Molecular docking studies were carried out using various test compounds with proteins such as acetyl cholinesterase (PDB ID: 4M0E), amyloid-beta (PDB ID: 2BEG), BACE1, (PDB ID: 5NN8), Tau/ GSK -3β (PDB ID: 1J1B), TrkA ,(PDB ID: 4PMS) and phosphoinositide 3-kinase (PI3K; PDB ID: 5JHA). Missing hydrogen atoms were added and correct bond orders were assigned, and then formal charges and orientations were fixed. The amino acid flips were assigned and H-bonds were optimized. Non-hydrogen atoms were minimized; the root mean square (RMS) reached default value of 0.3 Å. Sitemap 2.3 was used to investigate the binding site in the docking studies.

The test compounds were built using builder panel in Maestro. The compounds were taken for ligand preparation by Ligprep 2.3 module (14) which performed addition of hydrogen, 2D to 3D conversion, realistic bond lengths and bond angles, low energy structure with correct chiralities, ionization states, tautomers, stereo chemistries and ring conformations.

Induced fit docking (IFD) was studied by the method of Trott et al., (15). The prepared protein was loaded in the workspace and the sitemap predicted active site was specified for IFD. The grid was calculated for about 20 Å to cover the active site residues. The van der Waal's radii of the non- polar receptor and ligand atoms were scaled by a default factor of 0.50. IFD was calculated for the test compounds against AD drug targets. 20 conformational poses were calculated where the best conformational pose was selected based on the docking score, glide energy, hydrogen bonding and hydrophobic bonding interactions.

The PC12 cells were purchased from National Centre for Cell Science (NCCS), Pune and grown on poly styrene tissue-culture dishes Dulbecco’s modified Eagle’s medium (DMEM) containing 10% of foetal bovine serum, 5% of horse serum supplemented with 1mM of pyruvate, 2mM of glutamine, 100 units/ml of penicillin, and 100 units/ml of streptomycin with 90 % of air, and 5% of CO₂. Cells were sub-cultured in 96-well plates at a cellular density of 10⁴ cells per well and allowed to adhere and grow. When cells reached the 95% of confluence, they were placed in serum-free medium.

The inhibition of AChE and BuChE were assessed by using the method of Ellman et al., (18). The 96-well plates were incubated with 0.01ml of different concentrations (0.001 to 100µM) of test compounds at 37 ^oC for 6min, followed by the addition of to 0.03 ml of acetylthiocholine iodide or butyrylthiocholine iodide. Kinetic characterization of the hydrolysis of ACh and BuCh catalyzed by cholinesterase was performed. The absorbance was read at different time intervals (0.0, 70, 140, and 210 s) at 405nm. The concentration of test compounds that produced 50% inhibition (IC₅₀) of cholinesterase activities was calculated by nonlinear regression analysis of the response-concentration (log) curve.

To study the MAOs inhibition, briefly, the five different concentrations (0.0, 0.120, 0.320, 0.620µg) of 0.1 ml of test compounds and 0.9ml of MAO were incubated at 37 ^oC for different time intervals (0, 15, 30, 60min) in 96-well microplate. The substrate concentrations (0.03, 0.1, 0.23, 0.36, 0.5, and 1.0µg) of p-tyramine were used and the initial catalytic rates of MAO were estimated in the presence and absence of different concentrations of test compounds. The reaction was started by adding 0.1 ml of Amplex Red reagent, 0.2 ml of horseradish peroxidase, and 0.5 ml of p-tyramine and pargyline for 20 min at 37 ^oC. The H₂O₂ production was read at 590nm. The MAO inhibition was estimated by constructing the Liner weaver-Burk plots and the kinetic data were expressed in K_m and V_max values.

Cell viability was measured by the method of Mosmann (16) using the MTT reagent. The toxicity effects of test compounds on PC12 cells were evaluated. Briefly, cells in 24-well plates were incubated with different concentrations of test compounds for 24 h and 48 h. The cells were rinsed with 1.0 ml of 1M PBS (pH 7.4); 0. 2 ml of MTT was added to wells and incubated for 30 min at 37°C. After removing the medium with MTT, living cells containing MTT formazan crystals were solubilized with 0.2ml of DMSO and read at 570 nm.

