FITOTE-03103; No of Pages 9 Fitoterapia xxx (2015) xxx–xxx
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Fitoterapia
3Q2 4 5 6 7
Judith Wittenauer a,b,⁎, Sonja Mäckle a,b, Daniela Sußmann a, Ute Schweiggert-Weisz a, Reinhold Carle b,c a b c
Fraunhofer Institute for Process Engineering and Packaging, Department of Process Development for Plant Raw Materials, Giggenhauser Str. 35, D-85354 Freising, Germany Hohenheim University, Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology, Garbenstraße 25, D-70599 Stuttgart, Germany King Abdulaziz University, Biological Science Department, Faculty of Science, P.O. Box 80257, Jeddah 21589, Saudi Arabia
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Article history: Received 21 October 2014 Accepted in revised form 6 January 2015 Accepted 8 January 2015 Available online xxxx
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Chemical compounds studied in this article: Gallic acid (PubChem CID: 370) (+)-Catechin (PubChem CID: 9064) Caftaric acid (PubChem CID: 6440397) Quercetin 3-O-glucoside (PubChem CID: 5280804) Quercetin 3-O-glucuronid (PubChem CID: 12004528) Procyanidin B1 (PubChem CID: 11250133) Procyanidin B2 (PubChem CID: 122738) Trans-resveratrol (PubChem CID: 445154)
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Keywords: Grape pomace Vitis vinifera Polyphenols Collagenase Elastase Skin aging
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1. Introduction
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Skin aging is a complex biological phenomenon influenced by several factors including genetics, environmental exposure, hormonal changes, and metabolic processes. As the skin is the
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 enzymes such as collagenases and elastases. Inhibition of their enzymatic activities by natural plant compounds might be a promising approach to prevent extrinsic skin aging. Especially polyphenols are supposed to interact with those enzymes due to their molecular nature. In our investigation, extracts of pomace from Riesling grapes were analyzed for their inhibitory properties on collagenase as well as elastase. Crude grape pomace extract showed a dosedependent inhibitory activity against both enzymes with IC50-values of 20.3 μg/ml and 14.7 μg/ml for collagenase and elastase activity, respectively. The extracts were fractionated into four fractions containing phenolic compounds differing in chemical structure and polarity. Except for the stilbene containing fraction, all other fractions showed inhibitory effects on both enzyme activities. The most pronounced impact was found for the hydrophilic low molecular weight polyphenols containing the free phenolic acids. In particular, gallic acid showed considerable inhibition values. EGCG was used as a positive control and showed a dose-dependent inhibition of collagenase activity (IC50 = 0.9 mM). © 2015 Published by Elsevier B.V.
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only organ directly exposed to the environment, aging processes resulting from environmental damage are of considerable relevance. Particularly solar UV radiation adversely impacts skin health, due to, amongst others, generation of reactive oxygen species (ROS) [1–3]. ROS are able to initiate complex molecular pathways including the activation of enzymes that degrade extracellular matrix (ECM) proteins in the dermis such as collagen and elastin ensuring the skin's threedimensional integrity [1,2]. As a consequence of protease activation, breakdown, fragmentation and disorganisation of
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Inhibitory effects of polyphenols from grape pomace extract on collagenase and elastase activity
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journal homepage: www.elsevier.com/locate/fitote
⁎ Corresponding author at: Fraunhofer Institute for Process Engineering and Packaging, Department of Process Development for Plant Raw Materials, Giggenhauser Str. 35, D-85354 Freising, Germany. Tel.: +49 8161 491 440. E-mail address: [email protected] (J. Wittenauer).
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
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2.1. Materials
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Grape pomace from white wine (Vitis vinifera L. cv. ‘Weisser Riesling’) of vintage 2010 was kindly provided by the Baden-Badener Winzergenossenschaft (Baden-BadenNeuweier, Germany). Pomace samples (voucher specimen #600385.1 as deposited at the Fraunhofer IVV, Freising, Germany) were collected after pressing the mash, freeze dried, sealed in polyethylene bags, and kept at 5 °C until further use. C18 reversed-phase cartridges (Chromabond, 2000 mg) were obtained from Macherey-Nagel (Düren, Germany). C. histolyticum collagenase type IA (ChC), N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) and porcine pancreas elastase type III (PPE) were purchased from Sigma (St. Louis, MO). N-Succ-Ala-Ala-Ala-p-nitroanilide (AAAPVN) was obtained from Serva Electrophoresis (Heidelberg, Germany). Polyphenols used as standards for the inhibition studies were gallic acid (≥97%), catechin (≥99%), epigallocatechin gallate (≥95%) (Sigma, St. Louis, MO); caftaric acid (≥97%), quercetin 3-O-glucoside (≥98%), quercetin 3-Oglucuronid (≥95%), procyanidin B1 (≥90%), procyanidin B2 (≥90%) (Fluka, Buchs, Switzerland), and trans-resveratrol (98%) (ABCR, Karlsruhe, Germany). All reagents and chemicals used were of analytical grade and purchased from VWR (Darmstadt, Germany).
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2.2. Preparation of polyphenol rich extracts
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2.2.1. Crude extract Extraction of polyphenols from grape pomace was carried out according to Kammerer, Claus, Carle and Schieber [8]. Briefly, lyophilised grape pomace was ground using a ZM 100 centrifugal mill with a 1 mm ring sieve (Retsch, Haan, Germany). Aliquots of 2 g of the powdered samples were weighted into Erlenmeyer flasks, flushed with nitrogen and extracted with 50 ml of methanol for 60 min under shaking, using a KS 500 laboratory shaker (Janke & Kunkel, IKA Labortechnik, Staufen, Germany). Extracts were filtered through a paper filter (Munktell, grade 3hw); the residue was reextracted with 50 ml of the solvent for 30 min to ensure an exhaustive extraction. Supernatants were combined, and the organic solvent was removed by evaporation in vacuo at 40 °C. Residues obtained were dissolved in 10 ml water and filtered through a 0.2 μm syringe filter (Sartorius, Göttingen, Germany), and the pH was adjusted to 7.0 with sodium hydroxide solution (1 M).
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promoting properties, such investigations would be of basic interest. In the present study, the in vitro inhibitory potential of phenolic compounds obtained from white grape pomace against the activity of Clostridium histolyticum collagenase (ChC) and porcine pancreatic elastase (PPE) was investigated. The polyphenolic profile of the grape pomace extract was determined, moreover, different fractions of the extract as well as individual polyphenols were assayed for their inhibitory activity against ChC and PPE, respectively.
