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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 4  |  Issue : 4  |  Page : 305-311

Phytochemical and antioxidant assessments of Dioscorea bulbifera stem tuber


1 Department of Biochemistry, Faculty of Science, Madonna University, Rivers State, Nigeria
2 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Niger Delta University, Bayelsa, Nigeria

Date of Submission10-Jun-2020
Date of Acceptance27-Jun-2020
Date of Web Publication30-Dec-2020

Correspondence Address:
Dr. Othuke Bensandy Odeghe
Department of Biochemistry, Faculty of Science, Madonna University, Rivers State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_96_20

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  Abstract 


Background: Dioscorea bulbifera (D. bulbifera) is used in traditional medicine for the treatment of many disease conditions. However, there is a paucity of information on the antioxidant potential of its stem tuber. Methods: The antioxidant activity of the methanolic extract of D. bulbifera stem tuber was evaluated by measuring its ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide anion radical (O2-) and nitric oxide (NO) radical. It was also analyzed for bioactive secondary metabolites using standard qualitative and quantitative spectrophotometric methods. Results: The extract showed total phenolic content (0.243 ± 0.052 mg) gallic acid equivalent compared to tannins content (0.259 ± 0.034 mg). Low flavonoid contents (0.060 ± 0.025 mg) quercetin equivalent (QE) and high flavonol contents (1.399 ± 0.075 mg) QE were found in the extract. The extract showed a potent concentration-dependent DPPH radical inhibitory potential with maximal inhibition activity (69.39 ± 1.62%) at 5000 μg/mL compared to ascorbic acid with maximal inhibition activity (88.9 ± 2.67%) at the same concentration. The extract produced maximal O2- anion inhibitory activity (52.86 ± 0.68%) at 2500 μg/mL compared to quercetin with maximal inhibitory activity (68.23 ± 0.41%) at the same concentration. The extract exhibited NO activity in a dose-depend fashion when compared to α-tocopherol. Conclusion: These results showed that D. bulbifera stem tuber extract contains bioactive secondary metabolites with potent-free radical scavenging activity, which could be extracted and standardized for use as food, medicine, and in industries.

Keywords: Analyses, antioxidants, Dioscorea bulbifera, free radicals, phytochemicals


How to cite this article:
Odeghe OB, Adikwu E, Ojiego CC. Phytochemical and antioxidant assessments of Dioscorea bulbifera stem tuber. Biomed Biotechnol Res J 2020;4:305-11

How to cite this URL:
Odeghe OB, Adikwu E, Ojiego CC. Phytochemical and antioxidant assessments of Dioscorea bulbifera stem tuber. Biomed Biotechnol Res J [serial online] 2020 [cited 2021 Jan 22];4:305-11. Available from: https://www.bmbtrj.org/text.asp?2020/4/4/305/305650




  Introduction Top


Oxidative stress is initiated by free radicals, which seek stability through electron pairing with biological macromolecules such as proteins, lipids, and DNA in healthy human cells and causing biomolecular damage.[1] Antioxidants are essential substances that can retard or prevent the oxidation of biomolecules. They counteract the effect of free radicals thereby preventing oxidative damage.[2] Many medicinal plants have been extensively examined for their antioxidant activities in recent decades. Antioxidants from spicy, aromatic, medicinal, and other plants are used to produce natural antioxidant preparations for cosmetic, food, and other important applications.[3] The intake of natural antioxidants has been associated with lower risks of diseases such as cardiovascular diseases and cancer.[4] Most plants contain terpenoids, phenolic, and alkaloids.[5] Phenolic compounds can scavenge excess free radicals and effectively reduce oxidative stress in plants. Phenolics also protect protein, DNA and lipids in humans from oxidative damage. They have been successfully used as antioxidants by humans for the prevention and treatment of diseases. The search for new and safe antioxidants from plants is imperative for applications as antioxidants, foods, and medicine. Phytochemical screening is an essential and primary method experimentally used to search for antioxidant compounds in plants.[6]

