Int J Pharm Pharm Sci, Vol 8, Issue 8, 189-193Original Article


REACTIVE OXYGEN SPECIES GENERATION IN THE ANTIBACTERIAL ACTIVITY OF LITSEA SALICIFOLIA LEAF EXTRACT

NITUMANI KALITA*a, MOHAN CHANDRA KALITAa, MADHUCHANDA BANERJEEb

aDepartment of Biotechnology, Gauhati University, GNB Road, Guwahati 781 014, Assam, India, bDepartment of Zoology, Midnapore College, Raja Bazar Main Rd, Medinipur, West Bengal 721101, India
Email: nitumani.kalita86@gmail.com

Received: 13 Apr 2016 Revised and Accepted: 20 June 2016

ABSTRACT

Objective: The present work was carried out to investigate the antibacterial activity of Litsea salicifolia leaf extract and to study whether there is a generation of oxidative stress in its mechanism of antibacterial action.

Methods: L salicifolia was screened for its antibacterial activity against the bacterial strains collected from the Microbial Type Culture Collection and gene bank (MTCC) viz. Escherichia coli MTCC 443 and Staphylococcus aureus MTCC 96. Disc diffusion method was used for screening. The preliminary screening was done with petroleum ether (PE), chloroform (CHF), methanol (MT) and aqueous (AQ) extracts of the L. salicifolia leaf. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using macro-broth dilution method. In this work, oxidative stress on bacterial cells after exposure to plant extract was measured using nitroblue tetrazolium method (NBT).

Results: Experimental evidence indicated that the CHF extract is more efficient against S. aureus compared to the other extracts with MIC value of 0.076 mg/ml and MBC value of 0.4 mg/ml. Our results revealed that there was a generation of reactive oxygen species (ROS) in the treated bacterial cell cytoplasm. Transmission electron microscopy (TEM) revealed considerable damage in the cell envelope as well as morphological changes in the extract treated bacterial cells. There were also changes in DNA isolated from treated cells.

Conclusion: From the present study, we can conclude that the active constituents in the plant extract contribute in cell killing involving generation of free radical-induced oxidative stress, which possibly the cause or the consequence of the alteration of some other cellular mechanisms ultimately leading to cell death.

Keywords: Medicinal plant, Antibacterial activity, Oxidative stress

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

In recent years, although various antimicrobial agents have been newly introduced the increasing development of drug resistance of the infectious microbes has limited the lifespan of these new antimicrobial agents [1]. Clinical microbiologists are applying various strategies to combat drug-resistant microbes, including the search for novel antimicrobial agents from plants. Because from the recent past, plants have been an integral part of traditional medicine system and indigenous people of the world rely on plant-based medicines to cure various diseases. Besides, plant-derived antimicrobial agents are gaining popularity because of easy availability and less side effects [2]. Furthermore, antimicrobial drugs derived from plants will be new to the microbes as antimicrobial drugs of present times are mostly bacterial, or fungal origin [2] and therefore, microbes are less likely to develop resistance against plant-derived antimicrobial drugs.

Plants contain a variety of secondary metabolites such as phenols, quinones, flavonoids, alkaloids, tannins, terpenoids that have been proven to possess antimicrobial activity [3-6]. In most cases, these secondary metabolites are synthesized by plants in response to plant pathogen attack. The mechanism of plant antimicrobial activity is poorly understood, although there are some reports on the probable mechanism of action of phytochemicals such as flavonoids have been reported to inhibit DNA synthesis in Proteus vulgaris [7], alkaloids like berberine have been proposed to be potent inhibitors of various enzymes like lactate and malate dehydrogenase [8]. Phenolics were thought to exert their antimicrobial effect by inhibition of enzymes through reacting with sulfhydryl groups containing compounds [9]. Quinone, a class of phytochemicals is known to provide a source of stable free radicals as well as bind to proteins irreversibly inhibiting bacterial growth [10].

