Igalan from Inula helenium (L.) suppresses the atopic dermatitis-like response in stimulated HaCaT keratinocytes via JAK/STAT3 signaling.

OBJECTIVE: This study aimed to evaluate the protective effect of igalan, a sesquiterpene lactone isolated from Inula helenium (L.), on inhibiting inflammation, regulating the epidermal differentiation gene expression, and reactive oxygen species scavenging in atopic dermatitis (AD)-like inflammatory keratinocytes. METHODS: HaCaT human keratinocytes were treated with igalan at indicated concentrations before being activated by a combination of TNF-α and IFN-γ or IL-4 representative for T-helper 1 and T-helper 2 cell cytokines, which are associated with AD pathogenesis. RESULTS: By inhibiting the NF-κB pathway as well as the STAT activation, igalan could downregulate several marker inflammatory genes in AD, such as TARC/CCL17, MDC/CCL22, and RANTES/CCL5. In contrast, igalan, acting as JAK inhibitor, could promote the mRNA expression levels of the genes FLG, LOR, KRT10, and DSC1, which encode for essential proteins responsible for keratinocyte differentiation, by inhibiting STAT3 signaling. Furthermore, igalan exerts its antioxidant effect through activating the Nrf2 pathway, triggering the expression of some enzymes that contribute to preventing intracellular ROS generation during inflammation. CONCLUSION: These findings indicate that igalan, via suppressing JAK/STAT3 signaling, could impair the production of pro-inflammatory chemokines and enhance expression levels of several genes involved in keratinocyte differentiation in AD-like stimulated keratinocytes.

Cellular Target Proteome in Breast Cancer Cells of an Oplopane Sesquiterpenoid Isolated from Tussilago farfara.

Tussilago farfara is a traditional herbal medicine used to treat coughs, bronchitis, and asthma. Its bioactive compounds include sesquiterpenoids with anti-inflammatory, antiproliferative, neuroprotective, and other effects. Biochemical studies have highlighted the mechanisms of action, but the investigations of related molecular pathways have not specified direct molecular targets. Therefore, this study profiled cellular target proteins of a sesquiterpenoid isolated from T. farfara using quantitative chemical proteomics in MDA-MB-231 and MCF-7 human breast cancer cells. Compound 8, 7ß-(3'-ethyl-cis-crotonoyloxy)-1α-(2'-methyl butyryloxy)-3,14-dehydro-Z-notonipetranone, exhibited potent antiproliferative activity based on its α,ß-unsaturated carbonyl moiety, and its potential cellular target proteins were identified using a compound 8-based clickable probe. Among >200 identified proteins, 17 showed enrichment ratios of >3 in both cell lines, while recombinant 14-3-3 protein zeta and peroxiredoxin-1 were verified using isothermic calorimetry and their alkylation sites. Considering the interaction between the α,ß-unsaturated carbonyl moiety of compound 8 and cysteine residues of the proteins, peptides containing Cys25 and Cys94 of 14-3-3 protein zeta and Cys83 of peroxiredoxin-1 were significantly reduced by this sesquiterpene ester. Although the results did not elucidate the effects of compound 8 in breast cancer cells, identification of potential target proteins contributes to enhanced understanding of its antiproliferative and anti-inflammatory effects.

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Method for Pharmacokinetic Evaluation of 7ß-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranon in Rats.

Recently, potent neuroprotective and anti-diabetic effects of 7ß-(3-Ethyl-cis-crotonoyloxy)-1α-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (ECN), a sesquiterpenoid isolated from Tussilago farfara Linnaeus, have been elucidated. To facilitate further pre-clinical evaluation in rats, an analytical method for the determination of ECN in rat plasma was developed and optimized by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples were pretreated by the protein precipitation method with an acetonitrile solution of losartan (LST) as the internal standard. Chromatographic separation was performed using a an Octadecyl-silica (ODS) column (2.6 µm, 100 x 4.6 mm) in the isocratic mode. The mobile phase, comprising 10 mM ammonium formate in water pH 5.75) and acetonitrile (11:89, v/v), was eluted at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed in the multiple reaction monitoring mode with positive electrospray ionization, and the mass transitions of ECN and LST were m/z 431.3 to 97.3 and m/z 423.1 to 207.2, respectively. The calibration curves of spiked plasma samples were linear in the 10.0-10,000 ng/mL range (r2 > 0.996). The lower limit of quantification (LLOQ) was determined as 10.0 ng/mL. Validation was conducted in the LLOQ, and three quality control (QC) sample levels (10.0, 25.0, 3750, and 7500 ng/mL) were studied. Among them, the relative standard deviation for the within- and between-run precisions was under 9.90%, and the relative error of the accuracies was within the -8.13% to 0.42% range. The validated method was successfully employed to investigate the pharmacokinetic properties of ECN in rats, which revealed the linear pharmacokinetic behavior of ECN for the first time.

