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Garlic has been reported to have chemopreventive effects against a variety of cancers. However, different garlic preparations contain different constituents. We investigated the chemopreventive effect of aged garlic extract (AGE), an odorless product from prolonged extraction of fresh garlic, on colon carcinogenesis and cell proliferation in 1,2-dimethylhydrazine (DMH)-induced colon neoplastic rats. Rats were given weekly subcutaneous injections of DMH (20mg/kg) for 20 wk, and fed either a basal diet or one containing 4% AGE. Serum from AGE-treated rats contained detectable S-allylcysteine. The AGE diet significantly reduced the number of colon tumors and aberrant crypt foci compared to the basal diet. Cell proliferation of normal-appearing colonic mucosa was assessed by MIB-5 immunohistochemistry. AGE treatment significantly decreased the mean MIB-5-1abeling index. These findings suggest AGE has a chemopreventive effect on colon carcinogenesis through suppression of cell proliferation. KEY WORDS: * aged garlic extract * S-allylcysteine * colon carcinogenesis * chemoprevention * cell proliferation

Because colorectal cancer is likely to develop in many people at some point during their lives, prevention has become a high priority. Diet and nutrition play an important role during the multistep colon carcinogenic process. Garlic has been traditionally used as a spice and is well known for its medicinal properties; several studies have indicated its pharmacologic functions, including its anticarcinogenic properties. However, the mechanisms by which garlic can prevent colorectal cancer remain to be elucidated. This study investigated the effect of aged garlic extract (AGE) on the growth of colorectal cancer cells and their angiogenesis, which are important microenvironmental factors in carcinogenesis. AGE suppressed the proliferation of 3 different colorectal cancer cell lines--HT29, SW480, and SW620--in the same way, but its effects on the invasive activities of these 3 cell lines were different, the invasive activities of SW480 and SW620 cells were inhibited by AGE, whereas AGE had no effect on the invasive activity of Ht29 cells. The action of AGE appears to be dependent on the type of cancer cell. On the other hand, AGE enhanced the adhesion of endothelial cells to collagen and fibronectin and suppressed cell motility and invasion. AGE also inhibited the proliferation and tube formation of endothelial cells potently. These results suggest that AGE could prevent tumor formation by inhibiting angiogenesis through the suppression of endothelial cell motility, proliferation, and tube formation. AGE would be a good chemopreventive agent for colorectal cancer because of its antiproliferative action on colorectal carcinoma cells and inhibitory activity on angiogenesis. KEY WORDS: * aged garlic extract * colon cancer * angiogenesis * chemoprevention

A unique garlic preparation, aged garlic extract (AGE), was examined for its modifying effect on diethylnitrosamine (DEN)-induced neoplasia of the liver in male F344 rats, using the medium-term bioassay system based on the 2-step model of hepatocarcinogenesis. Carcinogenic potential was scored by comparing the numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci. GST-P-positive foci were significantly decreased in rats treated with AGE at doses of 2, 5, and 10 mL/kg, i.g., 5 times per week during the promotion phase. In addition, to clarify the mechanism underlying the inhibitory effect of AGE, the effect of AGE on hepatocellular proliferation was evaluated using partially hepatectomized rats as a liver-regeneration model. The bromodeoxyuridine-labeling indices in the livers of the AGE group were significantly lower than those in the control group at 24 h, the maximum proliferation period after partial hepatectomy. These findings indicate that AGE inhibited the development of putative preneoplastic lesions in rat hepatocarcinogenesis, involving a slowing in the proliferation rate of liver cells after partial hepatectomy. KEY WORDS: * hepatocarcinogenesis * aged garlic extract * chemoprevention * medium-term bioassay system * glutathione S-transferase

S-Alk(en)yl-L-cysteine sulfoxides are pharmaceutically important secondary metabolites produced by plants that belong to the genus Allium. Biosynthesis of S-alk(en)yl-L-cysteine sulfoxides is initiated by S-alk(en)ylation of glutathione, which is followed by the removal of glycyl and γ-glutamyl groups and S-oxygenation. However, most of the enzymes involved in the biosynthesis of S-alk(en)yl-L-cysteine sulfoxides in Allium plants have not been identified. In this study, we identified three genes, AsGGT1, AsGGT2, and AsGGT3, from garlic (Allium sativum) that encode γ-glutamyl transpeptidases (GGTs) catalyzing the removal of the γ-glutamyl moiety from a putative biosynthetic intermediate of S-allyl-L-cysteine sulfoxide (alliin). The recombinant proteins of AsGGT1, AsGGT2, and AsGGT3 exhibited considerable deglutamylation activity toward a putative alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine, whereas these proteins showed very low deglutamylation activity toward another possible alliin biosynthetic intermediate, γ-glutamyl-S-allyl-L-cysteine sulfoxide. The deglutamylation activities of AsGGT1, AsGGT2, and AsGGT3 toward γ-glutamyl-S-allyl-L-cysteine were elevated in the presence of the dipeptide glycylglycine as a γ-glutamyl acceptor substrate, although these proteins can act as hydrolases in the absence of a proper acceptor substrate, except water. The apparent K m values of AsGGT1, AsGGT2, and AsGGT3 for γ-glutamyl-S-allyl-L-cysteine were 86 μM, 1.1 mM, and 9.4 mM, respectively. Subcellular distribution of GFP-fusion proteins transiently expressed in onion cells suggested that AsGGT2 localizes in the vacuole, whereas AsGGT1 and AsGGT3 possess no apparent transit peptide for localization to intracellular organelles. The different kinetic properties and subcellular localizations of AsGGT1, AsGGT2, and AsGGT3 suggest that these three GGTs may contribute differently to the biosynthesis of alliin in garlic.

1,2,3,4-Tetrahydro-beta-carboline derivatives (THbetaCs) are formed through Pictet-Spengler chemical condensation between tryptophan and aldehydes during food production, storage and processing. In the present study, in order to identify the antioxidants in aged garlic extract (AGE), we fractionated it and identified four THbetaCs; 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acids (MTCC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-1,3-dicarboxylic acid (MTCdiC) in both diastereoisomers using liquid chromatography mass spectrometry (LC-MS). Interestingly, these compounds were not detected in raw garlic, but the contents increased during the natural aging process of garlic. In in vitro assay systems, all of these compounds have shown strong hydrogen peroxide scavenging activities. (1S, 3S)-MTCdiC was found to be stronger than the common antioxidant, ascorbic acid. MTCC and MTCdiC inhibited AAPH-induced lipid peroxidation. Both MTCdiCs also inhibited LPS-induced nitrite production from murine macrophages at 10-100 microM. Our data suggest that these compounds are potent antioxidants in AGE, and thus may be useful for prevention of disorders associated with oxidative stress.