Dioscorea bulbifera L (D. bulbifera) also known as air potato is a medicinal plant that belongs to the family Dioscoreaceae. It is one of the most widely consumed yam species. It can grow extremely quickly, roughly 8 inches/day, and eventually reach over 60 feet long. D. bulbifera which has a bitter-salty taste and a faint odor has higher nutritional values in relation to other species of Dioscorea.[7] Its leaves are used majorly as a staple food by hunters-gatherers living in areas such as Abayanda in Uganda and Pygmies in Central Africa.[8] D. bulbifera is used as a folk remedy to treat conjunctivitis, diarrhea, and dysentery.[9] Its preparation has been used for constipation, memory enhancement, fever, and anti-aging.[10] It is also used as an infusion for the treatment of sores and cuts due to its high tannin composition which can facilitate wound healing in an inflamed membrane.[7] In Madagascar and Cameroon, the pounded bulbils are used for the treatment of boils, abscesses, and wound infections.[11] It is commonly used in traditional Indian, Chinese, and African medicine for the treatment of sore throat,[12] type II diabetes mellitus[13] and breast cancer.[14] Despite the use of its stem tuber, there is a paucity of information on its antioxidant activity. This study, screened the methanolic extract of the stem tuber of D. bulbifera for phytochemical constituents and antioxidant activity. The finding in this study will add to knowledge, and the economic importance of D. bulbifera through standardization for use as food, medicine, and in industries.


  Methods Top


Materials

Plant material and plant selection: D. bulbifera stem tuber was classified, identified, and authenticated by a Chief Plant Taxononmist Mr. A. Ozioko in a reputable research laboratory known as International Centre for Ethnomedicine and Drug Development in Nsukka, Enugu State, Nigeria with a herbarium number of Intercedd/1580. It was selected for this study based on ethnomedicinal uses and the existing literature on its biological activities.

Chemicals

All the chemicals used in this study are of analytical grade. The solvents ethanol, ethyl acetate, and hexane were purchased from EMD Biosciences (Gibbstown, NJ). L-ascorbic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, sulfuric acid, sodium nitroprusside (SNP), sodium nitrite, Sulfanilamide, phosphoric acid, naphthylethylenediamine dihydrochloride, potassium dihydrogen phosphate, potassium hydroxide, acetic acid, gallic acid, quercetin, ferric chloride (FeCl3+), ethylenediaminetetraacetic acid (EDTA), phosphate buffered saline (PBS), sodium carbonate (Na2CO3), perchloric acid (HClO4), polyvinylpolypyrrolidone, riboflavin, ferrous sulphate (FeSO4.7H2O), hydrogen peroxide (H2O2), thiobarbituric acid, Folin–Ciocalteu's reagent (FCR), and trichloroacetic acid were all purchased from Sigma Chemical Co.(St. Louis, MO).

Preparation of extracts

D. bulbifera stem tuber was air-dried at room temperature and reduced to fine powder by milling. The powdered material was subjected to extraction with 80% methanol. The methanolic extract was concentrated using a water bath set at 45°C and then stored at 4°C until used. The extract yield was found to be 1.24%. It was dissolved in the appropriate solvent for the antioxidant assay.

Rapid 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay using dot-blot

Qualitative screening of D. bulbifera extract for anti-oxidant activity was performed using DPPH radical according to the method of Soler-Rivas et al.[15] with slight modifications by Awah et al., 2010.[16] Briefly, an aliquot (5 μL) of each dilution of the plant extract and standard anti-oxidant was carefully loaded on a piece of thin layer chromatography (TLC) plates (silica gel 60 F254, Merck) and allowed to air dry. The sheet was then sprayed with DPPH (0.2% (w/v) in methanol to reveal the anti-oxidant activity of the extract. The intensity of the yellow color and the rate at which the color of the extract spots changed from purple to yellow indicated the scavenging potential of the extract and its phenolic content.[17]

Qualitative phytochemical analyses

Tests for flavonoids, tannins, carbohydrates, glycosides, saponins, resins, terpenoids, and alkaloids were carried out using standard methods[18],[19] as described below:

Test for tannins

Pulverized sample (0.5 g) of each plant was boiled in 20 mL of distilled water in a test tube and then filtered with Whatman No. 1 filter paper. Then, 0.1% FeCl3 was added to the filtrate and observed for brownish green or a blue black coloration, which shows the presence of tannins.

