development and application of a methodology to …

DEVELOPMENT AND APPLICATION OF A METHODOLOGY TO DETERMINE

DIFFERENT FERULIC ACID POPULATIONS IN CEREALS AND CEREAL BASED

PRODUCTS

A THESIS

SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL

OF THE UNIVERSITY OF MINNESOTA

BY

SHARMILA VAIDYANATHAN

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

MASTER OF SCIENCE

Dr. MIRKO BUNZEL

January 2012

i

Acknowledgements

I thank my advisor, Dr.Mirko Bunzel, for his sustained support, guidance, and

encouragement. I have learned a lot from him about my work and general approach to

research. I will always be grateful to him for giving me this opportunity.

I thank my lab mates Catrin Tyl, Rachel Kyllo and Bridget McClatchey, for all the

interactions, help, and for creating a wonderful work atmosphere. I would also like to

acknowledge the contributions of Maggie Jilek, for extending help and support.

I have had the opportunity to interact with Dr. Ismail and Dr. Gallaher as both professors

and committee members. I thank them for being so approachable and for their valuable

suggestions.

Partial funding for this project was provided by USDA. Their contribution is highly

appreciated.

I thank Kampffmeyer Food Innovation GmbH for kindly providing samples.

I thank my parents for all the encouragement and making their presence felt throughout.

A special note of thanks belongs to my husband who has shared all the “ups” and

“downs” of this journey as if it were his own.

ii

Dedication

To my parents and my husband

iii

Acknowledgements………………………………………………………………… i

Dedication…………………………………………………………………………. ii

List of Tables………………………………………………………………………. vi

List of Figures……………………………………………………………………… vii

Chapter 1: Introduction…………………………………………………………… 1

Chapter 2: Literature Review and Study Objectives……………………………… 4

2.1 Nature and occurrence of ferulic acid………………………………………….. 5

2.2 Potential health beneficial effects of ferulic acid……………………………..... 9

2.3 Metabolism and bioavailability of ferulic acid………………………………… 11

2.4 Current methods of ferulic acid analysis in cereal grains……………………… 16

2.5 Objectives of this study………………………………………………………… 22

Chapter 3: Manuscript…………………………………………………………….. 23

3.1 Abstract………………………………………………………………………… 25

3.2 Introduction…………………………………………………………………….. 26

3.3 Materials and methods…………………………………………………………. 28

3.3.1 Isolation of standard materials representing ferulic acid ester-linked to

mono-/oligosaccharides, to soluble polysaccharides and to

insoluble polysaccharides………………………………………………………….. 29

3.3.2 Chemical characterization of the feruloylated standard materials…………… 31

3.3.3 Method to analyze free ferulic acid (FFA), ferulic acid ester-linked to

mono-/oligosaccharides (OF), ferulic acid ester-linked to soluble polysaccharides

(SPF) and ferulic acid ester-linked to insoluble polysaccharides (IPF)……………. 33

3.3.4 Method optimization…………………………………………………………. 35

3.3.5 Application of the developed methodology to processed and unprocessed

cereal samples……………………………………………………………………….37

iv

3.4 Results and discussion…………………………………………………………. 37

3.4.1 Characterization of standard compounds……………………………………. 38

3.4.2 Method development………………………………………………………… 39

3.4.3 Determination of recovery rates………………………………………………42

3.4.4 Application of the method to cereal samples………………………………… 43

3.5 Literature cited………………………………………………………………… 47

Chapter 4: Supplementary Data…………………………………………………... 58

4.1 Carbohydrate analysis………………………………………………………….. 59

4.2 Analysis of trans-p-coumaric acid, trans-sinapic acid, and cis-ferulic acid…… 61

4.3 Analysis of ferulic acid dehydrodimers and dehydrotrimers…………………... 62

4.4 Sample screening to optimize process conditions……………………………... 65

References………………………………………………………………………….66

Appendices…………………………………………………………………………77

Appendix A. List of Instruments…………………………………………………... 78

Appendix B. List of Chemicals…………………………………………………….. 80

Appendix C. Detailed Procedures…………………………………………………. 82

C-1. Determination of the carbohydrate composition of the control bran (CB),

optimized bran (OP), insoluble optimized bran (ISOP) and soluble

optimized bran (SOP)……........................................................................................ 82

C-1.1. Polysaccharide hydrolysis (cellulose analysis)……………………………... 82

C-1.2. Polysaccharide hydrolysis (analysis of the non-cellulosic polysaccharides).. 82

C-1.3. Reduction and acetylation of the monosaccharides, extraction of alditol

acetates……………………………………………………………………………... 83

C-1.4. Analysis of neutral sugars to determine correction factors………………… 83

C-2. Separation and detection of the alditol acetates by GC-FID…………………. 84

C-3. Alkaline extraction and analysis of trans-p-coumaric acid, cis-ferulic acid,

and trans-sinapic acid……………………………………………………………… 85

C-4. Analysis of trans-p-coumaric acid, cis-ferulic acid, and trans-sinapic acid by

RP-HPLC…………………………………………………………………………... 85

C-5. Extraction and analysis of dehydrodiferulic and dehydrotriferulic acids…….. 86

v

C-6. Analysis of dehydrodiferulic and dehydrotriferulic acids by RP-HPLC…….. 87

Appendix D. Chromatograms…………………………………………………….... 89

Appendix E. Standard Curves……………………………………………………... 104

vi

List of Tables

Table Page No.

Table 1. Recovery data for the proposed methodology 55

Table 2. Ferulic acid analysis of sample set 1 (processed and non processed

wheat bran) 56

Table 3. Ferulic acid analysis of sample set 2 (processed and non processed

wheat bran) 57

Table 4. Neutral monosaccharide composition of control bran (CB), optimized

bran (OP), soluble optimized bran (SOP) and insoluble optimized bran (ISOP)

polysaccharides 60

Table 5. Total contents (free and ester-linked forms) of some hydroxycinnamic

acids in the control bran (CB), optimized bran (OP), soluble optimized bran

(SOP), and insoluble optimized bran (ISOP) 62

Table 6. Contents of ester-linked dehydrodiferulic and dehydrotriferulic

acids in the control bran (CB), optimized bran (OP), soluble optimized

bran (SOP) and insoluble optimized bran (ISOP) 64

Table 7. Ferulic acid contents of different ferulic acid containing populations

(ferulic acid ester-linked to insoluble polysaccharides (IPF), to soluble

polysaccharides (SPF), to oligosaccharides (OF) as well as free ferulic acid

(FFA)) separated from differently processed wheat bran samples 65

Table 8. Correction factors for the analysis of monosaccharides in form of their

alditolacetates against acetylated inositol 84

Table 9. Gradient to analyze hydroxycinnamic acid monomers by RP-HPLC 86

Table 10. Gradient to analyze dehydrodiferulic and dehydrotriferulic acids by

RP-HPLC 87

Table 11. Correction factors used for ferulate dimers and trimers

quantification using 5-5(methylated)-dehydrodiferulic acid as internal standard 88

vii

List of Figures Page No.

Figure 1. Common hydroxycinnamic acids in plant based foods 6

Figure 2. Feruloylated arabinoxylan–oligosaccharides isolated from different

cereal grains after partial enzymatic degradation of the arabinoxylan backbone 8

Figure 3. Resonance structures of a ferulic acid radical 9

Figure 4. Feruloylated mono-/oligosaccharides used as standard compounds for

method development and partial validation 52

Figure 5. Proposed methodology to separate free ferulic acid (FFA), ferulic acid

ester-linked to mono-/oligosaccharides (OF), to soluble polysaccharides (SPF)

and to insoluble polysaccharides (IPF) 53

Figure 6a. Liberation of ferulic acid from feruloylated insoluble polysaccharides

by alkaline hydrolysis (2M NaOH, room temperature, 0-27 h) 54

Figure 6b. Stability of ferulic acid during alkaline hydrolysis (2M NaOH, room

temperature, 0-27 h) 54

Figure 7. trans-Ferulic acid standard chromatogram at 325 nm 89

Figure 8. Ferulic acid obtained from saponification of the IPF population (ferulic

acid ester-linked to insoluble polysaccharides) from the control bran (monitored

at 325 nm) 89

Figure 9. Ferulic acid obtained from saponification of the IPF population (ferulic

acid ester-linked to insoluble polysaccharides) from the soluble optimized bran

(monitored at 325 nm) 90

Figure 10. Free ferulic acid extracted from the control bran (monitored at

325 nm) 91

Figure 11. Free ferulic acid extracted from soluble optimized bran


Figure 12. Ferulic acid obtained from saponification of the OF population

(ferulic acid ester-linked to mono-/oligosaccharides) from the control bran


viii

Figure 13. Ferulic acid obtained from saponification of the SPF population

(ferulic acid ester-linked to soluble polysaccharides) from the control bran



(ferulic acid ester-linked to mono-/oligosaccharides) from the sample B1



(ferulic acid ester-linked to soluble polysaccharides) from the sample B1



(ferulic acid ester-linked to mono-/oligosaccharides) from sample 2.8



(ferulic acid ester-linked to soluble polysaccharides) from the sample 2.8


Figure 18. trans-Sinapic acid standard chromatogram (monitored at 325 nm) 95

Figure 19. trans-p-Coumaric acid standard chromatogram (monitored at 325 nm) 95

Figure 20. cis-Ferulic acid standard chromatogram (monitored at 325 nm) 96

Figure 21. trans-Ferulic acid, cis-ferulic acid trans-sinapic and trans-p-coumaric

acid obtained from saponification of control bran (monitored at 325 nm) 97

Figure 22. Analysis of dehydrodiferulic (DFA) dehydrotriferulic (TFA) acids

after saponification of the optimized bran (monitored at 280nm) 97

Figure 23. GC-FID standard chromatogram showing the analysis of

monosaccharides in form of their alditol acetates 98

Figure 24. Separation of cellulosic and hemicellulosic monosaccharides from

the isolated feruloylated insoluble polysaccharide standard in form of

their alditol acetates 99

Figure 25. Separation of hemicellulosic monosaccharides from the isolated

feruloylated insoluble polysaccharide standard in form of their alditol acetates 100

ix

Figure 26. Separation of hemicellulosic monosaccharides from isolated

feruloylated soluble polysaccharide standard in form of their alditol acetates 101

Figure 27. Separation of cellulosic and hemicellulosic monosaccharides from the

optimized bran in form of their alditol acetates 102

Figure 28. Carbohydrate analysis of hemicellulosic monosaccharides from the

optimized bran in form of their alditol acetates 103

Figure 29. trans-Ferulic acid standard curve (external calibration, RP-HPLC) 104

Figure 30. trans-Sinapic acid standard curve (external calibration, RP-HPLC) 104

Figure 31. trans-p-Coumaric acid standard curve (external calibration,

RP-HPLC) 105

Figure 32. cis-Ferulic acid standard curve (external calibration, RP-HPLC) 105

1

Chapter 1

Introduction

2

Introduction

Hydroxycinnamic acids are natural components of commonly consumed plant based

foods. Ferulic acid is the predominant hydroxycinnamic acid in cereals and cereal based

products. Several studies have demonstrated the antioxidant potential of ferulic acid both

in vivo and in vitro, thus raising interest in this compound to protect against oxidative

stress in living organisms including humans. Numerous studies focusing on the

antioxidant activity of ferulic acid and ferulic acid derivatives, their possible

contributions to health beneficial effects, and their bioavailability from food matrices are

found in literature. Although ferulic acid is of wide interest in the research community,

there is a lack of standardized and validated procedures to analyze ferulic acid and its

derivatives. Especially in cereals and cereal based products this becomes an issue.

Different ferulic acid populations exist in these products and comparisons are made

between studies from research groups often using very different analytical approaches

with unknown specificity and accuracy. In cereals, several populations of ferulic acid

need to be differentiated since ferulic acid not only occurs in the free form but it is also

found covalently linked (ester and ether linkages) to plant cell wall polymers and, to a

lesser degree, low molecular weight compounds. Especially the free acid and three ester-

linked ferulic acid populations are of interest for potential health beneficial effects in

humans. The proportions between these populations can be altered by food processing,

thus potentially changing ferulic acid bioavailability. Hence, their analysis is a key

element to study both bioavailability of different ferulic acid populations and processing

3

conditions to influence their composition. Development and application of a method to

analyze these ferulic acid populations in cereal based products is described in this thesis.

4

Chapter 2

Literature Review and Study Objectives

5

Literature Review

Hydroxycinnamic acids are plant secondary metabolites that are formed in the phenyl

propanoid pathway. Studies having their focus on whole grain diets showed that the

hydroxycinnamic acid enriched bran fraction contributes significantly to the health

benefits. While the (non-starch) polysaccharide fraction of the bran received wide

attention pertaining to health benefits in the past, the focus has shifted more recently to

phytochemicals associated with non-starch polysaccharides, especially hydroxycinnamic

acids such as ferulic acid. This literature review focuses on the predominant

hydroxycinnamic acid in cereal grains, ferulic acid, its occurrence, nature, potential

health benefits, bioavailability, and metabolism as well as on available methodologies to

quantify various ferulic acid populations in cereals.

2.1 Nature and occurrence of ferulic acid

Hydroxycinnamic acids are ubiquitous in plants. They are formed from phenylalanine

and, in case of grasses, from tyrosine both of which are formed in the shikimic acid

pathway. These aromatic amino acids are then metabolized in the phenlypropanoid

pathway into hydroxycinnamic acids, aldehydes, and alcohols. p-Coumaric acid, caffeic

acid, ferulic acid, and sinapic acid (Figure 1) are the most important hydroxycinnamic

acids in plant based food products.

6

Ferulic acid is commonly found in cereal grains, coffee, spinach, beetroot, citrus fruits

etc. Based on the consumption pattern of an individual, an average daily intake between

150 - 250 mg can be estimated (Zhao and Moghadasian 2008). In cereals grains, ferulic

acid is concentrated in the cell walls of the aleurone, pericarp, and the embryo (Parker et

al. 2005). Thus, ferulic acid is concentrated in the bran fraction which is separated from

the starchy endosperm during milling. Highest ferulic acid concentrations are found in

corn bran (ranging between 2610 - 3300 mg/ 100 g), followed by wheat bran, rye bran,

and oat bran (Zhao and Moghadasian 2008).

