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Potent antibacterial property of APC protein from curry leaves (Murraya koenigii L.)
Food Chemistry 118 (2010) 747–750
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Potent antibacterial property of APC protein from curry leaves (Murraya koenigii L.) Mylarappa B. Ningappa, B.L. Dhananjaya, R. Dinesha, R. Harsha, Leela Srinivas * Adichunchanagiri Biotechnology and Cancer Research Institute, Balagangadharanatha Nagara, Mandya District, Karnataka 571 448, India
a r t i c l e
i n f o
Article history: Received 19 January 2009 Received in revised form 12 May 2009 Accepted 19 May 2009
Keywords: Curry leaves (Murraya koenigii) Antibacterial protein Human pathogenic bacteria
a b s t r a c t A monomeric protein with molecular mass of 35 kDa, isolated from Murraya Koenigii L. (curry leaves) shows potent antibacterial activity. The protein designated as APC (antioxidant protein from curry leaves) demonstrated potent antibacterial activity against all the human pathogenic strains tested. APC effectively inhibited Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Klebsiella pneumoniae, Salmonella typhi and Bacillus subtilis. The inhibition is comparable to that of commercial antibiotics chloramphenicol, streptomycin and gentamycin. APC inhibited bacterial growth, with MIC values ranging from 13 to 24 lg/ ml, which are comparable to MIC values of standard antibiotics. APC is devoid of ribonuclease/deoxyribonuclease and protease activity. APC is non-toxic at tested doses. These results encourage further studies of APC as a potent therapeutic agent. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Numerous antimicrobial molecules, such as antimicrobial peptides, proteins and small molecular weight organic substances are present in plants, acting as host defence mechanisms (Broekaert, Terras, Cammue, & Osborn, 1995; Selitrennikoff, 2001). Numerous compounds with broad spectrum of inhibitory activity against pathogenic bacteria and fungi have been isolated and their mechanism of action demonstrated (Ng, 2004a, 2004b; Selitrennikoff, 2001). Very few proteins with antibacterial activity has been reported to date, from radish seeds (Terras et al., 1992), WSG from Withania sominifera (Girish et al., 2006), WJAMP-1 from Wasabia japonica (Kiba, Saitoh, Nishihara, Omiya, & Yamamura, 2003) and AceAMP1, isolated from onion (Allium cepa L.) seeds (Cammue et al., 1995). Recently, hevein, a small (4.7 kDa) cysteine-rich protein with antibacterial activity was isolated from the latex of Hevea brasiliensis (Kanokwiroon, Teanpaisan, Wititsuwannakul, Hooper, & Wititsuwannakul, 2008). Murraya koenigii L. (curry leaf) belonging to family Rutaceae is used as a spice for its characteristic flavour and aroma. It is reported to have antioxidant, anti-diabetic, anticarcinogenic, antidysenteric, stimulant, hypoglycaemic and antimicrobial activities (Khanum, Anilakumar, Sudarshana Krishna, Viswanathan, & Santhanam, 2000; Ningappa, Dinesha, & Srinivas, 2008; Ningappa & Srinivas, 2008; Yadav, Vats, Dhunnoo, & Grover, 2002). Biologically active carbazole alkaloids are reported to have antimicrobial properties (Ramsewak, Nair, Strasburg, De Witt, & Nitiss, 1999). Curry leaves have been reported to contain tocopherol, b-carotene, lutein * Corresponding author. Tel.: +91 8234 287850; fax: +91 8234 287984. E-mail address: [email protected] (L. Srinivas). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.05.059
and alkaloids (Khanum et al., 2000). In this study, an aqueous solution of the antioxidant protein (APC) isolated from curry leaves (Ningappa & Srinivas, 2008) is tested for antibacterial activity against various human pathogenic bacteria. 2. Materials and methods Curry leaves (Murraya koenigii L.) were obtained from a garden maintained by Adichunchanagiri Biotechnology and Cancer Research Institute (ABCRI), B.G. Nagara, Mandya district, India. Prof. G.R. Shivamurthy, taxonomist, University of Mysore, India, authenticated the plant. The plant was deposited at ABCRI against voucher No. ABCRI 7/2007. Agar, beef extract, yeast extract and peptone were purchased from Hi Media Private Ltd., Mumbai, India. Authentic pure cultures of human pathogenic bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi, Vibrio cholerae, Klebsiella pneumoniae and Salmonella paratyphi were obtained from the Microbiology Department, Adichunchanagiri Institute of Medical Sciences (AIMS), B.G. Nagara, Karnataka, India. Bacteria were multiplied in nutrient agar at 36 ± 2 °C. After two days’ cultures were harvested and prepared at a final concentration of 1 108 cfu/ml and used for in vitro inhibition assay. 2.1. Isolation of APC from curry leaves The APC was purified according to the method of Ningappa and Srinivas (2008). Briefly, curry leaves were washed, shade dried and powdered. Five grams of curry leaves powder were homogenised in 20 ml of 10 mM Tris buffer, pH 7.0, with addition of polyvinylpyrrolidone to remove polyphenols. The suspension was incubated overnight at 4 °C with constant stirring, then filtered and centrifuged at 13,000 rpm at 4 °C for 20 min. The supernatant was
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treated with 0.1% polyethyleneimine to precipitate nucleotides. The resulting pellet was discarded and supernatant brought to 65% saturation with ammonium sulphate. The pellet was dissolved in 20 mM Tris buffer, pH 7.4 (NH4 SO4 extract). The NH4 SO4 extract (21 mg) was loaded onto a Sephadex G-75 column (14 73 cm), pre-equilibrated and eluted with 20 mM Tris buffer, pH 7.4, at a flow rate of 1.5 ml/5 min. Protein elution was monitored at 280 nm using a spectrophotometer. The antibacterial activities of eluted samples were tested by agar diffusion method on nutrient agar. Active peak II (8 mg), pooled and lyophilised was further fractionated on a Sephadex G-75 column by eluting with 20 mM Tris buffer, pH 7.4, to give a homogenous preparation, APC, which showed antibacterial activity.