Test for saponins

A quantity (0.5 g) of plant extract was boiled with distilled water (20 mL) in a water bath and filtered. Then, 10 mL of the filtered sample was mixed with 5 mL of distilled water in a test tube and shaken vigorously to obtain a stable persistent froth. The frothing was mixed with olive oil (3drops) and observed for emulsion formation, which indicates the presence of saponins.

Test for resins

Pulverized plant material (0.5 g) was extracted with 15 mL of 96% ethanol. The ethanol extract was then poured into 20 mL of distilled water in a beaker. A formation of resinous precipitate indicates the presence of resins. Furthermore, 0.12 g of the extract was extracted with chloroform and the extract concentrated to dryness. The residue was re-dissolved in 3 mL of acetone and 3 mL of HCl added. The mixture was heated in a water bath for 30 min. A pink color that changes to magnets red confirmed the presence of resins.

Test for alkaloids

To 0.5 g of pulverized plant material, 5 mL of 1% HCl was added and boiled for 5 min in a steam bath. This was filtered and 1 mL of the filtrate was treated with a few drops of Dragendorff's reagent, a second 1 mL portion was treated similarly with Wagner's reagent and a third portion with Mayer's reagent. The formation of red, reddish-brown, and creamy white precipitates respectively was an indication of the presence of alkaloids.

Test for glycosides

To 0.5 g of pulverized plant samples, 10 mL of distilled water was added and boiled for 5 min. It was filtered and the filtrate (2 mL) was hydrolyzed with some drops of concentrated HCl and rendered alkaline with some drops of ammonia solution. Five drops of this solution was mixed with 2 mL of Benedict's reagent and boiled. A reddish–brown precipitate shows the presence of glycosides.

Test for flavonoids

Pulverized plant samples (0.2 g) were heated with 10 mL of ethyl acetate in boiling water for 3 min. The mixture was filtered and 4 mL of the filtrate was shaken with 1 mL of 1% aluminum chloride solution and observed. A yellowish coloration in the ethyl acetate layer indicates the presence of flavonoids.

Test for steroids and terpenoids

To 9 mL of ethanol, was added 0.5 g of pulverized plant materials and refluxed for a few minutes. The filtrate was concentrated to 2.5 mL in a boiling water bath and 5 mL of hot water added. The mixture was allowed to stand for 1 h and the waxy matter filtered off. The filtrate was extracted with 2.5 mL of chloroform using separating funnel. To 0.5 mL of the chloroform extract in a test tube was added 1 mL of concentrated sulfuric acid to form a lower layer. A reddish brown interface shows the presence of steroids. Another 0.5 mL of the chloroform extract was evaporated to dryness on a water bath and heated with 3 mL of concentrated sulfuric acid for 10 min on a water bath. A gray color indicates the presence of terpenoids.

Test for proteins

Distilled water (5 mL) was added to 0.1 g of the pulverized samples and left to stand for 3 h and then filtered. To 2 mL portion of the filtrate was added 0.1 mL of Million's reagent, shaken and kept for observation. The formation of yellow precipitate shows the presence of proteins.

Carbohydrates

To 2 mL of the sample in a test tube, 2 drops of α-naphthol solution was added then 1 mL of conc. H2SO4 was carefully poured down the sides of the tube to form two layers. A color change to brown at the junction of the two layers indicates the presence of carbohydrates.

