Dipasperoside A, a Novel Pyridine Alkaloid-Coupled Iridoid Glucoside from the Roots of Dipsacus asper

Abstract

A new pyridine alkaloid-coupled iridoid glucoside, dipasperoside A (1), and 20 known compounds (2–21) were isolated from a water extract of Dipsacus asper roots. Compound 1 possessed a unique structural feature with a nicotinic acid nucleus coupled through C-5 with C-7 of a secoiridoid/iridoid glucoside dimer, and esterified with a C-7 hydroxyl group of an iridoid glucoside monomer. All isolates were evaluated for their inhibitory activity against nitric oxide (NO) production in a lipopolysaccharide (LPS)-activated murine macrophage cell line, RAW264.7.

Dipsacus asper WALL (Dipsacaceae), a perennial herb, is distributed in mountainous regions of China, Korea, and Japan.¹⁾ The roots of Dipsacus asper is a well-known traditional medicine and has been used as a tonic, an analgesic, and an anti-inflammatory agent for treating spermatorrhea and pain.²⁾ It is also a common ingredient in formulations widely used for treating bone fractures and enhancement of liver and kidney functions.²⁾ This plant contains iridoid glycosides, triterpenoid saponins, alkaloids, and phenolic compounds,¹⁾ and has been reported to possess various biological activities such as anti-inflammatory,³⁾ antioxidant,⁴⁾ anticomplementary,⁵⁾ anticancer,^6,7) inhibition of Alzheimer’s disease,^8,9) antinociceptive,¹⁰⁾ cardioprotective,¹¹⁾ and osteoprotective^12,13) effects. In our screening for the anti-inflammatory activity of traditional medicinal plants, we found that a water extract of D. asper roots showed significantly inhibitory activity against the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells with an IC₅₀ value of 45.1 µg/mL. Bioassay-guided fractionation led to the isolation of a new pyridine alkaloid-coupled iridoid glucoside, dipasperoside A (1), and 20 known compounds [vanillic acid (2),¹⁴⁾ 3,4-dihydroxybenzoic acid (3),¹⁴⁾ 3,4-dihydroxybenzaldehyde (4),¹⁵⁾ caffeic acid (5),¹⁴⁾ 3β,5α-tetrahydrodesoxycordifoline lactam (6),¹⁶⁾ 4,5-dicaffeoylquinic acid (7),¹⁷⁾ 3,5-dicaffeoylquinic acid (8),¹⁷⁾ 3,4-dicaffeoylquinic acid (9),¹⁷⁾ sweroside (10),¹⁸⁾ loganin (11),¹⁸⁾ cantleyoside (12),¹⁹⁾ dipsanoside A (13),²⁰⁾ dipsanoside B (14),²⁰⁾ triplostoside A (15),¹⁹⁾ akebia saponin D (16),²¹⁾ loganic acid (17),¹⁹⁾ sylvestroside I (18),¹⁹⁾ 4′-O-acetyl-akebia saponin D (19),²¹⁾ dipsacus saponin A (20),²²⁾ and 3-O-α-L-arabinopyranosyl-28-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyloleanolic acid (21)²³⁾] (Chart 1). In this study, we report the structural elucidation of 1 together with the NO production inhibitory activity of the isolates in LPS-activated RAW264.7 cells.

