The materials used in this study included: bovine milk obtained from cattle farmers in Enrekang Regency, South Sulawesi, M. oleifera leaf from Topoyo Subdistrict, West Sulawesi Province (2°02'17.21"S, 114°15'30.36"E), CH₃OH_(pa), NaCl, (NH₄)₂SO₄, NaOH_(pa), HCl, xanthine substrate and allopurinol were purchase from Sigma Aldrich. The instruments used in this study were autoclave, centrifuge (Hermle Z336K), Alu-Lid rotor (Hermle 220.87 V20), rotary evaporator, vortex, stirrer magnetic, UV-Vis 1800 (Shimadzu-Japan), FTIR Spectrophotometer (Shimadzu-Japan), GCMS-QP2010 Ultra (Shimadzu-Japan).

The XO isolation process is a modified method from Bou-Salah (Bou-Salah et al. 2020), in which 500 mL fresh bovine milk was heated to a temperature of 30 °C, combined with 178.5 g of NaCl, then centrifuged at a speed of 3000 rpm for 30 minutes. The supernatant was fractionated with ammonium sulfate at 4 °C using an ice bath, then centrifuged at 8000 rpm at 4 °C for 20 minutes using a Alu-Lid rotor (RFC 21.379/24.325 xg; angle rotor 24 × 1.5/2.0 mL; angle 45°, maximum speed 15.000/16.000 rpm). The precipitate was dissolved in 0.05 M potassium phosphate buffer pH 7.5 to 250 mL.

Xanthine substrate of about 15.21 mg was added to the measuring flask and then added with five drops of 1 M NaOH, shaken until dissolved. The solution was diluted with CO₂-free demineralized water to 100.0 mL (1 mM concentration). The xanthine substrate was prepared by diluting the stock solution to obtain a standard solution, with a concentration of 0.05; 0.1; 0.15; 0.2, and 0.25 mM (Kostić et al. 2015).

Allopurinol 1000 μg/mL stock solution was prepared by weighing 10 mg of allopurinol and dissolving it in 5 drops of 1 M NaOH. The solution was transferred to a volumetric flask with a volume of 10 mL and then diluted with CO₂-free demineralized water. The standard allopurinol solution was prepared by diluting the stock solution to get a series of allopurinol standard solutions, with a concentration of 0.1; 0.2; 0.5; 1.0 and 2.0 μg/mL (Gong et al. 2020).

The crude XO extract was weighed about 22.17 mg using a 25 mL weighing bottle, then the extract was added into a volumetric flask and diluted with phosphate buffer solution. The volume was diluted to the limit mark to obtain an XO solution of 0.1 U/mL (Kostić et al. 2015).

The crude extract of the enzyme was characterized to determine the optimum conditions of the enzyme, such as pH, substrate concentration, and temperature effect (Natsir et al. 2002; Kostić et al. 2015). The optimum conditions were determined by analyzing the optimum activity of the enzyme. It was calculated by equation 1:

$\mathrm{Ea}=\frac{(\mathrm{Ab}-\mathrm{Ac})\mathrm{V}\times \mathrm{df}}{12.2\times 0,1}$ (1)

Where Ea is enzyme activity (mU/mL); Ab is the absorbance of blank; Ac is the absorbance of control; V is total volume assay (mL); df is dilution factor; 12.2 is uric acid extinction coefficient at 290 nm (mM); and 0.1 is the volume of XO used in milliliter (mL).

Phosphate buffer solutions of 0.2 M (3.9 mL) with a pH variation of 6; 6.5; 7; 7.5 and 8 were added 2 mL of 0.15 mM xanthine substrate solution, then pre-incubated for 10 minutes at 25 °C. 0.1 mL of XO was added to the mixtures and then incubated for 30 minutes at 25 °C. The absorption of the sample was measured at λ_max 232 nm using a UV-Vis spectrophotometer (Natsir et al. 2002; Kostić et al. 2015; Sharma et al. 2016).

The optimum substrate concentration was determined by adding 2 mL of phosphate buffer solution at the optimum pH, with a xanthine substrate concentration of 0.05; 0.10; 0.15; 0.20, and 0.25 mM. After pre-incubation, 0.1 mL of XO was added to the solution, and the mixture was incubated at 25 °C for 30 minutes. A similar procedure was applied for control by replacing the crude enzyme extract using 0.1 mL of distilled water (Natsir et al. 2002; Kostić et al. 2015).

