N. X. Nhiem et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1777–1781
1781
14. Yun, K. J.; Kim, J. Y.; Kim, J. B.; Lee, K. W.; Jeong, S. Y.; Park, H. J.; Jung, H. J.; Cho,
Y. W.; Yun, K.; Lee, K. T. Int. Immunopharmacol. 2008, 8, 431.
CA2E1ꢁCA2E3. Fraction CA2E1 was chromatographed on a silica gel column
eluting with CHCl3/MeOH/H2O (55:10:1, v/v/v) to yield compound 9 (5.0 mg).
Fraction CA2E2 was chromatographed on a silica gel column eluting with
CHCl3/MeOH/H2O (35:10:1, v/v/v), yielding compound 10 (7.0 mg).
15. Guo, J. S.; Cheng, C. L.; Koo, M. W. L. Planta Med. 2004, 70, 1150.
16. Aguirre, M. C.; Delporte, C.; Backhouse, N.; Erazo, S.; Letelier, M. E.; Cassels, B.
K.; Silva, X.; Alegria, S.; Negrete, R. Bioorg. Med. Chem. 2006, 14, 5673.
17. Adnyana, I. K.; Tezuka, Y.; Banskota, A. H.; Xiong, Q.; Tran, K. Q.; Kadota, S. J.
Nat. Prod. 2000, 63, 496.
21. Ahmad, V. U.; Bano, S.; Bano, N. Phytochemistry 1986, 25, 1487.
22. Kojima, H.; Tominaga, H.; Sato, S.; Ogura, H. Phytochemistry 1987, 26, 1107.
23. Maeda, C.; Ohtani, K.; Kasai, R.; Yamasaki, K.; Duc, N. M.; Nham, N. T.; Cu, N. K.
Q. Phytochemistry 1994, 37, 1131.
24. Each compound (2.0 mg) was dissolved in 1.0 N HCl (dioxane/H2O, 1:1, v/v,
1.0 mL) and then heated to 80 °C in a water bath for 3 h. The acidic solution
was neutralized with silver carbonate and the solvent thoroughly driven out
under N2 gas overnight. After extraction with CHCl3, the aqueous layer was
concentrated to dryness using N2 gas. The residue was dissolved in 0.1 mL of
18. Han, J. T.; Bang, M. H.; Chun, O. K.; Kim, D. O.; Lee, C. Y.; Baek, N. I. Arch.
Pharmacol. Res. 2004, 27, 390.
19. The sample of Centella asiatica (L.) was collected at Tam Dao National Park,
Vinh Phuc, Vietnam, during June 2009 and identified by Dr. Ninh Khac Ban
(Institute of Ecology and Biological Resources, VAST). A voucher specimen
(IMBC CA-0609) was deposited at the Herbarium of Institute of Marine
Biochemistry, VAST.
dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine
20. The dried leaves of C. asiatica (3.0 kg) were extracted with MeOH three times
under reflux for 15 h to yield 250 g of a dark solid extract. This extract was
suspended in water and partitioned with ethyl acetate to yield ethyl acetate
extract (CA1, 100 g) and water extract (CA2, 150 g). The ethyl acetate extract
(CA1) was then subjected to chromatography on a silica gel column eluting
with a gradient of CHCl3/MeOH (from 50:1 to 5:1 v/v) yielded five fractions
CA1AꢁCA1E. Fraction CA1A was chromatographed on an LH-20 column eluting
with CHCl3/MeOH (1:1 v/v) to yield 7 (30.5 mg) and 8 (84.7 mg). Fraction CA1C
was chromatographed on a silica gel column using CHCl3/MeOH (10:1, v/v) as
an eluent to give fractions CA1C1ꢁCA1C4. The CA1C3 fraction was
chromatographed on an YMC RP-18 column eluting with MeOH/H2O (5:1 v/
v) to give 4 (60.0 mg). Fraction CA1C4 was chromatographed on an YMC RP-18
column eluting with MeOH/H2O (4:1 v/v) to yield 5 (10.5 mg). The H2O soluble
fraction (CA2) was chromatographed on a Diaion HP-20P column (Mitshubishi
Chemical Co., Japan) eluted with a step gradient of MeOH in water (0%, 25%,
50%, 75%, and 100% MeOH) yielding the five fractions, CA2AꢁCA2E. The
fraction CA2B was chromatographed on a silica gel column eluting with CHCl3/
MeOH/H2O (75:20:3, v/v/v) yielding the four fractions, CA2B1ꢁCA2B4. Fraction
CA2B2 was chromatographed on a silica gel column eluting with CHCl3/MeOH/
(0.06 M, 0.1 mL) was added to the solution. The reaction mixture was heated at
60 °C for 2 h, and 0.1 mL of trimethylsilylimidazole solution was added,
followed by heating at 60 °C for 1.5 h. The dried product was partitioned with
n-hexane and H2O (0.1 mL, each), and the organic layer was analyzed by gas
liquid chromatography (GC): Column: column SPB-1 (0.25 mm ꢀ 30 m);
detector FID, column temp 210 °C, injector temp 270 °C, detector temp
300 °C, carrier gas He (2.0 mL/min). The retention times of persilylated
glucose and rhamnose were founded to be 14.11 and 4.50 min, respectively,
when compared with the standard solutions prepared by the same reaction
from the standard monosaccharides. (The retention times of persilylated
D-
glucose, -glucose, and -rhamnose were 14.11, 14.26, and 4.50 min,
L
L
respectively).
25. White amorphous powder; ½a D25
ꢂ
ꢁ18° (c 0.1, MeOH); UV kmax (log
e
, MeOH)
;
211 (1.75) nm; IR (KBr) nmax 3407, 2954, 1728, 1231, and 1064 cmꢁ1
1H and
13C NMR are given in Table 1; ESI-MS m/z 975 [M+H]+, 997 [M+Na]+;
HR-ESI-MS m/z 975.5160 [M+H]+ (calcd for C48H79O20: 975.5165).
26. The nitrite, which accumulated in the culture medium, was measured as an
indicator of NO production by means of the Griess reaction. Briefly, 100 mL of
cell culture medium (without phenol red) was mixed with an equal volume of
Griess reagent (equal volumes of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric
acid and 0.1% (w/v) naphthylethylenediamine-HCl), incubated at room
temperature for 10 min, and then the absorbance was measured at 550 nm
using a microplate reader. Fresh culture medium was used as the blank in all
experiments. The amount of nitrite in the samples was obtained by means of
the NaNO2 serial dilution standard curve and the nitrite production was
measured.
H2O (75:20:3, v/v/v) yielded compound
6 (120.9 mg). Fraction CA2C was
chromatographed on an YMC RP-18 column eluting with acetone/H2O (2:1, v/
v) to yield four sub-fractions, CA2C1ꢁCA2C4. Fraction CA2C1 was
chromatographed on
a
silica gel column (50 g, 2 ꢀ 50 cm) eluting with
CHCl3/MeOH/H2O (30:10:1, v/v/v), yielding compound 1 (7.1 mg). Fraction
CA2C2 was further chromatographed on a silica gel column (50 g, 2 ꢀ 50 cm)
eluting with CH2Cl2/MeOH/H2O (35:10:1, v/v/v), yielding compounds
2
(10.2 mg) and 3 (8.0 mg). Fraction CA2E was chromatographed on an YMC
27. TNF-a production in the supernatant of RAW 264.7 cells was quantified using
RP-18 column eluting with acetone/H2O (1:1, v:v) to yield three fractions,
an OptEIA™ assay kits according to the manufacturer’s instructions.