Cells were seeded (10⁴ cells/ cm²) in 6-well plates and after 80% confluence was attained, fucoidan was added in test groups at concentrations ranging from 5 to 100 µg in DMEM and maintained in CO₂ incubator. The cells which did not receive fucoidan served as control and 0.1 % of vehicle controls were treated alone. After incubation with fucoidan at the end of experimental period, cells were washed with medium and further studies were carried out.

BBB permeation assay was assessed by the method of Pardridge (17). Brain penetrations of test compounds were evaluated using a parallel artificial membrane permeation assay (PAMPA). The donor 96-well microplate (PVDF membrane pore size 0.45 μm, diam. 25 mm) and the acceptor microplate were both from Millipore (Sigma-Aldrich). The acceptor microplates (Costar®) were filled with 0.3 ml of PBS: ethanol (7:3), and the filter membranes were impregnated with 0.004ml of porcine brain lipid (PBL) in dodecane. Test compounds at the concentrations of 0.2 ml were added to the donor wells. The acceptor filter plates were placed on the donor plates and incubated for 16 hours at 25°C (degree Celsius). After incubation, the donor plates were removed and the concentration of test compounds in the acceptor wells were estimated using a UV plate reader (FlexStation®3). Samples were analyzed at five wavelengths and the results were tabulated.

To analyze the percentage of neurite length, cells were observed using a light microscope and images captured using a camera connected to the microscope at 50x. 50 % of cells per field were counted and the neurite length was estimated using Magnus Pro software on the images. The µm of neurite lengths was converted into percentage of neurite length. The percentage of neurite length was calculated as the total of neurite length cells divided by the number of cells multiplied by 100

D. melanogaster is ‘non-regulated insect model and therefore this research did not require the approval of our Institutional Animal Ethical Committee.

Drosophila melanogaster diet was prepared from 100 ml of semi-solid diet which contained 5.5 gm of corn flour, 3.5 gm of sucrose, 2.0 gm of dextrose, 1.5 gm of agar, 1.0 ml of propionic acid and 1.0 g of yeast extract. After preparation, flies were transferred to Erlenmeyer flask containing media.

Drosophila melanogater (adult male flies, red eye) was authenticated and stock cultures were maintained at Entomology Research Institute (ERI), Loyola College, Chennai, Tamil Nadu, India. The flies were maintained at 25^oC (80 % relative humidity) with 12:12 hours of light and dark cycle.

Flies were divided into six experimental groups as given below. The total number of flies in each group (n) was 50.

Group 1: Served as control D. melanogaster

Group 2: D. melanogaster exposed to 25 µg of monocrotophos

Group 3: D. melanogaster exposed to 25 µg monocrotophos plus50 µg fucoidan

Group 4: D. melanogaster exposed to 50 µg of fucoidan

Group 5: D. melanogaster exposed to 50 µg of donepezile

After 10 day period, behavioural studies were analyzed; then their brains were dissected, and further studies were carried out.

Quantification of food intake was studied by the method of Ja et al., (19) using Drosophila melanogaster. Adult flies (20/vial) were placed in food vials and one capillary feeding tube per vial by a plastic pipette tip (5 mm) was placed. Flies consumed liquid medium (5% sucrose and 1% blue 1dye in water) from the capillary tubes for 8h; the amount of liquid medium consumed from each tube was recorded, and flies were then provided with fresh capillary feeding tubes containing 5% sucrose medium without blue dye for 18h. Flies were housed in the same vials while consuming medium from both the first (containing blue dye) and second (without blue dye 1) capillary tubes. Excreted blue dye of vials was collected by adding 3ml of water to each group of insects followed by vortexing. The optical density of dye was read at 630nm and absorbance values were converted to volumes by interpolation from standard curves of dye. Vials without flies were used to control evaporation of liquid medium from capillary tubes. The descent of the top meniscus of liquid was monitored for 24 hours and the volume consumed was expressed in units of μl per fly.

D. melanogaster were anesthetized at-40 °C and brains were dissected by using dissection microscopy. The heads were decapitated and placed in a glass dissection dish well with 1.5 ml of 1M phosphate buffer saline (pH, 7.4). The petri dish was coated with sylgard transparent resin containing drops of PBS. The head was held and the maxillary palps were removed on both sides with two pairs of forceps to open the head cuticle. Then the brain was carefully removed from the head cuticle and excess surrounding tissue was removed using fine mounted pin and the brain lobes were stored at -80 ^oC.

