Summary of Chemistry doctoral thesis: Study on chemical constituents and biological activities of Tacca Vietnamensis and Tacca chantrieri growing in Vietnam

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The aim of the thesis: Study on chemical constituents of two Tacca species including Tacca vietnamensis and Tacca chantrierri growing in Vietnam; evaluate cytotoxic and inflammatory activities of isolates to find out bioactive compounds.

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Nội dung Text: Summary of Chemistry doctoral thesis: Study on chemical constituents and biological activities of Tacca Vietnamensis and Tacca chantrieri growing in Vietnam

MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING 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: 9.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Hanoi - 2018
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 1st Reviewer: 2nd Reviewer: 3rd Reviewer: The thesis will be defended at Graduate University of Science and Technology - Vietnam Academy of Science and Technology, at 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.
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.
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. 1.1. 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]. 1.2. 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, …
3 1.3. Introduction to cancer Introduction to cancer and some treatments; Some types of cancer drugs are naturally derived. 1.4. 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. 2.2. Methods 2.2.1. Methods for isolation Chromatographic methods such as thin layer chromatography (TLC), column chromatography (CC). 2.2.2. 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). 2.2.3. 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. 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 Tacca vietnanensis:
4 This section presents the process of isolating the compounds from Tacca vietnamensis. Figure 2.1. Isolation of compounds from Tacca vietnamensis 2.3.2. 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
5 2.4. Physical properties and spectroscopic data of the isolated compounds 2.4.1. 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. 2.4.2. 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. 2.5. Results on biological activities of isolated compounds 2.5.1. 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 (%) TV1 45.1 ± 2.2 TV5 72.0 ± 2.5 TV8 42.2 ± 1.8 TV2 43.2 ± 1.8 TV6 40.0 ± 2.0 TV9 40.1 ± 3.0 TV3 63.2 ± 1.5 TV7 46.9 ± 2.2 Butein* 90.0 ± 5.0 TV4 67.5 ± 2.1 (10 µM) Table 2.2. Inhibitory NO effects of compounds TV3-TV5 in the LPS-stimulated BV2 cells Comp. IC50 (µM) Comp. IC50 (µM) TV3 52.1 ± 3.6 TV5 43.7 ± 4.2 TV4 47.3 ± 6.0 Butein* 4.3 ± 0.5 - 13 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 (%) TC1 85.1 ± 4.5 TC6 47.4 ± 2.5 TC11 40.8 ± 2.0 TC2 63.8 ± 3.6 TC7 42.0 ± 2.1 TC12 36.8 ± 2.8 TC3 43.2 ± 2.4 TC8 42.0 ± 3.0 TC13 28.7 ± 1.9 TC4 47.1 ± 2.5 TC9 45.7 ± 2.2 Butein (10 78.0 ± 4.2 TC5 46.5 ± 3.3 TC10 44.3 ± 2.1 µM)
