Inhibitory effects of constituents from the aerial parts of rosmarinus officinalis L. on triglyceride accumulation

Article abstract of DOI:10.3390/molecules22010110

Sixteen flavonoids (1-16) including two new ones, named officinoflavonosides A (1) and B (2) were obtained from the aerial parts of Rosmarinus officinalis. Among the known ones, 6, 10, and 13 were isolated from the rosmarinus genus for the first time. The

Full text of DOI:10.3390/molecules22010110

molecules
Article
Inhibitory Effects of Constituents from the Aerial
Parts of Rosmarinus officinalis L. on
Triglyceride Accumulation
Jian Li 1, Tiwalade Adegoke Adelakun 1, Sijian Wang 2, Jingya Ruan 1, Shengcai Yang 1,
Xiaoxia Li 1, Yi Zhang 1,2,* and Tao Wang 1,2,
*
1
Tianjin State Key Laboratory of Modern Chinese Medicine, 312 Anshanxi Road, Nankai District,
Tianjin 300193, China; beyondwill@126.com (J.L.); tiwa_ade@yahoo.com (T.A.A.);
Ruanjy19930919@163.com (J.R.); 15122473723@163.com (S.Y.); huifeidedouzi@yeah.net (X.L.)
Tianjin Key Laboratory of TCM Chemistry and Analysis, Institute of Traditional Chinese Medicine,
Tianjin University of Traditional Chinese Medicine, 312 Anshanxi Road, Nankai District, Tianjin 300193,
China; 15122587883@163.com
2
*
Correspondence: zhwwxzh@263.net (Y.Z.); wangtao@tjutcm.edu.cn (T.W.); Tel./Fax: +86-22-5959-6163 (T.W.)
Academic Editors: Celestino Santos-Buelga and Arturo San Feliciano
Received: 10 November 2016; Accepted: 8 January 2017; Published: 17 January 2017
Abstract: Sixteen flavonoids ( ) and B
1–
16) including two new ones, named officinoflavonosides A (
1
(
2) were obtained from the aerial parts of Rosmarinus officinalis. Among the known ones, 6, 10, and
13 were isolated from the rosmarinus genus for the first time. Their structures were elucidated by
chemical and spectroscopic methods. Moreover, the effects on sodium oleate-induced triglyceride
accumulation (TG) in HepG2 cells of the above-mentioned compounds and 16 other isolates (17
reported previously to have been obtained in the plant were analyzed. Results show that eight kinds
of flavonoids (compounds and 11) and seven kinds of other known isolates (compounds
17 20 23 26 and 31) possessed significant inhibitory effects on intracellular TG content in HepG2
cells. Among them, the activities of compounds and 20 were comparable to that of orlistat, which
–32)
1, 2, 3, 6–9
–
,
,
1
suggested that these compounds in this plant might be involved in lipid metabolism.
Keywords: Rosmarinus officinalis; flavonoids; terpenoids; phenolic acids; triglyceride accumulation
inhibitory effects; HepG2 cells
1. Introduction
Rosmarinus officinalis L., is a perennial edible herb, belonging to Labiatae, commonly known as
rosemary. R. officinalis extract has been reported to have antioxidant, anti-inflammatory, antidiabetic
and anticancer properties [
from the aerial parts of R. officinalis, the isolation and structure elucidation of 16 terpenoids including
normonoterpenoid, diterpenoid and triterpenoid glycosides [ ], had been reported by us. Their structures
were elucidated by chemical and spectroscopic methods. As a continuing study on the plant, we
obtained 16 flavonoids including two new ones, named officinoflavonosides A ( ) and B ( ). In this
1]. In the course of our characterization studies on bioactive constituents
2
1
2
paper, we describe the isolation and structure elucidation of these new flavanoids, along with the
triglyceride (TG) accumulation inhibitory effects of the above-mentioned 45 compounds in HepG2 cells.
2. Results and Discussion
During the course of our continuous studies on bioactive constituents from 95% EtOH eluate
of D101 CC and CHCl3 layer [
compounds, named officinoflavonosides A (
luteolin-7-O- -D-glucoside ( ) [ ], luteolin-7-O-
2
,
3
] from R. officinalis aerial parts, 16 flavonoids, including two new
) and B ( ) (Figure 1), together with 14 known ones,
-D-lutinoside ( ) [ -D-glucuronide
], luteolin 30-O-
1
2
β
3
3
β
4
4
β
Molecules 2017, 22, 110; doi:10.3390/molecules22010110
www.mdpi.com/journal/molecules

Molecules 2017, 22, 110
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(
β
5
) [
-D-glucuronide (
10) [ ], apigenin-7-O-
13), 600-O-(E)-feruloylnepitrin (14), 6-hydroxy luteolin-7-O-
5
], 5,7,40-trihydroxy-30-O-
) [
], luteolin 30-O-(400-O-acetyl)-
-D-lutinoside (11) [ ], nepitrin (12) [
-D-glucopyranoside (15) [10], and
10, and
β
-D-glucuronic acid-600-methyl ester (
-D-glucuronide (
], 600-O-(E)-p-coumaroylnepitrin
6
) [
6
], luteolin 30-O-(300-O-acetyl)-
7
5
β
8) [5], acacetin (9) [7], tiliadin
(
(
8
β
4
9
β
homoplantaginin (16) [11] (Figure 2) were further obtained. Among the known ones, 6,
13 were isolated from the rosmarinus genus 14 for the first time, and the NMR data of 13 and 14 in
DMSO-d6 were reported for the first time.
