Maclura cochinchinensis (Lour.) Corner Heartwood Extracts Containing Resveratrol and Oxyresveratrol Inhibit Melanogenesis in B16F10 Melanoma Cells.

This study aimed to isolate and purify resveratrol and oxyresveratrol from the heartwoods of Maclura cochinchinensis, and to evaluate their inhibitory effects on melanogenesis in B16F10 murine melanoma cells. A methanol maceration process yielded a crude extract comprising 24.86% of the initial mass, which was subsequently analyzed through HPTLC, HPLC, and LC-MS/MS. These analyses revealed the presence of resveratrol and oxyresveratrol at concentrations of 4.32 mg/g and 33.6 mg/g in the extract, respectively. Initial purification employing food-grade silica gel column chromatography separated the extract into two fractions: FA, exhibiting potent inhibition of both tyrosinase activity and melanogenesis, and FM, showing no such inhibitory activity. Further purification processes led to the isolation of fractions Y11 and Gn12 with enhanced concentrations of resveratrol (94.9 and 110.21 mg/g, respectively) and fractions Gn15 and Gn16 with elevated levels of oxyresveratrol (321.93 and 274.59 mg/g, respectively), all of which significantly reduced melanin synthesis. These outcomes affirm the substantial presence of resveratrol and oxyresveratrol in the heartwood of M. cochinchinensis, indicating their promising role as natural agents for skin lightening.

α-Glucosidase inhibition of prenylated pyranocoumarins: Kinetics, in silico prediction of their physicochemical properties and molecular docking.

Three naturally occurring prenylated pyranocoumarins, nordentatin (1), dentatin (2), and clausarin (3), isolated from the roots of Clausena excavata (Family Rutaceae), and O-methylclausarin (4) which was obtained by methylation of 3, were investigated for their α-glucosidase inhibitory activity. The mechanism of action and the in silico prediction of their physicochemical and ADMET properties as well as the molecular docking were also studied. Compounds 1-4 exhibited stronger α-glucosidase inhibitory activity than the positive control, acarbose, through a non-competitive mechanism. Among them, 3 exhibited the highest activity, with an IC50 of 8.36 µM, which is significantly stronger than that of acarbose (IC50 = 430.35 µM). The prenyl group on C-3 and the hydroxyl group on C-5 in 3 may play important roles in enhancing the activity. Calculated physicochemical and ADMET parameters of 1-4 satisfied the Lipinski's and Veber's rules. Molecular simulation analysis indicated they are promising drug candidates with no hepatotoxicity. Compound 3 exhibited potent activity in the experiment and demonstrated good drug properties based on the calculations. A molecular docking study revealed that 3 showed H-bonding and π-π stacking interactions with selective Phe321, as well as interactions with thirteen other amino acid residues of the α-glucosidase.

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 four­khrinone (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.

Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63­73%) and CsCPR I (79­83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected].

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.

Expression of CA125 and cisplatin susceptibility of pleural effusion-derived human lung cancer cells from a Thai patient.

Advances in understanding lung cancer biology and tumor markers aid clinicians in managing the disease. Cancer-associated antigen (CA)125 has garnered increasing attention in lung cancer research and may benefit the treatment and follow-up of this type of cancer. In Thai lung cancer patients, knowledge regarding ethnic differences in cancer cell biology is largely absent. We generated lung cancer cells from the pleural effusion fluids of a Thai patient and designated these as P1 cells. P1 cells were assessed for growth rate, response to chemotherapy, and the presence of tumor markers, in particular CA125 expression. Results of immunofluorescence indicated that P1 cells exhibited strong expression levels of CA125, comparable to that of established H460 lung cancer cells. Furthermore, P1 cells were analyzed for the expression of additional markers. Results revealed that H460 cells exhibited strong immunofluorescent signals from cytokeratin-19 fragments (CYFRA 21-1) and squamous cell carcinoma antigen (SCCA) while P1 presented only CYFRA 21-1 signals. We also found evidence of relative cisplatin resistance in P1 compared to the susceptibility level of established lung cancer cells. Thus, the results and methodology described in this study may aid the development of lung cancer diagnostic and therapeutic approaches and, in particular, advance understanding of ethnic differences.

Isolation and characterization of proline/betaine transporter gene from oil palm.

