Qian Lin, Lina Yang,*, Lin Han, Ziyi Wang, Mingshuo Luo,Danshi Zhu, He Liu,*, Xin Li, Yu Feng

a College of Food Science and Technology, Bohai University, Jinzhou 121013, China

b National & Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products, Jinzhou 121013, China

Keywords:

Soy hull polysaccharide

Antihyperlipidemic

Gut microbiota

Short-chain fatty acids

A B S T R A C T

The present work aimed at investigating the effects of soy hull polysaccharide (SHP) in alleviating adverse effects in rats fed a high-fat-high-sucrose diet.After SHP feeding for 4 weeks, the fasting blood glucose(FBG), serum triglyceride (TG), serum high-density lipoprotein cholesterol (HDL-C), short chain fatty acids(SCFAs), and 16S rDNA gene sequence were determined.Administration of SHP significantly decreased body fat content and TG levels, and increased water intake and HDL-C levels after 4 weeks of treatment.The antihyperglycemic effect of SHP at a dose of 400 mg/kg mb had the most significant effects among the three dosage groups.SHP notably restored the FBG in rats fed a high-fat-high-sucrose diet (P < 0.05).Furthermore,SHP at 400 mg/kg mb increased the abundance of Bacteroidetes and decreased that of Firmicutes and Actinobacteria at the phylum level.The polysaccharide treated groups had significantly higher content of total SCFAs, and the main fermentation products were acetic, propionic, n-valeric and i-valeric acids.Thus, SHP restores blood lipid levels in rats fed a high-fat-high sucrose diet through regulation of the gut microbiota.

1.Introduction

In recent decades, in most of the developed world and in developing countries, the prevalence of obesity has steadily risen with the adoption of a sedentary lifestyle combined with consumption of energy-dense foods [1].Obesity, sedentary lifestyles and energydense diets are the main drivers of the global epidemic of type 2 diabetes mellitus [2].

Polysaccharide, a natural product comprising polydisperse polymers, has been demonstrated to have antihyperglycemic and antihyperlipidemic activities according to numerous studies.For example,Morus albafruit polysaccharides,Cordycepsmilitaryacidic-extractable polysaccharides, corn silk polysaccharides andSiraitia grosvenoriipolysaccharides have been found to have antihyperglycemic and antihyperlipidemic effects that clearly relieve diabetes symptoms in high-fat-high-sucrose diets and streptozotocin(STZ)-induced diabetic animal models [3-6].Notably, Nie et al.[7]has shown thatPlantago asiaticaL.polysaccharide not only has the above activities, but also may be associated with regulation of the gut microbiota and increased levels of short chain fatty acids (SCFAs).The gut microbiota contains trillions of microbes whose populations are orders of magnitude larger than those of all eukaryotic cells in the host [8].Thousands of microbes live in the human gut, helping to break down food that is otherwise difficult to digest [9].Previous studies have indicated that intestinal microorganisms are closely associated with some metabolic diseases in humans, such as obesity and diabetes; therefore, functional foods can regulate metabolism by improving profiles of intestinal microbes in the body [10-12].Recent research has indicated that alterations in the activity or composition of gut microbiota are linked to the pathogenesis of obesity and related disorders in humans [8,13,14].Currently, dietary interventions in some clinical studies have successfully been linked to beneficial phenotypic changes through manipulation of the gut microbiota [15].Metagenomic high-throughput sequencing has revealed the effects of polysaccharides on the gut microbiota and established the relationships among intestinal flora, obesity and diabetes.High-throughput sequencing technology has many advantages in research on microbial structure.The application of high-throughput sequencing allows for more comprehensive study of gut microbiota composition [16].The gut microbiota can influence the calories absorbed by the body,thus, affecting body weight.Human enzymes can convert starches into simple sugars, but cannot digest many dietary polysaccharides.However, microbial enzymes can turn those polysaccharides into digestible sources of energy, such as monosaccharides and SCFAs.SCFAs are bioactive metabolites produced by the microbiota, include acetic, propionic, isobutyric, butyric, isovaleric and pentanoic acid.Many human studies and numerous animal studies have shown beneficial roles of these metabolites in the prevention and treatment of obesity and its comorbidities.Among them, acetic acid can be oxidized by the brain, heart and peripheral tissues, and simultaneously promote β-cell insulin secretion.Propionic acid can affect liver and cholesterol metabolism.Butyric acid has substantial benefits for the human body, specifically, it can provide energy to colonic epithelial cells, regulate the growth and apoptosis of epithelial cells and immune cells, inhibit colitis and colon cancer, modulate oxidative stress and affect the composition of the mucus layer [17].Propionic acid has been reported to decrease fatty acid levels in the liver and serum, reduce food intake in humans, exert immunosuppressive effects in the body and improve tissue insulin sensitivity [18].The anti-inflammatory and anti-cancer effects ofn-butyrate have attracted widespread attention [19].In addition, butyric acid can provide energy to intestinal epithelial cells, maintain the integrity of the intestinal barrier and enhance intestinal immunity [20].Additionally, SCFAs also play an important role in maintaining the water and electrolyte balance, resisting pathogenic microorganisms, regulating intestinal flora balance, improving intestinal function, and regulating immunity,anti-inflammatory, anti-tumor effects and gene expression.

