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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 2  |  Page : 139-148

Appraising the neuroprotective competence of nitrogen-enriched Arthrospira platensis in comparison to commercial resource in depressed mice models


1 Department of Biotechnology and Bioinformatics, Bishop Heber College, Tiruchirappalli, Tamil Nadu, India
2 Department of Zoology, Holy Cross College, Tiruchirappalli, Tamil Nadu, India

Date of Web Publication30-Oct-2019

Correspondence Address:
Dr. Arockiya Anita Margret
Department of Biotechnology and Bioinformatics, Bishop Heber College, Tiruchirappalli-620 017, Tamil Nadu.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jrptps.JRPTPS_54_18

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  Abstract 

Back ground: Arthrospira platensis encompasses vital nutrients and is commercialized globally. It comprises of imperative resource that has the potential to combat neurological deformities caused due to stress and anxiety. Objective: This work evaluates the neuroprotective effect of spirulina cultivated on nitrogen enriched medium and compares it with the commercial samples. Materials and Methods: The study is authenticated with an antioxidant assay and the vital compounds are relatively profiled by GC-MS study. Furthermore, a molecular docking analysis is implemented to investigate the therapeutic potentials of the phytocompounds against Monoamine Oxidase –A and establish them as inhibitors. The ethanolic extract of spirullina as are fed on depressed mice models to assay its neuroprotective effect and rehabilitation of brain cells by a histopathological study. Results: The antioxidant content of the augmented sample was consistent on par with the commercial sample. The in silico assay was performed with 10 extricated compounds of both the samples where, Butanoic acid, 3-hydroxy- furnished a minimum binding affinity energy value of -56.24 kcal/mol and dodecanamide was efficient to bind with the active site of the amino acid residue TYR 69 with a minimum energy of -87.8 kcal/mol. The histopathological examination upholds the refurbished parameter of vital phytocompounds with placid cellular edema and perivascular infiltration. Conclusion: There is a wide range of need to develop research against stress and anxiety and the study fortifies the restorative efficacy of the phytocompounds as a neuroprotective drug.

Keywords: Arthrospira platensis, butanoic acid 3-hydroxy-, GC-MS, molecular docking, monoamine oxidase A, neuroprotectants


How to cite this article:
Anita Margret A, Kalaiyarasan M, Avila Jerley A, Jeevaraj T. Appraising the neuroprotective competence of nitrogen-enriched Arthrospira platensis in comparison to commercial resource in depressed mice models. J Rep Pharma Sci 2019;8:139-48

How to cite this URL:
Anita Margret A, Kalaiyarasan M, Avila Jerley A, Jeevaraj T. Appraising the neuroprotective competence of nitrogen-enriched Arthrospira platensis in comparison to commercial resource in depressed mice models. J Rep Pharma Sci [serial online] 2019 [cited 2019 Nov 17];8:139-48. Available from: http://www.jrpsjournal.com/text.asp?2019/8/2/139/269945




  Introduction Top


Arthrospira platensis (commonly known as spirulina) is a unicellular planktonic microalga that grows in aquatic habitat such as fresh, salt, and brackish bodies of water.[1] It is a free-floating cylindrically shaped filamentous photosynthetic species. It received its scientific term based on the unified morphology of septa arrangement and helical form. Spirulina [Figure 1] is renowned for its nutritional benefits and termed as a super food owing to the intense presence of vital nutrients such as vitamin B12. It is cultivated and marketed commercially worldwide both as a food supplement and as an active ingredient in functional foods.[2] It has attained considerable promotion in health sector and bestows pharmaceutical benefits with compounds such as carotene and omega 3 and 6 polyunsaturated fatty acids.[3]
Figure 1: Pictorial representation of Anthrospira plantensis (Adapted from King Dnarmsa, Philippines, posted online)

