University of Ghana http://ugspace.ug.edu.gh UNIVERSITY OF GHANA COLLEGE OF HEALTH SCIENCES EFFECT OF SWEDISH BITTERS ON SELECTED RAT CYTOCHROME P450 ENZYME ACTIVITY AND HEPATIC ANTIOXIDANT LEVELS BY ABIGAIL ANING (10220960) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL IN PHARMACOLOGY DEGREE DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY JULY, 2019 i University of Ghana http://ugspace.ug.edu.gh DECLARATION I, Abigail Aning, hereby declare that this project, aside other cited works, is the result of research carried out by me under the supervision of Prof. Regina Appiah-Opong and Dr. Seth Kwabena Amponsah. This work has not been submitted in part or whole elsewhere for the award of any degree. 01-05-2020 …………………. ……………………. Abigail Aning Date (Student) 04-05-2020 …………………. ..……………………. Prof. Regina Appiah-Opong Date (Supervisor) 01-05-2020 …………………. ……………………….. Dr. Seth Kwabena Amponsah Date (Co-Supervisor) ii University of Ghana http://ugspace.ug.edu.gh ABSTRACT Background: The use of herbal products has gained popularity especially in developing countries. A number of patients are known to take these herbal products concurrently with conventional drugs. Concomitant use of herbal preparations and conventional drugs may result in herb-drug interactions, often via modulation of drug metabolizing enzymes, in particular, Cytochrome P450 (CYP) enzymes. Swedish bitters is one of such herbal preparations on the market which may be concomitantly administered with other conventional drugs because of its use as a digestif. Aims: The aims of the current study were to determine the effect of Swedish bitters on the activities of rat liver microsomal CYP1A2, CYP3A4, CYP2B6, CYP2C9 and CYP2D6 enzymes, and also its effect on hepatic antioxidant levels. The effect of Swedish bitters on rat hematological and biochemical parameters was also assessed. Methods: Male Sprague-Dawley rats 6-8 weeks old were put into 5 groups (5 rats/group). The groups consisted of a positive control (15 mg/kg/day of phenobarbital), a negative control (distilled water), and low (5 mL/kg/day), medium (10 mL/kg/day) and high (20 mL/kg/day) doses of Swedish bitters, respectively. After a 7-day administration period of the aforementioned treatments, the rats were euthanized and blood obtained by cardiac puncture. The livers were isolated, immediately placed on ice and stored at -80oC until use. Microsomal preparations were obtained from harvested livers by homogenization and differential centrifugation. The substrates of each specific CYP was added to microsomal preparations in phosphate buffer (pH 7.4) at 37oC and their metabolites measured using spectrophotometric and chromatographic assays. Effect of Swedish bitters on enzyme activity was determined based on metabolite levels. Antioxidant levels/reactions such as iii University of Ghana http://ugspace.ug.edu.gh Glutathione (GSH), Lipid peroxidation (LPO), Superoxide Dismutase (SOD) and Catalase (CAT) were evaluated in liver cytosol using standard methods. Additionally, hematological and biochemical parameters in the various groups from blood collected were determined using automated analyzers. Results: Results showed that Swedish bitters increased the activities of CYP2B1/2B2, CYP3A4, CYP2C9 and CYP2D6; with CYP2C9 significantly increased (p < 0.01). The activity of CYP1A1/1A2 did not differ significantly compared to the non-treated groups. There was a marginal increase in CYP1A2 activity in treatment groups, however, this was not statistically significant. Furthermore, with antioxidant levels/reactions assayed in liver cytosol, there was a dose dependent decrease in GSH levels and increase in SOD activity, however, both were not statistically significant. Catalase activity was found to have decreased dose-dependently, with the rats that received high dose of Swedish bitters showing a significant decrease compared to the untreated group. Lipid peroxidation did not alter significantly in treated groups compared to the untreated group. For hematological and biochemical parameters, monocytes and alkaline phosphatase (ALP) levels decreased significantly in the rats that received high doses of Swedish bitters. Conclusion: Swedish bitters increased the activity of rat CYP2C9 significantly. Swedish bitters also altered the levels of catalase enzyme activity and reduced ALP levels, significantly. Findings suggest that Swedish bitters may interact with other drugs that are metabolized by these CYPs especially when taken over longer periods. iv University of Ghana http://ugspace.ug.edu.gh DEDICATION This work is dedicated to my family, especially my mother, for their encouragement and support throughout this period of study. v University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT I am grateful to the Almighty God for His mercies, divine favor, guidance and protection during the entire period of this study. I wish to express profound thanks to my supervisors, Prof. (Mrs.) Regina Appiah-Opong and Dr. Seth Kwabena Amponsah, whose laboratory resources helped lessen the financial burden on me. Also, their constant academic guidance and motivation led to successful completion of this work. I also appreciate their insightful reviews that enhanced the quality of this study. God richly bless you all. A special appreciation goes to my lecturers and the entire staff of the Department of Pharmacology and Toxicology, School of Pharmacy, University of Ghana. Special thanks go to Mr. Believe Ahedor and staff of the Department of Animal Experimentation, Noguchi Memorial Institute for Medical Research (NMIMR), for their invaluable technical assistance. I also appreciate all staff of the Clinical Pathology Department, NMIMR, for their unwavering support. Special acknowledgement goes to all my friends especially, Mr. Elvis Nelson Adam, Mr. Isaac Tuffour, Miss. Eunice Dotse, Miss Trudy Philips, Miss Benessa Acquah, Mr. Ebenezer Ofori- Attah, Mrs. Eunice Ampem Danso and Mr. Martin Akandawen for their prayers and diverse support. vi University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS DECLARATION ................................................................................................................ ii ABSTRACT ....................................................................................................................... iii DEDICATION .................................................................................................................... v ACKNOWLEDGEMENT ................................................................................................. vi TABLE OF CONTENTS .................................................................................................. vii LIST OF FIGURES .......................................................................................................... xii LIST OF TABLES ........................................................................................................... xiii LIST OF ABBREVIATIONS .......................................................................................... xiv CHAPTER ONE ................................................................................................................. 1 1.0 INTRODUCTION ........................................................................................................ 1 1.1 BACKGROUND .......................................................................................................... 1 1.2 JUSTIFICATION ......................................................................................................... 3 1.3 HYPOTHESIS .............................................................................................................. 5 1.4 AIM ............................................................................................................................... 5 1.5 SPECIFIC OBJECTIVES ............................................................................................. 5 CHAPTER TWO ................................................................................................................ 6 2.0 LITERATURE REVIEW ............................................................................................. 6 2.1 COMPLEMENTARY AND ALTERNATIVE MEDICINE........................................ 6 2.1.1 Biology-based practices ............................................................................................. 8 vii University of Ghana http://ugspace.ug.edu.gh 2.1.2 Herbs and herbal products ......................................................................................... 9 2.2 HERBAL BITTERS ................................................................................................... 11 2.2.1 Swedish Bitters ........................................................................................................ 12 2.3 XENOBIOTIC METABOLIZING ENZYMES ......................................................... 18 2.3.1 Drug Interactions ..................................................................................................... 22 2.4 HERBS/HERBAL PRODUCT AND LIVER HEALTH ........................................... 25 2.5 REVIEW OF METHODS........................................................................................... 29 2.5.1 Protein Determination .............................................................................................. 29 2.5.2 CYP Enzyme Activity.............................................................................................. 31 2.5.3 GSH determination .................................................................................................. 32 2.5.4 SOD Activity ........................................................................................................... 33 2.5.5 Catalase Activity ...................................................................................................... 34 2.5.6 Lipid Peroxidation ................................................................................................... 34 CHAPTER THREE .......................................................................................................... 35 3.0 MATERIALS AND METHODS ................................................................................ 35 3.1 STUDY DESIGN........................................................................................................ 35 3.1.1 Inclusion/exclusion criteria ...................................................................................... 35 3.2 ETHICAL CONSIDERATIONS ................................................................................ 35 3.3 CHEMICALS AND REAGENTS .............................................................................. 35 3.4 FINGERPRINTING OF SWEDISH BITTERS USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)....................................................................... 36 viii University of Ghana http://ugspace.ug.edu.gh 3.5 EXPERIMENTAL ANIMALS ................................................................................... 37 3.5.1 Rat grouping and treatment administration .............................................................. 37 3.6 PREPARATION OF MICROSOMAL FRACTIONS................................................ 38 3.6.1 Protein Determination ............................................................................................. 38 3.7 CYP ENZYME ASSAYS ........................................................................................... 39 3.7.1 Methoxy- Ethoxy- Benzyloxy- and Pentoxy-resorufin O-dealkylation (MROD, EROD, BROD and PROD) ............................................................................................... 39 3.7.2 Diclofenac Hydroxylation ........................................................................................ 40 3.7.3 Dextromethorphan O-demethylation ....................................................................... 40 3.8 BIOCHEMICAL AND HEMATOLOGICAL ANALYSIS OF BLOOD COLLECTED FROM SD RATS .............................................................................................................. 41 3.9 ANTIOXIDANT ASSAYS OF CYTOSOL FROM HOMOGENIZED LIVERS OF SD RATS ................................................................................................................................ 42 3.9.1 GSH determination .................................................................................................. 42 3.9.2 SOD Activity ........................................................................................................... 42 3.9.3 Catalase Activity ...................................................................................................... 43 3.9.4 Lipid Peroxidation ................................................................................................... 43 3.10 STATISTICAL ANALYSIS .................................................................................... 44 CHAPTER FOUR ............................................................................................................. 45 4.0 RESULTS ................................................................................................................... 45 4.1 CHEMICAL FINGERPRINTING.............................................................................. 45 ix University of Ghana http://ugspace.ug.edu.gh 4.2 PROTEIN CONTENT OF MICROSOMES .............................................................. 46 4.3 CYP ENZYME ASSAYS ........................................................................................... 47 4.3.1 CYP1A1/1A2 Activity ............................................................................................. 47 4.3.2 CYP1A2 Activity ..................................................................................................... 48 4.3.3 CYP2B1/2B2 Activity ............................................................................................. 48 4.3.4 CYP3A4 Activity ..................................................................................................... 49 4.3.5 CYP2C9 Activity ..................................................................................................... 50 4.3.6 CYP2D6 Activity ..................................................................................................... 51 4.3.7 Overall Effect of Swedish bitters on Rat CYP Enzyme Activity ............................ 52 4.4 BIOCHEMICAL AND HEMATOLOGICAL PARAMETERS ................................ 53 4.5 ANTIOXIDANT ASSAYS OF CYTOSOL FROM HOMOGENIZED LIVERS OF SD RATS ................................................................................................................................ 56 4.5.1 GSH content ............................................................................................................. 56 4.5.2 SOD Activity ........................................................................................................... 56 4.5.3 Catalase Activity ...................................................................................................... 57 4.5.4 Lipid Peroxidation ................................................................................................... 58 CHAPTER FIVE .............................................................................................................. 