Determination of the Major Flavonoid from Qina (Eucalyptus Globules L.) Using Shift Reagent, UV and IR Spectrophotometry

Determination Of The Major Flavonoid From Qina (Eucalyptus globules L.) Using Shift Reagent, UV And IR Spectrophotometry. BY Leni Ali Edris Adam B.Sc. Science and Education, El-Zeim Alazhari University( 2003) Postgraduate Diploma in Applied Chemistry, Gezira University( 2009) A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Chemistry Department of Applied Chemistry and Chemical Technology Faculty of Engineering and Technology Supervisor: Prof.Mohamed Abdel Karim Mohamed Co-supervisor:Dr. Mohamed Osman Babiker October -2012 ~1~ Determination Of The Major Flavonoid From Qina (Eucalyptus globules L.) Using Shift Reagent, UV And IR Spectrophotometry. BY Leni Ali Edris Adam Examination Committee: Name Position Signature Prof.Mohamed Abdel Karim Mohame Chairperson ................. Dr. Abo Bakr Khidir Ziada Intarnal Examiner …………. Dr. Abd Elsalam Abdalla Dafa Alla Extarnal Examiner …….. Date OF Examination: 6\10\2012 ~2~ Dedication This work is dedicated to My father who Deserved all respect, my mother For her care and passion, my husband for his help and support, my family and Friends. ~3~ Acknowledgements I thank Allah, Almighty for help. I wish to express my deep gratitude to my supervisor Prof. Mohamed Abdel Karim Mohamed for supervision and advice. I am grateful to all those who helped me to finish this thesis. My thanks are also extended to my colleagues for kind support. ~4~ Abstract Qina bark (Eucalyptus globules.L) is used in ethnomedicine as anti- inflammatory and antimalarial remedy.This study was aimed to extract and determine the physiochemical properties of the major flavonoid of quina bark. The plant material was collected from northern Kordofan and extracted with ethanol. The solvent was removed under reduced pressure . The crude product was fractionated over silica gel plates to give pure component flavonoid . The plates were developed with butanol:acetic acid:water (1:3:1) solvent sytem . UV studies using shift reagents indicated that flavonoid is either : a 5-hydroxyflavanone, ( C15H12O3 ) or 2`-hydroxydihydrochalcone, (C15H14O2 ) . Further UV studies using UV shift reagents indicated that the flavonoid is substituted by a 5-OH function. In conclusion : the UV and I.R spectrum of the isolated flavonoid indicaed that it is either a flavanone or a dihydrochalcone. It is therefore recommended that other structural determinatin techniques such as NMR may be carried out to confirm the structure. ~5~ الخـــــﻻصــة نبات الكينا من النباتات التي تحتوي على الفﻻفونويدات والتي تعتبر من مضادات اﻷكسدة القوية ومضادات اﻹلتهابات الطبيعية ويستخدمها الجسم للتخلص من السموم .تهدف الدراسة الي تحليل نبات الكينا لتحديد الخصائص الفيزيائيه والكيميائيه للفﻻفونيدات في لحاء الكينا. تم جمع النبات من منطقة شمال كردفان و استخلصت باﻹيثانول و تم تبخير المذيب تحت ضغط منخفض. هذا الناتج الخام لكروموتواغرفيا الطبقة الرقيقة التي طبقت في الواح السيلكاجل الزجاجية نتجت فﻻفونويد نقي وارتفعت نقاط المحلول باستخدام البيوتانول: حمض الخليك: ماء )1:3:1( ا وضحت الدرسات الطيفيه التي استخدمت فيها مطيافية اﻻشعه فوق البنفسجيه \ وتحت االحمرء ان الناتج الخام اما : 5هيدروكسي فﻻفانون ( C15H12O3 ) او2 هايدروكسي داي هايدرو جالكون ( C15H14O2) . دلت دراسه مطيافيه UV باستخدام الكواشف ان الفﻻفونويد يحمل الزمره OH-5 .وأوصت الدراسه بأستخدام تقنيه NMR اﻻكثر تطوار للتمييز بين الشكلين . ~6~ Contents Page page NO Dedication i Acknowledgment ii Abstract iii Abstract (Arabic) IV List of contents V Chapter one 1 Introduction 1 1.1 General approach 1 1.2 Classes of flavonoid 2 1.3 Biological role 8 1.4 Antioxidant activity of flavonoids and reactivity 10 with peroxy radical 1.5 Example flavonoids 15 1.5.1 Quercetin 15 1.5.2 Epecatechin 16 1.6 Dietary sources 17 1.6.1 Citrus 17 1.6.2 Green tea 18 1.6.3 Dark chocolate 18 1.7 Synthesis of flavonoids 19 ~7~ 1.8 Sources and role of phenolic acids and flavonoids 20 in plant 1.9 Separation – detection 21 1.10 Spectrophotomeric detection 25 1.11.1 Eucalyptus 27 1.11.2 Size and habitat 28 1.12 Aim of this work 28 Chapter two 2 Materials and methods 29 2.1 Materials 29 2.2 Methods 29 2.2.1 Preparation of test reagent 29 2.3 Plant Extract 29 2.4 Photochemical screening 29 2.5 Extraction of flavonoids 29 ~8~ 2.6 Thin-layer chromatography of the cru product 30 2.7 Preparative thin – layer chromatography 30 Chapter three 3 Result and Discussion 31 3.1 Spectral data of compound I 31 Fig (1) I R spectrum of compound I 32 Fig (2) The UV spectrum in methanolic solution of 35 compound I Fig (3) sodium methoxide with a methanolic solution 37 of compound I Fig (4) shift reagent aluminum chloride 38 Fig (5) drops of hydrochloric acid with a 39 methanolic solution of compound I Fig (6) sodium acetate with a methanolic solution of 41 compound I Fig (7) boric acid with methanolic