Effect of tartary buckwheat, rutin, and quercetin on lipid metabolism in rats during high dietary fat intake

Abstract Tartary buckwheat is rich in flavonoids. However, the health‐promoting effect of these flavonoids has not been adequately studied. In the present study, we investigated the impact of rutin, quercetin, and Tartary buckwheat on the lipid metabolism of rats on a high‐fat diet. Quercetin could significantly reduce body weight, serum triacylglycerol, low‐density lipoprotein cholesterol, TNF‐α, insulin, and ameliorate glucose tolerance. It was surprising that Tartary buckwheat significantly increased the weight of the rats. Rutin, quercetin, and Tartary buckwheat tended to decreased fat deposition in the liver of rats but have little effect on short‐chain fatty acid production. The changes in the structure and diversity of the microbiota were found to be modulated by these diets. It was concluded that quercetin could attenuate high‐fat diet‐induced obesity, rutin, quercetin, and Tartary buckwheat can shape the specific structure of gut microbiota. Mechanism of Tartary buckwheat on lipid metabolism needs further systematic research.

. Intestinal flora in the host plays an important role in energy metabolism and balance through adjusting the molecular signals released. The contents of signaling molecules such as ghrelin, leptin, and glucagon peptide (glp-1) have shown excess or insufficiency on the body's energy (van de Wouw et al., 2017). Dietary or drug intervention to regulate the structure of intestinal microbes has been demonstrated to have a positive effect on both humans and animals (Li, Watanabe, & Kimura, 2017;Sonnenburg et al., 2016).The metabolites of gut microbes with food, such as short-chain fatty acids (SCFAs), bile acid, GABA, dopamine, and norepinephrine, play an important role in changing dietary habits and host metabolism, and in maintaining weight and energy balance by affecting signaling molecules through gut-brain axis adjustment (van de Wouw et al., 2017). SCFAs, produced by GM degrading nondigestible carbohydrates, have a potentially inhibitory effect on inflammation and can be an important regulatory factor in human metabolism. However, the relationship between GM, SCFAs, and glucolipid metabolism is still unclear, so more research is required.
Epidemiological data have indicated that increasing the intake of grains, vegetables, and fruit can significantly reduce the risk of metabolic syndromes (MS) such as obesity (Miller et al., 2017). Flavonoids exist widely in fruits, vegetables, and whole grains, and exhibit a variety of physiological activities such as regulating gut microbiota, and antioxidative and anti-inflammatory effects (D'Andrea, 2015). Previous reports have indicated the modulatory capacity of flavonoids on the gut microbial community (Gil-Cardoso et al., 2016). Recent studies have indicated that quercetin and rutin can modify the gut microbial balance, and alleviate obesity and liver disease (D'Andrea, 2015;Panchal, Hemant, Arumugam, & Brown, 2011;Porras et al., 2016). Thus, the use of food rich in flavonoids, such as quercetin, to metabolize lipids might be a potential strategy for modulating the composition of intestinal bacteria. Tartary buckwheat, belonging to the genus Fagopyrum of the Polygonaceae family, has been reported to help control blood glucose, blood pressure, and blood lipid levels (Zhu, 2016). Tartary buckwheat is rich in flavonoids, mainly rutin (Zhu, 2016), and has therefore been attracting more research attention.
However, it needs to be clarified whether dietary rutin or quercetin in buckwheat can ameliorate lipid metabolism, because the starch and protein in buckwheat flour may also have beneficial effects on lipid metabolism. Meanwhile, our previous study indicated that the nutritive active components of Tartary buckwheat were related to different parts and processing methods (Peng et al., 2017(Peng et al., , 2015. Thus, the real effect of these flavonoids present in buckwheat needs further studies to promote the development and utilization of Tartary buckwheat.
The present study aims to investigate the potential benefits of an experimental treatment with quercetin, rutin, and Tartary buckwheat on high-fat diet fed rats on regulating lipid metabolism, restoring host microbial balance, and regulating the SCFAs signaling mechanisms for MS. The results will improve the understanding of the functional properties of buckwheat, and also the design of therapeutic approaches to manipulate gut microbiota for treating MS or malnutrition.
All other chemicals and solvents used in the study were of analytical grade.