2.6. Assay for deoxyribonuclease activity The deoxyribonuclease activity of APC was estimated according to the method of Wang and Ng (2001). Briefly, sperm DNA (1 mg) in 0.2 ml of ammonium acetate buffer (0.1 M), pH 5.5, and 100 ll of APC (0–100 lg) were incubated at 25 °C for 15 min. The reaction was terminated by adding 0.3 ml of ice-cold 20 mM lanthanum nitrate in 1.2% (v/v) perchloric acid, and incubated for 20 min at room temperature. Then, samples were centrifuged at 3000g for 5 min at 0 °C. The supernatant was diluted three-fold with water and the optical density was read at 260 nm against a blank without APC. One unit of enzymatic activity is defined as the amount of enzyme required to increase the absorbance at 260 nm of 0.001 min 1 ml 1 at pH 5.5 and 37 °C using sperm DNA.
2.2. Antibacterial activity of APC 2.7. Assay of protease activity Antibacterial activity was evaluated by the well diffusion method on nutrient agar medium (Forbes, Sahm, Weissfeld, & Trevino, 1990). This was confirmed by the inhibitory effect on bacterial growth as reflected by the inhibition zone, compared to that of known antibiotics. The sterile nutrient agar medium (20 ml) in Petri dishes was uniformly smeared using sterile cotton swabs with test pure cultures of human pathogenic bacteria S. aureus, B. subtilis, E. coli, S. typhi, V. cholerae, K. pneumoniae and S. paratyphi. The nutrient agar media was prepared by dissolving 0.3% beef extract, 0.3% yeast extract, 0.5% peptone, 0.5% NaCl and 1.5% agar in 1 l of distilled water. The wells of 5 mm diameter were made using a sterile cork borer in each petri dish and the buffer extract (0–100 lg), NH4SO4 extract (0–74 lg), G-75 eluted samples (0–50 lg) and isolated APC (0–24 lg) were added; a blank well loaded without test compound was regarded as control. For each treatment 10 replicates were prepared. The plates were incubated at 37 °C for 24 h and the resulting zone of inhibition was measured by comparing control and the standard antibiotic. 2.3. Determination of minimum inhibitory concentration (MIC) The minimum inhibitory concentration of isolated APC were determined by serial dilution in the nutrient agar, with concentrations ranging from 5, 10, 20, 25, 50, 75 and 100 lg/ml. The inoculum was prepared from fresh overnight broth culture in nutrient broth. Plates were incubated for 24 h at 37 °C. MIC was recorded as the lowest extract concentration demonstrating no visible growth in the broth (Prescot, Harley, & Klein, 1996). 2.4. Estimation of protein The protein estimation was determined by Bradford’s method (Bradford, 1976) using bovine serum albumin as standard.
Proteolytic activity was assayed according to the method of Murata, Satake, and Suzuki (1963), using 2% fat-free casein in Tris–HCl buffer pH 8.5 as substrate. APC sample (0–100 lg) was incubated with casein substrate (0.5 ml) for 120 min at 37 °C. Adding 1 ml of 0.44 M trichloroacetic acid terminated the reaction. After standing for 30 min the mixture was then centrifuged. Sodium carbonate (5 ml, 0.4 M) and Folin–Ciocalteu’s reagent (0.5 ml diluted to 1/3 of its original strength) were added to 1 ml of supernatant and the absorbance read at 660 nm. One unit of enzyme activity is defined as the amount of enzyme required to cause an increase per min in OD of 0.01 at 660 nm. Enzyme activity is expressed in terms of specific activity. 2.8. Haemolytic activity assay Haemolytic (direct/indirect) activity of APC was determined according to the method of Boman and Kaletta (1957), using packed human erythrocytes (blood group A). The direct and indirect haemolytic assays were carried out using washed erythrocytes. For the direct haemolytic assay, packed erythrocytes (1 ml) were suspended in nine volumes of phosphate-buffered saline (PBS), which formed the stock. The stock (1 ml) was incubated with various concentrations of APC (0–250 lg) for 30 min at 37 °C. For the indirect haemolytic assay, stock was prepared by mixing packed erythrocytes (1 ml), egg yolk (1 ml) and phosphate-buffered saline (8 ml). One millilitre of suspension from stock was incubated with various concentrations of APC (0–250 lg) for 30 min at 37 °C. The reaction was terminated by adding 10 ml of ice-cold PBS and then centrifuged at 4 °C and 800g. The amount of haemoglobin released in the supernatant was measured at 540 nm. One millilitre of stock erythrocytes with 10 ml ice-cold PBS alone was considered as 0% lysis.
2.5. Assay for ribonuclease activity The ribonuclease activity of APC was estimated according to the method of Lam and Ng (2001), by measuring the production of acid-soluble, UV-absorbing species. Briefly, yeast tRNA (200 lg) was incubated with the APC (0–100 lg) in 150 ll 100 mM MES buffer (pH 6.0) at 37 °C for 15 min. The reaction was terminated by adding 350 ll of ice-cold 3.4% (v/v) perchloric acid. After standing on ice for 15 min, the mixture was centrifuged at 15,000g for 15 min at 4 °C. The absorbance of the supernatant, after suitable dilution, was measured at 260 nm. One unit of ribonuclease activity is defined as the amount of ribonuclease that produces an absorbance increase at 260 nm of 1 min 1 in the acid-soluble fraction per ml of reaction mixture under the specified conditions.
