Pharmacogn. Commn. 2016; 6(2) 93-99



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Pharmacogn. Commn. 2016; 6(2) 93-99

A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products

www.phcogcommn.org

Original Article



Pharmacognosy Communications, 

Vol 6, Issue 2, Apr-Jun, 2016

 

93

Original Article



Growth Inhibitory Activity of Selected High Antioxidant  

Australian Syzygium Species Against the Food Poisoning and  

Tissue Necrotic Pathogen Clostridium perfringens

ABSTRACT

Introduction: 

Clostridium perfringens is a gram positive pathogen which 

is an etiological agent in Clostridial myonecrosis and enteritis necroticans.  

Unless promptly treated, C. perfringens infections may result in tissue  

necrosis and death. Syzygium australe (brush cherry) and Syzygium lu-

ehmannii (riberry) fruit and leaves have documented therapeutic proper-

ties as general antiseptic agents against an extensive panel of bacteria. 

Despite this, studies are yet to test the growth inhibitory activity of these 

species against C. perfringens.  Methods:  S. australe and S. luehmannii 

fruit and leaf extracts were investigated by disc diffusion assay for growth 

inhibitory activity against a clinical strain of Clostridium perfringens. Their 

MIC values were determined to quantify and compare their efficacies. 

Toxicity was determined using the Artemia franciscana nauplii bioassay. 

Results: Methanolic and aqueous 

S. australe and S. luehmannii fruit and 

leaf extracts as well as the corresponding fruit ethyl acetate extracts,  

displayed growth inhibitory activity in the disc diffusion assay against  

C. perfringens. The aqueous and methanolic extracts were particularly  

potent growth inhibitors, each with MIC values substantially <500 µg/mL. The 

S. australe fruit extracts were nontoxic in the Artemia franciscana bioassay 

(LC


50

 values <1000 µg/mL). All ethyl acetate extracts were also nontoxic. In 

contrast, the S. luehmannii aqueous and methanolic extracts (fruit and leaf), 

as well as the S. australe leaf extracts displayed substantial toxicity in the 

assay. Conclusion: The potent growth inhibitory bioactivity of the fruit and 

leaf aqueous and methanolic Syzygium spp. extracts against C. perfringens  

indicates their potential as medicinal agents in the treatment and prevention 

of clostridial myonecrosis and enteritis necroticans.



Key words: 

Syzygium australe, Syzygium luehmannii, Riberry, Brush cher-

ry, antioxidant, Myonecrosis, Enteritis necroticans, Gas gangrene.

Correspondence:

Ian Edwin Cock, 

School of Natural Sciences, Nathan Campus,

Griffith University, 170 Kessels Rd, Nathan,

Queensland 4111, AUSTRALIA.

Tel no.: +61 7 37357637; Fax no: +61 7 37355282.

E-mail: i.cock@griffith.edu.au (I. E. Cock).

DOI : 10.5530/pc.2016.2.7

INTRODUCTION

The genus Clostridium comprises a large group of sporulating, Gram-

positive, fermentative saprophytes. Generally existing in nature as part 

of the natural bacterial flora, the genus is clinically significant as several  

members of the genus have been linked to a number of diseases in  

humans.These include C. botulinum (botulism), C. difficile (pseudomem-

branous colitis), C. sordellii (pneumonia, endocarditis) and C. perfrin-

gens (food poisoning, clostridial myonecrosis).

1,2

 Although non-invasive, 



pathogenic clostridia are opportunistic and under suitable conditions 

can infect a host and cause disease through the production of various 

exotoxins.

3

 The diseases vary greatly in both associated symptoms and  



degree of lethality, and some species can cause multiple diseases  

depending on the route of infection. The best known example within the 

genus is C. perfringens, which can cause food poisoning if ingested, or 

clostridial myonecrosis (gas gangrene) through the infection of deep-

tissue wounds.

4

 



C. perfringens is one of the most common causes of food poisoning,  

accounting for up to a million infections annually in the United States.

5

 

Present in air, soil, water, faeces and as part of the human gut microbiota,  



the bacterium commonly infects food and at ambient temperatures  

can propagate to infectious levels. Whilst proper food preparation 

can significantly reduce the likelihood of infection, the sporulating 

nature of C. perfringens can result in the organism’s survival.

