Effects of Astaxanthin from Shrimp Shell on Oxidative Stress and Behavior in Animal Model of Alzheimer’s Disease

This study aimed to investigate the effect of astaxanthin (ASX) extracted and ASX powder from shrimp (Litopenaeus vannamei) shells on Wistar rats with Alzheimer’s disease, induced by amyloid-β (1-42) peptides. In this task, the rats were divided into eight groups: (1) Control, (2) sham operate, (3) negative control (vehicle) + Aβ1-42, (4) ASX extract+Aβ1-42, (5) commercial ASX + Aβ1-42, (6) ASX powder + Aβ1-42, (7) blank powder + Aβ1-42, and (8) vitamin E + Aβ1-42. All treatments were orally administrated for 30 days. At 14- and 29-days post injection, animals were observed in behavioral tests. On the 31st day, animals were sacrificed; the hippocampus and cortex were collected. Those two brain areas were then homogenized and stored for biochemical and histological analysis. The results showed that the Aβ1-42 infused group significantly reduced cognitive ability and increased memory loss, as assessed by the Morris water maze test, novel object recognition test, and novel object location test. Moreover, the Aβ1-42 infused group exhibited a deterioration of oxidative markers, including glutathione peroxidase enzymes (GPx), lipid peroxidation (MDA), products of protein oxidation, and superoxide anion in the cortex and the hippocampus. Meanwhile, ASX powder (10 mg/kg body weight) showed a significant reduction in cognitive and memory impairments and oxidative stress which is greater than ASX extract in the same dose of compound or vitamin E (100 mg/kg body weight). Our study indicates the beneficial properties of ASX in alleviation of cognitive functions and reducing neurodegeneration in Wistar rats induced by amyloid-β (1-42) peptides.

. These showed that the memory impairment was induced by Aβ1-42. Treatments with vitamin E (VE), ASX extract (AE), commercial ASX (AC), and ASX powder (AP) significantly decreased escape latency and increased time spent in target quadrant (p < 0.05) for all treatments. Interestingly, the AC and AP groups had significantly lower escape latency and higher time spent in target quadrant than the AE and VE treatment groups (p < 0.05).
For the object recognition and location memory tests, the discrimination index was lower in Aβ1-42 treated rats compared with a sham control group (SO), while the groups treated with VE, AE, AC, and AP had significantly higher discrimination index. With the blank powder encapsulation, the discrimination index was not increased. In contrast, the discrimination index for the AC and AP treatments was significantly higher than the AE group for both the object recognition and location tests ( Figure 2 A-D). The aforementioned results confirm that treatments with ASX from white shrimp shells significantly improved learning and reduced memory dysfunction in the AD model induced by Aβ1-42 (p < 0.05). The AP treatment showed better learning and memory than AE (p < 0.05) and was equivalent with AC. Notably, the AE treated group did not improve the learning and memory functions than those of AP and AC treated mice.