60 5.0 DISCUSSION ............................................................................................................. 60 CHAPTER SIX ................................................................................................................. 67 6.0 CONCLUSION, LIMITATIONS AND RECOMMENDATION .............................. 67 REFERENCES ................................................................................................................. 68 x University of Ghana http://ugspace.ug.edu.gh APPENDICES .................................................................................................................. 86 APPENDIX I .................................................................................................................... 86 APPENDIX II ................................................................................................................... 87 APPENDIX III .................................................................................................................. 88 APPENDIX IV.................................................................................................................. 89 APPENDIX V ................................................................................................................... 89 APPENDIX VI.................................................................................................................. 90 xi University of Ghana http://ugspace.ug.edu.gh LIST OF FIGURES Figure 2.1. Types of Complementary and Alternative Medicine ...................................... 8 Figure 2.2. Swedish Bitters by NatureWorks .................................................................. 18 Figure 2.3. Drug transporters and metabolizing enzymes that function in the liver, intestine and kidneys. ...................................................................................................................... 20 Figure 2.4. Proportion of drugs metabolized by different CYPs ..................................... 22 Figure 2.5. Production of ROS by internal and external sources. .................................... 29 Figure 2.6. Reaction schematic for Bradford Protein Assay............................................ 30 Figure 2.7. Alkoxyresorufin O-dealkylation .................................................................... 31 Figure 2.8. Hydroxylation of diclofenac to 4’hydroxydiclofenac ................................... 32 Figure 2.9. Dextromethorphan O-demethylation ............................................................. 32 Figure 2.10. Chemical reaction between Glutathione and O-phtalaldehyde ................... 33 Figure 4.1. Chromatographic fingerprint of Swedish Bitters........................................... 45 Figure 4.2. Effect of Swedish bitters on CYP1A1/1A2 activity in rat liver microsomes 47 Figure 4.3. Effect of Swedish bitters on CYP1A2 activity in rat liver microsomes. ....... 48 Figure 4.4. Effect of Swedish bitters on CYP2B1/2B2 activity in rat liver microsomes. 49 Figure 4.5. Effect of Swedish bitters on CYP3A4 activity in rat liver microsomes. ....... 50 Figure 4.6. Effect of Swedish bitters on CYP2C9 activity in rat liver microsomes. ....... 51 Figure 4.7. Effect of Swedish bitters on CYP2D6 activity in rat liver microsomes. ....... 52 Figure 4.8. GSH levels in Swedish bitters treated rat groups compared with untreated. 56 Figure 4.9. SOD activity in Swedish bitters treated groups compared with untreated. ... 57 Figure 4.10. Catalase activity of Swedish bitters treatment groups compared with untreated. ........................................................................................................................... 58 Figure 4.11. Lipid peroxidation of Swedish bitters treatment groups compared with untreated ............................................................................................................................ 59 xii University of Ghana http://ugspace.ug.edu.gh LIST OF TABLES Table 3.1. Experimental conditions for fluorescence CYP enzyme assays ..................... 39 Table 4.1. Peak retention times and area .......................................................................... 46 Table 4.2. Summary of the effect of Swedish bitters on selected rat CYP enzyme activity ........................................................................................................................................... 52 Table 4.3. Effect of Swedish bitters on rat hematological parameters ............................. 54 Table 4.4. Effect of Swedish bitters on rat serum biochemical parameters ..................... 55 xiii University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS 4’HD 4’Hydroxydiclofenac A/G Albumin/Globulin ratio AED Animal Equivalent Dose ALB Albumin ALT Alanine aminotransferase AO Aldehyde oxidase AST Aspartate aminotransferase BASO Basophil BROD Benzyloxyresorufin O-dealkylation BSA Bovine Serum Albumin CAM Complementary and Alternative medicine CAT Catalase CBBG Coomassie Brilliant Blue G-250 COX-1 Cyclooxygenase-1 CYP Cytochrome P-450 DBil Direct Bilirubin DILI Drug-induced Liver Injury DN Diabetic nephropathy xiv University of Ghana http://ugspace.ug.edu.gh DPPH Diphenylpicrylhydrazyl DXM Dextromethorphan DXT Dextrorphan EDTA Ethylenediaminetetraacetic acid EOS Eosinophil EROD Ethoxyresorufin O-deethylation FDA Food and Drugs Administration FMO Flavin-containing monooxygenases FRAP Ferric reducing-antioxidant power GC Gas chromatography GGT Gamma glutamyl transferase GLB Globulin GPx Glutathione peroxidase GR Glutathione reductase GSH Reduced glutathione GST Glutathione S-transferase GTE Green Tea Extract HCT Hematocrit HGB Hemoglobin xv University of Ghana http://ugspace.ug.edu.gh HILI Herb-induced Liver Injury HL-60 Human Leukemia cell line HPLC High Performance Liquid Chromatography LPO Lipid Peroxidation LYM Lymphocytes MAO Monoamine oxidases MCH Mean Corpuscular Hemoglobin MCV Mean Corpuscular Volume MDA Malondialdehyde MON Monocytes MPV Mean Platelet Volume MROD Methoxyresorufin O-demethylation NADPH Reduced Nicotinamide Adenine Dinucleotide Phosphate NAT N-acetyl Transferase NBT Nitroblue tetrazolium NEU Neutrophils NMIMR Noguchi Memorial Institute for Medical Research NSAID Nonsteroidal Anti-inflammatory drug OPA O-phthalaldehyde xvi University of Ghana http://ugspace.ug.edu.gh OPT O-phthalaldehyde OVCAR-3 Human Ovarian cancer cell line P450 Cytochrome P-450 PCT Procalcitonin PDW Platelet Distribution Width P-gp P-glycoprotein PLT Platelets PROD Pentoxyresorufin O-dealkylation RBC Red Blood Cells RDW Red Cell Distribution Width RDW_SD RDW expressed as a standard deviation ROS Reactive oxygen species SD Sprague-Dawley SH Sulfhydryl SOD Superoxide Dismutase SULT Sulfotransferase T/CAM Traditional Complementary and Alternative Medicine TBA Thiobarbituric acid TBARS Thiobarbituric acid Reactive Substances xvii University of Ghana http://ugspace.ug.edu.gh TBil Total Bilirubin TLC Thin-Layer Chromatography TM Traditional Medicine TP Total Protein UDP Uridine 5'-diphospho UG-IACUC University of Ghana – Institutional Animal Care and Use Committee UGT Uridine 5'-diphospho (UDP)–glucuronosyl transferase WBC White Blood Cells WHO World Health Organization XME Xenobiotic Metabolic Enzymes XO Xanthine oxidase xviii University of Ghana http://ugspace.ug.edu.gh CHAPTER ONE 1.0 INTRODUCTION 1.1 BACKGROUND Herbal preparations have been used since time immemorial for the treatment of numerous diseases (Tugume and Nyakoojo, 2019). These herbs and herbal preparations often consist of different plants or plant parts. Currently, up-to-date scientific investigations are used to elucidate purported pharmacological activities of these herbal preparations. Additionally, some of the herbal preparations that are on the market now are in processed dosage forms (Olumese and Adegbolagun, 2015). Reports suggest that a large number of individuals mostly in Africa, China and India rely on herbal medicines for treatment of various ailments (Wachtel-Galor and Benzie, 2011, Bodeker and Kronenberg, 2002). Among the common herbal products on the market are herbal bitters. Herbal bitters are prepared using infusion or distillation methods (Awa and James, 2013). Aromatic herbs, barks, roots and/or fruits are some of the constituents used in the preparation of these bitters because of their flavor and medicinal potential (Awa and James, 2013). Some these constituents include; goldenseal rhizome, angelica root, artichoke leaf, gentian root, wormwood leaves, and bitter orange peel (Awa and James, 2013). These herbal bitters are used as digestive stimulants, antibacterial agents, and detoxifiers (Olumese and Adegbolagun, 2015). It has also been reported that these herbal bitters boost the body’s immune system (Olumese and Adegbolagun, 2015). It is noteworthy that, a number of herbal bitters were once sold as medicines but are now generally considered as digestifs: drinks consumed after meals to aid digestion (Awa and James, 2013). 1 University of Ghana http://ugspace.ug.edu.gh Swedish bitters is one of the many brands of herbal bitters currently available on the market. The bitters is a mixture of 11 herbs which include: theriaca venezian, angelica root, myrrh, saffron, rhubarb root, senna leaf, camphor, carline root manna, zedoary and aloe in fixed concentrations. Swedish bitters is known to promote biliary, pancreatic and gastric secretion; relieve bloating, flatulence and gastrointestinal cramps; improve liver function; and boost the immune system (Awa and James, 2013). Despite the health benefits of these herbs or herbal products, it has been found that some can modulate the activity of xenobiotic metabolizing enzymes, especially the Cytochrome P450 (CYP) enzymes (Miller, 1998, Wang et al., 2001). CYP enzymes are a large group of heme-thiolated proteins primarily found in smooth endoplasmic reticulum of liver cells and epithelial cells of small intestines. Most of these enzymes can be found in the liver, however, the kidneys, lungs, intestines and the skin also contain amounts of these isozymes (Ogu and Maxa, 2000). CYP enzymes are essential to the synthesis of many endogenous molecules including cholesterol, thromboxane A2, prostacyclins, and steroids (Lynch and Price, 2007). CYP enzymes are also important in the biotransformation of xenobiotics. Biotransformation makes xenobiotics water-soluble and easily excreted by the kidney. Although there are numerous isoforms of CYP enzymes exist in humans, six of them; CYP2D6, CYP3A4, CYP1A2, CYP2C9, CYP2C19, and CYP3A5 are the most important as they metabolize about 90 percent of all drugs available on the market (Lynch and Price, 2007). Herb-drug interaction may occur when there is concomitant administration of herbal medicines and conventional drugs. The potential effect of this could be sub-therapeutic or adverse effects of the conventional drug. Clinical case reports of herb-drug interactions 2 University of Ghana http://ugspace.ug.edu.gh involving top-selling herbs or herbal products such as Panax ginseng (Ginseng), Ginkgo biloba (Ginkgo), and Hypericum perforatum (St. John’s wort) have been reported in the United States of America (Iwata et al., 2004). Co-administration of St. John’s wort is known to decrease blood levels of cyclosporine, warfarin, digoxin, and many other drugs metabolized by CYP3A4 (Iwata et al., 2004, Hu et al., 2012). To avoid possible interactions between herbs and conventional drugs, it is necessary identify modulatory potential of commonly used herbs or herbal products (Hu et al., 2012). In addition, herbs and herbal products can be beneficial or detrimental to liver function (Guan and He, 2015), hence the need to monitor liver function in the presence of herbs. Furthermore, there is the tendency for herb-drug interactions to occur because a known majority use herbs or herbal products for various medical conditions (Wachtel-Galor and Benzie, 2011). There is, however, a paucity of scientific data on herb-drug interactions, and possible impact of these herbal preparations on hepatic and hematological parameters in many of these settings, i.e. developing countries. 1.2 JUSTIFICATION The use of conventional and herbal formulations is a common practice particularly in patients with several disease conditions (HemaIswarya and Doble, 2006, Nadler et al., 2004). Many patients take herbal medicines together with conventional drugs because they believe that the combination has synergistic effect. However, herbal products can alter the absorption and/or elimination of concomitantly administered conventional drugs (Arhewoh et al., 2017). These herbal products often induce/inhibit drug metabolizing enzymes, the commonest being the Cytochrome P450 (CYP) enzymes (Appiah-Opong et al., 2008). Herb-drug interactions involving CYPs have been identified as a potential risk factor for 3 University of Ghana http://ugspace.ug.edu.gh adverse drug effects and ultimately, therapeutic failure (Federico and Mario, 2001). For example, dihydropyridine-type of calcium-channel blockers levels in circulation are known to be elevated when there is concomitant administration of these dihydropyridine-type of calcium-channel blockers with grapefruit juice (Alabi et al., 2013). Swedish bitters is used by many, both healthy and sick, mostly after meals to aid digestion. It is a blend of 11 herbs: saffron, camphor, senna leaf, theriaca venezian, rhubarb root, zedoary, manna, angelica root, carline root, aloe, and myrrh. Some constituents of Swedish bitters have been found to have modulatory effect on drug metabolizing enzymes. Aloe vera has been reported to induce CYP reductase and some Phase II biotransformation enzymes (Singh et al., 2000). The bioactive compounds crocin and safranal found in saffron have shown CYP modulatory activity (Dovrtelova et al., 2015). Crocin decreased the activity of CYP2B, CYP2A, CYP2C11 and CYP3A enzymes, whereas safranal increased the activities of CYP2C11, CYP3A and CYP2B enzymes (Dovrtelova et al., 2015). Rhubarb, another constituent of Swedish bitters, has shown inhibitory effect on CYP2C6 and CYP3A4 (Gao et al., 2013, Iwata et al., 2004). Furthermore, some species of Angelica have been reported to cause induction/inhibition activity against CYP2C, CYP3A, CYP2D1 and CYP2D6 (Yoo et al., 2007, Ishihara et al., 2000, Tang et al., 2006). Some bitters affect the levels of lipid profile measurements such as cholesterol and triglycerides and also, antioxidant parameters such as lipid peroxidation and catalase activities (Alabi et al., 2013, Anyasor et al., 2017). In addition, bitters have been found to affect hematological and chemical parameters of blood (Ekor et al., 2010). There was elevation in alanine and aspartate aminotransferase activities, while decreasing total protein content. There is, however, a lack of scientific information on the potential net effect of all 4 University of Ghana http://ugspace.ug.edu.gh these extracts found in Swedish bitters on drug metabolizing enzymes. The consequence of modulation of hepatic drug metabolizing enzymes could be changes in the blood concentration of concurrently administered conventional drugs, which could lead to adverse or sub-therapeutic effect. Therefore, this study seeks to determine the effect of Swedish bitters on hepatic drug metabolizing enzymes and hepatic antioxidant levels. 