Simultaneous determination of asarinin, ß-eudesmol, and wogonin in rats using ultraperformance liquid chromatography-tandem mass spectrometry and its application to pharmacokinetic studies following administration of standards and Gumiganghwal-tang.

Asarinin, ß-eudesmol, and wogonin have common antiangiogenic activities and have the potential for use in chemotherapy. Besides, they are multivalent substances that are combined in various herbal medicines. The purpose of this study was to develop a method for simultaneous analysis of asarinin, ß-eudesmol, and wogonin, which are representative pharmacological components of Asarum heterotropoides, Atractylodes lancea, and Scutellaria baicalensis, respectively, in rat biosamples using ultraperformance liquid chromatography-tandem mass spectrometry. The three components were separated using 5 mm aqueous ammonium acetate containing 0.1% formic acid and acetonitrile as a mobile phase, equipped with a KINETEX core-shell C18 column. The analysis was quantitated on a triple-quadrupole mass-spectrometer employing electrospray ionization, and operated in the multiple reaction monitoring mode. The chromatograms showed high resolution, sensitivity, and selectivity with no interference with plasma, urine, and feces constituents. The developed analytical method satisfied international guidance criteria and could be successfully applied to the pharmacokinetic (PK) studies evaluating oral bioavailability of asarinin, ß-eudesmol, and wogonin after oral and intravenous administration and their urinary and fecal excretion ratios after oral administration to rats. Furthermore, the analysis was extended to PK studies following oral administration of Gumiganghwal-tang. This study was the first simultaneous analysis of the aforesaid three constituents in rat plasma, urine, and feces that also determined their PK parameters.

Nanoformulated Codelivery of Quercetin and Alantolactone Promotes an Antitumor Response through Synergistic Immunogenic Cell Death for Microsatellite-Stable Colorectal Cancer.

Microsatellite-stable colorectal cancer (CRC) is known to be resistant to immunotherapy. The combination of quercetin (Q) and alantolactone (A) was found to induce synergistic immunogenic cell death (ICD) at a molar ratio of 1:4 (Q:A). To achieve ratiometric loading and delivery, the micellar delivery of Q and A (QA-M) was developed with high entrapment efficiency and drug loading at an optimal ratio. QA-M achieved prolonged blood circulation and increased tumor accumulation for both drugs. More importantly, QA-M retained the desired drug ratio (molar ratio of Q to A = 1:4) in tumors at 2 and 4 h after intravenous injection for synergistic immunotherapy. Tumor growth was significantly inhibited in murine orthotopic CRC by the treatment of QA-M compared to PBS and the combination of free drugs (p < 0.005). The combination of nanotherapy stimulated the host immune response to induce long-term tumor destruction and induced memory tumor surveillance with a 1.3-fold increase in survival median time compared to PBS (p < 0.0001) and a combination of free drugs (p < 0.0005). The synergistic therapeutic effect induced by codelivery of Q and A is capable of reactivating antitumor immunity by inducing ICD, causing cell toxicity and modulating the immune-suppressive tumor microenvironment. Such a combination of Q and A with synergistic effects entrapped in a simple and safe nanodelivery system may provide the potential for scale-up manufacturing and clinical applications as immunotherapeutic agents for CRC.

Intestinal Absorption of Isoalantolactone and Alantolactone, Two Sesquiterpene Lactones from Radix Inulae, Using Caco-2 Cells.

BACKGROUND: Isoalantolactone and alantolactone are the main sesquiterpene lactones in Radix Inulae (dried root of Inula helenium L. or I. racemosa Hook. F.), which is a frequently utilized herbal medicine. They also occur in several plants and have various pharmacologic effects. However, they have been found to have poor oral bioavailability in rats. OBJECTIVES: To understand the intestinal absorptive characteristics of isoalantolactone and alantolactone as well specific influx and efflux transporters in their absorption. METHODS: Bidirectional permeabilities of isoalantolactone and alantolactone were investigated across Caco-2 cell monolayers. Transport assays were performed using different concentrations of two lactones and specific inhibitors of ATP-binding cassette transporters and influx transporters. RESULTS: The absorption permeability of isoalantolactone and alantolactone was high at the tested concentrations (5, 20 and 80 µmol/l), and the major permeation mechanism of both lactones was found to be passive diffusion with active efflux mediated by multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP). CONCLUSION: Our results demonstrated that the absorption permeability of isoalantolactone and alantolactone was good in the Caco-2 cell model. The isoalantolactone and alantolactone absorption elucidated in this study provides useful information for further pharmacokinetics studies. Since low intestinal absorption can now be ruled out as a cause, further studies are needed to explain the low oral bioavailability of the two sesquiterpene lactones.

