Research Article Received: 3 March 2014
Revised: 23 July 2014
Accepted article published: 8 September 2014
Published online in Wiley Online Library: 8 October 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6902
Phytochemical profile, antioxidative and anti-inflammatory potentials of Gynura bicolor DC. Che-yi Chao,a Wen-hu Liu,b Jia-jiuan Wuc and Mei-chin Yina,c* Abstract BACKGROUND: The phytochemical composition of aqueous and ethanol extracts from Gynura bicolor DC., a vegetable, was determined. Human umbilical vein endothelial (HUVE) cells were used to examine the antioxidative and anti-inflammatory potentials of these extracts at 1, 2 or 4% (v/v) against high-glucose-induced injury. RESULTS: Both aqueous and ethanol extracts contained phenolic acids, flavonoids, carotenoids and anthocyanins in the ranges 1428–1569, 1934–2175, 921–1007 and 2135–2407 mg per 100 g dry weight respectively. Both extracts were rich in quercetin, lutein, malvidin and pelargonidin. Addition of these extracts at test doses decreased reactive oxygen species formation, preserved glutathione content and retained glutathione peroxide and catalase activities in high-glucose-treated HUVE cells (P < 0.05). Treatments with these extracts at 2 and 4% lowered interleukin-6, tumor necrosis factor-alpha and prostaglandin E2 production and reduced cyclooxygenase-2 activity (P < 0.05). CONCLUSION: These findings suggest that this vegetable could be considered as a functional food and might provide antioxidative and anti-inflammatory protection. © 2014 Society of Chemical Industry Keywords: Gynura bicolor DC; anthocyanins; lutein; HUVE cell; antioxidative
INTRODUCTION
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It is reported that vegetables with dark green color are good sources of phytochemicals and may possess nutritional benefits to prevent chronic diseases.1,2 The leaf part of Gynura bicolor DC. is commonly used as a vegetable in Asian countries including Taiwan, China and Japan. The characteristic of this plant food is a reddish purple color on the abaxial side and a green color on the adaxial side (Fig. 1). Gynura bicolor is also used in folk medicine for diabetes treatment in southern China.3 Tuekpe et al.4 reported that dietary intake of G. bicolor increased urinary potassium excretion and improved blood pressure management in healthy Japanese women. The study of Li et al.5 revealed that intragastrical administration of ethyl acetate and n-butanol extracts from dried G. bicolor attenuated hyperglycemia in diabetic mice. Those previous studies implied that this vegetable was a potent functional food. However, the possible active compounds of this plant food remain unknown. Our previous study found that G. bicolor contained several triterpenoids, one group of phytochemicals, but only in minute amounts.6 The study of Teoh et al.7 indicated that the total phenolic acid content of several extracts from G. bicolor leaves was in the range 0.28–24.7 mg g−1 extract, and these extracts exhibited cytotoxic effects toward several cancer cell lines. Clearly, further study regarding the phytochemical composition of G. bicolor is required in order to enhance its future application. Based on its purple and green color, it is reasonable to hypothesize that G. bicolor contains anthocyanins, flavonoids or carotenoids. Shimizu et al.8 J Sci Food Agric 2015; 95: 1088–1093
reported that the root of G. bicolor contained anthocyanins. However, the edible part of this plant food is the leaf, not the root. Thus the major purpose of our present study was to analyze the content of several known phenolic acids, flavonoids, carotenoids and anthocyanins in aqueous and ethanol extracts from G. bicolor leaves. Endothelial cell dysfunction plays a crucial role in the pathogenesis of vascular abnormalities, including diabetes. The human umbilical vein endothelial (HUVE) cell line is widely used as an endothelial cell model to investigate the antioxidative and/or anti-inflammatory protection of plant extracts on vascular endothelial cells against high-glucose-induced damage.9,10 In our present study, this cell line was used and the protective potentials of aqueous and ethanol extracts from G. bicolor against high-glucose-induced oxidative and inflammatory injury were examined. These results could help to explain the activity or action mode of this plant food.
∗
Correspondence to: Mei-chin Yin, Department of Nutrition, China Medical University, Taichung City, Taiwan. E-mail: [email protected]
a Department of Health and Nutrition Biotechnology, Asia University, Taichung City, Taiwan b Department of Nutrition, Chung Shan Medical University, Taichung City, Taiwan c Department of Nutrition, China Medical University, Taichung City, Taiwan
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Phytochemical profile of Gynura bicolor
www.soci.org RP-C18 column was used. UV spectra were recorded from 220 to 450 nm. Calibration curves of external standards were in the range 10–200 ng mL−1 , and R2 values were higher than 0.98 (peak areas vs concentration). Determination of carotenoids The UV–visible spectrophotometry method of Craft13 was used to determine total carotenoid content, expressed as lutein equivalent. The HPLC method described by Granado et al.14 was used to measure the content of known carotenoids such as lycopene, 𝛽-carotene, lutein and zeaxanthin. Gynura bicolor is a low-lipid vegetable and our preliminary experiment detected no carotenoid esters in it, so a saponification step was not necessary. A 1 g portion of aqueous or ethanol extract was mixed with 10 mL of tetrahydrofuran/methanol (1:1 v/v) containing 0.1 mL L−1 butylated hydroxytoluene and 100 mg of MgCO3 for 1 h under dim light. This procedure was repeated three times and the organic extracts were combined. After vacuum filtration, the sample was reconstituted with 10 mL of tetrahydrofuran/ethanol (1:2 v/v), followed by filtration through a 0.22 μm nylon filter. An HPLC system consisting of a photodiode array detector (Waters, Milford, MA, USA) and a Spheri-5 chromatographic column (ODS, 5 μm, 220 mm × 4.6 mm) with a guard column (Aquapore ODS type RP-C18) was used. The mobile phase was acetonitrile/methanol (85:15 v/v). Absorbance was measured at 290 and 450 nm. Quantification was based on the standard curve of each compound.
Figure 1. Picture of Gynura bicolor.
MATERIALS AND METHODS Materials Fresh G. bicolor harvested in spring 2013 was purchased from farms in Puli Town, Nanton County, Taiwan. The voucher specimen number of this vegetable was TAI177594 according to the record at the Herbarium of College of Life Science, National Taiwan University, Taipei City, Taiwan. A 50 g portion of leaves of G. bicolor was chopped, homogenized in a Waring blender and mixed with 150 mL of sterile distilled water or 750 mL L−1 ethanol at 25 ∘ C for 12 h. After filtration through Whatman No. 1 filter paper, the aqueous or ethanol extract was further freeze-dried to a fine powder. Pure standards of phenolic acids, flavonoids, carotenoids and anthocyanins were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA).
