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J IRAN CHEM SOC DOI 10.1007/s13738-017-1175-0

ORIGINAL PAPER

Inhibitory effect of safranal and crocin, two principle compounds of Crocus sativus, on fibrillation of lysozyme

Tahereh Naderi Joloudar1,2 · Ali Akbar Saboury1 · Marzieh Dehghan Shasaltaneh3 · Seifollah Bahramikia4 · Mohammad Ali Ebrahimi2 · Atiyeh Ghasemi1 

Received: 7 March 2017 / Accepted: 1 August 2017 © Iranian Chemical Society 2017

hydrophobic and hydrophilic groups contribute to lower lysozyme fibril accumulation.

Keywords Aggregation · Anti-amyloidogenic · Hen egg white lysozyme · Crocin · Safranal

AbbreviationsHEWL Hen egg white lysozymeThT Thioflavin TC. sativus Crocus sativus LANS Anilinonaphthalene-8-sulfonic acidTEM Transmission electron microscopyCD Circular dichroism

Introduction

It has been postulated that one of the main contributors to amyloid-related diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, type II diabe-tes and familial amyloidosis is aggregation of intra-and/or extracellular misfolded proteins into fibrillar depos-its [1–6]. Up to now, more than 25 different proteins and peptides (β-amyloid, tau, α-synuclein, huntingtin, amylin, β2-microglobulin, lysozyme, etc.) have been found to form amyloid aggregates in humans [7–9]. Different stud-ies showed that amyloid fibril formation is associated with not only disease-related proteins, but also with proteins not involved in any known amyloid diseases, suggesting [10–12] that nearly all proteins have the ability to form amyloid fibrils under certain conditions. Regarding these facts, the study of amyloid aggregation of disease-unassociated pro-teins could shed light on our understanding on the modes of aggregation and ways to disrupt them [10–12].

Abstract A key feature in more than twenty amyloid-related diseases is the aggregation of intra-and/or extra-cellular misfolded proteins as amyloid fibrils. Therefore, preventing or reversing amyloid aggregation by using of small molecules is considered as useful approaches to the treatment of these diseases. We have evaluated the ability of safranal and crocin, to inhibit amyloid self-assembly of hen egg white lysozyme (HEWL), as an in vitro model system. Structural properties of HEWL in the presence of these com-pounds were investigated individually using thioflavin T, anilinonaphthalene-8-sulfonic acid fluorescence assays, far-UV circular dichroism and scanning electron microscopy as well as docking method. Our results showed that incubation of HEWL with either crocin or safranal at various concen-trations leads a significant inhibition in the rate of amyloid formation. Docking analysis revealed crocin and safranal interact with the central hydrophobic region of lysozyme through van der Waals interaction. Hydroxyl group in crocin through hydrogen bonds connected to the several hydrophilic amino acids of lysozyme, while in safranal there are just one aldehyde group that through hydrogen bonds connected to aspartic acid in lysozyme. It can be concluded that both

* Ali Akbar Saboury saboury@ut.ac.ir

1 Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

2 Department of Biotechnology, Payame Noor University, Tehran Shargh, Tehran, Iran

3 Laboratory of Neuro-organic Chemistry, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran

4 Department of Biology, Faculty of Science, Lorestan University, Khorramabad, Iran

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Hen egg white lysozyme (HEWL), a monomeric protein composed of 129 amino acids with helix rich conformation, is one of the best non-amyloidogenic known model proteins with well characterized molecular structure and physiochem-ical properties, to study protein aggregation in vitro [13, 14]. This protein is homologous (40%) to human lysozyme whose different variants form massive amyloid deposits in the liver and kidneys of affected individuals [14]. The dis-ease, usually called autosomal hereditary systemic amyloi-dosis, is caused by a single-point mutation in the lysozyme-encoding gene (Ile56Thr and Asp67His) [15, 16]. Although the molecular mechanism of amyloid fibril formation and its direct correlation with diseases in vivo is still unknown, the cytotoxicity of fibril aggregates and mainly its oligomeric precursors have been confirmed by many research groups [17, 18].

