2
10
Vol. 57, No. 2
ꢁ1
Yantai Chemical Plant, Yantai, P. R. China. Silica gel H: 10—40 mm, Yantai IR (KBr) cm : 3417, 3068, 3036, 2917, 1734, 1248, 1079. UV l
max
1
Chemical Plant, Yantai, P. R. China. Sephadex LH-20: Pharmacia. TCL: (MeOH) nm (log e): 268 (0.91), 262 (1.02), 256 (1.14), 204 (4.03) nm. H-
1
3
HSGF254 precoated silica gel plates, 10—40 mm, Yantai Chemical Plant, NMR (600 MHz, DMSO) and C-NMR (150 MHz, DMSO): Table 2. HR-
ꢀ
Yantai, P. R. China.
ESI-MS m/z: 360.10552 ([MꢀNa] , C H NNaO ; Calcd 360.10592).
16 19 7
Plant Material Dried, whole plants of Dracocephalum peregrinum col-
Cell Lines Used RAW 264.7, 293, were obtained from Shanghai Insti-
lected in Xinjiang autonomous region, P. R. China, in August 2007. The tute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), and
plant material was authenticated by Prof. Han-min Zhang. Voucher speci- maintained in media recommended by the suppliers supplemented with 10%
mens (NO. DP070901) are deposited at the Department of Phytochemistry, fetal bovine serum (FBS) (Gibco, Paisley, U.K.), and streptomycin (100
School of Pharmacy, the Second Military Medical University, China.
mg/ml) in a humidified 5% CO atmosphere at 37 °C.
2
Extraction and Isolation The air-dried and powdered whole plants of
Measurement of NO in LPS Activated Macrophages RAW 264.7
5
Dracocephalum peregrinum (10 kg) were refluxed with ethanol (75% v/v) macrophages were seeded onto 24-well cell culture plates (10 cells/well).
three times, 2 h for each. After removal of the solvent under reduced pres- The cells were co-incubated with drugs and LPS (1 mg/ml) for 24 h. The
sure, the residue was partitioned sequentially with petroleum ether, chloro- amount of NO was assessed by determining the nitrite concentration in the
form, ethyl acetate, n-butanol to give four portions. The ethyl acetate portion
culture supernatants with Griess reagent in RAW 264.7. Aliquots of super-
(
1
5
98 g) was subjected to silica gel column chromatography (200—300 mesh, natants (100 ml) were incubated, in sequence, with 50 ml 1% sulphanilamide
.0 kg), eluting with the gradient CHCl : CH OH (30 : 1—15 : 1—10 : 1—
and 50 ml 0.1% naphthylethylenediamine in 2.5% phosphoric acid solution.
3
3
: 1—2 : 1—1 : 1), and gave six fractions: I (10 g), II (11 g), III (12 g), IV The absorbances at 570 nm were read using a microtiter plate reader.
(
15 g), V (13 g), VI (11 g).
NF-kB Activation Assay NF-kB activation was assayed using stable
Fraction III (12 g) was subject to silica gel column chromatography pNF-kB-luc-293 cells (Baran et al., 2007). The cells seeded in 96-well plate
5
(200—300 mesh, 100 g), eluting with the gradient CHCl : CH OH (30 : 1—
at 1.0ꢃ10 cells/well were pre-treated with test drugs for 15 min, and then
incubated with 10 mg/ml recombinant human tumor necrosis factor (TNF-a)
3
3
1
5 : 1—10 : 1—5 : 1—2 : 1—1 : 1), to afford six subfractions (Ea1—Ea6),
Ea4 was purified over silica gel column chromatography, eluting with for 6 h. The cells were lysed, and luciferase activity was measured with a lu-
CHCl : CH OH (20 : 1—10 : 1—5 : 1), to yield compound: 1 (12 mg). Frac- ciferase assay system (Promega, WI, U.S.A.).
3
3
tion IV (15 g) was purified over silica gel column chromatography, eluting
with CHCl : CH OH (15 : 1—8 : 1—4 : 1) to afford three subfractions
References
3
3
(
Eb1—Eb3), Eb2 subfraction purified over Sephadex LH-20 for two times,
1) Editorial Board of Flora of China of Chinese Academy of Sciences,
Flora of China, 65, 346—384 (1977).
to yield compounds: 2 (15 mg) and 3 (13 mg). Fraction V (13 g) was purified
over silica gel column chromatography, eluting with CHCl : CH OH (8 : 1—
2) Editorial Board of Zhong Hua Ben Cao of State Administration of
Traditional Chinese Medicine, ZhongHua BenCao, 7, 27—33 (1999).
