高效液相色谱-荧光检测法测定沉香中黄曲霉毒素B1、B2、G1、G2

目的建立高效液相色谱–光化学衍生–荧光检测法测定沉香药材中黄曲霉毒素B1、B2、G1、G2。方法采用高效液相色谱法,通过免疫亲和柱提取和净化,荧光检测器检测。Agilent Zorbax Ecilpse Plus C18色谱柱(250 mm×4.6 mm,5μm);流动相:甲醇–水(45∶55);体积流量:0.8 m L/min;柱温:30℃;进样盘温度:4℃;荧光激发波长为360 nm,发射波长为450 nm。结果黄曲霉毒素B1、B2、G1、G2分别在9.3~74.4、3.0~24.0、9.3~74.4、3.5~28.0 pg线性关系良好,r均大于0.998 0;检测限分别为1.86、0.60、1.86、0.70 pg,定量限分别为7.44、2.40、7.44、2.80 pg。平均回收率分别为78%、92%、82%、99%,RSD值分别为4.4%、3.0%、4.3%、2.8%。结论所建立的方法结果准确、重复性、稳定性均良好,可用于沉香药材中黄曲霉毒素的质量控制。     

超高效液相色谱-荧光法测定丹参饮片中黄曲霉毒素

目的 建立超高效液相色谱-荧光法测定丹参饮片中黄曲霉毒素B₁、B₂、G₁、G₂。方法 样品经70%甲醇提取,通过免疫亲和柱净化后,用超高效液相色谱-荧光法进行分析测定,WatersAcquity UPLC BEH C₁₈色谱柱（100 mm×2.1 mm,1.7 μm）;流动相为甲醇-乙腈（1∶1）、水,梯度洗脱;体积流量0.4 mL/min;柱温30℃;荧光检测器激发波长365 nm;发射波长456 nm;进样量2 μL。结果 黄曲霉毒素B₁、B₂、G₁、G₂分别在0.120 1～1.920 8、0.036 1～0.577 2、0.122 1～1.954 0、0.042 1～0.673 2 ng/mL内线性关系良好,r均大于0.99;定量限分别为0.10、0.03、0.39、0.03 ng/mL;黄曲霉毒素B₁的回收率为107%,RSD为6%;黄曲霉毒素总含量的回收率为80%,RSD为9%。专属性、重复性、精密度、稳定性试验均符合检测要求。结论 所建立的方法准确、可靠、专属性强,可准确地测定丹参饮片中黄曲霉毒素的含量。     

HPLC法测定150种中药材中黄曲霉毒素G2、G1、B2、B1的含量

目的:建立了HPLC法测定150种中药材中的黄曲霉毒素G₂、G₁、B₂、B₁含量。方法:样品经70%甲醇提取、免疫亲和色谱柱净化后,用HPLC-柱后衍生-荧光检测器测定结果:黄曲霉毒素G₁、B₂在0.15～6.00 ng·ml^-1范围内,黄曲霉毒素C₁、B₁在0.5～20.00 ng·ml^-1范围内线性关系良好回收率为85.6%～92.0%结论:本法操作简便,结果准确、重复性好,可用于中药材中黄曲霉毒素G₂、G₁、B₂、B₁的测定     

11种中药材中黄曲霉毒素G_2、G_1、B_2、B_1的HPLC法测定

建立了HPLC法测定11种中药材中的黄曲霉毒素G_2、G_1、B_2、B_1.样品经70%甲醇提取、免疫亲和色谱柱净化后,用HPLC-柱后衍生-荧光检测器测定.黄曲霉毒素G_2、B_2在1.5～60 Pg范围内,黄曲霉毒素G1、B1在5～200 Pg范围内线性关系良好.回收率为60%～120%.     