Effect of Astaxanthin in Reducing Brain Oxidative Stress
The lipid peroxidation product malondialdehyde (MDA), level of protein carbonyl, glutathaione peroxidase (GPx) assays, and percent inhibition of superoxide anion were used to evaluate the freeradical scavenging capacity of ASX in both the cortex and hippocampus to compare non-treated, Vtreated, VE-treated, and blank powder (BP)-treated groups. As shown in Figure 3    Effect of astaxanthin on discrimination index after 1 (A) and 24 h (B) of rats presented with object recognition test and effect of astaxanthin on discrimination index after 30 min (C) and 24 h (D) of rats presented with object location test. Control rats (C), sham operate (SO): Artificial cerebrospinal fluid (ACSF), vehicle plus Aβ 1-42 treated group (V), vitamin E: 100 mg/kg BW plus Aβ 1-42 treated group (VE), astaxanthin extract: 10 mg/kg BW plus Aβ 1-42 treated group (AE), commercial astaxanthin: 10 mg/kg BW plus Aβ 1-42 treated group (AC), astaxanthin powder: 10 mg/kg BW plus Aβ 1-42 treated group (AP), and blank powder: 10 mg/kg BW plus Aβ 1-42 treated group (BP). Values are expressed as mean ± SEM (n = 8). # -p < 0.05 compared with control group; * -p < 0.05 compared with vehicle and blank powder groups; • -p < 0.05 compared with astaxanthin extract group. Figure 1A,B, show that artificial cerebrospinal fluid (ACSF) produced no significant change in either escape latency or time spent in the target quadrant in the Morris water maze test. Intracerebroventricular administration of Aβ 1-42 significantly increased escape latency and decreased time spent in target  Figure 1A,B). These showed that the memory impairment was induced by Aβ  . Treatments with vitamin E (VE), ASX extract (AE), commercial ASX (AC), and ASX powder (AP) significantly decreased escape latency and increased time spent in target quadrant (p < 0.05) for all treatments. Interestingly, the AC and AP groups had significantly lower escape latency and higher time spent in target quadrant than the AE and VE treatment groups (p < 0.05).
For the object recognition and location memory tests, the discrimination index was lower in Aβ 1-42 treated rats compared with a sham control group (SO), while the groups treated with VE, AE, AC, and AP had significantly higher discrimination index. With the blank powder encapsulation, the discrimination index was not increased. In contrast, the discrimination index for the AC and AP treatments was significantly higher than the AE group for both the object recognition and location tests (Figure 2A-D). The aforementioned results confirm that treatments with ASX from white shrimp shells significantly improved learning and reduced memory dysfunction in the AD model induced by Aβ 1-42 (p < 0.05). The AP treatment showed better learning and memory than AE (p < 0.05) and was equivalent with AC. Notably, the AE treated group did not improve the learning and memory functions than those of AP and AC treated mice.

Effect of Astaxanthin in Reducing Brain Oxidative Stress
The lipid peroxidation product malondialdehyde (MDA), level of protein carbonyl, glutathaione peroxidase (GPx) assays, and percent inhibition of superoxide anion were used to evaluate the free-radical scavenging capacity of ASX in both the cortex and hippocampus to compare non-treated, V-treated, VE-treated, and blank powder (BP)-treated groups. As shown in Figures 3-6, AE, AC, AP, and VE treated groups had decreased MDA, and protein carbonyl, while increased percent inhibition of superoxide anion and GPx activity when compared to the V-treated and BP-treated groups (p < 0.05). These results indicate that the effectiveness of ASX from white shrimp shell was not significantly different (p ≥ 0.05) from commercial ASX.

Effect of Astaxanthin in Reducing Brain Oxidative Stress
The lipid peroxidation product malondialdehyde (MDA), level of protein carbonyl, glutathaione peroxidase (GPx) assays, and percent inhibition of superoxide anion were used to evaluate the freeradical scavenging capacity of ASX in both the cortex and hippocampus to compare non-treated, Vtreated, VE-treated, and blank powder (BP)-treated groups. As shown in Figure 3    group (AC), astaxanthin powder: 10 mg/kg BW plus Aβ1-42 treated group (AP), and blank powder: 10 mg/kg BW plus Aβ1-42 treated group (BP). Values are expressed as mean ± SEM (n = 5). # -p < 0.05 compared with control group; * -p < 0.05 compared with vehicle and blank powder groups; °-p < 0.05 compared with astaxanthin extract group.

Effects of ASX on Neuronal Survival and Amyloidosis in Hippocampal and Cortex Regions
The CA1 and CA3 in hippocampus, as well as the cortex regions were used as the reference zones because they are involved in memory and neuronal loss in these regions resulted in Alzheimer's disease. The low power views of the mouse hippocampus and cortex were shown in Figure S2. Cresyl violet staining was used to evaluate the surviving neurons in the cortex and hippocampal CA1, CA3. Figure 7 shows that surviving cells in the control group (a, I, and q) showed round and pale-stained nuclei in the cortex and hippocampus whereas the Aβ1-42 treated group showed serious cell death (c, k, and s). The SO treatment was shown as not significantly different in the surviving neurons when compared to the control group. AC and AP (10 mg/kg) treatments showed a significant decrease in neuronal degeneration in CA1 and CA3 regions of hippocampus and the cortex, whereas the AE group showed a significant decrease in neuronal degeneration in the cortex only (t). The VE group treatment showed a significant decrease in neuronal degeneration in CA1 of hippocampus and cortex (g and w). The BP treated group with alginate and modified starch vehicle did not show any protection against neuronal injury (h, p, and x).