Phosphate buffer solution 0.2 M (3.9 mL) of optimum pH was added to 2 mL of xanthine substrate with optimum concentrations, and then pre-incubated for 10 minutes. The enzyme XO (0.1 mL) was added, incubated for 30 minutes at 20; 25; 30; 35, and 40 °C. After the incubation process, the absorption was measured at λ_max of 232 nm using a UV-Vis spectrophotometer (Natsir et al. 2010; Monika et al. 2019).

M. oleifera leaves were harvested from the tree by manually collecting the 3^rd to 5^th petiole leaves. The leaves were washed and then dried for 7–10 days at room temperature. After drying, the leaves were then processed into a fine powder using a grinding machine. Dry M. oleifera leaves powder was mixed with methanol in a ratio of 1:20 (w/v). The extraction process was conducted at 45 °C for 20 minutes with constant stirring using a magnetic stirrer. The extract obtained was filtered and then evaporated using a rotary evaporator to obtain a thick methanol extract. The metabolomic profile of the methanol extract was analyzed using FTIR and GCMS (Natsir et al. 2018, 2019; Rocchetti et al. 2019).

The FTIR spectrum of M. oleifera leaves methanol extract was analyzed using an FTIR spectrophotometer (Shimadzu-Japan) at a wavenumber of 4000–250 cm^-1. The spectrum was recorded using approximately 1 mg of methanol extract (Meenakshi et al. 2020).

The methanol extract of M. oleifera leaves was analyzed using GCMS-QP2010 Ultra (Shimadzu), which was connected to a capillary column DB-1 (0.25 m film 0.25 mm I. d. 30 m length). The temperature of the injector was kept at 250 °C (constant). The column oven temperature was set at 50 °C for 3 minutes, then raised to 280 °C for 3 minutes, and finally held at 300 °C for 10 minutes. The chromatogram results were identified by comparing the obtained spectral configurations on mass spectral databases that were readily available (NIST libraries) (Ezhilan and Neelamegam. 2012).

The methanol extract of M. oleifera leaves were diluted to a 10, 20, 40, 80 and 160 μg/mL concentrations with 0.05 mM phosphate buffer solution pH 7.5. An aliquot of 3 mL extract solution was added to a reaction tube, followed by 2 mL of 0.15 mM xanthine and 0.2 mL of XO, and then incubated at room temperature for 45 minutes. After incubation, 1 mL HCl (0.58 M) was added to the mixtures to stop the enzymatic reaction. Water was used as the control solution for the negative control, and allopurinol as a positive control. The absorbance of the solution was measured using a UV-Vis spectrophotometer at λ_max of 232 nm. Calculation of inhibition ability was obtained from the linear equation of the time versus concentration of the XO curve (Fachriyah et al. 2018).

Molecular docking was performed using the AutoDock Vina package developed by Trott and co-workers to determine the ligand’s binding site into the receptor’s catalytic site (Trott and Olson 2010). In this study, the M. oleifera leaves extracts were indicated to treat gout disease induced by hyperuricemia. A study reported that 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, quinic acid, and 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran were identified as the primary compounds of M. oleifera and therefore used as the ligand molecules for the docking. The chemical structures of those ligands were retrieved from the PubChem database, as shown in Fig. 1. All ligands were downloaded and saved as sdf extensions. Open Babel 2.4.1 program packages were applied to convert sdf files to pdbqt extension (O’Boyle et al. 2011). As for the target molecule, XO was assigned as the receptor since this enzyme is related to gout disease. The receptor’s tertiary structure was obtained from a protein data bank (PDB ID: 1v97) at a resolution of 1.94 Å, as shown in Fig. 2 (Okamoto et al. 2004). The polar hydrogen and Kollman’s united atom charges were added to the receptor using AutoDock Tools 1.5.6 created by Morris and co-workers (Morris et al. 2009). Afterwards, the XO was saved in pdbqt format.

Figure 1. 

The compounds’ chemical structure of (a) 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside (PubChem ID: 537841), (b) Quinic acid (PubChem ID: 6508), (c) 2-Dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran (PubChem ID: 559105), and (d) Allopurinol (PubChem ID: 135401907) as the positive control.

In performing molecular docking, a grid box parameter is required to decide the positional and rotational of the ligand into the moiety of the receptor (Arwansyah et al. 2021a). The grid box was constructed using 24 × 24 26 points and a grid spacing of 1.00 Å. Meanwhile, the grid box’s central coordinates were set to x = 148.649, y = 43.411, and z = 26.399. The exhaustiveness was computed at 100. Other parameters were assigned as the default of AutoDock Vina. To determine the binding pose and conformation of the ligand within the receptor site, the Broyden-Fletcher-Goldfarb-Shanno (BFGS) algorithm was used as a search parameter. The docking protocols were set according to a similar procedure with our previous study provided in Ref. (Arwansyah et al. 2021b).

