A new ascorbic acid derivative and two new terpenoids from the leaves and twigs of Rhododendron decorum

Article abstract of DOI:10.1080/10286020.2018.1561669

The phytochemical study of the ethanol extract of the leaves and twigs of Rhododendron decorum afforded a new ascorbic acid derivative (1), a new ionone analogue (2), a new ursane-type triterpenoid glucoside (3), and four known compounds (4?7). The structures were elucidated by spectroscopic analyses, including HRESIMS, 1D, and 2D NMR. The anti-neuroinflammatory activities of the compounds were evaluated by measuring inhibitory effects of LPS-induced NO production in BV2 cells.

Full text of DOI:10.1080/10286020.2018.1561669

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: https://www.tandfonline.com/loi/ganp20
A new ascorbic acid derivative and two new
terpenoids from the leaves and twigs of
Rhododendron decorum
Yu-Xun Zhu, Zhao-Xin Zhang, Huan-Ping Zhang, Li-Sha Chai, Li Li, Shuang-
Gang Ma & Yong Li
To cite this article: Yu-Xun Zhu, Zhao-Xin Zhang, Huan-Ping Zhang, Li-Sha Chai, Li Li, Shuang-
Gang Ma & Yong Li (2019): A new ascorbic acid derivative and two new terpenoids from the
leaves and twigs of Rhododendronꢀdecorum, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2018.1561669
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JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
https://doi.org/10.1080/10286020.2018.1561669
A new ascorbic acid derivative and two new terpenoids
from the leaves and twigs of Rhododendron decorum
Yu-Xun Zhu, Zhao-Xin Zhang, Huan-Ping Zhang, Li-Sha Chai, Li Li,
Shuang-Gang Ma and Yong Li
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing
100050, China
ABSTRACT
ARTICLE HISTORY
The phytochemical study of the ethanol extract of the leaves and
twigs of Rhododendron decorum afforded a new ascorbic acid
derivative (1), a new ionone analogue (2), a new ursane-type tri-
terpenoid glucoside (3), and four known compounds (4ꢀ7). The
structures were elucidated by spectroscopic analyses, including
HRESIMS, 1D, and 2D NMR. The anti-neuroinflammatory activities
of the compounds were evaluated by measuring inhibitory effects
of LPS-induced NO production in BV2 cells.
Received 29 October 2018
Accepted 17 December 2018
KEYWORDS
Rhododendron decorum;
ascorbic acid
derivative; terpenoid
1. Introduction
Rhododendron decorum is an evergreen shrub that distributes mainly in southwestern
China and northeastern Burma. It is used as folk medicine in China to treat rheuma-
tism and pain [1]. The family of Ericaceae is rich in terpenoids, lignans, and flavo-
noids, some of which show valuable anti-inflammatory, antinociceptive, antiviral,
sodium channel antagonistic, and cytotoxic activities [2–3]. To explore the chemical
CONTACT Yong Li
liyong@imm.ac.cn
State Key Laboratory of Bioactive Substance and Function of Natural
Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China
Supplemental data for this article can be accessed at https://doi.org/10.1080/10286020.2018.1561669.
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
Y.-X. ZHU ET AL.
Figure 1. Structures of compounds 1ꢀ5.
diversity and bioactivity, leaves and twigs of R. decorum were collected from Yunnan
Province. In the present investigation, three new compounds along with five known
compounds were isolated (Figure 1). Herein, the isolation and structural elucidation
as well as the anti-neuroinflammatory activities of these compounds are described.
