bản tóm tắt tiếng anh nghiên cứu thành phần hóa học và hoạt tính sinh học của ba loài thuộc chi bách bộ (stemona) mọc ở lào

1
INTRODUCTION

1. Proposal
So far, we do not have medicine for cancer and AIDS as well as bacteria which
have good resistance to antimicrobial agents. Only few effective drugs are available
in the market, but they are too expensive. In addition, some of them are also harmful
to normal cells. Therefore, many scientists have been carrying out their research to
looking for new drugs. Some natural products, which have strong antimicrobial,
anticancer, antiimflammatory activity, are good sources for developing new drugs.
The Stemonaceae family widely grows in Southeast Asian countries such as
Vietnam, Laos, Thailand Stemona plants have been used for anticough, anti-
helminths, anti cancer remedies in the folk medicines. So far, chemical constituents
of Laos Stemona sp. are unknown. Only some studies have been done on their
classification and their biological activity. Thus, I select the title for my PhD thesis
as “Studies on the chemical constituents and the biological activity of three Stemona
species in Laos”.
2. Subjects of the research:
Studying on chemical constituents and biological activity of the roots of
Stemona plants, collected in Laos, including S. cochichinensis, S. pierrei and S.
tuberosa plants.
3. New results from the research
1. In the first time from the roots of S.cochinchinensis plants, collected in
Savannakhet province (Laos) were isolated and structurally identified 12
metabolites, including one new: 1-(3’-hydroxy-2’-methoxyphenyl)-2-(3’’-
hydroxy-5-methoxy-2,4-dimethylphenyl)ethane (K8) and 11 known compounds:
β-sitosterol (K1), Sesamin (K2), Methyl cis-4-hydroxycinnamate (K3), Methyl
trans-4-hydroxycinnamate (K4), 5’-methoxy-4’-hydroxy-1’-(1-methoxyprop-2-
enyl)benzen (K5), Methyl 4’-hydroxy-3’-methoxycinnamate (K6), Stemanthrene
C (K7), Stemanthrene A (K9), Pinoresinol (K10), Maistemonine (K11) and

Isomaistemonine (K12).
2. From the roots of S. pierrei plants, seven compounds have been isolated and
structurall identified as Sesamin (K2), Stemobenzofuran A (K13, new),
Stemanthrene B (K14), Syrigaresinol (K15), Benzoic acid (K16), p-Anisic acid (K17),
methyl 1’-hydroxy-1’-(4-hydroxy-3-methoxyphenyl)propylate (K18, new).
3. From the roots of S. tuberosa plants, six compounds have been isolated and
structurall identified as: Stemophenanthren A-D (K19-K22), Stemostemanthren E
(K23) and Stemobenzofuran B (K24). All six compounds are new ones.
4. Eight compounds were tested on a cytotoxicity towards for cancer lines, such as
KB, Hep-G2, Lu, MCF7. The results showed that K21, K22 and K23 obtained
good cytotoxicity towards 4 cancer cell lines with IC
50
values from 4.49 to 63.65
µg/ml. Five compounds ( K7, K8, K9, K19 and K20) have a middle cytotoxicity
towards KB cell line (IC
50
values from 20.67 to 82.30 µg/ml).
4. Structure of PhD thesis
PhD thesis contained 140 pages with 31 tables, 71 pictures, 101 references,
including Introduction (2 pages), Chapter 1: Overview of reseach (25 pages), Chapter

2
2: Materials and Methods (6 pages), Chapter 3: Experiments (20 pages), Chapter 4:
Results and discussion (77 pages), Conclusion (2 page).

CHAPTER 1: OVERVIEW OF RESEARCH
1.1. INTRODUCTION FOR STEMONACEAE FAMILY
1.1.1. Classification of Stemonaceae
Stemonaceae is belonging to Pandanales order. It is a family of
monocotyledonous flowering plants (Monocosts) and Angiosperms. It is devided into

four genera, including Croomia.Torr, Stemona. Lour, Stichoneuron Hook. F and
Pentastemona Steenis. The genus Stemona is biggest with about 30 species. These
plants grow everywhere, but concentrated in China, Vietnam, Laos, Cambodia and
Thailand.
Below are the classification of Stemonaceae.
Table 1.1: Classification of the genus Croomia based on WCSP (Word Checklist of
Selected Plant family) up to 1/1/2011
1. Croomia heterosepala (Baker) Okuyama
2. Croomia japonica Miq.
3. Croomia pauciflora (Nutt.) Torr.

Table 1.2: Classification of the genus Stemona Lour based on WCSP (Word
Checklist of Selected Plant family) up to 1/1/2011
1. Stemona angusta I.R.H.Telford
2. Stemona aphylla Craib
3. Stemona australiana (Benth.)
C.H.Wright
4. Stemona burkillii Prain
5. Stemona cochinchinensis Gagnep.
6. Stemona collinsiae Craib
7. Stemona curtisii Hook.f.
8. Stemona griffithiana Kurz
9. Stemona hutanguriana Chuakul
10. Stemona japonica (blume) Miq.
11. Stemona javanica (Kunth) Engl.
12. Stemona kerrii Prain
13. Stemona kurzii Prain
14. Stemona lucida (R.Br.) Duyfjes
15. Stemona mairei (H.Lév.) K.Krause
16. Stemona parviflora C.H.Wright

17. Stemona phyllantha Gagnep
18. Stemona pierrei Gagnep
19. Stemona prostrata I.R.H.Telford
20. Stemona sessilifolia (Miq.) Miq.
21. Stemona squamigera Gagnep
22. Stemona tuberosa Lour.
23. Stemona tuberosa var. moluccana
(Blume) ined.
24. Stemona tuberosa var. tuberosa

