A rapid HPLC method for determination of coniferyl ferulate in Angelica sinensis and Ligusticum chuanxiong

Aim A reliable and rapid HPLC method was developed for quantitative determination of coniferyl femlate, an ester of ferulic acid, with multiple pharmacological activities in Angelica sinensis and Ligusticum chuanxiong, two commonly used Chinese medicines. Methods The determination was achieved by using a Zorbax ODS C18 analytical column （250 mm×4.6 mm ID, 5 μm） at isocratic elution of 1% aqueous acetic acid and acetonitrile （1：1） with diode-array detection （318 nm）. The calibration curve of coniferyl femlate showed good linearity （r^2 = 0.9995） within the test range. Results The developed method showed good precision with intra- and inter-day variations of 0.22% - 1.16% and 0.86% - 2.62% between the levels of 0.380 - 0.038 mg·mL^-1, respectively. The repeatability represented as RSD of coniferyl femlate was less than 2.7% for three levels （0.2 - 1.0 g of Angelica sinensis）, and the recovery was 105.3% with RSD of 3.2%. Conclusion The validated method was successfully applied to quantify coniferyl femlate in 12 samples of Danggui and Chuanxiong.

Chemical Constituents of Angelica sinensis

Aim To investigate the active constituents responsible for thepharmacological activities of Angelica sinensis (Oliv) Diels. Methods Chromatography was used toisolate chemical components, and spectroscopy was used to identify their structures. Results Sevencompounds were isolated and their structures were identified as ferulic acid (1), conife-rylferukte(2) , bis (2-ethylhexyl) phthalate (3), dibutyl phthalate (4), lignoceric acid (5), palmitic acid(6), and Z-6, 7-cis-dihydroxyligustilide (7) Conclusion Bis (2-ethylhexyl) phthalate and dibutylphthalate were obtained from Angelica sinensis for the first time.

Effects of angelica sinensis polysaccharide-iron complex on hemolytic anemia and bone marrow injury in mice

To investigate the therapeutic effects of angelica sinensis polysaccharide-iron complex （APIC） on hemolytic anemia and bone marrow injury in mice models. The hemolytic anemia mouse model was established by i.p. of phenylhydrazine （PHZ）. Changes of the indices including red blood cell count （RBC）, hemoglobin （Hb） and hematocrit （HCT） were determined by blood analyzer, and reticulocytes were observed by brilliant cresol blue staining during administration. Bone marrow injured mouse model was established by i.p. of cytoxan （CY） and chloramphenicol （CH）, and the therapeutic effect was observed by H-E staining. The indices of APIC treated groups with the medium and high doses were higher than those of the model group significantly. Moreover, the Hb and HCT were restored to the normal level after drug treatments. In addition, APIC can promote the proliferation and differentiation of reticulocytes obviously in the early stage of anemia mice, decrease adipose cell proliferation in bone marrow of injured mice and hasten the recuperation. In conclusion, APIC has therapeutic efficacy on hemolytic anemia and bone marrow injury caused by chemicals, which is reported for the first time.

Polysaccharides from Angelica sinensis alleviate neuronal cell injury caused by oxidative stress

Angelica sinensis has antioxidative and neuroprotective effects. In the present study, we aimed to determine the neuroprotective effect of polysaccharides isolated from Angelica sinensis. In a pre-liminary experiment, Angelica sinensis polysaccharides not only protected PC12 neuronal cells from H202-induced cytotoxicity, but also reduced apoptosis and intracellular reactive oxygen species levels, and increased the mitochondrial membrane potential induced by H202 treatment. In a rat model of local cerebral ischemia, we further demonstrated that Angelica sinensis poly-saccharides enhanced the antioxidant activity in cerebral cortical neurons, increased the number of microvessels, and improved blood flow after ischemia. Our findings highlight the protective role of polysaccharides isolated from Angelica sinensis against nerve cell injury and impairment caused by oxidative stress.

