Plant Foods Hum Nutr DOI 10.1007/s11130-014-0461-6
ORIGINAL PAPER
Phenolic Acids of the Two Major Blueberry Species in the US Market and Their Antioxidant and Anti-inflammatory Activities Jie Kang & Keshari M. Thakali & Gitte S. Jensen & Xianli Wu
# Springer Science+Business Media New York 2014
Abstract Highbush (cultivated) and lowbush (wild) are the two major blueberry species in the US market. Eight phenolic acids were detected and quantified from these two species by HPLC-MS. Chlorogenic acid was found to be the predominant phenolic acid in both species, with 0.44 mg/g fresh weight in lowbush blueberries and 0.13 mg/g fresh weight in highbush blueberries. Total phenolic content in lowbush blueberries is over three times higher than that of highbush blueberries. The phenolic acid mixtures representing those in the two species were prepared by using authentic standards to assess their contribution to total antioxidant and antiinflammatory activities of the whole berries. Neither lowbush nor highbush blueberry phenolic acid mixture contributed significantly to the total antioxidant capacity of their relevant whole berries measured by oxygen radical absorbance This study was supported by USDA (6251-51000-005-02S). Electronic supplementary material The online version of this article (doi:10.1007/s11130-014-0461-6) contains supplementary material, which is available to authorized users. J. Kang : K. M. Thakali USDA Arkansas Children’s Nutrition Center, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 15 Children’s Way, Little Rock, AR 72202, USA G. S. Jensen NIS Labs, 1437 Esplanade, Klamath Falls, OR 97601, USA X. Wu (*) Food Research and Technology, The Hershey Company, 1025 Reese Avenue, Hershey, PA 17033, USA e-mail: [email protected] Present Address: J. Kang State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, People Republic of China
capacity (ORAC). Both phenolic acid mixtures were able to enter the cell and showed in cell antioxidant activities from the cell based antioxidant protection of erythrocytes (CAP-e) assay. Lowbush blueberry phenolic acid mixture was found to show anti-inflammatory activities by inhibiting the nuclear factor-κB (NF-κB) activation and the production of inflammatory cytokines (TNF-α and IL-6) at the high dose. Keywords Anti-inflammation . Antioxidant . Blueberries . HPLC-MS . Phenolic acid
Introduction The blueberry of the genus Vaccinium is one of the few fruits native to the North America [1]. There are two major blueberry species in the US market: V. corymbosum (highbush or cultivated blueberries) and V. angustifolium (lowbush or wild blueberries). Highbush blueberries are cultivated in almost all of the North America, while lowbush blueberries are only produced commercially in the Eastern Canada and the Northeastern US. For centuries, blueberries maintained popularity in the North America. Consumption of blueberries has increased rapidly over the past decade, largely driven by their health benefits [2, 3] . Blueberry is now one of the most commonly consumed berries in the US, ranks only second to strawberries in popularity. Blueberries were found to contain extremely high level of polyphenols and exhibit strong antioxidant activities and potential anti-inflammatory effects in vitro [4, 5] Studies on human subjects, though still limited, have reported that blueberries decreased cardiovascular risk factors in obese men and women with metabolic syndrome [6], and increased serum antioxidant status above the control level [7]. The health benefits of blueberries are likely due to the polyphenolic compounds, either directly or indirectly [8].
Plant Foods Hum Nutr
Several groups of polyphenols were found in the blueberries, including anthocyanins, proanthocyanidins, flavones, phenolic acids (PA) and stilbenes [9, 10]. They have been shown to be strong antioxidants by various in vitro assays [11–13]. Recently, high quantities of PA were detected in the gastrointestinal tract and in the animal bodies following berry consumption [14, 15]. In our previous studies, seven PA identified in the serum of rats fed 10 % lowbush blueberry diet were found to promote bone growth [16], to show potential athero-protective effects [17] and to attenuate mammosphere formation [18]. These studies implicated that PA may be important in vivo bioactive compounds in blueberries. In the past, bioactivities of PA in blueberries have been studied mostly as the extracts or fractions. Considering the fact that other polyphenolic compounds might co-exist in these extracts/fractions, it is hard to grant the observed biological effects to any single group of polyphenols. Therefore, a new approach was adopted in this study. PA were firstly analyzed and quantified in the two blueberry species by HPLC-MS/MS. Based on the analyses, the PA mixtures representing those in both species were prepared by using authentic standards according to their relevant profiles and quantities. These two PA mixtures were subjected to chemical-based oxygen radical absorbance capacity (ORAC) and cell-based antioxidant protection in erythrocytes (CAP-e) to evaluate their antioxidant activities. Numerous studies have implicated that the nuclear factor-κB (NF-κB) is an important nuclear factor that modulates the expression of immuneregulatory genes relevant in critical illness, inflammatory diseases, apoptosis, and cancer [19]. In this study, antiinflammatory effects of these two PA mixtures were investigated by measuring their inhibitory effects against NF-κB activation and the production the inflammatory cytokines TNF-α and IL-6, induced by lipopolysaccharide (LPS). We hope this approach would facilitate further understanding of the antioxidant and anti-inflammatory effects of PA in these two major blueberry species.
