Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.):
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| Format: | Thesis Book |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | VI, 282 S. Ill., graph. Darst. |
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| 100 | 1 | |a Kisseih, Emelia |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) |c vorgelegt von Emelia Kisseih |
| 264 | 1 | |c 2014 | |
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| adam_text | TABLE OF CONTENTS
ABBREVIATIONS I
ABSTRACT IV
1 INTRODUCTION 1
1.1 GENERAL INTRODUCTION 1
1.2 AIMS OF THIS RESEARCH 3
1.3 THE SKIN 4
1.3.1 EPIDERMIS 6
1.3.2 EPIDERMAL-DERMAL JUNCTION 8
1.3.3 DERMIS 9
1.3.4 SUBCUTANEOUS LAYER 9
1.3.5 HYPODERMIS 10
1.3.6 ADNEXAL STRUCTURES OF THE SKIN 10
1.4 WOUNDS 13
1.4.1 CLASSIFICATION OF WOUNDS 13
1.4.2 MECHANISM OF WOUND HEALING 15
1.5 SOME CRUCIAL ASPECTS OF WOUND HEALING 25
1.5.1 KERATINOCYTE DIFFERENTIATION IN WOUND HEALING 25
1.5.2 FIBROBLAST AND KERATINOCYTE MIGRATION IN WOUND HEALING 27
1.5.3 COLLAGEN SYNTHESIS IN WOUND HEALING 28
1.5.4 ANTIMICROBIAL ACTIVITY IN WOUND HEALING 30
1.6 SKIN CELLS USED FOR IN VIVO FUNCTIONAL ACTIVITY DETERMINATION 31
1.6.1 DERMAL FIBROBLASTS (PNHDF) 31
1.6.2 EPIDERMAL KERATINOCYTES (PNHEK) 33
HTTP://D-NB.INFO/1062792785
TABLE OFCONTENTS
1.63 HUMAN, ADULT, LOW CALCIUM, HIGH TEMPERATURE (HACAT) KERATINOCYTES
34
1.7 CHEMOTAXONOMY OF COMBRETACEAE 36
1.7.1 C. MUCRONATUM (SCHUM. & THONN.) 37
2 MATERIALS AND METHODS 40
2.1 GENERAL PHYTOCHEMICAL EQUIPMENT AND MATERIALS 40
2.1.1 PHYTOCHEMICAL EQUIPMENT 40
2.1.2 SOLVENTS AND CHEMICALS 41
2.2 CELL CULTURE EQUIPMENT AND MATERIALS 42
2.2.1 CELL CULTURE EQUIPMENT 42
2.2.2 GENERAL CONSUMABLES 48
2.2.3 SOFTWARE 49
2.3 PLANT MATERIALS 50
2.3.1 COLLECTION AND IDENTIFICATION OF SELECTED PLANT MATERIALS 50
23.2 EXTRACTION OF PLANT MATERIALS 50
2.3.3 PHYTOCHEMICAL SCREENING OF EXTRACTS BY THIN LAYER CHROMATOGRAPHY
(TLC)... 51
2.3.4 DETERMINATION OF TANNIN CONTENT 52
2.4 OPTIMIZATION OF SOLVENT EXTRACTION PROCEDURE FOR C. MUCRONATUM 53
2.4.1 DEFATTENING WITH PETROLEUM ETHER 53
2.4.2 EXTRACTION BY ULTRA TURRAX 54
2.5 EXTRACTION, ISOLATION AND CHARACTERIZATION OF COMPOUNDS FROM C.
MUCRONATUM
LEAVES 54
2.5.1 BULK EXTRACTION WITH ETHANOL 50 % FOR PREPARATIVE COLUMN
CHROMATOGRAPHY 54
2.5.2 ETHYL ACETATE-WATER PARTITIONING OF ETHANOL 50 % EXTRACT OF C.
MUCRONATUM.. 55
TABLE OF CONTENTS
2.5.3 FRACTIONATION OF ETOAC EXTRACT ON SEPHADEX LH-20 STATIONARY PHASE
55
2.5.4 ISOLATION OF PROCYANIDINS 56
2.6 IDENTIFICATION OF FLAVONOIDS BY SPIKING EXPERIMENTS BY UPLC-QDA-PDA
63
2.7 IDENTIFICATION OF FATTY ACIDS IN PETROLEUM ETHER EXTRACT BY GC-MS
AFTER SILYLATION
WITH BSTFA/TMS/PYRIDINE 64
2.7.1 FRACTIONATION OF PETROLEUM ETHER EXTRACT OF C. MUCRONATUM LEAVES
