universiti putra malaysia extraction of -oryzanol and ... · konsentrasi bagi -orizanol dan...

UNIVERSITI PUTRA MALAYSIA

EXTRACTION OF -ORYZANOL AND TOCOPHEROLS FROM RICE (Oryza sativa) BRAN

ROSNIZAM ISMAIL FSTM 2008 19

EXTRACTION OF -ORYZANOL AND TOCOPHEROLS

FROM RICE (Oryza sativa) BRAN

By

ROSNIZAM ISMAIL

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of Requirements for the Degree of Master of Science

July 2008

ii

Especially dedicated to……..

Emak….in memory, may Allah bless her wherever she is,

abah, family,

friends and whoever has blown up my spirit..

…thank you for the guidance, encouragement, and love given

throughout my life…..

iii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of

the requirement for the degree of Master of Science.

EXTRACTION OF -ORYZANOL AND TOCOPHEROLS

FROM RICE (Oryza sativa) BRAN

By

ROSNIZAM ISMAIL

July 2008

Chairman : Associate Professor Azizah Abdul Hamid, PhD

Faculty : Food Science and Technology

The objective of this study was to extract -oryzanol and tocopherols from rice bran.

Different extraction times and sequential extractions utilizing different solvents on -

oryzanol and tocopherols levels from crude rice bran oil (CRBO) were studied. Rice

bran was extracted using hexane with five different extraction times, while sequential

extractions was carried out utilizing hexane, hexane/dichloromethane,

dichloromethane and dichloromethane/methanol. Extraction of -oryzanol and

tocopherols was further studied using precipitation and liquid-liquid separation from

unsaponified fraction of CRBO. Precipitation was carried out by diluting the sample in

hexane overnight at -20˚C. Liquid-liquid separations were carried out using hexane-

methanol or hexane-acetonitrile. Solid and liquid portions formed from precipitation

were further separated by liquid-liquid separations. The final stage of the extraction

process was a combination of precipitation and single-stage chromatographic

iv

separation. Results showed that percentage of extracted oil was not significantly

different with the different extraction times used. However, 20 min extraction resulted

in the highest -oryzanol level (p<0.05). On the other hand, 30 min extraction gave the

highest tocopherols level (p<0.05). Sequential extractions resulted in higher -

oryzanol and tocopherols levels than that of hexane extraction alone (p<0.05). After

saponification of CRBO extraction (with hexane, hexane/dichoromethane and

dichloromethane), the unsaponified fraction was found to contain 1.4% -oryzanol and

0.02% tocopherols. In liquid-liquid separations on unsaponified fraction of CRBO

study, the process was able to concentrate 2.1% -oryzanol in methanol and 1.8% in

acetonitrile, respectively. On the other hand, 0.03% tocopherols was concentrated in

methanol and 0.04% in acetonitrile, respectively. In precipitation study, 4.04% -

oryzanol was concentrated in solid portion. In column chromatography study,

combining precipitation and single stage silica chromatography was able to

concentrate 10.99% -oryzanol and 0.02% tocopherols. This work proposed an

alternative method for -oryzanol and tocopherols extraction from rice bran.

v

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains.

PENGEKSTRAKAN -ORYZANOL DAN TOKOFEROLS DARIPADA

DEDAK BERAS (Oryza sativa)

Oleh

ROSNIZAM ISMAIL

Julai 2008

Pengerusi : Profesor Madya Azizah Abdul Hamid, PhD

Fakulti : Sains dan Teknologi Makanan

Kajian ini dijalankan dengan objektif untuk mengekstrak -orizanol dan tokoferol

daripada dedak beras. Perbandingan dalam masa pengekstrakan dan pengekstrakan

secara sekuel menggunakan pelarut yang berbeza telah dikaji ke atas jumlah -orizanol

