Thermal adaptation in the honeybee (Apis mellifera) via changes to the structure of malate dehydrogenase.

In honeybees there are three alleles of cytosolic malate dehydrogenase gene: F, M and S. Allele frequencies are correlated with environmental temperature, suggesting that the alleles have temperature-dependent fitness benefits. We determined the enzyme activity of each allele across a range of temperatures in vitro The F and S alleles have higher activity and are less sensitive to high temperatures than the M allele, which loses activity after incubation at temperatures found in the thorax of foraging bees in hot climates. Next, we predicted the protein structure of each allele and used molecular dynamics simulations to investigate their molecular flexibility. The M allozyme is more flexible than the S and F allozymes at 50°C, suggesting a plausible explanation for its loss of activity at high temperatures, and has the greatest structural flexibility at 15°C, suggesting that it can retain some enzyme activity at cooler temperatures. MM bees recovered from 2 h of cold narcosis significantly better than all other genotypes. Combined, these results explain clinal variation in malate dehydrogenase allele frequencies in the honeybee at the molecular level.

Anti-tyrosinase and anti-melanogenic effects of piperine isolated from Piper nigrum on B16F10 mouse melanoma cells.

The essential function of melanin is to protect our skin against harmful environmental factors. However, excessive melanin production can cause undesirable hyperpigmentation issues, such as freckles and melasma. Although several compounds are used to control melanin production by inhibiting tyrosinase (TYR), their efficacy is limited by skin-related adverse effects and cytotoxicity concerns. Consequently, searching for new natural compounds with an effective TYR inhibitor (TYR-I) activity but less harmful effects continues. Plant-based natural extracts are an alternative that are in great demand due to their safety and diverse biological properties. This study assessed ten isolated plant compounds for their TYR-I activities using an in vitro mushroom TYR inhibition assay. Among these compounds, piperine (400 µM) demonstrated the highest TYR-I activity, with a potency of 36.27 ± 1.96 %. Hence, this study examined the effect of piperine on melanogenesis in melanocyte stimulating hormone-treated B16F10 melanoma cells and using kojic acid as a positive reference. Cell viability was evaluated through the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Measurements of cellular TYR activity and melanin content were performed and related to changes in the transcriptional expression levels of melanogenesis-related genes, assessed via quantitative real-time reverse transcriptase (RT-q)PCR analysis. The results revealed that piperine at a concentration of 44 µM significantly reduced cellular TYR activity by 21.51 ± 2.00 % without causing cytotoxicity. Additionally, at the same concentration, piperine significantly decreased the intracellular melanin content by 37.52 ± 2.53 % through downregulating transcription levels of TYR and TYR-related protein 1 (TRP-1) but not TRP-2. Kojic acid, at a concentration of 1407 µM, induced a significant decrease in the melanin content and cellular TYR activity by suppressing all three melanogenesis-related genes. These findings suggest that piperine has potential as a potent depigmenting agent.

In vitro cytotoxic activity on KATO-III cancer cell lines of mangiferolic acid purified from Thai Tetragonula laeviceps propolis.

Gastric cancer is a global health concern, but current treatment with chemotherapy and surgery is often inadequate, prompting the exploration of alternative treatments. Propolis is a natural substance collected by bees known for its diverse properties linked to floral sources. The Dichloromethane Partitioned Extract (DPE) from Tetragonula laeviceps propolis, in Bankha district, Thailand was previously shown to possess significant cytotoxicity against KATO-III gastric cancer cells, while showing lower cytotoxicity toward WI-38 normal fibroblast cells. Here, the DPE was further fractionated by column chromatography, identified active fractions, and subjected to structural analysis using nuclear magnetic resonance spectroscopy. Cytotoxicity against KATO-III cells was reevaluated, and programmed cell death was analyzed using flow cytometry. Expression levels of cancer-related genes were measured using quantitative real-time reverse transcriptase PCR. Cardol C15:2 (compound 1) and mangiferolic acid (MF; compound 2) were discovered in the most active fractions following structural analysis. MF exhibited strong cytotoxicity against KATO-III cells (IC50 of 4.78-16.02 µg/mL), although this was less effective than doxorubicin (IC50 of 0.56-1.55 µg/mL). Morphological changes, including decreased cell density and increased debris, were observed in KATO-III cells treated with 30 µg/mL of MF. Significant induction of late-stage apoptosis and necrosis, particularly at 48 and 72 h, suggested potential DNA damage and cell cycle arrest, evidenced by an increased proportion of sub-G1 and S-phase cells. Doxorubicin, the positive control, triggered late apoptosis but caused more necrosis after 72 h. Furthermore, MF at 30 µg/mL significantly increased the expression level of COX2 and NFκB genes linked to inflammation and cell death pathways. This upregulation was consistent at later time points (48 and 72 h) and was accompanied by increased expression of CASP3 and CASP7 genes. These findings suggest MF effectively induces cell death in KATO-III cells through late apoptosis and necrosis, potentially mediated by upregulated inflammation-related genes.

Four new alkylamides from the fruits of Piper retrofractum and antityrosinase evaluation.

Four new alkylamides named retroframides A-D (1-4) together with twenty-two known compounds were isolated from the fruits of Piper rectrofractum. The structures of new compounds were elucidated on the basis of spectroscopic data including 2D NMR and chemical derivatization followed by GC-MS analysis. Of isolated compounds, piperine (25) and pellitorine (26) revealed moderate inhibition against tyrosinase with percentage inhibition of 36.1 and 40.7.