Neuropsychopharmacological profiling of scoparone in mice.

Scoparone (6,7-dimethoxycoumarin) is a simple coumarin from botanical drugs of Artemisia species used in Traditional Chinese Medicine and Génépi liquor. However, its bioavailability to the brain and potential central effects remain unexplored. We profiled the neuropharmacological effects of scoparone upon acute and subchronic intraperitoneal administration (2.5-25 mg/kg) in Swiss mice and determined its brain concentrations and its effects on the endocannabinoid system (ECS) and related lipids using LC-ESI-MS/MS. Scoparone showed no effect in the forced swimming test (FST) but, administered acutely, led to a bell-shaped anxiogenic-like behavior in the elevated plus-maze test and bell-shaped procognitive effects in the passive avoidance test when given subchronically and acutely. Scoparone rapidly but moderately accumulated in the brain (Cmax < 15 min) with an apparent first-order elimination (95% eliminated at 1 h). Acute scoparone administration (5 mg/kg) significantly increased brain arachidonic acid, prostaglandins, and N-acylethanolamines (NAEs) in the FST. Conversely, subchronic scoparone treatment (2.5 mg/kg) decreased NAEs and increased 2-arachidonoylglycerol. Scoparone differentially impacted ECS lipid remodeling in the brain independent of serine hydrolase modulation. Overall, the unexpectedly potent central effects of scoparone observed in mice could have toxicopharmacological implications for humans.

Screening and verification of CYP3A4 inhibitors from Bushen-Yizhi formula to enhance the bioavailability of osthole in rat plasma.

ETHNOPHARMACOLOGICAL RELEVANCE: With the features of multiple-components and targets as well as multifunction, traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of various diseases for a long time. During the application of TCM, the researches about bioavailability enhancement of the bioactive constituents in formula are flourishing. Bushen-Yizhi formula (BSYZ), a TCM prescription with osthole (OST) as one of the main bioactive ingredients, have been widely used to treat kidney deficiency, mental retardation and Alzheimer's disease. However, the underlying biological mechanism and compound-enzyme interaction mediated bioavailability enhancement of OST are still not clearly illuminated. AIM OF THE STUDY: The aim of this study is to explore the material basis and molecular mechanism from BSYZ in the bioavailability enhancement of OST. Screening the potential CYP3A4 inhibitors using theoretical prediction and then verifying them in vitro, and pharmacokinetics study of OST in rat plasma under co-administrated of screened CYP3A4 inhibitors and BSYZ were also scarcely reported. MATERIALS AND METHODS: Screening of CYP3A4 inhibitors from BSYZ was performed with molecular docking simulation from systems pharmacology database. The screened compounds were verified by using P450-Glo Screening Systems. A multiple reaction monitoring (MRM) mass spectrometry method was established for OST quantification. Male Sprague-Dawley rats divided into four groups and six rats in each group were employed in the pharmacokinetics study of OST. The administrated conditions were group I, OST (20 mg/kg); group II, BSYZ (containing OST 1 mg/mL, at the dose of 20 mg/kg OST in BSYZ); group III, co-administration of ketoconazole (Ket, 75 mg/kg) and OST (20 mg/kg); group IV, co-administration of CYP3A4 inhibitor (10 mg/kg) and OST (20 mg/kg). They were determined by using HPLC-MS/MS (MRM) and statistical analysis was performed using student's t-test with p < 0.05 as the level of significance. RESULTS: 21 potential CYP3A4 inhibitors were screened from BSYZ compounds library. From the results of verification in vitro, we found 4 compounds with better CYP3A4 inhibition efficiency including Oleic acid, 1,2,3,4,6-O-Pentagalloylglucose, Rutin, and Schisantherin B. Under further verification, Schisantherin B exhibited the best inhibitory effect on CYP3A4 (IC50 = 0.339 µM), and even better than the clinically used drug (Ket) at the concentration of 5 µM. In the study of pharmacokinetics, the area under the curve (AUC, ng/L*h) of OST after oral administration of BSYZ, Ket and Schisantherin B (2196.23 ± 581.33, 462.90 ± 92.30 and 1053.03 ± 263.62, respectively) were significantly higher than that of pure OST treatment (227.89 ± 107.90, p < 0.01). CONCLUSIONS: Schisantherin B, a profoundly effective CYP3A4 inhibitor screened from BSYZ antagonized the metabolism of CYP3A4 on OST via activity inhibition, therefore significantly enhanced the bioavailability of OST in rat plasma. The results of this study will be helpful to explain the rationality of the compatibility in TCM formula, and also to develop new TCM formula with more reasonable drug compatibility.

