small alpha peptides are the most expensive substances , and most of them are not easily available commercially . pharmacological studies have proved that many peptides , including those isolated from plants , have a potential antitumor effect . 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 . 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 . various plant extracts are reported to exhibit high antifungal activity due to proteins or peptides . 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 . research on a. indica has revealed the occurrence of various compounds such as terpenoids , and flavonoids [ 7 , 8 ] . 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 [ 911 ] . many of the shortcomings of ninhydrin have been met by the synthesis of a variety of ninhydrin analogs . all amino acids and their carboxyl group derivatives like esters and amides , including small peptides , produce a purple color with the classical ninhydrin reagent . this reagent was modified by us by adding cupric ion in order to distinguish qualitatively the carboxyl group derivatives of amino acids from the amino acids on paper after chromatography . amino acids produce a pink color , and their carboxyl derivatives like esters and amides , including small peptides , produce a yellow color with cu(ii)-ninhydrin reagent . the cu(ii)-ninhydrin method discussed here is a novel one because no other methods presently used can form two different coloured products with a single developing reagent . we have used this method for the detection and purification of amino acid derivatives from different plant products [ 13 , 14 ] . in this paper , we report the isolation and sequence determination of a small alpha peptide from the young leaves of a. indica . whatman no . 1 filter paper discs were obtained from whatman international ltd , maidstone , england . the young leaves of a. indica were homogenized ( 1  g/10  ml ) with warm 80% ethanol . the extraction was partitioned three times with equal volumes of petroleum ether to remove the pigments . the pigment - free alcohol fraction was evaporated to dryness over a boiling water bath . the resulting residue was treated with 2% polyvinylpyrrolidone ( one ml for each gram of leaf used as a starting material ) and centrifuged at 3000  g for 10  min at 4c to remove the phenolic compounds . the clear supernatant obtained was used as the crude source of the small alpha peptide . the cu(ii)-ninhydrin reagent was prepared by dissolving cupric nitrate ( 25  mmol / l ) and ninhydrin ( 1% w / v ) in a minimum quantity of a mixture of water and glacial acetic acid ( 3  :  1  v / v ) and diluted with required amount of acetone . the crude extract was spotted in the center of a circular whatman no . 1 filter paper on the arc of a small circle drawn with a pencil . depending upon the number of samples to be analyzed , the paper may be demarcated . the diameter of the samples spotted was restricted to 0.5  cm by intermittent use of a hot air dryer . the sample spotting may be repeated 15 to 20 times to ensure sufficient concentration of the component to be detected . the chromatography was carried out in an isopropanol  :  water ( 4  :  1 , v / v ) solvent system by connecting a filter paper wick to the solvent through a hole made at the center of the circular paper . after the run which required approximately 2040  min , the air - dried chromatogram was developed by spraying uniformly with cu(ii)-ninhydrin reagent followed by drying at 60c for 10 min . to subject the compound for various spectral studies , the corresponding region of the paper on the other chromatogram containing the cu(ii)-ninhydrin - positive compounds was cut into pieces and eluted in 80% ethanol . the compound , thus obtained , was used for conducting the spectral studies . 1  ml of the purified cu(ii)-ninhydrin - positive compound was added with 1  ml of cu(ii)-ninhydrin reagent , and the mixture was incubated at 40c for 5  min . the amount of these compounds was determined by using a standard graph constructed with l - glycyl glycine as the standard . the purified compound was scanned for its absorption properties , from 200  nm to 900  nm in a shimadzu , uv - vis spectrophotometer . the purified compound was also subjected to ft - ir analysis using a shimadzu model ftir-8300 infrared spectrometer . ir spectra were scanned between 500 and 4,000 wave numbers ( per centimeter ) . the purified cu(ii)-ninhydrin - positive compound was analyzed using the gc / ms instrument : trace ultra version 5.0 produced by thermo . the separation conditions were as follows : db-5 column 30  m  0.25  mm  0.25  m , mobile phase helium at flow rate 1.0  ml / min , injection chamber temperature 220c , and oven temperature starts at 80c raised to 250c at a rate of 8c per minute . the purified cu(ii)-ninhydrin - positive compound in deuterated acetone as a solvent was subjected to h and c nmr analysis using a bruker 500  mhz liquid - state nmr spectrometer .   for this experiment , 0.5  ml of this sample aliquot was mixed with 0.5  ml of concentrated hcl in a clean dry test tube ( the final concentration of hcl is 6  n ) . this was placed in an incubator at 110c for 24 hours after which the sample was reconstituted to 0.5  ml with distilled water . 1 filter paper and developed with the isopropanol  :  water ( 4  :  1  v / v ) system . the chromatograms were uniformly sprayed with ninhydrin reagent and were air dried and heated at 65c for 10  min .   