Chemical constituents from the leaves of Styrax annamensis Guill

Cite this paper: Vietnam J. Chem., 2020, 58(5), 630-636 Article 
DOI: 10.1002/vjch.202000052 
630 Wiley Online Library © 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH 
Chemical constituents from the leaves of Styrax annamensis Guill 
Nguyen Thanh Tra
1,2
, Tran Dang Anh
1
, Pham Van Cuong
3
, Nguyen Thi Thu Ha
1,2
, Le Thi Tu Anh
1
, 
Ba Thi Cham
1
, Ninh The Son
1* 
1
Institute of Chemistry, Vietnam Academy Science and Technology (VAST), 
18 Hoang Quoc Viet, Cau Giay district, Hanoi 10000, Viet Nam 
2
Graduate University of Science and Technology, VAST, 
18 Hoang Quoc Viet, Cau Giay district, Hanoi 10000, Viet Nam 
3
Institute of Marine Biochemistry, VAST, 18 Hoang Quoc Viet, Cau Giay district, Hanoi 10000, Viet Nam 
Received April 6, 2020; Accepted April 9, 2020 
Abstract 
A phytochemical study of the ethyl acetate extract (EtOAc) of Styrax annamensis leaves resulted in the isolation 
and determination of six known compounds, including three nor-neolignans type 2-phenylbenzofurans egonol (1), 
egonoic acid (2) and (–)-machicendiol (3), two lignans styraxin (4) and vladinol D (5), and a triterpenoid pomolic acid 
(6). The chemical structures of these compounds were determined by NMR spectroscopies, and MS data. This is the 
first time to isolate these compounds from S. annamensis species. Compounds 3 and 5 have also been observed from 
genus Styrax for the first time. 
Keywords. Styrax annamensis, leaves, nor-neolignan, lignan, triterpenoid. 
1. INTRODUCTION 
Genus Styrax, also known as Storax or Snowbell, is 
one of the largest genera in the family Styracaceae, 
comprising of about 130 species of small trees and 
shrubs.
[1]
 Styrax plants were native to tropical and 
subtropical areas, especially available in Eastern and 
Southeastern Asia.
[2]
 The dried exudative gums 
derived from pierced bark of the various Styrax 
species are currently seen as commercial products, 
e.g, for a long history, three species S. tonkinensis 
(Siam benzoin), S. benzoin (Sumatra benzoin), and 
S. benzoides were being well-known employed for 
perfume, incense, and medicine.
[3,4]
 Phytochemical 
investigations of Styrax species have resulted in the 
isolation and structural elucidation of various classes 
of compounds, and lignans and triterpenoids are 
major components. Crude extracts, fractions as well 
as isolated compounds obtained from this genus 
have a wide range of biomedical features. As 
examples, Park et al. (2009, 2010) used isolated 
lignans from Styrax japonica stem bark to combat 
the pathogenic fungus Candida albicans,
[5-7]
meanwhile glycoprotein from this plant caused for 
oxidative inhibitory and anti-inflammatory actions in 
HCT116 colonic epithelial cell.
[8]
 Oliveira et al. 
(2012) suggested that S. camporum extract can lead 
to significantly decrease the extent of DNA damage 
in liver cells induced by methanesulfonate.
[9] 
There are about thirteen Styrax plants in 
Vietnam,
[10]
 and several species have already been 
studied in both phytochemical and biological 
aspects. S. annamensis Guill, locally called Bo De 
Trung Bo, was mostly distributed from Hagiang to 
Khanhhoa.
[10]
 To the best of our knowledge, there 
were no documents to research phytochemistry for 
this species. In the current paper, we firstly report 
the isolation and structural determination of six 
compounds, including three nor-neolignans, two 
lignans, and a triterpenoid from the leaves of S. 
annamensis. 
2. MATERIALS AND METHODS 
2.1. General experimental procedures 
ESI-MS data were obtained with a Thermo Scientific 
LTQ Orbitrap XL spectrometer (USA). NMR spectra 
have been recorded on a Bruker 500 MHz 
spectrometer, operating at 125 MHz for 
13
C NMR and 
at 500 MHz for 
1
H NMR. Silica gel 40-63 µm and 
Sephadex LH-20 have been employed for column 
chromatography (CC). TLC silica gel 60 F254 (Merck) 
was used for thin-layer chromatography (TLC). 
Vietnam Journal of Chemistry Ninh The Son et al. 
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 631 
2.2. Plant material 
The leaves of S. annamensis species were collected 
in Dienbien province in March 2018. The plant 
material was identified by Dr. Nguyen Quoc Binh, 
Institute of Ecology and Biological Resources. A 
voucher specimen (VN-1547F) was deposited in 
Laboratory of Applied Biochemistry, Institute of 
Chemistry. 
