Antiobesity effect of brewer’s yeast biomass in animal model

Journal of Functional Foods 55 (2019) 255–262

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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff

Antiobesity effect of brewer’s yeast biomass in animal model Chih-Ling Chang, Tsai-Hua Kao

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Department of Food Science, Fu Jen Catholic University, New Taipei City 242, Taiwan

A R T I C LE I N FO

A B S T R A C T

Keywords: Antiobesity Brewer’s yeast biomass Prenylflavonoids Hopbitter acids

Brewer’s yeast biomass (BYB) is a byproduct of the beer-brewing process. This study explored the inhibition effect of BYB on obesity using an animal model. The prenylflavonoid and hop bitter acid contents of BYB were also evaluated to identify possible active substances. BYB ethanol extract contained four prenylflavonoids and six hop bitter acids. The content of hop bitter acids was higher than that of prenylflavonoids. Feeding Sprague–Dawley rats with 50 or 250 mg/kg BW doses of BYB ethanol extract and 250 mg/kg BW of BYB supernatant was found to significantly reduce tissue fat weight, serum triglyceride and hepatic triglyceride, serum insulin and HOMA-IR, as well as to increase liver antioxidant activity. Moreover, BYB ethanol extract could inhibit increased weight, feed efficiency, heparin-releasable lipoprotein lipase enzyme activity, and thiobarbituric acid reactive substance in liver. In particular, low doses of BYB ethanol extract could enhance lipolysis.

1. Introduction Obesity is increasingly a global problem. World Health Organization has reported that more than 1.6 billion adults (≥15 years) are with overweight (Ebrahimzadeh Attari et al., 2018). In particular, patients with central obesity may risk complications such as diabetes, hypertension, cardiovascular diseases, and some types of cancer (Ebrahimzadeh Attari et al., 2018). A high-fat diet, increased intestinal absorption and fat synthesis, and inhibition of lipolysis and fatty acid oxidation are all factors leading to obesity, and can increase serum lipid concentrations (Ahrens et al., 1957). Studies have reported a number of phytochemicals that can modulate concentrations of both high- and low-density lipoprotein cholesterols (HDL-C and LDL-C, respectively) and reduce the risk of hypertension (Carrera-Quintanar et al., 2018). A variety of by-products are produced in the beer brewing process, such as spent grains (SG) produced after milling, mashing, and lautering; spent hops (SH) produced by boiling wort with hops; and surplus yeasts (SY) produced by fermentation and aging after yeast inoculation (Mussatto & Roberto, 2006). The SH are frequently mixed with SY to form brewer’s yeast biomass (BYB), and the majority of the food-processing by-products are currently used as fodder. Because BYB is the byproducts produced after the addition of hops, they theoretically contain more functional components. Research conducted previously by the present authors has indicated that beer lee (mixture of brewing byproduct) is enriched with prenylflavonoids and hop bitter acids, compounds that have been demonstrated to exhibit numerous physiological effects. Xanthohumol in prenylflavonoids reduces the expression of

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peroxisome proliferator-activated receptor, inhibits the effects of enhanced biological phosphorus removal (C/EBPR) and diacylglycerol acyltransferase, and activates AMP-activated protein kinase (AMPK) signaling pathway, thereby reducing formation of adipose tissues (Casaschi et al., 2004; Kiyofuji, Yui, Takahashi, & Osada, 2014; Samuels, Shashidharamurthy, & Rayalam, 2018). Xanthohumol-rich extract from hop also could suppress the weight of rats with high-fat diets, reduce weight gain around the liver, and reduce the triglyceride content in their plasma and liver (Yui, Kiyofuji, & Osada, 2014). Isohumulone in hop bitter acids can increase mRNA expression of acylCoA oxidase, acyl-CoA synthetase, hydroxymethylglutaryl-CoA synthetase, lipoprotein lipase, and fatty acid transport protein, decrease mRNA activity of Apo CIII and Apo AI, and regulate hepatic lipid biosynthesis (Miura et al., 2005; Yang, Della-Fera, Rayalam, & Baile, 2007). Although these studies have shown that prenylflavonoids and hop bitter acids have antiobesity capabilities, most are based on reference materials, and no research has been conducted on food systems or food processing byproducts. However, if it could be demonstrated that food processing byproducts have the same physiological effects as reference materials, using such byproducts would be more practical and sustainable because of reduced waste and materials reuse. This study investigated the effect of BYB ethanol extract on obesity using a Sprague–Dawley (SD) rat model and analyzed prenylflavonoids and hop bitter acids in BYB ethanol extract to determine the relationship between the antiobesity effect of BYB and the content of these two components.

