Chemical Constituents Of The Leaves Of Cistanche

Abstract: Cistanche leaves have long been utilized as traditional medicine for treating 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-2,20 -casino-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. The interpretation of spectrometric and spectroscopic data determined their structures. 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. 

Keywords: Diospyros kaki; persimmon leaves; flavonoid; antioxidant

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1. 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 ability to help prevent cholesterol and melanin accumulation [1]. Recent research has suggested that the extracts of persimmon leaves possess a wide range of biological properties, including radical scavenging, neuroprotection, thrombosis inhibition, anti-atherosclerosis, and anti-allergy [2–6]. 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 [8–12]. 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 for treating 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 high-performance liquid chromatography-2,20 -casino-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), one new natural compound (3), and 25 previously known compounds. The application of spectroscopic and spectrometric methods characterized the structures. 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.

2. Results and Discussion 

2.1. 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-tetramethyl chroman-2-carboxylic acid) were used as internal standards to ensure the reliability of the results. It was inferred that most of these peaks were flflavonoid 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 (H2O) layer showed weak activity (Figure 1B,C). 

Figure 1. DPPH scavenging effects of the EtOH extract (A), EtOAc layer (B),and HO layer (C) HhO layer of the persimmon leaves: (D) online ABTS-HPLC chromatogram of the EtOH extract.

2.2. Phytochemical Investigation

To identify these antioxidative compounds, various chromatographic and spectroscopic methods were carried out for the isolation and structural characterization of the compounds. A new flflavonoid (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 (Figures 2 and 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 C3olO3 based on high-resolution mass spectrometry (HRMS) data. The spectrum exhibited absorption bands at 207 and 315 nm, indicating that compound1 had a flavonol backbone. The 'H nuclear magnetic resonance (NMR) data (Table 1Figure S1) showed a typical pattern of coumaroylated flavonol glycoside, showing two sets of AA'BB'-type signals (8 8.00 (2H, d, J= 8.5 Hz, H-2' and H-6 ),6.87 (2H, d, J= 8.5 HzH-3' and H-5')) in the B ring of kaempferol as well as the signals (0 7.45 (2H, d, J = 8.5 HzH-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 (8H 7.65 (1H, d, J = 15.5 Hz, H-7"") and  6.35 (1Hd, J = 16.0 Hz, H-8") were observed, indicating trans-olefinic protons of the country group. Additionally, an anomeric proton signal (0H 5.57 (1H, d, J = 8.0 Hz, H-1) was observed in the sugar region, suggesting the presence of the B-configurated cyclic sugar group. The 13C and distortionless enhancement by polarization transfer NMR data (Iable 1Figure 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 (8c 158.1, 134.9and 179.2) were characteristic signals of flavonol3-0-glycoside. Additionally, the carbonyl carbon signal at C-1" (8c 168.7) and two trans-olefinic carbon signals at C-2'" and C-3"(6 146.9, 115.2) were typical chemical shifts of the coumaroyl group. The anomeric carbon signal at C-1(6C 100.4) and other signals for the glycosyl moiety from C-2" to C-6"' (6c74.3, 73.4, 70.5, 77.4, and 62.0) were observed. These one-dimensional (1D) NMR data were superimposable to those of kaempferol-3-0-B-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 nuclearOverhauser enhancement spectroscopy (NOESY) NMR data (Figure S6). While the NOES 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 NOESYNMR data is an effective method to determine the type of glycosyl moiety. The locationof the galactose moiety was deduced to be at C-3 according to the downfield shift of C-2and C-4, as further evidenced by the heteronuclear multiple bond correlation (HMBCbetween 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 (0 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-3-0-B-2"-coumaroylgalactoside. Although compound 2 was previously isolated from various sources, including Ouercus suber (16] and Alliumporrum (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 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 (δH 3.91 (3H, s, 3000 -OCH3)). Additionally, an anomeric proton signal (δH 5.64 (1H, d, J = 8.0 Hz, H-100 )) 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-200 )) was shown in the sugar region, as with compound 1. The 13C NMR data (Table 1, Figure S11) showed 31 resonances comprising two trans-olefifinic 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-200 to C-600 (δC 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-100 and C-3 (δC 134.8) and H-200 and C-1000 (δC 168.4) (Figure S14). The position of an additional methoxy group was determined by the key correlation between 3000 -OCH3 and C-3000 (δC 149.4). The above results suggested the structure of compound 3 as kaempferol-3-O-β- 2”-feruloyl glucoside. 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-7,40 -di-O-β-glucosyl kaempferol, 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 AA0 BB0 -type signals (δH 8.06 (2H, d, J = 9.0 Hz, H-20 , H-60 ), 6.87 (2H, d, J = 9.0 Hz, H-30, H-50 )) in the B ring of kaempferol and a singlet signal at δH 7.02 (2H, s, H-3000 , H-7000 ) of a galloyl moiety in aromatic region, which is a characteristic signal of the allocated flavonol. An anomeric proton signal (δH 5.78 (1H, d, J = 8.0 Hz, H-100 )) indicated that the glycosyl linkage was a β-configuration. Furthermore, a downfield shifted proton signal (5.27 (1H, t, J = 9.5 Hz, H-200 )) suggested that the galloyl group was attached at the hydroxyl group of C-200 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 allocated flavonol glycoside. The carbon signals from C-200 to C-600 (δC 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-200 - 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

2.3. 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. 

Figure 4. Radical scavenging activities of the isolated compounds (4–8, 11, 13, 15, and 16) were assessed by online HPLC-ABTS assay.

2.4. 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 specifific 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 signifificant 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-200 -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. 

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