Analysis of volatile metabolite variations in strip green tea during processing and effect of rubbing degree using untargeted and targeted metabolomics.

Strip green tea (SGT) is widely distributed in China owing to its unique appearance and aroma but the evolution and formation mechanisms of volatile metabolites (VMs) during SGT processing, and especially in the unique process of rubbing, remain unclear. In this study, based on untargeted metabolomics, 217 VMs (8 categories) were identified, and fixation and rubbing processes were found to be key for SGT aroma formation. Moreover, targeted metabolomics was applied to obtain 38 differential VMs and their related substances, of which fatty acid-derived volatiles (14 VMs) and glycoside-derived volatiles (8 VMs) showed significant contributions to SGT aroma, and their derivation laws during SGT manufacturing were clarified. Furthermore, the effect of rubbing degree on volatile metabolite formation was explored, and 11 key differential VMs were screened by variable importance in projection, and odor activity value analyses. Appropriate rubbing promoted the loss of grassy VMs (such as 1-octanol and 2-pentyl-furan) and enrichment of floral/fruity VMs (such as trans-ß-ionone, nonanal, geraniol, citral, (Z)-3,7-dimethyl-2,6-octadien-1-ol, and (Z)-hexanoic acid, 3-hexenyl ester). Our study not only enriches the chemical theory of green tea processing but also provides technical support for the precision directional processing of high-quality SGT.

Effects of Lipophilicity and Structural Features on the Antiherpes Activity of Digitalis Cardenolides and Derivatives.

There is growing interest in exploring Digitalis cardenolides as potential antiviral agents. Hence, we herein investigated the influence of structural features and lipophilicity on the antiherpes activity of 65 natural and semisynthetic cardenolides assayed in vitro against HSV-1. The presence of an α,ß-unsaturated lactone ring at C-17, a ß-hydroxy group at C-14 and C-3ß-OR substituents were considered essential requirements for this biological activity. Glycosides were more active than their genins, especially monoglycosides containing a rhamnose residue. The activity enhanced in derivatives bearing an aldehyde group at C-19 instead of a methyl group, whereas inserting a C-5ß-OH improved the antiherpes effect significantly. The cardenolides lipophilicity was accessed by measuring experimentally their log P values (n-octanol-water partition coefficient) and disclosed a range of lipophilicity (log P 0.75±0.25) associated with the optimal antiherpes activity. In silico studies were carried out and resulted in the establishment of two predictive models potentially useful to identify and/or optimize novel antiherpes cardenolides. The effectiveness of the models was confirmed by retrospective analysis of the studied compounds. This is the first SAR study addressing the antiherpes activity of cardenolides. The developed computational models were able to predict the active cardenolides and their log P values.

1-Octanol emitted by Oecophylla smaragdina weaver ants repels and deters oviposition in Queensland fruit fly.

Humans have used weaver ants, Oecophylla smaragdina, as biological control agents to control insect pests in orchards for many centuries. Over recent decades, the effectiveness of weaver ants as biological control agents has been attributed in part to deterrent and oviposition inhibiting effects of kairomones produced by the ants, but the chemical identity of these kairomones has remained unknown. We have identified the kairomone responsible for deterrence and oviposition inhibition by O. smaragdina, providing a significant advance in understanding the chemical basis of their predator/prey interactions. Olfactometer assays with extracts from weaver ants demonstrated headspace volatiles to be highly repellent to Queensland fruit fly, Bactrocera tryoni. Using electrophysiology and bioassays, we demonstrate that this repellence is induced by a single compound, 1-octanol. Of 16 compounds identified in O. smaragdina headspace, only 1-octanol evoked an electrophysiological response from B. tryoni antennae. Flies had greatly reduced oviposition and spent significantly less time in an olfactometer arm in the presence of 1-octanol or a synthetic blend of headspace volatiles containing 1-octanol than in the presence of a synthetic blend of headspace volatiles without 1-octanol, or clean air. Taken together, our results demonstrate that 1-octanol is the functional kairomone component of O. smaragdina headspace that explains repellence and oviposition deterrence, and is hence an important contributor to the effectiveness of these ants as biological control agents.

Building Flexible Escherichia coli Modules for Bifunctionalizing n-Octanol: The Byproduct of Oleic Acid Biorefinery.

