Chlorogenic acid alleviates obesity and modulates gut microbiota in high‐fat‐fed mice

Abstract To evaluate the anti‐obesity effects of chlorogenic acid (CGA), the mice were fed a high‐fat diet (HFD) upon chlorogenic acid treatment for 6 weeks. The results showed administration of chlorogenic acid (150 mg per kg per day) remarkably promoted body loss, reduced lipid levels in plasma and altered mRNA expression of lipogenesis and lipolysis related genes in adipose tissue. Moreover, chlorogenic acid also reversed the HFD‐induced gut microbiota dysbiosis, including significantly inhibiting the growth of Desulfovibrionaceae, Ruminococcaceae, Lachnospiraceae, Erysipelotrichaceae, and raising the growth of Bacteroidaceae, Lactobacillaceae. Overall, the amelioration of HFD‐induced gut microbiota dysbiosis by chlorogenic acid may contribute, at least partially, to its beneficial effects on ameliorating HFD‐induced obesity.

be one of the underlying mechanisms by which chlorogenic acid exerts its anti-obesity effects. To test our hypothesis, we assessed the potential of chlorogenic acid in altering the gut microbiota composition as well as maintaining gut homeostasis upon HFD challenge in this study. Also, combining physiological and biochemical parameters, and relative genes expression to demonstrated chlorogenic acid is useful for the prevention and treatment of obesity.

| Animals and diets
Eighteen ICR male mice (clean grade, the Wu laboratory animal trading co., Ltd., Fuzhou, China), 5-to 6-weeks old, with body weights ranging from 29 to 31 g. Mice were housed up to six per cage with a 12-hr light/12-hr dark cycle (lights on from 8:00 a.m.to 8:00 p.m.) at 23 ± 1°C and 50 ± 10% humidity. All mice were fed a normal diet and adapt environmental one week. They were then randomly separated into three groups: normal diet group (ND), high-fat diet model group (HFD), and high-fat diet with 150 mg/kg bw/day of chlorogenic acid (CGA). The normal diet contained (in weight percent): 22.3% protein, 60.6% carbohydrate, and 4.0% fat, high-fat diet contained: 21.6% protein, 43.1% carbohydrate, and 18.4% fat. All mice were treated orally by gavage, the ND and HFD groups received an oral saline, and CGA group received chlorogenic acid dissolved in saline at the same volume. The food and water were available ad libitum during 6-week administration. Body weight was measured every three days at a time.

| Biochemical analysis
After 6 weeks of feeding, all animals were fasted 12 hr and weighed. The blood was collected by EDTA tubes and centrifuged at 1500 g for 10 min at 4°C. The levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate transaminase (AST), alanine transaminase (ALT), and blood urea nitrogen (BUN) in plasma were evaluated by using automatic biochemical analyzer (7080; Hitachi Co., Japan).

| Histological analysis
Epididymal white adipose tissues (WAT) and livers were fixed with 4% paraformaldehyde for 24 hr and embedded in paraffin. Then, 5μm sections were prepared and stained with hematoxylin and eosin (H&E). The physiology of epididymal WAT and livers were observed by inverted microscope (Motic BA210T, China).

| Quantitative RT-PCR
Total RNA was extracted from epididymal WAT using an Uniq-10 Trizol total RNA extraction kit (Sanggon Biotech Co., Ltd., Shanghai, China). cDNA was synthesized with 0.8 μg of total RNA by RevertAid First strand cDNA Synthesis kit. RT-PCR was performed using the SYBR Green Abstract PCR Mix (Sanggon Biotech Co., Ltd., Shanghai, China) and LightCycler 480 II system (Roche, Basel, Switzerland). The mRNA levels of target genes were normalized to β-actin. Primer sequences are shown in Supporting Information Table S1.

| Gut DNA extraction
The luminal contents of the cecum were isolated to extract the total bacterial community DNA using the DNeasy PowerSoil Kit (QIAGEN, Inc., Netherlands), following the manufacturer's instructions, and stored at −20°C prior to further analysis. The quantity and quality of extracted DNAs were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively.
After that, the 16s rDNA sequencing and analysis were performed as described previously (Yang, Dou, & An, 2018).

| Data analysis and statistics
All data were presented as the mean value ± standard deviation (SD) and comparisons of data were carried out using a Student's t test or a one-way analysis of variance (ANOVA) with Duncan's test. Values of p < 0.05 were considered to be statistically significant.