中华绒螯蟹(Eriocheir sinensis)血细胞生成的初步研究

为了揭示失血和LPS刺激后中华绒螯蟹血细胞的生成及成熟过程,利用流式细胞技术将中华绒螯蟹造血组织细胞和血细胞按大小进行分类,利用EdU标记检测失血刺激后中华绒螯蟹造血组织细胞的增殖情况,分析失血和LPS刺激后中华绒螯蟹不同类型血细胞比例的变化.结果显示:中华绒螯蟹造血组织细胞分布于106~384μm^(2)之间,而血细胞分布在119~434μm^(2)之间,其中,透明细胞和小颗粒细胞主要分布在200~300μm^(2)之间,中颗粒和大颗粒细胞主要分布在300~400μm^(2)之间,随着血细胞颗粒性的增加,细胞逐渐增大.在失血刺激6、12和24 h时,中华绒螯蟹造血组织细胞增殖加速,说明失血刺激可以促进造血组织细胞增殖以加速新血细胞的生成.失血刺激2 h后透明细胞比例显著降低,至12 h时,各类细胞基本恢复至正常水平.LPS刺激1、3、6和9 h后,透明细胞比例呈现先降低后升高再降低的变化,而小、中和大颗粒细胞比例变化与透明细胞比例变化趋势相反,LPS刺激24 h和48 h后,透明细胞比例显著升高,大颗粒细胞比例显著降低.由此可知,失血和LPS刺激引起了中华绒螯蟹机体不同细胞的免疫反应,可能通过不同方式影响血细胞的生成、分化和成熟.

Protective effect of Angelica sinensis on cerebral neurons from rat embryos under hypoxia

BACKGROUND： The enhanced expression of c-Fos protein in nerve cells after hypoxia is the marker for converting extracellular hypoxia information to intracellular changes at hypoxia, and it is suspected that the increase of c-Fos protein can lead to the synthesis and excretion of related neurotrophic factor and nerve growth factor. However, it is still unclear what functional changes of nerve cells are induced by the increase of c-Fos protein at hypoxia, and whether it is good for the survival of damaged neurons. OBJECTIVE： To observe the expression of c-Fos in the cerebral neurons from embryos of rats with hypoxia in uterus, and investigate the pathway for the protective effect of Angelica sinensis injection on the cerebral neurons from rat embryos under hypoxia. DESIGN： A completely randomized controlled study. SETTING： Department of Histology and Embryology, Luzhou Medical College. MATERIALS： Twelve female Wistar rats in oestrum and 1 male adult Wistar rat with body mass of 220 to 250 g were selected. Rabbit-anti-rat neuro-specific enolase （NSE） and rabbit-anti-rat c-Fos were purchased from Wuhan Boster Biological Technology Co., Ltd.; Double-staining kit was bought from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Angelica sinensis injection was produced by the Department of Pharmacy, the Second Affiliated Hospital of Hubei Medical University. METHODS： The experiments were completed in the experimental animal center and the Department of Histology and Embryology of Luzhou Medical College from December 2004 to December 2005. ①Twelve adult female Wistar rats in oestrum and 1 male Wistar rat were housed in one rearing cage. The appearance of vaginal embolus at 8：00 in the next morning was recorded as 0 day of pregnancy and the rats were recorded for 15 days, and they were divided randomly into three groups, control group （n =4）, hypoxia group （n =4） and Angelica group （n =4）. The pregnant rats in the hypoxia group were firstly injected with saline （8 mL/kg）, then put into 2 L wide-mouthed bottle containing 100 g sodalime, and then the lid of the bottle was closed tightly to induce hypotonic hypoxia for 1 hour followed by 1-hour re-oxygenation. The pregnant rats were killed under anesthesia, and then fetuses were taken out by rapid cesarean. Part of the brain tissues were exposed and then fixed in formaldehyde （40 g/L）. The pregnant rats in the Angelica group were treated the same as those in the hypoxia group except that saline was replaced by 250 g/L Angelica sinensis injection which was injected via caudal vein （8 mL/kg）. The rats in the control group were injected with saline （8 mL/kg） slowly via caudal vein, but not put into the wide-mouthed bottle for hypoxia, and then the brain tissues were removed and fixed as those in the hypoxia group after 1 hour. ②Twenty embryos from rats were chosen randomly in each group and then routinely embedded in paraffin. Paraffin sections of 4 μ m thick were prepared through the anterior fontanelle of head of the fetal rats. The sections were immunohistologically stained with c-Fos/NSE. ③The one-way analysis of variance （ANOVA） was used to compare the differences of measurement data among the groups, and the q test was applied in the two-two comparison. MAIN OUTCOME MEASURES： The numbers of c-Fos and c-Fos/NSE positive neurons in cerebrum from rat embryos were observed. RESULTS： ① Numbers of NSE positive neurons in cerebrum of rat embryos in the control group, hypoxia group and Angelica group were （84.3 ±9.0）, （90.2±12.5） and （86.7±9.7） cells/high power field （P 〉 0.05）. ②The number of c-Fos/NSE positive neurons was more in the hypoxia group than in the control group and Angelica group [（38.4±5.28）, （11.35±2.67）, （20.65±4.07） cells/high power field, q =29.17, 19.14, P 〈 0.05]. CONCLUSION： Hypoxia can stimulate the expression of c-Fos in cerebral neurons from rat embryos. Angelica sinensis injection could reducing the damage of hypoxia to neurons and play a neuroprotective role by decreasing the expression of c-Fos protein in hypoxic neurons.

