BIOASSAY GUIDED ISOLATION OF SECONDARY METABOLITES FOR ANTIDIABETIC AND ANTI-AMNESIC POTENTIAL OF ELAEAGNUS UMBELLATA THUNB.
PhD Thesis
By
Nausheen
DEPARTMENT OF CHEMISTRY UNIVERSITY OF MALAKAND 2019
BIOASSAY GUIDED ISOLATION OF SECONDARY METABOLITES FOR ANTIDIABETIC AND ANTI-AMNESIC POTENTIAL OF ELAEAGNUS UMBELLATA THUNB.
By
Nausheen
A THESIS SUBMITTED TO THE UNIVERSITY OF MALAKAND
IN
PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (PhD) IN BIOCHEMISTRY 2019
In the name of Allah, the most beneficent, the most merciful.
CERTIFICATE OF APPROVAL
This is to certify that the research work presented in this thesis entitled, “BIOASSAY
GUIDED ISOLATION OF SECONDARY METABOLITES FOR ANTIDIABETIC
AND ANTI-AMNESIC POTENTIAL OF ELAEAGNUS UMBELLATA THUNB.” conducted by Mrs. Nausheen under the supervision of Dr. Muhammad Zahoor, Assistant
Professor. No part of this thesis has been submitted anywhere else for any other degree. This thesis is submitted to the Department of Chemistry, in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in the field of Biochemistry, Department of
Chemistry, University of Malakand is hereby approved for submission.
Student Name: Mrs. Nausheen Signature------Examination Committee: External Examiner 1: Dr. Signature------External Examiner 2: Dr. Signature------Internal Examiner: Dr. Signature------
Supervisor: Dr. Muhammad Zahoor Assistant Professor, Signature------Department of Chemistry, University of Malakand.
Chairman: Dr. Manzoor Ahmad, Signature------Associate Professor, Department of Chemistry, University of Malakand.
AUTHOR'S DECLARATION
I Mrs. Nausheen hereby state that my PhD thesis titled “BIOASSAY GUIDED
ISOLATION OF SECONDARY METABOLITES FOR ANTIDIABETIC AND ANTI-
AMNESIC POTENTIAL OF ELAEAGNUS UMBELLATA THUNB.” is my own work and has not been submitted previously by me for taking any degree from University of
Malakand or anywhere else in the country/world.
At any time if my statement is found to be incorrect even after my graduation, the University has the right to withdraw my PhD degree.
Nausheen Date:
PLAGIARISM UNDERTAKING
I solemnly declare that research work presented in the thesis titled “BIOASSAY
GUIDED ISOLATION OF SECONDARY METABOLITES FOR ANTIDIABETIC
AND ANTI-AMNESIC POTENTIAL OF ELAEAGNUS UMBELLATA THUNB.” is solely my research work with no significant from any person. Small contribution/help wherever taken has been dually acknowledged and that complete thesis has been written by me. I understand the zero tolerance policy of the HEC and University of Malakand towards plagiarism. Therefore i as an author of the above titled thesis declare that no portion of my thesis has been plagiarized and any material used as reference is properly referred/cited.
I undertake that if i am found guilty of any formal plagiarism in the above titled thesis even after award of PhD degree, the University reserves the rights to withdraw/revoke my PhD degree and that HEC and the University has the right to publish my name on the
HEC/University website on which name of students are placed who submitted plagiarized thesis. (A copy of the plagiarism report performed at the time of thesis submission, by quality enhancement cell (QEC) of the University is also attached).
NAUSHEEN
I am dedicated my thesis to my beloved parents and husband, whose prayers, moral and financial support enabled me to conduct and complete this achievement.
Nausheen
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ACKNOWLEDGMENTS
Beginning with the greatest name of Al-Mighty Allah, the most gracious, sympathetic and ever merciful, who gave me mental and physical strength that enable me to complete this difficult task in time. It was great blessing of Allah that I got loving parents, inspiring and talented teachers and friends who provide me the intellectual guidance, moral support and all-round help for the fulfilment of my work.
I offer my humblest thanks from the core of heart to Holly prophet Muhammad (PBUH) who is forever a torch of guidance and knowledge for humanity as a whole. I have no words to express my deepest sense of gratitude to the Holly prophet Muhammad (PBUH), who has deep sense of love, affection, sympathy, kindness and sincerity for whole humanity and all creatures leaving on the earth.
I deem it utmost pleasure to avail this opportunity to express my heartiest gratitude to my respectable research supervisor, Dr. Muhammad Zahoor, Associate Professor, Department of Chemistry, University of Malakand, for his professional advice, valuable guidance, technical approach, consistent encouragement, sympathetic attitude, art of making useful suggestion and inspiring attitude made it very easy to undertake this work and to write these thesis.
I wish to express from the core of my heart very special thanks to Dean of Sciences Prof. Dr. Rasheed Ahmad Department of Chemistry and Dean of Biological Sciences Prof. Dr. Mir Azam Khan Department of Pharmacy, University of Malakand for their tremendous knowledge of the subject and incentive guidance throughout my research work.
I am greatly thankful to express heartiest gratitude to Dr. Manzoor Ahmad Chairman Department of Chemistry whose unwavering support, keep interest and encouragement due to which I was succeeded to complete this task and led to the completion of this scientific work.
I would like to express my special appreciation and thanks to Dr. Mohammad Nisar Chairman Department of Botany you have been a tremendous mentor for me. Your impeccable research skills, clear guidance, administrative support and advice on both research as well as on my career have been invaluable.
I would like to pay sincere thanks, to Dr. Waqar Ahmad Department of Pharmacy, Dr. Ezat Khan, Dr. Abdul Lateef, Dr. Mumtaz Ahmad, Dr. Sultan Alam, Dr. Naveed Umer, Dr. ii
Najeeb and all faculty members of Department of Chemistry, University of Malakand, for their useful comments, support, kind guidance and encouragement and helping during my research work. I also offer special thanks to Dr. Nasiara Karim, Dr. Shoaib, Dr. Rukhsana, Dr. Wadud Ali Shah and Dr. Farhat Ullah, Department of Pharmacy University of Malakand and Dr. Imran Khan, Department of Pharmacy, University of Swabi, Sajjad Ahmad Department of Pharmacy, Sarhad University of Information Technology, Peshawar, Zia Uddin Department of pharmacy, COMSATS University Islamabad, Abbottabad Campous whose encouragement, impeccable research skills and support help me throughout my research work. I am thankful to all the faculty members, technical and administrative staff of the Chemistry, Botany and Pharmacy departments, University of Malakand, who contributed to the successful completion of my research work and providing me lab facilities.
Finally, I pay my regards and duly acknowledge the co-operation, guidance and support of my loving Parents, Brothers and sister. Words are meaningless to what they have given me. It is, believe their prayers, sincere encouragement, and moral support which were a constant source of strength and inspiration to me that despite, many constraints, I was able to complete my Ph.D. work. And most of all for my loving, supportive, encouraging, and patient husband Muhammad Nazir whose faithful support during my Ph.D. is so appreciated. Thank you.
NAUSHEEN
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LIST OF ABBREVIATIONS
Abbreviations Full Name
% SAP % Spontaneous Alternation-performance
µg Micro gram
µL Micro Litre
2D Two dimensional
AAR Alternate Arm-returns
ABI Acquired Brain Injury
ABTS 2, 2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
ACh Acetylcholine
AChE Acetylcholine esterase
ACTH Adrenocorticotropic hormone
AD Alzheimer's disease
ADH Alcohol dehydrogenase
AGEs Advance Glycation end-products
ALDH Aldehyde dehydrogenase
ALP Serum alkaline-phosphatase
ANOVA Analysis Of Variance
APP Amyloid precursor protein
Aq. Ext Fraction Aqueous
As Peak area of standard
Ax Peak area of sample
Aβ β-amyloid
BBB Blood brain barrier
BChE Butyryl cholinesterase
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But. Ext Fraction n-Butanol
C13NMR Carbon-13 Nuclear Magnetic-resonance
Chf-Ext Fraction of chloroform
CNS Central nervous system
COX-2 Cyclooxygenase-2
CRF Corticotrophin releasing factor
CRP C-reactive protein
Cs Concentration of standard
CVA Cerebrovascular accident
Cx Concentration of sample
DI Discrimination index
DM Diabetes-mellitus
DMSO Di-methylsulphoxide
DNA Deoxyribonucleic Acid
DNSA 3, 5-dinitrosalicylic acid
DPPH 2, 2-Diphenyl,1,picrylhydrazyl
E. umbellata Elaeagnus umbellata
EDTA Ethylene Diamine Tetra Acetic acid
EI-MS Electron Ionization Mass Spectrometry
EtAc-Ext Fraction of ethyl acetate
FAB-MS Fast Atom Bombardment Mass Spectrum
FDA Food and drug administration
FTIR Fourier Transform Infrared-spectroscopy
GC Gas-chromatography
GC/MS Gas-chromatography/Mass-spectrometry
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GIP Glucose insulinotropic polypeptide
GLP-1 Glucagon like polypeptide-1
GLUT4 Glucose transporter 4
GSK3β Glycogen synthase kinase 3β
HDL High Density-lipoprotein
Hex. Ext Fraction n-hexane fraction
HFD High fat diet
HMBC Hetero-nuclear multiple-Bond Coherence
HNMR H+-Nuclear Magnetic-resonance
HPA Hypothalamic pituitary adrenal
HPLC-UV High performance liquid chromatography- ultra violet
HPTLC High Performance thin-liquid-chromatography
HSAM Highly-superior Autobiographical Memory
HSQC Hetero-nuclear single-quantum Coherence i.p. Intraperitoneal
IAPP Islet amyloid polypeptide
IC 50 Minimum inhibitory concentration
IDDM Insulin dependent diabetes mellitus
IDE Insulin-degrading enzyme
IL-1β Interleukin-1β
IR Insulin receptor
JNK Jun N-terminal kinase
LDL Low Density-lipoproteins
MCI Mild cognitive impairment
MDD Major depressive disorder
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Me-Ext Methanolic extract mm Meli meter
MMP-1 Matrix metalloproteinase-1
MS Mass spectroscopy
MTBI Mild traumatic brain injury
IR Insulin receptor
NFT Neurofibrillary tangles
NF-κB Nuclear factor-kappa B
NGF Nerve growth factor
NIDDM Non-insulin dependent diabetes mellitus
NMR Nuclear-magnetic-resonance
NO Nitric oxide
NOESY Nuclear Overhauser Effect Spectroscopy
NORT Novel object recognition Test p.o. Per oral, p38-MAPK p38 Mitogen-activated Protein-kinase
PD Parkinson's disease
PDB Protein Data Bank
PPHG Postprandial hyperglycemia
PTA Post traumatic Amnesia
PTSD Post-traumatic stress disorder
RA Retrograde Amnesia
RAGE Receptor advanced Glycation end-products
ROS Reactive oxygen species
SAR Same Arm-returns
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SARK Stress activated Protein-kinase
SDAM Severely-deficient Autobiographical Memory
SEM Standard error of the mean
SGOT Serum-glutamate-oxaloacetate-transaminase
SGPT Serum-glutamate-pyruvate-transaminase
SMBG Self monitoring of blood glucose
STM Short term memory
STZ Streptozotocin
T2D Type-2 diabetes
T2DM Type-2 diabetes mellitus
TBI Traumatic-brain injury
TC Total-cholesterol
TCM Traditional Chinese medicine
TF Time for familiar (object exploration)
TFC Total Flavonoid Contents
TG Triglycerides
TGA Transient global amnesia
TLC Thin-layer chromatography
TN Time for novel (object exploration)
TNF-α Tumor necrosis factor-α
TPC Total Phenolic Content
UV Ultra Violet
UVAD Ultraviolet array-detector
WHO World health organization
WKS Wernicke-Korsakoff syndrome
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List of Contents
1. Introduction ...... 1 1.1. Human health and medicinal plants ...... 1 1.2. Antidiabetic potential of phenolic compounds ...... 2 1.3. Link between berry fruits and type 2 diabetes ...... 4 1.4. Diabetes Mellitus (DM) ...... 5 1.5. Clinical manifestation of DM ...... 6 1.6. Diabetes mellitus classifications ...... 6 1.6.1. Diabetes Type I ...... 6 1.6.1.1. Pathogenesis of diabetes type I ...... 7 1.6.1.2. Prevention and new treatments ...... 7 1.6.2. Diabetes Type 2 ...... 8 1.6.2.1. Pathophysiology of type 2 diabetes ...... 9 1.6.2.2. Preventive measures and treatments of type 2 diabetes ...... 9 1.6.2.3. Lifestyle changes ...... 10 1.6.2.4. Pharmaceutical Therapy to reduce postprandial hyperglycemia ...... 10 1.6.2.5. Inhibition of carbohydrate digesting enzymes ...... 11 1.6.2.6. Treatment of hypertension ...... 12 1.6.2.7. Treatment of hyperlipidemia ...... 12 1.7. Memory ...... 13 1.7.1. Sensory memory ...... 13 1.7.2. Short term memory ...... 13 1.7.3. Long term memory ...... 14 1.8. Memory disorders ...... 14 1.8.1. Acquired Brain Injury ...... 14 1.8.2. Agnosia ...... 15 1.8.3. Alzheimer's disease (AD) ...... 15 1.8.4. Amnesia ...... 16 1.8.4.1. Traumatic brain injury ...... 16 1.8.5. Dementia ...... 17 1.8.6. Hyperthymestic syndrome...... 17 1.8.7. Huntington disease ...... 18 1.8.8. Parkinson’s disease (PD) ...... 18
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1.8.9. Stress ...... 19 1.8.10. Wernicke Korsakoff syndrome ...... 19 1.9. Amnesia and their different types ...... 20 1.9.1. Anterograde amnesia ...... 20 1.9.2. Retrograde amnesia ...... 20 1.9.3. Post traumatic amnesia ...... 20 1.9.4. Dissociative amnesia ...... 21 1.10. Causes of Amnesia ...... 21 1.11. Preventive measures for amnesia ...... 21 1.12. The Link between type 2 diabetes and neurodegeneration ...... 22 1.13. Mechanism that underlies cognitive impairments in T2DM ...... 24 1.13.1. Insulin Resistance ...... 24 1.13.2. Inflammatory mechanism ...... 24 1.13.3. High glucose concentration ...... 25 1.13.4. Oxidative Stress ...... 25 1.14. Aims and objectives of the study ...... 29 2. LITERATURE AND REVIEW ...... 30 2.1. Elaeagnaceae Family ...... 30 2.2. Elaeagnus Genus ...... 31 2.3. Pharmacological effects of some important medicinal species of Genus Elaeagnus ... 33 2.3.1. Pharmacological effects of Elaeagnus angustifolia ...... 33 2.3.2. Pharmacological effects of Elaeagnus oldhamii Maxim...... 34 2.3.3. Pharmacological effects of Elaeagnus pungens Thunb...... 35 2.3.4. Pharmacological effects of Elaeagnus philippinsis ...... 36 2.3.5. Pharmacological effects of Elaeagnus multiflora Thunb...... 36 2.3.6. Pharmacological effects of Elaeagnus parvifolia Wall...... 36 2.3.7. Pharmacological effects of Elaeagnus bockii Diels ...... 36 2.3.8. Pharmacological effects of Elaeagnus orientalis ...... 37 2.3.9. Pharmacological effects of Elaeagnus phillipinus ...... 37 2.3.10. Pharmacological effects of Elaeagnus pyriformis ...... 37 2.3.11. Pharmacological effects of Elaeagnus latifolia Linn...... 38 2.3.12. Pharmacological effects of Elaeagnus macrophylla Thunb...... 38 2.3.13. Pharmacological effects of Elaeagnus henryi Warb...... 38
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2.3.14. Pharmacological effects of Elaeagnus lanceolata Warb...... 39 2.3.15. Pharmacological effects of Elaeagnus gonyanthes Benth...... 39 2.3.16. Pharmacological effects of Elaeagnus conferta Roxb...... 39 2.3.17. Pharmacological effects of Elaeagnus x ebbingei L...... 39 2.3.18. Pharmacological effects of Elaeagnus caudata Schlecht...... 40 2.3.19. Pharmacological effects of Elaeagnus genus Elaeagnus kologa Schldl...... 40 2.3.20. Pharmacological effects of Elaeagnus glabra ...... 40 2.3.21. Elaeagnus umbellata Thunb...... 40 2.3.21.1. Elaeagnus umbellata Thunb. Description ...... 40 2.3.21.2. Elaeagnus umbellata Thunb. Distribution ...... 40 2.3.21.3. Taxonomic position of Elaeagnus umbellata Thunb...... 41 2.3.21.4. Pharmacological effects of E. umbellata Thunb...... 42 2.4. Antidiabetic effect of genus Elaeagnus species ...... 44 3. MATERIAL AND METHODS ...... 54 3.1. Chemicals ...... 54 3.2. Plant sample collection ...... 54 3.2.1. Extraction and Fractionation ...... 55 3.3. Preliminary phytochemical analysis ...... 56 3.3.1. Qualitative screening ...... 56 3.3.2. Dragendorff’s test ...... 56 3.3.3. Ferric chloride test ...... 57 3.3.4. Keller Killiani test ...... 57 3.3.5. Gelatin test ...... 57 3.3.6. Liebermann Burchard test ...... 57 3.3.7. Bontrager’s test ...... 57 3.4. Investigation of total phenolic content ...... 58 3.5. Investigation of total flavonoid contents ...... 58 3.6. HPLC-UV characterization ...... 58 3.8. Gas-chromatographic analysis and identification of components ...... 59 3.9. Isolation, purification and characterization of pure compounds ...... 59 3.10. Antioxidant scavenging assays ...... 60 3.10.1. DPPH scavenging assay ...... 60 3.10.2. ABTS scavenging assay ...... 60
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3.11. Antibacterial activity ...... 61 3.12. In vitro anti-diabetic assays ...... 61 3.12.1. α-amylase enzyme inhibition ...... 61 3.12.2. α –glucosidase enzyme inhibition ...... 61 3.13. In vitro Anti Alzheimer’s study ...... 62 3.13.1. Anti-cholinesterase Assays ...... 62 3.14. In vivo antidiabetic assay ...... 63 3.14.1. Animals ...... 63 3.14.2. Acute toxicity study of the fruit Me-Ext/fractions of Elaeagnus umbellata Thunb...... 63 3.14.3. Animal experimental design for inducing type 2 diabetes ...... 64 3.14.4. Treatment protocol ...... 65 3.14.5. Collection of blood and estimation of biochemical parameters ...... 65 3.15. Molecular docking validation for anti-diabetic enzymes ...... 65 3.16. In vivo anti-amnesic assays ...... 67 3.16.1. Experimental scheme ...... 67 3.16.2. Y-Maze test for Spontaneous Alternation ...... 68 3.16.3. Novel object recognition Test ...... 69 3.17. Molecular docking validation for antiamnesic activity ...... 69 3.18. Histopathology ...... 70 3.18.1. Histopathology technique ...... 70 3.19. Statistical analysis ...... 73
3.20. Assessment of IC 50 Values ...... 73 3.21. Regression and linear Correlation (R2) ...... 73
4. RESULTS ...... 74 4.1. Extraction and fractionation yield ...... 74 4.2. Preliminary phytochemical screening ...... 74 4.2.1. Qualitative screening ...... 74 4.2.2. Total Phenolic content (TPC) ...... 74 4.2.3. Total Flavonoid contents (TFC) ...... 75 4.2.4 Identification of phenolic compounds through HPLC-UV technique ...... 77 4.2.4. GC-MS examination for essential oil ...... 79 4.3. Compounds isolation and structural confirmations ...... 85
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4.3.1. Compound-I ...... 85 4.3.2. Compound-II ...... 90 4.3.3. Compound-III ...... 92 4.3.4. Compound-IV ...... 96 4.3.5. Compound-V ...... 99 1.1.1. Compound-VI ...... 103 1.1.2. Compound-VII ...... 106 1.1.3. Compound-VIII ...... 110 1.2. In vitro Antioxidant potential study ...... 114 1.2.1. Antioxidant potential of essential oil ...... 114 1.2.2. Antioxidant studies on extract/fractions ...... 115 1.2.2.1. DPPH scavenging potential ...... 115 1.2.2.2. ABTS free radical scavenging potential ...... 115 1.2.3. Antioxidant potential of isolated Compounds ...... 117 1.2.3.1. DPPH scavenging potential of isolated compounds ...... 117 1.2.3.2. ABTS free radical scavenging potential of isolated compounds ...... 117 1.3. In vitro Antibacterial Activity ...... 120 1.3.1. Antibacterial potential of extract/fractions of E. umbellata ...... 120 1.3.2. Antibacterial Activity of isolated compounds ...... 123 1.4. In vitro anti-diabetic studies on extracts/fractions ...... 125 1.4.1. In vitro α-amylase enzyme inhibitory assay ...... 125 1.4.2. In vitro α-glucosidase enzyme inhibitory assay ...... 125 1.5. In vitro anti-diabetic studies on essential oil ...... 127 1.5.1. In vitro α-amylase and α-glucosidase inhibitory assay ...... 127 1.6. In vitro antidiabetic studies of isolated compounds ...... 129 1.6.1. In vitro α-amylase enzyme inhibitory studies of isolated compounds ...... 129 1.6.2. In vitro α–glucosidase enzyme inhibitory studies of isolated compounds ...... 129 1.7. In vitro anticholinesterase assays ...... 132 1.7.1. Choline inhibition potential of extract/fractions ...... 132 1.7.2. Relationship of total phenolic/flavonoid contents versus cholinesterase inhibition assays ...... 134 1.7.3. Cholinesterase inhibition potential of essential oil ...... 135 1.7.4. AChE and BChE inhibition potential of pure compounds ...... 137
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1.8. In vivo antidiabetic studies of extract/fractions ...... 140 1.8.1. Toxicity study ...... 140 1.8.2. Estimation of biochemical parameters ...... 140 1.8.2.1. Effect of E. umbellata Thunb. methanolic fruit extract/fractions on glycemia ...... 140 1.8.2.2. Effect of E. umbellata Thunb. methanolic fruit extract/fractions on body weight in diabetic rats ...... 142 1.8.2.3. Measurement of serum lipid profile in diabetic rats ...... 143 1.8.2.4. Effect of E. umbellata Thunb. methanolic fruit extract/fractions on liver and renal functions in STZ-induced diabetic rats ...... 144 1.9. Molecular docking validation of antidiabetic enzymes ...... 145 1.9.1. α-Amylase ...... 145 1.9.2. α-Glucosidase...... 149 1.10. In vivo antidiabetic studies of isolated compound ...... 153 1.10.1. Acute toxicity of compound ...... 153 1.10.2. Estimation of biochemical parameters ...... 153 1.10.2.1. Effect of isolated compound of E. umbellata on blood glycemia ...... 153 1.10.2.2. Effects of isolated compound of E. umbellata on body weight in diabetic rats ...... 154 1.10.2.3. Effect of isolated compound of E. umbellata on lipid profile in streptozotocin induced diabetic rats...... 156 1.10.2.4. Effect of isolated compound of E. umbellata on liver and renal functions in streptozotocin-induced diabetic rats ...... 157 1.11. Effect of different extracts/fractions Compound on pancreas histopathology in STZ-induced diabetic rats ...... 158 1.12. In vivo antiamnesic study of active extract/fraction of E. umbellata ...... 163 1.12.1. Y-maze test ...... 163 1.12.2. Novel Object Recognition Test ...... 164 1.13. In vivo antiamnesic study of isolated compound of E. umbellata ...... 167 1.13.1. Y-maze test ...... 167 1.13.2. Novel Object Recognition Test ...... 169 1.14. Molecular docking validation of isolated compounds for anticholinesterases (AChE & BChE) ...... 172
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1.15. Molecular docking validation of HPLC detected compounds for anticholinesterases (AChE & BChE) ...... 180 DISCUSSION ...... 188 6. CONCLUSION ...... 198 REFERENCES ...... 201
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List of Figures
Figure 1.1: Structures of important plant polyphenols ...... 4
Figure 1.2: Various factors that link the association between T2DM and AD [309] ...... 26
Figure 1.3: Effect of chronic inflammation on the association between T2DM and AD [319]...... 27
Figure 1.4: Mechanistic overview that link cognitive impairments in T2DM & neurodegeneration ...... 28
Figure 1.5: Graphical overview of the whole work ...... 29
Figure 3. 1: Plant and fruits of E. umbellata Thunb a) E. umbellata Thunb. tree b) Berried shrubs c) Berries/fruits 55
Figure 3.2: Schematic diagram of extraction and fractionation yield 56
Figure 3.3: Phenolic Compounds identified in E. umbellata Thunb. fruit methanolic extract/fractions studied in molecular docking. 67
Figure 3. 1: Plant and fruits of E. umbellata Thunb a) E. umbellata Thunb. tree b) Berried shrubs c) Berries/fruits ...... 55
Figure 3.2: Schematic diagram of extraction and fractionation yield ...... 56
Figure 3.3: Phenolic Compounds identified in E. umbellata Thunb. fruit methanolic extract/fractions studied in molecular docking...... 67
Figure 4.1 (B): Total Flavonoid Content in extract/fractions of Elaeagnus umbellata Fruit .. 76
Figure 4.2: HPLC-UV Chromatograms of phenolic compounds in E. umbellata Thunb. fruit (A) Me.Ext, (B) Chf-Ext and (C) EtAc-Ext ...... 79
Figure 4.3: GC-MS chromatogram of E. umbellata fruit essential oil ...... 80
Figure 4.4: Major compounds of Elaeagnus umbellata Thunb...... 81
Figure 4.5: Active Antidiabetic and neuroprotective compounds of Elaeagnus umbellata Thunb. essential oil via GC-MS...... 82
Figure 4.6: Structural formula of compound-I ...... 86
Figure 4.7: FTIR spectrum of compound-I ...... 87
Figure 4.8: FAB-Mass of compound-I...... 88
Figure 4.9: 1H- (500 MHz.) NMR spectrum of compound-I ...... 89
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Figure 4.10: 13C-NMR (126 MHz) spectrum of compound-I ...... 90
Figure 4. 11: Structural formula of compound-II ...... 91
Figure 4.12: 1H NMR (CHLOROFORM-d, 500MHz) of compound-II ...... 91
Figure 4.13: 13C NMR (Chloroform-d, 100MHz) of compound-II ...... 92
Figure 4.14: EIMS of Compound-II ...... 92
Figure 4.15: Structural formula of compound-III ...... 93
Figure 4.16: FTIR spectra of compound-III ...... 94
Figure 4.17: 1H NMR (Acetone, 300MHz) of compound-III ...... 95
Figure 4.18: 13C NMR (Acetone, 75MHz) of compound-III ...... 95
Figure 4.19: EIMS of compound-III ...... 96
Figure 4.20: Structural formula of compound-IV ...... 97
1 Figure 4.21: H NMR (METHANOL-d4, 500MHz) of compound-IV ...... 98
13 Figure 4.22: C NMR (METHANOL-d4, 100MHz) of compound-IV ...... 98
Figure 4.23: EIMS spectra of compound-IV ...... 99
Figure 4.24: Structural formula of compound-V ...... 100
1 Figure 4.25: H NMR (METHANOL-d4, 300MHz) of compound-V ...... 101
13 Figure 4.26: C NMR (METHANOL-d4, 75MHz of compound-V ...... 101
Figure 4.27: EIMS of compound-V ...... 102
Figure 4.28: FTIR spectra of compound-V ...... 102
Figure 4.29: Structural formula of compound-VI ...... 103
Figure 4.30: FTIR spectra of compound-VI ...... 104
Figure 4.31: 1H- (500 MHz) of compound-VI ...... 105
Figure 4.32: 13C-NMR (126 MHz) of compound-VI ...... 105
Figure 4.33: EI-MS Mass of compound-VI ...... 106
Figure 4.34: Structural formula of compound-VII ...... 107
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1 Figure 4.35: H NMR (DMSO- H2O, 300MHz) of compound-VII ...... 108
Figure 4.36: 13C NMR (DMSO-d6, 75MHz) of compound-VII ...... 108
Figure 4.37: FTIR spectra of compound-VII ...... 109
Figure 4.38: EIMS of compound-VII ...... 110
Figure 4.39: Structural formula of compound-VIII ...... 111
Figure 4.40: FTIR spectra of compound-VIII ...... 112
1 Figure 4.41: H NMR (METHANOL-d4, 300MHz) of compound-VIII ...... 112
13 Figure 4.42: C NMR (METHANOL-d4, 75MHz) of compound-VIII ...... 113
Figure 4.43: EIMS of compound-VIII ...... 113
Figure 4.44: % DPPH and ABTS free radical Scavenging activity of essential oil of Elaeagnus umbellata at various concentrations...... 114
Figure 4.45: % DPPH and ABTS free radical Scavenging activity of Elaeagnus umbellate fruit/berries extract/fractions at various concentrations...... 117
Figure 4.46 (B): % ABTS inhibition potential of compounds I-VIII at various concentrations ...... 119
Figure 4.47: Antibacterial potential of extract/fractions of E. umbellata ...... 122
Figure 4.48: Antibacterial activity of the compounds I-VIII against various bacterial strains ...... 124
Figure 4.49: % α-amylase inhibition potential of E. umbellata fruit Me-Ext and subsequent fractions at various concentrations ...... 127
Figure 4.50: α-glucosidase inhibition potential of E. umbellata fruit Me-Ext and subsequent fractions at various concentrations...... 127
Figure 4.51: (A) % α-amylase (B) α-glucosidase inhibition potential of E. umbellata essential oil at various concentrations ...... 128
Figure 4.52: (B): α-glucosidase inhibition potential of isolated compounds from E. umbellata at various concentrations...... 131
Figure 4.53: (B): BChE inhibition potential of Elaeagnus umbellata Thunb. fruit hydro methanolic extract and subsequent fractions...... 134
Figure 4.54: (A) Linear correlation of TPC vs. % AChE inhibition and (B) % BChE inhibition And (C) TFC vs. % AChE inhibition and (D)% BChE inhibition ...... 135
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Figure 4.55: AChE inhibition potential of essential oil of E. umbellata at different concentration...... 136
Figure 4.56: BChE inhibition potential of essential oil of E. umbellata at different concentration ...... 137
Figure 4.57: AChE inhibition potential of isolated compounds from E. umbellata Thunb... 139
Figure 4.58: BChE inhibition potential of isolated compounds from E. umbellata Thunb. .. 139
Figure 4.59: Effect of E. umbellata fruit methanolic extracts/fractions and glibenclamide on blood glucose level in STZ-induced diabetic rats...... 141
Figure 4.60: Effects of E. umbellata fruit methanolic extracts/fractions on body weight in STZ-induced diabetic rats. Each value is Mean±SEM of 8 animals...... 143
Figure 4.61: Mode of binding of different compounds and acarbose in α-amylase enzyme active sites. a) Acarbose, b) Rutin, c) Quercetin, and Epigallocatechin gallate ...... 147
Figure 4.62: A) Stereo view of the docking pose of A) Acarbose, B) Rutin C) Quercetin D) Epigallocatechin gallate in the binding pocket of α-amylase enzyme active sites ...... 148
Figure 4.63: Mode of binding of different compounds and acarbose in α-glucosidase enzyme active sites. a) Acarbose, b) Epigallocatechin gallate, c) Quercetin and d) Rutin. The highlighted area in a) is the common area between acarbose and all compounds ...... 151
Figure 4.64: (A) Stereo view of the docking pose of A) Acarbose, B) Rutin C) Quercetin D) Epigallocatechin gallate in the binding pocket of α-glucosidase enzyme active sites ..... 152
Figure 4.65: Effect of extract/fractions on pancreas histopathology in STZ-induced diabetic rats ...... 161
Figure 4.66: Effect of Compound-V on pancreas histopathology in STZ-induced diabetic rats ...... 162
Figure 4.67: Effect of Chf. Ext (50, 100 and 200 mg/kg) of E. umbellata Thunb. on the Y- maze task...... 164
Figure 4.68: Effect of Chf. Ext (50, 100 and 200 mg/kg) of E. umbellata Thunb. in short-term memory NORT...... 165
Figure 4.69: Effect of Chf. Ext (50, 100 and 200 mg/kg body weight) of E. umbellata Thunb. in long term memory NORT. (A) Time spent in sample phase (B) Time spent in test phase (C) %DI...... 166
Figure 4.70: Effect of isolated compound CGA (1, 3, 10 and 30 mg/kg) of E. umbellata Thunb. on the Y-maze task ...... 168
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Figure 4.71: Effect of isolated compound CGA (3, 10 and 30 mg/kg) of E. umbellata Thunb. in short-term memory NORT. (A) Time spent in the sample phase (B) Time spent in the test phase (C) %DI...... 170
Figure 4.72: Effect of isolated compound CGA (3, 10 and 30 mg/kg) of E. umbellata Thunb. in long term memory NORT...... 171
Figure 4.73: Superimposed ribbon diagram for chlorogenic acid and donepezil ...... 173
Figure 4.74: (A) Stereo view of the docking posture of chlorogenic acid (green color stick model) in the binding pocket of AChE (B) 2D interactions of chlorogenic acid...... 174
Figure 4.75: (A) Stereo view of the docking posture of chlorogenic acid (purple color stick model) in the binding pocket of BChE (B) 2D interactions of chlorogenic acid...... 174
Figure 4.76: Superimposed ribbon diagram for ellagic acid and donepezil ...... 175
Figure 4.77: (A) Stereo view of the docking posture of ellagic acid (green color stick model) in the binding pocket of AChE (B) 2D interactions of ellagic acid...... 176
Figure 4.78: Superimposed ribbon diagram for Morin and donepezil...... 177
Figure 4.79: (A) Stereo view of the docking posture of Morin (green color stick model) in the binding pocket of AChE. (B) 2D interactions of Morin...... 177
Figure 4.80: Superimposed ribbon diagram for Catechin and donepezil ...... 178
Figure 4.81: (A) Stereo view of the docking posture of catechin (green color stick model) in the binding pocket of AChE (B) 2D interactions of catechin ...... 179
Figure 4.82: Superimposed ribbon diagram for rutin and donepezil ...... 180
Figure 4.83: (A) Stereo view of the docking posture of rutin (green color stick model) in the binding pocket of AChE (B) 2D interactions of rutin...... 181
Figure 4.84: Superimposed ribbon diagram for epigallocatechin gallate and donepezil ...... 182
Figure 4.85: (A) Stereo view of the docking posture of epigallocatechin gallate (green color stick model) in the binding pocket of AChE (B) 2D interactions of Epigallocatechin gallate...... 183
Figure 4.86: Superimposed ribbon diagram for Quercetin and donepezil ...... 184
Figure 4.87: (A) Stereo view of the docking posture of Quercetin (green color stick model) in the binding pocket of AChE (B) 2D interactions of Quercetin...... 184
Figure 4.88: Superimposed ribbon diagram for Catechin hydrate and donepezil ...... 185
Figure 4.89: (A) Stereo view of the docking posture of catechin hydrate (green color stick model) in the binding pocket of AChE (B) 2D interactions of catechin hydrate...... 186
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List of Tables
Table 2.1: Assessment of relative phytochemical constituent’s profiles and biological roles of Elaeagnaceae genera ...... 30 Table 2.2: Various Elaeagnus species and their biological uses ...... 45
Table 3. 1: Experimental design and various tretament groups used in the study ...... 64 Table 3.2: Experimental design and various tretament groups used in the study ...... 68 Table 3.3: Experimental design and various tretament groups used in the study ...... 68
Table 4. 1: Preliminary qualitative phytochemical screening of Met-Ext from E. umbellata Fruit ...... 74 Table 4.2: Total Phenolic and Flavonoid Contents in extract/fractions of Elaeagnus umbellata Fruit ...... 75 Table 4.3: Identification and Quantification of phenolic compounds in E. umbellata Thunb. fruit Me-Ext/fractions ...... 78 Table 4.4: Parameters of various components of essential oil of E. umbellata Thunb...... 83 Table 4.5: List of major components identified in essential oil of E. umbellata ...... 84 Table 4.6: Compound-I chemical shifts in solvent MeOD ...... 88 Table 4.7: The chemical shifts of compound-VI in solvent MeOD ...... 106 Table 4.8: % DPPH and ABTS free radical Scavenging activity of Essential oil of Elaeagnus umbellata at various concentrations...... 114 Table 4.9: %DPPH and ABTS free radical Scavenging activity of Elaeagnus umbellata fruit extract/fractions at various concentrations ...... 116 Table 4.10: % DPPH and ABTS free radical Scavenging activity of isolated compounds Elaeagnus umbellata at various concentrations ...... 118 Table 4.11: Antibacterial potential of extract/fractions of Elaeagnus umbellata ...... 121 Table 4.12: Antibacterial potential of isolated compounds I-VIII ...... 124 Table 4.13: % α-Glucosidase and α-Amylase inhibition of E. umbellata fruit methanolic extract and subsequent fractions at various concentrations ...... 126 Table 4.14: % α-amylase and α- glucosidase inhibition potential of essential oil of E. umbellata with IC50 at various concentrations ...... 128 Table 4.15: % α-Glucosidase and α-Amylase inhibition of isolated compounds of Elaeagnus umbellata fruit at various concentrations ...... 130 Table 4.16: Choline esterase inhibition potential of extract/fractions of E. umbellata ...... 133 Table 4.17: Cholinesterase inhibition potential of essential oil of E. umbellata ...... 136
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Table 4.18: Cholinesterase inhibition potential of compound I-VIII at various concentrations ...... 138 Table 4.19: Effect of E. umbellata fruit methanolic extract/fractions on blood glucose level in streptozotocin induced diabetic rats ...... 141 Table 4.20: Effects of E. umbellata fruit methanolic extract/fractions on body weight in STZ- induced diabetic rats ...... 142 Table 4.21: Effect of E. umbellata fruit methanolic extract/fractions on lipid profile in streptozotocin induced diabetic rats ...... 144 Table 4.22: Effect of E. umbellata fruit extract/fractions on liver and renal functions in streptozotocin-induced diabetic rats ...... 145
Table 4.23: The Glide Scores and IC50 values of acarbose and α-amylase inhibitors present in Elaeagnus umbellata Thunb...... 146
Table 4.24: The Glide Scores and IC50 values of acarbose and α-glucosidase inhibitors present in Elaeagnus umbellata Thunb...... 150 Table 4.25: Effect of daily oral administration of isolated compound of E. umbellata on glucose level in streptozotocin induced diabetic rats ...... 154 Table 4.26: Effects of isolated compound of E. umbellata on body weight in STZ-induced diabetic rats ...... 155 Table 4. 27: Effect of isolated compound of E. umbellata on lipid profile in streptozotocin induced diabetic rats ...... 156 Table 4.28: Effect of isolated compound of E. umbellata on liver and renal functions in streptozotocin-induced diabetic rats ...... 157
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Abstract
The creatures on the earth are in search of discovering new things since the start of life on earth. All these efforts are for making their lives better in all aspects of life. With the passage of time human being faced various diseases from minor to chronic and are still facing. In ancient civilization people used herbal products to treat human and animal diseases. Human are in search of discovering new potent and effective medicine from medicinal plants. All plants yield chemical compounds called primary metabolites required for their basic metabolic activities. Apart from primary metabolites they also produces secondary metabolites with more specific and are not used for energy productions but are used for attracting insects which help in pollination and some of them are even used for defence against invaders. They also have beneficial functions in humans if eaten/used as drugs. Such metabolites can be used to produce potent drugs for the modern era diseases. Due to the instinct nature of human from the day first he is behind in search of better in every aspect of life. Although 800 plants have been reported to have antidiabetic activities however none of them is perfect to resolve the issue completely. Due to the mentioned fact above we design this study in the hope that probably this plant; Elaeagnus umbellata (silver berry) will be more potent than the already reported plants. Our decision of selecting this plant as antidiabetic and antiamnesic is not random we have selected this plant due its phytochemical composition and inhibitory potential. To best of our knowledge the activities we carried have not been reported before. Although other berries fruits of the other families have already reported to have antidiabetic affects.
