Embelin and levodopa combination therapy for improved Parkinson’s disease treatment

2.1 Chemicals and reagents

Rabbit polyclonal β-actin and rabbit polyclonal Nurr1 primary antibodies were procured from Invitrogen, Thermo Fisher Scientific (Waltham, MA, USA). The main chemicals used in this study were procured from the following sources: rotenone (TCI Chemicals Pvt. Ltd., Chennai, India); the Abcam Urea Assay Kit ab83362 (Boston, MA, USA); thiobarbituric acid (TCI Chemicals Pvt. Ltd., Tamil Nadu, India); olive oil, Thuruthel Drug Lines (ERUMBA Pharmacy, Payyannur, Kerala, India); l-DOPA (product code: 77201; Sisco Research Laboratories Pvt. Ltd., Maharashtra, India); dimethyl sulfoxide (DMSO) (Pure Chemicals Co., Chennai, India); and hydroxypropyl cellulose (HiMedia, Mumbai, India). An authenticated sample of E. ribes (berries) was provided by Dr. V. Chelladurai, Botanist, Thirunelveli, Tamil Nadu, India. All other chemicals and reagents used were of analytical grade.

2.2 Preparation of rotenone

Fifty milligrams of rotenone was dissolved in 1 mL of DMSO. From this solution, 0.2 mL was mixed with 19.8 mL olive oil to prepare the stock solution.

2.3 Preparation of embelin

Coarsely powdered E. ribes berries (1.5 kg) were extracted three times with 2 L of n-hexane using the cold extraction method (24 h for each extraction). The solvent was decanted, pooled, and distilled in a boiling water bath. The extract was concentrated in vacuo and subjected to column chromatography over silica gel (100–200 mesh). Benzene was used to elute embelin from the column. This procedure yielded an orange powder that, when crystallized with diethyl ether, afforded orange plates of embelin (yield: 2 g) [52]. The structural characterization of embelin was confirmed in earlier work by Fourier transform infrared (FT-IR) spectroscopy [53].

2.4 Animal grouping and experimental design

Male Swiss albino mice (25–30 g) were procured from M/s Biogen Laboratory (Bengaluru, India). Animals were housed in ventilated polypropylene cages and acclimatized for 7 days to laboratory conditions before starting experiments. They were kept under ambient conditions with a natural light/dark cycle at 25 ± 2°C and 40–60% relative humidity and fed with standard pellet diet and water ad libitum.

The mice were randomly divided into seven groups (10 per group). The treatments were as follows: group I, vehicle control, namely olive oil (2 mL kg⁻¹ orally [p.o.]); groups II–VII, rotenone (2.5 mg kg⁻¹, intraperitoneally); group III, embelin (20 mg kg⁻¹, p.o.); group IV, embelin (40 mg kg⁻¹ p.o.); group V, embelin (20 mg kg⁻¹ p.o.) and LD (7.5 mg kg⁻¹ p.o.); group VI, embelin (40 mg kg⁻¹ p.o.) and LD (7.5 mg kg⁻¹ p.o.); and group VII, LD (7.5 mg kg⁻¹ p.o.). The treatment protocol in mice as indicated above was for a period of 21 days.

1. Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. All animal experimental procedures were performed according to the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India, and were approved by Institutional Animal Ethics Committee (reference number: SU/CLAR/RD/003/2019).

2.5 Preparation of the PD mouse model

Previous work has shown that chronic daily intraperitoneal injection of rotenone that is prepared using a natural oil causes behavioral (locomotor) deficits and neurochemical abnormalities characteristic of PD [54,55]. Therefore, in the present work, rotenone was prepared in a mixture of olive oil and DMSO (99:1) and administered intraperitoneally to Swiss mice for 21 days.

2.6 Cardiac perfusion and brain homogenate preparation

At the end of the experiment, the animals were anesthetized using isoflurane and subjected to cardiac perfusion using normal saline (0.9% NaCl) to eliminate the blood. The skull of the mice was opened and the midbrain carefully isolated and washed with ice-cold phosphate-buffered saline (PBS), pH 7.4. The whole midbrain tissue isolated from each mouse was kept on ice and homogenized with 0.1 M PBS (pH 7.0) using a Potter–Elvehjem PTFE-coated pestle and glass tube (PRO Scientific Inc., Oxford, CT, USA).

