Summary of chemistry doctoral thesis: Study on chemical constituents and biological activities of Tacca vietnmensis and Tacca chantrierispecies growing in Vietnam

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.

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

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

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

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, …

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

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

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 (%)

- 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 (%)

- 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

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-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 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 δH0.95 (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. 

13C-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

(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

and ROESY correlations of TV1

Figure 3.6 HR-ESI-MS of TV1

Table 3.1 NMR spectral data of TV1 and reference compound

5′′′ 70.7 70.5 4.75 (m)

6′′′ 18.5 18.3 1.62 (d, 6.0)

a Recorded in C 5 D 5 N, b 125 MHz, c 500 MHz, # δ C of taccasuboside C [ 19 ]

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 δH0.96 (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. 

13C-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 3.14 The important HMBC and

COSY correlations of TV2

Figure 3.15 HR-ESI-MS of

TV2