Soy hulls, which are the seed coat of soybeans (approximately 8%), are one of the major by-products released during the initial cracking process in soybean oil production.However, large amounts of soy hulls are underused [21].Moreover, soy hull polysaccharide(SHP) from soy hulls has been isolated by ammonium oxalate assisted with microwave treatment and its molecular weight, polysaccharide composition, fluidity and structural characteristics were studied in our team’s previous work [22].Hence, the present study used a high-fathigh-sucrose diet fed rats and aimed at investigating hypoglycemic and hypolipidemic effects of SHP, evaluating its functional effects and exploring its influence on obesityviamicrobial components, with potential therapeutic implications for the gut microbiota.

2.Materials and methods

2.1 Materials

Soy hull was acquired from Shandong Yuwang Co., Ltd;triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C)assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); blood glucose meters were purchased from Sinocare Biosensor Co., Ltd.(Changsha, China); high-fat-high-sucrose diet was purchased from Beijing Keao Xieli Feed Co., Ltd.(Beijing,China); all other chemicals of this experiment were analytical grade or high-performance liquid chromatography (HPLC) grade.

2.2 Microwave-assisted extraction

According to previous study [23], SHP was extracted by ammonium oxalate assisted in microwave radiation.In brief, dry soy hull was ground into powder (to pass a 60 mesh sieve).Add 1% ethanol solution at a ratio of soy hull powder to water 1:10, stirred for 30 min at room temperature, passed through 200 mesh filter cloth, and dried in the air dry oven at 65 °C.Soy hull residue (50 ×g)was dispersed in 1 L water at 85 °C which containing 0.6% ammonium oxalate, then maintained at 85 °C for 35 min in the microwave extractor (Qihuabo, LD-4, China).The dispersion was passed through 200 mesh filter cloth, and centrifuged at 4 500 ×gfor 10 min (Backman, Avanti J-25, USA).After that, the supernatant was concentrated by a vacuum rotary evaporator (Shanghai Yarong,RE-3000A, China), then turning pH of filtration to 4.0.Next, adding double ethanol (95%,V/V) to concentrated solution with slow mixing.The precipitate was separated and dried in the air dry oven at 65 °C and the SHP was obtained.

