Materials

The research was conducted from July to December 2020 at the Fish Pests and Diseases Laboratory, Faculty of Fisheries and Marine, Universitas Lambung Mangkurat, Banjarbaru, Indonesia. The Jeruju leaves (A. ilicifolius) were collected from the riverbank of Bunipah, Aluh-aluh District, Banjar Regency, South Kalimantan, Indonesia. The isolate of A. hydrophila was obtained from the Mandiangin Freshwater Aquaculture Center for Fisheries (BPBAT), Banjar, South Kalimantan, Indonesia. The materials used were tryptic soy agar (TSA, Merck), tryptic soy broth (TSB, Merck), glutamate starch phenol agar (GSP agar, Merck), agar (Merck), distilled water, ethanol (Merck), and 2,2-diphenyl-1-picrylhydrazyl (DPPH, Merck). The tools used were digital scales, Whatman filter paper No. 42, ovens (Tungtec Instruments TH-160F), rotary vacuum evaporators (IKA RV 10), petri dishes, and Becker glass.

Jeruju leaves’ extraction

The plant materials (Jeruju leaves) were cleaned, cut into small pieces, and dried in an oven at 40°C–50°C. The dried Jeruju leaves were crushed into a fine powder. A total of 100 g of fine powder of Jeruju leaves was macerated in 400 ml of ethanol for 24 hours. Ethanol is used as a solvent that is safe for the consumption for fish and humans. Ethanol is also a natural solvent for both food and natural medicine (Hikmawanti et al., 2021). Ethanol used in this study is of analytical grade (Merck) and safe for the environment, considering it will apply to a fish culture environment. Ethanol is a solvent used to extract compounds from natural materials with good results (Sultana et al., 2009). The extract obtained was then evaporated using a rotary vacuum evaporator. Then, the Jeruju extract was dried in a vacuum desiccator for 4–5 days. The Jeruju leaf extract was stored at 4°C before assaying.

Figure 1. Distribution of Jeruju (A. ilicifolius) in Indonesia. Source: Global Biodiversity Information Facility (2021). [Click here to view]

Phytochemical screening

The ethanol extract of Jeruju leaves was subjected to qualitative screening of chemical components to determine the presence of alkaloids, anthraquinones, flavonoids, steroids, terpenoids, saponins, and phenols using conventional standard protocols, as described by Harborne (1998).

Metabolomic profiling

Metabolomic profiling of the Jeruju leaf extract was conducted using liquid chromatography and high-resolution mass spectrometry (LC-HRMS Shimadzu-8040, Japan) with an injection volume of 1 µl. LC-HRMS was equipped with an autosampler, binary pump, column compartment, and a diode array detector for scanning spectroscopy. Chromatographic separation was performed using a C-18 column, Shim Pack FC-ODS (2 mm ø × 150 mm, 3 µm). Two solvents were prepared including solvent A (H₂O: MeOH, 8:2, with 0.1% formic acid) and solvent B (0.1% formic acid in acetonitrile). The two solvents were adjusted to 95:5 ratios, respectively, with an elution gradient of 0/0 at 0 minutes, 15/85 at 5 minutes, 20/80 at 20 minutes, and 90/10 at 24 minutes. Mass spectroscopy (MS) analysis was performed by electrospray ionization (ESI) with positive ions as the source. MS data were obtained through collision energy traps starting at 5.0 V. The ESI source parameters were regulated, including a capillary voltage of 3.0 kV, source temperature of 100°C, desolvation temperature of 350°C, sampling cone of 23 V, and desolvation gas flow of 6 l/hour. The chromatogram data obtained were compared with the data profile from the mzCloud Library system (Riyadi et al., 2020; 2021).

Prediction of biological activity

The metabolomic profile detected from the ethanol extract of Jeruju leaves with LC-HRMS was predicted for biological activity using the PASS server (http://www.pharmaexpert.ru/passonline/index.ph). The PASS server is a software that is useful for predicting the biological activity of a compound (Rumengan et al., 2021). The predicted biological activity requires a structural formula in the form of canonical SMILE obtained from the National Center for Biotechnology Information (https://pubchem.ncbi.nlm.nih.gov/) (Aisiah et al., 2020).