3) Li J. B., Ding Y., Chin. J. Chin. Mater. Med., 26, 697—698 (2001).
4) Gu H. F., Chen R. Y., Sun Y. H., Liu F., Chin. J. Chin. Mater. Med., 29,
232—234 (2004).
5) Janicsák G., Veres K., Kakasy A. Z., Máthé I., Biochem. System. Ecol.,
34, 392—396 (2006).
6) Saeidnia S., Gohari A. R., Uchiyama N., Ito M., Honda G., Kiuchi F.,
Chem. Pharm. Bull., 52, 1249—1250 (2004).
3
3
4
: 1—2 : 1—1 : 1) to afford four subfractions (Ec1—Ec4), and Ec3 was puri-
fied over Sephadex LH-20 several times, to yield compound: 4 (20 mg).
Peregrinumin A (ꢂAacacetin-7-O-(2,3-O-diacetyl-a-L-rhamnosyl)-(1→
2
5
6
(
)-b-D-glucoside, 1): Yellow amorphous powder (CH OH). [a] ꢂꢁ78°
3 D
ꢁ1
cꢂ0.1250, CH OH). IR (KBr) cm : 3421, 2923, 2852, 1741, 1656, 1605.
3
1
UV l
(
(MeOH) nm (log e): 205 (3.49), 269 (1.93), 323 (2.37). H-NMR
max
13
600 MHz, DMSO) and C-NMR (150 MHz, DMSO): Table 1. HR-ESI-MS
ꢀ
m/z: 699.18948 ([MꢀNa] C H NaO ; Calcd 699.1901).
3
2
36
16
Peregrinumin B (ꢂAcacetin-7-O-(2,4-O-diacetyl-a-L-rhamnosyl)-(1→6)-
7) Uchiyama N., Kiuchi F., Ito M., Honda G., Takeda Y., J. Nat. Prod.,
66, 128—131 (2003).
8) Dastmalchi K., Damien-Dorman H. J., Laakso I., Hiltunen R., LWT-
Food Sci. Technol., 40, 1655—1663 (2007).
9) Monsef-Esfahani H. R., Karamkhani F., Nickavar B., Abdi K., Fara-
marzi M. A., Chem. Nat. Comp., 43, 40—43 (2007).
10) Lu M., Tian X., Acta Pharm. Sinica, 34, 925—927 (1999).
11) Zeng J. W., Qian S. H., Wu J. Z., Yang N. Y., China Journal of Chinese
Meteria Medica., 31, 400—402 (2006).
2
5
b-D-glucoside, 2): Yellow amorphous powder (CH OH). [a] ꢂꢁ77°
3
D
ꢁ1
(
cꢂ0.1400, CH OH). IR (KBr) cm : 3421, 2924, 1733, 1655, 1605. UV
lmax (MeOH) nm (log e): 204 (3.64), 269 (1.93), 289 (2.37), 326 (2.34). H-
NMR (600 MHz, DMSO) and C-NMR (150 MHz, DMSO): Table 1. HR-
3
1
1
3
ꢀ
ESI-MS m/z: 699.18856 ([MꢀNa] , C H NaO ; Calcd 699.1901).
3
2
36
16
Peregrinumin C (ꢂAcacetin-7-O-(3,4-O-diacetyl-a-L-rhamnosyl)-(1→6)-
2
5
b-D-glucoside, 3): Yellow amorphous powder (CH OH). [a] ꢂꢁ90°
3
D
ꢁ1
(
cꢂ0.1500, CH OH). IR (KBr) cm : 3421, 2924, 1733, 1656, 1605. UV
lmax (MeOH) nm (log e): 203 (4.87), 268 (4.19), 323 (4.65), 360 (2.18). H- 12) Zhang L. Z., Guo Y. J., Tu G. Z., Shi R. B., Qin W., Ba Y. Y., Yang F.,
NMR (600 MHz, DMSO) and C-NMR (150 MHz, DMSO): Table 1. HR-
3
1
1
3
Acta Pharmaceutica Sinica, 43, 169—172 (2008).
13) Seigler D. S., Pauli G. F., Nahrstedt A., Leen R., Phytochemistry, 60,
873—882 (2002).
ꢀ
ESI-MS m/z: 699.18904 ([MꢀNa] C H NaO ; Calcd 699.1901).
3
2
36
16
Peregrinumcin A (ꢂ(2R)-b-D-(6-O-Acetyl)-glucosyl-2-phenylacetonitrile,
2
5
4
): Colourless needle crystal (CH OH). [a] ꢂꢁ35° (cꢂ0.1950, CH OH).
3
D
3