CHAPTER I

INTRODUCTION

1.1 Rice

Rice is the principle cereal food in Asia and staple food of nearly half of the

world’s population. Rice (Oryza sativa Linn.) is the most common crop, and very

important in the world. In Asia, rice is so central to the culture that the word is almost

synonymous with food.

Rice (Oryza sativa L.)1 is a short-lived plant related to the grass family, with a

life cycle of 3-7 months. Rice can grow to 1–1.8 m tall, occasionally more depending

on the variety and soil fertility. The grass has long, slender leaves 50–100 cm long

and 2–2.5 cm broad. The small wind-pollinated flowers are produced in a branched

arching to pendulous inflorescence 30–50 cm long. The edible seed is a grain

(caryopsis) of 5–12 mm long and 2–3 mm thick. The span of one cycle varies

depending on its type and the growing environment (Figure 1.1).

2

Leaf

Embryo

Influrescence

AleuronelayerRoot

Seed

Spikelet

Stamen anther

Stigma

Figure 1.1 Schematic of rice.2

Rice is roughly divided into two types, Japonica and Indica. There are three

main races of domesticated rice: Indica, or South Asian rice (Basmati type, center

above); Japonica, or East Asian rice (short-grain, right above); and Javanica, or

Southeast Asian rice. Indica and Javanica varieties are generally adapted to the

tropics, while Japonica rices are adapted to more temperate growing regions. Both

Japonica and Indica types of rice include non-glutinous and glutinous rice. Each type

of rice has its own special characteristics and each has its own place in rice cooking.

Non-glutinous rice is popularly used in general rice cooking. This rice is somewhat

transparent and when cooked it is less sticky than glutinous rice. It is usually cooked

in water and served plain. Glutinous rice tends to be white and opaque and is very

sticky when cooked. It is commonly used to make rice cakes and various kinds of

desserts, and processed to make rice snacks. There are different types of rice presently

grown and used in Thailand; white, black, and red rice.3

3

White rice is the name given to milled rice which has had its husk, bran, and

germ removed. This is done largely to prevent spoilage and to extend the storage life

of the grain. After milling, the rice is polished, resulting in a seed with a bright, white,

shiny appearance.

Red rice, also known as weedy rice, is a specie of rice that produces far fewer

grains per plant than cultivated rice and is therefore considered a pest. Because red

rice and cultivated rice are so closely related, herbicides that would kill red rice would

also kill cultivated rice. A genetically modified form of cultivated rice has been

developed that will resist a herbicide, but this form of rice has not been approved for

human consumption. This genetically modified form of cultivated rice has, however,

appeared on the rice market.

Brown rice is the least processed from of rice. It has only the outer hull removed.

It still retains the white, starchy interior (the endosperm) as well as the nutritious outer

layers (the bran) and the embryo (the germ). The bran layers, which are rich in

minerals and vitamins, especially the B-complex group, give it the characteristic tan

color and nut-like flavor. Because of the higher fiber and oil content of thses bran

layers, brown rice takes longer to cook than white rice.

Black rice is planted mainly in South Asia and other countries, such as Italy,

Greece and the United States. There are many varieties of black rice from China,

Thailand and Indonesia. In Thailand, black rice is the second most common rice and

grown in the Northeastern and Northern parts of country. It could be either medium

or long grain. It contains high amounts of protein, phytofats, cellulose, minerals,

vitamins, and niacin. The functional properties of black rice including carcinogenic,4

mutagenic,5 and antioxidative activities.6 Black rice is rich in anthocyanins such as

4

cyanidin-3-O-β-D-glucoside, delephinidin-3-O-β-D-glucoside and pelagonidin-3-O-

β-D-glucoside7, which are important to suppress oxidation in the body, and these

benefits are not found in white rice. Black rice also contains more vitamin B, niacin,

vitamin E, calcium, magnesium, iron and zinc than white rice.

Therefore the pigmented rice (e.g. red, purple, and black rice) are some varieties

of rice that have a color on the palea, lemma and another inside part such as pericarp

tegmen and aleurone layer. It contains phytochemicals that are reponsible for their

colors. Generally, these colored compounds or pigments fall into a number of large

groups such as chlorophylls, riboflavin, carotenoids, flavonoids and quinones. The

structure of the pigmented rice kernel is illustrated in Figure 1.2. Most of these

pigments are reported to form in plant for vital functions, which could benefit human

health in a meaningful way. Their impotant bioactivities include free-radical

scavenging,8 enhancement of the immune system9 and reduction risk of cancer10 and

heart disease.11 Pigmented rice is, thus, anticipated the greater functional dietary

potential than that of the white rice.12

5

Figure 1.2 The structure of rice kernel.13

Pigmented rice bran is the hard outer layer of grain and consists of combined

aleurone and pericarp. Along with germ, it is an integral part of whole grains, and is

often produced as a by-product of milling in the production of refined grains. When

bran is removed from grains, they lose a portion of their nutritional value. Bran is

present in and may be milled from any cereal grain, including rice, wheat, maize, oats,

and millet. Bran is particularly rich in dietary fiber, and omegas and contains

significant quantitative of starch, protein, vitamins, and dietary minerals.

Rice bran is the layer between the inner white rice grain and the outer hull.

While comprising just 8% of total weight, rice bran (which includes the germ)

accounts for 60% of the nutrients found in each rice kernel. Rice bran is an important

source of rice oil and other phytochemicals which possess antioxidative and disease-

fighting properties. Antioxidative polyphenols in rice bran include ferulic acids, its

esterified derivatives (oryzanols), tocopherols and other phenolic compounds.14 The

6

bran fraction, which includes the germ or embryo in most commercial milling

operations, represents only about 8% of paddy weight but contains about three-fourths

of the total oil. Containing about 15-20% oil (the same general range of soybeans),

rice bran is commercially feasible for oil extraction.

1.2 Antioxidant activity

Antioxidants are classified into two broad divisions, depending on whether they

are soluble in water (hydrophilic) or lipids (hydrophobic). In general, water-soluble

antioxidants react with oxidants in the cell cytoplasm and the blood plasma, while

lipid-soluble antioxidants protect cell membranes from lipid peroxidation.15 These

compounds may be synthesized in the body or obtained from the diet.16 The different

antioxidants are present at a wide range of concentrations in body fluids and tissues,

with some such as glutathione or ubiquinone mostly present within cells, while others

such as uric acid are more evenly distributed throughout the body. Plants, which are

sources of phytochemicals with strong antioxidant activity, have attracted a great deal

of attention in recent years.

Phytochemicals are non-nutritive plant chemicals that have protective or disease

preventive properties. There are more than thousand known phytochemicals. It is

well-known that plant produces these chemicals to protect itself, but recent researches

have demonstrated that many phytochemicals can protect humans against diseases.

Some of the well-known phytochemicals are lycopene in tomatoes, isoflavones in soy

and flavanoids in fruits (Table 1.1). Phytochemicals are not essential nutrients and are

not required by the human body for sustaining life. There are many phytochemicals

7

and each works differently. Some possible actions of phytochemicals are summarized

in Table 1.1.

Table 1.1 Some of the best known phytochemicals and their benefits and sources.17

Phytochemical Potential Health Benefits Food Source Anthocyanidins Reduce risk of heart disease Grapes, raspberries,

blueberries, cherries Carotenoids Encourage normal cell

growth, reduce risk of cancer Yellow-orange vegetables and fruits, red fruits, green leafy vegetables

Catechins Reduce risk of cancer Green tea Chalcones Reduce risk of cancer Licorice

Coumarins Reduce risk of cancer Carrots, caraway, celery, parsley

Curcumins Reduce risk of cancer, Reduce risk of heart disease Antimicrobial

Turmeric, ginger

Diallyl sulfide, disulfides, trisulfides Reduce risk of cancer Onions, garlic, chives, leeks

Dithiolthiones Reduce risk of cancer Cruciferous vegetables Ellagic acid Reduce risk of cancer Grapes, strawberries,

raspberries, nuts

Flavonoids Reduce risk of heart disease, Reduce risk of cancer Most fruits and vegetables

Glucarates Reduce risk of cancer Citrus, grains, tomatoes, bell peppers

Indoles, isothiocyanates

Reduce risk of cancer Broccoli, cabbage, cauliflower, radish

Isoflavones Lower blood cholesterol Reduce risk of cancer Reduce risk of heart disease Reduce risk of osteoporosis

Soy foods (soybeans, tofu, soy milk, soy protein powder)

Alpha-linolenic acid Lower blood cholesterol Reduce hypertension Reduce risk of heart disease Reduce risk of cancer Reduce inflammation Improve immune system

Vegetable oils (canola or soybean), flax seed

Lignans Lower cholesterol Reduce risk of cancer

Soybeans, flax seed, sesame

8

Table 1.1 (Continued)

Phytochemical Potential Health Benefits Food Source Liminiods Reduce risk of cancer Citrus

Phenolic acids Reduce risk of cancer Berries, grapes, nuts, whole grains

Phthalides, polyacetylenes

Reduce risk of cancer Caraway, celery, cumin, dill, fennel, parsley

Phytates Reduce risk of cancer Grains, legumes Phytosterols Reduce risk of cancer Nuts, seeds, legumes Saponins Reduce risk of cancer Beans, herbs, licorice root Terpenoids Reduce risk of cancer Cherries, citrus, herbs (basil,

oregano, thyme, sage)

The following Table 1.2 gives the phytochemicals or phytochemical classes

which provide the predominant source of coloring for the specified fruits or

vegetables.

Table 1.2 Dominant phytochemical pigments.18

Color Pigment Fruit or vegetable Red Anthocyanins

Lycopene Betacyanins

Strawberries, Raspberries, Cherries, Grapes Cranberries, Pomegranates, Apples, Red Tomatoes, Pink Grapefruit, Watermelon Beets

Orange Lycopene Carotenoids

Carrots, Mangoes, Apricots, Cantelope, Pumpkin, Sweet Potatoes, Oranges, Tangerines

Blue/Purple Betacyanins Blueberries, Plums, Eggplant, Concord grapes Yellow Zeaxantin

Curcumin Corn, Avocado Tumeric(curry)

Green Chlorophyll Broccoli, Kal, Spinach, Cabbage, Asparagus, Green tea

Black Thearubigins Anthocyanins

Black tea Blackberries

9

Many phytochemicals are polyphenol antioxidants that impart bright colors to

fruits and vegetables. Lutein makes corn yellow, lycopene makes tomatoes red,

carotene makes carrots orange and anthocyanin makes blueberries blue. Both the

bright colors and the antioxidant activities are due to alternating single-bonded and

double-bonded carbons. There is abundant evidence from epidemiological studies

showing that the phytochemicals in fruits and vegetables can significantly reduce the

risk of cancer, probably due to polyphenol antioxidant and anti-inflammatory effects.

1.2.1 Phenolic compounds

Phenolic compounds, or polyphenols, constitute one of the most numerous and

widely-distributed groups of substances in the plant kingdom, with more than 8,000

phenolic structures currently known.19 Polyphenols are products of the secondary

metabolism of plants. The expression "phenolic compounds" embraces a considerable

range of substances that possess an aromatic ring bearing one or more hydroxyl

substituents. Most of the major classes of plant polyphenols are listed in Table 1.3.

Table 1.3 The major classes of phenolic compounds in plants. 19

Number of carbon atoms

Basic skeleton Class Examples

6 C6 Simple phenols Benzoquinones

Catechol, Hydroquinone 2,6-Dimethoxybenzoquinone

7 C6-C1 Phenolic acids Gallic acid, Salicylic acid 8 C6-C2 Acetophenones

Tyrosine derivatives Phenylacetic acids

3-Acetyl-6-methoxybenzaldehyde Tyrosol p-Hydroxyphenylacetic acid

10

Table 1.3 (Continued)

Number of carbon atoms

Basic skeleton Class Examples

9 C6-C3 Hydroxycinnamic acids Phenylpropenes Coumarins Isocoumarins Chromones

Caffeic acid, Ferulic acid Myristicin, Eugenol Umbelliferone, Aesculitin Bergenon Eugenin

10 C6-C4 Naphthoquinones Juglone, Plumbagin 13 C6-C1-C6 Xanthones Mangiferin 14 C6-C2-C6 Stilbenes

Anthraquinones Resveratrol Emodin

15 C6-C3-C6 Flavonoids Isoflavonoids

Quercetin, Cyanidin Genistein

18 (C6-C3)2 Lignans Neolignans

Pinoresinol Eusiderin

30 (C6-C3-C6)2 Biflavonoids Amentoflavone n (C6-C3)n

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

Lignins Catechol melanins Flavolans (Condensed Tannins)

Lignins Catechol melanins Flavolans (Condensed Tannins)

