Effects of Different Treatment Methods of Dried Citrus Peel (Chenpi) on Intestinal Microflora and Short-Chain Fatty Acids in Healthy Mice

Chenpi is a kind of dried citrus peel from Citrus reticulata, and it is often used as traditional Chinese medicine to treat dyspepsia and respiratory tract inflammation. In this study, to determine which way of chenpi treatment plays a better effect on the prevention of obesity in healthy mice, we conducted 16S ribosomal RNA (rRNA) gene sequencing for intestinal microbiota and gas chromatography-mass spectrometry detector (GC/MSD) analysis for short-chain fatty acids (SCFAs) of female rats fed with either chenpi decoction or chenpi powder-based diet (n = 10 per group) for 3 weeks. Chenpi powder (CP) group significantly reduced abdominal adipose tissues, subcutaneous adipose tissue, and the serum level of total triacylglycerol (TG). At a deeper level, chenpi powder has a better tendency to increase the ratio of Bacteroidetes to Firmicutes. It alters the Muribaculaceae and Muribaculum in intestinal microbiota, though it is not significant. The concentrations of acetic acid, valeric acid, and butyric acid increased slightly but not significantly in the CP group. Chenpi decoction just reduced perirenal adipose tissues, but it shows better antioxidant activity. It has little effect on intestinal microbiota. No differences were found for SCFAs in the chenpi decoction (CD) group. The results indicated that chenpi powder has a better effect in preventing obesity in mice. It can provide a basis for the development of functional products related to chenpi powder.


INTRODUCTION
Dried citrus peel (chenpi) is the mature dry pericarp of Citrus reticulata. As a traditional Chinese medicine, it has a good effect on treating dyspepsia and improving respiratory tract inflammation. Chenpi contains many active components, such as essential oil (1), flavonoid (2), pectin (3), insoluble fiber (4), and so on. Citrus peel essential oils may ameliorate hypercholesterolemia and hepatic steatosis by modulating lipid and cholesterol homeostasis, and most of them have good antimicrobial and antioxidant activities (5,6). Polymethoxyflavones, a kind of flavonoid from citrus peel, have anti-obesity, anti-hyperglycemic, and antiviral activities; meanwhile, it may effectively prevent the progression of metabolic syndrome (7)(8)(9)(10). Pectin polysaccharide has in vitro intestinal immunomodulatory activity (11). In addition to the abovementioned active substances, pure chenpi powder also contains a large amount of dietary fiber. The composition and activity of intestinal microbiota and the production of short-chain fatty acids (SCFAs) were affected by dietary fiber (12). Meanwhile, the production of SCFAs (in particular, acetate, propionate, and butyrate) is closely related to intestinal health and function (13).
Intestinal microbiota are microorganisms colonized in the human digestive tract, which is closely related to age, obesity, and inflammation (14)(15)(16). In recent years, the study on intestinal microbiota is a hot spot. Diet has different effects on intestinal microflora. More and more evidence shows that intestinal microflora is closely related to metabolism, host gene expression, and other factors (17)(18)(19). Chenpi has been proven to have a modulation effect on the composition of intestinal microbiota species, the abundance of microbiota, fecal SCFAs, intestinal barrier function, and gastrointestinal inflammation (20)(21)(22).
Obesity as a thorny issue worldwide is caused by many factors. Obesity can cause a series of complications, such as hypertension, hyperlipidemia, metabolic diseases, and increasing organ burden (23,24). Several studies have observed the effects of extracts or natural products on intestinal microorganisms, SCFAs, glucose metabolism, and body weight of healthy mice model (25,26). Looking for natural products that can alleviate and treat obesity is a healthy and safe method. Although there are some studies on the effect of reducing weight and lipid of chenpi, there is no study on which way of chenpi treatment can play a better effect. In this experiment, we observed the effect of the chenpi on healthy mice. Traditionally, chenpi was infused with boiling water to extract their effective components such as "decoction." In this study, we added chenpi to the normal diet of mice in two forms, both chenpi decoction and chenpi powder. This study aimed to investigate the modulation effect of two different types of chenpi on the accumulation of adipose, intestinal microbiota, antioxidant capacity, and SCFAs to unvail their potential application for obesity prevention, which may also provide a basis for the use of chenpi as a kind of anti-obesity food in the food industry.

