Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20

Coffee silverskin: A possible valuable cosmetic ingredient Francisca Rodrigues, Ana Palmeira-de-Oliveira, José das Neves, Bruno Sarmento, M. Helena Amaral & M. Beatriz P. P. Oliveira To cite this article: Francisca Rodrigues, Ana Palmeira-de-Oliveira, José das Neves, Bruno Sarmento, M. Helena Amaral & M. Beatriz P. P. Oliveira (2015) Coffee silverskin: A possible valuable cosmetic ingredient, Pharmaceutical Biology, 53:3, 386-394, DOI: 10.3109/13880209.2014.922589 To link to this article: http://dx.doi.org/10.3109/13880209.2014.922589

Published online: 04 Dec 2014.

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Date: 07 November 2017, At: 12:32

http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, 2015; 53(3): 386–394 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.922589

ORIGINAL ARTICLE

Coffee silverskin: A possible valuable cosmetic ingredient Francisca Rodrigues1,2, Ana Palmeira-de-Oliveira3, Jose´ das Neves4, Bruno Sarmento4,5, M. Helena Amaral6, and M. Beatriz P. P. Oliveira1 Department of Chemical Sciences, Faculty of Pharmacy, REQUIMTE, University of Porto, Porto, Portugal, 2Fourmag Lda, Parque Industrial do Cruzeiro, Moreira de Co´negos, Portugal, 3CICS-UBI – Health Sciences Research Centre, University of Beira Interior, Covilha˜, Portugal, 4INEB – Instituto de Engenharia Biome´dica, Porto, Portugal, 5Department of Pharmaceutical Sciences, CICS – Centro de Investigac¸a˜o em Cieˆncias da Sau´de, Instituto Superior de Cieˆncias da Sau´de-Norte, CESPU, Gandra, Portugal, and 6Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal

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Abstract

Keywords

Context: Currently, there is a great tendency in cosmetic area to use natural extracts. Coffee silverskin (CS) is the most abundant solid by-product generated during roasting of coffee processing. Objectives: To evaluate different CS extracts as promising cosmetic ingredients, regarding antioxidant, antimicrobial, and cytotoxic properties. Materials and methods: Aqueous, hydroalcoholic and ethanolic CS extracts were obtained by an environmentally friendly procedure considering costs and pollution. Extracts were characterized for total phenolic and flavonoid contents (TPC and TFC, respectively), antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), antimicrobial activity expressed as minimal inhibitory concentration (MIC) and cytotoxicity using the 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and lactate dehydrogenase (LDH) assays in two skin cell lines (fibroblasts and keratinocytes). Results: The TPC of extracts was 18.33–35.25 mg of gallic acid equivalents per g of material on a dry basis (mg GAE/g db). The TFC of extracts was 1.08–2.47 mg cathechin equivalents per g dry material (mg CE/g db). The antioxidant activity was high, with values ranging between 95.95 and 216.40 mmol Fe2+/g for aqueous and alcoholic samples, respectively. Preliminary assays for antimicrobial potential showed that extracts display antibacterial activity. The MIC varied from 31.3 to 250 mg/mL for Gram-positive, and from 31.3 to 1000 mg/mL for Gram-negative. Extracts did not affect in vitro cell viability, with values near 100% in all concentrations tested. Conclusion: Results seem show that CS is a safe source of natural antioxidants with antifungal and antibacterial activity and no cytotoxicity, with potential usefulness for cosmetic applications.

Agro-industrial by-products, antimicrobial activity, antioxidant activity, coffee industry residues, cosmetics, cytotoxicity, innovative revalorization of coffee by-products, skin care

Introduction Alongside rising interest in natural products for health and personal care applications, there is a trend towards the use of environmentally friendly products obtained by sustainable resources. By-product valorization is a relatively new concept in the field of industrial residues management, promoting the principle of sustainable development. Application of agroindustrial residues in bioprocesses, on one hand, provides alternative substrates with potential market value, while, on the other hand, helps solving pollution problems, of otherwise disposable remainders (Pandey et al., 2000). Food processing by-products enable reduced waste and the recovery of extracts for the production of precious metabolites via chemical and Correspondence: Francisca Rodrigues, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Tel: + 351 220 428 500. Fax: +351 226 093 390. E-mail: [email protected]

History Received 16 October 2013 Revised 24 November 2013 Accepted 01 May 2014 Published online 4 December 2014

biotechnological processes. After specific pre-treatments with physical and biological agents followed by tailored recovery procedures, they might provide valuable natural antioxidants, antimicrobial agents, vitamins, etc., along with macromolecules, such as cellulose, starch, lipids, proteins, plant enzymes and pigments, of enormous interest to pharmaceutical, cosmetic, and food industries (Federici et al., 2009). In particular, agro-industrial by-products are good sources of phenolic compounds, and have also been explored as a source of natural antioxidants (Balasundram et al., 2006). While the use of naturally occurring phenolic compounds as food antioxidants is particularly interesting, practical aspects should be considered as the extraction efficiency, availability of raw material and safety. Coffee beans contain several classes of health-related chemicals such as phenolic compounds, diterpenes, xanthines, and vitamin precursors (Alves et al., 2009; Ludwig et al., 2012). Coffee phenolics have attracted much interest in recent