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Occurrences of stress-induced psychiatric disorders are prone more in the contemporary era of scientific developments that leads to stress-induced psychiatric disorders. Generalized anxiety disorder and depression (depressive episode) are commonly occurring psychiatric ailments that can overlap with similar symptoms. This menace is considered a threat and a burden that agonizes the world intensively.[4] Numerous psychotropic medications are designed to treat their pathology and symptoms. Monoamine inhibitors (MAO-I) are a class of antidepressants that subside deamination the action of monoamine oxidase and regulates the function of neurotransmitters.[5] Furthermore, the pathology of this mental ailment demands the nutritional condition of the body. There is an increased rate in the metabolism where the biochemical constitution of the body is utilized widely. There is a surplus change in the adrenal production and consumption of vitamins, minerals, and micronutrients which further accelerate metabolism of proteins, fats, and carbohydrates, producing quick energy to overcome this psychiatric condition.[6]

To manage this metabolic condition, there is a need of antioxidant defense in the biological system. Nonenzymatic antioxidants such as vitamin E (alphatocopherol), vitamin C (ascorbic acid), β-carotene, minerals, and trace elements can efficiently overcome the nutritional scarcity. The super food, spirulina, can facilitate this condition by bestowing its antioxidant potential. Furthermore, its credentials can be enhanced by culturing it in a nitrogen-enriched medium, as variation in its composition can increase its nutrient content and biomass.[7] This study persuades to evaluate the neuroprotective effect of spirulina cultivated on nitrogen-enriched medium and efforts a therapeutic remedy to hoist as monoamine inhibitors.


  Materials and Methods Top


Drugs and reagents

All chemicals used were of analytical grade purchased from Rankam Chemical Co.

Strain procurement, culture development, and cultivation on nitrogen-enriched media

Spirulina (Arthrospira platensis) National Collection of Industrial Microorganisms (NCIM -5412) (on solid media) was procured from the National Facility for Marine Cyanobacteria, Bharathidasan University, Tiruchirappalli, India. The strain was previously maintained on Zarrouk’s agar media slants at 4°C. Loop full of spirulina culture was inoculated in a 50mL flask containing 10mL sterile standard medium (Modified Zarrouk’s Medium) under sterile condition. Sodium carbonate was added after autoclaving, and pH was adjusted to 8.8–9.0. Growth and maintenance of the culture was done in an illuminated (4500 lux) growth room at 30 ± 2°C under 12/12h light-dark cycles. Manual shaking of cultures was done periodically thrice every day. To study the effect of the inorganic nitrogen concentration on the growth of the strain NCIM-5412 in batch culture, a modified Zarrouk’s medium was used where the concentration of sodium nitrate (NaNO3) was 0.625, 1.250, 1.875, or 2.500 gL−1

Preparation of ethanol extracts

The cultivated sample was shade dried and finely powdered. 10g of powdered nitrogen-enriched sample (1) and the commercial sample obtained (2) were extracted using Soxhlet apparatus. The samples were placed in the thimble and air dried at room temperature for 18h. Extraction was performed for 3h using 100mL solvent (95% ethanol) and the filtrate was consumed for further analysis.

Phytochemical determination

Determination of total phenolics

The total phenolic contents in the extracts were determined spectrophotometrically at an absorbance of 765 nm according to Folin-Ciocalteu method.[8] Tannic acid was used to set up the standard curve. The content of phenolic compounds of the samples was expressed as tannic acid equivalents in mg per gram dry weight. All the samples were analyzed in triplicates.

Determination of total flavonoids

Estimation of the total flavonoids in the extracts was carried out using the method of Ordon-Ez et al.,[9] where 0.5mL of 2% AlCl3 ethanol solution was added to 0.5mL of sample. After one hour at room temperature, the absorbance was measured at 420 nm. A yellow color indicated the presence of flavonoid, and the contents were expressed as quercetin equivalents in mg per gram dry material. All the determinations were performed in triplicates, and the mean absorption along with the regression value was assayed.

