Construction of ROS-Responsive Hyaluronic Acid Modified Paclitaxel and Diosgenin Liposomes and Study on Synergistic Enhancement of Anti-Ovarian Cancer Efficacy.

Purpose: Ovarian cancer is a fatal gynecologic malignancy with a high rate of abdominal metastasis. Chemotherapy still has a poor clinical prognosis for ovarian cancer patients, with cell proliferation and angiogenesis leading to invasion, migration, and recurrence. To overcome these obstacles, we constructed a novel HA-modified paclitaxel and diosgenin liposome (PEG-TK-HA-PDLPs) using two novel functional materials, DSPE-PEG2000-HA and DSPE-PEG2000-TK-PEG5000, to specifically deliver the drugs to the tumor site in order to reduce OC cell proliferation and anti-angiogenic generation, thereby inhibiting invasion and migration. Methods and Results: PEG-TK-HA-PDLPs were prepared by film dispersion, with ideal physicochemical properties and exhibits active targeting for enhanced cellular uptake. The ZIP synergy score for PTX and Dios was calculated using the online SynergyFinder software to be 3.15, indicating synergy. In vitro results showed that PEG-TK-HA-PDLPs were highly cytotoxic to ID8 cells, induced ID8 cell apoptosis, and inhibited ID8 cell migration and invasion. In vivo studies showed that PEG-TK-HA-PDLPs could prolong the circulation time in the blood, accumulate significantly in the tumor site, and effectively fight against angiogenesis with significant anti-tumor effects. Conclusion: The production of PEG-TK-HA-PDLPs is an effective strategy for the treatment of OC.

A novel UPLC-MS/MS method for simultaneous quantification of trigonelline, 4-hydroxyisoleucine, and diosgenin from Trigonella foenum-graecum extract: Application to pharmacokinetic study in healthy and type 2 diabetic rats.

Trigonelline (TR), 4-hydroxyisoleucine (4-HI), and diosgenin (DG) are the main bioactives of the purified standardized extract of the popular plant Trigonella foenum-graecum L. (TFG), and it has been proven effective for the treatment of various diseases. However, to the best of our knowledge, no study has investigated the pharmacokinetic parameters of purified standardized T. foenum-graecum extract in normal and diabetic Wistar rats. The present study has developed and validated a rapid, reliable, and sensitive simultaneous ultra-performance liquid chromatography MS method to estimate these bioactives. The chromatographic separation was achieved using methanol, acetonitrile, and 0.1% formic acid with the ideal gradient flow system on a BEH Shield RP 18 column. A positive electrospray ionization mode was selected to estimate m/z values of TR (138.14 > 94.63), 4-HI (148.19 > 74.08), and DG (415.54 > 271.33). The method was robust and reproducible over the linearity range of 60-5000, 6-5000, and 15-5000 ng/mL for TR, 4-HI, and DG, respectively. Using this novel validated method, we investigated the pharmacokinetic parameters of bioactives using Phoenix WinNonlin version 8.0 (Certera) in normal and diabetic rats. The assay was successfully applied for the estimation of pharmacokinetic parameters using noncompartmental analysis. This investigation shows that the absorption rate increased, whereas distribution and elimination processes slowed down in diabetic rats compared with normal rats.

Soluplus-Mediated Diosgenin Amorphous Solid Dispersion with High Solubility and High Stability: Development, Characterization and Oral Bioavailability.