2. Results and discussion
Compound 1 was obtained as white solid. Its molecular formula was determined to
þ
be C H O based on HRESIMS (m/z 377.0844 [M þ Na] , calcd 377.0843), corre-
1
6
18 9
sponding to 8 degrees of unsaturation. The IR spectrum showed typical signals of
hydroxy (3378 cm ) and ester carbonyl (1782 cm ) functionalities. The H NMR
ꢀ
1
ꢀ1
1
data (Table 1) showed resonances of an oxygenated methyl group (d 3.67), two oxy-
H
genated methylenes (dH 4.33, 4.58, 4.48, and 4.28), and three oxygenated methines
1
3
(
dH 4.69, 5.50, and 5.90). The C NMR (Table 2) coupled with DEPT spectra exhib-
ited 16 carbon resonances, including a methyl group, two methylenes, seven methines
3
2
3
2
(
four sp and three sp ), and six quaternary carbons (two sp and four sp ). The
1
1
H- H COSY (Figure 2) and HSQC spectra established three fragments: CH -
2
CH(OH)-CH, CH (OH)-CH-CH, and CH-CH.
2
13
1
The H and C NMR data of 1 were similar to those of amarusine A (4) [4],
which possesses a skeleton composed of a spirocyclic di-tetrahydrofuran fused to a
butyrolactone ring and an aromatic ring. NMR data of 1 indicated the existence of a
3
,4-dioxygenated aromatic ring, which is different from amarusine A (4). This was
confirmed by the HMBC correlations of H-5 (d 7.24) to C-1 (d 131.0) and C-4 (d
C
H
C
0
1
49.0). In the NOESY spectrum (Figure 3), the correlations of H-8/H-4 and H-7/H-8
0
0
0
indicated H-4 , H-7, and H-8 are a-oriented. H-7/H-8 and H-4 /H-5 were deduced to
be in the erythro and threo configuration, respectively, based on the coupling con-
stants of J7,8 ¼ 10.4 Hz and J 0 ¼ 2.0 Hz. In addition, the CD spectrum of 1 was con-
0
4 ,5
sistent with amarusine A (4). Thus, the structure of 1 was elucidated as depicted and
named rhododeonin A.
1
Compound 2 has a molecular formula of C H O based on HRESIMS. The H
20 34 9
NMR data showed resonances of five methyls (d 1.17, 1.33, 1.40, 2.27, and 3.30), six
H
oxygenated methines (d 4.01, 4.10, 4.30, 4.32, 4.50, and 5.07), and two olefinic protons
H

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
1
Table 1. H NMR spectroscopic data of compounds 1 ꢀ 3 in pyridine-d (d in ppm, J in Hz).
5
a
a
b
No
1
1
No
2
No
1
3
–
–
1
2
–
–
2.28 (dd, 12.5, 4.2)
1.27–1.33, (m)
4.30–4.35, (m)
2
7.35 (d, 1.7)
2.21 (dd, 13.8, 4.3)
2
–
2.00 (dd, 13.7, 7.8)
–
3
4
–
–
–
3
4
4.48–4.52, (m)
2.36 (dd, 14.3, 3.4)
2.31 (d, 7.3)
3
4
3.58 (d, 8.7)
–
–
5
6
7.23–7.25, (m)
7.26 (d, 1.9)
–
5
6
–
–
5
6
1.13–1.16, (m)
1.68 (d, 12.1)
1.43–1.45, (m)
–
7
5.90 (d, 10.4)
–
7
6.88 (d, 16.0)
–
7
1.51–1.58, (m)
1.40 (d, 11.8)
8
9
3.24–3.27, (m)
4.58 (dd, 11.6, 6.3)
8
9
7.73 (d, 16.0)
–
–
8
9
–
1.72–1.76, (m)
–
–
4
–
–
–
–
.33 (dd, 11.6, 4.2)
0
1
10
11
2.27, (s)
1.17, (s)
–
1.33, (s)
1.40, (s)
5.07 (d, 7.7)
–
4.10 (t, 8.1)
–
4.28–4.33, (m)
10
11
0
2
1.99–2.06, (m)
1.69–1.75, (m)
0
3
12
13
1
12
13
14
5.45, (brs)
–
–
0
4
5.50 (d, 2.0)
4.69–4.70, (m)
0
0
5
0
0
6
4.48 (dd, 9.5, 4.5)
2
15
16
2.43–2.50, (m)
1.17–1.20, (m)
2.06–2.13, (m)
4.28 (dd, 9.5, 5.3)
0
–
OCH
3
3.67, (s)
3
1
.96–2.01, (m)
0
4
4.28–4.33, (m)
4.00–4.03, (m)
4.59, (dd, 11.6, 1.8)
17
18
19
–
0
5
2.54 (d, 11.3)
1.36–1.51, (m)
0
6
4
.43 (dd, 11.6, 5.2)
–OCH
3
3.30, (s)
20
0.87–0.94, (m)
–
2
2
1
2
1.36–1.39, (m)
1
.25–1.30, (m)
1.92 (brd, 13.1)
.72–1.78, (m)
1
2
2
3
4
1.60, (s)
4.47 (d, 11.0)
3
.72–3.76, (m)
2
2
2
2
2
3
1
2
3
4
5
6
5
6
7
8
9
1.04, (s)
1.16, (s)
1.18, (s)
–
0.95 (d, 6.4)
0.91 (brs)
6.29 (d, 8.1)
4.20–4.25, (m)
4.29–4.33, (m)
4.36–4.40, (m)
4.02–4.08, (m)
4.47–4.49, (m)
0
0
0
0
0
0
0
4
.40–4.42, (m)
a
Recorded at 600 MHz.
