Tyrosinase inhibition
The tyrosinase inhibition using the extract of different plants and kojic acid as positive control has been examined with a fixed L-DOPA concentration and varying the inhibitor concentration. Linear regression has been developed by plotting tyrosinase activity inhibition versus the initial concentration of inhibitor [3, 4]. A strong inhibition of tyrosinase-diphenolase activity (TYR-Di) has been determined by using all examined plants extracts. All examined extracts displayed a concentration-dependent inhibition toward the activity of TYR-Di. The developed data were expressed as the concentration of inhibitor which can inhibit 50% of tyrosinase activity (IC50) as illustrated in (Fig. 2A). The order of IC50 values for TRY-Di inhibition using Moringa oleifera L., Ocimum basilicum L., Artemisia annua L., and Coriandrum sativum L. were respectively as follow: 98.93, 122.49, 136.01, and 193.67 µg/ml, however, the IC50 values for TRY-Di inhibition using kojic acid was 88.92 µg/ml. The extract of Moringa oleifera is a promising potent inhibitor for the inhibition of TYR-Di when compared with the other plant extracts, based on the values of IC50. This may be ascribed to the high contents of flavonoids and phenolics constituents. Among the investigated plants, Coriandrum sativum L. displayed the lowest flavonoids contents, however, the Moringa oleifera L. showed the highest contents of flavonoid compounds.
A bivariate correlation (Pearson coefficient) has been employed for the determination of the relation among the total flavonoids in the plant extract and IC50 of TYR-Di inhibition (Fig. 2B). In this study, the correlation coefficient (r) was found to be – 0.8443 among the two variables which designates a good negative correlation. Hence, the TYR-Di inhibition may be due to the flavonoids in the plant extract [24, 43]. Previous researchers have been described that the extract of different plants had a potent inhibitory effect on the tyrosinase activity [2, 43–45]. A remarkable inhibitory effect of the Moringa oleifera L. leaves extract on the tyrosinase activity have been reported to be due to a high content of flavonoid compounds [24, 28]. Derivatives of kaempferol, luteolin, and quercetin reported as major flavonoid compounds which are predominant in the plant extract and exhibited a significant inhibition on the catalytic reaction of tyrosinase [5, 28].
Type of tyrosinase inhibition
To determine the inhibition type for mushroom TYR-Di, the potent extract of the examined plant parts was selected after performing interpolations based on the low IC50 value. The values of Vmax and Km were calculated from the Lineweaver-Burk plot using L-DOPA as substrate. The results revealed that the enzyme catalytic activity followed Michaelis-Menten kinetics. The Vmax was 1.58 U/mg protein and Km of 0.89 mM for the diphenolase activity of TYR using the dataset obtained in the absence of an inhibitor. At various inhibitor concentrations, the kinetics and type of TYR-Di inhibition were clearly varied according to the type and concentration of examined plant. The developed Lineweaver-Burk plots of the TYR-Di activity with exposure to various inhibitor doses showed that the inhibition was mixed type (i.e. in which the enhancement of inhibitor concentration triggered increase in the value of Km and decreases in the Vmax value) in the presence of the extract of Ocimum basilicum L, and Artemisia annua L (Fig. 3A&B). A non-competitive type of inhibition on TYR-Di (i.e. in which the supplement of extra inhibitor triggered reduction in the value of Vmax in the presence of the extract of Coriandrum sativum L (Fig. 3D). However, the extract of Moringa oleifera L. leaves displayed competitive inhibition (i.e. in which the supplement of extra inhibitor triggered increment in the value of Km and kept the value of Vmax unaltered) on the TYR-Di activity. The values of Vmax were distinctly reduced by 19.3, 30.2, and 40.7% in the presence of 50, 100, and 150 µg/ml of Moringa oleifera L. leaves extract with respect to control without inhibitor represent 100%, respectively. While the Km values were sharply reduced in the range from 29.5–54.4% in the presence of inhibitor dose of 50–150 µg/ml, compared to the preparations without inhibitor (Fig. 3C). Similar results have been mentioned by [2, 44–46].
