Characterization and bioactivities of a novel polysaccharide obtained from Gracilariopsis lemaneiformis.

Gracilariopsis lemaneiformis is a type of red alga that contains seaweed polysaccharide agar. In this study, a novel non-agar seaweed polysaccharide fraction named GCP (short of crude polysaccharide obtained from Gracilariopsis lemaneiformis) was isolated from Gracilariopsis lemaneiformis. Structural analysis showed that GCP shows triple helical chain conformation when dissolved in water and has many branches and long side chains. Also, 1→3 linkage is the major linkage and the sugar structures are galactopyranose configurations linked by ß-type glycosidic linkages. Two macromolecular substance fractions (GCP-1 and GCP-2) were purified by DEAE Sepharose Fast Flow column chromatography. Moreover, a splenocyte damage assay and splenocyte proliferation assay were used to analyse the bioactivities of GCP, GCP-1 and GCP-2. It was demonstrated that polysaccharides could protect splenocyte damaged by H2O2; GCP-2 shows a greatest protection rate, that is, 92.8%, which significantly enhanced the splenocyte proliferation, and GCP showed the highest proliferation rate, 9.30%. The results suggested that this type of novel non-agar polysaccharide displayed remarkable antioxidant and immunomodulatory activities and early alkali treatment could decrease the activities. It may represent a potential material for health food and clinical medicines.

Immunomodulation of Proton-activated G Protein-coupled Receptors in Inflammation.

Proton-activated G protein-coupled receptors (GPCRs), initially discovered by Ludwig in 2003, are widely distributed in various tissues. These receptors have been found to modulate the immune system in several inflammatory diseases, including inflammatory bowel disease, atopic dermatitis, and asthma. Proton-activated GPCRs belong to the G protein-coupled receptor family and can detect alternations in extracellular pH. This detection triggers downstream signaling pathways within the cells, ultimately influencing the function of immune cells. In this review, we specifically focused on investigating the immune response of proton-activated GPCRs under inflammatory conditions.

A novel killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b.

In our previous study, it was found that the killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b has both killing activity and ß-1,3-glucanase activity and the molecular mass of it is 47.0 kDa. In this study, the same yeast strain was found to produce another killer toxin which only had killing activity against some yeast strains, but had no ß-1,3-glucanase activity and the molecular mass of the purified killer toxin was 67.0 kDa. The optimal pH, temperature and NaCl concentration for action of the purified killer toxin were 3.5, 16 °C and 4.0 % (w/v), respectively. The purified killer toxin could be bound by the whole sensitive yeast cells, but was not bound by manann, chitin and ß-1,3-glucan. The purified killer toxin had killing activity against Yarrowia lipolytica, Saccharomyces cerevisiae, Metschnikowia bicuspidata WCY, Candida tropicalis, Candida albicans and Kluyveromyces aestuartii. Lethality of the sensitive cells treated by the newly purified killer toxin from W. anomalus YF07b involved disruption of cellular integrity by permeabilizing cytoplasmic membrane function.

Enzyme-linked immunosorbent assay for okadaic acid: investigation of analytical conditions and sample matrix on assay performance.

Okadaic acid (OA) is a lipophilic marine biotoxin. In this study, OA was coupled with the carrier proteins keyhole limpet hemocyanin and bovine serum albumin as immunity and detection antigens by an active ester method. The polyclonal antibody against OA was prepared successfully, an indirect competitive ELISA (ciELISA) developed for the detection of OA in shellfish, and the reactive conditions of ciELISA optimized. The LOD (15% inhibition concentration) for the microwell plates was 1.28 +/- 0.38 ng/mL, corresponding to 12.8 +/- 3.8 ng/g. Two extraction methods were used to remove shellfish matrix interference with high recovery of spiked samples, and the methanol extraction of shellfish mussel was analyzed after dilution in phosphate-buffered saline. For validation of the optimized ciELISA, spiked and natural samples were analyzed by ciELISA, and HPLC with fluorescence detection. The correlation of linear regression equation was y = 1.0064x - 10.234, and the correlation coefficient was 0.9347. From the results of the comparative study, the established ciELISA assay using polyclonal antibody against OA could be used in preliminary screening of suspicious shellfish samples.

