Dispolongiosides A and B,Two New Fucose Containing Spirostanol Glycosides From the Rhizomes of Disporopsis longifolia Craib.,and Their Nitric Oxide Production Inhibitory Activities

Abstract

Two new fucose containing spirostanol glycosides, (25R)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranoside (1) and (25S)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranoside (2), together with five known spirostan glycosides, (25R)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (3), (25S)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (4), (25R)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (5), (25R)-26-[(β-D-glucopyranosyl)oxy]-22α-methoxyfurost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (6), and (25R)-26-[(β-D-glucopyranosyl)oxy]-22α-hydroxyfurost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (7) were isolated from the rhizomes of Disporopsis longifolia Craib. Their structures were determined by extensive analysis of HRESIMS and NMR spectral data, as well as by comparison of the spectral data with those reported in the literature. Compounds 1 to 7 inhibited NO production in LPS-activated RAW264.7 cells with IC₅₀ values in the range from 24.5 ± 2.5 μM to 81.3 ± 4.2 μM. The NO production inhibitory activities of compounds 1 and 2 with IC₅₀ values of 26.6 ± 1.9 μM and 24.5 ± 2.5 μM are as potent as that of the positive control of N^G-monomethyl-L-arginine acetate (L-NMMA) with an IC₅₀ value of 23.8 ± 2.1 μM.

Keywords

asparagaceae dispolongioside a dispolongioside b nitric oxide inhibitor spirostan glycoside

Introduction

The phytochemistry of plants belonging the genus Disporopsis (Asparagaceae) is mainly characterized by steroidal saponins and phenolic compounds, which have showed antitumor, anti-inflammatory, anti-diabetes, and anti-microbial activities.^1–6 Disporopsis longifolia Craib. is a herbaceous, perennial plant, which is distributed in Vietnam, Thailand, China, and Laos at a height about 400 to 1500 m. In Vietnam the rhizomes of this plant are used in folk medicine with effects of nourishing yin to lessen fire, and to invigorating liver and kidney.^1,2 However, so far, there have been no publications on the chemical constituents and biological activities of this medicinal plant, thus prompting this study. This paper reports the isolation, structure determination of two new (1 and 2) and five known (3-7) steroidal glycosides from the rhizomes of D. longifolia and their inhibition of nitric oxide (NO) production in LPS stimulated RAW 264.7 cells.

