A Superior All-Natural Antioxidant Biomaterial from Spent Coffee

Research Article pubs.acs.org/journal/ascecg

A Superior All-Natural Antioxidant Biomaterial from Spent Coffee Grounds for Polymer Stabilization, Cell Protection, and Food Lipid Preservation Lucia Panzella,*,† Pierfrancesco Cerruti,‡ Veronica Ambrogi,§ Sarai Agustin-Salazar,‡ Gerardino D’Errico,†,⊥ Cosimo Carfagna,‡,§ Luis Goya,∥ Sonia Ramos,∥ Maria A. Martín,∥ Alessandra Napolitano,† and Marco d’Ischia† †

Department of Chemical Sciences, University of Naples “Federico II”, Via Cintia 4, I-80126 Naples, Italy Institute for Polymers, Composites and Biomaterials (IPCB-CNR), Via Campi Flegrei 34, I-80078 Pozzuoli, Italy § Department of Chemical, Materials and Production Engineering, University of Naples “Federico II”, Piazzale Tecchio 80, I-80125 Naples, Italy ⊥ CSGI (Consorzio per lo Sviluppo dei Sistemi a Grande Interfase), Florence, Italy ∥ Department of Metabolism and Nutrition, ICTAN, CSIC, José Antonio Novais 10, 28040 Madrid, Spain ‡

ABSTRACT: Treatment with boiling 6 M HCl increases up to 30 times the intrinsic antioxidant potency of spent coffee grounds, leading to a versatile multifunctional material (hydrolyzed spent coffee grounds, HSCG). Spectral and morphological analyses suggest that the remarkable potentiation of the antioxidant activity is due to efficient removal of the hydrolyzable components, mainly carbohydrates, making the polyphenol-rich component available for interaction with free radicals and oxidizing species. HSCG efficiently protects hepatocarcinoma (HepG2) cells from oxidative stress-induced injury and delays lipid peroxidation in fish and soybean oils. Moreover, films made of polyethylene/2% HSCG blends display greater stability to thermal and photo-oxidative degradation. HSCG may thus represent an easily accessible and sustainable alternative to currently available biomaterials with intrinsic antioxidant properties for biomedical, industrial, and technological applications. KEYWORDS: Spent coffee grounds, Polyethylene, Antioxidant, HepG2 cells, Lipid peroxidation inhibition

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INTRODUCTION Biodegradable polymers endowed with antioxidant and free radical scavenging properties occupy an increasing segment in the current panorama of sustainable functional materials because of their potential applications in a broad variety of traditional and emerging sectors, such as biocompatible materials for biomedical devices. Since inflammation is usually the main factor that impairs the efficacy of medical devices,1 it would follow that biocompatible materials for use in the clinical setting must be capable of coping with the manifold adverse effects of oxidative stress and primary biochemical correlate of inflammatory reactions, such as generation of excessive reactive oxygen species (ROS).2−4 Engineering antioxidant properties into a material may therefore be an important goal to improve biocompatibility and to confer resilience to oxidative degradation.5−7 Besides biomedical devices, another major field of application of antioxidant materials is for incorporation into widely used polymers both for stabilization and functionalization. The photo- and thermooxidative degradations of polyethylene and © XXXX American Chemical Society

other conventional polymers are issues of considerable concern, which has spurred intense research aimed to increase stability of the material without undesirable effects associated with the release of toxic additives.8−11 Polymer functionalization is likewise an important focus for research especially for active food packaging, a crucial part of the food sector aimed to delay or prevent oxidative deterioration of packaged food. One straightforward means of endowing materials with antioxidant properties would be based on the incorporation of low molecular weight antioxidants,12−15 but critical limitations may derive from their possible leakage from the polymer. A viable approach to settle this issue is the development of easily accessible insoluble or macromolecular materials with intrinsic antioxidant properties that are less likely to leach from the polymer than small-sized additives.10 Several strategies have been reported to provide antioxidant functionality to Received: October 6, 2015 Revised: December 22, 2015

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DOI: 10.1021/acssuschemeng.5b01234 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

mide, pyrogallol, ethylendiamine tetracetic acid (EDTA), nitroblue tetrazolium (NBT), 1,1,3,3-tetramethoxypropane, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), trichloroacetic acid, thiobarbituric acid (TBA), xylenol orange, Triton X-100, tertbutylhydroperoxide (t-BuOOH), activated carbon, o-phthaldialdehyde (OPT), glutathione (GSH), 2,7-dichlorofluorescin diacetate (DCFHDA), phosphate buffered saline (PBS), reduced nicotine adenine dinucleotide (NADH), and Bradford reagent were used as obtained. An unstabilized grade of a butene copolymer linear low density polyethylene (LLDPE), DJM1826, with a melt flow index (MFI) of 2.5 g 10 min−1 was supplied as a powder and used as obtained. UV−vis spectra were performed using a Beckman DU 640 spectrophotometer. Morphological analysis was performed using a FEI Quanta 200 FEG scanning electron microscope (SEM). Samples were analyzed at 30 kV acceleration voltage. Gas sorption measurements were carried out with N2 at 77 K using an ASAP 2020 from Micromeritics. A 2.0 ± 0.1 g sample was degassed at 95 °C under vacuum for 10 h prior to analysis. The specific surface area (SSA) was determined by applying the Brunauer−Emmett−Teller (BET) equation31 to the relative pressure range of 0.01−0.99 for the adsorption branch of the isotherm. The BET calculations were performed with the ASAP 2020 V4.02 software from Micromeritics. 13 C solid-state magic angle spinning (MAS)-NMR measurements were performed on samples dried under vacuum at 60 °C for 24 h. Measurements were carried out using a Bruker Avance II 400 spectrometer operating at 100.47 MHz. Samples were spun at 9 kHz in 4 mm zirconium oxide rotors. Spectra were collected using a single pulse excitation sequence with a 13C 90° pulse width of 3.2 s, recycle delay of 2 s, and contact time of 2 ms, by averaging 16384 scans. FT-IR analysis was performed using a PerkinElmer Spectrum 100 spectrometer with an average of 32 scans in the range 4000−400 cm−1, with a resolution of 4 cm−1. SCG and HSCG spectra were recorded in attenuated total reflectance (ATR) mode, whereas films were characterized in transmission mode. In the latter case, each sample subjected to accelerated aging was analyzed periodically and then returned to the oven for continued aging. Chemiluminescence (CL) experiments were performed by means of a Lumipol 3 luminometer both in nitrogen and air flow. The samples were circular cuts of polymer film weighing about 6 mg. Each sample was heated from 30 to 230 °C at 5 °C min−1 and then cooled to room temperature. Tensile tests were performed at the selected aging times on eight specimens for each formulation, using an Instron model 5564 dynamometer equipped with a 1 kN load cell, at 23 ± 2 °C and 45 ± 5% RH, with a 10 mm min−1 clamp separation rate. Electron paramagnetic resonance (EPR) measurements were performed by following an experimental procedure recently set up for paraffin-embedded sections of melanoma samples and melanin samples.32,33 Samples were measured using an X-band (9 GHz) Bruker Elexys E-500 spectrometer equipped with a superhigh sensitivity probe head. SCG and HSCG samples were transferred to flame-sealed glass capillaries, which in turn were coaxially inserted in a standard 4 mm quartz sample tube. Measurements were performed at room temperature. The instrumental settings were as follows: sweep width, 100 G; resolution, 1024 points; modulation frequency, 100 kHz; modulation amplitude, 2.0 G. The amplitude of the field modulation was preventively checked to be low enough to avoid detectable signal overmodulation. EPR spectra were measured with a microwave power of ∼0.5 mW to avoid microwave saturation of resonance absorption curve. Sixteen scans were accumulated to improve the signal-to-noise ratio. For power saturation experiments, the microwave power was gradually incremented from 0.004 to 125 mW. The g value and the spin density were evaluated by means of an internal standard, Mn2+-doped MgO, prepared by a synthesis protocol reported in the literature.34 Since sample hydration was not controlled during the measurements, spin density values have to be considered as order of magnitude estimates.35

