Discordant Analytical Results Caused by Biotin Interference on Diagnostic Immunoassays in a Pediatric Hospital.

Recent studies have reported that biotin interferes with certain immunoassays. In this study, we evaluated the analytical interference of biotin on immunoassays that use streptavidin-biotin in our pediatric hospital. We tested the effect of different concentrations of biotin (1.5-200 ng/ml) on TSH, Prolactin, Ferritin, CK-MB, ß-hCG, Troponin I, LH, FSH, Cortisol, Anti-HAV antibody (IgG and IgM), assays on Ortho Clinical Diagnostic Vitros 5600 Analyzer. Biotin (up to 200 ng/mL) did not significantly affect Troponin I and HAV assays. Biotin (up to 12.5 ng/ml) resulted in <10% bias in CK-MB, ß-hCG, AFP, Cortisol, Ferritin assays and biotin >6.25 ng/mL significantly affected TSH (>20% bias) assay. Prolactin was significantly affected even at low levels (Biotin 1.5 ng/mL). Thus, we recommend educating physicians about biotin interference in common immunoassays and adding an electronic disclaimer.

High-Throughput Spheroid Screens Using Volume, Resazurin Reduction, and Acid Phosphatase Activity.

Mainstream adoption of physiologically relevant three-dimensional models has been slow in the last 50 years due to long, manual protocols with poor reproducibility, high price, and closed commercial platforms. This chapter describes high-throughput, low-cost, open methods for spheroid viability assessment which use readily available reagents and open-source software to analyze spheroid volume, metabolism, and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination-for both single images and images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyze plate uniformity (such as edge effects and systematic seeding errors), detect outliers, and calculate dose-response. The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time.

Dose-dependent artificial prolongation of prothrombin time by interaction between daptomycin and test reagents in patients receiving warfarin: a prospective in vivo clinical study.

BACKGROUND: Daptomycin has been reported to cause artificial prolongation of prothrombin time (PT) by interacting with some test reagents of PT. This prolongation was particularly prominent with high concentrations of daptomycin in vitro. However, whether this prolongation is important in clinical settings and the optimal timing to assess PT remain unclear. METHODS: A prospective clinical study was conducted with patients who received daptomycin for confirmed or suspected drug-resistant, gram-positive bacterial infection at a university hospital in Japan. PT at the peak and trough of daptomycin was tested using nine PT reagents. Linear regression analyses were used to examine the difference in daptomycin concentration and the relative change of PT-international normalized ratios (PT-INR). RESULTS: Thirty-five patients received daptomycin (6 mg/kg). The mean ± standard deviation of the trough and peak concentrations of daptomycin were 13.5 ± 6.3 and 55.1 ± 16.9 µg/mL, respectively. Twelve patients (34%) received warfarin. With five PT reagents, a significant proportion of participants experienced prolongation of PT-INR at the daptomycin peak concentration compared to the PT-INR at the trough, although the mean relative change was less than 10%. None of the participants clinically showed any signs of bleeding. A linear, dose-dependent prolongation of PT was observed for one reagent [unadjusted coefficient ß 3.1 × 10-3/µg/mL; 95% confidence interval (CI) 2.3 × 10-5-6.3 × 10-3; p = 0.048]. When patients were stratified based on warfarin use, this significant linear relationship was observed in warfarin users for two PT reagents (adjusted coefficient ß, 6.4 × 10-3/µg/mL; 95% CI 3.5 × 10-3-9.3 × 10-3; p < 0.001; and adjusted coefficient ß, 8.3 × 10-3/µg/mL; 95% CI 4.4 × 10-3-1.2 × 10-2; p < 0.001). In non-warfarin users, this linear relationship was not observed for any PT reagents. CONCLUSIONS: We found that a higher concentration of daptomycin could lead to artificial prolongation of PT-INR by interacting with some PT reagents. This change may not be clinically negligible, especially in warfarin users receiving a high dose of daptomycin. It may be better to measure PT at the trough rather than at the peak daptomycin concentration.

