Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method.

BACKGROUND AND AIMS: Many fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals ((•)OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to 'fingerprint' (•)OH-attacked polysaccharides. METHODS: We tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during (•)OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography. KEY RESULTS: Strawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA-pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC·UA-GalA disaccharides (where pAMAC·UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC·UA-GalA; pear produced it transiently. CONCLUSIONS: GalA-pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents ((•)OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC·UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by (•)OH. The evidence shows that (•)OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). (•)OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.

Fingerprinting of hydroxyl radical-attacked polysaccharides by N-isopropyl-2-aminoacridone labelling.

Hydroxyl radicals (•OH) cause non-enzymic scission of polysaccharides in diverse biological systems. Such reactions can be detrimental (e.g. causing rheumatic and arthritic diseases in mammals) or beneficial (e.g. promoting the softening of ripening fruit, and biomass saccharification). Here we present a method for documenting •OH action, based on fluorescent labelling of the oxo groups that are introduced as glycosulose residues when •OH attacks polysaccharides. The method was tested on several polysaccharides, especially pectin, after treatment with Fenton reagents. 2-Aminoacridone plus cyanoborohydride reductively aminated the oxo groups in treated polysaccharides; the product was then reacted with acetone plus cyanoborohydride, forming a stable tertiary amine with the carbohydrate linked to N-isopropyl-2-aminoacridone (pAMAC). Digestion of labelled pectin with 'Driselase' yielded several fluorescent products which on electrophoresis and HPLC provided a useful 'fingerprint' indicating •OH attack. The most diagnostic product was a disaccharide conjugate of the type pAMAC·UA-GalA (UA=unspecified uronic acid), whose UA-GalA bond was Driselase-resistant (product 2A). 2A was clearly distinguishable from GalA-GalA-pAMAC (disaccharide labelled at its reducing end), which was digestible to GalA-pAMAC. The methodology is applicable, with appropriate enzymes in place of Driselase, for detecting natural and artificial •OH attack in diverse plant, animal and microbial polysaccharides.

Mutated Shiitake extracts inhibit melanin-producing neural crest-derived cells in zebrafish embryo.

The ability of natural extracts to inhibit melanocyte activity is of great interest to researchers. This study evaluates and explores the ability of mutated Shiitake (A37) and wildtype Shiitake (WE) extract to inhibit this activity. Several properties such as total phenolic (TPC) and total flavonoid content (TFC), antioxidant activity, effect on cell and component profiling were conducted. While having no significant differences in total phenolic content, mutation resulted in A37 having a TFC content (1.04 ± 0.7 mg/100 ml) compared to WE (0.86 ± 0.9 mg/100 ml). Despite that, A37 extract has lower antioxidant activity (EC50, A37 = 549.6 ± 2.70 µg/ml) than WE (EC50 = 52.8 ± 1.19 µg/ml). Toxicity tests on zebrafish embryos show that both extracts, stop the embryogenesis process when the concentration used exceeds 900 µg/ml. Although both extracts showed pigmentation reduction in zebrafish embryos, A37 extract showed no effect on embryo heartbeat. Cell cycle studies revealed that WE significantly affect the cell cycle while A37 not. Further tests found that these extracts inhibit the phosphorylation of Glycogen synthase kinase 3 ß (pGSK3ß) in HS27 cell line, which may explain the activation of apoptosis in melanin-producing cells. It was found that from 19 known compounds, 14 compounds were present in both WE and A37 extracts. Interestingly, the presence of decitabine in A37 extract makes it very potential for use in the medical application such as treatment of melanoma, skin therapy and even cancer.