Oxidative Medicine and Cellular Longevity

Oxidative Medicine and Cellular Longevity / 2019 / Article
Special Issue

Applications of Antioxidants in Metabolic Disorders and Degenerative Diseases: Mechanistic Approach

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Research Article | Open Access

Volume 2019 |Article ID 5360560 | 23 pages | https://doi.org/10.1155/2019/5360560

Encapsulated Mulberry Fruit Extract Alleviates Changes in an Animal Model of Menopause with Metabolic Syndrome

Academic Editor: Simona Bungau
Received28 Oct 2018
Revised29 Jan 2019
Accepted20 Feb 2019
Published28 Apr 2019

Abstract

Currently, the therapeutic strategy against metabolic syndrome and its complications is required due to the increasing prevalence and its impact. Due to the benefits of both mulberry fruit extract and encapsulation technology, we hypothesized that encapsulated mulberry fruit extract (MME) could improve metabolic parameters and its complication risk in postmenopausal metabolic syndrome. To test this hypothesis, female Wistar rats were induced experimental menopause with metabolic syndrome by bilateral ovariectomy (OVX) and high-carbohydrate high-fat (HCHF) diet. Then, they were orally given MME at doses of 10, 50, and 250 mg/kg BW for 8 weeks and the parameters, such as percentage of body weight gain, total cholesterol, triglycerides, HDL-C, LDL-C, atherogenic index, fasting blood glucose, plasma glucose area under the curve, serum angiotensin-converting enzyme (ACE), oxidative stress status, histology, and protein expression of PPAR-γ, TNF-α, and NF-κB in adipose tissues were determined. MME improved body weight gain, adiposity index, glucose intolerance, lipid profiles, atherogenic index, ACE, oxidative stress status, and protein expression of TNF-α and NF-κB. Moreover, MME attenuated adipocyte hypertrophy and enhanced PPAR-γ expression. Taken altogether, MME decreased metabolic syndrome and its complication via the increased PPAR-γ expression. Therefore, MME is the potential candidate for improving metabolic syndrome and its related complications. However, further research in clinical trial is still necessary.

1. Introduction

Metabolic syndrome (MetS), one of the important noncommunicating diseases (NCDs), is continually increasing. It has been reported that the global prevalence of MetS in female is higher than that in male. It has been reported that the prevalence of MetS in male and female are around 23% and 29%, respectively [1]. This prevalence is also elevated in postmenopausal women [2, 3]. In addition, postmenopausal condition increases vulnerability to many metabolic disorders, including obesity, hypertension, insulin resistance, glucose intolerance, and dyslipidemia [4]. Moreover, it has been reported that postmenopausal metabolic syndrome is also associated with the development of adipose tissue oxidative stress and inflammation [5]. Even though the increasing rate of metabolic syndrome is alarming its importance to the world, the current therapeutic efficacy is still limited [68]. Therefore, a novel protective strategy against MetS that is cheap and easy to approach is required.

Recently, it has been demonstrated that substances that are rich in polyphenolic compounds, especially anthocyanins, can improve metabolic disorders in menopause [911]. Therefore, the application of anthocyanin-rich substances against menopause-related disorders, such as MetS and fatty liver, has gained much attention. Ripened mulberry fruits (Morus alba L.) are rich in anthocyanins and possess antioxidant [1214], antidyslipidemia [1517], antidiabetes [12, 1618], antiobesity [19], anti-inflammation [19], and antiartherosclerosis [20] effects. In addition, several studies have demonstrated that mulberry extract can also attenuate oxidative stress-related disorders [21, 22].

Despite numerous health benefits, active ingredients in mulberry fruit, such as polyphenolic compounds including anthocyanins and flavonol glycosides, are unstable and highly labile [23, 24]. In addition, most of these substances are poorly absorbed and are instable during food processing, distribution, or storage or in the gastrointestinal tract [2527]. All of these limitations could be solved by the formulation process. Interestingly, encapsulation technology which protects the core material by using a carrier wall can increase the stability by decreasing the decay induced by the environment, increase the solubility, and mask the undesirable taste [27, 28]. Due to the advantages of polyphenol-rich substances and mulberry fruits together with the benefit of encapsulation technology on the bioavailability mentioned earlier, we hypothesized that microencapsulated mulberry fruit extract could improve metabolic syndrome in menopause. To elucidate this issue, this study is aimed at determining the effect of the microencapsulated mulberry fruit extract on metabolic disorders in ovariectomized (OVX) rats fed with high-carbohydrate high-fat diet (HCHF), an animal model of menopausal women with metabolic syndrome.

2. Materials and Methods

2.1. Preparation of Encapsulated Mulberry Fruit Extract

Ripened mulberry fruits (Morus alba L. var. Chiangmai) were collected from the Queen Sirikit Department of Sericulture Center, Udon Thani Province. The fresh mulberry fruits were cleaned and dried in the oven (Memmert GmbH, USA) at 60°C for 72 hours. The dried mulberry was grounded to fine powder and prepared as 50% alcohol extract by the maceration technique. The extract was filtered with Whatman No.1 filter paper and dried in the oven (Memmert GmbH, USA) at 60°C for 24 hours. In this study, maltodextrin dextrose equivalent 10 (DE10) was selected as the encapsulation matrix. It was mixed with mulberry fruit extract at the ratio of 9 : 1 (). Then, the mixture was dissolved in warm distilled water at 50°C and stirred for 30 minutes. Following this process, the solution was frozen at -20°C for 18 hours in a freezer and subjected to a drying process in a freeze dryer (Labconco freeze dryer, Labconco Corporation, Kansas City, MO, USA) for 48 hours (−86°C, 0.008 mbar). The dry sample was packed and stored in a desiccator containing silica gel at 4°C.