The role of ferulic acid in the cell wall has been widely studied in the past. Ferulic acid in

the cell wall is involved in functions such as termination of cell elongation and protection

against pathogens which involves reduced degradation of the cell wall by microbial and

fungal enzymes (Buanafina 2009). Also, the involvement of ferulic acid in cell

aggregation was demonstrated for cultured rice callus cells (Kato et al. 1994). All these

functions of ferulic acid are based on the fact that it is bound to cell wall polysaccharides

which will be described in more detail below. Polysaccharide coupled ferulates are able

trans-p -

Coumaric acid

trans-Caffeic

acid

trans-Ferulic

acid

trans-Sinapic

acid

Figure 1. Common hydroxycinnamic acids in plant based foods

7

to form dimers through an oxidative mechanism (formation of ferulic acid

dehydrodimers) or through the action of UV light (formation of ferulic acid cyclobutane

dimers), thus cross-linking the polysaccharide chains. Higher oligomers of ferulic acid

such as dehydrotrimers and dehydrotetramers can be formed through oxidative

mechanism only, cross-linking up to four polysaccharide chains (Ralph et al. 2004a,

Buanafina 2009, Bunzel 2010). Also, polysaccharide bound ferulic acid can cross-couple

with monolignols (hydroxycinnamic alcohols), thus coupling polysaccharides to lignin in

cell walls (Ralph et al. 2004b).

As early as 1963 it was hypothesized that ferulic acid is linked to xylan chains via ester

bonds (Fausch et al. 1963). Ten years later, Geissmann and Neukom (1973) demonstrated

the presence of ester-linked ferulic acid in wheat flour and quantified its contents for the

first time. To demonstrate how ferulic acid is attached to cell wall polysaccharides in

detail, feruloylated oligosaccharides were liberated (either enzymatically or chemically)

and the structures of the feruloylated oligosaccharides were elucidated. By using an

enzymatic treatment, feruloylated wheat bran oligosaccharides were isolated and

identified as feruloylated arabinoxylan-oligosaccharides (Smith and Hartley 1983).

Feruloylated arabinoxylan-oligosaccharides were also isolated from corn bran (Saulnier

et al. 1995), barley (Gubler et al. 1985), rye (Steinhart and Bunzel 2003), wild rice

(Bunzel et al. 2002) etc. Figure 2 shows feruloylated arabinoxylan-oligosaccharides

demonstrating the ester-linkage of ferulic acid to the arabinose side-chains of the

arabinoxylans. In pseudo-cereals such as amaranth ferulic acid is linked to arabinans and

8

galactans as demonstrated by the isolation and identification of characteristic feruloylated

oligosaccharides (Bunzel et al. 2005a).

Polysaccharide bound ferulic acid was also identified as a nucleation site for lignin

biosynthesis (Ralph et al. 1995). Coupling of monolignols to ferulates results in the

formation of C-C-linkages or ether linkages. Ether-linked ferulic acid has been reported

in wheat straw (Scalbert et al. 1985). Also, cross-coupling products comprised of ferulic

acid and monolignols were isolated from wheat and oat straws (Jacquet et al. 1995) and

from several cereal grains (Bunzel et al. 2004).

Figure 2. Feruloylated arabinoxylan-oligosaccharides isolated from different cereal

grains after partial enzymatic degradation of the arabinoxylan backbone

9

Existing data therefore points out that ferulic acid in cereals exists mostly as ester-linked

or ether-linked ferulic acid and much less of the free ferulic acid is found.

2.2 Potential health beneficial effects of ferulic acid

The presence of hydroxycinnamic acids in whole grain diets is thought to be responsible

for many observed physiological effects (Slavin 2003). Especially the antioxidant

properties of hydroxycinnamic acids seem to contribute to their physiological effects.

Several studies have focused on the antioxidant potential of ferulic acid both in vitro and

in vivo (Shahidi and Chandrasekara 2010). The antioxidant properties of ferulic acid are

mainly based on its ability to donate a hydrogen atom forming a resonance stabilized

ferulic acid radical (Graf 1992) (Figure 3).

Some ferulic acid dimers also show antioxidant activity based on metal chelation

(Neudörffer et al. 2004). Pure hydroxycinnamic acids were tested in vitro for their

antioxidant activity using the human LDL model system (Andreasen et al. 2001a).

Caffeic acid was found to be most effective in protecting LDL against oxidative damage,

followed by sinapic acid, ferulic acid, and p-coumaric acid. It was also observed that rye

Figure 3. Resonance structures of a ferulic acid radical

10

bran extracts were more effective antioxidants when compared to (refined) flour extracts

(Andreasen et al. 2001a). These data are, however, predictable taking into account that

hydroxycinnamic acids are more concentrated in the bran fraction. In vitro antioxidant

assays were reported by other groups, too (Kanski et al. 2002, Cheng et al. 2007). Several

studies also focused on the in vivo antioxidant activity of ferulic acid. In an animal

experiment, Balasubashini et al. (2004) showed that low doses of ferulic acid (10 mg/kg

body weight) were effective in lowering blood glucose levels in rats. They also found

increased levels of superoxide dismutase, catalase, and glutathione peroxidase in the

tissues of diabetic rats treated with ferulic acid as compared to the rats treated with the

drug glibenclamide, an antidiabetic drug. It was observed that low doses of ferulic acid

were more capable of triggering these beneficial effects than higher doses (Balasubashini

et al. 2004). Another study looked at the effect of ferulic acid on β-amyloid peptide

toxicity in mice (Yan et al. 2001). The β-amyloid peptide is discussed to induce oxidative

stress and inflammation in the brain, thus being involved in the pathogenesis of

Alzheimer’s disease. It was observed that treatment with ferulic acid provided protection

against some of the adverse effects resulting from intracerebroventicular injection of a β-

amyloid peptide. The injection of this peptide produced Alzheimer like symptoms such as

behavior and learning impairments in mice. This study however failed to conclude, if on

withdrawal of treatment with ferulic acid these effects would persist. Ferulic acid was

also found to be effective against UV-induced oxidation of phosphatidylcholine from

liposomal membranes (Saija et al. 1999). The ability of ferulic acid to penetrate through

the outermost layer of the epidermis (stratum corneum) was demonstrated in this study,

11

too, thereby suggesting that ferulic acid can be an effective ingredient in skin lotion

products.

Besides the antioxidant activity of free ferulic acid the antioxidant capacity of

feruloylated oligosaccharides was also studied. Purified feruloylated oligosaccharides

from wheat bran showed activity in the DPPH antioxidant activity test and inhibited rat

erythrocyte hemolysis in vitro (Yuan et al. 2005). This inhibition was found to be dose

dependent. Ohta et al. (1997) showed that ferulic acid ester-linked to arabinose is more

effective against LDL oxidation than free ferulic acid and partially feruloylated soluble

polysaccharides. This may be due to the experimental conditions and the differences in

solubility of the tested components. Similar results were published by Baublis et al.

(2000), demonstrating that ester-linked phenolics from wheat bran were more effective

antioxidants than free phenolics when tested with an emulsion of salmon oil.

Also, anti-inflammatory effects of ferulic acid were demonstrated in the past by, for

example, showing a reduction of markers of inflammation in cell cultures subjected to

bacterial endotoxins. Other effects suggested for ferulic acid are, for example, anti-

apoptotic, hepato- and pulmonary protective effects (Srinivasan et al. 2007).

2.3 Metabolism and bioavailability of ferulic acid

As more studies established the antioxidant potential of ferulic acid and its potential role

in combating oxidative disorders, the focus shifted on understanding metabolism and

bioavailability of ferulic acid. As described earlier, ferulic acid can be found in different

forms in plant based food products. In cereal grains little free ferulic acid is found and

most of the ferulic acid is ester-linked to soluble and predominantly insoluble

12

arabinoxylans. During food processing, e.g. by using xylanases in breadmaking, or during

germination of the kernels it is also possible that feruloylated oligosaccharides are

formed. When these different ferulic acid populations are compared regarding their

bioavailability, all studies performed so far showed that free ferulic acid is most

bioavailable among these ferulic acid populations (Zhao and Moghadasian 2008).

Studies have demonstrated that phenolic compounds in general are absorbed mainly in

the jejunum. Actual mechanisms of absorption still remain to be elucidated. Data suggest

the involvement of monocarboxylic acid transporters, although other mechanisms of

absorption such as proton driven transport, sodium dependent transporters, and passive

diffusion have also been discussed (Zhao and Moghadasian 2010). Besides absorption

from the jejunum, ferulic acid seems to be absorbed form the stomach, too (Zhao et al.

2004). Injection of ferulic acid in a rat stomach in an in situ set up suggested that a major

proportion (ca. 74%) of the administered ferulic acid was absorbed from the stomach in

less than half an hour.

Rats fed with ferulic acid supplemented standard diets showed a maximum plasma

concentration of ferulic acid after 30 min (Rondini et al. 2002). The main ferulic acid

metabolite found in the plasma was the sulfated form. In the urine, sulfated and

sulfoglucuronidated ferulic acids were the dominant metabolites. In the before mentioned

study, in which ferulic acid was injected in a rat stomach (Zhao et al. 2004), free ferulic

acid was demonstrated to be the predominant form of ferulic acid in the portal vein.

However, in the celiac artery the sulfoglucuronidated from was found to be the dominant

form, suggesting that the liver is the major site of conjugation. Again, the major

13

metabolite found in urine was the sulfoglucuronidated form of ferulic acid. In both

studies a major portion of the initial ferulic acid was not recovered in the urine or bile

indicating a distribution to the tissues. The perfusion of ferulic acid through intestinal

tissues was also studied. Ferulic acid was found to be absorbed as such through intestinal

cells confirming the findings of the previous studies showing that the liver is the main

site of conjugation (Adam et al. 2002).

Summarizing the above mentioned studies, it can be postulated that free ferulic acid is

rapidly absorbed as such in the stomach and the small intestine, conjugated mainly in the

liver, and partly distributed to the tissues before being excreted through the urine, mainly

in its sulfoglucuronidated form.

Although the metabolism of ferulic acid in its free form is well established, the presence

of large amounts of ferulic acid derivatives bound to matrix compounds makes the food

matrix an important component in establishing the bioavailability of ferulic acid. In most

studies investigating the ferulic acid bioavailability from different food products the

excretion of ferulic acid and its metabolites via urine and feces is used to estimate ferulic

acid bioavailability. Fruits and vegetables have lower ferulic acid contents compared to

cereals (Manach et al. 2004, Zhao and Moghadasian 2008), but the bioavailability from

these products is generally better. The urinary excretion of ferulic acid after tomato

consumption by human subjects was analyzed to be 11 - 25% of the ingested dose

(Bourne and Rice-Evans 1998). Urinary excretion of ferulic acid after consumption of a

low alcohol beer was found to be higher than that obtained in the previous study for some

of the subjects. A wide variation of total ferulic acid (19 – 92% of the ingested amounts)

14

found in the urine was observed between subjects (Bourne et al. 2000). While the

bioavailability of ferulic acid from beer is good, the bioavailability from cereal grains and

other cereal based products is usually low. Several studies were performed to describe the

bioavailability of ferulic acid from cereal based matrices. To understand the role of

ferulic acid being ester-linked to cell wall polysaccharides in cereal grains, a comparison

was done by feeding rats with diets supplemented with free ferulic acid and diets

containing wheat bran. The fecal excretion of ferulic acid was enhanced with the intake

of bran enriched diets, which was in accordance with a reduced urinary excretion from

the same diet (Adam et al. 2002). Rats fed a refined corn bran diet showed even lower

ferulic acid absorption than rats fed wheat bran diets (based on urinary excretion) (Zhao

et al. 2005). A potential impact of adaptation to a refined corn bran diet was also

investigated in an animal study, but no effect was found. The urinary excretion of ferulic

acid did not change over an adaptation period of 10 days (Zhao et al. 2005). Ferulic acid

bioavailability after the consumption of bran enriched products was studied in human

subjects, too. Urinary excretion of ferulic acid after the consumption of rye bran enriched

foods by women in a randomized crossover study was surprisingly high with

approximately 28% of the ingested total ferulic acid (Harder et al. 2004). The

bioavailability of ferulic acid from rye bran seems to be much higher than the

bioavailability of ferulic acid from a wheat based high bran breakfast cereal (Kern et al.

2003a). After a single dose of the wheat bran breakfast cereal, approximately 3% of the

ingested ferulic acid was excreted with the urine within 24 h. Also, the predominant form

15

of plasma ferulic acid found in this study was the glucuronidated form and not the

sulfoglucuronidated form which was dominant in the above mentioned rat studies.

The above mentioned studies looked at the cereal matrix as a whole influencing the

bioavailability of ferulic acid. However, a recent study focused in more detail on the

influence of the ferulic acid being ester-linked to different types of carbohydrates (Zhao

et al. 2003). This study, which was performed in rats, demonstrated that free ferulic acid,

ferulic acid ester-linked to arabinose, and ferulic acid ester-linked to soluble

arabinoxylans have different bioavailabilities. As observed in previous studies, free

ferulic acid was rapidly absorbed in the foregut. Ferulic acid ester-linked to arabinose

showed a 60% reduction in the ileum, indicating absorption of this population in the

small intestine. Ferulic acid ester-linked to soluble polysaccharides was reduced by 44%

in the cecum, and 33% was found in the feces (Zhao et al. 2003).

A reduction of the respective ferulic acid population in the cecum may not be an

indication of absorption only. It can also indicate that ferulic acid either still attached to

the cell wall polymers or after release by microbial enzymes in the large intestine is

further metabolized and therefore not analyzed as ferulic acid in the mentioned

experiment. Fermentation of wheat bran using fecal cultures to simulate colon conditions

showed the release of ferulic acid ester-linked to insoluble polysaccharides (Kroon et al.

1997). However, the reduction of ester-linked ferulic acid did not match the free ferulic

acid concentrations in the fermentation medium at the end of the fermentation. This

reduction of ferulic acid concentrations in the fermentation medium indicates a possible

metabolism of the (liberated) ferulic acid by the microbial population. The ability of

16

microorganisms from the human colon to metabolize ferulic acid was also demonstrated

by another group. Russell et al. (2008) showed that metabolites formed from ferulic acid

by human and ruminant microbiota are fairly similar. Also, similar metabolites were

observed in the feces of different healthy human volunteers, although the fermentation

kinetics may differ in detail. While microbial esterases in the large intestine are one way

to liberate ester-linked ferulic acid in the human GI-tract, the presence of esterases in the

mucosa of duodenum, jejunum and ileum of rats was demonstrated by Andreasen et al.