OH
O
OH
OH
HO
O
O
O
O
O
CH3O
OH
HOOC
OH
O
O
O
OH
HO
O
O
HO
OH
HO
1
2
OH
OH
HO
O
Figure 1. The new compounds 1 and 2 obtained from the aerial parts of R. officinalis.
Figure 2. The known flavonoids (3–16) obtained from the aerial parts of R. officinalis.
This paper will elucidate the isolation and structure of new compounds, officinoflavonosides
A (
1
) and B (
2
). Meanwhile, the effects of 16 flavonoids and 16 previously isolated terpenoids [
2],
officinoterpenoside D (1
7
), (1S,4S,5S)-5-exo-hydrocamphor 5-O- -D-glucopyranoside (18), isorosmanol
β
(
(
(
19), rosmanol (20), 7-methoxyrosmanol (21), epirosmanol (22), officinoterpenosides A1 (23) and A2
24), along with ursolic acid (25), micromeric acid (26), glucosyl tormentate (27), officinoterpenoside B
28), niga-ichigoside F1 (29), oleanolic acid (30), officinoterpenoside C (31), asteryunnanoside B (32
)
(Figure 3) on the reduction of TG content in HepG2 cells were determined.

Molecules 2017, 22, 110
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Figure 3. The known terpenoids (17–32) [2,3] obtained from the aerial parts of R. officinalis
(Glc: β-D-glucopyranosyl).
Officinoflavonoside A (
1
) was isolated as an amorphous yellow powder with negative rotation
α]
25 –11.9 (c = 0.79, MeOH)}. Its molecular formula was determined to be C23H20O13 by negative-ion
D
◦
{[
HRESI-TOF-MS (m/z 503.0838 [M
yielded D-glucuronic acid, which was identified by comparing the Rf value on TLC plate with the
authentic sample. The 1H- and 13C-NMR (Table 1) spectra of
, which were assigned by various NMR
experiments including 1H-1H COSY, HSQC, and HMBC spectra, indicated there was a luteolin part
6.21 (1H, br. s, H-6), 6.51 (1H, br. s, H-8), 6.81 (1H, s, H-3), 7.01 (1H, d, J = 8.0 Hz, H-5 ), 7.66 (1H,
br. d, ca. J = 8 Hz, H-60), 7.69 (1H, br. s, H-20), 12.94 (1H, br. s, 5-OH)), a
-D-glucuronyl moiety
−
H]−, calcd for C23H19O13 503.0831). Acid hydrolysis of
1
1
(
δ
'
β
(δ
5.39 (1H, d, J = 8.0 Hz, H-100)), together with an acetyl group (δH 2.07 (3H, s); δC 20.9 (COCH3),
169.1 (COCH3)) in the structure. A downfield shift of 0.3 ppm f0or the C-200, and upfield shift by
00
00
2.0 ppm both for the C-1 and 3 compared with those of luteolin 3 -O-β-D-glucuronide (5) [5], which
indicated that the C-200 hydroxyl proton was substituted with an acetyl group. Finally, the linkages
of β-D-glucuronyl moiety and acetyl group were clarified on the basis of HMBC experiment, which
showed long-range correlations from δH 5.39 (H-100) to δC 144.9 (C-30); δH 4.89 (1H, dd, J = 8.0, 8.0 Hz,
00
H-2 ) to δC 169.1 (2-OCOCH3) (Figure 4). On the basis of the above-mentioned evidence, the structure
of officinoflavonoside A was determined as luteolin 30-O-(200-O-acetyl)-β-D-glucuronide (1).
1
Table 1. H- and 13C-NMR data for 1 in DMSO-d6.
No.
δC
δ
(J in Hz)
No.
δC
δ
(J in Hz)
H
H
0
2
3
4
5
6
7
8
9
163.2
103.1
181.7
161.4
98.8
-
4
5
6
1
2
3
4
5
151.7
116.8
122.4
98.8
73.2
73.4
71.5
75.2
170.3
169.1
20.9
-
-
0
6.81 (s)
7.01 (d, 8.0)
7.66 (br. d, ca. 8)
5.39 (d, 8.0)
4.89 (dd, 8.0, 8.0)
3.57 (dd, 8.0, 8.0)
3.55 (dd, 8.0, 8.0)
4.01 (d, 8.0 Hz)
-
0
-
-
00
00
6.21 (br. s)
00
164.2
94.0
-
00
6.51 (br. s)
00
157.2
103.6
121.3
115.7
144.9
-
-
-
00
10
6
0
00
00
1
2
3
2 -COCH3
2 -COCH3
-
0
7.69 (br. s)
-
2.07 (s)
12.94 (br. s)
0
5-OH

Molecules 2017, 22, 110
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Figure 4. The main 1H-1H COSY and HMBC correlations of 1 and 2.
25
Officinoflavonoside B (
2
) with negative optical rotation ([
α
]
−
47.4◦ in MeOH) was also isolated
was determined from
H]−, calcd for C28H31O16 623.1618), too. The IR
spectrum exhibited typical absorption bands of hydroxyl (3332 cm−1), ester carboxyl group (1658 cm−1),
aromatic ring (1603, 1514, 1462 cm−1), and an O-glycosidic linkage (1067 cm−1). The UV data of
showed the characteristic maxima absorption of luteolin aglycon at 340 nm (4.17) and 267 nm (4.11).