Table 1.3: Classification of the genus Stichoneuron Hook. F based on WCSP (Word
Checklist of Selected Plant family) up to 1/1/2011
1. Stichoneuron caudatum Ridl.
2. Stichoneuron membranaceum Hook.f.
Table 1.4: Classification of the genus Pentastemona based on WCSP (Word
Checklist of Selected Plant family) up to 1/1/2011
1. Pentastemona egregia (Schott) Steenis

3
2. Pentastemona sumatrana Steenis
1.1.2. Introduction to the genus Stemona
The genus Stemona has about 30 species. In Vietnam, six species belonging to
this genus have been collected and identified. They are Stemona cochinchinensis
Gagnep, Stemona collinsiae Craib, Stemona pierrei Gagnep, Stemona phyllantha
Gagnep, Stemona saxorum Gagnep and Stemona tuberosa. Lour.
Laos has 11 species of this genus. They are S.tuberosa., S. phyllantha Gagnep.,
S. squamigera Gagnep., S. cochinchinensis Gagnep., S. pierrei Gagnep., S. saxorum
Gagnep., S. collinsae Craib., S. aphylla Craib., S. burkillii Prain., S. griffithiana Kurz
and S. kerrii Craib. They distribute in the mountains in the north, delta in the middle and
the south of Laos.

Stemona genus has several trivial names such Samzip, Mưnang, Ìnhạng (Lao
and Thai). They have stems or clim in moist area, such as in south east Asia. Each
species has typical characters as below:
 Stemona sp have stems, mostly grow in moist area (near rivers, streams or lacks).
Normally, they are in delta.
 Stemona sp as climb plants mainly are available in forests. They are about 1.4 m
length.
1.1.3. Usage
Based on traditional medicines, Stemona sp is sweet, pungent. They have been
used as anticough and anti-helminths remedies. People in Laos use all plant parts to
make nematocidal medicines, insecticide.
1.2. CHEMICAL CONSTITUENTS OF STEMONA SP.
Stemona sp. is a rich source of secondary metabolites such as alkaloids,
stilbenoids, stemanthrenes, stemofurans.
Alkaloids isolated from Stemona sp have various carbon skeletons. However,
they have the same pehydroazaazulen core. So far, more than 90 alkaloids have been
purified from Stemona sp. They are devided into five skeleton as following:

N
O
O
N
N
O
N
O
O
O
N
O

O
N
O
O
V
IV
III
II
I

Figure 1.1. Five alkaloid skeletons from Stemona sp
CHAPTER 2: MATERIALS AND METHODS

4
2.1. MATERIALS
2.1.1. Plant materials
Stemona cochinchinensis was collected in Savannakhet province, Laos in
October 2010. It was identified by MSc. Nguyen The Anh, Institute of Chemistry,
Vietnamese Academy of Science and Technology. A voucher specimen (KPN-
01.2010) was deposited at the Hebarium of Faculty of Chemistry, Hanoi National
University of Education, Hanoi

Figure 2.1: Stemona cochinchinensis
S. peirrei were collected in Savannakhet Province, Southern Laos, in June 2011
by Mr. Khamko V. A. and identified by MSc. Nguyen The Anh, Institute of
Chemistry, Vietnamese Academy of Science and Technology. A voucher specimen
(KPN-02.2011) was deposited at the Hebarium of Faculty of Chemistry, Hanoi
National University of Education, Hanoi.

Figure 2.2: Stemona peirrei

Stemona tuberosa was collected in Attapu Province, Southern Laos, in July
2011 by one of the authors (Mr. Khamko V.A) and identified by Dr.Nguyen Thi Lien
(Faculty of Biology, HNUE, Hanoi) as Stemona tuberosa Lour. A voucher specimen
(KPN-03.2011) was deposited at the Hebarium of Faculty of Chemistry, HNUE,
Hanoi, Vietnam.

Figure 2.3: Stemona tuberosa

5
2.1.2. Chemicals:
In this PhD thesis all chemicals (MeOH, n-hexane, ethyl acetate…) are pure for
analytic purpose.
2.2. METHODS
2.2.1. Extraction and Isolation
Selected plants were treated by the same methods for researching natural
products. Compounds were isolated by several chromatographic methods such as thin
layer chromatography (TLC), column chromatograpgy (CC), reversed phase column
chromatography (RP-18), preparative HPLC (prep. HPLC).
2.2.2. Structural elucidation
Their structures will be elucidated by spectroscopic analysis such as Infrared
spectrum (IR), Ultra violet spectrum (UV-Vis), Mass spectrum (MS) and Nuclear
Magnetic Resonance (NMR) including 2D NMR. In particular cases, chemical
reactions might be employed to get crystals for X-ray crystallographic analysis.
IR spectrum was recorded on a JASCO FT/IR-5300, at Department of physical
chemistry, Faculty of Chemistry, Hanoi University of Education.
Uv-vis spectrum was measured on a Shimadzu UV-1650PC instrument in MeOH
at Department of physical chemistry, Faculty of Chemistry, Hanoi University of
Education.
FT-ICR-MS spectrum was recoded on a Variance 320-MS instrument.
1D and 2D NMR spectra were recorded on a Brucker Avance 500 MHz with

TMS (trimethylsilan) as standard at Institute of Chemistry, VAST.
Some compounds were measured their MS on Brucker Apex III (FT-ICR-MS)
at Leibniz Institute of Plant Biochemistry in Germany.