dan tokoferol daripada minyak dedak beras. Dedak beras yang telah distabilkan

diekstrak dengan heksana dengan lima masa pengekstrakan yang berbeza. Manakala

pengekstrakan sekuel dijalankan dengan menggunakan heksana,

heksana/diklorometana, diklorometana dan diklorometana/metanol. Kajian pra-

konsentrasi bagi -orizanol dan tokoferol selanjutnya telah dikaji dengan kaedah

pemendakan dan kaedah pemisahan fasa cecair-cecair daripada bahagian tidak-

tersaponifikasi (sebagai sampel) minyak dedak beras. Kaedah pemendakan telah

dijalankan dengan mencampurkan sampel dalam heksana dan dibiarkan semalaman

pada suhu -20˚C. Manakala kaedah pemisahan fasa cecair-cecair dijalankan dengan

mencampurkan sampel dalam heksana-metanol atau heksana-asetonitril. Bahagian

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pepejal dan cecair yang terbentuk daripada kaedah pemendakan telah selanjutnya

dikaji dengan kaedah pemisahan fasa cecair-cecair. Bahagian terakhir kajian

pengekstrakan bahan ini dijalankan dengan gabungan kaedah pemendakan dan kaedah

pemisahan kromatografi turus. Keputusan telah menunjukkan peratusan minyak dedak

beras yang terekstrak tidak berbeza secara signifikan dengan masa pengekstrakan yang

berbeza. Walau bagaimanapun, pengekstrakan selama 20 minit telah memberi jumlah

-orizanol yang tertinggi daripada masa pengekstrakan yang lain (p<0.05). Bagi

tokoferol, pengekstrakan selama 30 minit memberi jumlah tertinggi bagi bahan

tersebut (p<0.05). Pengekstrakan sekuel memberi jumlah lebih tinggi bagi kedua-dua

-orizanol dan tokoferol daripada pengekstrakan dengan mengunakan heksana sahaja

(p<0.05). Bahagian tidak-tersaponifikasi minyak dedak beras yang diekstrak dengan

menggunakan tiga peringkat pengekstrakan sekuel mengandungi 1.4% -orizanol dan

0.02% tokoferol. Dalam kajian pra-konsentrasi -orizanol dan tokoferol, kaedah

pemisahan fasa cecair-cecair ke atas bahagian tidak-tersaponifikasi minyak dedak

beras telah mengkonsentrasikan -orizanol kepada 2.1% dalam metanol dan 1.8%

dalam asetonitril (p<0.05). Tokoferol telah dikonsentrasikan kepada 0.03% dalam

metanol dan 0.04% dalam asetonitril (p<0.05). Dalam kajian pemendakan, -orizanol

telah dikonsentrasikan kepada 4.04% iaitu dalam fasa pepejal. Pemisahan fasa cecair-

cecair ke atas fasa pepejal (daripada pemendakan) menunjukkan -orizanol lebih

tinggi berada dalam methanol, sebaliknya, tokoferol berada lebih banyak dalam

asetonitril. Dalam kajian pemisahan kromatografi turus, gabungan kaedah

pemendakan dan kromatografi turus silica mengkonsentrasikan -orizanol kepada

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10.99% dan tokoferol kepada 0.02%. Kajian ini mencadangkan satu kaedah alternatif

dalam pengekstrakan -orizanol dan tokoferol daripada dedak beras.

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ACKNOWLEDGEMENTS

Alhamdulillah, praise to Allah s.w.t, The Almighty, for giving me strength, patience

and ability to complete this project and thesis.

Firstly, I’d like to express my appreciation and thank you so much to Associate

Professor Dr. Azizah Abdul Hamid of the Department of Food Science, Faculty of

Food Science and Technology, UPM, as the Chairman of my Supervisory Committee

for her guidance, patience, advice and encouragement and most important, give me the

chance to gain knowledge and experience through the challenging research. Precious

experiences gained from being her graduate student and her commitment to help the

success of my study will always be remembered in my heart.