N-Pyrazoloyl and N-thiopheneacetyl hydrazone of isatin exhibited potent anti-inflammatory and anti-nociceptive properties through suppression of NF-κB, MAPK and oxidative stress signaling in animal models of inflammation.

BACKGROUND: Hydrazide derivatives constitute an important class of compounds for new drug development as they are reported to possess good anti-inflammatory and analgesic activity. The present study was aimed to investigate the role of newly synthesized hydrazide derivatives N-pyrazoloyl hydrazone of isatin (PHI) and N-thiopheneacetyl hydrazone of isatin (THI) in acute and chronic inflammatory pain models induced by carrageenan and complete Freud's adjuvant (CFA). MATERIALS: PHI and THI (0.1, 1 and 10 mg/kg) pretreatments were provided intraperitoneally to male BALB/c mice prior to inflammatory inducers. Behavioral responses to inflammation and pain were evaluated by assessment of paw edema, mechanical allodynia, mechanical and thermal hyperalgesia. Cytokines production and NF-κB levels were evaluated by ELISA. Western blot analysis was performed for the detection of IκBα, p38, JNK and ERK. Hematoxylin and eosin (H&E) staining and radiographic analysis were performed to evaluate the effect of PHI and THI treatment on bone and soft tissues. Oxidative stress was determined by reduced glutathione, glutathione-S-transferase and catalase assays. Evans blue dye was used to monitor vascular protein leakage. RESULT: PHI and THI dose dependently (0.1, 1 and 10 mg/kg) reduced inflammation and pain in mice, however, the dose of 10 mg/kg exhibited significant activity. The anti-inflammatory and analgesic effects were attributed to suppression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) production levels. PHI and THI significantly blocked CFA-induced activation of NF-κB and MAPK signaling pathways. Oxidative stress and plasma nitrite levels were reduced remarkably. The PHI and THI (10 mg/kg) treatment did not exhibit any apparent toxicity on the liver, kidney, muscles strength, and motor co-ordination in mice. CONCLUSION: Both PHI and THI possess significant anti-inflammatory and analgesic activity via inhibition of inflammatory mediators.

Igalan induces detoxifying enzymes mediated by the Nrf2 pathway in HepG2 cells.

Igalan is one of the sesquiterpene lactones found in Inula helenium L., which is used as the traditional medicine to treat inflammatory diseases. However, the pharmacological effects of igalan have not been characterized. In this study, we isolated igalan from I. helenium L. and evaluated the effects of igalan on signaling pathways and expression of target genes in HepG2 cells. Igalan activated the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by increasing the inactive form of GSK3ß, the phosphorylated form of AKT, and the nuclear accumulation of Nrf2. Thus, target genes of Nrf2 such as HO-1 and NQO1 increased in HepG2 cells. Moreover, igalan inhibited the tumor necrosis factor-α (TNF-α)-induced nuclear factor-κB activation and suppressed the expression of its target genes, including TNF-α, interleukin (IL)-6, and IL-8 in HepG2 cells. Our results indicate the potential of igalan as an activator of cellular defense mechanisms and a detoxifying agent.

Antinociceptive properties of 25-methoxy hispidol A, a triterpinoid isolated from Poncirus trifoliata (Rutaceae) through inhibition of NF-κB signalling in mice.

The 25-methoxy hispidol A (25-MHA) is a triterpenoid, isolated from the immature fruit of Poncirus trifoliata (Rutaceae). The pretreatment with 25-MHA markedly (p < 0.001) attenuated the formalin-induced biphasic responses as well as acetic acid-induced writhing responses. The intraperitoneal administration of 25-MHA significantly attenuated the mechanical hyperalgesia (p < 0.001) and allodynia (p < 0.05). Similarly, 25-MHA also significantly attenuated (p < 0.001) complete Freund's adjuvant (CFA)-induced paw edema in mice. The 25-MHA treatment significantly attenuated the production of nuclear kappa B (NF-κB) (p65 nuclear subunit). The cytokines are the important mediators of inflammation and pain; however, treatment with 25-MHA exhibited significant inhibition (p < 0.001) on the mRNA expression levels of various inflammatory mediators. The 25-MHA administration also significantly enhanced antioxidant enzymes (p < 0.001) and inhibited the oxidative stress markers. The current study indicates that 25-MHA significantly (p < 0.001) inhibited the nitric oxide (NO) in mice plasma. Similarly, the haematoxylin and eosin (H&E) staining shows that 25-MHA administration significantly inhibited the inflammatory process in the mice paw tissue compared with the CFA-treated group. The 25-MHA treatment did not exhibited any toxicity on the liver, kidney, muscles strength, and motor co-ordination in mice. The 25-MHA was coadministered with the various drugs such as tramadol, piroxicam, and gabapentin to observe the synergistic effect.

