Studies On Metabolism Of Irisolidone In Vovo And In Votro Of Rats

Irisolidone, a major isoflavone found in Pueraria lobata flowers, exhibits a wide spectrum of bioactivities such as anti-inflammatory, antioxidative, antiviral, anti-tumor, and estrogenic effects and protecting against ethanol-induced damage and hepatic injury. In our previously study, the metabolism of its glucoside, kakkalide, has been comprehensively investigated, while its metabolic pathway in vivo and in vitro has not been studied yet. In present study, an ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the in vivo and in vitro metabolism of irisolidone in rats, which would laid theoretical and technology foundation for active metabolites for new drug development and provide scientific introduction for its clinical application. The investigations were as follows.1. Extraction, activated carbon adsorption, and recrystal were used to separate and purify irisolidone from the EtOH extraction of Pueraria lobata flowers. TLC、HPLC-UV、 UHPLC/Q-TOF MS. H-NMR method were used to identification or testify the purity of Ir. Results shows that the light yellow crystal was Ir, and its purity was more than95%, which was fundamental for the metabolism of Ir in vivo and in vitro of rats.2. An ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the in vivo metabolism of irisolidone in rats. Plasma, bile, urine, and feces were collected from rats after a single100mg/kg oral dose of irisolidone. Protein precipitation, solid phase extraction (SPE) and ultrasonic extraction were used to prepare samples of plasma, bile/urine, and feces, respectively. A total of46metabolites were detected and tentatively identified based on the mass spectral fragmentation patterns, elution order or confirmed using available reference standards. The metabolic pathways of irisolidone in rats included decarbonylation, reduction, demethylation, demethoxylation, dehydroxylation, hydroxylation, sulfation, and glucuronidation. The relative content of each metabolite was also determined to help understand the major metabolic pathways of irisolidone in rats. The parent drug was the most important excretion form with28%in the urine and80%in the feces. As a poly-hydroxyl compound, very little Ir was detected in plasma (2%), and it was mainly in the form of phase Ⅱ metabolites. The metabolite Ir-7G (more than65%in plasma, more than95%in bile) was the major metabolite of Ir in vivo. 3. Kakkalide and irisolidone, the main isoflavones of Flos Puerariae, exhibit a wide spectrum of bioactivities. Intestinal bacteria biotransformation plays an important role in the metabolic pathways of isoflavones, and is directly related to the bioactivities of the prodrugs after oral administration. To our knowledge, the metabolic pathways of kakkalide and irisolidone in vitro have not been comprehensively studied yet. This paper describes the strategy using ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) for the rapid analysis of the metabolic profiles of kakkalide and irisolidone after incubated with human and rat intestinal bacteria. Bacteria incubated samples were prepared and analyzed after incubated under anaerobic conditions for48h. A total of17metabolites, including parent compounds, were detected in human and rat intestinal bacteria incubated samples. The results obtained indicate that hydrolysis, dehydroxylation, demethoxylation, demethylation, hydroxylation, decarbonylation, and reduction were the detected metabolic pathways of kakkalide and irisolidone in vitro. The conversion rate of irisolidone in human and rat bacteria was8.57%and6.51%, respectively. Biochanin A was the relatively main metabolite of irisolidone, and the content of biochanin A in human and rat bacteria was3.68%and4.25%, respectively. The conversion rate of kakkalide in human and rat bacteria was99.92%and98.58%, respectively. Irisolidone was the main metabolite of kakkalide, and the content of irisolidone in human and rat bacteria was89.58%and89.38%, respectively. This work not only provides the evidence of kakkalide and irisolidone metabolites in vivo, but also demonstrates a simple, fast, sensitive, and inexpensive method for identification of metabolites of other compounds transformed by intestinal bacteria.4. An ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method was employed to investigate the metabolic pathways of irisolidone and the pharmacokinetics of its main metabolites in rats, after a single100mg/kg oral dose of irisolidone. Protein precipitation method was used to prepare plasma samples. A total of14metabolites included irisolidone were detected and tentatively identified based on the mass spectral fragmentation patterns, elution order or confirmed using available reference standards. The pharmacokinetics of main metabolites included three glucuronide metabolites tectorigenin-7-O-glucuronide (Te-7G),6-hydroxybiochanin A-6-O-glucuronide (6OH-BiA-6G), irisolidone-7-O-glucuronide (Ir-7G), and three sulfate metabolite tectorigenin-7-O-sulfate-4’-O-sulfate (Te-7S-4’S), tectorigenin-7-O-sulfate (Te-7S) and irisolidone-7-O-sulfate (Ir-7S), and aglycone tectorigenin (Te), and irisolidone (Ir) were evaluated. The plasma concentrations reached maximal values of0.297μmol/L at10.3h for Te-7S-4’S,0.199μmol/L at21.67h for Te-7S,0.154μmol/L at8.00h for Te-7S,4,10μmol/L at15.3h for6OH-BiA-6G,10.7μmol/L at9.71h for Ir-7G,0.918μmol/L at11.3h for Te,0.150μmol/L at8.67h for Ir-7S, and0.843μmol/L at9.67h for irisolidone, respectively. Since the total plasma concentrations of conjugatedmetabolites were much higher than that of the irisolidone aglycone, an extensive phase Ⅱ metabolism plays an important role in the pharmacokinetics of irisolidone in vivo. The major metabolites Ir-7G and6-OH-BiA-6G accounted for55.8%and31.5%, respectively, indicating that the metabolic pathways of KA and Ir in vivo of rat were similar. However, the Tmax of the metabolites of KA were34-38h, much longer than that in present study which were all less than21h. The aldose reductase inhibit ability of Ir-7G, Ir, Te and KA was evaluated. Results indicated that the aldose reductase inhibit ability of Te was stronger than the other three. We could infer that P. thomsonii flower may be a batter medicine for diabetes than P. Puerariae flower.In present study, the metabolism of Ir in vivo, the metabolism of Ir in vitro by intestinal bacteria and the pharmacokinetics of Ir in blood were comprehensively studied, which present the absorption, metabolism and excretion of Ir in rat after oral administration. It may provide the evidence about the new drug development of isoflavones and a metabolic method about other compounds.