Extracts of Scutellariae Radix inhibit low-density lipoprotein oxidation and the lipopolysaccharide-induced macrophage inflammatory response

  • Authors:
    • Ohn Soon Kim
    • Chang‑Seob Seo
    • Yeji Kim
    • Hyeun‑Kyoo Shin
    • Hyekyung Ha
  • View Affiliations

  • Published online on: March 17, 2015     https://doi.org/10.3892/mmr.2015.3502
  • Pages:1335-1341
0

Abstract

Traditional herbal formulas made from Scutellariae Radix (SR), the root of Scutellaria baicalensis, have previously been used in the treatment of inflammatory diseases, such as atherosclerosis. The aim of the present study was to investigate the effects of SR on low‑density lipoprotein (LDL) oxidation and inflammation in macrophages, which are early events in the development of atherosclerosis. High‑performance liquid chromatography photo‑diode array analysis was used to obtain a three‑dimensional chromatogram of SR. The antioxidative effects of SR were evaluated by determining its scavenging activities against ABTS and DPPH radicals. The inhibitory effect of SR on LDL oxidation was examined using a thiobarbituric acid‑reactive substance assay and a relative electrophoretic mobility assay. In addition, the anti‑inflammatory effects of SR were evaluated in lipopolysaccharide (LPS)‑induced RAW264.7 murine macrophage cells. The results showed that SR exhibited radical‑scavenging activities in a dose‑dependent manner; in addition, SR attenuated the Cu2+‑induced oxidation of LDL as well as significantly inhibited nitric oxide (NO) production and inducible NO synthase (iNOS) expression in LPS‑induced RAW264.7 cells. Furthermore, SR induced the protein expression of heme oxygenase‑1 (HO‑1) in RAW264.7 cells. In conclusion, the results of the present study demonstrated that SR decreased the oxidation of LDL and suppressed inflammatory responses in macrophages, which occurred at least in part via the induction of HO‑1. These results therefore suggested that SR may be a potential therapeutic agent for the treatment of atherosclerosis.

Introduction

Atherosclerosis is a complex vascular disorder involved in the pathogenesis of myocardial and cerebral infarction (1). The atherosclerotic process involves low-density lipoprotein (LDL) oxidation, oxidized LDL (oxLDL) uptake and inflammatory response induction in macrophages, the transformation of the lipid-laden macrophages into foam cells, proliferation of vascular smooth muscle cells (VSMCs) as well as foam cell accumulation resulting in fatty streaks and subsequent plaque formation (2,3). Modulation of these key atherogenic events may therefore be a promising therapeutic strategy for atherosclerotic management.

Scutellariae Radix (SR), the root of Scutellaria baicalensis, is one of the most widely used types of Traditional Chinese Herbal Medicines. SR, whose Chinese name is Huang-qin, forms the major ingredient of numerous herbal preparations and is used to treat bacterial infections as well as various inflammatory diseases. The principal constituents of SR contain diverse flavonoids, such as baicalein, baicalin and wogonin (4). In addition, previous studies have demonstrated that SR may have various beneficial pharmacological activities including antioxidative (5), anticancer (6,7) and anti-inflammatory (8,9) effects.

Heme oxygenase (HO), a rate-limiting enzyme in heme degradation (10), is an inducible form of HO, which was reported to have cytoprotective effects (11). These effects include pro-oxidative heme degradation, which results in biliverdin release and the subsequent conversion of biliverdin into bilirubin, both of which have antioxidant properties (12). In addition, heme degradation generates carbon monoxide, which has antiproliferative and anti-inflammatory properties (13,14). Numerous studies have suggested that HO-1 may be a potential therapeutic target for the treatment of inflammatory diseases, such as atherosclerosis.

In the present study, high-performance liquid chromatography (HPLC) was used to analyze the flavonoid components of SR. In addition, the present study investigated whether SR exhibited inhibitory effects on LDL oxidation and the macrophage inflammatory response, which are early events in the development of atherosclerosis.