2.7 Isolation of the postmitochondrial fraction

The midbrains were immediately immersed in 0.7 mL of ice-cold isolation buffer (IB) composed of 225 mm mannitol, 75 mm sucrose, 1 mm ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 5 mm HEPES-KOH (pH 7.2), and 1 mg mL⁻¹ of essential fatty acid-free bovine serum albumin (BSA) in a 5 mL plastic tube. The tissue was homogenized at 25,300g using a tissue homogenizer (REMI Lab Homogeniser RQ-127A/D, Maharashtra, India) and transferred into a 1.5 mL microcentrifuge tube. The brain homogenate was centrifuged at 1,100g for 2 min in a refrigerated centrifuge (Eppendorf, Model 5425R, Hamburg, Germany). The supernatant was placed aside and the pellet was resuspended in 0.2 mL of the buffer and centrifuged again at 1,100g for 2 min. The pellet was discarded and the supernatant was combined with that from the first centrifugation. Next, 0.5 mL of combined supernatants was mixed with 0.07 mL of 80 vol% Percoll solution, carefully layered on top of 0.7 mL of 10% Percoll solution, and centrifuged at 18,500g for 10 min. Then, 80% Percoll was diluted in buffer to prepare the 10% solution. The mitochondria-enriched fraction was collected at the bottom of the tube and resuspended in 0.7 mL of washing buffer composed of 250 mM sucrose, 5 mM HEPES-KOH (pH 7.2), 0.1 mM EGTA, and 1 mg mL⁻¹ BSA. The suspension was centrifuged at 10,000g for 5 min. The final mitochondrial pellet was resuspended in 0.07 mL of washing buffer and stored on ice. To isolate mitochondria, the same procedure was employed as described above, except that IB was supplemented with 0.01% digitonin at the tissue homogenization step, to release mitochondria. The mitochondrial protein content was measured with a Lowry assay kit (Bio-Rad, Haryana, India) according to the manufacturer’s instructions. After removing the pellet, the postmitochondrial supernatant fraction was used to estimate the levels of nitric oxide (NO), urea, peroxynitrite, and malondialdehyde (MDA), a measure of lipid peroxidation (LPO).

2.8 Measurement of LPO

The MDA content was determined by the thiobarbituric acid (TBA) reaction as described previously [56]. To 0.5 mL of homogenate, 1.5 mL of 20% acetic acid, 0.2 mL of sodium dodecyl sulfate (SDS), and 1.5 mL of TBA were added. The mixture was adjusted to 4.0 mL with distilled water and then heated for 60 min at 95°C using a glass ball as a condenser. After cooling, 4.0 mL of a butanol-pyridine mixture was added and the mixture was shaken well. After centrifugation at 1,792g for 10 min, the organic layer was removed and its absorbance was read at 532 nm. The standards and blanks were treated in a similar manner. The MDA concentration, an indicator of LPO, is expressed as nmol mL⁻¹.

2.9 Measurement of NO

Nitrite ( NO 2 − ) and nitrate ( NO 3 − ) are stable final products of NO metabolism and may be used as indirect markers for the presence of NO. The total NO concentration is commonly determined as the sum of the NO 2 − and NO 3 − concentrations. In the analyzed samples, NO 3 − was reduced to NO 2 − in the presence of cadmium and then converted to nitric acid; this process used Griess’s reagent o produce a colored product (Sigma-Aldrich, Stainheim, Germany). The NO 2 − concentration was determined by spectrophotometric analysis at 540 nm [57].

2.10 Measurement of peroxynitrite

Peroxynitrite levels were measured according to the method described by Beckman et al. [58] Peroxynitrite-mediated nitration of phenol was measured spectrophotometrically at 412 nm. In brief, 100 µL of homogenate was placed in a glass test tube, to which 5 mM phenol in 5 M sodium phosphate buffer (pH 7.4) was added to a final volume of 2 mL. After mixing, the solution was incubated for 2 h at room temperature and then 15 µL of 0.1 M sodium hydroxide was added and the absorbance was read at 412 nm. The method is based on the oxidation of o-phenylenediamine, a colorless substance, by peroxynitrite to yield a colored product. The absorbance increase of this chemical reaction is linearly related to the concentration of peroxynitrite in the range of 4.4 × 10⁻⁷ to 8.0 × 10⁻⁶ mol L⁻¹, with a detection limit of 1.7 × 10⁻⁷ mol L⁻¹ (3σ).

