Converting Spent Coffee Grounds into Bioactive Extracts with Potential

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Converting spent coffee grounds into bioactive extracts with potential skin anti-aging and lightening effects Helena M. Ribeiro, Margherita Allegro, Joana Marto, Bruno Pedras, Nuno G. Oliveira, Alexandre Paiva, Susana Barreiros, Lidia M. D. Goncalves, and Pedro Miguel Calado Simões ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00108 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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ACS Sustainable Chemistry & Engineering

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Converting spent coffee grounds into bioactive extracts with

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potential skin anti-aging and lightening effects

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Helena M Ribeiro a, Margherita Allegro a, Joana Marto a*, Bruno Pedras b, Nuno G

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Oliveira a, Alexandre Paiva b, Susana Barreiros b, Lídia M Gonçalves a, Pedro Simões b

5 6 7 8 9

a

Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade

de Lisboa, 1649-003 Lisboa, Portugal b

LAQV-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia,

Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal

10 11

*

Corresponding author. E-mail: [email protected]

1 ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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The aim of this study was to valorize spent coffee grounds (SCG) into bioactive extracts

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for improving skin health. These extracts were obtained by subcritical water extraction

15

(SWE) at 100 bar and temperatures up to 220 ºC, in a semi-continuous mode. They

16

were analyzed for phenolic acids, carbohydrates, antioxidant activity (AA) measured by

17

the DPPH assay, ROS scavenging activity in keratinocyte cells, and elastase and

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tyrosinase inhibitory activity. SCG extracts collected up to 140 ºC had higher phenolic

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acids content (19.9 mg GA/gdry SCG), higher AA (EC50 of 20.6 µg mL-1), and lower

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carbohydrate content (38 mg sugars/gdry SCG) than the fraction collected from 140 to 220

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ºC (5.7 mg GA/gdry SCG, EC50 of 132.2 µg mL-1, and 286 mg sugars/gdry SCG). The extracts

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were proven to have anti-aging and skin lightening effects by inhibiting elastase (99%

23

and 97.9%) and tyrosinase (78.6% and 92.1%) activity. The extracts were incorporated

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in shear thinning and acidic hydrogels for topical application. Release (70%) and

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permeation studies (65.3± 13.1µg/cm2) as well as cytotoxicity assays were performed to

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evaluate the release and permeation of bioactive compounds and the safety of

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hydrogels.

28 29

Keywords: Spent coffee grounds; subcritical water; topical formulation; anti-aging

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activity; anti-elastase; anti-tyrosinase

31 32 33

Introduction Agro-industrial wastes are a viable resource for bioactive substances, like

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polyphenols, polysaccharides and lipids that may find application in the pharma, food

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and cosmetics sectors. 1


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Coffee is an important food commodity in the world whose consumption has reached

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9 million ton in 2015. 2 Green coffee beans are a rich source of phenolic substances.

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They mainly comprise chlorogenic acids (ca. 12% of dry solid weight) that are the ester

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form of trans-cinnamic acids and quinic acids. 3 During roasting, the chlorogenic acid

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content of coffee beans decreases and Maillard reactions occur between proteins, sugars

41

and chlorogenic acids to form melanoidins and other complex substances. 3 Melanoidins

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are substances with relatively high molar mass and unidentified structure that present

43

antioxidant capacity.

44

Spent coffee grounds (SCG) is the residue that remains after roasted coffee beans are

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extracted with boiling water to obtain the coffee beverage. It is an important by-product,

46

reaching up to 6 million tons worldwide per year. SCG is currently valueless and

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normally treated as a waste or used in composting processes. Yet, SCG is a

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lignocellulosic matrix (approximately 40%, 10%, and 20% in hemicellulose, cellulose

49

and lignin, respectively) containing other useful compounds in addition to phenolics,

50

such as lipids and protein (approximately 10% and 14%, respectively). 4 In recent years,

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the search for possible applications of this by-product has increased, including its use as

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a source of dietary fiber with health benefits, 4-6 the production of SCG extracts with

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antioxidant capacity, 7 the incorporation of SCG oil in topical formulations leading to

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improved skin lipid levels (sebum) and hydration, 8-9 or even the use of SCG for the

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production of biodiesel. 10

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The use of clean and sustainable extraction technologies to obtain extracts with high

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bioactivity and no toxicity due to the solvent used has increased in the last years. Water

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is a renewable and environmentally friendly solvent. At a temperature range of 100-300