Materials and methods

Preparations of SR extract, standard and sample solution

The roots of S. baicalensis were purchased in April 2012 from Kwangmyungdang Medicinal Herbs (Ulsan, Korea) and were confirmed taxonomically by Professor Je-Hyun Lee of Dongkuk University (Gyeongju, Korea). A voucher specimen of SR (SR-2012-EBM91) was deposited at the Herbal Medicine Formulation Research Group, Korea Institute of Oriental Medicine (Daejeon, Korea). Dried roots of S. baicalensis were extracted using distilled water (1 liter) through reflux at 100°C for 2 h. The extracted solution was filtered through filter paper, evaporated to dryness and then freeze-dried (47.84 g). The yield of water extract obtained was 47.84%. For HPLC analysis, the 100 mg sample powder was dissolved in 100 ml 70% methanol (J.T. Baker, Phillipsburg, NJ, USA) and the solution was passed through a 0.2 mm syringe filter (Woongki Science Co. Ltd, Seoul, Korea) prior to HPLC injection. Baicalin, baicalein and wogonin were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). The purities of these compounds were >98.0%, as determined using HPLC analysis. Standard stock solutions of baicalin, baicalein and wogonin (all 1,000 μg/ml) were prepared in 100% methanol and stored at 4°C. Working standard solutions were prepared by serial dilution of 3.91–500 μg/ml baicalin, 2.34–300 μg/ml baicalein, and 1.53–200.00 μg/ml wogonin, with 100% methanol.

Chromatographic system

Analysis was performed using a Shimadzu LC-10A HPLC system (Shimadzu Corp., Kyoto, Japan), which was comprised of a solvent delivery unit, an online degasser, an autosampler and a photo-diode array (PDA) detector. The data processor used LCsolution software (version 1.24; Shimadzu Corp.). The analytical column used was a Gemini C18 (250×4.6 mm; particle size, 5 mm; Phenomenex, Inc., Torrance, CA, USA). The mobile phases were (A) 1.0% acetic acid (Merck KGaA, Darmstadt, Germany) in aqueous solution and (B) 1.0% acetic acid in acetonitrile-methanol (7:3; J.T. Baker) solution. Gradient elution of the two mobile phases were as follows: 25% B for 0–10 min, 25–45% B for 10–20 min, 45% B for 20–24 min, 45–48% B for 24–35 min, 48–25% B for 35–40 min and 25% B for 40–50 min. Analysis was performed at a flow rate of 1.0 ml/min with PDA detection at 277 nm. The injection volume was 10 μl.

Determination of antioxidant activity

The radical-scavenging activity of SR against 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Sigma-Aldrich, St. Louis, MO, USA) was determined using the method described by Re et al (15), with slight modifications. In brief, ABTS radical cations (ABTS+) were produced by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate (Sigma-Aldrich); the solution was stored in the dark at room temperature for 16 h. The solution was then diluted with phosphate-buffered saline (PBS; pH 7.4; Bio-Rad Laboratories, Inc., Hercules, CA, USA) to an absorbance of 0.7 at 734 nm using a microplate reader (Benchmark Plus; Bio-Rad Laboratories, Inc.). A total of 100 ml diluted ABTS+ solution was then added to a 96-well plate containing the samples (20, 40, 80 and 160 mg/ml SR, or 2.5, 5 and 10 mg/ml AA). Following incubation for 5 min, absorbance was measured at 734 nm using a Benchmark Plus microplate reader. The extent of decolorization was calculated as the percentage reduction in absorbance. The scavenging capacity of the test compounds was calculated using the following equation: ABTS radical-scavenging activity = (1-Asample/Acontrol) ×100, where Acontrol is the absorbance of the negative control and Asample is the absorbance of the standard antioxidant or extract. RC50 values (the concentration required for 50% reduction of ABTS radical) were calculated as the concentration at which the absorbance was diminished by 50%.

The 2,2-diphenyl-2-picrylhydrazyl (DPPH; Sigma-Aldrich) free radical-scavenging activity of SR was determined according to the method described by Moreno et al (16), with certain modifications. In brief, 100 μl sample at various concentrations (20, 40, 80 and 160 mg/ml SR, or 5, 10 and 20 mg/ml AA) was added to 100 μl DPPH solution (0.15 mM in ethanol) in a 96-well plate. Following incubation for 30 min in the dark at room temperature, the absorbance was measured at 517 nm. The scavenging activity (%) was calculated using the formula described in the preceding paragraph. Ascorbic acid (AA), a positive control for the antioxidant assay, was purchased from Sigma-Aldrich.