Elaeagnus umbellata belongs to Elaeagnaceae family are one of the wild spiny branched shrubs widely distributed in Himalayan areas of Pakistan. E. umbellata barriers are sources of phenolic and other bioactive compounds that scavenge the reactive oxygen species (ROS) formed during metabolic processes. ROS has been the causative agent of various chronic
i
ailments like diabetes mellitus, cancer, cardiovascular illness, arteriosclerosis, ischemic, nephritis, rheumatism, and neurodegenerative disorders like Alzheimer’s disease (AD).
Elaeagnus umbellata fruit methanolic extract and their subsequent fractions were evaluated for curing of diabetes and neurological complaints.
In the current study E. umbellata fruit berry have been evaluated for the mentioned diseases in form of crude methanolic extract, their subsequent fractions, essential oil and isolated pure compounds. The crude methanolic extract was produced by maceration process. The fractions were obtained by fractionation procedure based on solvent polarity beginning from lower to highly polar solvent (n-hexane, chloroform, ethyl-acetate, n-butanol and finally aqueous fraction was left yielding 95, 210, 115, 90 and 220 grams of extracts, respectively).
HPLC-UV was used for the identification of phenolics present in the extract/fractions and their distribution amongst the fractions (HPLC finger printing). Essential oil were obtained through hydro distillation using Clevenger apparatus. The extracted components/fractions were subjected to column isolation to isolate pure compounds. Thin layer chromatography was used to confirm the isolation of a compound in pure state. The isolated compounds were purified via pencil/pen gravity column with small diameter. The compounds were finally identified through various spectroscopic techniques like; Furrier transform infra-red (FTIR),
EI-MS, HNMR and C13NMR spectroscopic techniques while the essential oil was analysed through GC-MS for identification of phytoconstituents.
E. umbellata berries methanolic extract/fractions, essential oil and isolated compounds were examined for in vitro and in vivo inhibition against α- amylase and α- glucosidase enzymes and anti-hyperglycemic effects in type 2 diabetes mellitus. The acetyl cholinesterase and butyryl cholinesterase inhibition potential and in vivo antiamnesic potential were also determined for extract/fractions and isolated compounds. Phytochemical analysis,
ii
antibacterial activity, total phenolic and flavonoid contents were determined for methanolic extract and subsequent fractions.
On the basis of their antioxidant potential against DPPH (2, 20-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) free radicals, the Me-Ext, sub fractions (Chf-Ext, EtAc-Ext) and compound-I were found to be more active against α- amylase and α-glucosidase with IC50 values 400, 58, 200, 32 µg/ml and 200, 60, 140, 30
µg/ml respectively. Amongst different fractions of Elaeagnus umbellata fruit; the Chf-Ext and EtAc-Ext and compound-V showed maximum % inhibition against AChE (87±1.2,
84±1.0, 89±2.1) with IC50 values 33, 55, 31 µg/mL and BChE (89±0.3, 82±0.5, 90±2.1) at
1000 µg/mL concentration with IC50 values 35, 58, 30 µg/mL respectively.
Study of acute toxicity for Met-Ext/fractions (100-2000 mg/kg) and compound-V (10, 15, 40,
50 and 100 mg/kg/b/w) of E. umbellata were carried out which did not produce any significant behavioral alterations (respiratory aches, convulsions shortage, writhing, variations to reflex actions or mortality) in adults Sprague Dawley rats weighing 150-200 g.
An insignificant increase in petulance was detected at 2000 mg/kg dose for Met-Ext/fractions and 100 mg/kg dose for compound-V in three animals out of total eight. All animals appeared healthy at the end one solar day cycle i.e. 24 hour to 1 week with no noticeable variations in appearance or behavior. No mortality was noticed up to one week. All procedures related to animal were carried out according to the Animal Scientific Procedure Act; UK (1986). In vivo results indicates that the Met- Ext, Chf-Ext, EtAc-Ext fractions and compound-I were found to be more potent in curing the hyperglycemia in STZ- induced type 2 diabetes mellitus in rats as considerable reduction of glucose level was observed when compared to the standard glibenclamide drug (0.5 mg/kg, p.o.). Furthermore, considerable reduction in serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, total cholesterol, low density lipoproteins and triglycerides were observed
iii
indicating useful effects of extracts on secondary complications associated with type 2 diabetes mellitus.
In vivo antiamnesic activity was carried out to evaluate the short and long term memory using the behavioural trials; Y-Maze Spontaneous Alternation Behaviour and recognition test for novel object. In albino mice the Chf. Ext fraction and compound-I (CGA) overturned the amnesia prompted by scopolamine and significant increase in SAP for Chf. Ext (200 mg/kg) fraction and CGA (30 mg/Kg) compound analogous to donepezil (2 mg/kg) drug in the Y- maze procedure. Similarly, Chf. Ext (100 mg/Kg) and CGA (10 mg/Kg) shown significant (p
< 0.05) increase in %DI related to scopolamine (1 mg/kg).
Finally the molecular docking was applied to identify some common interactions observed between acarbose and all docked compounds in the active sites of both α-amylase and α- glucosidase enzymes that have shown the inhibitory effects of the mentioned enzymes. The mechanisms of binding were also analyzed on a GOLD suit v5.6.3 against AChE & BChE enzyme. The Analysis of binding modes indicated similar binding orientations for rutin, epigallocatechin gallate, chlorogenic acid, quercetin, catechin, morin, catechin hydrate, ellagic acid and donepezil in the active gorge of the receptor protein of AChE and BChE enzymes.
In conclusion, E. umbellata fruit can be potentially recommended for controlling type 2 diabetes mellitus, oxidative stress caused by reactive oxygen species, memory impairments and neurological disorders. To search more potent antidiabetic and antiamnesic phytochemicals, more studies are required to formulate potent drugs and evaluate their proper mechanisms.
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CHAPTER 01 INTRODUCTION
1. Introduction 1.1. Human health and medicinal plants
To endorse traditional medicine and herbal therapeutics, the knowledge regarding plant bioactive compounds would play a significant role in human healthcare. Their botanical characteristics and scientific confirmation are required along with their folkloric use, distribution, and their phytochemical and pharmacological activities. Since the start of human civilization, human beings are in search of finding remedies within their locality and have used different therapeutic approaches in order to circumvent various ailments. According to
WHO report almost 80% of world populations rely upon medicinal plants for their essential health requirements. Our country Pakistan has great diversity of medicinal plants with significant medicinal importance [1]. Medicinal plants have received much importance due to their effectiveness as antioxidant and antimicrobial agents due to their phytochemicals ingredients like carotenoids, phenolics, phenols and flavonoid [2]. Medicinal plants have the potential to cure a variety of diseases but inadequate scientific information reduce its marketability [3]. The uses of medicinal products are increasing even in the developed countries and now the international market accounts for over 60 billion USD annually for herbal medications. Thus, the educated community, health care specialists and even the
Western qualified physicians show great interests in using traditional medicinal plants by keeping the impact on their patients too [4].
In Pakistan, most of the people uses medicinal herbal products because of inadequate or unreachable basic health facilities [5]. Furthermore, about 10% of the national floras of
Pakistan are used for medicinal and therapeutic purposes. In Pakistan phytomedicinal research is a new area of interest and it required further work to identify and document new medicinal plants and proceeded it for isolation of potent drugs against various diseases [6].
Plants are the primary source of biologically active phyto constituent’s also known as secondary metabolites with essential health benefits especially in Asia. To develop and
1
CHAPTER 01 INTRODUCTION promote research in pharmaceutics it is very necessary to explore and isolate secondary metabolites having antioxidant and other biological potentials from medicinal herbs [7].
During metabolic processes various reactive species are produced that can be linked with various physiological and pathological diseases such as stroke, rheumatoid arthritis, diabetes, inflammation, aging, mutagenicity, carcinogenicity and neurodegenerative disorders [8].
Recent reported studies has shown that plants are a major source of polyphenol that scavenge reactive species and efficiently avert oxidative cell damage [9]. Polyphenolic compounds are mainly present in fruits, vegetables and tea which greatly help in curing the oxidative stress and related diseases [10]. Among the plant bioactive phytochemicals flavonoids and phenolic acids are considered essential secondary metabolites having strong scavenging potential with wide pharmacological activities like like anti-diabetic [11], anti-cancer [12], anti- atherosclerosis [13], antimicrobial [14], immunomodulatory [15], reno or hepatoprotective effects [16] and improve human immunity [17].
1.2. Antidiabetic potential of phenolic compounds
According to the WHO evaluation report medicinal plants are of great importance as they are sources of safer modern and effective drug with comparatively low side effects [18].
Furthermore, pharmacological and chemical investigations are necessary to explore potential antidiabetic agents from plants sources [19].
The phenolic phytochemicals (Figure 1) which comprises of flavonoids, phenolic acids, stilbenes and lignans has been recommended to be subjected for the isolation of effective and protective drugs for diabetes. Further investigations about human clinical examination and animal models are also needed to confirm the advantageous results of these phytoconstituents against diabetes [20].
Antidiabetic medicinal herbs and their compounds have been confirmed to have beneficial effect in case glucose intolerance in human and also help in relieving complications related
2
CHAPTER 01 INTRODUCTION with diabetes. Plant phenolics can inhibit α-amylase and α-glucosidase that have wide health benefits by decreasing the related complications of type 2 diabetes [21]. Phenolic compounds majorly found in many fruits and vegetables particularly, in berries, grapes and tomatoes.
Flavonoids possess biological effects like anti-hyperglycaemic anti-hyperlipidemic and anti- cancer with different mechanism of action [22]. Some experimental studies have shown that polyphenolics found in fruits and vegetables are very helpful in controlling the postprandial blood glucose level by stimulating insulin response [23]. The natural phenolic constituents of grape, green and white tea are catechins that inhibit α-amylase and α-glucosidase thus might control hyperglycemic level. Phenolic acids stimulate glucose uptake that is comparable with the effect of oral hypoglycemic agent metformin and thiazolidinedione [24]. Literature study on female described that consumptions of five or more fruits and vegetables can cause substantial decrease in the threat of type 2 diabetes [25]. Chlorogenic acid, the major constituent of coffee has shown beneficial effects on type 2 diabetic individuals, that might interact with intestinal glucose absorption [26]. Furthermore, similar mechanism of action has been shown by antidiabetic drug acarbose in treatment of type 2 diabetes [27].
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CHAPTER 01 INTRODUCTION
Figure 1.1: Structures of important plant polyphenols
1.3. Link between berry fruits and type 2 diabetes
Due to the scientific exploration on berries in the recent years it has gain much importance for human beings as they have rich phytoconstituents and have great health benefits. The key flavonoid found in berries are proanthocyanins, anthocyanin, catechins and flavonols, while the hydroxylated phenolic acids are cinnamic and benzoic acid [28]. Many literature studies revealed that regular consumption of flavonoid rich berries fruit can alleviate type 2 diabetes
[29], ischemic diseases [30], aging effects , Parkinson's and Alzheimer's disease [31].
Many foods, herbal extracts, fruits, vegetables and their active ingredients have shown significant effect on type 2 diabetes and its complications. The active ingredients found in various berries like strawberries, raspberries, blueberries and black currants can reduce hyperglycemia through regulation of α-glucosidase and α-amylase enzymes [32]. Many authors reported the beneficial effects of berries fruits (black currants, strawberries, blueberries and raspberries), apples, vegetables and fruits juices that have exhibited positive health benefits recommending it to be a good source of phytochemical that could be used for
4
CHAPTER 01 INTRODUCTION curing type 2 diabetes and associated hypertension [33]. Apostolidis reported that high antioxidant potential of cranberry extract might be due to rosmarinic acid and total phenolic contents, suggesting its potential importance on diabetes. Fruits and vegetables have taken great consideration globally due to extensive beneficial effects on human health and are suggested to be a protective source against obesity, cancer, diabetes and heart related diseases due to high phytochemicals; flavonoid content, catechins, β-carotene, tannins and vitamins
[34]. Several studies have demonstrated that berry fruits can greatly cure T2D, which is characterized by impairments in metabolic functions by producing reactive species which ultimately destroy cells and defence system [35]. To properly manage T2D further work is needed for isolation of potent compounds from medicinal plants with low side effects [36].
1.4. Diabetes Mellitus (DM)
DM is considered as a metabolic disorder that increase glucose level in blood which ultimately reduce life expectancy, diminished life quality and finally leads to mortality and morbidity [37]. Around 25% world population of the developing and developed countries are suffering from Diabetes mellitus. Diabetes mellitus produced abnormal postprandial increase in blood glucose level either an inherited or due to insufficient secretion of insulin [38].
Reactive species of oxygen generated during metabolic processes are harmful for bio- molecules and are involved in the progression of various chronic diseases like cancer, diabetes mellitus, cardiovascular diseases, atherosclerosis, rheumatism, ischemic, nephritis,
Parkinson’s and Alzheimer’s disease (AD) [39]. Diabetes mellitus is considered as a major chronic ailments after cancer and cardiovascular diseases in human which is caused either due to insufficient insulin secretion by pancreatic islet cells of Langerhans or due to insulin resistance that leads to hyperglycemia [40]. Increase in blood glucose is associated with the activities of two intestinal enzymes; α-amylase and α-glucosidase that cause degradation of carbohydrates into disaccharides and finally to monosaccharide. Plasma glucose can be
5
CHAPTER 01 INTRODUCTION regulated by inhibiting these two enzymes that would slowdown carbohydrate degradation into glucose [41].
1.5. Clinical manifestation of DM
Clinically diabetes mellitus can be characterized by unnecessary urine excretion, continuous appetite, loss in weight, thirst, fatigue and blurred vision. The major metabolic side effects of
DM includes keto acidosis, hyper-osmolar coma, neuropathy, renal failure, retinopathy, skin problems and increasing threats of cardiovascular complications [42]. DM is documented as the seventh cause of death worldwide that effect 100 million people every year [43]. It has been estimated that in 2003 diabetic people were 150 million and this number will be increased to 300 million by 2025. DM is the endocrine ailment mainly associated with hyperglycemia and hyperlipidemia due to impairments in metabolic pathways [44].
1.6. Diabetes mellitus classifications
Diabetes mellitus can be further categorise into two types. Diabetes Type I also stated as infantile onset diabetes that occurs due to pancreatic β-cells destruction and estimated around
5-10% cases worldwide [45]. The second category is diabetes type II also called adults onset diabetes that have been estimated to be approximately 90-95% globally. Type I diabetes is initiated by inadequate insulin secretion while type II diabetes is usually due to progressive rate of insulin resistance in liver and peripheral tissues [45].
1.6.1. Diabetes Type I
Diabetes type I is categorized into immune or nonimmune type I that is mostly prevalent amongst children. The clinical manifestations of immune-mediated type I diabetes are loss in weight, polyuria, polydipsia and ketosis. According to the American Diabetes Association the diabetes with immune mediated is designated as type IA which happens due to pancreatic
6
CHAPTER 01 INTRODUCTION
β-cells destruction while the nonimmune mediated is termed as type IB with severe insulin insufficiency [46].
1.6.1.1. Pathogenesis of diabetes type I
Diabetes type I occurs due to pancreatic β-cells damage. According to literature autoantibodies expression in young age is an indication of key threat of type I diabetes development. However, progression of type I diabetes due to autoantibodies in human has not been documented [47]. Generally, investigation on type I diabetic individuals represent the occurrence of CD-4 & 8, B-lymphocytes, T-lymphocytes and macrophages that leads to pancreatic islet cells damage. It has been confirmed through scientific investigation that type
I diabetes in animal can be cured with anti-CD3 [48].
1.6.1.2. Prevention and new treatments
In spite of significant progress in diabetes treatment by orally taken hypoglycemic agents, exploration for new antidiabetic herbal medications continues due to limitations of the current synthetic drugs [49].
Non-pharmacological diabetes treatment includes mineral supplementation, acupuncture and hydrotherapy, oral hypoglycemic agents, exogenous insulin and transplantation. These treatments are unaffordable and needs expertise in prescription and administration with many signs of complications [50]. Therefore, herbal preparations have received a wide consideration as an alternative way to recompense observed inadequacies in conventional pharmacotherapy [51]. World Health Organization reported 80% population in the developing countries used herbal medications prepared from medicinal plants along with great importance over the whole world to treat many ailments [4].
Up till now the introduction of insulin remains the key treatment for type I diabetes.
Furthermore, the administration of quickly absorbed insulin analogues and insulin glargine
7
CHAPTER 01 INTRODUCTION has been newly introduce to the insulin market can provide same results that has been gained through insulin pumps [52]. Metformin treatment together with insulin has also been recommended for type I diabetes. Transplantation of islet tissues has also beneficial effects on type I diabetes but it is limited due to auto immunity and less availability of donor tissues
[53].
1.6.2. Diabetes Type 2
Diabetes Type 2 has estimated 90% of non-insulin dependent cases that were most prevalent in the age range of ≤30 years. T2D is characterized by hyperglycemia however other complications are also closely associated with T2D like insulin resistance, low insulin secretion, hyperinsulinemia and consumption of carbohydrate rich food [54]. T2D is the major health problem both in the developing and underdeveloped countries. W.H.O has estimated that almost 350 million individuals were suffering from diabetes, globally. This increase in diabetes type 2 has counted 90% and it will be epidemic in the nearby future if strong preventive measures were not taken [55]. The pathophysiological procedures involved in the etiology of this disease is insufficient insulin that might be due to insulin resistance or autoimmune damage of islet β-cells [50]. Thus inadequate insulin or impaired insulin action on target tissues disrupts the carbohydrates, proteins and lipids metabolic pathways.
Hyperglycemia is associated with polyuria, polydipsia, fatigue, polyphagia, loss in weight
[51] and micro vascular impairments such as heart disease, peripheral vascular ailments, stroke, nephropathy, retinopathy and neuropathy [56]. There has been considerable discussions, that the main cause of T2D in adults is insulin resistance or hypo secretion while in children’s it is due to the deficits in insulin action which ultimately cause impairments in
β-cell functions that leads to over production of insulin which produces insulin resistance and finally diabetes. Furthermore, hyperglycemia further worsen the condition of hyperglycemia by itself that leads to a condition termed as glucose toxicity [57].
8
CHAPTER 01 INTRODUCTION
1.6.2.1. Pathophysiology of type 2 diabetes
The main pathophysiological cause of T2D is hypo secretion of insulin. Moreover, hyperglycemia occurs mainly due to deficits in insulin signaling that inactivate tyrosine kinase action of insulin receptor (IR) [58]. Hence, T2DM can be treated by pharmaceutical agents that activates IR receptors to reinstate auto phosphorylation process in insulin resistant cells [59]. Maintenance of glucose homeostasis required translocation of glucose transporter
(GLUT4) from intracellular vesicles, whose expression mostly occurs in adipose tissue, skeletal and cardiac muscle. Impairments in GLUT4 expression is strongly associated with deficits in insulin signaling that ultimately leads to high glucose in liver tissues [60].
1.6.2.2. Preventive measures and treatments of type 2 diabetes
The only way to control T2DM is the use of proper diet, insulin, anti-diabetic medications; thiozolidinediones, biguanides, sulfonylureas (glibenclamide, glimepiride), using specific enzyme inhibitors like acarbose and miglitol to reduce demands for insulin. However, these drugs cause some side effects like nausea, diarrhea, myocardial infarction, dyspepsia, peripheral edema and dizziness. Mostly anti-diabetic medications have been isolated from plants sources that reinstate insulin production and inhibiting intestinal glucose absorption
[61]. The tendency in screening the antidiabetic and hypoglycemic potential of medicinal plants has increased, as it provides an important source to isolate novel effective drugs.
However, the WHO has recommended healthy food consumption and daily exercise as an effective technique of controlling type 2 diabetes. The ideal aim to control T2D is an effective control of blood glucose level, HbA1c, hypertension and hyperlipidaemia. However insulin is the main medical therapy accepted by FDA for diabetic patients [62].
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CHAPTER 01 INTRODUCTION
1.6.2.3. Lifestyle changes
In diabetes management the important concern is changes in the life style. To educate the people about this serious problem one have to make their minds to monitor their daily blood glucose level by itself. To achieve this important task a national standards for diabetes have been planned in which education regarding self-management have given utmost importance
[63]. Good life style includes healthy eating habits, behavioural modification policies, reducing use of high caloric foods and whole family must encourage him to eat low carbohydrate food. Increasing daily physical activity and exercise are the main components of therapy in weight management that can decrease insulin resistance. However better treatment will requires combination therapy that must include antidiabetic agents, insulin along with better food and good lifestyle [64].
1.6.2.4. Pharmaceutical Therapy to reduce postprandial hyperglycemia
Presently, in US the following pharmaceutics are available for diabetes treatment both in children’s and in adults one [65]. The current pharmaceutics and their effective role are given as follow:
i. Biguanides: It includes metformin which effectively enhancing hepatic tissues
function and lowers glucose level.
ii. Sulfonylureas: It includes chlorpropamide, acetohexamide, tolazamide, glimepiride,
gliclazide, glipizide, tolbutamide and glyburide which stimulate secretion of insulin.
iii. Meglitinide: It includes repaglinide which stimulate insulin secretion.
iv. Glucosidase inhibitors: It includes miglitol and acarbose which slows down
carbohydrate digestion and absorption.
v. Thiazolidenediones: It includes rosiglitazone, troglitazone and pioglitazone which
improve insulin secretion.
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CHAPTER 01 INTRODUCTION
The first factor in T2DM management is the regulation of postprandial glucose level through herbal and synthetic medications. Furthermore, various side effects are associated with synthetic medicines, but the medicines obtained from plants sources are usually connected with least or no side effect and thus exert an increasing attention in diabetes medications in almost all human societies [66]. Various attempts have been made all around the world to isolate biologically active compounds from plants with broad range of biological activities.
Kirithikar and Basu have reported around 800 plants with antidiabetic potential with no documented side effects as compared to synthetic antidiabetic drugs [67]. Furthermore, search for new antidiabetic drugs from plants sources is still attractive due to presence of active natural compounds like glycosides, alkaloids, terpenoids, flavonoids, carotenoids, etc. which demonstrate alternative and safe effects on DM [68]. Because of great therapeutic values the herbal preparations make them the key player for all available therapies due to easy availability, low side effects and low cost, especially in rural areas. As every plant has a natural habitat due to which it is limited to that specific areas on the globe. Therefore, it is needed to study unexplored plants and discover the new antidiabetic plants in every part of the world [69].
1.6.2.5. Inhibition of carbohydrate digesting enzymes
In 1980 the α-amylase and α-glucosidase enzyme inhibitors were introduced as antidiabetic drug which effectively control glucose level by lowering carbohydrates metabolism [70]. The
α-glucosidase inhibitors were considered as an efficient therapeutic agent that considerably reduces postprandial glucose after carbohydrate rich diet. For controlling postprandial hyperglycemia (PPHG) acarbose inhibitor were used successfully to inhibit α-amylase and α- glucosidase, but miglitol and voglibose inhibited only α-glucosidase. Metformin the first oral agent that takes benefit on sulfonylureas in lowering of glucose and HbA1c levels [71]. It is necessary to explore medicinal herbs to isolate potent enzymes inhibitors with promising
11
CHAPTER 01 INTRODUCTION biological activities. Commercially, these inhibitors were formulated in such a way to target carbohydrate digesting enzymes in order to reduce the metabolic process that leads to control blood glucose [70].
It is assumed that about 80% diabetic people belong to middle income countries. Therefore, efforts have been made to search inhibitors of α-amylase and α- glucosidase from plants, marine algae, bacteria, and fungi because the in use drugs are quite expensive. Majority of plant extracts and pure compounds have been effectively used against either α-amylase or α- glucosidase enzymes [72].
The unwanted side effects of the presently available synthetic enzymes inhibitors drive the attention of scientists to explore new inhibitors from natural sources. Being a global lifestyle ailment that affects millions of people with variety genetic backgrounds, the search for alternate inhibitors from the pharmacogenetics point of view is needed. The side effects of acarbose and miglitol are intestinal troubles including diarrhea. Therefore plants are subjected to exploration of suitable inhibitors for α-amylase and α-glucosidase with effective protection
[73].
1.6.2.6. Treatment of hypertension
According to diabetologists hypertension is a life-threatening process in children which can be cured using ACE inhibitors, diuretics and calcium antagonists that prevent diabetic nephropathy [74].
1.6.2.7. Treatment of hyperlipidemia
Hyperlipidemia associated with T2D in children is a serious problem. Hyperlipidemia can be cured by taking good quality food, proper activity and medications that are helpful in stopping weight loss and maintaining lipid levels [75].
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CHAPTER 01 INTRODUCTION
1.7. Memory
The memories are stored in the brain portion called the limbic region. The limbic region comprises of the hippocampus, the para hippocampal area, the amygdala, portions of the cortex and the cerebellum [76]. The limbic region is involved in memory saving, coordination of sensation and motivation along with coordination of endocrine system. Hippocampus region manage behaviour, short and long term memory while the cerebral cortex handle processing of short to long term memory.
Amygdala is involved in processing of memory, social, emotional and sexual behaviour while cerebellum perform procedural memory [77]. There are two types of memory, short and long term memory. The first model about memory was developed in 1968 called
Atkinson shiffrin model which recommends three stages of memory like sensory (very short), short and long term memory [78].
1.7.1. Sensory memory
The duration of sensory memory is very short and last for less than second. It drops very speedily once observing a thing or item. After observation the stimuli were efficiently filter through attention and finally information are transferred from sensory to short term memory
[78].
1.7.2. Short term memory
The short term memory usually retained for seconds to hours. It accounts for such working memory that has been stored in our senses for longer time like memorising something. Peterson reported that short term memory retains for ≤ 30 seconds after observing anything [79]. Prefrontal cortex plays an essential part in memory while the consolidation of short to long term memory is managed in the hippocampus region. George
Miller reported that due to low capability of short term memory a normal human can holds
13
CHAPTER 01 INTRODUCTION information only for 5-9.5 seconds [80]. The Atkinson Shiffrin model recommends that processing of short memory to long term memory will only takes place if the learning info is sufficiently fast. This principally means that one would have enough capacity for accumulation and storing of new information [78].
1.7.3. Long term memory
The information in long term memory declines very slowly and can be retained for unlimited time. Short term memory changes to long term memory through consolidation which involves rehearsal and meaningful associations [78]. It should be noted that hippocampus only store information, cortex is involved in storage process while amygdala control emotional type of memory [81].
Long term memory is further categorised into explicit and implicit memory. Conscious information belongs to explicit memory, while information that you remember unconsciously and easily is known as implicit memory. Explicit memory can be intentionally and consciously recalled which means that the information can be explained or declared accomplished with various daily activities [82].
1.8. Memory disorders
Memory disorders are discussed in alphabetical order as follow:
1.8.1. Acquired Brain Injury
The main cause of acquired brain injury (ABI) is the damage of brain that proceeds after birth. The impairments related with ABI are cognitive deficits, behavioural, physical and emotional impairments that occurs in either traumatic or non-traumatic injuries to brain causing permanent or transitory changes in brain functioning [83].
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CHAPTER 01 INTRODUCTION
1.8.2. Agnosia
Agnosia is a condition that occurs after brain injury, specifically in the parietal or occipital region that accounts for inability to distinguish individuals, materials, or sounds. Recovery from agnosia depends on severity and position of brain injury, type of agnosia and neurological disorder [84].
1.8.3. Alzheimer's disease (AD)
AD is a chronic neurodegenerative condition of mental functional deficits leading to death.
World Alzheimer’s report declared that AD patients’ accounts for about 46.85 million in
2015 worldwide and this figure will be double in 2030 and might probably become three times in 2050. The rate of AD in female was more prevalent with severe cognitive impairment when compared to male sufferers [85]. The remarkable sign of AD includes memory impairments, cognitive functions deficit, alterations in learning, imprecise and inappropriate behaviour and amnesia. AD is characterized to be the most common form of dementia, which places an extensive and increasing burden on patients and society.
Deposition of βA peptide and neurofibrillary tangles (NFT), formation of hyper phosphorylated tau protein, low level of neurotransmitter ACh and loss of neurons are the pathological signs of AD [86], greatly associated with neuroinflammation, excitotoxicity and oxidative stress [87]. A cholinesterase’s; AChE & BChE enzyme causes the degradation of neurotransmitters ACh which produce cholinergic deficits. Inhibitions of these two enzymes might cure AD, Parkinson’s and Dementia disease. The fundamental root of AD is still uncertain, an imbalance in oxidative stress is strongly associated with early stages of AD
[88].
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CHAPTER 01 INTRODUCTION
1.8.4. Amnesia
Amnesia is a memory disorder comprising of anterograde and retrograde amnesia. Amnesic patients have disruptions in memory but no impairments in cognition, awareness and attention. In retrograde amnesia an individual is unable to remember the previous memories while in anterograde no new memories are formed due to failure in thalamus and hypothalamus regions [89]. Neurological ailments create a great burden on society which requires a therapeutic way to manage the threat of memory related disorder including amnesia, dysfunction in cognition and learning which might be due to exposure of brain cells to oxidative stress [90].
1.8.4.1. Traumatic brain injury
The pathological signs of traumatic brain injury (TBI) include cognitive dysfunction, amnesia and long term post-traumatic stress depending on severity of the injury [91]. It has been reported that a temporary or permanent amnesia condition occurs after head injury. An individual with head injury is unable to recall information during PTA condition. Instead, after PTA, forgetting process becomes normal. Along with deficits in memory TBI also progressively causes AD. TBI-associated death mostly occurs among elderly population due to high incidence of falls [92].
Oxygen deficiency can also causes brain injuries due to strokes and cerebrovascular accident
[93]. Traumatic brain injury can also cause the most common psychiatric illness called the major depressive disorder (MDD). Almost 50% of TBI patients are at risk of developing
MDD after injury. Although some researchers have found that people with more severe TBI have a greater possibility of experiencing worse depressive symptoms than people with less severe injuries [94].
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CHAPTER 01 INTRODUCTION
1.8.5. Dementia
Dementia is a neurodegenerative and memory disorder in which loss in thinking ability and language failure e.g., object designation, noun making and verb usage, occurs [95]. It has been reported that dementia patients were 47 million in 2015 globally, which might be triple in 2050. AD and aging represent the common causes of Dementia. Moreover Type 2 diabetes is also associated with vascular dementia, cognitive failure and AD [96].
Sleep is also a major factor to develop dementia, particularly in adults with age ≥60 years facing troubles in normal sleep that ultimately leads to cognitive failure and AD. The sleep disturbance is supported by evidence that it induces systemic inflammation, which is progressively observed in the course of Alzheimer’s disease. This Systemic inflammation finally produces variations in microglia cells of CNS which are observed in AD and cognitive aging. Literature study revealed that elderly schizophrenic patients are mostly associated with increased severity of AD and dementia [97].
1.8.6. Hyperthymestic syndrome
Patients with hyperthymestic syndrome also known as highly superior autobiographical memory (HSAM) can remember any event with high level of comprehensive autobiographical memory. This syndrome rarely occurs as there are few reported cases worldwide [98]. Parker et al. reported important features of hyperthymestic condition in which an individual spend a lot of time in thinking and recalling their past events.
Autobiographical memory includes the recollection of personal information in much detailed manner along with realistic information. However, the personal experiences varies considerably across the individuals [99]. Literature has been mainly focused on two syndromes; highly superior and severely deficient autobiographical memory (HSAM &
SDAM). In HSAM an individual remember a lot of information [100] while, SDAM individuals are unable to remember autobiographical memory with more detail and is
17
CHAPTER 01 INTRODUCTION considered to be as developmental conditions. The signs of SDAM are unknown but some neurological ailment may cause impairments in memory without any deficit in cognition
[101].
1.8.7. Huntington disease
Huntington disease is a genetic brain disease in which the patient facing unrestrained movements, loss in emotional and intellectual ability. Huntington parent have 50% chances that the incoming children would be Huntington due to inherited nature of the disease [102].
In adults the disease most probably appears in the age of 30-50 years. Individuals effected with Huntington disease experienced petulance, depression, unconscious trembling movements, poor coordination of the body parts, failure in making decisions, learning, taking a thing and intellectual capabilities [103].
A rare type of Huntington disease observable in infantile is characterized by continuous falling, troubles in waking, movement problems, inflexibility, indistinct communication, emotional and psychological alterations [104].
1.8.8. Parkinson’s disease (PD)
Parkinson is another neurodegenerative disorder which shares same symptoms that occurs in normal aging. Parkinson occurs when dopamine productive cells dying off, that control normal moments [105]. The clinical manifestations of this disease are; motor nerve dysfunctions, trembles, tardiness, impaired stability, exhaustion and muscular inflexibility while the non-motor signs includes impairment in in sensory, cognition and emotion, sleep, sexual and neuro psychiatric problems [106].
PD is also closely associated with memory impairments due to injury in frontal lobe of brain that might also be appears in normal aging but not strongly correlated. Cognitive impairment is the major cause of PD that comprised mild cognitive impairment (MCI) to dementia [107].
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CHAPTER 01 INTRODUCTION
Like AD, PD is also characterized by spreading through prion-like misfolded proteins [108].
Clinically spreading of misfolded proteins occurs by extracellular transmission and axonal transportation with neuronal connectivity [109]. This model suppose the spreading of prion- like protein viz βA and tau proteins in AD and α synuclein deposition in PD.
1.8.9. Stress
Stress is a sensation of emotional or physical tension that causes frustration, or nervousness due to any event or thought. In response to physical or mental stress an individual initiate secretion of stress hormones like epinephrine, corticoids, corticotrophin releasing factor and adrenocorticotropic hormone for fight or flight reactions [110].
However as a result of continuous stimulation of stress stimuli impairs functioning and episodic memories or permanent neuro physiological and psychiatric variations that leads to
AD [111], while acute stress might ultimately progress to post traumatic stress disorder (PTSD) [112]. Indeed, the emotional and intellectual variations as a result of stress will create a psychiatric and neurological illness that has also been detected in AD and PTSD
[113]. Furthermore the limbic regions control memories and intellectual functions along with regulation of stress receptors.
Opioid receptors are present in CNS stimulated in response to stress stimuli that regulate emotional responses, learning and memory. However alterations in opioids can ultimately causes stress stimulated memory disorders, AD and PTSD [114]. Reported research suggested the close interaction between opioid system and stress that perform important task in processing of memory under stress condition [110].
1.8.10. Wernicke Korsakoff syndrome
A severe vitamin deficient neurological ailment called Wernicke Korsakoff syndrome (WKS) occurs due to thiamine deficiency and has also shown close association with excessive
19
CHAPTER 01 INTRODUCTION alcohol intake [115]. Pronounced signs of WKS comprise misperception, inability of acquiring new information, profound amnesia, disorientation and memory deficits. The effects of alcohol intake are distinct from WKS symptoms which are long lasting [116].
1.9.Amnesia and their different types
Amnesia is a condition with comparatively great importance in the neuro psychological literature because memory has great importance in some one life to define itself and their place along with normal life working. Amnesia occurs when brain portion responsible for retrieving stored memories is not working properly as compared to healthy individuals [117]. Amnesia has the following types:
1.9.1. Anterograde amnesia
Anterograde is the common type in which an individual is unable to store new information after any event due to brain injury, however long term memory remains undisturbed. The pronounced signs of brain injury may be due to excessive alcohol intake, malnourishment, WKS, operation, cerebrovascular actions, brain stroke and shock. This type has also been observed in AD and dementia [118].
1.9.2. Retrograde amnesia
In retrograde type of amnesia the individual is unable to remember memories before the onset of events while the new information after the event becomes encoded. Common signs are cerebrovascular accident, alcohol intake, brain stroke, and encephalitis and brain injury [119].
1.9.3. Post traumatic amnesia
The main cause of post traumatic amnesia is brain injury due to accident or fall that leads to loss in memory either temporary or might be permanent with anterograde, retrograde, or both. The sufferer is unable to process memory from short to long term after head injury. It
20
CHAPTER 01 INTRODUCTION will possibly indicate that recovery takes more time from remaining amnesic symptoms
[120].
There is close association between post traumatic amnesia and traumatic stress disorder
(PTSD) due to mild brain injury leading to impairments in memory. In US, PTSD accounts the fifth chronic and most prevalent mental condition [121]. Signs of PTSD are; memory loss, hyper arousal and vigilance, and emotional shocking. However besides painful events the previous events in life and genetic family background may also be considered [122].
1.9.4. Dissociative amnesia
In the Second World War dissociative amnesia was categorized as hysteria which later on was considered as psychogenic and now dissociative amnesia. It was also termed as functional amnesia [123] and amnestic block syndrome in which the forgotten memories are blocked due to poor access to the consciousness but might be recovered later on [124] .
Dissociative amnesia may probably be due to brain damage, physical trauma and psychic stress [125]. Among other psychiatric diseases dissociative type is the rarer form of amnesia only accounts 0.2-7% [126].
1.10. Causes of Amnesia
Main causes of amnesia include AD, TBI, encephalitis, dementia, Parkinson’s disease, stroke or attacks [127, 128]. While tumor in brain schizophrenia, unlawful behaviour, brain surgery, head injury or trauma, lack of adequate oxygen in the brain, Wernicke-Korsakoff syndrome, drug (tranquilizers) induced form are considered as less common reasons to become amnesic [129-131].