Artificial biorefinery of oleic acid into 1,10-decanedioic acid represents a revolutionizing route to the sustainable production of chemically difficult-to-make bifunctional chemicals. However, the carbon atom economy is extremely low (56%) due to the formation of unifunctional n-octanol. Here, we report a panel of recombinant Escherichia coli modules for diverse bifunctionalization, where the desired genetic parts are well distributed into different modules that can be flexibly combined in a plug-and-play manner. The designed ω-functionalizing modules could achieve ω-hydroxylation, consecutive ω-oxidation, or ω-amination of n-octanoic acid. By integrating these advanced modules with the reported oleic acid-cleaving modules, high-value C8 and C10 products, including ω-hydroxy acid, ω-amino acid, and α,ω-dicarboxylic acid, were produced with 100% carbon atom economy. These ω-functionalizing modules enabled the complete use of all of the carbon atoms from oleic acid (released from plant oil) for the green synthesis of structurally diverse bifunctional chemicals.

Preparation and characterization of 3D human glioblastoma spheroids using an N-octanoyl glycol chitosan hydrogel.

The current 2D culture model systems developed for drug screening are not sufficient to reflect the characteristics of in vivo solid tumors. Therefore, more effective in vitro tumor model systems must be developed for translational studies on therapeutic drug screening and testing. Herein, we report a new ultra-low adhesion (ULA) hydrogel for generating 3D cancer cell spheroids as tumor models in vitro. N-octanoyl glycol chitosan (OGC) was synthesized and coated onto the surface of a typical cell culture dish. Cell spheroids were effectively formed on the OGC-coated surface, and phenotypes of the tumor cells were well maintained during culture. More importantly, U373-MG cells cultured on OGC-coated plates were more resistant to doxorubicin than cells cultured on typical plates. Our OGC-based ULA system may offer a convenient method for 3D cell culture to provide enhanced performance in cancer research, drug screening and toxicology.

Volatile 1-octanol of tea (Camellia sinensis L.) fuels cell division and indole-3-acetic acid production in phylloplane isolate Pseudomonas sp. NEEL19.

Tea leaves possess numerous volatile organic compounds (VOC) that contribute to tea's characteristic aroma. Some components of tea VOC were known to exhibit antimicrobial activity; however, their impact on bacteria remains elusive. Here, we showed that the VOC of fresh aqueous tea leaf extract, recovered through hydrodistillation, promoted cell division and tryptophan-dependent indole-3-acetic acid (IAA) production in Pseudomonas sp. NEEL19, a solvent-tolerant isolate of the tea phylloplane. 1-octanol was identified as one of the responsible volatiles stimulating cell division, metabolic change, swimming motility, putative pili/nanowire formation and IAA production, through gas chromatography-mass spectrometry, microscopy and partition petri dish culture analyses. The bacterial metabolic responses including IAA production increased under 1-octanol vapor in a dose-dependent manner, whereas direct-contact in liquid culture failed to elicit such response. Thus, volatile 1-octanol emitting from tea leaves is a potential modulator of cell division, colonization and phytohormone production in NEEL19, possibly influencing the tea aroma.

Novel SN38 derivative-based liposome as anticancer prodrug: an in vitro and in vivo study.

BACKGROUND: Many novel drug delivery systems have been extensively studied to exploit the full therapeutic potential of SN38, which is one of the most potent antitumor analogs of camptothecins (CPTs), whose clinical application is seriously hindered by poor water solubility, low plasmatic stability, and severe toxicity, but results are always unsatisfactory. METHODS: In this study, combining the advantages of prodrug and nanotechnology, a lipophilic prodrug of SN38, SN38-PA, was developed by conjugating palmitic acid to SN38 via ester bond at C10 position, and then the lipophilic prodrug was encapsulated into a long-circulating liposomal carrier by film dispersion method. RESULTS: The SN38-PA liposomes were characterized as follows: an average particle size of 80.13 nm, an average zeta potential of -33.53 mv, and the entrapment efficiency of 99%. Compared with CPT-11, SN38-PA liposome was more stable in close lactone form, more efficient in conversion rate to SN38, and more potent in cytotoxicity against tumor cells. Pharmacokinetic study showed that SN38-PA liposome had significantly enhanced plasma half-life (t1/2) value of SN38 and increased area under the curve (AUC) of SN38, which was 7.5-fold higher than that of CPT-11. Biodistribution study showed that SN38-PA liposome had more active metabolite SN38 in each tissue. Finally, the pharmacodynamic study showed that SN38-PA liposome had higher antitumor effect with the antitumor inhibition rate of 1.61 times than that of CPT-11. CONCLUSION: These encouraging data merit further investigation on this novel SN38-PA liposome.