The brain tissues were removed and 100 mg of tissues were homogenized with 1.0 of 1mM PBS using pestle and mortor at -4^oC. The cells were pelleted by centrifuging at 12,000rpm for 10 min and the supernatant was centrifuged at 10,000rpm for 15 min. The supernatant was used for ELISA analysis.

The levels of AChE, PKI3, TrkA, Tau, BAECl and APP were quantified using a sandwich enzyme-linked immunosorbent assay. The absorbance was determined at 450 nm using Spectra Max M3 microplate reader (Molecular Devices Corporation, Sunnyvale, CA, United States) in accordance with the manufacturer’s instructions. All experiments were performed in triplicate.

The contents of glutamate and GABA were estimated using paper chromatographic method of Papastamatis and Wilkinson, (24). 50 mg of brain were homogenized with l ml of 0.3 M triethanolamine buffer (pH 6.8) and then centrifuged at 15,000rpm for 20 min. The extract was re-suspended in 1.0 ml of 20 mM phosphate buffer saline (pH 7.4) and centrifuged at 10,000rpm for 10 min. The extraction was spotted on Whatman filter paper (No. 1) by using solvent phenol: H₂O system (80:20). The paper was air tired. GABA and glutamate were located by ninhydrin spray. GABA and glutamate spots were cut out, eluted and were quantified.

5mg of brain was dissected and homogenized with 0.5 ml of 0.1 M perchloric acid. After centrifugation at 15,000rpm for 30 min, the supernatant was collected. Biogenic amines were measured using paper chromatography. The levels of serotonin, dopamine, tryptamine and octopamine were estimated by the method of Erspamer and Boretti (25). The activities of AChE and BuchE were assessed by using the method of Ellman et al., (18).

The formation of thiobarbituric acid reactive substances (TBARS) was estimated by the method of Beige and Aust (20). In brief, the brains were homogenized with 0.5 ml of 15% trichloroacetic acid (TCA), 0.2ml of 0.35% tert-butyl alcohol (TBA) and 0.1 ml of 5N of hydrochloric acid (HCl); it was incubated at 95 °C for 15 min. The supernatant was kept for 20 min at 37^oC and centrifuged at 12,000 rpm for 5 min. The absorbance was read at 535 nm. The content of lipid peroxidation was expressed as a TBARS produced in nanomoles per mg of protein.

The activity of CAT was estimated by the method of Yumoto et al., (21). 1 ml of 50 mM phosphate buffer (pH, 7.4) containing 0.1 ml of 100 mMH₂O₂ was added to 0.1 ml of homogenized brain. It was incubated for 2 min at 37 °C and absorbance was read at 240 nm. The CAT activities were expressed in units per mg protein, and one unit of enzyme activity was defined as the amount of enzyme required to break down 1 mM of H₂O₂.

The activity of SOD was determined by the method of Marklund and Marklund (22) using the auto-oxidation of pyrogallol. Briefly, 1 ml of 62.5 mM tris–cacodylic acid buffer and 0.5 ml of 4 mM pyrogallol were added to 0.1 ml of homogenized brain. To observe the auto-oxidation of pyrogallol, the absorbance was measured at 420 nm.

The levels of GSH were estimated by the method of Moron et al., (23). 0.1 ml of homogenized brain was treated with 0.1 ml of 25% TCA and the precipitate was pelleted by centrifuging at 70, 000rpm for 10 min. The free endogenous sulfhydryl group was estimated in 3.0 ml of a reaction mixture containing 2 ml of 0.5 mM 5, 5′-Dithiobis (2-nitrobenzoic acid) (DTNB) and 1.0 ml of tissue supernatant. The GSH reacted with DTNB and absorbance was read at 412 nm.

The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were analyzed using ELISA kits (Molecular Devices Corporation, Sunnyvale, CA, United States) in accordance with the manufacturer’s instructions. The absorbance was determined at 520 nm using Spectra Max M3 microplate reader. All experiments were performed in triplicate.

The levels of B cell lymphoma 2 (Bcl-2) and caspase-3 (Casp-3) were analyzed using ELISA kits (Molecular Devices Corporation, Sunnyvale, CA, United States) in accordance with the manufacturer’s instructions. The absorbance was determined at 470 nm using Spectra Max M3 microplate reader. All experiments were performed in triplicate.

