*3.9. Inhibition of ROS Generation in H2O2-Induced HepG2 Cells*

H2O2-induced HepG2 cells were employed to determine the inhibitory effect on ROS production [3]. HepG2 cells (1.0 <sup>×</sup> <sup>10</sup><sup>5</sup> cells per well) were seeded in a 6-well plate and co-cultured with isolated compounds with 50 µg/mL and Vc (8 µg/mL). After incubation for 20 h, the medium was removed, and 2 µL of H2O<sup>2</sup> (0.5 mM) was added to each well for another 6 h. At the end of experiment, the cells were labeled with 2 µL DCFH-DA (10 mM) in the dark at 37 ◦C for 20 min. The absorbance was recorded by flow cytometry (Guava easyCyte 6-2L, Millipore, Billerica, Massachusetts, MA, USA).

### *3.10. Determination of Cell Apoptosis*

The protective effect of each compound on H2O2-induced apoptosis of HepG2 cells was determined using a human annexin VFITC/PI apoptosis kit [38]. HepG2 cells were pre-treated with or without isolated compounds for 48 h. After incubation, 100 µL of the binding buffer was added to the cells, and the cells reacted in the dark with 10 µL annexin V-FITC for 5 min at room temperature and with 10 µL propidium iodide (PI) in an ice bath for 5 min, successively. Cell apoptosis was immediately analyzed using flow cytometry.

### *3.11. Statistical Analysis*

All the experiments were performed in triplicate. All the values are expressed as mean ± standard deviation (SD). The differences within and between the groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's test. Difference was considered statistically significant at *p* < 0.05. All analyses were performed using Origin 2019b software (OriginLab, Northampton, MA, USA).

### **4. Conclusions**

In this study, different fractions from *A. fragrans* leaves were fractionated, and their TPC and TFC were analyzed. Under antioxidant activity guided isolation, compounds **1**−**7**, including four flavonoid glycosides (**1**–**4**) and two flavonoids (**5** and **6**), were isolated and identified from *A. fragrans* leaves, which suggested that this species is rich in flavonoid compounds. Compounds **2**, **5**, and **6** showed significant antioxidant activity in DPPH, ABTS radical scavenging, and FRAP assays. Furthermore, they visibly prevented the oxidative stress damage through a decrease in ROS content and cell apoptosis in H2O2 induced HepG2 cells. According to these results, polyphenol compounds, especially flavonoids, have considerable antioxidant capacity because of their phenolic hydroxyl groups. Furthermore, compound **2**, possessing the glycoside moiety and three phenolic hydroxyl groups, was the main antioxidant component with the highest content from *A. fragrans* leaves. Compound **6** displayed the best antioxidant activity, which may be a major contribution to the activity of *A. fragrans.* Furthermore, the extracts of *A. fragrans* could be served as a feasible natural source of antioxidants in promising health beverages. The study on the compounds from *A. fragrans* leaves suggest that these could be served as antioxidant healthy tea for treating oxidative stress-induced cell damage and could serve as nutritional supplements applied in the food and health industry.

**Author Contributions:** S.H.: investigation, methodology, data curation, visualization, writing original draft; X.C.: conceptualization, methodology, data curation, visualization, writing—original draft; A.K.: writing—review and editing; Y.L.: formal analysis, writing—review and editing, funding acquisition; Y.W.: visualization; Q.C.: software, data curation, supervision; T.Z.: software, validation; J.C.: resources, supervision; G.C.: conceptualization, methodology, validation, writing—review and editing, supervision, project administration, funding acquisition; All authors have read and agreed to the published version of the manuscript.

**Funding:** This project was financially supported by Yunnan Major Science and Technology Project in China (2019ZF010), the National Natural Science Foundation of China (31872676 and 32060542) and China Postdoctoral Science Foundation (2020M673586XB).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** This project was supported in parts by Yunnan Major Science and Technology Project (Grant No. 2019ZF010) and the National Natural Science Foundation of China (Grant No. 32060542).

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of the compounds are available from the authors.

### **References**

