FITOTE-03103; No of Pages 9

Fitoterapia xxx (2015) xxx–xxx

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Fitoterapia
journal homepage: www.elsevier.com/locate/fitote

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1Q1 Inhibitory effects of polyphenols from grape pomace extract on collagenase

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2 and elastase activity
Judith Wittenauer a,b,⁎, Sonja Mäckle a,b, Daniela Sußmann a,

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3Q2
4 Ute Schweiggert-Weisz a, Reinhold Carle b,c

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5 a
Fraunhofer Institute for Process Engineering and Packaging, Department of Process Development for Plant Raw Materials, Giggenhauser Str. 35, D-85354 Freising, Germany
6 b
Hohenheim University, Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology, Garbenstraße 25, D-70599 Stuttgart, Germany
7 c
King Abdulaziz University, Biological Science Department, Faculty of Science, P.O. Box 80257, Jeddah 21589, Saudi Arabia

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1 0 a r t i c l e i n f o a b s t r a c t

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12
Article history:
Received 21 October 2014 D
Breakdown and disorganization of extracellular matrix proteins like collagen, fibronectin and
elastin are main characteristics of skin aging due to the enhanced activation of proteolytic
16
17
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13 Accepted in revised form 6 January 2015 enzymes such as collagenases and elastases. Inhibition of their enzymatic activities by natural 18
14 Accepted 8 January 2015 plant compounds might be a promising approach to prevent extrinsic skin aging. Especially 19
15 Available online xxxx
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polyphenols are supposed to interact with those enzymes due to their molecular nature. In our 20
investigation, extracts of pomace from Riesling grapes were analyzed for their inhibitory 21
32 Chemical compounds studied in this article: properties on collagenase as well as elastase. Crude grape pomace extract showed a dose- 22
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33 Gallic acid (PubChem CID: 370) dependent inhibitory activity against both enzymes with IC50-values of 20.3 μg/ml and 14.7 μg/ml 23
34 (+)-Catechin (PubChem CID: 9064)
for collagenase and elastase activity, respectively. The extracts were fractionated into four 24
35 Caftaric acid (PubChem CID: 6440397)
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fractions containing phenolic compounds differing in chemical structure and polarity. Except for 25
36 Quercetin 3-O-glucoside
37 (PubChem CID: 5280804) the stilbene containing fraction, all other fractions showed inhibitory effects on both enzyme 26
38 Quercetin 3-O-glucuronid activities. The most pronounced impact was found for the hydrophilic low molecular weight 27
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39 (PubChem CID: 12004528) polyphenols containing the free phenolic acids. In particular, gallic acid showed considerable 28
40 Procyanidin B1 (PubChem CID: 11250133) inhibition values. EGCG was used as a positive control and showed a dose-dependent inhibition of 29
41 Procyanidin B2 (PubChem CID: 122738) collagenase activity (IC50 = 0.9 mM). 30
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31 Trans-resveratrol (PubChem CID: 445154) © 2015 Published by Elsevier B.V.

43 Keywords:
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44 Grape pomace
45 Vitis vinifera
46 Polyphenols
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47 Collagenase
48 Elastase
49 Skin aging
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50
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1. Introduction only organ directly exposed to the environment, aging processes 54

resulting from environmental damage are of considerable 55
51 Skin aging is a complex biological phenomenon influenced relevance. Particularly solar UV radiation adversely impacts 56
52 by several factors including genetics, environmental exposure, skin health, due to, amongst others, generation of reactive 57
53 hormonal changes, and metabolic processes. As the skin is the oxygen species (ROS) [1–3]. ROS are able to initiate complex 58
molecular pathways including the activation of enzymes 59
that degrade extracellular matrix (ECM) proteins in the 60
⁎ Corresponding author at: Fraunhofer Institute for Process Engineering and
dermis such as collagen and elastin ensuring the skin's three- 61
Packaging, Department of Process Development for Plant Raw Materials,
Giggenhauser Str. 35, D-85354 Freising, Germany. Tel.: +49 8161 491 440. dimensional integrity [1,2]. As a consequence of protease 62
E-mail address: judith.wittenauer@ivv.fraunhofer.de (J. Wittenauer). activation, breakdown, fragmentation and disorganisation of 63

http://dx.doi.org/10.1016/j.fitote.2015.01.005
0367-326X/© 2015 Published by Elsevier B.V.

Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005
 2 J. Wittenauer et al. / Fitoterapia xxx (2015) xxx–xxx

64 the ECM proteins occur, visibly manifested by the typical UV promoting properties, such investigations would be of basic 125
65 induced skin alterations such as deep wrinkles, loss of skin tone interest. 126
66 and resilience [3]. In the present study, the in vitro inhibitory potential of 127
67 Matrix metalloproteinases (MMPs), a family of multi- phenolic compounds obtained from white grape pomace against 128
68 domain zinc-containing endopeptidases with a broad range of the activity of Clostridium histolyticum collagenase (ChC) and 129
69 substrate specificities, constitute the major proteolytic enzyme porcine pancreatic elastase (PPE) was investigated. The poly- 130
70 group involved in the degradation of dermal connective tissue phenolic profile of the grape pomace extract was determined, 131
71 components. Under normal physiological condition, MMP moreover, different fractions of the extract as well as individual 132
72 activities are precisely regulated at the level of transcription polyphenols were assayed for their inhibitory activity against 133
73 and by endogenous protein inhibitors. In addition, MMPs are ChC and PPE, respectively. 134

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74 secreted from cells as an inactive precursor requiring cleavage
75 by extracellular proteinases to be activated. These regulation

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2. Materials and methods 135
76 processes enable the remodeling of connective tissue under
77 physiological and pathophysiological circumstances like wound
2.1. Materials 136
78

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healing, inflammation and cancer. The impairment of this
79 balance, due to oxidative stress, results in an excess of MMPs
Grape pomace from white wine (Vitis vinifera L. cv. 137
80 being a key feature of premature aging of the skin as well as
‘Weisser Riesling’) of vintage 2010 was kindly provided by 138

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81 various inflammatory and degenerative diseases.
the Baden-Badener Winzergenossenschaft (Baden-Baden- 139
82 In addition, serine proteases such as elastases are involved
Neuweier, Germany). Pomace samples (voucher specimen 140
83 in ECM degradation. Elastolytic enzymes are released from

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#600385.1 as deposited at the Fraunhofer IVV, Freising, 141
84 neutrophils and dermal fibroblasts in response to UV induced
Germany) were collected after pressing the mash, freeze 142
85 skin inflammation [4]. Due to their active side catalytic triad,
dried, sealed in polyethylene bags, and kept at 5 °C until 143
86 elastases are multi-specific and cleave several proteins within

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further use. 144
87 the extracellular matrix including collagen, fibronectin and
C18 reversed-phase cartridges (Chromabond, 2000 mg) 145
88 elastin. Elastin is an important ECM protein having the unique
were obtained from Macherey-Nagel (Düren, Germany). 146
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89 property of elastic recoil, which is vital for providing elasticity
C. histolyticum collagenase type IA (ChC), N-[3-(2-furyl) 147
90 to skin and other tissues like arteries, lungs and ligaments.
acryloyl]-Leu-Gly-Pro-Ala (FALGPA) and porcine pancreas 148
91 Several studies have shown that human neutrophil elastase
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elastase type III (PPE) were purchased from Sigma 149

92 is also involved in the degradation of ECM connective tissue
(St. Louis, MO). N-Succ-Ala-Ala-Ala-p-nitroanilide 150
93 through the activation of inactive MMP-1 and MMP-2
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(AAAPVN) was obtained from Serva Electrophoresis (Hei- 151

94 precursors [5–7].
delberg, Germany). Polyphenols used as standards for the 152
95 Inhibiting the activity of ECM degrading proteins like
inhibition studies were gallic acid (≥97%), catechin (≥99%), 153
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96 collagenases and elastases may be a useful approach to prevent

epigallocatechin gallate (≥95%) (Sigma, St. Louis, MO); caftaric 154
97 UV induced skin alterations and premature skin aging. Scaveng-
acid (≥97%), quercetin 3-O-glucoside (≥98%), quercetin 3-O- 155
98 ing of ROS by natural antioxidants might be one option to inhibit
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glucuronid (≥95%), procyanidin B1 (≥90%), procyanidin B2 156