Single cell protein production from yacon extract using a highly thermosensitive and permeable mutant of the marine yeast Cryptococcus aureus G7a and its nutritive analysis.

The intracellular protein in the highly thermosensitive and permeable mutant can be easily released when they are incubated both in the low-osmolarity water and at the non-permissive temperature (usually 37 degrees C). After the mutant was grown in the yacon extract for 45 h, the crude protein content in the highly thermosensitive and permeable mutant Z114 was 59.1% and over 61% of the total protein could be released from the cells treated at 37 degrees C. The mutant cells grown in the yacon extract still contained high level of essential amino acids and other nutrients. This means that the yacon extract could be used as the medium for growth of the highly thermosensitive and permeable mutant which contained high content of crude protein.

Evaluation of Volatile Compounds during the Fermentation Process of Yogurts by Streptococcus thermophilus Based on Odor Activity Value and Heat Map Analysis.

The volatile composition of yogurt produced by Streptococcus thermophilus fermentation at different time points was investigated by gas chromatography-mass spectrometry combined with simultaneous distillation and extraction. A total of 53 volatile compounds including 11 aldehydes, 10 ketones, 8 acids, 7 benzene derivatives, 13 hydrocarbons, and 4 other compounds were identified in all of the samples. Ketones and hydrocarbons were the predominant volatile components in the early stage, whereas acids were the predominant volatiles in the late stage. The importance of each volatile was evaluated based on odor, threshold, and odor activity values (OAVs). Twenty-nine volatiles were found to be odor-active compounds (OAV > 1), among which (E, E)-2,4-decadienal had the highest OAV (14623-22278). Other aldehydes and ketones such as octanal, dodecanal, 2-nonen-4-one, and 2-undecanone also showed high odor intensity during fermentation. Heat map analysis was employed to evaluate the differences during fermentation. The results demonstrated that the volatile profile based on the content and OAVs of volatile compounds enables the good differentiation of yogurt during fermentation.

Postmenopausal mild hirsutism and hyperandrogenemia due to ovarian Sertoli-Leydig cell tumor: A case report.

Among several types of ovarian tumors, Sertoli-Leydig cell tumors are considered significantly rare, accounting for less than 1% of all primary ovarian tumors. Hirsutism caused by ovarian tumors accounts for approximately 1% of all cases of hirsutism. We report a case of a woman with a ovarian Sertoli-Leydig cell tumor who presented with hirsutism. A 45-year-old woman (gravida 12, para 2) who experienced menopause when she was 43 years old had excessive hair on her face and lower abdomen since 2 years. Her body mass index was 24.3 kg/m2. She also had hair growth on her upper lip, submandibular area, lower abdomen, vulva, and bilateral tibia (front), and around her breast. She had a Ferriman-Gallwey score of 8. Ultrasound findings revealed a 4.8 × 3.5-cm left adnexal mass. Pelvic computed tomography (CT) findings revealed that her left accessory gland had a low-density mass (CT value, 25 Hu). Her serum testosterone level was 15.80 nmol/l. The patient underwent a laparoscopic left adnexectomy. Subsequently, she was diagnosed with ovarian Sertoli-Leydig cell tumor by immunohistochemical staining. A week after surgery, her serum testosterone level decreased from 15.80 nmol/l to 1.03 nmol/L. Her hirsutism almost completely disappeared 3 months after surgery. It is vitally important to establish the final diagnosis according to the clinical manifestations and laboratory values in addition to imaging studies and laparoscopic examination of a rare coexistence of hirsutism and hyperandrogenemia in a postmenopausal woman based on ovarian pathology.

Ozone Degradation of Prometryn in Ruditapes philippinarum: Response Surface Methodology Optimization and Toxicity Assessment.

