Polygokingiasides A and B: Two New Spirostane Glycosides From the Roots of Polygonatum kingianum With Nitric Oxide Inhibitory Activity

Abstract

Two new spirostane glycosides, named polygokingiaside A (1) and polygokingiaside B (2), and 6 known spirostane glycosides, (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (3), (25S)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (4), (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (5), (23S,25R)-spirostan-5-en-3β,23-dihydroxy-12-one-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (6), (24S,25R)-spirostan-5-en-3β,24-dihydroxy-12-one-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (7), and (25R)-spirostan-5-en-3β-hydroxy-12-one-3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (8), were isolated from the roots of Polygonatum kingianum Collett & Hemsl. (Asparagaceae). Their structures were determined by extensive analysis of high-resolution electron spray ionization mass spectrometry and nuclear magnetic resonance spectral data, as well as by comparison of the spectral data with those reported in the literature. Anti-inflammatory activity of compounds 1-8 was evaluated by their inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. At a concentration of 20 µM, compounds 1-8 exhibited a modest inhibitory effect on NO production in LPS-stimulated RAW246.7 cells with inhibitory values ranging from 9.5 ± 0.8% to 33.8 ± 2.1%.

The genus Polygonatum comprises 74 accepted species belonging to the Asparagaceae family. Polygonatum kingianum Collett & Hemsl has 13 synonyms: P. agglutinatum Hua, P. cavaleriei H. Lév., P. darrisii H. Lév., P. ericoideum H. Lév., P. esquirolii H. Lév., P. huanum H. Lév., P. kingianum var. cavaleriei (H. Lév.) C. Jeffrey & McEwan, P. kingianum var. ericoideum (H. Lév.) C. Jeffrey & McEwan, P. kingianum var. grandifolium D. M. Liu & W. Z. Zeng, P. kingianum var. uncinatum (Diels) C. Jeffrey & McEwan, P. uncinatum Diels, P. vietnamicum L. I. Abramova, and Galium tuberosum Lour. The species is a precious medicinal plant that is distributed in south-central China, southeast China, Myanmar, Thailand, and in the north of Vietnam.^1,2 The roots of P. kingianum are well known as a traditional medicinal herb and functional food in China and Vietnam for the treatment of lung diseases, back pain, and body weakness. ^{1 -3} As reported earlier, the roots of P. kingianum contain mainly steroidal saponins. ^{3 -7} Especially, saponins from P. kingianum may be used as adjuvant therapy to control blood glucose and insulin resistance in type 2 diabetic individuals. ⁸ With the aim of finding natural bioactive constituents from Vietnamese medicinal plants, we describe herein the isolation and chemical structure elucidation of 8 spirostane glycosides, including 2 that are new, from the methanol extract of the roots of P. kingianum. The anti-inflammatory activity of these compounds was evaluated by their inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.

