UNIVERSITI PUTRA MALAYSIA

TOTAL PHENOLIC CONTENT AND ANTIOXIDANT ACTIVITY OF FLAVONOIDS ISOLATED FROM LEAVES OF SELECTED CITRUS

SPECIES

SALFARINA BINTI RAMLI

FS 2006 61

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

SALFARINA BINTI RAMLI

MASTER OF SCIENCE

1

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

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: Associate Professor Umi Kalsom Yusuf, PhD Faculty: 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

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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, Rf values,

colour reactions under UV and UV+NH3, 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 60th 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.

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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: Profesor Madya Umi kalsom Yusuf, PhD Fakulti: 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+NH3,

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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.

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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

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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 2007

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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:

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TABLES OF CONTENTS

DEDICATION i ABSTRACT Ii ABSTRAK iv ACKNOWLEDGEMENTS vi APPROVAL vii DECLARATION ix LIST OF TABLES xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS

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

compound 45

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

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The comparison of the antioxidant activity of crude extracts and BHT

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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

citrus 72

Identification of flavonol from the leaves of citrus 72 Antioxidant activity of isolated flavonoid compound Inhibition of lipid peroxidation inhibition activity of isolated

flavonoid compound 78

The comparison of the lipid peroxidation inhibition activity of isolated flavonoids with markers

81

Free radical scavenging activity of isolated flavonoids 84 Structure activity relationship of flavonoids and their

antioxidant activity

87

V CONCLUSION 93

REFERENCES/BIBLIOGRAPHY 96

BIODATA OF THE AUTHOR 107

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LIST OF TABLES

Tables Page

2.1 Basic skeleton and classes of phenolics 6

2.2 Active oxygen and related species 16

2.3 Natural antioxidant for food use 19

2.4 Comparison of synthetic and natural antioxidant 19

2.5 Distribution of flavonoids in the genus citrus

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4.1 Mean antioxidant activity of citrus leaves extracts and BHT

53

4.2 Results of phenolic content in two different types of extracts and their antioxidant activity after 120 minutes

58

4.3 Spectral data of flavonoid components found in citrus leaves

65

4.4 Chromatographic data of flavonoids found in citrus leaves

66

4.5 Isolated flavonoid components from the citrus leaves

67

4.6 Lipid peroxidation inhibition activity of isolated flavonoids and markers at 20th to 120th minutes using a β-carotene bleaching method

79

4.7 The order of efficiency of isolated flavonoids in both antioxidant assays

91

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LIST OF FIGURES

Figures

Page

2.1 Phenol

5

2.2 Classes and examples of phenolic compounds

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2.3 The structure of some phenolic compounds and their number of basic skeleton

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2.4 Basic structure of flavonoids

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2.5 Structure of aglycone, methylated flavonoids and glycosylated flavonoid

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2.6 Major sources of free radicals in the body and the consequences of free radical damage

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3.1 Summary of methodology

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4.1 Total phenolic content of both citrus leaves extracts. Values are means ± standard deviation

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4.2 Antioxidant activity of citrus extracted with 80% methanol using β –carotene bleaching method

54

4.3 Antioxidant activity of citrus extracted with water using β -carotene bleaching method

54

4.4 The antioxidant activity of both citrus leaves extracts in comparison with BHT at 120th minutes

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4.5 Band I of flavone of apigenin from C. aurantifolia leaves

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4.6 Band I of flavonol spectrum of Quercetin 4’-O-glucoside from C. aurantifolia leaves

68

4.7 Spectral analysis of apigenin isolated from C. aurantifolia leaves with shift reagents

70

4.8 Structure of Apigenin and Apigenin glucoside 71

4.9 Structure of Luteolin derivatives

72

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4.10 Structure of Isovitexin

73

4.11 Structure of Vitexin 7-O-arabinoside

73

4.12 Shift and decreased in intensity at band I of Quercetin 4’-O-glucoside after the addition of NaOH compare to neutral spectrum

74

4.13 Structure of Quercetin 4’-O-glucoside

74

4.14 Structure of Kaempferol derivatives

75

4.15 Lipid peroxidation inhibition activity of isolated flavonoids and markers using a β-carotene bleaching method

80

4.16 Mean of lipid peroxidation inhibition activity ± standard deviation of isolated flavonoids and both markers at 60th minute

83

4.17 The DPPH free radical scavenging activity of the isolated flavonoids.

86

4.18 Structure quercetin

87

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LIST OF ABBREVIATIONS

AlCl3 Aluminium chloride

BAW n-nutanol:acetic acid:water; 4:1:5

BEW n-butanol:ethanol:water; 4:1:2:2

BHA Buthylated hydroxyanisole

BHT Buthylated hydroxyl toluene

CAW Chloroform:acetic acid:water; 30:15:2

Forestal Hydrochloric acid:acetic acid:water; 3:30:10

GAE Gallic acid

H3BO3 Boric acid

HCl Hydrochloric acid

HoAc Acetic acid

NaOAc Natrium acetate

NaOH Natrium hydroxide

NH3 Ammonia

Nm Nanometer

OH Hydroxyl

OMe Methyl ether

Sh Shoulder

TBPW Toluene:n-buthanol:pyridine:water; 1:5:3:1

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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

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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).

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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

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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.

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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

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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 C6 Simple phenols, benzoquinones, 7 C6-C1 Phenolic acids 8 C6-C2 Acetophenones, phenylacetic acid 9 C6-C3

(phenylpropanoid) Hydroxycinnamic acid, phenylpropenes, coumarins, isocoumarins

10 C6-C4 Naphthoquinones 11 C6-C1-C6 Xanthones 13 C6-C2-C6 Stilbenes 15 C6-C3-C6 Flavonoids, isoflavonoids 18 (C6-C3)2 Lignan 30 (C6-C3-C6)2 Biflavonoids n (C6-C3)n

(C6)n (C6-C3-C6)n

Lignins Catechol melanin Flavolans (condensed tannin)

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Phenolic compound

Phenolic acids and coumarin

Flavonoids and stilbenes

Polymeric phenolics

Tocols

Benzoic acid derivatives, eg: vanilic acid, syringic acid, salicylic acid

Cinnamic acid derivatives, eg: caffeic acid, ferulic acid, p-Coumaric

Coumarins, eg: aesculetin, scopoletin

Phenolic acid amino eg; aromatic amino acids phenylalanine, tyrosine

Lignans and lignins

Suberins and cutins

Tannins

Stilbene eg: resveratrol

Flavonoids eg: Flavone, flavonone, flavonol, flavanone, isoflavone, anthocyanidin

Tocopherols and tocotrienols

Anthocyanidin

Figure 2.2: classes and examples of phenolic compounds (sources: Shahidi and Naczk (2004) and Walker (1975).

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R1

R3

R2 COOH

R1

R3

R2 CH=CH-COOH

Benzoic acid (C6-C1) Cinnamic acid (C6-C3)

O

O

OH

Flavonol (C6-C3-C6)

O

OH

OH

OH

OH

OH

H

Catechin; a tannin (C6-C3-C6)

Figure 2.3: The structure of some phenolic compounds and their number of basic skeleton (sources: Shahidi and Naczk (2004))

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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 (C6-C1) and phenolic substances with a phenylpropane structure (C6-C3) 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

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