The potential of Sonneratia caseolaris mangrove leaves extract as a bioactive food ingredient using various water extract

Sampling method

The leaves of S. caseolaris were collected from a mangrove plant in Ujung Pangkah Gresik, East Java, Indonesia (6.8899940° S, 112.6081658° E). Sample was collected from the third to the fifth leaf, counted from the top of each plant. These leaves were thoroughly rinsed to remove salt using running water and then air-dried at room temperature for 7 days. Subsequently, the dried leaves were mashed into a fine powder for extraction.

Sample extraction

The extraction process utilized various solvent, including distilled water (A), bottled drinking from three different brands (Ax (B), Cx (C), Kx (D), and methanol (Cat. 179337, Sigma-Aldrich^®) (E) for control purposes. The bottle drinking water (B, C, and D) was sourced from the Indonesian market. and commonly used as a type of mineral water in Indonesia. Mangrove leaf powder was extracted with each solvent at ratio 1:3 (w/v). The extraction was conducted through maceration at room temperature for 24 hours, followed by filtration using Whatman^® quantitative filter paper, ashless, Grade 42 (Cat. WHA1442041, Merck). The extraction process was repeated three times for each solvent. The resulting filtrates were subsequently evaporated using a rotary evaporator (DLab rotary evaporator RG100-S, 40°C). Every 1000 grams of fresh leaves will become 200 grams of dried leaves. In this study, the third and fifth leaves are selected based on previous research findings (unpublished data). These leaves have shown higher total phenol, total alkaloid, and flavonoid content compared to other parts of the leaf. However, there is no significant difference in pH and viscosity.

Calculation of extraction yield and pH examination

The extraction yield was calculated by the following formula.¹ The pH of the extracts was measured with a pH meter (Toledo S-220-KIT). The analyzed sample is a working solution derived from the evaporator, consisting of 1 mL of solution mixed with 20 mL of double-distilled water.

Total phenol content

The total phenol test refers to Ref. 23 Total phenol content was measured by mixing 30 μL Folin-Ciocalteu (Cat. F9252, Sigma-Aldrich^®) 1.0 N reagent with 60 μL of the sample, placed on the microplate. Afterwards, 150 μL of 20% sodium carbonate (Cat. 223530, Sigma-Aldrich^®) solution was placed on a microplate, incubated for 15 minutes at room temperature and in a dark place. Samples were centrifuged for 8 minutes at 1600 rpm. The supernatants were measured on a Microplate reader at a wavelength. The total phenol was calculated using the gallic acid (Cat. 91215, Sigma-Aldrich^®) standard curve (mgGAE/100g).

Total flavonoid content

1 mL sample was mixed with 3 mL of ethanol 96% (Cat No. 159010, Sigma-Aldrich^®), 0.2 mL of 10% aluminium chloride (Cat. 8.01081, Sigma-Aldrich^®), 0.2 mL of 1 M potassium acetate (Cat No. 1.04820, Sigma-Aldrich^®), and 5.6 mL of distilled water. The mixture was incubated for 10 minutes at room temperature. The supernatant was measured on a Microplate reader at a wavelength of 415 nm. The total phenol was calculated using the standard curve of quercetin acid (mgQE/100g) (Lerck, Cat No. PHR1488).

Antioxidant activity

Antioxidant activity was measured using a microplate reader (96-flat bottom with lid).²³ The DPPH (Cat. 300267, Sigma-Aldrich^®) reagent was prepared at a concentration of 0.2 mM in absolute ethanol. The first column was filled with 200 μL of ethanol as a blank. Line A was filled with sample stocks. 100 μL of ethanol solvent was added to each well plate for serial dilution, except for line A. The second -11^th column in line A was filled with 200 μL of sample stock solution. Serial dilution was carried out by taking 100 μL from line A, and placing it in lines B, C, D, E, F, and G respectively. In line G, 100 μL solution was removed, resulting in each well containing 100 μL of the solution. Subsequently, 100 μL of DPPH was introduced into each well. Line H was negative control (100 μL solvent mixed with 100 μL of 0.2 mM DPPH reagent). The plate was covered and incubated at room temperature for 30 minutes. All samples were measured at 517 nm wavelength. DPPH absorption inhibition was calculated with the following formula.²

In order to obtain the percentage of inhibition (Inhibitory Concentration value, IC₅₀), a linear regression curve (Y=ax + b) was made with the x-axis as the concentration (μg/mL) and the y-axis as the percentage of inhibition. The IC₅₀ value is a calculation of how much of the sample concentration is needed to inhibit 50% of free radical activity.

Statistical analysis

The data were statistically tested using a completely randomized design with three replications. The normality of the data was tested using the Kolmogorov-Smirnov test and then subjected to one-way ANOVA. Differences between means were determined using Duncan’s multiple range test, with P<0.05 regarded as significant. Statistical analysis was conducted using SPSS version 17.0.