Results and Discussion

Compound 1 was obtained as a colorless amorphous powder. Its molecular formula was deduced to be C₄₅H₇₂O₁₉ based on the cluster of quasi-molecular ion peaks in the high-resolution electron spray ionization mass spectrometry (HR-ESI-MS), including those at m/z 951.4325 [M + ³⁵ Cl]⁻ (Calcd. for [C₄₅H₇₂O₁₉ ³⁵Cl]⁻, 951.4356), m/z 952.4373 [M + ³⁵⁺³⁷ Cl]⁻ (Calcd. for [C₄₅H₇₂O₁₉ ³⁵⁺³⁷ Cl], 952.4390), m/z 953.4361 [M + ³⁷ Cl]⁻ (Calcd. for [C₄₅H₇₂O₁₉ ³⁷Cl]⁻, 953.4327), and m/z 917.4749 [M + H]⁺ (Calcd. for [C₄₅H₇₃O₁₉]⁺, 917.4741) (Supplemental Figure S2). The ¹H-nuclear magnetic resonance spectrum of 1 exhibited 3 methyl singlets at δ _H 0.84, 1.07, and 1.25 (each, 3H), 1 methyl doublet at 1.02 (3H), and 1 olefinic broad singlet at δ _H 5.40 corresponding to a steroidal aglycon having a double bond at C-5/C-6.4 ^{4 -7,9} Besides, 3 sugar units were identified by 3 anomeric protons at δ _H 4.32, 4.37, and 4.53 (each, 1H, doublet, J = 7.5 Hz) (Supplemental Figure S3). The ¹³C-NMR spectrum of 1 (Supplemental Figure S4) indicated the presence of 45 carbon signals, which were then further classified from the heteronuclear single-quantum correlation spectroscopy (HSQC) (Supplemental Figure S5), including 27 carbons of the steroidal aglycon and 18 carbons of 3 sugar molecules. The ¹³C NMR spectrum of compound 1 indicated signals for C-3, C-5, C-6, C-22, C-25, and C-26 (δ _C 80.0, 142.0, 122.5, 121.8, 85.2, and 77.6, respectively) confirming that the aglycone of 1 was in good accordance with that of nuatigenin [(22S,25S)-22,25-epoxyfurost-5-en-3β,26-diol] ¹⁰ having a sugar moiety linked to C-3, as with chonglouoside SL-11, chonglouoside SL-12, chonglouoside SL-13, chonglouoside SL-14, chonglouoside SL-15, and chonglouoside SL-16. ⁹ Detailed analysis of the ¹H, ¹³C NMR, HSQC, heteronuclear multiple bond correlation (HMBC), and ¹H-¹H correlation spectroscopy (COSY) of 1 (Supplemental Figures S3-S7) and comparison of the sugar NMR data of 1 with the corresponding data of kinggianoside A (8), ⁷ (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (3), ⁷ chonglouoside SL-10, and chonglouoside SL-11 ⁹ suggested that 1 glucosyl unit was attached to C-26 and the other sugar moiety, including 1 glucose and 1 galactose, was linked to C-3, which were further confirmed by the HMBC spectrum (Supplemental Figure S6). In the ¹H-¹H COSY spectrum, cross-peaks were observed from H-2″ (δ _H 3.51) to H-1″ (δ _H 4.37)/H-3″ (δ _H 3.58), from H-4″ (δ _H 4.07) to H-3″/H-5″ (δ _H 3.53), and from H-5″ to H-6″ (δ _H 3.62/3.88). This evidence, together with the small coupling constant (J = 3.0 Hz) of H-4″, confirmed that H-4″ was in an equatorial orientation and the sugar was galactose. In the HMBC spectrum, the anomeric proton H-1′ (δ _H 4.32) correlated with C-26 (δ _C 77.6), while proton H-1″′ (δ _H 4.53) correlated with gal C-4 (δ _C 79.2), and gal H-1″ (δ _H 4.37) correlated with C-3 (δ _C 80.0) confirming that the sugar linkages of compound 1 are as shown in Figure 1. Furthermore, the coupling constant (J = 7.5 Hz) observed for the anomeric protons in the ¹H NMR spectrum of 1 indicated that the sugar linkages must be in the β-form. In the nuclear Overhauser effect spectroscopy (NOESY) (Supplemental Figure S8), cross-peaks from H-21 (1.02) to H-17 (1.79), H-18 (0.84) to H-23 (1.31/1.62), and from H-17 to H-16 (4.48) reconfirmed the aglycon structure of 1 as nuatigenin [(22S,25S)−22,25-epoxyfurost-5-en-3β,26-diol]. Finally, the presence of d-glucose and d-galactose in the acid hydrolysis products of compound 1 was confirmed by gas chromatographic (GC) analysis of their trimethylsilyl derivatives, as previously described. ¹¹ Consequently, compound 1 was determined to be 26-O-β-d-glucopyranosyl nuatigenin 3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside and named as polygokingiaside A.

Figure 1

Chemical structure of compounds 1‐8 isolated from the roots of Polygonatum kingianum.

