Characterization of Free and Cell-Wall-Bound Phenolic Compounds ...

Aus dem Institut für Humanernährung und Lebensmittelkunde
der Agrar- und Ernährungswissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel
Characterization of Free and
Cell-Wall-Bound Phenolic Compounds in
Chinese Brassica Vegetables
Dissertation
Zur Erlangung des Doktorgrades
der Agrar- und Ernährungswissenschaftlichen Fakultät
der Christian-Albrechts-Universität zu Kiel
Dekan: Prof. Dr. Joachim Krieter
vorgelegt von
M.Sc. Britta Harbaum
aus Itzehoe
Kiel, 2007
Erster Berichterstatter: Prof. Dr. Karin Schwarz
Zweiter Berichterstatter: Prof. Dr. Wolfgang Bilger
Tag der mündlichen Prüfung: 8. November 2007

Die vorliegende Arbeit wurde auf Anregung und unter der Leitung von
Frau Prof. Dr. Karin Schwarz
in der Abteilung Lebensmitteltechnologie am Institut für Humanernährung und
Lebensmittelkunde der Christian-Albrechts-Universität zu Kiel in der Zeit von April 2004 bis
September 2007 durchgeführt und entstand im Rahmen des DFG-Projektes „Investigation of
phenolic compounds and antioxidative potential in Chinese Brassica vegetables“.
Mein besonderer Dank gilt meiner Doktormutter Prof. Dr. Karin Schwarz, die mir die
Möglichkeit gegeben hat, die vorliegende Arbeit in ihrer Abteilung durchzuführen, sowie für
die Schaffung des sehr angenehmen und konstruktiven Arbeitsklimas.
Herrn Prof. Dr. Wolfgang Bilger danke ich für die Übernahme der zweiten Berichterstattung.
Mein weiterer Dank gilt meinen Betreuern Eva Maria Hubbermann und Heiko Stöckmann
sowie allen weiteren Mitarbeitern und Doktoranden aus der Abteilung
Lebensmitteltechnologie für die schöne gemeinsame Zeit. Ein ganz besonderer Dank gilt
Stephanie thor Straten, die unermüdlich am Chinakohl-Projekt mitgearbeitet hat und durch
ihren Fleiß und ihre Verlässlichkeit maßgeblich zum Gelingen der Arbeit beigetragen hat.
Mein weiterer Dank gilt Prof. Dr. Zhujun Zhu für die gute Zusammenarbeit und die
freundliche Aufnahme während meines Aufenthaltes in China, sowie Ni Xiaolei und Yang
Jing für die Pflanzenanzucht und –verarbeitung und die schöne Zeit in China.
Des Weiteren danke ich der Leitung und der spektroskopischen Abteilung des Otto-Diels-
Institutes für Organische Chemie der CAU Kiel, insbesondere Herrn Dr. Christian Wolff, für
die NMR Messungen und Auswertungen.
Schließlich danke ich meinem Verlobten Jan, meiner Mutter Erika, Wiebke, Frauke und Rolf
für die Liebe, seelische Unterstützung und den Ansporn während der gesamten Zeit.

TABLE OF CONTENTS
TABLE OF CONTENTS 1
GENERAL INTRODUCTION 3
1. INTRODUCTION 4
2. OBJECTIVES OF THE THESIS 5
3. THEORETICAL BACKGROUND 7
3.1. POLYPHENOLS 7
3.1.1. Free Phenolic Compounds 7
3.1.2. Cell-Wall-Bound Phenolic Compounds 9
3.2. BIOSYNTHESIS AND FUNCTION OF POLYPHENOLS 10
3.3. BIOAVAILABILITY OF POLYPHENOLS 13
3.4. POLYPHENOLS IN BRASSICA VEGETABLES 14
3.5. CHINESE BRASSICA VEGETABLES 16
3.5.1. Nutritional Relevance 16
3.5.2. Cultivation Conditions 17
3.5.3. Vegetable Processing 17
4. MATERIALS AND METHODS 19
4.1. PLANT MATERIAL 19
4.2. DETERMINATION OF PHENOLIC COMPOUNDS 21
4.2.1. Free Phenolic Compounds 22
4.2.1.1. Extraction 22
4.2.1.2. Qualitative Analysis by HPLC-ESI-MS n 22
4.2.1.3. Structural Elucidation 24
4.2.1.3.1. Compound Isolation from Plant Material 24
4.2.1.3.2. NMR-Spectroscopy 25
4.2.1.4. Quantitative Analysis by HPLC-DAD 25
4.2.1.5. Total Phenolic Content (Folin Ciocalteu) 26
4.2.2. Cell-Wall-Bound Phenolic Compounds 27
4.2.2.1. Extraction of the Cell Wall 27
4.2.2.2. Hydrolysis Reaction of Cell Wall and Extract Preparation 27
4.2.2.3. Qualitative and Quantitative Analysis by HPLC 28
4.3. DETERMINATION OF ASCORBIC ACID 28
1

4.4. ANTIOXIDATIVE CAPACITY ASSAYS 28
5. LITERATURE CITED 30
LIST OF PAPERS AND MANUSCRIPTS 37
CHAPTER I 39
IDENTIFICATION OF FLAVONOIDS AND HYDROXYCINNAMIC ACIDS IN PAK CHOI VARIETIES
(BRASSICA CAMPESTRIS L. SSP. CHINENSIS VAR. COMMUNIS) BY HPLC-ESI-MS N AND NMR
AND THEIR QUANTIFICATION BY HPLC-DAD
CHAPTER II 43
IMPACT OF FERMENTATION ON PHENOLIC COMPOUNDS IN LEAVES OF PAK CHOI (BRASSICA
CAMPESTRIS L. SSP. CHINENSIS VAR. COMMUNIS) AND CHINESE LEAF MUSTARD (BRASSICA
JUNCEA COSS)
CHAPTER III 47
CELL-WALL-BOUND PHENOLIC COMPOUNDS IN LEAVES OF PAK CHOI (BRASSICA
CAMPESTRIS L. SSP. CHINENSIS VAR. COMMUNIS)
CHAPTER IV 65
CELL-WALL-BOUND PHENOLIC COMPOUNDS IN LEAVES OF CHINESE BRASSICA VEGETABLES
AND THE INFLUENCE OF POST-HARVEST TREATMENTS
CHAPTER V 85
FREE AND BOUND PHENOLIC COMPOUNDS IN LEAVES OF PAK CHOI (BRASSICA CAMPESTRIS
L. SSP. CHINENSIS VAR. COMMUNIS) AND CHINESE LEAF MUSTARD (BRASSICA JUNCEA
COSS)
GENERAL DISCUSSION 89
GENERAL SUMMARY 93
ZUSAMMENFASSUNG 96
ABSTRACT 99
KURZDARSTELLUNG 100
2

GENERAL INTRODUCTION
Characterization of Free and
Cell-Wall-Bound Phenolic Compounds in
Chinese Brassica Vegetables
- Introduction
- Objectives of the Thesis
- Theoretical Background
- Materials and Methods
Dissertation
M. Sc. Britta Harbaum
Institut für Humanernährung und Lebensmittelkunde
der Christian-Albrechts-Universität zu Kiel
3

1. Introduction
A high intake of fruits and vegetables is associated with a reduced risk of coronary heart
disease and cancer. Plants are rich in polyphenols, which are considered to play a beneficial
role as bioactive compounds. Phenolcarboxylic acids and flavonoids are among the most
important polyphenols in plants. Polyphenols possess important antioxidative properties and
exert anticancerogenic, antimutagenic, and antiviral action. Antioxidants are currently in the
scientific limelight, and the significance of non-vitamin antioxidants has been recognized.
However, limited data are available on the phenolic content of different plant cultivars. More
information is necessary to calculate more precisely the dietary uptake in nutritional studies.
For food scientists and nutrition researchers, it is very important to identify the healthprotective
constituents in plants as well as these substances’ actions (e.g. antioxidative
potential). Therefore, the complexity and variety of these compounds in foods and vegetables
requires further investigation.
It has been shown that Brassica species potentially exert inhibitory activity against chronic
diseases like cancer due to their phytochemical content (1). Brassica vegetables are a
significant source of polyphenols. Several studies have investigated the qualitative and
quantitative contents of polyphenols. However, there is only scant knowledge about the
phenolic composition of Chinese Brassica vegetables (Chinese cabbage pak choi and Chinese
leaf mustard). In China, cabbages figure prominently in the human diet, but Chinese
cabbages, such as pak choi or Chinese leaf mustard, are not well known in Europe. However,
the depressed cabbage market in Germany may be offset by increasing consumption of
foreign produce such as Chinese cabbages (2).
Polyphenol content may vary in plants depending e.g. on climatic conditions and harvest
season. Moreover, differences in polyphenol content can be expected between different
cultivars as well as within individual plants (e.g. blades vs. stalks; outer and inner leaves).
Food processing may also affect polyphenol content.
Phenolic compounds have been found in significant quantities in the cell walls of plants.
These compounds are esterified with the cell wall components. Phenolic esters - particularly
hydroxybenzoic and hydroxycinnamic acids and aldehydes - may cross-link plant cell wall
carbohydrates by means of dimer formation. In addition, esterification and dimer formation
determines the mechanical properties and biodegradability of the cell walls. The presence of
esterified phenolic compounds may protect against pathogen infestation of the plants and may
be an important factor for the degradability of cell wall in the intestine.
4

2. Objectives of the Thesis
Vegetables from the Brassica species are among the most important constituents of the
Chinese and European diets. There is scant knowledge about the phenolic structures and
composition of the Chinese Brassica vegetables, however, particularly with respect to their
hydroxycinnamic acid spectrum and cell-wall-bound phenolic compounds. As the glycosides
and organic acid residues as well as the linkage to the cell wall may affect the bioavailability
and activity of phenolic compounds, it is important to differentiate between the bound
moieties.
The distribution of polyphenols in plants might vary according to plant part (i.e. blades,
stalks, etc.); the amount of light exposure the parts receive (cultivation condition), their
physical structure (habitus), and even food processing may affect polyphenolic
concentrations.
The overall objective of this thesis is to contribute to the existing knowledge on free and
bound polyphenols in Chinese Brassica vegetables. By providing information on the
polyphenols’ occurrence, structure, and concentrations, as well as qualitative and quantitative
changes they undergo by food processing, e.g., the thesis may help food researchers to
evaluate the health-promoting effects of vegetables from this important species.
To achieve these objectives, the following steps will be taken:
1. The development of a method for the comprehensive characterization of phenolic
compounds in various pak choi and Chinese leaf mustard cultivars. The Chinese
cabbages will be investigated for their spectrum and structural properties of flavonoid
and hydroxycinnamic acid derivatives by HPLC-ESI-MS n . The fragmentation pattern by
MS n should lead to clear structural information as well as to the discovery of unknown
polyphenols (Chapter I).
2. Unknown polyphenols that cannot be clearly identified by the MS n fragmentation
procedure will be isolated for structural elucidation by NMR (nuclear magnetic
resonance). The isolated compound will be obtained as natural reference compound for
the precise quantification of polyphenols in the plants (Chapter I).
5

3. With respect to their distribution in different plant parts and the influence of different
growing conditions, the total and individual concentrations of free polyphenols
(flavonoids and hydroxycinnamic acid derivatives) in the individual Chinese cabbage
cultivars will be determined by HPLC-DAD quantification. The results will be
discussed in the context of different climatic conditions and influencing factors as well
as in terms of the relevance of Chinese cabbages for human nutrition in comparison to
other Brassica specimens (Chapters I, II, and V).
4. The changes in free phenolic compounds in Chinese cabbage that occur during various
processes, such as storage and fermentation, will be demonstrated by HPLC-ESI-MS n
and HPLC-DAD. This is followed by further discussion of the qualitative and
quantitative changes with respect to the polyphenols’ changed bioavailability
(Chapter II).
5. The antioxidant capacity of the Chinese cabbage plant material will be determined and
the results will be compared to the quantitative content of polyphenols to assess the
qualitative and quantitative changes occurring during the traditional fermentation
procedure of Chinese cabbages (Chapter II).
6. A method for the release and extraction of the bound phenolic compounds from isolated
cell wall material of Chinese Brassica vegetables will be developed to estimate the
major influencing factors on the release of bound phenolics (Chapters III and IV).
7. A comprehensive characterization of the contents of bound phenolic compounds in
different plant parts and the influence of plant age will be presented (Chapters III and V).
8. The influence of post-harvest treatments on the content of bound phenolic compounds
and the possible changes occurring during the traditional Chinese fermentation will be
determined and evaluated, as these factors may change the bioavailability of bound
phenolic compounds in vivo (Chapter IV).
9. A comparison of the contents of free and bound phenolic compounds in the fresh plant
material will be conducted in order to estimate and compare the importance of free and
bound phenolic compounds for the human diet (Chapter V).
6

3. Theoretical Background
3.1. Polyphenols
Polyphenolic compounds act in plants as pigments (e.g. anthocyanins), UV protectants,
signaling transmitters, and constituents of the cell wall components (e.g. lignin). Polyphenols
are present in free (e.g. esters of aglycones with sugars and/or organic acids) and bound (cellwall-associated)
forms in plants. Flavonoids are the most abundant phenolic compounds in
human nutrition. Scalbert et al. (3) specified a daily intake of approximately 1 g of
polyphenols (one third phenolic acids, two thirds flavonoids) through fruit juice, tea, wine,
coffee, beer, vegetables, etc. The daily intake of flavonols was reviewed by Manach et al. (4)
and amounts to 20-35 mg/day.
Flavonoids are non-vitamin compounds. They are not essential but have the ability to act
antioxidatively as well as synergistically with vitamins, e.g. vitamin C. An inverse association
of the intake of flavonoids with coronary heart disease was observable in epidemiological
studies. The Zutphen Elderly Study was the first study to indicate a reduced risk of mortality
from coronary heart disease in men who ingested flavonoids in the form of fruits and
vegetables; the beneficial effect was correlated to the quercetin content in apples and onions
(5).
3.1.1. Free Phenolic Compounds
There are two main groups of polyphenols: phenolic acids (e.g. cinnamic and benzoic acids)
and flavonoids (flavanols, flavonols, flavanons, flavons, anthocyanins, and isoflavonoids).
The classification of flavonoids (C6-C3-C6 carbon skeleton) is based on variations of the
functional groups of the heterocyclic ring C and affirms the high diversity of the compounds
(Figure 1). The different flavonoids possess similar structures, but may have different
functions due to the different functional groups of the heterocyclic ring C and different
numbers of hydroxyl-groups, O-sugars, and methoxy groups. Flavonoids possess different
moieties, including glycosides and organic acids. The moieties of flavonols (e.g. kaempferol,
quercetin, isorhamnetin) are mainly bound at the hydroxyl-group at position 3 (and sometimes
at position 7 (Figure 2). The primary esterified sugar is D-glucose, but other sugar moieties
are also known (e.g. galactose, rhamnose). More than 6000 different flavonoids have been
identified (6).
7

Figure 1. Mean structure of flavonoids (flavan)
HO
O
A C
OH
OH
Figure 2. Structure of flavonols
O
7
6
R1
B
8
A
5
OH
R2
1
O
C
4
8
2'
1'
2
3
3'
B
6'
4'
5'
Flavonol R1 R2
Isorhamnetin OMe H
Kaempferol H H
Myricetin OH OH
Quercetin OH H
Hydroxycinnamic acids are simple polyphenols (C6-C3 carbon skeleton). Figure 3 presents
the main structures of hydroxycinnamic acids in plants, such as sinapic acid, ferulic acid,
p-coumaric acid, and caffeic acid. Hydroxyferulic acid, a main intermediate product of the
biosynthesis pathway of hydroxycinnamic acid derivatives, is less well known as a final
phenolic compound in plants.
MeO
OH
COOH
OMe
HO
OH
COOH
OMe
OH
COOH
OMe
OH
COOH
OH
OH
COOH
Sinapic acid Hydroxyferulic acid Ferulic acid Caffeic acid p-Coumaric acid
Figure 3. Structures of hydroxycinnamic acids in plants
Hydroxybenzoic acids (C6-C1 carbon skeleton) are identical in their phenolic structure to
hydroxycinnamic acids.

3.1.2. Cell-Wall-Bound Phenolic Compounds
Bound phenolic compounds are present as a component of plant cell walls. These compounds
are hydroxycinnamic acids and aldehydes, hydroxybenzoic acids, and hydroxybenzaldehydes.
Cell-wall-bound phenolic compounds are present as monomeric, dimeric, or oligomeric
compounds, which are esterified to the cell wall. Figure 4 demonstrates the bound ferulic acid
arabinoxylan and galactose in the cell wall. The feruloylated galactose is derived from the
galactan side-chains of pectins, e.g. (7).
HO
HO
O O
OH
OMe
O
OH
O
O
OH
O
A
OH
O
OH
O
HO
O
OH OH
9
HO
HO
O
O
OH
OH
HO
O
O O
Figure 4. Bound ferulic acid to (A) an arabinoxylan and (B) pectin galactose side-chain
OH
O
OMe
OH
HO
O
B
OH
OH
OH
The dimer and oligomer formation results in cross-links that form bridges between the cell
wall components (carbohydrates, lignins, pectins) and proteins (7-10). There are two possible
pathways for dimer formation: 1. radical generation (e.g. 8-O-4’-dehydroferulic acid) and
2. photochemical reaction (cycle formation: truxin and truxillic acids) (Figure 5) (11-13).

OH
COOR
OMe
Light
A
H 2 O 2
Peroxidase
.
O
COOR
OMe
OH
HO OMe
HO
OMe
B e.g. 8-O-4'-Dehydrodiferulic acid
COOR
.
O
COOR
COOR
OMe
OH
OMe
Figure 5. Biosynthesis of dimeric cell-wall-associated compounds: (A) radical pathway (B)
photochemical pathway; R = cell wall component (e.g. arabinoxylan) [according to Bunzel
(11)]
The radical coupling mechanism is the most important pathway for dimer formation and the
resulting dehydrodimers are widely present in plants (e.g. dehydrodiferulic acids). The
dimeric cyclic compounds, which are formed by photochemical reaction, are predominantly
present in grasses (13).
These bound dimeric or oligomeric phenolics influence the biodegradability of the cell walls
(14) and may act as chemical barriers for pathogen infestation (15).
3.2. Biosynthesis and Function of Polyphenols
The biosynthesis of the main polyphenol groups, i.e. flavonoids, hydroxycinnamic acids, cellwall-bound
phenolic compounds, and lignins, is presented in Figure 6.
The first step of polyphenolic biosynthesis is induced by the enzyme phenylalanine
ammonium-lyase (PAL) for the generation of cinnamic acid, which is further involved in the
biosynthesis of hydroxycinnamic acids and flavonoids in plants.
10
O
COOR
.
OMe
COOR
COOR
OH
OMe
H
RO
O
O
OMe
OMe
O
OR

Malonyl-CoA
CHS/CHR
Trihydroxy-
chalcone
IFS
Isoflavone
(Genistein)
p-Coumaroyl-CoA
Flavonol
(Kaempferol)
GT
IFS
Flavonolglycoside
Tetrahydroxy-
chalcone
Flavonone
(Naringenin)
Malonyl-CoA
CHS
CHI
FN3H
Dihydroflavonol
(Dihydrokaempferol)
FLS DFR
Flavandiol
Feruloyl-CoA
Feruloyl-
polysaccharide
complex
Feruloyl-
polysaccharideester
(Cell wall component)
ANS
Anthocyanidin
GT
Anthocyanin
4CL
FT
LAR
Flavanol
(Catechine)
Figure 6. Biosynthesis pathway of the main groups of polyphenols in plants
[according to Bunzel (11), Winkel-Shirley (16), Mock et al. (17), Duthie and Crozier (18),
and Dixon and Paiva (19)]
ANS anthocyanidin-synthase, CAD cinnamoyl-dehydrogenase, CCR 4-coumaroyl: CoA-reductase, C4H
cinnamate-4-hydroxylase, 4C3H 4-coumaroyl-3-hydroxylase, CHI chalcone-isomerase, 4CL 4-coumaroyl: CoAligase,
CHR chalcone-reductase, CHS chalcone-synthase, DFR dihydroflavonol-4-reductase, F5H feruloyl-5hydroxylase,
FL feruloyl: CoA-ligase, FLS flavonol-synthase, FN3H flavonone-3-hydroxylase, FT feruloyltransferase,
GT glycosyl-transferase, IFS isoflavone-synthase, LAR leucoanthocyanidin-4-reductase, OMT Omethyl-transferase,
PAL phenylalanine-ammoniumlyase, SCE sinapoylcholin-esterase, SCT sinapoylcholintransferase,
SGT sinapoylglycosyl-transferase, SMT sinapoylmalate-transferase, a reported for Arabidopsis
thaliana and Brassica rapa (17)
11
FL
Phenylalanine
Cinnamic acid
p-Coumaric acid
Caffeic acid
Ferulic acid
5-Hydroxyferulic acid
Sinapic acid
Sinapoylglucose
Sinapoylmalate
Proanthocyanidin
PAL
C4H
4C3H
OMT
F5H
OMT
SGT
SMT a
4CL, CCR,
CAD
p-Coumaroyl alcohol
+ Quinic
acid
SCT
Coniferyl
alcohol
4CL, CCR,
CAD
SCE
Gallic acid
Hydroxycinnamoylquinic
acid
4CL, CCR,
CAD
Sinapoyl alcohol
Sinapoylcholine
Lignin
(Cell wall
component)

Simple phenylpropanoids, such as p-coumaric acid, caffeic acid, ferulic acid, hydroxyferulic
acid, and sinapic acid (Figure 3), are formed by hydroxylation, methylation, and dehydration
reactions, and further conjugated by sugars, organic acids (e.g. quinic acid, malic acid, or
tartaric acid), or cell wall components. The formation of malate derivatives (e.g.
sinapoylmalate) is induced by the enzyme sinapoylmalate-transferase, which has been clearly
identified in Arabidopsis thaliana, rape (Brassica rapa), and radish (Raphanus sativus) plants
(20, 21).
Concerning the cell wall, activated ferulic acid is involved in the formation of
feruloylpolysaccharide esters via complexes and has been reported in monocotyledons
(arabinoxylans) and dicotyledons (pectins) (11, 22). Hydroxycinnamate amide formation in
elicited dicotyledons and secretion into the cell wall for further oxidative linkage to cell wall
components have also been reported (pathway not presented) (22). The further cross-link of
polysaccharides (such as hemicelluloses) to pectins or complex lignin polymers, resulting
from the radical coupling reactions (chapter 3.1.2.), affects the cell wall architecture (12). It is
known that monomeric and dimeric cell-wall-bound phenolic compounds are involved in the
synthesis of lignin and the cell wall stiffening mechanism, which is regulated by ascorbic acid
and hydrogen peroxide levels in the apoplast of the cell (23).
Lignin, a main structural element of plant cell wall carbohydrates, consists of phenyl
propanoid units, which are generated through hydroxycinnamic acid and identical
hydroxycinnamic alcohols, as seen in Figures 6 and 7 (24): Activated phenolic acids sinapic
acid, ferulic acid, and p-coumaric acid were reduced by the enzyme CoA-reductase; the
formed aldehydes were reduced to monolignols (identical alcohols) via alcoholdehydrogenase
(25). The formation of radicals by peroxidase resulted in polymerization and
the generated lignin confers rigidity to the cell wall.
MeO
OH
CH 2 OH
OMe
Sinapoyl alcohol
OH
12
CH 2 OH
OMe
Feruloyl alcohol
OH
CH 2 OH
p-Coumaroyl alcohol
Figure 7. Monomeric phenolic compounds of the cell wall polymer lignin (monolignols)

