Chemical constituents of Bergenia crassifolia roots and their growth inhibitory activity against Babesia bovis and B. bigemina

Article abstract of DOI:10.1016/j.phytol.2018.11.009

Bergenia crassifolia is a tea plant from in Northeast Asia, and it used to be a traditional medicine in Asian countries. From the roots of this plant, two new compounds, (2R,3S)-3-O-p-hydroxybenzoyl-5-O-galloylcatechin and 6-O-(3′′-O-methylgalloyl) arbutin, were isolated together with 20 known flavonoids, including catechins, kaemferols, arbutin derivatives, and bergenin derivatives. Their chemical structures were elucidated on the basis of spectroscopic data analyses. The anti-babesial activities of the isolated compounds were estimated using in vitro tests against Babesia bigemina and B. bovis, which are the most prevalent species associated with bovine babesiosis. The compounds with galloyl groups showed moderate growth-inhibition effects on B. bovis (IC₅₀ 0.80–10.3 μg/mL) and B. bigemina (IC₅₀ 5.69–8.60 μg/mL).

Full text of DOI:10.1016/j.phytol.2018.11.009

PhytochemistryLetters29(2019)79–83
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Chemical constituents of Bergenia crassifolia roots and their growth
inhibitory activity against Babesia bovis and B. bigemina
⁎
Orkhon Banzragchgarava, Toshihiro Murataa, , Bumduuren Tuvshintulgab, Keisuke Suganumab,c
,
Ikuo Igarashib, Noboru Inoued, Javzan Batkhuue, Kenroh Sasakia
a Department of Pharmacognosy, Tohoku Medical and Pharmaceutical University, 4-1 Komatsushima 4-chome, Aoba-ku, Sendai, 981-8558, Japan
b National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, 080-8555, Japan
c Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, 080-8555, Japan
d Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido, 080-8555, Japan
e School of Engineering and Applied Sciences, National University of Mongolia, POB-617, Ulaanbaatar-46A, Mongolia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bergenia crassifolia is a tea plant from in Northeast Asia, and it used to be a traditional medicine in Asian
countries. From the roots of this plant, two new compounds, (2R,3S)-3-O-p-hydroxybenzoyl-5-O-galloylcatechin
and 6-O-(3′′-O-methylgalloyl) arbutin, were isolated together with 20 known flavonoids, including catechins,
kaemferols, arbutin derivatives, and bergenin derivatives. Their chemical structures were elucidated on the basis
of spectroscopic data analyses. The anti-babesial activities of the isolated compounds were estimated using in
vitro tests against Babesia bigemina and B. bovis, which are the most prevalent species associated with bovine
babesiosis. The compounds with galloyl groups showed moderate growth-inhibition effects on B. bovis (IC50
0.80–10.3 μg/mL) and B. bigemina (IC50 5.69–8.60 μg/mL).
Bergenia crassifolia
Galloyl
Anti-babesial activity
Babesia bigemina
Babesia bovis
1. Introduction
bergenin derivatives, arbutin derivatives, catechin gallates, ellagi-
tannins, and hydrolysable tannins (Salminen et al., 2014; Shikov et al.,
Bergenia crassifolia (L). Fritsch. grows in China, Mongolia, Siberia,
Altai, and the Himalayan region (Shikov et al., 2012). B. crassifolia is
commonly known as a Mongolian and Siberian tea and as a European
ornamental plant (Kraujaliene et al., 2016), but it is also considered as a
traditional medicine in India (Ballabh et al., 2008), Tibet (Zhao et al.,
2006), China (Zhou et al., 2011), Russia (Shikov et al., 2014a), and
Mongolia (Ligaa, 1996; WHO, 2013). Furthermore, Russian Pharma-
copoeia lists rhizomes of this plant as hemostatic, astringent, anti-in-
flammatory, and antimicrobial agents (Shikov et al., 2014b). In Mon-
golia, the roots and leaves of B. crassifolia, for which the local crude
drug name is “Bagdgar badaan”, are used for the treatment of typhoid,
lung fever, stomach and intestine disorders, diarrhea, and lung in-
flammation (Ligaa, 1996; WHO, 2013). It has been reported that ex-
tracts of B. crassifolia possess a wide range of biological activities, in-
2014a).
