A study on puerarin in situ gel eye drops: Formulation optimization and pharmacokinetics on rabbits by microdialysis.

Puerarin (PUE), an isoflavonoid isolated from Pueraria lobata (Willd) Ohwi root, is a ß-adrenergic receptor inhibitor used in treating glaucoma. The concentration range of gellan gum was determined based on the formulation viscosity and gelling capacity. PVP-K30 and gellan gum were used as variables, with the viscosity of formulation: STF = 40: 21, the 4 h permeation rate of rabbit isolated sclera, and 2 h in vitro release rate as response values. The JMP software was used to optimize the results, presenting that gellan gum was the main factor influencing viscosity. The in vitro release and permeation rate were primarily influenced by PVP-K30. The optimal prescription was 0.45% gellan gum and 6.0% PVP-K30. The in vitro release and permeation characteristics of puerarin in situ gel (PUE-ISG) were investigated using PUE solution as a control. The dialysis bag method results indicated that the release of the solution group leveled off after 4 h, while the PUE-ISG group had been continuously releasing. However, the cumulative release rates of the two were no longer significantly different at 10 h. The cumulative permeation rates of the ISG and solution groups were not significantly different (P > 0.05) in the rabbit isolated sclera. The apparent permeability Papp and steady-state flux Jss of PUE-ISG were 0.950 ± 0.059 cm/h and 9.504 ± 0.587 mg·(cm·h), respectively. A sensitive and stable HPLC-MS/MS analytical method for quantifying aqueous humor concentrations of PUE was validated. A microdialysis technique was successfully used in the aqueous humor pharmacokinetics study to sample aqueous humor from rabbit eye continuously. The results revealed that PUE-ISG significantly increased the drug concentration in the aqueous humor, with Cmax and AUC(0-t) 3.77 and 4.40 times higher than those of the solution group, respectively. Tmax was also significantly prolonged, indicating good prospects for clinical application. The developed PUE-ISG preparation has the characteristics of rapid drug release and sustained permeation, and increase the drug concentration in aqueous humor, with all inactive ingredients remaining within the maximum allowable limits recommended by the FDA guideline.

UGT1A1 and UGT1A9 Are Responsible for Phase II Metabolism of Tectorigenin and Irigenin In Vitro.

Tectorigenin and irigenin are biologically active isoflavones of Belamcanda chinensis (L.) DC. Previous studies indicated that both compounds could be metabolized in vivo; however, the kinetic parameters of enzymes involved in the metabolization of tectorigenin and irigenin have not been identified. The aim of this study was to investigate UGTs involved in the glucuronidation of tectorigenin and irigenin and determine enzyme kinetic parameters using pooled human liver microsomes (HLMs) and recombinant UGTs. Glucuronides of tectorigenin and irigenin were identified using high-performance liquid chromatography (HPLC) coupled with mass spectrometry and quantified by HPLC using a response factor method. The results showed that tectorigenin and irigenin were modified by glucuronidation in HLMs. One metabolite of tectorigenin (M) and two metabolites of irigenin (M1 and M2) were detected. Chemical inhibition and recombinant enzyme experiments revealed that several enzymes could catalyze tectorigenin and irigenin glucuronidation. Among them, UGT1A1 and UGT1A9 were the primary enzymes for both tectorigenin and irigenin; however, the former mostly produced irigenin glucuronide M1, while the latter mostly produced irigenin glucuronide M2. These findings suggest that UGT1A1 and UGT1A9 were the primary isoforms metabolizing tectorigenin and irigenin in HLMs, which could be involved in drug-drug interactions and, therefore, should be monitored in clinical practice.

Elucidation of the Transport Mechanism of Puerarin and Gastrodin and Their Interaction on the Absorption in a Caco-2 Cell Monolayer Model.