biocompatible polymers by conjugating active components such as glutathione or antioxidant polymers like polyaniline.16,17 Representative examples include a biodegradable, thermoresponsive gel with intrinsic antioxidant properties, poly(polyethylene glycol citrate-co-N-isopropylacrylamide), that was obtained by copolymerization of citric acid, poly(ethylene glycol) (PEG), and poly-N-isopropylacrylamide.6 Catechol-modified chitosan films have also been produced to serve as a localized source or sink of electrons that can be transferred to soluble mediators.18,19 Poly(antioxidant β-amino ester) biodegradable hydrogels containing quercetin and curcumin as antioxidants were described and reported to protect endothelial cells from hydrogen peroxide-induced oxidative stress.7 In a similar manner, potentiation of the antioxidant capacity of carboxymethyl cellulose in wounds was achieved by modification with collagen peptides.1 Relatively less attention has been devoted to assess the potential of natural phenol-based polymers as active antioxidants for cell protection and polymer stabilization and functionalization. An attractive source of natural antioxidants is provided by phenol-rich waste products of the agricultural and food industry. One noticeable example is represented by grape pomace tannins (GPT), which are the subject of increasing investigations for manifold applications.20,21 Much less attention has so far been paid to spent coffee grounds (SCG) derived from the production of espresso beverages or soluble coffee. Coffee is by far one of the most consumed beverages in the world and the resulting SCG, because of their high content in caffeine, tannins, and polyphenols, can have negative effects on the environment, requiring proper management and disposal. Proposed use of SCG includes production of biofuel, removal of pollutants from water, and as a source of recovery of compounds of potential interest to the food and pharmaceutical industries.22−27 SCG contain mainly carbohydrates (38−42%), proteins (8%), and chlorogenic acids (3−4%).28 As a major outcome of the roasting process, SCG contain also melanoidins, which are usually quantified to account for up to around 25% (w/w) of the dry weight of roasted coffee beans.28 Although the presence of phenolic components confers a significant antioxidant activity to SCG, the possible conversion into value-added materials and chemical systems with specific properties has remained virtually unexplored. An interesting clue to this aim came from the recent finding that hydrolytic treatment of black sesame (Sesamum Indicum L.) seeds with 6 M HCl at 100 °C overnight leads to a black pigment with potent antioxidant efficiency.29 Prompted by this observation, we disclose herein an expedient chemical procedure to convert SCG into an allnatural biocompatible material, termed hydrolyzed spent coffee grounds (HSCG), endowed with potent antioxidant properties for diverse applications, including cell protection, food lipid preservation, and thermal and photo-oxidative stabilization of polymers.

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EXPERIMENTAL SECTION

General Experimental Methods. SCG were collected from a local coffee shop. Espresso coffee residues were chosen because of the higher pool of bioactivity retained with respect to the ones from the soluble coffee industries.30 Soybean and cod liver oils were purchased from local shops. DPPH, iron(III) chloride, ammonium iron(II) sulfate hexahydrate, iron(II) chloride tetrahydrate, 2,4,6-tris(2pirydyl)-s-triazine, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p-disulfonic acid monosodium salt hydrate (ferrozine), sodium nitroprusside (SNP), N-(1-naphthyl)ethylenediamine dihydrochloride, sulfanilaB