CHAPTER 3: EXPERIMENTS
3.1. Roots of S. cochinchinensis
The powder of dried roots of Stemona cochinchinensis (3.0 kg) was extracted as
shown in scheme 3.1.
Scheme 3.1. Isolation process of Stemona cochinchinensis










After running TLC with different solvent systems, we found a suitable solvent
systems for column chromatography. The crude extract was purified as follow:
Scheme 3.2. Separation of compounds from n-hexane extract of S. cochinchinensis
Dried sample

MeOH:H
2
O (80:20)

Extract

EtOAc extract

n-hexane
extract

n-buthanol
extract
n-hexane, EtOAc and n-buthanol


6







Scheme 3.3. Separation of compounds from EtOAc extract of S. cochinchinensis









3.2. Roots of S. pierrei
Scheme 3.4. Separation of compounds from n-hexane extract of S. pierrei

SKC / n:E /8:1

n-Hexan extract
(CH: 5,76g)

CH1(K1)
1,05g
CH2B3(K4)
7mg
CH2B2(K3)
4mg
CH2B1(K2)
9mg
CH2
1,3g
SKC / n: E/4:1
CH2B
78mg
CH2C
63mg
CH2C2(K5)

9mg
HPLC/ n:E /1:1

HPLC/ n:E /2:1

CE2
165mg
SKC / n: E/1:1
CE2B
77mg
CE2B1
(K10)
7mg
CE2B2
(K11)
9mg
CE2B3
(K12)
5mg
HPLC / n:
E/3:7
EtOAc extract
(CE:7.67g)
SKC/n:E/4:1
CE1
0,61g
CE1D1
(K9)
17mg
CE1C1

(K8)
4mg

SPD /C:M/1:1
HPLC/ n: E/3:2:1
SKC / n: E/ 3:1
CE1A
(K6)
88mg
CE1B
(K7)
83mg
CE1C
181mg

CE1D
72mg

SKC/ n: E/4:1
SKC/ n:E /8:1

n-Hexane extract
PH (3.17g)
PH3C
66mg
SKC/ n: E/3:1
PH3
322mg
PH3C1 (K13)
4mg

HPLC n: E/3:2
PH1
414mg
PH1B1 (K2)
5mg
PH1B
56mg
HPLC/ n: E/3:1

7
Scheme 3.5. Separation of compounds from EtOAc extract of S. pierrei









3.3. Roots of S. tuberosa
Scheme 2.6. Separation of compounds from n-hexane extract of S. tuberosa

3.4. Bioassays
Some isolated compounds were tested their antimicrobial, antifungal,
cytotoxicity and their results are described in tables 4.18.

CHAPTER 4: RESULTS AND DISCUSSION
4.1. DETERMINATION OF STRUCTURES OF ISOLATED COMPOUNDS
FROM S. COCHICHINENSIS
PE1
657mg
PE3
325mg
PE4
233mg
SKC n:E/5:1
SKC/n:E/3:1
SKC/n:E/2:1
HPLC/n:E/1:1
HPLC/n:E/1:1
HPLC/n:E/2:1
HPLC/n:E/3:2
PE4A
43mg
PE3B
53mg

PE1B
83mg
PE1D
69mg
PE4A2
(K18)
5mg
PE3B2
(K17)
5mg
PE3B1
(K16)
3mg
PE1D2
(K12)
6mg

PE1D3
(K15)
7mg

PE1D1
(K11)
7mg
PE1B1
(K14)
7mg
PE1B2
(K7)
5mg

EtOAc ext.
(PE: 6.67g)
SKC n:E/ 6:1
TH5
0.78g
SKC/n:E/3:2

HPLC/n:E/1:1
TH5B
57mg
TH5B4
(K24)
9mg
TH5B1
3mg
TH2
1.27g
SKC/ n:E/2:1
HPLC/n:E/3:2
TH2C1
(K19)
4mg
TH2C2
(K20)
8mg


TH2C

97mg
TH2C4
(K22)
12mg

TH2C5
(K23)
5mg

TH2C3
(K21)
6mg
n-Hexane ext.
(TH:14.76g)
SKC/ n:E/ 10:1

8
4.1.1. Chemical constituents of S. cochichinensis
Crude extract of S. Cochichinensis were separated as scheme 3.1 and followed
by schemes 3.2, 3.3 to give 12 compounds.
 K1 (β-sitosterol):
This compound K1 was purified by recrystallization as a white crystals, well-
dissolved in chloroform and n-hexane. It was determined as β-sitosterol, one of the
most popular sterols in plants.

Figure 4.1:
1
H NMR spectrum of K1 in CDCl
3

 K2(Sesamin):
NMR spectra and data of K2 are described in figure 4.2 and table 4.1.

Figure 4.2:
1
H and
13
C NMR spectra of K2 in CDCl
3

K2 is determined to be Sesamin, previously isolated from Lindera obtusiloba.
Table 4.1:
1
H and
13
C NMR spectral data of K2
N
o

1
H NMR, δ (ppm), J(Hz)
13
C NMR(δ
ppm)
1, 1’
-
135.1
2, 2’
6.84, d, J=2.0 Hz
106.5

3, 3’
-
148.0
4, 4’
-
147.1
5, 5’
6.80, m
108.2
6, 6’
6.80, m
119.4
7, 7’
4.72, d, J= 4.5 Hz
85.8
8, 8’
3.05, m
54.4
9, 9’
3.87, m, 4.23, m
71.7
10, 10’
5.95, s
101.1
O
O
H
H
7
9'

8'
7'
9
1
O
O
O
O
1'
2'
8
2
6
6'
4'
4

9
 K3 and K4 (Methyl cis- and trans-4-hydroxycinnamates)
Their NMR spectra and data are shown in figures 4.3., 4.4 and table 4.2.
HO
H
3
COOC
H
H
1
3
1'
2'

4'

Figure 4.3.
1
H NMR spectrum of K3 CDCl
3

HO
H
H
COOCH
3
1
2
3
1'
2'
4'

Figure 4.4.
1
H NMR spectrum of K4 CDCl
3
Table 4.2.
1
H and
13
C NMR spectral data of K3 and K4
No.
1