6 Table 2.4. Inhibitory NO effects of compounds TC1-TC2 in the LPS-stimulated BV2 cells Comp. IC50 (µM) Comp. IC50 (µM) TC1 12.4 ± 2.4 Butein 4.3 ± 0.8 TC2 59.0 ± 3.5 2.5.2. 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 TC2 24.5 ± 1.2 19.0 ± 1.5 20.9 ± 1.6 TC7 30.7 ± 1.5 19.1 ± 1.4 24.2 ± 1.5 TC9 30.8 ± 2.0 20.2 ± 1.2 49.3 ± 3.2 TC13 17.9 ± 1.8 18.8 ± 1.3 22.0 ± 2.0 Ellipticine 1.1 ± 0.1 0.7 ± 0.1 0.8 ± 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 E (TV5), (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) 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-
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-D- glucopyranosyl (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 3.1.1. Spectral characteristics of taccalonolide and withanolide compounds 3.1.2. Spectral characteristics of spirostanol saponin 3.1.3. Chemical structure of isolated compounds from Tacca vietnamensis: 3.1.3.1 Compound TV1: Taccavietnamoside A (new compound)
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 C45H72O18 on the basic of HR-ESI-MS pseudo-ion at m/z 923.4607 [M+Na]+ (Calcd for [C45H72O18Na]+, 923.4611). The 1H-NMR spectra of TV1 appeared signals including an olefinic protons at δH 5.28 (br s), four methyl groups at δH 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 δH 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 δC 37.0, 40.9, 68.5, 111.5 and 140.7; 24 methine carbons at δC 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, 103.7 and 121.7; 10 methylen carbons at δC 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 δC 14.5, 16.4, 18.3,18.6, 19.3 and 26.1. The HMBC correlations between H-4 (δH 2.64 and 2.70) and C-5 (δC 140.7)/C-6 (δC 121.7); between H-19 (δH 0.95) and C-5 (δC 140,7) confirmed the position of double bond at C-5/C-6. Moreover, the acetal group at C-22 was confirmed by 13C-NMR chemical shift of C-22 (δC 111.5) as well as the HMBC correlations between H-20 (δH 3.00)/H-21 (δH 1.20)/H-26 (δH 3.60 and 4.13) and C-22 (δC 111.5). Analysis the data of 1H-, 13C-NMR and DEPT spectra, chemical shift of C-22 (δC111.5- 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 δC 66.0, 45.1 and 68.5, respectively
9 (Taccasuboside C: δC 64.6, 43.6, and 70.0 [19], recorded in pyridine-d5), 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 (δH 1.20) and H-23 (δH 3.99); and between H-23 (δH 3.99) and H-27 (δH 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 13C 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-L- rhamnopyranosyl-(1→3)]-β-D-glucopyranoside and named taccavietnamoside A. Figure 3.5. The important HMBC Figure 3.6. HR-ESI-MS of TV1 and ROESY correlations of TV1 Table 3.1. NMR spectral data of TV1 and reference compound C C# Ca,b Ha,c(mult., J, Hz) Aglycone 1 37.5 37.4 0.91 (m)/1.66 (m) 2 30.1 30.0 1.80 (m)/2.06 (m) 3 77.9 77.8 3.88 (m) 4 38.7 38.6 2.64 (dd. 12.0, 12.0) 2.70 (br d, 12.0) 5 140.8 140.7 - 6 121.8 121.7 5.28 (br s) 7 32.4 32.2 1.45 (m)/1.81 (m) 8 31.6 31.5 1.48 (m) 9 50.3 50.2 0.85 (m)
