Enzyme-Treated Asparagus Extract Prevents'Hydrogen Peroxide-Induced Pro-Inflammatory Responses by Suppressing p65 Nuclear Translocation in Skin L929 Fibroblasts.

We recently reported that enzyme-treated asparagus extract (ETAS) attenuates hydrogen peroxide (H(2)0(2))-stimulated matrix metalloproteinase-9 expression in skin fibroblast L929 cells. To further elucidate the anti-aging effects of ETAS on skin, we examined whether ETAS has preventive effects on H202-induced pro-inflammatory responses of skin fibroblasts. H(2)0(2) induced Ser536 phosphorylation and nuclear accumulation of nuclear factor-κB (NF-κB) p65, and increased the mRNA levels .of interleukin-12α (IL-12α)-and inducible nitric oxide synthase (iNOS) in L929 cells. Pretreatment of the cells with JSH-23, an inhibitor of NF-κB nuclear translocation, abolished the H(2)(0(2)-induced expression of IL-12α and iNOS, indicating that the increased transcription is regulated by p65. The H(2)0(2)-stimulated nuclear accumulation of p65 and-induction of IL12a and iNOS mRNA were significantly attenuated after pretreatment with ETAS for 3 h, and these responses were completely abolished when the duration was extended to 24 h. However, ETAS did not affect the H(2)0(2)-stimulated degradation of IκBα and phosphorylation of p65. On the other hand, ETAS treatment for 24 h resulted in decreased protein levels of importin-α. These results suggest that ETAS prevents pro-inflammatory responses by suppressing the p65 nuclear translocation in skin fibroblasts induced by H202.

Isolation, structural elucidation, and biological evaluation of a 5-hydroxymethyl-2-furfural derivative, asfural, from enzyme-treated asparagus extract.

A novel 5-hydroxymethyl-2-furfural (HMF; 1) derivative, which is named asfural (compound 2), was isolated from enzyme-treated asparagus extract (ETAS) along with HMF (1) as a heat shock protein 70 (HSP70) inducible compound. The structure of compound 2 was elucidated on the basis of its spectroscopic data from HREIMS and NMR, whereas the absolute configuration was determined using chiral HPLC analysis, compared to two synthesized compounds, (S)- and (R)-asfural. As a result, compound 2 derived from ETAS was assigned as (S)-(2-formylfuran-5-yl)methyl 5-oxopyrrolidine-2-carboxylate. When compound 2, synthesized (S)- and (R)-asfural, and HMF (1) were evaluated in terms of HSP70 mRNA expression-enhancing activity in HL-60 cells, compound 2 and (S)-asfural significantly increased the expression level in a concentration-dependent manner. HMF (1) also showed significant activity at 0.25 mg/mL.

Structures and antioxidant and intestinal disaccharidase inhibitory activities of A-type proanthocyanidins from peanut skin.

Nine compounds including a new A-type proanthocyanidin trimer, epicatechin-(2ß→O→7,4ß→8)-[catechin-(6→4ß)]-epicatechin (8), and a known trimer, epicatechin-(4ß→8)-epicatechin-(2ß→O→7,4ß→8)-catechin (9), being reported for peanut skin for the first time, were isolated and purified. Their structures were determined by spectroscopic methods and by degradation reactions with L-cysteine in acidic conditions. The DPPH radical scavenging activity and the inhibitory activity on maltase and sucrase of the isolated compounds were investigated. All compounds showed strong DPPH scavenging activities (EC50 < 20 µg/mL). Compound 8 showed the strongest inhibitory activity on maltase with an IC50 value of 0.088 mg/mL, while compound 9 exhibited the strongest inhibition on sucrase with an IC50 value of 0.091 mg/mL.

Purification and characterization of fructan and fructansucrase from Lactobacillus fermentum AKJ15 isolated from Kodo ko jaanr, a fermented beverage from north-eastern Himalayas.

Fructansucrase and fructan produced from Lactobacillus fermentum AKJ15 were isolated from seeds of Kodo ko jaanr, a fermented mild-alcoholic beverage prepared in North East India. The strain was identified by 16S rRNA gene sequence analysis and biochemical characterization. The strain displayed maximum fructansucrase activity of 4.3 U/ml (1.02 U/mg) at 28°C at 180 rpm. The enzyme purified by polyethylene glycol-400 gave specific activity of 5 U/mg and showed 90 kDa band on non-denaturing Sodium Dodecyl Sulphate-Poly Acrylamide Gel Electrophoresis (SDS-PAGE). The purified enzyme confirmed the presence of fructan by periodic acid Schiff's staining which showed magenta colour bands with both sucrose and raffinose. The strain produced 10.2 mg/ml fructan in broth under optimized culture conditions. The purified fructansucrase displayed V(max) of 5.42 U/mg and K(m) of 16.65 mM. The enzyme showed maximum activity at 30°C and at pH 5. The structure of fructan was analysed by (1)H and (13)C NMR spectra confirming ß-(2-1) and ß-(2-6) linkages.

Fusion proteins comprising the catalytic domain of mutansucrase and a starch-binding domain can alter the morphology of amylose-free potato starch granules during biosynthesis.

It has been shown previously that mutan can be co-synthesized with starch when a truncated mutansucrase (GtfICAT) is directed to potato tuber amyloplasts. The mutan seemed to adhere to the isolated starch granules, but it was not incorporated in the starch granules. In this study, GtfICAT was fused to the N- or C-terminus of a starch-binding domain (SBD). These constructs were introduced into two genetically different potato backgrounds (cv. Kardal and amf), in order to bring GtfICAT in more intimate contact with growing starch granules, and to facilitate the incorporation of mutan polymers in starch. Fusion proteins of the appropriate size were evidenced in starch granules, particularly in the amf background. The starches from the various GtfICAT/SBD transformants seemed to contain less mutan than those from transformants with GtfICAT alone, suggesting that the appended SBD might inhibit the activity of GtfICAT in the engineered fusion proteins. Scanning electron microscopy showed that expression of SBD-GtfICAT resulted in alterations of granule morphology in both genetic backgrounds. Surprisingly, the amf starches containing SBD-GtfICAT had a spongeous appearance, i.e., the granule surface contained many small holes and grooves, suggesting that this fusion protein can interfere with the lateral interactions of amylopectin sidechains. No differences in physico-chemical properties of the transgenic starches were observed. Our results show that expression of granule-bound and "soluble" GtfICAT can affect starch biosynthesis differently.