Identification of Phenolic Constituents and Inhibitory Activity of Persimmon Calyx and Shiteito against Tumor Cell Proliferation

Morio Yoshimura; Akira Mochizuki; Yoshiaki Amakura

doi:10.1248/cpb.c20-00259

Abstract

The persistent calyx on the fruit of Diospyros kaki, called “Shitei” in Japanese, is reported to contain phenolic compounds including condensed tannins. In this study, we isolated and characterized a new compound, together with 26 phenolic components, from the 70% acetone extract of Shitei, with structural elucidation based on spectroscopic analyses. In addition, we confirmed the presence of condensed tannins by ¹³C-NMR spectra, and the weight-average molecular weight was estimated by gel permeation chromatography (GPC) analysis. Next, Shiteito, a Kampo medicine consisting of Shitei, ginger, and clove clinically used to treat chronic hiccoughs occurring in association with anticancer drug treatments, and hot-water extracts of each of its components, were analyzed by HPLC, which determined that the main ingredient in Shiteito was derived from clove. We therefore isolated the ingredients and investigated their anti-tumor cell proliferative activity, together with Shiteito and Shitei extracts. As a result, Shiteito showed weak inhibition of hepatocellular carcinoma (Hep3B) cell proliferation at a high concentration. In contrast, ellagic acid, one of the main constituents of Shiteito, showed significant cytotoxicity against Hep3B cells, and significant inhibition of gastric adenocarcinoma (AGS) cell proliferation in a concentration-dependent manner. The ethyl acetate (EtOAc) fraction of the 70% acetone extract of Shitei significantly inhibited the proliferation of colon adenocarcinoma (Caco-2) and AGS cells at low to middle concentration, while showing strong cytotoxicity against Hep3B. These data indicate that Shiteito and Shitei extracts could enhance cancer drug treatment by preventing the associated chronic hiccups, and have the potential to be adjuvant treatments as well.

Introduction

Diospyros kaki Thunb. (Ebenaceae) is widely distributed in East Asia and known to contain polyphenols including condensed tannins abundantly in its leaf, bark, and fruits.^1–3) The fresh fruit containing water-insoluble (highly polymerized) condensed tannins is edible, whereas fruit mainly containing water-soluble (low-molecular weight [MW]) condensed tannins is inedible because it is bitter without alcoholic or carbon dioxide (CO₂) treatment or drying. In addition, the persistent calyx on the fruit [Japanese name: Shitei (柿蔕)] morphologically resembles the leaves, which have been reported to contain phenolic compounds including condensed tannins.⁴⁾ Therefore, functional secondary metabolites are expected to be a constituent of Shitei. Furthermore, Shiteito, which is a Kampo medicine prepared with Shitei, ginger (rhizome of Zingiber officinale Roscoe) and clove (flower bud of Syzygium aromaticum Merrill et Perry), is clinically used for the treatment of chronic hiccups that persist for more than several days as a result of anticancer drug treatments.⁵⁾ In addition, the aqueous ethanol extract of Shitei is known to have colorectal cancer growth inhibitory effects,⁶⁾ and the antitumor activity of hydrolyzable tannins in clove have been reported.⁷⁾ Therefore, we hypothesized that a similar activity could be expected with Shiteito. Thus, it is meaningful if Shiteito, which is used to prevent hiccups occurring as a side effect of anticancer drugs, also has anticancer activity.

Here, we describe the ingredients of Shitei and Shiteito, as well as their antitumor cell proliferative activity, to determine the further usefulness of Shiteito in the prevention of chronic hiccups and as an adjuvant treatment in cancer chemotherapy.

Results and Discussion

Isolation and Structure Elucidation of Phenolic Compounds in Shitei

The 70% acetone extract of Shitei was partitioned into n-hexane-, ethyl acetate (EtOAc)-, and n-butanol (BuOH)-soluble fractions using solvent extraction. Column chromatographic separation of the EtOAc fraction yielded compound 1 and 26 known compounds, which were characterized as protocatechuic acid (2), vanillic acid (3), gallic acid (4), 3,5-dimethoxy-4-hydroxybenzoic acid (5), scopoletin (6), cis-p-coumaric acid (7), trans-p-coumaric acid (8), 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (9),⁸⁾ (+)-catechin (10), gallocatechin (11), aromadendrin (12), taxifolin (13), ampelopsin (14), kaempferol (15), quercetin (16), kaempferol 3-O-glucoside (17),⁹⁾ kaempferol 3-O-galactoside (18),⁹⁾ quercetin 3-O-glucoside (19),⁹⁾ quercetin 3-O-galactoside (20),⁹⁾ kaempferol 3-O-glucoside-2″-O-gallate (21),⁹⁾ kaempferol 3-O-galactoside-2″-O-gallate (22),⁹⁾ quercetin 3-O-glucoside-2″-O-gallate (23),⁹⁾ dehydroconiferyl alcohol (24),⁸⁾ 1-(4′-hydroxy-3′-methoxyphenyl)-2-[4″-(3-hydroxypropyl)-2″-6″-dimethoxyphenoxy]propane-1,3-diol (25),¹⁰⁾ procyanidin B1 (26),¹¹⁾ and procyanidin B3 (27)¹¹⁾ using NMR data or direct HPLC comparison with authentic samples (Figs. 1, 2). Among the isolates, compounds 7, 9, 12, 13, 14, 24, and 25 were characterized for the first time from D. kaki.

