University of Khartoum Graduate College Medical and Health Studies Board Activity
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University of Khartoum Graduate College Medical and Health Studies Board Activity- guided Isolation and Structure Determination of Antioxidant and Antidiabetic Compounds from Bauhinia rufescence L. By: Wadah Jamal Ahmed Osman B. Pharm. U of K. (2003) M. Pharm. King Saud University (2012) A thesis submitted in fulfillment of the requirement for the degree of PhD in Pharmacognosy Supervisor Prof.Abdelkhaleig Muddathir, (B.Pharm.M.pharm., PhD) Professor of Pharmacognosy, U.of K. 2014 I Co-Supervisor: Prof. Dr. Hassan Elsubki Khalid B.Pharm., PhD Professor of Pharmacognosy, U.of K. II DEDICATION First of all I thank Almighty Alla for his mercy and wide guidance on a completion of my study. This thesis is dedicated to my parents, who taught me the value of education, to my beloved wife and to my beautiful kids. I express my warmest gratitude to my supervisor Professor Dr Prof. Abdelkhaleig Muddathir and Prof. Dr. Hassan Elsubki for their support, valuable advice, excellent supervision and accurate and abundant comments on the manuscripts taught me a great deal of scientific thinking and writing. In addition, I would like to express my appreciation to all members of the Pharmacognosy Department for their encouragement, support and help throughout this study. Great thanks for Professor Kamal Eldeen El Tahir (King Saud University, Riyadh) and Prof. Sayeed Ahmed (Jamia Hamdard University, India) for their co-operation and scientific support during the laboratory work. Wadah jamal Ahmed July, 2018 III Contents 1. Introduction and Literature review 1.1.Oxidative Stress and Reactive Metabolites 1 1.2. Production Of reactive metabolites 1 1.3. Biological roles of Free radicals. 2 1.4.Antioxidants 3 1.5.Diabetes: 3 1.6. Complications of Diabetes mellitus 4 1.7. Available therapy for Diabetes mellitus 4 1.8. Oxidative Stress and diabetes 5 1.9.Oxidative stress and diabetic complications 5 1.10. Medicinal plants 6 1.11. Medicinal plants, antioxidants and 7 diabetics complications 1.12. Medicinal plants with ant diabetic activity 8 1.13. Phytochemicals and Management of 15 Diabetes mellitus. 1.14. Flavonoids 15 1.15. Alkaloids 21 1.16. Terpenoids and Steroids 26 1.17. Glycosides 33 2.1.Family Fabaceae 36 2.2 Bauhinia rufescens 36 2.3.Scientific classification: 36 2.4.Description 36 2.5.Medicinal uses of B. rufescens: 37 2.6.Local uses of B. rufescens: 38 2.7.Biological Activities of the Genus Bauhinia 38 2.8. Anti-diabetic Properties 38 2.9.Antimicrobial studies 39 IV 2.10.Analgesic Potential Studies 41 2.11.Anti-inflammatory activity 41 2.12.Pharmacological and Toxicological 43 Investigations of Genus Bauhinia 3. Materials and Methods 3.1. Plant material 47 3.2.Extraction procedure 47 3.3.In vivo anti diabetic assay of plant extracts 47 3.4. In vitro anti diabetic assay of most active 48 fraction using TLC bio autographic method for the detection Amylase enzyme inhibitors. 3.5.Antioxidant assay 49 3.6. Quantitative antioxidant assay using DPPH 49 radical scavenging assay 3.7. Qualitative antioxidant assay using 49 Bioautography method 3.8.LC-MS analysis of the active fraction 50 3.9.High performance thin layer chromatography 51 (HPTLC) analysis of active fraction 3.10. Isolation of some active compounds and 52 structures determination using Nuclear Magnetic Resonance (NMR). 3.11.In silico assessment of the compounds from 53 active fraction 4.Results and Discussion 4.1. Anti diabetic assay of plant extracts. 55 4.2.Quantitative antioxidant assay using DPPH 56 radical scavenging assay of plant extracts 4.3.Fractionation of active methanol extract 56 4.4. Quantitative antioxidant assay using DPPH 57 radical scavenging assay of methanolic fractions (ethyacetate, n-butanol, chlorororm and aqueous fractions). 4.5. In vitro anti diabetic assay and qualitative 58 antioxidant evaluation of most active fraction (ethyl acetate fraction). V 4.5. Isolation and structure determination of 60 compound (1) and compound (2) from ethyl acetate fraction. 5-1 4.6.1.isolation of the active constituents 60 4.6.2 Structure elucidation of compound (1) 60 4.6.2 Structure elucidation of compound (2) 66 7 4.7.Identification of Phytoconstituents from 69 ethyl acetate fraction by LC-MS 8. 4.8.In silico assessment of the identified 77 compounds as inhibitors for α-amylase and α- glucosidase enzyme. 9. 4.9.Standardization and High performance thin 91 layer chromatography (HPTLC) fingerprint analysis References 96 List of Figures Fig. (1): Some classes of flavonoids. 16 Fig. (2) Flavonoids for diabetes management 20 Fig. (3): Glycosides for diabetes management 35 Fig. (4): Bioautographic assay showing the inhibition of α-amylase by some 59 compounds developed on a silica gel G60 F254 plate Fig. (5): Bioautographic method showing the antioxidant compounds (yellow 59 color spots) after developing on a silica gel Fig. (6): 1H-NMR-spectrum of compound -1 (Rutin), (700 MHz, DMSO). 63 Fig. (7): 13C-NMR-spectrum of compound -2 (Rutin), (700 Hz, DMSO). 