2.3 Experimental animals and diets

Male SD rats (body mass (200 ± 20) g) were purchased from the Experimental Animal Center of Jinzhou Medical University.All animals were kept in the specific pathogen free (SPF) animal laboratory where ambient environmental conditions (12:12 h lightdark cycle, temperature (24 ± 1) °C, (60 ± 10)% relative humidity)were maintained, and free access to diet and water every day until the end of the study.After a week of adaptation, 9 rats were fed the normal diets as the normal control (NC) group.Another 9 rats as the model control group (DC) were fed high-fat-high-sucrose diet(15.6% protein, 31.1% adipose, 53.3% carbohydrate) for 4 weeks.At the same time, the other 27 rats were fed high dose (HS, 800 mg/kg,9 rats), medium dose (MS, 400 mg/kg, 9 rats) and low dose (LS,100 mg/kg, 9 rats) of SHP, respectively.During the experiment,collected blood samples from the tail vein and gut feces every week.Each treatment sustained for 4 weeks, and all rats had free access to food, water and clean cages.All animal experiments were approved by the Animal Care and Use Committee of Jinzhou Medical University (Animal experiment permission number: 2019015).

2.4 Growth parameters

During the experiment, body mass, daily water intake and food intake of all rats were recorded for 5 times at 0, 1, 2, 3, and 4 weeks,respectively.

2.5 Fasting blood glucose (FBG)

FBG of all rats was measured for 5 times at 0, 1, 2, 3, and 4 weeks, respectively.Blood samples were collected from the tail vein after overnight fasting and FBG levels were measured using blood glucose meters (Sinocare Biosensor, China).

2.6 Serum parameters analysis

Blood samples were collected from the tail vein and centrifuged(2 000 ×g, 1 min, 4 °C) to obtain the serum.Then serum index was analyzed through TG and HDL-C assay kit (Nanjing Jiancheng,China) based on manufacturer’s protocol.

2.7 Gut bacterial DNA extraction

Rat gut feces were collected and stored at –80 °C (SA NYO,MDF-382E(CN), Japan).The DNA of original gut microbiota was extracted by QIAamp DNA Stool Mini Kit from QIAGEN(Dusseldorf, Germany).All appropriate controls and standards as specified by manufacturer’s kit were used.In brief, the gut feces were suspended in inhibit EX buffer, and then the DNA was combined with QIAamp membrane for split.Residual inhibitors and contaminants were removed in the washing step and the purified complete DNA were eluted from the QIAamp spin column.

2.8 16S rDNA gene sequencing

Genomic DNA was accurately quantified using the Qubit 3.0 DNA Assay Kit (Q10212, Life) to determine the amount of DNA that should be added to the polymerase chain reaction (PCR).The primers used in PCR system (T100TM Thermal Cyeler,BIO-RAD) have been fused to the V3–V4 universal primers of the Miseq sequencing platform.The V3–V4 region of the 16S rDNA gene fragments was amplified using a set of primer pairs(341F: 5’-CCCTACACGACGCTCTTCCGATCTG (barcode)CCTACGGGNGGCWGCAG-3’; 805R: 5’-GACTGGAGTTCCTT GGCACCCGAGAATTCCAGACTACHVGGGTATCTAATCC-3’)fusion of the Miseq sequencing platform.The PCR mixture was prepared as follows: 10–20 ng of the purified DNA, 15 μL of 2 ×Taqmaster-Mix (P111-03, Vazyme), 1 μL of Bar-PCR primer F (10 μmol/L),1 μL of primer R (10 μmol/L), and double-distilled water to a final volume of 30 μL.PCR cycling conditions consisted of an initial denaturation of 3 min at 94 °C; 5 cycles of 30 s at 94 °C, 20 s at 45 °C, 30 s at 65 °C; 20 cycles of 20 s at 94 °C, 20 s at 55 °C,30 s at 72 °C; and 5 min at 72 °C.Then perform a second round of amplification, introducing Illumina bridge PCR-compatible primers.This PCR system was prepared as follows: 20 ng of the PCR products, 15 μL of 2 ×Taqmaster-Mix, 1 μL of Bar-PCR primer F (10 μmol/L),1 μL of Primer R (10 μmol/L), and double-distilled water to a final volume of 30 μL with PCR cycling conditions consisted of an initial denaturation of 3 min at 95 °C; 5 cycles of 20 s at 94 °C, 20 s at 55 °C, 30 s at 72 °C; and 5 min at 72 °C.PCR products were quantified 400 bp, pooled in equimolar ratios, 0.6 times magnetic beads (Agencourt AMPure XP), and then used for Illumina MiSeq sequencing.Statistical analysis of biological information is typically performed at operational taxonomic units(OTUs) at 97% similar levels.