BACKGROUND AND PURPOSE: The traditional Chinese medicine, diosgenin (Dio), has attracted increasing attention because it possesses various therapeutic effects, including anti-tumor, anti-infective and anti-allergic properties. However, the commercial application of Dio is limited by its extremely low aqueous solubility and inferior bioavailability in vivo. Soluplus, a novel excipient, has great solubilization and capacity of crystallization inhibition. The purpose of this study was to prepare Soluplus-mediated Dio amorphous solid dispersions (ASDs) to improve its solubility, bioavailability and stability. METHODS: The crystallization inhibition studies were firstly carried out to select excipients using a solvent shift method. According to solubility and dissolution results, the preparation methods and the ratios of drug to excipient were further optimized. The interaction between Dio and Soluplus was characterized by differential scanning calorimetry (DSC), fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), powder X-ray diffraction (PXRD) and molecular docking. The pharmacokinetic study was conducted to explore the potential of Dio ASDs for oral administration. Furthermore, the long-term stability of Dio ASDs was also investigated. RESULTS: Soluplus was preliminarily selected from various excipients because of its potential to improve solubility and stability. The optimized ASDs significantly improved the aqueous solubility of Dio due to its amorphization and the molecular interactions between Dio and Soluplus, as evidenced by dissolution test in vitro, DSC, FT-IR spectroscopy, SEM, PXRD and molecular docking technique. Furthermore, pharmacokinetic studies in rats revealed that the bioavailability of Dio from ASDs was improved about 5 times. In addition, Dio ASDs were stable when stored at 40°C and 75% humidity for 6 months. CONCLUSION: These results indicated that Dio ASDs, with its high solubility, high bioavailability and high stability, would open a promising way in pharmaceutical applications.

Diosgenin, a steroidal saponin, and its analogs: Effective therapies against different chronic diseases.

BACKGROUND: Chronic diseases are a major cause of mortality worldwide, and despite the recent development in treatment modalities, synthetic drugs have continued to show toxic side effects and development of chemoresistance, thereby limiting their application. The use of phytochemicals has gained attention as they show minimal side effects. Diosgenin is one such phytochemical which has gained importance for its efficacy against the life-threatening diseases, such as cardiovascular diseases, cancer, nervous system disorders, asthma, arthritis, diabetes, and many more. AIM: To evaluate the literature available on the potential of diosgenin and its analogs in modulating different molecular targets leading to the prevention and treatment of chronic diseases. METHOD: A detailed literature search has been carried out on PubMed for gathering information related to the sources, biosynthesis, physicochemical properties, biological activities, pharmacokinetics, bioavailability and toxicity of diosgenin and its analogs. KEY FINDINGS: The literature search resulted in many in vitro, in vivo and clinical trials that reported the efficacy of diosgenin and its analogs in modulating important molecular targets and signaling pathways such as PI3K/AKT/mTOR, JAK/STAT, NF-κB, MAPK, etc., which play a crucial role in the development of most of the diseases. Reports have also revealed the safety of the compound and the adaptation of nanotechnological approaches for enhancing its bioavailability and pharmacokinetic properties. SIGNIFICANCE: Thus, the review summarizes the efficacy of diosgenin and its analogs for developing as a potent drug against several chronic diseases.

Pickering stabilized nanocellulose-alginate: A diosgenin-mediated delivery of quinalizarin as a potent cyto-inhibitor in human lung/breast cancer cell lines.

The current study explores the facile fabrication of multilayer self-assembled electrostatic oil-in-water Pickering emulsions (PEs) using quaternized nanocellulose (Q-NC) and diosgenin-conjugate alginate (DGN-ALG) particles as stabilizers to form hydrocolloid nanocarriers. The conditions of formulation such as storage time, pH, temperature and salt effect on the emulsion stability were evaluated. The results deduced showed good emulsion droplet stability over a period of 30 days. Morphological analysis revealed the hydrodynamic sizes of the PE droplets to be spherically shaped with an average diameter of 150 ± 3.51 nm. Creaming index, wettability and critical aggregation concentrations (CAC) as well as chemical characterization of the PEs were examined. In vitro release kinetics of encapsulated quinalizarin as a model drug was investigated with a determined cumulative drug release (CDR) of 89 ± 1.21% in simulated pH blood medium of pH 7.4. In addition, cellular internalization of the PEs was studied via confocal microscopy imaging and showed high cellular uptake. Also, evaluated in vitro cytotoxicity by MTT assay demonstrated excellent anticancer activity in human lung (A549) and breast (MCF-7) cancer cell lines.

Therapeutic Potential of Diosgenin and Its Major Derivatives against Neurological Diseases: Recent Advances.