Beside, my deep sense of gratitude and respect goes to my co-supervisors, Associate

Professor Badlishah Sham Baharin and Dr. Quek Siew Young (previously one of the

supervisory committee, however, she was discontinued being a lecturer in UPM), for

their sincere advice and support. I’d like to extend my appreciation to Associate

Professor Dr. Nazamid Saari and Associate Professor Dr. Azizah Osman for their

guidance, advice and encouraging interest in my study.

My sincere thank also to all dedicated friend of my fellow graduate students and also

to all helpful staff of the Faculty of Food Science and Technology. Sincere gratitude is

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dedicated to Malaysian Government for the financial support provide through IRPA

fund and to Graduate School of UPM for providing me with Biasiswa Pasca Siswazah.

Finally, my deepest gratitude and appreciation goes to my beloved father, for his

continuous support and to all my brothers and sister, thank you so much for their love

and support who have been inspiring my life.

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I certify that an Examination Committee met on 1st of July 2008 to conduct the final

examination of Rosnizam Ismail on his Master of Science Thesis entitled “Extraction

of -oryzanol and tocopherols from rice (Oryza sativa) bran” in accordance with

Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian

Malaysia (Higher Degree) Regulations 1981. The committee recommends that the

candidate be awarded the relevant degree. Members of the Examination Committee

are as follows:

Azizah Osman, PhD

Associate Professor

Faculty of Food Science and Technology

Universiti Putra Malaysia

(Chairman)

Yaakob Che Man, PhD

Professor



(Internal Examiner)

Aabdul Azis Ariffin, PhD

Associate Professor



(Internal Examiner)

Osman Hassan, PhD Professor

Faculty of Science and Technology

Universiti Kebangsaan Malaysia

(External Examiner)

_______________________________

Hasanah Ghazali, PhD

Professor and Deputy Dean

School of Graduate Studies


Date: 25 September 2008

xi

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirements for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Azizah Abdul Hamid, PhD

Associate Professor



(Chairman)

Badlishah Sham Baharin, MSc

Associate Professor



(Member)

______________________

Aini Ideris, PhD

Professor and Dean

School of Graduate Studies


Date: 16 Oktober 2008

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DECLARATION

I declare that the thesis is based on my original work except for quotations and

citations, which have been duly acknowledged. I also declare that is not been

previously and is not concurrently submitted for any degree at UPM or at any other

institution.

__________________

Rosnizam Ismail

Date:

xiii

TABLE OF CONTENTS

Page

ABSTRACT iii

ABSTRAK v

ACKNOWLEDGEMENT viii

APPROVAL x

DECLARATION xii

LIST OF TABLES xvi

LIST OF FIGURES xviii

LIST OF PLATES xix

LIST OF ABBREVIATIONS xx

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 5

2.1 Overview of Rice 5

2.2 Rice Bran 5

2.2.1 Stabilization of Rice Bran 7

2.2.2 Chemical Composition of Rice Bran 8

2.2.3 Rice Bran Lipid 10

2.3 -oryzanol 11

2.3.1 Chemical Structure of -oryzanol 12

2.3.2 Physiological Activity of -oryzanol 13

2.4 Tocopherols 16

2.4.1 Chemical Structure of Tocopherols 17

2.4.2 Physiological Activity of Tocopherols 19

2.5 -oryzanol and Tocopherols Extraction 22

2.5.1 Sequential Chemical Extractions 23

2.5.2 -oryzanol and Tocopherols Separation 25

2.5.3 Liquid-liquid Separation and Precipitation Separation 27

2.5.4 Adsorption Column Chromatography 29

3 EXTRACTION OF CRUDE RICE BRAN OIL 31

3.1 Introduction 31

3.2 Methodology 32

3.2.1 Reagents and Solutions 32

3.2.2 Standard Solutions 33

3.2.3 Rice Bran 33

xiv

3.2.4 Effect of Different Extraction Times on

CRBO Yield, -oryzanol and Tocopherols Levels 34

3.2.5 Effect of Sequential Extractions on CRBO yield,

-oryzanol and Tocopherols Levels 34

3.2.6 Determination of -oryzanol and Tocopherols 35

3.2.7 Determination of -oryzanol and Tocopherols

High Performance Liquid Chromatography (HPLC) 38

3.2.8 Statistical Analysis 40

3.3 Results and Discussion 40

3.3.1 Extraction of CRBO 40

3.3.2 Effect of Extraction Times and Sequential Extraction

on -oryzanol 44

3.3.3 Effect of Extraction Times and Sequential Extraction

on Tocopherols 47

3.4 Conclusion 53

4 PRECONCENTRATION OF -ORYZANOL AND TOCOPHEROLS

FROM UNSAPONIFIED FRACTION OF CRBO 54

4.1 Introduction 54

4.2 Methodology 55


4.2.2 Extraction of CRBO 56

4.2.3 Preparation of Unsaponified Fraction of CRBO 56

4.2.4 Determination of -oryzanol and Tocopherols using HPLC 57

4.2.5 Liquid-liquid Separation of Unsaponified Fraction of CRBO 57

4.2.6 Precipitation of Unsaponified Fraction of CRBO 58

4.2.7 Liquid-liquid Separation on Precipitation Fractions

of Unsaponified Fraction of CRBO 58

4.2.8 HPLC Determination of -oryzanol and Tocopherols 58



4.3.1 -oryzanol and Tocopherols Content of

Unsaponified Fraction of CRBO 60

4.3.2 Liquid-liquid Separation of Unsaponified

Fraction of CRBO 61

4.3.3 -oryzanol and Tocopherols obtained from

Precipitation of Unsaponified Fraction of CRBO 66

4.3.4 Liquid-liquid Separation on Precipitation

Fractions of Unsaponified Fraction of CRBO 67

4.4 Conclusion 73

5 SEPARATION OF -ORYZANOL AND TOCOPHEROLS USING

ADSORPTION COLUMN CHROMATOGRAPHY 74

5.1 Introduction 74

xv

5.2 Methodology 75


5.2.2 Unsaponified Fraction of CRBO 75

5.2.3 Fractionation of -oryzanol and Tocopherol

Using Silica Column Chromatography 76

5.2.4 HPLC Determination of -oryzanol and Tocopherols 77



5.3.1 Adsorption Chromatographic Separation of -oryzanol 79

5.3.2 -oryzanol Levels in Eluted Fraction of

Chromatographic Separation 81

5.3.3 Adsorption Chromatographic Separation of Tocopherols 85

5.3.4 Tocopherols Levels in Eluted Fraction of

Chromatographic Separation 87

5.4 Conclusion 92

6 SUMMARY AND RECOMMENDATIONS 93

BIBLIOGRAPHY 96

APPENDICES 109

BIODATA OF STUDENT 120

LIST OF PUBLICATIONS 121

xvi

LIST OF TABLES

Table Page

2.1 Composition of paddy kernel 7

2.2 Proximate chemical composition of rice bran (dry basis), percentage 9

2.3 Lipids profile in crude rice bran oil 11

2.4 Name and structural interrelationship of natural tocopherols 18

3.1 Extracted CRBO levels with different extraction times

and sequential extraction 41

3.2 Effect of different extraction times and sequential extraction

on -oryzanol levels from rice bran 45

3.3 Effect of different extraction times on tocopherols levels

from rice bran 48

3.4 Effect of sequential extractions on tocopherols levels from rice bran 49

4.1 -oryzanol and tocopherols levels in unsaponified fraction of CRBO 60

