Two New Nitrogen-Containing Glycosides From Stixis scandens

Abstract

Phytochemical investigation of a methanol extract of Stixis scandens stems and leaves led to the isolation of 3 nitrogen-containing compounds (1-3) and 6 phenolics (4-9), which included 2 new glycosides, one a phenolic amide glucoside, scandemide (1), and the other an indole alkaloid glycoside, stixilenin (2). Their structures were elucidated by spectroscopic (1D- and 2D-NMR) and spectrometric (HR-ESI-MS) analyses, and acid hydrolysis. The isolated compounds were evaluated for their inhibitory effects on nitric oxide production in lipopolysaccharide-induced RAW264.7 cells.

Introduction

Stixis Lour. is a small genus of the family Capparaceae, consisting of 9 species and one subspecies endemic to Southeast Asia. ¹ Six Stixis species have been described in the Flora of Viet Nam, some of which are used as traditional medicines.^2,3 S. scandens Lour. is a medicinal plant, widely distributed in the northern regions of Viet Nam. The leaves, stems, and roots of this species are mainly used as folk medicine for the treatment of rheumatism, arthralgia, and eye diseases. There have been a few phytochemical investigations on Stixis species indicating the presence of alkaloids, L-stachydrine, and glucosinolates.^4,5 In previous studies, from S. suaveolens we isolated 2 new phenolic amides, stixilamides A and B, ⁶ together with 23 known compounds.^7‐9 To date, only one phytochemical report on S. scandens has been published, showing the presence of alkaloids. ⁴ This study describes the isolation and structural elucidation of 2 new nitrogen-containing glycosides (1 and 2), along with 7 known compounds (3-9), and their anti-inflammatory effect on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells.

Results and Discussion

By sequential Diaion HP-20 column chromatography (CC) (MeOH–H₂O), silica gel CC (CH₂Cl₂–MeOH), silica gel RP-18 CC (CH₂Cl₂–MeOH), and HPLC (J'sphere M-80 150 mm × 20 mm, eluting with ACN in H₂O, a flow rate of 3 mL/min), 2 new compounds named scandemide (1) and stixilenin (2) along with 7 known compounds, dihydroascorbigen-5′-O-β-glucopyranoside (3), ¹⁰ isoquercitrin (4), ¹¹ quercitrin (5), ¹² quercetin-3-O-β-glucopyranosyl-(1→2)-α-rhamnopyranoside (6), ¹³ kaempferol-3-O-β-glucopyranoside-7-O-α-rhamnopyranoside (7), ¹² kaempferol-3-O-β-glucopyranosyl-(1→2)-α-rhamnopyranoside (8), ¹³ and trans-syringin (9) ¹⁴ were isolated from the methanol extract of the stems and leaves of S. scandens.