Puerarin (PUR) and gastrodin (GAS) are often used in combined way for treating diseases caused by microcirculation disorders. The current study aimed to investigate the absorption and transportation mechanism of PUR and GAS and their interaction via Caco-2 monolayer cell model. In this work, the concentration in Caco-2 cell of PUR and GAS was determined by HPLC method. The bidirectional transport of PUR and GAS and the inhibition of drug efflux including verapamil and cyclosporine on the transport of these two components were studied. The mutual influence between PUR and GAS, especially the effect of the latter on the former of the bidirectional transport were also investigated. The transport of 50 µg·mL-1 PUR in Caco-2 cells has no obvious directionality. While the transport of 100 and 200 µg·mL-1 PUR presents a strong directionality, and this directionality can be inhibited by verapamil and cyclosporine. When PUR and GAS were used in combination, GAS could increase the absorption of PUR while PUR had no obvious influence on GAS. Therefore, the compatibility of PUR and GAS is reasonable, and GAS can promote the transmembrane transport of PUR, the effect of which is similar to that of verapamil.

Roles and mechanisms of puerarin on cardiovascular disease：A review.

Cardiovascular diseases (CVDs) are now the leading cause of mortality and morbidity worldwide，resulting in a large global economic burden. Recently, complementary and alternative medicine, such as traditional Chinese medicine (TCM) have received great attention. Puerarin (Pue) is an isoflavone isolated from the roots of Pueraria lobata (Willd.) Ohwi (also named "Ge gen" in China), and is a versatile TCM herb used for the treatment of fever, diarrhea, diabetes mellitus CVDs and cerebrovascular diseases. Numerous lines ofin vitro studies, as well as in vivo animal experiments have established that Pue offers beneficial roles against the progression of atherosclerosis, ischemic heart diseases, heart failure hypertension and arrhythmia by inhibiting pathological processes, such as the mitigation of endothelium injury, protection against inflammation, the disturbance of lipid metabolism, protection against ischemic reperfusion injury, anti-myocardial remodeling and other effects. Here, we provide a systematic overview of the pharmacological actions and molecular targets of Pue in cardiovascular disease prevention and treatment, to provide insights into the therapeutic potential of Pue in treating cardiovascular diseases.

Metabolite identification of iridin in rats by using UHPLC-MS/MS and pharmacokinetic study of its metabolite irigenin.

Iridin, one of the main bioactive components isolated from Belamcanda chinensis (L.) DC, exerts various pharmacological activities, such as anti-inflammation, antioxidant, and antitumor. However, the metabolism and pharmacokinetics of iridin are still unknown. After 100 mg/kg administration of iridin orally, the plasma, urine, and fecal bio-samples from Sprague-Dawley (SD) rats were collected and detected by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The pharmacokinetics of the major metabolite irigenin (aglycon of iridin) and a total of thirteen metabolites of iridin were identified, including five metabolites in plasma, ten metabolites in urine, and six metabolites in feces. The most principal metabolic pathway of iridin was glucuronidation after demethylation and was mediated by UDP-glucuronosyltransferases (UGTs) 1A7, 1A8, 1A9 and 1A10. This study highlights the first-time investigation of the metabolism of iridin in vivo, and the pharmacokinetics of irigenin (the major metabolite of iridin) in rats. These results provide robust evidence for further research and clinical application of iridin.

A rapid ultra high performance liquid chromatography-tandem mass spectrometry method for the quantification of daidzein, its valine carbamate prodrug, and glucuronide in rat plasma samples: Comparison of the pharmacokinetic behavior of daidzine valine carbamate prodrugs.