RNA samples were isolated from brain of flies using an RNeasy mini kit (QIAGEN) according to the manufacturer's instructions. RNA from brains was extracted using the Trizole reagent (Gibco). RNA was converted to cDNA using oligo (dT) and Superscript II reverse transcriptase (Invitrogen).

qRT-PCR was performed using real-time PCR system with SYBR Green PCR Master Mix (Takara's). Specific primers were used (Table 1). PCRs were denatured at 94°C for 5 min, followed by a 29-s denaturation at 94°C, annealing at 55°C for 33 s, and an extension at 72°C for 1min. Threshold cycle was used to assess relative levels of m-RNAs versus reference beta-actin. Quantification was performed in two independent experiments with five samples for each group set up in triplicates.

Qualitative RT-PCR was done using specific primers (Table 1). The PCR mixture contained 200 nM of reverse and forward primers with about 500 ng of cDNA. PCRs were denatured at 94°C for 3 min, annealed at 55°C for 30 s, and extended at 72°C for 2 min. Products were loaded and electrophoresed in 2 % agarose gel.

The behaviour was assessed by the method of Pendleton et al., (26) for locomotor ability in D. melanogater. Fifty flies were placed in 100 ml measuring cylinder and allowed 10 min to acclimatize. During the test, fifty flies of control and experimental groups were placed in the far left bottom tube at the loaded position. Flies were gently tapped down to the bottom of the tube three times with a force of one tap per second. The top receiver tubes were quickly moved to the left, allowing flies 20 sec to climb up into the top receiver tube from a started position in each trial. Flies that succeeded in climbing up to the receiver tube were transferred to the next bottom tube at the end of the 20 sec by moving the array of top tubes one slot to the right, followed by gentle tapping and prompt placement of top tubes back to the starting position. The flies were tapped down and allowed to climb up for 20 s and then tapped down again. This was recorded and repeated three more times. At the fourth time, the number of flies passing the tube (16.5 cm) was recorded. The percentage of flies that escaped was then calculated.

T Maze test was done by using the method of Versace and Rosenberger, (27). T Maze (31 × 17.5 cm) consisted of starting chamber and a central chamber (12 × 8 × 1.5 cm) connected on each side to a food chamber. The starting chamber (9.5 × 2.5 cm) contained the flies at the beginning of each time slot. Food chambers (9.5 × 2.5 cm) were filled with 4 ml of food. In each experimental phase, flies began the exploration of the apparatus from the starting chamber. The central chambers were connected to the food chambers with a funnel that prevented flies to re-enter the central chamber. A performance index (PI) was calculated using the following formula: The percentage distribution (%) for chamber = Number of flies in the chamber / Numbers of flies per test ×100.

T Maze test was done using the method of Simonnet et al., (28) with minor modification. The plastic T Maze consisted of three chambers. The control chamber was (13.5 x 9.3 x4.5 cm) connected to the left chamber (12 × 8 × 1.5 cm) and the right chamber (12 × 8 × 1.5 cm). The filter papers (6 mm diameter) were soaked in 40 μl of ether and placed in one of the chambers for 24 hours. The number of flies which entered the ether solution for 30 min interval was calculated. The olfactory index was calculated as follows: olfactory index = Number of flies in the without ether chamber - Number of flies in the with ether chamber/Total number of flies.

Aggression assays were studied by the method of Hoffman, (29). A customized acrylic was filled with 2% of agar to moisturize the chambers (1 cm wide, 4 cm long and 0.6 cm high). Flies were placed in the center of the spacer and their movements were recorded simultaneously on video and subsequently analyzed for behavior (chasing, touching, wing extension, or copulation). The aggression index (CI) was calculated by dividing the time spent in aggression divided by the total time until aggression.

