Schisandrosides A–D, Dibenzocyclooctadiene Lignan Glucosides from the Roots of Schisandra chinensis

Abstract

Four new dibenzocyclooctadiene lignan glucosides, schisandrosides A–D (1–4), as well as two known rare nortriterpenoids, micrandilactone C (5) and propindilactone Q (6), were isolated from the roots of Schisandra chinensis BAILLON (Schisandraceae). The structure of compounds 1–4 were elucidated by physical and spectroscopic data interpretation. To the best of our knowledge, schisandrosides A–D (1–4) represent the first example of a dibenzocyclooctadiene lignan glycoside.

The fruits of Schisandra chinensis BAILLON (Schisandraceae), known as Schisandrae Fructus, have traditionally been used in Korea, Japan, and China for the treatment of coughs, spontaneous sweating, dysentery and insomnia.¹⁾ Previous phytochemical investigation on the fruits of S. chinensis has resulted in the isolation of numerous lignans^2,3) and nortriterpenoids.⁴⁾ Although diverse pharmacological effects such as anti-hepatotoxic, anti-inflammatory, anti-oxidant and anti-tumor activities of the fruits of S. chinensis and dibenzocyclooctadiene lignans have been reported,^5,6) there are just few phytochemical or pharmacological investigations on the leaves, stems, or roots of S. chinensis.⁷⁾ Therefore, as part of our ongoing project to search novel secondary metabolites from medicinal plants, we chose the roots of S. chinensis for detailed phytochemical study.

Repeated chromatography of the ethyl acetate (EtOAc)- and butyl alcohol (BuOH)-soluble residues from the roots of S. chinensis led to the isolation and characterization of four new dibenzocyclooctadiene lignan glucosides, schisandrosides A–D (1–4), as well as two known nortriterpenoids, micrandilactone C (5)⁸⁾ and propindilactone Q (6).⁹⁾ To the best of our knowledge, this is the first report on the isolation of dibenzocyclooctadiene lignan glycosides from nature. The structure elucidation of 1–4 and the biological evaluation of the isolates are described herein (Fig. 1).

Fig. 1. Structures of 1–6 Isolated from the Roots of S. chinensis

Results and Discussion

Compound 1 was obtained as a white amorphous powder and its molecular formula was established as C₂₈H₃₆O₁₂ by high-resolution (HR) direct analysis in real time (DART) MS (m/z=582.2549 [M+NH₄]⁺; calcd for C₂₈H₄₀NO₁₂, 582.2551). Unambiguous NMR assignments (Table 1) were made by application of 1 dimensional (D) and 2D homo- and heteronuclear NMR experiments [¹H-NMR, ¹³C-NMR, distortionless enhancement by polarization transfer (DEPT), ¹H–¹H correlation spectroscopy (COSY), rotational nuclear overhauser effect spectroscopy (ROESY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond correlation (HMBC)]. The ¹H-NMR spectrum of 1 revealed the presence of two aromatic singlet signals (δ 7.64 and 6.76, 1H each), a methylenedioxy group (δ 5.96 and 6.04, 1H each), and three methoxy signals (δ 3.55, 3.99 and 4.02, 3H each). The ¹H-NMR spectrum of 1 also showed signals for two methyl groups, with one attached to a quaternary carbon (δ 1.34, s) and one to a tertiary carbon (δ 0.86, d, J=7.0 Hz). The ¹³C-NMR and DEPT spectra of 1 showed total 28 carbons, which included three methoxy groups, two methyls, four methylenes, eight methines, and eleven quaternary carbons. Comparison of the above data with those in the literatures^10–14) suggested that 1 is a dibenzocyclooctadiene lignan with a hexose. The presence of 7-hydroxy cyclooctadiene ring in 1 was confirmed by ¹H–¹H COSY correlations from δ 2.10 (H-8) to δ 2.48/3.31 (H-9 α/β) and 0.86 (H-17) and HMBC correlations for H-11 (δ 6.76)/C-9 (δ 34.9), C-10 (δ 134.3), and C-15 (δ 123.4); H-4 (δ 7.64)/C-5 (δ 135.2), C-6 (δ 42.2), and C-16 (δ 125.5); H-9 (δ 2.48 and 3.31)/C-8 (δ 42.8) and C-15 (δ 123.4); H-18 (δ 1.34)/C-7 (δ 72.0) and C-8 (δ 42.8); and H-6 (δ 2.80 and 2.62)/C-7 (δ 72.0) and C-16 (δ 125.5) (Fig. 2). The HMBC correlations for OCH₂O (δ 5.96 and 6.04)/C-12 (δ 148.7) and C-13 (δ 135.7); OCH₃-1 (δ 3.55)/C-1 (δ 152.5); OCH₃-2 (δ 3.99)/C-2 (δ 141.8); and OCH₃-14 (δ 4.02)/C-14 (δ 142.3) confirmed the assignments of the location of the methylenedioxy (C-12 and C-13) and methoxy groups (C-1, C-2, and C-14). The hexosyl group in 1 was inferred as a glucopyranosyl by careful analysis of the COSY, HMQC, HMBC, and ROESY spectra of 1 (Figs. 2, 3). The anomeric proton signal observed at δ 5.72 (J=7.5 Hz) in the ¹H-NMR spectrum indicated that the glucosyl linkage is β-configuration. The HMBC correlation from the anomeric proton signal (δ 5.72) to C-3 (δ 151.0) was indicated the glucosyl group to be located at C-3. The circular dichroism (CD) spectrum of 1 showed a negative Cotton effect at 220 nm and a positive Cotton effect at 254 nm, indicating that 1 has a R-biphenyl configuration.¹⁰⁾ The observed ROESY correlations (Fig. 3) of H-11/H-9α, H-11/CH₃-17, H-4/H-6β, and H-4/Glc-1′ implicated that two methyl groups are all α-configuration.^11,13) On the basis of the above data, it was inferred that 1 has twist-boat-chair (TBC) conformation for the cyclooctadiene ring and relative configurations of C-7 (R) and C-8 (S).¹³⁾ Thus, the structure of 1 was established to be as shown, which was assigned the trivial name, schisandroside A.