10 C C# C a,b Ha,c(mult., J, Hz) 10 37.2 37.0 - 11 21.1 21.0 1.38 (m) 12 40.2 40.0 1.11 (m)/1.71 (m) 13 41.1 40.9 - 14 56.7 56.5 1.05 (m) 15 32.3 31.9 1.45 (m)/1.97 (m) 16 81.9 81.8 4.60 (m) 17 62.6 62.3 1.88 (t,. 8.5) 18 16.6 16.4 0.99 (s) 19 19.4 19.3 0.95 (s) 20 35.8 35.8 3.00 (q, 7.0) 21 14.9 14.5 1.20 (d, 6.5) 22 112.2 111.5 - 23 64.6 66.0 3.99 (br d, 8.5) 24 43.6 45.1 2.47 (br d, 12.0) 2.57 (m) 25 70.0 68.5 - 26 69.3 69.1 3.60 (d, 10.5) 4.13 (d, 10.5) 27 26.9 26.1 1.59 (s) 3-O- Glc 1′ 99.9 99.8 4.85 (d, 7.5) 2′ 78.4 78.3 4.00 (dd, 7.5, 8.5) 3′ 87.5 87.2 4.12 (dd, 8.5, 9.0) 4′ 69.9 69.8 4.00 (dd, 8.5, 9.0) 5′ 78.1 77.9 3.77 (m) 6′ 62.3 62.2 4.29 (br d, 11.5) 4.41 (br d, 11.5) 2′-O- Rha 1′′ 102.7 102.5 5.81 (br s) 2′′ 72.5 72.3 4.72 (br s) 3′′ 72.9 72.7 4.46 (dd, 2.5, 9.0) 4′′ 73.9 73.7 4.29 (m) 5′′ 69.9 69.9 4.82 (m) 6′′ 18.7 18.6 1.72 (d, 6.0) 3′-O- Rha 1′′′ 103.9 103.7 5.71 (br s) 2′′′ 72.5 72.4 4.81 (br s) 3′′′ 72.7 72.5 4.48 (dd, 2.5, 9.0) 4′′′ 73.6 73.5 4.29 (m) 5′′′ 70.7 70.5 4.75 (m) 6′′′ 18.5 18.3 1.62 (d, 6.0) a Recorded in C5D5N, b125 MHz, c 500 MHz, # δC of taccasuboside C [19]
11 Figure 3.7. 1H-NMR spectrum of TV1 Figure 3.8. 13C-NMR spectrum of TV1 Figure 3.9. DEPT spectrum TV1 Figure 3.10. HSQC spectrum of TV1 Figure 3.11. HMBC spectrum của TV1 Figure 3.12. ROESY spectrum of TV1 3.1.3.2 Compound TV2: Taccavietnamoside B (new compound) Figure 3.13. Chemical structure of TV2 and reference compound TV1 Compound TV2 was obtained as a white amorphous powder and its molecular formula was determined as C51H82O23 on the basic of HR-ESI-MS pseudo-ion at m/z 1085.5133 [M+Na]+ (Calcd for [C51H82O23Na]+, 1085.5139). The 1H-NMR spectra of TV2 appeared signals including an olefinic protons at δH 5.27 (br s), four methyl groups at δH 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
12 anomeric protons at δH 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 δC 37.0, 41.0, 68.5, 111.5 and 140.7; 29 methine carbons at δC 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, 106.4 and 121.7; 11 methylen carbons at δC 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 δC 14.5, 16.5, 18.2, 18.6, 19.3, and 26.2. The NMR data and chemical shift at C-22 (δC111.5- spiro ring) on 13C-NMR spectrum, which suggested TV2 is a spirostanol saponin. The 1H- and 13C-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 δH 5.21 (d, J = 8.0) and 6 carbons at δC 62.5, 71.4, 76.3, 78.3, 78.6 and 106.4. 