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DOI: 10.3109/13880209.2014.922589

years due to their strong antioxidant and metal-chelating properties (Panusa et al., 2013). These properties are believed to provide in vivo protection against free radical damage and reduce the risk of degenerative diseases associated with oxidative stress. Advances in industrial biotechnology offer potential opportunities for economic utilization of agro-industrial residues of coffee such as coffee pulp, coffee husk, or coffee silverskin (Pandey et al., 2000). Usually, two coffee beans are found in each fruit, each bean covered with a thin closely fitting skin called silverskin (CS), outside of which is a looser, yellowish skin called the parchment, the whole being encased in a pulp which forms the flesh of the cherry (Saenger et al., 2001). The CS is a tegument of coffee beans that constitutes a by-product of the roasting procedure and is the most abundant solid by-product generated during the coffee process. CS has no commercial value and is currently discarded as a solid waste, in some cases, it is used as fertilizers or simply burned (Saenger et al., 2001). This has negative effects on the environment, and so, the disposal of CS requires proper management. Alternatively, the transformation of this by-product into valuable products for other purposes is attractive. However, few studies have been performed on the properties of CS, in particular the antioxidant capacity (Borrelli et al., 2004; Narita & Inouye, 2012; Pourfarzad et al., 2013). According to Narita and Inouye (2012), consumers generally prefer natural antioxidants to synthetic ones because of the higher safety. Recently, it has been reported that CS is a rich source of antioxidants constituents (Borrelli et al., 2004; Narita & Inouye, 2012), but to the best of our knowledge, there are no reports on the use of CS for cosmetic applications. A recent work carried out in our laboratory showed that antioxidant content of CS extracts depends of the temperature and time of extraction (Costa et al., 2014). We consider that lower temperatures may be more attractive from an economical and industrial point of view. Thus, our current goal involves the extraction of active compounds from biological waste at relatively low temperatures. The objective of this work was to evaluate the antioxidant activity aqueous, hydroalcoholic, and alcoholic of CS extracts obtained at 40  C. Also, the antimicrobial properties and cytotoxicity of extracts were assessed as relevant to the use of these ingredients in cosmetics. The present study was designed (a) to quantify the antioxidant activity of different CS extracts by DPPH and FRAP assay and evaluate the total polyphenol content and the total flavonoid content; (b) to evaluate the antimicrobial activity of CS extracts against bacteria and yeast; (c) to analyze the cytotoxicity profile of CS extracts in keratinocytes and fibroblasts from skin; (d) to choose the best extract to be used in cosmetic applications.

Materials and methods Materials Coffee silverskin samples were provided by Bicafe´ (BICAFE´ – Torrefacc¸a˜o e Come´rcio de Cafe´ Lda, Portugal), located in Porto, Portugal. Ascorbic acid, DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical, catechin, Folin–Ciocalteu’s reagent, gallic acid, iodine, Trolox (6-hydroxy-2,5,7,8-

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tetramethylchroman-2-carboxylic acid, a water-soluble derivative of vitamin E), and a-tocopherol were all purchased from Sigma-Aldrich (Steinheim, Germany). Reagent grade ethanol, sodium acetate, sodium carbonate decahydrate, sodium nitrite, aluminum chloride, and sodium hydroxide were purchased from Merck (Darmstadt, Germany). Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAXÔ-I, fetal bovine serum (FBS), streptomycin, penicillin, and amphotericin B were from Invitrogen (Carlsbad, CA). MTS assay kit (CellTiter 96Õ Aqueous One Solution Cell Proliferation Assay) was purchased from Promega Co. (Madison, WI). Lactate dehydrogenase (LDH) colorimetric cytotoxicity assay kit (LDH Cytotoxicity Detection Kit) was purchased from Takara Bio Inc. (Shiga, Japan). Deionized water was obtained using Mili-Q wa purification system (TGI Pure Water Systems, St Lincoln, NE). Preparation of coffee silverskin extracts CS was milled to particle size of approximately 0.1 mm using a A11 basic analysis mill (IKA Wearke, Staufen, Germany) and stored in silicone tubes at room temperature (25–28  C) until extraction. Three samples of 1 g were subjected to maceration for 30 min at 40  C with 20 mL of ethanol, ethanol:water (1:1) or distilled water. Extracts were filtered through Whatman No. 1 paper filter and the filtrates were collected. Aqueous filtrates were freeze-dried and hydroalcoholic and alcoholic filtrates were reduced to residue using a rotary evaporator set to 37  C. Samples were stored at 4  C until analysis. For determination of the chemical parameters, the extracts were reconstituted in the solvent used for extraction. Determination of total phenolic and flavonoid contents Total phenolic content (TPC) was spectrophotometrically determined according to the Folin–Ciocalteu procedure (Singleton & Rossi, 1965) with minor modifications. Briefly, 500 mL of extract (1 mg/mL) was mixed with 2.5 mL of the Folin–Ciocalteu reagent (10  dilution) and allowed to react for 5 min. Then, 2.5 mL of Na2CO3 7.5% (w/v) solution was added and after 15 min at 45  C, and 30 min at room temperature, the absorbance was measured at 765 nm using a microplate reader (Synergy HT, BioTek Instruments, Inc., Winooski, VT). A calibration curve of gallic acid with a linearity range of 5–100 mg/mL, and R240.998 was used. The TPC of the extract was expressed as mg of gallic acid equivalents per g of plant material on dry basis (mg GAE/g db). The assay was done in triplicate. Total flavonoid content (TFC) was determined by a colorimetric assay based on the formation of flavonoid– aluminum compound according to Barroso et al. (2011). Briefly, 1 mL of extract (1 mg/mL) was mixed with 4 mL of water and 300 mL of NaNO2 solution (5% w/v). Then, 300 mL of AlCl3 solution (0.1 g/mL) were added, and after 1 min, 2 mL of NaOH (1 M) and 2.4 mL of water. The absorbance was determined at 510 nm using a microplate reader. A calibration curve of catechin with a linearity range of 0400 mg/mL and R240.999 was used. The TFC of the