99 such skin deteriorative enzymes, as ROS play an important role
(≥90%) (Fluka, Buchs, Switzerland), and trans-resveratrol 157
100 in the activation of these enzymes. Phenolic compounds are an
(98%) (ABCR, Karlsruhe, Germany). 158
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101 important group of natural antioxidants. They belong to diverse

All reagents and chemicals used were of analytical grade 159
102 subclasses of secondary plant metabolites classified as phenolic
and purchased from VWR (Darmstadt, Germany). 160
103 acids, flavonoids, stilbenes and lignans and are ubiquitously
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104 found in the plant kingdom. In particular, red and white grapes
105 contain high amounts of phenolic acids and flavonoids such as 2.2. Preparation of polyphenol rich extracts 161
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106 gallic acid and catechin [8]. Due to their chemical structure,
107 polyphenols have powerful antioxidant activities being able 2.2.1. Crude extract 162
108 to scavenge a wide range of ROS such as hydroxyl radicals, Extraction of polyphenols from grape pomace was carried 163
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109 superoxide radical and O− 2 [9]. In addition, polyphenols may out according to Kammerer, Claus, Carle and Schieber [8]. 164
110 inhibit the activity of proteolytic enzymes in vitro by acting as Briefly, lyophilised grape pomace was ground using a ZM 100 165
111 166
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complexing or precipitating agents as indicated in literature centrifugal mill with a 1 mm ring sieve (Retsch, Haan,
112 [10,11]. Especially, green tea polyphenols such as catechin and Germany). Aliquots of 2 g of the powdered samples were 167
113 epigallocatechin gallate, commonly used as ingredients in anti- weighted into Erlenmeyer flasks, flushed with nitrogen and 168
114 aging skin care formulations, have been shown to exhibit extracted with 50 ml of methanol for 60 min under shaking, 169
115 moderate inhibitory effects against collagenase and elastase using a KS 500 laboratory shaker (Janke & Kunkel, IKA 170
116 activity, presumably through non-covalent binding [12–14]. Labortechnik, Staufen, Germany). Extracts were filtered through 171
117 However, the mechanism of action of polyphenols is not fully a paper filter (Munktell, grade 3hw); the residue was re- 172
118 understood, and studies about the inhibitory effects of natural extracted with 50 ml of the solvent for 30 min to ensure an 173
119 active ingredients on skin degrading enzymes are scarce. Most of exhaustive extraction. Supernatants were combined, and the 174
120 the studies have been performed with isolated compounds, thus organic solvent was removed by evaporation in vacuo at 40 °C. 175
121 not considering the complex nature of crude extracts. Addition- Residues obtained were dissolved in 10 ml water and filtered 176
122 ally, data about inhibitiory properties of further polyphenolic through a 0.2 μm syringe filter (Sartorius, Göttingen, Germany), 177
123 subclasses are scarce. However, in particular, for the systematic and the pH was adjusted to 7.0 with sodium hydroxide solution 178
124 development of natural formulations exerting skin health (1 M). 179

Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005
 J. Wittenauer et al. / Fitoterapia xxx (2015) xxx–xxx 3

180 2.2.2. Polyphenol fractions a) dilutions of crude grape pomace extract with water at 236
181 The fractionation of polyphenolic compounds of the grape concentrations of 35.3 ± 1.7 μg/ml (1:20), 23.5 ± 1. μg/ml 237
182 pomace extract was achieved by solid phase extraction (SPE). (1:30), 14.1 ± 0.3 μg/ml (1:50), 8.8 ± 0.4 (1:80) and 7.1 ± 238
183 Cartridges were first activated with 12 ml of methanol, washed 0.3 μg/ml (1:100) (extract: water); 239
184 with 20 ml of water and then loaded with 2 ml of the crude b) the individual fractions 1–4; and 240
185 extract. Elution of the fractions followed by subsequent c) aqueous solutions of the individual reference polyphenols 241
186 application of various solvent mixtures. Fraction 1 was eluted at concentrations of 250 μM, 500 μM, 750 μM and 1000 μM, 242
187 with 20 ml of acetic acid (2%). Fraction 2 was obtained next by respectively. 243
188 application of 20 ml of a 3:1 mixture of acetic acid (2%) and
189 acetonitrile/acetic acid (2%) (v/v). Elution with 20 ml of acetic First, 30 μl of the samples (a–c) were incubated with 10 μl of 244

