Chemical Components in Hedera rhombea Leaves and Their Cytotoxicity

Abstract

Two novel triterpene glycosides (1 and 2), 17 known triterpene glycosides (3–19), two known flavonoid glycosides (20 and 21), and two known norsesquiterpene glucosides (22 and 23) were isolated from Hedera rhombea (Araliaceae) leaves. The structures of 1 and 2 were determined by spectroscopic analysis, including two-dimensional NMR spectroscopy, and chromatographic analysis of the hydrolyzed products. The cytotoxicity of the isolated triterpene glycosides (1–19) against HL-60 human promyelocytic leukemia cells was evaluated. Compounds 9, 10, and 11 were cytotoxic to HL-60 cells with IC₅₀ values of 7.2, 21.9, and 32.8 µM, respectively. Other compounds isolated from the leaves were not cytotoxic at sample concentrations of 50 μM.

Introduction

The genus Hedera belongs to the Araliaceae family. Most of the approximately 300 species identified globally are cultivated as ornamental foliage plants. Hedera rhombea (Miq.) Bean, distributed in Eastern Asia, is a climbing plant with evergreen leaves.¹⁾ Triterpene glycosides (kizutasaponins,^2–5) triterpenes,⁶⁾ and polyacetylenes⁷⁾) have been reported as the chemical constituents of H. rhombea. However, no systematic phytochemical studies have been performed using this plant. As a series of our systematic investigations of biologically active compounds from natural sources, we examined the chemical components in the leaves of H. rhombea. Here, we describe the isolation and structural identification of 23 compounds. These include 19 triterpene glycosides (1–19), two of which are novel (1 and 2), two flavonoid glycosides (20 and 21), and two norsesquiterpene glucosides (22 and 23). The compounds were identified based on spectroscopic analysis, including two-dimensional NMR spectroscopy, and by chromatographic analysis of the hydrolyzed products. The isolated triterpene glycosides (1–19) were evaluated for their cytotoxicity against HL-60 human leukemia cells.

Results and Discussion

Methanol extracts of H. rhombea leaves (3.0 kg) were repeatedly subjected to column chromatography (CC). Compounds 1–23 were identified (Fig. 1).

Fig. 1. Chemical Structures of 1–23 Isolated from the Leaves of Hedera rhombea

Compound 1 was obtained as an amorphous solid. The molecular formula of C₄₈H₈₂O₂₀, was determined by high resolution electrospray-ionization time of flight mass spectroscopy (HR-ESI-TOF-MS) (m/z: 1001.5301 [M + Na]⁺) and ¹³C-NMR. The ¹H- and ¹³C-NMR spectra of 1 contained signals for six tertiary methyl groups [δ_H 2.03 (3H, s, H₃-29)/δ_C 16.8 (C-29), δ_H 1.82 (3H, s, H₃-27)/δ_C 14.3 (C-27), δ_H 1.53 (3H, s, H₃-28)/δ_C 31.7 (C-28), δ_H 1.07 (3H, s, H₃-18)/δ_C 17.3 (C-18), δ_H 1.02 (3H, s, H₃-30)/δ_C 16.7 (C-30), and δ_H 0.88 (3H, s, H₃-19)/δ_C 17.5 (C-19)] and an olefinic group [δ_H 5.77 (1H, t, J = 6.8 Hz, H-24)/δ_C 129.4 (C-24) and 132.1 (C-25)]. These signals were characteristic of the dammarane-24-ene-type triterpene skeleton. Signals for three anomeric protons and carbons [δ_H 5.44 (1H, d, J = 7.6 Hz, H-1″)/δ_C 106.0 (C-1″), δ_H 5.05 (1H, d, J = 7.4 Hz, H-1′)/δ_C 105.4 (C-1′), δ_H 4.91 (1H, d, J = 7.8 Hz, H-1‴)/δ_C 103.5 (C-1‴)] were also observed. Enzymatic hydrolysis of 1 with naringinase yielded 1a as the aglycone. The structure of 1a was identified as (20S)-dammar-24-ene-3β,6α,20,21,26-pentol (1a)⁴⁾ by comparing the ¹H- and ¹³C-NMR data with those reported previously. This was the same as that of the known triterpene glycoside 4, with D-glucose as the carbohydrate moiety. Identification of D-glucose was carried out by direct HPLC analysis of the hydrolysate performed using an aminopropyl-bonded silica gel (Si) column. The exact assignments for the ¹H- and ¹³C-NMR spectra of the glycosyl moieties composed of only D-glucose were solved by detailed analysis of the one-dimensional (1D) totally correlated spectroscopy (TOCSY) followed by interpretation of the ¹H–¹H shift correlation spectroscopy (COSY), ¹H-detected heteronuclear single-quantum coherence (HSQC), and HSQC-TOCSY spectra. The ¹H-NMR subspectra of individual glucosyl units were obtained by selective irradiation of each anomeric proton signal. Subsequent analysis of the ¹H–¹H COSY spectrum sequentially assigned the proton resonances for the three glucosyl units, helping us identifiy their multiplet patterns and coupling constants (Table 1). The HSQC and HSQC-TOCSY spectra correlated the proton signals with the corresponding one-bond coupled carbon shifts. The assigned ¹H- and ¹³C-NMR signals indicated that the glycosyl moieties of 1 comprised a C-2-substituted β-D-glucopyranosyl unit (δ_H-1′ 5.05/δ_C-1′ 105.4: Glc-I) and two terminal β-D-glucopyranosyl units (δ_H-1″ 5.44/δ_C-1″ 106.0: Glc-II; δ_H-1‴ 4.91/δ_C-1‴ 103.5: Glc-III) (Table 1). The anomeric configurations of the glucosyl moieties were ascertained as β by the relatively large ³J_H-1,H-2 values (7.4–7.8 Hz). In the ¹H-detected heteronuclear multiple-bond connectivity (HMBC) spectrum of 1, long-range correlations were observed between the anomeric proton (H-1′) of Glc-I and C-3 of the aglycone at δ_C 89.5, H-1″ of Glc-II and C-2 of Glc-I at δ_C 83.4, and between H-1‴ of Glc-III and C-26 of the aglycone at δ_C 75.2 (Fig. 2). Accordingly, 1 was characterized as (24E)-26-[(β-D-glucopyranosyl)oxy]-6α,20,21-trihydroxydammar-24-en-3β-yl O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside.