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extract was expressed as mg of catechin equivalents per g of plant material on dry basis (mg CE/g db). The assay was done in triplicate.

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Antioxidant capacity of coffee silverskin extracts The antioxidant activity of the samples was evaluated by DPPH radical-scavenging activity and ferric reducing antioxidant power (FRAP). DPPH is known as a stable free radical which possesses a characteristic maximum absorption between 515 and 517 nm. The reaction mixture on 96 wells plate consisted of one solution per well for different sample concentrations (30 mL) and methanol solution (270 mL) containing DPPH radicals (6  105 mol/L). The mixture was left to stand for 30 min in the dark. The reduction of the DPPH radical was determined by measuring the absorption at 517 nm (Guimara˜es et al., 2010) using the microplate reader. The antioxidant capacity based on the DPPH free radical scavenging ability of the extract was expressed as mmol Trolox equivalents per g of plant material on dry basis (mmol Trolox E/g db). A calibration curve of Trolox with a linearity range of 2.5–100 mg/mL and R240.996 was prepared. FRAP was carried out according Benzie and Strain (1999) with minor modifications. Briefly, 90 mL of different sample concentrations were added to 2.7 mL of the FRAP reagent (10:1:1 of 300 mM sodium acetate buffer at pH 3.6, 10 mM TPTZ, and 20 mM FeCl36H2O) and the reaction mixture was incubated in a water bath at 37  C. The increase in the absorbance at 592 nm was measured after 30 min using a micro-plate reader. A calibration curve of FeSO4–7H2O with a linearity range of 150–2000 mM and R240.996 was used. The results were expressed as mmol Fe2+ equivalents per g of material on dry basis (mmol Fe2+/g db). Antimicrobial activity of coffee silverskin extracts

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respectively (NCCLS, 2008). Briefly, two-fold serial dilutions of extracts were performed in Mueller Hinton broth (MH; Difco Laboratories, Detroit, MI) for bacteria and with RPMI (Biochrom, Berlin, Germany) for yeast. Concentrations ranging from 3.9 to 500 mg/mL were tested. Microorganism growth was visually compared for each concentration with the negative control (without extracts). Minimal inhibitory concentration (MIC) was defined as the lowest extract concentration able to completely inhibit microorganisms’ growth, corresponding to 100% MIC value. All determinations were performed in triplicate for each assay and three independent experiments were run with concordant results. Cytotoxicity and cell viability of coffee silverskin extracts Cell lines, primary cell isolation and culture conditions Human immortalized non-tumorigenic keratinocyte cell line (HaCaT) and human foreskin fibroblasts (HFF-1) were purchased from CLS Cell Lines Service (Eppelheim, Germany) and ATCC (Manassas, VA), respectively. HaCaT and HFF-1 cells were individually maintained in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAXÔ-I (Invitrogen, Grand Island, NY), 10% inactivated fetal calf serum (FBS; Invitrogen), 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen, Grand Island, NY), and 0.25 mg/mL amphotericin B (Invitrogen, Grand Island, NY), in a 5% CO2 environment at 37  C. The number of viable cells was periodically assessed by the trypan blue exclusion assay. After attachment, they were incubated in serum-free medium containing 1 mg/mL–1 mg/mL extracts for 24 h at 37  C. Plant extracts were dissolved in cell culture medium.

