Ho Sun Song

Whitening activity of luteolin related to the inhibition of cAMP pathway in α-MSH-stimulated B16 melanoma cells

To examine the possibility of luteolin as a whitening agent, we measured antioxidant activity using DPPH assay, NBT/XO assay and intracellular ROS scavengning assay and depigmenting activity using tyrosinase assay, α-MSH-induced melanin production in B-16 cells. Luteolin showed dose-dependent anti-oxidant activity in DPPH, NBT/XO and intracellular ROS assay. Also, luteolin directly inhibited xanthine oxidase activity in a dose-dependent manner. Although luteolin did not directly inhibit tyrosinase activity, it dose-dependently inhibited both tyrosinase activity and melanin production in B16 melanoma cells stimulated by 1 μM α-MSH. Luteolin dose-dependently inhibited cAMP levels in B16 melanoma cells stimulated by 1 μM α-MSH and 1 μM forskolin, which suggest that luteolin directly inhibits adenyl cyclase in B16 melanoma cells. Therefore, these results suggest that whitening activity of luteolin may be due to the inhibition of adenyl cyclase involved in the signal pathway of α-MSH in B16 melanoma cells.

Effect of coenzyme Q10 on cutaneous healing in skin-incised mice

Coenzyme Q10 (CoQ10) is a biosynthesized quinone with 10 isoprene side chains in humans. To investigate the anti-inflammatory and wound healing effect of CoQ10, we performed in vivo and in vitro experiments. In vivo studies, there were 3 groups; Naive (without skin incision), Control (with skin incision) and CoQ10 (100 mg/kg treatment with skin incision). Collagen-like polymer (CLP) level of CoQ10 group was increased significantly compared to the control group (p<0.05). Also, CoQ10 group showed significant inhibition on myeloperoxidase (MPO) and PLA2 level compared to the control group (p<0.05). These data show that CoQ10 may have an anti-inflammatory and a wound healing effect. CoQ10 showed significant antioxidant activity in vivo on malondialdehyde (MDA) and superoxide dismutase (SOD) levels compared to the control group (p<0.05). Although CoQ10 did not show antioxidant activity in cell free system of DPPH radical scavenge, it had a potent antioxidant activity in cell culture system of both silica- and zymosan-induced reactive oxygen species generation using Raw 264.7 cells. This result may be associated with the conversion of CoQ10 to the reduced form (CoQ10H2) in the presence of some kinds of intracellular reducing agents. In conclusion, it is considered that CoQ10 appears to have a cutaneous healing effect in vivo, which may be related to the secondary action of CoQ10.

Competitive inhibition of cytosolic Ca2+-dependent phospholipase A2 by acteoside in RBL-2H3 cells

The aim of this study was to investigate whether acteoside isolated from Clerodendron trichotomum Thunberg may act as a selective inhibitor of phospholipase A2 in RBL-2H3 cells. Acteoside dose-dependently inhibited 0.5 μM melittin-induced release of [3H]arachidonic acid, which was due to the inhibition of cytosolic Ca2+-dependent phospholipase A2 (cPLA2) rather than secretory PLA2 (sPLA2). In Dixon plots, the apparent Ki value of acteoside on cPLA2 was 5.9 μM and the inhibitory pattern appeared to be a competitive inhibitor. The above data, suggests that acteoside acts as a competitive inhibitor of cPLA2 in RBL-2H3 cells.

Lutein is a competitive inhibitor of cytosolic Ca2+-dependent phospholipase A2

Objectives  We have investigated the effect of lutein on phospholipase A2 (PLA2) isozymes.

Morinda citrifolia Inhibits Both Cytosolic Ca2+-dependent Phospholipase A2 and Secretory Ca2+-dependent Phospholipase A2

This study investigated the effects of the methanol extracts of Morinda citrifolia containing numerous anthraquinone and iridoid on phospholipase A(2) (PLA(2)) isozyme. PLA(2) activity was measured using various PLA(2) substrates, including 10-pyrene phosphatidylcholine, 1-palmitoyl-2-[(14)C]arachidonyl phosphatidylcholine ([(14)C]AA-PC), and [(3)H]arachidonic acid (AA). The methanol extracts suppressed melittin-induced [(3)H]AA release in a concentration-dependent manner in RAW 264.7 cells, and inhibited cPLA(2)/sPLA(2)-induced hydrolysis of [(14)C]AA-PC in a concentration- and time-dependent manner. A Dixon plot showed that the inhibition by methanol extracts on cPLA(2) and sPLA(2) appeared to be competitive with inhibition constants (K(i)) of 3.7microg/ml and 12.6microg/ml, respectively. These data suggest that methanol extracts of Morinda citrifolia inhibits both Ca(2+)-dependent PLA(2) such as, cPLA(2) and sPLA(2). Therefore, Morinda citrifolia may possess anti-inflammatory activity secondary to Ca(2+)-dependent PLA(2) inhibition.

Antioxidant Effect of CoQ10 on N-nitrosodiethylamine-induced Oxidative Stress in Mice

The antioxidant effect of CoQ(10) on N-nitrosodiethylamine (NDEA)-induced oxidative stress was investigated in mice. Food intake and body weight were similar in both CoQ(10) and control groups during the 3-week experimental period. NDEA significantly increased the activities of typical marker enzymes of liver function (AST, ALT and ALP) both in control and CoQ(10) groups. However, the increase of plasma aminotransferase activity was significantly reduced in the CoQ(10) group. Lipid peroxidation in various tissues, such as heart, lung, liver, kidney, spleen and plasma, was significantly increased by NDEA, but this increase was significantly reduced by 100 mg/kg of CoQ(10). Superoxide dismutase activity increased significantly upon NDEA-induced oxidative stress in both the control and CoQ(10) groups with the effect being less in the CoQ(10) group. Catalase activity decreased significantly in both the control and CoQ(10) groups treated with NDEA, again with the effect being less in the CoQ(10) group. The lesser effect on superoxide dismutase and catalase in the NDEA-treated CoQ(10) group is indicative of the protective effect CoQ(10). Thus, CoQ(10) can offer useful protection against NDEA-induced oxidative stress.

