in vitro antibacterial effects of cinnamomum extracts on common bacteria found in wound infections...

In vitro antibacterial effects of Cinnamomum extracts on commonbacteria found in wound infections with emphasison methicillin-resistant Staphylococcus aureus

Ayuba Sunday Buru a, Mallikarjuna Rao Pichika a,n, Vasanthakumari Neela b,Kavitha Mohandas a

a International Medical University, 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur, Malaysiab Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang Selangor,Malaysia

a r t i c l e i n f o

Article history:Received 14 August 2013Received in revised form7 December 2013Accepted 22 February 2014

Keywords:Antibacterial effectCinnamomumWound pathogensMalaysian traditional system of medicineMethicillin resistant Staphylococcus aureus(MRSA)

a b s t r a c t

Ethnopharmacological relevance: Cinnamomum species have been widely used in many traditionalsystems of medicine around the world. In the Malaysian traditional system of medicine, the leaves,stem bark and stem wood of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum andCinnamomum impressicostatum have been used to treat wound infections. To study the antibacterialeffects of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomumimpressicostatum against common bacteria found in wound infections with primary focus onmethicillin-resistant Staphylococcus aureus (MRSA).Materials and methods: The crude extracts from the leaves, stem-bark and stem-wood of Cinnamomuminers, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum wereobtained using sequential extraction with hexane, ethylacetate, methanol and water. The volatile oilswere obtained by hydro-distillation. The antibacterial activities of extracts were investigated using diskdiffusion assays and broth microdilution assays.Results: The volatile oils obtained from the stem-bark of Cinnamomum altissimum, Cinnamomumporrectum and Cinnamomum impressicostatum have shown significant antibacterial activity against awide range of Gram positive and Gram negative bacteria including MRSA. A few test extracts have shownbetter activity against MRSA as compared to methicillin sensitive Staphylococcus aureus (MSSA). Amongstall the test extracts, Cinnamomum impressicostatum stem-bark water extract produced the largestinhibition zone of 21.0 mm against MRSA while its inhibition zone against MSSA was only 8.5 mm.The minimum inhibitory concentration (MIC) of this extract against MRSA was 19.5 μg mL�1 and thecorresponding minimum bactericidal concentration (MBC) was 39.0 μg mL�1.Conclusions: This study has scientifically validated the traditional use of Cinnamomum species in treatingwound infections. Of high scientific interest was the observation that the antibacterial effect ofCinnamomum impressicostatum stem-bark crude water extract against MRSA was significantly higherthan its effect against MSSA, suggesting that the extract contains a compound(s) with higher specificneutralising activity against the drug resistance markers of MRSA.

& 2014 Published by Elsevier Ireland Ltd.

1. Introduction

The ethnomedicine and folklore have recorded the immensecontribution of medicinal plants and the vast repertoire of bior-esources as a source of traditional ethno-therapeutics for the

prevention, alleviation and cure of various diseases. Contemporarily,most drugs used to treat pathogenic, infectious microorganisms aresecondary metabolites isolated from natural resources. Importantly,these therapeutic secondary metabolites also serve as templates orlead compounds for the derivation of novel antibiotics. Plantsecondary metabolites have the important characteristic of beinghydrophobic (Cox et al., 2000; Lambert et al., 2001; Skandamis et al.,2001; Carson et al., 2002; Ultee et al., 2002), which enable them topartition the lipids of the bacterial cell membrane, thereby disrupt-ing the membrane structure and rendering them more permeable

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Journal of Ethnopharmacology

http://dx.doi.org/10.1016/j.jep.2014.02.0440378-8741 & 2014 Published by Elsevier Ireland Ltd.

n Correspondence to: Department of Pharmaceutical Chemistry, InternationalMedical University, 126, Jalan Jalil Perkasa 19, Bukit Jalil, 57000 Kuala Lumpur,Malaysia. Tel.: þ60 3 2731 7208.

E-mail address: [email protected] (M.R. Pichika).

Please cite this article as: Buru, A.S., et al., In vitro antibacterial effects of Cinnamomum extracts on common bacteria.... Journal ofEthnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.02.044i

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and subjecting them to the effects of bioactive antibacterial com-pounds. Plants contain a number of organic components includingalkaloids, flavones, phenols, quinones, terpenoids, glycosides andtannins known to possess antibacterial activity (Cowan, 1999;Erdogrul, 2002).

There are approximately 20,000 plant species being used asethno-medicines all over the world. The World Health Organisa-tion (WHO) estimated that 80% of the global population isdependent either directly or partly on plant based drugs as apanacea against communicable and non-communicable diseases(World Health Organisation (WHO), 2005). There has recentlybeen a surge in the emergence and re-emergence of drug resistantbacteria such as MRSA that are recalcitrant to treatment withhitherto effective antibacterial agents (Gibbons, 2004; Aqil et al.,2006; Jang-Gi et al., 2011). This is partly due to indiscriminate useof antibiotics for the treatment of infectious diseases and evolu-tionary adaptations by pathogenic microorganisms enabling themto avert the deleterious effects of antimicrobials.

MRSA is the cause of a wide-range of infectious complicationswhich can be life-threatening (Couto et al., 2008). It has beenimplicated as a dominant hospital-acquired pathogen whichcauses a substantial burden for health and economies in the worldtoday (Gibbons, 2004; Foster, 2004). Hospital-acquired infectionsare defined as infections that are likely to have been strictlyacquired in the hospital and which are not in incubationary phaseupon admission of the patient into hospital (Garner et al., 1988).Vancomycin is the reserved drug of choice for the treatment ofMRSA infections (Bailie and Neal, 1988). However, recent evidenceindicates the evolution of Vancomycin Resistant Staphylococcusaureus (VRSA) (Gould, 2008).