Microorganisms strains

Cell viability and toxicity assays

To test the antimicrobial activity, eight microorganisms (seven bacteria and one yeast) were enrolled in the study. Both American type culture collection (ATCC) and clinical isolates were selected, corresponding to two Staphylococcus aureus (ATCC 6538 and one clinical isolate), Staphylococcus epidermidis (clinical isolate), two Escherichia coli (ATCC 1576 and one clinical isolate), Klebsiella peumoniae (ATCC 4352), Pseudomonas aeruginosa (ATCC 9027), and Candida albicans (ATCC 10231). Clinical isolates were identified as to species-level with Vitek-2 identification cards (Biomerieux, Marcy l’Etoile, France) and all strains were kept frozen in Brain Heart Infusion (Difco Laboratories, Detroit, MI) with 20% glycerol (Sigma) at 70  C until testing. For each experiment, microorganisms were subcultured twice in Nutrient agar (Difco Laboratories, Detroit, MI) for bacteria and Sabouraud dextrose agar (SDA; Difco Laboratories, USA) for yeasts, to assess culture viability.

Two different assays were used to assess cell integrity and cytotoxicity of the extracts: (1) monitoring the uptake of the vital mitochondrial dye, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS) by cell mitochondria and (2) determining the leakage of the cytosolic enzyme, lactate dehydrogenase (LDH) into the cell medium (LDH assay). In both cases, cells (HFF-1 or HaCaT) were seeded in 96-well plates (5  103 cells/well) and incubated with medium for 24 h (37  C, 5% CO2). HFF-1 and HaCaT cells were used at passages 17–19 and 77–79, respectively. Then, triplicate wells were incubated with extracts dissolved in cell culture medium pre-filtered by 0.45 mm Millex GV filters (Millipore, Nepean, ON, Canada) at different concentrations (0.1–1000 mg/mL). Cells were exposed to extracts for 24 h. Medium without FBS was used in the case of LDH. Afterwards, MTS and LDH assays were performed according to manufacturers’ instructions. Briefly, cell supernatants were collected for the LDH assay and cells were washed twice with phosphate buffered saline pH 7.4 (PBS) before being incubated with 120 mL of MTS in medium for 4 h (37  C, 5% CO2). The absorbance was measured for each well at 490 nm with background subtraction at 630 nm using a microplate reader. In the case of LDH,

Determination of minimal inhibitory concentrations The antibacterial activity was tested according to CLSI M7-A7 microdilution (NCCLS, 2006) and the antifungal activity was tested according to M27-A2 microdilution from the same protocol, after 48 h of incubation at 37  C and 35  C,

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the absorbance after 30 min/room temperature incubation of cell supernatants with LDH reagent was measured at 490 nm with a background subtraction at 690 nm using a microplate reader. Also, the maximum release of this enzyme (high control) was determined by incubating cells with 1% (w/v) Triton X-100 (Boehringer, Mannheim, Germany), while spontaneous LDH release (low control) was assessed by incubating the cells with DMEM alone. In MTS assay, the positive control was the medium and the negative control was Triton X-100. In all cases, each concentration was tested in triplicate in three independent experiments.

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Statistical analysis Data were reported as mean ± standard deviation of at least triplicate experiments. Statistical analysis of the results was performed with SPSS 19.0 (SPSS Inc., Chicago, IL). One-way ANOVA was used to investigate the differences between different extracts for all assays. Post hoc comparisons were performed according to Tukey’s HSD test. In all cases, p50.05 was accepted as denoting significance.

Results Determination of total phenolic content (TPC) The broad range of effects of free radicals in biological systems has drawn the attention of many experimental studies. It has been proved that these mechanisms may be important in the pathogenesis of certain diseases and ageing. The TPC of the different CS extracts was determined using the Folin–Ciocalteu reagent. Results are presented in Table 1. The largest amount of total phenolic components (35.25 ± 0.25 mg) extracted from 1 g of CS was determined with ethanol as a solvent. It was almost two times higher than aqueous extract (18.33 ± 0.52 mg).

In vitro antioxidant activity The antioxidant potential of the CS extracts was determined by two methods based on different approaches, namely the DPPH radical scavenging method, and the FRAP assay, which have been extensively used to determine the antioxidant potential of various plant extracts and natural products (Rodrigues et al., 2013). Irrespective of the solvent used for extraction, all the extracts obtained from CS showed antioxidant potential (Table 1). Similar to the DPPH results, the highest antioxidant activity in FRAP was also obtained in alcoholic extract (Table 1). A range of 95.95, 134.0, and 216.40 mmol Fe2+/g for aqueous, hydroalcoholic, and alcoholic samples were obtained, respectively. Antimicrobial activity Microorganisms such as fungi and bacteria are important factors that cause oxidative processes during storage of cosmetic products and consequent deterioration. Despite several previous studies on the antimicrobial properties of coffee against many human pathogens, to date there is no research on the antifungal and antibacterial activity of CS (Martı´nez-Tome´ et al., 2011). The antibacterial activity of CS extracts was evaluated by the determination of MIC values (Table 2). In this study, microorganisms were selected to cover Gram-positive bacteria (S. aureus and S. epidermidis), Gram-negative bacteria (E. coli, K. pneumoniae, P. aeruginosa) and a yeast (Candida albicans). The consistent and reproducible results

Table 2. Antibacterial activity of different extracts of CS expressed as the minimal inhibitory concentration (MIC). Data expressed as mean of the replicates (n ¼ 3).