(2001b). The luminal contents of these enzymes were shown to be much lower than the

mucosal concentrations. Based on above mentioned studies it can be assumed that the

release of ferulic acid by esterases of the small intestine or microbial esterases in the

large intestine as well as the metabolism of ferulic acid by gut microbiota are also

important factors studying the bioavailability of ferulic acid and its derivatives.

2.4 Current methods of ferulic acid analysis in cereal grains

Contrary to what is sometimes claimed as recent discovery, the knowledge that most

ferulic acid in cereals is covalently linked to polysaccharides was found as early as 1963

when Fausch and coworkers liberated ferulic acid from unfractionated wheat flour

pentosans by alkaline hydrolysis (Fausch et al. 1963). Due to the susceptibility of ester

linkages to alkaline hydrolysis, saponification is still most often used to liberate ester-

linked ferulic acid before its analysis by different chromatographic methods. However, a

standard method of alkaline hydrolysis and extraction of ferulic acid does not exist as

recently reviewed by Barberousse et al. (2008). Besides saponification, hydrolysis of the

17

ester-linkage by using acidic conditions or esterases was attempted in the past, too. Since

a plethora of methods were published in the past, only a few methods are described here.

Saulnier et al. (2001) analyzed enzymatically destarched wheat bran for its ferulic acid

content after alkaline hydroysis with 2 M NaOH for 30 min at 35 °C. After acidification

of the hydrolysate, the liberated ferulic acid was extracted using diethyl ether as the

extraction solvent. Ferulic acid contents of different barley cultivars were analyzed by

treatment of the sample with 0.2 N sulfuric acid at 100 °C for 60 min, followed by α-

amylase treatment. The supernatants were then analyzed for the ferulic acid content

(Zupfer et al. 1998). Enzymatically treated wheat and rice bran samples were analyzed

for their ester-linked phenolic acid contents by first extracting the samples with 70%

ethanol to remove the free phenolics, followed by drying the residue using increasing

ethanol concentrations and diethyl ether. Hydrolysis of the dried residue was performed

with 2 M NaOH for 2 h. Hydrolysates were acidified and extracted with ethyl acetate and

analyzed for their ferulic acid contents (Hegde et al. 2006). Whole grain corn flour was

analyzed for covalently linked ferulic acid by alkaline hydrolysis using 2 M NaOH for 1

h, followed by extraction with ethyl acetate (Adom and Liu 2002). And finally, Bunzel et

al. (2001) liberated ester-linked ferulic acid monomers and dimers from different cereal

grain dietary fibers by using 2 M NaOH for 18 h at room temperature. After acidification

of the hydrolysates the ferulic acid monomers and dimers were extracted into diethyl

ether before being derivatized and analyzed by GC-MS/FID.

It is evident from this brief selection of methods that a commonly used approach to

liberate ester-linked ferulic acid and to extract the liberated acid from the alkaline

18

hydrolysate does not exist. It has to be noted, however, that all alkaline methodologies

should be performed in an oxygen-devoid environment to minimize degradation of the

ferulic acid side-chain. Oxidative degradation of the side-chain under alkaline condition

may lead to degradation of ferulic acid and the formation of vanillin (Bunzel et al.

2005b). Although not attempted in this study the cleavage of ether-linked ferulic acid

requires much harsher conditions, for example 4 M NaOH at 170 °C in pressure vessels

for 1 h (Renger and Steinhart 2000).

While ester-linked ferulic acid can be liberated by mucosa-bound or microbial esterases

in the (human) GI-tract, the place and degree of liberation is dependent on whether the

ferulic acid is attached to mono-/oligosaccharides, soluble polysaccharides or to insoluble

poylsaccharides in a complex matrix. Therefore, a methodology that is able to

differentiate these three ferulic acid populations next to the well bioavailable free ferulic

acid is required. Although such a detailed methodology does not exist, methods were

published in the past attempting to classify the different ferulic acid populations to some

degree.

Such an approach was first published by Krygier et al. (1982) to analyze rapeseed

samples. This method differentiates between the following ferulic acid populations: “free

phenolic acids”, “soluble phenolic acid esters”, and “insoluble-bound phenolic acids”.

The differentiation between insoluble-bound phenolic acids on the one hand and the free

phenolic acids and soluble phenolic acid esters on the other hand was performed by

extraction of the defatted rapeseed flour with 70% methanol/70% acetone (1/1 (v/v))

leaving the insoluble bound phenolic acids behind. The separation between the free

19

phenolic acids and the soluble phenolic acid esters was based on the partition between

acidified water (pH 2) and a diethyl ether/ethyl acetate (1/1 (v/v)) mixture. In an attempt

to liberate ester-linked ferulic acid after separation of the different populations they also

demonstrated that hydroxycinnamic acids were partially destroyed under the conditions

of acidic hydrolysis (1 N HCl, 30 min, reflux). Losses varied between different

hydoxycinnamic acids; sinapic acid showed the greatest loss (91.7%) while o-coumaric

acid (used as internal standard) was most stable (loss of 15.1%). Loss of trans-ferulic

acid was 78%. This method was also applied to potato, oats, rice, corn and wheat flours

(Sosulski et al. 1982).

In the recent years more studies attempted to differentiate ferulic acid populations, mostly

to characterize different cereal grains. “Free”, “soluble-conjugate” and “insoluble bound”

ferulic acid of whole rice, wheat, oats, and corn were analyzed (Adom and Liu 2002).

Both “free” and “soluble-conjugate” ferulic acid was extracted by using 80% ethanol.

Since the procedure is not well described it can only be assumed that the analysis of free

ferulic acid is based on the separation of free ferulic acid and “soluble conjugate” ferulic

acid by HPLC. The sum of “free” and “soluble conjugate” ferulic acid was determined

after saponification of an aliquot of the 80% ethanol extract and HPLC analysis. It is

assumed that the “soluble conjugate” ferulic acid is then determined by subtraction. By

using this method the free ferulic acid proportions in rice, wheat, oats, and corn were

determined to be 0.5%, 0.2%, 0.4% and 0.1% of the total ferulic acid contents in these

cereal samples. “Soluble conjugate” ferulic acid proportions were 7%, 1%, 2% and 1%,

of the total ferulic acid contents. “Insoluble bound“ ferulic acid contents were found to

20

make up the major portion of the total ferulic acid in the samples, with 93 %, 99%, 98%

and 99% of the total ferulic acid for rice, wheat, oats, and corn. A similar method was

used to characterize the ester-linked ferulic acid populations in 11 different wheat

varieties. Proportions for free ferulic acid ranged between 0.1% - 0.4% of the total ferulic

acid contents, proportions for “soluble conjugate” ferulic acid ranged between 0.5% -

2.3%, and proportions for “insoluble bound” ferulic acid ranged between 97.7% - 99.4%

(Adom et al. 2003). Different wheat varieties were also characterized for their ester-

linked ferulic acid populations following the method proposed by Sosulski et al. (1982).

Here the populations were reported as “soluble free”, “soluble conjugate” and “insoluble

bound” ferulic acid (Moore et al. 2005). The contributions of the different ferulic acid

populations to the total ferulic contents were as follows: “soluble free” ferulic acid 0.1 -

0.4 %,”soluble conjugate” ferulic acid 5.1 - 10.4 %, and “insoluble bound” ferulic acid

89.2 - 94.6%.

Another approach published by Kim et al. (2006) aimed to determine “extractable

phenolic acids” (“free” and “alkaline hydrolysable” hydroxycinnamic acids) and “bound

phenolic acids” in wheat bran from different sources. ”Extractable phenolic acids” were

extracted using 80% methanol. Separation of the “extractable ferulic acids” into “free”

and “alkaline hydrolysable” extractable ferulic acids was performed by drying the

methanol extract, and partition of the residue between acidified water and diethyl ether.

Finally, Kern et al. (2003b) described another liquid/liquid partition system to

differentiate between free ferulic acid and extractable ester-linked ferulic acid. Instead of

21

using acidified water and diethyl ether (Kim et al. 2006) or a mixture of dietyl ether and

ethyl acetate (Krygier et al. 1982) they used pure ethyl acetate as the organic solvent.

As can be seen from this brief overview, available methods do not clearly distinguish

between the ferulic acid populations analyzed. Moreover, the used terms are confusing

and often do not adequately describe the analyzed ferulic acid population. For example,

terms such as “bound” or “conjugate” do not specify if the ferulic acid is ester-linked or

ether-linked. Also, there is a wide range of methods used to liberate ferulic acid from its

ester (and sometimes ether) linkages. Finally, none of the approaches to differentiate

ferulic acid populations has been adequately tested for accuracy.

22

2.5 Objectives of this study

The aim of this thesis project was to develop, test and apply a methodology to separate

and quantify four different ferulic acid populations (free ferulic acid, ferulic acid ester-

linked to mono-oligosaccharides, ferulic acid ester-linked to soluble polysaccharides,

ferulic acid ester-linked to insoluble polysaccharides) in cereals and cereal based

products.

To reach this goal the following objectives were defined:

To isolate standard materials representing the four ferulic acid populations of

interest.

To develop a methodology to separate and quantify the mentioned four ferulic

acid populations. This methodology was aimed to be simple enough to be used in

laboratories world-wide thus not relying on very specialized laboratory

equipment.

To test the developed method for its selectivity and accuracy.

To apply the method on cereal grains and processed samples to observe whether

population changes can be adequately detected by this method.

23

Chapter 3

Manuscript

24

Development and application of a methodology to determine different ferulic acid

populations in cereal products

Sharmila Vaidyanathan and Mirko Bunzel*

University of Minnesota

Department of Food Science and Nutrition

*Corresponding Author

Department of Food Science and Nutrition – CFANS

University of Minnesota

1334 Eckles Avenue

St Paul, MN 55108

Phone: +1-612-624-1764

Fax: +1-612-625-5272

Email: [email protected]

25

3.1 Abstract

The bioavailability of ferulic acid is dependent on its form present in food. This

necessitates a methodology to quantify different ferulic acid populations in food products,

especially cereal based products. The aim of the proposed methodology is to separate and

quantify ferulic acid ester-linked to mono- and/or oligosaccharides, to soluble

polysaccharides, and to insoluble polysaccharides as well as in its free form.

Development and partial validation of this method, which is widely based on liquid/liquid

extraction and precipitation steps, was performed by using characterized standard

materials isolated from corn bran for each of the mentioned populations. As the

determination of ferulic acid ester-linked to mono-/oligosaccharides was one of the major

goals of this methodology, three different feruloylated mono-/oligosaccharides were used

for the method development and validation. To determine the accuracy of the method,

ferulic acid containing standard materials added to a starch matrix were extracted and

separated according to the developed protocol and the separated ferulic acid esters were

saponified before ferulic acid was analyzed by RP-HPLC. Recovery rates were generally

between 70 - 103% with lowest recovery rates for ferulic acid ester-linked to soluble

polysaccharides and highest recovery rates for ferulic acid ester-linked to insoluble

polysaccharides and ester-linked to oligosaccharides. Finally, the applicability of the

method on actual samples (bran and processed bran samples) was demonstrated.

26

3.2 Introduction

Ferulic acid is a plant secondary metabolite formed in the phenylpropanoid pathway. It

belongs to a group of compounds called hydroxycinnamic acids, which also includes, for

example, caffeic acid, p-coumaric acid, and sinapic acid. Interest in hydroxycinnamic

acids was mostly generated due to their ability to act as potent antioxidants as recently

reviewed by Shahidi and Chandrasekara (2010). Therefore, epidemiological studies have

looked into the possibility of ferulic acid being beneficial against disorders in which

oxidative processes play an important role, such as cancer, diabetes, Alzheimer’s disease

and cardiovascular disorders (Zhao and Moghadasian 2008). These studies focus on

ferulic acid itself or on ferulic acid as a part of whole grain diet (Slavin 2003).

Nature and occurrence of ferulic acid in cereals has long been revealed. In fact, as early

as 1973 it was shown that ferulic acid occurs ester-linked to insoluble polysaccharides in

wheat flour from which it can be liberated by alkaline treatment (Geissmann and Neukom

1973). It is now well known that ferulic acid in grasses occurs mostly ester-linked to cell

wall arabinoxylans (and oligosaccharides) (Smith and Hartley 1983) but also ether-

linked to lignin (Scalbert et al. 1985) or lignin like polymers (Bunzel et al. 2004).

However, most studies do not clearly specify the type of linkages in their materials. The

commonly used term “bound”, often found in studies determining ferulic acid

bioavailability, often refers to the ester-linked ferulic acid only. Also, most studies do not

differentiate between ferulic acid ester-linked to different polymers and/or oligomers

although application of an early published method could give at least some hints about

27

the different ferulic acid populations (Sosulski et al. 1982). This method enables the

classification of phenolic compounds in cereals as free, esterified and insoluble ester–

linked, but it was not validated for quantitative purposes.

Especially since there is a wide interest to better understand ferulic acid bioavailability

(Bourne and Rice-Evans 1998, Bourne et al. 2000, Manach et al. 2004, Zhao and

Moghadasian 2010) further classification of the varying ferulic acid derviatives in cereal

products would be desirable, namely a classification into free ferulic acid (FFA), ferulic

acid ester-linked to oligosaccharides (OF), ester-linked to soluble-polysaccharides (SPF)

and ester-linked to insoluble-polysaccharides (IPF). FFA is most bioavailable among the

mentioned populations (Zhao and Moghadasian 2008). It was found to be rapidly

absorbed from the stomach of rats, with 74% of the injected dose being absorbed after 25

min of incubation (Zhao et al. 2004). After the intake of a diet supplemented with pure

ferulic acid, it also appeared rapidly in the plasma of rats (Rondini et al. 2002). These

data suggest the absorption of the bulk of FFA before it comes extensively in contact

with the microbial population of the gut. Also in rats, ferulic acid ester-linked to

oligosaccharides is partially absorbed in the foregut (40%) (Zhao et al. 2003). The

remaining feruloylated oligosaccharides reach the cecum where further metabolism may

occur. In contrast, a 44% reduction (from the initial amount fed) of ferulic acid ester-

linked to soluble polysaccharides was found to occur in the cecum, and a further

reduction was observed in the feces (Zhao et al. 2003) . However, reduction should not be

confused with absorption as ferulic acid can also be metabolized by gut microorganisms

(Russell et al. 2008, Braune et al. 2009), thus lowering the amounts of detected ferulic

28

acid. The hydrolysis of ester-linked ferulates by feruloyl esterases in the large intestine, a

pre-requirement for reabsorption of these compounds from the large intestine, is

dependent on the physicochemical properties of the polysaccharides (Kroon et al. 1997),

highlighting the importance of differentiating between ferulic acid ester-linked to either

soluble or insoluble polysaccharides.