Compound 2 was obtained as a colorless amorphous powder, and its molecular formula was deduced to be C₄₅H₇₄O₁₉ based on the cluster of quasi-molecular ion peaks in the HR-ESI-MS at m/z 941.4717 [M + Na]⁺ (Calcd. for [C₄₅H₇₄O₁₉Na]⁺, 941.4717) (Supplemental Figure S8). The NMR spectra of 2 (Supplemental Figures S10-S16) were similar to those of compounds 1, 3, ⁷ and 4 ¹² suggesting that compound 2 was a spirostane glycoside. The sugar moieties of 2 were also found to be similar to 1 and 3, identified from anomeric protons and carbons at δ _H 4.27 (d, J = 7.5 Hz)/δ _C 104.7, 4.37 (d, J = 7.5 Hz)/δ _C 103.0, and 4.53 (d, J = 7.5 Hz)/δ _C 106.1. One glucose was attached to C-26 and a β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside moiety was linked to C-3, which were confirmed by ¹H-¹H COSY and HMBC spectra, as shown in Supplemental Figure S1. Comparing the NMR data of 2 with that of 3 showed that they were very similar except for the proton signals of C-26 at δ _H3.80 (1H, H_a-26) and δ _H 3.36 (1H, H_b-26) (Δ = 0.44) in compound 2, instead of proton signals at δ _H 3.75 (1H, H_a-26) and 3.41 (1H, H_b-26) (Δ = 0.34) in 3. This evidence indicated that the configuration of the methyl group at C-25 of 2 is S and that of 3 is R. ^5,13,14 In addition, NOE cross-peaks from H-18 (0.85) to H-23 (1.67/1.70) were observed, reconfirming the 22α-OH group (Supplemental Figure S16). Finally, the presence of d-glucose and d-galactose in the acid hydrolysis products of compound 2 were confirmed by GC analysis of their trimethylsilyl derivatives, as previously described. ¹¹ Consequently, compound 2 was determined to be (25S)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside, and named as polygokingiaside B.

Compounds 3-8 were identified as (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (3), ⁷ (25S)−26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside or dehydrotomatoside (4), ¹² (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (5), ⁷ (23S,25R)-spirostan-5-en-3β,23-dihydroxy-12-one-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (6), ⁶ (24S,25R)-spirostan-5-en-3β,24-dihydroxy-12-one-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (7), ¹⁵ and (25R)-spirostan-5-en-3β-hydroxy-12-one-3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (8). ⁷ The NMR spectral data of compounds 2-8 were consistent with those previously reported in the literature (Supplemental Figures S17-S38). Anti-inflammatory activity of compounds 1-8 was evaluated by their ability to inhibit NO production in LPS-stimulated RAW 264.7 cells. Each compound was assessed at a concentration of 20 µM. Compounds 1-8 moderately inhibited NO production with inhibitory rates from 9.5 ± 0.8% to 33.8 ± 2.1%. No cytotoxic effect on the RAW 264.7 cells was found after treating the cells with compounds 1-8 (20 µM). l-N^G-monomethyl-l-arginine was used as a positive control. Its NO inhibitory value was 83.1 ± 2.3% at a concentration of 20 µM (Table 1).

Table 1

Effect of Compounds 1-8 (20 µM) on Nitric Oxide Production in LPS-Stimulated RAW 264.7 cells.

Compounds	Inhibition (%)	Cell viability (%)
1	33.8 ± 2.1	97.5 ± 0.8
2	27.1 ± 1.3	93.6 ± 1.7
3	21.4 ± 1.7	99.1 ± 1.1
4	29.6 ± 1.3	93.8 ± 1.5
5	23.5 ± 0.7	98.2 ± 0.6
6	9.5 ± 0.8	98.5 ± 0.9
7	12.8 ± 0.9	94.1 ± 1.3
8	14.2 ± 1.6	99.4 ± 1.8
L-NMMA a	83.1 ± 2.3	94.5 ± 2.7

Abbreviations: L-NMMA, N^G-monomethyl-L-arginine; LPS, lipopolysaccharide.

^aPositive control compound.

