METHOD DEVELOPMENT FOR SEPARATION AND ANALYSIS OF PR-2 ANTIFUNGAL PROTEIN FROM PUMPKIN RINDS USING REVERSE PHASE CHROMATOGRAPHY

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1 Research Paper METHOD DEVELOPMENT FOR SEPARATION AND ANALYSIS OF PR-2 ANTIFUNGAL PROTEIN FROM PUMPKIN RINDS USING REVERSE PHASE CHROMATOGRAPHY Mr Shrikant R. Kulkarni Author for correspondence Department of Chemical Engineering, Vishwakarma Institute of Technology, Pune (M.S.), India ABSTRACT A novel antifungal protein (Pr-2) was separated from Pumpkin (Cucurbita pepo L.) rinds using Methanol soluble extraction, ultra filtration, and followed by separation of protein (Pr-2) using Reverse Phase Chromatography technique (RP-HPLC). Further characterization and thereby identification is done by using Mass Spectrometry (MS) and is found to be having molecular mass of the order of Da. Pr-2 is considered as good candidate for use as a natural antifungal agent. KEYWORDS: Pumpkin rind, Cucurbita pepo, antifungal protein INTRODUCTION Pumpkin, Cucurbita pepo L., (English against conventional antibiotics has led to the search for novel antibiotic agents. name; summer pumpkin) is a Plants serve as the major source of herbaceous, monoecious, annual plant of the Cucurbita-ceae family [1]. A number of studies have recently been undertaken to identify novel and potent antimicrobial proteins because these so called natural antibiotics hold a great potential to overcome antimicrobial resistance. The emergence of clinical protein and carbohydrates for humans and livestock. Many functional classes of proteins have been purified and characterized from plant food itself. Plant responses to pests and pathogens include de novo synthesis of proteins and peptides with antifungal activity. Antifungal proteins and peptides exert a bacterial strains exhibiting resistance protective activity against fungal

2 invasion and play an important role in defending crops against fungal attack [2]. Over the past decade or so, many studies have focused on screening antifungal proteins from various sources because they possess economical value due to their ability to protect important crops from fungal pathogens. In addition, it has been reported that transgenic plants expressing antifungal proteins have increased substantially to fungal diseases [3,4]. Till date, antifungal proteins have been identified from a large number of leguminous species [2]. Apart from physiological or mechanical functions, these proteins may further act as protective barriers or growth inhibitors against various pathogens as well. Novel antifungal proteins have the potential for wide range of applications in fields like, medicine, food safety, and agriculture etc. Pumpkin has been traditionally used for its medicinal value in many countries, including China, Yugoslavia, Argentina, India, Mexico, and Korea [5,6]. Its popular medicinal uses have been the focus of many studies over the last few decades, and the antibiotic effects of pumpkin have been analyzed, along with its anti-diabetic, anti- hypertension, anti-tumor [7], antibacterial [8,9], anti-inflammatory, and anti-mutagenic effects [10]. It has been reported that some proteins isolated from pumpkin have toxic effects particularly on phytopathogens. This study preferentially was conducted to isolate and further to identify pumpkin protein (Pr-2) from its rind. MATERIALS & METHODS Pumpkin (Cucurbita pepo L.) was obtained from local market. It is shredded first and is then peeled off and the given mass of peel (rind) on weighing was placed in a container containing a given volume of HPLC grade Methanol & Millipore water mixture (1:1 v/v) in a beaker. The homogenization was brought about by sonication using a sonicator bath for about 15 minutes. This has facilitated in getting an extract of pumpkin rind containing the potential anti fungal protein (Pr-2). The mixture was then filtered using a vacuum filter with the help of 0.2 µ filter such that extract can be separated from the homogenized and filtered mixture. The extract in the form of filtrate is then first tested for identifying wavelength of maximum absorption.(λ max ) using UV-