2.9 SCFA analysis

Take 0.1250 g of acetic, propionic, isobutyric, butyric, isovaleric,and pentanoic acid, andn-valeric acid standard and added to a 100 mL volumetric flask, and diethyl ether was added to make a volume as a stock solution.Take 1, 0.75, 0.5 and 0.25 mL stock solution and 100 mL volumetric flask, respectively, and make up to volume with diethyl ether.After configuration, analyze the injection and draw a standard curve.

After the dry faeces samples were shaken, the sample was added with 2 mL of water (1:3 aqueous solution of phosphoric acid), vortexed and homogenized for 2 min, extracted with 2 mL of diethyl ether for 10 min, centrifuged at 4 000 ×gfor 20 min (low temperature treatment, placed in an ice water bath for centrifugation).After centrifugation, the ether phase was taken out, and then extracted with 2 mL of diethyl ether for 10 min at 4 000 ×g.Finally, the ether phase was taken out again, and the two extracts were combined and volatilized to a volume of 2 mL for injection analysis.Contents of SCFAs were analyzed by gas chromatography-mass spectrometry(GC-MS) (Thermo Fisher Scientific Corp, GCMS ISQ LT, USA).GC conditions: TG WAX 30 m × 0.25 mm × 0.25 μm, carrier gas was He,velocity of flow was 1.0 mL/min, the inlet temperature was 240 °C,the programmed temperature was maintained at 100 °C for 5 min,then the temperature was raised to 150 °C at 5 °C/min, and then raised to 240 °C at 30 °C/min for 30 min.MS conditions: GC-MS interface temperature was 250 °C, ion source temperature was 200 °C,ionization mode was electron ionization, electron energy was 70 eV.

2.10 Induction of type 2 diabetes mellitus (T2DM)

After 5 weeks of dietary manipulation, all rats were fasted for 12 h and then injected intraperitoneally twice with 40 mg/kg STZ,dissolved in 0.1 mol/L citric acid/sodium citrate buffer (pH 4.5),while the NC group rats were injected intraperitoneally with isometric physiological saline.After feeding for 3 days, blood glucose levels of the rats were evaluated by taking a drop of blood from the tip of the tail with blood glucose meters.Rats with FBG concentrations exceeded 11.1 mmol/L were identified as the T2DM.

2.11 Statistical analysis

All data were expressed as a mean ± standard deviation(SD).The statistical analyses were performed using the SPSS 19.0.Differences between experimental groups were known as statistically significant ifP< 0.05 by one-way analysis of variance of Duncan’s multiple range tests.

3.Results and Discussion

3.1 Growth parameters

In the present study, we investigated the role of SHP in regulating rate of body mass growth, food intake and water intake in a model based on SHP gavage in high-fat-high-sucrose diet fed rats.Changes in rate of body mass growth, water intake and food intake among experimental groups are shown in Figs.1a–1c.All rats showed increased body mass during the obesity induction period.Compared with that in the DC group, the body mass of rats declined after SHP treatment.However, the rate of body mass decline was slightly improved in the groups with gavage of SHP.Among them, the rate of weight growth in the MS group was less than other groups.However,diet intake and water intake showed meaningful trends, in which the DC group was highest, the NC group was lowest, and the groups with SHP gavage increased gradually, followed by HS, MS and LS group.

