高效液相色谱-光化学衍生法测定湖南产莲子中黄曲霉毒素G1、G2、B1、B2

目的对不同来源的湖南产莲子中黄曲霉毒素G_1、G_2、B_2、B_1进行高效液相色谱-光化学衍生法测定。方法采用高效液相色谱–光化学衍生法,采用岛津GL Inertsil ODS-3色谱柱(250 mm×4.6 mm,5μm),流动相为甲醇–乙腈–水(35∶13∶52),柱温35℃;光化学衍生器(254 nm)激发波长λ_(ex)=360 nm,发射波长λ_(ex)=450 nm。结果黄曲霉毒素G_1、G_2、B_2、B_1分别在6.7～33.3、11.4～56.8、10.3～51.5、4.1～20.3 pg线性关系良好;平均回收率分别为98.07%、97.72%、96.11%、99.52%,RSD值分别为1.69%、1.40%、2.72%、1.34%(n=6)。9批莲子样品中,有6批未检出黄曲霉毒素,来自于农贸市场农户自存的2批次检出黄曲霉毒素B_1,实验室塑料袋包装储存1年的1批检出黄曲霉毒素B_1、B_2,但质量分数均低于法定标准限量。结论不同来源湖南产莲子中黄曲霉毒素质量分数均低于《中国药典》2020年版规定限量。该法测定结果准确、重复性良好,可为完善莲子安全性控制和质量标准提升提供实验依据。     

维生素B2和B6的同步荧光分析法及其在维生素复合制剂中的应用

本文建立了维生素B₂和B₆的同步荧光分析法。以△λ=58nm进行同步扫描所得的两个同步荧光峰(以发射波长表示,分别位于526nm和389nm)可用以同时分别定量维生素B₂和B₆。方法快速、灵敏。维生素B₂和B₆的工作曲线线性范围分别为0～1μg/ml和0～1.5μg/ml,检出限分别为0.5ng/ml和1ng/ml。方法已应用于三种复合维生素B制剂中维生素B₂和B₆的分析。     

人参皂苷Rg1及其肠内菌代谢产物Rh1对小鼠免疫细胞功能的影响

目的初探人参皂苷Rg₁及其肠内菌代谢产物Rh₁对正常小鼠免疫功能的影响。方法用Rh₁与Rg₁分别处理脾T细胞,B细胞及腹腔巨噬细胞(Mφ);MTT比色法测T和B细胞增殖能力;中性红比色法测Mφ的吞噬功能;Griess法测Mφ释放NO的水平。结果Rh₁能促进脾细胞增殖、下调Con A诱导的T细胞增殖;Rh₁与Rg₁对LPS诱导的B细胞增殖均无明显作用;Rg₁和Rh₁能提高Mφ的吞噬能力和促进NO的释放。结论Rg₁及其代谢产物Rh₁可共同作用于T细胞和Mφ而产生免疫调节作用。     

HPLC法测定复方吡拉西坦脑蛋白水解物片中维生素B1、B2和B6含量

摘 要 目的:建立HPLC法测定复方吡拉西坦脑蛋白水解物片中维生素B₁、维生素B₂和维生素B₆含量的方法。方法: 采用Insteril ODS-3色谱柱（250 mm×4.6 mm,5 μm）,流动相：0.01 mol·L^-1庚烷磺酸钠（含0.25%三乙胺,用冰醋酸调节pH至3.8)-甲醇（75∶25）,柱温30℃,检测波长为280 nm,流速：1.0 ml·min^-1。结果: 维生素B₁、维生素B₂、维生素B₆分别在3.98～99.40 μg·ml^-1（r=0.999 7）、4.08～101.91μg·ml^-1（r=0.999 9）、2.08～52.00 μg·ml^-1（r=0.999 9）范围内线性关系良好,平均回收率分别为99.18%、99.53%、99.27%,RSD分别为0.60%、0.67%、0.71%(n=9)。结论:本法简便、快速、准确,可用于复方吡拉西坦脑蛋白水解物片中维生素B₁、维生素B₂和维生素B₆的含量测定     

八角属植物中的C6-C3类化合物及其药理活性研究进展

八角属植物中富含C₆-C₃类化合物,结构类型多样,有苯丙素类、木质素类和独特新颖的植物醌类,并具有较好的抗炎、神经营养、抗癌和抗氧化等活性,药用开发前景广阔。本文根据国内外文献报道,对该属植物C₆-C₃类化合物及其药理活性研究进展进行综述,为进一步研究开发该属植物提供参考。     

63Ni-NiCl2在大鼠体内的药代动力学及其组织残留

镍是动物体必需的微量元素,参与核酸和蛋白质的代谢、激素的调节过程,具有刺激生血、促进红细胞再生的功能[1],与激素、蛋白质和脂类的代谢也密切相关.国内相关研究较少,尤其使用同位素标记法研究更少.     

