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

Sequence Determination of a Novel Tripeptide Isolated from the Young Leaves of Azadirachta indica... Hindawi Publishing Corporation International Journal of Peptides Volume 2013, Article ID 629549, 6 pages http://dx.doi.org/10.1155/2013/629549 Research Article Sequence Determination of a Novel Tripeptide Isolated from the Young Leaves of Azadirachta indica A. Juss M. Rajeswari Prabha and B. Ramachandramurty Department of Biochemistry, PSG College of Arts & Science, Civil Aerodrome Post, Coimbatore, Tamil Nadu 641014, India Correspondence should be addressed to M. Rajeswari Prabha; prabha11.bio@gmail.com Received 10 October 2012; Revised 16 December 2012; Accepted 12 January 2013 Academic Editor: Tzi Bun Ng Copyright © 2013 M. Rajeswari Prabha and B. Ramachandramurty. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 aer ft purification. 1. Introduction has revealed the occurrence of various compounds such as terpenoids, and flavonoids [ 7, 8]. But the presence of small Small alpha peptides are the most expensive substances, alpha peptides has not been reported so far. andmostofthemare noteasilyavailable commercially [1]. Ninhydrin reactions using manual and automated tech- Pharmacological studies have proved that many peptides, niques as well as ninhydrin spray reagents are widely used to including those isolated from plants, have a potential antitu- analyze and characterize amino acids, peptides, and proteins, mor eeff ct [ 2]. eTh se peptides have anumberofadvantages as well as numerous other ninhydrin-positive compounds in over other chemical agents including their low molecular biomedical, clinical, food, forensic, histochemical, microbi- weight,relativelysimplestructure,lower antigenicity,fewer ological, nutritional, and plant studies [9–11]. Many of the adverse actions, easy absorption, and a variety of routes shortcomings of ninhydrin have been met by the synthesis of administration [3]. Many antibacterial peptide families of a variety of ninhydrin analogs. All amino acids and their have been isolated from plants. Pp-thionin, for example, carboxyl group derivatives like esters and amides, including showed activity against Rhizobium meliloti, Xanthomonas small peptides, produce a purple color with the classical campestris, Micrococcus luteus.Circulins A-Band cyclopsy- ninhydrin reagent. This reagent was modified by us by adding chotride A from the cyclotides family showed antibacterial cupric ion in order to distinguish qualitatively the carboxyl eeff cts against human pathogens such as Staphylococcus group derivatives of amino acids from the amino acids on aureus, Micrococcus luteus, Escherichia coli, Pseudomonas paperaeft rchromatography[ 12]. Amino acids produce a pink aeruginosa, Proteus vulgaris, and Klebsiella oxytoca at micro- color, and their carboxyl derivatives like esters and amides, molar concentrations [4]. Various plant extracts are reported including small peptides, produce a yellow color with Cu(II)- to exhibit high antifungal activity due to proteins or peptides ninhydrin reagent. eTh Cu(II)-ninhydrin method discussed [5]. Cardiovascular activity of milk casein-derived tripeptides here is a novel one because no other methods presently has also been reported, where bioactive tripeptide-containing used can form two dieff rent coloured products with a single milk products attenuated the blood pressure development in developing reagent. We have used this method for the spontaneously hypertensive rats [6]. Research on A. indica detection and purification of amino acid derivatives from 2 International Journal of Peptides different plant products [ 13, 14]. In this paper, we report the 3.284 isolation and sequence determination of a small alpha peptide from the young leaves of A. indica. 2. Materials and Methods Ninhydrin was acquired from Pierce (Rockford, IL, USA). Cupric nitrate was of BDH, analytical grade (Mumbai, India). Organic solvents and acids used were of the highest purity available. Whatman No. 1 filter paper discs were obtained from Whatman International Ltd, Maidstone, England. Polyvinylpyrrolidone was purchased from Loba Chemie Pvt. −0.309 200 400 600 800 Ltd., Mumbai. (nm) 2.1. Preparation of the Crude Extract. The young leaves of Figure 1: UV-Vis spectrum of the small alpha peptide purified from A. indica were homogenized (1 g/10 mL) with warm 80% A. indica. ethanol. The extract was filtered through a Whatmann No. 1 filter paper. 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 4 C to remove the phenolic compounds. The clear supernatant obtained was used as the crude source of the small alpha peptide. 2.2. Preparation of Cu(II)-Ninhydrin Reagent. The Cu(II)- ninhydrin reagent was prepared by dissolving cupric nitrate (1/cm) (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 Figure 2: FT-IR spectrum of the small alpha peptide purified from diluted with required amount of acetone. A. indica. 