Isolation of lignans and neolignans from Pouzolzia sanguinea with their cytotoxic activity

Cite this paper: Vietnam J. Chem., 2021, 59(2), 146-152 Article 
DOI: 10.1002/vjch.202000120 
146 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH 
Isolation of lignans and neolignans from Pouzolzia sanguinea with their 
cytotoxic activity 
 Le Thi Hong Nhung
1,2
, Nguyen Thi Hoang Anh
1,3
, Bui Huu Tai
1,4
, Phan Van Kiem
1,4*
1
Graduate University of Science and Technology, Vietnam Academy of Science and Technology (VAST), 18 
Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam
2
Faculty of Chemical Technology, Hanoi University of Industry, Bac Tu Liem, Hanoi 10000, Viet Nam
3
Institute of Chemistry, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam
4
Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam
Submitted July 13, 2020; Accepted August 5, 2020 
Abstract 
One lignan (7′S,8′R,8S)-4,4′-dihydroxy-3,3′,5,5′-tetramethoxy-7′,9-epoxylignan-9′-ol-7-one (1) together with four 
neolignans (7α,8α)-dihydrodehydrodiconiferyl alcohol 9-O-β-D-glucopyranoside (2), (7α,8α)-dihydrodehydro-
diconiferyl alcohol 9′-O-β-D-glucopyranoside (3), icariside E3 (4), and icariside E5 (5) were isolated from Pouzolzia 
sanguinea. Their chemical structures were elucidated by ESI-MS, NMR spectra, as well as in comparison with the data 
reported in literature. At concentration of 30 µM, compounds 1-5 exhibited weak cytotoxic activity with cell viability 
percentages ranging from 59.9±0.98 % to 84.2±0.98 % and from 77.7±0.81 % to 100.3±0.78 % on CAL27 (oral 
adenosquamous carcinoma cell) and MDA-MB-321 (breast cancer cell) cell lines, respectively. 
Keywords. Pouzolzia sanguinea, lignan, neolignan, cytotoxicity. 
1. INTRODUCTION 
Pouzolzia species have been used to treat ulcers in 
traditional medicinal remedy in several countries such 
as India, China, Thailand, and Vietnam.
[1-3]
 Previous 
reports indicated that methanolic extract of P. indica 
significantly exhibited anti-proliferative effect and 
induced apoptotic process on NB4 and HT93A acute 
leukemic cell lines.
[3]
 Phytochemical studies on 
Pouzolzia genus revealed the presence of norlignans, 
prenylated isoflavones, triterpenes which have shown 
anti-inflammation and cytotoxic activities.
[4,5] 
In our 
previous report, several norlignans were identified 
from aerial parts of P. sanguinea. Their chemical 
structures were remarkable not only by the loss of one 
carbon in lignan skeleton but also the presence of an 
additional benzene ring.
[6]
 Continuously, herein, we 
report the isolation and identification of one lignan 
and four neoligans from P. sanguinea. Cytotoxic 
effects of the isolated compounds were evaluated on 
CAL27 and MDA-MB-231 cell lines using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 
bromide (MTT) assay. 
2. MATERIALS AND METHODS 
2.1. Plant materials 
Plant materials were collected at Da Lat, Lam 
Dong, Vietnam in March 2018. Plant taxonomy, 
Pouzolzia sanguinea (Blume) Merr. was identified 
by Dr. Nguyen The Cuong, Institute of Ecology 
and Biological Resources, VAST. Voucher 
specimen (NCCT0318) was kept at the Institute of 
Ecology and Biological Resources, VAST. 
2.2. General experimental procedures 
The used characterization techniques are the same as 
described elsewhere.
[14] 
2.3. Extraction and isolation 
The dried powdered P. sanguinea sample (5.0 kg) 
was ultrasonic extracted with MeOH for three times 
to get MeOH extract (350g). The MeOH extract was 
suspended with water and successively separated in 
n-hexane, dichloromethane, and ethyl acetate to give 
Vietnam Journal of Chemistry Phan Van Kiem et al. 
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 147 
corresponding soluble fractions and water layer. 