2.2. Measurement of Total Phenolic Compound Contents and Flavonoid Contents

The amount of total phenolic compounds in the sample was determined by using the Folin-Ciocalteu colorimetric method via a microplate reader (iMark™ Microplate Absorbance Reader) [29]. The freshly prepared reagent consisting of 158 μl of distilled water and 20 μl of 50% Folin-Ciocalteu reagent (Sigma-Aldrich, USA) was mixed with 20 μl of the extract and incubated for 8 minutes. Following this process, 30 μl of 20% sodium carbonate (Na2CO3) (Sigma-Aldrich, USA) was added and incubated at room temperature in a dark room for 2 hours. Then, absorbance was measured at 765 nm. Results were expressed as mg gallic acid equivalent (GAE)/mg extract. Various concentrations of gallic acid (Sigma-Aldrich, USA) were used as a standard calibration curve.

Flavonoid content was assessed by using the aluminum chloride method [30]. In brief, 100 μl of the extract at various concentrations was mixed with 100 μl of 2% methanolic aluminum chloride (Sigma-Aldrich, USA) and incubated at room temperature in a dark room for 30 minutes. At the end of the incubation period, absorbance at 415 nm was taken against the suitable blank. Various concentrations of quercetin (Sigma-Aldrich, USA) were used for the standard calibration curve preparation. Results were expressed as μg quercetin equivalent/mg extract.

2.3. Fingerprint Chromatogram Assessment

High-performance liquid chromatography (HPLC) analysis was used to determine the fingerprint chromatogram. Chromatography was performed by using a Waters® system equipped with a Waters® 2998 photodiode array detector. Chromatographic separation was performed using a Purospher® STAR, C-18 encapped column (5 μm) and LiChroCART® 250-4.6 HPLC cartridge, Sorbet Lot No. HX255346 (Merck, Germany). The mobile phase (HPLC-grade) consisted of 100% methanol (solvent A) (Fisher Scientific, USA), and 2.5% acetic acid (solvent B) (Fisher Scientific, USA) in deionized (DI) water was used to induce gradient elution. The gradient elution was carried out at a flow rate of 1.0 ml/min with the following gradient: 0-17 min, 70% A, 18-20 min, 100% A; 20.5-25 min, 10% A. The sample was filtered (0.45 μm, Millipore), and a direct injection of the tested sample at the volume of 20 μl on the column was performed. Chromatogram detection was performed at 280 nm using a UV detector, and data analysis was performed using EmpowerTM3.

2.4. Determination of Biological Activities
2.4.1. Antioxidant Activity Assessment

Antioxidant activity of the extracts was determined by using 1,1-diphenyl-2-picryl-hydrazyl (DPPH), ferric reducing antioxidant power (FRAP), and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. For the DPPH assay, an aliquot of 0.15 mM DPPH in methanol (180 μl) (Sigma-Aldrich, USA) was mixed with 20 μl of various concentrations of extracts and incubated for 30 minutes. The absorbance was measured against the blank at 517 nm via a microplate reader (iMark™ Microplate Absorbance Reader) [31].

FRAP assays were carried out based on the ability of the tested substance to convert ferric tripyridyltriazine (Fe3+-TPTZ) to ferrous tripyridyltriazine (Fe2+-TPTZ). The FRAP working solution was prepared by mixing 300 mM acetate buffer (Sigma-Aldrich, USA), 10 mM TPTZ (Sigma-Aldrich, USA), and 20 mM ferric chloride (FeCl3) (Sigma-Aldrich, USA) solutions at a ratio of 10 : 1 : 1, respectively. In brief, 190 μl of the FRAP reagent was mixed with 10 μl of extract and incubated at 37°C for 10 minutes. After the incubation, an absorbance at 593 nm was measured against the blank [32]. Ascorbic acid was used as positive control, and results were expressed as value.

2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was also used to determine the free radical scavenging activity of the microencapsulated mulberry fruit extract [33]. The ABTS solution was prepared by mixing 7 mM ABTS (Sigma-Aldrich, USA) and 2.45 mM potassium persulfate (K2S2O8) (Sigma-Aldrich, USA) at a ratio of 2 : 3 () and diluted with deionized water at a ratio of 1 : 20 () before use. In brief, 30 μl of various concentrations of extracts was mixed with 120 μl of distilled water and 30 μl of ethanol and reacted with 3 ml of ABTS solution. The absorbance was measured at 734 nm with a spectrophotometer (Pharmacia LKB-Biochrom 4060). Trolox was used as a standard. Results were expressed in terms of (concentration in micrograms per milliliter required to inhibit ABTS radical formation by 50%).

2.4.2. Measurement of Carbohydrate-Metabolizing Enzymes

The effects of extract on carbohydrate-metabolizing enzymes were assessed by focusing on the alterations of α-amylase, α-glucosidase, and aldose reductase enzymes. The assessment of α-amylase inhibition activity was measured using the spectrophotometric method with slight modification [34]. In brief, the substrate solution was prepared by dissolving 1 g of starch (Univar) and 0.01 M calcium chloride (CaCl2) (Sigma-Aldrich) in 100 ml of 0.5 M Tris-HCl buffer, pH 6.9 (Sigma-Aldrich), boiled for 5 minutes, and incubated at 37°C for 5 minutes before use. Then, the reaction mixture consisting of 25 μl of porcine pancreatic amylase (2 units/ml, Sigma-Aldrich) and 50 μl of substrate solution was mixed with 50 μl of various concentrations of extracts and incubated at 37°C for 10 minutes. The reaction was stopped by adding 125 μl of 50% acetic acid and centrifuged at 3000 rpm, 4°C, for 5 minutes. The supernatant was harvested and absorbance measured at 595 nm.

The inhibitory effect of extract on α-glucosidase was determined by the chromogenic method [35]. A stock solution of α-glucosidase was prepared by dissolving 2 units of α-glucosidase (Sigma-Aldrich) in 1 ml of 50 mM phosphate buffer, pH 6.8. The reaction mixture containing 20 μl of various concentrations of the sample, 60 μl of α-glucosidase enzyme, and 20 μl of 1 mM para-nitrophenyl-glucopyranoside (Sigma-Aldrich) was incubated at 37°C for 20 minutes. Following this process, the reaction was stopped by adding 50 μl of 1 M sodium carbonate (Sigma-Aldrich) and absorbance was measured at 405 nm.