Two valine carbamate prodrugs of daidzein were designed to improve its bioavailability. To compare the pharmacokinetic behavior of these prodrugs with different protected phenolic hydroxyl groups of daidzein, a rapid and sensitive method for simultaneous quantification of daidzein, its valine carbamate prodrug, and daidzein-7-O-glucuronide in rat plasma was developed and validated in this study. The samples were processed using a fast one-step protein precipitation method with methanol added to 50 µL of plasma and were analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry. To improve the selectivity, peak shape, and peak elution, several key factors, especially stationary phase and the composition of the mobile phase, were tested, and the analysis was performed using the Kinetex® C18 column (100 × 2.1 mm, 2.6 µm) within only 2.6 min under optimal conditions. The established method exhibited good linearity over the concentration range of 2.0-1000 ng/mL for daidzein, and 8.0-4000 ng/mL for the prodrug and daidzein-7-O-glucuronide. The accuracy of the quality control samples was between 95.5 and 110.2% with satisfactory intra- and interday precision (relative standard deviation values < 10.85%), respectively. This sensitive, rapid, low-cost, and high-throughput method was successfully applied to compare the pharmacokinetic behavior of different daidzein carbamate prodrugs.

LC-MS/MS analysis and pharmacokinetics of daidzein and its 7-O-glucuronide in rats after oral administration of 7-O-L-valyl carbamate prodrug.

Background: A valine carbamate prodrug (7-P) was designed to enhance the low bioavailability of daidzein due to its low water solubility and membrane permeability. Here, we developed a high-throughput HPLC-MS/MS method to measure daidzein and its 7-O-glucuronide after oral administration of daidzein or 7-P. Materials & methods: A HPLC-MS/MS method was validated and successfully applied to assess the pharmacokinetic behavior of daidzein and its 7-O-glucuronide after orally administrating daidzein or 7-P. The validated method on selectivity, linearity (r ≥ 0.995), precision (relative standard deviation <11.4%), accuracy (relative error <7.1%), extraction recovery (>92.4%), matrix effect (<8.2%) and stability were satisfied. Conclusion: The proposed economical, rapid and sensitive method will be an alternative analytical procedure for daidzein and its metabolite in biological samples.

A Novel Solid Nanocrystals Self-Stabilized Pickering Emulsion Prepared by Spray-Drying with Hydroxypropyl-ß-cyclodextrin as Carriers.

A drug nanocrystals self-stabilized Pickering emulsion (NSSPE) with a unique composition and microstructure has been proven to significantly increase the bioavailability of poorly soluble drugs. This study aimed to develop a new solid NSSPE of puerarin preserving the original microstructure of NSSPE by spray-drying. A series of water-soluble solid carriers were compared and then Box-Behnken design was used to optimize the parameters of spray-drying. The drug release and stability of the optimized solid NSSPE in vitro were also investigated. The results showed that hydroxypropyl-ß-cyclodextrin (HP-ß-CD), rather than solid carriers commonly used in solidification of traditional Pickering emulsions, was suitable for the solid NSSPE to retain the original appearance and size of emulsion droplets after reconstitution. The amount of HP-ß-CD had more influences on the solid NSSPE than the feed rate and the inlet air temperature. Fluorescence microscopy, confocal laser scanning microscopy and scanning electron microscopy showed that the reconstituted emulsion of the solid NSSPE prepared with HP-ß-CD had the same core-shell structure with a core of oil and a shell of puerarin nanocrystals as the liquid NSSPE. The particle size of puerarin nanocrystal sand interfacial adsorption rate also did not change significantly. The cumulative amount of released puerarin from the solid NSSPE had no significant difference compared with the liquid NSSPE, which were both significantly higher than that of puerarin crude material. The solid NSSPE was stable for 3 months under the accelerated condition of 75% relative humidity and 40 °C. Thus, it is possible todevelop the solid NSSPE preserving the unique microstructure and the superior properties in vitro of the liquid NSSPE for poorly soluble drugs.

Natural Isoflavones and Semisynthetic Derivatives as Pancreatic Lipase Inhibitors.

