生地黄中低聚糖的提取和纯化研究

目的研究从生地黄中提取分离低聚糖的工艺。方法以水苏糖的量为指标,采用正交试验优化水提取工艺,考察了除杂工艺中大孔树脂种类、洗脱液用量及活性炭脱色方法,并采用葡聚糖凝胶柱纯化得到生地黄低聚糖。结果优选的水提取工艺为:12倍量水,煎煮3次,每次0.5 h。水提取液经D-101大孔吸附树脂除杂,0.1%活性炭脱色3次,SephadexG15葡聚糖凝胶分离纯化获得生地黄低聚糖部位,其中水苏糖质量分数大于60%。结论该工艺从生地黄中提取分离低聚糖部位,工艺合理、可行,低聚糖部位收率达药材量的26%。     

Molecular basis for resistance of herpes simplex virus type 1 mutants to the sulfated oligosaccharide inhibitor PI-88

Herpes simplex virus type 1 variants selected by virus propagation in cultured cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to the presence of PI-88 during their initial infection of cells and/or their cell-to-cell spread. Nucleotide sequence analysis revealed that the deletion of amino acids 33-116 of gC but not lack of gC expression provided the virus with selective advantage to infect cells in the presence of PI-88. Purified gC (Delta33-116) was more resistant to PI-88 than unaltered protein in its binding to cells. Alterations that partly contributed to the virus resistance to PI-88 in its cell-to-cell spread activity were amino acid substitutions Q27R in gD and R770W in gB. These results suggest that PI-88 targets several distinct viral glycoproteins during the course of initial virus infection and cell-to-cell spread.     

Human anti-α-fucose antibodies are xenoreactive toward GGTA1/CMAH knockout pigs

Progress has been made in overcoming antibody-mediated rejection of porcine xenografts by deleting pig genes that produce unique carbohydrate epitopes. Pigs deficient in galactose α-1,3 galactose (gene modified: GGTA1) and neu5Gc (gene modified: CMAH) have reduced levels of human antibody binding. Previously we identified α-fucose as a glycan that was expressed in high levels on cells of GGTA1/CMAH KO pigs. To validate the α-fucose phenotype observed previously we compared lectin affinity toward human and pig serum glycoproteins by dot blot analysis and confocal microscopy. Human anti-fucose antibody isolated by affinity chromatography was tested for specificity to L-fucose by custom macroarray. The affinity and cytotoxicity of the isolated human anti-fucose antibody toward human and GGTA1/CMAH KO pig PBMCs was determined by flow cytometry. Dot blot and confocal analysis support out previous findings that α-fucose is more highly expressed in pigs than humans. Pig kidney glomeruli and tubules contain abundant α-fucose and may represent focal sites for anti-α-fucose antibody binding. The Isolated human anti-fucose IgA, IgG and IgM bound to GGTA1/CMAH KO pig PBMC and were cytotoxic. Interestingly, the isolated human IgG cross reacted with the methyl pentose, L-rhamnose. Human anti-fucose antibody bound and was cytotoxic to GGTA1/CMAH KO pig peripheral blood monocytes. We have shown that α-fucose is an abundant target for cytotoxic human antibody in the organs of genetically modified pigs important to xenotransplantation.     

表阿霉素卡拉胶寡糖-金纳米粒的制备和质量控制

目的　建立盐酸表阿霉素-卡拉胶寡糖-金纳米（EPI-CAO-AuNPs）中盐酸表阿霉素（EPI）的含量测定方法,并测定其包封率。方法　采用ZORBAX-Extend-C18色谱柱（4.6 ×150 mm,5 μm）,以乙腈和水为流动相,对检测波长、流动相比例及pH、流速和柱温等因素进行优化。结果　优化后最适色谱条件为：流动相为乙腈/水（30/70,V/V）,含0.1 %的三氟乙酸（TFA）,柱温25 ℃,流速1.0 mL/min,检测波长233 nm。经测定,EPI-CAO-AuNPs中EPI的载药量为12.5 %,包封率为94.3 %。结论　该测定方法操作简单,快速灵敏,重复性好,可在15 min内完成测定,适用于EPI-CAO-AuNPs材料中EPI的含量检测。     

Pre-clinical safety evaluation of the synthetic human milk,nature-identical,oligosaccharide 2′-O-Fucosyllactose (2′FL)

In order to match the composition of human breast milk more closely, it is now possible to supplement commercial infant formula (IF) with synthesised oligosaccharides that are chemically identical to human milk oligosaccharides. The safety data generated on a new human-identical milk oligosaccharide (HiMO), 2′-O-Fucosyllactose (2′FL), are presented. Standard in vitro genotoxicity tests were performed. To investigate the toxicological profile in a model representative of the intended target population, 2′FL was administered via gavage in a juvenile adapted sub-chronic rat study at dose levels of 0, 2000, 5000 and 6000 mg/kg bw/day. Fructooligosaccharide (FOS), currently acknowledged as safe and approved for use in IF, was used as a reference high-dose control at 6000 mg/kg bw/day. 2′FL was non-mutagenic in the in vitro assays. Oral administration up to 5000 mg/kg bw/day to rats over 90 days was not associated with any adverse effects based on clinical observations, body weight gain, food consumption, ophthalmoscopy, clinical pathology, organ weights and histopathology findings. Based on this 90-day study, a No Observed Adverse Effect Level (NOAEL) of 5000 mg/kg bw/day for both male and female rats was established for 2′FL. These findings support the safety of synthetic 2′FL for possible use in infant food.     