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with the FBS-free medium the following day. After 20 h, 5 μM DCFHDA was added to the wells at 37 °C for 30 min, cells were then washed with PBS, placed in fresh FBS-free medium with the different quantitites of SCG and HSCG, and ROS production was monitored for 120 min. For the protection assay, cells were seeded and left overnight before treating them with SCG or HSCG samples for 20 h. Then DCFH-DA was added for 30 min and cells washed with PBS prior to the addition of 400 μM t-BuOOH to every well but controls with further incubation for 2 h. Control cells without t-BuOOH treatment were used as negative control. Multiwell plates were measured in a fluorescent microplate reader at an excitation wavelength of 485 nm and emission wavelength of 528 nm. Results are expressed as percent of fluorescence units. GSH content was quantitated by a fluorometric assay.42 The method takes advantage of the reaction of GSH with OPT at pH 8.0. HepG2 cells were plated in 60 mm diameter plates at a concentration of 1.5 × 106 cells/plate. Cells were treated with the different quantities of the samples for 20 h, collected by scraping in 1.5 mL of PBS, and centrifuged (1500 rpm, 4 °C, 5 min). Cells were lysed by adding 110 μL of 5% w/v trichloroacetic acid containing 2 mM EDTA, and protein was measured by the Bradford reagent. Following centrifugation of cells at 7500 rpm and 4 °C for 30 min, 50 μL of the clear supernatant was transferred to wells in a 96-well plate. Then, 15 μL of 1 M NaOH was added to neutralize the sample followed by 175 μL of 0.1 M sodium phosphate buffer (pH 8.0) containing 5 mM EDTA, and finally, 10 μL per well of a stock solution of OPT (10 mg/ mL) in methanol was added. After 15 min at room temperature in the dark, fluorescence was measured at an emission wavelength of 460 nm and an excitation wavelength of 340 nm. The results of the treatments were referred to those of a standard calibration curve of GSH (5−1000 ng). Polyethylene Film Preparation and Aging. LLDPE films containing HSCG were obtained by extrusion, using a Collin TeachLine E20T single screw extruder equipped with a horizontal flat die and a Collin CR72T chill-roll and calendering unit. The temperature profile was as follows (from hopper to die): 150, 170, 180, 180, 170 °C. Prior to processing, HSCG was dried under vacuum for 24 h at 60 °C and successively mechanically mixed with the polyethylene (PE) powder in a 0.5% and 2.0% amount. The resulting films, referred to as PE + 0.5% HSCG and PE + 2% HSCG, respectively, were homogeneous, transparent, and light amber colored. The average thickness of all LLDPE-based films was 115 ± 10 μm. In order to evaluate the photo-oxidative stability at low temperature, the prepared films were aged at 40 °C up to 1000 h under dry conditions in an Angelantoni Sunrise environmental chamber equipped with a mercury lamp simulating solar irradiation (200 < λ < 700 nm, average incident light intensity 100 μW/cm2) and periodically examined by FT-IR and tensile tests. Accelerated Thermal Aging of Oil Samples. 17 HSCG (50 mg) were deposited on the bottom of a flask containing 10 mL of soybean or cod liver oils and incubated at 60 °C. Control samples without HSCG were also incubated. The hydroperoxide contents of the oil samples were determined after 6 days of incubation, using an adaptation of the ferrous oxidation xylenol orange (FOX) assay.44 A total of 10 mg of the oil samples was withdrawn and dissolved in 3 mL of chloroform, and 60 μL of each solution was mixed with 2 mL of reagent mixture containing 0.11 mM xylenol orange, 0.25 mM ammonium iron(II) sulfate hexahydrate, and 25 mM H2SO4 in methanol containing 3.88 mM BHA. After 30 min incubation in the dark at room temperature, absorbance at 560 nm was measured. A calibration curve was prepared with t-BuOOH. The concentration of hydroperoxides in the oil samples was expressed as mmol t-BuOOH per 1 kg of oil. Experiments were run in triplicate. Accelerated Iron-Induced Aging of Oil Samples. 45 Cod liver oil (10 mg) was suspended in 1 mL of a buffer solution (0.05 M TRIS, 0.15 M KCl, 1% Triton X-100, pH 5.0) by homogenization using a Tenbroeck glass/glass potter for 5 min in the presence or absence of HSCG (1−5 mg/mL). A total of 100 μL of 1 mM iron(II) chloride tetrahydrate was added, and the mixtures were taken under vigorous stirring at room temperature. After 3 h, thiobarbituric acid reactive

Preparation of HSCG. Air-dried SCG (3 g) were treated with 70 mL of 6 M HCl under stirring at 100 °C overnight. After cooling at room temperature, the mixture was centrifuged (7000 rpm, 4 °C, 30 min) and the precipitate washed with water until neutrality and freezedried to give a black powder (ca. 930 mg, 31% yield). DPPH Assay. 36,37 SCG or HSCG (final dose 0.05−0.5 mg/mL) were added to a freshly prepared 0.2 mM solution of DPPH in methanol, and the mixture was taken under vigorous stirring at room temperature. After 10 min, the absorbance at 515 nm was measured. Experiments were run in triplicate. Trolox was used as a reference antioxidant. Control experiments were performed on activated carbon (final dose 0.5−2 mg/mL). Ferric Reducing/Antioxidant Power (FRAP) Assay. 38 SCG or HSCG (final dose 0.02−0.1 mg/mL) were added to a solution of 1.7 mM FeCl3 and 0.83 mM 2,4,6-tris(2-pirydyl)-s-triazine in 0.3 M acetate buffer (pH 3.6), and the mixture was taken under vigorous stirring at room temperature. The reduction of Fe3+ to Fe2+ was monitored by measuring the absorbance at 593 nm after 10 min. Results were expressed as Trolox equivalents. Experiments were run in triplicate. NO Scavenging Assay. 39 SCG or HSCG (final dose 0.33 mg/ mL) were added to a freshly prepared 10 mM solution of SNP in 0.2 M phosphate buffer (pH 7.4), and the mixture was taken under vigorous stirring at room temperature. One milliliter of the mixture was periodically withdrawn and added to 1 mL of Griess reagent (0.5% sulfanilamide and 0.05% N-(1-naphthyl)ethylenediamine dihydrochloride in 2.5% phosphoric acid), and the absorbance at 540 nm was measured. Results were expressed as a percentage of reduction of the absorbance at 540 nm of a control mixture run in the absence of sample. When required, HSCG (final dose 0.33 mg/mL) was added to the control mixture after addition of the Griess reagent. Experiments were run in triplicate. Quercetin was used as reference antioxidant. Superoxide Scavenging Assay. 40 SCG or HSCG (final dose 0.07 mg/mL) were added to 0.05 M ammonium hydrogen carbonate buffer (pH 9.3) containing 0.33 mM EDTA, 0.01 mM NBT, and 3.3 mM pyrogallol. The mixture was taken under vigorous stirring, and after 5 min, the absorbance at 596 nm was measured. Results were expressed as a percentage of reduction of the absorbance at 596 nm of a control mixture run in the absence of sample. Experiments were run in triplicate. Quercetin was used as the reference antioxidant. Cellular Assays. Human hepatoma HepG2 cells were maintained in a humidified incubator containing 5% CO2 and 95% air at 37 °C. They were grown in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM F-12) from Biowhitaker, supplemented with 2.5% Biowhitaker fetal bovine serum (FBS) and 50 mg/L of each of the following antibiotics: gentamicin, penicillin, and streptomycin. To assay direct effect, cells were incubated with 0.5, 1, 5, and 10 μg/ mL of SCG or HSCG for 20 h. To assay for a protective effect, cells were pretreated with the same quantities of the two samples for 20 h, then the medium was discarded, the cells were washed with PBS, and medium containing 400 μM t-BuOOH was added for 2 h, after which the cell cultures were processed as detailed below for each assay. Cell viability was determined by using the crystal violet assay.41 HepG2 cells were seeded at low density (104 cells per well) in 96-well plates, grown for 20 h under the different conditions, and incubated with crystal violet (0.2% in ethanol) for 20 min. Plates were rinsed with water, allowed to dry, and 1% sodium dodecyl sulfate added. The absorbance of each well was measured using a microplate reader at 570 nm. At higher doses, cellular damage was evaluated by lactate dehydrogenase (LDH) leakage.42,43 Cells were seeded (2 × 105 cells per plate) in 60 mm plates, grown for 20 h with the different treatments, and then the cell culture medium was collected, and the cells were scraped off in PBS. LDH activity was determined by the disappearance of NADH at 340 nm. LDH leakage was estimated from the ratio between the LDH activities in the culture medium and the total activity (culture medium plus intracellular). Cellular ROS were quantified by the dichlorofluorescein assay using a microplate reader to screen the antioxidant effect of the different quantities of samples.42,43 Briefly, the cells were seeded in 24-well plates (2 × 105 cells per well) in medium containing FBS and replaced C