Phenolic compounds are important antioxidants, because of their high redox

potentials. They act as reducing agents, hydrogen donors, singlet oxygen quenchers

and as metal chelating agents.20 Health-related effects of phenolic compounds such as

antibacterial,21 antimutagenic,22 anticarcinogenic,23 antithrombotic and vasodilatory

activities24,25 have been reported. The cited beneficial effects have been related to their

antioxidant properties. The number, type and concentration of phenolics in plants

exhibit extreme diversity. Phenolic compounds vary in structure. Hydroxybenzoic and

hydroxycinnamic acids have a single-ring structure. However flavonoids comprise

three ring structures and can be further classified into anthocyanins, flavan 3-ols,

flavones, flavanones and flavonols. Some flavonoids such as flavan 3-ols can be

11

found in the form of dimers, trimers and polymers.26 In plants, phenolics mainly occur

as glycosylated forms through O-glycosidic bonds with a number of different sugars

such as glucose, galactose, rhamnose, arabinose, xylose and rutinose.27 In addition,

phenolic compounds are also present acylations with phenolic or aliphatic acids,

which complicates the identification task. Distinctions are thus made between the

phenolic acids, flavonoids, stilbenes, and lignans (Figure 1.3). Among these phenolic

substances, flavonoids, and in particular, anthocyanins are of interest because of their

high occurrence in foods, especially in fruits, vegetables, and green leafy vegetables

including green tea.28

R1

R2

R3

O

OH

Hydroxybenzoic acids

R1 = R2 = OH, R3 = H; Protocatechvic acidR1 = R2 = R3 = OH; Gallic acid

O

OH

R1

R2

Hydroxycinnamic acids

R1 = OH; Coumaric acidR1 = R2 = OH; Caffeic acidR1 = OCH3, R2 = OH; Ferulic acid

O

Flavonoids

OH

HO

HO

Stilbenes

Lignans

HO

CH3O

OH

OCH3

CH2OH

CH2OH

Figure 1.3 Chemical structures of some polyphenols.29

12

1.2.2 Carotenoids

Carotenoids are a class of natural fat-soluble pigments that are associated with

the lipidic fractions. They are a group of over 600 dyes found in plants that provide

color ranging from light yellow to red. Carotenoids are polyisoprenoid compounds

and can be divided into two main groups: (a) carotenes or hydrocarbon carotenoids,

which composed of only carbon and hydrogen atoms and (b) xanthophylls that are

oxygenated hydrocarbon derivatives that contain at least one oxygen function such as

hydroxy, keto, epoxy, methoxy or carboxylic acid groups (Figure 1.4).30 Their

structural characteristic is a conjugated double bond system, which influences their

chemical, biochemical and physical properties. This class of natural pigments occurs

widely in nature. Carotenoids are synthesized by plants and many microorganisms,

thus animals can obtain them from food. They are responsible for the beautiful colors

of many birds, insects and marine animals, as well as the colors of many flowers and

fruits.31 This attribute is of great importance in foods, since color is often a criterion

of quality and is typically modified by food processing.32 In addition, carotenoid

content in fruits and vegetables depends on several factors such as, genetic variety,

maturity, postharvest storage, processing and preparation. In humans, carotenoids

play two primary roles; most of them give antioxidant activity and some of them are

converted into vitamin A. Of the 600 carotenoids that have been identified, about 30

to 50 are believed to have vitamin A activity. Carotenoids that the body converts to

vitamin A are referred to as "provitamin A" carotenoids. The most well known of this

group are β-carotene and α-carotene. Some of the better known carotenoids without

provitamin A activity but with very high antioxidant activity are lutein, lycopene, and

zeaxanthin.

13

HOe

O

HO

dHO

f

O

HOca b

HO

gOH h

Q9

10

15

15'

10'

9'R

Figure 1.4 Structures of carotenoids; antheraxanthin; R=e, Q=c; auroxanthin: R=Q=f;

α-carotene: R=a, Q=b; β-carotene: R=Q=a; α-cryptoxanthin: R=c, Q=b; β-

cryptoxanthin: R=c, Q=a; flavoxanthin/chrysanthemaxanthin: R=f, Q=d; lutein: R=c,

Q=d; lutein 5,6-epoxide: R=e, Q=d; luteoxanthin: R=e, Q=f; lycopene: R=Q=h;

mutatoxanthin: R=f, Q=c; neoxanthin: R=g, Q=e; neochrome: R=g, Q=f;

violaxanthin: R=Q=e; zeaxanthin: R=Q=c.33

In human beings, carotenoids can serve several important functions. The most

widely studied and well-understood nutritional role for carotenoids is their provitamin

14

A activity. Deficiency of vitamin A is a major cause of premature death in developing

nations, particularly among children. Vitamin A, which has many vital systemic

functions in humans, can be produced within the body from certain carotenoids,

notably β-carotene.34 Dietary β-carotene is obtained from a number of fruits and

vegetables, such as carrots, spinach, peaches, apricots, and sweet potatoes.35 Other

provitamin A carotenoids include α-carotene (found in carrots, pumpkin, and red and

yellow peppers) and cryptoxanthin (from oranges, tangerines, peaches, nectarines, and

papayas). Carotenoids also play an important potential role in human health by acting

as biological antioxidants, protecting cells and tissues from the damaging effects of

free radicals and singlet oxygen. Lycopene, the hydrocarbon carotenoid that gives

tomatoes their red color, is particularly effective at quenching the destructive potential

of singlet oxygen.36 Lutein, zeaxanthin and xanthophylls found in corn and in leafy

greens such as kale and spinach, are believed to function as protective antioxidants in

the macular region of the human retina.37 Astaxanthin, a xanthophyll found in salmon,

shrimp, and other seafoods, is another naturally occurring xanthophyll with potent

antioxidant properties.38 Other health benefits of carotenoids that may be related to

their antioxidative potential include enhancement of immune system function,39

protection from sunburn,40 and inhibition of the development of certain types of

cancers.41 Carotene can be stored in the liver and converted to vitamin A as needed.

α-Carotene has the same kind of ring on the left end of the molecule, and a

slightly different kind of ring on the right end of the molecule. It is an example of a

non-symmetrical carotene molecule. This carotene is called a precursor to provitamin

A compound. Some food sources of α-carotene found in pumpkin carrots winter

squash and tangerines. It has been associated with reduced risk of lung cancer.42

15

β-Carotene can be found in yellow, orange and green leaft fruits and vegetable.

These are carrots, spinach, lettuce, tomatoes, sweet potatoes, broccoli, cantaloupe,

orange and winter squash. As a rule of thumb, the grater the intensity of the color of

the fruit or vegetable, the more β-carotene it contains. β-Carotene consists of the

lycopene 'backbone' with two rings on each end. The two rings are structurally

identical, but one of them is upside down and backwards relative to the other. β-

Carotene is the most common of the carotenes and is important as a precursor for

vitamin A. β-Carotene is an antioxidant and can be useful for curbing the excess of

damaging free radicals in the body. However, the usefulness of β-carotene as a

dietary supplement (i.e. taken as a pill) is still subject to debate.43 β-Carotene is fat

soluble, so a small amount of fat is needed to absord it into the body. In recent years,

carotenoids including β-carotene have received a tremendous amount of attention as

potential anti-cancer and anti-aging compounds. β-Carotene is a powerful antioxidant,

protecting the cells of the body from damage caused by free radicals. It is also one of

the carotenoids believed to enhance the function of the immune system.

γ-Carotene is a precursor of β-carotene. It has a ring like beta-carotene's rings on

one end and no ring on the other. Some food sources of γ-carotene are found in

carrots, sweet potatoes, corn, tomatoes, watermelon, and apricots. 44

δ-Carotene is a precursor of α-carotene. It has the same type of ring as α-

carotene on the right end, but the left end is not cyclized.45

16

1.2.3 Vitamin E isomers (Tocopherols and Tocotrienols)

Vitamin E is fat-soluble antioxidant vitamins46, which consists of eight different

forms or isomers; tocopherols (α- ,β-, γ-, δ-isomes) and tocotrienols (α-, β-, γ-, δ-

isomers). The unsaturated analogues of tocopherols and tocotrienols structures are

similar except the tocotrienol structure has double bonds on the isoprenoid units.

Many derivatives of these structures are due to the different substituents possible on

the aromatic ring at positions 5, 6, 7, and 8 (Figure 1.5). Some food sources

containing vitamin E include plant and seed oils, nuts, whole grains, green leafy

vegetables, eggs, liver and milk.

Tocopherol

O

HO

R2

R1

O

HO

R2

R1 Tocotrienol

1

2

345

6

7

8

8

7

6

5 43

2

1

R1 R2

α- CH3 CH3

β- CH3 H

γ- H CH3

δ- H H

Figure 1.5 Chemical structures of tocopherols and tocotrienols.

17

Both the tocopherols and tocotrienols occur in alpha, beta, gamma and delta

forms, determined by the number of methyl groups on the chromanol ring. Each form

has slightly different biological activity.47 The α-tocopherol form is the most

important lipid-soluble antioxidant and protects cell membranes against oxidation by

reacting with the lipid radicals produced in the lipid peroxidation chain reaction. This

removes the free radical intermediates and prevents the propagation reaction from

continuing. The oxidised α-tocopheroxyl radicals produced in this process may be

recycled back to the active reduced form through reduction by ascorbate, retinol or

ubiquinol. Tocotrienols have been reported to be involved with inhibition of

cholesterol synthesis, lowering serum-cholesterol levels in various animal models,

and suppressing tumor-cell proliferation, with the γ- and δ-homologs demonstrating

greater potency than the α-homolog.48

1.2.4 Antioxidants and Free Radicals

Free radicals are highly reactive compounds that are created in the body during

normal metabolic functions or introduced from the environment. Free radicals are

inherently unstable, since they contain “extra” energy. To reduce their energy load,

free radicals react with certain chemicals in the body, and in the process, interfere

with the cells’ ability to function normally. Antioxidants work in several ways; they

may reduce the energy of the free radical, stop the free radical from forming in the

first place, or interrupt an oxidizing chain reaction to minimize the damage caused by

free radicals.

Free radicals are believed to play a role in more than sixty different health

conditions, including the aging process, cancer, and atherosclerosis. Reducing

18

exposure to free radicals and increasing intake of antioxidant nutrients has the

potential to reduce the risk of free radical-related health problems.

There are numerous types of free radicals that can be formed within the body.

The most common is reactive oxygen species or ROS include; the superoxide anion

(O2-), the hydroxyl radical (OH·), singlet oxygen (1O2), and hydrogen peroxide

(H2O2). Superoxide anions are formed when oxygen (O2) acquires an additional

electron, leaving the molecule with only one unpaired electron. Within the

mitochondria O2-· is continuously being formed. The rate of formation depends on the

amount of oxygen flowing through the mitochondria at any given time. Hydroxyl

radicals are short-lived, but the most damaging radicals within the body. This type of

free radical can be formed from O2- and H2O2 via the Harber-Weiss reaction. The

interaction of copper or iron and H2O2 also produce OH · as first observed by Fenton.

These reactions are significant as the substrates are found within the body and could

easily interact.49 Hydrogen peroxide is produced in vivo by many reactions. Hydrogen

peroxide is unique in that it can be converted to the highly damaging hydroxyl radical

or be catalyzed and excreted harmlessly as water. Glutathione peroxidase is essential

for the conversion of glutathione to oxidized glutathione, during which H2O2 is

converted to water.50 If H2O2 is not converted into water, 1O2 is formed. Singlet

oxygen is not a free radical, but can be formed during radical reactions and also

causes further reactions. Singlet oxygen violates Hund's rule of electron filling in that

it has eight outer electrons existing in pairs leaving one orbital of the same energy

level empty. When oxygen is energetically excited one of the electrons can jump to

empty orbital creating unpaired electrons. Singlet oxygen can then transfer the energy

19

to a new molecule and act as a catalyst for free radical formation. The molecule can

also interact with other molecules leading to the formation of a new free radical.

Antioxidants are vital substances which possess the ability to protect the body

from damage caused by free radical induced oxidative stress. There is an increasing

interest in natural antioxidants, e.g., polyphenols, present in medicinal and dietary

plants, which might help prevent oxidative damage.51 In this study, the crude extracts

obtained from solvent extraction were used for the determination of relative

antioxidative activities in several tests (thiocyanate method, the H2O2-scavenging

activity chemiluminescence system (XYZ system), the Cu2+/bathocuproine

colorimetry (PAO) assay, and the 1,1-diphenyl-2-picrylhydrasyl (DPPH) free radical-

scavenging activity assay). The metal chelating activity is also determined using

ferrozine.

Thiocyanate method

Hydrogen peroxide oxidizes ferrous iron to the ferric state52 as shown in the

following reaction:

RCOOH + 2Fe2+ + 2H+ → 2Fe3+ + H2O + ROH

The concentration of Fe3+ ions formed during the hydroperoxide decomposition

can be determined by the thiocyanate method, which consists of ammonium

thiocyanate and ferrous ion in acid solution giving the formation of a red thiocyanate

complex. This method assumed that hydroperoxides are stoichiometrically consumed

in the oxidation of Fe2+ to Fe3+ ion. The Fe3+ ion are then quantitatively complexed

with SCN- ions, the concentration of hydroperoxides can be determined

20

spectrophotometrically by measuring the coloured [Fe(SCN)4]3- complex at its

absorbance at 500 nm as follow:

Fe+3 + 3 NH4SCN → Fe(SCN)4]3-

A visual comparison may be made between a prepared sample and a standard.

The color intensity of the standard is adjusted to match that of the sample by adding a

solution containing a known amount of iron. The volume of standard iron solution

required is used to calculate the quantity of iron in the sample. More commonly,

especially if iron is determined routinely, a spectrophotometer is utilized. A

calibration curve is constructed for the instrument using solutions of known iron

concentration.