Recorded at 500 MHz.
b
1
3
(
dH 6.88 and 7.73). The C NMR and DEPT spectra exhibited 20 carbon resonances,
including five methyls, three methylenes, eight methines, and four quaternary carbons.
The NMR data of 2 were consistent with an ionone-type glycoside and comparable to
those of pisumionoside (5) [5]. The HMBC correlation from H -14 (-OCH , d 3.30)
3
3
H
to C-5 (dC 81.2) indicated that 5-OH is methylated. In the NOESY spectrum, the

4
Y.-X. ZHU ET AL.
1
3
Table 2. C NMR spectroscopic data of compounds 1 ꢀ 3 in pyridine-d (d in ppm).
5
a
a
b
No
1
No
2
No
3
1
2
3
4
5
6
7
8
9
131.0
112.1
149.2
149.0
117.0
121.1
86.4
58.1
58.4
176.1
82.2
118.3
91.1
75.5
75.3
56.3
1
2
3
4
5
6
7
8
9
10
11
12
13
1
2
3
4
5
6
39.6
44.3
74.3
37.7
81.2
82.0
130.7
152.8
198.5
28.0
26.9
29.4
21.0
104.4
76.1
79.5
72.5
79.1
63.6
50.0
1
2
3
4
5
6
7
8
48.5
69.2
86.2
44.5
57.0
19.8
34.4
40.7
48.7
38.7
24.5
126.4
138.9
42.9
29.1
25.1
48.8
53.8
39.8
39.6
31.3
37.3
24.7
66.2
18.0
18.1
24.3
176.7
17.9
21.8
96.2
74.6
79.4
71.7
79.7
62.8
9
0
1
10
11
12
13
14
15
16
17
18
19
20
2
2
2
2
2
2
2
2
2
3
1
2
3
4
5
6
0
2
0
3
0
4
0
0
5
0
0
6
0
-OCH
3
0
0
0
-
OCH
3
1
2
3
4
5
6
7
8
9
0
0
0
0
0
0
0
a
Recorded at 150 MHz.
Recorded at 125 MHz.
b
1
1
Figure 2. H - H COSY and key HMBC correlations of compounds 1ꢀ3.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 3. Conformations and key NOESY correlations of compounds 1ꢀ3.
correlations of H -11/H-3, H -13/H-3, H-2a/H -11, H-2a/H-3, H-8/H -13, and H-7/
3
3
3
3
H -13 indicated that H-3 and H -13 are a-oriented and HO-6 is b-oriented. In add-
3
3
0
ition, the anomeric proton H-1 (d 5.07) was observed with a large coupling constant
H
(7.7 Hz), which indicated the b-configuration of the glucose. Acid hydrolysis and GC
analysis of the sugar moiety of 2 confirmed the sugar is -glucose. The absolute config-
D
uration of 2 was deduced to be the same as pisumionoside (5) due to the approximate
optical value (–4.4 for 2 in our test and –3.4 for pisumionoside in the literature). Thus,
the structure of 2 was elucidated as depicted and named rhododeoside A.