The mechanism of inhibition of the selected plant extract for the diphenolase activity of tyrosinase was further examined via the determination of the inhibition constants [43]. The (\({\text{K}}_{\text{i}}^{\text{a}\text{p}\text{p}}\)) which is the equilibrium constant of the inhibitor binding to the free tyrosinase and the (\({\text{K}}_{\text{i}\text{i}}^{\text{a}\text{p}\text{p}}\)) which is the constant for the binding of inhibitor for the enzyme-substrate complex were determined according to [28]. From the obtained results of the double reciprocal plots of Lineweaver-Burk, a secondary plot of the apparent slopes (Km/Vmax) and y-intercepts (1/Vmax) were performed against various inhibitor concentrations. The Vmax values were clearly reduced, and Km values were increased during the rise in the concentrations of the extract of Ocimum basilicum L. and Artemisia annua L. The results also revealed that the tested inhibitors (plants extract) could combine with the free enzyme and with the enzyme-substrate complex. The inhibition equilibrium constants (\({\text{K}}_{\text{i}}^{\text{a}\text{p}\text{p}}\)) of Ocimum basilicum L. and Artemisia annua L. on TYR-Di activity using L-DOPA substrate were respectively found to be 75, and 110 µg/ml (Fig. 4A-B). While the (\({\text{K}}_{\text{i}\text{i}}^{\text{a}\text{p}\text{p}}\)) of Ocimum basilicum L. and Artemisia annua L. on TYR-Di activity were 200, and 155 µg/ml when using L-DOPA substrate, respectively (Fig. 5A-B). In the previous circumstances, the values of \({\text{K}}_{\text{i}}^{\text{a}\text{p}\text{p}}\) were significantly lower than the values of \({\text{K}}_{\text{i}\text{i}}^{\text{a}\text{p}\text{p}}\), representing that the inhibitor displays as competitive-uncompetitive mixed-I type inhibitor [28, 43, 47]. This behavior indicates that the competitive influence was greater than the uncompetitive one, hence the free TYR inhibited more than the TYR-Di complex.
The inhibitory kinetics of the extract of Moringa oleifera L. leaves displayed a rise in the values of Km and retained Vmax unaltered indicating a competitive inhibition of the extract of Moringa oleifera L. leaves on the activity of TYR-Di using L-DOPA substrate (Fig. 4C). This inhibition type suggests that the extract can inhibit only the free enzyme. Hence, the value recorded for the \({\text{K}}_{\text{i}}^{\text{a}\text{p}\text{p}}\) constant (198 µg/ml) as well as the competitive type of inhibition proposed that the Moringa oleifera L. leaves extract contains a strong potent inhibitor on the TYR-Di activity.
The extract of Coriandrum sativum L showed a sharp reduction in the Vmax value, while the value of Km was unchanged, indicating a noncompetitive inhibition of the TYR-Di activity. Additionally, the \({\text{K}}_{\text{i}}^{\text{a}\text{p}\text{p}}\) value was the same as the value of \({\text{K}}_{\text{i}\text{i}}^{\text{a}\text{p}\text{p}}\). (Fig. 4D&5C). Therefore, the inhibitor can bind to both the free enzyme and the enzyme-substrate complex. From the previous results, it is significant that the extract of Moringa oleifera L. leaves with a competitive inhibition type which is rarely observed amongst different enzyme inhibitors and has a potent specific inhibitor in the extract toward the TYR-Di, is a more promising TYR-Di inhibitor than other examined ones. The influence of the existence or absence of non-ionic surfactant on the TYR-Di inhibition by the extract of Moringa oleifera L. leaves was evaluated. The results revealed the inhibition percentage is not significantly reduced by using various concentrations of Triton X-100. Hence, the used surfactant cannot unmask the hidden TYR catalytic site and subsequently affirmed the absence of Pan-Assay Interfering Substances (PAINS) using the tested extract. These findings are well agreed with those obtained by [24].
Assessment of antioxidant activity and flavonoid isolation from Moringa oleifera L leaves extract
The flavonoid constituents of Moringa oleifera leaf powder were fractionated using n-hexane, ethyl acetate, and n-butanol. The fraction of ethyl acetate for Moringa oleifera L leaf powder displayed not only the maximum antioxidant activity for hydrogen peroxide and DPPH radicals, but also the lowest value for IC50 (34.3 µg/ml) of tyrosinase inhibition as shown in (Fig. 6A). Hence, the obtained fraction of ethyl acetate was employed for the flavonoid’s purification. The highest enzyme inhibition and antioxidant potentiality by using the ethyl acetate fraction of Moringa oleifera L have been reported by [24], comparing to the other fractions. The promising antityrosinase and antioxidant activities of the ethyl acetate fraction of Moringa oleifera L. leaves may be attributed to its flavonoids constituents as detected by [46, 48, 49]. The antioxidant activity of rutin is linked to the presence of various chemical features including hydroxyl-, and oxo-functional groups, unsaturation, and catechol group that donate electron radical species [34]. After concentration in rotary evaporator, the developed flavonoids fractions exhibited a single peak at 510 nm (Fig. 6B), the fractions were then gathered in a tube and concentrated using rotary evaporator.