Micro-endoscopic system for functional assessment of neural circuits in deep brain regions: Simultaneous optical and electrical recordings of auditory responses in mouse's inferior colliculus.

In vivo Ca2+ imaging is a powerful method for the functional assessment of neural circuits. Although multi-photon excitation fluorescence microscopy has been widely used, observation of circuits in deep brain regions remains challenging. Recently, observing these deep regions has become possible via an endoscope consisting of an optical fiber bundle or gradient-index lens. We have designed a micro-endoscope system that enables simultaneous optical recording of fluorescence and electrical recording of neural activity. Using this system, we recorded auditory responses by simultaneously detecting changes in the fluorescence intensity of a Ca2+ indicator dye, multi-unit activities (MUA), and local field potentials (LFP) in the mouse's inferior colliculus (IC). Such simultaneous optical and electrical recordings enabled detailed comparison of electrically recorded phenomena (MUA and LFP) and optically recorded Ca2+ response. By systematically changing sound frequency and intensity, we determined the frequency tuning of the recording site. The best frequency shifted higher as the probe advanced more deeply, demonstrating that the system is capable of optically measuring the dorso-ventral organization of IC (i.e., tonotopicity). Thus, our new micro-endoscope system will be useful in the neurophysiological studies of a wide range of brain circuits, including those within the auditory system.

Carbon and silver nanoparticles in the fight against the filariasis vector Culex quinquefasciatus: genotoxicity and impact on behavioral traits of non-target aquatic organisms.

Mosquito-borne diseases represent a deadly threat for millions of people worldwide. The Culex genus, with special reference to Culex quinquefasciatus, comprises the most common vectors of filariasis across urban and semi-urban areas of Asia. In recent years, important efforts have been conducted to propose green-synthesized nanoparticles as a valuable alternative to synthetic insecticides. However, the mosquitocidal potential of carbon nanoparticles has been scarcely investigated. In this study, the larvicidal and pupicidal activity of carbon nanoparticle (CNP) and silver nanoparticle (AgNP) was tested against Cx. quinquefasciatus. UV-Vis spectrophotometry, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray (EDX) spectroscopy, and Raman analysis confirmed the rapid and cheap synthesis of carbon and silver nanoparticles. In laboratory assays, LC50 (lethal concentration that kills 50 % of the exposed organisms) values ranged from 8.752 ppm (first-instar larvae) to 18.676 ppm (pupae) for silver nanoparticles and from 6.373 ppm (first-instar larvae) to 14.849 ppm (pupae) for carbon nanoparticles. The predation efficiency of the water bug Lethocerus indicus after a single treatment with low doses of silver and carbon nanoparticles was not reduced. Moderate evidence of genotoxic effects induced by exposure to carbon nanoparticles was found on non-target goldfish, Carassius auratus. Lastly, the plant extract used for silver nanosynthesis was tested for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity. Overall, our results pointed out that AgNP and CNP can be a candidate for effective tools to reduce larval and pupal populations of filariasis vectors, with reduced genotoxicity and impact on behavioral traits of other aquatic organisms sharing the same ecological niche of Cx. quinquefasciatus.

Free radical scavenging, antidiarrheal and anthelmintic activity of Pistia stratiotes L. extracts and its phytochemical analysis.

In this phyto-pharmacological screening of Pistia stratiotes L leaf and root extracts each separately in two different solvents demonstrated its potential medicinal value. Apparent antioxidant value is demonstrated by DPPH, Nitric oxide scavenging and Ferric ion reducing method. Additionally, total flavonoid and phenolic compounds were measured. The leaf methanolic extract scavenged both nitric oxide (NO) and DPPH radical with a dose dependent manner. But the pet ether fraction of root was found to have highest efficacy in Fe(3±) reducing power assay. Flavonoid was found to contain highest in the pet ether fraction of root (411.35mg/g) in terms of quercetin equivalent, similarly highest amount (34.96mg/g) of total phenolic compounds (assayed as gallic acid equivalents) were found to contain in the same fraction. The methanolic fractions appeared less cytotoxic compared to pet ether extracts. The plant extracts caused a dose dependent decrease in faecal droppings in both castor oil and magnesium sulphate induced diarrhea, where as leaf extracts in each solvent appeared most effective. Also, the plant extracts showed anthelmintic activity in earthworm by inducing paralysis and death in a dose dependent manner. At highest doses (50 mg/ml) all fractions were almost effective as the positive control piperazine citrate (10 mg/ml). Thus, besides this cytotoxic effect it's traditional claim for therapeutic use can never be overlooked.

