Terpenoid and phenolic constituents from the roots of Ilex pubescens

Article abstract of DOI:10.1016/j.fitote.2019.104298

Five new metabolites, including two monoterpene glycosides Pubescenosides L–M (1–2) and three phenolic glycosides, Pubescenosides N-P (3–5), along with nineteen known ones, including liganoids, hemiterpenoids and caffeoylquinic acid derivates, were isolated from the roots of Ilex pubescens. Their structures were elucidated from extensive spectroscopic analysis, including 1D and 2D NMR experiments. This study is the first to report monoterpene glycosides with β-pinene aglycone in Aquifoliaceae. Nine of these compounds were evaluated in vitro for their anti-platelet aggregation activities. Among them, compounds 3 and 4 showed moderate inhibitory activities on ADP-induced blood platelet aggregation [inhibition (%): 32.3 and 33.6, respectively] as compared to aspirin.

Full text of DOI:10.1016/j.fitote.2019.104298

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Terpenoid and phenolic constituents from the roots of Ilex pubescens
⁎
Xu-Dong Zhoua, Xiang-Wei Xuc, Yi-Yuan Xic, Yuan Zhoub,
a TCM and Ethnomedicine Innovation & Development Laboratory, School of Pharmacy, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China
b Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji School of Pharmacy, Huazhong University of Science and Technology, 13
Hangkong Road, Wuhan 430030, China
c Pharmacy Department, Yongkang First People's Hospital, Yongkang, Jinhua, Zhejiang 321300, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Five new metabolites, including two monoterpene glycosides Pubescenosides L–M (1–2) and three phenolic
glycosides, Pubescenosides N-P (3–5), along with nineteen known ones, including liganoids, hemiterpenoids and
caffeoylquinic acid derivates, were isolated from the roots of Ilex pubescens. Their structures were elucidated
from extensive spectroscopic analysis, including 1D and 2D NMR experiments. This study is the first to report
monoterpene glycosides with β-pinene aglycone in Aquifoliaceae. Nine of these compounds were evaluated in
vitro for their anti-platelet aggregation activities. Among them, compounds 3 and 4 showed moderate inhibitory
activities on ADP-induced blood platelet aggregation [inhibition (%): 32.3 and 33.6, respectively] as compared
to aspirin.
Aquifoliaceae
Ilex pubescens
Pubescenosides L–P
β-Pinene glycoside
Phenolic glycoside
Anti-platelet aggregation
1. Introduction
paper describes the isolation and structural elucidation of these com-
pounds. In addition, ADP-induced blood platelet aggregation inhibitory
Ilex pubescens Hook. et Arn., known under the Chinese name as
‘Mao-dong-qing’, is an evergreen shrub belonging to Aquifoliaceae. The
roots of I. pubescens are commonly used in Chinese herbal medicine for
the treatment of cardiovascular disease in south China [1]. Upon un-
dergoing pharmacological evaluation, roots extracts of I. pubescens ex-
hibited a variety of bioactivities and were applied in treatment of
several human diseases including the abilities to dilate blood vessels,
improve microcirculation, reduce blood pressure, inhibit platelet ag-
gregation, prevent thrombosis, and decrease cardiac ischemia. The ex-
tracts can also act as a radiosensitizer, making tumor cells more sus-
ceptible to radiotherapy during cancer treatment [2]. Previous
phytochemical investigations into I. pubescens roots and leaves led to
the isolation of triterpene saponins [3–12], lignan glycosides [13],
phenylethanols [14,15], and other minor compounds [16–18], which
may account for its biological activities.
activities were evaluated in nine of these compounds.
2. Experimental section
2.1. General experimental procedures
HRESIMS were measured on a Bruker APEX IV FT_MS (7.0 T)
spectrometer. Optical rotations were measured on an Autopol-IV po-
larimeter (RudoPH Research analytical). IR spectra were obtained using
a NEXUS-470 FTIR (Nicolet) spectrometer. 1D and 2D NMR spectra
were recorded on Vnmrs-500 spectrometers. Semi-preparative HPLC
was performed on
a Waters model 2487 (Agilent ODS column,
250 × 9.4 mm i.d., 5 μm) with an Alltech evaporative light scattering
detector (ELSD). GC analysis was carried out on an Agilent 6890 N gas
chromatograph, with an HP-5 capillary column (28 m × 0.32 mm) and
an FID detector operated at 260 °C (column temp. 180 °C), 1.0 mL/min
N2 as carrier gas. Macroporous resin (HPD100) were purchased from
Hebei Bao-En Biotech Ltd.. Thin-layer and column chromatography was
performed using silica gel (Qingdao Haiyang Chemical Co. Ltd., GF254
or 200–300 mesh) and Sephadex LH-20 (Pharmacia Biotech Ltd.) and
ODS (Merck & Co., Inc. USA). All the solvents were of analytical grade
and were purchased from Beijing Chemical Company Ltd..
