JOURNAL OF CHEMICAL RESEARCH 2009
159
system EtOAc-pyridine-EtOH-HP (8: 1 : 1 :2; v/v), comparing with
galactopyranosy 1-(I~6)-0-13- D-galactopyranosyl]
glycerol.
monosaccharide staodards aod stained by aoiline/diphenylamine/
phosphoric acid at 100°C, which showed the sugar moiety of 1/2
contained only galactose.
The complete assignment
made by the use of two-dimensional
of their spectroscopic
data were
(2D) NMR techniques,
including TOCSY, HMQC, HMBC and NOESY spectra.
Compound had the molecular formula C39H6S019
2
Alkaline hydrolysis and GC-MS analysis of1 and 2
based on HR-ESI-MS
Calcd 863.4252),
analysis (m/z 863.4255 ([M + Na]+;
indicating the existence of six degrees
Compound 1 (5.0 mg) in dry MeOH (3 mL) was treated with 5%
NaOMe-MeOH (0.5 mL) at room temperature for 10 min. The
reaction mixture was neutralised with 0.1 M HCl aod diluted with
water (10 mL). The aqueous solution was extracted with n-hexaoe
for three times, aod the orgaoic phase was combined aod dried with
anhydrous Na2S04. The n-hexaoe layer was concentrated to yield the
fatty acid methyl ester, which was identified as linolenic acid methyl
ester based on the aoalysis of GC-MS by comparison with authentic
sample (tR, 9.078 min). The same procedure was used for 2, and the
fatty acid methyl ester which was obtained, was identified as linoleic
acid methyl ester (tR, 7.834 min).
of unsaturation,
one less degree of unsaturation
than that
of 1. The compound was also obtained as white amorphous
powder. The NMR data of the sugar and glycerol moiety were
almost identical with those of 1. The only difference was the
presence of one less double bond in the acyl part judging from
the existence of two less olefinic protons in 2 (Table). Alkaline
methanolysis of 2 yielded the fatty acid methyl ester, which
was identified as linoleic methyl ester by GC- MS analysis (see
experimental part). The structure of2 was thus 1-0-(9Z,12Z-
1-0-(9Z. 12Z. 15Z-octadecatrienoyl}-3-0- [~-D-galactopyranosyl-
(1->6)-O-~-D- galactopyranosyl-(1->6)-O-~-D-
glycerol (1): White amorphous powder. [aj2oo = -15.0° (c
MeOH). IR (KEr): Vrnox 3404,3007,2923, 1735, 1645, 1460, 1139,
1069. 'H and l3C NMR see Table 1 ESI-MS (pos.): 861 [M + Na]+.
HR-ESI-MS: 861.4101 ([M + Na]+, C39H66Na019+;calcd 861.4096).
1-0-(9Z.12Z-octadecadienoyl)-3-0-[~-D-galactopyranosyl-
galactopyranosyl)
octadecadienoy 1)- 3-0- [13-D-galactopyranosyl-(
galactopyranosy 1-(I~6)-0-13- D-galactopyranosyl]
The complete assignment of their spectroscopic data followed
from the interpretation of 2D- NMR techniques, including the
TOCSY, HMQC, HMBC, and NOESY spectra.
I~6)-0-13- D-
glycerol.
=
0.1,
(1->6)-O-~-D- galactopyranosyl-(1->6)-O-~-D-
glycerol (2): White amorphous powder. [aj2oo =
galactopyranosyl)
11.4° (c 0.1,
-
=
Experimental
MeOH). IR (KEr): Vrnox 3420,3010,2923,
1069. 'H and l3C NMR see Table 1 ESI-MS (pos.): 863 [M + Nat.
HR-ESI-MS: 863.4255 ([M + Na]+, C39H68Na019+;calcd 863.4252).
1729, 1633, 1453, 1130,
High-speed
counter-current
chromatography
(HSCCC)
was
performed on a GS-lOOO instrument (Institue of Food Biological
Engineering, Zhejiaog Gongshaog University, Haogzhou, People's
Republic of China). IR spectra were recorded on a Thermo Nicolet
6700 spectrometer (Thermo, America) with KEr disks. Optical
rotations were measured on a PerkinElmer 341 polarimeter at room
temperature. NMR spectra ('H, l3C, aod 2D NMR) were recorded
on a Broker AM-500 spectrometer with TMS as ao internal staodard.