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Cell culture HUVE cells obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1.5 g L−1 NaHCO3 , 5.5 mmol L−1 glucose, 100 mL L−1 fetal bovine serum, 100 U mL−1 penicillin and 100 U mL−1 streptomycin under 95% air/5% CO2 at 37 ∘ C. Phosphate buffer saline (PBS, pH 7.2) was used to adjust the cell number to 105 mL−1 for various experiments and analyses. HUVE cells were treated with PBS containing aqueous or ethanol extract at 1, 2 or 4% (v/v) for 12 h at 37 ∘ C. After washing with serum-free DMEM, cells were further treated with medium containing 33 mmol L−1 glucose for 24 h at 37 ∘ C. Control groups were cells treated with 5.5 mmol L−1 glucose and without any extract.
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Determination of phenolic acids and flavonoids The methods described by Sreelatha and Padma11 were used to measure total phenolic acid content, expressed as gallic acid equivalent, and total flavonoid content, expressed as quercetin equivalent. Nine known phenolic acids (caffeic, chlorogenic, cinnamic, coumaric, ellagic, ferulic, gallic, protocatechuic and rosmarinic acids) and eight known flavonoids (apigenin, epicatechin, kaempferol, luteolin, myricetin, naringenin, quercetin and rutin) were simultaneously quantified by the high-performance liquid chromatography (HPLC) method described by Sellappan et al.12 and compared with external standards of known concentrations. Briefly, 0.5 g of aqueous or ethanol extract was mixed with 5 mL of 6 mol L−1 HCl and 15 mg of ascorbic acid. Methanol was added to give a total volume of 25 mL. The sample was then refluxed at 95 ∘ C for 2 h to hydrolyze the flavonoid glycosides to aglycons, followed by filtration through a 1 μm syringe nylon filter. An HPLC system equipped with a diode array UV–visible detector and a Phenomenex Prodigy 5-m, ODS-2,
Determination of anthocyanins A pH differential method, in which absorbances at 510 and 700 nm in buffers with pH 1.0 and 4.5 were measured, was used to determine total anthocyanin content, expressed as cyanidin-3-glucoside equivalent.15 The HPLC method described by Ayranci and Erkan16 was used to analyze the content of known anthocyanin compounds including cyanidin, keracyanin, kuromanin, malvidin, pelargonidin, peonidin and petunidin. Briefly, 1 g of aqueous or ethanol extract was further extracted with 15 mL of methanol containing 10 mL L−1 formic acid, followed by shaking at 25 ∘ C for 1 h. An Agilent 1100 series HPLC instrument (Agilent Technologies, Redwood, CA, USA) equipped with a diode array detector and a Hypersil ODS C18 column (5 μm particle size, 250 mm × 4.6 mm) was used. The mobile phase consisted of 50 mL L−1 orthophosphoric acid in water (solvent A) and 50 mL L−1 orthophosphoric acid in methanol (solvent B). UV–visible spectra were recorded between 250 and 600 nm. Pure cyanidin, keracyanin, kuromanin, malvidin, pelargonidin, peonidin and petunidin were used as standards, and a calibration curve was created for each compound.
www.soci.org 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay MTT assay was performed to examine cell viability. Briefly, HUVE cells were incubated with 0.25 mg mL−1 MTT for 3 h at 37 ∘ C. The amount of MTT formazan product was determined by measuring the absorbance at 570 nm (630 nm as a reference) using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was expressed as a percentage of control groups. Analyses for glutathione (GSH), reactive oxygen species (ROS) and activity of glutathione peroxidase (GPX) and catalase (CAT) Cell homogenate was centrifuged at 3000 × g for 20 min at 4 ∘ C and the supernatant was used for these assays according to the manufacturers’ instructions. GSH content was quantified by a commercial colorimetric assay kit (OxisResearch, Portland, OR, USA). ROS level was determined using an oxidation-sensitive dye, 2′ ,7′ -dichlorofluorescein diacetate, and the fluorescence value was measured using a fluorescence microplate reader at excitation and emission wavelengths of 485 and 530 nm respectively. Relative fluorescence unit (RFU) was the difference in fluorescence values obtained at times 0 and 5 min. Results were expressed as RFU mg−1 protein. The activity (U mg−1 protein) of GPX and CAT in HUVE cells was assessed using assay kits (EMD Biosciences, San Diego, CA, USA). Protein concentration was measured using a Bio-Rad protein assay reagent. Interleukin (IL)-6, tumor necrosis factor-alpha (TNF-𝜶), prostaglandin E (PGE2 ) and cyclooxygenase (COX)-2 activity measurement After washing and homogenization, the released level of IL-6 or TNF-𝛼 in the supernatant of HUVE cells was measured using cytoscreen immunoassay kits (BioSource Int., Camarillo, CA, USA). The assay sensitivity, i.e. detection limit, was 5 pg mg−1 protein for IL-6 and 10 pg mg−1 protein for TNF-𝛼. PGE2 level and COX-2 activity were determined using commercial assay kits (Cayman Chemical Co., Ann Arbor, MI, USA). Statistical analysis The effect of each treatment was analyzed from ten different preparations (n = 10). Data were reported as mean ± standard deviation (SD) and subjected to analysis of variance. Differences among means were determined by the least significance difference test, with significance defined at P < 0.05.