Cinnamomum is a genus within the botanical family Lauraceae.Many plant species within this genus are used as spices. Much ofcinnamon bioactivity resides in its oil, which is about 90%cinnamaldehyde (Bown, 1995). Cinnamon as a plant possesseschemo-preventive, antispasmodic, anti-ulcer, choleretic, sedative,hypothermic, antifungal, antibacterial, antiviral, antipyretic, lipo-lytic, antiseptic, anaesthetic, anodyne, cytotoxic, hypolipidemicand antiplatelet properties and is also an immunostimulatoryagent. It may be useful in reducing the risk of cardiovasculardisease and cancer (Craig, 1999; Jayaprakasha et al., 2002). InMalaysia, there are approximately 13 documented Cinnamomumspecies currently being used in the Malaysian traditional system ofmedicine (Compendium of Medicinal Plants used in Malaysia,Volume I, 2002). Village folk in particular have been using theleaves, stem-bark and stem-wood of Cinnamomum iners, Cinna-momum porrectum, Cinnamomum altissimum and Cinnamomumimpressicostatum to treat wound infections. Therefore, in thisstudy we investigated the antibacterial activity of crude extractsof the aforementioned Cinnamomum species against commonbacteria causing wound infections, with emphasis on MRSA, inorder to validate their reputed ethnomedicinal/folklore claim.

2. Materials and methods

2.1. Plant materials

The plants, Cinnamomum iners (HTBP 2952), Cinnamomum porrec-tum (HTBP 2954), Cinnamomum altissimum (HTBP 2953) and Cinna-momum impressicostatum (HTBP 3152) collected in the months ofOctober and November 2010 from Kuala Lumpur, Kedah and Penangwere used in the study. The plants were authenticated by Mr. AhmadZainudin Bin Ibrahim, taxonomist, Putrajaya Botanical Graden Herbar-ium. The voucher specimens (numbers indicated in parentheses) weredeposited in the Putrajaya Botanical Graden Herbarium (HerbariumTaman Botani, Putrajaya).

2.2. Preparation of extracts

The plant materials (leaves, stem bark, and stem wood) werewashed thoroughly under running tap water to remove dirt and theywere then dried in a hot-air oven at a temperature of 4075 1C. Thedried plant material was then powdered using a conventional blender.One kilogram of each plant material was extracted sequentially using a

Table 1Percentage yields (w/w) of Cinnamomum extracts.

Plant Part Extracts Yield (% w/w)

Cinnamomum porrectum Leaves Volatile oil 3Hexane 6Ethyl acetate 12Methanol 8Water 15

Stem bark Volatile oil 4Hexane 7Ethyl acetate 14Methanol 9Water 18

Stem wood Volatile oil 3Hexane 6Ethyl acetate 12Methanol 18Water 15

Cinnamomum iners Leaves Volatile oil 2Hexane 4Ethyl acetate 7Methanol 9Water 11

Stem bark Volatile oil 1.5Hexane 8Ethyl acetate 11Methanol 13Water 22


Cinnamomum altissimum Leaves Volatile oil 1.5Hexane 7Ethyl acetate 11Methanol 14Water 22

Stem bark Volatile oil 2Hexane 4Ethyl acetate 7Methanol 9Water 11

Stem wood Volatile oil 1.5Hexane 9Ethyl acetate 12Methanol 15Water 22

Cinnamomum impressicostatum Leaves Volatile oil 2Hexane 4Ethyl acetate 7Methanol 9Water 11

Stem bark Volatile oil 1.5Hexane 8Ethyl acetate 13Methanol 16Water 22


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Soxhlet extractor (Favorits apparatus) with solvents of increasingpolarity starting with n-hexane, followed by ethyl acetate, methanoland water. The extracts were concentrated using a rotary evaporator(Buchis Rotavapor R-200 and R-215, Switzerland) at a temperature

not exceeding 60 1C and then completely freeze-dried (Labconcos

Freezone 6.0 and 4.5) where necessary. The dried extracts were storedin tightly capped containers at �80 1C until further experimentation.The volatile oils were obtained using hydro-distillation. All the extracts

Table 2Antibacterial activity of Cinnamomum extracts against gram positive bacteria commonly found in wound infections.

Plant Part Extract Mean7SD diameters (mm) of inhibition zones a

MSSA MRSA VRE SS SE BC EF

Cinnamomum iners Leaves Volatile oil – – – – – – –

Hexane – – – – – – –

Ethyl acetate – – – – – – –

Methanol – – – 9.570.7071 10.570.7071 – –

Water – – – – 8.571.4142 – –

Stem bark Volatile oil 7.570.7071 10.570.7071 7.570.7071 871.4142 – – –

Hexane – – – – – – –

Ethyl acetate 7.070.00 13.071.4142 7.570.7071 11.570.7071 – – 7.570.7071Methanol – 9.570.7071 – – – – –

Water – – – – – – 11.570.07071Stem wood Volatile oil – – – – – – –

Hexane – – – – – – –


Methanol – – – – – – –

Water – – – – – – –

Cinnamomum altissimum Leaves Volatile oil – – – – – – –

Hexane – – – – – – –


Methanol – – – 11.570.7071 13.570.7071 – 11.570.7071Water 11.570.7071 – – 8.570.7071 11.570.7071 – 7.570.7071