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″ (δH 5.76) to glc C-2′ (δC 78.5), from glc H-1″″ (δH 5.21) tới rha C-4‴ (δC 84.3), from rha H-1‴ (δH 5.71) to glc C-3′ (δC 86.2), and from glc H-1′ (δH 4.85) to C- 3 (δC 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-D- glucopyranoside 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-L- rhamnopyranosyl-(1→3)]-β-D-glucopyranoside and named taccavietnamoside B. Figure 3.15. HR-ESI-MS of Figure 3.14. The important HMBC and TV2 COSY correlations of TV2
13 Table 3.2. NMR spectral data of TV2 and reference compound C C# Ca,b DEPT Ha,c (mult., J, Hz) Aglycone 1 37.4 37.4 CH2 0.92 (m)/1.66 (m) 2 30.0 30.0 CH2 1.80 (m)/2.06 (m) 3 77.8 77.8 CH 3.86 (m) 4 38.6 38.8 CH2 2.63 (dd, 12.0, 12.0)/2.69 (dd, 4.5, 12.0) 5 140.7 140.7 C - 6 121.7 121.7 CH 5.27 (d, 4.5) 7 32.2 32.3 CH2 1.42 (m)/1.80 (m) 8 31.5 31.5 CH 1.48 (m) 9 50.2 50.2 CH 0.86 (m) 10 37.0 37.0 C - 11 21.0 21.0 CH2 1.38 (m) 12 40.0 40.1 CH2 1.11 (m)/1.71 (m) 13 40.9 41.0 C - 14 56.5 56.6 CH 1.05 (m) 15 31.9 32.0 CH2 1.43 (m)/1.97 (m) 16 81.8 81.8 CH 4.60 (m) 17 62.3 62.3 CH 1.88 (t, 7.5) 18 16.4 16.5 CH3 0.99 (s) 19 19.3 19.3 CH3 0.96 (s) 20 35.8 35.8 CH 3.00 (q, 7.0) 21 14.5 14.5 CH3 1.21 (d, 7.0) 22 111.5 111.5 C - 23 66.0 66.0 CH 3.97 (br d, 8.5) 24 45.1 45.2 CH2 2.47 (br d, 11.0)/2.54 (t, 11.0) 25 68.5 68.5 C - 26 69.1 69.2 CH2 3.60 (d, 10.5)/4.12 (d, 10.5) 27 26.1 26.2 CH3 1.59 (s) 3-O-Glc 1′ 99.8 99.8 CH 4.85 (d, 8.0) 2′ 78.3 78.5 CH 4.00 (t, 8.0) 3′ 87.2 86.2 CH 4.12 (m) 4′ 69.8 69.7 CH 4.05 (t, 8.5) 5′ 77.9 78.0 CH 3.76 (m) 6′ 62.2 62.1 CH2 4.29 (dd, 3.0, 12.0)/4.40 (dd, 5.0, 12.0) 2′-O-Rha 1′′ 102.5 102.5 CH 5.76 (br s) 2′′ 72.3 72.4 CH 4.69 (br s) 3′′ 72.7 72.7 CH 4.47 (dd, 3.0, 9.0) 4′′ 73.7 73.7 CH 4.25 (m) 5′′ 69.9 69.8 CH 4.80 (m) 6′′ 18.6 18.6 CH3 1.72 (d, 6.5) 3′-O-Rha 1′′′ 103.7 103.1 CH 5.71 (br s) 2′′′ 72.4 72.0 CH 4.82 (br s) 3′′′ 72.5 72.3 CH 4.54 (dd, 2.5, 9.0)
14 C C# Ca,b DEPT Ha,c (mult., J, Hz) 4′′′ 73.5 84.3 CH 4.39 (m) 5′′′ 70.5 68.7 CH 4.76 (m) 6′′′ 18.3 18.2 CH3 1.66 (d, 6.0) 4′′′-O-Glc 1′′′′ 106.4 CH 5.21 (d, 8.0) 2′′′′ 76.3 CH 4.05 (m) 3′′′′ 78.6 CH 4.02 (m) 4′′′′ 71.4 CH 4.23 (t, 9.0) 5′′′′ 78.3 CH 3.76 (m) 6′′′′ 62.5 CH2 4.29 (dd, 3.0, 12.0)/4.40 (dd, 5.0, 12.0) a Recorded in C5D5N, b125 MHz, c 500 MHz, #δC of taccavietnamoside A (TV1) Figure 3.16. 1H-NMR Figure 3.17. 13C-NMR spectrum of TV2 spectrum of TV2 Figure 3.18. DEPT spectrum Figure 3.19. HSQC spectrum of TV2 of TV2 Figure 3.20. HMBC spectrum Figure 3.21. COSY of Figure 3.22. ROESY of TV2 TV2 of TV2