Table 1. ¹H- and ¹³C-NMR Chemical Shift Assignments for 1 and 2^a)

1			2
Position	δH	δC	Position	δH	δC
1	1.51 m	39.1	1	1.26 m	39.9
	0.82 m			3.25 m
2	2.31 m	26.8	2	2.14 m	26.3
	1.91 m			2.26 m
3	3.40 m	89.5	3	4.29 m	81.0
4		40.7	4		43.9
5	1.14 d (10.4)b)	57.3	5	1.71 m	47.6
6	4.31 m	67.5	6	1.44 m	17.3
				1.75 m
7	1.88 m	48.1	7	1.75 m	32.9
8	1.99 m	41.4	8	1.29 m	45.5
9	1.47 m	50.5	9	2.60 m	62.4
10		38.9	10		37.4
11	1.49 m	21.9	11		200.0
	1.18 m
12	2.26 m	28.1	12	5.90 s	128.3
	1.46 m
13	2.13 m	41.8	13		168.7
14		50.7	14		43.9
15	1.14 m	31.7	15	1.14 m	28.2
	1.67 m			2.18 m
16	1.95 m	24.8	16	1.91 m	23.1
	1.90 m			2.03 m
17	2.29 m	46.3	17		46.5
18	1.07 s	17.3	18	3.18 dd (13.7, 3.5)	42.0
19	0.88 s	17.5	19	1.05 m	44.3
				1.60 m
20		76.5	20		30.6
21	4.05 d (10.6)	66.8	21	1.03 m	33.7
	4.00 d (10.6)			1.21 m
22	1.95 m	36.2	22	1.72 m	31.5
	2.09 m			1.75 m
23	2.47 m	22.8	23	4.16 d (9.1)	64.0
	2.57 m			3.75 d (9.1)
24	5.77 t (6.8)	129.4	24	1.10 s	13.9
25		132.1	25	1.37 s	17.2
26	4.24 m	75.2	26	1.29 s	19.5
	4.51 m
27	1.82 s	14.3	27	1.30 s	23.5
28	1.53 s	31.7	28		176.1
29	2.03 s	16.8	29	0.84 s	32.7
30	1.02 s	16.7	30	0.77 s	23.3
Glc-I 1′	5.05 d (7.4)	105.4	Ara 1′	5.10  d (6.1)	104.3
2′	4.30 dd (9.2, 7.4)	83.4	2′	4.56 dd (8.5, 6.1)	75.9
3′	4.33 dd (9.2, 9.2)	78.4	3′	4.10 dd (9.6, 8.5)	74.1
4′	4.17 dd (9.2, 9.2)	71.7	4′	4.16 m	69.2
5′	3.94 m	78.0	5′	4.24 dd (12.0, 3.8)	65.5
6′	4.59 br d (12.4)	62.9		3.66 dd (12.0, 10.1)
	4.39 dd (12.4, 3.0)
Glc-II 1″	5.44 d (7.6)	106.0	Rha-I 1″	6.22 brs	101.7
2″	4.08 dd (9.0, 7.6)	77.0	2″	4.73 br d (1.9)	72.3
3″	4.24 dd (9.0, 9.0)	78.1	3″	4.68 dd (8.4, 1.9)	72.6
4″	4.32 dd (9.0, 9.0)	71.8	4″	4.29 dd (8.4, 8.4)	74.0
5″	3.96 m	78.1	5″	4.64 m	69.7
6″	4.51 br d (11.5)	62.8	6″	1.63 d (6.2)	18.5
	4.43 dd (11.5, 3.0)
Glc-III 1‴	4.91 d (7.8)	103.5	Glc-I 1‴	6.23 d (8.3)	95.8
2‴	4.08 dd (9.0, 7.8)	75.2	2‴	4.08 dd (9.1, 8.3)	74.6
3‴	4.25 dd (9.0, 9.0)	78.6	3‴	4.21 dd (9.1, 9.1)	78.6
4‴	4.27 dd (9.0, 9.0)	71.7	4‴	4.32 dd (9.1, 9.1)	70.9
5‴	3.96 m	78.5	5‴	3.96 m	78.1
6‴	4.56 br d (11.9)	62.8	6‴	4.69 dd (12.0, 3.0)	69.2
	4.40 dd (11.9, 3.5)			4.30 br d (12.0)
			Glc-II 1″″	4.96 d (7.8)	104.9
			2″″	3.94 dd (9.0, 7.8)	75.3
			3″″	4.14 dd (9.3, 9.0)	76.5
			4″″	4.42 dd (9.3, 9.3)	78.3
			5″″	3.64 m	77.2
			6″″	4.19 br d (11.4)	61.3
				4.08 dd (11.4, 3.0)
			Rha-II 1″‴	5.86 br s	102.7
			2″‴	4.67 br d (3.2)	72.6
			3″‴	4.55 dd (9.4, 3.2)	72.8
			4″‴	4.33 dd (9.4,8.0)	74.0
			5″‴	4.98 m	70.3
			6″‴	1.70 d (6.2)	18.5

a) Spectra were measured in C₅D₅N. b) Coupling constants (J) in Hz are given in parentheses.