Anticancer Activity of Smallanthus sonchifolius Methanol Extract against Human Hepatocellular Carcinoma Cells.

Background: This research aimed to investigate the cytotoxicity of methanol extract of Smallanthus sonchifolius leaf (YLE) against a human hepatocellular carcinoma cell line (HepG2). This plant is currently used as a traditional herbal remedy in the treatment of liver diseases in some rural parts of Myanmar. Methods: The cytotoxic activity of the plant extract against the cancerous cell line was assessed using an MTT assay. YLE demonstrated a significant effect (IC50 = 58.2 ± 1.9 µg/mL) on anti-cancer activity, which was further investigated using various assays including an in vitro cell migration assay, a colony formation assay, cell cycle analysis, western blot analysis, and a ROS assay. The significance of the phytochemical constituents of YLE could be identified using LC/Q-TOF-MS techniques. Results: We putatively identified the active components in YLE, which were possibly melampolide-type sesquiterpenoids. YLE showed an inhibitory effect on HepG2 cell proliferation and cell migration. YLE also induced cell cycle arrest and necrosis in a dose-dependent manner. Additionally, YLE significantly suppressed ROS formation in HepG2 cells. Conclusions: These findings suggest that YLE is sufficient for application as a promising anti-liver drug in herbal medicine.

Sesquiterpene lactones-enriched fraction of Inula helenium L. induces apoptosis through inhibition of signal transducers and activators of transcription 3 signaling pathway in MDA-MB-231 breast cancer cells.

Inula helenium L., commonly known as Elecampane, has been extensively used for many countries in the folk medicine. Its root is a rich source of sesquiterpene lactones, which possess various pharmacological activities. To develop the phytomedicine including sesquiterpene lactones, we prepared hexane fraction from I. helenium (HFIH) and examined the inhibitory effect of HFIH on signal transducers and activators of transcription 3 (STAT3) activation in human breast cancer MDA-MB-231 cells. Additionally, detailed chemical investigation was done to pinpoint the most active sesquiterpene lactones responsible for its anticancer activity. HFIH selectively suppressed STAT3 phosphorylation at tyrosine 705, not affecting its upstream kinases. HFIH downregulated the expression of STAT3 target genes including cyclin D1 , c-myc, and bcl-2 and induced caspase-mediated apoptosis. Moreover, sesquiterpene lactones of HFIH clearly suppressed STAT3 activation. The in vivo results further supported that HFIH inhibits the growth of human breast xenograft tumors. Our results suggest that HFIH possesses potential anticancer activity, which is mainly mediated through STAT3 signaling pathway. These findings provide the potential of HFIH as a promising phytomedicine for the treatment and prevention of triple-negative breast cancer.

Correction to: Anti-inflammatory, anti-rheumatic and analgesic activities of 2-(5-mercapto-1,3,4-oxadiazol-2-yl)-N-propylbenzenesulphonamide (MOPBS) in rodents.

Unfortunately, Fig. 1 was incorrectly published in the original publication. The corrected Fig. 1 is given as below.

Anti-inflammatory, anti-rheumatic and analgesic activities of 2-(5-mercapto-1,3,4-oxadiazol-2-yl)-N-propylbenzenesulphonamide (MOPBS) in rodents.

