**Plasma Lipidomics Reveals Insights into Anti-Obesity E**ff**ect of** *Chrysanthemum morifolium* **Ramat Leaves and Its Constituent Luteolin in High-Fat Diet-Induced Dyslipidemic Mice**

**Jong Cheol Shon 1,2, Won Cheol Kim 2, Ri Ryu 3, Zhexue Wu 2, Jong-Su Seo 1, Myung-Sook Choi 4,\* and Kwang-Hyeon Liu 2,\***


Received: 2 September 2020; Accepted: 26 September 2020; Published: 29 September 2020

**Abstract:** The *Chrysanthemum morifolium* Ramat (CM) is widely used as a traditional medicine and herbal tea by the Asian population for its health benefits related to obesity. However, compared to the flowers of CM, detailed mechanisms underlying the beneficial effects of its leaves on obesity and dyslipidemia have not yet been elucidated. Therefore, to investigate the lipidomic biomarkers responsible for the pharmacological effects of CM leaf extract (CLE) in plasma of mice fed a high-fat diet (HFD), the plasma of mice fed a normal diet (ND), HFD, HFD plus CLE 1.5% diet, and HFD plus luteolin 0.003% diet (LU) for 16 weeks were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with multivariate analysis. In our analysis, the ND, HFD, CLE, and LU groups were clearly differentiated by partial least-squares discriminant analysis (PLS-DA) score plots. The major metabolites contributing to this differentiation were cholesteryl esters (CEs), lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs), ceramides (CERs), and sphingomyelins (SMs). The levels of plasma CEs, LPCs, PCs, SMs, and CERs were significantly increased in the HFD group compared to those in the ND group, and levels of these lipids recovered to normal after administration of CLE or LU. Furthermore, changes in hepatic mRNA expression levels involved in the Kennedy pathway and sphingolipid biosynthesis were also suppressed by treatment with CLE or LU. In conclusion, this study examined the beneficial effects of CLE and LU on obesity and dyslipidemia, which were demonstrated as reduced synthesis of lipotoxic intermediates. These results may provide valuable insights towards evaluating the therapeutic effects of CLE and LU and understanding obesity-related diseases.

**Keywords:** *Chrysanthemum morifolium* Ramat leaves; obesity; lipidomics; liquid chromatography tandem mass spectrometry; phospholipid; sphingolipid

#### **1. Introduction**

Obesity, one of the major worldwide health problems in recent years, is usually caused by an imbalance in food intake and energy expenditure, with overconsumption of energy intake or decrease in physical activity. Among the various models of inducing obesity in animals, the high-fat diet

(HFD)-induced obesity model alters diverse biochemical factors, such as insulin, eventually leading to abnormal lipid metabolism. HFD-induced obesity is characterized by abnormally high levels of blood lipids, such as cholesterol, low-density lipoprotein (LDL), and triacylglycerol (TAG), leading to progressive weight gain in various tissues of the body (e.g., liver and fat tissues) due to excess fat accumulation [1,2]. It is also associated with an increase in the prevalence and risk of metabolic syndrome-related diseases, including type 2 diabetes (T2D), dyslipidemia, cardiovascular diseases (CVD), hypertension, and fatty liver [3,4]. Especially, the development of obesity by HFD indicates that it is pathophysiologically very similar to obesity in humans [5,6].

*Chrysanthemum morifolium* Ramat (CM) has been used for almost a hundred years in the form of a traditional medicine, beverage, and herbal tea in many Asian countries, including Japan, China, Thailand, and Korea, due to its health benefits related to inflammation, hypertension, and arteriosclerosis. The main bioactive components in the flowers of CM are flavonoids and phenolic acids, which are composed of chlorogenic acid, acacetin, apigenin, and luteolin, along with its glucoside form [7]. Previous studies have shown that CM and its components possess various biological functions, including antioxidant [8], anti-inflammatory [9], anti-tumorigenic [10], and cardiovascular-protective functions [11]. Moreover, the CM flower extract has been shown to prevent hyperlipidemic fatty liver disease by increasing hepatic peroxisome proliferator-activated receptor (PPAR)-α expression in hyperlipidemic fatty liver disease caused by high-fat milk [12]. Such beneficial effects have also been reported in HFD-induced obese mice [13] and an alloxan-induced diabetes mellitus model [14]. Almost all the studies conducted on the efficacy of CM related to health-promoting effects have been focused on its flowers. However, although not used as a traditional medicine, unlike flowers, the leaves of CM have a large amount of potential bioactive components, such as flavonoids, galuteolin, quercetin, and chlorogenic acid [15,16]. Consequently, the leaves can be used for medicinal purposes because of their high content of bioactive components. Furthermore, a recent study revealed that weight gain, fat deposition, and plasma lipid levels were significantly reduced by the administration of CM leaf extract (CLE) in HFD-fed mice [17]. However, apart from the previous approach based on the major lipids, such as cholesterol, lipoprotein, and TAG, detailed studies on lipid profiles, including changes in individual lipid species, towards the pharmacological effects of CLE in obese mice have rarely been described.

Modern lipidomics technology based on mass spectrometry provides significant insights into the metabolism and alterations in lipids through the identification and quantification of individual lipid species in a broad range of lipids [18]. This technology has been used extensively in biomedical research to evaluate toxicity and pharmacological properties of drugs [19], to discover potential biomarkers for disease states [20], and to understand the pathological mechanism of the metabolic syndrome [21]. Previous studies have shown that changes in specific lipid profiles and composition, such as ceramides and sphingomyelin species, are closely related to obesity-related diseases and metabolic disorders [22, 23]. In addition, these techniques have also been used to explore the pharmacological effects of herbal medicines, such as *Cyclocarya paliurus* [24] and *Camellia sinensis* leaves [25], and *Panax ginseng* radix [26].

Thus, in this study, we conducted lipidomics analysis in HFD-induced metabolic disorders representing obesity and dyslipidemia as a pathology model. In addition, we also investigated the pharmacological effects of CLE and luteolin, which are closely related to the physiological functions and quality of CM [27], and its mechanisms against HFD-induced dyslipidemia through alterations of plasma lipid metabolites using the technique of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Methanol, isopropanol, and water were purchased from Merck (LC-MS grade, Darmstadt, Germany). Ammonium acetate, chloroform, methyl-*tert*-butyl ether (MTBE), butylhydroxytoluene

(BHT), and TAG (15:0/15:0/15:0) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lysophosphatidylcholine (LPC 17:1), lysophosphatidylethanolamine (LPE 17:1), phosphatidylcholine (PC 17:0/14:1), phosphatidylethanolamine (PE 17:0/14:1), diacylglycerol (DAG 8:0/8:0), sphingomyelin (SM d18:1/12:0), ceramide (CER d18:1/12:0), and cholesteryl ester (CE 15:0) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). The lipid standards mentioned above were used as internal standards for semi-quantification of each lipid.

#### *2.2. Preparation of Chrysanthemum morifolium Ramat Leaf Extract*

CM leaves (Haihang Industry, Jinan, China) were washed, crushed to 20 mesh, and extracted by adding 10 volumes of 70% ethanol to 10 kg CLE in an extraction vessel. The extraction was performed at 65–70 ◦C in the roof of the pot, which had a condenser. Vapor was condensed and returned back to the pot, which was recirculated three times, and each time-period lasted for 2 h. The extracted CLE liquid was subsequently concentrated and spray-dried at 120 ◦C to 80 mesh. The yield obtained was 10%. The contents of flavonoids in the CLE were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described in previous papers [28–30]. The contents (mg/g dry weight) of ten flavonoids in CLE were as follows: luteolin, 0.057; galuteolin, 0.47; luteolin-7-*O*-glucuronide, 0.28; luteolin-7-*O*-6-acetyl glucoside, 0.0047; diosmetin-7-*O*-rutinoside, 0.056; diosmetin-7-*O*-glucoside, 0.055; acacetin-7-*O*-rutinoside, 1.7; apigenin, 0.0062; quercetin, 0.0032; chlorogenic acid, 0.19 (Figure 1).

**Figure 1.** The total ion chromatogram (**A**) and selected reaction monitoring chromatogram (**B**) of individual flavonoids from *Chrysanthemum morifolium* Ramat leaf extracts (CLE).

