ALPHA-AMYLASE INHIBITORY COMPOUNDS FROM LANNEA SCHWEINFURTHII STEM BARK AND THEIR MODES OF INHIBITION

ABSTRACT

Management of hyperglycemia is an essential strategy in control of diabetes mellitus which remains a major health problem despite several therapeutic interventions particularly inhibitors of a-amylase. Use of ethnobotanical options has prominently been reported, for instance, Lannea schweinfurthii reported for management of hyperglycemia, although there is no scientific information to validate its anti-hyperglycemic potential. The study was to determine structures of compounds from Lannea schweinfurthii stem bark which had in-vitroa-amylase inhibitory activities and their modes of inhibition. Lannea schweinfurthii stem bark was collected from Kisumu County, Kisumu Karateng (34°45’0’’, 0°6’0’’), air dried under shade, ground into fine powder and sequentially extracted using n-hexane, dichloromethane, ethyl acetate and methanol. Crude n-hexane/DCM, EtOAc and MeOH extracts were subjected to in-vitroα-amylase inhibition assay where high inhibitory activities (p>0.05) were observed for EtOAc extract (IC50 = 0.578 mg/mL) and MeOH extract (IC50 = 0.497 mg/mL) while low inhibitory activity (P0.05) for n-hexane/DCM extract (IC50 = 1.024 mg/mL) relative to metformin (IC50 = 0.468 mg/mL) on α-amylase. Crude extracts were subjected to chromatographic separation techniques and purification on TLC, CC over silica gel and sephadex LH-20, that led to identification of  two alkenyl cyclohexenone derivatives; (4R,6S)-4,6-dihydroxy-6-((Z)-nonadec-14’-en-1-yl)cyclohex-2-en-1-one (28),(2S,4R,5S)-2,4,5-trihydroxy-2-((Z)-nonadec-14’-en-1-yl)cyclohexan-1-one (29), two prenylated flavonoids; 5-hydroxy-7,8-(2’’,2’’-dimethylchromene)-flavanone (31), 5-methoxy-7,8-(2’’,2’’-dimethylchromene)-flavanone (32) and two sterols; stigmasterol (30), 3-O-[β-glucopyranosyl-(1’’→2’)-O-β-xylopyranosyl]-β-stigmasterol (33). α-Amylase inhibition assay of isolated compounds showed high inhibitory activities (IC50 = 0.665 mM and 0.580 mM) for compounds 31 and 33,respectively, relative to (p>0.05) metformin (IC50 = 0.468 mM) while 32 showed moderate inhibitory activity (IC50 = 0.826 mM) and 28 showed (IC50 = 1.245 mM) low inhibitory activity (P0.05). Kinetic analysis through Lineweaver-Burk plots depicted compounds 31 and 32 as competitive inhibitors based on the inhibition constants of Ki = 24.29 mM and Ki= 37.9 mM, respectively, while 33 depicted both Ki = 1.186 mM and Ki' = 4.184 mMindicative of mixed inhibition. Compounds 31 and 32 bound to active sites of free enzyme while 33 bound on both free enzyme and enzyme-substrate complex though small Ki = 1.186 mM indicated stronger binding to free enzyme and most active. High inhibitory activity of 33 was attributed to hydroxyl groups that could have bound to α-amylase active sites. Compounds 31, 32 and 33 suppressed carbohydrates hydrolysis through modulating α-amylase activity could reduce post-prandial hyperglycemia. Potential of 31, 32 and 33 to inhibit α-amylase supports usage of Lannea schweinfurthii stem bark in management of post-prandial hyperglycemia.

TABLE OF CONTENTS
Title…………………………………………………………………………………………....................................................................................i
Declaration ..................................................................................................................................................................................... ii
Acknowledgement ..................................................................................................................................................................... iii
Dedication ..................................................................................................................................................................................... iv
Abstract ........................................................................................................................................................................................... v
Table of contents ....................................................................................................................................................................... vi
List of abbreviations and acronyms .................................................................................................................................... x
List of tables ............................................................................................................................................................................... xii
List of figures ............................................................................................................................................................................ xiii
List of appendices ................................................................................................................................................................... xiv
CHAPTER ONE: INTRODUCTION ......................................................................................................................................... 1
1.1 Background of the study ................................................................................................................................................. 1
1.2 Statement of the problem .............................................................................................................................................. 5
1.3 Objectives ............................................................................................................................................................................. 6
1.3.1 General objectives ......................................................................................................................................................... 6
1.3.2 Specific objectives ......................................................................................................................................................... 6
1.4 Hypothesis ........................................................................................................................................................................... 6
1.4.1 Null Hypothesis .............................................................................................................................................................. 6
1.5 Justification .......................................................................................................................................................................... 7
CHAPTER TWO: LITERATURE REVIEW .............................................................................................................................. 8