Acid hydrolysis of with 1.0 M HCl liberated L-rhamnose and D-glucose, which were identified by
HPLC analysis using an optical rotation detector [
]. The 1H- and 13C-NMR (Table 2) spectra of
indicated the presence of 6-methoxy luteolin (nepitrin) ( 6.70 (1H, s, H-3), 6.89 (1H, s, H-8), 6.90
(1H, d, J = 8.0 Hz, H-50), 7.40 (1H, dd, J = 2.0, 8.0 Hz, H-60), 7.41 (1H, d, J = 2.0 Hz, H-20)), and
-D-glucopyranosyl ( -L-rhamnopyranosyl moiety
5.13 (1H, d, J = 7.5 Hz, H-100)), together with an
1.05 (3H, d, J = 6.5 Hz, H-6 ), 4.57 (1H, br. s, H-1 )). The connectivity of oligoglycoside moieties to the
aglycon part was characterized by a HMBC experiment on . Thus, the HMBC experiment of showed
D
as an amorphous yellow powder. The molecular formula C28H30O16 of
negative-ion HRESI-TOF-MS (m/z 623.1595 [M
2
−
2
2
2
2
δ
a
β
δ
α
000
000
(δ
2
2
long-range correlations between the following proton and carbon pairs (δH 5.13 (100-H) and δC 156.3
(C-7); δH 4.57 (H-1000) and δC 65.8 (C-600)) (Figure 4). Consequently, the structure of officinoflavonoside
B was determined to be nepitrin 7-O-α-L-rhamnopyranosyl(1→6)-β-D-glucopyranoside (2).
1
Table 2. H- and 13C-NMR data for 2 in DMSO-d6.
No.
δC
δ
(J in Hz)
No.
δC
δ (J in Hz)
H
H
00
2
3
4
5
6
7
8
9
10
164.8
102.4
182.0
152.7
132.7
156.3
94.2
152.1
105.8
120.7
113.2
146.2
150.8
115.9
119.0
-
1
2
3
4
5
6
100.3
73.1
76.4
69.4
75.5
65.8
5.13 (d, 7.5)
3.32 (m, overlapped)
3.32 (m, overlapped)
3.21 (dd, 9.0, 9.0)
3.61 (m)
3.51 (dd, 4.5, 12.0)
3.85 (br. d, ca. 12)
4.57 (br. s)
3.64 (br. d, ca. 3)
3.46 (dd, 3.0, 9.0)
3.15 (dd, 9.0, 9.0)
3.42 (m)
00
6.70 (s)
00
-
-
-
-
00
00
00
6.89 (s)
000
-
-
-
1
2
3
4
5
6
100.3
70.3
70.7
71.9
68.2
17.7
60.2
-
000
0
000
1
2
3
4
5
6
0
000
7.41 (d, 2.0)
0
000
-
-
0
000
1.05 (d, 6.5)
3.77 (s)
13.06 (br. s)
0
6.90 (d, 8.0)
7.40 (dd, 2.0, 8.0)
6-OCH3
5-OH
0
TG accumulation of inhibitory effects of compounds isolated from aerial parts of R. officinalis
were screened in sophisticated sodium oleate (SO)-induced HepG2 cells. The results showed
that TG was accumulated as lipid droplets in the SO treated HepG2 cells monolayer and at least
a 5-fold increase (p < 0.001) in TG content was observed from 51.14
to 288.34 ± 5.72 mg/dL (model group). Compared to the SO group, orlistat significantly (p < 0.001)
decreased this value to 230.06 2.68 mg/dL by a 20.2% clearance ratio and eight of the 16 kinds
of flavonoids tested showed similar inhibitory effects. Compounds and 11 reduced the
±
4.70 mg/dL (normal group)
±
1, 2, 3, 6–9

Molecules 2017, 22, 110
5 of 11
intracellular TG content by 17.0%, 8.4%, 9.8%, 9.7%, 11.3%, 8.5%, 13.0% and 6.0%, respectively, as given
in Figure 5A. In addition, 16 terpenoids (17 32) were evaluated in the same method and seven kinds
showed significantly inhibitory activities. The clearance ratios of 17 20 23 26 and 31 were 6.3%, 8.3%,
–
–
,
,
8.4%, 23.3%, 5.8%, 8.4% and 8.5%, respectively, as given in Figure 5B.
A
7.0
6.5
6.0
*
*
**
**
**
**
7
**
5.5
5.0
4.5
4.0
1.5
1.0
0.5
0.0
***
***
***
Nor. Mod. Orli.
1
2
3
4
5
6
8
9
10 11 12 13 14 15 16
B
7.0
6.5
6.0
5.5
5.0
4.5
4.0
*
**
*
**
**
*
***
***
***
1.5
1.0
0.5
0.0
Nor. Mod. Orli. 17 18 19 20 21 22 23 24 25 26 27 28 30 29 31 32
Figure 5.
in HepG2 cells; (
cells. Cells were incubated with 200
and positive-controlled orlistat (5
(
A
) Effects of 16 kinds of flavonoids (
1
–
16) from R. officinalis on triglyceride (TG) accumulation
32) from R. officinalis on TG accumulation in HepG2
mol/L SO for 48 h. Meanwhile, tested compounds (30 mol/L)
mol/L, Orli.) were co-incubated to evaluate their inhibitory
B) Effects of 16 terpenoids (17–
µ
µ
µ
effects. Cells cultured in normal medium without sodium oleate (SO) were set as normal group (Nor.).
The intracellular TG content in each well was examined using a TG assay kit. Each value represents the
mean ± S.E.M., n = 5, *** p < 0.001, ** p < 0.01, * p < 0.05 vs. model group (Mod.).
