Plant Foods Hum Nutr DOI 10.1007/s11130-014-0459-0
ORIGINAL PAPER
Obtaining from Grape Pomace an Enzymatic Extract with Anti-inflammatory Properties B. Rodríguez-Morgado & M. Candiracci & C. Santa-María & E. Revilla & B. Gordillo & J. Parrado & A. Castaño
# Springer Science+Business Media New York 2014
Abstract Grape pomace, a winemaking industry by-product, is a rich source of bioactive dietary compounds. Using proteases we have developed an enzymatic process for obtaining a water-soluble extract (GP-EE) that contains biomolecules such as peptides, carbohydrates, lipids and polyphenols in soluble form. Of especial interest is its high polyphenol content (12 %), of which 77 % are flavonoids and 33 % are phenolic acids. The present study evaluates in vitro the potential anti-inflammatory effect of GP-EE by monitoring the expression of inflammatory molecules on N13 microglia cells stimulated with lipopolysaccharide (LPS). GP-EE decreases the mRNA levels of the inflammatory molecules studied. The molecules under study were as follows: inducible nitric oxide synthase (iNOS), tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), the ionized calcium binding adaptor molecule-1(Iba-1) and the Toll like receptor-4 (TLR-4), as well as the iNOS protein level in LPS-stimulated microglia. Our findings suggest that, as a result of its ability to regulate excessive microglial activation, GP-EE possesses antiinflammatory properties. Therefore, acting as a chemopreventive agent, it may be of therapeutic interest in neurodegenerative diseases involving neuroinflammation. We can,
therefore, propose GP-EE as a useful natural extract and one that would be beneficial to apply in the field of functional foods. Keywords Grape pomace . Enzymatic extract . Polyphenols . Anti-inflammatory . Neurodegeneration Abbreviations GP Grape pomace GP-EE Grape pomace enzymatic extract LPS Lipopolysaccharide iNOS Inducible nitric oxide synthase TNF-α Tumor necrosis factor- alpha IL-1β Interleukin-1β TLR-4 Toll like receptor-4 Iba-1 Ionized calcium binding adaptor molecule-1 UPLC Ultra-high performance liquid chromatography RT Reverse transcription
Introduction B. Rodríguez-Morgado : C. Santa-María : E. Revilla : J. Parrado : A. Castaño (*) Departamento de Bioquímica y Biología Molecular, Universidad de Sevillla, C/Profesor García González, 2, 41012 Sevilla, Spain e-mail: [email protected] B. Gordillo Food Colour & Quality Lab, Department of Nutrition & Food Science. Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain M. Candiracci Brigham and Women Hospital, Thorn 13, Anesthesia. 75 Francis St, Boston, MA 02115, USA
Grape pomace, a winemaking industry by-product, has traditionally been considered as an environmental problem. However, GP is being increasingly recognized as a source of bioactive dietary compounds such as polyphenols [1]. To date, its efficient recovery to convert it into a high value-added product has been accomplished by conventional solid–liquid extraction with organic solvents (ethanol, methanol, ethyl ether, ethyl acetate, etc.) or by heated sulphurated water. It is also recovered enzymatically with carbohydrate hydrolases (cellulase and pectinase [1–5] and by physical methods such as superheated liquid extraction and supercritical fluid extraction.
Plant Foods Hum Nutr
Grape polyphenols possess many biological activities, including anti-inflammatory properties [6] that can be beneficial to human health. It is now widely accepted that inflammation plays a pivotal role in neurodegenerative processes like Parkinson’s disease or Alzheimer’s disease. The hallmark of neuroinflammation is the activation of microglia, the brain’s major defense against immune challenge. However, activated microglia may also contribute to neurodegeneration through the release of pro-inflammatory and/or cytotoxic factors such as IL-1 β, TNF- α, nitric oxide (NO) and reactive oxygen intermediates, among others. It can therefore be assumed that a certain degree of brain inflammation is required to repair the damaged tissue yet excessive inflammation causes neuronal cell death. It would be interesting, therefore, to search for molecules that could help control inflammation in the CNS [7–10]. Natural polyphenols have been shown to exert neuroprotective properties by inhibiting the release of proinflammatory cytokines after LPS-activation of microglia [11–14]. This paper describes a new extraction method based on enzymatic technology for obtaining a valuable product from GP. The chemical and functional characterization of the enzymatic extract GP-EE, focusing on its anti-inflammatory properties, is also described.