Stem bark Volatile oil – 1271.4142 – – 9.570.7071 – 7.570.7071Hexane – – – – – – –

Ethyl acetate 7.570.7071 570.7071 14.570.7071 – 8.570.7071Methanol 8.570.7071 – – – – 7.570.7071 –

Water – – – – – – –

Stem wood Volatile oil – – – – – – –

Hexane – – – – – – –


Methanol – – – – – – –

Water – – – – – – –

Cinnamomum porrectum Leaves Volatile oil – – – – – – –

Hexane – – – – – – –


Methanol – 9.572.1213 – 12.570.7071 10.570.7071 – 1171.4142Water – – – – 7.570.7071 – –

Stem bark Volatile oil – 7.570.7071 – – 7.570.7071 – –

Hexane – – – – – 871.4142 –

Ethyl acetate – 7.570.7071 – 1071.4142 –

Methanol – 10.570.7071 7.570.7071 – 13.570.7071 16.570.7071 1171.4142Water – 7.570.7071 – – – 1171.4142 –

Stem wood Volatile oil – – – – – – –

Hexane – – – – – – –


Methanol – – – – – – –

Water – – – – – – –

Cinnamomum impressicostatum leaves Volatile oil – – – – – – –

Hexane – – – – – – –


Methanol – – – – – – –

Water – – – – – – –

Stem bark Volatile oil 8.570.7071 14.570.7071 – – 770.00 – –

Hexane – 7.570.7071 – – – – –

Ethyl acetate – 7.570.7071 – – 9.570.7071 – –

Methanol – – – – – – –

Water 871.4142 21.071.4142 – – 11.570.7071 – –

Stem wood Volatile oil – – – – – –

Hexane – – – – 7.570.7071 – –


Methanol – – – – 971.4142 – –

Water – – – – 1171.4142 – –

Organism(s) Control antibiotic used Zone of inhibition (mm)

Methicillin Susceptible Staphylococcus aureus Methicillin 2070.0000Methicillin Resistant Staphylococcus aureus Vancomycin 2371.4142Vancomycin Resistant enterococci Linezolid 2070.0000

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were dissolved in 100% dimethylsulphoxide (Mercks, Germany) to aconcentration of 100 mg/mL.

2.3. Bacterial strains

Stock cultures of bacteria used for this study were kindly providedby the Department of Medical Microbiology and Parasitology, Facultyof Medical and Health Science, Universiti Putra Malaysia. Theseinclude Methicillin Sensitive Staphylococcus aureus (MSSA) (ATCC700637), Methicillin Resistant Staphylococcus aureus (MRSA) (ATCC700689), Vancomycin Resistant Enterococci (VRE) (ATCC 51299),Enterococcus faecalis (ATCC 29212), Staphylococcus saprophyticus(ATCC 15305), Staphylococcus epidermidis (ATCC 12228), Bacilluscereus (ATCC 10876), Pseudomonas aeruginosa (ATCC 27853), Escher-ichia coli (ATCC 25922), Stenotrophomonas maltophilia (ATCC 13637),Enterobacter aerogenes (ATCC 13048), Burkholderia pseudomallei(Clinical strain), Acinetobacter baumannii (ATCC 17978), Aeromonashydrophilia (ATCC 7966), Proteus mirabilis (ATCC 29906), Yersiniaenterocolitica (ATCC 23715), Acinetobacter iwoffii (ATCC 17925) andKlebsiella pneumoniae (ATCC 700603). The working media used forsusceptibility assays were Mueller–Hinton Agar (MHA, Oxoid, UK)and Mueller–Hinton Broth (MHB, Oxoid, UK).

2.4. Preparation of the crude extracts

All the extracts and essential oils were dissolved in 10% (v/v)dimethylsulphoxide in water to obtain the working concentration of100 mg/mL. Twenty microliters (20 μL) of the test solution wasaseptically loaded onto sterile disks (6 mm dia. Whatmans, England)and then air-dried. Negative control disks were similarly preparedwith 10% dimethylsulphoxide and then air dried. Once prepared, thedisks were stored overnight in sterile Petri-dishes until their applica-tion the following day in disk diffusion assays. Standard antibioticdisks were used as positive controls for each organism.

2.5. Antibacterial disk diffusion assay

The antibacterial activities of extracts and volatile oils (VO) ofCinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimumand Cinnamomum impressicostatumwere determined using amodifiedagar disk diffusion method (Bauer et al., 1966; Ncube et al., 2008).Inocula from frozen stocks of bacteria were subcultured twice onMueller–Hinton agar. Suitable single colonies taken from these plateswere resuspended in Mueller–Hinton broth and incubated with rotaryagitation at 37 1C for 1.5–2.0 h until the optical density reached theequivalent of a 0.5 McFarland Standard. Using a sterile swab, aloopful of the inoculum was spread onto Mueller–Hinton Agar. Eachdisk impregnated with either extract or VO was placed on theprepared bacterial culture plates and incubated at 37 1C for 18–24 h.Each disk diffusion assay of extracts and controls (DMSO and

commercial antibiotic disks) was carried out in triplicate. The zonesof inhibition (mm) were measured and the results were reported asmean7standard deviation (SD).