Biological activity and safety profile of the essential oil from fruits of Heracleum mantegazzianum Sommier & Levier (Apiaceae).

A composition of essential oils obtained from Heracleum mantegazzianum (Apiaceae) was examined using a GC-MS method. n-Octyl acetate (19.92%), n-hexyl-2-methylbutanoate (10.84%), n-octanol (10.13%), n-octyl butanoate (8.88%), n-octyl-2-methylbutanoate (8.01%), n-hexyl acetate (7.11%), n-octyl isobutanoate (5.5%) and n-hexyl isobutanoate (5.43%) were the main compounds. The high-performance counter-current chromatography was applied for purification of aliphatic alcohols and esters. A mixture of n-hexane, acetonitrile and tetr-butyl methyl ether (1:1:0.1, v/v) allowed to obtain n-octanol, n-octyl acetate, n-hexyl-2- methylbutanoate, n-octyl isobutanoate and n-octyl-2-methylbutanoate, with the purity range of 94-99%, in one single 74 min run. The antimicrobial activity was also determined against plant and foodborne pathogens. While n-octanol shares responsibility for the antibacterial activity of the essential oil, n-octyl acetate determines its antifungal action. The cytotoxic activity assessed on two normal kidney fibroblast cell lines: Vero (animal) and HEK-293 (human embryonic), and two human cancer cell lines: FaDu (squamous cell carcinoma of the pharynx) and SCC25 (squamous cell carcinoma of the tongue), showed a moderate cytotoxicity with CC50 values ranging from 262.3 to 567.8 µg/mL. Results indicate that normal cell lines were more sensitive to the tested essential oil than cancer cell lines. The antioxidant activity of oil and pure compounds was not significant.

Salinity impacts on water solubility and n-octanol/water partition coefficients of selected pesticides and oil constituents.

Salinity has been reported to influence the water solubility of organic chemicals entering marine ecosystems. However, limited data are available on salinity impacts for chemicals potentially entering seawater. Impacts on water solubility would correspondingly impact chemical sorption as well as overall bioavailability and exposure estimates used in the regulatory assessment. The pesticides atrazine, fipronil, bifenthrin, and cypermethrin, as well as the crude oil constituent dibenzothiophene together with 3 of its alkyl derivatives, all have different polarities and were selected as model compounds to demonstrate the impact of salinity on their solubility and partitioning behavior. The n-octanol/water partition coefficient (KOW ) was measured in both distilled-deionized water and artificial seawater (3.2%). All compounds had diminished solubility and increased KOW values in artificial seawater compared with distilled-deionized water. A linear correlation curve estimated salinity may increase the log KOW value by 2.6%/1 log unit increase in distilled water (R2 = 0.97). Salinity appears to generally decrease the water solubility and increase the partitioning potential. Environmental fate estimates based on these parameters indicate elevated chemical sorption to sediment, overall bioavailability, and toxicity in artificial seawater. These dramatic differences suggest that salinity should be taken into account when exposure estimates are made for marine organisms. Environ Toxicol Chem 2017;36:2274-2280. © 2017 SETAC.

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.

α-Pinene, linalool, and 1-octanol contribute to the topical anti-inflammatory and analgesic activities of frankincense by inhibiting COX-2.

ETHNOPHARMACOLOGICAL RELEVANCE: Frankincense oil and water extracts (FOE, FWE) have long been used for external treatment of inflammation and pain. The present study was conducted to identify the active ingredients responsible for the anti-inflammatory and analgesic effects and to determine the underlying mechanisms. MATERIALS AND METHODS: The compositions of FOE and FWE were identified and compared by GC-MS. The anti-inflammatory and analgesic activities of the two extracts and their possible active ingredients (α-pinene, linalool, and 1-octanol) were evaluated and compared in a xylene-induced ear edema model and a formalin-inflamed hind paw model. Inflammatory infiltrates and cyclooxygenase-2 (COX-2) expression in hind paw skin were investigated by histological staining. RESULTS: The contents of α-pinene, linalool, and 1-octanol in FOE were much higher than those in FWE. Mice treated with FOE exhibited greater and faster lessening of swelling and pain than mice treated with FWE. The combination of the three components had more potent pharmacological effects on hind paw inflammation and COX-2 overexpression than the three components used alone. CONCLUSIONS: These findings suggest that topical application of FOE or its active ingredients (including α-pinene, linalool, and 1-octanol) exhibit significantly anti-inflammatory and analgesic effects through inhibiting nociceptive stimulus-induced inflammatory infiltrates and COX-2 overexpression.

