New Glycosides from the Fruits of Nicandra physaloides

Three new glycosides (1–3) and 15 known ones (4–18) were isolated and identified from the fruits of Nicandra physaloides. The structures of these compounds were established by 1D and 2D NMR spectra and HR-ESI-MS. The compounds (4–18) were the first time isolated from the Nicandra genus and they (except 8, 10, 14) exhibited inhibitions on the NO release of LPS-induced RAW 264.7 cells with IC50 values from 26.9 to 47.5 μM.


Structure Elucidation
Compound 1 possessed the molecular formula of C19H28O10 according to the HR-ESI-MS at m/z 417.1755 [M + H] + . The 1 H-NMR spectrum (Table 1) Figure 2) between H-1′ and C-8, H-1′′ and C-6′ suggested the attachment position of the galactosyl at C-8 and C-1′′ of arabinose at C-6′ of galactosyl [24]. Assignments of all groups of 1 were achieved by 1 H-1 H COSY, HSQC and HMBC ( Figure 2). The absolute configuration of the glycosyls group of compound 1 was determined by GC analysis according to the same tR at 12.9 and 32.5 min with standard L-arabinopyranose and D-galactopyranoside, respectively. From the above data, the structure of 1 was elucidated as 2-phenylethyl O-α-L-arabinopyranosyl-(1→6)-β-Dgalactopyranoside, named Nicglycoside A.    The HMBC correlations ( Figure 2) between H-1 and C-8, H-1 and C-6 suggested the attachment position of the galactosyl at C-8 and C-1 of arabinose at C-6 of galactosyl [24]. Assignments of all groups of 1 were achieved by 1 H-1 H COSY, HSQC and HMBC ( Figure 2). The absolute configuration of the glycosyls group of compound 1 was determined by GC analysis according to the same t R at 12.9 and 32.5 min with standard L-arabinopyranose and D-galactopyranoside, respectively. From the above data, the structure of 1 was elucidated as 2-phenylethyl O-α-L-arabinopyranosyl-(1→6)-β-Dgalactopyranoside, named Nicglycoside A. the phenyl-glycosides and fat glycosides from the fruits of Nicandra physaloides have been studied and reported. Here, the isolation and structural elucidation of compounds 1-18, as well as their anti-inflammatory activities, were elaborated and provided.    [24]. Assignments of all groups of 1 were achieved by 1 H-1 H COSY, HSQC and HMBC ( Figure 2). The absolute configuration of the glycosyls group of compound 1 was determined by GC analysis according to the same tR at 12.9 and 32.5 min with standard L-arabinopyranose and D-galactopyranoside, respectively. From the above data, the structure of 1 was elucidated as 2-phenylethyl O-α-L-arabinopyranosyl-(1→6)-β-Dgalactopyranoside, named Nicglycoside A.     Figure 2) between H-1 and C-6, H-1 and C-6 suggested the attachment position of the galactosyl was at C-6 and C-6 of galactosyl was substituted connecting with the C-1 of arabinose. Its absolute configurations of the glycosyls group were determined by GC analysis as with the compound 1. From the above data and combined with the literatures [24,25], the structure of 2 was elucidated as (Z)-hex-3-en-1-ol-α-L-arabinopyranosyl-(1→6)-β-D-galactopyranoside, named Nicglycoside B.   (Figure 2). The absolute configuration of the glycosyls group of compound 3 was determined by GC analysis according to the same t R at 16.8 min and 12.9 min with standard D-glucose and L-arabinose, respectively. From the above data and combined with the literature [26,27], the structure of 3 was elucidated as (Z)-3-hexenyl O-β-D-glucopyranosyl-(1→2)-O-α-L-arabinopyranosyl-(1→6)-O-β-D-glucopyranoside, named Nicglycoside C.