Flavonoids are formed by the enzyme chalcone synthase and are synthesized by the
condensation of activated p-coumaroyl acid and activated malonyl acid. Further
hydroxylation, methylation, dehydration, and isomerization reactions of the generated
tetrahydroxychalcone (trihydroxychalcone) lead to the large variety of different flavonoid
structures, which are further esterified to glycoside esters via glycosyl-transferase (GT) (16).
The biosynthesis of polyphenols in plants is influenced by a variety of factors, including UV
light, wounding, pathogen attacks, low temperatures, and nutritional stress (phosphate,
nitrogen, and iron) (19). The polyphenol synthesis of flavonoids is induced by UV-B radiation
(26, 27). Polyphenols have the ability to protect the plant from UV-induced damage and
absorb UV-A (320-400 nm: flavonoids) and UV-B radiation (280-320 nm: hydroxycinnamic
acids). The synthesis of polyphenols in plants is involved in the regulation of the production
of reactive oxygen species (ROS), which are generated by e.g. photosynthesis and respiration
in plants. Reactive oxygen species (ROS) play a significant role in plant stress due to their
signaling function as well as production by stress factors (28).
The formation of free radicals and ROS in cells may damage tissues, e.g. DNA, proteins, or
membranes. Dietary antioxidants have the ability to act synergistically or additively to reduce
oxidative stress and protect biological tissues. The antioxidative capacity is dependent on the
molecule structure of phenolic compounds, e.g. on their content of hydroxyl groups on the
aromatic ring and other moieties (29, 30).
3.3. Bioavailability of Polyphenols
The structure and biological properties of polyphenols affect their bioavailability (rate and
extent of intestinal absorption) as well as the occurrence of metabolites in plasma (3). The
simple phenolic acids can be absorbed via the gut barrier, but more complex polyphenols like
flavonoids with a larger molecular weight are poorly absorbed (31). The glycoside and ester
linkages of the polyphenols influence the absorption site in the human gut, e.g. by means of
specific sugar transporters in the small intestine or by microbiotic activity (esterase and
glucosidase activity) in the colon (3). The bioavailability of less glycosylated flavonoids such
as quercetinmonoglucoside is therefore higher than that of the quercetin aglycone as well as
the more highly glycosylated quercetin derivatives (e.g. rutin) (31, 32). Therefore, the
bioavailability of polyphenols varies according to different food sources and is dependent on
the type of polyphenolic glycosides that they contain.
Furthermore, the influence and bioavailability of cell-wall-bound phenolic compounds differs
from that of free phenolic compounds; therefore, their absorption and physiological functions
13

may also be different: Dietary fiber is an important constituent of the human diet. The
chemical structure and architecture of cell wall components (hemicelluloses, celluloses,
lignin, and pectin) influence the biodegradability and fermentation of dietary fibers by
microorganism activity in the human colon (33). The bound phenolic acids (e.g. ferulic acid)
and their cross-linkages cause a reduced rate of the degradation and fermentation of cell wall
components, such as arabinoxylans (34), which may also limit the release of these bound
compounds (35). The cell-wall-bound phenolic acids are released only by enzymatic activity
of colon microflora (xylanase and esterase) (36). The linkage to cell wall components limits
the bioavailability of bound phenolics, e.g. ferulic acid, and the excretion in the urine was
found to be distinctly lower than of non-bound phenolic acids (35).
3.4. Polyphenols in Brassica Vegetables
Several Brassica vegetables were investigated to determine their qualitative and quantitative
phenolic content. Table 1 gives an overview of the polyphenols identified in Brassica
vegetables. The main flavonoids are kaempferol- and quercetinglycosides (mono- to
pentaglycosides), which are sometimes acylated with organic acids (hydroxycinnamic acids).
Isorhamnetin derivatives were also detected (37-46).
The main identified hydroxycinnamic acids are quinic acid derivatives (chlorogenic acid,
feruloylquinic acid) and glycosides derivatives, e.g. sinapoylglucose, or two or three
hydroxycinnamic acids esterified with one gentiobiose unit: e.g. trisinapoylgentiobiose or
disinapoylgentiobiose (39, 44, 47, 48).
However, less is known about the qualitative and quantitative content of bound phenolic
compounds in Brassica species. Beveridge et al. (49) reported on the content of cell-wallbound
phenolic compounds in broccoli and identified vanillin, p-coumaric acid, benzoic acid,
and p-hydroxybenzaldehyde with a combined total content of approximately 90 µg/g cell
wall.
14

Table 1. Overview of Identified Polyphenols in Different Cultivars of Brassica Vegetables
Vegetable Botanical synonym Polyphenols Contents a Reference Year
Pak Choi Brassica campestris L. ssp chinensis caffeic acid, chlorogenic acid, kaempferolglycosides flavonoids: 5.3-13.3 µmol/g dm
hydroxycinnamic acids: 2.3-8.9 µmol/g dm
Pak Choi Brassica rapa L. ssp. chinensis L.
(Hanelt.)
acylated kaempferol- and quercetindi- and triglucosides,
isorhamnetinglucosides
15
Sakakibara et al. (45) 2003
0.7-1.2 mg/g dm (aglycone content) Rochefort et al. (38) 2006
Chinese Cabbage Brassica campestris var. perviridis L. caffeic acid, chlorogenic acid, kaempferolglycosides flavonoids: 5.4-11.5 µmol/g dm
hydroxycinnamic acids: 2.4-7.6 µmol/g dm
Cabbage Brassica oleracea L. convar. capitata (L.)
Alef. var. alba DC.
Cabbage Brassica oleracea L. convar. capitata (L.)
Alef. var. alba DC.
Sakakibara et al. (45) 2003
acylated kaempferol- and quercetintriglucosides not quantified Nielsen et al. (41) 1993
acylated kaempferoltetraglucosides not quantified Nielsen et al. (40) 1998
Cabbage Brassica oleracea var. capitata L. kaempferolglycosides, luteolinglycosides, quercetin flavonoids: 0.32 µmol/g dm
hydroxycinnamic acids: 1.82 µmol/g dm
Sakakibara et al. (45) 2003
Cabbage Brassica oleracea var. capitata kaempferol and quercetin derivatives 0.1-0.8 mg/g dm (aglycone content) Kim et al. (42) 2004
Broccoli Brassica oleracea L. var. italica kaempferol- and quercetinglycosides, chlorogenic acids,
hydroxycinnamoyldiglycosides
(gentiobiose derivatives)
Broccoli Brassica oleracea var. botyris L. kaempferol- and luteolinglycosides, caffeic acid,
chlorogenic acid
Tronchuda cabbage
(inner leaves)
Tronchuda cabbage
(external leaves)
Brassica oleracea L. var. costata DC kaempferoltriglycosides, quercetintriglycosides,
hydroxycinnamoyldiglycosides (gentiobiose derivatives)
Brassica oleracea L. var. costata DC kaempferoltetra-, tri-, and diglycosides,
hydroxycinnamoylquinic acids
Cauliflower Brassica oleracea L. var. botrytis quercetintriglucosides, kaempferoldi-, -tri-, -tetra-, and
pentaglucosides (sometimes acylated with one or two
hydroxycinnamic acids), hydroxycinnamoyldiglycosides
a Calculated on dry material basis (dm): 1/10 of fresh material.
flavonoids: 1-10 mg/g dm
hydroxycinnamic acids: 0.2- 1.5 mg/g dm
flavonoids: 2 µmol/g dm
hydroxycinnamic acids: 1.2 µmol/g dm
Vallejo et al. (48) 2003
Sakakibara et al. (45) 2003
approx. 0.1-1 mg/g dm Sousa et al. (37)
Ferreres et al. (39)
approx.1-10 mg/g dm Ferreres et al. (46)
Ferreres et al. (39)
2005
2006
2005
2006
not quantified Llorach et al. (44) 2003

3.5. Chinese Brassica Vegetables
Chinese cabbages such as pak choi (Brassica campestris L. ssp. chinensis var. communis) or
leaf mustard (Brassica juncea Coss) are leafy headless cabbages and belong to the
Brassicaceae family (50, 51).
Pak choi is also known as paksoi, buk choy, or bai cai (Chinese) and represents a variety of
cultivars of green pak choi, white pak choi, and purple and flowering cabbages.
The main countries for cultivation are China, Korea, Taiwan, and Japan, but the plants are
also grown in some western countries, including the Netherlands, Great Britain, and Germany
(52). Pak choi is often consumed in steamed or fermented form. The leaves are very soft and
sensitive to pressure (50).
3.5.1. Nutritional Relevance
Brassica vegetables are the most important vegetables in the human diet throughout the
world. Their nutritional relevance results from their high calcium, vitamin C, thiamin,
riboflavin, niacin, and vitamin B6 content (53). Pak choi is rich in carbohydrates, proteins,
minerals, B-vitamins, and vitamin C. The main constituents of pak choi and Chinese cabbage
were reviewed by Herrmann (51) and are presented in Table 2.
Table 2. Constituents of Pak Choi and Chinese Cabbage per 100 g Fresh Plant Material (51)
Pak choi Chinese cabbage
Water 95.3 g 94.4 g
Proteins 1.5 g 1.2 g
Lipids 0.2 g 0.2 g
Minerals 0.8 g 1 g
Potassium 252 mg 238 mg
Sodium 65 mg 9 mg
Calcium 105 mg 77 mg
Magnesium 19 mg 13 mg
Phosphorus 37 mg 29 mg
Vitamin C 45mg 27mg
Table 3 presents the composition of the dietary fiber (cell wall components) of pak choi (54).
The main compounds are insoluble components, such as pectins, celluloses, and
hemicelluloses. Lignins were present in smaller amounts (i.e. 4.8% of the insoluble cell wall
components). Soluble compounds represent only a small portion of cell wall components
(approx. 6%).
16

Table 3. Soluble and Insoluble Cell Wall Components of Pak Choi per 100 g Dry Material
(dm) (54)
Component [g/100g dm]
Soluble Hemicellulose 0.5
Pectin 0.9
Insoluble Cellulose 9.5
Hemicellulose 4.8
Pectin 7.7
Lignin 1.1
3.5.2. Cultivation Conditions
The preferred ground for the cultivation of Chinese Brassica vegetables like pak choi and
Chinese leaf mustard is loamy soil. These cultivars need plenty of water, but water-logging
has to be avoided (55).
Chinese Brassica vegetables are long-day plants and the optimum growing temperature is
20°C (52). A daytime length of approx. 10-14 hours or temperatures above 10-12°C results in
flowering (56, 57). Chinese cabbage is therefore cultivated under warm climatic conditions
and under field conditions in cool seasons (spring and autumn) with shorter daytimes, like the
Zhejiang region in China, where the temperatures are approx. 2-8°C in January and 27-30°C
in July; the mean value for the whole year is 15-19°C. Cultivation is also possible in Central
Europe, but resulted in the abovementioned flowering due to the long days in the summer
season. In the cooler seasons (winter, autumn, spring), cultivation is carried out under
greenhouse conditions due to controlled temperatures.
Cultivation involves many different conditions (e.g. differences in light supply in greenhouse
or field conditions) that might influence the occurrence (quantitative and qualitative) of
polyphenols in the plant.
3.5.3. Vegetable Processing
The fermentation process is normally used to preserve foods and vegetables, like cabbages,
olives, or cucumbers. The traditional Chinese fermentation procedure includes the withering
of the whole Chinese cabbage plant (unshredded), the addition of salt (approx. 100g/5kg fresh
weight), and kneading until sap leaks from the plants. The fermentation procedure then
continues under pressure in clay pots at ambient temperatures and resulted in a decrease in pH
from 7 to 4 due to activity by microorganisms (lactobacilli) that are already present in the
plant material. In the case of white cabbage fermentation (sauerkraut), salt is added to the
shredded plant material and placed in a jar under pressure. Several microorganisms are
responsible for the fermentation of white cabbage, including different strains of lactobacilli
17

(L. mesenteroides, L. plantarum, L. cucumeris, L. pentoaceticus), which naturally occur in the
plant material and are also added as starter culture. These microorganisms are active at
different stages of the process and produce lactic acid, propionic acid, and acetic acid (from
fermentable sugars). Therefore, a proportion of approx. 2% acids results in a strong decrease
in pH (to 3.5). This microbial-chemical fermentation process is dependent on salt
concentration, air elimination, and the temperature (58).
Food processing (e.g. fermentation, storage, or cooking) may alter the free and bound
phenolic content as result of the transformation or degradation that takes place during
processing (59-64). The oxidation and structural changes that occur in polyphenols via
vegetable or food processing may lead to newly formed compounds in vegetables and foods,
e.g. brown pigments (65) or phenolic aglycones (59). The impact of fermentation on phenolic
compounds in cabbages is controversial. Chun et al. (66) reported lower total phenolic content
in processed cabbage compared to raw cabbage. However, Ciska et al. (61) detected higher
total phenolic content in sauerkraut extracts compared to white cabbage extracts (Folin
Ciocalteu assay). Another study found constant amounts of kaempferol in cabbage throughout
the fermentation process (analysis based on the determination of flavonoid aglycones) (67).
Qualitative changes in the polyphenolic pattern of the individually glycosylated and
acetylated derivatives have not been investigated. The changes in the glycoside and organic
acid residues are of interest with respect to bioavailability and metabolism in humans.
However, nothing is known about the influence of cabbage fermentation on the content of
cell-wall-bound phenolic compounds.
18

4. Materials and Methods
4.1. Plant Material
Figure 8 presents the 11 white and green pak choi cultivars used in this investigation. This
variety of cabbage is about 20 to 50 cm high. The 11 pak choi cultivars differ in terms of their
leaf blade/stalk ratio (Chapters I and V), the habitus of the blade, the stalk thickness, and the
blade and stalk color.
Cv. Ai Kang Qing Cv. Lu Xiu Cv. Suzhou Qing
Round light green blade (spoon), Round grass-green plane blade, Round dark green plane blade,
non-sulcated, thick whitish non-sulcated, thick light non-sulcated, thick light stalk,
stalk, headless green stalk, slight head slight head
Cv. Shanghai Qing Cv. Nanjing Zhong Gan Bai Cv. Hei You Bai Cai
Small round light green blade Round, wavy light green Round, wavy dark green blade,
(spoon), non-sulcated, short blade, non-sulcated, whitish non-sulcated, grass-green thin
thick whitish stalk, slight head thin stalk, headless stalk, headless
Figure 8. Eleven cultivars of pak choi plants (Brassica campestris L. ssp. chinensis var.
communis) cultivated in China, autumn 2005
19

Cv. Ai Jiao Huang Cv. Si Yue Man Cv. Si Ji Xiao Bai Cai
Round light green plane blade, Round light green plane blade, Terete sulcated wavy grass-
non-sulcated, thick whitish non-sulcated, whitish stalk, green blade, thin whitish
stalk, slight head headless stalk, headless
Cv. Huang Xin Cai Cv. Hangzhou You Dong Er
Small round dark green wavy Round grass-green plane blade,
blade, non-sulcated, light green non-sulcated, thick light green
stalk, headless stalk, slight head
Figure 8 (continued). Eleven cultivars of pak choi plants (Brassica campestris L. ssp.
chinensis var. communis) cultivated in China, autumn 2005
Leaf mustard (Brassica juncea Coss) is an important Chinese leaf mustard specimen. This
Chinese cabbage especially varies in its leaf form. Figure 9 presents the two leaf mustard
cultivars Xue Li Hong and Bao Bao Qing Cai. Their size is comparable to pak choi and their
leaves are longer.
20

cv. Xue Li Hong cv. Bao Bao Qing Cai
longish sulcated light green wavy sulcated light green
blade, thin light green stalk, blade, thick whitish stalk,
headless headless
Figure 9. Two cultivars of Chinese leaf mustard (Brassica juncea Coss) cultivated in China,
autumn 2005
4.2. Determination of Phenolic Compounds
The extraction procedure of free and bound phenolic compounds of Chinese Brassica
vegetables is schematically presented in Figure 10.
Water/methanol extraction,
column chromatography,
prep. HPLC
Purified compound
NMR
spectroscopy
Free phenolic compounds Bound phenolic compounds
HPLC-ESI-MS n
Freeze-drying
and milling
Dried plant material
Extract
Fresh plant material
Extraction by ultrasonic with
50% MeOH containing
1% m-phosphoric acid,
0,5% oxalic acid dihydrate
HPLC-DAD - Folin Ciocalteu
- Assays for
antioxidative
capacity
Figure 10. Flowchart of the extraction procedure of free and bound phenolic compounds in
Chinese Brassica vegetables
21
White residue
Washing procedure with SDS and
Na2S2O5 solutions (Ultra Turrax)
Freeze drying
and ball milling
Dried powdered residue
(cell wall)
Extract
HPLC-ESI-MS
96 h hydrolysis period with 1M
NaOH, acidification, extraction
by ethylacetate, solvent removal,
residue solubilized in 25% MeOH
HPLC-DAD

4.2.1. Free Phenolic Compounds
4.2.1.1. Extraction
The extraction was carried out by ultrasonic treatment of freeze-dried and ground plant
material with 50% acidic aqueous methanolic solution (Chapters I, II, and V).
The extraction method is a combined technique for the determination of all polyphenols
(flavonoids and hydroxycinnamic acids as well as anthocyanins) and ascorbic acid in pak choi
and Chinese leaf mustard. m-Phosphoric and oxalic acids were added to determine ascorbic
acid content in extracts; the latter is only stable at a low pH (chapter 4.3).
This method was developed for the rapid and simple extraction of freeze-dried plant samples
from the Chinese cabbages.
4.2.1.2. Qualitative Analysis by HPLC-ESI-MS n
The high performance liquid chromatography (chapter 4.2.1.4.) coupled with an electrospray
ionization (ESI) ion trap mass spectrometer (MS n ) allows the analysis of compounds directly
from the liquid eluent of the HPLC. The determination of the mass spectrometer is related to
the HPLC conditions, such as flow rate, pH of solvent, or polarity of the eluent, which
influence the ionization of the analyt molecule. The ESI enables soft ion production. The
liquid eluent has to be more volatile than the analyt molecule in order for evaporation of the
droplets. The droplets were formed by nebulizing through the nitrogen gas, e.g. via spray
capillary in the chamber. The loss of solvent created by the heating gas stream leads to a
higher charge density of the droplets, which are divided into smaller droplets by closer,
charged analyt molecules and the final formation of single ions (Coulomb explosion).
Another possible process is the emission of ions directly from the droplets when the charged
field generated by the ions overruns the highly charged surface of the droplets (ion
evaporation) (68-70).
22

Figure 11. Illustration of the fragmentation procedure in the ion trap (71)
The extent of ionized analyt molecules and the further focusing and transport of the ions in
the ion trap mass analyzer due to the presented voltages [a combination of electrostatic lenses
and a split RF octopole ion guide (70)] are responsible for the sensitivity of the MS n system.
The generated ions accumulate in the ion trap, which consists of a ring electrode between two
end-cap electrodes (Figure 11). These electrodes with oscillating potentials are responsible
for the formation of a quadrupol field for the trapping of ions. The ions in the trap, which pass
in through the first end-cap, conserve a high level of energy, which results in their exit out the
other end-cap. For the accumulation of ions, the presence of a collision gas is necessary,
which extracts the energy from the ions to further retain the ions in the trap. The ions are
confined in the trap and oscillate by means of a periodic motion, which is characterized by
axials and radial motion. The motions were excited by resonant irradiation at these
frequencies by an oscillating potential across the end-cap electrodes (dipolar mode) (72).
Furthermore, the energy of the ions is taken up when the frequency of the dipolar field is
identical with the frequency of the ions (resonance), and results in the movement away from
the center and further exiting from the ion trap. The mass spectrum can be generated by the
resonance conditions. The advantage of this procedure is the combination of the high scan
speed and mass resolution (70).
In MS n analysis, the first step is the isolation procedure of the analyt molecule. That requires
the removal of all the other ions from the trap, as these may interfere. The system therefore
generates a wide broadband of frequencies with the exception of the one that matches the
resonance of the desired ion. The second step is the fragmentation procedure (Figure 11). The
isolated precursor ions take up energy from the dipolar field, collide with the helium
23

ackground gas, and split in product ions; the mass spectra can now be further generated.
Because of the small amounts of energy remaining in the product ion, a subsequent
fragmentation takes place. The fragmentation procedure can be repeated several times (MS n )
by isolation of the product ions (now precursor ions) and further collision with the
background gas to obtain newly formed product ions in the ion trap that can be further
analyzed (70).
The electrospray ionization is usually used to determine polyphenols due to the gentle
ionization of the analyt molecule (73). The analyt molecule could be systematically
fragmented (Chapters I and II). Thus, it is possible to obtain a specific fragmentation pattern
by MS n , particularly for the flavonoid derivatives whose specific position of bound moieties
like glycosides and organic acids is known (74). It is therefore possible to tentatively identify
the phenolic structures and the number of bound moieties. Polyphenols are predominantly
transferred to anions in the ion source due to their ability to easily release protons from the
presented hydroxyl groups of their molecular structure (acidic protons); however,
anthocyanins e.g. are predominantly measured in the positive ionization modus due to their
positive charge (75, 76).
For Brassica species, it was shown that the sugar moieties like glucose of the detected
flavonoid derivatives are predominantly bound to positions 7 and 3 of the aglycone (Figure 1,
page 8). In previous studies, the preferred cleavage of moieties at position 7 compared to
moieties at position 3 were reported, which enables the specific characterization of the
molecular structures of these polyphenols (Chapters I and II).
However, the ESI-MS method only allows the identification of molecular masses and their
fragment masses for further characterization of the molecule derivatives; for a detailed
structural elucidation, NMR spectroscopy has to be used.
4.2.1.3. Structural Elucidation
4.2.1.3.1. Compound Isolation from Plant Material
The isolation of free phenolic compounds was carried out by aqueous and methanolic
extraction of freeze-dried plant material, compound separation by column chromatography
(Amberlite XAD-7), and further purification by preparative HPLC (Chapter I). The separation
by preparative HPLC under pressure led to highly purified defined products. The higher
dimension of the column enables the purification of higher compound dimensions, which is
different from the analytical HPLC (chapter 4.2.1.4.).
24

4.2.1.3.2. NMR-Spectroscopy
A detailed structural elucidation of isolated compounds was carried out by nuclear magnetic
resonance spectroscopy (NMR) (Chapter I). NMR spectroscopy includes the determination of
chemical shifts (δ in parts per million) for carbons ( 13 C NMR) and protons ( 1 H NMR) and
enables the characterization of chemical structures by determination of signal correlations
(cross peaks of the two-dimensional NMR spectroscopy):
1 1
H- H COSY: Correlated Spectroscopy
This spectroscopy describes the simple correlation and cross peaks of 1 H-NMR frequencies
presented on two axes and indicates the adjacencies of protons in the molecular structure.
HSQC: Heteronuclear Single Quantum Correlation
HSQC spectroscopy results in the two-dimensional spectra of 1 H-NMR and 13 C-NMR. The
obtained signals and cross peaks showed significant correlations of the carbon and the directly
attached protons and represent one-bond couplings.
HMBC: Heteronuclear Multiple Bonds Correlation
HMBC spectroscopy represents the correlation of multiple-bond couplings of protons and
carbons by cross peaks ( 1 H-NMR and 13 C-NMR), which are more than one bond removed
within the molecular structure (predominantly two or three bonds).
4.2.1.4. Quantitative Analysis by HPLC-DAD
Reverse-phased high performance liquid chromatography (analytical RP-HPLC) in
combination with UV-VIS spectroscopy (diode array detection) is mostly used for the
determination of free polyphenols, such as flavonoids and hydroxycinnamic acids (Chapters I,
II, and V). HPLC is a precise and fast method for the determination of several polyphenols in
plant extracts. This analytical method includes low column dimensions and solvent flows (in
contrast to the preparative HPLC). The possibility to develop an elution via a gradient of the
solvents leads to the separation and determination of individual compounds in a highly
precise way. The use of different wavelengths in DAD-determination enables the detection of
different polyphenolic classes (e.g. anthocyanins at 516 nm, both flavonoids and
hydroxycinnamic acid derivatives at 330 nm, and hydroxybenzoic acids at 280 nm). The
resulting peak area provides direct information about the quantitative content of the detected
compound in extract. Figure 12 presents the calibration curves of the isolated compound
25

kaempferol-3-O-hydroxyferuloyldiglucoside-7-O-glucoside and of the standard compound
sinapic acid (detection limits Chapter I).
Peak area
Kaempferol-3-O-hydroxyferuloyldiglucoside-
7-O-glucoside
y = 17845x - 15.553
R 2 3500
3000
2500
2000
1500
1000
500
0
= 1
0.000 0.050 0.100
[mg/ml]
0.150 0.200
26
Peak area
6000
5000
4000
3000
2000
1000
0
y = 120836x - 23.591
R 2 = 0.9998
Sinapic acid
0.000 0.010 0.020 0.030 0.040 0.050
[mg/ml]
Figure 12. HPLC calibration curves of kaempferol-3-O-hydroxyferuloyldiglucoside-7-Oglucoside
and sinapic acid
4.2.1.5. Total Phenolic Content (Folin Ciocalteu)
The Folin Ciocalteu reagent was first presented in 1927 for the determination of proteins by
oxidation of the phenol moiety of tyrosin (77) and further improved by Singleton and Rossi
(78) in 1965 for the determination of polyphenols.
This method is based on an oxidation/reduction reaction of a molybdotungstate reagent. The
electron transfer reaction leads to the formation of the blue color, which can be simply
quantified by spectrophotometry at 725 nm (79, 80).
The reaction was as follows: Na2WO4/Na2MoO4 → (phenol-MoW11O40) -4
Mo(VI) (yellow) + e - → Mo(V) (blue) (80)
The calibration was normally carried out by gallic acid equivalents, but other standards were
also reported. This technique is simple and fast and often used as screening method for the
determination of polyphenols in plant extracts. However, other compounds that act as
reductons may interfere and render the assay imprecise (Chapter II) (80).
In the present study, the Folin Ciocalteu assay was modified by the use of 0.033 M NaOH
(instead of water) to buffer the prepared acidic extracts as described above.

