Journal of Natural Products
Article
from Sigma-Aldrich, Inc. (St. Louis, MO, USA), and a GLUT-4 ELISA
kit and a PPARγ luciferase assay kit were purchased from eBioscience
(San Diego, CA, USA) and Promega (Beijing, China), respectively.
Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum
(FBS) were purchased from Gibco BRL (NY, USA) and were used for
the culture and differentiation of the C2C12 mouse muscle myoblasts.
The organic solvents (analytical grade) used for isolation, purification,
and biological evaluations were purchased from Tianjin DaMao
Chemical Reagent (Tianjin, China).
Plant Material. Samples of T. triquetrum were collected in August
2014 from the town of Changliu in Haikou, China. The plant samples
were authenticated by Prof. Naikai Zeng (School of Pharmaceutical
Science, Hainan Medical University), and a voucher specimen (No.
TT201408) was deposited.
Extraction and Isolation. Air-dried and pulverized aerial parts of
T. triquetrum (5.0 kg) were extracted with an EtOH−H2O (70:30)
mixture (3 × 50.0 L) at room temperature. After filtration and
evaporation of the solvent, the extract (500 g) was dissolved and
partitioned with EtOAc (3× 3.0 L) and n-BuOH (3× 4.0 L). The n-
BuOH extract (180.5 g) was subjected to a silica gel column, eluting
with different ratios of CH2Cl2−MeOH, and seven fractions (A−G)
were collected. Fraction B was purified by HPLC, eluting with
MeOH−H2O (52:48), to afford a mixture of 9 and 10 (8.0 mg).
Fraction C (12.5 g) was further separated to afford subfractions 1−5.
Compounds 5 (3.5 mg) and 11 (25.0 mg) were obtained from fraction
C using different column chromatography methods, particularly HPLC
eluting with MeOH−H2O (45:55). Subfraction 4 was further
fractionated by HPLC, eluting with MeOH−H2O (40:60), to afford
6 (3.0 mg) and a mixture of 7 and 8 (5.0 mg). Compounds 1 (15.0
mg) and 2 (22.0 mg) were isolated from fraction F (1.3 g) using a
Sephadex LH-20 column and HPLC eluting with MeOH−H2O
(38:62). Fraction G (1.3 g) was further purified using a Sephadex LH-
20 column and HPLC, eluting with MeOH−H2O (35:65), to afford 3
(12.0 mg) and 4 (8.0 mg).
Therefore, the structures of 9 and 10, tadehaginosides I and J,
were defined as shown in Figure 1.
It has been well documented that Z/E isomeric compounds
commonly coexist in nature and are difficult to separate.21
From a biosynthesis perspective, tadehaginosides A−D may
have formed from tadehaginoside via different phytochemical
cyclization processes. Compounds with bicyclo[2.2.2]octene or
cyclobutane frameworks are known natural products.22,23
The abilities of the isolated compounds to stimulate glucose
uptake in C2C12 mouse skeletal muscle myotubes, which have
frequently been used to evaluate the hypoglycemic activities of
natural agents, were tested.24,25 The C2C12 myotubes were
treated with the isolated compounds (10 μM) for 6 h.
Compounds 3−11 significantly increased the glucose uptake of
the C2C12 myotubes, and 4 exhibited the most potent effect,
with an efficacy comparable to that of 100 nM insulin (Figure
5A). Because of its interesting structure and potent activity, the
time- and dose-dependent effects of 4 were subsequently
evaluated in detail. The C2C12 myotubes were treated with 4
(10 μM) at the indicated time points. Compound 4
significantly increased glucose uptake from 2 to 8 h with a
maximum enhancement at 4 h, as shown in Figure 5B. Thus,
the dose-dependent effect of 4 was evaluated at 4 h, and the
results revealed that 4 at concentrations of 5−20 μM increased
the basal and insulin-elicited glucose uptake of C2C12
myotubes. Similar effects were observed at concentrations of
10 and 20 μM (Figure 5C). The effect of 4 (10 and 20 μM) on
glucose uptake was comparable to that of 20 μM rosiglitazone.
Moreover, the MTT assay revealed that 4 (1−20 μM) was not
cytotoxic toward C2C12 myotubes after a 4 h incubation
period (Figure 5D). Molecular docking experiments demon-
strated that 3 and 4 bind tightly to PPARγ, a key regulator of
glucose homeostasis that regulates the expression of GLUT-4.
Ile281, His323, and Tyr473 were identified as the key residues
involved in the interactions between 4 and PPARγ, as shown in
Figure 6A. Luciferase assays and enzyme-linked immunosorb-
ent assays (ELISAs) demonstrated that 4 significantly enhanced
the transcriptional activity of PPARγ (Figure 6C) and increased
the GLUT-4 protein level (Figure 6D). Collectively, these
results suggested that 4 could stimulate basal and insulin-
elicited glucose uptake through upregulation of PPARγ and
GLUT-4.