Polysaccharide from Angelica Sinensis Suppresses Inflammation and Reverses Anemia in Complete Freund's Adjuvant-induced Rats

The antinlammatory and antianemic activities of Angelica sinensis polysaccharide(ASP)isolated from roots of Angelica sinensis(AS)was investigated in a complete Freund's adjuvant(CFA)-induced arthritic rat model.It was observed that serum iron(SI)and total iron binding capacity(TIBC)levels were elevated after 4-week oral administration of ASP.Red blood cell(RBC)count and hemoglobin(Hb)concentrations were ameliorated as well.Moreover,infammatory cytokines IL-6 and TNF-a were decreased strikingly in CFA-induced arthritic rats after treatment of ASP.Evidence also showed that ASP strongly inhibited hepcidin expression through the Janus kinase/signal transducers and activators of transcription(JAK2/STAT3)pathway.Furthermore,ASP exhibited reduced primary and secondary lesions in adjuvant arthritis,attenuating synovitis and inflammatory joint damage.Data presented in this article collectively indicated that ASP significantly decreased proinflammatory cytokines(TNF-a,IL-6),which might play a crucial role in the CFA-induced arthritic rats,and had a therapeutic effect on adjuvant arthritis in rats.Results of Western blot analysis indicated that ASP inhibited the activation of IL-6/JAK2/STAT3 signaling pathway in the CFA-induced arthritic rats.

Preparation and Identification of Angelica sinensis Polysaccharide-iron Complex

Angelica sinensis polysaccharide（ASP） was extracted from Angelica sinensis by boiling water. An Angelica sinensis polysaccharide-iron complex（APC） was prepared under the alkaline condition by adding a ferric chloride solution to the ASP solution. Then some identifiable properties of the complex were studied. The content of iron（ Ⅲ ） in the complex was determined with iodometry. The thermal property, the microscopic structure, the spectral characteristics, and N, C, H contents of the complex were examined by a variety of techniques including DSC, TEM, IR, NMR, and elemental analysis. The content of iron（ Ⅲ ） in the complex ranges from 10% to 40%. The DSC result shows that the melting point of the complex is about 450 ℃. The TEM result shows that the complex has an iron（ Ⅲ ） core（β-FeOOH core） linked by hydroxy and oxy bridges, with the polysaccharide chains attached to the surface of the core. The IR and NMR results also show that there is a β-FeOOH core in the complex. The elemental analysis shows that the contents of N, C, H in the complex are, respectively, lower than those of N, C, H in ASP. All our studies indicate that the APC consists of a β-FeOOH core surrounded by ASP.

A New Dimeric Phthalide from Angelica sinensis

A new dimeric phthalide named Z, Z＇-3.3＇a, 7.7＇a-diligustilide was isolated from the roots of Angelica sinensis. Its structure was determined using spectroscopic methods and X-ray crystallographic diffraction analysis.