Results and Discussion

Compound 1 (Figure 1) was obtained as a white amorphous powder. Its molecular formula was deduced to be C₃₉H₆₂O₁₂ by the pseudo-molecular ion peaks at m/z 745.4130 [M + Na]⁺ (calcd. for [C₃₉H₆₂O₁₂Na]⁺, 745.4134, Δ = −0.5 ppm) and m/z 723.4313 [M + H]⁺ (calcd. for [C₃₉H₆₃O₁₂]⁺, 723.4320, Δ = −1.0 ppm) in the high resolution electron spray ionization mass spectrum (HRESIMS), indicating 9 indices of hydrogen deficiency. The ¹H NMR and HSQC spectra of 1 contained signals corresponding to an olefinic proton [δ_H 5.39 (1H, br s)], two anomeric protons [δ_H 4.34 and 4.56 (each 1H, d, J = 7.8 Hz)], two secondary methyl groups [δ_H 0.98 and 1.32 (each 3H, d, J = 7.0 Hz)], and two tertiary methyl groups [δ_H 0.84 and 1.07 (each 3H, s)]. The ¹³C NMR and HSQC spectra of 1 showed signals of 39 carbon atoms. Of these, two anomeric carbons (δ_C 103.0 and 106.0) were demonstrated for the two monosaccharide units, acquiring two indices of hydrogen deficiency. Signals of two olefinic carbons (δ_C 122.5 and 142.1), an acetal carbon [δ_C 110.6] and the remaining seven indices of hydrogen deficiency suggested the aglycone moiety of 1 to be a spirostan steroid containing one double bond. The spirostan skeleton was also confirmed by HMBC correlations between H₃ to 19 (δ_H 1.07) and C-1 (δ_C 38.5)/C-5 (δ_C 142.1)/C-9 (δ_C 51.7)/C-10 (δ_C 38.0), H₃ to 18 (δ_H 0.84) and C-12 (δ_C 40.9)/C-13 (δ_C 41.4)/C-14 (δ_C 57.8)/C-17 (δ_C 63.8), H₃ to 21 (δ_H 0.98) and C-17 (δ_C 63.8)/C-20 (δ_C 42.9)/C-22 (δ_C 110.6), and H₃ to 27 (δ_H 0.81) and C-24 (δ_C 29.9)/C-25 (δ_C 31.4)/C-26 (δ_C 67.9) (Figure 2). The COSY cross peaks of H₂ to 1 (δ_H 1.10 and 1.90)/H₂ to 2 (δ_H 1.60 and 1.93)/H-3 (δ_H 3.52)/ H₂ to 4 (δ_H 2.28 and 2.44), and HMBC correlations between H₂ to 4 (δ_H 2.28 and 2.44) and C-5 δ_C 142.1)/C-6 (δ_C 122.5) suggested the location of the oxygenated group at C-3, and double bonds at C-5/C-6, and hence the aglycone of 1 was determined to be spirost-5-en-3-ol, one of the major steroidal skeletons in Disporopsis species.³ The splitting pattern of protons H₂ to 4 [δ_H 2.28 (dd, J_H−4 _ax/ _H−4 _eq = 11.5 Hz, J_H−4 _ax/ _H−3 _ax = 11.0 Hz) and 2.44 (dd, J_H−4 _eq/ _H−4 _ax = 11.5 Hz, J_H−4 _eq/ _H−3 _ax = 4.0 Hz) indicated an axial orientation of H-3 (β-configuration at C-3). Additionally, the splitting pattern of protons H₂ to 26 in the ¹H-NMR spectrum of 1 [δ_H 3.33 (dd, J_H−26 _ax/ _H−26 _eq = 10.8 Hz, J_H−26 _ax/ _H−25 _ax = 10.8 Hz), and 3.46 (dd, J_H−26 _eq/ _H−26 _ax = 10.8 Hz, J_H−26 _eq/ _H−25 _ax = 2.8 Hz)] suggested the axial orientation of H-25 (25R-configuration). The 25R-configuration in 1 was also supported by NOE correlation between H-25 (δ_H 1.61) and H _ax -23 (δ_H 1.72) (Figure 3). Signals of the doublet methyl group (δ_C 17.5 and δ_H 1.32) in the sugar moiety of 1 suggested the presence of a 6-deoxy-hexose unit. Furthermore, the HMBC correlation of H₃ to 6′ (δ_H 1.32)/C-4′ (δ_C 82.4) and the broad doublet multiplicity of H-4′ [δ_H 3.84 (brd, J = 3.0 Hz)] indicated that 1 contains a fucose unit instead of either a galactose (as in 3 and 5) or rhamnose unit (as in uttronin B).⁷ Six carbon signals of the remaining monosaccharide (106.0, 76.0, 78.3, 71.6, 78.1, and 62.9) were assigned for a glucopyranosyl group. The HMBC correlation between Glc H-1″ (δ_H 4.56) and Fuc C-4′ (δ_C 82.4) confirmed the presence of a glucopyranosyl-(1→4)-fucopyranosyl disaccharide. Later, acid hydrolysis of 1 obtained D-glucose and D-fucose, which were identified by their positive sign for optical rotations and TLC analysis in comparison with authentic monosaccharides.⁸ The coupling constants of the anomeric protons (J_{H−1′/H−2′} = 7.8 Hz and J_{H−1″/H−2″} = 7.8 Hz) indicated β-D-glucopyranosyl and β-D-fucopyranosyl linkages. Thus, compound 1 was determined to be (25R)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranoside and named dispolongioside A (Figure 1 and Supplemental Figures S1–S8).

Figure 1.

Chemical structure of compounds 1 to 7.

Figure 2.

The ¹H-¹H COSY and key HMBC correlations of compounds 1 and 2.

Figure 3.

The key NOESY correlations of compounds 1 and 2.