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190 acid (2%) and acetonitrile/acetic acid (2%) (v/v) at a ratio of 3:2 ChC solution and 60 μl tricine buffer for 20 min at 37 °C. 245
191 resulted in fraction 3. The last fraction (fraction 4) was obtained Subsequently, 20 μl of the FALGPA solution was added to 246

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192 by using 20 ml of acetonitrile/acetic acid (2%) (v/v). initiate the reaction. For negative controls water was added 247
instead of FALGPA solution. The ChC inhibitory activities of the 248
individual samples were measured by continuously monitoring 249

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193 2.3. HPLC analysis
the decrease in absorbance of FALGPA at 335 nm for 20 min 250
after starting the reaction. Initial velocities were calculated from 251
194 Separation of polyphenols was performed using an Agilent

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the slope of the absorbance change during the first 10 min of 252
195 HPLC series 1200 system (Agilent, Waldbronn, Germany)
hydrolysis. The relative inhibition was calculated according to 253
196 with ChemStation software, a model G1379 degasser, a
Eq. (1). IC50 values were determined from dose–effect curves. 254
197

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model G1312B binary gradient pump, a model G1367D
198 thermoautosampler, a model G1316B column oven and a
199 model G1315C diode array detector. The column used was a C ChC inhibition activity ð%Þ ð1Þ
200 18 Synergi-Hydro column (150 × 3.0 mm i.d.; 4 μm particle initial velocitycontrol ‐ initial velocitysample

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201 size) from Phenomenex (Torrance, CA) operated at 25 °C. ¼  100
initial velocitycontrol
202 The mobile phase consisted of 2% (v/v) acetic acid in water
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203 (eluent A) and 0.5% acetic acid in water and acetonitrile (50:50, 256
204 v/v, eluent B) using the following gradient program: 0–5% B
205 (35 min), 5–20% B (45 min), 20–100% B (30 min), 100% B
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2.5. Elastase assay 257

206 isocratic (3 min),100–0% B (10 min) at a flow rate of 0.8 ml/min.
207 Total run time was 123 min. The injection volume varied
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Porcine pancreatic elastase (PPE) inhibition of the individ- 258

208 between 10 and 30 μl. Polyphenols were monitored at 280 nm,
ual samples a–c (see Section2.4 2.4) was determined spectro- 259
209 and UV/Vis spectra were recorded in the range of 200 to
photometrically using AAAPVN as the substrate by monitoring 260
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210 600 nm.

the release of p-nitroaniline at 410 nm. PPE was dissolved in 261
211 Polyphenols were identified by UV–vis spectra and compar-
2 mM Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) buff- 262
212 ison with literature data and reference compounds. The
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er (pH = 8,0). Thereof, an aliquot of 10 μl was taken and 263

213 quantitation of individual polyphenols was performed using
loaded in the wells of the microtiter plates together with 100 μl 264
214 calibration curves of the corresponding reference compounds.
of the Tris buffer and 30 μl of the individual sample solutions. 265
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215 Gallic acid, caftaric acid, (+)-catechin, epigallocatechin gallate,

After 20 min of pre-incubation at 25 °C, 40 μl of the substrate 266
216 procyanidin B1, procyanidin B2, quercetin 3-O-glucoside,
AAAPVN (dissolved in 2 mM Tris buffer at a concentration of 267
217 quercetin 3-O-glucuronid, kaempferol 3-O-glucosid, and trans-
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0.25 mg/ml) was added. Absorbance was measured for 20 min 268
218 resveratrol served as reference compounds. The quantitation of
after the addition of AAAPVN. Initial velocities, the inhibition 269
219 epicatechin was performed applying the catechin calibration
activity and IC50 values were calculated in accordance with the 270
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220 curve. All determinations were carried out in quadruplicate.

