*Article* **Antioxidant, Anti-Inflammatory and Attenuating Intracellular Reactive Oxygen Species Activities of** *Nicotiana tabacum* **var. Virginia Leaf Extract Phytosomes and Shape Memory Gel Formulation**

**Chuda Chittasupho 1,2 , Kunyakorn Chaobankrang <sup>1</sup> , Araya Sarawungkad <sup>1</sup> , Weerasak Samee <sup>3</sup> , Sudarshan Singh <sup>1</sup> , Kirachuda Hemsuwimon <sup>4</sup> , Siriporn Okonogi 1,2, Kantaporn Kheawfu 1,2 , Kanokwan Kiattisin <sup>1</sup> and Wantida Chaiyana 1,2,\***


**Abstract:** Oxidative stress is one of the major causes of skin aging. In this study, the shape memory gels containing phytosomes were developed as a delivery system for *Nicotiana tabacum* var. Virginia fresh (VFL) and dry (VDL) leaf extracts. The extracts were loaded in the phytosomes by a solvent displacement method. The physical and chemical characteristics and stability of phytosomes were evaluated by dynamic light scattering and phytochemistry, respectively. The in vitro antioxidant activity and intracellular reactive oxygen species reduction of phytosomes and/or extracts were investigated by the DPPH and ABTS radical scavenging assays, FRAP assay, and DCFH-DA fluorescent probe. The cytotoxicity and anti-inflammatory activity of VDL and VFL phytosomes were studied by an MTT and a nitric oxide assay, respectively. Here, we first reported the total phenolic content in the dry leaf extract of *N. tabacum* var. Virginia was significantly greater than that of the fresh leaf extract. The HPLC analysis results revealed that VDL and VFL extracts contained 4.94 ± 0.04 and 3.13 ± 0.01 µg/mL of chlorogenic acid and 0.89 ± 0.00 and 0.24 ± 0.00 µg/mL of rutin, respectively. The phytosomes of the VDL and VFL extracts displayed stable size, polydispersity index, zeta potential values, and good chemical stability. VDL and VDL phytosomes showed higher phenolic and flavonoid contents which showed stronger DPPH and ABTS radical scavenging effects and reduced the intracellular ROS. The results suggested that the phenolic compounds are the main factor in their antioxidant activity. Both VDL and VFL phytosomes inhibited nitric oxide production induced by LPS, suggesting the anti-inflammatory activity of the phytosomes. The shape memory gel containing VDL and VFL phytosomes had good physical stability in terms of pH and viscosity. The VDL and VFL phytosomes dispersed in the shape memory gels can be considered as a promising therapeutic delivery system for protecting the skin from oxidation and reactive oxygen species.

**Keywords:** phytosomes; tobacco leaf; polyurethane-62; reactive oxygen species; shape memory gel