4.2.2. Cell-Wall-Bound Phenolic Compounds
4.2.2.1. Extraction of the Cell Wall
The cell wall isolation procedure was accomplished by several washings of fresh plant
material with solutions containing different concentrations of sodium dodecylsulphate (SDS)
and sodium metabisulphite. This procedure leads to a water insoluble residue, which was
freeze-dried and powdered by ball milling for homogenization (Chapter III).
Sodium metabisulphite was used to prevent oxidation during the washing procedure.
However, the disadvantage of this method is the loss of water-soluble pectic polysaccharides
(81). The cell wall isolation procedure was carried out according to Beveridge et al. (49) for
broccoli florets and to Selvendran and Ryden for young cabbage leaves (81). This method is
often used for the cell wall isolation of plants in the literature (49, 82-85). Figure 13 shows
the light microscopy picture of isolated dried cell wall material (particle size 5-20 µm) that
was free of cellular contents. The extraction of the isolated dried and powdered cell wall with
50% aqueous methanolic solution and further analysis by HPLC (chapter 4.2.2.3) verified the
clear absence of free phenolic compounds in the cell wall material.
Figure 13. Light microscopy picture of isolated dried powdered cell wall (whole leaf)
4.2.2.2. Hydrolysis Reaction of Cell Wall and Extract Preparation
The white powdered residue received from the cell wall isolation procedure was treated with
sodium hydroxide and caused the hydrolysis reaction of the esterified bound phenolic
compounds. Hydrolysis reaction with sodium hydroxide is usually used for the qualitative and
quantitative determination of bound phenolic compounds in monocotyledons and
dicotyledons in the literature (13, 82, 84, 85).
The hydrolysis conditions were optimized (e.g. by the concentration of sodium hydroxide
solution and hydrolysis time, Chapters III and IV), which results in a simple and fast method
27

enabling the determination of bound phenolic compounds in a large number of isolated cell
wall samples.
4.2.2.3. Qualitative and Quantitative Analysis by HPLC
The qualitative determination by HPLC-DAD was carried out by a comparison of the
retention times of phenolic compounds with standard compounds, UV-spectra of the DAD,
and HPLC-ESI-MS (isolation modus) (Chapters III, IV, and V). The quantification was carried
out by HPLC-DAD: hydroxybenzaldehydes (vanillin and p-hydroxybenzaldehyde) and
hydroxybenzoic acids (vanillic acid) monitored at 280 nm, and hydroxycinnamic acids (pcoumaric
acid, sinapic acid, trans- and cis-ferulic acids) monitored at 330 nm (chapter
4.2.1.4).
4.3. Determination of Ascorbic Acid
Ascorbic acid was also measured in the prepared plant extract (chapter 4.2.1.1.). The extract
was diluted 1:9 with water, which contains 1% m-phosphoric acid and 0.5% oxalic acid
dihydrate. Dilution was carried out to minimize the methanol proportion in the analysis
solution. m-Phosphoric acid and oxalic acid were added for the stabilization of ascorbic acid,
which was further directly analyzed by HPLC-DAD at 245 nm (Chapter II). The ascorbic acid
content could be eliminated as an interfering compound in Folin Ciocalteu assay and assays
for the determination of antioxidative potential.
4.4. Antioxidative Capacity Assays
The antioxidant capacity was determined by radical scavenging assays (Chapter II): The
DPPH assay (2,2-diphenyl-1-picrylhydrazyl radical) and TEAC assay [2,2´-azino-bis-3ethylbenzo-thiazoline-6-sulfonic
acid diammonium salt (ABTS) radical].
The stable DPPH radical (violet, absorbance at 516 nm, Figure 14) was reduced by presented
antioxidants (such as polyphenols and vitamin C) and leads to the decolorization of the DPPH
molecule monitored at 516 nm. Due to the reaction of the polyphenols, the electron-transfer
process is a fast reaction from the phenoxide anion to the DPPH radical (86). The decrease of
absorbance at 516 nm was measured and expressed as Trolox (tocopherol-acetate: 6-hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalents. The DPPH radical scavenging
activity is usually determined in alcohol solvents (e.g. ethanol). For the determination of
antioxidative capacity of acidic 50% methanolic extracts, the solvent was modified by using
50% aqueous methanolic solution containing 0.1 M PBS-buffer.
28

O 2 N
N
N
NO 2
NO 2
Figure 14. DPPH radical reaction
+ RH
NH
+
O2N The blue-green ABTS radical (Figure 15) was generated by the reaction of ABTS with
potassium peroxodisulphate and further solubilized in 0.1 M PBS buffer. The TEAC assay is
based on the reduction of the radical by electron transfer as presented for the DPPH radical
(79). The decrease of the absorbance at 734 nm was monitored and expressed as Trolox
equivalents.
O 3 S
S
N
Et
N N
Figure 15. The blue-green ABTS radical
Et
S
N
29
N
NO 2
SO 3
NO 2
+
R

5. Literature cited
(1) Verhoeven, D. T. H.; Verhagen, H.; Goldbohm, R. A.; van den Brandt, P. A.; van Poppel,
G. A review of mechanisms underlying anticarcinogenicity by Brassica vegetables.
Chem. Biol. Interact. 1997, 79, 79-129.
(2) Bayrische Landesanstalt für Landwirtschaft. Agrarmärkte Jahresheft 2006, Teilauszug
Gemüse.
(3) Scalbert, A.; Williamson, G. Dietary intake and bioavailability of polyphenols. J. Nutr.
2000, 130, 2073-2085.
(4) Manach, C.; Williamson, G.; Morand, C.; Scalbert, A.; Remesy, C. Biovailability and
bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am. J.
Clin. Nutr. 2005, 81, 230-241.
(5) Hertog, M. G. L.; Feskens, E. J. M.; Hollmann, P. C. H.; Katan, M. B.; Kroumhout, D.
Dietary antioxidant flavonoids and risk of coronary heart disease: The Zutphen
Elderly Study. Lancet 1993, 342, 1007-1011.
(6) Schijen, E. G. W. M.; de Vos, C. H. R.; van Tunen, A. J.; Bovy, A. G. Modification of
flavonoid biosynthesis in crop plants. Phytochemistry 2004, 65, 2631-2648.
(7) Saulnier, L.; Thibault, J. F. Ferulic acid and diferulic acids as components of sugar-beet
pectins and maize bran heteroxylans. J. Sci. Food Agric. 1999, 79, 396-402.
(8) Bunzel, M.; Ralph, J.; Kim, H.; Lu, F.; Ralph, S. A.; Marita, J. M.; Hatfield, R. D.;
Steinhart, H. Sinapate dehydrodimers and sinapate-ferulate heterodimers in cereal
dietary fiber. J. Agric. Food Chem. 2003, 51, 1427-1434.
(9) Piber, M.; Koehler, P. Identification of dehydro-ferulic acid-tyrosine in rye and wheat:
Evidence for a covalent cross-link between arabinoxylans and proteins. J. Agric. Food
Chem. 2005, 53, 5276-5284.
(10) Grabber, J. H.; Ralph, J.; Hatfield, R. D. Cross-linking of maize walls by ferulate
dimerization and incorporation into lignin. J. Agric. Food Chem. 2000, 48, 6106-6113.
(11) Bunzel, M. Monomere und dimere Phenolcarbonsäuren als strukturbildende Elemente in
löslichen und unlöslichen Getreideballaststoffen. Dissertation, University of Hamburg
2001.
(12) Ralph, J.; Bunzel, M.; Marita, J. M.; Hatfield, R. D.; Lu, F.; Kim, H. K.; Schatz, P. F.;
Grabber, J. H.; Steinhart, H. Peroxidase-dependent cross-linking reactions of phydroxycinnamates
in plant cell walls. Phytochem. Reviews 2004, 3, 79-96.
30

(13) Hartley, R. D.; Morrison, W. H. Monomeric and dimeric phenolic acids released from
cell walls of grasses by sequential treatment with sodium hydroxide. J. Sci. Food
Agric. 1991, 55, 365-375.
(14) Hatfield, R. D.; Ralph, J.; Grabber, J. H. Cell wall cross-linking by ferulates and
diferulates in grasses. J. Sci. Food Agric. 1999, 79, 403-407.
(15) Takenaka, S.; Nishio, Z.; Nakamura, Y. Induction of defense reactions in sugar beet and
wheat by treatment with cell wall protein fractions from the mycoparasite Pythium
oligandrum. Phytopathology 2003, 93, 1228-1232.
(16) Winkel-Shirley, B. Flavonoid biosynthesis. A colorful model for genetics, biochemistry,
cell biology, and biotechnology. Plant Physiol. 2001, 126, 485-493.
(17) Mock, H. P.; Vogt, T.; Strack, D. Sinapoylglucose: Malate sinapoyltransferase activity in
Arabidopsis thaliana and Brassica rapa. Z. Naturforsch. 1992, 47C, 680-682.
(18) Duthie, G.; Crozier, A. Plant-derived phenolic antioxidants. Curr. Opin. Clin. Nutr.
Metab. Care 2000, 3, 447-451.
(19) Dixon, R. A.; Paiva, N. L. Stress induced phenylpropanoid metabolism. Plant Cell 1995,
7, 1085-1097.
(20) Milkowski, C.; Baumert, A.; Schmidt, D.; Nehlin, L.; Strack, D. Molecular regulation of
sinapate ester metabolism in Brassica napus: expression of genes, properties of the
encoded proteins and correlation of enzyme activities with metabolite accumulation.
Plant J. 2004, 38, 80-92.
(21) Nielsen, J. K.; Olsen, O.; Haastrup Petersen, L.; Sorensen, H. 2-O-(p-coumaryl)-Lmalate,
2-O-caffeoyl-L-malate and 2-O-feruloyl-L-malate in Raphanus sativus.
Phytochemistry 1984, 23, 1741-1743.
(22) Brett, C. T.; Wende, G.; Smith, A. C.; Waldron, K. W. Biosynthesis of cell-wall ferulate
and diferulates. J. Sci. Food Agric. 1999, 79, 421-424.
(23) Zarra, I.; Sanchez, M.; Queijeiro, E.; Pena, M. J.; Revilla, G. The cell wall stiffening
mechanism in Pinus pinaster Aiton: Regulation by apoplastic levels of ascorbate and
hydrogen peroxide. J. Sci. Food Agric. 1999, 79, 416-420.
(24) Vaya, J.; Aviram, M. Nutritional antioxidants: Mechanisms of action, analyses of
activities and medical applications.
http://www.bentham.org/cmciema/sample/cmciema1-1/vaya/vaya-ms.htm.
(25) Walter, M. H.; Grima-Pettenati, J.; Grand, C.; Boudet, A. M.; Lamb, C. J. Cinnamoylalcohol
dehydrogenase, a molekular marker specific for lignin synthesis: cDNA
31

cloning and mRNA induction by fungal elicitor. Proc. Nat. Acad. Sci. 1988, 85, 5546-
5550.
(26) Copper-Driver, G. A.; Bhattacharya, M. Role of phenolics in plant evolution.
Phytochemistry 1998, 49, 1165-1174.
(27) Lois, R. Accumulation of UV-absorbing flavonoids induced by UV-B radiation in
Arabidopsis thaliana L. Planta 1994, 194, 498-503.
(28) Mittler, R. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 2002, 7,
405-410.
(29) Miller, N. J.; Rice-Evans, C. A. Cinnamates and hydroxybenzoates in the diet:
antioxidant activity assessed using ABTS + radical cation. British Food J. 1998, 99,
57-62.
(30) Rice-Evans, C. A.; Miller, N. J.; Bolwell, P. G.; Bramley, P. M.; Pridham, J. B. The
relative antioxidant activities of plant-derived polyphenolic flavonoids. Free Radic.
Res. 1995, 22, 375-383.
(31) Scalbert, A.; Morand, C.; Manach, C.; Remesy, C. Absorption and metabolism of
polyphenols in the gut and impact on health. Biomed. Pharmacother. 2002, 56, 276-
282.
(32) Cermak, R.; Landgraf, S.; Wolffram, S. The bioavailability of quercetin in pigs depends
on the glycoside moiety and on dietary factors. J. Nutr. 2003, 133, 2802-2807.
(33) The definition of dietary fibre. AACC REPORT 2001, 46, 112-126.
(34) Hopkins, M. J.; Englyst, H. N.; Macfarlane, S.; Furrie, E.; Macfarlane, G. T.; McBain, A.
J. Degradation of cross-linked and non-cross-linked arabinoxylans by the intestinal
microbiota in children. Appl. Environ. Microbiol. 2003, 69, 6354-6360.
(35) Adam, A.; Crespy, V.; Levrat-Verny, M. A.; Leenhardt, F.; Leuillet, M.; Demigne, C.;
Remesy, C. The bioavailability of ferulic acid is governed primarily by the food
matrix rather than its metabolism in intestine and liver rats. J. Nutr. 2002, 132, 1962-
1968.
(36) Kroon, P. A.; Faulds, C. B.; Ryden, P.; Robertson, J. A.; Williamson, G. Release of
covalently bound ferulic acid from fiber in the human colon. J. Agric. Food Chem.
1997, 45, 661-667.
(37) Sousa, C.; Valentao, P.; Rangel, J.; Lopes, G.; Pereira, J. A.; Ferreres, F.; Seabra, R. M.;
Andrade, P. B. Influence of two fertilization regimes on the amounts of organic acids
and phenolic compounds of tronchuda cabbage (Brassica oleracea L. var. costata
DC). J. Agric. Food Chem. 2005, 53, 9128-9132.
32

(38) Rochfort, S. J.; Imsic, M.; Jones, R.; Trenerry, V. C.; Tomkins, B. Characterization of
flavonol conjugates in immature leaves of pak choi [Brassica rapa L. ssp. chinensis L.
(Hanelt.)] by HPLC-DAD and LC-MS/MS. J. Agric. Food Chem. 2006, 54, 4855-
4860.
(39) Ferreres, F.; Sousa, C.; Vrchovska, V.; Valentao, P.; Pereira, J. A.; Seabra, R. M.;
Andrade, P. B. Chemical composition and antioxidant activity of tronchuda cabbage
internal leaves. Eur. Food Res. Technol. 2006, 222, 88-98.
(40) Nielsen, J. K.; Norbaek, R.; Olsen, C. E. Kaempferol tetraglucosides from cabbage
leaves. Phytochemistry 1998, 49, 2171-2176.
(41) Nielsen, J. K.; Olsen, C. E.; Petersen, M. K. Acylated flavonol glycosides from cabbage
leaves. Phytochemistry 1993, 34, 539-544.
(42) Kim, D. O.; Padilla-Zakour, O. I.; Griffiths, P. D. Flavonoids and antioxidant capacity of
various cabbage genotypes at juvenile stage. J. Food Sci. 2004, 69, 685-689.
(43) Wildanger, W.; Herrmann, K. Flavonole und Flavone der Gemüsearten I. Flavonole der
Kohlarten. Z. Lebensm. Unters.-Forsch. 1973, 152, 134-137.
(44) Llorach, R.; Gil-Izquierdo, A.; Ferreres, F.; Tomas-Barberan, F. A. HPLC-DAD-MS/MS
ESI characterization of unusual highly glycosylated acylated flavonoids from
cauliflower (Brassica oleracea L. var. botrytis) agroindustrial byproducts. J. Agric.
Food Chem. 2003, 51, 3895-3899.
(45) Sakakibara, H.; Honda, Y.; Nakagawa, S.; Ashida, H.; Kanazawa, K. Simultaneous
determination of all polyphenols in vegetables, fruits, and teas. J. Agric. Food Chem.
2003, 51, 571-581.
(46) Ferreres, F.; Valentao, P.; Llorach, R.; Pinheiro, C.; Cardoso, L.; Pereira, J. A.; Sousa,
C.; Seabra, R. M.; Andrade, P. B. Phenolic compounds in external leaves of tronchuda
cabbage (Brassica oleracea L. var. costata DC). J. Agric. Food Chem. 2005, 53,
2901-2907.
(47) Price, K. R.; Casuscelli, F.; Colquhoun, I. J.; Rhodes, M. J. C. Hydroxycinnamic acid
esters from broccoli florets. Phytochemistry 1997, 45, 1683-1687.
(48) Vallejo, F.; Tomas-Barberan, F. A.; Garcia-Viguera, C. Effect of climatic and sulphur
fertilisation conditions, on phenolic compounds and vitamin C, in the inflorescences
of eight broccoli cultivars. Eur. Food Res. Technol. 2003, 216, 395-401.
(49) Beveridge, T.; Loubert, E.; Harrison, J. E. Simple measurement of phenolic esters in
plant cell walls. Food Res. Intern. 2000, 33, 775-783.
33

(50) Seidemann, J. Pak-Choi, ein wenig bekanntes Gemüse. Die industrielle Obst und
Gemüseverwertung 1996, 10, 339-340.
(51) Herrmann, K. Inhaltsstoffe des Chinakohl und Pak-Choi. Die industrielle Obst und
Gemüseverwertung 1999, 2, 40-44.
(52) Kollabis-Rippel, K. Untersuchungen zu Pak Choi (Brassica rapa L. ssp. chinensis (L.)
Hanelt): Anbau und Genußeigenschaften. Dissertation 2000, Institut für
landwirtschaftlichen und gärtnerischen Pflanzenbau der Technischen Universität
München, Herbert Utz Verlag.
(53) Herrmann, K. Inhaltsstoffe der Kohlarten Teil I: Allgemeine chemische
Zusammensetzung einschließlich der Mineralstoffe, Spurenelemente und Vitamine.
Die industrielle Obst und Gemüseverwertung 1994, 7, 244-252.
(54) Vollendorf, N. W.; Marlett, J. A. Comparison of the two methods of fiber analysis of 58
foods. J. Food Compos. Analysis 1993, 6, 203-214.
(55) Merkblatt 2156 über Pak Choi, Bayerische Gartenakademie, Landesanstalt für Weinbau
und Gartenbau, Januar 2004.
(56) Keller, F. Pak-Choi, eine neue Kohlart aus dem fernen Osten. Der Gemüsebau 1986, 13,
15-16.
(57) Wonnerberger, C.; Keller, F. Gemüsebau. 1. Auflage, Ulmer Verlag, Stuttgart. 2004.
(58) Ternes, W. Naturwissenschaftliche Grundlagen der Lebensmittelzubereitung, 2. Auflage,
B. Behr's Verlag GmbH &Co., Hamburg 1994.
(59) Romero, C.; Brenes, M.; Garcia, P.; Garcia, A.; Garrido, A. Polyphenol changes during
fermentation of naturally black olives. J. Agric. Food Chem. 2004, 52, 1973-1979.
(60) Beecher, G. R. Overview of dietary flavonoids: Nomenclature, occurence and intake. J.
Nutr. 2003, 133, 3248S-3254S.
(61) Ciska, E.; Karamac, M.; Kosinska, A. Antioxidant activity of extracts of white cabbage
and sauerkraut. Pol. J. Food Nutr. Sci. 2005, 14, 367-373.
(62) Hollman, P. C. H.; Arts, I. C. W. Flavonols, flavones and flavonols - nature, occurence
and dietary burden. J. Sci. Food Agric. 2000, 80, 1081-1093.
(63) Gil, M. I.; Ferreres, F.; Tomas-Barberan, F. A. Effect of postharvest storage and
processing on the antioxidant constituents (flavonoids and vitamin C) of fresh cut
spinach. J. Agric. Food Chem. 1999, 47, 2213-2217.
(64) Price, K. R.; Casuscelli, F.; Colquhoun, I. J.; Rhodes, M. J. C. Composition and content
of flavonol glycosides in broccoli florets (Brassica oleracea) and their fate during
cooking. J. Sci. Food Agric. 1998, 77, 468-472.
34

(65) Pourcel, L.; Routaboul, J.-M.; Cheynier, V.; Lepiniec, L.; Debeaujon, I. Flavonoid
oxidation in plants: From biochemical properties to physiological functions. Trends
Plant Sci. 2006, 12, 29-36.
(66) Chun, O. K.; Smith, N.; Sakagawa, A.; Lee, C. Y. Antioxidant properties of raw and
processed cabbages. Int. J. Food Sci. Nutr. 2004, 55, 191-199.
(67) Tolonen, M.; Taipale, M.; Viander, B.; Pihlava, J.-M.; Korhonen, H.; Ryhänen, E.-L.
Plant-derived biomolecules in fermented cabbage. J. Agric. Food Chem. 2002, 50,
6798-6803.
(68) Iribarne, J. V.; Thomson, B. A. On the evaporation of small ions from charged droplets.
J. Chem. Phys. 1976, 64, 2287-2294.
(69) Kebarle, P.; Peschke, M. On the mechanisms by which the charged droplets produced by
electrospray lead to gas phase ions. Anal. Chimica Acta 2000, 406, 11-35.
(70) Agilent 1100 Series LC/MSD Trap Operations Maunual. January 2002, Version 4.1.
(71) GdCH Fortbildung, „Spezielle Techniken der HPLC und HPLC-MS“, Saarbrücken,19.-
23.09.2005.
(72) March, R. E. An introduction to quadrupole ion trap mass spectrometry. J. Mass
Spectrom. 1997, 32, 351-369.
(73) Cuyckens, F.; Claeys, M. Optimization of a liquid chromatography method based on
simultaneous electrospray ionization mass spectrometry and ultraviolet photodiode
array detection for analysis of flavonoid glycosides. Rapid Commun. Mass Spectrom.
2002, 16, 2341-2348.
(74) Mauri, P.; Pietta, P. Electrospray characterization of selected medicinal plant extracts. J.
Pharm. Biomed. Anal. 2000, 23, 61-68.
(75) Kammerer, D.; Claus, A.; Carle, R.; Schieberle, A. Polyphenol screening of pomace
from red and white grape varieties (Vitis vinifera L.) by HPLC-DAD-MS/MS. J.
Agric. Food Chem. 2004, 52, 4360-4367.
(76) Favretto, D.; Flamini, R. Application of electrospray ionization mass spectrometry to the
study of grape anthocyanins. Am. J. Enol. Vitic. 2000, 51, 55-64.
(77) Folin, O.; Ciocalteu, V. On tyrosine and tryptophan determinations in proteins. J. Biol.
Chem. 1927, 73, 627-650.
(78) Singleton, V. L.; Rossi, J. A. Colorimetry of total phenolics with phosphomolybdicphosphotungstic
acid reagent. Am. J. Enol. Vitic. 1965, 16, 144-158.
(79) Huang, D.; Ou, B.; Prior, R. L. The chemistry behind antioxidant capacity assays. J.
Agric. Food Chem. 2005, 53, 1841-1856.
35

(80) Prior, R. L.; Wu, X.; Schaich, K. Standardized methods for the determination of
antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food
Chem. 2005, 53, 4290-4302.
(81) Selvendran, R. R.; Ryden, P. Isolation and analysis of plant cell walls. Methods Plant
Biochem. 1990, 2, 549-579.
(82) Parr, A. J.; Ng, A.; Waldron, K. W. Ester-linked phenolic components of carrot cell
walls. J. Agric. Food Chem. 1997, 45, 2468-2471.
(83) Parker, M. L.; Ng, A.; Smith, A. C.; Waldron, K. W. Esterified phenolics of the cell
walls of chufa (Cyperus esculentus L.) tubers and their role in texture. J. Agric. Food
Chem. 2000, 48, 6284-6291.
(84) Rodriguez-Arcos, R. C.; Smith, A. C.; Waldron, K. W. Ferulic acid crosslinks in
asparagus cell walls in relation to texture. J. Agric. Food Chem. 2004, 52, 4740-4750.
(85) Waldron, K. W.; Parr, A. J.; Ng, A.; Ralph, J. Cell wall esterified phenolic dimers:
Identification and quantification by reverse phase high performance liquid
chromatography and diode array detection. Phytochem. Anal. 1996, 7, 305-312.
(86) Foti, M. C.; Daquino, C.; Geraci, C. Electron-transfer reaction of cinnamic acids and
their methyl esters with the DPPH radical in alcoholic solutions. J. Org. Chem. 2003,
69, 2309-2314.
36

LIST OF PAPERS AND MANUSCRIPTS
Chapter I
Identification of Flavonoids and Hydroxycinnamic Acids in Pak Choi Varieties
(Brassica campestris L. ssp. chinensis var. communis) by HPLC-ESI-MS n and NMR
and Their Quantification by HPLC-DAD
Chapter II
Impact of Fermentation on Phenolic Compounds in Leaves of Pak Choi (Brassica
campestris L. ssp. chinensis var. communis) and Chinese Leaf Mustard (Brassica
juncea Coss)
Chapter III
Cell-Wall-Bound Phenolic Compounds in Leaves of Pak Choi (Brassica campestris L.
ssp. chinensis var. communis)
Chapter IV
Cell-Wall-Bound Phenolic Compounds in Leaves of Chinese Brassica Vegetables
and the Influence of Post-Harvest Treatments
Chapter V
Free and Bound Phenolic Compounds in Leaves of Pak Choi (Brassica campestris L.
ssp. chinensis var. communis) and Chinese Leaf Mustard (Brassica juncea Coss)
37

