恩诺沙星淀粉微球的制备及缓释性能的研究

为了优选恩诺沙星淀粉微球的最佳制备工艺,试验将反相乳液聚合法与包埋载药法结合起来,以可溶性淀粉为载体、环氧氯丙烷为交联剂,制备恩诺沙星淀粉微球;以载药量和包封率为指标,通过L9(34)正交试验对制备工艺进行优化,采用扫描电镜观察载药微球的粒径大小及形态。结果表明:最佳工艺条件为淀粉4 g、恩诺沙星0.04 g、交联剂1.0 g、乳化剂0.8 g、反应时间2 h;按照此优化工艺参数制得的载药微球的总载药量为2.53%、包封率为89.72%;恩诺沙星淀粉微球大小较均匀,形态圆整,分散性较好,粒径为60μm左右;在最初2.5小时时释药量约为43.12%,至第8小时时释药量达80.69%,之后逐步释放,到第10小时时累积释药量占总药量的82.51%。说明此制备工艺可行,所制得的恩诺沙星淀粉微球具有一定的缓释效果。     

堆型艾美耳球虫3-1E重组质粒微球的制备及体外释放试验

用聚乳酸羟基乙酸共聚物（PLGA）将鸡堆型艾美耳球虫重组表达质粒pcDNA3.1-3-1E包封,采用水包油包水（W/O/W）双重乳化方法制备pcDNA3-3-1E/PLGA微球。通过正交试验设计优化PLGA微球制备工艺,考察PLGA浓度、聚乙烯醇（PVA）浓度、超声功率、复乳搅拌时间对评价指标（即微球粒径大小、包封率、载药量）的影响,确定制备微球的最佳工艺条件。测定微球的形态、粒径、完整性、质粒DNA包封率、载药量和释放程度;进行体外模拟鸡胃液和肠液试验,观察微球体外释药效果。结果显示,当PLGA浓度为8%、PVA浓度为1.5%、超声功率为60W、复乳搅拌6h为微球制备的最佳工艺参数,在光镜下呈散在圆形,粒径小于12μm。微球的包封率、载药量分别为84.25%和4.46%,裸质粒与微球中质粒超螺旋比例差距不显著,这表明在包被过程中的超螺旋质粒未发生明显的降解。在模拟鸡的胃肠液累积释放试验中,它的累积释放能力依次为pH 3.0〉pH 7.4,载药微球在模拟鸡的胃肠液中24h释放26.8%,模拟肠液中24h释放11.2%,30d时体外累积释放率为81.7%,表明微球具有一定的缓释效果。     

槲皮素纳米粒的制备及其体外评价

为了考察槲皮素纳米粒的体外特性,试验采用薄膜分散法制备了槲皮素纳米粒,采用透射电镜、X射线衍射法(XRD)对其进行表征,采用动态膜透析法考察载药纳米粒的体外释药特性。结果表明:薄膜分散法制备的纳米粒呈球形或类球形,平均粒径为(51.5±5.9)nm,平均包封率为(89.13±3.97)%,平均载药量为(2.87±1.91)%;槲皮素以无定型状态或分子状态分散在纳米粒中;槲皮素纳米粒体外释放具有明显的缓释作用。说明槲皮素纳米粒制备工艺简单,其粒径、包封率、载药量可控,具有缓释作用。     

黄芪多糖MPEG-PLA嵌段共聚纳米微球制备及体外释放研究

[目的]制备黄芪多糖MPEG-PLA嵌段共聚纳米微球,优选出最佳工艺条件,共考察微球的形态、粒径、载药量、包封率以及体外释放特性。[方法]选用聚乳酸-聚乙二醇(PLA-PEG)嵌段共聚物作为包载材料,采用复乳法制备黄芪多糖MPEG-PLA嵌段共聚纳米微球,优选最佳工艺条件。透射电子显微镜观察微球的形态、激光粒度分析仪测定其粒径及分布。采用紫外分光光度法测定微球的载药量和包封率,并研究其体外释放性质。[结果]采用复乳法制备的载药球电镜扫描结果外形圆滑,颗粒大小均匀,平均粒径873nm,包封率78%,释放率高达79.17%,释放时间较长为10天,且释放平缓,突释率较低。[结论]采用MPEG-PLA二氯甲烷溶液作为油相、黄芪多糖水溶液作为内水相,PVA水溶液作为外水相制备黄芪多糖MPEG-PLA嵌段共聚纳米微球的工艺稳定可行。     

