562
H.-K. Liu et al. / Phytochemistry 70 (2009) 558–563
solution, was mounted on a Nonius CCD diffractometer equipped
with Mo radiation (k = 0.71073 Å). Crystal data: C31H50O2,
Mr = 454.71 g/mol, triclinic, P1, a = 7.3237(2) Å, b = 12.1169(3) Å,
c = 16.1701(4) Å, V = 1381.95(6) Å3, Z = 2, Dcalc = 1.093 mg/m3,
F(000) = 504. A total of 12303 reflections were collected (12303
unique, Rint = 0.0000) in the range 2.53° < h < 28.91°.The structure
was solved using direct methods and refined by Full-matrix
least-squares on F2 values. The non-hydrogen atoms were refined
anisotropically. All hydrogen atoms were fixed at calculated posi-
tions. The final indices were R1 = 0.0478, wR2 = 0.1211 with good-
ness-of-fit = 0.868. Atomic coordinates for this structure have
been deposited with the Cambridge Crystallographic Data Centre
as supplementary publication No. CCDC 688411. These data can
cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting
The Cambridge Crystallographic Data Centre, 12, Union Road, Cam-
bridge CB2 1EZ, UK; fax: +44 1223 336033.
(R)-MTPA derivative (1a): To a solution of 1 (9 mg) in dry CH2Cl2
(5 mL) were added 1,3-dicyclohexyl-carbodiimide (30 mg), 4-
dimethylaminopyridine (23 mg), and (R)-MTPA acid (20 mg). After
being stirred at room temperature for 20 h, the reaction mixture
was evaporated to dryness. The residue was purified by silica gel
column (EtOAc: n-hexane, 1:9, v/v), to yield the (R)-MTPA ester
1a: 1H NMR (CDCl3) d 0.72 (3H, s, Me-18), 0.87 (3H, d, J = 7.0 Hz,
Me-21), 0.88 (3H, s, Me-30), 0.92/0.95 (each 3H, d, J = 7.5 Hz, Me-
26/-27), 1.05/1.07/1.10 (each 3H, s), 4.58/4.65 (H2-240), 5.35 (1H,
d, J = 9.0 Hz, H-22).
(v/v) fetal calf serum (GIBCO, Carlsbad, CA) and maintained at
37 °C in an atmosphere of 5% CO2 and 95% air.
3.9. Measurement of gene expression
Based on the procedure previously described (Chen et al., 2008)
total RNA was extracted using TRI-reagent according to the manu-
facturer’s instructions. 50 ng of reverse transcribed cDNA was em-
ployed for further PCR. PCR products were separated by gel
electrophoresis, visualized and photographed with a digital cam-
era, and quantified with Genetools 3.06 (Syngene).
3.10. Preparation of cell extract for SDS PAGE and immunoblotting
Based on the procedure previously described (Chen et al., 2008),
cells were washed with ice-cold PBS and scraped into ice-cold lysis
buffer. Equal amounts of protein (40 lg) were separated using SDS
10% polyacrylamide gels. Following transfer to nitrocellulose mem-
brane, blots were blocked with 5% (w/v) non-fat milk in Tris-buf-
fered saline containing 0.1% (v/v) Tween 20 for 1 h and incubated
with primary antibodies prior to incubation with the secondary
antibody. Finally, results were visualized after development of
films with the aid of ECL kit (Amersham Biosciences).
3.11. Statistics
(S)-MTPA derivative (1b): As above, except for use of (S)-MTPA
acid (20 mg). (S)-MTPA ester 1b: 1H NMR (CDCl3) d 0.71 (3H, s, Me-
18), 0.86 (3H, d, J = 7.0 Hz, Me-21), 0.87 (3H, s, Me-30), 1.00/1.01
(each 3H, d, J = 7.0 Hz, Me-26/-27), 1.05/1.07/1.10 (each 3H, s),
4.76/4.83 (H2-240), 5.36(1H, d, J = 9.5 Hz, H-22).
The significance of various treatments was determined by the
Student’s t-test. The results were expressed as mean S.E.M. Dif-
ferences were considered significant if P < 0.05.