2.3. Extraction and isolation 
The dried leaves powder of S. annamensis (1.0 kg) 
was ultrasonically extracted with n-hexane (10 L × 3 
times) for 1h at 45 °C. After that, the powder was 
then extracted with EtOAc (10 L × 3 times) for 1h at 
45 °C, and concentrated under vacuum (20~30 
mbar) at 50 °C to give a EtOAc residue (55.0 g). 
This residue was subjected to silica gel (10 × 50 cm, 
350.0 g) column chromatography (CC), eluting with 
a gradient of n-hexane-CH3COCH3 (9:1→ 0:10, v/v) 
and CH3COCH3-MeOH (9:1→ 0:10, v/v) to yield 13 
fractions (Fr.1-Fr.13). 
Figure 1: Compounds 1-6 isolated from S. annamensis leaves and their key HMBC and COSY correlations 
The Fr.3 (3.8 g) was subjected for Sephadex LH-
20 CC [MeOH-CH2Cl2 (9:1, v/v)] to yield 4 
fractions (Fr.3.1-Fr.3.4). Compound 6 (5.0 mg) was 
obtained from the fraction Fr.3.4 (0.7 g) by using 
Sephadex LH-20 CC [MeOH-CH2Cl2 (9:1, v/v)]. 
The Fr.4 (3.2 g) was subjected for CC on silica 
gel eluting with a gradient solvent of CH2Cl2-
CH3COCH3 (9:1, v/v) to yield 3 fractions (Fr.4.1-
Fr.4.3). Compound 1 (7.0 mg) was isolated from the 
fraction Fr.4.2 (1.2 g) by using Sephadex LH-20 CC 
[MeOH-CH2Cl2 (9:1, v/v)]. The fraction Fr.5 (4.8 g) 
was divided into 4 fractions (Fr.5.1-Fr.5.4) by CC 
on silica gel eluting with a gradient solvent of 
CH2Cl2-CH3COCH3 (9:1, v/v). Using Sephadex LH-
20 CC [MeOH-CH2Cl2 (9:1, v/v)], compounds 2 
(6.0 mg) and 5 (7.0 mg) were separated from the 
 ... idative,
[12]
 anti-cancer,
[13]
 and 
anti-bacterial treatments.
[14]
Compound 2 was isolated as white amorphous 
powders, and its molecular formula C19H16O6 was 
identified basing on the ion loses a proton at m/z 339 
[M–H]– in the negative ESI-MS spectrum. The 1H 
and 
13
C-NMR data of 2 were similar to those of 1, 
except for hydroxypropyl side chain in 1 was 
replaced by carboxyethyl unit [H-1'' (δH 2.99, t, 7.5 
Hz) and C-1'' (δC 33.5); H-2'' (δH 2.58, t, 7.5 Hz) and 
C-2'' (δC 39.8); and C-3'' (δC 179.8)] in 2 (table 1). 
The HMBC evidence H-2''/C-1'', and C-3'', and H-
1''/C-4, C-5, and C-6 (figure 1) also affirmed that 
carboxyethyl unit substituted at carbon C-5 in 2. The 
structure of 2 was further confirmed by comparison 
with literature data as known compound egonoic 
acid.
[15]
Compound 3 was separated as white amorphous 
powders. The 
1
H and 
13
C-NMR data of 3 were very 
similar to those of 1. The difference between 1 and 3 
occurred at carbon C-1'' since a proton of methylene 
H-1'' in 1 was replaced by hydroxy group in 3. This 
phenomenon has been so far confirmed by 2D-NMR 
(figure 1), especially MS data. The molecular mass 
of 3 is higher than that of 1 by 12 Da. From these 
findings and comparison with literature data, 
compound 3 was elucidated as (–)-machicendiol.[16] 
This is the first time to observe this compound in 
genus Styrax, to date. 
