OF COLOURS, COLOURANTS I - AND COLOURED SUBSTANCES: A PHARMACIST'S TRIPODAL TRAWL UNIVERSITY OF IBADAN LIBRARY OF COLOURS, COLOURANTS AND COLOURED SUBSTANCES: A PHARMACIST'S TRIPODAL TRAWL An inaugural lecture delivered at the University of l b h on Thursday, 05 May, 2016 AREMU OLAJIRE ADEGOKE Professor of Pharmaceutical Chenristry Faculty of Pharmacy University of Ibadan Ibadan, Nigeria UNIVERSITY OF IBADAN UNIVERSITY OF IBADAN LIBRARY I b h U niversity Ress Publishing House University of Ibadan I-, Nigeria @ University of Ibadan, 20 16 Ibadan, Nigeria First Published 201 6 All Rights Reserved ISBN: 978 - 978 - 54291 - 2 - 1 Printed by: Ibc1dan University Printery UNIVERSITY OF IBADAN LIBRARY l7w Vice-Chancellor, Deputy Vice-Cl~tmcellro (Admini- stmrion), D e p u ~V ice-C lmn ce llor (AcadeI ~ic~), Registrar, LibrarianJ Provost of tl2e College of Medicine, Deat~o f the Fwirlty of P h a m c y I Dean of the Postgraduate School, Dems of other Fucgtlties nnd of Studertts, Directors of Institutes, Distinguished Ladies and Gent lern er 1. W b l e I owe a debt of gratitude to God, The Almighty, for the privilege to stand in this auditorium to present this inaugural lecture on behalf of the Faculty of Pharmacy. This is the 12" inaugural lecture coming from the Faculty since the first one pmentwl in 1984 by my mentor and indefatigable, indisputable and indushi ous postgraduate studies supervisor, who over the years has doubled as a father to me. Emeritus Professor Ajibola Akinyerni Olaniyi. This is the fourth from the Department of Pharmaceutical Chemistry. I consider myself as one who has merely and solely received the grace of God to stand in the same boat as others of noble birth and upbringing to contribute to the history of Pharmacy Education at Ibadan. Born with no hope, no future, no class, no inheritance, no nobility and nothhg by worldly standards to be reckoned with. But that it might be by the purest crystalline grace, God has brought this base thing of the world and things that are despised to confound the wise, the rich and the endowed so that 1 may boldly declare that 1 am truly a trophy of God's grace. The story of my life is one that testifies of the mercy, love and grace of God. Left without a father from the age of one; a vegetable hawker till 10; a bar waiter till 15; a shop tender till 25 and becoming a Professor at 42 can only come through the endowment of God, Most High. Thus, this evening, I return all the glory. for all I have been and hope to be, to God, The Almighty. God granted me the wisdom to cany out all you will hear today and the strength and grace to have "laboured more ab~i~idanttllyw n they dl: yet nor I, but the grace of God whiclt was wifh me". To the apologetics of the existence of a mighty hand who UNIVERSITY OF IBADAN LIBRARY rules in the affairs of men and the supposed atheist, I declare God can take any soul from obscurity to the limelight of His design for "He raiseth itp the poor out of the dztst, and lzpeth rlz needy our of the dinglzill; That he moy set him with princes, even with the princes of his people". The topic for this inaugural lecture was conceived as far back as 2008 when the picture of the various contributions I was making to Pharmaceutical Chemistry became apparent. This topic was not confirmed until about two years ago when my last daughter insisted "I have a gift for you" instead of joining us at the family altar at around 10 pm. Out came a paper beautifully painted with multiple colours resembling that of a rainbow. Hence, by the mouths of two witnesses the topic for this inaugural was confirmed Phapacy as a Profession The profession of Pharmacy is one saddled with the responsibility of providing appropriate medicines, drug information and pharmaceutical care for the patients. The Physician is left to no choice following diagnoses to seek for drugs that will be used for the treatment of the patient. The drug itself has been defined as any substance or mixture of substances that is manufactured, sold and offered for sale, for use in the treatment, mitigation, prevention or diagnosis of diseases, either in man or animal. It could also be used for restoration, correction or modification of organic functions in man or animal. On the other hand, a drug product is a finished dosage form, for instance, tablet, capsule, solution or suppository which contains the drug (commonly called active- pharmaceutical or drug in,gedient) usually, but not neces- sarily, in association with inactive ingredients (exMpients). Pharmacy has several sub-speci alties, including Pharmacognosy, Pharmaceutical Chemistry, Pharmaceutics and Industrial Phmacy, Pharmacology, Pharmaceutical Microbiology, Clinical Pharmacy and Pharmacy Admini- stratibn. All these sub-specialties are designed towards optimising the benefits derivable from the administration of a given drug and in particular towards a holistic undestanding UNIVERSITY OF IBADAN LIBRARY of drug. In addition, the Pharmacist is also trained in the rudiments of management, forensic science and accounting. Wannaceutical Chemistry is an important component of Applied Chemistry; it deals with the study of chemistry of drugs. Since all drugs are chemical compounds (whether organic or inorganic), Phmnaeutical Chemistry dwells on the physical, chemical, biopharmaceutical, medicinal p m p t ~ e sof drugs as well as drug designs and discoveries. The two main areas of Pharmaceutical Chemistry are Medicinal Cheniistry and Pharmaceutical Analysis. M ~ d M Cd h emisby, a multidisciplinary subject, has been defined as a basic science which bring$ together the knowledge and skills gained from ' the disciplines of chemistry, biology and biochemistry and apply them to the problem of drug chemistry and activity (Olani yi 2005). Phmamatka l Adys i s deals with the adoptiqn of iarious physical and chemical test pro&- to ensure that a given drug or dosage form contains what it purports to contain (Kar 2005). It emphasizes all the issues relating to quality of medicines. The= are conventional agreements compiled in Pharmacopoeias and other official Compendia on the level of purity expected of each drug, excipient and drug product. The discipline of Pharm$ceutical Analysis has been burdened with the choice of an appropriate method for the purity check and determination of quality of drugs and drug products. The choice of a particular method of analysis is based on sukh &tors as ph$sicochemi8d, of the drugs, sample type (qM&) and Size :(lot ); data qa C ~ ~ X ~ ~ ~ &&j(~s) ~of ~ ~ ~ J ; ' analyte(s); precision and accuracy expected; likely inter- ferenms; number and frequency of samples for analysis and, of course, analyst preference. Two bioad categories of methods adopted are classical and instrumental methods of analysis. The classical methods utilise the age-long titrimetric and gravimetric methods, while instmmmtal techniques adopt the physical properties resulting from the chemistry of the compounds. Hence, the latter is often r e f d to as physicochemical methods of drug UNIVERSITY OF IBADAN LIBRARY analysis. This category includes refractometry, electro- chemical techniques, chromatographic techniques and spectroscopic techniques. Modern design of most techniques utilises amalgamation of the separation power of chromatography with the sensitivity of electrochemical and spectroscopic techniques. . . * , , Spectroscopy Of the instrumental techniques available for the analyst, spectroscopy has been the subject of much improvement over - the years. Spectroscopy is the-study of interactions of - - electromagnetic radiation with matter. The various com- ponents of the electromagnetic spectrum are presented in figure 1. Fig. 1: Components of electromagnetic spectrum. I have focused my research career on the utilisation of various spectroscopic techniques for the quality assessment of pharmleuticals. Since spectroscopy is also an ' important t&hnique available to the organic and medicinal chemists, I have had the privilege of contributing to the structure UNIVERSITY OF IBADAN LIBRARY elucidations of both natural products and synthetic com- pounds. The interactions of energy with organic (and inorganic alike) molecules lead to changes in the electronic, vibrational or rotational energies inherent in the molecules. Combi- nations of these energies constitute the internal energy of molecules. I will attempt as much as possible to reduce to the barest minimum a discussion on spectroscopy as many of my students in time past have wondered if such interactions are possible and often 'asked; "how are you sure of what you are discussing"? Mr. Vice-Chancellor Sir, I must state that to understand spectroscopy takes some guts and some imaginative thinking. I have had to demonsme and indeed "gyrate" in class to describe what this supposed "abstract" science is all h u t . Molecules and indeed matter can take OH two orientations in space; it can be in the ground or excited state. We all kaow that humans can be in the excited r n d a nd this is often associated with some degree of elation and profound joy resulting from several causes that are positively incorporated and interjected into n o d - d a y occurrences. An elated individual feels on top of the world, and he can utter statements or carry out some actions which he may 1-r rejoice about or regret. In such moods, Kings in the Biblical times had promised and even offered "up to half their kingdom" if a riddle could be solved or when he was just excited by wine or display of beauty or loyalty. However, as everyone of us in this auditorium will realise and agree with me, such elatd states do not last forever; we are s w n back to our normal-day activities and if not guided by some higher devotion and powers, depression may set in (the morning afer syndrome). Different events lead to different levels of elation in humans. And often, we may not be able to generalize what excites all humans equally alike. Are there. some individuals who don't seem excited by anything? If there are, it may mean that what will excite them has not been provided or has not occurred. It is the same with molecules. UNIVERSITY OF IBADAN LIBRARY Molecules will prefer to occupy the ground state which corresponds to a state of lowest energy. It is not at rest, as supposed originally, but it is involved in some flip-flop movement hoping to be excited. This state is referred to as the mghest Occupied Molecular Orbital (HOMO) state. Appropriate mergy that separates the &und state from the excited state must be supplied to allow the excitation to bccut. 'Ihis energy is usually inq uantized forms and ordered. No transition will occur except energies of appropriate values are supplied that counterbalance the energy differential k w e e n the states. On excitation, molecules occupy the Lowest Unoccupied Molecular Orbital (LUMO) state (fig. 2). Various spectroscopic techniques take advantage of the internal energies of atoms and molecules. Such spectroscopic techniques include atomic a b q t i o n and emission spectroscopy; ultraviolet-visible; i n f r a d ; nuclear magnetic resonance spectroscopy and spectxafluorimetry. Fig. 2: U-nic t r a n s h a and energy states. S i g M € S a n d M ~ e n t r s The analytical process is d procedure of gaining information. At least, samples contain only latent information on the composition and stmcture, namely by their intrinsic UNIVERSITY OF IBADAN LIBRARY properties. By interactions between the sample and the measuring system, this infomation is transformed stepby- step into signals, measured results md useful. chemical information @ a m 2 004). Information is always connected with signals. In general, signals are definite states or processes of material systems. They can, therefore, be differentiated into static and dynamic signals. Examples of static signals are script, colours, images, figures and buildings. On the other hand, dynamic signals result from electrical, thermal, Optical, acoustic, or chemical interactions. Nowadays, these signals are converted in each case into electric signals, in which form they may be eeated and transmitted Finally, the essential signal characteristics are recorded in a suitable form. The process of signal genenition and evaluation is given by the chemical measurement prmss (Danzer 2007). Analytical signals are geherated by interactions between species of the anal*, to be precise, between certain f o m of intrinsic enera of them and an external system of matter and energy, respectively. The systems and resulting interactions are summarized in table 1. The signals often generated are in some instances minute and an subjected to sane degree of transduction and enhancement to make them visible and in essence make sense. But to the uninitiated, they appear nothing more than spikes or lines (fig. 3). The typical signals generated by some of the spectroscopic and spectrometric techniques carried out by Adegoke (2005) are presented iri figure 4. 'Herein lies the confusion and the allusion to the fact that spectroscopy is an abstract science. But I must confess I have been .at home with the subject of signal generation and interpretation since my introduction to the subject matter during Medical Laboratory Sciences training. UNIVERSITY OF IBADAN LIBRARY Table I: O w m k w d V.rbus Forms of Inbmctlom taking pbce between Meamring Samples and merent forna d Matter and Energy to prdoee Anrlgtlerl Swab . I Molecults Atom Chexnical Rcacdon rons (Solvated) 13lectrons Wccffonbulms Ion btams Particlematter 7a) Imlastic interaction interactions (energy m k r w ith sample metromagut tic Radiation-matter species: ( S ~ O S C O P y ) Radiation in-ons (b) Elastic interactions [energy transfer): DIFFRACTION, MICROSCOPY) Heat Tbcrmal i n m o n s Dhctcd move& h&om betwen p- (partitim, adsorption) Fig, 3: Diffmt types of signals mordings (schematic) obtained by various instmmmts and registering. UNIVERSITY OF IBADAN LIBRARY Fig* 4: Typical W, IR, NMR and MS spectra. Ultraviolet and Visible Spectroscopy Mr. Vice-Chancellor, I started my research career mostly in the realm of the applications of Ultra-Violet and Visible spectroscopy (UV-Vis), and I will give a very brief description here. In UV-VIS absorption spectroscopy, many organic compounds absorb-quites trongly with only a Limited number of inorganic ions doing same (Willard et al. 1988). The practice is to use reagents to make majority of inorganic ions without inherent colours to absorb radiation. The entire process involved in majority of UV-Vis spectroscopy has to do with absorption of radiation to produce electronic transition. In reality, the molecules are as energetic as he- modem teenagers. They invariably rock, roll, twist, jerk h d bend, and if the music is of the right rhythm, choice and frequency, the electrons within the molecule shall move from the 'ground state' to the 'excited state'. Electrons generally UNIVERSITY OF IBADAN LIBRARY found in the conjugated dohble bonds invhriably give rise to spectra in the W and visible regions of the electromagnetic spectrum. It is pertinent to mention here that an excited electron nonnally returns to the ground state in about 10-9 to 10d seconds (Kar 2005a). The absorption of radiant energy by molecules is influenced by several factors, including absorbing p u p s (or chmmophores), solvent effects, effect of temperature, and . inorganic ions. Adegoke and Idowu (2010) demonstrated comphensively the effects of chromophore, auxochromes, solvents and temperature on the W absorption patterns of a group of four novel dyes. Other functional p u p s that either increase or reduce the wavelength or intensity of absorption of the cwmophore are referred to as auxochromes. These species in themselves do not absorb light, but when present in preferred positions on molecules dramatically modify the absorption of such molecules. The applications of W-VTS spectmscopy fall under two main categories, qualitative and quantitative analyses. In structure determination, UV-VIS spectroscopy is used to detect the presence of chromophm like dimes, aromatics, polyenes, conjugated ketones and the likes. The technique used to be popular in &&mining identity and purity of drug molecules until the utilization of inh-red spectroscopy came to the fore. Adegoke (2005) copiously adopted W-VIS spectro~opy for the quantitative analyses of pharmaceutical phenol ethers using two main laws, Lambert's (equatiod 1) and Beer's (equation 2) laws. The quantum of the absorption is designated in tern of the absorption, & that is represented by equations 1-3. where, Io = Intensity of radiation passing into the absorbing hym, and I = Intensity of radiation passing out of the M m g la yer & i s a constant and I is the thickness (path length). UNIVERSITY OF IBADAN LIBRARY Combining equations 1 and 2 gives 3 which is Beer- b b e r t ' s law or simply Beer's law when I = lcm. u is called the molar absorptivity, E (when c is in mol d d ) and A=ic n (when c is in % w/v). Bearing in mind the ease in calculations and also the convenience of reference, the