Compound 1 was isolated as a white solid. Its molecular formula was determined as C₂₆H₃₃NO₁₁ on the basis of HR-ESI-MS molecular ion peaks at m/z 536.2124 [M + H]⁺, (C₂₆H₃₄NO₁₁⁺_, calcd. for 536.2132) and m/z 558.1945 [M + Na]⁺, (C₂₆H₃₃NNaO₁₁⁺_, calcd. for 558.1951). The ¹H NMR spectrum of 1 revealed the presence of 4 aromatic protons, an A₂X₂ system, for a 1,4-disubstituted benzene ring [δ_H 7.15 (2H, dd, J = 8.4, 1.8 Hz, H-2, H-6); 6.73 (2H, dd, J = 8.4, 1.8 Hz, H-3, H-5)], a two-proton singlet for a tetrasubstituted benzene ring [δ_H 6.93 (2H, s, H-2′, H-6′)], 2 olefinic protons [δ_H 6.69 (1H, d, J = 12.6, H-7′), 5.95 (1H, d, J = 12.6 Hz, H-8′)], and 2 methoxy groups attached to the aromatic systems at δ_H 3.86 (6H, s). The signals of 2 aromatic protons at δ_H 6.93 and 2 methoxy groups at δ_H 3.86 indicated the presence of a symmetrically substituted benzene ring. In addition, a monosaccharide unit with an anomeric proton signal at δ_H 4.94 (1H, d, J = 7.8 Hz, H-1′′), 2 methine protons δ_H 4.60 (1H, d, J = 5.4 Hz, H-7), 4.16 (1H, qd, J = 7.0, 5.4 Hz, H-8) and one methyl group at δ_H 1.05 (3H, d, J = 7.2 Hz, H-9) were also observed. The ¹³C NMR spectrum of 1 showed 26 carbon signals, including one amide group (δ_C 169.1), 2 olefin carbons (δ_C 137.8, 124.6), 12 aromatic carbons, 1 methyl carbon, 2 methine carbons, 2 methoxy group, and 6 carbons of a monosaccharide. The coupling constant (J = 7.8 Hz) of the anomeric proton H-1′′and the carbon signals of the sugar moiety [δ_C 105.1 (CH), 78.4 (CH), 77.8 (CH), 75.7 (CH), 71.3 (CH), and 62.5 (CH₂)] suggested that the monosaccharide was β-glucopyranose. The presence of a D-glucose moiety in 1 was determined by acid hydrolysis and GC analysis. The HMBC correlations between the protons of 2 methoxy groups (δ_H 3.86) and C-3′/C-5′ (δ_C 153.9), between H-7′ (δ_H 6.69), H-8′ (δ_H 5.95) and C-2′/C-6′ (δ_C 108.7), between H-2′/H-6′ (δ_H 6.93) and C-4′ (δ_C 136.6); and between H-1′′ (δ_H 4.94) and C-4′ (δ_C 136.6) suggested that the 2 methoxy groups and the β-D-glucopyranosyl unit were located at C-3′, C-5′, and C-4′, respectively. In addition, the structure of 1 was also supported by other HMBC correlations. The HMBC correlations from H-8 (δ_H 4.16) to C-9 (δ_C 15.0) and C-9′ (δ_C 169.1); from H-7 (δ_H 4.60) to C-1 (δ_C 134.1), C-2/C-6 (δ_C 128.6) and C-8 (δ_C 52.1); from H-2/H-6 (δ_H 7.15), H-3/H-5 (δ_H 6.73) to C-4 (δ_C 157.8) showed that the methyl and 2 hydroxyl groups were linked at C-8, C-4, and C-7, respectively. The relative configuration of C-7/C-8 and geometry of the olefinic group C-7′/C-8′ were determined to be erythro and Z-forms, respectively, based on the coupling constants of ³J_H-7/H-8 = 5.4 Hz and ³J_{H-7′/H-8′} = 12.6 Hz. ^15,16 Based on these results, compound 1 was determined as a new phenolic glycoside and named scandemide (1) (Figures 1 and 2).