Tadehaginoside A (1): amorphous powder (MeOH); [α]2D5 −28 (c
0.1, MeOH); UV (MeOH) λmax (log ε) 270 (2.81), 307 (0.15) nm;
ECD (0.008 M, MeOH) 268.5 (Δε = −28.2), 310.5 (Δε = +12.3) nm;
IR (film) νmax 3405, 3392, 1717, 1709, 1612, 1607, 1515, 1167, 1074,
828 cm−1; 1H and 13C NMR (methanol-d4) data, see Table 1;
(+)-HRESIMS m/z 891.2327 [M + Na]+ (calcd for C42H44O20Na,
891.2324).
Tadehaginoside B (2): Aamorphous powder (MeOH); [α]2D5 −30
(c 0.1, MeOH); UV (MeOH) λmax (log ε) 269 (2.81), 308 (0.15) nm;
ECD (0.008 M, MeOH) 268.0 (Δε = +26.3), 310.5 (Δε = −13.5) nm;
1
IR (film) νmax 3428, 1718, 1620, 1514, 1189, 1070, 830 cm−1; H and
13C NMR (methanol-d4) data, see Table 1; (+)-HRESIMS m/z
891.2318 [M + Na]+ (calcd for C42H44O20Na, 891.2324).
EXPERIMENTAL SECTION
Tadehaginoside C (3): amorphous powder (MeOH); [α]2D5 −108
(c 0.03, MeOH); UV (MeOH) λmax (log ε) 225 (1.02), 277 (0.15)
nm; IR (film) νmax 3356, 1723, 1715, 1609, 1516, 1072, 827 cm−1; 1H
and 13C NMR (DMSO-d6) data, see Table 1; (+)-HRESIMS m/z
891.2322 [M + Na]+ (calcd for C42H44O20Na, 891.2324).
■
General Experimental Procedures. Optical rotations and UV
spectrometric data were recorded using a PerkinElmer 341 (San
Diego, CA, USA) and a Shimadzu UV2550 (Kyoto, Japan)
spectrometer, respectively. ECD spectroscopic data were measured
on a JASCO J-815 instrument (Kyoto, Japan). IR data were recorded
using a Shimadzu FTIR-8400S spectrometer (Kyoto, Japan). NMR
experiments were performed using a Bruker AV III 600 spectrometer
(Bruker BioSpin, Germany). HRESIMS data were obtained using an
LTQ-Orbitrap (Thermo Scientific, MA, USA) and a Q-TOF (Agilent
6540, CA, USA) instrument. A silica gel (200−300 mesh, Qingdao
Marine Chemistry Co. Ltd., Qingdao, China) column was used to
isolate the n-BuOH extract, and a Sephadex LH-20 (GE Healthcare
Biosciences AB, Uppsala, Sweden) column was used to purify the
compounds. HPLC was conducted using a Shimadzu LC-6AD system
equipped with an SPD-10A detector (Kyoto, Japan). Molecular
Operating Environment (MOE) 2014.09 (Chem Comp Group,
Canada) was applied for the molecular docking studies. Fluorescence
intensities were determined using an M1000 PRO apparatus (Tecan,
Switzerland). Centrifugation was performed using a SIGMA 3-18K
centrifuge (Sigma, Germany). GC data were recorded on a Shimadzu
GC-14C instrument (Kyoto, Japan). Rosiglitazone was purchased
Tadehaginoside D (4): amorphous powder (MeOH); [α]2D5 −58 (c
0.1, MeOH); UV (MeOH) λmax (log ε) 226 (1.02), 277 (0.15) nm; IR
(film) νmax 3407, 1717, 1612, 1515, 1072, 828 cm−1; 1H and 13C NMR
(DMSO-d6) data, see Table 1; (+)-HRESIMS m/z 891.2321 [M +
Na]+ (calcd for C42H44O20Na, 891.2324).
Tadehaginoside E (5): amorphous powder (MeOH); [α]2D5 −30 (c
0.1, MeOH); UV (MeOH) λmax (log ε) 204 (2.81), 226 (1.02), 277
(0.15) nm; IR (film) νmax 3356, 1690, 1612, 1560, 1072, 828 cm−1; 1H
and 13C NMR (methanol-d4) data, see Table 2; (+)-HRESIMS m/z
457.1104 [M + Na]+ (calcd for C21H22O10Na, 457.1111).
Tadehaginoside F (6): amorphous powder (MeOH); [α]2D5 −22 (c
0.1, MeOH); UV (MeOH) λmax (log ε) 204 (2.62), 227 (1.34), 275
(0.18) nm; IR (film) νmax 3356, 1691, 1614, 1562, 1070, 827 cm−1; 1H
and 13C NMR (methanol-d4) data, see Table 2; (+)-HRESIMS m/z
457.1112 [M + Na]+ (calcd for C21H22O10Na, 457.1111).
Tadehaginosides G and H (7 and 8): amorphous powder
(MeOH); 1H and 13C NMR (methanol-d4) data, see Table 2;
H
J. Nat. Prod. XXXX, XXX, XXX−XXX