In order to interpret data from bioavailability studies dealing with cereal products, it is

necessary to determine ferulic acid in its different forms. Although free, “soluble bound”

and “insoluble bound” ferulic acid populations were analyzed in cereals in the past

(Sosulski et al. 1982, Adom et al. 2003, Moore et al. 2005), a procedure which is able to

reliably quantify these populations and which is able to differentiate between ferulic acid

ester-linked to mono-/oligosaccharides and ester-linked to soluble polysaccharides does

not exist to the best of our knowledge. The aim of this study was to develop such a

methodology and demonstrate its applicability on cereal products.

3.3 Materials and methods

If not otherwise specified, chemicals were from Sigma-Aldrich (St. Louis, MO, USA) or

Fisher Scientific (Pittsburgh, PA, USA). All chemicals were of appropriate purity for

analytical applications. Diethyl ether was periodically tested for the absence of peroxides

by using peroxide test strips (Peroxide Test, EM Science, Gibbstown, NJ, USA).

29

3.3.1 Isolation of standard materials representing ferulic acid ester-linked to mono-

/oligosaccharides, to soluble polysaccharides and to insoluble polysaccharides.

Ferulic acid in its trans-configuration was commercially available (MP Biomedicals Inc.,

Solon, OH, USA), whereas the other standard materials had to be isolated from corn bran.

Corn bran was obtained from Cargill, Inc. (Indianapolis, IN, USA). Bran was milled to

pass a 0.5 mm sieve and defatted for 2 h using acetone (bran/acetone ratio, 1/10 (w/v)) at

60 °C. The procedure was repeated twice and the acetone was discarded after each

extraction. The extracted bran was then dried under vacuum.

Preparation of the insoluble feruloylated polysaccharide standard material involved

destarching of the milled, defatted corn bran. In brief, 200 mL of phosphate buffer (0.08

M, pH 6.0) and 1.5 mL of thermostable α-amylase (Amylex 4T, ≥24540 LU/g, Danisco,

Louvain-La-Neuve, Belgium) were added to 20 g of milled defatted bran. The suspension

was placed in a water bath at 95 °C and contents were mixed every 15 min. After 1 h, the

sample was cooled to room temperature and the pH was adjusted to 4.5 with HCl (37%).

Amyloglucosidase (AMG 300, ≥300 AGU/mL, Novozymes, Franklinton, NC, USA) was

added (700 µL), and the flasks were incubated for 45 min in a water bath at 60 °C. The

residue after centrifugation was washed twice with water (40 mL each), twice with 80%

(v/v) ethanol (40 mL each) and twice with acetone (40 mL each) (Bunzel et al. 2001).

The residue was dried at 40° C to remove traces of acetone and then freeze dried. The

above procedure was repeated three times and the residues were pooled and analyzed for

their ferulic acid contents as described below.

30

Soluble polysaccharides were obtained by treating the milled, defatted bran with α-

amylase as described above. However, the bran/buffer ratio was reduced to 4.5 mL

buffer/g bran, resulting in a more concentrated polysaccharide solution. The supernatant

obtained after α-amylase treatment was filtered through a Fisherbrand P-5 filter

(Pittsburgh, PA, USA). The residue was washed twice with 5 mL of water. The washes

were filtered and pooled with the first filtrate. The above procedure was performed four

times and all supernatants were pooled. The pooled solution was dialyzed for 48 h (10

kDa membrane, Spectrum Laboratories Inc., Rancho Dominguez, CA, USA) against

water at 4°C. The water was changed every 4 h, except for the last change of the day,

when the water was left unchanged for 12 h. The resulting high molecular weight

material was concentrated under vacuum (rotary evaporation <40° C) and used as the

feruloylated soluble polysaccharide standard.

Feruloylated mono- and oligosaccharide standards were isolated previously Dr. Bunzel’s

group (Allerdings et al. 2006). In brief, feruloylated mono-/oligosaccharides were

liberated from destarched and partially deproteinated corn bran (insoluble corn fiber) by

acid hydrolysis (50 mM trifluoroacetic acid (TFA), 3 h, 100 °C). Hydolysates were

further purified using Amberlite XAD-2 fractionation, Sephadex LH-20 chromatography,

and RP-HPLC. Structural characterization of the isolated feruloylated mono-

/oligosaccharides was performed by using one and two dimensional NMR. The purity of

the compounds was estimated by 1H-NMR in D2O and was found to be >93%. Three

different feruloylated mono-/oligosaccharides were used for the method validation,

namely 5-O-trans-feruloyl-L-arabinofuranose (F-A), β-D-xylopyranosyl-(1→2)-5-O-

31

trans-feruloyl-L-arabinofuranose (F-A-X), and α-L-galactopyranosyl-(1→2)-β-D-

xylopyranosyl-(1→2)-5-O-trans-feruloyl-L-arabinofuranose (F-A-X-G) (Figure 4).

3.3.2 Chemical characterization of the feruloylated standard materials.

To characterize the isolated high molecular weight standard materials both ferulic acid

contents and carbohydrate compositions were analyzed. Insoluble feruloylated

polysaccharides (100 mg) were saponified with 5 mL of 2 M NaOH solution (previously

purged with nitrogen). The headspace of the tube was purged with nitrogen and samples

were hydrolyzed for 18 h in the dark. The samples were acidified to a pH <2.0 using ca. 1

mL HCl (37% solution). Extraction of the acidified solution was performed three times

with diethyl ether (5 mL each). Extracts were pooled and dried under a stream of

nitrogen. The residue was redissolved in methanol (MeOH)/H2O (50/50 (v/v), 0.5 mL).

The reconstituted sample was filtered through a 0.45 µm filter (Fisherbrand, Pittsburgh,

PA, USA). Appropriately diluted samples were analyzed for their ferulic acid content by

RP-HPLC using a photo diode array detector monitoring the effluent at 325 nm. A Luna

phenyl–hexyl column (250 × 4.6 mm i.d., 5 μm particle size) was used for separation.

Solvents used for the gradient were 1 mM TFA (A), MeOH/1 mM TFA 90/10 (v/v) (B)

and acetonitrile/1 mM TFA 90/10 (v/v) (C), and the gradient was run at 45 °C. The

following gradient program was used (Dobberstein and Bunzel 2010): 87% A and 13 %

C for 10 min, changed over 10 min to 77% A, 3% B, 20% C, over 5 min to 70% A, 5%

B , 25% C, and over 5 min to 25% A, 25% B, 50% C. The gradient was completed by a

10 min equilibration step. Quantification was performed using an external calibration

32

curve. Dialyzed and concentrated feruloylated soluble polysaccharides (1 mL) were

freeze dried. Hydrolysis of the freeze dried residue was performed as mentioned above,

with the exception that 2 mL NaOH solution were used and extractions were performed

with 2 mL diethyl ether per extraction. Dried extracts were redissolved in 0.25 mL

MeOH/H2O (50/50 (v/v)) and analyzed by using RP-HPLC as described above.

Carbohydrate analysis of both the soluble and insoluble feruloylated polysaccharides was

performed by using a modification of the method described by Blakeney et al. (1983).

Monosaccharides were released by a two-step acid hydrolysis (12 M sulfuric acid for 2 h

at room temperature, and 2 M sulfuric acid for 1 h at 100 °C), followed by reduction with

sodium borohydride and acetylation of the formed sugar alcohols. Hydrolysis of the

feruloylated soluble polysaccharides did, however, not require the initial 12 M sulfuric

acid hydrolysis step. Analysis of the alditol acetates was performed using GC-FID

(Finnigan Focus GC Thermo Scientific Inc., Milan, Italy ) on a DB -225 capillary column

(0.25 mm × 30 m, film thickness 0.15 µm) (J&W Scientific, Folsom, CA, USA). The

column temperature was maintained at 180 °C for 5 min, increased to 186 °C (1 °C/min),

increased to 210 °C (4°C/min) and held for 8 min, increased to 220 °C (10 °C/min) held

for 2 min. Helium (3 mL/min) was used as carrier gas. Quantification was performed by

addition of inositol as internal standard. A standard mixture containing arabinose, xylose,

glucose, galacatose, fucose, rhamnose, and mannose was analyzed and response factors

against inositol were calculated for the individual neutral monsaccharides.

33

3.3.3 Method to analyze free ferulic acid (FFA), ferulic acid ester-linked to mono-

/oligosaccharides (OF), ferulic acid ester-linked to soluble polysaccharides (SPF)

and ferulic acid ester-linked to insoluble polysaccharides (IPF)

A scheme of the developed methodology is shown in Figure 5. Phosphate buffer (7 mL,

0.08 M, pH 6.0) and thermostable α-amylase (50 µL) were added to 200 mg of a cereal

sample. The tubes were placed in a boiling water bath and the samples were mixed every

15 min. After 1 h, samples were cooled to room temperature and centrifuged at 500 × g

for 4 min. The residue and supernatant were separated. The residue was washed twice

with 2.5 mL water each and washes were pooled with the supernatant. The volume of the

pooled supernatant/washes was made up to 12 mL and absolute ethanol was added to get

an ethanol concentration of 80% (v/v), resulting in the precipitation of polysaccharides

(80% ethanol suspension). The residue after the water wash was extracted twice with 4

mL 80% ethanol (v/v) each. Each extraction was performed by vortexing (3 times, 15 sec

each) followed by centrifugation at 500 × g for 3 min. The extracts were pooled with the

80% ethanol suspension described above. The residue after water and 80% ethanol

extraction was dried under a stream of nitrogen until visibly dry and hydrolyzed using 5

mL of 2M NaOH (previously purged with nitrogen). The head space of the tube was

purged with nitrogen and hydrolysis was performed for 18 h in the dark. After 18 h,

hydrolysis was stopped by adding ca. 1 mL of 37% HCl to obtain a pH of <2 (more HCl

was added if needed). The solution was extracted three times with diethyl ether (10 mL, 2

34

x 5 mL). The ether fractions were pooled and dried under a stream of nitrogen. The

analyzed ferulic acid represents the IPF.

The pooled 80% ethanol suspension was made up to 70 mL, transferred to centrifuge

tubes, and complete precipitation was allowed to occur overnight. Samples were

centrifuged to separate the precipitate (mostly soluble polysaccharides including SPF)

from the supernatant (mono- and oligosaccharides etc. including OF and FFA). The

supernatant was transferred to a round bottom flask and the precipitate was washed twice

with 5 mL 80% ethanol (v/v) each. The washes were pooled with the supernatant. The

precipitate was dried under a stream of nitrogen, saponified and further treated as

described for the ferulic acid determination of the feruloylated soluble polysaccharide

standard materials. The ferulic acid analyzed in this fraction represents SPF.

The supernatant after centrifugation of the 80% ethanol suspension (80% soluble

material) was evaporated to dryness under vacuum and reconstituted in 5 mL acidified

water (pH 2.0). Reconstitution was performed by first adding 2 mL of pH 2.0 water

(which was transferred to a 25 mL Kimax® tube) followed by rinsing the flask twice

with 2 mL and 1 mL of pH 2.0 water, respectively. The pooled acidified water was

extracted three times with diethyl ether (10 mL, 2 x 5 mL). The ether was first used to

rinse the flask before it was added to the Kimax® tube containing the reconstituted

acidified water. Ether extracts were dried and analyzed for FFA (reconstitution in

MeOH/H2O 50/50 (v/v), RP-HPLC). 4 M NaOH (5 mL, previously purged with nitrogen)

was added to the 5 mL of extracted pH 2.0 water. Hydrolysis, extraction (10 mL, 2 x 5

35

mL diethyl ether) and ferulic acid analysis was performed as described for the standard

materials. The analyzed ferulic acid represents OF.

3.3.4 Method optimization

Optimization and validation of the described method was performed by using the isolated

standards materials. While each step of the methodology was tested for its efficiency,

three key steps were evaluated in-depth before the entire method was tested. The first

critical step to be optimized was the solvent based separation of OF and FFA. F-A

solution was prepared by dissolving 2 mg in 10 mL of distilled water and adding 0.25 mL

of ethanol. Addition of a small amount of ethanol reflects the worst case scenario that,

accidentally, ethanol was not completely removed in the prior drying step mentioned in

the method. The pH of the solution was reduced to <2. The sample was extracted twice

with ethyl acetate (5 mL each). Extracts were pooled and washed with 5 mL water. The

organic phase was dried under vacuum and the residues were redissolved in 0.5 mL

MeOH/H2O (50/50 (v/v)). Samples were analyzed for F-A using RP-HPLC with the same

gradient used for ferulic acid analysis. F-A was found to elute separately and was

quantified using an external F-A calibration curve. This procedure was repeated to test

diethyl ether as the extraction solvent. The second key step to be tested was the stability

of FFA during the treatment of the sample with phosphate buffer and α-amylase at 95 °C

for 1 h. This step was tested by adding α-amylase and FFA to the buffer under the

mentioned conditions and testing the recovery of FFA at 15 min, 30 min and 60 min. The

third key step to be tested was completeness of ferulic acid ester hydrolysis with 2 M

36

NaOH for 18 h and the stability of liberated ferulic acid under these conditions. The

alkaline hydrolysis was tested by using IPF and saponification times of 3 h, 9 h, 18 h, and

27 h. Hydrolysis was stopped by lowering the pH to <2 using HCl (37%) and the

hydrolyzates were analyzed for ferulic acid as described for the standard materials. Also,

ferulic acid was dissolved in 2 M NaOH, extracted at 3 h, 9 h, 18 h, and 27 h and

analyzed by RP-HPLC.