Table 1 Total protein yield and antibacterial activities of fractions from different chromatographic steps of isolation procedure. Chromatographic fraction
Yield (%)
1. Buffer extract 100 53 ± 4 2. 65% (NH4)2 SO4 Precipitation 3. Gel filtration on Sephadex G-75 Peak I 25 ± 3 Peak II 22 ± 2 Peak III 14 ± 2 4. Rechromatography on Sephadex G-75 APC 17 ± 2 The symbols + and respectively.
Antibacterial activity + +
+
+
denote the presence and absence of antibacterial activity,
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2.9. Cytotoxicity assay Cytotoxicity was determined according to the method of Chwetzoff, Tsunasawa, Sakiyama, and Ménez (1989). EAT cells were suspended in Tyrode’s solution (5 106 cells/2 ml) and incubated with APC (0–250 lg) for 30 min. Trypan blue saline (1%, 100 ll) solution was then added, and unstained (viable) cells were counted using a haemocytometer. The percentage of viable cells was determined by comparing with the number of viable cells in the control (designated as 100%).
Increase of zone size (mm)
35 30 25
Staphylococcus aureus Bacillus subtilis Escherichia coli Salmonella typhi Vibrio cholerae Klebsiella pneumoniae Salmonella paratyphi b
B. subtilis S. typhi K. pneumoniae
MIC (lg/ml) APC
G
Cp
Sm
24.5 ± 3 25.2 ± 3 14.7 ± 1 15.2 ± 1 13.6 ± 1 13.3 ± 2 19.2 ± 3
20.8 ± 0.7 20.8 ± 0.4 24.2v0.1 19.2 ± 0.4 18.4 ± 0.3 16.8 ± 0.1 16.2 ± 0.2
14.4 ± 0.3 14.4 ± 0.5 14.4 ± 0.3 12.8 ± 0.7 12.8 ± 0.5 12.8 ± 0.6 12.4 ± 0.4
13.6 ± 0.4 16.8 ± 0.3 16.3 ± 0.7 14.4 ± 0.5 14.4 ± 0.3 14.4 ± 0.4 11.2 ± 0.4
Values are expressed as mean ± SD (n = 10). G, gentamycin; Cp, chloramphenicol; Sm, streptomycin.
2.10. Statistical analysis Statistical analysis was done using SPSS (Windows version 10.0.1; SPSS Inc., Chicago, IL) using a one-way student’s t-test; p < 0.05 was considered as statistically significant, when comparing with relevant controls. All results refer to mean ± SD.
20 15 10
3. Results and discussion
5
0
0.05
0.1
0.015
APC (µg) Fig. 1. Dose-dependent antibacterial activity of APC against different human pathogenic strains in agar diffusion assays. The diameter of the clear zone was measured and plotted after subtracting the diameter of the well (5 mm). Results are mean ± SD for three independent assays each performed in triplicate.
Table 2 Antibacterial activity of APC from curry leaves and antibioticsa,b. Microorganisms
Staphylococcus aureus Bacillus subtilis Escherichia coli Salmonella typhi Vibrio cholerae Klebsiella pneumoniae Salmonella paratyphi a
Microorganisms
a
S. aureus E. coli V. cholerae S. paratyphi
0
b
Table 3 Minimum inhibitory concentrations (MIC) of APC and antibioticsa,b in serial dilution method.
Diameter of inhibition zone (mm) APC
G
Cp
Sm
25 ± 3 14 ± 1 30 ± 3 15 ± 1 13 ± 1 13 ± 2 19 ± 3
18 ± 1 18 ± 2 18 ± 1 16 ± 1 16 ± 2 16 ± 2 18 ± 2
21 ± 2 17 ± 3 17 ± 1 18 ± 1 18 ± 1 18 ± 2 18 ± 2
26 ± 3 30 ± 2 26 ± 3 24 ± 1 23 ± 3 21 ± 1 20 ± 2
The results are mean ± SD (n = 6). G, gentamycin; Cp, chloramphenicol; Sm, streptomycin.
Murraya koenigii is a common plant with medicinal properties. In our study, when the aqueous extract of M. koenigii was tested for antibacterial activity against human pathogenic bacteria, it showed inhibition of bacterial growth. This initial observation prompted us to systematically evaluate the active principle. The Tris buffer extract (39 mg) of curry leaves upon 65% ammonium sulphate saturation yielded 21 mg of protein with antibacterial activity. The ammonium sulphate extract was fractionated with Sephadex G-75 column chromatography, into three peaks (Peaks I, II and III), amongst which peak II showed antibacterial activity. Peak II upon rechromatography on Sephadex G-75 column resulted in homogenous preparation with antibacterial activity referred to as antioxidant protein from curry leaves (APC) (Table 1). The preparation was homogeneous with a single peak with molecular mass of 35 kDa according to mass spectrometry (Ningappa & Srinivas, 2008). Antimicrobial protein WSG isolated from W. sominifera has a molecular mass of 28 kDa (Girish et al., 2006). An antimicrobial protein of about 10 kDa, called Ace-AMP1, was isolated from onion (A. cepa L.) seeds (Cammue et al., 1995). Recently, hevein, a small (4.7 kDa) cysteine-rich protein with antibacterial activity was isolated from latex of H. brasiliensis (Kanokwiroon et al., 2008). The observed molecular mass of 35 kDa is well within the range of antibacterial proteins reported.