A phytochemical investigation carried out on the roots of I. pub-
escens was undertaken in previous studies, which led to the determi-
nation of the absolute structure of ilexpublesnins C–R [19,20]. As an
ongoing program toward the discovery of novel bioactive constituents
in I. pubescens, two new monoterpeniod glycosides and three phenolic
glycosides, namely pubescenoside L–P (1–5) (Fig. 1), and nineteen
known ones (6–24) (see Supplementary materials) were isolated. This
⁎ Corresponding author.
E-mail address: zhouyuan@hust.edu.cn (Y. Zhou).
https://doi.org/10.1016/j.fitote.2019.104298
Received 17 May 2019; Received in revised form 6 August 2019; Accepted 6 August 2019
Availableonline07August2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

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X.-D. Zhou, et al.
Fitoterapia138(2019)104298
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Fig. 1. Structures of compounds 1–5.
2.2. Plant material
57:43, 2.0 mL/min) yield compounds 12 (9 mg) and 17 (8 mg). Fr.J-3
(2.8 g) was chromatographed on Sephadex LH-20 (50 × 3 cm) in MeOH
afforded three fractions (Fr.J-3-1–Fr.J-3-3). Fr.J-3-3 (1.7 g) was sub-
jected to RP-C18 CC (40 × 3 cm) eluted with MeOH-H2O (70:30) and
further purified by semi-preparative HPLC (MeOH/H2O 41:59, 2.0 mL/
min) yield compounds 6 (15 mg) and 14 (160 mg). Fr.K (14 g) was
subjected to CC on silica gel (400 g, 50 × 4 cm) eluted with CHCl3-
MeOH-H2O (60:30:10, low layer) yield six fractions (Fr.K-1–Fr.K-6).
Fr.K-3 (2.5 g) was chromatographed on Sephadex LH-20 (50 × 3 cm) in
MeOH afforded two fractions (Fr.K-3-1–Fr.K-3-2). Fr.K-3-1 (510 mg)
was separated by semi-preparative HPLC (MeOH/H2O 35:65, 2.0 mL/
min) yield compounds 23 (220 mg) and 24 (20 mg). Fr.K-4 (2.5 g) was
chromatographed on Sephadex LH-20 (50 × 3 cm) in MeOH yield three
fractions (Fr.K-4-1–Fr.K-4-3). Fr.K-4-1 (1.1 g) was subjected to RP-C18
CC (40 × 3 cm) eluted with MeOH-H2O (70:30) and further purified by
semi-preparative HPLC (MeOH/H2O 38:62, 2.0 mL/min) yield com-
pounds 3 (23 mg) and 15 (250 mg). Fr.K-4-3 (510 mg) was separated by
semi-preparative HPLC (MeOH/H2O 60:40, 2.0 mL/min) yield com-
pounds 2 (5 mg), 11 (10 mg) and 7 (50 mg). Fr.L (19 g) was chroma-
tographed on RP-C18 CC (50 × 4 cm) using a gradient of MeOH-H2O
(20:80, 40:60, 60:40, 80:20) afforded five fractions (Fr.L-1–Fr.L-5).
Fr.L-3 (2 g) was subjected to CC on Sephadex LH-20 (50 × 3 cm) in
MeOH-H2O (70:30) afforded three fractions (Fr.L-3-1–Fr.L-3-3).