Chart 1. Structures of Compounds (1–21) and Compound 1a

Compound 1 was obtained as a yellowish, amorphous solid with [α]_D²² −72.6 (c=0.1, CH₃OH), and its molecular formula was determined to be C₅₆H₇₃NO₂₉ by high-resolution time-of-flight electrospray ionization-mass spectra (HR-TOF-ESI-MS). The IR spectrum of 1 showed absorption bands of hydroxyl (3392 cm⁻¹) and α,β-unsaturated ester carbonyl (1697 cm⁻¹) groups. The ¹H-NMR spectrum (Table 1) displayed characteristic signals ascribable to two sets of the loganin (11) moieties^20,24) (units A and B). The down-field shift of H-7a (δ_H 5.44) and H-7b (δ_H 5.13) relative to that of 11 (δ_H 4.03)¹⁸⁾ indicated that the hydroxyl group at C-7 in both loganin moieties of 1 was esterified. The ¹³C-NMR data (Table 1) also supported the presence of the C-7 esterified loganin moieties, which were further confirmed by analysis of the ¹H–¹H shift correlation (COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) correlations (Fig. 1). In addition, the ¹H-NMR spectrum displayed the signals owing to a 3,5-disubstituted pyridine group (δ_H 8.91, d, J=2.0 Hz, H-2″, δ_H 8.34, t, J=2.0 Hz, H-4″, δ_H 8.75, d, J=2.0 Hz, H-6″), an isolated olefin (δ_H 7.64, s, H-3), a trans-coupled olefin (δ_H 6.43, dd, J=16.0, 8.8 Hz, H-6, δ_H 6.56, d, J=16.0 Hz, H-7), a vinyl group (δ_H 5.77, H-8, δ_H 5.28, 5.25, H-10), an acetal (δ_H 5.62, d, J=6.8 Hz, H-1), two methines (δ_H 3.64, H-5, δ_H 2.76, ddd, J=8.4, 7.3, 6.8 Hz, H-9), and a terminal β-glucopyranosyl unit (δ_H 4.75, d, J=7.8 Hz, H-1′, δ_H 3.23, H-2′, δ_H 3.39, H-3′, δ_H 3.29, H-4′; δ_H 3.34, H-5′, δ_H 3.66, 3.91, H-6′) (Table 1). Whereas, with the exception of 34 carbon signals for two loganin moieties, the ¹³C-NMR spectrum of 1 (Table 1) displayed 22 carbon signals including those of the above mentioned groups and an ester carbonyl carbon (δ_C 165.9, C-7″). These data were similar to those of lonijaposide A isolated from the flower buds of Lonicera japonica,²⁵⁾ except for the absence of O-methyl and carboxypropyl groups, and a high-field shift of C-7″ (1: δ_C 165.9; lonijaposide A: δ_C 170.5). Further analysis of the COSY, HMQC, and HMBC correlations (Fig. 1) confirmed the presence of the moiety, a nicotinic acid nucleus coupled through C-5 with C-7 of secologan-6-enic acid (unit C). Thus, compound 1 is proposed to be a structure consisting of a nicotinic acid nucleus-coupled secologan-6-enic acid and two loganin moieties. The connectivity of three moieties was determined by the analysis of the HMBC correlations. The long-range correlations of H-7a with C-7″ and H-7b with C-11 observed in the HMBC spectrum indicated that units A and C should be connected through an ester bond between C-7a and C-7″, while unit B was linked with unit C through an ester bond between C-7b and C-11. The rotating frame Overhauser effect spectroscopy (ROESY) correlations between H-1 and H-8, and between H-5 and H-9 (Fig. 1), together with the coupling constants of J_H-1,H-9 (6.8 Hz) and J_H-5,H-9 (7.3 Hz), suggested H-9 was trans relationship to H-1, and cis relationship to H-5, the same as those in secologanin unit of the reported compounds.^{18,20,24–26)} Furthermore, compound 1 was reacted with sodium methoxide to afford compound 1a and loganin (11).²⁷⁾ The product, loganin, possessed the optical rotation value ([α]_D²⁵ −89.1) identical to the reported data ([α]_D²⁰ −82.7).¹⁸⁾ The structure of 1a was assigned as the methyl ester of unit C (Chart 1) on the basis of ¹H-NMR data.²⁷⁾ The absolute stereochemistry of 1a was determined to be the same as those of lonijaposide A and its other related compounds by comparing circular dichroism (CD) data of 1a with those of the reported pyridinium alkaloid-coupled secoiridoids.^25–27) Finally, acid hydrolysis of 1 gave D-glucose, which was identified by the HPLC analysis of a chiral derivative of the hydrolysate.^28,29) Thus, the structure of 1 was assigned as shown in Chart 1. To the best of our knowledge, the occurrence of pyridine alkaloid-coupled secoiridoids in plants is rare. They have so far been reported only from L. japonica.^25,26) This is the first report on the presence of pyridine alkaloid-coupled secoiridoids in D. asper.

