Two novel flavonoids from the leaves of Rhododendron dauricum L. with their inhibition of TNF-α production in LPS-induced RAW 264.7 cells

Article abstract of DOI:10.1080/14786419.2019.1648455

Two new flavonoids, (2S)-6,8-dimethyl-5,7,3′,4′-tetrahydroxyflavanone 4′-O-β-D-glucopyranoside (1) and quercetin 3-O-β-D-(6′′-p-methoxybenzoyl)-galactopyranoside (2), together with ten known flavonoids (3–12) were isolated from the leaves of Rhododendron dauricum L. The structures of the flavonoids were characterized from spectroscopic data (1D and 2D NMR and HR-ESI-MS). The isolated flavonoids were evaluated for their inhibitory effects on the production of tumour necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 cells. Compound 11 exhibited inhibitory activity against TNF-α production with an IC₅₀ value of 46.2 ± 1.2 μM.

Full text of DOI:10.1080/14786419.2019.1648455

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Two novel flavonoids from the leaves of
Rhododendron dauricum L. with their inhibition of
TNF-α production in LPS-induced RAW 264.7 cells
Chao Ye, Mei Jin, Chunshi Jin, Rongshen Wang, Jiaming Wang, Ying Zhang,
Sainan Li, Jinfeng Sun, Wei Zhou & Gao Li
To cite this article: Chao Ye, Mei Jin, Chunshi Jin, Rongshen Wang, Jiaming Wang, Ying Zhang,
Sainan Li, Jinfeng Sun, Wei Zhou & Gao Li (2019): Two novel flavonoids from the leaves of
Rhododendronꢀdauricum L. with their inhibition of TNF-α production in LPS-induced RAW 264.7
cells, Natural Product Research, DOI: 10.1080/14786419.2019.1648455
To link to this article: https://doi.org/10.1080/14786419.2019.1648455
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2019.1648455
Two novel flavonoids from the leaves of Rhododendron
dauricum L. with their inhibition of TNF-a production in
LPS-induced RAW 264.7 cells
a
ꢀ
a,b
ꢀ
a
a
a
Chao Ye , Mei Jin , Chunshi Jin , Rongshen Wang , Jiaming Wang , Ying
a
a
a
a
a
Zhang , Sainan Li , Jinfeng Sun , Wei Zhou and Gao Li
a
Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of
b
Education, College of Pharmacy, Yanbian University, Yanji, P. R. China; Department of Pharmacy,
Yanbian University Hospital, Yanji, P. R. China
ABSTRACT
ARTICLE HISTORY
Received 12 March 2019
Accepted 20 July 2019
0
0
Two new flavonoids, (2S)-6,8-dimethyl-5,7,3 ,4 -tetrahydroxyflava-
0
00
none 4 -O-b-D-glucopyranoside (1) and quercetin 3-O-b-D-(6 -p-
methoxybenzoyl)-galactopyranoside (2), together with ten known
flavonoids (3–12) were isolated from the leaves of Rhododendron
dauricum L. The structures of the flavonoids were characterized
from spectroscopic data (1D and 2D NMR and HR-ESI-MS). The
isolated flavonoids were evaluated for their inhibitory effects on
the production of tumour necrosis factor (TNF)-a in LPS-stimu-
lated RAW 264.7 cells. Compound 11 exhibited inhibitory activity
against TNF-a production with an IC50 value of 46.2 ± 1.2 mM.
KEYWORDS
Rhododendron dauricum;
Ericaceae; flavonoid; TNF-a
1. Introduction
Rhododendron dauricum L. belongs to the Ericaceae family and is mainly distributed in
northeast China, north Mongolia, Korea, Japan and Russia. The dried leaves and
CONTACT Gao Li
These authors contributed equally to the work.
gli@ybu.edu.cn; Wei Zhou
zhouwei8452@163.com
ꢀ
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2019.1648455.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
C. YE ET AL.
flowers of R. dauricum have been used as traditional Korean ethnic medicines to treat
acute and chronic bronchitis, asthma and cough (Popescu and Kopp 2013). Previous
chemical studies have verified that plants from the Rhododendron genus are enriched
with a large number of terpenoids, steroids, coumarins, phenols, volatile oils and flavo-
noids (Iwata et al. 2004; Qiang et al. 2011; Shakeel et al. 2013). Flavonoids and terpe-
noids are considered to be the major chemical components from the species of the
Rhododendron genus. We have previously discovered 13 triterpenoids, including one
new pentacyclic triterpenoid from the leaves of R. dauricum (Li et al. 2019).