Compound 3 has a molecular formula of C H O based on the HRESIMS. The
H and C NMR data of 3 resembled those of 2a,3a,24-trihydroxyurs-12-en-28-oic
36
58 10
1
13
acid-28-O-b- -glucopyranosyl ester [6], with the exception of the relative configur-
D
ation of C-3. Compound 3 contains a b-OH at C-3 instead of an a-OH at C-3 in the
known compound, which was supported by the downfield chemical shifts of C-3 and
the NOESY correlations of H-3/H-5 and H-3/H -23. The NOESY correlations of H -
3
3
2
5/H -24, H-2/H -25, H -30/H-18 indicated that H-2 is b-oriented and CH -23 and
2 3 3 3
0
CH -30 are a-oriented. In addition, the anomeric proton at H-1 (d 6.29) was
3
H
observed with a large coupling constant (8.1 Hz), which indicated the b-configuration
of the glucose. Acid hydrolysis and GC analysis of the sugar moiety of 3 confirmed
the sugar is -glucose. Thus, the structure of 3 was elucidated as depicted and named
D
rhododeoside B.
Other known compounds isolated from the plant are amarusine A (4) [4], pisu-
mionoside (5) [5], linarionoside A (6) [7], and grasshopper ketone (7) [8]. These
compounds were identified by comparing their NMR data with published literature.
Compounds were assayed for their anti-neuroinflammatory activities by measuring
inhibitory effects of LPS-induced NO production in BV2 cells. The inhibition rates of
compounds 5ꢀ7 (10 lM) are 18.08%, 16.67%, and 17.37%, respectively.
3. Experimental
3.1. General experimental procedures
IR data were measured on a Nicolet 5700 FT-IR spectrometer (Thermo Fisher
Scientific, Waltham, MA). Optical rotations were acquired with a Rudolph automatic
polarimeter (DKSH, Zurich, Switzerland). HRESIMS were carried out on an Agilent

6
Y.-X. ZHU ET AL.
6
520 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent, Polo Alto, CA). NMR
spectra were obtained on INOVA-500 (Varian, Polo Alto, CA) and Bruker AV600-III
Bruker Corporation, Karlsruhe, Germany) spectrometers. A Shimadazu LC-6AD
(
instrument (SPD-20A and RID-10A detectors) was used for preparative HPLC sepa-
rations. D101 type macroporous resin, Baoen Corporation (Cangzhou, China);
Sephadex LH-20, GE Chemical Corporation (Waupaca); silica gel and GF254 TLC
plates, Jiangyou Corporation (Yantai, China); and ODS (50 lm), Merck (Darmstadt,
Germany) were used for column chromatography.
3
.2. Plant material
Twigs and leaves of Rhododendron decorum were collected from Chuxiong, Yunnan
Province in April 2014. The plant was authenticated by associate Prof. Lin Ma of
Peking Union Medical College. A voucher specimen (ID-s-2603) was deposited in the
herbarium of our institute.
3
.3. Extraction and isolation
Twigs and leaves of R. decorum (100 kg) were extracted twice with EtOH at reflux.
