Anti-inflammatory Tirucallane Saponins from Paramignya scandens

Abstract

Five new tirucallane saponins, paramignyosides A–E (1–5), were isolated from the water fraction of the Paramignya scandens stem and leaves. Their structures were elucidated on the basis of spectroscopic evidence including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and one dimensional (1D)- and 2D-NMR. The effects of isolated compounds on pro-inflammatory cytokines were evaluated by measuring the production of interleukin (IL)-12 p40, IL-6, and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs). Paramignyoside C (3) exhibited selective and potent inhibitory effect (IC₅₀=5.03±0.19 µM) on the production of IL-12 p40 comparable to that of the positive control, SB203580 (IC₅₀=5.00±0.16 µM). Further studies are required to confirm efficacy in vivo and the mechanism of anti-inflammatory effects.

Tirucallane triterpenoids are metabolically altered triterpenes and have a structure either containing or derived from a precursor with a (13S,14R,17S,20S)-lanostane skeleton. Recently, tirucallane-type triterpenoids have received much attention from scientists because of their unique structures and various biological activities. Published investigations demonstrated that this type of compound possessed numerous interesting biological effects, such as cytotoxic,^1–6) anti-platelet aggregation,^7,8) anti-inflammatory,⁹⁾ antitubercular,¹⁰⁾ vasodilative¹¹⁾ activities, and inhibition of mouse 11β-hydroxysteroid dehydrogenase type 1¹²⁾ and human immunodeficiency virus (HIV-1) protease.¹³⁾ Previously, we reported two new tirucallane triterpenes from Paramignya (P.) scandens and evaluation of their cytotoxic effects.¹⁴⁾

As a part of our ongoing investigations on chemical constituents of Vietnamese marine organisms and medicinal plants possessing inhibitory effects on production of pro-inflammatory cytokines,^15–19) the current paper addresses the isolation, structural elucidation, and evaluation of the inhibitory effects on the production of interleukin (IL)-12 p40, IL-6, and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells (BMDCs) of five rare tirucallane saponins, paramignyosides A–E (1–5, see Fig. 1) from the P. scandens stem and leaves.

Fig. 1. Structures of 1–5

Results and Discussion

The dried stem and leaves of P. scandens were extracted with methanol. The organic extract was concentrated to dryness, suspended in water, and partitioned in turn with n-hexane and CH₂Cl₂. The water layer was passed through a Diaion HP-20 column chromatography (CC) and further purified by repeated silica gel, YMC RP-18, and Sephadex LH-20 CC to yield metabolites 1–5.

Paramignyoside A (1) was obtained as a white powder with the molecular formula, C₄₂H₆₆O₁₇, determined by a quasi-molecular ion peak at m/z 865.4233 [M+Na]⁺ in high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The ¹H-NMR spectrum exhibited typical signals of six tertiary methyl groups [δ_H 0.91 (H-18), 0.74 (H-19), 1.26 (H-26), 1.24 (H-27), 1.31 (H-28), and 1.07 (H-30), each 3H, s] and one olefinic proton [δ_H 5.31 (1H, d, J=2.5 Hz, H-7)]. In addition, two anomeric protons at δ_H 5.88 (1H, d, J=8.0 Hz, H-1′) and 4.59 (1H, d, J=8.0 Hz, H-1″), which correlated with the relevant anomeric carbons at δ_C 95.0 (C-1′) and 105.1 (C-1″) on heteronuclear single quantum coherence (HSQC) spectrum, indicated the presence of two sugar moieties. The ¹³C-NMR spectrum of 1 revealed 42 signals of a triterpene aglycon and a disaccharide moiety. Comparison of the ¹³C-NMR data of the disaccharide chain (see Table 1) with the corresponding values of (−)-4-[β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyloxy]benzyl alcohol²⁰⁾ and combination with acid hydrolysis followed by derivatization and GC analysis (see Experimental) as well as analysis of ¹H–¹H correlation spectroscopy (COSY) and heteronuclear multiple-bond correlation (HMBC) cross-peaks (see Fig. 2) indicated a β-D-glucopyranosyl-(1→3)-β-D-glucopyranoside structure. The ¹³C-NMR data for the aglycon showed typical signals of six methyl groups [δ_C 23.8 (C-18), 12.8 (C-19), 26.7 (C-26), 26.4 (C-27), 24.3 (C-28), and 27.9 (C-30)], one trisubstituted double bond [δ_C 119.5 (d, C-7) and 145.6 (s, C-8)], three oxymethine groups [δ_C 71.4 (C-3), 80.2 (C-23), and 78.1 (C-24)], one oxygenated quaternary carbon [δ_C 72.8 (C-25)], and two carbonyl carbons [δ_C 181.3 (C-21) and 176.6 (C-29)]. The NMR data for the aglycon of 1 were similar to those of mesendanin M,²¹⁾ except for the presence of a carbonyl group in 1 instead of an oxymethylene in mesendanin M. The position of the additional carbonyl group at C-29 we determined by the HMBC correlations of H-5 (δ_H 2.06) and H-28 (δ_H 1.31) with C-29 (δ_C 176.6). The anomeric proton H-1′ (δ_H 5.88) had a strong HMBC correlation with C-29 (δ_C 176.6) confirming the attachment of the disaccharide moiety at this carbon. Detailed analysis of other COSY and HMBC correlations clearly indicated the planar structure of 1 as shown in Fig. 2.