Design, synthesis and biological evaluation of selective hybrid coumarin-thiazolidinedione aldose reductase-II inhibitors as potential antidiabetics.

Thiazolidinediones (TZD), benzopyrans are the proven scaffolds for inhibiting Aldose reductase (ALR2) activity and their structural confluence with the retention of necessary fragments helped in designing a series of hybrid compounds 2-(5-cycloalkylidene-2,4-dioxothiazolidin-3-yl)-N-(2-oxo-2H-chromen-3-yl)acetamide (10a-n) for better ALR2 inhibition. The compounds were synthesized by treating substituted 3-(N-bromoacetyl amino)coumarins (9a-d) with potassium salt of 5-cyclo alkylidene-1,3-thiazolidine-2,4-diones (4a-d). The inhibition activity against ALR2 with IC50 values range from 0.012 ± 0.001 to 0.056 ± 0.007 µM. N-[(6-Bromo-3-coumarinyl)-2-(5-cyclopentylidene-2,4-dioxothiazolidin-3-yl)] acetamide (10c) with cyclopentylidene group on one end and the 6-bromo group on the other end showed better inhibitory property (IC50 = 0.012 µM) and selectivity index (324.166) against the ALR2, a forty fold superiority over sorbinil, a better molecule over epalrestat and rest of the analogues exhibited a far superior response over sorbinil and slightly better as compared with epalrestat. It was further confirmed by the insilico studies that compound 10c showed best inhibition activity among the synthesized compounds with a high selectivity index against the ALR2. In invivo experiments, supplementation of compound 10c to STZ induced rats delayed the progression of cataract in a dose-dependent manner warranting its further development as a potential agent to treat thediabetic secondary complications especially cataract.

Pharmacokinetics and tissue distribution study of 15 ingredients of Polygonum chinense Linn extract in rats by UHPLC-MS/MS.

A rapid and sensitive ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of 15 bioactive ingredients in rat plasma and tissues after oral administration of Polygonum chinense Linn extract (PCE). After addition of internal standards (ISs; rutin and danshensu), plasma and tissue samples were pre-treated by protein precipitation with acetonitrile-ethanol. The chromatographic separation was performed on an Agilent ZORBAX RRHD Eclipse Plus C18 column with gradient elution using a mobile phase composed of methanol and water (containing 0.2% acetic acid) at a flow rate of 0.3 mL min-1 . Mass spectrometric detection was carried out using a mass spectrometer in both positive and negative ion electrospray ionization modes by multiple reaction monitoring. The method provided excellent linearity, and the lower limit of quantification range 0.5-30 ng mL-1 for all analytes. The intra- and inter-day precision were less than 9.12% and the accuracy ranged from -4.02% to 6.32%, respectively. The mean extraction recovery and matrix effect of analytes and ISs ranged from 83.65% to 109.20%. The method was successfully applied to the pharmacokinetics and tissue distribution study of 15 ingredients of PCE in rats.

Scoparone as a therapeutic drug in liver diseases: Pharmacology, pharmacokinetics and molecular mechanisms of action.

Scoparone is an active and efficious ingredient of herbal medicine Artemisia capillaris Thunb, which has been used clinically in traditional Chinese medicine formula (e.g. Yin-Chen-Hao decoction) for the treatment of hepatic dysfunction, cholestasis and jaundice for over thousand years. More recently, scoparone has received increasing attention due to its multiple properties. In this comprehensive review, we provide the first summary of the pharmacological effects and pharmacokinetic characteristics of scoparone, and discuss future research prospects. The results implicated that scoparone possesses a wide spectrum of pharmacological activities, including anti-inflammatory, antioxidant, anti-apoptotic, anti-fibrotic and hypolipidemic properties. Pharmacokinetic studies have addressed that isoscopoletin and scopoletin are major primary metabolites of scoparone. Moreover, hepatic dysfunction might promote bioavailability of scoparone due to limited intrinsic clearance. On the other hand, the bioavailability of multi-component including scoparone in certain TCM formula can also be enhanced by applying this formula at a high dose on account of their interacted effects. In view of good pharmacological actions, scoparone is anticipated to be a potential drug candidate for various liver diseases, such as acute liver injury, fulminant hepatitis, alcohol-induced hepatotoxicity, non-alcoholic fatty liver disease and fibrosis. However, further studies are warranted to clarify its molecular mechanisms and targets, elucidate its toxicity, and identify its interplay with other active ingredients of classical TCM formula in clinical settings.

