الفهرس 
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Abstract
Family Fabaceae (Leguminosae, Legume, Pea, Or Bean Family) Is The Second Largest Family Of Flowering Plants Containing About 700 Genera And 20,000 Species.
Plants Of Family Fabaceae Are Growing In Many Different Environments And Climates Throughout The World. The Family Includes More Important Drugs Than Any Other Family.
The Studied Plant Sprouts Included:
Cicer Arietinum L. [Chickpea].
Lens Esculenta L. [Lentil].
Trigonella Foenum-Greacum L. [Fenugreek].
Vicia Faba L. [Faba Bean].
Literature Survey On Legume Sprouts
The Different Phytoconstituents Reported In The Several Studied Legume Sprouts, As Well As The Reported Biological Activities And Taxonomical Features Of The Family “Fabaceae” And The Genera, I.E., Cicer, Lens, Trigonella, And Vicia Have Been Reviewed.
Part I: Phytochemical Investigation
Chapter 1: Legume Sprouts UPLC-MS Profiling
The Aim Of This Work Was Planned To Define And Compare The Phytochemical Composition Of The Different Legume Sprouts, I.E., Cicer Arietinum, Lens Esculenta, Trigonella Foenum-Greacum, And Vicia Faba Using UPLC-PDA-ESI-Qtof-Msn. Extracts Were Analyzed In Positive And Negative Electrospray Ion (ESI) Modes To Provide A Greater Coverage Of The Metabolome. The Approach Allowed For The Tentative Identification Of More Than 300 Metabolites. The Identified Metabolites Belonged To Different Classes (Hydroxycinnamic And Hydroxybenzoic Acids, Flavonoids Viz., Flavones, Flavonols, Isoflavones, Flavanones And Dihydroflavonol, Coumestans, Saponins Viz., Triterpenoid/Steroidal, Fatty Acids, And Sphingolipids).
Unsupervised PCA Data Analysis Of Sprouts Metabolites Profile Via UPLC-MS
UPLC-MS Data Set Obtained In Negative And Positive Ionization Modes Of The Four Studied Sprouts Were Subjected To PCA Analysis. In Negative Mode, Three Major Distinct Clusters Were Formed, Corresponding To The 4 Studied Legume Sprouts (Cicer, Lens, Trigonella, And Vicia). The First Corresponded To The MS Signals For Isoflavones, Contributing Negatively To PC1 And Found More Enriched In Cicer. The Second, from MS Signals Assigned For Flavone-C-Glycosides Having A Positive Effect On PC1 And Found Abundant In Trigonella. Metabolites Showing Less Influential Loading Score Emanated from MS Signals Of Oxylipids Having A Positive Effect On PC1 And Found More Abundant In Both Vicia And Lens.
The Same Segregation Pattern Was Obtained In Positive Ionization Mode PCA Analysis. Segregation Obtained from Positive Mode Data Showed That Trigonella Was Enriched In N-Octadecanoyl-2-Methylamino Acetic Acid And Flavone-C-Glycosides, Whereas Lens And Vicia Were More Abundant In Unknown Terpenes And Contributing To Their Discrimination from Trigonella.
Chapter 2: Legume Sprouts Primary Metabolites GC-MS Analysis
Legume Sprouts GC-MS Analysis Led To The Identification Of 78 Metabolites Including Alcohols (2), Amino Acids (21), Aromatics (1), Fatty Acids (3), Inorganic Acids (1), Nitrogenous Compounds (5), Organic Acids (16), Steroids (1) And Sugars (28). Sugars Amounted For The Largest Percentile Levels Ca. 82-89% Of Detected Metabolites. Amino Acids Were Detected At 4-8% Of The Total Primary Metabolites, Whereas Fatty Acids Were Detected Though At Trace Levels.
Unsupervised Multivariate PCA Data Analysis Of Sprouts Primary Metabolite Profiles Using GC-MS
PCA Analysis Of Legume Sprouts GC-MS Data Failed To Classify Sprouts According To Their Primary Metabolite Profiles, Owing To The Similarity Between The Studied Sprouts Profiles Except For Slight Variation In Cicer Sprouts With Different Sugar Profile Than Other Sprouts.