Quantitative phytochemical analyses

Determination of total phenolic contents

Total phenolics were determined using Folin-Ciocalteau Reagent (FCR) as described by Velioglu et al., (1998),[20] with slight modifications according to Awah et al., 2012a.[21] FCR is a yellow acidic solution that has complex polymeric ions formed from phosphotungstic heteropoly and phosphomolybdic acids. The disintegration of a phenolic proton in a basic medium forms a phenolate anion that reduces FCR thereby forming a blue-colored molybdenum oxide whose color intensity is directly proportional to the phenolic contents. Briefly, the extract (100 μL) was dissolved in methanol (1 mg/mL) and mixed with Folin–Ciocalteu reagent (750 μL) and allowed to stand at 22°C for 5 min. Na2CO3 (750 μL) solution was then added to the mixture. Absorbance was measured at 725 nm after 90 min. Results were expressed as gallic acid equivalents.

Determination of tannin contents

Tannin content was measured using insoluble polyvinyl-polypyrrolidone (PVPP), which binds tannins as described by Makker et al.[22] The extract (1 mL) was dissolved in methanol (1 mg/mL) and the total phenolic content was determined. It was mixed with 100 PVPP (100 mg) for 15 min at 4°C and centrifuged (3000 rpm for 10 min). The clear supernatant was collected and the nontannin phenolics were determined same as the total phenolics. Tannin content was obtained from the difference between total and nontannin phenolic content.

Determination of flavonoids

The flavonoids content was determined according to the method described by Kumaran and KarunakaWran (2006)[23] with slight modifications according to Awah et al., 2010a.[24] This is based on the principle of the formation of a flavonoid-aluminum complex that has a maximum absorbance at 415 nm. The plant extract (100 μl) in methanol (10 mg/mL) was mixed with 100 μl of 20% aluminum trichloride in methanol. A few drops of acetic acid were added and diluted with methanol to 5 mL. After 40 min the absorption was read at 415 nm. Blank samples were prepared from 100 μl of plant extracts and a drop of acetic acid, and then diluted to 5 mL with methanol. The absorption of standard quercetin solution (0.5 mg/mL) in methanol was measured under the same conditions. The amount of flavonoids in the plant extract in quercetin equivalents (QE) was calculated by the following formula:



where Ao is the absorption of standard quercetin solution, A is the absorption of plant the extract, m is the weight of plant extract, and mo is the weight of quercetin in the solution. The flavonoid content obtained is expressed in mg quercetin equivalents (QE)/mg plant extract.

Determination of flavonols

The content of flavonol was also determined as described by Kumaran and Karunakaran[22] with slight modifications by Awah et al., 2010b.[24] Briefly, 1 mL of each methanolic plant extracts (10 mg/mL) was mixed with 1 ml of aluminum trichloride (20 mg/mL) and 3 mL of sodium acetate (50 mg/mL). The absorbance at 440 nm was read after 2.5 h. The absorption of standard quercetin solution (0.5 mg/mL) in methanol was also measured under the same condition. The amount of flavonols in the plant extract in quercetin equivalents (QE) was calculated using the same formula with flavonoids.

In-vitro anti-oxidant assays

2,2-diphenyl-1-picrylhydrazyl radical-scavenging assay using thin-layer chromatography

Qualitative screening for anti-oxidant activity was done using the DPPH radical according to the method of Takao et al.[25] Briefly, a thin layer chromatogram of the extract on silica gel plates (Merck) was developed using methanol–ethyl acetate (50:50, v/v) as mobile phase. DPPH radical test was performed directly on TLC plates by spraying with DPPH (0.2% (w/v) in methanol to reveal the anti-oxidant activity of the extract.

Quantitative DPPH radical-scavenging assay

Scavenging activity on DPPH free radicals by the extract was assessed according to the method reported by Gyamfi et al.[26] Briefly, a 2.0 mL solution of the extract at different concentrations diluted two-fold (2–250 μg/ml) in methanol was mixed with 1.0 mL of 0.3 mM DPPH in methanol. The mixture was shaken and allowed at room temperature in a dark place for 25 min. Blank solutions were produced with each test sample solution (2.0 mL) and 1.0 mL of methanol. The negative control contained 1.0 ml of 0.3 mM DPPH solution plus 2.0 mL of methanol. L-ascorbic acid was used as the positive control. Thereafter, at 518 nm, the absorbance of the assay mixture was measured against each blank (Agilent 8453E ultraviolet-visible spectrophotometer). Lower absorbance of the mixture indicates higher radical scavenging activity. DPPH radical scavenging activity was calculated using the equation:



where As is the absorbance of the test sample and A0 is the absorbance of the control. The IC50 value showed the concentration of the extract, which produced 50% inhibition of DPPH radical. It was calculated using linear regression of plots, where the abscissa represented the concentration of tested sample and the ordinate the average percent of inhibitory activity from three replicates.