岩藻黄素抗H2O2诱导WI-38细胞早衰的作用研究D*

目的：探讨岩藻黄素是否具有抑制H₂O₂引起的WI-38细胞早衰的活性。方法：WI-38细胞在添加岩藻黄素的培养基中培养一定时间后经H₂O₂处理诱导发生早衰,用MTT法检测细胞活力,SA-β-半乳糖苷酶染色法检测细胞内衰老相关的β-半乳糖苷酶活性。 结果：300 μM H₂O₂处理WI-38细胞20 min可成功建立早衰细胞模型。与空白对照组相比,H₂O₂处理组细胞活力明显降低,SA-β-半乳糖苷酶阳性细胞数明显增多。5 μM和10 μM岩藻黄素组细胞活力明显高于H₂O₂处理组,SA-β-半乳糖苷酶阳性细胞数较H₂O₂组明显减少。 结论：岩藻黄素能够抑制由H₂O₂处理引起的细胞早衰,具有一定的抗衰老作用。     

UFLC法测定复方丹参片中三七皂苷R1、人参皂苷Rg1及Rb1

目的 探索建立超快速液相色谱（UFLC）法测定复方丹参片中三七皂苷R₁、人参皂苷Rg₁及Rb₁。方法 采用SHIMADZU Shim-pack XR-ODS Ⅲ（2.0 mm×75 mm, 1.6 μm）色谱柱;以乙腈-水作为流动相进行梯度洗脱;体积流量为0.4 mL/min;检测波长为203 nm;进样体积为3 μL。结果 三七皂苷R₁、人参皂苷Rg₁及Rb₁分别在0.025 7～0.257 0、0.101 2～1.012 0、0.104 4～1.044 0 μg与峰面积呈良好的线性关系,平均加样回收率分别为96.7%、98.1%、98.8%。结论 本方法在15min内可以将三七皂苷R₁、人参皂苷Rg₁及Rb₁有效分离,节省了大量人力和流动相的消耗,为中药的质量控制技术提供参考方法。     

超临界CO2萃取薏苡仁油工艺条件优化

目的　应用超临界CO₂萃取技术提取薏苡仁中的薏苡仁油,优化工艺条件。方法　利用1L与600L的超临界萃取装置,考察原料水份、压力、时间与温度等参数对薏苡仁油提取得率的影响。结果　优化得到用超临界CO₂萃取技术从薏苡仁中提取薏苡仁油的工艺参数。结论　超临界CO₂萃取技术可用于薏苡仁油的提取,适用于工业化生产。     

Expression and bioactivity analysis of staphylococcal enterotoxin G and staphylococcal enterotoxin I

Context: The filtrate of Staphylococcus aureus culture, named staphylococcal enterotoxin C injection, has been used for 10 years in China. SEC2 has been claimed to be the only staphylococcal enterotoxins (SEs) without certifiable evidence.

Objectives: To present an efficient procedure for the expression and purification of recombinant proteins SEG and SEI, from S. aureus.

Materials and methods: In present work, we extracted total DNA from S. aureus (FRI 1230) and the recombinant proteins of SEG and SEI were then cloned, expressed and purified using E. coli. Splenic lymphocytes were used as effector cells and K562 and B16 cells were used as target cells to evaluate the inhibitory and stimulatory abilities of purified rSEG and rSEI on in vitro proliferation.

Results: The size of amplified products of SEG and SEI genes were found to be about 400 and 467?bp, respectively. pGEX-SEG and pGEX-SEI were constructed successfully. SEG and SEI were demonstrated to be active stimulators of T-cell proliferation; moreover, they inhibited the proliferation of K562 cells and B16 cells.