2.11 Measurement of urea

Elevated brain urea levels in neurodegenerative Huntington’s disease patients were reported as a major metabolic defect [59]; hence, brain urea levels were measured. In a 96-well plate, a 50 μL reaction mix containing urea assay buffer (42 μL), oxiRed probe (2 μL), enzyme mix (2 μL), developer (2 μL), and converter enzyme (2 μL) was added to each well containing 50 µL of urea standards, test samples, or blank control. The reaction mixture was mixed well and incubated for 60 min at 37°C (protected from light). The absorbance at 570 nm was measured using spectrophotometer (Elico, B-200, Hyderabad, India).

2.12 Western blot analysis

The SN tissue homogenate was prepared in lysis buffer containing 1% protease inhibitor (HiMedia Laboratories) for 1 h at 4°C. Samples were then centrifuged at 10,000g for 15 min at 4°C. Supernatants were collected and the protein concentration was determined using a Bradford protein assay kit. Equal amounts of protein (40 μg) were separated on 10% acrylamide gels using SDS–polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane. Membranes were incubated with 5% nonfat milk for 1 h at room temperature (to block nonspecific protein binding) prior to incubation overnight at 4°C in a solution of rabbit polyclonal anti-β-actin (1:1,000) and rabbit polyclonal anti-Nurr1 (1:1,000). The next day, membranes were washed three times with Tris-buffered saline with 0.5% Tween 20 (TBST), incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5,000) for 1 h at room temperature, and then washed three times with TBST. The target proteins were visualized using ECL reagent and exposed to a piece of X-ray film. The densities of specific protein bands were determined using Adobe Photoshop CS6 software (version 12.0). The Nurr1 (66 kDa) protein level was normalized to the β-actin (42 kDa) protein level (loading control).

2.13 Gut histopathology and brain immunohistochemical staining

Gut tissue was fixed in 10% neutral buffered formalin, dehydrated, embedded in paraffin, and then sectioned at a thickness of 5 μm and stained with hematoxylin and eosin (H&E) for histopathological investigation. For histomorphometry analysis, the gut tissue samples were well-oriented with longitudinally cut crypts to precisely assess alterations in the overall tissue architecture. Regarding gut tissue samples, at least 4–5 consecutive villi well distended from the base to tip were rated. The number of goblet cells per villus was counted in 10 well-oriented and adjacent crypt villi on each section stained with H&E. All measurements were made at 100× magnification. On each slide, one section that represented the best view of the villus and crypts was selected for analysis. The slides were examined under an Olympus light microscope and photomicrographs were taken with a Sony camera.

For brain tissue, mice in each group were anesthetized with isoflurane and then transcardially perfused with 0.9% normal saline to clear the blood from the brain. The brain from each mouse was immersed in 10% neutral buffered formalin (the fixative solution) for 4 h. The tissues were cryoprotected in 30% sucrose, embedded in tissue-freezing medium with liquid nitrogen, and sectioned at 3–5 µm using a cryostat. Sections were stored under anti-freeze buffer. Parallel free-floating sections were subjected to endogenous peroxidase quenching with 1% hydrogen peroxide in PBS, followed by treatment with blocking buffer (5% normal chicken serum and 0.3% Triton X-100 in PBS overnight at 4°C) to block nonspecific protein binding. Sections were incubated overnight at 4°C with mouse monoclonal anti-tyrosine hydroxylase (TH; diluted 1:3,000; Thermo Fisher Scientific, Invitrogen). After washing with PBS, tissues were incubated for 2 h at room temperature with affinity-purified rabbit anti-mouse IgG antibody (diluted 1:50 dilution; Sigma-Aldrich). Then, the sections were exposed to an avidin–biotin peroxidase complex for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. All immunoreactions were observed using a compound light microscope and these results were quantified using the ImageJ 1.46 (National Institutes of Health, Bethesda, MD, USA). For quantification, fragmentation (F) reflects that TH expression (brown patches in the image) that appeared as either single, homogeneous, and continuous or complex, heterogeneous, and discontinuous patches. Based on this protein expression classification, F was calculated as: F = N/A, where N is the number of protein expression patches in the image and A is the total area (in pixels) of the protein signal.