So far, several therapeutic approaches have been proposed to deal with amyloidogenic diseases. These approaches mainly include direct inhibition of the self-assembly pro-cesses, diminishing the production of amyloidogenic forms, increasing the stability of the native structure of proteins and finally raising the clearance rate of aggregated proteins

[19]. In recent years, some attention has been devoted to small molecule inhibitors of amyloid fibril formation. Small-molecule inhibitor approach was initially based on the long known finding that molecules such as Congo red (CR) and thioflavin T (ThT), having aromatic structures, interact specifically with amyloid fibrils and inhibit their formation [20–22].

Saffron or Crocus sativus L. (C. sativus) has been widely used as a medicinal plant to promote human health, espe-cially in Asia. From ancient times, the old-aged spice saffron has been used for flavoring and coloring food preparations. Different pharmacological studies on ethanolic and aqueous extracts of saffron as well as active ingredients including safranal and crocin have shown several biological activities including anticancer, antioxidant, cardioprotective effects, antidepressant, anti-anxiety, memory improvement prop-erties, anti-nociceptive and anti-inflammatory properties [23–32] (Fig. 1).

In addition, neuroprotective activities of saffron against focal cerebral ischemia, Alzheimer’s disease, as well as anticonvulsant effects have been verified [30–33]. These beneficial medicinal effects of extracts typically result from

Fig. 1 Chemical structure of the main saffron components. a Safranal, b gentiobiose, c crocin

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the main components of saffron namely crocin, picrocrocin and safranal. Moreover, the inhibitory effect of saffron and crocin on amyloid beta peptide fibrillogenesis and its protec-tive action against H2O2-induced toxicity in human neuro-blastoma cells in in vitro had been established [34].

Since fibrils achieved from human lysozyme in vitro were detected to be like with those obtained from patients of Alzheimer, on the other hands, fibrils resulting from human lysozyme bear a remarkable resemblance with HEWL fibrils, we chose HEWL as the best Alzheimer model to achieve the results similar to in vivo. So in this investiga-tion, to better understand the inhibitory influence of effective materials of saffron on amyloid fibrillation of HEWL, sev-eral analysis methods were applied including ThT and ANS fluorescence spectroscopy, circular dichroism, transmission electron microscopy and docking method.

Materials and methods

Protein and chemicals

HEWL (EC 3.2.1.17), anilinonaphthalene-8-sulfonic acid (ANS) and all salts and organic solvents were obtained from Merck (Darmstadt, Germany). Thioflavin T was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemi-cals used were analytical grade.

Preparation of HEWL fibrillar aggregates

Lysozyme fibrils were prepared according to Arnaudov and de Vries method [23]. HEWL sample solutions (1 mM) were prepared in glycine–HCl buffer (30 mM, pH 2) containing 0.02% NaN3. To induce the production of the amyloid struc-ture, HEWL solutions were incubated for 6 days at 57 °C in a water bath without agitation [35]. The formation of lysozyme aggregates was monitored by ThT and ANS fluo-rescence assays far-UV CD spectroscopy and transmission electron microscopy.

Anti‑aggregating activity of compounds

To study the anti-aggregating effects, compounds at vari-ous concentrations (0.1, 0.5, 1 and 5 mM), were added to lysozyme solution (1 mM) prepared in glycine buffer and incubated for 6 days at 57  °C. The mixtures were then assayed at indicated periods for the residual aggregates.

Thioflavin T (ThT) fluorescence assay

Lysozyme fibril formation was monitored by characteristic changes in ThT fluorescence intensity. Five microliters of HEWL samples (1 mM) was added to 445 µL of 15 µM ThT

solution (from 1 mM ThT stock solution in 10 mM sodium phosphate, 150 mM NaCl, pH 7.0, passed through a 0.45-µm filter paper), mixed thoroughly and incubated for 1 min. The fluorescence intensity was measured using a Cary Eclipse VARIAN fluorescence spectrophotometer (Mulgrave, Aus-tralia), with an excitation wavelength of 440 nm. Emission spectra were recorded at from 450 to 600 nm, and the fluo-rescence intensity at 485 nm was employed for the determi-nation of the relative content of fibrils in the sample. Slits were adjusted to 5 and 10 nm for the excitation and emis-sion, respectively [36]. To eliminate the probability of fluo-rescence quenching by the individual drugs, we subtracted the fluorescence intensity of pre-formed amyloid fibrils in the presence of various drugs at the zero time and the indi-cated times after treatment with the compounds.