Table 1. ¹H- and ¹³C-NMR Data for New Compound 1 in MeOH-d₄ (J Values in Parentheses)

No.	δH	δC	No.	δH	δC	No.	δH	δC
1	5.62 d (6.8)	97.6	1a	5.34 d (4.6)	97.9	1b	5.17 d (5.4)	97.5
3	7.64 s	154.6	3a	7.46 s	152.5	3b	7.31 s	152.7
4		109.4	4a		113.1	4b		112.8
5	3.64 ma)	39.7	5a	3.23 ma	32.8	5b	2.80 ddd (8.5, 8.0, 7.8)	33.0
6	6.43 dd (16.0, 8.8)	135.7	6a	2.46 dd (14.1, 8.0)	40.4	6b	2.23 ma)	40.7
				1.88 ma)			1.56 ddd (14.1, 7.8, 4.9)
7	6.56 d (16.0)	128.5	7a	5.44 br t (5.0)	80.3	7b	5.13 br t (4.9)	78.8
8	5.77 dddd (17.2, 10.4, 8.4, 1.5)	135.4	8a	2.25 dqd (8.0, 6.6, 5.0)	41.2	8b	2.11 dqd (8.0, 6.8, 4.9)	41.2
9	2.76 ddd (8.4, 7.3, 6.8)	46.9	9a	2.19 ddd (8.5, 8.0, 4.6)	46.8	9b	1.86 ddd (8.5, 8.0, 5.4)	47.1
10	5.28 dd (17.2, 1.5)	119.6	10a	1.15 d (6.6)	13.9	10b	1.09 d (6.8)	14.1
	5.25 dd (10.4, 1.5)
11		168.1	11a		169.3	11b		168.9
			11a-OMe	3.58 s	51.8	11b-OMe	3.70 s	51.7
1′	4.75 d (7.8)	100.3	1′a	4.68 d (7.8)	100.2	1′b	4.63 d (7.8)	100.2
2′	3.23 ma)	74.7	2′a	3.22 ma)	74.7	2′b	3.22 ma)	74.7
3′	3.39 t (9.0)	78.0	3′a	3.38 t (9.0)	78.0	3′b	3.38 t (9.0)	78.0
4′	3.29 m	71.6	4′a	3.29 m	71.6	4′b	3.29 m	71.6
5′	3.34 m	78.4	5′a	3.34 m	78.4	5′b	3.34 m	78.4
6′	3.91 dd (11.7, 2.0)	62.8	6′a	3.89 dd (12.0, 2.0)	62.8	6′b	3.89 dd (12.0, 2.0)	62.8
	3.66 dd (11.7, 5.6)			3.65 dd (12.0, 6.0)			3.65 dd (12.0, 6.0)
2″	8.91 d (2.0)	152.2
3″		128.1
4″	8.34 t (2.0)	135.2
5″		135.0
6″	8.75 d (2.0)	149.2
7″		165.9

a) Chemical shifts were deduced on the basis of cross peaks in COSY and HMQC spectra.

Fig. 1. COSY (Bold Lines) and Significant HMBC (Arrows: ¹H→¹³C) Correlations and Selected ROESY Correlations (Dashed Arrows) in 1

All the isolates (1–21) were then tested for their inhibitory activity against NO production in LPS-activated RAW264.7 macrophage cells.³⁰⁾ Two alkaloid-coupled iridoids (1, 6), caffeic acid (5), two iridoid tetramers (13, 14), and four saponins (16, 19–21) showed inhibitory activity against NO production (Table 2). Akebia saponin D (16), a major constituent of the roots of D. asper, exhibited the strongest activity with an IC₅₀ value of 12.7 µM, while 1 inhibited NO production with an IC₅₀ value of 15.2 µM. Their activities were more potent than that of a positive control, N^G-monomethyl-L-arginine (L-NMMA, IC₅₀ 22.6 µM), a nonselective NOS inhibitor.³¹⁾ 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that the tested compounds had no significant cytotoxicity to the LPS-activated macrophage RAW264.7 cells at concentrations of up to 50 µM. These results demonstrated that compounds 1 and 16 might be the important anti-inflammatory components of D. asper roots and also supported the traditional utility of this plant for treating inflammatory diseases.