Obesity, now widespread all over the world, is frequently associated with some chronic diseases. Thus, there is a growing interest in the prevention and treatment of obesity. To date, the only antiobesity drug is orlistat, a natural product-derived pancreatic lipase (PL) inhibitor with some undesired side effects. In the last decades, many natural compounds or derivatives have been evaluated as potential PL inhibitors, and natural polyphenols are among the most promising for possible exploitation as antiobesity agents. However, few studies have been devoted to isoflavones. In this work, we report a study on the PL inhibitory properties of a small library of semisynthetic isoflavone derivatives together with the natural leads daidzein (1), genistein (2), and formononetin (3). In vitro lipase inhibition assay showed that 2 is the most promising PL inhibitor. Among synthetic isoflavones, the hydroxylated and brominated derivatives were more potent than their natural leads. Detailed studies through fluorescence measurements and kinetics of lipase inhibition showed that 2 and the bromoderivatives 10 and 11 have the greatest affinity for PL. Docking studies corroborated these findings highlighting the interactions between isoflavones and the enzyme, confirming that hydroxylation and bromination are useful modifications.

Metabolic tracking of isoflavones in soybean products and biosamples from healthy adults after fermented soybean consumption.

Fermentation may enhance the nutritional properties of foods by increasing metabolite bioactivity or bioavailability. This study explored the effect of fermentation on isoflavone bioavailability and metabolism. Isoflavone metabolites were tracked in foods and biospecimens of healthy adults after fermented soybean (FS) or non-fermented soybean (NFS) consumption in a randomized, controlled, crossover intervention study. The change in soybean isoflavones caused by fermentation resulted in faster absorption and higher bioavailability after consumption of FS. Although the urinary level of total isoflavone metabolites was similar after the consumption of the two diets, urinary genistein 7-O-sulfate was derived as a discriminant metabolite for the FS diet by partial least squares discriminant analysis. This study suggests that an isoflavone conjugate profile might be a more appropriate marker than total isoflavone levels for discriminating between the consumption of FS and NFS diets.

Developing neobavaisoflavone nanoemulsion suppresses lung cancer progression by regulating tumor microenvironment.

It is necessary to create novel, efficacious and harmless therapeutic strategy for lung cancer treatment. The application of nanoemulsion to specifically suppress cancer progression in the tumor microenvironment would be an effective therapy. Neobavaisoflavone (Neo) is an isoflavone isolated from Psoralea corylifolia L, possesses striking anti-inflammatory and anti-cancer effects. In our stduy, Neo significantly reduced reactive oxygen species (ROS) generation in the activated myofibroblast. Furthermore, a novel Neo nanoemulsion (nano-Neo) was prepared to improve Neo solubility and bioavailability. Nano-Neo showed more effectively anti-proliferative role in lung cancer cells. In addition, in vivo analysis further demonstrated that nano-Neo could effectively suppress tumor growth compared to the free Neo-treated mice without noticeable damage to major organs. Furthermore, nano-Neo treatment markedly reduced extracellular matrix (ECM) deposition in tumor samples by repressing transforming growth factor-ß (TGF-ß)/SMADs signaling pathway. Meanwhile, the activated immune microenvironment in tumor tissues was dramatically improved by nano-Neo through reducing regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) infiltration, as well as improving the count of natural killer (NK) cells and M2 macrophage phenotype switch to pro-inflammatory M1. In addition, we found that the prepared nano-Neo exerted promising tumor targeting efficiency with improved pharmacokinetic properties. Therefore, the novel approach to prepare nano-Neo introduced here might provide an effective strategy for lung cancer treatment with few adverse effects.

Effects of puerarin on the pharmacokinetics of astragaloside IV in rats and its potential mechanism.