Corresponding author. E-mail address: [email protected] (T.-H. Kao).

https://doi.org/10.1016/j.jff.2019.02.027 Received 10 October 2018; Received in revised form 14 February 2019; Accepted 15 February 2019 Available online 25 February 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

Journal of Functional Foods 55 (2019) 255–262

C.-L. Chang and T.-H. Kao

2. Materials and methods

60% B at the 15th min, increased to 70% B at the 20th min, 75% B at the 30th min. The column temperature was at 35 °C, quantity injected was 20 μL, flow rate at 1.5 mL/min. The identification of prenylflavonoids and hop bitter acids were carried out by comparing the retention times and adsorption spectra with standards, where as the compound without commercial standard (6-prenylnaringenin) were identified with literature reference (Kao & Wu, 2013). The calibration curve with internal standard trans-chalcone (1 μg/mL) was used for quantification. Standards were analyzed in triplicate using HPLC and the peak areas were collected at different wavelength for quantitation of iso-α-acids (270 nm), 8-prenylnaringenin, 6-prenylnaringenin and isoxanthohumol (292 nm), α-acids and β-acids (330 nm) and xanthohumol (368 nm), respectively. Calibration curves were obtained by plotting concentration ratio against its area ratio, with the regression equation and correlation coefficient (r2) being calculated automatically. 6-prenylnaringenin was quantified using the standard curve for 8-prenylnaringenin as similarities in λmax have been reported (Kao & Wu, 2013). The contents of prenylflavonoids and hop bitter acids in BYB (μg/g) were quantified using the following formula:

2.1. Materials BYB was provided by local beer brewing company (Taipei, Tiawan). BYB was first centrifuged (6000g, 25 °C) for 20 min, than collected precipitate and supernatant respectively. The precipitate BYB was subsequently stored at −20 °C in vacuum packs after it was freeze-dried (−40 °C, 60 millitorr), whereas the supernatant was evaporated to dryness under vacuum. 2.2. Chemicals and reagents α-acids and β-acids mixture standard (ICE-2) and iso-α-acids mixture standard (ICS-I3) were from Labor Veritas Co. (Zürich, Switzerland). Xanthohumol was from Extrasynthese Co. (Genay, France). Isoxanthohumol was from ChromaDex Co. (Irvine, CA, USA). 8-prenylnaringenin and trans-chalcone (internal standard) were from Sigma-Aldrich Co. (Billerica, MA, USA). 95% ethanol was purchased from Taiwan Tobacco and Liquor Co. (Taipei, Taiwan). Deionized water was obtained using the Milli-Q water purification system of Millipore Co. (MA, USA). HPLC-grade solvents were purchased from Merck (Darmstadt, Germany). Serum biochemical index kits were from Fujifilm Co. (Saitama, Japan). Insulin test kit was from Mercodia AB Co. (Uppsala, Sweden). Triglyceride and total cholesterol test kits were from Randox Laboratories Ltd CO. (Antrim, UK).

C(μg/g) = [( A− b) ÷ a] × V× f÷ recovery ÷ Ws A: peak area of prenylflavonoid and hop bitter acid a: slope of calibration curve b: intercept of calibration curve V: volume of extract f: dilution factor Ws: weight of BYB (g)

2.3. Instrumentation

2.6. Anti-obesity activity of BYB ethanol extract and BYB supernatant

The HPLC-DAD system was from Jasco Co. (Tokyo, Japan), being composed of PU-2089 Plus pump, COLBOX column temperature controller, PU-2089 Plus photodiode-array detector. The HyPURITY C18 column (150 mm × 4.6 mm I.D., 5 μm) used to analyze prenylflavonoids and hop bitter acids was from Thermo Hypersil-Keystone Co. (Bellefonte, PA, USA). The freeze dryer (FD24) was from Gin-Ming Co. (Taipei, Taiwan). The Sorvall RC5C high-speed centrifuge was from Du Pont Co. (Wilmington, DE, USA). The VersaMax ELISA microplate reader was from Molecular Devices Co. (Sunnyvale, CA, USA). The clinical analyzer (FUJI DRI-CHEM 4000i) was from Fujifilm Co. (Saitama, Japan).

2.6.1. Experimental animals This animal test was permitted by Institutional Animal Care and Use Committee established in Fu Jen Catholic University. All the operations complied with National Institutes of Health guide for the care and use of Laboratory animals. A total of 50 SPF (specific pathogen free) male SD rats (5 week old) were purchased from BioLASCO (Taipei, Taiwan). They were housed in individual ventilation cages (IVC) in the animal center of Fu Jen University with a temperature at 22 ± 2 °C and relative humidity at 50–60%. After 1-week adaptation, rats were divided into 5 groups with 10 each and administered with water ad libitum and the following diet for 8 weeks: (1) Normal group- laboratory rodent diet 5001 (LabDiet Co, St. Louis, MO, USA) ad libitum and 1 mL of water (sample solvent) by tube feeding for each rat. (2) High fat diet group (HF)- high fat diet ad libitum and 1 mL of water (sample solvent) by tube feeding for each rat. (3) High fat diet with low dose of BYB ethanol extract group (HF + L)- high fat diet ad libitum and 50 mg/kg of BYB ethanol extract by tube feeding for each rat. (4) high fat diet with high dose of BYB ethanol extract group (HF + H)- high fat diet ad libitum and 250 mg/kg of BYB ethanol extract by tube feeding for each rat. (5) High fat diet with BYB supernatant group (HF + S)- high fat diet ad libitum and 250 mg/kg of BYB supernatant by tube feeding for each rat. The high fat diet was prepared by mixing 73.3% of Chow diet 5001 with 26.7% of butter powder so that contained 30% of fat and calorie density was 4.85 kcal/g. Rats were weighed weekly, and feed intake was recorded every 2 days to calculate changes in body weight, food intake, and feed efficiency.