盐酸多西环素壳聚糖微球的制备与评价

制备盐酸多西环素(doxycycline hydrochloride,DH)缓释微球,并对其进行评价。通过Design-expert软件进行试验设计,以载药量和包封率为考察标准进行优化,采用乳化交联的方法制备壳聚糖包载DH缓释微球,运用紫外-可见分光光度计(UV-VIS Spectrophotometer)、扫描电子显微镜(SEM)、傅里叶变换红外光谱(FT-IR)、热重分析(TGA)、X射线衍射(XRD)及拉曼光谱(Raman spectra)对微球的结构、性能和形态进行评价。结果显示,在最佳制备条件下,即壳聚糖为20 g/L,体系内DH质量为0.3 g,转速为910 r/min,液体石蜡12 mL时,所制备载药微球的载药量为56.49%,包封率为61.41%。FT-IR表明壳聚糖包载DH主要以物理作用为主;热失重表明微球物理包合后热稳定性较差;XRD结果表明DH被包载后晶体结构未发生变化。结果表明,本试验成功制备表面光滑、粒径整齐、载药量和包封率较高的DH壳聚糖微球。     

恩诺沙星聚合物胶束的制备及体外释放

通过比较3种不同质量比的两嵌段共聚物制备的聚合物胶束,优化恩诺沙星聚合物胶束的制备工艺,并考察该胶束的理化性质和体外释药特性。以自乳化溶剂挥发法制备恩诺沙星聚合物胶束,并采用单因素法优化处方;HPLC法测定其载药量、包封率、体外释药特性,激光粒度仪测定其粒径及分布,红外分光光度法（IR）确证含药胶束特征。结果显示,采用PLA16000-mPEG2000（聚乳酸-聚乙二醇单甲醚）共聚物为载体,以丙酮为有机溶剂,所制备胶束平均粒径为（117.2±8.2）nm,载药量为（3.3±0.17）%,包封率为（32.3±1.70）%;相对于另外2种材料制备的胶束有较好的载药量,且IR确证药物已被包封在胶束中。恩诺沙星胶束体外释药试验表明其具有一定的缓释特性。     

替米考星淀粉微球的制备及缓释性能的研究

以可溶性淀粉为原料,N,N′-亚甲基双丙烯酰胺为交联剂,采用包埋法制备了替米考星淀粉微球,通过L9(34)正交试验设计,以载药量和包封率的综合得分为指标,优化了替米考星淀粉微球的制备工艺;分别用激光粒度分布仪、扫描电镜和综合热分析仪对载药微球进行了表征。结果表明最佳工艺条件为：淀粉4 g、替米考星0.02 g、交联剂0.95 g、乳化剂0.75 g、反应时间1 h;影响因素的大小依次为：交联剂的质量＞替米考星和淀粉的投料质量比＞反应时间＞乳化剂的质量;按优化工艺参数制得的载药微球的总载药量2.24%,包封率为89.6%;替米考星载药微球具有一定缓释效果,其制备方法合理可行。     

恩诺沙星肺靶向明胶微球的制备及质量评价

研制恩诺沙星肺靶向明胶微球(ENR-GMS)并对其进行质量评价。采用乳化-化学交联法制备恩诺沙星肺靶向明胶微球,油镜下观察微球粒径大小及形态,并用显微照相机(Olym-pusDP12-2)拍照做记录。通过紫外分光光度法检测微球中恩诺沙星(ENR)的含量,进而计算得出其载药量和包封率,并通过溶出度测定法探讨其体外释放规律。制成的恩诺沙星肺靶向明胶微球为淡黄色粉末,油镜下大小较均匀,形态圆整、分散性较好,其内可见被包裹的黄色ENR粉末,平均直径约10.26μm。就粒径分布而言,5～12μm的微球占86.66%,载药量为4.51%,包封率为31%,在pH7.4的PBS缓冲液中的释药符合缓释特征,t1/2约为3.8h,比单纯的ENR延长3个多小时。体外释放实验显示前2.5h时释药量约37.02%,至第5.0h之间释药量达80.87%,之后逐步释放,至10h时药物释放累积量占总药量的87.96%。结果表明,制备出的恩诺沙星明胶微球符合肺靶向给药系统的要求,具有较好的缓释性和肺靶向性。     