6

 In fact,  



C. perfringens is reportedly the second most common cause of bacterial 

food poisoning, with only by nontyphoidal Salmonella  causing more  

cases.

5

 Antibiotic therapy can offer an effective means of treatment,  



however due to the potential development of bacterial resistance, the 

search is ongoing for alternative antimicrobial agents. In addition to 

the design and synthesis of new agents, the probing of natural resources 

for antimicrobial agents offers an appealing alternative to traditional 

means of drug development. The geographical isolation and unique flora  

positions Australia to be an ideal environment for such investigations 

with several previous studies reporting antimicrobial activity against 

a number of other microorganisms.

7

 Syzygium (family Myrtaceae) is a 



genus of flowering plants which comprises approximately 500 species.

8

  



Syzygium spp. are widespread globally, occurring in tropical and sub-

tropical regions of Africa, Southern and South-East Asia, and throughout 

Australia. The greatest diversity of Syzygium spp. occurs in South-East 

Asia and Northern Australia. Most species are evergreen trees or large  

shrubs, with glossy green foliage. Several species have culinary and  

therapeutic uses. In the commercially most important species Syzygium  

aromaticum (clove), the unopened flower bud is used as a spice. This 

plant also has uses in traditional medicine due to its anaesthetic  

properties.

9

 The antibacterial activity of S. aromaticum is also well 



known. Numerous studies have reported on the antibacterial

10

 and anti-



fungal

11

 activities of oils and extracts from this plant.



Several other Syzygium spp. (eg. Syzygium australe, Syzygium jambos, 

Syzygium luehmannii) produce edible fruit which have very high antioxi-

dant capacities.

12-14


 Consumption of high levels of antioxidants has been  

associated with a decreased incidence of chronic diseases.

12,15,16 

High  


antioxidant levels have also been shown to act as a preventative against 

Mitchell Henry Wright,

1

 Cameron Jay Lee,



Charmaine Estelle Pollock,



Anthony Carlson Greene,

1

 Ian Edwin Cock,

1,2*

1

School of Natural Sciences, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111, AUSTRALIA.



2

Environmental Futures Research Institute, Nathan Campus, Griffith University, 170 Kessels Rd, Nathan, Queensland 4111, AUSTRALIA.



WRIGHT et al.: Antibacterial activity of Syzygium spp. against C. perfringens 

94

 



Pharmacognosy Communications, 

Vol 6, Issue 2, Apr-Jun, 2016

the development of degenerative diseases such as cancer,

17

 cardiovascular 



diseases,

18

 neural degeneration,



19

 diabetes and obesity.

20

 Furthermore, 



recent studies have also reported antibacterial activity in extracts from  

high antioxidant Australian plants.

21-24

 Particularly noteworthy, the  



Australian species S. australe (Bush Cherry) and S. luehmannii (Riberry)  

have potent growth inhibitory activity against a broad panel of  

bacteria.

25,26


 However, despite the reported broad spectrum growth  

inhibitory activity of Australian Syzygium spp., numerous pathogens are  

yet to be evaluated for their susceptibility to Australian Syzygium spp.  

extracts. The current study was undertaken to test the ability of to  

S. australe and S. luehmannii fruit and leaf extracts to inhibit the growth 

of the Gram-positive anaerobic bacterium Clostridium perfringens.



MATERIALS AND METHODS

Plant source and extraction 

S. luehmannii and S. australe fruit and leaves were collected from verified 

trees in suburban regions of Brisbane, Australia and voucher specimens  

deposited in the School of Natural Sciences, Griffith University, Aus-

tralia. The plant materials were comprehensively dried using a Sun-

beam food dehydrator and the dried material stored at -30

o

C until 



required. Prior to use, the plant materials were thawed and ground 

into a coarse powder. Individual 1 g quantities of the ground plant ma-

terial were weighed into separate tubes and 50 mL of deionised water, 

methanol or ethyl acetate were added. All solvents were obtained from 

Ajax, Australia and were AR grade. The ground plant materials were 

independently extracted in each solvent for 24 h at 4

o

C through gentle 



shaking. The extracts were subsequently filtered through filter paper 

(Whatman No. 54) under vacuum, followed by drying using rotary 

evaporation in an Eppendorf concentrator 5301. The resultant dry ex-

tract was weighed and dissolved in 10 mL deionised water (containing 

1% DMSO).