The present results are similar to those from other researchers reporting that polysaccharide affects body mass, diet intake and water intake.Pan et al.[6]and Nie et al.[7]have found that corn silk polysaccharide and polysaccharide fromPlantago asiaticaL.alter growth parameter index effects in high-fat diet and STZ induced mouse or rat models of type 2 diabetes mellitus.Chang et al.[24]have also found that a water extract ofGanoderma lucidummycelium(WEGL) decreases body mass, inflammation and insulin resistance in mice fed a high-fat diet.

3.2 Serum parameters

A high-fat-high-sucrose diet causes high FBG and dyslipidemia,which is usually characterized by high levels of TC and TG and low levels of HDL-C.FBG and dyslipidemia are common in stages of diabetes and prediabetes [25].As shown in Fig.1d, the FBG levels in the NC group were relatively stable, but those in the DC group rose.The DC groups showed differences in the 4th week, as compared with the NC group.The FBG levels in the SHP group were lower than those in the DC group.Moreover, the FBG levels in MS and HS groups gradually decreased from the 2nd week, and the FBG levels in MS and HS groups were 12.38% and 8.36% lower than those in the DC group in the 4th week (P< 0.05).The reason for these results might be that SHP has viscous and gel characteristics, which might have slowed the diffusion of glucose and decreased the fasting blood glucose level [26,27].Ou et al.[28]have reported that polysaccharides can decrease the rate of glucose diffusion because of their viscosity.Thus, the results indicated that the SHP intervention lowered FBG.

Fig.1 Effect of SHP on the rate of body mass growth (a), diet intake (b), water intake (c) serum FBG levels (d), serum TG levels (e), and the ratio of HDL-C (f)in each group of rats with feeding high-fat-high-sucrose diet.Results are shown as means ± SEM, n = 9 per group.The different lowercase letter means significant difference (P < 0.05).

As shown in Figs.1e and 1f, compared with that in the NC group,the serum TG concentration in the MS group significantly increased from 1st to 3rd week (P< 0.05).However, the serum TG level of the MS group was lower than that of the NC group in the 4th week.Compared with that in the DC group, the TG concentration in the MS gavage group increased first and then decreased, and the TG concentration in the MS group returned to the levels in the NC group in the 4th week.The results indicated that the MS group showed the best effect, followed by the HS group.MS significantly increased the HDL-C concentration in serum, and the HDL-C level in the MS group was higher than that in the NC group (P< 0.05).The results were almost consistent with the data reported by Lin et al.[5], in whichSiraitia grosvenoriipolysaccharide showed clear glucose-lowering effects in hyperglycemia, amelioration of lipid metabolism and restoration of blood lipid levels in rabbits fed high-fat-high-sucrose diet.Furthermore, Nie et al.[7]have found that polysaccharide fromP.asiaticaL.causes significant decreases in the concentrations of blood glucose and TG, and significant increases in the levels of HDL-C, as compared with those in diabetic rats after 4 weeks’treatment.Notably, the polysaccharides in the middle-dose group had the best effect.The reason for this may be that the macronutrients (i.e.,carbohydrates, fats and proteins) are not fully interconvertible at the metabolic level, thus affecting systemic metabolism [29].

3.3 SHP modulates the structure of the gut microbiota

The effects of SHP on the gut microbiota in rats fed a highfat-high-sucrose diet was demonstrated by using high-throughput sequencing spanning the 16S rDNA V3–V4 hypervariable regions.To estimate the gut microbial community richness and diversity, we calculated the Ace, Chao1, Shannon and Simpson indices.As shown in Table 1, the OTUs and Ace and Chao1 indices of the MS group approached those of the NC group.In addition, the Simpson and Shannon indices of the MS groups were also very close to those of the NC group.These results indicated that the intake of SHP recobered the gut microbiota reach the normal diversity levels.

Table 1Diversity of gut microbiota in SHP-treated rats with feeding high-fat-highsucrose diet.

