Effect of white wheat bread and white wheat bread added with bioactive compounds on hypercholesterolemic and steatotic mice fed a high fat diet

Luisa Pozzoa,*, Laura Puccia, Guglielmo Buonamicib, Lucia Giorgettia, Maristella Maltintic, Vincenzo Longoa

* Corrispondence to: IBBA-CNR, Via Moruzzi 1, 56124 Pisa, Italy. E-mail: [email protected] a Istituto di Biotecnologia e Biotecnologia Agraria (IBBA), CNR, Pisa, Italy b Agriproject, Calci, Pisa, Italy c Fondazione G. Monasterio CNR-Regione Toscana, Pisa, Italy

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/jsfa.6972

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Abstract BACKGROUND: The effects of white wheat bread and white wheat bread added with a mixture (Cyclantera pedata, Glycine max, Monascus-fermented red mold rice, Cynaria scolymus and Medicago sativa) were examined on hypercholesterolemic and steatotic mice, divided into 4 groups: control diet (CTR), high fat diet (HFD), high fat diet with white wheat bread added with 1.5 g kg-1 of mixture (HFD+AB) and high fat diet with white wheat bread (HFD+B). RESULTS: The total serum cholesterol in the HFD+AB and HFD+B groups and the hepatic triglycerides in the HFD+AB group decreased compared to the HFD group. Liver histology confirmed a lower lipid drops accumulation in HFD+AB group compared to HFD and HFD+B groups. HFD+AB caused a 7.0- fold increase and a 3.5- fold reduction in the CYP7A1 and SREBP-1c gene expression, respectively, compared to HFD group. Moreover, HFD+B group showed a 2.2-, 8.4- and 1.5- fold increase in the HMG CoA reductase, CYP7A1 and LDLr gene expression compared to HFD group. CONCLUSIONS: The white wheat bread and added white wheat bread induced a cholesterol reduction by increasing CYP7A1. Moreover, the added white wheat bread improved steatosis decreasing SREBP-1c gene expression.

Keywords: white wheat bread, added white wheat bread, hypercholesterolemia, steatosis, hepatic mRNA

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Introduction Nonalcoholic fatty liver disease (NAFLD) comprises a range of pathologies from fat in the liver (simple steatosis), necroinflammatory disorder of non-alcoholic steatohepatitis (NASH) to cirrhosis and liver cancer. Overnutrition and underactivity, insulin resistance and genetic factor are the major risk factors which can cause

hepatic steatosis in view of the fact that

NAFLD/NASH is related with diabetes mellitus1, hypertension2, and obesity3. An excessive and inappropriate dietary-fat intake, combined with peripheral insulin resistance, continued triglycerides hydrolysis via lipoprotein lipase, and other genetic alterations in the key lipid metabolic pathways, results in increased blood free-fatty-acid (FFA) concentration and hepatic de novo lipogenesis, leading to increased triglycerides concentration in the liver .4,5 SREBP-1c (Sterol Regulatory Element Binding Protein-1c) plays a key role in the lipid metabolism and, in particular, regulates enzymes involved in fatty acids and triglycerides biosynthesis.6 The process of cholesterol synthesis and clearance mainly involved: 3-hydroxy-3methylglutaryl CoA (HMG CoA) reductase, a rate-limiting enzyme responsible of cholesterol biosybthesis, low density lipoprotein receptor (LDLr), responsible of plasma cholesterol uptake,7 and the rate-limiting enzyme cholesterol 7α-hydroxylase (CYP7A1), by which the liver eliminates cholesterol excess from the body, either by direct secretion into the bile or after its conversion into bile acids8. Recently there has been a growing interest to evaluate plant constituents on pharmacological basis to identify natural, more effective and safe agents able to protect against steatosis and dyslipidemia. Several studies already report positive effects of many plants in reducing metabolic disorders giving evidences that herbal combination can be particularly effective.9 In this perspective the present study aims to investigate the effects of a new herbal combination composed by five bioactive compounds (Cyclantera pedata, Glycine max, Monascus-fermented red mold rice, Cynaria scolymus and Medicago sativa). All the five plants are already known for their beneficial effects on human and animal health when separately administered since all of them can positively influence the lipids and glucidic metabolism. Caigua (Cyclantera pedata) is a slender tropical fruit, indigenous to South America known to contain triterpenoid saponins and