#### *2.3. Animal Experiments*

Male C57BL/6J mice (4-week-old) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All the mice were individually housed under constant temperature (24 ◦C) and a 12 h light/dark cycle, fed a normal chow diet for a one-week acclimation period, and subsequently, randomly divided into four groups. The mice in the different groups were fed a normal diet (ND, 12% kcal from fat, *n* = 6), high-fat diet (HFD, 45% kcal from fat, *n* = 6), HFD with 1.5% CLE (*w*/*w*) (*n* = 6), and HFD with 0.003% luteolin (*w*/*w*) (LU, *n* = 6) for 16 weeks. The mice were given free access to food and distilled water, and food intake and body weight were measured weekly. At the end of the mice experimental period, all the mice were anesthetized with isoflurane after 12 h of fasting. Blood samples were collected from the inferior vena cava in heparin-coated tubes, and plasma samples were obtained by centrifuging blood at 4000× *g* for 15 min at 4 ◦C. The liver, epididymal white adipose tissue (WAT), perirenal WAT, retroperitoneal WAT, mesentery WAT, subcutaneous WAT, interscapular WAT, interscapular brown adipose tissue, and skeletal muscle were removed, weighed, and immediately frozen in liquid nitrogen and stored at 80 ◦C. Animal studies were performed using protocols approved by the Kyungpook National University Industry Foundation (Approval No. KNU-2016-49).

#### *2.4. Lipid Extraction*

Lipids were extracted from mouse plasma according to the Matyash method with slight modifications [31]. In brief, a 50 μL internal standard (IS) mixture (LPC 17:1 400 ng/mL; LPE 17:1 400 ng/mL; PC 17:0/14:1 400 ng/mL; PE 17:0/14:1 400 ng/mL; DAG 8:0/8:0 40 ng/mL; SM d18:1/12:0 400 ng/mL; CER d18:1/12:0 40 ng/mL; CE 15:0 4 μg/mL; TAG 15:0/15:0/15:0 40 ng/mL) was transferred to a 2 mL Eppendorf tube and dried. Later, 5 μL plasma, 300 μL cold methanol, 100 μL water, and 1 mL MTBE along with 0.1% BHT were added to the tube. The mixture was shaken for 1 h at room temperature. Next, phase separation was induced by adding 250 μL water and incubating for 10 min at room temperature. After centrifugation (14,000× *g* for 15 min at 4 ◦C), the two phases were separately collected in 1.5 mL tubes. The upper (220 μL) and lower (110 μL) fractions were pooled and dried in a vacuum centrifuge (LABCONCO, Kansas City, MO, USA) for lipid profiling. The dried lipid extracts were reconstituted in 100 μL chloroform/methanol (1:9, *v*/*v*) prior to lipidomics analysis. Using the above method, hepatic lipids were extracted from the lyophilized samples (10 mg).

#### *2.5. Lipidomics Study*

Analysis of lipid extracts was performed using a high-performance liquid chromatography (HPLC) apparatus coupled to a Shimadzu 8040 tandem mass spectrometer (Shimadzu Corporation, Kyoto, Japan), based on a previously reported method with some modifications [32,33]. Semi-quantitative analysis of the extracted lipids from the plasma of mice was carried out using a Kinetex C18 column (100 × 2.1 mm, 2.6 μm, Phenomenex, Torrance, CA, USA) with a flow rate of 200 μL/min in positive mode. Mobile phase A was 10 mM ammonium acetate in water:methanol (1:9, *v*/*v*) and mobile phase B was 10 mM ammonium acetate in methanol:isopropanol (1:1, *v*/*v*). To achieve the best separation of the lipids, gradient elution was conducted as follows: 30% B (0 min), 95% B (15 min), 95% B (20 min), and 30% B (20–25 min). Each lipid quantitation was performed by selected reaction monitoring (SRM) of the precursor ions, ([M + H]<sup>+</sup> or [M + NH4] <sup>+</sup>), and the related product ion for each lipid. To calculate the concentration of each target lipid species, the ratio of target analyte and internal standard (IS) was multiplied by the concentration of one specific IS representing each lipid species based on single-point calibrations [34–36]. IS and SRM transition ions for each lipid class are described in a previously published paper [33].

#### *2.6. Hepatic Gene Expression Analyses*

Total RNA from the liver was extracted from three representative samples in each group according to the manufacturer's instructions (Invitrogen Life Technologies, Grand Island, NY, USA). The libraries of mRNA sequences were prepared as paired-end reads with a length of 100 bases using the TruSeq RNA Sample Preparation Kit (Illumina, CA, USA). After cDNA synthesis, the quality of these cDNA libraries was evaluated employing the Agilent 2100 Bioanalyzer (Agilent, CA, USA), and then sequenced as paired-ends using Illumina HiSeq2500 (Illumina, CA, USA). Quality control was performed using FastQC v. 0.11.2. Transcripts were assembled in Cufflinks v2.2.1 with TopHat Aligner [17,37]. Gene expression levels were estimated as fragments per kilobase of transcript per million mapped reads. Normalization factors were calculated using the differentially expressed genes (DEGES)/Empirical analysis of digital gene expression data in R (edgeR) method. Differentially expressed genes were identified based on a q-value threshold of less than 0.05. The RNA-sequence data is available at the National Center for Biotechnology Information (NCBI)'s Gene Expression Omnibus Database (http://www.ncbi.nlm.nih.gov/geo/): accession number GSE124777.

#### *2.7. Data Processing and Statistical Analyses*

To confirm stability and reproducibility of the mass spectrometric analysis, a quality control (QC) sample [National Institute of Standards and Technology (NIST) Standard Reference Material (SRM 1950 plasma)] was loaded at regular intervals (ten samples) [38,39]. Unsupervised principal component analysis (PCA) and supervised partial least squares-discriminant analysis (PLS-DA) multivariate analyses were used to construct pattern recognition models based on plasma lipidomics data with Pareto scaling, to explore the extent of differences among the four groups using the software SIMCA-P+ (version 13.0, Umetrics, Umea, Sweden). All data were reported as mean ± standard deviation

(SD). Statistically significant differences between all the groups were calculated by one-way analysis of variance (ANOVA) and Tukey's multiple comparison test using Statistica 7 (StatSoft Inc., Tulsa, OK, USA).

#### **3. Results and Discussion**

#### *3.1. Animal Characteristics*

Male C57BL/6J mice were fed an ND, HFD, HFD + CLE, and HFD + LU for 16 weeks. Changes in biological characteristics of these mice are shown in Figure 2. The values of body weight, liver weight, total adipose tissue weight, and total cholesterol were 28.1 ± 1.4 g, 1.0 ± 0.1 g, 1.8 ± 0.2 g, and 4.1 ± 0.6 mmol/L in the ND group, respectively; however, due to administration of HFD, these levels were significantly elevated to 46.6 ± 1.5 g, 2.8 ± 0.3 g, 7.7 ± 0.6 g, and 7.8 ± 1.7 mmol/L respectively, indicating that the obese mouse model was well-established. In contrast, supplementation with CLE or LU significantly lowered body weight (*p* < 0.001), liver weight (*p* < 0.01), and total adipose tissue weight (*p* < 0.01) compared to that in the HFD group. Recently, CLE was shown to ameliorate insulin resistance, thermogenesis, and energy expenditure along with a decrease in weight gain in white adipose tissue [17]. Liver histological analysis using Hemotoxylin and Eosin (H&E) staining was employed to evaluate the preventive effects of CLE on HFD-induced obese mice (Figure 2f). The livers of mice in the ND group did not exhibit lipid deposition and signs of steatosis, whereas those in the HFD group displayed accumulation of lipid droplets and developed hepatic steatosis. However, the CLE- or LU-treated groups exhibited a significant reduction of hepatic lipid droplets. While LU also decreased the number of lipid droplets in the liver as CLE, this effect tended to be less (Figure 2f). The hepatic TAG contents in each group were calculated by summing the contents of all quantified TAGs. The hepatic TAG contents in the ND, HFD, CLE, and LU groups were 222.3 ± 21.2, 707.0 ± 76.5, 531.6 ± 57.2, and 587.2 ± 31.0 μg/mg, respectively (Figure 2e). As expected, the hepatic TAG contents in the CLE and LU groups were lower than those in the HFD group (*p* < 0.01), and most individual TAG species in CLE tended to decrease slightly more than those in the LU group (*p* > 0.05) (Supplementary Figure S1). The more effective decrease of lipid droplet number and total hepatic triacylglycerols in the CLE treatment group compared to the LU treatment may be due to bioactive compounds such as quercetin and chlorogenic acid present with luteolin in CM leaves. Energy expenditure was significantly increased during a 12 h light/dark cycle after CLE and LU treatment when compared with the HFD group. Considering the suppressed hepatic lipogenesis and enhanced energy expenditure by CLE and LU treatment [17], it was evident that CLE and LU have a potential to attenuate obesity in mice.

