Antimicrobial activity of Terminalia catappa brown leaf extracts against Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853

The aim of this study was to determine the effects of various concentration of Terminalia catappa brown leaves extract which can inhibit the growth of Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853. The crushed-brown leaves of Terminalia catappa was extracted using 95% ethanol, filtered, and evaporated. The dried T. catappa extract was used to identify phytochemical content qualitatively. Total phenolic and flavonoid contents were also measured quantitatively from dried extract. The dried extracts were also dissolved in sterile aquadest and serial dilutions were prepared to final concentration of 30, 60 and 90%. A disc diffusion method was used to evaluate the antibacterial activity of various concentrations of ethanol extract of brown leaves of T. catappa. Inhibition zone diameter was measured to determine antibacterial activity. Gentamycin sulfate and distilled water were used as positive and negative controls, respectively. Dried ethanolic extract of brown T. catappa leaves contained flavonoid, quinon, phenolic, triterpenoid, and tannin. A total of 208.722 mg gallic acid equivalent/g extract of total phenolic and 35.7671 mg quercetin equivalent/g extract of total flavonoid were also found in the dried extract. The inhibition zone diameters of ethanolic extracts ranged from 1.73 to 9.06 mm ( S. aureus) and from 1.83 to 6.5 mm ( P. aeruginosa). The higher concentration of extract, the wider the inhibition zone diameters for both bacteria. P. aeruginosa was more resistant to high concentrations of extract (90%) than S. aureus. Ethanolic extracts of the brown leaves of T. catappa had different antibacterial effects against S. aureus and P. aeruginosa. The higher the concentration of extract, the wider the inhibition zone diameter for both bacteria. P. aeruginosa was more resistant to high concentrations of ethanolic extracts of the brown leaves of T. catappa.

Staphylococcus and Pseudomonas species have been identified as causative agents of disease and serious pathogens in many aquatic animals, including fish [1][2][3][4] , resulting in high mortality rates in many commercially farmed fish. Among various Staphylococcus and Pseudomonas species, Staphylococcus aureus and Pseudomonas aeruginosa are known to cause disease in Oreochromis niloticus and Oreochromis mossambicus 5 . To reduce high mortality rates in farmed fish, aquaculturists and researcher used chemical agents and antibiotics to promote growth or prevent S. aureus and P. aeruginosa infection 6 .
However, the use of antibiotics to prevent and cure common infectious diseases in fish is becoming increasingly limited due to environmental concern, and increasingly expensive and ineffective because of microbial resistance [7][8][9] . As alternatives, various plant extracts, such as those of Boesenbergia pandurata, Zingiber zerumbet and Solanum ferox, have been tested and used as an alternative to antibiotics [10][11][12] . Another potential plant extract that can be used as an antimicrobial is that of Terminalia catappa, which is widely distributed in tropical and sub-tropical regions, including Indonesia 13,14 .
Terminalia catappa L., belonging to the family Combretaceae, is a large deciduous tree. The aqueous extract of Terminalia catappa leaves has been known as a folk medicine for antipyretic, hemostatic, hepatitis and liver-related diseases purposes in the Philippines, Malaysia and Indonesia 15,16 . Past research revealed that the extract of T. catappa leaves can be used to improve a resistance to Aeromonas hydrophila in Betta sp 17 , remedy against tilapia (Oreochromis niloticus) parasites and bacterial pathogen 18,19 . Nevertheless, scientific literature concerning the antibacterial potency of T. catappa against Staphylococcus aureus and Pseudomonas aeruginosa is limited.
Thus, the aim of the study was to evaluate the effects of various concentration of T. catappa brown leaf extract on the growth of Staphylococcus aureus and Pseudomonas aeruginosa by calculating inhibition zone diameters. The phytochemical content of the extract was also qualitatively determined and the flavonoid and phenolic concentrations in the extract was quantified.

Site and time
The research was performed from March to May 2018 at the Animal Physiology, Development and Molecular Laboratory for extracting T. catappa leaves. Meanwhile, assay study was done at microbiology and molecular genetic laboratory.
Bacterial strains and culture condition Bacterial strains were obtained from a Microbiology Laboratory, Faculty of Pharmacy, Sumatera Utara University, Indonesia. Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 were used to investigate the antibacterial activity. Both bacteria were sub-cultured on nutrient agar and stored at 4°C until use.