The beneficial Firmicutes and the Bacteroidetes bacteria are dominant in the human gut microbiota [30].As previously shown,we found that SHP significantly changed the gut microbiota composition.We found that the abundance of Bacteroidetes increased and abundance of Firmicutes and Actinobacteria decreased at the phylum level in MS group, compared the gut microbiota composition of the DC group rats (Fig.2a).To determine the composition changes in the gut microbiota, we compared the relative abundance of the predominant taxa identified from the 4 groups (Figs.2a and 2b).There are significant differences in the composition of the gut microbiota at all taxonomic levels among the groups.Current research from the Mayo Medical Center indicates that body mass loss efficiently depends on the bacterial combinations in the intestines, and the level of the genusPhascolarctobacteriumis the key.Further, at the genus level, SHP treatment decreased the abundance of bacteria,includingBlautia,Prevotella,ClostridiumXIVa,Romboutsia,BarnesiellaandLachnospiracea incertae sedis, and up-regulated the population of bacteria includingBacteroides,Alloprevotella,Pseudoflavonifractor,Flavonifractor,Phascolarctobacterium,FalsiporphyromonasandAkkermansia, relative to that in DC group,while restoring the abundance of bacteria includingClostridiumIV,Intestinimonas,RuminococcusandHelicobacterto normal levels (Fig.2b).We believe that SHP, like other prebiotics such as xylooligosaccharides, can provide a better growing environment for Bacteroidetes rather than Firmicutes [31].Previous studies have found increased proportions ofRuminococcusin obesity or metabolic syndrome, and we found the same result in this study [32].Studies have also found that chitosan oligosaccharide treatment inhibits the reduction of occludin and relieves gut dysbiosis in diabetic mice by promotingAkkermansiaand suppressingHelicobacter[2].In our study, we found the same result, SHP promotedAkkermansiaand restoredHelicobacterto normal levels in the MS group.Some studies have reported thatPrevotellais beneficial [33-35], whereas others have reported it to be disadvantageous in obesity [36,37].In our study,Prevotellawas lower in the high-fat diet group than the NC group, and was significantly decreased by SHP.Previous studies have found that the intake of polysaccharides could reduce the risk factors of obesity and hyperlipidemia by regulating gut microbiota [38,39].High fat and high sugar diet without dietary fiber induced imbalance of intestinal flora [40].Gut microbiota degraded intestinal mucus [40]to increase the absorption of lipids and glucose, so the TG increased and FBG decreased.After the addition of appropriate amount of SHP, the gut microbiota degraded SHP to maintain the balance of flora [41].The integrated mucus barrier and SHP with high viscosity and hydrogel characteristics prevented the rapid absorption of lipids and glucose [42], so the FBG and TG values decreased and the FBG values increased.

Fig.2 Response of the gut microbiota to SHP treatment at the phylum (a) and genus (b) level.PCA of gut microbiota at the OUT level (c).Heatmaps for principal coordinate analysis of gut microbiota based on weighted UniFrac (d).

Fig.2(Continued)

To assess beta diversity, we performed principal component analysis (PCA) and weighted UniFrac distance-based principal coordinate analysis (PCoA) between the gut samples.The PCA score plot illustrated both the similarity and variance among the NC,DC, HS, MS, LS groups, and the first three components explained 91% of the total variance (53%, 29% and 9% for PC1, PC2 and PC3, respectively).PCA was performed to distinctly classify the microorganism compositions among different treatment groups.As shown in Fig.2c, a statistically significant separation appeared among the 5 groups.The first two axes explained 62% of the total variation for different groups.There was no overlap among the 5 groups of gut bacteria.Moreover, the similarity among the samples was visually seen in terms of the degree of aggregation, thus indicating that the carbon source affected the gut microbial community.In addition,PCoA based on weighted UniFrac (Fig.2d) was carried out to illustrate how SHP altered the gut microbiota composition at the OTU level.Notably, both PCA and PCoA graphs demonstrated that SHP played a role in regulating the structure of the gut microbiota.Further analysis confirmed the effects of various carbon sources on the gut microbiota with respect to relative abundance and composition.A heatmap was generated, using colors to reflect information on abundance and function, and intuitively display the functional abundance with a defined color depth (Fig.3).The hierarchical clustering heatmap of the top 27 ranked OTUs revealed that the DC group rats had a microbial profile distinct from that of NC group, and supplementation with SHP changed the microbial composition.The composition was not similar to that of the NC group but was close in some aspects.According to Ding et al.[19],Lycium barbarumpolysaccharides regulate the structure of the gut microbiota; Orlistat also improves obesity and metabolic alterations in high-fat diet fed mice and modulates the composition of the gut microbiota.