**Figure 2.** Effect of *Chrysanthemum morifolium* Ramat leaf extracts (CLE; 1.5%) and luteolin (LU; 0.003%) on body weight (**a**), liver weight (**b**), total adipose tissue weight (**c**), total cholesterol level (**d**), total hepatic triacylglycerol contents (**e**), and hepatic morphology (original magnification × 100) (**f**) in C57BL/6J mice fed a high-fat diet (HFD). The data are shown as mean ± standard deviation (SD) (*n* = 6). \*, # different letters indicate significant difference compared with normal diet (ND) group (\*) and HFD group (#), as determined by Tukey's multiple comparisons test. (\*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001, # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001).

#### *3.2. Multivariate Statistical Analysis of Mice Plasma Lipid Levels*

Representative pooled mice plasma samples were used to identify lipids therein. Approximately 140 individual lipid molecular species covering 9 lipid classes, comprising 16 LPC, 27 PC, 6 LPE, 11 PE, 41 TAG, 5 DAG, 14 SM, 14 CE, and 6 CER, were identified from pooled plasma samples of the mice [26]. The lipid profiles thus obtained from the ND, HFD, CLE, and LU groups were analyzed by multivariate statistical analysis to identify the extent of differences among the four groups. In the PCA score plot (Figure 3a), QC samples (NIST SRM1950 standard blood plasma, [40]) were tightly clustered, suggesting that the mass spectrometric analysis data has excellent stability and reproducibility. Performance of the constructed principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) model was assessed by fitness (*R*2) and predictability (*Q*2), and the performance levels of the good models are higher than 50%. The significance of the PLS-DA model was evaluated using cross-validated analysis of variance (CV-ANOVA) [41]. Each group showed a clear distinction in the PCA (*R*2X = 95%, *Q*<sup>2</sup> = 85%) and PLS-DA (*R*2X = 93%, *Q*<sup>2</sup> = 37%, *p* = 0.038) score plots with a good model quality. Clusters of lipid species from the CLE and LU groups were observed as separate clusters from those of the HFD group (Figure 3b,c).

**Figure 3.** Principal component analysis (PCA) three-dimensional (3D) score plots (**a**), PCA 3D score

plots excluding quality control (QC) (**b**), and partial least-squares discriminant analysis (PLS-DA) 3D score plots (**c**) based on the serum lipidomic profiling among four groups. (Circle (-): normal diet (ND); Box (-): high-fat diet (HFD); Inverted triangle (): HFD plus *Chrysanthemum morifolium* Ramat leaf extract 1.5% diet (CLE); Triangle (): HFD plus luteolin 0.003% diet (LU); Diamond (-): Quality control (QC)).

#### *3.3. E*ff*ect of a High-Fat Diet on Mice*

The sum (total) levels of the lipid species quantified in the plasma samples of mice were significantly elevated by HFD, and the lipid species contributing to these changes were LPC, PC, CE, SM, and CER. In contrast, the levels of LPE, PE, TAG, and DAG did not differ between the groups (Figure 4). The levels of 13 of 14 CE species, 12 of 27 PC species, 12 of 16 LPC species, all SM species, and 5 of 6 CER species were significantly increased by HFD, as shown in Supplementary Table S1. Similar to our results, accumulation of CE, PC, LPC, and sphingolipids (CER and SM) in plasma of HFD-induced obese and dyslipidemic mice has been reported [23,24,26,42–44]. The PC/PE ratio is a well-known marker associated with health and disease and regulates a variety of cellular processes [45]. Recently, an increase in these indicators has also been observed in the plasma of dyslipidemic and diabetic mice [24] and WAT macrophages in leptin-deficient (*ob*/*ob*) mice [46]. Consistent with this result, our results also showed a significant elevation in the levels of PC and PC/PE ratio in the HFD group compared with those in the ND group (Figure 4).

**Figure 4.** Overview of the effects of *Chrysanthemum morifolium* Ramat leaf extracts and luteolin on the serum lipidome in obese mice. The total lipid contents are expressed as the sum of contents of all identified lipids in mice plasma. Lipid classes are indicated on the *x*-axis: LPC, lysophosphatidylcholine; PC, phosphatidylcholine; LPE, lysophosphatidylethanolamine; PE, phosphatidylethanolamine; TAG, triacylglycerol; DAG, diacylglycerol; SM, sphingomyelin; CER, ceramide; CE, cholesteryl ester. The data are shown as mean ± standard deviation (SD) (*n* = 6). \*, # different letters indicate significant difference compared with the normal diet (ND) group (\*) and high-fat diet (HFD) group (#), as determined by Tukey's multiple comparisons test. CLE: HFD plus *Chrysanthemum morifolium* Ramat leaf extract 1.5% diet; LU: HFD plus luteolin 0.003% diet (\*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001, # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001).

#### *3.4. E*ff*ect of Chrysanthemum morifolium Ramat leaf Extract (CLE) and Luteolin (LU) in Mice*

We found that the sum (total) levels of LPC, PC, CE, SM, and CER were elevated in the HFD group compared to those in the ND group. Compared with the HFD group, CLE or LU treatment significantly reduced the sum total of the LPC and CE (*p* < 0.05), and also showed a tendency to decrease the SM and CER (*p* = 0.08) (Figure 4). However, even though CLE or LU had little effect on altering the sum total of the PC, the PC/PE ratio completely recovered to that of the ND group (Figure 4). The level of PC can be modulated by converting PE to PC by phosphatidylethanolamine *N*-methyltransferase (PEMT) as well as the Kennedy pathway that comprises Chpt1 and Cept1. The depletion of PC content by knockout of PEMT led to an increase in energy expenditure, weight loss, and insulin sensitivity, thereby preventing HFD-induced obesity and atherosclerosis [47–49]. Importantly, significantly higher hepatic expression of Chpt1 was observed in HFD-induced obese mice, however, CLE treatment significantly suppressed both the levels of Chpt1 and Cept1, while LU treatment significantly reduced only Cept1 (Figure 5a). In contrast, no statistically significant change in the hepatic expression of PEMT was noted between the groups (data not shown). Thus, CLE or LU treatment appears to have an inhibitory effect on the Kennedy pathway rather than through the PEMT pathway.

**Figure 5.** Scheme of the phospholipid synthesis (**a**) and sphingolipid synthesis (**b**) by which *Chrysanthemum morifolium* Ramat leaf extracts and luteolin regulates serum lipids and related hepatic gene-expression. The features of these compared to the ND group are color-coded by row, with red indicating high levels and green indicating low levels. ND: normal diet; HFD: high-fat diet; CLE: HFD plus *Chrysanthemum morifolium* Ramat leaf extract 1.5% diet; LU: HFD plus luteolin 0.003% diet; FA: fatty acid; DAG: diacylglycerol; PC: phosphatidylcholine; LPC: lysophosphatidylcholine; CE: cholesteryl ester; CER: ceramide; SM: sphingomyelin.

LPC is generated from the PC either via deacylation by phospholipase A2 (PLA2) isozymes or conversion by lecithin-cholesterol acyltransferase (LCAT) and is well known to be one of the major lipotoxic intermediates that promotes metabolic diseases through specific cell signaling, such as endoplasmic reticulum (ER) stress, chronic inflammation, apoptosis, and necrosis [50–52]. Consistent with our results, many previous studies have reported that the levels of most LPCs, including LPC 18:0, LPC 18:1, LPC 20:3, and LPC 22:6, were significantly increased in plasma of obese mice [26,42,44]. In addition to LPC, high plasma levels of CE are strongly recognized as a key marker to assess the risk and prediction of CVD [53,54]. Several studies have demonstrated that accumulation of high levels of CEs in plasma was observed in HFD-streptozotocin-induced diabetic (*db*/*db*) mice and HFD-induced obese mice, which are consistent with our findings [23,24,26,42]. In our study, one of the interesting findings was that significantly reduced levels of most LPC and CE were observed after CLE or LU treatment. However, no significant changes in the hepatic gene expressions, including LCAT, PLA2 isozymes, and lysophosphatidylcholine acyltransferase (LPCAT) isozymes that are involved in synthesis of these lipids, were observed between all the groups (*p* > 0.05; data not shown). Therefore, CLE or LU treatment might have a potential therapeutic effect through reduced levels of lipotoxic intermediates (LPC and CE).

Among the 63 lipid metabolites increased by the HFD, we identified 22 potential markers associated with anti-obesity effects of CLE or LU in the plasma of mice using Tukey's multiple comparison test. In particular, the levels of 6 CE (20:2, 20:3, 20:4, 20:5, 22:4, and 22:6), 7 LPC (16:0, 18:0, 18:1, 20:2, 20:3, 20:4, and 22:6), 5 PC (34:0 (16:0/18:0), 38:4 (18:0/20:4), 38:5 (18:1/20:4), 40:6 (18:0/22:6), and 40:7 (18:1/22:6)), and 4 sphingolipid (CER 34:1 (d18:1/16:0), SM 34:1 (d18:1/16:0), SM 36:1 (d18:1/18:0), and SM 36:2 (d18:2/18:0)) were significantly lowered in the HFD group treated with CLE or LU (Supplementary Table S1).