Table 1. The ¹H- (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) Spectroscopic Data of 1–3

Position	1		2		3
	δC	δH mult. (J in Hz)	δC	δH mult. (J in Hz)	δC	δH mult. (J in Hz)
1	33.0	1.42 m/1.54 m	32.9	1.41 m/1.52 m	33.0	1.42 m/1.55 m
2	27. 8	1.67 m/2.28 m	27.9	1.62 m/2.00 m	28.0	1.66 m/2.29 m
3	71.4	4.10 d (2.5)	71.1	4.02 br s	71.5	4.10 br s
4	49.5	—	49.4	—	49.6	—
5	46.5	2.06 m	46.3	2.03 m	46.5	2.05 m
6	25.4	2.20 m/2.84 m	25.3	2.26 m/2.78 m	25.5	2.10 m/2.85 m
7	119.5	5.31 d (2.5)	119.5	5.30 d (2.5)	119.6	5.30 br s
8	145.6	—	145.6	—	145.7	—
9	49.1	2.41 m	49.1	2.41 m	49.6	2.41 m
10	36.3	—	36.3	—	36.4	—
11	18.9	1.56 m/1.69 m	18.9	1.57 m/1.68 m	18.9	1.58 m/1.69 m
12	32.4	1.79 m	32.4	1.79 m	32.5	1.80 m
13	44.8	—	44.8	—	44.8	—
14	51.7	—	51.7	—	51.7	—
15	34.9	1.57 m/1.64 m	34.9	1.57 m/1.63 m	34.9	1.57 m/1.64 m
16	24.8	1.60 m/1.82 m	24.7	1.60 m/1.82 m	25.0	1.60 m/1.80 m
17	48.7	2.38 m	48.6	2.38 m	49.4	2.38 m
18	23.8	0.91 s	23.8	0.91 s	23.9	0.93 s
19	12.8	0.74 s	12.9	0.70 s	12.9	0.74 s
20	42.0	2.83 m	42.0	2.83 m	41.9	2.81 m
21	181.3	—	181.4	—	181.4	—
22	29.4	2.29 m/2.31 m	29.4	2.29 m/2.31 m	29.6	2.32 m/2.60 m
23	80.2	4.68 m	80.2	4.68 m	80.1	4.77 m
24	78.1	3.63 d (2.5)	78.1	3.63 d (3.5)	77.7	3.78 d (3.0)
25	72.8	—	72.8	—	80.2	—
26	26.7	1.26 s	26.7	1.25 s	24.5	1.36 s
27	26.4	1.24 s	26.4	1.23 s	23.4	1.33 s
28	24.3	1.31 s	24.0	1.23 s	24.2	1.31 s
29	176.6	—	176.5	—	176.9	—
30	27.9	1.07 s	27.9	1.07 s	27.9	1.07 s
	Glc I		GlcOAc		Glc I
1′	95.0	5.88 d (8.0)	93.1	5.69 (8.0)	95.5	5.50 (d, 8.0)
2′	73.4	3.58 dd (8.0, 9.0)	72.4	5.10 dd (8.0, 9.0)	74.1	3.38*
3′	88.2	3.64 t (9.0)	85.4	3.83 t (9.0)	78.5	3.44*
4′	69.5	3.52 t (9.0)	69.6	3.60 t (9.0)	71.2	3.40*
5′	78.5	3.44 m	78.7	3.50 m	78.0	3.29*
6′	62.3	3.74 dd (4.5, 12.0)3.86 br d (12.0)	62.1	3.77 dd (4.5, 12.0)3.88 br d (12.0)	62.8	3.67/3.89*
OAc			171.7	—
OAc			21.2	2.10 s
	Glc II		Glc		Glc II
1″	105.1	4.59 d (8.0)	105.4	4.40 d (8.0)	98.4	4.52 d (7.5)
2″	75.4	3.32*	74.9	3.21 dd (8.0, 9.0)	75.4	3.16*
3″	77.8	3.41 t (9.0)	77.9	3.37*	78.8	3.39*
4″	71.5	3.31*	71.4	3.31*	71.7	3.29*
5″	78.1	3.37 m	78.1	3.37 m	78.0	3.39*
6″	62.6	3.67 dd (6.0, 12.0)3.92 dd (1.5, 12.0)	62.5	3.67 dd (6.0, 12.0)3.91 dd (1.5, 12.0)	62.7	3.70/3.85*