Determination of total flavonoids content (TFC)

MIC (mg/mL)

Flavonoids, which constitute the largest group of plant phenolics accounting for over half of the eight thousand naturally occurring phenolic compounds (Balasundram et al., 2006), were present in all CS extracts. Results varied between 1.08 mg (water extract) and 2.61 mg (ethanol extract) of Quercetin Equivalents (QE)/g of CS (Table 1). The results clearly showed that the highest content of flavonoids was obtained with ethanol.

Bacterial strains

Aqueous extract

Hydro-alcoholic extract

Alcoholic extract

S. aureus ATCC 6538 S. aureus MRSA S. epidermidis B21 E. coli ATCC 1576 E. coli B26 K. pneumoniae ATCC 4352 P. aeruginosa ATCC 9027 C. albicans ATCC 10231

31.3 250 62.5 62.5 125 31.3 41000 41000

125 250 62.5 125 125 31.3 41000 41000

125 250 62.5 125 125 31.3 41000 41000

Data expressed as mean of the replicates. Table 1. Total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant potential assay (FRAP), and 2,2diphenyl-2-picrylhydrazyl hydrate (DPPH) scavenging assay of coffee silverskin extracts (CS). Aqueous extract Mean TPC (mg gallic acid equivalent/g) TFC (mg cathechin equivalent/g) DPPH (mmol trolox equivalent/g) FRAP (mmol Fe2+/g)

b,c

18.33 1.08b,c 206.00b,c 95.95c

389

SD 0.52 0.23 3.72 1.50

Hydroalcoholic extract Mean a,c

30.50 2.47a 242.28a,c 134.0

SD 0.25 0.06 5.42 1.50

Alcoholic extract Mean a,b

35.25 2.61a 386.35a,b 216.40a

SD 0.25 0.15 5.10 2.70

‘‘a–c’’ denote a significant difference between mean values, where ‘‘a’’ denotes a significant difference from CS aqueous extract; ‘‘b’’ a significant difference from CS hydroalcoholic extracts; and ‘‘c’’ a significant difference from CS alcoholic extracts. Repeated measures ANOVA followed by Turkey’s post-hoc test, n ¼ 3 independent experiments. SD, standard deviation.

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obtained showed that CS extracts exhibited interesting antimicrobial potentials (Table 2). Aqueous extracts of CS presented MICs in the range of 31.3–250 mg/mL against S. aureus, S. epidermidis, E. coli, and K. pneumoniae. The hydroalcoholic and alcoholic extracts showed similar activity against the microorganisms tested. Both presented MICs in the range of 31.3–250 mg/mL against those tested microorganisms. The values varied from 31.3 to 1000 mg/mL of culture medium, and the lowest and highest mean MICs among the three groups were observed against K. pneumoniae (31.3 mg/mL) and C. albicans/P. aeruginosa (41000 mg/mL), respectively. Concerning antifungal test, the analyzed extracts failed to show any activity against C. albicans.

Table 3. Effect on the metabolic activity of HaCaT cells after the exposure to CS extracts at different concentrations, measured by the MTS assay. Cell viability (%) Aqueous extract Concentration (mg/mL) 0.1 1.0 10 100 1000

Mean 99.79 91.31c 96.11b,c 99.51b.c 103.60

SD

Hydroalcoholic extract Mean

SD

1.09 96.11 3.14 93.43c 0.46 92.58a,c 4.56 97.67a 5.89 101.55

3.90 2.82 1.11 1.04 4.04

Alcoholic extract Mean (2,3)

103.46 103.46(1,3,4,5)a,b 100.78(1,2)a,b 100.78(2)a 101.91(2)

SD 2.21 2.49 0.4 1.5 0.42

Values are expressed as the means ± SD (n ¼ 6). ‘‘a–c’’ denote a significant difference between mean values, where ‘‘a’’ denotes a significant difference from aqueous extract; ‘‘b’’ a significant difference from hydro-alcoholic extract; and ‘‘c’’ a significant difference from alcoholic extracts. Repeated measures ANOVA followed by Turkey’s post-doc test, n ¼ 3 independent experiments. ‘‘1–5’’ denotes a significant difference between mean values, where 1 denotes a significant difference from concentration 0.1 mg/mL inside an extract and 2 denotes a significant difference from concentration 1.0 mg/mL inside the same extract, etc. Repeated measures ANOVA followed by Turkey’s post-doc test, n ¼ 3 independent experiments. SD, standard deviation.