ESI-MS was recorded on a Finnigao LCQOECAMass spectrometer.
GC-MS was performed on a Agilent 5975/6890 gas chromatograph-
mass spectrometer in the electron impact (EI) mode (ionising
potential, 70 eV) with ao HP-INNOWax capillary column (30 m x
0.25 mm i.d.,0.25 flm) (Agilent, America). Helium was used as carrier
gas, aod the column temperature was ramped from 220 to 240°C at
1°C/min. TLC aoalysis was carried out on silica gel GF254 aluminum
plates (Merk, Germaoy). All solvents used were of aoalytical grade
(Haogzhou Gaojing Fine Chemical Plaot, Haogzhou, P. R. China)
The financial support by a Grant (GZ051-4 (148)) from the
Sino-German
Centre for Research Promotion
is gratefully
acknowledged.
Received 17December 2008; accepted 5 January 2009
Published online: 6 April 2009
References
1
2
V.Reshef,E. Mizraehi,T. Maretzki,C. Silberstein,S. Loya,A. Hizi and
K. Nakata,C.T.Guo,M.Matsufuji,A. Yoshimoto,M. Inagaki,R. Higuchi
D. Colombo, F. Compostella, F. Ronehetti, A. Scala, L. Toma,
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C.C. Hou, Y.P.Chen, J.H. Wu, C.C. Huang, S.Y. Wang,N.S. Yangand
Plant material
The plaot material used for this study was collected from Haogzhou
City, Zhejiaog Province, P. R. China, in May 2007, aod identified by
Prof. Xiao-Qin Hu of Haogzhou Botaoical Garden where a voucher
specimen (No.20070623H) was deposited in the Herbarium.
4
6
7
Extraction and isolation
The freeze-dried aod powdered pumpkin (3 kg) was extracted with
95% ethaool (3 x 10 L) at room temperature. After removal of the
solvent, the crude extract (350 g) was suspended in Hp (3 L),
then extracted with EtOAc aod n-BuOH (5 x 500 mL) to afford two
extracts. The BuOH-soluble fraction (100 g) was subjected to MCI
CHP20P gel CC by eluting with MeOH/Hp (2:8-8:2, v/v) to give
two major fractions, Fr.l (3.2 g) aod Fr.2 (7.5 g). Fr. 2 was purified
by HSCCC with a two-phase solvent system composed of CH2Cl/
9
E. Larsen, A. Kharazmi, L.P. Christensenand S.B. Christensen,J. Nat.
10 A. Brooo,C. Rossi,G. Mareolongo,A.D.Lena,A. Venzo,C.P.Berrieand
11 Y. Matsumoto, H. Sahara, T. Fujita, S. Hanashima, T. Yamazaki,
S. Takahashi, F. Sugawara, Y. Mizushina, K. Ohta, N. Takshashi,
12 Q.H.Li, C.L.Fu,Y.K.Rui, G.H.Hu andT.Y.Cai,Plant Food Hum. Nutr.,
2005,60, 13.
14 M. Murkovie, V. Piironen,A.M. Lampi, T. Kraushofer and G. Sontag,
n-hexaoe/EtOH/Hp
(6:2:2:4) by eluting the lower aqueous phase
at 1.5 mUmin aod 800 rpm to give a mixture of 1 and 2 (200 mg),
which was further applied to preparative RP-18 HPLC (CH3CN/H20
5.5 :4.5, v/v) to afford 1 (40 mg) aod 2 (50 mg).
Acid hydrolysis of1 and 2
Purified glyceroglycolipid was hydrolysed with 0.1 M H2S04 (3 mL)
in a sealed tube for 24 h in a boiling water bath. The reaction
mixture was neutralised with BaC03 aod filtered. The filtrate was
concentrated, aod then aoalysed by TLC on silica gel with a solvent