CER, a precursor and major molecule of sphingolipids, and SM, which may act as a pool for the generation of CER, are the most abundant sphingolipids in lipoproteins and constitute about 3% and 87% of plasma sphingolipids, respectively [55]. Similar to our study, several studies have reported that CER and SM levels were significantly increased in plasma and liver of animal models caused by a HFD or leptin deficiency [56–59]. In addition, upregulation of genes involved in sphingolipid biosynthesis has been frequently observed in obesity and dyslipidemia. The synthesis of sphingolipids is initiated by serine palmitoyl-coenzyme A (CoA) transferase (SPT) using serine and palmitoyl-CoA as substrates, and CER is generated through a two-step process by ceramide synthase (CerS) and dihydroceramide desaturase (DES1), which is then converted to SM by a transfer of the head group of PC via the action of sphingomyelin synthase (Sgms) [60]. The gene expression of the SPT isozymes, 3-ketodihydrosphingosine reductase (kdsr), CerS isozymes, and Sgms isozymes, which are involved in regulating the de novo sphingolipid biosynthetic pathway and the levels of pro-ceramide gene expression, including sptlc2, CerS6, and sgms1, were significantly increased in the HFD group compared to those in the ND group (*p* < 0.05). Similar to our results, a previous study has reported that sptlc2, which is responsible for the initial steps in sphingolipid biosynthesis, was significantly elevated in HFD-induced insulin-resistant mice relative to a low-fat diet group [61]. In addition, upregulated expression levels of ceramide synthases (CerS1, CerS2, CerS4, and CerS6) have been observed in HFD-induced insulin resistance [61–63].

In the present study, CLE or LU treatment markedly decreased the levels of sphingolipids (CER and SM) in plasma (Figure 5b). As a result, the hepatic mRNA expression levels of CerS4, CerS6, Sgms1, and Sgms2 were significantly reduced after CLE or LU treatment (*p* < 0.05) (Figure 5b). CER 34:1 (d18:1/16:0) synthesized by CerS6 is specifically distinguished as a key CER compared to other CERs in obesity-related diseases, which worsens the overall health status through disruption of insulin signaling, induction of apoptosis, and suppression of energy expenditure or fatty acid oxidation [63–66]. Similar to our study (Figure 6), Raichur et al. also showed that treatment with antisense oligonucleotides as a selective CerS6 inhibitor prevented the development of obesity and T2D through improved insulin sensitivity and reduced body weight gain, together with approximately

a 50% reduction of CER 34:1 (d18:1/16:0) levels in both the plasma and liver [64]. In addition, sgms1 gene knockout mice showed dramatic attenuation of SM levels in plasma and liver with suppression of inflammatory cytokines [67]. Additionally, some studies reported that Sgms2 knockout could prevent the development of liver steatosis [68,69]. These results indicated that CLE or LU treatment ameliorated HFD-induced dyslipidemia in mice by lowering the lipotoxic intermediates (CER and SM) in the liver and plasma through inhibition of de novo synthesis of sphingolipids.

**Figure 6.** Scatter plot showing significant differences in the levels of the lipid biomarkers with the same lipid compositions among the normal diet (ND), high-fat diet (HFD), HFD plus *Chrysanthemum morifolium* Ramat leaf extract 1.5% diet (HFD + CLE), and HFD plus luteolin 0.003% diet (HFD + LU) groups. Each symbol represents an individual mouse (Circle (-): ND; Box (-): HFD; Inverted triangle (): CLE; Triangle (): LU; Diamond (-): Quality control (QC)). \*, # different letters indicate significant difference compared with the normal diet (ND) group (\*) and the HFD group (#), as determined by Tukey's multiple comparisons test. (\*\*\* *p* < 0.001, # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001). LPC: lysophosphatidylcholine; PC: phosphatidylcholine; CE: cholesteryl ester; CER: ceramide; SM: sphingomyelin.

Taken together, our study demonstrated that treatment with CLE significantly reduced lipotoxic intermediates, such as PC, LPC, CER, SM, and CE, in the plasma of HFD-induced obesity and dyslipidemia model. Especially, six metabolites, LPC 18:1, LPC 22:6, PC 40:7, CE 22:6, CER 34:1, and SM 34:1, might be used as biomarker candidates which could explain the pharmacological effects of CLE or LU on dyslipidemic mice (Figure 6). Elevated hepatic expression levels of genes involved in the Kennedy pathway and sphingolipid metabolism related to synthesis of lipotoxic intermediates in the group of HFD-induced obese mice were dramatically recovered to the levels of the ND group after CLE or LU treatment. Consequently, the supplementation of CLE and LU was shown to ameliorate HFD-induced dyslipidemia and these lipid biomarkers can be used to better understand obesity-related metabolic disorders and to investigate their beneficial effects associated with such disorders.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2072-6643/12/10/2973/s1, Figure S1: Effect of *Chrysanthemum morifolium* Ramat leaf extracts (CLE) and Luteolin (LU) supplements for 16 weeks on individual TAG species in the liver. \*, # different letters indicate significant difference compared with normal diet (ND) group (\*) and HFD groups (#), as determined by Tukey's multiple comparisons test (\*\*\* *p* < 0.001, # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001). Table S1: Identified potential lipid biomarkers in plasma indicating statistically significant difference among sampling groups.

**Author Contributions:** Conceptualization, K.-H.L.; Project administration, K.-H.L. and M.-S.C.; Funding acquisition, K.-H.L. and M.-S.C.; Supervision, K.-H.L. and M.-S.C.; Methodology, J.C.S. and W.C.K.; Formal analysis, J.C.S. and R.R.; Software, J.C.S. and Z.W.; Investigation, J.C.S. and R.R.; Writing—Original Draft, J.C.S.; Writing—Review and Editing, J.-S.S. and Z.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Bio-Synergy Research Project (NRF-2014M3A9C4066462 and NRF-2014M3A9C4066459) of the Ministry of Science and ICT through the National Research Foundation (NRF).

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

#### **Abbreviations**



#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Red Propolis and Its Dyslipidemic Regulator Formononetin: Evaluation of Antioxidant Activity and Gastroprotective E**ff**ects in Rat Model of Gastric Ulcer**


Received: 31 August 2020; Accepted: 24 September 2020; Published: 26 September 2020

**Abstract:** Propolis has various pharmacological properties of clinical interest, and is also considered a functional food. In particular, hydroalcoholic extracts of red propolis (HERP), together with its isoflavonoid formononetin, have recognized antioxidant and anti-inflammatory properties, with known added value against dyslipidemia. In this study, we report the gastroprotective effects of HERP (50–500 mg/kg, p.o.) and formononetin (10 mg/kg, p.o.) in ethanol and non-steroidal anti-inflammatory drug-induced models of rat ulcer. The volume, pH, and total acidity were the evaluated gastric secretion parameters using the pylorus ligature model, together with the assessment of gastric mucus contents. The anti-*Helicobacter pylori* activities of HERP were evaluated using the agar-well diffusion method. In our experiments, HERP (250 and 500 mg/kg) and formononetin (10 mg/kg) reduced (*p* < 0.001) total lesion areas in the ethanol-induced rat ulcer model, and reduced (*p* < 0.05) ulcer indices in the indomethacin-induced rat ulcer model. Administration of HERP and formononetin to pylorus ligature models significantly decreased (*p* < 0.01) gastric secretion volumes and increased (*p* < 0.05) mucus production. We have also shown the antioxidant and anti-*Helicobacter pylori* activities of HERP. The obtained results indicate that HERP and formononetin are gastroprotective in acute ulcer models, suggesting a prominent role of formononetin in the effects of HERP.

**Keywords:** propolis; formononetin; dyslipidemia; gastric ulcer; rats

#### **1. Introduction**

Dyslipidaemia is a condition defined either by the high levels of total or low-density lipoprotein (LDL) cholesterol, and/or by the low levels of high-density lipoprotein (HDL). Such lipid metabolic dysfunction, resulting from, e.g., lipid peroxidation or oxidative stress, plays an important role in the pathogenesis of atherosclerosis and other chronic diseases [1]. Changes in lifestyle (e.g., adoption of a heart-healthy diet and exercise) and the use of drugs (e.g., fibrates to reduce triacylglycerols, statins to reduce bad cholesterol (LDL), and niacin to increase the overall (HDL) cholesterol) are commonly implemented to ameliorate these metabolic diseases. Atherogenesis results from the imbalance between the antioxidant capacity and the activity of oxidative species (reactive oxygen, nitrogen, and halogen). The oxidizing cellular protein, lipid, and DNA directly injure cells or these can even die from disturbances in signaling pathways. Reactive oxygen species (ROS) participate as signaling molecules in fundamental cellular functions in ambient conditions. Oxidative stress, and activation of ROS, is triggered when a cell is depleted in small molecule antioxidants or when antioxidase systems are overloaded or not operating at all.

