Megastigmane and abscisic acid glycosides from the leaves of Laurus nobilis L.

Article abstract of DOI:10.1016/j.phytol.2019.06.011

Two new megastigmane glycosides, laurusides E-F (1 and 3) and three known ones (2, 5, and 7), together with a new abscisic acid glycoside, lauruside G (4) and a known one (6), were isolated from a methanolic extract of Laurus nobilis L. leaves using various chromatographic techniques. The structures of these compounds were fully characterized using a combination of spectroscopic techniques including multinuclear and multidimensional NMR and mass spectrometry. All of the isolates were evaluated for their inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 cells.

Full text of DOI:10.1016/j.phytol.2019.06.011

Phytochemistry Letters 33 (2019) 1–5
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Megastigmane and abscisic acid glycosides from the leaves of
Laurus nobilis L.
1
,2
1
5
5
6
Le Duc Dat , Ngo Viet Duc , Bui Thi Thuy Luyen , Ha Van Oanh , Hyun Jae Jang ,
7
4,⁎
3,⁎
Tran Thu Huong , Young Ho Kim , Nguyen Phuong Thao
1
Division of Computational Physics, Institute for Computational Science, Ton Duc Thang University, Ho Chi Minh City, Viet Nam
Faculty of Pharmacy, Ton Duc Thang University, Ho Chi Minh City, Viet Nam
2
3
4
5
6
7
Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam
College of Pharmacy, Chungnam National University, Daejeon 34134, Republic of Korea
Hanoi Univerisity of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Viet Nam
Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk 28116, Republic of Korea
School of Chemical Engineering, Hanoi University of Science and Technology, Hanoi, Viet Nam
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two new megastigmane glycosides, laurusides E-F (1 and 3) and three known ones (2, 5, and 7), together with a
new abscisic acid glycoside, lauruside G (4) and a known one (6), were isolated from a methanolic extract of
Laurus nobilis L. leaves using various chromatographic techniques. The structures of these compounds were fully
characterized using a combination of spectroscopic techniques including multinuclear and multidimensional
NMR and mass spectrometry. All of the isolates were evaluated for their inhibition of nitric oxide (NO) pro-
duction in lipopolysaccharide (LPS)-induced RAW264.7 cells.
Laurus nobilis
Lauraceae
Laurusides E-G
Anti-inflammatory effect
1
. Introduction
Laurus nobilis L. (family Lauraceae) was known as a folk medicine
inflammatory proteins and pro-inflammatory chemokines, cytokines,
and nitric oxide (Abarikwu, 2014). Macrophages are highly sensitive to
initiators of inflammation as lipopolysaccharide (LPS) which respond
by the release of mediators such as interleukins and cytokines inter-
feron gamma. Nitric oxide (NO), inducible NO synthase (iNOS), and
inducing the inflammatory gene expression where each is associated
somehow with the pathophysiological of the inflammation. Hence,
modulation of macrophage activation is a good strategy to prevent
these diseases (Valledor et al., 2010).
for the treatment of antiseptic, asthma, cardiac, diarrhea, digestive
disorder diseases, and rheumatic pains (Dall’Acqua et al., 2006; Loi
et al., 2004). The various parts of L. nobilis have been used for diff ;erent
purposes; for example, the leaves and fruits were used as serving
against emmenagogue and hysteria. Especially, the Iranian used L. no-
bilis leaves to treat epilepsy (khorasani, 1992). Powdered fruit in the
form of infusion exhibited carminative properties and diuretic (Simić
et al., 2003). Fatty oil from the fruits is used for treating bruises, fur-
uncles, rheumatism, sprains, and as an insect repellent (Chahal et al.,
In the present study, we described the isolation and structural elu-
cidation of seven secondary metabolites, including two new mega-
stigmane derivatives, laurusides E (1) and F (3), and a new abscisic acid
glycoside, lauruside G (4, Fig. 1), together with four known compounds
(2 and 5–7, Supplementary information) from a methanolic extract of L.
nobilisleaves. Furthermore, all the isolates were screened for their in-
hibition of NO production in LPS-induced RAW264.7 cells.