59

°C and above its vapor pressure as to remain in the liquid state, so-called subcritical



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water (SWE) presents unusual properties that makes it interesting as extraction and

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reaction solvent medium. For example, the water dielectric constant decreases sharply

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with increasing temperature, from ca. 80 at 20 ºC to about 30 at 200 ºC, as exhibited by

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methanol at ambient temperature. As a consequence, SWE gains the ability to dissolve

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less polar compounds. 11 In addition, the dissociation constant of water increases by 3

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orders of magnitude, which makes SWE suitable as a catalyst for hydrolysis reactions

66

that normally require an added acid or base. 11 Therefore, this environmentally friendly

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solvent has been proposed as an adequate solvent to extract bioactive polar substances

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from plants. 12

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The use of phenol-rich plant extracts has been shown to be a valuable strategy to

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prevent oxidative stress, due to their biological activity. Skin, which is constantly

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exposed to detrimental agents that continuously produce reactive species leading to

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ageing and diseases, 13 has been one of the targets of treatments with phenolics.

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Attributes such as stability, safety, cosmetic acceptability and drug administration are

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required in order to fulfil the development of skin preparations. 14 However, the stratum

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corneum (SC) and the its lipid matrix avoid drug permeation. This layer must be

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modified with chemical enhancers to allow the permeation of substances at a suitable

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rate to the desired target. 15 Chemical permeation enhancers can cause bilayers’

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structure disruption benefiting skin permeation. In addition to this mechanism, the use

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of solvents, such as ethanol, may increase the solubility of the drug, enhancing its

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permeation to the matrix.16

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Among different kinds of topical formulations for enhancing percutaneous permeation

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and maintaining the stability of ingredients, hydrogels are valuable vehicles of active


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substances, thanks to their high compatibility with many different species,

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biocompatibility, and enhancement of wounds healing. 17

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One goal of this research was to use SWE to obtain coffee extracts from spent coffee

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grounds, to perform the chemical characterization of these extracts, and to assess their

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properties, such as the antioxidant capacity, as well as the elastase and tyrosinase

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inhibitory activity. Another goal was to incorporate the spent coffee grounds extracts

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into hydrogels for topical application, and to assess those hydrogels in terms of their

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rheological profile, in vitro release, permeation, and cytotoxicity.

91 92

Material and Methods

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Materials

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Spent coffee grounds (SCG) were obtained at LAQV-REQUIMTE, Portugal, from

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Delta® Q capsules. SCG was oven dried at a temperature of 105 ºC for 24 hours with

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continuous circulation of air to remove humidity. The final water content of the dried

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spent coffee grounds was determined by Karl Fischer titration (Metrohm 831

98

Coulmeter, Portugal). A value of 1 wt% was obtained on average.

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The standards for sulfuric acid (96%), phenol (99%), D-glucose, caffeine,

100

trigonelline, chlorogenic acid, caffeic acid and p-coumaric acid (98-99% purity) were

101

obtained from SIGMA Aldrich (Lisbon, Portugal). Sodium carboxymethyl cellulose

102

was obtained from Panreac, acetonitrile and ethanol from Carlo Erba. Hydrophilic

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polysulfone membrane filters, 0.45 µm (Tuffryn®), were purchased from Pall

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Corporation. 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCFDA) was from Thermo

105

Fisher Scientific and spontaneously immortalized human keratinocyte cell lines HaCaT



106

(CLS, Germany) were used for antioxidant activity assays with cells. Cell culture

107

medium and supplements were from Gibco (Thermo Fisher Scientific, UK).

108

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The enzymes and substrates used on the enzymatic assays were mushroom tyrosinase

109

and tyrosine from SIGMA Aldrich (Lisbon, Portugal), Human neutrophil elastase

110

(HNE) and fluorogenic substrate (MeO-Suc-Ala-Ala-Pro-Val-AMC) from Calbiochem

111

(Merck, Germany).

112 113

Methods

114

Subcritical water extraction of SCG

115

SWE extraction of SCG was carried out at 220 ºC and 100 bar in a semi-continuous

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apparatus available at LAQV-REQUIMTE, Portugal, and described elsewhere. 18 The

117

high-pressure reactor (7 mL internal volume) was first filled with ca. 3 g of SCG and

118

then water was pumped through the reactor with a 1 mL/min constant flowrate during

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ca. 90 min.

