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

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

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

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

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

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

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
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

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


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'

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

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

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’
-

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

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’
-
-
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

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

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’
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
-


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’

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

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

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

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"

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

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
-

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

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

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

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

17


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

-
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

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

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

-
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)

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

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


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’
-
-

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.

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’

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

-
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”
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

>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-

(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:http://www.tandfonline.com/doi/full/10.1080/14786419.2013.832677#.Ujh
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