The Chemical Composition of Brazilian Green Propolis and Its Protective Effects on Mouse Aortic Endothelial Cells against Inflammatory Injury

Propolis has a very complex composition, with antibacterial, anti-inflammatory and other properties. To determine the composition of ethanol extracts of Brazilian green propolis (EEP-B) and their protective effect on mouse aortic endothelial cells (MAECs), the chemical composition of EEP-B was analysed by UPLC/Q-TOF-MS/MS, and the protective effect of EEP-B on the proliferation of lipopolysaccharide (LPS)-induced MAECs was determined by Cell Counting Kit-8 (CCK-8) assays. The protein levels of inflammatory cytokines tumour necrosis factor-α (TNF-α) and interleukin- 6 (IL-6) were measured by enzyme-linked immunosorbent assay (ELISA), and ICAM-1, VCAM-1 and MCP-1 expressions were analysed by western blotting. The results showed that a total of 24 compounds belonging to cinnamic acids and flavonoids, including 3,5-diisopentenyl-4-hydroxycinnamic acid (artepillin C), kaempferide, 3-isoprenyl p-coumaric acid, pinocembrin and 4′-methoxy pinobanksin, were identified in EEP-B. Among them, a new component, suggested to be 5-isoprenyl caffeic acid p-coumaric acid ester, was reported for the first time. The LPS-induced levels of TNF-α, IL-6, ICAM-1, VCAM-1 and MCP-1 were downregulated in response to 5, 10 and 20 μg/mL EEP-B. This study revealed that EEP-B could reduce LPS-induced inflammatory reactions, improve cell survival, and protect MAECs by regulating ICAM-1, VCAM-1 and MCP-1 expression. These findings could provide a theoretical basis for MAEC treatment using EEP-B.


Chemical Composition
The levels of total polyphenols and flavonoids in EEP-B were 218.67 mg/g and 227.20 mg/g, respectively.
Chromatographic profiles and chemical compositions were determined by UPLC-Q-TOF-MS, and are shown in Figure 1 and Table 1. A total of 28 constituents was identified in EEP-B. These components mainly belong to cinnamic acids, of which artepillin C was the most abundant component (relative content 35.68%). In addition, there were four triterpenes detected, but their structures have not been identified.

Effect of EEP-B on MACE Proliferation
As shown in Figure 4, the survival rate of each group was measured by CCK-8 assays, and there were significant differences among all groups. The survival rate of the LPS-stimulated group was 67.35%, which was significantly lower than that of the control group. The survival rates of the cells were significantly increased when the EEP-B was at the concentrations of 5, 10 and 20 μg/mL, and the survival rate was the highest at 20 μg/mL.

Effect of EEP-B on MACE Proliferation
As shown in Figure 4, the survival rate of each group was measured by CCK-8 assays, and there were significant differences among all groups. The survival rate of the LPS-stimulated group was 67.35%, which was significantly lower than that of the control group. The survival rates of the cells were significantly increased when the EEP-B was at the concentrations of 5, 10 and 20 µg/mL, and the survival rate was the highest at 20 µg/mL.

Effect of EEP-B on the Levels of IL-6 and TNF-α
The expression levels of IL-6 and TNF-α were measured by ELISA. As shown in Figure 5, compared with those of the control group, the amounts of IL-6 and TNF-α secreted in the LPSstimulated group were increased by 3.23-and 2.31-fold, respectively. After EEP-B treatment, the levels of IL-6 and TNF-α decreased significantly. At a concentration of 20 μg/mL EEP-B, the expression levels of these cytokines were the lowest. Influence of EEP-B on LPS-stimulated MACE growth. ## means extremely significant difference (p < 0.01) between LPS-stimulated group and control and ** means extremely significant difference (p < 0.01) between propolis treatment group and LPS-stimulated group.