Fig. 2. Agar diffusion assay. Effect of APC on growth of (A) Escherichia coli and (B) Staphylococcus aureus. Sm, 20 lg streptomycin (positive control); C, 10 mM Tris–HCl buffer (negative control).
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The dose-dependent antibacterial activity of APC against human pathogenic bacteria is shown in Fig. 1. APC showed a broad spectrum of very significant antibacterial activity by producing a clear zone of inhibition (20–25 mm) against E. coli and S. aureus (Table 2). V. cholerae, K. pneumoniae, S. typhi and B. subtilis were found to be moderately sensitive to APC, which showed an inhibition zone of 12–16 mm of (Table 2). It was interesting to observe that inhibition was comparable to that of the standards; chloramphenicol, streptomycin and gentamycin. Fig. 2 shows inhibition of E. coli and S. aureus growth by APC at 0.15 lg as studied by agar diffusion method. These results are well comparable with the antibacterial proteins listed earlier. When APC was tested, using the agar dilution assay for determining minimum inhibitory concentration (MIC), it was observed that it inhibited bacterial growth, with MIC values ranging from 13 to 24 lg/ml APC. APC showed comparable MIC values with standard antibiotics, which ranged from 11.2 to 20 lg/ml (Table 3). Thus APC is as potent as standard antibiotics in inhibiting the growth of bacterial strains. It is known that some ribonucleases/deoxyribonucleases/proteases exhibit antibacterial activity (Girish et al., 2006; Lam & Ng, 2001; Ng, 2004a; Wang & Ng, 2001). In order to know which class APC belongs to, it was screened for different enzyme activities. The results indicated that APC was devoid of ribonuclease, deoxyribonuclease and protease activity when tested at 50 and 100 lg amounts. This observation ruled out the possibility that APC is a ribonuclease, deoxyribonuclease or protease. In our study we also investigated the toxicity of APC using EAT cells. It was interesting to observe that there was no decrease in the number of viable cells when APC was added. Further, no haemolysis occurred, either directly or indirectly, when APC was added to human erythrocytes up to 0.25 mg. These results clearly indicate that APC is a non-toxic protein having antibacterial activity. Similarly, antimicrobial protein WSG isolated from W. sominifera, is also reported to be non-toxic (Girish et al., 2006). Further studies on the mechanism of action by which APC exhibits antibacterial activity are in progress. In conclusion, the 35 kDa APC isolated from M. koenigii exhibited a broad spectrum of antibacterial activity against human pathogenic bacteria, comparable to commercial antibiotics. As it is nontoxic, it appears to be a promising candidate for development of an effective antioxidant antibiotic.
Acknowledgements The authors acknowledge the Adichunchanagiri Mahasamstana Mutt and Shikshana Trust for providing facilities in the Adichunchanagiri Biotechnology and Cancer Research Institute (ABCRI) and Adichunchanagiri Institute of Technology (AIT) for providing continuous Internet facilities for carrying out this work. We acknowledge microbiology department, Adichunchanagiri Institute of Medical Sciences (AIMS), B.G. Nagara, Karnataka, India for providing bacterial strains.
References Boman, H. G., & Kaletta, U. (1957). Chromatography of rattlesnake: A separation of three phosphodiesterases. Biochimica et Biophysica Acta, 24, 619–623. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 7, 248–254. Broekaert, W. F., Terras, F. R., Cammue, B. P., & Osborn, R. W. (1995). Plant defensins: Novel antimicrobial peptides as components of the host defense system. Plant Physiology, 108, 1353–1358. Cammue, B. P., Thevissen, K., Hendriks, M., Eggermont, K., Goderis, I. J., Proost, P., et al. (1995). A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins. Plant and Cell Physiology, 109, 445–455. Chwetzoff, S., Tsunasawa, S., Sakiyama, F., & Ménez, A. (1989). Nigexine, a phospholipase A2 from cobra venom with cytotoxic properties not related to esterase activity. Purification, amino acid sequence and biological properties. The Journal of Biological Chemistry, 264, 13289–13297. Forbes, B. A., Sahm, D. F., Weissfeld, A. S., & Trevino, E. A. (1990). Methods for testing antimicrobial effectiveness. In E. J. Baron, L. R. Petrson, & S. M. Finegold (Eds.), Bailey and Scott’s diagnostics microbiology (pp. 171–194). St. Louis, Missouri: Mosby Co. Girish, K. S., Machiah, K. D., Ushanandini, S., Harish Kumar, K., Nagaraju, S., Govindappa, M., et al. (2006). Antimicrobial properties of a non-toxic glycoprotein (WSG) from Withania somnifera (Ashwagandha). Journal of Basic Microbiology, 46, 365–374. Kanokwiroon, K., Teanpaisan, R., Wititsuwannakul, D., Hooper, A. B., & Wititsuwannakul, R. (2008). Antimicrobial activity of a protein purified from the latex of Hevea brasiliensis on oral microorganisms. Mycoses, 51, 301–307. Khanum, F., Anilakumar, K. R., Sudarshana Krishna, K. R., Viswanathan, K. R., & Santhanam, K. (2000). Anticarcinogenic effects of curry leaves in dimethylhydrazine-treated rats. Plant Foods for Human Nutrition, 55, 347–355. Kiba, A., Saitoh, H., Nishihara, M., Omiya, K., & Yamamura, S. (2003). C-terminal domain of a hevein-like protein from Wasabia japonica has potent antimicrobial activity. Plant