Compounds 1 (8 mg) and 10 (28 mg) were obtained from Fr.L-3-1
(850 mg) by semi-preparative HPLC (MeOH/H2O 30:70). Fr.M (9 g) was
chromatographed on Sephadex LH-20 (50 × 3 cm) in MeOH afforded
three fractions (Fr.M-1–Fr.M-4). Fr.M-3 (2.1 g) was chromatographed
on RP-C18 CC (50 × 4 cm) using a gradient of MeOH-H2O (20:80,
40:60, 60:40) afforded four fractions (Fr.M-3-1–Fr.M-3-4). Fr.M-3-3
(260 mg) was separated by semi-preparative HPLC (MeOH/H2O 55:45,
2.0 mL/min) yield compounds 19 (24 mg) and 20 (22 mg).
The roots of Ilex pubescens were purchased from Guilin San-Jin
Pharmaceutical Co. Ltd., and were originally collected from Guangxi
province, China. The plants were identified by Prof. Peng-Fei Tu. A
voucher specimen (No. 091005) is deposited in the Herbarium of
Modern Research Center for Traditional Chinese Medicine (TCM),
Peking University, Beijing.
2.3. Extraction and isolation
The dried roots of I. pubescens (18 kg) were grinded and extracted
with 70% EtOH at the temperature of 70 °C. After the retrieval of the
ethanol, the residue suspended in water (50 L) was subjected to column
chromatograph (CC) on macroporous resin with an EtOH-H2O gradient
(30:70, 70:30, 90:10) to yield three fractions (Frs.1–3). Fr.1 (50 g) was
chromatographed on silica gel (1.0 kg, 40 × 10 cm) with a gradient of
CHCl3-MeOH (10:1–1:2) elution to yield three fractions (Frs. A−C).
Then these fractions were applied to CC on silica gel, ODS, and semi-
preparative HPLC to afford compounds 5 (10 mg), 6 (14 mg), 7 (25 mg),
8 (24 mg), 9 (22 mg), 22 (15 mg). Fr.2 (160 g) was chromatographed on
silica gel (2.5 kg, 100 × 10 cm) with a gradient of CHCl3-MeOH
(40:1–1:1) elution to yield thirteen fractions (Frs. A−M). Fr.G (25 g)
was subjected to CC on silica gel (500 g, 50 × 4 cm) eluted with CHCl3-
MeOH (10:1, 5:1, 2:1) yield seven fractions (Fr.G-1–Fr.G-7). Fr.G-5
(2.2 g) was chromatographed on Sephadex LH-20 (50 × 3 cm) in MeOH
afforded three fractions (Fr.G-5-1–Fr.G-5-3). Fr.G-5-3 (780 mg) was
separated by semi-preparative HPLC (MeOH/H2O 52:48, 2.0 mL/min)
yield compounds 4 (6 mg) and 13 (120 mg). Fr·I (3.6 g) was subjected to
CC on silica gel (300 g, 50 × 3 cm) eluted with CHCl3-MeOH (5:1, 2:1)
yield two fractions (Fr.I-1–Fr.I-2). Fr.I-1 (2.5 g) was chromatographed
on Sephadex LH-20 (50 × 3 cm) in MeOH afforded three fractions (Fr.I-
1-1–Fr.I-1-3). Fr.I-1-2 (1.4 g) was subjected to RP-C18 CC (40 × 3 cm)
eluted with MeOH-H2O (70:30) and further purified by semi-pre-
parative HPLC (MeOH/H2O 56:44, 2.0 mL/min) yield compounds 18
(25 mg) and 21 (500 mg). Fr.I-2 (400 mg) was chromatographed on
Sephadex LH-20 (100 × 2 cm) in MeOH afforded three fractions (Fr.I-2-
1–Fr.I-2-3). Fr.I-2-2 (130 mg) was separated by semi-preparative HPLC
(MeOH/H2O 36:64, 2.0 mL/min) yield compound 16 (10 mg). Fr.J
(18 g) was subjected to CC on silica gel (400 g, 50 × 4 cm) eluted with
CHCl3-MeOH-H2O (60:30:10, low layer) yield three fractions (Fr.J-
1–Fr.J-3). Fr.J-2 (3.1 g) was chromatographed on Sephadex LH-20
(50 × 3 cm) in MeOH afforded three fractions (Fr.J-2-1–Fr.J-2-3). Fr.J-
2-2 (120 mg) was separated by semi-preparative HPLC (MeOH/H2O
2.3.1. Data for pubescenoside L (1)
20
White, amorphous powder; [α]D – 2.60 (c 0.14, MeOH); IR (KBr)
νmax 3410, 2924, 1646, 1076 cm−1; 1H and 13C NMR data (pyridine-d5,
500/125 MHz), see Table 1; HRESIMS m/z 464.24856 [M + NH4]+
(calcd. for C21H34O10NH4, 464.24902).