2.6. Determination of minimum inhibitory concentration (MIC)and minimum bactericidal concentration (MBC)

The minimum inhibitory concentration (MIC) of Cinnamomumextracts that showed bioactivity against MRSA was determinedaccording to Clinical and Laboratory Standard Institute (CLSI) (2011)guidelines and the modified resazurin method described by Sarkeret al. (2007). The extract (100 mL) was subjected to two-fold serialdilutions with broth to achieve final concentrations ranging from0.00122 to 5 mg/mL. These extracts were loaded into the wells of asterile microtitre plate. Bacterial suspensions were compared to a0.5 McFarland turbidity standard to obtain 107–108 CFU/mL and weresubsequently diluted accordingly to achieve the final concentrationof 106 CFU/mL. Sterility and growth controls were included. Cellsuspensions were added into all wells except outer wells which wereused as sterility controls. The plates were prepared in triplicate andthen incubated at 37 1C for 18–24 h. Resazurin at 0.02% (w/v) wasprepared by dissolving resazurin sodium salt (Sigma-Aldrich, USA) indistilled water. The final concentration of resazurin was 0.002% (w/v)and this was applied to the wells of the microtitre plates. The colourchange was assessed visually. Colour changes from purple to pink orcolourless were recorded as positive. The lowest concentration atwhich the colour change did not occur (indicative of non-viability ofall cells present) was taken as the MIC. The MIC value was calculatedas the mean of three replicate treatments.

The minimum bactericidal concentration was determined byremoving 0.1 mL aliquots of Mueller–Hinton Broth (MHB) suspen-sion carrying no colour change (indicating no growth of bacteria)and then spread-plating the aliquots onto MHA followed byincubation at 37 1C for 24 h. The MBC determinations were carriedout in triplicate. MHA plates displaying no bacterial colonies weretaken as representative of the minimal bactericidal concentration.

3. Results

3.1. Percentage yield of each Cinnamomum species

The yields of extracts and volatile oils were calculated using thefollowing formula:

Percentage yield¼ ðmass of dried extract or volatile oil=massof the dried plant materialÞ � 100

The yields are shown in Table 1. In general, the yields were highfromwater extracts indicating that the plants are very rich in polarcompounds.

Table 2 (continued )

Organism(s) Control antibiotic used Zone of inhibition (mm)

Staphylococcus saprophyticus Cloxacillin 21.570.7071Staphylococcus epidermidis Tobramycin 21.570.7071Bacillus cereus Ciprofloxacin 22.570.7071Enterococci feacalis Ciprofloxacin 1770.0000

MSSA¼Methicillin-susceptible Staphylococcus aureus; MRSA¼Methicillin-resistant Staphylococcus aureus; VRE¼Vancomycin resistant enterococci; SS¼Staphylococcussaprophyticus; SE¼Staphylococcus epidermidis; BC¼Bacillus cereus; EF¼Enterococci feacalis.Gram positive organisms screened for antibacterial activity using Modified Kirby–Bauer Disk Diffusion Assays (zone diameters are given in mm7standard deviation).Largest zone diameters for each bacterium is underlined and given in bold face. The first and second largest zones respectively were obtained for MRSA challenged withstem bark water extract of Cinnamomum impressicostatum (21.071.4142mm) and Bacillus cereus challenged with stem bark methanolic extract of Cinnamomumporrectumy (16.570.7071mm) and Staphylococcus epidermidis challenged with stem bark methanolic extract of Cinnamomum porrectum (16.570.7071mm). Disks wereimpregnated with 20 μL of 100 mg/mL test solution).

a Each value represent the mean of three replicates, SD of all means (20 μL of 100 mg mL�1 solution).






Table 3Antibacterial activity of Cinnamomum extracts against gram positive bacteria commonly found in would infections.


EC AI AB KP PA SM AH PM EA YE BP

Cinnamomum iners Leaves Volatile oil – – – – – – – – – – –

Hexane – – – – – – – – – – –

Ethyl acetate – – – – – – – – – – –

Methanol – – – – – – – – – – –

Water – – – – – – – – – – –

Stem bark Volatile oil – 8.570.7071 – – – – – 7.570.7071 – – –

Hexane – – – – – – – – – – –

Ethyl acetate 8.570.7071 7.570.7071 7.570.7071 7.570.7071 871.4142 770.7071 – – – – 7.570.7071Methanol – – – – – – – – – – –

Water – – – – – – – – – – –

Stem wood Volatile oil – – – – – – – – – – –

Hexane – – – – – – – – – – –

Ethyl acetate – – – – – – – – – – –

Methanol – – – – – – – – – – –

Water – – – – – – – – – – –

Cinnamomum altissimum Leaves Volatile oil – – – – – – – – – – –

Hexane – – – – – – – – – – –

Ethyl acetate – – – – – – – – – – –

Methanol – – – – – 9.570.7071 – – – – –

Water – – – – – – – – – – –

Stem bark Volatile oil 7.570.7071 7.570.7071 – 7.570.7071 – 9.570.7071 1171.4142 – 7.570.7071 770.0Hexane – – – – – – – – – –

