Effects of gamma irradiation on the shoot length of Cicer seeds

The effects of radiation on the shoot and root lengths of germinated seedling of irradiated seeds of Cicer species, i.e. three kabuli types and four desi types of cultivated chickpea (Cicer arietinum Ladiz.) and 2 annual wild types (C. reticulatum Ladiz. and C. bijugum K.H. Rech.) were investigated. The seeds were irradiated with a ⁶⁰Co gamma source using 0, 200, 300 and 400 Gy doses at 1.66 kGy h⁻¹. At 200 Gy minor effects could be observed, but at 400 Gy an obvious depression of shoot length was observed. The kabuli types were more affected than the desi ones. The critical dose that prevented the shoot and root elongation varied among species and also ranged from genotypes to genotype within species.     

In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large‐scale production of functional food and nutraceutical ingredients.     

Chickpeas from a Chilean Region Affected by a Climate-Related Catastrophe: Effects of Water Stress on Grain Yield and Flavonoid Composition

The Valparaiso region in Chile was decreed a zone affected by catastrophe in 2019 as a consequence of one of the driest seasons of the last 50 years. In this study, three varieties (‘Alfa-INIA’, ‘California-INIA’, and one landrace, ‘Local Navidad’) of kabuli-type chickpea seeds produced in 2018 (control) and 2019 (climate-related catastrophe, hereafter named water stress) were evaluated for their grain yield. Furthermore, the flavonoid profile of both free and esterified phenolic extracts was determined using liquid chromatography-mass spectrometry, and the concentration of the main flavonoid, biochanin A, was determined using liquid chromatography with diode array detection. The grain yield was decreased by up to 25 times in 2019. The concentration of biochanin A was up to 3.2 times higher in samples from the second season (water stress). This study demonstrates that water stress induces biosynthesis of biochanin A. However, positive changes in the biochanin A concentration are overshadowed by negative changes in the grain yield. Therefore, water stress, which may be worsened by climate change in the upcoming years, may jeopardize both the production of chickpeas and the supply of biochanin A, a bioactive compound that can be used to produce dietary supplements and/or nutraceuticals.     

鹰嘴豆β-半乳糖苷酶的分离纯化与表征

从鹰嘴豆中分离得到了三种β－半乳糖苷酶（酶Ⅰ、酶Ⅱ和酶Ⅲ）。将酶Ⅰ和酶Ⅱ进一步纯化,其比活力分别提高了19倍和48倍．酶活力回收率分别为16％和18％,测得它们的表观分子里分别为2．4×104和5.8×104;最适pH分别5.9和5.0,最适温度分别为55℃和45℃．酶Ⅰ水解ONPG和PNPG的KM分别为33×10-2mol·dm-3和60×10-3mol·dm－3;酶Ⅱ水解ONPG和PNPG的KM分别为30×10－3mol·dm－3和60×10－4mol·dm－3.乳糖和半乳糖为该酶的竞争性可逆抑制剂,棉子糖为非竞争性可逆抑制剂．该酶受Hg2＋和PCMB强烈抑制和NEM明显抑制,而Mg2＋、Zn2＋和Ca2＋具有激活作用,推知巯基（－SH)是酶活性中心必须基团．     

植物毒素茄格孢吡喃酮A的研究

致病真菌Ascochyta rabiei感染鹰嘴豆植物体产生三种植物毒索．茄格孢毗喃酮A．茄格孢毗喃酮B和茄格孢毗喃酮C．其中茄格孢毗喃酮A的毒性最大．往往在感染的植物体中表现出植物体变黄．植物茎部变脆．易折断等病理现象。严重时导致整个植物体因枯萎病而死亡．体外培养Ascochyta rabiei研究已知．该致病菌产生一些叫做茄格孢毗喃酮的植物毒索．当用分离提纯的茄格孢毗喃酮A毒素喂养植物体时虽然出现了与病菌感染植物体完全一样的病理现象．但研究者均不能从感染植物体或者体外喂养毒索的植物体中重新分离到该毒索。很可能这种毒索通过某种机制被鹰嘴豆植物细胞代谢转换成另外一种物质．为了研究这一过程，我们提取鹰嘴豆植物的蛋白质提取物(30％-70％硫氨饱和沉淀法)于实验室条件下分离提取并用HPLC方法鉴定植物毒索茄格孢毗喃酮A反应．不管还原物质NADPH存在或者不存在．发现植物毒素茄格孢吡喃酮A消失．即使在鹰嘴豆蛋白制备物加热沸腾处理后与毒索的反应中也出现植物毒素茄格孢毗喃酮A的大量丢失．说明蛋白质制备物很可能不是酶．HPLC(高压液相色谱)检测反应产物时出现了一个新物质．NMR(核磁共振)确定该物质为脱甲基的茄格孢毗喃酮A．进一步的研究得知．该脱甲基的茄格孢毗喃酮A不是鹰嘴豆植物蛋白质提取物的作用下产生的．而是分离蛋白质用的硫氨作用于该植物毒素产生的．用透析方法除掉硫氨后的鹰嘴豆植物蛋白质制备物与该毒素反应时．脱甲基茄格孢吡喃酮A不再出现．但仍有茄格孢吡喃酮A植物毒素的丢失．最后发现，分离提纯蛋白质制备物所用的离子交换柱(FPLC)试剂Tris-HCI和NaCl也会导致茄格孢吡喃酮A植物毒素的丢失．与一些非特异性蛋白质化学物反应时．茄格孢吡喃酮A植物毒索还是存在大量的丢失．所有这些研究结果显示．这一种植物毒素茄格孢吡喃酮A似乎具有较高的反应活性．很可能与植物体中的一些物质结合或者局部反应．正因为是这一原因不能从感染植物体或者体外喂养毒素的植物体中重新分离到该毒素，而感染植物或体外喂养毒素的植物仍然表现出明显的病理现象．植物毒素茄格孢吡喃酮A的这种易反应特性．可能就是它对植物体具有较强毒性的原因之一．     

Isolation,cytotoxic evaluation,and simultaneous quantification of eight bioactive secondary metabolites from Cicer microphyllum by high‐performance thin‐layer chromatography

Chemical investigation of Cicer microphyllum resulted in the isolation and characterization of eight natural products viz. Stigmasterol, Oleanolic acid‐3‐acetate, Oleanolic acid, Biochanin A, Genistein, Pratensein, Chrysoeriol, and Luteolin. Herein, we report a novel, accurate, and cost‐effective high‐performance thin‐layer chromatography method for the simultaneous quantification of the isolated natural products on silica‐gel 60F₂₅₄ plates using the solvent system n‐hexane/ethyl acetate/formic acid (9.0:6.5:0.8, v/v/v). Natural products were quantified after postchromatographic derivatization with ceric ammonium sulfate. The method was validated as per the International Conference on Harmonization guidelines. All calibration curves showed a good linear relationship (r > 0.9943) within the test range. Precision was assessed by intra‐ and interday tests with relative standard deviations <1.82%, accuracy validation recovery 98.38–99.57% with relative standard deviations <1.00%. On quantification, Pratensein was a major constituent (0.921%). The screening for cytotoxic activity using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay resulted into identification of Luteolin as potent molecule with IC₅₀ 3.5 and 25.6 μg/mL against murine melanoma and human epidermoid carcinoma cell lines, respectively.