ANS binding assay

Five microliters of each HEWL sample solution (1 mM) was mixed with 100 µL ANS working solution (200 µM), and then, the mixture was incubated for 1 min at room tempera-ture. ANS fluorescence intensity was recorded on a Cary Eclipse VARIAN fluorescence spectrophotometer (Mul-grave, Australia) by exciting each sample at 350 nm, and the emission was recorded between 400 and 600 nm [37].

Circular dichroism (CD) spectroscopy measurement

Far-UV CD spectra were recorded using an AVIV 215 Spectropolarimeter (Aviv Associates, Lakewood, NJ, USA), at 25 °C. A 1-mm quartz cell was used for far-UV (190–260 nm) measurements. Three scans of each duplicate sample were recorded and averaged. Control buffer scans were run in duplicate, averaged and then subtracted from the sample spectra. The results were plotted as molar ellipticity versus wavelength (nm).

Transmission electron microscopy

For TEM analyses, 5 µL of each diluted sample (40-fold) (1  mg/ml) was placed on a glow-discharged 400-mesh carbon-coated copper grid. After adsorption for 2 min, the sample was washed with distilled water and air-dried. Each grid was then stained with 2% (w/v) uranyl acetate for 2 min. Excess stain was removed, and the sample was again allowed to air-dry. Finally, each grid was viewed with a CEM 902A Zeiss microscope (Oberkochen, Germany) [38].

Docking studies

The initial structure of lysozyme for docking studies was obtained from Protein Data Bank (PDB code 3IJU). The software of Autodock Vina, as a new generation of

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Autodock, was applied for all docking experiments. The mentioned software has more accuracy for the predic-tion of binding mode than Autodock 4 and has a new improved scoring function as well as it makes grid map

automatically and invisible from user. The partial charge of lysozyme residues was computed by the Gasteiger par-tial charge algorithm. To analyze hydrophobic interaction

Fig. 2 Inhibition of HEWL aggregation by a crocin and b safranal based on ThT fluorescence emission for 6 days. HEWL sample solu-tions (1  mM) were prepared in glycine–HCl buffer (30  mM, pH 2) in the absence or presence of compounds at various concentrations

(0.1, 0.5, 1 and 5  mM). Aliquots were withdrawn at the indicated time, ThT (15 μM) was added, and the fluorescence intensities were measured

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and hydrogen bond in the final model, Ligplot [39] and VMD 1.9.2 were applied to visualize our model [40].

Statistical analyses

All data are presented as mean ± SD. The mean values were calculated based on the data taken from at least three inde-pendent experiments using freshly prepared reagents. The statistical significances were accepted at P < 0.05.

Results

Effects of compounds on the kinetics of HEWL fibril formation

In order to assess the influence of each compound on HEWL amyloid fibrillation, we first measured the changes in ThT fluorescence intensity. As illustrated in Figs. 1b and 2a, simultaneous incubation of crocin or safranal at various

Fig. 3 Inhibition of HEWL aggregation by a crocin and b safranal based on ANS fluo-rescence emission for 6 days. Fluorescence emission was used to monitor the surface hydrophobicity of HEWL samples. HEWL sample solu-tions (1 mM) were prepared in glycine–HCl buffer (30 mM, pH 2) in the absence or presence of compounds at various concen-trations (0.1, 0.5, 1 and 5 mM). Aliquots were withdrawn at the indicated times, ANS (20 μM) was added, and the fluorescence intensities were measured

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concentrations with HEWL attenuated the ThT fluorescence intensity relative to the control sample.

Effects of compounds on HEWL tertiary structure

In exploring the effect of compounds on tertiary structure changes and surface hydrophobicity in HEWL, we examined the ANS fluorescence emission spectrum following exci-tation at 380 nm. As shown in Fig. 3, a marked increase in ANS fluorescence intensity was observed after fibrilli-zation of HEWL for 6 days, suggesting the exposure of hydrophobic patches on the surface of the protein. Simulta-neous incubation of crocin or safranal at various doses with HEWL attenuated the ANS fluorescence intensity relative to the control sample (Fig. 3a, b), indicating the reduction of exposed hydrophobic regions on the fibrils.