1.3 HYPOTHESIS Swedish bitters could modulate the activity of cytochrome P450 metabolizing enzymes and alter hepatic antioxidant levels. 1.4 AIM To determine the effect of Swedish bitters on the activity of rat liver Cytochrome P450 isoforms, and potential impact on antioxidant levels. 1.5 SPECIFIC OBJECTIVES 1. To determine the inhibition or induction potential of Swedish bitters on rat CYP1A2, CYP3A4, CYP2B1/2, CYP2C9 and CYP2D6. 2. To determine the effect of Swedish bitters on lipid peroxidation (LPO), and the levels of selected hepatic antioxidants: superoxide dismutase (SOD), reduced glutathione (GSH) and catalase (CAT). 3. To perform fingerprinting of Swedish bitters using high performance liquid chromatography (HPLC). 5 University of Ghana http://ugspace.ug.edu.gh CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 COMPLEMENTARY AND ALTERNATIVE MEDICINE Traditional medicine (TM), also referred to as “ethno-medicine, folk medicine, native healing, or complementary and alternative medicine (CAM)”, is one of the oldest forms of health care (Abdullahi, 2011). Traditional medicine involves the use of health practices and beliefs that incorporate mineral and/or animal based medicines, plants, exercises, manual techniques and spiritual therapies (Zakaryan and Martin, 2012). These are often applied individually or together to diagnose, maintain good health, as well as to avoid or treat diseases (WHO, 2002). Societies have developed several indigenous ways of healing that fall within the category of TM, for example the traditional African, Chinese and Indian medicines (Abdullahi, 2011). There is well documented information on the use of these traditional/complementary and alternative medicines (T/CAM). As much as 70% of people tend to use herbs as complementary and/or alternative medicine (Fasinu et al., 2012). In particular, nearly half of people in developed countries frequently use some form of T/CAM (e.g. Australia, 48% and France, 49%), and a widespread use occurs in many developing countries including Colombia, 40%; China, 40%; Chile, 71%; and as much as 80% of people in Africa (Bodeker and Kronenberg, 2002). In Ghana, a percentage use of about 70% of the population has been recorded (WHO, 2001). Certain social, cultural, as well as socioeconomic factors, impact T/CAM use in industrialized societies. In developing countries, affordability, accessibility, and cultural familiarity play a role in the use of T/CAM. 6 University of Ghana http://ugspace.ug.edu.gh The awareness of and use of CAM are multifaceted phenomena that have grown exponentially in the past few years. This apparently insatiable need for alternative approaches to medical care by a number of people seems particularly interesting as it comes at a time of extraordinary therapeutic and technological improvements. A major reason for this phenomenon undoubtedly, is the colossal rise in public access to information through the internet and widespread news media coverage. Numerous advertisements and an unending exposure through the lay media, ranging from sensationalist newspapers to magazines, medical journals, and books, have strongly encouraged the notion of disease prevention and treatment by unconventional means, striking a delicate and highly profitable chord in a truly global populace (Neldner, 2000). Another reason for the popularity of CAM is the escalating cost of current allopathic medical care. The expense and the ensuing rationing of new modalities by managed care programs have placed them out of reach of a large proportion of the population (Neldner, 2000). The resultant effect is the creation of a strong public desire for many of these complementary and alternative therapies to prevent and treat human diseases (Neldner, 2000). It can be noted that Neldner classified CAM products into two main groups; herbal and non-herbal (Neldner, 2000). Herbal therapy as the name implies, involves the use of a variety of plant species. Non-herbal therapies on the other hand include methods such as homeopathy, relaxation techniques, aromatherapy, chiropractic, support groups, acupuncture, prayer, light therapy, massage and many others (Neldner, 2000). CAM has been further classified into five groups (Figure 1); manipulative body-based practices, mind–body interventions, whole medical systems, energy medicine, and biology-based practices (Subramani and Lakshmanaswamy, 2017). 7 University of Ghana http://ugspace.ug.edu.gh Figure 2.1. Types of Complementary and Alternative Medicine (Subramani and Lakshmanaswamy, 2017) 2.1.1 Biology-based practices The use of naturally occurring substances, such as herbs, vitamins, and foods to heal, maintain good health and homeostasis is considered a biologically-based practice (Bower, 2009, Romm, 2010). They are sometimes classified broadly as dietary supplements (Romm, 2010). Other examples include herbal products, fatty acids, amino acids, probiotics, prebiotics, and functional foods. Conventional drugs are subjected to intense scrutiny and should have evidence for their safety and efficacy, before obtaining approval from the Food and Drugs Administration (FDA). However, the same requirements do not apply to natural remedies, which means that claims about their efficacy are generally unverified (Tachjian et al., 2010). Herbs and herbal products are the most frequently used biologically-based products (Romm, 2010). 8 University of Ghana http://ugspace.ug.edu.gh 2.1.2 Herbs and herbal products Herbs and herbal products are one of the most common forms of CAM (Patrick- Iwuanyanwu et al., 2012, Tachjian et al., 2010). Some herbal products are defined as medicines in many European countries, while in the United States they are classified as dietary supplements (Iwata et al., 2004). These herbs/herbal products were in use long before the introduction of modern medicine (Fasinu et al., 2012). They usually consist of different plants or plant parts (crude state or as plant preparations). Herbs and herbal products are made of active phytochemicals, mostly consisting of secondary metabolites produced through pathways such as the acetate–mevalonate, acetate–malonate, and shikimate (Fasinu et al., 2012). These metabolites include phenolics (e.g. salicylates, lignins, quinolones, and tannins), terpenoids (e.g. saponins, sesquiterpenes, iridoids, carotenoids, and steroids) and phenolic glycosides (e.g. glucosinolates, cyanogens, and flavonoids). The herbs and herbal products may also contain other phytochemicals including peptides, alkaloids, polysaccharides (e.g. gums and mucilages), resins, and essential oils making herbs highly susceptible for drug interactions (Fasinu et al., 2012). Secondary metabolites from plants have been effective sources of commercially important pharmaceutical compounds (HemaIswarya and Doble, 2006). Over all, plants have been a good source of novel drug compounds. About 41% of newly approved drugs within the period 1983–1994 were of natural product origin. However, within the same period, 60% of these products were found as anti-infective and anticancer compounds (Cragg et al., 1997). Initially, plant products were used in unmodified forms, as concentrated herbal 9 University of Ghana http://ugspace.ug.edu.gh extracts. As time went on, and with technological advancement, plant products exist in different formulations: capsules, tablets, suspensions, etc. A majority of herbs and herbal products have little or no toxicity assessment conducted on them, however, many exist on the market and are often self-prescribed (Arhewoh et al., 2017). Reports suggest that some herbs and herbal products may contain inorganic impurities such as arsenic, lead, and mercury; or intentionally added pharmaceuticals, which may be potentially harmful to humans (HemaIswarya and Doble, 2006). Thus, the need for studies to evaluate the safety of many of these herbal products. Furthermore, herbal medicines are popular amongst many patients who have been diagnosed with chronic diseases such as hypertension, diabetes and others. Reports suggest that about 72.8% of diabetic patients use herbs/herbal products as alternative therapies (Vaghela et al., 2017). The use of herbs in diabetes treatment is attributed to the presence of phytoconstituents which can preserve the function of β-cells (Vaghela et al., 2017). Some of the very common herbal medicines include chamomile, Echinacea, Gingko biloba, ginger (Zingiber officinale), ginseng and St John’s wort. Chamomile is used for its mild sedative effects but also has antispasmodic and antiseptic activity (Miller, 1998). Ginkgo biloba is has been shown to be very potent in dementia treatment, ginger is effective as an anti-nauseant and antispasmodic agent, while St John’s wort is indicated for anxiety, sleep disorders, and depression (Miller, 1998). Indeed, recognition of the efficacy of natural products as therapeutic agents is on the rise, hence the need for extensive studies (efficacy, toxicity and interaction) on these agents (Teiten et al., 2013). 10 University of Ghana http://ugspace.ug.edu.gh 2.2 HERBAL BITTERS Herbal bitters are blends of botanicals in water or alcohol (tincture) base (Olumese and Adegbolagun, 2015). The process of extracting alcohol from plants/herbs dates back to the “Hippocratic wine of the Greeks” (Tonutti and Liddle, 2010). As time went on, some of these alcohol extractions from plants were used for medicinal purposes. Herbal bitters, containing blended components in a base of alcohol (tincture) or water, were primarily sold as digestive aids on account of their capacity to increase the secretion of saliva and digestive juices (Olumese and Adegbolagun, 2015). An herbal extraction bitters containing gentian root in a distilled-alcohol base, famously known as “Stoughton’s Great Cordial Elixir”, became commercially available in 1690 (Johnson et al., 2015). This patented herbal formulation was advertised and sold for its therapeutic properties, however, it is the closest ancestor of what we know today as bitters. Currently, there are several bitters commercially available, even though most present-day manufacturers provide ingredients lists, they do not provide actual formulations (Johnson et al., 2015). Herbal bitters contain several secondary constituents including phenols, flavonoids, alkaloids and polyphenols. These constituents make them good candidates for scavenging free radicals (Olumese and Adegbolagun, 2015). Herbal bitters are known to be effect immune boosters, thus, they are proposed to effectively maintain overall health status and well-being (Olumese and Adegbolagun, 2015). Bitters have been used since antiquity to treat dyspeptic disorders by stimulating the secretion of digestive juices and strengthening the smooth muscles of the gastrointestinal tract (Saller et al., 2001). Herbal bitters may also be used as antidiabetic agents (Jimmy and Udofia, 2014), as well as treatment against cervical cancer (Onyeaghala et al., 2015). Reports also suggest that some bitters induce 11 University of Ghana http://ugspace.ug.edu.gh oxidative stress and inflammatory response (Oyewo, 2013) and affect lipid peroxidation, SOD and GSH levels (Adeyemi et al., 2012). Some common herbal bitters available on the market include Angostura Bitters, Hopped Grapefruit Bitters, Mole Bitters, Celery Bitters, Living Bitters and Swedish Bitters. 2.2.1 Swedish Bitters Swedish bitters is a liquid polyherbal tonic rediscovered in the eighteenth century by Dr. Claus Samst and Dr. Urban Hiärne (Gomez-Flores et al., 2011). The original Swedish name, Hiärne’s Testamente (Hiärne’s Testament) is accompanied by a folklore that says the medicine is Hiärne’s gift to mankind (Ahnfelt and Fors, 2016). Swedish bitters garnered universal appeal as a remedy for a wide range of disorders in the 18th century and was added to a number of pharmacopoeia and recipe collections in the 19th century (Ahnfelt and Fors, 2016). The origin of Swedish bitters however is attributed to Paracelsus, who at the beginning of the 16th century developed an “Elixir ad longam vitam” (“medicine for a long life”) containing aloe, myrrh and saffron (Theiss and Theiss, 1993). It has been widely used in Europe from the 1730s until its re-popularization by Maria Treben (Theiss and Theiss, 1993). Swedish bitters as the name implies has a bitter taste, brownish-black color with a fragrant smell. Although the recipe has changed over time, it mainly consists of Aloe vera (aloe), Angelica archangelica (angelica), Carlina acaulis (carline thistle), Rheum rhabarbarum (rhubarb), Senna alexandrina (senna), Curcuma zedoaria (zedoary), Cinnamomum camphora (camphor), Fraxinus ornus (manna), Commiphora myrrha (myrrh), Sassafras albidum (saffron) and other less defined ingredients (Teiten et al., 2013). 12 University of Ghana http://ugspace.ug.edu.gh Most of the constituents of Swedish bitters have some purported medicinal uses. Aloe vera has been used extensively as an external burn treatment and also to help relieve constipation by oral consumption (Manvitha and Bidya, 2014). Furthermore, Aloe vera is known to have anti-inflammatory and wound healing properties, by accelerating the growth of epithelial cells (Eshun and He, 2004). Additionally, Aloe vera has been found to anticancer properties with tumour growth suppression activity (Wolfgang, 1995). In the pharmaceutical industry, Aloe vera is widely used in the production of medicines, such as ointments, burn treatments, medicated creams and lotions to combat a variety of skin conditions (Eshun and He, 2004). Furthermore, it is used for the manufacture of health drinks in the food industry (Eshun and He, 2004). Aloe vera reportedly induced CYP reductase and some Phase II biotransformation enzymes (Singh et al., 2000). Other reports have also indicated inhibitory activities against CYP3A4 and CYP2D6 enzymes (Djuv and Nilsen, 2012). Studies also indicate that Aloe vera gel extract significantly increased the levels of reduced glutathione, glutathione-S-transferase (GST), superoxide dismutase, lipid peroxidation and catalase in diabetic rats’ kidney and liver (Rajasekaran et al., 2005). The leaves, fruits and roots of Angelica archangelica are ingredients found amongst traditional medicine practitioners, and are recognized as one of the most esteemed medicinal plants in Nordic countries (Sigurdsson et al., 2013). Extensive investigation has revealed that A. archangelica contains quite a lot of vital bioactive compounds, including terpenes, flavonoids, polyphenols, coumarins and polysaccharides, with a number of biological effects (Sigurdsson et al., 2013). For example, the root extract has shown antitumor potential against breast cancer cells in vitro and in vivo (Oliveira et al., 2019). The root essential oil has some antibacterial property mainly against Escherichia coli and 13 University of Ghana http://ugspace.ug.edu.gh Staphylococcus aureus and can be utilized as a natural preservative in foods (Acimovic et al., 2017). Antiviral potential of the fruit extract and five isolated compounds from the fruit have been identified against Herpes simplex virus-1 (Rajtar et al., 2017). Internally it is used in the treatment of digestive conditions, flatulence and also as a remedy for cold and other respiratory disorders (Kumar et al., 2011). Reports suggest