CuSO4-mediated LDL oxidation

A CuSO4-mediated method (17) was used to induce the oxidation of LDL. LDL samples (500 μg protein/ml; Biomedical Technologies, Inc., Stoughton, MA, USA) were prepared at 37°C for 5 min in a medium containing 10 mM phosphate buffer (pH 7.4; Sigma-Aldrich) with various sample concentrations. Oxidation was then initiated by the addition of 25 μM CuSO4 at 37°C for 6 h. The oxidation, lipid peroxidation and electrophoretic mobility of the LDL were then measured using the thiobarbituric acid-reactive substance (TBARS) and relative electrophoretic mobility (REM) assays, as described below.

Determination of TBARS

Lipid peroxidation of LDL was estimated by measuring malondialdehyde (MDA) concentration using a TBARS assay kit (BioAssay Systems, Hayward, CA, USA) according to the manufacturer’s instructions (18). In brief, following oxidation, 50 μg LDL was mixed with 200 μl thiobarbituric acid and incubated at 100°C for 30 min. When the reaction was complete, the absorbance was measured at 535 nm using a Benchmark Plus microplate reader.

REM assay

The electrophoretic mobility of LDLs was measured using agarose gel electrophoresis (0.8% agarose in Tris-acetate-EDTA buffer; Bio-Rad Laboratories, Inc.) and Coomassie brilliant blue R-250 (Bio-Rad Laboratories, Inc.) staining. Electrophoresis was performed at 100 V for 30 min (Wide Mini-Sub Cell GT systems; Bio-Rad Laboratory, Inc.). REM was defined as the ratio of the distance migrated from the origin by oxLDL versus that of native LDL (19).

Cell culture

A RAW264.7 murine macrophage cell line was purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were incubated at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Grand Island, NY, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 5.5% fetal bovine serum (Gibco-BRL).

In vitro cytotoxicity assay

Cells were seeded at 3×103 cells/well in a 96-well plate and incubated with various concentrations of SR (20, 40, 80 and 160 μg/ml) for 24 h. CCK-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to cells, which were then incubated at 37°C for 4 h. Following incubation, the absorbance was measured at 450 nm using a Benchmark Plus microplate reader.

Measurement of nitric oxide (NO) production

RAW264.7 cells were seeded at a density of 2.5×105 cells/well in a 48-well plate and incubated with lipopolysaccharide (LPS; 1 μg/ml; Sigma-Aldrich) for 24 h in the presence of various concentrations of SR (20, 40, 80 and 160 μg/ml). NG-Methyl-L-arginine acetate salt (NMMA), a positive control for the NO assay, was purchased from Sigma-Aldrich. Nitrite accumulated in the culture medium was quantified as an indicator of NO production. In brief, 50 μl cell culture medium was mixed with 100 μl Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid; Promega, Madison, WI, USA). The mixture was incubated at room temperature for 30 min and absorbance was then measured at 535 nm using a Benchmark Plus microplate reader. Fresh culture medium was used as a blank control for each experiment. The quantity of nitrite was determined from sodium nitrite standard curves produced in the present study.