ABSTRACT: This study optimized the method for ozone (O3) degradation of prometryn in the clam Ruditapes philippinarum and evaluated toxicity changes during ozone degradation. The gas chromatography method for the detection of prometryn was appropriately improved. The linear range was 5 to 500 ng/mL, with a correlation coefficient of 0.9964. The addition concentration of prometryn was 0.025 to 0.100 mg/kg, the recovery was 77.97 to 85.00%, the relative standard deviation (n = 6) was 2.36 to 7.86%, and the limit of detection was 0.3 µg/kg. Using the central composite design in two experiments, ozone as gas and ozone dissolved in water, the effect of degradation rate was studied on three variables: ozone concentration, temperature, and exposure time. Ozone as gas and ozone dissolved in water have the same degradation effect on prometryn. The O3 concentration was 4.2 mg/L, the temperature was 40°C, the exposure time was 10 min, and the maximum degradation rate was 89.94 and 89.69% for the two experiments, respectively. In addition, the toxicity of ozone degradation products was evaluated using buffalo rat liver cells. After ozone treatment for 30 min, the toxicity of the ozone degradation products was reduced to 52.15% compared with that of prometryn itself. The toxicity of the ozone degradation products increased slightly when the ozonation time was prolonged; the toxicity at 180 min was greater than that of the parent compound prometryn. Overall, the application of ozone degradation of pesticide residues is a promising new technology. This study determined better degradation conditions for prometryn in R. philippinarum and also provided a theoretical basis for the widespread use of ozone technology in the future.

Degradation of prometryn in Ruditapes philippinarum using ozonation: Influencing factors, degradation mechanism, pathway and toxicity assessment.

In recent years, prometryn was utilized as watergrass remover in the aquaculture industry, resulting in the accumulated residual in the aquatic products. The present study focuses on the ozone degradation of prometryn in the Ruditapes philippinarum. The ozone concentration in water increased along with the injection time (60min). The contents of hydroxyl (·OH) and superoxide (O2·-) radicals increased along with the ozone injection time. The effects of temperature, pH, prometryn initial concentration and ozone concentration on the removal efficiency of prometryn were evaluated. The maximum removal efficiency of 86.12% was obtained under the conditions of pH 7, prometryn initial concentration 0.05 mg/kg and the ozone concentration 4.2 mg/L at 28 °C for 30 min. Ion chromatography (IC) and Fourier transform infrared (FT-IR) spectroscopy results show that the S and N atoms in the outer layer of the triazine ring during the prometryn degradation process were oxidized and removed. A total of 30 intermediate compounds were identified using the gas chromatography-mass spectrometry (GC-MS) method. Combined with the IC and FT-IR results, three possible degradation pathways of prometryn were proposed. The prometryn was finally degraded into some small molecules with reduced toxicity by 63.16% for 120 min ozonization treatment. Overall, our work provides a novel approach for prometryn degradation in Ruditapes philippinarum, which can be extended for removing the residues of agricultural and veterinary drugs in other aquatic products.

Purification and molecular characterization of exo-beta-1,3-glucanases from the marine yeast Williopsis saturnus WC91-2.

The extracellular beta-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific beta-1,3-glucanase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The optimal pH and temperature of the purified enzyme were 4.0 and 40 degrees C, respectively. The enzyme was significantly stimulated by Li+, Ni2+, and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1,10-phenanthroline. The Km and Vmax values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLDAFEKR which is the conserved motif of the beta-1,3-glucanases from other yeasts.

Purification, characterization, and functional groups of an extracellular aflatoxin M1 -detoxifizyme from Bacillus pumilus E-1-1-1.

The experiment was conducted to purify high activity extracellular enzymes, which were produced by a strain that we previously screened was able to degrade aflatoxin effectively, and speculate the functional groups of the enzyme associated with degradation. An extracellular aflatoxin-detoxifizyme (DAFE) from Bacillus pumilus E-1-1-1 was purified through a process including ammonium sulfate precipitation, ultrafiltration, Sephadex chromatography, and ion exchange chromatography. The molecular mass of the enzyme assessed by SDS-PAGE was found to be approximately 58 kDa. The optimum reaction temperature and pH for the purified enzyme were 45°C and pH 7, respectively. The enzyme showed temperature stability of up to 60°C. Ba2+ , Ca2+ Na+ , Mn2+ , EDTA, and ß-mercaptoethanol showed inhibitory effects on the enzyme activity. Mg2+ , Fe3+ , Zn2+ and K+ were the activators of enzymes. This enzyme was composed of at least 15 kinds of amino acids. Lysine, tryptophan, and histidine residues were necessary and major functional groups to maintain enzyme activity, disulfide bonds were observed, serine residues had little effect on the enzyme activity, so it was not the necessary group to reflect the enzyme activity, and arginine had no effect on enzyme activity.

Inactivation of ID-1 gene induces sensitivity of prostate cancer cells to chemotherapeutic drugs.