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flavone glycosides and which can have antioxidant effects.5,10,11 Moreover, it has been previously reported that caigua reduces cholesterol levels in humans.12 Soybean (Glycine max) contains high quality proteins, dietary fiber and phytochemicals, all of which exert beneficial effects on human health.13,14 The effect of soy proteins and isoflavones on lipid metabolism involved gene expression of cholesterol and fatty acid synthesis,15,16 reducing the concentrations of serum lipids.17,18 Monascus-fermented rice, known as Red Mold Rice, is a Chinese dietary supplement and has gained popularity because of its properties as a natural statin. Recent studies demonstrated that RMR was effective against hypercholesterolemia and hypertriglyceridemia, obesity-related inflammation, insulin resistance and nonalcoholic fatty liver disease19-22. It has been demonstrated that monacolin K, a statin synthesized during Monascus purpureus fermentation,23

lowers

cholesterol by inhibiting HMG–CoA reductase.24 Artichoke (Cynara scolymus) is a perennial thistle originating in southern Europe around the Mediterranean.25 The major bioactive components contained in artichoke leaf extract are hydroxycinnamic acids, such as chlorogenic acid and cynarin, and flavonoids such as luteolinglycoside.26 Hydroxycinnamic acids and flavonoids possess cholesterol-reducing effect on hypercholesterolemic subjects and antioxidant properties via reduction in de novo cholesterol synthesis and free radical scavenging activity.27-29 Alfalfa (Medicago sativa) has a long history of dietary and medicinal uses in traditional herbal medicine. Many pharmacological activities of alfalfa have been reported to be beneficial in liver disorders,30 diabetes,31 hypercholesterolemia, atherosclerosis32 and cardiac disorders,33 probably due to the high contents of phytoestrogens and saponins. No studies have been found about synergic effects of a mix composed by Cyclantera pedata, Glycine max, Monascus-fermented red mold rice, Cynaria scolymus and Medicago sativa either about the effect of this mix added to white wheat bread on lipid metabolism. This research focused on the effects of white wheat bread and white wheat bread containing 1.5 g kg-1 of a mixture composed by five bioactive compounds (Cyclantera pedata, Glycine max, Monascus-fermented

red

mold

rice,

Cynaria

scolymus

and

Medicago

sativa)

on

hypercholesterolemic on steatotic mice. White bread has been chosen in this study since it is the most widely consumed bread in western country even though the prevailing recommendation

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based on epidemiological findings indicate that white wheat consumption should be replace with whole grain products.34 HMG CoA reductase, CYP7A1 and LDLr genes expression as markers of cholesterol metabolism and SREBP-1c gene expression as marker of the fatty acids metabolism have been investigated.