ChC assay 271
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221 2.4. Collagenase assay 3. Results and discussion 272

222 Inhibition effects of the grape pomace crude extract, the 3.1. Identification of polyphenolic compounds in the crude grape 273
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223 fractions derived thereof as well as the standards mentioned pomace extract and the fractions derived thereof 274
224 above against C. histolyticum collagenase (ChC) were measured
225 spectrophotometrically according to a modified procedure 3.1.1. Crude extract 275
226 described by van Wart and Steinbrink [15] using a BioTek Prior to the enzymatic inhibition studies, the identification 276
227 Synergi H1 multi-mode microplate reader (BioTek, Bad and quantitation of the polyphenolic pattern by HPLC was 277
228 Friedrichshall, Germany) and appropriate data analysis software a prerequisite to identify the compounds being primarily 278
229 (Gen 5, version 2.04, BioTek, Bad Friedrichshall, Germany). responsible for the enzyme inhibitory effects. Major polyphe- 279
230 ChC (1.1 U/ml) and FALGPA (1 mM), respectively, nols of the grape pomace were the flavanols catechin (4) and 280
231 were dissolved in 0.05 M tricine [N-(2-hydroxy-1,1- epicatechin (6) accompanied by the procyanidins B1 (3) and 281
232 bis(hydroxymethyl)ethyl)glycine] buffer containing 0.4 M B2 (5), while the phenolic acids gallic acid (1) and caftaric 282
233 NaCl and 0.01 M CaCl2; the pH was adjusted to 7.5 with 1 M acid (2), the flavonol glycosides quercetin-3-O-glucuronide 283
234 NaOH. The inhibitory effects of the following samples were (7) and quercetin-3-O-glucoside (8) as well as the stilbene trans- 284
235 investigated: resveratrol (9) were found to be minor compounds (Fig. 1). 285

Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005
 4 J. Wittenauer et al. / Fitoterapia xxx (2015) xxx–xxx

286 3.1.2. Polyphenol fractions 3.3. Collagenase and elastase inhibitory activity of white grape 322
287 To rank the compounds from specific polyphenolic classes pomace extracts 323
288 according to their relative enzyme inhibitory effects, the crude
289 extract was separated into four fractions, being composed of To investigate the inhibition exerted by the grape pomace 324
290 polyphenols having similar chemical structure and polarity, extract on ChC and PPE, the respective enzymes were incubated 325
291 and the fractions were analyzed by HPLC. As shown in Fig. 2, with the extract and the appropriate substrates. The assays 326
292 fraction 1 contained the free phenolic acids gallic acid and were performed with synthetic peptides as substrate mole- 327
293 caftaric acid, while the main components of the crude extract, cules, and enzyme preparations isolated from C. histolyticum 328
294 catechin and epicatechin as well as the procyanidins B1 and B2, and porcine pancreas, respectively. As reported in literature, 329
295 were found in fraction 2. The flavone glycosides quercetin-3-O- PPE is topologically very similar to human neutrophil elastase 330

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296 glucuronide and quercetin-3-O-glucoside could be detected and inhibition mechanisms for some phenolic substances were 331
297 in fraction 3, whereas the stilbene resveratrol was the only found to be equal [17,18]. Therefore, it can be assumed that the 332

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298 polyphenolic constituent detectable in fraction 4. in vitro assays performed with the ChC and PPE and synthetic 333
peptides are a reliable tool to get first indications of the 334
335

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capability of grape pomace polyphenols to prevent extracellular
299 3.2. Quantitation of polyphenolic compounds in the crude extract matrix degradation as a skin aging phenomenon. The inhibitory 336
300 and the four fractions potential of the grape pomace extract was examined for 337

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increasing dilutions, in order to establish dose-dependent 338
301 Quantitated amounts of major polyphenolic constituents in relationships, and to calculate the half maximal inhibitory 339
302 grape pomace extract and fractions thereof are summarized in concentrations (IC50). 340

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303 Table 1. In accordance with literature data [8,16], the flavanols Crude grape pomace extract was incubated with collage- 341
304 catechin and epicatechin were the predominant components in nase and elastase at dilution-ratios ranging from 1:20 to 1:100, 342
305 the crude extract with concentrations of 210.7 and 180.7 μg/ml, resulting in total polyphenolic contents ranging from 35.3 to 343