Studies of dose dependency of six selected compounds (compounds
comparatively showed the best TG-lowering activities, were then conducted. In this system, orlistat,
a lipase inhibitor showed significant TG accumulation effects at concentration of 5 mol/L. As shown
1, 2, 9, 20, 26 and 31), which
µ
in Figure 6, the results revealed that all the above isolates could inhibit the SO-induced intracellular TG
accumulation in a dose dependent manner. Among them, compound 20 exhibited the strongest activity
that it had demonstrated a clearance ratio of more than 5.6% from 1 µmol/L. Then, Oil red O staining
further visually confirmed the results that a large amount of lipid droplets were seen in SO-treated
HepG2 cells, indicating lipid accumulation, but the accumulation was inhibited by compound 20 in
a dose dependent manner (shown in Figure 7). Although the colorimetric assay results showed that
the concentration of 1 µmol/L exhibited no significant difference compared to the model group, the
possible reasons for this might be the lower precision of this detection method or the activity-instability
at this low concentration.
Although a limited number of compounds were tested for inhibitory effects on TG accumulation
in HepG2 cells, we can summarize the structure-activities relationship as follows: in flavonoids,
5,7-dihydroxyl substitution showed strong activities (compounds
1
,
6
–
9), and the activities would
be reduced when 7-hydroxyl group was glucosylated (Compound
9
> 10). Among the diterpenes,
compound 20 showed strongest inhibitory effect on TG accumulation. Methylation or configuration
changing at position 20 would quench the activity, which indicated that 20
inhibitory activity.
α-hydroxy is important for

Molecules 2017, 22, 110
6 of 11
A
B
Figure 6. Dose dependency of TG-lowering effects of compounds
HepG2 cells were treated with 0, 0.1, 1, 10 and 30 mol/L of indicated compounds, respectively in the
presence of SO for 48 h. Rate of TG lowering is given according to a formula: Rate of TG lowering
(%) = ((TG concentration of untreated groups TG concentration of treated groups)/TG concentration of
untreated groups) 100%. Each value represents the mean S.E.M., n = 5, *** p < 0.001 ** p < 0.01, * p < 0.05
1, 2, 9, 20, 26 and 31 in HepG2 cells.
µ
−
×
±
vs. untreated controls. (A) effects of Compounds 1, 2 and 9; (B) effects of Compounds 20, 26 and 31.
A
Normal
Model
Orlistat
Com. 20-1µmol/l
Com. 20-10 µmol/l
Com. 20-30 µmol/l
B
0.5
0.4
0.3
0.2
0.1
0.0
*
**
***
***
Nor.
Mod.
Orli.
1
10
30
Compoun 20 (μmol/l)
Figure 7. Effect of compound 20 from R. officinalis on TG accumulation in HepG2 cells. HepG2 cells
were treated with indicated concentrations of orlistat (orlistat group, Orli.) or compound 20 in the
absence (normal group, Nor.) or presence of SO for 48 h. Cells were stained with Oil Red O (
and analyzed by colorimetric assay at 492 nm ( ). Each value represents the mean S.E.M., n = 4,
*** p < 0.001, ** p < 0.01, * p < 0.05 vs. model group (Mod.).
A)
B
±

Molecules 2017, 22, 110
7 of 11
3. Experimental Section
3.1. General
Optical rotations were determined on a Rudolph Autopol® IV automatic polarimeter (l = 50 mm).
IR spectra were recorded on a Varian 640-IR FT-IR spectrophotometer (Varian Australia Pty Ltd.,
Mulgrave, Australia). UV spectra were obtained on a Varian Cary 50 UV-Vis spectrophotometer
(Varian, Inc., Hubbardsdon, MA, USA). NMR spectra were measured at a Bruker 500 MHz NMR
spectrometer (Bruker BioSpin AG Industriestrasse 26 CH-8117, Fällanden, Switzerland) at 500 MHz for
1H- and 125 MHz for 13C-NMR, with TMS as an internal standard. Positive- and Negative-ion
HRESI-TOF-MS were recorded on an Agilent Technologies 6520 Accurate-Mass Q-Tof LC/MS
spectrometer (Agilent Corp., Santa Clara, CA, USA).
Column chromatographies (CC) were conducted on macroporous resin D101 (Haiguang
Chemical Co., Ltd., Tianjin, China), Silica gel (74–149
Qingdao, China), and ODS (50 m, YMC Co., Ltd., Tokyo, Japan). Preparative HPLC (PHPLC) column,
250 mm, Nakalai Tesque, Inc., Tokyo, Japan) was used to purify
µm, Qingdao Haiyang Chemical Co., Ltd.,
µ
Cosmosil 5C18-MS-II (20 mm i.d.
the constituents.
×
3.2. Plant Material
The dried aerial parts of R. officinalis were collected from Butarie, Rwanda, and identified
by Dr. Li Tianxiang (Experiment Teaching Department, Tianjin University of Traditional Chinese
Medicine). The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of
Tianjin University of TCM (No. 20110910).
3.3. Extraction and Isolation
The dried aerial parts of R. officinalis (2.5 kg) were dealt by using the same method as reported
before [
were obtained.
The EtOH fraction (36 g) was subjected to normal phase silica gel CC (CHCl3
(100:3 100:5 100:7, v/v) CHCl3–MeOH–H2O (10:3:1 7:3:1, v/v/v)
eleven fractions (Fr. 1–11). Fraction 8 (5480.0 mg) was subjected to PHPLC through gradient
elution (MeOH–H2O (30:70 50:50 70:30 100:0, v/v)) to yield 22 fractions (Fr. 8-1–8-22).