中药污染黄曲霉毒素B1检测分析

目的:了解中药污染黄曲霉毒素B₁(AFB₁)情况。方法:采用间接竞争酶联免疫吸附法(ELISA)对3 7种中药材和22批中成药污染AFB₁进行检测。结果:有34种中药材污染AFB₁,占92 % ;有19批中成药污染AFB1,占86%。结论:中药材和中成药都不同程度地污染AFB₁。     

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     

LC-MS / MS 检测莲子中黄曲霉毒素 B1、B2、G1、G2的方法探讨简

目的 建立LC-MS/MS检测莲子中的黄曲霉毒素B1、B2、G1、G2的方法.方法 收集5批莲子,采用高效液相色谱-串联四极杆质谱联用技术,多反应监测（MRM）模式进行定量检测.结果 1批发霉的莲子中检出黄曲霉毒素B1、B2、G1、G2.结论 该方法专属性强、灵敏度高,可用于莲子中的黄曲霉毒素B1、B2、G1、G2的同时测定.     

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。     

HPLC-柱后光化学衍生法测定清火片(胶囊)中黄曲霉毒素G_2、G_1、B_2、B_1的含量及其安全性评价

目的:建立测定清火片(胶囊)中黄曲霉毒素(AF)G_2、G_1、B_2、B_1的方法,并评价该制剂的安全性。方法:采用高效液相色谱(HPLC)-柱后光化学衍生法,并以全国37个厂家生产的266批清火片(胶囊)为样品。色谱柱为Agilent C_(18);流动相为水-乙腈-甲醇(V/V/V),梯度洗脱;流速为1.0 mL/min;柱温为40℃;进样量为10μL;荧光检测器激发波长为360 nm、发射波长为450 nm。结果:AF G_2、AF G_1、AF B_2、AF B_1分别在进样量为10.197～101.97(r=0.999 7)、10.197～101.97(r=0.999 6)、9.958 6～99.586(r=0.999 1)、9.999 0～99.990(r=0.998 3)pg范围内线性关系良好;精密度(n=6)、重复性(n=6)、稳定性(12 h,n=5)试验的RSD均小于3.0%;检测限分别为0.80、4.00、0.80、4.00 pg;定量限分别为1.60、8.00、1.60、8.00 pg;加样回收率分别为85%～90%、85%～90%、55%～65%、65%～75%(RSD为1.8%～4.7%,n=6)。在266批样品中均未检测出AF G_2、AF G_1、AF B_2、AF B_1。结论:该方法可用于清火片(胶囊)中AF的检测;虽在抽检样品中未检测出AF,但建议增订AF检查项,以提高其安全性。     

HPLC柱后光化学衍生荧光检测法检测3种含土鳖虫中成药的黄曲霉毒素

目的：考察免疫亲和净化HPLC柱后光化学衍生荧光检测法在中成药中黄曲霉毒素测定的可行性,并对其污染情况进行筛查,为中成药黄曲霉毒素污染监管提供依据。方法：采用免疫亲和净化 HPLC柱后光化学衍生荧光检测法对含有土鳖虫的中成药中黄曲霉毒素的含量进行测定。样品经有机溶剂提取、免疫亲和柱净化后,利用高效液相色谱-光化学衍生-荧光检测器进行分析测定。对3种含土鳖虫的中成药,考察加样回收率,测定黄曲霉毒素残留量,并对测定结果进行分析。采用高效液相色谱- 串联质谱法对部分超出限度批次进行结果确认。结果：3种中成药中黄曲霉毒素B1、B2、G1、G2的回收率均在80%~113%。3种24批中成药中,21批检出黄曲霉毒素,检出率为87.5%,部分批次黄曲霉毒素含量明显偏高。结论：免疫亲和净化HPLC柱后光衍生荧光检测法结果准确,重现性好,可用于中成药中黄曲霉毒素的测定。含土鳖虫药材的中成药,个别品种黄曲霉毒素污染情况较为严重,存在安全隐患,应引起生产企业的重视,保障用药安全。     

HPLC法测定四维钙片中维生素C、B1、B2的含量

目的：建立高效液相色谱法测定四维钙片中维生素C、维生素B1和维生素B2含量的方法.方法：采用SHISEIDOC18（250 mm×4.6 mm,5μm）色谱柱,以0.05％磷酸溶液（含0.005 mol·L-1己烷磺酸钠）-乙腈（88：12）为流动相,流速：1.0ml· min-1,检测波长：244 nm,柱温：30℃,进样量：20μm.结果：维生素C、维生素B1和维生素B2的线性范围分别为7.52～90.24 μg· ml-1（r=1.000 0）,1.32 ～15.82 μg·ml-1（r=1.000 0）和1.27～15.24μg·ml-1 （r=1.000 0）;平均回收率分别为101.63％、99.77％和100.25％,RSD分别为1.51％、1.48％和1.84％（n=9）.结论：本法操作简便,结果准确、可靠,可用于四维钙片中维生素C、B1、B2的含量测定.     

