Two new galloyl glucosides from the leaves of Castanopsis fordii

Article abstract of DOI:10.3987/COM-17-S(T)5

Two new galloyl glucosides, furan-2-carbonyl 6-C-β-D-(6'-O-galloyl)glucopyranoside (1), grasshopper ketone 5-O-β-D-(6'-O-galloyl)glucopyranoside (2), and one known compound 4-quinolone-2-carboxylic acid, were isolated from ethanol extract of the fresh leaves of Castanopsis fordii (Fagaceae). The chemical structures of these compounds were determined by one and two dimensional (1D and 2D)-NMR, liquid chromatography/time-of-flight/mass spectrometry (LC/TOF/MS), chemical evidence, and comparison with the literature.

Full text of DOI:10.3987/COM-17-S(T)5

HETEROCYCLES, Vol. 97, No. , 2018, pp. -. © 2018 The Japan Institute of Heterocyclic Chemistry
Received, 16th November, 2017, Accepted, 10th April, 2018, Published online, 14th May, 2018
DOI: 10.3987/COM-17-S(T)5
TWO NEW GALLOYL GLUCOSIDES FROM THE LEAVES OF
CASTANOPSIS FORDII
Yong-Lin Huang,*,a Zhang-Bin Liu,a Ya-Feng Wang,a Li Ge,b Rui-Jie He,a
Jin-Lei Liu,a Ke-Di Yang,*, b and Dian-Peng Li a
aGuangxi Key Laboratory of Functional Phytochemicals Research and Utilization,
Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese
b
Academy of Sciences, Guilin 541006, China. Gaungxi university, Nanning
530004, China
Abstract
–
Two
new
galloyl
glucosides,
furan-2-carbonyl
ketone
6-C-β-D-(6'-O-galloyl)glucopyranoside
(1), grasshopper
5-O-β-D-(6'-O-galloyl)glucopyranoside (2), and one known compound
4-quinolone-2-carboxylic acid, were isolated from ethanol extract of the fresh
leaves of Castanopsis fordii (Fagaceae). The chemical structures of these
compounds were determined by one and two dimensional (1D and 2D)-NMR,
liquid chromatography/time-of-flight/mass spectrometry (LC/TOF/MS), chemical
evidence, and comparison with the literature.
Castanopsis is a genus belonging to the Fagaceae family. In previous studies, the main of the secondary
metabolites from Castanopsis were found to be tannins and related compounds,1-5 including polyphenols
and flavonoids from C. fordii.6,7 In the course of a chemical study on tannins and related compounds from
Castanopsis species, the ethanol extract of the fresh leaves of C. fordii was subjected to a more careful
investigation leading to the isolation of three compounds including two new ones. In this paper, we
describe the isolation, structure determination of the two new galloyl glucosides from the fresh leaves of C.
fordii.
Fresh leaves of C. fordii were extracted with 80% aqueous ethanol, and the extract was subjected to a
combination of column chromatography using Sephadex LH-20, MCI gel CHP 20P, Toyopearl HW-40F,
and Toyoperal Butyl-650C to afford three compounds, including the two new galloyl glucosides 1 ,
2, and one known compound 3 (Figure 1). The known compound 3 was elucidated on the basis of
spectroscopic and comparison with literature data as 4-quinolone-2-carboxylic acid.8

O
1'
11 12
6'
O
10
2
7
RO
HO
O
5
C
6
1
5
13
O
OH
HO
HO 3
6'
O
1 R=G
1a R=H
RO
HO
O
2 R=G
2a R=H
1'
OH
HO
H
N
CO2H
OH
OH
OH
O
G=
O
3
Figure 1. Structures of compounds 1, 1a, 2, 2a and 3
Compound 1 was isolated as an amorphous powder and gave a dark blue coloration after reaction with
ethanolic FeCl3, suggesting the presence of a pyrogallol ring in the molecule. The molecular formula was
determined to be C18H18O11 by the liquid chromatography/time-of-flight/mass spectrometry
(LC/TOF/MS), which showed the [M-H]- peak at m/z 409.0728 (Calcd for C18H17O11, 409.0776). In the 1H
NMR spectra (Table 1) showed the presence of one 2-furanyl group from the proton signals at δH 7.73
13
(1H, d, J = 1.7 Hz), 6.50 (1H, dd, J = 3.7, 1.7 Hz), and 7.48 (1H, d, J = 3.7 Hz), and analysis of the C
NMR spectra data showed that this compound contained a furan-2-carbonyl moiety from signals at δC
1
150.9, 123.9, 113.4 and 150.1, and one carbonyl group at δC 185.5.9 The H NMR spectra showed one
anomeric proton singlet appeared at δH 4.43 (1H, d, J = 9.6 Hz) and six proton signals appeared at δH 3.63
13
to 4.50 were suggested to be one sugar moiety, and this was supported by C NMR signals at δC 80.1,
71.6, 70.2, 77.4, 78.3, and 64.3. In addition, this sugar moiety was confirmed as a C-β-glucopyranosyl
unit by comparing the chemical shift and coupling constant of those compounds isoorientin and
vitexin.10,11 A two-proton singlet at δH 6.99 (2H, s) suggested that the presence one galloyl group, and
supported by
C
O
HO
HO
HO
HO
HO
O
O
O
O
O
HO
O
HO
O
O
O
HO
HO
HO
HO
OH
OH
1
2
Figure 2. Selected HMBC correlations (H to C) 1 and 2