2
017). In addition, this herbal is a famous industrial material used in
cosmetics, drugs, foods, or seasoning of meat/fishes products and
soups. Recently, phytochemical studies of L. nobilis have revealed that
additional constituents are alkaloids (Pech and Bruneton, 1982) and
sesquiterpenes (Cisero et al., 1992), while other compounds including
megastigmanes and phenolics have also been identified (De Marino
et al., 2004).
2. Results and discussion
The inflammation is considered to be a major risk factor in the
pathogenesis of chronic diseases where the macrophages are important
immune cells. It may regulate inflammation producing expression of
The methanolic extract of L. nobilis was partitioned into fractions
and isolated by multiple chromatographic steps over silica gel,
Sephadex LH-20, and YMC RP-18 column chromatography (CC) to yield
⁎
Corresponding authors.
E-mail addresses: yhk@cnu.ac.kr (Y. Ho Kim), thaonp@imbc.vast.vn (N.P. Thao).
https://doi.org/10.1016/j.phytol.2019.06.011
Received 19 April 2019; Received in revised form 21 June 2019; Accepted 25 June 2019
1874-3900/ © 2019 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

L. Duc Dat, et al.
Phytochemistry Letters 33 (2019) 1–5
Table 1
1
H and 13C NMR spectroscopic data for laurusides E–G (in CD
3
OD).
Position Lauruside E (1)
Lauruside F (3)
Lauruside G (4)
a
b
a
b
a
b
δ
C
δ
H
mult.
δ
C
δ
H
mult.
δ
C
H
δ mult.
(
J in Hz)
(J in Hz)
(J in Hz)
1
2
43.9
52.4
–
37.7
38.4
–
49.8
43.0
–
2.88 d (13.5)
1.79 m
1.94 m/
1.75 m
1
1
.83 dd (2.8,
3.5)
3
4
78.0
46.1
3.93 ddd (2.8, 35.1
2.43 t (6.7)
–
74.2
43.0
4.21 m
1
0.5, 13.5)
2.45 brd
13.5)
201.6
2.15 dd (7.1,
(
16.6)
2
.13 m
1.83 m
5
6
7
8
37.9
78.1
2.29 m
–
131.5
168.9
28.0
–
87.6
83.1
–
–
–
134.2 5.75 d (15.8)
134.6 5.90 dd (6.3,
2.53 m
130.8 6.26 d (15.8)
37.2
1.64 dd (6.3, 133.0 7.75 d (15.8)
15.8)
1
5.8)
9
77.6
4.44 dq (6.3,
76.0
3.90 m
142.2
–
1
2.4)
1
1
1
0
1
2
21.4
25.1
25.3
1.33 d (6.3)
0.93 s
20.0
27.2
27.3
1.21 d (6.3)
1.18 s
127.2 5.82 s
16.4
77.1
0.92 s
1.00 s
1.18 s
3.78 m/
3
.72 m
1
1
3
4
16.5
0.91 d (6.7)
11.8
1.74 s
19.8
20.8
175.1
1.16 s
1.97 s
–
COOH
9-Glucose
1
2
'
'
102.6 4.35 d (7.8)
102.3 4.33 d (7.8)
102.1 4.41 d (7.5)
3.46 dd (7.5,
9.0)
75.3
78.0
3.19 dd (7.8,
.0)
3.91
75.1
78.0
3.15 dd (7.8, 79.3
9
9.0)
*
3.34*
3
'
77.8
3.26 dd (9.0,
9
.0)
*
4'
5'
6'
71.5
76.9
68.5
3.33*
72.1
76.7
68.2
3.25*
71.6
77.9
3.27
3.34 m
3.43 m
3.94 m
3.96 dd (2.4,
3.99 dd (2.4, 62.7
11.5)
3.83 dd (2.4,
11.5)
1
3
1
1.5)
.59 dd (5.4,
1.5)
3.58 dd (5.4,
11.5)
3.66 dd (5.4,
11.5)
6
'-Apiose
6'-Arabinose
2'-Apiose
1”
2”
3”
4”
111.0 4.99 d (2.4)
109.9 4.97 brs
110.8 5.35 brs
*
*
*
*
78.0
80.5
75.3
3.92
83.1
78.9
85.8
3.96
3.79
78.5
80.6
75.3
3.29
–
–
3.68 dd (9.0)
3.93 m
3.69 dd (9.0)
4.02 dd (9.0)
3.60 dd
(10.8)
4
.01 dd (9.0)
5
”
65.6
3.59 dd (10.8) 63.0
.54 dd (10.8)
3.69 m/
3.61 m
66.0
Fig. 1. Structures of new compounds (1, 3, and 4) isolated from L. nobilis.