| Chlorogenic acid reduced HFD-induced body weight and fat weight increase
The body weight of mice in CGA group changed slowly during a 6-week feeding period (Figure 1a), and the mice fed HFD supplemented with chlorogenic acid (150 mg/kg) had significantly lower body weight in comparison to HFD-fed mice (Table 1). Further, the weight gain, liver weight, fat weight were also markedly decreased by chlorogenic acid treatment (p < 0.05). Histological section from epididymal WAT indicated fat mass and adipocyte size were greatly attenuated by CGA administration (Figure 1b,d).
Similarly, liver histological section in HFD-fed mice also showed abnormal hepatic steatosis and large amounts of lipid droplets, but chlorogenic acid can diminish these adverse changes ( Figure 1c). Thus, these results confirmed that chlorogenic acid has anti-obesity effects.

| Chlorogenic acid improved lipid profile and reduced toxicity in plasma
To examine the dyslipidemia-preventing effect of chlorogenic acid in HFD-fed mice, plasma lipid levels were analyzed. Table 2 shows the plasma biochemical variables in mice after 6 weeks of treatment with chlorogenic acid. The TG, TC, LDL-C levels in the HFD group were significantly increased compared to that in the ND group. However, treatment with chlorogenic acid significantly reduced TC, TG, LDL-C levels and increased HDL-C level as compared with the HFD group. Moreover, the hepatic toxicity and renal toxicity were investigated by measuring plasma AST, ALT, and BUN levels, respectively. ALT, AST, BUN levels increased in the HFD group, but chlorogenic acid administration significantly decreased AST and BUN levels compared to the HFD group (p < 0.05).

| Effects of chlorogenic acid on transcription of genes involved in lipid metabolism
Based on qPCR results in Figure  . Liver morphology (c) and epididymal WAT morphology (d) in different groups. HE staining (×200). Significant differences between HFD versus ND are indicated: *p < 0.05; **p < 0.01. Significant differences between CGA versus HFD are indicated: # p < 0.05; ## p < 0.01 TA B L E 2 Effects of CGA on plasma biochemical indicators in different groups at the end of 6-week feeding F I G U R E 2 Effect of CGA on mRNA expression of lipid metabolism-related genes in epididymal adipose tissue. The values of genes levels include peroxisome proliferator-activated receptor α (PPARα) (a), adponectin (b), FAS (c), LPL (d), PPARγ (e), SREBP-1c (f), AP2 (g), C/EBPα (h), and G protein-coupled receptor 43(GPR43) (i) were normalized to the value of β-actin, which was set to 1. *p < 0.05, **p < 0.01 compared with the HFD group by the student's t test

| Chlorogenic acid modulated gut microbiota at different taxonomic levels
High-throughput sequencing was applied to explore the effect of chlorogenic acid treatment on the richness and diversity of the gut microbiota. As shown in In addition, to profile the specific changes in the gut microbiota, the microbial community at the phylum level is shown in (Figure 3a and 34.5% (in the CGA group). In addition, as shown in Figure 3d, the Bacteroidetes-to-Firmicutes ratio was modestly increased in the HFD group compared with that in the ND group. In contrast, after chronical administration chlorogenic acid for 6 weeks, a relatively lower Firmicutes: Bacteroides ratio was observed in the CGA group.
At the family level, S24-7, Unclassified_Clostridiales, and Desulfovibrionaceae accounted for a high proportion in three groups ( Figure 4a). Moreover, the relative abundance of Desulfovibrionaceae in the HFD group was significantly higher than that in the ND group, but after chlorogenic acid treatment, the relative abundance of this bacteria decreased (Figure 4b). Lachnospiraceae, belongs to Firmicutes phylum, showed a slight drop in HFD group, but no significant difference compared with ND group (p < 0.05), however, the flora community in chlorogenic acid group was decreased compared TA B L E 3 Diversity and richness of gut microbiota in controls and chlorogenic acid-treated groups of mice F I G U R E 3 Distribution of the gut microbiota composition. ND group (a); HFD group (b); CGA group (c) at the phylum level and the ratio of Firmicutes to Bacteroidetes at different groups (d) to that in the HFD group (Figure 4e). Additionally, incremental microbiota such as Ruminococcaceae, Lactobacillaceae, Bacteroidaceaee, and Erysipelotrichaceae were observed in the CGA group compared to HFD group (Figure 4c,d,f,g).
The classification of the microbiota community structure at the genus level was assessed by a heat map ( Figure 5). Apparently, genera were showed at different levels in three groups. Obviously, the relative abundance of Oscillospira, Coprococcus, Anaerotruncus, Allobacterium, Bifidobacterium, Turicibacter were increased, and a lower relative abundance of Bacteroides and Ruminococcus were exhibited in the HFD, but the changes of these species could be reversed by CGA treatment. Collectively, these results indicated , Bacteroidaceae (f) and Erysipeiotrichaceae (g) was expressed as the mean + SD. Significant differences between HFD versus ND are indicated: *p < 0.05; **p < 0.01. Significant differences between CGA versus HFD are indicated: # p < 0.05; ## p < 0.01 The heat map of 20 genera with the highest frequency and relative abundance in groups that gut microbiota in HFD-fed mice were modulated by chlorogenic acid.