RESULTS
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The content of phenolic acids and flavonoids in aqueous and ethanol extracts of G. bicolor is presented in Table 1. Total phenolic acids and total flavonoids in both extracts were in the ranges 1428–1569 and 1934–2175 mg per 100 g dry weight respectively. Apart from cinnamic acid, eight phenolic acids could be detected in both extracts in the range 20–174 mg per 100 g dry weight. Ethanol extract had higher ellagic acid content than aqueous extract. Apart from luteolin, seven flavonoids could be detected in both extracts in the range 18–269 mg per 100 g dry weight. Ethanol extract had more apigenin and quercetin, but aqueous extract had more myricetin. Total carotenoids and total anthocyanins in both extracts were in the ranges 921–1007 and 2135–2407 mg per 100 g dry weight respectively (Table 2). Lycopene and 𝛽-carotene could not be
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Table 1. Content of phenolic acids and flavonoids in Gynura bicolor aqueous extract (AE) and ethanol extract (EE) Compound (mg per 100 g dry weight) Total phenolic acids Caffeic acid Chlorogenic acid Coumaric acid Ellagic acid Ferulic acid Gallic acid Protocatechuic acid Rosmarinic acid Total flavonoids Apigenin Epicatechin Kaempferol Myricetin Naringenin Quercetin Rutin
AE 1428 ± 137a 83 ± 9a 146 ± 13a 72 ± 8a 92 ± 6a 152 ± 10a 28 ± 5a 20 ± 2a 53 ± 5a 1934 ± 108a 160 ± 14a 18 ± 2a 57 ± 7a 127 ± 9b 93 ± 9a 221 ± 16a 131 ± 11a
EE 1569 ± 95a 105 ± 8a 138 ± 15a 65 ± 4a 123 ± 7b 174 ± 12a 44 ± 6a 32 ± 5a 75 ± 6a 2175 ± 135a 203 ± 8b 24 ± 3a 80 ± 9a 87 ± 7a 85 ± 5a 269 ± 12b 156 ± 13a
Data are expressed as mean ± SD (n = 10). Means in a row without a common letter differ, P < 0.05.
Table 2. Content of carotenoids and anthocyanins in Gynura bicolor aqueous extract (AE) and ethanol extract (EE) Compound (mg per 100 g dry weight) Total carotenoids Lutein Zeaxanthin Anthocyanins Cyanidin Kuromanin Malvidin Pelargonidin Peonidin Petunidin
AE
EE
921 ± 47a 215 ± 19a 71 ± 8a 2135 ± 42a 201 ± 14a 29 ± 3a 263 ± 13a 305 ± 17a 58 ± 5a 191 ± 10b
1007 ± 65a 264 ± 22b 83 ± 12a 2407 ± 50b 186 ± 10a 36 ± 4a 311 ± 15b 285 ± 12a 73 ± 8a 152 ± 12a
Data are expressed as mean ± SD (n = 10). Means in a row without a common letter differ, P < 0.05.
detected in either aqueous or ethanol extract. The contents of leutin and zeaxanthin were in the ranges 215–264 and 71–83 mg per 100 g dry weight respectively. Ethanol extract had higher lutein content than aqueous extract. Apart from keracyanin, six anthocyanins were detected in both extracts in the range 29–311 mg per 100 g dry weight. Ethanol extract had more malvidin, but aqueous extract had more petunidin. The effects of aqueous or ethanol extract from G. bicolor on the viability of HUVE cells against high glucose are shown in Fig. 2. High glucose led to 47% viability; however, pre-treatments with aqueous or ethanol extract from G. bicolor dose-dependently enhanced cell survival (P < 0.05). As shown in Table 3, high glucose increased ROS generation, lowered GSH level and reduced GPX and CAT activities in HUVE cells (P < 0.05). However, pre-treatments with aqueous or ethanol extract from G. bicolor dose-dependently
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Figure 2. Effects of Gynura bicolor aqueous extract (AE) and ethanol extract (EE) on cell viability determined by MTT assay. HUVE cells were treated with AE or EE at 1, 2 or 4%, followed by 33 mmol L−1 glucose (G33) treatment. Cells containing neither AE nor EE and treated with 5.5 mmol L−1 glucose (G5.5) were used as control. Data are expressed as mean ± SD (n = 10). Means without a common letter differ, P < 0.05. Table 3. Effects of Gynura bicolor aqueous extract (AE) and ethanol extract (EE) on ROS (nmol mg−1 protein) and GSH (ng mg−1 protein) levels and GPX and CAT activities (U mg−1 protein) in HUVE cellsa Treatment Control (G5.5) G33 AE, 1 + G33 AE, 2 + G33 AE, 4 + G33 EE, 1 + G33 EE, 2 + G33 EE, 4 + G33
ROS
GSH
0.12 ± 0.04a 1.29 ± 0.10e 0.97 ± 0.08d 0.70 ± 0.05c 0.41 ± 0.09b 1.01 ± 0.12d 0.72 ± 0.07c 0.43 ± 0.06b
97 ± 5e 45 ± 2a 55 ± 3b 68 ± 4c 84 ± 6d 53 ± 5b 65 ± 4c 80 ± 3d
GPX 71.3 ± 2.1d 35.2 ± 0.8a 36.5 ± 1.1a 42.9 ± 1.3b 56.5 ± 1.0c 36.1 ± 0.6a 43.0 ± 0.9b 58.1 ± 1.4c
CAT 70.4 ± 1.9d 31.7 ± 1.0a 33.0 ± 0.8a 40.4 ± 1.2b 52.7 ± 1.4c 32.9 ± 0.5a 41.7 ± 0.9b 54.2 ± 1.1c
Data are expressed as mean ± SD (n = 10). Means in a column without a common letter differ, P < 0.05. a HUVE cells were treated with AE or EE at 1, 2 or 4%, followed by 33 mmol L−1 glucose (G33) treatment. Cells containing neither AE nor EE and treated with 5.5 mmol L−1 glucose (G5.5) were used as control.
decreased ROS level and preserved GSH content (P < 0.05) and at 2 and 4% retained GPX and CAT activities (P < 0.05) in high-glucose-treated HUVE cells. High glucose also increased IL-6, TNF-𝛼 and PGE2 production and raised COX-2 activity (P < 0.05) (Table 4). Pre-treatments with aqueous or ethanol extract of G. bicolor at 2 and 4% lowered IL-6, TNF-𝛼 and PGE2 formation and reduced COX-2 activity (P < 0.05).