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306 respectively, followed by the procyanidins B1 (97.5 μg/ml) and 7.1 μg/ml. The effect of different extract concentrations on the 344
307 B2 (85.9 μg/ml). The flavonol-glycosides, the phenolic acids as enzyme activity is shown in Fig. 3a and b for ChC and PPE, 345
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308 well as resveratrol were present in minor amounts. respectively. As expected the slope of the digestion curves 346
309 Due to the high absorption values at the detected wave- of the collagenase assay decreases with increasing extract 347
310 length, the grape pomace extract and the fractions had to be concentration, which is due to the lowered enzyme activity, 348
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311 diluted 20-fold for the subsequent enzyme assays. Amounts of thus resulting in higher amounts of non-cleaved substrate 349
312 individual polyphenols detected in fractions 1–3 were slightly molecules. The higher initial absorption with increasing extract 350
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313 lower than in the crude extract, which probably resulted concentration probably results from self-absorption of grape 351
314 from losses owing to the fractionation process with the pomace extract components. Since the elastase assay was 352
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315 exception of resveratrol in fraction 4. This could be due to performed with N-Succ-Ala-Ala-Ala-p-nitroanilide as the sub- 353
316 further overlapping constituents contained in the crude extract strate peptide, the enzyme activity can be deduced from the 354
317 (Fig. 1) which also absorb light at the detected wavelength, thus release of p-nitroaniline resulting in increased absorption 355
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318 interfering the chromatographic analysis. Therefore, integration values. At increased extract concentrations slopes in digestion 356
319 of resveratrol in the chromatograms of the crude extract curves were less steep representing an increased enzyme 357
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320 resulted in apparently lower amounts than in the purified inhibition. 358
321 fraction. The grape pomace extract inhibited both enzymes in a dose- 359
dependent manner. Highest polyphenol concentrations of 360
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35.3 μg/ml resulted in an 80% and 73% relative inhibition of 361

ChC and PPE activity, respectively (Fig. 4). Inhibitory capacities 362
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declined with decreasing concentrations of the extract. Relative 363

inhibition of ChC was 55% at a polyphenol concentration of 364
23.5 μg/ml, and the inhibition capacity further decreased at 365
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lower polyphenol concentrations in a nearly linear manner to 366

40% and 28% for 14.1 μg/ml and 8.8 μg/ml, respectively. Further 367
dilution of the extract to 7.1 μg/ml resulted in a negligible 368
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decrease of the inhibition value. The relative inhibition of PPE at 369

a concentration of 23.5 μg/ml was found to be 63%. At lower 370
grape pomace extract concentrations, inhibitory activities 371
markedly declined with relative inhibition values of 49%, 36% 372
and 20%, for 14.1 μg/ml, 8.8 μg/ml and 7.1 μg/ml, respectively. 373
From the dose-response relationships, IC50 values of 20.3 μg/ml 374
for ChC and 14.7 μg/ml for PPE activity were calculated. 375
Comparison of our present findings with those reported 376
for other plant extracts is rather difficult, due to varying 377
assay conditions, particularly incubation times and types 378
Fig. 1. HPLC separation of polyphenols from crude grape pomace extract
of substrates and enzymes used for the determination of 379
(280 nm). Peak assignment: (1) gallic acid, (2) caftaric acid, (3) procyanidine B1,
(4) catechin, (5) procyanidin B2, (6) epicatechin, (7) quercetin-3-O-glucuronide, the enzymatic activities. Additionally, the extracts are often 380
(8) quercetin-3-O-glucoside, (9) resveratrol. obtained using different extraction methods. The polarity of the 381

Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005
 J. Wittenauer et al. / Fitoterapia xxx (2015) xxx–xxx 5