Table 2. Inhibitory Activity of Compounds 1–21 against NO Production in LPS-Activated Macrophage RAW264.7 Cells

Compounds	IC50 (µM)a)	Compounds	IC50 (µM)a)
1	15.2	16	12.7
2–4	>50	17, 18	>50
5	45.2	19	29.1
6	38.6	20	29.3
7–12	>50	21	23.9
13	30.9	L-NMMAb)	22.6
14	32.1

a) The inhibitory effects are represented as the molar concentration (µM) giving IC₅₀ relative to the vehicle control. b) Positive control.

Experimental

General Experimental Procedures

Optical rotations were recorded on a JASCO DIP-140 digital polarimeter. CD measurements were performed on a JASCO J-805 spectropolarimeter. IR spectra were measured with a Shimadzu IR-408 spectrophotometer. NMR spectra were measured using a JEOL JNM-LA400 spectrometer with tetramethylsilane (TMS) as the internal standard, and chemical shifts are expressed in δ values (ppm). HR-MS measurements were performed on a Shimadzu TOF mass spectrometer equipped with an ESI interface. Column chromatography was performed using silica gel (silica gel 60N, spherical, neutral, 40–50 µm, Kanto Chemical Co., Inc.) and reversed-phase silica gel (Cosmosil 75C₁₈-OPN, Nacalai Tesque Inc.). Medium-pressure liquid chromatography (MPLC) was performed using a Buchi double pump module C-605 system. Preparative HPLC was performed on a Discovery® C18 column (10×250 mm i.d., 5 µm particle size, Supelco, U.S.A., flow rate: 2 mL/min) using a Waters 600 pump (Waters, U.S.A.) and Waters 2998 photodiode array detector (Waters, U.S.A.).

Biological Material

The roots of D. asper WALL (Lot No. 09S3248) were commercially purchased from and authenticated by Uchida Wakanyaku, Ltd. (Tokyo, Japan) in February 2011. A voucher specimen (TMPW 27265) was deposited at the Museum of Materia Medica, Research Center for Ethnomedicines, Institute of Natural Medicine, University of Toyama, Japan.