Ethyl acetate extract (34 g) was separated on a silica 
gel column, eluting with gradient solvent system of 
dichloromethane/MeOH (0-100 % volume of 
methanol) to give 6 fractions, PSE1-PSE6. Fraction 
PSE2 was chromatographed on a RP-18 column and 
eluted with MeOH/water (1/1, v/v) to give 3 
fractions, PSE2A-PSE2C. PSE2B was purified by 
preparative HPLC using isocratic mobile phase 25 % 
acetonitrile in water to give compound 1 (4.6 mg, tR 
49.2 min). Water layer was loaded on diaion HP-20 
column, washed with water, and then eluted with 
water/methanol (25 %, 50 %, 75 %, and 100 % 
volume of methanol) to give four fractions PSW1-
PSW4. Fraction PSW3 (8 g) was separated on a 
silica gel column chromatography, eluting with 
dichloromethane/methanol/water (6/1/0.1, v/v/v) to 
give three fractions PSW3A- PSW3C. Fraction 
PSW3B was purified by preparative HPLC using 
isocratic mobile phase 21 % acetonitrile in water to 
give compounds 3 (8.4 mg, tR 39.6 min) and 2 (3.6 
mg, tR 42.2 min). Finally, fraction PSW3C was 
purified by preparative HPLC using isocratic mobile 
phase 18 % acetonitrile in water to obtain 
compounds 4 (10.6 mg, tR 40.3 min) and 5 (23.9 mg, 
tR 42.7 min). 
Figure 1: Chemical structures of compounds 1-5 
(7′S,8′R,8S)-4,4′-Dihydroxy-3,3′,5,5′-
tetramethoxy-7′,9-epoxylignan-9′-ol-7-one (1): 
Yellow gum; [ ] 
 -11.4° (c 0.1, MeOH); ESI-MS: 
m/z 435 [M+H]
+
; 
1
H-NMR (CD3OD, 500 MHz) δH 
7.41 (2H, s, H-2 and H-6), 4.30 (1H, m, H-8), 4.20 
(1H, dd, J = 8.0, 8.5 Hz, Ha-9), 4.27 (1H, dd, J = 4.5, 
8.5 Hz, Hb-9), 6.75 (2H, s, H-2′ and H-6′), 4.67 (1H, 
d, J = 8.0 Hz, H-7′), 2.67 (1H, m, H-8′), 3.71 (1H, 
dd, J = 4.5 and 11.5 Hz, Ha-9′), 3.67 (1H, dd, J = 
4.5, 11.5 Hz, Hb-9′), 3.94 (6H, s, 3,5-OCH3), 3.88 
(6H, s, 3′,5′-OCH3); 
13
C-NMR (CD3OD, 125 MHz) 
δC 128.5 (C-1), 107.8 (C-2), 149.2 (C-3), 143.4 (C-
4), 149.2 (C-5), 107.8 (C-6), 200.3 (C-7), 50.2 (C-8), 
71.6 (C-9), 132.9 (C-1′), 105.3 (C-2′), 149.3 (C-3′), 
136.4 (C-4′), 149.3 (C-5′), 105.3 (C-6′), 85.5 (C-7′), 
55.1 (C-8′), 61.4 (C-9′), 56.9 (3,5-OCH3), and 56.8 
(3′,5′-OCH3 ...  8.0 Hz)], two oxygenated 
methylene groups [δH 4.27 (1H, dd, J = 4.5, 8.5 Hz) 
and 4.20 (1H, dd, J = 8.0, 8.5 Hz), 3.71 and 3.67 
(each 1H, dd, J = 4.5, 11.5 Hz)], and two aliphatic 
methine groups [δH 4.30 and 2.67 (each, 1H, m)]. 