2.3. Circular Paper Chromatography. The crude extract was spottedinthe center of acircularWhatman No.1filterpaper cut into pieces and eluted in 80% ethanol. eTh compound, on the arc of a small circle drawn with a pencil. Depending thus obtained, was used for conducting the spectral studies. upon thenumberofsamples to be analyzed,the papermay be demarcated. The diameter of the samples spotted was 2.5. Colorimetric Determination of the Concentration of restricted to 0.5 cm by intermittent use of a hot air dryer. Cu(II)-Ninhydrin-Positive Compounds. 1mL of the puriefi d The sample spotting may be repeated 15 to 20 times to ensure Cu(II)-ninhydrin-positive compound was added with 1 mL sufficient concentration of the component to be detected. The of Cu(II)-ninhydrin reagent, and the mixture was incubated chromatography wascarried outinanisopropanol:water at 40 C for 5 min. eTh yellow color produced was read at (4 : 1, v/v) solvent system by connecting a filter paper wick to 420 nm. The amount of these compounds was determined by the solvent through a hole made at the center of the circular using a standard graph constructed with L-glycyl glycine as paper. After the run which required approximately 20– the standard. 40 min, the chromatogram was dried at ambient temperature for 30 min. eTh air-dried chromatogram was developed by 2.6. UV-Vis Spectrophotometry. The purified compound was spraying uniformly with Cu(II)-ninhydrin reagent followed ∘ scanned for its absorption properties, from 200 nm to 900 nm by drying at 60 Cfor 10 min. in a Shimadzu, UV-Vis spectrophotometer. 2.4. Purification of the Alpha Peptide. To subject the com- 2.7. FT-IR Spectrometry. The purified compound was also pound for various spectral studies, a simple and inexpensive subjected to FT-IR analysis using a Shimadzu model FTIR- purification procedure was followed. Two circular chro- 8300 infrared spectrometer. IR spectra were scanned between matograms of these compounds were run simultaneously 500 and 4,000 wave numbers (per centimeter). using Whatman No. 1 filter paper discs (12 cm). One was developed with the Cu(II)-ninhydrin reagent. The corre- sponding region of the paper on the other chromatogram 2.8. GC-MS Analysis. The purified Cu(II)-ninhydrin- containing the Cu(II)-ninhydrin-positive compounds was positive compound was analyzed using the GC/MS Abs. (%) 400 International Journal of Peptides 3 RT: 6.05–39.25 NL: 1.237𝐸 Ala TIC MS Asp Gly Ser Leu Glu Cys Pro Val 8.43 11.5 His 12.54 21.38 18.3 24.77 Phe Lys 15.61 10.58 35.57 8.97 39.01 Om 10.75 Thr 32.66 Ile 35.84 21.46 Tyr 35.52 37.01 25.14 10.15 27.63 12.71 35.35 24.39 20 34.56 17.8 18.61 25.74 6.27 29.07 20.3 23.84 13.62 22.57 32.44 30.28 7.9 14.87 16.99 10.84 8 101214161820222426283032343638 Time (min) Figure 3: Gas chromatography retention spectra of the standard amino acids. instrument: Trace Ultra version 5.0 produced by er Th mo. in 0.1% trifluoroacetic acid bueff r and spotted with CHCA The separation conditions were as follows: DB-5 Column (alpha-cyanohydroxycinnamic acid) matrix onto MALDI 30 m× 0.25 mm× 0.25𝜇 m, mobile phaseheliumatflow rate plateand allowedtodry.Amodel4800PlusMALDI 1.0 mL/min, injection chamber temperature 220 C, and oven TOF/TOF analyzer (Applied Biosystems Inc., Foster City, CA, ∘ ∘ ∘ temperature starts at 80 Craisedto250 Catarate of 8 C USA) was used for direct profiling and MS/MS fragmentation per minute. The ionization mode of the mass detector was at study. Acquisitions were performed in positive ion reflectron 70 eV. mode. MS spectra were accumulated in mass range 400– 4000 m/z.Spectra areobtainedfor themajor peptideions in MS mode, and sequence data are obtained when the 2.9. NMR Studies. The purified Cu(II)-ninhydrin-positive spectrometers automatically revert to MS/MS mode. MS/MS compound in deuterated acetone as a solvent was subjected 1 13 was achieved by 2 kV collision-induced dissociation (CID) in to Hand C NMR analysis using a Bruker 500 MHz liquid- positive ion mode. De novo sequencing analysis was used to state NMR spectrometer. determine the primary sequence structure for peptides that are not present in currently available databases. 2.10. Acid Hydrolysis of the Purified Compound and Separation by Paper Chromatography. For this experiment, the same sample purified using circular paper chromatography tech- 3. Results and Discussion nique was employed. 0.5 mL of this sample aliquot was mixed with 0.5 mL of concentrated HCl in a clean dry test tube (the The plant extract was run on circular paper chromatog- final concentration of HCl is 6 N). The tube was subsequently raphy using isopropanol : water (4 : 1) solvent system and sealed with the help of a glass blower. This was placed in an developed with Cu(II)-ninhydrin reagent. The production incubator at 110 C for 24 hours after which the sample was of yellow chromaphore indicated the presence of Cu(II)- reconstituted to 0.5 mL with distilled water. eTh hydrolyzed ninhydrin-positive compound. eTh detected compound was sample was spotted on circular Whatman No. 1 filter paper purified using inexpensive paper chromatographic method, and developed with the isopropanol : water (4 : 1 v/v) system. as described. 