Triterpenoid Saponins from the Seeds of Aesculus chinensis and Their Cytotoxicities

Abstract Six new triterpenoid saponins, aesculusosides A–F (1–6), together with 19 known ones, were isolated from the seeds of Aesculus chinensis. The new structures were elucidated through extensive spectroscopic analyses and by comparison with previously reported data. Some of the isolates were evaluated for their cytotoxic activities against MCF-7 cell line by an MTT assay, and compounds 15, 16, 19, and 23–25 exhibited inhibitory activities against MCF-7 with IC50 values ranging from 7.1 to 31.3 μM. Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-017-0148-4) contains supplementary material, which is available to authorized users.

Hippocastanaceae is known to be a rich source of escins, a group of structurally diverse natural products characterized by a pentacyclic triterpenoid framework combined with a oligoglycoside chain [1]. Modern pharmacological studies show that some of these triterpenoid saponins possess diverse activities, including anti-inflammatory [2,3], Jin-Tang Cheng and Shi-Tao Chen have contributed equally to this work.
& An Liu la62@163.com antitumor [4][5][6][7], antiviral [8], antioxidative [9,10] and antigenotoxic properties [10]. Aesculus chinensis Bge. (Hippocastanaceae), abundant in the northwestern China, is a medicinal plant and its dried ripe seeds have been used as a stomachic and analgesic in the treatment of ditension and pain in the chest and abdomen [8]. Previous investigations on the chemical constituents of the seeds led to the isolation of an array of triterpenoid saponins [2,8,[11][12][13]. In order to search for bioactive constituents from natural sources, we conducted the phytochemical investigation on the seeds of A. chinensis and identified six new (1-6) and 19 known triterpenoid saponins (7-25) (Fig. 1). Reported herein are the isolation and structure elucidation of compounds 1-6, as well as the cytotoxicities of some isolates.