Table 1. ¹H- (500 MHz) and ¹³C-NMR (125 MHz) Data of Compounds 1–4 in Pyridine-d₅^a)

Position	1		2		3		4
	δC	δH Multi (J in Hz)	δC	δH Multi (J in Hz)	δC	δH Multi (J in Hz)	δC	δH Multi (J in Hz)
1	152.5		152.7		147.8		152.5
2	141.8		142.0		140.9		142.5
3	151.0		151.2		146.5		150.3
4	116.1	7.64 s	115.9	7.69 s	118.1	7.26 s	116.3	7.81 s
5	135.2		135.1		129.5		133.7
6	42.2	2.80 d (13.5)	42.8	2.81 d (13.5)	38.9	2.54 dd (13.5, 1.5)	86.1	6.18 s
		2.62 d (13.5)		2.67 d (13.5)		2.49 dd (13.5, 6.5)
7	72.0		72.3		34.5	1.75 overlap	72.8
8	42.8	2.10 quintet (7.0)	42.6	2.15 quintet (7.0)	41.5	1.75 overlap	43.6	2.13 quintet (7.0)
9	34.9	2.48 dd (14.0, 7.5)	35.6	2.51 dd (14.0, 8.0)	36.0	2.02 dd (13.5, 1.5)	37.3	2.27 d (13.5)
		3.31 dd (14.0, 1.5)		3.35 dd (14.0, 1.5)		2.33 dd (13.0, 4.0)		3.01 dd (14.0, 9.5)
10	134.3		135.8		138.8		137.0
11	106.7	6.76 s	111.6	6.63 s	103.8	6.69 s	103.6	6.75 s
12	148.7		152.9		149.7		149.6
13	135.7		141.2		135.4		135.0
14	142.3		152.5		142.1		141.6
15	123.4		124.3		122.6		122.6
16	125.5		125.8		126.8		123.6
17	16.4	0.86 d (7.0)	16.6	0.89 d (7.0)	13.1	0.68 d (7.0)	19.9	1.32 d (7.0)
18	31.2	1.34 s	30.8	1.37 s	22.1	0.88 d (6.5)	29.5	1.51 s
OCH2O	101.7	5.96 s			101.6	5.93 d (1.0)	101.4	5.94 d (1.0)
		6.04 s				6.00 d (1.5)		6.00 d (1.0)
1-OCH3	61.0	3.55 s	61.2	3.76 s	60.4	3.78 s	61.0	3.62 s
2-OCH3	61.5	3.99 s	61.5	4.00 s			61.2	3.84 s
12-OCH3			56.3	3.87 s
13-OCH3			61.3	4.00 s
14-OCH3	59.9	4.02 s	61.0	3.65 s	59.9	3.94 s	59.5	3.95 s
7-OH				5.14 s
Glc-1′	102.2	5.72 d (7.5)	102.5	5.73 d (8.0)	105.0	5.49 d (7.5)	102.1	5.69 d (7.0)
2′	75.3	4.32 dd (9.0, 7.5)	75.3	4.33 dd (9.0, 8.5)	75.5	4.31 d (8.0)	75.4	4.33 dd (9.0, 7.5)
3′	78.8	4.14 dd (9.0, 9.0)	78.9	4.16 ddd (9.0, 8.5, 2.0)	78.8	4.31 overlap	79.2	4.29 t (8.5)
4′	71.3	4.32 dd (10.0, 9.0)	71.4	4.34 dd (9.5, 9.0)	71.4	4.42 m	71.8	4.20 dd (9.5, 8.5)
5′	78.9	3.82 ddd (9.5, 5.0, 2.5)	79.1	3.85 ddd (9.5, 5.5, 2.5)	79.3	3.98 ddd (9.5, 4.5, 2.5)	79.3	3.97 ddd (9.5, 4.5, 2.0)
6′	62.4	4.35 dd (12.0, 5.0)	62.6	4.31 dd (10.0, 5.0)	62.6	4.49 overlap	62.9	4.15 dd (12.0, 6.0)
		4.47 dd (12.0, 2.5)		4.50 dd (12.0, 3.0)		4.54 overlap		4.36 dd (12.0, 2.0)
Ang-1″							166.2
2″							128.3
3″							139.4	5.93 qq (7.0, 1.5)
4″							16.2	1.97 dq (7.0, 1.5)
5″							20.5	1.57 dq (1.5, 1.5)