Effects of ASX on Neuronal Survival and Amyloidosis in Hippocampal and Cortex Regions
The CA1 and CA3 in hippocampus, as well as the cortex regions were used as the reference zones because they are involved in memory and neuronal loss in these regions resulted in Alzheimer's disease. The low power views of the mouse hippocampus and cortex were shown in Figure S2. Cresyl violet staining was used to evaluate the surviving neurons in the cortex and hippocampal CA1, CA3. Figure 7 shows that surviving cells in the control group (a, I, and q) showed round and pale-stained nuclei in the cortex and hippocampus whereas the Aβ 1-42 treated group showed serious cell death (c, k, and s). The SO treatment was shown as not significantly different in the surviving neurons when compared to the control group. AC and AP (10 mg/kg) treatments showed a significant decrease in neuronal degeneration in CA1 and CA3 regions of hippocampus and the cortex, whereas the AE group showed a significant decrease in neuronal degeneration in the cortex only (t). The VE group treatment showed a significant decrease in neuronal degeneration in CA1 of hippocampus and cortex (g and w). The BP treated group with alginate and modified starch vehicle did not show any protection against neuronal injury (h, p, and x).

Effects of ASX on Neuronal Survival and Amyloidosis in Hippocampal and Cortex Regions
The CA1 and CA3 in hippocampus, as well as the cortex regions were used as the reference zones because they are involved in memory and neuronal loss in these regions resulted in Alzheimer's disease. The low power views of the mouse hippocampus and cortex were shown in Figure S2. Cresyl violet staining was used to evaluate the surviving neurons in the cortex and hippocampal CA1, CA3. Figure 7 shows that surviving cells in the control group (a, I, and q) showed round and pale-stained nuclei in the cortex and hippocampus whereas the Aβ1-42 treated group showed serious cell death (c, k, and s). The SO treatment was shown as not significantly different in the surviving neurons when compared to the control group. AC and AP (10 mg/kg) treatments showed a significant decrease in neuronal degeneration in CA1 and CA3 regions of hippocampus and the cortex, whereas the AE group showed a significant decrease in neuronal degeneration in the cortex only (t). The VE group treatment showed a significant decrease in neuronal degeneration in CA1 of hippocampus and cortex (g and w). The BP treated group with alginate and modified starch vehicle did not show any protection against neuronal injury (h, p, and x).  On the other hand, significant accumulation of positive staining of β-amyloid (% area) was observed in the hippocampal and cortex of Aβ 1-42 peptide infused rats using immunohistochemistry staining. Hippocampal CA1, CA3, and cortex of rats treated with ASX in various forms (AE, AC, AP, and VE) for 30 days showed a reduction of positive staining of β-amyloid when compared with other Aβ 1-42 treated groups. Interestingly, AC and AP showed better biological activity than other treatments (Figure 8).

Discussion
In recent years, ASX has gained attention for its potential therapeutic role in neurodegenerative diseases. Numerous reports that ASX treatment is effective at protecting neurons from various forms of CNS damage in models of specific neuronal damage and neurodegenerative disease [17][18][19]. There are few studies investigating the benefits of ASX from Pacific white shrimp (Litopenaeus vannamei) shell in neurodegenerative disease model, while there are many using Haematococcus pluvialis [9,20,21]. In fact, our ASX extract from Pacific white shrimp shell processing waste contains higher proportion of ASX diester and higher PUFA content (61.74%) [22] than Arabian red shrimp (PUFA content of 29.85%) and Haematococus pluvialis [23]. This implies that the powerful antioxidant properties of carotenoid extract of Litopenaeus vannamai may be attributed to the antioxidant synergism of ASX and PUFAs contained when in compared to other sources of ASX [22,24].
In this study, we determined the effects of ASX from white shrimp shells on spatial and nonspatial memory, and on neurodegeneration in various sub-regions of brain in the animal model of AD induced by Aβ1-42. The results clearly demonstrated that ASX from white shrimp shell significantly improved spatial memory ( Figure 1; Figure 2C,D) as indicated by Morris water maze and object recognition tests, improved non-spatial memory (Figures 2A,B) as indicated by object recognition test, and reduced neurodegeneration, as indicated by surviving neurons and amyloid beta plaque level ( Figure 8).
Recently, oxidative stress has been detected in the early stage of AD through Aβ accumulation and progression via mitochondrial dysfunction, caused by the generation of reactive oxygen species (ROS) and reduction in the level of detoxifying enzymes, including superoxide dismutase (SOD), catalase (CAT), and GPx [25,26]. ROS destroys the polyunsaturated fatty acids of cellular membranes to generate lipid peroxidation products such as MDA, which may serve as an indicator of the oxidative damage level and induce neuronal deterioration [27]. In addition, protein carbonyl is another hallmark of oxidative damage [28]. As expected, the observed neuroprotection by ASX from white shrimp shell was due to its antioxidant property through support of the activity of GPx, and percent inhibition of superoxide anion, as well as reduction of MDA and protein carbonyl levels, compared to vehicle-AD groups (p < 0.05) as shown in Figures 3-6. This is consistent with our previous study, where we showed that the ASX from white shrimp shells eliminates superoxide