[Solubilization characteristics of licorice based on supramolecular "imprinted template" theory].

To investigate the effect of the ingredient group of traditional Chinese medicine (TCM) with different "imprinted template" on the supramolecular solubilization ability of licorice, in order to lay a theoretical foundation for explaining the solubilization phenomenon of the components of TCM. Based on the independent supramolecular "imprinted template" rules, molecular connectivity index (MCI) and the correlation of n-octanol-water partition coefficient (logP) were used to indicate hydrophilic lipophilic capacity of TCM, and the extractum rate was used to indicate the solubilization effect of licoriceon single TCM herbs or compounds. The solubilization ability of licorice was evaluated based on MCI, logP and the extractum rate. According to the results, the correlation coefficient between MCI and logP for single herbs was 0.942, and that for single herb adding licorice was 0.916. The extractum rate of most herbs was increased after adding licorice. The correlation coefficient among the extractum rate as well as MCI and logP change values before and after adding licorice were respectively 0.837, 0.405. The correlation coefficient between MCI and logP for eight compounds was 0.937. Meanwhile, licorice had a solubilization effect on the remaining 7 compound except for Huangqi decoction. Therefore, licorice shows the solubilization effect through the independent supramolecular "imprinted template", so as to improve the hydrophilic lipophilic ability. There was a high positive correlation between the MCI and logP in ingredients for TCM, which could be used as important parameters to indicate the "imprinted template" feature for components of TCM. The study on the solubilizing effect of TCM with the supramolecular "imprinted template" theory was feasible, and will lay a foundation for the reform of single-herb dosage form.

Age-associated potency decline in bovine oocytes is delayed by blocking extracellular Ca(2+) influx.

Oocyte aging due to delayed fertilization is associated with declining quality and developmental potential. Intracellular calcium (Ca(2+)) concentration ([Ca(2+)]i) regulates oocyte growth, maturation, and fertilization and has also been implicated in aging. Using bovine oocytes, we tested the hypothesis that oocyte aging could be delayed by reducing [Ca(2+)]ivia blocking the influx of extracellular Ca(2+) or chelating ooplasmic free Ca(2+). After IVM, cumulus-oocyte complexes or denuded oocytes were cultured in medium supplemented with 1-octanol, phorbol 12-myristate 13-acetate, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis-acetoxymethyl ester (BAPTA-AM) to manipulate [Ca(2+)]i. Addition of 1-mM 1-octanol increased blastocyst development rates in the cumulus-oocyte complexes aged for 6 hours by IVF and for 6, 12, and 24 hours by parthenoactivation, and this effect was independent of the presence of cumulus cells. The intracellular levels of ATP, Glutathione, and Glutathione disulfide were not affected by 1-octanol, but [Ca(2+)]i was significantly decreased. When oocytes were cultured in Ca(2+)-free medium for 12 hours, the blastocyst development rate was greater and the beneficial effects of 1-octanol on oocyte aging were abolished. However, when the medium was supplemented with phorbol 12-myristate 13-acetate, [Ca(2+)]i increased and the blastocyst development rate decreased. Moreover, BAPTA-AM reduced [Ca(2+)]i and increased blastocyst development rates after IVF or parthenoactivation. We conclude that the age-associated developmental potency decline was delayed by blocking the influx of extracellular Ca(2+) or reducing ooplasmic free Ca(2+). 1-Octanol, BAPTA-AM, or Ca(2+)-free medium could be used to lengthen the fertilization windows of aged bovine oocytes.

[Determination of equilibrium solubility and n-octanol/water partition coefficient of pulchinenosiden D by HPLC].