Stixilenin (2) was isolated as a white solid. Its negative HR-ESI-MS gave a molecular ion peak at 484.1457 [M-H]⁻, corresponding to the molecular formula C₂₁H₂₇NO₁₂ (calcd. for C₂₁H₂₆NO₁₂⁻, 484.1455). The ¹H and ¹³C NMR spectra of 2 showed the characteristic signals of an indol backbone, 1 methyl ester group, and 2 saccharide units. The ¹³C NMR and HSQC spectra of 2 showed 21 carbon signals, in which 8 carbon signals of an indole skeleton with 2 substitutions [4 × CH (δ_C 132.9, 122.4, 113.9, 100.4), 4 × Cq (δ_C 156.0, 138.6, 122.8, 108.3], 2 carbon signals of one methyl ester group (δ_C 167.8, 51.4) and 11 carbon signals of a disaccharide unit were observed. The ¹H NMR spectrum of 2 showed proton signals due to an ABX system at δ_H 7.94 (1H, d, J = 9.0 Hz, H-4), 7.20 (1H, d, J = 2.4 Hz, H-7), and 7.02 (1H, dd, J = 9.0, 2.4 Hz, H-5); and a one-proton singlet at δ_H 7.90 (1H, s, H-2), which were assignable to a disubstituted indol backbone. The signals of 2 anomeric protons at δ_H 5.52 (1H, d, J = 1.2 Hz, H-1′′) and 5.00 (1H, d, J = 7.8 Hz, H-1′), in addition to 11 carbon signals of 2 sugar units [δ_C 102.0, 78.8, 78.7, 78.1, 71.5 (5 × CH) and 62.6 (CH₂); 110.8, 78.1 (2 × CH), 80.8 (Cq), and 75.5, 66.1 (2 × CH₂)] indicated the presence of one β-glucopyranosyl and one β-apiofuranosyl moiety in 2. ¹⁷ The existence of D-glucose and D-apiofuranose units were confirmed based on hydrolysis experiments and GC/MS analysis. The NMR data of 2 were similar to those of 6-[(β-D-apiofuranosyl-(1→2)-β-D-glucopyranosyl)oxy]-1H-indol-3-yl carboxylic acid methyl ester, except for the additional appearance of the β-D-apiofuranosyl moiety in 2. This was confirmed by the HMBC correlations of H-2 (δ_H 7.90) to C-3 (δ_C 108.3), C-9 (δ_C 138.6), and C-10 (δ_C 167.8); from H-4 (δ_H 7.94) to C-3 (δ_C 108.3); from the methoxy proton at δ_H 3.89 to C-10 (δ_C 167.8); from H-1′ (δ_H 5.00), H-4 (δ_H 7.94), H-5 (δ_H 7.02), and H-7 (δ_H 7.20) to C-6 (δ_C 156.0). The β-apiofuranosyl moiety was linked to a β-D-glucopyranosyl unit at C-2′ based on HMBC cross-peak of H-1′′ (δ_H 5.52) to C-2′ (δ_C 78.8). ¹⁸ Thus, the structure of 2 was confirmed to be 6-[(β-D-apiofuranosyl-(1→2)-β-D-glucopyranosyl)oxy]-1H-indol-3-yl carboxylic acid methyl ester (Figures 1 and 2).

Figure 1.

Structures of compounds 1 to 9.

Figure 2.

Key HMBC and COSY correlations of compounds 1 and 2.

The biological activities of S. suaveolens, as well as of other Stixis species, are not well known, but anti-inflammation appears to be the most common biological activity found for phenolic glycosides. ¹⁹ Therefore, we screened and evaluated the inhibitory activity of NO production of the 9 phenolic glycosides (1-9). All isolates were examined for their inhibitory effects on NO production in LPS-stimulated RAW264.7 cells. N-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthase was used as a positive control (IC₅₀ = 32.7 ± 2.3 µM). The compounds displayed either no or weak inhibition against NO production (Table 1). The survival rates measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay indicated that these compounds had no significant cytotoxicity to RAW264.7 cells at the experimental concentrations.

Table 1.

Inhibition of NO Production in Macrophage RAW264.7 Cells of Compounds 1 to 9.

Compound	IC50 (μM)	Compound	IC50 (μM)
1	>100	6	147.3 ± 6.6
2	197.7 ± 3.9	7	>100
3	>100	8	>100
4	>100	9	>100
5	>100	L-NMMA a	32.7 ± 2.3

Abbreviation: L-NMMA, N-monomethyl-L-arginine; NO, nitric oxide.

^a

Positive control.

Conclusion

Two new glycosides (1 and 2) along with 7 known compounds (3-9) were isolated from the stems and leaves of Stixis scandens, which showed either no or weak inhibitory activity against NO production in LPS-induced RAW264.7 cells.

Materials and Methods

General Experimental Procedures

HR-ESI-MS were run on a Sciex X500R QTOF mass spectrometer (Framingham, MA, USA). The ¹H NMR (600 MHz) and ¹³C NMR (125 MHz) spectra were recorded on a Bruker Avance III 600 spectrometer with tetramethylsilane (TMS) as the internal standard and values were given in ppm (δ). Optical rotation was measured on a Jasco P-2000 polarimeter, and IR spectra on a PerkinElmer Spectrum Two FT-IR Spectrometer by dispersion of the sample in KBr. Gas chromatographic (GC) spectra were recorded on a Shimadzu-2010 spectrometer. Column chromatography was carried out on silica gel 60 (0.040-0.063 mm, Merck), Diaion HP-20 resins, RP-18 (30-50 μm, Fuji Silysia Chemical), and Sephadex LH-20 (Amersham Pharmacia Biotech). HPLC for column chromatography J'sphere ODS-H80, 250 × 20.0 mm I.D. Thin layer chromatography was performed on silica gel 60F₂₅₄ (0.25 mm, Merck) and RP-18 F_254S plates (1.15685.0001, Merck). The compounds on the plates were observed under UV light (at 254 and 365 nm) and by spraying with a solution of vanillin/sulfuric acid and heating at 150 °C to 200 °C. All other chemicals and reagents were of analytical grade. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L-glutamine, 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES), penicillin, streptomycin, fungizone (PSF), LPS, MTT, and N^G-monomethyl-L-arginine (L-NMMA) were purchased from Sigma-Aldrich. The Griess reagents were obtained from Promega.

