Litchi (Litchi chinensis Sonn.) fruit is known for its rich source of phenolics. Litchi pericarp contains high levels of epicatechin that may form oligomers of various lengths. Except for several A or B type epicatechin dimers, other soluble oligomers have rarely been identified in the pericarp. Here, bioassay-guided column fractionation was applied to isolate bioactive phenolics from aqueous pericarp extract. A fraction (S3) was obtained by two rounds of Sephadex LH-20 column chromatography, and showed higher antioxidant activity and inhibition on the proliferation of human lung cancer cells (A549) than Litchi anthocyanins. S3 was further separated to isolate fractions P1–P4, which all showed higher antioxidant activity than vitamin C. P3 showed 32.9% inhibition on A549 cells at 30 μg/mL, higher than other fractions and cis-Dichlorodiamineplatinum (DDP, 0.5 μg/mL), but not as high as the combination of the four fractions. Using HPLC-Q-TOF-MS/MS, one B-type and complex A/B type epicatechin trimers were identified in P3; another B-type and two A/B-type trimers were identified in P4. P1 and P2, containing epicatechin and proanthocyanidin B2, respectively, showed no cell inhibition at 30 μg/mL. It is the first time that the two B type trimers of epicatechins (Litchitannin B1 and B2), have been found in Litchi species. The identified proanthocyanidins were detected in the pericarp of the young fruit, and the levels of the compounds decreased as the fruit developed, correlating to the decreasing patterns of the expression of LcLAR and LcANR, two key genes in the catechin biosynthesis pathway.

Anthocyanin degradation decreases ornamental or nutritional values of horticultural products. To investigate factors that may influence colour change in flower development, anthocyanin degradation was compared between the flowers of Brunfelsia calycina and Rosa chinensis, which show rapid and slow degradation, respectively. In-gel activity assays, high performance liquid chromatography (HPLC) analysis of tannins, enzyme kinetics measurement and immune-detection of anthocyanin degradation related-perioxidases (PODs) were carried out for the comparison. Rose petals possessed significantly lower anthocyanin degradation-related POD activities than Brunfelsia petals, which may be related to the high tannin contents. Epicatechin gallate (ECG) and gallocatechin gallate (GCG) were detected in rose as 161.3 ± 12.34 and 273.56 ± 41.23 μg/g FW (Fresh Weight) respectively, while not detected in Brunfelsia. ECG and GCG inhibited the activities of the Brunfelsia POD with half maximal inhibitory concentrations (IC50s) as 21.5 and 29.7 μM respectively, and increased the colour intensities of the anthocyanins. Catechin and epicatechin did not inhibit the POD activity, while serving as POD substrates, with Km (the Michaelis constant) as 0.48 and 1.23 mM. Similar protein levels of the anthocyanin degradation-related 40-kDa PODs were detected in Brunfelsia and rose. In summary, high amount of tannins, particularly ECG and GCG, in red rose petals may inhibit the degradation-related enzymes, leading to the maintenance of anthocyanins in vivo.

Brunfelsia calycina flowers lose anthocyanins rapidly and are therefore well suited for the study of anthocyanin degradation mechanisms, which are unclear in planta. Here, we isolated an anthocyanin-β-glycosidase from B. calycina petals. The MS/MS (Mass Spectrometry) peptide sequencing showed that the enzyme (72 kDa) was a β-xylosidase (BcXyl). The enzyme showed high activity to p-Nitrophenyl-β-d-galactopyranoside (pNPGa) and p-Nitrophenyl-β-d-xylopyranoside (pNPX), while no activity to p-Nitrophenyl-β-d-glucopyranoside (pNPG) or p-Nitrophenyl-β-D-mannopyranoside (pNPM) was seen. The optimum temperature of BcXyl was 40 °C and the optimum pH was 5.0. The enzyme was strongly inhibited by 1 mM D-gluconate and Ag⁺. HPLC (High Performance Liquid Chromatography) analysis showed that BcXyl catalyzed the degradation of an anthocyanin component of B. calycina, and the release of xylose and galactose due to hydrolysis of glycosidic bonds by BcXyl was detected by GC (Gas Chromatography) /MS. A full-length mRNA sequence (2358 bp) of BcXyl (NCBI No. MK411219) was obtained and the deduced protein sequence shared conserved domains with two anthocyanin-β-glycosidases (Bgln and BadGluc, characterized in fungi). BcXyl, Bgln and BadGluc belong to AB subfamily of Glycoside hydrolase family 3. Similar to BcPrx01, an anthocyanin-degradation-related Peroxidase (POD), BcXyl was dramatically activated at the stage at which the rapid anthocyanin degradation occurred. Taken together, we suggest that BcXyl may be the first anthocyanin-β-glycosidase identified in higher plants.

