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perature under pH 3.6 where the reduction of ferric cent yield. The ethanol extract was obtained as a
3+
tripyridyltriazine (Fe -TPTZ) to the ferrous form (blue dark reddish-brown mass in a 5.9 per cent yield.
color) can be monitored for 0-4 min absorbance change 2. Separation of biologically active compound A
at 595 nm with a plate reader (Biorad model 550). Chromatographic separation of ethanol extract
The FRAP reagent contained 10 mM TPTZ (Fluka), 40 (20.0 g) of C. comosa rhizome yielded the fractions
mM HCl (Merck), and 20 mM FeCl .6H O (Merck) at Zcc(R)-E-1 and Zcc(R)-E-2 of 11.40 g and 3.61 g, re-
2
3
the ratio of 10:1:1. The standard solution of spectively. Repeated separation of fraction Zcc(R)-E-
FeSO .7H O (Merck) and antioxidant ascorbic acid 1 using chromatograph techniques afforded active frac-
2
4
(Sigma) was used as the standard. The antioxidant tion Zcc(R)-E-15. In continuation of recrystallization,
capacity of the plant extracts was presented as μM the crystalline compound A was obtained (0.16 g).
vitamin C equivalence. The HPLC -retention time of compound A was

5. Cell Culture and Treatment Protocol 3.403 min [mobile phase: 40% MeOH:H O, wavelength
2
Human cervix adenocarcinoma cells (HeLa) were of detection 254 nm].
obtained from the National Cancer Institute (Thai- 3. Structure elucidation of the active compound
land) and cultured in minimum essential medium A
(MEM) supplemented with 10% fetal bovine serum, Compound A was obtained as a colorless needle,
1.5 g/L sodium bicarbonate, 1.0 mM sodium pyru- mp 210-212 ˚C
vate, 2 mM L-glutamine, 100 units/mL penicillin, and 1 H-NMR : δ (CD3OD) 5.95(1H, d, J= 2.0 Hz), 6.17
100 μg/ml streptomycin (Gibco BRL). The cultures ( H, d, J= 2.0 Hz), 2.69(3H, s, OCCH ).
1
3
were maintained at 37˚C in a 5 per cent CO humidi- 13 C-NMR: δ (CD3OD): 32.7(CH3), 63.0, 67.4, 70.9, 73.9,
2
a
fied atmosphere. At subconfluence, HeLa cells were 74.6, 102.2(C-6′,C-4′,C-3′,C-2′,C-5′,C-1′) , 91.6(C-3),
trypsinized and seeded to a 96-well plate at 1 × 10 5 94.9(C-5), 104.5(C-1), 160.3(C-6), 165.1(C-2), 165.4(C-
cells/well and allowed to attach overnight. Cells were 4), 202.7(CO).
then exposed to increasing concentrations of doxoru- IR: ν KBr 3434, 2920, 1637, 1604, 1466, 1367, 1288,
max
-9
-5
-1
bicin (DOX) (10 M to 10 M) or plant extracts for 48 1077, 834 cm .
hours, in triplicate. Based on spectroscopic data, compound A
6. Cytotoxicity assay was identified as 4,6-dihydroxy-2-O-(β-D-gluco-
HeLa cytotoxicity was determined by crystal vio- pyranosyl)acetophenone (1), Figure 1.
let method, as described by Takahashi . Briefly, af-
13
ter 48 hours incubation, cells were washed twice with O C CH 3 HO 6′
PBS, and fixed with 10 per cent buffered formalin. HO 1 O 1′ O 5′ 4′ OH
Prefiltered 0.1 per cent crystal violet solution in wa- 2′ OH
ter/MeOH was used to stain the live cells. Cell sur- 5 3 HO
vival was quantified by lysing the cells in 50 mM OH
sodium citrate solution in water/EtOH and measured (1)
the absorbance at 595 nM with the plate reader. To Figure 1 Compound A, 4,6-dihydroxy-2-O-(β-D-
determine the cytotoxic effect of the medicinal plant glucopyranosyl)acetophenone
extracts, IC of doxorubicin (Sigma) were evaluated
50
in comparison with the plant extracts. 4. Ferric Reducing/Antioxidant Power (FRAP)

Results Assay
The total antioxidant capacity of the plant ex-
1. Extraction of C. comosa rhizomes tracts (1 mg/mL) presented in a vitamin C equivalent
The n-hexane extract of C. comosa rhizomes was unit is shown in Table 1. Crude extract-Zcc(R) showed
obtained as a pale yellowish viscous oil in a 4.14 per the highest antioxidant power among the samples