UNIVERSITI PUTRA MALAYSIA. EXTRACTION OF -ORYZANOL AND TOCOPHEROLS FROM RICE (Oryza sativa) BRAN ROSNIZAM ISMAIL FSTM

Transcription

1 UNIVERSITI PUTRA MALAYSIA EXTRACTION OF -ORYZANOL AND TOCOPHEROLS FROM RICE (Oryza sativa) BRAN ROSNIZAM ISMAIL FSTM

2 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

3 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.. ii

4 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 hexanemethanol 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 iii

5 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. iv

6 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 prakonsentrasi bagi -orizanol dan tokoferol selanjutnya telah dikaji dengan kaedah pemendakan dan kaedah pemisahan fasa cecair-cecair daripada bahagian tidaktersaponifikasi (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 v

7 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 cecaircecair 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 vi

8 10.99% dan tokoferol kepada 0.02%. Kajian ini mencadangkan satu kaedah alternatif dalam pengekstrakan -orizanol dan tokoferol daripada dedak beras. vii

9 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 viii

10 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. ix

11 I certify that an Examination Committee met on 1 st 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 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 x

12 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 xi

13 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: xii

14 TABLE OF CONTENTS ABSTRACT ABSTRAK ACKNOWLEDGEMENT APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF ABBREVIATIONS Page iii v viii x xii xvi xviii xix xx CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW Overview of Rice Rice Bran Stabilization of Rice Bran Chemical Composition of Rice Bran Rice Bran Lipid oryzanol Chemical Structure of -oryzanol Physiological Activity of -oryzanol Tocopherols Chemical Structure of Tocopherols Physiological Activity of Tocopherols oryzanol and Tocopherols Extraction Sequential Chemical Extractions oryzanol and Tocopherols Separation Liquid-liquid Separation and Precipitation Separation Adsorption Column Chromatography 29 3 EXTRACTION OF CRUDE RICE BRAN OIL Introduction Methodology Reagents and Solutions Standard Solutions Rice Bran 33 xiii

15 3.2.4 Effect of Different Extraction Times on CRBO Yield, -oryzanol and Tocopherols Levels Effect of Sequential Extractions on CRBO yield, -oryzanol and Tocopherols Levels Determination of -oryzanol and Tocopherols Determination of -oryzanol and Tocopherols High Performance Liquid Chromatography (HPLC) Statistical Analysis Results and Discussion Extraction of CRBO Effect of Extraction Times and Sequential Extraction on -oryzanol Effect of Extraction Times and Sequential Extraction on Tocopherols Conclusion 53 4 PRECONCENTRATION OF -ORYZANOL AND TOCOPHEROLS FROM UNSAPONIFIED FRACTION OF CRBO Introduction Methodology Reagents and Solutions Extraction of CRBO Preparation of Unsaponified Fraction of CRBO Determination of -oryzanol and Tocopherols using HPLC Liquid-liquid Separation of Unsaponified Fraction of CRBO Precipitation of Unsaponified Fraction of CRBO Liquid-liquid Separation on Precipitation Fractions of Unsaponified Fraction of CRBO HPLC Determination of -oryzanol and Tocopherols Statistical Analysis Results and Discussion oryzanol and Tocopherols Content of Unsaponified Fraction of CRBO Liquid-liquid Separation of Unsaponified Fraction of CRBO oryzanol and Tocopherols obtained from Precipitation of Unsaponified Fraction of CRBO Liquid-liquid Separation on Precipitation Fractions of Unsaponified Fraction of CRBO Conclusion 73 5 SEPARATION OF -ORYZANOL AND TOCOPHEROLS USING ADSORPTION COLUMN CHROMATOGRAPHY Introduction 74 xiv

16 5.2 Methodology Reagents and Solutions Unsaponified Fraction of CRBO Fractionation of -oryzanol and Tocopherol Using Silica Column Chromatography HPLC Determination of -oryzanol and Tocopherols Statistical Analysis Results and Discussion Adsorption Chromatographic Separation of -oryzanol oryzanol Levels in Eluted Fraction of Chromatographic Separation Adsorption Chromatographic Separation of Tocopherols Tocopherols Levels in Eluted Fraction of Chromatographic Separation Conclusion 92 6 SUMMARY AND RECOMMENDATIONS 93 BIBLIOGRAPHY 96 APPENDICES 109 BIODATA OF STUDENT 120 LIST OF PUBLICATIONS 121 xv