* Overlapped signals. Assignments were confirmed by HSQC, HMBC, COSY, and ROESY experiments.

Fig. 2. Key COSY, HMBC, and ROESY Correlations of 1

The stereochemistry of 1 was established by comparison of its ¹H- and ¹³C-NMR chemical shifts and the coupling constants with those of similar compounds,^21–23) and further confirmed by a rotating frame Overhauser enhancement spectroscopy (ROESY) experiment. The configurations in the side chain of 1 were determined to be the same as those of mesendanin M²¹⁾ by the essential agreement of the ¹H- and ¹³C-NMR data between these two compounds. The resonance of proton H-3 at δ_H 4.10 (1H, d, J=2.5 Hz) and carbon C-3 at δ_C 71.4 is typical for a β-orientation of H-3.^21,22) The α-orientation of the methyl group C-28 was suggested by the ¹³C-NMR of C-28 at δ_C 24.3, which was quite different from that of reference compound with β-orientation of the methyl group C-28 at δ_C 17.5,²³⁾ and further confirmed by the spatial proximities observed of H-28 (δ_H 1.31) with H-5 (δ_H 2.06)/H_α-6 (δ_H 2.20) and that of H_β-6 (δ_H 2.84) with H-19 (δ_H 0.74) from the ROESY (see Fig. 2).

The ¹H- and ¹³C-NMR data of 2 were similar to those of 1 (see Table 1) except for an additional presence of an acetyl group at δ_C 171.7 and δ_C 21.2 (q)/δ_H 2.10 (3H, s) as also confirmed by HR-ESI-MS at m/z 907.4315 [M+Na]⁺. Proton H-2′ was strongly shifted downfield at δ_H 5.10 suggested for the esterification at C-2′,²⁴⁾ which was confirmed by an HMBC correlation of H-2′ (δ_H 5.10) with the carbonyl carbon at δ_C 171.7. Thus, the structure of paramignyoside B (2) was clearly elucidated.

Paramignyoside C (3) was also obtained as a white powder. Its molecular formula was determined to be the same as that of 1 by HR-ESI-MS at m/z 865.4213 [M+Na]⁺. The ¹H- and ¹³C-NMR data of 3 were similar to those of 1 (see Table 1) except for difference in the data of the side chain and sugar moieties. The oxygenated quaternary carbon C-25 was strongly shifted downfield at δ_C 80.2 suggested for the glycosylation at this carbon, which was confirmed by an HMBC cross-peak of H-1″ (δ_H 4.52) with C-25 (δ_C 80.2). The attachment of the other glucose moiety at C-29 was determined by an HMBC correlation of H-1′ (δ_H 5.50) with C-29 (δ_C 176.9).