120

The temperature of the outlet stream took ca. 30 min to attain 220 ºC. The total

121

amount of aqueous extract leaving the reactor was collected in two vials for subsequent

122

analysis. The aqueous extract that exited the reactor from the beginning of the assay

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until the outlet temperature reached 140 ºC was collected in a first vial (S1), while the

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rest of the aqueous extract was collected in a second vial (S2), as temperature increased

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from 140 to 220 ºC and was maintained at the latter temperature. Both samples were

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filtered through paper filters (FILTER-LAB, FILTROS ANOIA S.A., Barcelona,

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Spain), and lyophilized for 24 hours. Three samples of each extract were taken for

128

analysis. The SWE extraction of SCG was performed in duplicate.


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The yield of extraction was determined by measuring the mass of the SCG extract

130

after lyophilization and dividing it by the total mass of dry SCG loaded into the reactor.

131 132

Chemical Characterization of SCG extracts

133

Total phenolic content

134

The total phenolic content (TPC) of SCG extracts was determined by the Folin-

135

Ciocalteu method, using gallic acid (GA) as the standard. 7 The results were calculated

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as milligram of GA equivalent per gram of dry SCG (mg GA/g dry SCG).

137 138

Total carbohydrate content

139

The total carbohydrates content of SCG extracts was determined by the phenol-

140

sulphuric acid method, using D-glucose as the standard. 18

141 142

Analysis of phenolic acids and alkaloids

143

Phenolic acids, namely chlorogenic acid, caffeic acid and p-coumaric acid, and

144

alkaloids, namely trigonelline and caffeine, present in the SCG extracts were quantified

145

by HPLC with a Thermo Scientific Finnigan Surveyor Plus HPLC system

146

(Massachusetts, USA). The method used was taken from the literature. 19

147 148

Melanoidins content

149

The melanoidins content in SCG extracts was obtained according to a protocol taken

150

from the literature, 20 where the absorbance of the extracts was measured at 420 nm.

151 152

Antioxidant activity of SCG extracts



The free radical scavenging capacity of the extracts was assessed by the DPPH assay.

153 154

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The extracts were analyzed at final concentrations ranging from 1-250 µg/mL (1, 5,

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10, 25, 50, 100, 150, 200 and 250 µg/mL). Ascorbic acid (100 µM, positive control) was

156

also evaluated in the same way as described for extracts. Three independent

157

experiments were carried out for all the extracts and controls used.

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The antioxidant activity was expressed as the half effective concentration, EC50, i.e.,

159

the concentration of the extract solution necessary to decrease by 50% the absorbance

160

compared to that of the blank solution and expressed in µg of extract/mL DPPH

161

solution.

162 163 164

Reactive oxygen species scavenging activity of SCG extracts The intracellular production of reactive oxygen species (ROS) within HaCaT cell line

165

of SCG extracts was assessed with a fluorimetric technique previously published. 22

166

Samples (at concentrations of 1 mg/mL and 0.1 mg/mL) or ascorbic acid (1 mg/mL) were

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tested with hydrogen peroxide solution (500 µM) used for induction of ROS in cells.

168 169 170 171

SCG anti-aging Activity of SCG extracts The HNE inhibition assay was performed according to the technique previously published. 23

172 173 174

SCG lightening Activity of SCG extracts The mushroom tyrosinase inhibitory activity was assessed by a former technique

175

with modifications. 24 The reaction was performed in 96-well microplate with 151 µL of

176

0.1M sodium phosphate buffer (pH 6.5), 5 µL of dissolved extracts in buffer (1 mg/mL),


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8 µL of mushroom tyrosinase (2,500 unit/mL), and 36 µL of 3.75 mM L-tyrosine. The

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reaction was incubated at 37ºC in a microplate reader (FLUOstar Omega, BMGLabtech,

179

Germany) and the absorbance measured at 280nm each minute for 30 minutes. The

180

activity of samples tested was determined by the initial rate of formation of dopachrome

181

from the reaction mixture and compared with activity of buffer solution.

182 183 184

Hydrogels The hydrogels composition was: 2% CMC, 86% water, 2% dry extract and 10%

185

ethanol. Gels were prepared using a cold process, by dispersing the aqueous thickening

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agent, 2 wt.% sodium carboxymethyl cellulose (CMC) in 86 wt.% water, at 1000 rpm

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magnetic stirring for about 2 hours. Previously the SCG extract, S1, (2 wt.% on dry

188

basis) was dissolved in 10 wt.% ethanol under magnetic stirring at 700 rpm for about 10

189

minutes. This phase was added to the aqueous solution and the resulting mixture was

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homogenized until a transparent greenish-brownish or white homogeneous gel was

191

achieved (SCG gel). A gel with caffeine was used as control and had the same

192

composition of the other gel, except that the extract was replaced by 2 wt.% of caffeine

193

(CAF gel).