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Fig. 2. HPLC separation of fractions obtained from grape pomace extract by means of solid phase extraction (280 nm) and related structures.
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382 solvent has a strong influence on the inhibitory effects of a 3.4. Collagenase and elastase inhibitory activity of grape pomace 396
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383 plant extract [19], indicating that individual constituents may extract fractions 397
384 differ regarding their inhibitory effects. Using more lipophilic
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385 extraction media could result in extracts with high inhibition of In order to determine the most potent polyphenols in the 398
386 collagenase and elastase activities [19–21]. Methanol is an grape pomace extract inhibiting ChC and PPE activities, fractions 399
387 400
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efficient extraction media for a broad spectrum of polyphenols containing individual polyphenolic compounds exerting differ-
388 [8,22,23]. Since methanol is an intolerable ingredient for ent polarities were analyzed. As shown in Fig. 5, fraction 2, 401
389 cosmetic formulations, the extracts used in our study were enriched in catechins and procyanidins at a concentration of 402
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390 dissolved in water. Therefore, lower inhibition values than 27.4 μg/ml was the most potent in ChC inhibition (43%). Effects 403
391 described in literature due to lowered extraction yields could be on ChC activity were lower for fraction 1 (26%) comprising the 404
392 associated. In addition, some studies investigated plant extracts polar phenolic acids, and also for fraction 3 (24%) containing 405
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393 without explicit identification and quantitation of the active the flavonol-glycosides. Apparently, fractions 1 and 3 exhibited 406
394 compounds, and thus the exact concentrations of bioactives approximately identical inhibiting effect, although the polyphe- 407
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395 required for enzyme inhibition remain unclear [20,24]. nol concentration of fraction 1 (1.0 μg/ml) was about four times 408
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t1:1 Table 1
t1:2 Polyphenol contents of undiluted and diluted grape pomace extract and fractions thereof. Values represent mean ± standard deviation, n = 3.
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t1:3 Concentration (mg/kg Concentration (μg/ml)

grape pomace (DM))

t1:4 No. Retention time Identity Crude extract Diluted crude extract ⁎ Fraction 1 ⁎ Fraction 2 ⁎ Fraction 3 ⁎ Fraction 4 ⁎

t1:5 1 4.2 Gallic acid 596.36 ± 37.49 0.60 ± 0.03 0.47 ± 0.01 – – –
t1:6 2 11.1 Caftaric acid 735.32 ± 40.88 0.74 ± 0.04 0.55 ± 0.01 – – –
t1:7 3 22.6 Procyanidin B1 4858.58 ± 305.58 4.88 ± 0.17 – 4.72 ± 0.22 – –
t1:8 4 24.0 Catechin 10496.63 ± 1059.35 10.53 ± 0.72 – 10.42 ± 0.13 – –
t1:9 5 34.4 Procyanidin B2 4277.04 ± 261.87 4.30 ± 0.17 – 3.40 ± 0.16 – –
t1:10 6 38.7 Epicatechin 8994.93 ± 672.30 9.04 ± 0.39 – 8.84 ± 0.37 – –
t1:11 7 75.2 Quercetin-3-O-glucuronide 2432.29 ± 164.15 2.46 ± 0.10 – – 1.91 ± 0.08 –
t1:12 8 76.0 Quercetin-3-O-glucoside 2374.32 ± 169.64 2.40 ± 0.04 – – 1.96 ± 0.08 –
t1:13 9 83.4 Resveratrol 297.78 ± 37.88 0.30 ± 0.01 – – – 0.50 ± 0.02
t1:14 Total amount 35063.24 ± 2749.15 35.25 ± 1.67 1.02 ± 0.03 27.38 ± 0.88 3.77 ± 0.16 0.50 ± 0.02

t1:15 ⁎ Dilution applied in the enzyme assays (1:20).

Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005
 6 J. Wittenauer et al. / Fitoterapia xxx (2015) xxx–xxx

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Fig. 3. Activities of collagenase (a) and elastase (b) depending on four concentrations of grape pomace extract compared to control (n = 3).

409 lower than of fraction 3 (3.8 μg/ml). In addition, compared to [18,25]. Therefore, attaining the active center of the enzyme is 432

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410 the crude extract, fractions 1 and 3 exhibited relatively high obviously an important premise for inhibition which is probably 433
411 inhibition values at low polyphenol concentrations. Fraction 4, easier for smaller molecules like the phenolic acids of fraction 1 434
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412 containing resveratrol, did not show any inhibitory effects on than for the higher molecular polyphenols of fraction 2. 435
413 ChC activity. According to literature, inhibition of ChC commonly The summation of the relative ChC and PPE inhibitions of 436
414 results from unspecific non-covalent interactions like hydrogen all polyphenol fractions led to a total inhibition of 93% and 437
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415 bonding and hydrophobic interactions with collagenase side 85%, respectively. The lower inhibition value of the crude 438
416 chain groups leading to conformational changes [13,14]. Such an extract, compared to the calculated sum of the fractions, may 439
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417 unspecific inhibition possibly largely depends on the absolute be attributed to interfering non-polyphenolic compounds 440
418 number of hydroxyl groups and benzene rings which is reflected contained, the latter being devoid of the purified fractions. 441
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419 in the molecular weight of the polyphenolic substances but

