Teucrium plant species as natural sources of novel anticancer compounds: antiproliferative, proapoptotic and antioxidant properties

This study deals with total phenolic content, antiproliferative and proapoptotic activity of methanolic extracts from different Teucrium species and the effect on the prooxidant/antioxidant status in HCT-116 cells. The total phenolic content of the extracts was measured spectrophotometricaly and the obtained results ranged from 56.62 mg/g to 172.50 mg GA/g. The antiproliferative activity of methanolic extracts from different Teucrium species was determined using MTT cell viability assay, where IC(50) value was used as a parameter for cytotoxicity. The type of cell death was explored by fluorescence microscopy using the acridin orange/ethidium bromide method. MTT assay showed that all extracts significantly reduced cell viability in a dose-dependent manner, with very low IC(50) values. The highest content of phenolic compounds and the best cytotoxic activity on HCT-116 cells after 24 h of exposure was in T. chamaedrys extract, with IC(50) values of 5.48 × 10(-9) μg/mL. After 72 h, methanolic extract of T. arduini appeared to have the best cytotoxic activity on HCT-116, with IC(50) values of 0.37 μg/mL. Treatments caused typical apoptotic morphological changes in HCT-116 cells and showed a high percentage of apoptotic cells. The results of the presented research indicate that some Teucrium extracts are a very rich source of phenols, which may directly contribute to high antiproliferative and proapoptotic activity.     

Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic substrate enhancement is described targeting the secondary binding sites of PTPs. By screening four different PTPs from bacterial (MptpA) and human origin (PTP1B, HePtp, Shp2) with this assay, specific fragments were identified. One highly specific fragment that binds to the secondary site of Mycobacterium tuberculosis protein tyrosine phosphatase A (MptpA) was characterized in order to validate the assay concept. Finally by covalently linking the secondary fragment to a phosphotyrosine mimetic, a moderately active but highly specific inhibitor of MptpA was obtained.     

狂犬病病毒抗体竞争ELISA检测方法的建立及初步应用

目的建立狂犬病病毒抗体竞争ELISA检测方法,用于不同株狂犬病疫苗免疫后抗体水平的检测。方法用市售的不同狂犬病疫苗株抗原免疫NIH小鼠,制备免疫血清,用不同的疫苗株抗原包被酶标板,分别测定小鼠血清抗体效价,并通过不同株抗原抗体的交叉中和实验,分析抗原的差异性;在此基础上,将不同毒株抗原按不同比例混合包被酶标板,分别测定阳性血清样本,并与快速免疫荧光抑制试验(RFFIT)检测结果比较,确定包被抗原模式,建立狂犬病病毒抗体竞争ELISA检测方法,绘制标准曲线,确定最佳定量范围及灵敏度,确定Cut-off值;并对其特异性、精密性、准确性及稳定性进行验证;用建立的方法与其他市售狂犬病病毒抗体检测试剂分别检测血清样品,分析检测结果的一致性及相关性。结果狂犬病病毒抗原AG∶CTN-1=4∶1为最适包被抗原模式,试剂经优化后,检测抗体效价范围在535.00～33.44 mIU/ml之间,具有良好的线性关系(r>0.99),最低检出限为8.36 mIU/ml,Cut-off值为0.735 mIU。该方法检测人血清白蛋白、破伤风阳性血清、乙肝表面抗原阳性血清、白喉阳性质控血清均未发生反应;试验内变异系数在6.68%～7.84%之间;回收率在97.25%～104.50%之间;试剂置37℃3 d,与4℃保存试剂测定结果差异无统计学意义(P>0.05)。该方法检测血清样品与市售狂犬病病毒抗体检测试剂比较,均具有良好的一致性;与RFFIT测定结果比较,二者差异无统计学意义(P>0.05),回归方程为Y=-0.475+3.246 X,相关系数为0.801。结论已建立了狂犬病病毒抗体竞争ELISA检测方法,可用于大规模狂犬病病毒抗体的筛查。     