The binding modes of the test compounds with GSK-3β/Tau, A β, AchE, Tau, PIK3, TrkA and BACE1 which are involved in the neuropathology were obtained using molecular docking. Six potential compounds, i.e donepezil (3152), dermatansulfate (32756) manasamine-A (12020457), graciline (15384996), galantamine (92023653), fucoidan (92023653), macrolectin-A (92023653) interacted with important catalytic sites of acetylcholine esterase exhibiting good binding energy. The catalytic sites were PHE 295, HIP 447 and SER 203; TYR 72 , THR 75, ARG 296, ASP 74, SER 293 and PHE 295; ARG 296, PHE 295 and GLY122; PHE 295, SER 293, HIS 287, ARG 296; ASP 74 and HIS 447; ASP 74, THR 75, LEU 76, PHE 295 and ARG 296; at a distance of 3.2 Å, 2.8 Å and 3.0 Å; 3.1 Å, 2.7 Å, 3.0 Å, 2.8 Å, 3.0 Å and 3.2 Å; 3.2 Å, 3.2 Å and 3.1 Å; 3.1 Å, 3.2 Å, 2.9 Å and 2.3 Å; 3.1 Å and 3.2 Å; 2.7 Å, 3.0 Å, 2.4 Å, 3.2 Å and 3.0 Å (Table 2 and Figure 1). Amyloid-beta protein bound to dermatan sulphate by interacting with ASP 23, GLY 25 and LYS 28; it bound to manasamine-A by interacting with MET 35; it bound to galantamine by interacting with MET 35; it bound to fucoidan by interacting with ALA 21 and GLU 22; it bound to macolectin- A by interacting with MET 35 and GLU 22. Interactions of hydrogen bonds, distances and binding energy values are shown in Table 3 and Figure 2. BACE1 bound to donepezil by interacting with GLN 73 and THR 72; it bound to dermatan sulphate by interacting with GLY 11, ARG 235, GLN 73, THR 232 and LYS 321; it bound to manasamine –A by interacting with GLY 230; it bound to graciline by interacting with ARG235; it bound to galantamine by interacting with GLY 230, THR 232, GLY 11, GLN 73 and ASP 32; it bound to fucoidan by interacting with ARG 235, THR 72, TYR 198, GLY 34, ARG 128, ASP 32 and GLY 230; it bound to macrolectin -A by interacting with GLN 12 and PHE 108 (Table 4 and Figure 3).

Figure 1

(A) Donepezil , (B) dermatansulfate (C) manasamine-A , (D) graciline , (E) galantamine and (F) fucoidan interaction with AchE.

Figure 2

(A) Donepezil , (B) dermatansulfate (C) manasamine-A , (D) graciline , (E) galantamine and (F) fucoidan interaction with Aβ.

Figure 3

(A) Donepezil , (B) dermatansulfate (C) manasamine-A , (D) graciline , (E) galantamine and (F) fucoidan interaction with BACE-1.

Tau/GSK-3β target protein bound to donepezil by interacting with ARG 220; it bound to dermatan sulphate by interacting with CYS 218, ARG 220, GLN265 and ARG 22; it bound to manasamine-A by interacting with GLY 262; it bound to graciline by interacting with GLY 262; it bound to galantamine by interacting with CYS 218, ARG 223, ILE 228 and TYR 288; it bound to fucoidan by interacting with TYR 288, VAL 263 and GLU 290; it bound to macrolectin-A by interacting with ASN 64, ASP 200 and VAL 135. The glide energy and docking score of tested compounds are given in Table 5 and the interactions are shown in Figure 4. Figure 5 shows the interaction of TrkA target protein with test compounds. TrkA bound to donepezil by interacting with ARG 599 and MET 592; it bound to dermatan sulphate by interacting with GLU 590, ASP 668, GLU 560, LYS 544, GLY 670 and MET 67; it bound to manasamine-A by interacting with GLY 670; it bound to graciline by interacting with MET 592; it bound to galantamine by interacting with GLU 560; it bound to fucoidan by interacting with MET 671 and ASP 668; it bound to macrolectin- A by interacting with MET 592, TYR 591, ARG 599 and SER 673. Interaction of hydrophobic and glide energy is shown in Table 6. PI3K bound to donepezil by interacting with VAL 882, TYR 867 and SER 806; it bound to dermatan sulphate by interacting with LYS 890, SER 806, LYS 807, LYS 808, LYS 833, ASP 964 and GLU 880; it bound to manasamine- A by interacting with SER 806; it bound to graciline by interacting with THR 887, ASP 950, ASP 964 and SER 806; it bound to galantamine by interacting with THR 887, ASP 950, ASP 964 and SER 806; it bound to fucoidan by interacting with ASP 950, LYS 807, ASN 951, ASP 964, LYS 808 and LYS 833;it bound to macrolectin-A by interacting with ASP 950, ASP 954 and LYS 83 (Figure 6). Interaction of hydrophobic and glide energy is shown in Table 7. Macrolectein interacted with GSK-3β/Tau, A β, AchE, Tau, PIK3, TrkA and BACE1 (Figure 7). Detailed intermolecular interactions and binding energy are given in Tables 8-10. They show the docking results of all target proteins with respect to all the compounds with estimated inhibitor concentration (ki). The above docking results suggested that fucoidan bound to AD target proteins.