Fig. 1. Structure of Characterized Compounds (1–27)

Fig. 2. Key Heteronuclear Multiple Bond Connectivity (HMBC) and Nuclear Overhauser Effect Spectroscopy (NOESY) Correlations of Compound 1

(Color figure can be accessed in the online version.)

Compound 1 was obtained as pale brown amorphous powder and assigned the molecular formula C₁₇H₁₈O₆ using high-resolution electrospray ionization (HR-ESI)-MS data [m/z 317.1024 (M−H)⁻, calcd. for 317.1031]. The ¹H-NMR (¹H–¹H correlation spectroscopy (COSY)) identified a 1,3,4-trisubstituted phenyl group in the aromatic region in combination with heteronuclear single quantum coherence (HSQC) in the aliphatic region. The ¹³C-NMR showed 17 signals, including a carbonyl carbon (δ: 199.7), methylene carbon (δ 65.5), an oxygenated methine carbon (δ 56.32), and two methoxy carbons (δ 56.43, 56.38), which indicated that two phenyl groups are linked to 3-hydroxy-1-propanone. The location of each unit and functional group was determined using heteronuclear multiple bond connectivity (HMBC) key correlations from H-2 and H-6 to C-7, from H-2′ and H-6′ to C-9, and from respective methoxy protons to C-3 and C-3′, as shown in Fig. 2. The assignment of each methoxy proton was also confirmed using nuclear Overhauser effect spectroscopy (NOESY) correlations among H-2 and 3-OMe, and H-2′ and 3′-OMe (Fig. 2). To determine the absolute configuration of 1, we attempted the methylation of two phenolic hydroxy groups, followed by the modified Mosher’s method. However, methylation with an alkylation reagent under alkaline conditions resulted in quinone methide formation, and thereby racemization at C-9. The employment of trimethylsilyldiazomethane afforded a new TLC spot with a larger R_f value (n-hexane : EtOAc, 1 : 2), but the MS data (m/z 315 in the negative mode) suggested oxidation of the C-9 hydroxy group instead of methylation. Furthermore, the small value of optical rotation (+2.5°, c = 0.1, methanol [MeOH]) made it difficult to determine the absolute configuration of C-9. Based on these data, the structure of compound 1 was elucidated as depicted in Fig. 1.

The leaf, bark, and fruit of D. kaki is known to contain abundant condensed tannins, and we confirmed their presence using ¹³C-NMR analysis; the spectrum of the H₂O residue of Shitei showed broadening signals corresponding to a catechin unit with the following spectral data,¹²⁾ as shown in Fig. 3: ¹³C-NMR δ: 155 (C-5, 7, 8a), 145 (C-3′, 4′), 130 (C-1′), 115 (C-6′), 105 (C-2′, 5′), 95–105 (C-4a, 6, 8), 70–80 (C-2, 3, overlapped), 40 (C-4). These data indicated the presence of condensed tannins, and from the result we estimated the molecular weight (MW) of the condensed tannin in Shitei. The 70% acetone extract of Shitei and its fractions were analyzed using a gel permeation chromatography (GPC) column equipped with an HPLC system guided using a polystyrene standard curve (MW: 580, 3180, 21800, 139000, 333000, and 609000). The weight-average MW values of the extract and fractions were calculated as 46041 (70% acetone extract of Shitei), 3446 (n-hexane fraction), 7474 (EtOAc fraction), 22197 (n-BuOH fraction), and 75739 (H₂O residue). These data show that the Shitei extract contains highly polymerized condensed tannins, which were abundant in the H₂O residue.

Fig. 3. ¹³C-NMR Spectrum (126 MHz, Acetone-d₆: D₂O [7: 3]) of H₂O Residue of Shitei

(Color figure can be accessed in the online version.)

Analysis of Ingredients of Shiteito

To examine the active ingredients in Shiteito, hot-water extracts of Shiteito and each of its components (clove, ginger, and Shitei) were prepared and analyzed using HPLC (Fig. 4). The results showed main peaks that confirmed the Shiteito-contained clove; therefore, the hot-water extract of clove was used to isolate the main ingredients. The hot-water extract of clove was partitioned into EtOAc and n-BuOH fractions using solvent extraction, and the fractions were then separated using column chromatography. Subsequent structural analyses using MS and NMR identified the main peaks as gallic acid, ellagic acid, biflorin, and isobiflorin (t_R: 6.8, 42.0, 30.8, and 29.5 min, respectively). Therefore, we investigated the inhibitory activity of each isolate, alone or with other samples, on tumor cell proliferation. Additionally, the difference between Shiteito and the extract product after the manufacturing process was the absence of eugenol, which is the main ingredient in the essential oil of clove.

Fig. 4. HPLC Charts of Shiteito, Shiteito Extract Product, and Hot-Water Extract of Shiteito Components (Clove, Ginger, and Shitei) at UV 254 nm

The HPLC condition is described in Experimental. (Color figure can be accessed in the online version.)