17 LIST OF TABLES Table Page 2.1 Composition of paddy kernel Proximate chemical composition of rice bran (dry basis), percentage Lipids profile in crude rice bran oil Name and structural interrelationship of natural tocopherols Extracted CRBO levels with different extraction times and sequential extraction Effect of different extraction times and sequential extraction on -oryzanol levels from rice bran Effect of different extraction times on tocopherols levels from rice bran Effect of sequential extractions on tocopherols levels from rice bran oryzanol and tocopherols levels in unsaponified fraction of CRBO oryzanol and tocopherols levels of unsaponified fraction of CRBO obtained from liquid-liquid separation at 4 C oryzanol and tocopherols levels in unsaponified fraction of CRBO obtained from precipitation separation at -20 C oryzanol and tocopherols levels obtained from liquid-liquid separation (at 4 C) on liquid fraction of unsaponified fraction of CRBO Precipitation oryzanol and tocopherols levels obtained from liquid-liquid separation (at 4 C) on solid fraction of unsaponified fraction of CRBO precipitation Recovered samples weight obtained from eluted fraction of chromatographic separation of -oryzanol oryzanol levels obtained using silica column chromatography 82 xvi

18 5.3 Recovered samples weight obtained from eluted fraction of chromatographic separation of tocopherols Tocopherols obtained using silica column chromatography 89 xvii

19 LIST OF FIGURES Figure Page 2.1 Diagram of Paddy Structure Molecular Structure of Individual -oryzanol According to Xu et al. ( Diagram of Tocopherols Molecular Structure Flow Chart of Sequential Extractions of Rice Bran with Different Solvent Polarity Flow Chart of Saponification of CRBO for Determination of -oryzanol and Tocopherols Flow Chart of Sample Filtration Prior to HPLC Determination of -oryzanol and Tocopherols Chromatogram of -oryzanol Standard detected using C 18 Novapak column Chromatogram of Tocopherols Standard detected using C 18 Symmetry Column Experimental Layout of -oryzanol and Tocopherols Separation of Unsaponified Fraction of CRBO by Precipitation and Liquid-liquid Separation Experimental Layout of -oryzanol and Tocopherols Separation using Silica Column oryzanol Absorbance obtained from Silica Column Chromatography Elution of Three Different Types of Samples Tocopherols Absorbance obtained from Silica Column Chromatography Elution of Three Different Types of Samples oryzanol and Tocopherols Extraction from Rice bran 91 xviii

20 LIST OF PLATES Figure Page 1.1 BERNAS factory that rice bran was taken at Kuala Selangor Unstabilized rice bran from rice milling was packed and to be sent to animal feed company Immediate rice bran stabilization process by using microwave in the factory Stabilized rice bran Crude rice bran oil and unsaponified fraction of crude rice bran oil 111 xix

21 LIST OF ABBREVIATIONS C GC/MS HPLC IU TE ROS CVD LDL HDL CRBO NaOH BHT mg g kg ppm ml MHz min m mm Degree Celsius Gas chromatography/ mass spectroscopy High pressure liquid chromatography International Unit Tocopherol Equivalent Reactive oxygen species Cardiovascular disease Low-density lipoprotein High-density lipoprotein Crude rice bran oil Sodium hydroxide Butylated hydroxytoluene Milligram Gram Kilogram Part per million Milliliter Mega Hertz minutes Micrometer Millimeter xx

22 nm i.d. UV/Vis UV SAS MeOH MeCl 2 rpm mol. wt CaCl 2 atm Nanometer Internal diameter Ultraviolet/visible Ultraviolet Statistical Analysis System Methanol Dichloromethane Revolution per minute Molecular weight Calcium chloride atmosphere P empirical solvent polarity xxi

23 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

24 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. 2

25 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). 3