13C NMR signals at δC 120.5, 110.0 (2C), 145.2 (2C), 138.7, and 167.8. The ester of furan-2-carbonyl
group is located at glc-C-1, and the galloyl group is located at glc-C-6, respectively. These were
confirmed by the HMBC correlations of H-3 and H-1' with C-6 (δC 185.5), and H-6' with galloyl-C-7 (δC
167.8). Finally, enzymatic hydrolysis of 1 with tannase, which yielded gallic acid and 1a (scleropentaside
A).9 Based on these spectroscopic and chemical results, the structure of 1 was concluded to be
furan-2-carbonyl C-β-D-(6'-O-galloyl)glucopyranoside.
Table 1. 1H-(500 MHz) and 13C-(125 MHz) NMR data for 1 (in acetone-d6) and 2 (in DMSO-d6)Dꢀ
1
2
1H
13C
1H
13C
35.5
19.6
Position
Position
1
2
1
2
150.9
1.80 (ddd, 12.4, 3.6, 1.7)
1.15 (dd, 12.4, 11.0)
4.39 (m)
3
4
7.73 (d, 3.7)
123.9
113.4
3
4
61.2
46.4
6.50 (dd, 3.7, 1.7)
2.37 (m)
1.19 (m)
5
6
7.48 (d, 1.7)
150.1
185.5
5
6
77.1
117.4
210.5
99.8
197.5
26.3
29.0
26.4
31.7
97.0
73.5
77.0
70.0
73.7
63.6
7
8
5.78 (s)
9
10
11
12
13
1'
2'
3'
4'
5'
6'
2.08 (s)
1.22 (s)
1.29 (s)
0.96 (s)
1'
2'
3'
4'
5'
6'
4.43 (d, 9.6)
80.1
71.6
70.2
77.4
78.3
64.3
4.43 (d, 8.2)
3.97 (dd, 9.7, 8.2)
4.14 (t, 9.7)
3.25 (t, 9.7)
3.76 (m)
3.63 (dd, 9.6, 9.0)
3.48 (dd, 9.0, 8.5)
3.56 (dd, 9.0, 8.5)
3.78 (m)
4.30 (dd, 12.2, 6.2)
4.50 (dd, 12.2, 2.2)
4.12 (dd, 11.8, 6.3)
4.40 (dd, 11.8, 2.0)
galloyl
galloyl
1
2, 6
3, 5
4
120.5
110.0
145.2
138.7
167.8
1
2, 6
3, 5
4
119.5
108.7
145.5
138.4
165.8
6.99 (s)
6.93 (s)
ꢀ
7
7
ɑChemical shifts are given in δ values; multiplicities and coupling constants (J in Hz) in parentheses.

