UNIVERSITI PUTRA MALAYSIA TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM LEAVES OF SELECTED CITRUS SPECIES

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1 UNIVERSITI PUTRA MALAYSIA TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM LEAVES OF SELECTED CITRUS SPECIES SALFARINA BINTI RAMLI FS

2 TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM LEAVES OF SELECTED CITRUS SPECIES SALFARINA BINTI RAMLI MASTER OF SCIENCE 1

3 UNIVERSITI PUTRA MALAYSIA 2006 TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM LEAVES OF SELECTED CITRUS SPECIES By SALFARINA BINTI RAMLI Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the degree of Master Science 2

4 November 2006 Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM LEAVES OF SELECTED CITRUS SPECIES By SALFARINA RAMLI November 2006 Chairman: Faculty: Associate Professor Umi Kalsom Yusuf, PhD Science Water and 80% methanol crude extracts from the leaves of Citrus aurantifolia, C. hystrix, C. grandis, and Citrofortunella microcarpa were investigated for their total phenolic content and its relationship with total antioxidant activity. The total phenolic content was determined using a Folin-Ciocalteu reagent whereas determination of total antioxidant activity was carried out using a β-carotene bleaching method. Among the citrus leaves extracts, the highest total phenolic content was found from the water extract of Citrofortunella microcarpa whereas the water extract of C. maxima and Citrofortunella microcarpa gave the highest total antioxidant activity and was comparable with the antioxidant activity of marker BHT. However, no clear correlation was found between the total phenolic content and the total antioxidant activity of citrus leaves in both crude extract. Eleven flavonoid components were isolated and identified from the 80% methanolic extract of the leaves of Citrus aurantifolia, C. hystrix, C. grandis and 3

5 Citrofortunella microcarpa. The isolation and purification of all flavonoid components were carried out using standard paper chromatography technique. The isolated components were identified basis of their spectral analysis with shift reagents, R f values, colour reactions under UV and UV+NH 3, hydrolysis with acid and co-chromatogram with standard markers. The successfully isolated flavonoids were consisted from the group of flavone, flavone C-glycoside and flavonol. The methylated and glycosylated flavonoids were also isolated. The antioxidant capacity of isolated flavonoids was measured by two assays; a β-carotene bleaching method and a DPPH scavenging method. Statistical analysis showed that the total antioxidant and free radical scavenging activity of the isolated flavonoids was significantly low compared to the markers. Among the isolated flavonoids, results showed that at the 60 th minute, quercetin 4 -O-glucoside exhibited the highest total antioxidant activity. Quercetin 4 -O-glucoside also showed a significantly higher free radical scavenging activity in DPPH scavenging method than the other isolated flavonoids. The lowest activities in both assays were exhibited by vitexin 7-O-arabinoside and isovitexin. 4

6 Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains JUMLAH KANDUNGAN FENOLIK DAN AKTIVITI ANTIOKSIDAN FLAVONOID YANG DIPENCILKAN DARIPADA DAUN CITRUS TERPILIH Oleh SALFARINA BINTI RAMLI November 2006 Pengerusi: Fakulti: Profesor Madya Umi kalsom Yusuf, PhD Sains Jumlah kandungan fenolik dan aktiviti antioksidan daripada ekstrak mentah air dan 80% metanol daun Citrus aurantifolia, C. hystrix, C. grandis, dan Citrofortunella microcarpa telah dikaji. Penentuan jumlah kandungan fenolik dilakukan menggunakan bahan reagen Folin-Ciocalteu manakala jumlah aktiviti antioksidan dijalankan melalui kaedah pelunturan β-carotene. Daripada kesemua ekstrak mentah daun citrus, jumlah kandungan fenolik yang tertinggi diperolehi daripada ekstrak air daun Citrofortunella microcarpa, manakala ekstrak air daun C. maxima dan Citrofortunella microcarpa menunjukkan aktiviti antioksidan yang paling tinggi dan setara dengan aktiviti antioksidan penunjuk BHT. Walaubagaimanapun, tiada hubungan jelas yang dapat mengaitkan antara nilai aktiviti antioksidan dengan jumlah kandungan fenolik ekstrak ektraks daun citrus tersebut. Sebelas komponen flavonoid telah berjaya diasingkan daripada ekstrak 80% metanol daun Citrus aurantifolia, C. hystrix, C. grandis dan Citrofortunella microcarpa. Pemencilan dan penulenan komponen flavonoid dilakukan menggunakan teknik kertas kromatografi. Pengenalpastian komponen flavonoid dibuat melalui analisis spektrum menggunakan bahan reagen, perubahan warnanya di bawah sinaran UV dan UV+NH 3, 5