The molecular formula of paramignyoside D (4) was identified as C₄₈H₇₆O₂₂ by HR-ESI-MS at m/z 1027.4787 [M+Na]⁺. Its ¹H- and ¹³C-NMR data were also similar to those of 1 (see Table 2) except for an additional presence of a glucose moiety. Similar as in case of compound 3, the downfield shifted signal of C-25 at δ_C 81.1 indicating the attachment of the additional glucose at this carbon, which was further confirmed by an HMBC correlation of H-1‴ (δ_H 4.55) with C-25 (δ_C 81.1). Detailed analysis of HSQC, HMBC, COSY, and ROESY data clearly elucidated the structure of 4.

Table 2. The ¹H- (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) Spectroscopic Data of 4 and 5

Position	4		5
	δC	δH mult. (J in Hz)	δC	δH mult. (J in Hz)
1	33.0	1.42 m/1.55 m	32.9	1.41 m/1.53 m
2	27.8	1.66 m/2.28 m	27.9	1.62 m/2.00 m
3	71.3	4.11 br s	71.1	4.02 br s
4	49.5	—	49.7	—
5	46.6	2.06 m	46.3	2.03 m
6	25.4	2.20 m/2.85 m	25.4	2.15 m/2.80 m
7	119.6	5.30 d (3.0)	119.6	5.31 d (3.0)
8	145.6	—	145.6	—
9	48.8	2.41 m	49.4	2.41 m
10	36.3	—	36.3	—
11	18.9	1.57 m/1.70 m	18.9	1.58 m/1.68 m
12	32.4	1.79 m	32.4	1.79 m
13	44.8	—	44.8	—
14	51.7	—	51.7	—
15	34.9	1.58 m/1.63 m	34.9	1.58 m/1.63 m
16	24.7	1.66 m/2.28 m	24.7	1.60 m/1.82 m
17	48.5	2.40 m	49.3	2.40 m
18	23.9	0.91 s	23.9	0.91 s
19	12.8	0.75 s	12.9	0.70 s
20	41.6	2.80 m	41.6	2.80 m
21	181.5	—	181.5	—
22	31.6	2.27 m/2.33 m	31.7	2.27 m/2.33 m
23	78.5	4.78 m	78.4	4.77 m
24	77.7	3.50 d (2.0)	77.7	3.50 d (2.0)
25	81.1	—	81.1	—
26	23.6	1.38 s	23.7	1.35 s
27	23.3	1.34 s	23.3	1.35 s
28	24.3	1.31 s	24.0	1.23 s
29	176.6	—	176.4	—
30	27.9	1.07 s	27.9	1.08 s
	Glc I		GlcOAc
1′	95.0	5.58 d (8.0)	93.1	5.69 d (8.0)
2′	73.4	3.58 dd (8.0, 9.0)	72.4	5.10 dd (8.0, 9.0)
3′	88.1	3.64 t (9.0)	85.4	3.83 t (9.0)
4′	69.5	3.52 t (9.0)	69.6	3.60 t (9.0)
5′	78.4	3.43 m	78.7	3.49 m
6′	62.3	3.75 dd (4.5, 12.0)/3.87 dd (2.5, 12.0)	62.1	3.77 dd (4.5, 12.0)/3.88 dd (2.5, 12.0)
OAc			171.6	—
OAc			21.2	2.09 s
	Glc II		Glc I
1″	105.0	4.58 d (8.0)	105.4	4.39 d (8.0)
2″	75.4	3.32*	74.7	3.21 dd (8.0, 9.0)
3″	78.1	3.37*	78.0	3.37*
4″	71.5	3.31*	71.4	3.31*
5″	77.6	3.28 m	77.6	3.27*
6″	62.6	3.67*/3.85 dd (2.5, 12.0)	62.7	3.66/3.85*
	Glc III		Glc II
1‴	98.5	4.55 d (7.5)	98.5	4.55 d (7.5)
2‴	75.1	3.18 dd (7.5, 9.0)	75.2	3.17 dd (7.5, 9.0)
3‴	77.8	3.41 t (9.0)	78.1	3.39*
4‴	71.7	3.30*	71.7	3.34*
5‴	78.1	3.37*	78.1	3.35*
6‴	62.7	3.67*/3.91 dd (2.5, 12.0)	62.5	3.67/3.90*

* Overlapped signals. Assignments were confirmed by HSQC, HMBC, COSY, and ROESY experiments.