and Cell Physiology, 44, 296–303. Lam, S. K., & Ng, T. B. (2001). Isolation of a novel thermolabile heterodimeric ribonuclease with antifungal and antiproliferative activities from roots of the sanchi ginseng Panax notoginseng. Biochemical and Biophysical Research Communications, 285, 419–423. Murata, J., Satake, M., & Suzuki, T. (1963). Studies on snake venom. XII. Distribution of proteinase activities among Japanese and Formosan snake venoms. Journal of Biochemistry, 53, 431–443. Ng, T. B. (2004a). Proteins and peptides from fungi. Peptides, 25, 1055–1073. Ng, T. B. (2004b). Antifungal proteins and peptides of leguminous and nonleguminous origins. Peptides, 25, 1215–1222. Ningappa, M. B., Dinesha, R., & Srinivas, L. (2008). Antioxidant and free radical scavenging activities of polyphenol-enriched curry leaf (Murraya koenigii L.) extracts. Food Chemistry, 106, 720–728. Ningappa, M. B., & Srinivas, L. (2008). Purification and characterization of approximately 35 kDa antioxidant protein from curry leaves (Murraya koenigii L.). Toxicology in Vitro, 22, 699–709. Prescot, L. M., Harley, J. P., & Klein, D. A. (1996). Introduction to microbiology (5th ed.). The Benjamin Cummins Publishing Co. Inc (pp. 681–684). Ramsewak, R. S., Nair, M. G., Strasburg, G. M., De Witt, D. L., & Nitiss, J. L. (1999). Biologically active carbazole alkaloids from Murraya koenigii. Journal of Agricultural and Food Chemistry, 47, 444–447. Selitrennikoff, C. L. (2001). Antifungal proteins. Applied and Environmental Microbiology, 67, 2883–2894. Terras, F. R., Schoofs, H. M., De Bolle, M. F., Van Leuven, F., Rees, S. B., Vanderleyden, J., et al. (1992). Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus) seeds. Journal of Biological Chemistry, 267, 15301–15309. Wang, H. X., & Ng, T. B. (2001). Isolation of a novel deoxyribonuclease with antifungal activity from Asparagus officinalis seeds. Biochemical and Biophysical Research Communications, 289, 120–124. Yadav, S., Vats, V., Dhunnoo, Y., & Grover, J. K. (2002). Hypoglycemic and antihyperglycemic activity of Murraya koenigii leaves in diabetic rats. Journal of Ethnopharmacology, 82, 111–116.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Potent antibacterial property of APC protein from curry leaves (Murraya koenigii L.) Mylarappa B. Ningappa, B.L. Dhananjaya, R. Dinesha, R. Harsha, Leela Srinivas * Adichunchanagiri Biotechnology and Cancer Research Institute, Balagangadharanatha Nagara, Mandya District, Karnataka 571 448, India
a r t i c l e
i n f o
Article history: Received 19 January 2009 Received in revised form 12 May 2009 Accepted 19 May 2009
Keywords: Curry leaves (Murraya koenigii) Antibacterial protein Human pathogenic bacteria
a b s t r a c t A monomeric protein with molecular mass of 35 kDa, isolated from Murraya Koenigii L. (curry leaves) shows potent antibacterial activity. The protein designated as APC (antioxidant protein from curry leaves) demonstrated potent antibacterial activity against all the human pathogenic strains tested. APC effectively inhibited Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Klebsiella pneumoniae, Salmonella typhi and Bacillus subtilis. The inhibition is comparable to that of commercial antibiotics chloramphenicol, streptomycin and gentamycin. APC inhibited bacterial growth, with MIC values ranging from 13 to 24 lg/ ml, which are comparable to MIC values of standard antibiotics. APC is devoid of ribonuclease/deoxyribonuclease and protease activity. APC is non-toxic at tested doses. These results encourage further studies of APC as a potent therapeutic agent. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Numerous antimicrobial molecules, such as antimicrobial peptides, proteins and small molecular weight organic substances are present in plants, acting as host defence mechanisms (Broekaert, Terras, Cammue, & Osborn, 1995; Selitrennikoff, 2001). Numerous compounds with broad spectrum of inhibitory activity against pathogenic bacteria and fungi have been isolated and their mechanism of action demonstrated (Ng, 2004a, 2004b; Selitrennikoff, 2001). Very few proteins with antibacterial activity has been reported to date, from radish seeds (Terras et al., 1992), WSG from Withania sominifera (Girish et al., 2006), WJAMP-1 from Wasabia japonica (Kiba, Saitoh, Nishihara, Omiya, & Yamamura, 2003) and AceAMP1, isolated from onion (Allium cepa L.) seeds (Cammue et al., 1995). Recently, hevein, a small (4.7 kDa) cysteine-rich protein with antibacterial activity was isolated from the latex of Hevea brasiliensis (Kanokwiroon, Teanpaisan, Wititsuwannakul, Hooper, & Wititsuwannakul, 2008). Murraya koenigii L. (curry leaf) belonging to family Rutaceae is used as a spice for its characteristic flavour and aroma. It is reported to have antioxidant, anti-diabetic, anticarcinogenic, antidysenteric, stimulant, hypoglycaemic and antimicrobial activities (Khanum, Anilakumar, Sudarshana Krishna, Viswanathan, & Santhanam, 2000; Ningappa, Dinesha, & Srinivas, 2008; Ningappa & Srinivas, 2008; Yadav, Vats, Dhunnoo, & Grover, 2002). Biologically active carbazole alkaloids are reported to have antimicrobial properties (Ramsewak, Nair, Strasburg, De Witt, & Nitiss, 1999). Curry leaves have been reported to contain tocopherol, b-carotene, lutein * Corresponding author. Tel.: +91 8234 287850; fax: +91 8234 287984. E-mail address: [email protected] (L. Srinivas). 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.05.059
