hexane elution (fractions 3±12) as colorless crystals; m. p. References
171 ± 172 8C, [a]D20: ±109.68 (c 1.0, CHCl3) {lit. [10] m.p. 170.1 8C,
1
[a]D25: ±103.58 (c 6, CHCl3)} after purification by recrystalliza-
tion from ethyl acetate in hexane. The structure of compound
1 was also confirmed by 2D-NMR and X-ray crystallographic
analysis (data not shown) which are in agreement to those pre-
viously reported [12], [13].
Cantley LC. Structure and mechanism of the (Na,K)-ATPase. Curr Top
Bioenerg1981; 11: 201±37
2 Alberts B, Bray D, Johnson A, Lewis J, Raff M, Roberts K et al. Essential
cell biology: An introduction to the molecular biology of the cell. In:
Robertson M, Lawrence E, Neal V, Vinnicombe A, editors. Garland
Publishing, Inc, New York: 1997: pp. 371±406
3 Urayama O, Nakao M. Organ specificity of rat sodium- and potassium-
activated adenosine triphosphatase. J Biochem 1979; 86: 1371±81
4 Satoh K, Yasuda I, Nagai F, Ushiyama K, Akiyama K, Kano I. The effects
of crude drugs using diuretic on horse kidney (Na+,K+)-adenosine tri-
phosphatase. Yakugaku Zasshi 1991; 111: 138±45
Compound 1 (530 mg) was methylated with CH2N2 in CH2Cl2 to
give the methyl ester 2, quantitatively as a transparent oil; [a]D20
:
±91.98 (c 1.0, CHCl3), {lit. [10] m.p. 72±75 8C, [a]D25: ±91.98 (c 7.93,
CHCl3)}. Compound 2 (100 mg) was treated with excess LiAlH4 in
dry ether to obtain compound 3 as a white solid; m.p. 133±
5 Satoh K, Nagai F, Ushiyama K, Kano I. Specific inhibition of Na+,K+-
ATPase activity by atractylon, a major component of Byaku-jutsu, by
interaction with enzyme in the E2 state. Biochem Pharmacol 1996;
51: 339±43
134 8C, [a]D20: ±51.68 (c 1, CHCl3), {lit. [10] m.p. 134±138 8C, [a]D25
±51.648 (c 1.46, CHCl3)}.
:
6 Satoh K, Nagai F, Ushiyama K, Yasuda I, Akiyama K, Kano I. Inhibition of
Na+,K+-ATPase activity by b-eudesmol, a major component of Atracty-
lodis lanceae rhizoma, due to the interaction with enzyme in the Na E1
state. Biochem Pharmacol 1992; 44: 373±8
Compound 1 (500 mg) was treated with m-CPBA in CH2Cl2 at
room temperature for 15 hours to give 16b,17-epoxykauran-19-
oic acid (4) as a white solid; m.p. 154±156 8C, [a]D20: ±98.48 (c 1,
CHCl3), {lit. [14] m.p. 157±161 8C} and 17-hydroxykaur-15-en-
19-oic acid (5) as a white solid; m.p. 187±188 8C, [a]D20: ±101.68
(c 1, CHCl3) {lit [15] m.p. 193±194 8C}. Compounds 1, 4 and 5
were previously reported in Mikania species and many genera
[16], [17]. The physical properties and spectral data of com-
pounds 1±5 were in agreement with those reported in the litera-
ture. Copies of the original spectra are obtainable from the au-
thor of correspondence.
7 Satoh K, Nagai F. Interaction of Na+,K+-ATPase by 1,2,3,4,6-penta-O-
galloyl-b-D-glucose, a major constituent of both moutan cortex and
paeoniae radix. Biochem Pharmacol 1997; 53: 611±4
8 Blatter E, Caius JF, Mhasskar KS. Indian Medicinal Plants.; Vol. III, 2nd
ed Delhi: Jayyed Press, 1975: pp. 120±1
9 Roengsumran S, Musikul K, Petsom A, Vilaivan T, Sangvanich P, Porn-
pakakul S et al. Croblongifolin, a new anticancer clerodane from
Croton oblongifolius. Planta Med 2002; 68: 271±4
10
da Costa FB, Albuquerque S, Vichnewski W. Diterpenes and synthetic
derivatives from Viguira aspillioides with trypanomicidal activity.