solution of 42 compound I 3.2 Conclusion 44 Chapter Four ~9~ 4 REFERENCES 45 ~10~ 1- INTRODUCTION 1.1-GENERAL APPROACH Flavonoids are polyphenolic phytochemicals found in almost all types of plants and in all parts of the plant, from stems to roots to fruit. Although not considered nutrients and thus essential for life, flavonoids have gained increasing recognition for their health-related qualities. Flavonoids have potent antioxidant properties that contribute to their cholesterol lowering effects and cardiovascular protection . For example, naringenin and fisetin have been shown to be able to replace α-tocopherol as antioxidants, protective of cell membranes. In addition to their antioxidant effects, flavonoids are being studied for their anticarcinogenic properties. Citrus flavones have been implicated as inhibitors of Epstein– Barr virus activation and two-stage carcinogenesis of skin tumors1. The structure/activity relationships of flavonoids have been studied extensively to better understand what structural features are most important in various functions . However, their ubiquitous nature, multiple forms, and the metabolic changes of flavonoids result in difficulties in elucidation of the structure/activity relationships. For example, although flavonoids most commonly occur as glycosides in plants, upon digestion cleavage of the glycoside often occurs resulting in absorption of the flavonoid aglycone. Further metabolic changes of the flavonoid molecule occur in vivo, from conjugation to flavonoid glucuronides to degradation to phenolic acids. Most structure/activity relationship studies have concentrated on the in vitro effects of the flavonoid aglycones, such as the antioxidant and prooxidant behavior of flavones, isoflavones, and flavanones, which have been found to be dependent on the number and position of the hydroxyl substituent on the ~11~ aglycone backbone To establish structure-activity correlations the structures must be properly verified, which is why the previously mentioned studies have all employed specific flavonoids with known structures. Based on the ever-increasing number of flavonoids discovered and synthesized, elucidation of novel structures and related metabolites is of primary importance2. Mass spectrometric detection of flavonoids has become one of the most popular methods of analysis, often in conjunction with high performance liquid chromatography (HPLC) . Dissociation of the protonated and deprotonated flavonoid molecules yields some structural information , yet the signal intensities may not be significant enough for favorable results, especially in on-line methods. In fact, detection of the protonated or deprotonated flavonoid is not successful for some types of flavonoids. An additional problem is differentiation of the multiple isomers of flavonoids3. 1-2-Classes of flavonoid Vascular plants are able to synthesize a multitude of organic molecules phytochemicals, referred to as “secondary metabolites” . These molecules are involved in a variety of roles in the life span of plants, ranging from structural ones to protection. Phenolic compounds are regarded as one such group that are synthesized by plants during development and in response to conditions such as infection, wounding, UV radiation, etc4. Approximately 8000 naturally occurring compounds belong to the category of “phenolics”, all of which share a common structural feature: an aromatic ring bearing at least one hydroxyl substituent, i. e. a phenol . A straightforward classification attempts to divide the broad category of phenolics into simple phenols and polyphenols, based exclusively on the number of phenol subunits present Thus, the term “plant phenolics” ~12~ encompasses simple phenols, phenolic acids, coumarins, flavonoids, stilbenes, up to hydrolysable and condensed tannins, lignans, and lignins. Phenolic acids are aromatic secondary plant metabolites widely distributed throughout the plant kingdom .The term “phenolic acids”, in general, designates phenols that possess one carboxylic acid functionality. However, when talking about plant metabolites, it refers to a distinct group of organic acids. These naturally occurring phenolic acids contain two distinctive carbon frameworks: the hydroxycinnamic and hydroxybenzoic structures. Although the basic skeleton remains the same, the numbers and positions of the hydroxyl groups on the aromatic ring make the difference and establish the variety. Caffeic, p-coumaric, vanillic, ferulic, and protocatechuic are acids present in nearly all plants . Other acids are found in selected natural sources (e. g., gentisic, syringic)5. The polyphenols, to which the flavonoids belong, possess at least two phenol subunits; compounds possessing three or more phenol subunits are referred to as

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