(sodium salt) (FL), Trolox, MeOH, DMSO, formic acid, phosphate-buffered saline (PBS), 3,4-dihydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, 1,3,4, 5-tetrahydroxycyclohexanecarboxylic acid 3-(3,4dihydroxycinnamate), 3-(3,4-dihydroxycinnamoyl) quinic acid (chlorogenic acid), salicylic acid and gallic acid were all obtained from Sigma-Aldrich (Milwaukee, WI). Plant Materials and Extraction Procedure Freeze-dried whole lowbush BB (V. angustifolium) powder and freeze-dried whole highbush BB (V. corymbosum) powder were kindly provided by Future Ceuticals (Momence, IL). The powders (4.05 g) were supersonic-extracted twice with 25 mL of mixed solvent (MeOH: H2O: acetic acid = 70: 29.5:0.5) at 50 °C for 15 min as described in our previous report [15]. After extraction, the extracts were diluted appropriately with extraction solvent and filtered with 0.22 μm syringe filter for HPLC-MS/MS analyses. HPLC-MS/MS Analysis The HPLC-MS/MS analyses were carried out using an Agilent 1100 HPLC system including an autosampler, a binary pump and a diode array detector (Agilent Technologies, Palo Alto, CA), which coupled to the 4000 Q TRAPTM mass spectrometer (Applied Biosystems, Forest City, CA). Please refer to Electronic Supplementary Materials for detailed method description. ORAC Assay The ORAC assay was conducted based on the method reported by our group [5]. Please refer to Electronic Supplementary Materials for detailed method description. CAP-e Assay
Materials and Methods Chemicals and Reagents 2,2′-Azobis(2-amidinopropane)dihydrochloride (AAPH) was purchased from Wako Chemicals (Richmond, VA). Hydroethidine fluorescent stain (HE) was obtained from Polysciences (Warrington, PA). Fetal bovine serum (FBS) was bought from Hyclone (Logan, UT). Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 culture medium, 2′,7′dichlorofluorescein diacetate (DCFDA), were obtained from Invitrogen (Carlsbad, CA). Zeocineosin and RAW-Blue cells were purchased from Invivogen (San Diego, CA). Fluorescein
CAP-e assay was carried out based on the method published by Honzel et al. [20, 21], with modification using an accelerated and more sensitive microplate-based protocol [22]. Please refer to Electronic Supplementary Materials for detailed method description. Secreted Alkaline Phosphatase (SEAP) Reporter Assay Secreted Alkaline Phosphatase (SEAP) reporter assay was performed based on a recent report from our group [17]. Please refer to Electronic Supplementary Materials for detailed method description.
Plant Foods Hum Nutr
TNF-α and IL-6 Elisa Pro-inflammatory cytokines TNF-α and IL-6 in the supernatant was determined by ELISA using Duoset ELISA kits (R&D, Minneapolis, MN) according to a recent paper from our group [17]. Please refer to Electronic Supplementary Materials for detailed method description.