Effects of compounds on HEWL secondary structure

Concerning the fact that aggregation of proteins into amy-loid fibrils is accompanied by a conformational change from α-helical mode to β-sheet, far-UV CD was also used as another approach to confirm amyloid fibril formation. CD analysis revealed that 6-day incubation of HEWL sample under defined experimental conditions led to a transition from α-helical to β-sheet conformation (a deep at 217 nm) (Fig. 4). However, under the influence of crocin or safranal, no structural transition from the native α-helical-rich con-formation to amyloidogenic β-sheet was evident after 6 days, indicating the potential anti-amyloidogenic effects of these compounds (Fig. 4a, b) (Table 1).

Effects of compounds on HEWL morphology

In order to verify amyloid formation and to view aggregate morphology, samples of HEWL treated with crocin and safranal were viewed using electron microscopy (Fig. 5). The electron microscopy images indicated that in contrast to the dense morphology of the fibrils formed after 6 days (Fig. 5a), the formation of fibrils was reduced in samples

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Fig. 4 Influence of a crocin and b safranal on inhibition of HEWL aggregation using circular dichroism (CD) after 6 days. HEWL sam-ple solutions (1 mM) were prepared in glycine–HCl buffer (30 mM, pH 2) in the absence or presence of compounds at various concentra-tions (0.1, 0.5, 1 and 5 mM). Samples diluted tenfold with buffer and the far-UV CD spectra were recorded

Table 1 Quantitative analysis of CD spectra

Secondary structure analysis (alpha helix, beta sheet and random coil) in the presence of different treatments (crocin and safranal)

Control Crocin 0.5 mM Crocin 1 mM Crocin 5 mM Safranal 0.5 mM

Safranal 1 mM Safranal 5 mM

Alpha helix 94.8 94.3 95.3 95.2 94.7 93.8 95.6Beta sheet 4.6 5.2 4.4 4.3 4.8 5.5 4.2Random coil 0.6 0.5 0.3 0.5 0.5 0.7 0.2

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co-incubated with crocin and safranal for 6 days, so the growth of fibrils was inhibited during the processes (Fig. 5b, c).

Docking data

By using Autodock Vina, the binding site of the crocin and safranal compounds on the lysozyme protein was investi-gated. Based on the docking results, it was found that crocin and safranal bonded to the hydrophobic parts of lysozyme by van der Waals interactions. Hydroxyl groups of crocin connected to the hydrophilic amino acids of the mentioned protein, i.e., Asp, Lys, Ser and Thr by hydrogen bonds, while safranal contains a hydroxyl group which is bonded to the Asp of lysozyme by hydrogen bond (Fig. 6a, b).

In addition, our results showed crocin was bonded to two residues (Asp52 and Glu35) of active site of lysozyme and safranal was connected to one of them (Asp52).

Discussion

Intercellular interactions in fibrillation compounds play an important role in the design of inhibitors. More studies have reported to explain the molecular events involved in fibril formation, which has so far remained unresolved. It is believed that aggregation occurs as the result of intermo-lecular hydrophobic interactions between the protein mol-ecules [2–6]. In addition to these hydrophobic interactions, the core hydrophobic structure of the fibrils seems to be stabilized by hydrogen bond interactions of the polypeptide main chain [18]. In this regard, compounds which disturb in the mentioned interactions, it may be playing a suppresser role in the formation of amyloid aggregation [18]. Up to now, diverse small molecules including synthetic and natural

compounds have been examined and found to suppress or reduce the aggregation of various proteins, particularly in relation to Aβ deposition and aggregation of lysozyme and transthyretin [1, 19, 22]. HEWL is known to possess well-defined thermodynamic properties, folding mechanism and 3D structure. Human lysozyme and HEWL are involved in an amyloidosis disease [41].

In this study, the effect of two compounds extracted from saffron, namely crocin and safranal, which was applied to inhibit amyloid self-assembly of hen egg white lysozyme (HEWL), an ideal model system to study in vitro fibrilla-tion, was surveyed. Crocin contains gentiobiose moieties, which have hydroxyl groups. Safranal includes an aldehyde group binding to the ring. Due to the presence of cyclohex-ane rings and hydroxyl groups in crocin structure and also the presence of numerous aromatic residues in lysozyme protein, we predicted that the cyclohexane ring of the men-tioned compound might interact with the aromatic residues within the protein leading to the stabilization of their native or partially folded intermediates. Aromatic residues in the lysozyme protein induce hydrophobic and hydrophilic inter-actions with the crocin structure. These effects inhibit their self-assembly. On the other hand, aldehyde group of safra-nal interacts with several residues in the protein molecule using hydrogen bonds to prohibit the formation of amyloid self-assembly.