that some angelica species cause induction/inhibition activity against CYP2C, CYP3A, CYP2D1 and CYP2D6 (Yoo et al., 2007, Ishihara et al., 2000, Tang et al., 2006). In addition, Yeh et al. (2003), reported the hepatoprotective effect of angelica. They indicated that this effect is a consequence of the inhibition of reactive oxygen species that cause lipid peroxidation, thus reducing oxidative stress (Yeh et al., 2003). Carlina acaulis is a plant originating from the central and southern parts of Europe (Link et al., 2016). It is used conventionally as astringents and diuretics as well as for the treatment and management of all manner of skin disorders, pain, spasms and fevers (Jaiswal et al., 2011). The leaf extracts of C. acaulis subsp. Caulescens for example, has demonstrated anticancer activity against human melanoma cell lines in vitro, making it a potential candidate for skin cancer (Strzemski et al., 2017). It also has antioxidant activity, anti-inflammatory, anti-ulcer, antitrypanosomal and antimicrobial properties (Herrmann et al., 2011, Dordevic et al., 2007). Rheum rhabarbarum, commonly known as rhubarb, is a well-known ancient Chinese traditional medicine used in the treatment of a range of ailments (Nizioł et al., 2017). The root extracts of rhubarb has shown antimicrobial activity against a variety of microorganisms (Canli et al., 2016). Other studies have also demonstrated that rhubarb has wide-ranging in vitro biological activities such as cathartic, anti-inflammatory, anticancer, 14 University of Ghana http://ugspace.ug.edu.gh antibacterial, analgesic, hepatoprotective, antimutagenic, and anti-oxidative effects (Zheng et al., 2013). In addition, rhubarb has demonstrated good activity towards diabetic nephropathy (DN) clinically (Zheng et al., 2013). Rhubarb has demonstrated inhibitory effect on CYP2C6 and CYP3A4 (Gao et al., 2013, Iwata et al., 2004). Rhubarb exhibited significant enzyme lowering effect and liver protecting effect by decreasing levels of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST) (Xing et al., 2011). Senna alexandrina is used in contemporary medicines as a laxative, and it is an ingredient in many herbal remedies and tonics (Pansa, 2011). Curcuma zedoaria Roscoe, from the Zingiberaceae family and commonly referred to as zedoary is grown on a large scale usually as a vegetable, but also as a spice and perfumery material mostly in South-east Asian countries (Makabe et al., 2006). Conventionally, the dried rhizome of zedoary is carefully chosen to make drinks or to be processed as medicine (Lai et al., 2004). One notable feature of zedoary is its dark orange fleshed tubers which is similar to Curcuma longa (common turmeric) (Wilson et al., 2005). Zedoary is reported to have antimicrobial activity, anti-inflammatory activity, antioxidant activity, and anticancer activity against human ovarian cancer OVCAR-3 cells and human promyelocytic leukemia HL-60 cells (Lai et al., 2004, Mau et al., 2003, Makabe et al., 2006, Syu et al., 1998). Sun et al. (2010) reported a strong inhibitory effect of curcumenol, a major constituent of zedoary oil, on CYP3A4 activity (Sun et al., 2010). Cinnamomum camphora, commonly referred to as camphor, is highly recommended in traditional medicinal settings for the treatment and management of a variety of disease 15 University of Ghana http://ugspace.ug.edu.gh conditions. Among these, C. camphora is active as an antioxidant, anti-inflammatory, antibacterial and antifungal agent (Lee et al., 2006, Pragadheesh et al., 2013, Li et al., 2018, Zhou et al., 2017). Orally, camphor is administered for the treatment and management of hysteria, neuralgia, nervousness and also serious diarrhea. It is used effectively in the treatment of colds and chills (Lee et al., 2006). Camphor has been shown to affect the levels of alanine, and aspartate aminotransferase (ALT and AST respectively) liver enzymes in rats (Johari et al., 2015). Fraxinus ornus, manna ash, is found growing naturally in the wild mostly in the Mediterranean region as well as in south-central Europe, most notably in Romania and the south of the Czech Republic (Kostova, 2001). The ethanolic extract of the bark has shown antioxidative activity (Marinova et al., 1994). The stem bark of manna ash has been used for the treatment of arthritis, dysentery, inflammation and wounds (Kostova and Iossifova, 2002). Commiphora myrrha, a small tree or a large shrub, belongs to the Commiphora genus in the Burseraceae family, and it can be found in some African and Asian countries (Zhu et al., 2003). It produces a yellow non-volatile gum resin, called myrrh (Zhu et al., 2003). Extracts of Commiphora myrrha possess antioxidant, anti-inflammatory, antimicrobial, as well as analgesic effects (Su et al., 2011, Mohamed et al., 2014). Sassafras albidum sometimes known as white sassafras, is a medium-sized aromatic tree, which grows moderately fast on moist, well-drained, sandy soils (Griggs, 1990). Isolated compounds obtained from the chloroform bark extract of S. albidum has shown some antileishmanial activity (Pulivarthi et al., 2015). It has also been used traditionally as a 16 University of Ghana http://ugspace.ug.edu.gh blood and kidney cleanser (Cavender, 2006). However, reports indicate that the bioactive compounds crocin and safranal found in saffron alter CYP activity (Dovrtelova et al., 2015). Crocin significantly decreased the activity of CYP3A, CYP2A, CYP2C11 and CYP2B enzymes, on the contrary, the activity of CYP2B, CYP3A and CYP2C11 enzymes were significantly increased by safranal (Dovrtelova et al., 2015). Swedish bitters is marketed as a digestif although it is claimed to have other pharmacotherapeutic properties. Some studies have shown that it detoxifies the body, aids in respiratory health, joint mobility, healthy bladder and helps with the elimination of toxins from the skin (Awa and James, 2013). Furthermore, the bitters has been shown to reduce cholesterol levels as well as regulate blood glucose level, making it a potential therapeutic candidate for diabetes and obesity (Awa and James, 2013). In addition, it has shown antimicrobial and anti-inflammatory potential (Anyasor et al., 2017, Gomez-Flores et al., 2011). 17 University of Ghana http://ugspace.ug.edu.gh Figure 2.2. Swedish Bitters by NatureWorks (Luckyvitamin, 2018) 2.3 XENOBIOTIC METABOLIZING ENZYMES Xenobiotic metabolizing enzymes (XMEs) refers to an assorted group of proteins that breakdown a wide range of substances including drugs, food toxicants, pesticides, carcinogens, pollutants, and endogenous substances like bile acids, prostaglandins, and steroids (Penner et al., 2012). Metabolism of xenobiotics often leads to inactive or active and readily excretable metabolites (Brown et al., 2008). These metabolic reactions including oxidation, reduction, and hydrolysis are categorized as Phase I reactions while Phase II involves conjugation reactions (Wu and Lin, 2019). A third phase, Phase III, has been attributed to the function of membrane transporters on the efflux of compounds across plasma or intercellular membranes (Penner et al., 2012, Wu and Lin, 2019). Phase I reactions attach or reveal functional groups (e.g., - OH, - CO2H, - NH2, or - SH) on xenobiotics to enhance their hydrophilicity (Huang et al., 2018). Phase I XMEs include 18 University of Ghana http://ugspace.ug.edu.gh flavin-containing monooxygenases (FMOs), xanthine oxidase/aldehyde oxidase (XO/AO), monoamine oxidases (MAOs) and cytochrome P450s (CYPs or P450s) (Penner et al., 2012). Some phase II metabolism reactions include acetylation, sulfonation, methylation glutathione (GSH) and amino acids (e.g. glutamic acid, glycine, and taurine) conjugation, as well as glucuronidation (Penner et al., 2012). Some Phase II XMEs include N-acetyl Transferases (NATs), Uridine 5'-diphospho (UDP)–glucuronosyl transferases (UGTs), sulfotransferases (SULTs), methyl (N-methyl - , thiomethyl - , and thiopurinemethyl - ) transferases and Glutathione S-transferases (GSTs), (Wu and Lin, 2019, Penner et al., 2012). 19 University of Ghana http://ugspace.ug.edu.gh Figure 2.3. Drug transporters and metabolizing enzymes that function in the liver, intestine and kidneys. (Yeung et al., 2013) 20 University of Ghana http://ugspace.ug.edu.gh One of the major XMEs involved in Phase I reactions are the Cytochrome P450 (CYP) enzymes. The name “cytochrome P450” originated from the fact that “they are bound to membranes within a cell (cyto) and contain a heme pigment (chrome and P) that absorbs light at a wavelength of 450 nm when exposed to carbon monoxide” (Lynch and Price, 2007). The CYPs are a closely related group of enzymes that metabolize numerous drugs via oxidation (Ogu and Maxa, 2000). CYPs were first isolated by Klingenberg and Garfinkel from the liver (microsomes) of rats (Sychev et al., 2018). CYPs are heme- containing membrane proteins situated in the smooth endoplasmic reticulum of a number of cells. The majority of enzyme isoforms are found in the liver, however, the kidneys, lungs, intestines and the skin contain some amounts of this enzyme (Ogu and Maxa, 2000). With time, a recommended nomenclature system for the CYPs was established whereby Roman numerals (later changed to Arabic) denoted gene families, letters denoted subfamilies and Arabic numerals denoted individual genes (Pinto and Dolan, 2011). More than 1,000 isoenzymes exist, and of these, five are very essential in metabolism of 90% of all available drugs on the market (i.e. CYP1A2, CYP2C9, CYP2D6, CYP2C19 and CYP3A4) (Pinto and Dolan, 2011). 21 University of Ghana http://ugspace.ug.edu.gh Figure 2.4. Proportion of drugs metabolized by different CYPs (Häggström, 2014) 2.3.1 Drug Interactions Drug-drug interactions may be defined as the effect of one drug over another (Brody, 2018). Drug interactions could result in serious undesirable effects (toxicities) or a reduction/increase in the therapeutic properties of certain medicinal agents. Multidrug therapy, which is commonly observed in aged patients, increases the possibility of drug interactions substantially (Cascorbi, 2012). Drug interactions may occur at the pharmacodynamics level or at the pharmacokinetic level (Pai and Bertino, 2015). Pharmacokinetic drug interactions are more common and predictable more than pharmacodynamic interactions (Flynn, 2007). Pharmacodynamic interactions involve one drug influencing another drug’s effect directly (Cascorbi, 2012). For example, pharmacodynamic interactions can be observed where there is simultaneous administration of a nonsteroidal anti-inflammatory drug (NSAID) and phenprocoumon (an oral 22 University of Ghana http://ugspace.ug.edu.gh anticoagulant) (Tonkin and Lindon Wing, 1988, Cascorbi, 2012). An additive interaction is observed where there is more bleeding since phenprocoumon inhibits vitamin k (Cascorbi, 2012). Another example is the simultaneous administration of aspirin and ibuprofen where ibuprofen prevents the gastrointestinal bleeding side effect of aspirin by binding to cyclooxygenase-1 (COX-1) (antagonistic interaction) (Cascorbi, 2012). Pharmacokinetic drug interactions can take place at levels of drug absorption, elimination, metabolism (inhibition/induction of drug metabolizing enzymes) as well as drug transporters. Modulation of drug transporters and drug metabolizing enzymes are by far the most important interactions observed clinically (Pai and Bertino, 2015). The development of complexes can decrease the drugs’ bioavailability and hence affect absorption of the drug (Cascorbi, 2012). For example, bisphosphonates used in osteoporosis, for instance alendronate, with already low bioavailability of only 0.5% to 2%, can be markedly reduced by calcium ions in mineral water or milk (Cascorbi, 2012). Drug transporters interactions can be caused by alterations in the transporters’ expression levels or by substrates competing for binding sites (Vrbanac and Slauter, 2013). For example, verapamil and quinidine have been found to increase concentrations of digoxin, a cardiac glycoside, in plasma, because they block digoxin’s biliary and/or urinary excretion via the efflux transporter P-glycoprotein (P-gp) inhibition (Vrbanac and Slauter, 2013). Alternatively, P-gp induction can speed up efflux transport and decrease the bioavailability of drugs as seen with the simultaneous administration of cyclosporine and rifampicin leading to sub-therapeutic concentrations of cyclosporine (Cascorbi, 2012). Most metabolic interactions involve the cytochrome P450 (CYP) enzyme system, primarily expressed in the liver, and are involved in the Phase I oxidation of more than 23 University of Ghana http://ugspace.ug.edu.gh 50% of all drugs available on the market (Cascorbi, 2012). Reports suggest that a number of agents can modulate the metabolic activity of xenobiotic metabolizing enzymes (Ogu and Maxa, 2000). The most important isoforms involved in human drug metabolism include CYP1A2, CYP2C, CYP2D6, CYP2E1and CYP3A (Bertz and Granneman, 1997). CYP3A4 metabolizes over 50% of all clinically used drugs, whilst CYP2D6 metabolizes about 30% (Iwata et al., 2004, Pan et al., 2012). Clinical case reports of herbs that are known to modulate CYPs include Ginkgo, garlic, ginseng, and St John’s wort. Concomitant administration of St John’s wort is known to lower the blood concentration of digoxin, warfarin, cyclosporine and a host of other drugs metabolized by the CYP3A4 due to induction of CYP3A4 by St John’s wort (Iwata et al., 2004). Naringin, a flavonoid contained in citrus fruits, most especially grapefruit, is known to inhibit CYP3A4 therefore, resulting in the increase in bioavailability of other drugs (Cascorbi, 2012). For example, co-administration of grapefruit juice is found to increase the bioavailability of midazolam, cyclosporine, terfenidine and calcium channel blockers by CYP3A4 inhibition (Iwata et al., 2004). Fluoxetine, which is a selective serotonin reuptake inhibitor used for the management of major depression, is a strong inhibitor of CYP2D6. CYP2D6 is known to metabolize clozapine, which is an antipsychotic in the management of severe paranoid schizophrenia. Therefore, simultaneous administration of fluoxetine and clozapine results in increased levels of clozapine in plasma and augments clozapine's therapeutic effects and possible toxicity (Ferslew et al., 1998). The inhibition of CYP2D6 can also affect the biotransformation of codeine into its active metabolite morphine or the formation of O- desmethyltramadol from tramadol (Cascorbi, 2012). Ciprofloxacin exhibits an inhibitory potential for CYP1A2, resulting in an inhibition of theophylline metabolism leading to an 24 University of Ghana http://ugspace.ug.edu.gh increase in the plasma concentration of theophylline, with resultant cardiac and gastrointestinal side effects (Shakeri-Nejad and Stahlmann, 2006). Clopidogrel, which is a prodrug, requires biotransformation into its active metabolite by CYP2C19 for its antiplatelet effect (Mega et al., 2009). CYP2C19 is inhibited by proton-pump inhibitors (PPIs) such as pantoprazole, rabeprazole, lansoprazole, or omeprazole, therefore, simultaneous use of PPIs and clopidogrel leads to sub-therapeutic effect of clopidogrel (Mega et al., 2009, Ho et al., 2009). Studies on herb-drug/drug-drug/drug-food interactions have become an essential part of modern research (Ogu and Maxa, 2000). However at present, herb–drug interactions are not subjected to the same scrutiny as that of drug–drug interactions (Chan et al., 2016). It is therefore important to determine the effect of xenobiotics on CYPs in order to predict drug interactions. 