A novel UPLC/MS/MS method for rapid determination of murrayone in rat plasma and its pharmacokinetics.

Murraya paniculata (L.) is a traditional Chinese medicine (TCM) wildly grown in southeast China, and used for abortion in folk. Murrayone, a coumarin-containing compound extracted from M. paniculata, is the most bioactive substance in this species and is being developed as a novel cancer metastasis chemopreventive agent based on its unique pharmacological properties. In the present study, a novel rapid and sensitive method for quantitative analysis of murrayone in rat plasma and for determining its pharmacokinetics in rats was developed and validated using UPLC/MS/MS. Plasma samples were subjected to protein precipitation and then directly analyzed by UPLC/MS/MS. Both murrayone and coumarin as an internal standard (I.S.) were carried on a C18 column with a gradient mobile phase consisting of acetonitrile and water at a flow rate of 0.3 mL/min. Several gradient elution procedures were evaluated to achieve effective chromatography resolution and a sensitive response to murrayone and the I.S.. Mass spectrometry was carried out using a triple-quadrupole system via positive electrospray ionization and multiple reaction monitoring (MRM). Good linearity (r 2 = 0.9987) was achieved over a linear range of 4.0-1600 ng/mL with a lower limit of quantitation (LLOQ) of 4.0 ng/mL for murrayone. The inter- and intraday accuracy and precision ranged from 90.0 to 99.7% and 1.1 to 12.3% at four quality control concentrations, respectively. The average absolute recoveries of murrayone and the I.S. were determined to be 85.9-92.4% and 86.5-90.7%, respectively, at 10.0, 80.0, and 800 ng/mL. Murrayone was stable under a variety of storage and processing conditions that may be routinely encountered in laboratories based on all the stability tests. This newly developed method was successfully applied to the pharmacokinetic study of murrayone in rats for the first time, and the current assay methodology could provide important insights into potential therapeutics and facilitate further pharmacodynamic explorations of murrayone.

Pharmacokinetic study of precisely representative antidepressant, prokinetic, anti-inflammatory and anti-oxidative compounds from Fructus aurantii and Magnolia Bark.

BACKGROUND: Currently, few herbal pharmacokinetic (PK) parameters have been applied successfully for therapeutic monitoring because of the complexity of consistency when there are multiple chemicals and efficacies. PURPOSE: The present study aims to evaluate the herbal PK properties by investigating the PK parameters of the 8 absorbed bioactive compounds (ABCs), which can represent its parent herbal holistic efficacy, to achieve a PK therapeutic monitoring of herbs. METHOD: First, we tested the hypothesis that the antidepressant and prokinetic effects and related anti-inflammation and anti-oxidation activity (APIO) by Fructus aurantii-Magnolia Bark (FM) formula are related to 8 compounds according to the absorbable evidence and the determined contents. Subsequently, stable and representative APIO from 8ABCs allowed us to develop a sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of 8 compounds following the oral administration of FM decoction (20 g/kg) in rats. RESULT: 8 compounds either including Meranzin hydrate (MH) or MH alone almost identically (8 compounds: 91.62-108.82%)or nearly(MH: 65.38-88.41%) replicated the parent formula FM in terms of efficacy for inducing APIO. CONCLUSION: This unifying strategy shows how multi-herb formulas pharmacokinetic therapeutic monitoring can be achieved by the method we established.

Promotion of a quality standard for Porana sinensis Hemsl. based on the efficacy-oriented Effect-Constituent Index.

Multicompound determination for the quality control of traditional Chinese medicine (TCM) may often be inadequate, since these compounds may not be associated with, or fully represent, the clinical effects of TCM. Moreover, the individual contributions of each constituent to the pharmacological effect are often not considered. In China, Porana sinensis is widely used as a substitute for Erycibe sources to treat joint pain and rheumatoid arthritis. The existing quality control methods for P. sinensis neither consider the individual contributions of various compounds nor control the actual quality associated with different clinical efficacies. In the present study, a novel efficacy-oriented approach, named the effect-constituent index (ECI), was established for P. sinensis. Analyses of the spectrum-effect relationship and components in rat plasma were conducted to systematically and scientifically select quality markers. Quantitative analysis of multicomponents via a single marker method was introduced to enhance the practical application value of the established ECI. The established ECI shows a good ability to distinguish and predict the bioeffect-based quality of P. sinensis. The present study also provides a reference for the establishment and application of ECI as a quality control method for TCMs.