H NMR δ (ppm), J (Hz)
13
C NMR (δ ppm)
HMBC
K3
K4
K3
K4
1
-
-
165.8
167.9
-
2
5.75, d, J=12.5
6.30, d, J= 16.0
114.1
115.3
C-1/C-3
3
6.80, d, J=12.5
7.64, d, J= 16.0
114.3
115.9
C-2
1’
-
-
124.8

127.3
-
2’
7.63 d, J=8.5
7.43, d, J= 8.5
132.5
129.9
C-3’/C-4’
3’
6.78 d, J=8.5
6.85, d, J= 8.5
142.8
144.6
C-2’/C-4’
4’
-
-
157.9
157.7
-
5’
6.78 d, J=8.5
6.85, d, J= 8.5
142.8
144.6
C-4’/C-6’
6’
7.63 d, J=8.5
7.43, d, J= 8.5
132.5

129.9
C-4’/C-5’
1-OMe
3.80 s
3.80, s
50.1
51.7
-
 K5[5’-Methoxy-4’-hydroxy-1’-(1-methoxyprop-2-enyl)benzene]
NMR spectra of K5 are shown in figure 4.5 and table 4.3.

10

Figure 4.5.
1
H NMR spectrum of K5 CDCl
3

 K6 (Methyl 4’-hydroxy-3’-methoxycinnamate)
NMR spectra of K6 are in figure 4.6 and table 4.3.

Figure 4.6.
1
H and
13
C NMR spectra of K6 in CDCl
3

Table 4.3: NMR spectral data of K5 and K6

No.
1
H NMR δ (ppm), J (Hz)
13
C NMR δ (ppm)
K5

K6

K5

K6

1
4.07, d, J=6
-
73.2
167.7
2
6.14, m
6.28, d, J= 16.0
126.6
115.2
3
6.52, d, J=16.5
7.62, d, J= 16.0
132.6
144.9
1’
-

-
129.4
126.9
2’
6.88, m
7.07, m
120.4
123.0
3’
6.86, d, J=8.5
-
114.4
146.8
4’
-
-
145.6
148.0
5’
-
6.92, d, J= 8.5
146.6
114.7
6’
6.93, s
7.02, m
108.4
109.4
MeO-1
3.80, s

3.79, s
57.9
51.6
MeO-3’
-
3.92, s
-
55.9
MeO-5’
3.90, s
-
55.9
-
HO-4’
5.50, s
5.90, s
-
-

 K7 (Stemanthrene C) and K9 (Stemanthrene A)
NMR spectra of K7 are in figure 4.7 and table 4.4.
HO
COOCH
3
H
3
CO
1
2
3

1'
3'
5'
H
3
CO
HO
CH
2
OCH
3
1
2
3
1'
3'
5'

11

Figure 4.7.
1
H and
13
C NMR spectra of K7 in CDCl
3

NMR spectra of K9 are illustrated in figure 4.8. and table 4.4.

Figure 4.8.
1
H and
13
C NMR spectra of K9 in CDCl
3
Table 4.4.
1
H- and
13
C NMR spectral data of K7 and K9
Vị trí
1
H NMR δ (ppm), J (Hz)
13
C NMR δ (ppm)
K7
K9
K7
K9
1
-
-
130.5
130.8
2
-
-
143.2
143.5

3
-
-
146.9
147.0
4
6.85, d, J= 8.5
6.86, d, J=8.5
113.0
113.1
5
8.01, d, J= 8.5
8.04, d, J=8.5
124.3
124.1
6
-
-
126.6
126.3
1’
-
-
136.1
137.5
2’
-
6.1, s
116.2
110.5

3’
-
-
151.3
153.0
4’
-
-
114.9
116.2
5’
-
-
154.5
156.9
6’
-
-
120.1
120.1
1”
2.77, m
2.79, m
25.5
22.4
2”
2.69, m
2.65, m
22.2
29.6

2-OMe
3.81, s
3.80, s
61.4
61.3
3-OH
5.66, s
5.64, s
-
-
2’-Me
2.12, s
-
11.8
-
3’-OH
4.77, s
4.80, s
-
-
4’-Me
2.22, s
3.53, s
8.9
8.7
5’-OMe
3.50, s
3.53, s
59.9
59.6

1
2
4
5
1"
2"
1'
4'
6'
H
3
CO
HO OH
CH
3
H
3
CO
H
3
CO
HO
CH
3
OH
CH
3
H
3
CO

1" 2"
1
2
4
1'
2'
4'

12

 K8 [1-(3-hydroxy-2-methoxyphenyl)-2-(3’-hydroxy-5’-methoxy-2’,4’-
dimethylphenyl)etan]
NMR spectra and data of K8 are in figure 4.9 and table 4.5. Its ESI-MS has ion
peak [M+Na]
+
at m/z 135. Its molecular formula is C
18
H
20
O
4
. K8 is identified as 1-(3-
hydroxy-2-methoxyphenyl)-2-(3’-hydroxy-5’-methoxy-2’,4’-dimethylphenyl)etan.
Compound K8 is a new stilbenoid isolated from S. cochinchinensis in Laos.

