The abundance of epiphytic liverworts on the bark of Cryptomeria japonica in relation to different physical and biochemical attributes, found in Senchal Wildlife Sanctuary, Darjeeling, Eastern Himalaya

Background Maintenance of biodiversity is an integral part of sustainable forest management. Epiphytic bryophytes are an important element of biodiversity. Thus, this work aims to study the role of different physical and biochemical factors in affecting the growth and proliferation of epiphytic liverworts. Fifty trees in three different plots, distributed in Senchal wildlife sanctuary, Darjeeling, were surveyed. Factors such as light intensity, moisture, and diameter at breast height (DBH) of the tree were studied to evaluate their possible role in affecting epiphytic liverworts. The effect of bark biochemical characteristics on the abundance of epiphytic liverworts was also studied by undertaking a quantitative test of pH, phenol, flavonoid, ortho-dihydric phenol, terpene, total sugar, and tannin. Multiple regression analysis and principal component analysis (PCA) were carried out to test the effects of these parameters. Results Light intensity, moisture, and DBH highly influenced the abundance of liverworts. Old trees had higher epiphytic liverwort cover than younger ones. Bark biochemical properties like pH, phenol, flavonoid, ortho-dihydric phenol, tannin and sugar did not have a significant effect on the epiphytic liverwort cover, while the terpenoid content of the bark reduced liverworts cover. Conclusion To sustain the occurrence of epiphytic liverworts in ecosystems, forest management should ensure the presence of old trees. Light intensity and moisture had a large effect on the distribution and abundance of liverworts, so it is important to maintain tree cover, shrub layer, and tree density.

Tyrosinase kinetics: failure of the auto-activation mechanism of monohydric phenol oxidation by rapid formation of a quinomethane intermediate

When 3,4-dihydroxybenzylcyanide (DBC) is oxidized by mushroom tyrosinase, the first visible product, identified as the corresponding quinomethane, exhibits an absorption maximum at 480 nm. Pulse-radiolysis experiments, in which the o-quinone is formed by disproportionation of semiquinone radicals generated by single-electron oxidation of DBC, showed that the quinomethane (A480 6440 M-1·cm-1) is formed through the intermediacy of the o-quinone with a rate constant at neutral pH of 7.5 s-1. The oxygen stoichiometry of the formation of the quinomethane by tyrosinase-catalysed oxidation of DBC was 0.5:1. On the basis of oxygen utilization rates the calculated Vmax was 4900 nmol·min-1 and the apparent Km was 374 µM. The corresponding monohydric phenol, 4-hydroxybenzylcyanide (HBC), was not oxidized by tyrosinase unless the enzyme was pre-exposed to DBC, the maximum acceleration of HBC oxidation being obtained by approximately equimolar addition of DBC. These results are consistent with tyrosinase auto-activation on the basis of the indirect formation of the dihydric phenol-activating cofactor. The rapid conversion of the o-quinone to the quinomethane prevents the formation of the catechol by reduction of the o-quinone product of monohydric phenol oxidation from occurring in the case of the compounds studied. In the absence of auto-activation, the kinetic parameters for HBC oxidation by tyrosinase were estimated as Vmax 70 nmol·min-1 and Km 309 µM. The quinomethane was found to decay with a rate constant of 2k 38 M-1·s-1, as determined both by pulse-radiolysis and tyrosinase experiments. The second-order kinetics indicate that a dimer is formed. In the presence of tyrosinase, but not in the pulse-radiolysis experiments, the quinomethane decay was accompanied by a steady-state oxygen uptake concurrently with the generation of a melanoid product measured by its A650, which is ascribed to the formation of an oligomer incorporating the oxidized dimer.

Oxidation of monohydric phenol substrates by tyrosinase: effect of dithiothreitol on kinetics

The effect of thiol compounds on the monophenolase activity of tyrosinase was investigated using 4-hydroxyanisole as the substrate and dithiothreitol (DTT) as the model thiol compound. We have demonstrated three actions of DTT on tyrosinase-catalysed reactions: (1) direct reduction of the copper at the active site of the enzyme; (2) generation of secondary, oxidizable species by adduct formation with the o-quinone reaction product, 4-MOB, which leads to an increase in the total oxygen utilization by the reaction system; and (3) reversible inhibition of the enzyme. We confirm our previous observation that, at approx. 10 mol of DTT/mol of enzyme, the lag phase associated with monohydric phenol oxidation by tyrosinase is abolished. We suggest that this is due to reduction of the copper at the active site of the enzyme by DTT, since (a) reduction of active-site copper in situ by DTT was demonstrated by [Cu(I)]2-carbon monoxide complex formation and (b) abolition of the lag at low DTT concentration occurs without effect on the maximum rate of reaction or on the total amount of oxygen utilized. At concentrations of DTT above that required to abolish the lag, we found that the initial velocity of the reaction increased with increasing DTT, with a concomitant increase in the total oxygen utilization. This is due to the formation of DTT-4-methoxy-o-benzoquinone (4-MOB) adducts which provide additional dihydric phenol substrate either directly or by reducing nascent 4-MOB. We present n.m.r. evidence for the formation of mono- and di-aromatic DTT adducts with 4-MOB, consistent with a suggested reoxidation scheme in the presence of tyrosinase. Inhibition of the enzyme at concentrations of DTT above 300 pmol/unit of enzyme was released on exhaustion of DTT by adduct formation with 4-MOB as it was generated.

Diarylheptanoid and other phenolic constituents of Centrolobium species

The heartwood of a specimen of Centrolobium tomentosum Benth (Leguminosae) has given (–)-centrolobine (1a), (–)-de-O-methylcentrolobine (1b), (–)-centrolobol (2), piceatannol (3)and the isoflavone formononetin (4). The heartwood of a second Centrolobium species, which could not be identified further at the U.S. Forest Products Laboratory, yielded (+)-centrolobine, (+)-de-O-methylcentrolobine, (+)-centrolobol, formononetin, 4',7-dihydroxyflavanone (5), 3-hydroxy-9-methoxypterocarpan (6) and minor amounts of a new dihydric phenol. This was identified by n.m.r. and X-ray diffraction measurements as 2-(2'-hydroxy-4'-methoxyphenyl)benzofuran-6-ol (8).