2.3.2. Data for pubescenoside M (2)
20
White, amorphous powder; [α]D – 2.91 (c 0.13, MeOH); IR (KBr)
νmax 3424, 2923, 1695, 1076 cm−1; 1H and 13C NMR data (pyridine-d5,
500/125 MHz), see Table 1; HRESIMS m/z 464.24854 [M + NH4]+
(calcd. for C21H34O10NH4, 464.24902).
2

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X.-D. Zhou, et al.
Fitoterapia138(2019)104298
Table 1
of the methyl sugar peracetates. The aqueous layer was evaporated and
dissolved in anhydrous pyridine (100 μL), 0.1 mL cysteine methyl ester
hydrochloride (200 μL) was added, and the mixture was heated at 60 °C
for 1 h. The trimethysilylation reagent HMDS-TMCS (hexamethyldisi-
lazane-trimethylchlorosilanepyridine, 2:1:10) (Acros Organics, Geel
Belgium) was added and heated at 60 °C for 30 min. The thiazolidine
derivatives were analyzed by GC for sugar identification. The retention
times of L-arabinose (tR, 5.21 min), D-xylose (tR, 5.49 min) and D-glu-
cose (tR, 11.72 min) were confirmed by comparison with those of au-
thentic standards (Sigma, purities: ≥99%) [21].
1H NMR (500 MHz) and 13C NMR (125 MHz) data of 1–2 in pyridine-d5, (δ in
ppm, J in Hz).
Position
1
2
δH (J in Hz)
δC
δH (J in Hz)
δC
1
2
152.0
114.0
152.0
114.0
5.00, br s
5.00, br s
5.54, br s
5.56, br s
3
4
4.89, d (7.5)
2.15, m
72.2
32.5
4.92, d (7.5)
2.13, m
72.2
32.5
2.5. Anti-platelet aggregation induced by ADP
2.27, dd (14.0, 4.0)
1.84, m
2.27, dd (14.0, 4.0)
1.82, m
5
6
7
8
40.2
40.6
51.0
27.9
40.3
40.6
51.0
27.9
The blood platelet aggregation test was performed according to a
previously reported method [22]. Briefly, whole blood samples were
collected from the carotid artery of urethane-anesthetized rabbits. The
blood (8 mL) along with sodium citrate (3.8%, 1 mL) were transferred
into a plastic tube, and centrifuged at 800 r.p.m. for 15 min to obtain
platelet-rich plasma (PRP). The platelet concentration was adjusted to a
level of 2.5 × 108/μL by addition of homologous platelet-poor plasma
(PPP) obtained after further centrifugation of blood at 3000 r.p.m. for
15 min. In order to eliminate the effect of the solvent on the aggrega-
tion, the final concentration of DMSO was fixed at 0.5%. The light
transmission was set at 0% with PRP and at 100% with PPP. After
vehicle (0.5% DMSO, Model group) or different concentration of in-
dividual compounds (15 μL) along with PRP (275 μL) were incubated in
turbidimetric cups for 5 min at 37 °C, 10 μL ADP (adenosine dipho-
sphate) was added to make a final concentration of 10 μM. Changes in
the light transmittance of the reaction mixture were continuously re-
corded for 5 min and maximal aggregation was recorded by a LG-
PABER-I Platelet-Aggregometer (Beijing GTM-Steellex Instrument Co.
Ltd., China). Aspirin (No. 100113-201104, National institutes for Food
and Drug Control, China, purity: 99.7%) was used as positive control.
