First report of invasive Pomacea snails in Shandong Province / 中国血吸虫病防治杂志

Objective To characterize the species of invasive Pomacea snails that were discovered for the first time in Shandong Province. Methods Pomacea snails samples were collected in the field of Jining City, Shandong Province on October 2021 for morphological identification. Pomacea snails were randomly sampled and genomic DNA was extracted from foot muscle tissues of Pomacea snails for multiplex PCR amplification. The PCR amplification product was sequenced. Then, the sequence was aligned and a phylogenetic tree was created using the software MegAlign 7.1.0. In addition, Angiostongylus cantonensis infection was detected in Pomacea snails with the lung microscopy. Results A total of 104 living Pomacea snails were collected, and all were characterized as Pomacea spp. based on morphological features. Of 12 randomly selected adult Pomacea snails, multiplex PCR assay and sequencing identified eleven snails as P. canaliculata and one as P. maculata. No A. cantonensis infection was detected in 104 Pomacea snails. Conclusion This is the first report of invasive Pomacea snails in Shandong Province, where P. canaliculata and P. maculata are found.

Effects of water regulation on biosynthesis of calycosin-7-O-β-D-glucoside in Astragalus membranaceus var. mongholicus / 中国中药杂志

The effects of water regulation on the biosynthesis of calycosin-7-O-β-D-glucoside in 2-year-old Astragalus membranaceus var. mongholicus were studied,and the mechanism was explained from the aspects of key enzyme gene expression and antioxidant enzyme system. The content of calycosin-7-O-β-D-glucoside was determined by HPLC,and the expression levels of six key enzyme genes( PAL,4 CL,CHS,CHI,IFS,13'H) in the synthesis pathway were analyzed by q RT-PCR. The activities of protective enzymes and contents of osmoregulation substances and malondialdehyde were also determined. In the water deficit group,the maximum concentration of calycosin-7-O-β-D-glucoside was 0. 49 mg·g-1 on the 24 th day of treatment. In the whole water regulation,the water deficit group outweighed the water adequate group in osmoregulation substance and MDA contents. The activities of A. membranaceus var.mongholicus antioxidant enzymes SOD,POD,and CAT increased during the initial period of water regulation,but decreased with time.The expression of PAL,CHS,and 13'H in the water deficit group was at a low level,and the 4 CL had active expression,slightly lower than that in the water adequate group. The expression of CHI and IFS elevated rapidly when water deficit occurred. Correlation analysis showed that the content of calycosin-7-O-β-D-glucoside was positively correlated with CHI expression( P<0. 01) and IFS expression( P<0. 05). Therefore,water regulation can change the accumulation pattern of calycosin-7-O-β-D-glucoside,and water deficit may be an effective way to increase its content. CHI and IFS are the key genes in response to water deficit.

Short-term water changes response of saponin biosynthesis process in Astragalus membranceus / 中国中药杂志

The study is aimed to explore the effect of different water on the content of total saponins,astragaloside Ⅳ and gene expression in the growth of Astragalus membranceus. In this study, one-year-old A. membranaceus was used as the experimental material, by pot culture different water treatments were simulated at herbal garden in Jilin Agricultural University. The content of astragaloside Ⅳ was determined by HPLC and the total saponins by UV spectrophotometry. With 18 S RNA as a reference gene, fluorescent quantitative PCR was applied to analyze the eight key enzymes in astragalus saponin synthesis pathway AACT,HMGS,HMGR,IDI,FPS,SS,SE,CAS expression. With the decrease of soil water, the content of astragaloside Ⅳ in the root tissue of A. membranaceus showed an increasing trend, up to 1.46 mg·g~(-1). The total saponin content tended to increase, up to 6.80 mg·g~(-1). The results of relative expression of genes showed that the eight genes showed different effects at different water. With the change of soil water content, the amount of(AACT,IDI,SS) relative expression in drought stress group firstly increased and then decreased, then increased, and then decreased. The amount of(HMGS,HMGR,FPS) relative expression in drought stress group increased firstly and then decreased. The amount of(SE,CAS) relative expression in drought stress group increased firstly and then decreased, and continued to decrease after rehydration. The expression of key enzyme genes involved in the synthesis of astragaloside was influenced by each other, and the expression of key enzyme in roots showed a correlation with the content of astragaloside. Correlation analysis showed that there was a very significant positive correlation between HMGR gene and total saponins content in drought stress group and a significant negative correlation between content of CAS and total saponins. The contents of FPS,SE,CAS and astragaloside Ⅳ were very significantly and negative correlated. The relationship between other genes and quality was positive. Therefore, HMGR, FPS, SE and CAS genes have significant effects on the regulation of saponin content under water control. On the 15 th day after water regulation, the total amount of astragaloside and total saponins reached the highest value and could be harvested.