a) The assignments were based on COSY, HMQC, and HMBC experiments.

Fig. 2. Selected Correlations Observed in the COSY (▬) and HMBC (→) NMR Spectrum of 1–4 (in Pyridine-d₅)

Fig. 3. ROESY (↔) Correlations of 1–4 (in Pyridine-d₅)

Compound 2 was obtained as a white amorphous powder. Its HR-DART-MS exhibited [M+NH₄]⁺ signal at m/z=598.2861 (calcd for C₂₉H₄₄NO₁₂, 598.2864), consistent with an elemental formula of C₂₉H₄₀O₁₂. The proton and carbon signals in the ¹H- and ¹³C-NMR spectra of 2 (Table 1) exhibited strong similarities with those of 1 except for the absence of the methylenedioxy group and the presence of two additional methoxy groups. Thus, the structure of 2 was suggested as an analogue of 1 in which the methylenedioxy group was replaced to two methoxy groups. The positions of the five methoxy groups in 2 were assigned by observed HMBC correlations from δ_H 3.76, 4.00, 3.87, 4.00, and 3.65 to δ_C 152.7 (C-1), 142.0 (C-2), 152.9 (C-12), 141.2 (C-13), and 152.5 (C-14), respectively (Fig. 2). The sugar moiety of 2 was determined as β-glucopyranosyl by analysis of 1D and 2D-NMR spectral data and by comparison with those of 1. The location of the β-glucopyranosyl at C-3 was deduced from HMBC correlation for the anomeric proton H-1′ (δ 5.73) to C-3 (δ 151.2). The absolute stereochemistry of the biphenyl group of 2 was established as R, on the basis of the observation of a negative Cotton effect at 215 nm and a positive Cotton effect at 250 nm in its CD spectrum.¹⁰⁾ The observed ROESY correlations (Fig. 3) of 12-OCH₃/H-11, H-11/H-9α, H-11/CH₃-17, H-4/H-6β, and H-4/Glc-1′ indicated that two methyl groups are all α-configuration like 1.^11,13) From all of these data, the structure of the new dibenzocyclooctadiene lignan glycoside 2 was elucidated to be as shown and was named schisandroside B.

