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800-521-

SEPARATION OF ORYZANOL

FROM CRUDE RICE BRAN OIL

Arlene Karan

A thesis submitted in conformity with the requirements for the Degree of Master of Applied Science

Graduate Department of Chemical Engineering University of Toronto

0 Copyright by Arlene K m n 1998

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Separation of Oryzanol f?om Crude Rice Bran Oil Master of Applied Science, 1998 Arlene Karan Department of Chemical Engineering University of Toronto

ABSTRACT

The separation of oryranol from crude rice bran oil was investigated with the ukmate goal of industrial

appliaon. The oryzanol was concentrated from the crude oil and then isolated by silica gel column

chromatography. Reparation of the oryzanol concentrate was attempted by the extraction of

unsaponifiable matter, solvent extraction and step-wise elution chromatography. Methanol, ethanol and

dimethylformarnide were each evaluated for the solvent extraction. The column chromatographic

separation was optunized with respect to mobile phase, flowrate, column height, and column width.

The dimethylformarnide extraction of the crude oil proved to be the optimal method of oryzanol

concentration with respect to oryzanol recovery and cancentration in the extract. This method produced a

haion containing 8.4 * 0.6 % oryzanol, concentrating oryzanol by a fsctor of 9.7 0.6 fbm the crude

oil. The column chromatographic system, with the mobile phase toluenekthyl acetate 90: 10 by volume,

separated oryzanol from the less polar compounds m the extracted concentrate. However, oryzanol could

not be isolated Born the more polar components. Variations in flowrate, column height, and column width

did nat signlficaaty enhance the separation.

Scale-up of the ciimethyIfocmamide extraction did not afkt oryzanol recovery. Furthermore, extractions

with fresh and recycled solvent showed no difference in the extracted oryzanol ccmcentmte. Therdore,

pilotplaut scaleup of this extraction should be considered. Isolation of oryzanol &om the concentrate by

cu1~11lll chomatography should be fbther investigated with the use of reversephase silica as the

adsorbent.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Professor L.L. Diosady for his guidance, support,

and motivation throughout this challenging project. His ability to keep me focussed on the

bigger picture is a lesson I will always remember. I would also like to thank Joseph Alberti and

Professor Otto Meresz for their continuous assistance and invaluable advice.

I greatly appreciate the generosity of the Pulp and Paper Centre at the University of Toronto for

allowing me access to their High Performance Liquid Chromatograph. Special thanks to

Wenshan Zhuang for taking the time to instruct me on the use of the equipment. I kindly thank

Joy Cogson and the Ludwig Institute for the loan of the fiaction collector. I would also like to

express my thanks to David Balke and the members of the food engineering group for their

helpfbl input into my project. Finally, I would like to thank my family and Mark for their

support over the course of this thesis.

iii

TABLE OF CONTENTS

....................................................................................................................... 2.1 RICEBRAN 4

2.2 RICE BRAN OIL ................................................................................................................ 6

.................................................................................................................... 2.3 7-ORY~ANOL 9

2.3.1 Biological Effect of Oryzanol ................................................................................ 10

.......................................................................... 2.4 R E A C ~ ~ N S O F F A T S A N D F A ~ ACIDS 12

2.4.1 Saponification ........................................................................................................ 1 2

2.4.2 Esterification .......................................................................................................... 13

2.4.3 Hydrolysis ............................................................................................................. 13

2.4.4 Lipolytic Hydrolysis .............................................................................................. 13

2.4.5 Oxidation ............................................................................................................... 14

................................................................................................. 2.5 R E ~ ~ G O F C R U D E O I L 15

............................................................................................ 2.6 SEPARATIONOF~RYZANOL 17

2.6.1 Historical ............................................................................................................... 17

2.6.2 Saponification of Oil for Preparation of an Oryzanol Concentrate ......................... -18

2.7 CHROMATOGRAPHIC SEPARATION OF ORYZANOL ............................................................ 20

2.7.1 Chromatography Theory ........................................................................................ 20

2.7.1.1 General Theory ................................................................................................. -20

iv

2.7.1.2 HPLC Theory ..................................................................................................... 22

2.7.2 Separation of Oryzanol by HPLC ........................................................................... 23

2.7.3 Adsorption Column Chromatography of Oryzanoi Conceatrate .............................. 28

3.1 MATERIALS .................................................................................................................... 32

. 3 2 EXI'MCT?ON MJXHODS .................................................................................................. 32

3.2.1 Extraction of Unsaponifiable Matter ...................................................................... 32

..................................... 3.2.2 Solvent Extraction of Oryzanol from Crude Rice Bran Oil 33

3.2.2.1 Alcohol Extraction ............................................................................................ -33

3.2.2.2 Dimethylformamide Extraction .......................................................................... 34

3.2.2.3 Isolation of Oryzanol from Dimethylformamide Extract ..................................... 35

.................................................... 3.3 CHROMATOGRAPHIC METHODS ............................... ., -37

3 . 3.1 Column Chromatography ...................................................................................... 37

3.3.1 . 1 Packing the Column ........................................................................................... 37

. .............................................*......................................*......... 3.3 1. 2 Column Operation 37

3.3.2 Step-wise Elution .................................................................................................. -39

3.4 ANALYTICALMETHODS .................................................................................................. 40

3.4.1 Thin-Layer Chromatography .................................................................................. 40

3.4.1.1 Qualitative Methods ........................................................................................... 40

3.4.1.2 Semi-Quantitative Methods ................................................................................ 41

3.4.2 High Performance Liquid Chromatography ............................................................ 41

3.4.2.1 Operation .......................................................................................................... -41

3.4.2.2 Oryzanol Standards ........................................................................................... -43

3.4.2.3 Sample Preparation ............................................................................................ 44

3.4.3 Ultraviolet Spectroscopy ........................................................................................ 44

4.1 ORYZANOL CONTENT OF CRUDE RICE BRAN OIL ............................................................ 45

4.2 EXIIUCTION OF UNSAPONIFMLE MATTER .................................................................... 45

4.2.1 Saponification of Orylanol .................................................................................... 46

LIST OF FIGURES

Figure 2-1: Structure of the Mature E r e Grain ............................................................................ 5

................................................................................................. Figure 2-2 : Oryzanoi Structures 9

Figure 2-3 : HPLC Chromatogram of Crude Rice Bran Oil ..................................................... 26

Figure 2-4 : Overlay of Ultraviolet Spectra of Four Oryzanol Components ............................... -26

Figure 2-5 : HPLC Chromatognun of Saponified Rice Bran Oil ................................................ 27

Figure 3-1 : Dimethylformamide Extraction of Crude Rice Bran Oil ......................................... 34

Figure 3-2 : Preparation of Oryzanol Standards for HPLC ......................................................... 43

Figure 4- 1 : Saponification of Oryzanol ..................................................................................... 46

Figure 4-2 : Ionization of Phenol .............................................................................................. -47

Figure 4-3 : Schematic of TLC Plate ......................................................................................... 51

Figure 4-4: Effect of Mobile Phase .......................................................................................... 54

Figure 4-5 : Effect of Packing Height ........................................................................................ 55

.................................................................................................. Figure 4-6 : Effect of Flowrate 56

Figure 4-7 : HPLC Chromatogram of DMF Extract 6 x 30 rnL .................................................. 70

Figure 4-8 : HPLC Chromatognun of DMF Extract 6 x 120 mL: .............................................. -71

Figure 4-9 : HPLC Chromatogram of DMF 5 x 30 .................................................................... 81

Figure 4- 1 0 : HPLC Chromatogram of Run AW - Oryzanol Peak .......................................... -82

Figure 4-1 1 : HPLC Chromatognun of Run AW - Remaining Vials .......................................... 83

Figure 4-12 : HPLC Chromatognun of Run AW - Ethanol Wash ............................................. 84

Figure 4- 13 : HPLC Chromatogram of Run BG - Oryzanol Peak ............................................. -85

Figure 8- 1 :

Figure 8-2:

Binary Solvent Strength as a Function of % B ...................................................... 98

Calibration Curve for Oryzanol in TolueneEthyl Acetate 90: 10 ............................. 99

vii

...................... Figure 8-3 : Calibration Curve for Oryzanol in Hexane/Ethyl Acetate 80:20 v/v 100

Figure 8-4 : Calibration C w e for Oryzanol in HexaneEthyl Acetate 70: 3 0 1.k ...................... 10 1

Figure 8-5 : Calibration Curve for Oryzanol in HexanelDiethyl Ethel 70.30 .......................... -102

............... Figure 8-6 : HPLC Calibration Curve for Oryzanol(250 to 750 ppm) at h = 325 run 1 10

Figure 8-7 : HPLC Calibration Curve for Oryzanol(60 to 100 ppm) at h = 325 nm ................. I 1 1

Figure 8-8 : HPLC Standard Addition of Oryzanol to Rice Bran Oil ........................................ 1 14

viii

LIST OF TABLES

............................................................................ Table 2- 1 : Products fiom Crude Rice Bran OiI 6

Table 2-2 : Composition of Crude Rice Bran Oil ........................................................................ -8

Table 2-3 : Composition of Unsaponifiable Lipids ..................................................................... -8

Table 2-4: Unsaponifiable Content of Rice Bran Oil ................................................................ 16

Table 2-5 : Oryzanol and Tocotrienol Content of Rice Bran Oil .............................................. - 1 7

Table 3- 1 : Variables Optimized in Dimethylformamide Extraction .......................................... 35

Table 3-2 : Variables Optimized for Column Chromatographic Separation ................................ 39

Table 3-3 : Maximum Ultraviolet Absorbance Wavelengths for Oryzanol ............................... 4 4

Table 4- 1 : Effect of the pH of Saponified Oil on Oryzanol Recovery ....................................... 48

Table 4-2: Solvent Strengths of Mobile Phases ........................................................................ SO

Table 4-3 : Effect of Mobile Phase ............................................................................................ 52

Table 4-4 : Summary of Stepwise Elution of Rice Bran Oil ....................................................... 61

Table 4-5 : Alcohol Extraction of Crude Rice Bran Oil .............................................................. 62

Table 4-6 : EfTect of So1vent:Oil Ratio on Extractability of Oryzanol ........................................ 63

Table 4-7 : Effect of Hexane Wash on Oryzanol Recovery ........................................................ 65

Table 4-8: Summary of Dimethylformamide Extractions of Crude Rice Bran Oil ...................... 67

Table 4-9 : Comparison of Dimethylformamide Extract and Step-wise Elution ......................... 69

Table 4- 1 0 : Oryzanol Recovery from Preliminary Runs AQ - AV ........................................... 74

Table 4-1 1 : Oqmmol Recovery fiom Runs AW - BA .............................................................. 75

Table 4- 1 2 : Oryzanol Recovery from Runs BB - Run BG ....................................................... 76

Table 8- 1 : Solvent Strengths of Binary Mixtures ....................................................................... 97

........................................................................................ Table 8-2 : Oryzanol Content in Oil 113

ix

1 INTRODUCTION

Rice is a staple food upon which 60% of the total world population depends. In 199 1, world rice

production was 466 million metric tons (MMT) and has been increasing faster than any other

cereal grain (Sayre, 1991). This rapid growth has registered in many countries including China,

India, Brazil and the United States.

All rice is milled prior to consumption producing hull, bran, germ, and white rice. Rice hulls

have no food value whereas the bran and germ are rich in protein, lipids, vitamins, and trace

minerals (Saunders, 1985). The overall composition of rice bran, its nutritional profile, functional

characteristics and apparent hypoallergenicity offer it many applications in a healthy diet high in

dietary fiber and low in saturated fat (Marshall, 1994). In addition, Kahlon rt al. (1990) found

that rice bran was as effective as oat bran at lowering serum cholesterol in hypercholesteroiemic

hamsters.

Today, only 30% of rice is milled through two stage mills, which enable separation of the bran

fiom the hulls. As a result, only 8 MMT of rice bran suitable for food use or oil extraction is

produced annually (Sayre, 1991). The naturally occurring enzymatic activity of rice bran leads

to the rapid hydrolysis of the oil after milling. Therefore, immediate stabilization of the bran is

essential to prevent rancidity. Until recent developments of commercial stabilization processes

for rice bran, its predominant use and market in the United States had been as an ingredient in

livestock feed. One of the challenges facing the rice industry is to utilize the food value of rice

bran and its components more effectively (Marshall, 1994). Bran fiom rice is unique among

cereal grains in its high oil content of 16 - 32% (Marshall, 1994). In 1988, only 450,000 MT of

rice bran oil were produced despite the potential of 2 MMT. Japan is the leading producer of

rice bran oil with an average annual productinn of 100,000 MT. Extraction of rice bran oil began

in Korea with the hydraulic press method. Since then, technology hzs evolved to continuous

hexane extraction. There are ongoing investigations into various critical fluid extraction systems

(Sayre, 1988). Rice bran oil is generally considered to be one of the highest quality vegetable

oils available in terms of its cooking qualities, shelf life, and fatty acid composition (Sayre

199 1).

Rice bran oil has an excellent ability to adjust cholesterol serum level due to its low content of

l inolenic acid and its high antioxidant content, including both tocopherols and oryzanols. Rice

bran oil contains a higher level of unsaponifiable matter, 4.2%, than any of the other common

vegetable oils (Sayre, 199 1). The unsaponifiable matter is comprised mostly of sterols along with

oryzanol, both of which lend to the stability of the oil. The crude rice bran oil can be refined into

a number of high quality beneficial products including edible rice bran oil, free fatty acids,

glycerol, phospholipids, wax, sterols, triterpenes, tocopherols, and oryzanol.

y-Oryzanol is a mixture of the ferulic acid esters of triterpene alcohols and plant sterols (Rogers

et a/., 1993). It is found at concentration levels of 0.96 - 2 9 % in crude rice bran oil (Marshall,

1994). The oryzanols are antioxidants that are believed to possess curative functions for many

human illnesses. Most notably, y-oqnanol possesses hypocholesterolemic activity, enabling this

compound to aid in the reduction of cholesterol levels in humans (Seetharamaiah and

Chandrasekhara, 1988). It has been shown to be non-genotoxic and non-inhibitory of cellular

communication (Tamagawa et al., 1 992), making it safe for therapeutic use. As a result of its

properties, oryzanol is the most valuable component of rice bran oil in the Japanese market

(Sayre, 1988).

Agritech Inc. of Louisiana, MI, USA, has developed a process for the extraction of crude rice

bran oil from rice bran using a light hydrocarbon solvent. This process produces high quality oil

with a low free fatty acid content along with stable rice bran suitable for human consumption. In

light of the beneficial components in both the rice bran and its oil, Agritech has undertaken the

development of a comprehensive process capable of producing not only the bran and refined oil,

but also value-added components such as oryzanol. Accordingly, the separation of oryzanol

from crude rice bran oil with the perspective of future industrial application was the focus of this

work.

