Chemical Constituents Of The Leaves Of Cistanche Ⅱ

3. 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 flflavonoid and compound 2 was firstly isolated from D. kaki. Additionally, kaempferol-3-O-β-200 -feruloyl glucoside (3) was only reported as a hydrolyzed product of 3-O-β-(2-O-feruloyl)-glucosyl-7,40 -di-O-β-glucosyl kaempferol (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-200 -galloyl galactoside (11) has been previously reported in many sources, including D. kaki, but only the 1H NMR and MS have been reported due to the lack of detailed research. Hence, the 13C 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 flavonoid-rich 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, bioassay-guided 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 allocated kaempferol glycoside and allocated 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, the 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 the simultaneous determination of most components in the persimmon leaves. 

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4.1. Plant Material

The leaves of Diospyros kaki Thunb. (Ebenaceae) were purchased at a domestic Korean herbal market in Yeongcheon in March 2018. The leaves were harvested at the basin area surrounded by mountains at an altitude of 800–1200 m in Gyeongsangbuk-do in August 2017. The average amount of the annual precipitation in this area was 1050 mm, of which, half fell between June and August. The average annual temperature was about 12.5 ◦C and the average relative humidity was 69%. The temperature at harvest time was approximately 37–40 ◦C. The obtained leaves were dried at approximately 45 ◦C in a plant dryer. A voucher specimen (DIKA1-2018) has been deposited in the Laboratory of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Republic of Korea.

4.2. General Experimental Procedures

silica gel (ODS-A 12 nm S-150 µm, YMC, Tokyo, Japan). Flash chromatography was performed using a CombiFlash (Teledyne Isco, Lincoln, NE, USA) with pre-packed cartridges, RediSep-silica (12 g, 24 g, and 40 g), and RediSep-C18 (13 g, 26 g, 43 g, and 130 g). Preparative HPLC was performed using the Gilson purification system (Gilson, Middleton, WI, USA) with a YMC Pack ODS-A column (250 × 20.0 mm, 5.0 µm, YMC, Tokyo, Japan), a sphere ODS-M80 column (250 × 20.0 mm, 4.0 µm, YMC, Tokyo, Japan), and a Luna C18(2)column (250 × 21.2 mm, 10.0 µm, Phenomenex, Torrance, CA, USA). HPLC analysis was performed on a Youngin YL9100 HPLC system comprising an evaporative light scattering detector (Youngin, Anyang, Korea) with Luna C18(2) column (150 × 4.6 mm, 5.0 µm, Phenomenex, Torrance, CA, USA). The online HPLC-ABTS screening was performed on an Agilent 1200 HPLC system with a YMC Pack ODS-A column (150 × 4.6 mm, 5.0 µm). All solvents used for the chromatographic separations were distilled. 

4.3. Extraction and Isolation

The dried plant material (15.0 kg) was extracted with 216 L of ethanol (EtOH) in a water bath at 60 ◦C for 4 h, and the solvent was evaporated to obtain EtOH extract (1.2 kg, yield 8%). The extract was suspended in H2O (2.1 L) and partitioned with ethyl acetate (EtOAc, 4.9 L × 3) to give EtOAc- (321.9 g, yield 2.15%) and H2O-soluble layers (748.0 g, yield 4.99%), respectively. The EtOAc-soluble layer (321.9 g) was subjected to a silica gel column (φ 10.5 × 35.0 cm) with n- exane:EtOAc:methanol (MeOH) mixtures (from 8:1.8:0.2 to 0:0:1 v/v/v) to afford nine fractions (E1~E9).

Fraction E5 (14.2 g) was chromatographed over a Diaion HP-20 column (φ 5.0 × 29.0 cm) with acetone: H2O gradient (7:3 to 1:0) to afford seven fractions (E5-1~E5-7). Fraction E5-1 was subjected to a silica gel column with n-hexane:EtOAc: MeOH = 7:2.7:0.3 to 0:0:1 to afford 4 fractions (E5-1-1~E5-1-4). E5-1-1 (13.2 mg) and E5-1-2 (7.0 mg) were combined and purified by preparative (prep)-HPLC using an YMC Pack ODS-A column (H2O: MeOH = 27:23, 7 mL/min) to obtain compounds 19 (8.3 mg, tR 26.0 min) and 20 (2.5 mg, tR 24.0 min). 

