydroxy-1,7-bis(3,4- dihydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC7), (3R,5R)-3,5- dihydroxy-1,7-bis(4-hydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC8) and (3R,5R)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4- hydroxyphenyl)heptane 3-O-β-D-glucopyranoside (TC9); one megastigmane: (6S,9R)-roseoside (TC10); three compounds were isolated from Tacca genus for the first time: 2-hydroxyphenol-1-O-β-D-

25 23 glucopyranoside (TC11), 1-O-syringoyl-β-D-glucopyranoside (TC12) and benzyl-β-d-glucopyranosyl (1 6)-β-D-glucopyranoside (TC13). 3. Twenty-wo isolated compounds from Tacca vietnamensis and Tacca chantrieri were tested for their inhibitory activity on NO production in activated BV2 cells. As the results, spirostanol saponin compounds (TV3-TV5) were isolated from Tacca vietnamensis showed inhibitory activity on NO production in LPS-stimulated BV2 cells with IC 50 values of 52.1 ± 3.6 µm, 47.3 ± 6.0 µm, 43.7 ± 4.2 µm, respectively. Compounds chantriolide D (TC1) and chantriolide E (TC2) were isolated from Tacca chantrieri showed significant inhibitory activity on NO production in LPS-stimulated BV2 cells with IC 50 values of 12.4 ± 2.4 µm and 59.0 ± 3.5 μm, respectively. 4. Thirteen isolated compounds (TC1-TC13) from Tacca chantrieri species were evaluated for cytotoxic activity on four human cancer cell lines, including PC-3, LNCaP, MDA-MB-231 and HepG2. The chantriolide E (TC2) exhibited moderate activity on PC-3, LNCaP and MDA-MB-231 cell lines with IC 50 values of 24.5 ± 1.2 µm, 19.0 ± 1.5 µm, and 20.9 ± 1.6 µm, respectively. The results of the thesis also supplemented the claims of cytotoxic activity on the new cancer lines (PC-3, LNCaP, MDA-MB-231) of known compounds: Chantriolide A, two diaryl heptanoid glycoside (TC7, TC9) and one benzyl glycoside (TC13) in normal values of IC µm. RECOMMENDATIONS From the research results: Spirostanol saponin TV3-TV5, chantriolide D (TC1) and chantriolide E (TC2) showed significant inhibitory activity on NO production in LPS-stimulated BV2. Therefore, further research is needed on the applicability of these compounds in practice. Chantriolide E has demonstrated significant anti-inflammatory activity and demonstrated the inhibition activity of PC-3, LNCaP and MDA-MB-231 cancer cell lines. Therefore, there should be more research about the activity of this compound for use as medicines.

26 24 NEW FINDINGS OF THE THESIS compounds were isolated and identified from Tacca vietnamensis and Tacca chantrieri, including: - Seven new compounds: 5 spirostanol saponins: taccavietnamosides A-E; 1 taccalonolide: Chantriolide D; 1 withanolide glucoside: Chantriolide E. - 3 compounds were isolated for the first time from Tacca: 2- hydroxyphenol-1-o-β-d-glucopyranoside, 1-O-syringoyl-β-Dglucopyranoside, and benzyl-β-d-glucopyranosyl (1 6)-β-Dglucopyranoside compounds from Tacca vietnamensis and Tacca chantrieri were evaluated for their inhibitory activity on NO production in BV2 cells, LPS-stimulated for the first time. As results, taccavietnamosides C- E inhibited NO production in BV2 cells, LPS-stimulated, with IC 50 values of 52.1 ± 3.6 µm, 47.3 ± 6.0 µm, 43.7 ± 4.2 µm, respectively. Chantriolide D and chantriolide E inhibited NO production in BV2 cells, LPS-stimulated, with IC 50 of 12.4 ± 2.4 µm and 59.0 ± 3.5 μm compounds from Tacca chantrieri were evaluated for cytotoxic activities the first time toward four human cancer lines, including PC-3, LNCaP, MDA-MB-231 and HepG2 cells. The results showed that the new withanolide glucoside (chantriolide E) exhibited cytotoxic activities against three human cancer cell lines, PC-3, LNCaP, and MDA-MB-231 with IC 50 of 24.5 ± 1.2 µm, 19.0 ± 1.5 µm, 20.9 ± 1.6 µm, respectively. The cytotoxic activity of known compounds also reported for the first time. Chantriolide A, two diaryl heptanoid glycosides: (3R,5R)-3,5- dihydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane 3-O-β-D-glucopyranoside and (3R,5R)-3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl) heptane 3-O-β-D-glucopyranoside), and one benzyl glycoside, benzyl-β-dglucopyranosyl (1 6)-β-D-glucopyranoside showed moderate cytotoxic activity on 3 human cancer cell lines, PC-3, LNCaP, and MDA-MB-231 with IC 50 values ranging of μm.

27 25 PUBLICATIONS WITHIN THE SCOPE OF THESIS 1. Pham Hai Yen, Vu Thi Quynh Chi, Phan Van Kiem, Bui Huu Tai, Tran Hong Quang, Nguyen Xuan Nhiem, Hoang Le Tuan Anh, Ninh Khac Ban, Bui Van Thanh, Chau Van Minh, Seung Hyun Kim. Spirostanol saponins from Tacca vietnamensis and their anti-inflammatory activity. Bioorganic & Medicinal Chemistry Letters, 2016, 26, Pham Hai Yen, Vu Thi Quynh Chi, Dong-Cheol Kim, Wonmin Ko, Hyuncheol Oh, Youn-Chul Kim, Duong Thi Dung, Nguyen Thi Viet Thanh, Tran Hong Quang, Nguyen Thi Thanh Ngan, Nguyen Xuan Nhiem, Hoang Le Tuan Anh, Chau Van Minh, Phan Van Kiem. Steroidal glucosides from the rhizomes of Tacca chantrieri and their inhibitory activities of NO production in BV2 cells. Natural Product Communications. 2016, 11(1), Vu Thi Quynh Chi, Pham Hai Yen, Duong Thi Dung, Nguyen Xuan Nhiem, Hoang Le Tuan Anh, Dan Thi Thuy Hang, Chau Van Minh, Phan Van Kiem. Withanolide glucoside from the rhizomes of Tacca chantrieri. Vietnam Journal of Chemistry, 2015, 53(2e), Vu Thi Quynh Chi, Pham Hai Yen, Nguyen Xuan Nhiem, Bui Huu Tai, Hoang Le Tuan Anh, Nguyen Thi Viet Thanh, Chau Van Minh, Phan Van Kiem. Spirostanol saponins from Tacca vietnamensis. Vietnam Journal of Chemistry, 2015, 53(6e3), Vũ Thị Quỳnh Chi, Nguyễn Xuân Nhiệm, Dương Thị Dung, Đỗ Thanh Tuân, Hoàng Lê Tuấn Anh, Đỗ Thị Hà, Châu Văn Minh, Phan Văn Kiệm, Phạm Hải Yến. Nghiên cứu thành phần hóa học của thân rễ cây râu hùm (Tacca chantrieri). Tạp chí Dược liệu. 2015, 20(6), Vũ Thị Quỳnh Chi, Phạm Hải Yến, Nguyễn Xuân Nhiệm, Dương Thị Dung, Đan Thị Thúy Hằng, Bùi Hữu Tài, Hoàng Lê Tuấn Anh, Nguyễn Thị Việt Thanh, Châu Văn Minh, Phan Văn Kiệm. Các hợp chất diarylheptanoid phân lập từ thân rễ cây râu hùm (Tacca chantrieri). Tạp chí Hóa học. 2016, 54(2e),