In this thesis, the preparation of an oryzanol concentrate fiom crude rice bran oil and the fbrther

isolation of oryzanol by means of silica gel column chromatography were investigated. Three

methods were examined for the preparation of the oryzanol concentrate. As oryzanol has been

reported to be present in the unsaponifiable fiaction of crude rice bran oil, the initial method

consisted of extracting the unsaponifiable matter fiom saponified oil. In the second method, the

oryzanol fraction was concentrated ftom crude rice bran oil via solvent extraction. The solvents

assessed for this extraction were methanol, ethanol and dimethylformamide. Finally, step-wise

column chromatography was used to elute a concentrated oryzanol fiaction. Optimization of the

column chromatographic separation was attempted.

2 LITERATUIW REVIEW

2. t Rice Bran

Rice is one of the leading food crops of the world and is second only to wheat in terms of mnual

production for food use (Marshail, 1994). World rice production has seen a steady increase from

1980 to 1993 with consumption keeping pace. As a result of this constant growth in both

production and consumption, rice research and development activity has become increasingly

important.

The mature rough rice grain structure is composed of the hull, pericarp, seed coat, nucellus,

embryo, aleurone layer and endosperm as seen in Figure 2-1. The hull is removed from the

rough rice through the process of dehulling, exposing the caryopsis. The four layers of the

caryopsis - pericarp, seed coat, nucellus, and aleurone, along with much of the embryo comprise

the bran portion of the rice grain. This portion accounts for 5 - 8% of the brown rice weight and

contains almost all of the oil and most of the protein, ash, and fiber.

Hypocholesterolernic properties of rice bran, rice bran oil, and their fractions have been observed

in animal and human studies (Seethararnaiah and Chandrasekhara, 1988, 1989; Kahlon el al.,

1990; Nicolosi el al., 1991). Recent research has indicated that a diet supplemented with rice

bran may be an effective means of reducing serum cholesterol. As well, consumption of rice

bran may be specifidly beneficial in reducing the risk of cardiovascular disease and colon

cancer (Marshall, 1 994).

Figure 2-1: Structure of the Mature Rice Grain

(Marshall, 1 994)

Rice bran is potentially a rich source of nutrients for humans. Currently the vast majority of rice

bran is sold as animal feed. Numerous potential food uses for full-fat and defatted rice brans

have been well proven in laboratory tests but are not yet in commercial practice (Saunders,

1986). One of the challenges facing the rice industry is to utilize the food value of rice bran

more effectively (Marshall, 1994).

The greatest restriction to the use of rice bran as a food ingredient, or even as a source of edible

oil, is its naturally occurring enzymatic activity. This causes rapid hydrolytic rancidification of

the bran after milling, producing a soapy and/or bitter taste. In addition, oxidative rancidity

results in strong, unpleasant "rancid" odours. The mechanisms of hydrolytic and oxidative

rancidity are discussed in fbrther detail in Section 2.4. Stabilization of the bran soon after

milling or inactivation of the indigenous lipase enzyme beforehand is thus very important.

Extraction of the oil from rice bran should be done within a Few hours of milling to avoid a high

free fatty acid content.

2.2 Rice Bran Oil

Rice bran oil contains approximately 16 - 32% of rice bran (Marshall, 1994). It is extracted

from the stabilized bran after milling. The oil may be removed fiom the bran using hydraulic

pressing or solvent extraction. Hydraulic pressing results in only 50% oil recovery, therefore

solvent extraction, generally with hexane, is preferred. Once extracted, rice bran oil is stable and

may be stored for subsequent processing. Crude rice bran oil can be refined into a high quality

salad oil and contains several other components that may be isolated as commercial products as

itemized in Table 2- I .

Products fiom Crude Rice Bran Oil Edible Rice bran oil Free fatty acids Glycerol 0 ~ 0 1 Phospholipids wax Sterols, triterpenes tocopherols, tocotrienols

Table 2-1: Products from Crude Rice Bran Oil

(Sayre, 1989)

The composition of crude rice bran oil is shown in Table 2-2. The main component, the

triglycerides makes up approximately 80% of the oil. The triglyceride content varies but is

primarily dependent on the extent of hydrolysis that occurred prior to stabilization or the bran.

Partial esters present as mono- and diglycerides as well as the free fatty acids are a ~eflection of

the hydrolysis that has occurred. The fatty acid profile of rice bran oil reveals approximately

19% saturates (palmitic), 42% monounsaturates (oleic) and 39% polyunsaturates (linoleic). The

relatively even balance between linoleic and oleic acid, a low level of linolenic acid, and a high

level of antioxidants make rice bran oil stable under frylng conditions (Saunders, 1988).

Rice bran oil contains a high level of unsaponifiable matter, 4.2%, as compared to most other

vegetable oils. As seen in Table 2-3, the sterols make up the largest fiaction of the

unsaponifiables. The excellent resistance of rice bran oil to oxidation is due not only to its

tocopherol content but also to the ferulic acid esters of triterpene alcohols and phytosterols,

known as oryzanol. Oryzanol is present at 0.96 - 2.9% in rice bran oil (Marshall, 1994).

Numerous studies have shown that rice bran and its components are effective in lowering plasma

and liver cholesterol and the LDUHDL cholesterol ratio of plasma. It is the fir11 fat rice bran and

not the defatted bran that significantly lowers total cholesterol and low-density lipoprotein (LDL)

levels while maintaining the high-density lipoprotein @DL) levels. This indicates that the

hypocholesterolernic activity of rice bran is due primarily to the oil fiaction of the bran

(Marshall, 1 994).

I Triterpene Alcohols, Tocopherols

Composition of Crude Rice Bran Oil

Table 2-2 : Composition of Crude Rice Bran Oil (Sayre, 1988)

Sapoaifia bie Lipids Neutral Lipids

Trig1 yceridrs Dig1 ycerides Monoglycerides Free Fatty Acids Waxes

Glycolipids Phospholipids

Unsaponifirbk Lipids

-

Unsaponifiable Lipids in Cmde Rice Bran Oil

9046% 88-89?/0

83.86% 3 -4% 67% 24% 34%

6'7% 4 5 %

4.2%

(940 of Crude Oil) L

Total Unsawnifiable Lkids

Phytosterols, Sterolesters, Hydrocarbons

Campester01 Stipsnasterol

QMethyl Stmls C itrostadieno 1 Gramisterol Obtusifoliol

Triterpene Alcohols 24-Methylene-cycloartanol C ycloartenol C ycloartanol

Less Polar Compounds Aliphatic Alcohols Hydrocarbons : Squalene Tocotrieno Is Tocopherols

Table 2 -3 : Composition of Unsaponifiable Lipids (Sayre, 1988)

The fatty acid composition of a vegetable oil generally governs the extent of its effect on blood

cholesterol. However in the case of rice bran oil, its cholesterol-lowering properties cannot be

solely explained by its fatty acid composition. The University of Mczsachusetts-Lowell

laboratories have established that the unsaponifiables are likely the active ingredients. The

content of these active ingredients, which include plant sterols and oryzanol, is higher in rice

bran oil than any other vegetable oil (Campen, 1988).

Oryzanol was first separated from rice bran oil by Kaneko and Tsuchiya in 1954 and thought to

be a single compound. Subsequently, it was shown to be a mixture of ferulic acid esters of

triterpene alcohols and plant sterols (Shirnizu et a!., 1957; Ohta and Shimizu, 1957; Ohta and

Shimizu, 1958). The oryzanol compounds are shown in Figure 2-2 and have been identified as

cycloartenyl ferulate (ma), 24-methylene cycloartanyl ferulate (ILlb), campestryl fmlate (IIlc),

cycloartanyl ferulate (md), and P-sitosteryl ferulate (me).

Figure 2-2 : Oryzanol Structures

@iack and Saska, 1994)

y-Oryzanol is a white or slightly yellow crystal or crystalline powder, stable at room temperature

(Tarnagawa el al., 1992). !: has a melting point of 137.5"C: - 138. S°C and shows ultraviolet

absorption maxima at 23 1,290, and 3 I5 nm in heptane (Tsuchiya and Kaneko, 1954). Oryzanol

is soluble in solvents such as diethyl ether, methylene chloride, acetone and alcohol and is only

somewhat soluble in non-polar solvents like petroleum ether and hexane (Seetharamaiah and

Prab hakar, 1 986).

2.3.1 Biological Effect of Oryzanol

Oryzanol was initially introduced in 1962 in Japan for the treatment of vegetative neurosis

(Oguni, C. et al., 1962). In the 19703, oryzanol compounds were marketed for treatment of

symptoms due to climacteric disturbance, organ neurosis, gastro-intestinal disorder in irritable

bowel syndrome, and sequelae induced by head injury (Tarnagawa et a!., 1992). Oryzanol has

been shown to exhibit several other pharmacological effects including growth promoting activity

in animals (Sakama and Tsuchiya, 1959), the ability to maintain estrous cycles in rats (Sojiro and

Suetzu, 1980) and promotion of skin capillary action (Kamimura et a!., 1964).

Of much interest is the mild but significant cholesterol-lowering property of oryzanol.

Hypercholesterolernia is a major risk factor for coronary heart disease. However, treatment by

diet modification and/or administration of a hypolipidemic drug can reduce the risk (Yoshino el

a/., 1989). In 1986, y-orymol compounds were introduced for the treatment of hyperlipidemia

(Tamagawa et a/., 1992). Much research has been pursued to evaluate the benefits of oryzanol in

this capacity.

Seethararnaiah and Chandrasekhara (1988) studied the hypocholesterolemic activity of oryzanol

in rats and found that levels of total, free, and esterified cholesterol were significantly reduced in

groups fed oryzanol as compared to the control group. The addition of oryzanol to the diet at the

optimal level of 0.5%, did not lower HDL levels. Trials conducted by Yoshino et a!. (1989)

using patients with hyperlipidemia also showed a reduction in LDL-cholesterol levels while

maintaining HDL levels. Yoshino et a!. reported that oryzanol was found to inhibit not only

intestinal cholesterol absorption, but also hepatic cholesterol biosynthesis in experimental

animals.

There is considerable evidence to suggest that hypercholesterolemia, particularly increased LDL-

cholesterol levels, is associated with platelet aggregation. Platelet adhesion and aggregation are

key factors leading to arterial thrombosis (Carvalho el d, 1974). In view of the

hypocholesterolemic activity of oryzanol, its potential as an inhibitor of platelet aggregation was

studied (Seethararnaiah el a/., 1990). Oryzanol, when added to a high cholesterol diet, was found

to significantly inhibit the aggregation of platelets in rats. Further work is needed to understand

the mechanism of action of oryzanol.

Given the many therapeutic benefits of oryzanol, its safety as a drug must also be established. It

has been shown to be non-genotoxic and non-inhibitory of cellular communication (Tsushimoto

et al., 199 1). When administered orally, y-oryzanol is absorbed mainly into the portal venous

blood, mostly as intact form. However, during absorption it is partly rnetabdized to yield ferulic

acid in the intestine (Fugiwara et a!., 1983). Carcinogenicity studies of y-orytanol in B6C3FI

mice at dose levels 33.3, 100, 333 times the clinical dose, concluded that y-oryzanol is not

carcinogenic (Tamagawa el al. , 1 9%). However, in a rat wide-spectrum organ wcinogenesi s

model, y-oryzanol enhanced the induction of lung carcinomas but slightly inhibited the

development of pancreatic eosinophilic foci and hepatic lesions at doses 100 - 1 SO times the

clin~cal dose (Hirose et a!., 199 1). Nevertheless, oryzanol has had a safe clinical history since it

was first introduced in 1 962 (Yoshino et al., 1989).

2.4 Reactions of Fats and Fatty Acids

2.4.1 Saponification

Saponification involves the reaction of a fat (triglyceride) with an alkali to yield glycerol and a

salt (also called a soap). This reaction is hndamental in soap making.

C3H3(OOCR), + 3NaOH CP5(OH)3 + 3NaOOCR

Trig1 yceride Alkali Glycerol Fatty Acid Salt (Soap)

Under controlled conditions, reacting free fatty acids with an alkali results in the Formation of a

salt and sets fiee a water molecule.

RCOOH + NaOH -----+ RCOONa + H20

When fatty acids are used, soap formation may be effeeted with sodium carbonate. This reaction

produces a soap, water and carbon dioxide.

After neutralization, the batch is generally boiled with an excess of caustic soda to sapon@ the

small amount of neutral, unsplit fat which may still be present.

2.4.2 Esterification

Esterification of fatty acids generally occurs with alcohols such as glycerol. The reaction is

reversible and proceeds to completion only when water is removed. Acid catalysts promote this

reaction.

C3H5(Ok'i)3 + 3HOOCR ---+ C315(00CRh + 3 H2O

Glycerol Fatty Acid Trig1 yceride Water

2.43 Hydrolysis

The reverse reaction is known as hydrolysis and is similarly catalyzed. Tailor made triglycerides

of any desired composition may be made by hydrolyzing the fats to fatty acids, purifying the

fatty acids by fractional distillation, and recombining the fatty acids in desired proportions with

glycerol in the process of esterification. The products find many uses ranging from food

additives to non-edible industrial emulsifiers.

CJH~(OOCR)~ + 3 Hz0 CIH~(OH), + 3HOOCR

T rig1 y ceride Water Glycerol Fatty Acid

2.4.4 Li polytic Hydrolysis

During the dehulling of rice, the outer layers of the kernel are disrupted, allowing the oil to make

contact with lipases. The lipases catalyze the rapid hydrolysis of trigiycerides to fiee fktty acids.

Lipases produced by bacteria and mold on the surface of the kernel also come into contact with

the oil, further promoting the hydrolysis reaction. Therefore, the rate of free fatty acid formation

is dependent on the degree of disruption of the kernel, the quantity of lipase present, the moisture

content and temperature. In rice, approximately 30% of the oil can be converted to free fatty

acids in the period of one week, under high humidity, and temperature conditions (Enochian e!

a!. , 1980). High levels of free fatty acids in oil lead to a soapy taste.

2.4.5 Oxidation

Rancid off-flavours and off-odours in oil are the result of oxidative deterioration. The reaction

can be either enzyme catalyzed or non-enzymatic. Both reactions result in the production of

hydroperoxides that read to yield secondary oxidation products such as aldehydes and ketones.

These products produce the off-flavours and off-odours associated with rancidity.

Enzymatic oxidation is catalyzed by lipoxygenase, an enzyme found in the germ. It is

responsible for the oxidation of saturated free fatty acids. As lipoxygenase acts only on saturated

fiee fatty acids, the extent of oxidation by this mechanism depends on the amount of free fatty

acid made available by lipolytic hydrolysis.

Non-enzymatic oxidation may be catalyzed by metal ions, light, high-energy radiation, and heat.

The reaction can occur by t h e radical oxidation (auto-oxidation) or photoacidation routes. In

free radical oxidation, lipid molecules produce free radicals by their interaction with oxygen in

the presence of a catalyst. Hydroperoxides are the initial reaction products. In photo-oxidation,

a singlet oxygen is formed by the reaction of an excited photosensitive molecule (e.g. riboflavin,

heavy metal ions) with oxygen. The singlet oxygen goes on to react with fatty acids to produce

the hydro peroxides. Non-enzy matic oxidation, unlike other mechanisms, is inhibited by

antioxidants present in the rice bran.