Plant Material

The stems and leaves of Stixis scandens were collected in Xuan Dai Commune, Xuan Son District, Phu Tho Province in August 2020, and identified by Dr Nguyen Thi Kim Thanh, VNU University of Science. A voucher specimen (HNU-024662) was deposited at the HNU Herbarium at VNU University of Science.

Extraction and Isolation

The dried powder of S. scandens stems and leaves (5 kg) was extracted 3 times with MeOH-H₂O (95:5, v/v) at room temperature. The extract was filtered and concentrated under reduced pressure in a rotary evaporator at 45 °C to give a methanol residue (400 g). This was suspended in water and successively partitioned with n-hexane, and CH₂Cl₂. The remaining aqueous layer was fractionated by Diaion HP-20 CC by eluting with a gradient of MeOH in H₂O (25:1→100:0, v/v) to afford 4 fractions (W1-W4). Fraction W2 (24.5 g) was chromatographed on a silica gel column eluted with a gradient system of CH₂Cl₂–MeOH (30:1→1:1, v/v) to give 5 sub-fractions (W2.1-W2.5). Fraction W2.4 was re-chromatographed on a silica gel RP-18 column (MeOH–H₂O = 1:2.5, v/v) to afford 4 sub-fractions (W2.4.1-W2.4.4). Sub-fraction W2.4.1 was subjected to a silica gel column (CH₂Cl₂–MeOH = 12:1, v/v), and then purified by HPLC (J'sphere M-80, YMC, Kyoto, Japan; 150 mm × 20 mm column, eluting with 14% ACN in H₂O, a flow rate of 3 mL/min) to give compound 9 (8.2 mg) at the retention time of 30 min. Fraction W2.5 was re-chromatographed on a silica gel RP-18 column (MeOH–H₂O = 1:2, v/v) to yield 7 sub-fractions (W2.5.1-W2.5.7). Sub-fraction W2.5.2 was fractionated on a Sephadex LH-20 column (MeOH–H₂O = 1:1, v/v) and then subjected to HPLC (J'sphere M-80, 150 mm × 20 mm column, eluting with 14% ACN in H₂O, and a flow rate of 3 mL/min) to give compound 1 (5.0 mg) at the retention time of 52.1 min. Sub-fraction W2.5.6 was purified on a Sephadex LH-20 column (MeOH–H₂O = 1:1, v/v) and then applied to HPLC (J'sphere M-80 150 mm × 20 mm column, eluting with 20% ACN in H₂O, at a flow rate of 3 mL/min) to give compounds 2 (7.4 mg), 3 (11.3 mg), and 7 (6.4 mg) at retention times of 29.1, 34, and 43 min, respectively. Sub-fraction W2.5.7 was subjected to Sephadex LH-20 CC (MeOH–H₂O = 1:1, v/v) to afford compound 6 (20.0 mg). Fraction W3 (8 g) was chromatographed on a silica gel RP-18 column (MeOH–H₂O = 1:1.2, v/v) to yield 4 fractions (W3.1-W3.4). Fraction W3.2 was purified on a Sephadex LH-20 column (MeOH–H₂O = 1:1, v/v), then applied to HPLC (J'sphere M-80, 150 mm × 20 mm column, a flow rate of 3 mL/min, eluting with 25% and 27% ACN in H₂O) to give compounds 4 (4.0 mg, retention time of 28.7 min) and 5 (4.0 mg, retention time of 31.7 min). Purification of fraction W3.3 on a Sephadex LH-20 column (MeOH–H₂O = 1:1, v/v) resulted in the isolation of compound 8 (20.0 mg).