Compound 4 was obtained as white amorphous 
powders. The molecular formula C20H18O7 deduced 
from the proton adduct ion at m/z 371 [M+H]
+ 
in the 
positive ESI-MS. The 1D-NMR data (
1
H and 
13
C-
NMR) aided by the 2D-NMR spectroscopic data 
(HSQC and HMBC) of 4 revealed that secondary 
metabolite 4 is a lignan furofuran. In the 
1
H-NMR of 
4, the signals of furofuran nuclear resonated at δH 
3.45-5.29 ppm, while 3',4'-methylenedioxyphenyl 
unit was found to appear in the downfield [OCH2O 
(δH 5.97, s), H-2' (δH 7.34, d, 1.5 Hz), H-5' (δH 6.91, 
d, 8.0 Hz), and H-6' (δH 7.42, dd, 1.5, 8.0 Hz)]. The 
1
H-NMR of 4 also proved the appearance of a 1,3,4-
trisubstitutedphenyl unit [ABX spin system H-2'' (δH 
6.81, d, 2.0 Hz), H-6'' (δH 6.77, dd, 2.0, 7.5 Hz), and 
H-5'' (δH 6.98, d, 7.5 Hz)], and a methoxy group 3''-
OCH3 (δH 3.95, s). Meanwhile, the 
13
C-NMR data of 
lignan 4 contained twenty carbon signals, including 
six aromatic methines, four aliphatic methine 
carbons, two oxygenated methylene carbons, six 
quaternary carbons, one methoxy carbon 3''-OCH3 
(δC 56.0), and one carbonyl carbon C-8 (δC 176.8). 
The 2D-NMR data (HSQC and HMBC) is in 
accordance with this. As shown in figure 1, 
furofuran nuclear was established with the key HBC 
correlations H-1/C-2, and C-8, H-5/C-4, and C-6, H-
2/C-5, H-1/C-4, and H-6/C-1, and C-8. In the same 
manner, 3',4'-methylenedioxyphenyl unit has 
associated with the representative HMBC evidence 
OCH2O/C-3', and C-4', H-2'/C-3', and H-6'/C-2', and 
C-4'. The remaining 1,3,4-trisubstituted unit was 
characterized by the important HMBC correlations 
H-2''/C-3'', H-6''/C-2'', and C-4''. It turned out that 
OCH3 substituted at carbon C-3'' due to HMBC 
correlation OCH3/C-3''. The linkage between 
furofuran nuclear and 3',4'-methylenedioxyphenyl 
unit at carbon C-2 was identified by HMBC 
correlations H-2' and H-6'/C-2, as well as the 
connection between furofuran nuclear and 1,3,4-
trisubstituted phenyl unit was determined by H-2'' 
and H-6''/C-6. The structure of compound 4 has 
coincided with an isolated compound, namely 
styraxin.
[17]
 However, to the best of our knowledge, 
the NMR data of this compound have not yet 
assigned fully. This is the second time we isolated 
and provided full NMR results since it was only 
found in S. officinalis aerial part.
[17] 
Compound 5 was precipitated out of EtOAc 
extract as white amorphous powders. The molecular 
formula of 5 was to be C20H22O7, deducing from the 
proton adduct ion at m/z 375 [M+H]
+
 in the positive 
ESI-MS spectrum. The comprehensive analysis of 
1D-NMR data (
1
H and 
13
C) and 2D-NMR data 
(HSQC, HMBC, and COSY) can lead to identifying 
that secondary metabolite 5 was a tetrahydrofuran 
lignan, which named vladinol D.
[18,19]
 Considering 
the 
1
H-NMR data of 5, three aliphatic methines H-8, 
H-7' and H-8', and an oxygenated methylene H-9 of 
furan ring resonated in the region of 2.72-4.66 ppm, 
and induced serial COSY cross-peaks H-9/H-8/H-
8'/H-7'. Oxygenated methylene H-9' (δH 3.62 and δH 
3.67) correlated to C-8' (δC 54.6) suggests that this 
group substituted at carbon C-8'. The 
1
H-NMR data 
also showed the presence of a 1,3,4-trisubstituted 
benzoyl unit [ABX spin system H-2 (δH 7.61, d, 2.0 
Hz), H-6 (δH 7.63, dd, 2.0, 9.0 Hz), and H-5 (δH 
6.91, d, 9.0 Hz)], and a 1,3,4-trisubstituted phenyl 
unit [ABX spin system H-2' (δH 7.08, d, 2.0 Hz), H-
6' (δH 6.87, dd, 2.0, 8.0 Hz), and H-5' (δH 6.78, d, 8.0 
Hz)]. Two methoxy groups at δH 3.93 and δH 3.90 
directly substituted at carbons C-3 (δC 149.2) and C-
3' (δC 149.1) can be explained by the HMBC 
Vietnam Journal of Chemistry Chemical constituents from the leaves of 
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 634 
correlations 3-OCH3/C-3 and 3'-OCH3/C-3', 
respectively. Benzoyl moiety connecting to carbon 
C-8 of furan nuclear was confirmed by the key 
HMBC correlations H-8 and H-9/C-7, while phenyl 
unit linked to carbon C-7' due to the crucial HMBC 
correlations H-2' and H-6'/C-7'. Vladinol (5) has 
been isolated from various high plants, such as 
Vladimiria souliei or Forsythia suspensa,
[18,19]
 but 
this is the first time to observe in genus Styrax. 