4.2 -oryzanol and tocopherols levels of unsaponified fraction of CRBO obtained

from liquid-liquid separation at 4˚C 63

4.3 -oryzanol and tocopherols levels in unsaponified fraction of CRBO obtained

from precipitation separation at -20˚C 67

4.4 -oryzanol and tocopherols levels obtained from liquid-liquid

separation (at 4˚C) on liquid fraction of unsaponified fraction of CRBO

Precipitation 69

4.5 -oryzanol and tocopherols levels obtained from liquid-liquid

separation (at 4˚C) on solid fraction of unsaponified fraction of CRBO

precipitation 72

5.1 Recovered samples weight obtained from eluted fraction of

chromatographic separation of -oryzanol 81

5.2 -oryzanol levels obtained using silica column chromatography 82

xvii

5.3 Recovered samples weight obtained from eluted fraction of

chromatographic separation of tocopherols 87

5.4 Tocopherols obtained using silica column chromatography 89

xviii

LIST OF FIGURES

Figure Page

2.1 Diagram of Paddy Structure 6

2.2 Molecular Structure of Individual -oryzanol According to Xu et al. (1999 14

2.3 Diagram of Tocopherols Molecular Structure 18

3.1 Flow Chart of Sequential Extractions of Rice Bran with

Different Solvent Polarity 36

3.2 Flow Chart of Saponification of CRBO for Determination

of -oryzanol and Tocopherols 37

3.3 Flow Chart of Sample Filtration Prior to HPLC Determination

of -oryzanol and Tocopherols 38

3.4 Chromatogram of -oryzanol Standard detected using

C18 Novapak column 47

3.5 Chromatogram of Tocopherols Standard detected using

C18 Symmetry Column 51

4.1 Experimental Layout of -oryzanol and Tocopherols Separation

of Unsaponified Fraction of CRBO by Precipitation and

Liquid-liquid Separation 59

5.1 Experimental Layout of -oryzanol and Tocopherols Separation

using Silica Column 78

5.2 -oryzanol Absorbance obtained from Silica Column

Chromatography Elution of Three Different Types of Samples 80

5.3 Tocopherols Absorbance obtained from Silica Column

Chromatography Elution of Three Different Types of Samples 86

5.4 -oryzanol and Tocopherols Extraction from Rice bran 91

xix

LIST OF PLATES

Figure Page

1.1 BERNAS factory that rice bran was taken at Kuala Selangor 109

1.2 Unstabilized rice bran from rice milling was packed and to be

sent to animal feed company 109

1.3 Immediate rice bran stabilization process by using microwave

in the factory 110

1.4 Stabilized rice bran 110

1.5 Crude rice bran oil and unsaponified fraction of crude rice bran oil 111

xx

LIST OF ABBREVIATIONS

C Degree Celsius

GC/MS Gas chromatography/ mass spectroscopy

HPLC High pressure liquid chromatography

IU International Unit

TE Tocopherol Equivalent

ROS Reactive oxygen species

CVD Cardiovascular disease

LDL Low-density lipoprotein

HDL High-density lipoprotein

CRBO Crude rice bran oil

NaOH Sodium hydroxide

BHT Butylated hydroxytoluene

mg Milligram

g Gram

kg Kilogram

ppm Part per million

ml Milliliter

MHz Mega Hertz

min minutes

m Micrometer

mm Millimeter

xxi

nm Nanometer

i.d. Internal diameter

UV/Vis Ultraviolet/visible

UV Ultraviolet

SAS Statistical Analysis System

MeOH Methanol

MeCl2 Dichloromethane

rpm Revolution per minute

mol. wt Molecular weight

CaCl2 Calcium chloride

atm atmosphere

P’ empirical solvent polarity

CHAPTER 1

INTRODUCTION

Rice (Oryza sativa) bran has gained much interest in recent years because it serves as

an excellent source of beneficial -oryzanol, tocopherols and tocotrienols (Da Silva et

al., 2006; Iqbal et al., 2005; Jennings and Akoh, 2000; Dunford and King, 2000). It

has been viewed as a potential functional food and commercial source for use as food

additive, pharmaceutical and in cosmetic product (Parrado et al., 2006; Lloyd et al.,

2000). As an under-utilized by-product from paddy-milling, rice bran is potentially an

inexpensive raw material. The obstacle hindering the use of rice bran for human

consumption is its instability and rapid spoilage.