absorption of a l cm layer of a 1% wlv solution is usually recommended in most of the official compendia for many pharmaceutical substances and is evaluated by equation 4. where, c = Concentration of the absorbing substance represented as a parentage (w/v); and I = Thickness of the stbswbimg layer (cm). Mr. Vice-Chancellor Sir, majority of the pharmaceuticals do not absorb intensely in the region of less interference (visible region). Absorptions of most pharmaceutical com- pounds fall under the W region. Working in this region is often fraught with the difficulties of preventing non-specific absorption by most solvents, excipients, and since majority of substsulces, no matter how poorly endowed electronically will absofb here, scientists have over the years converted such molecules into newer ones through a variety of reactions. This conversion is however not limited to UV-VIS spec- photometry. When a compound is inherenay coloured, colorimeuic analysis is readily adoptable. This technique was developed UNIVERSITY OF IBADAN LIBRARY from the age-long visual comparative tests. However, with the development of simple colorimeters, reduction or complete elimination of visual emrs was made possible. The choice of a colorimetric procedure for the determination of a substance will depend upon such conditions as the concentration of the analyte in question, the condition under application and in particular the presence of interfering matrices (Basset et al. 1978). For compounds that are not inherently coloured, an initial reaction may be necessary to make applications of colori- metric methods possible. The challenge in such reaction(s) is the need to keep the various steps as simple as possible and to ensure that a complete conversion of the analyte of interest is made possible. The criteria for a satisfactory colorimetric analysis include specificity of the reaction, proportionality between colour and concentration, stability of the colour, reproducibility, clarity of the soiution and high sensitivity. Thus, in many instrumental techniques, it might be desirable to assay particular compounds in forms that are readily handled to improve sensitivity or selectivity. Usually, conversion of functional groups within the molecule to others more readily adaptable to the technique being adopted is preferred. This procedure called derivatization or derivative formation is applied in W-VIS spectroscopy, gas chroma- tography (GC) and high performance liquid chromatography (HPLC) (Adegoke 2012a). This has been the mainstay of my primary research focus. In such reactions involving applications of W-VIS spectrophotometry, colours are produced and having selected the & by a spectrophotometer, colorimeters are used for quantifying the amount of absorbing species. While this method has been popular for inorganic ions such as iron 0, Ni, Si, Zn and Co, colorimetric measurements of drugs and other organic compounds are now common after specific derivatization procedures. Although very few reactions are specific for a particular substance, many reactions are quite selective, or can be rendered selective through the UNIVERSITY OF IBADAN LIBRARY introduction of masking agents, control of pH, solvent extraction, adjustment of the oxidation state, or prior removal of interferents (Willard et al. 1988). Chemical derivatization fall under the category of indirect spectrophotometric analysis and the compounds of interest are often converted to those with different spectral properties. Chemical derivatization may be adopted in instances where the analyte absorbs weakly 'in the W region as is commqn with most drugs, .where interference by imlevan t absorption is present, where there is a need to improve selectivity of the m u r e o r when cost implications will favour adoption of a colorimetric method to that of a UV-VIS spectrophotometer (Davidsun 1997). GenemI Chwifxcation of Chemical Derivatbtion Reactions C A wide range of reactions have been adopted for the chemical derivatization of inorganic and organic pharmaceuticals. Majority of these reactions are colour-producing reactions (Adegoke 2012a). Colorimetric methods can selectively transform a h g , its impurity or metabolite so that the spectrum is shifted to the visible region and away from interference caused by mother drug, formulation components or biological substances, thereby conferring a further degree of specificity. Moreover, a drug with little or no useful absorption w be more sensitively determined by modifying it to a more highly absorptive chrrrmophote (Fell 1986). Some reactions have enjoyed wide applicability in chemical derivatization for the UV-VIS spectrophotometric deterrnination of pharmaceuticals. The major reactions are: ion-pair formation, complexation reactions, acid-base pro- cedures, enzymatic reactions, oxidation-reduction reactions and some miscellaneous methods as well as azo dye derivatization, hy drazine derivatization/Schiff base formation and organic charge transfer reactions/complexatim (Adegoke 2012a), UNIVERSITY OF IBADAN LIBRARY Ion Pair Analysis ion pair fomation, initially investigated in physical chemistry, was found extremely interesting for chemical analysis, including pharmaceutical analysis . Modem analytical methods.proved that the formation bf ion pairs is a consequence of the electrostatic, hydrophobic and charge transfer interactions and allowed optimal experimental conditions setting for their formation (Willard et al. 1988). Bmmocresol blue (BCB), brommmol purple (BCP) and bmmocresol green (BCG) have found great relevance'a s ion pair donors in most reported methods on pharmaceuticals, such as guanethidine sulphate, gumfacine hydrochloride, &usnoclor sulphate, guanoxan sulphate and debrisoquine sulphate (Wahbi et al. 1993); zohitriptan in tablets (Aydogmus and Inanli 2007) and phenothiazine derivdves in bulk drug and their pharmaceutical prepdons (Basavaiah and Krishnmurthy 1998). Complexation Resdom Complexation reactions have been adopted as an age-long approach for the analysis of metals and metalloids in water, pharmaceutical preparations and other matrices. The pro- cedure usually involves the selection of appropriate complexing agents, controlling the pH and appropriately selecting the temperature and solvents required. Once the metal ions bind the ligand, specific colour changes are obsemed, which can be quantitated as a function of the amount of metal ions present. This procedure is also utilized for the gravimetric analysis of metal ions. In recent times, the ability of some organic pharmaceuticals to serve as ligands has also been utilized' for the estimation of these drugs Complexation reactions have been used for the determinatior. of such metals as cerium subgroups (Zhen et al. 1999), lansoprazole with ~ e P~as+ava iah et al. 2007), some cephalosporins with I7e3+( Okoye et al. 2007). UNIVERSITY OF IBADAN LIBRARY Mdation-reduction Reactions Oxidation-reduction reactions have been used in some derivatization techniques. Amant adme HCI has been deter- mined by oxidation of the drug by ammoniuni molybdate parwish et al. 2006). Adegoke and Balogun (2010) demonstrated the ability of cerium ammonium sulphate to accurately deknnine some quinolones. Cerium ammonium sulphate has also found relevance in oxidation-reduction spectrophotomehy. It is commonly adopted for the oxidation of drugs that possess reducible moieties and, in turn, it gets oxidized to the cerate ion which has an intense yellow colour. In some determinations, the amount of residual cerium sulphate is determined by further reactions 'with such compounds as indigo carmine, methyl orange or p- dimeth y laminobenzaldehyde (Basavaiah et al. 2007). Other Methods Some other reagents have been used for the determination of a wide range of pharmaceuticals and include khloro-7-nitro- 2,1,3-benzoxadiazole (NBD-Cl), sodium 1,2-naphthe quinone4sulphonic acid (NQS),s odium nitroprusside and 3- methyl-2-betlzothiazolinone hydrazine (MBTH) HCI. Mr. Vice-Chancellor Sir, I have deliberateIy placed azo dye formation, h ydrazine derivatization and charge-transfer complexation last in this discussion on various techniques that have found usefulness in chemical derivatization procedures. This is because on this tripod have rested my long search for honours, and they have formed my tripodal trawl. A trawl when used as a verb refers to catching a fish or catching with a net or seine or more commonly. to search thoroughly, and when used as a noun connotes an act of drawing out fish or a catch. To me, bawl therefore means to search through a large amount of information or many possibilities; it also implies an investigation, hunt, runmiage, scan or thorough look. I have indeed put out my senses in all ramifications and set lines in preferred orders to catch possibilities of developing new methodologies from existing UNIVERSITY OF IBADAN LIBRARY and new reagents. I have focused on azo dye brivatization, Schiff base formation and charge transfer complexation for some eighteen years as a device to catch degrees and papers that served as pivots for my Masters' and Ph.D degrees and on which the declaration of my outstanding Professorial contributions rested. I must openly confess that in order to cany out any chemical derivatization procedure for an organic compound, a sound knowledge base of Organic Chemistry is important. I appreciate God for the wonderfd teachers I had in organic Chemistry in my undergraduate school days. I doff my hat for Professor T. Shambe who taught me Organic Chemistry (CHE 131) in 100 level and whose knowledge of the subject I admire so much that I used to feel 1 should have enrolled for B.Sc Organic Chemistry. Then, I passionately remember Professor V.C. Agwada in PCH 301 and 302 back then in University of Jos; he would come to class without notebook; he also ignited my interest in Organic Chemistry. Thus, while some run away from organic chemistry, these Chemists made me a lover of the complicated science involved in under- standing how reactions proceed and how electrons transpose themselves to ensure that a reaction is completed. The foundation in Organic Chemistry, Mr. Vice-Chancellor Sir, became the tunic required to know what reaction will proceed or which will not, and has made me, with all sense of modesty, the only pharmacist around who is not tired of trying out organic reactions for not on1 y colorimetric analysis but for synthesis of colours, colovants and coloured substan=. Organic chemists may not see the electrons moving, but by careful bderstmding of laiddown principles, we feel the impact in reactions and of course in observable manifestations of colours in many instances. The big question, Mr. Vice-Chancellor Sir, is: Do we see all things before we attest to their siflcance and relevance? Obviously Not! Hence, we may not see electrons and their disposition, but we obviously see the end results of their transposition. UNIVERSITY OF IBADAN LIBRARY Am Dye Derivatization Majority of the ckrivatization procedures I have camed out is based on formation of au, dyes. The azo dyes give rise to intensely coloured substances; they are characterized by the presence of the diazo linkage which brings two rings into conjugation and thereby extending the maximum wavelength L)to the visible region (fig. 5). As a result of such dye fornation, lower detection limits are obtained, while at the same time, some measure of selectivity is afforded. Azo dyes are produced by the diazo coupling reaction of a diazonium salt with a neutral, activated or deactivated skeleton. The formation of an azo dye proceeds by preparation of diazonium salt and coupling of the latter with a suitable compound. Dhmnium Sdts Diazonium salts are produced from amines. Each class of amines yields a different kind of product in its reaction with nitrous acid, HONO.T his unstable reagent is generated in the presence of the mine by the action of mineral acids on sodium nitrite. Peter Griess first discovered the original reaction in 1858 (Monison and Boyd 1992). When primary aromatic amines are treated with nitrous acid, diazonium salts are formed. Incidentally, structure I contributes more to the hybrid II, as shown by bond-distance measurements (March 1992a). UNIVERSITY OF IBADAN LIBRARY Fig. 5: Colorimetric determination of naproxen folloti ing am dye derivatization with C'DNBU (Idi~wue t a!. 20091, Prepamtion of Dhonium Salts The preparation of diazonium salts is by a pro&ss known as diazotization, and a salt of the ion results depending on the mineral acid used (Saunden 1949; Herbst and Hunger 1997). AT-NH, +' NaNQ +2HX cold r M - N ~ Y+Na X +2H@ A-nrb X could be HS04 or C1' if HsO+o r HCI is used. UNIVERSITY OF IBADAN LIBRARY Reactiotls of Diamnium Salts The large number of reactions undergone by diazonium salts may be divided into two classes: (i) Replacement reaction in which nitrogen is lost as N2 and some other atom or group becomes attached to the ring in its place. This is the best way to introduce F, CI, Br, I, CN, OH and H into aromatic rings (March 1992b). (ii) Diazo-coupling reaction in which the nitrogen is retained in the p d u c t (Momson and Boyd 1992b). Under the proper conditions, diazonium ions react with certain aromatic compounds to yield products of the general formula Ar-N=N-Ar, called am compounds or azo adducts. In this reaction, known as coupling readon, the nitrogen of the diazonium group is retained in the product, in contrast to the replacement reactions, in which nitrogen is lost. The .reaction on the aromatic substrate (coupler) proceeds by aromatic electrophilic substitution of the diazonium ion on sites on the ring predetermined by substituents present on the coupler as shown in Scheme 1 (March 1992b). The C-am, O-azo and S- am are equally possible. Scheme 1: Eiectrophilic substitution on benzene ring UNIVERSITY OF IBADAN LIBRARY ApplicaCions of A d y e F ormation in Colorimetric Analysis Two procedures are commonly adopted in the application of diazo coupling reaction for colorimetric analyses of pharmaceuticals: {a) Analysis of drugs involving preliminary di azotization of the dmg before coupling to a suitable reagent . (b) Analyses in which the diazonium salt is the reagent. Dhmtizstion of Dm9 befm Coupling In this method, the drug contains either a free primary amino group or other derivatives such as amide and nitro groups. I have alongside some of my students developed novel reactions through diszotization of drugs before coupling (Adegoke and Umoh 2009; Adegoke and Quadri 2012; Thomas and ~degike2 012; Thomas and Adegoke 2015). One of the reactions is presented in Scheme 2 for determination of cephalosporins. The earliest is the development of a new coupling component for sulpha- nilamide determination by Bmtton and Marshall (Bratton and Marshall 1939). The method involves colorimenic determination of sulphanilamide in blood or urine by first diazotizing the sulphpha drug after extraction and coupling with N-( 1- napht h y 1) eth ylenediamine dihydrochloride, a compound which later gained popularity as Bratton-Marshal reagent . UNIVERSITY OF IBADAN LIBRARY Sckm 2: Formation of Azo adduct between ceftriaxone and DM- (Adegolce & Q d r i 2 012) Mazotized Compound as the Reagent This was the main procedure I canied out during my Ph.D research (Adegoke 2005). This procedure has a broader application. Here, the reagent possessing a free primary m a t i c a mino group is diazotized using appropriate method. The magent is now coupled with h g s p ossessing appropriate activated aromatic skeleton. Earliest reagents in this category include diazotized pmitmaniline (Smith and King 1964) and diazotized sulphanilic acid (Kozlov et al. 1969). Diazotized p- nitromiline and dimtized sulphanilic acid have been used in an alkaline medium to determine ritodrine* HCI and its pharmaceutical preparation (Revanasiddappa and Manju 2001). Scheme 3 shows the reaction between aceciofenac and 4carboxyl-2,6-dinit~o~mndei azonium ion (CDNBD) as carried out by Adenbigbe et d. (20 12a). UNIVERSITY OF IBADAN LIBRARY Sehwc k Coupling reaction pattan bctween aceclofenac a d C DMD (Aderibigb et al. 2012a) ScW Base Formation M a n y colorimetric pmcedures are based on condensation reactions under suitable conditions between arnines and c h n y l s to generate Schiff's bases, hydrazones, semicarbazones or oximes (Scheme 4). Miwa, Yamamoto and Momose (1980) have extensively used 2- ni tmphen y l h ydrazine to estimate m h x y l i c acids including salicylic acid produced in hy dm1 y si s of aspirin. WhenRm=AUry iora fy ! , th8product iaa~s~ R- = N& (hyd-1, the product a hyd= R u NHCON ,b1- ( the product Is a =mica- ~*rO~(hydro>cy iamine ) ,Mprodudb~m Scbeme 4; Schiff Base Formation UNIVERSITY OF IBADAN LIBRARY The intensely coloured hydrazides produced are often determined colorimetrically . Prominent carbony1 group donors have been p-dimethylaminobenzaldehyde (Adegoke and Nwoke 2008) and pdimethylaminocinnmddeh; ie (Zawilla et al. 2002). Similarly, dihydralazine has been determined in pharmaceuticals