The 
13
C-NMR and HSQC spectra of 1 showed 
signals of 22 carbons including one carbonyl group 
(δC 200.3), twelve aromatic carbons (δC 
105.3~149.3), one oxygenated methine group (δC 
85.5), two oxygenated methylene groups (δC 71.6 
and 61.4), four methoxy groups [δC 56.9 and 56.8 
(each 2C)], and two aliphatic methine groups (δC 
55.1 and 50.2). Appearance of two pair of aromatic 
protons (δH 7.41 and 6.75) and four pair of aromatic 
carbons (δC 149.3, 149.2, 107.8, 105.3) magnetically 
equivalent indicated the presence of two symmetric 
1,3,4,5-tetrasubtitited benzene rings. The HMBC 
correlations between H2-9 (δH 4.27 and 4.20) and C-
8′ (δC 55.1)/C-8 (δC 50.2)/C-7′ (δC 85.5), H-7′ (δH 
4.67) and C-8′/C-8 /C-9 (δC 71.6) demonstrated the 
presence of tetrahydrofuran ring (C-ring, figures 1 
and 2). Next, HMBC correlations between H-2′/H-6′ 
(δH 6.75) and C-4′ (δC 136.4), methoxy protons (δH 
3.88) and C-3′/C-5′ (δC 149.3) supported assignment 
of the first 4′-hydroxy-3′,5′-dimethoxyphenyl group 
(A-benzene ring). Furthermore, HMBC correlations 
between H-2′/H-6′ (δH 6.75) and C-7′ (δC 85.5) 
indicated this 4′-hydroxy-3′,5′-dimethoxyphenyl 
connect to tetrahydrofuran ring at C-7′. Carbon 
Vietnam Journal of Chemistry Isolation of lignans and neolignans from 
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 150 
chemical shift value of C-9′ (δC 61.4), HMBC 
correlations between H2-9′ (δH 3.71 and 3.67) and C-
7′ (δC 85.5)/C-8′ (δC 55.1)/C-8 (δC 50.2) suggested 
hydroxymethylene group was at C-8′. Second 
benzene ring moiety (B-benzene ring) was deduced 
to be 4-hydroxy-3,5-dimethoxybenzoyl group which 
was confirmed by HMBC correlations between 
H-2/H-6 (δH 7.41) and C-4 (δC 143.4)/C-7 (δC 
200.3), between methoxy protons (δH 3.94) and C-
3/C-5 (δC 149.2). Additionally, HMBC correlations 
between H-8′ (δH 2.67)/H-8 (δH 4.30)/H2-9 (δH 4.27 
and 4.20) and C-7 (δC 200.3) indicated 4-hydroxy-
3,5-dimethoxybenzoyl group connected to 
tetrahydrofuran ring at C-8 to form a lignan 
backbone. Due to containing three chiral centers (C-
7′, C-8′, and C-8) relative configurations at those 
centers were examined by analysis of NOESY 
spectrum. NOESY correlations between H2-9′ (δH 
3.71 and 3.67) and H-7′ (δH 4.67)/H-8 (δH 4.30) 
indicated the close proximity of hydroxymethylene 
group (C-9′), H-7′, and H-8 as described in figure 1. 
Finally, absolute configurations at C-7′, C-8′, and C-
8 was determined to be 7′S, 8′R, and 8S by negative 
optical rotation [-11.4° (c 0.1, MeOH)] compared to 
previous literature [(7′S,8′R,8S)-enanthiomer: 
-1.5°
[7]: and (7′R,8′S,8R)-enanthiomer: +14°.[8] 
Furthermore, the ESI mass spectrum of 1 exhibited 
an ion peak at m/z 435 [M+H]
+
, corresponding to the 
molecular formula of C22H26O9. Thus, compound 1 
was determined to be (7′S,8′R,8S)-4,4′-dihydroxy-
3,3′,5,5′-tetramethoxy-7′,9-epoxylignan-9′-ol-7-one. 