Compound 2 was obtained as a white amorphous powder. HRESIMS analysis of 2 showed pseudo-molecular ion peaks at m/z 745.4126 [M + Na]⁺ (calcd. for [C₃₉H₆₂O₁₂Na]⁺, 745.4135, Δ = −1.2 ppm) and m/z 757.3921 [M + ³⁵Cl]⁻ (calcd. for [C₃₉H₆₂O₁₂³⁵Cl]⁻, 757.3935, Δ = −1.9 ppm), which indicated that 2 and 1 have the same molecular formula, C₃₉H₆₂O₁₂. As shown in Table 1, the ¹H- and ¹³C-NMR spectral data of 2 were close to those of 1 except for signals corresponding to the F-ring of the spirostan skeleton (from C-23 to C-27). In the ¹³C-NMR spectrum of 2, the carbon chemical shifts of C-23 (δ_C 27.0), C-24 (δ_C 26.8), C-25 (δ_C 28.5), C-26 (δ_C 66.1), and C-27 (δ_C 16.4) corresponded to those of 25S-stereoisomers such as neosibiricosides B and C [C-23 (δ_C 26.8), C-24 (δ_C 26.7), C-25 (δ_C 28.0), C-26 (δ_C 65.5), and C-27 (δ_C 16.7)].⁹ Additionally, the 25S-configuration (equatorial orientation of H-25) was further inferred by the splitting pattern of protons H₂ to 26 in the ¹H-NMR spectrum [δ_H 3.28 (dd, J_H−26 _eq/ _H−26 _ax = 11.4 Hz, J_H−26 _eq/ _H−25 _eq = 3.0 Hz), and 3.95 (dd, J_H−26 _ax/ _H−26 _eq = 11.4 Hz, J_H−26 _ax/ _H−25 _eq = 2.4 Hz)]. The splitting pattern of protons H₂ to 26 in the ¹H-NMR spectrum of 2 was also different from that of the 25R-configuration (axial orientation of H-25) as in 1 [δ_H 3.33 (dd, J_H−26 _ax/ _H−26 _eq = 10.8 Hz, J_H−26 _ax/ _H−25 _ax = 10.8 Hz), and 3.46 (dd, J_H−26 _eq/ _H−26 _ax = 10.8 Hz, J_H−26 _eq/ _H−25 _ax = 2.8 Hz)]. The 25S-configuration in 2 was agreed by NOE correlation between H₃ to 27 (δ_H 1.11) and H _ax -23 (δ_H 1.93) (Figure 3). Acid hydrolysis of 2 obtained D-glucose and D-fucose, which were identified by the positive sign of their optical rotations and TLC analysis in comparison with authentic monosaccharides.⁸ The coupling constants of the anomeric protons (J_{H−1′/H−2′} = 7.8 Hz and J_{H−1″/H−2″} = 7.8 Hz) indicated the β-D-glucopyranosyl and β-D-fucopyranosyl linkages. Therefore, compound 2 was determined to be (25S)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-fucopyranoside and named dispolongioside B (Figure 1 and Supplemental Figures S9–S18).

Table 1.

NMR Spectroscopic Data for Compounds 1, 2 and 5.

