Phenylethanol glycosides from the seeds of Aesculus chinensis var. chekiangensis

Three new phenylethanol glycosides (1-3) and one known analogue (4) were isolated from the seeds of Aesculus chinensis Bge. var. chekiangensis. To the best of our knowledge, this represents the first isolation of phenylethanol glycosides from the genus of Aesculus, which enriched its chemical composition. Structure elucidations were performed via extensive NMR and HRESIMS data together with comparison with literature data. Thereafter, the isolated compounds were assayed for their neuroprotective activities against CoCl2-induced cytotoxicity in PC12 cells and compound 3 exhibited moderate activity.


Introduction
The genus Aesculus, which belongs to the family Hippocastanaceae contains about 30 species found worldwide. The dried seeds of Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang, Aesculus chinensis Bge and Aesculus wilsonii Rehd are commonly used to treat chest and abdomen pain, dysentery and ague [1,2] in traditional Chinese medicine. Previous studies on the genus of Aesculus revealed the presences of diverse secondary metabolites such as triterpenoids [3][4][5][6][7], flavonoids [8,9], coumarins [10] and steroids [11]. And a number of pharmacological studies have suggested that A. chinensis exhibited beneficial effects on antitumor [12], neuroprotective [13], anti-inflammatory [14] and cardio-protective activities [15]. Nevertheless, compared to other species of Aesculus genus, the chemical investigation of Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang is limited. Our interests in cytotoxic and neuroprotective components from A. chinensis Bge. var. chekiangensis (Hu et Fang) Fang have led to the isolation of numerous new ones [16,17]. As a continuous search for structurally novel compounds with diverse bioactivities, three new phenylethanol glycosides (1-3) and one known analog (4) were obtained (Fig. 1), which represent the first examples of phenylethanol glycosides obtained from the genus of Aesculus. Herein, the isolation, structure identification and biological evaluation of 1-4 are described.
partitioned via D101 resin column eluting with a stepwise gradient of H 2 O-EtOH.

Hydrolysis and determination of absolute configuration of sugars
Compounds 1-3 (1.0 mg, respectively) was hydrolyzed with 2 M HCl (4.0 mL) at 90 °C for 2 h. Then the hydrolysed materials were disposed and tested by means of the procedure described in our previous work [16,17].

Neuroprotective effect assay
Compounds 1-4 were assayed for their neuroprotective effects against CoCl 2 -induced PC12 cell injury [18] by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) method with trolox as the positive control according to our previously reported procedure [16,17]. PC12 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum as well as 100 U/mL penicillin/ streptomycin and were incubated at 37 °C with 5% CO 2 . PC12 cells were placed into a 96-well plate at a density of 2 × 10 4 cells/well and kept there for 24 h. Cells were incubated with test compounds and trolox (10 μM) for 2 h. To induce an oxidative stress, 1 mM CoCl 2 was added to the cells and incubated for 24 h. Then, the supernatant was changed with 100 μL MTT solution (5 mg/mL) for 2.5 h, the plate was vibrated, and the absorbance at 490 nm was measured using a microplate reader.

Cytotoxicity assay
Cell viability was determined with the MTT method [19,20]. The human hepatocellular carcinomas cells (HepG2), the human colorectal carcinoma cells (HCT-116) and the human gastric carcinoma cells (MGC-803) were purchased from ATCC. HepG2, MGC-803 and HCT-116 were respectively cultured in DMEM and RPMI-1640 mediums, which were supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO 2 . HepG2, HCT-116, and MGC-803 cells (1 × 10 4 ) were seeded in 96-well tissue culture plates. Cells were treated in triplicate with five concentrations (50, 25, 12.5, 6.25 and 3.125 μM) of the tested compounds Subsequently, 100 μL of MTT (5 mg/mL) was added and the cells were incubated for additional 2.5 h. Thereafter, the supernatant was discarded and 0.15 ml of DMSO was added to each well, then the plate was mixed on a microshaker for 10 min and read on a microplate reader at 490 nm.
The two anomeric protons at δ 4.17 (1H, d, J = 7.7 Hz), 4.59 (1H, d, J = 1.2 Hz) correlated with carbons at δ 103.0 and 100.8 in heteronuclear single quantum coherence (HSQC) spectrum, respectively, indicated a disaccharide residue. Acid hydrolysis of 1 liberated d-glucose and l-rhamnose, which were identified by HPLC analysis after derivatization [21,22]. The β-orientation of the glucopyranosyl unit was deduced from the coupling Table 1  constant (J = 7.7 Hz, H-1′). The α-anomeric configuration of rhamnose was determined from the absence of nuclear overhauser effect spectroscopy (NOESY) correlations between protons H-1 and H-3/H-5. The β-dglucose was attached to the 4-methoxy-phenylethanol nucleus at C-8, evidenced by the HMBC correlation between H-1′ (δ H 4.17) to C-8 (δ C 69.9). In addition, the downfield chemical shift of C-6′ (δ C 67.0) of the glucose coupled with the cross peak of H-1′′ (δ H 4.59) to C-6′ (δ C 67.0) in HMBC spectrum suggesting the α-l-rhamnose was linked to C-6′. Based on these data, compound 1 was concluded to be 4-methoxy-phenylethanol-8-O-α-lrhamnopyranosyl-(1 → 6)-β-d-glucopyranoside. The elemental formula of compound 2 was confirmed to be C 21 H 32 O 12 with one oxygen more than that of 1 according to the [M + COOH] − ion peak at m/z 521.1870 in its HRESIMS spectrum. The 1 H and 13 C NMR data of 2 revealed a close resemblance to 1 except for the corresponding signals to the two sugar units. Careful analysis of the NMR data and the acid hydrolysis results affirmed the existence of two β-d-glucose groups in 2 instead of one β-d-glucose and one α-l-rhamnose in 1.  (Fig. 2). Consequently, compound 3 was assigned as 4-methoxy-
The neuroprotective effects of 1-4 were also evaluated in CoCl 2 -induced PC12 cell damage [24] by MTT assay. According to the references [25,26] and our study, the positive control, trolox, exhibited statistically significant neuroprotective effect at 10 μM (Fig. 3). Therefore, the concentration of 10 μM was selected for the cytotoxic and neuroprotective evaluation of these compounds. First, the cytotoxic activity of compounds 1-4 against PC12 cell line was tested and none of them showed cytotoxicity at 10 μM (Additional file 1: Fig.  S16). Subsequently, 10 µM compounds were bioassayed for their neuroprotective properties. And according to Fig. 3, compound 3 exhibited moderate activities against CoCl 2 -induced PC12 cell injury.

Conclusion
In this paper, three new phenylethanol glycosides (1-3) and one known compound (4) were obtained from the seeds of A. chinensis Bge. var. chekiangensis., which represents the first isolation of phenylethanol glycosides from the genus of Aesculus. The findings also against CoCl 2 -induced cell death in PC12 cells. The data (cell viability, measured by MTT assay) are expressed as mean ± SD. Three independent experiments were performed. Trolox was used as the positive control at 10 μM. Compared with CoCl 2 treated group, *P < 0.05, **P < 0.01