Sequence Determination of a Novel Tripeptide Isolated from the Young Leaves of Azadirachta indica A. Juss

The neem tree has long been recognized for its unique properties, both against insects and in improving human health. Every part of the tree has been used as a traditional medicine for household remedy against various human ailments, from antiquity. Although the occurrence of various phytochemicals in neem has been studied, we have identified the presence of a novel tripeptide in the young leaves of neem using a simple and inexpensive paper chromatographic method, detected by Cu(II)-ninhydrin reagent. The peptide nature of the isolated compound is confirmed by spectral studies. The sequence of the peptide is determined using de novo sequencing by tandem MS after purification. 1. Introduction Small alpha peptides are the most expensive substances, and most of them are not easily available commercially [1]. Pharmacological studies have proved that many peptides, including those isolated from plants, have a potential antitumor effect [2]. These peptides have a number of advantages over other chemical agents including their low molecular weight, relatively simple structure, lower antigenicity, fewer adverse actions, easy absorption, and a variety of routes of administration [3]. Many antibacterial peptide families have been isolated from plants. Pp-thionin, for example, showed activity against Rhizobium meliloti, Xanthomonas campestris, Micrococcus luteus. Circulins A-B and cyclopsychotride A from the cyclotides family showed antibacterial effects against human pathogens such as Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Klebsiella oxytoca at micromolar concentrations [4]. Various plant extracts are reported to exhibit high antifungal activity due to proteins or peptides [5]. Cardiovascular activity of milk casein-derived tripeptides has also been reported, where bioactive tripeptide-containing milk products attenuated the blood pressure development in spontaneously hypertensive rats [6]. Research on A. indica has revealed the occurrence of various compounds such as terpenoids, and flavonoids [7, 8]. But the presence of small alpha peptides has not been reported so far. Ninhydrin reactions using manual and automated techniques as well as ninhydrin spray reagents are widely used to analyze and characterize amino acids, peptides, and proteins, as well as numerous other ninhydrin-positive compounds in biomedical, clinical, food, forensic, histochemical, microbiological, nutritional, and plant studies [9–11]. Many of the shortcomings of ninhydrin have been met by the synthesis of a variety of