Various fractions of Hypericum x moserianum and Hypericum ericoides possess antiglycation, anti-lipid peroxidation, antioxidative activities and non-toxic effects in vitro.

In the present study, two species Hypericum x moserianum and Hypericum ericoides which belong to genus Hypericum were evaluated for their potential antiglycation, antioxidant, anti lipid peroxidation and cytotoxic activities. These species are widely used in folk medicine and to the best of our knowledge there were no previous reports regarding antioxidant, anti-glycation and cytotoxicity studies of these species. Among the crude methanol extracts and fractions of both the species, the ethyl acetate fraction of H. x moserianum exhibited promising antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 129.084±1.215µg/ml, followed by methanol extract (IC50=232.083 ± 1.215µg/ml) and aqueous fraction (IC50=266.962 ±2.213 µg/ml). The ethyl acetate fraction of H. ericoides exhibited IC50 value of 295.088 ± 2.320 µg/ml. In antiglycation assay, the ethyl acetate fraction of H. x moserianum showed 52.096% inhibition at 500µg/ml. For lipid peroxidation assay, the dichloromethane, aqueous and n-hexane fractions of H. x moserianum showed 67.241, 66.147 and 64.213% inhibition respectively, while aqueous fraction of H. ericoides exhibited 67.404% inhibition at 500µg/ml. In cytotoxicity assay, all fractions of both the species were found to be non-toxic on mouse fibroblast 3T3 cells with IC50 value greater than 30µg/ml as compared to cycloheximide with IC50 value 0.073±0.1µg/ml used as a standard. It was concluded from the study that among the two species, crude methanolic and ethyl acetate fractions were more active regarding the antioxidant, anti-glycation activities while dichloromethane, aqueous and n-hexane fractions possessed anti-lipid peroxidation activity.

Differences in the chemical profiles and biological activities of Paeonia lactiflora and Paeonia obovata.

Paeonia lactiflora and P. obovata are perennial herbs, each root of which has been consumed as a major oriental medicine, Paeoniae Radix and a famous folk medicine, Mountain Paeony Root, respectively. Although morphological studies have been performed comparing these two plants, there is insufficient scientific evidence that characterizes the differences in their chemical profiles and biological activities. Hence, the present study was undertaken to compare these two medicinal foods using a high-performance liquid chromatography-diode-array detector (HPLC-DAD) analysis and a gastric ulcer model in mice. HPLC analysis employed to assess the nine components revealed that P. lactiflora exhibited higher contents of phenolic compounds than P. obovata. Although a monoterpene glycoside, 6'-O-acetylpaeoniflorin was identified in P. obovata, it was not detected in P. lactiflora. Multivariate statistical analysis for HPLC data revealed that the orthogonal projections to latent structure-discriminant analysis is more appropriate than principal component analysis for differentiating the two groups. Moreover, the 50% methanol P. lactiflora extract (PL) was more effective against experimental gastric ulcer than P. obovata extract (PO) in the HCl/ethanol-induced ulcer model. In addition, PL displayed higher 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and lower nitric oxide production in a murine macrophage cell line, RAW 264.7, than PO. The DPPH radical scavenging activity of PL was as high as that of the positive control, butylated hydroxytoluene, at a concentration of 25 µg/mL.

A study on four antioxidation effects of lycium barbarum polysaccharides in vitro.