Figure 4.9.
1
H and
13
C NMR spectra of K8 in CDCl

3

Table 4.5:
1
H and
13
C NMR spectral data of K8
No.
1
H NMR δ (ppm), J
(Hz)
13
C NMR δ
(ppm)
HMBC
1
-

135.0

-
2
-
145.3
-
3
-
149.0
-
4

6.84, dd, J=1.5,7.5
113.7
2,3,5
5
6.96, t, J=7.5
124.9
3,4,6
6
6.77, dd, J=1.5, 7.5
121.5
4,1”
1’
-
138.5
-
2’
-
114.0
-
3’
-
152.7
-
4’
-
109.2
-
5’
-
156.0

-
6’
6.32, s
104.2
2”
1’’
2.85, m
31.1
6,2”
2’’
2.85, m
35.1
6’,1”
2-OMe
3.78, s
61.3
-
2’-Me
2.20, s
10.9
-
4’-Me
2.11, s
8.2
-
5’-OMe
3.77, s
55.7
-

 K10 (Pinoresenol):
NMR spectra and data of K10 are shown in figure 4.10 and table 4.6. It has
identical spectral data with Pinoresenol, thus K10 is Pinoresenol.
3
6
1"
2"
1
1'
4'
OCH
3
OH
CH
3
OH
CH
3
OCH
3

13

Figure 4.10.
1
H and
13
C NMR spectra of K10 in CDCl
3
Table 4.6.

1
H and
13
C NMR spectral data of K10
N
o

1
H NMRδ (ppm), J(Hz)
13
C NMR(δ ppm)
1. 1’
-
133.0
2. 2’
6.89, m
108.7
3. 3’
-
146.7
4. 4’
-
145.3
5. 5’
6.89, m
114.3
6. 6’
6.82, m
119.0
7. 7’

4.72, d, J=4.5
54.2
8. 8’
3.10, m
85.9
9. 9’
3.87, m; 4.23, m
71.7
3.3’-OCH
3

3.98, s
56.0
1. 1’
-
133.0


 K11 (Maistemonin) and K12 (Isomaistemonin)
NMR spectral data of two compounds K11 and K12 are presented in figure
4.11. and the table 4.7. They are maistemonine and isomaistemonine, those have the
same molecular formula of C
23
H
29
NO
6
. They are two isomers which were previously
isolated from Stemona mairei by Yang Ye and co-workers.

Figure 4.11.
1
H NMR spectra of K11 and K12 in CDCl
3

N
O
H
3
C
O
O
O
CH
3
OCH
3
H
H
CH
3
O


Maistemonine Isomaistemonine
O
O
OH
OCH
3

H
3
CO
HO
H
H
8'
7'
9
8
7
9'
1'
3'
4'
6'
6
5
3
1

14
Table 4.7.
1
H and
13
C NMR spectral data of K11 and K12
No.
1
H NMR δ (ppm). J (Hz)

13
C NMR δ (ppm)
K11
K12
K11
K12
1
1.88 m; 2.11 m
1.85 m; 2,08 m
35.8
35.3
2
1.50 m; 1.87 m
1.42 m; 1.85 m
26.6
25.9
3
3.39 m
3,36 m
63.6
63.0
5
2.93 m; 3.59 m
3.55 m; 2.89 m
47.2
46.7
6
1.46 m.1.82 m
1.85 m; 1.47 m
24.9

25.1
7
1.50 m; 1.95 m
1.35 m; 1.95 m
25.5
24.6
8
2.26, m; 2.89 m
2.24 m; 2.88 m
28.4
27.9
9
-
-
173.3
172.9
9a
-
-
79.3
78.9
10
-
-
136.4
135.7
11
-
-
197.9

179.3
12
-
-
91.7
91.2
13
-
-
172.6
172.2
14
-
-
97.0
96.6
15
-
-
175.0
174.1
16
2.02 s
1.98 s
8.9
8.4
17
1.79 s
1.77 s
8.4

7.7
18
3.86 m
3.83m
85.1
83.5
19
1.45 m, 2.31 m
1.45 m; 2.31 m
34.4
33.5
20
2.57 m
2.55 m
34.8
34.3
21
-
-
17.7
178.7
22
1.22 d, J=7.0
1.22 d, 7,0
14.9
14.5
OCH
3

4.00 s

3.97 s
58.8
58.5
4.2. ISOLATED COMPOUNDS FROM STEMONA PIERREI
4.2.1. Extraction and isolation
Extraction is illustrated in scheme 3.4 and 3.5. Seven compounds were isolated
from this plants by TLC, column chromatography and prep. HPLC.
4.2.2. Identification of compounds
*K13 (Stemobenzofuran A)
NMR spectra of K13 are shown in figure 4.12 and table 4.8.

Figure 4.12:
1
H and
13
C NMR spectra of K13 in CDCl
3

O
OCH
3
OH
CH
3
H
3
CO
OMe
1"
2"

1
3
1'
3'
5'
5

15
Table 4.8.
1
H and
13
C NMR spectral data of K13
No.
1
H NMR δ (ppm), J (Hz)
13
C NMR δ (ppm)
1
-
119.6
2
6.68, d, J = 9.0
103.2
3
7.22, t, J = 8.0
124.7
4
7.16, d, J = 8.5
104.3

5
-
153.4
6
-
155.4
1’
-
122.9
2’
7.17, s
106.9
3’
-
145.7
4’
-
139.2
5’
-
121.8
6’
-
145.6
1’’
6.89, s
101.8
2’’
-
154.2

5-OCH
3

3.97, s
55.6
3’-OCH
3

3.88, s
60.5
6’-OCH
3

3.96, s
56.4
5’-CH
3

2.44, s
13.5
4’-OH
5.70, s
-
K13 is identified as Stemobenzofuran A, a new stemofuran from S. pierrei in Laos.
 K14 (Stemanthren B)
NMR spectra of K14 are shown in figure 4.13. and table 4.9. It was determined
to be stemanthrene B.


Figure 4.13.

