الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 281 |  |  |
| | | from | 281 | | |
Abstract
Serenoa Repens W. Bartram Is The Sole Representative Of Serenoa (Arecaceae, Subfamily Coryphoideae, Tribe Coryphae, And Subtribe Livistoninae). It Is Distributed In The West India And The Southeastern Part Of The United States, Especially In South Carolina, Florida, And Southern California.
The Broad Spectrum Of The Traditional Uses Of Serenoa Repens Makes It Interesting To Study This Plant.
This Study Includes:
Genetic Profiling Of The Plant.
Chemical Study Of The Fruits Of Serenoa Repens W. Bartram Including; A Preliminary Phytochemical Screening, Investigation Of The Volatile Constituents, Study Of The Lipoid Content (Saponifiable And Unsaponifiable Matters), Qualitative And Quantitative Determination Of Phenolic And Flavonoid Compounds, HPLC/MS-MS Study Of Serenoa Repens W. Bartram Compounds, Investigation Of The Fruits Fractions (Methylene Chloride, Ethyl Acetate And N-Butanol).
Biological Study Of Different Extracts Of Serenoa Repens W. Bartram Including; Pharmacological Study (Anti-Inflammatory, Anti-Oxidant, Anti-Hyperglycemic, Anti-Peptic Ulcer And Hepatoprotective Activities), Cytotoxic And Antimicrobial Activities.
Part I: Genetic Study Of Serenoa Repens By DNA Fingerprinting
The RAPD Electrophoretic Profile Of The DNA Sample Of Serenoa Repens W. Bartram Amplified With The Ten Random Primers Showed Distinguishable Bands Generating 120 Fragments Pattern.
Part II: Phytochemical Study Of Serenoa Repens W. Bartram.
1. Preliminary Phytochemical Screening Of The Fruits Of Serenoa Repens W. Bartram
The Results Of Phytochemical Screening Showed The Presence Of Carbohydrates And/Or Glycosides, Sterols And/Or Triterpenes, Flavonoids And/Or Phenolic Acids, Volatile Components, Tannins. Absence Of Crystalline Sublimates, Cardiac Glycosides, Free And Combined Anthraquinones, Saponins, Oxidase Enzyme And Alkaloids And/Or Nitrogenous Bases In Serenoa Repens W. Bartram.
2. Investigation Of The Volatile Constituents Of The Fruits Of Serenoa Repens W. Bartram
The GC/MS Analysis Of The Hydrodistillated Volatile Constituents from The Fruits Of Serenoa Repens W. Bartram Originated from India Result In Identification Of 60 Compounds Including (Monoterpene And Sesquiterpene) Hydrocarbons And Oxygenated Compounds. Approximately 76.1% Of The Hydrodistillate Oil Was Oxygenated Compounds (15 Compounds), Of Which About 12 Monoterpens where 4-(1-Methylethyl)-Benzaldehyde (58.56%) Was Found To Be The Major Constituent Of The Oil Followed By 2-Caren-10-Al (11.83%) And 3 Sesquiterpenes Wherein 4-[Phenylethyl]-Acetophenone (0.14) Was The Major Compound.
Furthermore, 23.9% Of The Hydrodistillate Was Hydrocarbon (45 Compounds) Formed By A group Of 11 Monoterpenes Wherein Γ-Terpinene (0.67%) Showed The Highest Percentages, 34 Sesquiterpenes Identified where Trans-Caryophyllene (2.87%) Was The Major One. The Volatile Constituents Showed To Have About 53 Structures Reported For The First Time from Serenoa Repens W. Bartram.
3. Study Of The Lipoid Content Of The Fruits Of Serenoa Repens W. Bartram
The Lipoidal Matter Of The Fruits Of Serenoa Repens W. Bartram Was Extracted With N-Hexane And Subjected To Saponification Procedure. The Unsaponifiable Fractions As Well As The Fatty Acid Methyl Esters Were Analyzed By GC/MS.