2𝜇 g concentration of the peptide was obtained The chromatograms were uniformly sprayed with ninhydrin from 1 g of the leaf extract. eTh peptide nature of the purified reagent and were air dried and heated at 65 Cfor 10min. compound from the young leaves of A. indica was confirmed by UV spectrophotometer. Puriefi d alpha peptide from the 2.11. De Novo Sequencing Using MS/MS. Peptide sample young leaves of A. indica showed maximum absorption (purified Cu(II)-ninhydrin-positive compound) was evap- at 210 nm (Figure 1) confirming the peptide nature [ 15]. orated to dryness at room temperature and resuspended The peptide nature was further confirmed by identifying Relative abundance 4 International Journal of Peptides functional groups from the FT-IR studies. Figure 2 shows the NL: RT: 0–38.95 SM: 11G 1.92𝐸6 FT-IR spectrum of the puriefi d compound from the young 100 −1 TIC MS leaves of A. indica. eTh re were sharp peaks at 1600 cm and −1 90 EM-385 3380 cm indicating thepresenceofC=O andNHgroups 14.62 respectively, in the compound, confirming the peptide nature [14]. The Cu(II)-ninhydrin-positive compound purified from 70 21.5 the young leaves of A. indica was analyzed by GC/MS. eTh retention times of the compound were compared with those 11.5 of reference standard amino acids under the same conditions. 28.89 The identification of the amino acids in the sample was 8.5 based on direct comparison of the retention times and mass spectral data with those for standard compounds, and 34.72 by computer matching with the Wiley 229, Nist 107, and 21 Library, as well as by comparison of the fragmentation patterns of themassspectra with thosereportedinthe literature [16]. Figure 3 shows the GC-MS spectra of the standard amino acids. Figure 4 shows the GC-MS spectra of the Cu(II)-ninhydrin-positive compound purified from 0 the young leaves of A. indica. From the data obtained, it 0 5 10 15 20 25 30 35 canbeinferredthatthe peptideisolatedfromthe young Time (min) leaves of A. indica might contain alanine, cysteine, and phenyl Figure 4: Gas chromatography retention spectra of the the Cu(II)- alanine. ninhydrin-positive compound purified from A. indica. NMR studies indicated the presence of –CH ,–CH , 3 2 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 iso- lated compound was hydrolyzed with 6 N HCl. eTh chro- matograms 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. eTh 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 Figure 5: The purified small alpha peptide from A. indica was proteomics [17]. In spite of the continuously growing subjected to acid hydrolysis, spotted on a circular Whattman paper sequence databases, de novo sequencing of peptides, that is, and developed using isopropanol : water (4 : 1, v/v) system and sprayed with ninhydrin reagent. sequencing without assistance of a linear sequence database, is still essential in several analytical situations. Figure 6 represents the MS/MS spectra of the peptide isolated from the young leaves of A. indica.Table 1 represents the amino acid sequence of the peptide isolated from A. indica.The 321.4 sequence of amino acids was determined by the application of tandem MS. By referring to the mass unit of the respective 218.6 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). eTh peptide isolated from A. indica is a tripeptide, composed of three amino acid residues with alanine at the N-terminus and cysteine at the 100 200 300 400 500 600 700 800 900 C-terminus. The determined sequence data was submitted in 𝑚/𝑧 UniProt Knowledgebase database, and accession number was assigned for the sequence. eTh sequence data of the peptide Figure 6: MS/MS spectra of peptide isolated from A. indica. Relative abundance (%) Relative abundance International Journal of Peptides 5 Table 1: Sequence data summary of the peptide isolated from the Annona cherimola,” JournalofNatural Products,vol.67, no.9, young leaves of Azadirachta indica. pp. 1577–1579, 2004. [3] N. H. Tan and J. Zhou, “Plant cyclopeptides,” Chemical Reviews, Protein mass: 321.5 Da vol. 106, pp. 840–895, 2006. Fragment ion calculator results [4] P.Barbosa Pelegrini,R.P.Del Sarto, O. N. Silva, O. L. Franco, and M. F. Grossi-De-Sa, “Antibacterial peptides from plants: Sequence: N-Ala-Phe-Cys-C what they are and how they probably work,” Biochemistry Fragment ion table, monoisotopic masses Research International,vol.2011, ArticleID250349, 9pages, Seq No. 