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the ECM proteins occur, visibly manifested by the typical UV induced skin alterations such as deep wrinkles, loss of skin tone and resilience [3]. Matrix metalloproteinases (MMPs), a family of multidomain zinc-containing endopeptidases with a broad range of substrate specificities, constitute the major proteolytic enzyme group involved in the degradation of dermal connective tissue components. Under normal physiological condition, MMP activities are precisely regulated at the level of transcription and by endogenous protein inhibitors. In addition, MMPs are secreted from cells as an inactive precursor requiring cleavage by extracellular proteinases to be activated. These regulation processes enable the remodeling of connective tissue under physiological and pathophysiological circumstances like wound healing, inflammation and cancer. The impairment of this balance, due to oxidative stress, results in an excess of MMPs being a key feature of premature aging of the skin as well as various inflammatory and degenerative diseases. In addition, serine proteases such as elastases are involved in ECM degradation. Elastolytic enzymes are released from neutrophils and dermal fibroblasts in response to UV induced skin inflammation [4]. Due to their active side catalytic triad, elastases are multi-specific and cleave several proteins within the extracellular matrix including collagen, fibronectin and elastin. Elastin is an important ECM protein having the unique property of elastic recoil, which is vital for providing elasticity to skin and other tissues like arteries, lungs and ligaments. Several studies have shown that human neutrophil elastase is also involved in the degradation of ECM connective tissue through the activation of inactive MMP-1 and MMP-2 precursors [5–7]. Inhibiting the activity of ECM degrading proteins like collagenases and elastases may be a useful approach to prevent UV induced skin alterations and premature skin aging. Scavenging of ROS by natural antioxidants might be one option to inhibit such skin deteriorative enzymes, as ROS play an important role in the activation of these enzymes. Phenolic compounds are an important group of natural antioxidants. They belong to diverse subclasses of secondary plant metabolites classified as phenolic acids, flavonoids, stilbenes and lignans and are ubiquitously found in the plant kingdom. In particular, red and white grapes contain high amounts of phenolic acids and flavonoids such as gallic acid and catechin [8]. Due to their chemical structure, polyphenols have powerful antioxidant activities being able to scavenge a wide range of ROS such as hydroxyl radicals, superoxide radical and O− 2 [9]. In addition, polyphenols may inhibit the activity of proteolytic enzymes in vitro by acting as complexing or precipitating agents as indicated in literature [10,11]. Especially, green tea polyphenols such as catechin and epigallocatechin gallate, commonly used as ingredients in antiaging skin care formulations, have been shown to exhibit moderate inhibitory effects against collagenase and elastase activity, presumably through non-covalent binding [12–14]. However, the mechanism of action of polyphenols is not fully understood, and studies about the inhibitory effects of natural active ingredients on skin degrading enzymes are scarce. Most of the studies have been performed with isolated compounds, thus not considering the complex nature of crude extracts. Additionally, data about inhibitiory properties of further polyphenolic subclasses are scarce. However, in particular, for the systematic development of natural formulations exerting skin health
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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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2.3. HPLC analysis
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Separation of polyphenols was performed using an Agilent HPLC series 1200 system (Agilent, Waldbronn, Germany) with ChemStation software, a model G1379 degasser, a model G1312B binary gradient pump, a model G1367D thermoautosampler, a model G1316B column oven and a model G1315C diode array detector. The column used was a C 18 Synergi-Hydro column (150 × 3.0 mm i.d.; 4 μm particle size) from Phenomenex (Torrance, CA) operated at 25 °C. The mobile phase consisted of 2% (v/v) acetic acid in water (eluent A) and 0.5% acetic acid in water and acetonitrile (50:50, v/v, eluent B) using the following gradient program: 0–5% B (35 min), 5–20% B (45 min), 20–100% B (30 min), 100% B isocratic (3 min),100–0% B (10 min) at a flow rate of 0.8 ml/min. Total run time was 123 min. The injection volume varied between 10 and 30 μl. Polyphenols were monitored at 280 nm, and UV/Vis spectra were recorded in the range of 200 to 600 nm. Polyphenols were identified by UV–vis spectra and comparison with literature data and reference compounds. The quantitation of individual polyphenols was performed using calibration curves of the corresponding reference compounds. Gallic acid, caftaric acid, (+)-catechin, epigallocatechin gallate, procyanidin B1, procyanidin B2, quercetin 3-O-glucoside, quercetin 3-O-glucuronid, kaempferol 3-O-glucosid, and transresveratrol served as reference compounds. The quantitation of epicatechin was performed applying the catechin calibration curve. All determinations were carried out in quadruplicate.
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Porcine pancreatic elastase (PPE) inhibition of the individual samples a–c (see Section2.4 2.4) was determined spectrophotometrically using AAAPVN as the substrate by monitoring the release of p-nitroaniline at 410 nm. PPE was dissolved in 2 mM Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) buffer (pH = 8,0). Thereof, an aliquot of 10 μl was taken and loaded in the wells of the microtiter plates together with 100 μl of the Tris buffer and 30 μl of the individual sample solutions. After 20 min of pre-incubation at 25 °C, 40 μl of the substrate AAAPVN (dissolved in 2 mM Tris buffer at a concentration of 0.25 mg/ml) was added. Absorbance was measured for 20 min after the addition of AAAPVN. Initial velocities, the inhibition activity and IC50 values were calculated in accordance with the ChC assay
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2.4. Collagenase assay
3. Results and discussion
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Inhibition effects of the grape pomace crude extract, the fractions derived thereof as well as the standards mentioned above against C. histolyticum collagenase (ChC) were measured spectrophotometrically according to a modified procedure described by van Wart and Steinbrink [15] using a BioTek Synergi H1 multi-mode microplate reader (BioTek, Bad Friedrichshall, Germany) and appropriate data analysis software (Gen 5, version 2.04, BioTek, Bad Friedrichshall, Germany). ChC (1.1 U/ml) and FALGPA (1 mM), respectively, were dissolved in 0.05 M tricine [N-(2-hydroxy-1,1bis(hydroxymethyl)ethyl)glycine] buffer containing 0.4 M NaCl and 0.01 M CaCl2; the pH was adjusted to 7.5 with 1 M NaOH. The inhibitory effects of the following samples were investigated:
3.1. Identification of polyphenolic compounds in the crude grape 273 pomace extract and the fractions derived thereof 274
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2.5. Elastase assay
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ChC inhibition activity ð%Þ initial velocitycontrol ‐ initial velocitysample 100 ¼ initial velocitycontrol
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First, 30 μl of the samples (a–c) were incubated with 10 μl of ChC solution and 60 μl tricine buffer for 20 min at 37 °C. Subsequently, 20 μl of the FALGPA solution was added to initiate the reaction. For negative controls water was added instead of FALGPA solution. The ChC inhibitory activities of the individual samples were measured by continuously monitoring the decrease in absorbance of FALGPA at 335 nm for 20 min after starting the reaction. Initial velocities were calculated from the slope of the absorbance change during the first 10 min of hydrolysis. The relative inhibition was calculated according to Eq. (1). IC50 values were determined from dose–effect curves.
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a) dilutions of crude grape pomace extract with water at concentrations of 35.3 ± 1.7 μg/ml (1:20), 23.5 ± 1. μg/ml (1:30), 14.1 ± 0.3 μg/ml (1:50), 8.8 ± 0.4 (1:80) and 7.1 ± 0.3 μg/ml (1:100) (extract: water); b) the individual fractions 1–4; and c) aqueous solutions of the individual reference polyphenols at concentrations of 250 μM, 500 μM, 750 μM and 1000 μM, respectively.
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2.2.2. Polyphenol fractions The fractionation of polyphenolic compounds of the grape pomace extract was achieved by solid phase extraction (SPE). Cartridges were first activated with 12 ml of methanol, washed with 20 ml of water and then loaded with 2 ml of the crude extract. Elution of the fractions followed by subsequent application of various solvent mixtures. Fraction 1 was eluted with 20 ml of acetic acid (2%). Fraction 2 was obtained next by application of 20 ml of a 3:1 mixture of acetic acid (2%) and acetonitrile/acetic acid (2%) (v/v). Elution with 20 ml of acetic acid (2%) and acetonitrile/acetic acid (2%) (v/v) at a ratio of 3:2 resulted in fraction 3. The last fraction (fraction 4) was obtained by using 20 ml of acetonitrile/acetic acid (2%) (v/v).
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3.1.1. Crude extract Prior to the enzymatic inhibition studies, the identification and quantitation of the polyphenolic pattern by HPLC was a prerequisite to identify the compounds being primarily responsible for the enzyme inhibitory effects. Major polyphenols of the grape pomace were the flavanols catechin (4) and epicatechin (6) accompanied by the procyanidins B1 (3) and B2 (5), while the phenolic acids gallic acid (1) and caftaric acid (2), the flavonol glycosides quercetin-3-O-glucuronide (7) and quercetin-3-O-glucoside (8) as well as the stilbene transresveratrol (9) were found to be minor compounds (Fig. 1).
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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3.2. Quantitation of polyphenolic compounds in the crude extract and the four fractions
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Quantitated amounts of major polyphenolic constituents in grape pomace extract and fractions thereof are summarized in Table 1. In accordance with literature data [8,16], the flavanols catechin and epicatechin were the predominant components in the crude extract with concentrations of 210.7 and 180.7 μg/ml, respectively, followed by the procyanidins B1 (97.5 μg/ml) and B2 (85.9 μg/ml). The flavonol-glycosides, the phenolic acids as well as resveratrol were present in minor amounts. Due to the high absorption values at the detected wavelength, the grape pomace extract and the fractions had to be diluted 20-fold for the subsequent enzyme assays. Amounts of individual polyphenols detected in fractions 1–3 were slightly lower than in the crude extract, which probably resulted from losses owing to the fractionation process with the exception of resveratrol in fraction 4. This could be due to further overlapping constituents contained in the crude extract (Fig. 1) which also absorb light at the detected wavelength, thus interfering the chromatographic analysis. Therefore, integration of resveratrol in the chromatograms of the crude extract resulted in apparently lower amounts than in the purified fraction.