Resistance to anticancer drugs is one of the major reasons of treatment failure for androgen-independent prostate cancer (PC). Increase in expression of Id-1 has been reported in several types of advanced cancer including PC. It has been suggested that overexpression of Id-1 may provide an advantage for cancer cell survival and thus inactivation of Id-1 may be able to increase the susceptibility of cancer cells to apoptosis. In this study, using small RNA interfering (siRNA) technology, we inactivated the Id-1 gene in two androgen-independent PC cell lines, DU145 and PC3, and investigated whether down-regulation of Id-1 could lead to increased sensitivity of these PC cells to a commonly used anticancer drug, taxol (Tx). Our results showed that inactivation of Id-1 by siId-1 resulted in decrease in both colony forming ability and cell viability in prostate cancer cells after Tx treatment. Furthermore, the siId-1 induced sensitization to Tx was associated with activation of apoptotic pathway. In addition, c-Jun N-terminal kinase (JNK), one of the common pathways responsible for Tx-induced apoptosis, was also activated in the si-Id-1 transfected cells. Inhibition of JNK activity by a specific inhibitor, SP600125, blocked the siId-1-induced sensitivity to Tx. These results indicate that increased Id-1 expression in PC cells may play a protective role against apoptosis, and down-regulation of Id-1 may be a potential target to increase sensitivity of Tx-induced apoptosis in PC cells.

Sensors and Biosensors for the Determination of Small Molecule Biological Toxins.

The following review of sensors and biosensors focuses on the determination of commonly studied small molecule biological toxins, including mycotoxins and small molecule neurotoxins. Because of the high toxicity of small molecule toxins, an effective analysis technique for determining their toxicity is indispensable. Sensors and biosensors have emerged as sensitive and rapid techniques for toxicity analysis in the past decade. Several different sensors for the determination of mycotoxins and other small molecule neurotoxins have been reported in the literature, and many of these sensors such as tissue biosensors, enzyme sensors, optical immunosensors, electrochemical sensors, quartz crystal sensors, and surface plasmon resonance biosensors are reviewed in this paper. Sensors are a practical and convenient monitoring tool in the area of routine analysis, and their specificity, sensitivity, reproducibility and analysis stability should all be improved in future work. In addition, accuracy field portable sensing devices and multiplexing analysis devices will be important requirement for the future.

Regulation of angiogenesis by Id-1 through hypoxia-inducible factor-1alpha-mediated vascular endothelial growth factor up-regulation in hepatocellular carcinoma.

PURPOSE: Metastasis is commonly associated with poor prognosis of hepatocellular carcinoma (HCC). Being an important angiogenic factor, vascular endothelial growth factor (VEGF) plays a central role in HCC growth and metastasis. Recently, Id-1 (inhibitor of differentiation/DNA synthesis) has been suggested to be a key factor in cancer progression but the molecular mechanism remains unknown. EXPERIMENTAL DESIGN: We first showed that overexpression of Id-1 was correlated with HCC metastasis (P < 0.001) and its expression was significantly correlated with VEGF expression by tissue microarray. By ectopic transfection of Id-1 into HCC cells, Id-1 was able to induce VEGF secretion through activation of VEGF transcription. RESULTS: Increased VEGF secretion in Id-1 transfectants induced morphologic change and proliferation of human umbilical vascular endothelial cell resulting in promotion of angiogenesis. Id-1 induced transcriptional activation of VEGF by stabilizing hypoxia-inducible factor-1alpha protein. Down-regulation of Id-1 by antisense approach led to suppression of hypoxia-inducible factor-1alpha-mediated VEGF production. In addition, Id-1 suppression resulted in retardation of cell invasion through down-regulation of VEGF. CONCLUSIONS: Id-1 is a novel angiogenic factor for HCC metastasis and down-regulation of Id-1 may be a novel target to inhibit HCC metastasis through suppression of angiogenesis.

Twist overexpression correlates with hepatocellular carcinoma metastasis through induction of epithelial-mesenchymal transition.