194

A viscometer, Brookfield Rotation Viscosimeter®, RV DV-II, small sample adapter

195

(approximately 25 mL) with a spindle 27, at 25 ± 2 °C, (Brookfield Engineering

196

Laboratories, USA) was used to determine the apparent viscosity and the rheological

197

profile of hydrogels. A shear rate sweep from 0.6 to 60/s and up and down for 7.5 min

198

was assessed. pH values were determined using a pH-meter S20 Seven easy pH, Mettler

199

Toledo, Germany, as described elsewhere. 25

200



201 202 203 204

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Release and permeation assays of hydrogels The Franz diffusion cell apparatus was used to perform the in vitro release and permeation studies according to the technique previously published. 26 Release and permeation of caffeine from the hydrogels were measured using a

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hydrophilic polysulfone membrane 0.45µm (Tuffryn®, Pall Corporation) and a

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caucasian female human epidermis after ethical approval and informed consent. In order

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to ensure sink conditions phosphate buffer (pH 7.4) was used as receptor phase.

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The caffeine content was analyzed by HPLC. 27 The equipment used was LaChrom,

209

with Autosampler LaChrom L-7200 Merk Itachi, Diode array detector L-7450A and

210

pump L-7100 and column oven L-7350. Samples collected from permeation studies

211

were analyzed with microplate reader FLUOStar Omega Spectrophotometer, BGM

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Labtech, at 280 nm and the caffeine released or permeated was calculated. The gel CAF

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(2 wt.%) formulation was used as control. At least six replicated cells for each

214

formulation were used. The dissolution efficiency (DE) was determined from the

215

experimental data and fitted to several kinetic models. 28, 29

216 217

Cell viability of hydrogels

218

The cytotoxicity was evaluated using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-

219

diphenyl-2H-tetrazolium bromide) reduction as endpoint of the cell viability for 24h of

220

exposition according the previous published work. 22

221 222

Statistical analysis


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The GraphPad PRISM® 5 software was used to perform the ANOVA analysis and

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Tukey–Kramer post-hoc multiple comparison test. An α error of 5% was chosen to set

225

the significance level.

226 227

Results and discussion

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Characterization of SCG extracts

229

SWE extraction of SCG was carried out at a target temperature of 220 ºC, at 100 bar

230

and for 90 min. Other authors have used SWE to extract phenolic compounds from SCG

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within the temperature range 120-200 ºC. 8 On the other hand, previous reports show

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that at temperatures between 180 and 200 °C, practically all hemicellulose from

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biomass can be solubilized. 30 As explained in section 2.2.1, in each SWE assay two

234

samples were taken, as temperature increased from ambient to 140 ºC (S1), and as

235

temperature increased from 140 to 220 ºC and was kept at that temperature (S2). The

236

purpose was to know whether extracts with different properties could be obtained in a

237

single assay. In particular, the aim was to compare an extract expected to have a low

238

amount of (soluble) carbohydrates (S1), with an extract rich in oligomers and monomers

239

from the extraction and hydrolysis of structural hemicellulose (S2). Pressure in this

240

case was merely used to keep water in the liquid state. A visible difference in the color

241

of the aqueous extract exiting the high-pressure reactor was observed. At the beginning,

242

the collected aqueous phase was dark colored, gaining intensity and turbidity with the

243

increase in temperature; near the end of the assay, it turned back to pale. The two

244

samples collected were lyophilized and analyzed for their content in carbohydrates and

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phenolics. Regarding the latter, the Folin-Ciocalteu method was used to quantify TPC,

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whereas HPLC was used to quantify target compounds. The results obtained are shown



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in Table 1. The TPC values obtained in this work are within the range of other values

248

reported in the literature for SWE extracts from SCG. Ballesteros et al. 7 reported a TPC

249

of 17.4 mg GA/g dry SCG and 39.3 mg GA/g dry SCG for SCG extracts obtained in assays

250

performed at 160 ºC and 200 ºC, respectively, in batch reactors, and 50 min extraction

251

times. The extraction yields were, respectively, 14.7 and 26.1 g/100 g dry SCG. The

252

former value is in good agreement with the value obtained in this work for S1 samples,

253

whereas the second value is considerably lower than for S2 samples. Higher maximum

254

temperature and extraction time in the latter case may be behind the differences

255

observed.