Keratinocytes and fibroblasts cytotoxicity assay The effects of the extracts on the growth of keratinocyte (HaCaT) and fibroblast (HFF-1) cells were investigated by MTS and LDH assays. These cell lines are regarded as suitable in vitro models for testing the toxicity potential of substances or products intended for dermatological use (Pillai et al., 2010). According to the obtained results for the MTS assay, none of the extracts exhibited cytotoxicity against HaCaT cells (Table 3) at all concentrations up to 1000 mg/mL. Table 4 presents the cytotoxicity data of the three different extracts of CS against HFF-1 cells and as determined by the MTS assay. The number of viable fibroblast cells was similar for all extracts tested. The values ranged from 91.64% for aqueous extract (0.1 mg/mL), 96.92% for hydroalcoholic extract, and to 96.85% for alcoholic extract (1000 mg/mL). An important indicator of cell damage in oxidative stress is the increase in cell membrane permeability. In this study, potential cytotoxicity effects of different extracts of CS were investigated by examining membrane integrity of HaCaT and HFF-1 cells using a LDH release assay. In HaCaT cells, at all concentrations tested, CS extracts did not affect LDH leakage following 24 h incubation, presented for all tested concentrations in the absence of any toxicity, which means that all these extracts are safe on the concentration range studied. The evidence of possible absence of cytotoxicity of this extracts at five different concentrations, namely 0.1, 1.0, 10, 100, and 1000 mg/mL, implies that these extracts present low toxicity to these cells. Similarly, results for fibroblasts were comparable with those of HaCaT cells, with no significant increase in LDH being observed. The results between MTS and LDH are in concordance, which is in agreement with Issa et al. (2004) and Fotakis et al. (2006) who showed that both assays are inversely proportional.

Discussion In this work, a new procedure was proposed in order to maximize the extraction results and to define the best

Table 4. Effect on the metabolic activity of HFF-1 cells after the exposure to CS extracts at different concentrations, measured by the MTS assay. Cell viability (%) Aqueous extract Concentration (mg/mL) 0.1 1.0 10 100 1000

Mean (4,5)b,c

91.64 98.90(4,5)b,c 96.35(4,5)b 122.87(1,2,3)b,c 120.14(1,2,3)b,c

Hydro-alcoholic extract SD 7.90 5.87 8.60 1.05 5.38

Mean (2,3,5)a

130.53 147.08(1,4,5)a,c 144.00(1,5)a,c 128.63(2,5)a,c 96.92(1,2,3,4)a

SD 5.89 0.81 2.95 9.10 7.68

Alcoholic extract Mean (3,4,5)a

132.88 130.79(3,4,5)a,b 105.31(1,2,5)b 112.22(1,2,5)a,b 96.85(1,2,3,4)a

SD 3.08 4.23 5.06 4.36 5.36

Values are expressed as the means ± SD (n ¼ 6). ‘‘a–c’’ denote a significant difference between mean values, where ‘‘a’’ denotes a significant difference from aqueous extract; ‘‘b’’ denotes a significant difference from hydro-alcoholic extracts; and ‘‘c’’ denotes a significant difference from alcoholic extracts. Repeated measures ANOVA followed by Turkey’s post-doc test, n ¼ 3 independent experiments. ‘‘1–5’’ denote a significant difference between mean values, where 1 denotes a significant difference from concentration 0.1 mg/mL inside an extract and 2 denotes a significant difference from concentration 1.0 mg/mL inside an extract, etc. Repeated measures ANOVA followed by Turkey’s post-doc test, n ¼ 3 independent experiments. SD, standard deviation.

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conditions for antioxidant compounds extraction from CS to be used as new active ingredient for different kinds of industries, especially cosmetic industry. In particular, aqueous, hydroalcoholic, and alcoholic solvents were studied. Thirty minutes was fixed as the extraction time since the use of longer periods could not be economically advantageous. The extraction temperature was fixed at 40  C, considering also economical reasons and since no significant advantage in extraction efficiency was observed when using higher temperatures, as previously demonstrated by our group (Costa et al., 2014). Statistical data analysis revealed a significant influence of all the studied solvents on the total phenolics content and antioxidant activity, which were improved when the ethanol was the solvent. The TPC of the CS extracts obtained with different solvents increased with decreasing polarity of solvent systems. This might be due to the fact that phenolic compounds are often more soluble in organic solvents, less polar than water (Meneses et al., 2013). Moreover, the obtained values are in agreement with the content reported in other important antioxidant source by-products that are being studied for antioxidant activity, such as spent coffee grounds (13–35 mg gallic acid equivalents (GAE)/g dry matter) (Mussatto et al., 2011; Panusa et al., 2013), blackberry (50–80 mg GAE/g dry matter) (Aybastıer et al., 2013), brewer’s spent grains (9.9 mg GAE/g dry matter) (Meneses et al., 2013), almond shells (38.0 mg GAE/g dry matter) (Sfahlan et al., 2009), or apple pomace, which was below 15 mg GAE/g dry matter (Reis et al., 2012). When comparing the results obtained for CS with the ones for coffee, it is apparent that the coffee waste has a similar phenolic content (Naidu et al., 2008). Naidu et al. studied the antioxidant composition of green coffee and concluded that the total polyphenol content from extraction with isopropanol plus water was around 32 mg GAE/g. On an average, the flavonoid content was increased by about two-fold, when water was replaced by ethanol, which can be attributed to an on-average higher affinity of these compounds for ethanol than for pure water. Data examination reveals that the solvent had a significant effect on the extraction efficiency. According to some authors, the addition of water to polar organic solvents such as acetone, methanol, and ethanol creates a more polar medium that facilitates the extraction of phenolic compounds (Spigno et al., 2007). The content of total flavonoids was in high correlation with the content of total phenols (R2 ¼ 0.963). According to Hecˇimovic´ et al. (2011), the TFC in coffee varies between 12 and 20 mg GAE/g. Comparing with the flavonoid content in CS, the obtained results indicate that roasting affects the polyphenolic compounds of CS. Comparing with other coffee by-products like spent coffee grounds, the TFC is similar (2.11–8.03 mg QE/g dry waste) (Panusa et al., 2013). The DPPH radical scavenging activity of different solvents of the phenolic extract of each CS was measured and the mmol Trolox equivalents of each CS were determined. As indicated by the DPPH values in Table 1, for each waste material, the use of ethanol as an extraction solvent resulted in higher antioxidant activity. The results showed that DPPH radical scavenging activity efficiencies were in the order of alcoholic 4 hydroalcoholic 4 aqueous extract. The DPPH values range