| Animals and treatments
All experiments on animals were performed in strict compliance with the guides for the Care and Use of Laboratory Animals published by US National Institutes of Health and was approved by the Use Committee of Chengdu University. Seven-week-old male Sprague-Dawley (SD) rats (220 g ± 20 g, Permission Number: SCXK (Chuan) 2018-19) were fed with a standard diet to adapt to the environment then divided into five groups (ten rats per group): control (AIN 93-G), HFD (high-fat diet), HFDQ (HFD containing 0.1% quercetin), HFDR (HFD containing 0.2% rutin), and HFDB (HFD + Tartary buckwheat) (Table 1). Rats were fed the diets and water ad libitum, housed in standard polypropylene cages (five rats/cage), and maintained under a controlled room temperature (24 ± 2°C) and relative humidity (55 ± 5%) with a 12/12-hr light/dark cycle. The body weights were monitored weekly, and food intake was monitored daily. After 12 weeks, the rats were euthanized, and then, serum, liver, feces, and adipose tissue samples were collected and weighed. The right posterior lobe of the liver was fixed in 10% formalin. All experimental protocols were approved by the Animal Experimentation Ethics Committee of Chengdu University for the care and use of laboratory animals.

| Biochemical analysis
The serum samples were assayed for triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), lowdensity lipoprotein cholesterol (LDL-C), creatinine, and urea, and cystatin C levels were measured using assay kits (Changchun Huili Biotech Co., Ltd.). The fasting insulin, IL-6, and TNF-α levels were determined using commercial rat ELISA kits (ExCell Biology Inc., Taicang, China) according to the manufacturer's instructions. Oral glucose tolerance tests were performed after 11 weeks of feeding as follows: all the rats were fasted for 10 hr followed by an oral glucose load (2 g/kg of body weight); blood samples were collected from the tail vein, and glucose levels were determined using a Glucometer (Accu-chek ® Performa, Roche Diagnostics) at 0, 30, 60, and 120 min after glucose administration.

| Histopathology
Liver pathology was evaluated via hematoxylin and eosin (HE) staining.

| Identification and quantification of fatty acids
The fatty acids were extracted and quantified as described by Wang, Li, Muhammad, and Zhang (2016) with slight modification. One hundred µl of a 10 mg/ml internal standard (Tridecane acid) and 2 ml petroleum ether were added to 1-mL serum samples and, then, extracted using vortex oscillation for 3 min. After centrifugation at 10,000 g, the supernatant was collected. Extraction was repeated three times and the supernatants were combined, then dried using N 2 . After adding 3 ml 0.5 mol/L NaOH-methanol, the mixture was shaken, heated at 60°C for 6 min, and then, 3 ml 5% boron trifluoride-methanol was added and held at 60°C for 7 min. After cooling down to room temperature, 2 ml saturated NaCl and 1 ml n-hexane were added, vortexed, left for 30 min, and then, the supernatant was collected. FFA were identified and quantified using a gas chromatograph coupled with a flame ionization detector (Agilent 7890A, Agilent Technologies Inc.). The samples were separated using a SPTM-2560 capillary column (100 m × 0.25 mm × 0.2 μm; Supelco). The split ratio was set at 50:1, the injection volume was 1 μl, the injector temperature was 240°C, and the detector temperature was 250°C. The temperature program was as follows: initial temperature of 40°C, raised at 3°C/min to 220°C, raised at 1°C/min to 230°C, and then held there for 23 min. The flow rate of nitrogen was 1.0 ml/min.

| Identification and quantification of cecal shortchain fatty acids (SCFAs)
One hundred μl 15% H 3 PO 4 , 50 μg/ml internal standard (isocaproic acid), and 400 μl diethyl ether were added to 100-mg cecal were obtained with a certain threshold. Each OTU was selected as the representative sequence for taxonomical assignment using the UCLUST algorithm. The number of sequences per sample was corrected for differences in sequencing depth between samples by rarefication, that is, the same number of reads was randomly subsampled in each sample. Then, the absolute number of sequences of each OTU in each sample was converted to relative abundance to reduce the effect of differences in sequence reads. The representative sequences, together with the abundance data, were used for taxon-based analysis.