Mice and Housing
Forty four-week-old C57BL/six female mice (Tianqin Biotechnology Company, Changsha, China) were housed in a controlled room with a 12 h/day lighting cycle during the experimentation. Food and drinking water were freely available to mice. Following 1 week of acclimation, mice (n = 10) were randomly grouped to control (C), chenpi decoction (CD), control powder (P), chenpi powder (CP). They were all provided with a normal diet. The normal diet contained 54.9% corn, 5.6% casein, 18% soybean meal, 6.5% beer yeast, 0.7% lard, 0.8% bean oil, 0.5% salt, 1.4% fishmeal, and 1% premixture. The difference between granulated (C) and powder (P) groups is whether granulation is carried out. In the CD group and CP group, chenpi decoction and chenpi powder, respectively, were added to the normal diet. The body weight, food intake, and water intake were recorded once a week. After 3 weeks of administration, blood samples were collected by orbital bleeding. Liver, abdominal adipose tissues, subcutaneous adipose tissues, and perirenal adipose tissues were weighed and collected. Fecal samples were collected by 16S ribosomal RNA (rRNA) sequencing and analysis of SCFAs. The experimental protocol was approved by the Animal Care and Use Committee of Hunan Agricultural University.

Preparation of Chenpi Decoction and Chenpi Powder
Chenpi was purchased from Jiangmen Xinhui tangerine peel village market limited company, Guangdong Province. The variety of chenpi is red Pericarpium Citri Reticulatae, which is made by traditional sunlight drying. According to the traditional decocting method, 10 g chenpi was crushed into a coarse powder and 200 ml of water was added and boiled over 95 • C for 30 min. The filtrate was filtered out and then added 20 times of water to decoct again in the same way. The filtrate was combined, evaporated, and concentrated to 10 ml and stored at 4 • C. The concentration of chenpi decoction was 1 g/ml. CD group were administered 0.2 ml/day chenpi decoction by gavage. The mice in the C group were given distilled water at the same time. After grinding and sieving, the chenpi powder was sealed in vacuum and stored at 4 • C. The CP group were given 0.2 g/day chenpi powder in the diet.

Histopathological Observation
Paraformaldehyde solution in 4% was used to fix adipose tissues. Then, they were dehydrated by ethanol solution, embedded, and prepared. The subcutaneous adipose tissue was stained with H&E. Images were obtained using a Nikon Eclipse E100 Upright optical microscope from Nikon Corporation, Japan (27).

Measurement of Hepatic Malondialdehyde (MDA) and Superoxide Dismutase (SOD) Levels
About 0.5 g of each liver tissue was homogenized in 4.5 ml frozen normal saline and then centrifuged and collected supernatant at 2,000 rpm for 10 min at 4 • C for measurements. All these biochemical markers were measured using kits purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Coomassie Brilliant Blue was used to determine the concentration of protein (27). Each sample has a parallel sample.

16S Ribosomal RNA (rRNA) Gene Sequencing for Microbiota Profiling
Total genomic DNA was extracted from fecal samples and stored at −20 • C using the DNA kit according to the instructions for 16S rRNA gene pyrosequencing. Paired-end sequencing was performed on the Illumina MiSeq platform (29). The V3-V4 regions were amplified using a specific primer with the barcode by thermocycler PCR system. In summary, α-diversity, β-diversity, and principal coordinate analysis (PCoA) were calculated and generated by Quantitative Insights Into Microbial Ecology (QIIME). The measurement was repeated three times for each sample. The online platform of Majorbio Cloud (http:// www.majorbio.com/) was used to analyze data (30,31).

Detection of SCFAs
A total of 100 mg feces were dissolved in 0.9 ml water, then mixed, and then centrifuged at 13,200 g force for 10 min at 4 • C. A 1 µl supernatant of each sample was injected into the inlet for gas chromatography-mass spectrometry detector (GC/MSD) analysis. The levels of acetic, propionic, butyric, valeric, isobutyric, and isovaleric acids in SCFAs were measured using 8890B-5977B GC/MSD (Agilent Technologies Inc. CA, USA) (32,33). The measurement was repeated three times for each sample.

Statistical Analysis
The statistical analyses were completed using IBM SPSS Statistics 26.0. The t-test was performed to determine the difference between groups. Values of P < 0.05 mean statistically significant.