Discussion
In recent years, ASX has gained attention for its potential therapeutic role in neurodegenerative diseases. Numerous reports that ASX treatment is effective at protecting neurons from various forms of CNS damage in models of specific neuronal damage and neurodegenerative disease [17][18][19]. There are few studies investigating the benefits of ASX from Pacific white shrimp (Litopenaeus vannamei) shell in neurodegenerative disease model, while there are many using Haematococcus pluvialis [9,20,21]. In fact, our ASX extract from Pacific white shrimp shell processing waste contains higher proportion of ASX diester and higher PUFA content (61.74%) [22] than Arabian red shrimp (PUFA content of 29.85%) and Haematococus pluvialis [23]. This implies that the powerful antioxidant properties of carotenoid extract of Litopenaeus vannamai may be attributed to the antioxidant synergism of ASX and PUFAs contained when in compared to other sources of ASX [22,24].
In this study, we determined the effects of ASX from white shrimp shells on spatial and non-spatial memory, and on neurodegeneration in various sub-regions of brain in the animal model of AD induced by Aβ  . The results clearly demonstrated that ASX from white shrimp shell significantly improved spatial memory (Figure 1; Figure 2C,D) as indicated by Morris water maze and object recognition tests, improved non-spatial memory (Figure 2A,B) as indicated by object recognition test, and reduced neurodegeneration, as indicated by surviving neurons and amyloid beta plaque level (Figure 8).
Recently, oxidative stress has been detected in the early stage of AD through Aβ accumulation and progression via mitochondrial dysfunction, caused by the generation of reactive oxygen species (ROS) and reduction in the level of detoxifying enzymes, including superoxide dismutase (SOD), catalase (CAT), and GPx [25,26]. ROS destroys the polyunsaturated fatty acids of cellular membranes to generate lipid peroxidation products such as MDA, which may serve as an indicator of the oxidative damage level and induce neuronal deterioration [27]. In addition, protein carbonyl is another hallmark of oxidative damage [28]. As expected, the observed neuroprotection by ASX from white shrimp shell was due to its antioxidant property through support of the activity of GPx, and percent inhibition of superoxide anion, as well as reduction of MDA and protein carbonyl levels, compared to vehicle-AD groups (p < 0.05) as shown in Figures 3-6. This is consistent with our previous study, where we showed that the ASX from white shrimp shells eliminates superoxide anion, which causes lipid peroxidation [29]. It is also consistent with the effects of ASX from Haematococcus pluvialis [8,11,30].
ROS are associated with the formation of senile plaques in the brains of patients with AD, which may result in neuronal death [31,32]. Our results showed that scavenging of ROS by orally administered ASX from white shrimp shells significantly reduced neuronal loss in both cortex and hippocampus when compared to vehicle-AD group (p < 0.05) as shown in Figure 7.
We observed that amyloid plaque formation or aggregation via immunohistochemistry staining of amyloid beta peptide. The results shown in Figure 8, display that ASX significantly reduced positive staining of Aβ 1-42 when compared to the vehicle-AD group (p < 0.05). The results were consistent with Rahman et al. (2019) [8] who reported that ASX treatment showed significant protection against Aβ mediated neuronal damage and forming plaque in the hippocampus.
In the last decade, the encapsulation technique has been shown to be an effective method to protect bioactive compounds within a shell and provides many other advantages. ASX is a highly unsaturated molecule and naturally not stable, and thus its functional ability is easily degraded during processing and storage. Another disadvantage is that ASX from shrimp shells may change the color and flavor of food. Encapsulation is an effective means to protect the stability of ASX within the selected wall material also it can help increase the surface area for release to the intestine [33]. Our results showed that AP could improve learning both spatial and non-spatial memory compared with AE, as shown in Figures 1 and 2. In addition, AP showed significantly decreased oxidative damage and amyloid plaque level, and increased survival of neurons in both cortex and hippocampus areas when compared with those of the AE treatment groups. Therefore, use of encapsulation technology to improve the stability and efficacy of ASX extract in powder form is recommended for potential application in disease prevention of AD.