3
3.55 dd
(
10.8)
seven compounds (1–7).
Assignments were confirmed by HMQC, HMBC, and COSY experiments.
a
Lauruside E (1) was obtained as a white, amorphous powder with an
00 MHz.
2
1
b
optical rotation [ ]
22.0° (c 0.25, MeOH). The HR-QTOF-MS in-
400 MHz.
D
+
dicated a basic peak at m/z 545.2567 [M+Na] , confirming the mo-
* Overlapped signals.
lecular formula of C24
H
42
O12. The infrared (IR) spectrum of compound
–1
1
showed absorption peaks characteristic of hydroxyl (3387 cm ) and
–1
1
substituted double bond (1678 cm ) groups. The H NMR spectrum of
exhibited signals for four methyl protons [δ 1.33 (3H, d, J = 6.3 Hz,
(CH, C-5′), 68.5 (CH , C-6′), and 111.0 (CH, C-1′′), 78.0 (CH, C-2′′),
2
1
H
80.5 (C, C-3′′), 75.3 (CH , C-4′′), 65.6 (CH , C-5′′)]. Two sugar units
2
2
H-10), 0.93 (3H, s, H-11), 1.00 (3H, s, H-12), and 0.91 (3H, d, J =
including a glucopyranoside and an apiofuranoside moiety were sug-
gested. Indeed, the acid hydrolysis of 1 yielded D-glucose and D-apiose
products which were detected by comparison the retention time of their
6
.7 Hz, H-13)], a pair of methylene protons [δ
3.5 Hz, H-2a) and 1.83 (1H, dd, J = 2.8, 13.5 Hz, H-2b)], two olefinic
5.75 (1H, d, J = 15.8 Hz, H-7) and 5.90 (1H, dd, J = 6.3,
5.8 Hz, H-8)], and two anomeric [an β-glycoside δ 4.35 (1H, d, J =
.8 Hz, H-1′) and an α-glycoside δ 4.99 (1H, d, J = 2.4 Hz, H-1′′)]. The
C NMR and HSQC spectra of 1 (Table 1) showed 24 carbon signals
H
2.88 (1H, d, J =
1
protons [δ
H
hydrolysis products (t
authentic samples (t
= 22.6 min and t
= 27.5 min) with
R-D-api
R-D-glc
R-D-glc
1
7
H
= 22.9 min and t
= 27.7 min) using
R-D-api
H
analytical HPLC, respectively.