ON SILICA
GEL 65
2.7.2 IDENTIFICATION OF COMPOUNDS FROM THE MEOH SOLUBLE FRACTION OF THE
DICHLOROMETHANE EXTRACT OF C. MUCRONATUM BY GC-MS 65
2.7.3 CAPILLARY ZONE ELECTROPHORESIS (CZE) METHOD FOR THE IDENTIFICATION
OF SUGARS67
2.7.4 SCREENING OF C. MUCRONATUM BY ESI-MS FOR THE IDENTIFICATION OF
COMBRETASTATINS 67
2.7.5 QUALITY CONTROL OF C. MURONATUM SAMPLES 68
2.8 CELL CULTURE METHODS 69
2.8.1 ESTIMATION OF VIABLE CELLS BY CASY CELL COUNTING 69
2.8.2 PLANT EXTRACTS 69
2.8.3 TEST SOLUTIONS FOR FUNCTIONAL ACTIVITY DETERMINATION 69
2.8.4 PASSAGING OF HUMAN ADULT LOW CALCIUM HIGH TEMPERATURE (HACAT)
KERATINOCYTES 70
2.8.5 PASSAGING OF NORMAL HUMAN EPIDERMAL KERATINOCYTES (PNHEK) 71
2.8.6 PASSAGING OF FIBROBLASTS (PNHDF) 71
2.8.7 IN VITRO TESTS TO DETERMINE FUNCTIONAL ACTIVITY 72
2.8.8 METABOLIC ACTIVITY (MTT ASSAY) (MOSMANN, 1983) 72
2.8.9 CELL PROLIFERATION (BRDU ASSAY) (PORSTMANN ET AL., 1985) 73
2.8.10 CYTOTOXICITY (LDH ASSAY) (DECKER AND LOHMANN-MATTHES, 1998) 74
TABLE OF CONTENTS
2.8.11 MORPHOLOGICAL OBSERVATION OF CELLS WITH THE FLUORESCENCE
MICROSCOPY 75
2.8.12 DIFFERENTIATION ASSAYS 76
2.8.13 COLLAGEN QUANTIFICATION FROM PNHDF 82
2.8.14 CELL MIGRATION (SCRATCH) ASSAY (SPUR/.EM ET AL., 2002) 83
3 RESULTS 85
3.1 PHYTOCHEMICAL RESULTS 85
3.1.1 PHYTOCHEMICAL SCREENING OF PLANT MATERIAL BY THIN LAYER
CHROMATOGRAPHY
(II.C) 85
3.1.2 FRACTIONATION OF THE ETHYL ACETATE FRACTION ON SEPHADEX LH-20 90
3.1.3 ISOLATION AND IDENTIFICATION OF PROANTHOCYANIDINS 94
3.1.4 IDENTIFICATION OF FLAVONOIDS BY SPIKING EXPERIMENTS BY
UPLC-QDA-PDA ... 1 I 1
3.1.5 GC-MS IDENTIFICATION OF COMPOUNDS IN THE PETROLEUM ETHER EXTRACT
OF THE
LEAVES OF C. MUCRONATUM 115
3.1.6 IDENTIFICATION OF CARBOHYDRATES BY CAPILLARY ZONE ELECTROPHORESIS
(CZE).... 132
3.1.7 ESI-MS SCREENING OF C. MUCRONATUM EXTRACTS FOR COMBRETASTATINS 134
3.1.8 DEVELOPMENT OF A VALIDATED UPLC METHOD FOR THE ANALYSIS OF C.
MUCRONATUM
LEAVES EXTRACT (ETHANOL 50 %) ACCORDING TO THE ICH HARMONIZED TRIPARTITE
GUIDELINES (VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY
Q2, RL) 138
3.2 FUNCTIONAL ACTIVITIES OF C. MUCRONATUM EXTRACTS AND ISOLATED
PROCYANIDINS 156
3.2.1 METABOLIC ACTIVITY (MTT ASSAY) 156
3.2.2 CELL PROLIFERATION (BRDU ASSAY) 158
3.2.3 NECROTIC CYTOTOXICITY (LACTATE DEHYDROGENASE ASSAY) 160
3.2.4 MORPHOLOGICAL OBSERVATION OF HACAT KERATINOCYTES TREATED WITH
AQUEOUS
EXTRACT OF C. MUCRONATUM BY FLUORESCENCE MICROSCOPY 165
3.2.5 IMMUNOFLUORESCENCE STAINING FOR THE DETECTION OF INVOLUCRIN AND
CYTOKERATIN
10 EXPRESSION IN PNHEK BY CONFOCAL LASER SCANNING MICROSCOPY 166
TABLE OF CONTENTS
3.2.6 WESTERN BLOT ANALYSIS FOR INVOLUCRIN AND CYTOKERATIN 10 EXPRESSION
IN
PNHEK 170
3.2.7 CELL MIGRATION (SCRATCH) ASSAY 172