Qualitative phytochemical studies

Phytochemical analysis of the extracts for the presence of alkaloids, 

cardiac glycosides, tannins, saponins, phenolic compounds, flavonoids, 

polysteroids and triterpenoids was achieved as previously described.

27-29


 

Antioxidant capacity

The antioxidant capacity of each sample was assessed using a modified 

DPPH free radical scavenging method.

30,31

 Ascorbic acid (0-25 µg per 



well) was used as a reference and the absorbances were measured and 

recorded at 515 nm. All tests were completed alongside controls on each 

plate and all were performed in triplicate. The antioxidant capacity based 

on DPPH free radical scavenging ability was determined for each extract 

and expressed as µg ascorbic acid equivalents per gram of original plant 

material extracted.

Antibacterial screening

Clinical Clostridium perfringens screening

A clinical strain of Clostridium perfringens was supplied by Ms. Jane 

Gifkins (Griffith University) which was originally isolated and verified  

by Dr. John Bates (Department of Health, Queensland, Australia).  

Cultures were grown and maintained using a thioglycollate liquid media  

(Oxoid Ltd., Australia). All growth studies were performed using  

nutrient agar (Oxoid Ltd., Australia) under induced anaerobic conditions 

through the use of anaerobic jars and AnaeroGen™ 3.5 L atmospheric 

generation systems (Thermo Scientific). All studies were performed at 

30

o

C and the stock culture was subcultured and maintained in thiogly-



collate liquid media at 4

o

C.



Evaluation of antimicrobial activity

Antimicrobial activity of Syzygium  spp. extracts was determined  

using a modified disc diffusion assay.

23,30


 Briefly, 100 µL of C. perfringens 

was grown in 10 mL of fresh thioglycollate media until an approximate 

count of 10

8

 cells/mL was reached. A volume of 100 µL of the bacterial 



suspension was spread onto nutrient agar and extracts were tested for 

antibacterial activity using 6 mm sterilised filter paper discs. Discs were 

impregnated with 10 µL of the extracts, allowed to dry and placed onto 

the inoculated plates. The plates were left to stand at 4°C for 2 h before 

incubation at 30°C for 24 h. The diameters of the inhibition zones were 

measured to the closest whole millimetre. Each assay was performed 

in at least triplicate. Mean values (± SEM) are reported in this study. 

Standard discs of penicillin (2 µg) and ampicillin (10 µg) were obtained  

from Oxoid Ltd., Australia and used as positive controls to compare  

antibacterial activity. Filter discs impregnated with 10 µL of distilled water 

were used as a negative control.

Minimum inhibitory concentration (MIC) determination

The minimum inhibitory concentrations (MIC) of the extracts was deter-

mined as previously described.

32,33

 Briefly, the plant extracts were diluted 



in deionised water and tested across a range of concentrations. Discs 

were impregnated with 10 µL of the extract dilutions, allowed to dry and 

placed onto inoculated plates. The assay was performed as outlined above 

and graphs of the zone of inhibition versus concentration were plotted. 

Determination of MIC values were achieved using linear regression.

Toxicity screening

Reference toxin for toxicity screening

Potassium dichromate (K

2

Cr

2



O

7

) (AR grade, Chem-Supply, Australia) 



was prepared in deionised water (4 mg/mL) and serially diluted in artificial 

seawater for use in the Artemia franciscana nauplii bioassay. 

Artemia franciscana nauplii toxicity screening

Toxicity was tested using a modified Artermia franciscana nauplii  

lethality assay.