Chapter 3: NMR Fingerprinting Of Legume Sprouts
Nuclear Magnetic Resonance (NMR) Technique Was Utilized For Metabolites Fingerprinting Of The Legume Sprouts To Be Exploited In Their Quality Control Purposes And For Future Incorporation In Nutraceuticals. 32 Metabolites Were Identified Belonging To Different Classes, I.E., Fatty Acids, Sugars, Amino Acids, Nucleobases, Organic Acids, Sterols, Alkaloids, And Isoflavonoids. Isoflavones Were Detected Exclusively In Cicer Sprouts, Whereas Trigonella Sprouts Were characterized By 4-Hydroxyisoleucine. Vicia Sprouts Were Distinguished from Other Sprouts By The Presence Of L-Dopa. Trigonelline Alkaloid Was Detected In All Sprouts And Suggestive For Its Role In Sprouts Germination.
Quantification Of Major Metabolites Detected Via 1H-NMR
Quantitative NMR Was Employed For Assessing The Major Identified Metabolite Levels For Future Standardization Purposes. A Single Well-Resolved Signal In Each Identified Metabolite Was Used For The Unbiased Absolute Quantification In Sprout Samples. The Concentrations Were Expressed As µg/Mg Dry Powder In The Different Legume Sprout Samples.
1H-NMR Data Multivariate Data Analyses
Unsupervised Multivariate PCA Analysis Of Full Range 1H-NMR Data
Three Major Distinct Clusters Were Formed, Corresponding To The 4 Studied Legume Sprouts (Cicer, Lens, Trigonella, And Vicia). The First Corresponded To The 1H-NMR Signals For Isoflavones, Contributing Positively To PC1 And Found Exclusively In Cicer. The Second, from 1H-NMR Signals Assigned To Asparagine And 4-Hydroxy-Isoleucine Having A Negative Effect On PC1 And Abundant In Trigonella. Metabolites Showing Less Influential Loading Score Emanated from 1H-NMR Signals Of L-Dopa Which Found Exclusive In Vicia Sprouts And To Exhibit A Negative Effect On PC1 And A Positive One On PC2.
Unsupervised Multivariate PCA Analysis Of The Aromatic 1H-NMR Region Data
This Model Showed The Same Classification Pattern As The Full Range 1H-NMR Dataset With Higher Classification Power. Three Major Distinct Clusters Are Formed, Corresponding To The 4 Studied Legume Sprouts (Cicer, Lens, Trigonella, And Vicia). The First Corresponded To 1H-NMR Signals For Isoflavones, Contributing Negatively To PC1 And Found Exclusively In Cicer. The Second, from 1H-NMR Signals Were Assigned To L-Dopa Having A Positive Effect On PC1 And Abundant In Vicia. Metabolites Showing Less Influential Loading Score Emanated from The 1H-NMR Signals Of Tryptophan Found In Lens And Trigonella Sprouts And To Exhibit A Positive Effect On PC1 And A Negative One On PC2.
Supervised Multivariate OPLS-DA Analysis Of 1H-NMR Data
Both Full And Aromatic 1H-NMR Based PCA Analyses, Legume Metabolite Markers Were Further Confirmed By Constructing Several Supervised OPLS-DA Analyses. OPLS-DA Is More Powerful In The Identification Of Markers By Providing The Most Relevant Variables For The Differentiation Between Two Sample Groups. Firstly, Cicer Samples Were Modelled Against Other Sprout Samples And Analyzed Using Both 1H-NMR Full And Aromatic Region Data. The Results Confirmed The Unique Metabolic Profile Of Cicer, I.E., Isoflavonoids And Sucrose.
Trigonella Sprouts Were Modelled Against Other Sprout Samples And Analyzed Using Both 1H-NMR Full And Aromatic Region Dataset To Confirm Their Metabolic Markers. The Study Confirmed That The Trigonella Samples Exclusively Contained 4-Hydroxy-Isoleucine In Addition To Being The Richest In Trigonelline Alkaloid.
Vicia And Lens Full And Aromatic Region Data Were Modelled Against Each Other Using OPLS-DA Showing A Clear Separation Between Both Sprout Samples. Vicia Was Particularly Rich In Sugars Aside from The Exclusive Presence Of L-Dopa.
Part II: Biological Study
Chapter 1: In Vitro Antioxidant Assay
1. Antioxidant Activity Using 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Assay
Different Sprouts Methanol Extracts Ability To Scavenge 2,2-Diphenyl-1-Picrylhdrazyl (DPPH) Free Radical Was Evaluated. Values Were Expressed As IC50 (Concentration Which Causes 50% Inhibition Of DPPH Free Radical).