Similar as in case of compounds 1 and 2, paramignyoside E (5) is different from 4 only for an additional presence of an acetyl group [δ_C 171.6 and δ_C 21.2 (q)/δ_H 2.09 (3H, s)] as also confirmed by HR-ESI-MS at m/z 1069.4901 [M+Na]⁺. A strong HMBC cross-peak was observed between H-2′ (δ_H 5.10) and the carbonyl carbon at δ_C 171.6 confirming the attachment of the acetyl group at C-2′. Tirucallane saponins are relatively rare from natural sources.²⁵⁾ Noteworthy, to best of our knowledge, this is the first report of 25-glycosylated and/or O-acetylglycosylated derivatives of tirucallane triterpenoids to date.

All the isolated compounds were evaluated for their effects on pro-inflammatory cytokines by measuring the production of IL-12 p40, IL-6, and TNF-α in LPS-stimulated BMDCs. Interestingly, all the compounds show selective effects on the production of IL-12 p40 (see Fig. 3). Of which, paramignyoside C (3) exhibited most potent inhibitory effect on production of IL-12 p40 with an IC₅₀ value of 5.03±0.19 µM. The activity of 3 was comparable to that of the positive control, SB203580²⁶⁾ (IC₅₀=5.00±0.16 µM). In addition, paramignyosides D and E (4 and 5) showed significant inhibitory effects with IC₅₀ values of 19.59±0.97 and 14.09±0.65 µM, respectively. Moderate inhibitory effect on IL-12 p40 production was observed for paramignyoside B (2) with an IC₅₀ value of 29.15±1.33 µM, while weak activity (IC₅₀=48.68±1.89 µM) was observed for paramignyoside A (1). All the isolated compounds did not show significant inhibition of IL-6 and TNF-α in LPS-stimulated BMDCs (IC₅₀>100 µM). Among the various inflammatory cytokines, IL-12 plays a central role in the initiation and regulation of cellular immunity. It is involved in type-1 helper T-cell-mediated inflammation as a part of the normal immune response, as well as in inflammatory diseases, such as rheumatoid arthritis, asthma, psoriasis, and Crohn’s disease.^27–29) Although further studies are required to confirm efficacy in vivo and mechanism of anti-inflammatory effects, it is the contention of the authors that compounds 3–5 could be used in the development of therapeutic targets for modulation of IL-12 and related inflammatory diseases.

Fig. 3. Effect of Compounds 1–5 on IL-12 p40 Production in LPS-Stimulated BMDCs

The data were presented as inhibition rate (%) compared to the value of vehicle-treated DCs.

Experimental

General Experimental

High resolution mass spectra were recorded on a MicroQ-TOF III mass spectrometer (Bruker Daltonics, 255748 Germany). The ¹H-NMR (500 MHz) and 13C-NMR (125 MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer (Bruker, Billerica, MA, U.S.A.) using tetramethylsilane (TMS) as internal standard. Gas chromatography (GC) analysis was carried out on a Shimadzu-2010 spectrometer (Chiyoda-ku, Tokyo, Japan). CC was performed using a silica gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany) or YMC RP-18 resins (30–50 µm, Fuji Silysia Chemical, Aichi, Japan). Thin layer chromatography (TLC) used pre-coated silica gel 60 F₂₅₄ (1.05554.0001, Merck) and RP-18 F_254S plates (1.15685.0001, Merck). Compounds were visualized by spraying with aqueous 10% H₂SO₄ and heating for 3–5 min.

Plant Material

The stem and leaves of P. scandens (GRIFF.) CRAIB were collected in Da Lat, Lamdong, Vietnam during May 2013 and identified by Dr. Nong Van Duy (Tay Nguyen Institute of Scientific Research, VAST, Vietnam). A voucher specimen (TN3/055) was deposited at the Herbarium of Tay Nguyen Institute of Scientific Research.