Aflatoxin B1 and fumonisin B1 affect the oxidative status of bovine peripheral blood mononuclear cells

Mycotoxins are secondary metabolites having a high cytotoxic potential. They are produced by molds and released in food and feed. To date, the mechanisms underlying the mycotoxin-induced cytotoxicity have not been fully clarified. The induction of oxidative stress, as a possible mechanism, has been postulated. This in vitro study was focused on the effect of two widely occurring mycotoxins, aflatoxin B₁ (AFB₁) and fumonisin B₁ (FB₁), on the oxidative status of bovine peripheral blood mononuclear cells (PBMC) incubated for 2 and 7 days at different levels of AFB₁ (0, 5 and 20 μg/ml) and FB₁ (0, 35 and 70 μg/ml). Reactive oxygen metabolites (ROM), intracellular thiols (SH), malondialdehyde (MDA) and gene expression of cytoplasmic superoxide dismutase (SOD) and glutathione peroxidase (GSHPX-1) were measured on PBMC after incubation. The highest concentration of AFB₁ and all concentrations of FB₁ caused an increase (p < 0.05) of intracellular ROM without any time dependent effect. Intracellular SH decreased with 20 μgAFB₁/ml (p < 0.05) and the effect was particularly marked after 7 days of exposure. Intracellular SH were not affected by FB₁ even though a lower (p < 0.05) SH level after 2 days exposure than after 7 days was observed. MDA increased (p < 0.05) in AFB₁ or FB₁ treated PBMC. The exposure to FB₁ for 7 days increased MDA (p < 0.05) only in cells treated with 70 μg/ml. Exposure of PBMC to AFB₁ reduced SOD mRNA while FB₁ decreased both SOD and GSHPX-1 mRNA abundance. These results demonstrate that, even though by different mechanisms, AFB₁ and FB₁ may induce cytotoxicity through an impairment of the oxidative status of PBMC.     

Trp266 determines the binding specificity of a porcine aflatoxin B1 aldehyde reductase for aflatoxin B1-dialdehyde

Aflatoxin B₁ (AFB₁) is a severe threat to human and animal health. The aflatoxin B₁ aldehyde reductase (AFAR) family specifically catalyzes AFB₁-dialdehyde, a toxic metabolic intermediate of AFB₁, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB₁-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB₁-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB₁-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB₁-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π–π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB₁-dialdehyde.     

三七绒根中皂甙B1及B2的分离和鉴定

从三七Panax notoginseng(Burk.)F.H.Chen绒根中分得二种微量皂甙,三七皂甙B₁和B₂三七皂甙B₁为一种新皂甙,证明其结构为达玛20(22)-烯3β,12β,25三醇6-O-β-D-葡萄吡喃糖甙(Ⅰ),其皂甙元亦为一种新皂甙元,其结构为达玛20(22)-烯-3β,12β,6 C,25四醇。三七皂甙B₂经鉴定为已知皂甙人参皂甙(ginsenoside—Rh₁,Ⅱ)。     

Evidence for the participation of kinins in Freund's adjuvant-induced inflammatory and nociceptive responses in kinin B1 and B2 receptor knockout mice

