- Email: [email protected]
Antibacterial and antimutagenic activities of Dillenia indica extracts
Food Bioscience 5 (2014) 47 –53
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/fbio
Antibacterial and antimutagenic activities of Dillenia indica extracts Sweta Jaiswal, N. Mansa, M.S. Pallavi Prasad, Bhabani Sankar Jena1, Pradeep Singh Negin Fruit and Vegetable Technology, CSIR-Central Food Technological Research Institute, Mysore-570020, India
art i cle i nfo
ab st rac t
Article history:
In the present study, the antibacterial and antimutagenic activities of the fruit and bark
Received 21 May 2013
extracts of Dillenia indica were evaluated and the effect of their inhibitory concentrations on
Received in revised form
cell wall, nucleic acid leakage and pathogenic genes of the bacteria was studied. The fruit
9 October 2013
and bark extracts obtained by 70% aqueous acetone extraction showed minimum
Accepted 15 November 2013
inhibitory concentration (using agar dilution method) against different bacteria in the range of 2000–10,000 and 1250–5000 mg l 1, respectively, indicating higher antibacterial
Keywords:
activity for bark extract. At 500 μg/plate concentration, bark extract showed significantly
Antibacterial
(po0.01) higher antimutagenic activity in Ames test against the sodium azide induced
Antimutagenic
mutation in Salmonella tester strain (TA-1531). Both the extracts showed statistically
Dillenia indica
similar but strong antimutagenic activity at or above 1500 μg/plate concentration.
Minimum inhibitory concentration
The ability of these extracts to cause the disintegration of cell wall and leakage of genetic
Pathogenic genes
material is most likely to be the factor for their antibacterial activity. Further the extracts were able to inhibit the pathogenic genes present in tested bacteria. The fruit and bark extracts did not show any hemolysis at and below 2500 and 5000 mg l 1 concentration, respectively. The D. indica fruit and bark aqueous acetone extract may find application in foods and pharmaceuticals owing to their inhibitory properties. & 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
In recent years, there has been a dramatic increase in the number of reported cases of food borne illness (Scallan et al., 2011), consequently, there is considerable interest in finding out the ways to stop this upward trend and reduce the incidence of food poisoning. Due to negative consumer perceptions of artificial preservatives, attention is shifting towards alternatives that consumers perceive as natural, and in particular plant extracts, including their essential oils and essences are being used for preservation. It is well established
n
that several plant extracts have antimicrobial properties against bacteria, moulds and yeast, but their mechanism of antimicrobial action is not very clear. Membrane disruption by terpenoids and phenolics; metal chelation by phenols and flavonoids; and effect on genetic material by coumarin and alkaloids are reported to be responsible for inhibition of growth of microorganisms, and therefore, the versatile compositions of plant extract make them potential natural agents for food preservation (Negi, 2012). Plants also contain several compounds, which have been reported to be inhibitors of chemical carcinogenesis, and due
Corresponding author. Tel.: þ91 821 2515653; fax: þ91 821 2517233. E-mail address: [email protected] (P.S. Negi). 1 Present Address: Principal Scientist, RDPD and Bioresource Engineering, CSIR-Institute of Minerals and Materials Technology, Bhubaneswar- 751 013, India. 2212-4292/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fbio.2013.11.005
48
Food Bioscience 5 (2014) 47 –53
to the presence of these compounds several plant extracts are reported to be antimutagenic. The regular intake of antimutagenic agents can reduce genotoxic effects of mutagenic and carcinogenic factors, and use of antimutagens in diet is suggested as the most effective method for preventing cancer and genetic disorders (Ikken et al., 1999). Dillenia (Family: Dilleniaceae) is a small genus of trees found in Indo-Malaysian region extending to tropical Australia, and Dillenia indica is found in the forests of subHimalayan tract, from Uttarakhand eastwards to Assam. The ripe fruits are sour in taste and are used for flavouring of curies and preparation of jam and jelly. The acid juice is sweetened with sugar and used as cooling drink, and the fruits are also processed to commercial products such as ready-to-serve beverage and squash (Saikia & Saikia, 2002). Although fruits are used traditionally in some medicinal preparation by local population, to our knowledge, no systematic report exists about their antibacterial and antimutagenic properties. Therefore, in the present investigation, we have studied the antibacterial and antimutagenic properties of the fruit and bark extracts of D. indica. Further, the effect of the minimum inhibitory concentration on bacterial cell wall, nucleic acid leakage and pathogenic genes of the bacteria were also studied to find out the probable mode of their antibacterial action.
2.
Materials and methods
2.1.
Plant extract preparation
The fruits and bark of D. indica were obtained from Balasore, Odissa, India. The fruits were rinsed with distilled water and dried in shade. The dried fruit and bark of D. indica were powdered and the known quantity of the powder was extracted with 70% aqueous acetone. The extracts were dried under vacuum and the solvent free extracts were dissolved in propylene glycol (PG) for use in the present study.
2.2. (MIC)
Determination of minimum inhibitory concentration
The fruit and the bark extracts from the D. indica were tested against food-borne pathogens, Bacillus cereus (F 4810, Public Health Laboratory, London, UK), Staphylococcus aureus (FRI 722, Public Health Laboratory, The Netherlands), Escherichia coli (MTCC 108, Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India) and Yersinia enterocolitica (MTCC 859, Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India) by agar dilution method (Negi, Jayaprakasha, Rao, & Sakariah, 1999). Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration at which no bacterial growth was observed after incubation.
2.3.
Antimutagenic activity assay
The antimutagenic activity of fruit and the bark extracts of the D. indica was determined in terms of the inhibitions of mutagenic activity of the sodium azide using Salmonella
typhimurium (TA-1531, Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India) by standard plate incorporation test (Maron & Ames, 1983).