Extraction and Isolation

Commercially purchased D. asper roots (450 g) were extracted with H₂O under reflux (2 L×2) for 50 min to yield 62.8 g of extract. The extract (60 g) was dissolved in H₂O (500 mL) and successively partitioned with EtOAc and n-BuOH (each 400 mL×2) to give EtOAc (2.3 g), n-BuOH (14.4 g), and H₂O (42.3 g) fractions. Among them, the n-BuOH fraction was the most active against the production of NO in LPS-activated murine macrophage RAW264.7 cells. The n-BuOH fraction (14 g) was then chromatographed on silica gel with MPLC (5×25 cm, flow rate: 25 mL/min) using a solvent system of MeOH containing 5% H₂O (5, 10–40% linear gradient, 100% for 4 h) in CHCl₃, and the eluates (100 mL each) were combined according to TLC monitoring into seven fractions (fr. 1, 716 mg, fr. 2, 260 mg, fr. 3, 568 mg, fr. 4, 760 mg, fr. 5, 2.1 g, fr. 6, 1.1 g, fr. 7, 6.4 g). Fraction 1 was further subjected to normal-phase preparative TLC (silica gel 60F₂₅₄, Merck) with hexane–EtOAc (3 : 7) followed by reversed-phase preparative TLC (silica gel RP-18F₂₅₄, Merck) with H₂O–CH₃CN (2 : 3) to yield 2 (62.1 mg), 3 (14.5 mg), 4 (22.3 mg), and 5 (32.2 mg). Fraction 2 yielded crystals of 5 (182 mg). Fraction 3 was separated by normal-phase preparative TLC with CHCl₃–MeOH–H₂O (80 : 20 : 2) to yield 5 (123 mg), 6 (8.9 mg), and a mixture of dicaffeoylquinic acids that were further purified by preparative HPLC with 40% MeOH containing 0.2% formic acid to yield 7 (22.2 mg, t_R 11.9 min), 8 (19.5 mg, t_R 12.7 min), and 9 (26.1 mg, t_R 18.3 min). Fraction 4 was identified as sweroside (10). Fraction 5 was rechromatographed on reversed-phase silica gel (4×15 cm; 40–50 µm; flow rate, 15 mL/min) by MPLC using a H₂O–MeOH gradient system to afford four subfractions: 5-1, 94 mg; 5-2, 445 mg; 5-3, 930 mg; and 5-4, 224 mg. Fractions 5-2 and 5-4 were determined to be 11 and 10, respectively. Fraction 5-3 was further separated by normal-phase preparative TLC with CHCl₃–MeOH–H₂O (70 : 30 : 3) to yield 10 (34.1 mg), 11 (56.2 mg), 12 (237 mg), 15 (23.4 mg), and a mixture (134 mg). The mixture was then subjected to preparative HPLC eluted with H₂O–CH₃CN (73 : 27) to yield 13 (10.1 mg; t_R 12.1 min), 14 (2.1 mg, t_R 11.1 min), and 1 (3.4 mg, t_R 16.9 min). Fraction 6 was identified as 16. Fraction 7 was passed through a sephadex LH-20 (GE Healthcare, Sweden) column eluting with a H₂O–MeOH system (10 : 0→9 : 1→7 : 3→4 : 6→0 : 10) to afford five subfractions: 7-1, 345 mg; 7-2, 430 mg; 7-3, 1.1 g; 7-4, 454 mg; and 7-5, 2.1 g. The NMR data of subfractions 7-2 and 7-4 were identical with those of 17 and 16. Subfraction 7-3 (400 mg) was further subjected to preparative TLC with CHCl₃–MeOH–H₂O (60 : 40 : 5) to yield 17 (148 mg) and 18 (12.4 mg). Subfraction 7-5 (400 mg) was separated by preparative TLC with CHCl₃–MeOH–H₂O (60 : 40 : 5) to yield 16 (142 mg) and a mixture of saponins (123 mg). The mixture was purified by preparative HPLC eluted with H₂O–CH₃CN (62 : 38) to yield 16 (42.3 mg, t_R 9.8 min), 19 (14.1 mg, t_R 16.6 min), 20 (13.2 mg, t_R 19.9 min), and 21 (32.8 mg, t_R 21.9 min).

Dipasperoside A (1): Yellowish, amorphous solid; [α]_D²² −72.6 (c=0.1, CH₃OH); IR (KBr) ν_max 3392 (br), 2935, 2883, 1697, 1634, 1439, 1386, 1293, 1162, 1075 cm⁻¹; CD (c=8.17×10⁻⁵ M, EtOH) [θ]₂₆₂ −12983, [θ]₂₄₃ −2374; ¹H- and ¹³C-NMR, see Table 1; HR-TOF-ESI-MS m/z 1224.4341 (Calcd for C₅₆H₇₄NO₂₉ [M+H]⁺, 1224.4347).

