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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…..
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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
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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
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Internal Examiner)
Aabdul Azis Ariffin, PhD
Associate Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(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
Universiti Putra Malaysia
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
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Chairman)
Badlishah Sham Baharin, MSc
Associate Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Member)
______________________
Aini Ideris, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
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
4.2.1 Reagents and Solutions 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
4.2.9 Statistical Analysis 59
4.3 Results and Discussion 60
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
5.2.1 Reagents and Solutions 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
5.2.5 Statistical Analysis 77
5.3 Results and Discussion 79
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).