2,3]. As results, CHCl3 partition layer (269 g), H2O (47 g) and 95% EtOH (45 g) eluted fractions
→
CHCl3–MeOH
MeOH) to yield
→
→
→
→
→
→
→
→
Fraction 8-15 (254.6 mg) was separated by PHPLC (CH3CN–H2O (21:79, v/v)) to give fractions
8-15-1 and 8-15-2. Fraction 8-15-1 (59.6 mg) was purified by PHPLC (MeOH–H2O (44:56, v/v))
and (CH3CN–H2O (19:81, v/v)) to yield homoplantaginin (16, 23.0 mg). Fraction 8-19 (228.0 mg)
was purified by PHPLC (CH3CN–H2O (25:75, v/v)) to give 40,5,7-trihydroxy-30-O-
β
-D-glucuronic
acid-600-methyl ester (
6
, 36.1 mg), tiliadin (10, 6.3 mg), 600-O-(E)-p-coumaroylnepitrin (13, 21.4 mg),
00
and 6 -O-(E)-feruloylnepitrin (14, 9.5 mg). Fraction 9 (10.0 g) was separated by ODS CC (MeOH–H2O
(20:80
→
30:70
→
40:60
→
50:50
→
60:40
→
70:30
→
100:0, v/v)) to yield 14 fractions (Fr. 9-1–9-14).
Fraction 9-10 (1510.0 mg) was purified by Sephadex LH-20 CC (CHCl3–MeOH (1:1, v/v)) to
yield eight fractions (Fr. 9-10-1–9-10-8). Fraction 9-10-2 (512.2 mg) was subjected to PHPLC
(MeOH–1% CH3COOH (45:55, v/v)] to obtain 14 fractions (Fr. 9-10-2-1–9-10-2-14). Fractions 9-10-2-5
(21.5 mg) and 9-10-2-6 (14.5 mg) were combined and subjected to PHPLC (CH3CN–1% CH3COOH
(20:80, v/v)) to give homoplantaginin (16, 10.9 mg). Fraction 9-10-4 was separated by PHPLC
(MeOH–1% CH3COOH (45:55, v/v)) to yield 13 fractions (Fr. 9-10-4-1–9-10-4-13). Fraction 9-10-4-12
(116.0 mg) was subjected to silica gel CC (CHCl3-MeOH-H2O (20:3:1, v/v/v)
(CH3CN–1% CH3COOH (24:76, v/v)) to give luteolin 30-O-
-D-glucuronide (
30-O-(300-O-acetyl)- , 14.5 mg), and luteolin 30-O-(400-O-acetyl)-
-D-glucuronide (
, 9.3 mg). Fraction 10 (6.3 g) was isolated by PHPLC through gradient elution (MeOH–H2O
25:75 → 40:60 → 60:40→ 80:20 → 100:0, v/v)) to yield 35 fractions (Fr. 10-1–10-35). Fraction 10-24
→
MeOH) and PHPLC
, 14.9 mg), luteolin
-D-glucuronide
β
5
β
7
β
(8
(
(142.2 mg) was purified by Sephadex LH-20 CC (MeOH) and PHPLC (CH3CN–1% CH3COOH (16:84,

Molecules 2017, 22, 110
8 of 11
v/v)) to yield 6-hydroxy luteolin-7-O-β-D-glucopyranoside (15, 9.0 mg). Fraction 10-25 (149.4 mg) was
separated by PHPLC (CH3CN–1% CH3COOH (17:83, v/v)) to yield six fractions (Fr. 10-25-1–10-25-6).
Fraction 10-25-5 (20.8 mg) was further purified by Sephadex LH-20 (MeOH–H2O (50:50, v/v)), and
luteolin-7-O-
β-D-lutinoside (
4, 14.2 mg) was obtained. Fraction 10-26 (93.7 mg) was separated
by PHPLC (CH3CN–1% CH3COOH (16:84, v/v)) to yield luteolin-7-O-
β
-D-glucoside ( , 23.6 mg).
3
Fraction 10-27 (756.1 mg) was subjected to Sephadex LH-20 CC (MeOH) to yield nine fractions
(Fr. 10-27-1–10-27-9). Fraction 10-27-5 (114.3 mg) was isolated by PHPLC (CH3CN–1% CH3COOH
(17:83, v/v)) to give both officinoflavonoside B (2, 13.0 mg) and apigenin-7-O-β-D-lutinoside (11,
7.5 mg). Furthermore, fraction 10-27-7 (149.8 mg) was purified by PHPLC (CH3CN–1% CH3COOH
0
(20:80, v/v)) to yield luteolin 3 -O-
β-D-glucuronide (5, 28.6 mg) and nepitrin (12, 14.0 mg). Fraction 10-28
(473.3 mg) was also subjected to Sephadex LH-20 CC (MeOH) to yield seven fractions (Fr. 10-28-1–10-28-7).
Fraction 10-28-6 (222.0 mg) was prepared by PHPLC (CH3CN–1% CH3COOH (20:80, v/v)) to give
luteolin 30-O-
β-D-glucuronide (5, 13.5 mg). Furthermore, fraction 10-30 was isolated by Sephadex
LH-20 CC (MeOH) and PHPLC (CH3CN–1% CH3COOH (25:75, v/v)) to yield officinoflavonoside A (
1,
19.0 mg). Fraction 10-31 (198.5 mg) was separated by PHPLC (CH3CN–1% CH3COOH (26:74, v/v)) to
give 40,5,7-trihydroxy-30-O-β-D-glucuronic acid-600-methyl ester (6, 24.0 mg).