H2O2-scavenging activity chemiluminescence system (XYZ system)

The ROS/hydrogen donor/mediator system (XYZ system) is a new

chemiluminescence system for measurement of the ROS (X), hydrogen donor (Y) and

mediator (Z). This chemiluminescence can be observed by mixing three species, i.e. a

reactive oxygen species (X), a hydrogen donor (Y) and a mediator (Z) at room

temperature.53 The photon intensity in this system demonstrates the high

concentration depending on X, Y and Z species. Photon intensity from XYZ system

showed a linear correlation with the concentration of some hydroperoxides (H2O2,

tert-BuOOH and methyl ethyl ketone-OOH), hydrogen donors (samples) and

mediators (KHCO3, MeCHO and hemoglobin). The linear relationship between

photon intensity and concentration was observed in polyphenol-rich samples such as

teas and berries. Analysis of photon intensity can quantify the X, Y and Z contents

21

and/or activities in sample. In addition, photon intensity (P) in this system, shows

high concentration dependence on X, Y and Z, as indicated in the equation ;

[P] = k[X][Y][Z],

where k = photon constant

Potential Antioxidant (PAO assay)

In the PAO assay kit, an easy and convenient method to measure antioxidant

capacity is provided. Utilizing the reduction of cupric ion (Cu2+→ Cu+), antioxidant

capacity of samples can be detected in 5 minutes. Samples are mixed with Cu2+

solution. Cu2+are reduced by antioxidants to form Cu+. Reduced Cu+ react with

chromatic solution (Bathocuproine), and can be detected by absorbance at wavelength

480 to 490 nm. Antioxidant capacity can be calculated from the Cu+ formed. PAO can

detect not only hydrophilic antioxidants such as vitamin C, glutathione, but also can

detect hydrophobic antioxidants such as vitamin E, which is applicable for assessment

of total antioxidants of serum, foods and beverage samples.54

1,1-Diphenyl-2-picrylhydrasyl (DPPH) free radical-scavenging activity assay

DPPH assay is one of the most widely used methods for screening antioxidant

activity of plant extracts.55 DPPH is a stable, nitrogen-centered free radical which

produces violet colour in ethanol solution. It was reduced to a yellow coloured

product, diphenylpicryl hydrazine, with the addition of the fractions in a

concentration-dependent manner that can be easily monitored using a

spectrophotomer. The reduction in the number of DPPH molecules can be correlated

with the number of available hydroxyl groups. All the fractions showed significantly

22

higher inhibition percentage (stronger hydrogen-donating ability) and positively

correlated with total phenolic content.

Metal chelating activity

The metal chelating ability is measured by the formation of ferrous ion ferrozine

complex. Ferrozine combines with ferrous ions forming a red coloured complex

which absorbs at 562 nm.56 It was reported that the chelating agents which form σ

bond with a metal, were effective as secondary antioxidants, because they reduced the

redox potential, thereby stabilising the oxidised form of the metal ion.57 Chelation is

capture of positively-charged metal ions by a large molecule. The most widely used

chelating molecule is EDTA (ethylene diamine tetraacetic acid). EDTA has the

capacity to chelate almost every positive ion in the periodic table. EDTA is commonly

added to fatty, oily foods as an antioxidant that prevents metal ions that have entered

from metallic food-processing equipment from causing rancidity (i.e. metallic-

catalyzed oxidation of fat by oxygen).

1.2.5 Literature review

Recent studies have reported the antioxidant activities of rice as well as other

cereals as shown by the studies below.

In 1992, Santiago and Mori58 reported the therapeutic uses of rice bran and its

reaction with free radicals examined by spectrometry/spin trapping technique (ESR).

The scavenging action against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl and

superoxide radicals were examined by superoxide dismutase (SOD)-like activity and

quenching action on carbon-centered radicals generated in the brain of rats.

23

In 1998, Velioglu and co-workers59 determined the antioxidant activities and

total phenolic contents of 28 plant products, including sunflower seeds, flaxseeds,

wheat germ, buckwheat, and several fruits, vegetables, and medicinal plants. The total

phenolic content, determined according to the Folin-Ciocalteu method, varied from

169 to 10,548 mg GAE/100 g of dry product. Antioxidant activity of methanolic

extract evaluated according to the β-carotene bleaching method expressed as AOX

(Δlog A470/min), AA (percent inhibition relative to control), ORR (oxidation ratio),

and AAC (antioxidant activity coefficient) ranged from 0.05, 53.7, 0.009 and 51.7 to

0.26, 99.1, 0.46, and 969.3, respectively. The correlation coefficient between total

phenolics and antioxidative activities was statistically significant.

In 1999, Garry and co-workers60 found the phenolic-rich fractions of oats

possessed an antioxidant capacity that can be assessed quantitatively though their

ability to inhibit low-density lipoprotein (LDL) oxidation and protein oxidation in the

oxygen radical absorbance capacity (ORAC assay). The greatest degree of antioxidant

capacity was associated with compounds extracted with methanol from the aleuron.

These compounds included caffeic acid, ferulic acid, and avenanthramides.

In 1999, Osawa61 investigated the isolation and identification of antioxidative

pigments from black rice, cyanidin-3-O-β-D-glucoside together with delephinidin-3-

O-β-D-glucoside, and pelagonidin-3-O-β-D-glucoside. It was found that all three

anthocyanidin type pigments exhibited the strong antioxidative activity in the acidic

regions, however, only cyaniding-3-O-β-D-glucoside was found to be antioxidative

even in the neutral and basic pH conditions.

In 2000, Sun and co-workers62 studied the antioxidant activity of 12 varieties of

black rice and its correlation with flavonoids and pigments. Results indicated the

24

water extraction and 60% ethanol extaction of black rice had high antioxidant activity,

and significant positive correlation existed between the O2 scavenging rates and black

rice contents in the extraction. When the O2 scavenging rate is 50% (IC50) was

negative correlated with the flavonoids or pigment contents significantly.

Lloyd and co-workers63 investigated the changes in selected antioxidants in rice

bran from both long- and medium-grain rice during commercial milling and bran

processing. Rice bran collected from various milling breaks of a commercial system

had varying antioxidant levels. Bran collected after milling break had the highest

levels of tocopherol and tocotrienol. Oryzanol concentration was significantly higher

in outer bran layers. Results also indicated that the long-grain rice bran had average

15% more antioxidants than the medium-grain rice bran.

Zielinski and Kozlowsk64 examined the antioxidant properties of water and 80%

methanolic extracts of cereal grains and their different morphological fractions.

Wheat (Triticum aestivum L.) cv. Almari and cv. Henika, barley (Hordeum vulgare

L.) cv. Gregor and cv. Mobek, rye (Secale cereale L.) cv. Dan´ kowskie Zlote, oat

(Avena sativa L.) cv. Slawko and buckwheat (Fagopyrum esculentum Moench) cv.

Kora were used. PC (L-R-phosphatidylcholine) liposome system and

spectrophotometric assay of total antioxidant activity (TAA) were used to evaluate the

antioxidative activity of extracts. Among the water extracts, only the one prepared

from buckwheat exhibited antioxidant activity at the concentration analyzed. The

following hierarchy of antioxidant activity was provided for 80% methanolic extracts

originated from whole grain: buckwheat > barley > oat > wheat = rye. The

antioxidant activity was observed in extracts prepared from separated parts of

buckwheat and barley. In respect to hulls, the antioxidant hierarchy was as follows:

25

buckwheat > oat > barley. The correlation coefficient between total phenolic

compounds and total antioxidative activity of the extracts was -0.35 for water

extracts and 0.96, 0.99, 0.80, and 0.99 for 80% methanolic extracts originated from

whole grains, hulls, pericarb with testa fractions and endosperm with embryo

fractions, respectively.

Miller and co-workers65 compared the antioxidant activity of whole grains,

ready-to-eat (RTE) breakfast cereals to that of fruits and vegetables. Antioxidant

activity was detected by dispersing finely ground samples in a 50% methanol of the

stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). DPPH, which forms a deep

purple solution, reacts with antioxidants and color loss measured at 515 nm correlates

to antioxidant content, which is expressed as Trolox equivalent/100 g (TE). Whole

grain breakfast cereals analyzed contained between 2200-3500 TE. By comparison,

fruits generally ranged from 600-1700 TE, with a high of 2200 TE for red plums.

Berries averaged 3700 TE and vegetables averaged 450 TE with a high of 1400 TE

for red cabbage. A 41.0 g of average serving of RTE breakfast cereal provides 1120

TE, while an average 85.0 g serving of vegetables or fruits provides 380 and 1020 TE,

respectively. Whole grain breakfast cereals, fruits and vegetables are all important

dietary sources of antioxidants.

In 2001, Ling and co-workers66 investigated the influence of natural red or black

rice consumption on atherosclerotic plaque formation or development induced by high

cholesterol diet feeding in rabbits and to explore possible mechanisms by which

colored rice consumption decreases atherosclerotic plaque formation.

In 2002, Adom and Liu67 studied the complete phytochemical profiles in free,

conjugated, and insoluble bound forms, as well as their antioxidant activities in

26

uncooked whole grains. Corn had the highest total phenolic content (15.55-0.60 μmol

of gallic acid equivalent (GAE /g of grain) of the grains tested, followed by wheat

(7.99-0.39 μmol of GAE /g of grain), oats (6.53-0.19 μmol of GAE /g of grain), and

rice (5.56-0.17 μmol of GAE /g of grain). The major portion of phenolics in grains

existed in the bound form (85% in corn, 75% in oats and wheat, and 62% in rice),

although free phenolics were frequently reported in the literature. Ferulic acid was

the major phenolic compound in grains tested, with free, soluble-conjugated, and

bound ferulic acids present in the ratio 0.1:1:100. Corn had the highest total

antioxidant activity (181.42-0.86 μmol of vitamin C equivalent /g of grain), followed

by wheat (76.70 - 1.38 μmol of vitamin C equivalent /g of grain), oats (74.67-1.49

μmol of vitamin C equivalent /g of grain), and rice (55.77-1.62 μmol of vitamin C

equivalent /g of grain). Bound phytochemicals were the major contributors to the total

antioxidant activity; 90% in wheat, 87% in corn, 71% in rice, and 58% in oats.

Holasova and co-workers68 evaluated and compared the antioxidant activities of

buckwheat seeds, dehulled seeds, hulls, straws, and leaves with those of oats and

barley. The results showed the buckwheat seeds and leaves proved to be higher in

antioxidant activity when compared with those of oats, barley, buckwheat, straws, and

hulls. Antioxidant activities of buckwheat were derived mainly from methanol soluble

substances, while lipophilic substances showed only a slight antioxidant activity.

Statistically significant relationship between total phenolics as well as rytin content in

buckwheat and antioxidant activity was found.

Oki and co-workers69 demonstrated that polymeric procyanidins are the major

radical-scavenging components in red-hulled rice. The extracts from white-, black-,

and red-hull rice were prepared by sequential extraction with six different polar

27

solvents, and their radical-scavenging activities were measured by methods using 1,1-

diphenyl-2-picrylhydrazyl (DPPH) and tert-butyl hydroperoxyl (t-BuOO) assay. The

extracts prepared with methanol and deionized water, exhibited higher DPPH· and t-

BuOO· scavenging activities in all three cultivars. In addition, the acetone extract

from red-hulled rice exhibited a high DPPH· and t- BuOO·scavenging activity. The

major components responsible for radical scavenging activity in the acetone extract of

red-hulled rice were identified as procyanidins.

Itani and co-workers70 compared six rice cultivars (two red rices, two purple-

black rices and two white rices) for their antioxidative activities and differential

distribution of active substances. Ethanol extraction prepared from red and purple-

black hulled rice exhibited remarkably high superoxide anion- and radical-scavenging

activities compared with those from white-hulled rice. In addition, these activities

were localized mostly in pericarp and testa, namely, rice bran. Colored rice contained

polyphenols much more abundantly than white rice and their contents were mutually

related to their antioxidative potency. Spectrophotometric analyses showed that the

major active substances in red rice and purple-black rice were tannin and anthocyanin

pigments.

In 2003, Kaneda and co-workers71 studied the polyphenols in cereal grains,

especially barley malt by ESR using DPPH radical in ethanol solution. The difference

of peak height between the DPPH radical ethanol solution before and after mixing

with the pulverized cereal grain was used to calculate the amplitude of polyphenols in

the cereal grain. The method was fast and simple, and useful for brewing beer.

Zawistowski and co-workers72 extracted a compound comprising anthocyanins

from an outer layer from a starchy endosperm in de-hulled black rice (Oryza sativa).

28

The compounds comprised cyanidin-3-O-glucoside and peonidin-3-O-glucoside, and

additional comprised antioxidants, sterols, and stanols. This compound was useful in

enhancing and/or preserving the stability of high-density lipoprotein- cholesterol

(HPL-C) and the atherogenic lipoproteins such as low-density lipoprotein- cholesterol

(LDL-C), very low-density lipoprotein-cholesterol (VLDL-C), and intermediate-

density lipoprotein-cholesterol (IDL-C) from oxidation, in preventing, reducing,

eliminating or ameliorating injuries due to oxidative stress and inflammation.

Hu and co-workers73 showed the presence of anthocyanins in the aleurone layer

of black rice, in particular, cyaniding-3-glucoside and peonidin-3-glucoside. The

anthocyanins contributed to antioxidant activities in preventing DNA damage and

LDL deterioration in vitro. Black rice and specific anthocyanin components present in

black rice also suppressed the production of nitric oxide in the activated macrophage

without introducing cytotoxicity. These data suggested that black rice may have some

health benefits associated with relief of oxidative stress.