Investigation of the mechanism of bacterial killing by the novel antimicrobial agents may provide a better understanding of resistance mechanisms for the advancement of future drugs. Bactericidal antibiotics have been reported to kill bacteria by a common mechanism of cell death involving the induction of ROS, which subsequently results in DNA damage, protein oxidation and lipid peroxidation [11]. Antibiotic like quinolone, a gyrase inhibitor is known to interfere with the iron regulatory pathway resulting information of ROS which eventually cause cell death [12]. Plants are known to produce activated oxygen species to fight against pathogens [13]. The main objective of this study was to find out whether ROS generated oxidative stress is responsible for bacterial killing by L. salicifolia leaf extract. Furthermore, morphological changes in bacterial cells due to plant extract treatment were also demonstrated.

L. salicifolia (Roxb. ex Nees) Hook. f. commonly known as 'Dighloti' has been a part of traditional medicine systems in North East India from ancient time. The Apatani people of Ziro valley in Arunachal Pradesh are known to use the fruit of L. salicifolia in the treatment of bone fracture and stomach disorder [14]. The leaves of these plants are used in dysentery and pneumonia traditionally in Assam. L. salicifolia is cultivated for feeding Muga silk worms in North East India.

Rastogi and Borthakur [15] isolated two alkaloids dicentrinone and nordicentrine from leaves of L. salicifolia. There are reports on the antibacterial activity of these two alkaloids [16]. Phytochemical screening [17] of L. salicifolia leaf extract showed the presence of flavonoids, alkaloids, terpenoids, and peptides. There are few reports on the antibacterial activity of Litsea sp. [18, 19].

There is the previous report on the insecticidal activity of L. salicifolia [20]. This is the first report on antibacterial activity and mechanism of action of L. salicifolia leaf extract.

MATERIALS AND METHODS

Chemicals and reagents

Solvents (analytical grade) were obtained from Merck (Mumbai, Maharashtra). Chemicals including nitroblue tetrazolium chloride (NBT) and chloramphenicol were obtained from HiMedia Laboratories Pvt. Ltd. Dimethyl sulfoxide was obtained from HiMedia Laboratories Pvt. Ltd.

Preparation of extracts

Fresh leaves of L. salicifolia were collected locally from the foothills of Guwahati, Assam and the plant was identified at Botany Department, Gauhati University, Assam (voucher no. GUBH <17869>). The collected leaves were shade dried, and the dried leaves were ground to a fine powder using an electronic grinder. The powdered leaves were extracted with a series of solvents, PE, CHF and MT in the sequence of increasing polarity at room temperature using soxhlet apparatus and water extract was prepared by pouring double distilled water onto the fine powder and kept for 72 h at room temperature. The solvent extract and the filtrate of water extract were evaporated to dryness under reduced pressure using rotary vacuum evaporator [17].

Bacterial strain and culture condition

Two human pathogenic bacteria, one Gram positive (S. aureus MTCC 96) and one Gram-negative (E. coli MTCC 443) were obtained from MTCC and Gene Bank (MTCC). Strains were maintained at 4 °C on nutrient agar with periodical subculturing every month. The active culture required for bioassay was prepared by inoculation of a loopful of each strain into 10 ml of nutrient broth and incubated in a shaker at 37 °C for 18 h.

Preliminary antibacterial assay

The preliminary antibacterial assay was carried out using disc diffusion method [21]. With the help of a glass spreader standardized inoculums of approximately 1× 105CFU/ml (0.5 McFarland standard) were evenly spread over the surface of the plate containing Muller Hinton Agar media (MHA). Discs of 6 mm diameter prepared from Whatman filter paper No. 1, loaded with plant sample were placed on the surface of the medium and kept for 30 min at room temperature under laminar flow for compound diffusion. Chloramphenicol (10 µg per disc) was used as positive control and blank discs with dimethyl sulfoxide (DMSO) were used as negative control. The plates were incubated for 18 h at 37 °. Each experiment was performed thrice and zone of inhibition was recorded in millimeters.