Phenolic Composition by UPLC
Materials and Methods
The analysis of the individual phenolics was undertaken via ultra-high performance liquid chromatography (UPLC) using Agilent 1260 system equipment with a diode-array detector. Phenols were separated on a C18 Poroshell 120 column (2.7 μm particle size, 5 cm×4.6 mm) (Agilent, Palo Alto, CA) and maintained at 25 °C. The solvents used were waterformic acid (99:1, v/v) as solvent A, and acetonitrile as solvent B. The flow rate was 1.5 mL/min and the injection volume was 30 μl. The linear gradient elution was: 0 min, 100 % A; 5 min, 95 % A and 5 % B; 20 min, 50 % A and 50 % B; 22 min, 100 % A; 25 min, 100 % A. The detection wavelengths were 280 nm (flavanols and benzoic acids); 320 nm (cinnamic acids and their derivates) and 370 nm (flavonols). The analyses were performed in triplicate for each sample. Individual phenols were identified by comparing their retention time and spectra against standards. The external calibration method was used for quantification comparing the areas with standards of gallic, protocatechuic, caffeic acid, catechin, epicatechin, quercetin-3-O- glucoside and kaempferol-3-O-glucoside. All standards were HPLC-grade and purchased from Sigma-Aldrich (Madrid, Spain). Phenolic compounds which were not available as standards were identified by their retention time and spectra, according to the literature. These compounds have been assayed by assuming that their molar absorptivity is the same as that of the corresponding free standard molecule.
Enzymatic Hydrolysis
N13 Cell Culture and Immunostimulation Assays
GP was resuspended in water at a 20 % concentration. Enzymatic hydrolysis was performed using an endoprotease mixture (0,3 %v/v) in a bioreactor using the pH-stat method at 60 °C and pH 8. The solids were then removed by filtration and the final product (GP-EE) was concentrated using a rotary evaporator, obtaining a completely water-soluble syrup. The concentrated extract was lyophilized to obtain a fine red powder with a phenolic yield of 12 % of dry matter.
After stimulation with LPS, N13 microglia produces a repertoire of cytokines similar to primary microglia [18]. Cells were grown in RPMI 1640 (PAA, Linz, Austria) supplemented with 2 mM glutamine (PAA), 5 % (v/v) fetal bovine serum (PAA), 100 U/mL 131 penicillin, and 100 μg/mL streptomycin (PAA) at 37 °C and 5 % CO2. For subculture, cells were removed from the culture flask with a scraper, resuspended in the culture medium, and subcultured in 6-well plates for experiments (Nunc, Thermo Fisher Scientific, USA) in culture medium at a density of 5.0×105 cells/well/2 mL. After adhering, cells were stimulated with LPS (0.01 μg/mL) and simultaneously different concentrations of GP-EE were added (0, 1, 5 and 10 μg/mL of polyphenols). Finally, they were collected at different times after stimulation (1, 4 and 6 h), to extract RNA and proteins. Cells treated only with cell culture media but no GP-EE or LPS were used as control.
GP-EE Chemical Composition The chemical composition of GP-EE, as protein, fat, carbohydrates and fiber, was characterized by using AOAC standard protocols [15]. Molecular-mass protein distribution in GP-EE was determined by size-exclusion chromatography using an ÄKTA-purifier (GE Healthcare) according to the procedure described by Bautista [16], using a Superdex PeptideTM 10/300 GL column (optimum separation range 0.1–7 kDa). Total phenolics were determined using a modification of the Folin-Ciocalteau method [17].
Determination of Cell Viability After 24 h of GP-EE treatment (0, 1, 5 and 10 μg/mL of polyphenols), cells were washed with 1 mL of PBS and stained for 60 min with 1 mL of a 1 % crystal violet solution.
Plant Foods Hum Nutr
After careful aspiration of the crystal violet solution, the plates were washed with deionized water and dried prior to the solubilization of the bound dye with 1 mL of a 1 % aqueous SDS solution. The optical density of the plates was measured at 590 nm in a microplate spectrophotometer.