and alkaloids (Khanum et al., 2000). In this study, an aqueous solution of the antioxidant protein (APC) isolated from curry leaves (Ningappa & Srinivas, 2008) is tested for antibacterial activity against various human pathogenic bacteria. 2. Materials and methods Curry leaves (Murraya koenigii L.) were obtained from a garden maintained by Adichunchanagiri Biotechnology and Cancer Research Institute (ABCRI), B.G. Nagara, Mandya district, India. Prof. G.R. Shivamurthy, taxonomist, University of Mysore, India, authenticated the plant. The plant was deposited at ABCRI against voucher No. ABCRI 7/2007. Agar, beef extract, yeast extract and peptone were purchased from Hi Media Private Ltd., Mumbai, India. Authentic pure cultures of human pathogenic bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella typhi, Vibrio cholerae, Klebsiella pneumoniae and Salmonella paratyphi were obtained from the Microbiology Department, Adichunchanagiri Institute of Medical Sciences (AIMS), B.G. Nagara, Karnataka, India. Bacteria were multiplied in nutrient agar at 36 ± 2 °C. After two days’ cultures were harvested and prepared at a final concentration of 1 108 cfu/ml and used for in vitro inhibition assay. 2.1. Isolation of APC from curry leaves The APC was purified according to the method of Ningappa and Srinivas (2008). Briefly, curry leaves were washed, shade dried and powdered. Five grams of curry leaves powder were homogenised in 20 ml of 10 mM Tris buffer, pH 7.0, with addition of polyvinylpyrrolidone to remove polyphenols. The suspension was incubated overnight at 4 °C with constant stirring, then filtered and centrifuged at 13,000 rpm at 4 °C for 20 min. The supernatant was
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M.B. Ningappa et al. / Food Chemistry 118 (2010) 747–750
treated with 0.1% polyethyleneimine to precipitate nucleotides. The resulting pellet was discarded and supernatant brought to 65% saturation with ammonium sulphate. The pellet was dissolved in 20 mM Tris buffer, pH 7.4 (NH4 SO4 extract). The NH4 SO4 extract (21 mg) was loaded onto a Sephadex G-75 column (14 73 cm), pre-equilibrated and eluted with 20 mM Tris buffer, pH 7.4, at a flow rate of 1.5 ml/5 min. Protein elution was monitored at 280 nm using a spectrophotometer. The antibacterial activities of eluted samples were tested by agar diffusion method on nutrient agar. Active peak II (8 mg), pooled and lyophilised was further fractionated on a Sephadex G-75 column by eluting with 20 mM Tris buffer, pH 7.4, to give a homogenous preparation, APC, which showed antibacterial activity.
2.6. Assay for deoxyribonuclease activity The deoxyribonuclease activity of APC was estimated according to the method of Wang and Ng (2001). Briefly, sperm DNA (1 mg) in 0.2 ml of ammonium acetate buffer (0.1 M), pH 5.5, and 100 ll of APC (0–100 lg) were incubated at 25 °C for 15 min. The reaction was terminated by adding 0.3 ml of ice-cold 20 mM lanthanum nitrate in 1.2% (v/v) perchloric acid, and incubated for 20 min at room temperature. Then, samples were centrifuged at 3000g for 5 min at 0 °C. The supernatant was diluted three-fold with water and the optical density was read at 260 nm against a blank without APC. One unit of enzymatic activity is defined as the amount of enzyme required to increase the absorbance at 260 nm of 0.001 min 1 ml 1 at pH 5.5 and 37 °C using sperm DNA.
2.2. Antibacterial activity of APC 2.7. Assay of protease activity Antibacterial activity was evaluated by the well diffusion method on nutrient agar medium (Forbes, Sahm, Weissfeld, & Trevino, 1990). This was confirmed by the inhibitory effect on bacterial growth as reflected by the inhibition zone, compared to that of known antibiotics. The sterile nutrient agar medium (20 ml) in Petri dishes was uniformly smeared using sterile cotton swabs with test pure cultures of human pathogenic bacteria S. aureus, B. subtilis, E. coli, S. typhi, V. cholerae, K. pneumoniae and S. paratyphi. The nutrient agar media was prepared by dissolving 0.3% beef extract, 0.3% yeast extract, 0.5% peptone, 0.5% NaCl and 1.5% agar in 1 l of distilled water. The wells of 5 mm diameter were made using a sterile cork borer in each petri dish and the buffer extract (0–100 lg), NH4SO4 extract (0–74 lg), G-75 eluted samples (0–50 lg) and isolated APC (0–24 lg) were added; a blank well loaded without test compound was regarded as control. For each treatment 10 replicates were prepared. The plates were incubated at 37 °C for 24 h and the resulting zone of inhibition was measured by comparing control and the standard antibiotic. 2.3. Determination of minimum inhibitory concentration (MIC) The minimum inhibitory concentration of isolated APC were determined by serial dilution in the nutrient agar, with concentrations ranging from 5, 10, 20, 25, 50, 75 and 100 lg/ml. The inoculum was prepared from fresh overnight broth culture in nutrient broth. Plates were incubated for 24 h at 37 °C. MIC was recorded as the lowest extract concentration demonstrating no visible growth in the broth (Prescot, Harley, & Klein, 1996). 2.4. Estimation of protein The protein estimation was determined by Bradford’s method (Bradford, 1976) using bovine serum albumin as standard.