Inhibition of Tumor Cell Proliferation by Shiteito, Its Ingredients and Shitei Extract

Inhibition of the proliferation of human tumor oriented cell-lines (Hep3B: hepatocellular carcinoma, Caco-2: colon adenocarcinoma, and AGS: gastric adenocarcinoma) by the test substances was estimated by measuring the absorbance of formazan dye and lactose dehydrogenase (LDH) activity of the culture medium (Tables 1-1–1-3). Evidence of decreased cell viability and significantly high LDH activity following treatment with the test substances would suggest cytotoxicity. Shiteito is used for the treatment of chronic hiccups associated with anticancer drug treatment and, therefore, a cytotoxic agent would not be suitable for concomitant use. Shiteito and its extract product (50 and 100 µg/mL) showed weak inhibition of Hep3B cell proliferation (p < 0.05), and the ingredients, gallic acid, biflorin, and isobiflorin, also showed weak activity against Caco-2 cells at high concentrations (100 µM). In contrast, ellagic acid showed significant cytotoxicity against Hep3B cells, and significant inhibition of AGS cell proliferation in a concentration-dependent manner. In addition, the EtOAc fraction of the 70% acetone extract of Shitei significantly inhibited the proliferation of Caco-2 (at 12.5, 25, and 50 µM) and AGS (at 25 and 50 µM) cells, while showing strong cytotoxicity against Hep3B (>12.5 µg/mL), Caco-2, and AGS (both 100 µg/mL) cells. Exploration of the ingredients of Shitei led to the isolation of phenolic compounds, including flavonoids and their glycoside, from the EtOAc fraction, which would contribute to the observed activity. In particular, the n-hexane, n-BuOH, and H₂O fractions of the 70% acetone extract of Shitei significantly inhibited the proliferation of Hep3B cells, except for the 100 µg/mL concentration of the n-hexane fraction.

Table 1-1. Inhibitory Activity of Shiteito, Shiteito Extract Product, and Shitei Hot Water Extracts against Cell Line (Hep-3B, Caco-2, and AGS) Proliferation

Sample		% blank
Cell line		12.5 µg/mL	25 µg/mL	50 µg/mL	100 µg/mL
Shiteito
Hep3B	Cell viability	98.7 ± 1.9	99.7 ± 0.6	94.6 ± 1.6*	94.4 ± 0.1*
	LDH activity	93.5 ± 5.0	91.1 ± 3.9	91.9 ± 3.5	87.3 ± 5.9
Caco-2	Cell viability	99.5 ± 2.0	102.0 ± 2.4	101.6 ± 1.9	102.3 ± 1.5
	LDH activity	98.4 ± 5.4	87.3 ± 3.3	83.3 ± 8.8	78.3 ± 2.0
AGS	Cell viability	99.7 ± 1.8	101.0 ± 1.1	103.9 ± 1.9	103.2 ± 5.1
	LDH activity	86.4 ± 0.1	76.5 ± 1.6	71.7 ± 1.6	65.8 ± 2.2
Shiteito extract product
Hep3B	Cell viability	98.8 ± 1.1	98.0 ± 0.7	95.6 ± 0.2*	93.2 ± 3.4*
	LDH activity	84.8 ± 6.3	85.3 ± 3.0	82.4 ± 3.5	79.2 ± 3.0
Caco-2	Cell viability	98.6 ± 1.6	98.5 ± 1.5	100.6 ± 1.4	98.8 ± 2.2
	LDH activity	94.4 ± 8.0	88.9 ± 6.5	91.7 ± 8.2	83.7 ± 4.9
AGS	Cell viability	100.3 ± 1.5	102.0 ± 10.0	107.4 ± 3.6	106.7 ± 2.1
	LDH activity	85.4 ± 2.7	83.5 ± 1.9	78.8 ± 1.6	71.6 ± 1.8
Shitei hot water extract
Hep3B	Cell viability	97.9 ± 0.6	95.5 ± 1.7	93.2 ± 2.1*	91.8 ± 0.8**
	LDH activity	83.6 ± 4.2	79.3 ± 3.8	81.0 ± 8.8	93.1 ± 21.6
Caco-2	Cell viability	96.3 ± 1.0	99.1 ± 2.1	98.2 ± 1.0	98.5 ± 2.2
	LDH activity	95.7 ± 1.9	85.3 ± 5.5	85.3 ± 4.6	86.4 ± 2.1
AGS	Cell viability	103.9 ± 3.6	93.8 ± 14.6	102.1 ± 3.1	99.8 ± 7.7
	LDH activity	90.7 ± 2.7	82.2 ± 2.2	75.2 ± 3.0	72.4 ± 1.0

Upper and lower columns indicate cell viability estimated using Cell Count Reagent SF, and lactate dehydrogenase (LDH) activity measured using the cytotoxicity LDH assay kit-WST. Data points are means ± standard deviation (S.D.) (n = 3). Significant decreased cell viability compared with blank * p < 0.05 and ** p < 0.01. Significant increased LDH activity compared with blank ^# p < 0.05 and ^## p < 0.01.