A new pregnane glycoside and oligosaccharide from Parabarium huaitingii

Two new compounds, along with two known compounds, were isolated from the barks of Parabarium huaitingii, and their structures were determined as 5α-pregn-6-ene-3β,17α,20(S)-triol-20-O-β-d-digitoxopyranoside (1), cymaropyranurolactone 4-O-β-d-digitalopyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-oleandropyranosyl-(1 → 4)-O-β-d-cymaropyranoside (2), 3β,17α,20(S)-trihydroxy-5α-pregn-6-ene (3), and 5α-pregn-6-ene-3β,17α,20(S)-triol-3-O-β-d-digitalopyranoside (4) by spectroscopic methods.     

Enhanced cellular uptake of chlorine e6 mediated by stearic acid–grafted chitosan oligosaccharide micelles

Chlorines are attractive compounds for photodynamic therapy because of their high absorption in the red wavelength region. The stearic acid–grafted chitosan oligosaccharide (CSO-SA) micelles have been presented as potential candidates for intracellular drug delivery carrier because of their special structure. In this study, CSO-SA micelles were prepared to encapsulate chlorine e6 (Ce6). The physicochemical properties of synthesized CSO-SA micelles were characterized. The critical micelle concentration (CMC) of CSO-SA with 4.96% amino substituted degree (SD %) was about 36.27?±?1.51?μg/mL. The Ce6-loaded CSO-SA micelles were then prepared by a dialysis method, and the properties and drug release profiles of Ce6-loaded CSO-SA micelles (CSO-SA/Ce6) were investigated. The loading of Ce6 in the CSO-SA micelles could reach higher drug encapsulation efficiency (%), which was ~100%. The size of CSO-SA/Ce6 decreased after the loading of Ce6. The zeta potential of CSO-SA/Ce6 and the drug release rate decreased with the loading content of drug. After the Ce6 molecules were encapsulated into the micelles of CSO-SA, the cellular uptake percentage of Ce6 was much more than that of the free drug. And the cellular uptake percentage of CSO-SA/Ce6 micelles was increased with the incubation time in a short period.     

壳寡糖对体外培养软骨细胞增殖的影响及对IL-1β诱导凋亡的保护作用

目的 体外实验研究不同浓度壳寡糖对软骨细胞增殖及IL-Iβ诱导细胞凋亡的保护作用.方法 从8周龄SD大鼠乳鼠膝关节分离、培养原代软骨细胞,鉴定后选用第2代软骨细胞进行实验.分别加入不同浓度壳寡糖培养48 h,通过CCK-8细胞计数法检测软骨细胞的增殖情况;采用IL-1β诱导软骨细胞凋亡,同时加入不同浓度的壳寡糖处理,倒置相差显微镜下观察软骨细胞形态及核分裂象的变化,通过流式细胞仪检测细胞早期凋亡比例,并通过Hoechst33258荧光染色检测软骨细胞凋亡细胞核的形态学变化.结果 CCK-8检测结果表明壳寡糖作用软骨细胞不同浓度、不同时间的软骨细胞增殖率比较均无显著性差异（P＞0.05）;流式细胞检测结果表明壳寡糖不仅扭转了细胞活力和增殖活性的下降,而且改善了IL-1β诱导的软骨细胞核染色质的损害,同时对软骨细胞凋亡率也有不同程度的降低.结论 一定浓度的壳寡糖对软骨细胞增殖无明显影响;壳寡糖能明显抑制IL-1β诱导的软骨细胞凋亡,且呈剂量依赖性.     

江西建昌帮炆制地黄中辅料作用探索(Ⅰ)

目的:建立HPLC-ELSD测定熟地黄中单糖与低聚糖含量的方法,研究江西建昌帮炊制地黄过程中炮制辅料的作用.方法:采用HPLC测定地黄中单糖和低聚糖的含量,以蒸发光散射检测器进行检测,prevail carbohydrate ES色谱柱(4.6mm ×250 mm,5μm),考察辅料对建昌帮炊制地黄的影响.结果:炊制地黄中单糖含量高于各缺辅料炆制品,但其低聚糖含量低于各缺辅料炆制品.炆制地黄、炆制地黄(缺砂仁)、炆制地黄(缺砂仁与陈皮)、炆制地黄(缺砂仁、陈皮与黄酒)、炆制地黄(缺黄酒)、炆制地黄(缺陈皮)中单糖质量分数分别为(23.46±1.07)％,(21.81±0.94)％,(15.83±1.01)％,(14.14±0.58)％,(21.91±0.76)％,(21.53±0.84)％;低聚糖质量分数分别为(3.02±0.25)％,(3.29±0.17)％,(11.73±0.64)％,(14.33 ±0.83)％,(6.17 ±0.38)％,(3.77±0.19)％.结论:建立的含量测定方法操作简便、稳定、准确;辅料砂仁、陈皮与黄酒会影响熟地黄炮制过程中化学成分的变化,为阐明其炮制机制提供实验依据.