2.4.
Effect of the extracts on 260 nm absorbing material
For determination of loss of 260 nm absorbing material, the MIC and 0.5 MIC of the extracts were mixed with 100 μl of the cells (OD 0.3) and volume was made up to 1 ml using phosphate buffered saline (PBS). Immediately after addition of extract, the OD was measured at 260 nm against PBS blank (Carson, Mee, & Riley, 2002).
2.5.
Effect of the extracts on bacterial cell wall
Scanning Electron Microscopy (SEM) was used to investigate the effect of extracts on bacterial cell wall by the method described by Moosavy et al. (2008) with slight modifications. Overnight cultures were centrifuged at 7000 rpm for 10 min at 4 1C, washed twice with 0.1 M phosphate buffer (pH 6.5) and volume was made up to 0.5 ml with the same buffer. The MIC of the extracts was added and the final volume was made up to 1 ml using phosphate buffer. The above cell suspension was incubated for 1 h and cells were harvested at 6000 rpm for 10 min at 4 1C. The pellet was incubated in 1% glutaraldehyde overnight at 0 1C and the cells were harvested at 6000 rpm for 10 min at 4 1C. The cells were dehydrated in ethanol gradient (10–100%) and coated with a thin layer of gold using polaron SEM coating system. The cells were observed with a LEO 435 VP Scanning Electron Microscope at 20 kV attached to Mitsubishi Video copy processor. Photographs were taken using 35 mm Richo camera that was connected to monitor optically through fibre optics.
2.6.
Effect of extracts on pathogenic genes of bacteria
DNA from overnight grown B. cereus, E. coli, Y. enterocolitica cells was extracted by using Triton X 100 as a detergent, wherein culture grown in the presence of 0.5 MIC and MIC of the extracts was washed with 0.5% triton X100, heated at 95 1C for 10 min in 0.5% triton X, centrifuged at 6000 rpm at 4 1C for 15 min, and the supernatant was used as DNA (Ramesh, Padmapriya, Bharathi, & Varadaraj, 2003). For S. aureus, the DNA was extracted essentially by the method of Rohinishree & Negi (2012). Integrity of DNA was checked by agarose gel electrophoresis and it was quantified using absorbance reading at 260 nm. The DNA was used to check the presence of pathogenic genes in S. aureus (Staphylococcus Accessory Regulator, sarA), B. cereus (Phosphatidylinositol phospholipase, Pi-PLC), E. coli (E. coli exotoxin shiga toxin 2e, ECstx2e) and Y. enterocolitica (Attachment Invasion Loci, ail) using gene specific primers (Table 1) synthesized from Sigma Aldrich (Bangalore, India). The contents (2 ng DNA, 10 PCR buffer, 0.2 mM dNTP, 0.2 mM of both forward and reverse primers, I U Taq polymerase and water to make up 25 μl reaction mixture) were mixed a thin walled PCR tube and placed in a thermalcycler (Mastercycler, Eppendorf, Germany).
Food Bioscience 5 (2014) 47 –53
49
Table 1 – Primers and PCR conditions used to amplify various pathogenic genes in tested bacteria. Target gene
Primer sequence (5′–3′)
Amplicon size (bp)
Reaction conditions
sarA
F-TTAGCTTTGAAGAATTCGCTGT R-TTCAATTTCGTTGTTTGCTTC
275
Initial denaturation: 95 1C, 2 min; 35 cycles of: denaturation at 94 1C, 30 s annealing at 55 1C, 30 s and extension at 72 1C, 1 min; final extension: 72 1C, 10 min
Pi-PLC
F-AGTATGGGGAATGAC R-ACAATTTTCCCACGA
342
Initial denaturation: 94 1C, 5 min; 35 cycles of: denaturation at 94 1C, 1 min, annealing at 50 1C, 1 min and extension at 72 1C, 1 min; final extension: 72 1C, 8 min
ECstx2e
F-TGTGGCTGGGTTCGTTAATACGGC R-TCCGTTGTCATGGAAACCGTTGTC
101
Initial denaturation: 94 1C, 2 min; 35 cycles of: denaturation at 94 1C, 30 s, annealing at 52.6 1C, 30 s and extension at 72 1C, 1 min; final extension: 72 1C, 10 min
Ail
F-CTATTGGTTATGCGCAAAGC R-TGGAAGTGGGTTGAATTGCA
359
Initial denaturation: 95 1C, 2 min; 35 cycles of denaturation at 94 1C, 1 min, annealing at 55 1C, 1 min and extension at 72 1C, 1 min; final extension: 72 1C, 8 min
The resultant PCR products were analyzed by agarose gel electrophoresis.
2.7.
Haemolytic activity of extracts
To test whether the extracts contain factors that are toxic to higher animals, the haemolytic activity of extracts were determined using fresh rat red blood cells (Liu et al., 2007). The blood was collected in a tube containing ethylenediaminetetraacetic acid (EDTA). The erythrocytes were isolated by centrifugation at 5000 rpm for 10 min and washed twice with phosphate buffered saline (PBS, 100 mM, pH 7.4) to remove plasma and buffy coat. Cell pallet was resuspended in PBS to the original volume of the blood. An aliquot (0.1 ml) of the RBC was added to a test tube containing different concentrations of bark and fruit extract (625–10000 mg l 1 in 1.9 ml PBS). It was incubated for 1 h at 37 1C in shaking conditions (120 rpm), and then centrifuged at 5000 rpm for 5 min. The absorbance of the supernatant was measured at 550 nm. The baseline haemolysis and 100% haemolysis were defined as the amount of haemoglobin released in the presence of PBS and 0.1% Triton X-100, respectively. Only extracts without addition of RBC were also included in the study to eliminate the effect of extract colour.