Aldose reductase activity was measured via a spectrophotometric method by measuring the reduction in the absorption of NADPH at 390 nm over a 4-minute period with 10 mM DL-glyceraldehyde as the substrate [36]. The tissue sample was isolated and prepared as homogenate in 100 mM potassium phosphate buffer, pH 6.2, and subjected to a 10,000 rpm centrifugation at 4°C for 30 minutes. The supernatant was harvested and served as the source of aldose reductase. The assay mixture containing 100 μl of the aldose reductase enzyme, 300 μl of 0.1 M sodium phosphate buffer (pH 6.2), 100 μl of 0.15 mM NADPH (Sigma-Aldrich), 300 μl of 10 mM DL-glyceraldehyde, and 100 μl of different concentrations of the extract was recorded at the absorbance of 390 nm with a spectrophotometer (Pharmacia LKB-Biochrom 4060) as T0. After 4 minutes of incubation period, the absorbance was recorded as T4. Aldose reductase activity was calculated and expressed in terms of (concentration in micrograms per milliliter required to inhibit enzyme activity by 50%). Based on the suppression effect of quercetin on aldose reductase in the previous study, it was used as positive control [36].

2.4.3. The Suppression Effect on Angiotensin-Converting Enzyme

The assessment was performed based on the cleavage of the substrate hippuryl-glycyl-glycine by ACE and subsequent reaction with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to form 2,4,6-trinitrophenyl-glycyl-glycine [37]. In this study, the angiotensin-converting enzyme from rabbit lungs (Sigma, USA) was used as enzyme source and samples were prepared at various concentrations. An aliquot of the angiotensin-converting enzyme (0.05 units/ml, 10 μl) was mixed with various concentrations of the extract at the volume of 20 μl and served as an experimental sample or mixed with phosphate buffer (5 mM, pH 8.3) and served as control. Captopril was used as a positive control. Following this process, 50 μl of 100 mM Hip-Gly-Gly (Sigma, USA) was mixed with the reaction mixture and incubated at 37°C for 35 minutes. The reaction was stopped by adding 120 μl of 3 M sodium tungstate (Sigma, USA) and 0 .5M sulfuric acid (Sigma, USA) and centrifuged at 2500 rpm for 10 minutes. After the centrifugation, an aliquot of the supernatant was placed into a 96-well microtiter plate, mixed with 20 μl of 60 mM TNBS (Sigma, USA), and incubated in dark conditions for 20 minutes. At the end of the incubation period, an absorbance at 415 nm was recorded with a microplate reader (iMark™ Microplate Absorbance Reader). Results were expressed as .

2.4.4. Assessment of Pancreatic Lipase Activity

The working solution of lipase at a concentration of 10 mg/ml was prepared by dissolving lipase from porcine pancreas type II (Sigma, USA) in deionized (DI) water and subjected to a 16,000 rpm centrifugation for 5 minutes. The supernatant was harvested for further use. In this study, 100 mM Tris buffer pH 8.2 and p-nitrophenyl laurate (pNP laurate) were used as the substrate. The pNP laurate was dissolved in 5 mM sodium acetate (pH 5.0) containing 1% Triton X-100 to produce 0.08% substrate solution and served as stock solution. This solution was heated in boiling water for 1 minute, mixed well, and cooled down at room temperature. The reaction mixture containing 70 μl of assay buffer, 90 μl of substrate solution, 30 μl of lipase, and 10 μl of the extracts was mixed and incubated at 37°C for 2 hours. At the end of the incubation period, the solution was centrifuged at 16,000 rpm for 1 minute and absorbance was measured with a microplate reader (iMark™ Microplate Absorbance Reader) at 400 nm [38]. In this study, orlistat was used as positive control.

2.4.5. Assessment of Cyclooxygenase-2 (COX-2) Activity

COX-2 inhibition was measured by using a colorimetric COX-2 inhibitor screening assay kit (Cayman Chemical, USA). COX-2 inhibition activity was performed according to the manufacturer’s protocol. In brief, COX-2 working solution was prepared by dissolving COX-2 substance in 100 mM Tris-HCl buffer, pH 8.0, at a ratio of 1 : 100. The reaction mixture containing 150 μl of assay buffer, 10 μl of extracts, 10 μl of heme (Cayman Chemical, USA), 10 μl of COX-II working solution, 20 μl of 10 μM TMPD (N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride) (Sigma, USA), and 20 μl of 100 μM arachidonic acid (Cayman Chemical, USA) was added to 96-well microtiter plates and incubated at room temperature for 30 minutes. At the end of the incubation period, an absorbance at 590 nm was recorded and results were expressed as [39]. Indomethacin was used as a reference compound.

2.5. Experimental Protocol

A total of 48 female Wistar rats (weighing 200-250 g, 10 weeks old) were obtained from the National Laboratory Animal Center, Salaya, Nakhon Pathom, Thailand. The rats were kept under standard laboratory conditions with food and water ad libitum and housed in standard metal cages (6 per cage). Temperature was controlled at on a 12 : 12 h light-dark cycle. All procedures and experimental protocols were approved by the Institutional Animal Ethics Committee of Khon Kaen University (record no. AEKKU 27/2017). After 1 week of acclimatization, the animals were divided into 8 groups as the following. Group I:Normal diet (ND) + vehicle: all rats in this group received normal diet and were treated with vehicle.Group II:HCHF + vehicle: all rats in this group received high-carbohydrate high-fat diet (HCHF) diet and treated with vehicle.Group III:OVX-HCHF diet + vehicle: all animals in this group were subjected to bilateral ovariectomy (OVX), received HCHF diet, and treated with vehicle.Group IV:OVX-HCHF diet + isoflavone: rats in this group were subjected to bilateral ovariectomy, fed with HCHF diet, and treated with soy isoflavone extract (Fisiogen®) at dose of 15 mg/kg BW.Group V:OVX-HCHF diet + L-carnitine: rats in this group were subjected to bilateral ovariectomy, fed with HCHF diet, and treated with L-carnitine at a dose of 250 mg/kg BW.Group VI-VIII:OVX-HCHF diet + encapsulated mulberry fruit extract (MME): all rats in these groups were subjected to OVX, received HCHF diet, and treated with MME at various doses ranging from 10 to 50 to 250 mg/kg BW.