𝐵 A1 71.0203 [5] A. Jamil, M. Shahid, M. Masud-Ul-Haq Khan, and M. Ashraf, “Screening of some medicinal plants for isolation of antifungal F 2 218.6125 proteins and peptides,” Pakistan Journal of Botany,vol.39, no.1, C3 321.4521 pp. 211–221, 2007. [6] P. Jakala, E. Pere, R. Lehtinen, A. Turpeinen, R. Korpela, and H. Vapaatalo, “Cardiovascular activity of milk casein-derived tripeptides and plant sterols in spontaneously hypertensive isolated from the young leaves of A. indica,reportedinthis rats,” Journal of Physiology and Pharmacology,vol.60, no.4,pp. paper, will appear in the UniProt Knowledgebase under the 11–20, 2009. accession number B3EWR2. [7] A. Bose, K. Chakraborty, K. Sarkar et al., “Neem leaf glyco- The assessment of the biological role and applications protein directs T-bet-associated type 1 immune commitment,” of the purified tripeptide is under study. Compounds with Human Immunology,vol.70, no.1,pp. 6–15,2009. low molecular weight of 500 or less can function as efficient [8] M. K. Roy, M. Kobori, M. Takenaka et al., “Antiproliferative drug molecules [18]. Small peptides containing multifunc- effect on human cancer cell lines aeft r treatment with nimbolide tional amino acids like L-glutamic acid, L-aspartic acid, L- extracted from an edible part of the neem tree (Azadirachta lysine, L-histidine, L-cysteine, and L-serine can function as indica),” Phytotherapy Research,vol.21, no.3,pp. 245–250, 2007. potent chelating agents that can be employed in chelation [9] M. Friedman, J. Pang, and G. A. Smith, “Ninhydrin-reactive therapy [19]. Noveldrugs canalsobesynthesised by chemical lysine in food proteins,” Journal of Food Science,vol.49, pp.10– modification of these peptides. 13, 1984. [10] K. N. Pearce, D. Karahalios, and M. Friedman, “Ninhydrin assay 4. Conclusion forproteolysisinripeningcheese,” Journal of Food Science,vol. 53, pp. 432–438, 1988. From the overall results obtained from this work, it can be [11] M. Friedman, “Applications of the ninhydrin reaction for inferredthattheCu(II)-inhydrinpositivecompoundpuriefi d analysis of amino acids, peptides, and proteins to agricultural from the young leaves of A. indica is a tripeptide. Unlike and biomedical sciences,” Journal of Agricultural and Food amino acids, small peptides are highly expensive, and most Chemistry,vol.52, no.3,pp. 385–406, 2004. of them are not easily available commercially. Chemical [12] V. Ganapathy, B. Ramachandramurty, and A. N. Radhakrish- synthesis of peptides increases the cost almost exponentially nan, “Distinctive test with copper(II)-ninhydrin reagent for as the length of the peptide increases. If the separation small𝛼 -peptides separated by paper chromatography,” Journal of Chromatography,vol.213,no. 2, pp.307–316,1981. and characterization methods for specific small peptides from inexpensive biological sources are standardized, these [13] K. S. Nithya and B. Ramachandramurty, “Screening of some peptides can be easily isolated and supplied on demand selected spices with medicinal value for Cu (II)-ninhydrin pos- itive compounds,” International Journal of Biological Chemistry, for research as well as for commercial purposes. eTh small vol. 1, pp.62–68,2007. peptides may serve several purposes in the near future. [14] B. Ramachandramurty and V. N. Satakopan, “Isolation and partial characterization of a small alpha peptide from Cuminum Conflict of Interests cyminum L. seeds as detected by Cu(II)—ninhydrin reagent,” International Journal of Chemical Sciences,vol.7,no.4,pp.2872– eTh authors have only used the products of the commercial 2882, 2009. identities referred to in this paper. There is no secondary [15] A. R. Goldfarb, L. J. Saidel, and E. Mosovich, “eTh ultraviolet interest, conflict of interests or n fi ancial gain in this paper. absorption spectra of proteins,” eTh JournalofBiologicalChem- eTh ydonot have anysecondary rights.They arealsothe sole istry,vol.193,no. 1, pp.397–404,1951. authors of the paper. [16] M. Culea, “Amino acids quantitation in biological media,” Studia Universitatis Babesbolyai,vol.4,pp. 11–15, 2005. [17] A. M. Frank, M. M. Savitski, M. L. Nielsen, R. A. Zubarev, References and P. A. Pevzner, “De novo peptide sequencing and identifi- [1] B. Ramachandramurty, M. Rajeswari Prabha, and K. C. Raja, “A cation with precision mass spectrometry,” Journal of Proteome simple method for the production and detection of small alpha Research,vol.6,no. 1, pp.114–123,2007. peptides from pulses,” IUP Journal of Life Sciences,vol.4,pp. [18] A.R.Fersht,J.P.Shi,J.Knoll-Jones,D.M.Lowe,A.J.Wilkinson, 50–55, 2010. and D. M. Blow, “Hydrogen bonding and biological specificity ´ ´ [2] A. Wele, Y. Zhang, I. Ndoye, J. P. Brouard, J. L. Pousset, and analysed by protein engineering,” Nature,vol.314,pp. 235–238, B. Bodo, “A cytotoxic cyclic heptapeptide from the seeds of 1985. 6 International Journal of Peptides [19] T. Storr, M. Markel, G. X. Song-Zhao et al., “Synthesis, char- acterisation and metal coordinating ability to multifunctional carbohydrate containing compounds for Alzheimer’s therapy,” Journal of the American Chemical Society,vol.129,no. 23,pp. 4753–7463, 2007. 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Sequence Determination of a Novel Tripeptide Isolated from the Young Leaves of Azadirachta indica A. Juss