Extraction and Isolation

The dried stem and leaves of P. scandens (GRIFF.) CRAIB (1.5 kg) were ground and extracted three times with methanol (MeOH, 3×2.5 L) at temperature 45°C in an ultrasonic bath. The resulted solution was filtered and evaporated at reduced pressure to give 85 g residue. This residue was suspended in distilled water and partitioned in turn with n-hexane and CH₂Cl₂ to obtain corresponding extracts: n-hexane (PSh, 15.2 g), CH₂Cl₂ (PSc, 37.3 g), and water layer (PSw). The water layer was passed through Diaion HP-20 CC eluting with increasing concentration of MeOH in water (0, 25, 50, 75, and 100%) to obtain four fractions, PSw1–PSw4, after removal of the fraction eluted with water. Fraction PSw2 (22 g) was separated into eight subfractions, PSw2A–PSw2H, by silica gel CC using gradient eluation of CH₂Cl₂–MeOH (20 : 1–1 : 1, v/v). Subfraction PSw2G (2.5 g) was futher separted on YMC RP-18 CC eluting with MeOH–H₂O (1.5 : 1, v/v) to give five smaller fractions, PSw2G1–PSw2G5. Purification of fraction PSw2G3 (280 mg) by silica gel CC using CH₂Cl₂–MeOH–H₂O (4 : 1 : 0.1, v/v) as eluent furnished compounds 1 (5 mg), 2 (4 mg), and 3 (3.5 mg). Fraction PSw2G4 (450 mg) furnished compounds 4 (5 mg) and 5 (4.5 mg) after subjecting it to Sephadex LH-20 CC eluting with MeOH–H₂O (1 : 1, v/v), followed by silica gel CC eluting with CH₂Cl₂–MeOH–H₂O (3 : 1 : 0.1, v/v).

Paramignyoside A (1): White powder, [α]_D²⁵ −5.0 (c, 0.1, MeOH); HR-ESI-MS m/z 865.4233 [M+Na]⁺ (Calcd for C₄₂H₆₆NaO⁺₁₇, 865.4192); ¹H-NMR (CD₃OD 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) are given in Table 1.

Paramignyoside B (2): White powder, [α]_D²⁵ −12.3 (c, 0.1, MeOH); HR-ESI-MS m/z 907.4315 [M+Na]⁺ (Calcd for C₄₄H₆₈NaO⁺₁₈, 907.4298); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) are given in Table 1.

Paramignyoside C (3): White powder, [α]_D²⁵ −1.9 (c, 0.1, MeOH); HR-ESI-MS m/z 865.4213 [M+Na]⁺ (Calcd for C₄₂H₆₆NaO⁺₁₇, 865.4192); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) are given in Table 1.

Paramignyoside D (4): White powder, [α]_D²⁵ −6.5 (c, 0.1, MeOH); HR-ESI-MS m/z 1027.4787 [M+Na]⁺ (Calcd for C₄₈H₇₆NaO⁺₂₂, 1027.4720); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) are given in Table 2.

Paramignyoside E (5): White powder, [α]_D²⁵ −8.3 (c, 0.1, MeOH); HR-ESI-MS m/z 1069.4901 [M+Na]⁺ (Calcd for C₅₀H₇₈NaO⁺₂₃, 1069.4826); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) are given in Table 2.

Acid Hydrolysis of Compounds 1–5³⁰⁾

Each compound (2.0 mg) was dissolved in 1.0 N HCl (dioxane–H₂O, 1 : 1, v/v, 1.0 mL) and then heated to 80°C in a water bath for 3 h. The acidic solution was neutralized with silver carbonate and the solvent thoroughly driven out under N₂ gas overnight. After extraction with ethyl acetate, the aqueous layer was concentrated to dryness using N₂ gas. The residue was dissolved in 0.1 mL of dry pyridine, and then L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL) was added to the solution. The reaction mixture was heated at 60°C for 2 h, and 0.1 mL of trimethylsilylimidazole solution was added, followed by heating at 60°C for 1.5 h. The dried product was partitioned with n-hexane and H₂O (0.1 mL, each), and the organic layer was analyzed by gas liquid chromatography (GC): Column SPB-1 (0.25 mm×30 m); detector hydrogen flame ionization detecter (FID), column temp. 210°C, injector temp. 270°C, detector temp. 300°C, carrier gas He. The absolute configuration of the monosaccharides in compounds 1–5 was confirmed to be all D-glucose by comparison of the retention time of the monosaccharide derivative (t_R 14.14 min) with that of authentic sugar derivative samples prepared in the same manner (D-glucose derivative t_R 14.11 min, L-glucose derivative t_R 14.26 min).