peptide sample ( purified cu(ii)-ninhydrin - positive compound ) was evaporated to dryness at room temperature and resuspended in 0.1% trifluoroacetic acid buffer and spotted with chca ( alpha - cyanohydroxycinnamic acid ) matrix onto maldi plate and allowed to dry . a model 4800 plus maldi tof / tof analyzer ( applied biosystems inc . , foster city , ca , usa ) was used for direct profiling and ms / ms fragmentation study . spectra are obtained for the major peptide ions in ms mode , and sequence data are obtained when the spectrometers automatically revert to ms / ms mode . ms / ms was achieved by 2  kv collision - induced dissociation ( cid ) in positive ion mode . de novo sequencing analysis was used to determine the primary sequence structure for peptides that are not present in currently available databases . the plant extract was run on circular paper chromatography using isopropanol  :  water ( 4  :  1 ) solvent system and developed with cu(ii)-ninhydrin reagent . . 2  g concentration of the peptide was obtained from 1  g of the leaf extract . the peptide nature of the purified compound from the young leaves of a. indica was confirmed by uv spectrophotometer . purified alpha peptide from the young leaves of a. indica showed maximum absorption at 210  nm ( figure 1 ) confirming the peptide nature . the peptide nature was further confirmed by identifying functional groups from the ft - ir studies.figure 2 shows the ft - ir spectrum of the purified compound from the young leaves of a. indica . there were sharp peaks at 1600  cm and 3380  cm indicating the presence of c = o and nh groups respectively , in the compound , confirming the peptide nature . the cu(ii)-ninhydrin - positive compound purified from the young leaves of a. indica was analyzed by gc / ms . the retention times of the compound were compared with those of reference standard amino acids under the same conditions . the identification of the amino acids in the sample was based on direct comparison of the retention times and mass spectral data with those for standard compounds , and by computer matching with the wiley 229 , nist 107 , and 21 library , as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature . figure 4 shows the gc - ms spectra of the cu(ii)-ninhydrin - positive compound purified from the young leaves of a. indica . from the data obtained , it can be inferred that the peptide isolated from the young leaves of a. indica might contain alanine , cysteine , and phenyl alanine . nmr studies indicated the presence of ch3 , ch2 , and aromatic groups in proton spectra and the presence of c = o , ch , and sh / oh groups in carbon-13 spectra of the peptide purified from a. indica . this indicates the presence of aliphatic amino acid , sulphur containing amino acid , and aromatic amino acid in the isolated peptide . the peptide nature of the compound was also further confirmed from the acid hydrolysis experiment . the chromatograms here were developed with the ninhydrin reagent which can detect the amino acids produced by hydrolysis more effectively . figure 5 shows the acid - hydrolyzed a. indica peptide developed in isopropanol  :  water ( 4  :  1  v / v ) system by circular paper chromatography and sprayed with ninhydrin . the result indicated that there may be presence of three amino acids in the purified peptide . in the past decade , tandem mass spectrometry ( ms / ms ) has emerged as a technology of choice for high - throughput proteomics . in spite of the continuously growing sequence databases , de novo sequencing of peptides , that is , sequencing without assistance of a linear sequence database , figure 6 represents the ms / ms spectra of the peptide isolated from the young leaves of a. indica . the sequence of amino acids was determined by the application of tandem ms . by referring to the mass unit of the respective amino acids , the amino acid sequence of the peptide isolated from the young leaves of a. indica is found to be ala - phe - cys ( n - alanine - phenylalanine - cysteine - c ) . the peptide isolated from a. indica is a tripeptide , composed of three amino acid residues with alanine at the n - terminus and cysteine at the c - terminus . the determined sequence data was submitted in uniprot knowledgebase database , and accession number was assigned for the sequence . the sequence data of the peptide isolated from the young leaves of a. indica , reported in this paper , will appear in the uniprot knowledgebase under the accession number b3ewr2 . the assessment of the biological role and applications of the purified tripeptide is under study . compounds with low molecular weight of 500 or less can function as efficient drug molecules . small peptides containing multifunctional amino acids like l - glutamic acid , l - aspartic acid , l - lysine , l - histidine , l - cysteine , and l - serine can function as potent chelating agents that can be employed in chelation therapy . from the overall results obtained from this work , it can be inferred that the cu(ii)-inhydrin positive compound purified from the young leaves of a. indica is a tripeptide . unlike amino acids , small peptides are highly expensive , and most of them are not easily available commercially . chemical synthesis of peptides increases the cost almost exponentially as the length of the peptide increases . if the separation and characterization methods for specific small peptides from inexpensive biological sources are standardized , these peptides can be easily isolated and supplied on demand for research as well as for commercial purposes .
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 .