Vicia Sprout Extract Was The Most Effective In Inhibiting DPPH (IC50, 0.46 Mg/Ml), Whereas Cicer Sprout Extract Was The Least Effective With An IC50 Value Of 10.28 Mg/Ml.
2. Antioxidant Activity Using 2,2-Azinobis (3-Ethyl-Benzothiazoline-6-Sulfonic Acid (ABTS)
Different Legume Sprout Methanol Extracts Showed A Comparable Antioxidant Activity On 2,2-Azinobis (3-Ethyl-Benzothiazoline-6-Sulfonic Acid (ABTS) Free Radicals. Vicia Sprouts Showed The Highest Inhibition Of ABTS Free Radicals (IC50, 0.79 Mg/Ml). Trigonella Sprouts Showed Moderate ABTS Radicals Scavenging Activity (IC50, 4.88 Mg/Ml), Whereas Cicer And Lens Sprouts Were The Least Effective With An IC50 Value Of 9.33 And 8.40 Mg/Ml, Respectively.
3. Quantitative Estimation Of Total Flavonoid Content Using Alcl3 Colorimetric Assay
Different Legume Sprouts Total Flavonoid Contents Were Measured Using Aluminum Chloride Colorimetric Assay. Total Flavonoid Concentration Was Expressed As Mg Rutin Equivalent/G Dried Sprout Methanol Extract (Mg RE G-1 Extract). Vicia Sprouts Were The Richest In Total Flavonoids, Whereas Lens Showed The Lowest TF Content.
4. Antioxidant Effect Of Sprouts In Relation To Their Total Flavonoid (TF) Content
Both DPPH And ABTS Antioxidant Assays Showed Significant Correlations With Sprouts Flavonoid Contents, Suggestive Sprouts Antioxidant Activity Is Mostly Related To Their Total Flavonoid Contents. These Results Agree With Reports On Flavonoids As Direct Radical Scavenger Antioxidants.
Chapter 2: In Vivo Hypoglycemic And Hepatoprotective Activities Evaluation
1. Effect Of Different Treatments On Blood Glucose Level Of Diabetic Rats
Post Diabetic Induction By STZ, Significant Increase In Blood Glucose Level Of Diabetic Rats Left For Three Days, This Increase Amounted For 435.12% As Compared To The Control Group.
Treatment Of STZ-Diabetic Rats With Legume Sprout Methanol Extracts, I.E., Cicer, Lens, Trigonella, And Vicia Recorded Significant Decrease By 23.60, 36.28, 26.73, And 65.03 %, Respectively As Compared To Diabetic Group. Blood Glucose Level Showed A Significant Inverse Correlation With Legume Sprouts Flavonoid Content.
Vicia Sprouts Treatment Showed The Highest Decrease In Blood Glucose Level, Likely Due To Its Richness In Antioxidant Flavonoids. The Results Suggested That The Antidiabetic Effect Of Legume Sprouts Was Due Flavonoids Α-Glucosidase Inhibition And Pancreatic Cells Protection Against STZ-Induced Damage.
2. Effect Of Different Drugs On Liver Oxidative Stress Markers Of Diabetic Rats
2.1. Determination Of Superoxide Dismutase (SOD) Activity
Superoxide Dismutase (SOD) Activity Was Significantly Decreased In Liver Tissue Of STZ-Diabetic Rats Amounting For 65.79% As Compared With Control Group. Treatment Of STZ-Diabetic Rats With Legume Sprout Methanol Extracts, I.E., Cicer, Lens, Trigonella, And Vicia Showed Significant Elevation In SOD Activity At 27.52, 59.79, 21.95, And 80.82%, Respectively Compared To The Diabetic Group.
2.2. Determination Of Glutathione (GSH) Level
The Glutathione (GSH) Levels In STZ-Diabetic Rats Showed Significant Decrease By 64.60% As Compared With The Control Group. The STZ-Diabetic Rats Treated With Cicer, Lens, Trigonella, And Vicia Showed Significant Increase By 38.04, 33.15, 11.59, And 71.73% As Compared To The Diabetic Group, Respectively.
2.3. Determination Of Malondialdehyde (MDA) Level
A Significant Increase In Malondialdehyde (MDA) Level Amounting For 173.45% Was Showed After STZ Induction, Compared To The Control Group. The Diabetic Rats Treated With Cicer, Lens, Trigonella, And Vicia Recorded Significant Decrease In MDA Level By 34.62, 48.86, 44.98, And 52.75%, Respectively As Compared With The Diabetic Group.