Compound 3 was obtained as a white amorphous powder and its molecular formula was established as C₂₇H₃₄O₁₁ by HR-DART-MS (m/z=552.2433 [M+NH₄]⁺; calcd for C₂₇H₃₈NO₁₁, 552.2445). The proton and carbon signals in the ¹H- and ¹³C-NMR spectra of 3 were very similar to those of 1 (Table 1). However, preliminary inspection of the ¹H-NMR spectrum of 3 revealed the absence of one of the three methoxys in 1. Moreover, comparison of the ¹³C and DEPT NMR data for 1 and 3 indicated that the aliphatic quaternary carbon bearing oxygen (δ 72.0; C-7) of 1 was replaced by a methine carbon (δ 34.5; C-7) in 3. The positions of two methoxy groups and methylenedioxy group were assigned by HMBC correlations for δ_H 3.78 (3H, s)/C-1 (δ_C 147.8), δ_H 3.94 (3H, s)/C-14 (δ_C 142.1), δ_H 5.93 (1H, d, J=1.0 Hz)/C-12 (δ_C 149.7) and C-13 (δ_C 135.4), and δ_H 6.00 (1H, d, J=1.5 Hz)/C-12 (δ_C 149.7) and C-13 (δ_C 135.4) (Fig. 2). The sugar moiety and position were determined to be β-glucopyranosyl at C-3 in a similar manner to that of 1 or 2 (Figs. 2, 3). However, the absolute stereochemistry of the biphenyl group in 3 was determined to have S-configuration from its CD spectrum, which showed a negative Cotton effect at 240–250 nm and a positive Cotton effect at 222 nm.¹⁰⁾ The observed ROESY correlations (Fig. 3) of H-11/H-9β, H-9β/CH₃-18, H-4/H-6 and CH₃-17, and H-4/Glc-1′ supposed that CH₃-17 and CH₃-18 are α- and β-oriented, respectively.¹⁰⁾ Therefore, the structure of 3 was determined to be as shown and was named schisandroside C.

Compound 4 was obtained as a white amorphous powder. Its HR-DART-MS gave a molecular ion peak at m/z=680.2911 [M+NH₄]⁺ (calcd for C₃₃H₄₆NO₁₄, 680.2918), indicated a molecular formula of C₃₃H₄₂O₁₄. The proton and carbon signals in the ¹H- and ¹³C-NMR spectra of 4 (Table 1) were similar with those of 3. The major differences between 3 and 4 are the presence of additional methoxy group, two oxymethines, and a C-6 unit in 4. It was inferred that 4 has an angeloyl group from the observed signals at δ_H 1.57 (3H, dq, J=1.5, 1.5 Hz), 1.97 (3H, dq, J=7.0, 1.5 Hz), and 5.93 (1H, qq, J=7.0, 1.5 Hz)/δ_C 16.2, 20.5, 128.3, 139.4, and 166.2.¹⁰⁾ Thus, the structure of 4 was suggested as an angeloyl analogue of 3 with an additional methoxy group on the biphenyl ring and a hydroxyl group on the cyclooctadiene ring. The positions of the substituents were deduced as occurring at C-1, C-2, and C-14 (three methoxy groups); at C-12 and C-13 (methylenedioxy group); and at C-6 (angeloyl group) using HMBC NMR technique (Fig. 2). Moreover, it was deduced that the angeloyl and hydroxyl groups of 4 are β- and α-oriented, respectively, by ROESY correlations (Fig. 3). The CD spectrum exhibited a strong negative Cotton effect at λ_max 240–250 nm, which indicated that 4 is a dibenzocyclooctadiene lignan with S-biphenyl configuration like 3.¹⁰⁾ The ROESY spectrum also exhibited correlations between H-11/H-9β, H-4/H-6, H-6/CH₃-18, and H-4/Glc-1′, indicating that CH₃-17 and CH₃-18 are α- and β-oriented, respectively, the same as 3.¹⁰⁾ Therefore, the structure of this new dibenzocyclooctadiene lignan glycoside 4 was established to be as shown, which was assigned the trivial name, schisandroside D. The acid hydrolysis of 1–4 with 1.5 N aqueous HCl afforded D-glucose, which was identified by co-TLC and optical rotation (positive optical rotation) study, confirming their glucoside nature.

Even if numerous dibenzocyclooctadiene lignans have been isolated from plants belonging to Schisandraceae, to the best of our knowledge, this is the first report of the isolation of their glycosides from nature. In addition to the novel dibenzocyclooctadiene lignan glucosides, schisandrosides A–D (1–4), two known rare nortriterpenoids, micrandilactone C (5)⁸⁾ and propindilactone Q (6),⁹⁾ were also isolated in this study. The structures of these known compounds were identified by physical (mp, [α]_D) and spectroscopic (¹H-NMR, ¹³C-NMR, 2D-NMR, and MS) data measurement and by comparison with published values. Although micrandilactone C (5) was isolated from Schisandra micrantha,⁸⁾ it has not been reported from S. chinensis. Propindilactone Q (6) was recently isolated from the stem and leaves of this plant.¹⁵⁾

All isolates obtained in the present study were evaluated for their cytotoxicity against endometrial cancer cells (Ishikawa) using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assays. As a result, all compounds isolated, including new dibenzocyclooctadiene lignan glucosides 1–4, were found to be inactive (IC₅₀ values >50 µM) in this assay system. Although we did not find any activity for the isolates in the cytotoxicity assay, it is entirely possible these will be active in other types of test system.