Determination of constituents of essential oil from Angelica sinensis by gas chromatographymass spectrometry

Gas chromatographymass spectrometry (GC-MS) coupled with chemometric resolution upon two- dimensional data was employed to analyze the constituents of essential oils of Angelica sinensis. Constituents in essential oils of Angelica sinensis root were identified by GC-MS with the help of subwindow factor analysis (SFA) method resolving two-dimensional original data into mass spectra and chromatograms. 76 of 97 separated constituents in essential oil of Angelica sinensis root were identified and quantified, and they account for about 91.36% of the total content. The results show that ligustilide, butylene phthalide, 2-methoxy-4-vinylphenol, carvacrol, allo-ocimene,2,6,6-trimethylbicyclo-[3,1,1]hept-2-ene are the main constituents in essential oil of Angelica sinensis root.

Effect of Angelica sinensis on neural stem cell proliferation in neonatal rats following intrauterine hypoxia

BACKGROUND： Angelica sinensis is a widely used herb in Chinese traditional medicine. It has been shown to improve hypoxia in embryonic rats and reduce nestin expression in neural stem cells, resulting in proliferation of neural stem cells. OBJECTIVE： To study the protective effect of Angelica on neural stem cell proliferation in neonatal rats after intrauterine hypoxia. DESIGN, TIME AND SETTING： The randomized, controlled, experiment was performed at the Department of Histology and Embryology, Luzhou Medical College, China from July 2007 to January 2008. MATERIALS： Because gestational days 14-15 are a key stage in rat nervous system development, 21 healthy, pregnant Sprague Dawley rats （14 days after conception） were used for this study. Nestin monoclonal primary antibody was obtained from Chemicon, USA. Angelica parenteral solution （250 g/L） was obtained from Pharmaceutical Preparation Section, Second Affiliated Hospital of Wuhan University, China. METHODS： Rats were randomly divided into a control group （n = 5）, a hypoxia group （n = 8）, and an Angelica group （n = 8）. Saline （8 mL/kg） was injected into the caudal vein of rats in the hypoxia group once a day for seven consecutive days. Intrauterine hypotonic hypoxia was induced using 13% O2 for two hours per day on three consecutive days. Rats in the Angelica group received injections of Angelica parenteral solution （250 g/L）; all other protocols were the same as the hypoxia group. The control group procedures were identical to the hypoxia group, but under normal, non-hypoxic conditions. After birth, brain tissues were immediately obtained from neonatal rats and prepared for nestin immunohistochemistry. MAIN OUTCOME MEASURES： Nestin-positive cells in hippocampal CA3 area of neonatal rats in each group were quantified using image analysis to detect signal absorbance. RESULTS： The number of nestin-positive cells increased in the hippocampal CA3 area of neonatal rats in the hypoxia group. The number of nestin-positive cells was less in the Angelica group than in the hypoxia group. Integral absorbance of nestin-positive ceils in the hippocampal CA3 area of neonatal rats was significantly higher in the hypoxia group, compared with the control group （P 〈 0.05）. The integral absorbance of nestin positive cells was lower in the Angelica group, compared with the hypoxia group （P 〈 0.05）. CONCLUSION： Intrauterine hypoxia, induced for 2 hours daily for three consecutive days, with an oxygen concentration of 13%, stimulated the proliferation of neural stem cells. Angelica injection has a protective effect on neural stem cells from neonatal rats following intrauterine hypoxia by decreasing proliferation of neural stem cells.

Identification of the lysine and histidine transporter family in Camellia sinensis and the characterizations in nitrogen utilization

In plants,the lysine and histidine transporter(LHT)family represent a class of proteins that mediate the uptake,translocation,and utilization of amino acids.The tea plant(Camellia sinensis)is a perennial evergreen with a relatively high level of amino acids.However,systematic identification and molecular characterization of the LHT gene family has rarely been reported in tea plants.In this study,22 CsLHTs were identified from the‘Shuchazao’genome and classified into two groups.The modeled three-dimensional structure and the conserved domains presented a high similarity among the LHTs proteins.Moreover,it was predicted that a few genes were conserved through the analysis of the physiochemical characters,structures and cis-elements in promoters.The expression patterns in tea plants revealed that CsLHT7 was mainly expressed in the roots,and CsLHT4 and CsLHT11 exhibited relatively high expression in both the roots and leaves.Moreover,the expression of all three genes could be induced by organic nitrogen.Additionally,heterogeneous expression of CsLHT4,CsLHT7 and CsLHT11 in Arabidopsis thaliana decreased the aerial parts biomass compared with that in WT plants while significantly increased the rosette biomass only for CsLHT11transgenic plants versus WT plants.Overall,our results provide fundamental information about CsLHTs and potential genes in N utilization for further analysis in tea plants.