Chenpi Alleviates Accumulation of Adipose in Mice
To determine the anti-obesity effect of chenpi on mice, body weight, liver, abdominal adipose tissues, subcutaneous adipose tissues, and perirenal adipose tissues were weighed. Chenpi treatment has a tendency to regulate body weight, but the difference was not significant (P > 0.05) (Figure 1A). Liver weight has basically no change in every group (Figure 1B). Weight of abdominal adipose tissues and subcutaneous adipose tissue was significantly reduced at 0.13 and 0.15% in the CP group compared with the P group (P < 0.05) (Figures 1C,D). Perirenal adipose tissues were significantly reduced in CD (P < 0.05) compared with the C group ( Figure 1E). Serum concentrations of lipids were analyzed (Figure 2) to find out that chenpi powder can significantly reduce the serum level of TG by 24% compared to the P group (P < 0.05) (Figure 2B) but had no  remarkable effect on the serum levels of TC, LDL-C, and HDL-C. These items showed no significant changes in the CD group compared to the C group. The histopathological observation of adipose tissues showed that the CP group exhibited a strong inhibitory effect on the enlargement of adipocytes compared with the P group, while the difference was not significant in other groups (Figure 3). To sum up, compared with chenpi decoction, supplementation of chenpi powder in the diet significantly alleviated accumulation of lipid and serum TG metabolism, and it reduced the relative weight of abdominal adipose tissue and subcutaneous adipose tissue.

Chenpi Enhanced Antioxidant Capacity in the Liver
In order to test the antioxidant capacity of each group, the MDA index and SOD index of the liver were detected. The content of MDA was decreased in the CD group compared to the C group, while the content of MDA in the CP group was 1.35 nmol/mg higher than that of the P group (P < 0.05) (Figure 4). The activity of SOD was increased marginally in the CD group compared to the C group (P > 0.05).

Chenpi Modulated the Structural Composition of Intestinal Microbiota
Intestinal microbiota were known as a key factor in modulating obesity. Thus, to investigate whether chenpi influences the intestinal microbiota of mice, 16S rRNA sequencing was tested. We analyzed the composition and difference of intestinal microflora in different diet groups.
Microbial diversity and richness were evaluated by α-diversity and β-diversity. PCoA plot was applied to evaluate overall differences in β-diversity in unweighted UniFrac distance for the sample set (34,35). As shown in Figure 5, different diets have strong effects on the gut microbial composition revealed by a clear separation among four groups. Shannon and Simpson's indexes evaluated the diversity of the microbiota. ACE and Chao indexes described the richness of the microbiota (36). As shown in Figure 6, the CD group exhibited a higher richness of microbiota evidenced by the increased ACE and Chao indexes compared to C (P > 0.05) (Figures 6C,D) but with no significant difference. Simpson's index in the CP group significantly increased, but other indexes reduced.
As shown in Figure 7, there were differences in microbial composition among the four groups at phylum, family, and genus levels. Firmicutes and Bacteroidetes are the two majorities at the phylum level. CD group had a 51% higher ratio of Firmicutes to Bacteroidetes compared with the C group (P > 0.05) (Figures 7A, 8A). However, the CP group had a lower abundance of Firmicutes (P = 0.07) and a higher abundance of Bacteroidetes (P = 0.06) compared with the P group (Figures 7A,  8B). The difference is not significant. The dominant genera are Muribaculaceae, Lactobacillaceae, and Lachnobacterium at the family level. The relative abundance of Lactobacillaceae in the CD group is higher than in the C group (p > 0.05) (Figures 7B,  8C). The relative abundance of Muribaculaceae in two powder groups is higher than in two decoction groups (Figure 7B). The relative abundance of Muribaculaceae increased in the CP group compared with the P group (P = 0.086) (Figures 7B, 8D). Similar alterations were observed for norank_f_Muribaculaceae, Lactobacillus, and Lachnospiraceae _NK4A316_group at the genus level (Figures 7C, 8E). The relative abundance of Muribaculaceae (p = 0.09) and Muribaculum increased in the CP group compared with the P group (P = 0.08) (Figure 8F).

Chenpi Changed the Content of SCFAs in Feces
The content of SCFAs of feces is closely related to intestinal health. Here, the contents of acetic, propionic, butyric, valeric, isobutyric, and isovaleric acids were tested by GC/MSD. On the whole, the content of SCFAs in the two powder groups was higher than that in the decoction groups. There was no difference in the concentration of any SCFAs in feces in the CD group when compared with the control group. The group that consumed chenpi powder had higher concentrations of SCFAs than the P group, especially acetic, valeric, and butyric acids, but the difference was not significant (Figure 9).