Bovine babesiosis is a tick-transmitted disease caused by Babesia
bovis, B. bigemina, and B. divergens (Apicomplexa), which lead to serious
economic losses related to high morbidity and mortality in cattle industry
worldwide. In particular, B. bovis and B. bigemina are the most prevalent
and highly pathogenic species. Although a number of drugs and ther-
apeutics have been brought to the market, their beneficial effects have
shown a steady decline in recent decades due to their toxic side effects on
the host and the emergence of drug-resistant parasites and numerous
negative impacts, including toxicity (Mosqueda et al., 2012; Vial and
Gorenflot, 2006). Therefore, the development of a new class of drug
candidates is required urgently. Previous studies reported that B. bovis
and B. bigemina are endemic in Mongolia and these parasites have been
detected in cattle (Sivakumar et al., 2012; Altangerel et al., 2012).
Herein, we report the structure elucidations of compounds isolated from
B. crassifolia, which was collected in Mongolia, and their anti-babesial
activities against in vitro cultures of B. bovis and B. bigemina.
cluding
antimicrobial,
antiobesity,
antioxidant,
antiviral,
gastroprotective, diuretic, and hepatoprotective activities, and the
chemical compounds profile of B. crassifolia was characterized with
⁎ Corresponding author.
E-mail address: murata-t@tohoku-mpu.ac.jp (T. Murata).
https://doi.org/10.1016/j.phytol.2018.11.009
Received 24 July 2018; Received in revised form 25 October 2018; Accepted 8 November 2018
1874-3900/©2018PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

O. Banzragchgarav et al.
PhytochemistryLetters29(2019)79–83
2. Material and methods
Fr.1E (0.96 g) was separated by ODS-120 T column, eluting with
MeOH-H2O, 4:6 and 6:4 to partitions. Partitions were further separated
and purified by HPLCs [Column: ODS-120 T, Develosil C30-UG-5; mo-
bile phase: a gradient of acetonitrile–H2O (1:4 – 3:2, containing 0.2%
TFA)] to find compounds 9 (1.1 mg), 10 (3.1 mg), 12 (14.9 mg), and 13
(3.8 mg).
2.1. General experimental procedures
The UV spectra were recorded with a Shimadzu MPS-2450 (SHIM-
ADZU, Kyoto, Japan). The 1H NMR (400 MHz) and 13C NMR (100 MHz)
spectra were recorded using a Jeol JNM-AL400 FT-NMR spectrometer
(JEOL, Tokyo, Japan), and the chemical shifts were reported as δ values
with TMS as an internal standard at 30 °C (measured in methanol-d4).
Inverse-detected heteronuclear correlations were measured using
The diethyl ether-soluble fraction (3.73 g) was separated using a
silica gel column chromatography, eluting with a gradient of hexane-
acetone (100:0 – 0:100). Further purification by HPLCs with ODS-SM-
50C-M and Capcell Pak C8 columns eluted with acetonitrile–H2O (1:7 –
1
n
HMQC (optimized for JC-H = 145 Hz) and HMBC (optimized for JC-
H = 8 Hz) pulse sequences with a pulsed-field gradient. The HRFABMS
data were obtained using a Jeol JMS700 mass spectrometer (JEOL)
with a glycerol matrix. Preparative HPLC was performed using a Jasco
2089 with UV detection at 210 nm (JASCO, Tokyo, Japan), using the
following columns: Ultra Pack ODS-SM-50C-M (Yamazen, Osaka, Japan
1:3, containing 0.2% TFA) to give compounds 2 (36.8 mg), 3
(191.0 mg), and 5 (42.4 mg).
2.3.1. (2R,3S)-3-O-p-hydroxybenzoyl-5-O-galloylcatechin (5)
Colorless amorphous solid; [α]2D5 + 9 (c 2.1, MeOH); UV (MeOH)
max (log ε) 275 (4.39) nm; ECD (c 0.00021, MeOH) ([θ]) 219 (37,000),
λ
37 × 100 mm),
TSKgel
ODS-120 T
(Tosoh,
Tokyo,
Japan,
242 (11,900), 256 (18,000), 285 (–12,600), 401 (2500) nm; HRFABMS
21.5 × 300 mm), and Develosil C30-UG-5 (Nomura Chemical, Aichi,
Japan, 20 × 250 mm), Capcell Pak C8 (Shiseido, Japan, 20 × 250 mm),
and Mightysil RP-18 GP (Kanto Chemicalm Tokyo, Japan,
10 × 250 mm).