Fraction E8 (75.19 g) was fractionated into acetone-soluble (AS) and acetone-insoluble (AIS) fractions. Fraction AS (44.07 g) was chromatographed over a Diaion HP-20 column (φ 6.5 × 12.5 cm) with acetone: H2O mixtures (3:7 to 1:0) to afford 12 fractions (AS1~AS12). Fraction AS2 (2.5 g) was separated by a Sephadex LH-20 column (φ 4.7 × 51.0 cm) with MeOH to give nine fractions (AS2-1~AS2-9). Fraction AS2-2 (196.3 mg) was separated by flash chromatography (FC) using a RediSep-C18 cartridge (26 g, acetonitrile: H2O, 0:1 to 7:1) to yield compound 17 (28.6 mg). Fraction AS3 (3.1 g) was separated into 11 fractions using a Sephadex LH-20 column (φ 4.7 × 51.0 cm) with MeOH (AS3-1~AS3-11). 

Fraction AS3-6 (0.7 g) was separated by FC with RediSep-C18 (130 g, MeOH:H2O, 1:9 to 3:2) to give compounds 4 (51.8 mg), 5 (21.7 mg, tR 42.5 min), and 6 (20.2 mg, tR 47.0 min). Fraction AS4 (8.8 g) was subjected to a silica gel column (φ 5.2 × 21.0 cm) with MC:MeOH:H2O mixtures (from 8:1.8:0.2 to 7:2.7:0.3) to isolate Compounds 7 (5.0 mg), 8 (5.1 mg), 9 (20.0 mg), 10 (306.6 mg), and 12 (20.1 mg). Fraction AS5 was subjected to a silica gel column (φ 5.2 × 24.5 cm) with CH2Cl2:MeOH:H2O mixtures (8:1.8:0.2 to 7:2.7:0.3) to generate six fractions (AS5-1~AS5-6) to afford compounds 11 (3.0 mg) and 13 (7.4 mg). Fraction AS10 was separated into seven fractions using a Sephadex LH-20 column (φ 3.5 × 50.5 cm) with MeOH (AS10- 1~AS10-7). Fraction AS10-4 was separated by FC with a RediSep-C18 (43 g, MeOH:H2O,

0:1 to 3:2) cartridge to purify compounds 1 (5.3 mg), 2 (21.4 mg), 14 (15.9 mg), and 15 (40.4 mg). Fraction AS12 was separated into fifive fractions using a Sephadex LH-20 column (φ 3.5 × 50.5 cm) with MeOH (AS12-1~AS12-5). Compound 16 (20.1 mg) was obtained by recrystallization with MeOH from fraction AS12-5.

Fraction Als (31.1 g) was chromatographed silica gel column with h hexane EtOAc: MeOH mixtures (8:1.8:0.2 to 0:0:1) as a mobile phase to afford 20 fractions (AIS1-AIS20)Fraction AlS5 was subjected to a silica gel column with n-hexane:EtOAc:MeOH mixtures(8:1.8:0.2 to 0:0:1) to afford compound 23 (224.7 g). Fraction AIS6 was separated by FCwith a RediSep-C18 (43 g, MeOH:HO, 0:1 to 9:1) cartridge to give compound 25 (25.6 mg)Fraction AIS7 was subjected to a silica gel column with n-hexane:EtOAc: MeOH mixtures(8:1.8:0.2 to 0:0:1) to afford compounds 24 (100.0 mg) and 27 (214.1 mg). Fraction AIS10 was subjected to a silica gel column with n-hexane:EtOAc: MeOH mixtures (7:2.7:0.3 to 0:0:1)to isolate compounds 18 (5.0 mg) and 23 (16.7 mg). Fraction AIS11 was separated into11 subfractions (AIS11-1-AIS11-11) by FC with a RediSep-C18 (130 g, MeOH:HO, 1:1 to 4:1)cartridge. Compound 22 (37.8 mg) was obtained from fraction AS11-4 by prep-HPLC with a sphere column. Fraction AIS12 was subjected to a silica gel column (o 5.2 x 28.0 cm) with HCl: acetone mixtures (from 4:1 to 3:2) to afford compound 21 (188.0 mg). Finally, fractionAIS16 was separated using a Lichroprep RP-18 column (1.99 g, MeOH: H2O, 3:2 to 13:7) toobtain compound 3 (2.3 mg).

4.3.1. kaempferol-3-O-β-D-200 -coumaroylgalactoside (1

) Yellowish powder; m.p. 244.5 ◦C; [α] 22 D -59.1◦ (c 0.1, MeOH); UV (MeOH) λmax (log ε) 207 nm (3.98), 315 nm (3.92); IR (ATR) νmax 3458, 2922, 1650, 1588, 1364, 1260, 1175, 1076, 834 cm−1 ; 1H and 13C NMR data, see Table 1; HRMS (positive mode) m/z 595.1447 [M + H]+ (calcd for C30H27O13, 595.1452). 