Metabolism and pharmacokinetics of alantolactone and isoalantolactone in rats: Thiol conjugation as a potential metabolic pathway.

Alantolactone (AL) and isoalantolactone (IAL), two major active sesquiterpene lactones isolated from Radix Inulae extract, have a wide range of pharmacological activities. The predominant metabolic pathway of AL and IAL observed was glutathione (GSH) conjugation in vitro, which could occur in the absence of metabolic enzymes. Non-enzymatic conjugation with cysteine (Cys) couldalso be observed. Four metabolites (AL-GSH, AL-Cys, IAL-GSH, IAL-Cys) were subsequently isolated and confirmed by nuclear magnetic resonance (NMR). The results indicated that the thiol of GSH or Cys can be reacted with the exomethylene carbon atoms of α, ß-unsaturated carbonyl of AL and IAL. After intravenous administration in rats, AL and IAL were extensively metabolized, and the exposure, as measured by area under the concentration-time curve (AUC), for AL-GSH, AL-Cys, IAL-GSH, and IAL-Cys was approximately 1.54-, 0.96-, 1.50-, and 0.91-fold that of the parent drug, respectively. The AUC ratio of metabolites to parent compounds of oral administration was 3.66-, 9.19-, 12.97-, and 9.92-fold that of the parent drug for the above metabolites, respectively. The bioavailability of AL-total (AL, AL-GSH, AL-Cys) and IAL-total (IAL, IAL-GSH, IAL-Cys) was, respectively, 8.39% and 13.07%, which was 3.62- and 6.95- fold that of AL (2.32%) and IAL (1.88%), respectively. The oral exposure will be underestimated if the parent drugs are tested alone. These findings provide useful information for preclinical safety evaluation, and for predicting AL and IAL metabolism in humans.

Quantification of ß-eudesmol in rat plasma using LC-MS/MS and its application to a pharmacokinetic study.

A sensitive and specific LC-MS/MS assay for determination of ß-eudesmol in rat plasma was developed and validated. After liquid-liquid extraction with ethyl ether, the analyte and IS were separated on a Capcell Pak C18 column (50 × 2.0 mm, 5 µm) by isocratic elution with acetonitrile-water-formic acid (77.5:22.5:0.1, v/v/v) as the mobile phase at a flow rate of 0.4 mL/min. An ESI source was applied and operated in positive ion mode; a selected reaction monitoring scan was used for quantification by monitoring the precursor-product ion transitions of m/z 245.1 → 163.1 for ß-eudesmol and m/z 273.4 → 81.2 for IS. Good linearity was observed in the concentration range of 3-900 ng/mL for ß-eudesmol in rat plasma. Intra- and inter-day precision and accuracy were both within ±14.3%. This method was applied for pharmacokinetic studies after intravenous bolus of 2.0 mg/kg or intragastric administration of 50 mg/kg ß-eudesmol in rats.

Sesquiterpenes from the whole plants of Parasenecio roborowskii.

