Page 27 - วารสารกรมการแพทย์แผนไทยฯ ปีที่ 5 ฉบับที่ 1

Journal of Thai Traditional & Alternative Medicine Vol. 5 No. 1 January - April 2007 Úı

found that the hexane extract was effective in the umn chromatography was used.
uterotropic activity by increasing uterine weight and 3. Instruments
glycogen content in rats, whereas the ethylacetate 3.1 Ultraviolet (UV) spectrophotometer (Jasco,
extract exhibited the hypolipidemic activity in mice. Uvidec 650)
The isolation and initial assessment of the 3.2 Gemini 2000 NMR Spectrometer (Varian,
13
1
hypolipidemic effect of two new phenolic USA) for H and C-NMR
diarylheptanoids of C. comosa was reported in 1997. 3.3 JASCO A 302 Spectrophotometer for IR
Since C. comosa expresses assorted activities, determination
the search for new biological activities with respon- 3.4 Seisakusho micro melting point
sible compounds should be actively continued. In this 3.5 High performance liquid chromato-
paper, we report a bioassay-guided isolation of the graphy : Waters 600 Controller
cytotoxic and anti-oxidative constituent from the rhi- Detector: Waters 996 Photodiode Array Detector
zome of C. comosa. 3.6 Plate reader (Biorad model 550)

Methods
Materials and Methods
Materials 1. Extraction of C. comosa rhizomes
Pulverized rhizome (1 kg) was extracted succes-

1. Plant material sively with n-hexane and with ethanol in a Soxhlet
Fresh rhizomes of Waan chak mod luuk were extractor to yield dried extract of 41.42 g and 59.05 g,
collected from a plantation in Nakhon Pathom prov- respectively.
ince, Thailand in November 2000 and identified, based 2. Separation of biologically active compound A
on the plant taxonomy, as Curcuma comosa Roxb. 1,9,10 The ethanol extract (20 g) was chromatographed
The voucher specimens (Department of Medical Sci- on silica gel 60 using a gradient of ethylacetate-metha-
ences Herbarium No. 1464) were deposited at the nol as the eluting solvent to yield two fractions of
Department of Medical Sciences Herbarium. The fresh Zcc(R)-E-1 and Zcc(R)-E-2. Biologically guided sepa-
rhizomes were washed, sliced and dried in an oven ration led to the re-chromatography of fraction Zcc(R)-
at 40-50˚C. The dried rhizomes were ground and E-1 (11.40 g) with silica gel 60 twice and finally with
kept at room temperature in well-sealed closed ves- Cosmosil 75C18-OPN reversed-phase column chroma-
sels. tography. The active compound was obtained in

2. Chemicals methanol water fraction. After recrystallization in
2.1 Solvents -chloroform, ethyl acetate, etha- methanol, the purification of the active compound
nol were of commercial grade and were purified by was confirmed by subjecting it to HPLC using X
distillation before use. Terra RP column (5μm, 4.6 × 150 mm), 40 per
TM
18
-methanol was of analytical re- cent methanol in water as the eluent and the Waters
agent grade. 996 photodiode array as the detector.
2.2 Chemicals used - anisaldehyde-sulfuric 3. Structure elucidation of the active compound
11
acid spraying reagent (0.5% ethanolic solution of A
anisaldehyde with 5% sulfuric acid). The structure of the active compound was elu-
1
2.3 Siliga gel 60 for column chromatography cidated by spectroscopic techniques using H-NMR,
with particle sizes 0.063-0.200 mm, siliga gel GF 254 13 C-NMR and IR. The melting point of the compound
precoated plate for thin-layer chromatography and was also determined and uncorrected.
siliga gel PF for preparative-layer chromatography 4. Free Reducing/Antioxidant Power (FRAP)
254
were obtained from E. Merck, Germany. Cosmosil Assay
12
75C -OPN (nacalai tesque) for reversed-phase col- The FRAP assay was conducted at room tem-
18