15 3.1.4. Chemical structure of isolated compounds from Tacca chantrieri 3.1.4.1 Compound TC1: Chantriolide D (new compound) Figure 3.23. Chemical structure of TC1 and taccanlonolide M (13) Compound TC1 was obtained as a white amorphous powder. Its molecular formula was assigned as C35H50O15 on the basic of HR-ESI-MS pseudo-ion at m/z 711.3237 [M+H]+ (Calcd for [C35H51O15]+, 711.3222) and m/z 733.3055 [M+Na]+ (Calcd for [C35H50O15Na]+, 733.3042). The 1H-NMR spectra of TC1 exhibited signals for four methyl groups at δH 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 δH 1.91 (3H, s) and 2.06 (3H, s), which suggested the structure a steroid with two acetyl groups. In addition, anomeric protons at δH 4.20 (d, J = 8.0 Hz), indicated the presence of a sugar unit. The 13C-NMR and DEPT spectra of TC1 revealed the presence of 35 carbons, including: 2 ketone carbons at δC 206.0 and 211.7; 2 acetyl carbonyl carbons at δC 170.2 and 170.5, 3 non-protonated carbons at δC 41.9, 42.6, and 81.0; 18 methine carbons at δC 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 δC 25.2, 29.6, 44.5, and 61.9 and 6 methyl carbons at δC 13.3, 15.3, 15.3, 19.8, 20.2 and 21.0. All these data coupled with a literature survey indicated that TC1 was a steroidal glucoside [15]. The HMBC correlations HMBC between H-6 (δH 4.22) and C-5 (δC 81.0)/C-7 (δC 206.0)/C-10 (δC 42.6); between H-14 (δH 2.74)/H-16 (δH 1.46)/H-17 (δH 1.65) and C-15 (δC 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 13C-NMR chemical shift to a higher field at C-2 (δC 51.0), C-3 (δC 54.9) and correlation HMBC from H-4 (δH 2.37) to C-2 (δC 50.1)/C-3 (δC 54.9) suggested the epoxy group at C-2/C-3. Two
16 acetoxy groups at C-1 and C-12 were confirmed by HMBC correlations from HMBC to H-1 (δH 4.67) and H-12 (δH 4.93) to acetyl carbonyls (δC 170.2 and 170.5), respectively. The HMBC correlations between H-19 (δH 1.13) and C- 1(δC 72.7)/C-5 (δC 81.0)/C-9 (δC 37.1)/C-10 (δC 42.6); between H-18 (δH 0.76) and C-12 (δC 74.3), C-13 (δC 41.9), C-14 (δC 54.3), C-17 (δC 51.8); between H-21 (δH 0.80) and C-17 (δC 51.8), C-20 (δC 31.7), C-22 (δC 44.5); between H-25 (δH 1.17) and C-16 (δC 53.7), C-23 (δC 86.0), C-24 (δC 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′ (δH 4.20) to aglycone C-23 (δC 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 13C-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 (δH 0.76) and H-12 (δH 4.93)/H-8 (δH 2.59); H-19 (δH 1.13) and H-1 (δH 4.67)/H-2 (δH 3.57)/H-6 (δH 4.22)/H-8 (δH 2.59). The α-orientation of oxygenated group at C-23 was based on the ROE correlations between H-23 (δH 3.10) and H-16 (δH 1.46)/H- 25 (δH 1.17). Thus, the structure of TC1 was determined and named chantriolide D. Figure 3.24. The important HMBC correlations of TC1 Table 3.9. NMR spectral data of TC1 and reference compound C C# Ca,b DEPT Ha,c (mult., J, Hz) Aglycone 1 73.0 72.7 CH 4.67 (d, 5.0) 2 49.1 51.0 CH 3.57 (t, 5.0) 3 55.3 54.9 CH 3.51 (m) 4 29.7 29.6 CH2 2.37 (d, 16.0)/2.07* 5 81.3 81.0 C -
