Centre of Excellence in Microbiology, Dr. Ram Manohar Lohia Avadh University, Faizabad-224001, Uttar Pradesh, India.
BMC Complement Altern Med. 2011 Jul 12;11:56. doi: 10.1186/1472-6882-11-56.
Proteases play an important role in virulence of many human, plant and insect pathogens. The proteinaceous protease inhibitors of plant origin have been reported widely from many plant species. The inhibitors may potentially be used for multiple therapeutic applications in viral, bacterial, fungal diseases and physiological disorders. In traditional Indian medicine system, Cassia tora (Senna tora) is reportedly effective in treatment of skin and gastrointestinal disorders. The present study explores the protease inhibitory activity of the above plant seeds against trypsin, Aspergillus flavus and Bacillus sp. proteases.
The crushed seeds of Cassia tora were washed thoroughly with acetone and hexane for depigmentation and defatting. The proteins were fractionated by ammonium sulphate (0-30, 30-60, 60-90%) followed by dialysis and size exclusion chromatography (SEC). The inhibitory potential of crude seed extract and most active dialyzed fraction against trypsin and proteases was established by spot test using unprocessed x-ray film and casein digestion methods, respectively. Electrophoretic analysis of most active fraction (30-60%) and SEC elutes were carried employing Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Gelatin SDS-PAGE. Inhibition of fungal spore germination was studied in the presence of dialyzed active inhibitor fraction. Standard deviation (SD) and ANOVA were employed as statistical tools.
The crude seeds' extract displayed strong antitryptic, bacterial and fungal protease inhibitory activity on x-ray film. The seed protein fraction 30-60% was found most active for trypsin inhibition in caseinolytic assay (P < 0.001). The inhibition of caseinolytic activity of the proteases increased with increasing ratio of seed extract. The residual activity of trypsin, Aspergillus flavus and Bacillus sp. proteases remained only 4, 7 and 3.1%, respectively when proteases were incubated with 3 mg ml-1 seed protein extract for 60 min. The inhibitory activity was evident in gelatin SDS-PAGE where a major band (~17-19 kD) of protease inhibitor (PI) was detected in dialyzed and SEC elute. The conidial germination of Aspergillus flavus was moderately inhibited (30%) by the dialyzed seed extract.
Cassia tora seed extract has strong protease inhibitory activity against trypsin, Aspergillus flavus and Bacillus sp. proteases. The inhibitor in Cassia tora may attenuate microbial proteases and also might be used as phytoprotecting agent.
蛋白酶在许多人类、植物和昆虫病原体的毒力中起着重要作用。植物来源的蛋白质蛋白酶抑制剂已广泛报道于许多植物物种中。这些抑制剂可能具有多种治疗用途，可用于病毒、细菌、真菌疾病和生理紊乱。在传统的印度医学体系中，决明子（番泻叶）被报道对皮肤和胃肠道疾病有效。本研究探索了上述植物种子对胰蛋白酶、黄曲霉和芽孢杆菌蛋白酶的蛋白酶抑制活性。
用丙酮和正己烷彻底清洗决明子种子，以进行脱色和脱脂。用硫酸铵（0-30%、30-60%、60-90%）分级分离蛋白质，然后透析和分子筛层析（SEC）。通过使用未经处理的 X 射线胶片和酪蛋白消化方法分别对粗种子提取物和最活跃的透析级分对胰蛋白酶和蛋白酶的抑制潜力进行点测试。采用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）和明胶 SDS-PAGE 对最活跃的 30-60%级分和 SEC 洗脱物进行电泳分析。在存在透析活性抑制剂级分的情况下研究真菌孢子萌发的抑制作用。采用标准偏差（SD）和方差分析（ANOVA）作为统计工具。
粗种子提取物在 X 射线胶片上显示出强烈的抗胰蛋白酶、细菌和真菌蛋白酶抑制活性。在酪蛋白水解测定中，发现种子蛋白 30-60%级分对胰蛋白酶抑制最活跃（P < 0.001）。随着种子提取物比例的增加，蛋白酶的酪蛋白水解活性抑制增加。当蛋白酶与 3mg ml-1种子蛋白提取物孵育 60min 时，残留的胰蛋白酶、黄曲霉和芽孢杆菌蛋白酶的活性仅分别为 4%、7%和 3.1%。在明胶 SDS-PAGE 中，抑制剂（PI）的主要条带（~17-19kD）在透析和 SEC 洗脱物中均被检测到，这表明抑制活性明显。黄曲霉的孢子萌发受到种子提取物的中度抑制（30%）。
决明子种子提取物对胰蛋白酶、黄曲霉和芽孢杆菌蛋白酶具有强烈的蛋白酶抑制活性。决明子中的抑制剂可能减弱微生物蛋白酶，也可用作植物保护剂。