Figure 4

(A) Donepezil , (B) dermatansulfate (C) manasamine-A , (D) graciline , (E) galantamine and (F) fucoidan interaction with Tau protein.

Figure 5

(A) Donepezil , (B) dermatansulfate (C) manasamine-A , (D) graciline , (E) galantamine and (F) fucoidan interaction with TrkA.

Figure 6

(A) Donepezil , (B) dermatansulfate (C) manasamine-A , (D) graciline , (E) galantamine and (F) fucoidan interaction with PI3K.

Figure 7

Macrolactin –A interaction with (A) Amyloid-beta protein , (B) AchE , (C) TrkA , (D) PI3K, (E) Tau /GSK-3β and (F) BACE-1.

The morphological, differentiated and neuritent growth changes of PC12 cells treated with different concentrations of facoidan with monochrotophos for 7 days are given in Figures 8 and 9.

Figure 8

The neuroprotective effects of fucoidan in PC12 cells. Cells were treated with different concentrations of fucoidan and then incubated with or without monochrotophos for 7 days (n=3).

Figure 9

Effects of fucoidan on neuritent growth of neurons in PC12 cells Cells were treated with 6.25-12.5µg concentration of fucoidan (C-E) and then incubated with or without monocrotophosfor 7 days (n=3).

The fucoidan exhibited molecules permeability values of 3.3x10^-6cms^-1when compared to other test compounds (Table 11).

The inhibitory concentration (IC₅₀) values of the tested compounds and their selectivity indexes for cholinesterase (AChE and BuChE) and MAO are summarized in Table 12. Fucoidan showed potential effects and selective inhibition for MAOA; the other test compounds showed lower potential when compared to reference compounds such as lazabemine and iproniazide. Graphical data of the Line weaver–Burk reciprocal plots are given in Figure 10A. The overlaid reciprocal Line weaver–Burk plots (Figure 10B) showed that all plots for different concentrations of fucoidan were linear and intersected at the y-axis. Fucoidan reversed MAO-A inhibition in a time-dependent manner (Figure 10C). The AChE inhibitory activity is summarized in Table 12. There were both increased slopes and intercepts with increased inhibitory concentrations. The inhibition of AchE by fucoidan showed competitive inhibition constant (Ki) of 0.220mM. Manasamine–A exhibited potential inhibitory activity for Buch (IC₅₀=0.363mM) and fucoidan had excellent potential for inhibitory activity against AChE and MAO-A with IC₅₀ values of 0.64 and 2.15 mM respectively. These results highlight that fucoidan exhibited selective MAOA and AChE inhibition activities. PC12 cells were exposed to different concentrations of test compound (20 to 80µg/ml) for 24 hr and IC₅₀ values were calculated. Fucoidan at 100μg/ ml did not show significant effect on cell viability when compared to other test compounds (Figure 10D).

Figure 10

Kinetic studies on the mechanism of (A) AChE and (B) MAO-B inhibition by fucoidan. (C) Reversibility studies of MAO-A inhibition by fucoidan and pargyline. (D) Cell viability of PC12 cells and Cells were treated with different concentrations of fucoidan and then incubated with or without monochrotophos for 7 days (n=3). Data were expressed as percentage of cell viability. Values indicate mean ± SEM (n=3) and those having same alphabet did not deviate significantly by at p<0.05.

A significant increase in percentage of neurite length was observed in fucoidan supplemented cells when compared to that of control PC12 cells that did not receive fucoidan supplementation (Figure 11 A-F). A 5 µg of fucoidan enhanced percentage of neurite length significantly when compared to that of control cells at day 7 while a similar significant (p<0.05) when the cells were subjected to 10 µg of fucoidan cells were supplemented with 20 µg of fucoidan a significant increase (p<0.05) was seen in neurite outgrowth when compared to those of control cells on 1 day. On exposure to 50 µg, 75 µg, 100 µg fucoidan, the percentage of neurite length was significantly increased (p<0.05) when compared to that of control PC12 cells that of 20 µg fucoidan supplementation on 7^th day. DMSO treated cells did not show significance variation from their control cells even after 7 days.