Intracellular Ca2+ Mobilization and Beta-hexosaminidase Release Are Not Influenced by 60 Hz-electromagnetic Fields (EMF) in RBL 2H3 Cells

The effects of extremely low frequency electromagnetic fields (EMF) on intracellular Ca(2+) mobilization and cellular function in RBL 2H3 cells were investigated. Exposure to EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h did not produce any cytotoxic effects in RBL 2H3 cells. Melittin, ionomycin and thapsigargin each dose-dependently increased the intracellular Ca(2+) concentration. The increase of intracellular Ca(2+) induced by these three agents was not affected by exposure to EMF (60 Hz, 1 mT) for 4 or 16 h in RBL 2H3 cells. To investigate the effect of EMF on exocytosis, we measured beta-hexosaminidase release in RBL 2H3 cells. Basal release of beta-hexosaminidase was 12.3±2.3% in RBL 2H3 cells. Exposure to EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h did not affect the basal or 1 µM melittin-induced beta-hexosaminidase release in RBL 2H3 cells. This study suggests that exposure to EMF (60 Hz, 0.1 or 1 mT), which is the limit of occupational exposure, has no influence on intracellular Ca(2+) mobilization and cellular function in RBL 2H3 cells.

Inhibitory effect of acteoside on melittin-induced catecholamine exocytosis through inhibition of Ca2+-dependent phospholipase A2 and extracellular Ca2+ influx in PC12 cells

To investigate the inhibitory effect of acteoside on the process of exocytosis induced by melittin, we measured Ca2+ mobilization, arachidonic acid (AA) release and catecholamine exocytosis in PC12 chromaffin cells. Melittin significantly increased the intracellular Ca2+ mobilization via receptor-operated calcium channel but not the intracellular Ca2+ release. It caused AA release via activation of Ca2+-dependent phospholipase A2 (PLA2) and catecholamine secretion in a dose-dependent manner. Acteoside dose-dependently inhibited the release of AA and intracellular Ca2+ mobilization induced by melittin. Acteoside reduced the catecholamine release and raised the amount of intracellular chromogranin A which is co-released with catecholamine from melittin-stimulated PC12 cells. Taken together, our results suggest that acteoside could suppress the exocytosis via inhibition of Ca2+-dependent PLA2 and extracellular Ca2+ influx in PC12 cells stimulated by melittin.

Effects of C18 Fatty Acids on Intracellular Ca(2+) Mobilization and Histamine Release in RBL-2H3 Cells.

To investigate the underlying mechanisms of C18 fatty acids (stearic acid, oleic acid, linoleic acid and α-linolenic acid) on mast cells, we measured the effect of C18 fatty acids on intracellular Ca2+ mobilization and histamine release in RBL-2H3 mast cells. Stearic acid rapidly increased initial peak of intracellular Ca2+ mobilization, whereas linoleic acid and α-linolenic acid gradually increased this mobilization. In the absence of extracellular Ca2+, stearic acid (100 µM) did not cause any increase of intracellular Ca2+ mobilization. Both linoleic acid and α-linolenic acid increased intracellular Ca2+ mobilization, but the increase was smaller than that in the presence of extracellular Ca2+. These results suggest that C18 fatty acid-induced intracellular Ca2+ mobilization is mainly dependent on extracellular Ca2+ influx. Verapamil dose-dependently inhibited stearic acid-induced intracellular Ca2+ mobilization, but did not affect both linoleic acid and α-linolenic acid-induced intracellular Ca2+ mobilization. These data suggest that the underlying mechanism of stearic acid, linoleic acid and α-linolenic acid on intracellular Ca2+ mobilization may differ. Linoleic acid and α-linolenic acid significantly increased histamine release. Linoleic acid (C18:2: ω-6)-induced intracellular Ca2+ mobilization and histamine release were more prominent than α-linolenic acid (C18:3: ω-3). These data support the view that the intake of more α-linolenic acid than linoleic acid is useful in preventing inflammation.

Effect of Extremely Low Frequency Electromagnetic Fields (EMF) on Phospholipase Activity in the Cultured Cells

This study was conducted to investigate the effects of extremely low frequency electromagnetic fields (EMF) on signal pathway in plasma membrane of cultured cells (RAW 264.7 cells and RBL 2H3 cells), by measuring the activity of phospholipase A(2) (PLA(2)), phospholipase C (PLC) and phospholipase D (PLD). The cells were exposed to the EMF (60 Hz, 0.1 or 1 mT) for 4 or 16 h. The basal and 0.5 µM melittin-induced arachidonic acid release was not affected by EMF in both cells. In cell-free PLA(2) assay, we failed to observe the change of cPLA(2) and sPLA(2) activity. Also both PLC and PLD activities did not show any change in the two cell lines exposed to EMF. This study suggests that the exposure condition of EMF (60 Hz, 0.1 or 1 mT) which is 2.4 fold higher than the limit of occupational exposure does not induce phospholipases-associated signal pathway in RAW 264.7 cells and RBL 2H3 cells.