Effect of Lycopus lucidus Turcz. leaves on matrix metalloproteinase activity and antioxidant

Abstract

Lycopus lucidus Turcz. from Lamiaceae family, is a traditional eastern Asian medicinal plant, which is abundant in natural bioactive flavonoids, coumarins, terpenoids and tannins with potential antioxidant activity. In the present study, L. Turcz. leaves were extracted with acetone/methylene chloride and methanol solution, then sequentially partitioned with solvents (n-Hexane, 85% aqueous methanol, and water) in different polarities. Thus, two crude extracts (A+M and MeOH), as well as resulting four fractions (n-Hex, 85% aq. MeOH, n-BuOH and Water) was prepared to investigate the biological activities of L. Turcz. leaves. The effect of six samples on H2O2-induced injury in SH-SY5Y human neuroblastoma cells were evaluated by MTT assay at different doses (0.025 mg/L-0.25 mg/L). To study antioxidant effects of the crude extracts and fractions, reactive oxygen species (ROS) on SH-SY5Y human neuroblastoma cells and changes on intracellular glutathione (GSH) production were determined. In addition, nitric oxide (NO) as a mediator involved in the inflammation reaction, the inhibitory effects of NO production from samples were measured by using the in vitro lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cell line. Western blotting experiment was performed to compare the expression level of matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) in the phorbol 12-myristate 13-acetate (PMA)-induced HT-1080 cells. Finally, radical mediated DNA damage in HT-1080 cells were performed to investigate the protection ability from L. lucidus Turcz. leaves on DNA oxidative damage. In MTT assay, the pretreatment of MeOH extracts and four fractions from L. lucidus Turcz. leaves significantly increased the viability of H2O2 induced SH-SY5Y cells (p<0.05). But there was not any positive effect on SH-SY5Y cell viability after the treatment of A+M sample, which may be explained by the high cytotoxic effect from A+M sample. In western blotting experiment, these extracts and fractions led to a reduction of the secretion levels of MMP-2 and MMP-9 in HT-1080 cells. Due to the cell cytotoxicity of A+M extract, MMP-2 cannot be detected in this group, and only MMP-9 bands were found in control and Water fractions groups. In DNA oxidation analysis, the maximum inhibition of the genomic DNA damage caused by excessive oxidative stress was 80.89%, which is due to the present of A+M extracts. In antioxidant activity, intracellular ROS generation was significantly attenuated in two cells after treatment of crude extracts and fractions (p<0.05), 85% aq. MeOH and n-BuOH fractions showed a better scavenging ability than others. As for the effect of GSH production, A+M extract showed a more powerful ability in enhancing the GSH production by 15.81% than MeOH extract (13.53%) at the concentration of 1 mg/mL; meanwhile, among the fractions at the concentration of 0.5 mg/mL, 85% aq. MeOH fraction increased the GSH level most by 15.30% and followed by the n-BuOH fraction (11.80%). In regard to the effect of production of NO in RAW 264.7 cells, groups of treatment with samples all showed a significant decrease of NO level in comparison with control group (p<0.05). Both extracts efficiently decreased the NO production of cells at the inhibition of more than 80%. However, among the fractions, Water and n-Hex fractions showed better inhibitory effects in NO production than those of 85% aq. MeOH and n-BuOH fractions. Results obtained collectively indicate that the good biological activities from L. lucidus Turcz. leaves, which can be exploited for plant based anticancer and antioxidant agents in the pharmaceutical, cosmetic, nutraceutical and food industries.