Discussion: The current findings suggest that SEC2 might not be the only active component of staphylococcal enterotoxin C injection and may involve other essential proteins like SEG and SEI in its clinical efficacy.

Conclusion: This efficient procedure for the expression and purification of SEG and SEI and may be useful for mass production of therapeutically important proteins. In the future, proteins acting as active stimulators of T-cell proliferation may help in developing effective cancer therapy.     

重组金葡菌肠毒素O的克隆表达及生物学活性分析

克隆金葡菌肠毒素O(SEO)的全长基因，实现其可溶性表达,并对纯化的表达产物进行生物学活性分析。从金葡菌FRI 100菌株基因组中得到SEO基因，克隆至谷胱甘肽S-转移酶(GST)融合表达载体pGEX-4T-1，转化大肠杆菌，获高效表达。融合蛋白GST-SEO经Glutathione Sepharose 4B亲和纯化和凝血酶消化获重组SEO(rSEO)后，MTT法检测脾淋巴细胞的增殖作用，分析纯化后rSEO的生物学活性。测序结果表明，得到正确的肠毒素SEO基因序列，并获高效表达的融合蛋白；MTT结果表明，rSEO具有与SEC相当的显著的促淋巴细胞增殖以及抑制肿瘤细胞生长的能力。本研究成功克隆、表达、纯化了具有抗肿瘤生物学活性的rSEO蛋白，为进一步研究该蛋白的抗肿瘤机制奠定了基础，并有望成为一种新的超抗原制剂用于肿瘤的临床治疗。     

积雪草酸C2、C3、C23、C28衍生物的合成及其体外抗肿瘤活性研究

目的设计并合成积雪草酸C2、C3、C23、C28衍生物,并对其体外抗肿瘤活性进行研究。方法以天然产物积雪草酸为先导化合物,对C2、C3、C23位羟基及C28位羧基进行结构改造,并采用SRB法对目标化合物进行初步的体外抗肿瘤活性研究。结果设计并合成了目标化合物,利用MS及1H-NMR确证了结构;体外实验中,积雪草酸衍生物的抗肿瘤活性明显高于积雪草酸,并优于对照药吉非替尼。结论积雪草酸衍生物具有良好的抗肿瘤活性,值得进一步研究。     

一种快速分离纯化金葡菌肠毒素C2的方法

Objective To develop a simple and convenient method to purify staphylococcal enterotoxin C₂ (SEC₂) from the supernatant of staphylococcus aureus culture. Methods SEC2was purified by ultrafiltration, anion ion-exchange chromatography, and cation ion-exchange chromatography sequentially. The molecular weight of SEC₂ was detected by the SDS-PAGE and flight time mass-spectrum . The purity of SEC₂ was detected by the SDS-PAGE and high performance liquid chromatography. The purified SEC2 was verified by amino acids sequence analysis. Results The purity of purified SEC₂ was greater than 98% (w)which was consistent with literature, including microheterogeneity of isoelectric point, pI 7.49 and 6.74, the molecular weigh 27.58 KD.The amino acids sequence of purified SEC2 was consistent with what was reported in literatures（ESQPDPTPDELHKSS）. The maximum absorption wavelength of purified SEC2 was 277.5 nm. Conclusions The method is rapid, simple and convenient with high SEC₂ purity, the recovery is more than 50 %(w).This method is suitable to large scale preparation of SEC₂.     

齐墩果酸和熊果酸C3、C28衍生物的合成研究

目的设计合成齐墩果酸和熊果酸C_3、C_(28)衍生物。方法先将葡萄糖和半乳糖通过Schmidit法制备三氯乙酰亚胺酯糖给体;然后分别以齐墩果酸和熊果酸为起始原料,经苄基保护,再进行糖苷化、苄基脱保护、酰胺化、苯甲酰基脱保护,得到10个酰胺衍生物F_1~F_(10)。其中F_1、F_3通过四甲基哌啶氧化物(TEMPO)及NaClO/NaClO_2氧化体系得到糖醛酸化合物F_(11)、F_(12)。结果合成了5个熊果酸C_3位葡萄糖苷C_(28)位酰胺衍生物、5个齐墩果酸C_3位半乳糖苷C_(28)位酰胺衍生物及2个熊果酸C_3位葡萄糖醛酸苷C_(28)位酰胺衍生物,并分别通过1~H-NMR、(13)~C-NMR和MS确认结构。结论 12个化合物均未见报道,为三萜皂苷的构效关系及生物活性研究奠定基础。     