Experimental

General Experimental Procedures

Optical rotations were obtained using a JASCO P-2000 digital polarimeter. UV and CD spectra were recorded with a PerkinElmer, Inc. Lambda 25 UV/Vis spectrometer and Applied Photophysics Chirascan plus spectrometers, respectively. IR spectra were recorded using a JASCO FT/IR-4200 spectrophotometer. A mass spectrometer was an AccuTOF-TLC single-reflectron time-of-flight mass spectrometer equipped with a DART-SVP ion source (IonSense, Saugus, MA, U.S.A.). NMR experiments were conducted on a Bruker 400 MHz or Varian 500 MHz, and the chemical shifts were referenced to the residual solvent signals. TLC analysis was performed on Kieselgel 60 F₂₅₄ (Merck) plates (silica gel, 0.25 mm layer thickness); compounds were visualized by dipping plates into 20% (v/v) H₂SO₄ reagent (Aldrich) and then heat treated at 110°C for 5–10 min. Silica gel (Merck 60A, 70–230 or 230–400 mesh ASTM), Diaion HP-20, Sephadex LH-20 (Amersham Pharmacia Biotech), and reversed-phase silica gel (YMC Co., ODS-A 12 nm S-150 µm) were used for column chromatography. All solvents used for the chromatographic separations were distilled before use.

Plant Material

The roots of S. chinensis were collected in Mt. Jangan, Jangsu-gun, Chungcheongbuk-do, Korea (35°37′39N, 127°34′21E), in July, 2012 by two of us (B. R. and D. S. J.), and were identified by one of us (D. S. J). A voucher specimen (No. 2012-SCCH02) has been deposited in the Lab. of Natural Product Medicine, College of Pharmacy, Kyung Hee University, Republic of Korea.

Extraction and Isolation

The dried roots of S. chinensis (1.72 kg) were extracted with 70% aqueous EtOH (20 L) by maceration. The extract was concentrated in vacuo at 40°C to give 70% EtOH extract (235.75 g, 13.7%) and was suspended in distilled water and then partitioned with EtOAc and BuOH, successively. A portion of the EtOAc-soluble layer (48.09 g) was subjected to silica gel column chromatography (CC) and eluted with n-hexane–EtOAc–MeOH–H₂O mixture (9 : 1 : 0 : 0, 4 : 1 : 0 : 0, 3 : 2 : 0 : 0, 2 : 3 : 0 : 0, 0 : 1 : 0 : 0, 0 : 9 : 1 : 0.1, 0 : 8 : 2 : 0.2, and 0 : 7 : 3 : 0.3, final 100% MeOH) to afford 17 fractions (E1–E17). Fraction E13 (3.16 g) was fractionated through Sephadex CC (MeOH) to produce 4 fractions (E13-1–E13-4). Compound 6 (24.6 mg) was purified by recrystallization (in MeOH) from fraction E13-2 (1.15 g). Fraction E14 (7.73 g) was subjected to a silica gel CC with n-hexane–EtOAc–MeOH mixture (6 : 3 : 1) to produce 8 subfractions (E14-1–E14-8). The fraction E14-4 (1.04 g) was purified further over a Sephadex LH-20 CC with MeOH, yielding 3 (2.0 mg), 4 (16.3 mg), and 5 (220 mg). Compound 1 (48.2 mg) was obtained from fraction E14-5 (790 mg) through reversed-phase CC (MeOH–H₂O=3 : 2). The BuOH-soluble extract (34.54 g) was subjected to Diaion HP-20 CC and eluted with a stepwise gradient of MeOH–H₂O system (0 : 1 to 1 : 0) to yielded 8 fractions (B1–B8). The fraction B7 was further fractionated using a Sephadex LH-20 CC with CH₂Cl₂–MeOH mixture (1 : 1) to afford 2 (12.2 mg).