1
3
The NMR data of 1 (Table 1) was similar to those of alangioside B
with characteristic signals of a megastigmane skeleton including a
(2) (Otsuka et al., 1994) except for the downfield shift at C-3 (δ 78.0)
C
substituted double bond with two olefinic methine [δ
C
134.2 (C-7) and
43.9 (C-1) and 78.1 (C-
)], three methines [78.0 (C-3), 37.9 (C-5), and 77.6 (C-9)], two me-
52.4 (C-2) and 46.1 (C-4)], four methyls [δ 21.4 (C-10),
5.1 (C-11), 25.3 (C-12), and 16.5 (C-13)], and 11 aliphatic carbons [δ
02.6 (CH, C-1′), 75.3 (CH, C-2′), 78.0 (CH, C-3′), 71.5 (CH, C-4′), 76.9
in 1 respect to its chemical shift (δ 67.5) in 2. This information sug-
C
1
6
34.6 (C-8)], two none-protonated carbons [δ
C
gested for the difference of geometry at C-3. Additionally, the HMBC
cross-peaks of other anomeric H-1′′ (δ 4.99) to C-6′ (δ 68.5) of glu-
H
C
thylenes [δ
C
C
cose indicated the connection of second sugar moiety to its mega-
2
1
C
stigmane glucopyranoside framework via C-1′′→C-6′. This information
was clearly expressed the downfield shift of C-6′ (δ 68.5) in
C
2

L. Duc Dat, et al.
Phytochemistry Letters 33 (2019) 1–5
7
(8)
H-7 and H-8 indicates that the geometry of the Δ
double bond is E.
Therefore, the structure of lauruside
E (1) was established as
(
(
3R,6S,9S,7E)-megastigman-7-en-3,6,9-triol-9-O-β-D-apiofuranosyl-
1′′→6′)-O-β-D-glucopyranoside.
Lauruside F (3) was found to be a white, amorphous powder, and
the molecular formula C24
H
40
O
11 was determined by HR-QTOF-MS,
+
with a sodium adduct molecular ion peak at m/z 527.2464 [M + Na]
.
The NMR spectroscopic data of 3 exhibited for a megastigmane glyco-
side framework including 13 carbon signals for megastigmane skeleton,
six carbon signals attributed to a β-glucopyranosyl [δ 4.33 (d, J =
H
7
.8 Hz, H-1′)] moiety, and five carbon signals for an α-arabinofuranosyl
[
δ 4.97 (brs, H-1′)] moiety. Further examination of the 2D NMR data
H
of 3 (Supplementary information) indicated that it is structurally si-
milar to those of platanionoside G (Otsuka and Tamaki, 2002), except
for difference from an α-arabinofuranoside in 3 instead of an β-xylo-
pyranoside in platanionoside G, and the configuration of C-9 position.
These conclusions were confirmed via the HMBC cross-peaks from H-2
(
δ
H
1.79) and H-3 (δ
H
2.43) to ketone group at δ
C
201.6, and those of δ
H
1
.74 (H-13) to C-4/C-5/C-6, clearly identified the ketone group at C-4
5
and Δ at C-5/C-6, respectively. The HMBC correlations of an anomeric
proton at δ
proton at δ
H
H
4.33 (H-1′) to C-9 (δ
C
76.0) and secondary anomeric
4.97 (H-1′′) to C-6′ (δ 68.2) indicated a arabinofuranosyl-
C
(
1′′→6′)-glucopyranosyl moiety as the disaccharide sugar chain which
was allocated to C-9 of its megastigmane skeleton. Furthermore, acid
hydrolysis of 3 yielded glucose and arabinose products which were
detected by comparison the retention time of their hydrolysis products
(tR-L-ara = 24.4 min and tR-D-glc = 27.3 min) with authentic samples (tR-
L-ara = 24.4 min and tR-D-glc = 27.6 min) using analytical HPLC, re-
spectively. Further assignments by COSY, HMQC and HMBC correla-
tions of 3 were displayed in Fig. 2. The appearance of C-9 (δ 76.0)
C
relatively confirmed the S-form of C-9 position (Otsuka et al., 1994).
According to the above evidence, the structure of 3 was established as a
new compound as (9S)-9-hydroxymengastigman-5-en-4-one-9-ol-O-β-
D-(α-L-arabinofuranosyl-(1′′→6′)-glucopyranoside.
