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between δ 3.18–1.22. The13C NMRspectrum of2exhibitedaromaticcarbon signalsbetween δ154.43–111.85, anomericcarbon
at δ 100.8 (C-1′), sugar carbon signals between δ 84.46–61.23, ester carbon at δ 171.61 (C-17″), methoxy carbon at δ 56.15,
methyl carbons at δ 29.15 (C-11), 26.87 (C-12), 29.93 (C-15″), 29.59 (C-16″), 26.59 (C-18″), and 13.31 (C-20″), and the
remaining methylene and methine carbons in the range δ 56.48–22.86. The shifting of the C-4′ signal to the downfield range
suggested attachment ofthe diterpenic moietyat C-4′. The 1H and 13C NMRchemical shifts ofcompound 2 were compared with
the related pimarane-type diterpenes [13–17]. The multiplicity of each carbon was determined by analysis of DEPT spectrum.
The 1H–1H COSY spectrum of 2 showed correlation of H-3 with H-4; H-1′ with H-2′ and H-3′; H-15″ with H2-3″ and H-16″;
and H3-18″ with H2-12″, H2-14″, and H2-19″. The 1H–13C HETCOR spectrum of 2 exhibited correlation of H3-11 with C-6;
H3-12 with C-9; H-3 with C-4; H-2′ with C-3′ and C-4′; and H-4′ with C-17″. The HSQC spectrum of 2 showed long-range
correlations observed as H-1′ with C-4 and H-15″ with C-9″. Acid hydrolysis of 2 yielded β-L-arabinose as a glycone moiety
(TLC comparable). The structure of 2 has been formulated as 1-methoxy-6, 8-dimethyl-β-naphthyl-β-L-arabinopyranosyl-4′-
pimaran-17″-oic acid ester. This is a new phytoconstituent isolated from Catharanthus roseus roots.
EXPERIMENTAL
Chemicals. All chemicals used were of analytical grade: Hexane, ethyl acetate, methanol, ethanol, sulfuric acid, and
vanillin were purchased from Daejung Chemicals and Metals Co. Ltd, Korea. Pre-coated TLC plates (layer thickness 0.25 mm),
silica gel for column chromatography(70–230 mesh ASTM), and LiChroprep RP-18 (40–63 μm) were from Merck (Darmstadt,
Germany). Authentic standards of β-sitosterol and D-glucose were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Instrumentation. Melting points were determined on an Electrochemical Engineering melting point apparatus
(Electrochemical Engineering Ltd., model No. IA9100 Electrothermal, Seoul, South Korea). Optical rotation was measured on
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an AA-10 model polarimeter (Instruments Ltd., Seoul, South Korea). Both H and 13C-NMR spectra were obtained with a
Bruker Avance model DRX-500 spectrometer operating at 500 and 125 MHz, respectively. This NMR machine is available
at Seoul National University (SNU), Seoul, South Korea, and all NMR spectra were obtained at SNU. NMR spectra were
obtained in deuterated chloroform, pyridine, and methanol using tetramethylsilane (TMS) as internal standard, with chemical
shifts expressed in ppm (δ) and coupling constants (J) in Hz. EIMS were recorded on a JEOL JMS-SX 102 A spectrometer and
FAB/MS on a JEOL JMS-AX 505 WA. This mass machine is available at Seoul National University, Seoul, South Korea, and
all mass spectra were obtained at SNU. IR spectra were recorded on a Thermo Mattson, Infinity Gold FT-IR (German) model
60-AR spectrophotometer. This IR machine is available at the Korea Institute of Science and Technology (KIST) Seoul, South
Korea.
Culture Conditions. The hairy root line used in this study was previously generated byinfection of C. roseus seedling
with Agrobacterium rhizogenes 15834 [18]. The culture media consisted of a filter-sterilized solution of 3% sucrose, half-
strength Gamborg’s B5 salts, and full-strength Gamborg’s vitamins with the pHadjusted to5.7. The 50-mL cultures weregrown
in 250-mL Erlenmeyer flasks to the late exponential phase in the dark at 26°C at 100 rpm.
Extraction of Hairy Roots. The powdered hairy roots of C. roseus (200 g) were immersed in methanol (1.5 liters) for
three days at room temperature and then the supernatant was concentrated under vacuum to yield 22.5 g of the extract. This
material was suspended in water and extracted with ethyl acetate and n-butanol successively to produce 11.2 g of ethyl acetate
extract and 7.4 g of n-butanol extract.
Isolation of the Compounds from Ethyl Acetate Extract. The entire ethyl acetate extract was subjected to normal
phase CC over silica gel (400 g) to yield 26 fractions (each of 250 mL) with the following eluants: fractions 1–2 with n-hexane,
fractions 3–4 with n-hexane:ethyl acetate (9:1), fractions 5–6 with n-hexane:ethyl acetate (8:2), fractions 7–8 with
n-hexane:ethyl acetate (7:3), fractions 9–10 with n-hexane:ethyl acetate (1:1), fractions 11–12 with hexane : ethyl acetate (3:7),
fraction 13–14 with ethyl acetate, fractions 15–16 with ethyl acetate:methanol (9.5:0.5), fraction 17–18 with ethyl
acetate:methanol (9:1), fractions 19–20 with ethyl acetate:methanol (7:3), fractions 21–22 with ethyl acetate:methanol (1:1),
fractions 23–24 with ethyl acetate:methanol (3:7), and fractions 25–26 in methanol. All fractions were examined by TLC.
Fractions 1–4 were not further separated due to the low amount of substance. Fractions 5–6 (0.8 g) were crystallized after
purification by CC to yielded β-sitosterol (20 mg), whose identity was confirmed through the comparison of TLC and
spectroscopic data with those of an authentic sample. Fractions 7–8 (0.6 g) were further purified by CC over silica gel (100 g;
each fraction of 100 mL) eluting with chloroform and chloroform:methanol mixtures (99:1, 98.5:1.5, 98.2, 97.5:2.5 and 97:3)
to afford 3-epibetulinic acid (120 mg). Fractions 11–12 with hexane:ethyl acetate (3:7), after re-separation with
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