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To investigate the inhibition exerted by the grape pomace extract on ChC and PPE, the respective enzymes were incubated with the extract and the appropriate substrates. The assays were performed with synthetic peptides as substrate molecules, and enzyme preparations isolated from C. histolyticum and porcine pancreas, respectively. As reported in literature, PPE is topologically very similar to human neutrophil elastase and inhibition mechanisms for some phenolic substances were found to be equal [17,18]. Therefore, it can be assumed that the in vitro assays performed with the ChC and PPE and synthetic peptides are a reliable tool to get first indications of the capability of grape pomace polyphenols to prevent extracellular matrix degradation as a skin aging phenomenon. The inhibitory potential of the grape pomace extract was examined for increasing dilutions, in order to establish dose-dependent relationships, and to calculate the half maximal inhibitory concentrations (IC50). Crude grape pomace extract was incubated with collagenase and elastase at dilution-ratios ranging from 1:20 to 1:100, resulting in total polyphenolic contents ranging from 35.3 to 7.1 μg/ml. The effect of different extract concentrations on the enzyme activity is shown in Fig. 3a and b for ChC and PPE, respectively. As expected the slope of the digestion curves of the collagenase assay decreases with increasing extract concentration, which is due to the lowered enzyme activity, thus resulting in higher amounts of non-cleaved substrate molecules. The higher initial absorption with increasing extract concentration probably results from self-absorption of grape pomace extract components. Since the elastase assay was performed with N-Succ-Ala-Ala-Ala-p-nitroanilide as the substrate peptide, the enzyme activity can be deduced from the release of p-nitroaniline resulting in increased absorption values. At increased extract concentrations slopes in digestion curves were less steep representing an increased enzyme inhibition. The grape pomace extract inhibited both enzymes in a dosedependent manner. Highest polyphenol concentrations of 35.3 μg/ml resulted in an 80% and 73% relative inhibition of ChC and PPE activity, respectively (Fig. 4). Inhibitory capacities declined with decreasing concentrations of the extract. Relative inhibition of ChC was 55% at a polyphenol concentration of 23.5 μg/ml, and the inhibition capacity further decreased at lower polyphenol concentrations in a nearly linear manner to 40% and 28% for 14.1 μg/ml and 8.8 μg/ml, respectively. Further dilution of the extract to 7.1 μg/ml resulted in a negligible decrease of the inhibition value. The relative inhibition of PPE at a concentration of 23.5 μg/ml was found to be 63%. At lower grape pomace extract concentrations, inhibitory activities markedly declined with relative inhibition values of 49%, 36% and 20%, for 14.1 μg/ml, 8.8 μg/ml and 7.1 μg/ml, respectively. From the dose-response relationships, IC50 values of 20.3 μg/ml for ChC and 14.7 μg/ml for PPE activity were calculated. Comparison of our present findings with those reported for other plant extracts is rather difficult, due to varying assay conditions, particularly incubation times and types of substrates and enzymes used for the determination of the enzymatic activities. Additionally, the extracts are often obtained using different extraction methods. The polarity of the
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3.3. Collagenase and elastase inhibitory activity of white grape 322 pomace extracts 323
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3.1.2. Polyphenol fractions To rank the compounds from specific polyphenolic classes according to their relative enzyme inhibitory effects, the crude extract was separated into four fractions, being composed of polyphenols having similar chemical structure and polarity, and the fractions were analyzed by HPLC. As shown in Fig. 2, fraction 1 contained the free phenolic acids gallic acid and caftaric acid, while the main components of the crude extract, catechin and epicatechin as well as the procyanidins B1 and B2, were found in fraction 2. The flavone glycosides quercetin-3-Oglucuronide and quercetin-3-O-glucoside could be detected in fraction 3, whereas the stilbene resveratrol was the only polyphenolic constituent detectable in fraction 4.
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Fig. 1. HPLC separation of polyphenols from crude grape pomace extract (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, (8) quercetin-3-O-glucoside, (9) resveratrol.
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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Fig. 2. HPLC separation of fractions obtained from grape pomace extract by means of solid phase extraction (280 nm) and related structures.
3.4. Collagenase and elastase inhibitory activity of grape pomace 396 extract fractions 397
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Table 1 Polyphenol contents of undiluted and diluted grape pomace extract and fractions thereof. Values represent mean ± standard deviation, n = 3.
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In order to determine the most potent polyphenols in the grape pomace extract inhibiting ChC and PPE activities, fractions containing individual polyphenolic compounds exerting different polarities were analyzed. As shown in Fig. 5, fraction 2, enriched in catechins and procyanidins at a concentration of 27.4 μg/ml was the most potent in ChC inhibition (43%). Effects on ChC activity were lower for fraction 1 (26%) comprising the polar phenolic acids, and also for fraction 3 (24%) containing the flavonol-glycosides. Apparently, fractions 1 and 3 exhibited approximately identical inhibiting effect, although the polyphenol concentration of fraction 1 (1.0 μg/ml) was about four times
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solvent has a strong influence on the inhibitory effects of a plant extract [19], indicating that individual constituents may differ regarding their inhibitory effects. Using more lipophilic extraction media could result in extracts with high inhibition of collagenase and elastase activities [19–21]. Methanol is an efficient extraction media for a broad spectrum of polyphenols [8,22,23]. Since methanol is an intolerable ingredient for cosmetic formulations, the extracts used in our study were dissolved in water. Therefore, lower inhibition values than described in literature due to lowered extraction yields could be associated. In addition, some studies investigated plant extracts without explicit identification and quantitation of the active compounds, and thus the exact concentrations of bioactives required for enzyme inhibition remain unclear [20,24].
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Concentration (μg/ml)
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Crude extract
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Gallic acid Caftaric acid Procyanidin B1 Catechin Procyanidin B2 Epicatechin Quercetin-3-O-glucuronide Quercetin-3-O-glucoside Resveratrol Total amount
596.36 735.32 4858.58 10496.63 4277.04 8994.93 2432.29 2374.32 297.78 35063.24
0.60 0.74 4.88 10.53 4.30 9.04 2.46 2.40 0.30 35.25
0.47 ± 0.01 0.55 ± 0.01 – – – – – – – 1.02 ± 0.03
– – 4.72 ± 0.22 10.42 ± 0.13 3.40 ± 0.16 8.84 ± 0.37 – – – 27.38 ± 0.88
– – – – – – 1.91 ± 0.08 1.96 ± 0.08 – 3.77 ± 0.16
– – – – – – – – 0.50 ± 0.02 0.50 ± 0.02
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⁎ Dilution applied in the enzyme assays (1:20).
± ± ± ± ± ± ± ± ± ±
37.49 40.88 305.58 1059.35 261.87 672.30 164.15 169.64 37.88 2749.15
± ± ± ± ± ± ± ± ± ±
0.03 0.04 0.17 0.72 0.17 0.39 0.10 0.04 0.01 1.67
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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Fig. 3. Activities of collagenase (a) and elastase (b) depending on four concentrations of grape pomace extract compared to control (n = 3).
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[18,25]. Therefore, attaining the active center of the enzyme is obviously an important premise for inhibition which is probably easier for smaller molecules like the phenolic acids of fraction 1 than for the higher molecular polyphenols of fraction 2. The summation of the relative ChC and PPE inhibitions of all polyphenol fractions led to a total inhibition of 93% and 85%, respectively. The lower inhibition value of the crude extract, compared to the calculated sum of the fractions, may be attributed to interfering non-polyphenolic compounds contained, the latter being devoid of the purified fractions.
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lower than of fraction 3 (3.8 μg/ml). In addition, compared to the crude extract, fractions 1 and 3 exhibited relatively high inhibition values at low polyphenol concentrations. Fraction 4, containing resveratrol, did not show any inhibitory effects on ChC activity. According to literature, inhibition of ChC commonly results from unspecific non-covalent interactions like hydrogen bonding and hydrophobic interactions with collagenase side chain groups leading to conformational changes [13,14]. Such an unspecific inhibition possibly largely depends on the absolute number of hydroxyl groups and benzene rings which is reflected in the molecular weight of the polyphenolic substances but also in absolute polyphenol concentration applied in the enzyme assay. Hence, the superior inhibiting effect of fraction 2 against ChC activity may possibly be attributed to the higher concentration of individual polyphenols as well as the contained procyanidins. Relative inhibition of PPE caused by fractions 2 and 3 amounted to 17% and 19%, respectively. In contrast to ChC, highest inhibitory effects were observed for fraction 1 (47%) containing the phenolic acids. Notably, fraction 4 showed a weak interaction with PPE, resulting in a relative inhibition of 2%. Polyphenol interaction with PPE was reported to depend on rather specific interactions between polyphenolic hydroxyl groups and amino acid residues of the catalytic triad
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3.5. Collagenase and elastase inhibitory activity of individual 442 polyphenols 443 Since the grape pomace extract and the fractions derived thereof affected collagenase and elastase activity differently, polyphenols with different structural characteristics seem to be involved in the inhibition of the enzymes. To get further insight into their structure dependent activity, polyphenols identified by HPLC analysis were tested individually in the ChC and PPE assays at concentrations ranging from 250 to 1000 μmol/L. Additionally, epigallocatechin gallate (EGCG) was tested as a positive control [11], while epicatechin, the isomeric form of
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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
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Fig. 5. Relative inhibitory properties of polyphenol fractions from grape pomace on collagenase and elastase activity (n = 3).