Ethyl acetate – 7.570.7071 – – – – – – – – –

Methanol – 1171.4142 – – 11.570.7071 10.570.7071 – – – – –

Water – – – 8.570.7071 7.570.7071 770.0 – – – – –


Hexane – – – – – – – – – – –

Ethyl acetate – – – – – – – – – – –

Methanol – – – – – – – – – – –

Water – – – – – – – – – – –

Cinnamomum porrectum Leaves Volatile oil – – – – – – – – – – –

Hexane 10.570.7071 – – – – – – – – – –

Ethyl acetate – – – – – – – – – – –

Methanol – – – – – 7.570.7071 – – – – –

Water – – – – – – – – – – –

Stem bark Volatile oil – – – – – – 7.570.7071 – 7.570.7071 – –

Hexane – – – – – – – – – – –

Ethyl acetate – – – – – – – – – – –

Methanol – – – – – – – – – 9.570.7071 –

Water – – – – – – – – – – –


Hexane – – – – – – – – – – –

Ethyl acetate – – – – – – – – – – –

Methanol – – – – – – – – – – –

Water – – – – – – – – – – –

Cinnamomum impressicostatum leaves Volatile oil – – – – – – – – – – –

Hexane – – – – – – – – – – –

Ethyl acetate – – – – – – – – – – –

Methanol – – – – – – – – – – –

Water – – – – – – – – – – –

Stem bark Volatile oil – 1371.4142 – – – 7.570.7071 8.570.7071 770.00

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articleas:

Buru,A.S.,

etal.,

Invitro

antibacterial

effectsof

Cinnamom

umextracts

oncom

mon

bacteria....Journalof

Ethnopharm

acology(2014),http

://dx.doi.o

rg/10.1016/j.jep

.2014.02.044i




Table 3 (continued )


EC AI AB KP PA SM AH PM EA YE BP

Hexane – – – – – – – – – – –

Ethyl acetate – – – – – – 7.570.7071 – – – –

Methanol – – – – – – – – – – –

Water – – – – – 8.570.7071 9.570.7071 – – – –


Hexane – – – – – – 10.570.7071 – – – –

Ethyl acetate – – – – – – 8.570.7071 – – – –

Methanol – – – – – – – – – – –

Water – – – – – – – – – – –

Organisms Control antibiotic used Zone of inhibition (mm)

Escherichia coli Erythromycin 2170.0000Acinetobacter Iwoffii Imipenem 30.570.7071Acinetobacter baumannii Imipenem 35.570.7071Klebsiella pneumonia Gentamicin 15.570.7071Pseudomonas aeroginosa Imipenem 21.570.7071Stenotrophomonas moltipholia Cotrimoxazole 2170.0000Aeromonas Hydrophila Tobramycin 2170.0000Proteus mirabilis Ciprofloxacin 2270.0000Enterobacter aerogenes Imipenem 2770.0000Yersinia enterocolitica Tobramycin 2270.0000Bulkholdia pseudomallei Ceftazidine 1770.0000

EC¼Escherichia coli; AI¼Acinetobacter iwoffii; AB¼Acinetobacter baumanii; KP¼Klebsiella pneumoniae; PA¼Pseudomonas aeruginosa, SM¼Stenotrophomonas maltophilia; AH¼Aeromonas hydrophilia; PM¼Proteus mirabilis;EA¼Enterobacter aerogenes; YE¼Yersinia enterocolitica; BP¼Burkholderia pseudomallei.Gram negative organisms screened for antibacterial activity using Modified Kirby–Bauer Disk Diffusion Assays (zone diameters are given in mm7standard deviation). Largest zone diameters for each bacterium are underlined andgiven in bold face. The first and second largest zones respectively were obtained for Acinetobacter iwoffii challenged with the volatile oils of the stem bark of Cinnamomum impressicostatum (1371.4142 mm) and Pseudomonasaeruginosa challenged with the methanolic extract of the stem bark of Cinnamomum altissimum (11.570.7071 mm). Disks were impregnated with 20 μL containing 2 mg extract.

a Each value represents the means of three replicates, SD of all means (20 μL of 100 mg mL�1solution).

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Buru,A.S.,

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effectsof

Cinnamom

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bacteria....Journalof

Ethnopharm

acology(2014),http

://dx.doi.o

rg/10.1016/j.jep

.2014.02.044i




3.2. Antibacterial disk diffusion assays

The zones of inhibition produced by all the test compoundsagainst a wide range of bacteria are shown in Table 2 (Gram-positive bacteria) and Table 3 (Gram-negative bacteria). In general,the test compounds have shown better activity against Gram-positive bacteria than Gram-negative bacteria. From the results, itis evident that the stem-bark of all the Cinnamomum species hasshown better activity than other parts of the corresponding plants.It is also evident that the polar extracts (methanol and water) haveshown better antibacterial activity than other extracts.

The volatile oils obtained from the leaves and stem-wood of allspecies do not show antibacterial activity whilst the volatile oilsobtained from the stem-barks of Cinnamomum altissimum, Cinna-momum porrectum and Cinnamomum impressicostatum haveshown significant antibacterial activity. It is interesting to notethat a few test compounds (volatile oil and ethylacetate extractsfrom Cinnamomum iners stem-bark, volatile oil from Cinnamomumaltissimum stem-bark; methanolic extract from Cinnamomumporrectum stem-bark, volatile oil and water extracts from Cinna-momum impressicostatum stem-bark) have shown better activityagainst MRSA as compared to MSSA. Amongst all the test extracts,the Cinnamomum impressicostatum water extract of stem-barkproduced the largest inhibition zone of 21.071.4 mm againstMRSA while its inhibition zone against MSSA was only8.570.7 mm. The zones of inhibition of test extracts against MRSAare summarised in Table 4.

3.3. Minimum inhibitory concentration (MIC) and minimumbactericidal concentration (MBC) of Cinnamomum extracts

The Cinnamomum impressicostatum stem-bark water extractshowing the most potent antibacterial activities in disk diffusionassays was found to have an MIC and MBC of 19.531270.7071μg/mL and 39.062570.7071 μg/mL respectively (Table 4).