Chapter I
39

Identification of Flavonoids and Hydroxy-
cinnamic Acids in Pak Choi Varieties
(Brassica campestris L. ssp. chinensis var.
communis) by HPLC-ESI-MS n and NMR
and Their Quantification by HPLC-DAD
BRITTA HARBAUM, *,† EVA MARIA HUBBERMANN, † CHRISTIAN WOLFF, ‡
RAINER HERGES, ‡ ZHUJUN ZHU ‡ , and KARIN SCHWARZ †
Journal of Agricultural and Food Chemistry 2007,
55(20); 8251-8260. DOI: 10.1021/jf071314+
http://pubs.acs.org/cgi-bin/article.cgi/jafcau/2007/55/i20/pdf/jf071314+.pdf
Department of Food Technology, Institute of Human Nutrition and Food Science, University
of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany, Otto Diels Institute of Organic
Chemistry, University of Kiel, Germany, and Department of Horticulture, School of
Agriculture and Food Science, Zhejiang Forestry University, Lin'an, Hangzhou, Zhejiang
311300, China
* Author to whom correspondence should be addressed (telephone +49 431 880 5034; e-mail
info@foodtech.uni-kiel.de).
†
Institute of Human Nutrition and Food Science, Kiel
‡
Otto Diels Institute of Organic Chemistry, University of Kiel
§ Zhejiang Forestry University
40

SUMMARY
This article reports the identification and quantification of twenty-eight polyphenols in
different cultivars of the Chinese cabbage variety pak choi (Brassica campestris L. ssp.
chinensis var. communis) by HPLC-DAD-ESI-MS n . Eleven flavonoid derivatives and
seventeen hydroxycinnamic acid derivatives were detected; the major flavonoid identified in
pak choi was kaempferol, glycosylated and acylated with different compounds such as
glucose and hydroxycinnamic acids. Smaller amounts of the flavonoid isorhamnetin were also
detected in pak choi. The presented HPLC-MS n method for identification gives a high
compound separation, soft electrospray ionization (negative mode) and fragmentation
procedure of the analyt molecules. The obtained [M-H] - and MS n values enabled a detailed
structure elucidation in this study. Furthermore, an isolation procedure by extraction and
preparative HPLC for two specific polyphenols is presented; therefore, additional structural
determination was carried out by 1 H and 13 C NMR spectroscopy for the main flavonoid
derivative kaempferol-3-O-hydroxyferuloylsophoroside-7-O-glucoside and the main
hydroxycinnamic acid derivative sinapoylmalate. The results of the NMR experiments
verified the tentatively identified phenolic compounds by ESI-MS n . The hydroxycinnamic
acid hydroxyferulic acid as moiety of flavonoids was clearly identified by NMR spectroscopy
for the first time. Overall, hydroxycinnamic acid derivatives were identified as different esters
of quinic acid, hexoses, and malic acid. The latter ones (caffeoylmalate,
hydroxyferuloylmalate, sinapoylmalate, feruloylmalate, and p-coumaroylmalate) are
described for the first time in cabbages. The total and individual contents of flavonoid and
hydroxycinnamic acid derivatives were determined in eleven cultivars of pak choi, with
higher concentrations present in the leaf blade than in the leaf stalk. Hydroxycinnamic acid
esters, particularly malic acid derivatives such as sinapoylmalate, are present in both the leaf
blade and leaf stalk, whereas flavonoids were only detected in the leaf blade. The total
flavonoid contents ranged from 4.68 to 16.67 mg/g dry matter in the leaf blades. The total
hydroxycinnamic acid contents ranged from 1.48 to 5.83 mg/g dry matter in the leaf blades
and 0.33 to 1.36 mg/g dry matter in the leaf stalks. Concentrations of the individual
polyphenols presented for eight flavonoid and seven hydroxycinnamic acid derivatives in the
leaf blades differed significantly among the cultivars.
41

Chapter II
43

Impact of Fermentation on Phenolic
Compounds in Leaves of Pak Choi
(Brassica campestris L. ssp. chinensis var.
communis) and Chinese Leaf Mustard
(Brassica juncea Coss)
BRITTA HARBAUM, *,† EVA MARIA HUBBERMANN, †
ZHUJUN ZHU ‡ , and KARIN SCHWARZ †
Journal of Agricultural and Food Chemistry 2008,
56(1); 148-157. DOI: 10.1021/jf072428o
http://pubs.acs.org/cgi-bin/sample.cgi/jafcau/2008/56/i01/pdf/jf072428o.pdf
Department of Food Technology, Institute of Human Nutrition and Food Science, University
of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany, and Department of Horticulture,
School of Agriculture and Food Science, Zhejiang Forestry University, Lin'an, Hangzhou,
Zhejiang 311300, China
* Author to whom correspondence should be addressed (telephone +49 431 880 5034; e-mail
info@foodtech.uni-kiel.de).
†
Institute of Human Nutrition and Food Science, Kiel
‡ Zhejiang Forestry University
44

SUMMARY
This article focuses on the influence of fermentation on flavonoids and hydroxycinnamic
acids in Chinese Brassica vegetables. Four different cultivars (two pak choi cultivars
Hangzhou You Dong Er and Shanghai Qing and two Chinese leaf mustard cultivars Xue Li
Hong and Bao Bao Qing Cai) were fermented in a traditional Chinese method called pickling.
The plant material was investigated by HPLC-DAD-ESI-MS n before and after the
fermentation procedure in order to determine the qualitative and quantitative changes in
polyphenols and the antioxidative potential. A detailed description of the identified phenolic
compounds of leaf mustard by HPLC-ESI-MS n is presented here for the first time, including
hydroxycinnamic acid mono- and diglycosides (gentiobioses) and flavonoidtetraglycosides
(particularly cv. Xue Li Hong).
Structural changes in polyphenols occurred during the fermentation process. Flavonoid
derivatives with a lower molecular mass (di- and triglycosides) and aglycones of flavonoids
and hydroxycinnamic acids, which do not normally occur in fresh or untreated plant material
of the Chinese cabbages, were detected in fermented cabbages compared to the main
compounds detected in non-fermented cabbages (tri- and tetraglycosides of flavonoids, and
hydroxycinnamic acid derivatives of malic acid, hexoses, and quinic acid). This indicates the
degradation of polyphenols into smaller molecules by fermentation. Microbial activity and the
changed bioavailability of polyphenols due to the changes in glycosilation of the phenol
structure were discussed in this article. Furthermore, contents of flavonoid and some
hydroxycinnamic acid derivatives were found to decrease during the fermentation process
(HPLC-DAD analysis). Some marginal losses of polyphenols were already observed in the
kneading step of the plant material prior to fermentation. Additionally, the Chinese cabbage
extracts were characterized by the Folin Ciocalteu and antioxidative capacity assays with
regard to the influence of ascorbic acid, which was also present in extracts. The antioxidative
potential in the TEAC assay as well as the values for the total phenolic content (TPC; Folin
Ciocalteu assay) were much higher in fermented cabbages compared to non-fermented.
However, this was not observable in the DPPH assay. The increase of the antioxidative
potential detected in the TEAC assay as well as the Folin Ciocalteu assay (high correlation of
TEAC and TPC values) was attributed to the qualitative changes of polyphenols [formation of
free hydroxyl group(s) in the fermentation procedure] as well as other reductons potentially
present.
45

Chapter III
47

Cell-Wall-Bound Phenolic Compounds in
Leaves of Pak Choi (Brassica campestris
L. ssp. chinensis var. communis)
BRITTA HARBAUM, *,† EVA MARIA HUBBERMANN, †
ZHUJUN ZHU ‡ , and KARIN SCHWARZ †
Department of Food Technology, Institute of Human Nutrition and Food Science, University
of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany; and Department of Horticulture,
School of Agriculture and Food Science, Zhejiang Forestry University, Lin'an, Hangzhou,
Zhejiang 311300, China
* Author to whom correspondence should be addressed (telephone +49 431 880 5034; e-mail
harbaum@foodtech.uni-kiel.de).
†
Institute of Human Nutrition and Food Science, Kiel
‡ Zhejiang Forestry University
48

ABSTRACT
Following alkaline hydrolysis, seven different bound phenolic compounds were identified in
the cell wall material of the Chinese cabbage pak choi (Brassica campestris L. ssp. chinensis
var. communis) by HPLC-DAD: trans-Ferulic acid, cis-ferulic acid, p-coumaric acid, sinapic
acid, vanillic acid, vanillin, and p-hydroxybenzaldehyde. Dimeric or oligomeric phenolic
compounds were not present. trans-Ferulic acid was the major bound phenolic compound in
the leaf blade as well as the leaf stalk. The total content of the bound phenolic compounds
was significantly higher in the leaf blade (total: 79.9 µg/g cell wall) than in the leaf stalk
(total: 37.5 µg/g cell wall) in 8-week-old plants. Older plants exhibited lower concentrations
of cell-wall-bound phenolic compounds, observed for the leaf blade as well as the whole leaf,
but the levels did not change in the stalk.
Keywords: pak choi, bound phenolic compounds, leaf blade, leaf stalk, vegetation period
49

1. INTRODUCTION
Dietary fiber is an important constituent of the human diet and consists of several substances,
including polysaccharides, oligosaccharides, and lignin (1). Bound phenolic compounds,
minor compounds present in the cell wall, may influence the chemical properties and
degradability of dietary fiber.
Several studies have investigated the qualitative and quantitative content of cell-wall-bound
phenolic compounds. These compounds are esterified monomers, dimers, and oligomers of
phenolic acids (hydroxycinnamic and hydroxybenzoic acids and aldehydes) in plant cell walls
and may cross-link the different cell wall components, such as arabinoxylans, pectins, lignins,
other carbohydrates, and proteins (2-6). These bound phenolic compounds are mainly bound
to arabinoxylans in grasses and cereals and to pectic polysaccharides in dicotyledons (7-10).
The highest amounts of bound phenolics are present in monocotyledons (approximately
1-5 mg/g cell wall) (11-13). Ferulic acid is the most abundant phenolic acid (12). Total dimer
concentrations found ranged from 0.1 to 1 µg/g dm (11, 12). These dimers are formed by
oxidative coupling via radical formation (4) or to a lesser extent via photochemical processes
triggered by a light supply (photochemical reaction leads to truxillic and truxin acids) (14-16).
Dimers are predominantly generated via the radical coupling of ferulic acids; dimers of other
hydroxycinnamic acids, e.g. sinapic acid, are also known (17), as well as oligomers, such as
dehydrotriferulic acids and dehydrotetraferulic acids, to exist in maize bran (18).
Less is known about the biosynthesis of bound phenolic compounds in the cell wall. The
acylation of polysaccharides via feruloyl-CoA and coumaroyl-CoA was reported by Brett et
al. (19). They also reported the secretion of phenolic precursors, such as hydroxycinnamates
amides and esters into the cell wall of dicotyledons, which were oxidatively linked to the cell
wall polymers. The presence of esterified phenolic compounds may protect the plant against
pathogen infestation and generate a chemical barrier that improves disease resistance (20).
Furthermore, increases in dimeric and monomeric compound content following exposure to
light were reported. These compounds influence the mechanical properties of the cell walls,
such as rigidity during plant growth (21, 22). A reduced biosynthesis of cell wall phenolic
compounds was observed under osmotic stress conditions in wheat (23), and Weber et al. (24)
reported decreasing levels of cell wall phenols during leaf development.
Esterified phenolic compounds may be an important factor in the digestability of the cell wall
in the animal intestine as well as for enzymatic degradation. Several studies have investigated
the influence of enzymes with esterase activity on the bound phenolic compounds both in
vitro and in vivo. The release of the ferulic acid of soluble feruloylated oligosaccharides by
50

microbial ferulic acid esterase in the human colon was reported, although the rate and degree
of the release is dependent on the substrate (e.g. sugar beet fiber or wheat bran) (25).
Furthermore, other hydroxycinnamic acids (such as sinapic acid and p-coumaric acid from
wheat bran) were released by human colonic cinnamoyl esterase (26). The ability to release
bound phenolic acids from the cell wall with different enzyme preparations, e.g.
feruloylesterase and commercial enzyme preparations, were also reported (27-30).
The main identified phenolic compounds in the cell walls of Brassica vegetables like broccoli
are p-hydroxybenzaldehyde, vanillin, and p-coumaric acid (31). Nothing is known about the
content of cell-wall-bound phenolic compounds of the Chinese Brassica vegetable pak choi.
This cabbage possesses a variety of free phenolic compounds, such as flavonoids and
hydroxycinnamic acid (32, 33). Several hydroxycinnamic acid derivatives of malate,
glycosides, and quinic acid were identified, as well as additional hydroxycinnamic acid
moieties of flavonoid derivatives.
The aim of this study was to identify and quantify the bound phenolic compounds in the cell
walls of pak choi leaves and to pinpoint the various influencing factors, e.g. plant parts and
plant age.
2. MATERIALS AND METHODS
2.1. Chemicals
Acetonitrile (HPLC grade, Fisher Scientific), ascorbic acid (Carl Roth GmbH), caffeic acid
(Carl Roth GmbH), p-coumaric acid (Sigma), Driselase (Fluka), EDTA (Carl Roth GmbH),
ethylacetate (Carl Roth GmbH), ferulic acid (Carl Roth GmbH), formic acid (Carl Roth
GmbH), conc. HCl (Carl Roth GmbH), p-hydroxybenzaldehyde (Carl Roth GmbH), methanol
(HPLC grade, Fisher Scientific), sinapic acid (Carl Roth GmbH), sodium dodecylsulphate
(Carl Roth GmbH), sodium hydroxide (Carl Roth GmbH), sodium metabisulphite (Carl Roth
GmbH), vanillic acid (Carl Roth GmbH), vanillin (Carl Roth GmbH), and Viscozyme L
(Sigma) were utilized.
2.2. Plant Material and Sampling
Pak choi (Brassica campestris L. ssp. chinensis var. communis, cv. Hangzhou You Dong Er)
was cultivated in a greenhouse. Material from 8-week-old plants was harvested and cell walls
were isolated for the method development of hydrolysis reactions. For the vegetation assay,
whole leaves of 4-, 6-, and 8-week-old plants were harvested and further separated into leaf
51

stalks and blades. Cell wall isolation was carried out for blade, stalk (single; n = 1), and whole
leaf (duplicate, n = 2) in each specimen.
2.3. Isolation of Cell Wall Material
The isolation of cell wall material was carried out with a method described by Beveridge et al.
(31) for Brassica vegetables and with some modifications of the procedure presented for
young cabbage leaves (34). Fresh plant material was first reduced to small pieces and then
comminuted in a crushing machine. Twofold 1.5% SDS solution containing 5 mM sodium
metabisulphite (approximately 600 mL) was added to the plant material (approximately 300
g) and disrupted by ultra turrax treatment (12000 min -1 ) for 5 min. The suspension was then
stirred for 15 min and filtrated through a nylon mesh. The residue was washed again with
0.5% SDS solution containing 3 mM sodium metabisulphite (3 times). Afterwards, the
residue was rinsed three times with 3 mM sodium metabisulphite solution and three times
with distilled water. The white residue was washed with ethanol three times to remove any
free phenolic compounds or other interfering substances. The freeze-dried residue was ball
milled and kept dry at an ambient temperature in the dark until further analysis.
2.4. Hydrolysis Reaction and Extraction
Approximately 200 mg of the isolated and dried cell wall material was hydrolyzed with 25
mL 1 M NaOH for 24 h under N2 in the dark to release the bound phenolic compounds
(saponification). The suspension was filtrated and the filtrate was further acidified to pH < 2
with conc. HCl. The acidified solution was extracted three times with ethyl acetate and the
combined organic phases were evaporated to dryness under N2. The residue was dissolved in
500 µL 25% aqueous methanol and analyzed by HPLC-DAD. Each cell wall hydrolysis
reaction was performed in triplicate (m = 3).
2.5. Compound Stability under Alkaline Conditions
The defined contents of phenolic standards of hydroxycinnamic acids, hydroxybenzoic acids
and hydroxybenzaldehydes were added to the 1 M NaOH solution. After 24 h an aliquot was
diluted 1:1 with 2 M HCl and directly analyzed by HPLC-DAD.
2.6. Enzymatic Treatment
For enzymatic treatment, 20 mg of the enzyme Driselase was added to approx. 200 mg of the
isolated cell wall in 12.5 mL water and stirred at 37°C for 48 h according to Bunzel et al.
(13). Afterwards, the hydrolysis reaction was carried out as described above (addition of 12.5
52

mL 2M NaOH to obtain a final concentration of 1M NaOH). For Viscozyme L treatment, 200
mg of the enzyme solution was added to 200 mg of the cell wall in 12.5 mL water and stirred
at 30°C for 24 h. The hydrolysis reaction was carried out as described above (addition of
12.5 mL 2M NaOH to obtain a final concentration of 1M NaOH).
2.7. HPLC Analysis
The HPLC analyses were carried out with an HP1100 HPLC (Agilent Tech. Waldbronn,
Germany) equipped with a diode array detector (DAD). Separation was carried out on a 250 x
4 mm (inside diameter), 5 µm, RP-18 Nucleodur column equipped with an 8 x 4 mm
Nucleodur guard column at 20°C. Eluent A consisted of 0.1% formic acid in water (HPLCgrade)
and eluent B of 100% acetonitrile at a flow rate of 0.7 mL/min. Gradient elution was
started with 0% B and reached 10% B after 7 min, 19% B after 20 min, 22% B after 25 min,
23% B after 26 min, 24 % B after 35 min, 50% B after 50 min, and 100% B after 64 min.
Compounds were detected and quantified at 280 nm for hydroxybenzoic acids and
hydroxybenzaldehydes and at 330 nm for hydroxycinnamic acids. Injection volume was set to
100 µL. Calibration was carried out with the analogous standard compounds for each
identified phenolic compound. cis-Ferulic acid was calculated by the standard curve of transferulic
acid.
2.8. Identification of Phenolic Compounds
Phenolic compounds were identified using the UV-spectra of the DAD, via the comparison of
the retention times of standard compounds and by coupled electrospray ionization mass
spectrometry analysis (HPLC-ESI-MS; isolation modus, negative ionisation). ESI-MS
analysis was carried out with the same settings as described by Harbaum et al. (32) for free
phenolic compounds.
2.9. Synthesis of 5-5´Dehydrodiferulic Acid
The synthesis was carried out according to Bunzel (35).
2.10. Preparation of Cell-Wall-Bound Diferulic Acid from Wheat
Freeze-dried and ground wheat material was hydrolyzed and extracted as described above
without prior cell wall isolation to obtain dimeric compounds in wheat extract, which can be
identified by HPLC-ESI-MS analysis.
53

2.11. Statistical Analysis
Standard deviations and significant differences were calculated by SPSS 15.0 (one-way
ANOVA, Bonferroni, p < 0.05).
3. RESULTS AND DISCUSSION
3.1. Cell Wall Isolation and Identification of Cell-Wall-Bound Phenolic Compounds
The cell wall isolation procedure produces a water insoluble residue. The cell wall residue of
the whole ball-milled leaf, which shows a particle size of approx. 5-20 µm (light microscopy),
was hydrolyzed with different concentrations of sodium hydroxide as shown in Table 1. The
main phenolic compounds esterified with the cell wall of pak choi were trans-ferulic acid and
vanillin (approximately 25 µg/g, and 16 µg/g; hydrolysis reaction with 1 M NaOH, Table 1).
Other compounds identified were cis-ferulic acid, sinapic acid, vanillic acid, p-coumaric acid,
and p-hydroxybenzaldehyde by comparison with reference compounds, UV spectra, and mass
spectrometry (HPLC-ESI-MS). An HPLC-DAD chromatogram at 280 nm and 330 nm is
presented in Figure 1. The concentrations of detected bound phenolic compounds were in the
magnitude of µg/g dried cell wall material.
A concentration of 1 M NaOH and 24 h hydrolysis time was necessary for a hydrolysis
reaction, and the content of total hydrolyzed phenolics was higher compared to that obtained
with 0.1 M NaOH (Table 1) after 24 h hydrolysis. A concentration of 2 M NaOH did not
result in a significantly higher total amount of detectable phenolic compounds after a
hydrolysis period of 24 h. The quantification of bound phenolic compounds have often been
described by different hydrolysis steps (0.1 M, 1 M, and 2 M NaOH) in the literature (16, 36-
38). In order to facilitate rapid hydrolysis, the reaction was carried out in this study with
1 M NaOH for 24 h hydrolysis as described by Beveridge et al. (31).
54

Table 1. Influence of Different Sodium Hydroxide Concentrations on the Detectable Content
of Bound Phenolic Compounds in Cell Walls of Pak Choi (Cell Walls of Whole Leaves)
(m = 3)
Phenolic compound
Vanillic acid 4.1 ± 0.4 4.7 ± 0.4 4.8 ± 0.5
p -Hydroxybenzaldehyde 5.2 * ± 1.1 6.3 *,§ ± 0.7 7.8 § ± 0.9
Vanillin 11.8 * ± 0.5 16.2 *,§ ± 2.9 20.9 § ± 2.5
p -Coumaric acid 1.3 ± 0.2 3.5
*
± 0.0 4.2
*
± 0.4
Sinapic acid 1.7 ± 1.2 3.1 ± 0.3 2.9 ± 1.0
trans -Ferulic acid 17.4 ± 2.6 25.2 * ± 0.8 27.9 * ± 1.9
cis -Ferulic acid 2.5 ± 0.1 5.2
*
± 0.5 3.4
§
± 0.3
Total content b
0.1 M NaOH 1 M NaOH 2 M NaOH
[µg/g] a
[µg/g] a
44.1 ± 4.6 64.2 * ± 4.9 71.9 * ± 5.5
55
[µg/g] a
a Content in dried cell wall; significant differences are indicated by the ( * , § ) symbols.
b Total content of all individual compounds combined.
Figure 1. HPLC chromatogram of detected cell-wall-bound phenolic compounds in whole
leaves of pak choi, monitored at 280 nm and 330 nm
In the present study, dimeric or oligomeric phenolic compounds were not detected in the cell
wall material of pak choi. Beveridge et al. (31) have reported the absence of dimers as well as
oligomers in broccoli. These compounds are mostly present in the cell walls of grasses;
significant contents were reported for carrots (31, 36, 39), green asparagus (40), and other
dicotyledons. The synthesized standard compound 5-5`-dehydrodiferulic acid possesses a

etention time of tR = 44.4 min; HPLC-ESI-MS analysis showed the molecule mass [M-H] - at
m/z 385 and the UV spectra was in accordance with the literature data (37). Alkaline
hydrolysis products of wheat material showed a retention time of tR = 46.8 min and the
molecule mass was [M-H] - at m/z 385. As presented in Figure 1, no dimeric compounds were
identified in the cell wall extracts of pak choi in the range of identified retention times for
these compounds. The dimeric compounds were also not present in the stalk material of pak
choi, although the firmer texture and the influence of light exposure led to the assumption that
crosslinks had been generated during plant growth.
Experiments on the stability of hydroxycinnamic acids and hydroxybenzoic acids and
aldehydes in 1 M NaOH during 24 hours revealed recoveries of 98-102% (vanillic acid,
102%; p-hydroxybenzaldehyde, 102%; vanillin, 98%; p-coumaric acid, 100%; sinapic acid,
86%; ferulic acid, 98%). Only sinapic acid showed a reduced recovery of 86% and caffeic
acid was completely degraded. The instability of caffeic acid was also reported by Sun et al.
(41), but information on caffeic acid as a bound phenolic compound is scarce in the literature.
The occurrence of caffeic acid as a bound compound in beer was observed under protective
conditions by Nardini et al. (42). In order to test whether caffeic acid is present in the cell
wall, the hydrolysis reaction was carried out in the presence of 1% ascorbic acid and 10 mM
EDTA in the 1 M NaOH hydrolysis solution as described by Nardini et al. (42). Under these
conditions, caffeic acid added as a reference compound was quite stable (recovery of approx.
75%). However, no caffeic acid was found in the cell wall material. Furthermore,
hydroxyferulic acid, which had previously been identified as a moiety of free phenolic
compounds (such as flavonoids and esters of malates and glycosides in pak choi), was not
found in the cell wall material of pak choi.
3.2. Enzymatic Treatment of the Cell Wall
Enzymatic treatments were used to enhance the solubilization of the cell wall and to reduce
the cell wall material into smaller fragments. Driselase derived from Aspergillus niger
exhibits cellulase, xylanase, galactanase, arabinanase, and polygalacturonase activity but no
ferulic acid esterase activity (13). The possible contamination of the Driselase enzyme with
phenolic compounds has to be considered. Viscozyme L is an enzyme preparation that is used
during mashing. It was reported that Viscozyme L possesses ferulic acid esterase activity in
addition to xylanase, β-glucanase, and cellulase activity (43). The two different enzymes were
used before the hydrolysis reaction with 1 M NaOH. The washed and dried final residue after
enzymatic and additional alkaline treatment was much lower compared to residue that had not
been subjected to enzymatic treatment (2.8% for Viscozyme L and 12.6% for Driselase in
56

elation to the utilized cell wall, see Table 2) and resulted in stronger degradation of the cell
wall. The losses in cell wall mass (60% residue; solubilization of appr. 40%) during the
hydrolysis reaction without enzymatic treatment may have resulted from the solubilization of
hemicelluloses by sodium hydroxide treatment, as reported by Bunzel and Steinhart (44). The
use of these two enzyme preparations did not result in higher concentrations of released
phenolic compounds (Table 2), although there are differences in the hydrolyzed content of
the individual compounds (e.g. vanillin and trans-ferulic acid; data not shown). Additionally,
the use of Viscozyme L without alkaline hydrolysis (due to ferulic esterase activity) resulted
in lower levels of detectable phenolic compounds compared to the use of the enzyme with
alkaline hydrolysis (data not shown). Caffeic acid, which is degraded by alkaline treatment,
was not detected by enzymatic treatment without alkaline hydrolysis.
Table 2. Influence of Enzymatic Treatment on the Detectable Total Content of Bound
Phenolic Compounds in Cell Walls of Pak Choi
Total content b
Residue c
Without enzyme Viscozyme L Driselase
[µg/g] a
[µg/g] a
69.4 ± 8.3 66.7 ± 2.9 64.0 ± 1.8
60.0% 2.8% 12.6%
57
[µg/g] a
a Content in dried cell wall (m = 3).
b Total content of all individual compounds combined.
c Insoluble cell wall fraction after hydrolysis reaction in relation to utilized cell wall in %.
3.3. Quantitative Contents of Cell-Wall-Bound Phenolic Compounds during the
Vegetation Period
Figure 2 presents the changes during the vegetation in total bound phenolic content
differentiated by leaf blade, stalk, and whole leaf. A decrease of detectable bound phenolic
compounds during the vegetation period was observed for pak choi, particularly for the leaf
blade. The decrease was also observed for the detected compounds in the whole leaf.
However, changes in concentrations in the leaf stalk were marginal, which might explain the
less pronounced decrease in the whole leaf. The decrease of cell-wall-bound phenolic
compounds is in accordance with the literature data on grapevine leaves. Weber et al. (24)
detected a reduction of the wall bound compounds protocatechuic acid and aldehyde with
increasing plant age, and they suggested a relationship to age-dependent fungal disease
resistance. Furthermore, a decrease in monomeric compounds can be assumed due to dimer
formation in the plant with increasing age (e.g. by light); however, no formed dimeric

compounds were detected in older pak choi plants (in blade, stalk, or whole leaf).
Additionally, fluctuations in the contents of bound phenolic compounds in the cell wall
material of plants as well as the blade/stalk ratio have to be considered. A decreasing
blade/stalk ratio (Table 3) from 4-week-old plants (0.47) to 6-week-old plants (0.26) may
result in lower total content of bound phenolic compounds in the whole leaves compared to
the whole leaves of 4-week-old plants; however, the changes in the contents of the whole
leaves from 4 to 6 weeks were marginal. A distinct decrease in the total contents in 6-weekto
8-week-old plants was observable for both the leaf blade and the whole leaf with respect to
the marginal change of the blade/stalk ratio (6 weeks: 0.26; 8 weeks: 0.23).
Total content of
bound phenolic compounds
[µg/g cell wall]
180
160
140
120
100
80
60
40
20
0
4 Weeks 6 Weeks 8 Weeks
Plant age
58
Stalk
Blade
Whole leaf
Figure 2. Changes of total content of bound phenolic compounds in leaf blades, stalks, and
whole leaves of pak choi dependent on plant age (whole leaf n = 2, blade and stalk n = 1;
m = 3)