Pharmacological research has suggested that the flavonoids isolated from R. dauricum
exhibit broad biological activity, including anti-oxidative, anti-inflammatory and neuro-
protective effect, as well as vasodilation and myocardial preservation effects (Zhang
et al. 2010; Lou et al. 2015; Cui et al. 2019). Tumour necrosis factor (TNF)-a, a pro-
inflammatory cytokine, is generally recognised as one of the key players involved in
immunity and inflammation. Overexpression of the pro-inflammatory cytokine TNF-a is
associated with several diseases, including diabetes, septic shock, tumorigenesis, car-
diovascular diseases, rheumatoid arthritis and inflammatory bowel disease (Du et al.
2014). Thus, inhibition of TNF-a production may help to prevent inflammatory dis-
eases. In our continued search for novel flavonoids and active anti-inflammatory com-
pounds from the leaves of R. dauricum, two new flavonoids (1–2) together with ten
known flavonoids (3–12) were isolated from the ethanol extract of this plant. Herein,
we describe the isolation and structure elucidation of these twelve compounds (1–12),
as well as the inhibitory activity against TNF-a production in LPS-induced RAW
264.7 cells.
2. Results and discussion
Compound 1 was obtained as a yellow amorphous powder, with a molecular formula
þ
of C H O based on positive HR-ESI-MS (obsd. [M þ H] at m/z 479.1535, theor.
2
3 26 11
1
C H O at m/z 479.1548). The H-NMR spectrum of compound 1 showed proton sig-
2
3 27 11
0
nals for an 1,3,4-trisubstituted aromatic ring at dH 7.32 (1H, d, J ¼ 2.0 Hz, H-2 ), 6.99
0
0
(
1H, dd, J ¼ 8.0, 2.0 Hz, H-6 ) and 6.78 (1H, d, J ¼ 8.0 Hz, H-5 ), an oxygenated methine
signal at d 5.64 (1H, dd, J ¼ 10.2, 5.3 Hz, H-2), one methylene proton at d 2.91 (2H,
H
H
m, H-3), two methyl protons at d 2.06 (3H, s, 8-CH ) and 2.02 (3H, s, 6-CH ) and a
H
3
3
0
0
00
sugar moiety at d 4.80 (1H, d, J ¼ 7.9 Hz, H-1 ), 3.85 (1H, m, H-6 a), 3.72 (1H, m, H-
H
0
0
00 00 00 00
13
6
b), 3.45 (2H, m, H-2 , H-3 ), 3.40 (1H, m, H-5 ) and 3.38 (1H, m, H-4 ). The C-NMR
spectrum showed 23 carbon signals, including a carbonyl group at d 198.5 (C-4), 12
C
0
carbons from two benzene rings at d 164.2 (C-7), 160.3 (C-5), 159.4 (C-9), 152.4 (C-4 ),
1
C
0 0 0 0 0
50.5 (C-3 ), 128.0 (C-1 ), 118.8 (C-6 ), 116.7 (C-5 ), 116.4 (C-2 ), 104.8 (C-6), 104.2 (C-8)
and 103.2 (C-10), an oxygenated methine carbon at d 75.5 (C-2), a methylene carbon
C
at 42.9 (C-3) and two methyl carbons at d 8.2 (6-CH ) and 7.4 (8-CH ). The remaining
C
3
3
six carbon resonances were characteristic of a glucopyranosyl moiety at d 103.5 (C-
C
0
0
00 00 00 00 00
1
1
), 78.1 (C-5 ), 78.0 (C-3 ), 75.0 (C-2 ), 71.4 (C-4 ) and 62.5 (C-6 ). The observed H
1
3
and C-NMR data suggested that the skeleton of compound 1 was a glycosylated fla-
vanone. The absolute configuration of the sugar was determined to be D-glucose by
comparison of the retention time and optical rotation value with an authentic sample

NATURAL PRODUCT RESEARCH
3
of D-glucose after acid hydrolysis of compound 1 (Zong et al. 2016; Zhang et al.