4. Discussion

Plants have long been known to be a repository of therapeuticcompounds with immense benefits to man (Ncube et al., 2008).Phytochemical extracts from plants hold promise for use inallopathic medicine as they are a potential source of antiviral,antitumor and antimicrobial agents (Nair et al., 2005; Ramyaet al., 2008).

The importance and significance of the intense quest forantimicrobial agents from natural resources for the treatment ofinfectious diseases in the past 10 years cannot be underscored.With the rapid emergence of resistant, multidrug resistant andextremely drug resistant pathogenic strains, and the adverse sideeffects due to use of conventional antibiotics, the discovery of newantimicrobial agents is a vital aspect of research and developmentin the realm of public health. MRSA is one of the establishedcauses of complicated hospital and community acquired infections(McCarthy et al., 2010). Antibiotic resistance has been shown to beimplicated in about 70% of hospital-acquired infections in the USA(Li et al., 2010).

The results of this study support reports in the literature thatdocument the positive antibacterial activity of Cinnamomumspecies against a wide range of bacteria (Chang et al., 2001,2008). The highest antibacterial effect was observed against MRSAtreated with 2 mg of the water extract of the stem-bark ofCinnamomum impressicostatum (21.000071.4142 mm). Interest-ingly, challenging MSSA with the same extract yielded a signifi-cantly smaller inhibition zone (8.000071.4142 mm). Jensen andLyon (2009) state two mechanisms of resistance to the β-lactamantibiotics i.e. possession of the blaZ gene encoding for Class A β-lactamases and possession of the mecA gene encoding for the lowaffinity penicillin binding protein, PBP2a. The proven mechanismof β-lactam resistance in MRSA is the possession of the mecA geneencoding for PBP2a (Chambers, 2001). Bear in mind that manybacteria, including Staphylococcus aureus, innately produce peni-cillin binding proteins. The fact that the antibiotic compound(s) present in the stem bark of Cinnamomum impressicostatumwere effective against both MSSA and MRSA, but had a higher levelof efficacy against MRSA is suggestive that this compound(s) mayhave higher binding affinity for and neutralising activity againstPBP2a rather than the innate penicillin binding proteins present inMSSA. If this hypothesis holds true, it would make these naturalcompounds present in Cinnamomum impressicostatum stem-bark,and their derivatives ideal candidates for development into effi-cacious anti-MRSA drugs. Selected extracts from other plantsources have also demonstrated relatively higher antibacterialactivity against MRSA than MSSA (Kosowska-Shick et al., 2010;Park et al., 2011). In combination with Cinnamomum impressicos-tatum stem-bark water extract, the bioactive compound(s) in allthese plants hold immense promise as important lead compoundsfor further research and development.

While it initially seemed unlikely that superficial plant tissuesuch as the stem-bark would yield important therapeutic meta-bolites, this study has proven otherwise. It should be noted that

Table 4Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of active Cinnamomum extracts against MRSA.

Extract/control Part Extract ZI (mm) MIC (μg mL�1) MBC (μg mL�1)

Cinnamomum iners Stem bark Volatile oil 10.570.7071 625.0070 125070Ethyl acetate 13.071.4142 156.2570 312.570Methanol 9.570.7071 625.0070 125070

Cinnamomum altissimum Stem bark Volatile oil 12.071.4142 156.2570 312.570Ethyl acetate 7.570.7071 250070 500070

Cinnamomum porrectum Leaves Methanol 9.572.1213 62570 125070Stem bark Volatile oil 7.570.7071 250070 500070

Ethyl acetate 7.570.7071 250070 500070Methanol 10.570.7071 62570 125070Water 7.570.7071 250070 500070

Cinnamomum impressicostatum Stem bark Volatile oil 14.570.7071 156.2570 312.570Hexane 7.570.7071 250070 500070Ethyl acetate 7.570.7071 250070 500070Water 21.071.4142 19.5312570 39.062570

Vancomycin 2371.4142 4.882870 9.765670






other reports in the literature support the importance of stem-bark as a repository of therapeutic antimicrobial compounds(Doughari, 2006).

For the Gram-positives, the largest inhibition zones wereobtained from the water and methanolic extracts of cinnamonwhilst the second largest inhibition zones were obtained fromStaphylococcus epidermidis and Bacillus cereus challenged with themethanolic extracts of Cinnamomum porrectum. Clearly, the high-est antibacterial activity against the Gram-positives resides in thepolar and semi-polar fractions of cinnamon. Being water soluble,such compounds would be easily formulated as drugs.

Acinetobacter iwoffii is a documented opportunistic Gram-negative coccobacillus that is a cause of hospital-acquired infec-tions such as pneumonia, skin and wound infections, bacteraemiaand meningitis (Van Looveren et al., 2004). Acinetobacter speciesare intrinsically resistant to a wide range of antibiotics, includingthe penicillins, chloramphenicol, aminoglycosides and fluoroqui-nolones (Hawkey, 2008). Pseudomonas aeruginosa is a Gram-negative rod that is an opportunistic pathogen of both humansand plants. It has been widely implicated as a cause of pneumoniain cyctic fibrosis patients, septic shock and urinary tract infectionin neutropenic patients, gastrointestinal infection in prematureinfants and neutropenic cancer patients and skin and soft tissueinfections of burns and wounds (Moffett, 2010). Like Acinetobacterspecies, Pseudomonas is also a cause of hospital-acquired anddevice related infections (Leid, 2009). Pseudomonas aeruginosa isinnately resistant to many antibiotics (Shahid et al., 2003). Due tothe significance and wide-spread presence of these opportunisticand hospital-acquired Gram-negative pathogens and their intrin-sic drug resistance, development of suitable antibacterial thera-peutics, such as that from the volatile oils of the stem-bark ofCinnamomum impressicostatum for Acinetobacter iwoffii infectionsand the methanolic extract of the stem-bark of Cinnamomumaltissimum for Pseudomonas aeruginosa infections, would be atimely and valuable effort.