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Table 1. Antioxidant Properties of HSCG and SCGa

substances (TBARS) were quantified as follows: 100 μL of 2% BHT in ethanol was added to all the mixtures to stop the oxidation process, followed by addition of 1 mL of TBA reagent (15% w/v trichloroacetic acid and 0.375% w/v TBA in 0.25 M HCl). The reaction mixtures were taken under stirring at 80 °C for 15 min, and after that, the samples were brought to room temperature and centrifuged at 2000 rpm for 10 min. The absorbance of the supernatant was then measured at 532 nm. A standard curve was obtained using 1,1,3,3tetramethoxypropane as a precursor of malonaldehyde. The results were expressed as μmol of malondialdehyde per g of oil. Experiments were run in triplicate. t-BuOOH Scavenging Assay. HSCG (5 mg) were incubated with a 0.75 mM solution of t-BuOOH in chloroform (1 mL). Control experiments were performed in the absence of HSCG. After 30 min, the residual t-BuOOH was measured by the FOX assay as described above for the accelerated termal aging of the oil samples. Iron Chelation Assay. 46 HSCG (9 mg) were incubated with a 100 μM iron(II) chloride tetrahydrate solution in water (1 mL). Control experiments were performed in the absence of HSCG. After 5 min, the mixtures were centrifuged at 2000 rpm for 10 min, and 100 μL of a 5 mM ferrozine aqueous solution was added to 1.5 mL of the supernatant. The absorbance at 562 nm was measured after 5 min. Experiments were run in triplicate.

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sample

EC50 (DPPH assay) (mg/mL)b

Trolox equivs (× 103) (FRAP assay)

NO scavenging (%)c

superoxide scavenging (%)d

HSCG SCG Trolox quercetin

0.16 ± 0.02 5.00 ± 0.04 0.006 ± 0.001 npe

5.3 ± 1.5 11.3 ± 0.3 − npe

91.1 ± 0.9 55.5 ± 0.5 npe 44.0 ± 0.9

51 ± 5 3±2 npe 74 ± 3

Reported are the mean ± SD values of at least three experiments. EC50 is the dose of the material at which a 50% DPPH reduction is observed. cDetermined after 6 h with HSCG and SCG at 0.33 mg/mL dose and quercetin at 0.033 mg/mL. dDetermined after 5 min with HSCG and SCG at 0.07 mg/mL dose and quercetin at 0.013 mg/mL. e Not performed. a b

turned into pale yellow as a result of DPPH reduction, just a slight decrease in the purple color intensity was observed with activated carbon, with no color changing, suggesting that in this case a simple absorption rather than a reduction mechanism of scavenging was operative. In addition, in control NO scavenging experiments, HSCG was added to the SNP mixture after the addition of the Griess reagent, and no abatement of the absorbance at 540 nm was observed, ruling out a dye absorption contribution to the observed radical scavenging activity. Structural and Morphological Characterization of HSCG. To gain an insight into the processes that account for the observed potentiation of SCG antioxidant activity, in subsequent experiments, the morphological changes induced by the acidic treatment were investigated. Scanning electron microscopy (SEM) images revealed significant differences between SCG and HSCG. As shown in Figure 1a, c, and e, SCG features irregularly shaped particles with large size dispersion, ranging from few to hundreds of micrometers. Some fibrous material is also clearly visible. HSCG particles (Figure 1b,d,f) show comparable size to SCG, although the morphology is less compact and surfaces are smoother, suggesting the partial degradation and removal of the hydrolyzable polysaccharide fraction deeply embedded into the particles. Polysaccharides are the most abundant components in SCG. Cellulose and hemicellulose correspond to about 50 wt % on a dry weight basis, with hemicellulose being more abundant (about 40 wt %).47 As to the composition of hemicellulose sugars, SCG contains mainly mannose (ca. 37%) and galactose (ca. 32%), followed by glucose (ca. 24. %) and arabinose (ca. 7%).47 Figure 2 shows the N2 sorption isotherm for SCG and HSCG, which exhibits a typical absorption on macroporous absorbents with rather weak absorbate−adsorbent interactions, classified as Type III shape according to the IUPAC classification48 Specific surface area (SSA) values of 0.30 and 0.25 m2 g−1 for SCG and HSCG, respectively, were derived from such isotherms according to the Brunauer−Emmet− Teller (BET) equation.31 It is noticed that both samples show very low SSA, and the hydrolytic treatment had no effect on the surface area of SCG, supporting the idea that the observed antioxidant activity is not simply ascribable to a reactive surface effect. The conclusions based on morphological analysis were confirmed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and solid phase NMR spectra (Figure 3), which indicated removal of polysaccharides