1.11. Preventive measures for amnesia
Due to less availability of proper treatment for amnesic patients to improve their memory only the psychological counselling and the occupational therapy can be help to recover
21
CHAPTER 01 INTRODUCTION memory after developing strategies that how to understand and remember information related to daily events. However technical support like use of digital device can also help in retrieving of loss memory. For amnesia management there is no proper medicine available however, therapeutically effective pharmaceutical compositions are used to treat amnesia
[132]. However, some forms of amnesia like WKS can be cured by taking food rich in thiamine while alcoholism can be managed by prevention of alcohol drinks and illegal drug.
Brain injury is considered to be the main cause of amnesia so essential measures should be taken to reduce further complications [133].
Along with pharmacological treatments, social and emotional ways can also be helpful for anterograde amnesic patients. However main psychotic depression can be treated by applying electroconvulsive therapy (ECT) when pharmacotherapy failed. The neurocognitive side- effects such as anterograde amnesia regularly occur during the treatment [134]. The retrograde amnesiac type can recovered by consolidation procedure if carried for longer period of time in cases of damage in hippocampal area and impairments in recollecting memory prior to the time they became amnesic [135].
Currently researchers and investigators are searching out certain neurotransmitters like γ- amino butyric acid (GABA), glutamate (neuronal glutamate transporter), dopamine (DAT) and serotonin (SERT) that are related with memory recall in some kind of medication that might help in treating amnesia and memory loss [136].
1.12. The Link between type 2 diabetes and neurodegeneration
Literatures studies have strongly correlated T2DM and AD. The pronounced signs of both
T2DM and AD includes brain degeneration, low rate of cerebral glucose metabolism and insulin resistance in brain cells [137]. However loss of neurons and deposition of amyloid
(Aβ protein) and hyper phosphorylated tau protein tangles formation have been considered to be the major signs of AD [138]. Mounting evidences recommends the importance of
22
CHAPTER 01 INTRODUCTION amyloidogenic proteins in stimulating insulin resistance or destruction of β-cell, as it is required to drive the pathogenesis of these chronic diseases including neuroinflammation
[139]. Reported evidences suggest that amyloidogenic and amylin (islet amyloid) proteins accumulation finally leads to toxicity of β-cell and chronic T2DM [140]. It has been observed that amylin protein can also be accumulated in AD brain along with diabetic patients that strongly correlate with Aβ and tau proteins to promote amyloid deposition that contribute to cognitive deficits, impaired mitochondrial function, neuroinflammation, and neurodegenerative process [137]. Neuroinflammation finally stimulate the microglia cells which ultimately produce inflammatory cytokines and toxicity that leads to loss in neuronal functions [141]. The link between T2DM and AD has been presented in Figure 1.2 & 1.3.
Literature studies have confirmed that T2DM has strong correlation with cognitive impairments like dementia [142]. Neurological and neurons damage can be observed in around two-thirds of T2DM cases. Diabetes and deficits in cognition are closely related caused due to damages in cortical regions and particularly the hippocampus [142] that leads to impairments in insulin sensitivity, inflammatory mechanism , tau protein signaling, and mitochondrial functions [143]. Insulin resistance in T2DM causes hyperglycemia that mediates vascular damage and reduce the supply of oxygen and nutrients to the brain that leads to worse memory and neuronal function [144].
Mechanism that underlies T2DM can also be correlated with more progressive cognitive conditions like dementia [142]. The progression of T2DM into dementia is very serious problem and makes a huge burden on society due to severe chronic mental complications that leads to high mortality rates [145].
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CHAPTER 01 INTRODUCTION
1.13. Mechanism that underlies cognitive impairments in T2DM
1.13.1. Insulin Resistance
Insulin resistance is the major cause of T2DM has also been involved in AD, cognitive dysfunction or dementia (Fig. 1.2). Furthermore, AD is designated as type 3 diabetes due to their close association with DM [146]. In the hippocampus region of brain the IR are responsible for accelerating insulin signaling that in turn help memory enhancement [147].
Impairments of hippocampal region signaling can lead to events that promote neurodegeneration. In AD pathology the IR located in the hippocampus region of CNS has greatly affected [148]. Impairments in insulin binding with IR may cause reduction in insulin signaling and inactivation of IRS-1 which in turn reduce phosphatidyl-inositol-3-kinase and
AKT activity that leads to improved activation of glycogen synthase-kinase-3β which ultimately causes deposition of Aβ, TNF-α, and hyper phosphorylation of tau protein.
Furthermore, insulin resistance greatly affect the affinity of insulin degrading enzyme for Aβ protein that leads to their accumulation [149]. Literature studies have also reported low level of insulin both in AD and cognitive impaired patient along with T2DM [150].
1.13.2. Inflammatory mechanism
Impairments in insulin secretion may produce inflammatory cytokines that cause apoptosis and β-cell injury that has been observed both in T2DM and AD (Fig. 1.3). Previous studies reported that pro-inflammatory cytokines can cross BBB and reduced secretion of ACh and hippocampal nerve growth factor expression that leads to AD and memory impairments
[149].
Advance glycation end (AGEs) product can also link diabetes and AD which are produced in response to hyperglycemia [151]. AGEs are involved in activation of inflammatory mediators like nuclear factor-kappa B (NF-κB), IL-6, TNF-α and C-reactive protein. Previous observations suggested that AD patient’s brain tissue have shown disruption in BBB that has
24
CHAPTER 01 INTRODUCTION also been observed in diabetes and dementia [149]. Additionally there is close association between Aβ and AGEs receptor that stimulates Aβ inflow through the BBB and microglia cells activation that leads to neuronal death [152]. Reported studies showed that deficit in vascular function may also leak the BBB and thus increase accumulation of Aβ in the brain
[153].
1.13.3. High glucose concentration
High glucose concentration is the major pathological characteristic of diabetes mellitus that exert a negative effect on cognition. Production of AGEs product in response to high glucose may exert harmful effects on neurons and mitochondrial functions [154]. Furthermore the normal functions of microglial brain cells has been greatly affected by AGEs product that causes neurons injury and leads to progression of brain related complaints [155].
1.13.4. Oxidative Stress
Defects in antioxidant defence mechanism causes over production of ROS and RNS that leads to AD and T2D [156]. ROS production in response to high glucose concentrations may cause lipid peroxidation, impaired biochemical pathways, deficits in mitochondrial and β-cell functions can induce T2DM and AD complications. Mitochondrial dysfunction and oxidative stress may cause neuron injury due to insufficient ATP and calcium supply that leads to AD
[157]. Furthermore, ROS has been closely associated with tau protein hyper phosphorylation, accumulation of TNF and Aβ protein that ultimately causing oxidative stress like AD and
T2DM diseases [158]. All these mechanism are summarised in Figure 1.4.
Conclusive statement
It is concluded that E. umbellata is a rich source of potential bioactive materials which exhibited potent in vitro and in vivo antidiabetic and neuro-protective activities. Due to richness of polyphenolic compounds the plant exhibited multi-functionality. E. umbellata fruits
25
CHAPTER 01 INTRODUCTION could be recommended for controlling diabetes, memory impairments and neurological disorders. However, further work is required to investigate their exact mechanism and cellular pathways which are contributing in this cascade. The overall work has been shown in
Figure 1.5.
Figure 1.2: Various factors that link the association between T2DM and AD [309]
26
CHAPTER 01 INTRODUCTION
Figure 1.3: Effect of chronic inflammation on the association between T2DM and AD [319].
27
CHAPTER 01 INTRODUCTION
Figure 1.4: Mechanistic overview that link cognitive impairments in T2DM & neurodegeneration
28
CHAPTER 01 INTRODUCTION
Figure 1.5: Graphical overview of the whole work
1.14. Aims and objectives of the study
i. To explore the antioxidant potential of E. umbellata Thunb. fruit extract/fraction and
isolated compounds.
ii. Isolation of active antidiabetic and antiamnesic secondary metabolites from E.
umbellata Thunb. and their phytochemical studies. iii. To validate the antidiabetic and antiamnesic potential by molecular docking
mechanism.
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CHAPTER 02 LITERATURE AND REVIEW
2. LITERATURE AND REVIEW 2.1. Elaeagnaceae Family
Elaeagnaceae is also known as plant oleaster, of the order Rosales that are composed of shrubs and small trees. The Elaeagnaceae family is native to Australia and Northern to southern regions of tropical Asia. Elaeagnaceae family has three genera: Hippophae,
Shepherdia and Elaeagnus, and almost sixty species [159]. They are flowering thorny plants, with hairs or scales coated simple leaves. Some xerophytes and halophytes species of
Elaeagnaceae have nitrogen fixation ability that might them useful for soil repossession
[160]. The relative phytochemical constituent’s profiles and therapeutic functions of
Elaeagnaceae genera have been shown in Table 2.1.
Table 2.1: Assessment of relative phytochemical constituent’s profiles and biological roles of Elaeagnaceae genera
S.No Genera Phytoconstituents Biological role References I Carotenoid Provide Protection Quercetin [161] from nicotine abuse Isorhamnetin Hippophae cerebroside Cytoprotective [162] Kaempferol Ursolic acid Increase Hippophae Oleanolic acid concentration of [163] Rhamnoides α-linolenic acid Counterbalance hydroxyursolic acid [164] arsenic toxicity
Palmitic acid-1-O-Hexadecanolenin Amend dyspepsia [165]
Acylated flavonol glycosides Antioxidant activity [166] II Carotenoids Inhibit HIV-1 Leucoanthocyanins [167] reverse transcriptase Vitamin C Relieve Shepherdia complications of argentea Catechol diabetes (buffalo berry) Flavonols impairments [168, 169] Tannins
30
CHAPTER 02 LITERATURE AND REVIEW
III Phenolics Antioxidant [170] Antioxidant and Phytoconstituents DNA protective [171] potential Antinociceptive and Flavonoid anti-inflammatory [172, 173] activities Monoterpenes Sesquiterpenes Ursolic acid [174] Elaeagnus sp. Oleanolic acid (silverberry) Lupeol Anti-inflammation Botulin Syringic acid Kaempferol Elaeagnoside β-sitosterol Inhibition of enzyme 12-hydroxy-8,10-Octadecadienoic chymotrypsin [175] acid anti-carcinogenic Carotenoid (lycopene) activity, as well as [176, 177] hepatoprotective effect
2.2. Elaeagnus Genus
Elaeagnus species have shown a lot of pharmacological effects due to their folkloric therapeutic uses, diverse chemical composition and health benefits. Genus Elaeagnus is composed of around ninety species, distributed in Asia, North America South and Europe while almost 67 species (55 endemic) in China. Elaeagnus species including 1. Elaeagnus macrantha, 2. Elaeagnus obovatifolia, 3. Elaeagnus luxiensis, 4. Elaeagnus griffithii, 4a.
Elaeagnus griffithii variety griffithii, 4b. Elaeagnus griffithii variety pauciflora, 4c.
Elaeagnus griffithii variety multiflora, 5. Elaeagnus pingnanensis, 6. Elaeagnus loureiroi, 7.
Elaeagnus gonyanthes Benth., 8. Elaeagnus geniculate, 9. Elaeagnus obtuse, 10. Elaeagnus formosana, 11. Elaeagnus tonkinensis, 12. Elaeagnus schlechtendalii, 13. Elaeagnus oldhamii Maxim., 14. Elaeagnus conferta Roxburgh, 15. Elaeagnus macrophylla Thunberg,
16. Elaeagnus pallidiflora, 17. Elaeagnus thunbergii, 18. Elaeagnus wenshanensis, 19.
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CHAPTER 02 LITERATURE AND REVIEW
Elaeagnus taliensis, 20. Elaeagnus luoxiangensis, 21. Elaeagnus lanpingensis, 22. Elaeagnus pilostyla, 23. Elaeagnus xizangensis, 24. Elaeagnus liuzhouensis, 25. Elaeagnus heterophylla, 26. Elaeagnus tubiflora, 27. Elaeagnus bockii Diels, 27a. Elaeagnus bockii variety bockii, 27b. Elaeagnus bockii variety muliensis, 28. Elaeagnus davidii Franchet, 29.
Elaeagnus retrostyla, 30. Elaeagnus lanceolata Warburg ex Diels, 31. Elaeagnus viridis
Servettaz, 32. Elaeagnus longiloba, 33. Elaeagnus xichouensis, 34. Elaeagnus delavayi
Lecomte, 35. Elaeagnus xingwenensis, 36. Elaeagnus sarmentosa, 37. Elaeagnus cinnamomifolia, 38. Elaeagnus glabra Thunberg, 39. Elaeagnus pungens Thunberg, 40.
Elaeagnus tutcheri Dunn, 41. Elaeagnus henryi Warburg ex Diels, 42. Elaeagnus difficilis
Servettaz, 42a. Elaeagnus difficilis variety difficilis, 42b. Elaeagnus difficilis variety brevistyla, 43. Elaeagnus yunnanensis Servettaz, 44. Elaeagnus angustifolia Linnaeus, 44a.
Elaeagnus angustifolia variety angustifolia, 44b. Elaeagnus angustifolia variety orientalis
(Linnaeus, Kuntze) 45. Elaeagnus oxycarpa Schlechtendal, 46. Elaeagnus mollis Diels, 47.
Elaeagnus grijsii Hance, 48. Elaeagnus calcarea, 49. Elaeagnus stellipila, 50. Elaeagnus jingdongensis, 51. Elaeagnus argyi, 52. Elaeagnus micrantha, 53. Elaeagnus bambusetorum,
54. Elaeagnus guizhouensis, 55. Elaeagnus jiangxiensis, 56. Elaeagnus courtoisii Belval, 57.
Elaeagnus umbellata Thunberg, 58. Elaeagnus magna (Servettaz), 59. Elaeagnus nanchuanensis, 60. Elaeagnus wushanensis, 61. Elaeagnus multiflora Thunberg, 61a.
Elaeagnus multiflora variety multiflora, 61b. Elaeagnus multiflora variety tenuipes, 61c.
Elaeagnus multiflora var. obovoidea, 61d. Elaeagnus multiflora variety siphonantha (Nakai),
62. Elaeagnus angustata (Rehder), 62a. Elaeagnus angustata variety angustata, 62b.
Elaeagnus angustata variety songmingensis, 63. Elaeagnus tarokoensis, 64. Elaeagnus formosensis Hatusima, 65. Elaeagnus grandifolia Hayata, 66. Elaeagnus triflora Roxburgh,
67. Elaeagnus ovata Servettaz, 68. Elaeagnus philippinsis, 69. Elaeagnus parvifolia Wall. ex
Royle, 70. Elaeagnus phillipinus, 71. Elaeagnus pyriformis, 72. Elaeagnus latifolia Linn, 73.
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CHAPTER 02 LITERATURE AND REVIEW
Elaeagnus x ebbingei L., 74. Elaeagnus caudata Schlecht, 75. Elaeagnus kologa Schldl.
[178].
2.3. Pharmacological effects of some important medicinal species of Genus Elaeagnus
2.3.1. Pharmacological effects of Elaeagnus angustifolia
Elaeagnus angustifolia L. (Russian olive, oleaster) is an autumn fruit belonging to genus
Elaeagnus L. of family Elaeagnaceae. In Europe and Middle East, E. angustifolia is used as an ornamental plant [179]. The flowers and leaves are used in folkoric medicine as antipyretic, diuretic and also due to phenolic and flavonoids compounds protect cells from oxidative damage while fruits are used as appetiser [180, 181]. The fruit ripes in September, reddish to brown in colour, elliptic in shape and almost 12 mm long and 10 mm wide. E. angustifolia fruit is generally taken both in fresh and dried form and is dietary products having health benefits [182]. Fruits are a rich source of chemical compounds that has a high mineral content, protein, total soluble sugar, fat, crude fibre and total polyphenols and their antioxidant potential have also been assessed [182]. The herbal tea “Zhourat” which is a mixture of herbs and E. angustifolia flowers is used as a sedative, digestive and expectorant
[183]. E. angustifolia flour is used in ice cream to enhance the sensory value and antioxidant potential of the product [184].
Some recent reports indicates that in traditional Turkish medication the aerial parts of this plant are used as tonic, antipyretic, diuretic, antidiarrheal, anti-inflammatory, antinociceptive and analgesics and used as remedy to cure dysentery, jaundice, tetanus, fever, asthma, rheumatoid arthritis, antimicrobial and anticancer [185-191]. Daily fruits consumption is helpful to prevent certain prenatal complications, cataract, neoplastic illnesses, rheumatoid arthritis, and major non-communicable disorders like AD and PD, cardiovascular diseases and certain cancers. High fruits consumption can replace foods that are high in fats, sugar, salt and micronutrients, such as elements and dietary fibre [192-194]. E. angustifolia also
33
CHAPTER 02 LITERATURE AND REVIEW have shown some other properties like astringent, antipyretic effects [195], anti-ulcerogenic activity [196], wound healing effect [197] and muscle relaxant effect [198]. Literature study revealed isolation of various bioactive compounds from mature fruits like caffeic acid and isorhamnetin-3-O-b-galactopyranoside [199], phenolic acids and sugar [200], fatty acid composition of phospholipids and glycolipids, flavonoids, saponins, coumarins, phenolcarboxylic acids, polysaccharides, carotenoids and tannins [201], palmitic acid and linoleic acid from seeds and palmitoleic acid from pericarps [202], while trans-ethyl cinnamate ethyl cinnamate, cyclohexanecarboxylic acid, ethenyl ester, isobutyl cinnamate, palmitic acid, buthyl cinnamate, ethanol and methyl 9,9- dideutero- octadecanoate from fresh flower essential oil [203, 204]. Isolation of ascorbic acid, flavonol glycosides
(elaeagnosides), protein, carotene and magnesium ion were reported from flowers [205], phenolic acids from fruit [206], phenolic, flavonoid, terpenoid, and cytosterol compounds from leaves [181, 207], fats and proteins like linoleic acid, oleic acid, stearic acid globulin and albumin in E. angustifolia seed [208]. Major sugar like fructose and glucose were isolated from the fruit [209], while two polysaccharides were extracted from the pulp of E. angustifolia that have exerted a strong free radicals scavenging potential [210]. The fruit powders of E. angustifolia L. exerted anti-inflammatory potential by reducing the proinflammatory cytokine like TNF-α and increasing interleukin-10 [211, 212]. The ripe fruit composed of magnificent amount of antioxidant lycopene that regulate gene expression
[213]. Flavonoids have been considered one of the substantial components in E. angustifolia plant and their biological activities like antinociceptive and anti-inflammatory activities have been reported previously [172, 173].
2.3.2. Pharmacological effects of Elaeagnus oldhamii Maxim.
Pharmacological studies reported that E. oldhamii Maxim. has great traditional and medicinal values in treating various health related complaints such as rheumatoid arthritis, analgesia
34
CHAPTER 02 LITERATURE AND REVIEW and inflammations [214]. Literatures studies revealed that various compounds have been isolated from this plant such as lignanoid (isoamericanol-B), triterpenoid (cis and trans forms of 3-O-p-hydroxy cinnamoyl-oleanolic acid and 3-O-phydroxy cinnamoyl-ursolic acid), several monoterpenes and sesquiterpenes like kaempferol, caffeoyl and coumaroyl oleanolic acid, transtiliroside, betulin, caffeoylursolic acid, lupeol and syringic acid that considerably repress NO (nitric oxide) expression and inflammatory effects also exert effective cytotoxicity against lung cancer cells (A549) [215, 216].
2.3.3. Pharmacological effects of Elaeagnus pungens Thunb.
E. pungens leaf extracts are considered as folk medicinal herb in traditional Chinese medicine to treat severe cough, bronchitis, asthma and other respiratory ailments [217-220]. Reported studies revealed that the plant has maximum nutritive value, health benefits and immense marketplace due to the presence of essential amino acid, trace mineral elements (Fe, Mg, Zn,
Cu, Mn ect.), vitamins, sugar and organic acid [221]. The chemical constituent flavonoid, lignanoids, organic acids and terpenoids in Elaeagnus leaves possess pharmacological effects like antinociceptive, antiasthmatic, antitussive, expectorant activities and can treat asthma and chronic bronchitis [222], smooth muscle relaxant effect [223]. Previous studies have shown the isolation of phytoconstituents like 3,3′-dimethoxy-quercetin, methyl-3,4- dihydroxy-benzoate, 4-hydroxybenzoic acid, spingic acid, caffeic acid methylester, 4- methoxyl-benzoic acid, kaempferol, 3-O-βD glucoside, 3-methylkaempferol, dausosterol possessing anti-inflammatory and cytotoxicity activity [224], flavonol glycosides [225, 226], flavonoid glycosides that have shown no proliferating activity, suggesting no irritating effect on smooth muscle cells [227].
35
CHAPTER 02 LITERATURE AND REVIEW
2.3.4. Pharmacological effects of Elaeagnus philippinsis
E. philippinsis a medicinal plant that is present in East and Southeast Asia composed of phytochemicals used to treat amoebic dysentery [228].
2.3.5. Pharmacological effects of Elaeagnus multiflora Thunb.
In Korea and China, E. multiflora Thunb. (Cherry Elaeagnus, cherry silver berry) has been traditionally used in curing of diarrhea, itching, foul sours, cough and cancer due to more phenolics in leaves extracts [229]. The phytochemical contents reported in E. multiflora fruits were phenolic acids, flavan-3-ols and flavonoids that were used to reverse the action of α- glucosidase enzyme [230, 231]. E. multiflora heat treated juice exhibited significantly higher antioxidant activity than untreated juice due to presence of high phenolic and flavonoid contents. The fermented wine prepared from E. multiflora juice after heat treatment at 120°C for 30 min contained free sugars (glucose and fructose) , major organic acids, flavanols and phenolics like catechin, epicatechin, epigallocatechin, salicylic acid and gallic acid [232].
Previous studies reported that E. multiflora seed and flesh extracts has shown cancer preventive capacity against colon cancer cells (HT-29) and also exert anti-oxidative, anti- inflammatory and anti-proliferative effects [233].
2.3.6. Pharmacological effects of Elaeagnus parvifolia Wall.
E. parvifolia is the flora of Nepal also distributed in Afghanistan, Himalaya (Kashmir to
Bhutan), Assam and China. Isolation of steroids, flavonoids and triterpenoids have been documented from several species of Elaeagnus including E. parvifolia and are considered as folk medicinal plant [234].
2.3.7. Pharmacological effects of Elaeagnus bockii Diels
E. bockii diels have folk medicinal uses due to chemical constituents like triterpenoids, steroids and flavonoids [235]. Reported studies revealed the antidiabetic potential of E. bockii
36
CHAPTER 02 LITERATURE AND REVIEW diels by reducing ROS, malondialdehyde (MDA), serum lipid, glucose and anti-lipid peroxidation potential [236]. E. bockii Diels polysaccharide (EbD) has shown protective effect on small intestine from irradiation (~60 Co γ ray) induced damage by promoting the synthesis of intestinal mucosal protein and DNA, controlling the abnormality of cytokines and eliminating reactive oxygen free radical [237].
2.3.8. Pharmacological effects of Elaeagnus orientalis
Like other Elaeagnus species, E. orientalis has a lot of medicinal utilization and local physicians uses it for curing tumours, cancer and viral diseases [238]. In folkoric medicine, the fruit and flower are used as a tonic and antipyretic agent. Flowers of E. orientalis was chemically analysed for volatile constituents like ethyl cinnamate, ethanol, tetra hydrogeranyl acetone and phenantrenol. Ethyl cinnamate is the common constituent amongst all Elaeagnus species but their content was higher in the essential oils of E. orientalis was considered to be a cost-effective source for isolation Ethyl cinnamate for medicinal purposes [204]. In another study a new steroidal glucoside (Elaeagnoside) has been isolated along with β-sitosterol and
12-hydroxy-8,10-octadecadienoic acid that have shown significant inhibition potential against chymotrypsin enzyme [175].
2.3.9. Pharmacological effects of Elaeagnus phillipinus
Medicinally the E. phillipinus fruits has used against flatulence, amoebic dysentery, nausea and vomiting [189].
2.3.10. Pharmacological effects of Elaeagnus pyriformis
Molecular analyses, nutritional and biochemical screening of the endemic and endogenous fruits of E. pyriformis indicates highest content of vitamin C with highest antioxidant potential and isolation of best quality genomic DNA which has been standardized as well
[239]. The phytochemical fingerprints of fruit juices of actinorhizal plants like E. pyriformis
37
CHAPTER 02 LITERATURE AND REVIEW revealed the presence of various phyto-compounds with great medicinal and therapeutic values against diabetes, cancer, inflammation and liver related diseases [240]. E. pyriformis serve as functional food that is traditionally important due to highest antioxidant potential.
The fruits are used to relieve constipation [241, 242].
2.3.11. Pharmacological effects of Elaeagnus latifolia Linn.
E. latifolia Linn. (Bastard Oleaster) belongs to Elaeagnaceae family commonly distributed in the hilly parts of northeast India, Sri Lanka, Thailand, Vietnam and China. The wild edible astringent fruits of E. latifolia is a major source of phytochemicals, vitamins, minerals, essential fatty acids, carbohydrates, tannins, phenolics, flavonoids and other bioactive compounds like tannic acid, rutin, purpurin, quercetin, reserpine and catechin that have exhibited antioxidant, anti-cancerous and DNA protecting abilities [171, 238]. Phytochemical fingerprint of E. latifolia flowers has shown the presence of phytosterols, glycoside and saponins can cure cancer and cardiac ailments. These HPTLC fingerprint images are helpful for isolation and characterization of these active constituents [243, 244].
2.3.12. Pharmacological effects of Elaeagnus macrophylla Thunb.
Previous studies on E. macrophylla Thunb reported the phytochemicals like terpene, triterpenes (or steroids), glucose, glycosides, organic acid, protein, amino acid, saponins, steroid, cardiac glycosides, volatile oils, phenols, anthraquinones, flavonoids, coumarins, lactones and saccharides [245]. These pharmacologically active constituents has shown strong inhibition potential on nitrosation reactions, also exert antibacterial, anti-inflammatory effects and preventive ability against cancer and respiratory pathogens [245-249].
2.3.13. Pharmacological effects of Elaeagnus henryi Warb.
E. henryi Warb. have medicinal value and are usually used for curing cough and breath shortness. Previous investigations have shown that Elaeagnus plants contain some
38
CHAPTER 02 LITERATURE AND REVIEW phytochemical components including flavonoid, lignanoids, terpenoids and organic acids with strong anti-inflammatory, antinociceptive and cytotoxic actions [250].
2.3.14. Pharmacological effects of Elaeagnus lanceolata Warb.
Leaf extract of E. henryi is used in the preparation of Chinese patent medicines due to their medicinal value. The chemical constituent like flavonol glycosides of E. henryi extract are pharmacologically used for the treatment of breath shortness, cough and bronchitis [251].
2.3.15. Pharmacological effects of Elaeagnus gonyanthes Benth.
Phytochemical screening of E. gonyanthes Benth. were carried out using spectrophotometric and RP-HPLC method and phytoconstituents like flavonoids, oleanolic acid and ursolic acid having pharmacological importance were confirmed [252, 253].
2.3.16. Pharmacological effects of Elaeagnus conferta Roxb.
Owing to the global needs of E. conferta the approximate chemical composition of their berries/fruits, seeds and pulp indicates a rich source of carotene, ascorbic acid, protein, minerals (Ca, Mg, K, Mn, P), phenolics, flavonoids, lipids, carotenoids and carbohydrates possessing strong antioxidant activities, medicinal and nutritional values [254-257]. E. conferta dry fruit powder shown hepatoprotective effect by activating hepatic enzymes (ADH and ALDH) [258].
2.3.17. Pharmacological effects of Elaeagnus x ebbingei L.
Many perennial shrub species including E. x ebbingei L. has a lot of application in planning roadside greening in the urban areas of Southern Europe mainly for CO2 assimilation, carbon allocation, air pollution alleviation and seasonal leaf depositions of metals; Cd, Cu, Ni, Pb and Zn [259].
39
CHAPTER 02 LITERATURE AND REVIEW
2.3.18. Pharmacological effects of Elaeagnus caudata Schlecht
Previous studies suggested that the stem, bark and fruit extracts of E. caudata Schlecht can cure jaundice and liver related distresses due to pharmacologically active constituents; alkaloids, tannins, saponins, steroids and flavonoids [260, 261].
2.3.19. Pharmacological effects of Elaeagnus genus Elaeagnus kologa Schldl.
E. kologa Schldl. aerial parts extract have been assessed for phenolics and flavonoid compounds [170], that have shown high antioxidant and antibacterial action [262].
2.3.20. Pharmacological effects of Elaeagnus glabra
E. glabra control cancer incursion and metastasis by reducing the aggressiveness of human fibro sarcoma HT1080 cells in serum and plasma [263]. The extract also possess tumor suppressive ability by depressing the level of MMP-2 and MMP9 human sarcoma cell lines
[264].
2.3.21. Elaeagnus umbellata Thunb. 2.3.21.1. Elaeagnus umbellata Thunb. Description
E. umbellata Thunb. is a deciduous shrub with spiny branches covered with white silver colour scales and elliptic to ovate-oblong clusters of leaves . The bark is 2-5 m tall and 10 cm wide showing below the white hard wood. The flowers are fragranced and appear in groups with white to light yellow in colour. A single bush can produce fruits approximately 650 g. In an immature stage the fruits are silver in colour with brownish scales and ripen to freckled red in September and October. The fruits are sweet to acidic and might be stored at room temperature for almost 15 days. The fruits are good for eating along with its seeds [160, 265].
2.3.21.2. Elaeagnus umbellata Thunb. Distribution
E. umbellata Thunb. belongs to family Elaeagnacea native to Central Asia and South Europe and was presented as an ornamental shrub to USA. It is also distributed in Himalayan areas of
40
CHAPTER 02 LITERATURE AND REVIEW
Pakistan [266]. It is present at elevation of 6070 feet above sea level with location of N= 35o
23.826, E= 072o 36.312 in Kalam (Swat) Malakand Division, Khyber Pakhtunkhwa, Pakistan.
E. umbellata is native to temperate and tropical regions of Asia, Afghanistan, Japan, India,
China and different areas of Azad Kashmir at elevation of almost 6000 feet [267]. In Europe,
North America and Australia it is planted on the roads margins to avert soil erosion [268].
Due to silvery foliage leaves and attractive flowers the plant is used for ornamental purposes.
It was planted successfully in dry conditions as a protective hedge around fields, houses and gardens due to its hard nature. E. umbellata extensively grows in different regions of USA that offer food in seasonal food shortage to the wildlife [269]. E. umbellata can fix nitrogen and have tolerance to salty winds. It is planted in Japan for fixation of coastline sand mounds and eroded areas of hilly regions to grow vegetation [270]. In China it is periodically cultivated in gardens and their medicinal products are used to treat asthma, pulmonary infections, cardiac ailments and diarrhoea [271].
2.3.21.3. Taxonomic position of Elaeagnus umbellata Thunb.
Kingdom: Plantae
Phylum: Spermatophyta
Subphylum: Angiospermae
Class: Dicotyledonae
Order: Elaeagnales
Family: Elaeagnaceae
Genus: Elaeagnus
Species: Elaeagnus umbellate
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CHAPTER 02 LITERATURE AND REVIEW
2.3.21.4. Pharmacological effects of E. umbellata Thunb.
Previous studies reported that Elaeagnus berries extracts showed presence phytochemicals like of organic acids, soluble sugars (glucose and fructose), total protein contents, an excellent amount of vitamins, minerals, alkaloids, flavonoids, phenolics, saponin, carotinoids, tannins and other bioactive compounds along with essential fatty acids that possesses anti-oxidant and anti-lipid peroxidation [238, 244, 272-274]. The aerial parts extract showed moderate antimicrobial, antibacterial and antioxidant action due to saponins, steroids, alkaloids and terpenoids [275-277]. Terpenoids exhibits antiviral and antibacterial activities [278], alkaloids exhibited anthelmintic activity [279], aphrodisiac activity [280], curing of venereal diseases [281] and anti-malarial activity [282].
The fruit is identified as a new and prospective source of lycopene, β-carotene, lutein, phytofuluene, phytoene, β-cryptoxanthin and α-cryptoxanthin with therapeutic significances.
The lycopene is 17 times greater in quantity than in fresh tomato that can greatly cure cancer and myocardial infarction [283-287]. Anti-oxidant like β-carotenes are effective in curing of cancers while lutein is smooth muscle relaxant [288] and phytoenes are anti-inflammatory
[289]. The berry/fruit, flowers, seeds extracts and seeds oil have beentherapeutically and traditionally used as anti-oxidant, anti-diarrheal, as a tonic to cure coughs and pulmonary complications due to presence phenolics, flavonoids, terpenoids and tannins [160, 274, 286,
290]. Literature studies on E. umbellata revealed that flavonoids triterpenoids, steroids, coumarins, isoflavone and phenolic compounds have been isolated which have exhibited anti- oxidant, anti-plasmodial and antiproliferative action against cell lines (HT29 and HeLa) and also reverse the action of urease, cholinesterase’s (AChE and BuChE) enzymes [234, 291,
292]. Potter et al., isolated 84 constituents from floral volaties of E. umbellata Thunb. fruit. in which palmitic acid, eugenol, 4-methoxyanisole, 3-hexenyl acetate, phenyl acetaldehyde, fatty acid methyl esters, 4-methyl phenol, 2-nonenal, 2-hexenal and methyl palmitate are the
42
CHAPTER 02 LITERATURE AND REVIEW major one [293]. E. umbellata has great potential as a raw material for food industries and for commercial activities in unfavourable societies of hilly regions of Pakistan to evaluate this for physiochemical and sensory characteristics. For physical characteristics like fruit and seed weight, length diameter and pulp recovery have been evaluated, biochemically fruit has been analysed for total soluble solid , acidity, pH, sugar acid, vitamin C, fat and minerals content while for sensory evaluation fruit have been tested for flavour, taste, mouth feel and overall adequacy [294]. Different plant parts extracts of E. umbellata were evaluated for polyphenols, free amino acids contents, antioxidant and anti-proliferative activities against
HepG2 cells [295].
Chemical analysis of some commercial E. umbellata cultivars indicates the presence of polyphenols, α- and γ-tocopherols and total proanthocyanidin content in berry fruit and leaf extract. Polyphenols and vitamin E content (α-, γ-tocopherols) are the functional components of autumn olive leaf and berry are expected to be incorporated into the food products [296,
297]. The berries are astringent in flavour due to high contents of carotenoids, lycopene and phenolics among six varieties of autumn olive [298]. Nutritionally and pharmacologically genus Elaeagnus has got essential importance due to presence lycopene, flavanols, phenolics acids and flavonoids that have shown the high health benefits like wound healing, blood alcohol elimination, pain relief, anticancer and antimicrobial [283]. AOB extract is a source of polyphenols, flavonols, flavones, proanthocyanidins, anthocyanidins, catechins, and glycosides contribute highest in vitro antioxidant activity, oxygen quenching and enzyme inhibition potential [299, 300]. AOB has been marketed as a useful ingredient in the western,
Chinese, Koreans and Japanese diets because of their curative ability against cancer, liver related disorders, hepatitis, fracture, injury and diarrhea [176, 177, 301]. Previous studies revealed the anti-cancerous effect of fruit extracts inhibiting the human leukemia cancer cells
(HL-60) and lung cancer cells (A549) [302].
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CHAPTER 02 LITERATURE AND REVIEW
2.4. Antidiabetic effect of genus Elaeagnus species
Elaeagnus species has great folkloric uses in management of various diseases including the worldwide the most prevalent diabetes disease. Among them E. bockii Diels is a medicinal plant has reported antidiabetic potential along with anti-lipid peroxidation, lowering of serum lipid and serum glucose levels. Both high-lipid-feed induced and alloxan induced diabetes have been cured by treating with Elaeagnus bockii diels extracts [236].
The phytoconstituents found in E. pyriformis have shown strong antidiabetic potential [240].
Due to great medicinal significance of Elaeagnus species the E. umbellata were evaluated for their antidiabetic potential in this study. A summary of various Elaeagnus species and their biological uses have been presented in table 2.2.