Determination of MIC and MBC

For determination of MIC and MBC macro-broth dilution method was used [22]. The bacteria (105CFUs/ml) were grown in tubes containing a different concentration of plant extracts for 18h. MIC was considered to be the lowest concentration of the extract at which there was no visible turbidity. MBC was taken as the lowest concentration at which there was no visible bacterial growth of cultures after re inoculation in fresh media.

Assay of oxidative stress on bacterial cells

Oxidative stress on the bacterial cell was determined using the NBT method [23]. To the bacterial suspensions (105 CFU/ml) 0.1 ml of Hanks' balanced salt solution (HBSS) was added and incubated with plant extract for 5, 30, 60, 90 and 120 min time intervals, respectively at 37 °C. To each of the solutions, 0.5 ml of NBT at a concentration of 1 mg/ml was added and incubated at 37 °C for 30 min. 0.1 M HCL was added to each of the solutions to stop the reaction, and bacterial pellet was obtained by centrifugation. The reduced NBT is extracted out with DMSO and the pellet was diluted with HBSS. Formation of Formazan blue was measured at an absorption spectrum of 575 nm using a spectrophotometer.

Preparation of cells for TEM analysis

Changes in morphology and cell envelope were studied using a high-resolution TEM (JEM 2100; Jeol) with an accelerating voltage of 200 KeV. Bacterial culture of cell density mentioned above was treated with the plant extract for 1 h at 37 °C. Untreated control cells were cultured in nutrient broth in the absence of extract. Cell pellets obtained from the treated and control cell suspension were diluted with 0.1M sodium cacodylate buffer and mixed thoroughly with an equal volume of 2% glutaraldehyde and then left for 15 min at 4 °C.

The pellet obtained after centrifugation (10000 rpm/10 min) was refixed with 2% glutaraldehyde for 1 h at 4 °C. The pellet was then rinsed with 0.1M sodium cacodylate buffer, and post fixation was carried out with 2% osmium tetroxide (OsO4). The samples were dehydrated with graded acetone series and embedding in the pure embedding medium using Beem capsules. Ultrathin sections were prepared in copper grids and allowed to dry. The sections on the grid were stained using a double staining technique involving uranyl acetate and lead citrate staining.

DNA isolation

Plasmid DNA was isolated [24] from the control and extract treated S. aureus and E. coli cells in order to understand the effect of the extract on bacterial DNA. The bacterial cells were treated for 30 min, 60 min and 90 min. The isolated DNA was analyzed using gel electrophoresis.

Statistical analysis

Student’s t-Test was used to study the level of significance (p value<0.05 was considered significant). Each experiment was carried out in triplicate (mean±SD).

RESULTS

Antibacterial activity of L. salicifolia leaf extract

In the preliminary screening, all the four extract of L. salicifolia leaf showed antibacterial activity against S. aureus with CHF extract showing a zone of inhibition equal to that of the positive control (chloramphenicol). E. coli appeared to be sensitive only against chloroform extract (table 1).

The MIC and MBC values were depicted in table 2 and table 3 respectively. The MIC values of CHF extract were 0.076±0.02 mg/ml for S. aureus and 0.096±0.041 mg/ml for E. coli. AQ extract exhibited highest MIC value for S. aureus. Although all the four extracts appeared to be inhibitory against S. aureus, CHF extract was found to be most effective showing lowest MIC and MBC values.

