Ruticarpsides A–C, three new ester glycosides from the fruits of Tetradium ruticarpum

Article abstract of DOI:10.1080/10286020.2016.1278366

In the course of our ongoing phytochemical investigation on the n-butanol extract of the fruits of Tetradium ruticarpum (Rutaceae), three new compounds, ruticarpsides A–C (1–3), were obtained and their structures were elucidated by a comprehensive analysis of NMR and MS data. Compound 3 showed a weak inhibition effect on nitric oxide production in BV-2 microglial cells stimulated with lipopolysaccharide.

Full text of DOI:10.1080/10286020.2016.1278366

Journal of Asian Natural Products Research
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Ruticarpsides A–C, three new ester glycosides from
the fruits of Tetradium ruticarpum
Wei He, Yong Jiang, Ming-Bo Zhao, Ke-Wu Zeng & Peng-Fei Tu
To cite this article: Wei He, Yong Jiang, Ming-Bo Zhao, Ke-Wu Zeng & Peng-Fei Tu (2017):
Ruticarpsides A–C, three new ester glycosides from the fruits of Tetradium ruticarpum, Journal of
Asian Natural Products Research, DOI: 10.1080/10286020.2016.1278366
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Date: 06 March 2017, At: 04:35

Journal of asian natural Products research, 2017
http://dx.doi.org/10.1080/10286020.2016.1278366
Ruticarpsides A–C, three new ester glycosides from the fruits
of Tetradium ruticarpum
Wei He, Yong Jiang, Ming-Bo Zhao, Ke-Wu Zeng and Peng-Fei Tu
state Key laboratory of natural and Biomimetic drugs, school of Pharmaceutical sciences, Peking university
health science center, Beijing 100191, china
ABSTRACT
ARTICLE HISTORY
received 30 september 2016
accepted 29 december 2016
In the course of our ongoing phytochemical investigation on the n-
butanol extract of the fruits of Tetradium ruticarpum (Rutaceae), three
new compounds, ruticarpsides A–C (1–3), were obtained and their
structures were elucidated by a comprehensive analysis of NMR and
MS data. Compound 3 showed a weak inhibition effect on nitric oxide
production in BV-2 microglial cells stimulated with lipopolysaccharide.
KEYWORDS
Tetradium ruticarpum;
ruticarpsides a–c; no
inhibition
Tetradium ruticarpum
1. Introduction
e dried and nearly ripe fruits of Tetradium ruticarpum (A. Juss.) Hartley (Rutaceae),
known as “Wuzhuyu” in Chinese, are used as a traditional Chinese medicine for the
treatment of headache, abdominal pain, migraines, chill limbs, postpartum hemorrhage,
dysmenorrhea, diarrhea, nausea, and hypertension [1]. Various compounds, including
quinolones and indole alkaloids [2–6], limonins [7,8], flavonoids [9], caffeoylgluconic acids
[10], and essential oil components [11], have been isolated and identified from T. ruticar-
pum. Continued phytochemical research on this important medicinal plant obtained three
CONTACT Yong Jiang and Peng-fei tu
yongjiang@bjmu.edu.cn, pengfeitu@vip.163.com
© 2017 informa uK limited, trading as taylor & francis Group