34-36


 Briefly, 400 µL of seawater containing ~43 (mean 43.2, 

n=155, SD 14.5) A. franciscana nauplii were added to wells of a 48 well 

plate and immediately used in the bioassay. Volumes of 400 µL of refer-

ence toxin or the diluted plant extracts were transferred to the wells and 

incubated at 25 ± 1°C under artificial light (1000 Lux). For each plate, a 

negative control (400 µL seawater) was run in triplicate. All treatments  

were also performed in at least triplicate. The wells were assessed at  

regular intervals and the number of dead counted. The nauplii were 

deemed dead if no movement of the appendages was detected within  

10 sec. After 24 h, all nauplii were sacrificed and counted to determine 

the total % mortality per well. The LC

50

 with 95% confidence limits for 



each treatment was calculated using probit analysis. 

Statistical analysis

Data is expressed as the mean ± SEM of at least three independent  

experiments. 



RESULTS

Liquid extraction yields and qualitative phytochemical 

screening 

Extraction of Australian Syzygium spp. plant materials (1 g) with various 

solvents yielded dried plant extracts ranging from 62 mg (S. luehmannii  

leaf ethyl acetate extract) to 560 mg (S. luehmannii fruit methanolic  

extract) (Table 1). Methanolic extracts provided significantly greater 

yields of extracted material relative to the corresponding aqueous and  



WRIGHT et al.: Antibacterial activity of Syzygium spp. against C. perfringens 

Pharmacognosy Communications, 

Vol 6, Issue 2, Apr-Jun, 2016

 

95

Table 1:



 The mass of dried extracted material, the concentration after resuspension in deionised water, qualitative phytochemical 

screenings and antioxidant capacities (mg equivalence of ascorbic acid/g dried plant material) of plant extracts

 

 

S. luehmannii fruit

S. luehmannii leaf

S. australe fruit

S. australe leaf

W

M

E

W

M

E

W

M

E

W

M

E

Mass of dried extract (mg)

120

560


130

88

190



62

240


360

110


180

360


110

Concentration of 

resuspended extract (mg/

ml)


12

56

13



9

19

6



24

36

11



18

36

11



Phe

no

lics

Total phenolics

 +++

 +++


 ++

 +++


 +++

 ++


 +++

 +++


 ++

 +++


 +++

 ++


Water soluble phenolics

 +++


 +++

 -

 +++



 +++

 -

 +++



 +++

 -

 +++



 +++

 -

Water insoluble phenolics



 ++

 +++


 ++

 ++


 +++

 ++


 ++

 +++


 ++

 ++


 +++

 ++


Cardiac glycosides

 -

 -



 -

 -

 -



 -

 -

 -



 -

 -

 -



 -

Saponins


 +

 +

 -



 +

 +

 -



 +

 -

 -



 +

 +

 -



Triterpenes

 +

 +



 -

 +

 +



 -

 -

 +



 -

 +

 +



 -

Polysteroids

 -

 -

 -



 -

 -

 -



 -

 -

 -



 -

 -

 -



Al

ka

lo

ids

Meyer test

 -

 -

 -



 -

 -

 -



 -

 -

 -



 -

 -

 -



Wagner test

 -

 -



 -

 -

 -



 -

 -

 -



 -

 -

 -



 -

Flavonoids

 +++

 +++


 +

 ++


 +++

 ++


 +++

 +++


 +

 ++


 +++

 ++


Tannins

 +

 +



 -

 +

 +



 -

 +

 +



 -

 +

 +



 -

A

nthr

aq

uino

nes

Free


 -

 -

 -



 -

 -

 -



 -

 -

 -



 -

 -

 -



Combined

 -

 -



 -

 -

 -



 -

 -

 -



 -

 -

 -



 -

Antioxidant content 

by DPPH reduction 

(expressed as mg AA 

equivalence per 1 g plant 

material extracted)

59.2

94.6


1.5

44.7


43.4

5.5


40.7

55.2


9.2

25.3


40.2

2.58


+++ indicates a large response; ++ indicates a moderate response; + indicates a minor response; - indicates no response in the assay. W=aqueous extract; 

M=methanolic extract; E=ethyl acetate extract; AA=ascorbic acid.