Materials and Methods

General Experimental Procedures

Optical rotation was measured on a Jasco P-2000 polarimeter, NMR spectra on a Bruker 500 MHz spectrometer, and HR-ESI-MS on an Agilent 6530 Accurate Mass Q-TOF LC/MS. The QTOF instrument was set at 2 GHz extended dynamic range resolution mode, with a negative ESI capillary voltage of 3500 V, fragmentor voltage of 175 V, MS scan ranging from m/z 100-1700, and an MS acquisition rate of 1.0 spectra/second. 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 precoated silica gel 60 F₂₅₄ and/or RP-18 F_254S plates. Compounds were visualized by ultraviolet irradiation (254 and 365 nm) and by spraying with a sulfuric acid solution (5%) followed by heating with a heat gun. High-performance liquid chromatography (HPLC) was carried out using an AGILENT 1100 HPLC system.

Plant Material

The roots of P. kingianum Collett & Hemsl. (Asparagaceae) were collected at Ho Thau Commune, Hoang Su Phi District, Ha Giang Province, Vietnam, in September 2020, and identified by Professor Dang Kim Vui at the Institute of Forestry Research and Development, Thai Nguyen University of Agriculture and Forestry, Thai Nguyen City, Vietnam. A voucher specimen (HTD.09.2020) is kept at the Institute of Forestry Research and Development.

Extraction and Isolation

Dried, powdered P. kingianum roots (3.4 kg) were ultrasonically extracted with hot methanol (MeOH), 3 times. The extracts were filtered through filter paper, and the filtrate was evaporated to dryness to yield 550 g of a dark solid extract. This extract was suspended in water (H₂O) and successively partitioned with dichloromethane (CH₂Cl₂) and ethyl acetate giving the CH₂Cl₂ extract (IPK1A, 22 g), ethyl acetate extract (IPK1B, 1.4 g), and H₂O layer (IPK1C). The IPK1A fraction was chromatographed on a silica gel column, eluting with CH₂Cl₂/MeOH (100:0→ 0:1, v/v), to give 7 subfractions (IPK5A-IPK5G). The IPK5F (3.5 g) and IPK5G (1.5 g) fractions were combined and chromatographed on a silica gel column eluting with CH₂Cl₂/MeOH/H₂O (6/1/0.1, v/v/v) to give 8 smaller fractions (IPK9A-IPK9H). IPK9E (300 mg) was chromatographed on an RP-18 column eluting with acetone/H₂O (2/1, v/v) to give 2 fractions, IPK10A and IPK10B. Compound 8 (7.0 mg, t _R = 35.52′) was obtained from IPK10A by HPLC using a J’sphere ODS H-80, 250 mm × 2 0 mm column, acetonitrile (ACN) in H₂O (50%, v/v), and a flow rate of 3 mL/min. Continuously, the H₂O layer was chromatographed on a Diaion HP-20 column eluting with H₂O to remove sugar, then with increasing concentration of MeOH in H₂O (25%, 50%, 75%, and 100%) to obtain 4 fractions, IPK2A (12.0 g), IPK2B (6.3 g), IPK2C (5.3 g), and IPK2D (1.6 g). Fraction IPK2D was chromatographed on a silica gel column eluting with CH₂Cl₂/MeOH/H₂O (4/1/0.1,v/v/v) to give 6 subfractions IPK3A-IPK3F. IPK3B was further chromatographed by HPLC using a J’sphere ODS H-80, 250 mm × 20 mm column, ACN in H₂O (35%, v/v), and a flow rate of 3 mL/min to yield compound 6 (12.0 mg, t _R=30.82′). The IPK3C fraction was chromatographed on HPLC using the same conditions to yield 7 (4.6 mg, t _R = 29.10′)). IPK3D was chromatographed on a Sephadex column eluting with MeOH/H₂O (1/1, v/v) to give 2 smaller fractions, IPK4A-IPK4B. Compound 1 (7.0 mg, t _R = 52.62′) was obtained from IPK4A chromatographed on an RP-18 column eluting with acetone/H₂O (1/1.2, v/v). IPK4B was further chromatographed by HPLC using a J’sphere ODS H-80, 250 mm × 20 mm column, ACN in H₂O (27 %) to yield 2 (11.4 mg, t _R = 62.14′) and 3 (20.0 mg, t _R = 65.79′). Finally, the IPK3F fraction was chromatographed on HPLC using the same conditions to yield 4 (8.0 mg, t _R = 47.03′) and 5 (7.0 mg, t _R = 48.99′).