Effect of EEP-B on the Levels of IL-6 and TNF-α
The expression levels of IL-6 and TNF-α were measured by ELISA. As shown in Figure 5, compared with those of the control group, the amounts of IL-6 and TNF-α secreted in the LPS-stimulated group were increased by 3.23-and 2.31-fold, respectively. After EEP-B treatment, the levels of IL-6 and TNF-α decreased significantly. At a concentration of 20 µg/mL EEP-B, the expression levels of these cytokines were the lowest.
The expression levels of IL-6 and TNF-α were measured by ELISA. As shown in Figure 5, compared with those of the control group, the amounts of IL-6 and TNF-α secreted in the LPSstimulated group were increased by 3.23-and 2.31-fold, respectively. After EEP-B treatment, the levels of IL-6 and TNF-α decreased significantly. At a concentration of 20 μg/mL EEP-B, the expression levels of these cytokines were the lowest. Effects of EEP-B on the levels of IL-6 and TNF-α. ## means extremely significant difference (p < 0.01) between LPS-stimulated group and control and ** means extremely significant difference (p < 0.01) between propolis treatment group and LPS-stimulated group.

Effect of Propolis on the Levels of MCP-1, ICAM-1 and VCAM-1
The levels of MCP-1, ICAM-1 and VCAM-1 were measured by western blotting (Figure 6). The results showed that the expression of MCP-1, ICAM-1 and VCAM-1 in the LPS group was significantly higher than that in the control group, and increased by 3.95-, 7.76-and 9.11-fold, respectively. In the EEP-B treatment group, the levels of MCP-1, ICAM-1 and VCAM-1 also decreased significantly, especially at a concentration of 20 μg/mL. The levels of MCP-1, ICAM-1 and VCAM-1 were measured by western blotting (Figure 6). The results showed that the expression of MCP-1, ICAM-1 and VCAM-1 in the LPS group was significantly higher than that in the control group, and increased by 3.95-, 7.76-and 9.11-fold, respectively. In the EEP-B treatment group, the levels of MCP-1, ICAM-1 and VCAM-1 also decreased significantly, especially at a concentration of 20 µg/mL.

Discussion
The chemical constituents of propolis vary in different plant sources. Brazilian propolis can be classified into 13 different types according to different geographical sources, resulting in the complexity of the components [35,36]. The main chemical constituents of Brazilian green propolis are coumaric acids. In the current study, prenylated derivatives of p-coumaric acid, diterpenes and flavonoids were detected. Among these components, the level of artepillin C was much higher than that of the others, which is one of the characteristics of Brazilian propolis [37]. The level of artepillin C in propolis varies greatly depending on the geographical origin, and the samples collected from southeast Brazil are more abundant (usually 5-11%) in this component than those from other regions [37]. In addition, the components with high levels were kaempferide (7.06%), 3-isoprenyl p-coumaric acid (6.19%), pinocembrin (5.56%), diisoprenyl-p-coumaric acid isomer (4.49%), 4′-methoxy pinobanksin (4.10%) and 3-hydroxy-2,2-dimethyl-8-prenylchromane-6-propenoic (3.39%), which was consistent with previous studies [12].