Table 3. Influence of Plant Age on the Individual Detectable Contents of Bound Phenolic
Compounds in Cell Walls of Whole Leaves of Pak Choi
Phenolic compound
Vanillic acid 6.4 ± 1.0 4.3 * ± 0.6 3.6 * ± 0.4
p -Hydroxybenzaldehyde 3.7 ± 1.0 2.2 * ± 0.2 1.2 * ± 0.3
Vanillin 23.3 ± 2.2 23.0 ± 3.5 14.6 * ± 1.8
p -Coumaric acid 2.4 ± 0.3 2.7 ± 0.2 4.6 * ± 0.5
Sinapic acid 2.3 ± 0.2 0.9 * ± 0.1 0.5 § ± 0.1
trans -Ferulic acid 26.8 ± 1.5 28.1 ± 2.7 16.4 * ± 1.4
cis -Ferulic acid 9.0 ± 1.0 8.9 ± 1.0 5.6 * ± 0.9
Total content b
Blade/stalk ratio c
4 Weeks
Plant age
6 Weeks 8 Weeks
[µg/g] a
73.7 ± 4.5 70.2 ± 5.8 46.6 * ± 4.6
0.47 0.26 0.23
59
[µg/g] a
[µg/g] a
a * §
Content in dried cell wall (n = 2, m = 3); significant differences are indicated by the ( , )
symbols.
b
Total content of all individual compounds combined.
c
Ratio of blade to stalk based on fresh plant material.
3.4. Quantitative Contents of Individual Cell-Wall-Bound Phenolic Compounds
The detected individual concentrations of bound phenolic compounds during the vegetation
period of the whole leaves are presented in Table 3. The decrease presented for the total
contents was also observed for the individual compounds in whole leaves (particularly from 6
weeks to 8 weeks), e.g. vanillin and trans-ferulic acid. p-Coumaric acid was the only
compound to increase with plant age (2.7 µg/g for 6-week-old plants to 4.6 µg/g for 8-weekold
plants).
The concentrations of trans-ferulic acid and vanillin, which ranged from 16.4-28.2 µg/g and
14.6-23.3 µg/g in the cell walls over the vegetation period, are the main phenolic compounds
in pak choi (Table 3). The detected contents for vanillin were in the same range as reported in
the literature for broccoli florets: 26 µg/g vanillin, 19 µg/g p-hydroxybenzaldehyde and
28 µg/g p-coumaric acid. The contents for p-hydroxybenzaldehyde (1.2-3.7 µg/g cell wall)
and p-coumaric acid (2.4-4.6 µg/g cell wall) were much lower in the present study for pak
choi. However, trans-ferulic acid, the main compound in pak choi, was not detected in
broccoli florets by Beveridge et al. (31).
The total levels of bound phenolic compounds were much lower in the stalks than in the
blades of 4-, 6-, as well as 8-week-old plants (Figure 2). The total phenolic content in pak
choi is twice as high in the leaf blade (79.9 µg/g) than in the leaf stalk (37.5 µg/g) for

8-week-old plants. Table 4 shows the differences in the individual components of phenolic
compounds in the leaf stalk and leaf blade. The concentrations of p-coumaric acid, sinapic
acid, trans-ferulic acid, and cis-ferulic acid were distinct higher in the leaf blade than in the
leaf stalk. The high differences between the blades and stalks were significant regarding the
main phenolic compound trans-ferulic acid, whose concentrations were four times as high in
leaf blades (38.3 µg/g) than in leaf stalks (9.9 µg/g). Renard et al. (45) found trans-ferulic
acid concentrations of 2 mg/g in the cell walls of blades and 0.5 mg/g in the cell walls of
quinoa stalks. The high differences between blades and stalks of pak choi are also pronounced
for
p-coumaric acid and cis-ferulic acid in the present study. Sun et al. (41) reported higher
concentrations of monomeric bound phenolic compounds in cereal straws and stalks. Eraso
and Hartley (11) identified higher concentrations of cell-wall-bound monomers in sorghum
stalks (19 mg/g) than in blades (15 mg/g), but the concentrations of the detected dimeric
compounds in sorghum are approximately twofold higher in leaf blades (2.7 mg/g) than in
leaf stalks (1.3 mg/g).
Table 4. Influence of Different Plant Parts on the Detectable Content of Bound Phenolic
Compounds in Cell Walls (Greenhouses, 8-week-old Plants)
Phenolic compound
Vanillic acid 4.4 ± 0.5 4.2 ± 0.2
p -Hydroxybenzaldehyde 1.5 ± 0.1 1.9 ± 0.3
Vanillin 15.8 ± 1.6 12.5 * ± 0.9
p -Coumaric acid 2.9 ± 0.3 8.0 * ± 0.4
Sinapic acid 0.4 ± 0.1 1.0 * ± 0.1
trans -Ferulic acid 9.9 ± 0.5 38.3 * ± 4.1
cis -Ferulic acid 2.6 ± 0.3 13.9 * ± 2.5
Total content b
Stalk Blade
[µg/g] a
[µg/g] a
37.5 ± 2.9 79.9 * ± 3.9
a *
Content in dried cell wall (n = 1, m = 3); significant differences are indicated by the ( )
symbol.
b
Total content of all individual compounds combined.
Overall, pak choi possesses small amounts of cell-wall-bound phenolic compounds compared
to other vegetables like as carrots, sugar beets, or asparagus (39, 40, 46). For the
characterization of bound phenolic compounds, it is important to consider the individual plant
part as well as the age of the investigated plants. Moreover, other possible influences on
bound phenolic compounds during plant growth need to be taken into account, such as light
60

exposure or stresses, pathogen infestation, or agricultural conditions as well as post-harvest
effects, such as storage or cooking (9, 20, 22, 23, 36, 40).
4. ACKNOWLEDGMENT
We thank Stephanie thor Straten for her technical assistance.
5. LITERATURE CITED
(1) The definition of dietary fibre. AACC REPORT 2001, 46, 112-126.
(2) Bunzel, M.; Ralph, J.; Lu, F.; Hatfield, R. D.; Steinhart, H. Lignins and ferulate-coniferyl
alcohol cross-coupling products in cereal grains. J. Agric. Food Chem. 2004, 52,
6496-6502.
(3) Hatfield, R. D.; Ralph, J.; Grabber, J. H. Cell wall cross-linking by ferulates and
diferulates in grasses. J. Sci. Food Agric. 1999, 79, 403-407.
(4) Ralph, J.; Bunzel, M.; Marita, J. M.; Hatfield, R. D.; Lu, F.; Kim, H. K.; Schatz, P. F.;
Grabber, J. H.; Steinhart, H. Peroxidase-dependent cross-linking reactions of phydroxycinnamates
in plant cell walls. Phytochem. Reviews 2004, 3, 79-96.
(5) Piber, M.; Koehler, P. Identification of dehydro-ferulic acid-tyrosine in rye and wheat:
Evidence for a covalent cross-link between arabinoxylans and proteins. J. Agric. Food
Chem. 2005, 53, 5276-5284.
(6) Grabber, J. H.; Ralph, J.; Hatfield, R. D. Cross-linking of maize walls by ferulate
dimerization and incorporation into lignin. J. Agric. Food Chem. 2000, 48, 6106-6113.
(7) Fry, S. C. Intracellular feruloylation of pectic polysaccharides. Planta 1987, 171, 205-211.
(8) Rombouts, F. M.; Thibault, J. F. Feruloylated pectic substances from sugar-beet pulp.
Carbohydr. Res. 1986, 154, 177-187.
(9) Garcia, E.; Filisetti, T. M. C. C.; Udaeta, J. E. M.; Lajolo, F. M. Hard-to-cook-beans
(Phaseolus vulgaris): Involvement of phenolic compounds and pectates. J. Agric.
Food Chem. 1998, 46, 2110-2116.
(10) Saulnier, L.; Thibault, J. F. Ferulic acid and diferulic acids as components of sugar-beet
pectins and maize bran heteroxylans. J. Sci. Food Agric. 1999, 79, 396-402.
(11) Eraso, F.; Hartley, R. D. Monomeric and dimeric phenolic constituents of plant cell walls
- possible factors influencing wall biodegradability. J. Sci. Food Agric. 1990, 51, 163-
170.
61

(12) Andreasen, M. F.; Christensen, L. P.; Meyer, A. S.; Hansen, A. Content of phenolic acids
and ferulic acid dehydrodimers in 17 rye (Secale cereale L.) varieties. J. Agric. Food
Chem. 2000, 48, 2837-2842.
(13) Bunzel, M.; Allerdings, E.; Sinwell, V.; Ralph, J.; Steinhart, H. Cell wall
hydroxycinnamates in wild rice (Zizania aquatica L.) insoluble dietary fibre. Eur.
Food Res. Technol. 2001, 214, 482-488.
(14) Ford, C. W.; Hartley, R. D. Cyclodimers of p-coumaric and ferulic acids in the cell walls
of tropical grasses. J. Sci. Food Agric. 1990, 50, 29-43.
(15) Turner, L. B.; Mueller-Harvey, I.; McAllan, A. B. Light induced isomerization and
dimerization of cinnamic acid derivatives in cell walls. Phytochemistry 1993, 33, 791-
796.
(16) Hartley, R. D.; Morrison, W. H. Monomeric and dimeric phenolic acids released from
cell walls of grasses by sequential treatment with sodium hydroxide. J. Sci. Food
Agric. 1991, 55, 365-375.
(17) Bunzel, M.; Ralph, J.; Kim, H.; Lu, F.; Ralph, S. A.; Marita, J. M.; Hatfield, R. D.;
Steinhart, H. Sinapate dehydrodimers and sinapate-ferulate heterodimers in cereal
dietary fiber. J. Agric. Food Chem. 2003, 51, 1427-1434.
(18) Bunzel, M.; Ralph, J.; Brüning, P.; Steinhart, H. Structural identification of
dehydrotriferulic and dehydrotetraferulic acids isolated from insoluble maize bran
fiber. J. Agric. Food Chem. 2006, 54, 6409-6418.
(19) Brett, C. T.; Wende, G.; Smith, A. C.; Waldron, K. W. Biosynthesis of cell-wall ferulate
and diferulates. J. Sci. Food Agric. 1999, 79, 421-424.
(20) Santiago, R.; Butron, A.; Reid, L. M.; Arnason, J. T.; Sandoya, G.; Souto, X. C.; Malvar,
R. A. Diferulate content of maize sheaths is associated with resistance to the
Mediterranean corn borer sesamia nonagrioides (Lepidoptera: Noctuidae). J. Agric.
Food Chem. 2006, 54, 9140-9144.
(21) Miyamoto, K.; Ueda, J.; Takeda, S.; Ida, K.; Hoson, T.; Masuda, Y.; Kamisaka, S. Lightinduced
increase in the contents of ferulic and diferulic acids in cell walls of Avena
coleoptiles: Its relationship to growth inhibition by light. Physiol. Plantarium 1994,
92, 350-355.
(22) Tan, K. S.; Hoson, T.; Masuda, Y.; Kamisaka, S. Involvement of cell wall-bound
diferulic acid in light-induced decrease in growth rate and cell wall extensibility of
Oryza coleoptiles. Plant Cell Physiol. 1992, 33, 103-108.
62

(23) Wakabayashi, K.; Hoson, T.; Kamisaka, S. Osmotic stress suppresses cell wall stiffening
and the increase in cell wall bound ferulic and diferulic acids in wheat coleoptiles.
Plant Physiol. 1997, 113, 967-973.
(24) Weber, B.; Hoesch, L.; Rast, D. M. Protocatechualdehyde and other phenols as cell wall
components of grapevine leaves. Phytochemistry 1995, 40, 433-437.
(25) Kroon, P. A.; Faulds, C. B.; Ryden, P.; Robertson, J. A.; Williamson, G. Release of
covalently bound ferulic acid from fiber in the human colon. J. Agric. Food Chem.
1997, 45, 661-667.
(26) Andreasen, M. F.; Kroon, P. A.; Williamson, G.; Garcia-Conesa, M.-T. Esterase activity
able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the
intestine of mammals. J. Agric. Food Chem. 2001, 49, 5679-5684.
(27) Yu, P.; McKinnon, J. J.; Maenz, D. D.; Racz, V. J.; Christensen, D. A. The specificity
and the ability of Aspergillus feruloyl esterase to release p-coumaric acid from
complex cell walls of oat hulls. J. Chem. Technol. Biotechnol. 2004, 79, 729-733.
(28) Andreasen, M. F.; Christensen, L. P.; Meyer, A. S.; Hansen, A. Release of
hydroxycinnamic and hydroxybenzoic acids in rye by commercial plant cell wall
degrading enzyme preparations. J. Sci. Food Agric. 1999, 79, 411-413.
(29) Szwajgier, D.; Targonski, Z. Release of free ferulic acid and feruloylated arabinoxylans
from brewery's spent grain by commercial enzyme preparation. Electr. J. Pol. Agric.
Univers. 2006, 9, http://www.ejpau.media.pl/volume9/issue1/abs-26.html.
(30) Kroon, P. A.; Garcia-Conesa, M.-T.; Fillingham, I. J.; Hazlewood, G. P.; Williamson, G.
Release of ferulic acid dehydrodimers from plant cell walls by feruloyl esterases. J.
Sci. Food Agric. 1999, 79, 428-434.
(31) Beveridge, T.; Loubert, E.; Harrison, J. E. Simple measurement of phenolic esters in
plant cell walls. Food Res. Technol. 2000, 33, 775-783.
(32) Harbaum, B.; Hubbermann, E. M.; Wolff, C.; Herges, R.; Zhu, Z., Schwarz, K.
Identification of flavonoids and hydroxycinnamic acids in pak choi varieties (Brassica
campestris L. ssp. chinensis var. communis) by HPLC-ESI-MS n and NMR and their
quantification by HPLC-DAD. J. Agric. Food Chem. 2007, 55, 8251-8260.
(33) Rochfort, S. J.; Imsic, M.; Jones, R.; Trenerry, V. C.; Tomkins, B. Characterization of
flavonol conjugates in immature leaves of pak choi [Brassica rapa L. ssp. chinensis L.
(Hanelt.)] by HPLC-DAD and LC-MS/MS. J. Agric. Food Chem. 2006, 54, 4855-
4860.
(34) Selvendran, R. R.; Ryden, P. Isolation and analysis of plant cell walls. Methods Plant
Biochem. 1990, 2, 549-579.
63

(35) Bunzel, M. Monomere und dimere Phenolcarbonsäuren als strukturbildende Elemente in
löslichen und unlöslichen Getreideballaststoffen. Dissertation, University of Hamburg
2001.
(36) Ng, A.; Parr, A. J.; Ingham, M. L.; Rigby, N. M.; Waldron, K. W. Cell wall chemistry of
carrots (Daucus carota cv. Amstrong) during maturation and storage. J. Agric. Food
Chem. 1998, 46, 2933-2939.
(37) Waldron, K. W.; Parr, A. J.; Ng, A.; Ralph, J. Cell wall esterified phenolic dimers:
Identification and quantification by reverse phase high performance liquid
chromatography and diode array detection. Phytochem. Anal. 1996, 7, 305-312.
(38) Rodriguez-Arcos, R. C.; Smith, A. C.; Waldron, K. W. Effect of storage on wall-bound
phenolics in green asparagus. J. Agric. Food Chem. 2002, 50, 3197-3203.
(39) Parr, A. J.; Ng, A.; Waldron, K. W. Ester-linked phenolic components of carrot cell
walls. J. Agric. Food Chem. 1997, 45, 2468-2471.
(40) Rodriguez-Arcos, R. C.; Smith, A. C.; Waldron, K. W. Ferulic acid crosslinks in
asparagus cell walls in relation to texture. J. Agric. Food Chem. 2004, 52, 4740-4750.
(41) Sun, R. C.; Sun, X. F.; Zhang, S. H. Quantitative determination of hydroxycinnamic
acids in wheat, rice, rye, and barley straws, maize stalks, oil palm frond fiber, and fastgrowing
poplar wood. J. Agric. Food Chem. 2001, 49, 5122-5129.
(42) Nardini, M.; Ghiselli, A. Determination of free and bound phenolic acids in beer. Food
Chem. 2004, 84, 137-143.
(43) Szwajgier, D.; Pielecki, J.; Targonski, Z. The release of ferulic acid and feruloylated
oligosaccharides during wort and beer production. J. Inst. Brew. 2005, 111, 372-379.
(44) Bunzel, M.; Steinhart, H. Ballaststoffe aus Pflanzenzellwänden. Ernährungs-Umschau
2003, 12, 469-475.
(45) Renard, C. M. G. C.; Wende, G.; Booth, E. J. Cell wall phenolics and polysaccharides in
different tissues of quinoa (Chenopodium quinoa Willd). J. Sci. Food Agric. 1999, 79,
2029-2034.
(46) Parr, A. J.; Waldron, K. W.; Ng, A.; Parker, M. L. The wall bound phenolics of Chinese
water chestnut (Eleocharis dulcis). J. Sci. Food Agric. 1996, 71, 501-507.
This work was supported by DFG 592-5-1.
64

Chapter IV
65

Cell-Wall-Bound Phenolic Compounds
in Leaves of Chinese Brassica Vegetables
and the Influence of
Post-Harvest Treatments
BRITTA HARBAUM, *,† EVA MARIA HUBBERMANN, †
ZHUJUN ZHU ‡ , and KARIN SCHWARZ †
Department of Food Technology, Institute of Human Nutrition and Food Science, University
of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany; and Department of Horticulture,
School of Agriculture and Food Science, Zhejiang Forestry University, Lin'an, Hangzhou,
Zhejiang 311300, China
* Author to whom correspondence should be addressed (telephone +49 431 880 5034; e-mail
info@foodtech.uni-kiel.de).
†
Institute of Human Nutrition and Food Science, Kiel
‡ Zhejiang Forestry University
66

ABSTRACT
The release of cell-wall-bound phenolic compounds in Chinese cabbages according to various
durations of alkaline hydrolysis time is reported in this article. Monomeric vanillin and
trans-ferulic acid, the predominant bound phenolic compounds in Chinese cabbages, were
investigated over a seven-day hydrolysis period and triggered the complete release of bound
phenolics in all plant parts (cell wall of leaf blade or leaf stalk) after four days.
The concentrations of bound phenolics were found to be of the same magnitude in both
German- (greenhouse, 6-week-old plants) and Chinese-grown (field, 10-week-old plants)
specimens of the four investigated Chinese cabbages (pak choi: cv. Hangzhou You Dong Er
and cv. Shanghai Qing; and leaf mustard: cv. Xue Li Hong and cv. Bao Bao Qing Cai).
Furthermore, the influence of the traditional fermentation process on the content of cell-wallbound
phenolic compounds in Chinese cabbages was investigated for the four cultivars. Only
small changes in the cell-wall-bound phenolic content (decreases and increases of individual
compounds) occurred as a result of the fermentation procedure. In other words, there was no
clear indication that phenolic compounds had been released by either microorganism or
enzymatic activity (e.g. feruloyl esterase) or changes in pH.
Keywords: cell-wall-bound phenolic compounds, pak choi, leaf mustard, traditional Chinese
fermentation procedure
67