The crude extract was then loaded on a Soxhlet extractor and extracted successively
with petroleum ether, CH Cl , EtOAc, and MeOH. The EtOAc extract (1.4 kg) was
2
2
separated on a macroporous resin column eluted sequentially with 70:30, 40:60, and
:95 (v/v) H O-EtOH solutions. Then, a silica gel CC was used to separate the 30%
5
2
EtOH fraction (376 g). The column was eluted with a step gradient of CH Cl /MeOH
2
2
(
(
(
20:1, 10:1, 5:1, and 1:1, v/v) and yielded fractions E G -E G . Fraction E G
30 1 30 6 30 3
20.1 g) was further separated via Sephadex LH-20 column eluted with MeOH ꢀ H O
2
60:40, v/v) and yielded six fractions (E G L ꢀE G L ). Fraction E G L (12.1 g)
3
0
3 1
30 3 6
30 3 1
was separated via ODS column and eluted with a step gradient of MeOH/H O
2
(
10:90, 20:80, 30:70, 50:50, 30:70, and 100:0, v/v) to yield 7 fractions, E G L M to
30 3 1 1
E G L M . Fraction E G L M (2.3 g) was then separated via preparative HPLC
3
0
3 1
7
30 3 1
4
(
MeOH-H O, 24:76, v/v, 5 ml/min) and yielded five fractions, E G L M -1 to
2 30 3 1 4
E G L M -5. Fraction M -2 (103.2 mg) was purified via semi-preparative HPLC
3
0
3 1
4
4
(
MeCN-H O, 9:91, v/v, 3.5 ml/min) to afford 1 (2.2 mg, t ¼ 20.4 min) and 4 (29.6mg,
2
R
t ¼ 24.2 min). Fraction E G L M (1.2 g) was then separated via preparative HPLC
R
30 3 1
6
(MeOH-H O, 24:76, v/v, 5 ml/min) and yielded eight fractions, E G L M -1 to
2 30 3 1 6
E G L M -8. Fraction M -3 (35.2 mg) was purified via semi-preparative HPLC
3
0
3 1
6
6
(
MeCN-H O, 14:86, v/v, 3.5 ml/min) to afford 7 (4.5 mg, t ¼ 24.8 min). Fraction
2
R
E G (37.3 g) was separated via Sephadex LH-20 column and yielded two fractions
3
0 4
(
E G L and E G L ). Fraction E G L (24.1 g) was separated via ODS column to
30 4 1 30 4 2 30 4 1
yield 6 fractions, E G L M to E G L M . Compound 2 (11.6 mg, t ¼ 22.4 min), 5
3
0
4 1
1
30 4 1
6
R
(
7.8mg, t ¼ 20.7 min) and 6 (1.9 mg, t ¼ 32.0 min) were isolated from E G L M
R
R
30 4 1
3
using HPLC with MeCN ꢀ H O (14:86, v/v, 3.5 ml/min). Compound 3 (7.4 mg,
2
t ¼ 37.3 min) was isolated from the 60% EtOH fraction of the macroporous resin
R
column using HPLC with MeCN ꢀ H O (40:60, v/v, 3.5 ml/min).
2

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
3
.3.1. Rhododeonin A
2
0
White powder; [a] D–28.8 (c 0.03, MeOH); IR vmax 3378, 2958, 1782, 1679, 1610,
ꢀ
1
1
1521, 1462, 1390, 1279, 1201, 1131, 1076, 1040, 971, 821, 724 cm ; H NMR
1
3
5 5
(600 MHz, Pyridine-d ) and C NMR (150 MHz, Pyridine-d ) spectral data, see
þ
Tables 1 and 2; HRESIMS m/z: 377.0844 [M þ Na] (calcd for C H O
16
18 9
Na, 377.0843)
3
.3.2. Rhododeoside A
2
0
White powder; [a] D–4.4 (c 0.12, MeOH); IR vmax 3399, 2935, 1671, 1644, 1621,
ꢀ
1
1
1514, 1460, 1424, 1365, 1274, 1078, 1035, 900, 832 cm ; H NMR (600 MHz,
1
3
Pyridine-d ) and C NMR (150 MHz, Pyridine-d ) spectral data, see Tables 1 and 2;
HRESIMS m/z: 441.2115 [M þ Na] (calcd for C H O Na, 441.2095).