General Experimental Procedures
UV spectra were recorded on a Shimadzu UV-1601 instrument. HR-ESI-MS was performed on a Waters Xevo-TOF-MSTM.1D and 2D NMR spectra using a Bruker DPX 400 instrument with TMS as an internal standard. Preparative HPLC (Waters, Milford MA, USA, 515-2414) was performed on Sunfire (10 µm, 19 × 250 mm, Waters). ODS was obtained from YMC Company Ltd., Japan. Silica gel was used Qingdao Marine Chemical Ltd., Qingdao, China. All the solvents were of analytical grade and were purchased from Tianjinfuyu Company Ltd., Tianjin, China. ELISA reader was used from PerkinElmer

Plant Material
The fruits of Nicandra physaloides (L.) Gaertn were harvested from Harbin, Heilongjiang Province of China, in September 2014, which was identified by Prof. Ruifeng Fan of Heilongjiang University of Chinese Medicine. The voucher specimen (20140911) had been deposited at Heilongjiang University of Chinese Medicine.

Acid Hydrolysis and GC Analysis
The isolated glycosides (1-18) (2.0 mg) were refluxed with 2 mL H 2 O and 2 N aqueous 1 mL HCl for water bath (3 h). Then, the reaction mixtures were extracted with ethyl acetate for 3 times (5 mL). The aqueous layer was neutralized and evaporated with MeOH and then dissolved in anhydrous pyridine (5 mL) and treated with L-cysteine methyl ester hydrochloride (1.5 mg). After being stirred for 1 h at 60 • C, the mixture was added into 150 µL of HMDS-TMCS (3:1) and then stirred for another 30 min at 60 • C. The supernatant was concentrated under N 2 stream after being centrifuged off. The residue was partitioned between n-hexane and H 2 O (0.1 mL each), and the hexane layer (1 µL) was analyzed by GC [30,31], respectively. The configurations of D-glucose for compounds 3-18 were determined by the same t R of standard D-glucose (t R = 16.8 min), L-arabinose for compounds 1-5, 16, 18 (t R = 12.9 min), D-galactose for compound 1-2 (t R = 32.5 min), and L-rhamnose for compound 7, 10 (t R = 14.8 min).

Anti-Inflammatory Assays
RAW 264.7 cells were cultivated at densities of 5 × 10 5 cell/wells in 96-well for 24 h, then discarded the supernatants and stimulated by LPS (100 µL, 1 µg/mL) to generate NO for cultivating 24 h. Following incubation of the demonstrated time, the amount of sable nitrite, the end product of NO generation by activated cells, were determined by a modification of the Griess reaction [32,33]. The cells were treated with 100 µL of the compounds by the final concentration of 5, 25, 50, 100 and 200 µg/mL. Briefly, 100 µL of culture supernatants from control or stimulated macrophages were transferred to 96-well plates. Supernatants were mixed with 50 µL of 1% sulfanilic acid anhydrous in 85% phosphoric acid, incubated for 10 min at room temperature, shielded from light, followed by 50 µL of 1 mg/mL N-(1-naphthyl) ethylenediamine dihydrochloride for 10 min incubation in light proof. The absorbance was measured at 540 nm using an ELISA reader, and nitrite concentration was determined by comparison with a sodium nitrite standard curve. NMMA was used as a positive control. No isolates did showed an effect on the assay systems with the final concentration 0.2 (v/v) in DMSO and the MTT assay revealed no significant cytotoxic effects (over 90% cells survival) on cells treated with above compounds at concentrations up to 200 µg/mL.

Conclusions
As described in the introduction, Nicandra physaloides possessed many kinds of bioactivities such as heat-clearing, detoxifying, antipyresis, and anti-inflammation, and was applied to the treatments of rheumatoid arthritis, and so on. This study obtained the 18 glycosides compounds from the Nicandra physaloides fruits, including phenyl-glycosides and fat glycosides. Meanwhile, many researchers have reported that the aromatic glycosides and phenyl-glycosides show significant anti-inflammatory activities [34][35][36][37]. Thus, the anti-inflammatory activities of the compounds were evaluated, some of which showed significant activities. These results indicated that these glycosides compounds could be the pharmacodynamic material basis for anti-inflammation from the Nicandra physaloides fruits, and played important roles in the treatments of inflammatory diseases such as rheumatoid arthritis, nasosinusitis, influenza, urinary tract infection, sore and furuncle. The systematic studies on the composition in this manuscript will be the foundations and references of further research. We have made contributions to discovering active ingredients and leading compounds and provided experimental and scientific basis of drug design and drug discovery of the Nicandra physaloides.
Supplementary Materials: The following are available online: Figures S1-S6 and Table S1: The 13 C-NMR data of Compounds 4-18.