Compound 2 also gave dark blue coloration with ethanolic FeCl3 reagent, and the molecular formula
C26H34O12 was decided from the [M-H]- peak at m/z 537.1931 in the LC/TOF/MS (Calcd for C26H33O12,
1
13
537.1978). The H and C NMR exhibited a two-proton singlet at δH 6.93 (2H, s), and δC 119.5, 108.7
1
(2C), 145.5 (2C), 138.4, and 165.8 indicated that 2 had a galloyl group. The H NMR showed an
anomeric proton signal at δH 4.43 (1H, d, J = 8.2 Hz) suggest that 2 had a β-glucopyranose moiety, and
this was supported by 13C NMR signals at δC 97.0, 73.5, 77.0, 70.0, 73.7, and 63.6. As the aglycone, the
appearance of signals four tertiary methyl groups [δH 2.08, 1.22, 0.96, and 1.29; δC 26.3, 29.0, 31.7, and
26.4], one oxy-bearing methine [δH 4.39; δC 61.2], two sp3 quaternary carbons [δC 35.5 and 77.1], two
methylenes [δH 1.80 (1H, ddd, J = 12.4, 3.6, 1.7 Hz), 1.15 (1H, dd, J = 12.4, 11.0 Hz), 2.37 (1H, m), and
1.19 (1H, m), δC 19.6 and 46.4], an allenic unit [δH 5.78 (1H, s), δC 117.4, 210.5, and 99.8], and a
carbonyl carbon (δC 197.5), suggested that the aglycone was grasshopper ketone.12 Enzymatic hydrolysis
with tannase 2 yielded gallic acid and 2a (citroside A), confirming that 2 is a galloyl ester of citroside A.12
The galloyl moiety was determined to be located at glc-C-6 by the HMBC correlations of H-6 (δH 4.12
and 4.40) with galloyl-C-7 (δC 165.8). Based on these spectroscopic and chemical results, the structure of
2 was concluded to be grasshopper ketone 5-O-β-D-(6'-O-galloyl)glucopyranoside.
The known compound of 3 was elucidated on the basis of spectroscopic data analysis as
4-quinolone-2-carboxylic acid.
EXPERIMENTAL
The NMR spectra were measured in acetone-d6 or DMSO-d6 at 27 °C, using a Bruker Avance 500
1
spectrometer (500 MHz for H and 125 MHz for 13C) (Bruker Biospin AG, Fällanden, Switzerland).
Coupling constants and chemical shifts were given in Hz and on a δ (ppm) scale, respectively. Infrared
(IR) spectra were obtained by a Nicolet 6700 spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA) with KBr pellets. Liquid chromatography/time-of-flight/mass spectrometry (LC/TOF/MS)
was obtained using a JEOL JMS-T100TD spectrometer (JOL Ltd., Tokyo, Japan). Optical rotations were
recorded on a Perkin-Elmer 341 digital polarimeter (Perkin-Elmer, Norwalk, CT, USA). Column
chromatography (CC) was performed using Sephadex LH-20 (25–100 μm; GE Healthcare Bio-Science
AB, Uppsala, Sweden), MCI gel CHP 20P (75–150 μm; Mitsubishi Chemical), Diaion HP20SS
(Mitsubishi Chemical, Tokyo, Japan), and Toyopearl Butyl-650C (TOSH Co., Tokyo, Japan) columns
with H2O containing increasing proportion of MeOH (0̢100%). The enzymatic hydrolysis used tannase
(Shanghai Rui Qi Biology Technology Co., Ltd, China). TLC was performed on precoated Kieselgel 60
F254 plates (0.2 mm thick; Merck, Darmstadt, Germany) with toluene-HCO2Et-HCO2H (1:7:1, v/v) as the
solvent, and spots were detected by praying with a 2% ethanolic FeCl3.