Cell Culture

BMDCs were grown from wild-type C57BL/6 mice (Orient Bio Inc.). All animal procedures were approved and performed according to the guidelines of the Institutional Animal Care and Use Committee of Jeju National University (#2010-0028). Briefly, the mouse tibia and femur were obtained by flushing with Dulbecco’s modified Eagle’s medium to yield bone marrow cells. The cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS; Gibco), 50.0 µM β-ME, 2 mM glutamine supplemented with 3% J558L hybridoma cell culture supernatant containing granulocyte-macrophage colony stimulating factor. The culture medium was replaced with fresh medium every second day. At day 6 of culture, non-adherent cells and loosely adherent dendritic cell (DC) aggregates were harvested, washed, and resuspended in RPMI 1640 supplemented with 5% FBS.

Cytokine Production Measurements

BMDCs were incubated in 48-well plates in 0.5 mL containing 1×10⁵ cells per well, and then treated with the isolated compounds 1–5 at the indicated concentration for 1 h before stimulation with 10.0 ng/mL LPS from Salmonella minnesota (ALEXIS). Supernatants were harvested 18 h after stimulation. Concentrations of murine TNF-α, IL-6, and IL-12 p40 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA) (BD PharMingen) according to the manufacturer’s instructions. The data are presented as means±standard deviation (S.D.) of at least three independent experiments performed in triplicate.

Acknowledgments

This work was supported by a Vietnam national project of the Tay Nguyen 3 Program (code: TN3/T14) and the Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2009–0093815), Republic of Korea. The authors are grateful to Dr. Nong Van Duy, Tay Nguyen Institute of Scientific Research, VAST for the plant identification; and the Institute of Chemistry, VAST for measurement of the NMR spectra.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials, 1D- and 2D-NMR spectra for the new compounds 1–5.