Diosgenin (DG), a well-known steroidal sapogenin, is present abundantly in medicinal herbs such as Dioscorea rhizome, Dioscorea villosa, Trigonella foenum-graecum, Smilax China, and Rhizoma polgonati. DG is utilized as a major starting material for the production of steroidal drugs in the pharmaceutical industry. Due to its wide range of pharmacological activities and medicinal properties, it has been used in the treatment of cancers, hyperlipidemia, inflammation, and infections. Numerous studies have reported that DG is useful in the prevention and treatment of neurological diseases. Its therapeutic mechanisms are based on the mediation of different signaling pathways, and targeting these pathways might lead to the development of effective therapeutic agents for neurological diseases. The present review mainly summarizes recent progress using DG and its derivatives as therapeutic agents for multiple neurological disorders along with their various mechanisms in the central nervous system. In particular, those related to therapeutic efficacy for Parkinson's disease, Alzheimer's disease, brain injury, neuroinflammation, and ischemia are discussed. This review article also critically evaluates existing limitations associated with the solubility and bioavailability of DG and discusses imperatives for translational clinical research. It briefly recapitulates recent advances in structural modification and novel formulations to increase the therapeutic efficacy and brain levels of DG. In the present review, databases of PubMed, Web of Science, and Scopus were used for studies of DG and its derivatives in the treatment of central nervous system diseases published in English until December 10, 2019. Three independent researchers examined articles for eligibility. A total of 150 articles were screened from the above scientific literature databases. Finally, a total of 46 articles were extracted and included in this review. Keywords related to glioma, ischemia, memory, aging, cognitive impairment, Alzheimer, Parkinson, and neurodegenerative disorders were searched in the databases based on DG and its derivatives.

Polyphyllin I activates AMPK to suppress the growth of non-small-cell lung cancer via induction of autophagy.

Polyphyllin I (PPI), a bioactive constituent extracted from the rhizomes of Paris polyphylla, is cytotoxic to several cancer types. This study was designed to explore whether PPI prevents non-small-cell lung cancer (NSCLC) growth and to investigate the molecular mechanism. AMP-activated protein kinase (AMPK) has been implicated in the activation of autophagy in distinct tissues. In cultured human NSCLC cell lines, PPI induces autophagy by activating AMPK and then inhibiting mTOR signaling in a concentration-dependent manner. Furthermore, the activation of autophagy induced by PPI was reversed by the AMPK inhibitor compound C. Computational docking showed that PPI directly interacted with the allosteric drug and metabolite site of AMPK to stabilize its activation. Microscale thermophoresis and Drug Affinity Responsive Targeting Stability (DARTS) assay further confirmed the high affinity between PPI and AMPK. In vivo studies indicated that PPI suppressed the growth of NSCLC and increased the levels of LC3-II and phosphorylated AMPK in tumors isolated from a xenograft model of NSCLC in mice. Moreover, PPI exhibited favorable pharmacokinetics in rats. In summary, PPI conclusively acts as a direct AMPK activator to induce cell autophagy which inhibits the growth of NSCLC cells. In the future, PPI therapy should be applied to treat patients with NSCLC.

Polyphosphazene-based nanocarriers for the release of agrochemicals and potential anticancer drugs.

The synthesis and characterisation of novel polyphosphazene nanocarriers, based on hydrophilic polyalkylene oxide Jeffamine M1000 and hydrophobic steroids with a glycinate linker for pH-controlled release of diosgenin and two brassinosteroids (DI31 and S7) with agrochemical and potential anticancer activity, is hereby described. Polyphosphazenes carrying approximately 17 wt% of DI31 or S7 self-assembled in water to form 120-150 nm nanoaggregates, which showed an excellent plant growth effect on radish cotyledons due to sustained delivery of approximately 30% of the agrochemicals after 4 days. Cytotoxic evaluation showed that all polymers carrying steroids and Jeffamine M1000 resulted in strong to moderate toxicity to MCF-7 cancer cells and were non-toxic to primary human lung fibroblast cells at 0.1 to 0.025 mg mL-1. Thus, DI31 and S7 bearing polymers applied at 10-4 to 10-6 mg mL-1 for delivery of recommended DI31 or S7 quantities to crops should be harmless to humans. Particularly, DI31 and S7 bearing polymers with strong cytotoxicity on MCF-7 and non-toxicity on primary human lung fibroblasts, good cell uptake after 6 hours, proper hydrodynamic sizes between 100 and 200 nm, and slow sustained release of cytotoxic drugs (DI31, S7) in acidic conditions might potentiate their accumulation in cancer tissues with good antitumour effects and minor side effects. These results demonstrated that preparation of brassinosteroid bearing polymers is a promising strategy for the preparation of better agrochemicals with reduced pollutant impact on sustainable agriculture and potential anticancer formulations based on analogues of brassinosteroids.

