Der Pharma Chemica, 2015, 7(11):354-357
Chemical constituents of Raphanus sativus
Chemical investigation of the dichloromethane extract of the freeze-dried roots of Raphanus sativus afforded 3-(E)-
(methylthio)methylene-2-pyrrolidinethione (1), a mixture of 4-methylthio-3-butenyl isothiocyanate (2) and 4-
(methylthio)butyl isothiocyanate (3), β-sitosterol (4), β-sitosteryl-3β-glucopyranoside-6'-O-palmitate (5),
monoacylglycerols (6), and a mixture of α-linolenic acid (7)and linoleic acid (8). The structures of 1-3 were
elucidated by extensive 1D and 2D NMR spectroscopy, while those of 4-8 were identified by comparison of their
NMR data with those reported in the literature.
isothiocyanate, 4-(methylthio)butyl isothiocyanate, β-sitosterol, β-sitosteryl-3β-glucopyranoside-6'-O-palmitate,
monoacylglycerols, α-linolenic acid, linoleic acid
medicinal properties. The aqueous extract of the bark of R. sativus has been reported to significantly decrease the
weight of kidney stones and shown an increase in the urine volume of rats . The fresh juice of radish exhibited
gastroprotective potential , while the radish sprout exhibited hypoglycemic activity in rats  andhas also shown
antioxidant properties in rats . The methanolic and water extracts of the radish reduced the hepatotoxicity in
albino rats , while the aqueous extract of radish seeds exhibited antibacterial properties .Another study
reported that 4-methylthio-3-butenyl isothiocyanate obtained from the radish shows antimutagenic activity ,
induced detoxification enzymes in HepG2 human hepatoma cell line , reduced cell proliferation in a dose-
dependent manner and apoptosis in colon carcinoma cell lines . Furthermore, 4-methylthiobutyl isothiocyanate
isolated from the radishincreased significantly the p21 protein expression and ERK1/2 phosphorylation in a dose-
dependent manner to inhibit PC3 cell proliferation(P≤0.01) andselectively affected cell-cycle progression and
apoptosis induction of human leukemia cells. Glucosinolates, isothiocyanates, phenolics and anthocyanins were
reported as the chemical constituents of the radish sprouts and mature taproot . The major fatty acids in seed
lipids of the radish were reported to be erucic, oleic, linoleic, and linolenic acids,while the major fatty acids in the
radish family lipids were linolenic acid (52–55%), erucic acid (30–33%), and palmiticacid (20–22%).
We earlier reported the isolation of β-sitosterol (4), unsaturated triglycerides and the essential fatty acids, linoleic
a mixture of 4-methylthio-3-butenyl isothiocyanate (2)and 4-methylthiobutyl isothiocyanate (3), and 4 from radish
roots . Furthermore, β-sitosteryl-3β-glucopyranoside-6'-O-palmitate (5), monoacylglycerols (6), a mixture of 7
and 8, and triacylglycerolswere isolated from the partially hydrolyzed radish roots . In addition to compounds
H (500 MHz) and
C (125 MHz) NMR spectra were acquired in CDCl
on a 500 MHz Agilent DD2 NMR
performedwith silica gel 60 (70-230 mesh). Thin layer chromatography was performed with plastic backed plates
coated with silica gel F
solution followed by
General Isolation Procedure
A glass column 18 inches in height and 1.0 inch internal diameter was used for the fractionation of the crude
extracts. Ten milliliter fractions were collected. Fractions with spots of the same Rf values were combined and
rechromatographed in appropriate solvent systems until TLC pure isolates were obtained. A glass column 12 inches
in height and 0.5 inch internal diameter was used for the rechromatography. Five milliliter fractions were collected.
Final purifications were conducted using Pasteur pipettes as columns. One milliliter fractions were collected.
Three (14.77) kg of radish roots was purchased from the Arranque market, Manila, Philippines inJanuary 2015.
This was identified as Raphanus sativus at the Botany Division, Philippine National Museum.
dried samples (817.87 g) were incubated with freshly blended radish (3.53 kg) and two liters of distilled water for
three hours. Two liters of CH
was added to the mixture and left in a closed vessel for three days. After filtering,
the residue was washed with one liter of CH
. The washings and supernatant were combined for concentration
and eventual drying of the sample using a rotary evaporator, which afforded an 8.5 g of crude extract.