7 hidrolisis asid serta perbandingan ko-kromatografi dengan bahan penanda piawai. Komponen flavonoid yang telah dipencilkan terdiri daripada kumpulan flavone, flavone C-glycoside dan flavonol. Komponen flavonoid yang terikat dengan kumpulan metal dan gula juga berjaya dipencilkan. Kapasiti antioksidan bagi komponen flavonoid yang telah dipencilkan diuji melalui dua kaedah; pelunturan β-carotene dan kebolehan menghalang tindakan radikal bebas DPPH. Daripada ujian statistik yang dijalankan didapati aktiviti antioksidan dan tindakan menghalang radikal bebas komponen flavonoid yang telah dipencilkan adalah lebih rendah berbanding aktiviti penunjuk. Hasil kajian menunjukkan pada minit ke 60, quercetin 4 -O-glucoside menunjukkan aktiviti antioksidan yang tertinggi. Quercetin 4 -O-glucoside juga merupakan penghalang radikal bebas yang paling baik berbanding komponen flavonoid yang lain. Aktiviti yang terendah dalam kedua dua kaedah ujian antioksidan telah ditunjukkan oleh vitexin 7-O-arabinoside dan isovitexin. 6

8 ACKNOWLEDGEMENTS First of all, thank you Allah S.W.T for blessing me with many wonderful things and the opportunity to live with almost everything that I had wished for in life. Next, I would like to express my gratitude to my supervisor; Associate Professor Umi Kalsom Yusuf and the members of the supervisory committee; Associate Professor Asmah Rahmat and Professor Mawardi Rahmani for their guidance throughout my study. I truly adore your passion, commitment and dedication in research. To my parents; Haji Ramli Udin and Hajah Sahimah Ali, sisters and brother; thank you for always praying for my success and supporting me in every step that I take, all the way. Your endless love keeps me going. A special appreciation goes to all my wonderful friends. Thank you for always being there for me. Our friendship has rewarded me with valuable memories to learn from everyday. Each of you has given me so much strength to enable me to go through my journey to a great extent that I never thought I would be. Every moment we spent together will be an everlasting moment that I will always cherish. Thank you 7

9 This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows: Umi Kalsom Yusuf, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Chairman) Asmah Ramat, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member) Mawardi Rahmani, PhD Professor Faculty of Science Universiti Putra Malaysia (Member) AINI IDERIS, PhD Professor/ Dean School of Graduate Studies Universiti Putra Malaysia Date: 12 APRIL

10 DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been dully acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions. SALFARINA BINTI RAMLI Date: 9

11 TABLES OF CONTENTS DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS i Ii iv vi vii ix xii xiii xv CHAPTER I INTRODUCTION 1 II LITERATURE REVIEW Phenolic compound 5 Flavonoids 11 Biological activities of flavonoids 14 Antioxidant 16 Antioxidant activity of phenolics and flavonoids 20 Botany, distribution and ethnobotany of citrus species 23 Previous work on citrus Antioxidant activity of citrus 31 Previously isolated compound from citrus 34 III MATERIALS AND METHODS Total phenolic content and total antioxidant activity of the citrus leaves in the crude extracts Collection of samples and preparation of extracts 37 Determination of total phenolics content 38 Determination of total antioxidant activity 38 Isolation and identification of flavonoids from citrus leaves Plant material 40 Extraction, isolation and purification of flavonoids 40 Identification of flavonoids 42 Determination of total antioxidant activity of isolated flavonoids 45 compound Free radical scavenging activity of isolated flavonoids compound 46 Summary of methodology 47 IV RESULT AND DISCUSSION Total phenolic content 48 Total antioxidant activity of crude extract 52 10