Rice bran represents 10% of paddy (rough rice) weight fraction and reported to

contain up to 17% protein, 23% lipid, 27% dietary fibre, vitamins (thiamine,

riboflavin, niacin, tocopherols and tocotrienols), minerals (calcium, phosphorus and

magnesium) and other bioactive compounds, in particular, -oryzanol (Parrado et al.,

2006; Da Silva et al., 2006; Shin et al., 1997, Salunkhe et al., 1992; Saunders, 1990;

Fedeli, 1983).

Up to 28%, crude rice bran oil (CRBO) can be extracted from bran, making rice bran

oil the richest oil from cereal (Da Silva et al., 2006). Several countries including Japan

and India used the oil as speciality oil (Saunders, 1990; Prabhakar et al., 1986;

Bhattacharyya et al., 1983). Furthermore, CRBO is one of the nature’s richest sources

2

of potent antioxidant including tocopherols, tocotrienols and -oryzanol, which are

unique compared to other plants oil (Da Silva et al., 2006; Lloyd et al., 2000; Jennings

et al., 2000; Das et al., 1998). These compounds in CRBO are believed to be

responsible for hypocholesterolemic effect and reduction of aortic fatty streak

(Pszczola, 2001; Kahlon et al., 1996; Rogers et al., 1993).

-oryzanol has been shown to be a potent antioxidant and inhibits cholesterol

oxidation in vitro better than that of tocopherols and tocotrienols. 24-methylene

cycloartanyl ferulate exhibited the highest cholesterol antioxidation compared to other

-oryzanol members in rice bran oil (Xu et al., 2001). It was reported that -oryzanol

exhibited hypocholesterolemic action, decreased cholesterol absorption and plasma

cholesterol level (Wilson et al, 2007; Vissers et al., 2000). In technological uses, the

compound contributed to the rice bran oil stabilization at frying temperature and use as

special cosmetic ingredient (Miller et al., 2003).

Tocopherols is well known as an active free radical and reactive singlet oxygen

scavenger that prevent peroxidation of lipid either in vivo or in vitro systems.

Tocopherols has been repeatedly shown to have anticancer and anti-thrombosis

properties; able to reduce cardiovascular disease (CVD) and offer protection against

coronary heart disease by arresting free radical damages (Sylvester, 2002; Pszczola,

2001; Cicero and Gaddi, 2001; Eintenmiller and Landen, 1999). Both -oryzanol and

tocopherols are liposoluble compounds that are extracted in CRBO.

3

Various methods were used to separate or isolate both -oryzanol and tocopherols

either from rice bran or other samples. Extraction methods used commonly involved

solvent extraction followed by various concentration techniques such as adsorption

column chromatography, preparative HPLC, chemical separation (such as

esterification and transesterification and aminoalkylation), hydrogenation,

crystallization, molecular distillation, supercritical extraction and others. Some

methods involved sophisticated and expensive equipment and have certain drawbacks

(Abidi, 2001; Sumner Jr. et al., 2001; Akihisa et al., 2000; Qureshi et al., 2000; Xu et

al., 1999; Cheruckuri et al., 1999; Das et al., 1998; Hunt, 1997; Fizet, 1996; Baldwin

et al., 1990; Takagi et al., 1984).

Column chromatography was reported to be effective in separating desired

compounds. Selective separation of the compounds of interest from rice bran may

involve several complex and intensive unit operations. Beside, many procedures were

time demanding procedures which enhance oxidation compounds and thermal

degradation of the target compounds (Dunford et al., 2003; Guiochon and Lin, 2003;

Diack et al., 1994; Dondi and Guiochon, 1992).

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