Antibacterial activity assay

The antibacterial activity assay was conducted using the well-diffusion method on a petri dish with modifications (Balouiri et al., 2016; Tanod et al., 2019a). The well-diffusion method used two media layers, namely the base media and the seed media layer. The modification was carried out by adding TSA composition (2 g) with 2 g of bacto agar in 100 ml of distilled water as the base medium. The seedling layer was made from 70% TSA in 100 ml of distilled water, then put into a tube containing 9 ml of seed media and sterilized. Furthermore, 1 ml of A. hydrophila isolate was added to warm seedling with a density of 1 × 10⁷ colony/ml (bacterial solution compared to the McFarland, HiMedia standard). The A. hydrophila isolates used pure culture from BPBAT Mandiangin. The seeding medium that was added with A. hydrophila was vortexed, and then poured onto the base media layer. After the media hardens slightly, a well hole was made at a certain distance, using a 5 mm diameter glass tube. Each well was filled with 50 µl of Jeruju leaves’ ethanol extracts with concentrations of 50, 100, 150, 200, and 300 mg/ml and incubated at 37°C for 24 hours. Cefadroxil and tetracycline (1 mg/ml each) were used as comparison controls. After that, the zone of inhibition was observed and measured. All experimental measurement data were carried out in three replications and expressed as mean ± standard deviation (n = 3).

Antibacterial activity was evaluated using the broth dilution method based on the guidelines (EUCAST, 2000; Wiegand et al., 2008) with modifications. Exactly 10 ml of TSB was inoculated with 100 µl of A. hydrophila (density 1 × 10⁷ colony/ml), then incubated at 37°C for 24 hours. After that, 100 µl of the Jeruju leaves’ ethanol extract (200 mg/ml) was added. Aeromonas hydrophila culture with aquadest was used as the negative control, and cefadroxil and tetracycline (1 mg/ml each) were used as positive controls. The total colony count was carried out on GSP agar, based on the total plate count method, following the Indonesian National Standard No. 01-2332.3 of 2006 with modifications (Indonesian National Standardization Agency—BSN, 2006). Modifications made using TSB on broth media and solid media using GSP selective media. If the GSP is red, it indicates A. hydrophila is not growing, whereas if the GSP is yellow, it indicates A. hydrophila growth.

Antioxidant activity assay

Antioxidant activity was determined using the DPPH radical scavenging method (Molyneux, 2004; Tanod et al., 2019a). The ethanol extract of Jeruju leaves was added with ethanol so that the concentration was 100 µg/ml; then serial dilutions were made (6, 12, 24, 48, and 96 µg/ml). A 2 ml aliquot of each concentration’s extract solution was added to 2 ml of the 50 μM DPPH solution. The mixture was homogenized and left for 30 minutes in a dark room at room temperature. Then, the mixture measured the free radical scavenging at a wavelength of 517 nm with a spectrophotometer (UV-VIS spectrophotometer T90 + PG Instruments Ltd).

The absorbance value of the DPPH solution was also measured and determined by IC₅₀ (half-maximal inhibitory concentration). Ascorbic acid was used as a positive control. IC₅₀ was determined as the concentration of the extract solution required to scavenge 50% DPPH free radicals (Dewanto et al., 2021). The assay was carried out in three repetitions, and the measurement results were expressed with a standard deviation. The DPPH scavenging effects were calculated using the following equation:

$\mathrm{DPPH}\mathrm{scavenging}\mathrm{effect}\mathrm{(}\mathrm{\%)}=\frac{\mathrm{Blank}\mathrm{Absorbance}\mathrm{\times }\mathrm{Sample}\mathrm{Absorbance}}{\mathrm{Blank}\mathrm{Absorbance}}\times 100$