1
H NMR and
13
C NMR spectra of K14 in CDCl
3
Table 4.9.
1
H and
13
C NMR spectral data of K14 and Stemanthrene B
No.
1
H NMR δ (ppm)
13
C NMR δ
(ppm)
HMBC
K14

[42]

K14
[42]

1
-
-
131.5
131.5
-

2
-
-
144.5
144.4
-
H
3
CO
HO
HO CH
3
OCH
3
1
3
5
1"
2"
1'
3'
5'

16
3
-
-
147.1
147.0

4
6.90, d, J=8.5Hz
6.90, d,
J=8.5Hz
113.0
113.0
C6/C2/C3
5
7.71, d, J=8.5Hz
7.72, d,
J=8.5Hz
122.3
122.3
C6’/C1/C3
6
-

126.3
126.3
-
1’
-
-
136.9
136.9
-
2’
6.43, s
6.43, s
102.9

102.8
C2”/C3’/C4
’
3’
-
-
156.7
156.6
-
4’
-
-
110.7
110.6
-
5’
-
-
151.3
151.3
-
6’
-
-
114.8
114.7
-
1”
2.77, t, J=13
2.79, m

30.1
30.0
C2” /C2/C6
’
2”
2.69, q, J=13
2.70, m
22.7
22.7
C1”/C2’/C6
2-OCH
3

3.80, s
3.80, s
61.4
61.4
C2
3-OH
5.66, s
5.66, s
-

C2/C3/C4
3’-
OCH
3

3.85, s
3.85, s

55.7
55.7
C3’
4’-CH
3

2.16, s
2.16, s
8.2
8.1
C3’/C4’/C5
’
5’-OH
5.36, s
5.36, s
-

C4’/C5’/C6
’

 K15 (Syringaresinol):
NMR spectra and data of K15 are shown in figure 4.14. and table 4.10. It was
suggested to be syringaresinol.

Figure 4.14.
1
H and
13
C NMR of K15 in CDCl
3

Table 4.10.
1
H and
13
C NMR spectral data of K15 in comparison with those of
Syringaresinol (SSn)

N
o

1
H NMRδ (ppm), J(Hz)
13
C NMR(δ ppm)
HMBC
K15
SSn
K15
SSn
1, 5
3.10 dd, 4.5, 6.0
3.12 m
54.4
54.3
-
2, 6
4.73 d, 4.0
4.76 d,
8.1
86.1

86.0
-
4, 8
4.28 dd, 2.0, 6.5
4.30 m
71.9
71.8
2/6

O
O
OCH
3
OH
OCH
3
H
3
CO
H
3
CO
HO
H
H
1
2
3
4
5

6
8
1
'
2
'
3
'
4
'
5
'
6
'
1
''
2
''
3
''
4
''
5
''
6
''
7

17

3.92 m
3.94 m
1’, 1”
-
-
132.2
132.0
-
2’, 2”, 6’, 6”
6.59 s
6.61 s
102.8
102.7
2/6/4’
4”/3’/3”
5’/5”
3’, 3”, 5’, 5”
-
-
147.2
147.1
-
4’, 4”
-
-
134.4
134.3
-
3’, 5’, 3”, 5”-
OCH

3
3.91 s
3.92 s
56.4
56.3
3’/3”
5’/5”
4’, 4”-OH
5.91 s
5.57 s
-
-
-
*K16 (Benzoic acid)
NMR spectra (figure 4.15) suggested that K16 is benzoic acid.
C
OH
O
1
2
3
6
5
4

Figure 4.15.
1
H NMR spectrum of K16
 K17 (p-anisic acid)
NMR spectra and data of K17 are shown in figure 4.16. and table 4.11. K17 is

identified as p-anisic acid.

Figure 4.16.
1
H and
13
C NMR spectra of K17 in CDCl
3
Table 4.11.
1
H and
13
C NMR spectral data of K17
N
o

1
H NMRδ (ppm), J (Hz)
13
C NMR(δ ppm)
1
-
121.6
2; 6
8.06, d, 9.0
132.3
3; 5
6.95, d, 9.0
113.8
4

-
164.0
-COOH
-
173.7
-OCH
3

3.88, s
55.5
* K18 [(Methyl 1’-hydroxy-1’-(4-hydroxy-3-methoxyphenyl)propylate]

CH
3
O COOH
Axit p-anisic

18
NMR spectra and data of K18 are shown in figure 4.17. and table 4.12.

Figure 4.17.
1
H and
13
C NMR spectra of K18 in CDCl
3
Table 4.12.
1
H and
13

C NMR spectral data of K18
N
o

1
H NMRδ (ppm), J(Hz)
13
C NMR(δ ppm)
1
-
134.6
2
6.95 d, J = 2.0
108.2
3
-
146.7
4
-
145.3
5
6.88 d, J = 8.0
114.3
6
6.83 dd, J = 2.0, 8.5
118.6
1’
5.08 dd, J = 3.5, 8.5
70.3
2’

2.69 dd, J = 3.5, 16.5
43.3
2.77 dd, J=8.5, 16.5
3’
-
172.8
Thus, K18 is identified as methyl 1’-hydroxy-1’-(4-hydroxy-3-
methoxyphenyl)propylate - a new compound isolated from S. pierrei collected in
Laos. The left compound, isolated from S.pierrei, was identified as Sesamin (K2).
4.3. COMPOUNDS ISOLATED FROM STEMONA TUBEROSA
 K19 (Stemophenanthrene A)
NMR spectra and data of K19 are shown in Figure 4.18 and table 4.13.