2.4. Preparation of BYB ethanol extract and BYB supernatant for animal test Lyophilized BYB powder was mixed with 95% ethanol under the ration of 1:20 (w/v), and the mixture was subjected to ultrasonic extraction for 10 min followed by shaking extraction for 20 min. Subsequently, the mixture was centrifuged at 3500g for 5 min, and the supernatant was collected for dryness under vacuum. A total of 86.16 g of BYB ethanol extract was obtained from 1915 g of lyophilized BYB powder. For BYB supernatant, a total of 75 g solid was obtained from 1875 g BYB supernatant after evaporation. Both BYB ethanol extract and BYB supernatant were dissolved in deionized water for functional component analysis, and also dissolved in sterilized water for animal test. 2.5. Analysis of prenylflavonoids and hop bitter acids in BYB ethanol extract and BYB supernatant by HPLC-DAD (diodearray detector)

2.6.2. Sample collection Fecal samples were collected 3 days before sacrifice and dried at 65 °C. After fasting for 12 h, the rats were sacrificed using CO2. Blood was collected from the heart, and serum was separated from the blood samples. The organs and fat tissues were collected and washed using 0.9% saline, dried using filter papers, and weighed individually. The livers and fat tissues were stored at temperatures of −20 and 4 °C, respectively before test.

Contents of prenylflavonoids and hop bitter acids in BYB ethanol extract and BYB supernatant prepared by Section 2.4. were determined by a method based on our previous study (Kao & Wu, 2013) with slightly modification. A binary solvent system of deionized water at pH 1.6 adjusted with phosphoric acid (A) and acetonitrile (B) with the following gradient elution was developed: 38% B initially, maintained for 3 min, increased to 55% B at the 6th min and maintained for 4 min, 256

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and stored at −20 °C before analysis. A standard trolox solution in concentrations of 0, 200, 400, 600, 800, 1000, and 1500 μM was prepared. Each solution (30 μL) was added to 1 mL of ABTS‧+ (2,2′Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) solution, evenly mixed for 30 s, and allowed to rest for 30 s. The absorbance values at 734 nm were measured, and the absorbance values and concentrations were plotted as a quantitative curve. Then, 30 μL of serum and liver extract were sampled, and the absorbance at 734 nm was measured using the same method. The measured absorbance values were substituted into the trolox quantitative equation to calculate the relative trolox concentration (mM) of each sample; the lower the concentration, the higher the degree of oxidation.

2.6.3. Activity of lipolysis and heparin-releasable lipoprotein lipase (HRLPL) 0.1 g of the epididymal fat tissues were precisely weighed and cut into pieces. Then, 1 mL of Krebs–Ringer Bicarbonate buffer was added, and the sample was kept in a shaking water bath at 37 °C for 1 h. The glycerol concentration in the collected solution was analyzed using a commercial kit (GY105, Randox), and the lipolysis efficiency was determined through the glycerol content released from per unit weight of tissues (Berger & Barnard, 1999). Simultaneously, 90 μL of the collected solution and 900 μL of ρ-nitrophenyl butyrate (ρNPB) enzyme reaction solvent (0.15 M or 1 M NaCl) were preheated in an oven at 37 °C. Then, 10 μL of ρNPB substrate solution was added for reaction at 37 °C for 10 min. The reaction was terminated by adding a methanol–chloroform–heptane solution (10:9:7, v/v/v), after which the mixture was evenly mixed and centrifuged (1500g, 5 min). The mixture was then heated at 42 °C for 3 min, and 200 μL was taken and analyzed for absorbance at 400 nm. The activity of HR-LPL was calculated using the following formula:

2.6.9. Analysis of TBARS (thiobarbituric acid reactive substance) in serum and liver A solution of 1,1,3,3-tetramethoxypropane was prepared in concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 μM, as calibrators of 3,4-Methylenedioxyamphetamine. Each of the standards (100 μL) was added to 200 μL of 10% trichloroacetic acid. The solution rested on ice for 15 min for protein precipitation and then centrifuged at 5000g for 10 min. The supernatant liquid (200 μL) was added to 200 μL of 0.8% thiobarbituric acid and then allowed to rest in a 95 °C water bath for 10 min. Subsequently, the mixture was cooled in an ice bath for 2 min, after which 200 μL of it was sampled to measure the absorbance at 532 nm, and the absorbance values and concentrations were used to plot a quantitative curve. Then, 100 μL of serum or liver extract was measured the absorbance at 532 nm using the method described above. The absorbance of the sample were substituted into the standard curve to obtain serum and liver TBARS values.

C (μM) = (A 0.15M − A1M)/0.0148 A0.15 M: absorbance of 0.15 M NaCl reaction solution at 400 nm A1 M: absorbance of 1 M NaCl reaction solution at 400 nm 0.0148: extinction coefficient 2.6.4. Serum biochemical index The serum (10 μL) was instilled into a test strip, and the concentrations of glucose, triglyceride, total cholesterol, high-density lipoprotein, Na+, K+, Cl−, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, creatinine, and uric acid were measured using a clinical analyzer (FUJI DRI-CHEM 4000i).

2.6.10. Histopathological analysis The right upper lobes of the livers were removed and immersed in 10% formalin along with epididymal fat tissues, aortae, and kidneys, for paraffin embedding, hematoxylin and eosin (H&E) staining, and histopathological analysis.