Differentiation of human adipose-derived stem cells into neuron-like cells by Radix Angelicae Sinensis

Human adipose tissues are an ideal source of stem cells. It is important to find inducers that can safely and effectively differentiate stem cells into functional neurons for clinical use. In this study, we investigate the use of Radix Angelicae Sinensis as an inducer of neuronal differentiation. Primary human adipose-derived stem cells were obtained from adult subcutaneous fatty tissue, then pre-induced with 10% Radix Angelicae Sinensis injection for 24 hours, and incubated in serum-free Dulbecco＇s modified Eagle＇s medium/Nutrient Mixture F-12 containing 40% Radix Angelicae Sinensis to induce its differentiation into neuron-like cells. Butylated hydroxyanisole, a common in- ducer for neuronal differentiation, was used as the control. After human adipose-derived stem cells differentiated into neuron-like cells under the induction of Radix Angelicae Sinensis for 24 hours, the positive expression of neuron-specific enolase was lower than that of the butylated hydroxyani- sole-induced group, and the expression of glial fibrillary acidic protein was negative. Alter they were induced for 48 hours, the positive expression of neuron specific enolase in human adipose-derived stem cells was significantly higher than that of the butylated hydroxyanisole-induced group. Our experimental findings indicate that Radix Angelicae Sinensis can induce human adipose-derived stem cell differentiation into neuron-like cells and produce less cytotoxicity.

Effect of intrauterine hypoxia and Angelica sinensis injection on dentate gyrus neurons and learning and memory in juvenile rats

BACKGROUND： The present study analyzed the effect of 3 days （2 h/d） intrauterine hypoxia on learning and memory in juvenile rats, as well as the therapeutic effects of Angelica sinensis on dentate gyrus neurons, as well as learning and memory. OBJECTIVE： To explore the effects of intrauterine hypoxia on hippocampal dentate gyrus neurons, as well as learning and memory, in juvenile rats; to explore N-methyI-D-aspartate receptor-1 （NMDAR1） expression in the dentate gyrus of neonatal rats following intrauterine hypoxia, as well as prolonged hypoxia; to investigate the regulatory mechanisms of Angelica sinensis. DESIGN, TIME AND SETTING： A randomized and controlled experiment based on developmental neurobiology was performed at the Department of Histology and Embryology in Luzhou Medical College from October 2007 to October 2008. MATERIALS： Angelica sinensis solution （250 g/L） was obtained from Central South Hospital of Wuhan University, China. Neuron-specific enolase and NMDAR1 mRNA in situ hybridization reagents were provided by Wuhan Boster Biological Technology, China. Image-Pro Plus 6.0 analysis system was purchased from Media Cybernetics, USA. METHODS： Healthy pregnant Sprague Dawley rats （n = 30） were randomly divided into control （n = 10）, hypoxia （n = 10）, and Angelica （n = 10） groups. The Angelica and hypoxia pregnant rats were placed in a three-gas incubator （oxygen concentration： 13%） starting with day 14 of pregnancy for 2 hours/day for 5 consecutive days to establish a fetal rat intrauterine hypoxia model. One hour prior to modeling, the pregnant rats from the Angelica and hypoxia groups received Angelica sinensis and normal saline （8 mL/kg） injections, respectively, through the caudal vein. The control group procedures were identical to the hypoxia group, but lacked the hypoxic conditions. MAIN OUTCOME MEASURES： Brain tissues of neonatal rats were used to detect expression of NMDAR1 mRNA, and brain tissues of juvenile rats aged 30 days were used to determine neuron-specific enolase mRNA expression by in situ hybridization. Microscopic images （400x） of the hippocampal dentate gyrus were collected. The integral optical density （IOD） value of positive NMDAR1 mRNA cells in the dentate gyrus of neonatal rats, as well as the quantity and the IOD value of positive neuron-specific enolase mRNA cells in the dentate gyrus of juvenile rats, were analyzed with Image-Pro IPP6.0 software. At 30 days after birth, learning and memory parameters were measured in the juvenile rats using Morris water maze. RESULTS： The quantity and the IOD value of positive neuron-specific enolase mRNA cells in the dentate gyrus of the hypoxia group juvenile rats were significantly less than the control group （P 〈 0.05）, and also less than the Angelica group （P 〈 0.05）. The IOD value of positive NMDAR1 mRNA cells in the dentate gyrus of the hypoxia group neonatal rats was significantly greater than the control group, and also greater than the Angelica group （P 〈 0.05）. In the Morris water maze, the searching time during the probe trial and reversal probe trial was shorter in the hypoxia group juvenile rats compared with the control group, and the Angelica group was prolonged compared with the hypoxia group （P 〈 0.05）. CONCLUSION： Intrauterine hypoxia increased expression of NMDAR1 mRNA in the dentate gyrus of neonatal rats, reduced the number of dentate gyrus neurons, and negatively affected learning and memory in juvenile rats. In contrast, Angelica sinensis injection improved the intrauterine hypoxic condition, increased the number of dentate gyrus neurons, and improved the learning and memory deficits of the juvenile rats.