Pos.	1 (CD₃OD)		2 (CD₃OD)		5 (Py-d₅)
Pos.	δ_C	δ_H (mult., J in Hz)	δ_C	δ_H (mult., J in Hz)	δ_C
1	38.5	1.10 (m), 1.90 (m)	38.5	1.09 (m), 1.90 (m)	37.4
2	30.7	1.60 (m), 1.93 (m)	30.7	1.61 (m), 1.91 (m)	30.1
3	80.0	3.52 (m)	80.0	3.52 (m)	78.0
4	39.8	2.28 (dd, 11.5, 11.0) 2.44 (dd, 11.5, 4.0)	39.8	2.28 (dd, 11.5, 10.0) 2.44 (dd, 11.5, 4.8)	39.1
5	142.1	-	142.1	-	140.9
6	122.5	5.39 (br s)	122.5	5.39 (t, 2.5)	121.6
7	33.2	1.58 (m), 2.03 (m)	33.2	1.58 (m), 2.03 (m)	32.2
8	32.7	1.70 (m)	32.7	1.68 (m)	31.5
9	51.7	0.99 (m)	51.7	1.00 (m)	50.2
10	38.0	-	38.0	-	36.9
11	22.0	1.53 (m), 1.58 (m)	22.0	1.53 (m), 1.59 (m)	21.0
12	40.9	1.21 (m), 1.78 (m)	40.9	1.22 (m), 1.78 (m)	39.8
13	41.4	-	41.4	-	40.3
14	57.8	1.17 (m)	57.8	1.17 (m)	56.5
15	32.8	1.30 (m), 2.00 (m)	32.8	1.31 (m), 2.00 (m)	32.1
16	82.2	4.42 (dd, 7.2, 7.2)	82.3	4.42 (dd, 7.2, 7.2)	81.0
17	63.8	1.77 (m)	63.7	1.77 (m)	62.8
18	16.8	0.84 (s)	16.8	0.83 (s)	16.2
19	19.8	1.07 (s)	19.8	1.07 (s)	19.3
20	42.9	1.91 (m)	43.5	1.89 (m)	41.9
21	14.9	0.98 (d, 7.0)	14.7	1.02 (d, 7.0)	14.9
22	110.6	-	111.1	-	109.2
23	32.4	1.58 (m), 1.72 (m)	27.0	1.37 (m), 1.93 (m)	31.7
24	29.9	1.42 (m), 1.63 (m)	26.8	1.45 (m), 2.04 (m)	29.1
25	31.4	1.61 (m)	28.5	1.70 (m)	30.5
26	67.9	3.33 (dd, 10.8, 10.8) 3.46 (dd, 10.8, 2.8)	66.1	3.28 (dd, 11.4, 3.0) 3.95 (dd, 11.4, 2.4)	66.8
27	17.3	0.81 (d, 7.0)	16.4	1.11 (d, 7.0)	17.2
	3-O-Fuc		3-O-Fuc		3-O-Gal
1′	103.0	4.34 (d, 7.8)	102.9	4.34 (d, 7.8)	102.8
2′	73.0	3.43 (dd, 9.0, 7.8)	73.0	3.48 (dd, 9.0, 7.8)	73.2
3′	75.7	3.57 (dd, 9.0, 3.0)	75.7	3.57 (dd, 9.0, 3.0)	75.3
4′	82.4	3.84 (br d, 3.0)	82.4	3.85 (br d, 3.0)	79.8
5′	71.4	3.67 (m)	71.4	3.67 (m)	75.0
6′	17.5	1.32 (d, 7.0)	17.3	1.33 (d, 7.0)	60.7
	4′-O-Glc		4′-O-Glc		4′-O-Glc
1″	106.0	4.56 (d, 7.8)	105.9	4.57 (d, 7.8)	106.8
2″	76.0	3.28 (dd, 9.0, 7.8)	76.0	3.28 (dd, 9.0, 7.8)	75.7
3″	78.3	3.37 (dd, 9.0, 9.0)	78.3	3.38 (dd, 9.0, 9.0)	78.4
4″	71.6	3.29 (dd, 9.0, 9.0)	71.6	3.30 (dd, 9.0, 9.0)	71.1
5″	78.1	3.28 (m)	78.2	3.28 (m)	78.3
6″	62.9	3.68 (dd, 12.0, 5.0) 3.87 (dd, 12.0, 2.0)	62.9	3.68 (dd, 12.0, 5.0) 3.87 (dd, 12.0, 2.0)	62.9

The other compounds were identified to be (25R)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (3),¹⁰ (25S)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (4),¹⁰ (25R)-spirost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (5),¹¹ (25R)-26-[(β-D-glucopyranosyl)oxy]-22α-methoxyfurost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (6),¹¹ and (25R)-26-[(β-D-glucopyranosyl)oxy]-22α-hydroxyfurost-5-en-3β-yl O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside (7) (Supplemental Figures S19–S28).¹¹ Steroidal glycosides in terms of two frames spirostan and furostan have been reported to be major chemical components of the Disporopsis and Polygonatum genera.^12,13 However, compounds containing fucose moiety directly linked to the aglycone as in 1 and 2 are very rare. The spirostan and furostan saponins have been reviewed for their potential anti-inflammatory activity.^13,14 Therefore, compounds 1 to 7 were evaluated for anti-inflammatory activity by their ability to inhibit NO production in LPS stimulated RAW 264.7 cells. At a concentration as high as 100 μM, compounds 1 to 7 did not significantly show cytotoxic activity by MTT assay (data not shown). Therefore, the levels of NO production in the cell medium were measured in the presence of compounds 1 to 7 at serial diluted concentrations (0-100 μM). As shown in Table 3, compounds 1 to 7 exhibited NO inhibitory activity with IC₅₀ values in the range from 24.5 ± 2.5 μM to 81.3 ± 4.2 μM (Table 2). Compounds 1 and 2 were the best NO inhibitors, with IC₅₀ values (26.6 ± 1.9 μM and 24.5 ± 2.5 μM, respectively) similar to that of the positive control L-NMMA (23.8 ± 2.1 μM). Regarding structure activity relationship, our results showed that the spirostan saponins (1-5) could be better NO inhibitors than the furostan saponins (6 and 7). Among the spirostan saponins, the stereochemistry at C-25 did not clearly show any change in NO inhibitory activity between the two pairs of 25-epimers (1 and 2, 3 and 4).

Table 2.

Inhibitory Activity of Compounds 1 to 7 on NO Production in the LPS-Activated RAW264.7 Cells.