2.5 Refining of Crude Oil

Food applications of vegetable oils generally require only the triglyceride porticr! of the crude

oil. Some components, such as unsaponifiables, pigments, and partial esters, are more difficult

to remove but do not detract the oil from food utilization. Processing crude oil to food grade

edible oil involves dewaxing, degumming, neutralization, bleaching, winterization, and

deodorization, which are described below.

Dewaxing of rice bran oil is done while still in miscella form by cooling, crystallization of

waxes, and centrifugation or filtration for their removal. Gums, which are polar lipids having

surface-active properties, are removed by degurnming agents such as phosphoric or citric acid

that hydrolyze the gums. The hydrolyzed gums are separated fiom the oil by centrifbgation.

Neutralization, also known as refining, involves the removal of free fatty acids by caustic soda.

The acids are converted to sodium soaps, which are hydrated and also removed by

centrifugation. Large refining losses are often encountered with rice bran oil due to its high free

fatty acid content.

Acid-activated bleaching clays are added to the oil to remove pigments, oxidized lipids and polar

components ftom the oil. The clay is then removed from the oil by filtration. Deodorization is a

vacuum steam distillation process that removes odours, flavours, and tiee fatty acids. Volatile

compounds found in the deodorizer distillate include aldehydes ketones, peroxides, and a

portion of the tocopherols and sterols present in the oil. Winterization, generally performed

before deodorization, removes the high melting point triglycerides fiom the hction of the oil

that remains liquid at refigeration temperature. The triglycerides are crystallized out of the oil

by chilling and subsequently sepmted via filtration.

Processing of rice bran oil causes significant variation in the levels of unsaponifiables present in

commercial rice bran oils. Table 2-4 reveals the loss of up to SO?/, of the unsaponifiables

throughout the processing steps. More strikingly, up to 90% of the oryzanol and tocotrienol

content of crude oil can be lost as seen in Table 2-5. From these observations, it is evident that

new techniques in the processing of crude oil must be developed in order to preserve the active

components in rice bran oil. Isolation of these valuable components should begin with the crude

oil to ensure maximum recovery.

Table 2-4: Unsaponifiable Content of Rice Bran Oil (Marshall, 1994)

Oil Type Percent Crude Refined and bleached Refined, bleached and deodorized Refined, bleached, deodorized and winterized

Table 2-5 : OryzanoI and Tocotrienol Content of Rice Bran Oil (Marshall, 1994)

3.98 2.85 2.5 1 1.99

Content in Rice Bran Oil (ppm)

Crude Commercially Available:

Brand I Brand 2 Brand 3 Brand 4

Oryzanol 12 221

lo00 2000 720 200

Tocotrienoi 713

500 595 353 78

Crude rice bran oil possesses excellent oxidative stability due to its high content of oryzanols and

tocopherols. Refining of oil diminishes its stability due to the removal of antioxidant

components. Oryzanol and tocopherol content in crude oil was Found to remain almost constant

during ten weeks of storage. However, in refined oil, oryzanol content decreased slowly until 38

days and decreased rapidly thereafter (Yoon and Kim, 1994). Therefore, crude oil is the most

advantageous for storage as it has the best resistance to oxidation.

2.6 Separation of Oryzanol

2.6.1 Historical

Tsuchiya and Kaneko (1954) first detected oryzanol during their studies on vegetable oils. Rice

bran oil exhibited a characteristic absorption in the ultraviolet region and searching for its source,

a crystalline substance was isolated and named oryzanol (Shimizu ef a/., 1957). To separate

oryzanol, the researchers removed the free fatty acids tiom rice bran oil with sodium carbonate,

esterified the fats, and removed the fatty acid esters using low-pressure distillation. Repeated

chromatography of the residue and recrystallization afforded oryzanol.

Shimizu el ai. (1957) later extracted orytanol from rice bran oil utilizing the phenolic nature of

its hydroxyl group. Free fktty acids were removed with sodium carbonate and the deacidified oil

was passed through an alumina column. The adsorbed phenolic compounds were eluted &om

the column and oryzanol was recrystallized fiom the eluted hc t ion Acetylation of oryzanol

revealed that it was not a single unity as thought by Tsuchiya and Kaneko, but three separate

substances which were designed orymol-4 4 3 , and 4. Shirnizu and Ohta (1957) identified

oryzanol-A as cycloartenyl ferulate and oryzanol-(: as 24-methylene cycloartanyl ferulate

18

(Shimizu and Ohta, 1958). Oryzanol-B was later found to be a mixture of the remaining three

oryzanol compounds: campestryl ferulate, cycloartanyl fernlate, and $-sitosteryl ferulate.

2.6.2 Saponification of Oil for Preparation of an Oryzanol Concentrate

Oryzanol is reported to be found in the unsaponifiable hction of rice bran oil (Saunders, 1985).

Utilizing this fact the separation of oryzanol, in many cases, began with the saponification of rice

bran oil. This was followed by liquid-liquid extraction from the soap stock to prepare a

concentrated oryzano 1 fraction.

Tsuchiya et ai. (1958) separated a 2.5% yellow solid containing 60% oryzanol f?om rice bran oil.

The oil was saponified with 171.30?! NaOH and centrifuged to produce a soapstock. The

soapstock was decomposed with HCI, washed with hot water and made alkaline with NaOH. The

unsaponifiable matter was extracted with diethyl ether and the extract was discarded. The NaOH

layer was then treated with HCI and the final product was extracted with diethyl ether.

Seethararnaiah and Prabhakar (1986) extracted oryzanol from the soap stock of rice bran oil.

The oil was heated to 6S°C and 20% NaOH added with stirring for 20 minutes. The mixture was

cooled and allowed to settle overnight. The supernatant was decanted and the sediment was

centrifirged to obtain solid soap stock, containing 1.3 - 3.1% oryzanol. To prepare the oryzanol

concentrate, water was added to the soap stock, thoroughly mixed, and then shaken with

methanol and ether in a sepmtory funnel. The ether phase was separated and extracted with 10

mL portions of 5% KOH until the KOH layer was colourless. The combined KOH extracts were

neutralized with 1 N acetic acid and extraaed with diethyl ether. The ether extracts were washed

with water, dried over anhydrous Na2S04 and the solvent was evaporated. This achieved a

concentrate containing 16.3% oryzanol. The process recovered 76.5% of the oryzanol from the

soap stock. The oryzanol content was found to increase as the pH of the soap stock increase

from 8 to 1 0.5; however, overall recovery of oryzanol decreased at higher pH values. At pH 9.5

an emulsion formed during the extraction procedure, therefore pH 9 was chosen as optimal.

Alumina column chromatography and recrystallization, as discussed in Section 2.7.3, purified

the oryzanol concentrate.

It is difficult to compare the effectiveness of the separation methods by Tsuchiya et al. (1957)

and Seetharamaiah and Prabhakar (1 986) due to the lack of mass balances presented. The

overall recovery of oryzanol from rice bran oil was not indicated, nor was the loss of oryzanol

during saponification of the oil. The saponification reaction involves the cleavage of the ester

linkage in the tiglyceride by the hydroxide ion. Oryzanol also contains an ester linkage that

combines the ferulic acid with plant sterols and triterpene alcohols. Under appropriate

conditions, saponification may result in the cleavage of the ester linkage in the oryzanol

compound, splitting it into its two fi-actions.

Sakama and Tsuchiya (Chem abstracts, 1957) saponified oryzanol by warming it for two hours

with 5% KOH in ethanol along with water for the purpose of separating the esters of cyclic

alcohols Eom oryzanol. Hy Wells, Shin and Godber (1995) also noted the loss of oryzanol due

to saponification. The researchers prepared an i so pro panol-crude oil miscella from stabilized

rice bran oil. The miscella was saponified with 80% KOH wlw for complete saponification of

the glycerides. It was then further extracted using hexane and water. After one and a half hours,

the vitamin E and oryzanol content of both the aqueous and hexane layers were determined using

high performance liquid chromatography. Results indicated that under optimum saponification

and liquid-liquid extraction conditions, there was negligible loss of vitamin E during

saponification. Under the same conditions, 30% oryzanol was lost during saponification, while

the remaining 70% was recovered in the aqueous layer.

Evidently, saponification of the oil has been effective in preparing an oryzanol concentrate from

which oryzanol can later be isolated. However, the literature is unclear concerning the amount

of oryzanol recovered by these methods and the conditions leading to the saponification of this

compound. Furthermore, saponification converts the potentially edible rice bran oil into a

soapstock, which cannot be used for food applications. Given the excellent stability of rice bran

oil as a frying oil and its healthfid fatty acid composition, other methods of oryzanol separation

have also been investigated.

2.7 Chromatographic Separation of Oryzanol

2.7.1 Chromatogmphy Theory

2.7. I . 1 General Tkemy

Chromatography is the continuous equilibration of solute between two phases, the stationary

phase and the mobile phase. The partitioning of solutes between the two phases accounts for

their separation. Adsorption chromatography makes use of a solid stationary phase (adsorbent)

and a liquid or gaseous mobile phase. The adsorbent is either poured into an open tube, known

as column chromatography (CC) or spread on a plate, known as thin-layer chromatography

(TLC). The sample is introduced at one end of the adsorbent bed and induced to move through

the bed by means of pressured flow or capillary action. As the sample moves through the bed,

the solutes are adsorbed onto the adsorbent surface to a greater or lesser extent, depending on the

chemical nature of the solute. The more strongly adsorbed solutes will be retained longer on the

adsorbent surface and consequently move slowly through the bed. Conversely, weakly adsorbed

solutes will spend most of their time in the mobile phase and move quickly through the bed. It is

the variation in the migration rates of different solutes that effects their separation on the

chromatographic bed.

The success of a chromatographic separation is dependent on a number of parameten. The

choice of adsorbent and solvent are key factors along with column height, width, mobile phase

flowrate and temperature. In adsorption chromatography the solvent competes with the solute

for active adsorption sites on the adsorbent surface. A solvent's eluent strength (go) is a measure

of the solvent adsorption energy, with the value for pentane defined as zero. Therefore, the

greater a solvent's ability to displace solutes From a given adsorbent, the higher its eluent

strength and the more rapidly solutes will be eluted from the chromatographic bed. When

combining solvents, their resultant eluent strength is not a linear function of the relative

proportions of each solvent. A small amount of polar solvent will markedly increase the eluent

strength of a non-polar solvent (Harris, 1991). Snyder (1968) has developed a procedure for the

estimation of the eluent strength of binary mixtures as outlined in the Appendix (Section 8.2).

There are several adsorbents that have been employed in adsorption chromatography; however,

only a limited number find wide commercial application. Among these, silica gel and alumina

have proven to be especialiy usefbl.

Silica gel is the most universally used chromatographic adsorbent (Hams, 199 1 ; Snyder, 1968).

It is widely commercially available, has an immense capacity for both linear and non-linear

isotherm separations, and is almost completely inert towards labile samples. Silica is an

amorphous, porous solid that can be prepared in a wide range of surface areas and average pore

diameters. It is classified as a polar adsorbent. The active adsorption sites are surface Si-0-H

(silanol) groups, which can adsorb water from the air and are thereby slowly deactivated. The

gel can be re-activated by heating at 200°C to drive off the water. Higher temperatures will

cause irreversible damage to the silica gel. Due to the many advantages of working with silica, it

is a logical first choice as a general-purpose adsorbent (Snyder, 1968).

Alumina, like silica, is a good all-purpose polar adsorbent. Nevertheless, it can alter some

m p l e types, most notably strongly acidic samples (pKa <5) that are chemisorbed onto the

alumina. Active aluminas are markedly sensitive to the differing shapes of various aromatic

hydrocarbons permitting the excellent separation of many aromatic isomers.

2.1.1.2 HPLC Theory

High performance liquid chromatography (HPLC) is based on the principles of classical

chromatography but achieves a higher resolution of solutes. This is accomplished by reducing

the particle size of the stationary phase. The smaller particle size allows the solute to diffise

rapidly between the mobile and stationary phases. This increases the rate of equilibrium between

the two phases and results in more uniform migration paths. The use of finer particles creates a

much higher resistance to solvent flow. It is therefore necessary to use high pressure,

approximately 7 - 40 MPa, to force the solvent through the column.

Normal-phase chromatography refers to the use of a polar adsorbent, such as silica, and a less

polar solvent for the mobile phase. Also commonly used, reverse- ;?ax chromatography

employs a non-polar adsorbent and a more polar mobile phase. The adsorbent is generally a

bonded phase, such as C18, attached to the surface o f the silanol groups. Reverse-phase

chromatography provides excellent separations. It eliminates tailing problems associated with

adsorption of polar compounds by polar packings and is less sensitive to polar impurities, such as

water, in the eluent (Hams, 199 1 ).

2.7.2 Separation of Oryzanol by HPLC

Many researchers have employed chromatographic methods for the separation of oryzanol from

rice bran oil. It is an eflFective method of purifying oryzanol from the extracted concentrate.

Wells (1997) proposed that one approach for the separation and purification of tocopherols,

tocotrienols, and oryzanol from rice bran oil would be to produce an oil fiaction enriched with

the target compounds, suitable for HPLC separation. Wells' proposed process includes alkali

saponification of the oil, liquid-liquid extraction of target compounds from the saponified

mixture, partial vacuum evaporation of solvent from the target compounds, followed by HPLC

for purification. The effectiveness of HPLC for the separation of these target compounds has

been previously proven.

Rogers e! af. (1993) reported the identification and quantitation of individual y-oryzanol

components and simultaneous assessment of tocopherols and tocotrienols in rice bran oil. A

method was developed for their separation by reverse-phase HPLC. Oryzanol was quantified by

photodiode array (PDA) detection while tocopherols and tocotrienols by fluorescence detection.

Crude rice bran oil samples were prepared for HPLC wslysis by diluting the oil in mobile phase

at 4% concentration. The mobile phase used was acetonitrile/ methanol/ isopropanoV water

(45:45:5:5, by volume). The sample was vigorously mixed with a vortex mixer allowing for the

extraction of the tocol and oryzanol components from the oil into the mobile phase. The

remaining oil droplet at the bottom of the tube contained high concentrations of the more non-

polar triglycerides. This prevented their carry-over to the reverse-phase column.

A typical HPLC chromatogram obtained from a crude rice bran oil extract is shown in Figure

2-3. The sample was separated on a Hewlett Packard 200 x 2.1 mm narrow-bore analytical

column, packed with C 18 5pm Hypersil silica. Rogers et al. determined that the separation of

both the oryzanol and tocol components was most effective with a mobile phase consisting of

acetonitrile/ methanol/ isopropanou water (494533, by volume), which was programmed as a

linear gradient to acetonitrilel methanoV isopropanol (50:45:5, by volume) over six to ten

minutes. The final mobile phase conditions were maintained for 15 minutes before returning to

the original conditions. Absorption spectra for the four oryzanol peaks were acquired by PDA as

shown in Figure 2-4. The spectra of the four peaks were essentially identical and their maxima

corresponded to reported literature values (Tsuchiya and Kaneko, 1954).