Plant materials
The fresh leaves of C. fordii were collected at Yangshuo County, Guangxi Province, China, in August
2014, and identified by Prof. Shi-Hong Lu, Guangxi Institute of Botany. The voucher specimen
(20140821) as deposited in the Guangxi Key Laboratory of Functional Phytochemicals Research and
Utilization, Guangxi Institute of Botany, China.
Extraction and separation
The fresh bark of C. fordii (5.0 kg) was chipped into small pieces and extracted two times with
EtOH/H2O (8:2, v/v, 20 L) by maceration at room temperature. After filtration, the filtrate was combined
and concentrated under reduced pressure to give an aqueous solution. The solution was subjected to
Sephadex LH-20 (8 cm i.d.×40 cm, 0-100% MeOH in H2O) column chromatography to give nine
fractions. Fraction 2 (19.61 g) was separated by MCI gel CHP 20P (6.5 cm i.d.×35 cm, 0-100% MeOH in
H2O) column chromatography to give three fractions : frs 2-1 (8.91 g), 2 (9.04 g), and 3 (1.53 g). The
fraction 2-1 was further separated by Diaion HP20SS (4.0 cm i.d.×32 cm, 0-100% MeOH in H2O) and
Toyopearl Butyl-650C (2.0 cm i.d.×27 cm, 0-100% MeOH in H2O) column chromatography to give
compound 1 (25 mg). Fraction 2-2 was purified using Diaion HP20SS (4.5 cm i.d.×27 cm, 0-100%
MeOH in H2O) column chromatography to yield five subfractions: frs 22-1 (2.75 g), 2 (1.69 g), 3
(1.72 g), 4 (0.88 g), 5 (1.28 g), and 6 (0.29 g). The fraction 22-2 was separated by Sephadex LH-20
(2.5 cm i.d.×32 cm, 0-100% MeOH in H2O) and Diaion HP20SS (2.5 cm i.d.×27 cm, 0-100% MeOH in
H2O) column chromatography to afford compound 3 (31 mg). Fraction 22-4 was purified using
Toyopearl Butyl-650C (2.0 cm i.d.×27 cm, 0-100% MeOH in H2O) column chromatography to yield
compound 2 (30 mg).
28
Compound 1, white amorphous powder, [D]D -30.6 (c 0.6, MeOH); LC-MS/IT-TOF m/z: 409.0728
[M-H]- (Calcd for C18H17O11, 409.0776); IR Qmaxcm-1: 3321, 1806, 1769, 1618, 1557, 1498; UV Omax
1
(MeOH) nm (log H): 282 (4.89), 218 (4.35); H-NMR (125 MHz, acetone-d6) and 13C (500 MHz,
acetone-d6). See Table 1.
28
Compound 2, white amorphous powder, [D]D -76.1 (c 0.4, MeOH); LC-MS/IT-TOF m/z: 537.1931
[M-H]- (Calcd for C26H33O12, 537.1978); IR Qmaxcm-1: 3389, 1900, 1712, 1318, 1186; UV Omax (MeOH)
1
13
nm (log H): 268 (4.42), 225 (5.18); H-NMR (125 MHz, DMSO-d6) and C (500 MHz, DMSO-d6). See
Table 1.
Tannase hydrolysis of 1 and 2

A solution of 1 (5.1 mg) in water (1.0 mL) was incubated with tannase at room temperature for 2 h. The
reaction mixture was filtrated and the filtrate concentrated to dryness under reduced pressure. The residue
was dissolved in EtOH and applied to a column of Sephadex LH-20 with EtOH to give gallic acid (1.7 mg)
and 1a (scleropentaside A, 2.3 mg); 1H-NMR (125 MHz, DMSO-d6) δ: 7.61 (1H, d, J = 3.7 Hz, H-3),
6.71 (1H, dd, J = 3.7, 1.7 Hz, H-4), 8.02 (1H, d, J = 1.7 Hz, H-5), 4.28 (1H, d, J = 9.6 Hz, H-1'), 3.45 (1H,
dd, J = 9.6, 9.0 Hz, H-2'), 3.28 (1H, dd, J = 9.0, 8.5 Hz, H-3'), 3.13 (1H, dd, J = 9.0, 8.5 Hz, H-4'), 3.24
(1H, m, H-5'), 3.65 (1H, dd, J = 12.2, 2.2 Hz, H-6a'), 3.43 (1H, dd, J = 12.2, 6.2 Hz, H-6b'). Hydrolysis of
2 (6.0 mg) was also achieved in a similar manner to give gallic acid (1.6 mg) and 2a (citroside A, 3.5 mg);
1H-NMR (125 MHz, pyrdine-d5) δ: 1.78 (1H, ddd, J = 12.4, 3.6, 1.7 Hz, H-2a), 1.13 (1H, dd, J = 12.4,
11.0 Hz, H-2b). 2.33 (1H, m, H-4a), 2.00 (1H, m, H-4b), 6.13 (3H, s, H-8), 2.25 (3H, s, H-10), 1.22 (3H,
s, H-11), 1.65 (3H, s, H-12), 1.65 (3H, s, H-13), 4.43 (1H, d, J = 7.8 Hz, anomeric). The gallic acid wasꢀ
confirmed by silica gel TLC comparison with the standard [Rf 0.8, toluene:HCO2Et:HCO2H (1:7:1),
characteristic blue coloration with FeCl3 reagent].
ACKNOWLEDGEMENTS
This work was financially supports by the National Natural Science Foundation China (21562008);
Guangxi Natural Science Foundation (2014GXNSFCB118001); Program for Bagui Scholars of Guangxi
Institute of Botany (The third batch).
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