Polygokingiaside A (1)

Colorless amorphous powder, $\left[\alpha \right]{}_D^{25}:$ − 45.0° (c 0.1, MeOH); HR-ESI-MS m/z 951.4325 [M + ³⁵Cl]⁻ (Calcd. for [C₄₅H₇₂O₁₉ ³⁵Cl]⁻, 951.4356), m/z 952.4373 [M + ³⁵⁺³⁷Cl]⁻ (Calcd. for [C₄₅H₇₂O₁₉ ³⁵⁺³⁷Cl], 952.4390), m/z 953.4361 [M + ³⁷Cl]⁻ (Calcd. for [C₄₅H₇₂O₁₉ ³⁷Cl]⁻, 953.4327), and m/z 917.4749 [M + H]⁺ (Calcd. for [C₄₅H₇₃O₁₉]⁺, 917.4741); ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) data are given in Table 2.

Table 2

¹H-NMR and ¹³C-NMR Spectroscopic Data for Compounds 1 and 2 in Deuterated Methanol.

Position	δ C a	δ H (mult., J in Hz) b	δ C a	δ H (mult., J in Hz) b
1	38.5	1.10 (m), 1.90 (m)	38.5	1.10 (m), 1.89 (m)
2	30.7	1.62 (m), 1.92 (m)	30.7	1.62 (m), 1.94 (m)
3	80.0	3.55 (m)	80.0	3.56 (m)
4	39.7	2.28 (dd, 12.5, 11.5) 2.44 (brd, 11.5)	39.7	2.28 (dd, 12.5, 11.5) 2.44 (dd, 12.5, 2.5)
5	142.0	-	142.0	-
6	122.5	5.40 (brs)	122.5	5.39 (brs)
7	33.2	1.57 (m), 2.00 (m)	33.2	1.58 (m), 2.02 (m)
8	32.8	1.68 (m)	32.8	1.68 (m),
9	51.7	1.00 (m)	51.7	1.00 (m)
10	38.0	-	38.0	-
11	22.0	1.58 (m)	22.0	1.57 (m)
12	40.9	1.23 (m), 1.80 (m)	40.9	1.22 (m), 1.80 (m)
13	41.6	-	41.8	-
14	57.7	1.17 (m)	57.7	1.17 (m)
15	33.6	1.97 (m), 2.03 (m)	32.9	1.29 (m), 2.00 (m)
16	82.2	4.50 (m)	82.2	4.60 (m)
17	63.3	1.79 (m)	64.1	1.80 (m)
18	16.6	0.84 (s)	16.8	0.85 (s)
19	19.8	1.07 (s)	19.9	1.07 (s)
20	39.4	2.20 (t, 6.5)	41.0	2.10 (t, 7.0)
21	15.1	1.02 (d, 6.5)	16.0	1.03 (d, 7.0)
22	121.8	-	111.9	-
23	30.7	1.31 (m), 1.62 (m)	37.0	1.67 (m), 1.77 (m)
24	33.8	1.68 (m), 2.06 (m)	28.6	1.32 (m), 1.65 (m)
25	85.2	-	35.0	1.78 (m)
26	77.6	3.50 (m), 3.87 (m)	76.0	3.36 (m), 3.80 (dd, 10.0, 6.0)
27	24.3	1.25 (s)	17.4	0.98 (d, 6.5)
1′	105.0	4.32 (d, 7.5)	104.7	4.27 (d, 7.5)
2′	75.3	3.24*	75.2	3.20*
3′	77.8	3.30*	78.3	3.38*
4′	71.7	3.30*	71.7	3.29*
5′	78.0	3.37 (m)	77.9	3.25 (m)
6′	62.8	3.69 (dd, 12.0, 5.0) 3.88 (dd. 12.0, 2.0)	62.8	3.69 (dd, 12.0, 5.0) 3.88 (dd, 12.0, 2.0)
1″	103.0	4.37 (d, 7.5)	103.0	4.37 (d, 7.5)
2″	73.2	3.51*	73.2	3.52*
3″	75.3	3.53*	75.5	3.55*
4″	79.2	4.07 (d, 3.0)	79.2	4.06 (d, 3.0)
5″	75.5	3.53 (m)	75.7	3.30 (m)
6″	61.3	3.62 (dd, 11.5, 5.0) 3.88 dd, 11.5, 2.0)	61.3	3.62 (dd, 11.5, 5.0) 3.89 dd, 11.5, 2.0)
1″′	106.1	4.53 (d, 7.5)	106.1	4.53 (d, 7.5)
2″′	75.7	3.29*	75.2	3.58*
3″′	78.0	3.30*	78.1	3.35*
4″′	72.0	3.23*	72.0	3.23*
5″′	78.3	3.37 (m)	78.0	3.30 (m)
6″′	63.2	3.60 (dd, 11.5, 5.0) 3.90 (dd, 11.5, 2.0)	63.2	3.61 (dd, 11.5, 5.0) 3.91 (dd, 11.5, 2.0)

