Article pubs.acs.org/JAFC
Carnosic Acid as a Major Bioactive Component in Rosemary Extract Ameliorates High-Fat-Diet-Induced Obesity and Metabolic Syndrome in Mice Yantao Zhao, Rashin Sedighi, Pei Wang, Huadong Chen, Yingdong Zhu, and Shengmin Sang* Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, North Carolina 28081, United States ABSTRACT: In this study, we investigated the preventive effects of carnosic acid (CA) as a major bioactive component in rosemary extract (RE) on high-fat-diet-induced obesity and metabolic syndrome in mice. The mice were given a low-fat diet, a high-fat diet or a high-fat diet supplemented with either 0.14% or 0.28% (w/w) CA-enriched RE (containing 80% CA, RE#1L and RE#1H), or 0.5% (w/w) RE (containing 45% CA, RE#2), for a period of 16 weeks. There was the same CA content in the RE#1H and RE#2 diets and half of this amount in the RE#1L diet. The dietary RE supplementation significantly reduced body weight gain, percent of fat, plasma ALT, AST, glucose, insulin levels, liver weight, liver triglyceride, and free fatty acid levels in comparison with the mice fed with a HF diet without RE treatment. RE administration also decreased the levels of plasma and liver malondialdehyde, advanced glycation end products (AGEs), and the liver expression of receptor for AGE (RAGE) in comparison with those for mice of the HF group. Histological analyses of liver samples showed decreased lipid accumulation in hepatocytes in mice administrated with RE in comparison with that of HF-diet-fed mice. Meanwhile, RE administration enhanced fecal lipid excretion to inhibit lipid absorption and increased the liver GSH/GSSG ratio to perform antioxidant activity compared with HF group. Our results demonstrate that rosemary is a promising dietary agent to reduce the risk of obesity and metabolic syndrome. KEYWORDS: rosemary, carnosic acid, obesity, metabolic syndrome, high-fat diet, oxidative stress
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INTRODUCTION Obesity is now a global epidemic and is a high risk factor for chronic diseases such as type 2 diabetes, cardiovascular disease, hypertension, nonalcoholic fatty liver disease (NAFLD), osteoarthritis, cancer, and stroke.1 More than one-third (34.9%, more than 78 million) of adults in the United States were obese in 2011−2012.2 In addition, 90% of type 2 diabetic patients are overweight or obese.3,4 A poor dietary habit is one of the most common causes of obesity and type 2 diabetes.5,6 Dietary intervention with functional food has been proven to be an effective approach to reduce the risk of obesity and related type 2 diabetes.7,8 Much research is currently being devoted to the bioactive food components to improve metabolic syndrome. Rosemary (Rosmarinus officinalis L.) is well-known for its antioxidant properties and has been widely employed and commercialized as a preserving agent in the food industry, cosmetic additives, and nutritional supplements.9−12 The rosemary extract (RE) has been approved by the European Union (EU) for use in food preservation and has been categorized by the FDA as “generally recognized as safe”. Rosemary itself contains a variety of polyphenolic compounds, including carnosic acid (CA), carnosol, rosemanol, rosmarinic acid (RA), and many others.13−15 CA has been used as a principal component for the standardization of commercial rosemary extract in food or supplements.11 Only a few studies have investigated the beneficial effects of RE on obesity and metabolic disorders in various rodent models of high-fat (HF)-diet-induced16,17 and genetic obesity.18,19 Ibarra et al.17 demonstrated that RE (containing 20% CA) supplement at 500 mg of RE/kg of body weight for 16 weeks inhibited weight © XXXX American Chemical Society
gain and improved glucose and lipid homeostasis in HF-diet-fed male C57BL/6J mice. In another study, the male C57BL/6J mice were fed HF diet for 11 weeks and then given RE (containing 2.5% CA, 4% RA, and 5.6% carnosol) for 50 days. This 200 mg/ kg of RE administration significantly reduced weight and fat mass gain and protected obesity-induced liver steatosis.16 In those two HF-diet-induced obesity studies, even though the body weight was limited by oral RE administration, two different kinds of rosemary extract were used and it is hard to determine which component is the major active compound in rosemary. The objective of the present study was to determine whether CA is a major active component in RE and the dose−response effect of CA against HF-induced obesity and related metabolic syndrome in mice.