after derivatization with 2- hydroxy- 1- naphthaldehy& (Pous Miralles et al. 1993). Charge T W e r C omplexstion Certain substances combine in a 1:l molar ratio to form addition products. The molecular addition compound is held together by weak forces, such as Van der Waals. The two new molecular orbitals f o d a re illustrated in the figure 6. Since the formation of these complexes involves transfer of electronic charge from an 'electron-rich' molecule (a k w i s - base dongr) to an 'electron-deficient' molecule (a Lewis-acid acceptor), they are called charge-tmsfer complexes (Kemp 1984). The structure of most charge-transfer complexes can be visualized as a face-to-face association on a 1: 1 donof: acceptor basis: only thus, for example, can maximum overlap of m a t i c r r-orbitals take place. Fi& 6: Electronic transitions for charge-transfer complexes. Ihnor and acceptor orbitals combine to f m t wo orbitals (a and b) for the mmplex. New electronic transitions for long I are then possible &ween a and b. UNIVERSITY OF IBADAN LIBRARY This kind of structure is difficult to draw, and most representations use one or other of the conventions shown below (Kemp 1984). Chloranilic acid CNoranilic acid (2, 5-dichlofo-3, Mhydroxy-p- benzoquinone) has enjoyed the widest application as a ~r- electron acceptor, while drugs possessing excess lz-electrons or those with non-bonding n-electrons readily pair with it b fum brilliantly coloured purple or pink adducts which are measured coloiimesically as a means of quantitation of the amount of analyte pment. SeverPll reactions of chloranilic acid with drugs have been reporzed Benesi-Hildebrand equation (Equation 5 ) has found particular usefulness in the estimation of the f o d m - constant of molecular complexes and it is commonly utilized for this estimation. Where, [AJo is the initial concentration of the acceptor (eharge-transfer reagent), A is the absorbance of the charge traosfer band, [Dlo is the initial cmcentraxi,on of the donor (drug or chemical), Km is the famation constant of the new charge transfer band and ECT is the molar absorptivity. A plat of [AJO/A against l/[Dlo will yield intercept as 1 / ~an d the slope as IlK e from where the formation constant and the molar absorptivity are obtained CT complexes are associated UNIVERSITY OF IBADAN LIBRARY with the appearance of new W-VIS absorption bands (Mulliken and Persun 1%9). complexes are sometimes produced as naction inttmediates (Ross and Kuntz 1954, Coloter 1963; Khan and Ahmad 2009), and most often they exist as stable donor-acceptor ducts ( A n b w s 1954, A L Attas 2009; Y w d 19 73). One common mechanism for the CT complex formation c d e d out by Adegoke et al. (2014) for the &-nation of trimethopriml sulphamethoxazole is illustrated in Scheme 5. % CT eomplexation fmt ion between CAA and ttilrstthoptitnl s u l (Adegok~e et al. 2~014) Mr. Vice-Chancellor Sir, the application of the tripodal reactions of am dye famation. Schiff base formation and charge tmsfcr complexation, among other reactions, give rise to mlom. Calm could m e as c o l ~ t sa,nd their application in colorimetric analyses has led to the utilization of colound ~~ for simple, sensitive, selective and determination of compounds of phmwceutical UNIVERSITY OF IBADAN LIBRARY interest. In the next few paragraphs, I will give a brief overview of colours and colourants before considming my specific contr,i butions to colour chemistry. . )!!A( . , I I ,- .;~#;* 5(1 - q i ~ ' J\ w Coiours Colours have been with man from the ancient times. h e to its attractiveness, everyone is thrilled by the brilliance and alluring look of coloured substances. *A life without colom appears bland and blank. Thus, the appropriate aesthetics to any object in nature is judged by the allure or the ~ 0 1 i0t ~ ~ possesses and gives out. Many people are obviously fascinated by colours. The word translated "colour" in The Holy Bible literally connotes "an eye". Colom arise from white light. "White" light kontains the -re range of wavelengths within the visible region of the spectrum and if shone through a prism splits into a rainbow of respctive colours. This rainbow of colours has been popularly given the acronym; ROYGBIV (Red, Orange, Yellow, Green, Blue, ' Indigo and Violet), and the colours constitute $he visible spectrum of the electromagnetic radiation. .The origin of the names of the colours has been an intriguing subject. Dating back to centuries, the names of our everyday colours have origins in the earliest known languages. According to linguists: There was a time when there were no coIour- names as such . . . and that not very remote in many cases, when the present colour-words were terms that could be used in describing quite different qualities [inclu dashy, loud, gaudy . . .d ing] gay, lively, smart, dull, deaQ dreary . . . tarnished, stained, spotted, dirty, smeared . . . faint, faded [and feeble] (Melissa 2014). Recent research in this area has demonstrated that this hierarchy matches human's reaction to different frequencies in the visible spectrum; that is, the -stronger our reaction to that colour's frequency, the earlier it was named in the culture or as Loreto et a1. (2012) put it: UNIVERSITY OF IBADAN LIBRARY The colour spectrum clearly exists at a physical level of wavelengths, h u m s tend toreact most saliently to certain parts of this spectrum often - selecting exemplars for them, and finally comes the process of linguistic colour naming, which adheres to universal pattms resulting in a neat hierarchy.. . Similar to other cultures, English words for the colours generally followed that same pattern, with black and white coming first, and purple, orange and pink coming last (Melissa 2014). The names given to different colours have their origin from the parents of modem English language such as Proto-Indo-European (PIE), Proto-Germanic and Old English languages. Black derives from .wordsinvariably meaning the colour black, as well as dark, ink and 90 bum." Originally meaning, buming, blazing, glowing and shining, in PIE was bhleg: This was changed to bl& in Proto-Germanic, to blaken in Dutch and blnec; in Old English. This last word, blaec, also meant ink, as did blak (Old Saxon) and black (Swedish). White began its life in PIE as hintos and meant simply as white or bright. This had changed b khwitz in Proto- Germanic, and later languages bansfomed it into hvitr (Old Nm), hwit (Old Saxon) and wit Dutch). By the time Old English developed, the word was h i t . For red colour, in PIE, red was reudh and meant red and ruddy. In Proto-Germanic, red was rut-, and in its derivative. languages r d r ( Old Norse), rod (Old Saxon) and r@d (Danish). In Old English, it was written read. Oreen.means grow, in PIE, it was ghre. Subsequent languages wrote it grene (Old Frisian), g r m n (Old Norse) and grown (Dutch). In Old English, it was grene and meant the colour green as well as young and immature. Thousands of yeam . ago, yellow was considered to be closely related to green, and in PIE, it was ghel and meant both yellow and green. In Proto-Germanic, the word was gelwaz. Subsequent incamations of German had the word as UNIVERSITY OF IBADAN LIBRARY girlr (Old Norse), gel (Middle High G e m ) a nd gelo (Old High G e m ) . As late as Ofd English, yellow was written geolzr and geol~tuB. lue was also often confused with yellow back in the day. The PIE word was bhle-was and meant "lighttoloured, blue, blond yellow" and had its root as bhel which meant to shine. In Proto-Germanic, the word was blae~t*~an:,d in Old English, it was blaw. English also gets some of its words from French; and blue is one of them. In Old French (one of the vulgar Latin dialects whose height was between the 91h and 13' centuries AD),b lue was-written bleic and blew and meant a variety of things including the colour blue. Brown is derived from the Old Germanic for either or both a dark colour and a shining darkness (btunoz and bnma), brown is a fecent addition to English language. In Old English, it was bniri or brune, and its earliest known writing was in about 1000 AD. Purple also skipped the PIE and seems to have sprung up in the grnc entury AD,i n Old English as pztrplil. Borrowed from the Latin word pupura, purple originally meant alternately, "purple colour, purpledyed cloak, purple dye . . . a shellfish from which purple was made . . . [and] splendid attire generally." Orange colour's name derives from the Sanskrit word for the fruit naranga. Thus, the colour orange was named after the fruit, not the other way around. This transformed into the Arabic and Pers'an narculj, and by the time of Old French to pomrrte d'orenge. It was originally recorded in English as the name of the colour in 1512. Before then, the English speaking wodd referred to the orange colour as geoluhread, which literally trans1 ates to "y ellow-red." Pink is one of the most recent colours to gain a name, pink was first recorded as describing the "pale rose colour" in 1733. In the 16" century, pink was the common name to descdbe a plant whose ' petals had a variety of colours (Dianthus), and it originally may have come from a Dutch word of the same spelling that meant small (Melissa 2014). The values of a colour are recognised by making its tints and shades. Tints are light values that are made by mixing a UNIVERSITY OF IBADAN LIBRARY colour with white. For example, pink is a tint of red, and light blue is a tint of blue. Shades are dark values that are made by mixing a colour with black. Maroon is a shade of red, and navy is a shade of blue. In addition, colours are evaluated based on the hue (the percentage of primary colours); the chroma (richness of the colour) and the lightness (this refers to the amount of light reflected). The desire of humans to have any object they appreciate to be coloured led to the use of coloufants* c010urants Colourants are pigments or dyes used in giving acceptable aesthetic value to an object. A dye by definition is a coloured compound that adheres to cloth and retains its colour against the attack of light, moisture and soap under normal conditions of wear (Nathan and Mwth y 1968). Pigments are inorganic or organic, coIoured, white or black materials which are practically insoluble in the medium in which they are incorporated. Dyes, unlike pigments, dissolve during their application, and in the process lose their crystal or particulate structw. It is thus by physical characteristics, rather than by chemical composition, that pigments are differentiated from dyes (Herbst and Hunger 1997). The history of pigment application dates back to pre- historic cave paintings. which provides evidence of the use of ocher, hematite, brown im ore and other mineral-based pigments more than 30,000 years ago. Cinnabar, azurite. malachite and lapis lazuli have been traced back to the third- millennium BC in China and Egypt (Herbst and Hunger 1997). The beginning of organic pigment application dates back to antiquity. It is certain that the art of using plant or animal 'cpigments" to extend the specrral range of available inorganic colourants by a selection of more brilliant shades had been practised thousands of years ago. Uses of dyes are diverse. They have been used as indicators of solvent polarity, chemical equilibria, and molecular environment as well as indicators of aggregation and molecular order UNIVERSITY OF IBADAN LIBRARY (Olaniyi and Ogungbamila 1991; -Herbst and Hunger 1997; Buss and Eggers UWX); Rageh 2004). C ld f iea t too~f~ O q@c Pigments and Dyes Clmification of organic pigments is commonly done either by chemicai constitution or by colouristic properties. Srrict separation .of the two classification systems is however not very practical. Synthetic- organic dyes span a wide range of varying chemical smctures including among others, azo-, mtlquinones, carbonyls, nitros, rriphenylmethane and phthalocyanines. About 50% of dl the dyes manufactured come under the class of azo dyes prepared from diazonium salts. Azoammatic compounds have attracted much attention in recent times due to their emerging applicatiuns in dye stuff industry, for being excellent photo-aligning sub st^^^ for liquid crystals, highly efficient photorefmctive media, acid- base, ndox and metallochromic indicators, optical activators and opticalstorage media; Am dyes have also been used in photuatalytic reactions as catalyser exposing to ultraviolet light (Rau 1990; Kumar 1992; Nakamura et al. 2000: Yaroschchuk et al, 2001; Salahivel et al. 2003; Woud et d. 2004; Muruganandham and Swaminathan 2004; Muruganandham et al. 2006). They have also been widely employed as histological stains, such as: acid alizarin violet and Sudan series (Buss and Eggem 2000). One other major application of dyes, in particular azo dyes, is their use as colour additives in food, cosmetics and pharmaceuticals (Sasaki et al. 2002). It is said that we "eat with our eyes as much as with our mouths," and that's certainly the case when we walk down the aisles of a supemaiket (fig. 7) or shopping malls (Kobylewski and Jacobson 2010). UNIVERSITY OF IBADAN LIBRARY Fig 7:V arious consumer goods made aesthetfeally awptable by coloumn@ (FDA 2007). Despite extensive public awareness against artificial colouring, colomts have found usefulness in improving the aesthetic value of products. A variety of consumer goods are dyed with azo dyes. The consumer goods include foods, beverages, lipsticks, stick-on, ' tattoos, toothpastes, hair products; and the like. Some of these dyes present potential - risk to man and his environment..T he adverse effects of certain azo dyes on human health were first apparent when . . . cases of bladder cancer were detected in workers involved in the manufacturing and use of bemidinebased dyes (Oh et aI. 1997; Boms et al. 1999). Prior to that period, many bemidine-based dyes with disazo and t iam structures were used as direct dyes. Nowadays, however, dyes based on bemidine add its congeners can no longernbem anufachmd for textiles in the United States due to safety concerns (Bae and Freeman 2002). Two prominent terms associated with the assessment of the toxicological properties of am dyes are mutagenicity and genotoxicity. Mutagenicity refers to ' the induction of permanent transmissible changes in the amount oi structure of the genetic material of cells andlor organisms. Genotqxicity, UNIVERSITY OF IBADAN LIBRARY on the other hand, is a broader term which mfen to potentially harmful effects on genetic material which are not necessarily associated with mutagenicity. Thus, tests for genotoxici ty include indication of induced damage to DNA, such as unscheduled DNA synthesis, sister chromatid exchange and DNA strand 'breaks (European Chemical Bureau 2003). Unti 1 recently, these tests were frequently used. However,. a more useful approach for assessing DNA damage is the single-cell gel (SCG) or Comet assay et al. 2000). The ~ - i m s" SCG" 'or 'kcornet" are used interchangeably, ihd it refers to the assessment of individual cell DNA migiation pattems produced after treatment with candidate genotoxins when compared with negative and positive controls. The mutagenicity of azo dyes has been said to arise from the reduction of the azo group by the action of intestinal anaerobic bacteria or the hepatic am reductases, which releases aromatic ami nes. Therefore, dye mutagenicity is often related to the mutagenic properties of the aromatic amines employed in the synthetic steps or present as degradation products (Oh et al. 1997; Boms et al. 1999). With regards to the genotoxicity of aromatic amines, it is known that the presence of ring substituents in the position ortho to the amino-( -NH2) group reduces mutagenicity (Gong et al. 2002). These studies have led to non-mutagenic benzidine analogs containing bulky alkoxyl groups ortho to the amino group and amino derivatives of the dihydmphenophosphazine ring systems (Bello et al. 2000). Thc few azo dyes that have been certified as approved colourants for preparations intended for systemic and topical ' use me those that have been shown to be sufficiently safe in animal and clinical studies (Bello et al. 2000). The dyes include sunset yellow (1V) and allura red (V). The two dyes shun: u common p e n t stwture; sulfonated phenylazo- hy drox ynrphthulenes. UNIVERSITY OF IBADAN LIBRARY Due to the extensive awareness. and publications of toxicological data yearly, attention is now shifting to naturally obtained colourants for incorporation into products likely to gain contact with man through food. inhalation. skin and mucous membrane (Francis and Markakis 2009; Wissgott and Bortlik 1996: Espin et at. 2000). The natural colourant area can be subdivided into anthocyanins, betalains, chloro- phyll~, carotenoids, flavonoids, polyphenols. Monascus. hems, quinones, bili proteins, safflower, turmeric and ~s.c .