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strongest inhibiting activity of 60%, followed by gallic acid (51%). Both polyphenols differed markedly regarding their dose-dependency. In accordance with literature data, EGCG showed dose-dependent inhibitory properties [14], whereas gallic acid already inhibited ChC activity at lower concentrations in a highly efficient manner exerting relative inhibition of 42% at 250 μmol/L. As previously reported, higher hydroxylation of the molecules enhances their inhibitory effect due to stronger hydrogen bonding interactions with functional side chain groups of the enzyme [14]. Bras, Goncalves, Fernandes, Mateus, Ramos and de Freitas [18] referred that galloyl groups greatly contribute to higher binding activity towards proteins due to the three neighboring hydroxyl groups and the benzene ring facilitating both hydrogen and hydrophobic binding to
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catechin was not individually applied, due to its largely structural accordance to the latter. Among the analyzed polyphenols, gallic acid, catechin, procyanidin B1 and B2 exhibited inhibitory activities against both enzymes. As previously shown by Hrenn, Steinbrecher, Labahn, Schwager, Schempp and Merfort [26], resveratrol had no effects within the concentration ranges applied what might be a possible explanation for the lacking or low inhibition effects of fraction 4. The hydroxycinnamate caftaric acid did not show inhibition effects as well. This substance was not analyzed before. As depicted in Fig. 6a, the inhibition of ChC by individual polyphenols showed a dose-dependent relationship for gallic acid, procyanidin B1 and B2 as well as the EGCG control. At concentrations of 1 mmol/L, EGCG exhibited the
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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
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This study was financially supported by the Fraunhofer Gesellschaft zur Förderung der angewandten Forschung (FhG). The authors are grateful to Baden-Badener Winzergenossenschaft, Baden-Baden-Neuweier, Germany, for providing the grape pomace.
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In conclusion, crude grape pomace extract revealed its potential for cosmetic applications as strong dose-dependent inhibitory effects of both ChC and PPE were observed. Due to the high inhibition values at low total polyphenol concentrations, fraction 1, containing the polar free phenolic acids exhibited the most potent inhibitory activity. In particular, gallic acid carrying three neighboring hydroxyl and a carboxyl functional group seems to be decisively involved in the inhibitory effects of fraction 1. Since caftaric acid did not show any inhibitory effect, synergistic interactions between polyphenols and the enzyme could also play an important role regarding the inhibition mechanism. In accordance with earlier findings, also the catechin- and procyanidin-rich fraction 2 had inhibitory properties, in particular, against ChC. Due to the high polyphenol concentrations required for inhibition and the higher molecular weight and polymerisation degree of the molecules, their application for cosmetic purposes could be limited, since the size of polyphenols restricts their permeation into the epidermal and corium layers and the concentrations that are achieved in these layers are limited [27,28].
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[1] Brenneisen P, Wenk J, Klotz LO, Wlaschek M, Briviba K, Krieg T, et al. Central role of ferrous/ferric iron in the ultraviolet B irradiation-mediated signaling pathway leading to increased interstitial collagenase (matrixdegrading metalloprotease (MMP)-1) and stromelysin-1 (MMP-3) mRNA levels in cultured human dermal fibroblasts. J Biol Chem 1998;273: 5279–87. [2] Rittie L, Fisher GJ. UV-light-induced signal cascades and skin aging. Ageing Res Rev 2002;1:705–20. [3] Scharffetter-Kochanek K, Brenneisen P, Wenk J, Herrmann G, Ma WJ, Kuhr L, et al. Photoaging of the skin from phenotype to mechanisms. Exp Gerontol 2000;35:307–16. [4] Rijken F, Kiekens RCM, van den Worm E, Lee PL, van Weelden H, Bruijnzeel PLB. Pathophysiology of photoaging of human skin: focus on neutrophils. Photochem Photobiol Sci 2006;5:184–9. [5] Takeuchi H, Gomi T, Shishido M, Watanabe H, Suenobu N. Neutrophil elastase contributes to extracellular matrix damage induced by chronic low-dose UV irradiation in a hairless mouse photoaging model. J Dermatol Sci 2010;60:151–8. [6] Saunders WB, Bayless KJ, Davis GE. MMP-1 activation by serine proteases and MMP-10 induces human capillary tubular network collapse and regression in 3D collagen matrices. J Cell Sci 2005;118:2325–40. [7] Shamamian P, Schwartz JD, Pocock BJZ, Monea S, Whiting D, Marcus SG, et al. Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis. J Cell Physiol 2001;189:197–206. [8] Kammerer D, Claus A, Carle R, Schieber A. Polyphenol screening of pomace from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS. J Agric Food Chem 2004;52:4360–7. [9] Quideau S, Deffieux D, Douat-Casassus C, Pouysegu L. Plant polyphenols: chemical properties, biological activities, and synthesis. Angew Chem Int Ed 2011;50:586–621. [10] Bras NF, Goncalves R, Mateus N, Fernandes PA, Ramos MJ, Do Freitas V. Inhibition of pancreatic elastase by polyphenolic compounds. J Agric Food Chem 2010;58:10668–76. [11] Siedle B, Hrenn A, Merfort I. Natural compounds as inhibitors of human neutrophil elastase. Planta Med 2007;73:401–20. [12] Sartor L, Pezzato E, Dell'Aica I, Caniato R, Biggin S, Garbisa S. Inhibition of matrix-proteases by polyphenols: chemical insights for anti-inflammatory and anti-invasion drug design. Biochem Pharmacol 2002;64:229–37. [13] Kim YJ, Uyama H, Kobayashi S. Inhibition effects of (+)-catechin-aldehyde polycondensates on proteinases causing proteolytic degradation of extracellular matrix. Biochem Biophys Res Commun 2004;320:256–61. [14] Madhan B, Krishnamoorthy G, Rao JR, Nair BU. Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase. Int J Biol Macromol 2007;41:16–22. [15] van Wart HE, Steinbrink DR. A continuous spectrophotometric assay for Clostridium histolyticum collagenase. Anal Biochem 1981;113:356–65. [16] Maier T, Schieber A, Kammerer DR, Carle R. Residues of grape (Vitis vinifera L.) seed oil production as a valuable source of phenolic antioxidants. Food Chem 2009;112:551–9. [17] Lee KK, Kim JH, Cho JJ, Choi JD. Inhibitory effects of 150 plant extracts on elastase activity, and their anti-inflammatory effects. Int J Cosmet Sci 1999;21:71–82. [18] Bras NF, Goncalves R, Fernandes PA, Mateus N, Ramos MJ, de Freitas V. Understanding the binding of procyanidins to pancreatic elastase by experimental and computational methods. Biochemistry-Us 2010;49: 5097–108. [19] Alasbahi R, Melzig MF. The in vitro inhibition of human neutrophil elastase activity by some Yemeni medicinal plants. Planta Med 2008;74: 981-981. [20] Lee KK, Kim JH, Cho JJ, Choi JD. Inhibitory effects of 150 plant extracts on elastase activity, and their anti-inflammatory effects. Int J Cosmet Sci 1999;21:71–82. [21] Chiang H-M, Lin T-J, Chiu C-Y, Chang C-W, Hsu K-C, Fan P-C, et al. Coffea arabica extract and its constituents prevent photoaging by suppressing MMPs expression and MAP kinase pathway. Food Chem Toxicol 2011;49: 309–18. [22] Pinelo M, Rubilar M, Jerez M, Sineiro J, Nunez MJ. Effect of solvent, temperature, and solvent-to-solid ratio on the total phenolic content and antiradical activity of extracts from different components of grape pomace. J Agric Food Chem 2005;53:2111–7. [23] Lapornik B, Prosek M, Wondra AG. Comparison of extracts prepared from plant by-products using different solvents and extraction time. J Food Eng 2005;71:214–22. [24] Sumantran VN, Kulkarni AA, Harsulkar A, Wele A, Koppikar SJ, Chandwaskar R, et al. Hyaluronidase and collagenase inhibitory activities of the herbal formulation Triphala guggulu. J Biosci 2007;32:755–61.