Table 1-2. Inhibitory Activity of a 70% Acetone Extract of Shiteito and Its Fractions (n-Hexane, EtOAc, n-BuOH, and H₂O) against Cell Line (Hep-3B, Caco-2, and AGS) Proliferation

Sample		% blank
Cell line		12.5 µg/mL	25 µg/mL	50 µg/mL	100 µg/mL
Shitei_70% acetone extract
Hep3B	Cell viability	97.3 ± 1.5*	96.9 ± 1.6*	94.1 ± 1.2**	86.9 ± 1.9**
	LDH activity	85.5 ± 1.6	83.3 ± 4.0	82.8 ± 2.9	100.9 ± 1.4
Caco-2	Cell viability	95.1 ± 1.9*	96.2 ± 0.7	95.2 ± 1.0*	96.8 ± 1.1
	LDH activity	78.1 ± 1.4	74.1 ± 3.7	73.0 ± 1.7	74.2 ± 8.6
AGS	Cell viability	105.6 ± 5.9	105.0 ± 2.4	101.8 ± 2.2	100.7 ± 4.6
	LDH activity	94.8 ± 2.3	84.5 ± 1.2	78.9 ± 0.3	74.6 ± 0.8
Shitei_n-hexane fraction
Hep3B	Cell viability	96.1 ± 1.5**	93.8 ± 2.0**	85.9 ± 1.6**	68.9 ± 1.2**
	LDH activity	81.3 ± 6.0	78.6 ± 2.2	80.5 ± 2.8	114.3 ± 9.3^#
Caco-2	Cell viability	97.8 ± 0.7	101.0 ± 2.8	97.3 ± 1.8	89.1 ± 0.8**
	LDH activity	82.5 ± 3.3	77.4 ± 2.2	75.4 ± 2.3	91.6 ± 3.2
AGS	Cell viability	116.2 ± 2.0	113.9 ± 5.9	109.5 ± 8.9	6.6 ± 0.6**
	LDH activity	96.5 ± 3.6	89.0 ± 2.8	87.1 ± 1.6	124.4 ± 6.7^##
Shitei_EtOAc fraction
Hep3B	Cell viability	80.9 ± 1.4**	66.5 ± 1.0**	53.7 ± 0.8**	27.6 ± 5.0**
	LDH activity	158.4 ± 21.3^##	199.9 ± 41.2^##	233.5 ± 34.6^##	482.8 ± 87.8^##
Caco-2	Cell viability	91.4 ± 1.6**	77.7 ± 3.5**	70.9 ± 1.1**	55.7 ± 3.4**
	LDH activity	82.2 ± 6.6	84.2 ± 8.7	84.5 ± 4.3	139.0 ± 4.2^##
AGS	Cell viability	96.7 ± 1.6	91.2 ± 2.9**	76.7 ± 4.1**	5.8 ± 0.3**
	LDH activity	76.8 ± 0.7	72.1 ± 0.4	77.1 ± 2.2	144.9 ± 3.6^##
Shitei_n-BuOH fraction
Hep3B	Cell viability	95.7 ± 1.1**	94.5 ± 1.6**	92.2 ± 1.0**	86.6 ± 0.2**
	LDH activity	81.4 ± 1.9	82.4 ± 1.2	84.9 ± 1.3	84.7 ± 1.3
Caco-2	Cell viability	99.4 ± 1.2	98.9 ± 0.2	94.4 ± 0.9*	91.2 ± 0.4**
	LDH activity	77.6 ± 0.8	76.2 ± 2.9	77.6 ± 0.5	77.6 ± 3.2
AGS	Cell viability	99.5 ± 2.4	99.4 ± 5.8	98.5 ± 3.9	101.4 ± 4.9
	LDH activity	86.4 ± 2.6	82.7 ± 1.4	82.2 ± 1.3	83.2 ± 1.4
Shitei_H₂O residue
Hep3B	Cell viability	91.7 ± 1.8**	91.7 ± 1.8**	89.5 ± 1.0**	85.3 ± 0.6**
	LDH activity	80.8 ± 7.2	82.4 ± 8.4	82.5 ± 5.1	88.2 ± 5.2
Caco-2	Cell viability	94.9 ± 3.7	96.7 ± 3.1	96.1 ± 2.1	90.2 ± 0.8**
	LDH activity	83.3 ± 6.3	77.5 ± 1.3	80.4 ± 1.2	85.5 ± 5.1
AGS	Cell viability	102.6 ± 3.9	101.9 ± 3.0	101.9 ± 1.5	102.0 ± 1.5
	LDH activity	92.7 ± 3.0	88.4 ± 1.8	87.8 ± 2.9	87.6 ± 1.3

Upper and lower columns indicate cell viability estimated using Cell Count Reagent SF, and lactate dehydrogenase (LDH) activity measured using the cytotoxicity LDH assay kit-WST. Data points are means ± S.D. (n = 3). Significant decreased cell viability compared with blank * p < 0.05 and ** p < 0.01. Significant increased LDH activity compared with blank ^# p < 0.05 and ^## p < 0.01.