The anti-platelet aggregation activity of tested compound was eval-
uated as inhibition rate (%) which was determined by using the fol-
lowing formula:
2.46, t (5.5)
1.85, m
2.45, t (5.5)
1.85, m
2.20, m
2.20, m
9
1.11, s
25.9
72.2
Glc
1.11, s
25.9
22.2
Glc
10
0.62, s
0.61, s
Sugar
Glc
Glc
1
2
3
4
5
6
4.92, d (8.0)
3.94, d (8.0)
4.20, t (8.5)
4.16, dd (11.0, 3.0)
4.08, m
102.2
74.9
78.6
71.9
77.1
69.6
4.94, d (8.0)
3.94, d (8.0)
4.21, t (8.5)
4.13, dd (11.0, 2.0)
4.08, m
102.2
74.9
78.6
71.7
78.2
69.9
4.32, dd (11.5, 5.5)
4.79, dd (11.0, 3.0)
Ara
4.32, dd (11.5, 5.5)
4.79, dd (11.5, 2.0)
Xyl
Ara
Xyl
1
2
3
4
5
4.99, d (6.5)
4.45, dd (8.5, 6.5)
4.13, t (7.5)
4.30, m
105.4
72.1
74.4
69.1
66.5
5.06, d (7.5)
4.02, t (7.5)
4.12, m
105.9
74.9
77.3
71.1
67.1
4.19, m
3.73, dd (13.0, 3.0)
4.28, dd (13.5, 3.0)
3.67, t (11.0)
4.31, dd (11.5, 5.5)
2.3.3. Data for pubescenoside N (3)
20
Colorless gelatinous solid; [α]D – 30.0 (c 0.12, MeOH); IR (KBr)
νmax 3515, 1701, 1598, 1516, 1463, 1124 cm−1; UV (MeOH) λmax: 220,
275 nm; 1H and 13C NMR data (DMSO-d6, 500/125 MHz), see Table 2;
HRESIMS m/z 1190.41666 [2 M + NH4]+ (calcd. for C52H68O30NH4,
1190.41336).
Inhibition rate (%) = (1 − the maximal aggregation percent of
compound/the maximal aggregation percent of model group) × 100.
3. Results and discussion
2.3.4. Data for pubescenoside O (4)
20
Colorless gelatinous solid; [α]D – 45.7 (c 0.14, MeOH); IR (KBr)
Dry crude materials (18 kg) were grinded and extracted with 70%
EtOH at a temperature of 70 °C. After retrieval of ethanol, the residue
suspended in water (50 L) was subjected to column chromatograph
(CC) on macroporous resin with an EtOH-H2O gradient (30:70, 70:30,
90:10) to yield three fractions (Frs.1–3). Fr.1 (50 g) was chromato-
graphed on silica gel (1.0 kg, 40 × 10 cm) with a gradient of CHCl3-
MeOH (10:1–1:2) elution to yield three fractions (Frs. A−C). These
fractions were then applied to CC on silica gel, ODS, and semi-pre-
parative HPLC to afford pubescenoside P (5,10 mg), (7R, 8R)-syr-
ingylglycerol 9-O-β-D-glucopyranoside (6, 14 mg) [23], (7S, 8S)-syr-
ingylglycerol 9-O-β-D-glucopyranoside (7, 25 mg) [24], (7R, 8R)-
syringylglycerol (8, 24 mg) [24], (7S, 8S)-syringylglycerol (9, 22 mg)
[24] and kelampayoside A (22, 15 mg) [25]. Fr.2 (160 g) was chro-
matographed on silica gel (2.5 kg, 100 × 10 cm) with a gradient of
CHCl3-MeOH (40:1–1:1) elution to yield thirteen fractions (Frs. A−M).
Then Frs. D, E, G, I, L were applied to CC on silica gel, Sephadex LH-20,
ODS, and semi-preparative HPLC to afford pubescenoside L (1, 8 mg),
pubescenoside M (2, 5 mg), pubescenoside N (3, 6 mg), pubescenoside
O (4, 6 mg), syringin (10, 28 mg) [26], dihydrosyringin (11, 10 mg)
[27], tortoside A (12, 800 mg) [28], liriodendrin (13, 120 mg) [29],
(+)-syringaresinol (14, 160 mg) [30], (+)-fraxiresinol-1-O-β-D-gluco-
pyranoside (15, 250 mg) [31], eleutheroside E (16, 10 mg) [32], 4, 5-O-
diocaffeoylquinic acid (17, 8 mg) [33], 3, 5-O-diocaffeoylquinic acid
(18, 25 mg) [34], 3,4, 5-trimethyloxyphenol-O-β-D-glucopyranoside
(19, 24 mg) [34], kelamapayoside B (20, 22 mg) [24], koaburaside (21,
νmax 3449, 1706, 1599, 1514, 1463, 1124 cm−1; UV (MeOH) λmax: 220,
275 nm; 1H and 13C NMR data (DMSO-d6, 500/125 MHz), see Table 2;
HRESIMS m/z 1190.41223 [2 M + NH4]+ (calcd. for C52H68O30NH4,
1190.41336).