44
CHAPTER 02 LITERATURE AND REVIEW
Table 2.2: Various Elaeagnus species and their biological uses
S.No. Botanical References Used part Phytochemicals Folk Uses name Total moisture, ash,
protein, soluble sugar, fats, crude fibre, Antioxidant capacity [182] 1 Fruit titratable acidity and major mineral content Diuretic, tonic,
antipyretic, antidiarrheal and as a [185] Fruit NR medication against kidney disorders Used for dysentery and Fruit NR [186] diarrhea Anti-inflammatory, anti-nociceptive, analgesic and [187] Fruit NR medication for asthma, fever, tetanus, jaundice
and rheumatoid arthritis E. Its prevent major non- angustifoli communicable ailments a L. Antioxidants, elements (oleaster) (sodium, potassium, such as cancers, cardiovascular diseases, magnesium, calcium [192] prenatal difficulties, Fruit and phosphorus) and dietary fibre cataract, neoplastic, inflammatory,
Alzheimer's and Parkinson's diseases Traditionally used against oxidative stress Antioxidant like related disorders, Flowers, phenolics and flavonoid asthma, cough, nausea, leaves compounds diarrhea, fever, [181]
jaundice, rheumatoid
arthritis and wounds healing Anticancer, antioxidant, Fruits, [188] NR anti-inflammatory and Flowers antimicrobial Anti-diuretic, Flowers, antipyretic and fruits [180] Phytochemicals leaves are eaten as an appetiser Isorhamnetin, caffeic Fruits acid and isorhamnetin- 3-O-b- NR [303] galactopyranoside Fruits, seeds Palmitic, Linoleic and and palmitoleic acids [304] pericarps NR
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CHAPTER 02 LITERATURE AND REVIEW
Trans-ethyl cinnamate, methyl cinnamate , isopropyl cinnamate, isobutyl cinnamate, isopentyl cinnamate. Benzenoids were phenylethanol, phenylethyl acetate , Fresh benzyl alcohol, As a tonic and flowers phenylethyl benzoate , antipyretic agent [203] benzaldehyde, ethyl benzoate, phenyl acetaldehyde, benzyl acetate, hexahydrofarnesyl acetone, 2-hydroxy-5- methylacetophenone, heptanal, nonanal, decanal and undecanal Ethyl cinnamate, Cyclohexane carboxylic acid, ethenyl ester, As a tonic and
Isobutyl cinnamate, antipyretic agent Palmitic acid, Buthyl [204] Flowers cinnamate, Ethanol and Methyl 9,9- Dideutero- Octadecanoate Anti-diarrheal, anti- Fruit NR [186] diuretic and antipyretic Asthma, jaundice, Fruits [195] NR fever, tetanus and infusion rheumatoid arthritis
Leaves Flavonoid, terpenoid, and cytosterol NR [207]
compounds 4-hydroxybenzoic acid, Fruit NR [206] and caffeic acid Fruit seeds Flavonoid Anti-inflammatory, [172] and fruit Anti-nociceptive
extracts Seeds Fatty acids and proteins NR [208] (globulin and albumin) 4-hydroxybenzoic acid Preserved caffeic acid, Fructose NR [209] fruit and glucose Flavonoids, phenol carboxylic acids, [201] Fruit coumarins, carotenoids, NR
saponins, tannins and polysaccharides Berries and Flavonoids, carotene, Antioxidant effect [205] flowers vitamin C, magnesium,
46
CHAPTER 02 LITERATURE AND REVIEW
acylated flavonol glycosides (elaeagnosides A-G) and protein Fruit [212] powders NR Anti-inflammatory
Vitamin C, carotene, Fruit pulp protein, magnesium, Antioxidant effect acylated flavonol [210]
glycosides and flavonoids Fruits and Flavonoids, terpenoids Anti-nociceptive effect [172] seed extracts and cardiac glycosides Flavonoids Fruit seeds Muscle relaxant effect [198]
Antimicrobial and Bark NR [190] antifungal effect Monoterpenes, 2 E. sesquiterpenes, betulin, Analgesic and anti- oldhamii lupeol, kaempferol, inflammatory Maxim. [214] Leaves oleanolic, syringic, and
ursolic acids Iso-americanol-B, cis inhibition of nitric and trans forms of 3-O- oxide expression and
p-hydroxy cinnamoyl anti-inflammatory Leaves oleanolic acid and 3-O- effects [305] phydroxy cinnamoyl
ursolic acid Anti-inflammatory Leaves Isoamericanol-B [215] effect Trans-tiliroside, Treating lung disorders Leaf extract Oleanolic and ursolic and [216] acids of coumaroyl and effective in cytotoxic Caffeoyl agents Anti-asthmatic, 3 Leaf NR antitussive and [217] extracts expectorant activities 3,3′-dimethoxy quercetin, methyl-3,4- dihydroxy benzoate, Bark, caffeic acid, methyl- Cytotoxic activity [306] Leaves ester, dausosterol,
kaempferol-glucoside, E. pungens spingic acid, 4- methoxyl-benzoic acid, 3-methyl- kaempferol Amino acid, trace Nutritive value and mineral elements (Fe, [221] Seeds Mg, Zn, Cu, Mn), health effect
vitamines, sugar and organic acid Leaves Flavonoids compounds Antiasthmatic, [307]
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CHAPTER 02 LITERATURE AND REVIEW
antitussive, and expectorant activities Antiasthmatic, Flavonoid, lignanoids, antinociceptive, anti- Leaves organic acids and inflammatory, and terpenoids cytotoxic activities Smooth muscle Leaves Glycosylated flavonoid [227] relaxant effect Relaxant effect and Leaf NR prevent asthma and [223] extracts chronic bronchitis Antinociceptive, anti- Leaf Flavonol glycosides inflammatory, and cure breath shortness, [250]
cough, bronchitis and cytotoxic actions 4 E. Cure amoebic Fruit NR [228] philippinsis dysentery Antioxidant and α- 5 Flavan-3-ols, phenolic Fruit glucosidase inhibitory [230] acids and flavonoids action Treatment of cough, Leaf [229] Phenolic diarrhea, itching and extracts foul sores Fructose, glucose, major organic acids (lactic malic, succinic and oxalic acids), major Antioxidant action Fruit flavanols and phenolic [232]
E. acids (catechin, multiflora epicatechin, Thunb. epigallocatechin, gallic acid and salicylic acid) Anti- inflammatory, Fruit pulp, anti- oxidant and NR [233] seed repressed tumor cell (HT-29) Anti-inflammatory, Phenolic anti- oxidant, Fruit antidiarrheal, [308]
anticanerous and useful for cough and itch 6 Triterpenoids, steroids E. ungens Plant Folk medicinal uses [234] and flavonoids Triterpenoids, steroids 7 Plant Folk medicinal uses [235] and flavonoids Prevent cardiovascular Plant NR [236] E. bockii disease and diabetes Diels Protect the small intestine from Plant Polysaccharide [237] irradiation-induced injury 8 E. Plant extract Elaeagnoside, Chymotrypsin enzyme [175]
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CHAPTER 02 LITERATURE AND REVIEW
orientalis β-sitosterol and 12- inhibition hydroxy-8,10- octadecadienoic acid Ethyl cinnamate, Used as a tonic and Fruits and Ethanol, Tetra antipyretic agent [204] flowers hydrogeranyl acetone
and Phenantrenol Used for the treatment of tumours, certain type [238] Plant extract NR of cancer and viral diseases 9 E. Fruits Cure amoebic NR dysentery, flatulence, [189] phillipinu s nausea and vomiting 10 Anticancer, antidiabetic, anti- Fruits Phytochemical inflammatory and [240] E. constituents hepatoprotective pyriformis activities Phytochemical Antioxidant activity Fruits [241] constituents and relieve constipation 11 Carbohydrates, Antioxidant and DNA essential fatty acids, [171] minerals, vitamins, protector
tannins, flavonoids and phenolics E. latifolia Quality control of the Flowers Phytosterols, glycoside Linn drug and ensure [243] and saponins therapeutic efficiency Flavanoids , , essential Fruits Reducing the incidence fatty acids, vitamins [238] of cancer and minerals
12 Stem NR Antibacterial activities [247]
Stem NR Antimicrobial effects [248] Antimicrobial anti- Leaf NR [245] inflammatory Steroids, triterpenoids and volatile oils, cardiac Inhibit nitrosation glyosides, organic acids, reaction, scavenging E. Stem phenols, [309] effect and anti- macrophyl anthraquinones, cancerous effect la Thunb. flavonoids, coumarins and terpene lactones Antibacterial and Leaves Antimicrobial effects [249] antipyrotic drug Triterpenes (or steroids), Root and glucose and glycosides, stem organic acid, protein, NR [310] amino acid, saponins, steroid and cardiac
49
CHAPTER 02 LITERATURE AND REVIEW
glycosides Antinociceptive, anti- 13 E. henryi Flavonoid, lignanoids, inflammatory and cure Warb. Ex Leave organic acids, and [250] cough, breath shortness Diels. terpenoids and cytotoxic activities 14 E. Treatment of breath lanceolata Leaves Flavonol glycosides shortness, cough, or [251] Warb. bronchitis 15 E. Total flavonoids and phenolic acids gonyanthes Berries NR [252] (oleanolic and ursolic Benth acids) 16 carotene, ascorbic acid, Antioxidant activity Berries [254] protein, and magnesium Phenolics, flavonoids, carotenoids , lipids, Berries carbohydrates, ascorbic NR [255] E. conferta acid and titratable Roxb. acididity Fruits/berrie Minerals Antioxidant activity [257] s Antioxidant activity Dry fruits NR and clearance of blood [258] powder alcohol 17 Assessed CO2 E. x assimilation, air pollution mitigation [259] ebbingei L. Shrub NR and metals (Cd, Cu, Ni, Pb, and Zn) leaf deposition 18 E. caudata Alkaloids, tannins, Stem bark To cure jaundice and Schlecht saponins, steroids and [261] and fruit other liver troubles flavonoids Polyphenolic contents Cells protection and 19 Fruits [170] E. kologa antioxidant capacity Schldl. Leaf Antibacterial Antibacterial action [262] extracts components 20 Tumor suppressive Solvent NR ability by lowering [263] extract MMP2 and MMP9 levels E. glabra Control cancer invasion and metastasis by Solvent NR reducing human fibro [264] extract sarcoma (HT1080 cells) 21 To treat asthma, diarrhoea, pulmonary Fruits Medicinal products [270] E. infections and cardiac umbellata ailments Carotenoids and Thunb. Fruits Avert prostate cancer [311] lycopene Berries/fruit Gallic, vanillic, Anti-oxidant and anti- [312]
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CHAPTER 02 LITERATURE AND REVIEW
s coumaric, ferulic, lipid peroxidation sinapic, and caffeic acids Antioxidants (β- Antioxidant, anti- Berries carotene, lycopene, [285] cancerous and cure phytoene, lutein and myocardial infarction phytofuluene High phenolic contents, mineral content vitamin Berries C, flavonoid, Antioxidant activities [274] carotenoid, tannin, alkaloid and saponin contents Antibacterial activity Alkaloids, steroids, Aerial parts and free-radical [275] terpenoids, saponins scavenging activity Berries Flavonoids, essential Antioxidant property [244] fatty acids and vitamins Saponins, alkaloids, Flowers, Cure coughs and steroids, terpenoids and [286] Seeds pulmonary affections phenolics Solvent Antiviral and [278] Terpenoids extracts antibacterial activities Anthelmintic activity, aphrodisiac activity, [280] Solvent Alkaloids treatment of venereal extracts diseases and anti- malarial activity Solvent Triterpenoids, steroids [234] Folk medicinal uses extracts and flavonoids Aerial parts Phenolics and tannins Anti-bacterial activity [276] Coumarins (anthraquinone, 7- Hydroxy-chromen-2- Solvent one, 3-2,2,3,4,5-Penta Antiplasmodial activity extracts [300] hydroxy-hexyloxy-
chromen-2-one and 7-8- Dihyroxy-chromen-2- one) Leaves and Bioactive molecules Antimicrobial activities [277] root Phenolics, flavonoid, lycopene, quercetin-3-β- Antioxidant, D-glucoside, hesperidin antiproliferative and Solvent anthocyanin, ascorbic reserve the action of [292] extracts acid, β- carotene, urease and fumaric acid, 4- cholinesterases (AChE, hydroxybenzoic acid, BuChE) enzyme neohesperidin and rutin Solvent Flavonoids, terpenoids Anti-diarrheal effect [290] extracts and tannins and spasmolytic effect Phytochemicals, such as Smooth muscle [289] Plant extract lutein, phytofluene, and relaxant, anti-
phytoene inflammatory
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CHAPTER 02 LITERATURE AND REVIEW
Seeds and Mitigate coughs and [160] flowers NR pulmonary infections Extract Polyphenols, Amino Highest anti- Leaves acids (asparagine, amino proliferative activity stem and butyric acid and against HepG2 cells [295] fruits proline), radical gallic acid, (+)-catechin Polyphenols and α- and Fruits Functional foods [296] γ-tocopherols, Berry proanthocyanidins Lycopene, phenolic compounds, carotenoids. flavanols, Berries Inhibit cancer [298] hydroxybenzoic acids, and seeds hydroxycinnamic acids and hydroxybenzoic acids Malic acid, lycopene, Anti-oxidant action and Fruits [273] glucose, fructose and regulate metabolic Berry novel proteins pathways anti-cancer, Benzoic acid, cinnamic antimicrobial, Fruits acids, lycopene, expectorant, blood myricetin and alcohol elimination, [283] Epigallocatechin gallate curative for pain and wounds Isoflavone and phenol compounds (Stigmasterol, 3- hydroxy-methyl)-4- Plant NR [313] methoxy-phenol and 5- 7, dihydroxy-3-(2- hydroxy phenyl-4.H- chromen-4-one Fatty acid (C14-20), 2- hexenal, eugenol, methyl esters, methyl- Floral phenol, phenyl- Essential oil possess volatiles aeetaldehyde, nonenal, marketable value [314] 3-hexenyl acetate, methyl phenol, methyl anisole and methoxy anisole Glycosides, flavonols, minerals, Vitamins, Berry fruits [315] flavones, catechins, Anti-oxidant action
proanthocyanidins and anthocyanidins Antioxidant activity AOB Polyphenols [299] and bioactivity
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CHAPTER 02 LITERATURE AND REVIEW
Cure hepatitis, diarrhea, [301] AOB NR fracture and injury
[272] AOB Ascorbic acid and oil NR
Antioxidant activity Fruit Lycopene [213] and gene regulation Anti-carcinogenic Lycopene AOB, leaf activity, [176]
hepatoprotective effect Cure leukemia and Fruit Anti-cancerous agents lung cancer (HL-60, [302] A549)
Abbreviations: Elaeagnus: E; Autumn olive berry: AOB; Not reported: NR
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CHAPTER 03 MATERIAL AND METHODS
3. MATERIAL AND METHODS 3.1. Chemicals
DPPH (Sigma-Aldrich, CHEMIE GmbH; USA), ABTS, DTNB (lot No STBC0047V) &
Streptozotocin (Sigma-Aldrich; Germany), Type I α-Glucosidase (Saccharomyces cerevisiae), Type VI α-amylase (porcine pancreas, lot No SLBM2655V), PNPG, Potassium phosphate buffer (Sigma-Aldrich; Paris, France), Glucose estimation kits (S.D. Chek-Gold;
Germany), glibenclamide (Sanofi Aventis Pharma; Pakistan), methanol, n-butanol, n-hexane, ethyl acetate and chloroform (Merck; Germany), Tween-80 (Scharlau Chem; Spain); normal saline solution (Utsoka-Pharma; Pakistan), Lipid profile tests kits (Human; Germany), Renal profile tests kits (Bioneed diagnostic; Germany), Galanthamine (Lycoris Sp.), Acetyl cholinesterase (Electron-eel Type, VI-S), buterly cholinesterase (lot No SLBPO912V), 3,5-
Dinitrosalicylic acid (lot No D2401QEI) & Ascorbic acid (Sigma-Aldrich; USA),
Acetylcholine-iodide, Gallic acid & Folin-Ciocalteu regent (Sigma Aldrich; UK),
Butyrylcholine-iodide (lot No BCBS5091V, Sigma Aldrich; Switzerland). All the chemical used were of analytical grade with the exception of HPLC solvents that were of HPLC grade.
3.2. Plant sample collection
The fruits of E. umbellata Thunb. (Figure 3.1) were collected from the hilly region of Kalam,
Malakand Division of Khyber Pakhtunkhwa, Pakistan in August-September 2016. The plant sample was identified by plant taxonomist; Prof. Mehboob-UR-Rahman, PGC. Swat, Khyber
Pakhtunkhwa, Pakistan. The plant specimens were deposited in the Botanical Garden
Herbarium, University of Malakand, Pakistan with voucher number BGH.UOM.154. Before extraction, the aerial parts of the plant were cleaned and dried for 20 days in shade.
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CHAPTER 03 MATERIAL AND METHODS
Figure 3. 1: Plant and fruits of E. umbellata Thunb a) E. umbellata Thunb. tree b) Berried shrubs c) Berries/fruits
3.2.1. Extraction and Fractionation
The dried fruits (10 kg) were crushed through a grinder before maceration in 80% methanol.
The subsequent mixture was preserved for 14 days with periodical shaking and was then filtered. The filtrates were converted into semisolid mass under reduced pressure at 40°C in the rotary evaporator (Schwabach: 4000; Heidolph-Laborota-Germany). The semisolid mass obtained was solidified in open air (final mass = 750 g). The crude extract was subjected to fractionation by solvent-solvent extraction method starting from a low to high polarity (n- hexane, chloroform, ethyl acetate, and n-butanol) solvent. About 95, 210, 115, 90 and 220 g solid extracts were obtained from n-hexane, chloroform, ethyl acetate, n-butanol, and
55
CHAPTER 03 MATERIAL AND METHODS aqueous fractions respectively after evaporation [316]. The schematic diagram of extraction and fractionation yield is summarised in Figure 3.2.
Figure 3.2: Schematic diagram of extraction and fractionation yield
3.3. Preliminary phytochemical analysis
3.3.1. Qualitative screening
Hydro methanolic extract (Met-Ext) from E. umbellata fruit was investigated to identify major phytoconstituents like flavonoids, alkaloids, glycosides, terpenoids, anthraquinone, tannins and pigments. Various chemical test were performed on Met-Ext using reported assays [317].
3.3.2. Dragendorff’s test
About 0.2 g of hydro methanolic extract and 2% sulphuric acid was mixed in conical flask and kept for two min on a hot water bath. After cooling the sample was filtered and mixed with Dragendorff’s reagent. From the red colourationthe presence of alkaloids were confirmed [318].
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CHAPTER 03 MATERIAL AND METHODS
3.3.3. Ferric chloride test
Dried Met-Ext powder was taken in flask containing distilled water (10 ml) and boiled on hot water bath for 5 min. Filtration was done after cooling the mixture. Appearance of yellow colour upon addition of sodium hydroxide (20%) drop wise into 1 ml filtrate confirm flavonoids. The yellow colour of the mixture disappear after adding 2% HCl [319].
3.3.4. Keller Killiani test
According to the reported assay Met-Ext (5 ml), 2 mL glacial acetic acid and concentrated sulphuric acid (1 mL) were mixed and some drops of ferric chloride were also added. In the mixture precipitation after sulphuric acid addition was observed which appeared as brown ring at the bottom of the flask indicating glycosides [320].
3.3.5. Gelatin test
About 2 g extract sample was dissolved in 20 mL distilled water and kept in hot water bath for around 5 min. After cooling few drops of 10% ferric chloride was added to 1 mL filtrate.
Appearance of brownish to green color precipitate indicated existence of tannins [320].
3.3.6. Liebermann Burchard test
Met-Ext in 5 mL distilled water was kept in water bath till boiling. The filtration was carried out after cooling the solution. Formation of reddish-brown interface occurs after addition of chloroform (2 ml) and concentrated sulphuric acid (3 ml) which showed that terpenoids are present [320].
3.3.7. Bontrager’s test
About 2 g hydro methanolic extract was macerated with shaking in ether. Formation of reddish pink colour confirm the presence of anthraquinone [318].
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CHAPTER 03 MATERIAL AND METHODS
3.4. Investigation of total phenolic content
According to the previously reported method [321] Met-Ext (100 µL), distilled water (500
µL), Folin-Ciocalteu reagent (100 µL) and 1000 µL Sodium Carbonate (7%) were mixed and allowed to stand for 90 minutes and finally at 760 nm absorbance was recorded through UV-
Spectrophotometer. Calibration curve was formed by taking the standard Gallic acid. From calibration the TPC in samples were calculated and reported as mg GAE/g of dry sample.
Results were taken in triplicate.
3.5. Investigation of total flavonoid contents
According to previously reported method [322] the TFC was measured as mg QE/g of dry sample. A calibration curve was made by taking the dilution of standard Quercetin. From each sample dilutions 100 µL were taken and mixed with distilled water (500 µL), 100 µL of
Sodium nitrate (5%), 150 µL Aluminium chloride (10%) and 200 µL Sodium hydroxide
(1M). After 5 min absorbance were recorded at 510 nm by UV-Spectrophotometer. Results were taken in triplicate.
3.6. HPLC-UV characterization
To prepare extract for HPLC analysis 1 g powdered sample was added into mixture of methanol and water according to reported method [323] and heated at 50°C for one hour. The mixture was then subjected to filtration two times and then poured into HPLC vials.
HPLC Agilent 1260 system was used for the identification and separation of phenolic compounds, with basics parts like ultraviolet detector, a quaternary pump, degasser and auto sampler. The separation was carried out by C18 column. The wavelength used was 280 nm to record the absorbance of phytochemicals present [323]. Identification of phenolic compounds was done using their retention times, and available standards while % peak areas were used for quantitative estimation of a component.
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CHAPTER 03 MATERIAL AND METHODS
3.7. Essential oil isolation
The collected fresh fruits of E. umbellata were subjected isolation of essential oil through hydro distillation process using the Clevenger apparatus using the reported procedure [324].
Finally the essential oil obtained were kept at -20°C until required further for biological activities.
3.8. Gas-chromatographic analysis and identification of components
Essential oil of E. umbellata were analysed by means GC/MS (Agilent Technologies; USA) equipped with FID detector for phytoconstituents identification. The essential oil major constituents were identified by comparing their retention times and fragmentations of mass spectra with already reported published data. Further documentation was carried out using spectra information achieved from Wiley and NIST libraries [325].
3.9. Isolation, purification and characterization of pure compounds
Amongst various fractions (Met-Ext, Hex-Ext, Chf-Ext, EtAc-Ext, But-Ext and Aq-Ext) of crude extract, the Chf-Ext fraction was selected for further investigation to isolate bioactive compound from it in pure state as it was biologically the most potent one. This selection was based on the in vitro analysis results. The chloroform extract was mixed with silica gel slurry and then allowed to dry in air. The sample loaded silica was then carefully loaded to large silica gel column and eluted with solvent system. The elutions from column were collected in glass vials and were then loaded on thin layer chromatography (TLC) plates to combine identical fractions. Finally the narrow pen column was used to separate the active sub fraction and collect it into small vials. From all vials spot were tested on TLC plates and placed in suitable solvent system. The vials which have the same Rf were finally mixed and solidified.
After purification and solidification the compound were further characterized and identified
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CHAPTER 03 MATERIAL AND METHODS by spectroscopic techniques like FTIR, HNMR, 13CNMR and Mass spectroscopy (MS). The pure compounds, were screened for antidiabetic and antiamnesic activity [326].
3.10. Antioxidant scavenging assays
3.10.1. DPPH scavenging assay
Brand-Williams assay [327] was used to find out DPPH free radical scavenging capability of the extracts. About 24 mg, DPPH was dissolved in 100 mL methanol. Plant sample stock solutions (1 mg/mL) were also prepared in methanol. Using serial dilutions working solutions with the following concentrations: 1000, 500, 250, 125, 62.5 and 31.05 µg/mL were prepared. About 0.1 mL of each working dilution was mixed with DPPH (3.0 mL) and incubated at 25°C for 30 minutes. Absorbance was measured at 517 nm via UV- spectrophotometer (Thermo Electron Corporation: USA). Ascorbic acid was used as a standard. Results were presented as Mean SEM. % DPPH scavenging potential was calculated by the following formula:
(3.1)
3.10.2. ABTS scavenging assay
Antioxidant potential of berry extracts were also determined against ABTS free radical by method described by Re et al [328]. ABTS (7 mM) and potassium persulfate (2.45 mM) solutions were mixed thoroughly and were incubated overnight in dark for the production of
ABTS free radical. The absorption of this mixture was adjusted by adding methanol to 0.7 at
745 nm. About 300 µL extract working dilutions and 3.0 mL ABTS solutions were mixed and incubated for 6 minutes. Finally the absorbance was measured via UV spectrophotometer. Ascorbic acid was used as positive control. % ABTS scavenging potential was calculated using the following formula:
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CHAPTER 03 MATERIAL AND METHODS
(3.2)
3.11. Antibacterial activity
Antibacterial activity of all the extract/fractions and isolated compounds were assessed against various bacterial strains using reported assay [329]
3.12. In vitro anti-diabetic assays
3.12.1. α-amylase enzyme inhibition
The extracts solutions were prepared in normal saline with Tween-80 (5%) using the reported method [330]. The α-amylase enzyme inhibition potential was evaluated using 3,5- dinitrosalicylic acid assay [29]. The Me-Ext and subsequent fractions of E. umbellata were dissolved in DMSO (10%), 0.02 M Na2HPO4/NaH2PO4 buffer and 0.006M NaCl at pH 6.9.
Through serial dilutions the working solutions; 31.05, 62.5, 125, 250, 500 and 1000 µg/mL were prepared. 200 μl of α-amylase (2 units/ml) solution was mixed with working dilutions
(200 μl) and incubated at 30°C for 10 min. Subsequently 200 μl starch (1% in water: (w/v)) solution was added to each sample dilution followed by incubation for 3 min. The reaction was stopped by adding of 200 μl sodium potassium tartrate tetrahydrate (DNSA) reagent (12 gm) dissolve in 8.0 mL, 2M NaOH and 20 mL of 96 mM 3, 5 dinitrosalicylic acid solution.
The reaction mixture was boiled for 10 min in a water bath at 85–90°C. After cooling, dilution was done with 5 mL distilled water and finally, the absorbance was noted at 540 nm.
A blank solution was prepared containing only plant extract but no enzyme. Standard acarbose (100 μg/ml–2 μg/ml) was used as positive control (without plant extract). The α- amylase enzyme inhibitory potential was calculated by the following formula:
(3.3)
3.12.2. α –glucosidase enzyme inhibition
The α-glucosidase inhibition by Me-Ext and subsequent fractions were carried out according to the reported method of Ranilla et al. with minor changes [331]. The reaction mixture was
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CHAPTER 03 MATERIAL AND METHODS formulated by adding 100 μl α-glucosidase enzyme (0.5 unit/ml), 0.1 M phosphate buffer
(600 μl) at pH 6.9 and 50 μl each sample dilutions (31.05, 62.5, 125, 250, 500 and 1000
µg/mL). The mixture was incubated for 15 min at 37°C. The enzymatic reaction was started by adding 100 μl p-nitro-phenyl-α-D-glucopyranoside (5 mM) solution in 0.1 M phosphate buffer at pH 6.9 followed by 15 min incubation at 37°C. The reaction was stopped by adding
400 μl sodium carbonate (0.2 M) solution. The absorbance of the final reaction mixture was recorded at 405 nm. The reaction mixture with no plant extract was used as positive control while the blank solution was prepared without enzyme α-glucosidase. The α-glucosidase % inhibition was calculated using formula:
(3.4)
3.13. In vitro Anti Alzheimer’s study
3.13.1. Anti-cholinesterase Assays
Ellman assay [332] was used to evaluate E. umbellata fruit extract/fractions for anticholinesterase potentials i.e. Acetyl cholinesterase and Buteryl cholinesterase inhibition potential. According to this method 205 µL extract/fractions solutions and 5µL of AChE
(0.03 U/mL) and BChE (0.01 U/mL) enzymes along with catalyst DTNB (5 µL) were taken in a cuvette and kept at 30˚C in hot water bath. After 15 minutes of incubation both Acetyl choline iodide and Butyryl choline iodide were added as a substrate (5 µL) that resulted in the formation of yellow colour anion (5-Thio-2-nitro benzoate). After 4 minutes absorbance was recorded at 412 nm through double beam spectrophotometer (Thermo electron-corporation;
USA). A blank solution was considered as a control having no extract/fractions. Activity and inhibition potential of both enzymes was measured by the following formulas:
(3.5)
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CHAPTER 03 MATERIAL AND METHODS
(3.6)
(3.7)
V is inhibitor dependent rate of reaction while, Vmax is inhibitor independent rate of reaction.
3.14. In vivo antidiabetic assay
3.14.1. Animals
Sprague Dawley adult rats (150 to 170 g body weight) were purchased from Rifah Institute of
Pharmaceutical Sciences Islamabad. Animal’s acclimatization was carried out for one week in the laboratory animal house. The animals were provided with standard food as ad libitum fresh water. The animals were kept at room temperature around 22-25°C with light and dark cycle of about 12 hours each. All procedures related to the animals were carried out according to the Animal Scientific Procedure Act; UK (1986) and approval was taken from the Departmental Animal Ethical Committee (DAEC/PHARM/2018/1) of University of
Swabi.
3.14.2. Acute toxicity study of the fruit Me-Ext/fractions of Elaeagnus umbellata Thunb.
The acute toxicity of the Me-Ext/fractions of E. umbellata were evaluated according to the protocol described by Karim et al. [333] using adults Sprague Dawley rats weighing 150-170 g. All animals were divided into nine groups and each group composed of 8 animals. The control group animals received tween-80 suspension, orally. All animals were then treated orally with different doses of extract/fractions 100, 200, 400, 500, 1000, 1500 and 2000 mg/kg. After administration, the animals were observed for 0, 0.5, 1.0, 24, 48, 72 and 168 hours for physical, behavioral and pharmacological lethal effects. The extracts did not produce any drug-induced harmful physical signs and no mortality was detected. The extract remained safe and nontoxic up to 2000 mg/kg dose range. Therefore, according to OECD guidelines, 200 mg/kg extract dose that is 1/10th of 2000 mg/kg dose (maximum tested dose)
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CHAPTER 03 MATERIAL AND METHODS was selected to evaluate the in vivo antidiabetic activity [334]. All the doses of the extracts/fractions (200 mg/kg) were made by dissolving it in tween-80 suspension and standard glibenclamide drug (0.5 mg/kg, p.o) in normal saline and were administered orally.
3.14.3. Animal experimental design for inducing type 2 diabetes
T2D was induced according to the method previously described by Gopalakrishnan et al.
[335]. Animals were divided into two major groups. One group was given normal pellet diet and the other animal group was fed with high fat diet (HFD) (40% raw beef fat + 30% casein
+ 10% glucose + 7% wheat flour + 6% barn + 4% vitamin mixture and 3% salt mixture) for two weeks before commencing the experiment. After two weeks, induction of hyperglycemia was carried out in HFD Sprague Dawley rats via a single intraperitoneal (i.p) injection of
STZ (50 mg/kg) prepared in 0.9% normal saline solution after an overnight fast.
Subsequently 72 hours after administration of STZ, blood samples were collected from the tail vein via Glucometer strips by means of SD glucometer (Germany) and blood glucose level was measured [336]. Rats having fasting blood glucose level ≥300 mg/dl were considered hyperglycemic and were included in the study (Table 3.1).
Table 3. 1: Experimental design and various tretament groups used in the study Group Group Category Treatment given Route I Normal control Normal saline 8 mL/kg p.o. II Diabetic control STZ (50 mg/kg) i.p. III Positive control Glibenclamide 0.5 mg/kg p.o IV Me-Ext 100 mg/kg p.o V Me-Ext 200 mg/kg p.o VI Chf-Ext 100 mg/kg p.o VII Chf-Ext 200 mg/kg p.o VIII EtAc-Ext 100 mg/kg p.o IX EtAc-Ext 200 mg/kg p.o Me-Ext ; Methanolic extract, Chf-Ext ; Chloroform extract fraction, EtAc-Ext ; Ethyl acetate extract fraction , STZ; Streptozotocin, p.o. ; per oral, i.p.; intraperitoneal
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CHAPTER 03 MATERIAL AND METHODS
3.14.4. Treatment protocol
Rats were divided into 9 groups (n꞊8) after an overnight fast for about 12 hours. The first group was labelled as a normal control and was given normal saline orally while the rest of the eight groups were considered HFD groups. The second group categorized as diabetic control and was given normal saline. Standard 0.5 mg/kg glibenclamide drug (p.o) was given to the third group. The fourth group received the crude Me-Ext of E. umbellata (100 and 200 mg/kg; p.o), while the fifth, sixth, seventh, eighth and ninth groups were given Chf-Ext and
EtAc-Ext fraction of E. umbellata (100 and 200 mg/kg; p.o), respectively.
The treatment of plant extracts was continued for 21 days (daily at 09:00 am). The level of blood glucose and body weights were measured on 0, 4th, 7th, 10th, 15th, 21st day of treatment according to the previous protocol described by Bhat et al [337].
3.14.5. Collection of blood and estimation of biochemical parameters
At the completion of in vivo antidiabetic activity on 21st day, all animals were anesthetized via 35 mg/kg pentobarbital sodium and euthanized by cervical decapitation using previous procedure illustrated in schedule-1 of UK, animal scientific procedure act; 1986. Blood collection was carried out via cardiac puncture for studying the biochemical parameters
[338]. The blood samples were centrifuged for serum separation at 3500 rpm for 10 minutes.
The serum was analysed through spectrophotometer for investigation of biochemical parameters like SGPT, SGOT and ALP. TC, TG, LDL, HDL and serum creatinine were measured by CHOD-PAP and GPO-PAP procedure (Human kit; Germany) using UV-
Spectrophotometer [338].
3.15. Molecular docking validation for anti-diabetic enzymes
The minimization was achieved up to the average root-mean-square deviation of all the non- hydrogen atoms extended 0.3 Å. Selection of ligands used in docking were based on the
65
CHAPTER 03 MATERIAL AND METHODS natural constituents found in the Chf-Ext and EtAc-Ext layers: quercetin, rutin, chlorogenic acid, epigallocatechin gallate, morin, catechin hydrate, pyrogallol, ellagic acid and gallic acid, detected through HPLC analysis. To validate our docking results acarbose and epigallocatechin were added to the list (Fig. 3.2).
All compounds were built using the fragment library (Maestro; 10.6) and were set via Lig-
Prep module. Optimization of ligands was carried out by an OPLS-2005 force field in the
Macro-Model module [339].
The docking procedure for α-amylase created the production of a grid box and the docking site was designated as the centroid of the acarbose molecule. However, as the α-glucosidase enzyme is crystallized without any ligand, the binding site was determined using Sitemap
[340] and the grid generation proceeded by a grid box formation that is the centroid of the amino acids surrounding this binding site Arg407, Asp326, Arg197, and Asn258.
For both α-glucosidase and α-amylase enzymes, the defaulting grid size was taken from the
Glide program [341]. Consequently, the docking of ligands occurred into the definite binding site by means of Grid-Based docking and flexible glide docking (Glide-XP) using the default parameters of docking with no constraints. Docking of Ligands occurred into the stiff receptor lacking ligand nonpolar atoms or scaling-down the Vander Waals radii of receptor atoms.
The best-docked structures that have more favorable binding were selected using the Glide-
Score function with more negative Glide-Score. After visualization of the ligand-protein complex, the interactions were studied among different ligand-receptor.
.
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CHAPTER 03 MATERIAL AND METHODS
Figure 3.3: Phenolic Compounds identified in E. umbellata Thunb. fruit methanolic extract/fractions studied in molecular docking. a) Acarbose, b) Rutin, c) Epigallocatechin gallate, d) Epigallocatechin, e) Quercetin, f) Morin, g) Ellagic acid, h) Catechin, i) Chlorogenic acid, j) Pyrogallol
3.16. In vivo anti-amnesic assays
3.16.1. Experimental scheme
Six groups of Swiss Albino male mice were designed for administration of Chf-Ext and seven groups for administration of compound CGA. The doses of all tested solutions are presented in Table 3.2 & 3.3. In behavioural experiment normal saline, Chf-Ext, isolated compound CGA and donepezil were given to Albino mice before 1 h and scopolamine before 30 min of each experimental trial.
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CHAPTER 03 MATERIAL AND METHODS
Table 3.2: Experimental design and various tretament groups used in the study Group Group Category Treatment given Route I Normal control Normal saline (8 mL/kg) p.o. II Negative control scopolamine (1 mg/kg) i.p. III Positive control Donepezil (2mg/kg) p.o, i.p + scopolamine (1 mg/kg) IV Treatment group Chf-Ext (50 mg/kg) + p.o scopolamine (1 mg/kg) V Treatment group Chf-Ext (100 mg/kg) + p.o scopolamine (1 mg/kg) VI Treatment group Chf-Ext (200 mg/kg) + p.o scopolamine (1mg/kg)
Table 3.3: Experimental design and various tretament groups used in the study Group Group Category Treatment given Route I Normal control Normal saline (8 mL/kg) p.o. II Negative control Scopolamine (1 mg/kg) i.p. III Positive control Donepezil (2mg/kg) p.o. + scopolamine (1 mg/kg) i.p. IV Treatment group CGA (1 mg/kg) + p.o Scopolamine (1 mg/kg) V Treatment group CGA (3 mg/kg) p.o + scopolamine (1 mg/kg) VI Treatment group CGA (10 mg/kg) p.o + scopolamine (1 mg/kg) VII Treatment group CGA (30mg/kg) p.o + scopolamine (1 mg/kg)
3.16.2. Y-Maze test for Spontaneous Alternation
According to reported [342, 343] assay the Y-Maze test was designed to evaluate the short term memory (STM) in experimental mice. Y-maze apparatus is Y shaped comprised of three
68
CHAPTER 03 MATERIAL AND METHODS arms. The apparatus is handled through digital camera which recorded the entries of mice in various arms in a session of 5 min. To start behavioural test the mice was set in one arm and recorded the total entries and returns to tall arm, same arm and alternate arm. %SAP was calculated by the following formula:
(3.8)
3.16.3. Novel object recognition Test
According to previously published methods [342, 343] the novel object recognition (NORT) test was carried out which is composed of two phases; sample and test. Both the phases were done in an open field box of fly wood. In the sample phase both the objects were same
(plastic ball) however in test phase the novel object like Plastic Square was positioned. The mouse was subjected to both the phases with 5 minutes of intervals to evaluate the STM. To assess long term memory NORT were used after five days. Both the phases were repeated for long term memory except that the test phase for which time interval was 24 h after exposure to the sample phase. According to the method the time consumed in seconds was calculated manually for exploring the familiar object (TFO), novel object (TNO), and the time explored for both the objects (TFO+TNO). % of discrimination index (DI) was calculated by formula:
(3.9)
3.17. Molecular docking validation for antiamnesic activity
For a theoretical study towards anticholinesterase activity, two dimensional structures of molecules were drawn on Chem Draw Professional 16.0 (PerkinElmer Inc.) and finally adapted to 3 D conformations followed by energy minimizations using Chimera 1.13. 1rc.
Docking simulations were performed on a GOLD (Genetic Optimizations for Ligands) software (GOLD suit 5.6.3, Cambridge Crystallographic Data Center) [344]. Gold score was
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CHAPTER 03 MATERIAL AND METHODS selected as a fitness function for the ligand molecules. A search area of 6 Å radius was fixed for docking simulations in the active sites of the reference ligands. Acetyl cholinesterase and butyryl cholinesterase (PDB ID: 4BDS) were selected as receptor enzymes with well- established crystal structures recovered from the website of Protein Data Bank
(www.rcsb.org). These structures of protein were subsequently prepared by hydrogen addition and water and co-crystallized ligands elimination. Default settings were adopted for all parameters. Areas where reference ligands were bound to enzymes were designated as active sites. The HPLC detected and isolated compounds were docked into designated active gorges to see their possible interactions with different amino acids. Docking accuracy was also validated by redocking of the reference ligands. The different images (2 D & 3 D) were visualized and processed by using Discovery Studio Visualizer software [345].
3.18. Histopathology
To undergo histopathology of the dissected rat’s tissue collected from liver and kidney and finally were stored formalin (10%) solution. The tissue samples were processed according to standard protocol [346].
3.18.1. Histopathology technique
Before processing 1cm of liver and kidney tissue were cut and washed with running tap water. The washing process was continued overnight by keeping the condition that the tissue samples might not be damaged. The tissue samples were placed by using automatic tissue processor (Tissue-Tek®, Sakura; Japan). Dehydration process in ascending grade of alcohol was carried out for tissue placement with decreasing time period as follows;
Dehydration Reagent Time period (hrs.) Alcohol (30%) 3-4 Alcohol (50%) 2
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Alcohol (70%) 2 Alcohol (80%) 1.5 Alcohol (90%) 1.5 Absolute alcohol-I 1 Absolute alcohol-II 1 Clearing Reagent Time period (min.) Mixture of Alcohol & Xylene 45 Xylene-I 30 Xylene -I 15
Impregnation
Paraffin I & II liquefied at 72ºC was used for 2 hrs. to impregnate tissues sample
Implanting
Automatic tissue implanting assembly (Tissue-Tek®-TEC™; Sakura) was used for blocks preparation after processing of tissues. Blocks were prepared by placing the tissue sample in plastic cases and molten paraffin was poured into it. After cooling the blocks of tissue plates were allowed to dry.