Rogers et of- used semi-preparative HPLC to hctionate rice bran oil oryzanol components for

the purpose of individual component analysis by chemical ionization-mass spectrometry. TLC

was employed to further e ~ c h the crude oryzaaol fiaction on 500 p plates developed with

toluendethyl acetate 90: 10 v/v. The separation was visualized under ultraviolet light as blue

fluoresceat spots.

Simul~eous w o n of tocotrienols, tocopherols. and y-oryzanol components from a cnde rice bran oil sample. A) Ultmiolet detection of yayzanol components. Peaks: 1 = cycloartenyl ferulate; 2 = 24methylene cycloartarryl feculate; 3 = campesteryl fenrlate; 4 = fllsitosteryi fenrlate and cycioartanyl fedate. Fluomcent (B) vs. ultraviolet (C) detection of tocotrienols and tocopherols. Peaks: 5 = &ocophml; 6 = i3 and y-tmtrienols; 7 = a-tmtrienol; 8 = &tocopherol; 9 = f3 and y-tocopherols; and 10 = a-tocopherol

Figure 2-3 : HPU: Chromatogram of Crude Rice Bran Oil

(Rogers et al., 1993)

Figure 2-4 : Overlay of Ultraviolet Spec- of Four Oryzaaol Components

(Rogers et al., 1 993)

Diack and Saska (1994) separated vitamin E and y-oryzanol compounds from saponified rice

bran extracts, with the perspective of future large-scale purification of the various compounds.

After saponification hexane and water were added. The reaction mixture was allowed to settle

and was then filtered through a cellulose filter disk. The hexane layer was collected and washed

three times with deionized water. The resulting hexane solution was filtered through a layer of

anhydrous Na2S04 and the volume was reduced in a glass rotary vacuum evaporator. The

product was used for HPLC analysis. Diack and Saska found that normal-phase silica did not

give full resolution of the five oryzanol compounds. Only the Nova-Pak column resolved the

oryzanols as distinct fractions in rice bran oil. Figure 2-5 shows a typical chromatogram using

the Nova-Pak silica column with isooctandethyl acetate 9752.5 v/v as the mobile phase. The

selectivity between the two oryzanol hctions, peak 10 and 11, remained nearly constant

between 5 O C and 75OC. Elution order in reverse phase gives cycloartenyl ferulate, 24methylene

cycloartanyl ferulate, and campestryl ferulate (Figure 2-2, IIIa,b,c) followed by cycloartanyl

ferulate and P-sitosteryl ferulate (Figure 2-2, IIId,e) in one peak. Cycloartenyl ferulate and 24-

methylene cycloartanyl ferulate are likely the most polar due to the terminal double bond

susceptible to polarization.

---

0 5 10 15 20 25 30 35 40

Time 1 min)

Components: 1 = aiglyarides + toluene (imernal marker). 2 = oxidation pducts, 3 = a-tocopherol, I = a- tocouienol. 5 = not identified, 6 = y-tocopherol, 7 = y~otrienol, 8 = GtocopheroL 9 = Gtmtrienol. 10. I 1 = y-

Figure 2-5 : EPLC Chromatogram o f Saponified Rice Bran Oil

(Diack and Saska, 1994)

Evidently, HPLC can be an effective means of separating and purifying orymol on a small scale

&om either rice bran oil or saponified extracts. Rogers et d. (1993) proved the superiority of

reversephase HPLC over normal phase for the separation of both oqmmol and tocol

components. As an analytical technique, HPLC provides excellent resolution and quantitation.

However, it not routinely available for large-scale production. From the perspective of industrial

application, it is not technically feasible or economically sound to maintain 2 large throughput of

rice bran oil in an HPLC for the purpose of separating these componezts. In many cases,

researchers have turned to classical column adsorption chromatography for the purpose of

isolating oryzanol. Column chromatography requires no applied pressure, is more readily

scalable and can be operated in semi-continuous mode.

2.7.3 Adsorption Column Chromatography of Oryzrnol Concentrate

Adsorption column chromatography has been employed in the separation of oryzanol from rice

bran oil since the discovery of oryzanol. A variety of adsorbents have been used with varied

results. Shimizu et al. (1957) used an alumina column to isolate the phenolic compounds in

deacidified oil. The column was developed with petroleum ether and the adsorbed phenolic

compounds were eluted with glacial acetic acid/ benzene (I :3 v/v). Non-phenolic fractions were

extracted from the phenolic portion of the eluate, which was then passed through another

alumina column. This column was developed with benzendether (10: 1 and 1: 1 vlv), ether and

finally ethedmethanol (10:l v/v). The oryzanol fraction was eluted fiom the column with

benzendglacial acetic acid (1 0: 1 vlv). Fractional recrystallization &om ether/ethanol ( 1 : 1 v/v)

afforded 2.42 g of oryzanol kom the original 2 kg of crude oil. Shimizu ef a!. did not indicate

the original oryzanol content of the oil. Crude rice bran oil contains 0.96 - 2.9% oryzanol

(Marshall, 1994), therefore the maximum oryzanol recovery could only be 1 I% by this method.

Seetharamaiah and Prabhakar (1986) also used alumina for the isolation and purification of the

oryzanol concentrate extracted fiom the saponified oil. The concentrate was loaded onto a

neutral alumina column and eluted with hexane, petroleum ethedmethanol 9: 1 vlv and finally

with diethyl ethedglacial acetic acid 20: 1 v/v. TLC was carried out on each fraction to !ccate

oryzanol, which was detected under W light as blue fluorescent spots. The platcs were

developed in hexane/diethyl ether 9: 1 v/v followed by benzenddiethyl ether 4: 1 vlv in the same

direction. The column chromatography had enriched the oryzanol tiaction to 51%. The

oryzanol was crystallized fiom this concentrate using methanol and then recrystallized with

methanol-acetone 2: 1 v/v. The final recovery of oryzanol was not reported.

Tanaka et al. (1971) isolated an unknown ferulate in rice bran oil using silica gel as the adsorbent

for column chromatography. The ferulate, identified as methyl ferulate, was separated from the

soapstock of crude rice bran oil with a 5cm x 67.5cm column of 100 mesh silica gel. The

separation was performed in batches of 200g of the oil with the solvent system diethyl etherh-

hexane (5:95 and 30:70 vlv). The ferulate fiaction, which fluoresced blue when irradiated with

UV light on the column, eluted with diethyl ether-hexane 30:70 v/v. From this fraction, an

unknown ferulate was separated with a 5crn x 66cm column of 100 mesh silica gel containing

10% water. The column was eluted with diethyl etherhexme in the following order: IO:90,

20:80 and 30:70 vlv. The methyl ferulate was identified by its blue fluorescence under UV light

in the 30:70 vlv fiaction. This method was used to determine the methyl ferulate content of rice

bran oil and can therefore be considered effective in recovering the targeted product.

Column chromatography has been proven to be effective in the separation of oryamol f?om a

concentrate extracted fiom crude rice bran oil. Although the work of Shimizu et al. (1957)

successfhlly produced an isolated oryzanol hction using an alumina column, its oryzanol

recovery fiom oil is too low for industrial application. The losses can most likely be attributed to

the numerous extractions and the two column passes required for purification. Seetharamaiah

and Prabhakar (1986) also purified oryulnol on an alumina column, however lack of mass

balance data inhibits evaluation of the method for the purpose of industrial appiication. Tanaka

et al. (1 971 ) successhlly separated and recovered methyl ferulate from rice bran oil. Although

this method does not focus on oryzanol, both oryzanol and methyl ferulate have the ferulate

moiety common to their structures. It is the hydroxyl group of the ferulic acid that gives

oryzanol its phenolic characteristics. Shimizu et a/. (1957) used this phenolic nature to separate

oryzanol from rice bran oil by eluting it from an alumina column. It is therefore reasonable to

assume that the chromatographic method developed by Tanaka et al. (1971) may be adapted to

effect a similar separation of oryzanol fiom crude rice bran oil.

It is evident from the literature that silica gel column chromatography is a potential method of

isolation of the oryzanol compound. The effectiveness of saponification of rice bran oil for the

extraction of an oryzanol concentrate has not been definitively established in literature with

respect to potential oryzanol recovery. This thesis investigatesthe preparation of an oryzanol

concentrate f?om crude rice bran oil by three different methods. The saponification of the oil for

the extraction of the unsaponifiable matter, solvent extraction of a concentrated oryzanol fiaction

from crude oil and stepwise elution of an oryzanol concentrate were examined. Methanol,

ethanol and dirnethylformamide wexe each assessed for their ability to extract an oryzanol

fiation ftom the crude oil. The isolation of oryzanol from the concentrate was attempted by

silica gel column chromatography. Optimization of the chromatographic separation was

investigated with respect to mobile phase, flowrate and column dimensions.

3 EXPERIMENTAL, METHODS

3.1 Materials

Agritech Inc., Louisiana, USA, supplied the crude rice bran oil. The oryzenol standard, which

had a melting point of 135 - 137"C, was donated by the University of Louisiana, Department of

Food Science. The HPLC chromatogram and ultraviolet spectra of the oryzanol standard were in

agreement with literature. The silica gel used for column chromatography was purchased fiom

Sigma Aldrich (Oakville, Ontario) and had the following properties: 70-230 mesh, 60 A pore

size, 500m2 surface area. Silica Gel G, 250~x11, 20cm x 20cm aluminum backed, thin-layer

chromatography plates with fluorescence indicator were obtained from Whatman (Oakville,

Ontario). Solvents used for experimental work were analytical grade, distilled in glass while

those used for analytical work were HPLC grade. A11 solvents were purchased fiom Cdedon

(Georgetown, Ontario).

3.2 Extraction Methods

3.2.1 Extraction of Unsapooifir ble Matter

The unsaponifiable matter of the crude rice bran oil was extracted according to the American Oil

Chemists' Society (A.O.C.S.) Official Method Ca 6a-40. (A.O.C. S., 1980) Crude rice bran oil

was saponified while blanketed with nitrogen to avoid oxidation of oryzanol. The saponified oil

was extracted with hexane, which was later washed with alcohol. The hexane was evaporated

off using a Buchi RllO rotary vacuum evaporator. The residue was dried in a 80°C vacuum

oven for one hour, then cooled in a desiccator.

The effect of the soapstock pH on oryzanol recovery was also investigated. The saponified oil

was acidified to pH 7, 8, and 9 with 10% HCI prior to hexane extraction. Extraction of

unsaponifiable matter was continued according to the A.O.C. S. method described above.

33.2 Solvent Extraction of Oryanol from Crude Rice Bran Oil

3.2.2. I Alcohol Eklhction

The extractability of oryzanol from rice bran oil using methanol with 10% water and pure

ethanol was evaluated. For each solvent, the oil was extracted using a solvent-to-oil ratio of five.

In the case of ethanol, a solvent-to-oil ratio of twenty (i-e. tive extractions, each at a solvent-to-

oil ratio of four) was also examined. The following extraction scheme was used for both

solvents:

Crude rice bran oil was extracted with solvent at a solvent-to-oil ratio of one. The mixture was

centrifuged at 2000 rpm using an EC clinical centrifuge for ten minutes to separate the phases.

The alcohol layer was drawn off and the oil re-extracted with an equal volume of alcohol. This

procedure was repeated for a total of five extractions. The alcohol extracts were combined and

the solvent evaporated using a rotary vacuum evaporator. The residue was dried in a vacuum

oven at 80°C overnight and weighed. Oryzanol was separated by column chromatography

(Section 3.3.1.2) using toluenekthyl acetate 90: 10 v/v as the mobile phase. TLC was used to

estimate the oryzanol content of the extracted residue as discussed in Section 3.4.1.2.

3.2. 2.2 Dimetky~ormami& Extradon

Crude rice bran oil was extracted with dimethylfomamide @MF) according to the extraction

scheme shown in Figure 3- 1. The oil was dissolved in hexane and repeatedly extracted with

DMF. The combined DMF layers were washed back with he~ane to remove any non-polar

components that had migrated to the DMF layer during the initial extraction. For each extraction

step, the solution was mixed vigorously for one minute. All three layers, Hexane I , Hexane 2,

and DMF, were transferred to individual flasks and the solvent was evaporated using a rotary

vacuum evaporator. The samples were dried in a vacuum oven overnight at approximately

1 00°C. The residue was weighed and anal ymd by HPLC (Section 3.4.2) for oryzanol content.

10.Og crude rice bran oil dissolved in hexane solventoil ratio = X

Wash with Yx30mL DMF

DMF Layer + Z x 300 rnL hexane

I Final DMF Layer (

Figure 3-1 : Dimethylformamide Extraction of Crude Rice Bran Oil

Investigation of the variables listed in Table 3-1 optimized the DMF extraction of crude rice bran

oil. The effect of the soknt:oil ratio (X) was determined by dissolving the cruds rice bran oil in

hexane at so1vent:oil ratios of 10: 1 and 20: 1 . The oil was then extracted with 3 x 30 mL DMF

and the oryzanol content of the combined DMF layers was analyzed. The optimum number of

hexane washes (2) was determined by dissolving the oil in hexane at a so1vent:oil ratio of 20: 1,

extracting with 3 x 30 mL DMF and then washing with fresh hexane. For determination of the

optimum number of DMF extractions (Y), a so1vent:oil ratio of 20: 1 and one wash with hexane

was used.

Table 3-1 : Variables Optimized in Dimethylformamide Extraction

3* 2* 2.3 Is0latio~ of Ory~anolfnnn Dimethy&ionn& Extract

The DMF extract was tiactionated by column chromatography as described in section X.

Toluene/ethyI acetate 90: 10 v/v was used as the mobile phase. The eluted oryzanol peak was

identified by TLC analysis (Section 3.4.1.1). Wals containing the eluted oryzanol fiaction were

combined in a flask. The mobile phase was removed by vacuum distillation, using the Kuderna-

Danish apparatus. The final residue, concentrated in oryzanol, was weighed and prepared for

HPLC analysis as d e s c r i i in Section 3.4.2.

Parameter lO:l, 20:l

3,4, 5,6,7 1 ,3

Variable X Y Z

Solvent : Oil Ratio Number of DMF extractions Number of hexane washes

3 3 Chromatographic Methods

3.3.1 Column Chromatography

3-3- I. 1 P d n g the Cdumn

The chromatographic column was packed with silica gel, which had been dried in a forced air oven

at 20O0C overnight. The silica gel was slurried with 1Wh ethanol and allowed to equilibrate for

two hours prior to use. The silica gel-ethanol slurry was heated in a water bath at 75°C for 30

minutes before packing of the column. This forced out any air bubbles in the s h y thus increasing

uniformity of column packing. Glass wool, soaked in 1 OOO! ahanol, was placed at the bottom of

the column as a support for the silica gel. The slurry was poured down the column and as the silica

began to settle, the stopcock was opened to allow the excess ethanol to drain out. This resulted in

an even and tightly packed column. Once the column was packed to the desired height, a glass fiber

filter was placed on top of the silica gel to minimize disruption during column operation. In

prepamtion for chromatographic separation, the ethanol was displaced by the mobile phase.