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from 206.0 to 386.35 mmol Trolox equivalents, for water and ethanol. This can be attributed to the higher total polyphenolic content of CS. A good correlation between the total polyphenol content and the DPPH scavenging radical activity was expected. In addition, plotting the DPPH value of the various extracts against their corresponding total phenolic content showed that these values were correlated. However, a mean R2 value of 0.69 was determined. This discrepancy could be due to a larger natural variation in the polyphenolic content. Also, the low correlation observed may be due to compounds that do not respond to the used reagent but also have antioxidant activity, such as caffeine or isoflavones. Furthermore, DPPH radical scavenging activity depends on the hydrogen-donating ability of the extracted antioxidants (Prior et al., 2005). Comparing the total phenolic content and ferric reducing capacity of the samples a positive correlation (R2 ¼ 0.80) was observed as expected. Even though both the FRAP and DPPH assays were used to determine the antioxidant activity of the CS extracts, results are different due to the nature of each antioxidant assay. DPPH is a method based on the measurement of the reducing ability of antioxidants toward 1,1-diphenyl-2-picrylhydrazyl (DPPH). This ability can be evaluated by electron spin resonance or by measuring the decrease of its absorbance. Although both DPPH and FRAP have been widely used to measure the antioxidant capacity of natural extracts, the reaction of DPPH with free radical scavengers present in the test sample occurs rapidly and can be assessed by following the absorbance decrease at 517 nm. The reaction time of the improved DPPH assay is only 30 min, while the FRAP assay measures the reducing capability by increased sample absorbance based on the ferrous ions released, being thus more time consuming. In contrast, the DPPH assay is preferred to the ABTS assay because it is commercially available and does not have to be generated before assay like ABTS+ (Prior et al., 2005). It is worth noting that the extracts produced with water presented low antioxidant potential and also low content of total phenols when compared with most of the extracts produced with organic solvents like ethanol. Similar results have been reported during the extraction of antioxidant compounds from other raw materials such as spent coffee grounds (Mussatto et al., 2011; Panusa et al., 2013), grape by-products (Mendoza et al., 2013), or almond (Sfahlan et al., 2009). The use of ethanol as an extraction solvent is very interesting if it is desired the extract application in cosmetic products, for example. The use of organic solvents in the manufacturing process to obtain cosmetic ingredients is regulated, and from the perspective of cosmetic safety, it is preferable to use solvents such as ethanol or ethanol:water mixtures since they are in compliance with good manufacturing practice. Methanol is usually a good extraction solvent but it has a toxic character. Therefore, the extracts produced with methanol arises serious issues for the application in food and pharmaceutical products, according to the present legislation (Mussatto et al., 2011). Analyzing the antimicrobial activity, the inefficacy of the extracts against P. aeruginosa may be related with the presence of the extracellular matrix. For C. albicans, the complex yeast wall is responsible for preventing extracts to