Detection of active compound using LC-HRMS

LC-HRMS used HPLC (Thermo Scientific Diorex Ultimate 3000 RSLCnano, Japan) with solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in Acetonitrileà Acetonitrile), column Hypersil GOLD, 5 μm, 150 × 4.6 mm, flow rate 40 μL/min, column temperature 30°C. Mass Spectrometer used Scientific Q Exactive™ with software compound discovery with mzCloud™ MS/MS library. Samples were diluted to a volume of 1500 μL, and vortexed at 2000 rpm, for 2 min. The supernatant was filtered with a 0.22 μm syringe filter. The vial was inserted into the LC-HRMS autosampler.

Ligand and protein preparation

This study used three compounds that had been identified through LC-HRMS analysis, namely Betaine (CID 247), Choline (CID 305), TEMPO (CID 549976), and one control compound (CID 135263934) which acts as the original inhibitor of the Keap1 protein. Ligand was obtained from the PubChem web server (https://pubchem.ncbi.nlm.nih.gov/) in Sybil Data Files (SDF) format. Betaine, Choline, and TEMPO are compounds that are known to have antioxidant activity. In this study, it is anticipated that they may bind to Keap1 (6TYP), which serves as an endogenous Nrf2 inhibitor. as an endogenous Nrf2 inhibitor. The Keap1 3D structure was obtained from the RCSB PDB web server (https://www.rcsb.org) Keap1 was then prepared to remove water molecules and ligands with BIOVIA Discovery Studio 2019.

Molecular analysis of docking and ligand-receptor interactions

Ligands-Keap1 interactions were analysed by molecular docking using AutoDock Vina integrated in PyRx version 0.9.5. The molecular docking process is carried out using a specific docking method based on the active site of the Keap1 protein.^17,22 Docking results, bond positions, and amino acid residues formed between ligands-receptor were analysed using PyMol software and BIOVIA Discovery Studio 2019.

Molecular dynamic simulation

Molecular dynamic simulations were carried out using Yet Another Scientific Artificial Reality Application (YASARA). The molecular dynamic simulation aims to compare the interaction complexes of Betaine, Choline, TEMPO, and Keap1-binding inhibitors. The parameters in the simulation correspond to the physiological conditions of the cells, namely temperature 37 °C, 1 atm, pH 7.4, and 0.9% salt content for 50 ns with autosaved every 25 ps. The simulation is run by the md_run macro program, and the results are displayed by the md_analyze and md_analyzeres programs.^24–26

Phytochemical properties and antioxidant activity of S. caseolaris leave extract

The mass yield percentage, pH, total flavonoids, total phenols, and antioxidant activity of S. caseolaris mangrove leaves extracted using various solvents yielded different results. Mangrove leaves of S. caseolaris extracted with methanol (E) and distilled water (A) exhibited high antioxidant activity, with IC₅₀ values of 63.72 ± 1.27 mg/mL and 89.29 ± 006 mg/mL respectively. The high antioxidant activity was supported by the total amount of flavonoids and total phenol of both extracts, which were higher than the other extracts. In contrast, mangrove leaves extracted with bottled drinking water (Ax, Cx, and Kx) showed low antioxidant activity. Mangrove leaves extracted with distilled water (A), Ax brand bottled mineral water (B), and methanol (E) have a pH of less than 7 ( Table 1).⁴⁸

The content of bioactive compounds in mangrove leaf extract of S. caseolaris was analyzed using LC-HRMS. The analysis results revealed the presence of 16 identical active compounds in all S. caseolaris mangrove leaf extracts obtained with different solvents ( Table 2). The bioactive compound content in mangrove leaf extract from S. caseolaris was assessed through LC-HRMS. The findings from the analysis indicated the presence of 16 identical active compounds in all S. caseolaris mangrove leaf extracts, regardless of the solvents used (refer to Table 2). From the comprehensive selection, three compounds were identified exhibiting antioxidant activity: TEMPO, Betaine, and Choline. Figure 1 Displays the 2D and 3D representations of these substances interacting with the Keap1 protein.

Figure 1. Ligands-Keap1 interactions and the amino acid residues involved.

A-D. Visualization of molecular docking results between inhibitors, TEMPO, Betaine, and Choline with Keap1 protein. The top row shows each ligand's position that binds to the Keap1 protein. The bottom row shows the amino acid residues involved in the interactions of the inhibitor, TEMPO, Betaine, and Choline with the Keap1 protein. Keap1 protein is presented as a pink ribbon. The compounds are presented in the form of colored sticks, namely inhibitors (blue), TEMPO (orange), Betaine (gray), and Choline (yellow).

Ligand-Keap1 molecular interactions

This study compared the binding activities of Betaine, Choline, TEMPO, and inhibitors to the Keap1 protein. The inhibitor used is a natural inhibitor that binds to the active site of the Keap1 protein and serves as a control. Molecular docking results were visualized with PyMol and Discovery Studio to identify the binding site of Ligand-Keap1.