(positive-ion mode) m/z 563.1198 [M+H]+ (calcd for C29H23O12
,
563.1189); 1H NMR (methanol-d4, 400 MHz), δ 2.68 (1H, dd, J = 16.5,
6.5 Hz, H-4), 2.83 (1H, dd, J = 16.5, 5.0 Hz, H-4), 5.16 (1H, d,
J = 6.0 Hz, H-2), 5.38 (1H, m, H-3), 6.28 (1H, d, J = 2.5 Hz, H-6), 6.36
(1H, d, J = 2.5 Hz, H-8), 6.75 (2H, overlapping, H-5′ and 6′), 6.76 (2H,
d, J = 8.5 Hz, H-3′′ and 5′′), 6.85 (1H, br s, H-2′), 7.17 (2H, s, H-2′′′ and
6′′′), 7.74 (2H, d, J = 8.5 Hz, H-2′′ and 6′′); 13C NMR (methanol-d4,
100 MHz), δ 24.7 (C-4), 70.6 (C-3), 79.7 (C-2), 101.7 (C-8), 103.7 (C-6),
105.3 (C-4a), 110.6 (C-2′′′ and 6′′′), 114.4 (C-2′), 116.1 (C-3′′ and 5′′),
116.3 (C-5′), 119.2 (C-6′), 120.1 (C-1′′′), 121.9 (C-1′′), 131.0 (C-1′),
132.9 (C-2′′ and 6′′), 140.6 (C-4′′′), 146.4 (C-3′), 146.4 (C-4′), 146.7 (C-
3′′′ and 5′′′), 151.7 (C-5), 156.6 (C-8a), 158.4 (C-7), 163.6 (C-4′′), 166.4
(C-7′′′), 167.2 (C-7′′).
2.2. Plant material
Bergenia crassifolia roots were collected in September 2015 from
Gatsuurt area, Ulaanbaatar, Mongolia, asl; latitude: 48 51′416″ N,
longitude: 107 13′352″ E. A voucher specimen has been deposited in
the herbarium sector of National University of Mongolia. This plant
species was identified by Dr. R. Molor, Mamba Datsan university,
Ulaanbaatar, Mongolia.
2.3. Extraction and isolation
2.3.2. 6′-O-(3′′-O-methylgalloyl) arbutin (15)
Colorless amorphous solid; HRFABMS (positive-ion mode) m/z
439.1247 [M+H]+ (calcd for C20H23O11, 439.1240). 1H NMR (me-
thanol-d4, 400 MHz), δ 3.43 (3H, overlapping, H-Glc-2, Glc-3, and Glc-
4), 3.70 (1H, m, H-Glc-5), 3.85 (3H, s, H-OMe), 4.39 (1H, dd, J = 12.0,
7.0 Hz, H-Glc-6), 4.64 (1H, dd, J = 12.0, 2.5 Hz, H-Glc-6), 4.72 (1H, d,
J = 7.0 Hz, H-Glc-1), 6.58 (2H, d, J = 8.5 Hz, H-3 and 5), 6.92 (2H, d,
J = 8.5 Hz, H-2 and 6), 7.19 (1H, d, J = 2.0 Hz, H-2′), 7.25 (1H, d,
J = 2.0 Hz, H-6′); 13C NMR (methanol-d4, 100 MHz), δ 56.7 (C-OMe),
65.2 (C-Glc-6), 72.0 (C-Glc-4), 75.0 (C-Glc-2), 75.6 (C-Glc-5), 78.0 (C-
Glc-3), 103.8 (C-Glc-1), 106.3 (C-2′), 109.7 (C-6′), 116.7 (C-3 and 5),
119.5 (C-2 and 6), 121.4 (C-1′), 140.8 (C-4′), 146.4 (C-5′), 149.2 (C-3′),
152.4 (C-1), 153.9 (C-4), 168.1 (C-7′).
Air-dried B. crassifolia roots (756 g) were extracted four times with
acetone-H2O (4:1, v/v) at 60°C. Evaporation of the solvent under re-
duced pressure provided a acetone-H2O extract (196 g, 26% from the
roots). The initial separation was performed by means of liquid liquid
partition by resuspending the extract in H2O and the partitioned into
H2O-soluble (195 g), diethyl ether-soluble (3.73 g), and insoluble
(1.28 g) parts. The H2O-soluble fraction was subjected to porous
®
polymer gel (DIAION HP-20, Mitsubishi, Japan) column chromato-
graphy, eluted with H2O-MeOH in a gradient, and obtained Fr.1 A
(H2O, 95 g), Fr.1B (MeOH-H2O, 1:4; 38 g), Fr.1C (MeOH-H2O, 2:3,
35 g), Fr.1D (MeOH-H2O, 3:2, 11 g), Fr.1E (MeOH-H2O, 4:1, 0.96 g),
and Fr.1 F (MeOH, 0.38 g).