Table 1: Growth inhibition of E. coli and S. aureus by PE, CHF, MT and AQ extract of L. salicifolia

Plant name Pathogens Inhibition zone (mm)
PE
Litsea salicifolia (Roxb. ex Nees) Hook. f. E. coli NI
Staphylococcus aureus 15.5±0.4

NI: no inhibition; Cont*: positive control; cont**: Negative control, each experiment was carried out in triplicate (mean±SD)

Table 2: MIC of PE, CHF, MT and AQ extract against S. aureus and MIC of CHF extract against E. coli

Extract MIC (mg/ml)
S. aureus
PE 0.25±0.05
CHF 0.076±0.02
MT 0.38±0.076
AQ 1.33±0.57

NI: No inhibition, each experiment was carried out in triplicate (mean±SD)

Table 3: MBC of PE, CHF, MT and AQ extract against S. aureus and MBC of CHF extract against E. coli

Extract MBC (mg/ml)
S. aureus
PE 0.92±0.09
CHF 0.4±0.08
MT 2.3±0.5
AQ 4±0.5

NI: No inhibition, each experiment was carried out in triplicate (mean±SD)

Effect of plant extracts on oxidative stress

To investigate the role of oxidative stress on the antibacterial action of the CHF extract we studied the possible involvement of ROS pathway in the present system by performing NBT assay. The results of NBT assay revealed that there is an increase in ROS production with increasing time and reaches maximum in 90 min, after which there was no further increase in the case of S. aureus (fig. 1a).

a

b

Fig. 1: Effect of chloroform extract on the intracellular ROS concentration of: (a) S. aureus (b) E. coli
each bar represents mean±SD (n=3), Means of treated are significant (p<0.05) compared to control

In case of E. coli also there was an increase in the formation of Formazan blue confirming enhanced ROS production with time, showing the maximum optical density (OD) in 60 min. The control cells showed a negligible increase in oxidative stress in both S. aureus and E. coli (fig. 1b). Means of treated bacterial cells are significant compared to untreated control, as analyzed by Student’s t-test (p<0.05).

Changes in morphology in S. aureus and E. coli cells treated with L. salicifolia CHF extract

Analysis of the external morphological features of the two treated bacterial strains showed significant morphological changes. Untreated E. coli cells appeared to be normal in shape with a uniform inner membrane, an undamaged, intact outer membrane and thin periplasmic space (fig. 2a). The treated E. coli cells incubated with CHF extract showed swollen periplasmic space and some electron dense material got accumulated in the periplasmic space from the cytosol (fig. 2b and fig. 2c). There was leakage of the cytoplasmic content due to damage of the outer and inner membrane and finally complete disintegration of both the membranes (fig. 2d).

The untreated S. aureus cells showed normal coccal morphology under TEM micrographs with intact cell envelope (fig. 3a). The treated S. aureus cells showed damaged cell wall and membranes releasing electron dense particles in the surrounding environment (fig. 3b) and some cells appeared to undergo complete lysis (fig. 3c). Some cells were seen without a cell wall (fig. 3d and fig. 3e).

DNA analysis

The plasmid DNA isolated from the control S. aureus cells showed two bands on the gel, one band for supercoiled circular DNA and the other for nicked DNA and no DNA could be seen in the loading wells.

However, there was the formation of single-stranded breaks in the plasmid DNA isolated from the treated S. aureus and there are negligible, or no supercoiled DNA band could be seen in the gel (fig. 4). DNA could be seen on the loading well in case of treated cells.

Fig. 2: TEM micrographs of E. coli:

(a) Untreated bacteria. (b) and (c) after treatment with CHF extract there is a separation of inner and outer membrane and periplasmic space is swollen, accumulation of some electron dense particle in the periplasmic space (Arrow indicate swollen periplasmic space containing electron dense materials). (d) Leakage of cytoplasmic content due to membrane damage (indicated by an arrow) after treatment

Fig. 3: TEM micrographs of S. aureus.