Preliminary study on response and its mechanism of ginsenoside biosynthesis in Panax ginseng to water regulation / 中国中药杂志

The study is aimed to explore the effect of soil moisture content on ginsenoside biosynthesis and explain its mechanism from the perspectives of antioxidant enzyme system and gene expression of key enzymes in the pathway of ginsenoside synthesis. In the study,two years old Panax ginseng was used as the experimental material and three moisture gradient,40% of saturated water content( W1),60%( W2),80%( W3) were set up. The content of 11 monomeric saponins were determined by HPLC. With GAPDH as a reference gene,six key enzymes( HMGR,SS,β-AS,CYP716 A47,CYP716 A52 v2,CYP716 A53 v2) in ginseng saponin synthesis pathway expression were analyzed by fluorescent quantitative PCR and the activities of superoxide dismutase( SOD),peroxidase( POD),catalase( CAT) activity and MDA content were also determined. With the increase of soil water,the content of ginseng saponin and biomass showed an increasing trend. PPD( Rb1,Rc,Rb2,Rd,Rh2,Rb3,Rg3),PPT( Rg1,Re,Rf) ginsenoside,Ro and total ginsenoside reached the maximum value on August 30,were 9.92,5.48,0.63 mg·g-1,respectively. During the whole regulation period,the antioxidant activity of W3 was greater than that of W1,and the MDA content was less than that of W1. At W3,expression levels of β-AS,CYP716 A47 and CYP716 A53 v2 showed an increasing trend,while HMGR and SS genes showed relatively stable expression levels under various water conditions. According to the correlation analysis,HMGR and SS genes in the W3 treatment group were significantly positively correlated with PPD,PPT ginsenoside and Ro,CYP716 A52 v2 gene was significantly positively correlated with Ro,and CYP716 A47 gene was significantly positively correlated with PPD ginsenoside. There was a significant positive correlation between β-AS gene and PPD ginsenoside in W1 and W2 treatment. Therefore,W3 is the optimum moisture content,ginseng total saponins and monomer saponin content is the highest,the gene closely correlation with content of saponins and more conducive to the accumulation of ginsenosides.

Effect of temperature on content of baicalin and expression of key enzyme genes in callus of Scutellaria baicalensis / 中国中药杂志

The study is aimed to explore the effect of different temperature on the content of baicalin and gene expression in the growth of Scutellaria baicalensis. Four culture temperatures were used to establish the callus culture of S. baicalensis under dark conditions for 40 days and once every 5 days. The growth and baicalin contents were determined. 18S RNA was used as a reference gene to analyze the five key factors in baicalin biosynthesis pathway (PAL), cinnamic acid 4-hydroxylase (C4H), chalcone synthase (CHS), β-glucuronidase (GUS), baicalein-7--glucuronosyltransferase (UBGAT) gene expression levels. The results showed that biomass, baicalin content and accumulation increased with the increase of temperature. 25 °C and 30 °C were more suitable for the growth of S. baicalensis. The content and accumulation of baicalin at 25 °C reached the highest level at 30 days, reaching 2.75% and 12.44 mg, respectively. The relative expression levels of C4H, CHS, GUS and UGBAT genes at 15 °C were higher than those at other treatments. The correlation between the relative expression levels of each key enzyme and the content of baicalin was negatively correlated with the increase of incubation temperature. The relative expression levels of PAL, C4H and CHS genes at 25 °C and 30 °C were significantly correlated with the contents of baicalin and reached a highly significant or significant level. Relative low temperature conditions were not conducive to the growth of S. baicalensis and the accumulation of baicalin. The accumulation of baicalin by PAL and C4H in the upstream of the synthetic pathway was significant.