420 also in absolute polyphenol concentration applied in the 3.5. Collagenase and elastase inhibitory activity of individual 442
421 enzyme assay. Hence, the superior inhibiting effect of fraction polyphenols 443
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422 2 against ChC activity may possibly be attributed to the higher

423 concentration of individual polyphenols as well as the Since the grape pomace extract and the fractions derived 444
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424 contained procyanidins. Relative inhibition of PPE caused by thereof affected collagenase and elastase activity differently, 445
425 fractions 2 and 3 amounted to 17% and 19%, respectively. In polyphenols with different structural characteristics seem to be 446
426 contrast to ChC, highest inhibitory effects were observed for involved in the inhibition of the enzymes. To get further insight 447
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427 fraction 1 (47%) containing the phenolic acids. Notably, fraction into their structure dependent activity, polyphenols identified 448
428 4 showed a weak interaction with PPE, resulting in a relative by HPLC analysis were tested individually in the ChC and PPE 449
assays at concentrations ranging from 250 to 1000 μmol/L.
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429 inhibition of 2%. Polyphenol interaction with PPE was reported 450
430 to depend on rather specific interactions between polyphenolic Additionally, epigallocatechin gallate (EGCG) was tested as a 451
431 hydroxyl groups and amino acid residues of the catalytic triad positive control [11], while epicatechin, the isomeric form of 452
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Fig. 4. Dose-dependent inhibition of collagenase and elastase activity by grape pomace extract (n = 3).

Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005
 J. Wittenauer et al. / Fitoterapia xxx (2015) xxx–xxx 7

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Fig. 5. Relative inhibitory properties of polyphenol fractions from grape pomace on collagenase and elastase activity (n = 3).

453 catechin was not individually applied, due to its largely strongest inhibiting activity of 60%, followed by gallic acid 467
454
455
structural accordance to the latter.
Among the analyzed polyphenols, gallic acid, catechin,
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(51%). Both polyphenols differed markedly regarding their
dose-dependency. In accordance with literature data, EGCG
468
469
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456 procyanidin B1 and B2 exhibited inhibitory activities against showed dose-dependent inhibitory properties [14], whereas 470
457 both enzymes. As previously shown by Hrenn, Steinbrecher, gallic acid already inhibited ChC activity at lower concentra- 471
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458 Labahn, Schwager, Schempp and Merfort [26], resveratrol had tions in a highly efficient manner exerting relative inhibition of 472
459 no effects within the concentration ranges applied what might 42% at 250 μmol/L. As previously reported, higher hydroxyl- 473
460 be a possible explanation for the lacking or low inhibition ation of the molecules enhances their inhibitory effect due to 474
C

461 effects of fraction 4. The hydroxycinnamate caftaric acid did stronger hydrogen bonding interactions with functional side 475
462 not show inhibition effects as well. This substance was not chain groups of the enzyme [14]. Bras, Goncalves, Fernandes, 476
E

463 analyzed before. As depicted in Fig. 6a, the inhibition of ChC by Mateus, Ramos and de Freitas [18] referred that galloyl groups 477
464 individual polyphenols showed a dose-dependent relationship greatly contribute to higher binding activity towards proteins 478
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465 for gallic acid, procyanidin B1 and B2 as well as the EGCG due to the three neighboring hydroxyl groups and the benzene 479
466 control. At concentrations of 1 mmol/L, EGCG exhibited the ring facilitating both hydrogen and hydrophobic binding to 480
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Fig. 6. Dose-dependent inhibition of Clostridium histolyticum collagenase (a) and porcine pancreatic elastase (b) activity by individual grape pomace polyphenols (n = 3).

Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005
 8 J. Wittenauer et al. / Fitoterapia xxx (2015) xxx–xxx

481 proteins. Additionally to these non-specific interactions, the References 536

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Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005
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Please cite this article as: Wittenauer J, et al, Inhibitory effects of polyphenols from grape pomace extract on collagenase and
elastase activity, Fitoterapia (2015), http://dx.doi.org/10.1016/j.fitote.2015.01.005