*4.1. Plant Extraction and Standardization*

Plant extraction and standardization were performed as described previously [22]. Briefly, the *L. obtusiloba* stems were collected in the vicinity of Hongcheon, Korea, and the voucher specimen (no. YJP-14) was stored at the Herbarium of KIST Gangneung Institute (Gangneung, Korea). *L. obtusiloba* was identified by Dr. Sang Hoon Jung, KIST Gangneung Institute, Gangneung, Korea. Short and dried branches were segmented into small slices and pulverized with a commercially available food mixer. Using 50% ethanol, 15.8 kg of *L. obtusiloba* powder was extracted four times for 4 h at 70 ◦C. The ethanolic solution was vaporized to dryness under vacuum to obtain 1.7 kg of the total extract (yield 7.0%). The polyphenol content in the LOE was evaluated via the Folin–Ciocalteau assay and was approximately 23.7% described as (−)-epicatechin equivalents. Previous study has reported that the major compounds in LOE were hyperin, isoquercitrin, guaijaverin, avicularin, and quercitrin [22,23]. Among them, hyperin and isoquercitrin were determined as standard substances for standardization of LOE. An HPLC analysis of LOE yielded a linear calibration curve for the standard compounds, which include hyperin and isoquercitrin. The main compounds including hyperin and isoquercitrin were identified in *L. obtusiloba* stems and designated as standard compounds by the Korea Food and Drug Administration (KFDA). LOE (10 µL) in 50% aqueous methanol (10 mg/mL), hyperin (16 µg/mL), and isoquercitrin (7 µg/mL) was loaded onto an Agilent 1200 series HPLC system. Separation was performed using a C18 reverse-phase column (4.6 × 250 mm, 10 µm, Shiseido, Tokyo, Japan) at 35 ◦C and eluted at a flow rate of 1.0 mL/min using a mobile phase of 17% aqueous acetonitrile acidified with 0.1% trifluoroacetic acid. Chromatographic profiles were recorded at 254 nm. The correlation coefficient was 0.998 for the respective standard curve. Hyperin and isoquercitrin in the LOE were identified and quantified at 15.33 min and 16.48 min, respectively, as shown in Supplementary Figure S1. The results revealed that 100 mg of LOE contains 0.138 mg of hyperin and 0.055 mg of isoquercitrin. These concentrations are within the appropriate ranges approved by the KFDA. All other quality control test results including heavy metals and pesticide residues were within acceptable limits.

### *4.2. Animals*

This study complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the IACUC of Gyeonggi Biocenter (Approval No. 2009-09-09). For this study, 14 male C57BL/6 mice (6 weeks old, 21–24 g) and 42 male apoE−/<sup>−</sup> mice (6 weeks old, 20–25 g) were purchased from Central Lab (Seoul, Korea) and acclimated with normal chow diet for 2 weeks. At the age of 8 weeks, apoE−/<sup>−</sup> mice were randomized into 3 groups; fed a western diet (WD: Research Diets, Inc. (New Brunswick, NJ, USA) composed of (wt/wt) 20% protein, 50% carbohydrate, 21% fat, and 0.15% cholesterol); and administered the vehicle (0.5% carboxymethylcellulose (CMC); apoE−/<sup>−</sup> vehicle group, *n* = 14), LOE (100 mg/kg per day; apoE−/<sup>−</sup> LOE group, *n* = 14), or losartan (30 mg/kg per day; apoE−/<sup>−</sup> losartan group, *n* = 14) by gavage until the age of 28 weeks. C57BL/6 mice exposed to a normal chow diet and treated with 0.5% CMC by gavage were used as

negative controls. The dose of losartan was selected based on previous studies indicating that a maximal functional effect without hemodynamic compromise can be obtained with this dose (data not shown). After 20 weeks of treatment, blood was collected under pentobarbital (50 mg/kg, i.p.) anesthesia, and the aorta and the heart were removed. The blood was centrifuged (3000 rpm, 10 min), and serum was obtained and stored at −70 ◦C until use.

### *4.3. Vascular Reactivity*

Aortic rings (3–4 mm in length) were obtained and mounted in myographs and placed in organ baths containing Krebs bicarbonate solution (in mM: 119 NaCl, 4.7 KCl, 1.18 KH2PO4, 1.18 MgSO4, 1.25 CaCl2, 25 NaHCO3, and 11 D-glucose, pH 7.4), which was oxygenated (95% O2; 5% CO2) and warmed to 37 ◦C to measure isometric tension. Following equilibration for 60 min under a resting tension of 1.0 g, the maximal contraction was measured by monitoring vasoconstriction evoked using a potassium-rich Krebs solution (80 mM) for 10 min. Subsequently, the rings were washed for 60 min and contracted with phenylephrine (100 nM). After a washout and a 30-min equilibration period, the rings were contracted again with increasing concentrations of phenylephrine to approximately 80% of the maximal contraction. The relaxation induced by cumulative treatment with Ach (1 nM–10 µM) on a half-logarithmic scale was measured to yield a concentrationrelaxation curve to Ach. Concentration response is expressed as a percentage of contraction by phenylephrine.