2
1
Lauruside G (4) was obtained as a white, amorphous powder {[ ]
D
1
2.6° (c 0.25, MeOH)}. The molecular formula of 4 was established as
C
26
H O15 according to the HR-QTOF-MS peak at m/z 617.2420
+ 1
42
[
M + Na] . The H NMR spectrum of 4 exhibited the presence of
characteristic signals of a trans olefilic protons [δ
H
6.26 (1H, d, J =
1
5.8 Hz, H-7), 7.75 (1H, d, J = 15.8 Hz, H-8)], a vinyl group [δ
H
H
5.82
5.35
(
(
1H, s, H-10), and 1.97 (3H, s, H-14)], two anomeric protons [δ
1H, brs, H-1′′) and 4.41 (1H, d, J = 7.5 Hz, H-1′)], an oxymethine [δ
H
Fig. 2. Key HMBC (
and 4.
), COSY (
), and NOESY (
) correlations of 1, 3,
4
3
1
.21 (1H, m, H-3)], two oxymethylenes [δ
H
3.78 (1H, m, H-12a) and
0.92 (3H, H-
1) and 1.16 (3H, H-13)]. The C NMR data of 4 exhibited 26 carbons
175.1 (COOH)], a substituted double bond
130.8 (C-7) and 133.0 (C-8)], a vinyl [δ 142.2 (C-9), 127.2 (C-10),
and 20.8 (C-14)], an oxymethine [δ 74.2 (C-3)], three none-protonated
carbons [δ 49.8 (C-1), 87.6 (C-5), and 83.1 (C-6)], one oxymethylene
[δ
.72 (1H, m, H-12b)], and two singlet methyl protons [δ
H
1
3
glucopyranoside part. Moreover, the HMBC cross-peak of an anomeric
including one carboxyl [δ
C
proton at δ
H
4.35 (H-1′) to C-9 (δ
C
77.6) was also observed. Hence, an
[δ
C
C
apiofuranosyl-(1′′→6′)-glucopyranosyl moiety as the disaccharide
sugar chain was allocated to C-9 of an ionol moiety. Further assign-
ments for COSY, HMQC and HMBC correlations of 1 were displayed in
Fig. 2.
C
C
C
77.1 (C-12)], two methyl carbons [δ
C
16.4 (C-11) and 19.8 (C-13)],
and 11 carbons of two sugar moieties (Table 1). Compound 4 was de-
ducted with a monocyclofarnesane sesquiterpene (Fraga, 2013) con-
taining a conjugated carbonyl group.
The relative configuration of 1 was determined by analyzing proton-
proton coupling constants and spectroscopic data as well as NOESY
correlations. The hydroxyl group at C-3 (δ
C
78.0) was assigned to R-
The NMR spectroscopic data of 4 was similar to monaspilosuslin
(Cheng et al., 2010), except for differences with more two sugar units
orientation by comparing its chemical shift value to respect of reported
data [δ
C
67.4 (S-form, C-3) and 78.1 (R-form, C-3)] (Shu et al., 2013;
and an 12-oxymethylene in 4. The HMBC correlations of δ
H
0.92 (H-11)
1.16
83.1), indicated the
OH-12 and OH-5 positions, respectively. Moreover, the anomeric
Zhang et al., 2010) as well as large coupling constant (J = 13.5 Hz)
to C-1 (δ
(H-13) to C-4 (δ
CH
proton at δ
C
49.8)/C-6 (δ
C
83.1)/C-12 (δ
C
77.1) as well as those of δ
H
between H-3 and H-2α/H-2β, H-4. In the same manner, the Me-
C
43.0)/C-5 (δ
C
87.6)/C-6 (δ
C
1
3 group was suggested to S-orientation with the similar chemical shift
2
value [δ
Me-13)] (Shu et al., 2013). The appearance of C-9 (δ
to that of alangioside B (Otsuka et al., 1994) and different from R-form
74.0–75.5) (Marino et al., 2004), confirming the configuration of C-
C
16.8–17.5 ppm (S-form, Me-13) and 13.1–13.4 ppm (R-form,
H
4.41 (d, J = 7.5 Hz, H-1′) correlated to C-3 (δ 74.2)
C
C
77.6) was similar
confirmed the glucopyranosyl moiety was connected to its skeleton
through C-3. Furthermore, acid hydrolysis of 4 yielded glucose and
apiose products which were detected by comparison the retention time
of its hydrolysis product with authentic samples using analytical HPLC.