The objective of the study was to investigate the in vitro antioxidation activity of lycium barbarum polysaccharides (LBP). Ultraviolet spectrophotometry was adopted to determine the capability of LBP to clear superoxide anions, hydroxyl radicals, DPPH free radicals and ABTS free radicals. The result showed that the law for LBP to clear superoxide anions, hydroxyl radicals and DPPH free radicals was that the clearance rate increased gradually with the increase of the concentration, and when the concentration reached a certain value, the clearance rate leveled off, while the IC50 for clearing ABTS free radicals was 47.158 ± 6.231 µg/ml. The study concluded that LBP is a good in vitro antioxidant.

Induction of cytochrome P450 enzymes in primary equine hepatocyte culture.

In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC50=1.20 µM and 6.06 µM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.

A simple method to quickly and simultaneously purify and enrich intact rat brain microcapillaries and endothelial and glial cells for ex vivo studies and cell culture.

The blood-brain barrier is morphologically composed of cerebral microcapillary endothelium through its tight junctions. It serves as a mechanical, metabolic and cellular barrier and can also protect the brain from pathogen invasion. Many brain diseases involve a disturbance of blood-brain barrier function either as a consequence of a noxa or as primary failure. In vitro models of the blood-brain barrier are suitable tools to study drug transport, pathogen transmigration and leukocyte diapedesis across the cerebral endothelium. Such models have previously been derived mainly from porcine or bovine brain tissues. We describe here a simple method by which rat cerebral microcapillaries and cells of glial origin can be quickly and simultaneously purified. By using a capillary fragment size restriction method based on glass bead columns different fractions can be separated: vital, long capillary fragments for ex vivo uptake studies and smaller capillary fragments for endothelial culture. Furthermore, fractions can be obtained for astroglial and oligodendroglial cell cultures. With this method both microcapillary enrichment and glial cell purification are quickly achieved, which reduces expenditure, number of required animals and laboratory working time.

D-Isomer of gly-tyr-pro-cys-pro-his-pro peptide: a novel and sensitive in vitro trapping agent to detect reactive metabolites by electrospray mass spectrometry.

This paper describes a D-peptide isomer-based trapping assay using an LC/MS ion-trap spectrometer with an electrospray ionization (ESI) source as the analytical tool to study bioactivation of xenobiotics. Reactive metabolites were generated from parent compounds in in vitro incubations with different sources of CYP enzymes. A short D-isomer of gly-tyr-pro-cys-pro-his-pro proved to be a sensitive trapping agent and resistant to proteases. This method was tested with 16 probe substances. Acetaminophen, 1-chloro 2,4-dinitrobenzene, clozapine, diclofenac, imipramine, menthofuran, propranolol, pulegone and ticlopidine all formed D-peptide adducts, which were analogous to the GSH adducts previously described in the literature. New adducts were identified with clopidogrel (-Cl+peptide), nicotine (-CH(3+)H+peptide), nimesulide (+peptide) and tolcapone (+peptide), i.e., no GSH adducts of those drugs have been described in the literature. No adducts were identified with ciprofloxacin, ketoconazole and verapamil. In the literature no GSH adducts have been described with ciprofloxacin and verapamil. D-Peptide-based trapping proved to be a reliable and reproducible method to identify bioactivated intermediates. D-Peptide is a new and convenient protein trapping agent for use in early phase screening of bioactivation of new chemical entities and evaluation of toxic properties of chemicals.

Capillary electrophoresis-based sirtuin assay using non-peptide substrates.

Sirtuins are NAD(+)-dependent class III histone deacetylases, which catalyze the deacetylation of acetyl-lysine residues of histones and other protein substrates yielding the deacetylated protein, nicotinamide and 2'-O-acetyl-ADP-ribose. Two lysine amide derivatives containing dansyl (Dns) or 7-dimethylaminocoumarin (DMAC) residues, i.e. Dns-K(Ac)-NH(2) and DMAC-K(Ac)-NH(2), were synthesized and evaluated as substrates for human sirtuin 1. A CZE method with field amplified sample injection and a MEKC method with sweeping were established and validated for monitoring the deacetylation process of Dns-K(Ac)-NH(2) and DMAC-K(Ac)-NH(2), respectively. Deacetylation by sirtuin 1 was demonstrated for both of the substrates. The Michaelis-Menten constants, K(m), were 88.0µM for Dns-K(Ac)-NH(2) and 42.9µM for DMAC-K(Ac)-NH(2). The applicability of the methods was demonstrated using known sirtuin inhibitors. Resveratrol did not activate sirtuin 1 using the present CE-based enzyme assay. The results indicated that the lysine derivatives can be used in sirtuin assays instead of peptide substrates.