3-3.2.2 Column Operalion

In order to ensure minimal disruption and constant flowrate, a constant solvent head was

maintained on top of the coiumn throughout the chromatographic separation. This was done

using a separatory funnel which was filled with the mobile phase and mounted above the

chromatographic column. The tip of the separatory &me1 was placed at the desired solvent level

in the column while the top of the f i i ~ e l was sealed with a stopper and the stopcock opened. In

this manner, the mobile phase could only flow out of from the stern of the funnel if displaced by

air. Therefore, once the solvent level in the column reached the tip of the separatory h e 1 air

could no longer enter at the stem of the funnel and equilibrium was established. During column

operation the solvent level in the column declines, which allowing air to enter at the tip of the

separatory finel. The air then continually displaced the mobile phase in the hnnel until the

solvent level in the column once again rei&ed the tip of the funnel. The column was oper~tal at

room temperature and atmospheric pressure.

The flowrate of mobile phase through the column was set before each separation began. Prior to

applying a sample, the solvent level in the column was drained to the top of the silica gel.

Samples dissolved in mobile phase were delivered to the top of the column by pipette. The

sample was allowed to adsorb onto the silica before fresh mobile phase was added. This ensured

that no back mixing of the sample occurred as the mobile phase was fed from the separatory

funnel. Eluted fractions were collected in glass vials using a LKB Bromma 2070 Ultorac I1

collector. The fiaction collector controlled the collection time per vial, with the vial volume

depending on the flowrate of the mobile phase. Afler each chromatcygaphic separation, the

column was washed with approximately 100 mL of 100% ethanol to remove residual

components adsorbed on the silica.

The selection of mobile phase, flowrate, packing height and width was investigated in an attempt

to optimize the isolation of oryzanol. The flowrate was varied from 2.1 mL/min to 4.6 mL/min.

The dimensions of the three columns used in the separations as well as the mobile phases

examined are listed in Table 3-2.

I Variables I Parameters Column Dimensions (width x packing height)

Mobile Phase

1.5 crnx43 crn 2.0 cm x 69.5 cm 2.5 cm x 52 cm Ethyl Acetate Hexane Toluene I Ethyl Acetate 90: 10 v/v Hexane I Ethyl Acetate 80: 20 vlv Hexane I Ethyl Acetate 70: 30 v/v Hexane 1 Diethvl Ether 70: 30 v/v

- - -

Table 3-2 : Variables Optimized for Column Chromatographic Separation

The elution of oryzanol from the chromatographic column was monitored by periodic

measurement of the ultraviolet absorbance of the eluted fractions. Each fraction was analyzed by

TLC as discussed in section 3.4.1 . I . This allowed qualitative evaluation of the separation on the

column and identification of the oryzanol peak.

3.3.2 Step-wise Elution

Step-wise elution of crude rice bran oil was effected on a 2.5 cm diameter column packed with

silica gel to a height of 52 cm. The silica gel was slurried with 1000/o ethanol for the purpose of

packing. The ethanol was displaced with hexane to begin the separation. Crude rice bran oil was

dissolved in hexane and applied to the top ofthe column. Eluting the column with varying strengths

of mobile phase at each step eactionated the oil. Hexane was used as the first step in the elution to

remove all non-polar components fiom the oil. Succeeding steps increased in elutropic strength

through the addition of ethyl acetate to hexaae. Each step was collected, the solvent evaporated

using a rotary vacuum evaporator, and the residue dried in a vacuum oven at 80°C overnight. TLC

was used to analyze the residue from each step for estimation oforyzanol content.

The separation of an oryzanol hction by stepwise elution was optimized by varying the

percentage increase of ethyl acetate in each step of the elution. The mass of crude oil qplied to the

column, 10 g or 20 g, as well as the volume of mobile phase us@ 500 mL, 750 rnL or 1000rnL,

were investigated to determine their effect on oryzanol recovery. In addition, the use of hexane in

place of ethanol as a packing solvent was examined.

3.4 Analytical Methods

3*4* 1 Thin-Layer Chromatography

3.4.1.1 Qualitative M d A

Thin-layer chromatography (TLC) was used to detect the presence of oryzanol in various

samples. TLC also determined whether other compounds were present alongside oryzanol in the

fractions collected from the column chromatographic separation. TLC plates were activated in

an oven at 100°C for one hour prior to use. Samples were applied to the TLC plates with a

Hamilton 901 lo@ syringe in volumes of 5 - 20 pL, depending on the concentration of the

sample. Samples were spotted manually onto the plate, allowing each spot to dry between

successive applications. A heat gun was used to accelerate the evaporation of solvent between

applications. It was determined that a minimum of lpg of oryzanol was required in order to

visualize oryzanol on the developed plate. The origin line, where samples were applied, was

lightly marked with a pencil 2cm from the bottom of the plate. The solvent front was marked

parallel to the origin, 16cm above. This allowed for 2cm of haadling space above the solvent

line. Samples were placed lcm apart on the plate and 2cm away from the edge to avoid edge

effects.

The glass, Cwin-trough, developing chamber was lined with filter paper and the lid sealed with

vacuum grease. The chamber, with the mobile phase, was allowed to stand for one hour before

the TLC plate was inserted. This ensured that the inside atmosphere of the chamber was fblly

saturated with the solvent vapour and at equilibrium. The mobile phese used was toluene/ethyl

acetate (90: 10 vlv), which was reported in literature by Rogers, et al.. (1 993). It was found to

provide good separation of the oryzanol compound. Once the solvent reached the solvent fiont,

the plate was removed tiom the developing chamber and the mobile phase was allowed to

evaporate in the fumehood. Oryzanol was visualized as blue spots under ultraviolet light.

Phosphomolybdic acid was used as a developing reagent on some plates to detect non-polar

compounds not visible under ultraviolet light.

3.4.2.2 SemkQuantitative Mdhods

TLC was used to estimate the quantity of oryzanol in a given sample. Samples were applied to

the plate, as described in the previous section, in known volumes alongside standard oryzanol

samples of known concentrations. The concentration of oryzanol standard used was 1000 m g L

in toluenekhyl acetate 90: 10 v/v. Once the plate was developed, visual comparison of the

intensity of the sample and standard oryvlnol spots under ultraviolet light allowed for the

estimation of oryvlnol concentration of the sample.

3.4.2 High Peflommce Liquid Chromatography

3.4.2 I @eratr*on

Samples were analyzed and oryzanol content quantified by high performance liquid

chroma~ography (HPLC). The method of analysis was adapted 60m Rogers et al. (1993). The

system consisted of a Waters 600E high performance liquid chromatograph connected to a U6K

injector with a variable sample loop. Oryzanol components were detected at 325 nrn with a Waters

Model 991 programmable photodiode array deteztor. A Waters Model 600E controller capable of a

four solvent gradient controlled the HPLC system. The separation was effected on a Hewlett

Packard 200 x 2.1 mm narrow-bore analytical column (Toronto, Ontario) packed with 5 pm ODs

(C 1 8) Hypersil silica. The mobile phase consisted of acetonitrile/rnethanoVi~~propanoVwater

(45 : 45 : 5 : 5 v/v/v/v) which was programmed as a linear gradient to acetonitriidmet hanoVisopropano1

(50:45:5 v/v/v) from six to ten minutes. The final mobile phase conditions were maintained for 15

minutes before returning to the original conditions (Rogers et al, 1993). The flowrate was 0.7

mL/rnin and the separation was carried out at room temperature. The solvents were sparged with

helium (BOC Gaz, Toronto, Ontario) at 1 00 W m i n for 1 5 minutes before priming the pumps.

During analysis, the sparge rate was maintained at 30 a m i n as recommended by Waters. Data

acquisition and analysis display was accomplished with a NEC Powermate SX Plus computer

co~ected to a Waters model 990 plotter.

Samples were injected in 50 pL volumes for HPLC analysis with a Hamilton HPLC syringe. After

every 25 injections, the column was flushed with acetonitrile at 1.0 mUmin while 10 - IS, 50 pL

volumes of dimethyl sulfoxide were injected to remove any fats adsorbed onto the stationary phase.

The column was re-equilibrated with the mobile phase at 0.7 mL/min for one hour prior to analysis.

After 50 injections, the injection port was flushed with approximately 500 jL of dimethyl sulfoxide

followed by 500 pL of mobile phase.

3.4.2.2 OrytrurdStmdards

Chromic acid-washed glassware was used for the preparation of all $mdards. The stock standard

solution of oryzanol was prepared by weighmg orytanol on a Mettler AE 260 microbalance,

transferring to a 10 mL volumetric flask and diluting with rnethylene chloride. Calibration

standards were prepared by sequential dilution from the stock solution as illustrated in Figure 3-2.

Standards were diluted with HPLC mobile phase acetonitrile/rnethanoVi~~propanoVwater

(4545: 5 : 5 v/v/v/v). ThreemL aliquots of the oryzanol standards were transferred to glass vials

sealed with teflon lined caps and stored at 4OC.

10 000 ppm m00 PPm

1.25 mt 2500 ppm

I m L - 750ppm 1 mL - 500pprn

In& - 250 ppm

5 rnL solution methytene chloride

Figure 3-2 : Preparation of Oryzanol Standards for EPLC

10 mL solution 10% methyiene chloride in HPLC mobile phase

The calibration curve for oryzanol at 325 nm is given in the Appendix (Section 8.5). To

determine the accuracy of oryzanol quantitation by the HPLC system, a standard addition curve

was prepared as outlined in the Appendix (Section 8.6).

3.4.2.3 Sample fiepariation

Oryzanol is only slightly soluble in the HPLC mobile phase. Therefore, samples were dissolved

in rnethylene chloride to ensure that all the oryzanol stayed in solution. Samples were diluted

with mobile phase until the oryzanol concentration fell within the bounds of the calibmtion

curve. The final sample composition was 10% - 30% methylene chloride in the HPLC mobile

phase.

3.4.3 Ultraviolet Spectroscopy

U V spectroscopy was used to identify the presence of oryzanol and to estimate its concentration in

solution. Absorbance was measured on a Beckman DU-7 spectrophotometer at the wavelength of

maximum absorbance as shown in Table 3 3 . The correlation of ultraviolet absorbance to oryzanol

concentration is given in the Appendix (Section 8.3).

Solvent Toluene I Ethyl Acetate 90: 10 vlv Hexane I Ethyl Acetate 80:20 v/v Hexane I Ethyl Acetate 70: 30 v/v Hexane / Diethyl Ether 70:30 v/v Hexane I Diethyl Ether 50: 50 v/v

Table 3-3 : Maximum Ultraviolet Absorbance Wavelengths for Oryzanol

4 RESULTS AND DISCUSSION

4.1 Oryzanol Content of Crude Rice Bran Oil

The oryzanol content of the crude rice bran oil used in this work was 0.871 * 0.006 %. This

value was determined by the method of standard addition. Aliquots of oryzanol standard were

added to crude rice bran oil and the samples were analyzed by HPLC. Details of this method are

provided in the Appendix (Section 8.6).

4.2 Extraction of Uosaponifiable Matter

Oryzanol has been reported to be present in the unsaponifiable fiaction of rice bran oil (Sayre,

1988). In an effort to concentrate oryzanol from the starting crude rice bran oil, the extraction of

the unsaponifiable matter from rice bran oil was investigated. Once extracted, the oryzanol

could then be isolated fiom the unsaponifiable matter by column chromatography.

In previous work by Karan (1996), the recovery of oryzanol from the unsaponifiable fraction of

crude rice bran oil was shown to be minimal. The unsaponifiable matter was extracted from

saponified crude oil with hexane, according the A.O.C.S. method Ca 6a-40 (A.O.C.S, 1980). As

oryzanol is relatively insoluble in hexane, the extraction solvent was substituted with a more

polar solvent, toluene. Results by TLC analysis indicated a higher oryzanol content in the

unsaponifiable matter with toluene. However, the recovery of oryzanol from the oil was still

very low. TLC was used to analyze each phase of the extraction process to detect the missing

oryzanol. The developed plate identified the presence of oryzanol in only the unsaponifiable

matter. This indicated that it had undergone a change during the saponification reaction, which

made it undetectable by TLC. Therefore, the possibility of saponificatioo or ionization of

oryzanol during the saponification reaction was examined.

4.2.1 Saponification of Oryzanol

Literature defines oryzanol as a component of the unsaponifiable matter in rice bran oil.

However, Sakarna and Tsuchiya (1959) have been able to saponify the compound into its two

main components. The saponification reaction, as discussed in Section 2.4.1, cleaves the

triglycerides at the ester linkage. Oryzanol also contains an ester that links ferulic acid to the

plant sterols and triterpene alcohols. Therefore, it is possible that this compound could undergo

saponification. The suggested reaction is shown in Figure 4-1.

Oryzanol

(cycloart eny 1 ferulate)

Ferulic Acid Salt C ycloartenol

Figure 44: Saponification of Oryzanol

4.2.2 Ionization of Oryzanol

The saponification reaction converts fats (triglycerides) into glycerol and potassium fatty acid

salts. Extraction of the sapnified oil with hexane should separate the unsaponifiable matter,

including oryzanol, fkom the saponification products. Oryzanol has been shown to exhibit

phenolic properties (Shimizu and 0 hta, 1957). Therefore, its behaviour during saponification can

be investigated by examining reactions involving phenols. In the presence of sodium or

potassium hydroxide, phenol is converted to the phenoxide ion as seen in Figure 4-2. In order to

revert back to the neutral form of the phenol an acidification step is required (Carey, 1992). The

fatty acid salts produced as a result of saponification are carboxylic acids, which are soluble in

water and converted to the free acid form upon acidification.

Phenol Hydroxide Ion Phenoxide Ion Water

Figure 4-2 : Ionization of Phenol

The saponification reaction is carried out in potassium hydroxide. Therefore, the resulting

saponified oil is strongly basic. At this pH, phenols would exist as the phenoxide ion and

carboxylic acids as their salts. Acidification of the saponified oil would convert the phenoxide

ion back to the neutral phenol. Phenols have a pKa value of approximately 10, whereas the pKa

for carboxylic acids is roughly five. Hence, if the solution were acidified between these two pKa

values, the phenoxide ion should return to the neutral phenol form while the carboxylic acid

would remain as its salt. Upon extraction with hexane, the carboxylic acid salt would remain in

the aqueous phase while the phenol would be extracted into the hexane phase. In this manner,

oryzanol (the phenol) would be separated from the fktty acid salts (the carboxylic acid salt) by

means of extraction. In its neutral form TLC would readily detect the presence of oryzanal.

46

Acidification was carried out through the addition of 10Y0 HCl to the saponified mixture. The

unsaponifiable matter was then extracted with hexane and the oryzanol content of the collected

extracts was determined. Tabie 4-1 shows the effect of acidification on the extraction of

oryzanol from the unsaponifiable matter. Standard deviations were not calculated due t lack of

data. Acidifying the extraction solution to pH 8 resulted in a significant increase in the mass of

the unsaponifiable matter, which has been reported at 4.2% (Sayre, 1988). This was due to the

migration of compounds other than the unsaponifiables to the hexane phase. Acidification

increased the percentage of extracted oryzanol by a factor of ten. However, the yield from the

acidification process, 0.092%, was only one-tenth of the oryzanol content of the crude oil,

0.87 1 %. Qualitative trials at pH 7 and 9 revealed similar results.