RESULTS AND DISCUSSION

Preparation and Antioxidant Properties of HSCG. HSCG were prepared by treating SCG for 16 h in 6 M HCl at 100 °C. The material was collected by centrifugation, extensively washed with water, and freeze-dried to give eventually a black powder in ca. 30% yield on a weight basis. The antioxidant and free radical scavenging properties of HSCG were investigated with respect to SCG as reference material based on four widely used assays, i.e., (a) DPPH assay (following the “QUENCHER” method36), according to which the H-donor capacity of solid samples is straightforwardly determined by mixing them with the DPPH solution followed by the spectrophotometric measurement at 515 nm,37 (b) ferric reducing/antioxidant power (FRAP) assay, which measures the ability of the antioxidant to reduce a Fe3+-tripyridyltriazine complex to a dark blue Fe2+ complex with absorption maximum at 593 nm,38 (c) NO scavenging assay, based on determination of residual NO produced by decomposition of sodium nitroprusside (SNP),39 and (d) superoxide scavenging assay, based on the inhibition of blue diformazan formation from the reduction of nitroblue tetrazolium (NBT) by the superoxide produced from pyrogallol autoxidation.40 The DPPH assay indicated an impressive 30-fold higher efficiency of HSCG in comparison with the untreated sample, while an about doubled Trolox equivalent value was measured in the FRAP assay. A very high efficiency in the nitric oxide and superoxide scavenging assays was also observed (Table 1). For comparison, data relative to prototype antioxidants for each assay, that is, Trolox for DPPH and quercetin for NO and superoxide scavenging assays, are reported. Overall, these data demonstrate that HSCG is far more active than SCG as a multipotent antioxidant, serving as free radical quencher toward DPPH, as scavenger of superoxide and NO, and as ferric ion reductant. To rule out any contribution of an absorption mechanism in the antioxidant assays performed, in control experiments, the DPPH assay was repeated on a sample of activated carbon from a commercial source. An EC50 value of 1.11 ± 0.03 mg/mL was observed, suggesting that HSCG was almost seven times more efficient than activated carbon in scavenging the DPPH reactive molecule. Moreover, while in the case of HSCG, the starting deep purple-colored solution D


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Figure 1. SEM images of (a,c,e) SCG and (b,d,f) HSCG at different magnifications.

Figure 3. Structural characterization of HSCG and SCG: (a) ATRFTIR and (b) solid-state 13C NMR spectra.

CC vibration of aromatic rings from lignin moieties, were not significantly affected by the hydrolytic treatment, suggesting that these compounds were not extensively modified. Lignin is present in SCG in amounts as high as 25 wt %, with hydroxycinnamic acids as the most relevant precursors.47 The 13C magic angle spinning (MAS)-NMR spectrum of SCG (Figure 3b) showed main signals at around 105, 75, 60, and 30 ppm due to the cellulose and hemicellulose components.50 Minor broad resonance bands around 170, 150, 130, 115, and 25 ppm may be assigned at least in part to the aromatic carbons of syringyl and guaiacyl units of lignin and tannin constituents, these latter deriving mainly from catechin, gallocatechin, chebulic acid, and gallocatechin-3-O-gallate.51,52 The 13C MAS NMR spectrum of HSCG exhibited a resonance pattern close to that of SCG; however, line broadening was observed. As shown from the subtracted spectrum (HSCGSCG), indicating a significant reduction in the resonances at 105, 75, and 62 ppm, the majority of hemicellulose was removed following acidic treatment. On the other hand, a slight increase in carbonyl (170 ppm) and aromatic (around 145 ppm) regions was noticed. It has been reported that acid treatment of lignocellulosic biomass results in lignin depolyme-

Figure 2. N2 sorption isotherm for SCG and HSCG.

as the main modification induced by the acid treatment on SCG. As evident also from the subtracted spectrum (HSCG-SCG) (Figure 3a), the bands at 3300 and 1155−1035 cm−1 due to the O−H and C−O and C−O−H bonds stretching vibration, respectively, of cellulose and hemicellulose47 were greatly reduced in the ATR-FTIR spectrum of HSCG. On the contrary, the two sharp peaks at 2920 and 2850 cm−1 likely associated with the two C−H stretching vibration of lignins,49 together with the bands at 1700−1520 cm−1 mainly due to the E


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Scheme 1. Schematic Illustration of Main Reaction Routes Likely Involved in the Hydrolytic Procedure Leading to HSCG55,56

Biocompatibility and Cytoprotective Effects of HSCG. The biocompatibility and cytoprotective properties of HSCG were investigated in comparison with SCG by exposing hepatocarcinoma (HepG2) cells to both materials at doses of 1−10 μg/mL. Higher doses were tested to evaluate cytotoxicity of the samples, and significant cell damage was observed after treatment with 500−1000 μg/mL SCG and 1000 μg/mL HSCG. The requirement of such high concentrations to evoke cell injury indicates a low cytotoxicity of both samples. Notably, at all doses, the toxicity proved higher in the case of SCG (Table 2). In our experimental conditions, oxidative stress as apparent from decreased cell viability and glutathione (GSH) levels and overproduction of ROS (Figure 5a,b,d), was induced by 400 μM t-BOOH, and pretreatment with 1−10 μg/mL of HSCG significantly reduced ROS overproduced by the stress, whereas only the highest quantity of untreated SCG (10 μg/mL) showed a beneficial effect (Figure 5b). These results are in line with the antioxidant effects previously reported for SCG in human cells (HeLa).57 The same applies to GSH, with only 5−10 μg/mL of untreated SCG vs 1−10 μg/mL of HSCG significantly recovering the stress-induced depletion of GSH, the degree of recovery by HSCG being statistically higher than that of SCG at all doses (Figure 5d). It is worth noting that the direct effect with some doses showed a slight but significant reduction of GSH (Figure 5c); both an increase in steady state GSH after treatment with chlorogenic acid41 and a decrease in GSH after treatment with coffee melanoidins42 have been previously