Figure 4.18.
1
H and
13
C NMR spectra of K19 in CDCl
3

Analysis of its 2D NMR and FT-ICR-MS revealed that K19 is a new compound
isolated from S. tuberosa in Laos.
Table 4.13.
1
H and
13
C NMR spectral data of K19
No.
1

H NMR δ (ppm), J (Hz)
13
C NMR δ (ppm)
HMBC
1
-
129.4
-
2
7.87, d, J = 7.5
128.7
1,3
H
H
H
H
3
CO
OH
1
2
3
4
5
6
1'
3'
2'
HO
COOCH

3
O
O
OH
Me
1
3
5
1"
2"
1'
3'
5'

19
3
7.70, m
129.9
2, 4
4
7.62, m
128.7
3.5
5
8.10, d, J = 8.5
134.1
1,4,6’
6
-
137.4

-
1’
-
128.6
-
2’
-
130.4
-
3’
-
188.7
-
4’
-
182.2
-
5’
8.16, d, J = 8.5
121.2
3’,6’
6’
-
128.6
-
1”
9.59, d, J = 9.0
128.8
2”,6
2”

-
162.8
2’,6’, 1”
2’-Me
2.14, s
9.0
-
 K20 (Stemophenenthrene B)
NMR spectra and data of compound K20 are shown in figure 4.19 and table
4.14. It is identified as Stemophenanthrene B, a new stemanthrene isolated from Laos
S. tuberosa.


Figure 4.19.
1
H and
13
C NMR spectra of K20 in CDCl
3

Table 4.14.
1
H and
13
C NMR spectral data of K20
No.
1
H NMR δ (ppm), J (Hz)
13
C NMR δ (ppm)

HMBC
1
-
122.1
-
2
-
156.1
-
3
-
147.4
-
4
7.10 s
102.2
2,3,5
5
-
135.7
-
6
-
129.4
-
1’
-
100.1
-
2’

7.51 d, J=8.0
111.0
2”,3’
3’
7.23 t, J= 7.0
122.9
2’,4’2’
4’
7.26 m
123.9
3’,5’
5’
7.55 t, J=7.0
120.6
3’,4’
6’
-
103.6
-
1”
6.91 s
100.1
2”,2
2”
-
154.7
1”,2’
OMe
MeO
HO

OH
1
3
5
1" 2"
1'
3'
5'

20
2-OMe
4.00 s
56.5
-
2”-OMe
4.00 s
56.5
-

 K21 (Stemophenanthrene C) and K22 (Stemophenanthrene D)
NMR spectra and data of K21 are in figure 4.20 and table 4.15.

Figure 4.20.
1
H and
13
C NMR spectra of K21 in CDCl
3

K21 was characterized as a new stemophenanthrene C from S. tuberosa in Laos.

NMR spectra and data of K22 are in figure 4.21 and table 4.15.


Figure 4.21.
1
H and
13
C NMR spectra of K22 in CDCl
3

K22 is determined to be stemophenanthrene D, a new stilbenoid firtly isolated from
S. tuberosa collected in Laos.
Table 4.15.
1
H and
13
C NMR spectral data of K21 and K22
N
o

1
H NMR (δ ppm), J (Hz)
13
C NMR(δ ppm)
K21

K22

K21

K22

1
-
-
124.6
129.2
2
7.28 d, J=16.5
7.23 m
122.7
111.6
3
7.51 dd, J=1.5, 8.0
-
127.2
142.3
4
7.12 m
-
128.6
146.1
5
-
-
153.4
155.4
6
-
-

138.0
137.1
1’
-
-
133.5
128.8
2’
-
-
153.1
154.3
3’
7.04, d, J= 16.5
7.26, m
103.7
122.7
4’
7.12, m
7.57, d, J=7.5
129.9
120.7
5’
7.04, d, J=16.5
-
103.7
154.3
6’
-
-

153.1
124.0
Me
OMeHO HO
OH
1
3
5
1" 2"
1'
3'
5'
OMe
OH
MeO
OMe
1
3
5
1"
2"
1'
3'
5'

21
1”
6.94, t, J=7.5
7.49, d, J=7.5
121.1

111.0
2”
6.81, d, J=8.0
6.76, m
116.0
104.4
5-OMe
3.87, s
3.77, s
61.0
61.0
2’-OMe
3.91, s
-
56.2
-
6’-OMe
3.91, s
-
56.2
-
3-Me
-
2.41, s
-
13.6
 K23 (Stemophenanthrene E)
NMR spectra and data of K23 are in Figure 4.22. and table 4.16.

Figure 4.22.

1
H and
13
C NMR spectra of K23 in CDCl
3

K23 is identified as Stemophenanthrene E, a new stilbenoid purified from roots
of S. tuberosa collected in Laos.
Table 4.16.
1
H and
13
C NMR of K23
No.
1
H NMR δ (ppm), J (Hz)
13
C NMR δ (ppm)
HMBC
1
-
128.6
-
2
-
116.7
-
3
7.51, d, J= 8.0
111.3

2,4,5
4
7.27, m
122.8
3,5,6
5
-
132.7
-
6
-
152.3
-
1’
-
128.6
-
2’
-
154.8
-
3’
7.63, m
120.9
2’,4’
4’
7.30, m
123.9
2’,3’,5’,6’
5’

-
154.8
4’,6’
6’
-
154.3
-
1”
6.46, s
103.8
2,2”
2”
6.64, s
106.5
2’,6’,1”
2-Me
2.03, s
12.7
-
5-Me
2.03, s
12.7
-

 K24 (Stemobenzofuran B)
NMR spectra and data of K24 are illustrated in figure 4.23 and table 4.17.
Me
OH
HO
OH

OH
Me
1
3
5
1'
3'
5'
1"
2"

22

Figre 4.23.
1
H and
13
C NMR spectra of K24 in CDCl
3

In comparison with the NMR spectral data of Stemofuran in previous
publications we saw that they have some different signals. Thus, it is identified as
Stemobenzofuran B, a new compound isolated from S. tuberosa collected in Laos for
the first time.
Table 4.17.
1
H and
13
C NMR spectral data for K24
N

o

1
H NMR, δ (ppm), J(Hz)
13
C NMR(δ ppm)
HMBC
1
-
119.5
-
2
7.14, d, J=8.5
104.4
3,4
3
6.67, d, J=7.5
103.2
2,4
4
-
155.5
-
5
7.20, s
124.7
4
6
-
142.2