Table 2:

 MIC against C. perfringens growth (µg/

mL) and Artemia nauplii bioassay LC

50

 values 

(µg/mL) of S. australe and S. luehmannii fruit and 

leaf extracts

Extract

MIC

LC

50

SAFW

94

3310



SAFM

306


1879

SAFE

> 10,000


 -

SALW

> 10,000


244

SALM

881


294

SALE

 -

 -



SLFW

275


478

SLFM

161


414

SLFE

> 10,000


 -

SLLW

341


813

SLLM

462


450

SLLE

 -

 -



Numbers indicate the mean MIC or LC

50 


values of 

triplicate determinations. - indicates that MIC or LC

50

 

values were not obtained as the bacterial growth inhibition 



or % mortality did exceed 50% at any dose tested. 

ethyl acetate extracts, which gave low to moderate yields. The dried  

extracts were resuspended in 10 mL of deionised water (containing 1% 

DMSO), resulting in the concentrations presented in Table 1. 

Antioxidant content

Antioxidant capacity for the plant extracts (Table 1) ranged from 1.5 mg 

(S. luehmannii fruit ethyl acetate extract) to a high of 94.6 mg ascorbic 

acid equivalence per gram of dried plant material extracted (S. luehman-

nii fruit methanolic extract). The antioxidant capacities for the aqueous 

and methanolic extracts of each plant part were generally similar for both 

species and were substantially higher than the corresponding ethyl acetate 

extracts. 

Antimicrobial activity

To determine the ability of the crude fruit and leaf extracts to inhibit 

C. perfringens growth, 10 µL of each extract were screened using a disc 

diffusion assay. Bacterial growth was inhibited by 10 of the 12 extracts 

screened (83%) (Figure 1). The methanolic extracts were the most po-

tent inhibitor of growth (as judged by zone of inhibition), with inhibition 

zones as much as 19.3 ± 0.6 mm (S. australe fruit methanolic extract).  

This compares favourably with the penicillin (2 µg) and ampicillin  

controls (10 µg), with the zones of inhibition of 12.3 ± 0.3 and 13 ± 1.0 mm  

respectively. Indeed, the methanolic fruit and leaf extracts of both  

Syzygium spp. displayed inhibition zones > 12 mm. The aqueous S. australe  


WRIGHT et al.: Antibacterial activity of Syzygium spp. against C. perfringens 

96

 



Pharmacognosy Communications, 

Vol 6, Issue 2, Apr-Jun, 2016



Figure 1:

 Growth inhibitory activity of S. australe and S. Luehmannii fruit and leaf extracts against the C. perfringens environmental isolate measured as 

zones of inhibition (mm). SAFW=aqueous S. australe fruit extract; SAFM=methanolic S. australe fruit extract; SAFE=ethyl acetate S. australe fruit extract; 

SALW=aqueous S. australe leaf extract; SALM=methanolic S. australe leaf extract; SALE=ethyl acetate S. australe leaf extract; SLFW=aqueous S. luehman-

nii fruit extract; SLFM=methanolic S. luehmannii fruit extract; SLFE=ethyl acetate S. luehmannii fruit extract; SLLW=aqueous S. luehmannii leaf extract

SLLM=methanolic S. luehmannii leaf extract; SLLE=ethyl acetate S. luehmannii leaf extract; PC=penicillin (2 µg); AMP=ampicillin (10 µg). Results are expressed 

as mean zones of inhibition ± SEM.

Figure 2:

 The lethality of the S. australe and S. luehmannii fruit and leaf extracts (2000 µg/mL) and the potassium dichromate control (1000 µg/mL) towards  

Artemia franciscana nauplii after 24 h exposure. SAFW=aqueous S. australe fruit extract; SAFM=methanolic S. australe fruit extract; SAFE=ethyl acetate  

S. australe fruit extract; SALW=aqueous S. australe leaf extract; SALM=methanolic S. australe leaf extract; SALE=ethyl acetate S. australe leaf extract; 

SLFW=aqueous  S. luehmannii fruit extract; SLFM=methanolic S. luehmannii fruit extract; SLFE=ethyl acetate S. luehmannii fruit extract; SLLW=aqueous 

S. luehmannii leaf extract; SLLM=methanolic S. luehmannii leaf extract; SLLE=ethyl acetate S. luehmannii leaf extract; PC=potassium dichromate control; 

SW=seawater control. Results are expressed as mean % mortality ± SEM.