2.6.5. Analysis of triglyceride and cholesterol in liver and feces Shredded liver or dried feces (0.1 g) were added to 1 mL of chloroform–methanol (2:1, v/v) and centrifuged at 3000g for 10 min. The supernatant was filtered using a 0.45-μm filter membrane. Before analysis, the organic solvent was drained off and the remaining sample dissolved in dimethyl sulfoxide (DMSO) and then reacted with a color reagent following the test kit instructions. Absorbance values for the liver extract, fecal extract, standard (triglyceride or cholesterol), and blank (DMSO) were measured at 500 nm and determined using the following formula.

2.7. Statistical analysis All the analyses were done in duplicate and the data were subjected to analysis of variance (ANOVA) and Duncan’s multiple range test for mean comparison (α = 0.05) by using SAS (2016). 3. Results and discussion

Triglyceride orchloresterol (mg/dL) = [(A samle − Ablank) /(A standard − Ablank)] × 195

3.1. Distribution of prenylflavonoids and hop bitter acids in BYB Table 1 shows that BYB ethanol extract contained four prenylflavonoids (isoxanthohumol, 8-prenylnaringenin, 6-prenylnaringenin, and xanthohumol) and six hop bitter acids (cohumulone, humulone, adhumulone, colupulone, lupulone, and adlupulone). The content of

2.6.6. Analysis of serum insulin Serum (10 μL) was analyzed following the instructions provided with the Mercodia Rat Insulin Kit. 2.6.7. Analysis of homeostasis model assessment insulin resistance (HOMAIR) Fasting blood glucose and insulin data were analyzed and substituted into the following formula to calculate insulin resistance in vivo; the higher the HOMA-IR value, the lower the insulin sensitivity and the higher the insulin resistance.

Table 1 Content (mg/g) of prenylflavonoids and hop bitter acids in brewer’s yeast biomass (BYB) ethanol extract.

HOMA − IR value = fasting insulin (mU/mL) × fasting glucose (mmol/L)/22.5

2.6.8. Analysis of TEAC (trolox equivalent antioxidant capacity) in serum and liver A liver sample (0.1 g) was added to 1 mL of 1.15% KCl and 1 g of zirconium beads (0.1 mm in diameter) and centrifuged at 3000g for 10 min. The supernatant was sampled, filtered through a filter paper, 257

Compound

BYB ethanol extract

Isoxanthohumol 8-prenylnaringenin 6-prenylnaringenin Xanthohumol Cohumulone Humulone Adhumulone Colupulone Lupulone Adlupulone Total

0.61 0.16 0.26 0.40 1.89 4.98 0.93 1.11 0.74 0.25 11.3

± ± ± ± ± ± ± ± ± ± ±

0.07 0.01 0.02 0.03 0.19 0.21 0.08 0.15 0.01 0.03 0.7

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Table 2 Effect of ethanol extract and supernatant from brewer’s yeast biomass (BYB) on body weight, food intake, calorie intake, feed efficiency, body fat mass, and organ weight in SD rats fed with high fat diet.a Group

Normal

HF

HF + L

HF + H

HF + S

Initial body weight (g) Final body weight (g) Weight gain Food intake (g/d) Calorie intake (kcal/d) Feed efficiency (%) Perirenal fat pad(mg/g) Epididymal fat pad(mg/g)

212 ± 8A 512 ± 12BC 300 ± 10BC 30.1 ± 3.9A 100 ± 13B 18.5 ± 0.4C 15.1 ± 2.3D 11.9 ± 2.1C

215 ± 11A 553 ± 25A 338 ± 29A 31.4 ± 2.1A 152 ± 10A 21.9 ± 0.7A 26.9 ± 3.7A 22.4 ± 3.6A

211 ± 6A 500 ± 35C 289 ± 36C 29.6 ± 1.5A 150 ± 7A 19.1 ± 0.8C 18.2 ± 2.9CD 18.0 ± 2.1B

213 ± 6A 515 ± 46BC 302 ± 46BC 31.1 ± 0.2A 154 ± 13A 19.9 ± 0.8C 20.4 ± 3.4BC 17.7 ± 2.2B

211 ± 5A 535 ± 34AB 324 ± 34AB 30.6 ± 2.1A 151 ± 10A 20.6 ± 0.2B 21.8 ± 4.3B 18.6 ± 3.7B

Organ weight (mg/g) Heart Liver Spleen Lung Kidney Genital

4.15 29.3 1.43 2.83 7.43 9.87

± ± ± ± ± ±

0.22A 1.3A 0.13A 0.08A 0.30A 0.95A

4.24 26.7 1.38 2.88 7.17 9.51

± ± ± ± ± ±

0.25A 1.5B 0.13A 0.27A 0.48A 0.86A

4.13 26.1 1.36 2.76 7.30 9.31

± ± ± ± ± ±

0.27A 2.1B 0.11A 0.18A 0.51A 0.94A

4.31 26.1 1.40 2.69 7.14 9.32

± ± ± ± ± ±

0.19A 1.4B 0.17A 0.20A 0.33A 0.69A

4.18 25.5 1.35 2.78 7.10 9.51

± ± ± ± ± ±

0.17A 1.8B 0.06A 0.17A 0.34A 0.70A

a Each value is expressed as the means ± SD (n = 10). Data with different letters (A–D) in the same row are significantly different at p < 0.05. Normal: normal diet; HF: 30% high fat diet; HF + L: 30% high fat diet + 50 mg/kg/day BYB ethanol extract; HF + H: 30% high fat diet + 250 mg/kg/day BYB ethanol extract; HF + S: 30% high fat diet + 250 mg/kg/day BYB supernatant.

thus concluded that BYB inhibited lipogenesis because it contained these two functional components.

hop bitter acids was higher than that of prenylflavonoids. Humulone had the highest content, whereas the content of total functional compounds accounted for approximately 1.13% of the extract. Prenylflavonoids and hop bitter acids were not detected in the BYB supernatant.