3.2.8 COLLAGEN QUANTIFICATION FROM PNHDF 176
4 DISCUSSION 213
4.1 PHYTOCHEMICAL ANALYSIS OF C. MUCRONATUM 213
4.2 INFLUENCE OF C. MUCRONATUM EXTRACTS ON PHYSIOLOGY OF HACAT
KERATINOCYTES 217
43 MORPHOLOGICAL EFFECT OF AQUEOUS EXTRACT ON HACAT KERATINOCYTES 219
4.4 INFLUENCE OF AQUEOUS EXTRACT OF C. MUCRONATUM ON PHYSIOLOGY AND
DIFFERENTIATION
OF PNHEK 219
4.5 INFLUENCE OF AQUEOUS EXTRACT ON PHYSIOLOGY OF PNHDF 223
4.6 INFLUENCE OF AQUEOUS EXTRACT ON MIGRATION OF PNHEK AND PNHDF 225
4.7 INFLUENCE OF EPICATECHIN ON THE PHYSIOLOGY OF HACAT AND
DIFFERENTIATION OF
PNHEK 226
4.8 INFLUENCE OF PROCYANIDIN B2 ON THE PHYSIOLOGY OF HACAT AND
DIFFERENTIATION OF
PNHEK 227
4.9 INFLUENCE OF PROCYANIDIN B5 ON THE PHYSIOLOGY OF HACAT AND
DIFFERENTIATION OF
PNHEK 229
4.10 INFLUENCE OF PROCYANIDIN CI ON THE PHYSIOLOGY OF HACAT AND
DIFFERENTIATION OF
PNHEK 229
4.11 INFLUENCE OF PROCYANIDIN D1 ON THE PHYSIOLOGY OF HACAT AND
DIFFERENTIATION OF
PNHEK 231
4.12 INFLUENCE OF EQUIMOLAR CONCENTRATIONS OF PROCYANIDINS ON PNHDF 232
4.13 POTENTIAL USES OF AQUEOUS EXTRACT OF C. MUCRONATUM AND PROCYANIDIN
B2 233
TABLE OF CONTENTS
4.14 QUALITY CONTROL OF EPICATECHIN, PROCYANIDIN B2, VITEXIN AND
ISOVITEXIN VITEXIN IN C.
MUCRONATUM SAMPLES 234
4.15 CONCLUSION 237
5 REFERENCES 257
CURRICULUM VITAE 280
PUBLICATIONS 282
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| spelling | Kisseih, Emelia Verfasser aut Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) vorgelegt von Emelia Kisseih 2014 VI, 282 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Münster (Westfalen), Univ., Diss., 2014 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027625957&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Kisseih, Emelia Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) |
| subject_GND | (DE-588)4113937-9 |
| title | Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) |
| title_auth | Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) |
| title_exact_search | Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) |
| title_full | Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) vorgelegt von Emelia Kisseih |
| title_fullStr | Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) vorgelegt von Emelia Kisseih |
| title_full_unstemmed | Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) vorgelegt von Emelia Kisseih |
| title_short | Phytochemical characterization and in vitro wound healing activities of extracts and compounds from Combretum mucronatum (Schum. & Thonn.) |
| title_sort | phytochemical characterization and in vitro wound healing activities of extracts and compounds from combretum mucronatum schum thonn |
| topic_facet | Hochschulschrift |
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