Determination of antioxidant activity

Reducing ability

The antioxidant capacity of the extracts was estimated spectrophotometrically (ferric reducing antioxidant power [FRAP] assay) following the procedure of Benzie and Strain.[10] The method is based on the reduction of Fe3+ tripyridyl triazine (TPTZ) complex (colorless complex) to Fe2+ TPTZ (blue colored complex) formed by the action of electron donating antioxidants at low pH. This reaction is monitored by measuring the change in absorbance at 593 nm. FRAP reagent was prepared by mixing 300mM acetate buffer, 10mL TPTZ in 40mM HCl and 20mM FeCl3·6H2O in the proportion of 10:1:1 at 37°C. Freshly prepared working FRAP reagent was pipetted using 1–5mL variable micropipette (3.995mL) and mixed with 5 μL of the appropriately diluted plant sample and mixed thoroughly. An intense blue color complex was formed when ferric TPTZ (Fe3+ TPTZ) complex was reduced to ferrous (Fe2+) form and the absorbance at 593 nm was recorded against a reagent blank (3.995mL FRAP reagent + 5 μL distilled water) after 30min incubation at 37°C. All the determinations were performed in triplicates. The standard curve was linear between 200 and 1000 μM FeSO4. Results are expressed in μM Fe (II)/g dry mass and compared with that of butylated hydroxytoluene, ascorbic acid, quercetin, and catechin.

Statistical analysis

Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 17.0. Data obtained were expressed as mean ± standard deviation. One-way analysis of variance followed by multiple range test (Tukey post hoc test) was used to determine significant differences between the test groups and control. Differences at P < 0.05 were considered to be statistically significant.

Gas chromatography–mass spectrometry analysis

Gas chromatography–mass spectrometry (GC-MS) analysis of the samples was performed using a Perkin–Elmer Clarus 500 system comprising an AOC-20i auto-sampler. The GC is interfaced to a MS equipped with a column (Id: 250 µM) Elite-5MS (5% diphenyl/95% dimethyl poly siloxane) fused extended to a length of 30 m. For GC-MS detection, an electron ionization system was operated in electron impact mode with ionization energy of 70eV. Helium gas (99.999%) was used as a carrier gas at a constant flow rate of 1mL/min, and a sample injection volume of 1.6 μL was employed (a split ratio of 10:1). The inlet temperature was maintained at 280°C. The oven temperature was programmed initially at 60°C for 8 min, which further increased to 200°C for 5 min. This program extended at a rate of 20°C ending with 280°C for 15 min. Mass spectra were taken at 70eV at a scan interval of 0.5s and fragments from 45 to 450Da. The relative percentage amount of each component was calculated by comparing its average peak area to the total areas. The mass-detector used in this analysis was Turbo-Mass Gold-Perkin-Elmer, and the software adopted to handle mass spectra and chromatograms was a Turbo-Mass ver-5.2.

Identification of phytocomponents

Interpretation of mass-spectrum was conducted using the database of National Institute of Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown components was compared with the spectrum of known components stored in the NIST library. The name, molecular weight, and structure of the components of the test materials were ascertained

Molecular docking

Graphical-Automatic Drug Design System for Docking, Screening and Post-Analysis program iGEMDOCK was used to gain the docking results of the listed compounds with the target. The binding site of the target was prepared and the energy-minimized compounds were imported.

Preparation of ligands

The three-dimensional structures of the phytocompounds considered as ligands were retrieved and downloaded in structural data format and converted to Protein Data Bank (PDB) format using the online tool smile translator (cactus.nci.nih.gov/translate). The compound structures were energy minimized and considered for docking studies

Preparation of protein target

The target protein monoamine oxidase (MAO A) was retrieved from PDB. Water molecules were removed, and a single chain was selected between two chains. Generally, all waters (except those coordinated to metals) are deleted, but water that connects between the ligand and the protein is sometimes retained. Problems in the PDB protein structure were repaired by adjusting the protein, metal ions, and cofactors. The structure-forming bonds from the ligand or a cofactor to a protein metal were deleted by adjusting the ligand bond orders and formal charges. The minimization was done to restrain the input protein coordinates by a selected root mean square deviation tolerance.