PURPOSE: Hepatocellular carcinoma (HCC) is a rapidly growing tumor associated with a high propensity for vascular invasion and metastasis. Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process. Recently, Twist has been identified to play an important role in EMT-mediated metastasis through the regulation of E-cadherin expression. However, the actual role of Twist in tumor invasiveness remains unclear. The purpose of this study is to investigate the expression and possible role of Twist in HCC. EXPERIMENTAL DESIGN: We evaluated Twist and E-cadherin expression in HCC tissue microarray of paired primary and metastatic HCC by immunohistochemical staining. The role of Twist in EMT-mediated invasiveness was also evaluated in vitro in HCC cell lines. RESULTS: We first showed that overexpression of Twist was correlated with HCC metastasis (P=0.001) and its expression was negatively correlated with E-cadherin expression (P=0.001, r=-0.443) by tissue microarray. A significant increase of Twist at the mRNA level was also found in metastatic HCC cell lines MHCC-97H, MHCC-97L, and H2M when compared with nonmetastatic Huh-7, H2P, and PLC cell lines. The MHCC-97H cell line, which has a higher metastatic ability, was found to have a higher level of Twist than MHCC-97L. Accompanied with increased Twist expression in the metastatic HCC cell lines when compared with the nonmetastatic primary ones, we found decreased E-cadherin mRNA expression in the metastatic HCC cell lines. By ectopic transfection of Twist into PLC cells, Twist was able to suppress E-cadherin expression and induce EMT changes, which was correlated with increased HCC cell invasiveness. CONCLUSION: This study shows that Twist overexpression was correlated with HCC metastasis through induction of EMT changes and HCC cell invasiveness.

[Spectral analysis and synthesis of artificial antigen of the organophosphors pesticide methamidophos].

In the present paper, a hapten of methamidophos was synthesized and conjugated with KLH by active ester method, thus the first artificial antigen was obtained. By diazotization method methamidophos conjugated with BSA, and the second artificial antigen was obtained. The synthesized haptens were characterized by MS, IR and 1H NMR, and the two artificial antigens were determined by the method of IR spectrum. The result implied that both the artificial antigens have absorbance peaks of hapten and protein, indicating that they were prepared successfully. This could provide evidence that the method of IR spectrum can be used to determine whether the artificial antigens are synthesized successfully.

[Study of HPLC-DAD fingerprint on complex traditional Chinese medicine proprietary preparation-Baoji pills].

OBJECTIVE: Based on 'Back-tracking' method, identification and quality evaluation of complex traditional Chinese medicine (TCM) preparation of Baoji pills (BJP) were carried out by HPLC fingerprint analysis. METHOD: HPLC-DAD fingerprint of BJP was conducted with Zorbax SB-C18 column and non-linear elution with the mobile phase consisted of acetonitrile-0.5% glacial acetic acid at column temperature 30 degrees C and detective wavelengths of 250 nm and 283 nm. From the established chromatographic pattern of BJP, track backward to the corresponding crude herbal drugs in the formula, attribution ofmost peaks in the BJP fingerprint can be disclosed. RESULT: The BJP HPLC fingerprint consisted of 44 peaks among which 35 peaks were assigned by parallel comparison with the fingerprint of the 10 corresponding crude drugs in the formula such as pueraria, pummelo peel, and magnolia bark, etc. and 22 peaks we reidentified by comparison with the chemical reference substances. CONCLUSION: The established HPLC fingerprint represents the whole character of BJP, which enhanced the specialty for control and assessment of the product quality. It exemplified much more effective for quality control than selecting any marker for qualitative or quantitative testing target. And the Back-tracking' experimental method extended the study mentality for complex formula TCM products chromatographic fingerprinting analysis.

FTY720: a promising agent for treatment of metastatic hepatocellular carcinoma.

PURPOSE: Recurrence after resection and metastasis are common in hepatocellular carcinoma and are associated with poor prognosis. Therefore, effective treatment is urgently needed for improvement of patients' survival. Previously, we reported that FTY720 has an antimetastatic effect on hepatocellular carcinoma cell line through down-regulation of Rac signaling pathway. This study aims to investigate the in vivo antimetastatic potential of FTY720 in an orthotopic nude mice model using metastatic human hepatocellular carcinoma cell lines MHCC-97L (lower metastatic potential) and MHCC-97H (higher metastatic potential). EXPERIMENTAL DESIGN: The nude mice bearing liver tumors were randomized into a treatment group and a control group, each with 12 mice. FTY720 was administered at a dosage of 5 or 10 mg/kg via i.p. injection after 7 days of tumor inoculation. Thirty-five days later, the mice were sacrificed for record of intrahepatic and pulmonary metastases. RESULTS: After 35 days of FTY720 treatment at the dosages of 5 and 10 mg/kg, all 12 mice in the treatment group were alive and well. FTY720 at the dosages of 5 and 10 mg/kg significantly suppressed the tumor volume and intrahepatic and pulmonary metastases in the metastatic nude mice model. FTY720 suppressed intrahepatic and pulmonary metastases by inhibition of Rac expression, which at least in part down-regulated the vascular endothelial growth factor expression and CD34 staining in a dose-dependent manner. CONCLUSION: FTY720 is a promising novel therapeutic drug for treatment of hepatocellular carcinoma metastasis.