b型流感嗜血杆菌多糖ELISA检测方法的建立

目的建立b型流感嗜血杆菌(Haemophilus influenzae type b,Hib)多糖(Polyribosylribitolphosphate,PRP)的ELISA检测方法。方法用灭活的Hib菌免疫家兔,1周免疫4次,共免疫6周,采血,检测血清抗体滴度。对兔免疫血清进行非特异性抗体免疫吸附,纯化兔抗PRP抗体,用纯化的兔抗PRP抗体包被酶标板,HRP标记的羊抗兔IgG作为二抗,建立双抗体夹心ELISA法检测PRP。确定标准曲线的最佳线性范围,并对方法进行特异性、准确性和精密性验证。结果兔免疫血清的抗体效价可达1∶2 800。该方法的线性检测范围为0.030～0.500 ng/ml,检测限为0.030 ng/ml。该方法检测大肠杆菌上清蛋白、牛血清白蛋白、LB培养基及破伤风-乙脑结合疫苗均无特异性反应;检测0.500、0.250、0.150 ng/ml的PRP标准品试验内变异系数(CV)在1.16%～2.40%之间,回收率在93.2%～107.2%之间;试验间CV在1.57%～4.11%之间,回收率在101.3%～101.6%之间。结论该方法特异性、准确性和精密性均较好,可以特异性检测b型流感嗜血杆菌多糖。     

Mechanistic Investigation of ROS-Induced DNA Damage by Oestrogenic Compounds in Lymphocytes and Sperm Using the Comet Assay

Past research has demonstrated that oestrogenic compounds produce strand breaks in the DNA of sperm and lymphocytes via reactive oxygen species (ROS). In the current investigation, sperm and lymphocytes were treated in vitro with oestrogenic compounds (diethylstilboestrol, progesterone, 17β-oestradiol, noradrenaline and triiodotyronine) and several aspects of DNA damage were investigated. Firstly, mediation of DNA damage by lipid peroxidation was investigated in the presence of BHA (a lipid peroxidation blocker). BHA reduced the DNA damage generated by 17β-oestradiol and diethylstilboestrol in a statistically significant manner. No effects were observed for sperm. Secondly, the presence of oxidized bases employing FPG and EndoIII were detected for lymphocytes and sperm in the negative control and after 24 h recovery in lymphocytes but not immediately after treatment for both cell types. The successful detection of oxidized bases in the negative control (untreated) of sperm provides an opportunity for its application in biomonitoring studies. DNA repair at 24 h after exposure was also studied. A nearly complete recovery to negative control levels was shown in lymphocytes 24 h recovery after oestrogenic exposure and this was statistically significant in all cases. Rapid rejoining of DNA, in a matter of hours, is a characteristic of DNA damaged by ROS.     

Effect of CO(2) Enrichment on Synthesis of Some Primary and Secondary Metabolites in Ginger (Zingiber officinale Roscoe)

The effect of two different CO(2) concentrations (400 and 800 μmol mol(-1)) on the photosynthesis rate, primary and secondary metabolite syntheses and the antioxidant activities of the leaves, stems and rhizomes of two Zingiber officinale varieties (Halia Bentong and Halia Bara) were assessed in an effort to compare and validate the medicinal potential of the subterranean part of the young ginger. High photosynthesis rate (10.05 μmol CO(2) m(-2)s(-1) in Halia Bara) and plant biomass (83.4 g in Halia Bentong) were observed at 800 μmol mol(-1) CO(2). Stomatal conductance decreased and water use efficiency increased with elevated CO(2) concentration. Total flavonoids (TF), total phenolics (TP), total soluble carbohydrates (TSC), starch and plant biomass increased significantly (P ≤ 0.05) in all parts of the ginger varieties under elevated CO(2) (800 μmol mol(-1)). The order of the TF and TP increment in the parts of the plant was rhizomes > stems > leaves. More specifically, Halia Bara had a greater increase of TF (2.05 mg/g dry weight) and TP (14.31 mg/g dry weight) compared to Halia Bentong (TF: 1.42 mg/g dry weight; TP: 9.11 mg/g dry weight) in average over the whole plant. Furthermore, plants with the highest rate of photosynthesis had the highest TSC and phenolics content. Significant differences between treatments and species were observed for TF and TP production. Correlation coefficient showed that TSC and TP content are positively correlated in both varieties. The antioxidant activity, as determined by the ferric reducing/antioxidant potential (FRAP) activity, increased in young ginger grown under elevated CO(2). The FRAP values for the leaves, rhizomes and stems extracts of both varieties grown under two different CO(2) concentrations (400 and 800 μmol mol(-1)) were significantly lower than those of vitamin C (3107.28 μmol Fe (II)/g) and α-tocopherol (953 μmol Fe (II)/g), but higher than that of BHT (74.31 μmol Fe (II)/g). These results indicate that the plant biomass, primary and secondary metabolite synthesis, and following that, antioxidant activities of Malaysian young ginger varieties can be enhanced through controlled environment (CE) and CO(2) enrichment.     