2.14 Molecular docking studies

Molecular docking studies were executed with Autodock4.2 software [60] using the solid-state nuclear magnetic resonance of α-syn fibrils retrieved from the RCSB Protein Data Bank (PDB code 2N0A). This structure holds 10 units of the peptide (140 amino acids), which are arranged in parallel by presenting features that commonly stabilize amyloid folds (such as intermolecular salt bridges, a glutamine ladder, steric zippers involving hydrophobic residues, and in-register parallel-β-sheet hydrogen-bonding with a Greek key motif) [61]. The embelin structure was downloaded from the PubChem database. Before performing docking, all water molecules were removed and polar hydrogen was added to α-syn fibrils by using the hydrogen module in AutoDock Tools, followed by assigning Kollman united atom partial charges. The grid maps representing the proteins in the actual docking process were calculated with AutoGrid. The grids were chosen to be sufficiently large to include the active site. The dimensions of the grids were thus 126 Å × 126 Å × 126 Å, with the spacing of 0.375 Å between the grid points. The in silico bonding mode of ligand with the receptor was carried out using the empirical free energy function and the Lamarckian genetic algorithm, applying the following standard protocol with an initial population of 150 randomly placed individuals, a maximum of 2.5 × 10⁷ energy evaluations, GA crossover mode = two points. A genetic algorithm of 100 independent docking cycles was carried out for the ligand. A genetic algorithm of 100 independent docking conformers was carried out for the compound, and the best conformer was taken based on their lowest binding energy for further analysis. PyMOL (https://pymol.org/2/) and LigPlot 2.2 tools were used to visualize the docking results [62]. The entire structure of embelin bound to the target protein was examined for binding energy and also for specific and nonspecific interacting residues.

2.15 Statistical analysis

The data are presented as the mean ± standard error of the mean. Differences among the groups were compared with a one-way analysis of variance (ANOVA), followed by the Bonferroni test for multiple comparisons. The F and P values for the one-way ANOVA are shown in each panel of each figure. Significant differences between groups are denoted by asterisks, with details indicated in each figure legend. SigmaPlot 14.5 (Systat, USA) was used for statistical analysis.

3.1 Changes in brain MDA, NO, peroxynitrite, and urea levels

The mean MDA level in rotenone-treated mice was more than two times higher than in the control mice (11.36 ± 0.40 and 5.48 ± 0.17 nmol mL⁻¹, respectively; Figure 1a). The mice exposed to rotenone plus embelin and/or LD showed markedly reduced MDA compared with rotenone treatment alone (Figure 1a; P < 0.001). Notably, the combination of 40 mg kg⁻¹ embelin and 7.5 mg kg⁻¹ LD rendered even better protection against LPO. The mean peroxynitrite level in the rotenone-treated mice (group ІI) was markedly higher than the control mice (0.19 ± 0.001 and 0.13 ± 0.007 nmol mL⁻¹, respectively, P < 0.001; Figure 1b). The addition of embelin and/or LD to rotenone-treated mice significantly reduced the peroxynitrite level compared with the rotenone-treated mice (P < 0.001). Rotenone-treated mice that received 40 mg kg⁻¹ embelin or 40 mg kg⁻¹ embelin plus 7.5 mg kg⁻¹ LD showed almost similar peroxynitrite levels to the control mice (Figure 1b).

Figure 1

Antioxidant potential of embelin (E) and embelin plus LD therapies in mice treated with rotenone, based on the measurement of brain (a) LPO, denoted by MDA, (b) NO, (c) peroxynitrite (PN), and (d) brain urea (BU) levels. The values represent the mean ± standard error (n = 6). For the embelin groups, the number indicates the dose (in mg kg⁻¹ body weight). The F and P values are based on the one-way analysis of variance. The level of significance was found to be P < 0.001. The superscript letters indicate the results of the Bonferroni multiple comparison test. ^aSignificantly different from the control group. ^bSignificantly different from the rotenone group. ^cSignificantly different from the rotenone + LD group.

The mean NO level in rotenone-treated mice was significantly higher than in the control mice (19.0 ± 1.1 and 11.2 ± 1.9 µmol L⁻¹, respectively, P < 0.001; Figure 1c). Administering embelin and/or LD to rotenone-treated mice markedly reduced NO levels compared with mice that only received rotenone (P < 0.001). In particular, the NO levels in the rotenone- and drug-treated mice (40 mg kg⁻¹ embelin plus 7.5 mg kg⁻¹ LD) were not different from the control group.

