Osamu Negishi

Antioxidative Compounds from Crotalaria sessiliflora

Seven antioxidative compounds were isolated from the EtOAc extract of the aerial part of C. sessiliflora (Japanese name, tanukimame) by activity-guided fractionation with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Among the isolated compounds, hydroxyeucomic acid showed the strongest free radical-scavenging activity, which was almost identical to that of epigallocatechin gallate, against DPPH. Orientin and isoorientin showed strong anti-peroxidative activities toward linoleic acid and protective effects against the bactericidal action of the tert-butyl peroxyl radical. Their activities were nearly equal to that of epigallocatechin gallate.

Biosynthesis of Vanillin via Ferulic Acid in Vanilla planifolia

(14)C-Labeled phenylalanine, 4-coumaric acid, 4-hydroxybenzaldehyde, 4-hydroxybenzyl alcohol, ferulic acid, and methionine were applied to disks of green vanilla pods 3 and 6 months after pollination (immature and mature pods), and the conversion of these compounds to vanillin or glucovanillin was investigated. In mature green vanilla pods, radioactivities of 11, 15, 29, and 24% from (14)C-labeled phenylalanine, 4-coumaric acid, ferulic acid, and methionine, respectively, were incorporated into glucovanillin within 24 h. In the incorporation processes of methionine and phenylalanine into glucovanillin, some of the (14)C labels were also trapped by the unlabeled ferulic acid. However, (14)C-labeled 4-hydroxybenzaldehyde and 4-hydroxybenzyl alcohol were not converted to glucovanillin. On the other hand, in immature green vanilla pods radioactivities of the above six compounds were not incorporated into glucovanillin. Although 4-coumaric acid, ferulic acid, 4-hydroxybenzaldehyde, and 4-hydroxybenzyl alcohol were converted to the respective glucose esters or glucosides and vanillin was converted to glucovanillin, their conversions were believed to be from the detoxication of the aglycones. These results suggest that the biosynthetic pathway for vanillin is 4-coumaric acid --> --> ferulic acid --> --> vanillin --> glucovanillin in mature vanilla pods.

Effect of Polyphenol Oxidase on Deodorization

A mixture of purified polyphenol oxidases (PPO), or acetone powders prepared from fruits and vegetables, and polyphenolic compounds (PPs) totally eliminated a methylmercaptan odor. 2-Methyl-thiochlorogenic acid was isolated from the reaction mixture of methylmercaptan and chlorogenic acid with burdock acetone powder. Further, the formation of 5-methylthiochlorogenic acid and 2,5-bis(methylthio)-chlorogenic acid was suggested. These facts demonstrate that the o-quinone compounds formed from o-diphenols by PPO rapidly reacted with methylmercaptan. The oxidation reaction of PPs by using acetone powder containing PPO or peroxidase is considered to be more effective for removing bad smells from our mouths and from the environment.

Determination of hydroxycinnamic acids, hydroxybenzoic acids, hydroxybenzaldehydes, hydroxybenzyl alcohols and their glucosides by high-performance liquid chromatography

Abstract Three 4-hydroxycinnamic acids, three 4-hydroxybenzoic acids, three 4-hydroxybenzaldehydes, three 4-hydroxybenzyl alcohols and thier glucosides were separated by HPLC. HPLC analyses were performed with an octadecyl silica gel column and a gradient elution with methanol and water adjusted to pH 4.6 or 3.1. Compounds extracted from Vanilla planifolia green beans were analyzed by this method and 4-hydroxybenzaldehyde, vanillin and their respective glucosides were determined.

Biosynthesis of Caffeine from Purine Nucleotides in Tea Plant

The radioactivity of (14)C-labeled adenosine, inosine, and guanosine fed to excised tea shoots was incorporated into caffeine at a high rate (about 50% within 24 hr), after the rapid labeling of free nucleotides, while the radioactivity of xanthosine was directly incorporated into 7-methylxanthosine, and then into 7-methylxanthine, theobromine, and caffeine. Cell-free extracts from tea leaves also phosphorylated four purine nucleosides into the respective nucleotides. The phosphorylation rate was adenosine»inosine≧guanosine» xanthosine. A small amount of enzyme activity involved in the reaction from guanosine to xanthosine was detected, but the reaction from inosine to xanthosine was not observed. Based on these results, it is considered that the pathway leading to the formation of xanthosine from adenine nucleotides in caffeine biosynthesis is via AMP→IMP→XMP→xanthosine in tea plants, while there is a possibility that guanine nucleotides are converted to xanthosine via guanosine.