### *4.4. Determination of Vascular ROS Formation*

The in situ ROS synthesis was measured using the oxidative fluorescent dye DHE (Sigma–Aldrich, Milwaukee, WI, USA) as previously described [4]. Thoracic aortas from all groups were embedded into an optimal cutting temperature (OCT) compound (O.C.T. Tissue-Tek, Sakura Finetek, Torrance, CA, USA) and then frozen in liquid nitrogen for cryostat sectioning. The frozen aortas were sliced into 5 µm thick sections, followed by incubation with DHE (2.5 µM) in a humidified light-protected chamber for 30 min at 37 ◦C. Images were examined using a confocal microscope (LSM 510 META, Carl Zeiss, Inc., Overkochen, Germany) with a 20× epifluorescence objective. Mean intensities are expressed as arbitrary densitometric units.

### *4.5. Immunohistochemical Analysis of NADPH Oxidase Subunit Expression*

Thoracic aorta sections (5 µm) were sliced from paraffin blocks fixed with 4% paraformaldehyde. Antigen retrieval was performed by heating the sections in a 60 ◦C oven with a 10 mM citrate buffer overnight. After cooling, the sections were incubated with 3% hydrogen peroxide for 10 min to inhibit endogenous peroxidases. A rabbit polyclonal anti-p22phox antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a 1:50 dilution for p22phox immunostaining. An Alexa Fluor 488-conjugated secondary antibody (Invitrogen Corp., Carlsbad, CA, USA) was used at a 1:2000 dilution. For p47phox immunostaining, the sections were incubated with a rabbit polyclonal anti-p47phox antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted at 1:50 in 0.5 M TBST at 4 ◦C overnight. An Alexa Fluor 555-conjugated secondary antibody (Invitrogen Corp., Carlsbad, CA, USA) was used at a 1:2000 dilution. Nuclear counterstaining was accomplished with DAPI at a 1:10,000 dilution. The sections were stored in the dark until they were analyzed using a confocal microscope (LSM 510 META, Carl Zeiss, Inc., Overkochen, Germany).

### *4.6. Macroscopic Fluorescence Reflectance Imaging of Plaque Inflammation*

To investigate the ability of LOE to reduce plaque inflammation in apoE−/<sup>−</sup> mice, we employed ex vivo FRI to evaluate aortic plaque inflammation. Twenty-four hours before imaging, molecular imaging agents targeted against inflammatory endothelial cells (atherosclerotic plaque-homing peptide (AP)-hydrophobically modified glycol chitosan (HGC)-Cy5.5 nanoparticles; kindly provided by Dr. KM Kim from KIST (Korea)) were

intravenously injected via the tail vein (10 mg/kg) [39]. The extent of plaque inflammation in apoE−/<sup>−</sup> mice compared with control C57BL/6 mice and the plaque inflammationmodulating effects of LOE compared with losartan were determined via the ex vivo FRI of the extracted aortas. Immediately before imaging, the mice were sacrificed via cervical dislocation and perfused with 20 mL saline. The aortas, which were connected to the hearts, were then excised and imaged in the Cy5.5 channel on an IVIS-200 FRI system (Xenogen Corp., Alameda, CA, USA).

### *4.7. Measurement of H&E Staining and the Aortic Atherosclerotic Plaque Area*

After sacrifice, the mice were perfused with PBS through the left ventricle. For histopathological analysis, isolated aortic roots and right carotid arteries were embedded in an OCT compound. The frozen sections of the embedded aortic roots and carotid arteries were obtained. Adjacent sections were stained with H&E for general morphological analysis. The aortic lesion area (mm<sup>2</sup> ) was quantified using ImageJ software (NIH). The images of aortic plaque were calibrated using a hemocytometer, with 1 mm considered equal to 1600 pixels. The aortic plaque area was estimated as the area between the internal elastic lamina and the lumen on H&E sections.

### *4.8. Statistical Analysis*

Statistical analysis was performed using SPSS software (version 11, SPSS, Inc., Chicago, IL, USA). All values are reported as the mean ± SEM. Differences in the measured values among multiple groups were analyzed via analysis of variance, followed by Bonferroni's multiple comparison. For all statistical analyses, a *p*-value less than 0.05 was considered statistically significant.

### **5. Conclusions**

Treating WD-fed apoE−/<sup>−</sup> mice with LOE improves endothelial dysfunction by reducing NADPH oxidase expression and the formation of ROS. Consequently, LOE treatment is associated with the prevention of atherosclerotic inflammation and plaque development in apoE−/<sup>−</sup> mice. Altogether, the present findings indicate that LOE might be an attractive herbal candidate for the development of atherosclerosis associated with endothelial dysfunction and vascular oxidative stress.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/plants10112493/s1, Figure S1: Representative HPLC chromatograms of hyperin and isoquercitrin (a), and LOE (b) under conditions described in the Materials and Methods section. Peaks for hyperin and isoquercitrin are indicated.

**Author Contributions:** Conceptualization, methodology, validation, investigation, and writing review and editing, S.-H.I.; methodology, software, validation, formal analysis, data curation, writing—original draft preparation, and visualization, S.-H.P.; methodology, investigation, and formal analysis, O.-R.K. and E.-H.P.; funding acquisition, project administration, and software, J.-O.L., K.-R.K., and J.-H.K.; validation, and writing—review and editing, B.-H.H. and H.-J.Y.; conceptualization, writing—review and editing, supervision, and funding acquisition, M.-H.O. and K.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded in part by the Ministry of Knowledge Economy (MKE), Korea, under the Leading Industry Development for Gangwon Economic Region (LIDGER) program (No. 70007579) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07049375).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the IACUC of Gyeonggi Biocenter (Approval No. 2009-09-09).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All the data generated and analyzed during this study are included in this article.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

### **References**