Proteolytic activity was assayed according to the method of Murata, Satake, and Suzuki (1963), using 2% fat-free casein in Tris–HCl buffer pH 8.5 as substrate. APC sample (0–100 lg) was incubated with casein substrate (0.5 ml) for 120 min at 37 °C. Adding 1 ml of 0.44 M trichloroacetic acid terminated the reaction. After standing for 30 min the mixture was then centrifuged. Sodium carbonate (5 ml, 0.4 M) and Folin–Ciocalteu’s reagent (0.5 ml diluted to 1/3 of its original strength) were added to 1 ml of supernatant and the absorbance read at 660 nm. One unit of enzyme activity is defined as the amount of enzyme required to cause an increase per min in OD of 0.01 at 660 nm. Enzyme activity is expressed in terms of specific activity. 2.8. Haemolytic activity assay Haemolytic (direct/indirect) activity of APC was determined according to the method of Boman and Kaletta (1957), using packed human erythrocytes (blood group A). The direct and indirect haemolytic assays were carried out using washed erythrocytes. For the direct haemolytic assay, packed erythrocytes (1 ml) were suspended in nine volumes of phosphate-buffered saline (PBS), which formed the stock. The stock (1 ml) was incubated with various concentrations of APC (0–250 lg) for 30 min at 37 °C. For the indirect haemolytic assay, stock was prepared by mixing packed erythrocytes (1 ml), egg yolk (1 ml) and phosphate-buffered saline (8 ml). One millilitre of suspension from stock was incubated with various concentrations of APC (0–250 lg) for 30 min at 37 °C. The reaction was terminated by adding 10 ml of ice-cold PBS and then centrifuged at 4 °C and 800g. The amount of haemoglobin released in the supernatant was measured at 540 nm. One millilitre of stock erythrocytes with 10 ml ice-cold PBS alone was considered as 0% lysis.
2.5. Assay for ribonuclease activity The ribonuclease activity of APC was estimated according to the method of Lam and Ng (2001), by measuring the production of acid-soluble, UV-absorbing species. Briefly, yeast tRNA (200 lg) was incubated with the APC (0–100 lg) in 150 ll 100 mM MES buffer (pH 6.0) at 37 °C for 15 min. The reaction was terminated by adding 350 ll of ice-cold 3.4% (v/v) perchloric acid. After standing on ice for 15 min, the mixture was centrifuged at 15,000g for 15 min at 4 °C. The absorbance of the supernatant, after suitable dilution, was measured at 260 nm. One unit of ribonuclease activity is defined as the amount of ribonuclease that produces an absorbance increase at 260 nm of 1 min 1 in the acid-soluble fraction per ml of reaction mixture under the specified conditions.
Table 1 Total protein yield and antibacterial activities of fractions from different chromatographic steps of isolation procedure. Chromatographic fraction
Yield (%)
1. Buffer extract 100 53 ± 4 2. 65% (NH4)2 SO4 Precipitation 3. Gel filtration on Sephadex G-75 Peak I 25 ± 3 Peak II 22 ± 2 Peak III 14 ± 2 4. Rechromatography on Sephadex G-75 APC 17 ± 2 The symbols + and respectively.
Antibacterial activity + +
+
+
denote the presence and absence of antibacterial activity,
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2.9. Cytotoxicity assay Cytotoxicity was determined according to the method of Chwetzoff, Tsunasawa, Sakiyama, and Ménez (1989). EAT cells were suspended in Tyrode’s solution (5 106 cells/2 ml) and incubated with APC (0–250 lg) for 30 min. Trypan blue saline (1%, 100 ll) solution was then added, and unstained (viable) cells were counted using a haemocytometer. The percentage of viable cells was determined by comparing with the number of viable cells in the control (designated as 100%).
Increase of zone size (mm)
35 30 25
Staphylococcus aureus Bacillus subtilis Escherichia coli Salmonella typhi Vibrio cholerae Klebsiella pneumoniae Salmonella paratyphi b
B. subtilis S. typhi K. pneumoniae
MIC (lg/ml) APC
G
Cp
Sm
24.5 ± 3 25.2 ± 3 14.7 ± 1 15.2 ± 1 13.6 ± 1 13.3 ± 2 19.2 ± 3
20.8 ± 0.7 20.8 ± 0.4 24.2v0.1 19.2 ± 0.4 18.4 ± 0.3 16.8 ± 0.1 16.2 ± 0.2
14.4 ± 0.3 14.4 ± 0.5 14.4 ± 0.3 12.8 ± 0.7 12.8 ± 0.5 12.8 ± 0.6 12.4 ± 0.4
13.6 ± 0.4 16.8 ± 0.3 16.3 ± 0.7 14.4 ± 0.5 14.4 ± 0.3 14.4 ± 0.4 11.2 ± 0.4
Values are expressed as mean ± SD (n = 10). G, gentamycin; Cp, chloramphenicol; Sm, streptomycin.
2.10. Statistical analysis Statistical analysis was done using SPSS (Windows version 10.0.1; SPSS Inc., Chicago, IL) using a one-way student’s t-test; p < 0.05 was considered as statistically significant, when comparing with relevant controls. All results refer to mean ± SD.
20 15 10
3. Results and discussion
5
0
0.05
0.1
0.015
APC (µg) Fig. 1. Dose-dependent antibacterial activity of APC against different human pathogenic strains in agar diffusion assays. The diameter of the clear zone was measured and plotted after subtracting the diameter of the well (5 mm). Results are mean ± SD for three independent assays each performed in triplicate.