A part of Fr.1B (5.0 g) was suspended in MeOH-H2O (1:9) and
centrifuged. Supernatant resulted in 16 subfractions after it was sub-
jected to ODS-SM-50C-M column chromatography, eluting with a gra-
dient of acetonitrile–H2O [1:9 – 2:8, containing 0.2% trifluoroacetic
acid (TFA)]. Then the subfractions were purified by repeated high
performance liquid chromatography [HPLC; Column: ODS-120 T,
Develosil C30-UG-5; and Mightysil RP-18 GP, mobile phase: a gradient
of acetonitrile–H2O (1:19 – 1:3, containing 0.2% TFA)] and isolated
compounds 1 (14.4 mg), 7 (67.2 mg), 8 (1.9 mg), 11 (2.2 mg), 14
(10.9 mg), 16 (44.2 mg), 17 (4.5 mg), 18 (204.9 mg), and 20 (27.8 mg).
A part of Fr.1C (5.0 g) was subjected to ODS-SM-50C-M column
(MeOH- H2O, 1:4) to produce 11 subfractions (Frs. 2 A – 2 K). The
mixture of Frs.2C – 2I was purified by HPLCs [columns: ODS-120 T,
Develosil C30-UG-5, mobile phase: a gradient of acetonitrile–H2O (3:17
– 1:4, containing 0.2% TFA)] to obtain compounds 6 (46.2 mg), 15
(1.3 mg), 21 (54.8 mg), and 22 (18.3 mg).
2.4. Sugar identification
The previous procedure using HPLC for glycosyl moiety identifica-
tion was carried (Badral et al., 2017). That is 15 (0.5 mg) was hydro-
lyzed with 7% HCl (0.5 mL) at 90 °C for 1 h, and the each reaction
mixture was concentrated after going through an HP-20 column
(5 × 20 mm) with water (1 mL). Then, each residue was stirred with L-
cysteine methyl ester (5 mg) in pyridine (0.5 mL) (60℃, 1 h), and then
o-tolyl isothiocyanate (10 μL) was added (60℃, 1 h). The reaction
mixtures were analyzed by HPLC (column, Capcelpak C18 UG120, Si-
seido, Tokyo, Japan, 4.6 × 250 mm; mobile phase, acetonitrile-0.2%
TFA in H2O (25:75), 1.0 mL/min; detector, UV at 250 nm), and the
peaks of reactants from 15 (tR 18.3 min), D-glucose (tR 18.3 min), and
L-glucose (tR 16.7 min) were detected.
2.5. In vitro cultivation of the parasites, B. bovis and B. bigemina
Fr.1D (11.0 g) was subjected to ODS-120 T column, eluting with a
gradient of MeOH-H2O (1:3 – 6:4), and then purified by Develosil C30
-
2.5.1. Texas strain of B. bovis and the Argentine strain of B. bigemina
The B. bovis Texas strain (Hines et al., 1992) and B. bigemina Ar-
gentina strain (Hotzel et al., 1997) were maintained as described pre-
viously (Igarashi et al., 1998; Bork et al., 2003). These strains were
UG-5 column chromatography, eluting with gradient of
acetonitrile–H2O (1:7 – 1:3, containing 0.2% TFA) to yield compounds
4 (17.5 mg) and 19 (11.3 mg).
80

O. Banzragchgarav et al.
PhytochemistryLetters29(2019)79–83
Fig. 1. Structure of compounds 5–7 and 15.
obtained from Washington State University (Pullman, WA, USA).
Briefly, the culture was grown in a 24-well culture plate in a total vo-
lume of 1 mL with 100 μL Babesia-infected red blood cells (iRBCs) and
900 μL of M199 culture medium containing 40% cattle serum, 60 U/mL
of penicillin, 60 μg/mL of streptomycin, 0.15 μg/mL of amphotericin.