3.1. GC/MS Analysis Of The Unsaponifiable Matter:
The GC Analysis Of The Unsaponifiable Matter Of The Fruits Of Serenoa Repens W. Bartram Revealed That Campesterol And Stigmast-5-En-3-Ol (Β-Sitosterol) Represented The Identified Sterols In The Sample Under Investigation And There Are About 5.6% Of The Total Identified Components Also Squalene Represented The Identified Terpenoid And The Percentage Of Hydrocarbons Are Higher Than Sterols.
3.2. GC/MS Analysis Of Fatty Acid Methyl Esters (FAME):
The Saturated Fatty Acid 9-Octadecenoic Acid Represents The Highest Relative Percentage Of The Identified Saturated Fatty Acids Which Is Was About 26.01% While The Unsaturated Fatty Acid Oleoic Acid Ethyl Ester Represents The Highest Relative Percentage Of The Identified Unsaturated Fatty Acids Which Is Was About 13.48%.
The Presence Of The Unsaturated Fatty Acids Oleoic Acid Ethyl Ester, 8,11-Octadecadienoic Acid, 9,12-Octadecadienoic Acid And 9-Octadecenoic Acid In The Fruits Of Serenoa Repens W. Bartram Are Of Great Importance And May Explain The Use Of The Plant As Anti-Inflammatory And Anti-Oxidant.
4. Qualitative And Quantitative Determination Of Phenolic And Flavonoid Compounds Of The Fruits Of Serenoa Repens W. Bartram
HPLC Analysis Of Phenolic Compounds In Serenoa Repens W. Bartram Fruits Enabled The Identification And Quantification Of 33 Phenolic Compounds (11 Flavonoids And 22 Phenolic Acids).
The Percentage Of Total Identified Flavonoids Was 12.42%. Hesperidin Was The Major Identified Flavonoid With Concentration 4.2946 Ppm Of The Dried Powdered Fruit. The Percentage Of Total Identified Phenolic Acids Was 24.42%. Protocatechuic Acid And P-OH-Benzoic Acid Were The Major Identified Phenolic Acids With Concentration 3.9282 And 3.1368 Ppm Respectively, Of The Dried Powdered Fruit.
5. HPLC/MS-MS Study Of Serenoa Repens W. Bartram Compounds
About 15 Phenolic Compounds, (11 Flavonoids And Flavonoid Glycosides) And (4 Phenolic Acids), Have Been Identified By Both The Negative And Positive Ionization Mode Based On Mass Measurements Of The Pseudomelocular [M-H] Ions, Fragment Ions And Retention Time Of The Compounds Detected As Well As Comparison With Reported Data And By Searching Phytochemical Dictionary Of Natural Products Database (CRC).
Mass Spectra Obtained By HPLC-DAD-ESI/MS Analysis Revealed That Both O- And C- Glycosides Of Flavonoids Were Present. The Identified Compounds Were Belonging Mainly To The Following Classes; Flavonols (Quercetin, Isorhamnetin And Kaempferol), Flavone (Apigenin) And Flavanones (Hesperidin And Naringin), And The Derivatives Of Some Of Them.
6. Phytochemical Investigation Of Different Fractions Of Serenoa Repens W. Bartram Fruits
The Fruits (3 Kg) Were Powdered And Extracted By Cold Maceration With Ethanol (70%) Till Exhaustion. The Ethanolic Extract Of Fruits Was Evaporated Under Reduced Pressure At 40°C Till Dryness To Yield 400 G Residue. About 300 G Of The Residue Of The Fruits Were Suspended In 300 Ml Distilled Water And Partitioned Successively With N-Hexane (7 X 500 Ml), Methylene Chloride (6 X 500 Ml), Ethyl Acetate (5 X 500 Ml) And N-Butanol Saturated With Water (8 X 500 Ml).
Different Fractions Were Evaporated Separately Under Reduced Pressure At 40°C Till Dryness. The Fruits Yielded 10.5, 6.8, 4.8 And 36 G Residue Of The N-Hexane, Methylene Chloride, Ethyl Acetate And N-Butanol Fractions, Respectively.