17 C  C # C a,b DEPT Ha,c (mult., J, Hz) 6 78.4 78.2 CH 4.22* 7 205.8 206.0 C - 8 42.0 41.6 CH 2.59 (dd, 11.5, 12.0) 9 37.5 37.1 CH 2.21 (dd, 4.0, 12.0) 10 42.2 42.6 C - 11 25.7 25.2 CH2 1.41 (dd, 4.0, 15.0)/1.79 (br d, 15.0) 12 73.9 74.3 CH 4.93 (br s) 13 42.1 41.9 C - 14 55.4 54.3 CH 2.74 (d, 11.5) 15 210.8 211.7 C - 16 53.2 53.7 CH 1.46 (dd, 11.5, 11.5) 17 51.4 51.8 CH 1.65 (dd, 11.5, 11.5) 18 13.4 13.3 CH3 0.76 (s) 19 15.5 15.3 CH3 1.13 (s) 20 31.0 31.7 CH 1.52 (m) 21 19.4 19.8 CH3 0.80 (d, 6.0) 22 43.8 44.5 CH2 1.13*/2.13 (m) 23 86.4 86.0 CH 3.10* 24 42.0 41.8 CH 1.63 (m) 25 15.3 CH3 1.17 (d, 6.0) 1-OAc 170.3 170.2 - 20.7 20.2 CH3 1.91 (s) 12-OAc 170.6 170.5 - 21.0 21.0 CH3 2.06 (s) 23-OGlc 1′ 105.5 CH 4.20 (d, 8.0) 2′ 74.6 CH 2.98 (t, 8.0) 3′ 77.4 CH 3.17* 4′ 70.8 CH 3.10* 5′ 77.1 CH 3.10* 6′ 61.9 CH2 3.47 (dd, 4.0, 11.5)/3.66 (br d, 11.5) a Recorded in CD OD, b125MHz, c 500MHz, #  of taccanlonolide M [15], * Overlapped 3 C signals 3.1.4.1 Compound TC2: Chantriolide E (new) Compound TC2 molecular formula was assigned as C36H51O15+Cl on the basic of HR-ESI-MS pseudo-ion at m/z 781.2854 [M+Na]+ (Calcd for [C36H51O15ClNa]+, 781.2809). The 1H-NMR spectra of TC2 appeared signals of four methyl group protons: three tertiary methyl groups at δH 0.94 (3H, s), 1.09 (3H, s) and 2.14 (3H, s), one second methyl group at δH 1.01 (3H, d, J = 7.0 Hz); one methyl acetyl group δH 2.13 (H, br s); one anomeric proton at δH 4.36 (H, d, J = 8.0 Hz). The 13C-NMR and DEPT spectra of TC2 showed the signals of 36 carbons including 3 carbonyl carbons at δC 167.9, 172.3, 2 and 218.1; 5 non-protonated carbon at δC 42.0, 47.9, 74.7, 123.8, and 159.6; 17 methine carbons at δC 30.5, 35.4,
18 36.5, 41.3, 56.4, 57.3, 57.4, 60.4, 71.6, 74.6, 75.1×2, 76.7, 77.9, 78.0, 78.7 and 103.9; 6 methylene carbons at δC 25.4, 33.1, 38.1, 43.8, 62.8 and 63.5; 5 methyl carbons at δC 13.4, 14.8, 15.5, 20.7 and 21.4. Figure 3.25. 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 (δC 76.7)/C-5 (δC 74.7)/C-9 (δC 30.5)/C-10 (δC 42.0); H-18 (δH 1.09) and C-12 (δC 75.1)/C-13 (δC 47.9)/C-14 (δC 41.3)/C-17 (δC 57.4); H-21 (δH 1.01) and C-17 (δC 57.4)/C-20 (δC 36.5)/C-22 (δC 78.7); H-28 (δH 2.14) and C-23 (δC 33.1)/C-24 (δC 159.6)/C-25 (δC 123.8) showed position of 4 methyl groups at C-10, C-13, C-20 and C-24. The HMBC correlation from methyl proton (δH 2.13), aglycone H-12 (δH 5.18) to acetoxy carbonyl groups (δC 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 (δC 57.3), C-7 (δC 56.4)] and the HMBC correlation from H-6 (δH 2.99) to C- 5 (δC 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 (δH 4.65) to C-24 (δC 159.6)/C-25 (δC 123.8)/C-26 (δC 167.9) showed position of carbonyl group at C-26 and double bond at C-24/C-25. The HMBC correlation from H-15 (δH 2.49)/H-17 (δH 2.72) to C-16 (δC 218.1), suggesting the presence of oxo group at C-16. Acid hydrolysis of TC2 gave D-glucose