256

The S1 sample had a higher TPC and was richer in phenolic acids and alkaloids than

257

the S2 sample, as shown in Table 1. In addition, the S1 sample had an amount of

258

carbohydrates corresponding to less than 4 g/100 g SCG, consistent with a previous

259

water extraction of coffee beans in the process of making coffee. On the other hand, the

260

S2 sample had an amount of carbohydrates corresponding to around 30 g/100 g SCG,

261

consistent with the release of hemicellulose components. Moreover, an increase of ca.

262

20% of the relative content of melanoidins was observed in sample S2 relative to

263

sample S1.

264

SWE has also been used to extract polyphenols from other coffee residues, such as

265

coffee silverskin (CS), obtained during the coffee bean roasting step. 31 At 120 ºC, an

266

extract with 19.17 mg GA/g dry CS was obtained, which compares well with the TPC value

267

of 19.9 mg GA/g dry SCG for S1 samples in this work. Furthermore, Narita and Inouye 32

268

reported a total phenolics content of SWE extracts from silverskin residues ranging

269

from 22 to 28 mg/g dry CS at temperatures between 180 and 270 ºC.


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270

The antioxidant activity (DPPH) of the two SCG extracts was measured and

271

expressed as the half effective concentration (EC₅₀) (Table 1). The SCG extracts were

272

also tested for their reactive oxygen species (ROS) scavenging activity (Figure 1).

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Although both extracts showed a dose-response scavenging effect on the free radical

274

DPPH and exhibited a ROS scavenging effect, extract S1 had a higher antioxidant

275

capacity (indicated by a lower EC50). S1 extracts are comparatively less rich in

276

melanoidins than S2 extracts, but on the other hand have a higher TPC content. Also,

277

because they were collected in a temperature range at which SWE has a higher

278

dielectric constant, S1 extracts contain more polar phenolic compounds than S2

279

extracts. The combination of the last two factors must account for the higher antioxidant

280

activity exhibited by S1 extracts.

281 282 283

Most of the reports in the literature indicate that the antioxidant activity of roasted coffee is similar to that of tea, cocoa, and red wine. 33 According to literature, the roasting process has a contradictory effect on the

284

antioxidant capacity. The concentration of chlorogenic acids decreases significantly

285

with roasting. However, during roasting and through Maillard reactions, melanoidins

286

and other compounds are formed that show antioxidant capacity. 20, 34 Stennert and

287

Maier 35 evidenced the role of N-methylpyridinium, formed during the roasting process,

288

through degradation of trigonelline, as a major contributor to the antioxidant activity in

289

roasted coffee.

290

To evaluate the inhibitory activity of SCG extracts, in vitro assays were performed

291

against HNE and this was shown to be in the micromolar range with IC50 value of 13.7

292

± 0.9 and 24.3 ± 2.3 nM for SCG extracts S1 and S2, respectively. The ability of SCG

293

extracts to inhibit the elastin degrading enzyme (elastase) was explored, and very high



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anti-elastase activities were exhibited by the SCG extracts collected up to 140 ºC and

295

between 140 and 220 ºC, which inhibited over 99.0 ± 0.2% and 97.9 ± 0.3% of enzyme

296

activity respectively, because phenolic compounds have been reported to have anti-

297

elastase activities, i.e., they can inhibit the enzymes responsible for the hydrolysis

298

of elastin in the dermis. 36 According to the literature tyrosinase is a key enzyme in

299

melanin biosynthesis. This enzyme directly regulates the amount of melanin produced,

300

being involved in the formation of intermediates of the melanogenesis process, such as

301

L-Dopa and dopaquinone. Inhibition of tyrosinase activity decreases melanin

302

production and deposition. 24, 37

303

The results obtained revealed that the inhibition of mushroom tyrosinase activity

304

was 78.6 ± 1.4% and 92.1 ± 10.2% of the control for the SCG extracts S1 and S2,

305

respectively. Chang et al. 37 have shown that extracts with relatively high content of

306

phenolic substances were able to inhibit the tyrosinase activity and attributed this to the

307

antioxidant activity of the extracts. The anti-tyrosinase activity of extracts have been

308

also associated with the presence of caffeine and polyphenols in those extracts. 38

309 310 311

Characterization of SCG gels During the application of skin products, it is important to evaluate their resistance to

312

structure breakdown. Representative flow curves are represented in Figure 2 for SCG

313

hydrogel as well as control CAF gel. These hydrogels are considered shear thinning

314

(pseudoplastic) because the apparent viscosity values decrease as shear rate increases.