Compound 2 was isolated as pale-yellow amorphous 
powder. The 
1
H-NMR spectrum of 2 contained 
signals corresponding to an ABX coupled spin 
system [δH 7.01 (1H, d, J = 1.5 Hz), 6.89 (1H, dd, J 
= 1.5, 8.0 Hz), 6.77 (1H, d, J = 8.0 Hz)], an AX 
coupled spin system [δH 6.80 and 6.74 (each 1H, br 
s)], an anomeric proton [δH 4.37 (1H, d, J = 8.0 Hz)], 
an oxygenated methine group (δH 5.62 (1H, d, J = 
6.5 Hz)], and two methoxy groups (δH 3.87 and 3.84 
(each 3H, s)]. The 
13
C-NMR spectrum of 2 showed 
signals corresponding to 26 carbon atoms. Among 
them, six oxygenated carbons (δC 104.6, 78.3, 78.1, 
75.2, 71.7, 62.8) and J value of anomeric proton (δH 
4.37, d, J = 8.0 Hz) were assigned for a β-D-
glucopyranosyl group. The presence of two methoxy 
groups was agreed by two carbon signals at δC 56.8 
and 56.4. Additionally, three sp
3
-hybridized carbon 
atoms [δC 62.2 (C-9′), 32.9 (C-8′), 35.8 (C-7′)] and 
their bearing protons [δH 3.59 (t, J = 6.5 Hz, H2-9′), 
1.84 (m, H2-8′), 2.64 (t, J = 7.5 Hz, H2-7′), 
respectively] suggested the presence of 3-
hydroxypropyl group. The HMBC correlations 
between H2-7′ (δH 2.64) and C-1′ (δC 136.9)/ C-2′ (δC 
114.3)/ C-6′ (δC 118.2) indicated this hydroxypropyl 
group linked to C-1′. HMBC correlations between 
H-6′ (δH 6.80) and C-8 (δC 53.3)/C-4′ (δC 147.4), H-7 
(δH 5.62)/H-8 (δH 3.67) and C-5′ (δC 129.7)/C-4′ (δC 
147.4) established benzofuran moiety (A and C 
rings, Fig. 1). Other benzene ring (B-ring) was 
established to be 3-methoxy-4-hydroxyphenyl group 
which was supported by HMBC correlations 
between H-2 (δH 7.01)/H-6 (δH 6.89) and C-4 (δC 
147.7), H-5 (δH 6.77)/3-OCH3 (δH 3.84) and C-3 (δC 
149.0). Furthermore, HMBC correlations between 
H-2/H-6 and C-7 (δC 89.0) indicated 3-methoxy-4-
hydroxyphenyl group connect to C-7 of benzofuran 
moiety. Other methoxy group at C-3′ was also 
confirmed by HMBC correlations between 3′-OCH3 
(δH 3.87)/ H-2′ (δH 6.74) and C-3′ (δC 145.2). HMBC 
correlations between H2-9 (δH 4.23 and 3.78) and C-
7 (δC 89.0)/ C-8 (δC 53.3)/C-5′ (δC 129.7)/Glc C-1″ 
(δC 104.6) proved O-glucosidic linkage at C-9. The 
sugar linkage must be in the β-form identified by glc 
JH-1/H-2 = 7.5 Hz. Relative configurations at C-7 and 
C-8 were deduced to be 7α and 8α, respectively, by 
comparison their carbon chemical shifts (δC-7 89.0 
and δC-8 53.3) with that reported in the literature 
(relative 7α,8α isomer[9]: δC-7 89.0 and δC-8 53.3), 
relative 7β,8α isomer[10]: δC-7 83.3 and δC-8 54.1). 
Furthermore, the ESI mass spectrum of 2 exhibited 
an ion peak at m/z 545 [M+Na]
+
, corresponding to 
the molecular formula of C26H34O11. Consequently, 
compound 2 was determined as (7α,8α)-
dihydrodehydrodiconiferyl alcohol 9-O-β-D-
glucopyranoside. 
The 
1
H- and 
13
C-NMR data of compound 3 were 
found very similar with compound 2, except signals 
of two oxygenated methylene groups [δC 65.0 (C-9) 
and 69.9 (C-9′), table 1]. HMBC correlations 
between H-7 (δH 5.51) and C-9 (δC 65.0), H2-7′ (δH 
2.70) and C-9′ (δC 69.9) confirmed assignments of 
C-9 and C-9′ at chemical shift values of δC 65.0 and 
δC 69.9, respectively. Therefore, in compound 3, 
upfield movement at carbon chemical shift of C-9 
(δC 65.0) demonstrated a hydroxy group at C-9 
meanwhile downfield movement at carbon chemical 
shift of C-9′ (δC 69.9) suggested O-glucopyranosyl 
group at C-9′. The presence of O-glucopyranosyl 
group at C-9′ was also confirmed by HMBC 
correlations between Glc H-1″ (δH 4.27) and C-9′ (δC 
69.9), H2-9′ (δH 3.55 and 3.94) and Glc C-1″ (δC 
104.5). Carbon chemical shift values at C-7 (δC 89.0) 
and C-8 (δC 55.4) indicated 7α,8α relative 
configurations as shown in compound 2. The 
coupling constant (J = 7.5 Hz) observed for the 
anomeric proton in the 
1
H-NMR spectrum indicated 
the β-glucoside linkage of the O-glucose moiety. 
Furthermore, the ESI mass spectrum of 3 exhibited 
an ion peak at m/z 545 [M+Na]
+
, corresponding to 
Vietnam Journal of Chemistry Phan Van Kiem et al. 