酶联免疫法快速检测贝类中腹泻性贝类毒素

应用美国ABRAXIS腹泻性贝类毒素(DSP)试剂盒对广西钦州和防城港海域近江牡蛎各20个样品、北海海域文蛤20个样品进行了ELISA法DSP检测.全部检测过程在2h内完成,检测限为0.1 ppb,灵敏度为1.7 ppb.ELISA法检测DSP探作方便、成本低,检测限和灵敏度均满足要求.ELISA法有望作为理想的DSP...     

人细小病毒B19感染与早期自然流产关系的探讨

目的探讨人细小病毒B19(HPVB19)感染与早期自然流产的关系。方法用酶联免疫吸附试验(ELISA)法检测52例早期自然流产患者(研究组)和45例无异常人工流产孕妇(对照组)血清中HPVB19-IgM和IgG。结果研究组血中B19V-IgM阳性率为23.08%,而对照组阳性率为6.67%,2组间具有显著性差异(P<0.05);研究组近期HPVB19感染的概率是对照组的4.20倍(95%CI 1.10～15.99)。研究组B19V-IgG阳性率为32.69%,对照组为28.89%,2组差异无统计学意义(P>0.05)。结论 HPVB19感染可能是导致早期自然流产的原因之一。     

In vitro antioxidant properties of different parts of pomegranate flowers

The present study was designed to compare the antioxidant capacity of 70% ethanol extracts of petal, stamen and receptacle of pomegranate flowers (PF) by DPPH, reducing power, hydroxyl radicals (OH), DNA degradation and DNA breaking assays, as well as the peroxide value (POV). The different parts of PF showed significant activities in all the free radical scavenging tests. Among the parts, receptacle exhibited the highest inhibition of 44.86%, 61.21% and 44.47% in DPPH, OH and DNA degradation assays, respectively, and 63.96% reduction of ferricyanide to give Prussian blue colored complex in reducing power assay at maximum concentration tested, and showed the lowest POV of 68.51 meq/kg in experimental model of soybean oil after 12 days. Protection against oxidative damage to DNA molecules of various parts of flower was confirmed visually by DNA breaking assay. Total phenolic contents of the parts of flower and peel and leaves were estimated by Folin–Ciocalteu method and receptacle was found to contain the highest amount (148.41 ± 6.25 mg/g) of phenolics among the parts of flower, but the phenolic content of flower (petal, stamen and receptacle) was lower than peel, higher than leaves. Linear correlation (R² = 0.7139–0.9987) between antioxidant potential of various parts in different assays and phenolic contents was observed. This preliminary study indicates the antioxidant activity of PF, and moreover the results showed correlation with the amount of phenolic content present in different parts. Therefore, all parts of PF could be considered as a significant natural antioxidant source.     

氢供体组与辣根过氧化酶活性光谱研究

对邻苯二酚-苯胺和苯酚-氨基安替吡啉构成的两组测定HRP酶活性的体系进行了研究和比较。紫外可见光光谱和高效液相色谱分析表明,在HRP催化过程中,邻苯二酚-苯胺氢供体对会生成为一种粉红色产物,该产物最大吸收波长在510咖,可用于HRP的活性测量。且与常规使用的苯酚-氨基安替吡啉氢供体对的情况相比,具有更高的灵敏度、更低的检测限值和很好的重复性,在对HRP分别进行20次酶活测量,所得相对标准偏差仅为2．9％。使用邻苯二酚-苯胺氢供体对的方法可计算得到酶动力学参数Km（12．5mM）和Vmax（12．2mMmin^-1mg^-1）。