References

• 1)  Zhao Y. M., Shi Y. P., Chem. Biodivers., 8, 414–430 (2011).
• 2)  Namba T., “The Encyclopedia of Wakan-Yaku (Traditional Sino-Japanese Medicines) with Color Pictures,” Vol. I, Hoikusha, Osaka, 1993, p. 185.
• 3)  Hwang S. A., Hwang I. Y., Jung J., Kang H. K., Son K. H., Jeong C. S., Food and Nutrition Sciences, 3, 931–941 (2012).
• 4)  Hung T. M., Na M., Thuong P. T., Su N. D., Sok D., Song K. S., Seong Y. H., Bae K., J. Ethnopharmacol., 108, 188–192 (2006).
• 5)  Oh S. R., Jung K. Y., Son K. H., Park S. H., Lee I. S., Ahn K. S., Lee H. K., Arch. Pharm. Res., 22, 317–319 (1999).
• 6)  Jeong S. I., Zhou B., Bae J. B., Kim N. S., Kim S. G., Kwon J., Kim D. K., Shin T. Y., Jeon H., Lim J. P., Kim H., Kim H. K., Oh C. H., Arch. Pharm. Res., 31, 1399–1404 (2008).
• 7)  Hung T. M., Jin W., Thuong P. T., Song K. S., Seong Y. H., Bae K., Arch. Pharm. Res., 28, 1053–1056 (2005).
• 8)  Ji D., Wu Y., Zhang B., Zhang C. F., Yang Z. L., Fitoterapia, 83, 843–848 (2012).
• 9)  Zhang Z. J., Qian Y. H., Hu H. T., Yang J., Yang G. D., Life Sci., 73, 2443–2454 (2003).
• 10)  Suh H., Song D., Huh S., Son K., Kim Y., J. Ethnopharmacol., 71, 211–218 (2000).
• 11)  Jiang W. L., Zhang S. P., Zhu H. B., Hou J., Int. J. Cardiol., 155, 430–436 (2012).
• 12)  Kim B. S., Kim Y. C., Zadeh H., Park Y. J., Pi S. H., Shin H. S., You H. K., Biosci. Biotechnol. Biochem., 75, 13–19 (2011).
• 13)  Liu Z. G., Zhang R., Li C., Ma X., Liu L., Wang J. P., Mei Q. B., J. Ethnopharmacol., 123, 74–81 (2009).
• 14)  DellaGreca M., Monaco P., Pinto G., Pollio A., Previtera L., Temussi F., Bull. Environ. Contam. Toxicol., 67, 352–359 (2001).
• 15)  Syafni N., Putra D. P., Arbain D., Indo. J. Chem., 12, 273–278 (2012).
• 16)  Gülcemal D., Masullo M., Alankus-Caliskan O., Karayildirim T., Senol S. G., Piacente S., Bedir E., Magn. Reson. Chem., 48, 239–243 (2010).
• 17)  Timmermann B. N., Hoffmann J. J., Jolad S. D., Schram K. H., Klenck R. E., Bates R. B., J. Nat. Prod., 46, 365–368 (1983).
• 18)  Prasad D., Juyal V., Singh R., Singh V., Pant G., Rawat M. S. M., Fitoterapia, 71, 420–424 (2000).
• 19)  Ma W. G., Wang D. Z., Zeng Y. L., Yang C. R., Acta Botanica Yunnanica, 14, 92–96 (1992).
• 20)  Tian X. Y., Wang Y. H., Yu S. S., Fang W. S., Org. Lett., 8, 2179–2182 (2006).
• 21)  Kauno I., Tsuboi A., Nanri M., Kawano N., Phytochemistry, 29, 338–339 (1990).
• 22)  Jung K. Y., Do J. C., Son K. H., J. Nat. Prod., 56, 1912–1916 (1993).
• 23)  Kawai H., Kuroyanagi M., Umehara K., Ueno A., Satake M., Chem. Pharm. Bull., 36, 4769–4775 (1988).
• 24)  Tian X. Y., Wang Y. H., Liu H. Y., Yu S. S., Fang W. S., Chem. Pharm. Bull., 55, 1677–1681 (2007).
• 25)  Song W., Li S., Wang S., Wu Y., Zi J., Gan M., Zhang Y., Liu M., Lin S., Yang Y., Shi J., J. Nat. Prod., 71, 922–925 (2008).
• 26)  Yu Y., Song W., Zhu C., Lin S., Zhao F., Wu X., Yue Z., Liu B., Wang S., Yuan S., Hou Q., Shi J., J. Nat. Prod., 74, 2151–2160 (2011).