2.8.
Statistical analysis
Since the MIC values were the same in 4 experiments, the values were represented as such. The data of all other experiments were expressed as mean7SD (n ¼3). The effect of bark and fruit extracts on antimutagenic activity
and nucleic acid leakage was compared by t-test using Microsoft Excel.
3.
Results and discussion
Many herbs and plant extracts possess antimicrobial activity against a wide range of bacteria, yeast and molds and have been used in traditional medicines worldwide. In the present study, the 70% aqueous acetone extracts from the bark and fruit rinds of D. indica showed antibacterial activity against all the tested foodborne pathogens (Table 2), however, the bark extract was found to be the more effective against growth of the foodborne pathogens studied. The minimum concentration required to inhibit the complete growth by fruit extracts were 2000–10000 mg l 1 against different bacteria, whereas for similar effect only 1250–5000 mg l 1 of bark extract were required. The antimicrobial activity of the plant products can be attributed to the phenolic compounds that are present within them. Phenolics, when incorporated into packaging films, also showed a good antibacterial activity (Aguirre, Borneo, & Leon, 2013). The phenolic content of aqueous acetone extract of bark was found to be 54.17% as tannic acid equivalent (Deepa & Jena, 2011), whereas lower phenolics in fruit extracts were reported (Abdille, Singh, Jayaprakasha, & Jena, 2005). Probably these phenolics were responsible for variable antibacterial effect of fruit and bark extracts as higher antibacterial effect was observed for bark extracts having higher phenolics. The antimicrobial mechanism of phenolics is linked to their molecules structures (hydroxyl groups or phenolic ring) and is related to their capacity to complex with proteins and
50
Food Bioscience 5 (2014) 47 –53
4
Microorganism
3
Bacillus Cereus Yersinia enterocolitica Staphylococcus aureus Escherichia coli a
Minimum Inhibitory concentration (mg l 1)a Bark extract
Fruit extract
2500 1250 2500 5000
5,000 2,000 5,000 10,000
OD at 260 nm
Table 2 – Antibacterial activity of Dillenia indica extracts against tested bacteria.
B. cereus
Y. enterocolitica
S. aureus
E. coli
2
1
0
Values are from 4 experiments where no growth was observed.
B MIC
B .5 MIC
F MIC
F .5 MIC
Fig. 2 – The loss of genetic material from bacterial cells immediately after treatment with D. indica extracts.
% inhibition
80
40
0 500
1000
1500
2000
Concentration (µg/ plate)
Fig. 1 – Antimutagenic activity of Dillenia indica extracts ( Bark Fruit) against sodium azide induced mutation in Salmonella typhimurium strain TA 1531.
bacterial membrane (Zongo, Savadogo, Somda, Koudou, & Traore, 2011). The mechanism involves the alteration of the permeability of the cell membrane, binding to the active site of cellular enzymes, dissipation of the proton motive force, and disruption of the lipid structures (Negi, 2012). Treatment with phenolic compounds may also result in disintegration of cell wall of the bacteria leading to cell death (Borneman, Akin, & Vaneseltine, 1986). Fruits and vegetables are known to be the major sources of antimutagens and/or anticarcinogens. The bark and fruit extracts of D. indica also showed antimutagenic activity against sodium azide induced mutation in Salmonella tester strain TA 1531 (Fig. 1). Bark extract was more effective (po0.01) antimutagen as compare to fruit extract at 500 μg/ plate concentration, and showed moderate antimutagenic activity, whereas fruit extract had weak activity at similar concentration. Both the extracts showed moderate activity at 1000 μg/plate concentration and strong activity at 1500 and 2000 μg/plate concentration, but their antimutagenic activities were statistically similar (p40.05) at these concentrations. Dillenia fruit and bark extracts showed variable antimutagenicity, which probably may be attributed to the quantity of phenolics present in them as was seen earlier with extracts of fruits and vegetables (Ikken et al., 1999). In the present study, a concentration dependent loss of 260 nm absorbing material was observed for all the bacteria immediately after treatment with bark and fruit extracts of D.
indica (Fig. 2), and the leakage was directly proportional to the concentration of extract. Bark extracts caused significantly (po0.05) higher leakage than fruit extracts in all the bacteria at similar concentration and E. coli showed highest leakage, whereas Y. enterocolitica showed least leakage among all the bacteria tested. Plant phenolics are known to interact with DNA (Chadfield & Hinton, 2004); and similar to present study, a significant loss of OD260 absorbing material in Candida albicans treated with tea tree oil was reported (Hammer, Carson, & Riley, 2004). The changes in the cell wall of the bacteria after treatments with the extracts were clearly visible in SEM (Fig. 3). The cell wall exhibited slimy appearance and rupture of cell wall was also observed at MIC of fruit extract in S. aureus, MIC of bark extract in B. cereus and Y. enterocolitica. In E. coli, a decrease in size of cells was observed after treatment with both fruit and bark extracts. In general, the bacteria after treatment with extracts showed a lot of adhered material around the cell wall. Structural alterations were induced by exposure to increasing concentrations of nitrofuran, which resulted in gross structural abnormalities, and extracellular surface structures were also visible in the form of blebs on the cell wall (Chadfield & Hinton, 2004). Tea tree oil treated cells of S. aureus contained multilamellar mesosomes like structures that were not seen in untreated cells and content of some cells appeared depleted (Carson et al., 2002). The DNA extracted from cells treated with extracts were analysed for the presence of virulence genes of the respective bacterium, and no hybridization for cultures treated with 0.5 MIC and MIC of bark and fruit extract was observed (Fig. 4), whereas the untreated samples showed the presence of sarA gene in S. aureus (275 bp), Pi-PLC gene in B. cereus (342 bp), ECstx2e gene in E. coli (101 bp) and ail gene in Y. enterocolitica (359 bp). Vattem, Mihalik, Crixell, & McLean, (2007) postulated that the dietary phytochemicals may lower the level of virulence factors/ functions in bacteria. In the present study, the virulence genes could not be detected in all the tested bacteria when treated with MIC and 0.5 MIC of Dillenia extracts. Perilla oil is also reported to suppress the alphatoxin, SEA, SEB, and TSST-1 expression in S. aureus in a dosedependent manner (Qiu, Zhang, Luo, Li, & Dong 2011). Haemolytic activity of extracts was determined using rat blood at concentration ranging from 625 to 5000 mg l 1 for bark extracts and 1250–10000 mg l 1 for fruit extract. No haemolysis was observed for bark extract even at 5000 mg l 1
Food Bioscience 5 (2014) 47 –53
51
Fig. 3 – Changes in bacterial cell wall after treatment with D. indica extracts. (A) Staphylococcus aureus control, (B) S. aureus treated with MIC of bark extract, (C) S. aureus treated with MIC of fruit extract, (D) Escherichia coli control, (E) E. coli treated with MIC of bark extract, (F) E. coli treated with MIC of fruit extract, (G) Bacillus cereus control, (H) B. cereus treated with MIC of bark extract, (I) B. cereus treated with MIC of fruit extract, (J) Yersinia enterocolitica control, (K) Y. enterocolitica treated with MIC of bark extract, (L) Y. enterocolitica treated with MIC of fruit extract.