The CHCl3 partition (200 g) of the rosemary extract was subjected to silica gel CC (CHCl3
CHCl3–MeOH (100:1 100:3 100:5 100:7, v/v) CHCl3–MeOH–H2O (10:3:1 7:3:1, v/v/v)
MeOH) to yield 23 fractions (Fr. 1–23). Fraction 9 (56.3 g) was further subjected to silica gel CC
→
→
→
→
→
→
→
(Pet. Ether (PE)
→
PE-EtOAc (20:1
→
15:1
→
10:1
→
5:1
→
3:1, v/v)
→
EtOAc) to yield 19 fractions
(Fr. 9-1–9-19). Fraction 9-16 (5024.0 mg) was purified by PHPLC (MeOH–H2O (90:10, v/v)), and
13 fractions (Fr. 9-16-1–9-16-13) were given. Fraction 9-16-4 (629.8 mg) was subjected to PHPLC
(MeOH–H2O (75:25, v/v)) to give acacetin (9, 2.6 mg).
25
D
◦
Officinoflavonoside A (
1
): Yellow powder; [
α
]
−
11.9 (c = 0.79, MeOH); IR
ν
max (KBr) cm−1: 3209, 2927,
max (MeOH) nm (log ): 335
2861, 1736, 1656, 1607, 1505, 1435, 1356, 1301, 1255, 1167, 1081, 1039; UV
λ
ε
(4.20), 265 (4.15). H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data,
1
see Table 1; HRESI-TOF-MS: Negative-ion mode m/z 503.0838 [M – H]− (calcd for C23H19O13 503.0831).
25
D
◦
Officinoflavonoside B (
2
): Yellow powder; [
α
]
−
47.4 (c = 0.59, MeOH); IR
ν
max (KBr) cm−1: 3332, 2923,
max (MeOH) nm (log ): 340 (4.17), 267
1658, 1603, 1570, 1514, 1462, 1355, 1276, 1192, 1067, 1014; UV
λ
ε
(4.11). H-NMR (500 MHz, DMSO-d6) and 13C-NMR (125 MHz, DMSO-d6) spectroscopic data, see
1
Table 2; HRESI-TOF-MS: Negative-ion mode m/z 6231595 [M – H]− (calcd for C28H31O16 623.1618).
600-O-(E)-p-Coumaroylnepitrin (13): Yellow powder; The NMR data of 13 in DMSO-d6 are reported for
1
the first time. H-NMR (DMSO-d6, 500 MHz):
δ
6.70 (1H, s, H-3), 6.96 (1H, s, H-8), 7.45 (2H, m, H-20
and 60), 6.89 (1H, d, J = 9.0 Hz, H-50), 5.23 (1H, d, J = 7.0 Hz, H-100), 3.41 (1H, dd, J = 7.0, 9.0 Hz, H-200),
3.39 (1H, dd, J = 9.0, 9.0 Hz, H-300), 3.30 (1H, dd, J = 9.0, 9.0 Hz, H-400), 3.86 (1H, m, H-500), (4.25 (1H,
dd, J = 7.0, 12.0 Hz), 4.43 (1H, br. d, ca. J = 12 Hz), H2-600), 7.27 (2H, d, J = 8.5 Hz, H-2000,6000), 6.60 (2H,
d, J = 8.5 Hz, H-3000,5000), 7.46 (1H, d, J = 16.0 Hz, H-7000), 6.29 (1H, d, J = 16.0 Hz, H-8000), 3.77 (3H, s,
6-OCH3), 13.03 (1H, br. s, 5-OH). 13C-NMR (DMSO-d6, 125 MHz):
δ 164.5 (C-2), 102.3 (C-3), 182.0 (C-4),
152.5 (C-5), 132.5 (C-6), 156.1 (C-7), 94.0 (0C-8), 152.0 (C-9), 105.7 0(0C-10), 120.8 (C-10), 113.1 (C-20), 145.9
0
0
0
00
00
00
00
(C-3 ), 150.5 (C-4 ), 115.9 (C-5 ), 119.0 (C-6 ), 99.8 (C-1 ), 72.9 (C-2 ), 76.3 (C-3 ), 69.9 (C-4 ), 73.7 (C-5 ),
63.4 (C-600), 124.6 (C-1000), 115.5 (C-2000,6000), 129.9 (C-3000,5000), 159.7 (C-4000), 144.9 (C-7000), 113.4 (C-8000),
000
−
H] (calcd for C31H27O14 623.1406).