DISCUSSION
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The data of this study revealed that the leaves, an edible part, of G. bicolor contained eight phenolic acids, seven flavonoids, two carotenoids and six anthocyanins, indicating that G. bicolor was rich in at least four groups of phytochemicals. Furthermore, the presence of anthocyanins such as pelargonidin, cyanidin, kuromanin, peonidin, petunidin and malvidin in aqueous and ethanol extracts of this vegetable explained its reddish purple appearance. Teoh et al.7 reported that the content of total phenolic acids in G. bicolor aqueous extract was 28 mg per 100 g extract. However, our data indicated that the content of total phenolic acids in G.
bicolor aqueous extract was 1428 mg per 100 g dry weight. The discrepancy between the two studies may be due to the different cultural environment and season for this vegetable. In the present study the addition of G. bicolor aqueous and ethanol extracts at 2 and 4% effectively protected HUVE cells against subsequent high-glucose-induced oxidative and inflammatory stress, which in turn enhanced cell survival. Antidiabetic effects of ferulic acid, quercetin, lutein, apigenin and pelargonidin in mice or rats have been reported;17 – 21 the authors indicated that these compounds exhibited antidiabetic effects through their antioxidative and/or anti-inflammatory activities. Our data revealed that the content of these five compounds in aqueous and ethanol extracts from G. bicolor was in the range 152–305 mg per 100 g dry weight. Thus the antioxidative and anti-inflammatory protection from these extracts for HUVE cells that we observed could be substantially ascribed to the presence of these five agents. In addition, Bognar et al.22 reported that malvidin provided antioxidative and/or anti-inflammatory activities for RAW264.7 macrophages. In our study, malvidin content was higher than 200 mg per 100 g dry weight in both aqueous and ethanol
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Table 4. Effects of Gynura bicolor aqueous extract (AE) and ethanol extract (EE) on IL-6 (pg mg−1 protein), TNF-𝛼 (pg mg−1 protein) and PGE2 (pg g−1 protein) levels and COX-2 activity (U mg−1 protein) in HUVE cellsa Treatment Control (G5.5) G33 AE, 1 + G33 AE, 2 + G33 AE, 4 + G33 EE, 1 + G33 EE, 2 + G33 EE, 4 + G33
IL-6 11 ± 2a 89 ± 9d 83 ± 6d 67 ± 5c 46 ± 5b 81 ± 8d 64 ± 6c 41 ± 4b
TNF-𝛼
PGE2
16 ± 3a 102 ± 10d 96 ± 8d 78 ± 5c 50 ± 4b 93 ± 6d 76 ± 5c 47 ± 6b
COX-2
87 ± 8a 314 ± 18d 302 ± 15d 257 ± 12c 182 ± 10b 307 ± 14d 261 ± 13c 175 ± 9b
0.21 ± 0.08a 1.17 ± 0.11d 1.13 ± 0.07d 0.90 ± 0.05c 0.61 ± 0.08b 1.15 ± 0.13d 0.89 ± 0.10c 0.59 ± 0.06b
Data are expressed as mean ± SD (n = 10). Means in a column without a common letter differ, P < 0.05. a HUVE cells were treated with AE or EE at 1, 2 or 4%, followed by 33 mmol L−1 glucose (G33) treatment. Cells containing neither AE nor EE and treated with 5.5 mmol L−1 glucose (G5.5) were used as control.
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extracts from G. bicolor. Since malvidin was also a predominant component in G. bicolor aqueous and ethanol extracts, the antioxidative and anti-inflammatory contribution from this compound for HUVE cells against high glucose could not be ignored. These findings suggest that G. bicolor is rich in these active compounds and might be considered as a potent antidiabetic functional food. Although ethanol extract contained more total flavonoids and total anthocyanins than aqueous extract, it did not exhibit greater antioxidative and anti-inflammatory effects in HUVE cells against high-glucose-induced injury. Obviously, other unknown compounds and/or interactions between these compounds were involved in the protective activities of these extracts for HUVE cells. The study of Teoh et al.7 indicated that G. bicolor aqueous extract could scavenge free radicals such as 1,1-diphenyl-2-picrylhydrazyl. Our study further found that 1% aqueous or ethanol extract markedly decreased ROS production and preserved GSH content but did not affect GPX and CAT activities in high-glucose-treated HUVE cells. It is highly possible that the phytochemicals present in 1% extracts could scavenge free radicals such as ROS via their non-enzymatic antioxidant action, which consequently spared GSH and protected HUVE cells. In addition, we noted that these extracts at 2 and 4% were able to affect GPX, CAT and COX-2 activities in HUVE cells. It seems that the components present in these extracts could penetrate cells and mediate these enzymes. These results implied that G. bicolor extracts could exhibit enzymatic antioxidative and anti-inflammatory actions against high-glucose-induced damage in HUVE cells. It has been reported that lutein and zeaxanthin could protect eyes against vision loss and prevent the occurrence of ocular diseases including glaucoma, diabetic retinopathy and cataract.23,24 These carotenoids exerted these effects by blocking blue light damage and quenching oxygen free radicals such as singlet oxygen.25 Our present study found that both aqueous and ethanol extracts from G. bicolor contained lutein and zeaxanthin. Clearly, G. bicolor is a good source of these two carotenoids. These results suggest that this vegetable may benefit eye health. In addition, G. bicolor also contained substantial levels of malvidin and pelargonidin. It has been reported that malvidin and pelargonidin could offer neuroprotection against amyloid 𝛽 protein-induced damage or delay the progression of parkinsonism.26,27 Therefore this vegetable might also offer nutritional benefits to prevent the development of neurodegenerative disorders.
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In conclusion, G. bicolor DC. was found to be rich in phytochemicals, including phenolic acids, flavonoids, carotenoids and anthocyanins. Both aqueous and ethanol extracts from this plant food markedly protected HUVE cells against high-glucose-induced oxidative and inflammatory injury. These findings suggest that this vegetable could be considered as a functional food and might provide antioxidative and anti-inflammatory protection.
ACKNOWLEDGEMENT This study was partially supported by a grant from the Ministry of Science and Technology, Taipei City, Taiwan (NSC 101-2320-B-468-001).
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11 Sreelatha S and Padma PR, Antioxidant activity and total phenolic content of Moringa oleifera leaves in two stages of maturity. Plant Foods Hum Nutr 64:303–311 (2009). 12 Sellappan S, Akoh CC and Krewer G, Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. J Agric Food Chem 50:2432–2438 (2002). 13 Craft NE, Relative solubility, stability, and absorptivity of lutein and 𝛽-carotene in organic solvents. J Agric Food Chem 40:431–434 (1992). 14 Granado F, Olmedilla B, Blanco I and Rojas-Hidalgo E, Carotenoid composition in raw and cooked Spanish vegetables. J Agric Food Chem 40:2135–2140 (1992). 15 Cheng GW and Breen PJ, Activity of phenylalanine ammonialyase (PAL) and concentrations of anthocyanins and phenolics in developing strawberry fruit. J Am Soc Hort Sci 117:946–950 (1991). 16 Ayranci E and Erkan N, Radical scavenging capacity of methanolic Phillyrea latifolia L. extract: anthocyanin and phenolic acids composition of fruits. Molecules 18:1798–1810 (2013). 17 Muriach M, Bosch-Morell F, Alexander G, Blomhoff R, Barcia J, Arnal E, et al., Lutein effect on retina and hippocampus of diabetic mice. Free Radic Biol Med 41:979–984 (2006). 18 Srinivasan M, Sudheer AR and Menon VP, Ferulic acid: therapeutic potential through its antioxidant property. J Clin Biochem Nutr 40:92–100 (2007). 19 Mirshekar M, Roghani M, Khalili M, Baluchnejadmojarad T and Arab Moazzen S, Chronic oral pelargonidin alleviates streptozotocin-induced diabetic neuropathic hyperalgesia in rat: involvement of oxidative stress. Iran Biomed J 14:33–39 (2010).