315

Such flow curves are typical for hydrogels prepared with cellulose polymers, where

316

shear thinning behavior is caused by the disentanglement of the polymer in solution or

317

by increasing the polymer orientation in the direction of the flow. 39


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318 319 320

On the other hand, apparent viscosity and loop areas values offer comparative results between formulations concerning resistance to structural loss. Placebo gel presents a hysteresis loop while the other two gels show shear thinning

321

behavior (the apparent viscosity decreased concomitantly with the increase in shear

322

rate). This characteristic is required to achieve a good spreadability and, in the case of

323

topical products, a good “skin feel”. 40

324

The inclusion of SCG extract in the gel significantly decreased the resistance of the

325

formulations to structural breakdown. The SCG gel showed a lower apparent viscosity

326

(125 Pa s) than the apparent viscosity of the control, the CAF gel (4680 Pa s). These

327

apparent viscosities were calculated at the apex of the both curves at a shear rate of 60

328

/s. This phenomenon was already described in literature and explained as a possible

329

consequence of –OH groups of polyphenols acting as space producers between

330

polymeric chains, thus increasing the free volume and mobility of the matrix. 41,42

331

The pH value of the formulation prepared with the SCG extract was 5.73 ±0.01 and

332

that of the control gel of 6.8 ± 0.1. An antioxidant hydrogel with 2 wt.% of coffee

333

extracts for skin administration was developed. To obtain a gel, cellulose polymers were

334

used to achieve ideal viscosity. The final pH of topical products should be acidic as skin

335

barrier physiological pH. In this work, the formulations developed presented acid pH

336

values that follow topical application products.

337 338 339

In vitro release and permeation assays The release profiles through synthetic membrane obtained for CAF and SCG

340

hydrogels performed for 6 hours, are shown in Figure 3. Both gels showed high release

341

fluxes. Analysis of variance (ANOVA) showed that no significant differences were



Page 16 of 33

342

observed (p0.05). In the permeation profiles it is

380

showed an approximately constant slope from the beginning of permeation to the end of

381

the assay (24 h) for all the formulations, without dramatic fluxes variation.

382 383

Cell viability

384

The SCG extract S1 as well as the respective SCG gel (Figure 4) showed no

385

significant difference (p>0.05) to culture medium used as negative control, and

386

consequently they can be preliminarily regarded as safe and suitable for topical

387

application.

388



389 390

Page 18 of 33

Conclusions In this work, skin care products were developed based on bioactive compounds

391

obtained from spent coffee grounds through a green process using subcritical water

392

extraction. SWE extraction/hydrolysis of SCG yielded bioactive extracts rich in

393

phenolic acids, especially those collected as temperature varied up to 140 °C, which

394

exhibited an EC50 of 21 µg mL-1. These extracts were thus chosen as bioactive

395

ingredient for topical formulation. The resulting hydrogels presented shear thinning

396

rheological behavior and acidic pH that allowed the delivery and permeation of

397

bioactive substances with antioxidant activity to the epidermis. From these studies, and

398

their maximum safe concentrations, SCG extracts appears to be promising candidates

399

for incorporation into anti-aging and skin lightening sustainable cosmetic products.

400 401

Acknowledgments

402

This project was funded by the strategic projects PEst-OE/SAU/UI4013/2011 and

403

LAQV, REQUIMTE - UID/QUI/50006/2013, FCT/MCTES (PIDDAC) and by FEDER.

404

The authors acknowledge the drawing support of Inês Marques Ribeiro.

405 406

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Figure Captions

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Figure 1. Percentage of ROS reduction after exposure to H₂O₂ of HaCaT cells, in RPMI

537

medium containing different concentrations of SCG extract. Extract concentration: 1 mg

538

mL-1 and 0.1 mg mL-1. Ascorbic acid was used as positive control, and the RPMI

539

culture medium as negative control. SCG S1 stand for extracts collected at temperatures

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up to 140 ºC, while SCG S2 stand for extracts collected at temperatures between 140

541

and 220 ºC. AA = Ascorbic acid. The data are expressed as the percentage of the ROS

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level in the control group, as the mean of at least 6 replicate experiments ± standard

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deviation. Significance: (*) p < 0.05 versus positive control cells (H2O2), (**) p

Converting Spent Coffee Grounds into Bioactive Extracts with Potential

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