| D ISCUSS I ON
In the previous study, chlorogenic acid has exhibited anti-obesity property with improvements of lipid metabolism in HFD-induced mice. Evidence suggests that chlorogenic acid (400 mg/kg) can achieve a deciline in TC, TG, and LDL-C levels in plasma (Wu et al., 2014), in addition to this, chlorogenic acid (100 mg/kg) treatment also attenuated obesity-related hepatic steatosis (Ma, Gao, & Liu, 2015). The present study confirmed this effect that chlorogenic acid (150 mg/kg) led to weight loss (p < 0.05), suppressed lipogenesis, and ameliorated hepatic steatosis. There are evidences that obesity individuals are closely associated with higher TG, TC, LDL-C levels, and lower HDL-C level (Ko, Cockram, Woo, & Chan, 2001;Li, Huang, & Chen, 2008). Moreover, the declining ratio LDL-C/HDL-C, which is often considered to attenuate coronary heart disease risk related to obesity (Hwang et al., 2016). Here, it was revealed that chlorogenic acid could reverse plasma lipid changes altered by the HFD feeding, such as TG, TC, HDL-C, LDL-C, and LDL-C/HDL-C.
The adipose tissue is the most important organ for lipogenesis and metabolism of lipids and energy (Cariou et al., 2004). Here, we com- Obesity and related to metabolic disease are closed to the changes in gut microbial composition. Gut microbiota, particularly Firmicutes and Bacteroidetes are two major phyla in mice and human gut microbiota (Eckburg et al., 2005;Ley, Turnbaugh, Klein, & Gordon, 2006), and this phenomenon was also found in our study.
Previous studies have suggested the obesity individuals owned a smaller number of Bacteroidetes and higher proportion of Firmicutes compared to lean individual (Rastmanesh, 2011;Turnbaugh, Backhed, Fulton, & Gordon, 2008). Here, the results showed that HFD-induced mice had a relatively higher Firmicutes/Bacteroidetes ratio compared to the ND-fed mice, but these could be inverted by administering chlorogenic acid.
Increasing studies have pointed to a positive link between other bacterial phyla or special families and obesity (Table 4). In view of this, these species involved in energy metabolism also require further consideration, In this study, the increased relative abundance of Ruminococcaceae as well as its genus Oscillospira were found in HFD-induced obesity mice, however, but CGA could significantly reverse the change of this species (p < 0.05). Lachnospiraceae, a kind of digestive tract-associated bacteria, correlates with increased fat mass and lipid level (Kameyama & Itoh, 2014;Murugesan et al., 2016;Pataky et al., 2016). However, in our study, a large decrease in Lachnospiraceae when HFD-induced mice were simultaneously administrated chlorogenic acid. Meanwhile, our research also suggested chlorogenic acid promoted increase in the relative abundance of Bacteroidaceae, and Lactobacillaceae might prevent the negative metabolic phenotype correlated with obesitydriven dysbiosis. It has been reported that a high abundance of Erysipelotrichaceae was observed in HFD-induced mice, and they are strongly responsible for obesity (Hui et al., 2015). Interestingly, the relative abundance of Erysipelotrichaceae induced by high-fat diet can be alleviated by CGA treatment. Intriguingly, the family Desulfovibrionaceae (Proteobacteria phyla) was thought to be positively associated with obesity , but a lower abundance was observed in the chlorogenic acid group, which may contribute to alleviating the development of obesity. Taken together, in line with the previous research that polyphenol-induced intestinal microbiota homeostasis, we have reason to believe that chlorogenic acid shows anti-obesity effect through beneficial modulation of the gut microbiota.

| CON CLUS IONS
The present study demonstrated that 6 weeks of chlorogenic acid administration could reduce the body weight, improve plasma lipid associated with HFD-induced obesity and regulate lipogenesis and lipolysis genes expression in epididymal WAT. Moreover, chlorogenic acid treatment dramatically adjust the gut microbiota composition associated with obesity, such as decreasing Ruminococcaceae, Desulfovibrionaceae, Lachnospiraceae, Erysipelotrichaceae, and increasing Bacteroidaceaea and Lactobacillaceae with their genus members of the Bacteriodes and Lactobacillus, respectively. Our results demonstrated the potential possibility that chlorogenic acid in the prevention and treatment of obesity may closely rely on its role in regulation of gut microbiota.

ACK N OWLED G M ENTS
The authors are grateful to the China Postdoctoral Science

Foundation (2018M63072) and Research Fund for Taiwan-Straits
Postdoctoral Exchange Program (2018B003) for financial support.
The author also thanks Shanghai Personal Biotechnology Limited Company for the analysis of data.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interests.

E TH I C A L A PPROVA L
Animal experiment was conducted with the approval of the