rization/repolymerization, with an increase in condensed lignin.53−56 Both spectral and morphological analysis concur to suggest that the remarkable potentiation of the antioxidant activity of SCG is due to efficient removal of the hydrolyzable components, mainly carbohydrates (Scheme 1), making the polyphenol-rich component available for interaction with free radicals and oxidizing species. SCG and HSCG samples were characterized also by electron paramagnetic resonance (EPR). The spectra (Figure 4a) show a similar line shape, with a single signal at a g value of ∼ 2.0033, consistent with the presence of carbon-centered radicals.35 The main difference between the two spectra is the weightnormalized intensity, which is apparently much higher for HSCG than for SCG. This is confirmed by the spin density estimates, which differ for almost one order of magnitudes (going from ∼4 × 1016 spin/g for SCG to ∼2 × 1017 spin/g for HSCG). Moreover, the normalized power saturation profiles, reported in Figure 4b, presents an evident maximum for the SCG sample, while just a slope change in a monotonously increasing trend is observed for HSCG. This trend indicates a higher degree of molecular heterogeneity of the paramagnetic centers in HSCG compared to SCG. This change in paramagnetic properties is likely to account at least in part for the enhanced free radical scavenging capacity of HSCG relative to SCG, but the detailed mechanisms cannot be addressed given the high degree of molecular complexity represented in both materials. F


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air provide insight about the long-term antioxidant-stabilizing activity of HSCG on polyethylene. From Figure 6a, it is apparent that CL emission increased with increasing temperature due to the thermal decomposition of peroxides formed upon polymer processing. Nonetheless, the films containing HSCG exhibited much lower CL intensity, accounting for a very good performance under processing conditions, even at a weight content as low as 0.5%. Concerning the experiments performed under air (Figure 6b), a dramatic increase in CL intensity is noticed for all samples since the polymer backbone is oxidized during the test, so that a greater amount of peroxide species is decomposed upon heating. Also under these conditions, the antioxidant effect of HSCG is clear when the CL intensity is compared with that of the additive-free PE film. To inquire into the effect of HSGC on the photostability of PE at temperatures close to those of service life, the prepared films were subjected to UV irradiation at 40 °C for a prolonged time up to 1000 h and periodically analyzed by FTIR spectroscopy and mechanical tests throughout the aging process. The evaluation of chemical changes due to photooxidation was performed by monitoring the absorption intensity changes of carbonyl groups in the wavelength region between 1670 and 1800 cm−1 in the FTIR spectra. The peaks in the selected range were related to the formation and accumulation in the polymer of γ-lactones, esters, ketones, and associated carboxylic acids. Spectra of PE + 0.5% HSCG and PE + 2% HSCG were analyzed in comparison with neat PE. Figure 6c shows the accumulation rate of CO groups for all samples as a function of aging time. The carbonyl groups content remained low and almost constant up to 300 h irradiation for all samples, as carbonyls are secondary photoproducts of hydroperoxide homolysis. Subsequently, their formation rate significantly increased for PE due to the autoaccelerating nature of the oxidative reactions of polyolefins. On the other hand, the carbonyl formation rate of the films containing HSCG was lower, and no accelerating stage was observed after 300 h. The increase in carbonyl groups observed by spectroscopic analysis was correlated to the changes in tensile properties of the UV-irradiated films. Figure 6d reports the relative change of strain at break for LLDPE-based films as a function of irradiation time. All tested films show a drop in the tensile strain since the aging treatment resulted in a molecular weight reduction of the polymer matrix. In particular, PE showed a decrease in strain at break soon after 100 h, while a slight increase was detected for the stabilized samples since the aging treatment was responsible for thermal annealing of the polymer, which improved the degree of perfection of the crystals and favored crystalline phase aggregation.15 Interestingly, PE + 0.5% HSCG and PE + 2% HSCG retained about

Figure 4. (a) EPR spectra of SCG (dashed line) and HSCG (continuous line). (b) Power saturation profiles (amplitude vs power intensity) of SCG (□) and HSCG (■).

reported in HepG2 cells. It seems likely that in this case GSHbinding by melanoidin-type products overcomes the protective action of chlorogenic acid derived units. Polyethylene (PE) Stabilization by HSCG. Chemiluminescence (CL) emission of polyolefins is a well-established analytical technique capable of detecting the presence of very small amounts of peroxidic structures on polymer backbone, thus allowing evaluation of the efficiency of different antioxidant packages in a fast and reliable manner.10 The CL emission is related to the rate of polymer oxidation, and antioxidants reduce the CL intensity proportionally to their respective stabilizing efficiency. Homogeneous, transparent, light amber, linear, low density polyethylene (LLDPE) films containing 0.5% or 2% HSCG were obtained by extrusion. CL emission under nitrogen upon temperature ramping was studied to evaluate the content of peroxides formed in these films during processing at high temperature. Moreover, CL measurements on the films under

Table 2. Effect of High Doses of SCG and HSCG on HepG2 Cell Viabilitya LDH leakage (%)b sample

0 μg/mL

50 μg/mL

100 μg/mL

500 μg/mL

1000 μg/mL

SCG HSCG

2.79 ± 0.03a 2.79 ± 0.03a

3.55 ± 0.48a 3.04 ± 0.40a

4.24 ± 0.66a 3.43 ± 0.45a

32.46 ± 1.00c 4.06 ± 0.41a

91.67 ± 0.31d 24.03 ± 0.55b

a Lactate dehydrogenase (LDH) leakage was used as index of cell viability. bResults are expressed as percent of LDH activity in the culture medium with respect to the total activity (culture medium plus intracellular). Reported are the mean ± SD values of four experiments. Different letters indicate statistically significant differences (p < 0.05) among data.