Schisandroside A (1)

White amorphous powder. [α]_D²⁵ +12.25° (c=0.2, MeOH). CD λ_max (c=0.05, MeOH) nm (∆ε): 220 (−4.25), 254 (+5.32). UV λ_max (MeOH) nm (log ε): 218 (4.69), 257 (4.20), 292 (3.52). IR (attenuated total reflectance (ATR)) cm⁻¹: 3469, 2953, 1622, 1456, 1407, 1274, 1226, 1083, 1041, 935, 876. ¹H- and ¹³C-NMR data (C₅D₅N, 500 MHz and 125 MHz), see Table 1. HR-DART-MS (positive mode) m/z 582.2549 [M+NH₄]⁺ (calcd for C₂₈H₄₀NO₁₂: 582.2551).

Schisandroside B (2)

White amorphous powder. [α]_D²⁵ +6.17° (c=0.3, MeOH). CD λ_max (c=0.03, MeOH) nm (∆ε): 215 (−20.35), 234 (+14.49), 250 (+20.63). UV λ_max (MeOH) nm (log ε): 217 (5.11), 250 (4.81), 285 (3.89). IR (ATR) cm⁻¹: 3401, 2935, 1594, 1489, 1457, 1403, 1279, 1196, 1117, 1077, 927. ¹H- and ¹³C-NMR data (C₅D₅N, 500 MHz and 125 MHz), see Table 1. HR-DART-MS (positive mode) m/z 598.2861 [M+NH₄]⁺ (calcd for C₂₉H₄₄NO₁₂, 598.2864).

Schisandroside C (3)

White amorphous powder. [α]_D²⁵ −25.68° (c=0.25, MeOH); CD λ_max (c=0.025, MeOH) nm (∆ε): 222 (+7.23), 241 (−7.13), 251 (−7.06). UV λ_max (MeOH) nm (log ε): 219 (4.91), 254 (4.28), 281 (4.01). IR (ATR) cm⁻¹: 3359, 2927, 1747, 1715, 1680, 1594, 1473, 1269, 1048, 935. ¹H- and ¹³C-NMR data (C₅D₅N, 500 MHz and 125 MHz), see Table 1. HR-DART-MS (positive mode) m/z=552.2433 [M+NH₄]⁺ (calcd for C₂₇H₃₈NO₁₁, 552.2445).

Schisandroside D (4)

White amorphous powder. [α]_D²⁵ −36.40° (c=0.2, MeOH). CD λ_max (c=0.04, MeOH) nm (∆ε): 218 (+7.08), 242 (−7.66), 255 (−8.03). UV λ_max (MeOH) nm (log ε): 220 (4.86), 257 (4.23), 292 (3.72) nm. IR (ATR) cm⁻¹: 3384, 2927, 1747, 1715, 1689, 1594, 1473, 1269, 1046. ¹H- and ¹³C-NMR data (C₅D₅N, 500 MHz and 125 MHz), see Table 1. HR-DART-MS (positive mode) m/z 680.2911 [M+NH₄]⁺ (calcd for C₃₃H₄₆NO₁₄, 680.2918).

Acid Hydrolysis of 1–4:

Each compound (1 mg) was individually hydrolyzed with 1.5 N aqueous HCl (2 mL) at 90°C for 2h. After cooling, each reaction mixture was diluted with H₂O (20 mL) and extracted with EtOAc (2×20 mL), and then the aqueous layer was concentrated in vacuo at 40°C to give a sugar fraction. D-Glucose in the sugar fractions was identified by silica gel co-TLC (Rf 0.53, n-BuOH–acetic acid–H₂O, 2 : 1 : 1) with an authentic sample. The sugar fractions were separately subjected to silica gel CC and eluted with EtOAc–EtOH–H₂O mixture (7 : 4 : 1) to afford glucose (0.24 mg) from 1, [α]_D²⁵ +45° (c=0.02, H₂O), glucose (0.21 mg) from 2, [α]_D²⁵ +50° (c=0.02, H₂O), glucose (0.22 mg) from 3, [α]_D²⁵ +42° (c=0.02, H₂O), and glucose (0.23 mg) from 4, [α]_D²⁵ +46° (c=0.02, H₂O), respectively.

Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2004398) and by a Grant from the Bio-Synergy Research Project (NRF-2014M3A9C4066594) of the Ministry of Science, ICT and Future Planning through the National Research Foundation. We thank Korea Basic Science Institute (KBSI) for running NMR and MS experiments.

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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