52
Food Bioscience 5 (2014) 47 –53
Fig. 4 – Agarose gel electrophoresis for the PCR product. (A) SarA gene (275 bp) of S. aureus, (B) Pi-PLC gene (342 bp) of B. cereus, (C) ail gene (359 bp) of Y. enterocolitica and (D) ECVT2 gene (101 bp) of E. coli Ln 2,7,12 and 17- treated with MIC of bark extract; 3, 8, 13 and 18-treated with 0.5 MIC of bark extract; 4,9,14 and 19- treated with MIC of fruit extract; 5 10, 11 and 20- treated with 0.5 MIC of fruit extract; 1, 6, 15 and 16- untreated DNA; M is 100 bp DNA ladder
and for fruit extract at and below 2500 mg l 1 concentration, whereas fruit extract showed 21.3771.30 and 29.8071.00% (n ¼3) haemolytic activity at 5000 and 10000 mg l 1, respectively. Dillenia extracts seems to cause less haemolysis as compare to tick defensing (Nakajima et al., 2003), and a low haemolytic activity against erythrocytes is desirable from pharmaceutical point of view.
4.
Conclusions
The results of present study show that the bark and fruit extracts of D. indica possess antibacterial and antimutagenic activities. The strong antimutagenic activity observed in the present study indicates potential of these extracts for cancer chemoprevention, which needs to be exploited. The antibacterial activity of extracts may probably be due to the disintegration of cell wall and leakage of 260 nm absorbing material. Further, the treatment with plant extracts suppressed the virulence in tested bacteria. It is possible that the isolation of bioactive constituents from these extracts might lead to the development of new class of antimicrobials and reveal the exact mechanism of their action.
Acknowledgements The authors wish to express sincere thanks to Mr. K. Anbalagan, Senior Technician, Central Instruments Facility and Services, CSIR-CFTRI, Mysore for help in SEM studies.
r e f e r e n c e s
Abdille, M. H., Singh, R. P., Jayaprakasha, G. K., & Jena, B. S. (2005). Antioxidant activity of the extracts from Dillenia indica fruits. Food Chemistry, 90, 891–896. Aguirrea, A., Borneo, R., & Leon, A. E. (2013). Antimicrobial, mechanical and barrier properties of triticale protein films incorporated with oregano essential oil. Food Bioscience, 1, 2–9. Borneman, W. S., Akin, D. E., & Vaneseltine, W. P. (1986). Effect of phenolic monomers on ruminal bacteria. Applied and Environmental Microbiology, 52, 1331–1339. Carson, C. F., Mee, B. J., & Riley, T. V. (2002). Mechanism of action of Melaleuca alternifolia (tea tree) oil Staphylococcus aureus determined by time kill lysis, leakage and salt tolerance assays and electron microscopy. Antimicrobial Agents and Chemotherapy, 46, 1914–1920. Chadfield, M. S., & Hinton, M. H. (2004). In vitro activity of nitrofuran derivatives on growth and morphology of
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Salmonella enteric serotypre Enteritidis. Journal of Applied Microbiology, 96, 1002–1012. Deepa, N., & Jena, B. S. (2011). Antioxidant fraction from bark of Dillenia Indica. International Journal of Food Properties, 14, 1152–1159. Hammer, K. A., Carson, C. F., & Riley, T. V. (2004). Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. Journal of Antimicrobial Chemotherapy, 53, 1081–1085. Ikken, Y., Morales, P., Martinez, A., Marin, M. L., Haza, A. I., & Cambero, M. I. (1999). Antimutagenic effect of fruit and vegetable ethanolic extracts against N-nitrosamines evaluated by the Ames test. Journal of Agricultural and Food Chemistry, 47, 3257–3264. Liu, Z., Brady, A., Young, A., Rasimick, B., Chen, K., Zhou, C., & Kallenbach, N. (2007). Length effects in antimicrobial peptides of the (RW)n series. Antimicrobial Agents and Chemotherapy, 51, 597–603. Maron, D. M., & Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutation Research, 113, 173–215. Moosavy, M. H., Basti, A. A., Misaghi, A., Salehi, T. Z., Abbasifar, R., Mousavi, H. A. E., et al., (2008). Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes. Food Research International, 41, 1050–1057. Nakajima, Y., Ishibashi, J., Yukuhiro, F., Asaoka, A., Taylor, D. M., & Yamakawa, M. (2003). Antibacterial activity and mechanism of action of tick defensin against Gram-positive bacteria. Biochimica et Biophysica Acta (BBA)—General Subject, 1624, 125–130. Negi, P. S. (2012). Plant extracts for the control of bacterial growth: Efficacy, stability and safety issues for food application. International Journal of Food Microbiology, 156, 7–17.