Joseph and co-workers74 screened three methods to measure antioxidant activity

of sorghum, their bran, and baked and extruded products. These methods were oxygen

radical absorbance capacity (ORAC), 2,2′-azinobis(3-ethyl-bnzothiazoline-6-sulfonic

acid (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH). All sorghum samples were

also analyzed for phenolic contents. Both ABTS and DPPH correlated highly with

ORAC. Phenolic contens of the sorghum correlated highly with their antioxidant

activity when measured by the three methods (R2 ≥ 0.96).

Yen and co-workers75 studied the antioxidant activity of ethyl acetate extracts

from rice koji (EAERK) fermented with Aspergillus candidus CCRC 31543. EAERK

had a strong scavenging effect on the DPPH radical. Silica gel column

29

chromatography was used to separate EAERK into eight fractions. The antioxidants

of these Aspergillus metabolites were evaluated and compared with BHA and

EAERK.

In 2004, Kaneda and co-workers76 studied the superoxide dismutase (SOD)-like

activity and the ROS (superoxide anion, hydroxyl radical, singlet oxygen,

t-butylperoxyl radical) scavenging activities were evaluated for the extracts from 2

varieties (black and red rices) of ancient rice brans (8 types) by the nitro-blue

tetrazolium (NBT) and ESR (electron spin resonance)-spin trapping methods. All the

extracts from ancient rice brans (black and red rice) had SOD-like activity, which was

stronger than those from present-day rice brans. The ancient rice brans have

remarkably strong ROS scavenging activities compared with those of the present-day

rice brans. The ROS scavenging activities and SOD-like activities of the extracts

varied depending on the rice species and the planting region. In addition, the extracts

from ancient rice brans inhibited the Maillard reaction, which is known to be involved

in physiology aging processes. Thus, the results suggested the utility of the extracts

from ancient rice brans as antioxidative materials.

Joseph and co-workers77 analyzed anthocyanins from black sorghum. The

samples were also analyzed for antioxidant activity using the 2, 2′-azinobis (3-

ethylbenzothiaziline-6-sulfonic acid) method. The sorghum grains and their brans had

high antioxidant activity (52-40 μmol trolox equivalent /g) compared to other cereals

(0.1-34 mg trolox equivalent /g).

In 2005, Iqbal and co-workers78 studied the antioxidant activity of five

indigenous rice bran varieties, i.e. rice bran-super kernel (RB-kr), rice bran-super

2000 (RB-s2), rice bran-super basmati (RB-bm), rice bran-super 386 (RB-86)and rice

30

bran-super fine (RB-sf). The order of antioxidant activity was evaluated by

measurement of antioxidant activity in linoleic acid system, reducing power, metal

chelating ability, scavenging capacity by DPPH radicals, ABTS cation radicals and

conjugated dienes. The overall order of antioxidant activity was RB-kr > RB-s2 >

RB-bm > RB-86 > RB-sf.

Yan and co-workers79 studied the natural antioxidant enriched rice bran oil

extracts which were obtained by extracting rice bran oil with ethanol, isopropanol and

ethyl acetate. The yields of extraction were 26.8%, 67.1% and 82.8%, respectively.

The extracts were added into conjugated linoleic acid (CLA) for the oxidation test at

60°C. The results showed that although different extracts had different antioxidative

activity, for CLA, they all had higher activity than BHT (Buthylatehydroxytoluene)

and among them the isopropanol extract had the highest activity. It was clear by this

study that the isopropanol extract could be used as a good natural antioxidant to

protect CLA from oxidation.

Kim80 studied the radical scavenging capacity and antioxidant activity of an E

vitamer fraction from rice bran. The E vitamer fraction was prepared by a liquid-

liquid extraction method. The free radical scavenging capacity of the E vitamer

fraction was measured by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) method

at the concentration range of 2.5 to 640 ppm, whereas the antioxidant activities were

measured by both the reducing power and ferric thiocyanate (FTC) methods at

different concentrations (0, 2.5, 10, 40, and 160 ppm). Radical scavening capacity of

the fraction was effective up to 160 ppm, then remained stable. Also, at a

concentation of 160 ppm, it was highly effective in inhibiting linoleic acid

peroxidation. Similarly, superoxide scavenging and antioxidant activities of the E

31

vitamer fraction were compared with those of the synthetic phenol compds. Results

showed that with a concentration of the E vitamer fraction at 160 ppm, the antioxidant

activity was comparable to both butylated hydroxytoluence (BHT, 160 ppm) and

butylated hydroxyanisole (BHA, 160 ppm). No significant differences (P > 0.05) were

found among them. The E vitamer fraction may be a good substitute for the synthetic

phenol antioxidants currently used in industry because the former is more natural and

comparatively effective in its radical scavenging capacity and antioxidant activity.

Nam and co-workers81 evaluated antioxidative, antimutagenic, antitumor

promoting, and anticarcinogenic activities in mammalian cells of bran extracts from a

brown rice variety used in the home and two pigmented experimental rice cultivars.

The extracts from the pigmented rice seeds had generally higher activities in all tests

than did the extract from the nonpigmented variety. The results further demonstate

the health-promoting potential of the pigmented rice cultivars.

Ha and co-workers82 studied the bioactive components in rice bran oil which

improved lipid profiles in rats fed a high-cholesterol diet. The liver cholesterol and

triacylglycerol contents were higher in rats fed the high-cholesterol diet than the

normal group but significantly decreased by bioactive components in rice bran oil

supplementation. Similarly, hepatic thiobarbituric acid–reactive substances were

increased by a high-cholesterol diet and reduced by bioactive components in rice bran

oil supplementation in rats.

In 2006, Macias Francisco and co-workers83 isolated the bioactive steroids from

Oryza sativa L. Fifteen bioactive compounds were obtained and identified by

spectroscopic methods. Eight of these compounds were obtained for the first time in

Oryza sativa. They were β-sitosterol, 7-oxositosterol, stigmasterol, 7-oxostigmasterol,

32

(6α, 22E)-hydroxy-stigmata-4, 22-dien-3-one,(6β, 22E)-hydroxy-stigmata-4, 22-dien-

3-one, ergo-sterol peroxide, and 5α, 8α-pidioxy-24(R)-methylcolesta-6-en-3β-ol. It

was found that the most phytotoxic compounds on E. Crus-galli were ergo-sterol

peroxide and 7-oxostigmasterol.

Ragaee and co-workers84 studied four cereals including barley, pearl millet, rye,

and sorghum which were adapted to the growing conditions in the United Arab

Emirates. They were evaluated in terms of their composition of dietary fiber, resistant

starch, minerals and total phenols and antioxidant properties. Antioxidant activity was

evaluated on the basis of DPPH and ABTS assay. Sorghum was exceptionally high in

antioxidant activities followed by millet, barley and rye.

Zhang and co-workers85 found that the water fraction and normal-butyl alcohol

fraction of antioxidative extracts of black rice had the strongest antioxidative

capacities and their total antioxidant capacity (TACs) reached 383 kilounit (ku)/g and

392 ku/g, respectively. Four main antioxidative components were separated from the

water fraction and their TACs reached 976 ku/g, 878 ku/g, 1 134 ku/g and 1 087 ku/g,

respectively. The spectroscopic analysis indicated that the four active components of

the extracts of black rice were malvidin, pelargonidin-3,5-diglucoside, cyaniding-3-

glucoside and cyaniding-3,5-diglucoside. It is concluded that the anthocyanin

compounds are the most important substance basis for antioxidation.

Kaneda and co-workers86 found that extracts from ancient rice brans, especially

those from black rice bran, possess strong scavenging activities for reactive oxygen

species (ROS). In this study, they examined the origin of the ROS-scavenging

activities in the black rice bran extracs, and identified candidate scavengers such as

cyanidin-3-glucoside (Cy-3-glu) and cyanidin. Although ferulic acid is known to be

33

an antioxidative component of bran in currently available common white rice

varieties, it was not found in the black rice bran extracts. The ROS-scavenging

activities of Cy-3-glu and cyanidin, which were identified in this study, were

examined using the ESR-spin traping method and in terms of protective activity

against effects of uv (UVB) irradation on an epidermal cell line (HaCaT cell). These

anthocyanin compounds were found to possess both strong ROS-scavenging activities

and to suppress cell-damaging effects of UVB, indicating that both Cy-3-glu and

cyanidin were the active components involved in the antioxidative activity of black

rice bran exracts.

Chen and co-workers87 provided molecular evidence associated with the anti-

metastatic effects of peonidin-3-glucoside and cyaniding-3-glucoside, major

anthocyanins extracted from black rice (Oryza sativa L. indica), by showing a marked

inhibition on the invasion and motility of SKHep-1 cells. This effect was associated

with a reduced expression of matrix metalloproteinase (MMP)-9 and urokinase-type

plasminogen activator (u-PA). Peonidin 3-glucoside and cyanidin 3-glucoside also

exerted an inhibitory effect on the DNA binding activity and the nuclear translocation

of AP-1. Furthermore, these compounds also exerted an inhibitory effect of cell

invasion on various cancer cells (SCC-4, Huh-7, and HeLa). Finally, anthocyanins

from O. sativa L. indica (OAs) were evidenced by its inhibition on the growth of

SKHep-1 cells in vivo.

Nam and co-workers88 studied the use of ethanol-water (70:30, v/v) to extract

bran of rice seeds from twenty one pigmented and one nonpigmented rice cultivars,

which were evaluated for antioxidative activities using the following tests: inhibition

of peroxidation of linoleic acid; inhibition of peroxidation of rabbit lipid erythrocyte

34

membranes; reduction of potassium ferricyanide, and scavenging of superoxide

anions and hydroxyl radicals. With some exceptions, extracts from the pigmented rice

seeds had higher antioxidative activity than did the nonpigmented variety. The

following pigmented cultivars had the highest antioxidative activities in all tests:

Jumlalocal-1, Parnkhari 203, DZ78, LK1-3-6-12-1-1, and Elwee. A significant

correlation was also noted between reducing power, inhibition of erythrocyte ghost

membrane peroxidation, and superoxide anion and hydroxyl radical scavenging. The

results suggested that; (a) the ferricyanide test of reducing power might be a useful

and simple index for large-scale evaluation of antioxidative potencies of natural

products present in rice; (b) pigmented rice varieties with high antioxidative activities

provide a source of antioxidants and a genetic resource to develop new health-

promoting rice cultivars.

Yang and co-workers89 evaluated the antioxidant properties of methanolic

extracts from inoculated rice products (monascal polished rice (MPR) and monascal

dehulled rice (MDR)) as compared to uninoculated rice products (polished rice (PR)

and dehulled rice (DR)). With regard to EC50 values (mg extract/mL) of methanolic

extracts, inhibitions of peroxidation were excellent and in the descending order of

PR>DR>MDR>MPR. Effectiveness in reducing powers was in the descending order

of MPR>MDR>DR>PR. Scavenging abilities on DPPH radicals were in the

descending order of MPR>MDR>PR=DR. Chelating abilities on ferrous ions were in

the descending order of MPR=MDR>PR>DR. Phenolics were the major naturally

occurring antioxidant components found. Overall, monascal rice products were better

in reducing power, scavenging and chelating abilities and higher in total phenolic

content than uninoculated rice products.

35

In 2007, Carlos and co-workers90 evaluated the relationship between antioxidant

capacity and levels of various antioxidants in rice bran and brown rice powder. Three

different varieties of Venezuelan rice, namely, Cimarrón, Zeta 15 and FONAIAP-1,

were studied using ferric reducing antioxidant power (FRAP), 2,2′-azinobis-3-

ethylbenzotiazoline-6-sulphonic acid (ABTS), and oxygen radical absorbance

capacity (ORAC) to measure antioxidant capacity. The results showed that rice

varieties contained different levels and combinations of total polyphenols, γ-oryzanol,

α- and γ-tocopherols and α-, γ- and δ-tocotrienols. Compared to brown rice powder,

rice bran contained most of the antioxidants and had correspondingly higher values of

antioxidant capacity. Principal components analysis and multiple regression on the

data indicate that FRAP was sensitive to polyphenols and total tocotrienols, while

ORAC was sensitive to polyphenols and total tocopherols. ABTS was the least

sensitive of all assays tested.

Finocchiaro and co-workers91 compared the total antioxidant capacity (TAC) and

the antioxidant chemical components, namely, tocols, γ-oryzanols, and polyphenols,

of red and white rices. In addition, the effect of milling and cooking on antioxidants

was investigated in both types of rices. Dehulled red rice showed a TAC more than

three times greater than dehulled white rice and its high TAC was essentially

characterized by the presence of proanthocyanidins (PA) and associated phenolics.

Milling caused a significant loss of TAC, even if red rice maintained a higher TAC.

Cooking caused a further loss of antioxidants, but when there was a full uptake of

cooking water by the grains ("risotto, that literally means 'little rice'. In Italy, risottos

which is an Italian rice dish are eaten almost as often as pasta") this loss was limited.

36

Thus, the consumption of whole or partially milled rice would be preferred to

preserve its nutritional properties.