伊维菌素聚酯酸酐微球的制备

将2种聚酯酸酐(P(SA∶RA 20∶80),P(SA∶RA 30∶70))分别与伊维菌素溶于三氯甲烷中作为有机相,以聚乙烯醇水溶液为水相,用乳化溶剂挥发法制备出2种伊维菌素聚酯酸酐微球。采用光学显微镜考察所制备微球的形态及粒径,紫外分光光度法测定载药量和包封率。结果显示,制备出的P(SA∶RA 30∶70)/IVM微球的平均粒径为(72.240±24.747)μm,载药量为19.67%,包封率为87.70%。P(SA∶RA 20∶80)/IVM微球的平均粒径为(64.18±26.14)μm,载药量为17.72%,包封率为87.27%。这表明采用乳化溶剂挥发法成功制备出2种伊维菌素聚酯酸酐微球。     

磷酸替米考星缓释微球的制备工艺研究

为解决磷酸替米考星在养殖生产中适口性的问题,本试验以丙烯酸树脂为囊材,采用乳液-溶剂蒸发法制备磷酸替米考星微球,并采用高效液相色谱法对药物含量进行测定。将3批磷酸替米考星微球置于冰箱(4℃)、室温(25℃)与40℃(RH 75%)放置3个月,进行外观形态、粒径及其分布、载药量、体外释药特性的考察。结果表明:在该试验方法下制备的磷酸替米考星微球的形态良好,平均粒径为10.65μm,粒径7～30μm范围内的微球占总数的96.7%,载药量和包封率分别是28.36%、99.25%,分散性好,具有较好的热稳定性。     

Changes in rat uterine and cervical phospholipase A2 activity following progesterone agonist or antagonist administration at term

Our previous study revealed that a fall in plasma progesterone (P(4)) level was associated with a transient increase in cytosolic phospholipase A(2) (PLA(2)) activity and prostaglandin F(2)alpha level in the rat uterus and cervix during natural parturition. This study determined the changes in the PLA(2) activities during modulated occurrence of delivery by P(4) antagonist or agonist late in pregnancy. In rats undergoing P(4) antagonist-induced preterm delivery, the PLA(2) activities of both uterine and cervical cytosol significantly decreased 12 h after the challenge and tended to be attenuated within 72 h. The plasma P(4) level altered in a similar pattern. Blockade of delivery by chronic treatment with P(4) agonist was not associated with changes in uterine PLA(2) activity compared with that in normally delivering rats, although there was a persistent rise in cervical PLA(2) activity. The obtained data indicates that the PLA(2) activities in rat uterine and cervical cytosol are not regulated solely by P(4) and that delivery can occur without activation of this enzyme.     

Use of progesterone in microspheres for maintenance of pregnancy in mares.

Administration of progesterone in poly(d-,l-lactide) microspheres was used to maintain pregnancy in mares after luteolysis was induced by treatment with prostaglandin F2 alpha at day 14 of pregnancy. Mares were given vehicle only (control, n = 6) or 0.75 g (n = 7), 1.5 g (n = 8), or 2.25 g (n = 5) of microencapsulated progesterone at days 12 and 22 of pregnancy. Serum progesterone concentrations were determined daily, and pregnancy was evaluated by transrectal ultrasonography on alternate days. Significantly (P less than 0.05) more mares given 1.5 or 2.25 g of progesterone (6 of 8 and 4 of 5 mares, respectively), but not those given 0.75 g (3 of 7 mares), maintained pregnancy through day 32, compared with control mares (0 of 6). Progesterone concentrations decreased significantly (P less than 0.025) in all groups after administration of prostaglandin F2 alpha at day 14, and significant (P less than 0.05) effects of time and treatment on progesterone concentrations were found between days 12 and 22, and 22 and 32. Although treatment with 1.5-g and 2.25-g doses of microencapsulated progesterone improved maintenance of pregnancy, compared with that of vehicle-treated controls, administration of 2.25 g of microencapsulated progesterone appeared to be most efficacious in maintenance of pregnancy during the study interval.     