EFFECT OF ANGELICA SINENSIS ON AFFERENT DISCHARGE OF SINGLE MUSCLE SPINDLE IN TO ADS

Objective In drugs for i nvigorating blood circulation, to find a herb that can stimulate afferent discha rge of muscle spindle. Methods A single muscle spindle was isolated from sartorial mus cle of toad. Using air-gap technique, afferent discharge of the muscle spindle was recorded. Effects of Angelica Sinensis, Salvia Miltiorrhiza, and Safflower o n afferent discharge of the muscle spindle were observed. Results Angelica Sinensis could distinctly increase afferent di scharge frequency of the muscle spindle, and this increase was dose-dependent. But Salvia Miltiorrhiza and Safflower had no this excitatory effect. Conclusion It is known that Angelica Sinensis can invigorate bl ood circulation, and we have found its excitatory effect on muscle spindle which makes it possible to serve people with muscle atrophy if more evidences from cl inical experiments are available.

EFFECTS OF REINFORCED DECOCTION OF ANGELICAE SINENSIS FOR ENRICHING BLOOD ON THE IMMUNITY OF IMMUNOSUPPRESSED MICE

Objective To investigate the effect of reinforced Decoction of Angelicae Sinensis for enriching blood (RDAEB) on the immunity of immunosuppressed mice induced by cyclophosphamide (Cy). Methods Mice were given RDAEB through stomach perfusion for 10 d (50 mg/d). Then, RBC-C3bR rate,RBC-IC rate (as the index- es of erythrocyte immunity)and E-rosette forming rate,acidic a-naphthyl acetate esterase positive rate, lymphocyte transformation rate (as the indexes of cellular immunity) of mice were tested. Results RBC-C3Br rate, RBC-IC rat- e,E-rosette forming rate, acidic α-naphthyl acetate esterase positive rate and lymphocyte transformation rate in the Cy-RDAEB group were markedly higher than those in the Cy group (P<0.0l),and returned to the levels of normal group. Conclusion RDAEB is effective in recovering and enhancing cellular and erythrocyte immunity of immuno- suppressed mice.

Optimal Formula of Angelica sinensis Ameliorates Memory Deficits in β-amyloid Protein-induced Alzheimer's Disease Rat Model

Objective:Angelica(A.)sinensis is used as a traditional medical herb for the treatment of neurodegeneration,aging,and inflammation in Asia.A.sinensis optimal formula(AOF)is the best combination in A.sinensis that has been screened to rescue the cognitive ability in P-amyloid peptide(Ap25-35)-treated Alzheimer’s disease(AD)rats.The objective of this study was to investigate the effect of AOF on the learning and memory of AD rats as well as to explore the underlying mechanisms.

