Effects of Natural Flavonoid Isoorientin on Growth Performance and

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Food Safety and Toxicology

Effects of Natural Flavonoid-Isoorientin on Growth Performance and Gut Microbiota of Mice Li Yuan, Xueyi Li, Shengyuan He, chunxia gao, Chengtao Wang, and Yuyu Shao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03568 • Publication Date (Web): 30 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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Journal of Agricultural and Food Chemistry

Grown performance and health benefit

Gut microbiota

ALT AST

Colon inflammation

SOD GSH-Px Pathogenic genera

MDA H2O2 BUN UA CRE ACS Paragon Plus Environment


Effects of Natural Flavonoid-Isoorientin on Growth Performance and Gut

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Microbiota of Mice

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Li Yuan*†, Xueyi Li†, Shengyuan He†, Chunxia Gao†, Chengtao Wang*‡,

5

Yuyu Shao*†

6 †

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University, Shaanxi, Xi’an, 710119, People’s Republic of China

8 9

College of Food Engineering and Nutritional Science, Shaanxi Normal

‡

Beijing Engineering and Technology Research Center of Food Additives, Beijing

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Technology & Business University (BTBU), Beijing, 100048, People’s Republic of

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China

12 13

Corresponding authors:

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* E-mail: [email protected]

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17 18

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Abbreviations: ISO, isoorientin; Cont, control; ALT, alanine aminotransferase; AST,

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aspartate aminotransferase; BUN, blood urea nitrogen; UA, uric acid; CRE, creatinine;

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T-SOD, total superoxide dismutase; GSH-Px, glutathione peroxidase; H2O2, hydrogen

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peroxide; MDA, malondialdehyde; LPS, lipopolysaccharide.

ACS Paragon Plus Environment

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ABSTRACT

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Isoorientin (ISO) is a natural flavonoid which is a 6-C-glucoside of luteolin and

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has been demonstrated to possess the multiple biological properties. In this study, the

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effects of ISO on the growth performance and gut microbiota of BALB/c mice were

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investigated. The results showed that ISO could promote food intake and body weight

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gain, increase the digestibility of crude proteins and utilization of the gross energy,

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and strengthen antioxidant capacity of mice. We also demonstrated it has no

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side-effects on the hepatic and renal function. Moreover, ISO inhibited growth of the

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most bacteria in gut microbiota, especially the pathogenic genera of Alistipes,

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Helicobacter and Oscillibacter which could lead to inflammation. Metabolisms of

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epithelial cell signaling in Helicobacter pylori infection, Lipopolysaccharide (LPS)

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biosynthesis, and LPS biosynthesis proteins in gut microbiota of control group were

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more abundant than that in ISO group, while lipid metabolism and Vitamin B6

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metabolism were enriched in ISO group. We found the changes in enrichments of

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metabolic pathways of the gut microbiota along with the ISO application were

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positively correlated with the anti-oxidation, anti-inflammation, and antibiosis. This

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work provided a fundamental basis for the future development of ISO-functional

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foods used for resistance to oxidation, inflammation, and pathogens.

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KEYWORDS: isoorientin, growth performance, gut microbiota, hepatic and renal

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function



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INTRODUCTION

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Isoorientin (3',4',5,7-tetrahydroxy-6-C-glucopyranosyl flavone, ISO, Fig. 1)

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widely exists in foods and the edible plants, including buckwheat,1 corn silks,2

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passion fruit (Passiflora caerulea L.),3 Pueraria tuberosa4 and Patrinia5. ISO

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exhibits the significant anti-oxidant, anti-nociceptive and anti-inﬂammatory activities

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on mice.6-8 The hepatoprotective effect of ISO against CCl4-induced oxidative

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damages in rats9 and hydrogen peroxide-induced oxidation stress in HL-7702 liver

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cells10 were reported. Our previous study also found that ISO inhibited the release of

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inflammatory cytokine (i.e., TNF-α, IL-1, IL-6) in high-fructose-treated mice, and

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have improved immunity of mice.11

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Many studies have shown that the gut microbiota plays a significant role on the

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maintenance of health. On the one hand, gut microbiota helps to improve the

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absorption of nutrients and energy.12 On the other hand, gut microorganisms are

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involved in regulating a number of human diseases, such as obesity, diabetes,

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metabolic syndrome, cardiovascular diseases, colorectal cancer, allergy, asthma,

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neurological diseases, depression and anxiety.13-16

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It has been confirmed that 90-95% of dietary polyphenols can reach the colon

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and modulate the colonic microbial compositions and functions, which are not

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absorbed in the small intestine.17 For example, pterostilbene had the antiobesity

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effects by improving insulin sensitivity, modifying gut microbiota, decreasing

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Firmicutes and increasing Verrucomicrobia.18 Carvacrol could potentially be used to

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control gut dysbiosis and reduce C. difficile infection through increasing Firmicutes

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and reducing the proportion of Proteobacteria.19 In addition, trans-resveratrol and

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quercetin,20 EGCG,21 ellagitannins,22 wine polyphenols23 were all reported to impact

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the compositions and functions of gut microbiota.


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Nevertheless, the effects of ISO on the growth performance and gut microbiota

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of mice remain unknown. The objectives of the present study are to investigate the

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effects of ISO on body weight, apparent digestibility of nutrients, hepatic and renal

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functions, antioxidant capacity and gut microbiota in BALB/c mice.

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MATERIAL AND METHODS

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Chemicals and Reagents

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Isoorientin (purity ≥ 98 %) was purchased from Forever Biotechnology, Ltd.