1. INTRODUCTION
Bound phenolic compounds are mainly present in monocotyledons such as barley, rye, or
maize (1-3). Cell-wall-bound phenolic compounds occur in vegetables, e.g. in asparagus,
spinach, carrots, and broccoli (2, 4-7). Monomeric, dimeric, or oligomeric compounds, e.g.
trans-ferulic acid and its dehydrodimers, are esterified to the cell wall. Dimeric and
oligomeric compounds have the ability to cross-link cell wall components (1, 8).
The investigation of phenolic compounds via several hydrolysis steps consisting of 0.1 M,
1 M, and 2 M NaOH and lasting 24 hours each has been described for different plants (e.g.
asparagus or carrots); the objective was to stimulate the successive release of bound phenolic
compounds by inducing increasingly rigorous conditions (5, 9-11).
Food processing and vegetable storage can affect the composition of bound phenolic
compounds as well as generate the cross-links that occur naturally in plants (particularly
monocotyledons) and influence the texture and biodegradability of plants. A post-harvest
hardening of asparagus spears, the increasing crispness of Chinese waterchestnut, or the
hardening of beans by cooking were related to the increase of monomeric and dimeric bound
phenolic compounds in the cell walls (8, 12, 13). Ng et al. (9) reported an increase of
esterified ferulic acid cross-links during the maturation of carrots. Furthermore, the degree of
cross-links influences the gelation and hydration properties of sugar beet pectins (14).
Beveridge et al. (4) detected the compounds p-hydroxybenzaldehyde, vanillin, and pcoumaric
acid as the main bound phenolics in the cell walls of the Brassica vegetable
broccoli. Nothing is known about the qualitative or quantitative changes that take place in the
bound phenolic compounds of Brassica vegetables as a result of e.g. storage conditions or the
fermentation process. The traditional fermentation procedure for Chinese cabbages, which is
conducted without starter culture (and therefore differs from sauerkraut production in
Germany), is presented by Harbaum et al. (15). The plant material is withered, kneaded, and
fermented over a period of approximately four weeks. Lactobacillus streams are responsible
for the fermentation. Ciska et al. (16) attributed the release of cell-wall-bound phenolic
compounds during the fermentation of white cabbage (sauerkraut) to the higher antioxidative
potential and total phenolic content (by Folin Ciocalteu) present after fermentation. However,
the authors did not quantify the contents of the cell-wall-bound phenolics. Boskov Hansen
(17) reported a decrease in the contents of ester-bound phenolic compounds during breadmaking
as a result of enzymatic activity and changes in pH. Coghe et al. (18) described the
release of ferulic acid during brewing and fermentation due to the feruloyl esterase activity of
brewer’s yeast. Enzymes such as esterases (e.g. feruloylesterase) are able to release bound
68

phenolic compounds (19-21), e.g. during beer production (22). These enzymes are also
present in Lactobacillus streams (23-25). The esterase activity of microbiotics in the human
colon are able to release the ester-linked hydroxycinnamoyl acids from dietary fiber (26, 27).
The aim of this study was to determine the cell-wall-bound phenolic compounds in pak choi
and to evaluate the factors influencing the release of phenolic compounds by chemical
hydrolysis. A further objective was to investigate the influence of growing conditions and the
traditional Chinese fermentation procedure on the content of bound phenolics in Chinese
cabbage cultivars.
2. MATERIALS AND METHODS
2.1. Chemicals
Acetonitrile (HPLC grade, Fisher Scientific), p-coumaric acid (Sigma), ethylacetate (Carl
Roth GmbH), ferulic acid (Carl Roth GmbH), formic acid (Carl Roth GmbH), methanol
(HPLC grade, Fisher Scientific), conc. HCl (Carl Roth GmbH), p-hydroxybenzaldehyde (Carl
Roth GmbH), sinapic acid (Carl Roth GmbH), sodium dodecylsulphate (Carl Roth GmbH),
sodium hydroxide (Carl Roth GmbH), sodium metabisulphite (Carl Roth GmbH), vanillic
acid (Carl Roth GmbH), vanillin (Carl Roth GmbH), and Viscozyme L (Sigma) were utilized.
2.2. Plant Material and Sampling
Pak choi (cv. Hangzhou You Dong Er) plants were cultivated in a greenhouse and harvested
after eight weeks for the purpose of conducting time-dependent hydrolysis assays. Cell wall
isolation was carried out from whole leaves (i.e. blade and stalk intact) as well as on leaves
separated into leaf stalks and leaf blades.
For the fermentation procedure, two pak choi cultivars (cv. Hangzhou You Dong Er and cv.
Shanghai Qing) and two leaf mustard cultivars (cv. Xue Li Hong and cv. Bao Bao Qing Cai)
were cultivated under field conditions and harvested after ten weeks in China (whole leaf).
Cell wall isolation was carried out in duplicate for each cultivar (n = 2) prior to the
fermentation procedure. The same four Chinese cabbage cultivars were also grown in
Germany under greenhouse conditions and harvested after six weeks (whole leaf). Cell wall
isolation was carried out for each cultivar (single, n = 1).
69

2.3. Fermentation Procedure
The fermentation procedure of Chinese-grown plants was carried out as described by
Harbaum et al. (15). The fresh plant material was withered in the sun for two days in order to
reduce the moisture content by 50%. Afterwards the plants were kneaded with salt (100 g/5
kg fresh weight) until sap began to leak from the plant material. The wet plant material was
then fermented under pressure in clay pots for four weeks under ambient conditions. Cell
walls were isolated in duplicate (n = 2) for each cultivar after the fermentation procedure.
2.4. Isolation of Cell Wall Material
The isolation of cell wall material was carried out as described by Harbaum et al. (28) and
according to Beveridge et al. (4) for Brassica vegetables.
2.5. Time-Dependent Hydrolysis Reaction
6 mL of hydrolysis solution (1 M NaOH or 2 M NaOH) were added to 300 mg of isolated cell
walls, flushed by N2, and stirred in the dark. After each time point of hydrolysis reaction, an
aliquot of the suspension was sampled and centrifuged. 150 µL of the supernatant were
diluted, acidified with 75 µL of conc. HCl, and measured directly by HPLC-DAD (diode
array detector). Additionally, the time-dependent hydrolysis reaction was carried out after
enzymatic treatment of the cell wall with the cell-wall-degrading enzyme Viscozyme L as
presented by Harbaum et al. (28).
2.6. Compound Stability Under Alkaline Conditions
The defined contents of phenolic standards (vanillic acid, p-hydroxybenzaldehyde, vanillin,
p-coumaric acid, sinapic acid, and ferulic acid) were added to the 1 M NaOH hydrolysis
solution. After 96 h (4 days), an aliquot was diluted 1:1 with 2 M HCl and analyzed directly
by HPLC-DAD as described by Harbaum et al. (28).
2.7. Hydrolysis Reaction and Extraction
Hydrolysis reaction and extraction for the determination of bound phenolic compounds was
carried out according to Harbaum et al. (28). The hydrolysis time was set to 96 h (4 days)
with 1M NaOH.
2.8. HPLC-DAD Analysis
The HPLC analyses were carried out with a HP1100 HPLC (Agilent Tech. Waldbronn,
Germany) equipped with a diode array detector. Separation was carried out on a 250 x 4 mm
70

(inside diameter), 5 µm, RP-18 Nucleodur column equipped with an 8 x 4 mm Nucleodur
guard column at 20°C. Gradient elution and compound detection was carried out according to
Harbaum et al. (28).
2.9. Statistical Analysis
Standard deviations and significant differences were calculated by SPSS 15.0 (one-way
ANOVA, Bonferroni, p < 0.05).
3. RESULTS AND DISCUSSION
3.1. Release of Cell-Wall-Bound Phenolic Compounds
The cell wall isolation was carried out according to Harbaum et al. (28) based on the method
described by Beveridge et al. (4). The investigation and identification of bound phenolic
compounds of pak choi cell walls was carried out by an alkaline treatment (sodium
hydroxide). Cell wall material of the pak choi cv. Hangzhou You Dong Er was hydrolyzed
using 1 M NaOH and 2 M NaOH solutions. The detectable contents of hydrolyzed vanillin
and trans-ferulic acid within 48 hours are presented in Figure 1. The release of bound
phenolics and the direct detection of individual compounds during hydrolysis (i.e. direct
acidification of an extract aliquot and HPLC analysis without further solvent extraction), e.g.
trans-ferulic acid and vanillin, enable a precise analysis of the released bound phenolics over
the time of the hydrolysis reaction. trans-Ferulic acid and vanillin are the main bound
phenolics in pak choi and are stable under alkaline conditions as reported in a previous study
(28). Figure 1 shows no differences in the release of bound phenolics in plant material treated
with 1 M NaOH or 2 M NaOH. Therefore, there is no indication of differences in the strength
of cell-wall-bound linkages of phenolics. In contrast, other studies reported that the use of
sequential hydrolysis with alkaline solutions of varying concentrations and several steps may
reflect differences in the strength of the linkages, e.g. ester linkages of bound phenolic
compounds. Ng et al. (9) reported the main release of ferulic acid by 1 M NaOH of carrot cell
wall, but dimers are also released in significant amounts from the cell wall by 2 M NaOH.
Rodriguez-Arcos et al. (5) observed differences in the release by means of using several steps
depending upon the part of asparagus under investigation.
71

Released vanillin
[µg/g cell wall]
Released ferulic acid
[µg/g cell wall]
40
35
30
25
20
15
10
35
30
25
20
15
10
A
1M NaOH
2M NaOH
0 20 40 60
Hydrolysis time [h]
B
1M NaOH
2M NaOH
0 20 40 60
Hydrolysis time [h]
Figure 1. Release of individual cell-wall-bound phenolic compounds in 8-week-old pak choi
plants (cv. Hangzhou You Dong Er) dependent on hydrolysis time within 48 hours; (A)
vanillin, (B) trans-ferulic acid
The release of bound phenolics from the cell wall was strongly correlated with hydrolysis
time. The main contents of bound phenolic compounds in the cell walls of pak choi after 24 h
of hydrolysis were approximately 27 µg/g cell wall for vanillin and 27 µg/g cell wall for
trans-ferulic acid. But an increased release for both compounds was observed over the course
of an extended hydrolysis time (to 48 h) and indicates that the reaction was not complete after
24 h. The release of trans-ferulic acid showed a marginal increase by hour 48, but it is
possible that residual amounts of trans-ferulic acid may have remained in the cell wall;
72

vanillin showed a distinct increase during hydrolysis reaction by hour 48. The major portions
of bound vanillin and trans-ferulic acid were released from the cell walls (whole leaf) within
the first hour of hydrolysis, i.e. approximately half of the amount that was detected for
vanillin and trans-ferulic acid after 48 h. These results are in accordance with Beveridge et al.
(4), who characterized the release of total bound phenolic compounds from broccoli and
carrot cell walls by means of a simple and fast spectrophotometric method during a cell wall
hydrolysis time of 48 hours.
The pronounced increase in the release of vanillin from 24 h to 48 h hydrolysis time
compared to trans-ferulic acid may lead to the assumption that vanillin is potentially
generated from the cell wall constituent lignin during the hydrolysis reaction. Lignin consists
of polymerized monolignols (coumaroylalcohol, coniferylalcohol, and sinapoylalcohol, linked
via ether bonds) and constitutes 4.8% of the insoluble cell wall components of the whole pak
choi leaf (29). Figure 2 presents the release of bound vanillin and trans-ferulic acid
differentiated for cell wall material during a seven-day hydrolysis period. In order to compare
the release of bound phenolics in different plant parts [also described by Rodriguez-Arcos et
al. (5)], the plant material was divided into leaf blades and leaf stalks. Increasing detectable
contents of trans-ferulic acid were shown by day four (blade and stalk) (Figure 2B). Vanillin
also showed a distinct increase by day three for the cell wall samples as presented in Figure
2A. The increase of released vanillin from day two to day three is less pronounced for the
stalk than for the blade. This is in contrast to the expectation that the lignin portion, which
may have an influence, would be greater in the cell wall material of the stalk than of the leaf
blade. Additionally, the hydrolysis of ether bonds requires more rigorous conditions (4 M
NaOH and heat, e.g. 170°C) (30, 31). It can therefore be concluded that there is no remaining
bound vanillin content in the cell walls of pak choi after three days of hydrolysis and no
indication of lignin influence.
73

Released vanillin
[µg/g cell wall]
Released ferulic acid
[µg/g cell wall of blade]
60
50
40
30
20
10
0
80
78
76
74
72
70
68
66
64
62
60
A
1 2 3 4 5 6 7
Hydrolysis time [d]
B
74
blade
stalk
1 2 3 4 5 6 7
Hydrolysis time [d]
stalk
blade
20
19
18
17
16
15
14
13
12
Released ferulic acid
[µg/g cell wall of stalk]
Figure 2. Release of individual cell wall bound phenolic compounds in 8-week-old pak choi
plants separated into leaf blades and leaf stalks dependent on hydrolysis time within seven
days; (A) vanillin, (B) trans-ferulic acid
The release of bound phenolic compounds by means of alkaline hydrolysis within seven days
after enzymatic treatment was investigated. The enzyme Viscozyme L resulted in a higher
degradation of cell wall material [as presented in a previous work by Harbaum et al. (28)].
However, no improvement of bound phenolic release was observable within the seven-day
hydrolysis period after cell wall degradation by the enzyme Viscozyme L compared to the
same procedure without enzymatic treatment (data not shown). The degradation of the cell

walls prior to alkaline hydrolysis did not enhance the release of bound phenolic compounds
during the hydrolysis.
Table 1. Release of Bound Phenolic Compounds in the Cell Walls of Pak Choi Plants (Whole
Leaves) after One, Four, and Seven Days Hydrolysis Time (m = 3)
Phenolic compound
Hydrolysis time
[µg/g cell wall] a
[µg/g cell wall] a
[µg/g cell wall] a
1 Day 4 Days 7 Days
Vanillic acid 11.9 ± 2.0 17.9 *
± 1.5 17.2 * ± 0.6
p -Hydroxybenzaldehyde 4.1 ± 0.6 5.0 ± 0.9 5.1 ± 0.6
Vanillin 25.4 * ± 4.0 42.0 *,§ ± 9.8 48.9 § ± 5.8
p -Coumaric acid 2.7 ± 0.5 4.0 ± 0.4 3.9 ± 0.6
Sinapic acid 2.8 ± 1.2 2.1 ± 0.2 2.5 ± 0.6
trans -Ferulic acid 18.8 ± 2.1 19.9 ± 1.2 23.3 ± 2.5
cis -Ferulic acid 3.7 ± 0.2 3.3 ± 0.6 3.5 ± 0.3
Total content b
69.4 * ± 8.3 94.4 *,§ ± 13.3 104.5 § ± 9.3
a Contents in dried cell wall; significant differences are indicated by the ( * , § ) symbols.
b Total content of all individual compounds combined.
The influence of the hydrolysis duration (one, four, and seven days; without enzymatic
treatment) for all identified bound compounds is shown in Table 1 (whole leaves of pak choi
cv. Hangzhou You Dong Er; preparation by solvent extraction and further concentration). An
increase in detectable contents was also found for the individual compounds vanillic acid, phydroxybenzaldehyde,
and p-coumaric acid within four days of hydrolysis time, but no
further significant increase was observed until day seven. Sinapic acid and cis-ferulic acid
showed no significant changes within seven days of hydrolysis. The mean standard deviation
ranges from 5-15% of the detected content; higher standard deviations may result from the
variation of the content of individual compounds in the cell wall, e.g. vanillin contents. The
higher standard deviation for released vanillin during a prolonged hydrolysis time is also
shown in Figure 2A (stalk material in particular). Additionally, the sum of all bound phenolic
compounds was distinct higher after four days compared to one day of hydrolysis time but not
significantly higher after seven days. A hydrolysis duration of three or four days for the
complete detection of bound phenolic compounds is in accordance with studies conducting
the hydrolysis in four steps (0.1 M for 1 h, 0.1 M for 24 h, 1 M for 24 h, and 2 M for 24 h;
approximately three days total).
75

In this study, the duration time of the alkaline hydrolysis was found to be the most important
factor for the release of bound phenolic compounds from the cell wall. Therefore, the
hydrolysis reaction was conducted for 96 h (4 days) in the subsequent experiments to identify
the contents of bound phenolic compounds in the cell walls of Chinese cabbages. The
recoveries of reference compounds detected after treatment for 96 h with 1 M NaOH were as
follows: vanillic acid, 100%; p-hydroxybenzaldehyde, 103%; vanillin, 98%; p-coumaric acid,
100%; sinapic acid, 71%; and ferulic acid, 98%. These values indicate sufficient stability
during the elongated hydrolysis time. Only sinapic acid was degraded, which might explain
the lack of a detectable increase in its content when the hydrolysis time was increased
(Figure 3).
3.2. Contents of Bound Phenolic Compounds
Table 2 presents the contents of bound phenolic compounds in the four cultivars of Chinese
cabbage (pak choi: cv. Hangzhou You Dong Er and cv. Shanghai Qing; Chinese leaf mustard:
cv. Xue Li Hong and cv. Bao Bao Qing Cai) grown under greenhouse conditions in Germany.
Table 3 displays the contents of these cultivars grown in China under field conditions (nonfermented
plants in particular).
The contents in the cell walls of leaf mustard plants were higher compared to pak choi plants
cultivated in greenhouses in Germany: 73.6 µg/g for cv. Hangzhou You Dong Er and 69.6
µg/g for cv. Shanghai Qing as well as 95.5 µg/g for cv. Xue Li Hong and 120.8 µg/g for cv.
Bao Bao Qing Cai. trans-Ferulic acid and vanillin were the most abundant bound phenolic
compounds in the cell walls of each cultivar. The total contents of bound phenolic compounds
in non-fermented whole plants cultivated in China amounted to 101.4 µg/g for cv. Hangzhou
You Dong Er, 94.1 for cv. Shanghai Qing, 111.8 µg/g for cv. Xue Li Hong, and 89.4 µg/g for
cv. Bao Bao Qing Cai after 96 h of hydrolysis (Table 3). These contents were in the same
magnitude as those presented for whole plants cultivated under greenhouse conditions in
Germany (Table 2).
76

Table 2. Contents of Bound Phenolic Compounds in Different Cultivars of Chinese Brassica
Vegetables Cultivated in Germany under Greenhouse Conditions (Whole Leaf; n = 1, m = 3)
Phenolic compound
Vanillic acid 7.7 ± 0.4 5.4 ± 1.1
p -Hydroxybenzaldehyde 2.8 ± 1.0 2.0 ± 0.2
Vanillin 30.8 ± 11.2 21.5 ± 3.3
p -Coumaric acid 2.5 ± 1.1 2.1 ± 0.6
Sinapic acid 2.8 ± 0.3 1.0 ± 0.2
trans -Ferulic acid 23.8 ± 0.5 32.1 ± 2.7
cis -Ferulic acid 3.3 ± 0.6 5.4 ± 0.4
Total content b
73.6 ± 14.4 69.6 ± 4.9
Phenolic compound
Cv. Hangzhou You Dong Er Cv. Shanghai Qing
[µg/g cell wall] a
[µg/g cell wall] a
Cv. Xue Li Hong Cv. Bao Bao Qing Cai
[µg/g cell wall] a
[µg/g cell wall] a
Vanillic acid 7.4 ± 0.5 9.3 ± 0.3
p -Hydroxybenzaldehyde 2.6 ± 0.4 3.2 ± 0.2
Vanillin 35.8 ± 6.3 41.7 ± 1.9
p -Coumaric acid 4.3 ± 0.9 3.1 ± 0.1
Sinapic acid 2.4 ± 0.3 4.9 ± 0.8
trans -Ferulic acid 36.4 ± 2.1 51.2 ± 4.3
cis -Ferulic acid 6.5 ± 0.1 7.5 ± 0.3
Total content b
95.5 ± 8.4 120.8 ± 3.5
a Contents in dried cell wall.
b Total content of all individual compounds combined.
trans-Ferulic acid accounted for 32-46% of the total detected contents in plants cultivated in
Germany and 18-31% of plants cultivated in China, which indicates a higher proportion of
trans-ferulic acid as the main compound of the total bound phenolic content in Germany
compared to China. By contrast, the percentage of vanillin in the total content ranged from 31
to 41% in Germany and 41 to 49 % in China, which indicates an overall higher quantity of
vanillin in cell wall of plants grown in China than in Germany; vanillin and trans-ferulic acid
are compounds possessing a close structural relationship (32) and the reported differences
might be explained by differences in the biosynthesis of cell-wall-bound phenolic compounds
due to the plants’ age (China ten weeks, Germany six weeks) as well as radiation (field vs.
greenhouse). The influence of light supply on the cell wall phenolics has been reported in the
literature (33, 34).
77

The determined standard deviations of plant cell walls cultivated in Germany (Table 2) are
predominantly 5-15% of the detected contents (sometimes higher in detected low contents,
e.g. 2.8 ± 1.0 µg/g for p-hydroxybenzaldehyde of the cv. Hangzhou You Dong Er). The high
standard deviation of vanillin (cv. Hangzhou You Dong Er: 30.8 ± 11.2 µg/g) seems to be
related to the hydrolysis time; a low standard deviation was observed within the first few
days, whereas the standard deviation increased strongly after three days (Figure 2A).
3.3. Influence of Traditional Fermentation Procedure on the Content of Bound Phenolic
Compounds in Chinese Cabbages
The isolated cell wall material from whole leaves of non-fermented and fermented cabbages
was saponified to quantify the bound compounds in the cell wall after fermentation and to
characterize possible changes in the contents of bound phenolics during the fermentation
procedure. The results for the fermentation procedure of the two pak choi and two leaf
mustard cultivars are presented in Table 3.
Table 3. Influence of Fermentation Procedure on the Contents of Bound Phenolic
Compounds in Different Cultivars of Chinese Brassica Vegetables Cultivated in China under
Field Conditions (n = 2, m = 3)
Phenolic compound
Vanillic acid 10.3 ± 1.2 10.0 ± 1.5 7.7 ± 0.8 23.9 * ± 2.2
p -Hydroxybenzaldehyde 8.5 ± 0.8 4.2 * ± 1.6 7.0 ± 0.8 5.6 * ± 0.7
Vanillin 50.2 ± 6.2 29.8 * ± 8.7 38.4 ± 3.6 31.6 * ± 4.3
p -Coumaric acid 3.6 ± 0.5 2.7 * ± 0.8 2.5 ± 0.5 3.2 ± 0.5
Sinapic acid 5.7 ± 0.6 7.2 ± 3.8 3.6 ± 0.6 6.4 * ± 0.4
trans -Ferulic acid 18.0 ± 5.0 21.9 ± 3.6 28.9 ± 2.6 26.4 ± 1.9
cis -Ferulic acid 5.0 ± 0.9 3.0 * ± 0.2 6.0 ± 1.0 4.4 * ± 0.5
Total content b
101.4 ± 11.4 78.8 ± 18.8 94.1 ± 8.6 94.8 ± 15.6
Phenolic compound
[µg/g cell wall] a
[µg/g cell wall] a
[µg/g cell wall] a
[µg/g cell wall] a
Cv. Hangzhou You Dong Er Cv. Shanghai Qing
Non-fermented Fermented Non-fermented Fermented
[µg/g cell wall] a
[µg/g cell wall] a
[µg/g cell wall] a
[µg/g cell wall] a
Cv. Xue Li Hong Cv. Bao Bao Qing Cai
Non-fermented Fermented Non-fermented Fermented
Vanillic acid 9.1 ± 2.3 16.1 * ± 1.8 12.9 ± 6.3 14.6 ± 2.2
p -Hydroxybenzaldehyde 11.9 ± 2.2 9.5 ± 6.5 7.2 ± 4.1 6.3 ± 1.8
Vanillin 54.0 ± 9.8 41.0 ± 22.6 40.6 ± 21.6 36.1 ± 10.5
p -Coumaric acid 5.6 ± 0.7 5.3 ± 2.5 3.6 ± 2.9 5.5 ± 1.6
Sinapic acid 6.6 ± 0.8 10.2 * ± 2.7 3.4 ± 0.3 10.6 * ± 1.3
trans -Ferulic acid 21.1 ± 2.0 22.5 ± 1.6 18.8 ± 5.3 22.8 ± 2.4
cis -Ferulic acid 3.5 ± 0.3 3.1 ± 0.5 2.9 ± 0.8 3.2 ± 0.5
Total content b
111.8 ± 16.3 107.7 ± 36.6 89.4 ± 40.4 99.0 ± 17.5
a Contents in dried cell wall; significant differences are indicated by the ( * ) symbol.
b Total content of all individual compounds combined.
78

The content of bound phenolic compounds did not change markedly during the fermentation
process in China for the two pak choi cultivars Hangzhou You Dong Er and Shanghai Qing
and the two leaf mustard cultivars Xue Li Hong and Bao Bao Qing Cai. A slight decrease in
vanillin, p-hydroxybenzaldehyde, and cis-ferulic acid content was observed after
fermentation, which is significant for the Hangzhou You Dong Er and Shanghai Qing pak
choi cultivars. However, the contents of sinapic acid increased in all cultivars following
fermentation, significantly for the Shanghai Qing, Xue Li Hong, and Bao Bao Qing Cai
cultivars. Overall, the changes in bound phenolic contents are minimal during fermentation.
The high standard deviations also reflect the large variation of bound phenolic content in the
cell wall material. For example, the analysis of duplicates of the Bao Bao Qing Cai cultivar
(non-fermented) resulted in vanillin concentrations of 59.8 ± 5.6 µg/g and 21.5 ± 5.5 µg/g in
the cell wall materials isolated from two independent samples. Thus, it is difficult to detect
the small changes caused by the fermentation procedure.
The significant increase e.g. in bound vanillic acid after the fermentation of the pak choi cv.
Shanghai Qing, as well as the increase in sinapic acid in all cultivars requires clarification
(Table 3). Ng et al. (9) reported the sharp increase in p-hydroxybenzoic acid monomers in
carrots during storage; this was related to pathogen-elicitors as presented in the literature (35).
However, the storage of the carrots did not affect ferulic acid content or its dimers (9). There
was also no evidence of dimeric or oligomeric compound formation as a result of the
fermentation of the Chinese cabbages in our study. The formation of dimers was shown to
occur during the storage of other vegetables, however (such as asparagus) (5).
While bound phenolics are known to increase during pathogen infestation (35, 36) to improve
disease resistance, various pathogens, parasites, and fungi have been shown to exhibit
esterase activity (18, 37, 38). The growth of lactobacilli, enterobacteria, and fungi during
fermentation were reported (39). The pH decreased during fermentation from 7 to 4 due to
microorganism activity (e.g. lactobacillus) (15, 40, 41) caused by the formation of lactic acid
and acetate (40). The spontaneous fermentation of Brassica rapa (turnip) was reported by
Maifreni et al. (41) and was caused by various natural microorganisms: lactic acid bacteria
(main bacteria: Lactobacillus ssp. and Pediococcus ssp.) and yeast (main yeast: Candida).
The activity of esterases (e.g. feruloyl esterase) from Lactobacillus strains and yeast (e.g.
Candida or Saccharomyces) have been described in the literature (18, 23-25, 42).
Boskov Hansen et al. (17) observed changes in the bound phenolic content during breadmaking,
and suggested that the decrease might be indicative of endogenous enzymatic
activity, mechanical disaggregation that occurs during bread-making as well as from
hydrolysis due to the changes in pH that take place in sour dough. Coghe et al. (18) reported
79

the release of ferulic acid during brewing fermentation, which they attributed to the activity of
the feruloyl esterase in brewer’s yeast. In a recent study by Harbaum et al. (15), the
qualitative and quantitative changes in free phenolic compounds resulting from traditional
Chinese fermentation as presented in this study were investigated. The cleavage of ester
bonds of glycosides and organic acids of free hydroxycinnamic acids and flavonoids was
reported and attributed to microorganism and enzymatic activities (e.g. esterase). Ciska et al.
(16) and Harbaum et al. (15) observed an increase in the total phenolic content (determined
by Folin Ciocalteu) and the antioxidative potential of fermented plant material compared to
non-fermented plant material as presented for white cabbages (sauerkraut) and Chinese
cabbages. Ciska et al. (16) assumed that the release of bound phenolic compounds during the
fermentation procedure due to bacterial action might account for the higher antioxidative
potential of extracts. However, the minor changes as presented in Table 3 (e.g. for vanillin,
p-hydroxybenzaldehyde, and cis-ferulic acid content) and the high variation of bound
phenolic compounds in different isolated cell wall materials (see above) failed to provide
clear evidence that phenolic compound linkages to the cell wall components were cleaved
during fermentation, and did not indicate any enzymatic activity (microorganism or
pathogenic) or changes by pH.
However, large cell wall compounds, which were potentially degraded to smaller
hydroxycinnamoyl oligosaccharides, such as feruloylated arabinoxylan or pectin fragments,
may occur from fermentation caused by enzymatic activity. The occurrence of xylanase
activity or other hydrolyzing enzymes in plants has been reported in the literature (17, 43-45).
These soluble fragments may be lost during the cell wall isolation procedure, but may occur
in extracts for the determination of free phenolic compounds as presented by Ciska et al. (16)
and Harbaum et al. (15). These fragments showed a slightly stronger antioxidative potential
compared to free ferulic acid (46).
Overall, the content of bound phenolic compounds is very low in the plant cell walls as well
as in fresh plant material (approx. 1 µg/g fresh plant material) as compared to approx. 1 mg/g
free phenolics in the whole fresh plant material (15). This leads to the assumption that the
release of bound phenolics or the potentially presented cell wall fragments of bound phenolic
compounds would not significantly alter the contents in extracts of free phenolic compounds.
4. ACKNOWLEDGMENT
We thank Stephanie thor Straten, Ni Xiaolei, Yang Jing, and Silke Rühl for their technical
assistance.
80