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MATERIALS AND METHODS
Chemicals and Reagents. Commercial RE with 45% CA was purchased from Changsha Yaying Biotechnology Inc. (Changsha, People’s Republic of China). The CA content of rosemary extracts was determined by high-performance liquid chromatography (HPLC), and the main compounds of RE were determined by liquid chromatography/mass spectrometry (LC/MS). Antibodies against RAGE were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against β-actin and horseradish peroxidase (HRP) conjugated donkey antirabbit IgG were purchased from Cell Signaling Received: March 10, 2015 Revised: April 30, 2015 Accepted: April 30, 2015
A
DOI: 10.1021/acs.jafc.5b01246 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Table 1. Compositions of the Experimental Low-Fat Diet (LF), High-Fat Diet (HF), and HF plus Rosemary Extract (RE) Diets (RE#1L, RE#1H, and RE#2) LF protein carbohydrate fat total kcal/g
HF
casein/80 mesh L-cystine
corn starch maltodextrin 10 sucrose cellulose, BW200 soybean oil lard mineral mix, S10026 D calcium phosphate calcium carbonate potassium citrate, 1 H2O vitamin mix, V10001 choline biartrate RE#1 RE#2 dye (different color) total
RE#1H
RE#2
kcal %
g%
kcal %
g%
kcal %
g%
g%
g%
g%
19.2 67.3 4.3
20 70 10 100
26.2 26.3 34.9
20 20 60 100
26.2 26.3 34.8
20 20 60 100
26.2 26.3 34.8
20 20 60 100
26.1 26.2 34.7
20 20 60 100
3.85
5.24
5.24
LF ingredient
RE#1L
g%
5.23
HF
RE#1L
5.22 RE#1H
RE#2
g
kcal
g
kcal
g
kcal
g
kcal
g
kcal
200 3 506.2 125 68.8 50 25 20 10 13 5.5 16.5 10 2 0 0 0.05 1055.05
800 12 2024.8 500 275.2 0 225 180 0 0 0 0 40 0 0 0
200 3 0 125 68.8 50 25 245 10 13 5.5 16.5 10 2 0 0 0.05 773.85
800 12 0 500 275.2 0 225 2205 0 0 0 0 40 0 0 0
200 3 0 125 68.8 50 25 245 10 13 5.5 16.5 10 2 1.09 0 0.05 774.94
800 12 0 500 275.2 0 225 2205 0 0 0 0 40 0 0 0
200 3 0 125 68.8 50 25 245 10 13 5.5 16.5 10 2 2.18 0 0.05 776.03
800 12 0 500 275.2 0 225 2205 0 0 0 0 40 0 0 0
200 3 0 125 68.8 50 25 245 10 13 5.5 16.5 10 2 0 3.9 0.05 777.75
800 12 0 500 275.2 0 225 2205 0 0 0 0 40 0 0 0
4057
4057
4057
4057
4057
performed with Xcalibur version 2.0 (Thermo Electron, San Jose, CA, USA). Animals, Diets, and Experimental Design. Male C57BL/6J mice (5 weeks old) were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and were acclimated for 1 week before being randomly assigned to different experimental groups. All of the mice were maintained on a 12 h light/dark cycle on a standard chow diet until experimental analysis. Control low-fat diet (10% kcal energy from fat, D12450J, LF), high-fat diet (60% kcal energy from fat, D12492, HF), and HF mixed with 0.14% or 0.28% of CA-enriched RE (80% CA, RE#1L and RE#1H) and 0.5% of commercial RE (45% CA, RE#2) were produced from Research Diets (New Brunswick, NJ). All diets were pelleted, and the composition of diets is shown in Table 1. The experimental animal protocol was approved by the Institutional Animal Care and Use Committee of the North Carolina Research Campus (No. 12-012). After adaptation for 1 week, mice were randomly divided into five groups (n = 15 in each group, 5 mice per cage): (1) control low fat (LF); (2) control high fat (HF); (3) HF + 0.14% (w/w) CA-enriched RE (RE#1L); (4) HF + 0.28% (w/w) CA-enriched RE (RE#1H); (5) HF + 0.5% (w/w) commercial RE (RE#2). The RE#1H and RE#2 diets contain the same concentration of CA, and the RE#1L diet has half the amount of CA in comparison with those in RE#1H and RE#2 diets. Water and food were available to the animals ad libitum. Body weight and food intake were recorded weekly. At the end of the experiment, the animals were sacrificed by euthanization with isofluorane after fasting for 8 h, and plasma, liver, kidney, and adipose tissue (epididymal, retroperitoneal, and mesenteric) were collected, weighed, frozen under liquid nitrogen, and stored at −80 °C until further analyses. Body Composition-MRI Analysis. Whole body composition (fat and lean tissues) was determined using nuclear magnetic resonance technology with an EchoMRI Analyzer system (Model MRI-100, Houston, TX, USA) at week 15 (day 102). The EchoMRI-100 instrument provides measurements of whole body composition parameters: total fat, lean mass, and total body water in live mice without the need for anesthesia or sedation in about 2 min.