Additionally, the HMBC cross-peaks of the second anomeric proton at
(
δ
C
9
as S-form. Moreover, the 3-hydroxy-1-butenyl side chain at C-6 was
determined to be β-oriented based on the NOESY cross-peaks of H-7 to
H-5 and Me-12β. The larger coupling constant (J = 15.8 Hz) between
δ
H
5.35 (brs, H-1′′) to C-2′ (δ 79.3) was also observed. This finding was
C
3

L. Duc Dat, et al.
Phytochemistry Letters 33 (2019) 1–5
reasonable to resonate to the downfield shift of C-2′. Thus, an apio-
furanosyl-(1′′→2′)-glucopyranosyl moiety of the sugar side chain was
allocated to C-3 of the skeleton.
times with 95% MeOH at room temperature for 2 days each to give a
MeOH residue (120 g) after removal of the solvent under reduced pres-
sure. This residue was suspended in water (1.5 L) and partitioned in turn
The NOESY cross-peaks of δ
H-7) as well as those of δ
1), suggested the axial positions of H-3, H-7, H
spectively (Cheng et al., 2010). This finding and the chemical shift of C-
H
1.16 (H
3
-13) to δ
5.82 (H-10) and 0.92 (H
-11, and H -13, re-
H
4.21 (H-3) and 6.26
with CH
extracts: CH
The water layer has removed the solvent and subjected to Dianion
2
Cl
2
(3 × 1.5 L) and EtOAc (3 × 1.5 L) to furnish corresponding
(
H
6.26 (H-7) to δ
H
3
-
2
Cl (B, 51 g), EtOAc (C, 8 g) and water layer (D, 1 L).
2
1
3
3
HP-20 open CC using concentration of MeOH in H O (from 0% to
2
9
(10)
1
4 (δ
C
20.8) was also expressed the Z-configuration of Δ
. Whereas,
100%) to give four fractions (D1-D4). These fractions were collected
the appearance of C-5 (δ
C
87.6) and C-6 (δ
C
83.1) were similar to those
according to their TLC profiles. Fraction D2 (1.5 g) was separated on a
of monaspilosuslin (Cheng et al., 2010), allowed to assign R-form of
OH-5 and S-form of OH-6, respectively. Therefore, the structure of 4
was established as lauruside G.
silica gel CC using stepwise elution with CH
2
Cl
2
-MeOH-H O (15/1/
2
0.01–1/1/0.01, v/v/v) and further purified by silica gel CC, YMC RP-18
columm eluting with solvent system of MeOH-H O (1/2.5, v/v), and
O (1/1, v/v)
2
The other compounds were identified as alangionoside B (2)
Sephadex LH-20 eluting with solvent system of MeOH-H
2
(
Otsuka et al., 1994), (6S,9R)-vomifoliol-9-O-β-D-apiofuranosyl-(1′′→
to obtain 1 (7.2 mg), 3 (6.5 mg), and 5 (8.5 mg) from subfraction D2A
(0.7 g) and D2D (0.3 g), respectively. Next, fraction D3 (1.2 g) afforded
4 (9.0 mg) and 6 (10.2 mg) after subject it to YMC RP-18 CC with
6
′)-O-β-D-glucopyranoside (5) (Ito et al., 2001), (1′S,6′R)-8′-hydro-
xyabscisic acid O-β-D-glucoside (6) (del Refugio Ramos et al., 2004),
and (6S,9R)-roseoside (7) (Yajima et al., 2009) (Supplementary in-
formation) by comparing their spectroscopic data with published lit-
erature values.