Context: Puerarin and astragaloside IV (AS-IV) are sometimes used together for the treatment of disease in Chinese clinics, however, the drug-drug interaction between puerarin and AS-IV is still unknown.Objective: This study investigates the effects of puerarin on the pharmacokinetics of astragaloside IV in rats and clarifies its main mechanism.Materials and methods: The pharmacokinetic profiles of oral administration of astragaloside IV (20 mg/kg) in Sprague-Dawley rats, with or without pre-treatment of puerarin (100 mg/kg/day for 7 days) were investigated. The effects of puerarin on the transport and metabolic stability of AS-IV were also investigated using Caco-2 cell transwell model and rat liver microsomes.Results: The results showed that puerarin could significantly increase the peak plasma concentration (from 48.58 ± 7.26 to 72.71 ± 0.62 ng/mL), and decrease the oral clearance (from 47.5 ± 8.91 to 27.15 ± 9.27 L/h/kg) of AS-IV. The Caco-2 cell transwell experiments indicated that puerarin could decrease the efflux ratio of astragaloside IV from 1.89 to 1.26, and the intrinsic clearance rate of astragaloside IV was decreased by the pre-treatment with puerarin (34.8 ± 2.9 vs. 41.5 ± 3.8 µL/min/mg protein).Discussion and conclusions: These results indicated that puerarin could significantly change the pharmacokinetic profiles of astragaloside IV, via increasing the absorption of astragaloside IV or inhibiting the metabolism of astragaloside IV in rats.

Simultaneous quantification and rat pharmacokinetics of formononetin-7-O-ß-d-glucoside and its metabolite formononetin by high-performance liquid chromatography-tandem mass spectrometry.

Formononetin-7-O-ß-d-glucoside has been proved to have significant anti-inflammatory effect. To evaluate its rat pharmacokinetics, a rapid, sensitive, and specific liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of formononetin-7-O-ß-d-glucoside and its main metabolite formononetin in rat plasma. Samples were pretreated using a simple protein precipitation and the chromatographic separation was performed on a C18 column by a gradient elution using a mobile phase consisting of water and acetonitrile both containing 0.1% formic acid. Both analytes were detected using a tandem mass spectrometer in positive multiple reaction monitoring mode. The assay showed wide linear dynamic ranges of both 0.10-100 ng/mL, with acceptable intra- and inter-batch accuracy and precision. The lower limits of quantification were both 0.10 ng/mL using 50 µL of rat plasma for two analytes. The method has been successfully used to investigate the oral pharmacokinetic profiles of both analytes in rats. After oral administration of formononetin-7-O-ß-d-glucoside at the dose of 50 mg/kg, it was rapidly absorbed in vivo and metabolized to its metabolite formononetin. The plasma concentration-time profiles both showed double-peak phenomena, which would be attributed to the strong enterohepatic circulation of formononetin-7-O-ß-d-glucoside.

Pharmacokinetics, Tissue Distribution, and Druggability Prediction of the Natural Anticancer Active Compound Cytisine N-Isoflavones Combined with Computer Simulation.

Cytisine N-methylene-(5,7-dihydroxy-4'-methoxy)-isoflavone (CNF2) is a new compound isolated from the Chinese herbal medicine Sophora alopecuroides. Preliminary pharmacodynamic studies demonstrated its activity in inhibiting breast cancer cell metastasis. This study examined the pharmacokinetics, absolute bioavailability, and tissue distribution of CNF2 in rats, and combined computer-aided technology to predict the druggability of CNF2. The binding site of CNF2 and the breast cancer target human epidermal growth factor receptor-2 (HER2) were examined with molecular docking technology. Next, ACD/Percepta software was used to predict the druggability of CNF2 based on the quantitative structure-activity relationship (QSAR). Finally, a simple and effective HPLC method was used to determine plasma pharmacokinetics and tissue distribution of CNF2 in rats. Prediction and experimental results show that compared with the positive control HER2 inhibitor SYR127063, CNF2 has a stronger binding affinity with HER2, suggesting that its efficacy is stronger; and the structure of CNF2 complies with the Lipinski's Rule of Five and has good drug-likeness. The residence time of CNF2 in rats is less than 4 h, and the metabolic rate is relatively fast; But the absolute bioavailability of CNF2 in rats was 6.6%, mainly distributed in the stomach, intestine, and lung tissues, where the CNF2 contents were 401.20, 144.01, and 245.82 µg/g, respectively. This study constructed rapid screening and preliminary evaluation of active compounds, which provided important references for the development and further research of such compounds.