Stratil and co-workers92 used three methods, Folin–Ciocalteu (FCM) method, the

ferricyanide method according to Price and Butler (PBM)93 and a method using 4-

aminoantipyrine (AAPM) for assessment of phenolic compounds. Spectrophotometric

methods, TEAC (Trolox equivalent antioxidant capacity), DPPH (with diphenyl-

picrylhydrazyl radical), and FRAP (ferric reducing antioxidant power) were used for

evaluation of antioxidant capacity of 17 kinds of fruit and 6 kinds of cereal. The

results showed the relatively less commonly used PBM method was the most reactive

of the three methods (FCM, PBM and AAPM) used for the estimation of phenolic

compounds concentration. The commonly used FCM reacts only with the more

reactive phenolic compound. AAPM has significantly different mechanism of

response and reacts only with phenolic substances that are able to create a quinoidal

structure and thus with only a smaller part of the total content of phenolic compounds.

Three most often used methods for estimation of antioxidant capacity, TEAC, FRAP

and DPPH were modified and applied for the evaluation of antioxidant capacity to the

same set of standards, interferents and plant extracts. The methods enabled fast and

reproducible assessment of the equivalent antioxidant capacity of selected standard

(e.g. Trolox). The TEAC method was the most reactive and the FRAP method was

essentially the least. The DPPH radical was relatively stable and therefore less

reactive, so that it reacted only with more reactive reducing (phenolic) substances.

Results obtained using DPPH method evidently correlated with the low reactivity

(high stability) of the radical.

37

Youngmin and co-workers94 determined antioxidant activity of the methanolic

extracts from some grains and investigated relationships between antioxidant

activities and antioxidant contents in the extracts. DPPH radical scavenging activities,

and ABTS radical cation scavenging activities inhibitory effect on lipid peroxidation,

chelating activity and reducing power had been used to investigate the relative

antioxidant activities of the extracts from grains. The concentrations of total

polyphenolics and carotenoids in the extracts were measured by spectrophotometric

methods and vitamin E analysis was carried out by HPLC. The methanolic extracts

prepared from red sorghum and black rice showed significantly higher antioxidant

activities and contained higher polyphenolic contents than other grains such as white

rice, brown rice, mungbean, foxtail millet, prosomillet, barley, and adlay.

Polyphenolic compounds were the major naturally occurring antioxidants in grains.

The correlation coefficient between total polyphenolic content and ABTS radical

cation scavenging activity in the extracts was > 0.99. However, no relationship was

found between antioxidant activities and carotenoids and vitamin E derivatives.

Gorinstein and co-workers95 determined polyphenols, phenolic acids, fibres and

antioxidant capacity in water, acetone and methanol extracts of buckwheat, rice,

soybean, quinoa, and 3 amaranth cultivars. Their antioxidant activities were

comparatively assessed by total radical-trapping antioxidative potential (TRAP),

ferric ion-reducing antioxidant power (FRAP), cupric-reducing antioxidant capacity

(CU antioxidant capacityPRAC) and nitric oxide (NO) assays, which comprised of

contribution from polyphenols and phenolic acids. All the applied methods showed

that pseudocereals have higher antioxidant activity than some cereals (rice and

buckwheat) and they can be successfully replaced by cereals in case of allergy.

38

Yawadio and co-workers96 isolated two anthocyanins (cyanidin-3-O-glucoside

and peonidin-3-O-glucoside) and other phenolics (ferulic acids) from black and

pigmented brown rices (Oryza sativa L. japonica) and their complete structures were

identified by spectroscopic analysis (H-NMR, C-NMR and MALDI-MS).

Chung and Shin97 characterized alkaloids and phenolic acids from pigmented

rice (Oryza sativa cv. Heugjinjubyeo). 4-Carboethoxy-6-hydroxy-2-quinolone, ethyl-

3,4-dihydroxybenzoic acid, 4-hydroxy-3-methoxyphenylacetic acid, 3,4-

dihydroxybenzoic acid, and 4-hydroxy-3-methoxy cinnamic acid were extracted from

the ethyl acetate-soluble fraction of the aleurone layer of Oryza sativa. These

compounds showed significant antioxidant activity.

1.3 Supercritical Fluid Extraction (SFE)

A supercritical fluid (SF) is a material that can be either liquid or gas, used in a

state about the critical temperature (Tc) and critical pressure (Pc) where gases and

liquids can coexist. It shows unique properties that are different from those of either

gases or liquids under standard conditions. A gas, when compressed isothermally to

pressure more than its critical pressure, exhibits enhanced solvent power in the

vicinity of its critical temperature. Such fluids are called supercritical fluids (SCF).

Figure 1.6 is an example of the phase diagram of a single substance.

39

Figure 1.6 The phase diagram of a single substance.98

A typical phase diagram for a pure substance (Figure 1.6) shows temperature and

pressure regions when the substance occurs as a single phase (solid, liquid, and gas).

Such regions are bounded by curves indicating the coexistence of two phases (solid-

gas, solid-liquid and liquid-gas), which are involved in sublimation, melting and

vaporization equilibia, respectively. The three curves intersect at the so-called triple

point (TP), where the solid, liquid and gas phases coexist in equilibrium.

The coexistence curve representing the equilibrium between two phases with a

different internal symmetry (e.g. solid-liquid or solid-gas) tend to infinity or

eventually intercepts another coexistence curve. This is not the case with the liquid-

gas equilibrium since the vapor pressure curve suddenly breaks at a point called the

40

critical point (CP), which can thus be defined as a point in the phase diagram

designated by a critical temperature (Tc) and a critical pressure (Pc) above which (a)

no liquefaction will take place on raising the pressure and (b) no gas will be formed

on increasing temperature. This latter property allows for a new definition of

supercritical fluid, a substance that is above its critical pressure and temperature.99

Increasing the temperature also increase the pressure at which the liquid and

vapor phase coexist on the vapor pressure curve. The increase in the vapor pressure is

concomitant with a decrease in the difference between the density of the liquid and

gaseous phase. At a given pressure and temperature, the density of the liquid and gas

are identical so the two phases are indistinguishable. Above such a temperature or

pressure, liquid and gas occur as a single phase. This region of pressure and

temperature above Pc and Tc is called the supercritical region. A supercritical fluid is

thus a gas which has been heated above its critical temperature and simultaneously

compressed above its critical pressure.

The critical point (C) is marked at the end of the gas-liquid equilibrium curve,

and the shaded area indicates the supercritical fluid region. It can be shown that by

using a combination of isobaric changes in temperature with isothermal changes in

pressure, it is possible to convert a pure component from a liquid to a gas (and vice

versa) via the supercritical region without incurring a phase transition. Compounds,

which have been used in their supercritical state, consist mainly of carbon dioxide,

nitrous oxide, ethane, propane, n-pentane, ammonia, fluoroform, sulphur hexafluoride

and water. The choice of the SFE solvent is similar to the regular extraction. Principle

considerations are as follow:

41

a. Good solving property

b. Inert to the product

c. Easy separation from the product

d. Cheap

Carbon dioxide is the most commonly used SCF, due primarily to its low critical

parameters (31.1°C, 73.8 bar), low cost and non-toxicity. However, several other

SCFs have been used in both commercial and development processes. The critical

properties of some commonly used SCFs are listed in Table 1.4.

Table 1.4 Features of various solvents at the critical points.100

Solvents

Critical temperature

(°C)

Critical pressure

(bar)

Critical density (g/mL)

Inorganic 1. CO2 2. N2O 3. NO2 4. Ammonia 5. Water 6. Sulphur hexafluoride 7. Helium 8. Hydrogen 9. Xenon 10. Hydrogen chloride 11. Sulphur dioxide

Hydrocarbons 12. Methane 13. Ethane 14. Propane 15. n-Butane 16. n-Pentane 17. n-Hexane 18. 2,3-Dimethylbutane

31.1 36.5 158.0 132.5 347.2 45.5

-268.0 -240.0 17.0 51.0 157.0

-82.0 32.3 96.7 152.0 196.0 234.2 226.8

72.0 70.6 98.7 109.8 214.8 38.0 2.2 12.6 56.9 83.3 76.8

46.0 47.6 42.4 70.6 32.9 28.9 42.4

0.470 0.450 0.270 0.230 0.320

- 0.070 0.030 1.110 0.450 0.520

0.169 0.200 0.220 0.228 0.320 0.230 0.241

42

Table 1.4 (Continued).

Solvents

Critical temperature

(°C)

Critical pressure

(bar)

Critical density (g/mL)

19. Ethylene 20. Propylene 21. Benzene 22. Toluene

Alcohol 23. Methanol 24. Ethanol 25. Isopropyl alcohol

Ethers 26. Diethyl ether 27. Ethyl methyl ether 28. Tetrahydrofuran

Halides 29. Trifluoromethane 30. Dichlorodifluoromethane 31. Dichlorofluoromethane 32. Chlorotrifluoromethane 33. Trichlorofluoromethane 34. 1,2-Dichlorotetrafluoroethane

Miscellaneous 35. Acetone 36. Acetonitrile 37. Pyridine

11.0 92.0 288.9 319.0

239.0 243.4 235.3

193.6 164.7 267.0

26.0 111.7 178.5 28.8 196.6 146.1

235.0 275.0

347.0

50.6 45.4 98.7 41.1

78.9 72.0 47.6

63.8 47.6 50.5

46.9

109.8 32.9

214.8 28.9 78.9

47.0 47.0

56.3

0.200 0.220 0.302 0.292

0.270 0.276 0.273

0.267 0.272 0.320

0.520 0.558 0.522 0.580 0.554 0.582

0.279 0.250

0.312

Supercritical fluid extraction (SFE) is a technique in which CO2 is used under

extremely high pressure to separate materials (e.g., removing caffeine from coffee).

This technique resembles soxhlet extraction except that the solvent used is a

supercritical fluid, substance above its critical temperature and pressure.This fluid

provides a broad range of useful properties. One main advantage of using SFE is the

43

elimination of organic solvents, thus reducing the problems of their storage and

disposal in the lipidologist laboratory.

A flow schematic of the basic SFE equipment is shown in Figure 1.7. A

supercritical-fluid extractor consists of a tank of the mobile phase, usually CO2, a

pump to pressurize the gas, an oven containing the extraction vessel, a restrictor to

maintain a high pressure in the extraction line, and a trapping vessel. Analytes are

trapped by letting the solute-containing supercritical fluid decompress into an empty

vial, through a solvent, or onto a solid sorbent material. Extractions are done in

dynamic, static, or combination modes. In a dynamic extraction, the supercritical

fluid continuously flows through the sample in the extraction vessel and out the

restrictor to the trapping vessel. In static mode the supercritical fluid circulates in a

loop containing the extraction vessel for some period of time before being released

through the restrictor to the trapping vessel. In the combination mode, a static

extraction is performed for some period of time, followed by a dynamic extraction.

44

Figure 1.7 Schematic diagram of supercritical fluid extraction system.

The off-line mode is preferred when a deep knowledge of the features of the

extraction process and the wide variety of variables that affect supercritical fluid

extraction (SFE) can readily be altered for specific purpose (e.g. achieving rapid,

selective, efficient or precise extraction) by optimizing the process for one or more

parameters. The most influential variables on extraction quality parameters are as

follows:

1) The supercritical fluid used as extractant, which can be chosen from among a

large variety of options, used at variable pressures and temperatures (i.e. densities), as

well as flow-rates, and modified with suitable agents in order to alter its polarity, all

of which determine its solvent properties.

45

2) The extraction time, which once the extractant is chosen, allows such

parameters as throughput and efficiency to be alter at will.

3) The particle size, amount of sample, pore size and unknown material

thickness, on which efficient extraction in a reasonably short time relies heavily. The

size of heterogeneous samples can have a decisive effect on reproducibility.

4) The nature of the matrix, which influences the selectivity, quantitativeness and

analyte separation rate achieved in the extraction process through matrix-analyte

interactions.

5) The volume and dimensions of the extraction chamber and hence the time

required to achieve a preset efficiency.

The properties of supercritical fluids also provide some advantages for analytical

extractions. Supercritical fluids can have solvating powers similar to organic solvents,

but with higher diffusivities, lower viscosity, and lower surface tension. The solvating

power can be adjusted by changing the pressure or temperature, or by adding

modifiers to the supercritical fluid. A common modifier is methanol (typically 1-10%)

which increases the polarity of supercritical CO2. In the field of SFE, various

researchers proposed the use of SC-CO2 in order to separate carotenoids, polyphenols,

and vitamin E in rice as well as other cereals as shown by the studies below.

In 1996, Garcia and co-workers101 performed the separation of waxes and long

chain fatty acids from rice bran at 280 bar, with the temperature varying from 40 to

70°C. When comparing to hexane extraction, the total yield was up to 50% lower

under these conditions.

Shen and co-workers102 studied the effects of temperature, pressure and solvent

flow rates on the fractionation of rice bran oil (RBO) and showed oryzanol and δ-

46

tocopherol concentrations in raffinate were not reduced by fractionation, but the sterol

concentration was reduced under conditions favoring free fatty acid (FFA) removal.

The fractionation reduced the free fatty acid (FFA) concentration in raffinate by up to

50%.

