Phaselic Acid Research Articles

Modification of the Lowry assay to measure proteins and phenols in covalently bound complexes

It is well established that phenols interfere with many routine protein assays and a number of protocols have been developed to overcome this. One such method is based on the differences in response obtained with the Lowry assay in the presence and absence of copper. This assumes that the phenol response with the Lowry assay is not affected by copper. However ortho-diphenols such as catechol, methylcatechol, caffeic acid, chlorogenic acid, and phaselic acid show decreased responses in the presence of copper. Three methods of estimating protein were compared for their accuracy in measuring proteins in the presence of covalently bound ortho-diphenols; the Lowry assay, the modified Lowry assay, and a new method including a calculation to take into account differences in ortho-diphenol response in the presence and absence of copper. The ortho-diphenols were caffeic acid and phaselic acid, which were bound to bovine serum albumin and red clover protein either chemically or enzymatically. For all assays, the new method gave values within 4 to 8% of control values for protein (without bound phenols) as determined by the modified Lowry method. Values for the Lowry and modified Lowry methods varied by 20–50% from control protein values. The new method also gave a good approximation of protein-bound phenol content.

Subulatin, an antioxidic caffeic acid derivative isolated from the in vitro cultured liverworts, Jungermannia subulata, Lophocolea heterophylla, and Scapania parvitexta.

The new caffeic acid derivative, subulatin (1), was isolated from in vitro cultured liverworts, Jungermannia subulata, Lophocolea heterophylla, and Scapania parvitexta. The structure of 1 involved two caffeic acids, D-glucose, and 2-carboxy-6-(1,2-dihydroxy-ethyl)-4,5-dihydroxy-5,6-dihydro-4H-pyran. The connectivity of those and the absolute stereochemistry of 1 were elucidated on the basis of spectroscopic evidence. The antioxidative activity of 1 was comparable to that of alpha-tocopherol. (2'R)-Phaselic acid (2a) and (-)-9,2''-epiphylloyl-L-malic acid (4) were also isolated from J. subulata and L. heterophylla, respectively. A chiral HPLC analysis of the p-bromobenzoyl-malic acids derived from 2a showed that 2a from J. subulata was unusual (+)-trans-caffeoyl-D-malic acid.

Bioactive Isoflavonols and Other Components from Trifolium subterraneum

Two new isoflavonols (1, 2) with insect antifeedant activity have been isolated from the trifoliates of Trifolium subterraneum (Leguminosae). The structures of the compounds were determined by spectroscopic analyses. In addition, oct-1-en-3-yl arabinopyranosyl-(1→6)-β-glucopyranoside (3), a new metabolite, and phaselic acid (4), previously obtained from Trifolium repens, were isolated and characterized.

New amino acid derivatives conjugated with caffeic acid and DOPA from red clover (Trifolium pratense).

The polyphenols in red clover were examined from the standpoint of disease resistance to the northern anthracnose. The polyphenols were identified as trans-clovamide [N-trans-caffeoyl-3-(3, 4-dihydroxyphenyl)-L-alanine] (VI), cis-clovamide [N-cis-caffeoyl-3-(3, 4-dihydroxyphenyl)-L-alanine] (X) and phaselic acid (trans-caffeoyl-L-malic acid) (II). trans-Clovamide and cis-clovamide are new compounds.