-e ~taneous. The debate however is-still on-going. One dear fact is that there is a need for international harmonization of various standards and acceptable admissible dai 1y exposure to colour additives. It inust be emphasized that approval of one colour additive for a product does not necessarily transfer such approvals to other colourants. Everyone must be educated with regards to what can be tolerated, as idiosyncrasies have been associated with the use of some products containing coloumts* Mr. Vice-Chancel lor Sir, having examined the basis of colours, coloumts and eoloured substances. 1 hereby. present my contributions to Colour Chemistry. My contributions to Colour Chemistry run through the main tripodal triiwl of azo dye deri vati zation, Schiff base formation and c hmge transfer complexation. I will along the. line describe some other extensions to this story. Old Reageat, New Applications I Pnm-dirnethylaminobemI&hyde, DMAB (Ehrlich' s reagent), has found usefulness in a wide range of rppliclttions from analytical, biochemical to synthetic procedures and processes alongside other rniscel laneous nppliciltions. The unique structural fentuk contilining a pcaru-dimet hy lamino UNIVERSITY OF IBADAN LIBRARY substituent to an aldehyde moiety renders it highly reactive towards a wide range of compomds, and this has made it useful as a condensation m k n t and oxidizable and reducible reactant in many reactions. Adegok (2011a) presented a comprehensive review of the diverse applications of DMAE spanning over a century with a prospect that this compound will be more relevant in years to come in mimbiology, chemical -pathology and organic synthesis, as well as phamaceutical and analytical chemistry. The diverse potentials of this compound led to my mwling into its applications, and, indeed, I have found new applications for the compound. The ability of DMAB to fom condensation products with both primary and secondary amino groups have been explored for the development of simple and rapid analytical techniques for clinically useful drugs. DMAB was used for the assay of hydralazine hydrochloride in bulk and dosage f o m (fig. 8) based on mndensatibn reaction to generate an instant greenish-yellow coloured product (Adegoke and Nwoke 2008). ' Rg. 8: Formation of hydrazone m a hydr alazim and DM-. In another report, a new, simple; costeffective spectro- photometric method was developed for the determination of olanzapine in phamaceuticals (Adegoke et al. 2014a). The UNIVERSITY OF IBADAN LIBRARY ability of DMAB to be reduced and oxidized through the aldehyde group has also been utilized for accurate determination of some drugs. Adegoke and Osoye (201 1) developed an alternative simple, accurate and precise method for the determination of artesunate and di h ydroartemisinin in bulk samples and @sag,g forms. The method involves the reaction of the reactive methylene centres generated in situ from the acid decomposition of the artemisinin derivatives with p-dimethylaminobenzaIdehyde @MAE). DMAB was reduced to the purplecoloured alcohol, and this was quantitatively used to estimate the concentrations of the artemisinin derivatives (fig. 9). This reaction represents the first ever report of a full colorimetric determination of arkmisinin derivatives in literature. Fig. 9: F~rmationo f a purple colowed product from DMAB following reaction with artemisidin derivatives. A simple, accurate and sensitive spectrophotornetric method was also developed for the determination of ci profloxacin, pefloxacin and sparfloxacin. The method was based on the oxidation of these drugs with cerium (N)in the presence of perchloric acid and subsequent measurement of the excess Ce (IV) by its reaction with DMAB to give a brownish coloured product. The decrease in the absorption intensity (AA)o f the coloured product due to the presence of UNIVERSITY OF IBADAN LIBRARY the drug was correlated with drug concentration in the sample solution (A&goke and BaIogun 2010). Old Reagent, Novel AppIications Of the varied applications that have been reported for the utilization of DMAB in analytical and synthetic chemistry, its application as a coupling component has not been reported. The compound has a peculiar structure, and its ability' to direct incoming electmphile is determined by its 1& disubstituents; the aldehyde group and the dimethylamino substituent. By theoretical considerations, positions 3 and 5 (mowed) should be able to accept an electmphile to give an azo adduct. This is made possible by the deactivating effect of the aldehyde group (directs to nreta positions; 3 and 5 ) and the p i t iv e inductive effect of the dimethylamino fragment (directs ortho positions; 3 and 5); themfore, both substituents augment each other. As sound as this theory may seem, in practice, azo adducts, if formed, are not stable and readily decompose. Thus, Adegoke and Umoh (2009) discovered a new approach to the application of DMAB in azo adduct formation. Since the aldehyde group withdraws electron and possesses an internal mesomeric effect, any factor that will diminish this inherent behaviour will dramaticany enhance and pronounce the orthodirecting influence of the dimethylamino fragment. This mesomeric effect, we found wt, gets pronounced in aqueous systems and is destroyed in polar aprotic solvents like methanol. Thus, the ,first ever report in literature of the ability of DMAB to function in this regard was reported by my group. The mechanism envisaged was that coupling of the diazotized skeletons with DMAB in UNIVERSITY OF IBADAN LIBRARY methanol (as opposed to water) destroys the deactivating internal mesomeric effect of the aldehyde functional group thus permitting optimal activating influence of the dimethylami~0g roup. The end result is usually the formation of coloured azo adducts that are determined using the visible wavelength range. This opened wide spheres for spectro- photometric analyses of drugs. . . I I The first application of this mechanism was fo; the determination of reduced and diazotized metronidazole and tinidazole (A&goke and Umoh 2009). The procedure involved coupling of diezotized nitroimidazoles with p dimethylaminobenzaidehyde @MAB) to form a p n i s h - yellow solution. The persistent yellowish-green wlour produced was stable for seven days. This colour is completely different from the salmon-pink p r o d d after condensation d o n between reduced nitroimidazoles and D M . Figure 10 presents the structures of the new compounds produced between DMAB and MZ and 'IZ The possibility of steric hindrance on substitution by diazotid nitmimidamles is eliminated as the dimethylamino group is staggered from the diazo linkage (bond angle between N8, C14 and C12 for the rZ adduct is 120,456" and that of the MZ adduct between N18, C10 and C4 is 122.665'). This shows that the angles around a main chain nitrogen atom (the dim linkage) are all approximately equal to 120 degms: consequently, the group is planar. Likewise, the torsion angles are 0.94 and 0.33g0, respectively, for the TZDMAB and MZDMAB adducts. Since the torsion is between 0 and +W, the stereochemical arrangement is of the syn type. Both molecules are therefore of the trigonal planar skeleton. Both the bond and torsion angles favour the formations of a stable adduct. UNIVERSITY OF IBADAN LIBRARY . F4& 1Q=S- ofh-addmfbrrreed ww€mDMAlB and (8) - ~ i d a m 1aend (b) TtnidamIt, In-anothero f such applications, a new simple, accurate and cost-effective ~ p h o ~metihodc was also developed for the analysis of some cephdosporins (mftriaxone, &azi&me5 cefixime, cefo@xime and wfuraxime) in hulk samples and pharmaceutical dosage fams (Adegoke and Quadri 2012). The spectra me pmentd in figure 11. Two antiviral agents have also bcen successfully betamined using this reactid: acyclovir (Thomas and Adeg~ke2 012) and ganciclovir in figure 12 (Thomas and i$degoh 2015). Both methods are simple, inexpensive, 'repXOducibIe and fast. The mction has also been applied the simultaneous detenrdnation of airnethoprim and sulphamethoxazole using solid phase extraction as a preliminary selective step (Olaifa 2014). Similarly, the -ti011 has been successfully h p W fbr the accurate determination of gaqentin in bulk and dogage forms (Aiyenale 2016). UNIVERSITY OF IBADAN LIBRARY 3043 400 So0 ' Wavelength lnm) Fig. 11: Overlaid absorption spectra of am adducts formed between the cephalosporins and DMAB . '& Fig, 12: Overlaid absorption s w t m of ganciclovir, diazo[ tncd ganc IC IUV~T and su, adduct formed \;A& DMM. . UNIVERSITY OF IBADAN LIBRARY Old Reagent., Old Applications, New Devises Chloranilic acid ranks the highest in the order of priority of reagents that have been used in the application of charge- transfer complexation (CTC)r eaction of acceptor molecules. While this reagent is an old one and the applications well established, I have, along with my students, devised some new ways to evaluate the possibility of CTC formation or othewise through the applications of chemometrics. I have found out that there is a relationship among several factors and parameters which will determine and support charge transfer complexation. Thus, CTC reactions have been applied for the accurate dekrmination of metronidazole, tinidazole (Adegoke et al. 2010a; Adegoke 2011b), lumefantrine (Adegoke et al. 2011), trimethoprim and sulphmethoxazole (Adegoke et al. 2014), as well as olanzapine (Adegoke et al. 20 15). I We have discovered that the estimation of the formation constant as a function of temperature gives an 'indication of the stability or otherwise of the new CTC. In addition, more practical approach has been the ph ysicochemical parameterization of the factors that can account fur the stability of the CTC and thus permitting ready and accurate determination of the drug molecules in complex matrices. In the determination of olanzapine, OLP (Adegoke et aI. 2015), chemometrics were conducted and provided accurate explanation to the formed complex. The first ph y sicochemical parameter calculated was the transition energy of the complex which is obtained from the expression hvcT where h is Planck's constant and vcr is the wavenumber of the absorption peak of the CT complex. The transition energy was found to be 2.303 eV. Two other physicochemical parameters estimated were oscillator strength and transition dipole 'moment. The oscillator strength V) is a dimensionless quantity used to express the transition probability of the CT band and the transition dipole moment (pm) of the CT complex (Martin et al. 1983). Both parameters are obtained from equations 6 and 7 mpectivel y. UNIVERSITY OF IBADAN LIBRARY whm, Avln is the half-width i,e, the width of the band at the half the maximum absorption, and Av --wavenumber at the absorption maximum. The oscillator strength, f and the transition dipole moment obtained ere 7.289 and 0.892 Debye mpectively. The ionization potential, lo, of the donor in the charge transfer complex was smother ph ysicochemical parameter calculated using the empirical equation derived by Aloisi and' Pigantru, 1973 @resented in equation 8). Where, vm is the wavenumber of the CT band in cm"'.b was found to be 6.054 eV. The resonance energy of the complex (RN)i n the ground state is obtained from the theomtical equation derived by Brieglab (1% 1) given in equation 9. where, sm is the molar abmptivity of the complex at the maximum of the CT hvcr is the k i t i o n e nergy of the complex. The mnance energy was calculated as 4.687 eV for the new molecular wmplex. The dissociation energy (W) of the formed complex between OLP and CAA was calculated from the transition energy (hvn), ionization potential of the donor (lo) and the electron affinity. of CAA (EA= 1.1) using the relationship in equation 10 (McConnel et al. 1953). UNIVERSITY OF IBADAN LIBRARY The dissociation energy was fdund to be 2.65 1 eV. From the results presented in table 2, some .ob&rvable trends are clearly evident from these ph y'sicochemical parameters. The values obtained point to the good stability of the complex formed between -CAA as acceptor and olanzapine as donor. The ionization potential of the donor gave a high value of 6.054 eV denoting that O W is a good n- e l m n d onor and making the electrons readily available for donation to an acceptor such as CAA. Table 2: PhysiCOCBemicaI Parametem for the Fommtbn of CT- complex between Ohmpine and Chloradic Acid The secondary amino group found in olanzapine has proven to be a useful electron donor from the results of the ID obtained. The transition energy is about two times less than this ionization energy of the CI' band; hence, the energy banier required for elwtronic transition is readily surmounted, and the complex is produced readily. This further confirms the avidity at which the complex was produced. The .dissociation energy (W)w as 'also found to be far lesser than ID. Thus, the spontaneous decomposition of the CT complex will be minimal especially at room temperature where these values were obtained from. The high resonance energy of the formed complex will produce a stabilization effect on the complex, and this is expected considering bulky nature of O W and the tendency to be stable ui s donation of a lone pair electron to CAA. In addition to the foregoing, we have also estimated the thermodynamic parameters, and they have given us opportunity to adequately discover the propensity or UNIVERSITY OF IBADAN LIBRARY othenvise of CTC formation. The thermodynamic functions, standard free energy change (A&), the enthalpy change (AH) aid -the'e ntropy change (AS) were obtained from the well- established equations 11,12 and 13 respectively. The enthdpy of the CT formation was obtained by plotting the Log of formation constant against the reciprocal of absolute temperature. The plot is presented in figure 13. Table 3 contains the various thermodynamic parameters obtained alongside the molar absorptivit ies and formation constants at the four temperature levels. The standard free energy gave a negative value at all the four temperature conditions considered in this experimental design. This result points to the exothermic nature of the complex fornation. This thus explains why higher temperature values led to .decrease in the absorbance of the complex. The Gibbs free energy became increasingly higher with increase in temperature denoting that it becomes dificul t to generate the complex at higher temperatures. Since it is established that the formation of the 'complex occurs through an exothermic process, higher temperature will prevent the avidity of reaction and the spontaneity of the charge transfer complexation. The high Gibbs free energy obtained even at room temperature however attests to the ease of formation of the complex. Though relatively small, the enthalpy change - also points to the possibilities of ease of formation of the CT complexes. The enthalpy ' of formation for the molecular complex was found to be -0.8803 KJMO~-l. The entropy also gave relatively high values with the value reducing as temperature increased. UNIVERSITY OF IBADAN LIBRARY Fk. W: Plot of Log K of OLPCAA complexes as a funetion of i/f (IC'). A@ is thefie energy M g e : AH is is enthdpy change and AS is the - w Y change With the suasses recorded adopting this oldbr eagent, c h l d l i c acid, we are currently developing some new nitronaphthalenes as charge transfer acceptors. This is the thrust of the Ph.D. research of one of my students, Offiong Umoh, who has made tremendous p r o p s o n the Aption of novel h y h y l nitronaphthalenes of varying structms as charge transfer -tor for clinically useful drugs. UNIVERSITY OF IBADAN LIBRARY New Rea.pnt,'NovelA pplications Mr. Vice-Chancellor Sir, following the'successfulc ompletion of my,MSc degree in 1999 with a project utilizing diazotized 4-amino-3,5-dinitrobenzoic acid (ADBA) developed by Idowu (1998). I M v e d a 'terrible discouragement as journal outlets rejected the suppod new methodology reported for aspirin. The comment was that there was no distinction between the spectnun of aspirin and that of salicylic acid following reaction with dimtized ADBA. This same report was received for halofantrine ,repond by Idowu (1998), and no success was recorded for pamcetamol. With a sound knowledge base in organic chemistry, I threw my haw1 line into literature and sought for drugs that possess activated skeletons. Also, from a thorough knowledge of drug metabolism, I opined that sites liable to deactivation through functional group addition ifi dmg molecules during metablism could be possible sites for attack by an electrophile. The thorough search of Literature pruduced about 250 drug molecules that could be likely candidates. About 50 compounds were eventually mmed in the local market. The challenge I was left with was to produce the precursor mine (ADBA) in a high state of purity. This was accomplished and attested to by Tdowu'S c o l l a ~ o r isn Japan with the ADBA producecl having a 99.99% purity. Several refrnernents of the diazotizafun process led to the possibility of a reagent grade diazonium ion. This provided the platform to be involved in the mining of several B. P h m , PGD and M.Sc. students over the years, while it made a platform available