Schneiderman and co-workers103 extracted vitamin A palmitate (retinol

hexadecanoate, retinol palmitate) from cereal products using supercritical CO2 at 55

MPa and 60°C. Quantitative extraction required only 20 min. Retinol palmitate in the

extract was determined by reversed-phase liquid chromatography (LC) using an

oxidative mode electrochemical detector. The LC run time was 12 min. The detection

limit was 0.17 ng for a 20 μl injection, and response was linear over at least three

orders of magnitude. For corn, wheat and oat cereals fortified with retinol palmitate at

four levels in the 10-125 μg/g range, the overall average recovery was 95% with an

overall R.S.D. of 5%. For a wheat sample spiked at three levels in the 25-100 μg/g

level, the within-day average recovery was 101% with 3% R.S.D., and the between-

day recovery was 100% with 6% R.S.D. Sample of lipid in the form of added soybean

oil had no adverse effect on the extraction or the LC analysis.

In 1999, Kim and co-workers104 studied the use of SC-CO2 to enrich the rice bran

oil in essential fatty acids; palmitic acid, linolenic acid, linoleic acid, oleic acid,

stearic acid, tocopherol, squalene, etc. The oil rich essential fatty acid (EFA) was

extracted from the domestic brown rice bran using supercritical carbon dioxide

(SCC), and the extracts were analyzed with gas chromatography-mass spectrometry

(GC-MS). The extracted amount of rice bran oil was dependent upon the operating

pressure and temperature, and the fatty acid composition of oil was varied with the

reduced density of the SCC. About 70–80% of rice bran oil was extracted in 4 hours.

47

Especially, squalene which was not found in solvent extract phase, it was identified in

supercritical fluid extraction (SFE) phase only.

In 2000, Xu and Godber105 studied the advantage of SC-CO2 extracted γ-oryzanol

from rice bran in comparison with other organic extracting solvents. The experiments

were carried out at 680 bar and the results showed that the amount of γ-oryzanol

presented in the supercritical fluid extraction (SFE) extract was up to 80 times higher

than the amount obtained through hexane extraction.

Dunford and King106 studied the use of a fractionation column and SC-CO2 for

selective enrichment in lipid and sterol fractions of rice bran oil (RBO). They found

that the phytosterol content, specially oryzanol content, of deacidified RBO was about

three times higher than that found in a commercially available high-oryzanol of RBO.

In 2001, Dunford and King107 compared the effects of an isothermal or thermal

gradient supercritical fractionation column operation on the crude of rice bran oil

(RBO) deacidification efficiency and examined the effect of CO2 flow rate and

fractionation time on the extract and raffinate compositions. The essential fatty acid

(EFA) concentrations varied from 26.9 to 52.0% (extract) and 4.4 to 5.4% (raffinate).

The crude rice bran oil (RBO) contained 7% essential fatty acid (HPLC area) and the

experiments were carried out at 205 bar and at temperatures varying from 45 to 95°C

for 180 min.

In 2003, Dunford and co-workers108 examined the potential of a continuous

countercurrent supercritical carbon dioxide fractionation technique for deacidification

of crude rice bran oil. A pilot scale packed column was utilized for the experiments. It

was shown that fractionation at low pressure, 138 Bar, and high temperature, 80°C,

effectively removed free fatty acids from crude rice bran oil without any oryzanol loss

48

in the extracted fraction. Oryzanol content of the raffinate fraction was three times

higher than that of the feed material. Phytosterol fatty acid ester content of the

raffinate fraction was also increased during the deacidification process, however the

enrichment of these moieties was not as high as that found for oryzanols.

Perretti and co-workers109 studied the use of SFE for recovery of rice processing

byproducts, such as hulls, rice bran, broken and discoloured rice grains and

developmental research in novel conversion processes to manufacture value-added

food products. Conditions were studied to extract oil from products and by-products

of rice processing chain, and to increase the concentration of antioxidants

(tocochromanols and oryzanols) in oil. High pressure and temperature, compatible

with natural products, enable high yield and efficacious CO2 usage. The extraction

conducted at 10,000 psi and 80°C gave the highest extraction yield, and the initial

analyses indicated that the oil quality is as suitable for human consumption as the

traditionally extracted one. By-products may be valuable sources of antioxidants, and

preliminary results indicate that it is possible to improve extraction conditions for

their enrichment.

In 2004, Imsanguana and co-workers110 compared the efficiency of three

extraction methods; supercritical carbondioxide extraction (SC-CO2), solvent

extraction and soxhlet extraction for extraction of α-tocopherol and γ-oryzanol from

rice bran. The results showed that none of the solvents could extract α-tocopherol.

However, ethanol was suitable for γ-oryzanol extraction. In summary, SC-CO2 was

found to be the best solvent for extracting both α-tocopherols and γ-oryzanols from

rice bran, because it provided higher yields and extraction rate.

49

In 2005, Danielski and co-workers111 performed the supercritical fluid extraction

(SFE) of rice bran with CO2 at different operational conditions (from 100 to 400 bar,

50 and 60 °C) and the extract yields were in the range of 20%. The next step

corresponded to the deacidification of the obtained oil in a countercurrent (CC)

column, where the experiments were carried out at 250 bar and 67 °C. The results

have shown that the free fatty acids (FFA) removal from the crude rice bran oil

(RBO) was successfully achieved. Deacidified RBO with <1% FFA could be obtained

by applying the described process.

1.4 High-Performance Liquid Chromatography (HPLC)

High-performance liquid chromatography (HPLC) is unquestionably the most

widely used of all of the analytical separations. The reasons for the popularity of the

method are its sensitivity, its ready adaptability to accurate quantitative determination,

its suitability for separating nonvolatile species or thermally fragile ones and its

widespreaded applicability to substances that are of prime interest to industry.

1.4.1 Normal Phase Chromatography

Normal phase HPLC (NP-HPLC) was the first kind of HPLC setup used, and

retains analyte based on polarity. This method uses a polar stationary phase and a

non-polar mobile phase, and is used when the analyte of interest has a polar nature.

The polar analyte associates with and is retained by the polar stationary phase.

Adsorption strengths increase with increased analyte polarity, and the interaction

between the polar analyte and the polar stationary phase (relative to the mobile phase)

increases the elution time. The interaction strength not only depends on the functional

50

groups in the analyte molecule, but also on steric factors. The affect of sterics on

interaction strength allows this method to resolve or separate structural isomers. Use

of more polar solvents in the mobile phase will decrease the retention time of the

analytes while more hydrophobic solvents tend to increase retention times. Very polar

solvents in a mixture tend to deactivate the column by occupying the stationary phase

surface. This is somewhat particular to normal phase because it is most purely an

adsorptive mechanism as the interactions are with a hard surface rather than a soft

layer on a surface.

NP-HPLC had fallen out of favor in the 1970's with the development of

reversed-phase HPLC because of a lack of reproducibility of retention times as water

or protic organic solvents changed the hydration state of the silica or alumina

chromatographic media. Recently it has become useful again with the development of

hydrophillic interaction liquid chromatography (HILC) bonded phases which utilize a

partition mechanism which provides reproducibility.

1.4.2 Reversed Phase Chromatography

The reversed phase HPLC (RP-HPLC) consists of a nonpolar stationary phase

and a polar mobile phase, and was developed due to the increasing interest in large

nonpolar biomolecules. One common stationary phase is a silica which has been

treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or

C8H17. The retention time is therefore longer for molecules which are more non-polar

in nature, allowing polar molecules to elute more readily. Increasing of retention time

can be done by adding a polar solvent to the mobile phase, or decrease retention time

by adding a more hydrophobic solvent. Reversed phase chromatography is so

51

commonly used that it is not uncommon for it to be incorrectly referred to as "HPLC"

without further specification. Structural properties of the analyte molecule play an

important role in its retention characteristics. In general, an analyte with a larger

hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as

S-S and others) results in a longer retention time because it increases the molecule's

non-polar surface area, which is non-interacting with the water structure. On the other

hand, polar groups, such as -OH, -NH2, COO- or -NH3+ reduce retention as they are

well integrated into water. Very large molecules, however, can result in an incomplete

interaction between the large analyte surface and the ligands alkyl chains and can

have problems entering the pores of the stationary phase. Presently, RP-HPLC is the

most popular mode of liquid chromatography for determining phenolic compounds

and other natural products in plant extracts.

1.4.3 HPLC Instrumentation

High-performance liquid chromatography (HPLC) is a form of liquid

chromatography to separate compounds that are dissolved in solution. HPLC

instruments consist of a reservoir of mobile phase, a pump, an injector, a separation

column, and a detector. Compounds are separated by injecting a plug of the sample

mixture onto the column. The different components in the mixture pass through the

column at different rates due to differences in their partitioning behavior between the

mobile liquid phase and the stationary phase. Solvents must be degassed to eliminate

formation of bubbles. The pumps provide a steady high pressure with no pulsating,

and can be programmed to vary the composition of the solvent during the course of

the separation. Detectors rely on a change in refractive index, UV-Vis absorption, or

52

fluorescence after excitation with a suitable wavelength.112 Diagram of high

performance liquid chromatography is shown in Figure 1.8.

Figure 1.8 Diagram of high performance liquid chromatography (HPLC).113

1.4.4 Detectors for HPLC

The function of the detector in HPLC is to monitor the column effluent and

afford a means to many different principles but add output an electrical signal which

is proportional to some property of the analyte. The choice of detedtor is often

dictated by the chemical characteristics of the analyte species and this choice may

subsequently determine which eluent is used and also possibly which stationary phase

and mode of chromatography. The detector response will be related to the amount of

the analyte in the column effluent though different analytes will respond to differing

extents and hence the detector must be calibrated with respect to each of the analytical

species of interest.

53

1.4.4.1 UV-VIS Detector

UV-Visible absorption detectors are the most widely used detectors in liquid

chromatography. As most organic compounds have some useful absorption in the UV

region, these detectors are fairly universal in application, however, in practice, since

absorption maxima can differ greatly between compounds, wavelengths are set for the

best overall detection of all components. The operation of spectrophotometric

detectors is based on the measurement of the absorbance according to the well-known

Beer-Lambert law. Most detectors provide an output in absorbance units which is

linearly related to sample concentration over a rang of 104 to 105. Detection limits are

low to subnanogram range in favorable circumstances.114

1.4.4.2 Diode Array Detector

A diode array consists of a number of photosensitive diodes place side by side

and insulated from one another in the form of a multi-layer sandwich. Each diode may

be only a few thousands of an inch thick and the output from each diode can be

scanned, stored and subsequently processed by a computer in a number of different

ways. The common use of a diode array is to monitor light that has passed through a

liquid sensor cell as in a multi-wavelength liquid chromatography detector. The light

source is usually polychromatic (e.g. light from a deuterium lamp) and after passing

through the cell, the light is dispersed by a quartz prism or a diffraction grating onto

the surface of the diode array. Thus, each diode will receive light of a slightly

different wavelength to that received by its neighbor. Those wavelengths most useful

in liquid chromatography range from about 210 nm to 330 nm (i.e. UV light) and,

thus, a sufficient number of diodes must be incorporated in the array to (at least) cover

54

this range of wavelengths. Many organic compounds have characteristic spectra in the

UV which can be used to help identifying the substance passing though the sensor

cell. Thus, when a given substance is eluted through the sensor cell, all the outputs

from the array can be acquired and the result used to construct an absorption spectra

that can be compared with standard spectra for identification purposes. Alternatively,

by selecting the appropriate diode, the wavelength of the light at which there is

maximum absorption can be selectively monitored to provide maximum detector

sensitivity for that substance.115

1.4.5 Liquid Chromatography-Mass Spectrometry (LC-MS)

Mass spectrometry (MS) is a microanalytical technique requiring only a few

picomoles of sample to obtain characteristic information regarding the molecular

weight and sometimes the structure of the analyte. In all cases, energy is transferred

to the analyte molecules to affect ionization. In the classical technique of electron

ionization, some of the molecular ions of the analyte explode into a variety of

fragment ions. The resulting fragmentation pattern together with residual molecular

ions constitutes the mass spectrum. In principle, the mass spectrum of each compound

is unique and can be used as a chemical “fingerprint” to characterize the analyte.

Mass spectrometry is one of the physico-chemical methods applied to the structural

determination of organic compounds. The high sensitivity and possibilities of

hyphenation with chromatographic techniques sets MS among the most appropriate

physico- chemical methods for study of natural products from biological materials.

The characteristic features of MS is the use of different physical principles, both for

sample ionization and separation of the ions.

55

The combination of the electrospray (ES) ion source with HPLC has without a

doubt become the LC-MS interface in recent year. It is a particularly powerful

combination, since this ionization technique covers a wide range of samples that are

commonly separated by HPLC. In ES, ionization takes place at atmospheric, but the

technique differs significantly from APCI in that nebulization and ionization of the

mobile phase is effected by an electric field applied to the end of a restricted inlet

nozzle. In two variations on this interface, nebulization is assisted by a stream of

nitrogen introduced coaxially with the mobile phase (commonly called ion spray) or

by ultrasonication. A major advantage to ES is its ability to form multiply charged

ions that have high masses but low mass-to-charge (m/z) values and can be detected

using inexpensive quadrupole (low-mass-range) mass analyzers.

All ions in an ionization chamber are to be analyzed according to mass-to-charge

ratio (m/z). Ions have an electrical charge that permits them to be controlled by

various electrical fields. They are separated by their m/z values in a mass analyzer.

There are several types of mass analyzer: magnetic, transmission quadrupole,

quadrupole ion trap, magnetic ion trap, or time-of-flight analyzer. Ions are analyzed

according to their abundance along an m/z scale.