12 The comparison of the antioxidant activity of crude extracts and 55 BHT Relationship between the antioxidant activity 58 Flavonoids from the leaves of citrus 64 Identification of flavones from the leaves of citrus 68 Identification of flavones C-glycoside from the leaves of 72 citrus Identification of flavonol from the leaves of citrus 72 Antioxidant activity of isolated flavonoid compound Inhibition of lipid peroxidation inhibition activity of isolated 78 flavonoid compound The comparison of the lipid peroxidation inhibition activity 81 of isolated flavonoids with markers Free radical scavenging activity of isolated flavonoids 84 Structure activity relationship of flavonoids and their 87 antioxidant activity V CONCLUSION 93 REFERENCES/BIBLIOGRAPHY 96 BIODATA OF THE AUTHOR

13 LIST OF TABLES Tables Page 2.1 Basic skeleton and classes of phenolics Active oxygen and related species Natural antioxidant for food use Comparison of synthetic and natural antioxidant Distribution of flavonoids in the genus citrus Mean antioxidant activity of citrus leaves extracts and BHT Results of phenolic content in two different types of extracts and their antioxidant activity after 120 minutes Spectral data of flavonoid components found in citrus leaves Chromatographic data of flavonoids found in citrus leaves Isolated flavonoid components from the citrus leaves Lipid peroxidation inhibition activity of isolated flavonoids and markers at 20 th to 120 th minutes using a β-carotene bleaching method 4.7 The order of efficiency of isolated flavonoids in both antioxidant assays

14 LIST OF FIGURES Figures Page 2.1 Phenol Classes and examples of phenolic compounds The structure of some phenolic compounds and their number of basic skeleton Basic structure of flavonoids Structure of aglycone, methylated flavonoids and glycosylated flavonoid 2.6 Major sources of free radicals in the body and the consequences of free radical damage Summary of methodology Total phenolic content of both citrus leaves extracts. Values are means ± standard deviation 4.2 Antioxidant activity of citrus extracted with 80% methanol using β carotene bleaching method 4.3 Antioxidant activity of citrus extracted with water using β - carotene bleaching method 4.4 The antioxidant activity of both citrus leaves extracts in comparison with BHT at 120 th minutes Band I of flavone of apigenin from C. aurantifolia leaves Band I of flavonol spectrum of Quercetin 4 -O-glucoside from C. aurantifolia leaves 4.7 Spectral analysis of apigenin isolated from C. aurantifolia leaves with shift reagents Structure of Apigenin and Apigenin glucoside Structure of Luteolin derivatives 72 13

15 4.10 Structure of Isovitexin Structure of Vitexin 7-O-arabinoside Shift and decreased in intensity at band I of Quercetin 4 -Oglucoside after the addition of NaOH compare to neutral spectrum Structure of Quercetin 4 -O-glucoside Structure of Kaempferol derivatives Lipid peroxidation inhibition activity of isolated flavonoids and markers using a β-carotene bleaching method 4.16 Mean of lipid peroxidation inhibition activity ± standard deviation of isolated flavonoids and both markers at 60 th minute 4.17 The DPPH free radical scavenging activity of the isolated flavonoids Structure quercetin 87 14

16 LIST OF ABBREVIATIONS AlCl 3 Aluminium chloride BAW n-nutanol:acetic acid:water; 4:1:5 BEW n-butanol:ethanol:water; 4:1:2:2 BHA BHT Buthylated hydroxyanisole Buthylated hydroxyl toluene CAW Chloroform:acetic acid:water; 30:15:2 Forestal Hydrochloric acid:acetic acid:water; 3:30:10 GAE H 3 BO 3 HCl HoAc NaOAc NaOH NH 3 Nm OH OMe Sh Gallic acid Boric acid Hydrochloric acid Acetic acid Natrium acetate Natrium hydroxide Ammonia Nanometer Hydroxyl Methyl ether Shoulder TBPW Toluene:n-buthanol:pyridine:water; 1:5:3:1 15