Acknowledgments
3.5. Gilvsin B (2)
This work was supported by National Institute of Chinese Med-
icine (NRICM96-DHM-01 and NRICM96-DHM-04), Taipei, Taiwan,
ROC.
White powder (MeOH), mp 238–240 °C; ½a D24
ꢂ
25.0 (c 0.24,
CHCl3); IR (KBr)mmax 3435 (OH), 1704 (C@O), 1640 (C@C), 1470,
1370, 1260 cmꢀ1; for 1H and 13C NMR spectroscopic data (CDCl3),
see Tables 1 and 2, respectively. EIMS (30 eV) m/z (rel. Int.) 486
References
+
[M]+(20), 387 [M ꢀ CH3–C6H12
]
(100), 369 [387-H2O]+ (38), 341
[369-CO]+ (38), 329 [M ꢀ CH3–C9H18O]+ (47); HREIMS m/z
Ahmaol, S., Hussain, G., Razaq, S., 1976. Triterpenoids of Phellinus gilvus.
Phytochemistry 15, 2000.
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Baumert, A., Schumann, B., Porzel, A., Schmidt, J., Strack, D., 1997. Triterpenoids
from Pisolithus tinctorius isolates and Ectomycorrhizas. Phytochemistry 45,
499–504.
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Gomez-Valades, A.G., Vidal-Alabro, A., Molas, M., Boada, J., Bermudez, J., Bartrons, R.,
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inhibition of hepatic phosphoenolpyruvate carboxykinase (GTP) with RNAi.
Mol. Ther. 13, 401–410.
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486.3708 [M]+ (calcd 486.3709 for C31H50O4).
3.6. Gilvsin C (3)
Amorphous powder; ½a D24
ꢂ
29.2 (c 0.24, CHCl3); IR (KBr)mmax
3434 (OH), 1698 (C@O), 1635 (C@C), 1456, 1374, 1127 cmꢀ1; for
1H and 13C NMR spectroscopic data (CDCl3), see Tables 1 and 2,
respectively. EIMS (30 eV) m/z (rel. Int.) 440 [M]+(27), 356
+
+
[M ꢀ C6H12
]
(17), 341 [M ꢀ CH3–C6H12
]
(100), 323 [341-H2O]+
(52), 283 [M ꢀ CH3–C9H18O]+ (18); HREIMS m/z 440.3648 [M]+
(calcd 440.3655 for C30H48O2).
3.7. Gilvsin D (4)
Amorphous powder; ½a D24
ꢂ
6.5 (c 0.31, CHCl3); for 1H and 13C
NMR spectroscopic data (CDCl3), see Tables 1 and 2, respectively.
EIMS (30 eV) m/z (rel. Int.) 456 [M]+ (21), 357 [M ꢀ CH3–C6H12
]
+
(100), 311 [357 ꢀ H2O–CO]+ (48), 299 [M ꢀ CH3–C9H18O]+ (40),
253 [299 - HCOOH]+ (22); HREIMS m/z 456.3600 [M]+ (calcd
456.3604 for C30H48O3).
Hosoe, T., Iizuka, T., Chiba, Y., Itabashi, T., Morita, H., Ishizaki, T., Kawai, K-I., 2006.
Relaxing effects of Phellinus gilvus extract and purified ebricoic acid on rat aortic
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Hwang, H.J., Kim, S.W., Lim, J.M., Joo, J.H., Kim, H.O., Kim, H.M., Yun, J.W., 2005.
Hypoglycemic effect of crude exopolysaccharides produced by a medicinal
mushroom Phellinus baumii in streptozotocin-induced diabetic rats. Life Sci. 76,
3069–3080.
Knight, S.A., 1974. Carbon-13 NMR spectra of some tetra- and pentacyclic
triterpenoids. Org. Magn. Reson. 6, 603–611.
3.8. H4IIE cell culture
The rat liver cell line H4IIE was cultured with Dulbecco’s Mod-
ified Eagle’s Medium (DMEM) containing 1000 mg/L glucose, 5%