Therapeutic Evaluation of Synthetic Peucedanocoumarin III in an Animal Model of Parkinson's Disease.

The motor and nonmotor symptoms of Parkinson's disease (PD) correlate with the formation and propagation of aberrant α-synuclein aggregation. This protein accumulation is a pathological hallmark of the disease. Our group recently showed that peucedanocoumarin III (PCIII) possesses the ability to disaggregate ß sheet aggregate structures, including α-synuclein fibrils. This finding suggests that PCIII could be a therapeutic lead compound in PD treatment. However, the translational value of PCIII and its safety information have never been explored in relevant animal models of PD. Therefore, we first designed and validated a sequence of chemical reactions for the large scale organic synthesis of pure PCIII in a racemic mixture. The synthetic PCIII racemate facilitated clearance of repeated ß sheet aggregate (ß23), and prevented ß23-induced cell toxicity to a similar extent to that of purified PCIII. Given these properties, the synthetic PCIII's neuroprotective function was assessed in 6-hydroxydopamine (6-OHDA)-induced PD mouse models. The PCIII treatment (1 mg/kg/day) in a 6-OHDA-induced PD mouse model markedly suppressed Lewy-like inclusions and prevented dopaminergic neuron loss. To evaluate the safety profiles of PCIII, high dose PCIII (10 mg/kg/day) was administered intraperitoneally to two-month-old mice. Following 7 days of PCIII treatment, PCIII distributed to various tissues, with substantial penetration into brains. The mice that were treated with high dose PCIII had no structural abnormalities in the major organs or neuroinflammation. In addition, high dose PCIII (10 mg/kg/day) in mice had no adverse impact on motor function. These findings suggest that PCIII has a relatively high therapeutic index. Given the favorable safety features of PCIII and neuroprotective function in the PD mouse model, it may become a promising disease-modifying therapy in PD to regulate pathogenic α-synuclein aggregation.

Tissue distribution study of Angelica dahurica cv. Yubaizhi in rat by ultra-performance liquid chromatography with tandem mass spectrometry.

A sensitive and specific ultra-performance liquid chromatographic-tandem mass (UPLC-MS/MS) spectrometric method was established to investigate tissue distribution of fourteen coumarins of Angelica Dahurica cv. Yubaizhi roots (ADYR) in rat tissues, including isoimperatorin (1), imperatorin (2), isooxypeucedanin (3), byakangelicin (4), oxypeucedanin hydrate (5), bergapten (6), 2"R-neobyakangelicol (7), phellopterin (8), xanthotoxin (9), isopimpinellin (10), oxypeucedanin ethanolate (11), isobyakangelicol (12), columbianetin (13), (-)-marmesin (14). Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction-mode (MRM). The method established in this assay was successfully applied to tissue distribution study of the selected 14 coumarins after oral administration of the extract of ADYR in rat tissues, including heart, liver, spleen, lung, kidney, stomach, small intestine, muscle, testis, and brain. Tissue distribution characteristics of the fourteen coumarins were clearly elucidated, and the results of this study indicated that the fourteen coumarins were distributed to rat tissues rapidly and could be detected in all of the selected tissues after oral administration. Concentrations of the coumarins were obviously higher in kidney, liver and stomach tissues, and lower in testis, brain and muscle tissues. As an important part of ADMET/Act. study on ADYR, the tissue distribution of multiple coumarins of ADYR in rats provides a significant basis for better evaluation of the metabolism and disposition process in vivo of the herb medicine. The information provided in this research is very useful for further understanding of the metabolic mechanism of ADYR in vivo.

TCM-ADMEpred: A novel strategy for poly-pharmacokinetics prediction of traditional Chinese medicine based on single constituent pharmacokinetics, structural similarity, and mathematical modeling.