Influence of Nutritional Status on the Absorption of Polyphyllin I, an Anticancer Candidate from Paris polyphylla in Rats.

BACKGROUND AND OBJECTIVES: Protein-calorie malnutrition (PCM) is one of the most suffered complications in cancer patients. Polyphyllin I (PPI), a saponin isolated from rhizome of Paris polyphylla, is a potential candidate in cancer therapy. In this study, the influence of nutritional status on the absorption of PPI in rats was explored after oral administration. METHODS: PCM rats, namely mal-nourished (MN) rats, were induced from well-nourished (WN) rats by caloric restriction protocol. Intestinal absorption of PPI in WN and MN rats was evaluated by pharmacokinetic and intestinal perfusion methods. The potential mechanisms between two groups were investigated on the basis of intestinal permeability, intestinal efflux and PPI's depletions in vivo. The intestinal permeability was analyzed by determining the concentration of paracellular marker transport in serum and the expression of junction proteins in intestine. The intestinal efflux was evaluated through comparing the protein level of P-glycoprotein (P-gp) in intestine, and the depletions of PPI and/or generation of its metabolites in liver and intestines were analyzed by liquid chromatography triple quadrupole mass spectrometry (LC-MS/MS) method. RESULTS: Compared to WN rats, the oral systemic exposure of PPI was significantly increased in MN rats, evidenced by significant enhancement of maximum plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC0-60h) by more than 2.51- and 3.71-folds as well as terminal elimination half-life (t1/2) prolonged from to 7.3 to 14.1 h. Further studies revealed that the potential mechanism might be associated with combined contribution of improved intestinal absorption and depressed deglycosylation of PPI in MN rats. Furthermore, enhanced intestinal absorption of PPI was benefited from increased intestinal permeability and decreased intestinal efflux in MN rats. Meanwhile, the former manifested as increased transport of paracellular marker and decreased junction proteins levels, while the later evidenced by reduced P-gp expression. CONCLUSIONS: The oral exposure of PPI was enhanced in MN rats, which suggested that nutritional status alters the absorption of PPI, and thus the dosage of PPI should be modified during the treatment of cancer patient with PCM.

Plasma Creatine Kinetics After Ingestion of Microencapsulated Creatine Monohydrate with Enhanced Stability in Aqueous Solutions.

Creatine monohydrate represents one of the largest sports supplement markets. Enhancing creatine (CRE) stability in aqueous solutions, such as with microencapsulation, represents innovation potential. Ten physically active male volunteers were randomly assigned in a double-blind design to either placebo (PLA) (3-g maltodextrin; n = 5) or microencapsulated CRE (3-g creatine monohydrate; n = 5) conditions. Experimental conditions involved ingestion of the samples in a 70-mL ready-to-drink format. CRE was delivered in a novel microencapsulation matrix material consisting entirely of hydrolyzed milk protein. Three hours after ingestion, plasma creatine concentrations were unchanged during PLA, and averaged ∼45 µM. During CRE, plasma creatine concentration peaked after 30 min at 101.6 ± 14.9 µM (p < 0.05), representing a 2.3-fold increase over PLA. Thereafter, plasma creatine concentration gradually trended downwards but remained significantly elevated (∼50% above resting levels) 3 hr after ingestion. These results demonstrate that the microencapsulated form of creatine monohydrate reported herein remains bioavailable when delivered in aqueous conditions, and has potential utility in ready-to-drink formulations for creatine supplementation.

Metabolic profile and pharmacokinetics of polyphyllin I, an anticancer candidate, in rats by UPLC-QTOF-MS/MS and LC-TQ-MS/MS.