2.3.5. Data for pubescenoside P (5)
20
Colorless gelatinous solid; [α]D – 7.6 (c 0.10, MeOH); IR (KBr)
νmax 3434, 1660, 1055, 1028, 1008 cm−1; UV (MeOH) λmax: 255,
295 nm; 1H and 13C NMR data (DMSO-d5, 500/125 MHz), see Table 2;
HRESIMS m/z 391.07145 [M
–
H]− (calcd. for C15H19O11S,
391.06989).
2.4. Acid hydrolysis
Compounds 1–5 (2 mg) were heated in 3 mL of 10% HCl-dioxane
(1:1) at 90 °C for 4 h in a sealed amp. After the dioxane was removed,
the solution was extracted with EtOAc (3 mL × 3) to yield aglycon and
sugar. The sugar components in the aqueous layer left after acid hy-
drolysis of 1–5 were neutralized with NaHCO3 and analyzed by silica
gel TLC by comparison with standard sugars. The solvent system was n-
BuOH-TEA-H2O (60:0.7:30), and spots were visualized by spraying with
Phenylamine-Diphenylamine, and then heated at 200 °C for 1 min. For
sugars of 1–5, the Rf of glucose, arabinose and xylose by TLC was 0.57,
0.62 and 0.66 respectively. The results were confirmed by GC analysis
3

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X.-D. Zhou, et al.
Fitoterapia138(2019)104298
Table 2
1H NMR (500 MHz) and 13C NMR (125 MHz) data of 3–5 in DMSO-d6 (δ in ppm, J in Hz).
Position
3
4
5
δH J in Hz
6.61 s
δC
δH J in Hz
6.60 s
δC
δH J in Hz
δC
1
2
3
4
5
139.5
104.9
152.1
133.2
153.1
139.6
104.8
152.1
133.2
153.1
146.5
149.1
109.9
131.3
119.1
7.09 d (2.0)
6.92 dd (8.5,
2.0)
6
7
6.61 s
104.9
72.5
6.60 s
104.8
72.6
7.02 d (8.5)
6.34 dd
115.3
136.4
4.44 d (4.0)
4.43 d (4.0)
(18.0, 11.0)
5.12 dd
8
3.46 dd
75.5
3.47 dd
75.3
112.5
(10.5, 7.5)
(10.5, 7.5)
(11.0, 1.0)
5.70 dd
(18.0, 1.0)
9
3.17 dd
62.6
3.17 dd
(11.0, 6.0)
3.36a
62.6
(11.0, 6.0)
3.36 dd
(10.5, 5.0)
1′
2′
3′
4′
5′
6′
7′
Sugar
1
119.4
107.1
147.5
140.8
147.5
107.1
166.4
Glc
119.4
106.9
147.5
140.7
147.5
106.9
165.5
Glc
7.16 s
7.15 s
7.16 s
7.15 s
Glc
Glc
Glc
Glc
4.82 d (7.0)
3.23 m
3.26 m
3.28 m
3.40 m
4.20 dd
(12.0, 5.5)
4.46 dd
(12.0, 1.0)
3.66 s
103.2
74.1
4.82 d (7.5)
3.26 m
3.26 m
3.28 m
3.40 m
4.19 dd
(12.0, 5.5)
4.46 dd
(12.0, 2.0)
3.66 s
103.2
74.2
4.87 d (7.0)
3.24 m
100.0
73.2
76.5
69.9
74.8
65.7
2
3
76.1
76.1
3.25 m
4
69.9
69.9
3.15 m
5
74.0
74.0
3.48 m
6
63.8
63.8
3.74 dd
(11.0, 6.5)
4.02 dd
(11.0, 2.0)
3,5-OCH3
3′,5′-OCH3
2-OCH3
56.1
56.3
3.78 s
3.78 s
3.78 s
55.7
a
This signal under the superscript was overlapped.