Total phenol content assay

The ethanol extract of Jeruju leaves was evaluated for total phenol content according to the Folin–Ciocalteu method (Blainski et al., 2013; Lamuela-Raventós, 2017). Exactly 25 mg of Jeruju leaves’ ethanol extract was dissolved in 25 ml of ethanol:aquadest (1:1) solution. Then, 1 ml from the extract solution and 10 ml of distilled water + 1 ml of Folin–Ciocalteu reagent (homogenization) were added. After that, it was let to stand for 8 minutes and 3 ml of 20% Na₂CO₃ was added (which was let to stand for 2 hour at room temperature). Then, the absorption with a UV-Vis spectrophotometer at a wavelength of 750 nm was measured, which gave a blue color. In the same way, a gallic acid solution was prepared, i.e., 25 mg of gallic acid was dissolved in ethanol: water (1:1) to a volume of 25 ml. Then, the gallic acid solution in a series of dilutions of 5, 20, 40, 60, 80, and 100 g/ml was made. The standard curve of gallic acid was prepared with the concentration of gallic acid (µg/ml) against the absorbance value. Total phenolic was determined using the standard curve regression equation for gallic acid. The total phenol content was expressed in mg GAE/100 g dry extract (Muliadin et al., 2021; Riyadi et al., 2021b).

RESULTS AND DISCUSSION

Phytochemicals of Jeruju leaves’ ethanol extract

The Jeruju leaves were extracted using ethanol solvent to make the resulting extract more environment-friendly and safe to use for fish and humans. The extraction of 100 g of simplicia Jeruju leaves with 400 ml of ethanol solvent (analytical grade) obtained an extract weight of 21.30 g (extract yield of 21.30%). Phytochemical analysis was carried out to determine the type of natural products in the ethanol extract of Jeruju leaves. The phytochemicals’ screening of the ethanol extract of Jeruju leaves (A. ilicifolius) is presented in Table 1.

Table 1 shows the phytochemical screening of the ethanol extract of Jeruju leaves, indicating the presence of flavonoids, alkaloids, tannins, phenolics, steroids, and terpenoids. Jeruju was also reported to contain lignans (Kanchanapoom et al., 2001). Acanthus ilicifolius was also reported to contain alcohol, alkanes, fatty acids, lignans, steroids, and terpenoids (Wöstmann and Liebezeit, 2008). Acanthus ilicifolius collected from Kollam, Kerala, India, detected phytochemical components of saponins, tannins, terpenoids, flavonoids, alkaloids, and anthraquinones (Chundakkadu et al., 2011). The leaf extract of A. ilicifolius isolated 4-coumaric acid compounds, including coumarin compounds, lignans, flavonoids, and phenylethanoid (Ravikumar et al., 2012). Leaves of Jeruju collected from Sungai Tekong, Kubu Raya, West Kalimantan, Indonesia, detected phytochemical components of alkaloids, saponins, flavonoids, terpenoids, and phenol (Ernianingsih et al., 2014). Jeruju leaves collected from Kaligawe, Semarang, and the coastal area of Teluk Awur, Jepara, Central Java, Indonesia, were reported to contain saponins, quinone, and tannins compounds (Ardiantami et al., 2015). In A. ilicifolius, there were also found terpenes and flavonoids (Sreenivasa et al., 2015). Phytochemical studies of A. ilicifolius reported chemical constituents of triterpenoids, alkaloids, saponins glycosides, flavonoids, steroids, phenols, and coumarins (Bora et al., 2017). The methanol extract of A. ilicifolius leaves contained flavonoids, alkaloids, and phenols (Handayani et al., 2018). Jeruju also produces compounds of steroids, flavonoids, and tannins (Pringgenies et al., 2020).

The use of methanol, ethanol, and water solvents in plant extraction did not significantly affect the results of qualitative screening of phytochemical components. The statement is according to the results of Godwill et al. (2013), Chigayo et al. (2016), and Nurdyansyah and Widyastuti (2020). However, if quantitative screening of phytochemical components is carried out, the use of methanol showed higher amounts (especially for phenols, flavonoids, alkaloids, and terpenoids) than ethanol and water (Truong et al., 2019). So, it is indicated that methanol can extract better than ethanol and water.