金葡菌肠毒素SEC2的抗体制备及应用

生产厂家以金葡菌肠毒素C2(SEC2)的标示量作为金葡素注射液的质量控制标准，但其真正的含量由于滤液成分复杂而很难检测，本实验试图建立一种方便的鉴定金葡素注射液中的SEC2的方法。首先将重组的SEC2分别免疫BALB/c小鼠和新西兰兔，制备并纯化了单克隆及多克隆抗体。采用自制的抗体建立了检测SEC2的生物素-链霉亲和素酶联免疫吸附实验，检测目标蛋白的质量浓度范围为2～20 ng·mL^-1，在检测范围内检测值的平均变异系数为5.08%，采用本法检测质量浓度分别为100 ng·mL^-1的重组肠毒素(SEA/SEO/SEM/SEN/SEG/SEI)，均呈现阴性反应，说明此检测方法的专一性较好。采用此方法对金葡素注射液中的SEC2含量进行了检测，发现SEC2标识量与实际含量存在一定的差距。     

Comparative cytotoxicity study of enniatins A, A1, A2, B, B1, B4 and J3 on Caco-2 cells, Hep-G2 and HT-29

Enniatins (ENs) are ionophoric, phytotoxic, antihelminthic, and antibiotic compounds of hexadepsipeptidic structure produced by several strains of Fusarium spp. The cytotoxicity effect of the ENs A, A₁, A₂, B, B₁, B₄ and J₃ was compared on three tumor cell lines, the human epithelial colorectal adenocarcinoma (Caco-2), the human colon carcinoma (HT-29), and the human liver carcinoma (Hep-G2). The endpoint evaluated was the mitochondrial integrity by using the MTT assays, after 24 and 48 h of incubation. The IC₅₀ value for EN A₂ on Caco-2 cells, after 24 h exposure, was 18.7 ± 4.5 μM and decrease to 2.6 ± 0.7 μM at 48 h of incubation. However, ENs A, A₁, B₁ and B₄ exert pronounced cytotoxic effects in all the cell lines tested by the MTT assay after 24 and 48 h of incubation. The EN A₁ demonstrated to be the most cytotoxic ENs tested. Moreover, no statistical differences were found between the IC₅₀ values obtained for EN A₁ on Caco-2, HT-29 and Hep-G2, with IC₅₀ values ranging from 9.1 ± 2.2 μM to 12.3 ± 4.3 μM at 24 h and decreasing in a range variable from 1.4 ± 0.7 μM to 2.7 ± 0.8 μM at 48 h. On the other hand, EN A, B₁ and B₄ showed lower cytotoxicity, but in a similar range as the IC₅₀ values reported on HT-29 (IC₅₀ values (24 h): 16.8 ± 4.3-26.2 ± 6.7 μM), Caco-2 (IC₅₀ values (24 h): 19.5 ± 4.1 μM) and Hep-G2 (IC₅₀ values (24 h): 23.4 ± 5.6-26.2 ± 7.6 μM) cells. Cytotoxic effect with a 48 h of incubation revealed also a significant toxicity of ENs A (IC₅₀ values ranged from 8.2 ± 1.8 to 11.4 ± 4.6 μM), B₁ (IC₅₀ values variables from 3.7 ± 0.7 to 11.5 ± 5.3 μM) and B₄ (IC₅₀ of 4.5 ± 2.9-15.0 ± 4.0 μM). In summary, this study demonstrated that ENs can exert toxic activity at low micromolar concentrations in mammalian cells.     