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 151 
the molecular formula of C26H34O11. Therefore, 
compound 3 was determined as (7α,8α)-
dihydrodehydrodiconiferyl alcohol 9′-O-β-D-
glucopyranoside. 
Compound 4 was isolated as pale-yellow 
amorphous powder. The 
1
H-NMR and HSQC 
spectra of 4 showed an ABX coupled spin system 
[δH 6.59 (1H, d, J = 8.0 Hz), 6.57 (1H, d, J = 1.5 
Hz), 6.49 (1H, dd, J = 1.5, 8.0 Hz)], an AX coupled 
spin system [δH 6.73 (2H, overlapped, br s)], an 
anomeric proton [δH 4.63 (1H, d, J = 7.5 Hz)], and 
two methoxy groups [δH 3.84 and 3.71 (each 3H, s)]. 
Different with 
1
H-NMR spectra of compounds 2 and 
3, a doublet oxygenated methine signal was not 
observed in the 
1
H-NMR of 4, suggesting the 
absence of furan ring. Additionally, carbon signal of 
C-7 (δC 39.2), its bearing protons (δH 2.99 and 2.72), 
HMBC correlations between H-2 (δH 6.57)/H-6 (δH 
6.49) and C-7 indicating oxygenated methine group 
(C-7) in compounds 2-3 was replaced by methylene 
group in compound 4. Carbon chemical shift values 
of C-9 (δC 67.1) and C-9′ (δC 62.2) indicated the 
presence of hydroxy group at C-9 and C-9′, 
respectively. HMBC correlations between H-2′ / H-
6′ (δH 6.73)/Glc H-1″ (δH 4.63) and C-4′ (δC 143.6) 
indicated that O-glucopyranosyl group connect to C-
4′. The sugar linkage must be in the β-form indicated 
by glc JH-1/H-2 = 7.5 Hz. Furthermore, the ESI mass 
spectrum of 4 exhibited an ion peak at m/z 547 
[M+Na]
+
, corresponding to the molecular formula of 
C26H36O11. Thus, compound 4 was determined to be 
icariside E3 as previously reported by Sadhu and co-
authors (table 2).
[11]
The 
1
H- and 
13
C-NMR spectra of compound 5 
were similar with compound 4 except the 
appearance of vinyl group (-CH=CH-) instead of 
ethylene group (-CH2-CH2-). The presence of vinyl 
group at C-7′/C-8′ was also agreed with doublet 
signals of methylene proton H2-9′. Furthermore, 
value of J coupling constant between H-7′ and H-8′ 
(J = 15.5 Hz) indicated geometric configuration of 
double bond at C-7′/C-8′ to be E-configuration. 
Furthermore, the ESI mass spectrum of 5 exhibited 
an ion peak at m/z 545 [M+Na]
+
, corresponding to 
the molecular formula of C26H34O11. Consequently, 
compound 5 was determined to be icariside E5 as 
previously reported by Lee and co-authors (table 
2).
[12]
Compounds 1-5 were evaluated their cytotoxic 
effects on CAL27 and MDA-MB-231 cells using 
MTT assay.
[13]
 At concentration of 30 µM, 
compounds 1-5 exhibited weak cytotoxic activity 
with cell viability percentages ranging from 
59.9±0.98 % to 84.2±0.98 % and from 77.7±0.81 % 
to 100.3±0.78 % on CAL27 and MDA-MB-321 cell 
lines, respectively (table 3). Because of cell viability 
percentages all over 50 % in the presence of 
compounds 1-5 (30 µM), further cytotoxic study was 
not investigated as well as dose-dependent study. 
Table 3: Cytotoxic activity of 1-5 (30 µM) 
Compound 
Cell viability (%) 
CAL27 MDA-MB-231 
1 84.2±0.98 100.3±0.78 
2 78.2±0.88 91.4±1.14 
3 71.8±0.88 77.7±0.81 
4 59.9±0.98 97.0±0.93 
5 65.8±1.03 91.1±0.81 
Acknowledgment. This research is funded by 
Graduate University of Science and Technology, 
Vietnam Academy of Science and Technology under 
grant number: GUST.STS.ĐT2019/HH03. 
REFERENCES 
1. V. V. Chi. Dictionary of Vietnamese Medicinal 
Plants, Hanoi: Medicine Publishing House, Vol. 1.p. 
191, 2012. 