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Revised: 23 July 2014
Accepted article published: 8 September 2014
Published online in Wiley Online Library: 8 October 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6902
Phytochemical profile, antioxidative and anti-inflammatory potentials of Gynura bicolor DC. Che-yi Chao,a Wen-hu Liu,b Jia-jiuan Wuc and Mei-chin Yina,c* Abstract BACKGROUND: The phytochemical composition of aqueous and ethanol extracts from Gynura bicolor DC., a vegetable, was determined. Human umbilical vein endothelial (HUVE) cells were used to examine the antioxidative and anti-inflammatory potentials of these extracts at 1, 2 or 4% (v/v) against high-glucose-induced injury. RESULTS: Both aqueous and ethanol extracts contained phenolic acids, flavonoids, carotenoids and anthocyanins in the ranges 1428–1569, 1934–2175, 921–1007 and 2135–2407 mg per 100 g dry weight respectively. Both extracts were rich in quercetin, lutein, malvidin and pelargonidin. Addition of these extracts at test doses decreased reactive oxygen species formation, preserved glutathione content and retained glutathione peroxide and catalase activities in high-glucose-treated HUVE cells (P < 0.05). Treatments with these extracts at 2 and 4% lowered interleukin-6, tumor necrosis factor-alpha and prostaglandin E2 production and reduced cyclooxygenase-2 activity (P < 0.05). CONCLUSION: These findings suggest that this vegetable could be considered as a functional food and might provide antioxidative and anti-inflammatory protection. © 2014 Society of Chemical Industry Keywords: Gynura bicolor DC; anthocyanins; lutein; HUVE cell; antioxidative
INTRODUCTION
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It is reported that vegetables with dark green color are good sources of phytochemicals and may possess nutritional benefits to prevent chronic diseases.1,2 The leaf part of Gynura bicolor DC. is commonly used as a vegetable in Asian countries including Taiwan, China and Japan. The characteristic of this plant food is a reddish purple color on the abaxial side and a green color on the adaxial side (Fig. 1). Gynura bicolor is also used in folk medicine for diabetes treatment in southern China.3 Tuekpe et al.4 reported that dietary intake of G. bicolor increased urinary potassium excretion and improved blood pressure management in healthy Japanese women. The study of Li et al.5 revealed that intragastrical administration of ethyl acetate and n-butanol extracts from dried G. bicolor attenuated hyperglycemia in diabetic mice. Those previous studies implied that this vegetable was a potent functional food. However, the possible active compounds of this plant food remain unknown. Our previous study found that G. bicolor contained several triterpenoids, one group of phytochemicals, but only in minute amounts.6 The study of Teoh et al.7 indicated that the total phenolic acid content of several extracts from G. bicolor leaves was in the range 0.28–24.7 mg g−1 extract, and these extracts exhibited cytotoxic effects toward several cancer cell lines. Clearly, further study regarding the phytochemical composition of G. bicolor is required in order to enhance its future application. Based on its purple and green color, it is reasonable to hypothesize that G. bicolor contains anthocyanins, flavonoids or carotenoids. Shimizu et al.8 J Sci Food Agric 2015; 95: 1088–1093
reported that the root of G. bicolor contained anthocyanins. However, the edible part of this plant food is the leaf, not the root. Thus the major purpose of our present study was to analyze the content of several known phenolic acids, flavonoids, carotenoids and anthocyanins in aqueous and ethanol extracts from G. bicolor leaves. Endothelial cell dysfunction plays a crucial role in the pathogenesis of vascular abnormalities, including diabetes. The human umbilical vein endothelial (HUVE) cell line is widely used as an endothelial cell model to investigate the antioxidative and/or anti-inflammatory protection of plant extracts on vascular endothelial cells against high-glucose-induced damage.9,10 In our present study, this cell line was used and the protective potentials of aqueous and ethanol extracts from G. bicolor against high-glucose-induced oxidative and inflammatory injury were examined. These results could help to explain the activity or action mode of this plant food.
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Correspondence to: Mei-chin Yin, Department of Nutrition, China Medical University, Taichung City, Taiwan. E-mail: [email protected]
a Department of Health and Nutrition Biotechnology, Asia University, Taichung City, Taiwan b Department of Nutrition, Chung Shan Medical University, Taichung City, Taiwan c Department of Nutrition, China Medical University, Taichung City, Taiwan
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Phytochemical profile of Gynura bicolor
www.soci.org RP-C18 column was used. UV spectra were recorded from 220 to 450 nm. Calibration curves of external standards were in the range 10–200 ng mL−1 , and R2 values were higher than 0.98 (peak areas vs concentration). Determination of carotenoids The UV–visible spectrophotometry method of Craft13 was used to determine total carotenoid content, expressed as lutein equivalent. The HPLC method described by Granado et al.14 was used to measure the content of known carotenoids such as lycopene, 𝛽-carotene, lutein and zeaxanthin. Gynura bicolor is a low-lipid vegetable and our preliminary experiment detected no carotenoid esters in it, so a saponification step was not necessary. A 1 g portion of aqueous or ethanol extract was mixed with 10 mL of tetrahydrofuran/methanol (1:1 v/v) containing 0.1 mL L−1 butylated hydroxytoluene and 100 mg of MgCO3 for 1 h under dim light. This procedure was repeated three times and the organic extracts were combined. After vacuum filtration, the sample was reconstituted with 10 mL of tetrahydrofuran/ethanol (1:2 v/v), followed by filtration through a 0.22 μm nylon filter. An HPLC system consisting of a photodiode array detector (Waters, Milford, MA, USA) and a Spheri-5 chromatographic column (ODS, 5 μm, 220 mm × 4.6 mm) with a guard column (Aquapore ODS type RP-C18) was used. The mobile phase was acetonitrile/methanol (85:15 v/v). Absorbance was measured at 290 and 450 nm. Quantification was based on the standard curve of each compound.
