Biosci. Biotechnol. Biochem., 68 (12), 2616–2618, 2004
Anti-Oxidative Compounds in Barley Tea Hideo E TOH,1; y Kazushi M URAKAMI,1 Tokiyasu Y OGOH,1 Hajime I SHIKAWA,2 Yoshiyasu F UKUYAMA,3 and Hitoshi T ANAKA4 1
Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan Living Environmental Division, Aichi Environmental Research Center, 7-6 Nagare, Tsuji-machi, Kita, Nagoya 462-0032, Japan 3 Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan 4 Faculty of Pharmacy, Meijo University, Yagotoyama, Tempaku, Nagoya 468-8503, Japan 2
Received May 25, 2004; Accepted August 17, 2004
Five phenolic compounds, p-hydroxyacetophenone, 5,7-dihydroxychromone, naringenin, quercetin, and isoamericanol A, were found ﬁrst time in the barley tea, together with the known compounds, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, and p-coumaric acid. The antioxidative properties were evaluated by measuring their peroxynitrite-scavenging activities. Among these compounds, 3,4-dihydroxybenzaldehyde, p-coumaric acid, quercetin, and isoamericanol A showed stronger activities than that of BHT (butylated hydroxytoluene) at 400 M. Key words:
barley tea; peroxynitrite; antioxidative compound; quercetin; isoamericanol A
Our past work has established the useful eﬀects of widely consumed food products on human health. We have reported the isolation of several anti-oxidants from some food products as konnyaku and tomato puree.1,2) There have been a few studies on the chemical components of barley tea which is a well-known drink during the summer season in Japan.3) We now describe the presence of p-hydroxyacetophenone, 5,7-dihydroxychromone, naringenin, quercetin, and isoamericanol A in barley tea. All the isolated compounds were evaluated for their peroxynitrite-scavenging activities. Extraction and isolation of phenolic compounds from barley tea. Barley tea (1 kg) was extracted with 3-liter of MeOH, and then 1.25-liter of H2 O was added to the extract. The aqueous extract was successively extracted with 3-liter each of hexane, CHCl3 , and EtOAc. A portion of the EtOAc extract (2.7 g) was chromatographed in a silica-gel column (8 70 cm) eluted with hexane–EtOAc (1:1) (fractions 1–5). Fraction 1 was puriﬁed by HPLC (ODS, 10 250 and 20 250 mm), eluting with 30–60% MeOH, to provide compouds 1 y
Fig. 1. Isoamericanol A and Its Derivatives.
(13.7 mg) and 2 (4.6 mg). Fraction 2 was chromatographed in an ODS column, using 20–50% and then 50– 100% MeOH gradient elution, to give compounds 3 (5.7 mg) and 4 (1.0 mg). Fraction 3 was chromatographed in the column, eluting with 5–50% MeOH, to give compounds 5 (2.6 mg), 6 (4.2 mg), 7 (7.5 mg), and 8 (103 mg). Fractions 4 and 5 were ﬁnally respectively chromatographed, eluting with 20–60% MeOH to give compound 9 (23.2 mg) and with 5–50% MeOH to give compound 10 (1.5 mg). Anti-oxidative assay. Each sample solution (1 ml with ﬁnal concentrations of 400, 200 and 100 M in MeOH) was added to 1 ml of a 100 mM phosphate buﬀer solution (pH 6.4) containing 2 mM tyrosine, and then mixed with an Fe(III)–EDTA solution to make a ﬁnal concentration
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Anti-Oxidative Compounds in Barley Tea
of 20 M. This mixture was added to a peroxynitrite solution (1 mM ﬁnal concentration) that had been prepared according to the method in the literature4) and kept at room temperature for 5 min. The reaction mixture was analyzed three times by HPLC, using the detection of 3-nitrotyrosine at 200–500 nm absorbance. The same procedure was used for BHT (butylated hydroxytoluene) instead of a sample solution in order to compare the activity of the sample solution. Inhibition of the generated nitrotyrosine was measured by comparing the absorbance of a sample solution with the BHT standard solution. The injected sample was 20 l, and the tyrosine solution (1 mM) was used as a negative control. Compounds 1–9 were identiﬁed from the barley tea extract as p-hydroxybenzaldehyde (1),5) p-hydroxyacetophenone (2), 5,7-dihydroxychromone (3),6) naringenin (4), 3,4-dihydroxybenzaldehyde (5),5) p-hydroxybenzoic acid (6),5) vanillic acid (7),5) p-coumaric acid (8),5) and quercetin (9). The identiﬁcation of these compounds was established by comparing the physical and spectral data with literature values. Compound 10 was identiﬁed as isoamericanol A by comparing with an authentic sample7) belonging to unique neolignans like americanol A (11), americanin A (12), and isoamericanin A (13). Compounds 10 and 11 exhibited interesting neurotrophic properties.7–10) Compounds 10, 11 and 13 have also recently been isolated from a methanolic extract of the seeds of Brazilian Joannesia princeps.11) We also measured in this study the peroxynitrite-scavenging activities of all the compounds (1–10) isolated from barley tea together with those of compounds (11–13) (Table 1). Among these compounds, 3,4-dihydroxybenzaldehyde, p-coumaric acid, quercetin, isoamericanol A and its derivatives (11–13) had peroxynitrite-scavenging activities that were higher than that of BHT at 400 M. These results support our previous ﬁnding that catechol
Table 1. Inhibition by Isolated Compounds (1–10), Americanol A (11), Americanin A (12), Isoamericanin A (13), and BHT of the Reaction of L-Tyrosine with Peroxynitrite
derivative exhibited strong activities. It has already been elucidated that the activity of p-coumaric acid derivatives was mainly inﬂuenced by the functional groups at the 3-position on the aromatic ring and less by the conjugated side chain.4) The various substituents at the 1-position on the aromatic ring might also aﬀect on their antioxidative activities. In conclusion, quercetin (23.2 mg/kg) and p-coumaric acid (103 mg/kg), which were collected in relatively large quantity showed strong antioxidative activities in barley tea. Isoamericanol A is also a unique compound. Thus, barley tea powder (hattai-ko or mugi-kogashi) has natural antioxidants that might be turned into useful food material for human health. Further studies on the use of barley tea are in progress.
Acknowledgments This work was supported by ‘‘the cooperation of innovative technology and advanced research in evolutional area (city area)’’ Ministry of Education, Culture, Sports, Science and Technology of Japan and the Mugicha Association of Japan.
Concentration (M) Compound 100 1 2 3 4 5 6 7 8 9 10 11 12 13 BHT a
3:7 8:2 1:9 1:5 5:4 5:0 6:2 3:6 45:4 1:7 6:2 7:7 7:2 4:9 30:0 4:0 67:0 15:9 54:5 2:4 52:1 2:7 43:6 1:9 57:0 14:0 23:6 0:4
Mean value (%) for three values.
5:2 10:7 2:3 2:8 10:3 4:4 10:8 2:6 83:9 1:1 4:1 13:0 9:3 1:4 50:8 1:9 94:3 4:8 75:3 2:3 67:8 11:2 77:7 1:9 80:1 2:8 29:5 1:2
6:0 10:0 0:1 1:7 13:7 4:4 12:2 2:2 94:0 1:6 7:1 5:4 12:1 2:7 69:0 0:7 99:7 0:2 84:9 5:5 77:5 10:3 85:8 5:4 79:0 9:1 62:7 4:9
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