Figure 2. 

The tertiary structure of the created model of XO (PDB: 1v97) (Okamoto et al. 2004). The structures of α-helix, β-sheet, and turn are presented by cartoon models’ red, yellow, and green colours.

Determination of the optimum pH of the enzyme was carried out by conditioning the enzyme at a certain pH in the reaction between the enzyme and the substrate, as shown in Fig. 3a. Each type of enzyme has an optimum pH range, where the enzyme offers maximum activity in high stability. Generally, enzymes are amphiphilic, which means they can be acidic and base due to their active ability to provide functional groups of specific amino acid residues that are donor and acceptor proton (Singh et al. 2017). The XO activity showed that the optimum pH was at 6.5 with the activity of 32.47 mU/mL, and after pH increased at pH 7.0, XO activity decreased to 18.10 mU/mL. It was unveiled that the increase of enzyme activity at the optimum pH can be related to changes in ionization of the enzyme ionic group on the active site. Thus the conformation of the active site becomes more effective in binding and changing the substrate during the catalysis process (Huang et al. 2017).

Figure 3. 

Characterization of XO (a) pH; (b) Substrate concentration; (c) Temperature.

The effect of substrate concentration was assessed to determine the optimum substrate concentration suitable for the enzyme. The substrate concentration used was 0.05; 0.1; 0.15; 0.2; 0.25 mM. The results obtained are shown in Fig. 3b. The results demonstrated that higher enzyme activity was achieved at elevated substrate concentration. However, when thereafter it reached the optimum substrate concentration, the activity tended to decrease. We reported that the highest enzyme activity was at 0.1 mM substrate concentration, with an activity of 21.55 U/mL. The increase in substrate concentration is directly related to the reaction rate until it reaches a maximum value of V_max. If the substrate concentration is increased, there will be no increase in the reaction rate because the substrate has saturated the enzyme’s active site (Sharma et al. 2016).

Temperature is critical in enzymatic reactions because enzymes are proteins that are easily denatured against changing environmental conditions. The change in environmental temperature will affect enzyme activity (Claaßen et al. 2019). The enzyme will show optimal catalytic activity at a specific temperature and denatured when exposed to extreme temperatures (Roche and Royer 2018). When the temperature increases to optimal, the reaction rate would be accelerated because kinetic energy increases (Marañón et al. 2018). Increased kinetic energy will accelerate the motion of vibration, translation, and rotation of both enzymes and substrates. It will increase the frequency of collisions between enzymes and substrates (Zhang et al. 2016). In this study, the determination of the optimum temperature of the XO enzyme used a variation of incubation temperature in the range of 20; 25; 30; 35 and 40 °C. The results obtained are shown in Fig. 3c. From the graph, it is clearly seen that the optimum temperature was reached at 35 °C with an activity of 21.94 mU/mL.

FT-IR spectroscopic analysis of M. oleifera leaves methanol extract was used to analyze the phytoconstituents in the sample based on spectral data (Fig. 4).

Figure 4. 

FTIR spectrum of methanol extract of M. oleifera leaves.

The results of the FTIR analysis of the methanol extract of M. oleifera leaves (Table 1) showed the presence of flavonoids and phenolics from the O-H and C=O groups at 3435 cm^-1 and 1732, 1714 cm^-1 which indicated the presence of C-H stretching. Alkaloids in the C-N band at 1460, 1411 cm^-1 and N-H in the fingerprint region of 1635 cm^-1 (Maobe et al. 2013).

Tannins were discovered in the form of free phenol by stretching O-H at 3435 cm^-1 and C-O at 1238 cm^-1. The C=O band is represented by the peak found at 1732, 1714 cm, and the C-O band is represented by the peak found at 1238 cm^-1.

GCMS profiling data showed three main compounds in the methanol extract of M. oleifera leaves, namely 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, quinic acid, and 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran. The presence of 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside was identified from the O-H group at 3435 cm^-1, C-H at 2924, 2854 cm^-1, C=O at 1732, 1714 cm^-1 and C-O at 1238 cm^-1 which are stretch in cyclic ethers. The presence of the C-S group at 596 cm^-1 is a specific band of the 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside compound. Quinic acid was identified from the typical strain O-H at 3435 cm^-1, C-O at 1238 cm^-1, C-C=O at 528 cm^-1 and C-OH at 921 cm^-1 which is a typical band of carboxylic acid groups. The compound 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran was identified from the presence of C-H band at 2924, 2854 cm^-1, C-O at 1238 cm^-1, and Si-O-C at 1053 cm^-1 which are typical bands of this compound.