ETHNOPHARMACOLOGICAL RELEVANCE: Yuanhu Zhitong prescription (YZP) is a commonly used and relatively simple clinical herb preparation recorded in the China Pharmacopoeia. It contains Corydalis yanhusuo (Chinese name, Yanhusuo [YH]) and Angelica dahurica (Hoffm.) (Chinese name, Baizhi [BZ]), and has a long history of use in traditional Chinese medicine (TCM) for the treatment of stomach pain, hypochondriac pain, headache, and dysmenorrhea. AIM OF THE STUDY: A TCM-ADMEpred method is developed for novel strategy for poly-pharmacokinetics prediction of TCM. To predict the pharmacokinetic characteristics of the main YZP constituents in rat plasma using in silico models, based on the theory that structurally similar constituents show similar pharmacokinetic properties. This approach may facilitate in silico prediction of the pharmacokinetics of TCM. MATERIALS AND METHODS: A robust platform using ultra-performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) was developed and validated for simultaneous determination of seven active YZP constituents in rat plasma. These seven compounds were divided into two structural classes, alkaloids and coumarins. The correlation between AUC profiles within a structural class was expressed as Γ+, and this variable was used to develop two novel in silico models to predict constituent AUC values. The pharmacokinetics of tetrahydropalmatine, tetrahydroberberine, and corydaline following YZP administration were predicted using the Γ+-values of α-allocryptopine observed following YH administration, while those of imperatorin and isoimperatorin following BZ administration were predicted using the Γ+-values of byakangelicin observed following YZP administration. RESULTS: The UPLC-ESI-MS/MS method was successfully used to evaluate pharmacokinetic parameters after oral YZP, YH, or BZ administration. Our findings showed that co-administration of YH and BZ increased the AUC of four alkaloid constituents and reduced the AUC of three coumarin constituents, which might provide a scientific rationale for co-administering these herbs clinically as a YZP preparation, thus increasing their efficacy and reducing toxicity. The AUC values of imperatorin and isoimperatorin were predicted 3 h after oral BZ administration, with the bias ratios between the theoretical values and the observed experimental values ranging from 0.61% to 11.4%, and average bias ratios of 5.8% and 8.0%, respectively. The AUC values of tetrahydropalmatine, tetrahydroberberine, and corydaline were predicted 3 h after oral YZP administration, with bias ratios ranging from 3.7% to 46.4%, and average bias ratios of 23.8%, 15.4%, and 25.8%, respectively. CONCLUSION: The UPLC-ESI-MS/MS method was successfully applied to pharmacokinetic evaluations after oral administration of YZP, YH, and BZ to rats. The Γ+ variable was used to express the correlation between the AUC profiles of structurally similar compounds. This facilitated the development of an in silico model that was used to predict the AUC of three alkaloids in YZP and of two coumarins in BZ. Calculation of the bias ratios between the predicted and experimental values suggested that this in silico model provided a viable approach for the prediction of TCM pharmacokinetics.

Development and Validation of an Ultra-Performance Liquid Chromatography Method for the Determination of Wedelolactone in Rat Plasma and its Application in a Pharmacokinetic Study.

Wedelolactone is a coumarin ether with significant hepatoprotective effects. However, there are few pharmacokinetic studies of wedelolactone, which will affect the studies of its efficacy and potential toxicity. In this study, a selective ultra-performance liquid chromatography (UPLC) method was developed to confirm the pharmacokinetic parameters of wedelolactone in rat plasma. The chromatographic separation was carried out on a Kromasil C18 UPLC column (250 × 4.6 mm; 5.0 µm) by gradient mobile phase of methanol-water containing 0.5% acetic acid (v/v). Perfect linearity was obtained and the samples were stable under different conditions. The intra-day and inter-day precisions (relative standard deviation, %) were within 3.81% and accuracies (relative error, %) ranged from -4.01% to 7.12%. The extraction recoveries in rat plasma ranged from 95.98% to 108.93%. This rapid method was successfully applied in the pharmacokinetic study of wedelolactone in rat plasma. Following the oral administration of 5.00 mg/kg wedelolactone, the wedelolactone was rapidly absorbed. Pharmacokinetic parameters were used to quantitatively describe the dynamic changes of wedelolactone in vivo, providing a theoretical basis for pharmacological research on drugs and preclinical medication. The study of wedelolactone can provide a theoretical basis and quick analysis for the study of other traditional Chinese medicine. This may lead to breakthroughs in the pharmacokinetic study of complex Chinese medicines.

Enhanced identification of the in vivo metabolites of Ecliptae Herba in rat plasma by integrating untargeted data-dependent MS2 and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan.