Animals
Adult male Wistar rats, eight weeks old, weighing 250-300 g were obtained from Nomura Siam International Co., Ltd. Rats were maintained (at room temperature: (22 ± 3 • C) with humidity of: 55% ± 10%), on a 12 h light-dark cycle (lights on 06.00-18.00), and with ad libitum access to water and foods. This study was conducted in accordance with the recommendations in the guide for the care and use of animal care outlined by the Faculty of Science, Prince of Songkla University (MOE 0521.11/105).

Extraction of ASX from Shrimp Shells and Preparation of Encapsulated ASX (ASX-powder)
ASX extraction was prepared according to a previously described method [13]. To produce the ASX-powder, the emulsions were prepared by homogenizing the crude ASX (2 g/100 mL of wall material solution) with the wall material solution (alginate (2.0 g/100 mL) and modified starch (20 g/100 mL), mass ratio of 1:1) using a homogenizer (IKA T-25-Werke Gmbh & Co.KG, Staufen, Germany) at a rotational speed of 10,000 rpm for 20 min. The emulsion was converted into powders using cryogenic incorporated with freeze drying. The ASX-powder was collected and, stored in amber bottles.

Experimental Procedures
The rats were randomly divided into eight groups, each group consisted of eight rats, as follows: Group 1 was the control group (C); group 2 served as a sham control group (SO) and received artificial cerebrospinal fluid (ACSF); groups 3-8 were Aβ 1-42 infused groups in that they were injected intracerebroventricularly (i.c.v.) with amyloid beta peptides Aβ 1-42 by using stereotaxic apparatus. To produce neurotoxicity in groups 3-8, 10 µL of normal saline-dissolved Aβ 1-42 at a concentration of 2 mg/mL, was injected bilaterally on the first day. After injection of Aβ 1-42 , infused group 3 received propylene glycol (negative control; V), infused group 4 received ASX extract at 10 mg/kg/day p.o. (AE), infused group 5 received commercial ASX at 10 mg/kg/day p.o. (AC), infused group 6 received ASX powder at 10 mg/kg/day p.o. (AP), infused group 7 received powder blank at 10 mg/kg/day p.o. (BP), and infused group 8 received vitamin E at 100 mg/kg/day p.o. (VE). All treatments were orally administered for 30 days after Aβ 1-42 infused. On days 14 and 29 post injection, animals were observed in the following behavioral tests: Morris water maze test, object location test, and object recognition test. The animals in all groups were assessed on spontaneous locomotor behaviors before doing all experiment.

Morris Water Maze Test
The Morris water maze test (MWM) was developed by Richard Morris in 1981 [34], and is a commonly used cognitive and behavioral assessment tool to evaluate both working and long-term spatial memory. Results of the test are thought to reflect the subject's hippocampus function. MWM as shown in Figure S3, was performed on days 1-5 for training phases and on 14 and 29 day after oral administration compound of our experimental protocol, for testing phases. In the training phase, each rat was given four swimming trials per day for five consecutive days. In the testing phase, each rat receives the swimming trial or probe trial for the searching hidden platform, and the escape latency time was recoded. Twenty-four h later, the swimming trials or probe trial were conducted again, in which the platform was removed and rats were free to swim in the pool for 60 s [35]. Time spent by the rat in the target quadrant (target quadrant occupancy or located that hidden platform stand) for the search of the removed platform were recorded. Data were recorded by using video-image camera connected with a computer analysis system to observe swimming time.