Chronic inflammation is pathologically associated with various clinical conditions such as rheumatoid arthritis. Several anti-inflammatory and analgesic drugs currently available in market presents a wide range of problems. Therefore, the current study was aimed to evaluate anti-inflammatory and analgesic activities of newly synthesized compound 2-(5-mercapto-1,3,4-oxadiazol-2-yl)-N-propylbenzenesulphonamide (MOPBS). Carrageenan and CFA-induced models were developed for evaluation of anti-inflammatory and analgesic activity. Quantitative real-time PCR (qRT-PCR) was performed to determine the mRNA expression levels of inflammatory mediators. Pain behaviours were evaluated by performing Von Frey, Randall Selitto, cold acetone and hot plate test respectively. X-ray imaging and haematoxylin and eosin (H&E) staining were performed for examination of soft tissues of treated mice paw. Additionally, Kodzeila's screen test and weight test were performed for determination of any side effects on motor function and muscle strength. Acute pretreatment of animals with MOPBS (1, 10, 50 and 100 mg/kg, i.p.) produced a significant reduction of paw oedema against carrageenan-induced acute inflammation as well as notable inhibition of mechanical hyperalgesia, allodynia and thermal hyperalgesia. Similarly, in chronic inflammation model, administration of MOPBS (50 mg/kg, i.p.) produced a remarkable reduction of paw oedema. Additionally, MOPBS pretreatment showed a significant inhibition of thermal hyperalgesia, mechanical allodynia, and mechanical hyperalgesia in chronic arthritis model. Several pro-inflammatory mediators such as nitric oxide (NO), vascular endothelial growth factor (VEGF), interleukins (IL-1ß, IL-6) and tumor necrosis factor-α (TNF-α) were inhibited by MOPBS treatment in blood plasma and paw tissues, respectively. MOPBS also enhanced the mRNA expression levels of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), superoxide dismutase (SOD2) and heme oxygenase (HO-1) and in turn reduced arthritis severity and inflammation. Furthermore, anti-inflammatory data were confirmed by X-rays and histological analysis. MOPBS pretreatment did not produce any apparent toxic effect on gastric, kidney and liver function and on muscle strength and motor function. Hence, the present data suggest that MOPBS might be a candidate for several chronic inflammatory diseases such RA and other auto-immune diseases.

Identification of galiellalactone-based novel STAT3-selective inhibitors with cytotoxic activities against triple-negative breast cancer cell lines.

Signal transducer and activator of transcription 3 (STAT3) is phosphorylated in breast cancer cells, particularly triple-negative breast cancers (TNBCs). Therefore, the inhibition of constitutive phosphorylated STAT3 is a promising therapeutic for TNBC treatment. Recently, a series of novel STAT3 inhibitors based on natural (-)-galiellalactone have been identified to inhibit STAT3 phosphorylation at the Tyr705 residue. Interestingly, the truncation of the cyclohexene moiety of (-)-galiellalactone to [3.3] bicyclic lactone as a pharmacophoric core produced improved cytotoxic effects against TNBCs. The potent analogues 16 and 17, identified from a STAT3-mediated luciferase reporter assay, selectively inhibited the STAT3 signaling pathway without affecting STAT1 or STAT5.

Application of linear gradient elution in countercurrent chromatography for the separation of triterpenoid saponins from the roots of Pulsatilla koreana Nakai.

A linear gradient elution method using countercurrent chromatography was developed for the separation of four triterpenoid saponins from the roots of Pulsatilla koreana Nakai, including hederacolchiside E, which is responsible for the neuroprotective activity of this plant. The target fraction was obtained by 80% methanol elution of solid phase column chromatography. The partition coefficients of the target compounds were very different, which means they are difficult to separate with a single biphasic solvent system. Several important parameters for gradient elution, such as addition of alcohol content to the solvent system, starting point of the second mobile phase, and the time for the gradient change were logically determined and optimized. Four triterpenoid saponins could ultimately be separated, analyzed by high-performance liquid chromatography, and their structures were identified by comparing the mass spectra and NMR spectra with the literature data. The compounds and yields were: hederasaponin B (1; 21.3 mg/100 mg), hederacolchiside E (2; 19.8 mg/100 mg), cernuoside A (3; 18.4 mg/100 mg), and cernuoside B (4; 17.3 mg/100 mg). Gradient-elution countercurrent chromatography allows the effective separation of compounds with a wide polarity range.

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.

Metabolic interactions of magnolol with cytochrome P450 enzymes: uncompetitive inhibition of CYP1A and competitive inhibition of CYP2C.