DISCUSSION
We present the results of a study investigating the effects of different supplementation treatments with chenpi on various Frontiers in Nutrition | www.frontiersin.org health parameters, microbial composition, and content of SCFAs. In healthy mice, chenpi supplement changed the accumulation of fat. In particular, chenpi powder can effectively reduce the weight of abdominal adipose tissues, subcutaneous adipose tissue, and the serum level of TG. Other studies also have shown that chenpi can reduce the gain of body weight, organ weight, and accumulation of lipid (37). Obesity is closely related to hyperlipidemia, and reducing the content of serum triglyceride can effectively alleviate hyperlipidemia (38). There was no significant change in body weight and liver weight in our study, perhaps because the feeding time was not long enough. The effect of chenpi on the antioxidant activity of the liver was analyzed. MDA is the most frequently measured biomarkers of lipid peroxidation and oxidative stress that is considered hazardous to health (39). Oxidative damage can lead to a decrease in the content of SOD (40,41). Here, the decoction of chenpi shows stronger antioxidant activity, which might be explained as more antioxidants are released from chenpi after decoction treatment using a high temperature (42).
Chenpi and its main active substances can affect the composition and richness of intestinal microorganisms. Hesperidin can increase the proportion of Lactobacillus in healthy mice. Citrus polymethoxyflavones can greatly enrich the bacterium Bacteroides in high-fat diet (HFD) mice (43)(44)(45). The abundance of Proteobacteria and the ratio of Firmicutes to Bacteroidetes were decreased by the chenpi extract in HFD mice. Although the addition of chenpi supplement did not significantly increase the abundance and diversity of intestinal microbiota in our study, it shows that chenpi powder has  a better tendency to increase the ratio of Bacteroidetes to Firmicutes. This may be because the decoction does not extract the active ingredients of chenpi very well and contains fewer ingredients than chenpi powder. Although active compounds such as hesperidin, naringenin, and nobiletin can be detected in the water decoction of chenpi, some components cannot be fully and effectively extracted because of their poor water solubility (46). A study showed that the water solubility of 5-demethylnobiletin and hesperidin in chenpi was low (47,48). ACE and Chao indexes reduced in the CP group. This may be related to the reduction in harmful bacteria. Studies show that the abundance of Bacteroidetes was reduced by 50%, but Firmicutes was increased about 18% (49,50), the abundance ratio of Bacteroidetes to Firmicutes will decrease in fat mice compared to lean mice (51, 52), and our results are consistent with them. In the control group, Muribaculaceae, Lactobacillaceae, and Lachnospiraceae are the dominant strain at the family level. Chenpi powder increased the abundance of Muribaculaceae significantly at family and genus levels. A high abundance of Muribaculaceae is associated with longevity in mice (53). Chenpi decoction can increase the abundance of Lactobacillaceae that are intestinal beneficial bacteria (54). It has correlation coefficients between bacterial abundances and serum lipid oxidative. The correlations between the abundance of Lactobacillaceae, serum TG, and MDA levels were negative (55). Chenpi increased intestinal beneficial bacteria and reduced microbial abundance associated with obesity. Chenpi powder is more outstanding in the regulation of intestinal microbiota.
The content of SCFAs is closely related to the diet structure. Chenpi contains not only many active ingredients but also a lot of dietary fiber. A fiber-rich diet can increase the content of SCFAs in mice. Dietary fiber can be fermented by colonic microbiota to produce SCFAs. Many studies have shown that a diet rich in dietary fiber can change the content of SCFAs. Passion fruit peel can increase the concentrations of butyrate and acetate in cecal content (56). Salami with citrus fiber increased the production of acetate, propionate, and butyrate (57). Dietary fibers from papayas promoted the production of SCFAs (58). Intestinal microorganisms are closely related to SCFAs. Lachnospiraceae plays an important role in the production of butyrate (59,60). Muribaculaceae are helpful to the production of propionate (61). Escherichia coli could produce acetic acid (62). No significant changes in SCFAs were observed in our study, perhaps due to our shorter feeding cycle. In conclusion, daily consumption of chenpi has a certain effect on reducing weight and lipid. Compared with chenpi decoction, chenpi powder has a better effect in preventing obesity. Chenpi powder may be developed as supplementary functional food to prevent obesity in the future. In this study, we focused on the effect of different treatment methods of chenpi on healthy mice to predict the preventive effect on obesity. In the future, a highfat model would be established to observe this effect in depth.
It is our next direction to research study to develop a variety of popular chenpi functional foods.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: PRJNA729616.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Care and Use Committee of Hunan Agriculture University.