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(Shanghai, China). The total superoxide dismutase (T-SOD), glutathione peroxidase

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(GSH-Px), hydrogen peroxide (H2O2) and malondialdehyde (MDA) assay kits were

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purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).

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The BCA (Bicinchoninic Acid) Protein Quantification Kit was obtained from Thermo

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Scientific (Rockford, IL, USA). All other chemicals were analytical grade and

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produced in China.

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Animals, Diets and Experimental Design

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Specific-pathogen free male five-week-old BALB/c mice were obtained from the

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Experimental Animal Center of Xi’an Jiaotong University (Xi’an, China). All mice

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were housed in particulate air-filtered, standard temperature (25 ± 2°C) and

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humidity-controlled (60 ± 5 %) room with 12:12h light-dark cycle, and were allowed

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free access to autoclaved chow and water. After a week adjustable acclimation, mice

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were randomly divided into two groups, and 9 mice per group (n=9). In the

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ISO-treated

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carboxymethylcellulose aqueous solution and applied by intubation gavaging at 15

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mg/kg·BW (10 mL/kg of BW) once daily for 30 consecutive days. For the control

group

(ISO),

ISO

was

suspended


in

the

1%

sodium


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group (Cont), mice were given the same volume of vehicle via the intubation

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gavaging for 30 consecutive days. The body weight (BW) of all the groups of mice

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was measured once a week, and the average food consumption of each mouse was

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calculated. After 2 h of the last administration, mice were allowed free access to water

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for 12 h except for food, and then were sacrificed to contribute blood and organs,

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including liver and colon. The blood samples were centrifuged at 1200×g for 20 min

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and were stored at 4 °C, and the liver samples were frozen at -80°C. The colon of

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each mouse was immediately harvested and washed with phosphate-buffered saline,

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and 1.5 cm sections of the proximal and distal colon were fixed in 4%

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paraformaldehyde solution for histological analysis. The fecal samples were collected

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and frozen at -80 °C until DNA extraction. Animal protocols were approved by the

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Committee for Research and Ethical Issues of Shaanxi Normal University (Xi’an,

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China) and adhered to the guidelines of the Animal Ethical Committee.The animals

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were treated humanely and with regard for the alleviation of suffering.

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Digestibility Determination

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Fecal samples from each mouse and fodder samples were collected at day 0 and

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30 (14 hours after the final administration of ISO), and were immediately stored at

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-80 °C. Samples were oven-dried at 60 °C for 24h before the determination, and then

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were crushed and sieved with 40-mesh size. Fodder and fecal samples were analyzed

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for the apparent total tract digestibility of dry matter (method 930.15) and protein

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(method 984.13) using the AOAC (Association of Official Analytical Chemists)

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method.24 Gross energy was determined by measuring the heat of combustion in the


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samples, using a bomb calorimeter (ZDHW-8, Haotian Electric Co., Ltd, Guangdong,

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Zhongshan, China). Acid insoluble ash (AIA) contents of fodder and feces were

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determined using the method of Fan et al.25 The apparent tract digestibility of protein

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and gross energy was calculated using the following formula:

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Apparent tract digestibility (%) =100 – [(%AIA in fodder / % AIA in feces) ×

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(%nutrient in feces / %nutrient in fodder)] ×100

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Estimation of Serum Biochemical Indices of Mice

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The levels of total protein (TP), alanine aminotransferase (ALT), aspartate

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aminotransferase (AST), blood urea nitrogen (BUN), uric acid (UA) and creatinine

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(CRE) in blood were tested by the Affiliated Hospital of Shaanxi Normal University.

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Protein concentrations in the serum samples were measured using BCA assay kit.

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Estimation of Antioxidant Properties of the Serum

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The proteins concentrations in serum samples were measured using BCA assay

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kit. The levels of T-SOD, GSH-Px, H2O2 and MDA in the serum were determined

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using commercial kits according to the manufacturer's instructions.

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Metagenomic DNA Extraction and Amplicon Sequencing

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Metagenomic DNA of gut microbiota in fecal samples was extracted using the

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QIAamp DNA Stool Mini Kit (ID: 51504, QIAGEN China Co. Ltd., Shanghai,

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China). The metagenomic DNA was diluted to 1 ng/µL as the template for PCR using

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the primer 338F and 806R to amplify the V3-V4 regions of 16S rRNA gene.

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High-throughput sequencing of the amplicons was performed by Novogene

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Corporation using the Illumina HiSeq 2500 System.



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Bioinformatics Analysis

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The bioinformatics analysis was fully described in previous studies.26 Briefly, the

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sequencing data analysis including taxonomic annotation, alpha and beta diversity

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was conducted using Qiime pipeline. Statistical analysis and plotting were performed

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using R software. The significant difference was confirmed when p-value < 0.05.

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Colon Histopathology Observation

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Histopathology of the colon was studied using H&E staining. The colon tissue

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samples were fixed with 10% neutral formalin solution, embedded in paraffin, cut into

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slices (5-6µm thickness), and then stained with H&E dye. Next, the slides were

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examined under a light microscope (Olympus Optical Co., Ltd., Tokyo, Japan) for

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observations and photography.

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Statistical Analysis for Biochemical Indices

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All results of biochemical indices were presented as the means ± standard

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deviation (SD), and were analysed using the one-way factorial analysis of variance

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(ANOVA) and Duncan’s post-hoc test (SPSS v16.0).

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RESULTS

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Effects of ISO on Body Weight

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Compared with the Cont group, there was no significant difference of the initial

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body weight (BW) in ISO group (Tab. 1). However, ISO supplementation effectively

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(p

Effects of Natural Flavonoid Isoorientin on Growth Performance and

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