In this study, all OVX rats were anesthetized with thiopental sodium at a dose of 40 mg/kg BW prior to the induction of experimental menopause by bilateral ovariectomy. In brief, rats were placed in prone position and fixed to the operating table. Ovariectomy was performed by two dorsolateral incisions, approximately 1 cm long above the ovaries. The bulged area on the back was shaved bilaterally, and the skin and muscle were dissected. After the muscle dissection, the peritoneal space and adipose tissue surrounding the ovary were exposed. After identifying the ovary and uterine horn, ligation was performed at the distal uterine horn to remove the ovarian tissue completely in one action. The horn was returned to the abdominal cavity, and the muscle and skin were sutured [40, 41]. Then, the OVX rats were returned to their cage, after postoperation care. After 1 week of operation, animals were fed with a normal diet (a standard laboratory diet No. 082, C.P. Company, Bangkok, Thailand) which contained total energy around 4.02 kcal/g (fat 19.77%, protein 28.24%, and carbohydrate 51.99%) or high-carbohydrate high-fat (HCHF) diet which contained total energy around 4.62 kcal/g (fat 31.54%, protein 20.25%, and carbohydrate 48.21%). All animals were fed with either normal diet or HCHF diet for 20 weeks, and rats received HCHF diet which showed a percent change of body weight more than 40 percent; the homeostasis model assessment-estimated insulin resistance (HOMA-IR) index higher than that in the control group was selected for further study. Then, the recruited animals were randomly assigned to various treatment groups, including vehicle or distilled water, isoflavone, L-carnitine, and MME at various doses ranging from 10 to 50 to 250 mg/kg BW (the total volume of administration is 0.5 ml). All animals’ food intake and body weight changes were monitored every week. In addition, the parameters of metabolic syndrome, including total plasma cholesterol, triglycerides, low-density lipoprotein (LDL), high-density lipoprotein (HDL), atherogenic index (AI index), and fasting plasma glucose were determined, and an oral glucose tolerance test (OGTT) was performed both at 4 weeks and at 8 weeks. At the end of the study period, all animals were sacrificed and fat pads were isolated from various areas, including subcutaneous fat and intra-abdominal fat (gonadal, mesenteric, and retroperitoneal fat). The adipose tissue was kept cool in ice buckets and stored at -80°C until used. The adiposity index and histology of adipocytes were determined. Serum biochemicals, including angiotensin-converting enzyme, were also assessed. In addition, the oxidative stress status of adipose tissue was also determined using the malondialdehyde (MDA) level and the activities of main scavenging enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT). Moreover, the expression of PPAR-γ, TNF-α, and NF-κB in adipose tissues was also determined.

2.6. Biochemical Assays
2.6.1. Plasma Lipid Profile Assessment

Total plasma cholesterol was determined by using the “CHOD-PAP” enzymatic photometric test [42]. An aliquot of the sample or calibrator at the volume of 10 μl was mixed with 1,000 μl of cholesterol FS Reagent (DiaSys Diagnostic Systems GmbH, Germany) and incubated at 25°C in a dark room for 20 minutes. Following an incubation period, an absorbance at 500 nm was measured within 60 minutes by using a UV spectrophotometer (Pharmacia LKB-Biochrom 4060). Total cholesterol was expressed as mg/dl and calculated as follows:

Plasma triglyceride level was determined with the enzymatic colorimetric test by using glycerol-3-phosphate oxidase (GPO) reagent (DiaSys Diagnostic Systems GmbH, Germany). In brief, 10 μl of the plasma or calibrator and 1,000 μl of reagent were mixed and incubated at 25°C in the dark room for 10 minutes. The absorbance was measured at 500 nm within 60 minutes by using a UV spectrophotometer (Pharmacia LKB-Biochrom 4060). Triglyceride level was expressed as mg/dl and calculated as follows:

Plasma LDL-C was determined with the Friedewald equation [43] by using LDL-C select FS Reagent 1 and 2 (DiaSys Diagnostic Systems GmbH, Germany). In brief, 5 μl of plasma or TruLab L calibrator and 280 μl of reagent 1 were mixed and incubated at 37°C for 5 minutes. After incubation, an absorbance at 595 nm (A1) was measured by using the microplate reader (iMark™ Microplate Absorbance Reader). Then, 70 μl of reagent 2 was added to the mixture and incubated at 37°C for 5 minutes. After incubation, the absorbance at 595 nm (A2) was measured. LDL-C was expressed as mg/dl and calculated as follows:

Plasma HDL was determined by using HDL-C select FS Reagent 1 and 2 (DiaSys Diagnostic Systems GmbH, Germany) based on the same basic principle as that of cholesterol determination [44]. In brief, 5 μl of plasma or TruLab L calibrator and 240 μl of reagent 1 were mixed and incubated at 37°C for 5 minutes. After incubation, an absorbance at 595 nm (A1) was measured by using the microplate reader (iMark™ Microplate Absorbance Reader). Then, 60 μl of reagent 2 was added to the mixture and incubated at 37°C for 5 minutes. After incubation, the absorbance at 595 nm (A2) was measured. HDL-C was expressed as mg/dl and calculated as follows:

2.6.2. Determination of Atherogenic Index (AI)

The atherogenic index (AI index) is the most reliable indicator for the prediction of cardiovascular disease risk. The AI index was calculated by the total cholesterol (TC)/high-density lipoprotein cholesterol (HDL-C) ratio [45].

2.6.3. Assessment of Fasting Plasma Glucose and Oral Glucose Tolerance Test (OGTT)

To determine the fasting plasma glucose level, all animals were fasted for 12 hours. After the food deprivation period, basal blood glucose concentrations were measured by collected blood samples from the tail vein using the Accu-Chek® Performa blood glucose meter.