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Negi, P. S., Jayaprakasha, G. K., Rao, L. J. M., & Sakariah, K. K. (1999). Antibacterial activity of turmeric oil—A byproduct from curcumin manufacture. Journal of Agricultural and Food Chemistry, 47, 4297–4300. Qiu, J., Zhang, X., Luo, M., Li, H., Dong, J., et al., (2011). Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus. PLoS One, 6, e16160. Ramesh, A., Padmapriya, B. P., Bharathi, S., & Varadaraj, M. C. (2003). Yersinia enterocolitica detection and treatment. In B. L. Cabellero, C Trugo, & P. M. Finglas (Eds.), Encyclopedia of food science and nutrition. New York: Academic Press/ Elsevier. Rohinishree, Y. S., & Negi, P. S. (2012). Multiplex reverse transcription polymerase chain reaction to study the expression of virulence and stress response genes in Staphylococcus aureus. Journal of Food Science, 77, M95–M101. Saikia, L., & Saikia, J. (2002). Processing of Dillenia indica fruits for preparation of squash and its quality changes during storage. Journal of Food Science and Technology, 39, 149–151. Scallan, E., Hoekstra, R. M., Angulo, F. J., Tauxe, R. V., Widdowson, M. A., Roy, S. L., Jones, J. L., & Griffin, P. M. (2011). Foodborne illness acquired in the United States-major pathogens. Emerging Infectious Diseases, 17, 7–15. Vattem, D. A., Mihalik, K., Crixell, S. H., & McLean, R. J. C. (2007). Dietary phytochemicals as quorum sensing inhibitors. Fitoterapia, 78, 302–310. Zongo, C., Savadogo, A., Somda, K. M., Koudou, J., & Traore, A. S. (2011). In-vitro evaluation of the antimicrobial and antioxidant properties of extracts from whole plant of Alternanthera pungens H.B. & K. and leaves of Combretum sericeum G. Don. International Journal of Phytomedicine, 3, 182–191.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/fbio
Antibacterial and antimutagenic activities of Dillenia indica extracts Sweta Jaiswal, N. Mansa, M.S. Pallavi Prasad, Bhabani Sankar Jena1, Pradeep Singh Negin Fruit and Vegetable Technology, CSIR-Central Food Technological Research Institute, Mysore-570020, India
art i cle i nfo
ab st rac t
Article history:
In the present study, the antibacterial and antimutagenic activities of the fruit and bark
Received 21 May 2013
extracts of Dillenia indica were evaluated and the effect of their inhibitory concentrations on
Received in revised form
cell wall, nucleic acid leakage and pathogenic genes of the bacteria was studied. The fruit
9 October 2013
and bark extracts obtained by 70% aqueous acetone extraction showed minimum
Accepted 15 November 2013
inhibitory concentration (using agar dilution method) against different bacteria in the range of 2000–10,000 and 1250–5000 mg l 1, respectively, indicating higher antibacterial
Keywords:
activity for bark extract. At 500 μg/plate concentration, bark extract showed significantly
Antibacterial
(po0.01) higher antimutagenic activity in Ames test against the sodium azide induced
Antimutagenic
mutation in Salmonella tester strain (TA-1531). Both the extracts showed statistically
Dillenia indica
similar but strong antimutagenic activity at or above 1500 μg/plate concentration.
Minimum inhibitory concentration
The ability of these extracts to cause the disintegration of cell wall and leakage of genetic
Pathogenic genes
material is most likely to be the factor for their antibacterial activity. Further the extracts were able to inhibit the pathogenic genes present in tested bacteria. The fruit and bark extracts did not show any hemolysis at and below 2500 and 5000 mg l 1 concentration, respectively. The D. indica fruit and bark aqueous acetone extract may find application in foods and pharmaceuticals owing to their inhibitory properties. & 2013 Elsevier Ltd. All rights reserved.
1.
Introduction
In recent years, there has been a dramatic increase in the number of reported cases of food borne illness (Scallan et al., 2011), consequently, there is considerable interest in finding out the ways to stop this upward trend and reduce the incidence of food poisoning. Due to negative consumer perceptions of artificial preservatives, attention is shifting towards alternatives that consumers perceive as natural, and in particular plant extracts, including their essential oils and essences are being used for preservation. It is well established
n
that several plant extracts have antimicrobial properties against bacteria, moulds and yeast, but their mechanism of antimicrobial action is not very clear. Membrane disruption by terpenoids and phenolics; metal chelation by phenols and flavonoids; and effect on genetic material by coumarin and alkaloids are reported to be responsible for inhibition of growth of microorganisms, and therefore, the versatile compositions of plant extract make them potential natural agents for food preservation (Negi, 2012). Plants also contain several compounds, which have been reported to be inhibitors of chemical carcinogenesis, and due
Corresponding author. Tel.: þ91 821 2515653; fax: þ91 821 2517233. E-mail address: [email protected] (P.S. Negi). 1 Present Address: Principal Scientist, RDPD and Bioresource Engineering, CSIR-Institute of Minerals and Materials Technology, Bhubaneswar- 751 013, India. 2212-4292/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fbio.2013.11.005
48
Food Bioscience 5 (2014) 47 –53
to the presence of these compounds several plant extracts are reported to be antimutagenic. The regular intake of antimutagenic agents can reduce genotoxic effects of mutagenic and carcinogenic factors, and use of antimutagens in diet is suggested as the most effective method for preventing cancer and genetic disorders (Ikken et al., 1999). Dillenia (Family: Dilleniaceae) is a small genus of trees found in Indo-Malaysian region extending to tropical Australia, and Dillenia indica is found in the forests of subHimalayan tract, from Uttarakhand eastwards to Assam. The ripe fruits are sour in taste and are used for flavouring of curies and preparation of jam and jelly. The acid juice is sweetened with sugar and used as cooling drink, and the fruits are also processed to commercial products such as ready-to-serve beverage and squash (Saikia & Saikia, 2002). Although fruits are used traditionally in some medicinal preparation by local population, to our knowledge, no systematic report exists about their antibacterial and antimutagenic properties. Therefore, in the present investigation, we have studied the antibacterial and antimutagenic properties of the fruit and bark extracts of D. indica. Further, the effect of the minimum inhibitory concentration on bacterial cell wall, nucleic acid leakage and pathogenic genes of the bacteria were also studied to find out the probable mode of their antibacterial action.