In mass spectrometry, a substance is bombarded with an electron beam having

sufficient energy to fragment the molecule. The positive fragments which are

produced (cations and radical cations) are accelerated in a vacuum through a magnetic

field and are sorted on the basis of mass-to-charge ratio. Since the bulk of the ions

produced in the mass spectrometer carry a unit positive charge, the value m/z is

equivalent to the molecular weight of the fragment. The analysis of mass

spectroscopy information involves the re-assembling of fragments, working

56

backwards to generate the original molecule.116 A schematic representation of a mass

spectrometer is shown in Figure 1.9.

Figure 1.9 A schematic of a mass spectrometer.117

Quadrupole Mass Spectrometer

The quadrupole mass analyzer is a "mass filter". Combined direct-current (DC)

and radiofrequency (RF) potentials on the quadrupole rods can be set to pass only a

selected mass-to-charge ratio. All other ions do not have a stable trajectory through

the quadrupole mass analyzer and will collide with the quadrupole rods, never

reaching the detector. A crude schematic of a quadrupole mass filter is shown in

Figure 1.10.

57

Figure 1.10 Schematic diagram showing arrangement of quadrupole rods.118

Ions are extracted from the ion source and are accelerated (5-15 V) into the

central space that constitutes the quadrupole field along the longitudinal axis toward

the detector. Miller and Denton118 have described the concept of a quadrupole mass

filter as the combination or overlap of a low-pass and a high-pass filter. At least for

relatively light ions (i.e. < 300 Da), m/z analysis is not affected by structure of the ion.

The quadrupole m/z filter is scanned by ramping the magnitude of RF amplitude and

DC voltages at a fixed ratio. The resolving power of the instrument is established by

the ratio of the RF to DC voltage.

Time-of-Flight Mass Spectrometer

A time-of-flight (TOF) mass spectrometer119 involes measuring the time required

for an ion to travel from an ion source to a detector located 1-2 m from the source. All

the ion receive the same kinetic energy during instantaneous acceleration (e.g. 3000

eV), but because they may have different m/z values, they separate into groups

according to velocity (and hence m/z) as they travers the field-free region between the

58

ion source and detector. The ions sequentially strike the detector in order of increasing

m/z values, creating a time-based waveform, or simply a transient. Ion of low m/z

reach the detector before those of high m/z because the later have a lower velocity, as

indicated schematically in Figure 1.11.

Figure 1.11 Schematic diagramof a tim-of-flight (TOF) mass spectrometer.120

A very low concentration of sample molecules is allowed to leak into the

ionization chamber (which is under a very high vacuum) where they are bombarded

by a high-energy electron beam. The molecules fragment and the positive ions

produced are accelerated through a charged array into an analyzing tube. The path of

the charged molecules is bent by an applied magnetic field. Ions having low mass

(low momentum) will be deflected most by this field and will collide with the walls of

the analyzer. Likewise, high momentum ions will not be deflected enough and will

also collide with the analyzer wall. Ions having the proper mass-to-charge ratio,

however, will follow the path of the analyzer, exit through the slit and collide with the

59

collector. This generates an electric current, which is then amplified and detected. By

varying the strength of the magnetic field, the mass-to-charge ratio which is analyzed

can be continuously varied. The output of the mass spectrometer shows a plot of

relative intensity vs the mass-to-charge ratio (m/z). The most intense peak in the

spectrum is termed the base peak and all others are reported relative to it's intensity.

The peaks themselves are typically very sharp, and are often simply represented as

vertical lines.

HPLC-MS Interfaces

High performance liquid chromatography-mass spectrometry (HPLC-MS) is an

extemely versatile instrumental technique whose roots lie in the application of more

traditional liquid chromatography to theories and instrumentation that were originally

developed for gas chromatography (GC). As the name suggest the instrumentation

comprises a high performance liquid chromatograph (HPLC) attached, via a suitable

interface, to a mass spectrometer (MS). The primary advantage HPLC-MS has over

GC-MS is that it is capable of analysing a much wider range of components.

Compounds that are thermally labile, exhibit high polarity or have a high molecular

mass may all be analysed using HPLC-MS, even proteins may be routinely analysed.

Solutions derived from samples of interest are injected onto an HPLC column that

comprises a narrow stainless steel tube (usually 150 mm length and 2 mm internal

diameter, or smaller) packed with fine, chemically modified silica particles.

Compounds are separated on the basis of their relative interaction with the chemical

coating of these particles (stationary phase) and the solvent eluting througn the

column (mobile phase). Components eluting from the chromatographic column are

60

then introduced to the mass spectrometer via a specialised interface. The direct inlet,

moving-belt (MB), particle-beam (PB), thermospray, continuous-flow fast atom

bombardment (FAB), and atmospheric pressure ionization interfaces have been used

for LC-MS combination. Among the various LC-MS interfaces designed for

achieving solute enrichment, MB and PB have been the most successful systems.

With the two most common interfaces used for HPLC-MS are the electrospray

ionisation and the atmospheric pressure chemical ionisation interfaces.

Atmospheric Pressure Ionization

Two different sample introduction approaches are used in combination with

atmospheric pressure ionization (API) devices (Figure 1.12 (A) and (B)). They

primarily differ in the nebulization principle and in the application range they cover.

In a heated nebulizer or APCI interface, the column effluent is pneumatically

nebulized into a heated (quartz or stainless steel) tube, where the solvent evaporation

is almost completed.121

A

61

Figure 1.12 Schematic diagram of the Agilent Technologies atmospheric-pressure

ionization electrospray ion source (A) orthogonal nebulizer and (B) main

components.122

Atmospheric pressure chemical ionisation (APCI) is an analogous ionisation

method to chemical ionisation (CI). The significant difference is that APCI occurs at

atmospheric pressure and has its primary applications in the areas of ionisation of low

mass compounds, it is not suitable for the analysis of thermally labile compounds. In

APCI (Figure 1.13), the ionisation process initiated by electrons from a corona

discharge needle, is achieved in the same region. Subsequently, the ions generated are

sampled into the high vacuum of the mass spectrometer for mass analysis. In an

electrospray interface, the column effluent is nebulized into the atmospheric-pressure

region as a result of the action of a high electric field resulting from a 3 kV potential

difference between the narrow-bore spray capillary breaks into fine threads which

subsequently disintegrate in small droplets.

B

62

Figure 1.13 Schematic diagram of APCI ionisation process.123

Electrospray ionisation

An electrospray ionisation (ESI) is generally accomplished by forcing a solution

of the analyte through a small biased capillary such the fluid sprays into very fine

droplets. Effective spraying action and often the ionisation itself require the presence

of a high electric field. The electric field is usually imposed between the tip of the

spraying capillary and a cylindrical electrod, as indicated in Figure 1.14. The imposed

electric field is important for several reasons, one being that it keeps the droplets from

freezing, during endothermic loss of solvent by evaporation, by causing the charged

droplet to endure many collisions through which some translational energy is

converted to internal energy, thereby warming the droplet.124

63

Figure 1.14 A schematic of an ESI interface.125

In electrospray ionisation the analyte is introduced to the source at flow

rates typically of the order of 1µl min-1. The analyte solution flow passes through the

electrospray needle that has a high potential difference (with respect to the counter

electrode) applied to it (typically in the range from 2.5 to 4 kV). This forces the

spraying of charged droplets from the needle with a surface charge of the same

polarity to the charge on the needle. The droplets are repelled from the needle towards

the source sampling cone on the counter electrode. As the droplets traverse the space

between the needle tip and the cone, solvent evaporation occurs. This is enlarged

upon in Figure 1.15. As the solvent evaporation occurs, the droplet shrinks until it

reaches the point that the surface tension can no longer sustain the charge at which

point a "Coulombic explosion" occurs and the droplet is ripped apart. This produces

smaller droplets that can repeat the process as well as naked charged analyte

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molecules. These charged analyte molecules, not strictly ions, can be singly or

multiply charged. This is a very soft method of ionisation as very little residual energy

is retained by the analyte upon ionisation.

Figure 1.15 A schematic of the mechanism of ion formation in electrospray

ionization.126

The development and application of ESI to mass spectrometry are important for

a number of reasons:

(a) ESI is a means of production of ions from nonvolatile compounds.

(b) ESI can produce multiply charged ions, the mass-to-charge (m/z) value of

ions of macromolecules may fall within the mass range of most commonly used mass

spectrometer.

(c) Because of the redundant assessment of the mass to the intact molecule

through detection of electrosprayed ions differing in charge, it is possible to determine

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the molecular weight of the analyte to 1 part in 10,000 or better, depending on the

type of mass analyzer.

(d) ESI serves as one of the most effective and successful interface for liquid

chromatography-mass spectrometry (LC-MS) that has been developed.

(e) ESI is a soft ionization technique that permits investigations of noncovalent

associations of macromolecules such as proteins.

(f) ESI allows direct analysis of inorganic cations and anions, providing

information on valence state and molecular formulation.

1.4.6 Mass spectrometry-Mass spectrometry (MS/MS)

A tandem mass spectrometer (MS/MS) is one of several types of analytical

instruments known as mass spectrometers. It is perhaps the most significant advance

in newborn screening in the past 30 years. A tandem mass spectrometer is a

specialized instrument that detects molecules by measuring their weight (mass). Mass

spectrometers measure weight electronically and display results in the form of a mass

spectrum. A mass spectrum is a graph that shows each specific molecule by weight

and how much of each molecule is present. A tandem mass spectrometer can be

thought of as two mass spectrometers in series connected by a chamber that can break

a molecule into pieces perhaps like a puzzle. This chamber is known as a collision

cell. A sample is “sorted” and “weighed” in the first mass spectrometer, then broken

into pieces in the collision cell, and a piece or pieces sorted and weighed in the second

mass spectrometer. A tandem mass spectrometer is often abbreviated as “Tandem”

MS or “MS/MS”.127

In the "classical" ionisation methods for mass spectrometry, like electron impack

ionization (EI) and chemical ionization (CI), spectra usually contain a good amount of

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fragment ions that can be used to help confirm or elucidate chemical structures. In the

more modern methods of ionisation, like ESI or MALDI, spectra often only contain

the ionised molecule with very little fragmentation data and consequently the spectra

are of little use for structural characterisation. In these cases, induced fragmentation is

required using collision induced dissociation (CID) and tandem mass spectrometry

(MS/MS). One of the most commonly available tandem mass spectrometers is the

triple quadrupole (QQQ) instrument. Schematic diagram of an MS/MS is shown in

Figure 1.16.

Figure 1.16 Schematic diagram of a tandem mass spectrometer (MS/MS).128

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The Q-Tof is a tandem mass spectrometer (MS/MS) with two analysers: the first

being a quadrupole analyser that is used as an ion guide in MS mode, but as a

resolving analyser in MS/MS mode. The second analyser is a reflectron time-of-flight

analyser placed orthogonally to the quadrupole. The final detector is a microchannel

plate detector for high sensitivity. The accuracy and reproducibility of the time-of-

flight analyzer enable accurate mass measurements to be carried out with small

organic molecules and the excellent resolution makes charge state identification

routine. In MS/MS mode, the two analysers are used together for structural studies by

monitoring fragmentation patterns in molecules. The fragmentation patterns of

peptides are particularly well documented and MS/MS is used extensively in protein

chemistry for this purpose.

Numerous methods of HPLC and HPLC-MS as well as HPLC-MS/MS had been

employed for determining carotenoids and their analogues in foods as reported in the

literature. Some of the examples recently published are shown in the following

papers.

In 2000, Kamal-Eldin and co-workers129 were using normal-phase HPLC of

tocopherols and tocotrienols comparison of different chromatographic columns.

Normal-phase HPLC separations of vitamin E compounds in a prepared mixture

(containing oat extracts, palm oil and tocopherol standards) were tried on six silica,

three amino and one diol columns. As shown by calculations of retention factors (k),

separation factors (a), numbers of theoretical plates (N) and resolutions (R ), the best

separations were obtained on three silica columns and two amino columns using 4 or

5% dioxane in hexane as the mobile phase as well as on a diol column using 4% tert .-

butyl methyl ether in hexane as the mobile phase.

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In 2002, Susanne and Volker130 were using HPLC determination of carotenoids

from cereals with Special Reference to Durum Wheat (Triticum durum Desf.). To

optimize the extraction procedure, several factors with influence on extractability of

carotenoids were investigated. Finally, it was shown that soaking of samples in water

for 5 min prior to extraction with organic solvents had the strongest impact on

extraction yield and led to the most rapid and gentle method. Contents of carotenoids

in the extracts of several durum wheat and corn samples were doubled by soaking in

water before extracting with methanol/tetrahydrofuran (1/1, vol./vol.). In light of

these findings, literature data on contents of carotenoids in cereal grains have to be

viewed critically regarding the extraction procedures employed.