MeOH-H
2
O (1/2, v/v) and acetone-H O (1/3, v/v), respectively.
2
Finally, subfraction D4 (0.6 g) were purified on YMC RP-18 CC eluting
with acetonitrile-H O (1/1.8, v/v) to obtain compound 7 (9.5 mg).
2
NO is a multifunctional molecule in the regulation of host defense
mechanisms, apoptosis, neurotransmission, acute and chronic in-
flammation. The enhanced NO levels associated with acute or chronic
inflammatory diseases indicate that NO is related to pro-inflammatory
agents for inflammation-mediated pathogenesis. To assess the anti-in-
flammatory activity, all the isolated compounds (1–7) were evaluated
for NO production inhibitory activity in LPS-stimulated RAW 264.7
cells. Unfortunately, all the isolated compounds showed the weak in-
hibitory effects (IC50S > 50 μM).
3.4. Physical and spectroscopic data of new compounds
3.4.1. Lauruside E (1)
White, amorphous powder; [ ] D2 1 22.0 (c 0.25, MeOH); UV (MeOH)
λ
max (log ε) nm: 205 (3.56) and 267 (3.03); IR (KBr) νmax: 3387, 2942,
–1
2835, 1678, 1424, and 1011 cm ; positive-ion HR-QTOF-MS m/z
+
1
545.2567 [M + Na] (calcd. for C24
H
42NaO12, 545.2574); H NMR
OD) and 1 C NMR (100 MHz, CD
3
OD) data, see Table 1.
(400 MHz, CD
3
3
3
. Experimental
3.4.2. Lauruside F (3)
2
1
White, amorphous powder; [ ]
15.6 (c 0.4, MeOH); UV (MeOH)
D
3.1. General experimental procedures
λ
max (log ε) nm: 191 (3.66) and 266 (2.97); IR (KBr) νmax: 3329, 2940,
–
1
2
841, 1687, and 1022 cm ; positive-ion HR-QTOF-MS m/z 527.2464
+
1
Optical rotations were determined on a JASCO P-2000 polarimeter.
[M + Na] (calcd. for C24
H
40NaO11, 527.2468); H NMR (400 MHz,
CD
3
OD) and 13C NMR (100 MHz, CD
OD) data, see Table 1.
3
The UV spectrum was recorded on a JASCO V-630 spectrophotometer.
IR spectra were obtained on a Bruker TENSOR 37 FT-IR spectrometer.
The NMR spectra were recorded on a JEOL JNM-AL 400 MHz spectro-
meter, chemical shift (δ) are expressed in ppm with reference to the
TMS signals. The HR-QTOF-MS was acquired on an Agilent 6530
Accurate-Mass Q-TOF LC/MS system (Emeryville, CA, USA).
Preparative HPLC was performed on an Agilent 1260 apparatus
3.4.3. Lauruside G (4)
White, amorphous powder; [ ] D2 1 12.6 (c 0.25, MeOH); UV (MeOH)
max (log ε) nm: 202 (3.21) and 266 (2.98); IR (KBr) νmax: 3366, 2939,
λ
–
1
2831, 1715, 1419, and 1025 cm ; positive-ion HR-QTOF-MS m/z
+
1
617.2420 [M + Na] (calcd. for C26
H
42NaO15, 617.2421); H NMR
(400 MHz, CD
3
OD) and 1 C NMR (100 MHz, CD
3
3
OD) data, see Table 1.
