Correlation between the Potency of Flavonoids on Mushroom Tyrosinase Inhibitory Activity and Melanin Synthesis in Melanocytes

Twenty-seven flavonoids isolated from Dalbergia parviflora with vast structural diversity were screened for inhibitory activity against mushroom and murine tyrosinases using l-DOPA as the substrate. Among the flavonoids tested, only four—khrinone (5), cajanin (9), (3RS)-3′-hydroxy-8-methoxy vestitol (21), and (6aR,11aR)-3,8-dihydroxy-9-methoxy pterocarpan (27)—reacted with mushroom tyrosinase, with IC50 values of 54.0, 67.9, 67.8, and 16.7 μM, respectively, and only compound 27 showed inhibitory activity against murine tyrosinase. With cell-based assays, only compounds 9 and 27 effectively inhibited melanogenesis in B16-F10 melanoma cells (by 34% and 59%, respectively), at a concentration of 15 μM, without being significantly toxic to the cells. However, the crude extract of D. parviflora and some of the flavonoid constituents appeared to increase melanin production in B16-F10 cells, suggesting that there are flavonoids with both inhibitory and stimulatory melanogenesis in the crude extract. Studies on the correlation between the enzyme-based and cell-based assays showed that only the flavonoids with IC50 values below 50 μM against mushroom tyrosinase could inhibit the mammalian tyrosinase, and thus, reduce melanogenesis in B16-F10. Flavonoids with the IC50 values greater than 50 μM, on the other hand, could not inhibit the mammalian tyrosinase, and had either no effect or enhancement of melanogenesis. In conclusion, the tyrosinase enzyme from mushroom is not as selective as the one from mammalian source for the enzyme-based melanogenesis inhibitory screening, and the mammalian cell-based assay appears to be a more reliable model for screening than the enzyme-based one

Correlation between the Potency of Flavonoids on Mushroom Tyrosinase Inhibitory Activity and Melanin Synthesis in Melanocytes

Twenty-seven flavonoids isolated from Dalbergia parviflora with vast structural diversity were screened for inhibitory activity against mushroom and murine tyrosinases using l-DOPA as the substrate. Among the flavonoids tested, only four—khrinone (5), cajanin (9), (3RS)-3′-hydroxy-8-methoxy vestitol (21), and (6aR,11aR)-3,8-dihydroxy-9-methoxy pterocarpan (27)—reacted with mushroom tyrosinase, with IC50 values of 54.0, 67.9, 67.8, and 16.7 μM, respectively, and only compound 27 showed inhibitory activity against murine tyrosinase. With cell-based assays, only compounds 9 and 27 effectively inhibited melanogenesis in B16-F10 melanoma cells (by 34% and 59%, respectively), at a concentration of 15 μM, without being significantly toxic to the cells. However, the crude extract of D. parviflora and some of the flavonoid constituents appeared to increase melanin production in B16-F10 cells, suggesting that there are flavonoids with both inhibitory and stimulatory melanogenesis in the crude extract. Studies on the correlation between the enzyme-based and cell-based assays showed that only the flavonoids with IC50 values below 50 μM against mushroom tyrosinase could inhibit the mammalian tyrosinase, and thus, reduce melanogenesis in B16-F10. Flavonoids with the IC50 values greater than 50 μM, on the other hand, could not inhibit the mammalian tyrosinase, and had either no effect or enhancement of melanogenesis. In conclusion, the tyrosinase enzyme from mushroom is not as selective as the one from mammalian source for the enzyme-based melanogenesis inhibitory screening, and the mammalian cell-based assay appears to be a more reliable model for screening than the enzyme-based one

Structure and Antioxidant Activity Relationships of Isoflavonoids from Dalbergia parviflora

The antioxidant activities of 24 isoflavonoids that were previously isolated as pure compounds from Dalbergia parviflora were evaluated using three different in vitro antioxidant-based assay systems: xanthine/xanthine oxidase (X/XO), ORAC, and DPPH. The isolates consisted of three subgroups, namely isoflavones, isoflavanones, and isoflavans, each of which appeared to have diversified substituents, and were thus ideal for the study of their structure-activity relationships (SARs). The SAR analysis was performed using the results obtained from both the inter-subgroup isoflavonoids with the same substitution pattern and the intra-subgroup compounds with different substitution patterns. The inter-subgroup comparison showed that the isoflavones exhibited the highest antioxidant activities based on all three assays. The intra-subgroup analysis showed that the additional presence of an OH group in Ring B at either R3′ or R5′ from the basic common structure of the R7-OH of Ring A and the R4′-OH (or -OMe) of Ring B greatly increased the antioxidant activities of all of the isoflavonoid subgroups and that other positions of OH and OMe substitutions exerted different effects on the activities depending on the subgroup and assay type. Therefore, based on the structural diversity of the isoflavonoids in D. parviflora, the present study provides the first clarification of the detailed antioxidant SARs of isoflavonoids

An Mrp-Like Cluster in the Halotolerant Cyanobacterium Aphanothece halophytica Functions as a Na+/H+ Antiporter▿

The mrp homolog gene cluster mrpCD1D2EFGAB (Ap-mrp) was found in a halotolerant cyanobacterium, Aphanothece halophytica, amplified, and expressed in Escherichia coli mutant TO114. Ap-mrp complemented the salt-sensitive phenotype of TO114 and exhibited Na+/H+ and Li+/H+ exchange activities, indicating that Ap-Mrp functions as a Na+/H+ antiporter

Halotolerant Cyanobacterium Aphanothece halophytica Contains an Na+-dependent F1F0-ATP Synthase with a Potential Role in Salt-stress Tolerance*

Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium that can grow in media of up to 3.0 m NaCl and pH 11. Here, we show that in addition to a typical H+-ATP synthase, Aphanothece halophytica contains a putative F1F0-type Na+-ATP synthase (ApNa+-ATPase) operon (ApNa+-atp). The operon consists of nine genes organized in the order of putative subunits β, ϵ, I, hypothetical protein, a, c, b, α, and γ. Homologous operons could also be found in some cyanobacteria such as Synechococcus sp. PCC 7002 and Acaryochloris marina MBIC11017. The ApNa+-atp operon was isolated from the A. halophytica genome and transferred into an Escherichia coli mutant DK8 (Δatp) deficient in ATP synthase. The inverted membrane vesicles of E. coli DK8 expressing ApNa+-ATPase exhibited Na+-dependent ATP hydrolysis activity, which was inhibited by monensin and tributyltin chloride, but not by the protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The Na+ ion protected the inhibition of ApNa+-ATPase by N,N′-dicyclohexylcarbodiimide. The ATP synthesis activity was also observed using the Na+-loaded inverted membrane vesicles. Expression of the ApNa+-atp operon in the heterologous cyanobacterium Synechococcus sp. PCC 7942 showed its localization in the cytoplasmic membrane fractions and increased tolerance to salt stress. These results indicate that A. halophytica has additional Na+-dependent F1F0-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance