Z.-Z. Fu et al. / Phytochemistry Letters 14 (2015) 35–38
37
on a Bruker ARX-600 and ARX-150 MHz NMR spectrometer
equipped with a CH dual 5 probe. Samples were dissolved in
’
0
.6 ml DMSO-d or pyridine-d , and transferred into a 5 mm NMR
6
5
tubes. All chemical shifts are expressed as
d (ppm) relative to the
internal standard TMS ( = 0 ppm), and scalar coupling constants
d
1
1
are reported in Hz. DEPT, H– H COSY, HSQC, HMBC and NOESY
spectra were recorded using conventional pulse sequences.
Thin-layer chromatography was performed with silica gel
GF254 pre-coated plates (Qingdao Haiyang). Silica gel (200–
3
00 mesh, Qingdao Haiyang Chemical Co. Ltd., China) and
Sephadex LH-20 (Amersham Pharmacia Biotech) were used for
column chromatography. The water used in the experiment was
doubly distilled in the laboratory. Other chemicals and solvents
were of analytical grade.
3
.2. Plant material
Fig. 3. X-ray crystal structure of compound 1.
The fruits of D. carota L. were purchased in September 2007 from
Hangzhou, Zhejiang Province, PR of China, and identified by one
of the authors (Lin Zhang). A voucher specimen was deposited in
the Herbarium of the College of Biomedical Engineering and
Instrument Sciences, Zhejiang University, PR China.
glycosylated. The HMBC correlation achieved between the
0
anomeric proton at
d
H
5.05 (H-1 ) and C-11 (
d
C
81.6) also supported
a C-11 location for the sugar unit. Thus, the structure of compound
was established as (1 H, 3 H, 5 H, 7 H)-4 ,10 -dimethyl-7
isopropyl-1 -trihydroxy decahydronaphthalene-11-O-
-glucopyranoside.
Compound 1 showed no significant cytotoxicity on HUVEC
Fig. 4 left). However, it showed significant protection on H
2
b
b
b
a
a
a
b-
3
.3. Extraction and isolation
a, 3a, 4b
b-D
The air-dried fruits of D. carota L. (2 kg) were refluxed two
times with 95% aqueous EtOH. The combined EtOH extracts were
concentrated, suspended in H O, and then partitioned with
petroleum ether, CHCl , EtOAc and n-BuOH successively to give
four different polar parts. The CHCl layer (40.5 g) was fractionated
(
2 2
O -
2
induced cytotoxicity in HUVEC (Fig. 4 right). Reactive oxygen
species (ROS) are generated under various physiological and
pathological conditions such as aging, carcinogenesis, and
inflammation. An increase in intracellular ROS level has been
shown to damage tissues and cells. Therefore, the results suggest
that compound 1 could possess the ability to protect oxidative
damages for further applications.
3
3
by silica gel CC with a gradient of petroleum ether/EtOAc (7:1–1:7)
to obtain ten fractions (1–10). Fraction 8 (2.5 g) was chromato-
graphed on silica gel CC eluted with petroleum ether/EtOAc
(
3:1–1:2) to give six fractions (A1–A6). Fraction A5 was separated
by Sephadex LH-20CC with MeOH followed by repeated silica gel
CC with CHCl /EtOAc (7:3) to yield the compound 1 (78 mg).
n-BuOH layer (4.2 g) was subjected to silica gel CC with a gradient
of CHCl / MeOH (15:1–8:1) to afford eight fractions (1–8). Fraction
B7 was loaded onto a silica gel CC with CHCl /MeOH (88:12) to
3
3. Experimental
3
3.1. General experimental procedures
3
provide four fractions (B1–B4). Fraction B3 was separated by
repeated Sephadex LH-20CC with MeOH to afford compound 2
The melting points (uncorrected) were determined on a
Fisher–Johns melting point apparatus (Fisher Scientific, U.S.A.).
HR–ESI–MS and ESI–MS spectra were taken on a Bruker Daltonics
Apex III mass spectrometer. All NMR spectra were recorded
(
43.5 mg).
ꢀ
Compound 1 colorless needle crystals (MeOH); mp 112–114 C;
H NMR and C NMR (DMSO-d ) see Table 1; Positive HR–ESI–MS
6
1
13
+
m/z 295.1879 (calcd. for C15
28 4
H O Na, [M + Na] , 295.1885).
ꢀ
1
Compound 2 amorphous powder (MeOH); mp 155–157 C; H
13
NMR and C NMR (pyridine-d
m/z 457.2425 (calcd. for C21H O Na, [M + Na] , 457.2413), Positive
5
) see Table 1; Positive HR–ESI–MS
+
38 9
+
+
ESI–MS m/z 435.5 ([M + H] ), 273.5 ([M + H-162] ).
3
.4. X-ray crystallographic data of compound 1
Compound 1 was crystallized in MeOH. A monocrystal was
selected and mounted on a Gemini A Ultra CCD diffractometer
using Cu K radiation ( = 1.541 84 Å). The structure was solved
a
l
using the SHELXS-97 program and refined with SHELXL-97.
Refinement resulted in R = 0.0351 for 1. Resonant scattering,
principally from the O atoms, resulted in a Flack (Flack, 1983)
parameter X = 0.07 (19) and a Hooft 50 parameter Y = 0.06 (7) based
on 1125 Friedel pairs for 1.
3
.5. Enzymatic hydrolysis and determination of the absolute
configuration of the monosaccharide
Fig. 4. Examination of cytotoxic effect of compound 1 on HUVEC (left) and
A solution of 2 in 0.1 M acetate buffer (pH 4.0, 1.0 ml) was
2 2
protection of HUVEC cells from H O -induced cell death (right), **p < 0.01 indicates
a significant difference compared with control group.
treated with naringinase (Sigma Chemical Co., 3.0 mg) and then the