Testing of the complete method was performed by adding known amounts of isolated

standard materials representing the different ferulic acid populations to a starch matrix,

and performing the whole procedure. Sodium dihydrogen phosphate (57 mg) and

disodium hydrogen phosphate (8 mg) were added to a 25 mL Kimax® tube. After

addition of the dissolved standard materials, these buffer salts formed the required 0.08

M phosphate buffer (pH 6.0). Following the addition of 200 mg corn starch (Megazyme,

Bray, Wicklow, Ireland), insoluble feruloylated polysaccharides, free ferulic acid stock

(100 µL of a stock solution in 50/50 MeOH/H2O (v/v)), and F-A (1 mL of a stock

solution in water) were added to the buffer salts such that each of the standard materials

contributed ca. 1.5 mg of ferulic acid to the mixture. Finally, 6 mL of the soluble

feruloylated polysaccharide solution was added. The volume of the feruloylated

polysaccharide solution was added such that it contributes ca. 250 µg of ferulic acid to

the mixture. The reaction mixture was subjected to the described method, and the ferulic

acid concentration of each population was analyzed. Recovery rates were calculated

based on the amounts of added ferulic acid for each population. The ferulic acid contents

of the standard materials and of the separated populations were analyzed by alkaline

37

hydrolysis, extraction and HPLC analysis as described earlier. Additionally, the method

was tested by adding F-A-X and F-A-X-G instead of F-A.

3.3.5 Application of the developed methodology to processed and unprocessed cereal

samples

The developed methodology was applied in triplicate to two sets of processed and

unprocessed wheat bran samples. The first set of wheat bran samples were prepared by

Keith Petrofsky, Moonyeon Youn and Dr. Roger Ruan, University of Minnesota. These

samples were treated for 24 h with 0.1 N NaOH (5% (w/v)) while shaking. Samples were

neutralized by the addition of HCl, and slurry of “optimized bran” (OP) containing a 2 %

solid concentration was created by the addition of distilled water. This slurry was

homogenized, and the resulting suspension was passed through a separator to obtain

“soluble” (SOP) and “insoluble” optimized bran (ISOP) fractions, which were freeze

dried. Data were compared to unprocessed “control” wheat bran (CB).

The second set of wheat bran samples, were obtained from Kampffmeyer Food

Innovation GmbH, Hamburg, Germany. These samples contained control wheat bran

(B1), and three processed samples (B2-B4).

3.4 Results and Discussion

Various approaches for the analysis of ferulic acid and its derivatives in cereal products

were published earlier (Adom and Liu 2002, Moore et al. 2005, Kim et al. 2006, Cuevas

Montilla et al. 2011). None of these methods, however, is able to analyze all four ferulic

38

acid populations of interest (IPF, SPF, OF and FFA). In addition, these methods were not

tested for recovery rates which may lead to incorrect data if used for quantitative

purposes. The fact that IPF, SPF, OF and FFA may differ in their bioavailability makes it,

however, imperative to reliably differentiate and quantify these ferulic acid populations.

In order to develop such a methodology, standard materials representing the ferulic acid

populations of interest were isolated from corn bran (with the exception of the

commercially available ferulic acid). The isolation of a standard material adequately

representing SPF was challenging. Initial attempts to isolate soluble feruloylated

polysaccharides included their precipitation (obtained after de-starching corn bran) with

80% ethanol (v/v) and usage of the dried precipitate as standard material. However, this

material could not be used for method optimization and the determination of recovery

rates because the dried precipitate could not be fully redissolved in an aqueous solution.

Therefore, partially feruloylated soluble polysaccharides were separated from mono-

/oligosaccharides and other low molecular weight materials by dialysis. This was

followed by concentration of the feruloylated polysaccharide solution by partial

evaporation under vacuum, avoiding, however, complete removal of the water.

3.4.1 Characterization of standard compounds

Ferulic acid contents and carbohydrate compositions of the high molecular weight

standard materials were analyzed. The predominant monosaccharide in the feruloylated

soluble polysaccharide standard was glucose (54.4 ± 0.6%), followed by arabinose (19.5

± 1.9%), galactose (15.8 ± 1.2%), and xylose (10.2 ± 0.2%). Thus, the arabinose to

39

xylose ratio (1.9) of the feruloylated soluble polysaccharide standard would be very high

if these monomers were liberated from extracted arabinoxylans only. The unexpected

carbohydrate composition could, however, potentially be explained by the contribution of

carbohydrates from the enzyme preparation. Also, starch fragments may contribute to this

material and explain the high amounts of glucose. In order to prepare the feruloylated

soluble polysaccharide material from corn bran, only α-amylase was used to destarch the

bran. This enzyme cleaves α-D-(1→4)-glycosidic linkages of the starch, thereby usually

generating a mix of oligosaccharides of different chain lengths (van der Maarel et al.

2002). The presence of higher than expected amounts of glucose in the feruloylated

soluble polysaccharide material may be explained by starch fragments large enough to be

retained in the 10 kDa molecular weight cut off dialysis tubing. Insoluble partially

feruloylated polysaccharides, which were isolated from corn bran after destarching and

removal of soluble polysaccharides and other soluble compounds, showed an arabinose to

xylose ratio of 0.5. This is a common ratio for cereal insoluble arabinoxylans (Saulnier et

al. 1995), just as the carbohydrate composition in general is not unusual (32.5 ± 2.1%

glucose (89.9 ± 1.2 % there of cellulosic), 19.2 ± 0.7% arabinose, 40.7 ± 0.4% xylose, 7.5

± 1.1% galactose).

3.4.2 Method development

Published methods to determine ferulic acid derivatives include different ferulic acid

populations and differ in key steps to reliably differentiate between these populations.

One of the first methodologies to differentiate between free, soluble and insoluble

40

populations of ferulic acid was published by Krygier et al. (1982) and was originally

developed to analyze rapeseeds. This method was then also applied to wheat, corn, rice,

oats, and potato flour (Sosulski et al. 1982). Moore et al. (2005) used a similar

methodology to identify the phenolic acids in soft wheat varieties. A different approach

to quantify “free”, “soluble conjugate” and “bound” ferulic acid in corn, wheat, oats, and

rice was published by Adom and Liu (2002). The mentioned methods differ, for example,

in the choice of extraction solvents with, for example, using either ethyl acetate/diethyl

ether (1/1) (Sosulski et al. 1982, Krygier et al. 1982) or just ethyl acetate (Kern et al.

2003) to extract FFA. A recent review on ferulic acid analysis showed the different

conditions used to hydrolyze ester-linked ferulic acid in cereal samples, varying in

hydrolysis time, temperature, and NaOH concentrations (Barberousse et al. 2008). Also,

acidic conditions were applied in the past instead of alkaline saponification.

During the development of this method three essential steps were tested in depth to

ensure that the method reliably differentiates between different (potentially) bioavailable

ferulic acid populations and delivers accurate quantitative results. The first step to test

was the choice of the organic solvent to separate FFA and OF. Use of a suitable solvent is

critical because the polarity of the solvent is the only parameter differentiating between

these two populations. Ethyl acetate and diethyl ether, both used in previously published

studies, were tested for this purpose. Both solvents quantitatively extract FFA, but a co-

extraction of OF had to be minimized. By using F-A as an OF standard compound it was

found that ethyl acetate resulted in a loss of 59.5 ± 6.4% F-A, whereas the use of diethyl

41

ether resulted only in a loss of 3.5 ± 0.7%. Thus, diethyl ether was used to differentiate

between FFA and OF.

With the exception of the FFA fraction, ester-linked ferulic acid in the separated fractions

(OF, SPF, and IPF) needs to be liberated in order to be subsequently analyzed by RP-

HPLC. The standard conditions used in our laboratory, 2 M NaOH for 18 h, were tested

to determine whether all ester-linked ferulic acid is liberated and whether these

conditions contribute to the degradation of already liberated ferulic acid. The results of

these experiments are shown in Figure 6. Maximum liberation of ester-linked ferulic acid

from the partially feruloylated insoluble polysaccharides, which represents the most

complex matrix into which ferulic acid is incorporated in cereal grains, was already

achieved after 3 h (Figure 6a). Thus, a shorter hydrolysis time than used in our

laboratory may be used. Prolonging hydrolysis time to 18 h is, however, not

disadvantageous. Ferulic acid was not degraded within 18 h of hydrolysis (Figure 6b) if

hydrolysis is carried out taking precautions such as degassing the NaOH solution,

purging the headspace with nitrogen etc.

Finally, another step that may be involved in ferulic acid degradation was tested for its

suitability. The first step in the proposed methodology involves heating the sample at 95

°C for 1 h in phosphate buffer in order to destarch cereal based products by using heat-

stable α-amylase. Since ferulic acid can be decarboxylated under harsh conditions, ferulic

acid recovery at 15 min, 30 min, and 60 min was analyzed to ensure that decarboxylation

does not or only minimally occur under these conditions. The recovery rates obtained at

15 min, 30 min, and 60 min were 100.1 ± 3.1 %, 94.6 ± 0.3%, and 96.2 ± 6.7%,

42

respectively, and were not statistically (p < 0.05) different. These data are comparable to

data from Graf (1992) where decarboxylation of ferulic acid when heated at 100 °C for 1

h at pH 4.0 was found to be 5.2%. These results are also in line with data published by

Walter (1967) demonstrating that higher temperatures (>200 °C) are required to

decarboxylate larger amounts of ferulic acid.

3.4.3 Determination of recovery rates

Recovery rates for the different ferulic acid populations were determined by adding

defined amounts of the isolated standard materials representing FFA, OF, SPF, and IPF to

a starch matrix. The methodology was performed as described in Figure 2 and the

recovery of each population was calculated. Results are provided in Table 1. They

demonstrate good accuracy for the determination of IPF, FFA, and, depending on the

compound tested, OF. If F-A was tested as a compound representing OF, recovery rates

were only 79%. However, with increasing number of sugar moieties in the OF standard

compounds (F-A-X and F-A-X-G), the recovery rates went up. The recovery rates for

FFA are indicated with 89%. However, it has to be noted that initial recovery rates were

lower (ca. 75%). Minor modifications of certain steps and, more importantly, getting

more familiar with the complex method increased the recovery rates to 89%. The

recovery rates for soluble polysaccharides were only 70%. This recovery rate is not fully

satisfactory but could not be increased despite of multiple method modifications. One

problem with determining the recovery rate for this fraction was also that a standard

material with defined and consistent characteristics is hard to isolate. For the same reason

43

we could test this ferulic acid population using lower ferulic acid contents only (250 μg

as compared to 1.5 mg for the other fractions).

3.4.4 Application of the method to cereal samples

The applicability of the proposed methodology was tested by using two sets of samples

both containing non-processed and processed wheat bran samples (Tables 2 and 3).

Processing of the first set of wheat bran samples included alkaline treatment (0.1 N

NaOH, 24 h) as a key step to modify the bran’s properties. The processed bran (OP) was

also roughly fractionated into a soluble (SOP) and insoluble (ISOP) portion. Processing

of the bran resulted in the expected large increase of FFA (Table 2). This is in

accordance with data from Parker et al. (2005) who performed sequential hydrolysis on

wheat bran with NaOH solutions of increasing molarity. They found that most of the

ester-linked ferulic acid was released when the bran sample was treated with 0.1 M

NaOH for 24 h. Processing did not result in a large overall loss of ferulic acid as shown

by the comparable amounts of total ferulic acid obtained in CB and OP. It is obvious

from comparing the data for SOP and ISOP that most of the liberated ferulic acid is

present in the SOP fraction. This is somewhat surprising since ferulic acid is not highly

soluble in water.

A second set of unprocessed and processed bran samples was obtained from a grain

processing company. The processing conditions for these samples were based on

fermentation. Detailed processing parameters remain unknown. The data for these

samples are presented in Table 3. In particular, processed sample B4 is worthwhile to

44

discuss since the FFA content in B4 is ca. 20 times higher than in B1 and SPF and OF

were significantly higher than in the control bran B1, too. Thus, the applied processing is

capable of increasing all three populations mentioned above, creating a set of ferulic acid

derivatives that are potentially absorbed at different places in the GI-tract and also

throughout an extended period of time.

Characterization of wheat samples to quantify different ferulic acid populations has been

performed in the past (Adom and Liu 2002, Adom et al. 2003, Moore et al. 2005).

However, the differentiation between all four ferulic acid populations described here was

not possible yet, especially the differentiation between ferulic acid attached to either

water soluble or insoluble polysaccharides. Adom and Liu (2002) found for ground

whole grain wheat that soluble ester-linked ferulic acid populations (“soluble

conjugates”) contributed 1 % to the total ferulic acid content. The vast majority (98.8%)

of the ferulic acid was found in the “bound” population, with only 0.2% FFA contributing

to the ferulic acid pool (Adom and Liu 2002). Data for “soluble conjugates” and FFA

have to be interpreted carefully though since the calculation and methodological details

are not fully described and vary between publications (e.g. redissolving in different

solvents before the HPLC analysis). These data, showing that the majority of ferulic acid

is bound to polymers, confirmed data initially published several decades earlier

(Geissmann and Neukom 1973, Fausch et al. 1963) and described numerous times since.

Moore et al.(2005) compared the ferulic acid content of eight soft wheat varieties (whole

grain, ground) and found FFA values ranging between 0.10 and 0.44 % contributing to

the total ferulic acid content. Between 5.1 - 10.4 % of the total ferulic acid content had its

45

origin from “soluble conjugated ferulic acid”, roughly comparable to our OF fraction, and

89.2 - 94.6% of the ferulic acid was ester-linked to polymers, non-extractable by using

acetone/MeOH/H2O in a 7/7/6 (v/v/v) ratio. Kim et al. (2006) studied the ferulic acid

composition of commercial red and white wheat bran using a similar approach as

described here to differentiate between FFA and OF. Red wheat bran/white wheat bran

contained 0.1%/0.2% FFA, 2.1%/2.1% of “methanol extractable” ester-linked ferulic

acid, roughly comparable to our OF fraction, and 97.8%/97.6% of ferulic acid ester-

linked to non-methanol soluble materials.

In our study the ferulic acid compositions of the unprocessed wheat bran samples did not

show much of a difference. They contained 0.7%/1.2% FFA, 2.7%/2.8% OF, 5.4/7.2%

SPF, and 91.3%/88.8% IPF. The data for FFA and OF are roughly comparable to those

reported by Moore et al. (2005). As reported earlier for ferulic acid but also for ferulic

acid dimers (Renger and Steinhart 2000, Bunzel et al. 2001), a greater portion of the

ferulic acid was found in IPF than in SPF.