At least three independent experiments were conducted and analyzed statistically using t-student Bonferroni or analysis. Different levels of significance (*p
ORIGINAL PAPER
Obtaining from Grape Pomace an Enzymatic Extract with Anti-inflammatory Properties B. Rodríguez-Morgado & M. Candiracci & C. Santa-María & E. Revilla & B. Gordillo & J. Parrado & A. Castaño
# Springer Science+Business Media New York 2014
Abstract Grape pomace, a winemaking industry by-product, is a rich source of bioactive dietary compounds. Using proteases we have developed an enzymatic process for obtaining a water-soluble extract (GP-EE) that contains biomolecules such as peptides, carbohydrates, lipids and polyphenols in soluble form. Of especial interest is its high polyphenol content (12 %), of which 77 % are flavonoids and 33 % are phenolic acids. The present study evaluates in vitro the potential anti-inflammatory effect of GP-EE by monitoring the expression of inflammatory molecules on N13 microglia cells stimulated with lipopolysaccharide (LPS). GP-EE decreases the mRNA levels of the inflammatory molecules studied. The molecules under study were as follows: inducible nitric oxide synthase (iNOS), tumor necrosis factor- α (TNF-α), interleukin-1β (IL-1β), the ionized calcium binding adaptor molecule-1(Iba-1) and the Toll like receptor-4 (TLR-4), as well as the iNOS protein level in LPS-stimulated microglia. Our findings suggest that, as a result of its ability to regulate excessive microglial activation, GP-EE possesses antiinflammatory properties. Therefore, acting as a chemopreventive agent, it may be of therapeutic interest in neurodegenerative diseases involving neuroinflammation. We can,
therefore, propose GP-EE as a useful natural extract and one that would be beneficial to apply in the field of functional foods. Keywords Grape pomace . Enzymatic extract . Polyphenols . Anti-inflammatory . Neurodegeneration Abbreviations GP Grape pomace GP-EE Grape pomace enzymatic extract LPS Lipopolysaccharide iNOS Inducible nitric oxide synthase TNF-α Tumor necrosis factor- alpha IL-1β Interleukin-1β TLR-4 Toll like receptor-4 Iba-1 Ionized calcium binding adaptor molecule-1 UPLC Ultra-high performance liquid chromatography RT Reverse transcription
Introduction B. Rodríguez-Morgado : C. Santa-María : E. Revilla : J. Parrado : A. Castaño (*) Departamento de Bioquímica y Biología Molecular, Universidad de Sevillla, C/Profesor García González, 2, 41012 Sevilla, Spain e-mail: [email protected] B. Gordillo Food Colour & Quality Lab, Department of Nutrition & Food Science. Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain M. Candiracci Brigham and Women Hospital, Thorn 13, Anesthesia. 75 Francis St, Boston, MA 02115, USA
Grape pomace, a winemaking industry by-product, has traditionally been considered as an environmental problem. However, GP is being increasingly recognized as a source of bioactive dietary compounds such as polyphenols [1]. To date, its efficient recovery to convert it into a high value-added product has been accomplished by conventional solid–liquid extraction with organic solvents (ethanol, methanol, ethyl ether, ethyl acetate, etc.) or by heated sulphurated water. It is also recovered enzymatically with carbohydrate hydrolases (cellulase and pectinase [1–5] and by physical methods such as superheated liquid extraction and supercritical fluid extraction.
Plant Foods Hum Nutr
Grape polyphenols possess many biological activities, including anti-inflammatory properties [6] that can be beneficial to human health. It is now widely accepted that inflammation plays a pivotal role in neurodegenerative processes like Parkinson’s disease or Alzheimer’s disease. The hallmark of neuroinflammation is the activation of microglia, the brain’s major defense against immune challenge. However, activated microglia may also contribute to neurodegeneration through the release of pro-inflammatory and/or cytotoxic factors such as IL-1 β, TNF- α, nitric oxide (NO) and reactive oxygen intermediates, among others. It can therefore be assumed that a certain degree of brain inflammation is required to repair the damaged tissue yet excessive inflammation causes neuronal cell death. It would be interesting, therefore, to search for molecules that could help control inflammation in the CNS [7–10]. Natural polyphenols have been shown to exert neuroprotective properties by inhibiting the release of proinflammatory cytokines after LPS-activation of microglia [11–14]. This paper describes a new extraction method based on enzymatic technology for obtaining a valuable product from GP. The chemical and functional characterization of the enzymatic extract GP-EE, focusing on its anti-inflammatory properties, is also described.