2
W. HE ET AL.
Figure 1. structures of compounds 1–3.
new ester glycosides, ruticarpsides A–C (1–3) (Figure 1). Herein, the isolation, structure
elucidation, and biological evaluation of these three new compounds are reported.
2. Results and discussion
Ruticarpside A (1) was isolated as a colorless oil. e molecular formula of 1 was determined
as C24H40O10 from the HR-ESI-MS data at m/z 511.2522 [M + Na]+, indicating 5 indices of
hydrogen deficiency. e IR spectrum showed the presence of hydroxy (3385 cm−1), ester
(1715 cm−1), and double bond (1645 cm−1) functionalities. e 1H NMR data (Table 1) of
1 exhibited four C=C double bond proton signals at δH 6.82 (1H, t, J = 7.5 Hz, H-3″), 5.92
(1H, dd, J = 10.8 and 17.4 Hz, H-7″), 5.23 (1H, dd, J = 1.6 and 17.4 Hz, H-8″a), and 5.06
(1H, dd, J = 1.6 and 10.8 Hz, H-8″b); a group of n-butyl signals at δH 0.95 (3H, t, J = 7.4 Hz,
H-8), 1.39 (2H, m, H-7), 1.62 (2H, m, H-6), and 4.09 (2H, m, H-5); four methyl protons
[δH 1.46 (3H, s, H-3), 1.48 (3H, s, H-4), 1.82 (3H, s, H-9″), and 1.28 (3H, s, H-10″)], and
two methylene protons [δH 2.25 (2H, m, H-4″) and 1.62 (2H, m, H-5″)]. e 13C NMR
data (Table 1) exhibited two carbonyl signals [(δC 176.4 (C-1), 169.6 (C-1″)], four olefinic
carbon signals [δC 128.6 (C-2″), 144.6 (C-3″), 146.1 (C-7″), and 112.6 (C-8″)], and two
1
quaternary carbon signals [(δC 79.3 (C-2) and 73.7 (C-6″)]. In addition, the H and 13C
NMR data showed a typical glucosyl moiety in 1 [δH 4.51 (1H, d, J = 7.7 Hz, H-1′), δC 99.8
(C-1′)]. Acid hydrolysis of 1 yielded the sugar part, which was identified as d-glucose by
the arylthiocarbamate derivatives and high-performance liquid chromatography (HPLC)
analysis [12]. e coupling constant of the anomeric proton (J = 7.7 Hz) confirmed a β
configuration of the d-glucose.
e HMBC correlations of the H-3, H-4, and H-5 protons at δH 1.46, 1.48, and 4.09
with C-1 indicated that the n-butyl was connected to the methylpropionic acid moiety
(Figure 2). e 1H-1H COSY correlations of H-3″ (δH 6.82)/H-4″ (δH 2.25)/H-5″ (δH 1.62),
together with the HMBC correlation analyses, suggested the existence of (2E)-linalool-1-
oyl monoterpene moiety in 1 [13]. Acid hydrolysis of 1 yielded the (2E,6S)-2,6-dimethyl-
6-hydroxy-2,7-octadienoic acid by comparison of its specific optical rotation ([α]20 +22.4)
with the natural and synthetic ones [13]. e HMBC correlations of the anomeriDc proton
of glucosyl at δH 4.51 with C-2 indicated that the glucose unit was linked at C-2, and the