2
018). The coupling constant (J ¼ 7.9 Hz) of the anomeric proton confirmed a b-gluco-
0
0
sidic linkage (Orhan 2003). The aglycone was identified as 5,7,3 ,4 -tetrahydroxy-6,8-
dimethyl flavanone based on the close similarity of the NMR data with myrciacitrin III
(3, Matsuda et al. 2002), except for the relative positions of the OH groups in the B
ring, and sugar moiety. The glycosidic linkage was established based on the key
HMBC correlation (Supplementary material, Figure S1) between the anomeric proton
0
0
0
signal H-1 (dH 4.80) and the oxygenated C-4 (d 152.4), which indicated that the
C
0
b-D-glucose linkage was at the C-4 position of the B-ring. The HMBC correlations
from the methyl group protons 6–CH (d 2.02) to C-5 (d 160.3), C-6 (d 104.8) and
3
H
C
C
C-7 (d 164.2), and 8–CH (d 2.06) to C-7 (d 164.2), C-8 (d 104.2) and C-9 (d 159.4)
C
3
H
C
C
C
indicated the location of the two methyl groups at C-6 and C-8. The configuration of
compound 1 was considered to be the same as myrciacitrin III ([a]25D : –104.2, EtOH)
13
(
Matsuda et al. 2002) based on the similarity of chemical shifts in the C-NMR data
and specific optical rotation ([a]25D : –197.6, MeOH). According to the overall analysis,
0
0
the structure of compound 1 was identified as (2S)-6,8-dimethyl-5,7,3 ,4 -tetrahydroxy-
0
flavanone 4 -O-b-D-glucopyranoside.
Compound 2 was obtained as a yellow amorphous powder, with a molecular
þ
formula of C H O based on HR-ESI-MS (obsd. [M þ H] at m/z 599.1396, theor.
2
9 26 14
1
C H O at m/z 599.1395). The H-NMR spectrum showed that compound 2 had a
2
9 27 14
trisubstituted aromatic ring with an ABX-spin coupling system at d 7.73 (1H, d,
H
0
0
0
J ¼ 2.1 Hz, H-2 ), 7.58 (1H, dd, J ¼ 8.5, 2.1 Hz, H-6 ) and 6.86 (1H, d, J ¼ 8.5 Hz, H-5 ),
and an aromatic ring with an AX spin coupling system at 6.35 (1H, d, J ¼ 2.0 Hz, H-
8
) and 6.20 (1H, d, J ¼ 2.0 Hz, H-6). In addition, an 1,4-disubstituted aromatic ring
0
00
000
000
with protons at d 7.67 (2H, d, J ¼ 8.8 Hz, H-2 , 6 ), 6.78 (2H, d, J ¼ 8.8 Hz, H-3 ,
H
0
00
000
) and a methoxy group protons at d 3.86 (3H, s, 4 -OCH ) suggested the pres-
H 3
5
ence of a p-methoxybenzoyl group. A sugar moiety was present as indicated by
0
0
00
proton signals at d 5.31 (1H, d, J ¼ 7.9 Hz, H-1 ), 4.47 (1H, m, H-6 a), 4.33 (1H, m,
H
0
0
00
00
00
H-6 b), 3.90 (1H, m, H-4 ), 3.89 (1H, m, H-2 ), 3.87 (1H, m, H-5 ) and 3.63 (1H, m,
H-3 ). The C-NMR spectrum revealed 29 carbon signals, including signals from two
carbonyl groups at d 179.5 (C-4) and d 167.5 (C-7 ), 18 carbons from three ben-
0
0
13
0
00
C
C
0
00
0
zene rings at 165.8 (C-7), 164.9 (C-4 ), 163.0 (C-5), 158.3 (C-9), 149.8 (C-4 ), 145.8 (C-
0
0
000 000 0 000 0 0 0
3
3
1
1
), 132.3 (C-2 , 6 ), 123.1 (C-6 , 1 ), 122.8 (C-1 ), 117.4 (C-2 ), 116.0 (C-5 ), 114.5 (C-
00
000
, 5 ), 105.5 (C-10), 99.9 (C-6), and 94.7 (C-8), a double bond at 158.7 (C-2) and
0
00
35.3 (C-3), a methoxy group at d 55.9 (4 -OCH ) and a galactose moiety at d
C
C
3
0
0
00
00
00
00
00
04.4 (C-1 ), 75.0 (C-5 ), 74.8 (C-3 ), 73.0 (C-2 ), 70.4 (C-4 ), and 64.6 (C-6 ). The
coupling constant (J ¼ 7.9 Hz) of the anomeric proton confirmed a b-galactosidic
linkage (Lou et al. 2015). Acid hydrolysis of compound 2 gave D-galactose, which
1
13
was identified by the same method as for compound 1. The H and C-NMR spec-
tra suggested that the skeleton of compound 2 was a glycosylated flavonoid, and
the NMR spectroscopic data were found to be similar to quercetin 3-O-b-D-galacto-
pyranoside (7, Ye and Huang 2006). Thus, compound 2 was identified as a quer-
cetin-3-O-b-D-galactopyranoside derivative containing a p-methoxybenzoyl group. In
the HMBC spectrum of compound 2 (Supplementary material, Figure S1), connectiv-
ity between the p-methoxybenzoyl moiety and the D-galactose unit was indicated

