Bioactive Triterpenoid Saponins From the Seeds of Aesculus chinensis Bge. var. chekiangensis

Phytochemical investigation of Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang obtained 33 triterpenoid saponins, including 14 new ones, aesculiside C–P (1–14). The structure elucidations were performed through comprehensive MS, 1D and 2D-NMR analysis, and their absolute configuration was unambiguously determined by X-ray diffraction analysis as well as Mo2(OAc)4-induced ECD method for the first time. All the substances were examined for their cytotoxic activities against three tumor cell lines, Hep G2, HCT-116, and MGC-803. Of these, compounds 8, 9, 14–16, 18, and 22 exhibited potent cytotoxicities against all cell lines with IC50 of 2–21 μM, while compounds 3, 6, 7, 17–19, 20, 24, and 28 depicted moderate activity (IC50 13 to >40 μM). On these bases, the preliminary structure-activity correlations were also discussed. Meanwhile the neuroprotective properties of triterpenoid saponins from Aesculus genus were evaluated for the first time. Among them, compounds 1, 4, 12, 20, 22, 25, 29, and 31 exhibited moderate activities against COCl2-induced PC12 cell injury.


INTRODUCTION
Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang is a shrubby or small tree belonging to the Hippocastanaceae family which is widely distributed in China. The dry seeds of this plant, together with Aesculus chinensis Bge and Aesculus wilsonii Rehd, are the major sources of the traditional Chinese medicine "Suo Luo Zi." Traditionally, it has been exploited to treat chest and abdominal pain, dysentery and ague (Yang et al., 1999a;Zhang et al., 2006). Earlier phytochemical study of Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang obtained various types of isolates, for example, triterpenoids (Yuan et al., 2013), flavonoids (Kapusta et al., 2007), coumarins (Niu et al., 2015) together with steroids (Zhang et al., 2009). Polyhydroxylated triterpenoid saponins, isolated from Aesculus genus (Wei et al., 2004;Kim et al., 2017) with great structural diversity, have been proved to be the major bioactive principles including anticancer (Patlolla et al., 2006), neuroprotective (Cheng et al., 2016), anti-inflammatory (Matsuda et al., 1997), antioxidative (Küçükkurt et al., 2010), and antiedematous activities (Piller, 1976). As part of our continuous research to screen cytotoxic and neuroprotective compounds of this type, a series of new triterpenoids (1-14) along with 19 reported analogs (15-33) from the seeds of Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang were obtained. Their cytotoxic activity and neuroprotective activity were also examined. Herein, the isolation, structural elucidation, cytotoxic activity, and neuroprotective activities of these isolates are described.

Plant Material
Seeds of Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang were purchased from the Anguo Chinese medicine market (Hebei Province, P.R. China) in August 2015 and identified by professor Lijuan Zhang (Tianjin University of Traditional Chinese Medicine). The specimen was kept at the School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine.

Extraction and Isolation
The dried seeds of A. chinensis Bge. (8.8 kg) were extracted with 70% ethanol (10 L) under reflux for three times (3 h) at 70 • C. After the solvent was removed under reduced pressure at <45 • C, a dark residue (2,100 g) was obtained. The residue was adsorbed onto D101 resin and then sequentially eluted with H 2 O, a gradient of EtOH in water to give the corresponding fractions. The 60% EtOH-H 2 O part was chromatographed on silica gel, eluting with a gradient of 0-100% CH 2 Cl 2 /CH 3 OH to yield four fractions (A-D).

Hydrolysis and Determination of Absolute Configuration of Sugars
A solution of 1-14 (1.0 mg, respectively) in 2 M HCl (4.0 ml) was heated at 90 • C for 2 h. The reaction mixture was extracted with EtOAc (2 × 4 ml), and the aqueous phase was evaporated to dryness using a stream of N 2 . The residues and authentic sugar samples (D/L-galactose, D/L-glucose, D/L-xylose, and Dglucuronic acid) were, respectively, dissolved in pyridine (1.0 ml) containing L-cysteine methyl ester (1.0 mg) and heated at 60 • C for 1 h, and then o-tolyisothiocyanate (1.0 ml) was added to the mixture and heated further for 1 h. Then each reaction mixture was analyzed by the Waters e2695 HPLC system using a 2998 PDA detector (at 250 nm). Analytical HPLC was performed on the YMC-Pack-ODS-A column (250 × 4.6 mm, 5 µm) eluting with A (0.1% formic acid): B (acetonitrile) = 80:20 (v/v) at 1.0 ml/min. The absolute configuration of sugars in each compound was established by a comparison of the retention times with the standards where the time differences ( δ D-L) of one kind of sugar were sufficient to distinguish between D-and L-enantiomers (Tanaka et al., 2007;.