Identification of the extracted flavonoids from Moringa oleifera leaves
The HPLC chromatogram of the purified flavonoids in the extract of Moringa oleifera L leaf powder is illustrated in Fig. 7 and Table 1. This chromatogram revealed the presence of multiple peaks, which were detected at various retention times. Five peaks were noticed with a 33.11, 41.86, 46.10, 64.11, and 68.51 min retention time. The chemical identity of the purified flavonoids was determined based on the comparison with the available authenticate sample. From the HPLC chromatogram, three major chemical components were determined namely, rutin, kaempferol, and myricetin at retention time 41.86, 46.10, and 64.11 min, respectively. The putative area for rutin, kaempferol, and myricetin was respectively found to be 38.56, 22.97, and 19.01%. There are additional peaks of anonymous compounds observed in the chromatogram. Hence, the results signify that rutin is the putative predominant flavonoid compound in the Moringa oleifera L leaves extract with a primary antityrosinase activity which is highly comparable to a commercial inhibitor (kojic acid) for melanin biosynthesis. The HPLC-based chromatogram is clearly similar to that obtained from the examined samples of Moringa oleifera L leaf extract, where rutin is uniquely major component in the investigated extract [31]; however, the glucosinolates, quercetin-3-O-glucoside, kaempferol, quercetin-3-acetyl-glucoside and other phenolic constituents are identified by HPLC analysis as the major component in Moringa oleifera L leaves [24, 50]. The quantity of extractible rutin from Moringa oleifera L leaves is obviously enhanced by successive extraction using methanol [31]. On contrary, flavonoids compounds and their glucosides (not rutin) are identified as the predominant constituents in the M. oleifera L leaves extract as reported by [26, 50].
Table 1
Compound identification of the methanolic extract of M. oleifera L using HPLC data based on their retention times.
Peak No. | Retention time (min) | [M-H]− (m/z) | Expected compound identification | Reference |
1 | 33.11 | 88.3 | Gallic acid | [24, 28, 51] |
2 | 41.86 | 195.9 | Rutin |
3 | 46.10 | 123.1 | Kaempferol |
4 | 64.11 | 102.8 | Myricetin |
5 | 68.51 | 41.0 | Apigenin |
Chemical identity of putative rutin from Moringa oleifera
The ethyl acetate extract was subject to column chromatography to isolate and purify the putative rutin. The flavonoid rutin is then chemically identified by UV spectrophotometer, FTIR, mass spectrum, and 1H NMR spectroscopic analyses. The putative rutin extracted from M. oleifera L leaves displayed the λmax wavelength at 215, 256, and 370 nm as revealed from the UV absorption spectrum (Fig. 8A) which are similar to the reference sample as reported by [51]. Hence, the developed absorbance peaks affirmed the structural identity of the putative sample as rutin.
The FTIR analysis was used to reveal the structure of rutin and is illustrated by Fig. 8B. From the FTIR chromatogram, a broad absorption peak was observed at 3335 cm− 1 which corresponds to the O–H overlapping stretching vibrations of the sugar and phenolic groups [29, 52]. A peak with low intensity was determined at 1658 cm− 1 was assigned for the C = O stretching vibration. The absorption peaks at 1596, 1450, and 1461 cm− 1 related to the C = C stretching vibration of the aromatic ring [34]. The peaks appeared in the range of 1000–1360 cm− 1 could be attributed to C–H, C–O–C, C–C, and C = O vibrations [53].
The mass spectrum analysis enables the characterization of the unidentified bioactive compounds depends on the detection of the exact mass and the diagnostic parent and product fragment of the compound using MS/MS fragmentations [28, 31, 54]. Previous reports on the quantitative and qualitative identification of phenolic compounds in the crude extract of various plant extracts are performed using high performance liquid chromatography-MS (HPLC) [28], gas chromatography mass spectrometry (GC-MS) [54], and liquid chromatography-mass spectrometry (LC-MS) [55]. The mass spectrum of the putative compound was tentatively characterized as rutin with a characteristic product fragment ion at 300.55 m/z and at 274 m/z (Fig. 8C). Similar results have been determined for the rutin from leaves extract of Moringa stenopetala [31].
The 1H NMR analysis shows the molecular structure of the purified rutin (Fig. 8D), which were compared with the results from the previous literature. From the 1H NMR data, signals at δ4-5.5 ppm of aliphatic -OH groups of rutinose were detected. Signals at δ5.07–4.38 ppm were individually observed, hinting the presence of aliphatic CH of the rutinose units. Additionally, the peaks at δ3–4 ppm were recorded as the residual signals of C–H of rutinose. The chemical peaks of aromatic OH groups were reported at δ9–13 ppm; however, the aromatic CH resonances were noticed at δ6–8 ppm. The 1H NMR spectrum affirmed the presence of resonances for rutinose moiety, aromatic and aliphatic CH and OH groups of sugar, and hence the substance is unambiguously identified as rutin when compared with typical in the literature data [34, 53, 56].
