Aromatic Constituents from the Leaves of Actinidia arguta with Antioxidant and α-Glucosidase Inhibitory Activity

As the leaf of Actinidia arguta has shown antioxidant activity, a study was conducted to identify the active ingredients. Forty-eight compounds were isolated from the leaves of A. arguta through various chromatographic techniques. Further characterization of the structures on the basis of 1D and 2D NMR and MS data identified several aromatic compounds, including phenylpropanoid derivatives, phenolics, coumarins, flavonoids and lignans. Among them, five compounds were newly reported, naturally occurring, and named argutosides A–D (1–4), which consist of phenylpropanoid glycosides that are conjugated with a phenolic moiety, and argutoside E (5), which is a coumarin glycoside that is conjugated with a phenylpropanoid unit. The isolated compounds showed good antioxidant and α-glucosidase inhibitory activity with differences in activity depending on the structures. Molecular docking analysis demonstrated the interaction between the hydroxyl and carbonyl groups of compounds 1 and 5 with α-glucosidase. Taken together, the leaves of A. arguta are rich in aromatic compounds with diverse structures. Therefore, the leaves of A. arguta and their aromatic components might be beneficial for oxidative stress and glucose-related diseases.


Introduction
Oxidative stress is caused by the excessive production of reactive oxygen species (ROS) which bind to molecules in vivo and consequently alter their structures and functions. An antioxidant-related defense mechanism exists to protect against generated ROS. However, persistent oxidative stress by excessive production of ROS eventually leads to diverse severe diseases such as cancer, inflammation and metabolic diseases [1][2][3].
Diabetes is a metabolic disease with a high incidence worldwide. In diabetes, the blood glucose level increases due to the abnormal operation of insulin, which causes various complications and develops into a serious disease [4]. Various factors are known to be involved in the onset and progression of diabetes; oxidative stress is one such mediator [5,6]. The increased ROS attack the pancreas and interfere with the normal function of insulin [7,8]. In other words, oxidative stress and diabetes are mutually detrimental to each other [9,10].
Accordingly, research into the development of a therapeutic agent for diabetes is being actively conducted. α-Glucosidase is an intestinal enzyme which converts carbohydrates into single monosaccharides. Therefore, α-glucosidase inhibitors are used for the treatment of diabetes and carbohydrate-mediated diseases [11,12]. Antioxidants are also used for the prevention and treatment of diabetes. Natural products are good sources for antioxidants and are widely used in the prevention and treatment of various diseases [13]. In particular, polyphenols are representative components with antioxidant action and are present in various plants. In addition, they also have therapeutic potential for metabolic diseases and have shown excellent results in various diabetes models [14,15].
Actinidia arguta, also called hardy kiwifruit or kiwiberry, has small fruits with a smooth green skin. Due to its cold-resistant characteristic, it can be cultivated in Northeast Asia [16,17]. The grains of A. arguta are mainly consumed as fresh fruits while the cooked leaves are used in the treatment of various diseases with antioxidant, antibacterial, antidiabetic and anti-inflammatory effects [18][19][20]. We recognized the importance of A. arguta as a native plant together with its biological activities and, thus, investigated the efficacy and ingredients of A. arguta. As a follow-up study on A. arguta leaves [21], the antioxidant and anti-diabetic effects of the extracts were confirmed. Further investigation into the bioactive constituents of A. arguta leaves resulted in the isolation of 48 compounds, including five new compounds. On the basis of 1D and 2D NMR and MS data, the structures of the isolated compounds were determined to be aromatic and included phenylpropanoid derivatives, phenolics, coumarins, flavonoids and lignans. The antioxidant and α-glucosidase inhibitory activity of the isolated compounds were measured and their mechanism of action was analyzed using molecular docking analysis.

Plant Material
The leaves of A. arguta were obtained from a farm in Gwangyang, South Korea (GPS: DD 34.990714, 127.591508) in August 2016. After identification by the herbarium of the College of Pharmacy Chungbuk National University, voucher specimens (CBNU2016-AAL) were deposited in a specimen room of the herbarium.