Therefore, it strongly suspected that the difference in the phytochemical components in A. ilicifolius was due to environmental influences. The factors that influence differences in the production of chemical components are environmental conditions (Dewanto et al., 2019). Chemical components produced by organisms play a role in the defense system in maintaining life (Gallo et al., 2004), then organisms will produce more phytochemical components if they live in extreme environments. Liu et al. (2016) reported the difference of total tannin, flavonoid, rutin, and phenol in the same sample extract from different locations. Differences in phenol content were also reported in Mykhailenko et al. (2020); environmental factors affect the accumulation of phenolic compounds and their derivatives such as flavonoids, isoflavonoids, and xanthones in plants.

The phytochemical components in Jeruju leaves, like flavonoids, alkaloids, tannins, phenols, steroids, and terpenoids, reported having antibacterial and antioxidant properties. The flavonoid action mechanism as an antibacterial damaged bacterial cell walls and membranes, binding to cell adhesions and deactivating enzymes (Cowan, 1999). Flavonoid compounds act as scavengers for free radicals that arise due to bacterial infection to protect cells from negative enzymatic reactions (Xie et al., 2015). Alkaloids are chemical components that act as scaffolds for antibacterial drugs (Cushnie et al., 2014). The alkaloid action mechanism as an antibacterial is by inhibiting reductase dihydrofolate and topoisomerase type I enzymes, which play a role in DNA synthesis (Kittakoop et al., 2014; Samoylenko et al., 2009). Alkaloids have also been reported as inhibiting bacterial virulence without affecting growth or survival (LaSarre and Federle, 2013). An N group in the alkaloid structure can be an antioxidant because it acts as a free radical scavenger (Neganova et al., 2012). Alkaloid components were reported to increase phenolic compounds’ performance to provide a more potent antioxidant effect in plant extracts (Gan et al., 2017).

Table 1. Phytochemical screening of Jeruju leaves’ ethanol extract (A. ilicifolius). [Click here to view]

Tannin antibacterial mechanism was conducted by inactivating microbial adhesion cells and inhibiting iron availability for microorganisms. The antibacterial mechanism of tannins showed phenolic damage the bacterial cell wall of polypeptides (Akiyama et al., 2001; Ogbuagu, 2008). Tannins are known as antioxidants because they have the ?OH(hydroxyl) group attached to aromatic rings. Tannins effectively scavenged free radicals as an electron donor and a source of hydrogen atoms, active in metal chelation because of the ?OH groups and conjugated double bonds that allow the formation of electron delocalization (Dewanto et al., 2018). The phenol antibacterial mechanism deactivated proteins (enzymes) in the bacterial cell membrane. Phenol binds to proteins through hydrogen bonds resulting in a damaged protein structure where most of the cell wall and cytoplasmic membrane structures of bacteria contain protein and fat (Susanti et al., 2008). Phenolic components have the potential as antioxidants because they have –OH(hydroxyl) group on the aromatic ring (Agati et al., 2009). The hydroxyl group can donate H atoms to free radical compounds to reduce free radicals. Phenol hydroquinone and its derivatives act as oxidative inhibitors that bind to free radicals and react with reactive oxygen species (ROS) molecules to form more stable compounds (Harborne, 1998).

Steroids have been reported to fight Gram-positive and Gram-negative bacteria (Polat et al., 2011). Steroid components can disrupt the cell membrane of bacteria such as S. aureus, E. coli, and K. pneumoniae via the quaternary amine groups carried by the conjugate (Figueroa-Valverde et al., 2009). Steroids also exert cytotoxic effects on bacterial cells (Dogan et al., 2012). Steroid components were reported to play a role in enhancing endogenous antioxidants (Mooradian, 1993). The antibacterial mechanism of terpenoids is by damaging the bacterial cell membrane and dissolving the constituent membrane lipids (Cowan, 1999). The low concentrations of the terpenoid components only affect the enzymes involved in energy production, whereas the high concentrations of the terpenoids can lyse the membrane (Jasmine et al., 2011). Terpenoids have a relatively complex cyclic structure (consisting of alcohol, aldehyde, or carboxylic acid), so they have a hydroxyl group that could act as an antioxidant (Dewanto et al., 2019).