(a) Untreated bacteria. (b) After treatment with CHF extract, there is the disintegration of cell wall and cell membrane, leakage of cytoplasmic content. (c) Complete lysis of some cells after treatment. (d) and (e) some cells are lacking cell wall after treatment

Fig. 4: Plasmid DNA bands analyzed by gel electrophoresis. Lane 1: Control DNA isolated from S. aureus, Lane 2: DNA isolated from S. aureus cells treated with L. salicifolia extracts for 30 min, Lane 3: DNA isolated from S. aureus cells treated with extracts for 60 min and Lane 4: DNA isolated from S. aureus cells treated with extracts for 90 min

DISCUSSION

Although various plants have been studied for their antibacterial activity [25], their mechanism of action on bacteria is not clearly understood in most cases. In the present work, it is evident from the results that the L. salicifolia leaf extracts are more effective against S. aureus than E. coli. This is due to structural differences in the cell envelope of these two bacteria [26]. Gram-positive bacteria lack the additional outer membrane present in Gram-negative bacteria which is selectively permeable and does not allow some drugs and antibiotics to penetrate the cell. CHF extract was found to be effective against both E. coli and S. aureus. CHF extracts from plants have been previously reported to provide good antibacterial activity [27-29]. The broad spectrum activity of CHF extract is probably because of its ability to extract phytochemicals active against both Gram-positive and Gram-negative strains.

To detect the level of ROS production in the treated bacterial cells, NBT dye was used. Production of ROS in aerobic cells is a natural process that involves various intracellular and extracellular sources and cells have their own defense mechanism to handle free radical-induced oxidative stress. If the level of oxidative stress exceeds the normal, it can pose threat to the cell. Most of the bactericidal antibiotic such as ciprofloxacin [30] kill bacteria by stimulation of ROS-induced oxidative damage [11]. In the present work, extract treated S. aureus and E. coli showed a significant increase in ROS production compared to the untreated bacterial cells. The crude plant extract contains a variety of chemical constituents that act synergistically on bacterial cells, leading to the formation of free radicals. Phytochemicals like flavonoids, quinones and peptides are known to form complex with bacterial cell walls and create pores [2]. Possibly the formation of pores in the bacterial cell wall allowed various other chemical constituents in the extracts to penetrate the cell that in turn triggers various biochemical pathways resulting in the production of excessive ROS in the bacterial cytoplasm leading to cell death.

TEM micrographs showed considerable changes in morphology of treated cells. The extract treated E. coli cells showed separation of inner membrane from the cell wall and there was disruption of the outer and inner membrane. Some cells were with swollen periplasmic space containing cytoplasmic materials. The innate immune system of plants produces antimicrobial peptides as a first line of defense in response to pathogen attack that is known to interact with membrane lipids [31]. The interaction of the chemical constituents of the crude extract with the outer membrane of Gram-negative bacteria might have caused disruption and thereby destabilizing the outer surface which eventually cause splitting of the inner membrane. Probably disruption of the membranes led to the accumulation of cytoplasmic content in the periplasmic space. Treated S. aureus cells also showed cell wall damage. Some cells completely lost their cell wall. The probable reason could be the ability of some plant constituents to interfere with the process of bacterial cell wall formation, thereby causing disruption of cell wall [2]. Similar phenomena were also observed in Magnolia officinalis extract treated S. aureus cells [32]. The Aquilaria crassna leaf extract also reported exerting its antibacterial effect against S. epidermidis by disruption of cell wall [33]. There is the previous report that oxidative stress due to ROS production causes alteration of expression of bacterial membrane transporters [34]. Therefore, structural changes and cell membrane damage caused due to the interaction between plant extract and bacterial cells could be the possible cause or consequence of cell death in the present system. The possible reason for the increase in nicked DNA formation and loss of supercoiled DNA in case of CHF extract treated S. aureus cells could be due to binding of plant constituents to bacterial DNA or enzymes involved in DNA replication, which resulted in bacterial DNA breakage forming relaxed circular DNA. Plant constituents might have bound to DNA, thereby interfering movement in the gel and thus forming faint bands. There are previous reports on flavonoids and alkaloids showing antibacterial action by binding to DNA [7]. In case of E. coli also, there were significant changes in the banding pattern of control and treated cells in the gel.