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Table 1. the 1h and 13c nMr spectroscopic data of compounds 1–3 in cd3od (δ in ppm, J in hz).
1
2
3
No.
δC
δH
δC
δH
δC
δH
1
2
3
4
5
6
7
8
1′
2′
3′
4′
5′
6′
176.4
79.3
25.3
25.6
66.5
31.8
20.4
14.2
99.8
75.3
78.2
71.9
75.6
65.2
176.3
79.1
24.8
25.9
66.4
31.7
20.3
14.1
99.8
75.3
78.2
72.1
75.7
65.7
176.4
79.2
25.0
25.8
66.5
31.7
20.3
14.2
99.8
75.5
78.2
71.8
75.2
64.9
1.46 (s)a
1.47 (s)
1.47 (s)
4.03 (t, 6.7)
1.47 (s)a
1.49 (s)a
4.03 (td, 2.36.6)
1.57–1.63 (m)
1.34–1.39 (m)
0.90 (t, 7.3)
4.53 (d, 7.7)
3.26–3.29 (m)
3.40 (t, 9.0)
1.48 (s)a
4.08–4.10 (m)
1.61–1.64 (m)
1.37–1.42 (m)
0.95 (t, 7.4)
1.52–1.58 (m)
1.27–1.35 (m)
0.88 (t, 7.4)
4.55 (d, 7.7)
3.26–3.28 (m)
3.41 (t, 9.0)
3.34–3.36 (m)
3.55–3.59 (m)
4.24 (dd, 7.3, 11.8)
4.62 (dd, 2.0, 11.8)
4.51 (d, 7.7)
3.23–3.26 (m)
3.37 (dd, 8.0, 9.0)
3.27–3.29 (m)
3.45–3.48 (m)
4.42 (dd, 11.9, 2.1)
4.12–4.16 (m)
3.34 (d, 9.3)
3.50 (ddd, 2.1, 6.5, 9.1)
4.24 (dd, 6.5, 11.9)
4.46 (dd, 2.1, 11.9)
1″
2″
3″
4″
5″
6″
7″
8″
169.6
128.6
144.6
24.7
41.9
73.7
121.3
108.6
149.1
142.4
149.1
108.6
127.8
111.9
149.5
150.8
116.7
124.2
147.2
115.4
7.37 (s)
7.19 (d, 2.0)
6.82 (t,7.5)
2.18–2.32 (m)
1.61–1.64 (m)
6.82 (d, 8.2)
7.37 (s)
7.08 (dd, 2.0, 8.2)
7.65 (d, 15.9)
6.35 (d, 15.9)
146.1
112.6
5.92 (dd, 17.4, 10.8) 168.1
5.23 (dd, 17.4, 1.6)
5.06 (dd, 10.8, 1.6)
1.82 (s)
9″
10″
–och3
12.5
28.0
169.0
56.6
1.28 (s)
57.1
3.90 (s)
3.89 (s)
athe signals could be interchangeable.
Figure 2. Key hMBc and 1h-1h cosY correlations of compounds 1–3.
HMBC correlations of the H-6′ protons at δH 4.42 and 4.13 with C-1″ indicated that the
monoterpene unit was linked to the C-6′ of glucose. erefore, the structure of ruticarpside
A (1) was elucidated as shown.
Ruticarpside B (2) was isolated as a brown oil. Its molecular formula was established as
C23H34O12 from the HR-ESI-MS data at m/z 525.1955 [M + Na]+. Comparative analyses

4
W. HE ET AL.
of the 1H and 13C NMR data (Table 1) of 2 with those of 1 indicated that the (2E,6S)-2,6-
dimethyl-6-hydroxy-2,7-octadienoyl group in 1 was replaced by a 4-hydroxy-3,5-dimeth-
oxybenzoyl group in 2 [δH 7.37 (2H, s, H-2″, 6″), 3.90 (6H, s, 3″, 5″-OCH3)]. e relative
configuration of the glucose was determined as β according to the large J value of the ano-
meric proton [δH 4.55 (1H, d, J = 7.7 Hz, H-1′)]. e absolute configuration of the glucose
was determined as D configuration according to a procedure described previously [12]. e
HMBC correlation of H-6′ (δH 4.24, 4.62) with C-7″ (δC 168.1), H-1′ (δH 4.55) with C-2 (δC
79.1), and H-5 (δH 4.03) with C-1 (δC 176.3) further supported the above elucidation. us,
the structure of ruticarpside B (2) was assigned as shown.
Ruticarpside C (3) was isolated as a brown oil. Its molecular formula was C24H34O11,
established by the HR-ESI-MS data at m/z 521.2000 [M + Na]+. Comparative analyses of the
1H and 13C NMR data (Table 1) with those of 2 indicated that 3 has a feruloyl group, instead
of the 4-hydroxy-3,5- dimethoxybenzoyl group in 2 [δH 7.19 (1H, d, J = 2.0 Hz, H-2″), 6.82
(1H, d, J = 8.2 Hz, H-5″), 7.08 (1H, dd, J = 8.2, 2.0 Hz, H-6″), 7.65 (1H, d, J = 15.9 Hz, H-7″),
6.35 (1H, d, J = 15.9 Hz, H-8″), and 3.89 (3H, s)]. e feruloyl was deduced to be attached
to C-6′ by HMBC correlation of H-6′ (δH 4.24, 4.46) with C-9″ (δC 169.0). erefore, the
structure of ruticarpside C (3) was determined as shown.
Compounds 1–3 were evaluated for their inhibitory effects on NO production in LPS-
stimulated BV-2 microglial cells using the Griess assay [14,15]. Only weak inhibition effect
was observed for compound 3, with an IC50 value of 81.0 μM.
All of the compounds 1–3 have n-butyl group. Concerning they were isolated from
the n-butanol extract, they might be the artifacts during the extraction. To assess whether
these three compounds are natural compounds or artifacts produced during the extraction
process, a rapid extraction with water was performed, and the extract was immediately
examined by LC-MS. e resulting data unambiguously demonstrated that none of the three
compounds were detected in the rapid extraction sample, thus, they could be the artifacts.
3. Experimental
3.1. General experimental procedures
UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Kyoto,
Japan). IR spectra were recorded on a ermo Nicolet Nexus 470 FT-IR spectrometer
(ermo Fisher, Madison, WI, USA). NMR spectra were recorded on a Varian INOVA-500
NMR spectrometer (Varian, Palo Alto, CA, USA), using CD3OD as the solvent, and the
chemical shifs were referenced to the solvent residual peak. HRMS data were acquired on
a Bruker APEX II FT-ICR mass spectrometer (Bruker, Fllanden, Switzerland). Optical rota-
tions were recorded on a Rudolph RS Autopol VI automatic polarimeter (Rudolph Research
Analytical, Flanders, NJ, USA). Silica gel (200–300 mesh, Qingdao Marine Chemical, Co.
Ltd., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden)
were used for open column chromatography (CC). Semipreparative RP-HPLC was per-
formed on an Agilent 1260 system (Agilent Technologies, Santa Clara, USA) with a Zorbax
Eclipse XDB-C18 (9.4 × 250 mm, 5 μM) column.e analytical high-performance liquid
chromatography (HPLC) was performed on an Agilent 1260 system, using an ODS col-
umn (Agilent Zorbax SB-Aq, 4.6 × 250 mm, 5 μM), and the detected wavelength was set at
250 nm. TLC analyses were carried out on the pre-coated silica gel GF254 plates (Qingdao