Table 1-3. Inhibitory Activity of the Main Ingredients of Shiteito (Gallic Acid, Ellagic Acid, Biflorin, and Isobiflorin) and Camptothecin (Positive Control) against Cell Line (Hep-3B, Caco-2, and AGS) Proliferation

Sample		% blank
Cell line		12.5 µM	25 µM	50 µM	100 µM
Gallic acid
Hep3B	Cell viability	98.3 ± 1.0	97.3 ± 1.3	95.9 ± 2.4	96.2 ± 2.0
	LDH activity	100.3 ± 2.4	98.1 ± 4.9	107.9 ± 10.5	114.8 ± 3.3^#
Caco-2	Cell viability	96.8 ± 1.6	99.1 ± 5.4	95.9 ± 4.0	95.1 ± 1.8*
	LDH activity	95.5 ± 4.1	94.8 ± 8.4	102.0 ± 8.6	104.6 ± 3.0
AGS	Cell viability	98.4 ± 3.0	97.8 ± 7.1	104.0 ± 2.7	94.5 ± 9.5
	LDH activity	85.1 ± 1.0	84.4 ± 2.9	88.7 ± 3.8	100.2 ± 2.4
Ellagic acid
Hep3B	Cell viability	93.1 ± 2.4*	82.2 ± 2.1**	74.5 ± 1.8**	62.1 ± 1.4**
	LDH activity	117.0 ± 11.5	224.2 ± 27.8^##	325.2 ± 40.8^##	490.2 ± 10.8^##
Caco-2	Cell viability	99.5 ± 1.7	98.0 ± 2.6	98.0 ± 3.2	97.6 ± 3.3
	LDH activity	82.7 ± 5.8	91.6 ± 6.1	99.2 ± 1.8	95.2 ± 0.8
AGS	Cell viability	96.5 ± 11.7	82.9 ± 0.5**	53.6 ± 0.8**	39.1 ± 1.4**
	LDH activity	76.7 ± 1.5	73.2 ± 3.2	75.1 ± 2.2	87.7 ± 3.1
Biflorin
Hep3B	Cell viability	97.8 ± 1.4	96.5 ± 2.4	97.5 ± 1.1	96.8 ± 0.8
	LDH activity	80.9 ± 3.1	88.4 ± 4.0	90.1 ± 3.9	99.1 ± 2.8
Caco-2	Cell viability	98.3 ± 2.2	98.2 ± 1.0	96.4 ± 1.0	94.4 ± 1.5*
	LDH activity	90.2 ± 3.7	91.6 ± 3.3	89.9 ± 4.8	101.5 ± 1.7
AGS	Cell viability	105.5 ± 2.0	105.0 ± 3.0	101.1 ± 6.0	97.2 ± 7.0
	LDH activity	88.8 ± 0.8	88.6 ± 0.4	86.9 ± 3.7	91.1 ± 3.8
Isobiflorin
Hep3B	Cell viability	96.2 ± 1.7	93.8 ± 2.1*	94.7 ± 3.1	95.3 ± 1.7*
	LDH activity	83.1 ± 3.8	83.4 ± 7.9	107.6 ± 46.7	116.3 ± 31.0
Caco-2	Cell viability	97.1 ± 2.4	96.9 ± 0.7	95.6 ± 2.9	94.2 ± 2.6*
	LDH activity	93.8 ± 5.1	91.7 ± 1.6	95.3 ± 6.1	99.3 ± 5.6
AGS	Cell viability	103.1 ± 3.4	97.0 ± 2.9	101.5 ± 1.7	96.5 ± 2.1
	LDH activity	89.2 ± 3.9	87.6 ± 1.1	89.5 ± 3.3	89.3 ± 0.8
Sample		% blank
Cell line		1.25 µM	2.5 µM	5 µM	10 µM
Camptothecin
Hep3B	Cell viability	46.5 ± 1.4**	38.9 ± 0.8**	35.6 ± 0.6**	33.8 ± 0.2**
	LDH activity	149.0 ± 9.9^##	170.5 ± 1.0^##	175.0 ± 9.0^##	199.8 ± 10.2^##
Caco-2	Cell viability	43.4 ± 0.6**	49.4 ± 1.6**	47.8 ± 1.0**	49.7 ± 1.2**
	LDH activity	234.1 ± 4.9^##	128.4 ± 5.4^##	88.2 ± 2.0	88.4 ± 1.4
AGS	Cell viability	12.8 ± 1.3**	8.2 ± 0.1**	10.8 ± 0.4**	11.5 ± 0.7**
	LDH activity	166.3 ± 6.1^##	126.6 ± 4.0^##	110.0 ± 0.3^##	105.7 ± 2.6^#

Upper and lower columns indicate cell viability estimated using Cell Count Reagent SF, and lactate dehydrogenase (LDH) activity measured using the cytotoxicity LDH assay kit-WST. Data points are means ± S.D. (n = 3). Significant decreased cell viability compared with blank * p < 0.05 and ** p < 0.01. Significant increased LDH activity compared with blank ^# p < 0.05 and ^## p < 0.01.