Fig. 2. HMBC Correlations of the Glycoside Moieties of 1 and 2

Compound 2 was determined to be C₅₉H₉₄O₂₇ by HR-ESI-TOF-MS (m/z: 1257.5878 [M + Na]⁺) and ¹³C-NMR data. The UV spectrum showed absorption maximum owing to a conjugated carbonyl group (250.0 nm). The ¹H- and ¹³C-NMR spectra of 2 displyed signals for five anomeric protons and carbons [δ_H 6.23 (1H, d, J = 8.3 Hz, H-1‴)/δ_C 95.8 (C-1‴), δ_H 6.22 (1H, br s, H-1″)/δ_C 101.7 (C-1″), δ_H 5.86 (1H, br s, H-1‴″)/δ_C 102.7 (C-1‴″), δ_H 5.10 (1H, d, J = 6.1 Hz, H-1′)/δ_C 104.3 (C-1′), and δ_H 4.96 (1H, d, J = 7.8 Hz, H-1″″)/δ_C 104.9 (C-1″″)] and the methyl groups of two 6-deoxyhexoses [δ_H 1.70 (d, J = 6.2 Hz, H₃-6‴″)/δ_C 18.5 (C-1‴″) and δ_H 1.63 (d, J = 6.2 Hz, H₃-6″)/δ_C 18.5 (C-1″)], as well as six triterpene methyl groups [δ_H 1.37 (3H, s, H₃-25)/δ_C 17.2 (C-25), δ_H 1.30 (3H, s, H₃-27)/δ_C 23.5 (C-27), δ_H 1.29 (3H, s, H₃-26)/δ_C 19.5 (C-26), δ_H 1.10 (3H, s, H₃-24)/δ_C 13.9 (C-24), δ_H 0.84 (3H, s, H₃-29)/δ_C 32.7 (C-29), and δ_H 0.77 (3H, s, H₃-30)/δ_C 23.3 (C-30)], an olefinic group [δ_H 5.90 (1H, s, H-12)/δ_C 128.3 (C-12) and 168.7 (C-13)], and two carbonyl carbons [δ_C 200.0 (C-11) and 176.1 (C-28)]. Acid hydrolysis of 2 with 0.5 M HCl yielded an aglycone (2a), and D-glucose, D-arabinose, and L-rhamnose as the monosaccharide moieties. The structure of 2a was identified as 11-oxohederagenin⁸⁾ by comparing the ¹H- and ¹³C-NMR data with those reported previously. The C-3 oxymethine carbon and C-28 carbonyl carbon were observed at δ_C 81.0 and 176.1, respectively, in the ¹³C-NMR of 2, suggesting that 2 is a 3,28-bisdesmoside of 11-oxohederagenin. The ¹H- and ¹³C-NMR signals for the glycosyl moieties of 2 were assigned by the same procedures used for 1, using the ¹H–¹H COSY, 1D-TOCSY, HSQC, and HSQC-TOCSY spectral data. The findings indicated that the glycosyl moieties of 2 comprised two terminal α-L-rhamnopyranosyl units (Rha-I; Rha-II), an α-L-arabinopyranosyl unit glycosylated at C-2 (δ_C-2′ 75.9) (Ara), a β-D-glucopyranosyl unit glycosylated at C-6 (δ_C-6‴ 69.2) (Glc-I), and a β-D-glucopyranosyl unit glycosylated at C-4 (δ_C-4″″ 78.3) (Glc-II) (Table 1). The anomeric configurations of the Ara, Glc-I, and Glc-II moieties were ascertained by the relatively large ³J_{H-1, H-2} values (6.1–8.3 Hz). The anomeric protons of Rha-I and Rha-II were confirmed to be equatorial, and possessed an α-pyranoid anomeric form (¹C₄) due to the large ¹J_C-1,H-1 values (169.9 Hz for Rha-I and 168.4 Hz for Rha-II).⁹⁾ In the HMBC spectrum of 2, long-range correlations were observed between H-1″ of Rha-II and C-4″″ of Glc-II at δ_C 78.3, H-1″″ of Glc-II and C-6‴ of Glc-I at δ_C 69.2, H-1‴ of Glc-I and C-28 of the aglycone at δ_C 176.1, H-1″ of Rha-I and C-2″ of Ara at δ_C 75.9, and between H-1′ of Ara and C-3 of the aglycone at δ_C 81.0 (Fig. 2). Thus, 2 was determined to be 23-hydroxy-3β-[(O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl)oxy]-11-oxo-olean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester.

Compounds 3–23 were identified as kizutasaponin K₇ (3),³⁾ kizutasaponin K_7b (4),⁴⁾ kizutasaponin K₉ (5),⁴⁾ kizutasaponin K₁₃ (6),⁴⁾ kizutasaponin K_7a (7),⁴⁾ kizutasaponin K₅ (8),³⁾ kizutasaponin K₆ (9),⁵⁾ eleutheoside K (10),¹⁰⁾ akeboside Stb (11),¹¹⁾ HN-saponin F (12),¹²⁾ kizutasaponin K₈ (13),⁵⁾ kizutasaponin K₁₁ (14),⁵⁾ kizutasaponin K₁₀ (15),⁵⁾ kizutasaponin K₁₂ (16),⁵⁾ hederasaponin I (17),¹³⁾ glycoside L-H₃ (18),¹⁴⁾ glycoside L-E₂ (19),¹⁴⁾ rutin (20),¹⁵⁾ nicotiflorin (21),¹⁵⁾ (6R,9R)-3-oxo-α-ionol β-D-glucopyranoside (22),¹⁶⁾ and (6R,9S)-3-oxo-α-ionol β-D-glucopyranoside (23)¹⁶⁾ by comparing the physiochemical and spectroscopic data of 3–23 with those in the literature. This is the first report concerning the isolation of triterpene glycosides 17–19, flavonoid glycoside 21, and norsesquiterpene glucosides 22 and 23 from H. rhombea.