Materials and Methods

Animals and diet Eight-week-old C57BL/6J mice were purchased from Harlan. All mice were maintained at a controlled temperature of 21-25°C and 50-60% humidity under a 12 h light/dark cycle and fed a diet of commercial chow for 4 weeks. After this period of acclimatization, the trial started and the mice were randomly divided into two groups and fed the following diets: a control group fed the commercial chow (CTR, n=8; analytical contents: 190 g kg-1 crude protein, 60 g kg-1 crude fibers, 40 g kg-1 crude oils and fat and 70 g kg-1 crude ash, table 1) and another group fed a diet with high fat content (HFD, n=24; analytical contents: 210 g kg-1 crude protein, 45 g kg-1 crude fibers, 230 g kg-1 crude oils and fat and 50 g kg-1 crude ash, table 1). After 14 weeks from the start of the trial, two mice per group were sacrificed, after a 4 h of fasting; blood and liver samples were collected in order to verify the hypercholesterolemic and steatotic status. The mice of HFD group (n=24) was then randomly divided into three groups (8 mice in each group): a group that continued to feed a HFD diet (HFD), a group that fed a HFD diet containing the added white bread (HFD-AB) and a group that fed a HFD diet containing the bread (HFD-B). The control group continued to feed the commercial chow (CTR). The HFD-AB and HFD-B groups were fed high fat diet where the 350 g kg-1 of α-cornstarch was replaced with white wheat bread or added white wheat bread contained 1.5 g kg-1 of the mixture specified in table 2. The mix was supplied by International Agriproject (Calci, Pisa, Italy). The white wheat bread and the added white wheat bread were produced by Cerealia (Montaione, Firenze, Italy). The mice were fed the diets for 6 weeks. At the end of the treatment, six mice per group were fasted 4 h, weighed and sacrificed. Blood samples were obtained by cardiac puncture under general anesthesia prior to mouse sacrifice. Serum samples were obtained by centrifugation at 3500 xg for 15 min and used for glucose, cholesterol and triglycerides analysis. After liver weight recording, samples were stored

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at -80°C for total RNA extraction. Other liver samples were collected and stored in formalin at 4°C until the histological analysis. All animal procedures were performed with the approval of the Local Ethical Committee and in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

Serum glucose and lipid profile and liver triglycerides Serum analysis were performed using standard enzymatic techniques (Synchron CX9 Pro, Beckman Coulter Inc., Fullerton, USA) for triglycerides, glucose and total cholesterol. Triglyceride content was measured in liver homogenates using a kit (Giesse Diagnostics, Rome, Italy).

Isolation of total RNA and cDNA synthesis Total RNA was isolated from frozen mouse liver tissues (about 20 mg of tissue) with the RNeasy Mini Kit (Qiagen, Valencia, CA), following the supplied protocol. Genomic DNA elimination and reverse transcription of total RNA were performed using the QuantiTech Reverse Transcription Kit (Qiagen, Valencia, CA). RNA was quantified using NanoDrop (Celbio, Milan, Italy); its purity and integrity were evaluated by checking the absorbance ratio at 260–280 nm and assessing the sharpness of 18S and 28S ribosomal RNA bands on GelRed™-stained 10 g l-1 agarose gel and visualized by transillumination with ultra-violet light.

RT-PCR and sequence analysis Two μl of cDNA were added to SsoFast™ EvaGreen Supermix and were performed using 300 μM of forward–reverse primers specific for HMGCoA reductase, CYP7A1, LDLr, SREBP-1c and GAPDH (Table 3). The RT-PCR conditions and primers have been previously described.35 The cDNA fragments were separated on GelRed™-stained 10 g ml-1 agarose gel and visualized by transillumination with ultra-violet light. Primer pairs of HMGCoA reductase (3-hydroxy-3methyl-glutaryl-CoA reductase), CYP7A1 (cholesterol 7alpha-hydroxylase), LDLr (low density lipoprotein receptor) and SREBP-1c (sterol regulatory element-binding protein-1c) and for the endogenous reference GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were designed with

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Beacon Designer™ 5 (Premier Biosoft, International, Palo Alto, CA) using the mouse cDNA sequences available in GenBank.

Histopathological examination Liver (n=4 for each experimental group) were collected for histological examination and fixed in 10% neutral buffered formalin. The tissues were routinely embedded in paraffin wax blocks sectioned at a thickness of 5 µm and mounted on slides. Then sections were stained with haematoxylin and eosin (H-E) and examined under a light microscope. The liver degeneration severity was classified using the following semiquantitative scoring system: 0, when < 5% of hepatocytes are affected by fat vacuoles; 0.5, fat vacuoles are seen in 5%-15%; 1, when fat vacuoles are seen in 15%-30%; 2, when 30%-60% of hepatocytes are affected by fat vacuoles; 3, when fat vacuoles are found in 60%-80% of hepatocytes; 4, when