2.4 HERBS/HERBAL PRODUCT AND LIVER HEALTH The liver is an essential organ in the body responsible for detoxification, thus any damage to it will result in the weakening of its functions (Guan and He, 2015). In the face of growing interest in the use of herbs and herbal products, issues concerning their safety is on the ascendancy (Amadi and Orisakwe, 2018). Apart from herb-drug interactions, usage of these herbs is fraught with medical problems including liver damage where patients can record abnormal liver function tests while being asymptomatic and as well, an unexpected and severe liver failure occurring (Amadi and Orisakwe, 2018). Current estimates suggest that 15% of drug-induced liver injuries (DILI) are caused by herbs i.e. herb-induced liver injuries (HILI) (Raschi and De Ponti, 2015). Determining its true occurrence remains an 25 University of Ghana http://ugspace.ug.edu.gh uphill task, mainly because of the lack of legal controls and regulatory guidelines (Valdivia-Correa et al., 2016). However, there are certain factors contributing to liver toxicity by herbal medicines including misidentification or collection of wrong part of a medicinal plant, poor storage leading to modified products, adulteration during processing, as well as mislabeling of the final product (Larrey and Faure, 2011). Liver injury diagnosis commences with detailed information about the consumed herb/herbal product and the elimination of other possible causes of injury, including autoimmune diseases and viral hepatitis (Valdivia-Correa et al., 2016). Liver enzymes are significant biomarkers used to measure the extent of liver damage and are easily obtainable for the monitoring of individuals who have liver disease (Amadi and Orisakwe, 2018). Elevated levels of transaminase enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are important markers for chronic liver disease diagnosis (Amadi and Orisakwe, 2018). According to a database managed by the US National Library of Medicine, LiverTox, in excess of 30 herbal medicines have been reported to cause DILI (Liu et al., 2016). Herbal medicines with the tendency to cause major hepatotoxicity have been found to contain pyrrolizidine alkaloids (Larrey and Faure, 2011). Pyrrolizidine alkaloids are phytochemicals that are naturally produced by plants as secondary metabolites and these serve as defense mechanisms against insect herbivores and are toxic to most vertebrates (Fu et al., 2007). They require metabolic activation to the “pyrrolic” metabolites to exert their toxicity (Fu et al., 2007). Some of the known plant species having these alkaloids include Crotalaria, Heliotroprium, Symphytum (Comfrey), and Senecio as well as Gynura segetum. Others are germander (Teucrium chamaedris), Atractylis gummifera, kava–kava (Piper methysticum), Hedeoma pulegioides, great celandine 26 University of Ghana http://ugspace.ug.edu.gh (Chelidonium majus), and Mentha pulegium (Larrey and Faure, 2011). Red Yeast rice (Monascus purpureus), black cohosh (Actaea racemosa), Garcinia cambogia, green tea extract (GTE), obtained from the leaves of the Camellia sinensis plant, have all been linked to herb induced liver injury (Navarro et al., 2017). Some constituents of Swedish bitters have shown herb-induced liver toxicities. Case reports suggest that Aloe vera induced hepatotoxicity indicated by high liver enzyme levels. However, liver enzyme levels were normalized after discontinuation of the aloe preparations (Yang et al., 2010, Rabe et al., 2005). Free radicals are also implicated in the pathogenesis of toxic liver injuries which compromise the membranes of hepatocytes resulting in the seepage of enzymes and elevation in liver biomarkers (Spencer et al., 2011). Free radicals as well as other reactive oxygen species (ROS) are produced from the regular metabolic processes occurring in the body or from sources outside the body including industrial chemicals, air pollutants, radiation, certain drugs, and cigarette smoking (Lobo et al., 2010). ROS consist of both non-radical and radical oxygen species comprising of singlet oxygen (1O2), hydrogen peroxide (H2O2), hydroxyl radical (•OH) and superoxide anion (O •− 2 ) (Sharma et al., 2012). Normal levels of ROS are vital to physiological processes including cellular signaling pathways and reactions to infectious agents (Abdel lateif et al., 2016). On the contrary, high ROS concentrations are very dangerous to organisms, and when the amount of ROS surpasses the defense mechanisms of the organism, a cell is said to be in a state of “oxidative stress” (Sharma et al., 2012). Oxidative stress leads to direct or indirect ROS- mediated destruction to molecular species including lipids, proteins, and nucleic acids and has been implicated in conditions such as inflammation, certain cancers, antherosclerosis, 27 University of Ghana http://ugspace.ug.edu.gh diabetes, and the aging process (Lobo et al., 2010, Ray et al., 2012). Cells contain several defenses against ROS induced damage that include non-enzymatic and enzymatic antioxidants (Mesa-Herrera et al., 2019). A balance between the levels of ROS and antioxidants favors cellular homeostasis. “Antioxidants act as radical scavenger, hydrogen donor, electron donor, peroxide decomposer, singlet oxygen quencher, enzyme inhibitor, synergist, and metal-chelating agents” (Lobo et al., 2010). Non-enzymatic constituents of the antioxidant defense system include glutathione, tocopherol, and carotenoids. The enzymatic antioxidants include glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidases (GPx), catalase (CAT), and glutathione S-transferases (GSTs) (Lobo et al., 2010, Sharma et al., 2012). Several methods are employed in the evaluation of the antioxidant property of samples including in vitro and in vivo antioxidant models. Unlike the in vitro methods, samples to be tested for in vivo methods, are administered to the testing animals, and biological samples (blood and/or tissues) are frequently used for the assay (Alam et al., 2013). A number of in vitro methods utilize the free radical scavenging mechanisms; including diphenylpicrylhydrazyl (DPPH) radical scavenging and nitric oxide scavenging assays (Alam et al., 2013). In vivo methods may include but not limited to Superoxide dismutase (SOD), Ferric reducing-antioxidant power (FRAP), Glutathione-S-transferase (GST), and Catalase (CAT) activity (Alam et al., 2013). 28 University of Ghana http://ugspace.ug.edu.gh Figure 2.5. Production of ROS by internal and external sources. (Abdel lateif et al., 2016) 2.5 REVIEW OF METHODS 2.5.1 Protein Determination Several methods are used for protein content determination. In this study, the Bradford method (Bradford, 1976) was employed. This is a fast, relatively cheap and highly specific method for protein determination. It is very sensitive, detecting in the range of 1-20 µg for micro assays, and 20-200 µg for macro assays, and therefore, suitable for a wide range of substances. It is a colorimetric assay that detects the change in absorption of Bradford reagent when it binds to proteins. Bradford reagent contains Coomassie Brilliant Blue G- 29 University of Ghana http://ugspace.ug.edu.gh 250 (CBBG) dye (Figure 5) that binds to aromatic and basic amino acid residues (arginine, phenylalanine, tryptophan and proline). When the CBBG dye binds to proteins, its brown color is converted to blue and the amount of this blue form is detected at 595 nm to quantify the concentration of proteins (Figure 5). The unbound dye in solution is in its cationic form, with absorbance maximum at the wavelength of 470nm (brownish red). When it binds to protein it is found in its anionic form with absorbance maximum at the wavelength of 595nm (blue). Standards are prepared to construct a calibration curve. These standards are samples with known concentration of protein. Bovine serum albumin (BSA) dissolved in water is routinely used. BSA works effectively as a protein standard because it is extensively obtainable in high purity and relatively cheap. Dilution of the sample with unknown concentration is done to fit into the concentration range of the calibration curve. The protein concentration in the sample is determined using the calibration curve. Figure 2.6. Reaction schematic for Bradford Protein Assay (Krohn, 2005) 30 University of Ghana http://ugspace.ug.edu.gh 2.5.2 CYP Enzyme Activity The O-dealkylations of alkylresorufins are commonly used activity probes for assessing some CYP isoforms. The CYP enzyme activity was measured using probe substrates specific for each of the enzymes in the presence of NADPH cofactor. This study employed the method as described by Appiah-Opong et.al. (2007). Ethoxyresorufin, Methoxyresorufin, Benzyloxyresorufin and Pentoxyresorufin were used as substrates for CYP1A1/1A2, CYP1A2, CYP3A4 and CYP2B2/2B2 respectively. This assay measures the formation of the metabolite (resorufin) from the substrates. In the presence of the enzymes, there is dealkylation of the substrates to form resorufin which fluoresces at 530 nm excitation and 586 nm emission (Figure 6). Figure 2.7. Alkoxyresorufin O-dealkylation R is: H (methoxyresorufin), CH3 (ethoxyresorufin), C2H5 (n-propoxyresorufin), C3H7 (n- butoxyresorufin), C4H9 (n-pentoxyresorufin), C5H11 (n-hexoxyresorufin), C6H13 (n- heptoxyresorufin), C7H15 (n-octoxyresorufin), C6H5 (benzyloxyresorufin). (Vottero et al., 2011) For CYP2C9 and CYP2D6, the substrates employed were diclofenac and dextromethorphan, respectively. In the presence of the enzyme, there is hydroxylation of the diclofenac to 4-hydroxydiclofenac which is detected at a wavelength of 280 nm. Dextromethorphan (DXM) on the other hand is demethylated into dextrorphan (DXT) which is measured at 280 nm excitation and 310 nm emission. 31 University of Ghana http://ugspace.ug.edu.gh Figure 2.8. Hydroxylation of diclofenac to 4’hydroxydiclofenac (Othman et al., 2000) Figure 2.9. Dextromethorphan O-demethylation (DuBois and Mehvar, 2018) 2.5.3 GSH determination Many assays used for GSH determination are centered on the reaction of GSH with a fluorophore or chromophore such as O-phthalaldehyde (OPA or OPT) (Shetty et al., 2006). OPA is a dialdehyde which consists of two formyl groups bonded to adjacent carbon centers on a benzene ring. Due to the presence of a sulfhydryl (SH) group in its structure, GSH reacts freely with OPA to produce a highly stable and fluorescent iso-indole derivative (GSH-OPA) (Michaelsen et al., 2009). This reaction allows for sensitive and precise quantitative assessment of GSH in biological systems (Michaelsen et al., 2009). 32 University of Ghana http://ugspace.ug.edu.gh The highly fluorescent iso-indole GSH conjugate can be measured at 340 nm (excitation) and 420nm (emission) (Singh et al., 2017). Healthy or normal cells produce higher fluorescence as an indication of their high GSH content. Oxidative stressed cells on the other hand, produce a lower fluorescence due to their relatively low GSH concentration. Figure 2.10. Chemical reaction between Glutathione and O-phtalaldehyde (Singh et al., 2017) 2.5.4 SOD Activity Many different assays are currently in use for SOD activity measurement. SOD is responsible for the breakdown of harmful superoxide anions into hydrogen peroxide and oxygen (Spanou et al., 2011). In some assays, superoxide radicals/anions are produced by xanthine oxidase or by autoxidation of photo reduced riboflavin, and an indicator such as cytochrome c, nitroblue tetrazolium (NBT), pyrogallol, or epinephrine is monitored for a color change (Roth and Gilbert, 1984). In some of these experiments, the indicator compounds themselves produce the needed superoxide anions without the need for an added source. Autoxidation of this nature can occur with pyrogallol and epinephrine (Gao et al., 1998). In this study, the method by Marklund and Marklund (1974) was used with slight modifications. Pyrogallol (1,2,3-benzenetriol) has for a long time, been identified to rapidly autoxidize, particularly in alkaline solution and its autoxidation reaction has been 33 University of Ghana http://ugspace.ug.edu.gh used for the separation of oxygen from gases (Marklund and Marklund, 1974). The oxidation of pyrogallol leads to the formation of a yellow-colored product called purpurogallin (Mesa-Herrera et al., 2019). SOD inhibits almost entirely the pyrogallol autoxidation by competing very proficiently for the superoxide radicals, and so SOD activity is proportional to the rate of inhibition of pyrogallol autoxidation (Semsei and Nagy, 1984). 2.5.5 Catalase Activity Catalase (CAT) is an essential enzyme that is required to breakdown hydrogen peroxide (H2O2), a by-product of SOD activity, into water (H2O) and molecular oxygen (O2) (Shetty et al., 2006, Hadwan, 2018). A number of methods are available for CAT activity. In this study, CAT activity was assessed by a potassium dichromate colorimetric assay as described by Sinha (1972) with slight modifications. This method works on the principle that, in the presence of H2O2 and heat, dichromate in acetic acid reduces to chromic acetate with the production of perchloric acid as an unstable intermediate. The chromic acetate that is produced is measured colorimetrically at the wavelength of 570 nm (Sinha, 1972). 2.5.6 Lipid Peroxidation Oxidation of lipids in biological systems leads to the formation of lipid peroxidation (LPO) products, and these products can function as important biomarkers for oxidative stress and antioxidant functionality (Shetty et al., 2006). In this study, LPO was determined by the thiobarbituric acid reactive substances (TBARS) method as described by Okhawa et al. (1979). The assay involves the reaction of thiobarbituric acid (TBA) with LPO products in particular, malondialdehyde (MDA) which is a secondary oxidation product of lipids. The 34 University of Ghana http://ugspace.ug.edu.gh reaction products can be measured spectrophotometrically at wavelengths of 532-535 nm (Shetty et al., 2006). CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 STUDY DESIGN An experimental design using animal models, where a control group (untreated) was compared to treatment groups, and in vitro assays were employed. Animals were randomly selected for the various groups. 3.1.1 Inclusion/exclusion criteria For animal models, only healthy male Sprague-Dawley rats were used. Co-morbid and knock-out animals were excluded from the study. 