The interest in natural products as sources of new drugs and therapeutic strategies for chronic diseases is increasingly growing. Among those sources, natural antioxidants from different plants (e.g., *Dirmophandra mollis* Benth, *Ruta graveolens* L., *Ginkgo biloba* L., *Vitis vinifera* L, *Thymus zygis*, *Croton argyrophyllus* Kunth, *Hordeum vulgare, Dalbergia ecastophyllum, Origanum vulgare* L.) are receiving particular attention [2–7].

Propolis (or bee glue) is a complex mixture of molecules obtained from several resinous secretions (e.g., resins, mucilage, gums, lattices, leaf buds) collected by worker-bees from different plant species [8]. Its composition is thus strongly influenced by the diversity of local flora, harvesting place and season, and by the genetics of the bees [9]. Briefly, these plant resinous secretions are mixed with salivary and enzymatic secretions of honey bees, producing a cementing material, used to close open cracks occurring in beehives. Propolis is used for sealing the spaces in beehives and to smooth their internal walls, providing antiseptic activity, as it protects the bees' larvae and comb from microbial invasions [8].

Among several species, red propolis obtained from *Dalbergia ecastophyllum*(L.) Taub. (Fabaceae, [10]) shows various pharmacological properties of clinical interest, also being considered a functional food [11–13]. In particular, and due to its significant antioxidant activity [14–16], Brazilian red propolis has been proposed for dermal healing [17,18], and for the treatment of cancer cells [14,15,19,20], as an antimicrobial [16], antiproliferative [21,22], antihypertensive [23], and as a neuroprotective [11]. Several compounds have been identified in this resin, including the isoflavonoids formononetin, biochanin A, medicarpin, and pinocembrin [24,25]. Reported biological activities of these isolated compounds include the anti-inflammatory activities of biochanin A [26], pinocembrin [27], neovestitol, and vestitol [28], the antioxidant activities of formononetin [29], biochanin A [30], pinocembrin [31], the gastroprotective properties of biochanin A [32], the antifungal and dyslipidemic activities of formononetin [33,34], and the antimicrobial actions of neovestitol and vestitol [28].

Inhibitors of the HMG-CoA (3-hydroxy-3-methyl-glutaryl-coenzyme A) reductase play an active role in the treatment of hypercholesterolemia and have been found to play a protective role in several bacteria-associated diseases such as *Helicobacter pylori*-induced gastric ulcer. Association between *Helicobacter pylori* infection and dyslipidemia has been reported significantly [35]. Gastric ulcer is a chronic disease of high worldwide prevalence, associated to unexpected complications such as bleeding, perforation, and stenosis [36]. The imbalances of gastric mucosal defense mechanisms (prostaglandins, mucus, mucosal blood flow, bicarbonate, nitric oxide, and sulfhydryl compounds, and ATP sensitive K<sup>+</sup> channels) against exposure to endogenous and exogenous factors (e.g., non-steroidal anti-inflammatory drugs (NSAIDs), pepsin, bile acids, alcohol, stress, and trauma, *Helicobacter pylori* infection, hemorrhagic shock, sepsis, and burns) promote the development of gastric ulcers [36,37].

It is also known that a long-term use of NSAID, and/or the presence of *Helicobacter pylori* infection, are the main causes of incidence and recurrence of gastric ulcers [38–40]. Furthermore, excessive

alcohol consumption is an independent risk factor for the set-up of gastric ulcer, being thus a serious public health concern [41,42].

Using a rat model of inflammatory and neurogenic pain, without emotional or motor side effects, we have already demonstrated that hydroalcoholic extracts of red propolis (HERP) have significant anti-inflammatory and antinociceptive activities [43]. We have also described the anti-inflammatory activity of the dyslipidemic isoflavonoid formononetin [11,43]. In the present work, we proposed the in vivo characterization of the gastroprotective effects of HERP and formononetin, using an experimental rat model of gastric ulcer, as a suitable approach for phytomedical uses.

#### **2. Materials and Methods**

#### *2.1. Drugs and Reagents*

Alcian blue (Cas. No: 33864-99-2), carbenoxolone (Cas. No: 5697-56-3), cimetidine (Cas. No: 51481-61-9), formononetin (Cas. No: 485-72-3), indomethacin (Cas. No: 53-86-1), and omeprazole (Cas. No: 73590-58-6) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other used reagents were of analytical grade. Test substances were dissolved in 0.9% NaCl solution containing 2% Tween 80 (polysorbate 80), while indomethacin was dissolved in 2% sodium bicarbonate, all from Sigma-Aldrich (Darmstadt, Germany). HERP and formononetin doses were based on our preliminary experiments [5].

#### *2.2. Red Propolis Collection, Hydroalcoholic Extracts Preparation and Characterization*

Red propolis was collected from species of Brejo Grande, Sergipe, Brazil (10◦28 25 S, 36◦26 12 W). The material was labeled, and stored in sterile and refrigerated containers. For the extraction, a volume of 12.5 mL of ethanol (70%) was used to extract 1 g of propolis samples using an ultrasound bath for 1 h, at room temperature as described by Pinheiro et al. [22]. After extraction, the obtained mixture was centrifuged, and the obtained supernatant evaporated under low pressure resulting in HERP, which was stored at refrigerated temperature until further use. The obtained extraction yield was 34% (*w*/*w*). Analysis of hydroalcoholic extracts of red propolis (HERP) was performed using HPLC as described by Cavendish et al. [43], and formononetin (ca. 2%) was confirmed as the major component.

#### *2.3. Antioxidant Activity*

The antioxidant activity of HERP was determined as the capacity of the antioxidants present in the sample to scavenge the stable radical 2 2-diphenyl-1-picrylhydrazyl (DPPH·), applying a previously described methodology [44,45]. A volume of 3 mL of extract, at concentrations between 2.5 and 15.0 mg/mL, was homogenized with 750 μL of DPPH solution (200 mM). After 15 min at room temperature, the spectrophotometric reading was done at 517 nm. Ethanol 70% was used as control and subject to the same procedure. Experiments were done in triplicate. To determine the % of free radical inhibition (%I), the following equation was used:

$$\% \text{I} = 100 - (\text{A}\_{\text{C}}/\text{A}\_{\text{A}}) \times 100 \text{ A}$$

where AC is the absorbance of the control and AA is the absorbance of the sample. The mean inhibition index (IC50) was obtained from the %I values at each tested concentration by non-linear regression by computing the data in Prism GraphPad software (San Diego, CA, USA).

#### *2.4. Microorganism*

*Helicobacter pylori* strain ATCC 43504 was obtained from the Instituto Nacional de Controle de Qualidade em Saúde (INCQS—Fundação Oswaldo Cruz—Fiocruz/RJ, Brazil). The stock cultures were kept in Brucella broth at −20 ◦C until further use.

#### *2.5. Animals*

Wistar rats of both sexes, of 280–320 g mean weight, were received from the animal facilities of the Tiradentes University biotherium (Aracaju, Brazil). Animals were housed at 21 ± 2 ◦C in plastic cages, with free access to food (Purina®) and water ad libitum, under a 12-h light/12-h dark cycle. Twenty-four hours prior to the experiments, rats were provided with water ad libitum. All experiments and protocols were approved by the Institutional Ethics Committee (250608) of the Tiradentes University (Aracaju, Brazil) and were performed according to the EEC Directive of 1986, following the principles for laboratory animal use and care.

#### *2.6. Antiulcerogenic Activity*

#### 2.6.1. Ethanol-Induced Ulcers

These experiments were conducted according to the method described by Robert et al. [46]. Briefly, rats (*n* = 6/group) were fasted for 24 h and were then pre-treated orally with 50, 250, or 500 mg/kg HERP, 10 mg/kg formononetin, 100 mg/kg omeprazole, or vehicle (2% Tween 80, 10 mL/kg). Thirty minutes after treatment, all rats received 1 mL of absolute ethanol to induce gastric ulcers and were anaesthetized (3% halothane) and one hour later were euthanized by cervical dislocation.

The stomachs of the animals were then removed and opened along the greater curvature. To remove gastric contents and blood clots, the stomachs were gently rinsed with water. Tissues images were recorded as described by Pinto et al. [47], and total lesion areas were measured (mm2). Stomach samples from 50, 250, and 500 mg/kg HERP, formononetin, omeprazole, and vehicle treated animals were cut into serial 5 μm thick sections and were stained with hematoxylin-eosin (HE) according to laboratorial techniques.