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Marine Chemical Co. Ltd., Qingdao, China). Spots were visualized either under UV light
(254 and 365 nm) or by spraying with 2% vanillin-H2SO4 solution and heating. All of the
solvents were of analytical grade.
3.2. Plant material
e fruits of Tetradium ruticarpum were purchased from Anguo traditional Chinese medi-
cine market (Hebei Province, China), in October 2010. e plant material was identified by
one of the authors (P.-F. Tu). e voucher specimen (No. MT20101010) has been deposited
at the Herbarium of the Peking University Modern Research Center for Traditional Chinese
Medicine.
3.3. Extraction and isolation
e fruits of T. ruticarpum (10.0 kg) were extracted with 90% aqueous ethanol. Afer
removal of the ethanol by vacuum, the residue (800 g) was suspended in H2O and parti-
tioned with petroleum ether, CHCl3, and n-butanol. e n-butanol-soluble portion (300 g)
was subjected to silica gel CC, eluting with CHCl3–acetone (100:1–1:1 gradient system)
to afford 16 fractions (labeled A–P). Fraction C (5 g) was separated on an ODS column
using MeOH–water (1:9–1:0, v/v) as the mobile phase to give 18 subfractions (C1–C18).
Subfraction C15 (35.4 mg) was purified by semi-preparative reversed phase (RP)-HPLC
using a mobile phase of MeCN–H2O (45:55, v/v) to yield compound 1 (15.2 mg, tR 19.3 min).
Subfraction C13 (54.2 mg) was then subjected to a Sephadex LH-20 column using MeOH
as eluent to afford four fractions (Frs. C13a–C13d). Fr. C13b (14.5 mg) was further purified
by semi-preparative RP-HPLC using a mobile phase of MeCN–H2O (50:50, v/v) to yield
compound 2 (1.3 mg, tR 14.8 min). Subfraction C14 (73.7 mg) was separated on a Sephadex
LH-20 column using MeOH as the mobile phase to obtain 3 subfractions (Frs. C14a–C14c).
Compound 3 (34.7 mg, tR 13.7 min) was purified from the C14b (55.2 mg) subfraction by
semi-preparative RP-HPLC using a mobile phase of MeCN–H2O (40:60, v/v).
3.3.1. Ruticarpside A (1)
Colorless oil;[α]2D0 −0.45 (c 0.4, MeOH); UV (MeOH) λmax (log ε): 219 (1.40) nm. IR (KBr)
ν
max: 3385, 2961, 2934, 2872, 1715, 1645, 1462, 1387, 1282, 1164, 1066, and 922 cm−1; 1H
and 13C NMR spectroscopic data, see Table 1. Positive HR-ESI-MS: m/z 511.2522 [M + Na]+
(calcd for C24H40O10Na, 511.2519).
3.3.2. Ruticarpside B (2)
Brown oil; [α]2D0 −3.08 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 217 (0.90) and 276 (0.41)
nm. IR (KBr) νmax: 3363, 2940, 2870, 1716, 1612, 1515, 1462, 1425, 1337, 1283, 1226, 1055,
1
1033, 1014.0, and 766 cm−1; H and 13C NMR spectroscopic data, see Table 1. Positive
HR-ESI-MS: m/z 525.1955 [M + Na]+ (calcd for C23H34O12Na, 525.1948).
3.3.3. Ruticarpside C (3)
Brown oil; [α]2D0 −16.53 (c 0.5, MeOH); UV (MeOH) λmax (log ε): 204 (0.84), 214 (0.73),
234 (0.59), and 325 (0.86) nm. IR (KBr) νmax: 3393, 2960, 2872, 1723, 1631, 1597, 1516,
1462, 1431, 1372, 1279, 1166, 1127, 1056, 1034, 935, and 818 cm−1; for 1H and 13C NMR