Superoxide radical-scavenging assay

This assay is based on the ability of the extract to prevent the photochemical reduction of nitro blue tetrazolium (NBT)[27] as described by Martinez (2001).[28] Each 3.0 mL reaction mixture contained 0.05 M PBS, 2 μM riboflavin, 13 mM methionine, 100 μM EDTA, NBT (75 μM), and 1.0 ml of test sample solutions (10-250 μg/mL). The tubes were held in front of a fluorescent light (725 lumens and 34 watts) and after 20 min absorbance were read at 560 nm. The entire reaction assembly was placed in a box covered with aluminum foil. Identical tubes containing reaction mixtures were kept in the dark and served as blanks. The percentage inhibition of superoxide generation was estimated by comparing the absorbance of the reaction mixture containing test sample to the control using the equation below:





where A0 is the absorbance of the control, and As is the absorbance of the tested sample.

Nitric oxide radical scavenging assay

Nitric oxide (NO) generated from SNP was measured according to the method of? Marcocci et al. (2005).[29] The reaction mixture (5.0 mL) containing SNP (5 mM) buffer (pH 7.3), with and without the plant extract was incubated at 25°C for 180 min in front of a visible polychromatic light source (25 W tungsten lamp). The NO radical thus generated interacted with oxygen to produce the nitrite ion (NO2) which was assayed at 30 min intervals by mixing 1.0 ml of incubation mixture with an equal amount of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromophore (purple azo dye) formed during the diazotization of nitrite ions with sulfanilamide and subsequent coupling with naphthylethylenediamine dihydrochloride was measured at 546 nm. The nitrite generated in the presence or absence of the plant extract was estimated using a standard curve based on sodium nitrite solutions of known concentrations. Each experiment was carried out at least three times and the data presented as an average of three independent determinations.

Statistical analysis

Data were analyzed and graphs plotted using? Microsoft excel 2010 for Windows 8 (Microsoft Corporation, Redmond, Washington, USA). All results are expressed as mean ± standard error of mean.


  Results Top


Dot blot for 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity by thin layer chromatography

D. bulbifera extract showed significant scavenging of DPPH radical by changing the extract spots from purple to yellow. The faster the color of the spot changes to yellow and the intensity of the yellow spot indicate higher anti-oxidant activity [Figure 1].
Figure 1: Dot blot of radical scavenging capacity of Dioscorea bulbifera extract

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Qualitative and quantitative analyses of phytochemical constituents

Qualitative analysis of D. bulbifera extract shows the presence of important phytochemical constituents as summarized in [Table 1]. Phenolic (tannins and flavonoids), saponnin, resins, and alkaloids were the major phytochemical constituents present in the plant in relatively high amount. Quantitative analysis of D. bulbifera extract shows the highest level of nontannins than tannins and phenols. In addition, the extract has high flavonols content than flavonoid content [Table 1] and [Table 2].
Table 1: Phytochemical constituents in Dioscorea bulbifera extract

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Table 2: Quantitative phytochemical constituents

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Effect of extract on 2,2-diphenyl-1-picrylhydrazyl radicals

D. bulbifera shows significant dose-dependent DPPH radical scavenging capacity [Table 3]. D. bulbifera was efficient at the concentration of 500 μg/mL maximally inhibiting 69.39 ± 1.62% of the DPPH radical compared to ascorbic acid which maximally inhibited 88.9 ± 2.67%. The IC50 values for DPPH radical inhibition was 261.9 μg/mL compared to ascorbate which produced 1 10.69 μg/mL [Table 3].
Table 3: 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging activity of extracts compared to ascorbic acid