4
C. YE ET AL.
Figure 1. Structure of compounds 1-12 from Rhododendron dauricum.
0
0
000
by the cross peak between H-6 (d 4.47, 4.33) and C-7 (d 167.5). The HMBC cor-
H
C
0
0
relation between H-1 (dH 5.31) and C-3 (dC 135.3) revealed that the D-galactose
moiety was attached to the C-3. According to the overall analysis, the structure of
00
compound
2
was identified as quercetin 3-O-b-D-(6 -p-methoxybenzoyl)-
galactopyranoside.
The structures of the ten known flavonoids were determined to be myrciacitrin III (3,
Matsuda et al. 2002), (2S,3S)-taxifolin 3-O-b-D-glucoside (4, Seo et al. 2017), trans-taxifolin
3-O-a-L-arabinopyranoside (5, De Abreu et al. 2011), avicularin (6, Marzouk et al. 2007),
quercetin 3-O-b-D-galactopyranoside (7, Ye and Huang 2006), kaempferol 3-O-a-L-arabino-
furanoside (8, Alvarez et al. 2016), myricetin 3-O-a-L-arabinofuranoside (9, Kadota et al.
0
0
1
990), quercetin (10, Bitis et al. 2010), quercetin 3-O-a-L-(2 -O-acetyl) arabinofuranoside
(
11, Camacho-Luis et al. 2008) and (þ)-catechin (12, Badral et al. 2017) (Figure 1). The
structures were elucidated by comparing the experimental spectroscopic data with
reported values.
All the isolated flavonoids were screened for inhibitory effects on the level of
TNF-a in LPS-activated RAW 264.7 cells. First, cell viability was determined by the
MTT assay. Compounds 1–12 did not display significant cytotoxicity toward RAW
264.7 cells at 100 mM (Supplementary material, Figure S12). These compounds were
examined further at concentrations of 10, 30 and 100 mM to explore the effects on
the production of TNF-a. Compound 11 displayed inhibitory activity with an IC50
value of 46.2 ± 1.2 mM (Table 1). Compound 11 showed better inhibitory activity
than the other flavonoids tested. Analysis of the structure-activity relationship
0
0
showed that the acetyl group at the C-2 position of compound 11 was likely
responsible for the improved inhibitory of TNF-a production compared with the
other compounds.

NATURAL PRODUCT RESEARCH
5
Table 1. Inhibitory effects of compounds 1–12 on TNF-a levels
a
in LPS-stimulated RAW 264.7 cells .
Compounds
IC50 (mM)
1
2
3
4
5
6
7
8
9
76.9 ± 3.0
>100
72.75 ± 1.7
69.5 ± 1.9
49.6 ± 2.3
>100
>100
93.7 ± 2.1
>100
1
1
1
0
1
2
54.7 ± 1.8
46.2 ± 1.2
>100
b
Dexamethasone
3.4 ± 0.4
a
Data are the mean ± SD of three experiments.
Dexamethasone was used as the positive control.
b
3
. Experimental
.1. General experimental procedures
Optical rotations were measured with a Rudolph AutopolIautomatic polarimeter.
3
1
13
The H and C NMR spectra were recorded on Bruker AV 300 MHz and AV 500 MHz
spectrometers (Burker, F €a llanden, Switzerland) using TMS as an internal standard.