3.2 ETHICAL CONSIDERATIONS The study was approved by the Scientific and Technical Committee of the NMIMR with approval number STC paper 3(2) 2018-19 (Appendix I) and the Institutional Animal Care and Use Committee (IACUC) with approval number UG-IACUC 004/18-19 (Appendix II) 3.3 CHEMICALS AND REAGENTS NatureWorks® Swedish Bitters was purchased from Relish Health Foods, Osu, Accra, Ghana. Diclofenac, dextromethorphan, pentoxyresorufin and ethoxyresorufin were purchased from the Sigma-Aldrich company (St Louis, MO, USA). Other reagents include Bovine serum albumin (BSA) (Wako Pure Chemical Industries, Japan), reduced 35 University of Ghana http://ugspace.ug.edu.gh nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma-Aldrich, USA), reduced glutathione (GSH) (Sigma-Aldrich, Japan), o-phthalaldehyde (OPA) (Wako Pure Chemical Industries, Japan), zinc sulphate (Sigma-Aldrich, USA), pyrogallol (Wako Pure Chemical Industries, Japan), dichromate (BDH Chemicals Ltd, England), sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, Japan), thiobarbituric acid (TBA) (Wako Pure Chemical Industries, Japan), pyridine (Sigma-Aldrich, USA), and butanol (Wako Pure Chemical Industries, Japan). All additional chemicals and reagents used were of analytical grade and obtained from standard suppliers. 3.4 FINGERPRINTING OF SWEDISH BITTERS USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Fingerprinting of Swedish bitters was determined using an HPLC method as follows. Analyses was performed utilizing an Agilent 1100 system (Santa Clara, CA, USA), consisting of a quaternary pump, auto sampler, diode array detector (DAD), and HP ChemStation Software. Chromatographic separation was carried out on a Tskgel ODS C18 (250 x 4.6 mm i.d., 5 μm particle size) analytical column maintained at 30°C. The injection volume was 20 μL and the eluents, water in 0.1% phosphoric acid (A) and methanol (B) as mobile phase at a flow rate of 1 mL/min. The linear gradient program used was set as follows: 0–10 min, 10–30% B; 10–15 min, 30–50% B; 15–25 min, 70–90% B; 25–35 min, 90–90% B; 35–38 min, 90–10% B; 38–40 min, 10–10% B. Ultra-violet (UV) detection was performed at 280 nm. 36 University of Ghana http://ugspace.ug.edu.gh 3.5 EXPERIMENTAL ANIMALS Male Sprague Dawley (SD) rats, ranging from 6 – 8 weeks old, were obtained from the Department of Animal Experimentation, Noguchi Memorial Institute for Medical Research (NMIMR), University of Ghana, Legon. The rats were kept in groups of five in stainless steel cages (34 cm x 47 cm x 18 cm) and with bedding made of soft wood shavings. The SD rats were fed standard diet consisting of normal pellet (AGRIMAT, Kumasi, Ghana), given water ad libitum, and kept under standard laboratory conditions (temperature 25 ± 1°C, relative humidity 60-70%, and 12 h light-dark cycle). The rats were made to acclimatize with laboratory conditions for 7 days before commencing of experiment, similar to one reported by Alabi et al. (2013). 3.5.1 Rat grouping and treatment administration The SD rats were put into 5 groups (5 rats/group). Group 1 was administered the vehicle (distilled water), and this was the negative control. Group 2 was administered 15 mg/kg/day phenobarbital, and this was the positive control. Groups 3 - 5 were administered Swedish bitters at a low dose (5 mL/kg/day), medium dose (10 mL/kg/day) and high dose (20 mL/kg/day), respectively. The doses of Swedish bitters were animal equivalent doses (AED) calculated as described by Shin et al. (2010). The dose calculations are shown at Appendix III. All administrations were done by oral gavage for a period of 7 days. After the treatment period, the rats were subjected to euthanasia in a chloroform chamber, blood obtained by cardiac puncture and their livers harvested and placed on ice immediately. The livers were stored at -80°C until use. Blood from each animal was collected into tubes with 37 University of Ghana http://ugspace.ug.edu.gh or without ethylenediaminetetraacetic acid (EDTA), an anticoagulant, for biochemical and hematological analysis respectively. 3.6 PREPARATION OF MICROSOMAL FRACTIONS The excised livers stored at -80°C were thawed and homogenized individually in two volumes of potassium phosphate buffer (pH 7.4) using a mortar and pestle. Microsomal fractions from the livers were obtained by the method of ultracentrifugation as described by Appiah-Opong et al. (2018), with slight modification. Homogenates were centrifuged at 4,500 rpm for 20 min at 4°C (Eppendorf Centrifuge 5810R, Germany) and the supernatant further centrifuged at 25,000 rpm for 2 h at 4°C with an ultra-centrifuge (Beckman Avanti J-25, USA). After ultra-centrifugation, the resultant supernatant (cytosol) was separated from the pellet (microsomes) and stored separately at -80°C. The microsomes obtained were then homogenized in potassium phosphate buffer (pH 7.4), aliquoted and stored at -80°C until use. 3.6.1 Protein Determination The protein content of both the microsomes and cytosol was determined using the Bradford assay with Bovine Serum Albumin (BSA; St. Louis, MO, USA) as standard. Serial dilutions of the microsomes, cytosol and BSA were made. A volume of 200 μL of Biorad reagent dye (Bio-Rad Laboratories Inc., USA) was added to 10 μL of each microsomal, cytosolic and BSA dilution respectively in a 96-well plate in triplicates. The reactions were incubated for 5 min at room temperature, and absorbance read at a wavelength of 590 nm with a spectrophotometer (Tecan Infinite M200 Pro, Austria). The protein content was then calculated based on the BSA standard curve (Appendix IV). 38 University of Ghana http://ugspace.ug.edu.gh 3.7 CYP ENZYME ASSAYS 3.7.1 Methoxy- Ethoxy- Benzyloxy- and Pentoxy-resorufin O-dealkylation (MROD, EROD, BROD and PROD) The inhibition or induction of CYP 1A1/1A2, 1A2, 3A4 and 2B1/2B2 enzymes by the Swedish bitters was carried out using fluorimetric assays as described by Appiah-Opong et al. (2007). Assays consisted of 0.1 M phosphate buffer (pH 7.4), the substrates ethoxyresorufin, methoxyresorufin, benzyloxyresorufin and pentoxyresorufin (5 µM), and microsomal protein (0.1 mg protein/ml). All substrates were from Sigma-Aldrich (St Louis, MO, USA). The reaction mixtures were pre-incubated at 37°C for 5 min and then initiated by adding 100 µM of nicotinamide adenine dinucleotide phosphate, NADPH (in 0.1 M phosphate buffer). The reactions were allowed to proceed for 10 min (EROD and MROD), 20 min (PROD), 30 min (BROD) at 37°C. Table 1 shows the experimental conditions for these assays. Reactions were terminated with a solution of 80% acetonitrile and 20% 0.5 M Tris. Fluorescence measurements were done using a plate reader (Tecan Infinite Pro M200) at wavelengths of 530 nm excitation and 586 nm emission. All experiments were done in triplicate. Table 3.1. Experimental conditions for fluorescence CYP enzyme assays Enzyme Incubation Substrate Excitation Emission Substrate CYP concentration time (min) concentration wavelength wavelength (mg/mL) (µM) (nm) (nm) 1A1/1A2 1 10 ERes 5 530 586 1A2 1 10 MRes 5 530 586 3A4 1 30 BRes 5 530 586 2B1/2B2 1 20 Pres 5 530 586 Abbreviations: ERes: ethoxyresorufin; MRes: methoxyresorufin; BRes: benzyloxyresorufin; PRes: pentoxyresorufin. 39 University of Ghana http://ugspace.ug.edu.gh 3.7.2 Diclofenac Hydroxylation Inhibition or induction of CYP2C9 enzyme activity was assayed by measuring the formation of the metabolite 4-hydroxydiclofenac from diclofenac (Appiah-Opong et al., 2007). Reaction mixtures comprised of 0.1 M phosphate buffer (pH 7.4), microsomal protein (0.1 mg protein/ml) and the substrate, diclofenac (6 µM). Pre-incubation of the reaction mixture was done at 37°C for 5 min and reaction continued in the presence of 100 µM NADPH, for 10 min at 37oC. The reaction was terminated by the addition of 200 μL of ice cold methanol (stopping solution), after which centrifugation was done at 12,000 rpm for 5 min at room temperature. Supernatants after centrifugation were analyzed using an isocratic HPLC method with a C18 column, and at a carrier flow rate of 0.6 ml/min. A mobile phase consisting of 60% (v/v) 20 mM potassium phosphate buffer (pH 7.4), 22.5% (v/v) methanol, and 17.5% (v/v) acetonitrile was employed. Chromatographic peaks were monitored at a wavelength of 280 nm. Duplicate experiments were performed. 3.7.3 Dextromethorphan O-demethylation The effect of Swedish bitters on rat liver microsomal CYP2D6 levels was evaluated by measuring the formation of dextrorphan, the metabolite, from dextromethorphan, as described by Appiah-Opong et al.., (2007). The reaction mixtures constituted 0.1 M potassium phosphate buffer, 4.5 µM dextromethorphan and rat liver microsomal fraction (0.1 mg protein/ml). Pre-incubation was done at 37°C for 5 min before the addition of 100 µM NADPH and the reaction allowed to progress at 37°C for 45 min. Aliquots of 100 μL 300 mM zinc sulphate heptahydrate (stopping solution) were added and each reaction mixture centrifuged at 4,000 rpm for 15 min at room temperature. Consequently, the 40 University of Ghana http://ugspace.ug.edu.gh supernatant obtained was collected into vials and analyzed using an isocratic HPLC method with a C18 column. The mobile phase consisted of 24% (v/v) acetonitrile and 0.1% (v/v) triethylamine adjusted to pH 3 with perchloric acid. A carrier flow rate of 0.6 ml/min was employed and the metabolite formed monitored at wavelengths of 280 nm excitation and 310 nm emission. Duplicate experiments were done. 3.8 BIOCHEMICAL AND HEMATOLOGICAL ANALYSIS OF BLOOD COLLECTED FROM SD RATS Whole blood (in EDTA tubes) was analyzed for erythrocyte count (RBC), mean corpuscular volume (MCV), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), platelet count (PLT), procalcitonin (PCT), hematocrit (HCT), mean platelet volume (MPV), red cell distribution width (RDW), RDW expressed as a standard deviation (RDW_SD), platelet distribution width (PDW), lymphocyte (LYM), eosinophils (EOS), monocyte (MON), neutrophils (NEU), basophils (BASO) and total white blood cells (WBCs). For biochemical analysis, blood samples obtained from the SD rats were centrifuged at 1500 rpm for 10 min to obtain serum. The serum was analyzed for the levels of alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), alanine aminotransferase (ALT), Total bilirubin (TBil), direct bilirubin (DBil), total protein (TP), albumin (ALB), globulin (GLB), and albumin/globulin ratio (A/G). Measurements were done using a clinical chemistry analyzer (URIT-8021AVet Automatic Chemistry Analyzer) for serum and an automated hematology analyzer (URIT- 5250Vet Hematology Analyzer) for whole blood. 41 University of Ghana http://ugspace.ug.edu.gh 3.9 ANTIOXIDANT ASSAYS OF CYTOSOL FROM HOMOGENIZED LIVERS OF SD RATS 3.9.1 GSH determination Estimation of GSH levels in the cytosol was done using the method of OPA conjugation as described by Tuffour et al. (2018). Briefly, a stock standard GSH (1 mg/mL) was prepared and 7 concentrations obtained by serial dilution (0.125, 0.063, 0.031, 0.016, 0.008, 0.004, 0.002 mg/mL) to plot a standard curve (Appendix V). A volume of 50 µL of cytosol or GSH standard was aliquoted into a 96 well plate in triplicate. A blank of no cytosol was also set up in triplicate. A volume of 50 µL of 0.1M sodium phosphate buffer (pH 8.0) was added to each well followed by an additional 10 µL of 10 mg/mL ortho- phthalaldehyde. The plate was incubated for 15 min in the dark at room temperature after which fluorescence measurements were carried out at 340 nm (excitation) and 460 nm (emission) using a spectrophotometer (Tecan Infinite M200 Pro). The amount of GSH in the cytosol was calculated from the equation of the standard curve (Appendix V). 3.9.2 SOD Activity SOD activity was assessed using the method as described by Marklund and Marklund (1974). An aliquot of 20 µL cytosol (10 mg/mL) was placed into a 96 well plate in triplicate. Blanks containing no cytosol were also set up in triplicate. A volume of 200 µL 75 mM Tris-HCl buffer (pH 8.2) containing 30 mM EDTA was added to each well. In addition, 30 µL of 2mM pyrogallol was added. Absorbance readings were carried out at time intervals of 0 and 5 min at the wavelength of 420 nm. The activity of SOD was expressed as percent inhibition of pyrogallol autoxidation. 42 University of Ghana http://ugspace.ug.edu.gh 3.9.3 Catalase Activity Catalase activity was measured using the method as described by Sinha (1972). Briefly, aliquots of 50 µL cytosol (10 mg/mL) were put into 1.5 mL Eppendorf tubes. A sample blank was also set up with no cytosol. In addition 500 µL of freshly prepared 65 mM hydrogen peroxide in 50 mmol/L sodium potassium phosphate buffer (pH 7.4) was added to the samples. Negative controls were set up with no hydrogen peroxide. The mixtures were vortexed and incubated for 3 min at 37°C. A volume of 1 mL dichromate/ acetic acid was added to the mixtures and the tubes re-incubated at 100°C for 10 min. After cooling with water, the tubes were centrifuged at 2500 rpm for 5 min to remove precipitated protein. The changes in absorbance values were measured at the wavelength of 570 nm against the reagent blank. 3.9.4 Lipid Peroxidation Lipid peroxidation was assessed using the method described by Okhawa et al. (1979). A volume of 20 µL cytosol (10 mg/mL) was aliquoted into Eppendorf tubes. To the cytosol, 20 µL of 8.1% sodium dodecyl sulphate was added. This was followed by the addition of 150 µL 20% acetic acid. Also, 150 µL of 8% TBA was added after which the mixture was topped up with 60 µL of distilled water. The tubes were then incubated in a water bath at 95°C for 60 min. After which the incubated tubes were allowed to cool to room temperature and the resulting mixture topped up with 100 µL of distilled water. A volume of 150 µL 1:15 pyridine: butanol mixture was finally added and the tubes were vortexed for the thorough mixing of the contents for a minute. The resultant mixture was centrifuged at 3000 rpm for 10 min. Aliquots of the supernatant were put into 96 well plates and the 43 University of Ghana http://ugspace.ug.edu.gh absorbance read at the wavelength of 532nm using a spectrophotometer (Tecan Infinite M200 Pro). All experiments were done in triplicate. 3.10 STATISTICAL ANALYSIS All values were stated as mean ± standard error of the mean (SEM). Group differences were tested for significance by means of a one-way analysis of variance (ANOVA) followed by post hoc analysis using the Tukey’s multiple comparison test. P-values ˂ 0.05 were considered to be statistically significant. All graphs and analyses were done using Microsoft Excel 2013 and GraphPad prism software version 5.01. 44 University of Ghana http://ugspace.ug.edu.gh CHAPTER FOUR 4.0 RESULTS 4.1 CHEMICAL FINGERPRINTING The HPLC chromatogram of Swedish bitters is shown in Figure 4.1. A total of 13 peaks were identified. The various peaks obtained are indicated in Table 4.1. Peaks 2, 6, 7, and 8 showed the largest areas under the curve and had values of 1.99083e4, 1.25567e4, 2.13501e4, and 3.29723e4, respectively. Figure 4.1. Chromatographic fingerprint of Swedish Bitters 45 University of Ghana http://ugspace.ug.edu.gh Table 4.1. Peak retention times and area Peak Retention time (min) Area (mAU) 1 1.868 111.33876 2 5.732 1.99083e4 3 9.401 1994.87793 4 12.732 55.48267 5 16.316 1451.75110 6 18.905 1.25567e4 7 20.602 2.13501e4 8 22.158 3.29723e4 9 25.441 1686.14624 10 27.858 2338.13452 11 29.777 257.78955 12 30.551 504.33902 13 31.801 109.43531 4.2 PROTEIN CONTENT OF MICROSOMES Using the Bradford method, the microsomal protein content for each sample was calculated from the BSA standard curve (Appendix IV). The protein content ranged between 6 and 107 mg/mL. From this result, working concentrations of 1 mg/mL were prepared for the enzyme assays. 