The crude extract (8.5 g) was chromatographed by gradient elution using increasing proportions of acetone in
(10% increments) as eluents. The CH
petroleum ether to afford a mixture of 2 and 3 (9 mg) after washing with petroleum ether. The 50% acetone in
fraction was rechromatographed using 15% EtOAc in petroleum ether, followed by 20% EtOAc in
petroleum ether. The fractions eluted with 15% EtOAc in petroleum ether were combined and rechromatographed
(2 ×) using less 15% EtOAc in petroleum ether to afford 4 (25 mg) after washing with petroleum ether. The
more polar fractions were combined and rechromatographed using CH
yield a mixture of 7 and 8 (8 mg). The 60% acetone in CH
rechromatographed (3 ×) using CH
with petroleum ether.
, 500 MHz): δ 2.79 (dt, J = 2.5, 7.0 Hz, H
4), 3.67 (t, J = 7.5 Hz, H
-5), 7.58 (t, J = 2.5 Hz, H-6),2.51 (s, Me-7), 7.62 (br s, NH);
C NMR (CDCl
4-Methylthio-3-butenyl isothiocyanate (2): colorless oil.
H NMR (CDCl
, 500 MHz): δ 2.25 (s, Me), 3.53 (t, J =
6.6Hz, H2-1), 2.50 (dt, J = 7.2, 6.6 Hz, H
-2), 5.32 (dt, J =15.0, 7.2 Hz, H-3), and 6.18 (d, J =15.0 Hz, H-4);
, 125 MHz): δ 14.73 (Me), 45.13 (C-1), 33.88 (C-2), 120.04 (C-3), 129.15 (C-4), 131.39 (SCN).
4-(Methylthio)butyl isothiocyanate (3): colorless oil.
, 500 MHz): δ 2.09 (s, Me), 3.56 (t, J = 6.6
-1), 2.52 (t, J = 7.2 Hz, H
-2), 1.72 (m, H
-3), 1.80 (m, H2-4);
C NMR (CDCl
, 125 MHz): δ 15.43 (Me),
RESULTS AND DISCUSSION
Silica gel chromatography of the dichloromethane extract of R. sativus roots afforded 3-(E)-(methylthio)methylene-
2-pyrrolidinethione (1) , a mixture of 4-methylthio-3-butenyl isothiocyanate (2)  and 4-(methylthio)butyl
isothiocyanate (3) , β-sitosterol (4) , β-sitosteryl-3β-glucopyranoside-6'-O-palmitate (5) ,
monoacylglycerols (6) , and a mixture of α-linolenic acid (7)and linoleic acid (8) . The structures of 1-3
were elucidated by extensive 1D and 2D NMR spectroscopy, while those of 4-8 were identified by comparison of
their NMR data with those reported in the literature.
The pyrrolidine alkaloid, 3-(E)-(methylthio)methylene-2-pyrrolidinethione (1) previously isolated from radish
.4-Methylthio-3-butenyl isothiocyanate (2) was reported to be the principal antimutagen of the radish ,
exhibited chemopreventive effects against pancreatic carcinogenesis in hamster , and showed inhibition of
genotoxicity in in vivo and in vitro assay systems [21, 23]. It was also reported to possess antimicrobial activity
, exert free radical scavenging effects [25, 26], inhibit cell proliferation [23, 27, 28] and induce apoptosis in
human cancer cells [24, 29]. 4-(Methylthio)butyl isothiocyanate(3) exhibited in vitro antineoplastic activity and
selectivity toward leukemia cells , increased in a dose-dependent manner p21 protein expression and ERK1/2
phosphorylation to inhibit prostate adenocarcinoma cells (PC3) cell proliferation , demonstrated anti-cancer
effects (32-35], selectively affected cancer cell growth , and showed potential anti proliferative activity in
several cultured cancer cell lines [32, 36-38].
A research grant from the De La Salle University Science Foundation through the University Research Coordination
Office is gratefully acknowledged.
G. Stuart, 2014. Raphanus sativus–StuartXchange. Downloaded from www.stuartxchange.com /Labanos. html
on Sept. 7, 2014.
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