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The protective effect of crocin on the amyloid fibril formation of aβ42 peptide in vitro

Aβ is the main constituent of the amyloid plaque found in the brains of patients with Alzheimer’s disease. There are two common isoforms of Aβ: the more common form, Aβ40, and the less common but more amyloidogenic form, Aβ42. Crocin is a carotenoid from the stigma of the saffron flower and it has many medicinal properties, including antioxidant effects. In this study, we examined the potential of crocin as a drug candidate against Aβ42 amyloid formation. The thioflavin T-binding assay and electron microscopy were used to examine the effects of crocin on the extension and disruption of Aβ42 amyloids. To further investigate the relationship between crocin and Aβ42 structure, we analyzed peptide conformation using the ANS-binding assay and circular dichroism (CD) spectroscopy. An increase in the thioflavin T fluorescence intensity upon incubation revealed amyloid formation in Aβ42. It was found that crocin has the ability to prevent amyloid formation by decreasing the fluorescence intensity. Electron microscopy data also indicated that crocin decreased the amyloid fibril content of Aβ. The ANS-binding assay showed that crocin decreased the hydrophobic area in incubated Aβ42. CD spectroscopy results also showed that the peptide undergoes a structural change to α-helical and β-turn. Our study shows that the anti-amyloidogenic effect of crocin may be exerted not only by the inhibition of Aβ amyloid formation but also by the disruption of amyloid aggregates. Therefore, crocin could be essential in the search for therapies inhibiting aggregation or disrupting aggregation.

repair oxidized molecules. These stresses can cause injuries, such as the oxidation of membrane lipids and damage to DNA and cytoplasmic proteins [11,12]. A common factor in the proposed mechanisms of Aβ 42 toxicity is amyloid oligomerization [13,14]. The first phase of the oligomerization of Aβ 42 monomers includes the formation of pentamer/hexamer units that are called paranuclei. Paranuclei are the most basic and smallest structures that can be oligomerized into larger forms involving large oligomers, protofibrils and fibrils. Monomers, paranuclei and large oligomers are predominately unstructured with small amounts of β-sheet/β-turn and α-helix. During the formation of protofibrils, specific conformational changes occur causing the unknown structures of α-helix and β stranded structures to change into β-sheet/β-turn structures. A comparison of 34 physiologically relevant Aβ alloforms also revealed that an important feature controlling rapid oligomerization is the area of the C terminal. The side chain of residue 41 in Aβ 42 is important both for paranucleus formation and the accumulation and formation of large oligomers. The carboxyl groups of the C terminal of the side chain of residue 42 also affect paranucleus aggregation and oligomer formation [15]. Drugs that have been discovered with some potential to treat AD include acetyl cholinesterase enzyme inhibitors such as tacrine, donepezil, galantamine and rivastigmine, but they are very limited [16]. All these drugs have many potentially adverse side effects so attempting to discover new drugs with fewer side effects is an important area of research. One of the materials that has been clinically evaluated for its effect on AD is crocin. Saffron is a good source of crocin. Saffron has three main chemical components: carotenoids with their bright red color, picrocrocin, and saffron. Crocin is a glycosylated carotenoid that is among the few water-soluble carotenoids with antioxidant properties. Thus, it is one of the chemicals that could be appropriate in the treatment of oxidative stress diseases such as AD [17,18]. The purpose of this study was to evaluate the effectiveness, function and mechanism of crocin in preventing the Aβ 42 formation associated with early onset AD [19]. Spectroscopic studies of thioflavin proved that there was amyloid formation in the Aβ 42 peptide, but this was reduced in the presence of crocin. This finding was supported by the results of TEM imaging. ANS-binding experiments showed that the fluorescence intensity of Aβ 42 decreased in the presence of crocin, implying that the exposed hydrophobic region of Aβ 42 interacted with crocin. The data obtained from circular dichroism (CD) spectroscopy also proved that in both the fresh and incubated peptide, in the presence of crocin, the stability of the helical regions of the peptide increased. Results from this study provide helpful clues regarding the potential role of crocin in AD therapy and contribute to the development of an anti-amyloid therapeutic strategy.

ANS-binding experiments of the Aβ 42 peptide
An ANS-binding assay of Aβ 42 (0.22 mM) in the presence and absence of crocin (15.4 μM) was done using a Varian spectrofluorometer at 37ºC for one hour. The experiment was done in a 10 mM phosphate buffer along with 150 mM NaCl at pH 7.4. The wavelengths of excitation and emission were respectively set to 446 and 400-600 nm with respective slit widths of 5 and 10 nm. The fluorescence emission intensity was measured in a 10-mm path length quartz cuvette in 350 µl samples titrated with 1 μl aliquots of a 10 mM ANS (1-anilino-8naphthalene sulfonic acid) stock solution in a 50 mM phosphate buffer (pH 7.4).