Sectioning
Microtome (Accu-Cut® SRM™ 200 Sakura) was used to sectioned tissue blocks in about 4-5
µm in thickness and folds were removed at 56oC by placing it in water bath. Albumin was applied for proper cleaning and section sticking to the slides. Finally the sections were mounted on the slides and dried it in oven for 3-4 hrs.
Staining
After drying, the slides were placed for staining in automatic slide stainer (Tissue-Tek®
DRS™ (2000), Sakura; Japan) using Hematoxylin and Eosin staining solution according to standard protocol;
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CHAPTER 03 MATERIAL AND METHODS
Paraffin removal
Reagents Time period (min) Xylene 3 Xylene removal using alcohol Reagents Time period (min) Ethyl alcohol (100%) 1 Ethyl alcohol (100%) 1.30 Ethyl alcohol (50%) 1 Tap water 2 Distilled water 2 Staining Dye Reagents Time period (min) Hematoxylin (Annexure-3) 6 Tap water 2 Decolorization Reagents Time period (min) Acid alcohol (Annexure-4) 2 Tap water 1 Tissue Sections Mordanting Reagents Time period (min) Amino alcohol 5 Tap water 1 Ethyl alcohol (100%) 1 Counter Staining Reagents Time period (min) Eosin (Annexure-5) 1
Dehydration Reagents Time period (min) Ethyl alcohol (75%) 1 Ethyl alcohol (100%) 1 Clearing
Reagents Time period (min)
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Xylene 1.30 Xylene 1 Xylene 1.30 Mounting of Cover Slip
After completion of staining and cleaning process the slides were properly poured using DPX
(Scharlau) with great care in order to avoid bubbles formation.
Hematology and Serology
3 mL of blood samples were collected from rats in EDTA tubes and allowed to clot. Serum was collected 1 mL Eppendorf tubes by centrifugation at 3000 rpm for 10 min and was kept at 4°C. Serum was used for investigation of biochemical parameters like SGPT, SGOT and
ALP, TC, TG, LDL, HDL and serum creatinine by using Biochemistry analyzer (PS-520,
Shenzhen Procan Electronics; China).
3.19. Statistical analysis
All in vitro and in vivo experiments were performed in three replicates. The results were presented as Mean ± SEM, Student’s t-test, one way & two way ANOVA followed by
Dunnett’s posthoc multiple comparison & Bonferroni Post-test were used to determine the values of P. P < 0.05 were considered as significant.
3.20. Assessment of IC 50 Values
Linear regression was used to calculate IC50 for % DPPH, ABTS α-amylase, α-glucosidase,
AChE and BChE inhibition against the different concentration of test samples by means of
Excel program 2007.
3.21. Regression and linear Correlation (R2)
Regression and linear correlation (R2) were used to determine TPC, TFC, the antioxidant and enzyme inhibition potentials of samples using Excel 2007.
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4. RESULTS 4.1. Extraction and fractionation yield
After extraction the net yield of semisolid mass obtained was 750 g crude Me-Ext. While the fractionation yields were 95, 210, 115, 90 and 220 g solid extracts of n-hexane, chloroform, ethyl acetate, n-butanol, and aqueous fractions respectively. The extracts/fractions were screened for antidiabetic and antiamnesic potentials [316].
4.2. Preliminary phytochemical screening
4.2.1. Qualitative screening
The Me-Ext of E. umbellata Thunb. fruit give positive results for preliminary major phytochemical groups that are presented in Table 4.1.
Table 4. 1: Preliminary qualitative phytochemical screening of Met-Ext from E. umbellata Fruit
S. No Phytochemical Reagents used Analyses Results groups
1 Alkaloids Dragendorff’s Orange red color precipitate +
2 Flavonoids Ferric-chloride Yellow color and after HCL + addition becomes colorless
3 Glycosides Keller Killiani Formation of red to brown layer +
4 Tannins Gelatin Brownish-green precipitate +
5 Triterpenoids Liebermann Burchard Reddish brown boundary +
6 Anthraquinones Bontrager’s Formation of reddish color +
4.2.2. Total Phenolic content (TPC)
Results of TPC in hydro methanolic extract and various fractions of E. umbellata fruit are presented in Table 4.2. Standard Gallic acid curve was constructed by preparing the dilutions
20, 40, 60, 80 and 100 mg/mL to estimate the TPC in E. umbellata fruit samples by using regression equation. The phenolic contents of Me-Ext, Hex-Ext, Chf-Ext, EtAc-Ext, But-Ext and Aq-Ext were 26.81 ± 0.33, 36.28 ± 0.69, 56.97 ± 0.77, 49.15 ± 1.05, 44.95 ± 0.97 and
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20.54 ± 0.61. It was calculated that Chf-Ext and EtAc-Ext fractions showed highest percentage of total phenolic contents (Figure 4.1A).
Table 4.2: Total Phenolic and Flavonoid Contents in extract/fractions of Elaeagnus umbellata Fruit
Extracts sample TPC ( mg GAE/g) TFC (mg QE/g) Me-Ext 26.81 ± 0.33 38.94 ± 1.01 Hex-Ext 36.28 ± 0.69 41.05 ± 0.84 Chf-Ext 56.97 ± 0.77 75.53 ± 0.44 EtAc-Ext 49.15 ± 1.05 63.49 ± 1.03 But-Ext 44.95 ± 0.97 52.13 ± 0.68 Aq-Ext 20.54 ± 0.61 32.06 ± 0.69
4.2.3. Total Flavonoid contents (TFC)
To estimate the TFC in E. umbellata fruit hydro methanolic extract and various fractions, a regression curve of standard quercetin and test sample was constructed by preparing the dilutions 20, 40, 60, 80 and 100 mg/mL and the regression equation were used for TFC determination. Results show that total flavonoid contents of Me-Ext, Hex-Ext, Chf-Ext,
EtAc-Ext, But-Ext and Aq-Ext were 38.94 ± 1.01, 41.05 ± 0.84, 75.53 ± 0.44, 63.49 ± 1.03,
52.13 ± 0.68 and 32.06 ± 0.69 (Table 4.2). Results revealed that highest total flavonoid content was present in Chf-Ext and EtAc-Ext fractions (Figure 4.1B).
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Figure 4.1 (A): Total Phenolic Content in extract/fractions of Elaeagnus umbellata Fruit
Figure 4.1 (B): Total Flavonoid Content in extract/fractions of Elaeagnus umbellata Fruit
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4.2.4 Identification of phenolic compounds through HPLC-UV technique
Typical HPL-UV chromatograms of E. umbellata fruit Me-Ext/fractions are presented in
Figure 4.2. A total of twelve phenolic compounds (malic acid, gallic acid, vitamin C, chlorogenic acid, epigallocatechin gallate, quercetin, morin, ellagic acid, catechin hydrate, rutin, pyrogallol and mandelic acid) were identified in the Me-Ext while eight phenolic compounds; chlorogenic acid, epigallocatechin gallate, quercetin, morin, ellagic acid, catechin hydrate, rutin, and pyrogallol were identified in the Chf-Ext. In EtAc-Ext five phenolic compounds (gallic acid, quercetin, rutin, pyrogallol and mandelic acid) were identified (Figure 4.2). The Quantification and identification of each phenolic compound with their particular peak position and retention time (Rt) in chromatogram is presented in Table
4.3. All these phenolic compounds were identified with the help of standard phenolic compounds in fruit samples of E. umbellata. Quantification of antioxidants was done by formula:
(4.1)
Cx= Sample concentration; As= Standard peak area; Ax= Sample peak area; Cs= Standard concentration (0.09 µg/ml).
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Table 4.3: Identification and Quantification of phenolic compounds in E. umbellata Thunb. fruit Me-Ext/fractions
Sample No. Retention Phenolic HPLC- Peak Peak Concentration Identification Extract of time compounds UV Area Area of (µg/ml) Reference Peak (min) Identity λmax of standard (nm) sample 1 2.7 Malic acid 320 434.5 40.3 9.7 Ref. Stand. 2 4.3 Gallic acid 320 25.3 195.4 0.1 Ref. Stand.
3 4.6 Vitamin C 320 18.2 22.4 0.7 Ref. Stand.
4 6.0 Chlorogenic 320 331.6 12.9 23.1 Ref. Stand. Me.Ext acid 5 8.9 Epigallocatechin 320 972.0 72.6 12.1 Ref. Stand. gallate 6 10.3 Quercetin 320 1849.2 90.9 18.3 Ref. Stand. 7 12.3 Morin 320 25.7 2.0 11.5 Ref. Stand. 8 16.7 Ellagic acid 320 36.4 319.2 0.1 Ref. Stand. 9 20.0 Catechin 320 226.5 78.0 2.6 Ref. Stand. hydrate 10 22.7 Rutin 320 69.4 22.4 2.8 Ref. Stand. 11 28.1 Pyrogallol 320 11.8 1.0 10.5 Ref. Stand. 12 30.4 Mandelic acid 320 34.2 72.0 0.4 Ref. Stand. 1 6.0 Chlorogenic 320 126.5 12.9 8.8 Ref. Stand. acid 2 8.9 Epigallocatechin 320 4706.1 7261.5 58.3 Ref. Stand. Chf-Ext gallate 3 10.3 Quercetin 320 899.1 9089.3 8.9 Ref. Stand. 4 12.3 Morin 320 63.6 11.5 5.0 Ref. Stand. 5 16.7 Ellagic acid 320 148.0 319.2 0.4 Ref. Stand. 6 20.0 Catechin 320 3449.0 78.0 39.8 Ref. Stand. hydrate 7 22.7 Rutin 320 1126.2 2241.2 45.2 Ref. Stand. 8 28.1 Pyrogallol 320 58.5 1.0 52.1 Ref. Stand. 1 4.3 Gallic acid 320 966.3 195.4 4.5 Ref. Stand. EtAc- Ext 2 10.3 Quercetin 320 1302.0 90.9 12.9 Ref. Stand. 3 22.7 Rutin 320 355.0 22.4 14.3 Ref. Stand. 4 28.1 Pyrogallol 320 53.3 1.0 47.5 Ref. Stand. 5 30.4 Mandelic acid 320 488.7 72.0 6.1 Ref. Stand.
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Figure 4.2: HPLC-UV Chromatograms of phenolic compounds in E. umbellata Thunb. fruit (A) Me.Ext, (B) Chf-Ext and (C) EtAc-Ext
4.2.4. GC-MS examination for essential oil
The GC-MS chromatogram of E. umbellata fruit essential oil confirmed a total of 68 components and is presented in Figure 4.3 while their various parameters are listed in Table
4.4. Some of the most common components found in the essential oil of E. umbellata have also been shown in Table 4.5. Among these components some have been reported previously by other investigators that possess antioxidant and anticholinesterase potentials [557-559].
The major peaks given in the Table 4.5 shows various volatile compounds with retention times of 1.30, 1.39, 1.43, 1.59, 10.32, 10.38, 12.05, 15.02, 18.80, 19.21, 19.32, 24.05, 24.83,
25.03, 27.55, 30.85, 31.00, 32.01, 32.08, 32.21, 32.59, 43.03 min respectively. The structural formulas of these major compounds are given in Fig. 4.4. Some important active antidiabetic and neuroprotective phytochemicals in E. umbellata essential oil are presented in Figure 4.5.
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The important pharmacological potentials observed in this study are by virtue of presence of free radical, AChE, BChE, α- glucosidase and α- amylase enzymes inhibitors.
Figure 4.3: GC-MS chromatogram of E. umbellata fruit essential oil
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Figure 4.4: Major compounds of Elaeagnus umbellata Thunb. essential oil via GC-MS (A) 1,4-Dioxane-2,5-diol, (B) 2,3-dihydrobenzo furan (Coumaran), (C) 2-Methoxy-4-vinylphenol (p-Vinylguaiacol), (D) 6-methyl -2-Heptanone, (E) Decanoic acid, (F) Ethyl 3,3- dimethylbutyrate, (G) Dimethyl-3-vinyl-4-hexen,2-ol (H) Nonanoic acid, (I) (-)- Caryophyllene, (J) (-)Caryophyllene oxide, (K) Trimethyltricyclo [5.3.1.02.8] undecane-11- methanol, (L) 2-t-Butylpentanoic acid, (M) 9,12-Octadecadienoic acid, (N) Pentadecanoic acid, (O) n-hexadecanoic acid, (P) Octadecanoic acid
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Figure 4.5: Active Antidiabetic and neuroprotective compounds of Elaeagnus umbellata Thunb. essential oil via GC-MS (A) n-hexadecanoic acid, (B) 9,12-Octadecadienoic acid, (C) α-linolenic, (D) (-) Caryophyllene, (E) (-) Caryophyllene oxide, (F) Phytol, (G) Octadecanoic acid, (H) Tricosanoic acid, (I) 9-Octadecenal, (J) 9- octadecanoic acid, (K) 7- Tetradecanal, (L) 2-Methoxy-4-vinylphenol, (M) Tri-Octadecatrienoic acid, (N) 8,11- octadecadienoic acid, (O) Nonanoic acid, (P) Neophytadiene, (Q) Decanoic acid, (R) Pentadecanoic acid, (S) Decanal, (T) Humulene Epoxide, ((W) p-Vinylguaiacol, (X) (-)- Isolongifolol, (Y) (+)-Ascorbic acid 2,6-dihexadecanoate, (Z) 8, 11, 14-Eicosatrienoic acid
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Table 4.4: Parameters of various components of essential oil of E. umbellata Thunb.
S.No Retention Area Area Height Height Area Base Mass Compound name time (RT) % % Sum Peak peak % m/z
1 1.30 3383513 74.13 1917878 71.73 1.76 31.20 432 Methyl alcohol
2 1.39 22509 0.63 17454 0.76 1.29 43.10 455 Isopropyl alcohol 3 1.43 43059 1.41 32253 1.72 1.34 82.95 517 Acetic acid, methyl ester 4 1.59 286810 8.01 282261 12.37 1.02 83.05 515 Trichloromethane
5 10.32 11586984 100 14335131 100 33.04 120 560 Coumaran (2,3- dihydro 1- Benzofuran) 6 11.41 22956117 31.25 819738 32.24 7.79 120 534 5-methyl -3- Heptanol 7 12.05 70226678 48.69 66352765 46.85 17.04 150 555 2-Methoxy-4- vinylphenol (p- Vinylguaiacol) 8 15.02 5640019 38.99 55814679 55.51 13.35 94.91 512 2,5-Dimethyl,3- vinyl,4-hexen-2-ol
9 18.80 792137 5.71 582813 23.51 3.64 42.92 445 Humulene Oxide (3),11, 12 10 19.21 2284319 14.71 570779 23.25 4.22 67.01 480 (-) Caryophyllene oxide (3), 10. 11 11 19.32 2788109 13.41 546134 23.09 3.09 202.91 620 2H-Cyclopropa[g] benzofuran 12 24.05 9961820 100 118755 100 25.02 68.01 422 Neophytadiene
13 24.83 8504473 25.46 625592 25.98 6.46 81.12 516 3,7,11,15- Tetramethyl-2- hexadecen-1-ol 14 25.03 4490823 42.62 1924997 42.55 11.41 81.21 520 3,7,11,15- Tetramethyl-2- hexadecen-1 15 27.55 442310 8.45 85312 10.67 5.18 73.05 503 n-hexadecanoic acid 16 30.85 88179 1.93 26263 0.98 3.36 81.10 429 Cis-cis-9,12- Octadecanoic acid 17 31.00 186638 4.09 35911 1.34 5.20 79.10 458 8, 11, 14- Docosatrienoic acid 18 32.01 108272 2.37 36977 1.38 2.93 55.10 475 cis-9-Hexadecenal 19 32.08 2914832 55.66 260362 32.57 11.20 67.10 464 Cis-cis-9, 12- Octadecadienoic acid 20 32.21 1451139 27.71 278746 34.87 5.21 55.05 511 9-Octadecenoic acid, (E)- 21 32.59 272379 5.20 78422 9.81 3.47 43.10 530 Octadecanoic acid (44) 22 43.03 95010 2.56 12959 0.53 7.33 18.30 548 Tricosanoic acid
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Table 4.5: List of major components identified in essential oil of E. umbellata
S.No Retention Molecular Molecular Compound name Hits time (RT) mass formula (DB) (g/mol) 1 1.30 32 CH3OH Methyl alcohol 5 2 1.31 60 C2H4O2 Methyl formate 5 3 1.32 44 C2H4O Acetaldehyde 5 4 1.33 120 C4H8O4 1,4-Dioxane-2,5-diol 5 5 1.38 60 C3H8O Isopropyl alcohol 5 6 1.39 86 C5H10O 4-Penten-2-ol 5 7 1.40 88 C5H10O 2- Pentanol 5 8 1.41 74 C3H6O2 Methyl ethanoate 4 9 1.42 102 C4H6O3 Propanoic acid 5 10 1.43 74 C3H6O2 Acetic acid methyl ester 5 11 1.43 60 C2H4O2 Acetic acid 5 12 1.59 119 CHCl3 Chloroform (Trichloromethane) 5 13 1.60 182 C2H2Cl4O Bis -dichloromethyl -ether 5 14 1.68 74 C4H10O 1-Butanol 4 15 1.69 102 C5H10O2 n-Butyl-formate 5 16 7.47 132 C6H12OS Ethanethioic acid, S-(2- 5 methylpropyl) ester 17 6.66 116 C5H8O3 Pentanoic acid, 4-oxo 1 18 10.01 156 C10H20O Decanal 4 19 10.32 120 C8H8O Coumaran 5 20 11.41 130 C8H18O 5-methyl -3-Heptanol 5 21 12.05 212 C13H24O2 Cis- 11-methyl-2-dodecenoic acid 4 22 12.31 150.18 C9H10O2 2-Methoxy-4-vinylphenol (p- 5 Vinylguaiacol) 23 12.71 128 C8H16O 6-methyl -2-Heptanone 7 24 13.59 172 C10H20O2 Decanoic acid 5 25 14.23 144 C8H16O2 Ethyl 3,3-dimethylbutyrate 5 26 14.77 116 C5H8O3 4-hydroxy, 2-Pentenoic acid 10 27 15.02 154 C10H18O 2,5-Dimethyl,3-vinyl,4-hexen-2-ol 10 28 15.05 186 C10H18O3 Nonanoic acid, 9-oxo-methyl ester 10 29 18.01 158 C9H18O2 Nonanoic acid 10 30 18.21 204 C15H24 (-)-Caryophyllene 5 31 18.80 220.356 C15H24O Humulene Epoxide 5 32 19.31 220.350 C15H24O (-) Caryophyllene oxide 5 33 19.31 255 C16H17NO2 1-Phenyl-2-(4-methylphenyl)-3- 5 nitropropane 34 19.32 130 C9H6O 2H-Cyclopropa[g] benzofuran 5 35 20.51 218 C9H14O6 Dioxolane, dicarboxylic acid, 7 dimethyl ester 36 22.17 222 C14H22O2 Trimethyl-1-(3,methylbuta dienyl)- 7 7-oxabicyclo[4.1.0] heptan-3-ol 37 23.91 222 C15H26O (-)-Isolongifolol 4 38 24.05 278 C20H38 Trimethyl-3-methylidene 10 hexadec,1-ene (Neophytadiene) 39 24.82 242 C15H30O2 Pentadecanoic acid 10
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40 24.83 296 C20H40O Tetramethyl-2-hexa decen-1-ol 10 (Phytol) 41 25.03 280 C20H40 Tetramethyl-2-hexa decen 5 42 25.03 469 C32H52O2 Cycloeucalenyl acetate 5 43 25.30 234 C15H22O2 8-Keto,10-dehydrobrominated beta 5 snyderol 44 27.55 256 C16H32O2 Palmitic acid 5 (n-hexadecanoic acid) 45 27.70 653 C38H68O8 (+)-Ascorbic acid 2,6-dihexa 5 decanoate (L-Ascorbyl 2,6- Dipalmitate) 46 27.75 242 C15H30O2 Pentadecanoic acid 5 47 30.85 280 C18H32O2 Cis-cis-9,12- octadecadienoic acid 4 48 30.85 280 C18H32O2 10, 13- octadecadienoic acid 4 49 31.00 348 C23H40O2 8, 11, 14- Docosatrienoic acid 5 50 32.01 306 C20H34O2 8, 11, 14-Eicosatrienoic acid 5 51 31.05 278 C18H30O2 9, 12, 15-Octadecatrienoic acid 5 52 31.07 184 C11H20O2 Cyclo propane octanoic acid 5 53 32.08 280 C18H3202 -9, 12-Octadecadienoic acid 5 54 32.03 238 C16H30O Cis-9-Hexadecanal 5 55 32.07 210 C14H26O 7-Tetradecanal 5 56 32.08 306 C15H24Cl2O2 Dichloroacetic acid 5 57 32.09 266 C18H34O 9-Octadecenal 5 58 32.10 322 C21H38O2 Cis-11, 14-Eicosadienoic acid 5 59 32.21 511 9-Octadecenoic acid, (E)- 4 60 32.23 296 C19H36O2 10, 2-Hexacyclo propyl Decanoic 4 acid 61 32.59 530 Octadecanoic acid 1 62 32.60 242 C15H30O2 Pentadecanoic acid 3 63 32.72 C3H8N4 2-Hydrazino-2-imidazoline 3 64 43.00 250 C9H18N2O4S 2-Butanone,3,3-dimethyl-1-(methyl 5 sulfonyl) 65 43.03 548 C23H46O2 Tricosanoic acid 5 66 43.05 320 C16H17ClN2O3 4(4-Chlorophenyl)-3 morpholino 5 pyrrol-2-carboxylic acid 67 43.07 172 C10H20O2 2-t-Butylpentanoic acid 5 68 44.34 184 C13H28 3,8-dimethyl- Undecane 5
4.3. Compounds isolation and structural confirmations 4.3.1. Compound-I Experimental data of Compound-I:
IUPAC name: 3-(3, 4-dihydroxyphenyl) prop-2-enoyl] oxy}-1, 4, 5- trihydroxycyclohexanecarboxylic acid Common name: 3-O-caffeoyl quinic acid or chlorogenic acid
Appearance: amorphous colorless
Physical state: solid
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Chemical formula: C16H18O9
Molar mass (m/z): 353.3 gmol-1
FAB-MS (m/z): 355 [M] +
Melting point: 208 °C-209°C
Solvent scheme: n-Hexane and Ethyl acetate (3:7)
Rf. value: 0.45
Weight obtained: 3.4 gm
Solubility: Soluble in methanol
Figure 4.6: Structural formula of compound-I
The structure of compound-I is presented in Figure 4.6 while FTIR chromatogram of compound-I is presented in Figure 4.7. Peak at 3647.39 cm-1 demonstrating the –OH group while C-H bond stretching occurs at 3072.60 cm-1 peak. Aromatic nucleus has been detected from C=C strong absorption bands at 1687.71 cm-1. The carbonyl functionality (C=O) occurs at 1639.49 cm-1. The broad peak at 3329.14 cm-1 representing the carboxyl group (Figure 4.7).
The FAB-MS is represented in Figure 4.8.
Compound-I 1H-NMR spectra indicated singlet’s at 4.18 (dt, J = 5.2, 3.3 Hz, 1H), 3.74 (dd, J
= 8.6, 3.2 Hz, 1H), 5.34 (ddd, J = 9.6, 8.5, 4.4 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 6.78 (d, J =
8.1 Hz, 1H), 6.95 (dd, J = 8.2, 2.1 Hz, 1H), 7.56 (d, J = 15.9 Hz, 1H) and 6.26 (d, J = 15.9
Hz, 1H), corresponding to the H-3, H-4, H-5,H-2´, H-5´, H-6´, H-7´ and H-8´ position, while the two CH2 protons ( α and β) were assigns multiplets at δ 2.04 and 2.18 at position 2 and δ
2.24 and 2.07 at position 6, respectively (Figure 4. 9)
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The 13C-NMR spectrum showed sixteen signals, including eight methines, six quaternary and
13 two CH2 carbons (Figure 4.10). C NMR (126 MHz, MeOD) spectral data δ 76.11, 38.13,
71.27, 73.45, 71.89, 38.75, 176.99, 127.73, 115.19, 146.68, 149.46, 116.45, 122.97, 147.04,
115.17, 168.65, 48.98 are presented in Table 4.6.
Figure 4.7: FTIR spectrum of compound-I
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Figure 4.8: FAB-Mass of compound-I Table 4.6: Compound-I chemical shifts in solvent MeOD
Carbon No. 1H- (δ) 13C- (δ) Multiplicity 1 - 76.11 C
2α 2.04 38.13 CH2 2β 2.18 3 4.18 71.27 CH 4 3.74 73.45 CH 5 5.34 71.89 CH
6α 2.24 38.75 CH2 6β 2.07 7 - 176.99 C 1´ - 127.78 C 2´ 7.05 115.19 CH 3´ - 146.68 C 4´ - 149.46 C 5´ 6.78 116.49 CH 6´ 6.95 122.97 CH
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7´ 7.56 147.04 CH 8´ 6.26 115.17 CH 9´ ---- 168.70 C Solvent 3.74 48.98 -
Figure 4.9: 1H- (500 MHz.) NMR spectrum of compound-I
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Figure 4.10: 13C-NMR (126 MHz) spectrum of compound-I
4.3.2. Compound-II
Experimental data of Compound-II:
IUPAC name: 2, 3, 7, 8-Tetrahydroxy-chromeno-chromene-5,10-dione
Common name: Ellagic acid
Appearance: tan to gray color powder
Physical state: powder
Chemical formula: C14H6O8
Molar mass (m/z): 302.197 g/mol
Solvent scheme: n-Hexane and Ethyl acetate (2:8)
Solubility: Soluble in water, methanol and aqueous alkaline solution
Melting point: ≥350 °C Boiling point: 364°C
Rf. value: 0.57
Weight obtained: 30.4 mg
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Figure 4. 11: Structural formula of compound-II The structure of compound-II is presented in Figure 4.11. 1H NMR (CHLOROFORM-d
,500MHz): δ = 7.27 (s, 2 H) the chemical shift of 7.27 most probably represents the 2 protons attached on position 9 and 21 of the given structure, 1.56 ppm (s, 2 H), the peak on the high chemical shift of 1.56 might be hydroxyl group protons at position 6 and 18 of the given structure, 0.08 ppm (s, 2H) the singlet peak on very high chemical shift of 0.08 represents the proton of –OH group on position 8 and 20 of the given structure (Figure 4.12). 13C NMR
(Chloroform-d, 100MHz): δ = 108, 51.8, 48, 42, 37.6, 13.7 ppm (Figure 4.13). The EIMS mass of compound-II is given in Figure 4.14.
Figure 4.12: 1H NMR (CHLOROFORM-d, 500MHz) of compound-II
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Figure 4.13: 13C NMR (Chloroform-d, 100MHz) of compound-II
Figure 4.14: EIMS of Compound-II
4.3.3. Compound-III
Experimental data of Compound-III:
IUPAC name: 2-(2, 4-Dihydroxyphenyl)-3, 5, 7-trihydroxychromen-4-one
Common name: Pentahydroxy flavone or 7-Hydroxyflavonol or Morin
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Appearance: Light yellow powder
Physical state: Yellow solid
Chemical formula: C15H10O7
Molar mass (m/z): 302 g/mol
Solvent scheme: n-Hexane and Ethyl acetate (3:7)
Solubility: Soluble in alcohol (ethanol, methanol), ether and acetic acid
Melting point: 120 °C
Rf. value: 0.47
Weight obtained: 70 mg
Figure 4.15: Structural formula of compound-III The structural formula of compound-III is presented in Figure 4.15. The FTIR spectra shows aromatic functionality at 1612.49 cm-1 while, another broad peak at 3161.33 cm-1 demonstrating the C-H bond stretching representing in Figure 4.16.
1H NMR (Acetone, 300MHz): The HNMR spectrum has been given in Figure 4.17. The chemical shift from δ = 2 to 2.5 ppm shows the solvent peaks. δ = 12.18 (s, 1 H), this singlet peak could be attributed to -OH group proton attached to the Carbon number 2 of the said structure. δ= 9.73 (br. s., 1 H), this broad peak represent one proton attached to carbon number 22 of the provided structure. δ= 8.58 (br. s., 1 H), this singlet peak also represent the proton at position 21 of provided structure. δ= 8.36 (br. s., 1 H), this broad peak represent one proton attached on position number 18 of the given structure. δ= 8.01 (s, 1 H), this peak
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CHAPTER 04 RESULTS could probably represents the proton at position number 12 of given structure. δ= 7.84 (s, 1
H), this peak could be attributed to proton attached at carbon number 9 of the given structure.
δ= 7.69 (s, 1 H), δ= 6.99 (s, 1 H), δ= 6.53 (s, 1 H), δ= 6.27 ppm (s, 1 H), the last four peaks could be attributed to protons attached at position 8, 11, 17 and 20 of the given structure (Fig.
4.38). 13C NMR (Acetone, 75MHz): = 205.6, 175.7, 164.0, 161.4, 156.9, 147.4, 146.1,
144.9, 135.9, 122.9, 120.6, 115.3, 103.3, 98.3, 93.5 ppm (Figure 4.18). The EIMS is presented in Figure 4.19.
Figure 4.16: FTIR spectra of compound-III
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Figure 4.17: 1H NMR (Acetone, 300MHz) of compound-III
Figure 4.18: 13C NMR (Acetone, 75MHz) of compound-III
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Figure 4.19: EIMS of compound-III
4.3.4. Compound-IV
Experimental data of Compound-IV:
Common name: Gallic acid
IUPAC name: 3, 4, 5- Tri-hydroxy benzoic acid
Appearance: Light yellowish white crystalline powder
Physical state: Yellowish white
Chemical formula: C7H605
Molar mass (m/z): 170 g/mol
Solvent scheme: n-Hexane and Ethyl acetate (4:6)
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Solubility: Soluble in ethanol, methanol, ether, glycerol, ethyl acetate and acetone
Melting point: 260°C
Rf. value: 0.35
Weight obtained: 40 mg
Figure 4.20: Structural formula of compound-IV
1 The structure of compound-IV is presented in Figure 4.20. H NMR (METHANOL-d4,
500MHz): The proton NMR spectrum of the given compound has been provided in figure
4.21. The exploited solvent was Methanol-d, which has been detected at δ= 3.35 ppm as given in the spectrum. δ= 7.08 (s, 2 H) this singlet peak represents 2 protons attached on carbon number 3 and 10 of the given structure, δ= 4.93 ppm (br. s., 3 H) this broad singlet peak might be the proton attached as –OH group at carbon number 5, 7 and 9 of the identified
13 structure (Figure 4.21). C NMR (METHANOL-d4, 100MHz): δ = 169.0, 145.0, 138.2,
120.6, 108.9 ppm (Figure 4.22). The EIMS is represented in Figure 4.23.
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1 Figure 4.21: H NMR (METHANOL-d4, 500MHz) of compound-IV
13 Figure 4.22: C NMR (METHANOL-d4, 100MHz) of compound-IV
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Figure 4.23: EIMS spectra of compound-IV
4.3.5. Compound-V
Experimental data of Compound-V:
IUPAC name: 2-(3, 4-dihydroxyphenyl)-3, 4-dihydro-2H-chromene-3, 5, 7-triol
Common name: Cianidanol or catechin
Appearance: colourless solid
Physical state: solid
Chemical formula: C15H14O6
Molar mass (m/z): 290.1 g/mol
Solvent scheme: n-Hexane and Ethyl acetate (3:7)
Solubility: Soluble in ethanol, methanol, ethyl acetate, hexane and DMSO
Melting point: 175.5 °C
Rf. value: 0.42
Weight obtained: 2.5 gm
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Figure 4.24: Structural formula of compound-V
1 The structure of compound-V is presented in Figure 4.24. H NMR (METHANOL-d4
,300MHz): δ = 6.73-6.62, signifies the aromatic protons at 15th, 20th and 21th carbon of the given structure, δ= 5.83 (s, 1 H) this singlet could be the hydrogen atom at carbon number 11,
δ= 5.76 (s, 1 H) this singlet could be attributed to proton attached with carbon number 8 of the given structure, 4.76 (s, 4H) this broad singlet represents the four protons each one attached as a –OH group to the carbon number 6, 9, 16 and 18. δ= 4.46 (d, J=7.5 Hz, 1 H),
3.87 (d, J=5.1 Hz, 1 H) this duplet can be precisely represent by a proton attached to the carbon number 2 of the given structure, δ = 2.71 ppm (s, 1 H) this singlet has been contributed by the protons attached to the carbon number 4 of the given structure (Figure
13 4.25). C NMR (METHANOL-d4, 75MHz): δ = 156.4, 156.2, 155.5, 144.8, 130.8, 118.6,
113.9, 99.4, 94.9, 94.1, 81.5, 67.4, 27.1 ppm (Figure 4.26). The FTIR spectrum and EIMS is represented in Figure 4.27 & 4.28.
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1 Figure 4.25: H NMR (METHANOL-d4, 300MHz) of compound-V
13 Figure 4.26: C NMR (METHANOL-d4, 75MHz of compound-V
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Figure 4.27: EIMS of compound-V
Figure 4.28: FTIR spectra of compound-V
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1.1.1. Compound-VI
Experimental data of Compound-VI:
IUPAC name: Benzene-1, 3, 5-triol
Common name: Phloroglucinol
Appearance: colorless to beige solid
Physical state: solid
Chemical formula: C6H6O3
Molar mass (m/z): 127 g/mol
Melting point: 219 °C
Solvent scheme: n-Hexane and Ethyl acetate (6:4)
Solubility: Soluble in methanol, ethanol, and diethyl ether
Rf. value: 0.39
Weight obtained: 80 mg
Figure 4.29: Structural formula of compound-VI
The structure of compound-VI is presented in Figure 4.29 while FTIR spectrum in Figure
4.30. The absorption peaks at 3161.33 and 3479.58 cm-1 demonstrating the –OH group. Peak at 2887.44 cm-1 indicates C-H stretching. Aromatic nucleus can be recognized by C=C strong peak at 1612.49 cm-1. Band at 1739.79 cm-1 is due to functionality of 1, 3, 5 substitute benzene (Figure 4.30).
The NMR signals of compound-VI in methanol solvent are reported below: 1H NMR
(MeOD, 300 MHz) δ= 9.11 (s, 3H, OH), 5.83 (s, 3H, CH) (Figure 4.31, Table 4.7) while 13C
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NMR (MeOD, 75 MHz) δ160.0 ppm (CH2), 95.5 ppm (CH) (Figure 4.32, Table 4.7). The
EIMS is shown in Figure 4.33.
Figure 4.30: FTIR spectra of compound-VI
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Figure 4.31: 1H- (500 MHz) of compound-VI
Figure 4.32: 13C-NMR (126 MHz) of compound-VI
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Table 4.7: The chemical shifts of compound-VI in solvent MeOD
Carbon No. 1H (δ) 13C (δ) Multiplicity 1 - 160.0 C H
2 5.83 95.5 CH2 3 - 160.0 C H
4 5.83 95.5 CH2 5 - 160.0 CH
6 5.83 95.5 CH2 OH - 9.11 Solvent 3.74 48.98 -
Figure 4.33: EI-MS Mass of compound-VI
1.1.2. Compound-VII
Experimental data of Compound-VII:
IUPAC name: 1-hexylbenzene
Common name: n-Hexyl benzene, 1-Phenyl hexane
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Appearance: Colourless liquid
Physical state: Liquid
Chemical formula: C12H18
Molar mass (m/z): 162 g/mol
Solvent scheme: n-Hexane and Ethyl acetate (9:1)
Solubility: Soluble in aqueous solution
Melting point: -61.2°C
Rf. value: 0.27
Weight obtained: 40 mg
Figure 4.34: Structural formula of compound-VII
The structure of compound-VII is presented in Figure 4.34. The 1H NMR (DMSO- H2O,
300MHz) spectrum has been given in Figure 4.35. The solvents used were H2O and DMSO, the peaks of which are conspicuous in chemical shift 3.5 and 2.5 respectively. δ = 8.31 (s, 3
H) this singlet most probably represents 3 protons of Benzene ring on position 1, 2 and 3.
7.46 (s, 2 H), this chemical shift could possibly be represent by two protons on position 4 and
6 of the structure given. δ= 3.17 (t, 2 H, j=7.2 Hz), these two protons which have been represented by singlet peak could be attributed to position 7 of the structure. δ = 2.09 ppm
(m, 08 H), this singlet peak at the highest chemical shift could be attributed to the 08 protons at position 8, 9, 10 and 11 of the given structure. δ= 0.8 (t, 3H, j=7.8) present at position 12.
13C NMR (DMSO-d6, 75MHz): δ = 159.6, 148.6, 144.7, 140.1, 138.3, 136.8, 112.8, 112.2,
110.7, 108.1, 79.6, 31.1 ppm (Figure 4.36). The IR spectrum has been presented in Figure
4.37. The wave number above the finger print region represents the carbon-carbon double
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1 Figure 4.35: H NMR (DMSO- H2O, 300MHz) of compound-VII
Figure 4.36: 13C NMR (DMSO-d6, 75MHz) of compound-VII
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Figure 4.37: FTIR spectra of compound-VII
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Figure 4.38: EIMS of compound-VII
1.1.3. Compound-VIII
Experimental data of Compound-VIII:
IUPAC name: 2-Hydroxy-2-phenylacetic acid
Common name: Mandelic acid or Phenylglycolic acid
Appearance: White crystalline powder
Physical state: White crystalline
Chemical formula: C8H8O3
Molar mass (m/z): 152 g/mol
Solvent scheme: n-Hexane and Ethyl acetate (7:3)
Solubility: Soluble in ethanol, methanol, diethyl ether and isopropanol
Melting point: 120°C
Rf. value: 0.29
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Weight obtained: 70 mg
Figure 4.39: Structural formula of compound-VIII The structural formula of compound-VIII is represented in Figure 4.39. The FTIR spectra shows 1707 cm-1 strong peak which indicates carbonyl functionality (C=O) while another broad peak of 3392.79 cm-1 demonstrating C-H bond stretching representing in Figure 4.40.