In 2004, Ryynanen and co-workers131 optimized small-scale sample preparation

method including hot saponification in combination with a normal-phase high-

performance liquid chromatography with fluorescence detection NP-HPLC-FLD

procedure was shown to be reliable for the determination of tocopherols and

tocotrienols from cereals. Three critical factors were optimized for hot saponification:

time, temperature and amount of potassium hydroxide (KOH). Saponification under

carefully controlled conditions to avoid degradation of the vitamers was shown to be

an effective and sufficiently sensitive method for tocopherol and tocotrienol assay

from cereals. Using hot saponification of 25 min, the time needed for sample

preparation could significantly be reduced, and by scaling down the sample size to 0.5

g, the amounts of solvents needed were also reduced. Polar modification of n-hexane

with 20% ethyl acetate improved the extraction efficacy of the vitamers from the

saponification mixture. With the optimized method, total tocopherol and tocotrienol

content of rye flour was 27.8±0.1 mg/g, while those with cold saponification and

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direct extraction with hot 2-propanol and hexane were lower being 24.5±1.0 and

24.1±0.8 mg/g, respectively. Thus with an optimized hot saponification method,

higher amounts of tocopherols and tocotrienols were obtained than with the other

methods studied. The NP-HPLC-FLD method was verified to be sensitive and reliable

for the analysis of eight vitamers of tocopherols and tocotrienols. Finally, the

repeatability and accuracy of the optimized procedure was confirmed by analysing rye

flour and its applicability by analysing ten rye varieties for tocopherols and

tocotrienols. This study showed that rye grains possess a beneficial ratio of

tocotrienols to tocopherols as well as high amounts of tocopherols and tocotrienols,

although with evident variation between varieties.

Tian and co-workes132 determined 6-O-feruloylsucrose, 6-O-sinapoylsucrose,

ferulic acid, sinapinic acid, p-coumaric acid, chlorogenic (3-caffeoylquinic) acid,

caffeic acid, protocatechuic acid, hydroxybenzoic acid, vanillic acid, and syringic acid

in rice. The rice samples were extracted with 70% ethanol, filtered, and defatted. The

defatted aqueous solution was subjected to solid-phase extraction using a C18 silica

gel cartridge; no analyte was lost in this procedure. The 70% acidic methanol elution

was analyzed directly by HPLC and HPLC-ESI-MS (electrospray ionization mass).

Phenolic compounds were separated with a C18 reversed-phase column by gradient

elution using 0.025% trifluoroacetic acid in purified water (A) and acetonitrile (B) as

the mobile phase at a flow rate of 0.8 mL/min. Detection limits ranged from 0.10 to

0.35 ng per injection (5 μL). Relative standard deviations of 0.22–3.95% and

recoveries of 99–108% were obtained for simultaneous determination of these

phenolic compounds. The major soluble phenolic compounds in rice including 6′-O-

Feruloylsucrose and 6′-O-sinapoylsucrose. There were significant decreases (P < 0.01

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or P < 0.05) during germination for 24 h, while the levels of free ferulic acid and

sinapinic acid increased significantly (P < 0.01). In addition, the content of phenolic

compounds in the water used for soaking the rice was determined and revealed that

the decrease in 6′-O-feruloylsucrose and 6′-O-sinapoylsucrose during germination

was not due to loss in the soaking water but was probably caused by hydrolysis. The

increases in ferulic acid and sinapinic acid, both in the germinated rice and in the

soaking water at 12 and 24 h verify that germination causes metabolism of phenolic

compounds, especially as the bud appears. The two hydroxycinnamate glycosides

may participate in this metabolic conversion.

In 2005, Stoeggl and co-workers133 studied to establish methods and to compare

C18 and C30 silica stationary phases in order to seperate and detect tocopherols,

carotenoids, and γ-oryzanol in in crude rice bran oil. Comparing RP-LC on silica C18

and C30, higher resolution between all target compoundds was obtained using the

C30 stationary phase. Methanol was used as eluent and the elution strength was

increased by the addition of tert-buthylmethyl ether for highly hydrophobic analytes

such as γ-oryzanol. Detection was accomplished by diode array detection from 200 to

500 nm. Absorbance maxima were found at 295 nm for tocopherols, 324 nm for γ-

oryzanol, and 450 nm for carotenoids. Furthermore, compounds were characterized

and identified on the basis of their UV spectra. Both RP systems were coupled to MS

(LC-MS) by using an atmospher pressure chemical ionization interface.

Tian and co-workers134 isolated two hydroxycinnamate sucrose esters, 6-O-(E)-

feruloyl sucrose and 6-O-(E)-sinapoylsucrose from methanol extracts of rice bran.

Soluble and insoluble phenolic compounds as well as 6-O-(E)-feruloylsucrose and 6-

O-(E)-sinapoylsucrose from white rice, brown rice, and germinated brown rice were

71

analyzed using HPLC. The results demonstrated that the content of insoluble phenolic

compounds was significantly higher than that of soluble phenolics in rice, whereas

almost all compounds identified in germinated brown rice and brown rice were more

abundant than those in white rice. 6-O-(E)-Feruloylsucrose (1.09 mg/100 g of flour)

and 6-O-(E)-sinapoylsucrose (0.41 mg/100 g of flour) were found to be the major

soluble phenolic compounds in brown rice. During germination, the 70% decrease

was observed in the content of the two hydroxycinnamate sucrose esters, whereas free

phenolic acid content increased significantly. The ferulic acid content of brown rice

(0.32 mg/100 g of flour) increased to 0.48 mg/100 g of flour and became the most

abundant phenolic compound in germinated brown rice. The content of sinapinic acid

increased to 0.21 mg/100 g of flour, which was nearly 10 times as much as that in

brown rice (0.02 mg/100 g of flour). In addition, the total content of insoluble

phenolic compounds increased from 18.47 mg/100 g of flour in brown rice to 24.78

mg/100 g of flour in germinated brown rice. These data suggested that appropriate

germination of brown rice may be a method to improve health-related benefits.

Chen and co-workers135 developed and evaluated a rapid and relatively

inexpensive procedure for extracting tocopherols, tocotrienols, and g-oryzanol from

rice bran, and to quantify these compounds directly from the extract using reversed-

phase (RP)-HPLC. The one-min equilibrium extraction at a 1:60 (w/v) ratio of rice

bran to methanol recovered 92 to 102% of the target phytochemicals relative to those

of repeated, nonsaponified, direct solvent extraction methods. At this 1:60 ratio of

bran to solvent, isopropanol and methanol are superior extraction solvents relative to

hexane. A modified, mobile-phase gradient with 10% of aqueous phase for the first 3

min liminates all the methanol-soluble interfering compounds. This extraction method

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has the following advantages over the currently available methods: speed, no special

extraction instrumentation is needed; and the extraction solvent, methanol, is

compatible with subsequent quantification via RP-HPLC.

In 2006, Judprasong and co-workers136 determined soluble and total oxalate

contents in common vegetables, cereal grains and legume seeds and the effect of

household cooking on these substances. Each food sample was randomly purchased

from three main representative markets in metropolitan area of Bangkok, Thailand.

Oxalate content in various foods was determined by HPLC method. The

chromatographic separation was carried out on a 300 × 7.8 mm Biorad Aminex ion

exclusion column (HPX-87H), using an isocratic elution at 0.5 mL/min with 0.0125

M sulphuric acid as a mobile phase. UV detector was set at 210 nm. The amount of

oxalic acid in each sample was determined against a standard calibration curve of

oxalic acid (100–500 mg/mL) and expressed as mg oxalate in 100 g sample. The limit

of quantitation for the oxalates was 3 mg/100 g. All studied vegetables contained

relatively small amounts of total oxalate (< 100 mg/100 g), except chinese

convolvulus (Lpomoea reptans), acacia pennata (Acacia pennata), and cultivated

bamboo shoot (Bambusa spp.), contained total oxalate more than 150 mg/100 g which

can be significantly reduced after cooking by boiling. Among the legume seeds,

soybeans (Glycine max (L.) Merrill) and peanuts (Arachis hypogaea L.) contained

highest and moderate amounts of total oxalate, 204714mg and 142735 mg/100 g,

respectively. Rice contained negligible amount of total oxalate (< 3 mg/100 g). There

was significant reduction (P < 0:05) in total oxalate due to cooking by boiling,

percentage loss ranged form 18% in coconut heart top stems (Cocos nucifera Linn.) to

76% in A. pennata. Similar findings appeared in soluble oxalate, significant loss (P <

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0:05) ranged from 30% in cooked white stems swamp morning glory (Lpomoea

aquatica, Forsk) to 83% in cooked cultivated bamboo shoot (Bambusa spp.). Loss of

oxalates in various foods is likely due to their leaching loss in cooking water.

In 2007, Yu and co-workers137 were identification and quantification of the

vitamin E and γ-oryzanol components in rice bran and germ.Vitamin E and γ-oryzanol

components in rice bran and germ were analyzed by liquid chromatography and mass

spectrometry/mass spectrometry. The components were identified by electrospray

ionization mass spectrometry (ESI-MS) with both positive and negative ion modes.

Both deprotonated molecular ion [M - H]- and protonated molecular ion [M + H]+

found as the base peaks in spectra of vitamin E components made ESI-MS a valuable

analytic method in detecting vitamin E compounds, especially when they were at very

low levels in samples. UV absorption was used for quantification of vitamin E and γ-

oryzanol components. While the level of vitamin E in rice germ was 5 times greater

than in rice bran, the level of γ-oryzanol in rice germ was 5 times lower than in rice

bran. Also, the major vitamin E component was γ-tocopherol in rice germ and γ-

tocotrienol in rice bran. These data suggest that rice bran and germ have significantly

different profiles of vitamin E and γ-oryzanol components.

Devi and Renuka138 studied characterization of defatted rice bran (DRB)

employing HPLC for identifying the major phytochemicals in DRB and to examine its

commercial potential as a source of bioactive phytochemicals leading to value

addition of DRB otherwise used as cattle feed. Various solvent extracts showed the

presence of oryzanols, tocols, and ferulic acid. Methanol was the most effective

extractant under the optimized conditions of a material–solvent ratio of 1:15 (wt./vol.)

and a time of extraction of 10 h. The yields of total phenols, oryzanols and ferulic acid

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from DRB with methanol were 2204, 316, and 233 ppm, respectively. Enrichment of

antioxidants in the crude methanolic extract (CME) was achieved by sequential

extraction and fractionation, resulting in three enriched fractions, acetone extract

(AE), acetone extract-lipophilic fraction (AE-LP) and acetone extract-polar fraction

(AE-PP). While AE-LP was enriched in oryzanols and tocols by about 65 times, AE-

PP was enriched in ferulic acid by 70 times as compared to their contents in DRB.

Azizah and co-workers139 studied of the chemical composition of local stabilized

rice bran. The four rice-bran milling fractions, after stabilization by microwave

heating on site at the rice mill, were analyzed for their chemical composition. The

content of all fractions tested (in g/100 g) consisted of 8.7–18.9 fat, 8.8–15.2 protein,

8.5–12.6 moisture, 4.2–7.7 ash, 22.2–44.8 total carbohydrates (by difference) and

18.3–30.5 total dietary fibre. It is encouraging to note that total phenolic compounds

of all fractions were detected at 257–488 mg ferulic acid equivalent per 100 kg, while

quantitation of the major carotenoids (lycopene and β-carotene) and amino acid

composition were determined by using RP-HPLC. The conditions of carotenoids were

as follows: Bondapak C18 column (300mm × 3.9mm i.d), a flow rate of 1.5 mL/min,

aetonitrile:tetrahydrofuran: water in the ratio of 85:12.5:2.5 as a mobile phase, and

the diode array detector was performed at 450 nm. And amino acids analysis column

(3.9mm × 15 cm). A gradient mobile phase was employed. The mobile phases were:

A, acetonitrile:deionized water (60:40); and B, 19.0 g sodium acetate trihydrate in 800

ml deionized water, pH adjusted to 5.7, to which 0.5 ml glacial acetic acid was added.

The detector was performed at 254 nm. Carotenoid contents were found to be in the

range of 58.7–216 mg/100 g. The amino acid composition varied within wide limits

with proline, histidine and threonine as the amino acids. Higher concentrations of

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amino acids found (in decreasing order) were arginine, glutamic acid, aspartic acid

and serine. Phosphorus and potassium were among the major mineral constituents of

rice bran, with values up to 1,633 mg/100 g. The first rice bran fraction was found to

be highest in energy, fat and minerals and could be a very good source of dietary fiber

and other nutrients.

1.5 The Scope and Aims of This Research

The aims of this research work can be summarized as follows:

1.5.1 To investigate the antioxidant activities of some Thai black rice bran

extracts using a number of methods with different mechanisms. First, the total

phenolic content (TPC) of each black rice bran was measured following the Folin–

Ciocalteu method using gallic acid as a standard. The total antioxidant activity (TAA)

was determined by means of the ferric thiocyanate method, which is the measurement

of the inhibition of linoleic acid (LA) peroxidation. In this method, the amount of

peroxide produced during the initial stages of oxidation was determined. A method

employing spectroscopic detection of the reduced Cu+ complex formed after the

reduction of Cu2+ by the antioxidants (PAO assay) was also utilized. To measure the

radical and reactive oxygen scavenging capacity, a DPPH assay (2,2-diphenyl-1-

picrylhydrazyl), and a chemiluminescence system (XYZ system) were used. In

addition, the chelating activity against Fe2+ was also examined. Additionally, methods

employing high performance liquid chromatography (HPLC) with photodiode array

detector were utilized for the structural analysis of vitamin E components in bran of

the black rice cultivars.

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1.5.2 To apply a reliable analytical method employing supercritical fluid

extraction (SFE) having carbon dioxide as an extraction fluid as a fast extraction

technique, followed by the use of tandem mass spectrometer to accurately identify the

chemical structure of the carotenoid components in bran of some Thai black rice.

Additionally, a method employing liquid chromatography-mass spectrometry (LC-

MS) with electrospray ionization operating in a selected ion monitoring (SIM) mode

was utilized to quantify the identified carotenoid components.