Sensitive and selective detection of puerarin based on the hybrid of reduced graphene oxide and molecularly imprinted polymer.

Puerarin, an important isoflavone, has been widely used for the treatment of angina and hypertension. In this work, we developed a novel electrochemical sensor for the detection of puerarin based on the hybrid of reduced graphene oxide (RGO) and molecularly imprinted polymer (MIP). The RGO/MIP sensor functions by target puerarin recognition and electro-oxidization via a two-proton and two-electron process, enabling the detection of puerarin with good selectivity and high sensitivity. The MIP layer was integrated on the surface of RGO by the electro-co-polymerization of o-phenylenediamine (monomer) and puerarin (template), resulting in high surface area, binding capacity, good conductivity and faster mass transfer. The nanostructure of the RGO/MIP hybrid was demonstrated using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Experimental conditions involved in the sensor fabrication process were evaluated. Under the optimized condition, a wide linear range (0.02 µM ∼ 40 µM) and a low detection limit (0.006 µM) were achieved. The sensor was applied to detect puerarin in human urine and injection samples, and the result was comparable with that of the gold standard method of high-performance liquid chromatography (HPLC), indicating a promise in the further application to pharmacokinetics or therapeutic drug monitoring.

Simultaneous quantitation of glycyrrhetic acid and puerarin in plasma using ultra flow liquid chromatography tandem mass spectrometry and application in a pharmacokinetics study in healthy and alcoholic liver injury rats.

A rapid, sensitive, and accurate ultra flow liquid chromatography tandem mass spectrometry (UFLC-MS/MS ) method was developed and validated for simultaneous quantitation of glycyrrhetic acid and puerarin in plasma derived from healthy and alcoholic liver injury rats. Plasma samples from healthy and model rats were deproteinated with methanol using liquiritin as an internal standard. Chromatography separation was performed by a Waters BEH (ethylene-bridged hybrid) C18 column (2.1 × 50 mm; 1.7 µm) using a gradient elution from acetonitrile and water (containing 0.1% formic acid) and at a flow rate of 0.4 mL/min. Quantitation was performed on a Triple Quad 4500 tandem mass spectrometer coupled with an electrospray ionization source in negative multiple reaction monitoring mode. Specificity, carryover, dilution integrity, recovery, linearity, precision and accuracy, matrix effect, and stability were within acceptable limits. The newly established method was successfully applied to a pharmacokinetics study to investigate glycyrrhetic acid and puerarin in healthy and alcoholic liver injury rats.

Use of Physiologically Based Kinetic Modeling to Predict Rat Gut Microbial Metabolism of the Isoflavone Daidzein to S-Equol and Its Consequences for ERα Activation.

SCOPE: To predict gut microbial metabolism of xenobiotics and the resulting plasma concentrations of metabolites formed, an in vitro-in silico-based testing strategy is developed using the isoflavone daidzein and its gut microbial metabolite S-equol as model compounds. METHODS AND RESULTS: Anaerobic rat fecal incubations are optimized and performed to derive the apparent maximum velocities (Vmax ) and Michaelis-Menten constants (Km ) for gut microbial conversion of daidzein to dihydrodaidzein, S-equol, and O-desmethylangolensin, which are input as parameters for a physiologically based kinetic (PBK) model. The inclusion of gut microbiota in the PBK model allows prediction of S-equol concentrations and slightly reduced predicted maximal daidzein concentrations from 2.19 to 2.16 µm. The resulting predicted concentrations of daidzein and S-equol are comparable to in vivo concentrations reported. CONCLUSION: The optimized in vitro approach to quantify kinetics for gut microbial conversions, and the newly developed PBK model for rats that includes gut microbial metabolism, provide a unique tool to predict the in vivo consequences of daidzein microbial metabolism for systemic exposure of the host to daidzein and its metabolite S-equol. The predictions reveal a dominant role for daidzein in ERα-mediated estrogenicity despite the higher estrogenic potency of its microbial metabolite S-equol.