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proteins. Additionally to these non-specific interactions, the free carboxyl moiety in gallic acid could bind to the zinc site in the catalytic domain of ChC as possible inhibition mechanism as discussed by Kim, Uyama and Kobayashi [13]. However, to the best of our knowledge, the inhibitory effects of gallic acid have so far not been studied. Inhibition effects on ChC were only found at high concentrations of 1000 μmol/L for procyanidin B2 and procyanidin B1, respectively. Inhibitory activities of catechin were already observed at 250 μmol/L and, as an exception, inhibition did not increase with increasing concentration. The dose-dependent inhibitory effects of most of the polyphenols under investigation support our suggestion that ChC inhibition could be achieved by higher concentrations of individual polyphenols as earlier discussed. Inhibitions of PPE activities were significantly lower for the individual polyphenols (Fig. 6b), and dose-dependency was found to be poor. Inhibitory effects merely increased for polyphenol concentrations exceeding 750 μmol/L. At concentrations of 1 mmol/L, strongest inhibitory activities were observed for catechin (12.0%), EGCG (7.3%), and procyanidin B2 (6.4%). In agreement with Bras, Goncalves, Fernandes, Mateus, Ramos and de Freitas [18], the low inhibitory effects of individual polyphenols on PPE support that an unspecific complexation reaction may be excluded as the underlying inhibition mechanisms. Additionally, the low inhibition values of the individual compounds are in contrast to the inhibiting potency of the extract and its fractions, respectively, which may result from synergistic effects.
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[25] Siedle B, Murillo R, Hucke O, Labahn A, Merfort I. Structure activity relationship studies of cinnamic acid derivatives as inhibitors of human neutrophil elastase revealed by ligand docking calculations. Pharmazie 2003;58:337–9. [26] Hrenn A, Steinbrecher T, Labahn A, Schwager J, Schempp CM, Merfort I. Plant phenolics inhibit neutrophil elastase. Planta Med 2006;72: 1127–31.
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[27] Zillich OV, Schweiggert-Weisz U, Hasenkopf K, Eisner P, Kerscher M. Release and in vitro skin permeation of polyphenols from cosmetic emulsions. Int J Cosmet Sci 2013;35:491–501. [28] dal Belo SE, Gaspar LR, Campos PMBGM, Marty JP. Skin penetration of epigallocatechin-3-gallate and quercetin from green tea and Ginkgo biloba extracts vehiculated in cosmetic formulations. Skin Pharmacol Physiol 2009;22:299–304.
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Contents lists available at ScienceDirect
Fitoterapia
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Judith Wittenauer a,b,⁎, Sonja Mäckle a,b, Daniela Sußmann a, Ute Schweiggert-Weisz a, Reinhold Carle b,c a b c
Fraunhofer Institute for Process Engineering and Packaging, Department of Process Development for Plant Raw Materials, Giggenhauser Str. 35, D-85354 Freising, Germany Hohenheim University, Institute of Food Science and Biotechnology, Chair Plant Foodstuff Technology, Garbenstraße 25, D-70599 Stuttgart, Germany King Abdulaziz University, Biological Science Department, Faculty of Science, P.O. Box 80257, Jeddah 21589, Saudi Arabia
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Article history: Received 21 October 2014 Accepted in revised form 6 January 2015 Accepted 8 January 2015 Available online xxxx
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Chemical compounds studied in this article: Gallic acid (PubChem CID: 370) (+)-Catechin (PubChem CID: 9064) Caftaric acid (PubChem CID: 6440397) Quercetin 3-O-glucoside (PubChem CID: 5280804) Quercetin 3-O-glucuronid (PubChem CID: 12004528) Procyanidin B1 (PubChem CID: 11250133) Procyanidin B2 (PubChem CID: 122738) Trans-resveratrol (PubChem CID: 445154)
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Keywords: Grape pomace Vitis vinifera Polyphenols Collagenase Elastase Skin aging
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1. Introduction
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Skin aging is a complex biological phenomenon influenced by several factors including genetics, environmental exposure, hormonal changes, and metabolic processes. As the skin is the
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 enzymes such as collagenases and elastases. Inhibition of their enzymatic activities by natural plant compounds might be a promising approach to prevent extrinsic skin aging. Especially polyphenols are supposed to interact with those enzymes due to their molecular nature. In our investigation, extracts of pomace from Riesling grapes were analyzed for their inhibitory properties on collagenase as well as elastase. Crude grape pomace extract showed a dosedependent inhibitory activity against both enzymes with IC50-values of 20.3 μg/ml and 14.7 μg/ml for collagenase and elastase activity, respectively. The extracts were fractionated into four fractions containing phenolic compounds differing in chemical structure and polarity. Except for the stilbene containing fraction, all other fractions showed inhibitory effects on both enzyme activities. The most pronounced impact was found for the hydrophilic low molecular weight polyphenols containing the free phenolic acids. In particular, gallic acid showed considerable inhibition values. EGCG was used as a positive control and showed a dose-dependent inhibition of collagenase activity (IC50 = 0.9 mM). © 2015 Published by Elsevier B.V.
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only organ directly exposed to the environment, aging processes resulting from environmental damage are of considerable relevance. Particularly solar UV radiation adversely impacts skin health, due to, amongst others, generation of reactive oxygen species (ROS) [1–3]. ROS are able to initiate complex molecular pathways including the activation of enzymes that degrade extracellular matrix (ECM) proteins in the dermis such as collagen and elastin ensuring the skin's threedimensional integrity [1,2]. As a consequence of protease activation, breakdown, fragmentation and disorganisation of
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Inhibitory effects of polyphenols from grape pomace extract on collagenase and elastase activity
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journal homepage: www.elsevier.com/locate/fitote
⁎ Corresponding author at: Fraunhofer Institute for Process Engineering and Packaging, Department of Process Development for Plant Raw Materials, Giggenhauser Str. 35, D-85354 Freising, Germany. Tel.: +49 8161 491 440. E-mail address: [email protected] (J. Wittenauer).
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
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Grape pomace from white wine (Vitis vinifera L. cv. ‘Weisser Riesling’) of vintage 2010 was kindly provided by the Baden-Badener Winzergenossenschaft (Baden-BadenNeuweier, Germany). Pomace samples (voucher specimen #600385.1 as deposited at the Fraunhofer IVV, Freising, Germany) were collected after pressing the mash, freeze dried, sealed in polyethylene bags, and kept at 5 °C until further use. C18 reversed-phase cartridges (Chromabond, 2000 mg) were obtained from Macherey-Nagel (Düren, Germany). C. histolyticum collagenase type IA (ChC), N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA) and porcine pancreas elastase type III (PPE) were purchased from Sigma (St. Louis, MO). N-Succ-Ala-Ala-Ala-p-nitroanilide (AAAPVN) was obtained from Serva Electrophoresis (Heidelberg, Germany). Polyphenols used as standards for the inhibition studies were gallic acid (≥97%), catechin (≥99%), epigallocatechin gallate (≥95%) (Sigma, St. Louis, MO); caftaric acid (≥97%), quercetin 3-O-glucoside (≥98%), quercetin 3-Oglucuronid (≥95%), procyanidin B1 (≥90%), procyanidin B2 (≥90%) (Fluka, Buchs, Switzerland), and trans-resveratrol (98%) (ABCR, Karlsruhe, Germany). All reagents and chemicals used were of analytical grade and purchased from VWR (Darmstadt, Germany).
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2.2.1. Crude extract Extraction of polyphenols from grape pomace was carried out according to Kammerer, Claus, Carle and Schieber [8]. Briefly, lyophilised grape pomace was ground using a ZM 100 centrifugal mill with a 1 mm ring sieve (Retsch, Haan, Germany). Aliquots of 2 g of the powdered samples were weighted into Erlenmeyer flasks, flushed with nitrogen and extracted with 50 ml of methanol for 60 min under shaking, using a KS 500 laboratory shaker (Janke & Kunkel, IKA Labortechnik, Staufen, Germany). Extracts were filtered through a paper filter (Munktell, grade 3hw); the residue was reextracted with 50 ml of the solvent for 30 min to ensure an exhaustive extraction. Supernatants were combined, and the organic solvent was removed by evaporation in vacuo at 40 °C. Residues obtained were dissolved in 10 ml water and filtered through a 0.2 μm syringe filter (Sartorius, Göttingen, Germany), and the pH was adjusted to 7.0 with sodium hydroxide solution (1 M).