Whilst broad spectrum antibacterial activity against bothGram-positive and Gram-negative bacteria was observed for theCinnamomum species under study, overall, higher antibacterialactivity was observed against the Gram-positive target bacteria.

5. Conclusions

Village-folk have long used cinnamon for treatment of a varietyof health related disorders. The resulting benefits are likely to havevaried with the species of cinnamon they chanced upon. Thisstudy has clearly elucidated the relative efficacy of the variousspecies of cinnamon in terms of antibacterial efficacy against arange of Gram-positive and Gram-negative bacteria associatedwith wound infections.

Cinnamomum impressicostatum stem-bark water extract hasdemonstrated a strong antibacterial effect against MRSA. Whilstthis extract demonstrated activity against both MRSA and MSSA,significantly higher activity was observed against MRSA, suggest-ing that the stem-bark water extract may contain a compound(s) with higher neutralising activity against the drug resistancemarker, penicillin binding protein, PBP2a, than innate penicillinbinding proteins. Broad spectrum antibiotic activity was alsoexhibited against a range of other Gram-negative and Gram-positive bacteria, including the opportunistic and nosocomialpathogens, Acinetobacter iwoffii and Pseudomonas aeruginosa. Wethus conclude that compounds contained within the extracts ofthe Cinnamomum species under study are strong candidates forfurther development as efficacious antibacterial therapeutics.

Acknowledgements

We wish to express our gratitude to the International MedicalUniversity, Bukit Jalil, Kuala Lumpur, Malaysia, for funding thisproject (IMU 222/2010) and the Department of Medical Micro-biology and Parasitology, Faculty of Medicine and Health Sciences,Universiti Putra Malaysia, for providing lab facilities to carry outantimicrobial studies.

References

Aqil, F., Ahmad, I., Owais, M., 2006. Evaluation of anti-methicillin-resistantStaphylococcus aureus (MRSA) activity and synergy of bioactive plant extracts.Biotechnol. J. 1, 1093–1102.

Bailie, G.R., Neal, D., 1988. Vancomycin ototoxicity and nephrotoxicity. A review.Med. Toxicol. Advers. Drug Exp. 3, 376–386.

Bauer, A.W., Kirby, W.M., Sherris, J.C., Turck, M., 1966. Antibiotic susceptibilitytesting by a standardized single disc method. Am. J. Clin. Pathol. 45, 493–496.

Bown, D., 1995. Royal Horticultural Society Encyclopedia of Herbs and Their Uses(RHS), first ed. Dorling Kindersley, London

Carson, C.F., Mee, B.J., Riley, T.V., 2002. Mechanism of action of Melaleuca alternifolia(tea tree) oil on Staphylococcus aureus determined by time-kill, leakage, and salttolerance assays and electron microscopy. Antimicrob. Agents Chemother. 46,1914–1920.

Chambers, H.F., 2001. Methicillin-resistant Staphylococcus aureus. Mechanisms ofresistance and implications for treatment. Postgrad. Med. 109, 43–50.

Chang, S.T., Chen, P.F., Chang, S.C., 2001. Antibacterial activity of leaf essential oilsand their constituents from Cinnamomum osmophloeum. J. Ethnopharmacol. 77,123–127.

Chang, C.W., Chang, W.L., Chang, S.T., Cheng, S.S., 2008. Antibacterial activities ofplant essential oils against Legionella pneumophila. Water Res. 42, 278–286.

Clinical and Laboratory Standard Institute (CLSI), 2011. Performance Standards forAntimicrobial Susceptibility Testing: Twenty First Informational Supplement.CLSI document M100-S21.

Compendium of Medicinal Plants used in Malaysia, Volume I, 2002. HerbalMedicinal Research Centre, first ed. Malaysia.

Couto, I., Costa, S.S., Viveiros, M., Martins, M., Amaral, L., 2008. Efflux-mediatedresponse of Staphylococcus aureus exposed to ethidium bromide. J. Antimicrob.Chemother. 62, 504–513.

Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12,564–582.

Cox, S.D., Mann, C.M., Markham, J.L., Bell, H.C., Gustafson, J.E., Warmington, J.R.,Wyllie, S.G., 2000. The mode of antibacterial action of essential oil of Melaleucaalternifola (tea tree oil). J. Appl. Microbiol. 88, 170–175.

Craig, W.J., 1999. Health-promoting properties of common herbs. Am. J. Clin. Nutr.70, 491S–499S.

Doughari, J.H., 2006. Antimicrobial activity of Tamarindus indica Linn. Trop. J.Pharm. Res. 5, 592–603.

Erdogrul, Ö.T., 2002. Antibacterial activities of some plant extract used in folkmedicine. Pharm. Biol. 40, 269–273.

Foster, T.J., 2004. The Staphylococcus aureus “Superbug”. J. Clin. Investig. 114,1693–1696.