46 University of Ghana http://ugspace.ug.edu.gh 4.3 CYP ENZYME ASSAYS 4.3.1 CYP1A1/1A2 Activity Effect of Swedish bitters on CYP1A1/1A2 is presented in Figure 4.2. The results showed that there was a statistically significant difference between the negative control (NC) and the positive control (PC) groups (p < 0.05). However, no significant difference in activities was identified between the NC and the low, medium, and high dose Swedish bitters (LD, MD, and HD). 200 * 150 100 50 0 NC PC LD MD HD Treatment Groups Figure 4.2. Effect of Swedish bitters on CYP1A1/1A2 activity in rat liver microsomes Negative control (NC; distilled water), positive control (PC; phenobarbital 15mg/kg), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represents means and standard errors of the mean for triplicate experiments. ∗ represents values statistically different compared to the negative control as indicated with 𝑝 < 0.05. One-way ANOVA followed by Tukey`s multiple comparison test. 47 % CYP1A1/1A2 Activity University of Ghana http://ugspace.ug.edu.gh 4.3.2 CYP1A2 Activity For CYP1A2, there was no significant difference between the untreated (NC) and the Swedish bitters treatment groups as shown in Figure 4.3. However, there was significant difference between the PC and the NC group with p < 0.05. 400 * 300 200 100 0 NC PC LD MD HD Treatment Groups Figure 4.3. Effect of Swedish bitters on CYP1A2 activity in rat liver microsomes. Negative control (NC; distilled water), positive control (PC; phenobarbital 15mg/kg), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represents means and standard errors of the mean for triplicate experiments. ∗ represents values statistically different compared to the control experiments as indicated with 𝑝 < 0.05. One-way ANOVA followed by Tukey`s multiple comparison test. 4.3.3 CYP2B1/2B2 Activity The activity of CYP2B1/2B2 is shown in Figure 4.4. There was a significant difference between the NC and the PC groups (p < 0.001), no significant difference was found between the NC and the Swedish bitters treatment groups. 48 % CYP1A2 Activity University of Ghana http://ugspace.ug.edu.gh 1600 *** 1400 1200 1000 800 600 200 150 100 50 0 NC PC LD MD HD Treatment Groups Figure 4.4. Effect of Swedish bitters on CYP2B1/2B2 activity in rat liver microsomes. Negative control (NC; distilled water), positive control (PC; phenobarbital 15mg/kg), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represents means and standard errors of the mean for triplicate experiments. *** represents statistical difference compared to the control experiments as indicated with 𝑝 < 0.001. One-way ANOVA followed by Tukey`s multiple comparison test. 4.3.4 CYP3A4 Activity For CYP3A4 activity, no significant difference was found between the NC and treatment groups (Figure 4.5). However, there was significant difference between the NC and the PC at p < 0.001. 49 % CYP2B1/2B2 Activity University of Ghana http://ugspace.ug.edu.gh 4000 *** 3000 2000 1000 400 300 200 100 0 NC PC LD MD HD Treatment Groups Figure 4.5. Effect of Swedish bitters on CYP3A4 activity in rat liver microsomes. Negative control (NC; distilled water), positive control (PC; phenobarbital 15mg/kg), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represents means and standard errors of the mean for triplicate experiments. *** represent values statistically different compared to the control experiments as indicated with 𝑝 < 0.001. One-way ANOVA followed by Tukey`s multiple comparison test. 4.3.5 CYP2C9 Activity The effect of Swedish bitters on CYP2C9 activity is as shown in Figure 4.6. There was a significant (p < 0.001) increase in activity of CYP2C9, over 100%, for each of the given treatments compared to the NC. 50 % CYP3A4 Activity University of Ghana http://ugspace.ug.edu.gh *** 400 *** *** *** 300 200 100 0 NC PC LD MD HD Treatment Groups Figure 4.6. Effect of Swedish bitters on CYP2C9 activity in rat liver microsomes. Negative control (NC; distilled water), positive control (PC; phenobarbital 15mg/kg), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represent mean and standard error of the mean for duplicate experiments. *** represent values statistically different compared to the control experiments as indicated with 𝑝 < 0.001. One-way ANOVA followed by Tukey`s multiple comparison test. 4.3.6 CYP2D6 Activity The effect of Swedish bitters on CYP2D6 activity is shown in Figures 4.7. The activity of CYP2D6 was increased for all the doses of Swedish bitters administered to SD rats, but these increases were not found to be statistically significant. 51 % CYP2C9 Activity University of Ghana http://ugspace.ug.edu.gh 400 300 200 100 0 NC PC LD MD HD Treatment Groups Figure 4.7. Effect of Swedish bitters on CYP2D6 activity in rat liver microsomes. Negative control (NC; distilled water), positive control (PC; phenobarbital 15mg/kg), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represents means and standard errors of the mean for duplicate experiments. 4.3.7 Overall Effect of Swedish bitters on Rat CYP Enzyme Activity For the Swedish bitters treated groups, the activities of CYP1A1/2, CYP3A4, and CYP2B1/2 did not differ significantly compared to the negative control group. Although there was an increase in enzyme activity for CYP2D6, it was also not statistically significant. However, CYP2C9 enzyme activity in rats treated with Swedish bitters was found to be significantly increased compared to the negative control. The overall effect of Swedish bitters on selected rat CYP enzymes is shown in Table 4.2. Table 4.2. Summary of the effect of Swedish bitters on selected rat CYP enzyme activity 52 % CYP2D6 Activity University of Ghana http://ugspace.ug.edu.gh CYP ISOFORM ASSAY EFFECT OF SWEDISH BITTERS ON CYP ACTIVITY CYP1A1/2 MROD No significant effect CYP1A2 EROD No significant effect CYP2B1/2 PROD No significant effect CYP3A4 BROD No significant effect Significant increase in enzyme Diclofenac CYP2C9 activity Hydroxylation (LD/MD/HD: p < 0.001) Dextromethorphan O- CYP2D6 Demethylation No significant effect 4.4 BIOCHEMICAL AND HEMATOLOGICAL PARAMETERS The results for the hematological and biochemical analysis are shown in Table 4.3 and Table 4.4, respectively. Generally, there were no significant differences between the negative control and treatment groups (untreated and Swedish bitters administered SD rats). However, high dose Swedish bitters showed a significant decrease (8.44%) in monocytes and ALP levels (279.75U/L) compared with untreated group (21.43% and 532.41U/L, respectively). 53 University of Ghana http://ugspace.ug.edu.gh Table 4.3. Effect of Swedish bitters on rat hematological parameters Parameter Negative Control Positive Control Low dose Medium dose High dose (Distilled water) (Phenobarb) (5 mL/kg b.w.) (10 mL/kg b.w.) (20 mL/kg b.w.) WBC (x109/L) 6.47 (0.45) 8.78 (1.32) 8.83 (0.48) 7.98 (2.39) 6.46 (1.82) LYM (%) 31.91 (5.61) 47.18 (0.86) 37.86 (9.51) 17.71 (3.64) 26.81 (4.52) MON (%) 21.43 (2.67) 5.03 (0.88)** 12.09 (3.10) 6.95 (1.35)* 8.44 (1.37)* NEU (%) 51.63 (3.35) 47.95 (7.12) 46.57 (10.37) 68.44 (6.06) 61.99 (2.27) EOS (%) 3.97 (0.51) 4.14 (0.78) 3.28 (0.34) 2.19 (0.66) 2.48 (0.74) BASO (%) 0.22 (0.10) 0.40 (0.13) 0.21 (0.10) 0.36 (0.14) 0.30 (0.15) LYM (x109/L) 0.41 (0.10) 0.88 (0.21) 0.60 (0.23) 0.23 (0.03) 0.30 (0.10) MON (x109/L) 0.17 (0.06) 0.15 (0.05) 0.17 (0.07) 0.14 (0.03) 0.09 (0.00) NEU (x109/L) 0.47 (0.09) 1.01 (0.20) 0.54 (0.10) 0.99 (0.34) 0.68 (0.08) EOS (x109/L) 0.04 (0.01) 0.06 (0.01) 0.04 (0.01) 0.03 (0.01) 0.03 (0.00) BASO (x109/L) 0.002 (0.001) 0.009 (0.004) 0.002 (0.001) 0.006 (0.004) 0.003 (0.001) RBC (x1012/L) 9.16 (0.49) 8.50 (0.23) 9.88 (0.26) 9.36 (0.14) 9.69 (0.49) HGB (g/dL) 15.48 (0.63) 14.50 (0.51) 15.83 (0.31) 15.40 (0.25) 16.75 (1.05) HCT (%) 46.40 (2.95) 42.28 (1.45) 47.90 (0.84) 45.93 (0.48) 50.15 (3.65) MCV (fL) 50.66 (0.67) 49.80 (0.77) 48.63 (0.85) 49.17 (0.20) 51.75 (1.15) MCH (pg) 16.90 (0.19) 17.02 (0.24) 16.00 (0.60) 16.40 (0.30) 17.20 (0.20) RDW (%) 13.06 (0.99) 13.14 (0.46) 14.78 (1.37) 13.97 (1.48) 14.10 (0.30) RDW_SD (fL) 48.02 (1.97) 48.72 (1.93) 50.55 (3.06) 51.30 (5.08) 52.90 (0.00) PLT (x109/L) 1208.00 (162.30) 1250.40 (107.75) 1239.00 (94.62) 1462.33 (184.33) 1447.50 (117.50) MPV (fL) 5.30 (0.56) 4.60 (0.26) 4.38 (0.38) 4.60 (0.10) 4.35 (0.15) PDW (fL) 7.43 (0.63) 7.08 (0.45) 6.80 (0.60) 7.67 (0.23) 7.20 (0.00) PCT (%) 0.62 (0.82) 0.57 (0.06) 0.55 (0.09) 0.67 (0.07) 0.62 (0.03) Abbreviations: WBC, white blood cells; LYM, lymphocytes; MON, monocytes; NEU, neutrophils; EOS, eosinophils; BASO, basophils; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; RDW, red blood cell distribution; RDW_SD, RDW expressed as a standard deviation; PLT, platelets; MPV, mean platelet volume; PDW, platelet distribution width; PCT, platecrit. Values are group means. Numbers in bracket ( ) represent SEM. * represents statistically significant difference from negative control: ** P < 0.001; * P < 0.05 (One-way ANOVA followed by Tukey`s multiple comparison test). 54 University of Ghana http://ugspace.ug.edu.gh Table 4.4. Effect of Swedish bitters on rat serum biochemical parameters Parameter Control Positive Control Low dose Medium dose High dose (Distilled water) (Phenobarb) (5 mL/kg b.w.) (10 mL/kg b.w.) (20 mL/kg b.w.) TBil (umol/L) 2.22(0.12) 1.67(0.32) 2.25(0.30) 2.03(0.23) 2.80(0.20) DBil (umol/L) 0.86(0.08) 0.80(0.20) 0.93(0.22) 0.97(0.12) 0.65(0.35) ALT (U/L) 70.67(4.66) 110.78(20.08) 97.81(9.45) 54.31(4.62) 67.90(3.11) AST (U/L) 439.37(37.84) 351.71(74.50) 397.52(32.13) 401.81(31.43) 355.40(7.87) ALP (U/L) 532.41(40.83) 284.52(69.25)* 420.75(51.08) 471.17(42.13) 279.75(8.17)* GGT (U/L) 5.50(0.65) 5.00(0.58) 6.50(0.65) 6.50(2.50) 5.00(1.00) TP (g/L) 66.23(1.12) 66.27(0.26) 68.25(2.13) 68.23(3.15) 65.17(1.26) ALB (g/L) 31.00(0.94) 31.03(0.44) 29.80(1.32) 30.07(0.84) 30.03(0.68) GLB (g/L) 35.83(0.75) 35.23(0.67) 38.45(0.97) 38.17(2.31) 35.13(0.83) A/G 0.85(0.03) 0.88(0.03) 0.78(0.02) 0.79(0.03) 0.85(0.02) Abbreviations: TBil, total bilirubin; DBil, direct bilirubin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; GGT, gamma glutamyl transferase; TP, total protein; ALB, albumin; GLB, globulin; A/G, albumin/globulin ratio. Values are group means. Numbers in bracket ( ) represent SEM. * represents statistically significant difference from negative control: * P < 0.05 (One-way ANOVA followed by Tukey`s multiple comparison test). 55 University of Ghana http://ugspace.ug.edu.gh 4.5 ANTIOXIDANT ASSAYS OF CYTOSOL FROM HOMOGENIZED LIVERS OF SD RATS 4.5.1 GSH content The levels of GSH are presented in Figure 4.8. The treatment groups showed slight decrease in GSH levels (0.08 mg/mL for untreated, and approximately 0.05 mg/mL for treated groups) however, these decreases were not statistically significant. 0.010 0.008 0.006 0.004 0.002 0.000 NC PC LD MD HD Treatment Groups Figure 4.8. GSH levels in Swedish bitters treated rat groups compared with untreated. Negative control (NC; distilled water), positive control (PC; phenobarbital 15mg/kg), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represent means and standard errors of the mean for triplicate experiments. 4.5.2 SOD Activity The percentage SOD activity is shown in Figure 4.9. The positive control exhibited a significantly high SOD activity (60% more activity) compared to the negative control (untreated group). There was a dose dependent increment in SOD activity in SD rats administered Swedish bitters. This increase in SOD activity among Swedish bitters-treated groups were not statistically significant compared to negative control. 56 [GSH](mg/ml) University of Ghana http://ugspace.ug.edu.gh 200 * 150 100 50 0 NC PC LD MD HD Treatment Groups Figure 4.9. SOD activity in Swedish bitters treated groups compared with untreated. Negative control (NC; distilled water), positive control (PC; phenobarbital 15mg/kg), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represent means and standard errors of the mean for triplicate experiments. ∗ are values statistically different compared to the negative control as indicated with 𝑝 < 0.05. One-way ANOVA followed by Tukey`s multiple comparison test. 4.5.3 Catalase Activity Effect of Swedish bitters on catalase activity is shown in Figure 4.10. There was a dose dependent decrease in catalase activity, with the high dose treatment group statistically significant (p < 0.05) compared to untreated group. Catalase activity decreased from about 26kU (untreated) to about 12kU (high dose Swedish bitters treatment group). 57 %Inhibition of pyrogallol autoxidation University of Ghana http://ugspace.ug.edu.gh 40 30 20 * 10 0 NC LD MD HD Treatment Groups Figure 4.10. Catalase activity of Swedish bitters treatment groups compared with untreated. Negative control (NC; distilled water), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represent means and standard errors of the mean for triplicate experiments. ∗ are values statistically different compared to the negative control as indicated with 𝑝 < 0.05. One-way ANOVA followed by Tukey`s multiple comparison test 4.5.4 Lipid Peroxidation Effect of Swedish bitters on lipid peroxidation is shown in Figure 4.11. Thiobarbituric acid reactive substances (TBARS) was measured as a by-product of lipid peroxidation, and was expressed as moles/mg protein. The present results indicated no significant differences between the untreated and Swedish bitters treatment groups (with values between 350 and 400 n moles/mg protein). 58 Catalase Activity (kU) University of Ghana http://ugspace.ug.edu.gh 500 400 300 200 100 0 NC PC LD MD HD Treatment Groups Figure 4.11. Lipid peroxidation of Swedish bitters treatment groups compared with untreated Negative control (NC; distilled water), positive control (PC; 15mg/kg phenobarbital), low dose treatment (LD; 5mL/kg/day Swedish bitters), medium dose treatment (MD; 10mL/kg/day Swedish bitters), and high dose treatment (HD; 20mL/kg/day Swedish bitters). Data represent means and standard errors of the mean (SEM) for triplicate experiments. 