Antioxidant activity of selected plant species; potential new sources of natural antioxidants.

The aim of this study was to examine six plants from Serbia for their potential antioxidant activity. Therefore, six antioxidant activity assays were carried out, including: total antioxidant capacity, DPPH free-radical scavenging, the inhibitory activity toward lipid peroxidation, Fe(3+)- reducing power, Fe(2+)- chelating ability and hydroxyl radical scavenging activity. Total phenolic and flavonoid contents were also determined for each alcoholic extract. Cotinus coggygria extract contained the highest amount of total phenols (413mg GAE /g dry extract), while the highest proportion of flavonoids was found in the Echium vulgare methanol extract (105 mg RU/g). Cotinus coggygria and Halacsya sendtneri alcoholic extracts showed the highest total antioxidant capacity (313 and 231 mg AA/g dry extract), as well as DPPH free-radical scavenging (IC(50)=9 and 99 µg/ml), inhibitory activity toward lipid peroxidation (IC(50)=3 and 17 µg/ml) and reducing power. Whereas, the greatest hydroxyl radical scavenging activity, as well as ferrous ion chelating ability showed Echium vulgare, Echium rubrum and Halacsya sendtneri.

Chemical composition of the essential oil and hexane extract of Salvia chionantha and their antioxidant and anticholinesterase activities.

The essential oil and methyl ester of hexane extract of Salvia chionantha Boiss. were analysed by GC and GC-MS. Totally, 54 components were detected in the essential oil and all of them were fully determined. Germacrene D (25.03%), ß-caryophyllene (8.71%), spathulenol (5.86%) and α-humulene (4.82%) were identified as the major compounds. In the methylated hexane extract, 3-hydroxy hexadecanoic acid (39.39%), 3-hydroxy tetradecanoic acid (12.66%) and palmitic acid (12.02%) were the major fatty acids elucidated. The antioxidant activity of the essential oil and the hexane extract was determined by using four complementary test systems; namely, ß-carotene-linoleic acid, DPPH() scavenging, ABTS(+)* scavenging, and CUPRAC assays. In ß-carotene-linoleic acid assay, the extract showed 81.2±0.1% lipid peroxidation inhibition at 0.8 mg/mL concentration, while in ABTS(+)* assay the essential oil exhibited 77.4±0.5% inhibition at same concentration. Since, acetylcholinesterase and butyrylcholinesterase enzymes are taking place in pathogenesis of Alzheimer's disease, in vitro anticholinesterase activity of the essential oil and the extract was also studied spectrophotometrically. At 0.5mg/mL concentration, the essential oil showed moderate acetylcholinesterase (56.7±1.9%) and butyrylcholinesterase (41.7±2.9%) inhibitory activity, while the extract was only exhibited activity (63.1±0.8%) against butyrylcholinesterase enzyme. Hence, the essential oil may be useful as a moderate anticholinesterase agent, particularly against acetylcholinesterase.

Synergistic interaction of eugenol with antibiotics against Gram negative bacteria.

