Identification and pharmacokinetics of saponins in Rhizoma Anemarrhenae after oral administration to rats by HPLC-Q-TOF/MS and HPLC-MS/MS.

Rhizoma Anemarrhenae is a well-known herbal medicine with saponins as its commonly regarded major bioactive components. It is essential to classify the properties of saponins which are associated with their toxicity and efficacy. In this study, 25 compounds were identified by HPLC-Q-TOF/MS in the extract of Rhizoma Anemarrhenae and 8 saponins were detected in rat plasma by HPLC-MS/MS after oral administration of this extract. These were neomangiferin, mangiferin, timosaponin E1, timosaponin E, timosaponin B-II, timosaponin B-III, timosaponin A-III and timosaponin A-I. A sensitive and accurate HPLC-MS/MS method was developed and successfully applied to a pharmacokinetic study of the abovementioned eight saponins after oral administration of the Rhizoma Anemarrhenae extract to rats. The method validation, including specificity, linearity, precision, accuracy, recovery, matrix effect and robustness, met the requirements of the intended use. The pharmacokinetic parameter, T max value, ranged from 2 to 8 h for these eight saponins whereas their elimination half-life (t 1/2) ranged from 4.06 to 9.77 h, indicating slow excretion. The plasma concentrations of these eight saponins were all very low, indicating a relatively low oral bioavailability. All these results provide support for further clinical studies.

Biopharmaceutics and Pharmacokinetics of Timosaponin A-III by a Sensitive HPLC-MS/MS Method: Low Bioavailability Resulting from Poor Permeability and Solubility.

BACKGROUND: Timosaponin A-III is one of the most promising active saponins from Anemarrhena asphodeloides Bge. As an oral chemotherapeutic agent, there is an urgent need to clarify its biopharmaceutics and pharmacokinetics to improve its development potential. OBJECTIVE: This research explores the bioavailability of timosaponin A-III and clarifies its absorption and metabolism mechanisms by a sensitive and specific HPLC-MS/MS method. METHODS: Pharmacokinetics and bioavailability studies of timosaponin A-III were performed in Sprague- Dawley rats by oral (20 mg/kg) and intravenous administration (2 mg/kg). Control group was given the same volume of normal saline. The absorption of timosaponin A-III was investigated in a rat intestinal perfusion model in situ and a Caco-2 cell transport model in vitro. The metabolic rate of timosaponin A-III was determined in a rat liver microsome incubation system. RESULTS: After the oral administration, timosaponin A-III reached Cmax of 120.90 ± 24.97 ng/mL at 8 h, and the t1/2 was 9.94 h. The absolute oral bioavailability of timosaponin A-III was 9.18%. The permeability coefficients of timosaponin A-III in four intestinal segments ranged from 4.98 to 5.42 × 10-7 cm/s, indicating a difficult absorption. A strikingly high efflux transport of timosaponin A-III was found, PappBA 3.27 ± 0.64 × 10-6 cm/s, which was abolished by a P-gp inhibitor. Rat liver microsome incubation studies showed that timosaponin A-III could hardly be metabolized, with a t1/2 of over 12 h. In addition, the solubility test showed a low solubility in PBS solution, i.e. 30.58 µg/mL. CONCLUSION: Timosaponin A-III exhibited low oral bioavailability by oral and intravenous administration, which was probably caused by its low permeability and solubility. This study may provide a reference for its rational clinical use and further study on the pharmacology or toxicology of timosaponin A-III.

Identification of a novel BACE1 inhibitor, timosaponin A-III, for treatment of Alzheimer's disease by a cell extraction and chemogenomics target knowledgebase-guided method.

BACKGROUND: Rhizoma Anemarrhenae (RA) has been conventionally used for treatment of Alzheimer's disease (AD) in Traditional Chinese Medicine, and thus, the active components from RA can be screened. PURPOSE: This research aimed to identify the active components of RA and their targets and further clarify the molecular mechanisms underlying its anti-AD activity. METHODS: First, the potential active compounds from RA were screened by neurocyte extraction and micro-dialysis methods. Second, the potential targets were predicted by a chemogenomics target knowledgebase and further explored by surface plasmon resonance and enzyme activity assays. Third, the pharmacological effects were evaluated by employing APP/PS1 transgenic mice and SH-SY5Y-APP cells. ELISAs and Western blot analyses were used to evaluate the expression of key molecules in the amyloidogenic and NMDAR/ERK pathways. RESULTS: Timosaponin A-III (TA-III) was screened and identified as a potential active component for the anti-AD activity, and BACE1 was proven to be a potential high-affinity target. Enzyme kinetic analysis showed that TA-III had strong noncompetitive inhibitory activity against BACE1. The in vitro and in vivo assays indicated that TA-III had pharmacological effects through improving memory impairment, reducing Aß aggregation via the amyloidogenic pathway and preventing neuronal impairment through downregulating the NMDAR/ERK signaling pathway. CONCLUSION: TA-III targets BACE1 to reduce Aß aggregation through down-regulating the NMDAR/ERK pathway for treating AD.