3.3. Effect on lipolysis activity and HR-LPL activity Lipolysis activity can be measured using the glycerol release percentage. Under normal conditions, lipolysis is proportional to the amount of body fat; the higher the amount of body fat, the more vigorous the tissue metabolic activity to maintain metabolic balance. As shown in Table 3, the glycerol release percentage in the HF group was significantly higher than in the Normal, HF + H, and HF + S groups. However, no significant difference was found between the HF + L and HF groups, indicating that, compared with HF + H and HF + S, HF + L could release more glycerol by enhancing lipolysis and thereby reducing fat accumulation. Lipoprotein lipase (LPL) is a glycoprotein attached to the inner capillary wall, which hydrolyzes the TG in blood lipoproteins and releases nonesterified fatty acid to peripheral tissue or re-esterifies TG for fat tissue storage. The balance between these two mechanisms is related to weight loss and fat tissue formation. LPL in fat tissues is essential for storing lipids, whereas that in skeletal muscles improves fatty acid oxidation. Studies have shown that the LPL activity of fat tissues in obese people is higher than in those with healthy weight (Kusunoki, Tsutsumi, Sato, & Nakamura, 2012). This study found that HR-LPL activity in the HF group was significantly higher than in the Normal,

3.2. Effect on body weight, body fat mass, food intake and organ weight As shown in Table 2, after 8 weeks of feeding, the body weight of the high-fat (HF) group was 553 g, which was considerably higher than the 512 g in the Normal group. Weight gain induced by HF + H and HF + L treatments was effectively inhibited. However, body weight was not reduced in the group fed with BYB supernatant (HF + S), and the same trend was observed with regard to weight gain. No significant difference in total food intake between the groups was found. Regarding caloric intake, because the HF diet contained more calories per unit weight (HF: 4.85 kcal/g and Normal: 3.34 kcal/ g), the caloric intake of the HF group was accordingly higher. Compared with the Normal group, the feed efficiency of the HF group significantly increased, whereas that of the HF + L, HF + H and HF + S groups was reduced. In addition, feeding BYB ethanol extract and BYB supernatant (HF + L, HF + H and HF + S groups) also significantly reduced the perirenal fat pad weight by 32%, 24%, and 19%, respectively, and the epididymal fat pad weight by 20%, 21%, and 17%, respectively. These results indicated that, given the same amount of food intake, the increased weight gain, feed efficiency, and visceral fat formation induced by the HF diet were inhibited in rats fed with BYB samples. As seen in Table 2, feeding with BYB ethanol extract and supernatant did not affect heart, spleen, lung, kidney, and testicular tissue weight during the 8 feeding weeks, and the animals exhibited healthy growth. Studies have proposed that prenylflavonoids and hop bitter acids can inhibit lipogenesis. Nozawa (2005) reported that feeding KK-Ay mice with 1% xanthohumol for 31 days could effectively reduce the weight of retroperitoneal and epididymal white fat tissues. Yui et al. (2014) reported that adding 1% xanthohumol-rich hop extract to high fat diet could reduce body weight and liver weight in Wistar rats, however, the weight of perirenal fat pad and epididymal fat pad were not significantly different. Yajima et al. (2005) reported that adding 0.2% and 0.6% iso-α-acids to the diet of female C57BL/6N mice for 6 weeks could inhibit generation of subcutaneous, retroperitoneal, and parametrical fat tissues. Similarly, the present study found that BYB ethanol extract was rich in prenylflavonoids and hop bitter acids, and

Table 3 Effect of ethanol extract and supernatant from brewer’s yeast biomass (BYB) on lipolysis activity and heparin-releasable LPL of epididymal fat pads in SD rats fed with high fat diet.a Group

Glycerol release (% of HF treatment)

HR-LPL activity (% of HF treatment)

Normal HF HF + L HF + H HF + S

63.7 ± 16.5C 100 ± 15A 89.2 ± 8.9AB 81.9 ± 17.5B 77.7 ± 18.7BC

63.5 ± 16.2B 100 ± 24A 56.6 ± 16.6B 62.1 ± 19.8B 78.5 ± 32.8AB

a Each value is expressed as the means ± SD (n = 10). Data with different letters (A-C) in the same column are significantly different at p < 0.05. Normal: normal diet; Normal: normal diet; HF: 30% high fat diet; HF + L: 30% high fat diet + 50 mg/kg/day BYB ethanol extract; HF + H: 30% high fat diet + 250 mg/kg/day BYB ethanol extract; HF + S: 30% high fat diet + 250 mg/kg/day BYB supernatant.