for me to also complete my Doctoral research. he first successful. report for h e application of dimtized ADBA was one of the candidate drugs I trawIed into the chemical libmy of likely drugs for analysis by diazotized ADBA. Mefenamic acid was successfully determined by wlorimetric. means (Idowu et al. 2002). The diazonium ion was re-christened 4cIuboxyI-2,6- dinimbenzene diazonium ion (CDNBD) to emphasize the species involved in the reaction with activated skeletons. The UNIVERSITY OF IBADAN LIBRARY applications of ecarboxyl-2,6-dinitrobemene dimnium (CDNBD) ion as a derivatizing reagent was extensively adopted as an dff-shoot of my MSc research and for my Ph.D research endeavour. One chemical group that was analyzed with CDNBD is the heterogeneous skeletons referred to as pharmaceutical phenol ether ho~logues.T he use of the reagent led to the ready determination of pharmaceutical phenol ethers whose determination h i t h h as a chemical group was not possible by azo dye formation since phenol ethers being weakly activating do. not react with majority of diazonium ions. Also, CDNBD when compared with diazotized sulphanilic acid and para-nitroaniline gave clearly defined peaks for such pharmaceutical phenol ethers as indomthacin, pfopranolol, nimesulide, reserpine, pindolol, astemizole, naproxen, nabmetone, griseofulvin, nadolol and ciomiphene (Adegoke et al. 2005). The exceptionally high reactivity of CDNBD was demonstrated by the accurate determination - of ppranolol (Idowu et al. 2004), indomethacin (Adegoke et al. 2006a1, nadolol (Adegoke et al. m b ) , nserpine (Adegoke et al. 2007a) and nabwmtone (Adepke et al, 2007b). The spectra produced on coupling of these phenol ethers with CDNBD are presented in figure 14. Varying stoichiometric ratios were obtained and in particular for indomethacin, a 2:l mole ratio of CDNBD to indomethain was obmed. Spectroscopic characterizations using NMR and mass spectrometry of its azo adduct revealed that simultmus substimtion on the indomethacin molecule must have taken place. This may be due to the isolated nature of the rings present in indomethacin (fig. 15). This is however a remarkabIe discovery as substitution of an electrophile on a ring deactivates the ring and makes multiple substitutions diEficu1t. The high reactivity of CDNBD also led to the f o d o n o f azo dyes with artemisinin derivatives. This was also the first report of azo dye formation by artemisinin derivatives (Adegoke et al. 20 lob). UNIVERSITY OF IBADAN LIBRARY 15: Dim coupling reaction between indomethacin and CDNBD. UNIVERSITY OF IBADAN LIBRARY This once again repments a significant contribution as thk artemisinin derivatives do not belong to the class of skeletons for which am dye formation - might be plausible. The mechanism envisaged was that conversion of artemisinin derivatives takes place in the presence of strong acids, and this produces reactive methylme k t r e s . The reactive methylme centres are known to form azo adducts with highly reactive diazonium ions of which class CDNBD falls. The evidence for the formation of new am adducts was provided by the formation of hydrophobic brown coloured am adducts and proton NMR established the formation of the azo adducts. The reaction pathway is presented in figure 16. Once g o d detection limits are recorded in UV, pre-column derivatization is carried out to improve sensitivity in HPLC (Adegoke 2012b). The success recorded in the W spectrophotomeeric work led to the extension to liquid chmatopphic analyses (fig. 17) of artemisinin (ATS), artesunate (ART), artemether (ATM) and dihydroarkmisinin @HA) with good sensitivity recorded (Adegob et al. 2012~). This is the first practical application of CDNBD as a pre column derivatization reagent for the estimation of pharmaceuticals. * Flg. 16: Coupling reaction pattmn fm the formation of am duc t s between artemisinins and CDNBD. UNIVERSITY OF IBADAN LIBRARY Fig. 17: Qmsentative chn,miogramsu sing optimized c h r o m ~ i c separation conditions; (a) CDNBD, (b) ATS (CDNBD-ATS p a k 7.45 mia), (c) ART (CIDNBPART peak 7.45 min), (d) DHA (mr@D-ampe as 7.51 min) ar~d( el ATM (CDWD-ATM pealr 7.45 min). The CDNBD reagent has also been demonstrated to possess some venatility as it was found to couple readily with aceclofenac (Adefl bigbe et al. 201%) and reduced nifedipine (Aderibigbe et al. 2012b). New Chemical Entities Mr. ViceChanceUor Sir, a critical requirement for the completion of my Ph.D research was to demonstrate the mechanism underlying the dido coupling reaction betyeen CDNBD and the phamaamtical phenol ethers. Majority of phenol ethers do not couple \yith diazonium ion because they belong to the -class of moderately to weakly activating substituents. For over three years of working on the bench for my PhD degree, what I observed was the change of colours of the drugs upon contact with CDNBD (Adegoke 2005). As stated by Saunders (1949b), a notable characteristic of coupling of phenol ethers with highly reactive diazonium ions UNIVERSITY OF IBADAN LIBRARY is that the diazo coupling reaction proceeds with partial or complete removal of the dkyl group of the ether, a remarkable occurrence as phenol ethers are by no means easily hydrolyzed. They are hydrolyzed by the strong acid, hydroiodic acid (HI). While the proportion of the sidexhain removed depends on the diazocompound, it was hitherto not clear whether re- substitution takes place or that the diazonium displaces the . OH remaining. In actual fact, a dramatic experience occuned with ppranolol that had colour . changes observed in its coupling with CDMBD from blue to violet to reddish-pink which could therefore be attributed to the cleavage of the side chain. Thus, the blue colour may represent the azo adduct of intact pmpranolol and other colours to various stages of cleavage. From the NMR of propranolol-CDNBD azo adduct, it is clear that the re-substitution of the alkyloxamine side chain does not take place and the presence of a strong 0-I&& in the IR shows a residual OH on the ring. Literatures that I could not source and access in t h m years of P h 9 work became handy in three days! For this I remain grateful to God for the MacAlthur short duration support to visit the Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland in 2004. Mr. Vice-Chancellor Sir, I pray daily for our Universities in Nigeria that we will get to that advanced state of getting both ancient and timely literatures for research endeavours. I sincerely believe that fqum like this must uphold the truth and make proclamations about results of research that were generated to e m p ublications leading to Professorship. I can play to l e gallery to earn applause from a distinguished audience as this through well-crafted oratory proclamations of what was done and not done to attain achievements. But then we as academics must recognize that the worth of a man's Iife is not based on the abundance of attainments, but rather on the uniqueness, peculiarity and value he has been created with. The truth is that I never set out to design dyes during my Ph.D programme, but rather just laden with the burden and the requirements to cany out spectroscopic UNIVERSITY OF IBADAN LIBRARY investigations into the isolated dyes following reactions between CDNBD and some pharmaceutical phenol ethers. This however t d out to be a blessing. Thus, spectroscopic chhcterization of dl the azo adducts for the first time established the scission of the ether linkage on diazo coupling to generate naphthols. This, was a remarkable observation as confusion seems to exist in literature as to the exact mechanism of coupling arid if the ethers are formed again after coupling reaction. Adegoke et al. (2008) clearly demonstrated that the scission of ether linkage occurs without reconversion back to the ether but leaving behind a residual hydroxyl group. However, this led to the serendipitous discovery of a new class of azo dye series named phenylazohydroxynaphthalenes. These dyes have similar structures to approved colourants such as sunset yellow and allura red (fig. 18). The dyes were obtained from the &action of the diazonium ion, karbox y l-2,6-dini tmbemene diazonium ion (CDNBD) with naphthalene derivative obtained from pnaphthol, a-naphthol (and propranolol), naproxen and nabumetone respectively to produce AZ-01, AZ-02, A 2 4 3 and AU)4 (Scheme 6). Some properties of the Scheme 6 dyes were investigated and reported (Idowu et al. 2007). In particular, there are subtle differences in the hydrophobic properties of the dyes with the proximity effect of the hydroxyl group to the azo linkage in A Z O 1 , -03 and -04 confemng different migration patterns on TLC compared to AZ-02. The dyes were thereafter used as excellent solvent probes in the assessment of solvatochromic behavioum of solvents of varying polarities by A&goke and Idowu (2010). Some excellent structure- spectra correlations were afforded by the dyes. The results of the curve fitting coefficients for the solvatochromic assessments enabled the classification of the various interactions of solvents with the dyes and relate the soivatochromic behaviom to the substi tuent effects on the dye molecules. 'Charge-transfer complexation occuning between one of the congeners (AZ-02) and N,W- dimethylformamide was extensive1y studied and discovered UNIVERSITY OF IBADAN LIBRARY to be both concentration- and tempemuredependent. The electronic character and the chemical nature of the solvents as well as the chemical nature of the other substituents, apart from the eonlrnon hydroxyl group, are important factors-for the observed solvat0c:hrornic properties of the rlcarboxyl-2, 6-di~hophenylamhybxynaphthdrnes(A degoke and Idowu 2010). $ Amhydrazone tqutomerisrn (fig. 19) was iound by NMR studies to exist in AZ-01, -03 and -04 (Adegoke et al. 2008). The phenom€!n~no f d y d m z o n e tautoprism in azo dyes and other dye molecules has been studied by several authors. A hydrazone is a tautomet of an azo compound. Since the am and hydrazone tautomem show different optical and physical p p t i e s ; their tautomerization has been extensively studied regarding cul~ura nd .other related industries. The three dyes UNIVERSITY OF IBADAN LIBRARY gave two main bands in the 5050 mixture of DMF with other solvents consisting of a high energy band at 250-382 nm, while the low energ bands for the dyes occurred at 415-485 nk Specnal shifts in the binary solvent mixtures were related to the solvent &polarity, basicity of the less polar component 'relative to DMF, substituent type, molar transition energy, 'fohnation constant for the hydrogen-bonding solvated complexes and the standard free energy change for hydrogen bonding Gth DMF. The relative pdominance of the hyQazone tautowr bears a direct relationship to the basicity of {the solvent, presence d hydrogen bond donor substituent and was associated with high molar transition energies and .low f o d m c onstant The microenvironment surrounding the dye molecules played a major role in the stability of one tautomer relative to the other (Adegoke 201 lc). - El& I& New dye d e g ( R,=OH. RFR~=H [AZOl]: ' W ERI *R 3=H [=I; R I d H ,R pH, R3= CH(CH3)C00H [AZ03]; Rl=OI% R a .R ACHm-9 [W]al)ong side allura red and sun& yellow UNIV RSITY OF IBADAN LIBRARY Fig, 19: bhydrazone tautomerism in the three congeners AZ-Ol, AZ- 03 and AZ04. The prominent effects of solvated H-bonding complexes were observed for the low energy bands of the dyes in the binary solvent mixture. Table 4 shows the variation of formation constants (Kf)an d Gibbs free energy (AG)f or the dyes in the binary liquid pair. KJ shows a negative correlation with AG for AZ-Ol (Y = -07896X + 4.66907, It2 = 0.9942) for non- polar solvents implying that as the formation constant of the solvated complexes increases, the spontaneity of its formati on reduces. Similar effects were observed in polar solvents (Y = -0.3341X + 4.564, R~ = 0.9345). Mr. Vice-Chancellor Sir, an timicrobial properties of incorporated dyes in food, beverages, cosmetics and fabrics have become desirable as such potentials prolong the utility of such products by inhibiting bacterial and fungal growth. Thus, Adegoke et al. (2010~)i nvestigated the antimicrobial properties of these new dyes with a view to providing dud roles as dyes and preservatives. A remarkable contribution to knowledge in this field was the dernonsttatibn of the ability of these new investigative dyes to serve as antimicrobial agent again st mu1 tidrug resistant Staphylococcus aureus. UNIVERSITY OF IBADAN LIBRARY Table 4: Variation 01L ow En- band with Kfi- dG and &(30) The next sets of tests that were carried out on the four congeners were aimed at discovering the suitability of these dyes ~holeculesa s colourants for food, beverage or cosmetics* if they lack genotoxic effects. Combes and Haveland-Smith (1982) chronicled pub1 i shed data for azo, triphen ylmethane and xanthene dyes from short-term assays for muta- carcinogenicity. The extent of agreement between data from different tests and comlations with animal cancer assays were considered. Synthetic dyes from the three major structural classes exhibit genotoxicity, whilst only two natural colours have proved active to induce genotoxici ty. Activity may be due to the presence of certain functional groups, notably nitro- and amino-substi tuents which are metabolized to ultimate electrophiles that may be stabilized by electronic interaction with aryl rings. Metabolic processes, such as azo- reduction, may be activating or detoxifying as shown by reviewed literatures. The studies have' emphasized the need to control the level of these dyes in food products (Nevado et al. 1998; Opinion of Scientific panel 2005). Some other recent studies have indicated that some of the approved food colours such as tartmine and sunset yellow are not mutagenic or clastogenic (Ould . Elhkim et al. 2007; Poul et al. 2009, Thomas and Adegoke 2015). In another recent study (Mpountoukas et al. 2010), with amaranth, tartmine and UNIVERSITY OF IBADAN LIBRARY erythrosine, toxicity potentials to human lymphocytes in virro was suggested to be due to dimt binding to DNA. Therefore,, continuous evaluation of fwd additives is warranted to ensure safety of the users. The few azo dyes that have been cenified as approved colourants for preparations intended for systemic and topical use are those that have been shown to be sufficiently safe in animal and clinical studies. Based on this background, in vitro and in vivo genotoxicity assessments were carried out on our four dyes. Distinguished audience, these sets of tests led me deep into the realm ~f Biology and provided a platfond to advance in Chemical Biology. My collaborators at the Laboratory of Genetic Toxicology, University of Calcutta, Kolkata, India, could not believe a Chemist will prepare slides, stain, carry out singlecell gel electpphoresis and use the microscope with utmost proficiency. But I was -not surprised as my two-year' stay in Medical Laboratory Technoiogy came on handy. While I have nd certificate^ to show for those rigorous training in routine and. special diagnostics procedures in Haematology, Chemical pathology, Blood banking, Parasitolbgy, Microbiology and Histopathology, the skills q u i d a re still part of me till today. So I urge parents not to despise the few essential skills your wards can gain while searching for their dream course of study in a University. I The in vitro genotoxicity of the new monoam dye series was evaluated using human lymphocytes by alkaline comet assay. Cytotoxicity tests using trypan blue dye exclusion method were also carried out (Adegoke et al. 2012b). The viability test showed greater than 80% in all cases ensuring validity of the assay procedure. Varied results were obtained following exposure of the human lymphocytes to the five concentration levels of AZ-Ol, -02, -03 and -04. The influence of the dye concentrations on and tail extent mo&t and olive tail moment (OTM) are presented in table 5 relative to the negative control. I UNIVERSITY OF IBADAN LIBRARY *Signi- at p = 0.05, value for MMS for TEM (1 (IO FM)= 2.77d.34, value for MMS fop OTM (100 FM) = 16.11G.93 The effect of the dyes on % tail DNA following Comet assay is presented in figure 20. In general, two of the congeners (AZ-Ol and AZ-02) which are positional isomers in which the common OH group is ortho and para to the ezo linkage gave differing results in terms of the three parameters of DNA damage studied-% tail DNA, UTM and TEM. Flg. U): ~ f f & o f &Ul- A244 on % Tail DNA (* significant at p = 0.05). * Value for MMS (laOpM = 45.63 t8529). i I UNIVERSITY OF IBADAN LIBRARY The dye AZO1' produced a concentration-dependent incnase in DNA damage at all concentration levels with results obtained for the % tail DNA being statistically significant. The influence of structure on the biological properties of the test dyes became evident with the results obtained for AZ-02. This. congener did not produce any statistically significant DNA damage at concentrations of 