Microscopic scores were determined for the interstitial edema and for the disruption of superficial regions of the gastric gland with epithelial cell loss Intensities of epithelial cell loss and interstitial edema were categorized as follows: absence (0); focal limited to the upper third (1); focal beyond the upper third (2); and diffuse in the upper third (3). Three histological sections for each animal (*n* = 6/group) were randomly analyzed (without previous knowledge of the treatments).

#### 2.6.2. Non-Steroidal Anti-Inflammatory Drug (NSAID)-Induced Ulcers

These experiments were adopted from the method described by Djahanguiri, with a few modifications [48]. Briefly, rats (*n* = 6/group) were pre-treated orally with HERP (50, 250, or 500 mg/kg), formononetin (10 mg/kg), cimetidine (100 mg/kg), or vehicle (2% Tween 80) for 30 min, and were then treated with indomethacin (100 mg/kg, p.o.) to induce gastric ulcers. Animals were euthanized by cervical dislocation six hours later after anesthesia with 3% halothane. Stomachs were then removed and opened for the scoring of ulcer index as follows: (i) score 1, standing for loss of mucosal folding, mucosal discoloration, edema, or hemorrhage; (ii) score 2, standing for less than 10 petechiae; and (iii) score 3, standing for more than 10 petechiae [49].

#### *2.7. Determination of Gastric Juice Parameters Following Pyloric Ligature*

For the determination of the gastric juice parameters, the methods described by Shay et al. were followed with some modifications [50]. Animals were anaesthetized with ketamine/xylazine (60 and 10 mg/kg, respectively, i.p.) after 24 h of fasting, followed by laparotomy and pylorus ligation. Rats (*n* = 6/group) were then treated intraduodenally with HERP (50, 250, or 500 mg/kg), formononetin (10 mg/kg), cimetidine (100 mg/kg), or vehicle (2% Tween 80). Four hours later, animals were anesthetized with halothane (3%) and euthanized by cervical dislocation. Abdomens were then opened, stomachs removed, and gastric contents collected and centrifuged for 10 min at 8000× *g* (25 ◦C). The pH and volume of the gastric juice values were measured and total acid secretions in total gastric juice supernatants determined by titration with 0.01 N NaOH solution using phenolphthalein as indicator.

#### *2.8. Determination of Gastric Mucus Contents*

For the determination of the mucus contents the methods described by Sun et al. were followed, with some modifications [51]. Animals were anaesthetized with ketamine/xylazine (60 and 10 mg/kg, respectively, i.p.) after 24 h of fasting, followed by laparotomy and pylorus ligation. Rats (*n* = 6/group) were immediately treated intraduodenally with HERP (50, 250, or 500 mg/kg), formononetin (10 mg/kg), carbenoxolone (200 mg/kg), or vehicle (2% Tween 80). Four hours later, animals were anesthetized with halothane (3%) followed euthanasia by cervical dislocation. The removed stomach contents were immersed in 10 mL of solution containing 0.16-M sucrose, 0.02% Alcian blue, and 0.05-M sodium acetate (pH 5.8), and were kept at room temperature for 24 h. Alcian blue binding extracts were then centrifuged at 3000× *g* for 10 min and absorbances of supernatants were measured using spectrophotometry at 620 nm. Free gastric mucus contents were calculated from quantities of bound Alcian blue (mg/g tissue).

#### *2.9. Agar-Well Di*ff*usion Assays*

Antibacterial activity was investigated using a modified agar-well diffusion method [52]. Wells of 8 mm in diameter were sub-cultured with blood agar (Mueller-Hinton agar with 10% sheep blood) plates and inoculated with *Helicobacter pylori* suspensions of 6 <sup>×</sup> 10<sup>8</sup> CFU/mL (McFarland turbidity standard 0.5). Wells were then filled with 80 μL of 100 mg/mL HERP and tetracycline (0.03 mg/mL) was used as a standard compound. Plates were stored for 30 min at room temperature and then incubated for another 48 h at 37 ◦C under microaerophilic conditions. Subsequently, growth inhibition halos were measured using a digital pachymeter. The size of the inhibitory zones (diameter) was measured in triplicate and mean values of ≥08 mm considered active.

#### *2.10. Statistical Analysis*

Data are presented as the mean ± standard error of the mean (SEM) of n animals per group. Differences between the treated groups were identified using the one-way analysis of variance (ANOVA) and Bonferroni's test. Changes in microscopy parameters of indomethacin-induced ulcers (scores) were analyzed using Kruskal–Wallis test followed by Dunn's test. Differences were considered significant when *p* < 0.05.

#### **3. Results**

From the in vitro antioxidant activity test against free radical DPPH, an IC50 of 294 μg/mL was recorded for HERP. This result demonstrates the antioxidant potential of the variety of propolis used in this study. The antioxidant activity of the extract may be related to its ability to inhibit ethanol damage on the gastric mucosa. Ethanol is metabolized in the body, releasing superoxide anion and hydroperoxides. Such free radicals are known to be involved in the mechanism of acute and chronic ulceration. The sequestration of these chemical entities plays an important role in the healing process of gastric injury [53].

Free radicals are chemical entities capable of causing irreversible damage to the cell [4,54]. The antioxidant process includes the scavenging of these radicals (preventing their propagation), enzymatic hydrolysis of the ester linkage to remove peroxidized fatty acids from lipids, scavenging of transition metal ions, and reduction of peroxide catalyst enzymes. The pathogenesis of peptic ulcer is associated with endogenous and exogenous factors. Ethanol causes oxidative stress, which can happen by decreasing the release of nitric oxide, which has an important role in the metabolism of free radicals [55].

Following the induction of ulcers using ethanol, pre-treatments with the HERP at different doses (50, 250, or 500 mg/kg) inhibited the total lesion areas in a dose dependently fashion, when compared to the group treated with Tween 80 (2%) solution (*p* < 0.001; Figure 1). Formononetin (FOR, 10 mg/kg) and omeprazole (OMEP, 100 mg/kg) also reduced the areas of the total lesions significantly (*p* < 0.001) (Figure 1). Table 1 depicts the quantification effects of HERP and formononetin on ethanol-induced damage of gastric mucosa. Histopathological analyses of gastric mucosa after the administration of absolute ethanol are shown in Figure 2.

**Figure 1.** Effects of hydroalcoholic extracts of red propolis (HERP) and formononetin (FOR) on the ethanol-induced gastric damage; rats were pre-treated with a solution of 2% Tween 80 (vehicle), HERP (50–500 mg/kg), formononetin (10 mg/kg), or omeprazole (OMEP, 100 mg/kg) for 30 min prior to one hour treatment with absolute ethanol (1 mL). Total lesion areas were then determined and are presented as the means ± standard errors of the mean (SEM; *n* = 6/group); ANOVA followed by Bonferroni's test; \*\*\* *p* < 0.001 vs. vehicle group; ## *p* < 0.01 and ### *p* < 0.001 vs. OMEP group; && *p* < 0.01 vs. FOR group.



Data are presented as medians with minimum and maximal scores in brackets; differences were identified using Kruskal–Wallis followed by Dunn's test; \*\*\* *p* < 0.001 vs. vehicle group; and ### *p* < 0.001 vs. HERP groups at 250 and 500 mg/kg; three histological sections from each animal, *n* = 6/group.

The oral administration of absolute ethanol administration (vehicle-treated group) promoted microscopic damage, with a pronounced edema of the submucosa and a consistent epithelial cell loss, and presence of inflammatory cells. Ethanol-induced gastric ulcer resulted from the disruption of the vascular endothelium, increase of vascular permeability, formation of edema, and promotion of epithelial lifting. The gastric tissue of the animals treated with HERP (250 and 500 mg/kg, p.o.) showed less mucosal damage than those that were treated with the vehicle. The total lesion area was reduced in a concentration dependent manner, i.e., the increase of the HERP dose promoted a significant reduction of the total lesion area. Pre-treatment with formononetin and omeprazole also inhibited the formation of ethanol-induced lesions (vehicle group). Omeprazole is a proton-pump inhibitor thus it reduces the acid produced in the stomach. It is commonly used to promote the healing of erosive esophagitis and for the treatment of other gastrointestinal conditions (e.g., gastroesophageal reflux disease, peptic ulcer). Omeprazole treatment (OMEP, 100 mg/kg) for 30 min resulted in a total lesion area close to zero (mm2).