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Table 4 Effect of ethanol extract and supernatant from brewer’s yeast biomass (BYB) on lipidic parameters in male SD rats fed with high fat diet.a Normal

HF

HF + L

HF + H

HF + S

Serum (mg/dL) TG TC LDL-C HDL-C LDL-C/HDL-C

74.9 77.7 12.4 47.2 0.33

Fecal (mg/g) TG TC

13.6 ± 0.7B 2.47 ± 0.30B

14.8 ± 0.6 AB 2.68 ± 0.38AB

14.7 ± 1.8 AB 2.89 ± 0.40A

14.3 ± 2.1B 3.06 ± 0.41A

16.0 ± 1.7A 2.91 ± 0.44A

Liver (mg/g) TG TC

20.9 ± 0.9BC 3.51 ± 0.24A

25.3 ± 3.4A 3.60 ± 0.52A

21.6 ± 3.1B 3.53 ± 0.58A

19.5 ± 1.3C 3.37 ± 0.36A

22.6 ± 1.5B 3.67 ± 0.33A

± ± ± ± ±

18.3B 11.2A 5.9B 6.7A 0.08B

89.0 73.9 18.7 37.4 0.43

± ± ± ± ±

11.3A 5.9A 5.9A 4.1B 0.09A

52.3 73.6 13.1 46.4 0.31

± ± ± ± ±

11.1C 11.2A 4.7B 6.3A 0.11B

53.1 69.2 15.4 39.3 0.42

± ± ± ± ±

15.3C 6.9A 2.7AB 6.6B 0.07A

62.3 70.6 13.8 47.0 0.32

± ± ± ± ±

12.8C 8.8A 4.7AB 5.7A 0.09B

a Each value is expressed as the means ± SD (n = 10). Data with different letters (A–C) in the same row are significantly different at p < 0.05. Normal: normal diet; Normal: normal diet; HF: 30% high fat diet; HF + L: 30% high fat diet + 50 mg/kg/day BYB ethanol extract; HF + H: 30% high fat diet + 250 mg/kg/day BYB ethanol extract; HF + S: 30% high fat diet + 250 mg/kg/day BYB supernatant. TG: total triglyceride; TC: total cholesterol; LDL-C: low-density lipoprotein-cholesterol; HDL: high-density lipoprotein-cholesterol.

decrease in serum TG content was not caused by fecal discharge.

HF + L, and HF + H groups, indicating that BYB ethanol extract could effectively reduce HR-LPL activity in rat epididymal fat tissues, regardless of the dose. In summary, feeding 50 mg/kg BW of BYB ethanol extract could enhance lipolysis, whereas feeding 50 or 250 mg/kg BW of BYB ethanol extract could inhibit HR-LPL activity in fat tissues and reduce TG storage in cells, thereby reducing tissue weight and preventing obesity.

3.5. Effect on liver lipids As shown in Table 4, feeding with 50 or 250 mg/kg BW of BYB ethanol extract (HF + L and HF + H, respectively) and 250 mg/kg BW of BYB supernatant (HF + S) could effectively inhibit TG accumulation. No significant difference in TC content was found among the groups. Yui et al. (2014) reported that adding 1% xanthohumol-rich hop extract to high fat diet could lower liver TG and TC. Nozawa (2005) reported that feeding KK-Ay mice with 1% xanthohumol for 31 days significantly reduced TG accumulation in the liver. The same study also used real-time reverse transcription polymerase chain reaction to detect mRNAs involved in lipid and glucose metabolism in the liver. The results indicated that xanthohumol could reduce the content of sterol regulatory element-binding protein-1c (SREBP-1c) in mice, thereby inhibiting the gene synthesis of lipids such as the fatty acid synthase, malic enzyme, and ATP citrate-lyase, enhancing lipid oxidation, and reducing serum and liver TG content. Feeding female ApoE−/− mice with 300 mg/kg BW of xanthohumol for 8 weeks could inhibit the dephosphorylation of the acetyl-CoA carboxylase enzyme, regulate fatty acid and TG biosynthesis, and enhance mitochondrial fatty acid β-oxidation, thereby reducing liver TG content (Miura et al., 2005). Moreover, Apo CIII and LPL are essential in TG metabolism. Apo CIII can inhibit LPL from hydrolyzing TG in very low-density lipoprotein cholesterol. One study reported that adding 1.1 or 2.9 g/kg of iso-α-acids to the HF diet of female C57BL/6 mice reduced the mRNA expression for Apo CIII, enhanced mRNA expression for LPL, improved β-oxidation of liver cells, and reduced liver TG content, thereby reducing serum TG concentration (Miura et al., 2005).

