Tracking antiangiogenic components from Glycyrrhiza uralensis Fisch. based on zebrafish assays using high-speed countercurrent chromatography

Natural products are some of the most important sources of lead compounds for drug discovery. The advanced isolation technique of lead compounds of natural origin using therapeutically relevant bioassays is capable of enhancing work efficiency from complex multiconstituent extracts. In the present study, a bioassay-guided isolation strategy combined with bioactivity screening was used to identify novel angiogenesis inhibitors from licorice (Glycyrrhiza uralensis Fisch.) based on the zebrafish model and rapid preparative separation by high-speed countercurrent chromatography. Zebrafish embryos at 24 h postfertilization were chosen as the angiogenesis inhibition model for bioactivity screening. A solvent system (n-hexane-ethyl acetate-methanol-water) with different ratios was optimized and applied in the high-speed countercurrent chromatography separation of two fractions, Fr5 and Fr6, from the ethyl acetate extract of licorice. Blood circulation and vascular outgrowth in intersegmental vessels were found to be simultaneously inhibited by isoliquiritigenin and isolicoflavonol in a dose-dependent manner. Thus, these two compounds were identified and considered as active inhibitors against angiogenesis. These experimental results indicate that zebrafish bioassays combined with high-speed countercurrent chromatography may provide an alternative pathway for the rapid isolation of bioactive natural products.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of glycyrrhizic acid

Glycyrrhizic acid (GL) is a major active compound of licorice. The specific monoclonal antibody (MAb) (designated as 8F₈A₈H₄2H₇) against GL was produced with the immunogen GL–BSA conjugate. The dissociation constant (K _d) value of the MAb was approximately 9.96×10⁻¹⁰ M. The cross reactivity of the MAb with glycyrrhetic acid was approximately 2.6%. The conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA adapted with a modified procedure were established using the MAb. The IC₅₀ value and the detect range by the conventional icELISA were 1.1 ng mL⁻¹ and 0.2–5.1 ng mL⁻¹, respectively. The IC₅₀ value and the detect range by the simplified icELISA were 5.3 ng mL⁻¹ and 1.2–23.8 ng mL⁻¹, respectively. The two icELISA formats were used to analyze GL contents in the roots of wild licorice and different parts of cultivated licorice (Glycyrrhiza uralensis Fisch). The results obtained with the two icELISAs agreed well with those of the HPLC analysis. The correlation coefficient was more than 0.98 between HPLC and the two icELISAs. The two icELISAs were shown to be appropriate, simple, and effective for the quality control of raw licorice root materials.     

乌拉尔甘草种子硬实率的近红外光谱分析

以收获年份不同、收获地点不同的112份乌拉尔甘草种子为材料,其硬实率分布范围为0.3%～99.3%,根据3:1的比例划分校正集和检验集,采用近红外光谱技术结合定量偏最小二乘法对其硬实率进行了分析。研究结果表明,光谱范围采用5 000～6 000 cm~(-1),主成分数为6时,校正集和检验集的决定系数分别为90.85%和91.51%,相关系数分别为0.953 2和0.957 9,平均绝对误差分别为7.73%和6.96%,标准差分别为9.98和9.57。采用该方法建模时,即使采用不同的建模样品,校正集和检验集的决定系数均在90%以上,校正标准差和预测标准差在10.00左右,平均绝对误差在7.90%左右。该研究旨在利用近红外光谱法提出乌拉尔甘草种子硬实率的快速检测方法,以促进硬实种子在生产上的利用。     

Bioactive characterization of ultrasonicated ginger (Zingiber officinale) and licorice (Glycyrrhiza Glabra) freeze dried extracts

Ginger (Zingiber officinale) and Licorice (Glycyrrhiza glabra L.) are one of the most popular spices having a wide range of bioactive compounds that have varied biological and pharmacological properties. The study was aimed to extract polyphenols from Himalayan medicinal herbs ginger and licorice in different solvents using ultra-sonication technique. The extraction efficiency (EE) was determined, and the extracts were characterized for physical properties (particle size, colour values), total phenolics, flavonoids, antioxidant properties, and structural and morphological features. Ultra-sonicated ginger in aqueous phase had the highest EE of polyphenols (15.27%) as compared to other solvents. Similar trend was observed in licorice with EE of 30.52 % in aqueous phase followed by ethanol: water (50: 50), and methanol: water (50:50) with 28.52% and 26.39%, respectively. The preliminary screening showed the presence of tannins, phenolics, flavonoids, saponins and carbohydrates, steroids and alkaloids in all the extracts. The phenolic and flavonoid content of dried ginger was found higher in ethanolic extracts compared to fresh ones as revealed by HPLC. Similarly, for licorice, the ethanolic fractions had the highest polyphenolic content. The representative samples of ginger (ethanol: water 75:25 and ethylacetate: water 75:25) and licorice (ethanol: water 70:30 and methanol: water 50:50) were studied for FESEM and particle size. The results showed the agglomerated extract micro-particles with a diameter of 0.5–10 µm and increased particle size (ginger: 547 and 766 nm), and (licorice: 450 and 566 nm). The findings could be beneficial for the advancement of ginger and licorice processing, for the comprehension of these herbs as a source of natural antioxidants in different food formulations.     

微分电位溶出伏安法同时测定甘草中痕量铅和镉

建立了在NH4OAc HOAc介质中,用微分电位溶出伏安法测定中药甘草中痕量铅和镉的方法,利用该法对中药甘草煎煮前后及其煎液中铅和镉的含量进行测定。试验表明,镉和铅分别在-0.66V(vs.SCE)和-0.47V(vs.SCE)电位处形成两个灵敏的二次微分溶出峰;铅在0～0.8mg·L-1,镉在0～0.4mg·L-1范围,峰电流与浓度呈良好的线性关系;铅和镉检出限分别为0.15和0.1μg·L-1,相对标准偏差≤1.39%;铅和镉加标回收率在94%～103%之间。结果表明,该甘草样品中铅和镉含量均低于国家安全标准,甘草煎煮液中铅的浸出率较高,镉的浸出率则较低,方法也适用于其它类似中药。