Compounds	IC₅₀ (μM)
1	23.6 ± 1.9
2	20.7 ± 2.5
3	27.3 ± 2.6
4	29.1 ± 2.0
5	38.0 ± 3.3
6	74.4 ± 3.5
7	81.3 ± 4.2
L-NMMA ¹	23.8 ± 2.1

Positive control.

Materials and Methods

General Experimental Procedures

Optical rotation was measured on a Jasco P-2000 polarimeter, NMR spectra on a Bruker AvanceNEO 600 MHz spectrometer, and HRESIMS on a SCIEX X500 QTOF LC/MS. Flash column chromatography was performed using either silica gel or reversed phase (RP-18) resins as adsorbent. The ratio between the amount of silica gel and fraction was 20/1 (w/w). A fraction collector was set by volume per tube (15 mL/tube). Fractionation was monitored by thin layer chromatography (TLC) to combine test tubes showing similar TLC pattern. TLC was carried out on either pre-coated silica gel 60 F₂₅₄ and/or RP-18 F_254S plates. Compounds were visualized by UV irradiation (254 and 365 nm) and by spraying with H₂SO₄ solution (5%) followed by heating with a heat gun. HPLC was carried out using an AGILENT 1100 HPLC system.

Plant Material

The rhizomes of Disporopsis longifolia Craib (Asparagaceae) were collected in Hoang Su Phi District, Ha Giang Province, Vietnam, in September 2020, and identified by Dr Do Hoang Chung at the Institute of Forestry Research and Development, Thai Nguyen University of Agriculture and Forestry, Thai Nguyen City, Vietnam. A voucher specimen (NCCT-P129) is kept at the Institute of Forestry Research and Development.

Extraction and Isolation

Dried, powdered Disporopsis longifolia rhizomes (4.0 kg) were ultrasonically extracted with hot MeOH (45 °C), three times (each 15 L). The extracts were filtered through filter paper, and the filtrate was evaporated to dryness to yield 500 g of a dark solid extract. This was suspended in water (5.0 L) and successively partitioned with dichloromethane and ethyl acetate (each 5 L) giving the dichloromethane extract (IPL1, 20 g), ethyl acetate extract (IPL2, 1.2 g), and water layer (IPL1C). After removal of the solvent, the water layer was chromatographed on a Diaion HP-20 column eluting with water to remove sugar, then with increasing concentration of methanol in water (25, 50, 75, and 100%) to obtain four fractions, IPL3 (8.3 g), IPL4 (15.6 g), IPL5 (12.7 g) and IPL6 (0.7 g). Fraction IPL4 (15.6 g) was chromatographed on a silica gel column eluting with dichloromethane/methanol/water (5/1/0.1, v/v/v) to give five sub-fractions (IPL4A-IPL4E). IPL4D (4.3 g) was further chromatographed on a YMC column eluting with acetone/water (2/1, v/v) to give three sub-fractions (IPL4D1-IPL4D3). IPL4D3 (0.1 g) was further chromatographed by HPLC using a J'sphere ODS H-80, 250 mm × 20 mm column, acetonitrile in H₂O (75%, v/v) and a flow rate of 3 mL/min to yield compounds 1 (16.1 mg), 2 (15.7 mg) and 7 (5.0 mg). IPL5 (12.7 g) was chromatographed on a YMC column eluting with acetone/water (1.6/1, v/v) to give four sub-fractions (IPL5E1-IPL5E4). IPL5E4 (0.1 g) was further chromatographed by HPLC using a J'sphere ODS H-80, 250 mm × 20 mm column, MeOH in H₂O (90%, v/v) and a flow rate of 3 mL/min to yield compounds 3 (5.0 mg) and 4 (6.1 mg). The IPL6 fraction (0.7 g) was chromatographed on a silica gel column eluting with dichloromethane/methanol/water (3/1/0.1, v/v/v) to give three sub-fractions (IPL6A-IPL6C). IPL6C (0.2 g) was further chromatographed by HPLC using a J'sphere ODS H-80, 250 mm × 20 mm column, MeOH in H₂O (75%, v/v) and a flow rate of 3 mL/min to yield compounds 5 (6.2 mg) and 6 (8.1 mg).