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promoting properties, such investigations would be of basic interest. In the present study, the in vitro inhibitory potential of phenolic compounds obtained from white grape pomace against the activity of Clostridium histolyticum collagenase (ChC) and porcine pancreatic elastase (PPE) was investigated. The polyphenolic profile of the grape pomace extract was determined, moreover, different fractions of the extract as well as individual polyphenols were assayed for their inhibitory activity against ChC and PPE, respectively.
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the ECM proteins occur, visibly manifested by the typical UV induced skin alterations such as deep wrinkles, loss of skin tone and resilience [3]. Matrix metalloproteinases (MMPs), a family of multidomain zinc-containing endopeptidases with a broad range of substrate specificities, constitute the major proteolytic enzyme group involved in the degradation of dermal connective tissue components. Under normal physiological condition, MMP activities are precisely regulated at the level of transcription and by endogenous protein inhibitors. In addition, MMPs are secreted from cells as an inactive precursor requiring cleavage by extracellular proteinases to be activated. These regulation processes enable the remodeling of connective tissue under physiological and pathophysiological circumstances like wound healing, inflammation and cancer. The impairment of this balance, due to oxidative stress, results in an excess of MMPs being a key feature of premature aging of the skin as well as various inflammatory and degenerative diseases. In addition, serine proteases such as elastases are involved in ECM degradation. Elastolytic enzymes are released from neutrophils and dermal fibroblasts in response to UV induced skin inflammation [4]. Due to their active side catalytic triad, elastases are multi-specific and cleave several proteins within the extracellular matrix including collagen, fibronectin and elastin. Elastin is an important ECM protein having the unique property of elastic recoil, which is vital for providing elasticity to skin and other tissues like arteries, lungs and ligaments. Several studies have shown that human neutrophil elastase is also involved in the degradation of ECM connective tissue through the activation of inactive MMP-1 and MMP-2 precursors [5–7]. Inhibiting the activity of ECM degrading proteins like collagenases and elastases may be a useful approach to prevent UV induced skin alterations and premature skin aging. Scavenging of ROS by natural antioxidants might be one option to inhibit such skin deteriorative enzymes, as ROS play an important role in the activation of these enzymes. Phenolic compounds are an important group of natural antioxidants. They belong to diverse subclasses of secondary plant metabolites classified as phenolic acids, flavonoids, stilbenes and lignans and are ubiquitously found in the plant kingdom. In particular, red and white grapes contain high amounts of phenolic acids and flavonoids such as gallic acid and catechin [8]. Due to their chemical structure, polyphenols have powerful antioxidant activities being able to scavenge a wide range of ROS such as hydroxyl radicals, superoxide radical and O− 2 [9]. In addition, polyphenols may inhibit the activity of proteolytic enzymes in vitro by acting as complexing or precipitating agents as indicated in literature [10,11]. Especially, green tea polyphenols such as catechin and epigallocatechin gallate, commonly used as ingredients in antiaging skin care formulations, have been shown to exhibit moderate inhibitory effects against collagenase and elastase activity, presumably through non-covalent binding [12–14]. However, the mechanism of action of polyphenols is not fully understood, and studies about the inhibitory effects of natural active ingredients on skin degrading enzymes are scarce. Most of the studies have been performed with isolated compounds, thus not considering the complex nature of crude extracts. Additionally, data about inhibitiory properties of further polyphenolic subclasses are scarce. However, in particular, for the systematic development of natural formulations exerting skin health
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Separation of polyphenols was performed using an Agilent HPLC series 1200 system (Agilent, Waldbronn, Germany) with ChemStation software, a model G1379 degasser, a model G1312B binary gradient pump, a model G1367D thermoautosampler, a model G1316B column oven and a model G1315C diode array detector. The column used was a C 18 Synergi-Hydro column (150 × 3.0 mm i.d.; 4 μm particle size) from Phenomenex (Torrance, CA) operated at 25 °C. The mobile phase consisted of 2% (v/v) acetic acid in water (eluent A) and 0.5% acetic acid in water and acetonitrile (50:50, v/v, eluent B) using the following gradient program: 0–5% B (35 min), 5–20% B (45 min), 20–100% B (30 min), 100% B isocratic (3 min),100–0% B (10 min) at a flow rate of 0.8 ml/min. Total run time was 123 min. The injection volume varied between 10 and 30 μl. Polyphenols were monitored at 280 nm, and UV/Vis spectra were recorded in the range of 200 to 600 nm. Polyphenols were identified by UV–vis spectra and comparison with literature data and reference compounds. The quantitation of individual polyphenols was performed using calibration curves of the corresponding reference compounds. Gallic acid, caftaric acid, (+)-catechin, epigallocatechin gallate, procyanidin B1, procyanidin B2, quercetin 3-O-glucoside, quercetin 3-O-glucuronid, kaempferol 3-O-glucosid, and transresveratrol served as reference compounds. The quantitation of epicatechin was performed applying the catechin calibration curve. All determinations were carried out in quadruplicate.
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Porcine pancreatic elastase (PPE) inhibition of the individual samples a–c (see Section2.4 2.4) was determined spectrophotometrically using AAAPVN as the substrate by monitoring the release of p-nitroaniline at 410 nm. PPE was dissolved in 2 mM Tris (2-amino-2-hydroxymethyl-propane-1,3-diol) buffer (pH = 8,0). Thereof, an aliquot of 10 μl was taken and loaded in the wells of the microtiter plates together with 100 μl of the Tris buffer and 30 μl of the individual sample solutions. After 20 min of pre-incubation at 25 °C, 40 μl of the substrate AAAPVN (dissolved in 2 mM Tris buffer at a concentration of 0.25 mg/ml) was added. Absorbance was measured for 20 min after the addition of AAAPVN. Initial velocities, the inhibition activity and IC50 values were calculated in accordance with the ChC assay
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Inhibition effects of the grape pomace crude extract, the fractions derived thereof as well as the standards mentioned above against C. histolyticum collagenase (ChC) were measured spectrophotometrically according to a modified procedure described by van Wart and Steinbrink [15] using a BioTek Synergi H1 multi-mode microplate reader (BioTek, Bad Friedrichshall, Germany) and appropriate data analysis software (Gen 5, version 2.04, BioTek, Bad Friedrichshall, Germany). ChC (1.1 U/ml) and FALGPA (1 mM), respectively, were dissolved in 0.05 M tricine [N-(2-hydroxy-1,1bis(hydroxymethyl)ethyl)glycine] buffer containing 0.4 M NaCl and 0.01 M CaCl2; the pH was adjusted to 7.5 with 1 M NaOH. The inhibitory effects of the following samples were investigated:
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First, 30 μl of the samples (a–c) were incubated with 10 μl of ChC solution and 60 μl tricine buffer for 20 min at 37 °C. Subsequently, 20 μl of the FALGPA solution was added to initiate the reaction. For negative controls water was added instead of FALGPA solution. The ChC inhibitory activities of the individual samples were measured by continuously monitoring the decrease in absorbance of FALGPA at 335 nm for 20 min after starting the reaction. Initial velocities were calculated from the slope of the absorbance change during the first 10 min of hydrolysis. The relative inhibition was calculated according to Eq. (1). IC50 values were determined from dose–effect curves.
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a) dilutions of crude grape pomace extract with water at concentrations of 35.3 ± 1.7 μg/ml (1:20), 23.5 ± 1. μg/ml (1:30), 14.1 ± 0.3 μg/ml (1:50), 8.8 ± 0.4 (1:80) and 7.1 ± 0.3 μg/ml (1:100) (extract: water); b) the individual fractions 1–4; and c) aqueous solutions of the individual reference polyphenols at concentrations of 250 μM, 500 μM, 750 μM and 1000 μM, respectively.