According to the determination of the oral glucose tolerance test, the animals were administered 40% glucose solution at a dose of 2 g/kg of BW by oral gavage. The tail vein blood samples were taken at 30, 60, 90, and 120 minutes after the glucose administration [46]. The plasma glucose area under the curve (AUC) was calculated by trapezoidal approximation of plasma glucose levels and expressed as mg h/dl.

2.6.4. Determination of Plasma Angiotensin-Converting Enzyme Activity

Plasma angiotensin-converting enzyme assay was performed by using the modified method of Serra et al. [37]. The enzymatic reaction was started by adding 20 μl of plasma into 50 μl of substrate solution Hip-Gly-Gly (100 mmol/l) (Sigma, USA) and incubated at 37°C for 35 minutes. The reaction was stopped by adding 120 μl of 3 M sodium tungstate (Sigma, USA) and 0 .5M sulfuric acid (Sigma, USA) and centrifuged at 2500 rpm for 10 minutes. After the centrifugation, an aliquot of the supernatant was placed into a 96-well microtiter plate and mixed with 20 μl of 60 mM TNBS (Sigma, USA), incubated in dark conditions for 20 minutes. At the end of the incubation period, an absorbance at 415 nm was recorded with the microplate reader (iMark™ Microplate Absorbance Reader). The standard calibration curve was prepared by using the angiotensin-converting enzyme (Sigma-Aldrich, USA) at the concentration range of 0.001-1 units/ml. Results were expressed as units/mg protein.

2.7. Measurement of Serum Estradiol

The blood sample of each animal was collected. Then, it was centrifuged immediately at at 4°C for 15 min. The obtained serum was kept at −80°C until the time of measurement use. The estradiol level in the samples was measured using an Estradiol DSL-4400 Radioimmunoassay kit (Diagnostic Systems Laboratories Inc., Webster, TX, USA) according to the manufacturer’s instructions.

2.8. Histological Procedure and Adiposity Assessment

After the scarification, fat pads from various areas, including subcutaneous, gonadal, mesenteric, and retroperitoneal areas, were removed and immersed into a fixative solution containing 10% formalin (Sigma-Aldrich, USA) for 72 hours. Serial sections of tissues were cut frozen on a cryostat (Thermo Scientific™ HM 525 Cryostat) at 10 μm thickness. All sections were picked up on slides coated with 0.3% aqueous solution of gelatin containing 0.05% aluminum potassium sulfate (Sigma-Aldrich, USA). All assessments were observed after being hematoxylin-eosin- (H&E-) (Sigma-Aldrich, USA) stained and photographed [47]. Adipocyte cell diameter and density were estimated from 3 randomly selected different fields of each area by using Olympus light microscope model BH-2 (Japan) under 40x magnification with the PixeLINK PL-A6xx Capture and IT tool program. In addition, the adiposity index was calculated by the sum of intra-abdominal fat weight/body weight ratio × 100 and expressed as adiposity percentage.

2.9. Assessment of Oxidative Stress Status in Adipose Tissues

Adipose tissues were isolated and homogenized with 0 .1M potassium phosphate buffer solution, pH 7.4 (sample dilution 10 mg: PBS 50 μl). The derived homogenate was used for the determination of oxidative status, including malondialdehyde (MDA) level and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). The protein concentrations in adipose tissue homogenate were determined by using a Thermo Scientific NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware, USA), and the optical density at the wavelength of 280 nm was measured.

MDA level homogenate was assessed by thiobarbituric acid reaction [48]. The reaction mixture containing 50 μl of tissue homogenate, 50 μl of 8.1% sodium dodecyl sulfate (SDS) (Sigma-Aldrich, USA), 375 μl of 0.8% of thiobarbituric acid (TBA) (Sigma-Aldrich, USA), 375 μl of 20% acetic acid (Sigma-Aldrich, USA), and 150 μl of distilled water (DW) was boiled at 95°C in the water bath for 60 minutes. After boiling, it was cooled with tap water. Then, 250 μl of DW and 1,250 μl of the solution containing n-butanol and pyridine (Merck, Germany) at the ratio of 15 : 1 were added, mixed together, and centrifuged at 4000 rpm for 10 minutes. The upper layer was separated, and the absorbance was measured at 532 nm. TMP (1,1,3,3-tetramethoxypropane) (0-15 μM) (Sigma-Aldrich, USA) was served as standard, and the level of MDA was expressed as ng/mg protein.

The determination of SOD activity was performed according to the method of Sun et al. [49]. In brief, 20 μl of tissue sample was mixed with 200 μl of the reaction mixture containing 57 mM phosphate buffer solution (KH2PO4) (Sigma-Aldrich, USA), 0.1 mM EDTA (Sigma-Aldrich, USA), 10 mM cytochrome C (Sigma-Aldrich, USA) solution, 50 μM of xanthine (Sigma-Aldrich, USA), and 20 μl of xanthine oxidase (0.90 mU/ml) (Sigma-Aldrich, USA). The optical density at 415 nm was recorded. SOD enzyme (Sigma-Aldrich, USA) activities at the concentrations of 0-25 units/ml were used as standard, and the results were expressed as units/mg protein.

Catalase activity was assessed based on the ability of the enzyme to break down H2O2. In brief, the reaction mixture containing 50 μl of 30 mM hydrogen peroxide (in 50 mM phosphate buffer, pH 7.0) (BDH Chemicals Ltd., UK), 25 μl of 5 M H2SO4 (Sigma-Aldrich, USA), and 150 μl of 5 mM KMnO4 (Sigma-Aldrich, USA) was mixed with 10 μl of sample. After mixing thoroughly, an absorbance at 490 nm was measured [50]. CAT enzyme (Sigma-Aldrich, USA) at the concentration range between 10 and 100 units/ml was used as standard, and the result was expressed as units/mg protein.