The culture was incubated at 37 °C in 5% CO2 and 5% O2 in a humi-
dified multigas water-jacketed incubator. For continuous maintenance
of the culture, the hematocrit concentration was 10% and the culture
medium was replaced daily, and parasitemia was monitored every day
using Giemsa-stained RBC smears.
et al., 2011), catechin-3,7-di-O-gallate (7) (Malana, 1990), (+)-afz-
elechin (8) (Saijyo et al., 2008; Kashiwada et al., 1990), kaemferol-3-O-
β-D-galactopyranoside (9) (Scharbert et al., 2004), kaemferol-3-O-glu-
coside (10) (Kazuma et al., 2003), quercetin-3-O-β-D-glucopyranoside
(11) (Han et al., 2004), rhamnazin (12) (Subhadhirasakul et al., 2003),
arbutin-4-O-p-hydroxybenzoyl (13) (Morikawa et al., 2004), arbutin-6-
O-gallate (14) (Xin-Min et al., 1987), arbutin-4,6-di-O-gallate (16) (Xin-
Min et al., 1987), glucose-1,2,6-tri-O-gallate (17) (Nonaka et al., 1981),
bergenin (18) (Saijo et al., 1990; Jia et al., 1995), bergenin-11-O-p-
hydroxybenzoyl (19) (Fujii et al., 1996), bergenin-4-O-gallate (20)
(Saijo et al., 1990; Xin-Min et al., 1987), bergenin-11-O-gallate (21)
(Saijo et al., 1990; Jia et al., 1995), and bergenin-4,11-di-O-gallate (22)
(Saijo et al., 1990).
2.6. In vitro growth inhibition assay
This method was conducted as described previously (Rizk et al.,
2015). Briefly, 2.5 μL of infected RBCs were prepared with 1% initial
parasitemia and then added to 97.5 μL of medium containing various
concentrations of test compounds. Collectively, 2.5% hematocrit and 1%
initial parasitemia were prepared for each well in the 96-well culture
plate, in according with the previously described optimization using a
fluorescence-based inhibition assay (Rizk et al., 2015). Negative control
cultures were grown in medium containing the drug solvent [dimethyl
sulfoxide (DMSO)] without drugs. Cultures were incubated at 37 °C in
5% CO2 and 5% O2 without replacement of the medium for 4 days. On
day 4 of culturing, 100 μL of lysis buffer with 2 × SYBR Green I nucleic
acid stain was added to each well, and then incubated for 8 h in the dark
at room temperature. Parasite growth-relative fluorescence values were
Compound 5 was obtained as colorless amorphous solid. Its 1H NMR
spectrum suggested that 5 was a catechin derivative. Namely, the ali-
phatic coupling system proton resonances (δH 5.16, 1H, d, J = 6.0 Hz,
H-2; 5.38, 1H, m, H-3; 2.68, 1H, dd, J = 16.5, 6.5 Hz, H-4; 2.83, 1H, dd,
J = 16.5, 5.0 Hz, H-4) indicated the presence of a 3-hydroxy flavan
moiety as ring C, and a coupling system proton (δH 6.85, 1H, br s, H-2′;
6.75, 2H, overlapping, H-5′ and 6′) and a set of m-coupling proton (δH
6.28, 1H, d, J = 2.5 Hz, H-6; 6.36, 1H, d, J = 2.5 Hz, H-8) resonances
showed the presence of a catechol ring B and 5,7-dioxygenated ring A,
respectively. The 1H and 13C NMR spectra of 5 were simiar to those of
catechin-3,5-di-O-gallate (6) (Ivanov et al., 2011), except for the pre-
sence of a p-hydroxybenzoyl moiety resonances (δH 7.74, 2H, d, J
= 8.5 Hz, H-2′′ and 6′′; 6.76, 2H, d, J = 8.5 Hz, H-3′′ and 5′′; δc 121.9,
C-1′′; 132.9, C-2′′ and 6′′; 116.1, C-3′′ and 5′′ 163.6, C-4′′; 167.2, C-7′′)
of 5 instead of the O-gallyol group of 6. This suggested that 5 was a 3,5-
di-O-acylcatechin, and the 3-O-p-hydroxybenzoyl was established from
the long-range HMBC coupling between H-3 and C-7′′. The 2R*3S*-
relative configuration was determined by the NOE correlation between
H-3 and H-2′ and the broad coupling constant (6.0 Hz) of H-2 and H-3
(Watanabe, 1998). The negative Cotton effect around 285 nm in the
ECD spectrum of 5 showed a 2R-absolute configuration (Slade et al.,
determined using
a fluorescence plate reader (Fluoroskan Ascent,
Thermo Labsystem, USA) at 485 nm excitation and 518 nm emission
wavelengths. The experiment was repeated individually three times, and
each concentration was tested in triplicate. The half maximal inhibitory
concentration (IC50) was calculated using a curve-fitting method.