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Effects of extract on superoxide anion and nitric oxide radical

D. bulbifera inhibited the formation of superoxide anion (O2-) radical from reduced NBT in a dose-related manner [Table 4]. Maximal O2- anion inhibitory activity of 52.86 ± 0.68% at 250 μg/ml of the extract occurred when compared to quercetin which produced 68.23 ± 0.41% inhibition at the same concentration. This study shows that the extract in SNP solution decreased the level of nitrite, a stable oxidation product of NO liberated from SNP in a dose-related fashion [Figure 2]. The extract exhibited strong NO radical scavenging activity leading to the reduction of nitrite concentration in the assay medium. Comparatively, the extract produced dose-dependent scavenging of NO radical than quercetin [Figure 2].
Table 4: Superoxide anion radical scavenging activity of Dioscorea bulbifera compared to quercetin

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Figure 2: Effect of Dioscorea bulbifera on the accumulation of nitrite upon decomposition of SNP compared to α-tocopherol

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  Discussion Top


Plants are important sources of medicines from the inception of human civilization and are still the major sources of medicines used in modern and traditional systems of medicine.[30] Medicines from plant sources are due to the presence of phytochemicals which are biologically active chemical substances that work with nutrients and dietary fiber to protect against diseases. Depending on the origin, phytochemicals have several categories such as phenolics, alkaloids, steroids, saponins, and terpenes. Because of the presence of phytochemicals there is increase research attention on plants for medicinal purpose.[31] The phytochemical screening of D. bulbifera extract shows the presence of essential phytochemicals which include tannins, alkaloids, resins, glycosides and saponins. These phytochemicals are important bioactive agents which showed that D. bulbifera extract has potential therapeutic value. Alkaloid has been shown to have antimalarial effect whereas flavonoids have been reported to have anti-inflammatory, anti-tumor and antioxidant activities.[32] On the other hand, tannins are regarded as “health-promoting” components in beverages and food of plant origin. Tannins have been shown to have antimutagenic, anticarcinogenic and antimicrobial properties. Several studies have reported the antioxidant and antiradical activities of tannins.[33] The quantitative assay of D. bulbifera extract shows the presence of phenolics in sizeable quantity. Phenolic compounds are antioxidants; the antioxidant activity of phenols is mainly due to their redox properties, donation of hydrogen and the ability to quench singlet oxygen activity.[34] Dietary antioxidants can prevent food from lipid peroxidation and increase antioxidant intake in humans. Plant antioxidants can have a wide variety of activities including inhibition of oxidizing enzymes, increase enzymatic detoxification of reactive oxygen species, chelation of transition metals, singlet oxygen deactivation, and transfer of hydrogen or a single electron to radicals.[35]

The DPPH method was introduced by Blois, 1958,[36] about 50 years ago and it is acceptable and widely used for the assessments of compounds that have potential to scavenge-free radicals or donated hydrogen and to assess antioxidant capacity. In this study, D bulbifera shows significant dose-dependent DPPH radical scavenging capacity and produced higher effect when compared to ascorbate at the same concentration. In addition, D. bulbifera extract inhibited the formation of O2- radical from reduced NBT in a dose-related fashion. The extract exhibited strong NO radical scavenging activity leading to the reduction of NO concentration in the assay medium. Comparatively, the extract produced dose-dependent scavenging of NO radical than α-tocopherol. This shows that D. bulbifera extract has potential antioxidant effect which can be attributed to the presence of phenolic substances. D. bulbifera may be suitable for the treatment of cardiovascular diseases, cancer and others diseases, which have been associated with free radicals.


  Conclusion Top


Based on the findings in this study, D. bulbifera stem tuber extract contains bioactive secondary metabolites with potent free radical scavenging activity. It is recommended that further investigation on the isolation and characterization of the antioxidant constituents be done.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors declare no conflicts of interest.



 
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    Figures

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    Tables

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



 

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