5. LITERATURE CITED
(1) Grabber, J. H.; Ralph, J.; Hatfield, R. D. Cross-linking of maize walls by ferulate
dimerization and incorporation into lignin. J. Agric. Food Chem. 2000, 48, 6106-6113.
(2) Saulnier, L.; Thibault, J. F. Ferulic acid and diferulic acids as components of sugar-beet
pectins and maize bran heteroxylans. J. Sci. Food Agric. 1999, 79, 396-402.
(3) Andreasen, M. F.; Christensen, L. P.; Meyer, A. S.; Hansen, A. Content of phenolic acids
and ferulic acid dehydrodimers in 17 rye (Secale cereale L.) varieties. J. Agric. Food
Chem. 2000, 48, 2837-2842.
(4) Beveridge, T.; Loubert, E.; Harrison, J. E. Simple measurement of phenolic esters in plant
cell walls. Food Res. Technol. 2000, 33, 775-783.
(5) Rodriguez-Arcos, R. C.; Smith, A. C.; Waldron, K. W. Effect of storage on wall-bound
phenolics in green asparagus. J. Agric. Food Chem. 2002, 50, 3197-3203.
(6) Parr, A. J.; Ng, A.; Waldron, K. W. Ester-linked phenolic components of carrot cell walls.
J. Agric. Food Chem. 1997, 45, 2468-2471.
(7) Ishii, T. Feruloyl oligosaccharides from cell walls of suspension-cultured spinach cells
and sugar beet pulp. Plant Cell Physiol. 1994, 35, 701-704.
(8) Rodriguez-Arcos, R. C.; Smith, A. C.; Waldron, K. W. Ferulic acid crosslinks in
asparagus cell walls in relation to texture. J. Agric. Food Chem. 2004, 52, 4740-4750.
(9) Ng, A.; Parr, A. J.; Ingham, M. L.; Rigby, N. M.; Waldron, K. W. Cell wall chemistry of
carrots (Daucus carota cv. Amstrong) during maturation and storage. J. Agric. Food
Chem. 1998, 46, 2933-2939.
(10) Waldron, K. W.; Parr, A. J.; Ng, A.; Ralph, J. Cell wall esterified phenolic dimers:
Identification and quantification by reverse phase high performance liquid
chromatography and diode array detection. Phytochem. Anal. 1996, 7, 305-312.
(11) Hartley, R. D.; Morrison, W. H. Monomeric and dimeric phenolic acids released from
cell walls of grasses by sequential treatment with sodium hydroxide. J. Sci. Food
Agric. 1991, 55, 365-375.
(12) Garcia, E.; Filisetti, T. M. C. C.; Udaeta, J. E. M.; Lajolo, F. M. Hard-to-cook-beans
(Phaseolus vulgaris): Involvement of phenolic compounds and pectates. J. Agric.
Food Chem. 1998, 46, 2110-2116.
(13) Parker, C. C.; Parker, M. L.; Smith, A. C.; Waldron, K. W. Thermal stability of texture in
chinese waterchestnut may be dependent on 8,8´-diferulic acid (aryltetralyn form). J.
Agric. Food Chem. 2003, 2034-2039.
81

(14) Micard, V.; Thibault, J. F. Oxidative gelation of sugar-beet pectins: Use of laccases and
hydration properties of the cross-linked pectins. Carbohydr. Polymers 1999, 39, 265-
273.
(15) Harbaum, B.; Hubbermann, E. M.; Zhu, Z.; Schwarz, K. Impact of fermentation on
phenolic compounds in leaves of pak choi (Brassica campestris L. ssp. chinensis var.
communis) and Chinese leaf mustard (Brassica juncea Coss.). J. Agric. Food Chem.
2008, 56, 148-157.
(16) Ciska, E.; Karamac, M.; Kosinska, A. Antioxidant activity of extracts of white cabbage
and sauerkraut. Pol. J. Food Nutr. Sci. 2005, 14, 367-373.
(17) Boskov Hansen, H.; Andreasen, M. F.; Nielsen, M. M.; Larsen, L. M.; Bach Knudsen, K.
E.; Meyer, A. S.; Christensen, L. P.; Hansen, A. Changes in dietary fibre, phenolic
acids and activity of endogenous enzymes during bread-making. Eur. Food Res.
Technol. 2002, 214, 33-42.
(18) Coghe, S.; Benoot, K.; Delvaux, F.; Vanderheagen, B.; Delvaux, F. R. Ferulic acid
release and 4-vinylguaiacol formation during brewing and fermentation: Indications
for feruloyl esterase activity in Saccharomyces cerevisiae. J. Agric. Food Chem. 2004,
52, 602-608.
(19) Benoit, I.; Navarro, D.; Marnet, N.; Rakotomanomana, N.; Lesage-Meessen, L.;
Sigoillot, J.-C.; Asther, M.; Asther, M. Feruloyl esterases as a tool for the release of
phenolic compounds from agro-industrial by-products. Carbohydr. Res. 2006, 341,
1820-1827.
(20) Szwajgier, D.; Targonski, Z. Release of free ferulic acid and feruloylated arabinoxylans
from brewery's spent grain by commercial enzyme preparation. Electr. J. Pol. Agric.
Univers. 2006, 9, http://www.ejpau.media.pl/volume9/issue1/abs-26.html.
(21) Andreasen, M. F.; Christensen, L. P.; Meyer, A. S.; Hansen, A. Release of
hydroxycinnamic and hydroxybenzoic acids in rye by commercial plant cell wall
degrading enzyme preparations. J. Sci. Food Agric. 1999, 79, 411-413.
(22) Szwajgier, D.; Pielecki, J.; Targonski, Z. Feruloylated arabinoxylans as potential beer
antioxidants. Electr. J. Pol. Agric. 2005, 8,
http://www.ejpau.media.pl/volume8/issue4/art-38.html.
(23) Wang, X.; Geng, X.; Egashira, Y.; Sanada, H. Release of ferulic acid from wheat bran by
an inducible feruloyl esterase from an intestinal bacterium Lactobacillus acidophilus.
Food Sci. Technol. Res. 2005, 11, 241-247.
82

(24) Choi, Y. J.; Miguez, C. B.; Lee, B. H. Characterization and heterologous gene expression
of a novel esterase from Lactobacillus casei CL96. Applied Environ. Microbiol. 2004,
70, 3213-3221.
(25) Fenster, K. M.; Parkin, K. L.; Steele, J. L. Characterization of an arylesterase from
Lactobacillus helveticus CNRZ32. J. Appl. Microbiol. 2000, 88, 572-583.
(26) Andreasen, M. F.; Kroon, P. A.; Williamson, G.; Garcia-Conesa, M.-T. Esterase activity
able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the
intestine of mammals. J. Agric. Food Chem. 2001, 49, 5679-5684.
(27) Kroon, P. A.; Faulds, C. B.; Ryden, P.; Robertson, J. A.; Williamson, G. Release of
covalently bound ferulic acid from fiber in the human colon. J. Agric. Food Chem.
1997, 45, 661-667.
(28) Harbaum, B.; Hubbermann, E. M.; Zhu, Z.; Schwarz, K. Cell-wall-bound phenolic
compounds in leaves of pak choi (Brassica campestris L. ssp. chinensis var.
communis). unpublished.
(29) Vollendorf, N. W.; Marlett, J. A. Comparison of the two methods of fiber analysis of 58
foods. J. Food Compos. Analysis 1993, 6, 203-214.
(30) Morrison, W. H.; Akin, D. E.; Himmelsbach, D. S.; Gamble, G. R. Investigation of the
ester- and ether-linked phenolic constituents of cell wall types of normal and brown
midrib pearl millet using chemical isolation, microspectrophotometry and 13 C NMR
spectroscopy. J. Sci. Food Agric. 1993, 63, 329-337.
(31) Provan, G. J.; Scobbie, L.; Chesson, A. Determination of phenolic acids in plant cell
walls by microwave digestion. J. Sci. Food Agric. 1993, 64, 63-65.
(32) Gasson, M. J.; Kitamura, Y.; McLauchlan, W. R.; Narbad, A.; Parr, A. J.; Parsons, E. L.;
Payne, J.; Rhodes, M. J. C.; Walton, N. J. Metabolism of ferulic acid to vanillin. J.
Biol. Chem. 1998, 273, 4163-4170.
(33) Miyamoto, K.; Ueda, J.; Takeda, S.; Ida, K.; Hoson, T.; Masuda, Y.; Kamisaka, S. Lightinduced
increase in the contents of ferulic and diferulic acids in cell walls of Avena
coleoptiles: Its relationship to growth inhibition by light. Physiol. Plantarium 1994,
92, 350-355.
(34) Tan, K. S.; Hoson, T.; Masuda, Y.; Kamisaka, S. Involvement of cell wall-bound
diferulic acid in light-induced decrease in growth rate and cell wall extensibility of
Oryza coleoptiles. Plant Cell Physiol. 1992, 33, 103-108.
(35) Schnitzler, J. P.; Madlung, J.; Rose, A.; Seitz, H. U. Biosynthesis of p-hydroxybenzoic
acid in elicitor-treated carrot cell cultures. Planta 1992, 188, 594-600.
83

(36) Santiago, R.; Butron, A.; Reid, L. M.; Arnason, J. T.; Sandoya, G.; Souto, X. C.; Malvar,
R. A. Diferulate content of maize sheaths is associated with resistance to the
Mediterranean corn borer sesamia nonagrioides (Lepidoptera: Noctuidae). J. Agric.
Food Chem. 2006, 54, 9140-9144.
(37) Donaghy, J.; McKay, A. M. Purification and characterization of a feruloyl esterase from
the fungus Penicillium expansum. J. Appl. Microbiol. 1997, 83, 718-726.
(38) Yu, P.; McKinnon, J. J.; Maenz, D. D.; Racz, V. J.; Christensen, D. A. The specificity
and the ability of Aspergillus feruloyl esterase to release p-coumaric acid from
complex cell walls of oat hulls. J. Chem. Technol. Biotechnol. 2004, 79, 729-733.
(39) Ji, F.-D.; Ji, B.-P.; Li, B.; Han, B.-Z. Microbial changes during the salting process of
traditional pickled Chinese cabbage. Food Sci. Technol. Int. 2007, 13, 11-16.
(40) Erten, H. Fermentation of Glucose and Fructose by Leuconostoc mesenteroides. Turk. J.
Agric. For. 2000, 24, 527-532.
(41) Maifreni, M.; Marino, M.; Conte, L. Lactic acid fermentation of Brassica rapa:
Chemical and microbial evaluation of a typical Italian product (brovada). Eur. Food
Res. Technol. 2004, 218, 469-473.
(42) Basaran, P.; Hang, Y. D. Purification and characterization of acetyl esterase from
Candida guilliermondii. Lett. Appl. Microbiol. 2000, 30, 167-171.
(43) Suzuki, T.; Honda, Y.; Mukasa, Y. Effects of UV-B radiation, cold and dissication stress
on rutin concentration and turin glucosidase activity in tartary buckwheat (Fagopyrum
tataricum) leaves. Plant Sci. 2005, 168, 1303-1307.
(44) Dornez, E.; Joye, I. J.; Gebruers, K.; Lenartz, J.; Massaux, C.; Bodson, B.; Delcour, J.
A.; Courtin, C. M. Insight into variability of apparent endoxylanase and endoxylanase
inhibitor levels in wheat kernels. J. Sci. Food Agric. 2006, 86, 1610-1617.
(45) Debyser, W.; Delvaux, F.; Delcour, J. A. Activity of arabinoxylan hydrolyzing enzymes
during mashing with barley malt or barley malt and unmalted wheat. J. Agric. Food
Chem. 1998, 46, 4836-4841.
(46) Ohta, T.; Yamasaki, S.; Egashira, Y.; Sanada, H. Antioxidative activity of corn bran
hemicellulose fragments. J. Agric. Food Chem. 1994, 42, 653-656.
This work was supported by DFG 592-5-1.
84

Chapter V
85

Free and Bound Phenolic Compounds in
Leaves of Pak Choi (Brassica campestris
L. ssp. chinensis var. communis) and
Chinese Leaf Mustard
(Brassica juncea Coss)
BRITTA HARBAUM, *,† EVA MARIA HUBBERMANN, †
ZHUJUN ZHU ‡ , and KARIN SCHWARZ †
Food Chemistry 2008,
in press, DOI: 10.1016/j.foodchem.2008.02.069
http://dx.doi.org/10.1016/j.foodchem.2008.02.069
Department of Food Technology, Institute of Human Nutrition and Food Science, University
of Kiel, Heinrich-Hecht-Platz 10, 24118 Kiel, Germany; and Department of Horticulture,
School of Agriculture and Food Science, Zhejiang Forestry University, Lin'an, Hangzhou,
Zhejiang 311300, China
* Author to whom correspondence should be addressed (telephone +49 431 880 5034; e-mail
info@foodtech.uni-kiel.de).
†
Institute of Human Nutrition and Food Science, Kiel
‡ Zhejiang Forestry University
86

SUMMARY
This article deals with the quantification of both free and cell-wall-bound phenolic
compounds in pak choi and Chinese leaf mustard cultivars. Contents of free and bound
polyphenols in fresh plant material and their importance in the human diet were estimated and
discussed. Furthermore, the contents were compared with polyphenol concentrations in other
Brassica vegetables.
For the quantification of free phenolic compounds, eleven pak choi cultivars and two leaf
mustard cultivars grown under field conditions in China were investigated, separated in outer
and inner leaves as well as in leaf blades and leaf stalks. In most cases, there were no
significant differences between hydroxycinnamic acid derivative and flavonoid derivative
contents in outer and inner leaves for the 13 cultivars, which may be related to the habitus of
pak choi. Pak choi is a kind of leafy cabbage, i.e. less compact as compared to other cabbages
such as white cabbage, for example. However, the polyphenol content of blades and stalks
differed among all investigated cultivars: Hydroxycinnamic acids and flavonoids were present
in greater amounts in the leaf blade than in the leaf stalk. In contrast to hydroxycinnamic
acids trace or small amounts of flavonoids were detected in the pak choi and leaf mustard
stalks.
Additionally, comparison of Chinese cabbage plants grown in China and Germany under field
conditions showed flavonoid contents in the same magnitude, but lower hydroxycinnamic
acid contents for plants cultivated in Germany.
Moreover, the cell-wall-bound phenolic content of two pak choi cultivars and two leaf
mustard cultivars were investigated. The yield of isolated cell wall based on fresh plant
material is higher in the leaf blades (1.69-1.82%) than leaf stalks (0.82-1.25%). The
concentrations of bound phenolic compounds were higher in the leaf blade than in the leaf
stalk under field conditions in China. These compounds represent only a minor portion of the
total phenolic content (flavonoids and hydroxycinnamic acids) in leaf stalks (0.81-1.18%) and
leaf blades (0.05-0.08%) from fresh plant material. These findings indicate the marginal
importance of the cell-wall-bound phenolic compounds in these vegetables and their role in
human nutrition might therefore be fairly minor.
The post-harvest storage procedure of plant samples from four Chinese cabbage cultivars
under normal atmosphere in sheets resulted in most cases in an increase of phenolic content
within six days at 4°C and 20°C. The increase might have been triggered by post-harvest
plant stresses, which stimulate the biosynthesis of polyphenols.
87

GENERAL DISCUSSION
This thesis focused on the determination of free and cell-wall-bound phenolic compounds in
Chinese Brassica vegetables and investigated the influence of post-harvest treatments on
phenolic content. As significant health-protective substances, polyphenols are generating
increasing interest. The high variability of phenolic content in plants according to different
plant parts or cultivation conditions makes it difficult for food researchers to estimate the
uptake of polyphenols in human nutrition.
Several studies have identified the contents of free phenolic compounds (e.g. simple phenolic
aglycones) after hydrolysis reaction, but these data do not reflect the full content of the
complex phenolic structures. Simplified methods can characterize the levels and quantitative
changes of polyphenols, but they are not sophisticated enough to detect qualitative changes.
The presented HPLC-ESI-MS n method, which entails high compound separation by liquid
chromatography (HPLC), soft electrospray ionization (ESI), and an additional selective
fragmentation procedure by MS n of flavonoid and hydroxycinnamic acid derivatives, enables
detailed structural characterization, i.e. reveals bound moieties to the aglycone. Data
demonstrating the complexity of polyphenols were obtained (e.g. the various acylated
kaempferol-3-O-diglucosides-7-O-glucosides in pak choi and leaf mustard) and about 40
different phenolic derivatives were identified. However, while partial mass losses during the
fragmentation procedure enable the classification of bound moieties (glycosides or organic
acids), the description of isomerism requires additional structural elucidation. Therefore,
NMR spectroscopy was conducted to obtain unequivocal information about the isomers; the
presumed dihydroxymonomethoxycinnamic acid moiety of the flavonoid kaempferol was e.g.
clearly identified as hydroxyferulic acid. Information regarding the occurrence of
hydroxyferulic acid as a final compound in the plant material (and not merely an intermediate
product from plant phenolic biosynthesis) is scarce in the literature yet significant for
Brassica vegetables (e.g. Arabidopsis thaliana or Brassica rapa). Other identified
compounds, e.g. malate derivatives of hydroxycinnamic acids, were also described for
cabbages for the first time in this study. Overall, more than 6000 flavonoids are known, and
the determination of new phenolic compounds in Chinese cabbages, like malate derivatives or
flavonoid compounds esterified with hydroxyferulic acid, demonstrates the relevance of
structural elucidation. While many studies have focused on the bioavailability of polyphenols,
it is also important to identify and elucidate unknown polyphenolic structures in order to
89

obtain a better understanding of their uptake in human nutrition as well as their absorption
and biological effects in the human body, e.g. their antioxidative capacity.
On the one hand, the qualitative and quantitative content of free phenolic compounds is of
interest to food researchers for calculating the uptake of the substances in the human diet. On
the other hand, the detailed determination and exact calculation of the phenolic content is
difficult due to the high complexity of polyphenolic structures and lack of standard
compounds for method calibration, e.g. for HPLC-DAD. When simple polyphenols are used
as calibration standards, the true polyphenolic content is often underestimated in the plants.
The use of natural and directly obtained flavonoid derivatives from the pak choi plant material
resulted in more accurate values for the phenolic content in Chinese Brassica vegetables. The
high phenolic concentration in the leaf blades relative to the leaf stalks indicates the
importance of characterizing specific plant parts. The flavonoid content in the freeze-dried
plant material amounted to approx. 1-3% in the leaf blade; this finding identifies the leaf
blades as an important source of phenolic compounds. The major factor influencing the
plants’ phenolic content is the cultivation conditions, i.e. greenhouse vs. field conditions.
Light supply is an important variable in the phenolic characterization of plants. The Chinese
Brassica vegetables possess significantly higher phenolic content compared to other cabbage
cultivars (broccoli, white cabbage, tronchuda cabbage, and green cabbage). The difference is
attributed to the Chinese vegetables’ different constituents and the facilitated radiation of its
leaves during the growing period. In contrast, the leaves of other cabbage species (e.g. white
cabbage) exhibit relatively poor absorption of sunlight due to their more compact structure.
The absorption and bioavailability of polyphenols in the human body is dependent on their
structures and bound moieties. Qualitative changes in polyphenols, e.g. by food processing
(fermentation or storage), are therefore of interest. Structural changes were observed to have
occurred during the traditional fermentation procedure of Chinese cabbages and were
characterized by mass spectrometry and fragmentation procedures (MS n ). Marked differences
in the polyphenolic spectrum were found post-fermentation and indicate the degradation of
the compounds by fermentation into smaller molecules. The highly glycosylated flavonoid
compounds, for example, were transformed into less glycosylated compounds, which may
alter their bioavailability and antioxidative potential (due to the formation of free hydroxyl
groups at the aglycone). It is known that glycoside linkages influence the bioavailability of
polyphenols, and that less glycosylated compounds are better absorbed in the gastrointestinal
tract. Microorganism activity (like bacterial endospores, lactic acid bacteria, enterobacteria, or
fungi) might be responsible for the qualitative changes (esterase activity). Furthermore, the
90

increased antioxidative potential in vitro was attributed to the qualitative changes in the
polyphenols and may indicate enhanced health-promoting effects from the altered structures
in vivo via the consumption of fermented Chinese cabbages. However, during the
fermentation process, the concentration of flavonoid derivatives and some hydroxycinnamic
acid derivatives exhibited a decrease. This finding indicates that the flavonoid content had not
only been changed by the cleavage of the moieties but also degraded by the fermentation
process, which may impair the nutritional relevance of fermented cabbages. On the other
hand, storage conditions led to a distinct increase in phenolic content in the pak choi cultivars
in the first few days of storage (predominantly until day six at 4°C and 20°C).
It therefore appears possible to generate changed phenolic structures in fruits and vegetables
via the fermentation process and to increase phenolic content via certain storage conditions.
These methods of food processing could lead to improved nutrient uptake in human health
and increased health benefits, both of which may be of future interest to the pharmaceutical
and food industries.
Bound phenolic compounds are mainly present in the cell walls of monocotyledons, but can
also be found in dicotyledons to a lesser extent. Pak choi possesses relatively small amounts
of cell-wall-bound phenolic compounds compared to other vegetables (like carrots, sugar
beets, or asparagus).
For the accurate characterization of bound phenolic compounds in the cell wall material, it is
important to consider the several factors influencing the quantification as well as the
formation of bound phenolic compounds in the plant. The main influencing factor is the
alkaline hydrolysis period, which required 96 hours for both the leaf blade and leaf stalk in
this study. The concentration of the alkaline hydrolysis solution (0.1 M, 1 M, or 2 M NaOH
solution) also has an impact. Concerning the plant development, an influence factor on plants’
bound phenolic content was found to be the age of the Chinese cabbage plants. Growing and
climatic conditions did not produce markedly different effects in the plants, however
(according to a comparison of cultivation in China and Germany).
The proportion of cell-wall-bound phenolic compounds is less important with respect to the
total content (free and bound content combined) in dicotyledonic plants like Chinese Brassica
vegetables. Compared to the free phenolic compounds, they are present in only minor
amounts in fresh plant material (ca. 0.05-1.18% of the total phenolic content depending on the
plant part). These minor monomeric compounds may only exert a marginal effect on the
degradation and fermentation rate of dietary fiber in the human colon; their role in human
nutrition might therefore be fairly minor. Dimeric and oligomeric compounds are known to
91

influence the fermentation rate significantly. However, dimeric or oligomeric compounds are
not present in the Chinese Brassica vegetables. Furthermore, their different bioavailability
compared to free phenolic compounds with respect to their properties and effects in the
human body may be important. The accumulation and further possible concentration of
phenolic compounds bound to cell wall fragments in the gastrointestinal tract may have
beneficial health effects that differ from those exerted by free phenolic compounds.
Moreover, the possible changes to and release of bound phenolic compounds via food
processing may change the beneficial effects of these compounds, e.g. facilitate their
absorption in the intestine and colon. However, the type of food processing presented in this
study (i.e. the traditional Chinese fermentation procedure) has no influence on the linkages of
bound compounds, although other impacts of food processing (e.g. cleavage of bound
phenolic compounds in foods during bread-making) were reported in earlier studies. Due to
the non-cleavage of the bound phenolics by traditional Chinese fermentation, these
compounds did not appear to be involved in the changes to antioxidative capacity during
fermentation.
This thesis provides a detailed and complete characterization of polyphenols in Chinese
Brassica vegetables [structural elucidation for both free and bound compounds, total and
individual concentrations in the plants, and the qualitative and quantitative changes undergone
during food processing] that should contribute to a better understanding of the dietary uptake
and possible effects of polyphenols in vivo, i.e. their bioavailability and health-promoting
effects (antioxidant capacity).
92