6.1. Investigation Of Methylene Chloride Fraction Of Serenoa Repens W. Bartram Fruits:
This Study Includes Isolation And Identification Of The Major Compounds Of The Methylene Chloride Fraction Of Serenoa Repens W. Bartram Fruits. The Methylene Chloride Fraction (6.8 G) Was Fractionated On (250 G) Silica Gel H (E-Merk). Gradient Elution Was Carried Out Using N-Hexane 100%, Then With N-Hexane Containing 25% Increment Of Methylene Chloride Till 100% Methylene Chloride And Then With Methylene Chloride Containing 2% Increment Of Methanol Till 100% Methanol.
For Isolation And Purification Of Pure Compounds, The selected Subfractions Were Subjected To Further chromatographic Separation On Silica Gel 60 Column Using Gradient Elution And Sephadex LH-20 Using Isocratic Elution.
Identification Of The Compounds Was Based On Determination Of Their Physical characters, chromatographic Data, NMR Data And With Published Data. Compounds Isolated from This Fraction Were Identified As Compound M1 (Oleic Acid), Compound M2 (Triolein) And Compound M3 (Β-Sitosterol Glucoside).
6.2. Investigation Of Ethyl Acetate Fraction Of Serenoa Repens W. Bartram Fruits:
This Study Includes Isolation And Identification Of The Major Compounds Of The Ethyl Acetate Fraction Of Serenoa Repens W. Bartram Fruits. The Ethyl Acetate Fraction (4.8 G) Was Fractionated On (200 G) Silica Gel H (E-Merk). Gradient Elution Was Carried Out Using Methylene Chloride 100%, Then With Methylene Chloride Containing 5% Increment Of Methanol Till 100% Methanol.
For Isolation And Purification Of Pure Compounds, The selected Subfractions Were Subjected To Further chromatographic Separation On Silica Gel 60 Column Using Gradient Elution And Sephadex LH-20 Using Isocratic Elution. Identification Of The Compounds Was Based On Determination Of Their Physical characters, chromatographic Data, NMR Data And With Published Data.
Compounds Isolated from This Fraction Were Identified As Compound E4 Was Protocatecheuic Acid While Compound E5 Was P-Hydroxybenzoic Acid And Compound E6 Was (-)-Epicatechin.
6.3. Investigation Of Butanol Fraction Of Serenoa Repens W. Bartram Fruits
This Study Includes Isolation And Identification Of The Major Compounds Of The Ethyl Acetate Fraction Of Serenoa Repens W. Bartram Fruits. The N-Butanol Fraction (36 G) Was Fractionated On (125 G) Polyamide Column. Gradient Elution Was Carried Out Using Water 100%, Then With Water Containing 5% Increment Of Methanol Till 100% Methanol.
For Isolation And Purification Of Pure Compounds, The selected Subfractions Were Subjected To Further chromatographic Separation Sephadex LH-20 Using Isocratic Elution, Compound B7 (8 Mg) Was Obtained. Identification Of The Compound Was Based On Determination Of Their Physical characters, chromatographic Data, NMR Data And With Published Data. Compound Isolated from This Fraction Was Identified As Compound B7 (Quercitrin).
Part III: Biological Study Of Serenoa Repens W. Bartram.
1. Pharmacological Activities
1.1. Toxicological Study:
The Obtained Results Of The Acute Oral Toxicity Revealed No Signs Of Toxicity At The Dose Up To 5000 Mg/Kg Equivalent To 37.5 G Dry Herb. The Pharmacological Evaluation Of The Extracts Was Carried Out At A Fixed Daily Dose (100 Mg/Kg/Body Weight).
from These Results It Could Be Concluded That All The Tested Extracts Are Non-Toxic And Safe And These Agree With That Were Reported About The Extract Of Serenoa Repens W. Bartram, This May Explain Its Extensive Utilization In Traditional Medicine.
1.2. Acute Anti-Inflammatory Activity:
The Effect Of The Ethanolic And Aqueous Extracts Of The Fruits Of Serenoa Repens On The Rat Paw Oedema Induced By Carrageenan Was Determined According To The Method Described By (Winter Et Al., 1962). Both Hind Paws Were Excised And Oedema Measured By The Caliber And % Oedema Was Calculated. Furthermore, The Percentage Of Inhibition Of The Mean In Comparison With Control Was Estimated For Both Extracts And Calculated.