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contact with cell structures. Generally, Gram-negative are more resistant to bactericidal polyphenols than Gram-positive bacteria (Dhaouadi et al., 2013). According to Fabry et al. (1998), for crude extracts of plants to be considered as potentially useful therapeutically, they should have MIC values 58 mg/mL. Gibbons (2005) suggests that isolated phytochemicals should have MIC51 mg/mL. The MIC values herein determined for CS extracts were below these values, except for C. albicans and P. aeruginosa, thus indicating a potential antimicrobial effect which may be advantageous for cosmetic products. In particular, these extracts may provide self-preservative properties to the formulations in which they are incorporated. As CS is the outer layer of the roasted coffee beans, it is conceivable that some of the properties described for coffee brews, namely antimicrobial and antioxidant properties, are also maintained in CS. So, we expect that CS has similar antioxidant and antimicrobial activities to coffee brews. The latter is probably a consequence of the high contents of melanoidins generated during roasting, because CS has a lower content of free phenol compounds (Borrelli et al., 2004; Esquivel & Jime´nez, 2012; Saenger et al., 2001). Also, antibacterial activity of the extracts could be related to the presence of high amounts of phenolic and flavonoid compounds in their composition, as referred above. As shown, plants are rich in a wide variety of secondary metabolites, such as polyphenols and flavonoids, which have been found to have antimicrobial properties in vitro (Cowan, 1999). These compounds have been suggested to interact with microorganism cell membrane causing protein membrane damages and consequently inducing its disruption. This effect leads to leakage of cellular content and causes interference with active transport and metabolic enzymes, as also as cytoplasm coagulation (Burt, 2004; Cowan, 1999; Tiwari et al., 2009). This cascade effects result in cell death. According to Dorman and Deans (2000), the high phenolic antibacterial activity could also be explained in terms of phenol nucleus alkyl substitution. Its alkylation alters the distribution ratio between the aqueous/non-aqueous phases by reducing the surface tension or altering the species selectivity. This alkyl substituted phenolic compounds form phenoxyl radicals which interact with isomeric alkyl substituents (Pauli & Knobloch, 1987). The hydroxyl group relative position seems to exert an influence upon the components effectiveness against Gram-negative and Gram-positive bacteria (Dorman & Deans, 2000). So the results of previous reports about coffee, along with our findings in CS, can confirm the inhibitory effects of CS extracts on growth and development of fungi and bacteria. Further studies, to identify the responsible molecules for antibacterial activity observed, are being developed in our laboratory. The cytotoxicity results appear to indicate that fibroblasts are more sensitive to all extracts as compared with HaCaT cells, as previously mentioned by other authors to similar compounds (Neamnark et al., 2008; Zanatta et al., 2008). Furthermore, these results suggested that all extract of CS, at concentrations 51000 mg/mL, have no adverse effect on the viability of fibroblast cells in vitro. These results could indicate a probable protective effect of the extracts on the cell

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line, but further studies are needed to confirm that. Ekmekcioglu et al. (1999) studied the effect of coffee in Caco-2 cells and no cytotoxicity was observed. Other by-products, like brewers’ spent grain, a protein-rich by-product of the brewing industry, have been tested for their cytotoxicity effect (McCarthy et al., 2013). McCarthy et al. (2013) studied the cytotoxicity effect of Brewers’ spent grain, against U937 and Jurkat T cells and concluded that cytotoxicity is low. Over the three peel ethanolic extracts analyzed (Punica granatum (pomegranate), Nephelium lappaceum (rambutan), and Garcinia mangostana (mangosteen), only rambutan may be considered potentially useful as a source of natural antioxidants for food or drug product because of its high antioxidant activity and non-toxic property to normal cells, at concentrations above 100 mg/mL (Okonogi et al., 2007). In the same way, Khonkarn et al. (2010) studied the cytotoxicity of fruit peel extracts from rambutan, mangosteen, and coconut against KB (human epidermal carcinoma of the mouth with HeLa contaminant) and Caco-2 (human colorectal adenocarcinoma) cells and proved that coconut showed a high cytotoxic effect towards KB cells for concentration above 100 mg/mL. This means that most of the by-products extracts have some cytotoxicity. However, CS showed no cytotoxicity for concentrations above 1000 mg/mL. For the best of our knowledge, this is the first study that reports the effects of CS in skin cell lines. In contrast, most of the studies of cell toxicity of by-products are not done in fibroblast or keratinocytes. The unique study that reports the use of these cells with by-products was done by Rodrigues et al. (2013), who studied the effect of Medicago in fibroblast and HaCaT cells, and, as well, no cytotoxicity was observed.

Conclusion The CS extracts showed high polyphenol content, mainly in the ethanolic extract that presented a high flavonoid content. The antioxidant activity was high and positive correlations with polyphenol content were observed. Also, a good antimicrobial activity was observed against K. pneumoniae, S. epidermidis, and S. aureus, with no cytotoxicity of all extracts to fibroblasts and keratinocytes at tested concentrations. In conclusion, CS ethanol extract can be considered the best one, as it expressed high antioxidant and antimicrobial potential with no toxicity. In this way, CS extracts may constitute a promising source of good antioxidative agents. However, investigation of the activity constituents associated with further purification may provide useful comparative information in the future.

Acknowledgements The authors thank BICAFE´ for providing CS samples.

Declaration of interest The authors declare that there are no conflicts of interest. Francisca Rodrigues is thankful to Foundation for Science and Technology (Portugal) for the PhD Grant SFRH/BDE/51385/ 2011 financed by POPH-QREN and subsidized by European Science Foundation. This work was supported by the COMPETE program (PEst-C/SAU/UI0709/2011).

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