References

• 1) Chen H., Ma S. G., Fang Z. F., Bai J., Yu S. S., Chen X. G., Hou Q., Yuan S. P., Chen X., Chem. Biodivers., 10, 695–702 (2013).
• 2) Hu J., Song Y., Li H., Yang B., Mao X., Zhao Y., Shi X., Fitoterapia, 99, 86–91 (2014).
• 3) Kim K. H., Choi S. U., Kim Y. C., Lee K. R., J. Nat. Prod., 74, 54–59 (2011).
• 4) Kurimoto S., Takaishi Y., Ahmed F. A., Kashiwada Y., Fitoterapia, 92, 200–205 (2014).
• 5) Sichaem J., Siripong P., Tip-Pyang S., Phaopongthai J., Nat. Prod. Commun., 9, 367–368 (2014).
• 6) Zhao Q., Song Y., Feng C., Chen H., Arch. Pharm. Res., 35, 1903–1907 (2012).
• 7) Huang H. C., Tsai W. J., Morris-Natschke S. L., Tokuda H., Lee K. H., Wu Y. C., Kuo Y. H., J. Nat. Prod., 69, 763–767 (2006).
• 8) Huang H. C., Tsai W. J., Liaw C. C., Wu S. H., Wu Y. C., Kuo Y. H., Chem. Pharm. Bull., 55, 1412–1415 (2007).
• 9) Guo C., Wang J. S., Zhang Y., Yang L., Wang P. R., Kong L. Y., Chem. Pharm. Bull., 60, 1003–1010 (2012).
• 10) Tan M. A., Takayama H., Aimi N., Kitajima M., Franzblau S. G., Nonato M. G., J. Nat. Med., 62, 232–235 (2008).
• 11) Huang H.-L., Wang C.-M., Wang Z.-H., Yao M.-J., Han G.-T., Yuan J.-C., Gao K., Yuan C.-S., J. Nat. Prod., 74, 2235–2242 (2011).
• 12) Han M. L., Shen Y., Wang G. C., Leng Y., Zhang H., Yue J. M., J. Nat. Prod., 76, 1319–1327 (2013).
• 13) Ma C., Nakamura N., Hattori M., Kakuda H., Qiao J., Yu H., J. Nat. Prod., 63, 238–242 (2000).
• 14) Phan N. H. T., Thuan N. T. D., Ngoc N. T., Huong P. T. M., Thao N. P., Cuong N. X., Thanh N. V., Nam N. H., Kiem P. V., Minh C. V., Phytochem. Lett., 9, 78–81 (2014).
• 15) Quang T. H., Ngan N. T. T., Minh C. V., Kiem P. V., Nhiem N. X., Tai B. H., Thao N. P., Chae D., Mathema V. B., Koh Y.-S., Lee J. H., Yang S. Y., Kim Y. H., Arch. Pharm. Res., 36, 327–334 (2013).
• 16) Thao N. P., Dat L. D., Ngoc N. T., Tu V. A., Hanh T. T. H., Huong P. T. T., Nhiem N. X., Tai B. H., Cuong N. X., Nam N. H., Cuong P. V., Yang S. Y., Kim S., Chae D., Koh Y.-S., Kiem P. V., Minh C. V., Kim Y. H., Bioorg. Med. Chem. Lett., 23, 1823–1827 (2013).
• 17) Thao N. P., Cuong N. X., Luyen B. T., Thanh N. V., Nhiem N. X., Koh Y. S., Ly B. M., Nam N. H., Kiem P. V., Minh C. V., Kim Y. H., J. Nat. Prod., 76, 1764–1770 (2013).
• 18) Kiem P. V., Anh H. L. T., Nhiem N. X., Minh C. V., Thuy N. T., Yen P. H., Hang D. T., Tai B. H., Mathema V. B., Koh Y. S., Kim Y. H., Chem. Pharm. Bull., 60, 246–250 (2012).
• 19) Thao N. P., Cuong N. X., Luyen B. T. T., Quang T. H., Hanh T. T. H, Kim S., Koh Y.-S., Nam N. H., Kiem P. V., Minh C. V., Kim Y. H., Mar. Drugs, 11, 2917–2926 (2013).
• 20) Zi J., Li S., Liu M., Gan M., Lin S., Song W., Zhang Y., Fan X., Yang Y., Zhang J., Shi J., Di D., J. Nat. Prod., 71, 799–805 (2008).
• 21) Yuan C. M., Zhang Y., Tang G. H., Li Y., He H. P., Li S. F., Hou L., Li X. Y., Di Y. T., Li S. L., Hua H. M., Hao X. J., Planta Med., 79, 163–168 (2013).
• 22) Kurimoto S., Takaishi Y., Ahmed F. A., Kashiwada Y., Fitoterapia, 92, 200–205 (2014).
• 23) O’Neill M. J., Lewis J. A., Noble H. M., Holland S., Mansat C., Farthing J. E., Foster G., Noble D., Lane S. J., Sidebottom P. J., Lynn S. M., Hayes M. V., Dix C. J., J. Nat. Prod., 61, 1328–1331 (1998).
• 24) Swinny E. E., Z. Naturforsch. C, 56, 177–180 (2001).
• 25) Dinda B., Debnath S., Mohanta B. C., Harigaya Y., Chem. Biodivers., 7, 2327–2580 (2010).
• 26) Lee J. C., Laydon J. T., McDonnell P. C., Gallagher T. F., Kumar S., Green D., McNulty D., Blumenthal M. J., Keys J. R., Land vatter S. W., Strickler J. E., McLaughlin M. M., Siemens I. R., Fisher S. M., Livi G. P., White J. R., Adams J. L., Young P. R., Nature (London), 372, 739–746 (1994).
• 27) Gately M. K., Renzetti L. M., Magram J., Stern A. S., Adorini L., Gubler U., Presky D. H., Annu. Rev. Immunol., 16, 495–521 (1998).
• 28) Trinchieri G., Pflanz S., Kastelein R. A., Immunity, 19, 641–644 (2003).
• 29) Mannon P. J., Fuss I. J., Mayer L., Elson C. O., Sandborn W. J., Present D., Dolin B., Goodman N., Groden C., Hornung R. L., Quezado M., Neurath M. F., Salfeld J., Veldman G. M., Schwertschlag U., Strober W., Anti-IL-12 Crohn’s Disease Study Group, N. Engl. J. Med., 351, 2069–2079 (2004).
• 30) Hara S., Okabe H., Mihashi K., Chem. Pharm. Bull., 35, 501–506 (1987).