Biological activity prediction with PASS server

Furthermore, the list of compounds in Table 2 predicted their potential biological activity using the PASS server. The predicted value of the compound’s biological activity in the ethanolic extract of Jeruju leaves was expressed as a probability to be active (Pa) (Fig. 2). The prediction was carried out as an inhibitor of cell wall biosynthesis, peptidoglycan membrane inhibitor, protein synthesis inhibitor, nucleic acid synthesis inhibitor, and free radical scavenger (Madigan et al., 2019).

Figure 2 shows the potential of Jeruju leaves’ ethanol extract as an antibacterial against the growth of A. hydrophila, closely related to its mechanism, which is thought to be a peptidoglycan glycosyltransferase enzyme inhibitor, DNA synthesis inhibitor, and free radical scavenger. Peptidoglycan glycosyltransferase enzyme plays a role in peptidoglycan biosynthesis in bacterial cell wall formation (Derouaux et al., 2013). By inhibiting the peptidoglycan glycosyltransferase enzyme action, bacteria cannot synthesize peptidoglycan; so, bacteria cannot maintain their shape and protect themselves from osmotic pressure.

Biological activity prediction of the Jeruju leaves’ ethanol extract as an inhibitor of the peptidoglycan glycosyltransferase enzyme, DNA synthesis inhibitor, and free radical scavenger was supported by the results phytochemical screening and metabolomic profiling with LC-HRMS. The flavonoid and phenol compounds in the extract are thought to deactivate and inhibit the peptidoglycan glycosyltransferase enzyme’s performance. In addition, alkaloid components could inhibit DNA synthesis by inhibiting the enzyme’s dihydrofolate reductase and topoisomerase type I (Kittakoop et al., 2014). Metabolomic profiles shown in Table 2, which have a hydroxyl group (–OH), an amine group (–NH₂), and a cyclic nitrogen structure, are thought to act as free radical scavengers.

Table 2. Metabolomic profiles of the Jeruju leaves’ ethanol extract with LC-HRMS. [Click here to view]

Figure 2. Prediction of the biological activity of Jeruju leaves’ ethanol extract with PASS server. [Click here to view]

Antioxidant activity and total phenol content of the Jeruju leaf extract

This study also evaluated the antioxidant activity of the Jeruju leaf extract using the DPPH radical scavenging method. DPPH was a stable free radical and can accept electrons or hydrogen radicals to form a stable diamagnetic molecule (Tanod et al., 2019b). Antioxidant activity indicates chemical components’ ability to inhibit oxidation reactions, expressed as the percentage of DPPH radical scavenging. The percentage of DPPH radical scavenging for Jeruju leaf extract and ascorbic acid as a control is shown in Figure 3.

Table 3. Diameter of the inhibition zone from Jeruju leaves’ ethanol extracts against A. hydrophila. [Click here to view]

Table 4. Antibacterial activity of Jeruju leaves’ ethanol extract against A. hydrophila after 24 hours. [Click here to view]

Figure 3. DPPH scavenging effect of Jeruju leaves’ ethanol extract compared with ascorbic acid. [Click here to view]

Table 5. IC50 and total phenol content of Jeruju leaves’ ethanol extract (A. ilicifolius). [Click here to view]

Figure 3 shows an increase in the effect of DPPH radical scavenging, along with the increase in the concentration of Jeruju leaf extract. The antioxidant activity indicates the ability of an extract to scavenge free radicals (Tanod et al., 2019a). The ethanol extract of Jeruju leaf is thought to donate H atoms/electrons to interact with DPPH radicals. This study evaluated the IC₅₀ value and total phenol content of the Jeruju leaves’ ethanol extract (A. ilicifolius) using the Folin–Ciocalteu method (Table 5). The Jeruju leaves’ ethanol extract showed very strong potential as an antioxidant. Antioxidant activity was evaluated according to Blois (1958) and Riyadi et al.’s (2019) studies, namely very strong (IC₅₀ < 50 µg/ml), strong (IC₅₀ between 50 and 100 µg/ml), moderate (IC₅₀ between 100 and 150 µg/ml) and weak (IC₅₀ between 150 and 200 µg/ml).