Eugenol, the principal chemical component of clove oil from Eugenia aromatica has been long known for its analgesic, local anesthetic, anti-inflammatory, and antibacterial effects. The interaction of the eugenol with ten different hydrophobic and hydrophilic antibiotics was studied against five different Gram negative bacteria. The MIC of the combination was found to decrease by a factor of 5-1000 with respect to their individual MIC. This synergy is because of the membrane damaging nature of eugenol, where 1mM of its concentration is able to damage nearly 50% of the bacterial membrane. Eugenol was also able to enhance the activities of lysozyme, Triton X-100 and SDS in damaging the bacterial cell membrane. The hydrophilic antibiotics such as vancomycin and beta-lactam antibiotics which have a marginal activity on these gram negative bacteria exhibit an enhanced antibacterial activity when pretreated with eugenol. Reduced usage of antibiotics could be employed as a treatment strategy to slow down the onset of antibiotic resistance as well as decrease its toxicity. Experiments performed with human blood cells indicated that the concentration of eugenol used for the combination studies were below its cytotoxic values. Pharmacodynamic studies of the combinations need to be performed to decide on the effective dosage.

Identification of iNOS inhibitors using InteraX.

Inducible nitric oxide synthase (iNOS) is active as a homodimer. A cell-based assay suitable for high-throughput screening (HTS) was generated to identify inhibitors of iNOS dimerization using the InteraX enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins of complementing deletion mutants of beta-galactosidase, each fused to the oxygenase domain of human iNOS. The assay was characterized using known iNOS dimerization inhibitors, which gave a decrease in beta-galactosidase activity. Surprisingly, the assay was also able to identify compounds that have the same profile as known inhibitors of fully formed dimeric iNOS by causing an increase in beta-galactosidase activity. The iNOS InteraX assay was used to screen approximately 800,000 compounds in a 384-well format. After hit confirmation, 3359 compounds were taken forward for full IC50 determination in InteraX and cytotoxicity assays. Of these compounds 40.5% were confirmed as greater than 10-fold more active in InteraX compared to a cytotoxicity assay and were classified as potential iNOS dimerization inhibitors as they did not inhibit beta-galactosidase alone. In the same primary screen, 901 compounds gave a significant increase in beta-galactosidase activity. Many of these were known inhibitors of iNOS. After IC50 determination in InteraX and cytotoxicity assays, 182 novel compounds remained as potential arginine-competitive inhibitors of dimeric iNOS.

Thiol-disulfide organization in alliin lyase (alliinase) from garlic (Allium sativum).

Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5'-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S--S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored --SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two --SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free --SH groups. This allowed the oriented conjugation of alliinase, via the --SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.

Chemical composition and antioxidant activity of volatiles from Patrinia Villosa Juss obtained by optimized supercritical fluid extraction.

Supercritical CO(2) fluid extraction (SFE-CO(2)) was used to extract volatiles from Patrinia Villosa Juss. An orthogonal test L(9) (3)(4) including pressure, temperature, dynamic extraction time and modifier was performed to get the optimal conditions. Extract 1 was obtained by the optimal extraction condition 1: pressure=35 MPa, T=45 degrees C, dynamic extraction time=2.0 h and V(modifier (MeOH))=0% as guided by the index 1: the free radical scavenging activities in vitro against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) diammonium salt (ABTS). Extract 2 obtained by the optimal extraction condition 2: pressure=25 MPa, T=55 degrees C, dynamic time=2.5h and V(modifier (MeOH))=20% was guided by the index 2: the yield of the volatiles. The results showed that extract 1 possessed stronger antioxidant activity (EC(50)=32.01 microg/ml to DPPH and 50.90 microg/ml to ABTS(+)) than the extract 2 (EC(50)=95.62 microg/ml to DPPH and 99.78 microg/ml to ABTS(+)). Subsequently, the chemical compositions of the two extracts were identified by gas chromatography-mass spectrometry. Forty-six compounds were identified from extract 1, and the total volatile consisted of hydrocarbon (49.65%), aldehyde (16.66%), fatty acid (22.38%), terpene (9.04%) and little alcoholic. From extract 2, 32 compounds were identified, in which hydrocarbon, aldehyde, fatty acid and terpene possessed 58.21%, 5.97%, 13.19% and 21.79%, respectively. This is the first report of using SFE to extract the volatiles from P. Villosa Juss (a wild vegetable and medicine plant) and first time to systematically evaluate the volatiles' antioxidant activity and chemical composition.