Table 44 : Effeet of the pH of Saponified Oil on Oryzanol Recovery

Mass of unsaponifiable matter (g)

% Unsaponifiable extracted from oil % Oryzanol extracted &om oil

Therefore, ionization of the oryzanol compound was not solely responsible for the loss of

oryzanol during saponification. This implied that under the conditions of this experiment,

oryzanol was most likely saponified as shown in Figure 4- 1. As a result, this method of oryzanol

concentration was abandoned in favour of a more effective and efficient methodology. In

addition, the saponification of the crude oil renders it useless as an edible oil product.

pH 14

0.17 3.4%

0.008%

PB 8 0.72

15.0%

0.092%

4.3 Column Chromatography of Crude Rice Bran Oil

Tne sparation of oryzanol from crude rice bran oil was attempted by means of silica gel column

chromatography. In this thesis silica gel was used as the stationary phase as it is an all-purpose

adsorbent, widely available for industrial application at a reasonable cost. Tanaka el af. ( 1 970)

successfully recovered methyl ferulate, similar in structure to oryzanol, from crude rice bran oil

using silica gel column chromatography. The effect of temperature on separation was not

investigated in this work. Diack and Saska (1994) have shown that the selectivity between the

oryzanol fractions eluted from HPLC remained nearly constant between 5°C and 7S°C.

Furthermore, temperature control of the column would represent additional cost for the

prospective industrial process. The effects of column height, width, and flowrate on the

separation of oryzanol were examined. A detailed log of each column separation and its

parameters is provided in the Appendix (Section 8.4).

4 . Selection of Mobile Phase

The mobile phases considered were initially adapted &om literature separations. Toluendethyl

acetate 90: 10 vlv had successfblly separated concentrated oryzanol fractions on silica gel TLC

plates (Rogers et al., 199 1). Tanaka ef af. (1 970) used hexanddiethy1 ether 70:30 vlv to isolate

the methyl ferulate fraction fiom crude oil on a silica gel column. Hexane, a commonly used

solvent for fats and oils, was also investigated as a potential mobile phase in combination with

ethyl acetate. Table 4-2 lists the solvent strengths (&")of the mobile phases considered. These

values were calculated using the method developed by Snyder (1968) as outlined in the

Appendix (Section 8.2).

I Mobile Phase EO (on silica gel) Hexane Toluene Diethyl Ether Ethyl ~ c e t a t e 0.45 Hexane / Diethyl Ether 70: 30 v/v 0.176 Toluene I Ethyl Acetate 90: 10 vlv 0.3 18 Hexane / Ethyl Acetate 80:20 vlv 0.359 Hexane / Ethyl Acetate 70:30 v/v 0.38 1

Table 4-2: Solvent Strengths o f Mobik Phases

The separation of oryutnol From crude rice bran oil was effected on a 1 Scm x 43cm silica gel

column. One gram of crude oil, dissolved in the mobile phase, was applied to the column and

eluted with approximately 200 mL of the mobile phase. The flowrate was set at 2.50 d m i n and

5 mL fractions were collected for each sample vial. W spectroscopy was used to determine the

intensity of oryzanol concentration in the collected vials while TLC allowed qualitative analysis

of the separation.

In TLC, the ratio of the distance traveled by the solute to that traveled by the mobile phase is

designated as the & value. This value is constant for a given compound in a chromatographic

system. The & value of oryzanol was 0.42 in this system. For each chromatographic separation

TLC analysis resolved two key components in addition to oryzanol. For the purpose of

discussion, these will be referred to as the non-polar compounds (Rf = 0.68) and the polar

compounds (& = 0.24). A schematic of typical TLC plate prepared with samples £?om a

chromatographic run is shown in Figure 4-3.

Figure 4-3 : Schematic of TLC Plate

To ensure that the mobile phase systems were of appropriate solvent strength for the elution of

oryzanol, the potency of the strongest elution solvent, ethyl acetate, and the weakest elution

solvent, hexane, were evaluated. Chromatographic separation of crude rice bran oil with hexane

eluted only the non-polar compounds in a broad peak with no evidence of oryzanol. Separation

with ethyl acetate resulted in the co-elution of the non-polar compounds oryzanol and polar

compounds in a sharp elution peak. Accordingly, an intermediate strength mobile phase should

sufficiently elute oryzanol from the chromatographic column.

The eEkt of the selected mobile phases on the elution of oryzanol from rice bran oil is shown in

Figure 4-4 and summarized in Table 4-3. It is evident from the graph that hexanddiethy1 ether

70:30 v/v was uclsuccess~l in completely eluting oryzanol within the set elution volume. As

well, TLC analysis revealed no separation of the various compounds on the column. This can be

explained by the low solvent strength of this mobile phase. The solvent strength of diethyl ether,

0.29, is lower than the solvent strength of the weakest mobile phase, toluendethyl acetate 90: 10

v/v, 0.3 18. Therefore, increasing the percentage of diethyl ether in combination with hexane

would not be effective as a mobile phase in this separation.

I ( Start of I Total Peak I Separation of 1 Separation of 1 I Mobile Phase Volume Non-Polar I Polar Compounds I / \ZEf' I (mL) 1 Conpoudr from from Oryunol L

Toluene/Eth y 1 Acetate 90: 10 v/v HexanelDiethyl Ether 70:30 v/v

(mL)

60

HexaneEthyl Acetate 80:20 vlv

Table 4-3 : Effect of Mobile Phase

1 70

Hexand'thy 1 Acetate 70:30 v/v

Toluendethy 1 acetate 90: 1 0 v/v and hexanelethyl acetate 70: 30 v/v provided the sharpest

oryzanol peaks with each eluting oryzanol at similar elution volumes. TLC analysis proved that

both mobile phases separated the non-polar compounds fiom oryzanol. Hexandethyl acetate

70:30 v/v co-eluted the polar compounds with oryzanol. However, toluene/ethyl acetate 90: 10

V/V showed only an overlap of the two compounds, which indicated potential for their separation.

Hexandethyl acetate 80:20 v/v minimized the overlap of oryzanol and the polar compounds.

Therefore, although none of the mobile phases were able to isolate oryzanol, toluendethyl

acetate 90: 10 v/v and hexane/ethyl acetate 80:20 v/v demonstrated potential for the separation of

this component.

70

1

85

75

65

Oryunol

Yes

100

. Over lap

No

70

No

Yes Overlap

Yes No

Figure 4-4: Efftxt of Mobile Phase

400 -. - - - - (B) Hexane/Diethyl Ether 70:30

350 -- (C) HexanelEthyl Acetate 80:20

100 --

50 - .

Elution Volume (mL)

4.3.2 Effect of Packing Height and Width

Increasifig the height of the silica pacuing would extend the residence time of the solutes thereby

assisting the separation of components in the column. The dimensions of the two columns used

to test the effect of packing height were 1 .Scm x 43cm and 2.0cm x 69.5cm. Figure 4-5

illustrates the separation of one gram of rice bran oil with hexandethyl acetate 80:20 vlv on each

column. The 2.0cm x 69.5cm column produced a much broader oryzanol peak with an elution

volume of 170 mL. The oryzanol peak fiom the longer column as compared to the 1 Scm x 43cm

column had almost twice the elution volume and half the intensity. Similar results were

observed in the separation of the oil with hexandethyl acetate 70:30 v/v on both columns.

Analysis of the respective TLC plates indicated that separation of oryzanol fiom the polar

compounds did not benefit from the increased column height.

The effect of column width was determined by separating two grams of crude oil on a 2.5cm x

52cm column with hexandethyl acetate 80:20 v/v as the mobile phase. The oryzanol peak co-

eluted with the polar compounds over an elution volume of 70 mL. As expected, the increase in

column width did not augment the resolution of oryzanol from the polar compounds. However,

the separation did not deteriorate on the wider column, which had a larger starting mass of 2

grams of crude rice bran oil. These results suggested the feasibility for scale-up of a

chromatographic separation.

4.3.3 Effect of Flowrate

The increase in silica packing height in Section 4.3.2 may have also lead to the broadening of

bands within the column, thereby overlapping the oryzanol and polar compound peaks.

Increasing the flowrate of the mobile phase can diminish the broadening of bands, producing a

sharper elution peak. The effect of flowrate on this separation was investigated by separating

one gram of crude oil on the 2.0c1-n x 69.5cm column with hexandethyl acetate 8020 v/v.

Increasing the flowrate fiom 2.73 mUmin to 4.62 mUrnin decreased the elution time but did not

significantly change the elution peak as seen in Figure 4-6. TLC analysis revealed no further

separation of oryzanol fiom the polar compounds as a result of the increased flowrate.

Decreasing the flowrate to 2.0 d m i n did not improve the separation of the two peaks.

4.3.4 Outcome of Column Chromatography

Evidently the isolation of oryzanol Eom crude rice bran oil cannot be effected by the proposed

chromatographic system. The mobile phases, toluendethy 1 acetate 90: 1 0 vlv and hexandethyl

acetate 80:20 v/v, were successful in separating the non-polar compounds ftom oryzanol and

more polar components of the oil. Variations in column height, column width, and flowrate did

not enhance the separation of oryzanol fiom the more polar compounds.

Hexane readily separated the non-polar (triglyceride) compounds from the oil while leaving

o r p o l adsorbed to the silica. Accordingly, step-wise elution of an oryzanol &action from the

chromatographic column was investigated. The elution was initiated with hexane and the solvent

strength was increased in steps with the addition of ethyl acetate.

4.4 Stepwise Elution of Rice Bran Oil

Step-wise elution of crude rice bran oil was accomplished on a 2.5cm x 5 2 c ~ silica gel column.

The mobile phase was allc*..red to flow at the maximum flowrate. Fractionation of the oil was

governed only by the elution strength of the solvent and did not depend upon separation along

the length ofthe column. Hexane was used as the first step of the elution to remove all non-polar

compounds, mostly triglycerides, from the oil. Each subsequent step increased in elution

strength through the addition of increments of ethyl acetate, fiom 5 - 20%, to the hexane. Table

4-4 summarizes the results of the step-wise elution of rice bran oil.

The first step of the fractionation eluted approximately 81% of the crude oil. This step produced

clear, light yellow oil containing non-polar triglycerides as verified by TLC. Food applications

of vegetable oils generally require only the triglyceride portion of the crude oil. Therefore, this

fraction may be suitable in this regard.

TLC detected the olyzanol fraction of Run AB in the step eluted by hexandethyl acetate 90: 10

vlv. This step recovered 50% of the oryzanol and concentrated it by a factor of 4.1 fiom the

crude oil. In Run AE the addition of an intermediate solvent strength, hexane/ethyl acetate 955

VIV, succeeded only in distributing oryzanol among two separate steps. Increasing the volume of

solvent used in each step of the elution &om 500 mL to 750 rnL resulted in a 57% recovery of

oryzanol as seen in Run AF. However, the concentration factor for oryzanol did not rise.

Further increase of the solvent volume to 1000 mL in Run AI, using 20g of oil, recovered 62% of

the oryzanol and achieved the highest oryzanol concentration factor of 5.9. In Run AK the silica

gel was slurried with hexane in place of ethanol in an attempt to increase the selectivity of the

separation. Only 30% of oryzanol in the hexane/ethyl acetate 80:20 v/v step was recovered,

concentrating oryzanol by a factor of 3.2. This oryzanol recovery is approximately 20% lower

than the same separation in which the silica was slurried with ethanol.

Table 4-4 : Summary of Stepwhe Elution of Rice Bran Oil

Silica Run Packing

Solvent

AB Ethanol

AF Ethanol

A1 Ethanol

AK Hexane

Mass of Oil Elution Solvent (9)

10 Hexane Hex/Eth Ac 90: 10 Hex/Eth Ac 80: 20

10 Hexane HexIEth Ac 95:s HexIEth Ac 90: 10 HexlEth Ac 90: 10 Hex/Eth Ac 85: 15

10 Hexane H e a t h Ac 90: 10

10 Hexane HexEth Ac 90: 10 H e a t h Ac 80:20

% of Oil Eluted

% Oryzanol in Residue

% Oryuno' Recovered

Oryzanol Concentration

Factor

4.5 Solvent Extrrrction of an Oryzanol Concentrate from Crude Rice Bran Oil

4.5.1 Alcohol Extraction

The extraction of an oryzanol concentrate &om crude oil was pedormed with methanol at a

solvent ratio of 5: 1. Water, at 10% by volume, was added to the methanol to minimize oil

solubility. Oryzanol content was estimated by TLC analysis.

Methanohater 90: 10 v/v extracted only 3.34% of the oryzanol from the crude oil. As a result

of this low recovery the extraction was evaluated with ethanol, a less polar solvent. The results

are summarized in Table 4-5.

I Solvent : Oil Ratio I 5: 1 I 5: 1 I 20: 1 I

MethanoVWater 90: 10 v/v

I %ofOilExtractedinto Solvent 1 3.34*0.01% 1 40.10&0.01% 1 70.24* 0.01% 1

100% Ethanol I I

Table 4-5 : Alcohol Extraction of Crude Rice Bran Oil

% Oryzanol in Residue

% Oryzanol Recovered from Oil

At a so1vent:oil ratio of 20: 1, ethanol was able to extract 75% of the oryzanol tiom the m d e oil.

However, the final mass of the extracted fiaction accounted for 70.24% of the mass of the

starting oil. Therefore, the oryzanol concentration in this fiaction was 0.93%, only slightly

higher than its original concentration of 0.871% in the crude oil. From these results it is evident

that alcohol extraction of crude rice bran oil is not a viable method for the preparation of an

orytanol concentrate.

0.90 0.09%

3.4 * 0.3%

0.36 0.04%

I7 I%

0.93 =t 0.05%

75 k 4%

4.5.2 Dimethylformamide Extraction

Initial trials with N,N-dimethylfomamide @MF) indicated that most of the oryzanol was

extracted from the crude oil into the DMF phase. Therefore, the extraction was optimized as

outlined in the Experimental Methods (Section 3.2.2.2). The oryzanol content of all samples was

determined by HPLC analysis.

Crude rice bran oil was dissolved in hexane and extracted with 3 x 30 mL DMF portions. The

effect of the hexane:oil ratio on the extractability of oryzanol was determined. The ratios 10: I

and 20:l were investigated and the results are shown in Table 4-6. Increasing the so1vent:oil

ratio resulted in a 17% increase in oryzanol extraction. In addition, the concentration of oryzanol

in the DMF residue increased by 1 I%, confirming the advantage of the 20: 1 ratio in this

extraction. The hexane used to dissolve the oil can be recovered and reused for hture

extractions. Trials with both fresh and recycled hexane revealed no difference in oryzanol

extractability.