166.4 (C-9 ), 60.2 (6-OCH3). Negative-ion mode m/z 623.1413 [M
−
600-O-(E)-Feruloylnepitrin (14): Yellow powder; The NMR data of 14 in DMSO-d6 are reported for
1
the first time. H-NMR (DMSO-d6, 500 MHz):
δ 6.63 (1H, s, H-3), 6.95 (1H, s, H-8), 7.39 (1H, br. s,
H-20), 6.83 (1H, d, J = 8.0 Hz, H-50), 7.39 (1H, br. d, ca. J = 8 Hz, H-60), 5.23 (1H, d, J = 7.0 Hz, H-100),
3.40 (1H, dd, J = 7.0, 9.0 Hz, H-200), 3.38 (1H, dd, J = 9.0, 9.0 Hz, H-300), 3.32 (1H, dd, J = 9.0, 9.0 Hz,
H-400), 3.83 (1H, m, H-500), (4.24 (1H, dd, J = 6.5, 12.0 Hz), 4.44 (1H, br. d, ca. J = 12 Hz), H2-600), 7.13
(1H, br. s, H-2000), 6.60 (1H, d, J = 8.0 Hz, H-5000), 6.83 (1H, br. d, ca. J = 8 Hz, H-6000), 7.45 (1H, d,

Molecules 2017, 22, 110
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J = 16.0 Hz, H-7000), 6.37 (1H, d, J = 16.0 Hz, H-8000), 3.76 (3H, s, 6-OCH3), 13.07 (1H, br. s, 5-OH), 3.71
(3H, s, 3000-OCH3). 13C-NMR (DMSO-d6, 125 MHz): 164.6 (C-2), 102.1 (C-3), 182.0 (C-4), 152.4 (C-5),
132.4 (C-6), 156.1 (C-7), 94.0 (C-8), 152.00(0C-9), 105.7 0(0C-10), 120.6 (C-10), 113.000 (C-20), 1460.00 (C-30), 150.5
0
0
00
00
(C-4 ), 115.8 (C-5), 119.1 (C-6 ), 99.8 (C-1 ), 73.0 (C-2 ), 76.3 (C-3 ), 69.7 (C-4 ), 73.7 (C-5 ), 63.1 (C-6 ),
125.2 (C-1000), 111.0 (C-2000), 147.7 (C-3000), 149.3 (C-4000), 115.3 (C-5000), 122.7 (C-6000), 145.2 (C-7000), 113.8
(C-8000), 166.4 (C-9000), 60.2 (6-OCH3), 55.4 (3000-OCH3). Negative-ion mode m/z 653.1514 [M
(calcd for C32H29O15 653.1512).
−
H]−
Acid Hydrolysis of
1
and
2
. A solution of officinoflavonoside A (
1
, 2.0 mg) was hydrolyzed with
◦
10% H2SO4 in 50% EtOH (2 mL) at 10−0 C for 3 h. The reaction mixture was neutralized with ion
exchange resin Amberlite IRA-400 (OH form) and evaporated to dryness. The sugar was found to be
glucuronide by comparison of the Rf value (Rf = 0.21) on TLC plate (silica gel, CHCl3–MeOH–H2O
(6:4:1, v/v/v, lower layer)) with the authentic D-glucuronide.
Meanwhile, a solution of officinoflavonoside B (
reflux for 3 h, respectively. The reaction mixture was dealt with the similar methods as those used
to officinoterpenosides A–D [ ]. Identification of L-rhamnose (i) and D-glucose (ii) from presented
2, 2.0 mg) in 1 M HCl (1 mL) was heated under
2
2
in the aqueous was carried out by comparison of its retention time and optical rotation with that of
authentic samples, tR: (i) 9.1 min (negative, L-rhamnose), and (ii) 17.6 min (positive, D-glucose).
3.4. Evaluation of Effects on Sodium oleate-induced TG Accumulation in HepG2 Cells
Materials. A human hepatoma HepG2 cell line was obtained from Cell Resource Center of
Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical
College (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), penicillin and streptomycin
were purchased from Thermo SCIENTIFIC (Waltham, MA, USA). Fetal Bovine Serum (FBS) was
purchased from PAN-Biotech GmbH (Bavaria, Germany). TG assay kits were purchased from Wako
Pure Chemical Industries, Ltd. (Osaka, Japan). Sodium oleate and orlistat were obtained from
Sigma-Aldrich Corporation (St. Louis, MO, USA).
Cell culture. HepG2 cells were grown in DMEM supplemented with 10% FBS and 1% antibiotics
(100 unit/mL penicillin and 100 mg/mL streptomycin). Cells were maintained in an atmosphere of
95% air and 5% CO2 at 37 ◦C in subconfluent condition.
Sodium oleate-induced TG accumulation. TG accumulation inhibitory effects were screened as
previous report [12]. Briefly, Cells were seeded at a density of 100,000 cells/mL on Corning 48-multiwell
plates. After 24 h in culture, medium was exchanged for phenol red free-DMEM with or without
Sodium oleate (200
R. officinalis for another 48 h. Meanwhile, an anti-obesity drug, orlistat (5
a positive control. Cells were then rinsed with phosphate buffered saline twice and 200
µ
mol/L) in the presence or absence of the obtained isolates (30
mol/L), was used as
L deionized
µmol/L) from
µ
µ
water was added per well to get cell lysate by ultrasonication. Intracellular TG contents in the
lysates were finally evaluated using a GPO-POD method as the kit protocol provided. Briefly, the
method is based on the enzymatic hydrolysis of triglyceride to glycerol and free fatty acids by
lipoprotein lipase. The glycerol is phosphorylated by adenosine triphosphate in the presence of
glycerolkinase to form glycerol-3-phosphate and adenosine diphosphate. glycerol-3-phosphate is
oxidized by glycerophosphate oxidase (GPO) to form dihydroxyacetone phosphate and hydrogen
peroxide. Purple quinoneimine complex is produced by the peroxidase (POD) catalyzed coupling
of and 4AAP and ESPAS with hydrogen peroxide. The final absorbance was monitored at 492 nm
using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Concentration setting of test
sample and orlistat were established by pre-test results. Under the concentration, there were no
treatment-related changes in cell viability (Data not shown).