The macroscopic effects of HERP shown in Figure 2 clearly demonstrate a significant tissue recovery in the animals treated with HERP (250 and 500 mg/kg, *p* < 0.05) and formononetin (10 mg/kg, *p* < 0.001), against the positive control (vehicle). Pre-treatments with HERP doses (250 and 500 mg/kg, *p* < 0.05) and formononetin (10 mg/kg, *p* < 0.001) significantly reduced ulcer indices of indomethacin-induced ulcers in comparison with the those of the vehicle-treated group (Table 2). Cimetidine (100 mg/kg) also significantly reduced ulcer indices (*p* < 0.01, Table 2). Indomethacin is an indol derivative that is the first-choice approach for gastric-ulcer induction in laboratory. Cimetidine belongs to the histamine H2 receptor antagonists and acts by inhibiting the production of acid in the stomach. It is commonly used to treat and prevent certain types of stomach ulcer.

**Figure 2.** (**A**) Macroscopic effects of HERP, formononetin, and omeprazole in ethanol-induced gastric ulcers; (**B**) hematoxylin/eosin-stained histological sections of gastric mucosa specimens from ethanol treated rats; representative photomicrographs were generated from the same areas and were quantified (Table 1).


**Table 2.** Effects of HERP and formononetin on indomethacin-induced ulcers in rats.

Results are presented as means ± SEM (*n* = 6/group). Differences were identified using Kruskal–Wallis test followed by Dunn's test; \* *<sup>p</sup>* <sup>&</sup>lt; 0.05, \*\* *<sup>p</sup>* <sup>&</sup>lt; 0.01, and \*\*\* *<sup>p</sup>* <sup>&</sup>lt; 0.001 vs. vehicle group; ## *<sup>p</sup>* <sup>&</sup>lt; 0.01 vs. 500-mg/kg HERP group; & *<sup>p</sup>* <sup>&</sup>lt; 0.05 and && *<sup>p</sup>* <sup>&</sup>lt; 0.01 vs. 50-mg/kg HERP group.

The treatment with 50 and 250 mg/kg HERP in the pylorus ligature model of gastric secretion significantly reduced secretion volumes (*p* < 0.01) and increased H<sup>+</sup> concentrations (50 mg/kg, *p* < 0.01) (Table 3). Similarly, treatments with formononetin (10 mg/kg) significantly reduced secretion volumes (*p* < 0.01). However, HERP and formononetin treatments both did not succeed in significantly increasing the pH values compared to those in the group treated with the vehicle. In contrast, cimetidine affected all gastric juice parameters significantly (*p* < 0.01).

**Table 3.** Effects of intraduodenal treatments with HERP and formononetin on the biochemical parameters of gastric juice collected from the pylorus ligature rats.


Results are presented as means ± SEM (*n* = 6/group) and differences between treatment groups were identified using ANOVA followed by Bonferroni's test; \*\* *p* < 0.01 and \*\*\* *p* < 0.001 vs. vehicle group; # *p* < 0.05, ## *p* < 0.01, and ### *p* < 0.001 vs. 500-mg/kg HERP group.

Compared to the vehicle treated controls (Table 4), the production of mucus was significantly increased with HERP (500 mg/kg) treatment (*p* < 0.05). Treatments with formononetin (FOR, 10 mg/kg) and carbenoxolone (200 mg/kg) also increased the production of mucus (*p* < 0.05 and *p* < 0.001, respectively, when compared to the control).

**Table 4.** Effects of intraduodenal treatments with HERP and formononetin on Alcian blue binding to free gastric mucus from pylorus ligatures in rats.


Results are presented as means ± SEM (*n* = 6/group) and differences were identified using ANOVA followed by the Bonferroni's test; \* *p* < 0.05 and \*\*\* *p* < 0.001 vs. vehicle group.

In assessments of anti-*H. Pylori* activity, HERP treatments reduced the diameter of the inhibitory zone to 13.0 ± 2.0 mm at 100 mg/mL, whereas inhibition halos were 35.0 ± 0.5 mm in the presence of the standard tetracycline (0.03 mg/mL).

#### **4. Discussion**

Propolis is a product obtained from honey bee hives, characterized chiefly by a beeswax and a resin (obtained from apical buds, young leaves and exudates) [56]. Red propolis is ranked as the second most produced type of Brazilian propolis. Its characteristics (e.g., texture, odor, color) vary according to the source and type, but also harvesting season. The interest in Brazilian red propolis is attributed to its several beneficial effects for a range of health conditions, due to the presence of phytoestrogens. Phytoestrogens are naturally occurring compounds with a chemical structure similar to 17-β-estradiol, the mammalian estrogen. These compounds show the capacity to bind to estrogen receptors, either as agonists or antagonist, to promote, respectively, estrogenic or antiestrogenic activity [57]. They have been recommended for the treatment of estrogen-related diseases, as happens with dyslipidemia resulting from the high levels of estrogen in post-menopause women. On the other hand, during physiologic menstrual cycle bicarbonate secretion increases with estrogenic levels, which may contribute to reduce the risk of gastric ulcer.

In our study, two rat ulcer models were used (ethanol-induced and indomethacin-induced ulcers) and tested for the potential of hydroalcoholic extracts of red propolis (HERP), and its isoflavonoid formononetin, for gastric protection. The phytoestrogen formononetin is an O-methylated isoflavone (Figure 3) that was found to be present in hydroalcoholic extract of Brazilian red propolis.

**Figure 3.** Chemical structure of formononetin (C16H12O4, molecular weight 268.26 g/mol).

Our results demonstrate that HERP in the tested concentrations of 50, 250, and 500 mg/kg exhibited acute gastroprotective effects against ethanol- and indomethacin-induced ulcers, in a concentration dependent manner, with the highest dose increasing the mucus production significantly (Table 4). HERP and its major isoflavonoid formononetin (10 mg/kg) decreased gastric secretion volumes and increased mucus production in the present pylorus ligature model, while HERP depicted anti-*H. Pylori* activity. Oral administration of HERP decreased the total lesion areas (Figure 1) induced by absolute ethanol in a dose dependent fashion, and reduced the associated macroscopic and microscopic damage to mucosa (Figure 2), at doses which were considered safe, according to the toxicological study described by Silva et al. [58]. Similar reductions were also observed after formononetin treatment when compared to the vehicle-treated animals, which showed severe ulceration of gastric mucosa (vehicle group).

Ethanol has been previously used to induce ulcers in rodents, leading to gastric mucosal injury through the direct and indirect effects of reactive oxygen species (ROS) and cytokines [34]. Moreover, this process is reportedly mediated by activated neutrophils, and is associated with cellular lipid and protein peroxidation [59]. However, several studies have shown that natural products with antioxidant activity protect gastric mucosa against damage [60–63]. The advantages of HERP for human use have been extensively discussed in the literature, relying on the many identified beneficial effects, mostly attributed to its antioxidant properties [5,64–67]. Various phenolic antioxidants, such as flavonoids, coumarins, tannins, procyanidins, and xanthenes, act as radical scavengers, neutralizing ROS, inhibiting lipid peroxidation and other free radical-mediated pathologies, in a dose-dependent fashion. Plants enriched with antioxidants, such as isoflavonoid, may thus be considered as an alternative approach for the management and treatment of the chronic diseases related to oxidative

stress. Among flavonoids present in HERP, biochanin A has demonstrated gastroprotective effects by a strong induction of superoxide dismutase (SOD) and nitric oxide enzymes, and reduced release of malondialdehyde [32]. Asif et al. have reported a strong correlation between the antioxidant and antiglycation activities of biochanin A present in organic fractions of *Hordeum vulgare* [6].

The therapeutic potential of biochanin-A against myocardial infarction has also been proposed, and attributed to its capacity in compromising lipid peroxidation. Male Wistar rats were first subjected to isoproterenol-induced myocardial infarction [68], resulting in a significant increase of creatine kinase-MB and cardiac troponin, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, and lactate dehydrogenase, and reduction of the activity of antioxidant enzymes (i.e., SOD, catalase, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase) in the heart. Such antioxidant enzymes catalyze reactions to counterbalance free radicals and ROS, thus forming endogenous defense mechanisms. The authors reported the cardioprotective effects of biochanin-A by modulation of lipid peroxidation, through enhancing antioxidants and detoxifying enzyme systems [68]. The effect of biochanin-A on a high-fat diet-induced hyperlipidemia in mice has been studied by Xue et al. [69]. The authors have demonstrated the capacity of biochanin A in decreasing the LDL-cholesterol in about 85% and total cholesterol ca. 39% in a moderate dose, increasing the activity of lipoprotein lipase in 96% and of hepatic triglyceride lipase in 78%. The results also showed the increase of fecal lipid levels and decrease of epididymal fat index in hyperlipidemic mice in comparison to the control mice. Jia et al. demonstrated that formononetin attenuates hydrogen peroxide-induced apoptosis and nuclear factor-kappa B (NF-κB) activation in retinal ganglion cells, exposed to oxidative stress over a period of 24 h [70].