Polyphyllin I (PPI), a natural steroidal saponin originating from rihzome of Paris polyphylla, is a potential anticancer candidate. Previous pharmacokinetics study showed that the oral bioavailability of PPI was very low, which suggested that certain amount of PPI might be metabolized in vivo. However, to date, information regarding the final metabolic fates of PPI is very limited. In this study, metabolites of PPI and their pharmacokinetics in rats were investigated using UPLC-QTOF-MS/MS and LC-TQ-MS/MS. A total of seven putative metabolites, including six phase I and one phase II metabolites, were detected and identified with three exact structures by comparison with authentic standards for the first time. Oxidation, deglycosylation and glucuronidation were found to be the major metabolic processes of the compound in rats. The pharmacokinetics of prosapogenin A, trillin and diosgenin, three deglycosylation metabolites of PPI with definite anticancer effects, were further studied, which suggested that the metabolites underwent a prolonged absorption and slower elimination after intragastric administration of PPI at the dose of 500 mg/kg. This study provides valuable and new information on the metabolic fate of PPI, which will be helpful in further understanding its mechanism of action.

Dioscin protects against ANIT-induced cholestasis via regulating Oatps, Mrp2 and Bsep expression in rats.

Alpha-naphthylisothiocyanate (ANIT) is a toxicant that is widely used in rodents to model human intrahepatic cholestasis. The aim of the study is to investigate whether effects of dioscin on ANIT-induced cholestasis are related to changes in expression of hepatic transporters in rats. Effects of dioscin on cholestasis were examined by histology and biochemical marker levels. The functional changes of hepatic transporters were determined by in vitro, in situ and in vivo. qRT-PCR and western blot were used to assess the expression of hepatic transporters in cholestatic rats. Dioscin administration could ameliorate cholestasis, as evidenced by reduced biochemical markers as well as improved liver pathology. The uptakes of organic anion transporting polypeptide (Oatp) substrates were altered in liver uptake index in vivo, perfused rat liver in situ and isolated rat hepatocytes in vitro in cholestasis rats. qRT-PCR and western blot analysis indicated co-treatment of ANIT with dioscin prevented the adaptive down-regulation of Oatp1a1, 1b2, and prompted the up-regulation of Oatp1a4, multidrug resistance-associated protein (Mrp) 2 and bile salt export pump (Bsep). In addition, concerted effects on Mrp2 and Bsep occurred through up-regulation of small heterodimer partner by activating farnesoid X receptor. Dioscin might prevent impairment of hepatic function by restoring hepatic transporter expression.

A new quantitation method of protodioscin by HPLC-ESI-MS/MS in rat plasma and its application to the pharmacokinetic study.

A specific high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS method) was established for determining the concentration of protodioscin (PG) in rat plasma after intragastric administration of its standard form. Ginsenoside Rb1 was selected as the internal standard (IS). The plasma sample was prepared using one-step deproteinization procedure by adding three parts of acetonitrile to precipitate proteins. The chromatographic separation was accomplished on an Inersil ODS-3 C18 column (250 × 4.6 mm, 5 µm) with a mobile phase composed with acetonitrile and water containing 0.1% formic acid under a gradient elution mode at a flow rate of 1 mL min(-1). A 3:1 portion of the eluent after a microsplit was detected on a triple quadrupole tandem mass spectrometer coupled with electrospray ionization (ESI) in positive ion and multiple reaction monitoring (MRM) scanning modes. The mass transitions were selected as 888.1 → 1050.2 for PG and 948.2 → 1110.3 for IS, respectively. After careful validation, the plasma samples were always stable under different storage conditions. These analytical results rendered sensitive, selective, and reliable values by this established method which displayed high accuracy, adequate extracted recoveries, and almost negligible matrix effects. This method was applied to the pharmacokinetic studies on PG level in the rat plasma and its pharmacokinetic effect. The results of our studies suggest that the present method may be a useful tool for further clinical study of PG.

Transmembrane transport of steviol glucuronide and its potential interaction with selected drugs and natural compounds.