-
1’
-
122.9
-
2’
-
153.3
-
3’

146.1
-
4’
-
121.1
-
5’
-
154.0
-
6’
7.23, d, J=5.0
111.4
2’,5’
1”
6.87, s
101.9
1,1’,2’
2”

-
136.8
-
4-OMe
3.96, s
61.0
-
2’-OMe
3.84, s
55.6
-
4’-Me
2.42, s
19.7
-
3’-OH
-
-
-
5’-OH
-
-
-

4.4. BIOLOGICAL ACTIVITIES OF ISOLATED COMPOUNDS
Eight compounds have been tested their anticancer activity against four cancer
cell lines. Their results are shown in table 4.18.
Table 4.18. Anticancer properties

No

Samples

IC
50
values (µg/ml)
KB
Hep-G2
Lu
MCF7
1
K7
82.3
-
-
-
2
K8
59.90
-
-
-
O
H
3
CO
H
3
CO

CH
3
1
3
5
1"
2"
1'
3'
5'
OH
OH

23
3
K9
67.37
-
-
-
4
K11
>128
-
-
-
5
K20
51.02
>128

>128
>128
6
K21
45.15
47.5
63.65
59.78
7
K22
4.49
18.43
32.97
23.52
8
K23
14.2
39.13
39.75
16.66

CONCLUSIONS
1. This is the first time the roots of S. cochinchinensis, S. pierrei (collected in
Savannakhet province, Laos), S. tuberosa (collected in Attapu province, Laos)
were studied on chemical constituents and biological activities.
2. From the roots of S. cochinchinensis plants, twelve compounds have been
isolated and structurall identified as β-sitosterol (K1), Sesamin (K2), Methyl cis-
4-hydroxycinnamate (K3), Methyl trans-4-hydroxycinnamate (K4), 5’-methoxy-
4’-hydroxy-1’-(1-methoxyprop-2-enyl)benzen (K5), Methyl 4’-hydroxy-3’-
methoxycinnamate (K6), Stemanthrene C (K7), 1-(3-hydroxy-2-

methoxyphenyl)-2-(3’-hydroxy-5’-methoxy-2’,4’-dimethylphenyl)etan (K8,
new), Stemanthrene A (K9), Pinoresinol (K10), Maistemonine (K11) and
Isomaistemonine (K12).
3. From the roots of S. pierrei plants, seven compounds have been isolated and
structurall identified as Sesamin (K2), Stemobenzofuran A (K13, new),
Stemanthrene B (K14), Syrigaresinol (K15), Benzoic acid (K16), p-Anisic acid
(K17), methyl 1’-hydroxy-1’-(4-hydroxy-3-methoxyphenyl)propylate (K18, new).
4. From the roots of S. tuberosa plants, six compounds have been isolated and
structurall identified as: Stemophenanthren A-D (K19-K22), Stemostemanthren E
(K23) and Stemobenzofuran B (K24). All six compounds are new ones.
5. Eight compounds were tested on a cytotoxicity towards for cancer lines, such as
KB, Hep-G2, Lu, MCF7. The results showed that K21, K22 and K23 obtained
good cytotoxicity towards 4 cancer cell lines with IC
50
values from 4.49 to
63.65 µg/ml. Five compounds ( K7, K8, K9, K19 and K20) have a middle
cytotoxicity towards KB cell line (IC
50
values from 20.67 to 82.30 µg/ml).



24

LIST OF PUBLICATIONS

1. Vong Anatha Khamko, Dang Ngoc Quang, Pham Huu Dien (2012). A cytotoxic
stilbenoid and 4-hydroxycinnamates from Stemona cochinchinensis plants, growing in
Savannakhet Province (Laos). J. of Sciences of HNUE, Vol.57, No.7, pp.3-9.
2. Vonganatha Khamko, Pham Huu Dien, Jurgen Schmidt, Dang Ngoc Quang

(2012). Cytotoxic and anti-microbial constituents from the roots of Stemona
cochinchinensis in Laos. J. of Chemistry, VAST, Vietnam, Vol. 50, No.4A,
pp.203-206.
3. Nguyen Thi Thu Ha, Vong Anatha Khamko, Nguyen Thi Nhan, Sitha
Khemmarath, Pham Huu Dien, Dang Ngoc Quang (2013). Chemical constituents
from the ethyl acetate extract of roots of Stemona peirrei in Laos. J. of Chemistry,
Vol. 51, No.2C, pp.900-903.
4. Nguyen Thi Nhan, Vong Anatha Khamko, Nguyen Thu Ha, Dang Ngoc Quang,
Pham Huu Dien (2013). A cytotoxic stilbenoid and two benzoic acids from
Stemona peirrei plants, growing in Laos. J. of Sciences, Thainguyen University,
Vol.102, No.2, pp.151-154.
5. Vong Anatha Khamko, Dang Ngoc Quang, Pham Huu Dien (2013). Three new
phenanthrenes, a new stilbenoid isolated from the roots of Stemona tuberosa Lour.
and their cytotoxicity. Nat. Prod. Research, (was published
online: />35Mamhpk).

25

MINISTRY OF EDUCATION AND TRAINING
HANOI NATIONAL UNIVERSITY OF EDUCATION
===================

Vonganatha Khamko
Studies on the chemical constituents and the
biological activity of three Stemona species in Laos

Specialized in Organic Chemistry
Code: 62.44.01.14

ABSTRACT OF PhD THESIS

Supervisors: Assoc. Prof. Pham Huu Dien
Assoc. Prof. Dang Ngoc Quang






Hanoi, 2013