The difference in cytotoxicity of the test substances against the cell-lines was not clear; however, these data indicate that Shiteito and Shitei extracts could simultaneously assist in the cancer treatment by preventing chronic hiccups caused by some anticancer drugs.

Experimental

Materials and General Methods

NMR spectra were recorded using a Bruker AVANCE 500 (500 MHz for ¹H and 126 MHz for ¹³C) with chemical shifts given in δ (ppm) values relative to those of the solvent (MeOH-d₄ [δ_H 3.30, δ_C 49.0] and acetone-d₆ [δ_H 2.05, δ_C 29.8]) using a tetramethylsilane (TMS) scale. HR-ESI-MS spectra were obtained using a micrOTOF-Q (Bruker) mass spectrometer. Reversed-phase HPLC was performed using a Shimadzu LC-10Avp system (Kyoto, Japan), using a YMC-pack ODS-AQ (ϕ2.0 × 150 mm) column at 40 °C; the mobile phase was run at a flow rate of 0.25 mL/min.

The analyses were conducted using mobile phase A (100 µM phosphate buffer) and B (MeOH) at the following linear gradient: 0–50% B from 0–30 min, 50–60% B from 30–50 min, and 0% B from 50–75 min, monitored at 210–400 nm. GPC analysis was performed using the TSKgel super AW5000 column (ϕ6.0 × 150 mm, Tosoh Co., Tokyo, Japan) at 35 °C at a flow rate of 0.25 mL/min, and UV detection at a wavelength of 280 nm. The polystyrene standards (SL-105, MW: 580, 3180, 21800, 139000, 333000, and 609000) were purchased from Showa Denko K. K. (Tokyo, Japan). The GPC data was analyzed using Chromato-Pro-GPC software (Run Time Co., Tokyo, Japan).

Column chromatography was conducted using Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), Toyopearl HW-40 (coarse grade: Tosoh Co.), YMC-gel ODS-AQ12S50 (YMC Co., Ltd., Kyoto, Japan), MCI-gel CHP20P (Mitsubishi Chemical Co.), Chromatorex ODS (Fuji Silysia Chemical Ltd., Aichi, Japan), Develosil ODS (Nomura Chemical Co., Ltd., Aichi, Japan), and Sephadex LH-20 (GE Healthcare Japan, Tokyo, Japan). Shitei and Shiteito extract products were purchased from Tochimoto Tenkaido Co., Ltd., (Lot: 023415002, Osaka, Japan) and Kotaro Pharmaceutical Co., Ltd., (Lot: YH447, Osaka, Japan), respectively.

The Hep3B (human Negroid hepatocyte carcinoma, 86062703, Lot: 13C010), Caco-2 (human Caucasian colon adenocarcinoma, 86010202, Lot: 16F025), and AGS (human Caucasian gastric adenocarcinoma, 89090402, Lot: 12H012) were purchased from DS Pharma Biomedical (Osaka, Japan). Cell growth medium No.104 was purchased from DS Pharma Biomedical (Osaka, Japan). Ham’s F-12K was purchased from Thermo Fisher Scientific K. K. (Tokyo, Japan). The cytotoxicity LDH assay kit-WST and Cell Count Reagent SF were purchased form Dojindo (Kumamoto, Japan) and Nacalai Tesque (Kyoto, Japan), respectively.

Extraction and Isolation of Shitei

Shitei (1.5 kg) was macerated with 70% acetone (15 L) overnight at 20–25 °C, filtered, and then the extract was concentrated in vacuo to 2 L at <40 °C. The concentrate was further extracted three times with 2L each of n-hexane, EtOAc, and water-saturated n-BuOH to obtain the corresponding fractions at 2.4, 12.5, and 28.8 g yield, respectively, as well as the H₂O residue (118.1 g).

The EtOAc fraction (10.0 g) was suspended in aqueous MeOH, and the soluble portion was fractionated using Toyopearl HW-40 column chromatography (ϕ2.2 × 40 cm), eluted with the same aqueous MeOH (30→40→50→60→70%)→MeOH to yield each eluate (30%: 694.4 mg, 40%: 958.5 mg, 50%: 790.6 mg, 60%: 694.9 mg, 70%: 650.7 mg, and MeOH: 614.5 mg) and the insoluble residue (4.61 g).