Abaxially anthocyanic leaves of deeply-shaded understorey plants play important ecological significance for the environmental adaption. In contrast to the transient pigmentation in other plants, anthocyanins are permanently presented in these abaxially red leaves, however, the mechanism for the pigment maintenance remains unclear. In the present study, we investigated phenolic metabolites that may affect pigment stability and degradation in Excoecaria cochinchinensis (a bush of permanently abaxial-red leaves), via a comparison with Osmanthus fragrans (a bush of transiently red leaves). High levels of galloylated anthocyanins were identified in the Excoecaria but not in the Osmanthus plants. The galloylated anthocyanin showed slightly higher stability than two non-galloylated anthocyanins, while all the 3 pigments were rapidly degraded by peroxidase (POD) in vitro. High levels of hydrolysable tannins [mainly galloylglucoses/ellagitannins (GGs/ETs)] were identified in Excoecaria but none in Osmanthus. GGs/ETs showed inhibition effect on POD, with IC50 ranged from 35.55 to 83.27 μM, correlated to the markedly lower POD activities detected in Excoecaria than in Osmanthus. Strong copigmentation was observed for GGs/ETs and anthocyanins, with more than 30% increase in the red intensity of non-galloylated anthocyanin solutions. In the leaf tissue, the hydrolysable tannins were observed to be co-localized with anthocyanins at the abaxial layer of the Excoecaria leaves, correlated to the low POD activity, more acidity and increased red intensity of the tissue. The results suggest that the Excoecaria leaves accumulate a distinct group of phenolic metabolites, mainly GGs/ETs, at the abaxial layer, which prevent anthocyanin degradation and increase the pigment stability, and consequently lead to the permanent maintenance of the red leaves.

Here, we report amorphous carbon-supported 2-3 nm-sized TiB2 nanoparticles as highly active catalyst for hydrogen storage in NaAlH4. Nano-TiB2@C was synthesized by a facile calcination process at 550 °C with Cp2TiCl2 and MgB2 (molar ratio, 1:1) as precursors. The addition of 7 wt% nano-TiB2@C reduces the onset dehydrogenation temperature of NaAlH4 by 100 °C to 75 °C, and delivers a practically available hydrogen capacity of 5.04 wt%. The 7 wt% nano-TiB2@C-containing NaAlH4 sample releases 5.04 wt% of H2 at 140 °C within 60 min, and is completely hydrogenated at 100 °C within 25 min under 100 bar of hydrogen. More importantly, hydrogen capacity remained almost unchanged after 20 cycles, representing a stable cyclability, which is greatly better than those catalyzed by Ti halides or TiO2. The highly stable catalytic function is closely related to the in situ formed Ti-Al intermetallics, which greatly facilitates the dissociation and recombination of H-H and Al-H bonding.

Recent studies have confirmed that chlorophyllase (CLH), a long-found chlorophyll (Chl) dephytylation enzyme for initiating Chl catabolism, has no function in leaf senescence-related Chl breakdown. Yet, CLH is considered to be involved in fruit degreening and responds to external and hormonal stimuli. The purpose of this work was to elucidate in detail the biochemical, structural properties, and gene expression of four CLHs from the Solanum lycopersicum genome so as to understand the roles of Solanum lycopersicum chlorophyllases (SlCLHs). SlCLH1/4 were the predominantly expressed CLH genes during leaf and fruit development/ripening stages, and SlCLH1 in mature green fruit was modulated by light. SlCLH1/2/3/4 contained a highly conserved GHSXG lipase motif and a Ser-Asp-His catalytic triad. We identified Ser159, Asp226, and His258 as the essential catalytic triad by site-directed mutagenesis in recombinant SlCLH1. Kinetic analysis of the recombinant enzymes revealed that SlCLH1 had high hydrolysis activities against Chl a, Chl b, and pheophytin a (Phein a), but preferred Chl a and Chl b over Phein a; SlCLH2/3 only showed very low activity to Chl a and Chl b, while SlCLH4 showed no Chl dephytylation activity. The recombinant SlCLH1/2/3 had different pH stability and temperature optimum. Removal of the predicted N-terminal processing peptide caused a partial loss of activity in recombinant SlCLH1/2 but did not compromise SlCLH3 activity. These different characteristics among SlCLHs imply that they may have different physiological functions in tomato.