**1. Introduction**

Atherosclerosis is a narrowing of the arteries caused by the buildup of plaque. It is characterized by endothelial dysfunction, oxidative stress and chronic low-grade inflammation [1–3]. Endothelial dysfunction is the initial event leading to atherogenesis and is associated with increased generation of NADPH oxidase-derived reactive oxygen species (ROS) in vessel walls [4,5]. Increased vascular ROS generation, including hydrogen peroxide and superoxide anions, contributes to the formation of atherosclerotic plaque by inducing oxidative stress, reducing nitric oxide (NO) bioavailability, and

**Citation:** Ihm, S.-H.; Park, S.-H.; Lee, J.-O.; Kim, O.-R.; Park, E.-H.; Kim, K.-R.; Kim, J.-H.; Hwang, B.-H.; Youn, H.-J.; Oak, M.-H.; et al. A Standardized *Lindera obtusiloba* Extract Improves Endothelial Dysfunction and Attenuates Plaque Development in Hyperlipidemic ApoE-Knockout Mice. *Plants* **2021**, *10*, 2493. https://doi.org/10.3390/ plants10112493

Academic Editors: Juei-Tang Cheng, I-Min Liu and Szu-Chuan Shen

Received: 21 October 2021 Accepted: 11 November 2021 Published: 18 November 2021

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promoting proinflammatory responses [3,6,7]. Vascular NADPH oxidase represents an important source of ROS in numerous cardiovascular diseases involving the pathogenesis of atherosclerosis [8–10]. Indeed, Nox2 or p47phox protein deficiency reduced the formation of atherosclerotic plaque in high-fat diet-fed apolipoprotein E-deficient (apoE−/−) mice [11,12], indicating that NADPH oxidase was crucial for progression of atherosclerotic lesions. Furthermore, inflammation is related to all aspects of atherosclerosis, including the formation of foam cells, the progression and disruption of plaque, and the formation of thrombus [13]. Upregulation of adhesion molecules, cytokines, and chemokines expressed in endothelial cells and their complex interaction promotes leukocyte infiltration into the vascular wall, followed by transendothelial migration, which triggers atherogenesis [14,15].

*Lindera obtusiloba*, which is widely distributed in East Asian countries, has long been used as a traditional herbal medicine to augment circulation in the body, possibly via vasodilation; to suppress inflammation; and to prevent hepatic injury [16]. The extracts were derived from different parts of *L. obtusiloba* and their biologically active compounds such as butenolides, polyphenols, flavonoids, lignans, and neolignans exhibit antioxidant effects. The cytotoxic, anti-allergic, neuroprotective, antithrombotic, and anti-inflammatory effects of these bioactive compounds have been reported [17–20]. Thus, *L. obtusiloba* possesses an abundance of bioactive compounds, especially antioxidants, which have been studied in several diseases associated with oxidative stress. In addition, the *L. obtusiloba* extract (LOE) inhibits adipogenesis via persistent Wnt signaling and diminishes the tumor necrosis factor α- and lipopolysaccharide-induced IL-6 secretion by preadipocytes, suggesting its therapeutic potential in metabolic syndrome and obesity [21]. Our previous study demonstrated that LOE induces NO-mediated endothelium-dependent relaxation, reduces ROS generation in isolated aortic rings, and prevents hypertension and endothelial dysfunction induced by angiotensin II in rats [22]. In addition, we reported that LOE improves vascular oxidative stress and endothelial dysfunction most likely via normalization of the angiotensin system in diabetic mice [23]. Overall, these findings suggest that LOE has the potential to inhibit ROS generation, to improve endothelial dysfunction in vessel walls, and to reduce inflammatory cytokine production.

Therefore, the aim of the present study was to assess whether LOE improves endothelial dysfunction and prevents the development of atherosclerosis by reducing vascular ROS generation in an experimental model of atherosclerosis, the apoE−/<sup>−</sup> mice. In particular, the effect of the LOE intake was determined on (1) the endothelium-dependent vascular relaxation of mice aortic rings, (2) the vascular generation of ROS and NADPH oxidase subunits in aortic sections, and (3) the plaque inflammation of aortas and atherosclerotic plaque burden in the aortic sinus.

### **2. Results**