Cloning and bioinformatic analysis and expression analysis of beta-glucuronidase in Scutellaria baicalensis / 中国中药杂志

The β-Glucuronidase gene (sbGUS) cDNA firstly from Scutellari abaicalensis leaf was cloned by RT-PCR, with GenBank accession number KR364726. The full length cDNA of sbGUS was 1 584 bp with an open reading frame (ORF), encoding an unstable protein with 527 amino acids. The bioinformatic analysis showed that the sbGUS encoding protein had isoelectric point (pI) of 5.55 and a calculated molecular weight about 58.724 8 kDa, with a transmembrane regions and signal peptide, had conserved domains of glycoside hydrolase super family and unintegrated trans-glycosidase catalytic structure. In the secondary structure, the percentage of alpha helix, extended strand, β-extended and random coil were 25.62%, 28.84%, 13.28% and 32.26%, respectively. The homologous analysis indicated the nucleotide sequence 98.93% similarity and the amino acid sequence 98.29% similarity with S. baicalensis (BAA97804.1), in the nine positions were different. The expression level of sGUS was the highest in root based on a real-time PCR analysis, followed by flower and stem, and the lowest was in stem. The results provide a foundation for exploring the molecular function of sbGUS involved in baicalcin biosynthesis based on synthetic biology approach in S. baicalensis plants.

Effects of growing time on Panax ginseng rhizosphere soil microbial activity and biomass / 中国中药杂志

Using the field sampling and indoor soil cultivation methods, the dynamic of ginseng rhizosphere soil microbial activity and biomass with three cultivated ages was studied to provide a theory basis for illustrating mechanism of continuous cropping obstacles of ginseng. The results showed that ginseng rhizosphere soil microbial activity and biomass accumulation were inhibited observably by growing time. The soil respiration, soil cellulose decomposition and soil nitrification of ginseng rhizosphere soil microorganism were inhibited significantly (P <0.05), in contrast to the control soil uncultivated ginseng (R0). And the inhibition was gradual augmentation with the number of growing years. The soil microbial activity of 3a ginseng soil (R3) was the lowest, and its activity of soil respiration, soil cellulose decomposition, soil ammonification and soil nitrification was lower than that in R0 with 56.31%, 86.71% and 90. 53% , respectively. The soil ammonification of ginseng rhizosphere soil microbial was significantly promoted compared with R0. The promotion was improved during the early growing time, while the promotion was decreased with the number of growing years. The soil ammonification of R1, R2 and R3 were lower than that in R0 with 32.43%, 80.54% and 66.64% separately. The SMB-C and SMB-N in ginseng rhizosphere soil had a decreased tendency with the number of growing years. The SMB-C difference among 3 cultivated ages was significant, while the SMB-N was not. The SMB of R3 was the lowest. Compared with R0, the SMB-C and the SMB-N were significantly reduced 77.30% and 69.36%. It was considered by integrated analysis that the leading factor of continuous cropping obstacle in ginseng was the changes of the rhizosphere soil microbial species, number and activity as well as the micro-ecological imbalance of rhizosphere soil caused by the accumulation of ginseng rhizosphere secretions.

Effect of environmental factors on photosynthetic physiology and flavonoid constituent of Scutellaria baicalensis / 中国中药杂志

To discuss the effect of environmental factors and photosynthesis on the growing of plant and the content of active components in Scutellaria baicalensis, the photosynthetic physiology index and diurnal changes of flavonoid constituent of S. Baicalensis were observed and tested in flowering and fruiting stages, and in the meantime environmental parameters were recorded. The obtained data were analyzed data by using path analysis and gray correlation analysis. The results showed that PAR and SWC were important environmental factors impacting on photosynthesis of S. baicalensis. SWC, RH and Ca were important environmental factors impacting on baicalin content. PAR, Po and Ta were important environmental factors impacting on baicalein content.

Construction of prokaryotic expression vector, expression and purification of ginseng Cu/Zn superoxide dismutase / 中国中药杂志

The total RNA was extracted from ginseng leaves of Panax ginseng. The Cu/Zn-SOD gene was amplified via RT-PCR and the pET-28(a)-Cu/Zn-SOD expression vector was constructed. The pET-28 (a)-Cu/Zn-SOD recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells and was induced by IPTG in order to select optimal induction of expression conditions. The target protein was purified by the nickel ions (Ni ) affinity chromatography and the target protein enzyme activity was determinated by the xanthine oxidase method. The similarity of the Cu/Zn-SOD gene sequences and the Cu/Zn-SOD gene sequences of Korean ginseng in NCBI was 99. 00%. The target protein expression level was about 44.42%, and the molecular weight was 16.30 kDa after the pET-28(a)-Cu/Zn-SOD recombinants were induced by IPTG. The purified Cu/Zn-SOD protease activity reached 10,596.69 U x mg(-1). The P. ginseng pET-28(a)-Cu/Zn-SOD prokaryotic expression vector was built by the method of molecular biology, which provided the foundation for studying the Cu/Zn-SOD biology function.