Table 2: 
1
H and 
13
C-NMR data of compounds 4-2 
No 
4
 5 
δH (J in Hz) δC δH (J in Hz) δC 
1 3.45 (dd, 3.5, 9.5) 53.2 130.1 
2 5.33 (d, 4.0) 83.4 7.61 (d, 2.0) 112.5 
3 149.2 
4 
4.31 (dd, 3.5, 9.5) 
4.32 (dd, 7.0, 9.5) 
72.7 
153.5 
5 3.20 (m) 49.9 6.91 (d, 9.0) 115.9 
6 5.29 (d, 4.0) 84.5 7.63 (dd, 2.0, 9.0) 125.0 
7 200.3 
8 176.8 4.28 (m) 50.3 
9 4.19 (m) 71.8 
1' 132.3 133.6 
2' 6.90 (d, 1.5) 108.2 7.08 (d, 2.0) 111.5 
3' 148.0 149.1 
4' 148.4 147.5 
5' 6.76 (d, 8.0) 105.7 6.78 (d, 8.0) 116.0 
6' 6.86 (dd, 1.5, 8.0) 118.0 6.87 (dd, 2.0, 8.0) 121.0 
7' 4.66 (d, 9.0) 85.3 
8' 2.72 (m) 54.6 
9' 
3.62 (dd, 5.5, 11.5) 
3.67 (dd, 4.5, 11.5) 
61.3 
1'' 133.1 
2'' 6.81 (d, 2.0) 108.5 
3'' 146.8 
4'' 145.4 
5'' 6.98 (d, 7.5) 114.5 
6'' 6.77 (dd, 2.0, 7.5) 119.0 
OCH2O 5.97 (s) 101.5 
3-OCH3 3.93 (s) 56.47 
3'-OCH3 3.95 (s) 56.0 3.90 (s) 56.41 
 Compound 6 was obtained as white amorphous 
powders. On the basis of the proton adduct ion at 
m/z 473 [M+H]
+ 
in the positive ESI-MS 
spectroscopy, the molecular formula of 6 was to be 
C30H48O4. 1D-NMR data coulped with 2D-NMR 
data (HSQC and HMBC) of 6 corresponded to a 
well-known triterpenoid, namely pomolic acid.
[20-22]
1D-NMR revealed that compound 6 contained five 
methyl, one methine carbinol, one olefinic bond, one 
carbonyl carbon, and remaining aliphatic methines 
and methylenes. Hydroxygenation occurred at 
carbon carbinol C-3 (δC 80.1) with the key HMBC 
correlations H-1, H-23, and H-24/C-3. Double bond 
located at carbons C-12 (δC 129.3) and C-13 (δC 
140.2), with the characteristic HMBC correlations 
H-12/C-11 and C-13, H-18/C-12. Carboxyl group 
substituted at carbon C-17, which can be visible by 
HMBC correlations H-18 and H-22/C-28. The direct 
correlations H-23 and H-24/C-4, H-25/C-10, H-
26/C-9, H-27/C-14, H-29/C-19, and H-30/C-20 in 
the HMBC spectrum established the positions of 
methyl groups. Pomolic acid (6) is now abundant in 
Vietnam Journal of Chemistry Ninh The Son et al. 
© 2020 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 635 
nature, but regarding genus Styrax, here is the 
second time we have successfully isolated and 
identified since it was only found in the leaves of S. 
tonkinensis.
[ 23] 
4. CONCLUSION 
This is the first time that three nor-neolignans type 
2-phenylbenzofurans egonol (1), egonoic acid (2) 
and (–)-machicendiol (3), two lignans styraxin (4) 
and vladinol D (5), and a triterpenoid pomolic acid 
(6) were isolated and identified from the Vietnamese 
S. annamensis. Compounds 3 and 5 were also 
observed in the plants of genus Styrax for the first 
time. Our phytochemical result is also in agreement 
with previously various reports, in which lignans, 
neolignans, and triterpenoids are the main classes of 
isolated compounds in genus Styrax. 
Acknowledgments. This study is supported by a 
grant from Vietnamese National Foundation for 
Science and Technology Development (NAFOSTED, 
code: 104.01-2017.328) for financial support. 
. 
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Corresponding author: Ninh The Son 
Department of Applied Chemistry, Institute of Chemistry 
Vietnam Academy of Science and Technology 
18 Hoang Quoc Viet, Cau Giay district, Hanoi 10000, Viet Nam 
E-mail: yamantson@gmail.com; Tel: +84- 984423188.