| Statistical analysis
The data were expressed as mean ± SD and evaluated with oneway analysis of variance (ANOVA) and Student's t test. p < .05 was considered to be significant for a difference between mean values.
Alpha diversity and beta diversity were analyzed using a procedure in R. Principal coordinate analysis were using Bray-Curtis distances.
All statistical analyses were performed using IBM SPSS Statistics (version 22.0, IBM Corp.). and animal models used in these studies.

| Serum biochemicals
It was surprising that Tartary buckwheat bran significantly increased the weight of rats, perirenal fat, and epididymis fat content and increased food intake compared with HFD although HFDB was rich in rutin (0.27%). Another study reported no significant differences in the atherosclerosis index andoxidized LDL levels, and in lipid metabolism parameters between the Tartary buckwheat food and the placebo food groups (Nishimura et al., 2016). The rutin content of Tartary buckwheat is estimated to be approximately 100 times that of common buckwheat (Yasuda & Nakagawa, 1994), but no differences were reported between the two types of buckwheat F I G U R E 4 Effect of Tartary buckwheat and flavonoids on liver histology and lipid accumulation in HFD-fed rat, using Hematoxylin & Eosin staining method F I G U R E 5 Effects of Tartary buckwheat and flavonoids on fecal short-chain fatty acid production. ** p < .01 and * p < .05 denote statistically significant differences with control group; ## p < .01 and # p < .05 denote statistically significant differences with HFD group on the serum total cholesterol levels in another study (Wieslander et al., 2011). Thus, it might be deduced that there were effects caused by components in the buckwheat other than rutin. Our previous report has shown that Tartary buckwheat bran was rich in rutin, and contained much higher levels of fatty acids, and amino acids than Tartary flour (Peng et al., 2017). In the present study, the buckwheat and wheat flour were steamed to make them more similar to a human diet. Looking at these two studies, we can suggest that the nutrients available in Tartary buckwheat were connected to its components and processing method and that more attention should be paid to these differences.
The TNF-α and IL-6 contents increased significantly in the HFD, HFDR, and HFDB groups, but quercetin significantly reduced their contents relative to HFD, implying that quercetin may reduce fat accumulation by improving the state of inflammation in body. The high-fat diet also increased serum urea and creatinine levels, thus increasing the risk of overburdening the kidneys. It was interesting to note that buckwheat decreased the urea and creatinine levels, suggesting that buckwheat has the potential to protect renal function. Twelve weeks of quercetin dietary treatment improved glucose tolerance as shown by the reduced blood glucose levels in the oral glucose tolerance test (OGTT), but the rutin and Tartary buckwheat treatments had no similar effects. These findings suggested that quercetin administration could improve glucose tolerance and insulin resistance, and alleviate fat accumulation in diet-induced obese rats.
Regarding fatty acids (FAs), eight types of FA were detected in the serum of rats by gas chromatography at the 12th week for the control and HFD groups. Figure 3 shows that the concentrations of C16:0, C18:0, C18:1, C18:2, and C20:4 in rat serum were much higher than those of the other FAs, findings consistent with other reports (Wang et al., 2016;Zhukova, Novgorodtseva, & Denisenko, 2014). Saturated fatty acids, such as C14:0, C16:0, and C18:0, which can promote inflammation in the body, were higher in the HFD group. Administering flavonoids tended to reduce the contents of these FAs.