Glutathione peroxidase activity was also assessed. In brief, 20 μl of the sample solution was mixed with the reaction mixture consisting of 10 μl of 1 mM dithiothreitol (DTT) (Sigma-Aldrich, USA) and 10 mM monosodium phosphate (NaH2PO4) in DW, 100 μl of 1 mM sodium azide (Sigma-Aldrich, USA) in 40 mM potassium phosphate buffer (pH 7.0), 10 μl of 50 mM glutathione (Sigma-Aldrich, USA) solution, and 100 μl of 30% hydrogen peroxide (BDH Chemicals Ltd., UK) and incubated at room temperature for 10 minutes. Then, 10 μl of 10 mM DTNB (5,5-dithiobis-2-nitrobenzoic acid) (Sigma-Aldrich, USA) was added and an absorbance at 412 nm was recorded [51]. The standard calibration curve was prepared by using the GSH-Px enzyme (Sigma-Aldrich, USA) at the concentration range of 1-5 units/ml. GSH-Px activity was expressed as units/mg protein.

2.10. Western Blotting Analysis

Adipose tissue was homogenized and lysed in RIPA (radioimmunoprecipitation assay) buffer (1 : 5, ) (Cell Signaling Technology, USA) containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, and 1 mM phenylmethanesulfonyl fluoride (PMSF) (Cell Signaling Technology, USA). The tissue homogenate supernatant of the middle layer of fat samples was isolated after the centrifugation at 4°C for 10 minutes. Protein concentration was determined by using a Thermo Scientific NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware, USA). In addition, eighty micrograms of tissue lysates was adjusted to an appropriate concentration by using Tris-Glycine SDS-PAGE loading buffer and heated at 95°C for 10 minutes. The protein in the tissue sample was isolated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by loading 20 μl of the tissue sample on SDS-polyacrylamide gel. Then, the separated bands were transferred to a nitrocellulose membrane, washed with 0.05% TBS-T, and incubated in blocking buffer (5% skim milk in 0.1% TBS-T) at room temperature for 1 hour. After the blocking process, the nitrocellulose membrane was incubated with anti-PPAR gamma (Abcam, UK; dilution 1 : 1000), anti-NF-κB p65 (Cell Signaling Technology, USA; dilution 1 : 500), anti-TNF-α (Cell Signaling Technology, USA; dilution 1 : 500), and anti-β-actin (Cell Signaling Technology, USA; dilution 1 : 1000) antibodies at 4°C, overnight. Following the incubation period, the nitrocellulose membrane was rinsed with TBS-T (0.05%) again and incubated with anti-rabbit IgG, HRP-linked antibody (Cell Signaling Technology, USA; dilution 1 : 2000) at room temperature for 1 hour. The bands were visualized and quantitated by using the ECL detection systems (GE Healthcare) and LAS-4000 luminescent image analyzer (GE Healthcare). Band intensities were measured for statistical analysis using ImageQuant TL v.7.0 image analysis software (GE Healthcare). The expression was normalized using anti-β-actin. Data were presented as a relative density to the control normal diet group.

2.11. Statistical Analysis

All data are expressed as error of mean (SEM). Statistical significance was evaluated by using one-way analysis of variance (ANOVA), followed by Duncan’s post hoc test. Student’s test was used for comparison of the means for two groups. Statistical significance was evaluated at values <0.05. All statistical data analyses were performed using SPSS version 21.0 (IBM Corp. Released 2012. IBM SPSS Statistics for Windows).

3. Results

3.1. Assessment of Phenolic Compounds and Biological Activities

Table 1 shows that the contents of total phenolic compounds and flavonoid contents of the encapsulated mulberry fruit extract (MME) were GAE/mg sample extract and quercetin/mg sample extract, respectively, whereas the contents of the substances just mentioned in mulberry fruit extract were GAE/mg sample extract and quercetin/mg sample extract. The flavonoid content in MME was significantly higher than that in mulberry fruit extract ( value = 0.001). Phenolic compositions were identified using HPLC with UV-visible detection, and data are shown in Table 1 and Figure 1. It was found that the microencapsulated mulberry fruit extract (MME) at 200 milligrams contained cyanidin 3-glucoside, gallic acid, and quercetin-3-O-rutinoside, whereas the mulberry fruit extract at 200 milligram contained cyanidin-3-glucoside, gallic acid, and quercetin-3-O-rutinoside. No significant differences in gallic acid and quercetin-3-O-rutinoside contents between MME and mulberry fruit extract fruit extract were observed. Interestingly, the content of cyanidin-3-glucoside in MME was significantly higher than that in mulberry fruit extract ( value = 0.045). Since metabolic syndrome is comprised of the disorders of obesity, diabetes, hypertension, and dyslipidemia, the effects of the mulberry fruit extract and MME on the enzymes playing important roles on the mentioned conditions were also investigated and results are shown in Table 1. Regarding the suppression effects of the important enzymes in carbohydrate metabolism, including α-amylase, α-glucosidase, and aldose reductase, it was found that values of MME were , , and , respectively, whereas those in mulberry fruit extract were , , and . When compared to mulberry fruit extract, MME exerted a better effect on the suppression of aldose reductase activity ( value = 0.016). The suppression effects of the mulberry fruit extract and MME on ACE, lipase, and COX-2 activities were also assessed. values of the suppression effects of ACE, lipase, and COX-2 of MME were , , and whereas those of the mulberry fruit extract were , , and , respectively. MME showed the significant potent suppression effects of ACE and COX-2 activities than those in mulberry fruit extract ( value = 0.019 and 0.006, respectively). In addition to the enzymes mentioned earlier, the pathophysiology of MetS also involved oxidative stress and inflammation. Therefore, the effects of MME on antioxidant were also explored. values of antioxidant activity assessed via DPPH, FRAP, and ABTS of MME were , , and , respectively. values of antioxidant via the methods just mentioned on mulberry fruit extract were , , and . The antioxidant activities assessed via DPPH and ABTS of MME were significantly better than those in the mulberry fruit extract ( value = 0.016 and 0.048, respectively).