Discussion
The chemical constituents of propolis vary in different plant sources. Brazilian propolis can be classified into 13 different types according to different geographical sources, resulting in the complexity of the components [35,36]. The main chemical constituents of Brazilian green propolis are coumaric acids. In the current study, prenylated derivatives of p-coumaric acid, diterpenes and flavonoids were detected. Among these components, the level of artepillin C was much higher than that of the others, which is one of the characteristics of Brazilian propolis [37]. The level of artepillin C in propolis varies greatly depending on the geographical origin, and the samples collected from southeast Brazil are more abundant (usually 5-11%) in this component than those from other regions [37]. In addition, the components with high levels were kaempferide (7.06%), 3-isoprenyl p-coumaric acid (6.19%), pinocembrin (5.56%), diisoprenyl-p-coumaric acid isomer (4.49%), 4 -methoxy pinobanksin (4.10%) and 3-hydroxy-2,2-dimethyl-8-prenylchromane-6-propenoic (3.39%), which was consistent with previous studies [12].
Lipopolysaccharide (LPS) is involved in the development of atherosclerosis, especially in the initial process of inflammation in atherosclerotic vessels. LPS can activate and damage endothelial cells and cause changes in the morphology and function of vascular endothelial cells [29,30]. In atherosclerotic plaque formation, vascular endothelial cell apoptosis accelerates the process of atherosclerosis [41]. In this study, LPS was used to stimulate MAECs, resulting in increased IL-6 and TNF-α expression. TNF-α and IL-6 are important inflammatory cytokines whose secretion can be stimulated by hyperglycaemia, hyperlipidaemia and other metabolic abnormalities [42,43]. IL-6 can promote the expression of adhesion molecules and other inflammatory mediators in vascular endothelial cells, and enhance the local inflammatory response, and it is an important inflammatory factor involved in tissue lesions [44]. IL-6 expression was correlated with intimal thickening and decreased elasticity [45]. Tumour necrosis factor (TNF-α) can inhibit endothelial cell proliferation, activate the nuclear factor-κB (NF-κB) signalling pathway, induce the expression of intercellular adhesion molecules such as ICAM-1 and VCAM-1, and promote monocyte infiltration to participate in the inflammatory pathological process of atherosclerosis (AS) [46][47][48].
EEP-B inhibited the expression of IL-6 and TNF-α in MAECs, which was in accordance with a previous report, suggesting that Brazilian green propolis extract has good anti-inflammatory effects by inhibiting the proinflammatory cytokines IL-6 and TNF-α [17]. Both Chinese propolis and Brazilian propolis have good immunomodulatory effects on LPS-induced inflammation. Chinese propolis modulates the suppression of autophagy and the MAPK/NF-κB signalling pathway to protect vascular endothelial cells from LPS stimulation [49]. Brazilian green propolis exerts strong anti-inflammatory effects by inhibiting the production of many cytokines, such as IL-1α, IL-1β, IL-4, IL-6, TNF-α and MCP-1, in stimulated J774A cells [39]. Similar to green propolis, red propolis extract can also inhibit the release of inflammatory cytokines, including TNF-α, IL-1β, CXCL1/KC and CXCL2/MIP-2. In addition, Brazilian propolis can prevent neutrophil migration into the peritoneal cavity, and reduce leukocyte rolling and adhesion on the mesenteric microcirculation [50].
Propolis plays an important role in the prevention and treatment of cardiovascular diseases [20,51,52]. In initial atherosclerotic lesions, propolis polyphenol extracts improved the lipid profile and decreased the atherosclerotic lesion area [53], as well as reducing the total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), triglycerides and thiobarbituric acid-reactive substance concentrations [54]. The propolis component chrysin has been confirmed to inhibit plasma plasminogen activator inhibitor 1 (PAI-1) production, and could be used to treat or prevent thrombotic disorders [25]. The expressions of ICAM-1, VCAM-1 and MCP-1 were rapidly upregulated upon stimulation with the inflammatory factor LPS. The high expression of ICAM-1 enhances the adhesion of monocytes to endothelial cells, VCAM-1 can promote monocyte adhesion to endothelial cells, and monocyte chemotactic protein-1 (MCP-l) can activate monocytes by binding with receptors [55,56]. The current study revealed that Brazilian green propolis could reduce the expression levels of ICAM-1, VCAM-1 and MCP-1, in order to inhibit the migration of monocytes from the blood circulation to the intima of the blood vessels, reduce the aggregation of foam cells and lipids in the arterial wall, and inhibit the chemotaxis of monocytes to the lesion site, thereby reducing the accumulation of lipids in the intima and damage to the intima of blood vessels.

Preparation of Experimental Samples
Brazilian green propolis was supplied by Fujian Shenfeng Technology Development Co., Ltd. (Fuzhou, China). Propolis was extracted using ethanol (70%, v/v) according to a previously described method [38]. The ultrasonic wave method was used to extract propolis, and beeswax was removed. The ethanol extracts of Brazilian propolis were named EEP-B.