Fig.3 Hierarchical clustering function abundance heatmap of the gut microbiota of rats.

3.4 SCFAs

There is a potential association between the gut microbiota and the effects of obesity, diabetes and other diseases, owing to changes in SCFAs and other metabolites.SCFAs, dominated by acetate,propionates and butyrate, play important roles in energy metabolism,adipogenesis regulation and glucose homeostasis [7].The content of acetic, propionic,n-butyric,i-butyric,n-valeric andi-valeric acids is shown in Table 2.The polysaccharide treated groups contained significantly higher total SCFA contents than the DC group, except LS group (P< 0.05).Rats gavaged with SHP showed a recovery trend.Clearly, acetic, propionic,n-valeric andi-valeric acids were the main fermentation products in each model group, although their amounts varied among groups.Notably, the content of the acetate,n-butyric,n-valeric andi-valeric acids in the MS group was significantly higher than that in normal rats (P< 0.05).The MS group showed significantly improved production of SCFA.Our results indicated that the total SCFA content in the MS group was highest, and acetic, propionic, andi-butyric acids were the dominant components.The total SCFA content in the MS group approached that in the NC group and was approximately more than 2-fold higher than that in the DC group.

Table 2SCFA productions of rats with feeding high-fat-high-sucrose diet with/without SHP treatment.

Similar findings have indicated that the final metabolic products of marine polysaccharides are usually SCFA, which can influence gut microbiota ecology by altering the percentage of certain microorganisms and increase SCFA production by stimulating the growth of beneficial microbes (Ruminococcaceae, Bacteroidetes andLactobacillus) [43].Moreover, in rats administeredPolygonatum odoratumpolysaccharides and fed a high fat diet, we observed that isobutyric acid, butyric acid and valeric acid were upregulated,resulting in changes inRuminococcusandPrevotellaabundance[44].In addition, the SCFA altered gut microbiota can regulate blood glucose, lipid metabolism levels and other symptoms.

3.5 SHP decreases the rate of diabetes mellitus formation in rat models

After 3 days of injection of treatments or the same dose of STZ, as shown in Fig.4, the blood glucose levels of the rats were measured and found to be significantly lower in the SHP treatment groups than the DC group (P< 0.05); the MS group showed the better result.These findings indicate that SHP inhibits the formation of diabetes.Long-term intake of SHP may change the gut microbiota structure and metabolites, and SHP may potentially protect islet β-cells from destruction and prevent islet failure.Gowd et al.[45]have found that anthocyanins can lower blood glucose levels by protecting β-cells, improving insulin resistance, increasing insulin secretion, improving liver function and inhibiting carbohydrate hydrolyzing enzymes.

Fig.4 Effect of SHP on diabetes mellitus model amount of rats.

4.Conclusion

This study shows that SHP has antihyperglycemic activity and antihyperlipidemic effects, and regulates the gut microbiota.Administration of SHP significantly decreased body fat content, and improved polydipsia and the level of FBG.SHP also decreased the levels of serum lipids and increased fecal SCFA content.In addition,the structure of the gut microbiota was significantly altered, and the abundance ofBacteroidesin diabetic rats was increased by SHP treatment, an effect that may be associated with lipid metabolism and an increase in SCFA.This report lays a foundation for mechanistic research on anti-diabetic activity and protection against diabetes development.

Conflict of Interest

None.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No.31901680 and 31972031).