Various concentrations of enzyme were used in the inhibition assay to determine the relationship between increasing enzyme concentration and inhibition activities. We reported that the methanol extracts of M. oleifera leaves demonstrated in vitro XO inhibition activity, and the results are presented in Table 3.

The analysis inhibition showed that the effectiveness of inhibition was directly proportional to the increase in extract concentration. Methanol extract at a concentration of 10 mg/mL showed inhibition values of 5.73%, while at a 160 mg/mL concentration, the inhibition value was 21.35%. The increased inhibitory activity of the methanol extract of M. oleifera leaves was significantly linked with the metabolite content of the leaves. The presence of three main compounds in the methanol extract of M. oleifera leaves namely is 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, quinic acid, and 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran, become a constituent that works to prevent substrates from entering the enzyme’s active site.

The mechanism for binding the substituent to the active site of the XO enzyme occurs through the interaction of the O-H, C=O and C-H aliphatic functional groups of the three main compounds through hydrogen bonds and hydrophobic interactions. The interaction mechanism can be seen in Fig. 5. Another study investigating the potential of M. oleifera leaves constituents as XO inhibitors were conducted by Tian et al (2021) who studied the enzymatic hydrolysis of phenolic and peptide fractions of M. oleifera leaves. It was discovered that the hydrolysis process significantly increased the inhibitory activity of XO as well as the antioxidant activity. These findings in the present study showed methanol extract of M. oleifera leaves activity indicating a promising potential for its development as an XO inhibitor.

To analyze the molecular recognition concerning the inhibitory activity, an advanced experimental investigation by X-ray analysis is required to obtain insight into the molecular interaction, including binding energy between the extraction of M. oleifera leaves and the tested enzyme (XO). However, computational analysis using the molecular docking method can currently investigate the structural and conformational changes of the ligand-receptor complex (Qashqoosh et al. 2019). Therefore, this method is employed to find the viewpoints of physical and chemical properties regarding the binding action of M. oleifera into the moiety of XO.

In order to perform molecular docking, protein target (receptor) and promising drugs (ligand) are required to be prepared. As for receptor molecules, the crystal structure of XO was retrieved from the protein database (PDB: 1v97) (Okamoto et al. 2004). Meanwhile, the extracted M. oleifera was employed as the ligand molecules. From our docking simulations, the binding energies and the binding pose between ligands and receptors were obtained and presented in Table 4 and Fig. 7, respectively. Further, the details of molecular interaction consisting of hydrogen bonds and hydrophobic interactions between the ligands-receptor complexes were provided in Table 5 and Fig. 8, respectively.

Figure 7. 

Binding poses of ligand in complex with a receptor (XO). Complex 1 consisted of mixed compounds where the red, yellow, and cyan lines refer to 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, quinic acid, and 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran compounds, respectively, (b) complex 2 denoted to allopurinol (control). The PLIP program (Salentin et al. 2015) and Pymol v 2.3 program packages (DeLano. 2002) was used to display the conformational poses of each complex.

In this research, only the primary compounds of M. oleifera, i.e., 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, quinic acid, and 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran, were selected to become the ligand molecules. It was not easy to simulate three ligands simultaneously in docking protocols because ligands may overlap on each atom causing structural changes from the initial configuration. Molecular docking was performed separately for each ligand, and overall results were combined to create one configuration because of the similar receptor (XO) to overcome this issue.

Figure 8. 

Hydrophobic interaction of ligand in complex with the receptor. (a) 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, (b) Quinic acid, (c) 2-Dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran, (d) Allopurinol (control). The ligand refers to the stick model in magenta color. Redline is represented hydrophobic interaction between ligand and residues of the receptor.

Three ligands, i.e., 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside (PubChem ID: 537841), quinic acid (PubChem ID: 6508), 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran (PubChem ID: 559105) against XO are extracted from the PubChem database. The possibility of ligand binding to the receptor site is achieved when the binding energy of the ligand-receptor complex is a negative value. From our finding, three complexes were found with various binding energies, as listed in Table 4.