(Danvers, MA, USA). CA standard and other chemicals were purchased from Sigma (St. Louis, MO, USA). HPLC-grade solvents and other reagents were obtained from VWR Scientific (South Plainfield, NJ, USA). LC/MS-grade methanol and water were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Preparation of RE with 80% CA. RE with 80% CA was prepared from the commercial RE (45% CA) in our laboratory according to the method described in the US patent 5,256,70020 with modification. In brief, 50 g of RE (45% CA) was subjected to column chromatography over silica gel (230−400 mesh, Sorbent Technologies Inc., Atlanta, GA) and eluted with a gradient of hexane/acetone (50/2, 50/3, 50/4, 50/5, and 0/100) to give five fractions (Fr A−E). Fraction B (23.3 g) was applied to reverse-phase C18 silica gel medium-pressure column chromatography (35 mm × 400 mm, 60 Å, Sigma) eluted with 75% methanol/25% water containing 0.05% formic acid to give four subfractions (Fr. B1−B4). Fraction B2 (6.85 g) was identified as the CA-enriched fraction (80% CA) by HPLC. These steps were repeated five times in order to accumulate enough samples for the mouse study. Determination of the Major Components in RE by LC/MS. LC/MS was carried out with a Thermo-Finnigan Spectra System, consisting of an Accela high-speed MS pump, an Accela refrigerated autosampler, and an LTQ Velos ion trap mass detector (Thermo Electron, San Jose, CA, USA) incorporated with an electrospray ionization (ESI) interface. A Gemini C18 column (150 × 4.6 mm, 5 μm; Phenomenex, Torrance, CA) was used for separation at a flow rate of 200 μL/min. The column was eluted with 100% solvent A (5% aqueous methanol with 0.2% acetic acid) for 3 min, followed by linear increases in B (95% aqueous methanol with 0.1% acetic acid) to 60% from 3 to 8 min, then to 75% from 8 to 30 min to 82% from 30 to 48 min, and then with 100% from 48 to 55 min. The column was then re-equilibrated with 100% A for 5 min. The injection volume was 10 μL for each sample. Nitrogen gas was used as the sheath gas and auxiliary gas. The collisioninduced dissociation (CID) was conducted with an isolation width of 2 Da and normalized collision energy of 35 for MS2 and MS3. The mass range was measured from m/z 50 to 1000. Data acquisition was B
DOI: 10.1021/acs.jafc.5b01246 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chemical profiles of rosemary extracts (REs) used in this study and plasma carnosic acid (CA) concentrations in mice treated with REs. LC chromatograms of commercial RE (A1) and CA-enriched RE (A2) with negative ESI-MS as detector and MS/MS (negative) spectra of peaks I−III (A3−A5). Representative HPLC-ECD chromatograms of CA standard and the corresponding CA peak in plasma samples from mice in HF, RE#1L, RE#1H, and RE#2 groups (B1) and plasma CA concentrations of different groups (B2). Results are means ± standard deviations (n = 15), represented by vertical bars. Significant differences (p < 0.05, ANOVA) are identified by the different letters among HF, RE#1L, RE#1H, and RE#2 groups. linearity ranged from LOQ to 10 μg/mL (the upper limit was not tested) with a correlation coefficient of 0.999. Biochemical Analyses of Blood Samples. Blood samples were collected by cardiac puncture, and plasma was obtained by centrifuging the blood at 8000g for 15 min at 4 °C. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were colorimetrically measured using Infinity ALT and AST Reagent provided by Thermo Scientific (Waltham, MA). Plasma TNF-α was measured using a Mouse TNF-α ELISA kit (Millipore, Temecula, CA). To measure blood glucose, mice were fasted for 8 h before measurement. Blood was collected from the tail vein, and glucose concentrations were measured using a OneTouch Ultra2 Blood Glucose Meter (LifeScan, Milpitase, CA, USA) at weeks 0, 10, and 16. Insulin levels were determined using an Ultrasensitive Insulin ELISA kit (ALPCO, Salem, NH, USA). Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: [fasting glucose (mg/dL) × insulin (mU/L)]/405.22 Hepatic Lipid Analyses. Quantitative assay of lipids was conducted by measuring triglyceride and free fatty acids (FFA) concentrations in the liver. Hepatic lipids are extracted by homogenizing liver tissue in chloroform using 1% Triton X-100. The organic extracts were air-dried, vacuumed, and dissolved in Trition X-100. Triglyceride and FFA concentrations in extracts were determined using commercial kits (BioVision, Milpitase, CA). Fecal Lipid Excretion. The feces harvested during a 24 h period were collected at week 16, frozen at −80 °C, and pulverized. Total lipids were extracted from 100 mg of dried feces using the following method.23 The feces were cleaned and dried for 24 h by a freeze-dryer, incubated with 2 mL of chloroform/methanol (2/1) for 30 min at 60 °C with constant agitation, and then centrifuged. Water (1 mL) was added to the supernatant, and following vortexing, phase separation was induced by low-speed centrifugation (500g for 10 min). The lower chloroform
Plasma CA Concentrations Determined by HPLC Coupled with an Electrochemical Detector (ECD). Enzymatic deconjugation of the plasma samples was performed as described before, but with slight modification.21 In brief, plasma samples (100 μL) were treated with βglucuronidase (250 U) and sulfatase (3 U) for 45 min at 37 °C. This mixture was extracted with 2.5 mL of n-hexane by shaking for 5 min and centrifuged at 16.1 (×1000g) for 10 min at 4 °C, and then the supernatant was transferred to another tube and evaporated to dryness under a gentle stream of nitrogen at room temperature. The pellet was reconstituted in 150 μL of methanol containing 0.2% acetic acid and filtered through a 0.22 μm filter prior to injecting to the HPLC-ECD system. CA was quantified using an HPLC-ECD system (ESA, Chelmsford, MA, USA) consisting of an ESA Model 584 HPLC pump, an ESA Model 542 autosampler, an ESA organizer, and an ESA CoulArray detector coupled with two ESA Model 6210 four-sensor cells. A Gemini C18 column (150 × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) was used for chromatographic analysis at a flow rate of 1.0 mL/min. The mobile phases consisted of solvent A (30 mM sodium phosphate buffer containing 1.75% acetonitrile and 0.125% tetrahydrofuran, pH 3.35) and solvent B (15 mM sodium phosphate buffer containing 58.5% acetonitrile and 12.5% tetrahydrofuran, pH 3.45). The gradient elution had the following profile: 30% B from 0 to 3 min; 30−65% B from 3 to 10 min; 65−100% B from 10 to 40 min; 100% B from 40 to 45 min; and 30% B from 45 to 50 min. The cells were then cleaned at a potential of 1000 mV for 2 min. The injection volume of the samples was 10 μL. The eluent was monitored by the Coulochem electrode array system (CEAS) with potential settings at −50, 0, 100, 200, 300, 400, 500, and 600 mV. Data for Figure 2B1 was from the channel set at 200 mV of the CEAS. For CA, the limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 and 0.5 μg/mL, respectively. The C