The 50% MeOH eluate (700.0 mg) was separated using YMC-gel ODS-AQ column chromatography (ϕ1.1 × 40 cm) with aqueous MeOH (30→40%)→MeOH to obtain fractions A1–A22 (30%: A1–A16, 40%: A17–A21, and MeOH: A22) to yield the fractions A4, A10, A11, A13, and A15 as (+)-catechin (10, 67.7 mg), quercetin 3-O-galactoside (20, 11.0 mg), quercetin 3-O-glucoside (19, 47.5 mg), kaempferol 3-O-galactoside (18, 19.0 mg), kaempferol 3-O-glucoside (17, 19.1 mg), respectively. Fraction A2 (104.9 mg) was fractionated using MCI-gel CHP20P column chromatography (ϕ1.1 × 20 cm) and Sephadex LH-20 column chromatography (ϕ1.1 × 8 cm) to yield gallic acid (4, 4.3 mg), procyanidin B1 (26, 11.3 mg), and gallocatechin (11, 9.6 mg). Fraction A6 (12.1 mg) was fractionated using Sephadex LH-20 column chromatography (ϕ1.1 × 20 cm) eluted with EtOH, and the obtained fraction was further separated using preparative TLC to yield vanillic acid (3, 2.3 mg) and 3,5-dimethoxy-4-hydroxybenzoic acid (5, 0.9 mg). Fraction A12 was fractionated using Sephadex LH-20 column chromatography (ϕ1.1 × 20 cm) eluted with EtOH, and the obtained fraction was further purified using the Chromatorex ODS column (ϕ1.1 × 8 cm), which was eluted using H₂O→aqueous MeOH (10→20→30%)→MeOH to yield dehydroconiferyl alcohol (24, 7.9 mg) from the 30% MeOH eluate.

The 60% MeOH eluate (600.0 mg) was separated using YMC-gel ODS-AQ column chromatography (ϕ1.1 × 40 cm) with aqueous MeOH (30→40%)→MeOH to obtain fractions B1–B20 (30%: B1–B18, 40%: B19, and MeOH: B20) which yielded the fractions B9 and B14 as taxifolin (13, 6.4 mg) and quercetin 3-O-glucoside-2″-O-gallate (23, 7.0 mg), respectively. Fraction B2 (20.2 mg) was separated using Sephadex LH-20 column chromatography (ϕ1.1 × 8 cm) eluted with EtOH, and the obtained fraction was further purified using MCI-gel CHP20P column chromatography (ϕ1.1 × 8 cm) with H₂O→aqueous MeOH (10→20%)→MeOH to yield procyanidin B3 (27, 1.9 mg) from the 20% MeOH eluate. Fraction B3 (28.0 mg) was separated using MCI-gel CHP20P column chromatography (ϕ1.1 × 8 cm) with H₂O→aqueous MeOH (10→20%)→MeOH, and the 20% MeOH eluate (2.9 mg) was further separated using Sephadex LH-20 column chromatography (ϕ1.1 × 8 cm) eluted with EtOH to obtain 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (9, 0.2 mg) and (+)-catechin (10, 1.9 mg). Fraction B6 (18.2 mg) was separated using MCI-gel CHP20P column chromatography (ϕ1.1 × 8 cm) with H₂O→aqueous MeOH (10→20→30%)→MeOH, and the 30% MeOH eluate (2.9 mg) was further purified using Sephadex LH-20 column chromatography (ϕ1.1 × 8 cm) eluted with EtOH to obtain ampelopsin (14, 0.8 mg). Fraction B11 (9.0 mg) was separated using Sephadex LH-20 column chromatography (ϕ1.1 × 8 cm) eluted with EtOH to yield aromadendrin (12, 2.3 mg). Fraction B18 (128.1 mg) was separated using MCI-gel CHP20P (ϕ1.1 × 20 cm), Sephadex LH-20 (ϕ1.1 × 8 cm), and Chromatorex ODS column (ϕ1.1 × 8 cm) to yield kaempferol 3-O-glucoside-2″-O-gallate (21, 2.7 mg), and kaempferol 3-O-galactoside-2″-O-gallate (22, 2.7 mg).

The 40% MeOH eluate (958.5 mg) was separated using YMC-gel ODS-AQ column chromatography (ϕ1.1 × 40 cm) with aqueous MeOH (10→20→30→40→50%)→MeOH to obtain fractions C1–C22 (10%: C1, 20%: C2–C6, 30%: C7–C15, 40%: C16–C20, 50%: C21, and MeOH: C22), monitored using reversed-phase HPLC. Fraction C3 (6.3 mg) was separated using the MCI-gel CHP20P column (ϕ1.1 × 20 cm), and eluted with 10% MeOH to yield protocatechuic acid (2, 1.4 mg), and 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (9, 1.1 mg). Fractions C8 (36.2 mg) and C9 (367.8 mg) were separated in combination using MCI-gel CHP20P (ϕ1.1 × 20 cm), Sephadex LH-20 column chromatography (ϕ1.1 × 8 cm), and Chromatorex ODS column chromatography (ϕ1.1 × 6 cm) to obtain cis-p-coumaric acid (7, 0.4 mg), trans-p-coumaric acid (8, 0.8 mg), scopoletin (6, 2.3 mg), and compound 1 (2.1 mg). Fraction C10 (13.5 mg) was separated using Chromatorex ODS (ϕ1.1 × 6 cm), Sephadex LH-20 (ϕ1.1 × 7 cm), and subsequent MCI-gel CHP20P (ϕ1.1 × 5 cm) column chromatography eluted with 50% MeOH to obtain 1-(4′-hydroxy-3′-methoxyphenyl)-2-[4″-(3-hydroxypropyl)-2″-6″-dimethoxyphenoxy]propane-1,3-diol (25, 0.8 mg).

HPLC analysis of authentic samples of kaempferol (15) and quercetin (16) at UV 360 nm showed peaks at 50.70 and 46.63 min, respectively. The HPLC data of the EtOAc fraction also showed peaks at 50.70 and 46.63 min under the same condition, which indicated the presence of 15 and 16.