Steviol glucuronide (SVG) is the major metabolite derived from steviol, the aglycone of stevioside and rebaudioside A. After the ingestion of stevioside and rebaudioside A, SVG is formed and excreted into the urine in humans. In the present study, transporter mediated efflux and uptake of SVG was investigated in order to understand molecular mechanisms underlying its renal clearance. Results showed that SVG was not a substrate of efflux transporters BCRP, MRP2, MATE1 or P-gp. In contrast, OAT3 played a predominant role in the uptake of SVG in comparison to OATP1B1, OATP1B3, or OATP2B1. Quercetin, telmisartan, diclofenac, and mulberrin displayed a relatively strong inhibition against OAT3 mediated uptake of SVG with IC50 values of 1.8, 2.9, 8.0, and 10.0 µM, respectively. Because OAT3 is a major uptake transporter in the kidney, inhibition of OAT3 activity may alter SVG's renal clearance by drugs and natural compounds that are used concomitantly with stevia leaf extracts.

UPLC-QTOF-MS identification of metabolites in rat biosamples after oral administration of Dioscorea saponins: a comparative study.

ETHNOPHARMACOLOGICAL RELEVANCE: Among the 49 species of the genus Dioscorea distributed in China, Dioscorea nipponica Makino (DN), Dioscorea panthaica Prain et Burkill (DP), and Dioscorea zingiberensis C. H. Wright (DZ) possess more or less similar traditional therapeutic actions, such as activating blood, relieving pain, and dispersing swelling; they have been used as folk medicine in China since 1950s. The modern pharmaceutical industry has developed these three species as herbal medicines that have been used for decades for treating cardiovascular diseases. However, there is no available information in the literature explaining how their chemical components are converted and interrelated in vivo to support their efficacies. The present study aimed to a) compare the metabolic profiles of saponins from DN, DP and DZ, which are considered to be their bioactive components, and b) to compare the changes in sustained levels of metabolites from rat biosamples. MATERIAL AND METHODS: Total saponins (TS) from each of the three species, and four individual saponins, namely protodioscin (PD), pseudoprotodioscin (PSD), dioscin (DC) and diosgenin (DG), were given to rats by oral administration. Chemical profiles of the rats' plasma, urine and feces were monitored 1-36 h. A UPLC-QTOF-MS based method was performed to identify the absorbed constituents and their metabolic products in rat biosamples (i.e., blood, urine, and feces); the ratio of peak area of major saponins to that of internal standard was calculated and plotted versus time to characterize the sustained levels of saponins in biosamples. RESULTS: Totally 10 saponin-related compounds were detected in rat plasma, 10 in rat urine and 18 in rat feces. The results indicated that formation of diosgenin by desugarization was the main pathway by which steroidal glycosides were metabolized. Other types of bio-transformation were found among glycosides and aglycones, such as ring cyclization through loss of 26-O-glucosyl, substitution of ß-D-glucopyranosyl for α-L-rhamnopyrannosyl, hydrogenation of diosgenin at 5(6)-double bond, and hydration of 20(22)-double bond. Generally, the metabolic profiles of DN and DP were shown to be quite similar, but different from that of DZ. However, some particular similarities and connections were found among these three TS. Diosgenin was one of the main metabolites commonly found in plasma and feces (excluding urine), from all groups receiving different TS, as well as individual saponins; this is likely to be one of the bioactive constituents playing an essential role in cardioprotective efficacy. Furostane-type saponins in TS of DN, DP or DZ, such as PD, protogracillin, parvifloside, protodeltonin and protobioside, showed fast absorption into blood (<1h), but were maintained for a relatively short period (mostly<8h), while the spirostane-type saponin and sapogenin (DC and DG, respectively), were absorbed into circulation more slowly (>1h), but increased gradually and lasted longer (>36h). These two patterns suggest that the therapeutic effect of these Dioscorea saponins is achieved through a complex, multi-step process over time. In addition, it appears that PD, PSD, and DC contained in DN and DP were transformed into certain glycosides originally found in DZ but not in DN or DP (protodeltonin, deltonin, trillin, and progenin II), which might indicate another linkage among these three species. CONCLUSION: These similarities and connections described above constitute evidence supporting similarity in efficacy of these three herbs from the perspective of metabolism. The UPLC-QTOF-MS based method is accurate and efficient for analyzing metabolic changes in rat biosamples over time.