Dispolongioside A (1)

Colorless amorphous powder, $[α]_{D}^{25}$ : −54.0° (c 0.1, MeOH); IR (KBr) ν_max: 3396, 2924, 1644, 1455, 1052 cm⁻¹. HRESIMS m/z 745.4130 [M + Na]⁺ (calcd. for [C₃₉H₆₂O₁₂Na]⁺, 745.4134, Δ = −0.5 ppm), m/z 723.4313 [M + H]⁺ (calcd. for [₃₉H₆₃O₁₂]⁺, 723.4320, Δ = −1.0 ppm), m/z 757.3904 [M + ³⁵Cl]⁻ (calcd. for [₃₉H₆₂O₁₂³⁵Cl]⁻, 757.3935, Δ = −4.1 ppm). ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) data are given in Table 1.

Dispolongioside B (2)

Colorless amorphous powder, $[α]_{D}^{25}$ : −49.0° (c 0.1, MeOH); IR (KBr) ν_max: 3399, 2926, 1643, 1452, 1063 cm⁻¹. HRESIMS m/z 745.4126 [M + Na]⁺ (calcd. for [C₃₉H₆₂O₁₂Na]⁺, 745.4135, Δ = −1.2 ppm), m/z 757.3921 [M + ³⁵Cl]⁻ (calcd. for [₃₉H₆₂O₁₂³⁵Cl]⁻, 757.3935, Δ = −1.9 ppm). ¹H NMR (CD₃OD, 600 MHz) and ¹³C NMR (CD₃OD, 150 MHz) data are given in Table 1.

Acid Hydrolysis of Compounds 1 and 2

Compounds 1 and 2 (each, 8.0 mg) were dissolved in 0.5 mL of 2 M HCl and heated at 60 °C for 1.5 h. After cooling, the mixtures were extracted with EtOAc. The acid aqueous layer was neutralized with 2 M NaOH and freeze-dried. Two monosaccharides were confirmed to be glucose and fucose by TLC analysis in comparison with authentic samples. After preparative TLC of the sugar mixture (for each 1 and 2, 2.5 mg), each isolated sugar was filtered to eliminate silica gel residue. The optical rotation of each purified monosaccharide (1.2 mg of glucose and 1.0 mg of fucose) was measured to afford glucose and fucose as + 19.0 (c 0.1, H₂O) and + 16.2 (c 0.1, H₂O), respectively, which are consistent with the optical rotations of authentic D-glucose $[α]_{D}^{25}$ : + 18.1 (c 0.1, H₂O) and D-fucose $[α]_{D}^{25}$ : + 20.5 (c 0.1, H₂O).

Nitric Oxide Assay

Refer to Supplemental Material.

Conclusions

Five spirostan (1-5) and two furostan (6 and 7) glycosides were isolated from the rhizomes of Disporopsis longifolia Craib. Among them, compounds 1 and 2 are two fucose containing spirostanol glycosides, previously undescribed. Their structures were determined by extensive analysis of HRESIMS and NMR spectral data. Compounds 1 and 2 showed the best NO inhibition with IC₅₀ values of 26.6 ± 1.9 μM and 24.5 ± 2.5 μM, respectively. Additionally, spirostan-type saponins (1-5) showed smaller IC₅₀ values than those of furostan-type saponins (6 and 7).

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211055013 - Supplemental material for Dispolongiosides A and B, Two New Fucose Containing Spirostanol Glycosides From the Rhizomes of Disporopsis longifolia Craib., and Their Nitric Oxide Production Inhibitory Activities

Supplemental material, sj-docx-1-npx-10.1177_1934578X211055013 for Dispolongiosides A and B, Two New Fucose Containing Spirostanol Glycosides From the Rhizomes of Disporopsis longifolia Craib., and Their Nitric Oxide Production Inhibitory Activities by Tran Thi Thu Ha, Dang Kim Vui, Nguyen Huy Hoang, Bui Huu Tai and Phan Van Kiem in Natural Product Communications

Footnotes

Acknowledgments

We thank Dr Do Hoang Chung at The Institute of Forestry Research and Development, Thai Nguyen University of Agriculture and Forestry for the identification of the plant sample.

Conflict of Interest

The authors declare no conflict of interest.

Ethical Approval

Our institution does not require ethical approval for reporting individual cases or case series.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors would like to express their sincere thanks to the Ministry of Education and Training for supporting research funding through the topic: “Research on conservation and development of genetic resources of Disporopsis longifolia Craib. in Ha Giang province”, code: CT-2018-TNA-01.

Trial Registration

Not applicable, because this article does not contain any clinical trials.

ORCID iD

Phan Van Kiem

Supplemental material

Supplemental material for this article is available online.

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