• 27)  Compound 1 (3 mg) was reacted with sodium methoxide (3 mg, Wako Pure Chemical Industries, Ltd., Osaka, Japan) in methanol (1 mL) with stirring at the room temperature for 3 h. The reaction mixture was purified by reversed-phase preparative TLC (acetone–H₂O, 3 : 7) to afford compound 1a (0.6 mg) and loganin (1.1 mg, [α]_D²⁵ −89.1, c=0.015, MeOH). Compound 1a, [α]_D²⁵ −100.6 (c=0.03, MeOH); CD (c=1.97×10⁻⁴ M, EtOH) [θ]₂₆₂ −8547, [θ]₂₄₀ −709, [θ]₂₃₃ −1182, [θ]₂₂₀ −840, [θ]₂₂₀ −2619; ¹H-NMR (400 MHz, CD₃OD) δ_H: 8.93 (1H, d, J=2.0 Hz, H-2″), 8.66 (1H, d, J=2.0 Hz, H-6″), 8.37 (1H, t, J=2.0 Hz, H-4″), 7.41 (1H, s, H-3), 6.67 (1H, d, J=16.0 Hz, H-7), 6.27 (1H, dd, J=16.0, 9.2 Hz, H-6), 6.03 (1H, ddd, J=17.6, 10.8, 8.8 Hz, H-8) 5.32 (1H, overlap, H-1), 5.31 (1H, d, J=10.8 Hz, H-10), 5.22 (1H, d, J=17.6 Hz, H-10), 4.71 (1H, d, J=7.8 Hz, H-1′), 3.23–3.88 (6H, H-2′–6′), 3.45 (1H, overlap, H-5), 3.67 and 3.50 (s, 2×OMe), 2.52 (1H, dt, J=8.8, 7.2 Hz, H-9).
• 28)  Tanaka T., Nakashima T., Ueda T., Tomii K., Kouno I., Chem. Pharm. Bull., 55, 899–901 (2007).
• 29)  Sugar Analysis of Compound 1: Sugar configuration was determined using a convenient method described by Tanaka et al.²⁸⁾ Briefly, compound 1 (1 mg) was hydrolyzed with 0.5 M HCl (0.1 mL) at 75°C for 3 h and neutralized with Amberlite IRA400. After drying in vacuo, the residue was dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester hydrochloride (0.5 mg, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and heated at 60°C for 1 h. Then, phenyl isothiocyanate (0.5 mg, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was added, and the mixture was heated at 60°C for 1 h. The reaction mixture was directly analyzed by reserved-phase HPLC. Analytical HPLC was performed on a Discovery® C18 column (4.6×250 mm i.d., 5 µm particle size, Supelco, U.S.A., flow rate: 0.8 mL/min) at 35°C with isocratic elution of 25% CH₃CN in a 50 mM H₃PO₄ solution. Derivatives of standard D- and L-glucose (Wako Pure Chemical Industries, Ltd., Osaka, Japan) gave each peak at t_R 20.94 and 19.75 min, respectively.
• 30)  NO Production Inhibitory Assay: RAW264.7 cells were seeded in 96-well plates at a density of 1×10⁵ cells/well and allowed to adhere in humidified atmosphere containing 5% CO₂ and 95% air at 37°C for 4 h. Then, the cells were treated with Escherichia coli LPS (100 ng/mL, Sigma-Aldrich Co., St. Louis, MO, U.S.A.) with or without the test samples at five different concentrations for 24 h. NO production was determined by measuring the accumulation of nitrite in the supernatant of RAW264.7 cells using the Griess reagent. Briefly, 50 µL of the cell culture supernatant was mixed with 50 µL of the Griess reagent (1 : 1 of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthylethylenediamine dihydrochloride in H₂O, Wako Pure Chemical Industries, Ltd., Osaka, Japan) in a 96-well plate, followed by a reaction for 10 min at room temperature. The absorbance at 550 nm was measured with a Multiskan FC microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.), and the nitrite concentration in the supernatants was calculated using a standard curve obtained from different concentrations of sodium nitrite (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Cytotoxicity of the compounds tested against RAW264.7 cells was measured by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT; Sigma-Aldrich Inc., St. Louis, U.S.A.) assay method. L-NMMA (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was used as the positive control in this study, and all experiments were performed in three independent replicates.
• 31)  Rees D. D., Palmer R. M., Schulz R., Hodson H. F., Moncada S., Br. J. Pharmacol., 101, 746–752 (1990).