Preparation of the Aglycone of Compound 14
Compound 14 (15.0 mg) in 2 M HCl (10.0 ml) was heated at 50 • C for 4 h. The reaction mixture was extracted with EtOAc (2 × 10 ml), and the EtOAc phase was evaporated to dryness using a stream of N 2 . The residue was dissolved in THF (2.0 ml) and MeOH (1.00 ml), then NaOMe (2.00 mg, 2.2 eq) was added to the solution at 0 • C. The mixture was stirred at 25 • C for 4 h. The reaction was diluted with H 2 O (10.0 ml) and the mixture was extracted with ethyl acetate (3 × 3.00 ml). The ethyl acetate fraction was purified by a semipreparative RP HPLC (CH 3 CN-H 2 O, 45:65) to gain compound 14a.

Determination of the Absolute Configuration of the 21, 22-Diol Moieties in Compound 14a
First, Mo 2 (AcO) 4 (1.0 mg) dissolved in DMSO (1.0 ml) was subjected to ECD measurement as blank control. Then compound 14a (0.5 mg) and Mo 2 (AcO) 4 (1.0 mg) were added to DMSO (1.0 ml) and scanned directly. The CD spectrum was  recorded every 10 min until the Mo 2 (AcO) 4 -induced circular dichroism spectrum was stationary. The inherent ECD spectrum of 14a was subtracted. The absolute configuration was elucidated by the diagnostic band at approximately 310-340 nm in the induced ECD spectrum.

Cytotoxicity Assay
The in vitro cytotoxicity of compounds 1-33 was measured by MTT assay (El-Readi et al., 2013;Xia et al., 2015) with 5fluorouracil as the positive control. The human cancer cell lines, HepG2, HCT-116, and MGC-803 were purchased from ATCC. The tested cell lines were seeded in 96-well plates, and the plates were then incubated in a 37 • C incubator containing 5% CO 2 for 24 h. Subsequently, the tested compounds in DMSO were added to designated wells at a dosage of 3.125-50 µM. After 24 h, MTT was added to the culture medium and the absorbance at 490 nm was measured using a microplate reader.

Neuroprotective Effect Assay
The neuroprotective effects of compounds 1-33 were tested against C O Cl 2 -induced PC12 cell injury (Zou et al., 2002) with MTT method. Rat pheochromocytoma cell line (PC12) was cultured in 96-well plates with RPMI-1640 supplemented with 10% (v/v) inactivated fetal bovine serum and 100 U/ml penicillin/streptomycin. The cells were maintained at 37 • C in 5% CO 2 and 95% humidified air incubator. Cells were pre-treated for 2 h with or without compounds before incubation in a medium containing 1 mM CoCl 2 . After 24 h, MTT was added to the culture medium, and the absorbance at 490 nm was measured using a microplate reader.