Chemical Analysis of EEP-B
The quantification of total polyphenol and phenolic contents and chemical composition analysis were also performed according to a previously described method [28]. The quantification of total polyphenols was performed using the Folin-Ciocalteu method, and gallic acid was used as a standard. The total flavonoid content was determined as described in the national standards for propolis in China (GB/T 24283-2018), and rutin was used as a standard. UPLC-ESI-MS analysis was performed on a Waters UPLC system, and the mobile phases consisted of 0.1% formic acid in water (A) and acetonitrile (B). The mass spectra were obtained using a Waters definition accurate-mass quadrupole time-of-flight (Q-TOF) Xevo G2-XS mass spectrometer (Waters Ltd., Elstree, Hertfordshire, UK), which was equipped with an ESI source, in both positive and negative ion modes.

Cell Culture
MAECs and reagents for cell culture were purchased from Procell Life Science and Technology Co. (Wuhan, China). Cells were cultured to a density of 1 × 10 5 /mL at 37 • C in 5% CO 2 and then divided into the following five groups: cells not treated with LPS or propolis that were used as control, cells stimulated with LPS (10 µg/mL), and cells stimulated with LPS (10 µg/mL) that were treated with different concentrations of EEP-B (5, 10 and 20 µg/mL).

Cell Viability Assay
Cells were added to each well at a density of 1 × 10 5 /mL and cultured for 24 h, then 10 µL of Cell Counting Kit-8 (CCK-8) solution (Beyotime Biotechnology, Shanghai, China) was added and further cultured for 4 h at 37 • C in 5% CO 2 . Then, the absorbance was measured at 450 nm. The cell viability (%) was calculated by the following formula: (absorbance of experimental wells/absorbance of control wells) × 100%.

ELISA Analysis of IL-6 and TNF-α Expression
The cells were cultured for 24 h and used for enzyme-linked immunosorbent assay (ELISA) analysis. The supernatant of each group was analysed according to the procedures of the ELISA kit (Neobioscience, Shenzhen, China). The absorbance of each sample was measured at 450 nm by a microplate reader (Diatek, Wuxi, China), and the concentrations of IL-6 and TNF-α were calculated.

Western Blot Analysis of ICAM-l, VCAM-1 and MCP-1 Expression
The cells were cultured for 24 h and used for western blot analysis. Radio immunoprecipitation assay (RIPA) buffer was used to lyse the cells, and the protein concentration was measured by the bicinchoninic acid (BCA) method using the Pierce TM Rapid Gold BCA Protein Assay Kit (Elabscience, Wuhan, China). Total protein samples from each group were extracted for sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, incubated with the primary antibody against the target protein overnight at 4 • C and washed 5 times using Tris-buffered saline Tween-20 (TBST). Then, the secondary antibody was added and incubated for 1 h at room temperature, and the membrane was washed with TBST again. Finally, Clarity Western ECL Substrate was used for luminescence detection, and AlphaEaseFC was used to analyse the gel image. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Statistical Analysis
All experiments were performed in triplicate. The data are expressed as the mean ± standard deviation (n = 3). Statistical analysis was performed using t-tests and one-way ANOVA with SPSS software. Differences of p < 0.05 were considered statistically significant.

Conclusions
Propolis possesses rich chemical components and robust anti-inflammatory activity. In this study, the chemical composition of Brazilian green propolis was analysed by UPLC-Q-TOF-MS. A total of 28 compounds was identified, and a new component, 5-isoprenyl caffeic acid p-coumaric acid ester, was tentatively identified for the first time. In addition, EEP-B showed strong anti-inflammatory effects by inhibiting the levels of the cytokines TNF-α and IL-6, and protecting MAECs by regulating ICAM-1, VCAM-1 and MCP-1 expression. This study provides new insights into the chemical components and the anti-inflammatory activities of Brazilian green propolis.