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Copyright © 2013 M. Rajeswari Prabha and B. Ramachandramurty. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Publishing Corporation International Journal of Peptides Volume 2013, Article ID 629549, 6 pages http://dx.doi.org/10.1155/2013/629549 Research Article Sequence Determination of a Novel Tripeptide Isolated from the Young Leaves of Azadirachta indica A. Juss M. Rajeswari Prabha and B. Ramachandramurty Department of Biochemistry, PSG College of Arts & Science, Civil Aerodrome Post, Coimbatore, Tamil Nadu 641014, India Correspondence should be addressed to M. Rajeswari Prabha; prabha11.bio@gmail.com Received 10 October 2012; Revised 16 December 2012; Accepted 12 January 2013 Academic Editor: Tzi Bun Ng Copyright © 2013 M. Rajeswari Prabha and B. Ramachandramurty. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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 aer ft purification. 1. Introduction has revealed the occurrence of various compounds such as terpenoids, and flavonoids [ 7, 8]. But the presence of small Small alpha peptides are the most expensive substances, alpha peptides has not been reported so far. andmostofthemare noteasilyavailable commercially [1]. Ninhydrin reactions using manual and automated tech- Pharmacological studies have proved that many peptides, niques as well as ninhydrin spray reagents are widely used to including those isolated from plants, have a potential antitu- analyze and characterize amino acids, peptides, and proteins, mor eeff ct [ 2]. eTh se peptides have anumberofadvantages as well as numerous other ninhydrin-positive compounds in over other chemical agents including their low molecular biomedical, clinical, food, forensic, histochemical, microbi- weight,relativelysimplestructure,lower antigenicity,fewer ological, nutritional, and plant studies [9–11]. Many of the adverse actions, easy absorption, and a variety of routes shortcomings of ninhydrin have been met by the synthesis of administration [3]. Many antibacterial peptide families of a variety of ninhydrin analogs. All amino acids and their have been isolated from plants. Pp-thionin, for example, carboxyl group derivatives like esters and amides, including showed activity against Rhizobium meliloti, Xanthomonas small peptides, produce a purple color with the classical campestris, Micrococcus luteus.Circulins A-Band cyclopsy- ninhydrin reagent. This reagent was modified by us by adding chotride A from the cyclotides family showed antibacterial cupric ion in order to distinguish qualitatively the carboxyl eeff cts against human pathogens such as Staphylococcus group derivatives of amino acids from the amino acids on aureus, Micrococcus luteus, Escherichia coli, Pseudomonas paperaeft rchromatography[ 12]. Amino acids produce a pink aeruginosa, Proteus vulgaris, and Klebsiella oxytoca at micro- color, and their carboxyl derivatives like esters and amides, molar concentrations [4]. Various plant extracts are reported including small peptides, produce a yellow color with Cu(II)- to exhibit high antifungal activity due to proteins or peptides ninhydrin reagent. eTh Cu(II)-ninhydrin method discussed [5]. Cardiovascular activity of milk casein-derived tripeptides here is a novel one because no other methods presently has also been reported, where bioactive tripeptide-containing used can form two dieff rent coloured products with a single milk products attenuated the blood pressure development in developing reagent. We have used this method for the spontaneously hypertensive rats [6]. Research on A. indica detection and purification of amino acid derivatives from 2 International Journal of Peptides different plant products [ 13, 14]. In this paper, we report the 3.284 isolation and sequence determination of a small alpha peptide from the young leaves of A. indica. 2. Materials and Methods Ninhydrin was acquired from Pierce (Rockford, IL, USA). Cupric nitrate was of BDH, analytical grade (Mumbai, India). Organic solvents and acids used were of the highest purity available. Whatman No. 1 filter paper discs were obtained from Whatman International Ltd, Maidstone, England. Polyvinylpyrrolidone was purchased from Loba Chemie Pvt. −0.309 200 400 600 800 Ltd., Mumbai. (nm) 2.1. Preparation of the Crude Extract. The young leaves of Figure 1: UV-Vis spectrum of the small alpha peptide purified from A. indica were homogenized (1 g/10 mL) with warm 80% A. indica. ethanol. The extract was filtered through a Whatmann No. 1 filter paper. 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 4 C to remove the phenolic compounds. The clear supernatant obtained was used as the crude source of the small alpha peptide. 2.2. Preparation of Cu(II)-Ninhydrin Reagent. The Cu(II)- ninhydrin reagent was prepared by dissolving cupric nitrate (1/cm) (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 Figure 2: FT-IR spectrum of the small alpha peptide purified from diluted with required amount of acetone. A. indica. 