3 Visible Spectrophotometer (UV 1650PC) interfaced with the software UV Probe. The extract was further diluted in Methanol & Millipore water mixture (1:1 v/v) and the diluted extract was injected (20 µl) using a Hamilton Micro syringe to LC chromatograph (LC 10AT Vp). The system used LC-10AT vp) with a UV detector, is interfaced with a software Spinchrome. Replicate Instrumentation Spectrophotometer conditions System: UV 1650PC (Shimadzu Make) measurements were taken to test for the reproducibility in results. Both the instruments were calibrated using official methods prior to making measurements to ensure maximum accuracy. After getting the chromatogram for the separated protein (Pr-2) and the corresponding retention characteristics the identification of separated protein was done by further characterization using MS technique. Software: UV Probe Source: 50 W Deuterium Lamps Wavelength Range: nm Detector: Silicon Photodiode Chromatography Conditions System: LC 10AT vp (Shimadzu Make) Software: Spinchrome Column: Inertsil C-18 (ODS) (250 x 60 mm) Flow rate: 1 ml/min λ max : 280 nm. Mobile phase: Water: Methanol (1:1, v/v) (isocratic elution) Column Temperature: Ambient Run time: 5 min. Injection Volume: 20 µl of the prepared standard and sample solution

4 Fig. A is a chromatogram showing a solitary peak corresponding to Protein (Pr-2). Fig. B is a mass spectrum with a single peak for isolated Pr-2 protein with molecular mass of the order of Da having a single band. RESULTS AND DISCUSSION purified using vacuum filtration In this study, homogenous Pr-2 protein was successfully separated, and purified using multi step process. An antifungal followed by its dilution using mobile phase, Methanol & water (1:1, v/v) as a solvent for dilution of the extract, used protein was separated, and purified using in RP-HPLC. The extract first was vacuum filtration and C 18 RP-HPLC. tested for finding out absorption In the initial screening crude extract maximum and was found to be 280 nm. obtained from pumpkin rind was The extract (Sample) was then injected

5 onto a C 18 RP-HPLC column for getting chromatogram which is found to have a single but prominent peak as shown in Fig. 1. The confirmation of the separated component, antifungal protein (Pr-2) was done by further characterization using Mass Spectrometry (MS). To determine the precise molecular mass of the Pr-2 that was represented by the single band shown in (Fig. 2). In summary, Pr-2, a novel antifungal protein appears as a single band in mass spectrum. Earlier literature and research shows that the protein concerned has heat stable characteristic. It also exhibits growth inhibition against 10 species of harmful pathogenic fungi. Further research may throw more light on biological role of Pr-2 and could be developed as an antifungal agent. REFERENCES (1) Grosch, H.D., Belitz, W., Food Chemistry. Sprinmger-Verlag, Berlin. (2) Ng, T. B. Antifungal proteins and peptides of leguminous and nonleguminous origins. Peptides 2004, 25, (3) Oard, S.; Enright, F. Expression of the antimicrobial peptides in plants to control phytopathogenic bacteria and fungi. Plant Cell Rep. 2006, 25, (4) Tobias, D. J.; Manoharan, M.; Pritsch, C.; Dahleen, L. S. Cobombardment, integration and expression of rice chitinase and thaumatin-like protein genes in barley (Hordeum vulgare cv. Conlon). Plant Cell Rep. 2007, 26, (5) Jia, W.; Gao, W.; Tang, L. Antidiabetic herbal drugs officially approved in China. Phytother. Res. 2003, 17, (6) Adolfo, A. C.; Michael, H. Mexican plants with hypoglycaemic effect used in the treatment of diabetes. J. Ethnopharmacol. 2005, 99, (7) Vassiliou, A. G.; Neumann, G. M.; Condron, R.; Polya, G. M. Purification and mass spectrometry-assisted sequencing of basic antifungal proteins from seeds of pumpkin (Cucurbita maxima). Plant Sci. 1998, 134, (8) Wang, H. X.; Ng, T. B. Isolation of cucurmoschin, a novel antifungal peptide abundant in arginine, glutamate and glycine residues from black pumpkin seeds. Peptides 2003, 24, (9) Hammer, K. A.; Carson, C. F.; Riley, T. V. Antimicrobial activity of essential oils and other plant extracts. J. Appl. Microbiol. 1999, 86, (10) Caili, F.; Huan, S.; Quanhong, L. A review on pharmacological activities and utilization technologies of pumpkin. Plant Foods Hum. Nutr. 2006, 61,

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