(
Agilent Technologies, Palo Alto, CA, USA) equipped with a G1315D
Variable Wavelength Detector on collumn of Kinetex C18
(
250 × 4.6 mm, 5 μm). Column chromatography (CC) was performed
3.5. Acid hydrolysis of compounds
on silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck),
porous polymer gel (Diaion® HP-20, 20–60 mesh, Mitsubishi Chemical,
Tokyo, Japan), Sephadex™ LH-20 (GE Healthcare Bio-Sciences AB,
Uppsala, Sweden), octadecyl silica (ODS, 50 μm, Cosmosil 140 C18-
OPN, Nacalai Tesque), and YMC RP-18 resins (30–50 μm, Fuji Silysia
Chemical). Thin layer chromatography (TLC) used pre-coated silica gel
Compounds 1, 3 (2.5 mg, each), and 4 (2 mg) were hydrolyzed by
heating in 10% H SO aqueous solution at 80 °C for 3 h, neutralized
2
4
with barium carbonate, and then filtered, and the filtrate was extracted
with ethyl acetate (3 × 4 mL) to remove aglycone. The aqueous layer
was concentrated to obtain the sugar residue, which was dissolved in
1 mL of pyridine heated with 6 mg L-cysteinemethyl ester at 60 °C for
60 min, then phenylisothiocyanate (0.1 mL) was added to the reaction
mixture and further reacted at 60 °C for 60 min then the reaction mix-
ture was evaporated on a water bath, and then 1 mg of the product was
dissolved in acetonitrile and analyzed by standard C18 HPLC by stan-
dard C18 HPLC(Kinetex C18: 4.6 mm × 250 mm, 5 μm) at a flow rate of
0.8 mL/min with an ultra-violet (UV) detector at wavelength of 250 nm.
The The mobile phase was consisted as a gradient solvent system of
6
0 F254 (1.05554.0001, Merck) and RP-18 F254S plates (1.15685.0001,
Merck) and compounds were visualized by spraying with aqueous 10%
SO and heating for 1.5―2 min.
H
2
4
3.2. Plant material
The leaves of L. nobilis were collected in July 2016 at Sin Ho, Lai
Chau province, Vietnam, and identified by Prof. Tran Huy Thai,
Institute of Ecology and Biological Resources, VAST, Vietnam. A vou-
cher specimen (LN-1606) was deposited at the Herbarium of Institute of
Ecology and Biological Resources, VAST, Vietnam.
phase A (H
2
O) and phase B (ACN) as following the analytical condition:
20–80% (B) for 0–50 min.
3.6. In vitro NO assay
3.3. Extraction and isolation
3
.6.1. Cell culture
Fresh leaves of L. nobilis was dried under shiny for a dried sample. The
The RAW264.7 cells were obtained from Korean Cell Line Bank
dried leaves of L. nobilis (2.5 kg) were well ground and extracted three
(KCLB, Chongno-gu, Seoul, Korea) and maintained in Dulbecco’s
4

L. Duc Dat, et al.
Phytochemistry Letters 33 (2019) 1–5
Modified Essential (DMEM) supplemented with 10% fetal bovine serum
and Biological Resources, VAST, Vietnam) and Dr. Bui Huu Tai
(Institute of Marine Biochemistry, VAST, Vietnam) for identification of
plant and HR-QTOF-MS operation of compounds.
(
FBS), (100 units/mL) penicillin, and streptomycin sulphate (100 μg/
mL) at 37 °C in humidified incubator containing 5% CO . After pre-
2
incubator of RAW264.7 for 4 h, each compound at concentration of
3
–60 μM were added.
Appendix A. Supplementary data
3
.6.2. Cell viability
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2019.06.011.
Cell viability was evaluated by 3-(4,5 dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) methods (Carmichael et al., 1987),
in which the effects of tested compounds (1–7) were evaluated by de-
termining mitochondrial reductase function base on the reduction of
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Acknowledgments
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This work was financially supported by a grant from Ton Duc Thang
University. The authors thank Prof. Tran Huy Thai (Institute of Ecology
5