All ligands showed negative values binding energy which are -9.3 kcal/mol, -8.2 kcal/mol, and -10.6 kcal/mol for 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, quinic acid, and 2-dimethyl(trimethyl silylmethyl)silyloxymethyltetrahydrofuran consecutively, indicating that the ligands could bind to the receptor, forming a ligand-receptor complex. In addition to that, the ligands of all complexes have higher binding energies than allopurinol (a positive control), with the binding energy of -6.6 kcal/mol. This finding revealed that those 3 ligands found in the extract of M. oleifera leaves probably hold the potential as inhibitors for XO. Even though the inhibition activity of allopurinol is higher compared to the extract, the results still demonstrate that the M. oleifera leaves extracts containing those primary compounds could inhibit the XO enzyme. Thus, to identify the molecular interactions, including hydrogen bond and hydrophobic interaction between ligand and receptor, the snapshot structure of those ligands was analyzed using PLIP server (Salentin et al. 2015) and LigPlot v.4.5.3 program packages (Wallace et al. 1995).

Fig. 8 illustrates the binding site and geometrical pocket of the ligands in complex with the receptor. The result of complex 1 was visualized from multiple dockings of the several ligands, then combined into one configuration. The lines in red, yellow, and cyan colours correspond to the compounds of 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, quinic acid, and 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran, respectively. Meanwhile, complex 2 was denoted from the positive control into the site of XO. Of this figure, hydrogen bond and hydrophobic interactions contributed to the binding of ligand into the receptor. The detailed hydrogen bonds for those complexes are listed in Table 5. It was found that 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside of complex 1 participated in hydrogen bonds with residues Arg912, Gln1040, Ser1080, Ser1082 of the receptor. In quinic acid, the hydrogen bonds were made in the residues of Arg912, Arg912, Ala1079, Ser1080, Ser1080, Glu1261. For 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran, the hydrogen bonds were formed with the residues of Thr1083, Gly1260. Meanwhile, allopurinol shown in complex 2 participated in hydrogen bonds with residues Gly797, Phe798, Met1038, and Gln1194. On the other hand, the hydrophobic interactions between ligand and receptor are presented in Fig. 8. 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside of complex 1 formed the hydrophobic interaction with residues of the receptor, including Met1038, Arg912, Gly799, Glu1261, Phe911, Phe798, Ala1079, Ser1080, Gly1260, Ala1078, Thr1083, Lys1045, Val1259, Ser1082, Leu1042, Gly1039, Gln1040, Gln1194, Gln112, and Cys150. For quinic acid, the hydrophobic interaction was presented by the ligand’s interaction with residues of the receptor such as Met1038, Cys150, Gln112, Arg912, Phe798, Gly799, Phe911, Ala1078, Ala1079, Glu1261, Ser1080, Val1259, Lys104, Thr1083, Ser1082, Gln1010, Gln1194, and Gly1039. 2-Dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran participated in hydrophobic interaction with residues of Gly799, Arg912, Gly1260, Gln1040, Glu1261, Gln1194, Ser1080, Ala1079, Ala1078, Phe914, Phe1009, Arg880, Glu802, Phe798, Gln767, and Met1038. Meanwhile, for allopurinol, the hydrophobic interaction was observed between ligand and residues, including Gly1039, Phe798, Gly797, Gly796, Arg912, Gln1040, Gln112, Gln1194, Cys150, and Met1038. From these results, all primary ligands in the extracted M. oleifera leaves may become stable structures since the hydrogen bonds are coordinated between ligand and receptor. Also, the compounds of 5-O-acetyl-thio-octyl-β-L-rhamnofuranoside, quinic acid, and 2-dimethyl(trimethylsilylmethyl)silyloxymethyltetrahydrofuran bound to the similar residues of allopurinol, indicated that those compounds have similar activity as an inhibitor for XO. Furthermore, in the paper presented by Okamoto and co-workers (Okamoto et al. 2004), the catalytic site of XO provided and visualized in the UniProt database (https://www.ebi.ac.uk/pdbe/entry/pdb/1v97/bound/MTE), which includes residues Gly797, Met1038, Phe798, Arg912, Glu1261, Ala1079, Ala1078, Ser1080, Gln1040, Val1081, Ser1082, Gly1039, Gln1194, Cys150, and Gln112, is a crucial target for inhibiting the XO. Thus, the ligand that can bind to one of these residues is assumed to have a potential drug for treating goat disease. From our simulations, each ligand is bound to those residues by hydrogen bond and hydrophobic interaction, indicating those compounds can become inhibitor XO.