Western blot analysis

Following treatment, as mentioned previously, the cells were washed twice with cold PBS and then lysed with 1X Laemmli lysis buffer [60 mM Tris-Cl (pH 6.8), 2% SDS, 10% glycerol and 0.01% bromophenol blue; Bio-Rad Laboratories, Inc.] and boiled for 10 min. The protein content was measured using a bicinchoninic acid protein assay reagent (Pierce Biotechnology, Inc., Rockford, IL, USA). Reducing agent (2-mercaptoethanol; Sigma-Aldrich) was added to the samples to obtain a final concentration of 355 mM 2-mercaptoethanol. Equal quantities (20 μg) of total cellular protein were resolved using 10% SDS-polyacrylamide gel electrophoresis (Mini-Protean® Tetra Cell systems; Bio-Rad Laboratories, Inc.) and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Inc.). The immunoblot was incubated with blocking solution (5% skimmed milk; Bio-Rad Laboratories, Inc.) and then incubated overnight at 4°C with the following primary antibodies: Anti-β-actin (1:1,000; cat. no. sc-81178; Santa Cruz Biotechnology, Inc. Dallas, TX, USA), anti-inducible NO synthase (iNOS; 1:1,000; cat. no. sc-7271; Santa Cruz Biotechnology, Inc.) and anti-HO-1 (1:1,000; cat. no. ab13248; Abcam, Boston, MA, USA). The blots were washed three times with Tris-buffered saline containing Tween 20 (TBST; Bio-Rad Laboratories, Inc.) and then incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. 115-035-003; Jackson ImmunoResearch, West Grove, PA, USA) for 60 min at room temperature. The blots were washed three times with TBST and then developed using an enhanced chemiluminescence kit (cat. no. RPN2232; Amersham, GE Healthcare, Arlington Heights, IL, USA). The bands were visualized using the ChemiDoc™ XRS Imaging system (Bio-Rad Laboratories, Inc.).

Statistical analysis

Data were analyzed using the one-way analysis of variance followed by the Dunnett’s multiple comparison tests using GraphPad InStat 3.05 software (GraphPad Software Inc., La Jolla, CA, USA). Values are presented as the mean ± standard error of the mean. P<0.01 and P<0.05 were considered to indicate statistically significant differences between values.

Results

HPLC analysis

HPLC was used to confirm and quantify the standard components of SR, including baicalin, baicalein and wogonin. HPLC with a PDA detector were then used to produce a three-dimensional chromatogram (Fig. 1). Using optimized chromatography conditions, the three compounds were eluted in <45 min of sample analysis using the gradient elution of two mobile phases. The calibration curves of baicalin, baicalein and wogonin were: y=37580.44x+6967.22 (3.91–500.00 μg/ml), y=55300.58x–31507.13 (2.34–300.00 μg/ml) and y=67938.69x+11675.10 (1.53–200.00 μg/ml), respectively. The correlation coefficients of the three compounds were all 1.0000. The content of baicalin, baicalein and wogonin in SR were: 110.16±0.41 mg/g, 14.04±0.11 mg/g and 3.90±0.05 mg/g, respectively, with relative standard deviations of <1.50%.

Antioxidant activity of SR

In order to evaluate the antioxidant activity of SR, the scavenging activities of SR were tested against ABTS and DPPH radicals. The ABTS radical-scavenging activities of SR are presented in Table I. The results showed that the scavenging activities of SR increased in a dose-dependent manner. The RC50 against ABTS radicals was 51.53 μg/ml, whereas the RC50 of ascorbic acid, as a positive control, was 5.66 μg/ml. The antioxidant activities obtained using the DPPH method for SR are shown in Table II, the results of which were comparable to those of the ABTS assay. SR reduced the DPPH radical formation in a concentration-dependent manner. The RC50 of SR against DPPH radicals was 63.07 μg/ml, whereas the RC50 of ascorbic acid was 8.56 μg/ml.

Table I

Scavenging effects of SR on ABTS+.

Table I

Scavenging effects of SR on ABTS+.

AgentConcentration (μg/ml)Scavenging effect (%)RC50 (μg/ml)
SR20.023.95±2.3151.53±0.81
40.044.30±0.71
80.070.70±1.94
160.095.43±0.82
AA2.520.44±0.595.66±0.04
5.044.39±1.26
10.089.96±0.72

[i] Values are presented as the mean ± standard error of the mean of triplicate determinations. SR, Scutellariae Radix; ABTS+, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cations; RC50, concentration required for 50% reduction of ABTS+ at 5 min reaction; AA, ascorbic acid.

Table II

Scavenging effects of SR on DPPH.

Table II

Scavenging effects of SR on DPPH.