2.
Materials and methods
2.1.
Plant extract preparation
The fruits and bark of D. indica were obtained from Balasore, Odissa, India. The fruits were rinsed with distilled water and dried in shade. The dried fruit and bark of D. indica were powdered and the known quantity of the powder was extracted with 70% aqueous acetone. The extracts were dried under vacuum and the solvent free extracts were dissolved in propylene glycol (PG) for use in the present study.
2.2. (MIC)
Determination of minimum inhibitory concentration
The fruit and the bark extracts from the D. indica were tested against food-borne pathogens, Bacillus cereus (F 4810, Public Health Laboratory, London, UK), Staphylococcus aureus (FRI 722, Public Health Laboratory, The Netherlands), Escherichia coli (MTCC 108, Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India) and Yersinia enterocolitica (MTCC 859, Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India) by agar dilution method (Negi, Jayaprakasha, Rao, & Sakariah, 1999). Minimum Inhibitory Concentration (MIC) was defined as the lowest concentration at which no bacterial growth was observed after incubation.
2.3.
Antimutagenic activity assay
The antimutagenic activity of fruit and the bark extracts of the D. indica was determined in terms of the inhibitions of mutagenic activity of the sodium azide using Salmonella
typhimurium (TA-1531, Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh, India) by standard plate incorporation test (Maron & Ames, 1983).
2.4.
Effect of the extracts on 260 nm absorbing material
For determination of loss of 260 nm absorbing material, the MIC and 0.5 MIC of the extracts were mixed with 100 μl of the cells (OD 0.3) and volume was made up to 1 ml using phosphate buffered saline (PBS). Immediately after addition of extract, the OD was measured at 260 nm against PBS blank (Carson, Mee, & Riley, 2002).
2.5.
Effect of the extracts on bacterial cell wall
Scanning Electron Microscopy (SEM) was used to investigate the effect of extracts on bacterial cell wall by the method described by Moosavy et al. (2008) with slight modifications. Overnight cultures were centrifuged at 7000 rpm for 10 min at 4 1C, washed twice with 0.1 M phosphate buffer (pH 6.5) and volume was made up to 0.5 ml with the same buffer. The MIC of the extracts was added and the final volume was made up to 1 ml using phosphate buffer. The above cell suspension was incubated for 1 h and cells were harvested at 6000 rpm for 10 min at 4 1C. The pellet was incubated in 1% glutaraldehyde overnight at 0 1C and the cells were harvested at 6000 rpm for 10 min at 4 1C. The cells were dehydrated in ethanol gradient (10–100%) and coated with a thin layer of gold using polaron SEM coating system. The cells were observed with a LEO 435 VP Scanning Electron Microscope at 20 kV attached to Mitsubishi Video copy processor. Photographs were taken using 35 mm Richo camera that was connected to monitor optically through fibre optics.
2.6.
Effect of extracts on pathogenic genes of bacteria
DNA from overnight grown B. cereus, E. coli, Y. enterocolitica cells was extracted by using Triton X 100 as a detergent, wherein culture grown in the presence of 0.5 MIC and MIC of the extracts was washed with 0.5% triton X100, heated at 95 1C for 10 min in 0.5% triton X, centrifuged at 6000 rpm at 4 1C for 15 min, and the supernatant was used as DNA (Ramesh, Padmapriya, Bharathi, & Varadaraj, 2003). For S. aureus, the DNA was extracted essentially by the method of Rohinishree & Negi (2012). Integrity of DNA was checked by agarose gel electrophoresis and it was quantified using absorbance reading at 260 nm. The DNA was used to check the presence of pathogenic genes in S. aureus (Staphylococcus Accessory Regulator, sarA), B. cereus (Phosphatidylinositol phospholipase, Pi-PLC), E. coli (E. coli exotoxin shiga toxin 2e, ECstx2e) and Y. enterocolitica (Attachment Invasion Loci, ail) using gene specific primers (Table 1) synthesized from Sigma Aldrich (Bangalore, India). The contents (2 ng DNA, 10 PCR buffer, 0.2 mM dNTP, 0.2 mM of both forward and reverse primers, I U Taq polymerase and water to make up 25 μl reaction mixture) were mixed a thin walled PCR tube and placed in a thermalcycler (Mastercycler, Eppendorf, Germany).