2. C. M. Wilmot-Dear, I. Friis. The Old World species 
of Pouzolzia (Urticaceae, tribus Boehmerieae). A 
taxonomic revision, Nord. J. Bot., 2004, 24, 5-111. 
3. S. Chanyapat, J. Weena, U. Yaowalak, K. Tanawan. 
Antiproliferative effect and the isolated compounds 
of Pouzolzia indica. Evid-based Complement. 
Alternat. Med., 2013, Article ID 342352. 
DOI: 10.1155/2013/342352. 
4. Z. H. Chen, H. Zhang, S. H. Tao, Z. Luo, C. Q. 
Zhong, L. B. Guo. Norlignans from Pouzolzia 
Zeylanica var. Microphylla and their nitric oxide 
inhibitory activity, J. Asian Nat. Prod. Res., 2015, 17, 
959-966. 
5. D. Maiti, A. K. Singha, C. Sarkar, S. Sarkar, I. Sil 
Sarma, K. Manna, B. Dinda. Friedelane, isolated 
from Pouzolzia indica Gaud. exhibits toxic effect 
against melanoma, Cytotechnology, 2018, 70, 1111-
1120. 
6. L. T. H. Nhung, P. T. M. Huong, N. T. Anh, B. H. 
Tai, N. X. Nhiem, V. V. Doan, N. H. Hoang, Y. Seo, 
S. H. Kim, P. V. Kiem, Two new norlignans from the 
aerial parts of Pouzolzia sanguinea (Blume) Merr., 
Nat. Prod. Res., 2020, 1-8. DOI: 
10.1080/14786419.2020.1771707. 
7. L. Xiong, C. Zhu, Y. Li, Y. Tian, S. Lin, S. Yuan, J. 
Hu, Q. Hou, N. Chen, Y. Yang, J. Shi. Lignans and 
neolignans from Sinocalamus affinis and their 
absolute configurations, J. Nat. Prod., 2011, 74, 
1188-1200. 
8. S. Y. Lee, W. S. Suh, J. M. Cha, E. Moon, S. K. Ha, 
S. Y. Kim, K. R. Lee. Anti-neuroinflammatory 
constituents from Sinomenium acutum rhizomes, 
Vietnam Journal of Chemistry Isolation of lignans and neolignans from 
© 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 152 
Phytochem. Lett., 2016, 17, 79-84. 
9. S. Lee, I. H. Song, J. H. Lee, W. Y. Yang, K. B. Oh, 
J. Shin. Sortase A inhibitory metabolites from the 
roots of Pulsatilla koreana, Bioorg. Med. Chem. 
Lett., 2014, 24, 44-48. 
10. C. Wang Z. Jia. Lignan, phenylpropanoid and iridoid 
glycosides from Pedicularis torta, Phytochemistry, 
1997, 45, 159-166. 
11. S. K. Sadhu, A. Khatun, P. Phattanawasin, T. 
Ohtsuki, M. Ishibashi. Lignan glycosides and 
flavonoids from Saraca asoca with antioxidant 
activity, J. Nat. Med., 2007, 61, 480-482. 
12. D. Y. Lee, D. G. Lee, J. G. Cho, M. H. Bang, H. N. 
Lyu, Y. H. Lee, S. Y. Kim, N. I. Baek. Lignans from 
the fruits of the red pepper (Capsicum annuum L.) 
and their antioxidant effects, Arch. Pharm. Res., 
2009, 32, 1345-1349. 
13. T. Mosmann. Rapid colorimetric assay for cellular 
growth and survival: Application to proliferation and 
cytotoxicity assays, J. Immunol. Methods, 1983, 65, 
55-63. 
14. P. V. Kiem, D. T. H. Yen, N. V. Hung, N. X. Nhiem, 
B. H. Tai, D. T. Trang, P. H. Yen, T. M. Ngoc, C. V. 
Minh, S. J. Park, J. H. Lee, S. Y. Kim, S. H. Kim. 
Five new pregnane glycosides from Gymnema 
sylvestre and their α-glucosidase and α-amylase 
inhibitory activities, Molecules, 2020, 25, 2525. DOI: 
10.3390/molecules25112525. 
Corresponding author: Phan Van Kiem 
Institute of Marine Biochemistry 
Vietnam Academy of Science and Technology 
18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 
E-mail: phankiem@yahoo.com.