Vietnam Journal of Chemistry, 2018, 56(4), 521-524 Article

DOI: 10.1002/vjch.201800041

Serratene-type triterpenoids from Lycopodium cernuum

Pham Huu Dien1*, Nguyen Thi Thao1, Nguyen Thi Hai1, Le Thi Khanh Linh1,
Dang Ngoc Quang1, Bui Tuan Nam2, Nguyen Quang Tung3
1
Hanoi National University of Education, Cau Giay district, Hanoi, Viet Nam
2
Yen Minh High School, Yen Minh town, Ha Giang province, Viet Nam
3
Faculty of ChemicalTechnology, Hanoi University of Industry, Viet Nam
Received January 1st 2018; Accepted for publication April 26th 2018

Abstract
Four metabolites, serrat-14-en-3β,21β-diol (1), serrat-14-en-3β,21-diol (2), 3β,21β-dihydroxyserrat-14-en-21-yl
acetate (3), and nonacosanol (4) were isolated from the aerial parts of Lycopodium cernuum plant, collected in Hoang
Su Phi Dist., Ha Giang province. Their structures were elucidated by various spectroscopic analyses.
Keywords. Lycopodium cernuum, serrat-14-en-3β,21β-diol, serrat-14-en-3β,21-diol, 3β,21β-dihydroxyserrat-14-
en-21-yl acetate.

1. INTRODUCTION tetramethylsilane (TMS) as an internal standard,

Lycopodium cernuum L. plant, also called
Lycopodiella cernua (L.) Pic.-Serm. or “Thông
đất”,[1] “Thạch tùng nghiên”,[2] widely distributed in
high mountain areas of Vietnam, China, Thailand,
Indonesia, Phillipines etc. According to the
Vietnamese traditional medicine, the whole plant has
been used for the treatment of angina, dropsy,
rheumatoid arthritis, etc. Recently, it’s spreading a
rumour that Lycopodium cernuum L. plant can treat
Alzheimer’s disease, so the plant faces to be hunted
and exploited exhaustedly. Chemical investigations
on Lycopodium (also called Huperzia) genus have
resulted in the isolation of somelycopodium
alkaloids,[3,4] serratene-type triterpenoids,[5,6]
[7]
flavonoids ... Herein, we describe the isolation and
structural elucidation of some serratene-type
triterpenoids from Lycopodium cernuum plant,
growing in the Hoang Su Phi Distr. (Ha Giang
Province).
2. EXPERIMENTAL
2.1. General experimental procedures
IR spectra were recorded on a Shimadzu-FTIR
8101M spectrophotometer, using KBr disk. NMR
(1H, 13C NMR, DEPT, HSQC, HMBC) spectra were
recorded on a Bruker Avance 500 MHz. The
chemical shift (δ) values are given in ppm with
coupling constant J (by Hz). ESI-LC-MS spectra
were recorded on an Agilent LC mass spectrometer.
Silica gel (Merck Co., Germany) was used for flash
chromatography. TLC was carried out on precoated
Si gel GF254 (Merck Co., Germany) and TLC spots
were viewed at 254, 302 and 366 nm and visualized
by spraying with vanillin - 10 % H2SO4 solution.
2.2. Plant material
The aerial parts of Lycopodium cernuum were
collected in the Thang Tin Commun (Hoang Su Phi
Distr., Ha Giang Province) in October, 2016. The
plant material was identified by Prof. Do Huu Thu,
Institute of Ecology, Natural resource and Biology,
Vietnam Academy of Science and Technology,
Vietnam. A voucher specimen was deposited in the
Department of Organic Chemistry, HNUE
(TD10.2016).
2.3. Extraction and isolation
The air-dried aerial parts of L. cernuum (2.0 kg)
were ground into powder and extracted with 80 %
MeOH (10L x 3) at room temperature to give a
crude extract (175 g). The crude extract was
subjected to fractional extraction and then vacuum
evaporation, giving n-hexane (15 g), ethyl acetate
(EtOAc, 86 g), butanol (28 g), methanol (41 g)
extracts, respectively. The EtOAc extract was