G


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Figure 5. (a, b) Protective effect of SCG and HSCG on cell viability and ROS generation. HepG2 cells were treated with the noted quantitites of SCG and HSCG for 20 h, then the cultures were washed, and 400 μM tert-butylhydroperoxide (t-BuOOH) was added to all the cultures except controls for 2 h. Cells only submitted to t-BuOOH were used as positive controls. (a) Cell viability was determined as relative percent of crystal violet-stained control cells. (b) ROS production was expressed as relative percent of fluorescence units. (c, d) Direct and protective effect of SCG and HSCG on glutathione (GSH). (c) HepG2 cells were treated with the noted quantities of SCG and HSCG for 20 h, and intracellular concentration of GSH was determined and expressed as percent of fluorescent units relative to control. (d) HepG2 cells were treated with the noted quantities of SCG and HSCG for 20 h, then submitted to 400 μM t-BuOOH for 2 h, and GSH determined and expressed as above. C = control cells; t = t-BuOOH only treated cells. Data are means ± SD (n = 6−8). Different letters indicate statistically significant differences (P < 0.05) among different groups.

In other experiments, the lipid oxidation inhibition properties of HSCG were evaluated in aqueous emulsion (oil-in-water) model systems using Fe2+ as the oxidation inducer.45 HSCG was incorporated in different quantities into 1% aqueous emulsions of cod liver oil subjected to oxidation by 100 μM Fe2+ at room temperature, and after 3 h, thiobarbituric acid reactive substances (TBARS) were measured. No TBARS formation was observed under the same conditions using soybean oil, likely due to the antioxidant action of tocopherols. Figure 7 shows that inhibition of TBARS formation by HSCG was dose dependent. Lipid oxidation is a chain reaction proceeding through the formation of free radicals. Therefore, the lipid preservation capacity of HSCG may be attributed to the free radical scavenging property, which helps terminate oxidation at an early stage thus reducing the buildup of new radicals in the oxidation process. Inhibition of lipid peroxidation in the model emulsion system through a chelation mechanism is unlikely, as HSCG showed poor chelating properties toward Fe2+ ions in the ferrozine assay46 under conditions similar to those adopted in the oil oxidation experiments (not shown). On the other hand, reduction of hydroperoxides by HSCG cannot be excluded as ca. 40% abatement was observed in separate experiments in which HCSG was incubated with 0.75 mM tBuOOH in chloroform as a model compound. In conclusion, we report herein a simple, facile, and ingenuous chemical manipulation of SCG based on hydrolytic

75% deformation at break even after 300 h, under which conditions the undoped samples failed dramatically. Food Lipid Preservation by HSCG. The ability of HSCG to decrease lipid peroxidation in food models was investigated using bulk oil and aqueous emulsion model systems. Currently employed assays for food rancidity involve measurement of hydroperoxides and low molecular weight aldehydes. Reaction of oxygen with unsaturated lipids produces a wide range of compounds with hydroperoxides as the initial products.58 Lipid peroxidation is responsible for the changes in taste and odor of fats and oils by the production of secondary low molecular weight aldehydes. Occurrence of oxidized lipids in foods has been considered to be cytotoxic, atherogenic, and carcinogenic,59,60 and recently, consumer health consciousness has led to a demand for natural alternatives to synthetically produced food antioxidants to control the rate of oxidation of polyunsaturated fatty acids. In a first series of experiments, soybean and cod liver oil samples were subjected to accelerated aging at 60 °C17 in the presence and absence of HSCG deposited on the bottom of the flask (final dose 5 mg/mL). As shown in Table 3, all the oil samples showed an increase in hydroperoxide content as determined by the ferrous oxidation-xylenol orange (FOX) assay44 after a 6-day period. However, a more than 50% lower increase compared to the control samples was observed in the presence of HSCG. H


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Figure 6. Effect of HSCG on the thermal and photo-oxidative stability of PE films. (a) CL emission of films upon heating in nitrogen. (b) CL emission of films upon thermal oxidation in air. (c) Kinetic evolution of carbonyl groups of films exposed to UV irradiation at 40 °C. (d) Relative strain at break of films exposed to UV irradiation at 40 °C.

Table 3. Content of Hydroperoxides in Soybean and Cod Liver Oils Following Accelerated Aging at 60 °C in the Presence or Absence of 5 mg/mL HSCG hydroperoxides (mmol/kg)a sample soybean oil (control) soybean oil + HSCG cod liver oil (control) cod liver oil + HSCG

0 days 15 15 48 48

± ± ± ±

2 2 3 3

6 days 75 39 159 87

± ± ± ±

2 2 4 1

a

Determined by the FOX assay44 using a calibration curve obtained with t-BuOOH. The concentrations of hydroperoxides are expressed as mmol of t-BuOOH per 1 kg of oil. Reported are the mean ± SD values of three experiments.

Figure 7. Effect of HSCG on TBARS formation following ironinduced oxidation of the oil emulsions. TBARS are expressed as μmol of malondialdehyde per 1 g of oil. Reported are the mean ± SD values of three experiments.

treatment with acids under heating, which leads to HSCG as an all-natural polymer with multipotent intrinsic antioxidant properties, exhibiting up to more than 10 times stronger free radical scavenger and reducing potency compared to untreated SCG. HSCG displays a porous sponge-like morphology (SEM analysis) and consists mainly of phenolic-type polymers (13C solid-state NMR evidence) with intrinsic paramagnetic properties reflecting mainly EPR-detectable carbon-centered free radicals. HSCG can efficiently protect hepatocarcinoma cells from oxidative stress injury, can delay lipid peroxidation in soybean and fish oils, and can stabilize PE films from thermal

and photo-oxidative decomposition. Several key features would make HSCG a highly convenient and competitive solution compared to previously reported antioxidant polymers, including GPT. These several key features are listed below: (a) Easy accessibility as ubiquitous waste at little or no cost. (b) Cheap, efficient, and scalable chemical manipulation protocol with no derivatization or solvent/reagent-based treatment I