AgentConcentration (μg/ml)Scavenging effect (%)RC50 (μg/ml)
SR20.016.54±1.4863.07±3.00
40.037.70±1.87
80.060.74±2.36
160.078.68±1.64
AA5.015.54±0.438.56±0.04
10.063.66±0.25
20.093.23±0.00

[i] Values are presented as the mean ± standard error of the mean of triplicate determinations. SR, Scutellariae Radix; DPPH, 2,2-diphenyl-2-picrylhydrazyl; RC50, concentration required for 50% reduction of DPPH at 30 min reaction; AA, ascorbic acid.

Inhibitory effect of SR on CuSO4-mediated LDL oxidation

The degree of LDL oxidation and lipid peroxidation was evaluated using the TBARS assay to measure the concentration of the degradation byproduct MDA (20). As shown in Fig. 2A, CuSO4 increased the lipid peroxidation of LDL, which was then significantly inhibited following treatment with 80 or 160 μg/ml SR (P<0.01). Alteration in agarose gel electrophoretic mobility reflects an increase in the negative charge of LDL particles, which occurs during oxidation (19). In the present study, oxidation of LDL performed in the presence of SR significantly attenuated the increase in electrophoretic mobility of oxLDL following CuSO4 treatment (P<0.01) (Fig. 2B and C).

Cytotoxicity of SR in RAW264.7 cells

The cytotoxic effects of SR were investigated in RAW264.7 cells in order to establish the appropriate concentration range of SR treatment for subsequent experiments. The cytotoxicity of SR in this murine macrophage cell line was evaluated using the CCK-8 cell viability assay. RAW264.7 cells were exposed to various concentrations of SR (20, 40, 80 and 160 μg/ml) for 24 h. As shown in Fig. 3A, SR had no significant cytotoxic effects on RAW264.7 cells. Therefore, nontoxic concentrations of SR were used in all subsequent experiments.

Effects of SR on HO-1 expression

In order to examine the effects of SR on the protein expression of HO-1 in RAW264.7 cells, cells were treated with various concentrations of SR (40, 80 and 160 μg/ml) for 24 h. Protein levels of HO-1 were then determined using western blot analysis. The results showed that SR promoted HO-1 protein expression in a concentration-dependent manner (Fig. 3B).

Effects of SR on NO production and iNOS expression

RAW264.7 cells were treated with 1 μg/ml LPS for 24 h in the presence of SR (20, 40, 80 and 160 μg/ml). The quantity of NO produced was determined by measuring the amount of nitrite converted from NO in the media. As shown in Fig. 4A, SR significantly suppressed LPS-induced NO release in a concentration-dependent manner compared with that LPS-treated cells without SR (P<0.05 and 0.01). In addition, NMMA, an inhibitor of NO synthase, markedly reduced NO release in RAW264.7 cells (P<0.01) (Fig. 4A). The inhibitory effects of SR on NO release were 9.3, 18.58, 30.65 and 47.22% at 20, 40, 80 and 160 μg/ml, respectively. Furthermore, it was investigated whether SR affects the protein expression of iNOS in RAW264.7 cells. As shown in Fig. 4B, iNOS protein expression was markedly increased in LPS-stimulated cells compared with that of the untreated control cells; however, this increase was attenuated by the addition of SR at 40 or 160 μg/ml.

Discussion

Atherosclerosis comprises multiple events including LDL oxidation (21), inflammation at the injured area (22), foam cell formation (23) as well as VSMC proliferation and migration (24). Increasing evidence from in vitro and in vivo studies has suggested that oxidative modification of LDL, the major cholesterol carrier in the bloodstream, may be involved in the formation of early atherosclerotic lesions (22,25,26). Atherosclerosis is widely accepted to be an inflammatory disease (27); therefore, therefore it was suggested that reducing the processes involved in LDL oxidation and the macrophage inflammatory response may be crucial for protecting against atherosclerosis. The current study investigated the effects of SR, extracts of the root of S. baicalensis, on LDL oxidation and LPS-induced inflammation.