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Vietnam Journal of Chemistry
subjected to column chromatography over silica gel
and eluted with n-hexane - ethyl acetate gradient
from 10:1 to 1:100. Ten fractions were successively
obtained. Fraction 2 (1.20 g) were precipitated as
white crystals, recrystallized them to afford 1 (25
mg). Fraction 5 (520 mg) was separated by column
chromatography (SiO2) to afford 2 (12 mg). Fraction
8 (72 mg) was separated by column chromatography
(SiO2) to afford 3 (5 mg). Fraction 10 (32 mg) was
separated by column chromatography (SiO2) to
afford 4 (5 mg).
Compound 1 (Serrat-14-en-3β,21 -diol): White
powders; mp. 304-305 oC. ESI-LC-MS (m/z): 425.1
[M+H-H2O]+ IR (KBr, ν cm-1): 3494 (OH), 1463,
1385 (C=C); 1H NMR (500 MHz, CDCl3, TMS), H
(ppm): 5.33 (br s, H-15), 3.45 (br s, H-21), 3.19 (dd,
J = 11.5, 4.5 Hz, H-3), 2.21 (d, J = 14.5 Hz, H-27a),
1.71 (d, J = 14.5 Hz, H-27b), 0.97 (3H, s, H-23),
0.94 (3H, s, H-30), 0.88 (3H, s, H-29), 0.84 (3H, s,
26), 0.80 (3H, s, H-25), 0.77 (3H, s, C-24), 0.69 (3H,
s, H-28); 13C NMR (125 MHz, CDCl3, TMS), see
table 1.
Compound 2 (Serrat-14-en-3β,21-diol): White
powders; ESI-LC-MS (m/z): 424.6 [M+H-H2O]+; 1H
NMR (500 MHz, CDCl3, TMS), H (ppm): 5.33 (br
s, H-15), 3.22 (dd, J = 4.0, 11.5 Hz, H-3), 3.18 (dd, J
= 4.5, 11.0 Hz, H-21), 2.22 (d, J = 15.0 Hz, H-27a),
1.75 (d, J = 15.0 Hz, H-27b), 0.97 (3H, s, H-30),
0.96 (3H, s, H-23), 0.88 (3H, s, 29), 0.86 (3H, s, H-
24), 0.80 (3H, s, H-25), 0.77 (3H, s, H-26), 0.67
(3H, s, H-28); 13C NMR (125 MHz, CDCl3, TMS),
see table 1.
Compound 3 (3β,21β-dihydroxyserrat-14-en-21-
yl acetate): White powders, m.p.: 336-337 oC; well
dissolved in ethyl acetate, chloroform. ESI-LC-MS
(m/z): 424.8 [M+H-CH3COOH]+; 1H NMR (500
MHz, CDCl3, TMS): H (ppm): 5.33 (br s, H-15),
4.50 (dd, J = 11.5, 4.0 Hz, H-21), 3.18 (dd, J = 12.0,
5.0 Hz, H-3), 2.20 (d, J = 14.0 Hz, H-27a), 1.78 (d, J
= 14.0 Hz, H-27b), 2.05 (3H, s, CH3COOR), 0.97
(s), 0.90 (s), 0.84 (s), 0.82 (s), 0.80 (s) 0.77 (s), 0.69
(s) - 7 x CH3. 13C NMR (125 MHz, CDCl3, TMS),
see table 1.
Compound 4 (nonacosanol): White powders;
ESI-LC-MS: m/z 425.2 [M+H]+; 1H NMR (500
MHz, CDCl3 ), H (ppm): 3.64 (2H, t, J = 7.0 Hz, H-
1), 1.26-1.55 (54H, m, H-2÷H-28), 0.88 (3H, t, J =
6.5, H-29); 13C NMR (125 MHz, CDCl3, TMS), C
(ppm): 63.1 (C-1), 22.7-32.8 (27C, C-2÷C-28), 14.1
(C-29).
3. RESULTS AND DISCUSSION
Compound 1 was obtained as colourless amorphous
© 2018 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Pham Huu Dien et al.
powders. Its molecular formula was deduced to be
C30H50O2 on the basic of the pseudo-molecular ion