HR-ESI-MS was performed using a Bruker microTOF QIImass spectrometer (Bruker
Daltonics, Fremont, CA, USA). Column chromatography was carried out with normal-
phase silica gel (200–300 mesh, Branch of Qingdao Haiyang Chemical Co., Ltd,
Qingdao, P.R.China), and reversed-phase silica gel LiChroprep RP-18 gel (40–63 lm,
Merck KGaA, Darmstadt, Germany). Preparative MPLC was performed on an automatic
flash chromatography device (CHEETAH MP-200, Bonna-Agela Technologies Co., Ltd.)
and columns were packed with C18 (20–45 lm, 40–60 lm) silica. TLC was conducted
on pre-coated silica gel GF254 (200 ꢁ 200 mm, Qingdao Haiyang Chemical Co., Ltd.) or
RP-18 F254 (Merck) glass plates. All other chemicals and solvents were of analytical
grade and used without further purification.
3.2. Plant material
The leaves of R. dauricum L. were collected from the surrounding region of Yanji city,
Jilin province and identified by one of the authors (Prof. Gao Li). A voucher specimen
(YB-RD-1610) was deposited at the College of Pharmacy, Yanbian University.
3.3. Extraction and isolation
The dried leaves of R. dauricum (17.0 kg) were extracted with 95% EtOH (85 L ꢁ 3) at
room temperature for a week. The EtOH extract (3.8 kg) was suspended in H O and
2
partitioned with petroleum ether, EtOAc and n-BuOH. The EtOAc fraction (569.0 g) was
subjected to silica gel column chromatography with CH Cl –MeOH (25:1 to 5:1) to
2
2
obtain 11 fractions (Fr.E1–E11). Fraction E9 (104 g) was purified by silica gel column
chromatography and eluted with CH Cl –MeOH (18:1 to 10:1) to give seven sub-
2
2

6
C. YE ET AL.
fractions (Fr.E9-1–E9-7). Fraction E9-3 (3.2 g) was further separated by MPLC (C ,
18
4
0–60 lm) eluted with MeOH–H O (1:3 to 1:0) to give compounds 10 (12.6 mg) and
2
1
2 (2.3 mg). Fraction E9-7 (1.4 g) was purified by MPLC (C , 20–45 lm) with
18
MeOH–H O (1:4 to 1:0) as the elution solvent to afford compounds 2 (4.5 mg) and 11
2
(
(
16.4 mg). Fraction E10 (55 g) was purified by MPLC (C , 40–60 lm) with MeOH–H O
18 2
1:19 to 1:0) to give six sub-fractions (Fr.E10-1–E10-6). Fraction E10-3 (6 g) was purified
by silica gel column chromatography eluted with CH Cl –MeOH (20:1 to 5:1) followed
2
2
by MPLC (C , 20–45 lm) with MeOH–H O (1:5 to 1:0) to give compounds 5 (196.9 mg)
1
8
2
and 8 (8.4 mg). Fraction E10-5 (12.1 g) was separated using MPLC (C , 40–60 lm) with
18
MeOH–H O (1:4 to 1:0) to obtain compounds 9 (10.2 mg) and 7 (2.3 g). Fraction E11
2
(
9 g) was purified by MPLC (C , 40–60 lm) with MeOH–H O (1:9 to 3:2) to give three
18 2
sub-fractions (Fr.E11-1–E11-3). Fraction E11-2 (6.10 g) was subjected to silica gel col-
umn chromatography and eluted with CH Cl –MeOH (20:1 to 12:1) to yield compound
2
2
4
(122.1 mg) and three sub-fractions (Fr.E11-2-2–E11-2-4). Fraction E11-2-4 (4.51 g) was
purified by MPLC (C , 40–60 lm) with MeOH–H O (1:9 to 1:0) to give compounds 1
18
2
(
46.7 mg), 3 (1.9 mg), and 6 (3.42 g).