It Was Observed That Both Ethanolic And Aqueous Extracts Showed Significant Anti-Inflammatory Activity where Ethanolic Extract (% Of Inhibition 89.5%) Was More Powerful Than Aqueous Extract (% Of Inhibition 87.5%) But Both Were Lower Than Indomethacin (% Of Inhibition 92.5%), So The Ethanolic Extract Of Serenoa Repens Was Found To Have The Best Anti-Inflammatory Activity Compared To The Standard.
1.3. Anti-Hyperglycemic Activity:
Anti-Hyperglycemic Effect Of The Tested Extracts Was Evaluated And Compared With That Of Metformin Drug As Standard. Male Albino Rats Of Sprauge Dawely Strains (130-140 G) Were Injected Intra-Peritoneal With A Single Dose Alloxan (150 Mg/Kg B.Wt.) To Induce Diabetes Mellitus (Eliasson And Samet, 1969).
Both Ethanolic And Aqueous Extracts Showed Significant Anti-Hyperglycemic Activity As The Aqueous Extract Decreased The Elevated Serum Glucose Level That Induced By Alloxan By 54.76% While The Ethanolic Extract Decreased It By 44.95 In Compared With Metformin That Reduced It By 67.7 After Only 4 Weeks Interval.
1.4. Antioxidant Activity:
The Anti-Oxidant Activity Of The Ethanolic And Aqueous Extracts Of Fruits Of Serenoa Repens W. Bartram Under Investigation Was Calculated By The Determination Of Blood Glutathione Of Alloxan-Induced Diabetic Rats. Glutathione In Blood Was Determined According To The Method Described By (Beutler Et Al., 1963).
The Reduced Level Of Blood Glutathione In Diabetic Rats Was Restored After The Oral Administration Of (100 Mg/Kg) Of Ethanolic And Aqueous Extracts Of The Fruits Of Serenoa Repens W. Bartram. Both Ethanolic And Aqueous Extracts Showed Significant Anti-Oxidant Activity where Aqueous Extract (2.75% Change from Control) Was More Powerful Than Ethanolic Extract (5.22% Change from Control). The Relative Potency Of Aqueous Extract Was 98.6 While Ethanolic Extract Was 96.1 When Compared With Vitamin E As A Standard Drug (Potency = 100).
1.5. Anti-Peptic Ulcer Activity:
The Anti-Ulcer Activity Was Carried Out According To The Method Described By (Corell Et Al., 1979). The Ability Of The Aqueous And Ethanolic Extracts To Decrease The Number Of Stomach Ulcers Was Calculated As % Of Protection.
The Ethanolic And Aqueous Extracts Protect from Peptic Ulcer Induced By Indomethacin As The Ethanolic Extract Decreased The Number Of Ulcers By 48.39% While The Aqueous Extract Decreased It By 41.13% So The Ethanolic Extract Possessed Higher Anti-Ulcer Activity Than Aqueous Extract.
1.6. Hepatoprotective Activity:
Liver Damage In Rats Was Induced According To The Method Of (Klassen And Plaa, 1969) By Intra-Peritoneal Injection Of (5 Ml/Kg) Of 25% Carbon Tetrachloride (CCL4) In Liquid Paraffin.
Pretreatment Of Rats With Daily Oral Dose Of (100 Mg/Kg B.Wt.) Of Ethanolic And Aqueous Extracts Showed A Significant Activity In Decreasing The Liver Enzymes Levels In Ccl4 Induced Liver Damage As They Reduced The Percentage Of Liver Damage where The Ethanolic Extract Reduced ALP, AST And ALT To 67.26, 45.47 And 50.42, Respectively While The Aqueous Extract Reduced Them By 74.8, 52.8 And 51.25, Respectively.
Furthermore, After 7 Days Of Treatment Both Ethanolic And Aqueous Extracts Showed Reduction In ALP, AST And ALT Liver Enzymes As Ethanolic Extract Reduced ALP from 22.3±1.1 To 17.3±0.8, AST from 69.5±2.6 To 54.5±2.1 And ALT from 71.8±2.7 To 56.3±2.5 While Aqueous Extract Reduced ALP from 28.2±0.9 To 21.2±0.8, AST from 83.5±2.1 To 65.2±2.3 And ALT from 79.8±2.5 To 61.6±2.1 In Compared With Silymarin As A Standard Drug As It Reduced ALP from 18.9±0.6 To 7.6±0.1, AST from 53.2±2.8 To 37.2±1.3 And ALT from 53.7±1.8 To 39.9±1.4.