GENERAL SUMMARY
In the present thesis, free and cell-wall-bound phenolic compounds were investigated in the
Chinese Brassica vegetables pak choi and Chinese leaf mustard.
Chapter I deals with the investigation and structural determination of the mainly presented
polyphenol derivatives in the Chinese Brassica vegetable pak choi. Twenty-eight polyphenols
(11 flavonoid derivatives and 17 hydroxycinnamic acid derivatives) were detected in different
pak choi cultivars by HPLC-DAD-ESI-MS n and a selective fragmentation procedure.
Kaempferol was found to be the major flavonoid in pak choi, which were glycosylated
(mono-, di, and triclucosides) and acylated with different compounds. Isorhamnetin was
detected in smaller amounts. A structural determination was carried out by 1 H and 13 C NMR
spectroscopy for the main compound, kaempferol-3-O-hydroxyferuloylsophoroside-7-Oglucoside,
for the first time with respect to the organic acid moiety hydroxyferulic acid.
Furthermore, hydroxycinnamic acid derivatives were identified as esters of quinic acid,
glycosides, or malic acid. Malic acid derivatives, such as sinapoylmalate are described for the
first time in cabbages. Sinapoylmalate, the main hydroxycinnamic acid derivative in pak choi,
was also elucidated by 1 H and 13 C.
The content of polyphenols was determined in 11 cultivars of pak choi, with higher
concentrations present in the leaf blade than in the leaf stalk. Hydroxycinnamic acid esters,
particularly malic acid derivatives, are present in both the leaf blade and leaf stalk, whereas
flavonoids were only determined in the leaf blade under greenhouse conditions.
The objective of Chapter II was to characterize the influence of the traditional Chinese
fermentation procedure on the qualitative and quantitative polyphenol contents in Chinese
cabbages. Two pak choi cultivars and two Chinese leaf mustard cultivars were fermented in a
traditional Chinese method called pickling. The plant material was investigated before and
after the fermentation procedure in order to determine the qualitative and quantitative changes
in its polyphenols. A detailed description of the identified phenolic compounds of Chinese
leaf mustard by HPLC-ESI-MS n is presented here for the first time, including
hydroxycinnamic acid mono- and diglycosides (gentiobioses) and kaempferoltetraglycosides.
Flavonoid derivatives with a lower molecular mass (di- and triglycosides) and aglycones of
flavonoids and hydroxycinnamic acids were detected in fermented cabbages compared to the
main compounds detected in non-fermented cabbages. During the fermentation process,
93

contents of flavonoid derivatives and some hydroxycinnamic acid derivatives were found to
decrease.
The antioxidative potential as well as total phenolic content (Folin Ciocalteu) of fermented
cabbages was much higher compared to non-fermented cabbages, and these increases were
attributed to the qualitative changes of polyphenols as well as other reductones potentially
present after fermentation.
Chapter III focused on the determination of cell-wall-bound phenolic compounds in the
Chinese Brassica Vegetables. Following alkaline hydrolysis, seven different bound phenolic
compounds were identified in the cell wall material of the Chinese cabbage pak choi (cv.
Hangzhou You Dong Er) by HPLC-DAD: trans-Ferulic acid, cis-ferulic acid, p-coumaric
acid, sinapic acid, vanillic acid, vanillin, and p-hydroxybenzaldehyde. Dimeric or oligomeric
phenolic compounds were not present. The main bound phenolic compounds in the leaf blade
as well as the leaf stalk were trans-Ferulic acid and vanillin. The total as well as the
individual contents (vanillin, p-coumaric acid, sinapic acid, and cis-ferulic acid) of the bound
phenolic compounds were significantly higher in the leaf blade (total: 79.9 µg/g cell wall)
than in the leaf stalk (total: 37.5 µg/g cell wall) in 8-week-old plants. Older plants exhibited
lower concentrations of cell wall bound phenolic compounds, observed for the leaf blade as
well as the whole leaf, but the levels did not change in the stalk.
The release of cell-wall-bound phenolic compounds in Chinese cabbages according to various
durations of alkaline hydrolysis time is reported in Chapter IV. The predominant bound
phenolic compounds vanillin and trans-ferulic acid in Chinese cabbages were investigated
over a seven-day hydrolysis period and triggered the complete release of bound phenolics in
all plant parts (cell wall of leaf blade or leaf stalk) after four days.
Furthermore, the aim of Chapter IV the characterization of the influence of growing conditions
and the traditional Chinese fermentation procedure on the content of bound phenolics in
Chinese cabbage cultivars. The concentrations of bound phenolics were found to be of the
same magnitude in both German- (greenhouse, 6-week-old plants) and Chinese-grown (field,
10-week-old plants) specimens of the four investigated Chinese cabbages (pak choi: cv.
Hangzhou You Dong Er and cv. Shanghai Qing; and leaf mustard: cv. Xue Li Hong and cv.
Bao Bao Qing Cai). The influence of the traditional fermentation process on the content of
cell-wall-bound phenolic compounds in Chinese cabbages was investigated for the four
cultivars. Only small changes in the cell-wall-bound phenolic content (decreases and
94

increases of individual compounds) occurred as a result of the fermentation procedure. In
other words, there was no clear indication that phenolic compounds had been released by
either microorganism or enzymatic activity (e.g. feruloyl esterase) or changes in pH.
Chapter V focused on the investigation of both free and bound phenolic compounds in
Chinese cabbages grown under field conditions in Germany and China.
Eleven pak choi cultivars and two leaf mustard cultivars grown in China (field) were
investigated for the free polyphenol content in their outer and inner leaves as well as in their
leaf blades and leaf stalks. In most cases, there were no significant differences between the
hydroxycinnamic acid derivative and flavonoid derivative content in the outer and inner
leaves for the 13 cultivars. However, the content of blades and stalks differed:
Hydroxycinnamic acids and flavonoids were present in greater amounts in the leaf blade than
in the leaf stalk. Trace or small amounts of flavonoids were detected in the pak choi and leaf
mustard stalks. Additionally, the bound phenolic content of two pak choi cultivars and two
leaf mustard cultivars was investigated. The concentrations of cell-wall-bound phenolic
compounds were higher in the leaf blade than in the leaf stalk under field conditions in China.
These compounds represent only a minor portion of the total phenolic compounds (flavonoids
and hydroxycinnamic acids) in leaf stalks (0.81-1.18%) and leaf blades (0.05-0.08%) from
fresh plant material.
The storage of plant samples from four Chinese cabbage cultivars resulted in most cases in an
increase of phenolic content within six days at 4°C and 20°C. The increase might have been
triggered by post-harvest plant stresses, which stimulate the biosynthesis of polyphenols.
95

ZUSAMMENFASSUNG
In der vorliegenden Arbeit wurden die qualitativen und quantitativen Gehalte an freien und
zellwandgebundenen Phenolen in chinesischen Kohlgemüsen untersucht.
Das erste Kapitel handelt von der Identifizierung und Strukturauflärung der unterschiedlichen
Polyphenol-Derivate in chinesischem Kohlgemüse. 28 Polyphenole (elf Flavonoid-Derivate
und 17 Hydroxyzimtsäure-Derivate) konnten in unterschiedlichen Pak Choi Varietäten mittels
HPLC-DAD-ESI-MS n und einem anschließenden Fragmentierungs-Prozess in der Ionenfalle
des Massenspektrometers nachgewiesen werden. Das am häufigsten vorkommende Flavonoid
in Pak Choi ist Kämpferol, das acyliert und glukosiliert vorliegt (Mono-, Di, und
Triglukoside). Daneben wurden geringe Mengen an Isorhamnetin identifiziert. Für die
Hauptkomponente Kämpferol-3-O-hydroxyferuloylsophorosid-7-O-glucosid konnte erstmals
eine komplette Strukturaufklärung mittels 1 H and 13 C NMR-Spektroskopie durchgeführt
werden, insbesondere von der Hydroxyferulasäure als Teilkomponente eines Flavonoid-
Derivates. Weiterhin wurden unterschiedlichste Hydroxyzimtsäure-Derivate identifiziert, die
als Ester der Chinasäure, Äpfelsäure oder als Glykoside vorliegen. Äpfelsäure-Derivate, wie
z.B. Sinapinsäuremalat, konnten zum ersten Mal für Kohlgemüse beschrieben werden. Eine
Strukturaufklärung mittels 1 H and 13 C NMR-Spektroskopie wurde für Sinapoylmalat
durchgeführt.
Die Gehalte der identifizierten Polyphenole wurden für elf Pak Choi Varietäten unter
Gewächshausbedingungen angegeben, wobei höhere Gehalte in der Blattspreite im Vergleich
zum Blattstängel nachgewiesen werden konnten. Hydroxyzimtsäure-Derivate, insbesondere
Derivate der Äpfelsäure, waren in der Blattspreite wie auch im Blattstängel vorhanden.
Dagegen konnten Flavonoide nur in der Blattspreite nachgewiesen werden.
Das Ziel des zweiten Kapitels war die Charakterisierung der qualitativen und quantitativen
Veränderungen des Polyphenolspektrums in chinesischen Kohlgemüsen durch das
traditionelle chinesische Fermentationsverfahren. Zwei Pak Choi Varietäten und zwei
Senfkohl Varietäten wurden fermentiert (auch als sogenanntes „Pickeln“ bezeichnet). Das
Pflanzenmaterial wurde vor und nach dem Fermentationsverfahren untersucht um qualitative
und quantitative Veränderungen der Polyphenole auszumachen. Hierbei wurde das erste Mal
eine detaillierte Beschreibung der identifizierten Polyphenole in chinesischem Senfkohl
mittels HPLC-ESI-MS n vorgenommen, insbesondere für neu identifizierte Mono- und
96

Diglykosid-Derivate (Gentiobiosen) der Hydroxyzimtsäuren sowie für
Kämpferoltetraglykoside. Flavonoid-Derivate mit geringerer Molekülmasse (Di- und
Triglykoside) sowie Aglykone der Flavonoide und Hydroxyzimtsäuren wurden in
fermentierten Kohl im Vergleich zum unfermentierten Kohl nachgewiesen. Weiterhin konnte
festgestellt werden, dass die Gehalte der Flavonoid- und Hydroxyzimtsäure-Derivate durch
das Fermentationsverfahren abnehmen. Demgegenüber ergab sich, dass das antioxidative
Potential und der Gesamtphenolgehalt (Folin Ciocalteu) in fermentierten Kohl wesentlich
höhere Werte aufzeigten im Vergleich zum unfermentierten Kohl. Diese Anstiege wurden den
qualitativen Änderungen der Polyphenole sowie anderen möglicherweise vorhandenen
Reduktonen zugeordnet.
Das dritte Kapitel handelt von der Bestimmung von zellwandgebundenen Phenolen in
chinesischen Kohlgemüsen. Sieben unterschiedliche zellwandgebundene Phenole konnten
nach einer alkalischen Hydrolysereaktion in der Zellwand von Pak Choi (Varietät Hangzhou
You Dong Er) mittels HPLC-DAD nachgewiesen werden: trans-Ferulasäure, cis-Ferulasäure,
p-Cumarsäure, Sinapinsäure, Vanillinsäure, Vanillin und p-Hydroxybenzaldehyd. Dimere
oder oligomere phenolische Komponenten wurden nicht nachgewiesen. trans-Ferulasäure und
Vanillin waren die Hauptkomponenten der gebundenen Phenole in der Blattspreite wie auch
im Blattstängel. Der Gesamtgehalt wie auch die Gehalte einzelner gebundener Phenole
(Vanillin, p-Cumarsäure, Sinapinsäure, trans- und cis-Ferulasäure) in acht Wochen alten
Pflanzen waren signifikant höher in der Blattspreite (Gesamtgehalt: 79,9 µg/g Zellwand) im
Vergleich zum Blattstängel (Gesamtgehalt: 37,5 µg/g Zellwand). Ältere Pflanzen (acht
Wochen) wiesen im Vergleich zu jüngeren Pflanzen (vier und sechs Wochen) geringere
Gehalte an gebundenen Zellwandphenolen auf. Dies konnte für die Blattspreite, wie auch für
das gesamte Blatt beobachtet werden, jedoch nicht für den Blattstängel.
Die Freisetzung von zellwandgebundenen Phenolen hinsichtlich unterschiedlicher
Hydrolysezeiten ist im vierten Kapitel dargestellt. Die Freisetzung der Hauptkomponenten der
gebundenen Phenole in chinesischem Kohl (Vanillin und trans-Ferulasäure) wurde über eine
Hydrolysezeit von sieben Tagen beobachtet und ergab eine vollständige Freisetzung nach vier
Tagen in allen Pflanzenteilen (Zellwand der Blattspreite wie auch des Blattstängels).
Weiterhin war das Ziel des vierten Kapitels die Charakterisierung des Einflusses von
unterschiedlichen Anbaubedingungen der Pflanzen auf die Gehalte der gebundenen Phenole
in vier chinesischen Kohl Varietäten (Pak Choi Varietäten: Hangzhou You Dong Er und
97

Shanghai Qing; Senfkohl-Varietäten: Xue Li Hong und Bao Bao Qing Cai). Diese waren in
der gleichen Größenordnung beim Anbau in Deutschland (Gewächshaus, 6 Wochen alte
Pflanzen) wie auch in China (Feld, 10 Wochen alte Pflanzen). Weiterhin wurde der Einfluss
des traditionellen chinesischen Fermentationsverfahrens auf die Gehalte der gebundenen
Phenole untersucht. Nur geringe Veränderungen der Gehalte (Abnahmen und auch Zunahmen
von Einzelkomponenten) konnten durch das Fermentationsverfahren für die vier Kohl-
Varietäten nachgewiesen werden, so dass keine klaren Angaben gemacht werden können, ob
gebundene phenolische Komponenten der Zellwand durch die Aktivität von
Mikroorganismen oder Enzymen (z.B. Ferulasäure-Esterase) oder durch pH-Wert
Änderungen während der Fermentation freigesetzt werden.
Das fünfte Kapitel befasst sich mit der Untersuchung von freien und gebundenen Phenolen in
chinesischen Kohlgemüsen, die in China und Deutschland unter Feldbedingungen angebaut
wurden.
Elf Pak Choi Varietäten und zwei Senfkohl Varietäten wurden in China unter
Feldbedingungen angebaut und auf ihren Gehalt an freien Phenolen in äußeren und inneren
Blättern sowie in den Blattspreiten und Blattstängeln untersucht. Zwischen den äußeren und
inneren Blättern der 13 Varietäten konnten überwiegend keine signifikanten Unterschiede der
Konzentrationen an Hydroxyzimtsäure-Derivaten und Flavonoid-Derivaten festgestellt
werden. Die Gehalte zeigten jedoch Unterschiede zwischen der Blattspreite und dem
Blattstängel. Während Hydroxyzimtsäuren und Flavonoide in hohen Konzentrationen in der
Blattspreite vorlagen, konnten nur geringe Konzentrationen oder Spuren von Flavonoiden in
den Stängeln der Pak Choi und Senfkohl Varietäten gefunden werden. Weiterhin wurden die
Gehalte an gebundenen Phenolen in zwei Pak Choi Varietäten und Senfkohl Varietäten
untersucht. Diese waren höher in der Blattspreite als im Blattstängel in Pflanzen, die unter
Feldbedingungen in China angebaut wurden. Gebundene Phenole machen nur einen geringen
Anteil der gesamten Phenole im frischen Pflanzenmaterial aus: 0.81-1.18% im Blattstängel
und 0.05-0.08% in der Blattspreite.
Die Lagerung von Pflanzen von vier Varietäten des chinesischen Kohls zeigte eine Erhöhung
der Polyphenolgehalte bis zum sechsten Lagerungstag bei 4°C und 20°C. Der Anstieg kann
durch Pflanzenstress während der Nacherntebehandlung, der die Biosynthese der Polyphenole
stimuliert, verursacht worden sein.
98

ABSTRACT
About 40 flavonoids and hydroxycinnamic acid derivatives were identified in Chinese
cabbages (pak choi and Chinese leaf mustard) by HPLC-ESI-MS n . The detailed fragmentation
of phenolic compounds and their fragmentation pattern identified the main flavonoids as
kaempferol derivatives, glycosylated and esterified with different hydroxycinnamic acids.
Hydroxyferulic acid as a moiety of flavonoids was characterized for the first time by NMR
spectroscopy. The main hydroxycinnamic acids were shown to be malic acid derivatives,
which were identified for the first time in cabbages. Hydroxybenzoic acids were not detected
as moieties of free phenolic compounds. However, various cell-wall-bound
hydroxybenzaldehydes, hydroxybenzoic, and hydroxycinnamic acids were identified. These
compounds represent only minor amounts (stalk 0.81-1.18%; blade 0.05-0.08%) of the total
phenolic content in fresh plants cultivated in China. In pak choi and leaf mustard, the free
phenolic content is affected by cultivation conditions and varies quantitatively according to
plant part. The content differs among plants cultivated in China under field conditions and in
Germany under greenhouse conditions. The differences were attributed to the different
climatic conditions, e.g. light supply (plants exhibited approx. two- to threefold higher
flavonoid content in China). No tendency was observable vis-à-vis differences in the content
of free phenolic compounds among the outer and inner leaves cultivated in China. However,
the content in the leaf blade was much higher than in the leaf stalk, particularly for the
flavonoid derivatives, and the overall content in the plant was shown to be dependent on the
leaf blade/stalk ratio of each cultivar. Flavonoids were not detected in the leaf stalks of plants
cultivated under greenhouse conditions, but were found in trace amounts in the stalks of the
plants grown in China under field conditions. Additionally, the cell-wall-bound phenolic
content was also predominantly higher in the leaf blade than in the leaf stalk, for pak choi
cultivars in particular (approx. 140 µg/g cell wall of leaf blade and 70 µg/g cell wall of leaf
stalk).
Post-harvest treatments such as fermentation and storage resulted in qualitative and
quantitative polyphenolic changes. The qualitative changes resulting from fermentation were
analyzed in detail by HPLC-ESI-MS n . The analysis showed a partial loss of glycoside or
organic acid moieties of flavonoids and hydroxycinnamic acids as well as the formation of
aglycones, which was attributed to microorganism activity. An increase of the antioxidative
potential was observable for all fermented cultivars in the TEAC and total phenolic content
(Folin Ciocalteu) assays; the increase might be explained by the qualitative changes in the
99

polyphenolic structures (i.e. the formation of hydroxyl groups). Changes in the content of
cell-wall-bound phenolic compounds were marginal; the content did not change significantly
during the fermentation process and microorganism activity can be excluded.
Additionally, the storage of cabbages resulted in increased polyphenolic content within the
first few days (approx. 6 days).
KURZDARSTELLUNG
40 unterschiedliche Derivate der Flavonoide und Hydroxyzimtsäuren konnten in
chinesischem Kohlgemüse (Pak Choi und chinesischer Senfkohl) mittels HPLC-ESI-MS n
nachgewiesen werden. Durch das Fragmentierungsmuster (MS n ) wurde festgestellt, dass es
sich überwiegend um das Flavonoid Kämpferol handelt, welches glykosiliert und verestert mit
unterschiedlichen Hydroxyzimtsäuren vorliegt. Hydroxyferulasäure konnte zum ersten Mal
als Teilkomponente eines Flavonoid Derivates mittels NMR-Spektroskopie vollständig
aufgeklärt werden. Die Hydroxyzimtsäure Derivate liegen hauptsächlich als Ester der
Äpfelsäure vor und wurden bisher noch nicht für Kohlgemüse beschrieben. Weiterhin wurden
auch Polyphenole, die gebunden an die Zellwand vorliegen, nachgewiesen
(Hydroxybenzaldehyde, Hydroxybenzoe- und Hydroxyzimtsäuren). Die gebundenen Phenole
machen jedoch nur einen geringfügigen Teil der Gesamtphenole in der frischen Pflanze aus
(0,81-1,18% im Stängel; 0,05-0,08% in der Blattspreite; Anbau in China unter
Feldbedingungen).
Die Gehalte der Polyphenole in Pak Choi und chinesischem Senfkohl sind abhängig von den
Anbaubedingungen und dem untersuchten Pflanzenteil. Die identifizierten Gehalte in der
Blattspreite waren für Pflanzen die in China unter Feldbedingungen angebaut wurden ca.
dreimal so hoch wie in Deutschland unter Gewächshausbedingungen, was auf die
unterschiedlichen Anbaubedingungen, insbesondere die unterschiedlichen Lichtverhältnisse
im Gewächshaus und unter Feldbedingungen, zurückgeführt wurde. Keine Unterschiede
konnten zwischen äußeren und inneren Blättern in Bezug auf den Polyphenolgehalt
festgestellt werden (Anbau unter Feldbedingungen in China). Allerdings war der
Polyphenolgehalt der Blattspreite im Vergleich zum Blattstängel deutlich höher, insbesondere
für die Flavonoide, so dass der Gesamtgehalt in der Pflanze auch vom Blatt/Stängel-
Verhältnis der jeweiligen Varietät abhängig ist. Flavonoide wurden im Blattstängel der
Gewächshauspflanzen in Deutschland nicht nachgewiesen, jedoch unter Feldbedingungen in
China in geringen Gehalten oder Spuren. Auch die zellwandgebundenen Phenole konnten mit
100

höheren Konzentrationen in der Blattspreite im Vergleich zum Blattstängel nachgewiesen
werden. Qualitative und quantitative Veränderung der freien Polyphenolgehalte wurden bei
der Anwendung des traditonellen chinesischen Fermentationsverfahrens festgestellt. Die
qualitativen Veränderungen der Polyphenol-Strukturen wurden mittels HPLC-ESI-MS n
charakterisiert und zeigten die partielle Abspaltung von Glykosidresten und organischen
Säuren sowie die Bildung von Aglykonen. Diese strukturellen Änderungen werden auf die
Aktivität von Mikroorganismen während der Fermentation zurückgeführt. Während und nach
dem Fermentationsprozess wurde ein Anstieg des antioxidativen Potentials und des
Gesamtphenolgehaltes (Folin Ciocalteu) festgestellt, welcher durch die qualitativen
Strukturänderungen (Bildung von freien Hydroxylgruppen) erklärt sein kann. Veränderungen
im Gehalt der gebundenen Phenole sind marginal während der Fermentation, so dass eine
Aktivität von Mikroorganismen mit zellwandspaltenden Enzymen (Esterase-Aktivität)
ausgeschlossen werden kann. Eine Lagerung von chinesischem Kohlgemüse ergab einen
Anstieg der Polyphenolgehalte in den ersten Tagen, insbesondere bis zum sechsten Tag.
101

Curriculum Vitae
Personal Data
Name Britta Harbaum
Date of birth June 28 th ,1976
Place of birth Itzehoe
Nationality German
Professional Experience
04/04 – dto. Scientific employee at the Division of Food Technology, Institute of
Human Nutrition and Food Science, CAU Kiel, Germany
07/03 – 04/04,
03/99 – 02/03
& 06/97 – 10/98 Chemical-technical assistant at the Institute of Organic Chemistry,
CAU Kiel, Germany
Education
04/04 – 11/04 PhD student at the Division of Food Technology, Institute of Human
Nutrition and Food Science, CAU Kiel, Germany
24.03.2004 Master of Science (Nutritional science)
07/03 – 03/04 Master thesis at the Division of Food Technology, Institute of Human
Nutrition and Food Science, CAU Kiel, Germany
09/98 – 03/04 Studies in Nutritional Science and Home Economics, CAU Kiel,
Germany
08/95 – 06/97 Chemical-technical assistant (Off-the job training) at the Theodor-Litt-
Schule (Vocational school), Neumünster, Germany
16.06.1995 Abitur (Certificate of maturity), Kaiser-Karl-Schule, Itzehoe, Germany