Scandemide ( 1 ): white solid, [α]_D²⁵: −5.2° (c 0.0021, MeOH); UV (MeOH) λ_max 228, 298 nm. IR (KBr) ν_max: 3416.4, 2929.6, 2850.9, 1734.6, 1652.9, 1511.3, 1456.9, 1245.7, 1122.2, and 1070.8. HR-ESI-MS m/z 536.2124 [M + H]⁺, (C₂₆H₃₄NO₁₁⁺_, calcd. for 536.2132); m/z 558.1945 [M + Na]⁺, (C₂₆H₃₃NNaO₁₁⁺_, calcd for 558.1951). ¹H NMR (600 MHz, CD₃OD) and ¹³C-NMR (150 MHz, CD₃OD): see Supplemental Table S1.

Stixilenin ( 2 ): white solid. UV (MeOH) λ_max 218, 224, 276 nm. IR (KBr) ν_max: 3427.8, 2944.7, 2923.2, 1667.7, 1631.4, 1534.1, 1453.0, 1277.8, 1165.6, 1075.1, and 1048.9. HR-ESI-MS m/z 484.1457 [M-H]⁻, (C₂₁H₂₆NO₁₂⁻_, calcd. for 484.1455). ¹H NMR (600 MHz, CD₃OD) and ¹³C NMR (150 MHz, CD₃OD): see Supplemental Table S2.

Acid Hydrolysis

Each compound (1 and 2, 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 was thoroughly driven out under N₂ gas overnight. After extraction with CHCl₃, 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 chromatography: column of SPB-1 (0.25 mm × 30 mm); flame ionization detector, column temperature 210 °C, injector temperature 270 °C, detector temperature 300 °C, and carrier gas He (2.0 mL/min). The retention times of persilylated glucose and rhamnose were found to be 14.11 and 4.50 min, respectively, when compared with the standard solutions prepared by the same reaction from the standard monosaccharides. The retention times of persilylated D-glucose and D-apiose were 14.11 and 6.70 min, respectively.

Cell Culture

RAW264.7 cells were obtained from the American Type Culture Collection (ATCC). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, and 1% PSF. The cells were cultured in a humidified incubator with 5% CO₂ at 37 °C and subcultured every 3 days.

MTT Assay

Cytotoxicity of compounds was determined by the MTT method. Briefly, the cells were seeded in a 96-wells plate at a density of 5 × 10⁴ cells/well. After incubating overnight in the incubator, the medium was discarded and changed by FBS free medium. Then, the cells were treated with the compound at different concentrations for 2 h before being stimulated with 1 µg/mL LPS for 24 h. To determine the proliferation of the cells, MTT was added to the medium for 4 h. The medium was removed and the crystals were dissolved by adding 100 µL to the well. The optical density was measured by an enzyme-linked immunosorbent assay (ELISA) reader (Biotek) at 540 nm. The percentage of living cells was calculated related to the control group.

NO Inhibition Assay

RAW264.7 cells were cultured in the presence of the compounds at different concentrations while L-NMMA was used as the positive control in the same concentration range. After stimulation with LPS (1 µg/mL) for 24 h, NO production was indicated by the quantity of nitrite in the medium using the Griess assay. The cell medium and Griess reagent system were mixed together at a 1:1 ratio in a 96-well plate for 10 min. The optical density was measured at 540 nm using a microplate reader. A fresh culture medium was used as the blank in all experiments. The nitrite concentration in the medium was calculated from a sodium nitrite standard curve. The NO inhibition activities were calculated using the formula: % inhibition = 100% − [amount NO_sample/amount NO_LPS]*100.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221148621 - Supplemental material for Two New Nitrogen-Containing Glycosides From Stixis scandens

Supplemental material, sj-docx-1-npx-10.1177_1934578X221148621 for Two New Nitrogen-Containing Glycosides From Stixis scandens by Tran Thi Yen, Nguyen Thanh Tam, Nguyen Thanh Thi Kim and Vo Ngoc Binh, Nguyen Thao Kim Nu, Ngo Quoc Anh in Natural Product Communications