Figure 11

Morphological changes of neurite length in PC12 cell lines and cells were exposed to different concentrations (0-20µg) of fucoidan for 7 days. The percentage of neurite lengths was the highest in the experimental group supplemented with 20µg fucoidan on 7^th day. Data were expressed as mean ±SEM (n=3). p≤ 0.05 was considered significant when compared to that of cells at 0 day (before fucoidan supplementation).

Mortality rate was observed in control and experimental groups of flies (Figure 12A). Mortality rate was significantly increased (p<0.05) in monocrotophos fed flies when compared to those of control flies. Monocrotophos plus fucoidan fed group showed significantly reduced mortality rate (p<0.05) when compared to monocrotophos alone fed flies. Fucoidan (VI) and donepezil (IV) fed flies did not show any significant variation when compared to those of control flies.

Figure 12

The effects of fucoidan on (A) mortality, (B) food intake, (C) ROS level and (D) AchE and Buch in control and experimental groups of flies. Data are expressed as mean ± SEM (n=3). Statistical analysis was performed using one-way ANOVA followed by the least significant difference (LSD) test. Values not sharing same alphabets differ significantly by at p<0.05.

Food intake was observed in control and experimental groups of flies (Figure 12B). Food intake was significantly reduced (p<0.05) in monocrotophos fed flies when compared to those of control flies. Monocrotophos plus fucoidan fed group showed significantly increased food intake (p<0.05) when compared to monocrotophos alone fed flies. Fucoidan (VI) and donepezil (IV) fed flies did not show any significant variation when compared to those of control flies.

The levels of ROS in the brains of control and experimental groups were assessed (Figure 12C). Monocrotophos fed flies showed significant increased (p<0.05) in the levels of these enzymes when compared to those of control flies; monocrotophos plus fucoidan fed flies significantly decreased (p<0.05) these levels to near normal when compared to monocrotophos fed flies.

The activities of AchE and BuchE were assessed in the brain of control and experimental groups (Figure 12 D). The activity of AchE and BuchE were significantly reduced (p<0.05) in monocrotophos fed flies when compared with those of control. Monocrotophos plus fucoidan fed groups showed significant increase (p<0.05) in the activities of AchE and BuchE when compared to monocrotophos fed groups. Donepezil (IV) and fucoidan fed flies did not show any significant variation when compared to those of control flies.

The protein levels were analyzed in the brain of control and experimental groups (Figure 13A and E). The levels of Tau, PIK3, TrkA, AchE, APP and BAC1 were significantly increased (p<0.05) in the monocrotophos fed group when compared with those of control. The levels of these proteins were significantly reduced (p<0.05) on monocrotophos plus fucoidan fed flies when compared to monocrotophos fed flies. Donepezil and fucoidan fed flies did not show significant variation in the levels of proteins when compared to those of control flies.

Figure 13

Effects of fucoidan on (A) AchE, APP and BACE-1, (B) DA, GABA and GL , (C) Tau, PkI3 and TrkA, (D) TA, OA and 5-TH and U levels and activities in brain of control and experimental groups of D. melanogster. Data are mean ± SEM (n=3). Statistical analysis was performed using one-way ANOVA followed by the least significant difference (LSD) test. Values not sharing same alphabets differ significant by at p<0.05

The levels of dopamine, glutamate, GABA, octopamine, serotonin and tryptoamine were assessed in the brains of control and experimental groups (Figure 13B and D). The levels of these neurotransmitters in the monocrotophos fed flies were significantly reduced (p<0.05) when compared with those of control. However, the levels showed a comparative increase in monocrotophos plus fucoidan fed groups highlighting the effect of fucoidan on maintaining neurotransmission. In fucoidan (VI) and donepezil (IV) fed flies, there was no significant variation when compared to those of control brain.

The levels of SOD, CAT, GSH and LPO were assessed in the brains of control and experimental groups (Figure 14A and B). Monocrotophos fed flies showed significant changes (p<0.05) in the levels of these enzymes when compared to those of control flies; monocrotophos plus fucoidan fed flies significantly altered (p<0.05) these levels to near normal when compared to monocrotophos fed flies.

Figure 14

Effects of fucoidan on (A) SOD and CAT; (B) LPO and GSH , (C) TNF-α and IL-6 , (D) Bcl2 and caspase-3 levels in brain of control and experimental groups of D. melanogster. Data are mean ± SEM (n=3). Statistical analysis was performed using one-way ANOVA followed by the least significant difference (LSD) test. Values not sharing same alphabets differ significantly by at p<0.05.

