New spinosin derivatives from the seeds of Ziziphus mauritiana

Three new acylated flavonoid C-glycosides, 6‴-(-)-phaseoylspinosin (1), 6‴-(3″″,4″″,5″″-trimethoxyl)-(E)-cinnamoylspinosin (2), and 6‴-(4″″-O-β-D-gluco-pyranosyl)-benzoylspinosin (3), were isolated from the seeds of Ziziphus mauritiana (Rhamnaceae). A further 19 known compounds including eight spinosin analogues (4–11) were also isolated. Their structures were elucidated by means of spectroscopic analysis and chemical method. Among spinosin derivatives 1, 2, 4, 7, 8, and triterpenoid saponin 14, jujuboside A (14) displayed moderate acetylcholinesterase (AchE) inhibitory activity with an inhibition value of 46.2% at a concentration of 1 µM.

Compound 1 was isolated as a yellow amorphous powder. Its molecular formula was deduced to be C 43 H 50 O 19 on the basis of HREIMS (m/z 870.2999 [M] + ). The IR spectrum showed absorption bands at 3430 and 1708 cm -1 due to hydroxyl and carbonyl groups, respectively. The UV spectrum exhibited maximum absorptions at 210, 272, and 332 nm. All the protons and carbons of 1 appeared as pair signals in the 1 H and 13 C NMR spectra, which is characteristic of signals arising from a spinosin skeleton 5  In the ROESY spectrum of 1, correlations of H-7"" and H-8"" with both Me-12"" and Me-13"" were observed. In addition, the H-3α"" and H-3β"" were correlated with H-11"" and Me-12"", respectively. These data revealed that the unsaturated side chain -CH(7)=CH(8)-C(9)=CH(14)-COO(15)-of phaseic acid moiety in 1 oriented to the same side of both Me-12"" and Me-13"", while the CH 2 -11"" and O-C(6"") were oriented to the opposite side of Me-12"", Me-13"" and C-7"" side chain. Alkaline hydrolysis of 1 with 0.5% NaOH yielded spinosin (10) and phaseic acid (1a). Compound 1a showed the similar NMR spectroscopic data and optical rotation value ([α] 20 D -      It is note that the 1 H and 13 C NMR data of the spinosin (10) and its derivatives 1-9 exhibited doubling of signals at room temperature due to the rotational isomers produced by the rotational barriers 7-OCH 3 in flavones-6-C-glycoside. 4 As compared with the positive control, tacrine (59.0%), compounds 1, 2, 4, 7 and 14 displayed moderate AChE inhibitory activity with inhibition values of 24.9%, 33.9%, 26.9%, 28.8% and 46.2%, respectively, at a concentration of 1 µM, by the spectrophotometric method developed by Ellman et al. 35 with slight modification. Interstingly, AChE inhibition of compound 8 was only 7.8% at 1 µM concentration.

Experimental Section
General Experimental Procedures. Optical rotations were determined with a Jasco P-1020 polarimeter. UV (in MeOH) spectra were obtained with the Shimadzu UV-2401 PC spectraphotometer.
The Bruker Tensor-27 infrared spectrophotometer was used for IR spectra as KBr pellets. ESIMS and HREIMS spectra were recorded on API QSTAR Pulsar spectrometer and Waters Autospec Premier P776, respectively. 1D and 2D NMR spectra were performed on Bruker Avance III-600MHz spectrometers with TMS as an internal standard. Semi-preparative and preparative HPLC studies were performed on an Agilent 1260 and Gilson GX-27X liquid chromatograph. TLC was performed on precoated TLC plates (0.2-0.25 mm thickness, GF254 Si gel 60, Qingdao Marine Chemical Co., Ltd.) with compounds visualized by spraying the dried plates with 10% aqueous H 2 SO 4 followed by heating until the plate was dry.   Acid Hydrolysis of 1-3. Compounds 1-3 (each 10 mg) was dissolved in 2 N HCl (2 mL) and refluxed at 80 °C for 10 h. The reaction mixture was neutralized with Amberlit IRA-401 and a sugar residue was obtained. Identification of D-glucose was performed by comparison of OD (+) with authentic samples.

6'''-(-)-
Acetylcholinesterase (AChE) Inhibitory Activity. The AChE inhibitory activity was assayed using the spectrophotometric method developed by Ellman et al. 34 with slightly modification. In brief, the reaction mixture (200 μL in total) containing phosphate buffer (pH 8.0), testing compound (50 µM in DMSO), and AChE (0.02 U/mL), was incubated for 20 min (30 °C). Then, the reaction was initiated by the addition of 40 μL of solution containing DTNB (0.625 mM) and acetylthiocholine iodide (0.625 mM). The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 seconds for one hour. Tacrine was used as positive control with final concentration of 0.333 μM. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = (E -S) / E × 100 (E is the activity of the enzyme without test compound and S is the activity of enzyme with the test compound).

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-013-0028-5 and is accessible for authorized users.