6
W. HE ET AL.
spectroscopic data, see Table 1. Positive HR-ESI-MS: m/z 521.2000 [M + Na]+ (calcd for
C24H34O11Na, 521.1999).
3.4. Acid hydrolysis of compounds 1–3
d-Glucose (2.0 mg) and l-glucose (2.0 mg) were respectively heated with l-cycteine methyl
ester (2.5 mg) in pyridine (1 ml) at 60 °C for 60 min and then o-tolyl isothiocyanate (6 μl)
was added to the mixtures and heated further for 60 min [12]. e reaction mixtures were
analyzed by RP-HPLC and detected at 250 nm. Compounds 1–3 (4 mg, 1 mg, 4 mg, respec-
tively) were hydrolyzed by heating in 2 M CF3COOH (3 ml) for 12 h at 100 °C. Afer drying
under reduced pressure, the mixtures were extracted three times with isopyknic water-sat-
urated EtOAc. e EtOAc layer was dried under reduced pressure and dissolved in MeOH
for the optical rotation determination. Afer drying the H2O layer, the residual mixtures
were heated with l-cycteine methyl ester in pyridine at 60 °C for 60 min, and then o-tolyl
isothiocyanate (6 μl) was added to the mixtures and heated further for 60 min. e reaction
mixtures were analyzed by RP-HPLC and detected at 250 nm. e flow rate was 0.8 ml/min,
and the retention times (tR) of the glucose derivatives were as follows: d-glucose derivative
(tR 15.40 min) and l-glucose derivative (tR 14.20 min). By comparing the retention time
(compound 1: tR 15.38 min; compound 2: tR 15.41 min; compound 3: tR 15.38 min) with
the standard glucose derivative. e absolute configurations of the glucopyranosyl unit in
compounds 1–3 were determined as D.
3.5. NO inhibitory assay
BV-2 microglial cells were purchased from Peking Union Medical College (PUMC) Cell
Bank (Beijing, China). Cell maintenance, experimental procedures, and data determina-
tion for the inhibition of NO production and the viability assay are the same as previously
described [14,15]. Quercetin was used as a positive control (IC50 = 6.62 μM).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
is work was financially supported by National Key Technology R&D Program “New Drug
Innovation” of China [grant number 2012ZX09301002-002-002], [grant number 2012ZX09304-005].
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