3.4. Effect on serum lipids The effect of each treatment on serum lipids is shown in Table 4. Except for TC, significant differences could be found for all parameters. The content of TG in the BYB groups was lower than in the HF group. LDL-C content significantly decreased in the HF + L group, whereas HDL-C content significantly increased in the HF + L and HF + S groups. The LDL-C/HDL-C ratio in the HF group increased significantly, whereas in the HF + L and HF + S groups it decreased significantly. Nozawa (2005) reported that feeding KK-Ay mice with 0.3% or 1% xanthohumol for 31 days significantly increased serum adiponectin content and reduced TG concentration. In another study, two groups of male KK-Ay mice were fed with 1.8-g/kg of either isohumulone or isocohumulone for 2 weeks; compared with the HF group, the serum TG content in these two groups significantly decreased by 62.6% and 76.4%, respectively (Yajima et al., 2004). The presence of xanthohumol in BYB could possibly reduce TG content. Yui et al. (2014) reported that adding 1% xanthohumol-rich hop extract to high fat diet could lower plasma TG in Wistar rats, but no significant difference were found in plasma TC and HDL-C. Miura et al. (2005) indicated that the addition of 1.1 or 2.9 g/kg of iso-α-acids to the HF diet of female C57BL/6 mice could considerably increase serum HDL-C content and reduce the arteriosclerosis index. HDL-C content may be related to the regulation of cholesterol ester transfer protein (CETP), a hydrophobic glycoprotein synthesized by the liver. The primary function of CETP is to exchange cholesterol and TG between HDL-C and Apo B–containing lipoprotein. Inhibition of CETP activity could effectively increase serum HDL-C content (Tall, 1993). Hirata et al. (2012) supplemented the diet of CETP-transgenic mice with 0.05% xanthohumol. After 18 weeks, CETP performance was inhibited by 21%, Apo E apolipoprotein content was enhanced, and the serum HDL-C concentration increased significantly. The presence of prenylflavonoids and hop bitter acids in BYB could possibly regulate blood lipids. The fecal TG and TC concentrations in each BYB group were not significantly different from those in the HF group, indicating that the

3.6. Fasting blood sugar, serum insulin, and insulin resistance Both obesity and diabetes increase insulin resistance and insulin and glucose concentrations. HOMA-IR can be used to demonstrate insulin sensitivity; the lower its value, the higher the insulin sensitivity. As shown in Table 5, no significant difference in fasting blood glucose content was found among the treatment groups. Serum insulin concentration and HOMA-IR values significantly decreased in the experimental group, indicating that both BYB ethanol extract and BYB supernatant could effectively reduce insulin content in systemic circulation and improve the insulin sensitivity and blood glucose regulation in rats.

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Table 5 Effect of ethanol extract and supernatant from brewer’s yeast biomass (BYB) on serum glucose, insulin, HOMA-IR and serum parameters in male SD rats fed with high fat diet.a Normal

HF

HF + L

HF + H

HF + S

Glucose (mg/dL) Insulin (μg/L) HOMA-IR

263 ± 22A 0.47 ± 0.13C 7.47 ± 1.62C

278 ± 27A 0.93 ± 0.20A 19.1 ± 3.6A

270 ± 51A 0.58 ± 0.12BC 15.0 ± 2.9B

283 ± 50A 0.58 ± 0.23BC 15.7 ± 2.8B

267 ± 44A 0.79 ± 0.28B 15.2 ± 2.6B

Serum parameters GOT (U/L) GPT (U/L) CRE (mg/dL) UA (mg/dL) Na (mEq/L) K (mEq/L) Cl (mEq/L)

67.5 ± 5.9B 24.1 ± 5.4B 0.23 ± 0.05A 5.24 ± 2.00A 143 ± 1A 6.18 ± 0.83A 95.2 ± 1.9A

79.9 ± 13.9A 29.3 ± 5.8A 0.22 ± 0.04A 5.56 ± 0.62A 143 ± 1A 6.40 ± 0.95A 94.3 ± 2.3A

66.0 ± 10.4B 22.3 ± 3.8B 0.22 ± 0.04A 4.93 ± 1.68A 144 ± 3A 6.32 ± 1.69A 94.2 ± 1.9A

70.7 ± 10.8B 22.7 ± 4.3B 0.21 ± 0.03A 5.45 ± 1.84A 143 ± 2A 6.67 ± 1.08A 94.7 ± 2.2A

63.2 ± 6.9B 24.5 ± 4.4B 0.25 ± 0.07A 4.69 ± 1.21A 143 ± 1A 5.74 ± 1.01A 94.7 ± 3.1A

a

Each value is expressed as the means ± SD (n = 10). Data with different letters (A-B) in the same row are significantly different at p < 0.05. Normal: normal diet; Normal: normal diet; HF: 30% high fat diet; HF + L: 30% high fat diet + 50 mg/kg/day BYB ethanol extract; HF + H: 30% high fat diet + 250 mg/kg/day BYB ethanol extract; HF + S: 30% high fat diet + 250 mg/kg/day BYB supernatant.

respectively, than trolox. In addition to effectively inhibiting hydroxyl radical oxidation, the scavenging effect of 5 μM isoxanthohumol on lipid peroxidation free radicals was more favorable than that of xanthohumol in the same concentration. In this study, the relatively high liver TEAC might derive from the presence of isoxanthohumol and xanthohumol in the BYB samples. Regarding MDA concentrations, both doses of BYB ethanol extract effectively reduced the serum and liver MDA, whereas BYB supernatant did not have a significant effect. Rodriguez, Miranda, Stevens, and Deinzer (2001) reported that using the SD rat liver microsomes in the lipid peroxidation system induced by iron/ascorbate, 5–25 μM xanthohumol effectively inhibited lipid peroxidation in rat liver microsomes and exhibited a dose effect. In a Fe3+-ADP/NADPH environment, 25 μM xanthohumol could serve as an antioxidant. Miranda et al. (2000) found that, in the normal range of α-tocopherol in human serum (12.5–25 μM), the time that xanthohumol required to inhibit conjugated diene formation induced by Cu2+ was five hours longer than the same concentration of α-tocopherol. The abovementioned antioxidant evaluation indicated that BYB ethanol extract could contain xanthohumol and reduce MDA concentration by increasing the liver’s total antioxidant capacity.