Study on the pharmacokinetics profiles of polyphyllin I and its bioavailability enhancement through co-administration with P-glycoprotein inhibitors by LC-MS/MS method.

Polyphyllin I (PPI), one of the steroidal saponins in Paris polyphylla, is a promising natural anticancer candidate. Although the anticancer activity of PPI has been well demonstrated, information regarding the pharmacokinetics and bioavailability is limited. In this study, a series of reliable and rapid liquid chromatography-tandem mass spectrometry methods were developed and successfully applied to determinate PPI in rat plasma, cell incubation media and cell homogenate. Then the pharmacokinetics of PPI in rats was studied and the result revealed that PPI was slowly eliminated with low oral bioavailability (about 0.62%) at a dose of 50 mg/kg, and when co-administrated with verapamil (VPL) and cyclosporine A (CYA), the oral bioavailability of PPI could increase from 0.62% to 3.52% and 3.79% respectively. In addition, in vitro studies showed that with the presence of VPL and CYA in Caco-2 cells, the efflux ratio of PPI decreased from 12.5 to 2.96 and 2.22, and the intracellular concentrations increased 5.8- and 5.0-fold respectively. These results demonstrated that PPI, with poor oral bioavailability, is greatly impeded by P-gp efflux, and inhibition of P-gp can enhance its bioavailability.

Effect of liquid crystals with cyclodextrin on the bioavailability of a poorly water-soluble compound, diosgenin, after its oral administration to rats.

Diosgenin, found in wild yam (Dioscorea villosa), has been shown to ameliorate diabetes and hyperlipidemia, increase cell proliferation in a human 3D skin model, and inhibits melanin production in B16 melanoma cells. It is also an active element in cosmeceutical and dietary supplements. Although the bioavailability of diosgenin is low due to its poor solubility and intestinal permeability, it was subsequently improved using a ß-cyclodextrin (ß-CD) inclusion complex. Recently liquid crystals (LCs) were shown to enhance the bioavailability of poorly water-soluble drugs. The purpose in the present study was to prepare diosgenin LCs and investigate the interaction between LC and ß-CD in order to improve its bioavailability of diosgenin. Crystallinity and particle diameters of LCs in water were determined by small angle X-ray scattering (SAXS) and Zetasizer. Pharmacokinetic parameters were calculated using the plasma content of diosgenin after its oral administration to Wistar rats. Regarding the formation of glyceryl monooleate (GMO) and phytantriol (PHY) LC, SAXS patterns showed the hexagonal and cubic phases, respectively. Bioavailability was significantly enhanced after oral administration of LCs prepared by GMO than after diosgenin alone. The bioavailability was further improved with the combination of LC and ß-CD than LC and water.

Liquid chromatography tandem mass spectrometry in study of the pharmacokinetics of six steroidal saponins in rats.

A rapid, simple and sensitive high-performance liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous determination of six main steroidal saponins in Paris polyphylla in rat plasma. Ginsenoside Rg3 was selected as the internal standard (IS). Plasma samples were pretreated with protein precipitation, and the separation was achieved on a reverse phase Agilent poroshell120 EC-C18 column using a gradient mobile phase system of acetonitrile-water containing 0.1% formic acid. The triple quadruple mass spectrometer was set in negative electrospray ionization mode and multiple reaction monitoring (MRM) was used for six steroidal saponins quantification. The precursors to produce ion transitions monitored for polyphyllin I, polyphyllin II, polyphyllin VI, polyphyllin VII, dioscin, gracillin and IS were m/z 899.5>853.4, 1059.5>1013.5, 783.4>737.4, 1075.5>1029.5, 913.5>867.4, 929.5>883.4 and 819.5>783.4, respectively. The intra- and inter-day precisions (RSD%) were less than 13% and the average extraction recoveries ranged from 85% to 97.0% for each analyte. Six steroidal saponins were proved to be stable during sample storage, preparation and analytical procedures. The established method was employed for simultaneous quantification and successfully used for the first time for the pharmacokinetics evaluation of the six main compounds after intragastric administration of P. polyphylla extract in Sprague-Dawley rats.