Oil production from oil palm is adversely affected by drought and salt. Under drought and salt stress, proline content increases in oil palm; the mechanism for this is unknown. Here, an 8319-nucleotide sequence including cDNA, genomic DNA and the promoter region of proline transporter gene from oil palm Elaeis guineensis was determined. The transporter gene exhibited high similarity to Bet/ProT genes from several plants, but the highest homology was found with rice ProT1. The exon-intron structure of genomic DNA was unique, and numerous stress-response cis-elements were found in the promoter region. Expression of cDNA EgProT1 in Escherichia coli mutant exhibited uptake activities for glycinebetaine and choline as well as proline. Under salt-stressed conditions, exogenously applied glycinebetaine was taken up into the root more rapidly than the control. These data indicate that oil palm has a unique Pro/T1 gene. Nucleotide sequence data for the cDNA and genomic DNA of proline transporter gene from Elaeis guineensis are available in the DDJB database under accession numbers AB597035 and AB597036, respectively.

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 F(1)F(0)-type Na(+)-ATP synthase (ApNa(+)-ATPase) operon (ApNa(+)-atp). The operon consists of nine genes organized in the order of putative subunits ß, 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 F(1)F(0)-ATPase in the cytoplasmic membrane playing a potential role in salt-stress tolerance.

Preferential accumulation of betaine uncoupled to choline monooxygenase in young leaves of sugar beet--importance of long-distance translocation of betaine under normal and salt-stressed conditions.

It has been reported that glycinebetaine (betaine) is synthesized in response to abiotic stresses via a two-step oxidation of choline in which choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are involved. Here we show that significant amounts of betaine, > 20 micromol/gFW, accumulated in young leaves of Beta vulgaris even under normal growth conditions, whereas levels in old leaves, cotyledons, hypocotyls, and roots were low. Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. By contrast, the levels of BADH were high in all tissues. Exogenously supplied choline was converted into betaine in old leaves, but levels were significantly lower in young leaves under the same conditions. When d(11)-betaine was applied exogenously to old leaves, it was translocated preferentially into young leaves and roots. In response to salt stress, betaine levels increased in all tissues, but most significantly increased in young leaves. The levels of CMO increased in various tissues, but were low in young leaves. A betaine transporter gene was isolated. Its expression was more strongly induced in old leaves than in young leaves. Based on these data, we discussed the role of CMO and betaine transporter under stress and non-stress conditions.

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.

Analysis of the promoter activities of the genes encoding three quinoprotein alcohol dehydrogenases in Pseudomonas putida HK5.

The transcriptional regulation of three distinct alcohol oxidation systems, alcohol dehydrogenase (ADH)-I, ADH-IIB and ADH-IIG, in Pseudomonas putida HK5 was investigated under various induction conditions. The promoter activities of the genes involved in alcohol oxidation were determined using a transcriptional lacZ fusion promoter-probe vector. Ethanol was the best inducer for the divergent promoters of qedA and qedC, encoding ADH-I and a cytochrome c, respectively. Primary and secondary C3 and C4 alcohols and butyraldehyde specifically induced the divergent promoters of qbdBA and aldA, encoding ADH-IIB and an NAD-dependent aldehyde dehydrogenase, respectively. The qgdA promoter of ADH-IIG responded well to (S)-(+)-1,2-propanediol induction. In addition, the roles of genes encoding the response regulators exaE and agmR, located downstream of qedA, were inferred from the properties of exaE- or agmR-disrupted mutants and gene complementation tests. The gene products of both exaE and agmR were strictly necessary for qedA transcription. The mutation and complementation studies also suggested a role for AgmR, but not ExaE, in the transcriptional regulation of qbdBA (ADH-IIB) and qgdA (AGH-IIG). A hypothetical scheme describing a regulatory network, which directs expression of the three distinct alcohol oxidation systems in P. putida HK5, was derived.

Disruption of quinoprotein ethanol dehydrogenase gene and adjacent genes in Pseudomonas putida HK5.

Pseudomonas putida HK5 produces three different quinoprotein alcohol dehydrogenases: ADH-I, ADH-IIB and ADH-IIG. Gene organization of qedA, the gene for ADH-I, and other 10 genes in the cluster was related to the genome sequences of five other Pseudomonas strains. Insertion mutations in either qedA, exaE or agmR eliminated ADH-I activity, although the mutants were still able to grow on ethanol but more slowly than the wild-type strain. Mutant analysis demonstrated the requirement of agmR and exaE in ADH-I expression, and the tentative involvement of agmR, but not exaE, in the induction of ADH-IIB and ADH-IIG activities.