The docking protocol consisted of 25 generations per ligand and the population size of 100 random individuals. All the docking conformations were performed twice using genetic evolutionary algorithm, and the fitness of the docked structures were calculated. The hydrophobic and electrostatic preferences were set to 1.00. The binding site of the target was identified at a distance 8 Å. The empirical scoring function of iGEMDOCK was estimated as: Fitness = vdW + H bond + Elec.

Here, the vdW term is van der Waal energy; H bond and Elec terms are hydrogen bonding energy and electro statistic energy, respectively.

Animal study

All experiments of this study were performed in accordance with relevant guidelines and regulations of the institutional Ethical Committee for the Purpose of Control and Suspension of Experiments on Animals (CPCSEA) under the Ministry of Animal Welfare Division, with an approval ID: BDU/IAEC/2014/NE/39. Female Albino mice (5–6 weeks old, 22 ± 2g) were housed in standard environmental conditions (12h light-dark cycle, 50–70% humidity, and 20–25°C). Food and water were provided ad libitum. Seven mice were used in each group to minimize the number of experimental animals needed, while ensuring the validity of statistical analyses.

Experimental protocol and animal exposure

The aim of the study protocol was to induce depression and assay the neuroprotective effect by administering ethanol extracts of samples 1 and 2. The animals were randomly divided into four groups assigned as positive control (depressive without treatment), negative control (normal), and experimental groups 1 (nitrogen enriched [sample 1]) and 2 (commercial [sample 2]). The animals in positive control grouped as 1 were administered orally (p.o.) with saline as vehicle (1mL of 0.9%). On injecting methyl isobutyl ketone (100 mg/kg weight, i.p.) the animals were depressed. This was regularly practiced for two weeks and proceeded by a treatment phase that sustained for a week. Eventually the animals were sacrificed for histopathological studies.

Brain histological assay

The mice were sacrificed by cervical dislocation 24h after the final day of treatment phase. The brains were removed, sectioned, and put in a fixative solution of 10% formalin at room temperature. The cortex region of the brain was sectioned and counterstained with alum hematoxyl. The slices were dehydrated with alcohol followed by xylene and processed for paraffin embedding as tissue blocks (mp 58–60.C). Transverse sections of 5-μm thickness were cut on a rotary microtome and were stained by Ehrlich’s hematoxylin embedded with eosin in alcohol. The histopathological assay was executed under microscopic examination (DM 4000B, Leica Microsystems, Mumbai).


  Results Top


Spirulina cultivation in nitrogen-enriched media and analysis of dry weight

Spirulina sp. NCIM-5412 was successfully cultured in the nitrogen-enriched modified media and its dry weight was determined. The dry weight of 0.23g/L was achieved and considered significant to endure further product recovery studies.

Total phenolic and flavonoids contents

The phytochemical assay of both the samples of spirulina (sample 1: cultivated in nitrogen-enriched media; sample 2: commercial powder) was performed to expose the significance of phytocompounds and their applications in drug development. Results obtained in the present study showed an increased intensity of phenolic compounds in the enriched cultivated sample when compared with the standard, whereas the components of flavonoids were substantial in both the samples as shown in [Table 1].
Table 1: Total phenolic and flavonoid contents of both the sample extracts

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Antioxidant assay

Reducing ability (FRAP)

The reducing ability of both the extracts was in the range of 626.60–668.28 µM (Fe (II)/g) as shown in [Table 2]. The antioxidant potentials of both the extracts were estimated from their ability to reduce Ferric tripyridyltriazine (FeIII -TPTZ) complex to (FeII -TPTZ).
Table 2: Antioxidant assay both the sample extracts