2.3. Circular Paper Chromatography. The crude extract was spottedinthe center of acircularWhatman No.1filterpaper cut into pieces and eluted in 80% ethanol. eTh compound, on the arc of a small circle drawn with a pencil. Depending thus obtained, was used for conducting the spectral studies. upon thenumberofsamples to be analyzed,the papermay be demarcated. The diameter of the samples spotted was 2.5. Colorimetric Determination of the Concentration of restricted to 0.5 cm by intermittent use of a hot air dryer. Cu(II)-Ninhydrin-Positive Compounds. 1mL of the puriefi d The sample spotting may be repeated 15 to 20 times to ensure Cu(II)-ninhydrin-positive compound was added with 1 mL sufficient concentration of the component to be detected. The of Cu(II)-ninhydrin reagent, and the mixture was incubated chromatography wascarried outinanisopropanol:water at 40 C for 5 min. eTh yellow color produced was read at (4 : 1, v/v) solvent system by connecting a filter paper wick to 420 nm. The amount of these compounds was determined by the solvent through a hole made at the center of the circular using a standard graph constructed with L-glycyl glycine as paper. After the run which required approximately 20– the standard. 40 min, the chromatogram was dried at ambient temperature for 30 min. eTh air-dried chromatogram was developed by 2.6. UV-Vis Spectrophotometry. The purified compound was spraying uniformly with Cu(II)-ninhydrin reagent followed ∘ scanned for its absorption properties, from 200 nm to 900 nm by drying at 60 Cfor 10 min. in a Shimadzu, UV-Vis spectrophotometer. 2.4. Purification of the Alpha Peptide. To subject the com- 2.7. FT-IR Spectrometry. The purified compound was also pound for various spectral studies, a simple and inexpensive subjected to FT-IR analysis using a Shimadzu model FTIR- purification procedure was followed. Two circular chro- 8300 infrared spectrometer. IR spectra were scanned between matograms of these compounds were run simultaneously 500 and 4,000 wave numbers (per centimeter). using Whatman No. 1 filter paper discs (12 cm). One was developed with the Cu(II)-ninhydrin reagent. The corre- sponding region of the paper on the other chromatogram 2.8. GC-MS Analysis. The purified Cu(II)-ninhydrin- containing the Cu(II)-ninhydrin-positive compounds was positive compound was analyzed using the GC/MS Abs. (%) 400 International Journal of Peptides 3 RT: 6.05–39.25 NL: 1.237𝐸 Ala TIC MS Asp Gly Ser Leu Glu Cys Pro Val 8.43 11.5 His 12.54 21.38 18.3 24.77 Phe Lys 15.61 10.58 35.57 8.97 39.01 Om 10.75 Thr 32.66 Ile 35.84 21.46 Tyr 35.52 37.01 25.14 10.15 27.63 12.71 35.35 24.39 20 34.56 17.8 18.61 25.74 6.27 29.07 20.3 23.84 13.62 22.57 32.44 30.28 7.9 14.87 16.99 10.84 8 101214161820222426283032343638 Time (min) Figure 3: Gas chromatography retention spectra of the standard amino acids. instrument: Trace Ultra version 5.0 produced by er Th mo. in 0.1% trifluoroacetic acid bueff r and spotted with CHCA The separation conditions were as follows: DB-5 Column (alpha-cyanohydroxycinnamic acid) matrix onto MALDI 30 m× 0.25 mm× 0.25𝜇 m, mobile phaseheliumatflow rate plateand allowedtodry.Amodel4800PlusMALDI 1.0 mL/min, injection chamber temperature 220 C, and oven TOF/TOF analyzer (Applied Biosystems Inc., Foster City, CA, ∘ ∘ ∘ temperature starts at 80 Craisedto250 Catarate of 8 C USA) was used for direct profiling and MS/MS fragmentation per minute. The ionization mode of the mass detector was at study. Acquisitions were performed in positive ion reflectron 70 eV. mode. MS spectra were accumulated in mass range 400– 4000 m/z.Spectra areobtainedfor themajor peptideions in MS mode, and sequence data are obtained when the 2.9. NMR Studies. The purified Cu(II)-ninhydrin-positive spectrometers automatically revert to MS/MS mode. MS/MS compound in deuterated acetone as a solvent was subjected 1 13 was achieved by 2 kV collision-induced dissociation (CID) in to Hand C NMR analysis using a Bruker 500 MHz liquid- positive ion mode. De novo sequencing analysis was used to state NMR spectrometer. determine the primary sequence structure for peptides that are not present in currently available databases. 2.10. Acid Hydrolysis of the Purified Compound and Separation by Paper Chromatography. For this experiment, the same sample purified using circular paper chromatography tech- 3. Results and Discussion nique was employed. 0.5 mL of this sample aliquot was mixed with 0.5 mL of concentrated HCl in a clean dry test tube (the The plant extract was run on circular paper chromatog- final concentration of HCl is 6 N). The tube was subsequently raphy using isopropanol : water (4 : 1) solvent system and sealed with the help of a glass blower. This was placed in an developed with Cu(II)-ninhydrin reagent. The production incubator at 110 C for 24 hours after which the sample was of yellow chromaphore indicated the presence of Cu(II)- reconstituted to 0.5 mL with distilled water. eTh hydrolyzed ninhydrin-positive compound. eTh detected compound was sample was spotted on circular Whatman No. 1 filter paper purified using inexpensive paper chromatographic method, and developed with the isopropanol : water (4 : 1 v/v) system. as described. 