Previous research has also evaluated the percentage of DPPH radical scavenging from Jeruju leaf extract (A. ilicifolius). The percentage of DPPH radical scavenging from the ethanol extract of A. ilicifolius, collected from Poondiyankuppam, northeast coast of India, ranged from 50.55 ± 2.88 to 86.87 ± 5.04%, which was compared with DPPH radical scavenging of ascorbic acid ranging from 65.12 ± 5.40 to 90.32 ± 5.12% (concentration = 0.1–2 mg/ml, with a DPPH concentration of 0.1 mM). In addition, it was also reported that the total phenol content of the ethanolic extract of A. ilicifolius was 257 mg GAE/g (Thirunavukkarasu et al., 2011b).

The ethanol extract of A. ilicifolius leaves collected from Alapakkam, Tamil Nadu, India, detected flavonoid and phenol components, with DPPH scavenging activity ranging from 20.59 to 76.79%, which was compared with DPPH scavenging activity of ascorbic acid ranging from 91.18 to 96.43% (concentration = 200–1000 µg, with a DPPH concentration of 0.1 mM). This study also reported that the ethanol extract of A. ilicifolius leaves’ total phenol content was 17.22 mg/10 ml extract (Vani and Manikandan, 2018).

Previous studies also reported IC₅₀ of A. ilicifolius leaves’ ethanol extract of 78.90 ± 1.87 µg/ml and ascorbic acid of 10.08 ± 1.79 µg/ml (DPPH concentration = 0.2 mM), and total phenol content of 128.86 ± 0.01 mg GAE/g dry weight (Biswas et al., 2019). The methanol extract of A. ilicifolius leaves collected from Wonorejo, East Java, Indonesia, reported alkaloid components, flavonoids, glycosides, polyphenols, steroids, and tannins. In addition, it also reported an IC₅₀ value of 17.51 µg/ml of the methanol extract of A. ilicifolius leaves, with a DPPH concentration of 0.06 mM (Andriani et al., 2020).

CONCLUSION

These research findings provide a potential activity for Jeruju (A. ilicifolius) leaves’ ethanol extract as antioxidant and antibacterial for inhibiting A. hydrophila growth. The antibacterial action mechanism of Jeruju leaf extract is thought to be closely related to its antioxidant properties. Metabolomic profile structure indicates the alkaloid and flavonoid components that play a role in the antioxidant and antibacterial activity of the Jeruju extract. Further studies on an ethanol extract of Jeruju leaves in vivo on fish infected with A. hydrophila need to be carried out to observe the Jeruju leaf extract’s toxicity and stability.

ACKNOWLEDGMENTS

The authors acknowledge the Rector, Dean of Faculty of Fisheries and Marine, Head of Institute for Research and Community Service, Universitas Lambung Mangkurat, and Head of Mandiangin Freshwater Aquaculture Center for Fisheries (BPBAT) for providing research facilities.

AUTHORS’ CONTRIBUTIONS

All authors made substantial contributions to the conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agree to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.

FUNDING

PNBP funds funded this research through the Compulsory Research Lecturer Program with DIPA funding of 2020 (No.023.17.2.6777518/2020), Universitas Lambung Mangkurat, Indonesia.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

ETHICAL APPROVAL

This study does not involve experiments on animals or human subjects.

DATA AVAILABILITY

All data generated and analyzed are included within this research article.

PUBLISHER’S NOTE

This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.

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