is only 74.38 % of total PA in highbush blueberries (ESM Table 1). After quantification, the PA mixtures of two blueberries species were made according to the proportion and content of PA detected in the extract of two blueberries species using authentic standards. Antioxidant Activities of PA Mixtures by Chemical and Cell Based Assays
Statistical analysis The results were expressed as mean±SD (n=3). Data were subjected to ANOVA for statistical analyses and a value of p
ORIGINAL PAPER
Phenolic Acids of the Two Major Blueberry Species in the US Market and Their Antioxidant and Anti-inflammatory Activities Jie Kang & Keshari M. Thakali & Gitte S. Jensen & Xianli Wu
# Springer Science+Business Media New York 2014
Abstract Highbush (cultivated) and lowbush (wild) are the two major blueberry species in the US market. Eight phenolic acids were detected and quantified from these two species by HPLC-MS. Chlorogenic acid was found to be the predominant phenolic acid in both species, with 0.44 mg/g fresh weight in lowbush blueberries and 0.13 mg/g fresh weight in highbush blueberries. Total phenolic content in lowbush blueberries is over three times higher than that of highbush blueberries. The phenolic acid mixtures representing those in the two species were prepared by using authentic standards to assess their contribution to total antioxidant and antiinflammatory activities of the whole berries. Neither lowbush nor highbush blueberry phenolic acid mixture contributed significantly to the total antioxidant capacity of their relevant whole berries measured by oxygen radical absorbance This study was supported by USDA (6251-51000-005-02S). Electronic supplementary material The online version of this article (doi:10.1007/s11130-014-0461-6) contains supplementary material, which is available to authorized users. J. Kang : K. M. Thakali USDA Arkansas Children’s Nutrition Center, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 15 Children’s Way, Little Rock, AR 72202, USA G. S. Jensen NIS Labs, 1437 Esplanade, Klamath Falls, OR 97601, USA X. Wu (*) Food Research and Technology, The Hershey Company, 1025 Reese Avenue, Hershey, PA 17033, USA e-mail: [email protected] Present Address: J. Kang State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, People Republic of China
capacity (ORAC). Both phenolic acid mixtures were able to enter the cell and showed in cell antioxidant activities from the cell based antioxidant protection of erythrocytes (CAP-e) assay. Lowbush blueberry phenolic acid mixture was found to show anti-inflammatory activities by inhibiting the nuclear factor-κB (NF-κB) activation and the production of inflammatory cytokines (TNF-α and IL-6) at the high dose. Keywords Anti-inflammation . Antioxidant . Blueberries . HPLC-MS . Phenolic acid
Introduction The blueberry of the genus Vaccinium is one of the few fruits native to the North America [1]. There are two major blueberry species in the US market: V. corymbosum (highbush or cultivated blueberries) and V. angustifolium (lowbush or wild blueberries). Highbush blueberries are cultivated in almost all of the North America, while lowbush blueberries are only produced commercially in the Eastern Canada and the Northeastern US. For centuries, blueberries maintained popularity in the North America. Consumption of blueberries has increased rapidly over the past decade, largely driven by their health benefits [2, 3] . Blueberry is now one of the most commonly consumed berries in the US, ranks only second to strawberries in popularity. Blueberries were found to contain extremely high level of polyphenols and exhibit strong antioxidant activities and potential anti-inflammatory effects in vitro [4, 5] Studies on human subjects, though still limited, have reported that blueberries decreased cardiovascular risk factors in obese men and women with metabolic syndrome [6], and increased serum antioxidant status above the control level [7]. The health benefits of blueberries are likely due to the polyphenolic compounds, either directly or indirectly [8].
Plant Foods Hum Nutr
Several groups of polyphenols were found in the blueberries, including anthocyanins, proanthocyanidins, flavones, phenolic acids (PA) and stilbenes [9, 10]. They have been shown to be strong antioxidants by various in vitro assays [11–13]. Recently, high quantities of PA were detected in the gastrointestinal tract and in the animal bodies following berry consumption [14, 15]. In our previous studies, seven PA identified in the serum of rats fed 10 % lowbush blueberry diet were found to promote bone growth [16], to show potential athero-protective effects [17] and to attenuate mammosphere formation [18]. These studies implicated that PA may be important in vivo bioactive compounds in blueberries. In the past, bioactivities of PA in blueberries have been studied mostly as the extracts or fractions. Considering the fact that other polyphenolic compounds might co-exist in these extracts/fractions, it is hard to grant the observed biological effects to any single group of polyphenols. Therefore, a new approach was adopted in this study. PA were firstly analyzed and quantified in the two blueberry species by HPLC-MS/MS. Based on the analyses, the PA mixtures representing those in both species were prepared by using authentic standards according to their relevant profiles and quantities. These two PA mixtures were subjected to chemical-based oxygen radical absorbance capacity (ORAC) and cell-based antioxidant protection in erythrocytes (CAP-e) to evaluate their antioxidant activities. Numerous studies have implicated that the nuclear factor-κB (NF-κB) is an important nuclear factor that modulates the expression of immuneregulatory genes relevant in critical illness, inflammatory diseases, apoptosis, and cancer [19]. In this study, antiinflammatory effects of these two PA mixtures were investigated by measuring their inhibitory effects against NF-κB activation and the production the inflammatory cytokines TNF-α and IL-6, induced by lipopolysaccharide (LPS). We hope this approach would facilitate further understanding of the antioxidant and anti-inflammatory effects of PA in these two major blueberry species.