Role of histamine in airway remodeling of asthmatic guinea pig.

To investigate the role of histamine in airway remodeling, 50 healthy guinea pigs were divided into 5 groups: control group: nebulized inhalation of distilled water for 8 weeks; asthma model group: nebulized inhalation of ovalbumin (OVA) for 8 weeks after sensitization; continued asthma model group: nebulized inhalation of OVA for 14 weeks after sensitization; histamine group: nebulized inhalation of OVA for 14 weeks after sensitization and histamine was added in the last 6 weeks; antagonist group: nebulized inhalation of OVA for 14 weeks after sensitization and histamine receptor antagonists were added in the last 6 weeks. For each group, the concentration of histamine, sodium ion (Na(+)), chlorine ion (Cl(-)), arterial partial pressure of oxygen (PaO2), arterial partial pressure of carbon dioxide (PaCO2), pH, actual bicarbonate (AB), standard bicarbonate (SB) in serum, and thickness of airway mucosa, base membrane and smooth muscle were measured and compared with each other. The results showed that: (1) the concentration of histamine in serum and the thickness of airway increased, the following order was, the control group, the asthma model group, the continued asthma model group and histamine group (P<0.01); and the concentration of histamine in serum and the thickness of airway of antagonist group was lower than that of the continued asthma model group (P<0.05, 0.01). (2) PaO2 of the asthma model group was lower than that of the normal control group (P<0.01); PaO2, pH, AB, SB decreased, the following order was, the asthma model group, the continued asthma model group and the histamine group (P<0.01); and PaO2, pH, AB, SB of the antagonist group was higher than that of the continued asthma model group (P<0.01); but for PaCO2, the order was converse (P<0.01); For the concentration of Na(+) and Cl(-) in serum, there was no difference among these groups. It is concluded that: (1) Histamine is one of the mediators in the airway remodeling of asthma. (2) Histamine receptor antagonists may play a role in preventing and treating airway remodeling. (3) There is a negative correlation between the PaO2, pH and the wall thickness of the airway (P<0.01), while a positive correlation between the PaCO2, anion gap (AG) and the wall thickness of the airway (P<0.01).

Quantification of liensinine in rat plasma using ultra-performance liquid chromatography tandem mass spectrometry and its application to a pharmacokinetic study.

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed to determine liensinine in rat plasma using carbamazepine as the internal standard (IS). Sample preparation was accomplished through a protein precipitation procedure with acetonitrile to 0.1ml plasma sample. The analyte and IS were separated on an Acquity UPLC BEH C18 column (2.1mm×50mm, 1.7µm) with the mobile phase of acetonitrile and 0.1% formic acid in water with gradient elution at a flow rate of 0.40ml/min. The injection volume was 6µl. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 611.6→206.2 for liensinine and m/z 237.1→194.2 for IS. The linearity of this method was found to be within the concentration range of 10-1000ng/ml with a lower limit of quantification of 10ng/ml. Only 3.0min was needed for an analytical run. The matrix effect was 93.8-107.4% for liensinine. The intra- and inter-day precision (RSD %) were less than 9.9% and accuracy (RE %) was within ±10.5%. The recovery ranged from 76.2 to 86.8%. Liensinine was sufficiently stable under all relevant analytical conditions. The method was also successfully applied to the pharmacokinetic study of liensinine in rats. The pharmacokinetic parameters were demonstrated as followed: t1/2 was 8.2±3.3h, Cmax was 668.4±156.9ng/ml, and AUC0→∞ was 1802.9±466.4ng/mlh.