2. Cytotoxic Activity
The Ethanolic Extract Of The Fruits Of Serenoa Repens W. Bartram Was Tested Against Four Human Cell Lines; Human Hepatocellular (Hepg-2), Breast (MCF7), Colon (HCT11) And Prostate (PC3) Carcinoma Cell Lines. The Cytotoxic Assay Was Performed By MTT Method (Scudiero Et Al., 1988; Mosmann, 1983).
The Tested Samples Exerted Cytotoxic Activity Against Prostate Carcinoma And Breast Carcinoma Cell Lines With Different Concentrations But Showed Low Activity Against Colon Carcinoma And Inactive Against Hepatocellular Carcinoma When Compared With Doxorubicin.
The Ethanolic Extract Has Cytotoxic Activity Against Breast Carcinoma Cell Line (IC50 = 0.6 More Than Doxorubicin (IC50 = 1.72) However It Has Cytotoxic Activity Against Prostate Carcinoma Cell Line (IC50 = 0.82) Less Than Doxorubicin (IC50 = 0.78).
The Activity Of The Ethanolic Extract On Breast Cancer Cells Was Reported Here For The First Time As The Previously Reported Results Were On The Activity Of The Liposterolic Extract On Prostate Cancer Cells. This Activity May Be Due To The High Percentage Of Β-Sitosterol, Its Glycoside And Other Β-Sitosterol Derivatives.
3. Antimicrobial Activity
Anti-Microbial Activity Of The Tested Samples Was Determined Using A Modified Kirby-Bauer Disc Diffusion Method (Baccer Et Al., 1966). Plates Incubated With Filamentous Fungi As Asperagillius Fumigatus, Penicillium Italicum. Fusarium Oxysporum And Fusarium S. Cucurbitae At 25oc For 48 Hours; Gram (+) Bacteria As Bacillus Subtilis, And Staphylococcus Aureus; And Gram (-) Bacteria As Pseudomonas Aeruginosa, Escherichia Coli And Salmonella Typhimurium Were Incubated At 35-37oc For 24-48 Hours And Yeast As Candida Albicans Incubated As 30oc For 24-48 Hours And, Then The Diameters Of The Inhibition Zones Were Measured In Millimeters.
The Antimicrobial Activity Of The Investigated Samples Was Evaluated By Determination Of The Antimicrobial Sensitivity And MIC Values Against Two Gram-Positive And Three Gram-Negative Bacteria As Well As Five Fungal Strains. The Results Showed That The Ethanolic Extract And The Volatile Oil Had Moderate Anti-Microbial Activities Against Different Fungal And Bacterial Strains. The Activity Was Higher For Antifungal Followed By The Gram-Positive Strains, Gram-Negative Bacteria Strains Compared To The Positive Controls.
The MIC Was Detrmined Against selected Strains. The Samples Were Active As Anti-Fungal Against Fusarium S. Cucurbitae With MIC Of Oil 3.9 µg/Ml And Ethanolic Extract 7.81 µg/Ml, Followed By Fusarium Oxysporum With Concentration 7.81 µg/Ml For Both Samples And Low Activity Or Inactive Against Other Fungi Compared To The Positive Controls.
They Also Showed A Moderate Anti-Bacterial Activity Against Bacillis Subtilis, Gram +Ve, With Concentration 1.95 µg/Ml For Oil And 3.9 µg/Ml For The Ethanolic Extract, On The Other Hand The Other Bacterial Strains Were Poorly Active Or Inactive Compared To The Positive Controls.
The Volatile Oil Showed More Powerful Anti-Microbial Activity Than The Ethanolic Extract, These Activities May Be Attributed To The Unique Chemical Composition Of The Volatile Oil And The Presence Of The Major Isolate 4-(1-Methylethyl)-Benzaldehyde That Had Strong Fungistatic Activity As Reported In Previous Studies (Zhai Et Al., 2011; De Et Al., 2003).