Molecular docking of tyrosinase inhibited by Rutin
The structure of tyrosinase is characterized by the H2L2 tetramer structure. The 2H subunits comprise 392 amino acid residues and have 2 Cu site, however, the 2 L subunits contains 150 amino acid subunits. In the 2H subunits, the first Cu ion showed interaction with the histidine residues (HIS85, HIS61, and HIS94). While the second Cu ions demonstrate a coordination with HIS263, HIS259, and HIS296. Previous studies confirmed that the inhibition of tyrosinase (monophenolase and diphenolase) activity depends on the capability of the tested compound to interact with the histidine residues in the catalytic sites [8, 16].
Bioactive compounds in the leaves extract of M. oleifera L., including the derivatives of kaempferol, rutin, luteolin, apigenin and quercetin have been depicted to inhibit the tyrosinase activity [24, 28]. To evaluate the interactions and binding mode of the tested inhibitors with the amino acid residues in the tyrosinase catalytic site, the in-silico molecular docking analysis were performed using the crystallographic structure of the target protein for Agaricus bisporus (PDB code: 2Y9X) with native inhibitor (tropolone). Validation of the molecular simulation was performed by redocking the inherent ligand to tyrosinase (Fig. 9A,B) and then the developed rutin is docking to the target protein (Fig. 9C,D) using molecular operating environment (MOE) version 10 program tools. The preparation of the target protein generates the active sites without inherent ligand, hence the pockets of enzyme active sites with its accessible amino acids residues becomes available for the docking simulation [5, 8]. In the developed docked complexes, the bonding interaction and the binding energy values were used to predict the best docked position of the investigated compound against tyrosinase [5, 8]. The validation of the simulation process was reached when the RMSD (root mean square deviation) value is lower than 3 Å. The redocking of the co-crystallized native ligand with the 2Y9X tyrosinase displayed RMSD of 2.40Å and RMSD of 2.29Å for kojic acid, while the docking of rutin to 2Y9X tyrosinase was 1.38Å (Table 2), indicating the validity of the molecular simulation process [5].
Table 2
Binding energy, RMSD and hydrogen bond of various ligand against tyrosinase from Agaricus bisporus
Protein | Ligand | BE (kcal/mol) | RMSD (Å) | Amino acid residue |
Tyrosinase from Agaricus bisporus (2Y9X) | Rutin | -7.75 | 1.38 | HIS259, ASN260, SER282 |
Tropolone | -5.28 | 2.40 | MET280, VAL283, HIS263 |
Kojic acid | -4.69 | 2.29 | HIS85, GLU256, VAL283 |
* BE (Binding energy), RMSD (root mean square deviation) |
The bond strength, stability, and affinity of the examined compound against the target protein depends on the value of binding energy [5, 8, 57, 58]. The lower the value of binding energy, the higher the bond strength, stability, and affinity of the examined compound against the target protein [5, 18, 34, 59]. The docking pose of rutin against 2Y9X-tyrosinase with the lowest energy was selected for further studies and it was found to occupy the same position of the inherent ligand. Hence, rutin is efficaciously bind to the active site of tyrosinase. The value of the binding energy for the interaction between the tropolone ligand and tyrosinase was − 5.28 kcal/mol, however the binding energy value of rutin and tyrosinase was − 7.75 kcal/mol (Table 2). Additionally, the energy of binding for kojic acid against tyrosinase protein was − 4.69. The docking score (S) showed that rutin compound is able to tightly bind to the tested enzyme (2Y9X) with efficaciously inhibition for the target protein tyrosinase.
The hydrogen bonds are visualized in the MOE program software. On contrary, the aromatic interactions, hydrophobic interactions, Van der Walls interaction, and ionic bonds cannot be observed in the output data of MOE program, however, they still affect the binding energy of the ligand in the active site of tyrosinase. Due to the presence of -H and -OH groups in rutin, hydrogen bonding is the most observed in the docking process. The possible docking interaction mode of rutin within the tyrosinase active site is illustrated in Fig. 9. The hydrogen bonds formed between the hydroxyl groups of rutin and the plausible residues in the core of active site for tyrosinase namely, ASN260, HIS259, and SER282. While, the π-π bond was observed among the HIS263 in the tyrosinase active site and the tropolone (inherent ligand) as positive inhibitor (Fig. 9B). The kojic acid inhibitor could bind with the amino acid residues in the tyrosinase active site namely, HIS259, ASN260, SER282 as shown in Fig. 9E&F. The strength of hydrogen bond by rutin was found to be 0.8-4 kcal/mol and reversible. The molecular simulation model clearly revealed that rutin obtained from the leaves extract of Moringa oleifera L. is able to strongly inhibit tyrosinase via binding to the amino acid residues in the catalytic site, and hence reduces the melanogenesis. The obtained simulation results harmonized well with [1, 18, 21] who mentioned that rutin is successfully bound to only the amino acid residues in the enzymatic cavity; however, other docking search performed by [25, 60] detected the ability of rutin to coordinatively bind to Cu ion in the catalytic site.