The isolated triterpene glycosides (1–19) were evaluated for their cytotoxic activities against HL-60 cells.¹⁷⁾ Compounds 9, 10, and 11 were cytotoxic with IC₅₀ values of 7.2 ± 0.010, 21.9 ± 1.1, and 32.8 ± 0.056 µM, respectively. Etoposide had an IC₅₀ value of 0.70 ± 0.047 µM. Other compounds were not cytotoxic even at concentrations of 50  µM (< 50% growth inhibition).

Kizutasaponin K₆ (9) was the most potently cytotoxic of the isolated triterpene glycosides with an IC₅₀ value of 7.2 µM. Some triterpene glycosides reportedly induce apoptosis in cultured malignant tumor cells. However, no cell morphological changes characteristic of apoptotic cells (e.g., the nuclear chromatin condensation and formation of apoptotic bodies) were observed in HL-60 cells treated with 20 μM of 9 for 24 h (Fig. 3).

Fig. 3. Morphology of HL-60 Cells Treated with 9 or Etoposide HL-60 Cells Were Stained with DAPI after Treatment with Either 20 µM of 9 or 15 µM of Etoposide for 24 h and Observed by a Fluorescence Microscope

Inhibition of cancer cell growth by 9 was evaluated using JFCR39,¹⁸⁾ a panel screening against 39 cancer cell lines derived from various tissues of origin. The concentration of 9 that inhibited growth by 50% (GI₅₀) was measured in each of the cell line. The average logGI₅₀ (MG-MID) of 9 was −4.81 for the JFCR39 panel of screened cells, with values of −5.29 for renal ACHN cells, −5.17 for colon HCT-116 cells, and −5.13 for stomach St-4 cells, indicating sensitivety (Table 2). However, no significant differential cell growth inhibitory activities were observed, indicating that the cell growth inhibition by 9 (posessing surface-active properties) was partially associated with cell membrane interactions.

Table 2. Log GI₅₀ Values of 9 against a Panel Composed of 39 Human Cancer Cell Lines

Type of cancer	Cell line	log GI50 [M]a)
Breast	HBC-4	−4.73
	BSY-1	−4.77
	HBC-5	−4.72
	MCF-7	−4.74
	MDA-MB-231	−4.86
Central nervous system	U251	−4.76
	SF-268	−4.75
	SF-295	−4.82
	SF-539	−4.78
	SNB-75	−4.76
	SNB-78	−4.71
Colon	HCC2998	−4.76
	KM-12	−4.78
	HT-29	−4.78
	HCT-15	−4.71
	HCT-116	−5.17
Lung	NCI-H23	−4.77
	HCI-H226	−4.70
	NCI-H522	−4.85
	NCI-H460	−4.79
	A549	−4.75
	DMS273	−4.76
	DMS114	−4.78
Melanoma	LOX-IMVI	−4.87
Ovary	OVCAR-3	−4.78
	OVCAR-4	−4.89
	OVCAR-5	−4.77
	OVCAR-8	−4.76
	SK-OV-3	−4.75
Kidney	RXF-631 L	−4.79
	ACHN	−5.29
Stomach	St-4	−5.13
	MKN1	−4.84
	MKN-B	−4.76
	MKN-A	−4.76
	MKN45	−4.83
	MKN74	−4.81
Prostate	DU-145	−4.74
	PC-3	−4.85
MG-MIDb)		−4.81
Deltac)		0.48
Ranged)		0.59

a) Log concentration for inhibition of cell growth at 50% relative to control. b) Mean GI₅₀ value in all cell lines tested. c) Difference in the log GI₅₀ value between the most sensitive cells and the MG-MID value. d) Difference in the log GI₅₀ value between the most and least sensitive cells.

In summary, 23 compounds (1–23), including two unprecedented triterpene glycosides (1 and 2), were isolated from the leaves of H. rhombea. Among the isolated triterpene glycosides, kizutasaponin K₆ (9) was the most cytotoxic to HL-60 cells. In the JFCR39 panel screening, renal ACHN, colon HCT-116, and stomach St-4 cell lines were comparatively sensitive to 9. However, 9 did not display significant differential cell growth inhibitory activities.