17 CHAPTER 1 INTRODUCTION Plants synthesize diversity of chemical compound through networks of metabolic pathways. These individual chemicals made by plants called phytochemical, can be found accumulated at every parts of plant as in leaves, fruits, seeds, bark and others. Each phytochemical has different basic chemical properties that in turn gives special function to the plants that synthesized it. In common usage, many phytochemicals are associated with human benefits. Phenolic compounds are one of phytochemicals that has long history in the human industry as in tanning skin industry, ink manufacturing, in the fining of wines as well as to human health. Previously research showed the occurrence of phenolics in diet as in vegetables and fruits conferred protection against cardiovascular disease and certain forms of cancer (Hertog et al., 1992). Therefore, it was recommended to have at least 400 g or five portions of fruit and vegetables each day to protect against coronary heart disease (CHD), stroke and cancers (Jackson et al., 2005). Phenolics constitute of compounds with one or more hydroxyl groups (OH group) bounded to an aromatic ring. Numerous categories of these compounds exist, from the simple phenols to the complex structure of condensed tannin. Although phenolics are often classified as secondary metabolites which means not essential for the normal 16

18 growth, development or reproduction of plant, numerous studies have been dedicated to the benefits of plant phenolics and its antioxidant properties is among that received the highest attentions. Phenolic compounds such as flavonoids, catechin and anthocyanin from foods and beverages had conclusively exhibited antioxidant activities (Ghiselli et al., 1998), as a result, screening of antioxidant activity of food and plants extracts frequently accompanied with the total phenolics study. A number of studies reported a significant correlation between the phenolics content of the plant extracts and their antioxidant properties. Lipid peroxidation and free radicals are major cause and contributor to the oxidation process. Oxidation process has been implicated as commence in cancer and atherosclerosis as well as deterioration of fats and oils in food. An antioxidant has been used to help inhibiting oxidation generally by donating electron and hydrogen to the free radicals (Halliwell and Gutterridge, 1995). The flavonoids are the most studied phenolics related to the antioxidant ability of phenolics. The ability of flavonoids to act as antioxidant was due to their molecular structures that enable them to act against free radicals. The number of hydroxyl group, presence of aromatic ring, heterocyclic rings, and the other structural groups of flavonoids were factors that affected the antioxidant activity of flavonoids (Rice-Evans et al., 1996). 17

19 The action of phenolics especially flavonoids as substrates for oxidation is viewed as beneficial in both food study and human health. The isolation, identification of the active constituents and the study of their antioxidant activities has been carried out extensively. This knowledge will provide an identification of natural antioxidants that can be alternatives to synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), which has been debated for their safety in usage. Research by Benavente-García et al. (1997) found that flavonoids as flavanones, flavones, and flavonols present in the citrus fruits have shown powerful antioxidants and free radical scavenger activities. In Malaysia, citrus species are popular as the fruits and leaves are used as flavor in food and native medication. The plants are widely distributed and can be found in any part of Malaysia (Verheij and Coronel, 1992). However, from the previous researches and presence data, the antioxidant study and the isolation of flavonoids from leaves of citrus plants have never yet been done as data available so far were mainly focusing on the fruits, peels and seeds of citrus which were by-products of citrus juice-making industry. Therefore, in this study the leaves of four citrus species namely Citrus aurantifolia, C. hystrix, C. grandis, and Citrofortunella microcarpa were selected. Among the different parts of the plants, leaves deserve special attention owing to it copiousness and availability. Moreover, a study by Hyder et al. (2002) reported that leaves and green stems accumulated appreciable amounts of phenolics. Since the leaves of citrus are 18

20 abundant in Malaysia, this study could be a preliminary attempt of studying the citrus leaves as an economical natural source of antioxidant. Thus, the objectives of this study were: 1. to determine the total phenolic content of the citrus leaves in the organic and water extract 2. to study the relationship between total phenolic content and the antioxidant activity of the citrus leaves in both organic and water extract 3. to isolate and identify the flavonoid components from the leaves of selected citrus 4. to evaluate the antioxidant activity of the isolated flavonoid components using two assays; β-carotene bleaching and DPPH radical scavenging. 19

21 CHAPTER 2 LITERATURE REVIEW Phenolic compound According to Walker (1975), the term phenolic compound embraces a wide range of chemical compounds possessing an aromatic ring bearing one or more hydroxyl groups together with a number of other substituents. Therefore plant constituents that possesses a phenol group; an aromatic ring bonded with hydroxyl groups (OH) are collected as phenolic compound (Figure 2.1). OH Figure 2.1: Phenol (Source: Walker, 1975) Numerous plant constituents with phenol group existed. The existence of phenol in nature so diverse; from the simplest to the most complicated structures, thus a classification of phenols is very important specially in studying the structure activity relationship of the compounds. The classifications of phenolics were done according to the number of 20