Molecular docking results indicated that the TEMPO-Keap1 interaction possesses a lower binding affinity value compared to the Betaine-Keap1 and Choline-Keap1 interactions, which have values of -5.5 kcal/mol, -4.2 kcal/mol, and -3.7 kcal/mol, respectively. The lower binding affinity value observed for TEMPO-Keap1 interaction suggests its strength is greater than that of the Betaine-Keap1 and Choline-Keap1 interactions. Notably, the binding affinity value of TEMPO-Keap1 is higher than that of Inhibitor-Keap1, which exhibits a binding affinity value of -11.1 kcal/mol ( Table 3).

The visualization results revealed that Betaine shares the same binding site as the inhibitor (control), whereas TEMPO and Choline do not exhibit a specific binding site. They are overlap with the binding of the inhibitor ( Figure 1 on the top row). The visualization process continued with the identification of the amino acid residues involved in the Ligand-Keap1 interaction. It is observed that the Betaine-Keap1 interaction shares the same amino acid residues as the inhibitor-Keap1 interaction, and these interactions involve the highest number of residues when compared to the TEMPO-Keap1 and Choline-Keap1 interactions. Specifically, Ser363, Arg380, and Arg415 were identified as three amino acid residues engaged in hydrogen interactions on Betaine-Keap1. Furthermore, the visualization results indicated that Arg380 and Arg415 in Betaine-Keap1 interaction were also involved in hydrophobic interactions. Additionally, five other amino acid residues engaged in hydrophobic interactions in the Betaine-Keap1 were Tyr334, Gly364, Ala556, Ser602, and Gly603 ( Table 3 and Figure 1 in the bottom row).

Four amino acid residues are shared between the TEMPO-Keap1 and inhibitor-Keap1 interactions, namely Gly462, Gly509, Ala556, and Gly603, all of which are involved in hydrophobic interactions. The Choline-Keap1 interaction shares the same two amino acid residues as the inhibitor-Keap1 interaction, namely Gly509 and Ala556, and these interactions are also hydrophobic in nature. The presence of identical amino acid residues among TEMPO, Betaine, Choline, and the inhibitor in the Keap1 protein suggests the possibility that these compounds may exhibit similar activity to the inhibitor (control).

The binding affinity of the ligand-receptor complex increases when amino acid residues are involved in the interaction through hydrogen bonds and hydrophobic bonds.

Although, the Betaine-Keap1 interaction involves more amino acid residues than the TEMPO-Keap1 interaction, the binding affinity value of Betaine-Keap1 is higher than that of TEMPO-Keap1. It is possible that the amino acid residues Gly462 and Gly509 play a specific role in the TEMPO-Keap1 interaction, and these particular residues are not found in the Betaine-Keap1 interaction ( Table 3 and Figure 1 in the bottom row).

It’s important to note that a higher binding affinity value in the TEMPO-Keap1 interaction does not necessarily imply that it is weaker than the inhibitor-Keap1 interaction with its lower binding affinity value.

After molecular docking, molecular dynamics (MD) simulations were conducted to assess the stability of the Ligand-Keap1 interaction complex. The parameters employed in this simulation included the Root Mean Square Deviation (RMSD) of the Ligand-Keap1 complex, RMSD of Ligand movement, Root Mean Square Fluctuation (RMSF), and the number of hydrogen bonds within the Ligand-Keap1 complex.²²

The molecular dynamic analysis results indicated that both the TEMPO-Keap1 and Choline-Keap1 complexes exhibited similar stability to the Keap1 inhibitor throughout the simulation, as evidenced by an RMSD value of <2 Å. On the other hand, the Betaine-Keap1 complex at the beginning of the simulation up to 20 ns also showed the same stability, but after 20 ns the complex was unstable until the end of the simulation with an RMSD value of >3 Å. The RMDS Ligand movement results further supported the observation of Betaine’s instability after 20 ns, with significantly differing RMSD values when compared to TEMPO, Choline, and inhibitors ( Figures 2A and 2B). These findings align with previous studies, which reported that a stable ligand-receptor complex during the simulation typically maintain an RMSD value of 3 Å.^27,28

Figure 2. Molecular dynamic simulation.

The stability of the ligand-Keap1 interaction complex during the simulation was indicated by (A) the RMSD of the ligand-Keap1 complex value, (B) the RMSD of the Ligand movement value, (C) the RMSF value, and (C) the number of hydrogen bonds.

The fluctuation of the amino acid residues also serves as an indicator of the stability of the ligand-Keap1 complex. The results demonstrated that all complexes exhibited nearly identical fluctuations in amino acid residues ( Figure 2C). Previous research has indicated that a higher degree of fluctuation among amino acid residues correlates with increased instability in the e ligand-receptor complex.²⁹ In all interaction complexes, more than 200 hydrogen bonds were observed ( Figure 2D). The abundance of hydrogen bonds within these complexes indicates their stability. However, it’s important to note that the stability of the Betaine-Keap1 interaction requires further analysis using additional parameters to ensure a conclusive determination. This is because the results of the RMSD Ligand-Keap1 complex, RMSD Ligand movement, RMSF, and the number of hydrogen bonds of the Betaine-Keap1 complex exhibit variations.