Chemical Constituents of the Leaves of Diospyros kaki (Persimmon)

Diospyros kaki (persimmon) leaves have long been utilized as traditional medicine for the treatment of ischemic stroke, angina, and hypertension and as a healthy beverage and cosmetic for anti-aging. This study aimed to isolate as many compounds as possible from an ethanol extract of the persimmon leaves to identify the biologically active compounds. The antioxidative effect of the ethyl acetate layer from the ethanol extract of the persimmon leaves was demonstrated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and online high-performance liquid chromatography-2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (HPLC-ABTS) analysis. A new flavonoid, kaempferol-3-O-β-d-2″-coumaroylgalactoside (1), and a new natural compound, kaempferol-3-O-β-d-2″-feruloylglucoside (3) were isolated from the ethyl acetate layer, along with 25 previously known compounds, including fourteen flavonoids, one ionone, two coumarins, seven triterpenoids, and one acetophenone. Their structures were determined by the interpretation of spectrometric and spectroscopic data. All isolated compounds were rapidly evaluated using an online HPLC-ABTS assay, and of these, compounds 4–8, 11, 13, 15, and 16 clearly showed antioxidative effects. The amount of these compounds was 0.3–0.65% of the extract.


Introduction
Diospyros kaki Thunb. (persimmon) belongs to the family of Ebenaceae and is widely distributed in Korea, China, and Japan. Its fruit is eaten fresh or dry, while the leaves have long been used as a traditional medicine to treat ischemic stroke, angina, hypertension, atherosclerosis, and infectious diseases [1]. Furthermore, its leaves have been utilized as healthy beverages and cosmetics due to their anti-aging properties and abilities to help prevent cholesterol and melanin accumulation [1]. Recent research has suggested that the extracts of the persimmon leaves possess a wide range of biological properties, including radical scavenging, neuroprotection, thrombosis inhibition, anti-atherosclerosis, and anti-allergy [2][3][4][5][6]. A previous phytochemical investigation suggested that various types of flavonoids and terpenoids are the main constituents [7], and several tannins, naphthoquinones, coumarins, ionones, and fatty acids were also reported [8][9][10][11][12].
Reactive oxygen species (ROS) are reactive molecules produced in biological systems, and the balance between the generation and elimination of ROS is well controlled in normal cellular physiology [13]. However, excessive generation of ROS causes oxidative damage, and in turn, aging and age-related diseases including cancer, diabetes, and Parkinson's disease [14]. Hence, discovering antioxidants such as flavonoids and phenolic compounds could be a promising strategy to treat these diseases.
As part of our continuous project to find biologically active compounds [15], the antioxidative effect of the ethanol (EtOH) extract and solvent partitions from the persimmon leaves was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and online highperformance liquid chromatography-2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (HPLC-ABTS) analysis. A phytochemical study on the persimmon leaves led to the isolation of one new flavonoid (1) and one new natural compound (3), along with 25 previously known compounds. The structures were characterized by the application of spectroscopic and spectrometric methods. All isolated compounds were rapidly screened for their antioxidative effects using online HPLC-ABTS. Furthermore, the quantitative analysis of all isolated compounds was performed in the present study.

Antioxidative Effect of the Persimmon Leaves
The antioxidative effect of the ethanol (EtOH) extract of persimmon leaves was evaluated for a preliminary screening through DPPH ( Figure 1A). The 0.125 mg/mL of the extract scavenged approximately 80% of the DPPH radical, while 0.025 mg/mL of ascorbic acid made up 94% of the radical. The online HPLC-ABTS assay was carried out to rapidly ensure the reliability of these results ( Figure 1D). Gallic acid and Trolox were used as internal standards. The chromatogram at 734 nm (negative peak) suggested that approximately nine constituents could have antioxidative activities. Gallic acid and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) were used as internal standards to ensure the reliability of the results. It was inferred that most of these peaks were flavonoid derivatives such as flavonoid glycoside and flavanol, based on the dereplication analysis performed by comparing ultraviolet (UV) and mass spectra of the compounds with the published data. Bioassay-guided fractionation suggested that these antioxidative compounds were abundant in the ethyl acetate (EtOAc) layer, while the water (H 2 O) layer showed weak activity ( Figure 1B
Compound 11 was isolated as a yellow powder. The 1 H 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 13 C 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-2galloylgalactoside. Although compound 11 was previously isolated from various sources, including D. kaki [21,36], only the 1 H NMR and MS data were previously reported. Thus, the 13 C NMR data was reported for the first time in this study.

Antioxidative Activities of the Isolated Compounds
All isolated compounds were evaluated for their antioxidative effects using rapid online HPLC-ABTS analysis to determine which compounds contributed to the antioxidative effect of the persimmon leaves. Compounds 4-8, 11, 13, 15, and 16 showed potent antioxidative activities (Figure 4). Most of these compounds were kaempferol and quercetin derivatives, but some derivatives (1-3, 9, 10, 12, and 14) did not show activities. The structure-activity relationship was not fully determined but was partially revealed. In particular, quercetin and kaempferol with galloyl moieties (7, 8, 11, and 13) were found to have potent activities, but those with coumaroyl or feruloyl moieties (1-3 and 16) did not show any activities.