Plant materials
Brown leaves of T. catappa were collected from a region of Mulawarman university campus, Samarinda, East Kalimantan. Leaves were dried at room temperature for 2 days, crushed, transferred into a glass container and preserved until the extraction procedure.
Extraction procedure Approximately 1 kg of crushed leaves was soaked in 1 l of 95% ethanol for 5 days and shaken occasionally with a shaker. After 5 days, materials were filtered (Whatman No. 11 paper filter). The filtrate was evaporated using a rotary evaporator. Finally, the dried extracts were obtained and stored at 4°C in a dark bottle until use. The dried extracts were then dissolved in sterile distilled water and serial dilutions were prepared to give final concentrations of 30, 60 and 90%.

Phytochemical content
Dried extract samples were subjected to qualitative phytochemical analysis for flavonoids, quinon, alkaloids, phenolic, steroid, triterpenoid, saponins, and tannins using standard methods as previously described by Nugroho et al. 20 . Meanwhile, total phenolics and flavonoids were quantitatively measured, using the method described by Pourmorad et al. 21 .

Antibacterial activity assay
The antibacterial activity of T. catappa brown leaf ethanolic extract was evaluated using the disc diffusion method 22 . Three replicated agar plates were used for each different concentration and both controls (distilled water and 0.1% gentamycin sulfate). A total of 10 µl extract was added to a paper disc for each concentration and controls. Each disk was then placed in agar plate which had bacterial suspension in the plates. All plates were incubated at 37°C for 24 h. The diameter of inhibition zone created by each disc was measured (in mm) using a micrometer.

Data analysis
The inhibition zone data were expressed as means ± standard error. The data were subjected to ANOVA, followed by Duncan's post hoc test to evaluate significant differences among the groups of treatments. Meanwhile, the comparison between bacteria in each concentration was performed using a t-test. All significant tests were at P<0.05 levels and all analysis was done using SPSS 22 (SPSS, Inc., USA). The data of the phytochemical content and the concentration of flavonoid and phenolic were analyzed descriptively.

Results and discussion
The dried extract of T. catappa brown leaves contained flavonoids, quinon, phenolics, triterpenoids, and tannins. There were no alkaloids, steroids or saponin found in the dried extract. Total phenolic (208.722 mg gallic acid equivalent/g extract) and total flavonoid (35.7671 mg quercetin equivalent/g extract) were detected in the dried extract. The inhibition zone diameters of ethanolic extracts ranged from 1.73 to 9.06 mm for S. aureus, and from 1.83 to 6.5 mm for P. aeruginosa. Increasing the extract concentration increased the inhibition zone diameters for both bacteria ( Figure 1). P. aeruginosa was more resistant to high concentrations of extract (90%) than S. aureus (Table 1). According to Xie et al. 23 , flavonoids are known antibacterial agents against a wide range of pathogenic bacteria. In addition, Fu et al. 24 also revealed that phenolic extracts from some plants also have antibacterial effects against many kinds of bacteria. The data showing the inhibition zone diameters for both bacteria at each concentration of extract can be seen in Dataset 1 25 .

Conclusion
Ethanolic extracts of the brown leaves of T. catappa have potential antibacterial effects against S. aureus and P. aeruginosa, indicated by the inhibition zone formed around the extract. The inhibition zone diameter increased with increasing concentrations of T. catappa extract. P. aeruginosa exhibited more

Grant information
The author(s) declared that no grants were involved in supporting this work.
1. This study contributes information on the potential of T. catappa leaves extract as an antibacterial alternative to address antibacterial resistance problems in fish farming.

Methods
Site and time: Please specify the affiliation of the laboratory (whether it belongs to a department in a university or else).

Plant materials
Please correct the writing of Mulawarman University. How to preserve the crushed leaves? How long the preservation until the extraction?
Phytochemical contents What is the purpose of qualitative and quantitave phytochemical analysis in relation to the study objectives? Please elaborate in the introduction part.

Results
The inhibition zone in Figure 1 for the positive control is not very clear, therefore a zoom in figure is required to judge the result. I was expecting to see three discs in each plate of treatment and this experiment shall be replicated three times, before analyzing the data and arriving into a conclusion. Please comment on your replication method.

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others? Yes 1.

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes In the title, it should be mention ethanol extract, as the authors only evaluate ethanol extract of the leaves. In the figure 1, the quality is poor, so it seems the positive control do not have any inhibition zone! Only one strain of each bacteria was tested, it would be more authentic if more types of strains as well as some wild or environmental strains were tested. MIC/MBC of the extract against these bacteria should be determined. Data of this study should be compared with similar data published already. It is understood, that there may be no data of this particular species, but other species of terminalia can be consulted.

If applicable, is the statistical analysis and its interpretation appropriate? Not applicable
Are all the source data underlying the results available to ensure full reproducibility? Partly

Are the conclusions drawn adequately supported by the results?
No competing interests were disclosed.

Competing Interests:
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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