Phenolic constituents from twigs of Aleurites fordii and their biological activities

Three new neolignan glycosides (1–3), a new phenolic glycoside (15), and a new cyanoglycoside (16) were isolated and characterized from the twigs of Aleurites fordii together with 14 known analogues (4–14 and 17–19). The structural elucidation of the new compounds was performed through the analysis of their NMR, HRMS, and ECD spectra and by chemical methods. All isolated compounds were tested for their antineuroinflammatory and neuroprotective activities.

As an ongoing search for bioactive secondary metabolites from Korean medicinal sources, we investigated the methanolic extract of the twigs of A. fordii which resulted in the isolation and characterization of 14 lignan derivatives including three new neolignan glycosides (1)(2)(3), four phenolic glycosides including a new compound (15), and a new cyanoglycoside (16) from the organic extracts. The structures of the new compounds were established by NMR analysis ( 1 H and 13 C NMR, COSY, HSQC, HMBC, and NOESY), HRMS, and chemical methods. The isolated compounds 1-19 were evaluated for their antineuroinflammatory and neuroprotective activities. In this paper, we report the isolation and structural elucidation of these phytochemicals and their biological activity.
Compound 1 was obtained as a colorless gum. The molecular formula was determined to be C 25 H 32 O 11 from the [M + Na] + molecular ion peak in the positive mode HRFABMS. The  (Table 1)  that it is a typical dihydrobenzofuran neolignan glycoside [9][10][11]. The data for compound 1 were similar to those of glochidioboside isolated from Glochidion obovatum [12], except for the presence of a hydroxy group instead of the methoxy group at C-3′ in 1. The two-dimensional structure of 1 was elucidated via analysis of COSY, HSQC, and HMBC spectroscopic data ( Figure 2). The locations of the glucose unit and the methoxy group were confirmed from the observed HMBC correlations of H-1′′/C-9′ and 3-OCH 3 /C-3, respectively ( Figure 2). Acid hydrolysis of 1 was conducted to analyze the aglycone and sugar moiety. The structure of the aglycone (1a) was confirmed as demethyldihydrodehydrodiconiferyl alcohol based on the comparison of 1 H NMR and MS data [13]. The relatively large coupling constant of the anomeric proton (7.8 Hz) confirmed that the glucose is combined as β-form [14]. ᴅ-Glucose was identified by co-TLC with a standard sample [CHCl 3 /MeOH/H 2 O 2:1:0.1, R f = 0.
The molecular formula of compound 2, isolated as a colorless gum, was confirmed to be C 26 H 34 O 10 from the positive ion mode HRESIMS data. The 1 H and 13 C NMR spectra of 2 were very close to that of icariside E 4 (5) [17] with significant differences in the chemical shifts of C-1, C-8, C-9, C-5′, and C-6′ [2: δ C 134.3, 50.2, 63.6, 131.9, and 119.2; 5: δ C 138.9, 55.7, 65.1, 129.7, and 118.0, respectively], indicating that compound 2 could be a stereoisomer of 5 at C-7 and C-8. The inspection of the COSY, HSQC, and HMBC spectra confirmed the planar structure of 2. The HMBC correlation of H-1′′ to C-4 indicated that the rhamnose unit was linked to the oxygen at C-4 and the characteristic J value of the anomeric proton (1.5 Hz) confirmed the rhamnose as α-form ( Figure 2) [10]. Acid hydrolysis of compound 2 afforded the aglycone, dihydrodehydrodiconiferyl alcohol (2a) [18], and ʟ-rhamnose ([α] D 25 +9.0), which was identified in an identical manner to that of compound 1. The relatively large coupling constant (8.8 Hz) between H-7 and H-8 in 2, as opposed to the relatively small coupling constant (6.1 Hz) between H-7 and H-8 in 1, verified that H-7 and H-8 are cis-oriented [10,16], which was supported by the NOESY correlations of H-7/H-8, H-2/H-9, and H-6/H-9 ( Figure 2). The ECD spectrum of 2 showed negative CEs at 276 nm and 229 nm and a positive CE at 248 nm, indicating the absolute configuration of C-7 and C-8 as R ( Figure S15 in Supporting Information File 1) [19]. Therefore, the structure of compound 2 was determined to be (7R,8R)-dihydrodehydrodiconiferyl alcohol 4-O-α-ʟ-rhamnopyranoside and was named as aleuritiside B.
Compound 3 was obtained as a colorless gum after purification with a molecular formula of C 26   pos.  ]. The spectroscopic data resembled closely to those of icariside E 3 , isolated from Epimedium grandiflorum var. thunbergianum [20], indicating that compound 3 may have the identical planar structure to icariside E 3 , which was reported without assignment of the absolute configuration. The planar structure of 3 was further confirmed by analysis of 2D NMR data, including COSY, HSQC, and HMBC ( Figure 2). The determination of the stereochemistry for the sugar unit of 3 was conducted following the same method as for compound 2. The structure of the aglycone 3a obtained by acid hydrolysis of 3 was confirmed based on 1 H NMR and MS data [20]. The absolute configuration of 3a was established as 8S (a negative CE at 273 nm) based on the comparison of its ECD spectrum with the reported data [21]. Thus, the structure of compound 3 was determined as 8S-tetrahydrodehydrodiconiferyl alcohol 4-O-α-ʟ-rhamnopyranoside and was named aleuritiside C. . The location of the glucose unit was determined to be at C-4 by analysis of the HMBC data showing a correlation from H-1′ to C-4. The coupling constant (7.7 Hz) of the anomeric proton of glucose suggested that it was the β-form. Acid hydrolysis of 15 yielded 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-propanone (15a), whose 1 H NMR spectral data were in good agreement with the values reported previously [22], and ᴅ-glucopyranose was identified through co-TLC and the specific rotation value {[α] D 25 +86.0 (c 0.03, MeOH)}. Accordingly, the structure of compound 15 was identified as 2,6-dimethoxy-4-(1-oxo-3hydroxypropyl)phenyl β-ᴅ-glucopyranoside and named aleuriteoside A.
Compound 16 was isolated as a colorless gum and the molecular formula was determined to be C 24 [24]. The location of the glucose unit was determined to be at C-4 based on the analysis of the HMBC data showing a correlation from H-1′ to C-4 ( Figure 2). The HMBC cross-peaks of H-2′/C-7′′ and H-6′/C-7′′′ also indicated the presence of two galloyl groups at C-2′ and C-6′ of the glucose unit, respectively ( Figure 2). Alkaline hydrolysis of 16 yielded codiacyanogluco-side (16a) and gallic acid (16b). The identification of 16a and 16b was conducted by comparison of their 1 H NMR and MS data [24,25]. Consequently, the structure of 16 was determined to be codiacyano glucosyl-2′,6′-O-digallate, named aleucyanoglucoside.
The using deionized water to remove KOH. A portion of the reaction product was partitioned between EtOAc/H 2 O (each 1.0 mL) and the aglycone 16a was acquired from the EtOAcsoluble phase.
Measurement of nitric oxide production and cell viability. In a manner similar as described in [37], BV2 cells were used to test the inhibitory effect of the isolated compounds on LPSstimulated NO production [38,39]. The BV2 cells seeded on a 96-well plate (4 × 10 4 cells/well) were treated with and without various concentrations of the test compounds. The treated cells were stimulated with LPS (100 ng/mL) and incubated for 24 h. The level of nitrite (NO 2 , a soluble oxidation product of NO) in the culture medium was measured using the Griess reagent (0.1% N-1-naphthylethylenediamine dihydrochloride and 1% sulfanilamide in 5% phosphoric acid). The supernatant (50 μL) in each well was harvested and mixed with an equal volume of Griess reagent. After 10 min, the absorbance was measured at 570 nm with a microplate reader (Emax, Molecular Devices, Sunnyvale, CA, USA). Graded sodium nitrite solution was used as a standard to gauge NO 2 concentration. Cell viability was assessed by the MTT assay.