1 H NMR (METHANOL-d4 ,300MHz): δ = 7.48 - 7.51 (m, 2 H) this multiplet represents the 2 protons at position 5 and 9 of the given structure, 7.31 - 7.40 ppm (m, 3 H) this multiplet represents three protons at position 6, 7 and 8 of the given structure, 5.18 ppm (s, 1H) this singlet can be most probably attributed to the single proton on position 3 attached as hydroxyl
13 group (Figure 4.41) while, C NMR (METHANOL-d4 ,75MHz): δ = 174.8, 139.4, 128.1,
127.9, 126.6, 72.8 ppm Figure (4.42). The EIMS is presented in Figure 4.43.
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Figure 4.40: FTIR spectra of compound-VIII
1 Figure 4.41: H NMR (METHANOL-d4, 300MHz) of compound-VIII
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13 Figure 4.42: C NMR (METHANOL-d4, 75MHz) of compound-VIII
Figure 4.43: EIMS of compound-VIII
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1.2. In vitro Antioxidant potential study
1.2.1. Antioxidant potential of essential oil
In our current study, the free radicals scavenging potential of essential oil of E. umbellata fruit against DPPH and ABTS radicals were significant and comparable with the positive control. The % DPPH scavenging potential of essential oil of E. umbellata were 85.24 ± 0.63,
88.30 ± 0.81 against DPPH and ABTS with their IC50 value 70 and 105 µg/mL respectively at the highest concentration of 1000 µg/mL (Table 4.8 & Figure 4.44A). While standard ascorbic acid showed 91.56 ± 0.35% inhibition at 1000 µg/mL against DPPH and 92.63 ±
0.99 against ABTS having 32 and 29 µg/mL IC50 values (Table 4.8 & Figure 44B).
Table 4.8: % DPPH and ABTS free radical Scavenging activity of Essential oil of Elaeagnus umbellata at various concentrations
S.No Sample Concentration % DPPH DPPH % ABTS ABTS (µg/mL) Scavenging IC50 µg/mL Scavenging IC50 µg/mL Mean ± SEM Mean ± SEM 1000 85.24 ± 0.63 *** 88.30 ± 0.81 ns 500 82.44 ± 0.77 ** 79.85 ± 0.55 ** 250 71.14 ± 0.51 *** 74.82 ± 0.75 ** 1 E.O 125 59.29 ± 0.54 ** 70 52.51 ± 1.00 *** 105 62.5 49.31 ± 0.68 *** 41.39 ± 0.69 *** 31.05 44.03 ± 1.04 *** 36.24 ± 0.61 *** 1000 91.56 ± 0.35 92.63 ± 0.99 500 86.31 ± 0.67 83.82 ± 0.60 2 Ascorbic 250 77.11 ± 0.46 32 78.31 ± 0.34 29 acid 125 62.47 ± 0.63 70.59 ± 0.26 62.5 58.22 ± 0.49 63.44 ± 0.86 31.05 50.13 ± 0.31 51.86 ± 0.44
Figure 4.44: % DPPH and ABTS free radical Scavenging activity of essential oil of Elaeagnus umbellata at various concentrations.
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1.2.2. Antioxidant studies on extract/fractions 1.2.2.1. DPPH scavenging potential
The crude Me-Ext, Hex-Ext, Chf-Ext, EtAc-Ext, But-Ext and Aq-Ext inhibited DPPH by 55
± 1, 79 ± 1, 88 ± 1, 83 ± 1, 80 ± 1 and 40 ± 1 % with their IC50 values 550, 80, 40, 45, 60 and
1300 µg/mL respectively at the maximum concentration of 1000 µg/mL. The results indicated that Chf-Ext and EtAc-Ext caused significant inhibition with the lowest IC50 values comparable to standard ascorbic acid (Table 4.9 & Figure 4.45A). The standard ascorbic acid caused 95 ± 1% inhibition at 1000 µg/mL with an IC50 value of 30 against DPPH.
1.2.2.2. ABTS free radical scavenging potential
ABTS free radical scavenging of the Me-Ext and their subsequent fractions are presented in
Table 4.9 & Figure 4.45(B). The % ABTS inhibition of Me-Ext, Hex-Ext, Chf-Ext, EtAc-Ext,
But-Ext and Aq-Ext were 55 ± 1, 80 ± 1, 87 ± 1, 84 ± 1, 78 ± 1 and 43 ± 1 with their IC50 values 760, 135, 57, 70, 120 and 1175 µg/mL respectively at the maximum concentration of
1000 µg/mL. The results indicated that Chf-Ext and EtAc-Ext caused significant inhibition with lowest IC50 values (Table 4.9 & Figure 4.45B). Ascorbic acid caused 91 ± 1 inhibition at 1000 µg/mL with IC50 value of 32 µg/mL against ABTS.
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Table 4.9: %DPPH and ABTS free radical Scavenging activity of Elaeagnus umbellata fruit extract/fractions at various concentrations
S.No Sample Concentration % DPPH IC50 % ABTS IC50 (µg/mL) Scavenging Scavenging Mean±SEM Mean±SEM 1000 54.72±0.56 *** 55.19±1.08 *** 500 49.34±0.34 *** 43.09±0.50 *** 250 41.13±0.95 *** 39.53±0.28 *** 1 Met-Ext 125 33.62±0.84 *** 550 33.03±0.58 *** 760 62.5 20.03±0.51 *** 27.47±0.31 *** 31.05 17.94±0.31 *** 20.57±0.46 *** 1000 79.09±1.15 *** 80.28±0.63 *** 500 73.71±0.88 *** 71.49±0.98 *** 250 68.02±0.63 *** 64.09±0.64 *** 2 Hex-Ext 125 56.16±1.27 *** 80 48.92±0.84 *** 135 62.5 46.41±0.55 *** 42.58±1.11 *** 31.05 39.91±1.02 *** 31.27±0.83 *** 1000 88.20±1.16 *** 87.08±0.53 ** 500 78.38±0.92 *** 75.95±0.49 *** 250 69.26±1.07 *** 68.44±0.48 *** 3 Chf- Ext 125 60.22±1.00 *** 40 65.29±0.54 ns 57 62.5 54.60±0.85 *** 50.99±0.99 *** 31.05 48.32±0.91 ns 46.20±1.03 ** 1000 83.19±0.93 *** 84.30±0.81 *** 500 77.47±0.86 *** 79.39±0.69 ** 250 69.57±0.74 *** 66.82±0.75 *** 4 EtAc- 125 62.76±0.79 *** 45 54.06±0.59 *** 70 Ext 62.5 54.11±1.22 *** 49.24±0.61 *** 31.05 48.72±0.89 ns 44.51±1.00 *** 1000 80.38±0.87 *** 78.14±0.59 *** 500 74.49±0.86 *** 70.22±0.58 *** 250 69.87±1.14 *** 68.11±1.15 *** 5 But-Ext 125 64.16±1.01 *** 60 50.93±1.09 *** 120 62.5 51.20±0.83 *** 43.86±0.74 *** 31.05 46.39±0.60 *** 36.89±0.68 *** 1000 39.82±1.18 *** 43.07±0.57 *** 500 33.59±0.74 *** 36.74±0.36 *** 250 27.86±0.68 *** 31.24±0.59 *** 6 Aq-Ext 125 21.09±0.53 *** 1300 27.16±0.44 *** 1175 62.5 19.26±0.38 *** 19.15±0.18 *** 31.05 14.05±0.56 *** 17.25±0.38 *** 1000 94.88±0.96 91.11±0.68 500 86.59±0.63 83.42±0.43 7 Ascorbic 250 78.64±0.81 78.35±0.72 acid 125 69.14±0.34 30 65.15±0.61 32 62.5 62.87±0.45 57.14±0.33 31.05 51.11±0.71 50.35±0.46
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Figure 4.45: % DPPH and ABTS free radical Scavenging activity of Elaeagnus umbellate fruit/berries extract/fractions at various concentrations.
1.2.3. Antioxidant potential of isolated Compounds 1.2.3.1. DPPH scavenging potential of isolated compounds
DPPH inhibition potential of compounds I-VIII were 88±1.1, 89±0.4, 88±1.1, 85±0.5,
57±1.5, 67±0.4, 59±1.5, and 77±1.1, with their IC50 values 39, 35, 37, 52, 125, 105, 118 and 85 µg/mL respectively at the maximum concentration of 1000 µg/mL. The results indicate that compound-II, compound-I and compound-III causes significant % inhibition potential
with lowest IC50 values comparable to standard ascorbic acid (Table 4.10 & Figure 4.46A).
While standard ascorbic acid cause 91±0.5 inhibition at 1000 µg/mL concentration with IC50 value of 29 µg/mL against DPPH.
1.2.3.2. ABTS free radical scavenging potential of isolated compounds
ABTS free radicals scavenging of pure compounds are presented in Table 4.10 & Figure 4.46. ABTS inhibition potential of compounds I-VIII were 85±1.0, 88±0.5, 88±1.0, 86±2.1,
52±0.5, 64±1.1, 55±1.1, and 71±0.5 with their IC50 values 44, 38, 40, 54, 135, 112, 129 and 39 µg/mL respectively at the maximum concentration of 1000 µg/mL. The results indicate that compound-II, compound-III and compound-I causes significant % inhibition potential
with lowest IC50 values comparable to standard ascorbic acid (Table 4.10 & Figure 4.46 B).
While standard ascorbic acid causes 90±0.5 inhibition at 1000 µg/mL concentration with IC50 value of 32 µg/mL against ABTS.
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Table 4.10: % DPPH and ABTS free radical Scavenging activity of isolated compounds Elaeagnus umbellata at various concentrations
S.No Sample Concentration % DPPH DPPH % ABTS Scavenging ABTS (µg/mL) Scavenging IC50 µg/mL IC50 µg/mL Mean ± SEM Mean ± SEM 1000 88±1.1ns 85±1.0** 500 80±0.5*** 78±0.4** 1 Compound-I 250 75±1.2** 72±0.3** 125 63±0.4*** 39 65±1.0** 44 62.5 57±0.5*** 60±1.0 ns 31.05 44±1.4*** 55±0.5 ns 1000 89±0.4 ns 88±0.5 ns 500 84±1.1 ns 80±0.3 ns 2 Compound-II 250 80±0.4 ns 74±1.1 ns 125 73±0.3* 35 65±0.6 ** 38 62.5 66±0.5*** 57±1.0 *** 31.05 53±1.1*** 51±0.5 ** 1000 88±1.1 ns 88±1.0 ns 500 83±0.5 ns 80±1.0 ns 3 Compound-III 250 76±1.0* 75±0.5 ns 125 68±1.2*** 37 64±1.2 ** 40 62.5 59±1.3*** 57±1.4 *** 31.05 54±0.5*** 45±0.5 *** 1000 85±0.5*** 86±2.1 ns 500 78±1.1*** 77±0.5 *** 4 Compound-IV 250 70±1.5*** 70±1.1 *** 125 65±1.2*** 52 61±1.5 *** 54 62.5 57±0.5*** 52±0.4 *** 31.05 49±1.4*** 43±1.3 *** 1000 57±1.5*** 52±0.5 *** 500 50±1.1*** 46±2.1 *** 5 Compound-V 250 44±0.5*** 41±1.1 *** 125 37±0.4*** 125 36±1.4 *** 135 62.5 31±1.0*** 28±0.5 *** 31.05 29±1.4*** 21±1.0 *** 1000 67±0.4*** 64±1.1 *** 500 57±1.0*** 51±0.5 *** 6 Compound -VI 250 51±1.0*** 45±1.0 *** 125 40±0.5*** 105 39±1.2 *** 112 62.5 36±1.1*** 33±0.5 *** 31.05 32±0.5*** 25±1.0 *** 1000 59±1.5*** 55±1.1 *** 500 53±2.1*** 47±2.2 *** 7 Compound -VII 250 46±0.5*** 39±1.5 *** 125 40±1.1*** 118 31±1.0 *** 129 62.5 35±0.4*** 26±0.5 *** 31.05 28±1.1*** 19±1.4 *** 1000 77±1.1*** 71±0.5 *** 500 70±1.4*** 65±1.1 *** 250 64±0.5*** 59±2.1 *** 8 Compound-VIII 125 56±0.3*** 85 32±0.5 *** 96 62.5 48±1.0*** 45±1.1 *** 31.05 43±0.5*** 40±2.1 ***
1000 91±0.5 90±0.5 500 86±1.0 84±1.1 250 80±0.4 78±0.5 9 Ascorbic acid 125 77±0.4 29 70±0.5 32 62.5 73±0.5 64±1.0 31.05 69±0.4 56±1.1
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Figure 4.46(A): % DPPH inhibition potential of compounds I-VIII at various concentrations
Figure 4.46 (B): % ABTS inhibition potential of compounds I-VIII at various concentrations
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1.3. In vitro Antibacterial Activity
1.3.1. Antibacterial potential of extract/fractions of E. umbellata
The antibacterial potential of extract/fractions of E. umbellata is shown in Table 4.11 and graphically in Figure 4.47. The Chf-Ext, EtAc-Ext and Met-Ext extract/fractions showed significant effects against all bacterial strains whereas, Hex-Ext, But-Ext and Aq-Ext extract/fractions did not exhibit any significant effects. A broad spectrum imipenem standard was used as drug with high potential of zonal inhibition against all bacterial strains. The zone of inhibition of Chf-Ext was 15±1.5 mm followed by EtAc-Ext (15±1.5 mm), Met-Ext
(14±0.5 mm) against Escherichia coli. The high inhibition potential against Bacillus cereus was detected for the Chf-Ext, EtAc-Ext and Met-Ext with 14±0.5 mm, 14±0.4 mm and
12±1.5 mm zones of inhibition respectively. Against Salmonella typhi the EtAc-Ext has shown maximum zonal inhibition of 15±1.0 mm followed by Chf-Ext (12±1.6 mm) and Met-
Ext. (13±0.7 mm) while the But-Ext show the least zone of inhibition 2±0.2 mm. Both the
Chf-Ext and EtAc-Ext. has shown comparative zone of inhibition 11±0.8 mm and 11±1.3 mm against Klebsilla pneumnonia. The But-Ext fraction did not show any antibacterial effect while Met-Ext has shown maximum zone of inhibition 8±1.8 mm against Pseudomonas aeruginosa. The inhibition potential of Chf-Ext, EtAc-Ext and Met-Ext against Proteus mirabilis with values of 16±1.3, 12±0.5 and 11±2.0 mm. The Hex-Ext., But-Ext. and Aq-Ext. did not show any antibacterial effect against Staphylococcus aurous while, Chf-Ext. and Met-
Ext zone of inhibition with values of 8±0.6 and 6±1.4 mm.
The extract/fractions and compounds of E. umbellata displayed noticeable inhibition potential against the tested pathogens, therefore, it can be presumed as valuable source for isolation of active antibacterial agents.
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Table 4.11: Antibacterial potential of extract/fractions of Elaeagnus umbellata
% Zone of Inhibition (mm) Bacterial Met.-Ext Hex-Ext Chf- EtAc- But-Ext Aq-Ext Standard strains Ext Ext 14±0.5 5±0.2 15±1.5 16±0.3 5±0.2 9±0.2 33±0.2 Escherichia coli 12±1.5 8±1.0 14±0.5 14±0.4 4±0.5 5±0.5 30±0.5 Bacillus cereus 13±0.7 7±1.1 12±1.6 15±1.0 2±0.2 7±0.1 33±0.1 Salmonella typhi 10±2.5 6±1.5 11±0.8 11±1.3 6±0.3 5±0.2 29±0.5 Klebsiella pneumonia 8±1.8 2±0.5 6±0.2 7±1.5 0 1±0.1 27±1.1 Pseudomonas aeruginosa 11±2.0 5±2.1 16±1.3 12±0.5 2±0.1 4±0.1 30±0.5 Proteus mirabilis 6±1.4 0 8±0.6 5±2.2 0 0 25±0.3 Staphylococcus aurous
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Figure 4.47: Antibacterial potential of extract/fractions of E. umbellata
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1.3.2. Antibacterial Activity of isolated compounds
The antibacterial effect of isolated compounds is presented in Table 4.12 and Figure 4.48. All
the compounds showed significant effects against all the tested strains of bacteria. Imipenem
a broad spectrum drug was used as standard with noticeable zonal inhibition against the
tested bacterial strains. The compound-I show maximum zonal inhibition against all the gram
positive bacterial strains Escherichia coli, Bacillus cereus, Salmonella typhi, Klebsilla
pneumnonia and Pseudomonas aeruginosa with zonal inhibition of 18±1.0, 15±0.5, 13±0.7,
12±2.1 and 18±0.5 mm while zone of inhibition 17±2.1 mm followed by 15±1.2 mm against
gram negative bacteria Proteus mirabilis and Staphylococcus aureus. The inhibition potential
of compound-III was 18±1.1 mm followed by compound-II (17±0.3 mm), compound-IV
(15±0.3 mm), compound-VIII (15±0.5 mm), compound-VI (14±0.5 mm), compound-VI
(14±0.3 mm) and compound-V (10±0.4 mm). The maximum activity against Bacillus cereus
was observed for the compound-VI (16±1.5 mm) followed by compound-I, II and III with
15±0.5 mm, 15±1.2 mm and 14±0.1 mm zones of inhibition respectively, while compound-
III did not showed any activity. Against Salmonella typhi the compound-V, VI and VIII
showed similar sensitivity with the zone of inhibition of 14±1.1, 14±0.6 and 14±1.5 mm
followed by compound-I (13±0.7 mm), compound-III (12±1.2mm), compound-II (11±1.1
mm) and compound-IV (11±1.2 mm) while the compound-VII showed no zone of
inhibition. Compound-II showed highest zone of inhibition 18±0.5 mm while both the
compound-III and compound-VII has shown comparative zone of inhibition 16±0.5 mm and
16±1.5 mm followed by compound-VIII (15±0.5mm) against Klebsiella pneumonia. The
compound-I and VIII has shown similar zone of inhibition 18±0.5 mm against Pseudomonas
aeruginosa. The compound-VIII showed highest antibacterial activity against Proteus
mirabilis with the values of 19±1.4 mm followed by compound-I (17±2.1 mm), compound-
VI (17±0.1 mm) and compound-II (15±0.2 mm) respectively. The compound-II (17±2.1
mm) and compound-III (17±0.4 mm) has shown highest inhibition potential followed by
compound-I (15±1.2 mm) and compound-IV (14±1.1 mm) against Staphylococcus aureus
while, compound-V showed least zone of inhibition with values of 9±0.1 mm.
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Table 4.12: Antibacterial potential of isolated compounds I-VIII
Bacterial strains Zone of % Inhibition (mm) of compounds I II III IV V VI VII VIII Standard 18±1.0 17±0.3 18±1.1 15±0.3 10±0.4 14±0.5 14±0.3 15±0.5 33±0.2 Escherichia coli 15±0.5 15±1.2 14±0.1 11±2.1 9±0.5 16±1.5 11±0.5 0.0 30±0.5 Bacillus cereus 13±0.7 11±1.1 12±1.2 11±1.2 14±1.1 14±0.6 0.0 14±1.5 33±0.1 Salmonella typhi 12±2.1 18±0.5 16±0.5 14±1.1 12±0.4 11±0.2 16±1.5 15±0.5 29±0.5 Klebsiella pneumonia 18±0.5 14±1.1 14±0.5 10±1.5 17±0.5 10±1.1 17±1.6 18±0.5 27±1.1 Pseudomonas aeruginosa 17±2.1 15±0.2 12±1.5 13±0.5 10±0.1 17±0.1 11±2.1 19±1.4 30±0.5 Proteus mirabilis 15±1.2 17±2.1 17±0.4 14±1.1 9±0.1 10±0.2 10±2.2 16±2.5 25±0.3 Staphylococcus aurous
Figure 4.48: Antibacterial activity of the compounds I-VIII against various bacterial strains
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1.4. In vitro anti-diabetic studies on extracts/fractions
1.4.1. In vitro α-amylase enzyme inhibitory assay
The IC50 values were calculated by evaluating the plot of % α-amylase enzyme inhibition as
function of extract/fractions concentrations (Figure 4.49, Table 4.13). % α-amylase inhibition
potential of Me-Ext, Hex-Ext, Chf-Ext, EtAc-Ext, But-Ext and Aq-Ext were 59 ± 1, 57 ± 1,
81 ± 1, 72 ± 1, 47 ± 0.2 and 63 ± 1 with their IC50 values 400, 240, 58, 200, 620 and 360
µg/mL at the highest concentration (1000 µg/mL) respectively. The Chf-Ext was found to be
the most effective and potent fraction that showed the highest % α-amylase inhibition with
the lowest IC50 value. Acarbose was used as a standard which caused 86 ± 1% inhibition at
the maximum concentration of 1000 µg/mL with IC50 value 30 µg/mL.
1.4.2. In vitro α-glucosidase enzyme inhibitory assay
The IC50 values were determined by plotting % α-glucosidase enzyme inhibition as a function
of extract/fractions concentrations (Figure 4.50, Table 4.13). The % α-glucosidase inhibition
of Me-Ext, Hex-Ext, Chf-Ext, EtAc-Ext, But-Ext and Aq-Ext were 62 ± 1.1, 55 ± 1, 78 ± 1.0
, 70 ± 1, 57 ± 1.0 and 62 ± 1.2 with their IC50 values 400, 60, 140, 420, 240 240 µg/mL at the
highest concentration of 1000 µg/mL respectively. The Chf-Ext was the most potent fraction
and showed the highest % α-glucosidase inhibition potential with the lowest IC50 value.
Acarbose was used as a standard which caused 88 ± 1% inhibition at the maximum
concentration (1000 µg/mL) with IC50 value 32 µg/mL.
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Table 4.13: % α-Glucosidase and α-Amylase inhibition of E. umbellata fruit methanolic extract and subsequent fractions at various concentrations
S.No Sample Concentration % α-Glucosidase IC50 % α-Amylase IC 50 (µg/mL) inhibition inhibition Mean ± SEM Mean ± SEM 1000 62.42 ± 1.12 *** 59.33±0.56*** 500 57.76 ± 0.77 *** 52.36±0.77*** 250 52.11 ± 0.51 *** 45.21±1.01*** 1 Met-Ext 125 45.85± 0.62 *** 200 39.58±0.99*** 400
62.5 41.11 ± 1.01 *** 32.99±0.28***
31.05 38.09± 0.78 *** 29.88±0.98*** 1000 55.33 ± 0.68 *** 57.12±0.76*** 500 51.61 ± 0.89 *** 51.44±0.71*** 250 45.15 ± 0.62 *** 48.01±0.66*** 2 Hex-Ext 125 39.25 ± 0.58 *** 400 42.36±0.59*** 240 62.5 35.54 ± 0.31 *** 34.55±0.44*** 31.05 30.57 ± 0.99 *** 29.34±1.06*** 1000 78.19 ± 1.02 *** 81.21±0.51*** 500 67.22 ± 0.81 *** 77.23±0.47*** 250 61.64 ± 1.22 *** 65.12±0.33*** 3 Chf-Ext 125 53.08± 0.58 *** 60 58.11±0.97*** 58 62.5 50.51 ± 0.78 *** 50.51±1.02*** 31.05 48.49 ± 0.99 ns 47.25±1.11 ns 1000 70.43 ± 0.72 *** 72.11±0.77*** 500 61.81 ± 0.93 *** 65.12±0.68*** 250 50.17± 1.16 *** 52.14±0.47*** 4 EtAc-Ext 125 46.41 ± 0.75 *** 140 43.66±1.08*** 200 62.5 40.41 ± 0.78 *** 36.55±0.99*** 31.05 34.55± 0.99 *** 32.99±0.85*** 1000 56.89 ± 1.01*** 55.99±0.29*** 500 51.98± 1.06*** 47.33±0.91*** 250 47.53± 0.86*** 43.68±0.68*** 5 But-Ext 125 43.53± 0.74 *** 420 37.88±0.57*** 620 62.5 38.51± 0.99*** 33.11±1.03*** 31.05 31.01± 1.21*** 29.99±1.12*** 1000 62.36± 1.21*** 63.12±0.59*** 500 52.47 ± 0.76 *** 56.03±0.51*** 250 41.14 ± 0.61 *** 44.77±0.68*** 6 Aq-Ext 240 360 125 44.45 ± 0.55 *** 40.63±0.79*** 62.5 38.15± 0.78 *** 37.04±1.09*** 31.05 33.69± 0.98 *** 31.98±0.55*** 1000 87.65± 0.71 85.99± 0.44 500 83.05± 0.65 83.61 ± 0.58 250 78.90 ± 1.02 76.85 ± 0.96 7 Acarbose 30 32 125 71.83± 0.99 70.47 ± 0.78
62.5 65.15± 0.75 64.89± 0.71 31.05 50.01 ± 0.89 49.99 ± 0.66
.
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Figure 4.49: % α-amylase inhibition potential of E. umbellata fruit Me-Ext and subsequent fractions at various concentrations
Figure 4.50: α-glucosidase inhibition potential of E. umbellata fruit Me-Ext and subsequent fractions at various concentrations.
1.5. In vitro anti-diabetic studies on essential oil
1.5.1. In vitro α-amylase and α-glucosidase inhibitory assay
The % α-amylase and α-glucosidase enzyme inhibition potential of essential oil of E. umbellata are presented in Table 4.14. The % α-amylase enzyme inhibition potential were
88.30, 79.85, 74.82, 52.51, 41.39, 36.24 at 1000, 500, 250, 125, 62.5, 31.05 μg/ml concentrations with IC50 value 110 µg/mL (Figure 4.51A) while % α-glucosidase inhibition
127
CHAPTER 04 RESULTS potential of essential oil were 75.25, 69.61, 60.56, 52.51, 32.74, 30.61 at 1000, 500, 250, 125,
62.5, 31.05 μg/ml with IC50 value 120 µg/mL and is presented in Figure 4.51B. The IC50 values of essential oil were calculated by evaluating the plot of % α-amylase and % α- glucosidase enzymes inhibition as a function of concentrations. Standard Acarbose has shown
IC50 value 30 and 28 µg/mL against the mentioned enzymes.
Table 4.14: % α-amylase and α- glucosidase inhibition potential of essential oil of E. umbellata with IC50 at various concentrations
S.No Sample Concentration % α-amylase α-glucosidase % α-glucosidase α-amylase (µg/mL) inhibition IC50 µg/mL inhibition IC50 µg/mL Mean ± SEM Mean ± SEM 1000 88.30±0.81*** 75.25±0.77*** 500 79.85±0.55*** 69.61±0.61*** 250 74.82±0.75*** 60.56±0.52*** 1 E.O 125 52.51±1.00*** 110 52.51±1.01*** 120 62.5 41.39±0.69*** 32.74±0.68*** 31.05 36.24±0.61*** 30.61±0.63*** 1000 90.63±0.99 91.33±0.33 500 83.82±0.60 88.65±0.54 250 78.31±0.34 79.01±0.45 2 Acarbose 125 70.59±0.26 30 72.37±0.61 28 62.5 63.44±0.86 64.62±0.39 31.05 50.86±0.44 52.36±0.57
Figure 4.51: (A) % α-amylase (B) α-glucosidase inhibition potential of E. umbellata essential oil at various concentrations
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1.6. In vitro antidiabetic studies of isolated compounds
1.6.1. In vitro α-amylase enzyme inhibitory studies of isolated compounds
The IC50 values were calculated by evaluating the plot of % α-amylase enzyme inhibition as a function of extract/fractions concentrations (Figure 4.52A, Table 4.15). % α-amylase inhibition potential of compound-I, II, V, III, VIII, VI and VII were 85±1.0, 83±1.2,
83±0.5, 77±1.5, 75±1.5, 69±1.1, 63±0.5 and 55±1.5 with their IC50 values 32, 38, 38, 45, 55,
68, 95 and 115 µg/mL at highest concentration 1000 µg/mL respectively. The compound-I compound-I was the most potent fraction that has shown highest % α-amylase inhibition potential with lowest IC50 value. Acarbose was used as a standard which causes 90±0.5% inhibition at maximum concentration of 1000 µg/mL with IC50 value 25 µg/mL.
1.6.2. In vitro α–glucosidase enzyme inhibitory studies of isolated compounds
The IC50 values were determined from the plot of % α-glucosidase enzyme inhibition as a function of isolated compounds concentrations (Figure 4.52B, Table 4.15). % α-glucosidase inhibition potential of compound-I, V, II, III, VIII, IV, VII and VI were 87±1.0, 85±1.0,
82±1.0, 79±1.0, 66±1.1, 70±1.5, 58±2.1 and 55±2.1 with their IC50 values 30, 32, 35, 40, 62,
80, 105 and 110 µg/mL at highest concentration 1000 µg/mL respectively. The compound-I was the most potent fraction that has shown highest % α-glucosidase inhibition potential with lowest IC50 value. Acarbose was used as a standard which causes 89±0.5 inhibition at maximum concentration 1000 µg/mL with IC50 value 28 µg/mL.
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CHAPTER 04 RESULTS Table 4.15: % α-Glucosidase and α-Amylase inhibition of isolated compounds of Elaeagnus umbellata fruit at various concentrations
S.No Sample Concentration % α-amylase α-amylase % α-glucosidase α-glucosidase (µg/mL) inhibition IC50 µg/mL inhibition IC50 µg/mL Mean ± SEM Mean ± SEM 1000 85±1.0*** 87±1.0ns 500 79±1.1*** 80±0.5ns 1 Compound-I 250 71±0.5*** 74±0.5*** 125 66±0.4ns 32 65±0.4*** 30 62.5 54±1.2*** 56±0.3*** 31.05 49±1.1ns 49±1.0*** 1000 83±1.2** 82±1.0*** 500 75±0.5*** 75±0.5*** 2 Compound-II 250 68±1.3*** 69±0.5*** 125 56±0.5*** 38 61±0.4*** 35 62.5 50±0.4*** 54±0.3*** 31.05 44±1.4ns 47±1.1*** 1000 75±1.5*** 79±1.0*** 500 67±1.4*** 66±0.4*** 3 Compound-III 250 60±0.5*** 59±1.4*** 125 54±1.4*** 55 52±0.5*** 40 62.5 44±2.1*** 48±0.3*** 31.05 37±1.1*** 42±1.4*** 1000 77±1.5*** 70±1.5*** 500 70±0.5*** 63±1.2*** 4 Compound –IV 250 65±0.4*** 56±1.1*** 125 57±0.5*** 45 48±0.5*** 80 62.5 44±1.5*** 40±1.0*** 31.05 39±1.0*** 34±0.5*** 1000 83±0.5** 85±1.0* 500 77±0.4** 77±0.5ns 5 Compound-V 250 69±1.1** 70±1.2*** 125 63±1.2** 38 66±0. ** 32 62.5 54±0.5*** 58±0.3*** 31.05 45±1.3ns 50±1.2*** 1000 63±0.5*** 55±2.1*** 500 55±1.1*** 48±0.5*** 6 Compound-VI 250 42±0.4*** 43±0.4*** 125 39±1.5*** 95 37±1.5*** 110 62.5 32±1.4*** 30±1.4*** 31.05 29±1.0*** 27±1.0*** 1000 55±1.5*** 58±2.1*** 500 49±2.1*** 50±1.2*** 7 Compound-VII 250 44±0.5*** 45±0.5*** 125 37±1.4*** 115 39±1.1*** 105 62.5 28±2.1*** 32±0.4*** 31.05 22±1.1*** 28±1.2*** 1000 69±1.1*** 66±1.1*** 500 57±2.1*** 60±0.5*** 8 Compound-VIII 250 49±1.1*** 54±1.4*** 125 43±0.5*** 68 47±1.4*** 62 62.5 39±0.3*** 40±0.5*** 31.05 32±1.5*** 38±1.1*** 1000 90±0.5 89±0.5 500 83±0.5 82±0.4 9 Standard 250 75±1.1 77±1.0 Acarbose 125 69±0.5 25 71±0.5 28 62.5 62±0.5 68±0.5 31.05 48±0.4 60±0.4
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Figure 4.52 (A): % α-amylase inhibition potential of isolated compounds from E. umbellata at various concentrations.
Figure 4.52: (B): α-glucosidase inhibition potential of isolated compounds from E. umbellata at various concentrations.
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1.7. In vitro anticholinesterase assays
1.7.1. Choline inhibition potential of extract/fractions
Acetyl cholinesterase and butyryl cholinesterase inhibition of E. umbellata Thunb. fruit extract/fractions were determined at various concentrations and are presented in Table 4.16 along with their IC50 values. Among different fractions of E. umbellata fruit Chf-Ext and
EtAc-Ext showed prominent inhibition against AChE (87±1.2), (84±1.0) with IC50 values 33 and 55 µg/mL respectively. While, the fractions; But-Ext, Hex-Ext, Met-Ext and Aq-Ext showed % inhibition of 67±0.5, 70±0.2, 77±0.5 and 70±0.5 with IC50 values 90, 69, 70 and
74 µg/mL respectively (Figure 4.53A). Similarly, Chf-Ext and EtAc-Ext fractions also showed highest inhibition against, BChE which were 86±0.3, 82±0.5 with IC50 values 35 and
58 µg/mL respectively (Table 4.16 & Figure 4.53B). Other fractions like But-Ext, Hex-Ext,
Met-Ext and Aq-Ext showed % inhibition of 70±0.5, 69±0.6, 83±0.5 and 71±1.0 with their
IC50 values 62, 65, 80, and 78 µg/mL respectively. Methanolic extract and subsequent fractions of E. umbellata fruit showed concentration dependent activity and their results were compared with Donepezil standard, which was used as positive control which showed % inhibition of 92±0.3 with IC50 ; 28 µg/mL at concentration 1000 µg/mL.
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CHAPTER 04 RESULTS Table 4.16: Choline esterase inhibition potential of extract/fractions of E. umbellata
S.No Extract/fractions Concentration % AChE AChE % BChE BChE (µg/mL) Mean ± SEM IC50 µg/mL Mean ± SEM IC50 µg/mL 1000 77± 0.5*** 83±0.5*** 500 70± 1.0*** 75±0.4*** 250 66± 1.1*** 62±0.6*** 1 Met-Ext 125 57± 0.5*** 64 53±1.3*** 55
62.5 49± 0.3*** 48±0.5***
31.05 44± 1.2*** 43±1.2*** 1000 70±0.2*** 69±0.6*** 500 65±1.0*** 62±0.3*** 250 58±0.5*** 57±0.5*** 2 Hex-Ext 125 52±1.0*** 69 50±1.0*** 65 62.5 48±0.5*** 44±0.5*** 31.05 41±0.4*** 35±1.0*** 1000 87±1.2*** 86±0.3* 500 78±0.5*** 79±1.0*** 250 74±0.4*** 71±0.5*** 3 Chf-Ext 125 67±0.5*** 33 60±1.1*** 35
62.5 61±0.4 ns 55±0.5*** 31.05 49±0.5*** 48±0.2*** 1000 84±1.0 *** 82±0.5*** 500 72±1.1*** 76±1.1*** 250 69±0.5*** 68±0.5*** 4 EtAc-Ext 125 60±0.4*** 55 64±0.6*** 58 62.5 50±0.3*** 50±0.4*** 31.05 42±0.4*** 46±0.4*** 1000 67±0.5*** 70±0.5*** 500 60±0.3*** 65±0.5*** 5 But-Ext 250 54±0.4*** 59±0.4*** 125 49±1.1*** 90 54±1.1*** 62 62.5 43±0.3*** 49±0.4*** 31.05 34±1.1*** 40±0.3*** 1000 70±1.1*** 71±1.0*** 500 65±1.0*** 68±1.0*** 6 Aq-Ext 250 59±0.2*** 61±1.1*** 125 54±0.5*** 74 51±0.5*** 78
62.5 46±0.4*** 40±0.5*** 31.05 39±0.4*** 38±0.4*** 1000 93±0.5 92±0.3 500 89±1.1 90±0.2 7 Standard 250 81±0.5 86±0.5 Donepezil 125 78±0.4 25 78±1.1 62.5 66±0.3 69±0.5 28
31.05 54±0.5 53±0.4
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Figure 4.53: (A): AChE inhibition potential of Elaeagnus umbellata Thunb. fruit hydro methanolic extract and subsequent fractions.
Figure 4.53: (B): BChE inhibition potential of Elaeagnus umbellata Thunb. fruit hydro methanolic extract and subsequent fractions.
1.7.2. Relationship of total phenolic/flavonoid contents versus cholinesterase inhibition assays
Relationships between total phenolic/flavonoid contents and anti-cholinesterase studies have been presented in Figure 4.54 (A, B, C and D). Prominent correlation coefficient obtained by plotting AChE assay versus total contents of phenolic and flavonoid were; R2= 0.9776 and
R2= 0.9034 respectively. Regression line for AChE (y = 0.3125x + 69.146) goes parallel with 134
CHAPTER 04 RESULTS total phenolic contents and also is parallel for total flavonoid contents having regression line y= 0.2754x+ 73.871. Similarly, the correlation co-efficient of BChE assay was calculated as
0.9606 and 0.8736 for total phenolic and flavonoid contents respectively. While, their regression lines obtained were; y = 0.26x + 68.235 and y= 0.2272x + 73.162 which also goes parallel with the total phenolic and flavonoid contents.
Figure 4.54: (A) Linear correlation of TPC vs. % AChE inhibition and (B) % BChE inhibition And (C) TFC vs. % AChE inhibition and (D)% BChE inhibition
1.7.3. Cholinesterase inhibition potential of essential oil
The cholinesterase inhibition potential of essential oil of E. umbellata fruit is presented in
Table 4.17. The essential oil exhibited 85.44, 78.07, 71.86 67.59, 54.37, 47.37 μg/ml %AChE inhibition (Figure 4.55) while the % BChE inhibition potential were; 81.45, 76.08, 67.13,
56.82, 44.11, 40.66 μg/ml at various concentration respectively (Figure 4.56). The essential oil displays IC50 value of 48 and 90 μg/ml for AChE and BChE respectively. The standard
Galanthamine has shown % inhibition of 90.16 ± 0.67 against AChE and 91.22 ± 0.61 against
BChE with IC50 values of 25 and 30 µg/mL respectively.
