Mulberry (Morus australis Poir.) leaves have long been consumed in the form of tea or tincture especially in Asia, owing to their high antioxidant and blood pressure-regulating properties. Although it is thought that vascular abnormal contraction may be involved in the blood pressure-suppressing effect, the effect of mulberry on vascular abnormal contraction is still unknown. Therefore, we investigated mulberry leaves as a potential source of bioactive compounds that prevent vascular abnormal contraction. Mulberry leaves were divided into fresh leaves and tea leaves and further classified according to the age of the tree: more or less than 20 years old, into roasted and unroasted. Mulberry fruits were also evaluated.We assessed the preventive effect of mulberry extracts on vascular abnormal contraction. Extracts from mulberry leaves of trees more than 20 years old showed a strong preventive effect on vascular abnormal contraction of human coronary artery smooth muscle cells. Therefore, to identify the active components in mulberry leaves, we fractionated the active fractions by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The active fraction was further analyzed by mass spectrometry and nuclear magnetic resonance; an active component of the mulberry leaf extract was fisetin. In addition, our results indicated that the hydroxyl group at the C-3 position of fisetin is crucial for its activity. These results prove that fisetin is effective in preventing vascular abnormal contraction. Overall, mulberry leaves and fisetin are expected to be used in a wide range of fields such as functional foods, nutraceuticals, and drug targets.

This study aimed to determine the effects of vitamin C supplementation on antioxidant and inflammatory status and cell viability of bovine peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMCs) were isolated from eighteen clinically healthy Japanese Black calves in this study. PBMCs were cultured with vitamin C (vitamin C group) and without vitamin C (control group) and stimulated with or without lipopolysaccharide (LPS). As a result, the total antioxidant capacities, which are the reducing power of components from Fe3 + to Fe 2 + , in the cell culture supernatant with or without LPS stimulation were significantly higher in the vitamin C group than those in the control group (P < 0.05, P < 0.01, respectively). Tumor necrosis factor-alpha in the cell culture supernatant with LPS stimulation was significantly higher in the control group than that in the vitamin C group (P < 0.05). The viability of cells cultured with LPS stimulation was significantly higher in the vitamin C group than that in the control group after 72 h of culture (P < 0.05). These results suggested that vitamin C is related to the antioxidant and anti-inflammatory properties and cell viability of PBMCs obtained from calves.

The reaction field of abnormal vascular contraction induced by sphingosylphosphorylcholine (SPC) and the action point of SPC around the plasma membranes remain unknown. However, we found in a previous study that fisetin prevents SPC-induced vascular smooth muscle cells contraction, while the mechanism remains unknown. Therefore, in this study, we aimed to address the action point of SPC around the plasma membranes and the involvement of fisetin. We focused on microdomains and evaluated their markers flotillin-1 and caveolin-1 and the localization of SPC to investigate their action point. The results showed that microdomains of vascular smooth muscle cells were not involved in SPC-induced contraction. However, we found that after SPC had been affected on the plasma membrane, cells took up SPC via endocytosis. Moreover, SPC remained in the cells and did not undergo transcytosis, and SPC-induced contracting cells produced exosomes. These phenomena were similar to those observed in fisetin-treated cells. Thus, we speculated that, although not involved in the reaction field of SPC-induced contractions, the microdomain induced the endocytosis of SPCs, and fisetin prevented the contractions by directly targeting vascular smooth muscle cells. Notably, this preventive mechanism involves the cellular uptake of SPC via endocytosis.