Wild grapevines as rootstock regulate the oxidative defense system of in vitro grafted scion varieties under drought stress.

The narrow genetic base of modern cultivars is becoming a key bottleneck for crop improvement and the use of wild relatives is an appropriate approach to improve the genetic diversity of crops to manage the sustainable production under different abiotic and biotic constraints. In Pakistan, wild germplasm of grapevine viz Dakh, Toran, and Zarishk belong to Vitis vinifera subsp. sylvestris and Fatati belong to Vitis vinifera subsp. sativa is naturally present in humid and sub-humid areas of mountainous and sub-mountainous regions and showed varying level of tolerance against drought stress but have not been evaluated as rootstock. In this study, different tolerant behavior of wild grapevines as rootstock in grafted scion varieties were explored under different levels of PEG-6000 mediated drought stress i.e., -4.00, -6.00, and -8.00 bars. In response to drought stress, wild grapevines evoked several non-enzymatic and enzymatic activities. Among non-enzymatic activities, total chlorophyll contents of commercial varieties were sustained at higher level when grafted on wild grapevines Dakh and Fatati which subsequently reduced the damage of cell membrane via MDA. Whereas, to cope the membranous damage due to excessive cellular generation of ROS, wild grapevines triggered the enhanced activities of SOD to dismutase the free oxygen radicals into H2O2, then CAT enzyme convert the H2O2 into water molecules. Higher accumulation of ROS in commercial scion varieties were also coped by wild grapevines Dakh and Fatati through the upregulation of POD and APX enzymes activities. Based on these enzymatic and non-enzymatic indices, biplot and cluster analysis classified the wild grapevines as rootstock into three distinct categories comprises on relatively tolerant i.e., Dakh (Vitis vinifera subsp. sylvestris) and Fatati (Vitis vinifera subsp. sativa), moderate tolerant i.e., Toran (Vitis vinifera subsp. sylvestris) and relatively susceptible category i.e., Zarishk (Vitis vinifera subsp. sylvestris).

Novel Amide Derivatives as Potent Tyrosinase Inhibitors; In-vitro, In-vivo Antimelanogenic Activity and Computational Studies.

BACKGROUND: Tyrosinase is involved in the melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders. Controlling the melanogenesis could be an important strategy for treating abnormal pigmentation. METHODS: In the present study, a series of amide derivatives (3a-e and 5a-e) were synthesized aiming to inhibit tyrosinase activity and melanin production. All derivatives were screened for tyrosinase inhibition in a cell-free system. The possible interactions of amide derivatives with tyrosinase enzyme and effect of these interactions on tyrosinase structure were checked by molecular docking in silico and by Circular Dichroism (CD) studies, respectively. The most potent amide derivative (5c) based on cell-free experiments, was further tested for cellular ROS inhibition and for tyrosinase activity using mouse skin melanoma (B16F10) cells. RESULTS: The tyrosinase inhibitory concentration (IC50) for tested compounds was observed between the range of 68 to 0.0029 µg/ml with a lowest IC50 value of compound 5c which outperforms the reference arbutin and kojic acid. The cellular tyrosinase activity and melanin quantification assay demonstrate that 15µg/ml of 5c attenuates 36% tyrosinase, 24% melanin content of B16F10 cells without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that 5c effectively reduces melanogenesis without perceptible toxicity. Furthermore, the molecular docking demonstrates that compound 5c interacts with copper ions and multiple amino acids in the active site of tyrosinase with best glide score (-5.387 kcal/mol), essential for mushroom tyrosinase inhibition and the ability to diminish the melanin synthesis in-vitro and in-vivo. CONCLUSION: Thus, we propose compound 5c as a potential candidate to control tyrosinase rooted hyperpigmentation in the future.