WRIGHT et al.: Antibacterial activity of Syzygium spp. against C. perfringens 

Pharmacognosy Communications, 

Vol 6, Issue 2, Apr-Jun, 2016

 

97

fruit and S. luehmannii leaf extracts also displayed good inhibition of  



C. perfringens growth with >10 mm zones of inhibition. 

The antimicrobial efficacy of the extracts was further quantified through 

the determination of MIC values (Table 2). The aqueous and methanolic  

fruit extracts of both Syzygium  spp., were particularly effective at  

inhibiting C. perfringens growth with MIC values generally <300 µg/mL 

(<3 µg impregnated in the disc). Similarly, the aqueous and methanolic  

S. luehmannii leaf extracts, as well as the methanolic S. australe leaf 

extract, were good growth inhibitors with MIC values <900µg/mL  

(<9 µg infused into the disc). These results compare well with the growth 

inhibitory activity of the penicillin and ampicillin controls which were 

tested at 2 µg and 10 µg respectively. All other extracts were either of only 

low efficacy in the assay, or did not inhibit C. perfringens growth at all. 

Quantification of toxicity

All extracts were initially screened in the assay at 2000 µg/mL (Figure 2). 

As a reference toxin, potassium dichromate was also tested in the bio-

assay. The potassium dichromate reference toxin was rapid in its onset 

of mortality, inducing nauplii death within the first 3 h of exposure and 

100% mortality was evident within 4-5 h (unpublished results). With the 

exception of the ethyl acetate leaf extracts of both species, all extracts 

showed > 50% mortality rates at 24 h. 

To further quantify the effects of toxin concentration on Artemia nau-

plii mortality, the extracts were serially diluted in artificial seawater to 

test across a series of concentrations in the Artemia nauplii bioassay. The  

24 h LC


50

 values of the Syzygium spp. extracts towards A. franciscana 

are presented in Table 2. No LC

50

 values are reported for the fruit or leaf  



ethyl acetate extracts of either species as <50% mortality was seen  

in all tested concentrations. Extracts with an LC

50

>1000 μg/mL towards 



Artemia nauplii have been defined as being nontoxic.

37

 The S. australe  



fruit methanolic and aqueous extracts also both had LC

50

 values  



substantially >1000 μg/mL and were therefore deemed to be non-toxic. 

Interestingly, the aqueous and methanolic extracts of S. australe leaf 

and of S. luehmannii fruit and leaf, displayed LC

50

 values substantially  



<1000 μg/mL and were therefore considered toxic. This was a surprising 

result as S. australe and S. luehmannii fruit are considered nutritious, 

high antioxidant foods. However, it is noteworthy that the bioassay  

organism (Artemia franciscana) is sensitive to extreme pH ranges.  

Our study reported that all of the extracts with LC

50

 values < 1000 μg/mL  



had relatively high antioxidant capacities. Furthermore, previous 

 

studies



14

 reported that the high antioxidant capacities of these Syzygium 

spp. is largely due to their high ascorbic acid contents. Therefore, it is 

possible that the apparent toxicity reported in this study may instead be 

a result of the extracts high ascorbic acid contents. 

DISCUSSION

Previous studies have reported potent growth inhibitory activities for  

S. australe and S. leuhmannii fruit and leaf extracts against a panel of 

pathogenic bacterial species.

25,38,39

 Both Gram-positive and Gram- 

negative bacteria were screened in previous studies. Whilst the Syzygium 

spp. extracts had broad spectrum growth inhibitory activity, a greater 

susceptibility of Gram-positive bacteria was noted. Indeed, the previous  

studies reported that the aqueous and methanolic S. australe and S. leuh-

mannii  extracts inhibited the growth of 40-80% of the gram positive 

bacterial species tested, with the methanolic extracts generally having a 

broader specificity. In light of this broad spectrum gram positive growth 

inhibitory activity, the potent 

C. perfringens 

growth inhibition evident in 

our study is perhaps not surprising.

Whilst an investigation of the phytochemistry of the Syzygium spp.  

extracts was beyond the scope of our study, high flavonoid, terpenoid and 

tannin contents are characteristics of this genus.