F I G U R E 6
Effects of Tartary buckwheat and flavonoids on the alpha diversity of gut microbiota. ** p < .01 and * p < .05 denote statistically significant differences with control group; ## p < 0 0.01 and # p < .05 denote statistically significant differences with HFD group

| SCFAs production
SCFAs, such as acetate, propionate, and butyrate, are produced by the intestinal microbiota and are perhaps the most extensively studied molecules, which mainly influence the host's energy metabolism and appetite (Wong, Souza, Kendall, Emam, & Jenkins, 2006 (Schwiertz et al., 2010;Turnbaugh et al., 2006). However, SCFA supplementation tends to reduce body weight in both humans and rodents (Chambers et al., 2015;Lu et al., 2016). In mice, acetate in the colon has been shown to induce the secretion of glucagon-like peptide (GLP)-1, which reduces food intake and obesity (Tolhurst et al., 2012). Another study has proposed that circulating acetate enhances insulin secretion through a microbiome-brain-β-cell axis, and promotes obesity and the metabolic syndrome (Perry et al., 2016). Propionate is primarily used by hepatocytes, so gut-derived propionate has been shown to reduce liver fat in adults with NAFLD (Brass & Beyerinck, 1988 (Lin et al., 2012;Lu et al., 2016). In the present study, Figure 5 shows that high-fat diet rats exhibited significantly higher levels of acetate (1,424.63 μg/g) but lower levels of propionate (211.66 μg/g), butyrate (152.84 μg/g), and other SCFAs than the control group. These results agreed with the study of Perry et al. (2016), but were inconsistent with that of Porras (Porras et al., 2016) where HFD decreased the acetate, propionate, and butyrate contents. Differences in doses, patterns, species, timing, and composition of feedstuff are plausible explanations for these discrepancies.
Thus, more research is required to resolve these conflicts regarding the role of SCFAs in the metabolic syndrome. Rutin, quercetin, or  211.66, 193.87, 190.99, and 231.39 μg/g, respectively and 470.04 to 152.84, 167.36, 138.61, and 209.74 μg/g, respectively). These effects may have been mediated by the gut microbiota and increased steatosis in the liver and serum inflammation.

| Gut microbiota
To assess the effects of flavonoids and buckwheat treatments on the microbial composition of the gut microbiome, the V3-V4 hypervariable region of the 16S rDNA gene was sequenced. A total of 1,840,616 reads were obtained from the cecal samples. After denoising and filtering, 1,707,033 clean tags were used for subsequent analysis. Based

F I G U R E 7
Effects of Tartary buckwheat and flavonoids on gut microbiota balance in HFD-fed rats (phylum level). ** p < .01 and * p < .05 denote statistically significant differences with control group; ## p < .01 and # p < .05 denote statistically significant differences with HFD group on a 97% similarity level, all of the effective reads were clustered into OTU. Compared with the control group, the HFD treatment significantly reduced the number of OTUs. Figure 6 shows that other microbial diversity indices, the Shannon index, Chao1 index, Simpson index, and Observed species index decreased in the HFD group. Tartary buckwheat treatment increased Chao1 index significantly, and this may be an important potential beneficial effect for human. Quercetin tends to increased Chao1 index, but not reach significant.
Most of the identified reads from the cecal content of mice were classified within three phyla. The Firmicutes phylum was predominant, representing 49% of the 16S rDNA gene sequences, followed by Bacteroidetes (47%) and Proteobacteria (2%). These three phyla represented more than 95% of the sequences analyzed. Figure 7 shows the relative bacterial composition at the phylum level for each group. The relative abundance of the Bacteroidetes and Proteobacteria phyla was significantly higher when Firmicutes was lower in the HFD groups compared with the control groups.
These results were inconsistent with those of Porras et al (Porras et al., 2016). The review by Tagliabue and Elli discussed how obesity in humans has been associated with reduced bacterial diversity and an altered representation of bacterial species, but the identified differences were not consistent among the studies (Tagliabue & Elli, 2013). They concluded that the question still remains as to whether changes in the intestinal microbial community are one of the environmental causes of overweight and obesity or whether they are a consequence of obesity, specifically of the unbalanced diet that often accompanies the development of excess weight gain. These studies combined with the findings of the present study suggest that the relationship between bacterial composition at the phylum level and rats fed a high-fat diet need further clarification and that more confounding variables such as diet composition should be take into account. The diets in the HFDQ, HFDR, and HFDB groups showed little effect on changing these three phyla (Firmicutes, Bacteroidetes, and Proteobacteria).