3.7. Serum liver function index (GOT and GPT) As shown in Table 5, obesity induces excessive liver TG accumulation, leading to hepatocyte injury and fatty liver disease that result in significantly higher GOT and GPT levels in the HF group than in the four other groups. The GOT and GPT of rats fed with BYB ethanol extract and BYB supernatant for 8 weeks were effectively reduced, thereby also lowering the occurrence of non-alcoholic steatosis hepatitis caused by the HF diet. In a related study, when two groups of male KK-Ay mice were fed with 1.8 g/kg of isohumulone and isocohumulone for 8 weeks, their serum GOT and GPT values significantly decreased, as compared with the HF group (Yajima et al., 2004). 3.8. Serum renal indices and electrolytes As shown in Table 5, no significant difference in creatinine and uric acid concentrations was found among the groups, indicating that the BYB samples did not affect renal function. Similarly, no significant difference in Na+, K+, and Cl− concentrations was found. The results for serum renal indices and electrolytes indicated that neither BYB ethanol extract nor BYB supernatant adversely affected the rats’ physiological condition. Dorn, Bataille, Gaebele, Heilmann, and Hellerbrand (2010) reported that feeding female BALB/c mice with 1000 mg/kg of xanthohumol BW for 3 weeks caused no significant difference in creatinine, Na+, and K+ concentrations, compared with those of the Normal group.

3.10. Histopathological analysis Sections of fat tissue, liver, aorta, and kidney are illustrated in Fig. 2. The results indicated that both BYB ethanol extract and BYB supernatant could reduce the size of fat cells, and the BYB samples did not cause pathological changes to the liver, aorta, and kidney. Using pathological sections of the heart, lung, spleen, kidney, and thyroid, Dorn et al. (2010) showed that feeding female BALB/c mice with 1000 mg/kg BW of xanthohumol for 3 weeks did not cause organ toxicity. Liver tissue sections of ApoE−/− mice fed with 300 mg/kg BW of xanthohumol for 8 weeks revealed relatively small-diameter droplets of Oil Red O stain, and H&E-stained tissues showed no pathological changes (Doddapattar et al., 2013).

3.9. Antioxidant activity Obesity will induce inflammation, then several reactions including cytokine secretion, C-reactive protein activation and leukocyte infiltration will progress. These reactions lead formation of reactive oxygen species and resulting lipid oxidation and protein oxidation. Specifically, leptin, an adipocyte-derived hormone, also can induce oxidative stress (Huang et al., 2015). Compounds with antioxidant activity may reduce the obesity-related oxidative stress. In this study, BYB was found to contain prenylflavonoids which possess antioxidant activity and may reduce malondialdehyde production. Fig. 1 shows serum and liver TEAC values in SD rats after feeding with BYB. The results indicated that all of these, whether 50 and 250 mg/kg BW of BYB ethanol extract or 250 mg/kg BW of BYB supernatant, could increase total antioxidant capacity in the liver, but did not affect serum TEAC. In an experiment on oxygen radical adsorbing capacity, Gerhauser et al. (2002) found that the adsorbing capacity of 1-μM xanthohumol on hydroxyl and peroxy radicals was 8.9 and 2.9 times higher,

4. Conclusion The animal experiments demonstrated that feeding 50 and 250 mg/ kg BW of BYB ethanol extract and 250 mg/kg BW of BYB supernatant had potentials in anti-obesity. The effects of BYB ethanol extracts even in low dose of 50 mg/kg BW were better than BYB supernatant. BYB ethanol extract could significantly decrease in body weight, epididymal fat weight, feed efficiency, and liver TBARS, lower epididymal fat weight through reduced HR-LPL activity. Moreover, BYB ethanol extract could increase the liver TEAC value. After HPLC-DAD analysis, four prenylflavonoids, three α-acids and three β-acids were found in 260

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1.4

0.85

0.81

0.76

0.84

1.4

1.4

1.4

0.84 1.3

7.8 6.4

13 10

10

10

7.4 6.5

6.4

11

Fig. 1. Effect of ethanol extract and supernatant from brewer’s yeast biomass on TBARS amount of serum and liver in SD rats fed with high fat diet. Data are presented as mean ± SD (n = 10/group). Normal: normal diet; HF: 30% high fat diet; HF + L: 30% high fat diet + 50 mg/kg/day brewer’s yeast biomass ethanol extract; HF + H: 30% high fat diet + 250 mg/kg/day brewer’s yeast biomass ethanol extract; HF + S: 30% high fat diet + 250 mg/kg/day brewer’s yeast biomass supernatant.

Normal

HF

HF+L

HF+H

HF+S

adipose tissue

liver

aorta

kidney

Fig. 2. Effect of ethanol extract and supernatant from brewer’s yeast biomass on kidney in male SD rats fed with high fat diet. 261

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BYB ethanol, but were not found in BYB supernatant. Since many studies have focused on the effect of prenylflavonoids and hop bitter acids in inhibiting obesity, it is reasonable to infer that the inhibition effect in this animal experiment derived from these two components. The results of this study can serve as a reference in developing health supplements to regulate body fat formation, as well as in possible development of brewing industry byproducts as functional foods.

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