Characterization of in vitro ADME properties of diosgenin and dioscin from Dioscorea villosa.

Dioscorea villosa (wild yam) is native to North America and has been widely used as a natural alternative for estrogen replacement therapy to improve women's health as well as to treat inflammation, muscle spasm, and asthma. Diosgenin and dioscin (glycoside form of diosgenin) are reported to be the pharmacologically active compounds. Despite the reports of significant pharmacological properties of dioscin and diosgenin in conditions related to inflammation, cancer, diabetes, and gastrointestinal ailments, no reports are available on ADME properties of these compounds. This study was carried out to determine ADME properties of diosgenin and dioscin and their effects on major drug metabolizing enzymes (CYP 3A4, 2D6, 2C9, and 1A2). The stability was determined in simulated gastric and intestinal fluids (SGF, pH 1.2 and SIF, pH 6.8), and intestinal transport was evaluated in Caco-2 model. Phase I and phase II metabolic stability was determined in human liver microsomes and S9 fractions, respectively. Quantitative analysis of dioscin and diosgenin was performed by UPLC-MS system. Dioscin degraded up to 28.3 % in SGF and 12.4 % in SIF, which could be accounted for by its conversion to diosgenin (24.2 %. in SGF and 2.4 % in SIF). The depletion of diosgenin in SGF and SIF was < 10 %. Diosgenin was stable in HLM but disappeared in S9 fraction with a half-life of 11.3 min. In contrast, dioscin was stable in both HLM and S9 fractions. Dioscin showed higher permeability across Caco-2 monolayer with no significant efflux, while diosgenin was subjected to efflux mediated by P-glycoprotein. Diosgenin and dioscin inhibited CYP3A4 with IC50 values of 17 and 33 µM, respectively, while other CYP enzymes were not affected. In conclusion, dioscin showed better intestinal permeability. Conversion of dioscin to diosgenin was observed in both gastric and intestinal fluids. No phase I metabolism was detected for both compounds. The disappearance of diosgenin in S9 fraction indicated phase II metabolism.

Involvement of organic anion-transporting polypeptides in the hepatic uptake of dioscin in rats and humans.

The objective of this study was to clarify the mechanism underlying hepatic uptake of dioscin (diosgenyl 2,4-di-O-a-L-rhamnopyranosyl-p-D-glucopyranoside), an herbal ingredient with antihepatitis activity, in rats and humans. The liver uptake index (LUI) in vivo, perfused rat liver in situ, rat liver slices, isolated rat hepatocytes, and human organic anion-transporting polypeptide (OATP)-transfected cells in vitro were used to evaluate hepatic uptake of dioscin. Values of 11.9% ± 1.6% and 15.0% ± 0.9% of dose for uptake of dioscin were observed by LUI in vivo and perfused rat livers in situ, respectively. The time course of dioscin uptake by rat liver slices was temperature-dependent. Uptake of dioscin by rat liver slices and isolated rat hepatocytes was inhibited significantly by Oatp modulators, such as ibuprofen (Oatp1a1 inhibitor), digoxin (Oatp1a4 substrate), and glycyrrhizic acid (Oatp1b2 inhibitor), but not by TEA or p-aminohippurate. Uptake of dioscin in rat hepatocytes and OATP1B3-human embryonic kidney (HEK) 293 cells indicated a saturable process with a Km of 3.75 ± 0.51 µM and 2.08 ± 0.27 µM, respectively. (-)-Epigallocatechin gallate, cyclosporin A, rifampicin, and telmisartan inhibited transport of dioscin in OATP1B3-HEK293 cells. However, transcellular transport of dioscin in OATP1B1- or OATP1B1/multidrug resistance-associated protein 2-Madin-Darby canine kidney strain II cells was not observed. These results indicate that hepatic uptake of dioscin is involved in OATP1B3 in humans, and multiple Oatps might participate in this process in rats.