The mean brain urea in the rotenone-treated mice was nearly 75% higher than the control mice (57.60 ± 2.59 and 33.30 ± 7.51 nmol mg⁻¹, respectively, P < 0.001; Figure 1d). The addition of embelin and/or LD to rotenone-treated mice markedly reduced the brain urea levels compared with mice that only received rotenone (P < 0.001). Indeed, treatment returned the urea level to near what was found in control mice. Based on the above findings, treatment with embelin alone or embelin plus LD reversed the biochemical alterations caused by rotenone administration.

3.2 Nurr1 protein changes in the SN

Figure 2 shows the western blot analysis of Nurr1 (66 kDa) protein in the SN. Nurr1 expression in the rotenone-treated mice was significantly decreased (P < 0.05) compared with the control group. Nurr1 expression in the rotenone-treated mice administered LD standard therapy (7.5 mg kg⁻¹) was restored to the control level. Embelin (40 mg kg⁻¹) plus LD (7.5 mg kg⁻¹) combination therapy in rotenone-treated mice also reverted the Nurr1 protein expression to almost the control level. However, the effect of embelin alone (40 mg kg⁻¹) in restoring the Nurr1 protein level was comparatively less than the embelin plus LD combination therapy. Therefore, embelin plus LD combination therapy has a better protective effect on the brain dopaminergic system.

Figure 2

(a) Western blot analysis showing Nurr1 protein changes in the SN. Lane 1, molecular weight ladder; lane 2, control; lane 3, rotenone (R); lane 4, R + embelin (E, 40 mg kg⁻¹); lane 5, R + E (40 mg kg⁻¹) + LD (LD, 7.5 mg kg⁻¹); lane 6, R + LD (7.5 mg kg⁻¹). (b) The bar graph shows the relative expression of Nurr1 in various groups. The values present the mean ± standard error (n = 4). The F and P values are based on a one-way analysis of variance. The level of significance was found to be P < 0.001. The letters indicate the results of the Bonferroni multiple comparison test. ^aSignificantly different from the control group. ^bSignificantly different from the rotenone group. ^cSignificantly different from the rotenone + LD group.

3.3 Gut histopathological changes and histomorphometry

Micrographs of H&E-stained gut sections are shown in Figure 3. The control mice showed normal architecture of all three mucosa levels (Figure 3a, circle), with normal crypts and no inflammatory cell infiltration. In the rotenone-treated mice (Figure 3b), there was remarkable destruction of the mucosa and surface epithelia (arrow mark). Moreover, inflammatory cell infiltration (arrow head) was noted in the lamina propria. In the group treated with 10 mg kg⁻¹ embelin, there were epithelial destruction, mucosal edema (small arrow), intense inflammatory cell infiltration, and crypt distortion (Figure 3c), whereas in the group treated with 20 mg kg⁻¹ embelin, there was inflammation in the mucosal layer but no damage to the epithelial layer (Figure 3d). Reduced crypt damage, less edema, and less inflammation were seen in this group. In the group treated with 10 mg kg⁻¹ embelin plus LD (Figure 3e), there were epithelial destruction and moderate infiltration; normal arrangement of all the three mucosal layers is visible. However, in the groups treated with 20 mg kg⁻¹ embelin plus LD (Figure 3f) or LD alone (Figure 3g), there were less damage in gut tissues, no edema, proper arrangement of mucosal layers, and less inflammatory cell infiltration.

Figure 3

Representative micrographs of H&E staining of gut sections (n = 4). The control group (a) shows normal architecture of all three mucosal layers (circle), with normal crypts and no inflammatory cell infiltration. In the rotenone-treated group (c), there is destruction of the mucosa and surface epithelia (arrow mark). Moreover, inflammatory cell infiltration (small and thick arrow) is apparent in the lamina propria (b). In the rotenone + embelin (20 mg kg⁻¹) group (d), there is mucosal edema (small and thin arrow), with intense inflammatory infiltration and crypt distortion (e). In the rotenone + embelin (40 mg kg⁻¹) group, there were epithelial destruction and moderate inflammatory cell infiltration (f) apart from normal arrangement of all the three mucosal layers (g). In the rotenone + embelin (20 mg kg⁻¹) + LD (7.5 mg kg⁻¹) group, all three mucosal layers showed a normal arrangement (i, circle), although there were epithelial destruction and moderate inflammatory cell infiltration (h). In the rotenone + embelin (40 mg kg⁻¹) + LD (7.5 mg kg⁻¹) group, there were little damage, no edema, proper arrangement of mucosal layers, and limited inflammatory cell infiltration (j and k). The rotenone + LD (15 mg kg⁻¹) group showed similar features (l). The scale bar is 25 µm.