Planta Med. 1996; 62: 557±9
Alves TMA, Chaves PPG, Santos LMS, Nagem TJ, Murta SMF, Ceravolo IP
et al. A diterpene from Mikania obtusata active on Trypanosoma cruzi.
Planta Med 1995; 61: 85±7
Mukhopdhyay G, Mukherjee B, Patra A, Ghosh R, Roychowdhury P,
Lowe P. Refined NMR and X-ray crystallographic studies with a diter-
pene from Annona squamosa. Fitoterapia 1993; LXIV: 7±10
Brassy C, Bachet B, Wollenweber E. Acide decahydro-1,2,3,4,5,6,7,8,9,10
dimethyl-1,4a (methylene-1)ethano-7,8a phenanthrenecarboxylique-
1, acide (±)-kaurene-16 oique-19. Acta Cryst 1988; C44: 528±31
Fraga BM, Gonzalez P, Guillermo R, Hanson JR, Hernandez MG, Taka-
hashi JA. The microbiological transformation of two ent-16b,17-epo-
xykaurane derivatives by Gibberella fujikuroi. Phytochemistry 1994;
37: 717±21
Yahara S, Ishida M, Yamasaki K, Tanaka O, Mihashi S. 17-Hydroxy-ent-
kaur-15-en-oic acid and grandifloric acid. Chem Pharm Bull 1974; 22:
1629±31
Bohlmann F, Adler A, Schuster A, Gupta RK, KingRM, Robinson H. Di-
terpenes from Mikania species. Phytochemistry 1981; 20: 1899±902
Perez AL, Lara O, Devevar AR. Sesquiterpenoids and diterpenoids from
Tithonia longiradiata. Phytochemistry 1992; 31: 4227±31
Fiske C, Subbarow YH. The colorimetric determination of phosphorus.
J Biol Chem 1925; 66: 375±400
11
12
13
14
The bioassay of all compounds was carried out usingcrude rat
brain enzyme. The microsome of rat brain enzyme was prepared
accordingto Urayama [3]. Ouabain-sensitive (Na +,K+) and oua-
bain-insensitive (Mg2+)ATPase in rat brain microsome were de-
termined with and without 0.5 mM ouabain in a total volume of
420 mL for 30 min accordingto Satoh [5], [6], [7]. Ouabain, a car-
diac glycoside used as positive control, exhibits an IC50 value of
2.010±7 M against this crude enzyme preparation. The standard
assay mixture contained 3 mM ATP-tris, 5 mM MgCl2, 0.5 mM
EDTA, 140 mM NaCl, 14 mM KCl and 50 mM imidazole (pH 7.2)
with and without 0.5 mM ouabain. The stock solution of each
compound in DMSO was added to the assay mixture to give final
concentrations of 1, 0.1, and 0.01 mM for each compound. The
brain microsome sample (5 microgram/mL) was added and incu-
bation was carried out for 30 min at 37 8C. The reaction was stop-
ped by the addition of 50% trichloroacetic acid (TCA). Liberated
inorganic phosphate (Pi) was determined by the method of Fiske
and Subbarow [18]. One unit of specific activity is defined as the
liberation of 1 micromole of inorganic phosphate per mg protein
per min. The bioassay was performed in triplicate on a microtitre
plate reader (96 wells) which contained 420 mL of mixture/well.
Control was the microsomal enzyme containingDMSO in an
equivalent amount to that of the tested samples.
556
15
16
17
18
Acknowledgements
Financial support from the Thai-Japanese Joint Technical Trans-
fer Project (to NN) is gratefully acknowledged. We are also grate-
ful to Ms. Kanako Satoh and Ms. Takako Seto, Tokyo Metropolitan
Research Laboratory of Public Health, Japan for research facilities
and bioassay testing.
Letter¼ Planta Med 2003; 69: 555±556