Magnolol (MAG; 5,5'-diallyl-2,2'-biphenyldiol) is a major bioactive component of Magnolia officinalis. We investigated the metabolic interactions of MAG with hepatic cytochrome P450 monooxygenase (CYP) through in vitro microsomal metabolism study using human (HLM) and rat liver microsomes (RLM). CYP2C and 3A subfamilies were significantly involved in the metabolism of MAG, while CYP1A subfamily was not in HLM and RLM. The relative contribution of phase I enzymes including CYP to the metabolism of MAG was comparable to that of uridine diphosphate glucuronosyltransferase (UGT) in RLM. Moreover, MAG potently inhibited the metabolic activity of CYP1A (IC50 of 1.62 µM) and 2C (IC50 of 5.56 µM), while weakly CYP3A (IC50 of 35.0 µM) in HLM and RLM. By the construction of Dixon plot, the inhibition type of MAG on CYP activity in RLM was determined as follows: uncompetitive inhibitor for CYP1A (Ki of 1.09-12.0 µM); competitive inhibitor for CYP2C (Ki of 10.0-15.2 µM) and 3A (Ki of 93.7-183 µM). Based on the comparison of the current IC50 and Ki values with a previously reported liver concentration (about 13 µM) of MAG after its seven times oral administration at a dose of 50 mg/kg in rats, it is suggested that MAG could show significant inhibition of CYP1A and 2C, but not CYP3A, in the in vivo rat system. These results could lead to further studies in clinically significant metabolism-mediated MAG-drug interactions.

Heparosan-glucuronate 5-epimerase: Molecular cloning and characterization of a novel enzyme.

Iduronic acid (IdoA) is a critical component of heparan sulfate in its interaction with functional proteins. Heparosan-N-sulfate-glucuronate 5-epimerase (HNSG-5epi) converts d-glucuronic acid (GlcA) residues in N-sulfated heparosan (NS-heparosan), as an intermediate in heparan sulfate biosynthesis, to IdoA. In the present study, the authors discovered a different 5-epimerase, designated HG-5epi (heparosan-glucuronate 5-epimerase), that is involved in acharan sulfate biosynthesis and possesses novel substrate specificity. A candidate cDNA of HG-5epi was cloned from the cDNA library of Achatina fulica. The cloned cDNA contained a whole coding region that predicts a type II transmembrane protein composed of 601 amino acid residues. The amino acid sequence of HG-5epi is homologous to that of HNSG-5epi. Recombinant HG-5epi was expressed in insect cells and its enzymatic properties characterized. As expected, HG-5epi epimerizes GlcA residues in heparosan, but not in NS-heparosan. Conversion of IdoA to GlcA was also catalyzed by HG-5epi when completely desulfated N-acetylated heparin was used as the substrate, indicating a reversible reaction mechanism. At equilibrium of the epimerization, the proportion of IdoA in the reaction product reached up to 30% of total hexuronic acid. To our knowledge, this is the first report to describe an enzyme that catalyzes the epimerization of non-sulfated heparosan. This new enzyme may be applied to the study of synthetic heparan sulfate-related polysaccharides having certain biological and pharmacological activities. In addition, a new method using anion-exchange HPLC connected to a post-column fluorescent labeling system was developed for analyzing hexuronic acid isomers.

Platycodin D Blocks Breast Cancer-Induced Bone Destruction by Inhibiting Osteoclastogenesis and the Growth of Breast Cancer Cells.

BACKGROUND: Metastatic breast cancer cells are frequently associated with osteoclast-mediated bone resorption, resulting in severe bone destruction and increased mortality in patients. Platycodin D (PD) isolated from Platycodon grandiflorum is a triterpenoid saponin with anti-cancer and anti-angiogenic potential. METHODS: The in vivo activity was determined in mice with the intratibial injection of human metastatic breast cancer cells. Osteoclast formation and activity were detected using tartrate-resistant acid phosphatase staining and calcium phosphate-coated plates. The expression of osteoclastogenesis-inducing molecules was detected by RT-PCR and western blotting in RANKL-treated bone marrow macrophages (BMMs). Cell viability and DNA synthesis were measured with MTT and BrdU incorporation assays. The induction of apoptosis was estimated using TUNEL staining and a caspase-3 activity assay. RESULTS: The oral administration of PD inhibited MDA-MB-231 cell-induced osteolysis in an intratibial mouse model. PD treatment blocked RANKL-induced osteoclast formation by inhibiting the expression and nuclear translocation of NFATc1 and c-Fos in BMMs and consequently reduced osteoclast-mediated bone resorption. Furthermore, PD treatment induced apoptosis in osteoclasts and inhibited the growth of MDA-MB-231 cells. CONCLUSION: PD may block breast cancer-induced bone loss by suppressing the formation, activity, and survival of osteoclasts, as well as the growth of metastatic breast cancer cells.

Chikusetsusaponin IVa methyl ester induces cell cycle arrest by the inhibition of nuclear translocation of ß-catenin in HCT116 cells.