OBJECTIVE: To determine the equilibrium solubility of pulchinenosiden D in different solvents and its n-octanol/water partition coefficients. METHOD: Combining shaking flask method and high performance liquid chromatography (HPLC) to detect the n-octanol/water partition coefficients of pulchinenosiden D, the equilibrium solubility of pulchinenosiden D in six organic solvents and different pH buffer solution were determined by HPLC analysis. RESULT: n-Octanol/water partition coefficients of pulchinenosiden D in different pH were greater than zero, the equilibrium solubility of pulchinenosiden D was increased with increase the pH of the buffer solution. The maximum equilibrium solubility of pulchinenosiden D was 255.89 g x L(-1) in methanol, and minimum equilibrium solubility of pulchinenosiden D was 0.20 g x L(-1) in acetonitrile. CONCLUSION: Under gastrointestinal physiological conditions, pulchinenosiden D exists in molecular state and it has good absorption but poor water-solubility, so increasing the dissolution rate of pulchinenosiden D may enhance its bioavailability.

Parenteral formulation of larotaxel lipid microsphere tackling poor solubility and chemical instability.

The purpose of this study was to develop a parenteral larotaxel lipid microsphere (LTX-LM) and evaluate its stability. The preformulation study showed that LTX possessed poor solubility (0.057 µg/mL in aqueous phase) and chemical instability. LM was selected as the drug carrier due to its higher drug-loading capacities, higher physicochemical stability and reduced irritation and toxicity. High speed shear mixing and high-pressure homogenization were employed to prepare the LTX-LM. Particle size distribution (PSD), zeta-potential, drug content and entrapment efficiency (EE) were taken as indexes to optimize formulations. The dissolution studies were performed using a ZRS-8G dissolution apparatus according to the paddle method. Degradation kinetics test, freezing and thawing test and long term stability test were combined to evaluate the physicochemical stability of LTX-LM. From the degradation kinetics results, the shelf lives (T90%) of LTX in LM at 25 and 4°C (165, 555 days) were about 20 times as long as those in aqueous phase (200, 676 h), which were dramatically prolonged. The activation energies in aqueous solution and in LM calculated from the slopes were 41.93 and 42.25 kJ/mol. And its frequency factors (A) were 4.9 × 10(3)/s and 2.3 × 10(2)/s, respectively. Freezing and thawing test showed the PSD of LTX-LM became larger and wider increasing from 166.9 ± 53.2 nm to 257.4 ± 85.5 nm with more freeze-thaw cycles. From the long term stability test results, all the parameters changes were in qualified range.

Simultaneous determination of three purines in Alysicarpus vaginalis (L.) DC. by hollow fiber-based liquid-phase microextraction combined with high-performance liquid chromatography.

In this paper, a three-phase hollow fiber liquid-phase microextraction (HF-LPME) method combined with high-performance liquid chromatography (HPLC) was developed for the determination of hypoxanthine (HX), xanthine (Xan) and adenine (A) and then for the first time successfully applied to the analysis of HX, Xan and A in Alysicarpus vaginalis (L.) DC. medicinal materials. Different factors affecting the HF-LPME procedure were investigated and optimized. Under optimal extraction conditions (1-octanol as organic solvent, pH of the donor and acceptor phase 10.0 and 3.5, respectively, extraction time 40 min, stirring rate 800 rpm and salt addition 10%, w/v), HX, Xan and A could be determined within the test ranges with a good correlation coefficient (r(2) > 0.9992). The limit of detection for HX, Xan and A was 153, 173 and 97 ng/mL, respectively, and the intra- and inter-day relative standard deviations were no more than 9.8%. The content of HX, Xan and A in Alysicarpus vaginalis (L.) DC. medicinal materials was 120.40, 18.37 and 62.75 µg/g, respectively. This procedure afforded a convenient, sensitive, accurate and inexpensive method with a high extraction efficiency for determination of HX, Xan and A.