Six eremophilane-type (parasenolide A-F) and an eudesmane-type (parasenin) sesquiterpenoids, along with eight known sesquiterpenes, were isolated from the whole plants of Parasenecio roborowskii. The structures and absolute configurations of new compounds were elucidated using extensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR experiments, the CD exciton chirality methods, and single-crystal X-ray crystallography. All isolated compounds were evaluated for cytotoxicity against five human cancer (HeLa, HepG2, K562, MDA231, and NCI-H460) cell lines and a murine melanoma B16 F10 cell line by MTT assay. Compounds 1-15 showed cytotoxic activities, especially compounds 3, 4, 8, 10, and 12. These five compounds showed broad spectrum activities against all the tested cancer cell lines with IC50 ranging from 9.2 to 35.5µM. The study supports that eremophilenolides and eudesmane-type sesquiterpenes occur mainly in the genus Parasenecio and can be used as a chemosystematic marker of the genus.

High body clearance and low oral bioavailability of alantolactone, isolated from Inula helenium, in rats: extensive hepatic metabolism and low stability in gastrointestinal fluids.

Alantolactone (ALA) is a major bioactive sesquiterpene lactone present in the roots of Inula helenium L. (Asteraceae) which has been used widely in traditional medicine against various diseases such as asthma, cancer and tuberculosis. The pharmacologic activities of alantolactone have been well characterized, yet information on the physicochemical and pharmacokinetic properties of alantolactone and their mechanistic elucidation are still limited. Thus, this study aims to investigate the oral absorption and disposition of alantolactone and their relevant mechanisms. Log P values of alantolactone ranged from 1.52 to 1.84, and alantolactone was unstable in biological samples such as plasma, urine, bile, rat liver microsomes (RLM) and simulated gastrointestinal fluids. The metabolic rate of alantolactone was markedly higher in rat liver homogenates than in the other tissue homogenates. A saturable and concentration-dependent metabolic rate profile of alantolactone was observed in RLM, and rat cytochrome P450 (CYP) 1 A, 2C, 2D and 3 A subfamilies were significantly involved in its hepatic metabolism. Based on the well-stirred model, the hepatic extraction ratio (HER) was estimated to be 0.890-0.933, classifying alantolactone as a drug with high HER. Moreover, high total body clearance (111 ± 41 ml/min/kg) and low oral bioavailability (0.323%) of alantolactone were observed in rats. Taken together, the present study demonstrates that the extensive hepatic metabolism, at least partially mediated by CYP, is primarily responsible for the high total body clearance of alantolactone, and that the low oral bioavailability of alantolactone could be attributed to its low stability in gastrointestinal fluids and a hepatic first-pass effect in rats. Copyright © 2016 John Wiley & Sons, Ltd.

Pharmacokinetics, tissue distribution and excretion of isoalantolactone and alantolactone in rats after oral administration of Radix Inulae extract.

Radix Inulae is endemic to China and has been used in traditional medicine to treat upper body pain, emesis and diarrhoea, and to eliminate parasites. Here, an UPLC-MS/MS method was developed and applied to study the pharmacokinetics, distribution and excretion of isoalantolactone and alantolactone, which are two main active sesquiterpene lactones in Radix Inulae, in Sprague-Dawley rats following oral administration of total Radix Inulae extract. Isoalantolactone, alantolactone and osthole (internal standard) were prepared using acetonitrile precipitation, and the separation of isoalantolactone and alantolactone was achieved by isocratic elution using water (containing 0.1% formic acid) and acetonitrile as the mobile phase using a ZORBAX Eclipse Plus C18 column. The total run time was 6.4 min. The present study showed poor absorption of isoalantolactone and alantolactone in vivo. The apparent Cmax, Tmax, T1/2 and total exposure (AUC0-12h) in rat plasma were 37.8 ng/mL, 120 min, 351.7 min and 6112.3 ng-min/mL for isoalantolactone and 25.9 ng/mL, 90 min, 321.0 min and 4918.9 ng-min/mL for alantolactone, respectively. It was shown that the highest concentration was achieved in the small intestine and feces clearance was shown to be the dominant elimination pathway of the lactones.

Anticancer activity using positron emission tomography-computed tomography and pharmacokinetics of ß-eudesmol in human cholangiocarcinoma xenografted nude mouse model.