Transmission electron microscopy (TEM) of Aβ 42 peptide
Formvar and carbon-coated nickel TEM grids (SPI Supplies, Westchester, USA) were prepared by the application of a 2 μl protein sample at a concentration of 0.22 mM. The grids were then washed three times with 10 μl Milli-Q water and negatively stained with 10 μl uranyl acetate (2% w/v; Agar Scientific, UK). The grids were dried with filter paper between each step. The samples were viewed under 25-64 K magnifications at an excitation voltage of 100 kV using a ZEISS LE9910 transmission electron microscope. The dimensions of the fibrils were estimated by observing the images at 100% zoom in Paintbrush (Microsoft Corporation, USA).

CD spectroscopy of Aβ 42 peptide
Far-UV CD (190-260) was obtained to explore changes in the secondary structure of Aβ 42 in the absence and presence of crocin. The experiments were done in a 10 mM phosphate buffer (pH 7.4). CD spectra were obtained with a JASCO J-810 Spectropolarimeter (Jasco, Corporation, Japan) connected to a Desaga water bath. Spectra were recorded with a data interval of 1 nm, a response time of 4 seconds and a scan rate of 100 nm/min. Each spectrum was an average of 4 scans with a baseline scan subtracted.

RESULTS
Thioflavin T-binding assay Thioflavin T, which has fluorescent properties, is a dye that is generally used for observing and quantifying amyloid assembly under both in vivo and in vitro conditions in such a way that the fluorescence intensity is increased by connection to amyloid fibrils [22]. In this study, an enhancement in the ThT fluorescence following the incubation of Aβ 42 peptide was observed, implying amyloid formation in the peptide (Fig. 1A). However, in the presence of crocin, the fluorescence intensity was reduced, which means a reduction in the degree of amyloid assembly in the presence of crocin. This was also evident from the protective percentage: crocin provided 31% protection at the end point of 160 min.

ANS-binding study
ANS is a hydrophobic fluorescent molecular probe that has been used for examining the non-polar character of proteins and membranes [23][24][25]. ANS binding is used to study changes in exposed hydrophobicity in Aβ 42 upon incubation in the presence and absence of crocin. An increase in ANS fluorescence was observed in Aβ 42 until it maxed out and plateaued (data not shown). The ANS fluorescence maxima in the absence and presence of crocin Aβ 42 are shown in Fig. 1B. According to these data, Aβ 42 alone showed high ANS binding, which indicates hydrophobic protein surface exposure. In the presence of crocin, the ANS fluorescence intensity is reduced by 40% compared to the sum of the individual protein and crocin. The results show that hydrophobic surface exposure of Aβ 42 decreased in the presence of crocin, and this implies interaction between crocin and the protein and the formation of a complex between them.

Transmission electron microscopy (TEM)
TEM analysis was performed to obtain a morphological assessment of how crocin influenced Aβ 42 aggregation. Micrographs obtained for Aβ 42 incubated for 180 min displayed a large number of entangled fibril aggregates. In the presence of crocin it was apparent that the number of fibrils decreased ( Fig. 1C and D).

CD spectroscopy study
Circular dichroism (CD) spectroscopy is widely seen as a valuable technique for testing the protein structure in solution [26]. Fig. 2A and B show the CD spectra of native and incubated Aβ 42 in the absence and presence of crocin. As shown in Fig. 2A, the spectrum obtained for native Aβ 42 is characteristic of an α-helix conformation, showing negative ellipticity at around the 220 nm region. The CD spectrum of incubated Aβ 42 is typical of a peptide containing significant portions of β-sheet structure, as evidenced by the curve with a characteristic minimum at 217 nm (Fig. 2B). The evaluation of the secondary structure of native Aβ 42 revealed 21.2% β-sheet contents, which is similar to the observations of other researchers [27]. Adding crocin to Aβ 42 induced a change in the features of the Aβ spectrum, raising the intensity of the 220 nm band and increasing the α-helix content to 28.3%. After the addition of crocin to the incubated peptide, the CD spectrum indicates a conformational transition from a largely disordered structure to one highly enriched in α-helix. This is indicative of a decrease in the contribution from the unstructured peptide that was rearranged to form the α-helix. The values obtained from this analysis are listed in Table 1.