While the distribution of different ferulic acid populations in cereal grains has been well

studied over the last decades, the developed method can be used in the future to study the

composition of ferulic acid populations in processed cereal products. Knowledge of this

distribution is the key to understand ferulic acid bioavailiability and also the different

health beneficial effects of ferulic acid derivatives. In fact, feruloylated oligosaccharides

from wheat showed an inhibitory effect on rat erythrocyte hemolysis (Yuan et al. 2005).

Therefore, several studies have focused on the isolation of such oligosaccharides from

cereal matrices (Katapodis et al. 2003, Li et al. 2008, Katapodis and Christakopoulos

46

2008). Such oligosaccharides also have an advantage over FFA in terms of stability,

potentially protecting ferulic acid against decarboxylation reactions during processing.

With better understanding of the bioavailability of ferulic acid derivatives a method to

determine and distinguish different ferulic acid populations becomes vital to observe

effects of processing on cereal matrices.

47

3.5 Literature cited

Adom, K. K. and Liu, R. H. 2002. Antioxidant activity of grains. J. Agric. Food Chem.

50:6182-6187.

Adom, K. K., Sorrells, M. E. and Liu, R. H. 2003. Phytochemical profiles and antioxidant

activity of wheat varieties. J. Agric. Food Chem. 51:7825-7834.

Barberousse, H., Roiseux, O., Robert, C., Paquot, M., Deroanne, C. and Blecker, C. 2008.

Analytical methodologies for quantification of ferulic acid and its oligomers. J. Sci.

Food Agric. 88:1494-1511.

Blakeney, A. B., Harris, P. J., Henry, R. J. and Stone, B. A. 1983. A simple and rapid

preparation of alditol acetates for monosaccharide analysis. Carbohydr. Res. 113:291-

299.

Bourne, L. C. and Rice-Evans, C. 1998. Bioavailability of ferulic acid. Biochem.

Biophys. Res. Commun. 253:222-227.

Bourne, L., Paganga, G., Baxter, D., Hughes, P. and Rice-Evans, C. 2000. Absorption of

ferulic acid from low-alcohol beer. Free Rad. Res. 32:273-280.

Braune, A., Bunzel, M., Yonekura, R. and Blaut, M. 2009. Conversion of

dehydrodiferulic acids by human intestinal microbiota. J. Agric. Food Chem.

57:3356-3362.

Bunzel, M., Ralph, J., Marita, J. M., Hatfield, R. D. and Steinhart, H. 2001. Diferulates as

structural components in soluble and insoluble cereal dietary fibre. J. Sci. Food Agric.

81:653-660.

48

Bunzel, M., Ralph, J., Lu, F., Hatfield, R. D. and Steinhart, H. 2004. Lignins and ferulate-

coniferyl alcohol cross-coupling products in cereal grains. J. Agric. Food Chem.

52:6496-6502.

Cuevas Montilla, E., Hillebrand, S., Antezana, A. and Winterhalter, P. 2011. Soluble and

bound phenolic compounds in different Bolivian purple corn (Zea mays L.) cultivars.

J. Agric. Food Chem. 59:7068–7074.

Dobberstein, D. and Bunzel, M. 2010. Separation and detection of cell wall-bound ferulic

acid dehydrodimers and dehydrotrimers in cereals and other plant materials by

reversed phase high-performance liquid chromatography with ultraviolet detection. J.

Agric. Food Chem. 58:8927-8935.

Fausch, H., Kuendig, W. and Neukom, H. 1963. Ferulic acid as a component of a

glycoprotein from wheat flour. Nature 199:287.

Geissmann, T. and Neukom, H. 1973. A note on ferulic acid as a contsituent of the water-

insoluble pentosans of wheat flour. Cereal Chem. 50:414-416.

Graf, E. 1992. Antioxidant potential of ferulic acid. Free Radical Biol. Med. 13:435-448.

Katapodis, P., Vardakou, M., Kalogeris, E., Kekos, D., Macris, B. J. and

Christakopoulos, P. 2003. Enzymic production of a feruloylated oligosaccharide with

antioxidant activity from wheat flour arabinoxylan. Eur. J. Nutr. 42:55-60.

Katapodis, P. and Christakopoulos, P. 2008. Enzymic production of feruloyl xylo-

oligosaccharides from corn cobs by a family 10 xylanase from Thermoascus

aurantiacus. LWT - Food Sci. Technol. 41 1239-1243.

49

Kern, S. M., Bennett, R. N., Mellon, F. A., Kroon, P. A. and Garcia-Conesa, M. T. 2003.

Absorption of hydroxycinnamates in humans after high-bran cereal consumption. J.


Kim, K.-H., Tsao, R., Yang, R. and Cui, S. W. 2006. Phenolic acid profiles and

antioxidant activities of wheat bran extracts and the effect of hydrolysis conditions.

Food Chem. 95:466-473.

Kroon, P. A., Faulds, C. B., Ryden, P., Robertson, J. A. and Williamson, G. 1997.

Release of covalently bound ferulic acid from fiber in the human colon. J. Agric.

Food Chem. 45:661-667.

Krygier, K., Sosulski, F. and Hogge, L. 1982. Free, esterified, and insoluble-bound

phenolic acids. 1. Extraction and purification procedure. J. Agric. Food Chem.

30:330-334.

Li, K. Y., Lai, P., Lu, S., Fang, Y. T. and Chen, H. H. 2008. Optimization of acid

hydrolysis conditions for feruloylated oligosaccharides from rice bran through

response surface methodology. J. Agric. Food Chem. 56:8975-8978.

Manach, C., Scalbert, A., Morand, C., Remesy, C. and Jimenez, A. 2004.

Polyphenols:food sources and bioavailability. Am. J. Clin. Nutr. 79:727-747.

Moore, J., Hao, Z., Zhou, K., Luther, M., Costa, J. and Yu, L. 2005. Carotenoid,

tocopherol, phenolic acid, and antioxidant properties of Maryland-grown soft wheat.

J. Agric. Food Chem. 53:6649-6657.

Renger, A. and Steinhart, H. 2000. Ferulic acid dehydrodimers as structural elements in

cereal dietary fibre. Eur. Food Res. Technol. 211:422-428.

50

Rondini, L., Peyrat-Maillard, M.-N., Marsset-Baglieri, A. and Berset, C. 2002. Sulfated

ferulic acid is the main in vivo metabolite found after short-term ingestion of free

ferulic acid in rats. J. Agric. Food Chem. 50:3037-3041.

Russell, W. R., Scobbie, L., Chesson, A., Richardson, A. J., Stewart, C. S., Duncan, S.

H., Drew, J. E. and Duthie, G. G. 2008. Anti-inflammatory implications of the

microbial transformation of dietary phenolic compounds. Nutr. Cancer 60:636-642.

Saulnier, L., Vigouroux, J. and Thibault, J. F. 1995. Isolation and partial characterization

of feruloylated oligosaccharides from maize bran. Carbohydr. Res. 272:241-253.

Scalbert, A., Monties, B., Lallemand, J.-Y., Guittet, E. and Rolando, C. 1985. Ether

linkage between phenolic acids and lignin fractions from wheat straw.

Phytochemistry 24:1359-1362.

Shahidi, F. and Chandrasekara, A. 2010. Hydroxycinnamates and their in vitro and in

vivo antioxidant activities. Phytochem. Rev. 9:147-170.

Slavin, J. 2003. Why whole grains are protective: biological mechanisms. Proc. Nutr.

Soc. 62:129-134.

Smith, M. M. and Hartley, R. D. 1983. Occurrence and nature of ferulic acid substitution

of cell-wall polysaccharides in graminaceous plants. Carbohydr. Res. 118:65-80.

Sosulski, F., Krygier, K. and Hogge, L. 1982. Free, esterified, and insoluble-bound

phenolic acids. 3. Composition of phenolic acids in cereal and potato flours. J. Agric.

Food Chem. 30:337-340.

51

van der Maarel, M. J. E. C., van der Veen, B., Uitdehaag, J. C. M., Leemhuis, H. and

Dijkhuizen, L. 2002. Properties and applications of starch-converting enzymes of the

α-amylase family. J. Biotechnol. 94:137-155.

Walter, F., Parker, W. E., Wasserman, A. E. and Doerr, R. C. 1967. Thermal

decomposition of ferulic acid. J. Agric. Food Chem. 15:757-761.

Yuan, X., Wang, J. and Yao, H. 2005. Antioxidant activity of feruloylated

oligosaccharides from wheat bran. Food Chem. 90:759-764.

Zhao, Z., Egashira, Y. and Sanada, H. 2003. Digestion and absorption of ferulic acid

sugar esters in rat gastrointestinal tract. J. Agric. Food Chem. 51:5534-5539.

Zhao, Z., Egashira, Y. and Sanada, H. 2004. Ferulic acid is quickly absorbed from rat

stomach as the free form and then conjugated mainly in liver J. Nutr. 134:3083-3088.

Zhao, Z. and Moghadasian, M. H. 2008. Chemistry, natural sources, dietary intake and

pharmacokinetic properties of ferulic acid: A review. Food Chem. 109:691–702.

Zhao, Z. and Moghadasian, M. H. 2010. Bioavailability of hydroxycinnamates: a brief

review of in vivo and in vitro studies. Phytochem. Rev. 9:133-145.

52

5-O-trans-feruloyl-L-

arabinofuranose (F-A)

β-D-xylopyranosyl-(1→2)-5-O-trans-

feruloyl-L-arabinofuranose (F-A-X)

α-L-galactopyranosyl-(1→2)-β-D-

xylopyranosyl-(1→2)-5-O-trans-

feruloyl-L-arabinofuranose (F-A-X-G)

Figure 4. Feruloylated mono-/oligosaccharides used as standard compounds for

method development and partial validation

53

Hydrolysis with 2 M

NaOH for 18 h

Figure 5. Proposed methodology to separate free ferulic acid (FFA), ferulic acid

ester-linked to mono-/oligosaccharides (OF), to soluble polysaccharides (SPF) and to

insoluble polysaccharides (IPF)

Sample, phosphate buffer (0.08 M, pH 6.0), α-

amylase, 95 °C for 1 h

Residue

Supernatant

Washed with water,

washes combined with

supernatant

80% ethanol (v/v)

extraction

Ethanol addition to obtain a

final concentration of 80% (v/v)

Addition of 80%

ethanol extracts

Residue after 80%

ethanol (v/v)

extraction

Extraction of liberated

ferulic acid with

diethyl ether, analysis

of the dried and

redissolved extracts

for IPF

Overnight precipitation;

centrifugation to separate residue

and supernatant

Supernatant

Washed with 80% (v/v), washes

combined with supernatant

Alkaline hydrolysis,

extraction and

analysis for SPF

Dried under vacuum;

reconstituted in

acidified (pH 2.0)

water

Extraction with

diethyl ether

Analysis of the dried

and redissolved

extracts for FFA

Alkaline hydrolysis of

the aqueous phase,

extraction and analysis

for OF

Residue

54

0.0

10.0

20.0

30.0

40.0

50.0

0 5 10 15 20 25 30

µg

Feru

lic a

cid

/mg

IPF

Time [h]

0

100

200

300

400

500

0 5 10 15 20 25 30

Feru

lic a

cid

[µ

g]

Time [h]

Figure 6a. Liberation of ferulic acid from feruloylated insoluble

polysaccharides by alkaline hydrolysis (2 M NaOH, room temperature, 0 – 27 h)

Figure 6b. Stability of ferulic acid during alkaline hydrolysis (2M NaOH, room

temperature, 0 – 27 h)

55

Table 1. Recovery data for the proposed methodology

% Recovery Average ± SDa

Free ferulic acid (FFA) 89 ± 2%

Ferulic acid ester-linked to insoluble

polysaccharides (IPF) 100 ± 3%

Ferulic acid ester-linked to soluble

polysaccharides (SPF)

70 ± 4%

Ferulic acid ester-linked to mono-

/oligosaccharides (F-A) 79 ± 1%


/oligosaccharides (F-A-X) 101 ± 4%


/oligosaccharides (F-A-X-G) 103 ± 1%

a standard deviation, n=2

56

Table 2. Ferulic acid analysis of sample set 1 (processed and non-processed wheat bran)

Average ± SDa [µg/g ]

Sample IPFb SPF

c OF

d FFA

e Total

Control bran 2895±60 171 ± 9 85 ± 3 21 ± 1 3171 ± 62

Optimized bran 61±11 13 ± 2 89 ± 28 2687 ± 64 2849 ± 54

Insoluble optimized

bran 42±2 2 ± 1 4 ± 1 192 ± 25 241 ± 24

Soluble optimized bran 61±7 19 ± 1 74 ± 5 3631 ± 95 3785 ± 92


b IPF - ferulic acid ester-linked to insoluble polysaccharides

c SPF - ferulic acid ester-linked to soluble polysaccharides

d OF - ferulic acid ester-linked to mono-/oligosaccharides

e FFA - free ferulic acid

57

Table 3. Ferulic acid analysis of sample set 2 (processed and non-processed wheat bran)

Average ± SD

a [µg/g]

Sample IPFd SPF

e OF

f FFA

g Total

B1b 2908 ± 278 236 ± 31 93 ± 11 38 ± 17 3275 ± 271

B2c 3218 ± 234 239 ± 10 76 ± 13 22 ± 2 3554 ± 237

B3c 2527 ± 61 190 ± 10 48 ± 3 152 ± 4 2917 ± 57

B4c 1743 ± 55 558 ± 35 188 ± 13 786 ± 43 3276 ± 113


b B1- control Bran

c B2 - B4 - processed wheat bran samples

d IPF - ferulic acid ester-linked to insoluble polysaccharides

e SPF - ferulic acid ester-linked to soluble polysaccharides

f OF - ferulic acid ester-linked to mono-/oligosaccharides

g FFA - free ferulic acid

58

Chapter 4

Supplementary Data

59

Data provided in this chapter characterize the chemical composition of the samples

described in Chapter 3.3.5, (page37) in more detail. Further analysis was performed to

determine the polysaccharide composition as well as the trans-p-coumaric acid, trans-

sinapic acid, cis-ferulic acid and dehydrodiferulic contents of the first set of samples, i.e.

control bran (CB), optimized bran (OP), insoluble optimized bran (ISOP) and soluble

optimized (SOP).