Oil red O staining After a 48 h-treatment in 24-multiwell plates, cells were washed with
phosphate-buffered saline (PBS) and fixed in 10% paraformaldehyde for 30 min at room temperature,
followed by washing twice with deionized water and incubated in Oil Red O working solution (water at
a 3:2 ratio of stock solution, which prepared by 0.5% Oil Red O dye in isopropanol) for 1 h. The unbound

Molecules 2017, 22, 110
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dye was removed by washing with 75% ethyl alcohol for once and images were immediately captured
under an inverted phase contrast microscope (Leica, Wetzlar, Germany). For quantitative analysis,
stained cells were then dissolved in isopropanol and absorbance was measured at 492 nm.
3.5. Statistical Analysis
Statistical analysis was performed using one-way ANOVA with the Tukey’s test for multiple
comparisons. All data were expressed as mean
±
S.E.M. and values of p < 0.05 were considered significant.
4. Conclusions
Taken together, 16 flavonoids (
1–
16) including two new ones, officinoflavonosides A (
1) and B
(2
), were obtained from the aerial parts of R. officinalis. Among the known ones, 6,
10, and 13 were
isolated from the rosmarinus genus for the first time, and the NMR data of 13 and 14 in DMSO-d6 were
reported for the first time. Their structures were elucidated by chemical and spectroscopic methods
and their effects on TG accumulation were analyzed in HepG2 cells. The results showed that six
kinds of flavonoids (compounds
and ) and seven kinds of other known isolates (compounds 17
significant inhibitory activities on TG accumulation in vitro. Among them, the activities of compounds
and 20 were comparable to that of orlistat, which suggested that these compounds contained in the
3
,
6,
7
,
8,
9
and 11) together with the two novel ones (compounds
1
2
,
18 19 20 23 26 and 31) possessed
,
,
,
,
1
R. officinalis might be involved in the lipid metabolism.
Acknowledgments: Part of this research was supported by Programs for New Century Excellent Talents in
University (NCET-12-1069), and National Natural Science Foundation of China (81673688).
Author Contributions: Yi Zhang and Tao Wang designed the research and wrote the manuscript; Jian Li,
Tiwalade Adegoke Adelakun, and Sijian Wang performed the experimental work; Shengcai Yang and Xiaoxia Li
retrieved literature; Jingya Ruan perfected language. All authors discussed, edited and approved the final version.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Moore, J.; Yousef, M.; Tsiani, E. Anticancer Effects of Rosemary (Rosmarinus officinalis L.) Extract and
Rosemary Extract Polyphenols. Nutrients 2016, 8, 731. [CrossRef] [PubMed]
Zhang, Y.; Adelakun, T.A.; Qu, L.; Li, X.; Li, J.; Han, L.; Wang, T. New terpenoid glycosides obtained from
Rosmarinus officinalis L. aerial parts. Fitoterapia 2014, 99C, 78–85. [CrossRef] [PubMed]
Li, Z.; Chen, Z.; Li, X.; Xu, Q.; Yang, S. Chemical constituents in roots and stems of Physalis alkekengi var. franchetii.
Zhongcaoyao 2012, 43, 1910–1912.
Ren, Y.; Yang, J. Study on chemical constituents of Saussurea tridactyla Sch-Bip II. Zhongguo Yaoxue Zazhi 2001
,
36, 590–593.
Okamura, N.; Haraguchi, H.; Hashimoto, K.; Yagi, A. Flavonoids in Rosmarinus officinalis leaves. Phytochemistry
1994, 37, 1463–1466. [CrossRef]
Sathiamoorthy, B.; Gupta, P.; Kumar, M.; Chaturvedi, A.K.; Shukla, P.K.; Mauryam, R. New antifungal
flavonoid glycoside from Vitex negundo. Bioorg. Med. Chem. Lett. 2007, 17, 239–242. [CrossRef] [PubMed]
Na, Z.; Feng, Y.; Xu, Y. Chemical constituents from aerial parts of Eupatorium odoratum. Zhongcaoyao 2012, 43,
1896–1900.
Chen, S.; Wang, L.; Gao, G.; Liao, M.; Xiao, P. Studies on flavonoids from Aquilegia oxysepala. Zhongguo Zhongyao
Zazhi 1999, 24, 158–160. [PubMed]
Zheng, D.; Zhang, X.-Q.; Wang, Y.; Ye, W.-C. Chemical constituents of the aerial parts of Blumea riparia.
Zhongguo Tianran Yaowu 2007, 5, 421–424.
10. Pan, C.; Zhang, G.; Mi, W.; Chenm, L.; Wang, Q.; He, J. Flavoniod glycosides from whole plant of Leontopodium
leontopodioides (Wild.) Beauv. Shenyang Yaoke Daxue Xuebao 2009, 26, 886–888.
11. Wu, F.-H.; Liang, J.-Y.; Chen, R.; Wang, Q.-Z.; Li, W.-G. Chemical constituents and hepatoprotective activity
of Plantago depressa. Zhongguo Tianran Yaowu 2006, 4, 435–439.

Molecules 2017, 22, 110
11 of 11
12. Gao, J.; Li, J.; An, Y.; Liu, X.; Qian, Q.; Wu, Y.; Zhang, Y.; Wang, T. Increasing effect of Tangzhiqing formula
on IRS-1-dependent PI3K/AKT signaling in muscle. BMC Complement. Altern. Med. 2014, 14, 198. [CrossRef]
[PubMed]
Sample Availability: Sample Availability: Samples of all the compounds are available from the authors.
©
2017 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).