Chemical constituents of Chaenomeles sinensis twigs and their biological activity

A new megastigmane-type norsesquiterpenoid glycoside, chaemeloside (1), was isolated from the twigs of Chaenomeles sinensis together with 11 known phytochemicals through chromatographic methods. The chemical structure of the new isolate 1 was determined by conventional 1D and 2D NMR data analysis, ECD experiment, hydrolysis followed by a modified Mosher’s method, and LC–MS analysis. The characterized compounds’ biological effects including cytotoxicity against cancer cell lines, antineuroinflammatory activity, and potential neurotrophic effect were evaluated.

As a part of the continuing studies to identify bioactive constituents from the Korean medicinal plants [13,[16][17][18][19][20], previous phytochemical investigations on the MeOH extract of the twigs of C. sinensis have led to the isolation and characteri- zation of triterpenoids [13], biphenyls [14], lignans [15], and oxylipins [21] with cytotoxic, anti-inflammatory, or potential neuroprotective activities. In order to search for minor constituents of other structure classes in C. sinensis twigs, the MeOH extract and the four solvent-partitioned fractions were further investigated to afford a new megastigmane-type norsesquiterpenoid glycoside 1 along with 11 known compounds (2-12, Figure 1). The structure of the new compound 1 was established on the basis of spectroscopic and spectrometric data analysis, and chiral derivatization coupled with NMR or LC-MS experiments. All isolated compounds 1-12 were evaluated for their cytotoxicity against four human tumor cell lines, antineuroinflammatory activity using lipopolysaccharide (LPS)stimulated murine microglia BV-2 cell lines, and potential neurotrophic effects in C6 cells.

Results and Discussion
From the n-BuOH-soluble fraction of the MeOH extract of C. sinensis, the new compound 1 was isolated guided by the characteristic TLC spot detected under UV light or by heating after spraying anisaldehyde-sulfuric acid. Chaemeloside (1) was obtained as a colorless gum and its molecular formula was determined as C 19  NMR data of the aglycone were similar to those of a megastigmane-type norsesquiterpenoid, (6R,7E,9R)-9-hydroxy-4,7- megastigmadien-3-one (13, Figure 1) [22], with the significant difference of the downfield-shifted NMR resonances at H-2 [δ C 4.24 for 1; δ C 2.30 and 2.09 for 13] and C-2 [δ C 87.8 for 1; δ C 48.4 for 13] suggesting the presence of a hydroxy group at C-2 in compound 1. This initial proposal was supported by the HMBC correlations of H-4 and H-11 with C-2 and H-2 with C-3 ( Figure 2A). The glucopyranosyl unit was confirmed to be located at C-2 through a glycosidic bond by the HMBC crosspeak between H-2 and C-1′ ( Figure 2A). The intensive analysis of the 1D and 2D NMR data of compound 1 including COSY, HSQC, and HMBC analyses led to the elucidation of the planar structure of 1 (Figure 2A and Supporting Information File 1).
The strong NOESY cross-peaks of H-2 with H-6 and H-11 and H-6 with H-11 indicated that these three protons are co-facial ( Figure 2B and Supporting Information File 1). The absolute configuration of C-6 was assigned as S by a well-established ECD empirical rule [22]. In brief, a systemic analysis of the ECD data of 13 with its diastereomers and simple derivatives showed that a positive or negative Cotton effect around 240-245 nm is indicative of the 6R or 6S configuration, respectively. From a positive Cotton effect at 245 nm of compound 1 ( Figure 2C, left), the absolute configuration at C-6 was deduced as S (the stereochemical descriptor was flipped from R to S due to an O-glycosylation at C-2, see Figure 2C, right). The 9R con-figuration of 1 was determined by the modified Mosher's method [23][24][25]. The hydrolysis product of 1 (1a) was esterified with the Mosher reagents and the analysis of the Δδ S-R values of all protons in 1a indicated a 2S and 9R configuration ( Figure 2D). Finally, the absolute configuration of the glucopyranose was assigned as ᴅ by comparing the retention time of its chiral derivative with those of authentic samples [16,26]. Therefore, the structure of compound 1 was elucidated as (2S,6S,7E,9R)-2,9-dihydroxy-4,7-megastigmadiene-3-one-2-Oβ-ᴅ-glucopyranoside.
In the course of continuing search for cytotoxic, antineuroinflammatory, and neurotrophic secondary metabolites from C. sinensis [13][14][15]21], the isolates (1-12) were tested for these biological activities. The cytotoxicity was evaluated on the basis of the growth inhibitory effects of the isolated compounds 1-12 against four human tumor cell lines A549, SK-OV-3, SK-MEL-2, and BT549 using a sulforhodamine B (SRB) assay.
The potential antineuroinflammatory activity of the compounds 1-12 was tested by measuring the nitric oxide (NO) production levels in the LPS-stimulated murine microglia BV-2 cell line. Compound 11 (betulinal) showed a strong inhibitory effect on the NO production with an IC 50 value of 5.7 μM without cyto-  (9) and its derivatives showed antiviral and antitumor effects [41]. Interestingly, betulinal (11) has an advantage in view of toxicity as compared to betulinic acid (10). A previous report suggested that apigenin (8) can lower the amyloid beta-induced microglial activation to inflammatory phenotype and this potential could help to lower the amyloid beta-induced neuroinflammation in CNS [42]. Additionally, an apigenin-mediated improvement in memory dysfunction in an animal model further elaborated the neuroprotective potential of apigenin itself [43]. Altogether, apigenin (8) could be a potential candidate for the treatment strategies against neurodegenerative disorders. However, the potency of compound 10 could be attributed to the low cell viability (54.9 ± 2.8%). The other phytochemicals displayed mild or no NO inhibitory effects (IC 50 > 50 μM). Also, the potential neurotrophic effect of the isolated compounds 1-12 was evaluated by assessing their induction levels of nerve growth factor (NGF) secretion in C6 cells (Table 4). Among the tested compounds, only compound 8 (apigenin) exhibited a weak activity on the NGF release with a stimulation level of 127.8 ± 2.0% without displaying cell toxicity (100.9 ± 0.6%).