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2.2.2. Polyphenol fractions The fractionation of polyphenolic compounds of the grape pomace extract was achieved by solid phase extraction (SPE). Cartridges were first activated with 12 ml of methanol, washed with 20 ml of water and then loaded with 2 ml of the crude extract. Elution of the fractions followed by subsequent application of various solvent mixtures. Fraction 1 was eluted with 20 ml of acetic acid (2%). Fraction 2 was obtained next by application of 20 ml of a 3:1 mixture of acetic acid (2%) and acetonitrile/acetic acid (2%) (v/v). Elution with 20 ml of acetic acid (2%) and acetonitrile/acetic acid (2%) (v/v) at a ratio of 3:2 resulted in fraction 3. The last fraction (fraction 4) was obtained by using 20 ml of acetonitrile/acetic acid (2%) (v/v).
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3.1.1. Crude extract Prior to the enzymatic inhibition studies, the identification and quantitation of the polyphenolic pattern by HPLC was a prerequisite to identify the compounds being primarily responsible for the enzyme inhibitory effects. Major polyphenols of the grape pomace were the flavanols catechin (4) and epicatechin (6) accompanied by the procyanidins B1 (3) and B2 (5), while the phenolic acids gallic acid (1) and caftaric acid (2), the flavonol glycosides quercetin-3-O-glucuronide (7) and quercetin-3-O-glucoside (8) as well as the stilbene transresveratrol (9) were found to be minor compounds (Fig. 1).
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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3.2. Quantitation of polyphenolic compounds in the crude extract and the four fractions
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Quantitated amounts of major polyphenolic constituents in grape pomace extract and fractions thereof are summarized in Table 1. In accordance with literature data [8,16], the flavanols catechin and epicatechin were the predominant components in the crude extract with concentrations of 210.7 and 180.7 μg/ml, respectively, followed by the procyanidins B1 (97.5 μg/ml) and B2 (85.9 μg/ml). The flavonol-glycosides, the phenolic acids as well as resveratrol were present in minor amounts. Due to the high absorption values at the detected wavelength, the grape pomace extract and the fractions had to be diluted 20-fold for the subsequent enzyme assays. Amounts of individual polyphenols detected in fractions 1–3 were slightly lower than in the crude extract, which probably resulted from losses owing to the fractionation process with the exception of resveratrol in fraction 4. This could be due to further overlapping constituents contained in the crude extract (Fig. 1) which also absorb light at the detected wavelength, thus interfering the chromatographic analysis. Therefore, integration of resveratrol in the chromatograms of the crude extract resulted in apparently lower amounts than in the purified fraction.
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To investigate the inhibition exerted by the grape pomace extract on ChC and PPE, the respective enzymes were incubated with the extract and the appropriate substrates. The assays were performed with synthetic peptides as substrate molecules, and enzyme preparations isolated from C. histolyticum and porcine pancreas, respectively. As reported in literature, PPE is topologically very similar to human neutrophil elastase and inhibition mechanisms for some phenolic substances were found to be equal [17,18]. Therefore, it can be assumed that the in vitro assays performed with the ChC and PPE and synthetic peptides are a reliable tool to get first indications of the capability of grape pomace polyphenols to prevent extracellular matrix degradation as a skin aging phenomenon. The inhibitory potential of the grape pomace extract was examined for increasing dilutions, in order to establish dose-dependent relationships, and to calculate the half maximal inhibitory concentrations (IC50). Crude grape pomace extract was incubated with collagenase and elastase at dilution-ratios ranging from 1:20 to 1:100, resulting in total polyphenolic contents ranging from 35.3 to 7.1 μg/ml. The effect of different extract concentrations on the enzyme activity is shown in Fig. 3a and b for ChC and PPE, respectively. As expected the slope of the digestion curves of the collagenase assay decreases with increasing extract concentration, which is due to the lowered enzyme activity, thus resulting in higher amounts of non-cleaved substrate molecules. The higher initial absorption with increasing extract concentration probably results from self-absorption of grape pomace extract components. Since the elastase assay was performed with N-Succ-Ala-Ala-Ala-p-nitroanilide as the substrate peptide, the enzyme activity can be deduced from the release of p-nitroaniline resulting in increased absorption values. At increased extract concentrations slopes in digestion curves were less steep representing an increased enzyme inhibition. The grape pomace extract inhibited both enzymes in a dosedependent manner. Highest polyphenol concentrations of 35.3 μg/ml resulted in an 80% and 73% relative inhibition of ChC and PPE activity, respectively (Fig. 4). Inhibitory capacities declined with decreasing concentrations of the extract. Relative inhibition of ChC was 55% at a polyphenol concentration of 23.5 μg/ml, and the inhibition capacity further decreased at lower polyphenol concentrations in a nearly linear manner to 40% and 28% for 14.1 μg/ml and 8.8 μg/ml, respectively. Further dilution of the extract to 7.1 μg/ml resulted in a negligible decrease of the inhibition value. The relative inhibition of PPE at a concentration of 23.5 μg/ml was found to be 63%. At lower grape pomace extract concentrations, inhibitory activities markedly declined with relative inhibition values of 49%, 36% and 20%, for 14.1 μg/ml, 8.8 μg/ml and 7.1 μg/ml, respectively. From the dose-response relationships, IC50 values of 20.3 μg/ml for ChC and 14.7 μg/ml for PPE activity were calculated. Comparison of our present findings with those reported for other plant extracts is rather difficult, due to varying assay conditions, particularly incubation times and types of substrates and enzymes used for the determination of the enzymatic activities. Additionally, the extracts are often obtained using different extraction methods. The polarity of the
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3.1.2. Polyphenol fractions To rank the compounds from specific polyphenolic classes according to their relative enzyme inhibitory effects, the crude extract was separated into four fractions, being composed of polyphenols having similar chemical structure and polarity, and the fractions were analyzed by HPLC. As shown in Fig. 2, fraction 1 contained the free phenolic acids gallic acid and caftaric acid, while the main components of the crude extract, catechin and epicatechin as well as the procyanidins B1 and B2, were found in fraction 2. The flavone glycosides quercetin-3-Oglucuronide and quercetin-3-O-glucoside could be detected in fraction 3, whereas the stilbene resveratrol was the only polyphenolic constituent detectable in fraction 4.
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Fig. 1. HPLC separation of polyphenols from crude grape pomace extract (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, (8) quercetin-3-O-glucoside, (9) resveratrol.
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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Fig. 2. HPLC separation of fractions obtained from grape pomace extract by means of solid phase extraction (280 nm) and related structures.
3.4. Collagenase and elastase inhibitory activity of grape pomace 396 extract fractions 397
t1:1 t1:2
Table 1 Polyphenol contents of undiluted and diluted grape pomace extract and fractions thereof. Values represent mean ± standard deviation, n = 3.
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In order to determine the most potent polyphenols in the grape pomace extract inhibiting ChC and PPE activities, fractions containing individual polyphenolic compounds exerting different polarities were analyzed. As shown in Fig. 5, fraction 2, enriched in catechins and procyanidins at a concentration of 27.4 μg/ml was the most potent in ChC inhibition (43%). Effects on ChC activity were lower for fraction 1 (26%) comprising the polar phenolic acids, and also for fraction 3 (24%) containing the flavonol-glycosides. Apparently, fractions 1 and 3 exhibited approximately identical inhibiting effect, although the polyphenol concentration of fraction 1 (1.0 μg/ml) was about four times
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solvent has a strong influence on the inhibitory effects of a plant extract [19], indicating that individual constituents may differ regarding their inhibitory effects. Using more lipophilic extraction media could result in extracts with high inhibition of collagenase and elastase activities [19–21]. Methanol is an efficient extraction media for a broad spectrum of polyphenols [8,22,23]. Since methanol is an intolerable ingredient for cosmetic formulations, the extracts used in our study were dissolved in water. Therefore, lower inhibition values than described in literature due to lowered extraction yields could be associated. In addition, some studies investigated plant extracts without explicit identification and quantitation of the active compounds, and thus the exact concentrations of bioactives required for enzyme inhibition remain unclear [20,24].
383 384
t1:3
Concentration (mg/kg grape pomace (DM))
Concentration (μg/ml)
t1:4
No.