Effect of co-existent components in CO2 supercritical fluid extract of Angelica Sinensis Radix on metabolism of Z-ligustilide after oral administration in rats

Objective:To establish a basis for Angelica Sinensis Radix(ASR)as a dietary supplement for colorectal cancer chemoprevention,the effect of co-existent components in supercritical fluid extract(SFE)of ASR on the pharmacokinetics of Z-ligustilide after oral administration was investigated in vitro and in vivo.Methods:Incubation in gastrointestinal contents and incubation in rat liver tissue homogenates post-mitochondrial supernatant(PMS)experiments were used to study changes in the levels of Z-ligustilide in vitro.Results:Within 4 hours,the level of Z-ligustilide in SFE declined at a slower rate than in its pure form.Clearance of Z-ligustilide after administration in its pure form was significantly slower than that of SFE of ASR(CL,0.96±0.16 mL·min/kg versus 1.24±0.21 mL·min/kg P<0.05;AUC,243.37±16.84 versus 176.69±12.59 mg·min/L).Conclusion:These phenomena may be attributed to the interactions between the co-existent components in SFE of ASR and Z-ligustilide enhancing the stability of Z-ligustilide.These results suggest that the bioavailability of Z-ligustilide in SFE of ASR is improved.However,stabilization of plasma concentration was not sustained,so that the efficacy of active components could not be maintained.Thus,further processing of SFE of ASR is required.

中华乌塘鳢(Bostrychus sinensis)白细胞介素8基因的克隆及免疫应答表达分析

由细菌等感染引起的疾病对中华乌塘鳢(Bostrychus sinensis)养殖业的可持续发展造成严重威胁。白细胞介素8(Interleukin-8,IL-8)在调节炎症反应和抗感染免疫方面扮演着重要的作用。利用PCR扩增和克隆测序获得了中华乌塘鳢IL-8(命名为BsIL-8)cDNA序列。BsIL-8 cDNA序列全长1244 bp,包含576 bp的5’端非编码区序列(5’-UTR),312 bp的开放阅读框序列(ORF)和356 bp的3’端非编码区序列(3’-UTR)。BsIL-8共编码103个氨基酸,包含1个由18个氨基酸组成的信号肽和1个由62个氨基酸组成的SCY结构域。其中,SCY结构域包含1个CXC基序(Cys^(30)-Arg^(31)-Cys^(32))以及4个保守的半胱氨酸残基(Cys^(30)、Cys^(32)、Cys^(57)和Cys^(73))。序列分析结果表明,BsIL-8与大弹涂鱼(Boleophthalmus pectinirostris)的IL-8序列相似性和一致性最高(分别为92.4%和86.4%)。基因组织表达分布结果显示,BsIL-8基因在主要组织器官中呈泛在性表达,头肾BsIL-8基础表达量最高,肝脏次之,外周血最低。Poly(I:C)免疫刺激后,BsIL-8在4个重要免疫器官(脾脏、肝脏、头肾和外周血)中的表达量均发生了显著的上调(分别在刺激后3、12、24和24 h时表达量达到最高,P<0.01)。副溶血弧菌(Vibrio parahaemolyticus)感染后,BsIL-8基因在脾脏和外周血中的表达量均显著上调(均在感染后12 h时表达量达到最高,P<0.01),而在肝脏和头肾中的表达量均显著下调(均在感染后12 h时表达量达到最低,P<0.01)。综上所述,BsIL-8基因参与了中华乌塘鳢抗感染免疫应答过程。

Molecular cloning and functional analysis of two calcium associated cuticular protein genes in Neocaridina denticulata sinensis

The body surface of crustaceans is covered with a sturdy shell.The growth and development of crustaceans are realized through molting.Neocaridina denticulata sinensis is a suitable candidate for crustacean scientific research.Two calcium-associated cuticular protein genes,named NdCAP-1 and NdCAP-2,were obtained from N.denticulata sinensis.Molecular docking simulated the binding effect of both proteins and calcium ions.Semi-quantitative reverse transcription PCR results show that NdCAP-1 is expressed in D_(2-4) stage,NdCAP-2 expressed in D_(2-4) and A-B stages,and both were significantly expressed in the cephalothorax cuticle and pereiopods.Then,it was revealed that NdCAP-1 and NdCAP-2 are regulated by NdEcR-mediated 20 E signaling pathways.Knockdown of NdCAP-1 and NdCAP-2 was observed to cause surface defects.The recombinant proteins(rNdCAP-1 and rNdCAP-2),obtained by prokaryotic expression,had calcium-binding and chitin-binding ability,inhibited formation of calcium carbonate precipitate.These results show that calcium-associated cuticular proteins play important roles in cuticle formation and calcification.