40,41

 Flavonoids have well 



established bacterial growth inhibitory activities.

42

 Relatively high levels 



of kaempferol and myricetin have been reported in leaf extracts from the 

taxonomically related species Syzygium aromaticum (clove).

9

 The same 



study also reported potent growth inhibitory activity for pure kaemp-

ferol, myricetin and a methanolic S. aromaticum leaf extract against a  

panel of bacteria. Similarly, quercetin, rutin and their corresponding  

glycosides inhibit the growth of Pseudomonas maltophilia and Enterobacter 

cloacae.

43

 The antimicrobial activity of terpenoids hasbeen extensively 



documented. Monoterpenoids including α-pinene, β-pinene, sabinene, 

mycrene, terpinene, limonene, piperitone and β-phellandrene inhibit the 

growth of a panel of bacteria including several nitrofurantoin resistant 

strains of Enterobacteriaceae.

42

 Similarly, the antibacterial activities for  



several sesquiterpenoids including α-cubebene, copaene and caryophyl-

lene have been reported.

42

 Furthermore, many tannin compounds have 



bacterial growth inhibitory activity. Gallotannins inhibit the growth of a 

broad spectrum of bacterial species

44

 through a variety of mechanisms 



including binding cell surface molecules including lipotoichoic acid and 

proline-rich cell surface proteins,

45,46

 and by inhibiting glucosyltransferase 



enzymes.

47

 Elligitannins are also highly potent inhibitors of bacterial 



growth, with MIC values as low as 62.5 µg/ml.

44,46


 Ellagitannins have also 

been reported to function via several antibiotic mechanisms including  

interaction with cytoplasmic oxidoreductases and by disrupting bacterial cell 

walls.


44,46

 Thus, it is likely that multiple compounds within the Syzygium 

spp. extracts are contributing to the growth inhibition of C. perfringens. 

The findings reported here also indicate that the majority of the  

Syzygium  spp. extracts examined in this study displayed significant  

toxicity (<1000 µg/mL) in the Artemia nauplii bioassay. Indeed, the most 

promising aqueous and methanolic S. luehmannii fruit extracts (MIC 

values of 275 and 161 µg/mL respectively) had LC

50

 values of approxi-



mately 450 µg/mL, indicating moderate to high toxicity. This toxicity 

would impact on the usefulness of these extracts as a medicinal anti-

septic agent. Similar extracts prepared from fruits of these species have  

previously been reported to have high ascorbic acid levels.

13,14

 Whilst 


these ascorbic acid levels may have beneficial therapeutic effects, they 

may also be responsible (at least in part) for the toxicity reported here. 

Whilst A. franciscana have generally been reported to be a robust and 

hardy organism for toxicity screening, they are susceptible to pH changes.

37

  

The levels of ascorbic acid previously reported in S. australe and S. leuh-



mannii extracts

13,14


 would be expected to have a significant impact on the 

pH of the seawater in the tests and this change may be responsible for the 

mortality induction reported in our study.Indeed, studies in our labora-

tory have shown that testing pure ascorbic acid in the concentrations 

previously reported to be in these extracts results in mortality similar to 

that reported in our study (unpublished results). 

Whilst toxicity was assessed in this study with the test organism  

A. franciscana, toxicity towards A. franciscana has previously been 

shown to correlate well with toxicity towards human cells for many toxins.

37

 



However, further studies are required to determine whether this is also 

true for the Syzygium  spp. extracts examined in these studies. Toxic 

anti bacterial extracts may still be useful as non-medicinal antibacterial 

agents (eg. surface disinfectants and topical antiseptics). Likewise, toxic 

plant extracts may also still have medicinal potential even if they are not 

antimicrobial. Previous studies have demonstrated that toxicity in the 

A. franciscana bioassay may indicate anti-cancer potential.

37

 Indeed, the 



anti-proliferative activity of S.australe and S. luehmannii fruit and leaf 

extracts has been reported against CaCo2 and HeLa human cancer cell 

lines,

12

 indicating that these extracts may also have potential in the devel-



opment of new anticancer drugs.