We demonstrate that chikusetsusaponin IVa methyl ester (CME), a triterpenoid saponin from the root of Achyranthes japonica, has an anticancer activity. We investigate its molecular mechanism in depth in HCT116 cells. CME reduces the amount of ß-catenin in nucleus and inhibits the binding of ß-catenin to specific DNA sequences (TCF binding elements, TBE) in target gene promoters. Thus, CME appears to decrease the expression of cell cycle regulatory proteins such as Cyclin D1, as a representative target for ß-catenin, as well as CDK2 and CDK4. As a result of the decrease of the cell cycle regulatory proteins, CME inhibits cell proliferation by arresting the cell cycle at the G0/G1 phase. Therefore, we suggest that CME as a novel Wnt/ß-catenin inhibitor can be a putative agent for the treatment of colorectal cancers.

Simultaneous separation of three isomeric sennosides from senna leaf (Cassia acutifolia) using counter-current chromatography.

Senna leaf is widely consumed as tea to treat constipation or to aid in weight loss. Sennoside A, A1 , and B are dirheinanthrone glucosides that are abundant and the bioactive constituents in the plant. They are isomers that refer to the (R*R*), (S*S*), and (R*S*) forms of protons on C-10 and C-10' centers and it is difficult to refine them individually due to their structural similarities. The new separation method using counter-current chromatography successfully purified sennoside A, A1 , and B from senna leaf (Cassia acutifolia) while reversed-phase medium-pressure liquid chromatography yielded sennoside A only. n-Butanol/isopropanol/water (5:1:6, v/v/v) was selected as the solvent system for counter-current chromatography operation, and the partition coefficients were carefully determined by adding different concentrations of formic acid. High-resolution mass spectrometry and NMR spectroscopy were performed to verify the chemical properties of the compounds.

Modulation of Cytochrome P450 Activity by 18ß-Glycyrrhetic Acid and its Consequence on Buspirone Pharmacokinetics in Rats.

The aim of this study was to elucidate the inhibition mechanism of 18ß-glycyrrhetic acid (GLY) on cytochrome P450 (CYP) activity and in vivo pharmacokinetic consequences of single GLY dose in rats. An in vitro CYP inhibition study in rat liver microsomes (RLM) was conducted using probe substrates for CYPs. Then, an in vivo pharmacokinetics of intravenous and oral buspirone (BUS), a probe substrate for CYP3A, was studied with the concurrent administration of oral GLY in rats. In the in vitro CYP inhibition study, CYP3A was involved in the metabolism of GLY. Moreover, GLY inhibited CYP3A activity with an IC50 of 20.1 ± 10.7 µM via a mixed inhibition mechanism. In the in vivo rat pharmacokinetic study, single oral GLY dose enhanced the area under plasma concentration-time curve (AUC) of intravenous and oral BUS, but the extent of increase in AUC was only minimal (1.12-1.45 fold). These results indicate that GLY can inhibit the in vitro CYP3A-mediated drug metabolism in RLM via a mixed inhibition mechanism. However, the impact of single oral GLY dose on the pharmacokinetics of BUS in rats was limited, showing that GLY could function as merely a weak inhibitor for CYP3A-mediated drug metabolism in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

A comparative study on hulled adlay and unhulled adlay through evaluation of their LPS-induced anti-inflammatory effects, and isolation of pure compounds.

Coicis semen (=the hulled seed of Coix lacryma-jobi L. var. ma-yuen (Rom.Caill.) Stapf; Gramineae), commonly known as adlay and Job's tears, is widely used in traditional medicine and as a nutritious food. Bioassay-guided fractionation of the AcOEt fraction of unhulled adlays, using measurement of nitric oxide (NO) production on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells, led to the isolation and identification of two new stereoisomers, (+)-(7'S,8'R,7″S,8″R)-guaiacylglycerol ß-O-4'-dihydrodisinapyl ether (1) and (+)-(7'S,8'R,7″R,8″R)-guaiacylglycerol ß-O-4'-dihydrodisinapyl ether (2), together with six known compounds, 3-8. Compounds 3 and 4 exhibited inhibitory activities on LPS-induced NO production with IC50 values of 1.4 and 3.7 µM, respectively, and suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expressions in RAW 264.7 macrophage cells. Simple high-performance liquid chromatography with ultraviolet detection (HPLC/UV) was used to compare the AcOEt fraction of unhulled adlays responsible for the anti-inflammatory activity in RAW 264.7 cells and the inactive AcOEt fraction of hulled adlays.