CONCLUSION

The present work, for the first time provides information for L. salicifolia antibacterial activity and its mechanism of action on Gram positive and Gram negative bacteria. L. salicifolia possesses potent antibacterial activity and is able to kill bacteria involving generation of ROS induced oxidative stress. In the present study, we performed the antibacterial activity of PE, CHF, MT and AQ extract of L. salicifolia leaf. CHF extract appeared to have broad spectrum antibacterial activity. Further experiments to isolate specific compounds from the plant responsible for antibacterial activity are in progress. Cytotoxicity studies of the extracts will be required to provide information on the effect of the extract on the human body.

ACKNOWLEDGEMENT

This research work was supported by the Department of Science and technology (DST INSPIRE). Assistance from Sophisticated Analytical Instrument Facility (SAIF) North Eastern Hill University, Shillong for TEM analysis is gratefully acknowledged. Authors are also thankful to Dr. G. C. Sarma, Curator, Botany Department, Gauhati University for helping in the identification and preservation of voucher specimen of the plant.

CONFLICTS OF INTERESTS

Authors have no conflicts of interest to declare

REFERENCES

  1. Coates A, Hu Y, Bax R, Page C. The future challenges facing the development of new antimicrobial drugs. Nat Rev Drug Discovery 2002;1:895-910.
  2. Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564–82.
  3. Brantner A, Males Z, Pepeljnjak S, Antolic A. Antimicrobial activity of Paliurus spina-christi mill. J Ethnopharmacol 1996;52:119–22.
  4. Kazmi MH, Malik A, Hameed S, Akhtar N, Ali SN. An anthraquinone derivative from Cassia italica. Phytochemistry 1994;36:761–3.
  5. Souza AB, Martins CH, Souza MG, Furtado NA, Heleno VC, de Sousa JP, et al. Antimicrobial activity of terpenoids from Copaifera langsdorffii Desf. against cariogenic bacteria. Phytother Res 2011;25:215-20.
  6. Cushnie TPT, Lamb AJ. Antimicrobial activity of flavonoids. Int J Antimicrob Agents 2005;26:343–56.
  7. Mori A, Nishino C, Enoki N, Tawata S. Antibacterial activity and mode of action of plant flavonoids against Proteus vulgaris and Staphylococcus aureus. Phytochemistry 1987;26:2231–4.
  8. Kapp E, Whiteley C. Protein ligand interactions: isoquinoline alkaloids as inhibitors for lactate and malate dehydrogenase. J Enzyme Inhib 1991;4:233-43.
  9. Mason TL, Wasserman BP. Inactivation of red beet betaglucan synthase by native and oxidized phenolic compounds. Phytochemistry 1987;26:2197–202.
  10. Stern JL, Hagerman AE, Steinberg PD, Mason PK. Phlorotannin-protein interactions. J Chem Ecol 1996;22:1887–99.
  11. Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics. Cell 2007;130:797–810.
  12. Dwyer DJ, Kohanski MA, Hayete B, Collins JJ. Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. Mol Syst Biol 2007;3:91.
  13. Mehdy MC, Sharma YK, Sathasivan K, Bays NW. The role of activated oxygen species in plant disease resistance. Physiol Plant 1996;98:365-74.
  14. Kala CP. Ethnomedicinal botany of the apatani in the Eastern Himalayan region of India. J Ethnobiol Ethnomed 2005;1:1-8.
  15. Rastogi RC, Borthakur N. Alkaloids of Litsea laeta and L. salicifolia. Phytochemistry 1980;19:998-9.
  16. Barinas JA1, Suarez LE. Chemical constituents of Talauma arcabucoana (Magnoliaceae): their brine shrimp lethality and antimicrobial activity. Nat Prod Res 2011;25:1497-504.