Detection and identification of the in vivo metabolites of traditional Chinese medicine by untargeted profiling strategies are often confronted with severe interference from complex endogenous substances. Here we developed an integral approach, by combining untargeted data-dependent MS2 (dd-MS2) of Q-Orbitrap mass spectrometry and predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion scan (pMRM-IDA-EPI) of triple quadrupole-linear ion trap (QTRAP) mass spectrometry, aiming to detect and identify more extensive metabolites in bio-samples. Ecliptae Herba (EH) is a widely consumed medicinal herb with the effects of nourishing liver/kidney, but its metabolites in vivo have not been fully elucidated. Firstly, after UHPLC separation on an HSS T3 column, chemical fingerprinting of 70% ethanolic extract of EH was performed by untargeted dd-MS2 in negative ion mode. We could characterize 41 compounds from EH, and 24 were detectable in the plasma of rats (prototypes) after oral administration of EH extract (1 g/kg). Secondly, using echinocystic acid (triterpene), wedelolactone (coumarin), and apigenin (flavonoid) as the different parent templates, an MRM list containing 150 predicted ion-pairs was established to enhance MS2 scan by pMRM-IDA-EPI, which enabled the primary identification of up to 200 metabolites. The biotransformations mainly involve oxidation, hydrogenation, methylation, glucuronidation, sulfonation etc. Thirdly, the rat plasma samples obtained after oral administration of three pure compounds (echinocystic acid, wedelolactone and apigenin) were analyzed to verify the reliability of metabolites identification, and 11, 4, and 10 metabolites were found individually. This is the first comprehensive research on the metabolism of EH in vivo.

Metabolism of Scoparone in Experimental Animals and Humans.

Scoparone, a major constituent of the Chinese herbal medicine Yin Chen Hao, expresses beneficial effects in experimental models of various diseases. The intrinsic doses and effects of scoparone are dependent on its metabolism, both in humans and animals. We evaluated in detail the metabolism of scoparone in human, mouse, rat, pig, dog, and rabbit liver microsomes in vitro and in humans in vivo. Oxidation of scoparone to isoscopoletin via 6-O-demethylation was the major metabolic pathway in liver microsomes from humans, mouse, rat, pig and dog, whereas 7-O-demethylation to scopoletin was the main reaction in rabbit. The scoparone oxidation rates in liver microsomes were 0.8 - 1.2 µmol/(min*g protein) in mouse, pig, and rabbit, 0.2 - 0.4 µmol/(min*g protein) in man and dog, and less than 0.1 µmol/(min*g) in rat. In liver microsomes of all species, isoscopoletin was oxidized to 3-[4-methoxy-ρ-(3, 6)-benzoquinone]-2-propenoate and esculetin, which was formed also in the oxidation of scopoletin. Human CYP2A13 exhibited the highest rate of isoscopoletin and scopoletin oxidation, followed by CYP1A1 and CYP1A2. Glucuronidation of isoscopoletin and scopoletin was catalyzed by the human UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, and UGT2B17. Dog was most similar to man in scoparone metabolism. Isoscopoletin glucuronide and sulfate conjugates were the major scoparone in vivo metabolites in humans, and they were completely excreted within 24 h in urine. Scoparone and its metabolites did not activate key nuclear receptors regulating CYP and UGT enzymes. These results outline comprehensively the metabolic pathways of scoparone in man and key preclinical animal species.

Molecular Pathway of Psoralidin-Induced Apoptosis in HepG2 Cell Line.

OBJECTIVE: To test the role of psoralidin in human liver cancer HepG2 cells in vitro. METHODS: Cell viability was assessed by methylthiazolyldiphenyl-tetrazolum bromide assay and apoptotic cells were labeled by annexin V then sorted by flow cytometry. Protein expressions of caspase-3, caspase-8, caspase-9, Bax, Bid, Bcl-2, Bcl-xL and p53 were examined by western blot while activity of caspase-3, -8 and -9 were also determined. RESULTS: Psoralidin reduces cell viability greatly in a time dependent manner (64%, 40%, 21%, 12% at 2, 6, 24 and 48 h treatment with 64 µmol/L psoralidin respectively) and up-regulates activities of caspase-3, -8 and -9 in a concentration dependent manner (between 4 to 64 µmol/L). Psoralidin also increases the expression of pro-apoptosis genes Bax, Bid and p53 while decreases the expression of pro-survival genes Bcl-2 and Bcl-xL, both in a concentration dependent manner between 4 and 64 µmol/L (P<0.05 at 16 and 64 µmol/L). Caspase-3 inhibitor (Ac-DEVD-CHO at concentrations between 10 to 20 µmol/L), p53 inhibitor (pifithrin-α at 5 µmol/L) and cyclosporin A can attenuate the apoptotic effect of psoralidin. CONCLUSION: The cytotoxic role of psoralidin might work through both intrinsic and extrinsic apoptotic pathway.