59 [TBARS] n moles/mg protein University of Ghana http://ugspace.ug.edu.gh CHAPTER FIVE 5.0 DISCUSSION The safety of herbal products has turned out to be a major worry in public health as their acceptance and global market growth continues to rise (Kosalec et al., 2009). Herbal medicines, single or poly herbal formulations, contain a number of different compounds wherein no particular active component is accountable for the total efficacy. Quality control and quality assurance of herbal drugs remain a challenge for the reason that a high variability of chemical components are involved (Mohammad et al., 2010). Chromatographic fingerprinting is often used in the quality assessment of herbal preparations (Gunalan et al., 2012), and WHO has accepted fingerprint analysis as a suitable measure for the quality control of herbal preparations (Cieśla, 2012). A herbal sample fingerprint is defined as “a set of characteristic chromatographic or spectroscopic signals, whose comparison leads to an unambiguous sample recognition” (Cieśla, 2012). Some chromatographic methods for fingerprinting include gas chromatography (GC), thin layer chromatography (TLC), as well as high performance liquid chromatography (HPLC) which was utilized in this study. The HPLC chromatogram of Swedish bitters in this study recorded 13 peaks at the wavelength of 280 nm. The HPLC fingerprint of saffron, a constituent of Swedish bitters, has been established (Ahnfelt and Fors, 2016). However, no HPLC chromatogram of the bitters exists in literature. This chromatogram can therefore serve as a reference pattern for Swedish bitters. Generally, samples with comparable chromatographic fingerprints may show some similar properties (Fan et al., 2006). Cytochrome P450 enzymes are key Phase I drug metabolizing enzymes that catalyze biotransformation of lipophilic xenobiotics into polar forms that can be easily excreted via 60 University of Ghana http://ugspace.ug.edu.gh the kidneys. Modulation in the activities of these enzymes, particularly via drug/herb interactions may consequently affect levels of conventional drugs in circulation (Doligalski et al., 2012). A typical example of a drug/herb interaction is what is observed between grapefruit juice and drugs such as midazolam, cyclosporine, terfenidine and calcium channel blockers metabolized by CYP3A4. The grapefruit inhibits CYP3A4 enzyme activity leading to the increase in bioavailability of these drugs. In the present study, the activities of six CYPs including CYP1A1/1A2, CYP1A2, CYP2B1/2B2 and CYP3A4 were determined after a 7-day treatment with Swedish bitters. From the results obtained, there were no significant differences between the activities of rat CYP1A1/1A2 and CYP1A2 when compared to the negative control groups; suggesting that Swedish bitters did not modulate these enzymes over the treatment period. In addition, CYP2B1/2B2 and CYP3A4 activity in rats administered Swedish bitters did not show an increase in enzyme activity compared to the negative control, however, these were not statistically significant when compared to the negative control group. There have been reports of some of the constituents of Swedish bitters having modulatory effect on CYP enzymes. For example, Aloe vera juice has been found to have inhibitory effect against CYP3A4 and CYP2D6 (Djuv and Nilsen, 2012). Zedoary turmeric oil also has also been shown to inhibit CYP2C9 and CYP2D6 (Cheng et al., 2014). In addition, safranal, an active component of saffrom, has been shown to increase the activity of CYP2B, CYP3A and CYP2C11 enzymes (Dovrtelova et al., 2015). In the current study however, Swedish bitters was found to increase the activities of CYP2C9 and CYP2D6, with activity of CYP2C9 being significantly different compared to the untreated. So far CYP2D6 is known to be a non- inducible enzyme, therefore it is possible that Swedish bitters enhanced the activity of the 61 University of Ghana http://ugspace.ug.edu.gh enzyme. Since protein expressions of these enzymes were not assessed, it cannot be confirmed that CYP induction occurred due to the treatment with Swedish bitters. Studies on some constituents of Swedish bitters have shown modulatory activities however no reports have been made on the combined effect the components of Swedish bitters have on CYP enzymes in rats. The increased activity of CYP2C9 by Swedish Bitters may cause herb-drug interactions involving this enzyme. Drugs that are metabolized by this enzyme may be metabolized faster in the presence of Swedish bitters, and this may lead to sub- therapeutic effect of drugs. From these results, administration of Swedish bitters especially over long periods, may alter the pharmacokinetics of xenobiotics that are metabolized by CYP2C9. Furthermore, results from this study serves as a basis for human studies to ascertain effect of Swedish bitters on concomitantly administered conventional drugs (drug-herb interaction). The liver plays a vital role in storage, secretion, and metabolism, and is a major detoxification organ. Therefore, any injury or pathological condition to the liver (liver cirrhosis and hepatic failure) can be life threatening (Girish et al., 2009). Some common implicated agents of liver injuries include therapeutic drugs (e.g., antibiotics and anti- tubercular drugs), toxic compounds (e.g., aflatoxin and CCl4), alcohol, and parasites (e.g., leptospira, hepatitis virus and malarial parasites) (Subramoniam and Pushpangadan, 1999). In the current study, biochemical and hematological investigation on blood samples were performed to evaluate the toxicity of Swedish bitters on the liver. From the results, there appeared to be no significantly difference between the biochemical and hematological parameters of the Swedish bitters-treated groups and the negative control group (untreated). However, the high dose Swedish bitters treated group had a significant 62 University of Ghana http://ugspace.ug.edu.gh decrease in ALP as well as monocyte levels. Previous reports have indicated that Swedish bitters decreased total protein level and elevated white blood cell count (Ekor et al., 2010). In addition, Ekor et al. (2010) also showed that there was increase in alanine and aspartate aminotransferase activities and significant decrease in total protein. Awa and James (2013) also reported an increase in the level of total proteins when Swedish bitters was administered to rats. Contrary to the previous reports, findings from this study indicate that Swedish bitters decreased the monocyte count, although other white blood cell counts were not affected. Also, alanine and aspartate aminotransferases were not altered significantly, however, ALP levels were significantly lowered in Swedish bitters treated groups compared to the untreated group. In the study carried out by Ekor et al. (2010), administration of the bitters was done for a period of 30 days, however, in the present study treatment was done for 7 days. The duration of administration may have had a role in the difference in hematological and biochemical levels as stipulated by the principles of toxicity. In the current study, high dose Swedish bitters decreased ALP and monocytes in treatment rats after 7 days. It may, however, be prudent to ascertain this observed effect over a longer period (30 days). Glutathione is a major antioxidant of the body, which protects cells from oxidative stress/damage. Glutathione works as a scavenger of radicals, directly or indirectly using GSH-dependent enzymes (Kanďár et al., 2014). A decrease in GSH levels below normal may cause oxidative stress leading to cell damage. The concentration of GSH present in the cytosol of cells is usually in region of 1–10 mM (Meister, 1988). The GSH concentration in many cells is found to be 1–2 mM, and in hepatocytes, which transports GSH, the concentration can be as high as 10 mM (3.0733 mg/mL) (Forman et al., 2009). 63 University of Ghana http://ugspace.ug.edu.gh This study showed GSH concentrations in the range of 0.0049 mg/mL to 0.00789 mg/mL across treatment groups. There was a decrease in GSH concentrations for Swedish bitters treatment groups although not statistically significant compared to the negative control group. A study carried out by Awa and James (2013) showed that there was a slight reduction in the level of GSH in rats administered with Swedish bitters over a period of three weeks compared to the control, which was also shown in this study. Given that the rats were treated for only 7 days, it is not entirely clear what will happen to GSH levels after prolonged intake of the bitters. The SOD enzyme protects living cells against superoxide radicals. (Semsei and Nagy, 1984). SOD is responsible for the breakdown of the harmful superoxide radical (O •–2 ) into molecular oxygen (O2) and hydrogen peroxide (H2O2) (Spanou et al., 2011). The SOD activity was reported as the percentage inhibition of pyrogallol autoxidation. The enzyme inhibits almost entirely the pyrogallol autoxidation by competing effectively for the superoxide radicals, as a result, SOD activity is measured by the rate of inhibition of pyrogallol autoxidation. There was dose dependent increment in SOD activity in the Swedish bitters treatment groups compared to the untreated group. A study carried out by Awa and James (2013) showed that Swedish bitters reduced the levels of SOD in the test group compared to the negative control group. In this study, there was dose dependent increase in SOD activity. However, this increment was not statistically significant and so may not be clinically significant. Catalase enzyme is responsible for the decomposition of harmful H2O2 into water (H2O) and molecular oxygen (O2) (Spanou et al., 2011). A study by Alabi et al. (2013) showed significant increase in catalase activity after treatment with Swedish bitters over a period 64 University of Ghana http://ugspace.ug.edu.gh of 32 days. Contrary to this finding, the study carried out by Awa and James (2013) showed that there was a decrease in the levels of catalase enzyme in the Swedish bitters treatment group compared to the control. Results from the present study revealed a dose dependent decrease in catalase activity compared to the untreated group. These findings are in line with findings from studies conducted by Awa and James (2013) where treatment was for 3 weeks. From the current study, it can be postulated that administration of Swedish bitters over a long period may cause an accumulation of harmful H2O2, and this may lead to oxidative stress. The oxidation of lipids leads to the production of products which further propagates free radical reactions (Shetty et al., 2006). A study by Alabi et al. (2013) showed a significant decrease in lipid peroxidation in Swedish bitters treated rats. Another study by Awa and James (2013) indicated that the level of malondialdehyde (MDA) in their test group (Swedish bitters treated rats) was elevated when compared to the control, suggesting elevated levels of lipid peroxidation. These results were however, not statistically significant. Similarly, in this study, there was no significant difference found between the untreated and the Swedish bitters treatment groups of rats indicating that Swedish bitters did not affect lipid peroxidation. These findings differ from studies conducted by Alabi et al. (2013) as well as Awa and James (2013) as described previously. The differences may be due to the differences in strain of rats used as well as duration of administration. Alabi et al. used albino rats for a period of 32 days whilst Awa and James used albino rats for a period of 3 weeks. In addition, there are several brands of the bitters with possible differences in their constituents. Even though these studies did not state the brand of 65 University of Ghana http://ugspace.ug.edu.gh Swedish bitters used, they were sourced from different locations, which could account for the differences observed. 66 University of Ghana http://ugspace.ug.edu.gh CHAPTER SIX 6.0 CONCLUSION, LIMITATIONS AND RECOMMENDATION This study showed that Swedish bitters increased the activities of rat liver CYP2B1/2B2, CYP3A4, CYP2C9 and CYP2D6 however, the increase in activity was found to be significant for CYP2C9 compared to the negative control. No significant changes were found in rat CYP1A1/1A2, and 1A2 activities after treatment with the bitters. The bitters did not cause any significant changes in GSH level and lipid peroxidation activity. There was a statistically significant decrease in catalase activity in the high dose treatment group, and an increase in SOD activity across treatment groups. Generally, the bitters did not alter hematology and liver function as assessed by full blood count and the biochemistries, except for monocytes and ALP levels which decreased significantly at high doses of Swedish bitters. Limitations of the current study included; the lack of HPLC fingerprint of Swedish bitters from literature, making it difficult to have a comparison. In addition limited resources did not enable the acquisition of pure compounds for targeted analysis of the chemical constituents of the bitters. Animal studies cannot always be extrapolated to man and so some human recombinant CYPS could have been used. It is recommended that Swedish bitters be administered to rats over an extended period of times and the effect of this duration on CYP enzymes assessed. Furthermore, hematology and biochemistry parameters ought to be assayed over this extended period of administration of Swedish bitters. Further research should also be conducted to ascertain the effect of Swedish bitters on human recombinant CYPs, Phase II drug metabolizing enzymes or studies with human subjects. 67 University of Ghana http://ugspace.ug.edu.gh REFERENCES ABDEL LATEIF, K. S., MAGHRABI, I. A. & ELDEAB, H. A. 2016. The Plant Natural Products: Their Antioxidants, Free Radical Scavengers, DNA Protection and Antimicrobial Activities. Journal of Bioprocessing & Biotechniques, 06. ABDULLAHI, A. A. 2011. Trends and challenges of traditional medicine in Africa. African Journal of Traditional Complementary Alternative Medicine, 8, 115-123. ACIMOVIC, M. G., PAVLOVIC, S. D., VARGA, A. O., FILIPOVIC, V. M., CVETKOVIC, M. T., STANKOVIC, J. M. & CABARKAPA, I. S. 2017. Chemical Composition and Antibacterial Activity of Angelica archangelica Root Essential Oil. 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Isolation and characterization of several aromatic sesquiterpenes from Commiphora myrrha. Flavour and Fragrance Journal, 18, 282-285. 85 University of Ghana http://ugspace.ug.edu.gh APPENDICES APPENDIX I STC approval letter 86 University of Ghana http://ugspace.ug.edu.gh APPENDIX II Ethical clearance from UG-IACUC 87 University of Ghana http://ugspace.ug.edu.gh APPENDIX III Calculation of animal equivalent dose (AED) of Swedish bitters Pharmacologically active dose (PAD) = 10 mL PAD = 10 mL/day for an adult Human dose = 10 mL/60 kg = 0.167 mL/kg/day AED = PAD × Km ratio = 0.167 mL/kg/day × 6.2 = 1.03 mL/kg/day Multiply by safety factor (10) AED = 1.03 mL/kg/day x10 = 10.3 mL/kg/day Medium dose = 10 mL/kg/day Low dose = (10 mL/kg/day) /2 = 5 mL/kg/day High dose = (10 mL/kg/day) × 2 = 10 mL/kg/day Km: Correction factor estimated by dividing body weight of species to its body surface area. Km ratio = Human Km/Rat Km = 37/6 Reference: (Shin et al., 2010) 88 University of Ghana http://ugspace.ug.edu.gh APPENDIX IV Protein standard curve BSA STANDARD CURVE 1 y = 1.6923x - 0.0067 0.8 R² = 0.9991 0.6 0.4 0.2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 -0.2 [BSA] mg/mL APPENDIX V GSH standard curve 60000.0 50000.0 y = 808281x + 1431.5 40000.0 R² = 0.9859 30000.0 20000.0 10000.0 0.0 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 [GSH] mg/ml 89 Mean Fluorescence MEAN ABSORBANCE University of Ghana http://ugspace.ug.edu.gh APPENDIX VI Representative HPLC results for CYP2C9 and CYP2D6 CYP2C9 ASSAY Flow rate = 1 mL/min Injection Volume = 50 μL Wave length = 300 nm Mobile Phase = 20mM Kpi (60%): Methanol (25.5%): Acetonitrile (14.5%) Column TSKgel ODS 100v 5μm; 250mm x4.6mm M M= Metabolite (RT = 3.272 min) Diclofenac 90 University of Ghana http://ugspace.ug.edu.gh CYP2D6 Flow rate = 0.7 mL/min Injection Volume = 20 μL Detection 280 Excitation; 310 Emmission Mobile Phase = Water (75%): Acetonitrile (24%): TEA (1%) Column TSKgel ODS 100v 5 μm; 250mm x4.6mm RT of Metabolite = 9.3min mV Detector A:Ex:280nm,Em:310nm 150 125 DEX 100 75 50 25 0 -25 0.0 5.0 10.0 15.0 20.0 25.0 30.0 min 91 University of Ghana http://ugspace.ug.edu.gh mV 70 Detector A:Ex:280nm,Em:310nm 60 50 Metabolite 40 30 20 10 0 -10 -20 -30 0.0 5.0 10.0 15.0 20.0 25.0 30.0 min Data Comparison uV 100000 90000 80000 70000 60000 50000 NC 40000 30000 20000 Sample 10000 0 -10000 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min 92