ParametersUnitsME-50% hydroalcoholicMMEStandard reference

Total phenolicmg GAE/mg extract
Total flavonoidsμg quercetin/mg extract
Cyanidin 3-glucosideμg Cyn-3-glu/200 mg extract
Quercetin 3-O-rutinoside (rutin)μg quercetin-3-O-rutinoside/200 mg extract
Gallic acidμg gallic/200 mg extract
Antioxidant activities
 DPPH (mg/ml)0.43 ± 0.050.04 ± 0.010.03 ± 0.01, ascorbic acid
 FRAP (mg/ml)122.19 ± 12.82, ascorbic acid
 ABTS (mg/ml), Trolox
Antidiabetic markers
α-Amylase inhibition (mg/ml)
α-Glucosidase inhibition (mg/ml)
 Aldose reductase inhibition (mg/ml), quercetin
Cardiovascular marker
 ACE inhibition (mg/ml), Captopril
Obesity marker
 Lipase inhibition (mg/ml), Orlistat
Inflammatory marker
 COX-2 inhibition (mg/ml), Indomethacin

Data are presented as . Values are statistically significantly different by Student’s t test compared between MME and mulberry fruit extract. (, and 0.01, respectively).
3.2. Metabolic Parameter Changes
3.2.1. Effect on Food Intake, Energy Intake, and Body Fat

Table 2 shows that HCHF diet failed to produce significant changes in food intake, but it significantly enhanced energy intake in both normal and OVX rats ( value < 0.05 all; compared to normal rats which received normal diet and vehicle). In addition, HCHF diet also increased the weights of abdominal fat ( value<0.001 all; compared to normal rats which received normal diet and vehicle). Isoflavone, L-carnitine, and MME at a dose of 250 mg/kg BW could attenuate the increase in abdominal fat ( value < 0.05, 0.001, and 0.05, respectively; compared to OVX rats which received HCHF and vehicle) in OVX rats which received HCHF diet.


Treatment groupFood intake (g/day)Energy intake (kcal/g)Abdominal fat weight (g)

ND + vehicle
HCHF + vehicleaaaa
OVX + HCHF + vehicleaaaa
OVX + HCHF + isoflavone 15 mg/kg BW
OVX + HCHF + L-carnitine 250 mg/kg BW
OVX + HCHF + MME 10 mg/kg BW
OVX + HCHF + MME 50 mg/kg BW
OVX + HCHF + MME 250 mg/kg BW

Data are presented as (/group). a, aaa value < 0.05 and 0.001, respectively, compared to control rats which received normal diet and vehicle and , value < 0.05 and 0.001, respectively, compared to OVX rats which received HCHF and vehicle. ND: normal diet; HCHF: high-carbohydrate high-fat diet; OVX + HCHF: ovariectomized rats which received high-carbohydrate high-fat diet; MME10, 50, and 250: an encapsulated mulberry fruits extract at doses of 10, 50, and 250 mg/kg BW, respectively.
3.2.2. Effect on Body Weight Gain

The effects of MME on the metabolic parameters are shown in Table 3. It was found that HCHF significantly increased the percent of body weight gain both in normal and in OVX rats ( and 0.033, respectively; compared to normal rats, which received normal diet and vehicle). Both isoflavone and L-carnitine, which served as positive control in this study, could counteract the increase in the percent of body weight gain induced by HCHF in ovariectomized rats ( value = 0.009 and 0.006, respectively; compared to OVX rats, which received HCHF and vehicle). OVX rats which received MME at doses of 10 and 250 mg/kg BW also showed the significant reduction in percent of body weight gain ( value = 0.048 and 0.016, respectively; compared to OVX rats, which received HCHF and vehicle).


ParametersND + vehicleHCHF + vehicleOVX + HCHF + vehicleOVX + HCHF + isoflavone15OVX + HCHF + L-car250OVX + HCHF + MME10OVX + HCHF + MME50OVX + HCHF + MME250

Body weight gain (%)aa
TC (mg/dl)
4-weekaaa,bbb
8-weekaa,b
Triglycerides (mg/dl)
4-weekaaa,bbb
8-weekaaa,b
HDL-C (mg/dl)
4-weekaaaaaa
8-weekaaaaaa
LDL-C (mg/dl)
4-weekaaa
8-weeka
Atherogenic index
4-weekaaaa
8-weekaaaaaa
FBG (mg/dl)
4-weeka
8-weekaaaa
Plasma glucose AUC (mg h/dl)
4-weekaaa
8-weekaaaaa,bb
ACE (units/mg.protein)aaaaaa

Data are presented as (/group). a, aa, aaa value < 0.05, 0.01, and 0.001, respectively; compared to control rats, which received normal diet and vehicle, b, bb, bbb value <0.05, 0.01, and 0.001, respectively, compared to normal rats, which received HCHF diet and vehicle and , , value < 0.05, 0.01, and 0.001, respectively, compared to OVX rats, which received HCHF and vehicle.
3.3. Effect on Lipid Profiles and Atherogenic Index