[In vivo metabolism of three coumarins from Chimonanthi Radix based on UHPLC-QTOF-MS/MS].

Scopolin (SC-1), scopoletin (SC-2) and isofraxidin (IS-1) are the main active constituents in Chimonanthi Radix. However, the in vivo metabolism of SC-1, SC-2 and IS-1 have not been comprehensively clarified. In this study, the in vivo metabolic profiles of these three coumarins in the rat plasma, urine and feces were analyzed. Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS/MS) method was applied to characterize the prototypes and metabolites of SC-1, SC-2 and IS-1 in rat feces, urine, and plasma after intravenous administration. A total of 11 metabolites of the three parent compounds were tentatively identified. The main metabolic pathways were analyzed by identification of metabolites, and it was found that these three coumarins underwent multiple in vivo metabolic reactions including glucuronidation, sulfonation, isomerism and reduction. In this study, the analysis of metabolites of three coumarins basically demonstrated their in vivo metabolic process, providing basis for the further pharmacokinetics and pharmacological evaluations of SC-1, SC-2 and IS-1.

Preclinical Pharmacokinetics of Scoparone, Geniposide and Rhein in an Herbal Medicine Using a Validated LC-MS/MS Method.

The herbal formula Yin-Chen-Hao-Tang has been reported to have anti-fibrosis properties. The aim of this study was to reveal the pharmacokinetic characteristics of bioactive compounds in this herbal formula. A new high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for simultaneous determination of scoparone, geniposide and rhein in rat plasma. A pharmaceutical herbal powder was administered to rats at doses of 1 g/kg and 3 g/kg orally. The method showed excellent linearity (r² > 0.999) and validation was successfully conducted for the pharmacokinetic study. The results show that the Cmax values and areas under the curve of scoparone, geniposide and rhein were higher and not proportional to the dose in rat plasma, while the Tmax and half-life values were consistent in the group that received 1 g/kg. The clearance of the higher dose (3 g/kg) did not decrease proportionally to that of the low dose. The results showed the nonlinear pharmacokinetic properties of scoparone, geniposide and rhein in Yin-Chen-Hao-Tang that suggested possible accumulation of bioactive compounds through oral administration. This pharmacokinetic study reveals that an increased dose of this herbal formula would largely increase the maximum concentration and bioavailability of scoparone, geniposide and rhein.

Preparation and Pharmacokinetics Evaluation of Solid Self-Microemulsifying Drug Delivery System (S-SMEDDS) of Osthole.

The study was performed aiming to enhance the solubility and oral bioavailability of poorly water-soluble drug osthole by formulating solid self-microemulsifying drug delivery system (S-SMEDDS) via spherical crystallization technique. Firstly, the liquid self-microemulsifying drug delivery system (L-SMEDDS) of osthole was formulated with castor oil, Cremophor RH40, and 1,2-propylene glycol after screening various lipids and emulsifiers. The type and amount of polymeric materials, good solvents, bridging agents, and poor solvents in S-SMEDDS formulations were further determined by single-factor study. The optimal formulation contained 1:2 of ethyl cellulose (EC) and Eudragit S100, which served as matrix forming and enteric coating polymers respectively. Anhydrous ethanol and dichloromethane with a ratio of 5:3 are required to perform as good solvent and bridging agent, respectively, with the addition of 0.08% SDS aqueous solution as poor solvent. The optimized osthole S-SMEDDS had a high yield (83.91 ± 3.31%) and encapsulation efficiency (78.39 ± 2.25%). Secondly, osthole L-SMEDDS was solidified to osthole S-SMEDDS with no significant changes in terms of morphology, particle size, and zeta potential. In vitro release study demonstrated a sustained release of the drug from osthole S-SMEDDS. Moreover, in vivo pharmacokinetic study showed that the Tmax and mean residence time (MRT(0-t)) of osthole were significantly prolonged and further confirmed that osthole S-SMEDDS exhibited sustained release effect in rabbits. Comparing with osthole aqueous suspension and L-SMEDDS, osthole S-SMEDDS increased bioavailability by 205 and 152%, respectively. The results suggested that S-SMEDDS was an effective oral solid dosage form, which can improve the solubility and oral bioavailability of poorly water-soluble drug osthole.