Correlation study on lignin contents of Schisandra chinensis and ecological factors / 中国中药杂志

The contents of schisandrin, schizandrin A, B and C were determined by HPLC, and the effects of the climate factors and altitude on lignin contents were analyzed in order to select the optimal cultivation area of S. chinensis. The lignin contents were analyzed by HPLC using a ZORBAX SB-C18 column (4.6 mm x 250 mm, 5 microm). The column temperature and detection wave length were set at 35 degrees C and 254 nm, respectively. Methanol-water was used as the mobile phase in gradient elution mode and the flow-rate was 1.0 mL min(-1). The method had a good repeatability, stability and accuracy. The correlation of climate factors and lignins contents was analyzed by SPSS software. The results showed that the schizandrin A content in S. chinensis fruits were higher than 0.4% in Ji'an, Liuhe, Antu and Fusong in Jilin province, which met the quality requirement. It had significant linear negative correlation relationship between schisandrin, schizandrin A, B and altitude, the contents decreased with the increase of altitude. The significant negative linear fitting coefficient was 0.844 1 between schisandrin and altitude; but it had not significant correlation between schizandrin C and altitude. A significant positive correlation of climate factors and the contents of S. chinensis lignins were mainly the temperature factors (the average annual temperature, the highest temperature in July, the average temperature in July, the highest temperature in January, the average temperature in January) and precipitation factor (average annual precipitation), which reveals that higher temperature and precipitation were helpful to the formation and accumulation of lignins of S. chinensis. So the cultivation area of S. chinensis should be in the low elevations region with warm and rainy climate.

Physiological ecology responses of Scutellaria baicalensis to drought and rewatering / 中国中药杂志

To study the physiological ecology responses of Scutellaria baicalensis to drought and rewatering of short period, we tested and analyzed photosynthesis and chlorophyll fluorescence parameters of S. baicalensis leaves processed by different ways of water treatment in drought and rewatering period, characteristic indexes of physiology and biochemistry of root SOD, POD, PAL, C4H, etc. and accumulation dynamic change of root baicalin and baicalein. The result showed that along with the worsening drought, P(n), T(r), G(s) and F(v)/F(m) of S. baicalensis declined in different water supply, and F(o) increased. The response of SOD and POD's activity in S. baicalensis root to drought in I and II was earlier than it in III. The response time and increase range of baicalin accumulation existed differences in different water supply, and the indexes regained after rewatering. Therefore, photosynthesis of S. baicalensis changed and it destroyed the antioxidant metabolism balance when soil water content decreased resulting from drought. The synergistic effect of defence mechanism launched by S. baicalensis, SOD, POD, PAL, C4H, baicalin and baicalein reduced active oxygen's damage to the cell.

Effects of different treatments on seed germination of Dioscorea nipponica / 中草药

Objective: To study the effects of different temperature conditions (10, 15, 20, 25, and 30 °C) and different concentration of gibberellin (GA3) and naphthalene acetic acid (NAA) on the germination responses of Dioscorea nipponica seeds. Methods: The seed germination rate and germination vigor were recorded and analyzed statistically, through setting conditions including cold stratification and soaking treatment with different concentration of GA3/NAA in culture dishes. Results: After sand stratification at 4 °C, the germination percentage and germination vigor of D. nipponica seeds were the highest of 80.00% and 53.33% at 25 °C, respectively. After soaking in 100 mg/L GA3 for 24 h without stratification, the germination percentage and vigor of D. nipponica seeds were 66.65% and 48.35%, respectively; After soaking in 1 mg/L NAA for 12 h, the germination percentage and germination vigor were 58.35% and 3.35%, respectively. The germination process treated with low temperature sand stratification was the fastest. The seeds began to germinate at the day 6, and germinated completely within 12 d. Conclusion: The seeds should germinate at 25 °C after sand stratification at 4 °C for 60 d, or the seeds without stratification would germinate after being treated with 100 mg/L GA3.