By coupling several techniques such as thioflavin T (ThT) and anilinonaphthalene-8-sulfonic acid (ANS) fluorescence, far-UV circular dichroism (CD) and transmission electron microscopy, we demonstrated that both small molecules possessed anti-amyloidogenic activities due to an enhanced alpha helix structure in the protein.

Some studies have emphasized that the lysozyme amyloid fibril formation might be initiated by the partial unfolding of the central region of the protein encompassing the β-domain

Fig. 5 TEM images of HEWL (1 mM) samples incubated in a the absence and presence of b crocin (1 mM) and c safranal (1 mM) under acidic environment (pH 2) at 57 °C after 6 days

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and the C-helix located in the alpha domain [42]. This fact might increase the population of aggregation-prone partially unfolded species. In addition, this fact might expose the hydrophobic core and the aromatic amino acids much better than the native form. Thus, stabilization of the native state by small compounds having aromatic rings might increase the activation energy barrier, thereby slowing the aggrega-tion kinetics and departing from amyloidogenic state [14, 42]. Regarding these facts, and also the presence of numer-ous aromatic residues in lysozyme structure, we conclude that the rings of compounds probably interact with these residues leading to the stabilization of the unfolded structure

of HEWL. The reduction in ThT and ANS fluorescence intensity in the presence of the compounds might be due to HEWL stabilization in certain specific conformations. In addition, our CD measurements also indicated that in the presence of each compound, no structural transition from the native α-helical-rich conformation to amyloidogenic β-sheet was evident after 6 days. These results reconfirmed the inhibitory effects of compounds on native or partially folded intermediates conformations.

In all experiments, the strongest inhibitory effect was observed for crocin, bearing several hydroxyl groups and the lowest activity was observed for safranal, bearing methyl

Fig. 6 Binding mode of a crocin and b safranal compounds with HEWL by VMD (left) and Ligplot (right)

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substitution on the aromatic ring. It seems that hydroxyl groups could be formed new hydrogen bonds which prevent the formation of beta sheets. On the other hand, safranal has only one hydroxyl group which also leads to the inhibition of beta sheets, but these groups in safranal are lower than in crocin and it bonded to one residue of lysozyme in the active sites relative to crocin; thus, the inhibitory effect of safranal is lower than crocin. Our results are in accordance with the other studies in which many compounds with ring structures and hydroxyl groups are able to inhibit the amyloid aggrega-tion effectively [43, 44].

In order to verify these data, we analyzed theoretically the binding site of the crocin and safranal on the lysozyme pro-tein by docking studies. Based on the docking results, it was found that crocin and safranal bonded to the hydrophobic parts of lysozyme by van der Waals interactions. Hydroxyl groups of crocin connected to the hydrophilic amino acids of the mentioned protein, i.e., Asp, Lys, Ser and Thr by hydro-gen bonds, while safranal contains a hydroxyl group which is bonded to the Asp of lysozyme by hydrogen bond. Since Glu35 and Asp52 are two residues which are considered to be essential parts of the active site of lysozyme [3, 4], the binding sites of crocin and safranal were assessed in the vicinity of the lysozyme active site using docking results. Our results showed crocin was bonded to two residues (Asp52 and Glu35) of active site of lysozyme and safranal was connected to one of them (Asp52). The results of dock-ing confirmed the data resulting from experimental parts.

Conclusion

In conclusion, crocin and safranal prevent the formation of aggregation in the beta sheets which induce neurodegenera-tive diseases and also crocin have more potent than safra-nal. Nevertheless, the detailed mechanisms of the inhibi-tory action remain largely unknown. Further investigation is warranted to explore the underlying mechanism(s) of the interaction between HEWL and these compounds.

Acknowledgements The financial support of Research Council of University of Tehran and the Iranian National Science Foundation (INSF) is highly appreciated.

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