31.25 and 62.5 p M with respect to % tail DNA and at cancentrations of 31.25, 62.5 and 125 pM for TEM @ > 0.051 The results obtained for AZ03 and AZ04 showed a i ; l 8 f ~dq~x atuir; from that of A M i and m.A i all the five concentration levels, the two dyes (AZ-03 and -04) did not produce any cellular damage. The biological properties of these four dyes bear a good relationship with their structure. AZ-01 and AZ-02 are obtained from the diazo coupling reaction of p- and a- naphthols with CDNBD respectively. The naphthols and phenols having terminal hydroxyl p u p s could readily ionize. The ionization may in some instances generate reactive oxygen species ('OH)w hich is known to be genotoxic (Bandyopadhyay et al. 1999). However, both and a- naphthols have not been proven to be either mutagenic or genotoxic in vitro and in vivo though their presence in the environment, consumer goods and cosmetics are limited by the European Union (SCCNFP 2001; OECD SIDS 2006). Tntracellular hydrolysis of azo dyes has been reported to increase the toxicity of am dyes (Gottlieb et al. 2003). Thus, if AZ-Ol gets hydrolyzed or reduced within the cells, any of the observed DNA damage may have occurred in addition to the presence of the hydroxyl group. AZ-02, from our previous study (Adeguke and Idowu 2010), was found to have a high polarity and it readily ionizes to donate protons . to the environment. Thus, the lack of toxicity at lower concentrations may be due to the existence of the dyes in the ionic form which expectedly Limits intracellular crossing except if it is coupled to an ion channel in an active transport process. The other two congeners (AZ03 and AZ04) did not produce any statistically significant difference in DNA damage relative to the negative control at all , the 5- UNIVERSITY OF IBADAN LIBRARY concentration levels adopted in this study. These dyes, in addition to the common hydroxyl substituent, ~ o n t aait~ th e 7" positiis the propionic (AZ03) and butanone (AZ-04) substituents. Both dyes also exist in the hydrazone tautomer similar to AZ-O 1. However, ' presence of the additional C-7 substituents appears to . have completely modified their toxicity. In earlier developments of sourcing for dyes which contain skeletons different from the mutagenic and genotoxic bemidine molecules, it was discovered that the presence of ring substituents in the position ortho to the amino (em2) group of the perecursor amme re&icez Eaiagsnlciy (Gong e~ al. 2002). These studies have also led to non-mutagenic bcnzidine analogs containing bulky alkoxyl groups ortho to the amino group and amino derivatives of the dihydrophenophosphazine ring systems (Bello et al. 2000). The implication of these , differences in structure is that processes that will be responsible for the breakdown of the dyes to toxic end-products will be slowed down or completely eliminated with the consequence that the dyes become 'non-toxic. The results of the comet assays are presented in figure 21 for the lowest and highest dose. Fig. 21: Comet assay results with the four monoazo dyes using human lymphacytes at the two extreme dose Mr. Vice-Chancellor Sir, following these.r esults obtained in the in vitro assessment df the genotoxicity of these title dyes, a short-term in vkvo genotoxicity evaluation was carried out in mice. Aqueous colloidal solutions of the dyes were administered to mice on each day for 5 successive days using UNIVERSITY OF IBADAN LIBRARY gasttic gavages. Two end-point assessments of the genotoxicity potentials of the dyes were assessed, comet assay and chromosomal aberration studies (Adegoke et d. 2014b). The dyes were well tolerated at the doses investigated, as theni were no deaths or any adverse phannacotoxic events. Dosedependent DNA damage (in terms of percentage of tail DNA and Olive tail moment) occurred with AZOl and AZ02, although the effects were significant only with the highest doses. AZ03 gave similar patterns with those of AZ4l and A2-02. whi!e. repllrement with butanone in AZ04 altered the observed pattern. Minimal chromosomal damages were obtained for the four dyes, with AZOl and AZ02 giving non-significant damages, while the highest dose of AZ03 produced significant aberrations in terms of breaks. The results of the chromosomal abenation studies are presented in tables 6 and 7 for the four dyes. Some minor isochromatid breaks and gaps .were also noticed in the dye-treated mice. Mitotic indices in all cases were not significantly different from concomitantly administered vehicle control showing lack of cytotoxicity of the monoazo dyes at these doses. Representative plates for the chromosomal abemtion studies m presented in figure 22; One intriguing observation with the dyes is that A Z O l and AZ02 that gave some minute genotoxicity results in vitro had the effects reversed, while AZ03 and AZ04 that produC6d no DNA damage in vim gave some levels of genotoxicity in viva The few incidences of chromatid &b observed for both AZ03 and AZ-04 may be accounted for by the possibility of their metabolism into the p u m wc oupling components. The dyes, AZ03 and AZ04 were obtained from potent non-steroidal anti-inflammatory drugs nepmxen and naburntone, respectively (Adegoke et al. 2008). If these precmn are produced when AZ-03 and AZ04 an metabolized, then same level of DNA 'damage or CAs may be anticipated as most drugs ere known to be genotoxic at some doses. In order to further understand the kind of damage that UNIVERSITY OF IBADAN LIBRARY was taking place and possibly the mechanisms responsible, in v i m mechanistic-based binding studies of the dyes with calf- thymus (CT) double-strand DNA (&-DNA) and bovine sew albumin (BSA) were carried out to provide a clue to the ' made of interactions . of the new dyes with vSw in Bone M m w o f Mice Table 7: Chromosome Aberrations Induced byA2-03 a d A 244 in vivo in Bone Marrow of Mice Tm- T u m l C l t o m ~ m e . k r r r ~ Wd' %M? IW4W -kw C' G." 3' B" M e w - M-BJ) .- Lcgendsfiir Tablps 4 & 5: G', G**:c hromatid gap and ismhrmatid gaps; B', Be*:c h d d brtaks and bdwomatid lmaks; RR: chromatid remmgcments. " 100 metaphe celWanimal(3 mimalddose). Numba of CAdccll excluding gaps. ' % DE peccenfage of damaged mctaphase cells with at least one CA (excfuding gaps). * Mitotic Index (%). ' Distilled water (negative -1). * Significantly differeat from coacurreat c m d d m a t p 1 0.05 UNIVERSITY OF IBADAN LIBRARY Fig. 22: Represen tat iv e plates for chromosomal aberration studies (negative control; dye treatments and positive control; arrows show aberration;). For the DNA binding studies, the modes of interaction of the four congeneric monoazo dyes, with CT ds-DNA in aqueous medium was investigated spectrophotometricall y . Charac- teristic spectral changes observed at two temperatures (293 and 310 K) were assigned to either change in absorption intensity or change in wavelengths. The changes bear a direct relationship with the structures of the dyes. All the dyes gave good binding constants with DNA. Varying stoichiometric ratios were established between the dyes and DNA. Thermodynamic considerations enabled the delineation of the binding modes to be hydrogen bonding (AZ-01 and 43), electrustatic interactions (AZ-03) and hydrophobic bonding (AZ04). Correlation of the formation constant with Massieu- Planck's constant established the hydrophobic interaction between DNA and AZ-04 (Adegoke et al. 2012~). It was observed that the binding of A Z O l and A Z 0 2 with DNA involves a negative AG, A H 4 and A S d , while for AZ-03 negative AG, AH& and,AS>O were observed. For AZ-04, negative AG, and'hS>O were observed. The binding thermodynamics, as observed by Ahmadi et al (2009), reflect a subtle balance between the hydrogen, Van der Waals or multiple bonds and entropic effects. The change in entropy is governed by the release of counter ions and water from DNA and the dye molecules. For transfer of small molecules from polar to ion-polar environments, hydrophobic interactions usually give AFMand A S S with negative AG (Bekker and T.- I & UNIVERSITY OF IBADAN LIBRARY Norden 1997). For the interaction with bovine serum albumin, the focus was on the biophysical interactions of these monoazo dyes with bovine serum albumin @SA) that play an important role in drug transport and storage in vertebrates. The molecular interactions of the monoazo dyes with BSA were conducted by spectroscopic means in order to gain in-sight into the intermolecular binding forces responsible for the observed biological profile of the dyes. Adegoke et a1 (20124 observed trends in the spectra of unbound and bound monoazo dyes (tables 8 ahd 9) which suggest that the dye molecules are interacting with BSA through a wide range of-m echanisms and for which the structures of the dyes played key roles in determining the observed patterns. Table 8: High Energy Ektmnic Absorption -S Data of the Complexes Formed by the Moaosu, Dye with V e Ratio s of BSA *shul&rs at this wavelength UNIVERSITY OF IBADAN LIBRARY TsMe 9: Ekchnk Absorption Spctral Data for the Low Energy Tmwsltian of the CompZexa Formed between the Mumum Dyeg and Vary@ Ratios of BSA One congener (AZ-02) gave a minor peak characteristic of charge-transfer complexati on. The binding constants of the four monoazo dyes were estimated and found to v&y according to the dye structure and temperature of investigation. AZOl and -04 combined with BSA at approximately 1: 1 mole ratio, while the other two congeners with additional proton donors gave greater than this mole * ratio. Thermodynamic considerations established that the dyes utilized the various forms of binding modes; hydrogen bonding, hydrophobic bonding, van der Wads and A243 was particular1 y involved in electrostatic interactions giving positive entropy change for a small enthalpy change. Mr. Vice-Chancellor Sir, one structural defect obsewed with the new au, dye series is lack of water solubilizing agents. I proposed two approaches: use of sulphonated dye intermediates as coupling components for CDNBD and diazotization of amino-sulphonated intermediates prior to diazo coupling reaction with activated skeletons. The first approach yielded beautifully coloured azo dyes, but isolation and yield from the acidic media became a tough issue to handle. However, the second approach has produced about ten (10) novel azo dyes for which preliminary studies demonstrated their suitability as colourants and indicators of acid-base equilibria. Spectroscopic characterizations have yielded the structures of the novel ezo dyes. One of my Ph.D students and a colleague in the Department, Pharm. Segun Thomas, is presently in Kolkata, India not just for the fun of UNIVERSITY OF IBADAN LIBRARY enjoying Bengali cuisines, but primarily to establish the gemtoxicity potentials or othenvise of these new dye enti ties. New Reagents, Emerging Applications Mr. VicGChancellor Sit, one new emerging application that has fallen within our trawl ..line is the development and applications of new molecules as sensitive and selective colorimetric chcmosensom. Colorimetric sensors have become very popular in recent years due to their capability to detect and, in some instances, to semiquantitate analyte by naked eye detection without resorting to expensive instrumentation (Zhang et al. 2006). The colorimetric chemosenm are the~ foxc onsidered as one of the most effective anal yhcal methods for envimnmentd monitoring, particularly detection of major cationic and anionic species whose presence in the environment have deleterious con- sequences. Chemosensors include integrated chernosem~s, conjugated chemosensors and colorimetric chemosensors. Colorimetric chemosensors are designed in such a way that the meptor and signaling units are either fused together into one unit or connected by some unsaturated groups. Generally, electron donor-acceptor moieties connected by x- conjugation are chosen as suitable system for this purpose. Recognition of analyte by the chemosensor is expected to affect the conjugation between elecmn-donor and acceptor segment of the molecule substantially and thereby give rise to drastic'colour changes which can be recognized Three of the four investigative monoazo dyes, possess the azo-hydrazone tautomerism. This property makes the molecules capable of serving as colorimetric chemosensm provided we can find environmentally important inorganic. species that can carry out the intramolecular tautomeric switch and produce a stable colour. The first inorganic species candidate we were able to detect to carry out this conversion of the azo dyes was cyanide. The dyes were therefore evaluated as a high1y selective , colorimetric chemosensor for cyanide ion. Adegok et al. (2014~) discovered that presence of cyanide ion gave an obvious colour change to produce purple or liliac colour in acetone. UNIVERSITY OF IBADAN LIBRARY Under the optimum con& tions, linear relationships between the CN concentrations and light absorption were established. Using these azo-h ydrazone molecular switch enti ties, excellent selectivity towards the detection of CN' in aqueous soluti mi over miscellaneous competitive anions was observed. Such selectivity mainly results from the possibility of exclusive nucleophilic - attack on the azo-hy drazone chemosensors by cyanide anions in aqueous system, not afforded by other competing anions (fig. . 23). The nucleophi lie addition product was supported by the extension of chromophore and the brilliant colour produced. If proton abstraction had taken place, the colbur will not be intense as observed. Fig. 23: Colorimetric chemosemr detaminatign of cyanid;. with 4- carboxyl-2,6 4ini~phenylazohydmxynaph~denesa nd lack of interference by other anions.' Heavy metals are known to pose serious environmental and health hazards when present at levels exceeding the normal ' limits set by the , World Health Organization. In UNIVERSITY OF IBADAN LIBRARY addition, heavy metals arc known to catalyze the decom- position of pharmaceuticals, cosmetics and beverages. The current effort we are making is to develop specific azo and azomethine moieties for the rapid colorimetric chemosensing of these heavy metals. The.current design (fig. 24) is focused .onh ighly functionalized .tricyclic skeletons with the azo and &omt hine linkages providing conjugated skeletons for colour formation following binding of the metal ions. It. is hoped that novel molecules will be reported from these investigations soon. FPg, 2rl= Novel am and ~z0rnahiwtr icyclic skeletons as eoIorim&ic chemmson far metals. New Chemical Entities, Impmved hoperties Mr. vice-Chancellor ' Sir, di stinguishd audience, Schiff bases, known to tie good chelating agents, are easily prepared and characterid Little internst has been given to their uses fa analytical purposes because of two serious drawbacks; they are insoluble in quebus sorutions and they decompose easily in acidic solutions, limiting their use to basic conditions. In qynthaizing Schiff bases, two conditions are critical: the pH 'of the - medium and steric and electronic effects of the reactants, . . We sought to ' design novel Schiff b ' k t hat -will be soluble in water and at the same time provide analytical pleorms for the detection of some -environmentally UNIVERSITY OF IBADAN LIBRARY important inorganic cations and anions, while at the same time serving as excellent solvent probes. We made a great success designing, s ynt hetising and chamcterising nine novel Schi ff bases (fg, 25). This research was sponsored by the last ever Senate Research grant (2010) of the University of Ibadan. Well, I am still grateful at least for having my Uni versi ty sponsoring some ' ninemade-in-Universit y-of- Ibadan Novel Schiff bases. We went further to thoroughly investigate one of the molecules, and we found it to be an excellent solvent probe that wuld distinguish between ethanol and methanol, -and also serve as a highly selective coIorimetric chemosensor for nitrite in dried meat and cat fishes prepad by smoking. Mr. Vice-Chancellor Sir, distinguished audience,. you might be wondering why I have deliberately refused to divulge the structures of these new molecules; the document is ready to be submitted f& a patent! Fig. 25: KBr disks of the nine Npel Miff bases with improved properties. - Colours Arising from Storage of Pharmaceutical Roduets One of the professional duties of a Pharmacist is to provide medicines in-as tate of excellent physical, biopharmaceutical. . UNIVERSITY OF IBADAN LIBRARY microbiological and chemical status within the shelf life of the product. In many instances, a drug or its product is regarded as being stable if it maintains its integrity in terms of the aforementioned properties. Instability in pharmaceuticals manifest as caking of suspension, cracking of emulsions, mottling and colour separation of coloured tablets, excessive microbial con tamination among other properties. Modem-day formulation efforts are aimed at providing optimal conditions for the stability of pharmaceuticals. Some major factors that contribute