3. Results and discussion
From the acetone-H2O (4:1) extract of the air-dried roots of B.
crassifolia, (2R,3S)-3-O-p-hydroxybenzoyl-5-O-galloylcatechin and 6-O-
(3′′-O-methylgalloyl) arbutin were isolated together with 20 known
compounds using HP-20 and silica gel open columns and HPLC. All the
known compounds were identified by comparing their NMR and phy-
sical data with data in the literature: catechin (1) (Davis et al., 1996),
(−)-epicatechin-3-O-p-hydroxybenzoate (2) (Watanabe., 1998), ca-
techin-3-O-gallate (3) (Davis et al., 1996), catechin-5-O-gallate (4)
(Ramanandraibe et al., 2008), catechin-3,5-di-O-gallate (6) (Ivanov
2005). Accordingly, 5 was determined as (2R,3S)-3-O-p-hydro-
xybenzoyl-5-O-galloylcatechin as shown in Fig. 1.
Compound 15 was obtained as colorless amorphous solid with a
negative optical rotation ([α]2D4 –18). The molecular formula C20H22O11
for 15 was established by HRFABMS data, m/z 439.1247 [M+H]+
(calcd for C20H23O11, 439.1240). In the 1H and 13C NMR spectra of 15,
a set of AB type coupling system proton (δH 6.92, 2H, d, J = 8.5 Hz, H-
2 and 6; 6.58, 2H, d, J = 8.5 Hz, H-3 and 5), their corresponding
carbon (δC 119.5, C-2 and 6; 116.7, C-3 and 5) and the oxygenated
81

O. Banzragchgarav et al.
PhytochemistryLetters29(2019)79–83
Table 1
measurements. This work was supported by Grant-in-aid project from
Mongolian Foundation of Science and Technology (2018/58), the JICA
M-JEED project, and partially supported by the Kanno Foundation of
Japan, a Cooperative Research Grant (28-joint-12, 29-joint-6, 30-joint-
11) of National Research Center for Protozoan Diseases, Obihiro
University of Agriculture and Veterinary Medicine, and AMED/JICA
SATREPS.
Inhibitory activity of compounds isolated from B. crassifolia roots.
compoundb
IC50 (μg/mL)a
B. bovis
B. bigeminia
6
0.83
0.80
8.24
10.3
6.04
0.21
0.01
0.05
1.5
5.69
6.31
6.59
6.41
8.60
0.09
0.30
7
1.10
0.77
0.85
0.47
16
17
0.54
0.26
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a
b
c
Values are mean
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Reference compound (Rizk et al., 2015).
phenyl carbon (δC 152.4, C-1; 153.9, C-4) resonances were observed,
which suggested the presence of an arbutin moiety. The resonances of
the sugar moiety were also obserbed in the spectra, and an anomeric
proton resonance at δH 4.72 (1H, d, J = 7.0 Hz, H-Glc-1) was long
range coupled with the C-1 carbon in the HMBC spectrum of 15. The
lower shifted C-Glc-6 (δC 65.2) and H-Glc-6 (δH 4.39, 1H, dd, J = 12.0,
7.0 Hz; 4.64, 1H, dd, J = 12.0, 2.5 Hz) resonances suggested a 6-O-
acylated glycosyl moiety. These spectroscopic features were close to
those of the related compound, arbutin-6-O-gallate (14) (Xin-Min et al.,
1987) identified in this study. Sugar analysis using HPLC after acid
hydrolysis and the coupling constant of Glc-1 (7.0 Hz) showed that 15
had a β-D-glucosyl moiety (Badral et al., 2017). The spectra of 15
showed the presence of a modified galloyl aromatic ring (δH 7.19, 1H,
d, J = 2.0 Hz, H-2′; 7.25, 1H, d, J = 2.0 Hz, H-6′; δC 121.4, C-1′; 106.3,
C-2′; 149.2, C-3′; 140.8, C-4′; 146.4, C-5′; 109.7, C-6′; 168.1, C-7′) and
an O-methyl group (δH 3.85, 3H, s; δC 56.7), in place of an usual O-
galloyl group in 14. The long-range HMBC correlations between the O-
methyl proton and C-3′ indicated that the O-methyl group linked to C-
3′. Therefore, compound 15 was identified as 6-O-(3-O-methylgalloyl)
arbutin as shown in Fig. 1.
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We thank Mr. S. Sato and Mr. T. Matsuki, Tohoku Medical and
Pharmaceutical University, for assistance with the mass spectra
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