Conclusion
A new megastigmane-type norsesquiterpenoid glycoside 1 was isolated along with 11 known compounds from the MeOH extract of C. sinensis twigs and their structures were character-
Assessment of the NO generation and cell viability. Analogous as described in [45]. The BV-2 cells, developed by Dr. V. Bocchini at the University of Perugia (Perugia, Italy), were used for this study [46,47]. The cells were seeded in a 96-well plate (4 × 10 4 cells/well) and incubated in the presence or absence of various doses of the tested compounds. Lipopolysaccharide (LPS, 100 ng/mL) was added to all wells containing the pretreated cells except the one for control and grown for 1 d.
The produced levels of nitrite (NO 2 ), a soluble oxidized product of NO, was evaluated with 0.1% N-1-naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid, aka the Griess reagent. The supernatant (50 μL) was mixed with the Gries reagent (50 μL) and after 10 min the absorbance was gauged at 570 nm. For a positive control, the reported nitric oxide synthase (NOS) inhibitor ʟ-NMMA was employed. Graded sodium nitrite solutions were utilized to determine the nitrite concentrations. An MTT assay was used for the cell viability assay.
Nerve growth factor and cell viability assays. Analogous as described in [13]. The C6 glioma cells (Korean Cell Line Bank, Seoul, Republic of Korea) were used to assess the release of NGF into the culture medium. The test cells were seeded onto 24-well plates at a density of 1 × 10 5 cells/well. After 24 h, the cells were treated with serum-free DMEM and incubated with the designated concentrations of the compounds for an additional 24 h. The medium supernatant was collected from the culture plates and the NGF levels were evaluated using an ELISA development kit from R & D system (Minneapolis, MN, USA). The cell viability was also assessed with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay in which the results were expressed as a percentage of the control group (untreated cells).

Supporting Information
Supporting Information File 1 1D and 2D NMR, HRMS, and ECD spectra of compound 1, 1 H and 13 C NMR spectra of 1a, and 1 H NMR spectra of 1s and 1r.