[M+H-H2O]+ peak at m/z 425.1 in ESI-LC-MS
spectrum, with six degrees of unsaturation. Its IR
spectrum (KBr) showed the presence of free
hydroxyl group (3494 cm-1), a double bond >C=C<
(1463 and 1383 cm-1). The 1H NMR spectrum of 1
in CDCl3 displayed signals for seven tertiary
methyls, two axial protons of an oxygenated methine
at H 3.45 (br s, H-21), 3.19 (dd, J = 11.5, 4.5 Hz, H-
3), an olefinic proton at H 5.33 (br s, H-15). All
above results suggest that 1 belongs to serratenes – a
group of naturally occurring pentacyclic-
triterpenoids, which posses seven tertiary methyls
and seven-membered ring C. The 13C NMR, DEPT
and HSQC spectra of 1 presented 30 carbon signals
including seven methyl carbons, ten methylene
carbons, seven methine carbons and six quaternary
carbons (table 1). Among them, two carbons at C
138.5 (C-14) and 122.1 (C-15) were assigned to a
double bond >C=C< of pentacyclictriterpenoids; two
methine carbons at C 78.9 (C-3) and 76.3 (C-21)
were assigned to carbinol carbons; one methylene
carbon at C 56.3 (C-27) was assigned to seven-
membered ring C. From above analyses of NMR,
MS spectra, melting point of 1, in comparison with
those in,[7,8] we made a conclusion that 1 is serrat-
14-en-3β,21 -diol. This metabolite presented in
some plants, especially in Lycopedium genus such as
L. megastarchyum,[8] Huperzi aserrata Thunb.,[6,7]
now it is the first time 1 was isolated from L.
cernuum. According to,[10] 1 might be a potential
candidate for the development of anti-cancer drugs
for the treatment of leukemia.
Compound 2 was obtained as colourless
amorphous powders. Its molecular formula was
deduced to be C30H50O2 on the basic of the pseudo-
molecular ion [M+H-H2O]+ peak at m/z 424.6 in
ESI-LC-MS spectrum, with six degrees of
unsaturation. Its 1H NMR spectrum in CDCl3
resembles that of 1, displayed signals for seven
tertiary methyls, one axial proton of an oxygenated
methine at H 3.22 (dd, J = 4.0, 11.5 Hz, H-3), one
equatorial at 3.18 (dd, J = 4.5, 11.0 Hz, H-21); an
olefinic proton at H 5.33 (br s, H-15). It means that
2 should be an epimer of 1 at C-21. The 13C NMR,
DEPT and HSQC spectra of 2, like 1, presented 30
carbon signals including seven methyl carbons, ten
methylene carbons, seven methine carbons and six
quaternary carbons (table 1). Among them, two
carbons at C 138.2 (C-14) and 122.2 (C-15) were
assigned to a double bond >C=C< of
pentacyclictriterpenoids; two methine carbons at
C78.9 (C-3) and 79.2 (C-21) were assigned to
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Vietnam Journal of Chemistry Serratene-type triterpenoids from…