Ovarian Steroid Regulation of Endometrial Phospholipase A2 Isoforms in Horses

Real‐time PCR was used to investigate the role of progesterone (P4) and oestradiol (E2) in regulation of endometrial cytosolic, secretory and calcium‐independent phospholipase A2 (PLA2G4A, PLA2G2A and PLA2G6, respectively) gene expression. Ovariectomized mares underwent 6 days of E2 pre‐treatment followed by 14 days of P4 supplementation. At the start of P4 treatment (Day 1), mares were assigned in a 2 × 2 factorial design to receive either E2 or vehicle starting on Day 11 and endometrial biopsy collection on either Day 14 when P4 concentrations remained high (>4 ng/ml) or Day 16 when P4 concentrations had declined (0.5–2 ng/ml). Additional biopsies were collected from ovariectomized mares on Day 8, which served as control. Blood samples were collected for P4 determination. PLA2G4A expression was higher (p < 0.05) on Day 14 compared with Day 8. In contrast, PLA2G2A did not change significantly (p < 0.12). PLA2G4A and PLA2G2A gene expression increased (p < 0.05), as P4 concentration dropped, on Day 16. In contrast, PLA2G6 gene expression did not show differences between days. Treatment with oestradiol did not increase PLA2 isoforms expression when compared to treatment with the vehicle. PLA2G4A and PLA2G2A were positively correlated with each other and negatively correlated with P4 concentrations. In conclusion, P4 withdrawal upregulated PLA2G4A and PLA2G2A gene expression, and this was not affected by E2. PLA2G4A and PLA2G2A but not PLA2G6 gene expression may be involved in controlling prostaglandin F2 alpha synthesis and luteolysis.     

Lysophosphatidic acid expression in theca cells depends on the type of bovine ovarian follicle

Lysophosphatidic acid (LPA) exerts various actions on the mammalian reproductive system. In cows, LPA stimulates the synthesis and secretion of luteotropic factors in the ovary, which affects the growth and development of ovarian follicles. The role of LPA in granulosa cells, oocyte and oocyte‐cumulus complex (COC) has previously been investigated; but its role in the theca layer, which is an important structural and functional component of the ovarian follicle, is still unclear. The goal of this study was to investigate the expression of LPA in theca cells originating from different bovine ovarian follicle types. Theca cells were separated from healthy, transitional and atretic ovarian follicles, based on intrafollicular estradiol: progesterone ratios. LPA concentration in the follicular fluid (FF) in different follicle types was measured, and expression of the enzymes responsible for LPA synthesis (autotaxin [AX], phospholipase A2 [PLA2]) and receptors for LPA (LPAR1‐4) were determined. The obtained results confirmed the follicle‐type dependent presence of LPA in the FF of the bovine ovarian follicles. The highest concentration of LPA was detected in follicles classified as healthy and dominant. LPAR1‐4, PLA2 and AX expression in theca cells in all of the types of follicles examined were detected at mRNA and protein level. These results suggest that theca cells can be a source of LPA synthesis other than granulosa cells and COCs, as well as the target for its action in the bovine ovarian follicle, with PLA2 and LPAR4 playing major roles in LPA synthesis and action.     