to instability of. pharmaceutical products are chemistry of the drugs and excipients, light, humidity and oxygen. Two of the prominent factors - light and humidity - are extremely abundant in the tropical climate where Nigeria belongs. In the long run, instability leads to decomposition, and impurities may arise from decomposition or get carried through from raw materials. My panihlar interest in impurities profiling stems from the colous produced during improper storage of pharmaceutical products, and this had led me to get involved in impurity profiling of marketed pharmaceutical products in Nigeria. I have adopted a combination of chromatographic and once again azo dye derivatization in the profiling of impurities in pharmaceutical products. The first procedure reported in this regard is a simple, p i s e a nd robust reversed phase liquid chromatographic (LC) method which was developed and validated for the quantitative determination of griseofulvin (GF) and its impurities in drug substances and tablet dosage forms (Kahsay, Adegoke et al. 2013). Robustness study was performed by means of an experimental design and multivariate analysis using Mod& 5.0 software (Umetrics, Ume& Sweden). Satisfactory results were obtained from the validation studies. The use of volatile mobile phases allowed for the identification of three main impurities present above the identification threshold using tandem mass spectrometry (MS). The chromatographic conditions were selecied to accomplish optimal separation between griseofulvin its' major impurities (fig. 26), and various parameters that could influence resolution of peaks were optimized (fig. '27). UNIVERSITY OF IBADAN LIBRARY Fig. 26: ChmnabgramW ried from the thm C18 (250 mm x4.6 mm, 5 urn) edumm showing overall similar separations of GF and the irnpuritb from a 0.5 mglml solution uf gWmlWn drug substance using the final mathod. Chmmtqraphk conditions: mobile phase A (Water - 0.1 % formic acid pH 4.5,80:20v/v)and B (ACN-water-0.1 %formicadd pH4.5,85:15:TOvh/v), flow rab 1 mVmin, column temperakvle 30 "C, injection volume 10 ~la nd UV dedectfwr at 290 nm. Imp: fmpurtty and GF: GrbWvin. flg.27: Response sudhce plots showing the influence of temperature and acetmltrik on Rs 1 (A) and pH and tempemhim on Rs 2 (8) for the deDermimtb of griw&Mn (GF) and Impurides. The other paremetem am k e p t ~ a t t h e k ~ v a l u e s . UNIVERSITY OF IBADAN LIBRARY The strategy adopted in another study was to commence a comprehensive impurities profiling of ,some cephalosporins beginning k t h ceftriaxone. One expired brand and one brand still \;ithin its shelf life were investigated' The methodology -consisted of a modification of the British Pharmacopoeia 2007 method (Adegoke 2013). The obsewed c h r o r n a t o ~ are presented in f i w 2 8 . I n the typical chromatograms, the 'relative area % of the main impurity in the old brand. H;as 9.9 % reldve to 7.0 8 in the new brand. FIg = impurities pro6ling of cdbiaxone bands using i&tic high pmfomuw liquid chromatography. '7J UNIVERSITY OF IBADAN LIBRARY Mr. Vice-Chancellor Sir, one drug that has found regular use is paracetarn01 (PCM}, which is a commonly used analgesic consumed by an average Nigerian daily. But the drug is subjkt to the presence of impurities, the major impurity is toxic to the body. Para-aminophenol (PAP) is a degradation product of paracetamol upon hydrolysis of the acetamido group tomana mino. Phm-minophenol is the fmt intermediate in the industrial synthesis of paracetam01 and a degradation product of, paracetamol; thus, the main impurity in paracetamol. PAP is a metabolite of paracetamol and, like paracetamol, also induces hepatotoxicity. It is also nephrotoxic, being five times more potent than paracetam01 (Harmon 2006). PAP could be carried over into paracetam01 tablets as starting material or intermediate impurity; it could also be present as a degradation product. Therefore, its iden'tification, quantification and control are an important part of drug quality a s s h c e f or PCM. The presence of PAP gives a brownish tinge to paracetarno1 tablets. Recently, we designed a procedure which involves the diazotization of PAP and coupling with chromotropic acid. This project was executed by Agboola (2015). The method was found to be highly selective and sensitive and we were able to detect up to 0.14% PAP in commercial PCM tablets hawked in Bodija market of badan metropalis. This level was found higher than recommended by the Pharmacopoeias (0.1%) pointing to the need for adequate regulation of drug distribution. The react ion pathway and the W-Vis spectra produced for the reaction of PAP with chromotropic acid are presented in figure 29. UNIVERSITY OF IBADAN LIBRARY 29: Colorimetric micrr.'-termintion of PAP in Paracetamol Collabodve Nanopwticle Research Mr. Vice-Chancellor Sir, my interest in Nanopanicles Research eminated from two sides - the applications of nanopanicles in @ours and the ecot~xicologicalp rofiles of these new compounds. Nanotechnology and its applications have gained momentum over the past decade. Possessing unique elm-mechanical and thermal properties, carbon nanotubes (CNT) and other nanoparticles have numerous appliatiuns in iqdustrial and biomedical sectors. With my c o l l a ~ oat ~'th e h b m t s r y of Genetic Toxicology, University of Calcutta, India, we are currently investigating cytotoxicity, genotoxicity and in 'vim mechanistic-based binding studies of various nanopar.t icl.& using several tests and procedures. . Multi-Walled CNT (MWCNT) was examined in one of our studies (Ghosh et al. 2015). Over the past decade, a number of studies have investigated MWCW-induced toxicity and potential carcinogenesis in mammalian. systems. However, only limited infomation regarding the toxicity of MWCNT is available in the plant. ' system. With few exceptions, most of these studies have addressed the effect on UNIVERSITY OF IBADAN LIBRARY physiological parameters and germination rates. The.s tudy on AIlium cepn root tips was designed to understand 'the toxic potential of MWCNT in the plant system with a major ehphasis on cy totoxicity , genotoxicity, oxidative stress and global DNA methylation. Multiple cytotoxic (membrane integrity, tochondrial - . dehydrogenase activity, mito- chondrial membrane potential) and genot~xic( chromosome abrration, micronucleus formation, coatnet assay, - DNA laddaing 'and RAPD analysis) endpoints were evaluated to achieve a better understanding of MW CNT-induced toxicity in the plant system. The eff& sf MWCNT on oxidative stress and Cell cycle progression were also evaluated. The results (figs. 30 and 31) revealed that MWCNT uptake in root cells significantly altered cellular morphology. M e m e integrityind mitochondria1 function were also compmised. The nanotubes ' induced significant DNA damage, mimnucleu~sf ormation and - chromosome abemations. DNA laddering assay reveaied the f o d o n of internucleosomal fragments, which is indicative of apoptotic cell death. This ' finding was confirmed by an akumulation of cells in the sub- GO phase .of the cell cycle. An increase in CpG methylation was bbserved using; the isoschizomers MspYHpaII. HPLC analysis of DNA samples revealed a. significant increase in the levels of 5-meth yl-deox y c ytidine (5mdC). These results confirm the cyto-genotoxic effect of MWCNT in the plant system and simultaneously highlight the importance of this epigenetic study in naqoparticle toxicity. UNIVERSITY OF IBADAN LIBRARY SEM Fig. 3& SEM: Surface adhesion of MWCNT on Allium cepu root; (A)c ontrol - roots showing the absence of particle adhesion to the surface, (El and C) showing MWCNT adhered to the mt surface; TEM: TEM images of ultrathin sections of plant cells showing an effect of MWCNT mtuaent; (A) A. cepu root c d s in the absence of treatment with normal cellular organization, well defined nuclear and organellat smrctures, (B and C) MWCNT treated A, cepa root ~ l l ws it h extensive vacuolation, loss of nuclear orpiration, ruptured plasma membrane and shrinkage of the protoplast, (C)s howing particle deposition. Fig, 3 1: gesu 11, r)I hi N't '~'1'-i~lriucedc lrronlr~sume aberration and micronuckus assay-Allium test: bar graph showing the effect of MWCNT exposure on (A) &tic index (MI),@) % chromosome ' ~ t i m(%CA ), (C) ~cmtlucleus formation repmend as MNIlOOO cells; V < 0.05; figure in inset @) shows repmentative imagcs of akmtiolls scored- MN @I-El), anaphase bridge 0 3 ) and sady migration ofcfua-mt ill maphase. UNIVERSITY OF IBADAN LIBRARY The binding studies of W C N T with d-thymus DNA (Ghosh et al. 2016) were canied out to understand the mechanism by which specific DNA damage was produced in the ANim cepa root tip assay. MWCNT' was found to produce structural changei ip CT-DNA. The interaction or binding of MWCNT wasinvestigated in order to discover if it . brings about any significant changes of the DNA double helix using circular dichroism (CD)s pectra of the CT-DNA at two concentration levels of MWCNT ~ p s e n t i n gan increasing MWCNTlDNA molar ratio. In ,addition, spectrophoto~c titrations between MWCNT and CT-DNA were carried out in order to utilize specval changes as a means of detecting specific binding modes of either intercalation or d e w t i o n of DNA. Interactions of MWCNT induced significant changes in the CD spectra of the B-form of natural DNA. The intensities of the positive CD bandeat 280 nm decreased significantly. This decrease was found to be concen tration-dependent, Following spectrophotometiic titrations, specific subtle conformational changes were observed with n molar ratio combination of 2;l between MWCNT and CT-DNA; these were characterized by a funnation constant of the order of lo3 M' and a negative Gibbs free energy suggesting that MWCNT avidly binds to DNA. Thermodynamic considerations revealed that electrostatic interactions between the DNA base pairs, and the MWCNT are taking place accounting for the negative free energy change, posjtive enthalpy change with a small entropy change. The results obtained in the study of the binding interactions of MWCNT with DNA confirm that a cytogenetic effect of MWCNT with DNA is a possibility in vivo. The Greens, The Reddish-brown, The Oil One grand and glorious thing God has done for His creation is to give this world a variety of natural products and plants with diverse colours. While 'we make attempt to create colours, several plants have already been endowed with UNIVERSITY OF IBADAN LIBRARY diverse colom. I got fascinated about the beauty of coiourful plants in my first sojourn outside the shores of this country. At the. burst of spring in Glasgow, Scotland several plants came out in most brilliant colours I have ever seen, following close to five months of extreme snowy winter and its storms. I would not have h e n "relevant in natural products research but for a majw'sacrifice back then in L995. I started off as an Intern Phamacist at the Jos University Teaching Hospital, and barely one month later I was specially invited as the pioneer htem Pharmacist to the prestigious National Institute for ~h-eut icai Research and Development (NIPRD), Idu-Abuja. That meant a great pay cut as I earned N3,200 instead of W,500 that colleagues in Teaching Hospitals were earning. I guessed the times of training were not yet over. However, not only did the sojourn at NIPRD yield two papers, I also encountered another*g reat Chemist. Professor J. Okogun, of the Department of Medicinal Plant Research and Traditional Medicine, NIPRD (during my rotation in his department). He took me through the rudiments of chromatographic fractionation of natural products to the point of isolation of single components for spectroscopic characterization. I continue to thank God for that experience, which became a platform to assist friends and colleagues in the University of Ibadan. The skill on chromatographic fractionation and characterizations of isolated compounds has led to the successful completion of four Ph.D degrees in the University of Ibadan. Therefore, while I do not regard myself as a Natural Products Chemist, these collaborations have produced 26 publications. In one of such studies, Olayemi et al. (2010a, 2010b, 2011, 2013) provided the scientific basis for the folkloric uses of Cnesris femginea (de Candolle) Connanrceae by thorough studies of the haemamlogical, acute and chtonic toxicities and the reproductive effects of the brown crude methanolic extract and chromatographic fractions of the plant. In the collaborative studies with Oyedeji (20134 20 13b ), we established the scientific basis for theni ndigenous use of Portulaca oleracea through a UNIVERSITY OF IBADAN LIBRARY variety of procedures among which are assessment of the haematologicd and biochemical parameters following oral daily dosing of the pn-coloured crude extracts and chromatographic fractions to male albino rats, Bs well as anti- fertility and teratogenic effects on chromatographic pure fractions of the plant. A landmark achievement in the collaborative efforts was the isolation, characterization and evaluation of the anti-TB properties of crude extracts and isolated compounds from the plants; Eucalyptus camaldulensis Dehnh. (Myrtaceae) and Ewdyptus torelliana F. ~ u e l(lL awal et al. 20 12). The plants are used in Nigerian traditional medicine for the treatment of cough associated with tuberculosis (TB) and other respiratory infections. The hexane, chIoxoform, methanol extracts, and isolated compounds of E. camaldulemis and E. torel l ia~ were screened for activity against Mycobncteriwn tuberculosis H37Rv (MtbH37Rv) to authenticate the traditional use of these plants. The microplate alamar blue assay (MABA) method was used to investigate the anti-M. tuberculosis activities. Bioassa y-guided fractionation of the. hexane extract of E. mrelliann leaf was performed, and isolated compounds were characterized by MS, 1D- and 2D- NMR. Spectroscopic characterization led to the identification of two compounds, h y droxymyristic acid methylester (1) and a substituted pyrenyl ester, a sterol (2). Co1t~itlSion8 Has there been any reward from the trawling? The search has been long; the pursuits toilsome, and the labour hard, with unfettered sacrifices. The search goes on and on. The soul has been held in a long krfusion, to apprehend an expected end. Pursuing hard on a tripodal stand, nay, not a stand, but a trawl.. . For wide was the search, tortuous the pursuit, traversing oceans and miles, far, far away from motherland The comfort of -the home. to deny. Then, the Almighty God has always allowed torrents of grace and wisdom. Till... . . .in a proclamation of the Eureka the Phannwist longs. Then loo. UNIVERSITY OF IBADAN LIBRARY it was found. 'A big fish? A big catch? Nay; several molecules of chemical and biological significances. But and then,. .. : The story continues, till more pearls get within the trawl. Onward the labour must be and will be. Guided by the same denominator. The denominator of grace and wisdom from the Almighty, Who €iftsgthpeo or out of the dimghill and ssts among the princes of His people. What a wonder God performs in 'righteousness. Who can query Him? Thanking you faithful Father 'for His mercies ay endure' 'Everf aithful,e ver sure'. , ReuDlnmeqdatInm- Mr. ViceChancellor Sir, I will besliccinct and economical with my recommendations, and I will only mention two of them that are upptimost in my thoughts. (1) I wish to recommend the n&d for us to be careful with our consumption of coloured and flavoured foods and other artificially-prepared consumer goods. Apart from the fact that majority of these coloured subshces are deleterious in high doses, the ultimate effects on biological systems are yet to be adequately detedned and reported Womsome is the conflicting daily admissible dose set by major world regulatory agencies on colourants across several regions of the globe. (2) Mr. ViceChancellor Sir, I like to submit that the University *ofI badan is ripe for a functional (not less than 400 'MHz) nuclear ' magnetic .resonance spmqhotometer (NMR). In fact, the first University in Nigeria and one that still strives to be the best should be a hub for the spectroscopic characterizations of molecules of synthetic and natural origins. W e look forward to a &son in UI where our scientists will no longer need spectro~opicp ilgrimage abroad. 1 know and I am persuaded that we are nearer that season as you accede to this demand, Mr. Vice-Chancellor Sir.- UNIVERSITY OF IBADAN LIBRARY Acknowledgments My life is akin to a chemical product that has utilized several reagents, chemicals, solvents and reaction pathways to &hieve a thoroughly pure crystalline end-product free from exaggerated levels of impurities. 