5
5
þ
2
0
34
9
3
.3.3. Rhododeoside B
2
0
White powder; [a]
þ58.3 (c 0.14, MeOH); IR v
3383, 2923, 2867, 1730, 1645,
453, 1378, 1306, 1268, 1225, 1071, 1049, 1030, 964, 809 cm ; H NMR (500 MHz,
D
max
ꢀ
1 1
1
1
3
Pyridine-d ) and C NMR (125 MHz, Pyridine-d ) spectral data, see Tables 1 and 2;
5
5
þ
HRESIMS m/z: 673.395 [M þ Na] (calcd for C H O Na, 673.3922)
36
58 10
3
.4. Acid hydrolysis and GC analysis of the sugar moieties of compounds 2
and 3
Compounds 2 and 3 (1.0 mg) were added to 2 N HCl (2 ml). The solution was heated
ꢁ
at 90 C for 12 h. The reaction mixture was evaporated and partitioned with EtOAc
and H O. The aqueous layer was concentrated to furnish the sugar mixture, which
2
was dissolved in dry pyridine and reacted with -cysteine methyl ester hydrochloride
L
ꢁ
(
2 mg) at 80 C for 1 h. After removal of the solvent, N-trimethylsilylimidazole (1 ml)
ꢁ
was added, and the mixture was heated at 80 C for 0.5 h. The mixture was evapo-
rated to dryness, and the residue partitioned into n-hexane and H O. The sample was
2
ꢁ
analyzed on a GC system equipped with an FID (detector temperature, 300 C).
ꢁ
Chromatography conditions: injection temperature, 280 C; column, HP-5
ꢁ
(
60 m ꢂ 0.32 mm ꢂ 0.25 lm); initial column temperature, 200 C; column temperature
ꢁ
ꢁ
ꢁ
increased to 280 C (10 C/min) and kept at 280 C for 40 min under N carrier gas
2
(1.8 ml/min).
In the GC chromatogram, the derivatives of the acid hydrolysate of 2, 3 and the
D-glucose (standard) showed approximate retention times of 29.61, 29.61, and
9.62 min, respectively.
2
3.5. Anti-neuroinflammatory activity assays
Compounds (5ꢀ7) were tested for their anti-neuroinflammatory activity by measur-
ing inhibitory effects of LPS-induced NO production in BV2 cells. Curcumin was
used as the positive control. The BV2 macrophage cell line was obtained from the
Cell Culture Center at the Institute of Basic Medical Sciences, Peking Union Medical
College. LPS was bought from Sigma-Aldrich company. After preincubation for 24 h

8
Y.-X. ZHU ET AL.
ꢀ
5
in a 96-well plate, the cells were treated with the test compounds (10 mol/L), then
stimulated with LPS for 24 h. The production of NO was determined via measuring
the concentration of nitrite in the culture supernatant. NaNO was utilized to gener-
2
ate a standard curve. The absorbances at 550 nm were measured.
Acknowledgment
The authors are grateful to the Department of Instrumental Analysis of our institute for the
UV, IR, NMR and MS spectra measurements.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was financially supported by grants from the National Natural Science Foundation
of China (Nos. 21572274, 21732008, and 81630094) and the CAMS Innovation Fund for
Medical Sciences (No. 2016-I2M-3-012).
References
[1] Chinese Materia Medica Compilation Commission. Chinese Materia Medica (Shanghai
Scientific & Technical Publishers, Shanghai, 1999), pp. 52–60.
[2] Y. Li, Y.B. Liu, J.J. Zhang, Y. Liu, S.G. Ma, J. Qu, H.N. Lv, and S.S. Yu, J. Nat. Prod.
7
8, 2887 (2015).
[
[
3] Y. F. Liu and S. S. Yu, J. Asian Nat. Prod. Res. 19, 1047 (2017).
4] J. Sun, P. C. Zhang, Q. Wei, H. Xun, F. Tang, Y. D. Yue, L. Li, X. F. Guo, and
R. Zhang, Tetrahedron. Lett. 55, 4529 (2014).
[5] T. Murakami, K. Kohno, K. Ninomiya, H. Matsuda, and M. Yoshikawa, Chem. Pharm.
Bull. 49, 1003 (2001).
[
[
[
6] H. A. Jung, H. Y. Chung, J. H. Jung, and J. S. Choi, Chem. Pharm. Bull. 52, 157 (2004).
7] H. Otsuka, Phytochemistry 37, 461 (1994).
8] H. X. Kuang, B. Y. Yang, Y. G. Xia, and W. S. Feng, Arch. Pharm. Res. 31, 1094
(
2008).