Comparative pharmacokinetics of six coumarins in normal and breast cancer bone-metastatic mice after oral administration of Wenshen Zhuanggu Formula.

ETHNOPHARMACOLOGICAL RELEVANCE: Wenshen Zhuanggu Formula (WSZG) is a traditional Chinese medicine (TCM) prescription used in clinics for adjuvant treatment of breast cancer bone metastases in Longhua Hospital in China. WSZG has been reported to decrease the risk of bone metastases and alleviate the severity of bone lesions in a breast cancer xenograft model. AIM OF THE STUDY: The present study aimed at investigating the pharmacokinetic behaviors of six coumarins in normal and breast cancer bone-metastatic mice following oral administration of WSZG extract. MATERIALS AND METHODS: A bone-metastatic mouse model was established by intracardiac injection of MDA-MB-231BO breast cancer cells, and WSZG extract (1.60 g/kg) was given orally to the model and normal mice for 4 weeks. Then, the blood pharmacokinetic parameters of six bioactive components from WSZG (psoralen, isopsoralen, bergapten, xanthotoxin, osthole, and imperatorin) were analyzed by liquid chromatography tandem mass spectrometry. RESULTS: There were significant differences in pharmacokinetic behaviors between normal and pathological states. Compared with normal mice, the model mice showed significantly increased AUC0-t and AUC0-∞ of the bioactive compounds (P < 0.05) and significantly decreased total blood clearance (CLZ/F) (P < 0.05). CONCLUSIONS: The different pharmacokinetic behaviors might be partly ascribed to intestinal functional disorders and imbalance of gastrointestinal microbiota under the morbid state. The findings provide some valuable information to evaluate the clinical efficacy and safety of this TCM formula.

Evaluation of 99mTc-sulfonamide and sulfocoumarin derivatives for imaging carbonic anhydrase IX expression.

With the aim to prepare hypoxia tumor imaging agents, technetium(I) and rhenium(I) tricarbonyl complexes with dipyridylamine (L1 = N-{[1-(2,2-dioxido-1,2-benzoxathiin-6-yl)-1H-1,2,3-triazol-4-yl]methyl}-N-(2-pyridinylmethyl)-2-pyridinemethanamine; L3 = N-{[1-[N-(4-aminosulfonylphenyl)]-1H-1,2,3-triazol-4-yl]methyl}-N-(2-pyridinyl-methyl)-2-pyridinemethanamine), and iminodiacetate (H2L2 = N-{[1-(2,2-dioxido-1,2-benzoxathiin-6-yl)-1H-1,2,3-triazole-4-yl]methyl}-N-(carboxy-methyl)-glycine; H2L4 = N-{[1-[N-(4-aminosulfonylphenyl)]-1H-1,2,3-triazole-4-yl]methyl}-N-(carboxymethyl)-glycine) ligands appended to sulfonamide or sulfocoumarin carbonic anhydrase inhibitors were synthesized. The Re(I) complexes were characterized using 1H/13C NMR, MS, EA, and in one case the X-ray structure of [Et3NH][Re(CO)3(L2)] was obtained. As expected, the Re coordination geometry is distorted octahedral, with a tridentate iminodiacetate ligand in a fac arrangement dictated by the three strong-field CO ligands. Inhibition studies of human carbonic anhydrases (hCAs) showed that the Re sulfocoumarin derivatives were inactive against hCA-I, -II and -IV, but had moderate affinity for hCA-IX. The Re sulfonamides showed improved affinity against all tested hCAs, with [Re(CO)3(L4)]- being the most active and selective for the hCA-IX isoform. The corresponding 99mTc complexes were synthesized from fac-[99mTc(CO)3(H2O)3]+, purified by HPLC, and obtained with average 41-76% decay-corrected radiochemical yields and with >99% radiochemical purity. Uptake in HT-29 tumors at 1 h post-injection was highest for [99mTc(CO)3(L4)]- (0.14 ±â€¯0.10%ID/g) in comparison to [99mTc(CO)3(L1)]+ (0.06 ±â€¯0.01%ID/g), [99mTc(CO)3(L2)]- (0.03 ±â€¯0.00%ID/g), and [99mTc(CO)3(L3)]+ (0.07 ±â€¯0.03%ID/g). The uptake in tumors was further reduced at 4 h post-injection. For potential imaging application with single photon emission computed tomography, further optimization is needed to improve the affinity to hCA-IX and uptake in hCA-IX expressing tumors.