To identify these antioxidative compounds, various chromatographic and spectroscopic methods were carried out for the isolation and structural characterization of the compounds. A new flavonoid (1) and a new natural compound (3) were obtained from the ethyl acetate layer of the ethanol extract, together with 25 previously reported compounds, namely kaempferol-3-O-β-2″-coumaroylglucoside (2) [16], (+)-catechin (4) [17], hyperoside (5) [17], isoquercitrin (6) [18], quercetin-3-O-β-2″-galloylgalactoside (7) [19], quercetin-3-O-β-2″-galloylglucoside (8) [20], trifolin (9) [18], astragalin (10) [18], kaempferol-3-O-β-2″-galloylgalactoside (11) [21], kaempferol-3-O-α-arabinoside (12) [22], kaempferol-3-O-β-2″-galloylglucoside (13) [23], quercetin-3-O-β-2″-coumaroylglucoside (14) [24], quercetin (15) [15], kaempferol (16) [25], (6S,9S)-roseoside (17) [26], scopoletin (18) [27], umbelliferone (19) [28], 1-(2,4-dihydroxy-6-methylphenyl)ethanone (20) [29], barbinervic acid (21) [30], diospyric acid B (22) [7], rotungenic acid (23) [31], pomolic acid (24) [32], siaresinolic acid (25) [33], oleanolic acid (26) [25], and ursolic acid (27) [25] by using spectroscopic and spectrometric and physical data in comparison with the published data and also with thin layer chromatography (TLC) analysis (Figure 2 and Figure 3). Among these, compounds 2, 16, 17, 19, 20, and 24 were firstly isolated from D. kaki.

Compound 1 was obtained as a yellow powder, in which the molecular formula was established as C₃₀H₂₆O₁₃ based on high-resolution mass spectrometry (HRMS) data. The UV spectrum exhibited absorption bands at 207 and 315 nm, indicating that compound 1 had a flavonol backbone. The ¹H nuclear magnetic resonance (NMR) data (Table 1, Figure S1) showed a typical pattern of coumaroylated flavonol glycoside, showing two sets of AA′BB′-type signals (δ_H 8.00 (2H, d, J = 8.5 Hz, H-2′ and H-6′), 6.87 (2H, d, J = 8.5 Hz, H-3′ and H-5′)) in the B ring of kaempferol as well as the signals (δ_H 7.45 (2H, d, J = 8.5 Hz, H-2′′′ and H-6′′′), 6.81 (2H, d, J = 8.5 Hz, H-3′′′ and H-5′′′)) in the aromatic ring of the coumaroyl group. Two doublet signals (δ_H 7.65 (1H, d, J = 15.5 Hz, H-7′′′)) and δ_H 6.35 (1H, d, J = 16.0 Hz, H-8′′′) were observed, indicating trans-olefinic protons of the coumaroyl group. Additionaly, an anomeric proton signal (δ_H 5.57 (1H, d, J = 8.0 Hz, H-1′′)) was observed in the sugar region, suggesting the presence of the β-configurated cyclic sugar group. The ¹³C and distortionless enhancement by polarization transfer NMR data (Table 1, Figure S2) showed 30 resonances comprising two trans-olefinic carbons, ten aromatic carbons, and six glucosyl moiety carbons, and ten non-protonated carbons including two carbonyl carbons. In particular, the chemical shifts at C-2, C-3, and C-4 (δ_C 158.1, 134.9, and 179.2) were characteristic signals of flavonol 3-O-glycoside. Additionally, the carbonyl carbon signal at C-1′′′ (δ_C 168.7) and two trans-olefinic carbon signals at C-2′′′ and C-3′′′ (δ_C 146.9, 115.2) were typical chemical shifts of the coumaroyl group. The anomeric carbon signal at C-1′′(δ_C 100.4) and other signals for the glycosyl moiety from C-2′′ to C-6′′ (δ_C 74.3, 73.4, 70.5, 77.4, and 62.0) were observed. These one-dimensional (1D) NMR data were superimposable to those of kaempferol-3-O-β-2′′-coumaroylglucoside (2) [16]. However, the careful comparison of the ¹³C NMR data between the two compounds suggested that compound 1 had a galactose moiety, which was further demonstrated by the nuclear Overhauser enhancement spectroscopy (NOESY) NMR data (Figure S6). While the NOESY correlation between H-2′′ and H-4′′ was observed in compound 2, there was no correlation between these protons in compound 1. In general, interpreting ¹³C NMR and NOESY NMR data is an effective method to determine the type of glycosyl moiety. The location of the galactose moiety was deduced to be at C-3 according to the downfield shift of C-2 and C-4, as further evidenced by the heteronuclear multiple bond correlation (HMBC) between H-1′′ and C-3 (Figure S5). The position of the coumaroyl group was demonstrated to be at C-2′′ based on the downfield shift (δ_H 5.36 (1H, dd, J = 10.0, 8.0 Hz, H-2′′)) and the HMBC correlation between H-2′′ and C-1′′′. As a result, the structure of compound 1 was determined as kaempferol-3-O-β-2″-coumaroylgalactoside. Although compound 2 was previously isolated from various sources, including Quercus suber [16] and Allium porrum [34], compound 1 was isolated and structurally characterized for the first time.