DOI: 10.1021/acs.jafc.5b01246 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 2. Effects of long-term carnosic acid (CA) enriched rosemary extract (RE) (RE#1L, RE#1H, and RE#2) administration on body weight (BW) (A), weight gain (B), and body composition (C) in high-fat-diet-fed C57BL/6J mice. Results are means ± standard deviations (n = 15 for A and B; n = 5 for C) represented by vertical bars. The asterisk (*) denotes a significant difference of the HF group in comparison with the LF group: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. Significant differences (p < 0.05, ANOVA) are identified by the different letters among HF, RE#1L, RE#1H, and RE#2 groups. phase was then removed and transferred to a new tube, and the sample was evaporated to dryness by a nitrogen evaporator and weighed. Hepatic GSH/GSSG and Oxidative Stress Levels. The ratio of GSH/GSSG in the liver was determined by a commercial Glutathione Assay Kit (Trevigen, Gaithersburg, MD, USA). The lipid peroxidation product malondialdehyde (MDA) in the plasma and liver was measured by the thiobarbituric acid reactive substances (TBARS) method using a commercial kit (Cayman Chemical, Ann Arbor, MI, USA). Liver Histology. Liver tissues were fixed with 10% formalin and processed for paraffin embedding. Tissue sections were cut to 5 μm and stained with hematoxylin and eosin (H&E) for assessing histopathological changes. Plasma and Liver AGEs. The plasma AGE level was measured on the basis of the methods of Henle,24 Munch,25 and Raposeiras-Roubin.26 In brief, plasma was diluted 1/20 with PBS (pH 7.4), and the fluorescence intensity was recorded at 360 nm excitation and 460 nm emission wavelengths by a multimode microplate reader (Synergy 2, Biotek). Fluorescence intensity was expressed in arbitrary units (AU), and the fluorescence was normalized with the LF group. The liver AGE level was determined according to the methods of Nakayama27 and Yamabe.28 In brief, minced liver tissue was delipidated with chloroform and methanol (2/1, v/v) with overnight shaking. After it was washed with methanol and distilled water, the tissue was homogenized in 0.1 N NaOH, followed by centrifugation at 8000g for 15 min at 4 °C. The amounts of AGEs in these alkali-soluble samples were determined by measuring fluorescence at 360 nm excitation and 460 nm emission wavelengths. The fluorescence reading of 1 mg/mL of BSA was defined as 1 unit. The fluorescence values of samples were measured at a protein concentration of 1 mg/mL and expressed in arbitrary units (AU) normalized by the reading from 1 mg/mL of BSA. Western Blot Assay of Proteins. Whole protein lysates of liver tissue were extracted using RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, Thermo Fisher Scientific) supplemented with 1% protease inhibitor cocktail and 1% phenylmethylsulfonyl fluoride. Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). Aliquots containing 50 μg of protein were loaded onto a 10− 12% sodium dodecyl sulfatepolyacrylamide gel, transblotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), blocked with Tris-buffered saline with 1% Casein with 0.1% Tween-20, and then incubated with each of the primary antibodies of RAGE and β-actin. The membrane was then incubated with horseradish peroxidase conjugated
donkey antirabbit or goat antimouse IgG (Cell signaling, Danvers, MA, USA). The bound complexes were detected with SuperSignal Chemiluminescent Subtract (Thermo Scientific, Rockford, IL, USA). The immunoblot bands were quantified by densitometry analysis, and the ratio to β-actin was calculated and presented, setting the values of low-fat diet fed mice as 1. Statistical Analysis. All results are presented as means ± standard deviation. A Student’s t test was performed using GraphPad Prism version 5.04. An asterisk (*) means a significant difference of HF group in comparison with the LF group: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. The intergroup variation was measured by one-way ANOVA with Tukey’s test. Significant differences are identified by the different letters among HF, RE#1L, RE#1H, and RE#2 groups, and a p value of