500 mg) [35], 2-hydroxymethyl-3-hydroxyl-1-butene-4-O-β-D-(6′′-O-
caffeoyl)-glucopyranoside (23, 220 mg) [18], 2-caffeoyloxymethyl-3-
hydroxyl-1-butene-4-O-β-D-glucopyranoside (24. 20 mg) [18].
HMBC correlation from Ara-H-1 (δH 4.99) to inner-Glc-C-6 (δC 69.6).
The above information indicated 1 was a disaccharide of β-pinene. In
the NOESY spectrum, the NOE cross-peaks of H-5/H-4a, H-5/9-CH3, H-
7/9-CH3, H-4a/H-8a indicated that they were on the same face, as-
signed α-orienated as while the NOE correlations of H-3 with axial H-4b
(δH 2.27) and equatorial methyl 10-CH3 suggested H-3, H-4b, 10-CH3
were co-facial, assigned as β-orienated, conforming the configuration of
3-OHα (Fig. 2). On the basis of the above analysis, compound 1 was
elucidated as 3α-hydroxyl-β-pinene-3-O-β-D-glucopyranosyl-(1 → 6)-α-
L-arabinpyranoside.
Pubescenoside L (1) was assigned a molecular formula of C21H34O10
determined on the basis of its positive HRESIMS [M + NH4]+ ion peak
at m/z 464.24856 (calcd. 464.24902). The IR spectrum showed ab-
sorption bands for hydroxyl (3410 cm−1), methyl (2924 cm−1) and
olefinic (1646 cm−1) groups. Acid hydrolysis of 1 provided sugar
L-arabinose as identified by TLC and GC
analysis. TheD-glucose and
components
1H and 13C NMR data (Table 1) of 1 showed signals as-
signable to a pinene moiety [δH 0.62, 1.11 (3H each, both singlets, H-10
and H-9, respectively), 1.85, 2.20 (1H each, both m, H-8), 1.84 (1H, m,
H-5), 2.15, 2.27 (1H each, dd, J = 14.0, 4.0 Hz, H-4), 2.46 (1H, t,
J = 5.5 Hz, H-7), 4.89 (1H, d, J = 7.5 Hz, H-3), two olefinic proton
signals at δH 5.00, 5.54 (1H each, both br s, H-2a and 2b)]. The con-
figuration of the two sugar units were also determined to be β-glucosyl
and α-arabinosyl moieties by the respective coupling constants (J = 8.0
and 6.5 Hz) of the anomeric proton. 1H-1H COSY spectrum revealed the
partial structure shown in bold in Fig. 2: H-3/H-4, H-4/H-5, H-5/H-8,
H-8/H-7. Detailed HMBC correlations, including from H-2 to C-1, C-3
and C-7, from H-3 to C-1, C-4, from H-4 to C-3 and C-5, from H-7 to C-6
and C-8, from H-8 to C-5 and C-7 suggested 1 as a scaffold of 3-hydroxyl
β-pinene. The HMBC correlation (Fig. 2) from the inner-Glc-H-1 (δH
4.92) to C-3 (δC 72.2) determined its glycosylation site at 3-O-. The
linkage site between arabinose and glucose was determined by the
Pubescenoside M (2) was assigned a molecular formula of
C21H34O10 determined on the basis of its positive HRESIMS
[M + NH4]+ ion peak at m/z 464.24854 (calcd. 464.24902). The IR
spectrum showed absorption bands for hydroxyl (3424 cm−1), methyl
(2934 cm−1) and olefinic (1639 cm−1) groups. IR and UV spectra were
nearly the same as those of 1. Detail analysis for 1D and 2D NMR data
(Table 1) indicated that compound 2 possessed a similar structure to 1,
except for a difference appeared in the sugar component. The terminal
sugar was xylose in compound 2, whereas it was arabinose in 1. The
coupling constant (J = 7.5 Hz) of the anomeric proton indicated a β-
xylosyl moiety. This conclusion was also supported by acid hydrolysis
of 2, which provided sugar components D-glucose and D-xylose as
identified by TLC and GC analysis. Therefore, compound 2 was eluci-
dated as 3α-hydroxyl-β-pinene-3-O-β-D-glucopyranosyl-(1 → 6)-β-D-xyl-
pyranoside.
4