The levels of TNF-α and IL-6were assessed in the brains of control and experimental groups (Figure 14C). Monocrotophos fed flies showed significantly reduced levels (p<0.05) of these proteins when compared to those of control flies; in the monocrotophos plus fucoidan group there was an increased level (p<0.05) of TNF-α and IL-6 when compared to those of monocrotophos fed flies.

The levels of Bcl2 and caspase-3 proteins were assessed in the brains of control and experimental groups (Figure 14D). Monocrotophos fed flies showed significantly reduced levels (p<0.05) of these proteins when compared to those control flies; in the monocrotophos plus fucoidan group there was an increased level (p<0.05) of Bcl2 and casp-3 when compared to those of monocrotophos fed flies.

m-RNAs levels were analyzed in the brain of control and experimental groups (Figure 15A, B , C and D). The mRNA levels of Tau, PKI3,TrkA AchE, β-APP and BACE1, were significantly increased in monocrotophos fed group when compared with those of control. Monocrotophos plus fucoidan fed flies showed significant decrease (p<0.05) in the m-RNAs levels when compared to monocrotophos fed flies. Donepezil and fucoidan fed flies did not show any significant (p>0.05) variation in the level of m- RNA when compared to those of control flies.

Figure 15

Effects of fucoidan on (A) Tau, TrkA, PkI3, (B) AchE, APP and BAEE-1 in brain of control and experimental groups of D. melanogaster. (C and D) quantification of m-RNAs by using image J software. Data are mean ± SEM (n=3). Statistical analysis was performed using one-way ANOVA followed by the least significant difference (LSD) test. Values not sharing same alphabets differ significantly by at p<0.05.

Also m-RNAs levels were analyzed in the brain of control and experimental groups (Figure 16). The mRNA levels of mTOR, VMAT and SERT were significantly increased in the monocrotophos fed group when compared with those of control. Monocrotophos plus fucoidan fed flies showed significant decrease (p<0.05) in the m-RNAs levels when compared to monocrotophos fed flies. Donepezil and fucoidan fed flies did not show any significant variation in the level of m- RNAs when compared to those of control flies.

Figure 16

Effects of fucoidan on TOR, VMAT and SERT in brain of control and experimental groups of D. melanogster. Data are mean ± SEM (n=3) and statistical analysis was performed using one-way ANOVA followed by the least significant difference (LSD) test. Values not sharing same alphabets differ significantly by at p<0.05.

The latency of learning memory and aggressivity were assessed in D. melanogaster in control and experimental groups (Figure 17 A). There was significantly delayed acquisition latency (p<0.05) in monocrotophos fed flies when compared to those of control flies, indicating memory deficits. Monocrotophos plus fucoidan treated group significantly improved acquisition latency (p<0.05) when compared to those of control flies, indicating improved learning and memory. Fucoidan (VI) and donepezil (IV) fed flies did not show any significant variation when compared to control flies.

Figure 17

Effects of fucoidan on (A) learning and memory , (B) movement , (C) olfactory and (D) courtships behaviours in control and experimental groups of D. melanogster. Data are mean ± SEM (n=3). Statistical analysis was performed using one-way ANOVA followed by the least significant difference (LSD) test and values having same alphabet did not deviate significantly by at p<0.05.

The latency time of locomotor behavior was assessed in D. melanogaster in control and experimental groups (Figure 17B). The latency time of D. melanogaster was significantly reduced (p<0.05) in monocrotophos fed flies when compared to those of control flies. Monocrotophos plus fucoidan fed group showed significant increase (p<0.05) in the latency time when compared to monocrotophos fed alone flies. The fucoidan (VI) and donepezil (IV) fed flies did not show any significant variation when compared to control flies.

The index of olfactory and courtships behavior was assessed in D. melanogaster in control and experimental groups (Figure 17C and D). The olfactory and courtships of D. melanogaster was significantly reduced (p<0.05) in monocrotophos fed flies when compared to those of control flies. Monocrotophos plus fucoidan fed group showed significant increase (p<0.05) in the index time when compared to monocrotophos fed alone flies. The fucoidan (VI) and donepezil (IV) fed flies did not show any significant variation when compared to control flies.