Experimental

Optical rotations were measured by using a P-1030 (JASCO, Tokyo, Japan) automatic digital polarimeter. UV spectrum was measured with a V-630 UV-Vis spectrophotometer (JASCO). IR spectra were recorded on a FT-IR 620 spectrophotometer (JASCO). NMR spectral data were obtained on a DRX-600 (600 MHz for ¹H-NMR, Bruker, Karlsruhe, Germany) using standard Bruker pulse programs at 300 K. Chemical shifts were presented as δ values with reference to tetramethylsilane (TMS) as an internal standard. HR-ESI-TOF-MS data were recorded on an LCT mass spectrometer (Waters-Micromass, Manchester, U.K.). Diaion HP-20 (Mitsubishi-Chemical, Tokyo, Japan), silica gel Chromatorex BW-300 (300 mesh, Fuji-Silysia Chemical, Aichi, Japan), and octadecylsilanized (ODS) silica gel COSMOSIL 75C₁₈-OPN (75 µM particle size, Nacalai Tesque, Kyoto, Japan) were used for column chromatography (CC). TLC was performed on precoated silica gel 60 F₂₅₄ or RP₁₈ F_254S plates (0.25 mm thick, Merck, Darmstadt, Germany), and spots were visualized by spraying the plates with 10% H₂SO₄ aqueous solution, followed by heating. HPLC was performed with a system consisting of a LC-20AD pump (Shimadzu, Kyoto, Japan), a Shodex OR-2 detector (Showa-Denko, Tokyo, Japan), and a Rheodyne™ injection port (Thermo Fisher Scientific, Waltham, MA, U.S.A.). HL-60 cells (JCRB0085) were obtained from the Japanese Collection of Research Bioresources (JCRB) cell bank (Osaka, Japan). The following materials and reagents were used for the cell culture assays: SH-1300 microplate reader (Corona Electric, Ibaraki, Japan); 96-well flat-bottom plates (Iwaki Glass, Chiba, Japan); fetal bovine serum (FBS), RPMI 1640 medium, etoposide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and naringinase (EC 3.2.1.40) (Sigma-Aldrich, MO, U.S.A.); penicillin G sodium salt, streptomycin sulfate, and phosphate buffered saline (PBS) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). All other chemicals used were of biochemical reagent grade.

Plant Material

Dried H. rhombea leaves were collected from the medicinal plant garden of the Tokyo University of Pharmacy and Life Sciences in May, 2017. A voucher specimen has been deposited in our laboratory (voucher No. DL-2017-010, Department of Medicinal Pharmacognosy).