Based on a scoring system established by Okayasu et al. [63], inflammatory cell infiltrates, epithelial changes, and the mucosal architecture were scored based on specific criteria. The changes in the gut histomorphometry were analyzed and the overall scores for each group are presented in Table 1. Similar to the histopathological changes, gut from mice treated with 20 mg kg⁻¹ embelin plus LD showed substantial improvement compared with the other treated groups.

3.4 Effect of embelin and embelin plus LD treatments on the rotenone-induced reduction of the TH-positive cells in the SN

TH catalyzes the rate-limiting step in DA biosynthesis in dopaminergic neurons. The number of TH-positive cells was evaluated in the SN (Figure 4). Compared with the control group (Figure 4a), there was a marked reduction in TH-positive cells in the rotenone-treated mice (Figure 4b). By contrast, in the drug-treated groups (Figure 4c–g), there was protection of TH-positive cells. Figure 4h shows the percentage of TH-positive cells.

Figure 4

The effect of embelin and embelin + LD therapies on the TH protein expression level. TH immunostaining in the SN from the (a) control, (b) rotenone, (c) rotenone + embelin (20 mg kg⁻¹), (d) rotenone + embelin (40 mg kg⁻¹), (e) rotenone + embelin (20 mg kg⁻¹) + LD (7.5 mg kg⁻¹), (f) rotenone + embelin 40 mg kg⁻¹ + LD 7.5 mg kg⁻¹, and (g) rotenone + LD (7.5 mg kg⁻¹) groups. The arrows denote the TH-positive cells. (h) Quantitative analysis of TH protein expression. The results are expressed as the mean + standard error of the mean (n = 4). The F and P values are based on the one-way analysis of variance with Bonferroni multiple comparison test. The level of significance was found to be P < 0.001. ^aSignificantly different from the control group. ^bSignificantly different from the rotenone group. ^cSignificantly different from the rotenone + LD group.

3.5 In silico studies revealed the binding affinity of embelin for α-syn fibrils

The binding mode of embelin with α-syn fibrils was analyzed using the Autodock 4.2 suite. Thirteen putative binding sites were identified (Figure 5a). Site 2 (with residues Y39, S42, and T44), site 9 (with residues G86, F94, and K96), and site 3/13 (with residues L43, L45, V48, and H50) showed a high probability of interaction [64]. The embelin scoring energy (−4.18 kcal mol⁻¹) is most favorable for interaction with site 3/13. For this interaction, two hydrogen bonds are formed. The first hydrogen bond is between the second oxygen atom of embelin with the ND1 group of the polar residue His50(D), with a bond distance of 3.0 Å (Figure 5b and c). The second hydrogen bond is between the fourth oxygen atom of embelin and the NZ group of positively charged residue Lys45(D), with a bond distance of 2.9 Å (Figure 5b and c). In addition, residues Val48(C), Val48(D), Val48(F), His50(G), and His50(F) are involved in hydrophobic interactions to sustain the stability of the embelin–α-syn complex. Furthermore, van der Waals interactions occur with the residues Lys45(C), Val48(E), Val49(C), Val49(D), Val49(E), Val49(F), His50(C), and His50(E) (Figure 5b and c).

Figure 5

(a) An illustration of embelin (green structure) in the substrate-binding pocket of α-syn. The crucial residues of α-syn are shown as atom stick figures. (b) The secondary structure view of the embelin–α-syn complex. Hydrogen bonds are represented by the magenta dashed line. (c) A LigPlot view of the embelin–α-syn complex. The Lys45(D) and His50(D) residues of α-syn are highlighted. The image shows the hydrogen bonds and hydrophobic and van der Waals interactions that mediate embelin–α-syn binding.