Serum lipid profiles, including total cholesterol, triglycerides, HDL-C, and LDL-C, are shown in Table 3. The current data demonstrated that HCHF diet treatment for 4 weeks significantly decreased HDL-C but increased LDL-C and failed to produce the significant changes of total cholesterol and triglycerides ( value < 0.001 and 0.033, respectively, compared to normal rats, which received normal diet and vehicle) in normal rats. However, HCHF diet could increase total cholesterol, triglycerides, and LDL-C but decreased HDL-C in OVX rats at this duration of treatment ( value < 0.001, value < 0.001, value = 0.005, and value < 0.001, respectively, compared to normal rats which received normal diet and vehicle). OVX rats, which were treated with HCHF diet and received isoflavone, showed the significant decrease in total cholesterol, triglycerides, and LDL-C ( value < 0.001, value = 0.003, and value < 0.001, respectively, compared to OVX rats which received HCHF and vehicle) but failed to show the significant change in HDL-C. OVX rats, which received HCHF diet and received L-carnitine, significantly decreased total cholesterol, triglycerides, and LDL-C but increased decreased HDL-C in OVX rats at this duration of treatment ( value <0.001, value < 0.001, value = 0.023, and value < 0.001, respectively, compared to OVX rats which received HCHF and vehicle). At the 4-week intervention period, all doses of MME used in this study also significantly decreased total cholesterol ( value = 0.001, value < 0.001, and value < 0.001, respectively, compared to OVX rats, which received HCHF and vehicle), triglycerides ( value = 0.046, value = 0.006, and value = 0.001, respectively, compared to OVX rats which received HCHF and vehicle), and LDL-C ( value <0.001, value = 0.001, and value = 0.036, respectively, compared to OVX rats which received HCHF and vehicle) but increased HDL-C ( value = 0.003, value = 0.001, and value < 0.001, respectively, compared to OVX rats, which received HCHF and vehicle). When the treatment was prolonged to 8 weeks, HCHF diet still failed to change total cholesterol and triglyceride levels. The decreased HDL-C was still observed ( value < 0.001; compared to normal rats, which received normal diet) whereas the increased LDL-C did not present in normal rats anymore. In OVX rats, HCHF diet could increase total cholesterol, triglycerides, and LDL-C but increased HDL-C ( value = 0.001, value < 0.001, -value = 0.037, and value < 0.001, respectively, compared to normal rats which received normal diet and vehicle). At the 8-week intervention period, OVX rats which received HCHF diet and isoflavone, L-carnitine, and all doses of MME significantly mitigated the elevation of total cholesterol ( value < 0.001, value = 0.012, value = 0.007, value = 0.014, and value = 0.013, respectively, compared to OVX rats which received HCHF and vehicle), triglycerides ( value = 0.008, value < 0.001, value = 0.048, value = 0.022, and value = 0.005, respectively, compared to OVX rats which received HCHF and vehicle) and LDL-C ( value < 0.001 all; compared to OVX rats which received HCHF and vehicle). The reduction of HDL-C in OVX rats which received HCHF diet was also attenuated by L-carnitine and all doses of MME ( value < 0.001, value < 0.001, value = 0.004, and value = 0.009, respectively, compared to OVX rats which received HCHF and vehicle).

Atherogenic index (AI), one of the parameters used for indicating cardiovascular risk, was also investigated. The current data showed that at 4 and 8 weeks of the study period, HCHF diet could increase AI in both normal and OVX rats ( value = 0.003 and value = 0.005, respectively, and -value < 0.001 all, compared to normal rats which received normal diet and vehicle). At 4 weeks of intervention, the elevation of AI in OVX rats which received HCHF was attenuated by L-carnitine and MME at doses of 10, 50, and 250 mg/kg BW ( value = 0.041, value = 0.049, value = 0.020, and value = 0.038, respectively, compared to OVX rats which received HCHF and vehicle). When the treatments were prolonged to 8 weeks, the improved AI in OVX rats with HCHF diet treatment was still observed in those which received isoflavone, L-carnitine, and all doses of MME ( value = 0.020, value < 0.001, value < 0.001, value < 0.001, and value < 0.001, respectively, compared to OVX rats, which received HCHF and vehicle) as shown in Table 3.

3.4. Changes of Fasting Blood Sugar and Glucose Area under the Curve during Oral Glucose Tolerance Test (OGTT)

It was found that HCHF diet increased fasting blood sugar in normal rats at 8 weeks and in OVX rats at both 4 and 8 weeks of intervention ( value < 0.001, value = 0.026, and value = 0.018, respectively, compared to normal rats which received normal diet and vehicle). After 4 weeks of treatment, OVX rats which received MME at doses of 10, 50, and 250 mg/kg BW significantly mitigated the elevation of fasting blood sugar induced by HCHF diet in OVX rats ( value = 0.045, value = 0.001, and value = 0.017, respectively, compared to OVX rats which received HCHF and vehicle). However, at 8 weeks of intervention, the significant reduction in blood sugar was observed only in OVX rats which received medium and high doses of MME treatment ( value = 0.043 and value = 0.045, respectively, compared to OVX rats which received HCHF and vehicle) as shown in Table 3.

Glucose areas under the curve during the oral glucose tolerance test at 4 and 8 weeks of the treatment period are also shown in Table 3. The results showed that normal rats and OVX rats which received HCHF diet and vehicle significantly increased the plasma glucose area under the curve at 4 weeks and 8 weeks of experiments ( value = 0.008 and value = 0.002, respectively, and value = 0.032 and -value <0.001, respectively, compared to normal rats which received normal diet and vehicle group). At 4 weeks of the study period, only OVX rats, which were fed with HCHF diet and received MME at a dose of 50 mg/kg BW, significantly decreased the plasma glucose area under the curve ( value = 0.031, compared to OVX rats which received HCHF and vehicle). When the treatment was prolonged, isoflavone, L-carnitine, and all doses of MME could increase the plasma glucose area under the curve in OVX rats with HCHF diet ( value = 0.009, value = 0.022, value = 0.007, value = 0.006, and value = 0.042, respectively, compared to OVX rats which received HCHF and vehicle).

3.5. The Suppression of Angiotensin-Converting Enzyme (ACE) Activity

Table 3 shows that HCHF diet significantly increased the activity of ACE ( value < 0.001 all, compared to normal rats which received normal diet and vehicle). This elevation was mitigated by isoflavone, L-carnitine, and MME at doses of 10, 50, and 250 mg/kg BW ( value = 0.005, value = 0.001, value = 0.016, value <0.001, and value <0.001, respectively, compared to OVX rats which received HCHF and vehicle).

3.6. Changes of Serum Estradiol

Table 4 shows that HCHF diet failed to produce the significant change of serum estradiol in female rats. However, bilateral ovariectomy significantly decreased serum estradiol in female rats fed with HCHF diet ( value<0.05, compared to control rats which received normal diet and vehicle or ND + vehicle; value<0.01; compared to normal rats which received HCHF diet and vehicle). Isoflavone, L-carnitine, and all doses of MME failed to produce the significant changes of estradiol level in OVX rats which received HCHF diet.


Treatment groupEstradiol (pg/ml)

ND + vehicle
HCHF + vehicle
OVX + HCHF + vehiclea,bb
OVX + HCHF + isoflavone 15 mg/kg BW