RESULTS
For the target of isolation of triterpene saponins, the 70% ethanol extracts of air-dried seeds of Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang were chromatographed through a D101 column and eluted with a gradient of EtOH in water. The 60% EtOH part was separated consequently as it contains abundant triterpene saponins under the guidance of UPLC-Q/TOF-MS. Thereafter, 14 undescribed triterpenoid saponins (1-14, aesculiside C-P) (Figure 1) and 19 known analogs (15-33) were afforded and identified (Figures S1-S157). The full assignments of the NMR data of compounds 1-14 are recorded in Tables 1-4. Aesculiside C (1) was isolated as a white amorphous powder which exhibited an ion peak at m/z 1073.5149 [M-H] − (calcd. 1073.5169). Its molecular formula was confirmed as C 52 H 82 O 23 based on HR-ESI-MS as well as 13 C NMR spectroscopic data. The IR absorptions at 3,414 and 1,732 cm −1 implied the existence of the hydroxyl and carboxyl groups, respectively. The NMR data of 1 exhibited characteristic signals of a triterpenoid saponin.
Aesculiside E (3) was acquired as a white amorphous powder and its molecular formula was determined as C 53 H 84 O 22 (m/z 1071.5376 [M-H] − ; calcd. for C 53 H 83 O 22 , 1071.5376). Acid hydrolysis of 3 yielded D-galactose, D-glucose, and Dglucuronic acid. The NMR data of 3 showed a lot of resemblance with those of 2 except for the presence of a tigloyl moiety instead of an acetyl unit in 3, which was supported by the characteristic olefinic quartet at δ 7.00 in its 1 H-NMR spectrum. Moreover, the HSQC correlation signals of δ H 1.61 with δ C 14.0 and δ H 1.87 with δ C 12.3 confirmed the existence of a tigloyl group. The aforementioned data, together with the HMBC correlation from H-21 (δ 6.48) to C-1 ′′′′ (δ 168.5) confirmed the connection between the tigloyl group and C-21. Consequently, it was assigned as 3-O-[β-D-galactopyranosyl-(1→ 2)]-β-D-glucopyranosyl-(1→ 4)-β-D-glucuronopyranosyl-21β-tigloyl-3β, 16α, 21β, 22α, 28-pentahydroxyolean-12-ene. Aesculiside F (4) and 1 gave the same molecular formula, deduced as C 52 H 82 O 23 from its HR-ESI-MS and 13 C NMR spectroscopic data. Comparison of the NMR data of 4 with those of 1 indicated that both saponins are closely related, differing at trisaccharide moiety where the galactose in 1 was replaced by a glucose in 4, based on the distinction of their 13 C NMR data ( Table 3) (Yoshikawa et al., 1998), which was further verified by hydrolysis and derivatization as aforementioned. HMBC correlations revealed the position and sequences of the sugar moiety in 4 as described before. Hence, compound 4 was identified and named aesculiside F.
Aesculiside G (5), a white amorphous powder, was established to be an analog of 4 by HRESIMS and NMR spectrum interpretation. Careful analysis of their NMR data suggested that 4 possessed one more acetyl group compared to 5.
The absolute configurations of the aglycones of 1-13 were all deduced to be 3S, 16R, 21R, 22R based on the absolute configuration of 14 and their mutual biogenetic source.
The cytotoxic activities against three human cancer cell lines (Hep G2, HCT-116, and MGC-803) of compounds 1-33 were evaluated using the MTT method, with 5-fluorouracil (5-FU) as positive control (Table 5). Among them, compounds 8, 9, 14-16, 18, 22 showed potent cytotoxicity against all the tested human cancer cell lines with IC 50 ranging between 2 and 21 µM .  Compounds 3, 6, 7, 17-19, 20, 24, 28 were less active (IC 50 : 13 to >40 µM) whereas the other isolates displayed no toxicity in all cell lines at 50 µM. These results suggested that the compounds with acylations at both C-21 and C-22 exhibited stronger inhibitory activities than those with acylations at C-21 and C-28 or only at C-21. In addition, it seems that the presence of the tigloyl, angeloyl, methylbutyryl, and isobutyryl groups affects the inhibitory activity of these compounds on the tested cell lines positively.

CONCLUSION
Plants of the genus Aesculus have been proved to be rich in polyhydroxyoleanene triterpenoid saponins which have been characterized more than 100. When compared to the relatively extensive research on other species of Aesculus genus, little is known regarding the chemical constituents and the biological activity of the Aesculus chinensis Bge. var. chekiangensis (Hu et Fang) Fang species. The present paper reports 14 new polyhydroxy oleanene saponins (1-14) along with 19 known analogs from the seeds of A. chinensis Bge. var. chekiangensis. Structure elucidation was achieved via various techniques, and the absolute configuration of the aglycones was undoubtedly defined through X-ray diffraction analysis as well as Mo 2 (OAc) 4induced ECD method for the first time. Further cytotoxicity evaluation against three human tumor cell lines suggested that compounds 8, 9, 14-16, 18, 22 displayed strong inhibitory activities against all three cell lines; compounds 3, 6, 7, 17-19, 20, 24, 28 exhibited weak activities while the remaining isolates showed no toxicity at 50 µM. These results suggested that isolates with two acylations at C-21 and C-22 might be important for the cytotoxicity, especially substituted by tigloyl, angeloyl, methylbutyryl, and isobutyryl groups. In addition, the first test about the neuroprotective properties of triterpenoid saponins from Aesculus genus found that compounds 1, 4, 12, 20, 22, 25, 29, 31 exhibited moderate activities against CoCl 2 -induced PC12 cell injury.

DATA AVAILABILITY STATEMENT
The crystallographic dataset generated for this study can be found in the Cambridge Crystallographic Data Centre under the CCDC number 1957449. All other datasets generated for this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
NZ and SW was responsible for the isolation and elucidated of compounds. NZ tested cytotoxicity, neuroprotective effects of the compounds, interpreted the data, and wrote the paper. SC, QZ, and NK revised the manuscript. LD and FQ were the project leaders organizing and guiding the experiment. All authors read and approved the final manuscript.