1 have received bendt s From severai people, and I will only make attempts to mention as many is my finite mind can recollect, remember and recount. If you do not get mentioned by name and dispositions, just be assured that your labour of love has not been forgotten. Your specific labour of love on my life and career has been to the Lbrd of Harvest, who will surely grant you a glorious recompense for a11 your efforts. I am a product of sacrificial tutoring and tutelage by my teachers, right from my nursery school days. Openly, I acknowledge the love and sacrifices of my teachers frmn Nursery, Primary, Secondary, -Medical Laboratory Science School and University days. I particularly remember Miss Adegbite and Mr. Oduja (then in Rimary school). I also ackndwledge my teachers during the secondary school days - Mama Elizabeth Fadeji (Mama Marhs); Mr. Oyatope and Mr. AIegi (my Chemisq teachers); Mrs. Nike Ajayi (Physics) and Mr. Abraham Olaniyan (Mathematics and Additional Mathematics). The contributions of Mr. Afolabi to my armory of hymns and praises are acknowledged. And to my Rincipal, Mr. OwoIabi whose zeal, seriousness and commitment I cherish. I appreciate the contributions of my University teachers through whom 1 learnt what it takes to encourage young people into academics. We are still friends till today, and I appreciate your timely counsel at all times. Special appreciation to Professors E.N. Sokomba, T.A . Iranloye, V.C. Agwada, T.E. AIernika, Francis Okwuasaba, J.A. Kolawole and Pahick Olorunfemi, Drs. Afolabi and Sunday Ntiejumokwu and a host of others. I acknowledge with thanks the support, fatherly counsel and guidance I have received from Emeritus Professor A.A. Olaniyi all these years. I made up my mind that I must work with this man who seems to UNIVERSITY OF IBADAN LIBRARY have captured all principles of Pharmaceutical Chemistry and put them in great textbooks for all Pharmacy students across Africa to' read. I thank God that I met you Sir, and all the impacts you and Mama have made in my life will not be forgotten. My gratitude goes to the Dean of Faculty of Pharmacy, Professor + Chinedurn P. Babalola, who has provided several platforms for my career advancements. I acknowledge the Vice-Chancellor, Professor Idow u Olayinka, for timely encouragements. That pep talks similitude h m a coach to .m amateur from the Conference Centre to the Staff Club car park in December 2010 provided the needed stoutheartedness to apply as a high-flyer for Professorship. I have enjoyed working and relating with Professor and Professor (Mrs.) B.O. Oke on UI campus. Special appciation goes to the Deputy Vice-ChanCellot (Academic), Professor Gbemisola A. Oke, who gave the needed inspiring leadership at the Centre for Entrepreneurship and Innovation (CEI). My gratitude also goes to Professor B.O. Oke who is the Chairman Administrative Commit tee of the Multidisciplinary Central Research Laboratory (MCRL). Special appreciation to colleagues at the CEI - Dr. Ayotola Aremu Director), Dr. Adedapo, Dr. Oyedunni Arulogun, Dr. Diji, Dr. Ilori and Dr. Ornobowale. Special thanks to all members of the Administrative Committee of the MCRL and staff members with whom we have been building on the legacies of the founding fathers. My sincere appreciation goes to Deacon and Professor (Mrs.) Sola Odeku and the entire family for the love, helping hands and sup* at all times and for sharing in the vision of building a great career in academics. I have received true hands of fellowship from the following individuals and l remain grateful for the love and tutoring - Professors K.T. Jaiyeoba, O.A. Itiola, J.O. Moody, Edith Ajaiyeoba, Bolanle Adeniyi, H.A.B. Coker, A.O. Ogundaini, T.A . Olugbade, 0.0.B olaji , Cecilia Igwilo and Dr. Olusegun . Komolafe. Special thanks to my friends and colleagues - Frofessors Tunji Oyeshile, Funsho Olayemi and UNIVERSITY OF IBADAN LIBRARY Ayo Kehinde; Drs. T.O. Idowu, J.O. Soyinka, Bimbo Elusiyan, Phams Simon Iyanda, Abayomi Omotosho .and Omotaja Ilupeju, Drs. Dele Odeniyi. Titilayo Fakeye, Sina Oyedeji, Funke Akin-Ajwi, Tolulope Ajala, Funmi Adejumo, Philip Idowu and Mubo Sonibare, Phann. Mrs. Funmi Ologe, Rev. A b l OgungbemirO, Joseph Ajani and Debo Oyebamiji. I acknowledge the support of all the colleagues at the Schools of Pharmacy in Sagamu, Ilorin, Ile-Ife and Lagos. I acknowledge the support and friendship from all members of staff of the Department of Pharmaceutical Chemistry - Dr. Olayemi M. Adegbolagun (My HOD), Prof. S.O. Idowu, Dr. .LC. Umchukwu, Dr. B.. Samuel, Pharmacists Segun Aderibigbe, Yinka Kotila and Keji Adeyerno, Mrs. C. Ajibike Ademola, Mrs. Abiodun Iyiola, Mr. 0iugbenga Adeyemi, Mr. Dayo Adegboyega, Mrs. Rasheedat Ilori, Mr. Seun David, Mr. ~ 6 s i nA le, Mr. Razak Olawuqi, Mr. Bolaji Oseni, Mr. Mayowa Obitokun, Mr. Anifowose, Mr. Sunday Aniekan, Mrs. Nnena Ogunlabi Ad Mrs. Okewumi. Special appreciation to Emeritus h f . A .A. Olani yi, Professors Oluw atoyin Odeku, Chinedm Babalola anc! Ayo Kehinde for their kind and excellent editorial work on the draft of this inaugural lecture. - S&ial appreciation to the University of Jos 1995 set of ~harm&ists.T hey gave me the title of "Prof right from 1990 when our 100 level first semester results got r e l d T hank you for being God's mouthpiece as prophecy came to pass 17 years afier. graduation. You will also sail through to your aspirations in life. My special gratitude goes to all the agencies that have offered me scholmhips, -fellowships and grants from my undergraduate school days. Special thanks to .t he University of *bs,C hemical and Allied Products Plc, Mobil Producing Nigeria Unlimited, John D. and CatheJine T. MacArthur Foundation, Chinese Academy of Sticks, The World' Academy of Sciences, Indian Nation'd Science Association. CV R& Fellowship, eOIMBRA Universities assokiation, West African R e s k h Association, University of Ibadan for Senate ~ e & h grants and TETFUND. - UNIVERSITY OF IBADAN LIBRARY I achowledge all my foreign collaborators - Professors R.D. Waigh, Sandy Gray, Dr. Geoffrey Coxon, Professor Dr. Dong-ying Chen, Professors Anita Mukherjee and Harold S. ree em&, Dm. Maumita Bandyopadhyay, Manosij Ghosh, Sonali Sinha, Professor Drs. Ann Van Schepdael, W i n Adams, Deidre Cooboter, Dr. Getu Kahsay, Professor Reimmel Adosraku. Thfough these foreign collaborators, I have experienced the extreme cold and pleasant spring of Glasgow; the summer warmth and highly humid winter of Shanghai; the autuinn of metropolitan Leuven; Belgium; the monsoon and summer nights of cosmopolitan Kolkata; the beautiful nights of Boston and the rich Ashante tradition of ~ k iGh,ana It has been great to be connected to the best hands in Synthetic Chemistry, Spectroscopy, Chromato- graphy and Genotoxicity. I appreciate t!ie warmth of Christian fellowship I have enjoyed at ElShaddai Baptist Church, Agbowo, Ibadan. Special thanks to Rev. and Dr. @hF.a)la de; Rev. and Dr. (Mrs.) Abiodun; Pastor and Mrs. Job Adeyerno; other Pastors, Deacons and -dlc hurch members. Special appreciation to my neighburs at Ajibode - Professor and Mrs. Ayoola; Deacon. and Mrs. Owonikoko; Professor and Mrs. Olatunbosun; Pastor and Pastor (Mrs.) Olaoye and Mr. and Mrs. A&kala. To my wife, Adenike, the one God specifically and selectively derivatized, conserved, preserved and reserved for me! Thanks for your love, sacrifice, concern, care and mdtherly guidance and thank you for agreeing to marry a man that seemed to have no firture*I cherish your eyes of faith that saw the glory of a Professorial husband Mr. Vice-Chancellor Sir, I do colours, but Adenike my wife knows colours, and she has always added colours to our home. To thi derivatives of Aremu - Ifeoluwa, Iyanuoluwa and Ibukunoluwa - I see you daily as God's own special derivatization products and I rejoice. Thank you for prompting me in those early days to work on my assignments "so that your uncle will not beat you". And thank you fbr enduring the niany'moments of my globetrotting. You shall individually be water than I have been in Jesus name. UNIVERSITY OF IBADAN LIBRARY To my mum, Dorcas Abemu, and my siblings; David, Deborah, Oluniran, Olubisi and Kay& and their families, yqu are all important parts of this story, and I long to see the family &united at the throne where another family circle won't be broken anymore through the merit of the sacrificial Lamb of God Special appreciation to my in-laws - The Akanjis, The Onaolapos, The Fakuntes, The Orowales, The Onigbindes, The Olusundes and all others. I appreciate the warm welcome I have received from you all from the first day. To all my students over the years with whom I have become a partner with similar bawls in order to catch derivatives of chemical and biological importance, I say thank you. These field soldiers have never been tired of seeing their leader.a round them on the bench and often have never given up on my insistence and strict requirements that they try out procedures over and over again. Special appreciation to P h m . Segun Thomas, Offiong Umoh, Mukaram Adeni yi- Akee, P h m . Monsurat Quadri, Bose Balogun, Folake Osoye, P h m . LRkan Omkotan, Funke Olaifa, Ternitope Adesuji, Ronke Adewole, Pharm. Sefiu Olaley e, Stephen Emmanuel, Olutayo Ogunsan ya, Funbi D m o I a , Kemi Abiodun, Damilola Makanjuola, Agaba Otache, Abiola Oyebamiji, Elizabeth Aiyenale, Folake Adenmosun, Tosin Ayodele, Pharm. Chioma Anekwe, Christians Olayinka, Kafayat Abol aji, Solomon A m , A yopo Sotade, Yinka Adedim and several others. Special appreciation to the Faculty of Pharmacy Inaugural Lecture Committee headed by Dr. Omonike Ogbole and to other members - Drs. Adenike Okunlols, 0.A. 'Funke Akin- Ajani, Funmi Ayeni, Pharms. O.E. Thomas, Tmitayo Ajayi and Keji Adeyerno, Messers O.J. Adeyemi and BE. Osinuga as we11 as Mrs. Chika Orialcu and Mrs. A h l a Aladesae. To this great audience, thank you for your attention. Do you now love spectroscopy? May your lives remain colourful. Now unto the King eternal, immortal, invisible, the only wise God, be honour and glory forever and ever. Amen. UNIVERSITY OF IBADAN LIBRARY References Adegoke, A.O. (2005) Novel colorimetric assays of selected phimwutical phenol ether homologues using 4carboxyI -2,6- dinitrobeme dimniwn ion. PkD Thesis, Univemity of badan. (20 13) West ' AWan Research Association fall Newsletter p. 6. (20 11 a ) Analytical, Biochemical and Synthetic applications of para-Dimethylamin~benzaldehyde. 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Zhen, F.G., Mai, P.J., Liang, Z.W. and Lu, X.B. (1999) Study on the Spectrophotometric Determination of Rare Earths with a New Chromogenic Reagent Di bromo-pr methyl- chlorosulfanazo (IDBMCSA). Chinese Chemical Letters 10 (10): 85 1-854. UNIVERSITY OF IBADAN LIBRARY BIODATA OF PROFESSOR AREMU OL&jIRE ADEGOKF, Professor Aremu Olajire Adegoke was born on the 21 August, 1970 in Oghmoso, Oyo State, Nigeria He attended the Osupa Baptist Day Primary School; Ogbomoso and St. Joseph Primary School, Vom, Plateau State (1975-1981). After his primary education, .he proceeded to the Baptist Secondary Grammar School, Aho yaya, Ogbomoso (1 98 1- 1986) where he passed out as the overall best candidate (Eight Distinctions and a ~iedi t )a nd as the best graduating student in Additional Mathematics, Chemistry, Economics and Geography; he also served as the Health and Laboratory Prefect. For his higher education, he was at the Federal S c h d of Medical Laboratory Technology, Jos, Plateau State (19 87- 1989). He was on the Distinction roll at the s c h l t hroughout his studentship. He gained admission to study Phannacy at the Faculty of Pharmaceutical Sciences, University of Jos in 1989 and graduated in 1995 with an overall Distinction, which due to the &classification of the Bachelor of Phmacy dew, translates to a First Class degree. He was the best overall graduating student in the University of Jos in 1995, and the best student in Pharmaceutical Chemistry, Pharmaceutical Technology, Pharmaceutical Mimbiology , Pharmacology, Pharmacognusy and Clinical Phmacy. He had his internship aaining at the prestigious National Institute for Phmaceutical. Research and Development (NIPRD), Idu-Abuja, as the pioneer Intern Pharmacist (19 95- 1996), and the mandatory National Youth Service at the General Hospital, Dutse, Jigawa State (19 96- 1997). He was awarded the M.Sc. in Pharmaceutical Chemistry at the University of badan (1999) and Ph.D. in Pharmaceutical Analysis and Synthetic Chemistry (2005) from the same'University. Professor Adegoke has been a recipient of many scholaiships, prizes, fellowships, grants. He was a University of Jos Scholk from 1991-1995; Chemical and Allied Roducts Limited (1991-1994) and Mobil Producing Nigeria Unlimited (199 1- 1995) undergraduate UNIVERSITY OF IBADAN LIBRARY scholatships awardee. He was awarded eight out ot tne m e convocation prizes from the University of Jos in 1996. During his Ph.D' programme, he was awarded the John D. and Catherine T. Mac Arthur Foundation grant that enabled him to visit the Department of Phiimaceutical Sciences, University of Srrathclyde, Glasgow, UK:'He won four prizes (two each in 2007 and 2009) for publications of articles from his Ph.D Thesis, a scheme initiated by the Postgraduate School, University of Ibadan. Professor Adegoke has been a recipient of many Postdoctoral Fellowships and Orants, including; TETFUND Overseas Conference grant to attend Drug Discovery and Therapy World congress in USA (2014); West African Research Association Travel Grant (2013); CV Raman International Fellowship for Senior African Reseadhers (2013); University of Ibadan Senate Research grants (2007 and 2012); COIMBRA Short Stay fellowship for Young African Researchers (2011); INSA JRD TATA Fellowship (20 lo), and CAS-TWAS Postdoctoral fel Iowship in China (ZOOS). He was appointed Graduate Assistant (1998-1999); Lecturer II in Pharmaceutical Chemistry in February, 2000, promoted to Lecturer I in October, 2003; Senior k t u r e r in Octok, 2006 and Professor of Pharmaceutical Chemistry with effect h m O ctober 1,2012. He has sewed as Sub Dean (Postgraduate), Faculty of Pharmacy. University of Ibadan, Ibadm (2006-2009); Member, University of badan Zoological Garden Management Committee (2003-2007); Member, University of Ibadan Zoological Garden Finance Committee (200572007); Member, University- Private Sector Collahmtion Sub-Committee, Cenm for Entrepreneurship and Innovation, University of Ibadan, Ibadan (2007 to date); Acting Head, Department of Phannaceuticd Chemistry, Faculty of Pharmacy, University of Ibadan, Ibadan (2010 to 20 12); Member, University Senate (20 10 ti 11 date). He is also a member, Senate Cumculum Committee; Member, Administrative Committee for the Multidisciplinary. Central Research Laboratory, University of .I badan (July 20 14 to UNIVERSITY OF IBADAN LIBRARY date). He was Deputy Coordinator, Multidisciplinary Central Research Laboratory (MCRL), University of Ibadan (July 2014. to 'June 2015) and he is currently the Coordinator, MCRL. b m J uly 5,2015 to date. He has served as External Examiner to the Departments of Pharmaceutical Chemistry, Obafemi Awolowo University , Ile-Ife and University of Lagos. He was a Visiting Senior Lecturer, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife (2013 to 20 14). He is a Member, West African Postgraduate College of Pharmacists (2013 to date). He is a Lifetime Member, West African Research Association; Member, Pharmaceutical Society of Nigeria and Member, National Association of Pharmacists in Academia He is an Expert reviewer for over 25 journals focusing on Spectroscopy. Organic Chemistry, Food Chemistry, Analytical Chemistry, Nanoparticles Research, Pharmaceutical Analysis and Natural Roducts Chemistry. Pmfessor Adegoke has authored 94 publications (comprising of a cwdited book; a chapter in a book; 81 peer- reviewed joumal articles and 11 conference papers). He is cmnt1y supervising three Ph.D students and has successfuUy supervised 17 B. Pharm. and over 30 PGD and MSG students. Professor Adegoke is an ordained Deacon at El-Shad& Baptist Church, badan. He is married to his friend, Adenike Esther Adegoke (nee Akanji), and the marriage is blessed with Ifeoluwa, Iyanuoluwa and Ibukunoluwa. UNIVERSITY OF IBADAN LIBRARY NATIONAL ANTHEM Arise, 0 compatriots Nigeria's call obey To serve our fatherland With love and strength and faith The labour of our hems.' past Shall never be in vain To serve with heart and might One nation bound in f n d m Peace and unity 0 God of ~~eatiun Direct our noble cause Guide thou our leaders right Help our youths the truth to know In love and honesty to grow And living just and true Great lofty heights attain To build a nation where peace And justice shall reign UNIVERSITY OF IBADAN ANTHEM. Unibadan, Fountainhead Of hue learning, deep and sound Soothing spring for all who thirst Bounds of knowledge to advance Pledge to serve our cherished goals! Self-reliance, unity ' That our nation may with pride Help to build a world that ismtrulyfr ee Unibadan, first and best Raise true minds for a noble cause Social justice, equal chance Greatness won with honest toil Guide our people this tci know Wisdom's best to service turned - Help enshrine the right to learn For a tnind that knows is a mind that's free UNIVERSITY OF IBADAN LIBRARY UNIVERSITY OF IBADAN LIBRARY