(sodium salt) (FL), Trolox, MeOH, DMSO, formic acid, phosphate-buffered saline (PBS), 3,4-dihydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, 1,3,4, 5-tetrahydroxycyclohexanecarboxylic acid 3-(3,4dihydroxycinnamate), 3-(3,4-dihydroxycinnamoyl) quinic acid (chlorogenic acid), salicylic acid and gallic acid were all obtained from Sigma-Aldrich (Milwaukee, WI). Plant Materials and Extraction Procedure Freeze-dried whole lowbush BB (V. angustifolium) powder and freeze-dried whole highbush BB (V. corymbosum) powder were kindly provided by Future Ceuticals (Momence, IL). The powders (4.05 g) were supersonic-extracted twice with 25 mL of mixed solvent (MeOH: H2O: acetic acid = 70: 29.5:0.5) at 50 °C for 15 min as described in our previous report [15]. After extraction, the extracts were diluted appropriately with extraction solvent and filtered with 0.22 μm syringe filter for HPLC-MS/MS analyses. HPLC-MS/MS Analysis The HPLC-MS/MS analyses were carried out using an Agilent 1100 HPLC system including an autosampler, a binary pump and a diode array detector (Agilent Technologies, Palo Alto, CA), which coupled to the 4000 Q TRAPTM mass spectrometer (Applied Biosystems, Forest City, CA). Please refer to Electronic Supplementary Materials for detailed method description. ORAC Assay The ORAC assay was conducted based on the method reported by our group [5]. Please refer to Electronic Supplementary Materials for detailed method description. CAP-e Assay
Materials and Methods Chemicals and Reagents 2,2′-Azobis(2-amidinopropane)dihydrochloride (AAPH) was purchased from Wako Chemicals (Richmond, VA). Hydroethidine fluorescent stain (HE) was obtained from Polysciences (Warrington, PA). Fetal bovine serum (FBS) was bought from Hyclone (Logan, UT). Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 culture medium, 2′,7′dichlorofluorescein diacetate (DCFDA), were obtained from Invitrogen (Carlsbad, CA). Zeocineosin and RAW-Blue cells were purchased from Invivogen (San Diego, CA). Fluorescein
CAP-e assay was carried out based on the method published by Honzel et al. [20, 21], with modification using an accelerated and more sensitive microplate-based protocol [22]. Please refer to Electronic Supplementary Materials for detailed method description. Secreted Alkaline Phosphatase (SEAP) Reporter Assay Secreted Alkaline Phosphatase (SEAP) reporter assay was performed based on a recent report from our group [17]. Please refer to Electronic Supplementary Materials for detailed method description.
Plant Foods Hum Nutr
TNF-α and IL-6 Elisa Pro-inflammatory cytokines TNF-α and IL-6 in the supernatant was determined by ELISA using Duoset ELISA kits (R&D, Minneapolis, MN) according to a recent paper from our group [17]. Please refer to Electronic Supplementary Materials for detailed method description.
is only 74.38 % of total PA in highbush blueberries (ESM Table 1). After quantification, the PA mixtures of two blueberries species were made according to the proportion and content of PA detected in the extract of two blueberries species using authentic standards. Antioxidant Activities of PA Mixtures by Chemical and Cell Based Assays
Statistical analysis The results were expressed as mean±SD (n=3). Data were subjected to ANOVA for statistical analyses and a value of p