3. Hepatoprotective Activity
3.1. Determination Of Liver Function Enzymes
3.1.1. Serum Alanine Aminotransferase (ALT) Level
A Significant Increase In Alanine Aminotransferase (ALT) Of Diabetic group Amounted By 52.70%, As Compared To The Control Group. Treatment Of Diabetic Rats With Cicer, Lens, Trigonella, And Vicia Showed Decrease In ALT By 15.92, 23.00, 14.60, And 23.89%, Respectively As Compared To Diabetic Group.
3.1.2. Serum Aspartate Aminotransferase (AST) Level
Aspartate Aminotransferase (AST) Recorded Significant Increase In Diabetic group Amounted By 62.50%, As Compared To The Control Group. Treatment With Cicer, Lens, Trigonella, And Vicia Recorded Decrease In AST By 3.84, 12.63, 8.79, And 14.83%, Respectively As Compared To Diabetic Group.
3.1.3. Serum Aspartate Aminotransferase (AST) Level
Alkaline Phosphatase (ALP) Recorded Significant Increase In Diabetic group Amounted By 67.58%, As Compared To The Control Group. Diabetic Rats Treated With Cicer, Lens, Trigonella, And Vicia Recorded Significant Decrease In ALP By 25.25, 23.89, 26.37, 35.69%, Respectively As Compared To Diabetic Group.
4. Determination Of Lipid Profile
4.1. Determination Of Cholesterol
Cholesterol Level Showed Marked Increase In Diabetic Rats By 506.65% As Compared To Control Group. Treatment With Cicer, Lens, Trigonella, And Vicia Recorded Significant Decrease In Cholesterol Level By 78.55, 71.18, 47.67, And 79.09%, Respectively Comparing With The Diabetic Group.
4.2. Determination Of High-Density Lipoprotein - Cholesterol (HDL-C)
A Significant Decrease Was Noticed In HDL Level In Diabetic Rats By 54.81% As Compared To The Control Group. Treatment With Cicer, Lens, Trigonella, And Vicia Produce Significant Increase In HDL Level Reached To 17.41, 17.75, 28.91, And 19.38%, Respectively As Compared To The Diabetic Group.
4.3. Determination Of Low-Density Lipoprotein - Cholesterol (LDL-C)
The Results Recorded Significant Increase In LDL-C Level In Diabetic Rats By 208.54% As Compared With The Control Group. Treatment With Cicer, Lens, Trigonella, And Vicia Recorded Significant Decrease In LDL-C Level Amounted To 187.99, 168.24, 60.92, 54.52, 58.62, And 66.07%, Respectively As Compared To The Diabetic Group.
4.4. Determination Of Triglycerides
The Results Recorded Significant Increase In Triglycerides Level In Diabetic Rats Amounted To 107.56% As Compared With The Control Group. Treatment With Cicer, Lens, Trigonella, And Vicia Recorded Significant Decrease In Triglyceride Level Amounted To 48.29, 36.02, 36.65, And 46.62% Respectively As Compared To The Diabetic Group.
5. Histopathological Analysis
All Studied Legume Sprouts, I.E., Cicer, Lens, Trigonella, And Vicia Showed Preserved Lobular Hepatic Architecture, Normal Morphological Appearance, Improvement In Steatosis, And Mild Interlobular Inflammation Compared To The Diabetic Group. Vicia Sprouts Treatments Showed The Most Improvement In Liver Architecture With Vacuolization.
6. In Vivo Antihyperglycemic Activity Of Sprouts In Relation To Their Total Flavonoid (TF) Content
The Studied Legume Sprouts Showed A Comparable Hypoglycemic Action. The Antidiabetic Activity Is Significantly Correlated To Their Total Flavonoid Contents. The Main Mechanism Of The Antidiabetic Action May Be The Inhibition Of Carbohydrates Digestive Enzymes By Flavonoids.
The Antihyperglycemic Activity Of Sprouts Extract Was Associated With A Significant Improvment In The Hepatic Function And Architecture In STZ-Diabetic Rats. An Amelioration In The Oxidative Stress And The Dyslipidemia Associated With Hyperglycemia Was Observed.
Results Suggested That Legume Sprout Methanol Extract Especially Vicia Exhibits Insulin-Like Action To Correct Metabolic Alterations With Powerful Hepatoprotective Action And Has A Potential For Future Clinical Applications.