Antioxidant Phenolic Constituents from the Leaves of Acer ginnala var. aidzuense

The genus Acer, belonging to family Aceraceae has about 200 species worldwide and among them, 26 species are distributed in Japan1. Acer ginnala Maxim. var. aidzuense (Franch) Pax (syn. Acer aidzuense (Franch.) Nakai), commonly known as ‘Karakogi-kaede’ is a deciduous tree about 2-5 m mainly found in wetlands2. It is widely distributed in eastern parts of Japan but comparatively rare in western parts including Kyushu Island3. It was thought to be extinct for last 30 years in Kumamoto Prefecture, Japan until we recently reported it from Kumagun, Asagiri Town4. Many species of Acer are widely used as traditional medicines in Japan and other countries. For example, the stem bark of Acer nikoense is used for the treatment of hepatic disorders and eye diseases and also as health food5. The stem and leaves of Acer palmatum are used in treatment of stomach ache and joint pain6. The sap of A. ginnala is used as stomachic and for the treatment of diarrhea7. The tea made from young leaves of A. ginnala, known as Gao-Cha is used for the prevention of hypertension and inflammation8 and also used in treatment of eye problems and headache9. These traditional uses suggested that the different plant parts Acer ginnala var. aidzuense may also have some health beneficial effects. In this paper, we report the chemical constituents in the leaves of Acer ginnala var. aidzuense and the antioxidant activity of the extract and isolated compounds.


Introduction
The genus Acer, belonging to family Aceraceae has about 200 species worldwide and among them, 26 species are distributed in Japan 1 . Acer ginnala Maxim. var. aidzuense (Franch) Pax (syn. Acer aidzuense (Franch.) Nakai), commonly known as 'Karakogi-kaede' is a deciduous tree about 2-5 m mainly found in wetlands 2 . It is widely distributed in eastern parts of Japan but comparatively rare in western parts including Kyushu Island 3 . It was thought to be extinct for last 30 years in Kumamoto Prefecture, Japan until we recently reported it from Kumagun, Asagiri Town 4 .
Many species of Acer are widely used as traditional medicines in Japan and other countries. For example, the stem bark of Acer nikoense is used for the treatment of hepatic disorders and eye diseases and also as health food 5 . The stem and leaves of Acer palmatum are used in treatment of stomach ache and joint pain 6 . The sap of A. ginnala is used as stomachic and for the treatment of diarrhea 7 . The tea made from young leaves of A. ginnala, known as Gao-Cha is used for the prevention of hypertension and inflammation 8 and also used in treatment of eye problems and headache 9 . These traditional uses suggested that the different plant parts Acer ginnala var. aidzuense may also have some health beneficial effects. In this paper, we report the chemical constituents in the leaves of Acer ginnala var. aidzuense and the antioxidant activity of the extract and isolated compounds.

Plant Materials
The leaves of Acer ginnala var. aidzuense were collected from Asagiri Town, Kumamoto, Japan in May 2014 and shade dried for one month. The voucher specimens are deposited at the Museum of Traditional Medicines, School of Pharmacy, Kumamoto University.

Free Radical Scavenging Activity
The DPPH radical-scavenging activity of extract and isolated compounds was examined using the method reported previously 10 with slight modifications. Briefly, 50 μL of 200 mM MES [2-(N-morpholino) ethanesulphonic acid] buffer (pH 6.0), 100 μL of samples with different concentrations (in DMSO:Ethanol = 1:1) and 50 μL of 800 mM DPPH in ethanol solution were mixed in a 96well plate and kept in dark at room temperature for 20 minutes. The anti-oxidative activity corresponding to the scavenging of DPPH radicals was measured at 510 nm with UV spectrophotometer using following formula: Radical scavenging activity (%) = 100×(A−B)/A. Where, A is the control absorbance of DPPH radicals without samples and B is the absorbance after reacting with samples. Trolox was used as the positive control. The result is expressed as mean of three experiments. From these data, curve was plotted and concentration ((µg/mL or μM)) of the sample required for 50% reduction of the DPPH radical absorbance (EC 50 ) was calculated.

Results and Discussion
Since, we have reported Acer ginnala var. aidzuense from Kumamoto after many years, the main aim of this study Journal of Natural Remedies | ISSN: 2320-3358 www.informaticsjournals.com/index.php/jnr | Vol 17 (1) | January 2017 was to identify the chemical constituents in the leaves of title plant and evaluate the antioxidant activity. Three phenolic compounds namely, gallic acid (1) 11 , methyl gallate (2) 11 and acertannin (3) 12 ( Figure 1) were isolated from the 70% MeOH extract of the leaves. Structures of these compounds were elucidated on the basis of NMR spectra and comparison with literature data. The 70% MeOH extract and all of the isolated compounds were tested for their in vitro antioxidant activity towards DPPH free radical scavenging assay. The concentrations of the sample required for 50% reduction of the DPPH radical absorbance (EC 50 values) are given in Table 1. The results were compared with Trolox as positive control. The 70% MeOH extract (EC 50 , 7.0 μg) showed more potent activity than Trolox (EC 50 , 12.2 μg). Among the isolated compounds, the activity of acertannin (3) was the strongest (EC 50 , 8.6 µM) followed by gallic acid (1) (EC 50 , 12.9 µM) and methyl gallate (2) (EC 50 , 13.0 µM), and all of these compounds were more potent as compared to Trolox (EC 50 , 48.8 µM). These findings are similar to previous studies on Acer plants and their bioactivities 8, 12 . Thus, these results suggest that the leaves of Acer ginnala var. aidzuense contain highly antioxidant phenolic compounds including gallic acid, methyl gallate and acertannin. The consumption of the tea made from the leaves may have preventive effects against damage caused by oxidative stress. However, more studies are required for the quantitative estimation of these compounds and in vivo biological activities of extracts and individual compounds.