Quantitative Analysis of Isolated Compounds
The quantitative analysis of all isolated compounds in the EtOAc-soluble extract was performed to confirm that the antioxidative effects of the persimmon leaves could be caused by these active compounds. A specific HPLC method with diode array detection and evaporative light scattering detector was developed for the simultaneous determination of 27 compounds. The contents of all isolated triterpenoids were approximately 5.9% of the extract, and of these, those of siaresinolic acid (25), oleanolic acid (26), and ursolic acid (27) accounted for a significant portion, as reported in previous studies [7]. The con-

Quantitative Analysis of Isolated Compounds
The quantitative analysis of all isolated compounds in the EtOAc-soluble extract was performed to confirm that the antioxidative effects of the persimmon leaves could be caused by these active compounds. A specific HPLC method with diode array detection and evaporative light scattering detector was developed for the simultaneous determination of 27 compounds. The contents of all isolated triterpenoids were approximately 5.9% of the extract, and of these, those of siaresinolic acid (25), oleanolic acid (26), and ursolic acid (27) accounted for a significant portion, as reported in previous studies [7]. The contents of flavonoids were approximately 5.4%, and of these, those of active compounds were 3.2% (Table 2). In particular, the contents of kaempferol-3-O-β-2 -galloylgalactoside (9), kaempferol-3-O-β-2"-galloylglucoside (10), isoquercitrin (13), and quercetin-3-O-β-2"galloylglucoside (15) were more than 0.3%. The analytical method was verified using a simple validation procedure to ensure the relevance of the method, which showed adequate specificity, linearity, accuracy, and precision.

Discussion
Phytochemical investigations to identify biologically active compounds in persimmon leaves have been widely carried out. So far, a considerable number of triterpenoids and flavonoids, including kaempferol and quercetin derivatives, have been reported from D. kaki [1]. In this study, we obtained 27 compounds, including sixteen flavonoids, one ionone, two coumarins, seven triterpenoids, and one acetophenone. Of these, compound 1 was found to be a new flavonoid and compound 2 was firstly isolated from D. kaki. Additionally, kaempferol-3-O-β-2 -feruloylglucoside (3) was only reported as a hydrolyzed product of 3-O-β-(2-O-feruloyl)-glucosyl-7,4 -di-O-β-glucosylkaempferol (3), isolated from Allium tuberosum [35]. Compound 3 was not only obtained directly from a natural source for the first time but has also not been reported in D. kaki previously. Furthermore, kaempferol-3-O-β-2 -galloylgalactoside (11) has been previously reported in many sources, including D. kaki, but only the 1 H NMR and MS have been reported due to the lack of detailed research. Hence, the 13 C NMR data was reported for the first time here.
Until now, there have been few studies that demonstrated the antioxidative abilities of extracts or fractions of persimmon leaves [37,38]. Most studies used rapid assay methods such as DPPH or ABTS assays. In particular, in the previous paper, 200 µg/mL of flavonoidrich fraction exhibited 68.73% inhibition of DPPH radical. Aside from this result, however, this fraction also showed superoxide anion radical scavenging, hydroxyl radical scavenging, and metal chelating activities [38]. Although we did not evaluate these assays, bioassayguided isolation was carried out because the ethanol extract and ethyl acetate fraction in the present study showed comparable DPPH radical scavenging activity. Additionally, despite previous results, only a few studies to identify biologically active compounds have been carried out. A few secoiridoids and lignans showed radical scavenging activities [39]. In the case of flavonoids, there have been several reports that quercetin, kaempferol, and their glycosides have antioxidative properties [40]. Antioxidative properties of galloylated kaempferol glycoside and galloylated quercetin glycoside obtained from other sources have been reported [41]. As yet, there have been no reports that each of these compounds derived from the persimmon leaves has antioxidative effects, except that a mixture of these compounds exhibited an antioxidative effect [21].
Additionally, so far, simultaneous determination of only a few triterpenoids or flavonoids has been carried out for the quantitative analysis of these compounds [42,43]. However, the present study suggests a method for simultaneous determination of most components in the persimmon leaves.

Plant Material
The leaves of Diospyros kaki Thunb.

Extraction and Isolation
The dried plant material (15. The EtOH extract (10 mg) was dissolved in 10 ug/mL internal standard solution (1 mL). Simple method validation was carried out to ensure the relevance of the developed method and qualitative results. Five different solutions of each compound were analyzed to make each calibration curve. The intra-and inter-day precision and accuracy were confirmed through three replicates within a single day and three consecutive days. All samples were filtered through 0.2 µm membrane filters.

DPPH and Online HPLC-ABTS Analysis
The ability of samples to scavenge DPPH radicals was assessed based on a previous paper. Briefly, DPPH (0.1 mM) in methanol (100 µL) was mixed with various concentrations of samples (100 µL) for 1 h in the dark. The absorbance was recorded at 517 nm.
The online HPLC-ABTS analysis was performed based on the previous report with modifications. A mixed solution containing ABTS (0.08 mM) with potassium persulfate (0.12 mM) was made into an ABTS reagent. The reagent was stored at 4 • C for 12 h to stabilize radicals. All samples were analyzed by an Agilent HPLC system. The gradient conditions were the same as those used for quantitative analysis. The eluate was sent to a T-junction and reacted with ABTS reagent in a reaction coil at 40 • C. The chromatogram was visualized at 254 nm (positive peak), as well as at 734 nm (negative peak), to record the decrease in ABTS radicals.

Conclusions
In conclusion, this study presents a phytochemical investigation based on bioassayguided isolation. As a result, a new flavonoid, kaempferol-3-O-β-D-2"-coumaroylgalactoside (1), and a new natural compound, kaempferol-3-O-β-D-2"-feruloylglucoside (2), were isolated, along with 25 previously known compounds, including fourteen flavonoids, one ionone, two coumarins, seven triterpenoids, and one acetophenone. All compounds were evaluated on antioxidative effects, and of these, nine flavonoids were found to possess activities. Simultaneous quantitative analysis was performed to confirm that the persimmon leaves have antioxidative effects due to these compounds.