Extraction and Isolation

Dried leaves of H. rhombea (3.0 kg) were extracted with hot MeOH (20 L). The MeOH extract was concentrated under reduced pressure, and the viscous concentrate (830 g) was passed through a porous-polymer polystyrene resin column (Diaion HP-20 column) (80 mm i.d. × 400 mm) and successively eluted with 30% MeOH (12 L), 50% MeOH (5 L), MeOH (15 L), ethanol (EtOH) (9 L), and EtOAc (4 L). The 50% MeOH eluted fraction (130 g) was subjected to ODS silica gel (Si) CC (66 mm i.d. × 150 mm) and eluted with a stepwise gradient mixture of MeOH–H₂O (2 : 3; 1 : 1; 7 : 3), which produced 8 fractions (A–H). Fraction B was separated by ODS Si CC (66 mm i.d. × 200 mm) eluted with MeCN–H₂O (1 : 4; 1 : 3; and 3 : 7) to give subfractions B-1–B-8. Fraction B-2 was separated by Si CC (45 mm i.d. × 250 mm) eluted with CHCl₃–MeOH–H₂O (90 : 10 : 1; 60 : 10 : 1; 40 : 10 : 1; 20 : 10 : 1) to give further 18 subfractions B-2-1–B-2-18. Fraction B-2-4 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (2 : 3; 9 : 11; 1 : 1; 3 : 2), Si CC (20 mm i.d. × 250 mm) eluted with CHCl₃–MeOH–H₂O (70 : 10 : 1), and by preparative HPLC (10 mm i.d. × 250 mm) using acetonitrile (MeCN)–H₂O (2 : 5) as the mobile phase to yield 22 (7.5 mg) and 23 (4.2 mg). Fraction B-2-11 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (2 : 3; 9 : 11), Si CC (20 mm i.d. × 250 mm) eluted with CHCl₃–MeOH–H₂O (40 : 10 : 1), and by preparative HPLC (10 mm i.d. × 250 mm) using MeCN–H₂O (1 : 1) as the mobile phase to yield 21 (15.8 mg). Fraction B-2-14 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (9 : 1; 11 : 9; 3 : 2) to yield 20 (6.6 mg). Fraction B-2-15 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (1 : 1; 3 : 2; 4 : 1) to yield 6 (18.3 mg). Fraction B-2-17 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (1 : 4; 2 : 5) and MeOH–H₂O (2 : 3; 1 : 1), and by preparative HPLC (10 mm i.d. × 250 mm) using MeCN–H₂O (2 : 5) as the mobile phase to yield 1 (5.4 mg), 2 (6.6 mg), 6 (3.4 mg), 16 (9.7 mg) and 18 (10.1 mg). Fraction B-2-18 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (1 : 4; 1 : 3; 2 : 5; 2 : 3) and MeOH–H₂O (1 : 1) to yield 17 (12.4 mg). Fraction B-5 was purified by ODS Si CC (30 mm i.d. × 250 mm) eluted with MeOH–H₂O (3 : 2), and by preparative HPLC (10 mm i.d. × 250 mm) using MeCN–H₂O (3 : 7) as the mobile phase to yield 15 (5.6 mg). Fraction C was separated by ODS Si CC (66 mm i.d. × 200 mm) eluted with MeCN–H₂O (1 : 4; 1 : 3; 3 : 7) to give 13 subfractions C-1–C-13. Fraction C-8 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (1 : 1) and Si CC (20 mm i.d. × 250 mm) eluted with CHCl₃–MeOH–H₂O (60 : 10 : 1) to yield 4 (4.5 mg) and 5 (4.9 mg). Fraction F was purified by ODS Si CC (30 mm i.d. × 250 mm) eluted with MeOH–H₂O (6 : 1) and MeCN–H₂O (6 : 4) to yield 13 (15.1 mg), 14 (110.0 mg), and 16 (7.5 mg). The MeOH eluted fraction (295 g) was subjected to Si CC (66 mm i.d. × 150 mm) and eluted with a stepwise gradient mixture of EtOAc–MeOH–H₂O (90 : 10 : 1; 60 : 10 : 1; 40 : 10 : 1; 20 : 10 : 1), which produced 21 fractions (a–u). Fraction i was separated by ODS Si CC (66 mm i.d. × 200 mm) eluted with MeCN–H₂O (11 : 3; 1 : 2; 1 : 1) to give 26 subfractions i-1–i-26. Fraction i-5 was purified by ODS CC (30 mm i.d. × 250 mm) eluted with MeCN–H₂O (1 : 3; 1 : 1) to yield 12 (3.2 mg). Fraction i-8 was purified by ODS CC (30 mm i.d. × 250 mm) eluted with MeCN–H₂O (1 : 2) to yield 7 (4.6 mg) and 9 (20.0 mg). Fraction i-9 was purified by ODS CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (3 : 2; 7 : 3) to yield 7 (4.4 mg). Fraction i-12 was purified by Si CC (20 mm i.d. × 250 mm) eluted with CHCl₃–MeOH–H₂O (50 : 10 : 1; 40 : 10 : 1; 30 : 10 : 1; 20 : 10 : 1) and ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (7 : 4; 7 : 3; 8 : 2) to yield 3 (8.8 mg). Fraction i-14 was purified by Si CC (20 mm i.d. × 250 mm) eluted with CHCl₃–MeOH–H₂O (60 : 10 : 1; 20 : 10 : 1; 10 : 10 : 1) to yield 8 (21.4 mg). Fraction i-16 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (7 : 3) and MeCN–H₂O (2 : 3) to yield 12 (3.2 mg). Fraction i-17 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (7 : 3; 8 : 2) and MeCN–H₂O (2 : 3) and Si CC (20 mm i.d. × 250 mm) eluted with CHCl₃–MeOH–H₂O (40 : 10 : 1) to yield 19 (2.4 mg). Fraction i-21 was purified by ODS Si CC (20 mm i.d. × 250 mm) eluted with MeOH–H₂O (2 : 3; 1 : 1; 3 : 2) and Si CC (20 mm i.d. × 250 mm) eluted with CHCl₃–MeOH–H₂O (60 : 10 : 1; 50 : 10 : 1; 40 : 10 : 1) to yield 9 (79.3 mg) and 11 (8.1 mg). Fraction i-23 was purified by ODS CC (30 mm i.d. × 250 mm) eluted with MeOH–H₂O (8 : 2) to yield 10 (3.1 mg).