金黄色葡萄糖球菌肠毒素C2改构蛋白大鼠药动学研究

目的 研究金黄色葡萄糖球菌肠毒素C2改构蛋白（2M-118）在大鼠体内单次和多次给药后的药动学特征。方法 24只大鼠随机分为3组,每组8只,雌雄各半,分别单次iv低、中和高剂量（1、2和4 mg/kg）2M-118,高剂量组大鼠于单次给药后,继续每天给药1次,共给药8次。于给药前（0 h）,首次及末次给药后5、10、20、30、45 min和1.0、1.5、2.0、4.0、6.0、8.0 h采集眼静脉丛全血约0.5 mL,制备血清。采用双抗体夹心酶联免疫吸附测定（ELISA）法检测大鼠血清药物浓度,采用DAS 3.2.8药动程序计算药动学参数。结果 大鼠单次iv 2M-118后,在1～4 mg/kg剂量内,峰浓度（C_max）、初始浓度（C₀）和药时曲线下面积（AUC）均与剂量呈正相关;消除相半衰期（t_1/2Z）随剂量递增明显延后,平均t_1/2z分别为0.24、0.60和1.18 h;表观分布容积（V_z）随剂量递增而增大;各剂量组的清除率（CL_z）较为一致。与同剂量（4 mg/kg）单次给药相比,大鼠多次给药后的主要药动学参数基本保持一致,体内药物无蓄积倾向。结论 大鼠单次iv 2M-118后,在1～4 mg/kg剂量内,体内暴露量与剂量呈正相关,其清除可能呈现非线性动力学特征;单次与多次iv给予相同剂量2M-118后,药动学行为特征基本一致,无明显药物蓄积。     

Pipping success and liver mRNA expression in chicken embryos exposed in ovo to C8 and C11 perfluorinated carboxylic acids and C10 perfluorinated sulfonate

Several perfluoroalkyl compounds (PFCs) are ubiquitous environmental contaminants that can biomagnify in species at high trophic levels including wild birds. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been detected in wild birds and are known to reduce hatching success of laboratory-exposed chicken embryos at environmentally relevant concentrations. Limited toxicity data are available regarding avian exposure to PFCs of chain lengths greater than C₈, which are of increasing environmental relevance following the recent phase-out of PFOS and PFOA. In this study, linear PFOA, perfluoroundecanoic acid (PFUdA) and perfluorodecane sulfonate (PFDS) were injected into the air cell of white leghorn chicken eggs (Gallus gallus domesticus) prior to incubation to determine effects on embryo pipping success. Furthermore, mRNA expression of key genes involved in pathways implicated in PFC toxicity was monitored in liver tissue. PFOA, PFUdA or PFDS had no effect on embryonic pipping success at concentrations up to 10 μg/g. All PFCs accumulated in the liver to concentrations greater than the initial whole-egg concentration as determined by HPLC/MS/MS. Hepatic accumulation was highest for PFOA (4.5 times) compared to PFUdA and PFDS. Cytochrome P450 1A4 and liver fatty acid binding protein mRNA expression increased after exposure to PFUdA but was only statistically significant at 10 μg/g; several orders of magnitude higher than levels found in wild bird eggs. Based on the present results for white leghorn chickens, current environmental concentrations of PFOA, PFUdA and PFDS are unlikely to affect the hatching success of wild birds.     

A phospholipase A2 from Bothrops asper snake venom activates neutrophils in culture: Expression of cyclooxygenase-2 and PGE2 biosynthesis

In this study, the production of prostaglandin E₂ (PGE₂) and up-regulation in cyclooxygenase (COX) pathway induced by a phospholipase A₂ (PLA₂), myotoxin-III (MT-III), purified from Bothrops asper snake venom, in isolated neutrophils were investigated. The arachidonic acid (AA) production and the participation of intracellular PLA₂s (cytosolic PLA₂ and Ca²⁺-independent PLA₂) in these events were also evaluated. MT-III induced COX-2, but not COX-1 gene and protein expression in neutrophils and increased PGE₂ levels. Pretreatment of neutrophils with COX-2 and COX-1 inhibitors reduced PGE₂ production induced by MT-III. Arachidonyl trifluoromethyl ketone (AACOCF₃), an intracellular PLA₂ inhibitor, but not bromoenol lactone (BEL), an iPLA₂ inhibitor, suppressed the MT-III-induced AA and PGE₂ release. In conclusion, MT-III directly stimulates neutrophils inducing COX-2 mRNA and protein expression followed by production of PGE₂. COX-2 isoform is preeminent over COX-1 for production of PGE₂ stimulated by MT-III. PGE₂ and AA release by MT-III probably is related to cPLA₂ activation.