Table 2 Antibacterial activity of APC from curry leaves and antibioticsa,b. Microorganisms
Staphylococcus aureus Bacillus subtilis Escherichia coli Salmonella typhi Vibrio cholerae Klebsiella pneumoniae Salmonella paratyphi a
Microorganisms
a
S. aureus E. coli V. cholerae S. paratyphi
0
b
Table 3 Minimum inhibitory concentrations (MIC) of APC and antibioticsa,b in serial dilution method.
Diameter of inhibition zone (mm) APC
G
Cp
Sm
25 ± 3 14 ± 1 30 ± 3 15 ± 1 13 ± 1 13 ± 2 19 ± 3
18 ± 1 18 ± 2 18 ± 1 16 ± 1 16 ± 2 16 ± 2 18 ± 2
21 ± 2 17 ± 3 17 ± 1 18 ± 1 18 ± 1 18 ± 2 18 ± 2
26 ± 3 30 ± 2 26 ± 3 24 ± 1 23 ± 3 21 ± 1 20 ± 2
The results are mean ± SD (n = 6). G, gentamycin; Cp, chloramphenicol; Sm, streptomycin.
Murraya koenigii is a common plant with medicinal properties. In our study, when the aqueous extract of M. koenigii was tested for antibacterial activity against human pathogenic bacteria, it showed inhibition of bacterial growth. This initial observation prompted us to systematically evaluate the active principle. The Tris buffer extract (39 mg) of curry leaves upon 65% ammonium sulphate saturation yielded 21 mg of protein with antibacterial activity. The ammonium sulphate extract was fractionated with Sephadex G-75 column chromatography, into three peaks (Peaks I, II and III), amongst which peak II showed antibacterial activity. Peak II upon rechromatography on Sephadex G-75 column resulted in homogenous preparation with antibacterial activity referred to as antioxidant protein from curry leaves (APC) (Table 1). The preparation was homogeneous with a single peak with molecular mass of 35 kDa according to mass spectrometry (Ningappa & Srinivas, 2008). Antimicrobial protein WSG isolated from W. sominifera has a molecular mass of 28 kDa (Girish et al., 2006). An antimicrobial protein of about 10 kDa, called Ace-AMP1, was isolated from onion (A. cepa L.) seeds (Cammue et al., 1995). Recently, hevein, a small (4.7 kDa) cysteine-rich protein with antibacterial activity was isolated from latex of H. brasiliensis (Kanokwiroon et al., 2008). The observed molecular mass of 35 kDa is well within the range of antibacterial proteins reported.
Fig. 2. Agar diffusion assay. Effect of APC on growth of (A) Escherichia coli and (B) Staphylococcus aureus. Sm, 20 lg streptomycin (positive control); C, 10 mM Tris–HCl buffer (negative control).
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The dose-dependent antibacterial activity of APC against human pathogenic bacteria is shown in Fig. 1. APC showed a broad spectrum of very significant antibacterial activity by producing a clear zone of inhibition (20–25 mm) against E. coli and S. aureus (Table 2). V. cholerae, K. pneumoniae, S. typhi and B. subtilis were found to be moderately sensitive to APC, which showed an inhibition zone of 12–16 mm of (Table 2). It was interesting to observe that inhibition was comparable to that of the standards; chloramphenicol, streptomycin and gentamycin. Fig. 2 shows inhibition of E. coli and S. aureus growth by APC at 0.15 lg as studied by agar diffusion method. These results are well comparable with the antibacterial proteins listed earlier. When APC was tested, using the agar dilution assay for determining minimum inhibitory concentration (MIC), it was observed that it inhibited bacterial growth, with MIC values ranging from 13 to 24 lg/ml APC. APC showed comparable MIC values with standard antibiotics, which ranged from 11.2 to 20 lg/ml (Table 3). Thus APC is as potent as standard antibiotics in inhibiting the growth of bacterial strains. It is known that some ribonucleases/deoxyribonucleases/proteases exhibit antibacterial activity (Girish et al., 2006; Lam & Ng, 2001; Ng, 2004a; Wang & Ng, 2001). In order to know which class APC belongs to, it was screened for different enzyme activities. The results indicated that APC was devoid of ribonuclease, deoxyribonuclease and protease activity when tested at 50 and 100 lg amounts. This observation ruled out the possibility that APC is a ribonuclease, deoxyribonuclease or protease. In our study we also investigated the toxicity of APC using EAT cells. It was interesting to observe that there was no decrease in the number of viable cells when APC was added. Further, no haemolysis occurred, either directly or indirectly, when APC was added to human erythrocytes up to 0.25 mg. These results clearly indicate that APC is a non-toxic protein having antibacterial activity. Similarly, antimicrobial protein WSG isolated from W. sominifera, is also reported to be non-toxic (Girish et al., 2006). Further studies on the mechanism of action by which APC exhibits antibacterial activity are in progress. In conclusion, the 35 kDa APC isolated from M. koenigii exhibited a broad spectrum of antibacterial activity against human pathogenic bacteria, comparable to commercial antibiotics. As it is nontoxic, it appears to be a promising candidate for development of an effective antioxidant antibiotic.
Acknowledgements The authors acknowledge the Adichunchanagiri Mahasamstana Mutt and Shikshana Trust for providing facilities in the Adichunchanagiri Biotechnology and Cancer Research Institute (ABCRI) and Adichunchanagiri Institute of Technology (AIT) for providing continuous Internet facilities for carrying out this work. We acknowledge microbiology department, Adichunchanagiri Institute of Medical Sciences (AIMS), B.G. Nagara, Karnataka, India for providing bacterial strains.
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