2𝜇 g concentration of the peptide was obtained The chromatograms were uniformly sprayed with ninhydrin from 1 g of the leaf extract. eTh peptide nature of the purified reagent and were air dried and heated at 65 Cfor 10min. compound from the young leaves of A. indica was confirmed by UV spectrophotometer. Puriefi d alpha peptide from the 2.11. De Novo Sequencing Using MS/MS. Peptide sample young leaves of A. indica showed maximum absorption (purified Cu(II)-ninhydrin-positive compound) was evap- at 210 nm (Figure 1) confirming the peptide nature [ 15]. orated to dryness at room temperature and resuspended The peptide nature was further confirmed by identifying Relative abundance 4 International Journal of Peptides functional groups from the FT-IR studies. Figure 2 shows the NL: RT: 0–38.95 SM: 11G 1.92𝐸6 FT-IR spectrum of the puriefi d compound from the young 100 −1 TIC MS leaves of A. indica. eTh re were sharp peaks at 1600 cm and −1 90 EM-385 3380 cm indicating thepresenceofC=O andNHgroups 14.62 respectively, in the compound, confirming the peptide nature [14]. The Cu(II)-ninhydrin-positive compound purified from 70 21.5 the young leaves of A. indica was analyzed by GC/MS. eTh retention times of the compound were compared with those 11.5 of reference standard amino acids under the same conditions. 28.89 The identification of the amino acids in the sample was 8.5 based on direct comparison of the retention times and mass spectral data with those for standard compounds, and 34.72 by computer matching with the Wiley 229, Nist 107, and 21 Library, as well as by comparison of the fragmentation patterns of themassspectra with thosereportedinthe literature [16]. Figure 3 shows the GC-MS spectra of the standard amino acids. Figure 4 shows the GC-MS spectra of the Cu(II)-ninhydrin-positive compound purified from 0 the young leaves of A. indica. From the data obtained, it 0 5 10 15 20 25 30 35 canbeinferredthatthe peptideisolatedfromthe young Time (min) leaves of A. indica might contain alanine, cysteine, and phenyl Figure 4: Gas chromatography retention spectra of the the Cu(II)- alanine. ninhydrin-positive compound purified from A. indica. NMR studies indicated the presence of –CH ,–CH , 3 2 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 iso- lated compound was hydrolyzed with 6 N HCl. eTh chro- matograms 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. eTh 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 Figure 5: The purified small alpha peptide from A. indica was proteomics [17]. In spite of the continuously growing subjected to acid hydrolysis, spotted on a circular Whattman paper sequence databases, de novo sequencing of peptides, that is, and developed using isopropanol : water (4 : 1, v/v) system and sprayed with ninhydrin reagent. sequencing without assistance of a linear sequence database, is still essential in several analytical situations. Figure 6 represents the MS/MS spectra of the peptide isolated from the young leaves of A. indica.Table 1 represents the amino acid sequence of the peptide isolated from A. indica.The 321.4 sequence of amino acids was determined by the application of tandem MS. By referring to the mass unit of the respective 218.6 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). eTh peptide isolated from A. indica is a tripeptide, composed of three amino acid residues with alanine at the N-terminus and cysteine at the 100 200 300 400 500 600 700 800 900 C-terminus. The determined sequence data was submitted in 𝑚/𝑧 UniProt Knowledgebase database, and accession number was assigned for the sequence. eTh sequence data of the peptide Figure 6: MS/MS spectra of peptide isolated from A. indica. Relative abundance (%) Relative abundance International Journal of Peptides 5 Table 1: Sequence data summary of the peptide isolated from the Annona cherimola,” JournalofNatural Products,vol.67, no.9, young leaves of Azadirachta indica. pp. 1577–1579, 2004. [3] N. H. Tan and J. Zhou, “Plant cyclopeptides,” Chemical Reviews, Protein mass: 321.5 Da vol. 106, pp. 840–895, 2006. Fragment ion calculator results [4] P.Barbosa Pelegrini,R.P.Del Sarto, O. N. Silva, O. L. Franco, and M. F. Grossi-De-Sa, “Antibacterial peptides from plants: Sequence: N-Ala-Phe-Cys-C what they are and how they probably work,” Biochemistry Fragment ion table, monoisotopic masses Research International,vol.2011, ArticleID250349, 9pages, Seq No. 