The abovementioned beneficial effects of flavonoids (biochanin-A, formononetin) identified in HERP, substantiate the rationale for exploiting additional biological activities related to their antioxidant properties. Fukai et al. anticipated the potential use of these compounds as chemopreventive agents for peptic ulcer or gastric cancer in *Helicobacter pylori*-infected individuals [71].

*Helicobacter pylori* infections are associated with dyslipidemia. Epidemiologic and clinical data suggest that such an infection can be a contributing factor in the progression of atherosclerosis [35]. In 2010, Satoh et al. reported that *Helicobacter pylori* infection was significantly associated with high LDL-cholesteremia and low HDL-cholesteremia in Japanese male subjects [72]. The association of this infection with metabolic syndrome in the Japanese population, has also been reported in the past [73]. Thus, treating *Helicobacter pylori* infections and protecting gastric mucosa from ulceration may have a synergistic effect in prophylaxis of dyslipidemia as well.

From our results, the histological analyses obtained from ethanol-induced ulcers show that HERP and formononetin attenuate lymphocytic infiltration, suggesting immunomodulatory effects on the release of cytokines (Figure 2). Furthermore, bioactive fractions of geopropolis that were collected from the same region reportedly decreased neutrophil migration and reduced NO-related inflammatory interactions between leukocytes and endothelial cells [74].

Numerous studies have demonstrated enhanced immune responses in the presence of propolis [75–78]. Moreover, the isoflavonoid biochanin A inhibited lipopolysaccharide (LPS)-induced activation of microglia, and production of tumor necrosis factor (TNF)-α, NO, and SOD in mesencephalic neuron-glia cultures and microglia-enriched cultures [79]. Formononetin also inhibited TNF-α and interleukin (IL)-6 expression, and improved SOD activity in traumatic brain injury [29] and LPS-induced acute lung injury models [80]. These studies suggest that the gastroprotective properties of HERP are mediated, at least partially, by formononetin.

NSAIDs are the most commonly used treatment for inflammatory diseases, but are proven to cause gastric and duodenal ulcers in rats and humans [81]. The potent NSAID indomethacin causes gastric legions by inhibiting cyclo-oxygenase (COX), resulting in decreased prostaglandin synthesis [82]. The ensuing injury is characterized by reduced bicarbonate and mucus secretion and blood flow, increased acid back-diffusion, and inhibition of repair. In the present study, HERP and its flavonoid formononetin inhibited the formation of indomethacin-induced gastric lesions. Flavonoids were

previously shown to inhibit COX-1 and COX-2 enzymes [83], and the isoflavonoids genistein and daidzein significantly reduced COX activity [84]. Hence, the present observations of increased mucus production in the presence of HERP or formononetin were attributed to COX inhibition.

Flavonoid treatments reportedly increased the production of mucus and bicarbonate, and affected proton pump activities in parietal cells [85]. Hence, the present effects of HERP likely reflect formononetin contents. Hajrezaie et al. showed that biochanin A increases mucus secretion in ethanol-induced ulcer models [32], and De Barros et al. showed that quercitrin and afzelin increase the secretion of mucus and reduce H+- and K+-ATPase activity in vitro [86].

In further experiments, HERP and formononetin decreased gastric secretion volumes after pylorus ligature. As HERP and formononetin treatments were administered via a intraduodenal route, systemic effects are likely not to be related to the local neutralization of gastric acid or physical barriers. Moreover, physiological gastric acid secretion by parietal cells is activated by several stimuli. These include acetylcholine, which acts directly via M3 receptors and indirectly by M2 and M4 receptors, and is associated with the inhibition of somatostatin secretion, histamine, which acts directly via H2 receptors, and gastrin, which predominantly acts indirectly via cholecystokinin-2 (CCK-2) receptors on enterochromaffin-like cells and is associated with histamine release [87]. Flavonoids have been shown to induce the expression of acetylcholinesterase, which hydrolyzes acetylcholine in cultured osteoblast synapses [88]. Additionally, flavonoids reportedly increase mucosal prostaglandin levels and reduce histamine production by mast cells by inhibiting histidine decarboxylase [89].

Herein, we showed increased H<sup>+</sup> concentrations following treatments with HERP, likely reflecting the presence of acid phenolic compounds [16]. However, no significant changes in pH were observed under these conditions, whereas cimetidine treatment modified all biochemical parameters of gastric mucosa, suggesting distinct mechanisms of action. Similarly, formononetin treatments reduced gastric secretion volumes, but had no significant effects on pH or total acid secretion, indicating that changes in gastric acid secretions do not play an important role in the gastroprotective mechanisms of this compound. *H. Pylori* is associated with various gastric diseases and is a frequently encountered human pathogen [90]. In our study, we demonstrated that HERP treatment (100 mg/mL) inhibited the growth of *H. Pylori* in vitro, highlighted by the identification of an inhibitory zone of 13.0 ± 2.0 mm, in comparison to the 35.0 ± 0.5 mm inhibition halos in the presence of standard tetracycline (0.03 mg/mL). The anti-*H. Pylori* effect has been demonstrated for several propolis varieties. Using the same agar-well diffusion method, Boyanova et al. (2003) reported the increase of mean diameters of *H. Pylori* growth inhibition from 17.8, 21.2, 28.2 mm when treated, respectively, by 30, 60, and 90 mL of ethanolic propolis extract (30%), whereas the treatment with of ethanol alone (30 mL) resulted in a mean diameter of 8.5 mm [91]. More recently, Baltas et al. (2016) reported inhibition diameters ranging from 31.0 to 47.0 mm from 15 different ethanolic propolis extracts tested at 75 μg/mL [92]. The authors found a significant positive correlation between the reported anti-*H. Pylori* activity of the extracts and the total phenolic content.

#### **5. Conclusions**

In this work, we reported the gastroprotective effect of HERP, and its predominant isoflavonoid formononetin, known for its hypolipidemia effects. Our results demonstrated that treating animals with HERP resulted in the reduction of mucosal damage, in particular with doses as high as 200 and 500 mg/kg (p.o.), with a significant reduction in the loss of epithelial cells and on edema. The macroscopic effects of sample-treated rats corroborate these results. The treatment with HERP increased H<sup>+</sup> concentration, as well as the mucus production, both contributing to enhance the protection of gastrointestinal mucosa, and to reducing the aggressive effects in gastric mucosa, thus reducing the risk of ulcers. The antioxidant activity of HERP was also shown, reporting a IC50 of 294 μg/mL. Together with the prophylactic role in reducing the lipid accumulation, formononetin was shown to have a prominent role in increased mucus production. HERP can be further exploited as an alternative phytomedicine for the treatment of ulcers with hypolipidemic profile. HERP and formononetin have recognized

gastroprotective properties, as they significantly inhibit the development of acute ulcers in the presence of ethanol and indomethacin, ameliorated inflammatory cell infiltration and edema, reducing gastric secretion volumes, and increasing gastric mucus contents.

**Author Contributions:** M.A.A.d.M., A.R.S.R., A.K.d.L., G.B.B., M.S.P., R.L.C.d.A.-J. and M.Z.G. contributed to the methodology, formal analysis, investigation, resources, and data curation; red propolis collection, preparation of the hydroalcoholic extracts, and sample characterization have been carried out by F.F.P., S.M.T., A.S. and P.S.; in vivo experiments have been performed by R.L.C.d.A.-J., M.Z.G., F.F.P., S.M.T., P.S. and J.C.C.; quantification analyses and antibacterial activities have been run by E.B.S., L.K.H.; writing of the original manuscript was the contribution of M.A.A.d.M., E.B.S., A.S., and P.S.; conceptualization, review, and editing of the manuscript, as well as project administration, supervision, and funding acquisition was contributed to by A.S., P.S., E.B.S., E.N. and J.C.C. All authors have made a substantial contribution to the work and have approved its publication. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been funded by the Fundação de Apoio à Pesquisa e à Inovação Tecnológica do Estado de Sergipe (FAPITEC/SE), by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). R.L.C.d.A.-J., S.M.T., and J.C.C. received CNPq productivity grants. E.B.S. acknowledges the sponsorship of the project UIDB/04469/2020 (strategic fund), from the Portuguese Science and Technology Foundation, Ministry of Science and Education (FCT/MEC) through national funds, and was co-financed by FEDER, under the Partnership Agreement PT2020. E.N. and A.S. acknowledge the support of the research project: Nutraceutica come supporto nutrizionale nel paziente oncologico, CUP: B83D18000140007.

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

#### **References**


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