Table 46 : Effixt of So1vent:Oil Ratio on Extractability of Oryzanol

Solvent:Oil Ratio

M e r the DMF extraction of the oil, the combined DMF layers were washed with fiesh hexane to

remove any non-polar compounds. The effectiveness of the hexane wash was investigated by

extracting the oil, which was dissolved in hexane at a so1vent:oil ratio of 20: 1, with 3 x 30 rnL

Mass of DMF Residue (g)

Mws of Oryzanol in DMF reaidue (mg)

% Oryzanol in DMF Residue

DMF portions. The DMF layers were then washed with one or three hexane volumes of 150 mL

each. The results, which are summarized in Table 4-7, indicated that the first hexane wash

removed most of the non-polar materials alorg with some oryzanol.

In comparison to the DMF residue that had not been washed with hexane the single hexane wash

decreased the mass of the DMF residue by 79%, further concentrating oryzanol by 76%.

Implementing three hexane washes resulted in only a modest increase in oryzanol concentration

in the DMF residue. However, the three washes also extracted additional oryzanol from the DMF

layer into the hexane layer.

TLC analysis of the DMF layer before the hexane wash revealed the presence of non-polar

compounds. After the single hexane wash, these non-polar compounds were no longer visible in

the DMF layer. As expected, the compounds appeared in the hexane wash. There was no

discemable difference on the TLC plates between the DMF layers washed once or three times

with hexane. These results substantiated both the necessity and sufficiency of one wash with

hexane for the removal of non-polar components with minimal loss of oryzanol from the DMF

layer.

Table 4-7 : Effect of Besane Wash on Oryzanol Recovery

Number of Hexane Washes

M u s of DMP Residue

(R)

Mass of Oryunol in Wash L.~er (mg)

% Oryzanol in DMF ReJidue

The maximum amount of oryzanol extractable from the oil by DMF was determined by

increasing the number of extractions fiom three to seven. The oil was dissolved in hexane at a

so1vent:oil ratio of 20: 1. The combined DMF layers were washed once with hexane at a

hexane:DMF ratio of 2: 1. As seen in Table 4-8, the amount of 0 r y 7 ~ i ~ l extracted increased

significantly fiom three to four extractions. Above four extractions, the extractability of

oryzanol continued to steadily increase. Comparison of the means of each extraction showed

with 90% confidence that the oryzanol content achieved with five extractions is statistically

larger than that achieved with four extractions. However, increasing the number of extractions to

six or seven, did not produce a statistically significant gain in the amount of oryzanol extracted

from the oil.

The increase from five to six extractions resulted in the loss of approximately 50% more

oryzanol to the hexane wash layer. Furthermore, above five extractions, the mass of the DMF

residue increased as more components fiom the crude oil were extracted into the DMF layer.

Correspondingly, the percentage of oryzanol in the DMF layer began to decrease. Therefore, an

extraction scheme with five DMF extractions of the crude oil was determined to be optimal with

respect to oryzanol recovery and its concentration in the DMF residue. Under these conditions,

85.8% of the oqmmol in the crude oil was extracted. The percentage of oryzanol in this extract

reached its maximum of 8.4% * 0.6%, which corresponded to a concentration factor of 9.7 0.6

fiom the oryzanol concentration present in the original crude oil.

Table 4-8: Summary of Dimethylformamide Extractions of Crude Rice Bran Oil

DMF Extractions

3 x 3 0 m L Std. Dev. 4 x 3 0 m L Std. Dev. Sx3Om.L Std. Dev. 6 x 3 O m L Std. Dev. 7 x 3 0 m L Std. Dev.

Scale-up: 6 x 120mL, Std. Dev.

Oryzanol in DMF Oryunol in HEX2 Total Oryzanol Mass of DMF Oryzanol in Layer Layer Extracted Extract DMF Extract

(w) ("/.I (mg) ('w (mg) ("/.I (g) ("/.I 40.4 41.3 7.8 8.9 48.2 50 0.7 7 5.3 13.2 0.5 0.5 4.8 13 0.2 2

63.7 73.1 7.9 6.9 71.6 80.0 0.83 8.1 0.8 1 .O 1.8 1.6 1.3 1.1 0.05 0.5

70.2 80.7 11.5 10.8 81.8 91.5 1.13 6.6 3.9 4.5 0.3 1.6 3.9 5 .1 0.09 0.8

Oryzanol 1 Concentration 1 Factor

4.5.2. I W e - u p of Dhethyrfmamide Edr(IC11'0n

The DMF extradon of crude rice bran oil was scaled up by a factor of four anu compared to the

original extraction. The DMF 6 x 30 mL extraction using 10 grams of rice bran oil was scaled

up linearly to a 6 x 120 rnL extraction. The results are summarized in Table 4 -3. The oryzaaol

content in the DMF layer of the 6 x 30 mL and 6 x 120 rnL extractions are not statistically

different at the 90a/o confidence level. This suggested the potential for scale up of this extraction

scheme. However, qualitative analysis of HPLC chromatograms revealed that the non-polar

content of the 6 x 1 20 rnL DMF layer was much higher than that of the 6 x 30 rnL extraction.

Figure 4-7 and 4-8 represent the HPLC chromatogram of the DMF layer from the 6 x 30 mL and

6 x 120 mL extractions respectively. In each chromatogram, oryzanol is eluted from 11 - 21

minutes. The non-polar compounds eluted immediately after oryzanol from 22 - 35 minutes.

The 6 x 120 mL extraction shows four peaks in the non-polar section at 1.5 times the intensity of

the oryzanol peak, whereas the 6 x 30 mL extraction has only three peaks, each at lower

intensities. Therefore, scale-up of the DMF extraction would sustain oryzanol recovery levels

but would require brther washing of the DMF layer with kesh hexane to sufficiently remove the

non-polar compounds.

4.6 Comparison of Dimethylformamide Extraction and Stepwise Elution

It is evident fiom Table 4-8, that the DMF extraction process is super& to the step-wise elution

of oil for the preparation of an oryzanol concentrate. The DMF extraction recovered 23% more

oryzanol fiom the crude oil than the step-wise elution. As well, the DMF extraction produced a

more concentrated residue at 8.4 1%. This extraction scheme required only one-third the amount

of solvent used in the step-wise elution and is readily scaiable. The mass of oil extracted into the

otyzanol concentrate during DMF extraction is minimal, leaving 91.4% of the crude oil for

Mher processing of a refined product.

I I Stepwise Elution I DMF Extmction I

I Oryzanol Concentration Factor I 5.9 * 0. I I 9.7 k 0.6 I

Oryzanol Concentration in Extract

I % of Oil Remaining 1 82.8 * 0.9 % ! 9 1 & 9 % I

5.1 i O . 1 % 8.4 * 0.6 %

I Volume of Solvent Required

Table 4-9 : Comprrisoa of Dimethylformamide Extract and Stepwise Elution

2000 rnL 650 mL

Figure 4-7 : HPLC Chromatogram of DMF Extract 6 r 30 mL

Figure 4-8 : HPLC Chromatognm o f DMF Extract 6 x 120 mL:

4.7 Isolation of Oyunol from Dimethylformamide Extract by Column Chromatography

The isolation of oryzanol fiom DMF extracts was attempted on a 1.5crn x 42cm silica gel

column. The chromatographic column was eluted with the mobile phase and then washed with

100 mL of 100% ethanol. The oryzanol peak, the repining eluted vials (termed 'remains' for

the purpose of discussion), and the ethanol wash, were collected and analyzed for oryzanol

content by WLC. In preparation for HPLC analysis, the solvent was removed from the samples

using a rotary vacuum solvent evaporator, and then dried in a vacuum oven.

4.7.1 Loss of Oryzanol during Evaporation

Preliminary experimental trials shown in Table 4- 10 resulted in significant oryzanol losses of up

to 80%, which followed no particular trend. The final ethanol wash ensured that no oryzanol

remained on the column. Analysis of all eluted fi-actions allowed for a complete mass balance on

oryzanol. The compound could not have been lost on the column, as the separation was

physical, with no chemical reactions occurring. Thus, oryzanol must have been lost during

evaporation of the solvent &om the samples. Oryzanol, being a stable solid with a melting point

of 13 S°C - 13 7°C and a molecular weight of approximately 500 g/mol, would not be expected to

volatilize under these experimental conditions. The factors leading to the loss of oryzanol were

therefore investigated.

The greatest loss oforyzanol occurred in Run AR with the use of hexanelethyl acetate 80:20 vlv

as the mobile phase. This loss was minimized by 20% in Run AQ with the increase of the

starting mass applied to the column. As seen in Run AS, the mobile phase toluendethyl acetate P

90: 10 v/v combined with a lower vacuum oven temperature further reduced the loss of oryzanol

by 30%. Also noted was the increase in loss when the olyzanol standard was used as the starting

material on the column.

To determine the effect of solvent evaporation on the recovery of oryzanol, samples from Run

AW through Run BA were prepared without the use of the vacuum oven. The results are

summarized in Table 4- 1 1. The solvent was evaporated at 40 *C - 50°C under full water

vacuum. The samples were analyzed by HPLC before solvent evaporation to confirm the

presence of oryzanol. Samples were then spiked with an oryzanol standard in order to follow the

loss of oryzanol during solvent evaporation.

Dilute solutions of oryzanol in the solvent resulted in significantly higher losses. In the step-

wise elution of Run AZ oryzanol was eluted with 1000 mL of hexanekthyl acetate 90: 10 vlv,

which produced a 250 ppm solution. Almost all of the oryzanol was lost during evaporation of

solvent from this dilute solution. This effect is further substantiated by the results of Run AQ

and Run AR discussed above.

Table 4-10 : Oryzrnol Recovery from Preliminary Runs AQ - AV

(Rotavapor and Vacuum Oven used for Solvent Removal)

Mobile Phase Hex/Eth. Ac 80:20

I Oryzand Peak: Elution Volume I % Oryzanol Recovered 4 1 & 4 %

I Vacuum Oven Temperature eC) I

Orvzanol Std 1 Std + DMF 5x30 I Orvzanol Std I AR

DMFS x30 0.178 * 0.001

13.38 Hex(Eth.Ac

80: 20

AS DMF 5 x 3 0 0.178 * .001

13.38 ToVEt h. Ac

90: 10

Table 4-12 : Oryzanol Recovery from Runs BB - Run BG

(Kuderna-Danish distillation apparatus used for solvent recovery)

Starting Material Mass (8) Oryzanol (mg)

DMF 6 x 30 1-08 * 0.01 58.03 0.04

DMF5x30 0.99 * 0.01 55.63 * 0.4

Mass of Oryuaol (mg): 1 I 1 I

DMF5x30 0.99 * 0.01

49.26 * 0.03 Mobile Phase

Orymnd Peak Elution Volume (mL) After Solvent Evaporation % Oryunol Recovered Mass of Peak % Oryzanol in Peak

n c DMF 5 x 3 0 1.08 * 0.01

58.03 * 0.04 ToVEth. Ac 90: 10

DMF5x30 1.08 0.01

58.03 k 0.04 ToVEth. Ac

90: 10

Crude Oil 1.08 * 0.01

58.03 * 0.04 ToVEth. Ac

90: 10

Remaining Hds After Solvent Evaporation Mass of Peak

Etharnol Wmh After Solvent Evaporation

Ory~artol Mass Balance Total in System: Total Recovered: 5% Recovered

ToVEth. Ac 90: 10

0.27 k 0.01

0.05 =t 0.05

* all masses are reported in mg unless otherwise indicated

55.63 * 0.04 46k2

83*3%

ToVEth. Ac 90: 10

-

-

ToVEt h. Ac 90: 10

49.26 * 0.03 34.4 0.4 69.9 * 0.7 %

0.59 * 0.01

0.282 0.001

58.03 * 0.04 47 k 2

8 1 * 4 %

1.91 * 0.02

-

0.55 * 0.01

-

0.29 * 0.01

0.26 * 0.01

Hexane, which is more volatile than toluene, promoted the loss of oryzanol during evaporation.

Run AZ, for which hexandethyl acetate 90: 10 vfv was used as the mobile phase, resulted in the

recovery of only 1.4% of the total oryzanol. The mobile phase hexandethyl acetate 80:20 v/v

employed in Run AY yielded a recovery of 45.5%, substantially lower than similar b i d s run

with toluendethyl acetate 90: 10 v/v. These results are in agreement with observations from Run

AQ and Run AR.

Oryzanol, in its pure form, is more likely to evaporate with the solvent than oryzanol present in

an oil matrix. Trials run on the column with the oryzanol standard, such as Run AT, delivered

losses higher than those run with the DMF extract. Evaluation of Run BA illustrates this point.

The tiactions collected From the separation of the DMF extract in Run BA were divided into two

samples, a control and a spike, to which the oryzanol standard was added. Under the same

solvent evaporation conditions, 30% less oryzanol was recovered from the spiked sample as

compared to the control. This difference can only be attributed to the greater vulnerability of

pure oryzanol to evaporation during solvent recovery.

To overcome the problem of oryzanol loss during evaporation of solvent, the sample was

distilled under minimal vacuum using a Kuderna-Danish column and flask. This apparatus

minimized the possibility of co-distillation by refluxing the sample in the packed column. With

this setup, oryzanol must repeatedly volatilize before leaving with the solvent as distillate. Table

4-12 outlines the results from samples of Run BB to Run BG prepared by this distillation

method. Oryzanol standards were not added to any of the trials.

Mass balances performed on the fractions indicated that loss of the starting material was

minimal. Run BG, which was carried out on a fieshly packed column, reached the maximum

oryzanoi recovery of 99 4 04. Evidently, the Kuderna-Danish column improved the recovery

of oryzanol. It should be noted that the elution volume was very small in these runs thereby

increasing the oryzanol concentration in the sample. As well, the freshly packed silica seemed to

improve recovery.

Evaluation of all observations indicated that oryzanol most likely co-distilled with the solvent

under the appropriate conditions. The factors which promoted co-distillation include: low

starting mass for separation on the column, the use of hexane in the mobile phase, the purity of

oryzanol in the sample, and large elution volumes that resulted in dilute oryzanol concentrations

in the samples.

4.7.2 Recovery of Oryzanol from the Chromatographic Separation

The isolation of oryzanol by silica gel column chromatography was evaluated using results

shown in Tables 4-1 1 and 4-12, dong with TLC and HPLC analysis. In each of the runs using

toluew/ethyl acetate 90: 10 vlv, TLC plates confirmed that the oryzanol fiaction was separated

fiom the non-palar compounds on the column. However, there was no separation of the polar

compounds Rorn the oryzanol peak.

Examination of the HPLC chromatograms of DMF 5 x 30mL and Run AW shown in Figures 4-9

through 4-12 provided insight into the distribution of components initially present in the DMF

extract. The extract was fractionated using toluenekhyl acetate 90:10 vlv on the

chromatographic column. The reverse-phase HPLC column eluted the polar compounds fiom

1 - 8 minutes. the oryzanol peak fiom I2 - 21 minutes and the non-polar components fiom 24 -

3 5 minutes. The chromatograms are presented at four different W wavelengths to reveal all UV

active components in the separation. Oryzanol was detected at 325 nm, its wavelength of

maximum absorbance. As expected, the polar compounds were mostly eluted in the ethanol

wash. The chromatogram of the oryzanol peak reveals the strong presence of orymol along

with a relatively small concentration of polar and non-polar compounds. The majority of the

non-polar compounds are identified in the Remains fraction. The purity of the oryzanol peak

was determined fiom the mass of oryzanol recovered and the mass of the oryzanol peak residue.