DISCUSSION
Aβ fibrils are shown in the Alzheimer's Aβ peptide. In this sense, the alphahelical structural changes to the beta sheet are of physiological importance and lead to the formation of amyloid structures [28][29][30][31][32][33]. Aβ peptides are composed of two isomers: Aβ 40 and Aβ 42 , in which 10% of the total Aβ consists of Aβ 42 [30]. Aβ 42 is the most amyloidogenic form of the peptide because it is more hydrophobic than Aβ 40 [16,35].
In this study, we used a thioflavin T-binding assay to provide some evidence about amyloid formation and interaction with crocin. Incubation of Aβ 42 at a temperature of 37ºC for 3 h induced the formation and accumulation of amyloid fibrils, as shown by the increases in the fluorescence intensity. This is in agreement with the results of other studies [37][38][39]. Crocin effectively prevented amyloid formation by incubated Aβ 42 by reducing the amount of amyloid fibril. This was proved by the reduction in thioflavin T fluorescence intensity. TEM examination of incubated Aβ 42 at 37ºC for 3 h also showed a great amount of amyloid oligomer [39,40]. In addition, electron microscopy imaging revealed a decrease in the amount of amyloid fibrils in the presence of crocin ( Fig. 2C and D).
The ANS-binding assay of incubated Aβ 42 showed very high fluorescence intensity due to the exposure of the hydrophobic region because of change in the peptide conformation [32,41,42]. However, adding crocin to Aβ 42 significantly decreased the fluorescence intensity, probably due to the interaction between crocin and Aβ 42 .
Our CD results also showed that natively dissolved Aβ 42 develops a greater proportion of β-sheet structures upon incubation. Accordingly, as shown in Fig.  2A and B and Table 1, it is reasonable to conclude that after incubation with crocin, Aβ 42 develops a partial helical structure. The evaluation of the CD peak supports the idea that crocin causes Aβ 42 to form an alpha-helix structure that is a non-amyloidogenic conformer [25]. Possible reasons for this observation are provided below. NMR structural investigation into the secondary structure of Aβ 42 has shown that the peptide contains two amyloid helical regions, including residues 8-25 and 28-38, which have been connected by a β turn [43,44]. It has a hydrophilic N-terminal starting Asp and a hydrophobic C-terminal ending alanine in and it contains a central hydrophobic cluster (residues 17-21) that appears to be the main constituent of amyloid plaques as found in the brain of AD patients. In addition, residues 28-42 have been shown to have a high probability for β-sheet structure formation [43]. Presumably, incubation increases the interaction between hydrophobic areas and increases the β-sheet content (41.30%) which is important for amyloid formation [45]. These results are consistent with the aggregative ability and neurotoxicity. However, the β-sheet content in incubated Aβ 42 decreased to 4.8% in the presence of crocin. This is probably due to interaction between the crocin and Aβ 42 and stabilization of the protein structure.
Since the content of β-sheet in incubated Aβ 42 in the presence of crocin is even less than that in fresh Aβ 42 (Table 1), we conclude that crocin interacts both with the aggregation-prone hydrophobic core (residues 17-21) and the C-terminal residue (residues [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. It is likely that crocin interacts with the C-terminal region and prevents its interaction with other monomers, forms paranuclei and subsequently amyloid fibrils. In addition, the binding of crocin to the aggregation-prone hydrophobic core stabilizes this region and prevents conformational change from an α-helix or random coil structure to a β-sheet, which is the key to forming fibrils and inducing cytotoxic effects [43]. Another explanation of the effects of crocin on amyloid may be related to its antioxidant properties. Many studies on antioxidants and their effect on amyloid fibril formation have been conducted. In studies on vitamin E and its isomers (forms of tocopherols), it was found that these vitamins may reduce the severity of amyloid fibril formation by reducing oxidative metabolites. Studies by Mishima et al. [46] also showed that the antioxidant power of α-tocotrienol is greater than that of α-tocopherol. The difference is in the greater number of double bonds that the former compound has (3 double bonds in the lipophilic tail). If the antioxidant activity has a direct relationship with the number of double bonds, then crocin, having several double bonds, will have a beneficial antioxidant property [17]. Therefore, it can be concluded that crocin, like other antioxidants, will slow down the formation of amyloid fibrils in Aβ 42 .

CONCLUSIONS
The inhibitory effects of crocin clearly indicate that it could be used as a pharmacological agent to inhibit or retard amyloid fibril formation in Alzheimer's disease. We have shown that crocin controls Aβ 42 -mediated amyloid fibril formation in vitro, probably through the stabilization of the helical structure, which prevents fibril formation and dissolves previously formed aggregates. However, further studies, including molecular dynamic simulation, are needed into the hypothesis regarding the formation of a complex of crocin with protein to eliminate the free hydrophobic area or prevent interaction with the C-terminal region of amyloid fibrils.
The prevention of neuronal cell apoptosis has always been emphasized as a therapeutic strategy for the treatment of neuronal cell destruction. The main advantage for crocin as a drug for the treatment of AD appears to be that it has fewer side effects, it has economic benefits (it forms a greater percentage of saffron constituents and can be easily extracted) and it is water soluble. It is encouraging that crocin remains a candidate drug against AD. The experimental data described here may well indicate that the utilization of crocin can control amyloid fibril formation and contribute to the development of a therapeutic strategy against AD.