Figure 1. Picture of Gynura bicolor.
MATERIALS AND METHODS Materials Fresh G. bicolor harvested in spring 2013 was purchased from farms in Puli Town, Nanton County, Taiwan. The voucher specimen number of this vegetable was TAI177594 according to the record at the Herbarium of College of Life Science, National Taiwan University, Taipei City, Taiwan. A 50 g portion of leaves of G. bicolor was chopped, homogenized in a Waring blender and mixed with 150 mL of sterile distilled water or 750 mL L−1 ethanol at 25 ∘ C for 12 h. After filtration through Whatman No. 1 filter paper, the aqueous or ethanol extract was further freeze-dried to a fine powder. Pure standards of phenolic acids, flavonoids, carotenoids and anthocyanins were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA).
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Cell culture HUVE cells obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1.5 g L−1 NaHCO3 , 5.5 mmol L−1 glucose, 100 mL L−1 fetal bovine serum, 100 U mL−1 penicillin and 100 U mL−1 streptomycin under 95% air/5% CO2 at 37 ∘ C. Phosphate buffer saline (PBS, pH 7.2) was used to adjust the cell number to 105 mL−1 for various experiments and analyses. HUVE cells were treated with PBS containing aqueous or ethanol extract at 1, 2 or 4% (v/v) for 12 h at 37 ∘ C. After washing with serum-free DMEM, cells were further treated with medium containing 33 mmol L−1 glucose for 24 h at 37 ∘ C. Control groups were cells treated with 5.5 mmol L−1 glucose and without any extract.
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Determination of phenolic acids and flavonoids The methods described by Sreelatha and Padma11 were used to measure total phenolic acid content, expressed as gallic acid equivalent, and total flavonoid content, expressed as quercetin equivalent. Nine known phenolic acids (caffeic, chlorogenic, cinnamic, coumaric, ellagic, ferulic, gallic, protocatechuic and rosmarinic acids) and eight known flavonoids (apigenin, epicatechin, kaempferol, luteolin, myricetin, naringenin, quercetin and rutin) were simultaneously quantified by the high-performance liquid chromatography (HPLC) method described by Sellappan et al.12 and compared with external standards of known concentrations. Briefly, 0.5 g of aqueous or ethanol extract was mixed with 5 mL of 6 mol L−1 HCl and 15 mg of ascorbic acid. Methanol was added to give a total volume of 25 mL. The sample was then refluxed at 95 ∘ C for 2 h to hydrolyze the flavonoid glycosides to aglycons, followed by filtration through a 1 μm syringe nylon filter. An HPLC system equipped with a diode array UV–visible detector and a Phenomenex Prodigy 5-m, ODS-2,
Determination of anthocyanins A pH differential method, in which absorbances at 510 and 700 nm in buffers with pH 1.0 and 4.5 were measured, was used to determine total anthocyanin content, expressed as cyanidin-3-glucoside equivalent.15 The HPLC method described by Ayranci and Erkan16 was used to analyze the content of known anthocyanin compounds including cyanidin, keracyanin, kuromanin, malvidin, pelargonidin, peonidin and petunidin. Briefly, 1 g of aqueous or ethanol extract was further extracted with 15 mL of methanol containing 10 mL L−1 formic acid, followed by shaking at 25 ∘ C for 1 h. An Agilent 1100 series HPLC instrument (Agilent Technologies, Redwood, CA, USA) equipped with a diode array detector and a Hypersil ODS C18 column (5 μm particle size, 250 mm × 4.6 mm) was used. The mobile phase consisted of 50 mL L−1 orthophosphoric acid in water (solvent A) and 50 mL L−1 orthophosphoric acid in methanol (solvent B). UV–visible spectra were recorded between 250 and 600 nm. Pure cyanidin, keracyanin, kuromanin, malvidin, pelargonidin, peonidin and petunidin were used as standards, and a calibration curve was created for each compound.
www.soci.org 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay MTT assay was performed to examine cell viability. Briefly, HUVE cells were incubated with 0.25 mg mL−1 MTT for 3 h at 37 ∘ C. The amount of MTT formazan product was determined by measuring the absorbance at 570 nm (630 nm as a reference) using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability was expressed as a percentage of control groups. Analyses for glutathione (GSH), reactive oxygen species (ROS) and activity of glutathione peroxidase (GPX) and catalase (CAT) Cell homogenate was centrifuged at 3000 × g for 20 min at 4 ∘ C and the supernatant was used for these assays according to the manufacturers’ instructions. GSH content was quantified by a commercial colorimetric assay kit (OxisResearch, Portland, OR, USA). ROS level was determined using an oxidation-sensitive dye, 2′ ,7′ -dichlorofluorescein diacetate, and the fluorescence value was measured using a fluorescence microplate reader at excitation and emission wavelengths of 485 and 530 nm respectively. Relative fluorescence unit (RFU) was the difference in fluorescence values obtained at times 0 and 5 min. Results were expressed as RFU mg−1 protein. The activity (U mg−1 protein) of GPX and CAT in HUVE cells was assessed using assay kits (EMD Biosciences, San Diego, CA, USA). Protein concentration was measured using a Bio-Rad protein assay reagent. Interleukin (IL)-6, tumor necrosis factor-alpha (TNF-𝜶), prostaglandin E (PGE2 ) and cyclooxygenase (COX)-2 activity measurement After washing and homogenization, the released level of IL-6 or TNF-𝛼 in the supernatant of HUVE cells was measured using cytoscreen immunoassay kits (BioSource Int., Camarillo, CA, USA). The assay sensitivity, i.e. detection limit, was 5 pg mg−1 protein for IL-6 and 10 pg mg−1 protein for TNF-𝛼. PGE2 level and COX-2 activity were determined using commercial assay kits (Cayman Chemical Co., Ann Arbor, MI, USA). Statistical analysis The effect of each treatment was analyzed from ten different preparations (n = 10). Data were reported as mean ± standard deviation (SD) and subjected to analysis of variance. Differences among means were determined by the least significance difference test, with significance defined at P < 0.05.