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(13) del Mar Castro López, M.; López de Dicastillo, C.; López Vilariño, J. M.; González Rodríguez, M. V. Improving the capacity of polypropylene to be used in antioxidant active films: incorporation of plasticizer and natural antioxidants. J. Agric. Food Chem. 2013, 61, 8462−8470. (14) Persico, P.; Ambrogi, V.; Baroni, A.; Santagata, G.; Carfagna, C.; Malinconico, M.; Cerruti, P. Enhancement of poly(3-hydroxybutyrate) thermal and processing stability using a bio-waste derived additive. Int. J. Biol. Macromol. 2012, 51, 1151−1158. (15) Ambrogi, V.; Cerruti, P.; Carfagna, C.; Malinconico, M.; Marturano, V.; Perrotti, M.; Persico, P. Natural antioxidants for polypropylene stabilization. Polym. Degrad. Stab. 2011, 96, 2152− 2158. (16) Williams, S. R.; Lepene, B. S.; Thatcher, C. D.; Long, T. E. Synthesis and characterization of poly(ethylene glycol)−glutathione conjugate self-assembled nanoparticles for antioxidant delivery. Biomacromolecules 2009, 10, 155−161. (17) Nand, A. V.; Swift, S.; Uy, B.; Kilmartin, P. A. Evaluation of antioxidant and antimicrobial properties of biocompatible low density polyethylene/polyaniline blends. J. Food Eng. 2013, 116, 422−429. (18) Kim, E.; Liu, Y.; Shi, X. W.; Yang, X.; Bentley, W. E.; Payne, G. F. Biomimetic approach to confer redox activity to thin chitosan films. Adv. Funct. Mater. 2010, 20, 2683−2694. (19) Kim, E.; Liu, Y.; Baker, C. J.; Owens, R.; Xiao, S.; Bentley, W. E.; Payne, G. F. Redox-cycling and H2O2 generation by fabricated catecholic films in the absence of enzymes. Biomacromolecules 2011, 12, 880−888. (20) Olejar, K. J.; Ray, S.; Ricci, A.; Kilmartin, P. A. Superior antioxidant polymer films created through the incorporation of grape tannins in ethyl cellulose. Cellulose 2014, 21, 4545−4556. (21) González-Centeno, M. R.; Jourdes, M.; Femenia, A.; Simal, S.; Rosselló, C.; Teissedre, P. L. Characterization of polyphenols and antioxidant potential of white grape pomace byproducts (Vitis vinifera L.). J. Agric. Food Chem. 2013, 61, 11579−11587. (22) Mussatto, S. I.; Machado, E. M. S.; Martins, S.; Teixeira, J. A. Production, composition, and application of coffee and its industrial residues. Food Bioprocess Technol. 2011, 4, 661−672. (23) Campos-Vega, R.; Loarca-Piña, G.; Vergara-Castañeda, H. A.; Oomah, B. D. Spent coffee grounds: a review on current research and future prospects. Trends Food Sci. Technol. 2015, 45, 24−36. (24) Vardon, D. R.; Moser, B. R.; Zheng, W.; Witkin, K.; Evangelista, R. L.; Strathmann, T. J.; Rajagopalan, K.; Sharma, B. K. Complete utilization of spent coffee grounds to produce biodiesel, bio-oil, and biochar. ACS Sustainable Chem. Eng. 2013, 1, 1286−1294. (25) Jiménez-Zamora, A.; Pastoriza, S.; Rufián-Henares, J. A. Revalorization of coffee by-products. Prebiotic, antimicrobial and antioxidant properties. LWT-Food Sci. Technol. 2015, 61, 12−18. (26) Monente, C.; Bravo, J.; Vitas, A. I.; Arbillaga, L.; De Peña, M. P.; Cid, C. Coffee and spent coffee extracts protect against cell mutagens and inhibit growth of food-borne pathogen microorganisms. J. Funct. Foods 2015, 12, 365−374. (27) Zuorro, A.; Lavecchia, R. Spent coffee grounds as a valuable source of phenolic compounds and bioenergy. J. Cleaner Prod. 2012, 34, 49−56. (28) Moreira, A. S. P.; Nunes, F. M.; Domingues, M. R.; Coimbra, M. A. Coffee melanoidins: structures, mechanisms of formation and potential health impacts. Food Funct. 2012, 3, 903−915. (29) Panzella, L.; Eidenberger, T.; Napolitano, A.; d’Ischia, M. Black sesame pigment: DPPH assay-guided purification, antioxidant/ antinitrosating properties, and identification of a degradative structural marker. J. Agric. Food Chem. 2012, 60, 8895−8901. (30) Cruz, R.; Cardoso, M. M.; Fernandes, L.; Oliveira, M.; Mendes, E.; Baptista, P.; Morais, S.; Casal, S. Espresso coffee residues: a valuable source of unextracted compounds. J. Agric. Food Chem. 2012, 60, 7777−7784. (31) Brunauer, S.; Emmett, P. H.; Teller, E. Adsorption of gases in multimolecular layers. J. Am. Chem. Soc. 1938, 60, 309−319. (32) Cesareo, E.; Korkina, L.; D’Errico, G.; Vitiello, G.; Aguzzi, M. S.; Passarelli, F.; Pedersen, J. Z.; Facchiano, A. An endogenous electron

(c) Multipotent activity in different antioxidant assays against ROS and RNS (d) Insolubility ensuring lower leaking from polymer blends (e) Good biocompatibility Although we are aware that several issues still need to be addressed, for example, a detailed cost-benefit analysis including management of SCG recovery, evaluation of the hydrolysis costs at a large scale, and primarily the development of suitable devices for use in the food industry, we are confident that the results of the present work will stimulate further studies to promote exploitation of the new material in biomedicine, active food packaging, and materials technology.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS This work was partially supported by grants from Italian MIUR (PRIN 2010-2011 PROxi project). REFERENCES

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A Superior All-Natural Antioxidant Biomaterial from Spent Coffee

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