0
0
0
(2S)-6,8-Dimethyl-5,7,3 ,4 -tetrahydroxyflavanone 4 -O-b-D-glucopyranoside (1):
Yellow amorphous powder, [a]25D : –197.6 (c 0.06, MeOH). HR-ESI-MS: m/z 479.1535
þ
1
[
(
5
3
2
M þ H] (calcd. for C H O , m/z 479.1548) . H-NMR (300 MHz. methanol-d ); d: 7.32
2
0
3
27 11
4
0
0
1 H, d, J ¼ 2.0 Hz, H-2 ), 6.99 (1 H, dd, J ¼ 8.0, 2.0 Hz, H-6 ), 6.78 (1 H, d, J ¼ 8.0 Hz, H-5 ),
0
0
00
.64 (1 H, dd, J ¼ 10.2, 5.3 Hz, H-2), 4.80 (1 H, d, J ¼ 7.9 Hz, H-1 ), 3.85 (1 H, m, H-6 a),
0
0
00
00
00
00
.72 (1 H, m, H-6 b), 3.45 (2 H, m, H-2 , H-3 ), 3.40 (1 H, m, H-5 ), 3.38 (1 H, m, H-4 ),
13
.91 (2 H, m, H-3), 2.06 (3 H, s, 8-CH ), 2.02 (3 H, s, 6-CH ); C-NMR (75 MHz, methanol-
3
3
0
0
d ), d: 198.5 (C-4), 164.2 (C-7), 160.3 (C-5), 159.4 (C-9), 152.4 (C-4 ), 150.5 (C-3 ), 128.0
4
0
0
0
0
0
0
(
(
C-1 ), 118.8 (C-6 ), 116.7 (C-5 ), 116.4 (C-2 ), 104.8 (C-6), 104.2 (C-8), 103.5 (C-1 ), 103.2
0
0
00
00
00
00
C-10), 78.1 (C-5 ), 78.0 (C-3 ), 75.5 (C-2), 75.0 (C-2 ), 71.4 (C-4 ), 62.5 (C-6 ), 42.9 (C-3),
8
.2 (6-CH ), 7.4 (8-CH ).
3
3
0
0
Quercetin
3-O-b-D-(6 -p-methoxybenzoyl)-galactopyranoside
(2):
Yellow
þ
amorphous powder, [a]25D : þ16.7 (c 0.04, MeOH). HR-ESI-MS: m/z 599.1396 [M þ H]
1
(
calcd. for C H O , m/z 599.1395). H-NMR (500 MHz, methanol-d ) d: 7.73 (1 H, d,
29 27 14 4
0
000
000
0
J ¼ 2.1 Hz, H-2 ), 7.67 (2 H, d, J ¼ 8.8 Hz, H-2 , 6 ), 7.58 (1 H, dd, J ¼ 8.5, 2.1 Hz, H-6 ),
0
000
000
6
(
.86 (1 H, d, J ¼ 8.5 Hz, H-5 ), 6.78 (2 H, d, H-3 , 5 ), 6.35 (1 H, d, J ¼ 2.0 Hz, H-8), 6.20
0
0
00
00
1 H, d, J ¼ 2.0 Hz, H-6), 5.31 (1 H, d, J ¼ 7.9 Hz, H-1 ), 4.47 (1 H, m, H-6 a), 4.33 (1 H, m,
0
0
00
00
H-6 b), 3.90 (1 H, m, H-4 ), 3.89 (1 H, m, H-2 ), 3.87 (1 H, m, H-5 ), 3.86 (3 H, s,
0
00
00 13
-OCH ), 3.63 (1 H, m, H-3 ). C-NMR (125 MHz, methanol-d ) d: 179.5 (C-4), 167.5
3 4
4
(
(
(
0
0
0
0
0
0
0
C-7 ), 165.8 (C-7), 164.9 (C-4 ), 163.0 (C-5), 158.7 (C-2), 158.3 (C-9), 149.8 (C-4 ), 145.8
0 000 000 0 000 0 0
C-3 ), 135.3 (C-3), 132.3 (C-2 , 6 ), 123.1 (C-6 , 1 ), 122.8 (C-1 ), 117.4 (C-2 ), 116.0
0 000 000 00 00
C-5 ), 114.5 (C-3 , 5 ), 105.5 (C-10), 104.4 (C-1 ), 99.9 (C-6), 94.7 (C-8), 75.0 (C-5 ),
00 00 00 00 000
4.8 (C-3 ), 73.0 (C-2 ), 70.4 (C-4 ), 64.6 (C-6 ), 55.9 (4 -OCH ).