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CHAPTER 04 RESULTS Table 4.17: Cholinesterase inhibition potential of essential oil of E. umbellata
S.No Sample Concentration % AChE AChE % BChE BChE (µg/mL) Mean ± SEM IC50 µg/mL Mean ± SEM IC50 µg/mL 1000 85.44 ± 0.35** 81.45 ± 0.68*** 500 78.07 ± 1.20*** 76.08 ± 0.71*** 250 71.86 ± 1.29*** 71.13 ± 1.04*** 1 E.O 125 67.59 ± 1.22*** 48 62.82 ± 0.98*** 90 62.5 54.37 ± 0.90*** 44.11 ± 0.64*** 31.05 47.37 ± 0.32** 40.66 ± 0.72*** 1000 90.16 ± 0.67 91.22 ± 0.61 500 86.11 ± 0.23 85.36 ± 0.48 2 Standard 250 79.12 ± 0.58 80.56 ± 0.33 Galanthamine 125 77.55 ± 0.51 25 78.89 ± 0.65 30 62.5 64.29 ± 0.55 65.58 ± 0.33 31.05 51.66 ± 0.49 50.16 ± 0.56
Figure 4.55: AChE inhibition potential of essential oil of E. umbellata at different concentration.
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Figure 4.56: BChE inhibition potential of essential oil of E. umbellata at different concentration
1.7.4. AChE and BChE inhibition potential of pure compounds
Acetyl cholinesterase and butyryl cholinesterase inhibition of isolated compounds were determined at various concentrations and their IC50 values were calculated (Table 4.18).
Compound-I and III showed prominent inhibition against AChE (90±0.2ns), (89±2.1 ns) at
1000 µg/mL with same IC50 value 31 µg/mL respectively. While, compound-IV, II, VIII, V,
VI and VII showed % inhibition of 84±2.1, 81±0.5, 74±2.5, 70±2.1, 66±1.2 and 65±2.2
µg/mL with IC50 values 45, 59, 70, 75, 92, and 93 µg/mL respectively (Figure 4.57). Isolated compound-I and II showed comparable result with the standard drug Donepezil that showed
% inhibition = 93±0.5 against AChE with IC50 value 25 µg/mL at concentration 1000 µg/mL.
Similarly, isolated compounds also showed highest inhibition against, BChE enzymes. The compound-III and I showed highest % inhibition 90±2.1, 88±0.2 with IC50 of 30 and 32
µg/mL at 1000 µg/mL respectively (Table 4.18 & Figure 4.58). Other compounds like II,
VIII, V, IV, VII and VI showed % inhibition of 80±0.5, 77±2.2, 69±0.5, 66±1.5, 60±2.1 and
58±2.2 with their IC50 values 62, 65, 74, 83, 110 and 115 µg/mL respectively. Isolated compound of E. umbellata fruit showed concentration dependent activity and their results were compared with Donepezil standard, which was used as positive control showed % inhibition 92±0.3 and IC50 value 26 µg/mL. 137
CHAPTER 04 RESULTS Table 4.18: Cholinesterase inhibition potential of compound I-VIII at various concentrations
S.No Sample Concentration % AChE AChE % BChE BChE (µg/mL) Mean ± SEM IC50 µg/mL Mean ± SEM IC50 µg/mL 1000 90±0.2ns 88±0.2 ns 500 82±0.1*** 85±0.4** 1 Compound -I 250 76±0.5* 76±0.2*** 125 68±0.4*** 31 70±0.5*** 32 62.5 59±0.3*** 62±0.6*** 31.05 50±0.4 ns 50±0.4 ns 1000 81±0.5** 80±0.5*** 500 75±0.4*** 74±0.2*** 2 Compound –II 250 68±0.1*** 68±0.4*** 125 52±0.4*** 59 60±0.5*** 62 62.5 48±0.5*** 52±0.3*** 31.05 41±0.3** 45±0.4*** 1000 89±2.1 ns 90±2.1 ns 500 84±2.1* 87±0.5 ns 3 Compound -III 250 80±0.5 ns 80±0.6*** 125 77±1.5 ns 31 74±1.4 ** 30 62.5 70±0.5 ns 69±0.4 ns 31.05 62±1.1*** 61±1.4*** 1000 84±2.1*** 66±1.5*** 500 79±1.2*** 61±2.5*** 4 Compound –IV 250 70±0.3*** 54±1.1*** 125 64±1.4*** 45 47±0.5*** 83 62.5 59±1.1*** 42±1.1*** 31.05 44±0.5*** 36±0.5*** 1000 70±2.1*** 69±0.5*** 500 64±0.5*** 62±1.1*** 5 Compound –V 250 60±0.4*** 54±0.5*** 125 52±0.6*** 75 49±1.2*** 74 62.5 44±0.3*** 40±0.4*** 31.05 33±1.5*** 38±1.2*** 1000 66±1.2*** 58±2.2*** 500 52±1.6*** 50±2.1*** 6 Compound -VI 250 40±0.5*** 47±0.5*** 125 34±1.5*** 92 41±1.1*** 115 62.5 29±2.1*** 35±2.1*** 31.05 21±1.5*** 29±0.5*** 1000 65±2.2*** 60±2.1*** 500 59±1.5*** 55±0.5*** 7 Compound -VII 250 50±0.5*** 49±1.1*** 125 41±2.5*** 93 43±1.1*** 110 62.5 31±1.4*** 38±0.4*** 31.05 26±2.3*** 35±0.3*** 1000 74±2.5*** 77±2.2*** 500 66±2.1*** 62±0.5*** 8 Compound -VIII 250 53±1.6*** 58±1.1*** 125 49±0.5*** 70 50±0.5*** 65 62.5 43±0.4*** 48±0.4*** 31.05 39±1.1*** 40±1.1*** 1000 93±0.5 92±0.3 500 89±1.1 90±0.2 9 Standard 250 81±0.5 86±0.5 Donepezil 125 78±0.4 25 78±1.1 26 62.5 66±0.3 69±0.5 31.05 63±0.5 65±0.4
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Figure 4.57: AChE inhibition potential of isolated compounds from E. umbellata Thunb.
Figure 4.58: BChE inhibition potential of isolated compounds from E. umbellata Thunb.
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1.8. In vivo antidiabetic studies of extract/fractions
1.8.1. Toxicity study
Toxicity study revealed that the dose of Me-Ext/fractions in range of 100-2000 mg/kg did not produce any significant behavioural alterations (respiratory aches, convulsions shortage, writhing, variations to reflex actions or mortality) in animals. An insignificant increase in petulance was detected at 2000 mg/kg dose in three animals out of eight. All animals appeared healthy at 24 hours to 1 week with no noticeable variations in appearance or behaviour. No mortality was noticed up to one week.
1.8.2. Estimation of biochemical parameters 1.8.2.1. Effect of E. umbellata Thunb. methanolic fruit extract/fractions on glycemia
The effect of E. umbellata Me-Ext their subsequent fractions Chf-Ext, EtAc-Ext (100 and
200 mg/kg) and standard glibenclamide on variations in blood glucose in normal control group, diabetic control, and plant extracts treatment group are shown in Figure 4.59 and
Table 4.19. Oral administration of the Me-Ext and Chf-Ext (100 and 200 mg/kg) caused a significant decrease in blood glucose level compared to diabetic control at the end of 21st day treatment. Blood glucose level reduction was observable from the 5th day and onward. The
EtAc-Ext did not show any significant blood glucose reduction at 100 mg/kg, however, it showed significant reduction in blood glucose at 200 mg/kg but the effect was much weaker than the Chf-Ext at the end of treatment period. Furthermore, the onset of the effect was also delayed and significant lowering in blood glucose was seen from 10th day and onward.
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CHAPTER 04 RESULTS Table 4.19: Effect of E. umbellata fruit methanolic extract/fractions on blood glucose level in streptozotocin induced diabetic rats
S. Groups Dose day 1st day 5th day 8th day 10th day 15th day 21st No (mg/kg) 1 Normal 0.3ml 103± 22 110±13*** 108±12*** 102±11*** 109±10*** 104±15*** control 2 Diabetic 0.3ml 371±37 344±30 380.87±50 376±34 316±50 384±30 control 3 Glibenclamide 0.5 364±21 203±43** 207±35** 201±22** 152±31*** 136±21*** 4 cMe-Ext 100 361±43 273±54* 260±87* 240±67** 222±12** 150±32*** 5 cMe-Ext 200 370±23 286±45* 245±14* 210±21** 164±10** 140±20*** 6 cChf-Ext 100 372±19 285±13* 280±23* 255±15* 210±21** 168±18** 7 cChf-Ext 200 374±21 281±32* 260±33* 245±20** 190±31** 138±22*** 8 cEtAC-Ext 100 365±17 340±14ns 365±17 ns 356±14 ns 353±19 ns 333±22* 9 cEtAC-Ext 200 361±17 339±12* 325±37* 286±24* 233±29* 193±12*
Figure 4.59: Effect of E. umbellata fruit methanolic extracts/fractions and glibenclamide on blood glucose level in STZ-induced diabetic rats.
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CHAPTER 04 RESULTS 1.8.2.2. Effect of E. umbellata Thunb. methanolic fruit extract/fractions on body weight in diabetic rats
The effect of E. umbellata Me-Ext their subsequent fractions Chf-Ext, EtAc-Ext (100 and
200 mg/kg) and standard glibenclamide on changes in body weight in the normal control group, diabetic control, and plant extracts treatment group are shown in Figure 4.60 and
Table 4.20. STZ-induced diabetic rats showed significant reduction in body weight as compared to normal control rats during the experimental period. Loss in body weight was continued in diabetic control rats till the end of 21st-day treatment. The Me-Ext, Chf-Ext, and
EtAc-Ext (100 and 200 mg/kg) reversed the STZ-mediated reduction in body weight and caused significant increases in body weight at the end of 21 days treatment.
Table 4.20: Effects of E. umbellata fruit methanolic extract/fractions on body weight in STZ- induced diabetic rats
S.No Groups Dose day day day day day day 21st % change (mg/kg) 1st 5th 8th 10th 15th in b.w (g) 1 aNormal 0.3ml 164±2 163±3 169±1 170±2 172±3 173±2*** +5.4 control 2 bDiabetic 0.3ml 167±2 164±7 147±4 139±05 131±4 130±2 -22.15 control 3 cGlibenclamide 0.5 168±3 172±3 185±3 185±12 190±6 189±4** +12.5 4 cMe-Ext 100 167±2 171±2 174±2 179±4 181±6 178±3** +6.5 5 cMe-Ext 200 170±2 170±7 180±3 184±3 190±4 190±4** +11.7 6 cChf-Ext 100 168±3 169±3 170±2 173±4 173±2 175±3** +4.1 7 cChf-Ext 200 166±2 167±2 171±4 172±7 175±3 188±2** +13.2 8 cEtAC-Ext 100 165±3 166±4 157±2 146±3 158±4 169±4* +2.4 9 cEtAC-Ext 200 168±3 168±2 175±3 171±5 171±7 179±3** +6.5
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Figure 4.60: Effects of E. umbellata fruit methanolic extracts/fractions on body weight in STZ-induced diabetic rats. Each value is Mean±SEM of 8 animals.
1.8.2.3. Measurement of serum lipid profile in diabetic rats
The levels of parameters of lipid profiles including TC, TGs, LDL, HDL and cholesterol in normal control group, diabetic control group and plant extracts treated group are shown in
Table 4.21. Diabetic control group showed a significant increase in TC, TGs, LDL and cholesterol while a significant decrease was observed in HDL cholesterol when compared to normal control group (Table 4.21). The Me-Ext, Chf-Ext, and EtAc-Ext (100 and 200 mg/kg) showed a significant decrease in TC, TGs, LDL and cholesterol as compared to diabetic control group at the end of 21 days treatment. Furthermore, the Me-Ext, Chf-Ext, and EtAc-
Ext (100 and 200 mg/kg) also significantly increased HDL cholesterol in diabetic rats at the end of 21 days of treatment.
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CHAPTER 04 RESULTS Table 4.21: Effect of E. umbellata fruit methanolic extract/fractions on lipid profile in streptozotocin induced diabetic rats
S.No Groups Dose TC (mg/dl) TGs (mg/dl) HDL(mg/dl) LDL(mg/dl) (mg/kg) 1 aNormal control 0.3ml 125 ± 6.10** 123.6 ± 8.5** 37±3.1* 74± 5.5** 2 bDiabetic 0.3ml 163.3 ± 6.5 165.0 ± 7.9 25.2 ±2.2 170.4±8.9 control 3 cGlibenclamide 0.5 138.5± 6.3** 125.3 ± 5.5** 40.5 ±4.5** 89.3± 5.5*** c 4 Me-Ext 100 140.2± 5.3* 145.5± 7.7* 33.5 ±4.3* 145.6± 4.2* 5 cMe-Ext 200 131.5±7.5** 138.8±6.5** 36.6±5.5* 93.5±4.6** 6 cChf-Ext 100 145.4±5.5* 143.2±3.2* 34.2±2.5* 125.3±3.5* 7 cChf-Ext 200 135.4±5.7** 133.2±5.1** 37.2±3.5* 95.3±3.5** 8 cEtAC-Ext 100 147.2±5.6* 146.8 ±4.0* 30.1 ±4.5* 123.8±6.0* 9 cEtAC-Ext 200 137.2±7.6** 136.8 ±5.0** 36.1 ±5.5* 93.8±8.0**
1.8.2.4. Effect of E. umbellata Thunb. methanolic fruit extract/fractions on liver and renal functions in STZ-induced diabetic rats
The activity of hepatic enzymes like SGPT, SGOT and ALP and renal functions like serum
creatinine and blood urea nitrogen in the normal control group, diabetic control group, and
plant extracts treated group are shown in Table 4.22. STZ-induced diabetic rats showed a
significant increase in the levels of SGPT, SGOT and ALP as compared to the normal
control. The Me-Ext, Chf-Ext, and EtAc-Ext (100 and 200 mg/kg) significantly reduced the
SGPT, SGOT, and ALP in STZ-induced diabetic rats. The Me-Ext (100 and 200 mg/kg),
Chf-Ext and EtAc-Ext (200 mg/kg) also significantly reduced the serum creatinine and blood
urea nitrogen in STZ-induced diabetic rats. The standard glibenclamide drug also
significantly reduced the SGPT, SGOT, ALP serum creatinine and blood urea nitrogen in
STZ-induced diabetic rats.
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CHAPTER 04 RESULTS Table 4.22: Effect of E. umbellata fruit extract/fractions on liver and renal functions in streptozotocin-induced diabetic rats
S.No Treatment Dose SGPT (IU) SGOT (IU) ALP (IU) BUN Serum groups (mg/kg) (mg/dl) creatinine (mg/ml) 1 aNormal 0.3ml 20±5.6*** 16±3.5*** 141±7.2** 18.5±3.5** 0.527±0.2*** control 2 bDiabetic 0.3ml 62.47±7.5 62.27±6.1 272.57±8.3 35.7±4.5 2.57±0.2 control 3 cGlibenclamide 0.5 24.5±6.4*** 20.07±3.6*** 142.47±9.3*** 17.6±2.3** 0.56±0.2*** 4 cMe-Ext 100 44.56±6.0* 39.17±3.9** 197.39±10.33* 21.4±5.4** 1.50±0.3* 5 cMe-Ext 200 30.37±8.0** 21.97±5.6*** 160.19±12.23** 20.3±3.2** 0.85±0.2** 6 cChf-Ext 100 45.70±4.5** 34.37±3.5** 174.22±8.5* 24.6±4.5* 1.46±0.2* 7 cChf-Ext 200 28.80±3.5* 22.37±4.5** 154.32±11.5** 18.6±2.5** 0.76±0.2** 8 cEtAC-Ext 100 38.70±4.5* 45.37±2.8* 185.50±17.2* 25.5±5.2* 1.7±0.3* 9 cEtAC-Ext 200 28.85±3.5** 25.37±3.5** 165.50±11.2** 21.5±6.2** 1.2±0.2*
1.9. Molecular docking validation of antidiabetic enzymes
1.9.1. α-Amylase
To validate the molecular docking process, the docked acarbose molecule was superimposed
to the one obtained from the α-amylase crystal structure, RMSD value of 1.3 Å for all heavy
atoms (excluding the hydrogen atoms) was observed. Furthermore, docked acarbose molecule
showed similar interactions to those found in the crystal structure [347]. Both the docked
acarbose molecule and the crystal structure was shown to be embedded within the binding
site and surrounded by a number of hydrophobic residues. In addition, the protonated
acarbose amino group was forming an ionic interaction with Asp200. H-bonds were formed
with the following amino acid residues Glu240, Lys200, Glu233, and Thr163 (Figure 4.61).
All docked compounds occupied the same binding site occupied by acarbose. All compounds,
except acarbose, occupied a smaller part of the binding site and this ensued in the mixed type
to the non-competitive inhibitory effect of these compounds [348]. The Glide Score of all
compounds were consistent with the inhibitory activities of α-amylase as shown in Table 4.23
and were in the order of acarbose, rutin, quercetin, epigallocatechin gallate, epigallocatechin,
and catechin hydrate [349]. Figure 4.61 shows the compounds within the binding site and
highlighting the similar interactions with binding site residues. One of the major residues that
were found to interact is Asp300. Acarbose was found to form a salt bridge with this residue 145
CHAPTER 04 RESULTS while all other compounds lacked this interaction and formed H-bonds instead; this tighter interaction would explain the higher inhibitory activity observed for acarbose. In addition, the larger volume occupied by the acarbose molecule in comparison with the smaller compounds may explain the mixed type to the non-competitive inhibitory effect of these compounds
[349]. The stereo view of the docking pose of acarbose, rutin, quercetin and epigallocatechin gallate in the binding pocket of α-amylase enzyme active sites are presented in Figure 4.62.
Table 4.23: The Glide Scores and IC50 values of acarbose and α-amylase inhibitors present in Elaeagnus umbellata Thunb.
Compound α-amylase enzyme inhibition (%)* Glide Score Acarbose 83 -14.158 Rutin 50 -10.434 Quercetin 41 -8.840 Epigallocatechin gallate 21 -7.990 Epigallocatechin 5 -4.550 Catechin hydrate 4 -4.080
Glide Score is an empirical scoring function that estimates the ligand binding free energy, more negative values represent tighter binders. It has been optimized for docking accuracy and binding affinity prediction. Glide Score should be used to rank positions of different ligands in virtual screening. The GlideScore of all compounds were consistent with the inhibitory activities of α-amylase * [350].
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CHAPTER 04 RESULTS
Figure 4.61: Mode of binding of different compounds and acarbose in α-amylase enzyme active sites. a) Acarbose, b) Rutin, c) Quercetin, and Epigallocatechin gallate
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CHAPTER 04 RESULTS
Figure 4.62: A) Stereo view of the docking pose of A) Acarbose, B) Rutin C) Quercetin D) Epigallocatechin gallate in the binding pocket of α-amylase enzyme active sites
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1.9.2. α-Glucosidase
The α-glucosidase enzyme crystal structure locked any ligand within its binding site. In the search for the binding enzyme binding cavity, the Sitemap module [351] was used. Six binding cavities were found; the largest volume cavity was selected for docking so as to accommodate the acarbose large molecule. Subsequently, the grid box was set to be the centroid of the amino acids surrounding this binding site, namely: Arg407, Asp326, Arg197, and Asn258.
All docked compounds only occupied a part of the binding site occupied by acarbose (Figure
4.63). Some common interactions were observed between the different binding site residues in acarbose and other compounds used in the docking. Similar to the interactions observed in the α-amylase binding site, the docked test compounds occupied a smaller part of the binding site which also confers the mixed non-competitive inhibitory effect of these compounds on the α-amylase receptor, however, acarbose molecule extended through the full size of the binding site (Figure 4.63). The Glide-Scores of all these compounds were found to go in parallel with their experimental α-glucosidase inhibitory activities (Table 4.24) which followed the order of acarbose, epigallocatechin gallate, quercetin, rutin, epigallocatechin and catechin hydrate [349]. It is worth mentioning that all compounds showed weaker inhibitory activity on α-glucosidase than on α-amylase except for epigallocatechin gallate [348]. The stereo view of the docking pose of acarbose, rutin, quercetin and epigallocatechin gallate in the binding pocket of α-glucosidase enzyme active sites are presented in Figure 4.64.
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Table 4.24: The Glide Scores and IC50 values of acarbose and α-glucosidase inhibitors present in Elaeagnus umbellata Thunb.
Compound α-glucosidae enzyme inhibition (%)* Glide Score Acarbose - -7.725 Epigallocatechin gallate 32 -6.283 Quercetin, 28 -6.258 Rutin 15 -5.830 Epigallocatechin 7 -5.550 Catechin hydrate 1 -4.080
The Glide-Scores of compounds: acarbose, epigallocatechin gallate, quercetin, rutin, epigallocatechin and catechin hydrate were found to go in parallel with their experimental α- glucosidase inhibitory activities. All compounds showed weaker inhibitory activity on α- glucosidase except for epigallocatechin gallate. The more negative Glide Score values represent tighter binders * [350].
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Figure 4.63: Mode of binding of different compounds and acarbose in α-glucosidase enzyme active sites. a) Acarbose, b) Epigallocatechin gallate, c) Quercetin and d) Rutin. The highlighted area in a) is the common area between acarbose and all compounds
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Figure 4.64: (A) Stereo view of the docking pose of A) Acarbose, B) Rutin C) Quercetin D) Epigallocatechin gallate in the binding pocket of α-glucosidase enzyme active sites
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CHAPTER 04 RESULTS 1.10. In vivo antidiabetic studies of isolated compound
1.10.1. Acute toxicity of compound
Toxicity study revealed that the dose of compound-III (10, 15, 40, 50 and 100 mg/kg body weight) isolated from E. umbellata did not produce any significant behavioural alterations
(respiratory aches, convulsions shortage, writhing, variations to reflex actions or mortality) in animals. All animals appeared healthy at 24 hours to 1 week with no noticeable variations in appearance or behaviour. No mortality was noticed up to one week.
1.10.2. Estimation of biochemical parameters 1.10.2.1. Effect of isolated compound of E. umbellata on blood glycemia
The effect of isolated compound of E. umbellata (2, 5, 10, 15, 30 and 50 mg/kg body weight) and standard glibenclamide on blood glucose level in normal control group, diabetic control and compound treatment group are shown in Table 4.25. Oral administration of the compound (30, 15, 50 and 10 mg/kg body weight) caused a significant (p˂0.01) decrease in blood glucose level as compared to diabetic control at the end of 21st day treatment. Blood glucose level reduction was observable from the 7th day and onward. The compound at dose
2 mg/kg body weight has slowly reduce blood glucose level while compound at 15 mg/kg body weight dose displayed comparable decline in glucose level to that with standard drug
Glibenclamide (0.5 mg/kg). Furthermore, the onset of the effect was also delayed and significant lowering in blood glucose was seen from 10th day and onward.
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CHAPTER 04 RESULTS Table 4.25: Effect of daily oral administration of isolated compound of E. umbellata on glucose level in streptozotocin induced diabetic rats
S. Groups Dose day 1st day 4th day 7th day 10th day 15th day 21th No mg/kg
1 Normal 0.4 ml 115.2 ± 3.6 109.4 ± 103.5 ± 44.2 102.4 ± 100.3 ± 101.9 ± 2.8 controla 3.4 5.3 5.8
2 Diabetic 0.4 ml 460.2 ± 5.5 484.3 ± 493.3 ± 5.8 511.2 ± 517.3 ± 495.1 ± 4.7 controlb 5.8 4.3 6.1
3 Glibenclamidec 0.5 466.4 ± 3.5 312.5 ± 298.5 ± 4.3* 251.3 211.5 ± 190.4 ± 4.3* ±4.7** 4.5** 1.9**
4 Compoundc 2 476.6 ± 5.1 475.2 ± 345.1 ± 309.5± 243.4 ± 243.4 ± 2.5* 3.3** 5.5** 4.8** 4.8**
5 Compoundc 5 471.4 ± 3.1 489.6 ± 368.3± 3.5** 254.9± 241.5 ± 201.4 ± 4.7* 6.1** 3.5** 1.7**
6 Compoundc 10 467.1 ± 4.3 449.4 ± 332.4± 4.7** 291.2± 220.4 ± 193.6 ± 3.6* 4.3** 4.2** 3.8**
7 Compoundc 15 461.8 ± 2.9 418.4 ± 320.5 ± 253.1± 206.5± 190.3± 1.5** 3.9* 4.1** 4.6** 3.4**
8 Compoundc 30 431.8 ± 3.3 360.4 ± 322.4 ± 243.1± 213.5± 178.5± 1.5** 5.9* 4.1** 2.6** 1.5**
9 Compoundc 50 471.8 ± 1.9 428.4 ± 320.8 ± 233.1± 201.5± 192.5± 1.4** 2.8* 2.1** 3.5** 1.4**
1.10.2.2. Effects of isolated compound of E. umbellata on body weight in diabetic rats
The effect of isolated compound (2, 5, 10, 15, 30 and 50 mg/kg body weight) and standard
glibenclamide on changes in body weight in normal control group, diabetic control and
compound treatment group are shown in Table 4.26. STZ-induced diabetic rats revealed
significant (p˂ 0.001) reduction in body weight (15.9 ± 4.9) as compared to normal control
rats 21st day during the experimental study period. Loss in body weight was continued in
diabetic control rats till the end of 21st day treatment. The compound at dose 5, 30 and 50
mg/kg body weight reversed the STZ mediated reduction in body weight and caused
significant (P<0.01) increases in body weight comparable to standard drug glibenclamide at
the end of 21 days treatment respectively. While the compound at dose 10 mg/kg body
weight also showed significant (P<0.05) increases in body weight 21.4 ± 4.5. 154
CHAPTER 04 RESULTS Table 4.26: Effects of isolated compound of E. umbellata on body weight in STZ-induced diabetic rats
S. Groups Dose day day 4th day day day 15th day 21th changes No mg/kg 1st 7th 10th in b.w %
1 Normal 0.4 ml 25 ± 27 ± 3.2 27 ± 26.5 ± 26.9 ± 26 ± 3.8 controla 2.7 3.6 3.5 5.4
2 Diabetic 0.4 ml 22.8 ± 22.3 ± 21.9 ± 20.4 ± 20.1 ± 15.9 ± -11.8 controlb 4.6 2.8* 4.9** 6.1** 3.7** 4.9***
3 Glibenclamide 0.5 21.9 ± 22.5 ± 22.8 ± 23.4 ± 23.7 ± 24.8 ± +8.2 c 4.5 3.3* 4.6* 5.0** 3.8** 4.6**
4 Compoundc 2 22.0 ± 22.4 ± 23.0 ± 23.4 ± 23.4 ± 23.0 ± +6.3 4.3 6.0* 3.9* 4.6** 3.4** 3.9**
5 Compoundc 5 23.5 ± 23.7 ± 24.1 ± 23.8 ± 24.4 ± 24.1 ± +3.8 3.7 5.1* 4.5* 5.4** 4.7** 4.5**
6 Compoundc 10 20.9 ± 21.2 ± 21.4 ± 22.7 ± 22.5 ± 21.4 ± +7.6 5.1 4.7* 4.5** 3.8* 5.1** 4.5*
7 Compoundc 15 23.5 ± 23.7 ± 23.8 ± 24.0 ± 24.4 ± 23.8 ± +3.8 4.2 4.6* 5.1** 4.2** 4.7** 5.1**
8 Compoundc 30 21.2± 20.4 ± 24.5 ± 23.0 ± 21.4 ± 24.5 ± +3.8 2.1 2.5* 2.1** 2.2** 3.5** 2.1**
9 Compoundc 50 20.25± 20.4 ± 24.5 ± 23.0 ± 21.4 ± 24.5 ± 23.12 3.3 2.5* 2.1** 2.2** 3.5** 2.1**
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1.10.2.3. Effect of isolated compound of E. umbellata on lipid profile in streptozotocin induced diabetic rats
The parameters of lipid profiles like TC, TGs, LDL, HDL and cholesterol in normal control group, diabetic control group and compound treatment group are shown in Table 4.27.
Diabetic control group has shown a significant increase (p <0.01) in TC, TGs, LDL and cholesterol while a significant (P<0.05) decrease occurs in HDL cholesterol as compared to normal control group (Table 4.27). The compound at doses of 2, 5, 10 and 15 mg/kg body weight showed a significant decrease (P<0.05, P<0.01) in TC, TGs, LDL and cholesterol as compared to diabetic control group at the end of 21 days treatment. Furthermore, the compound (2, 5, 10 and 15 mg/kg body weight) also significantly increased (P<0.05, P<0.01)
HDL cholesterol in diabetic rats at the end of 21 days treatment. The compound treated groups showed comparable results with standard drug Glibenclamide.
Table 4. 27: Effect of isolated compound of E. umbellata on lipid profile in streptozotocin induced diabetic rats
S. No. Groups Dose TC (mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl) mg/kg
1 Normal 0.4 ml 131± 6.6 44 ± 4.5 92 ± 4.4 137 ± 7.5 controla
2 Diabetic 0.4 ml 197 ± 5.3** 40± 3.5* 187 ± 3.4** 178 ± 4.6** controlb
3 Glibenclamidec 0.5 146 ± 6.0** 48± 5.3** 98 ± 5.7*** 138 ± 3.6**
4 Compoundc 2 153 ± 3.8** 48 ± 4.8** 99 ± 4.6*** 148 ± 5.3**
5 Compoundc 5 149 ± 4.7** 45 ± 5.0** 93 ± 5.0*** 146 ± 6.1**
6 Compoundc 10 148 ± 4.2* 48 ± 6.2* 92 ± 4.5* 142 ± 4.9**
7 Compoundc 15 147 ± 2.9** 43 ± 3.8** 90 ± 4.8** 140 ± 5.6*
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1.10.2.4. Effect of isolated compound of E. umbellata on liver and renal functions in streptozotocin-induced diabetic rats
The activity of hepatic enzymes like SGPT, SGOT and ALP and renal functions like serum
creatinine in normal control group, diabetic control group and compound treatment group are
shown in Table 4.28. STZ-induced diabetic rats showed a significant (P<0.01) increase in the
levels of SGPT, SGOT and ALP as compared to the normal control. The compound at doses
of 2, 5, 10 and 15 mg/kg body weight significantly reduced (P<0.05, P<0.01) the SGPT,
SGOT and ALP in diabetic rats. The compound at doses of 2, 5, 10 and 15 mg/kg body
weight also significantly reduced the serum creatinine in diabetic rats (P<0.05, P<0.01,
P<0.001). The standard glibenclamide drug also significantly reduced (P<0.01) the SGPT,
SGOT, ALP serum creatinine (P<0.001) in STZ-induced diabetic rats.
Table 4.28: Effect of isolated compound of E. umbellata on liver and renal functions in streptozotocin-induced diabetic rats
S. Groups Dose ALP (IU) SGPT (IU) SGOT (IU) Serum creatinine No. mg/kg (mg/dl)
1 Normal controla 0.4 ml 188 ± 4.8 18 ± 5.1 23 ± 5.5 0.58 ± 3.2
2 Diabetic controlb -- 299 ± 5.5** 59 ± 3.8** 46 ± 4.3** 2.9 ± 2.5**
3 Glibenclamidec 0.5 206 ± 6.1** 23 ± 4.7** 26 ± 4.8** 0.70 ± 2.1***
4 Compoundc 2 204 ± 6.5** 23 ± 3.9** 22 ± 6.0** 0.89 ± 2.3***
5 Compoundc 5 202 ± 6.3** 19 ± 3.9** 20 ± 3.7** 0.68 ± 1.8***
6 Compoundc 10 200 ± 5.7** 22 ± 6.1* 21 ± 5.3** 0.65 ± 1.2**
7 Compoundc 15 193 ± 4.8** 21 ± 4.7** 19 ± 5.6* 0.59 ± 2.1*
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1.11. Effect of different extracts/fractions Compound on pancreas histopathology in STZ-induced diabetic rats
The effect of extract/fractions and compounds in different doses on pancreas in STZ-induced diabetic rats is represented in figure 4.65 & 4.66.
At the end of experimental duration, three Rats from each group were selected and then dissected. After extraction of Pancreas, histopathological examination was carried in the samples of Pancreas. The selected samples were fixed and stained with hematoxyline, eosin
(HE) and then examined under light microscope. The histological pattern of pancreas of normal control group has shown clear lobular architecture (Figure 4.65 (A). Islets of
Langerhans were of normal diameter and structure, acinar cells were clear with prominent and centrally placed nuclei and interlobular spaces were also visible. Besides, the central cellular integrity and lobular structure of pancreas was retained.
The pancreas of Diabetic control group (Figure 4.65 (B) showed a typical histological pattern with slight grade of congestion and dilated blood vessels with inflammatory cell infiltration.
The diameter of Islet of Langerhans was decreased with decreased cellular density. Shrined and pyknotic nuclei were also visible in the center of Islet. The boundary of endocrine and exocrine portion of pancreas was also indistinct. Presence of inflammatory cells can be observed in connective tissue spaces. Group C is showing histological pattern of pancreas of treated with glibenclamide (500g/kg/overly) (Figure 4.65). Pancreatic tissue of this group of rates did not show any violation from the normal histological pattern during experimental period. No congestion or deterioration was observed in the in acinar cells. There was no reduction in the diameter of Islet of Langerhans and nuclei were also centrally placed. The boundary of endocrine and exocrine portion of pancreas was distinct and overall normal histological pattern was retained. Histological pattern of Met-Ext treated Group of rats
(Figure 4.65 (D) showed a deterioration of pancreatic tissue. The diameter of Islet of
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CHAPTER 04 RESULTS Langerhans was declined with reduced cellular mass and the nuclei were not properly aligned. Presence of inflammatory cells and pyknotic nuclei were also observed to some extent, which showed that Met-Ext was least effective in treating STZ-induced diabetes.
Normal histological pattern of pancreas was shown by Chf. Ext treated Group of rats which is presented in Figure 4.65 (E). Acinar cells were clear with prominent and centrally placed nuclei and interlobular spaces were also visible. Islet of Langerhans was of normal diameter and structure and the overall cellular integrity and architecture was maintained. This result showed maximum efficacy of Chf. Ext Group for treatment of STZ-induced diabetes. Group
F is showing restoration of histological architecture of pancreas of EtAc. Ext treated Group
(Figure 4.65). The size and number of Islets of Langerhans were rejuvenated. The distinction of septa between Islets and Acinar had also got evident and clear. The inflammatory cells were also negligible. So it is concluded from our results that highest recovery of pancreas was shown by Rats Chf. Ext Group (E) followed by EtAc. Ext (F) while the overall recovery of different experimental groups was as follows:
Glibenclamide group C > Chf. Ext (E) > EtAc. Ext (F) > Met-Ext (D)
The effect of Compound-V (catechin) on pancreas histopathology in STZ-induced diabetic rats indicates that normal histological pattern of pancreas was shown by Normal control group of rats which is presented in (Figure 4.66 (A). Acinar cells were clear with prominent and centrally placed nuclei and interlobular spaces were also visible. Islet of Langerhans was of normal diameter and structure and the overall cellular integrity and architecture was maintained. The pancreas of Diabetic control group of rats (Figure 4.66 (B) showed a typical histological pattern with slight grade of congestion and dilated blood vessels with inflammatory cell infiltration. The diameter of Islet of Langerhans was decreased with decreased cellular density. Shrined and pyknotic nuclei were also visible in the center of Islet.
The boundary of endocrine and exocrine portion of pancreas was also indistinct. Presence of
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CHAPTER 04 RESULTS inflammatory cells can be observed in connective tissue spaces. The histological pattern of pancreas of group C rates treated with glibenclamide 500g/kg/overly (Figure 4.66).
Pancreatic tissue of this group of rates did not show any violation from the normal histological pattern during experimental period. No congestion or deterioration was observed in the in acinar cells. There was no reduction in the diameter of Islet of Langerhans and nuclei were also centrally placed. The boundary of endocrine and exocrine portion of pancreas was distinct and overall normal histological pattern was retained.
Histological pattern of Compound (2 mg/kg/orally) treated group of rats (Figure 4.66 (D) did not show restoration of pancreatic tissue. The diameter of Islet of Langerhans was declined with reduced cellular mass and the nuclei were not properly aligned. Presence of inflammatory cells and pyknotic nuclei were also observed to some extent, which showed that the Compound with dose 2 mg/kg/orally was not effective to treat STZ-induced diabetes.
Group (E) has shown a little bit restoration of histological architecture of pancreas (Figure
4.66). The size and number of Islet of Langerhans were recovered in group E (Compound 5 mg/kg/orally) have shown low grade rejuvenation but still the distinction of septa between
Islets and Acinar is not prominent. Presence of inflammatory cells was negligible and the overall cytoplasmic integrity was retained to some extent. Group (F & G) is showing normal histological pattern of pancreas with clear lobular architecture (Figure 4.66). Islet of
Langerhans was of normal diameter and structure, acinar cells were clear with prominent and centrally placed nuclei and interlobular spaces were also visible. Besides, the central cellular integrity and lobular structure of pancreas was retained. This result showed maximum efficacy of Compound at dose of 10 mg/kg/orally and 15 mg/kg/orally for treatment of STZ- induced diabetes.
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Figure 4.65: Effect of extract/fractions on pancreas histopathology in STZ-induced diabetic rats Note: (A) Normal control group; (B) Diabetic control group (C) glibenclamide (500 lg/kg/overly) (D) Met-Ext treated Group (E) Chf. Ext treated Group (F) EtAc. Ext treated Group
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Figure 4.66: Effect of Compound-V on pancreas histopathology in STZ-induced diabetic rats Note: (A) Normal control group; (B) Diabetic control group; (C) glibenclamide (500 lg/kg/overly) (D); Compound (2 mg/kg/orally) ;(E) Compound 5 mg/kg/orally) (F), Compound; (10 mg/kg/orally) (G), Compound (15 mg/kg/orally).
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1.12. In vivo antiamnesic study of active extract/fraction of E. umbellata
1.12.1. Y-maze test
Y maze test results are presented in Figure 4.67. Results indicated that no significant (p>0.05) reduction were observed in the total numbers of arm entries in both scopolamine and treatment groups compare to control (Figure 4.67A). Returns to the same arms were significantly (p < 0.01) high in group treated with scopolamine as compared to control group.
Chf. Ext 200 mg/kg body weight and donepezil (2 mg/kg) shown significant (p<0.01) reduction in the percentage of same arm returns comparable to control group (Figure 4.67B).
Similarly, Chf. Ext (200 mg/kg) and standard drug donepezil (2 mg/kg body weight) also have shown a significant (p < 0.01) increase as compared to scopolamine (Figure 4.67C).
Dose dependent increase was also observed in %SAP which is significant (p < 0.01) for Chf.
Ext (200 mg/kg) comparable to the donepezil (2 mg/kg body weight) (Figure 4.67D).
