Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
b
Research and Development Department, Bizen Chemical Co., Ltd., Kumayama-cho, Akaiwa-gun, Okayama 709-0716, Japan
c
Department of Interactable Diseases, International Medical Center of Japan, Shinjuku-ku, Tokyo 162-8655, Japan
d
Faculty of Pharmaceutical Sciences, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
e
High Technology Research Center, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan
f
Department of Radiation Oncology, Kawasaki Medical School, Kurashiki, Okayama 701-0192, Japan
g
Department of Applied Biological Science, Frontier Research Center for Genomic Drug Discovery, Tokyo University of Science,
Noda, Chiba 278-8510, Japan
Received 10 June 2004; accepted 9 August 2004
Abstract
We succeeded in purifying the major glycolipid fraction in the class of sulfoquinovosyl diacylglycerol, monogalactosyl diacylglycerol and
digalactosyl diacylglycerol (DGDG) from a green vegetable, spinach (Spinacia oleracea L.). This glycolipid fraction was an inhibitor of
DNA polymerases and a growth inhibitor of NUGC-3 human gastric cancer cells, and, interestingly, the activities were much stronger when
the fraction was hydrolyzed by lipase. Glycolipids in the hydrolyzed fraction consisted of sulfoquinovosyl monoacylglycerol (SQMG),
monogalactosyl monoacylglycerol (MGMG) and DGDG. In the in vivo antitumor assay using Greenes melanoma, the fraction containing
SQMG, MGMG and DGDG showed to be a promising suppressor of solid tumors. Spinach glycolipid fraction might be a potent antitumor
compound if directly injected into a tumor-carrying body, and this fraction may be a healthy food material that has antitumor activity.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Spinach; Glycolipids; DNA polymerase; Enzyme inhibitor; Antitumor effect; Cytotoxicity
1. Introduction
Abbreviations: SQDG, sulfoquinovosyl diacylglycerol; SQMG, sulfoquinovosyl monoacylglycerol; MGDG, monogalactosyl diacylglycerol;
MGMG, monogalactosyl monoacylglycerol; DGDG, digalactosyl diacylglycerol; pol, DNA-directed DNA polymerase (EC 2.7.7.7); dTTP, 2Vdeoxythymidine 5V-triphosphate; PBS, phosphate-buffered saline; TLC,
thin-layer chromatography.
B
This work was partly supported by a Grant-in-Aid for Kobe Gakuin
University Joint Research (B) (Y.M., C.M., H.I., Y.F. and H.Y.). Y.M.
acknowledges Grant-in-Aid from Hyogo Science and Technology Association, The Japan Food Chemical Research Foundation and Grant-in-Aid
16710161 for Scientific Research, The Ministry of Education, Culture,
Sports, Science and Technology, Japan.
* Corresponding author. Laboratory of Food & Nutritional Sciences,
Department of Nutritional Science, Kobe Gakuin University, Nishi-ku,
Kobe, Hyogo 651-2180, Japan. Tel.: +81 78 974 1551x3232; fax: +81 78
974 5689.
E-mail address: mizushin@nutr.kobegakuin.ac.jp (Y. Mizushina).
0955-2863/$ see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.jnutbio.2004.08.005
122
Fig. 1. Chemical structures of the glycolipids from spinach. (A) SQDG. (B)
SQMG. (C) MGDG. (D) MGMG. (E) DGDG. R1 to R7 in these structures
are fatty acids.
Fig. 2. The method of purification of glycolipid fraction (Fraction II) from dried spinach (S. oleracea L.).
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only the activity of pol a but also that of pol h in a dosedependent manner (Fig. 7). The IC50 values of pol a and rat
pol h were 21 and 91 Ag/ml, respectively. The inhibitory
effect was obviously enhanced after lipase treatment. The
inhibitory effect was strengthened about twofold. The dose
response curves in Fig. 7 did not change when activated
DNA was used as the DNA template primer instead of a
chemically synthesized DNA such as poly(dA)/oligo(dT)12
18 (data not shown). The monoglyceride forms such as
MGMG and SQMG were suggested to be better polymerase
inhibitors than the diglycerides (MGDG and SQDG). The
same tendency was observed in the cytotoxicity test
described as follows.
Fraction II could also suppress NUGC-3 cell growth in a
dose-dependent manner, and the LD50 value was 57 Ag/ml
(Fig. 8). The cell growth effect was clearly enhanced. As
described previously, some of the monoglycerides from
glycolipids showed antitumor activity [22]. We next focused
on the effects of the lipase-hydrolyzed Fraction II in in vivo
antitumor experiments.
126
Acknowledgments
We are grateful to Dr. Masaharu Takemura of the
Nagoya University and Dr. Akio Matsukage of the Japan
Womens University for preparing calf pol a and rat pol h,
respectively, and for their valuable discussions concerning
inhibitors. We thank Dr. Fumio Sugawara, Mr. Shinji
Kamisuki, Mr. Satoshi Nakazawa and Ms. Akitsu Ogawa
of the Tokyo University of Science for analyses of
NMR spectroscopy.
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