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Table 1 – Primers and PCR conditions used to amplify various pathogenic genes in tested bacteria. Target gene
Primer sequence (5′–3′)
Amplicon size (bp)
Reaction conditions
sarA
F-TTAGCTTTGAAGAATTCGCTGT R-TTCAATTTCGTTGTTTGCTTC
275
Initial denaturation: 95 1C, 2 min; 35 cycles of: denaturation at 94 1C, 30 s annealing at 55 1C, 30 s and extension at 72 1C, 1 min; final extension: 72 1C, 10 min
Pi-PLC
F-AGTATGGGGAATGAC R-ACAATTTTCCCACGA
342
Initial denaturation: 94 1C, 5 min; 35 cycles of: denaturation at 94 1C, 1 min, annealing at 50 1C, 1 min and extension at 72 1C, 1 min; final extension: 72 1C, 8 min
ECstx2e
F-TGTGGCTGGGTTCGTTAATACGGC R-TCCGTTGTCATGGAAACCGTTGTC
101
Initial denaturation: 94 1C, 2 min; 35 cycles of: denaturation at 94 1C, 30 s, annealing at 52.6 1C, 30 s and extension at 72 1C, 1 min; final extension: 72 1C, 10 min
Ail
F-CTATTGGTTATGCGCAAAGC R-TGGAAGTGGGTTGAATTGCA
359
Initial denaturation: 95 1C, 2 min; 35 cycles of denaturation at 94 1C, 1 min, annealing at 55 1C, 1 min and extension at 72 1C, 1 min; final extension: 72 1C, 8 min
The resultant PCR products were analyzed by agarose gel electrophoresis.
2.7.
Haemolytic activity of extracts
To test whether the extracts contain factors that are toxic to higher animals, the haemolytic activity of extracts were determined using fresh rat red blood cells (Liu et al., 2007). The blood was collected in a tube containing ethylenediaminetetraacetic acid (EDTA). The erythrocytes were isolated by centrifugation at 5000 rpm for 10 min and washed twice with phosphate buffered saline (PBS, 100 mM, pH 7.4) to remove plasma and buffy coat. Cell pallet was resuspended in PBS to the original volume of the blood. An aliquot (0.1 ml) of the RBC was added to a test tube containing different concentrations of bark and fruit extract (625–10000 mg l 1 in 1.9 ml PBS). It was incubated for 1 h at 37 1C in shaking conditions (120 rpm), and then centrifuged at 5000 rpm for 5 min. The absorbance of the supernatant was measured at 550 nm. The baseline haemolysis and 100% haemolysis were defined as the amount of haemoglobin released in the presence of PBS and 0.1% Triton X-100, respectively. Only extracts without addition of RBC were also included in the study to eliminate the effect of extract colour.
2.8.
Statistical analysis
Since the MIC values were the same in 4 experiments, the values were represented as such. The data of all other experiments were expressed as mean7SD (n ¼3). The effect of bark and fruit extracts on antimutagenic activity
and nucleic acid leakage was compared by t-test using Microsoft Excel.
3.
Results and discussion
Many herbs and plant extracts possess antimicrobial activity against a wide range of bacteria, yeast and molds and have been used in traditional medicines worldwide. In the present study, the 70% aqueous acetone extracts from the bark and fruit rinds of D. indica showed antibacterial activity against all the tested foodborne pathogens (Table 2), however, the bark extract was found to be the more effective against growth of the foodborne pathogens studied. The minimum concentration required to inhibit the complete growth by fruit extracts were 2000–10000 mg l 1 against different bacteria, whereas for similar effect only 1250–5000 mg l 1 of bark extract were required. The antimicrobial activity of the plant products can be attributed to the phenolic compounds that are present within them. Phenolics, when incorporated into packaging films, also showed a good antibacterial activity (Aguirre, Borneo, & Leon, 2013). The phenolic content of aqueous acetone extract of bark was found to be 54.17% as tannic acid equivalent (Deepa & Jena, 2011), whereas lower phenolics in fruit extracts were reported (Abdille, Singh, Jayaprakasha, & Jena, 2005). Probably these phenolics were responsible for variable antibacterial effect of fruit and bark extracts as higher antibacterial effect was observed for bark extracts having higher phenolics. The antimicrobial mechanism of phenolics is linked to their molecules structures (hydroxyl groups or phenolic ring) and is related to their capacity to complex with proteins and
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4
Microorganism
3
Bacillus Cereus Yersinia enterocolitica Staphylococcus aureus Escherichia coli a
Minimum Inhibitory concentration (mg l 1)a Bark extract
Fruit extract
2500 1250 2500 5000
5,000 2,000 5,000 10,000
OD at 260 nm
Table 2 – Antibacterial activity of Dillenia indica extracts against tested bacteria.
B. cereus
Y. enterocolitica
S. aureus
E. coli
2
1
0
Values are from 4 experiments where no growth was observed.
B MIC
B .5 MIC
F MIC
F .5 MIC
Fig. 2 – The loss of genetic material from bacterial cells immediately after treatment with D. indica extracts.
% inhibition
80
40
0 500
1000
1500
2000
Concentration (µg/ plate)
Fig. 1 – Antimutagenic activity of Dillenia indica extracts ( Bark Fruit) against sodium azide induced mutation in Salmonella typhimurium strain TA 1531.