Object Location Test
The object location memory test is used to assess cognition, particularly in spatial memory and discrimination, in rodent models of central nervous system disorders [36]. The fundamental concept of this test is that rodents are prone to give more time exploring a novel object than a familiar object and can remember when an object has been moved to a new location as shown in Figure S4. In our study, the test occurred in an open field arena, to which the animals were habituated beforehand. After habituation, two objects made of similar material were introduced to the arena. During the familiarization trial, the animals were allowed to explore the arena with the two objects for 3 min. After intervals of 30 min and 24 h, testing trials were done, in which one of the objects had been moved to a new location. Between trials, the objects and test areas were cleaned with 70% (v/v) ethanol to remove odor cues for later trials. The exploration activity, defined as sniffing while directing the nose toward and within 2 cm of the object, was separately scored for each object. Finally, the scores were used to calculate the discrimination index (ID) according to the formula: ID = (t [novel] − t [familiar])/ (t [novel] + t [familiar]) × 100.

Object Recognition Test
The object recognition test was used to assess the subject's perception of color, shape, and texture as shown in Figure S5. In this test, each rat received two consecutive 3 min object exploration trials, separated by 1 and 24 h inter-trial intervals. During the familiarization trial, rats were individually presented with two similar objects. In the second trial, one of the two objects was randomly selected and replaced with a third, novel object. During the two trials, exploration of each object was defined as sniffing, licking, chewing, or having moving vibrissae while directing the nose toward the object, while within 2 cm of the object. Time of exploration was recorded separately for each object. Between trials, the objects and test areas were cleaned with 70% (v/v) ethanol to remove odor cues. Finally, the discrimination index was calculated as time spent exploring the novel object compared with the familiar object relative to the total time spent exploring all objects, according to the formula:

Preparation of Brain (Cortex and Hippocampus) Tissues Homogenate and Protein Extraction
After completion of the experiment, the animals were sacrificed and the brains were quickly collected. The hippocampus and cortex (n = 5) were dissected and the tissues were frozen and stored at −80 • C. The cortex and hippocampus tissues were homogenized in 0.2 M phosphate buffer saline (PBS) followed by centrifugation at 13,000 rpm at 4 • C for 25 min. Supernatants were collected and stored at −80 • C for later determination of GPx activity and the levels of protein carbonyl, MDA, and superoxide anion.

Measurement of Glutathione Peroxidase (GPx) Activity
Glutathione peroxidase (GPx) activity was determined by previously published methods [37], in which the activity is measured indirectly by a coupled reaction with glutathione reductase. Oxidized glutathione, produced upon reduction of hydrogen peroxide by glutathione peroxidase, is recycled to its reduced state by glutathione reductase and NADPH. The oxidation of NADPH to NADP + is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in the A340 nm is directly proportional to the glutathione peroxidase activity. Our final 1 mL of system mixture contained 48 mM sodium phosphate, 0.38 mM EDTA, 0.12 mN β-NADPH, 0.95 mM sodium azide, 3.2 units of glutathione reductase, 1 mM glutathione (GSH), 0.02 mM DL-dithiothreitol, 0.0007% H 2 O 2 , and the standard enzyme glutathione peroxidase solution or a homogenate brain sample. The glutathione peroxidase solution was used as a standard for enzyme activity. The standard curve was plotted as the rate of A340 nm per min against GPx activity. One unit activity was defined as the amount of enzyme necessary to catalyze the oxidation by H 2 O 2 of 1 µmole of GSH to GSSG per min at pH 7 at 25 • C. Data were reported as units of GPx per mg protein.

Superoxide (O 2 − ) Anion Assay
The O 2 − level was determined by spectrophotometric procedure according to Ukeda et al. [38], and based on a xanthine/xanthine oxidase (XO) assay which is marked by a color change from nitro blue tetrazolium (NBT)-yellow to formazan-blue. The reagent mixture included ethylenediaminetetraacetic acid (EDTA), NBT, xanthine, and xanthine oxidase (XO), along with sample. Absorbance of formazan chromophore was measured at 560 nm against blank and standard curves of TEMPOL. The data were expressed as % inhibition, as calculated following the Equation (1).
A = OD of reagent only and B = OD of reagent and sample.

Measurement of Protein
Protein carbonyl, produced during protein oxidation in the biological sample, was measured by using a commercial assay kit (Sigma, USA).