4.1 Carbohydrate analysis

The neutral monosaccharide composition of the polysaccharides was analyzed by

cleaving the polysaccharides into the monosaccharides and analysis of the liberated

monosaccharides after reduction and derivatization. To cleave cellulose quantitatively

into glucose units a pre-hydrolysis using 12 M sulfuric acid is required to overcome the

strong hydrogen bonding in the crystalline cellulose regions.

In brief, polysaccharide hydrolysis of the samples was performed with sulfuric acid (two

step hydrolysis with 12 M sulfuric acid and 2 M sulfuric acid to hydrolyze cellulose, 2 M

sulfuric acid only to hydrolyze non-cellulosic polysaccharides), followed by reduction

with sodium borohydride and acetylation of the released monosaccharides. The formed

alditol acetates were extracted with chloroform and analyzed using GC-FID. Detailed

procedures are provided in Appendix C-1 and C-2.

The results of the carbohydrate analysis are given in Table 4.

60

Table 4. Neutral monosaccharide composition of control bran (CB), optimized bran

(OP), soluble optimized bran (SOP) and insoluble optimized bran (ISOP) polysaccharides

Average

a

[µg/mg] Standard deviation

[µg/mg]

CB Cb CB H

c

Arabinose 57 7

Xylose 97 5

Mannose 12 3

Galactose 18 9

Glucose 166 8

Glucose Cb 72 10

OP C OP H

Arabinose 49 1

Xylose 81 1

Mannose 7 1

Galactose 12 2

Glucose 144 4

Glucose C 49 6

SOP C SOP H

Arabinose 49 2

Xylose 57 3

Mannose 9 2

Galactose 13 3

Glucose 129 5

Glucose C 40 8

ISOP C ISOP H

Arabinose 33 5

Xylose 88 10

Mannose NDd -

Galactose 25 2

Glucose 127 9

Glucose C 91 2

a n=2;

b C - hellulosic content;

c H - hemicellulosic content;

d ND - not detected

61

4.2 Analysis of trans-p-coumaric acid, trans-sinapic acid and cis-ferulic acid

The esterified hydroxycinnamic acids had to be liberated as described for the trans-

ferulic acid. However, a pre-separation into different populations before saponification

was not attempted. Thus, samples were treated with 2 M NaOH solution (18 h, room

temperature) to liberate trans-p-coumaric acid, trans-sinapic acid, and cis-ferulic acid.

After acidification of the hydrolysate, the liberated hydroxycinnamic acids were extracted

using diethyl ether. Extractions were dried under a stream of nitrogen and reconstituted in

MeOH/H2O (50/50 (v/v)) before being analyzed by RP-HPLC. The detailed methods are

provided in Appendix C-3 and C-4 and sample chromatograms are provided in Appendix

D.

The results for the analysis of these hydroxycinnamic acids are given in Table 5.

62

Table 5. Total contents (free and ester-linked forms) of some hydroxycinnamic acids in

the control bran (CB), optimized bran (OP), soluble optimized bran (SOP), and insoluble

optimized bran (ISOP)

Sample trans-p-Coumaric

acid

trans-Sinapic acid cis-Ferulic acid

Average

a

[µg/g]

Standard

deviation

[µg/g]

Averagea

[µg/g]

Standard

deviation

[µg/g]

Averagea

[µg/g]

Standard

deviation

[µg/g]

CB 74 13 297 59 194 40

OP 91 1 142 9 192 2

SOP 106 9 222 3 256 2

ISOP 13 1 63 3 91 4 a n=2

4.3 Analysis of ferulic acid dehydrodimers and dehydrotrimers

Ferulic acid dehydrodimers and higher oligomers such dehydrotrimers are important

structural components of the plant cell wall but may also be involved in health beneficial

effects due to their antioxidant properties as described in Chapter 2. Since

dehydrodiferulic and dehydrotriferulic acids are formed from monomeric ester-linked

ferulic acid they are ester-linked to plant cell wall polysaccharides, too (and partially

ether-linked to lignin or lignin-like compounds). Therefore, they have to be liberated

before being analyzed by RP-HPLC. Liberation was achieved using alkaline hydrolysis

(2 M NaOH, 18 h, room temperature). The liberated dehydrodiferulic and

63

dehydrotriferulic acids and the internal standard, 5-5(methylated)-dehydrodiferulic acid,

were extracted with diethyl ether after acidification of the hydrolysate. The organic phase

was dried and the residue was reconstituted as described for the monomeric

hydroxycinnamic analysis described in Chapter 4.2. Samples were analyzed using RP-

HPLC. The detailed methods are provided in Appendix C-5 and C-6. Supporting

chromatograms are provided in Appendix D. The results of the analysis of ester-linked

dehydrodiferulic and dehydrotriferulic acids in the samples are given in Table 6.

64

Table 6. Contents of ester-linked dehydrodiferulic and dehydrotriferulic acids in the

control bran (CB), optimized bran (OP), soluble optimized bran (SOP) and insoluble

optimized bran (ISOP)

CB OP SOP ISOP

Dimer/Trimera Average

b

[µg/g]

SDc

[µg/g]

Average

[µg/g]

SD

[µg/g]

Average

[µg/g]

SD

[µg/g]

Average

[µg/g]

SD

[µg/g]

8-8c -DFA

139 5 137 19 160 5 72 11

8-8nc-DFA 59 2 67 7 72 4 46 5

8-8THF-DFA NDd - ND - ND - ND -

SUM 197

204

232

118

8-5nc

-DFA 104 4 49 6 58 2 23 3

8-5c-DFA 433 22 58 15 BQLe - BQL -

8-5dc-DFA 33 8 BQL - BQL - BQL -

Sum 570

108

58

0

5-5-DFA 224 8 199 1 224 5 82 5

8-O-4-DFA 156 8 151 3 172 4 68 4

5-5/8-O-4-TFA 123 12 174 8 174 6 78 20

8-O-4/8-O-4-

TFA +f - + - + - + -

8-8c/8-O-4- TFA ND - ND - ND - ND -

Σ DFAs + TFAs 1271 835 860 369 a DFA - dehydrodiferulic acid; TFA – dehydrotriferulic acid, c – cyclic; nc – noncyclic;

dc - decarboxylated; THF – tetrahydrofuran b n=3

c SD - standard deviation

d ND - not detected

e BQL - below quantification limit

f + - detected but since a correction factor is not available for this compound

quantification was not possible

65

4.4 Sample screening to optimize process conditions

Besides the industry samples which were described in Chapter 3 other samples from the

same company were screened to optimize their processing conditions. Since these data

were just required to steer the process, samples were not analyzed in duplicate or

triplicate. While these data cannot be linked to process conditions (processing parameters

are unknown) they demonstrate the applicability of the method to samples with large

variations among the different ferulic acid populations. The analytical procedure was

described in detail in Chapter 3. Supporting chromatograms are provided in Appendix D.

The results of this screening process can be found in Table 7.

Table 7. Ferulic acid contents of different ferulic acid containing populations (ferulic

acid ester-linked to insoluble polysaccharides (IPF), to soluble polysaccharides (SPF), to

oligosaccharides (OF) as well as free ferulic acid (FFA)) separated from differently

processed wheat bran samples

Ferulic acid [µg /g wheat bran sample]

Sample IPF SPF

OF

FFA

Total

2.6 1679 740 505 203 3128

2.7 1284 820 685 208 2997

2.8 1242 867 803 275 3186

2.9 1052 698 932 216 2899

3.3 1754 755 456 314 3279

3.4 1786 678 236 730 3430

3.5 1513 700 354 708 3275

3.6 1642 708 302 572 3225

66

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Appendices

78

Appendix A. List of Instruments

1) pH Meter: Corning

Corning, NY, USA

1) Magnetic stirrer: Chemglass

Vineland, NJ, USA

2) Analytical balance: Shimadzu

Columbia, MD,USA

3) Balance: H&C Weighing Systems™

Columbia, MD, USA

4) Centrifuge: BD Diagnostic Systems

Franklin Lakes, NJ, USA

5) Incubator shaker: New Brunswick Scientific Co.,INC.

Edison, NJ, USA

6) Shaker bath: Pegasus Scientific Inc.

Rockville, MD, USA

7) Vacuum oven: Stifflers Surplus, INC.

Chandler, AZ, USA

8) Rotovap: Büchi Corporation

New Castle, DE, USA

9) Freeze dryer: Labconco Corporation

Kansas City, MO, USA

10) Freeze dryer: SP Scientific

79

Gardiner, NY, USA

11) Sonicator: All Spec Industries

Wilmington, NC, USA

12) HPLC system: Shimadzu

Columbia, MD, USA

(HPLC Components: SPD M20A Photo Diode Array Detector, CBM 20A

Communication Bus Module DGU 20A Degasser, SIL 10AF Autosampler, CTO

20A Column Oven)

13) GC-FID system: Thermo Scientific Inc.,

Milan, Italy

80

Appendix B. List of Chemicals

Name of chemical Hazard symbol Manufacturer

details H

ealth F

ire R

eactivity

Personal

Protection

Acetic acid 3 2 0 H Fisher Scientific,

Pittsburgh, PA, USA

Acetic anhydride 3 2 0 - Sigma Aldrich,

St. Louis, MO, USA

Acetone 2 3 0 H Fisher Scientific,

Pittsburgh, PA, USA

Acetonitrile 2 3 0 H Fisher Scientific,

Pittsburgh, PA, USA

Ammonium

hydroxide solution

3 0 0 - Sigma Aldrich,

St. Louis, MO, USA

p-Coumaric acid 2 0 0 - Acros Organics,

Fairlawn, NJ, USA

Diethyl ether 2 2 0 H Sigma Aldrich,

St. Louis, MO, USA

meso-Erythritol 1 1 0 E Alfa Aesar,

Ward Hill, MA, USA

Ethanol 2 3 0 H Decon Labs, Inc.,

King of Prussia, PA,

USA

Ethyl acetate 2 3 0 G Sigma Aldrich,

St. Louis, MO, USA

trans-Ferulic acid 2 1 0 E MP Biomedicals Inc.,

Solon, OH, USA

Fucose 1 1 0 E Acros Organics,

Fairlawn, NJ, USA

Galactose 2 1 0 E Sigma Aldrich,

St. Louis, MO, USA

Glucose 0 0 0 - Sigma Aldrich,

St. Louis, MO, USA

Hydrochloric Acid

(37%)

3 0 1 - Sigma Aldrich,

St. Louis, MO, USA

Inositol 1 1 0 E Fluka,

St Louis, MO, USA

Methanol 2 3 0 H Sigma Aldrich,

St. Louis, MO, USA

Mannose 1 1 0 E Sigma Aldrich,

St. Louis, MO, USA

81

Name of Chemical

Hazard Symbol Manufacturer

Details

Health

Fire

Reactivity

P

ersonal

Protection

n-Methylimidazole 3 2 0 - MP Biomedicals Inc.,

Solon, OH, USA

Methyl sulfoxide

1 2 0 F Acros Organics,

Fairlawn, NJ, USA

Rhamnose 1 1 0 E Sigma Aldrich,

St. Louis, MO, USA

Sinapic acid 2 0 0 - Fluka,

St Louis, MO, USA

Sodium hydroxide 3 0 2 J Fisher Scientific,

Pittsburgh, PA, USA

Sodium phosphate

dibasic

1 0 0 E Sigma Aldrich,

St. Louis, MO, USA

Sodium phosphate

monobasic

1 0 0 E Sigma Aldrich,

St. Louis, MO, USA

Starch (maize) 1 1 0 E Bray, Wicklow,

Ireland

Sulfuric acid 3 1 2 - EM Science,

Gibbstown, NJ, USA

Trifluoroacetic acid 3 0 0 H Alfa Aesar,

Ward Hill, MA, USA

82

Appendix C. Detailed Procedures

C-1. Determination of the carbohydrate composition of the control bran (CB),

optimized bran (OP), insoluble optimized bran (ISOP) and soluble optimized bran

(SOP)

The carbohydrate compositions of these samples were analyzed using a modified method

described by Blakeney et al. (1983).

C-1.1. Polysaccharide hydrolysis (cellulose analysis)

Sulfuric acid (0.5 mL, 12 M) was added to the samples (30 mg) in small (5 mL) Kimax®

tubes. The tubes were placed in an ice bath for 30 min and vortex-mixed every 5 min.

The samples were then allowed to stand at room temperature for 2 h (vortexed every 15

min). The suspension was subsequently diluted to 3 mL with water resulting in a 2M

sulfuric acid concentration. The suspension was placed in a boiling water bath and mixed

after 20 and 40 min. After 1 h, the suspension was brought down to room temperature.

The pH of the suspension was adjusted to 8 - 9 using 25% ammonium hydroxide.

Samples were filtered and transferred into a 10 mL volumetric flask. The tubes were

rinsed with distilled water, and the washes were used to make up the volume to the 10

mL mark.

C-1.2. Polysaccharide hydrolysis (analysis of the non-cellulosic polysaccharides)

Sulfuric acid (3 mL, 2 M) was added to the samples (30 mg) in small (5mL) Kimax®

tubes. The suspension was placed in a boiling water bath and mixed after 20 and 40 min.

After 1 h, the suspension was brought down to room temperature. The pH of the

suspension was adjusted to 8 - 9 using 25 % ammonium hydroxide. Samples were filtered

83

and transferred into a 10 mL volumetric flask. The tubes were rinsed with distilled water,

and the washes were used to make up the volume to the 10 mL mark.

C-1.3. Reduction and acetylation of the monosaccharides, extraction of alditol

acetates

An aliquot of the filtered suspension (100 µL) was transferred into a 15 mL Kimax®

tube. Freshly prepared sodium borohydride in DMSO (2% (w/v), 100 µL per sample) was

added. The tubes were placed in a 60 °C incubator and shaken constantly at medium

speed. After 1 h, the samples were cooled to room temperature, and 100 µL of 80% acetic

acid (v/v) containing the internal standard inositol (0.8mg/mL) were added to the reduced

suspension. Acetylation of the suspension was initiated by adding 2 mL acetic anhydride

and 200 µL of the catalyst n-methylimidazole. The reaction was allowed to occur for 10

min. Samples were then cooled in an ice bath and 5 mL of water was added. The samples

were extracted with 2 mL chloroform. The chloroform phase was washed with water

twice and the washes were discarded. The extracts were placed in the freezer overnight to

freeze out any remaining water. The extracted alditol acetates were analyzed using gas

chromatography with flame ionization detection (GC-FID).