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GC-MS studies

GC-MS chromatogram analysis of the ethanolic extract of cultivated sample [Figure 2] extricated 28 different compounds [Table 3], which indicates the assortment of different phytoconstituents. The ethanolic extract of commercial sample [Figure 3] imparted 20 different compounds which were detected and catalogued in parallel to the NIST library as predicted in [Table 4]. Furthermore, these compounds were compared with the GC-MS spectrum of commercial sample. Eight compounds, namely, butanoic acid, glycerin, nonanal, 1, 4:3, 6-dianhydro-a-d-glucopyranose, phenol, 2, 4-bis(1,1-dimethyl), heptadecane, octadecanoic acid, and dodecanamide, were observed to be common in both the GC-MS profiles.
Figure 2: Pictorial representation of gas chromatography–mass spectrometry profile extricating phytocompounds from nitrogen-enriched cultivated sample of Arthrospira platensis

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,
Table 3: List of extricated phytocompounds from nitrogen-enriched cultivated sample

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,
Figure 3: Pictorial representation of gas chromatography–mass spectrometry profile extricating phytocompounds from commercial sample of Arthrospira platensis

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,
Table 4: List of extricated phytocompounds from commercial sample

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Molecular docking studies of the phytocompounds against the target proteins MAO A

An in silico assay was done to determine the best compound by docking against the depression-inducing proteins MAO A. The docking study was performed using the program Gem Dock considering the vital phytocompounds as ligands. The active site associated with the protein (MAO A) were studies that composed of amino acid residues, such as Tyr69, Tyr197, Phe208, Glu216, Tyr407, Phe352, and Tyr444.[11] The phytocompounds drawn in the study were used as ligands to target the mental illness–inducing proteins and therefore aspire the compounds as antidepressant agents. The assay was performed by relating 10 extricated compounds of both the samples from the GC-MS assay. These compounds were selected based on their resemblance with the extracted compounds of commercial sample, medicinal assets, and high retention time during extrication. The poses of all compounds are depicted in [Figure 4] and presented in [Table 5]. The predicted binding and docked energies are the sum of the intermolecular energy and the torsional free-energy penalty, and the docking ligand’s internal energy, respectively, and the inhibition constant (Ki) is calculated in the software as follows: Ki = exp (ΔG/(R*T). However, the top ranked pose was generated with the phytocompound butanoic acid, 3-hydroxy-, which was isoenergetic against the proteins with a minimum energy score of −56.24 kcal/mol, whereas dodecanamide was efficient to bind with the active site of the amino acid TYR 69 with a minimum energy of −87.8 kcal/mol.
Figure 4: Pictorial representation of the test compounds interaction with its targeted protein

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,
Table 5: Hits and docking score of phytocompounds against the targeted protein monoamine oxidase A

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Histopathological examination

Histological observations after administration of methyl isobutyl ketone showed sustained cell damage due to the oxygen exhaustion in the neural cells. The majority of cells in the nitrogen-enriched source group remained intact and stained homogeneously [Figure 5]. Although the commercial sample upshot a reduced damage when compared with negative control, cell gliosis was relatively lower in the nitrogen-enriched sample.
Figure 5: Histopathological examination in cerebral cortex regions of the (A) depression-incited mice showing cellular edema (CE), (B) depressionincited mice showing perivascular infiltration (PI) with increased gliosis (GL), (C) normal mice, and (D) treated mice with nitrogen-enriched ethanol extract of Anthrospira plantensis (x400)

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  Discussion Top


The neuroprotective capability of spirulina cultivated on nitrogen-enriched medium is endorsed in this study by an in silico assay substantiated with an in vivo analysis. Appearance of the cultivated culture was dark green in color when compared with the commercial sample that depicts the increasing cell mass. Furthermore, the influence of nitrogen in the media would increase the concentration of phenolic compounds. Miranda et al.[12] studied the antioxidant activity of carotenoids, phenolics, and tocopherols extracted from Spirulina maxima and found that the phenolic compounds are responsible for the antioxidant properties.