Content of Methylated Inositols in Familiar Edible Plants

Familiar plants contain large amounts of inositols; soybean, white clover, red clover, bush clover, locust tree, wisteria, and kudzu of the legume family contain pinitol (3-O-methyl-chiro-inositol) at approximately 200-600 mg/100 g fresh weight (FW). The contents of pinitol in other plants were 260 mg/100 g FW for sticky mouse-ear, 275 mg/100 g FW for chickweed, and 332 mg/100 g FW for ginkgo. chiro-Inositol of 191 and 156 mg/100 g FW was also found in dandelion and Japanese mallotus, respectively. Ononitol (4-O-methyl-myo-inositol) of 166 mg/100 g FW was found in sticky mouse-ear. Furthermore, young leaves of ginkgo contained sequoyitol (5-O-methyl-myo-inositol) of 287 mg/100 g FW. Hydroxyl radical scavenging activities of the methylated inositols were higher than those of the original inositols. Effective uses of these familiar edible plants are expected to promote good health.

Inositols affect the mating circadian rhythm of Drosophila melanogaster

Accumulating evidence indicates that the molecular circadian clock underlies the mating behavior of Drosophila melanogaster. However, information about which food components affect circadian mating behavior is scant. The ice plant, Mesembryanthemum crystallinum has recently become a popular functional food. Here, we showed that the close-proximity (CP) rhythm of D. melanogaster courtship behavior was damped under low-nutrient conditions, but significantly enhanced by feeding the flies with powdered ice plant. Among various components of ice plants, we found that myo-inositol increased the amplitude and slightly shortened the period of the CP rhythm. Real-time reporter assays showed that myo-inositol and D-pinitol shortened the period of the circadian reporter gene Per2-luc in NIH 3T3 cells. These data suggest that the ice plant is a useful functional food and that the ability of inositols to shorten rhythms is a general phenomenon in insects as well as mammals.

Phenylpropanoid 2,3-dioxygenase involved in the cleavage of the ferulic acid side chain to form vanillin and glyoxylic acid in Vanilla planifolia

Enzyme catalyzing the cleavage of the phenylpropanoid side chain was partially purified by ion exchange and gel filtration column chromatography after (NH4)2SO4 precipitation. Enzyme activities were dependent on the concentration of dithiothreitol (DTT) or glutathione (GSH) and activated by addition of 0.5 mM Fe2+. Enzyme activity for ferulic acid was as high as for 4-coumaric acid in the presence of GSH, suggesting that GSH acts as an endogenous reductant in vanillin biosynthesis. Analyses of the enzymatic reaction products with quantitative NMR (qNMR) indicated that an amount of glyoxylic acid (GA) proportional to vanillin was released from ferulic acid by the enzymatic reaction. These results suggest that phenylpropanoid 2,3-dioxygenase is involved in the cleavage of the ferulic acid side chain to form vanillin and GA in Vanilla planifolia. Phenylpropanoid 2,3-dioxygenase is involved in the cleavage of the ferulic acid side chain to form vanillin and glyoxylic acid in Vanilla planifolia.

In vivo deodorisation with caffeoylquinic acid derivatives

Ku-ding-cha contained a large amount of the caffeoyl quinic acid (CQA) derivatives, di-CQA, 16.7% in dry weight as well as mono-CQA, 9.2%. A mixture of 3,5-DiCQA and 5-CQA with an apple acetone powder (AP), containing polyphenol oxidase, showed high capturing activities towards methanethiol (MeSH), propanethiol (PrSH), and 2-propenethiol (AllSH). Two adducts from 3,5-diCQA and methanethiol (MeSH) were identified. Furthermore, drinking Ku-ding-cha and eating apple or prune reduced the amount of allyl methyl sulfide (AMS) and AllSH gas after garlic ingestion. These results indicate the good effects of CQA derivatives on in vivo deodorisation.