Garner, J.S., Jarvis, W.R., Emori, T.G., Horan, T.C., Hughes, J.M., 1988. CDC definitionsfor nosocomial infections. Am. J. Infect. Control 16, 128–140.

Gibbons, S., 2004. Anti-staphylococcal plant natural products. Nat. Prod. Rep. 21,263–277.

Gould, I.M., 2008. Clinical relevance of increasing glycopeptide MICs againstStaphylococcus aureus. Int. J. Antimicrob. Agents 31, 1–9.

Hawkey, P.M., 2008. Molecular epidemiology of clinically significant antibioticresistance genes. Br. J. Pharmacol. 153, S406–S413.

Jang-Gi, C., Ok-Hwa, K., Young-Seob, L., Hee-Sung, C., You-Chang, O., Obiang-Obounou, B., Min-San, K., Dong-Hwan, S., Hun-Soo, K., Hyun, P., Dong-Won,S., Jung-Rae, R., Dong-Yeul, K., 2011. In vitro and in vivo antibacterial activity ofPunica granatum peel ethanol extract against salmonella. Evid.-Based Comple-ment. Altern. Med. 2011, 1–8.

Jayaprakasha, G.K., Rao, L.J., Sakariah, K.K., 2002. Chemical composition of volatileoil from Cinnamomum zeylanicum buds. Z. Naturfor. C, J. Biosci. 57, 990–993.

Jensen, S.O., Lyon, B.R., 2009. Genetics of antimicrobial resistance in Staphylococcusaureus. Future Microbiol. 4, 565–582.

Kosowska-Shick, K., McGhee, P.L., Appelbaum, P.C., 2010. Affinity of ceftaroline andother β-lactams for penicillin-binding proteins from Staphylococcus aureus andStreptococcus pneumoniae. Antimicrob. Agents Chemother. 54, 1670–1677.

Lambert, R.J.W., Skandamis, P.N., Coote, P.J., Nychas, G.J.E., 2001. A study of theminimum inhibitory concentration and mode of action of oregano essential oil,thymol and carvacrol. J. Appl. Microbiol. 91, 453–462.

Leid, J.G., 2009. Bacterial biofilms resist key host defenses. Microbe magazine 4,66–70.

Li, M., Cheung, G.Y., Hu, J., Wang, D., Joo, H.S., Deleo, F.R., Otto, M., 2010.Comparative analysis of virulence and toxin expression of global community-associated methicillin-resistant Staphylococcus aureus strains. J. Infect. Dis. 202,1866–1876.



http://refhub.elsevier.com/S0378-8741(14)00157-3/sbref1




































































McCarthy, N.L., Sullivan, P.S., Gaynes, R., Rimland, D., 2010. Health care-associatedand community-associated methicillin-resistant Staphylococcus aureus infec-tions: a comparison of definitions. Am. J. Infect. Control 38, 600–606.

Moffett, K.S., 2010. Pseudomonas aeruginosa in patients with cystic fibrosis. Anti-microb. Ther. 1, 1.

Nair, R., Kalariya, T., Sumitra, C., 2005. Antibacterial activity of some selected Indianmedicinal flora. Turk. J. Biol. 29, 41–47.

Ncube, N.S., Afolayan, A.J., Okoh, A.I., 2008. Assessment techniques of antimicrobialproperties of natural compounds of plant origin: current methods and futuretrends. Afr. J. Biotechnol. 7, 1797–1806.

Park, S.C., Park, Y., Hahm, K.S., 2011. The role of antimicrobial peptides in preventingmultidrug-resistant bacterial infections and biofilm formation. Int. J. Mol. Sci.12, 5971–5992.

Ramya, S., Govindaraji, V., Kannan, N.K., Jayakumararaj, R., 2008. In vitro evaluationof antibacterial activity of phytochemical extracts from leaves of Aegle marme-los (L.) Corr. (Rutaceae). Ethnobot. Leafl. 12, 1124–1128.

Sarker, S.D., Nahar, L., Kumarasamy, Y., 2007. Microtitre plate-based antibacterialassay incorporating resazurin as an indicator of cell growth, and its applicationin the in vitro antibacterial screening of phytochemicals. Methods 42, 321–323.

Shahid, M., Malik, A., Sheeba, 2003. Multidrug-resistant Pseudomonas aeruginosastrains harbouring R-plasmids and AmpC β-lactamases isolated from hospita-lised burn patients in a tertiary care hospital of North India. FEMS Microbiol.Lett. 228, 181–186.

Skandamis, P., Koutsoumanis, K., Fasseas, K., Nychas, G.J.E., 2001. Evaluation of theinhibitory effect of oregano essential oil on E. coli 0157:H7, in broth culture withor without EDTA, using viable counts, turbidity and impedance. Int. J. Food Sci.Technol. 13, 65–75.

Ultee, A., Bennik, M.H., Moezelaar, R., 2002. The phenolic hydroxyl group ofcarvacrol is essential for action against the food-borne pathogen Bacillus cereus.Appl. Environ. Microbiol. 68, 1561–1568.

Van Looveren, M., Goossens, H., The ARPAC Steering Group, 2004. Antimicrobialresistance of Acinetobacter spp. in Europe. Clin. Microbiol. Infect. 10, 684–704.

World Health Organisation (WHO), 2005. WHO Global Atlas on Traditional,Complementary and Alternative Medicine. WHO Centre for Health and Devel-opment, pp. 41–49.






































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