Therefore the study substantiates phenolic compounds as vital source in the mechanism of reduction among intermediate chain reaction. Polyphenolic compounds are known to have antioxidant activity and are sourced due to their redox properties, which play a significant role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides.[13] This infers an immense advantage in increasing the therapeutic efficacy against several human ailments.[14] The phytochemical studies endorse this by an elevated intensity in the presence of phenols when evaluated with the commercial sample, and this was reported by Parvin et al.[15] Hence this study attests Miranda et al.[12] and accounts the augmentation of phenol compounds in nitrogen-enriched medium that functions as a good antioxidant. The FRAP values for the ethanol extracts of both the samples were significantly lower than that of the standard source ascorbic acid and can be enhanced further with improved cultural conditions. The FRAP is a widely used technique that evaluates the antioxidant activity exhibited by the presence of phytocompounds. The flavonoids and phenolic compounds exhibit a strong potency of binding with the metal complex, particularly iron and copper. Hence the reducing capacity of the extracts is based on the principle of increase in the absorbance of the reaction mixtures, and as the absorbance increases, the antioxidant activity also increases.[16] GC-MS is an incredible technique used for the analysis of nonpolar and volatile components.[17]

The study slots in two samples namely, ethanolic extract of cultivated sample and commercial powder. The target protein MAO A was counteracted by 10 different phytocompounds efficiently through amino acid residues. Flexible docking of all data sets used for the computational study was carried out on the active site of the protein. The lowest energy and maximum number of conformations per cluster were set as the criteria to predict the binding modes of the compounds. Based on the procedure explained in the experimental section, the binding affinity of the docked molecules was evaluated by binding energy, docked energy, calculated inhibition constants (calc.), and hydrogen bonds in addition to the hydrophobic interactions at the channel pocket, where ∆G is the docking energy, Rcal is 1.98719, and TK is 298.[18],[19],[20] The synchronized neuroprotective effect of the vital phytoconstituents was further assayed by an in vivo study that included histopathological analysis. Persistent diminution in cerebral blood flow and brain energy metabolism causes progressive dysfunction of neurons, resulting in degenerative changes in glial cells. This damage was characterized by loose and disordered arrangements of cells and swelling deformations in cell shape resulting in shortening and disappearance of apical dendrites. The restraint in blood flow further cause microvascular constriction and glucose and oxygen delivery [21],[22] that can develop cellular edema and perivascular infiltration [Figure 2]. These are considered as neural deformity that symbolizes pathological condition due to the stress induced to the animals, which reduced cognitive abilities with an increase in anxiety. Subsequently, damage was much less than that seen in treated samples.


  Conclusion Top


This study has an optimistic result that concludes with the potential of spirulina (Anthrospira platensis) as an effective antioxidant stress reliever that can act as a neuroprotectant. The variation in the culture conditions with nitrogen-enriched media increases the biomass content, and the considerable total phenolic content substantiates the antioxidant efficacy. Furthermore, the GC/MS assay reveals the specific phytocompounds present in the plant sample. Eight phytocompounds were common to the commercial sample, and this depicts that there was no deleterious change in the extrication with ethanol. The docking study delivers a note that the compounds can act as monoamine inhibitors. Butanoic acid, 3-hydroxy- exhibited a minimum binding energy score of −56.24 kcal/mol and dodecanamide furnished a value of −87.8 kcal/mol in the targeted active site. The central nervous system has a high oxygen consumption and low antioxidant defense activity. This study bestows a blend of vital compounds whose interactions are substantiated by in silico assay, which is endorsed by an in vivo study that examined the restoration ability of oxidative stress caused by the brain damage. Conversely, imperative research can elevate these compounds as psychoactive drugs with preclinical studies that can target various other mental ailments and pathological conditions. Downstream processing, purification, and standardization would bring in superior standard and claim its demand commercially.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Fujisawa T, Narikawa R, Okamoto S, Ehira S, Yoshimura H, Suzuki I, et al. Genomic structure of an economically important cyanobacterium, arthrospira (spirulina) platensis NIES-39. DNA Res 2010;17:85-103.  Back to cited text no. 1
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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