RESULTS
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The content of phenolic acids and flavonoids in aqueous and ethanol extracts of G. bicolor is presented in Table 1. Total phenolic acids and total flavonoids in both extracts were in the ranges 1428–1569 and 1934–2175 mg per 100 g dry weight respectively. Apart from cinnamic acid, eight phenolic acids could be detected in both extracts in the range 20–174 mg per 100 g dry weight. Ethanol extract had higher ellagic acid content than aqueous extract. Apart from luteolin, seven flavonoids could be detected in both extracts in the range 18–269 mg per 100 g dry weight. Ethanol extract had more apigenin and quercetin, but aqueous extract had more myricetin. Total carotenoids and total anthocyanins in both extracts were in the ranges 921–1007 and 2135–2407 mg per 100 g dry weight respectively (Table 2). Lycopene and 𝛽-carotene could not be
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Table 1. Content of phenolic acids and flavonoids in Gynura bicolor aqueous extract (AE) and ethanol extract (EE) Compound (mg per 100 g dry weight) Total phenolic acids Caffeic acid Chlorogenic acid Coumaric acid Ellagic acid Ferulic acid Gallic acid Protocatechuic acid Rosmarinic acid Total flavonoids Apigenin Epicatechin Kaempferol Myricetin Naringenin Quercetin Rutin
AE 1428 ± 137a 83 ± 9a 146 ± 13a 72 ± 8a 92 ± 6a 152 ± 10a 28 ± 5a 20 ± 2a 53 ± 5a 1934 ± 108a 160 ± 14a 18 ± 2a 57 ± 7a 127 ± 9b 93 ± 9a 221 ± 16a 131 ± 11a
EE 1569 ± 95a 105 ± 8a 138 ± 15a 65 ± 4a 123 ± 7b 174 ± 12a 44 ± 6a 32 ± 5a 75 ± 6a 2175 ± 135a 203 ± 8b 24 ± 3a 80 ± 9a 87 ± 7a 85 ± 5a 269 ± 12b 156 ± 13a
Data are expressed as mean ± SD (n = 10). Means in a row without a common letter differ, P < 0.05.
Table 2. Content of carotenoids and anthocyanins in Gynura bicolor aqueous extract (AE) and ethanol extract (EE) Compound (mg per 100 g dry weight) Total carotenoids Lutein Zeaxanthin Anthocyanins Cyanidin Kuromanin Malvidin Pelargonidin Peonidin Petunidin
AE
EE
921 ± 47a 215 ± 19a 71 ± 8a 2135 ± 42a 201 ± 14a 29 ± 3a 263 ± 13a 305 ± 17a 58 ± 5a 191 ± 10b
1007 ± 65a 264 ± 22b 83 ± 12a 2407 ± 50b 186 ± 10a 36 ± 4a 311 ± 15b 285 ± 12a 73 ± 8a 152 ± 12a
Data are expressed as mean ± SD (n = 10). Means in a row without a common letter differ, P < 0.05.
detected in either aqueous or ethanol extract. The contents of leutin and zeaxanthin were in the ranges 215–264 and 71–83 mg per 100 g dry weight respectively. Ethanol extract had higher lutein content than aqueous extract. Apart from keracyanin, six anthocyanins were detected in both extracts in the range 29–311 mg per 100 g dry weight. Ethanol extract had more malvidin, but aqueous extract had more petunidin. The effects of aqueous or ethanol extract from G. bicolor on the viability of HUVE cells against high glucose are shown in Fig. 2. High glucose led to 47% viability; however, pre-treatments with aqueous or ethanol extract from G. bicolor dose-dependently enhanced cell survival (P < 0.05). As shown in Table 3, high glucose increased ROS generation, lowered GSH level and reduced GPX and CAT activities in HUVE cells (P < 0.05). However, pre-treatments with aqueous or ethanol extract from G. bicolor dose-dependently
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J Sci Food Agric 2015; 95: 1088–1093
Phytochemical profile of Gynura bicolor
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Figure 2. Effects of Gynura bicolor aqueous extract (AE) and ethanol extract (EE) on cell viability determined by MTT assay. HUVE cells were treated with AE or EE at 1, 2 or 4%, followed by 33 mmol L−1 glucose (G33) treatment. Cells containing neither AE nor EE and treated with 5.5 mmol L−1 glucose (G5.5) were used as control. Data are expressed as mean ± SD (n = 10). Means without a common letter differ, P < 0.05. Table 3. Effects of Gynura bicolor aqueous extract (AE) and ethanol extract (EE) on ROS (nmol mg−1 protein) and GSH (ng mg−1 protein) levels and GPX and CAT activities (U mg−1 protein) in HUVE cellsa Treatment Control (G5.5) G33 AE, 1 + G33 AE, 2 + G33 AE, 4 + G33 EE, 1 + G33 EE, 2 + G33 EE, 4 + G33
ROS
GSH
0.12 ± 0.04a 1.29 ± 0.10e 0.97 ± 0.08d 0.70 ± 0.05c 0.41 ± 0.09b 1.01 ± 0.12d 0.72 ± 0.07c 0.43 ± 0.06b
97 ± 5e 45 ± 2a 55 ± 3b 68 ± 4c 84 ± 6d 53 ± 5b 65 ± 4c 80 ± 3d
GPX 71.3 ± 2.1d 35.2 ± 0.8a 36.5 ± 1.1a 42.9 ± 1.3b 56.5 ± 1.0c 36.1 ± 0.6a 43.0 ± 0.9b 58.1 ± 1.4c
CAT 70.4 ± 1.9d 31.7 ± 1.0a 33.0 ± 0.8a 40.4 ± 1.2b 52.7 ± 1.4c 32.9 ± 0.5a 41.7 ± 0.9b 54.2 ± 1.1c
Data are expressed as mean ± SD (n = 10). Means in a column without a common letter differ, P < 0.05. a HUVE cells were treated with AE or EE at 1, 2 or 4%, followed by 33 mmol L−1 glucose (G33) treatment. Cells containing neither AE nor EE and treated with 5.5 mmol L−1 glucose (G5.5) were used as control.
decreased ROS level and preserved GSH content (P < 0.05) and at 2 and 4% retained GPX and CAT activities (P < 0.05) in high-glucose-treated HUVE cells. High glucose also increased IL-6, TNF-𝛼 and PGE2 production and raised COX-2 activity (P < 0.05) (Table 4). Pre-treatments with aqueous or ethanol extract of G. bicolor at 2 and 4% lowered IL-6, TNF-𝛼 and PGE2 formation and reduced COX-2 activity (P < 0.05).