Association study of brain-derived neurotrophic factor Val66Met polymorphism and clinical characteristics of first episode schizophrenia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To assess the association between brain-derived neurotrophic factor (BDNF) gene Val66Met polymorphism and clinical characteristics of first episode schizophrenia in a Chinese Han population.</p><p><b>METHODS</b>Genotyping of BDNF Val66Met polymorphism was carried out for 135 schizophrenic patients and 483 healthy controls with TaqMan probe technology. The patients' psychotic symptoms were assessed using the positive and negative syndrome scale (PANSS).</p><p><b>RESULTS</b>A significant difference was found in genotype distribution and allelic frequency of the Val66Met polymorphism between the two groups (P< 0.01). In patients, Met homozygotes had a significantly higher score in anxiety/depression factor, cognitive factor and total score of PANSS than Val carriers.</p><p><b>CONCLUSION</b>BDNF gene Val66Met polymorphism is associated with the pathogenesis of schizophrenia. The Met/Met genotype of BDNF Val66Met variant may be a risk factor for symptoms in first episode schizophrenia patients.</p>

Study on fitting growth density and best harvest time of Tribulus terrestris / 中国中药杂志

<p><b>OBJECTIVE</b>To study the suitable growth density and the optimal harvest time of Tribulus terrestris.</p><p><b>METHOD</b>Four growth densities were set with 60 cm breadth ridge and individual distance of 10, 20, 30 and 50 cm. The yield per individual and per unit area under the different growth densities were determined. Using yam saponin as a standard substance, the total saponin of T. terrestris was determined by UV spectrophotometry.</p><p><b>RESULT</b>The individual yield decreased with the density increase, but the difference between 30 cm and 50 cm individual distance was not substantial. The yield per unit area increased with density increase, and the difference between all densities was significant. The yield peak was in the last ten-day of August. The best leaves area index was 1.4 at the growth peak time. The total saponins content reached peaks respectively in the last ten-day of June and August, but the peak in last ten-day of August was consistent with the one of yield per unit area, and the total ashes content was the lowest at the same time.</p><p><b>CONCLUSION</b>The suitable growth density is 10 cm individual distance on the 60 cm breadth ridge. The optimal harvest time is in the last ten-day of August. The yield per unit area is 1 400 - 2 000 kg x hm(-2).</p>

An epidemiological study on sexual transmission of human immunodeficiency virus among pre-marital group in Yining city, Xinjiang / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the human immunodeficiency virus (HIV) status through heterosexual transmission in Yining city and to provide information on effective intervention measures.</p><p><b>METHODS</b>Cohort of HIV sero-discordant couples identified from 1997 to 2000 was formed. Proportional risk model was used to analyze the time of HIV sero-conversion and the related factors. All the recruiters were under informed consent.</p><p><b>RESULTS</b>Through following on 22 sero-discordant couples, we found that the incidence density (ID) of HIV sero-conversion was 32.49/100 person-year (PY) with 33.74/100 PY for women. In the proportional hazard model, the course of sero-conversion was only 2.43 years and the frequency of sexual contact was statistically significant (>or= 3 times/week vs. < 3 time/week: RR = 1.984, 95% CI: 1.045 - 3.767), indicating this factor was related to the hazard of HIV sero-conversion. However, the viral load of HIV infections has no such effect on HIV sero-conversion of their spouses. In addition, the ratio of CD4(+)/CD8(+) was lower in spouses of HIV sero-conversion than that in spouses of HIV non-sero conversion (t test: t = 4.77, P < 0.01).</p><p><b>CONCLUSION</b>In order to control HIV transmission among general population, we suggested that HIV/AIDS counseling and testing be developed for pre-marital people in the region with high HIV prevalence.</p>

Lipopolysaccharide induces expression of macrophage inflammatory protein-1alpha in human umbilical vein endothelial cells / 中华病理学杂志