2.1 Epidemiological aspects of diabetes mellitus......................................................................................................... 8
2.2 Ethnobotanical management of diabetes mellitus ........................................................................................... 10
2.3 Common natural inhibitors of human intestinal glucosidases ..................................................................... 12
2.4 Modes of enzymatic inhibition for human intestinal glucosidases ............................................................ 14
2.5 Ethnomedicinal uses of Lannea species ................................................................................................................ 18
2.6 Phytochemistry of Lannea species ........................................................................................................................... 22
CHAPTER THREE: MATERIALS AND METHODS .......................................................................................................... 25
3.1 General experimental procedures, instrumentation, solvents and fine consumables ......................... 25
3.2 Collection of plant material ........................................................................................................................................ 26
3.3 Extraction of plant material ........................................................................................................................................ 26
3.4 Isolation of compounds from Lannea schweinfurthii stem bark ................................................................. 26
3.4.1 Fractionation of n-hexane/dichloromethane extract ................................................................................... 27
3.4.2 Fractionation of ethyl acetate extract ................................................................................................................ 28
3.4.3 Fractionation of methanol extract ....................................................................................................................... 29
3.5 Physical and spectroscopic data of isolated compounds .............................................................................. 29
3.5.1 Compound 28 .............................................................................................................................................................. 30
3.5.2 Compound 29 .............................................................................................................................................................. 30
3.5.3 Compound 30 .............................................................................................................................................................. 31
3.5.4 Compound 31 .............................................................................................................................................................. 31
3.5.5 Compound 32 .............................................................................................................................................................. 31
3.5.6 Compound 33 .............................................................................................................................................................. 32
3.6 In-vitro inhibition and kinetic analysis of modes of α-amylase inhibition .............................................. 33
3.6.1 Alpha-Amylase inhibition assay ............................................................................................................................ 33
3.6.2 Modes of α-amylase inhibition ............................................................................................................................. 33
3.7 Statistical analysis .......................................................................................................................................................... 35
CHAPTER FOUR: RESULTS AND DISCUSSION ........................................................................................................... 36
4.1 Crude extract yields ...................................................................................................................................................... 36
4.2 Bioassay results of crude extracts ........................................................................................................................... 36
4.2.1 In-vitro α-amylase inhibition IC50 by the crude extracts .......................................................................... 36
4.3 Structure elucidations ................................................................................................................................................. 37
4.3.1 (4R,6S)-4,6-dihydroxy-6-((Z)-nonadec-14’-en-1-yl)cyclohex-2-en-1-one (28) ................................ 37
4.3.2 (2S,4R,5S)-2,4,5-trihydroxy-2-((Z)-nonadec-14’-en-1-yl)cyclohexan-1-one (29) ............................ 40
4.3.3 Stigmasterol (30) ...................................................................................................................................................... 43
4.3.4 5-hydroxy-7,8-(2’’,2’’-dimethylchromene)-flavanone (31) ...................................................................... 46
4.3.5 5-methoxy-7,8-(2’’,2’’-dimethylchromene)-flavanone (32) .................................................................... 48
4.3.6 3-O-[β-Glucopyranosyl-(1’’→2’)-O-β-xylopyranosyl]-β-stigmasterol (33) ....................................... 51
4.4 Bioassay of isolated compounds ......................................................................................................................... 55
4.4.1 In-vitro α-amylase inhibition IC50 by the compounds ........................................................................... 55
4.4.2 In-vitro modes of α-amylase inhibition by the active compounds .................................................... 56
CHAPTER FIVE: SUMMARY, CONCLUSION AND RECOMMENDATIONS .................................................... 60
5.1 Summary ....................................................................................................................................................................... 60
5.2 Conclusion .................................................................................................................................................................... 61
5.3 Recommendations .................................................................................................................................................... 62
5.4 Significance of the study ........................................................................................................................................ 63
5.5 Limitations of the study........................................................................................................................................... 63
5.6 Suggestions for further studies ........................................................................................................................... 63
REFERENCES ....................................................................................................................................................................... 64
APPENDICES ....................................................................................................................................................................... 81