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Figure 4.67: Effect of Chf. Ext (50, 100 and 200 mg/kg) of E. umbellata Thunb. on the Y- maze task: (A) number of arm entries (B) Same arm returns (C) alternate arm returns (D) %SAP. *p < 0.05, **p < 0.01 versus control and #p < 0.05, ##p < 0.01 versus scopolamine (Scop) 1 mg/kg. (One way ANOVA followed by Dunnett's posthoc test).
1.12.2. Novel Object Recognition Test
To assess short term memory in the sample phase indicates that no significant difference were observed in consuming time for exploration of two objects between Chf. Ext treated groups and scopolamine treated group (Figure 4.68A). However, Chf. Ext (50, 100 and 200 mg/kg) and donepezil (2 mg/kg) groups consume more time with novel object in the test phase but not statistically significant. However, scopolamine (1 mg/kg) group spent more time with familiar object compared to novel object (p < 0.05) (Figure 4.68B). The % DI was significantly high for Chf. Ext 100 mg/kg body weight (p < 0.05), 200 mg/kg body weight (p
< 0.01) and donepezil (p < 0.01) group compares to scopolamine group. All groups show %
DI above 50% while scopolamine group has shown significantly low value as compares to
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CHAPTER 04 RESULTS vehicle control (p < 0.01) (Figure 4.68C). Similar results were observed in the sample phase in long term memory where none of the groups has shown significant (p > 0.05) increase in time spending for exploring the two identical objects like that in short term memory (Figure
4.69A). A significant (p < 0.05) increase occurs in the test phase of long term memory in spending the time for exploration of novel object with Chf. Ext 100-200 and donepezil 2 mg/kg treated groups (Figure 4.69B). However, scopolamine group has shown a significant
(p < 0.05) increase in exploration of time with the familiar object compared with novel one.
The Chf. Ext 100 mg/Kg (p < 0.05), 200 mg/kg (p < 0.01) and donepezil 2 mg/kg groups (p <
0.01) have shown increase in % DI, while scopolamine 1 mg/kg group has significantly (p <
0.01) lower % DI compared with vehicle control (Figure 4.69C).
Figure 4.68: Effect of Chf. Ext (50, 100 and 200 mg/kg) of E. umbellata Thunb. in short- term memory NORT. (A) Time spent in the sample phase (B) Time spent in the test phase (C) %DI. **p < 0.01 versus control and #p < 0.05, ##p < 0.01 versus Scop 1 mg/kg. A and B were analyzed using Two-way ANOVA repeated (Mixed model) followed by Bonferroni post-test and C was analyzed by One-way ANOVA followed by Dunnett's posthoc test. 165
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Figure 4.69: Effect of Chf. Ext (50, 100 and 200 mg/kg body weight) of E. umbellata Thunb. in long term memory NORT. (A) Time spent in sample phase (B) Time spent in test phase (C) %DI. **p < 0.01 versus control and #p < 0.05, ##p < 0.01 versus Scop 1 mg/kg. A and B were analyzed using Two-way ANOVA repeated (Mixed model) followed by Bonferroni post-test and C was analyzed by One-way ANOVA followed by Dunnett's posthoc test.
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1.13. In vivo antiamnesic study of isolated compound of E. umbellata
1.13.1. Y-maze test
Y maze test results are shown in Figure 4.70. Results indicated that no significant (p > 0.05) reduction were observed in the total numbers of arm entries in scopolamine along with all groups compared to control (Figure 4.70A). Scopolamine significantly increased the number of returns to same arms as compared to control (p < 0.05). The isolated compound CGA at the dose of 10 and 30 mg/kg body weight has shown significant reduction (p < 0.05; p <
0.01) in total returns to same arms as compared to scopolamine group. Donepezil also significantly decreased same arm returns compared to scopolamine treated group (p < 0.01)
(Figure 4.70B). Scopolamine significantly decreased the number of alternate arm returns as compared to control (p˂0.01) (Figure 4.70C). Similarly, CGA at the dose of 10 and 30 mg/kg body weight also significantly increased the number of returns to alternate arms as compared to the scopolamine group (p˂0.05; p˂0.01). The results obtained with donepezil at 2 mg/kg were comparable to CGA (30 mg/kg body weight). Similarly %SAP was also significantly increased with CGA at 10 and 30 mg/kg (p˂0.05; p˂0.01) as compared to scopolamine treated group (Figure 4.70D).
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Figure 4.70: Effect of isolated compound CGA (1, 3, 10 and 30 mg/kg) of E. umbellata Thunb. on the Y-maze task: (A) number of arm entries (B) Same arm returns (C) alternate arm returns (D) %SAP. *p < 0.05, **p < 0.01 versus control and #p < 0.05, ##p < 0.01 versus scopolamine (Scop) 1 mg/kg. (One way ANOVA followed by Dunnett's posthoc test).
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1.13.2. Novel Object Recognition Test
To assess short term memory in the sample phase no significant difference in spending time for exploring identical objects for CGA (1, 3, 10 and 30 mg/Kg) and scopolamine group
(Figure 4.71A) were observed. While in test phase, CGA (10 and 30 mg/Kg) treated groups and donepezil (2 mg/kg) spend more time with novel object while scopolamine (1 mg/kg) treated group spent significantly (p < 0.05) more time with familiar object (Figure 4.71B).
Results indicated that the %DI for CGA 10 mg/kg (p < 0.05), 30 mg/kg (p < 0.01) and donepezil (p < 0.01) groups was significantly high compares to scopolamine group that have significantly (p < 0.01) low value as compared to vehicle control (Figure 4.71C). Similarly, in the sample phase of long term memory, no significant difference were observed in time spending for exploring the two objects between the CGA and scopolamine treated groups
(Figure 4.72A). In the test phase of long term memory CGA (10 and 30 mg/Kg) and donepezil 2 mg/kg showed a significant (p < 0.05) increase in time spending for exploring the novel object (Figure 4.72B). However, scopolamine group has shown a significant (p < 0.05) increase in spending time with familiar object. The %DI was significantly (p < 0.01) higher for CGA (10 mg/Kg (p < 0.05), 30 mg/Kg) and donepezil (2 mg/kg) compared to scopolamine (1 mg/kg) that have significantly (p < 0.01) lower value as compared to the vehicle control (Figure 4.72C).
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Figure 4.71: Effect of isolated compound CGA (3, 10 and 30 mg/kg) of E. umbellata Thunb. in short-term memory NORT. (A) Time spent in the sample phase (B) Time spent in the test phase (C) %DI. **p < 0.01 versus control and #p < 0.05, ##p < 0.01 versus Scop 1 mg/kg. A and B were analyzed using Two-way ANOVA repeated (Mixed model) followed by Bonferroni post-test and C was analyzed by One-way ANOVA followed by Dunnett's posthoc test.
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Figure 4.72: Effect of isolated compound CGA (3, 10 and 30 mg/kg) of E. umbellata Thunb. in long term memory NORT. (A) Time spent in the sample phase (B) Time spent in the test phase (C) %DI. **p < 0.01 versus control and #p < 0.05, ##p < 0.01 versus Scop 1 mg/kg. A and B were analyzed by Two-way repeated (Mixed model) ANOVA followed by Bonferroni post test and C was analyzed by One-way ANOVA followed by Dunnett's posthoc test.
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1.14. Molecular docking validation of isolated compounds for anticholinesterases (AChE & BChE)
To study the molecular docking for of cholinesterase (AChE & BChE) inhibition; binding analyses of the isolated compounds were performed on a GOLD suit v5.6.3. For this purpose, crystal structures of TcAChE in complex with donepezil from Tetronarce californica (PDB
ID: 1EVE) and hBChE in complex with tacrine (PDB ID: 4BDS) from Homo sapiens were used as receptors. The docking pose of most active compound chlorogenic acid superimposed onto donepezil inside the binding cavity of 1EVE is shown in Figure 4.73. Analysis of binding modes indicates similar binding orientations for chlorogenic acid and donepezil in the active gorge of the receptor protein. Upon visual inspection (Figure 4.74A & B), we can observe interactions such as π-π stacking (TRP83 to phenolic ring), conventional hydrogen bonding (TYR129 to OH of phenolic ring), pi-lone pair (PHE329 to C=O group of caffeoyl moiety), and numerous van der Waals attractions for the best docking pose of chlorogenic acid (Gold fitness score = 62.69).
For studying the mechanism of inhibition for human BChE, the crystal structure with PDB
ID of 4BDS was used as target protein. The mechanism of BChE inhibition by chlorogenic acid was studied by viewing its interactions in the active gorge of human BChE (PDB ID:
4BDS) as the enzyme model. The structural features of the most active compound chlorogenic acid suggest that it must have interactions like hydrophilic and hydrophobic in the active site of the enzyme. Upon visual inspection of the best docked pose (Figure 4.75A
& B), chlorogenic acid can be seen to bind hydrophobically (π-π stacking) with amino acid residues LEU283, PHE326, and TRP228. Numerous hydrogen bonding interactions between amino acids (TYR125, SER195, and GLU194) and hydroxyl groups of chlorogenic acid also seems to contribute to stability of chlorogenic acid-BChE complex.
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Figure 4.73: Superimposed ribbon diagram for chlorogenic acid and donepezil
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A B
Figure 4.74: (A) Stereo view of the docking posture of chlorogenic acid (green color stick model) in the binding pocket of AChE (B) 2D interactions of chlorogenic acid.
A B
Figure 4.75: (A) Stereo view of the docking posture of chlorogenic acid (purple color stick model) in the binding pocket of BChE (B) 2D interactions of chlorogenic acid.
174
CHAPTER 04 RESULTS The docking posture of the active ellagic acid superimposed onto donepezil in the inner surface of the binding cavity of 1EVE is shown in Figure 4.76. Examination of different binding modes indicates similar binding coordination for ellagic acid and donepezil in the active pocket of the receptor protein. Upon visual inspection (Figure 4.77A & B), different interactions can be seen such as π-π stacking (TRP278, TYR333), conventional hydrogen bonding (TYR120, ARG288, PHE287, ASP71 to OH of phenolic ring), carbon hydrogen bond interactions (PHE330, ILE286), unfavorable donor-donor interaction (ARG288 to OH group) and various number of van der Waals attractions for the best docking pose of ellagic acid (Gold fitness score = 55.24).
Figure 4.76: Superimposed ribbon diagram for ellagic acid and donepezil
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A B
Figure 4.77: (A) Stereo view of the docking posture of ellagic acid (green color stick model) in the binding pocket of AChE (B) 2D interactions of ellagic acid. The docked pose of the morin superimposed onto donepezil inside the binding cavity of
1EVE is shown in Figure 4.78. Inspection of different binding modes shows resemblance in binding coordination for morin and donepezil in the active gorge of the receptor protein.
Upon visual inspection (Figure 4.79A & B), we can identify different types of interactions such as π-π stacking (TRP83 to phenolic ring), π-π T shaped interactions (PHE330, TYR333,
PHE329 to phenolic ring), conventional hydrogen bonding (TYR129, GLU198, GLY116,
HIS439, PHE329 to OH of phenolic ring), carbon-Hydrogen bond (GLY440 to C=O group), and various van der Waals attractions for the docking pose of morin (Gold fitness score =
59.49).
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Figure 4.78: Superimposed ribbon diagram for Morin and donepezil
A B
Figure 4.79: (A) Stereo view of the docking posture of Morin (green color stick model) in the binding pocket of AChE. (B) 2D interactions of Morin.
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CHAPTER 04 RESULTS The docked pose of catechin superimposed onto donepezil inside the binding cavity of 1EVE is shown in Figure 4.80. Different binding modes are investigated which shows resemblance in binding coordination for catechin and donepezil in the active gorge of the receptor protein.
Upon visual inspection (Figure 4.81A & B), different types of interactions can be identified such as π-π stacking (TRP83, PHE329 to phenolic ring), π-π T shaped interactions (PHE330,
TYR333 to OH of phenolic ring), conventional hydrogen bonding (GLU198, GLY116,
PHE329 to OH of phenolic ring), carbon-Hydrogen bond (HIS439 to Hydrogen), pi-lone pair interaction (TYR333 to OH of phenolic ring) and several van der Waals attractions for the docking pose of catechin (Gold fitness score = 59.66).
Figure 4.80: Superimposed ribbon diagram for Catechin and donepezil
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A B
Figure 4.81: (A) Stereo view of the docking posture of catechin (green color stick model) in the binding pocket of AChE (B) 2D interactions of catechin
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1.15. Molecular docking validation of HPLC detected compounds for anticholinesterases (AChE & BChE)
The best docking posture of the most active rutin compound superimposed onto donepezil in interior part of the binding pocket of 1EVE is shown in Figure 4.82. Investigation of binding poses shows similarities in binding orientations of rutin with donepezil in the active site of the receptor protein. Upon visual inspection (Figure 4.83A & B), different interactions can be witnessed like π-π stacking (TRP83 to phenolic ring), π-π T-shaped interactions (TYR120), pi alkyl interaction (TRP278), conventional hydrogen bonding (ARG288, PHE287, PHE329,
TYR120, TYR69, ASN84, GLU198), pi-lone pair (PHE329 to Oxygen) and carbon hydrogen bond interaction (GLY440) for the best docking pose of rutin (Gold fitness score = 95.35).
Figure 4.82: Superimposed ribbon diagram for rutin and donepezil
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A B
Figure 4.83: (A) Stereo view of the docking posture of rutin (green color stick model) in the binding pocket of AChE (B) 2D interactions of rutin. The best docking posture of one of the active epigallocatechin gallate superimposed onto donepezil inside the binding pocket of 1EVE is shown in Figure 4.84. Analysis of different binding ways shows resemblance in binding orientations for epigallocatechin gallate and donepezil in the active cavity of the receptor protein. Upon visual inspection (Figure 4.85A &
B), various interactions can be observed such as π-π stacking (TYR333), pi-pi T-shaped interactions (PHE330), pi-Anion interaction (Asp71), pi-sigma interaction (TYR333), conventional hydrogen bonding (PHE330. PHE287, ARG288, GLY118, GLY117, SER199 to OH group) and carbon hydrogen bond interaction (ILE286) for the docking pose of epigallocatechin gallate (Gold fitness score = 75.60).
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Figure 4.84: Superimposed ribbon diagram for epigallocatechin gallate and donepezil
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A B
Figure 4.85: (A) Stereo view of the docking posture of epigallocatechin gallate (green color stick model) in the binding pocket of AChE (B) 2D interactions of Epigallocatechin gallate. The docked pose of one of the active compound quercetin superimposed onto donepezil in
the inner surface of binding cavity of 1EVE is shown in Figure 4.86. Investigation of binding
ways shows similarities in binding coordination for quercetin and donepezil in the active
cavity of the receptor protein. Upon visual inspection (Figure 4.87A & B), various
interactions can be observed such as π-π stacking (TRP83), π-π T shaped interactions
(TYR120), pi-Anion interaction (ASP71), conventional hydrogen bonding (SER121,
GLU198, TYR120), unfavorable Acceptor-Acceptor interaction (TYR69), carbon-Hydrogen
interaction (VAL70) and numerous van der Waals attractions for the docking pose of
quercetin (Gold fitness score = 60.43).
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Figure 4.86: Superimposed ribbon diagram for Quercetin and donepezil
Figure 4.87: (A) Stereo view of the docking posture of Quercetin (green color stick model) in the binding pocket of AChE (B) 2D interactions of Quercetin.
The docking posture of the active catechin hydrate compound superimposed onto donepezil in interior part of the binding pocket of 1EVE is shown in Figure 4.88. Investigation of binding procedures shows similar binding directions for catechin hydrate and donepezil in the active gorge of the receptor protein. Upon visual inspection (Figure 4.89A & B), we can 184
CHAPTER 04 RESULTS observe different interactions like π-π stacking (TRP278 to phenolic ring), π-π T-shaped interactions (TYR120, PHE329), conventional hydrogen bonding (SER199, ARG288,
PHE287, to OH of phenolic ring) and several numbers of van dar waals interactions for the best docking pose of catechin hydrate (Gold fitness score = 52.53).
The Gold score values and energy of ligands of isolated and HPLC detected compounds are presented in Table 4.29. The rutin, epigallocatechin gallate, chlorogenic acid, quercetin, catechin, morin, catechin hydrate, ellagic acid have higher Gold score values with highest cholinesterase inhibitory activities. While compounds; gallic acid, mandelic acid, phloroglucinol and malic acid showed weaker inhibitory activity. The more Gold Score values represent tighter binders.
Figure 4.88: Superimposed ribbon diagram for Catechin hydrate and donepezil
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CHAPTER 04 RESULTS
Figure 4.89: (A) Stereo view of the docking posture of catechin hydrate (green color stick model) in the binding pocket of AChE (B) 2D interactions of catechin hydrate.
186
CHAPTER 04 RESULTS Table 4.29: The Gold Scores and energy of ligand values of cholinesterase inhibitors present in Elaeagnus umbellata Thunb.
Compound Compound name Gold Score Energy of ligand Compound-I Chlorogenic acid (CGA) 62.69 -10.556 Compound-II Ellagic acid 55.24 -6.971 Compound-III Morin 59.49 -13.929 Compound-IV Gallic acid 39.42 -6.917 Compound-V Catechin 59.66 -7.288 Compound-VI Phloroglucinol 33.28 -5.071 Compound-VII 1-Hexyl benzene 48.31 -0.655 Compound-VIII Mandelic acid 38.94 -2.355 HPLC detected Rutin 95.35 -27.018 HPLC detected Epigallocatechin gallate 75.60 -13.585 HPLC detected Quercetin 60.43 -14.310 HPLC detected Catechin hydrate 52.53 -6.743 HPLC detected Malic acid 31.26 -3.782
The Rutin, Epigallocatechin gallate, Chlorogenic acid, Ellagic acid, Catechin, Morin, Quercetin, Catechin hydrate, have higher Gold score values with highest cholinesterase inhibitory activities. While compounds; Gallic acid, Mandelic acid, Phloroglucinol and Malic acid showed weaker inhibitory activity. The more Gold Score values represent tighter binders [345] .
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CHAPTER 05 DISCUSSION DISCUSSION
In spite of the available antidiabetic medications, herbal remedies and extracts are of great importance for the ethnobotanical community as they are considered to be less toxic than the synthetic drugs [352]. There has been an increasing interest of the scientific community in traditional and herbal medicines due to their pharmacological and economic advantages
[353]. Medicinal plants received much attention due to the existence of indispensable bioactive compounds such as phenolics and flavonoids which have shown strong antioxidant properties [354].
The objective of the current investigation was to comprehensively estimate the antioxidant, antidiabetic and antiamnesic potential of E. umbellata fruit; an indigenous medicinal plant. In the current study, the Me-Ext of E. umbellata fruit, their subsequent fractions, isolated essential oil and compounds showed strong antioxidant potential, this might be due to the existence of phenolic and flavonoid compounds. HPLC-UV fingerprints of the Me-Ext and subsequent fractions of E. umbellata fruit also confirmed the presence of phenolic and flavonoid compounds, which is an agreement with a previously reported study showing the presence of phenolic acids (gallic acid, vanillic acid, coumaric acid, sinapic acid, ferulic acid and caffeic acids) in the hydro methanolic berry extracts [355].
Significant antioxidant potentials were exhibited by extracts/fractions of E. umbellata fruit, essential oil and isolated compounds against DPPH and ABTS. The results of current study revealed that highest % radical scavenging potential was exhibited by Chf-Ext and EtAc-Ext fraction out of which Chf-Ext. was further procedeed for isolation of compounds. % DPPH and ABTS inhibition potential of various plant test samples and isolated compounds were comparable with standard ascorbic acid (positive control) showing a concentration-dependent response also go parallel with reported studies [274, 312]. The plant samples and isolated compounds were also assessed against both gram positive and gram negative strains. The
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CHAPTER 05 DISCUSSION results indicated broad spectrum action of extracts/fractions and isolated compounds. The findings of this study specify that the plant E. umbellata can potentially be used against infectious diseases. Our findings were consistent with previous reported studies [277, 356,
357].
Essential oil was isolated for the first time from E. umbellata fresh fruits and characterization has been done through GC-MS. Results indicated that essential oil showing strong antioxidant, antidiabetic and cholinesterase inhibition potential that might be due to their strong phytochemical profile [358, 359]. Among the major compounds identified in the essential oil of Elaeagnus umbellata Thunb fruit the active antidiabetic and neuroprotective components were n-hexadecanoic acid, Octadecadienoic acid, α-linolenic, (-)
Caryophyllene, Caryophyllene oxide, Phytol, Octadecanoic acid, Tricosanoic acid, 9-
Octadecenal, octadecanoic acid, 7-Tetradecanal, 2-Methoxy-4-vinylphenol, 9, 12, 15-
Octadecatrienoic acid, 8,11- Octadecadienoic acid, Nonanoic acid, Neophytadiene, Decanoic acid, Pentadecanoic acid, Decanal, Humulene Epoxide, 2-Methoxy-4-vinylphenol (p-
Vinylguaiacol), Isolongifolol, (+)-Ascorbic acid 2,6-dihexadecanoate, 8, 11, 14-
Eicosatrienoic acid also have been reported by other authors [360-362].
Among the various metabolic disorders, diabetes mellitus is one of the common endocrine disorders. It is characterized by absolute or relative deficiency in insulin secretion, hyper- glycemia disturbances of protein, carbohydrate and fat metabolism [363]. This disease leads to retinopathy, atherosclerosis, neuropathy and nephropathy, hyperglycemia, hypertension and hyperlipidemia. Therefore, an effective therapeutic approach is demand of the day for prevention of dysfunctioning or degeneration of β- cells because Oral hypoglycemic agents and insulin, currently used have serious side effects [57, 364]. α-Amylase is the key enzyme in the human body that is responsible for the breaking down of polysaccharides starch into disaccharides. Then the α-glucosidase enzyme cause the hydrolysis of disaccharides into
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CHAPTER 05 DISCUSSION simple sugars which are absorbed through small intestines causing postprandial hyperglycemia [365]. Thus α-Amylase and α-glucosidase inhibitors prevent the absorption of dietary starch, thus decrease the postprandial glucose level. Inhibition of the breakdown of starch may have useful effects in dabetic people [265, 366]. Polyphenols of plant like phenolic acids and flavonoids are also advantageous in controlling postprandial glucose by inhibiting α-glucosidase [265]. In our study, we found that the Me-Ext, Chf-Ext and EtAc-
Ext fractions along with isolated compound-V (Catechin) of E. umbellata significantly inhibited α-amylase and α-glucosidase enzymes indicating antihyperglycemic effects which were in agreement with reported study on phenolic compounds [367]. The IC50 values of Chf-
Ext and EtAc-Ext were found comparable with positive control (acarbose) showing a concentration-dependent response. These data suggest that the antidiabetic agents are preferentially present in these extracts.
Furthermore, validation of molecular docking procedure, the superimposition of docked acarbose molecule to the one that has obtained from the α-amylase crystal structure and similar interactions were found in the α-amylase binding site. It was concluded from reported molecular docking study on phenolic compounds that comparable binding modes of all molecules within the vicinity of the acarbose binding site emphasized that the effects of Chf-
Ext and EtAc-Ext is due to their organic constituents [368].
The use of high-fat diet and Streptozotocin to induce T2D in rats has already been reported in the literature [335]. In this model, administration of HFD causes obesity in rats which leads to insulin resistance. Furthermore, low dose of STZ which is known as diabetogenic and is a
β- cell toxin, causes destruction and severe decline of β- cells [369, 370]. As a result, lacking of insulin also causes hyperglycemia [371]. Thus the hyperglycemia coupled with other metabolic irregularities including insulin resistance and hyperlipidemia closely depict the metabolic appearances of T2D in humans [372]. Furthermore, in normal metabolic
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CHAPTER 05 DISCUSSION condition, insulin causes lipid metabolism through activation of lipoprotein-lipase enzyme that breaks down triglycerides to fatty acids and glycerol. These fatty acids are used as energy source or re-esterified in the body tissues for storage. In T2D insulin insufficiency or resistance leads to inactivation of lipoprotein lipase causes a condition of hypertriglyceridemia. In this study, the major changes in lipid profile such as an high serum triglycerides, serum cholesterol, serum LDL cholesterol and low serum HDL cholesterol in
STZ- induced diabetic rats are in agreement with the lipid profiles alterations reported by other researchers [353]. High LDL level is characterized by transporting cholesterol to the tissues from the liver that leads to the development of coronary heart disease [373].
While, HDL cholesterol is considered as a valuable lipoprotein that transport endogenous cholesterol and cholesteryl esters to the liver and steroidogenic tissues from the body tissues and prevent deposition of cholesterol, thus inhibiting atherosclerosis [374].
In the current study, the extracts/fractions of E. umbellata significantly reduced blood glucose in STZ (50 mg/kg body weight) induced diabetogenic animal model. This reduction in blood glucose level by E. umbellata fruit extract/fractions might be due to isolated compound catechin present in E. umbellata. The antihyperglycemic effect of Me-Ext and Chf-Ext along with compound catechin of E. umbellata was equivalent to standard glibenclamide.
Furthermore, the phytoconstituents like catechin present in the active fraction Chf-Ext may increase the secretion of insulin from pancreatic βeta-cells, thus resulting in an improvement in glycemic control [375].
Weight loss is also a serious problem in STZ induced diabetes which may be due to hyperglycemia, hypoinsulinemia, loss of proteins and muscle wasting [376]. STZ-induced diabetic rats revealed significant reduction in the body weight as compared to the normal control rats during the experimental study period. The extract/fractions and compound-V of
E. umbellata significantly increase the STZ mediated reduction in body weight. This outcome is consistent with previous studies and could be due to the capability E. umbellata extracts to 191
CHAPTER 05 DISCUSSION reduce hyperglycemia [376, 377]. Moreover, the Me-Ext, Chf-Ext, EtAc-Ext and compound-
V showed a significant decrease in TC, TGs, LDL and cholesterol while significantly increased HDL cholesterol in diabetic control group at the end of experiment.
Studies have shown that STZ induces CYP2E1 dependent oxidative stress and causes the release of various liver microsomal enzymes including SGOT, SGPT and serum ALP in the blood that indicate liver damage or condition of T2D disease [378]. The extracts/fractions and isolated compound catechin of E. umbellata have significantly reduced the levels of SGPT,
SGOT and ALP in diabetic control group that indicates a possible hepatoprotective effect.
The standard glibenclamide drug also significantly reduced the levels of SGPT, SGOT and
ALP in diabetic control group. Furthermore, the Me-Ext, Chf-Ext and EtAc-Ext also caused a significant reduction in serum creatinine and blood urea nitrogen indicating protective effects on kidneys.
Hepatoprotective effect recommends that E. umbellata plant hold antioxidant potential due to phytoconstituents that assisted to combat the oxidative stress-related hepatotoxicity produced by the induction of CYP2E1 in STZ-induced diabetes comparable with reported study [379,
380]. STZ-induced diabetes is usually associated with impairment of renal function as mediated by significant increases in serum creatinine level and blood urea nitrogen. This is due to the interaction of STZ with glomerular tissues and glomerular filtrations [381]. In the current study, the Me-Ext, Chf-Ext, EtAc-Ext and standard antidiabetic drug glibenclamide also significantly reduced the serum creatinine level and blood urea nitrogen in diabetic control rats that revealed its renoprotective effect. Isolated compound catechin also observed the same renoprotective effect go parallel with reported study [382].
The overall antidiabetic activity of the Me-Ext, sub fractions and isolated compound catechin of E. umbellata may be due to their strong antioxidant potential. In addition to reducing carbohydrate metabolism by inhibiting α- amylase enzymes and α- glucosidase, the
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CHAPTER 05 DISCUSSION phenolic and flavonoids compounds are known to revealed antidiabetic effect by decreasing the intestinal carbohydrate absorption, insulin action or insulin secretion , increase in β- cell function and antioxidant effect [375, 383].
These data confirmed that the Me-Ext, sub fractions and catechin of E. umbellata have significant antidiabetic activity against α- glucosidase and α- amylase enzymes in STZ- induced diabetes mellitus supported by docking analysis. Furthermore, these extracts and isolated compound catechin have protective effects on the major tissues including liver, kidney and Pancreas and thus reduce the diabetes associated complications [24].
Histopathological analysis clearly showed improvement in different extract/fractions and compound-V (catechin) treated groups compared to STZ-induced diabetic group. The pancreatic tissue of diabetic rat demonstrated degeneration and vacuolization in the
Langerhan’s islet cells. The diameter of Islet of Langerhans was decreased with decreased cellular density. Shrined and pyknotic nuclei were also visible in the center of Islet. The boundary of endocrine and exocrine portion of pancreas was also indistinct, besides decrease in islets size and β-cell number was also observed. This data was in agreement with Ramadan et al. [384].
Current results indicated that histological pattern of pancreas of groups treated with glibenclamide (500g/kg/overly), Chf. Ext and Compound-V (10 & 15 mg/kg/orally) did not show any violation from the normal histological pattern during experimental period. These groups showing maximum pancreatic regeneration which were also in consistent with Hadi et al study [385]. Subhrojit Sen et al have also observed increase in number and diameter of
Islet of Langerhans in STZ-induced diabetic Rates after treatment with glycyrrhizin acid
[386]. Similarly Arzani Birgani et al have also reported an increase in β-cell function after treatment with betulinic acid, which is in agreement with our results [387]. It can be concluded that different extract possess significant anti-diabetic activity in diabetic mice.
Based on increased levels of insulin and histopathology studies of pancreatic tissue, anti-
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CHAPTER 05 DISCUSSION diabetic activity of those samples was believed to occur through the mechanism of increasing secretion of healthy pancreatic β-cells.
Acetyl cholinesterase (AChE) enzyme has a key role in the cholinergic nervous system.
Therapies planned to invalidate the cholinergic deficit in AD is generally based on AChE inhibitors, thus enhance cholinergic conduction in the brain. Several literature studies revealed that cholinesterase inhibitors might act on several therapeutic targets such as hydrolysis of AChE enzyme and prevention of β-amyloid plaques formation [388]. However, there is still a need of novel AChE inhibitors with low toxicity and high penetration rate to the central nervous system (CNS). The only standard drugs used as acetyl cholinesterase inhibitors for brain related disorders like AD are galanthamine, donepezil and rivastigmine
[389]. Natural products have already demonstrated to be promising sources of functional acetyl cholinesterase (AChE) inhibitors. The standard drugs for AD, galanthamine and rivastigmine are alkaloids derived from plants [390]. AChE inhibition potential of medicinal plant has provide a remedial approach for various brain related diseases like dementia and
Parkinson’s disease in addition to AD, increase cholinergic neurotransmissions [391]. There is still a need to explore new plants for new potent and enduring AChE inhibitors with low side effects. Many plant species from various parts of the world have been evaluated for anti- cholinesterase activity [392]. Literature studies reported reactive species were closely associated with oxidative stress and AD [393]. Antioxidant remedy has proved to be successful in the amelioration of cognitive dysfunction and behavioural insufficiency in patients with mild to moderate AD. Natural products are the major source of antioxidants which delay the development of AD and neuron injury induced by oxidative stress [394].
Medicinal plants received much attention due to presence of important bioactive secondary metabolite such as carotenoid, phenolic and flavonoid [320]. It is obvious of our current investigational study that E. umbellata fruit provide a significant source of secondary metabolites which play a role as cholinesterase inhibitors which finds parallel with previous
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CHAPTER 05 DISCUSSION study [395]. In in vivo study the Chf-Ext and EtAc-Ext extracts/fractions and isolated compound chlorogenic acid of fruit showed strong cholinesterase inhibitions with positive control Donepezil. Y maze spontaneous alteration is used for studying working memory and exploration of the animals. The test is based on the willing of the animals to explore new environment. The animals which are normal will explore the new arm, the animals whose memory is not working properly will again enter the old arm previously explore. This gave less number of spontaneous explorations as compared to normal animals [396]. In this study, the chloroform extract and chlorogenic acid significantly decreased the number of alternate arm returns as compared to control. Furthermore, the extract and CGA also significantly increased spontaneous alternation performance which was parallel with reported study [397].
Novel object recognition test is use for studying both short and long term memory [398]. The general principle of novel object recognition test is based on exploring new object. The rodents spend more time with unfamiliar object as compared to familiar object. During experiment animals are familiarized with two similar objects and after a wash out period one object in the closed box is replaced with a new unfamiliar object [396]. In this study, the chloroform extract and CGA caused increase exploration of the novel object compared to the familiar object. The increase in frequency of visiting novel object and time with novel object observed with compounds 10 and 30mg/kg was comparable to reference drug donepezil at
2mg/kg. These findings were further supported by their potential to inhibit AChE and BChE inhibition both in vitro and in in silico studies. Current results were supported by previous
Zahra R et al study [399]. These results indicate the isolated compound CGA may provide novel leads for the development of agents affecting memory in disorders such as Alzheimer’s disease. To our knowledge there was no reported neuroprotective study on E. umbellata however chlorogenic acid found in the fruit of this plant has shown neuroprotective effect
[400].
195
CHAPTER 05 DISCUSSION In this study Elaeagnus umbellata fruit extracts show presence of phytochemical groups including alkaloids, flavonoids, glycosides, tannins, terpenoids, anthraquinone and pigments which goes parallel with previously published work [401]. In contrast to our results, flavonoid was considered absent in the phytochemical analysis of the aerial parts of
Elaeagnus umbellata [402]. The astringent taste of the fruits/berries might be due to the presence of high total phenolic content, lycopene and flavanol [403].
In Linear correlation of total phenolic/flavonoid contents versus cholinesterase inhibition assays, the concentrations of phenolics and flavonoids in different fractions exhibited strong correlation with cholinesterase inhibition potential. Results of current study demonstrated the high efficiency of Chf-Ext and EtAc-Ext extracts/fraction for inhibition potential against acetyl cholinesterase and butyryl cholinesterase enzymes. The high potency and anti-amnesic potential of Chf-Ext fraction might be due to the presence of isolated compound; chlorogenic acid, quercetin, catechin and morin which have strong neuroprotective effects on learning and memory impairment [404] as validated by molecular docking study. Chlorogenic acid (CGA) is the key polyphenolic constituent that exhibits cardio protective effects, inhibitory potential of lipid hyper oxidation, anti-tumor activity and free radical scavenging potential [405]. It has been evidenced that the anti-anxiety effects of CGA is associated with its anti-oxidant potential [406] and neuroprotective potential [400]. Molecular docking study validated that the compounds; gallic acid, mandelic acid and malic acid showed weaker inhibitory activity while, HPLC detected compounds rutin and epigallocatechin gallate show the highest Gold score values with highest inhibitory potential. These effects may be associated with the use of
E. umbellata fruits for medicinal purposes, may be beneficial to human health and as a functional food with effectiveness for treatment of degenerative diseases and memory impairments. In the current study various important phytoconstituents like phenolic acids and flavonoids in E. umbellata fruits/berries has been investigated that are responsible for in vivo
196
CHAPTER 05 DISCUSSION anti-amnesic and antidiabetic potential but required more work to plan strategies for isolation of more bioactive and potent inhibitors against cholinesterase’s and diabetes.
197
CHAPTER 06 CONCLUSION 6. CONCLUSION
The Me-Ext and subsequent fractions, essential oil and isolated compounds of E. umbellata fruits/berries has significantly inhibited DPPH and ABTS free radicals. These effects might be due to the presence of phenolic and flavonoids phytoconstituents present in these extract/fractions. Among the sub fractions the Chf-Ext and EtAc-Ext were found more potent in which the Chf-Ext was further processed for isolation of antidiabetic and anti-amnesic secondary metabolites/compounds. Methanolic extract/fractions, essential oil and isolated compounds were examined for in vitro and in vivo inhibition against α- amylase and α- glucosidase enzymes and anti-hyperglycemic effects in high-fat diet and low dose STZ- induced diabetic rats. Moreover, considerable reduction in serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, total cholesterol, low density lipoproteins and triglycerides were detected designating the useful and beneficial effects of extracts/fraction and isolated compounds on secondary complications associated with type 2 diabetes mellitus. The acetyl cholinesterase and butyryl cholinesterase inhibition potential and in vivo antiamnesic potential were also determined for extract/fractions and isolated compounds. Antiamnesic activity was used to assess the short term and long term memory through behavioural trials; Y-Maze Spontaneous Alternation Performance and novel object recognition test using animal model of albino mice. Phytochemical analysis, antibacterial activity, total contents of phenolic and flavonoid were also analysed in methanolic extract and subsequent fractions.
Finally the molecular docking study was used to identify some common interactions observed between acarbose and all docked compounds in the active sites of both α-amylase and α- glucosidase enzyme that have shown their inhibitory effects. The mechanisms of binding validation were also performed on a GOLD suit v5.6.3 for identification of binding orientations for rutin, epigallocatechin gallate, chlorogenic acid, quercetin, catechin, morin, catechin hydrate, ellagic acid and donepezil in the active gorge of the receptor protein of 198
CHAPTER 06 CONCLUSION AChE and BChE enzymes. The anti-amnesic potential of E. umbellata fruits extract/fractions might be due to isolated compound chlorogenic acid, catechin, ellagic acid, morin and HPLC detected compounds like rutin, epigallocatechin gallate, quercetin and catechin hydrate. In conclusion, E. umbellata fruit can be potentially recommended for controlling type 2 diabetes mellitus, oxidative stress, memory impairments and neurological disorders. However, more work is required for isolation of these novel and safe bioactive antidiabetic and antiamnesic compounds and elucidate their mechanism of action.
Future recommendations
1. Due to the great importance and rare nature of the plant E. umbellata Thunb.
proper training should be given to the members of the local community
regarding the natural resources conservation.
2. Due to the instinct nature of human from the day first he is behind in search of
better in every aspect of life. Although 800 plants have been reported to have
antidiabetic activities however none of them is perfect to resolve the issue
completely. Due to the mentioned fact above we design this study in the hope
that probably this plant will be more potent than the already reported plants.
Our decision of selecting this plant is not random we have selected this plant
due its phytochemical composition. So E. umbellata Thunb. plant has been
recommended as antidiabetic plant for the first time due to the high
phytochemical contents in their fruit berry.
3. The link between diabetes and brain related diseases were assessed on
molecular bases.
4. Molecular docking study validated that some HPLC detected compounds have
shown highest Gold score values indicating their highest inhibition potential of
diabetic & cholinesterase enzymes. So proper assays should be developed to
199
CHAPTER 06 CONCLUSION isolate such potent phytochemicals and to develop specific drugs for brain
related disorders from plant sources.
5. Due to low side effects and easy availability of plant sources the antibacterial,
antidiabetic and antiamnesic drugs should be isolate and design with their
proper mechanism of actions.
6. More medicinal plants should be explored worldwide to replace synthetic
drugs.
200
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