Enrichment of Polyglucosylated Isoflavones from Soybean Isoflavone Aglycones Using Optimized Amylosucrase Transglycosylation.

Isoflavones in soybeans are well-known phytoestrogens. Soy isoflavones present in conjugated forms are converted to aglycone forms during processing and storage. Isoflavone aglycones (IFAs) of soybeans in human diets have poor solubility in water, resulting in low bioavailability and bioactivity. Enzyme-mediated glycosylation is an efficient and environmentally friendly way to modify the physicochemical properties of soy IFAs. In this study, we determined the optimal reaction conditions for Deinococcus geothermalis amylosucrase-mediated α-1,4 glycosylation of IFA-rich soybean extract to improve the bioaccessibility of IFAs. The conversion yields of soy IFAs were in decreasing order as follows: genistein > daidzein > glycitein. An enzyme quantity of 5 U and donor:acceptor ratios of 1000:1 (glycitein) and 400:1 (daidzein and genistein) resulted in high conversion yield (average 95.7%). These optimal reaction conditions for transglycosylation can be used to obtain transglycosylated IFA-rich functional ingredients from soybeans.

Fabrication of ultra-small nanocrystals by formation of hydrogen bonds: In vitro and in vivo evaluation.

Poor water solubility and low bioavailability hinder the clinical application of about 70% of newly synthesized compounds. Nanocrystal technology has become a preferred way to improve bioavailability by improving solubility. However, it remains challenging to produce nanocrystals with ultra-small particle sizes to further enhance the extent of bioavailability. Herein, we constructed ultra-small puerarin nanocrystals (Pue-NCs) (20-40 nm) via formation of hydrogen bond during HPH. We confirmed the formation of hydrogen bonds by 1H NMR and FTIR, and observed the distribution of polymer chains by SEM and TEM. The absorption mechanisms were studied in Caco-2 cell monolayers, and the results showed that the major transport mechanism for puerarin was passive diffusion, meanwhile, for Pue-NCs, the passive transport and micropinocytosis-mediated endocytosis coexisted. The absolute bioavailability of Pue-NCs was 35.28%, which was 11.54 folds compared to that of puerarin. Therapeutic equivalence was demonstrated between Pue-NCs and puerarin injection at 50 mg/kg and 15 mg/kg, respectively, in isoproterenol-induced myocardial ischemia model. This study provides a novel strategy for preparing ultra-small nanocrystals by HPH to increase bioavailability of poorly soluble drugs.

An ultra high performance liquid chromatography with tandem mass spectrometry method for simultaneous determination of thirteen components extracted from Radix Puerariae in rat plasma and tissues: Application to pharmacokinetic and tissue distribution study.

A rapid and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was established and validated for simultaneous determination of thirteen bioactive components (gallic acid, protocatechuic acid, puerarin, p-hydroxycinnamic acid, daidzin, ononin, daidzein, naringenin, genistein, apigenin, formononetin, biochanin A, and ß-sitosterol) of Radix Puerariae extract in rat plasma and tissues. The plasma and tissues samples were pretreated by protein precipitation extraction, and umbelliferone and rutin were used as internal standards. Sample separation was performed on a ZORBAX RRHD Eclipse plus C18 column (2.1 mm × 50 mm, 1.8 µm, Agilent) with a mobile phase consisting of methanol-water (containing 0.1% formic acid). The mass spectrometry analysis was conducted in positive and negative ionization modes with multiple reaction monitoring. The lower limit of quantitation range for the 13 analytes was 0.2-35 ng/mL. The intra- and inter-day precision of all the analytes were less than 10.92%, with an accuracy ranging from -13.10 to 11.96%. Both the recovery and matrix effect were within acceptable limits. This method was successfully applied to pharmacokinetic and tissue distribution study of the 13 bioactive components in rats after oral administration of R. Puerariae extract.