Table 4 4 2 shows that the value for the purity of the oryzanol peak is lower than expected,

ranging from 6.8% - 12.4%. This can be partially attributed to the fact that the mass of the

residue was recorded immediately after distillation of the solvent, without drying to constant

weight in an oven. As the mass of the residue was less than 0.5 grams, the residual solvent in the

flask would contribute a significant error. The residue was not dried in an oven in order to

eliminate further loss of oryzanol.

The larger mass of starting material used in Run BB through Run BG resulted in the interference

of non-polar compounds with the elution of oryzanol fiom the HPLC column. This can be seen

in the HPLC chromatogram of Run BG in Figure 4-1 3. In comparison to Run AW, the oryzanol

peak seen at 220 nm of Run BG shows another compound eluting at 23.5 minutes in the tail of

oryzmol. Evidently, the larger starting mass resulted in the co-elution of some non-polar

material with oryzaaol on the silica gel column. Nevertheless, the separation of oryzanol fiom

the more polar components in the DMF extract was not accomplished by this chromatographic

system.

Figure 4-9 : EPU: Chromatogram of DMF 5 s 30

Figure 4-10 : EPLC Chromatognm of Run A W - Oryzanol Peak

Figure 4-11 : BPLC Chromatogram of Run AW - Remaining Vials

Figure 4 4 2 : HPLC Chromatogram of Run AW - Ethanol Wash

Figure 4-13 : BPLC Chromatogram of Run BG - Oryzanoi Peak

5 CONCLUSIONS

I . Oryzanol is not recovered in the unsaponifiable matter of crude rice bran oil. Acidification

of the saponified oil did not significantly increase oryzanol recovery. The compound is most

likely saponified in this reaction. This implies that the literature is incorrect in identifjmg

oryzanol as a fraction of the unsaponifiable lipids. Furthermore, the methods for oryzanoi

separation reported in literature began with the saponification of the oil suggesting that fbll

recovery of oryzanol is not possible.

2. Oryzanol cannot be isolated from crude rice bran oil by silica gel column chromatography.

The mobile phase toluene/ethyl acetate 90: 10 v/v and hexane/ethyl acetate 80:20 vlv

successfully separated non-polar higlycerides from oryzanol on the silica gel column.

However, oryzanol could not be separated from more polar compounds in the oil.

3. Step-wise elution of crude rice bran oil on a silica gel column eluted 62 * 2 % of the

oryzanol with hexandethyl acetate 90: 1 0 vlv. The concentration of oryzanol in the residue

was 5.1 0.1%, which corresponded to an oryzanol concentration factor of 5.9 * 0.1.

4. Extraction of the oil with either methanouwater 90: 10 v/v or ethanol did not produce a

concentrated oryzanol fiaction. The DMF extraction of crude oil proved to be the superior

method of oryzanol concentration from the crude oil.

5. The optimum DMF extraction scheme involved dissolving oil in hexane at a so1vent:oil ratio

of 20: 1, which was extracted five times with DMF at a hexane:DMF ratio of 20:3. The

combined DMF layers were washed once with hexane at a hexane:DMF ratio oF2: 1.

6. The DMF extract recovered 86 * 2% of the oryzanol and produced a concentrate containing

8.4 0.6% oryzanol. This resulted in an oryzanol concentration factor of 9.7 * 0.6 from the

crude oil.

7. Silica gel column chromatography, using toluene/ethyl acetate 90: 10 v/v, did not successfidly

isolate oryzanol from the DMF extract. The maximum purity obtained in the eluted oryzanol

peak was 12.4 * 0.1 %.

8. Oryzanol co-distilled with the solvent during solvent evaporation under the conditions of

these experiments. Co-distillation was promoted by the following conditions: the use of

hexane as the solvent, dilute oryzanol concentrations in the samples, and the use of oryzanol

standard in the samples.

9. The use of the Kudema-Danish column for solvent recovery minimized the co-distillation of

oryumo 1.

1. This research showed that the DMF extraction scheme was favourable for the concentration

of oryzanol fiom crude rice bran oil. Scale-up the extraction by a factor of four sustained

olyzanol recovery levels. Furthermore, it was shown that the use of recycled solvent did not

adversely affect oryzanol recovery. Therefore, pilot plant scale-up of this extraction should

be investigated.

2. The DMF extraction removed less than 10% of the starting mass of crude rice bran oil. The

remaining 90% contained the non-polar triglycerides, which are desirable as edible oil

products. The composition and suitability of this fraction should be studied so that it may be

fblly utilized as a commercial product.

3. The column chromatographic system using silica gel was shown to be ineffective in isolating

oryzanol fiom the more polar components in either the crude oil or the DMF extract.

However, the reverse-p hase HPLC column successfil l y isolated oryzanol fkom these

compounds. Therefore, the use of a reverse-phase adsorbent in the column chromatographic

system should be investigated.

4. Oryzanol was found to co-distill with the solvent under the experimental conditions of this

work. The volatility of oryzanol has not been reported in literature. A more thorough

investigation of this property of oryzanol should be undertaken to fully understand its

behaviour.

5. DMF was shown to be a good solvent for the extraction of an oryzanol concentrate fiom the

crude oil. Unlike the alcohols tested, DMF is an aprotic solvent. Therefore, it is suggested

that the effectiveness of other aprotic solvents, such as dimethyl sulfoxide, be examined for

their potential in carrying out this extraction.

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20. Rukmini, C., (1 988) Chemical, Nutritional and Toxicological Studies of Rice Bran Oil, Fwd Chemistry, 30,257-268.

21. Saunden, R.M., (1985) Rice Bran: Composition and Potential Food Uses Western Regional Research Center, USDA, Albany, California.

22. Sayre, Robert N., (1988) Rice Bran as a Source of Edible Oil and Higher Value Chemicals, Western Regional Research Center, ARS, USDA.

23. Sayre, Robert, N., (1991) Rice Bran: Evolution From an Underutilized By-Product to a Source of Healthfbl Nutrients, Western Regional Research Center, ARS, USDA.

24. Seetharamaiah, G.S. and Chandrasekhara,N., (1988) Hypocholesterolemic Activity of Oryzanol in Rats, Nufr. Rep. Int., 38,927-93 5.

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27. Seetharamaiah,G.S. and Prabhakar,J.V., (1986) Orytanol Content of Indian Rice Bran Oil and Its Extraction fiom Soap Stock, Journal of Food Science rmd Technolog, 23,270-273.

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8 APPENDICES

8.1 Unsaponifiable Matter

The A.O.C. S. Official Method Ca 6a-40 (A.O.C.S., 1980) was used to extract the unsaponifiable

matter From crude rice bran oil with the goal of concentrating oryzanol. Unsaponifiable matter

includes those substances found dissolved in fats and oil, which cannot be saponified by the

caustic alkalies. Heme was substituted for petroleum ether.

Procedure:

Weigh accurately approximately 5 g of well-mixed sample into a flask. Add 30 mL of 95%

alcohol and 5 mL of aqueous KOH (50% by weight). Boil gently but steadily under a reflux

condenser for one hour or until completely saponified. Complete saponification is essential.

Transfer to a separatory fume1 and wash to the 40 mL mark with alcohol. Compiete transfer

with warm and then cold distilled water until the total volume is 80 rnL. Wash out the flask

with a little hexane and add to the funnel. Cool the separatory fbnnel and contents to room

temperature and then add SO mL of hexane.

Insert the stopper and shake vigorously for at least one minute and allow to settle until both

layers are clear. Remove the hexane layer.

The hexane layers are accumulated in a 500 mL separatory hmel.

Repeat the extraction using 50 mL portions of hexane each time, at least 6 more times,

shaking vigorously with each extraction.

Wash the combined hexane extracts in the separatory h e 1 3 times with 25 mL portions of

10% alcohol in distilled water, shaking vigorously and drawing off the alcohol layer after

each wash.

7. Transfer the hexane extract to a tared beaker and evaporate to dryness. Complete the drylng

to constant weight, preferably in a vacuum oven at 75°C - 80°C. Cool in a dessicator and

weigh.

8. Afler weighing, take up ihe residue in 50 mL of warm 95% alcohol containing

phenolphthalein indicator and previously neutralized to a faint pink colour. Titrate with

0.02N NaOH to the same colour.

Calculations:

Weight of fatty acids in the extract, in grams = rnL of 0.02 N NaOH x 0.0056

Unsaponifiable matter, % = (Weight of residue - weight of f a m acids) x 100 Weight of sample

8.2 Solvent Strengths of Binary Mixtures

A Rapid Procedure for Estimating the Solvent Strengths of a Bioary Mixtun (Snyder, 1968)

- -

Solvent s0(A1203) sO(silica) * nb

Toluene 0.94 0.29 0.22 6.8 Ethyl Acetate 1.02 0.58 0.45 10 Ethyl Ether 0.96 0.38 0.29 4.5 Hexane 0.8 0.01 0.0 I 6.7 Ethanol 1.71 0.88 0.68 I0 Met hano l 2.49 0.95 0.73 10 silica) = 0.77 x &"(&O3) (P. 196) For adsorption on silica, solvents with so > 0.38 have a = 10 (p. 195 j

% H20 a From Table 6-1 on p. 136 - 0 1. t2 Values are for narrow bore silica

(20-40 A pore diameter)

800m2 surface area Activated at 195°C in air for 2-4 hours

a = surface absorbent property Assume a = 0.69 as this method is based on water deactivated adsorbents

Formulas used in Calculation:

A E =

A € ' = &+ME

Table 8-1: SoIvent Strengths of Binary Mixtures

p p p p p

Solvent Solvent A B

Toluene Ethyl Acetate

Hexane Ethyl Acetate

Hexane EthylEther

8.3 Oryzanol Calibration Curves for UV Spectroscopy

Figure 8-2: Calibration Curve for Oryzanol in ToluenclEthyl Acetate 90: 10 hmax = 322 nrn

0.04 0.06 0.08

Concentration (mglL)

Figure 8-3 : Cali bration Curve for Oryzanol in HexanelEthyl Acetate 80:20 v/v hmax= 319 nm

0.02 0.04 0.06 0.08

Concentration (rnglL)

8.4 Summary of Column Chromatography Runs

Run

Run E-T

Run A

Run B

Run C

Run D

Run E

Run F

Run G

Run l

Run H

Run J

Run K

lobile Phase

Starting Material

1 Q oil, 1 g oil + 20 mg oryzanol

1 g rice bran oil

1 g rice bran oil

I g rice bran oil

1 ,l lg unsap matter from 5g oil, pH=8.01

1 g rice bran oil

I g rice bran oil + 10 mg oryzanol

1 g rice bran oil

1 g rice bran oil

1 g rice bran oil + 12 rng oryzanol

t g rice bran oil

0.17g unsap matter from 5g oil, N2

Solvent 2

Ethd Acetate

Ethyl Acetate

Diethyl Ether

Ethyl Acetate

Ethyl Acetate

Ethyl Acetate

Ethvl Acetate

Column Packing

Solvent

95% Ethanol

95% Ethanol

95% Ehanol

95Oh Ethanol

95% Ethanol

95% Ethanol

95Oh Ethanol

95% Ethanol

95% Ethanol

95% Ethanol

95Oh Ethanol

95Oh Ethanol

-

Ethyl Acetate

Ethyl Acetate

Ethyl Acetate

Ethyl Acetate

Ethyl Acetate

Solvent 1

Toluene

Toluene

Hexane

Hexane

Toluene

Hexane

Hexane

Hexane

Hexane

Hexane

Hexane

Toluene

Ratio (1 :2)

9O: lO

90:lO

70:30

I

5g rice bran oil

1 a rice bran OH

Run Q 11 g i c e bran oil ( Heated 95% EtOH I Hexane ( Ethyl Acetate ( 8020 I I I I I

95Oh Ethanol

95% Ethanol - - --

1 g rice bran oil

0.72g unsap matter from 5g oil,N2, pHs8

Run P

I RunR l lrrricebrano~ I Heated 95% EtOH 1 Hexane I Ethvl Acetate 1 80320 1

Hexane

Toluene

-

95% Ethanol

95% Ethanol

1 g rice bran oil Heated 95% EtOH -

tlexane

Hexane

1 g rice bran oil

Di8thg Ether

Ethyl Acetate

Ethyl Acetate

Diethvl Ether

60:40

90:10

Heated 95% EtOH

80:20

5050

Hexane Ethyl Acetate 80:20

Run

Run E-T

Run A

Run 6

Run C

Run D

Run E

Strength

0.318

0.31 8

Column Dimensions Elution Volume % Oryzanol Peak Volume % Oryzanol I I I I

Height (cm) 1 Diameter (cm) (mL) I in011 (mL) from Peak 1 I

0.359

0.359

Run l 0.358

0.359

0.359

Run K 0.31 8 2.50

Run N 1 0.193 1 2.50

Run 0 1 0.318 1 2.50

Run P 0.359 2.15

Run Q 0.359 2.25

Run R 0.359 2.25

Run S 0.450 2.50

O W O'Z

szz s- L

G* 1

OPZ S' L

S9Z S. b

082 S* b

sz

8.5 Oryzanol Calibration Curves for HPLC

Linear Regression: y = mx + b

y = Total Peak Area x = Oryzanol Concentration (pprn)

Range m b Rd s n t 50 - 150 ppm 0,001 SO 0.01 158 0.9678 1 0.00527 6 -0.130

95% Confidence Interval for the Prediction of Q, given yo:

Where: xo = Predicted oryzanol concentration of sample yo = Total peak area of sample - y = Average y value of standard samples

C (x, - ;); Sum of residuals h m regression s = Standard error of regression n = Number of observations in the regression b = y-intercept from regression line

tnp,d2 = t-statistic at a = 0.05, p =2

Calibration Curve Overlap: I

Y

0.30

0.25

0.24

0.23

0.20

0.15

X

50-100 ppm 191.64

158.42

15 1.78 145.13

125.20

91.97

250-750 ppm 179.20

1 56.19

151.59 146.99

133.18

110.18

4 9 = O O C

Table 8-2 : Oryzrnol Content in Oil

From Std Addition, at y=O Conc'n of HPLC sample ( m a ) Conc'n of RBO Stock (mg/L) Oryzanol in Stock (mg) % Oryzanol in Oil

From RB0300 Oryzanol in Stock (mg) % Oryzanol in Oil

Difference: ActuaVHPLC

Value

The oryzanol content of the crude rice bran oil is 0.87 1 0.006%

From this analysis, it can be concluded that HPLC analysis detects 95.1 * 0.9 %

of oryzanol present in the sample.

Figure 8-8 : HPLC Standard Addition of Oryzanol to Rice Bran Oil

-600 -500 -400 -300 -200 - 100 0 100 200 300 400 500 600

Concentration of Oryzanol Added (ppm)