DISCUSSION
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The data of this study revealed that the leaves, an edible part, of G. bicolor contained eight phenolic acids, seven flavonoids, two carotenoids and six anthocyanins, indicating that G. bicolor was rich in at least four groups of phytochemicals. Furthermore, the presence of anthocyanins such as pelargonidin, cyanidin, kuromanin, peonidin, petunidin and malvidin in aqueous and ethanol extracts of this vegetable explained its reddish purple appearance. Teoh et al.7 reported that the content of total phenolic acids in G. bicolor aqueous extract was 28 mg per 100 g extract. However, our data indicated that the content of total phenolic acids in G.
bicolor aqueous extract was 1428 mg per 100 g dry weight. The discrepancy between the two studies may be due to the different cultural environment and season for this vegetable. In the present study the addition of G. bicolor aqueous and ethanol extracts at 2 and 4% effectively protected HUVE cells against subsequent high-glucose-induced oxidative and inflammatory stress, which in turn enhanced cell survival. Antidiabetic effects of ferulic acid, quercetin, lutein, apigenin and pelargonidin in mice or rats have been reported;17 – 21 the authors indicated that these compounds exhibited antidiabetic effects through their antioxidative and/or anti-inflammatory activities. Our data revealed that the content of these five compounds in aqueous and ethanol extracts from G. bicolor was in the range 152–305 mg per 100 g dry weight. Thus the antioxidative and anti-inflammatory protection from these extracts for HUVE cells that we observed could be substantially ascribed to the presence of these five agents. In addition, Bognar et al.22 reported that malvidin provided antioxidative and/or anti-inflammatory activities for RAW264.7 macrophages. In our study, malvidin content was higher than 200 mg per 100 g dry weight in both aqueous and ethanol
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Table 4. Effects of Gynura bicolor aqueous extract (AE) and ethanol extract (EE) on IL-6 (pg mg−1 protein), TNF-𝛼 (pg mg−1 protein) and PGE2 (pg g−1 protein) levels and COX-2 activity (U mg−1 protein) in HUVE cellsa Treatment Control (G5.5) G33 AE, 1 + G33 AE, 2 + G33 AE, 4 + G33 EE, 1 + G33 EE, 2 + G33 EE, 4 + G33
IL-6 11 ± 2a 89 ± 9d 83 ± 6d 67 ± 5c 46 ± 5b 81 ± 8d 64 ± 6c 41 ± 4b
TNF-𝛼
PGE2
16 ± 3a 102 ± 10d 96 ± 8d 78 ± 5c 50 ± 4b 93 ± 6d 76 ± 5c 47 ± 6b
COX-2
87 ± 8a 314 ± 18d 302 ± 15d 257 ± 12c 182 ± 10b 307 ± 14d 261 ± 13c 175 ± 9b
0.21 ± 0.08a 1.17 ± 0.11d 1.13 ± 0.07d 0.90 ± 0.05c 0.61 ± 0.08b 1.15 ± 0.13d 0.89 ± 0.10c 0.59 ± 0.06b
Data are expressed as mean ± SD (n = 10). Means in a column without a common letter differ, P < 0.05. a HUVE cells were treated with AE or EE at 1, 2 or 4%, followed by 33 mmol L−1 glucose (G33) treatment. Cells containing neither AE nor EE and treated with 5.5 mmol L−1 glucose (G5.5) were used as control.
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extracts from G. bicolor. Since malvidin was also a predominant component in G. bicolor aqueous and ethanol extracts, the antioxidative and anti-inflammatory contribution from this compound for HUVE cells against high glucose could not be ignored. These findings suggest that G. bicolor is rich in these active compounds and might be considered as a potent antidiabetic functional food. Although ethanol extract contained more total flavonoids and total anthocyanins than aqueous extract, it did not exhibit greater antioxidative and anti-inflammatory effects in HUVE cells against high-glucose-induced injury. Obviously, other unknown compounds and/or interactions between these compounds were involved in the protective activities of these extracts for HUVE cells. The study of Teoh et al.7 indicated that G. bicolor aqueous extract could scavenge free radicals such as 1,1-diphenyl-2-picrylhydrazyl. Our study further found that 1% aqueous or ethanol extract markedly decreased ROS production and preserved GSH content but did not affect GPX and CAT activities in high-glucose-treated HUVE cells. It is highly possible that the phytochemicals present in 1% extracts could scavenge free radicals such as ROS via their non-enzymatic antioxidant action, which consequently spared GSH and protected HUVE cells. In addition, we noted that these extracts at 2 and 4% were able to affect GPX, CAT and COX-2 activities in HUVE cells. It seems that the components present in these extracts could penetrate cells and mediate these enzymes. These results implied that G. bicolor extracts could exhibit enzymatic antioxidative and anti-inflammatory actions against high-glucose-induced damage in HUVE cells. It has been reported that lutein and zeaxanthin could protect eyes against vision loss and prevent the occurrence of ocular diseases including glaucoma, diabetic retinopathy and cataract.23,24 These carotenoids exerted these effects by blocking blue light damage and quenching oxygen free radicals such as singlet oxygen.25 Our present study found that both aqueous and ethanol extracts from G. bicolor contained lutein and zeaxanthin. Clearly, G. bicolor is a good source of these two carotenoids. These results suggest that this vegetable may benefit eye health. In addition, G. bicolor also contained substantial levels of malvidin and pelargonidin. It has been reported that malvidin and pelargonidin could offer neuroprotection against amyloid 𝛽 protein-induced damage or delay the progression of parkinsonism.26,27 Therefore this vegetable might also offer nutritional benefits to prevent the development of neurodegenerative disorders.
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In conclusion, G. bicolor DC. was found to be rich in phytochemicals, including phenolic acids, flavonoids, carotenoids and anthocyanins. Both aqueous and ethanol extracts from this plant food markedly protected HUVE cells against high-glucose-induced oxidative and inflammatory injury. These findings suggest that this vegetable could be considered as a functional food and might provide antioxidative and anti-inflammatory protection.
ACKNOWLEDGEMENT This study was partially supported by a grant from the Ministry of Science and Technology, Taipei City, Taiwan (NSC 101-2320-B-468-001).
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