Measurement of Malondialdehyde (MDA)
Malondialdehyde (MDA) is produced during lipid peroxidation, and was measured using the methods of Ohkawa et al. [39]. A calibration curve was prepared using 1,1,3,3-tetramethoxypropane (TMP). A volume of 0.2 mL sample supernatant was mixed with 1.5 mL acetic acid (20%) at pH 3.5, 1.5 mL thiobarbituric acid (0.8%), and 0.2 mL sodium dodecyl sulphate (8.1%) and were added to processed tissue samples (0.1 mL), and then heated at 100 • C for 60 min, then cooled. A volume of 5 mL of n-butanol-pyridine (15:1) and 1 mL of distilled water were added and the mixture was vortexed vigorously. After centrifugation at 4000 rpm for 10 min, the organic layer was withdrawn and absorbance was measured at 532 nm using a spectrophotometer (ASYS UVM340, Biochrom Ltd., Cambridge, UK). Concentration of MDA was expressed as nmol/g tissue. The total brain protein assay was analyzed by the methods of Lowry et al. [40] with slight modification using Pierce BCA assay kit.

Cresyl Violet Staining for Nissl Substance
After test animals were sacrificed, the whole brains (n = 3) were fixed in Bouin solution (Bio-optica, Milano, Italy) for 72 h. The brains were washed with 0.1 M phosphate buffered saline (PBS) and dehydrated sequentially in 70%, 80%, 90%, and 100% ethanol and then transferred to toluene. They were infiltrated and embedded in paraffin, sliced to a thickness of 5 µm using a rotary microtome, Leica RM2235 (Leica Microsystems, Nussloch, Germany), and then placed on silane-coated slides. They were deparaffinized with xylene, rehydrated in 100%, 95%, 90%, 80%, and 70% alcohol, and finally stained with cresyl violet (0.5%) to determine the neuronal density [41] in hippocampal CA1, CA3, and cortex regions. Neurons morphology was observed and captured by a Nikon E600 microscope with a DXM 1200 digital camera, using ACT-1 software (Nikon Inc., Badhoevedorp, The Netherlands). For all experimental groups, the surviving neurons were counted and recorded as number of neuron cells (size greater than 7 µm) using ImageJ (Java 1.4.2, U. S. National Institutes of Health, Bethesda, MA, USA).

Immunohistochemistry Analysis
For immunohistochemistry, the brain sections from the different groups were deparaffinized with xylene and rehydrated in ethanol as above. Endogenous peroxidase was removed using 3% H 2 O 2 in 100% methanol for 90 min. After washing three times with 0.1 M PBS containing Tween-20 (PBST), the sections were incubated in 1% glycine in 0.1 M PBST for 15 min and then washed three times with 0.1 M PBST. The brain sections were then incubated in blocking solution containing bovine serum albumin (4%) and 2% normal goat serum in 0.1 M PBST for 2 h. The slides were then incubated with primary antibody: Anti-beta amyloid antibody (1:200; catalog no. ab 2529, Abcam, CA, USA) at room temperature overnight. They were washed three times for 5 min each with PBST and were then incubated with secondary antibody: HRP-conjugated goat anti-rabbit IgG (1:1000; catalog no. ab 6721, Abcam) at room temperature for 2 h. After washing three times with PBST, liquid DAB-Plus Substrate Kit (Invitrogen, Camarillo, CA, USA) was added onto the brain sections to develop the color for 2 min and the reaction was then stopped by RO water. The slides were counter-stained with Mayer's hematoxylin and mounted with permount. The results were recorded using a Nikon E600 microscope with a DXM 1200 digital camera, and ACT-1 software. Data were expressed as positive staining of β-amyloid (% area; total area covered with plaques relative to the total area) using ImageJ.

Statistical Analysis
All data are presented as the mean ± standard error of mean (SEM). Significant differences between groups were analyzed using one-way ANOVA followed by the LSD's post hoc test. Statistical significance was assumed at a p < 0.05. Statistical analysis was performed using the SPSS 17.0 software package for Windows.

Conclusions
Here, we observed that after oral administration for one month, ASX from shrimp (Litopenaeus vannamei) shells protected Wistar rats from Aβ 1-42 induced oxidative stress and resulted in improved learning and memory. Our study aimed to compare the effects of ASX in various forms, and we found that ASX powder showed better protection against cognitive dysfunction than the ASX extract at the same dose of compound. On the basis of these results, we suggest that ASX powder may be a promising candidate as a functional food product to protect from brain disorders, including in AD therapy.