The root of S. baicalensis is one of the primary ingredients in numerous traditional medicines in China, Japan and Korea (2830). Studies have reported that traditional herbal formulas containing SR have anti-atherosclerotic activities (3134). Ger-Gen-chyn-lian-tang was shown to reduce atherosclerotic progression in an apolipoprotein E−/− mouse model (31); in addition, Dahuang Zhechong, in pill form, inhibited VSMC proliferation and reversed the pathological changes observed in the aorta of atherosclerotic rabbit models (32,33). Furthermore, Huanglian-jie-du-tang was demonstrated to inhibit the progression of atherosclerosis in diet-induced hypercholesterolemic rabbits (34).

The LDL oxidation hypothesis of atherosclerosis development suggested that inhibition of LDL oxidation may be a potential target for anti-atherosclerotic therapy (25). Previous studies have reported that SR inhibited lipid peroxidation in the rat liver (35,36). In the present study, TBARS and REM assays were used to demonstrate that SR reduced LDL oxidation in a dose-dependent manner. Lipid hydroperoxides formation in LDL was previously reported to be an early event in LDL oxidation (37). In the present study, it was observed that SR decreased TBARS formation in Cu2+-induced oxLDL. In the REM assay, oxLDL migrated further compared with native LDL due to the increase in negative change; in addition, oxLDL was less visible following Coomassie blue staining due to partial degradation. In addition, SR inhibited Cu2+-induced LDL oxidation in a dose-dependent manner. These data suggested that SR possesses potent antioxidative abilities, which may be mediated through the inhibition of LDL oxidation.

In order to evaluate the anti-inflammatory effects of SR, a LPS-induced NO production model was used in the present study. iNOS expression and NO production were previously reported to be increased in atherosclerotic lesions (38). In the present study, nitrite concentrations in the culture medium were determined and the results indicated that SR significantly inhibited NO production in a concentration-dependent manner. In addition, western blot analysis revealed that SR treatment markedly inhibited iNOS expression. A previous study reported comparable anti-inflammatory effects of SR (9). Kim et al (9) reported that SR displayed significant anti-inflammatory effects in vitro and in vivo via suppression of mitogen-activated protein kinase signaling. The induction of HO-1 has been widely reported to be involved in the atherosclerosis process (3941); therefore, it is regarded as a major therapeutic target for the prevention or treatment of atherosclerosis (40). The results of the current study demonstrated that SR induced HO-1 expression and exhibited anti-inflammatory properties. In addition, these results suggested that the induction of HO-1 contributed to the anti-inflammatory effects of SR and that SR may have potential therapeutic value in the treatment or prevention of atherosclerosis.

In conclusion, the results of the present study demonstrated that SR was able to reduce the oxidation of LDL and suppress the macrophage inflammatory response. The antioxidant properties and inhibitory effects of SR on macrophage inflammatory responses suggested that SR may be effective in the prevention or treatment of atherosclerosis; however, further studies are required in order to confirm these results.

Acknowledgments

The present study was supported by grants from the Korean Institute of Oriental Medicine (nos. K13030 and K12271).

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July 2015
Volume 12 Issue 1

Print ISSN: 1791-2997
Online ISSN:1791-3004

2015 Impact Factor: 1.559
Ranked #48/123 Medicine Research and Experimental
(total number of cites)

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APA
Kim, O.S., Seo, C., Kim, Y., Shin, H., & Ha, H. (2015). Extracts of Scutellariae Radix inhibit low-density lipoprotein oxidation and the lipopolysaccharide-induced macrophage inflammatory response. Molecular Medicine Reports, 12, 1335-1341. https://doi.org/10.3892/mmr.2015.3502
MLA
Kim, O. S., Seo, C., Kim, Y., Shin, H., Ha, H."Extracts of Scutellariae Radix inhibit low-density lipoprotein oxidation and the lipopolysaccharide-induced macrophage inflammatory response". Molecular Medicine Reports 12.1 (2015): 1335-1341.
Chicago
Kim, O. S., Seo, C., Kim, Y., Shin, H., Ha, H."Extracts of Scutellariae Radix inhibit low-density lipoprotein oxidation and the lipopolysaccharide-induced macrophage inflammatory response". Molecular Medicine Reports 12, no. 1 (2015): 1335-1341. https://doi.org/10.3892/mmr.2015.3502