Compound 1: [α]²⁰_D +2.5° (c = 0.1, MeOH). UV λ_max (MeOH) nm (log ε): 229 (4.14), 280 (3.90), 305 (3.79). ¹H-NMR (500 MHz, MeOH-d₄) δ: 7.60 (1H, dd, J = 2.0, 8.5 Hz, H-6), 7.55 (1H, d, J = 2.0 Hz, H-2), 6.88 (1H, d, J = 2.0 Hz, H-2′), 6.79 (1H, d, J = 8.5 Hz, H-5), 6.75 (1H, dd, J = 2.0, 8.5 Hz, H-6′), 6.71 (1H, d, J = 8.5 Hz, H-5′), 4.74 (1H, dd, J = 5.5, 8.5 Hz, H-9), 4.24 (1H, dd, J = 8.5, 10.5 Hz, H-8), 3.85 (3H, s, 3-OMe), 3.81 (3H, s, 3′-OMe), 3.69 (1H, dd, J = 5.5, 10.5 Hz, H-8). ¹³C-NMR (126 MHz, MeOH-d₄) δ: 199.7 (C-7), 153.1 (C-4), 149.3 (C-3′), 149.0 (C-3), 147.0 (C-4′), 130.5 (C-1), 129.9 (C-1′), 125.2 (C-6), 122.3 (C-6′), 116.6 (C-5′), 115.7 (C-5), 112.9 (C-2′), 112.7 (C-2), 65.5 (C-8), 56.43, 56.38 (each 1C, 3-OMe, 3′-OMe), 56.32 (C-9). HR-ESI-MS: m/z 317.1024 (M−H)⁻ (calcd for C₁₇H₁₈O₆-H, 317.1031).

Isolation of Constituents of Clove

Clove (50 g) was boiled with hot water (500 mL) for 1 h, and the filtered extract was successively partitioned using EtOAc and n-BuOH (900 mL each) to obtain EtOAc (777.7 mg) and n-BuOH (852.5 mg) fractions and H₂O residue (3.38 g). The EtOAc fraction (749.2 mg) was subjected to Chromatorex ODS column chromatography (ϕ1.1 × 25 cm) eluted with aqueous MeOH (20→30→40%)→MeOH to obtain gallic acid (86.8 mg) and ellagic acid (171.3 mg) from the 20% MeOH and MeOH eluates, respectively. The n-BuOH fraction (820.7 mg) was also subjected to Chromatorex ODS column chromatography (ϕ1.1 × 25 cm) eluted with H₂O→10% MeOH→MeOH to yield isobiflorin (92.2 mg) and biflorin (51.4 mg) from the 10% MeOH eluate.

Cell Culture

Each human cell-line (Hep3B: hepatocellular carcinoma, Caco-2: colon adenocarcinoma, and AGS: gastric adenocarcinoma) was seeded in culture medium (Hep3B and Caco-2: cell growth medium No.104 plus 10% fetal bovine serum [FBS], AGS: Ham’s F-12K + 10% FBS) at a density of 1.0 × 10³ cells/well in 96-well plates. After incubation for 24 h at 37 °C in an atmosphere of 5% CO₂, the culture medium was supplemented with the various samples [Shiteito, Shiteito extract product, hot-water extract of Shitei, Shitei extracts (70% acetone extract and its n-hexane, EtOAc, and n-BuOH fractions and H₂O residue), and constituents of Shiteito (gallic acid, ellagic acid, biflorin, and isobiflorin)] at final concentrations of 12.5, 25, 50, and 100 µg/mL for the extract, and 12.5, 25, 50, and 100 µM for the isolated components. Camptothecin was used as the positive control to induce apoptosis, and was added at final concentrations of 1.25, 2.5, 5, and 10 µM. After 48 h culture, the culture medium and adherent cells were analyzed using the cytotoxicity LDH assay kit-WST and Cell Count Reagent SF, respectively.

LDH Assay

The culture medium from the previously grown cells was transferred to another 96-well microplate and analyzed using the cytotoxicity LDH assay kit-WST according to the manufacturer’s instruction. Briefly, 100 µL of working solution was added to each well, incubated for 30 min at room temperature, and the microplate was protected from light. After the addition of 50 µL stop solution to each well, the absorbance at 490 nm was measured using a microplate reader. Each experiment was conducted in triplicate, and the data are presented as averages compared with the blank. The significance was determined using Student’s t-test.

Cell Viability

The number of viable adherent cells was estimated using the Cell Count Reagent SF according to the manufacturer’s instruction. Briefly, the test solution (10 µL) was added to the cells and incubated for 1 h at 37 °C in an atmosphere of 5% CO₂, then the absorbance at 450 nm was measured using a microplate reader. Each experiment was conducted in triplicate, and the results are shown as averages compared with the blank. The significance was determined using Student’s t-test.

Acknowledgments

We thank Ms. Hidemi Sugiwaki of Matsuyama University for technical assistance. This work was supported by JSPS KAKENHI Grant Number JP16K18904.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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