Abbreviation: NMR, nuclear magnetic resonance.

Asterisk indicates overlapped signals.

^aMeasured at 125 MHz.

^bMeasured at 500 MHz.

Polygokingiaside B (2)

Colorless amorphous powder, $\left[\alpha \right]{}_D^{25}$ : − 61.2° (c 0.1, MeOH); HR-ESI-MS m/z 941.4711 [M + Na]⁺ (Calcd. for [C₄₅H₇₄O₁₉Na]⁺, 941.4717). ¹H-NMR (CD₃OD, 500 MHz) and ¹³C-NMR (CD₃OD, 125 MHz) data are given in Table 2.

Acid hydrolysis and confirmation of monosaccharide

Refer to Supplemental Material.

NO Assay

Refer to Supplemental Material.

Conclusions

Polygonatum kingianum Collett & Hemsl. (Asparagaceae) is a valuable medicinal plant, used in traditional remedies to treat some diseases. From the methanol extract of the roots of this plant, 2 new spirostane glycosides, named polygokingiaside A (1) and polygokingiaside B (2), and the 6 known spirostane glycosides (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (3), (25S)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (4), (25R)-26-O-(β-d-glucopyranosyl)-furost-5-en-3β,22α,26-triol 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (5), (23S,25R)-spirostan-5-en-3β,23-dihydroxy-12-one-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (6), (24S,25R)-spirostan-5-en-3β,24-dihydroxy-12-one-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (7), and (25R)-spirostan-5-en-3β-hydroxy-12-one-3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (8) were isolated. Their structures were determined by extensive analysis of HR-ESI-MS and NMR spectral data, as well as by comparison of the spectral data with those reported in the literature. Anti-inflammatory activity of compounds 1-8 was evaluated by their inhibition of NO production in LPS-stimulated RAW 264.7 cells. At a concentration of 20 µM, compounds 1-8 moderately inhibited NO production with inhibitory rates from 9.5 ± 0.8% to 33.8 ± 2.1%.

Supplemental Material

Figure S1 - Supplemental material for Polygokingiasides A and B: Two New Spirostane Glycosides From the Roots of Polygonatum kingianum With Nitric Oxide Inhibitory Activity

Supplemental material, Figure S1, for Polygokingiasides A and B: Two New Spirostane Glycosides From the Roots of Polygonatum kingianum With Nitric Oxide Inhibitory Activity by Tran Thi Thu Ha, Trinh Quang Huy, Pham The Chinh and Phan Van Kiem in Natural Product Communications