Results and Discussion
Compound 1 was isolated as an amorphous powder, with the molecular formula of C 50 H 80 O 23 as determined by its 13 C-NMR data and negative-ion HR-ESI-MS (m/z 1047.5035 ([M-H] -), calc. 1047.5018). The IR absorption bands at 3424 and 1721 nm -1 implied the presence of the hydroxyls and carboxyl groups, respectively. In the 1 H-NMR spectrum (Table 1), it displayed the occurrence of one olefinic proton (d H 5.28) and six methyls (d H 1.43, 1.21, 0.96, 0.93, 0.92, and 0.88), which was in conformity with the appearance of one olefinic C-atom (d C 129.9) and six methyls (d C 30. 3, 27.6, 23.0, 19.2, 17.5, and 16.4) in its 13 C-NMR data, characteristic of a triterpenoid skeleton [15]. In addition, the presence of three anomeric carbon signals at d C 104.9, 104.8 and 104.2, as well as other oxygenated carbon signals in the region of d C 83.0-61.9 in the 13 C-NMR spectrum, indicated that there existed a trisaccharide moiety. The above evidence suggested that the structure of 1 showed a close resemblance to that of aesculuside-B [15], except for the presence of an additional acetyl group. Careful analysis of its HSQC and 1 H-1 H COSY spectra revealed the presence of the following fragments, a (C-1/C-2/C-3), b (C-15/C-16), c (C-21/C-22), d (C-1 0 /C-2 0 /C-3 0 /C-4 0 ), e (C-1 00 /C-2 00 /C-3 00 /C-4 00 /C-5 00 /C-6 00 ), and f (C-1 000 /C-2 000 /C-3 000 /C-4 000 /C-5 000 /C-6 000 ) as shown in Fig. 2 In the ROESY spectrum of 1, there were no solid correlations which can be used to establish the relative configurations of C-3, C-16, C-21, and C-22. Thus, alkaline hydrolysis of 1 with 1% NaOMe, which liberated aesculuside-B, was conducted to assign the stereochemistry of chiral centers in 1 [15]. Furthermore, the coupling constant of H-21 and H-22 (J = 9.6 Hz) also supported the proposed stereochemistry of C-21 and C-22 [8]. Acid hydrolysis of 1 yielded glucose and glucuronic acid. The three monosaccharides were determined to be one b-D-glucuronopyranosyl acid and two b-D-glucopyranoses inferring from the coupling constants of the anomeric protons (d H 4.50, d, J = 7.6 Hz, H-1 0 ; d H 4.83, d, J = 7.8 Hz, H-1 00 ) and the typical carbon chemical shifts (d C 104.9, C-1 0 ; d C 104.2, C-1 00 ; d C 104.7, C-1 000 ). Based on the above evidence, compound 1 was characterized as 28-O-acetylprotoaesci- Compound 2, obtained as an amorphous powder, was assigned to have a molecular formula of C 52 H 82 O 24 by negative-ion HR-ESI-MS (m/z 1089.5107 ([M-H] -), calc. 1089.5123). The 1 H and 13 C NMR data of 2 (Tables 1, 2) exhibited an identical trisaccharide moiety and aglycone as 1. The only difference between them was an additional acetyl group in 2, with an extra characteristic proton signal at d H 2.08 in its 1 H-NMR spectrum and two additional carbon signals at d C 173.5, 21.4 in its 13 C-NMR spectrum. Detailed analysis of its 1D and 2D NMR spectra suggested that the additional acetyl group was attached to C-22, which could be deduced by the key HMBC cross-peak of H-22 (d H 5.21)/C-1 0000 (d C 173.5). The downfield shift of H-22 further supported the above conclusion. Therefore, 2 was established as 22 Compound 3 was determined to have the same molecular formula of C 50 H 80 O 23 as 1 by its negative-ion HR-ESI-MS (m/z 1047.5005 ([M-H] -), calc. 1047.5018). The characteristic NMR data suggested that 3 had the similar structure as 1. The significant differences in 1 H and 13 C-NMR spectra from those of 1 and 3 were chemical shifts of C-21(d C 82.8) and C-22 (d C 73.9) with corresponding protons at d H 5.50 and 3.97. The downfield shifts of C-21 implied that the acetyl group at the C-22 in 1 was transferred to C-21 in 3, which was further confirmed by the key HMBC cross-peak of H-21(d H 5.50)/C-1 0000 (d C 174.0). In addition, the alkaline hydrolysis of 3 yielded aesculuside-B [15]. Hence, the structure of 3 was determined as 21-O-   13 C-NMR spectra of 5 with those of 4 indicated a high degree of similarity. The differences between 5 and 4 were the absence of an acetic group at the C-28 and that the isopropyl group was transferred to C-21 other than C-22 in compound 4, as confirmed by the combinational analysis of 2D NMR data (Fig. 2). Thus,  Table 2), indicated that aglycone moiety in 6 was barringtogenol C [16]. Furthermore, the characteristic carbon resonances displayed in the region of d C 83.0-62.6 in its 13 C-NMR data ( Table 2), indicated that there also existed a trisaccharide moiety. Acid hydrolysis of 6 also yielded glucose and glucuronic acid. The HMBC correlations from H-21 to C-20/C-22/C-29/C-30/C-1 0000 , and from H-28 to C-1 0000 , allowed for the assignment of two acetyl groups as shown in Fig. 1   Triterpenoid Saponins from the Seeds of Aesculus chinensis 51   [11].

General Experimental Procedures
Optical rotations were measured with Perkin Elmer/Model-343 digital polarimeter. IR spectra were recorded on a JASCO FT/IR-480 spectrophotometer and reported as

Extraction and Isolation
The dried seeds of A. chinensis (10 kg) were extracted with 70% ethanol under reflux for three times (

Alkaline Hydrolysis of Compound 1
Compound 1 (5 mg) was added to a MeOH solution (5 ml) of NaOMe (1 mg). The mixture was stirred at room temperature for 4 h and then neutralized with 20% aqueous HCl. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (CH 2 Cl 2 /MeOH 2:1-1:1) to furnish aesculuside-B (1.2 mg, 46% yield), which was identical with authentic sample by TLC, 1 H-and 13 C-NMR spectra comparisons [15].