C-1.4. Analysis of neutral sugars to determine correction factors

A standard solution was prepared by dissolving arabinose, xylose, fucose, rhamnose,

glucose, mannose, and galactose in distilled water (0.5 mg/mL) each. A 100 µL aliquot of

this standard solution was treated (reduction, acetylation, and extraction of alditol

acetates) as described for the samples. The correction factors shown in Table 8 were used

for quantification

84

C-2. Separation and detection of the alditol acetates by GC-FID

Column Specifications:

DB-225 capillary column (0.25 mm × 30 m, film thickness 0.15 µm) (J&W Scientific,

Folsom, CA, USA).

Detector: FID

Carrier Gas: Helium (3 mL/min)

Make up gas: Nitrogen

Injector temperature: 225°C

Split ratio: 1/25

Equilibration time: 10 min

Temperature gradient:

The column temperature was maintained at 180 °C for 5 min, increased to 186 °C

(1°C/min), increased to 210 °C (4°C/min) and held for 8 min, increased to 220 °C (10

°C/min) held for 2 min.

Table 8. Correction factors for the analysis of monosaccharides in form of their

alditolacetates against acetylated inositol

Monosaccharide Calculated correction

factor

Rhamnose 0.76

Fucose 1.11

Arabinose 0.98

Xylose 1.06

Mannose 1.11

Galactose 0.94

Glucose 1.32

85

C-3. Alkaline extraction and analysis of trans-p-coumaric acid, cis-ferulic acid, and

trans-sinapic acid

100 mg of the sample were weighed into a medium sized Kimax® tube and 5 mL of 2 M

NaOH (previously purged for several minutes with nitrogen) were added. The headspace

of the tube was purged with nitrogen, and samples were hydrolyzed for 18 h in the dark.

The samples were acidified to a pH <2.0 using ca. 1 mL HCl (37% solution). Samples

were extracted three times with diethyl ether (10 mL, 2 × 5 mL). The ether fractions were

pooled and dried under a stream of nitrogen. The residue was reconstituted in

MeOH/H2O (50/50 (v/v), 0.5 mL). Reconstituted samples were filtered through a

membrane filter (0.45 µm, Fisherbrand, Pittsburgh, PA, USA). Appropriately diluted

samples were analyzed for trans-sinapic acid, trans-p coumaric acid, and cis-ferulic acid

using RP-HPLC. Chromatograms were monitored at 325 nm, and quantification was

done using an external calibration curve for the respective hydroxycinnamic acids.

C-4. Analysis of of trans-p-coumaric acid, cis-ferulic acid, and trans-sinapic acid by

RP-HPLC

The analysis of these hydroxycinnamic acid monomers was performed using RP-HPLC.

Column: Luna phenyl hexyl column (250 mm × 4.6 mm i.d., 5 μm particle size)

(Phenomenex, Torrace, CA, USA).

Guard column: Luna phenyl hexyl cartridge (3 mm × 4.6 mm i.d.)

Column temperature: 45°C

86

The following solvents were used to form a gradient (Table 9) at a flow rate of 1

mL/min:

Solvent A: 1 mM trifluoroacetic acid (TFA)

Solvent B: acetonitrile/ 1mM TFA (90/10 (v/v))

Solvent C: MeOH/1 mM TFA (90/10 (v/v))

Table 9. Gradient to analyze hydroxycinnamic acid monomers by RP-HPLC

Solvent concentrations Time [min] Action

A B C

87% 13% 0% 10 hold

77% 20% 3% 10 linear increase



87% 13% 0% 10 hold

C-5. Extraction and analysis of dehydrodiferulic and dehydrotriferulic acids

Alkaline hydrolysis, extraction, and reconstitution were performed as described for the

hydroxycinnamic acid monomers (C-3). However, before acidification, an internal

standard (5-5(methylated)- dehydrodiferulic acid in MeOH/H2O (v/v), 15 µg) was added.

The reconstituted sample was analyzed using RP-HPLC. Chromatograms were monitored

at 280 nm, and quantification was done using correction factors of the individual dimers

and trimers (Table 11) against the internal standard.

87

C-6. Analysis of dehydrodiferulic and dehydrotriferulic acids by RP-HPLC

The analysis of ferulic acid dimers was performed using RP-HPLC.

Column: Luna phenyl hexyl column (250 mm × 4.6 mm i.d., 5 μm particle size)

(Phenomenex, Torrace, CA, USA).

Guard column: Luna phenyl hexyl cartridge (3 mm × 4.6 mm i.d.)

Column temperature: 45°C

The following solvents were used to form a gradient (Table 10) at a flow rate of 1

mL/min:

Solvent A: 1 mM TFA

Solvent B: Acetonitrile/ 1mM TFA (90/10 (v/v))

Solvent C: MeOH/1 mM TFA (90/10 (v/v))

Table 10. Gradient to analyze dehydrodiferulic and dehydrotriferulic acids by RP-HPLC

Solvent Concentrations Time [min] Action

A B C

85% 15% 0% initial






65% 30% 5% 5 hold

55% 40% 5% 10 linear

88

Table 11: Correction factors used for ferulate dimers and trimers quantification using

5-5(methylated)-dehydrodiferulic acid as internal standard

Ferulate dimer/trimer Correction factor

8-8(cyclic)-dehydrodiferulic acid 4.350

8-8(noncyclic)-dehydrodiferulic acid 1.936

8-8(tetrahydrofuran)-dehydrodiferulic acid 6.040

8-5(noncyclic)-dehydrodiferulic acid 1.509

8-5(cyclic)-dehydrodiferulic acid 4.597

8-5(decarboxylated) -dehydrodiferulic acid 1.341

5-5-dehydrodiferulic acid 1.514

8-O-4-dehydrodiferulic acid 0.845

5-5/8-O-4-dehydrotriferulic acid 2.089

8-8(cyclic)/8-O-4-dehydrotriferulic acid 3.591

89

Appendix D. Chromatograms

Figure 7. trans-Ferulic acid standard chromatogram at 325 nm

Figure 8. Ferulic acid obtained from saponification of the IPF population (ferulic acid

ester-linked to insoluble polysaccharides) from the control bran (monitored at 325 nm)

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

0

100

200

300

400

500

6001: 325 nm, 4 nm

100 ug/mL

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

0

200

400

600

800

1000

1200

1400

1: 325 nm, 4 nm

Insol Bound 1:5

trans-ferulic acid

90

Figure 9. Ferulic acid obtained from saponification of the IPF population (ferulic acid

ester-linked to insoluble polysaccharides) from the soluble optimized bran (monitored at

325 nm)

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

-5

0

5

10

15

20

25

30

1: 325 nm, 4 nm

Insoluble Bound 1:5

trans-ferulic acid

91

Figure 10. Free ferulic acid extracted from the control bran (monitored at 325 nm)

Figure 11. Free ferulic acid extracted from the soluble optimized bran (monitored at 325

nm)

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

-10

0

10

20

30

40

50

1: 325 nm, 4 nm

FFA Undiluted

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

0

200

400

600

800

1000

1200

1400

1600

1: 325 nm, 4 nm

FFA 1:5

trans-ferulic acid

trans-ferulic acid

92

Figure 12. Ferulic acid obtained from saponification of the OF population (ferulic acid

ester-linked to mono-/oligosaccharides) from the control bran (monitored at 325 nm)

Figure 13. Ferulic acid obtained from saponification of the SPF population (ferulic acid

ester-linked to soluble polysaccharides) from the control bran (monitored at 325 nm)

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

0

50

100

150

200

250

300

350

1: 325 nm, 4 nm

Oligo Bound Undiluted

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

-50

0

50

100

150

200

250

300

350

400

450

1: 325 nm, 4 nm

Sol Pol Bound Undiluted

trans-ferulic acid

trans-ferulic acid

93

Figure 14. Ferulic acid obtained from saponification of the OF population (ferulic acid

ester-linked to mono-/oligosaccharides) from the sample B1 (monitored at 325 nm)


ester-linked to soluble polysaccharides) from the sample B1 (monitored at 325 nm)

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

-50

0

50

100

150

200

250

300

350

400

4501: 325 nm, 4 nm

Oligo Bound Bran undiluted

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

0

100

200

300

400

500

1: 325 nm, 4 nm

Sol Pol Bound Bran undiluted

trans-ferulic

acid

trans-ferulic

acid

94

Figure16. Ferulic acid obtained from saponification of the OF population (ferulic acid

ester-linked to mono-/oligosaccharides) from sample 2.8 (monitored at 325 nm)


ester-linked to soluble polysaccharides) from sample 2.8 (monitored at 325 nm)

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

0

200

400

600

800

10001: 325 nm, 4 nm

Oligo Bound 1:2

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

200

400

600

800

1000

1200

1400

1600

1800

2000

mA

u

0

200

400

600

800

1000

1: 325 nm, 4 nm

Sol Pol 1:2

trans-ferulic

acid

trans-ferulic

acid

95

Figure 18. trans-Sinapic acid standard chromatogram (monitored at 325 nm)

Figure 19. trans-p-Coumaric acid standard chromatogram (monitored at 325 nm)

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

50

100

150

200

250

300

350

400

mA

u

-20

0

20

40

60

80

100

120

140

160

180

1: 325 nm, 4 nm

58 ug/mL

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

50

100

150

200

250

300

350

400

mA

u

-10

0

10

20

30

40

50

60

1: 325 nm, 4 nm

Std 20 ug

trans-sinapic

acid

trans-p-coumaric

acid

96

Figure 20. cis-Ferulic acid standard chromatogram (monitored at 325 nm). Since cis-

ferulic acid is prepared from trans-ferulic acid and since it is used without further

purification the chromatogram contains trans-ferulic acid, too)

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

50

100

150

200

250

300

350

400

mA

u

0

20

40

60

80

100

1: 325 nm, 4 nm

19.62 ug/mL

trans-ferulic acid cis-ferulic acid

97

Figure 21. trans-Ferulic acid, cis-Ferulic acid trans-Sinapic and trans-p-Coumaric acid

obtained from saponification of control bran (monitored at 325 nm).

Figure 22. Analysis of dehydrodiferulic (DFA) and dehydrotriferulic (TFA) acids after

saponification of the optimized bran (monitored at 280 nm). c - cyclic, nc - noncyclic.

Minutes

15 16 17 18 19 20 21 22 23 24 25 26 27 28

mA

u

0

50

100

150

200

250

300

350

400

mA

u

0

50

100

150

200

250

300

350

1: 325 nm, 4 nm

Oligo Bound Undiluted

Minutes

20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0

mA

u

0

50

100

150

200

250

300

350

400

mA

u

0

500

1000

1500

2000

2500

3000

1: 325 nm, 4 nm

OP3

8-8c-

DFA

8-8nc-

DFA 8-5nc-

DFA

5-5-DFA 8-O-4-DFA

8-5c-DFA

5-5/8-O-4-TFA

Internal Standard

8-O-4/8-O-4-

TFA

trans-p-coumaric acid

trans-ferulic acid trans-sinapic acid

cis- ferulic acid

98

Figure 23. GC-FID standard chromatogram showing the analysis of monosaccharides in

form of their alditol acetates

Rh

amn

ose

Fuco

se

Ara

bin

ose

Xyl

ose

Man

no

se

Gal

acto

se

Glu

cose

Ino

sito

l

99


isolated feruloylated insoluble polysaccharide standard in form of their alditol acetates

(Chapter 3.3.2)

Ara

bin

ose

Xyl

ose

Gal

acto

se

Glu

cose

In

osi

tol

100

Figure 25. Separation of hemicellulosic monosaccharides from the isolated feruloylated

insoluble polysaccharide standard in form of their alditol acetates (Chapter 3.3.2)

Ino

sito

l

Ara

bin

ose

Xyl

ose

Gal

acto

se

Glu

cose

101

Figure 26. Separation of hemicellulosic monosaccharides from the isolated feruloylated

soluble polysaccharide standard in form of their alditol acetates (Chapter 3.3.2)

Ara

bin

ose

Xyl

ose

Gal

acto

se

Glu

cose

Ino

sito

l

102


optimized bran in form of their alditol acetates (Chapter 3.3.5)

Ara

bin

ose

Xyl

ose

Gal

acto

se

Glu

cose

Ino

sito

l

103

Figure 28. Separation of hemicellulosic monosaccharides from the optimized bran in

form of their alditol acetates (Chapter 3.3.5)

Ara

bin

ose

Xyl

ose

Glu

cose

Ino

sito

l

Gal

acto

se

104

Appendix E. Standard Curves

Figure 29. trans-Ferulic acid standard curve (external calibration , RP-HPLC)

Figure 30. trans-Sinapic acid standard curve (external calibration, RP-HPL

y = 96595x - 5820.6 R² = 0.9999

0

5000000

10000000

15000000

20000000

25000000

0.00 50.00 100.00 150.00 200.00 250.00

Are

a at

32

5 n

m

Concentration [µg/mL]

trans-Ferulic acid standard curve

y = 44780x + 60440 R² = 0.9999

0

2000000

4000000

6000000

8000000

10000000

12000000

0.00 50.00 100.00 150.00 200.00 250.00

Are

a at

32

5 n

m


trans-Sinapic acid standard curve

105

Figure 31. trans-p-Coumaric acid standard curve (external calibration, RP-HPLC)

Figure 32. cis–Ferulic acid standard curve (external calibration, RP-HPLC)

y = 88103x - 68.083 R² = 0.9999

0

500000

1000000

1500000

2000000

2500000

0.00 5.00 10.00 15.00 20.00 25.00

Are

a at

32

5 n

m


trans-p-Coumaric acid standard curve

y = 65813x + 149970 R² = 0.9989

0

1000000

2000000

3000000

4000000

5000000

6000000

0.00 20.00 40.00 60.00 80.00 100.00

Are

a at

32

5 n

m


cis-Ferulic acid standard curve

development and application of a methodology to …

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