<p><b>OBJECTIVE</b>To understand whether endotoxin lipopolysaccharide (LPS) is able to induce the expression of macrophage inflammatory protein-1alpha (MIP-1alpha) mRNA and protein in human umbilical vein endothelial cells (HUVECs).</p><p><b>METHODS</b>The expression of MIP-1alpha mRNA was determined by dot blotting analysis and by in situ hybridization using a digoxigenin-labeled MIP-1alpha cDNA probe after exposure of the cultured HUVECs to LPS at different concentrations. The expression of MIP-1alpha mRNA was determined by RT-PCR as well. In addition, the expression of MIP-1alpha protein was tested by cell enzyme-linked immunosorbent assay (ELISA) using a goat anti-human monoclonal MIP-1alpha antibody.</p><p><b>RESULTS</b>Dot blotting showed that the absorbance values of the dots on the nitrocellulose membrane were 1.490 and 3.310 when exposed to LPS at the concentrations of 1 micro g/ml and 10 micro g/ml which were 1.97- and 4.38-fold over that of the control group (0.775), respectively. In situ hybridization revealed that exposure to LPS at a concentration of 1 micro g/ml led to a significant increase in the MIP-1alpha mRNA expression in HUVECs as compared to the control group (F = 142.83, P < 0.01), whereas the MIP-1alpha mRNA in HUVECs was somewhat decreased when exposed to LPS at a concentration of 10 micro g/ml. RT-PCR revealed that the expression of MIP-1alpha mRNA in HUVECs were 1.65-, 2.86- and 1.26-fold over that of the control group when exposed to LPS at the concentrations of 1 micro g/ml, 5 micro g/ml and 10 micro g/ml respectively. Cell ELISA showed that after exposure of the HUVECs to LPS at the concentrations mentioned above, the expression of MIP-1alpha protein was strongly increased, especially in the 5 micro g/ml LPS group. Analysis of variance showed that there was a significant difference between groups (F = 15.36, P < 0.05).</p><p><b>CONCLUSIONS</b>LPS may induce a high level of MIP-1alpha mRNA and protein expression in HUVECs, and it might, hereby, play an important role in the recruitment of the monocytes/macrophages into the arterial intima.</p>

Inhibition of magnesium lithospermate B on the c-Jun N-terminal kinase 3 mRNA expression in cardiomyocytes encountered ischemia/reperfusion injury / 药学学报

<p><b>AIM</b>To study the function of c-Jun N-terminal kinase 3 (JNK3) in the process of ischemic/reperfused heart injury and the mechanism underlying the protective action of magnesium lithospermate B (MTB), a bioactive compound isolated from Danshen.</p><p><b>METHODS</b>By in situ hybridization, JNK3 mRNA was detected in the ventricular preparations of the Langendorff ischemic/reperfused rat heart. The inhibitory effect of MTB on the expression of JNK3 mRNA was also investigated.</p><p><b>RESULTS</b>The purple and blue hybridization signals were located in the cytoplasm of the cardiomyocytes, which were weaker in the non-perfused hearts and stronger in the hearts encountered 30 min of ischemia and 30 min of reperfusion. Image analysis showed that the expression of JNK3 mRNA in the cardiomyocytes increased after 30 min of ischemia and 30 min of reperfusion, which showed significant difference compared with that in the cardiomyocytes of the non-perfused heart and the control heart (P < 0.05). Treatment with of 0.1, 1 and 10 mumol.L-1 MTB abolished the elevation of JNK3 mRNA expression in the ischemic/reperfused heart (P < 0.05).</p><p><b>CONCLUSION</b>JNK3 may be another component in the signal transduction pathway of ischemia/reperfusion induced cardiomyocyte apoptosis. MTB may protect the heart from ischemia/reperfusion injury by reducing apoptosis through inhibition of the JNK3 activity.</p>

A HPLC Method for Determination of the Content of an analog of Baogongteng A-ATST in the Aqueous Humor of Rabbits / 上海第二医科大学学报

Objective A HPLC method was established for determination of the content of ATST in the aqueous humor of rabbits. MethodsThe mobile phase was consisting of methanol-1％ triethylamine(57:43) and omeprazole (OMZ) as internal standard. The detection was carried out with an ultraviolet detector operated at 235nm. ResultsThere was linearity over the range of 2. 056～41.12 ug/ml in the humor aquosus, r=0.9997. The average recovery of ATST was 94.58 ％. Intra-day and in- ter-day RSD were less than 5 ％ and 10 ％ ( n = 5), respectively. Conclusion The method is reliable. It can be used for the study on the pharmacokinetics of ATST.