Cholangiocarcinoma (CCA) is an important public health problem in several parts of South East Asia, particularly in Thailand. The limited availability of effective diagnostic tools for early stage CCA, including chemotherapeutic options, constitutes a major problem for treatment and control of CCA. The aim of the present study was to assess the anti-CCA activity and pharmacokinetics of ß-eudesmol in CCA-xenografted nude mouse model and healthy mice. Positron emission tomography-computed tomography (PET-CT) with (18)F-fluorodeoxyglucose was used for detecting and monitoring tumour development, and PET-CT with technetium-99m was used to investigate its pharmacokinetics property. Results support the role of PET-CT as a potential tool for detecting and monitoring the progress of lung metastasis. Tumour size and lung metastasis were significantly inhibited by 91.6% (of baseline) and 95% (of total lung mass), respectively, following treatment with high-dose ß-eudesmol (100 mg/kg body weight for 30 days). Survival time was prolonged by 64.4% compared with untreated controls. Systemic clearance of the compound was rapid, particularly during the first 60 min. The compound was distributed to the vital organs at maximum levels 2 h after oral administration and 15 min after intravenous injection. Results from the present study suggest the potential of ß-eudesmol as a promising candidate for further development as an anti-CCA drug with respect to its pharmacodynamics and pharmacokinetic properties. PET-CT, with radiotracers (18)F-fluorodeoxyglucose and technetium-99m, was shown to be a reliable tool in the investigation of anti-CCA and pharmacokinetic properties of ß-eudesmol in CCA-xenografted and healthy mice.

Simultaneous determination of sesquiterpene lactones isoalantolactone and alantolactone isomers in rat plasma by liquid chromatography with tandem mass spectrometry: application to a pharmacokinetic study.

A selective, sensitive, and accurate LC-MS/MS method for the simultaneous determination of isoalantolactone and alantolactone in rat plasma has been developed using psoralen as the internal standard. LC-MS/MS analysis was carried out on a Triple Quadrupole mass spectrometer using positive ion ESI and the selected reaction monitoring mode. The assays were linear in the range of 7.5-750 ng/mL for isoalantolactone and 5.5-550 ng/mL for alantolactone. The average recoveries in plasma samples both were better than 85%. The intra- and inter-day precision and accuracy values were found to be within the assay variability criteria limits according to the US FDA guidelines. The method was successfully applied to pharmacokinetic studies of the two structural isomers after an intravenous injection of Inula helenium formulation to rats.

Alantolactone induces apoptosis in glioblastoma cells via GSH depletion, ROS generation, and mitochondrial dysfunction.

Glioblastoma multiforme (GBM) is the most malignant and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies, the prognosis of glioblastoma remains very poor. Alantolactone, a sesquiterpene lactone compound, has been reported to exhibit antifungal, antibacteria, antihelminthic, and anticancer properties. In this study, we found that alantolactone effectively inhibits growth and triggers apoptosis in glioblastoma cells in a time- and dose-dependent manner. The alantolactone-induced apoptosis was found to be associated with glutathione (GSH) depletion, reactive oxygen species (ROS) generation, mitochondrial transmembrane potential dissipation, cardiolipin oxidation, upregulation of p53 and Bax, downregulation of Bcl-2, cytochrome c release, activation of caspases (caspase 9 and 3), and cleavage of poly (ADP-ribose) polymerase. This alantolactone-induced apoptosis and GSH depletion were effectively inhibited or abrogated by a thiol antioxidant, N-acetyl-L-cysteine, whereas other antioxidant (polyethylene glycol (PEG)-catalase and PEG-superoxide-dismutase) did not prevent apoptosis and GSH depletion. Alantolactone treatment inhibited the translocation of NF-κB into nucleus; however, NF-κB inhibitor, SN50 failed to potentiate alantolactone-induced apoptosis indicating that alantolactone induces NF-κB-independent apoptosis in glioma cells. These findings suggest that the sensitivity of tumor cells to alantolactone appears to results from GSH depletion and ROS production. Furthermore, our in vivo toxicity study demonstrated that alantolactone did not induce significant hepatotoxicity and nephrotoxicity in mice. Therefore, alantolactone may become a potential lead compound for future development of antiglioma therapy.