Measurement of α-Glucosidase Activity
The inhibitory effect on α-glucosidase was measured using α-glucosidase (from Saccharomyces cerevisiae (EC 3.2.1.20) [21]. A test sample was mixed with 80 µL enzyme buffer and 10 µL α-glucosidase and incubated for 15 min at 37 • C. Then, after the addition of 10 µL p-nitrophenyl α-D-glucopyranoside solution for enzyme reaction, the amount of p-nitrophenol that was cleaved by the enzyme was determined by measuring the absorbance at 405 nm in a 96-well microplate reader. Acarbose was used as a positive control.

Measurement of DPPH Radical Scavenging Activity
The antioxidant activity was evaluated by measuring the free radical scavenging activity using DPPH as previously reported [20]. In brief, freshly prepared DPPH solution was mixed with the samples. The mixture was reacted at room temperature for 10 min, and the absorbance was measured at 550 nm. Ascorbic acid was used as a positive control.

Structural Elucidation
Chromatographic separation of the EtOAc fraction of A. arguta resulted in the isolation of five new compounds (1-5) together with forty-three known compounds (6-48) (Figure 1). These signals were assigned to the trans-caffeoyl group and the trans-coumaroyl group based on the HMBC correlations between H-7/C-1, H-7/C-9 and H-2"/C-7", H-7"/C-9", respectively ( Figure 2). Therefore, compound 1 was suggested to consist of a glucose, a trans-caffeoyl group and a trans-coumaroyl group. The connections between these moieties were determined by HMBC correlation. The HMBC correlations from H-1 of a glucose to C-5 of a trans-caffeoyl group, and from H-6 of a glucose to C-9" of a trans-coumaroyl group suggested the linkages of a trans-caffeoyl group to a glucose and a glucose to a trans-coumaroyl group. Combined with the above-mentioned data, compound 1 was elucidated as shown and named argutoside A. Compound 2 (Tables 1 and 2) was purified as a brown syrup. The molecular formula was deduced as C 23 Figure 2. Conclusively, compound 4 was defined as shown and named argutoside D.
Compound 5 (Table 3) C-8a)] suggested the existence of one 6,7-disubstituted coumarin skeleton. Further analysis using the HMBC correlation together with a comparison to the previous data identified the coumarin moiety as 6,7-dihydroxycoumarin, esculetin [22]. Therefore, compound 1 was suggested to consist of a glucose, a trans-caffeoyl group and a 6,7-dihydroxycoumarin moiety. The HMBC correlations from H-1 of a glucose to C-7 of a 6,7-dihydroxycoumarin group suggested the linkage between 6,7-dihydroxycoumarin and a glucose, and the HMBC correlations from H-6 of a glucose to C-9" of trans-caffeoyl group suggested the linkage between a glucose and a trans-caffeoyl group. Based on these data, the structure of compound 5 was defined as esculetin 7-O-(6 -O-trans-caffeoyl)-βglucopyranoside and named argutoside E.

Molecular Docking Analysis
Further molecular docking analysis was conducted for two types of human maltaseglucoamylase (NtMGAM and CtMGAM) in order to propose the mechanisms of the αglucosidase inhibitory activity of active compounds. Consistent with experimental results, interactions with the α-glucosidase were suggested for active compounds. Hydrogen bonds were formed between compound 1 and NtMGAM and CtMGAM, respectively. Compound 5 exhibited the interaction by forming hydrogen bonds and Pi-alkyl interactions, as shown in Figure 4. These results indicate that compounds 1 and 5 could be inserted into the active site of the enzyme by different types of interactions and could inhibit α-glucosidase activity.

Conclusions
An investigation into the leaves of A. arguta led to the isolation of 48 aromatic compounds, including 5 new compounds. The structures of the isolated compounds were determined to be aromatic, including phenylpropanoid derivatives, phenolics, coumarins, flavonoids and lignans. Five new compounds were defined as argutosides A-D (1-4), which consist of phenylpropanoid glycosides conjugated with a phenolic compound, and argutosides E (5), which is a coumarin glycoside conjugated with a phenylpropanoid. The isolated compounds showed good antioxidant and α-glucosidase inhibitory activity with differences in activity depending on the structures. The analysis of the interactions between hydroxyl and carbonyl groups of active compounds 1 and 5 and α-glucosidase by molecular docking analysis supported the α-glucosidase inhibitory activity. In conclusion, the aromatic constituents of A. arguta leaves with α-glucosidase inhibitory activity might be beneficial to glucose-related diseases.