Stability and Structure Studies on Alisol A 24-Acetate

Abstract

Rhizoma Alismatis, the dried rhizome of Alisma orientalis (SAM.) JUZEP, is a famous Traditional Chinese Medicine (TCM) which has been widely used for diuretic, hypolipidemic, anti-inflammatory and anti-diabetic purposes in China for more than a thousand years. Protostane-type triterpenes are the principal active constituents of Rhizoma Alismatis and more than 50 unique protostane-type triterpenes, including alisols A, B and their monoacetates, have been isolated from this herbal drug. [1][2][3] As the bioactive "marker compounds" of Rhizoma Alismatis, protostane-type triterpenes, including alisol A 24-acetate, which is one of the main active triterpenoid compounds isolated from Rhizoma Alismatis, have been determined for the quality control of Rhizoma Alismatis. 4,5) However, during the course of our research on the quantification of alisol A 24-acetate, it was found that alisol A 24-acetate was unstable and could be transformed into other compounds in methanol.In this paper, the stability of alisol A 24-acetate in different solvents was described in detail. Furthermore, by using LC-MS, NMR, and single crystal X-ray diffraction techniques, the structures of the compounds transformed from alisol A 24-acetate and the single crystal X-ray structure of alisol A 24-acetate were elucidated for the first time. ExperimentalGeneral Experimental Procedures A Shimadzu 10A HPLC system (Tokyo, Japan), equipped with a quaternary pump, a diode array spectrophotometric detector (DAD) and an Altech ELSD-2000ES evaporative light scattering detector (ELSD), was used for HPLC analysis. NMR spectra were recorded on a Bruker DRX-500 NMR spectrometer equipped with 5 mm probes using TMS as internal standard. LC-MS analysis was performed on an Agilent 1000 HPLC system (Palo Alto, CA, U.S.A.) equipped with a MDS SCIEX QSTAR mass spectrometer with an ESI source. A single crystal was mounted on a Japan MAC DIP-2030K area detector diffraction meter (Tokyo, Japan). HPLC-grade acetonitrile (CH 3 CN) and methanol (MeOH) were purchased from Fisher Scientific (Fair Lawn, NJ, U.S.A.). HPLC-grade deionized water (H 2 O) was purified by Milli-Q Water purification system (Millipore, MA, U.S.A. C-and 2D-NMR spectra.HPLC Analysis HPLC analysis was performed on a YMC analytical column (Tokyo, Japan) with 5 mm C18-reversed phase material (250ϫ4.60 mm i.d.) using CH 3 CN/H 2 O (70% or 52%) as the mobile phase with the flow rate of 1.0 ml/min at room temperature. The injection volume was 20 ml. For UV detection, the detection wavelength were set at 210 nm, while for ELSD detection, the tube temperature was set at 90°C, and air was used as the gas with the flow rate of 2.5 l/min. LC-MS Analysis LC-MS analysis was performed on a YMC analytical column (Tokyo, Japan) with 5 mm C18-reversed phase material (250ϫ 4.60 mm i.d.) using CH 3 CN/H 2 O (52%, v/v) as the mobile phase. The flow rate was 1.0 ml/min and split 0.4 ml/min to mass spectrometer. The injection volume was 20 ml. Mass spectra were acquired using a MDS SCIEX QSTAR mass spectrometer equipped with ...

Protostane Triterpenoids from the Rhizome of Alisma orientale Exhibit Inhibitory Effects on Human Carboxylesterase 2

“…Makabel and co-workers reported the intramolecular transacetylation of alisol A 24-acetate. 15 Moreover, both alisol A 24-acetate and 23-acetate are susceptible to deacetylation into alisol A when stored in methanol for a long time. 14 The threo 23-OAc, 24-OH configuration in compound 3 corresponded to a stable structure without the transfer of an acetyl group from 23-OH to 24-OH.…”

“…On the basis of the 20R configuration in protostane triterpenoids isolated from A. orientale and the correlation of H-20/H-23 observed in its NOESY spectrum, the 23S and 24S configurations of compound 3 were established. 15,16 Therefore, the structure of compound 3 was assigned as shown, and the compound was given the trivial name alismanol C.…”

Qualitative and Quantitative Analysis of Major Triterpenoids in Alismatis Rhizoma by High Performance Liquid Chromatography/Diode-Array Detector/Quadrupole-Time-of-Flight Mass Spectrometry and Ultra-Performance Liquid Chromatography/Triple Quadrupole Mass Spectrometry

“…Deionized water was prepared using a system from Millipore (Bedford, MA, USA); other solvents were of analytical grade. Twenty-one standards were previously isolated and purified from AMR in our laboratory; their structures were confirmed by MS, 1 H-NMR spectrometry, and 13 C-NMR spectrometry [24,25]. Fourteen of them, including 16-oxo-alisol A 23-acetate (purity, 98.5%), 16-oxo-alisol A 24-acetate (purity, 99.0%), alisol C (purity, 98.5%), alisol F (purity, 99.6%), alisol C 23-acetate (purity, 99.5%), alisol L (purity, 99.0%), alisol F 24-acetate (purity, 99.2%), alisol A (purity, 99.3%), alisol A 23-acetate (purity, 98.7%), alisol A 24-acetate (purity, 99.2%), alisol G (purity, 99.0%), alisol B (purity, 99.7%), alisol B 23-acetate (purity, 99.8%) and 11-deoxy-alisol B (purity, 99.0%), were used for quantitation; their purity were determined by HPLC-DAD-ELSD.…”

Alisol B, a Novel Inhibitor of the Sarcoplasmic/Endoplasmic Reticulum Ca2+ ATPase Pump, Induces Autophagy, Endoplasmic Reticulum Stress, and Apoptosis

“…Finally, the compounds in the purified active fraction were deduced by comparing their 1 H and 13 C nuclear magnetic resonance spectroscopic data with those of known Alisma components (22,23) and were identified to be alisol A 23-acetate and alisol A 24-acetate (Fig. 1A), compounds that are known to interconvert rapidly (24). Cytotoxicity was also observed by these compounds upon incubation with the cells.…”