𝐵 A1 71.0203 [5] A. Jamil, M. Shahid, M. Masud-Ul-Haq Khan, and M. Ashraf, “Screening of some medicinal plants for isolation of antifungal F 2 218.6125 proteins and peptides,” Pakistan Journal of Botany,vol.39, no.1, C3 321.4521 pp. 211–221, 2007. [6] P. Jakala, E. Pere, R. Lehtinen, A. Turpeinen, R. Korpela, and H. Vapaatalo, “Cardiovascular activity of milk casein-derived tripeptides and plant sterols in spontaneously hypertensive isolated from the young leaves of A. indica,reportedinthis rats,” Journal of Physiology and Pharmacology,vol.60, no.4,pp. paper, will appear in the UniProt Knowledgebase under the 11–20, 2009. accession number B3EWR2. [7] A. Bose, K. Chakraborty, K. Sarkar et al., “Neem leaf glyco- The assessment of the biological role and applications protein directs T-bet-associated type 1 immune commitment,” of the purified tripeptide is under study. Compounds with Human Immunology,vol.70, no.1,pp. 6–15,2009. low molecular weight of 500 or less can function as efficient [8] M. K. Roy, M. Kobori, M. Takenaka et al., “Antiproliferative drug molecules [18]. Small peptides containing multifunc- effect on human cancer cell lines aeft r treatment with nimbolide tional amino acids like L-glutamic acid, L-aspartic acid, L- extracted from an edible part of the neem tree (Azadirachta lysine, L-histidine, L-cysteine, and L-serine can function as indica),” Phytotherapy Research,vol.21, no.3,pp. 245–250, 2007. potent chelating agents that can be employed in chelation [9] M. Friedman, J. Pang, and G. A. Smith, “Ninhydrin-reactive therapy [19]. Noveldrugs canalsobesynthesised by chemical lysine in food proteins,” Journal of Food Science,vol.49, pp.10– modification of these peptides. 13, 1984. [10] K. N. Pearce, D. Karahalios, and M. Friedman, “Ninhydrin assay 4. Conclusion forproteolysisinripeningcheese,” Journal of Food Science,vol. 53, pp. 432–438, 1988. From the overall results obtained from this work, it can be [11] M. Friedman, “Applications of the ninhydrin reaction for inferredthattheCu(II)-inhydrinpositivecompoundpuriefi d analysis of amino acids, peptides, and proteins to agricultural from the young leaves of A. indica is a tripeptide. Unlike and biomedical sciences,” Journal of Agricultural and Food amino acids, small peptides are highly expensive, and most Chemistry,vol.52, no.3,pp. 385–406, 2004. of them are not easily available commercially. Chemical [12] V. Ganapathy, B. Ramachandramurty, and A. N. Radhakrish- synthesis of peptides increases the cost almost exponentially nan, “Distinctive test with copper(II)-ninhydrin reagent for as the length of the peptide increases. If the separation small𝛼 -peptides separated by paper chromatography,” Journal of Chromatography,vol.213,no. 2, pp.307–316,1981. and characterization methods for specific small peptides from inexpensive biological sources are standardized, these [13] K. S. Nithya and B. Ramachandramurty, “Screening of some peptides can be easily isolated and supplied on demand selected spices with medicinal value for Cu (II)-ninhydrin pos- itive compounds,” International Journal of Biological Chemistry, for research as well as for commercial purposes. eTh small vol. 1, pp.62–68,2007. peptides may serve several purposes in the near future. [14] B. Ramachandramurty and V. N. Satakopan, “Isolation and partial characterization of a small alpha peptide from Cuminum Conflict of Interests cyminum L. seeds as detected by Cu(II)—ninhydrin reagent,” International Journal of Chemical Sciences,vol.7,no.4,pp.2872– eTh authors have only used the products of the commercial 2882, 2009. identities referred to in this paper. There is no secondary [15] A. R. Goldfarb, L. J. Saidel, and E. Mosovich, “eTh ultraviolet interest, conflict of interests or n fi ancial gain in this paper. absorption spectra of proteins,” eTh JournalofBiologicalChem- eTh ydonot have anysecondary rights.They arealsothe sole istry,vol.193,no. 1, pp.397–404,1951. authors of the paper. [16] M. Culea, “Amino acids quantitation in biological media,” Studia Universitatis Babesbolyai,vol.4,pp. 11–15, 2005. [17] A. M. Frank, M. M. Savitski, M. L. Nielsen, R. A. Zubarev, References and P. A. Pevzner, “De novo peptide sequencing and identifi- [1] B. Ramachandramurty, M. Rajeswari Prabha, and K. C. Raja, “A cation with precision mass spectrometry,” Journal of Proteome simple method for the production and detection of small alpha Research,vol.6,no. 1, pp.114–123,2007. peptides from pulses,” IUP Journal of Life Sciences,vol.4,pp. [18] A.R.Fersht,J.P.Shi,J.Knoll-Jones,D.M.Lowe,A.J.Wilkinson, 50–55, 2010. and D. M. Blow, “Hydrogen bonding and biological specificity ´ ´ [2] A. Wele, Y. Zhang, I. Ndoye, J. P. Brouard, J. L. Pousset, and analysed by protein engineering,” Nature,vol.314,pp. 235–238, B. Bodo, “A cytotoxic cyclic heptapeptide from the seeds of 1985. 6 International Journal of Peptides [19] T. Storr, M. Markel, G. X. Song-Zhao et al., “Synthesis, char- acterisation and metal coordinating ability to multifunctional carbohydrate containing compounds for Alzheimer’s therapy,” Journal of the American Chemical Society,vol.129,no. 23,pp. 4753–7463, 2007. 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