Compound 3 was isolated as a yellow powder, and the molecular formula was established as C₃₁H₂₈O₁₄ by analyzing HRMS data. The UV spectrum showed the UV absorption at 210 and 327 nm due to the same aglycone with compounds 1 and 2. The ¹H NMR data (Table 1, Figure S10) were similar to those of compound 2, but compound 3 had a feruloyl group instead of the coumaroyl group, as evidenced by the presence of an additional methoxy group (δ_H 3.91 (3H, s, 3′′′-OCH₃)). Additionally, an anomeric proton signal (δ_H 5.64 (1H, d, J = 8.0 Hz, H-1′′)) was observed, indicating that the glycosyl linkage was a β-configuration, and the downfield-shifted signal (5.03 (1H, t, J = 8.5 Hz, H-2′′)) was shown in the sugar region, as with compound 1. The ¹³C NMR data (Table 1, Figure S11) showed 31 resonances comprising two trans-olefinic carbons, ten quarternary carbons, ten aromatic carbons, six glucosyl moiety carbons, and one methoxy carbon, and ten non-protonated carbons, including two carbonyl carbons corresponding to kaempferol, feruloyl, and glucose groups. In particular, carbon signals from C-2′′ to C-6′′ (δ_C 75.8, 76.3, 71.5, 78.8 and 62.5) suggested the presence of a glucose moiety. The locations of the glucose moiety and feruloyl group were assigned by the long-range HMBC correlations between H-1′′ and C-3 (δ_C 134.8) and H-2′′ and C-1′′′ (δ_C 168.4) (Figure S14). The position of an additional methoxy group was determined by the key correlation between 3′′′-OCH₃ and C-3′′′ (δ_C 149.4). The above results suggested the structure of compound 3 as kaempferol-3-O-β-2”-feruloylglucoside. To the best of our knowledge, compound 3 was only reported as a product of the hydrolysis of 3-O-β-(2-O-feruloyl)-glucosyl-7,4′-di-O-β-glucosylkaempferol, isolated from Allium tuberosum [35]. Therefore, the structure of 3 was elucidated as a new natural compound.

Compound 11 was isolated as a yellow powder. The ¹H NMR data (Figure S19) displayed a set of AA′BB′-type signals (δ_H 8.06 (2H, d, J = 9.0 Hz, H-2′, H-6′), 6.87 (2H, d, J = 9.0 Hz, H-3′, H-5′)) in the B ring of kaempferol and a singlet signal at δ_H 7.02 (2H, s, H-3′′′, H-7′′′) of a galloyl moiety in aromatic region, which is a characteristic signal of galloylated flavonol. An anomeric proton signal (δ_H 5.78 (1H, d, J = 8.0 Hz, H-1′′)) indicated that the glycosyl linkage was a β-configuration. Furthermore, a downfield shifted proton signal (5.27 (1H, t, J = 9.5 Hz, H-2′′)) suggested that the galloyl group was attached at the hydroxyl group of C-2′′ because this shift could be attributed to the anisotropic influence of the O-galloyl moiety [21]. The ¹³C NMR data (Figure S20) exhibited 26 resonances, indicating galloylated flavonol glycoside. The carbon signals from C-2′′ to C-6′′ (δ_C 71.1, 72.7, 68.2, 76.0, and 60.1) suggested the presence of a galactose moiety. Therefore, the structure of compound 11 was confirmed as kaempferol-3-O-β-2′′-galloylgalactoside. Although compound 11 was previously isolated from various sources, including D. kaki [21,36], only the ¹H NMR and MS data were previously reported. Thus, the ¹³C NMR data was reported for the first time in this study.

4. Discussion