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J. Cai et al.
insufficient effectiveness and durability, some of the current agents have side effects, including
hypoglycemia, weight gain, edema, fractures, lactic acidosis, gastrointestinal intolerance and so
on (Zhang et al. 2009). Thus, there is an emerging necessity to explore novel agents for the
treatment of T2DM to prevent the spread of this global epidemic.
Insulin resistance in liver cells principally causes impaired glycogen synthesis and fails to
suppress glucose production, which is the major contribution to hyperglycemia (Kola et al.
2008). HepG2 cells are hepatocellular carcinoma cells and have been proven to be valuable in
investigating liver-derived functions. They maintain most functions of liver and are steady
through many passages (Gupta et al. 2007). HepG2 cells have been used to investigate T2DM
via an insulin-resistant model (Luo et al. 2009). In this study, we have also utilised insulin-
resistant HepG2 cells to investigate effects of the isolated compounds from Anoectochilus
elwesii on stimulating glucose uptake under high glucose condition.
Anoectochilus elwesii (Clarke ex Hook. f.) King et Pantl. has been used in Chinese folk
medicine in diabetes and nephropathy ailments (Flora Republicae Popularis . . . 1999).
Pharmacological evaluation of the antihyperglycemic activity of the ethonal extract of this plant
in rats confirmed the folk information. A preliminary phytochemical study on A. elwesii has
revealed the occurrence of flavonoids, triterpenoids and sterols. As part of our ongoing
investigation for the chemistry of A. elwesii, here we report on the isolation and characterisation
from the herbs of A. elwesii of some flavonoids, including a new flavonol triglycoside,
isorhamnetin-3-O-b-D-glucopyranosyl (1→2)-a-L-rhamnopyranosyl (1→6)-b-D-glucopyrano-
side (4).
2. Results and discussion
Compound 4, yellowish powder from MeOH, Rf 0.53 (CHCl3–MeOH–H2O 65:35:10, lower
phase). IRmax (KBr): 3327, 2934, 2890, 1660, 1610, 1567, 1511, 1452, 1364, 1283, 1210, 1183,
1
1
1080, 1016, 993, 836, 811 cm21. H and 13C NMR, see Table S1. The H NMR spectrum
displayed three aromatic protons at d 7.84 (1H, d, J ¼ 2 Hz), 7.52 (1H, dd, J ¼ 2 and 8 Hz) and
6.91 (1H, d, J ¼ 8 Hz) for H-20, H-60 and H-50, respectively. Two doublets at d 6.20 (1H, d,
J ¼ 2 Hz) and 6.42 (1H, d, J ¼ 2 Hz) integrating single protons were assigned to H-6 and H-8,
respectively. A signal at d 3.83 (3H, s) indicated the presence of a methoxyl substituent on the
flavonol skeleton. The number and characteristic shifts of the 13C glycosidic signals indicated the
presence of two hexoses and a methyl hexose system in the pyranose form. The signals at d 4.17
(1H, d, J ¼ 7.5 Hz), 5.45 (1H, d, J ¼ 7.5 Hz) and 4.73 (1H, s) were attributed to H-1 of two b-
glucosyl units and an a-rhamnosyl unit, respectively. The existing two glucose units had b-
glycosidic linkages, as concluded from the magnitude of the vicinal proton couplings (7.5 Hz) of
1
the anomeric protons in the H spectrum. The 13C NMR spectrum showed a singlet which
resonated at d 56.32, and its protons (d 3.83) showed a correlation with C-30 in HMBC spectrum,
which was assigned to the methoxyl substituent at C-30. The signal at d 178.02 was attributed to the
carbonyl carbon (C-4). The signals of the aglycone and saccharides were assigned by HMBC (as
Table S1). In the 13C NMR spectrum, signals of the two b-glucopyranosyl and an a-
rhamnopyranosyl moieties could be detected at 106.24, 101.77 and 100.22. Furthermore, the
glucose linked to the hydroxyl group at C-3 of the aglycone was unambiguously confirmed by a
long-range correlation between the proton of H-100 (d 5.45) and C-3 (d 133.76) in the HMBC
spectrum. The downfield00shift of the C-600 and the upfield shift of C-1000, and furthermore, the
correlation between H-6 and C-1000 (d 100.22) suggested the position of attachment of the
rhamnosyl moiety at C-600 of glucose. The anomeric proton H-1000 (d 4.73) showed a correlation
with C-600 (d 67.71), and similarly for the third sugar moiety, a long-range correlation between H-
10000 (d 4.18) and C-2000 (d 81.48) was observed. Hence, the glycosidic linkageswere proven to be the
two-linked glucopyranose and rhamnopyranose with a terminal glucopyranose. Acid hydrolysis