Antioxidant Activity of Aqueous Extracts from Eucommia ulmoides and Cistanche deserticola: An In Vitro Study

The plants Eucommia ulmoides and Cistanches deserticola have a long-standing history in traditional medicine due to their strong antioxidant properties and therapeutic potential in treating various diseases, such as kidney deficiency [1], impotence, and hypertension [2]. The compounds derived from E.ulmoides and C.deserticola exhibit antioxidant, anti-inflammatory, anti-aging, and cardioprotective properties through multiple pathways [3]. Oxidative stress is a well-known contributor to many chronic diseases, including cancer, cardiovascular diseases, and neurodegenerative disorders, and plays an important role in wound healing. Fibroblasts, which are also known as the critical cells in the wound healing process, are involved in the production of extracellular matrix and regulation of inflammation, angiogenesis, and tissue repair. In this study, we aimed to evaluate the antioxidant activity of the aqueous extracts from E. ulmoides and C. deserticola in NIH/3T3 fibroblasts by visualizing H2O2-induced intracellular reactive oxygen species (ROS) using confocal microscopy with DCFHDA (2',7'-dichlorodihydrofluorescein diacetate). DCFHDA is a non-fluorescent compound that is converted to the highly fluorescent DCF (2',7'-dichlorofluorescein) by ROS. The antioxidant activity of the extracts was evaluated in two variants of cell exposure: pretreatment of cells with extracts followed by exposure to 0.5 mM H2O2 for 15 minutes and simultaneous exposure to the extract and 0.5 mM H2O2 for 15 minutes. The concentration of the extracts used for treatment was determined according to 20μM of gallic acid in the DPPH test. Zeiss LSM-800 with objective PlanApo20x/08 was utilized for visualization. Both pretreatment with extracts groups showed a significant protective effect against cellular oxidative stress as no DCF (2',7'-dichlorofluorescein) signal was detected, and all cells survived after treatment with 0.5 mM H2O2 (Figure 1). In contrast, in the control and both simultaneous treatments with extracts and 0.5 mM H2O2 groups, a fluorescent signal of DCF was detected, and a large number of cells died and detached from the substrate. The mechanism by which the extracts protect cells from oxidative stress only by front-loading treatment may involve modulating the levels of intracellular antioxidant pathways. C. deserticola polysaccharide upregulates the NRF2/HO-1 antioxidant pathway [4], while E. ulmoides Oliv polysaccharide inhibits ROS by activating NF-κB, an inflammatory response pathway, to protect cells from oxidative stress damage polysaccharide [5]. In addition, the extract was also able to reduce the cellular inflammatory response by directly inhibiting the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β) and interleukin-6 (IL-6) [6]. Plant extracts may also protect cellular components by increasing the expression levels of intracellular antioxidant substances such as glutathione and β-carotene. Overall, the study suggests that the protective effects of E. ulmoides and C. deserticola extracts on cells under oxidative stress may involve complex interactions with cellular components and intracellular pathways [8]. Further research is needed to fully understand the mechanisms by which the extracts provide protection against oxidative stress and to determine the most effective ways to use these extracts for this purpose [8]. Detection of reactive oxygen species induced by H2O2 in NIH/3T3 fibroblasts by DCFHDA assay. Confocal and transmitted light images after 15 minutes of exposure to 0.5mM H2O2 NIH/3T3 fibroblast pretreated overnight with extracts from E.ulmoides and C.deserticola and NIH/3T3 fibroblast images after 15 minutes of exposure simultaneously to 0.5mM H2O2 and E.ulmoides and C.deserticola extrats. Cells without treatment with extracts were used as control. Scale bar 50μm.