Phenolic Composition by UPLC
Materials and Methods
The analysis of the individual phenolics was undertaken via ultra-high performance liquid chromatography (UPLC) using Agilent 1260 system equipment with a diode-array detector. Phenols were separated on a C18 Poroshell 120 column (2.7 μm particle size, 5 cm×4.6 mm) (Agilent, Palo Alto, CA) and maintained at 25 °C. The solvents used were waterformic acid (99:1, v/v) as solvent A, and acetonitrile as solvent B. The flow rate was 1.5 mL/min and the injection volume was 30 μl. The linear gradient elution was: 0 min, 100 % A; 5 min, 95 % A and 5 % B; 20 min, 50 % A and 50 % B; 22 min, 100 % A; 25 min, 100 % A. The detection wavelengths were 280 nm (flavanols and benzoic acids); 320 nm (cinnamic acids and their derivates) and 370 nm (flavonols). The analyses were performed in triplicate for each sample. Individual phenols were identified by comparing their retention time and spectra against standards. The external calibration method was used for quantification comparing the areas with standards of gallic, protocatechuic, caffeic acid, catechin, epicatechin, quercetin-3-O- glucoside and kaempferol-3-O-glucoside. All standards were HPLC-grade and purchased from Sigma-Aldrich (Madrid, Spain). Phenolic compounds which were not available as standards were identified by their retention time and spectra, according to the literature. These compounds have been assayed by assuming that their molar absorptivity is the same as that of the corresponding free standard molecule.
Enzymatic Hydrolysis
N13 Cell Culture and Immunostimulation Assays
GP was resuspended in water at a 20 % concentration. Enzymatic hydrolysis was performed using an endoprotease mixture (0,3 %v/v) in a bioreactor using the pH-stat method at 60 °C and pH 8. The solids were then removed by filtration and the final product (GP-EE) was concentrated using a rotary evaporator, obtaining a completely water-soluble syrup. The concentrated extract was lyophilized to obtain a fine red powder with a phenolic yield of 12 % of dry matter.
After stimulation with LPS, N13 microglia produces a repertoire of cytokines similar to primary microglia [18]. Cells were grown in RPMI 1640 (PAA, Linz, Austria) supplemented with 2 mM glutamine (PAA), 5 % (v/v) fetal bovine serum (PAA), 100 U/mL 131 penicillin, and 100 μg/mL streptomycin (PAA) at 37 °C and 5 % CO2. For subculture, cells were removed from the culture flask with a scraper, resuspended in the culture medium, and subcultured in 6-well plates for experiments (Nunc, Thermo Fisher Scientific, USA) in culture medium at a density of 5.0×105 cells/well/2 mL. After adhering, cells were stimulated with LPS (0.01 μg/mL) and simultaneously different concentrations of GP-EE were added (0, 1, 5 and 10 μg/mL of polyphenols). Finally, they were collected at different times after stimulation (1, 4 and 6 h), to extract RNA and proteins. Cells treated only with cell culture media but no GP-EE or LPS were used as control.
GP-EE Chemical Composition The chemical composition of GP-EE, as protein, fat, carbohydrates and fiber, was characterized by using AOAC standard protocols [15]. Molecular-mass protein distribution in GP-EE was determined by size-exclusion chromatography using an ÄKTA-purifier (GE Healthcare) according to the procedure described by Bautista [16], using a Superdex PeptideTM 10/300 GL column (optimum separation range 0.1–7 kDa). Total phenolics were determined using a modification of the Folin-Ciocalteau method [17].
Determination of Cell Viability After 24 h of GP-EE treatment (0, 1, 5 and 10 μg/mL of polyphenols), cells were washed with 1 mL of PBS and stained for 60 min with 1 mL of a 1 % crystal violet solution.
Plant Foods Hum Nutr
After careful aspiration of the crystal violet solution, the plates were washed with deionized water and dried prior to the solubilization of the bound dye with 1 mL of a 1 % aqueous SDS solution. The optical density of the plates was measured at 590 nm in a microplate spectrophotometer.
At least three independent experiments were conducted and analyzed statistically using t-student Bonferroni or analysis. Different levels of significance (*p