Experiments were designed to investigate the role of kinin B₁ and B₂ receptors in Freund's adjuvant (CFA)-induced inflammation and nociception responses by the use of B₁ and B₂ null mutant mice. Intradermal (i.d.) injection of CFA produced time-dependent and marked hyperalgesic responses in both ipsilateral and contralateral paws of wild-type mice. Gene disruption of the kinin B₂ receptor did not interfere with CFA-induced hyperalgesia, but ablation of the gene of the B₁ receptor reduced the hyperalgesia in both ipsilateral (48±13%, at 12 h) and contralateral (91±22%, at 12 h) paws. Treatment of wild-type mice with the selective B₁ antagonist des-Arg⁹-[Leu⁸]-BK (150 nmol/kg, s.c.) reduced CFA-evoked thermal hyperalgesia, to an extent which was similar to that observed in mice lacking kinin B₁ receptor. I.d. injection of CFA produced a time-related and long-lasting (up to 72 h) increase in paw volume in wild-type mice. A similar effect was observed in B₁ knockout mice. In mice lacking B₂ receptor, the earlier stage of the CFA-induced paw oedema (6 h) was significantly greater compared with the wild-type animals, an effect which was almost completely reversed (76±5%) by des-Arg⁹-[Leu⁸]-BK. This data demonstrates that kinin B₁ receptor, but not B₂ receptor, exerts a critical role in controlling the persistent inflammatory hyperalgesia induced by CFA in mice, while B₂ receptor appears to have only a minor role in the amplification of the earlier stage of CFA-induced paw oedema formation. The results of the present study, taken together with those of previous studies, suggest that B₁ receptor antagonists represent a potential target for the development of new drugs to treat persistent inflammatory pain.     

维生素B2片含量测定方法的改进

目的:快速测定维生素B2片的含量及含量均匀度.方法:以0.3%(V/V)氨溶液为溶剂,超声5min使维生素B2溶解,再用UV法测定.结果:维生素B2在1.5～24.3μg/ml的浓度范围内呈良好的线性关系(r=0.9999),高、中、低浓度的加样回收率分别为99.9%、99.8%、99.6%;RSD分别为0.2%、0.2%、0.6%(n=3).结论:本法简便、快速,有较好的精密度和准确度.     

Study of the Ochratoxin A effect on Aspergillus parasiticus growth and aflatoxin B1 production

Aflatoxin B1 (AFB1) is a carcinogenic metabolite produced by certain Aspergillus species. Ochratoxin A (OTA) is a metabolite of Aspergillus ochraceus and Penicillium verrucosum. AFB1 and OTA are amongst the most frequent combinations of mycotoxins found in plant products. Thus, synergistic effects or interactions between the two mycotoxins could be taking place. The aim of the present study was to investigate the effect of OTA on Aspergillus parasiticus growth and AFB1 production in yeast extract sucrose (YES) medium at concentrations of 0.16, 1.6 and 16 ng OTA flask(-1). The AFB1 extracted from cultures and purified with immunoaffinity columns was then quantitated by HPLC. The recovery and detection limit of the method were 95.3% and 0.02 ng AFB1 mL(-1), respectively. Maximum AFB1 productions in cultures with OTA were observed from 9 to 12 days (76.09-82.52 ng AFB1 flask(-1)) while in control cultures (without OTA) maximum production (197.2 ng AFB1 flask(-1)) was observed on 14th day. Maximum AFB1 levels in cultures with OTA were reduced by a percentage of 58-61% compared to control cultures. Furthermore AFB1 levels in cultures with OTA were practically (92%) degraded after 18 days of incubation. Conclusively when OTA is present the production of AFB1 by A. parasiticus in YES medium is inhibited.     

人参皂甙Rh2体外对小鼠黑色素瘤细胞的分化诱导作用

体外实验证明人参皂甙Rh₂在10μg·ml^-1的浓度下能明显抑制B₁₆细胞的生长,并呈浓度依赖性,其IC₅₀为4.1μg·ml^-1。Rh₂在10μg·ml^-1浓度下对B₁₆细胞有较强的分化诱导作用,表现为黑色素生成能力明显增加;形态向上皮样细胞分化;细胞呈网状结构;黑色素颗粒增多,生长变缓慢。细胞动力学研究结果表明,Rh₂可使B₁₆细胞阻断在G₁期。提示Rh₂对B₁₆细胞具有分化诱导作用。