carbinol carbons; one methylene carbon at C 56.1
(C-27) was assigned to seven-membered ring C.
Based on data derived from NMR, MS analyses,
melting point of 2, in comparison with those in
literature,[7] compound 2 is confirmed to be serrat-
14-en-3β,21 -diol. This metabolite was already
detected in some plants, especial in Lycopedium
genus such as L. megastarchyum,[8] Huperzi aserrata
Thunb.,[6,7] but was isolated from L. cernuum for the
first time in this work. According to,[9] compound 2
exhibited a remarkable inhibitory effect on skin
tumor promotion.

Table 1: 13C NMR (125 MHz, CDCl3, TMS) spectral data of compounds 1-3
No. 1 2 3 No. 1 2 3
1 38.6 38.6 38.6 17 43.4 49.5 49.6
2 25.2 25.3 24.2 18 37.2 38.9 37.1
3 78.9 78.9 78.8 19 31.2 37.1 36.8
4 38.2 38.2 38.2 20 27.6 27.6 27.6
5 55.8 55.8 55.7 21 76.3 79.2 81.1
6 18.9 18.9 18.9 22 37.5 37.2 37.8
7 45.2 45.2 45.2 23 15.7 15.8 15.8
8 39.0 39.0 39.0 24 19.8 19.8 19.8
9 56.9 57.2 57.1 25 28.1 28.1 28.1
10 36.0 36.1 36.0 26 15.4 15.4 15.4
11 25.5 27.2 25.3 27 56.3 56.1 56.0
12 27.2 27.6 27.2 28 13.3 13.4 13.5
13 62.9 62.9 62.9 29 27.7 27.7 27.5
14 138.5 138.2 138.3 30 21.8 21.1 19.8
15 122.1 122.2 122.0 CH3CO - - 21.3
16 24.0 24.1 23.9 CH3CO - - 171.0

R1
1: R1=OH, R2=H
21 R2 2: R1=H, R2=OH
H
29 3: R1=OOCCH3, R2=H
23 24 23
30
1
14 28

15
3 H
27 CH3[CH2]27CH2OH
HO
4
26 25
Figure 1: Structures of compounds 1-4

The molecular formula of compound 3 was
deduced to be C32H52O3 on the basic of the pseudo-
molecular ion [M+H-CH3COOH]+ peak at m/z 424.8
in ESI-LC-MS spectrum. Its 1H NMR spectrum
looks like of 1 except the presence of acetate methyl
at H 2.05 (3H, s) and one acetylated carbinol proton
at H 4.50 (dd, 11.5 4.0 Hz) that was assigned to H-
21. It means that 3 should be an acetylated
derivative of 1 at C-21. The 13C NMR, DEPT and
HSQC spectra of 3 presented 32 carbon signals
including eight methyl carbons (one from them is an
acetate methyl), ten methylene carbons, seven
methine carbons and seven quaternary carbons (table
1). Among them, there are two olefinic carbons at δC
138.3 and 122.0 ppm very typical for double bonds
at C14-C15, one carboxylate carbon at δC 171.0 ppm
assigned for acetate carbonyl; one carbinol carbon at
δC 78.8 ppm (C-3). By using HMBC it found that the
signal of carbinol carbon at δC 81.1 (C-21) has a
crosslink correlation with the acetate group. The
mass, 1H and 13C NMR spectral data identified 3 as
3β,21β-dihydroxyserrat-14-en-21-yl acetate.[6] This

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Vietnam Journal of Chemistry
is the first time that compound 3 was isolated from
L. cernuum.
For the case of compound 4, its molecular
formula was deduced to be C29H60O on the basic of
the pseudo-molecular ion [M+H]+ peak at m/z 425.2
in ESI-LC-MS spectrum. The 1H NMR signals
indicates the presence of 59 saturated protons of one
methyl [δH 0.90 (3H, t, J = 6.5 Hz, H-29)] and 27
methylenes [δH 1.26-1.55 (54H, m, H-2-H-28)] and
one carbinol methylene at H 3.64 (2H, t, J = 7.0 Hz,
H-1). The 13C NMR spectrum of 4 revealed 29
carbon signals with some overlaps at C 29.0-29.6
ppm; one carbinol carbon at C 63.1 ppm. The MS,
1 5.
H and 13C NMR spectral data identified 4 as
nonacosanol.[7]
4. CONCLUSION
From the aerial parts of Lycopodium cernuum plant,
collected in the Thang Tin Commun (Hoang Su Phi
Distr., Ha Giang Province) in October, 2016 we
isolated four compounds. By various spectral
methods, these compounds were elucidated as serrat-
14-en-3β,21β-diol, serrat-14-en-3β,21-diol, 3β,21β-
dihydroxyserrat-14-en-21-yl acetate, and
nonacosanol.
Acknowledgements. Authors thank Prof. Do Huu
Thu, Institute of Institute of Ecology, Natural
resource and Biology, VAST, for identifying the
sample.
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Corresponding author: Pham Huu Dien
Hanoi National University of Education
136, Xuan Thuy, Cau Giay district, Hanoi, Viet Nam
E-mail: dienph@hnue.edu.vn; Tel: +84- 912109836.
© 2018 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Pham Huu Dien et al.
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