64 Fig. (8): HMBC-spectrum of compound -1, showed some two and three bond 65 correlations, (700 MHz, DMSO). Fig. (9): HSQC-spectrum of compound -1 (Rutin), showed direct proton-carbon 69 correlation (700 MHz, DMSO). Fig. (10): 1H-NMR-spectrum of compound -2 (Kaempferol -3-O-rutinoside), 70 (700 MHz, DMSO). VI Fig. (11): 13C-NMR-spectrum of compound -2 (Kaempferol -3-O-rutinoside), 76 (700 Hz, DMSO). Fig. (12): Total MS chart represent molecular ion peaks of identified compounds from 77 active fraction analyzed by LC/MS/MS. Fig. (13): HPLC peaks of compounds identified by LC/MS/MS. 82 Fig. (14): In silico assessment of identified compounds against human α- 86 glucosidase enzyme Fig. (15): In silico assessment of identified compounds against human α-amylase 92 enzyme Fig. (16): HPLC analysis of ethyl acetate fraction of Bauhinia rufescense 92 Fig. (17): 3D graph of HPTLC chromatogram 93 Fig. (18 TLC chromatogram of ethyl acetate fraction as shown by TLC scanner 94 (254 and 366 nm), List of Tables Table 1: : List of plants having insulin mimetic or insulin secreatory activity 9 Table 2: List of important alkaloids used in the treatment of Diabetes mellitus 21 Table 3: List of important Terpenoids and Steroids used in the treatment of Diabetes 26 mellitus Table 4: Invivo- Anti diabetic assay of methanol, chloroform and pet.Ether extracts 55 of Bauhinia rufescens . Table 5: The results of antioxidant Activity of methanol ,chloroform and pet.ether 56 extracts Table 6: The results of antioxidant Activity of different methanolic fractions 57 Table 7: 1H- and 13C-NMR assignments of compound -2 (700 MHz, DMSO) in 62 comparison with reported data for Rutin (Quercetin-3-Orutinoside) (100 MHz, CD3OD) Table 8: 1H- and 13C-NMR assignments of compound -1 (500 MHz, DMSO) in 67 comparison with reported data for Kaempferol -3-O-rutinoside (100 MHz, CD3OD) Table 9: Identified compounds using LC-MS/MS 62 Table 10: : In silico assessment of identified compounds against human α- 78 glucosidase and α-amylase enzymes Table 11: HPTLC profile at 366 (nm) of ethyl acetate fraction 92 VII List of Abbreviations δ Chemical shift value 13C NMR Carbon 13-nuclear magnetic resonance 1HNMR proton nuclear magnetic resonance DEPT Distortionless Enhancment by Polarization Transfer Hz Hertz MS Mass spectrum [M]+ Molecular ion peak m/z Mass/ charge ratiomp nm Nanometer Rf Relative to solvent front TLC Thin layer chromatography ROS Reactive oxygen species HPTLC High performance Thin layer chromatography HMQC Heteronuclear Multiple Quantum Correlation HMBC Heteronuclear Multiple Bond Correlation PTLC Preparative thin layer chromatography VIII A b s t r a c t Background: Diabetes mellitus is a group of disorders of multiple etiologies resulting from a defect in insulin secretion, insulin action, or both. Diabetes remain one of the world’s major health problems and many repots have been supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes. This study aimed to evaluate the antidiabetic and antioxidant activity of Bauhinia rufescens a plant used in Sudanese folkloric medicine for treatment of diabetes. It was also aimed to identify isolates and characterize the bioactive antidiabetic and antioxidant compounds using bioactivity guided fractionation followed by High Performance Thin Layer Chromatography (HPTLC) autobiography, Liquid-Mass spectroscopy analysis and nuclear magnetic resonance (NMR). Method: The powdered plant leaves were exclusively extracted using petroleum ether, chloroform and methanol, successively. These extracts were tested in streptozotocin-induced diabetic Wistar rats for antidiabetic as well as DPPH antioxidant potential. Standard Propyl Gallate was used as (+ve) control and the difference in results was considered significant when P < 0.05. The best active extract was fractionated with chloroform, ethyl acetate and butanol along with the remaining aqueous fraction. The fractions are tested for antioxidant and antidiabetic activity using HPTLC autobiography (α amylase and α glucosidase). Further, bioactive compounds were isolated by Preparative Thin Layer Chromatography and identified using LC/MS and NMR spectroscopy. IX Results: The methanol extract significantly decreased the blood glucose level in normal fasted rats by 27 %; two hours following administration and decreased the blood glucose level in streptozotocin-treated rats by 48.9%; two hours following oral administration, while chloroform and pet. Ether extracts not significantly decreases the glucose level.Significantly the methanol extract also showed strong antioxidant activity (89%) compared with other extracts; thereforeit was further subjected for fractionation by using chloroform, ethyl acetate, n-butanol and water sequentially, and then tested for their antioxidant activity; where the ethyl acetate fraction was showed good activity compared with other fractions. Bioactivity guided fractionation of Bauhinia rufescensled to isolation and characterization of two compounds with potent antidiabetic and antioxidant activity namely; Quercetin-3-O- rutinoside and Kaempferol -3-O-rutinoside.