22 carbon in their basic skeleton (Table 2.1). Figure 2.2 and 2.3 showed the classes of phenolic compounds and the number of their basic skeleton. In nature, phenolic compounds do not occur in a free state but bound to glycosides in the form of esters. The nomenclatures of aglycones (phenolics without sugar group attached) are distinguished from their glycosides form by the insertion of id- into the name of aglycone, for example, cyanin for the glycosylated form and cyanidin for the aglycone (Riběreau-gayon, 1972). Table 2.1: Basic skeleton and classes of phenolics (source: Riběreau-gayon, 1972) No of carbon atoms Basic skeleton Class 6 C 6 Simple phenols, benzoquinones, 7 C 6 -C 1 Phenolic acids 8 C 6 -C 2 Acetophenones, phenylacetic acid 9 C 6 -C 3 (phenylpropanoid) 10 C 6 -C 4 Naphthoquinones 11 C 6 -C 1 -C 6 Xanthones 13 C 6 -C 2 -C 6 Stilbenes 15 C 6 -C 3 -C 6 Flavonoids, isoflavonoids 18 (C 6 -C 3 ) 2 Lignan 30 (C 6 -C 3 -C 6 ) 2 Biflavonoids n (C 6 -C 3 ) n (C 6 ) n (C 6 -C 3 -C 6 ) n Hydroxycinnamic acid, phenylpropenes, coumarins, isocoumarins Lignins Catechol melanin Flavolans (condensed tannin) 21

23 Phenolic compound Phenolic acids and coumarin Flavonoids and stilbenes Polymeric phenolics Tocols Benzoic acid derivatives, eg: vanilic acid, syringic acid, salicylic acid Stilbene eg: resveratrol Lignans and lignins Tocopherols and tocotrienols Cinnamic acid derivatives, eg: caffeic acid, ferulic acid, p- Coumaric Coumarins, eg: aesculetin, scopoletin Flavonoids eg: Flavone, flavonone, flavonol, flavanone, isoflavone, anthocyanidin Anthocyanidin Suberins and cutins Tannins Phenolic acid amino eg; aromatic amino acids phenylalanine, tyrosine Figure 2.2: classes and examples of phenolic compounds (sources: Shahidi and Naczk (2004) and Walker (1975). 22

24 R 1 R 1 R 2 COOH R 2 CH=CH-COOH R 3 R 3 Benzoic acid (C 6 -C 1 ) Cinnamic acid (C 6 -C 3 ) O OH O Flavonol (C 6 -C 3 -C 6 ) OH OH O OH OH OH H Catechin; a tannin (C 6 -C 3 -C 6 ) Figure 2.3: The structure of some phenolic compounds and their number of basic skeleton (sources: Shahidi and Naczk (2004)) 23

25 Phenolics found in both edible parts as fruits and vegetables and also in non edible parts. Chalcones and aurones are the yellow pigment of some flowers, whereas anthocyanins are another phenolic pigments that occur as glycoside which responsible for the red and blue colors. Betacyanins are the red pigments for the colour of red beet (Beta vulgaris). Benzoic acid (C 6 -C 1 ) and phenolic substances with a phenylpropane structure (C 6 -C 3 ) as eugenol and isoeugenol are common constituents of essential oils (Riběreau-gayon, 1972). Plant phenols are chemically reactive and usually acidic. However, they can be separated from other constituents by their solubility in aqueous sodium carbonate. Total phenolic assay using Folin-Ciocalteu reagent has been widely used to investigate the content of phenolics in the samples studied. Folin-Ciocalteu reagent is nonspecific to phenolic compounds because it can also be reduced by many nonphenolic compounds (e.g.,vitamin C, Cuprum (I)). However, after being adjusted by a sodium carbonate solution to ph ~10, only phenolic compounds react with Folin-Ciocalteu. In the Folin-Ciocalteu assay, the reducing activity of phenolic was evaluated from its transferring electrons ability. For this reason, Folin-Ciocalteu assay was once grouped into an antioxidant assay category. The blue compounds formed between phenolic and Folin-Ciocalteu reagent was proportional to the concentration of all phenolics in the extracts and was independent with the structure of phenolic compounds. The total phenols assay by Folin-Ciocalteu is convenient, simple, and reproducible. As a result, a 24