Uterine clearance mechanisms during the early postovulatory period in mares

Uterine response to inoculation with Streptococcus zooepidemicus organisms, 51Cr-labeled 15-microns microspheres, and charcoal was evaluated in 9 mares (4 resistant and 5 susceptible to endometritis) to determine mechanical and cellular clearance rates during the early postovulatory period. Mares were inoculated at estrus prior to ovulation during estrous cycles 1, 3, and 5. Uterine swab specimens for aerobic and anaerobic bacteriologic culture and serum for progesterone determination were obtained on postovulation day 3 during estrous cycle 1, on the day of ovulation during estrous cycle 3, and on postovulation day 5 during estrous cycle 5. Immediately thereafter, the uterus was irrigated with 50 ml of sterile physiologic saline solution containing tracer amounts of 125I-labeled human serum albumin. Streptococcus zooepidemicus was isolated from 10 of 15 (67%) uterine specimens collected from susceptible mares and incubated aerobically. Escherichia coli also was isolated from 2 of the 10 specimens incubated aerobically. Anaerobic bacteriologic culture of specimens from all mares yielded no growth. Chromium-labeled microspheres were recovered twice from 2 susceptible mares, on day 0 and day 5. Charcoal was retained in 5 specimens collected from 3 susceptible mares. Bacteriologic culture of specimens from resistant mares did not yield growth. On day 0, chromium-labeled microspheres and charcoal were recovered once from 1 resistant mare. Mares susceptible to endometritis accumulated more fluid within the uterine lumen after ovulation than did resistant mares (mean +/- SEM, 52.73 +/- 15.22 ml and 7.41 +/- 1.96 ml, respectively; P less than 0.01). From this study, it appeared that uterine cellular and bactericidal mechanisms are dysfunctional during the early postovulatory period. However, there appeared to be no disruption of the mechanisms responsible for mechanical clearance of materials inoculated in the uterus.     

载牛冠状病毒N蛋白壳聚糖微球的免疫效果评价

为了对壳聚糖分子作为佐剂的缓释效果进行免疫学评价,本研究合成了牛冠状病毒DB2毒株N蛋白基因的3'端第487位～1287位碱基,用大肠杆菌对其进行了高效表达,通过SDS-PAGE电泳分离、电洗脱纯化该牛冠状病毒N蛋白(BCV N).以戊二醛为交联剂,采用乳化交联的方法制备空白壳聚糖微球.利用吸附法制备载BCVN蛋白的壳聚糖微球,通过肌肉注射途径免疫BALB/c小鼠,用间接ELISA方法检测免疫鼠血清中的抗体水平,由此评价壳聚糖微球对BCVN的缓释作用和佐剂效应.结果表明,壳聚糖微球的形态较好、表面光滑、分散性较好,平均粒径为6.50±1.77μm,电势分布在36 mV,吸附BCV N蛋白的壳聚糖微球在体内所产生的免疫效果明显优于唯N蛋白组,说明载BCV N蛋白的壳聚糖微球在体内具有一定的缓释效果,使N蛋白在体内缓慢释放而长时间诱导抗体的产生,该研究结果为以壳聚糖微球作为疫苗佐剂积累了实验数据.     

阿莫西林脂质体的制备及质量评价

采用薄膜分散法制备阿莫西林脂质体,以粒径和包封率为指标评价其质量.前期以正交设计优化筛选出最佳制备工艺,以最优工艺制备阿莫西林脂质体,测定其平均粒径和包封率.实验所制得的脂质体的平均粒径为114.6 nm,包封率为64.89％.结果表明,采用薄膜分散法制备阿莫西林脂质体的方法可行,阿莫西林脂质体的制备为兽药新剂型的研究与应用提供了有价值的参考.     

Suitability of poultry litter ash as a feed supplement for broiler chickens

An experiment was conducted to evaluate poultry litter ash (PLA) at graded levels (0, 25, 50, 75, and 100%) as a dietary substitute for dicalcium phosphate (dicalP) for broiler chickens, where PLA was substituted for dicalP on a wt/wt basis. A decreased BW gain was observed at 21 d from PLA at the 100% substitution rate when compared with the 25% substitution rate. No effects on BW gain, feed consumption, or FCR were incurred by study termination at 41 d, and the 100% substitution rate of dicalP did not cause any detrimental effects on the live performance in market age broilers. Although femur ash percentages decreased with increasing levels of PLA, bone integrity was not compromised by the addition of PLA to the diet. Dry matter digestibility of dietary Ca (26.32–58.65%) and P (30.23–42.68%) increased with increasing levels of PLA (0–100%), respectively, inferring that the Ca and P component of the diet was more efficiently used as the level of PLA increased. In conclusion, PLA resulting from the combustion of poultry litter can be used as a P and Ca source for broilers.