3
7
3.4. Determination of absolute configuration of the sugars
Acid hydrolysis was carried out to obtain the free sugar residues. Compounds 1 and 2
ꢂ
(
3 mg each) were treated with 1.0 M HCl at 85 C for 2 h and the reaction mixture was
partitioned with EtOAc. The EtOAc residue was dissolved in dry pyridine after the

NATURAL PRODUCT RESEARCH
7
ꢂ
addition of L-cysteine methyl ester hydrochloride and heated at 60 C for 2 h.
ꢂ
Trimethylsilylimidazole was added followed by heating at 60 C for 2 h, and then the
mixture was evaporated. The dried product was partitioned with n-hexane and H O.
2
The n-hexane layer was analysed by GC, and the sugars were confirmed to be D-glu-
cose and D-galactose by comparison of the retention times with those of authentic
samples. The absolute configurations of the sugar units were also elucidated by ana-
lyzing the optical rotation values using a polarimeter after the acid hydrolysis. The
hydrolysates of compounds 1 and 2 were detected at t 21.83 (positive optical rota-
R
tion) and 22.74 min (positive optical rotation) indicating D-glucose and D-galactose,
respectively.
3.5. Cell viability assay
5
Cell viability was measured by the MTT assay. RAW 264.7 cells (1 ꢁ 10 cells/well) were
ꢂ
seeded into 96-well plates for 24 h at 37 C. After incubation, the cells were treated
with compounds 1–12 (100 lM) for 24 h, added with 20 lL MTT (0.5 mg/mL) was
added and the mixture was incubated for an additional 3 h. The absorbance was
measured at 540 nm on a micro-plate reader.
3.6. TNF-a production assay
The levels of TNF-a were analysed by the ELISA method using commercially available
5
kits. RAW 264.7 cells (1 ꢁ 10 cells/well) were incubated in 96-well plates and treated
with compounds 1–12 at 10, 30 and 100 lM for 2 h before stimulation with LPS (1 lg/
mL). After that, the supernatants were collected after 24 h stimulation. Dexamethasone
was used as a positive control. The absorbance was examined with a micro-plate
reader at 450 nm.
3.7. Statistical analysis
All data were expressed as the mean ± standard deviation (SD), and analysed using the
GraphPad Prism Software (San Diego, CA, USA); p values < 0.05 were deemed to indi-
cate statistical significance.
4. Conclusion
In summary, two new flavonoids (1–2) and ten known flavonoids (3–12) were isolated
and identified from the leaves of R. dauricum. Among the isolates, five compounds
(1–3, 9, 11) were found in the Ericaceae family for the first time. Compound 11 exhib-
ited TNF-a inhibitory activity with an IC50 value of 46.2 ± 1.2 mM. This study demon-
strated that new flavonoid compounds remain undiscovered in this plant and that
some of these isolates may have potential use in the treatment of inflamma-
tory diseases.

8
C. YE ET AL.
Disclosure statement
No potential conflict of interests was reported by the authors.
Funding
This work was supported by the National Natural Science Foundation of China under Grant
numbers 81660699, 81660579 and 81760627. We thank Renee Mosi, PhD, from Liwen Bianji,
Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of
this manuscript.
References
Alvarez JM, Cuca LE, Carrasco-Pancorbo A, Ruiz-Muelle AB, Fernandez I, Fernandez-Gutierrez A.
2016. Phenolic constituents of leaves from Persea caerulea Ruiz & Pav; Mez (Lauraceae).
Biochem Syst Ecol. 67:53–57.
Badral D, Odonbayar B, Murata T, Munkhjargal T, Tuvshintulga B, Igarashi I, Suganuma K, Inoue
N, Brantner AH, Odontuya G, et al. 2017. Flavonoid and galloyl glycosides isolated from
Saxifraga spinulosa and their antioxidative and inhibitory activities against species that cause
piroplasmosis. J Nat Prod. 80(9):2416–2423.
Bitis L, Kultur S, Melikoglu G, Ozsoy N, Can A. 2010. Flavonoids and antioxidant activity of Rosa
agrestis leaves. Nat Prod Res. 24(6):580–589.
Camacho-Luis A, Gayosso-De-Lucio JA, Torres-Valencia JM, Munoz-Sanchez JL, Alarcon-
Hernandez E, Lopez R, Barrion BL. 2008. Antioxidant constituents of Geranium bellum Rose.