The results of this study indicate that the Syzygium spp. extracts examined  

warrant further study due to their 

C. perfringens 

growth inhibitory 

activity. Conversely, the toxicity detected for these extracts indicates 



WRIGHT et al.: Antibacterial activity of Syzygium spp. against C. perfringens 

98

 



Pharmacognosy Communications, 

Vol 6, Issue 2, Apr-Jun, 2016

that further toxicity studies are required to evaluate the safety of these  

extracts for medicinal usage. Whilst the extracts examined in this report 

have potential as 

C. perfringens 

growth inhibitory agents, caution is 

needed before these compounds can be applied to medicinal purposes. 

Purification and identification of the bioactive components is needed to 

examine the mechanisms of action of these agents. 



CONCLUSION

The results of this study demonstrate the potential of S. australe and  

S. leuhmannii fruit and leaf extracts to inhibit C. perfringens growth.  

As the fruit and leaves of these Syzygium species are edible and are used 

as a culinary agent, they have potential in the prevention and treatment 

of clostridial myonecrosis and enteritis necroticans. However, before 

being acceptable for therapeutic uses, further cell line toxicity studies 

are required to verify the safety of these extracts. Furthermore, studies 

aimed at the purification and identification of the bioactive components 

arerequired to examine the mechanisms of action of these extracts. 



ACKNOWLEDGEMENTS

Financial support for this work was provided by the Environmental  

Futures Research Institute and the School of Natural Sciences, Griffith 

University, Australia. 



CONFLICTS OF INTEREST

The authors report no conflicts of interest.



ABBREVIATION USED

DMSO: Dimethyl sulfoxide; LC

50

: The concentration required to achieve 

50% mortality; MIC: Minimum inhibitory concentration.



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PICTORIAL ABSTRACT

• 

S. australe and S. luehmannii leaf and fruit extracts inhibit C. perfringens 



growth.

• 

Aqueous and methanolic extracts were particularly potent with MIC’s 



generally <500 µg/mL.

• 

S. australe fruit extracts and the ethyl acetate extracts from both Syzy-



gium spp. were nontoxic. (LC

50

’s>1000 µg/mL).



• 

All S. luehmannii aqueous and methanolic extracts as well as the S. aus-

trale leaf extracts, displayed substantial toxicity.

• 

Syzygium spp. extracts have therapeutic potential in the prevention and 



treatment of clostridial myonecrosis and food poisoning.

ABOUT AUTHORS

Dr. Mitchell Henry Wright: Received his PhD in 2014, for his work investigating the manganese reduction and 

oxidation characteristics of environmental bacteria. He is currently a postdoctoral researcher at Griffith University,  

Australia, where he is working on several projects both in the areas of Geomicrobiology and Pharmacognosy.  

His present research interests are the use of bacteriogenic manganese oxides in the bioremediation of metal-

contaminated sites as well as the use of Australian native plants in the treatment and prevention of various patho-

genic bacteria. Dr Wright’s interests include wearing silly hats and supporting bad football teams.



Dr. Anthony Greene: Is a senior lecturer and researcher at Griffith University, Brisbane Australia. He obtained 

his PhD in Microbiology from the University of New South Wales and focuses on extreme environments, bio-

remediation and Geomicrobiology. His specific interests include the microbial ecology of thermophilic, saline 

and alkaliphilic environments and the mechanisms and industrial potential of extremophilic bacteria contained 

therein.

Dr. Ian Cock: Leads a research team in the Environmental Futures Research Institute and the School of Natural 

Sciences at Griffith University, Australia. His research involves bioactivity and phytochemical studies into a 

variety of plant species of both Australian and international origin, including Aloe vera, South Asian and South 

American tropical fruits, as well as Australia plants including Scaevola spinescens, Pittosporum phylliraeoides, 

Terminalia ferdinandiana (Kakadu plum), Australian Acacias, Syzygiums, Petalostigmas and Xanthorrhoea john-

sonii (grass trees). This range of projects has resulted in nearly 200 scientific publications in a variety of peer 

reviewed journals. Unlike Dr Wright who enjoys wearing silly hats, Dr Cock is wearing the hat in this photo due 

to a lost wager.



SUMMARY


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