F I G U R E 8
Effects of Tartary buckwheat and flavonoids on gut microbiota balance in HFD-fed rats (family level). ** p < .01 and * p < .05 denote statistically significant differences with control group; ## p < .01 and # p < .05 denote statistically significant differences with HFD group Figure 8 shows the effects of Tartary buckwheat and flavonoids on the balance of gut microbiota at the family level in HFD-fed rats.
Lachnospiraceae, which may protect against colon cancer in humans by producing butyric acid (Meehan & Beiko, 2014), significantly decreased in the HFD, HFDQ, HFDR, and HFDB groups. These results were consistent with the changes in butyric acid content.
Lachnospiraceae has been found to cause diabetes in germ-free mice (Kameyama & Itoh, 2014). Prevotellaceae, composed of four genera, which can be opportunistic pathogens, increased significantly in the HFD group but decreased in the HFDB, HFDQ, and HFDR groups.
Consistent with results at the phylum level, the abundance of Bacteroidaceae increased significantly in the HFD, HFDB, and HFDQ groups. Bacteroidaceae increased significantly in the HFDR group compared with the HFD group. The abundance of Tannerellaceae, Desulfovibrionaceae, and Akkermansiaceae was much higher in the HFD group, but was significantly reduced by rutin treatment, but quercetin had no effect. Quercetin also increased the abundance of Ruminococcaceae to approach that of the control: These may be the important differences in the effect of rutin and quercetin. Figure 8 also shows the changes in the main microbiota at the family level.
At the genus level, most of the sequences belonged to the genera, F I G U R E 1 0 Effects of Tartary buckwheat and flavonoids on gut microbiota balance in HFD-fed rats (genus level). ** p < .01 and * p < .05 denote statistically significant differences with control group; ## p < .01 and # p < .05 denote statistically significant differences with HFD group buckwheat and flavonoids did not increase the relative abundance of Lactobacillus, which suggested a positive link with weight control.
The main change in the other genera is shown in Figures 9 and 10.
Beta diversity measurements were performed on the cecal content to determine whether there were any differences in global bacterial composition between the five treatment groups. A PCoA based on the Bray-Curtis distance was performed ( Figure 11) showing that the bacterial communities of rats fed with HFD clustered together according to the second axis (22.8%) based on the diet. For the HFD rats with or without treatment with Tartary buckwheat or flavonoids, the communities of the HFDR group were separate from the others on the second axis (22.8%). The HFD group could also be distinguished from the HFDB and HFDQ groups on the first axis (35.8%). However, the HFDQ and HFDB groups were dispersed along these two axes, although the weight of rats treated with Tartary buckwheat and quercetin was significantly different. This result implied that the relationship between weight gain and gut microbiota was complex, and more evidence was needed. One interesting finding was the similarity in the microbiota composition of rats from the same cage, with some differences from rats in other cages from the same treatment group. This also implied that the community of rats has an important effect on microbiota structure and may need to be taken into consideration in any further research.
In conclusion, our findings suggest that quercetin could attenuate HFD-induced obesity and related metabolic disorders in rats.
This may have been caused by its antioxidant activity and inhibitory effect on inflammation. Rutin had little effect on attenuating obesity, but tended to decrease fat deposition in the liver. It was surprising that Tartary buckwheat increased body weight and fat accumulation, despite showing the effect of attenuating hepatic steatosis. HFD increased the fecal acetate content but decreased other SCFAs. Tartary buckwheat, quercetin, and rutin had little effect on the production of SCFAs. Tartary buckwheat, quercetin, and rutin treatments all modulated the gut microbiota, but more research is needed to clarify the relationship between gut microbiota and obesity. Meanwhile, the role of quercetin and rutin in the diet needs more systematic assessment so that confounding variables can be taken into account and techniques can be standardized.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
All animals used in this study were reared and euthanized with the approval of the Use Committee of Chengdu University. All experiments were performed following the guidelines and regulations of "the instructive notions with respect to caring for laboratory animals" issued by the Ministry of Science and Technology of the People's Republic of China.