bacterial membrane (Zongo, Savadogo, Somda, Koudou, & Traore, 2011). The mechanism involves the alteration of the permeability of the cell membrane, binding to the active site of cellular enzymes, dissipation of the proton motive force, and disruption of the lipid structures (Negi, 2012). Treatment with phenolic compounds may also result in disintegration of cell wall of the bacteria leading to cell death (Borneman, Akin, & Vaneseltine, 1986). Fruits and vegetables are known to be the major sources of antimutagens and/or anticarcinogens. The bark and fruit extracts of D. indica also showed antimutagenic activity against sodium azide induced mutation in Salmonella tester strain TA 1531 (Fig. 1). Bark extract was more effective (po0.01) antimutagen as compare to fruit extract at 500 μg/ plate concentration, and showed moderate antimutagenic activity, whereas fruit extract had weak activity at similar concentration. Both the extracts showed moderate activity at 1000 μg/plate concentration and strong activity at 1500 and 2000 μg/plate concentration, but their antimutagenic activities were statistically similar (p40.05) at these concentrations. Dillenia fruit and bark extracts showed variable antimutagenicity, which probably may be attributed to the quantity of phenolics present in them as was seen earlier with extracts of fruits and vegetables (Ikken et al., 1999). In the present study, a concentration dependent loss of 260 nm absorbing material was observed for all the bacteria immediately after treatment with bark and fruit extracts of D.
indica (Fig. 2), and the leakage was directly proportional to the concentration of extract. Bark extracts caused significantly (po0.05) higher leakage than fruit extracts in all the bacteria at similar concentration and E. coli showed highest leakage, whereas Y. enterocolitica showed least leakage among all the bacteria tested. Plant phenolics are known to interact with DNA (Chadfield & Hinton, 2004); and similar to present study, a significant loss of OD260 absorbing material in Candida albicans treated with tea tree oil was reported (Hammer, Carson, & Riley, 2004). The changes in the cell wall of the bacteria after treatments with the extracts were clearly visible in SEM (Fig. 3). The cell wall exhibited slimy appearance and rupture of cell wall was also observed at MIC of fruit extract in S. aureus, MIC of bark extract in B. cereus and Y. enterocolitica. In E. coli, a decrease in size of cells was observed after treatment with both fruit and bark extracts. In general, the bacteria after treatment with extracts showed a lot of adhered material around the cell wall. Structural alterations were induced by exposure to increasing concentrations of nitrofuran, which resulted in gross structural abnormalities, and extracellular surface structures were also visible in the form of blebs on the cell wall (Chadfield & Hinton, 2004). Tea tree oil treated cells of S. aureus contained multilamellar mesosomes like structures that were not seen in untreated cells and content of some cells appeared depleted (Carson et al., 2002). The DNA extracted from cells treated with extracts were analysed for the presence of virulence genes of the respective bacterium, and no hybridization for cultures treated with 0.5 MIC and MIC of bark and fruit extract was observed (Fig. 4), whereas the untreated samples showed the presence of sarA gene in S. aureus (275 bp), Pi-PLC gene in B. cereus (342 bp), ECstx2e gene in E. coli (101 bp) and ail gene in Y. enterocolitica (359 bp). Vattem, Mihalik, Crixell, & McLean, (2007) postulated that the dietary phytochemicals may lower the level of virulence factors/ functions in bacteria. In the present study, the virulence genes could not be detected in all the tested bacteria when treated with MIC and 0.5 MIC of Dillenia extracts. Perilla oil is also reported to suppress the alphatoxin, SEA, SEB, and TSST-1 expression in S. aureus in a dosedependent manner (Qiu, Zhang, Luo, Li, & Dong 2011). Haemolytic activity of extracts was determined using rat blood at concentration ranging from 625 to 5000 mg l 1 for bark extracts and 1250–10000 mg l 1 for fruit extract. No haemolysis was observed for bark extract even at 5000 mg l 1
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Fig. 3 – Changes in bacterial cell wall after treatment with D. indica extracts. (A) Staphylococcus aureus control, (B) S. aureus treated with MIC of bark extract, (C) S. aureus treated with MIC of fruit extract, (D) Escherichia coli control, (E) E. coli treated with MIC of bark extract, (F) E. coli treated with MIC of fruit extract, (G) Bacillus cereus control, (H) B. cereus treated with MIC of bark extract, (I) B. cereus treated with MIC of fruit extract, (J) Yersinia enterocolitica control, (K) Y. enterocolitica treated with MIC of bark extract, (L) Y. enterocolitica treated with MIC of fruit extract.
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Fig. 4 – Agarose gel electrophoresis for the PCR product. (A) SarA gene (275 bp) of S. aureus, (B) Pi-PLC gene (342 bp) of B. cereus, (C) ail gene (359 bp) of Y. enterocolitica and (D) ECVT2 gene (101 bp) of E. coli Ln 2,7,12 and 17- treated with MIC of bark extract; 3, 8, 13 and 18-treated with 0.5 MIC of bark extract; 4,9,14 and 19- treated with MIC of fruit extract; 5 10, 11 and 20- treated with 0.5 MIC of fruit extract; 1, 6, 15 and 16- untreated DNA; M is 100 bp DNA ladder
and for fruit extract at and below 2500 mg l 1 concentration, whereas fruit extract showed 21.3771.30 and 29.8071.00% (n ¼3) haemolytic activity at 5000 and 10000 mg l 1, respectively. Dillenia extracts seems to cause less haemolysis as compare to tick defensing (Nakajima et al., 2003), and a low haemolytic activity against erythrocytes is desirable from pharmaceutical point of view.
4.
Conclusions
The results of present study show that the bark and fruit extracts of D. indica possess antibacterial and antimutagenic activities. The strong antimutagenic activity observed in the present study indicates potential of these extracts for cancer chemoprevention, which needs to be exploited. The antibacterial activity of extracts may probably be due to the disintegration of cell wall and leakage of 260 nm absorbing material. Further, the treatment with plant extracts suppressed the virulence in tested bacteria. It is possible that the isolation of bioactive constituents from these extracts might lead to the development of new class of antimicrobials and reveal the exact mechanism of their action.
Acknowledgements The authors wish to express sincere thanks to Mr. K. Anbalagan, Senior Technician, Central Instruments Facility and Services, CSIR-CFTRI, Mysore for help in SEM studies.
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