Retention time
Identity
Crude extract
Diluted crude extract ⁎
Fraction 1 ⁎
Fraction 2 ⁎
Fraction 3 ⁎
Fraction 4 ⁎
t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14
1 2 3 4 5 6 7 8 9
4.2 11.1 22.6 24.0 34.4 38.7 75.2 76.0 83.4
Gallic acid Caftaric acid Procyanidin B1 Catechin Procyanidin B2 Epicatechin Quercetin-3-O-glucuronide Quercetin-3-O-glucoside Resveratrol Total amount
596.36 735.32 4858.58 10496.63 4277.04 8994.93 2432.29 2374.32 297.78 35063.24
0.60 0.74 4.88 10.53 4.30 9.04 2.46 2.40 0.30 35.25
0.47 ± 0.01 0.55 ± 0.01 – – – – – – – 1.02 ± 0.03
– – 4.72 ± 0.22 10.42 ± 0.13 3.40 ± 0.16 8.84 ± 0.37 – – – 27.38 ± 0.88
– – – – – – 1.91 ± 0.08 1.96 ± 0.08 – 3.77 ± 0.16
– – – – – – – – 0.50 ± 0.02 0.50 ± 0.02
t1:15
⁎ Dilution applied in the enzyme assays (1:20).
± ± ± ± ± ± ± ± ± ±
37.49 40.88 305.58 1059.35 261.87 672.30 164.15 169.64 37.88 2749.15
± ± ± ± ± ± ± ± ± ±
0.03 0.04 0.17 0.72 0.17 0.39 0.10 0.04 0.01 1.67
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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Fig. 3. Activities of collagenase (a) and elastase (b) depending on four concentrations of grape pomace extract compared to control (n = 3).
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[18,25]. Therefore, attaining the active center of the enzyme is obviously an important premise for inhibition which is probably easier for smaller molecules like the phenolic acids of fraction 1 than for the higher molecular polyphenols of fraction 2. The summation of the relative ChC and PPE inhibitions of all polyphenol fractions led to a total inhibition of 93% and 85%, respectively. The lower inhibition value of the crude extract, compared to the calculated sum of the fractions, may be attributed to interfering non-polyphenolic compounds contained, the latter being devoid of the purified fractions.
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lower than of fraction 3 (3.8 μg/ml). In addition, compared to the crude extract, fractions 1 and 3 exhibited relatively high inhibition values at low polyphenol concentrations. Fraction 4, containing resveratrol, did not show any inhibitory effects on ChC activity. According to literature, inhibition of ChC commonly results from unspecific non-covalent interactions like hydrogen bonding and hydrophobic interactions with collagenase side chain groups leading to conformational changes [13,14]. Such an unspecific inhibition possibly largely depends on the absolute number of hydroxyl groups and benzene rings which is reflected in the molecular weight of the polyphenolic substances but also in absolute polyphenol concentration applied in the enzyme assay. Hence, the superior inhibiting effect of fraction 2 against ChC activity may possibly be attributed to the higher concentration of individual polyphenols as well as the contained procyanidins. Relative inhibition of PPE caused by fractions 2 and 3 amounted to 17% and 19%, respectively. In contrast to ChC, highest inhibitory effects were observed for fraction 1 (47%) containing the phenolic acids. Notably, fraction 4 showed a weak interaction with PPE, resulting in a relative inhibition of 2%. Polyphenol interaction with PPE was reported to depend on rather specific interactions between polyphenolic hydroxyl groups and amino acid residues of the catalytic triad
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3.5. Collagenase and elastase inhibitory activity of individual 442 polyphenols 443 Since the grape pomace extract and the fractions derived thereof affected collagenase and elastase activity differently, polyphenols with different structural characteristics seem to be involved in the inhibition of the enzymes. To get further insight into their structure dependent activity, polyphenols identified by HPLC analysis were tested individually in the ChC and PPE assays at concentrations ranging from 250 to 1000 μmol/L. Additionally, epigallocatechin gallate (EGCG) was tested as a positive control [11], while epicatechin, the isomeric form of
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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
444 445 446 447 448 449 450 451 452
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Fig. 5. Relative inhibitory properties of polyphenol fractions from grape pomace on collagenase and elastase activity (n = 3).
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strongest inhibiting activity of 60%, followed by gallic acid (51%). Both polyphenols differed markedly regarding their dose-dependency. In accordance with literature data, EGCG showed dose-dependent inhibitory properties [14], whereas gallic acid already inhibited ChC activity at lower concentrations in a highly efficient manner exerting relative inhibition of 42% at 250 μmol/L. As previously reported, higher hydroxylation of the molecules enhances their inhibitory effect due to stronger hydrogen bonding interactions with functional side chain groups of the enzyme [14]. Bras, Goncalves, Fernandes, Mateus, Ramos and de Freitas [18] referred that galloyl groups greatly contribute to higher binding activity towards proteins due to the three neighboring hydroxyl groups and the benzene ring facilitating both hydrogen and hydrophobic binding to
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catechin was not individually applied, due to its largely structural accordance to the latter. Among the analyzed polyphenols, gallic acid, catechin, procyanidin B1 and B2 exhibited inhibitory activities against both enzymes. As previously shown by Hrenn, Steinbrecher, Labahn, Schwager, Schempp and Merfort [26], resveratrol had no effects within the concentration ranges applied what might be a possible explanation for the lacking or low inhibition effects of fraction 4. The hydroxycinnamate caftaric acid did not show inhibition effects as well. This substance was not analyzed before. As depicted in Fig. 6a, the inhibition of ChC by individual polyphenols showed a dose-dependent relationship for gallic acid, procyanidin B1 and B2 as well as the EGCG control. At concentrations of 1 mmol/L, EGCG exhibited the
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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
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Acknowledgements
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This study was financially supported by the Fraunhofer Gesellschaft zur Förderung der angewandten Forschung (FhG). The authors are grateful to Baden-Badener Winzergenossenschaft, Baden-Baden-Neuweier, Germany, for providing the grape pomace.
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In conclusion, crude grape pomace extract revealed its potential for cosmetic applications as strong dose-dependent inhibitory effects of both ChC and PPE were observed. Due to the high inhibition values at low total polyphenol concentrations, fraction 1, containing the polar free phenolic acids exhibited the most potent inhibitory activity. In particular, gallic acid carrying three neighboring hydroxyl and a carboxyl functional group seems to be decisively involved in the inhibitory effects of fraction 1. Since caftaric acid did not show any inhibitory effect, synergistic interactions between polyphenols and the enzyme could also play an important role regarding the inhibition mechanism. In accordance with earlier findings, also the catechin- and procyanidin-rich fraction 2 had inhibitory properties, in particular, against ChC. Due to the high polyphenol concentrations required for inhibition and the higher molecular weight and polymerisation degree of the molecules, their application for cosmetic purposes could be limited, since the size of polyphenols restricts their permeation into the epidermal and corium layers and the concentrations that are achieved in these layers are limited [27,28].
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proteins. Additionally to these non-specific interactions, the free carboxyl moiety in gallic acid could bind to the zinc site in the catalytic domain of ChC as possible inhibition mechanism as discussed by Kim, Uyama and Kobayashi [13]. However, to the best of our knowledge, the inhibitory effects of gallic acid have so far not been studied. Inhibition effects on ChC were only found at high concentrations of 1000 μmol/L for procyanidin B2 and procyanidin B1, respectively. Inhibitory activities of catechin were already observed at 250 μmol/L and, as an exception, inhibition did not increase with increasing concentration. The dose-dependent inhibitory effects of most of the polyphenols under investigation support our suggestion that ChC inhibition could be achieved by higher concentrations of individual polyphenols as earlier discussed. Inhibitions of PPE activities were significantly lower for the individual polyphenols (Fig. 6b), and dose-dependency was found to be poor. Inhibitory effects merely increased for polyphenol concentrations exceeding 750 μmol/L. At concentrations of 1 mmol/L, strongest inhibitory activities were observed for catechin (12.0%), EGCG (7.3%), and procyanidin B2 (6.4%). In agreement with Bras, Goncalves, Fernandes, Mateus, Ramos and de Freitas [18], the low inhibitory effects of individual polyphenols on PPE support that an unspecific complexation reaction may be excluded as the underlying inhibition mechanisms. Additionally, the low inhibition values of the individual compounds are in contrast to the inhibiting potency of the extract and its fractions, respectively, which may result from synergistic effects.
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[25] Siedle B, Murillo R, Hucke O, Labahn A, Merfort I. Structure activity relationship studies of cinnamic acid derivatives as inhibitors of human neutrophil elastase revealed by ligand docking calculations. Pharmazie 2003;58:337–9. [26] Hrenn A, Steinbrecher T, Labahn A, Schwager J, Schempp CM, Merfort I. Plant phenolics inhibit neutrophil elastase. Planta Med 2006;72: 1127–31.
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