Compound 1: Amorphous solid; [α]_D²⁵ −35.2 (c = 0.075, MeOH); IR ν_max (film) cm⁻¹: 3389 (OH), 2925 (CH); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) spectra, see Table 1; HR-ESI-TOF-MS m/z: 1001.5301 [M + Na]⁺ (Calcd for C₄₈H₈₂NaO₂₀: 1001.5297).

Enzymatic Hydrolysis of 1

Compound 1 (1.2 mg) was treated with naringinase in AcOH/AcOK buffer (pH 4.3, 2.0 mL) and stirred at room temperature for 96 h. The reaction mixture was subjected to silica gel CC (10 mm i.d. × 150 mm) and eluted with CHCl₃–MeOH–H₂O (70 : 10 : 1), followed by EtOH–Me₂CO (1 : 1) to give 1a (0.5 mg) and a sugar fraction (0.4 mg). The sugar fraction was analyzed by HPLC under the following conditions: column, Capcell Pak NH2 UG80 (4.6 mm i.d. × 250 mm, 5 µm, Shiseido, Tokyo, Japan); solvent, MeCN–H₂O (17 : 3); detection, optical rotation (OR); and flow rate: 1.0 mL/min. HPLC analysis of the sugar fraction showed the presence of D-glucose (t_R = 16.02 min, positive OR).

Compound 2: Amorphous solid; [α]_D²⁵ −23.2 (c = 0.10, MeOH); UV λ_max nm (log ε): 250.0 (4.05), 203.5 (3.86); IR ν_max (film) cm⁻¹: 3384 (OH), 2923 (CH), 1739 and 1651 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) spectra, see Table 2; HR-ESI-TOF-MS m/z: 1257.5878 [M + Na]⁺ (Calcd for C₄₈H₈₂NaO₂₀: 1257.5880).

Acid Hydrolysis of 2

Compound 2 (2.8 mg) was dissolved in 1.0 M HCl (dioxane–H₂O, 1 : 1, 2.0 mL) and was heated at 95 °C for 1.5 h under an argon atmosphere. After cooling, the reaction mixture was neutralized by consective passage through an Amberlite IRA-96SB (Organo, Tokyo, Japan) column (10 mm i.d. × 100 mm) and a Diaion HP-20 column (10 mm i.d. × 100 mm) eluted with MeOH–H₂O (2 : 3) followed by EtOH–Me₂CO (1 : 1) to give 11-oxohederagenin (0.79 mg) and a sugar fraction (1.8 mg). HPLC analysis of the sugar fraction showed the presence of D-glucose (t_R = 16.39 min, positive OR), D-arabinose (t_R = 9.81 min, positive OR), and L-rhamnose (t_R = 7.95 min, negative OR).

Cell Culture and Assay for Cytotoxic Activity against HL-60 Cells

HL-60 cells (JCRB 0085) obtained from the Japanese Collection of Research Bioresources (Osaka, Japan) were cultured in RPMI 1640 medium (HL-60 cells) containing heat-inactivated 10% (v/v) FBS supplemented with L-glutamine, 100 unit/mL penicillin G sodium salt, and 100 µg/mL streptomycin sulfate in a humidified incubator at 37 °C with an atmosphere of 5% CO₂. The cells (HL-60: 4 × 10⁴ cells/mL) were continuously treated with each compound for 72 h, followed by the measurement of cell viability by using an MTT assay.¹⁴⁾

4′,6-Diamidino-2-phenylindole (DAPI) Staining

HL-60 cells (5 × 10⁵) were treated with either 20 µM of 9 or 15 µM of etoposide for 24 h, and collected by centrifugation at 500 × g for 5 min. The cells were fixed with 1% glutaraldehyde at room temperature for 30 min and centrifuged again for 5 min. The collected cells were stained with DAPI at room temperature and observed immediately by fluorescence microscopy.

Acknowledgments

This work was supported by JSPS KAKENHI Grant Number JP 16H06276 [Advanced Animal Model Support (AdAMS)] and JSPS KAKENHI Grant Number JP 18K06735.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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