4.3.2. kaempferol-3-O-β-D-200 -feruloylglucoside (3) 

Yellowish powder; m.p. 225.2 ◦C; [α] 22 D -119.6◦ (c 0.1, MeOH); UV (MeOH) λmax (log ε) 210 nm (4.17), 327 nm (3.94); IR (ATR) νmax 3369, 1652, 1599, 1512, 1360, 1264, 1177, 1076, 841 cm−1 ; 1H and 13C NMR data, see Table 1; HRMS (negative mode) m/z 623.1375 [M − H]− (calcd for C31H27O14, 623.1401).

4.3.3. kaempferol-3-0--D-2-galloylgalactoside (11)

Yellow powder; HNMR (500 MHZ, DMSO-) 6 H NMR 8.06 (2H, d,J= 9.0 Hz, H-2H-6'), 7.02 (2H, S, H-3, H-'), 6.87 (2H, d, I = 9.5 H, H-, H-5, 6.39 (1H, S, H-8), 6.16(1H s, H-6), 5.78 (1H, d, = 8.0 Hz, H-1%),5.27 (1H, t, =9.5 Hz H-2" 13C NMR (125 MHzDMSO-6) 8 177.1 (C-4), 165.4 (C-7), 165.4 (C-1), 161.2 (C-5), 160.1 (C-4, 156.3 (C-2),156.3(C-9), 145.5 (C-4"), 145.5 (C-6", 138.4 (C-5/), 132.5 (C-3), 131.0 (C-2'), 131.0 (C-6), 120.7 (C1), 119.8 (C-2'), 115.2 (C-3'), 115.2 (C-5, 108.9 (C-3), 108.9 (C-7), 103.8 (C-10), 98.8 (C-6)98.8 (C-1%), 93.7 (C-8), 76.0 (C-5), 72.7 (C-3), 71.1 (C-2%), 68.2 (C-4"), 60.1(C-6").

4.4. Ouantitative Analsis ofNine Compounds in the Persimmon Lenves

HPLC analysis was performed on a Waters HPLC system comprising 1525 pumps and 2996 photodiode array detectors (Waters, Milford, MA, USA). The UV wavelength was set at 260 nm. A PhenomenexLuna C18(2) column (150 x 4.6 mm,5.0 um, Phenomenex, Torrance, USA) was used, and the injection volume was 10 uL. The column temperature was set at 25 °C. The mobile phase consisted of 0.02% trifluoroacetic acid (TFA, Sigma-AldrichSt. Louis, MO, USA) in water (A) and acetonitrile (B) with a flow rate of 0.7 mL/min. The gradient conditions were as follows: 0-30 min, 15-20% B; 30-45 min, 20-35% B: 45-70 min35-100%; 70-80 min, 100%. 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 um membrane filters.

4.5. 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 uL) was mixed with various concentrations of samples (100 uL) 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-function 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 recordhe a decrease in aBIS radicals

5. ConclusionsIn conclusion, this study presents a phytochemical investigation based on bioassay-guided isolation. As a result, a new flavonoid, kaempferol-3-0-B-D-2"-coumaroylgalactoside(1), and a new natural compound, kaempferol-3-0--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 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.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10102032/s1, Figures S1–S9: The 1H, 13C, COSY, HSQC, HMBC, and ROESY NMR, UV, IR, and HRMS spectra of compound 1, Figures S10–S18: The 1H, 13C, COSY, HSQC, HMBC, ROESY NMR, UV, IR, and HRMS spectra of compound 3, Figures S19–S20: The 1H and 13C NMR spectra of compound 11, Table S1: The quantitative analysis of the other 18 compounds. 

Author Contributions: J.K. and J.-E.P. made equal contributions to this study; conceptualization, H.-C.K. and D.-S.J.; methodology and validation, J.K., J.-E.P., J.-S.L., J.-H.L. and H.H.; software, J.K. and J.-S.L.; validation, J.K. and H.H.; formal analysis, J.K. and J.-E.P.; investigation, J.K., J.-E.P., J.-S.L., J.-H.L., H.H., S.-H.J., H.-C.K. and D.-S.J.; resources, H.-C.K. and D.-S.J.; data curation, J.K., J.-E.P. and J.-S.L.; writing—original draft preparation, J.K., J.-E.P. and J.-S.L.; writing—review and editing, H.-C.K. and D.-S.J.; visualization, J.K. and J.-S.L.; supervision, H.-C.K. and D.-S.J.; project administration, H.-C.K. and D.-S.J.; funding acquisition, S.-H.J., H.-C.K. and D.-S.J. All authors have read and agreed to the published version of the manuscript.

Conflflicts of Interest: The authors declare no conflict of interest. 

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