J Mex Chem Soc. 52:103–107.
Cui B, Zhang S, Wang Y, Guo Y. 2019. Farrerol attenuates b-amyloid-induced oxidative stress
and inflammation through Nrf2/Keap1 pathway in a microglia cell line. Biomed Pharmacother.
109:112–119.
De Abreu MB, Temraz A, Malafronte N, Gonzalez-Mujica F, Duque S, Braca A. 2011. Phenolic
derivatives from Ruprechtia polystachya and their inhibitory activities on the glucose-6-phos-
phatase system. Chem Biodivers. 8(11):2126–2134.
Du W, Erden O, Pang Q. 2014. TNF-a signaling in Fanconi anemia. Blood Cells Mol Dis. 52(1):
2–11.
Iwata N, Wang N, Yao X, Kitanaka S. 2004. Structures and histamine release inhibitory effects of
prenylated orcinol derivatives from Rhododendron dauricum. J Nat Prod. 67(7):1106–1109.
Kadota S, Takamori Y, Khin N, Kikuchi T, Tanaka K, Ekimoto H. 1990. Constituents of the leaves
of Woodfordia fruticosa Kurz. I. Isolation, structure, and proton and carbon-13 nuclear mag-
netic resonance signal assignments of woodfruticosin (woodfordin C), an inhibitor of deoxy-
ribonucleic acid topoisomerase II. Chem Pharm Bull. 38(10):2687–2697.
Li G, Wang J, Jin M, Jin C, Ye C, Zhou Y, Wang R, Cui H, Zhou W. 2019. A new pentacyclic triter-
penoid from the leaves of Rhododendron dauricum L. with inhibition of NO production in
LPS-induced RAW 264.7 cells. Nat Prod Res. DOI: 10.1080/14786419.2019.1566822.
Lou XW, Lin QH, Zhang GY, Liu WY, Feng F, Qu W. 2015. Identification and characterization of
three new flavonoids from Rhododendron dauricum. Chin J Nat Med. 13(8):628–633.
Matsuda H, Nishida N, Yoshikawa M. 2002. Antidiabetic principles of natural medicines. V.
Aldose reductase inhibitors from Myrcia multiflora DC. (2): Structures of myrciacitrins III, IV,
and V. Chem Pharm Bull. 50(3):429–431.
Marzouk MS, Soliman FM, Shehata IA, Rabee M, Fawzy GA. 2007. Flavonoids and biological activ-
ities of Jussiaea repens. Nat Prod Res. 21(5):436–443.
Orhan DD. 2003. Novel flavanone glucoside with free radical scavenging properties from Galium
fissurense. Pharm Biol. 41(7):475–478.

NATURAL PRODUCT RESEARCH
9
Popescu R, Kopp B. 2013. The genus Rhododendron: an ethnopharmacological and toxicological
review. J Ethnopharmacol. 147(1):42–62.
Qiang Y, Zhou B, Gao K. 2011. ChemInform abstract: chemical constituents of plants from the
genus Rhododendron. Chem Biodivers. 8(5):792–815.
Seo UM, Nguyen DH, Zhao BT, Min BS, Woo MH. 2017. Flavanonol glucosides from the aerial
parts of Agrimonia pilosa Ledeb. and their acetylcholinesterase inhibitory effects. Carbohydr
Res. 445:75–79.
Shakeel UR, Sofi SN, Khuroo MA, Taneja SC, Bhat KA, Vishwakarma R. 2013. New natural com-
pounds from Rhododendron lepidotum. Nat Prod Res. 27:2033–2038.
Ye G, Huang C. 2006. Flavonoids of Limonium aureum. Chem Nat Compd. 42(2):232–234.
Zhang B, Hou Y, Liu B, Liu Q, Qiao G. 2010. The Rhododendron dauricum L. flavonoids exert vaso-
dilation and myocardial preservation. Iran J Pharm Res. 9(3):303–311.
Zhang N, Huang W, Xia G, Oppong MB, Ding L, Li P, Qiu F. 2018. Methods for determination of
absolute configuration of monosaccharides. Chin Herb Med. 10(1):14–22.
Zong J, Peng Y, Bao G, Hou R, Wan X. 2016. Two oleanane-type saponins with anti-proliferative
activity from Camellia oleifera Abel. seed cake. Molecules 21(2):188.