  17. Harborne JB. Phytochemical methods: a guide to modern technique of plant analysis. 3rd ed. London: Chapman and Hall; 1998.
  18. Mandal SC, Kumar CK, Majumder A, Majumder R, Maity BC. Antibacterial activity of Litsea glutinosa bark. Fitoterapia 2000;71:439-41.
  19. Zhang W, Hu FJ, Lv WW, Zhao CQ, Shi BG. Antibacterial, antifungal and cytotoxic isoquinoline alkaloids from Litsea cubeba. Molecules 2012;17:12950-60.
  20. Phukan S, Kalita MC. Phytopesticidal and repellent efficacy of Litsea salicifolia (Lauraceae) against Aedes aegypti and Culex quinquefasciatus. Indian J Exp Biol 2005;43:472-4.
  21. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk susceptibility tests. Approved Standard (M02–A11). Vol. 32. Wayne, PA; 2012.
  22. Ericsson JM, Sherris JC. Antibiotic sensitivity testing: report of an international collaborative study. Acta Pathol Microbiol Scand Sect B: Microbiol Immunol 1971; 217 Suppl 217:1–90.
  23. Banerjee M, Mallick S, Paul A, Chattopadhyay A, Ghosh SS. Heightened reactive oxygen species generation in the antimicrobial activity of a three component iodinated chitosan-silver nanoparticle composite. Langmuir 2010;26:5901-8.
  24. Sambrook J, Russell DW. Molecular cloning-A laboratory manual. 3rd ed. New York: Cold Spring Harbor Press; 2001.
  25. Silva NCC, Fernades Junior A. Biological properties of medicinal plants: a review of their antimicrobial activity. J Venomous Anim Toxins Incl Trop Dis 2010;16:402-13.
  26. Murray RGE, Steed P, Elson HE. The location of the mucopeptide in sections of the cell wall of Escherichia coli and other gram-negative bacteria. Can J Microbiol 1965;11:547-60.
  27. Kothari S, Mishra V, Bharat S, Tonpay SD. Antimicrobial activity and phytochemical screening of serial extracts from leaves of Aegle marmelos (linn.). Acta Pol Pharm 2011;68:687-92.
  28. Dhabi NAA, Balachandran C, Raj MK, Duraipandiyan V, Muthukumar C, Ignacimuthu S, et al. Antimicrobial, antimycobacterial and antibiofilm properties of Couroupita guianensis Aubl. fruit extract. BMC Complementary Altern Med 2012;12:1-8.
  29. Kamaraj C, Rahuman AA, Siva CD, Iyappan M, Kirthi AV. Evaluation of antibacterial activity of selected medicinal plant extracts from south India against human pathogens. Asian Pac J Trop Dis 2012;2 Suppl 1:296-301.
  30. Becerra MC, Sarmiento M, Paez PL, Argüello G, Albesa I. Light effect and reactive oxygen species in the action of ciprofloxacin on Staphylococcus aureus. J Photochem Photobiol 2004;76:13–8.
  31. Stotz HU, Waller F, Wang K. Innate Immunity in Plants: The role of antimicrobial peptides. In: Hiemstra PS, Zaat SAJ, editors. Antimicrobial Peptides and Innate Immunity. Switzerland: Springer Basel; 2013. p. 29-51.
  32. Hu Y, Qiao J, Zhang X, Ge C. Antimicrobial effect of Magnolia officinalis extract against Staphylococcus aureus. J Sci Food Agric 2011;91:1050-6.
  33. Kamonwannasit S, Nantapong N, Kumkrai P, Luecha P, Kupittayanant S, Chudapongse N. Antibacterial activity of Aquilaria crassna leaf extract against Staphylococcus epidermidis by disruption of cell wall. Ann Clin Microbiol Antimicrob 2013;12:1-7.
  34. Cao B, Liu J, Qin G, Tian S. Oxidative stress acts on special membrane proteins to reduce the viability of Pseudomonas syringae pv tomato. J Proteome Res 2012;11:4927-38.

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