Determination of equilibrium solubility and n-octanol/water partition coefficient of pulchinenosiden D by HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>To determine the equilibrium solubility of pulchinenosiden D in different solvents and its n-octanol/water partition coefficients.</p><p><b>METHOD</b>Combining shaking flask method and high performance liquid chromatography (HPLC) to detect the n-octanol/water partition coefficients of pulchinenosiden D, the equilibrium solubility of pulchinenosiden D in six organic solvents and different pH buffer solution were determined by HPLC analysis.</p><p><b>RESULT</b>n-Octanol/water partition coefficients of pulchinenosiden D in different pH were greater than zero, the equilibrium solubility of pulchinenosiden D was increased with increase the pH of the buffer solution. The maximum equilibrium solubility of pulchinenosiden D was 255.89 g x L(-1) in methanol, and minimum equilibrium solubility of pulchinenosiden D was 0.20 g x L(-1) in acetonitrile.</p><p><b>CONCLUSION</b>Under gastrointestinal physiological conditions, pulchinenosiden D exists in molecular state and it has good absorption but poor water-solubility, so increasing the dissolution rate of pulchinenosiden D may enhance its bioavailability.</p>

Physicochemical characterization and in vitro permeation of an indirubin derivative.

The active component of the traditional Chinese medicine, indirubin, exerts anticancer effect on different cancer cell lines. E804, a potent derivative of indirubin inhibits the activation of Stat3 and Stat5 in chronic myelocytic leukaemia (CML) cells. However, physicochemical properties and permeation rate of the compound relevant to the drug formulation have never been reported. Therefore, the ionization constant (pK(a)), lipophilicity (logD/P), aqueous and organic solubility of E804 and its permeation across Caco-2 cells were investigated. Both high throughput and traditional determinations were used in this study. The Caco-2 cell permeation assay was carried out in Poloxamer 188/HBSS++ solution in order to maintain the solubility of drug. The potential P-gp (P-glycoprotein) interaction for E804 was determined through Calcein-AM uptake assay. The results showed that E804 did not have a detectable pK(a) in the range of pH 2-11. Log D (distribution coefficient) and Log P (partition coefficient) were determined to be 3.54 ± 0.03. Aqueous solubility test revealed that E804 is practically insoluble in water. Among organic solvents E804 showed the highest solubility in DMSO. The P(app A→B) and P(app B→A) across Caco-2 cell monolayer were 2.0 ± 0.25 × 10(-6)cm/s and 1.14 ± 0.12 × 10(-6)cm/s respectively, and the calculated efflux ratio (ER) was 0.57. Calcein-AM uptake assay showed that E804 was not a strong substrate for P-gp. The results indicate that solubility is the major rate limiting step for the drug permeation. The high membrane permeability makes E804 promising for the oral delivery. Therefore, further investigation on solubility of E804 in lipid vehicles is needed to determine an appropriate formulation for the drug.

Identification of metabolites of Si-Ni-San, a traditional Chinese medicine formula, in rat plasma and urine using liquid chromatography/diode array detection/triple-quadrupole spectrometry.

Si-Ni-San (SNS) is a widely used traditional Chinese medicine formula (TCMF) in treating various diseases. However, the in vivo integrated metabolism of its multiple components remains unknown. In this paper, a liquid chromatography coupled with diode array detection and triple-quadrupole spectrometry (LC-DAD-MS/MS) method was developed for detection and identification of SNS metabolites in rat plasma and urine at a normal clinical dosage. Accurate structural elucidation was performed using MS/MS, UV data and n-octanol/water partition coefficient. Based on the proposed strategy, 36 absorbed compounds and 29 metabolites in plasma and 33 metabolites in urine were detected by a highly sensitive MRM method. Our results indicated that phase II reactions (e.g., methylation, glucuronidation and sulfation) were the main metabolic pathways of gallic acid and flavanones, while phase I reactions (e.g., hydroxylation) were the major metabolic reaction for triterpenoid saponins. The metabolite profile analysis of SNS provided a comprehensive understanding of the in vivo metabolic fates of constituents in SNS. Moreover, the results in this work demonstrated the present strategy based on the combination of chromatographic, spectrophotometric, mass-spectrometric, and software prediction to detect and identify metabolites was effective and reliable. And such a strategy may also be extended to investigate the metabolism of other TCMF.

A transmembrane amino acid in the GABAA receptor ß2 subunit critical for the actions of alcohols and anesthetics.

Alcohols and inhaled anesthetics enhance the function of GABA(A) receptors containing α, ß, and γ subunits. Molecular analysis has focused on the role of the α subunits; however, there is evidence that the ß subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the α subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABA(A) receptor ß(2) subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant α(1)ß(2)(N265C)γ(2S) receptors [but not α(1)ß(2)(I264C)γ(2S)], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the ß(2) subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant α(1)ß(2)(N265C)γ(2S) receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site.