Measurement of NGF secretion and cell viability assays.
C6 glioma cells (Korean Cell Line Bank, Seoul, Republic of Korea) were used to measure the release of nerve growth factor (NGF) into the culture medium. The C6 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 different concentrations of the test compounds for an additional 24 h. The NGF levels were evaluated in the medium supernatant using an ELISA development kit. Cell viability was measured using the MTT assay and the results were expressed as a percentage of the control group (untreated cells).
Cytotoxicity assessment. The SRB assay was performed to evaluate cytotoxicity of all the isolated compounds against four cultured human cancer cell lines. The cell lines (National Cancer Institute, Bethesda, MD, USA) were used A549 (nonsmall cell lung adenocarcinoma), SK-OV-3 (ovarian malignant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon adenocarcinoma). Each cell line was inoculated over standard 96-well flat-bottom microplates and incubated for 24 h at 37 °C in condition of a humidified atmosphere of 5% CO 2 . The attached cells were incubated with various concentrations of the test compounds and the cells were cultured for 48 h. Then, the culture medium was removed from each well and the cells were fixed with 10% cold trichloroacetic acid at 4 °C for 1 h. After the supernatant was discarded and the plates were washed with tap water, the cells were stained with 0.4% SRB solution and incubated for 30 min at room temperature. These cells were washed to remove the unbound dye and subsequently solubilized with 10 mM unbuffered Tris base solution (pH 10.5). The absorbance was measured spectrophotometrically at 520 nm with a microtiter plate reader. Etoposide (Sigma Chemical Co., ≥98%) was used as a positive control.

Supporting Information
Supporting Information File 1 Copies of NMR spectra including 1D and 2D NMR and HRMS data of compounds 1-3, 15, and 16 and ECD spectra of compounds 1-3.