Chlorophylls: Chemistry and Biological Functions: Chemistry and Human Health, 2nd Edition

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14
Chlorophylls: Chemistry and Biological Functions
Sunil Pareek,
1
∗Narshans Alok Sagar,
1
Sunil Sharma,
1
Vinay Kumar,
1
Tripti Agarwal,
1
Gustavo A.
Gonzalez-Aguilar,
2
and Elhadi M. Yahia
3
1
Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Plot No. 97, Sector
56, HSIIDC Industrial Estate, Kundli 131028, Sonepat, Haryana, India
2
Centro de Investigación en Alimentación y Desarrollo, Carretera a la Victoria Km 0.6, CP 83000, Hermosillo, Sonora, Mexico
3
Facultad de Ciencias Naturales, Universidad Autónoma de Querétaro, Avenida de las Ciencias s/n, Juriquilla, Querétaro, 76230 Qro., Mexico
14.1 Introduction
Chlorophylls are unique pigments with green color and are
found in diverse plants, algae, and cyanobacteria (Inanc,
2011). The term chlorophyll is derived from the Greek
chloros meaning “green”and phyllon meaning “leaf.”Iso-
lation and naming of the chlorophyll was first carried out
by Joseph Bienaimé Caventou (French pharmacist) and
Pierre-Joseph Pelletier (French chemist) in 1817 (Gopi
et al., 2014). Chlorophyll is made up of carbon and nitrogen
atoms along with a magnesium ion in central position.
However, chlorophyll is found in almost every greenpart of
plants, i.e. leaves and stem, within the chloroplast, the main
organelle which contains the highest amount.Chloroplasts
are found in the mesophyll layer, in the middle of plant
leaves. Chloroplasts possess thylakoid membranes which
contain green chlorophyll pigment. Chloroplast can be
referred to as the “food factory”of the plant cell because it
produces energy and glucose for the whole plant in asso-
ciation with CO
2
, water, and sunlight.
The name “chlorophyll”was first given to the chloro-
plast of higher plants only, but later it was extended to all
photosynthetic porphyrin pigments (Vernon and Seely,
1966). It comes under the special class of compounds
called tetrapyrroles because it contains four pyrrole rings
joined together with a covalent bond, as are vitamin B
12
and the heme molecule (Willows, 2004).
14.1.1 Importance
The main source of life on earth is the solar energy that is
captured by green plants, algae, and various photosynthetic
bacteria. Although there are different photosynthetic pig-
ments such as carotenoids and phycobilins which entrap
solar radiation, chlorophyll is the most important of these
molecules. It converts solar energy into chemical energy
that is used to build essential carbohydrate molecules
(glucose) which are used as food source for the whole plant
(Hynninen and Leppakases, 2002). The process can be
described by the equation shown in Figure 14.1.
Broadly, the conversion process of solar energy into
chemical energy is called photosynthesis. Chlorophyll
pigment absorbs blue light and red light of solar radiation
at 430 nm and 660 nm, respectively, and it reflects the
green spectrum (Inanc, 2011) (Figure 14.2).
14.1.2 Types and Distribution of Chlorophylls
The numbers of naturally occurring chlorophylls may not
yet be fully known. Chlorophyll aand chlorophyll bare
the main components of photosystems in photosynthetic
organisms. The types of different chlorophyll pigments
present in nature in decreasing order of importance are
given in Table 14.1.
Initially, chlorophyll was classified into four –chlorophyll
a, chlorophyll b,chlorophyllc, and chlorophyll d(Vernon
and Seely, 1966) –but later a new type of chlorophyll was
discovered within stromatolite (a hard rock structure made
by cyanobacteria) in western Australia, which was named
chlorophyll f. Thus eventually chlorophyll was divided into
five classes, a,b,c,d,andf.
14.1.2.1 Chlorophyll a
This type of chlorophyll is found in almost all photo-
synthetic organisms, i.e. plants, algae, cyanobacteria, and
∗
Corresponding author.
269
Fruit and Vegetable Phytochemicals: Chemistry and Human Health, Second Edition. Edited by Elhadi M. Yahia.
© 2017 John Wiley & Sons, Ltd. Published 2017 by John Wiley & Sons, Ltd.

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aquatic species (Jordan et al., 2001; Nakamura et al., 2003).
Previously it had been called chlorophyll α. It is found in all
light harvesting complexes (LHCs) and both reaction
centers (RCs) in organisms, photosystem I (PS I) and
photosystem II (PS II). It absorbs mainly red light from
the solar spectrum; the absorption peak is captured at
420 nm and 660 nm in organic solvents and at 453 nm and
670–480 nm in photosynthetic cells (in vivo). It works as
primary donor in the RCs of PS I and PS II (Scheer, 1991).
Subsequently, two types of chlorophyll a, Ca 670 and Ca
680, were identified which are responsible for absorption
of different wavelengths from the light spectrum (French
et al., 1972). This observation was confirmed by curve
analysis of the absorption spectra of many plants and
algae. Various experiments were conducted, and the
results reported were that Ca 670 possessed greater
half-width than Ca 680 in the fraction in PS I, and Ca
680 possessed greater half-width than Ca 670 in PS II
(French et al., 1969a, 1969b; French 1970).
14.1.2.2 Chlorophyll b
Previously, this was called chlorophyll β. Chlorophyll b
has been confirmed to be found in green algae and also in
higher plants. It assists chlorophyll ain the photo-
synthesis process. This pigment has yellow color in its
natural state but absorbs blue light from the whole solar
spectrum. For chlorophyll bthe characteristic absorption
peak was observed at 453 nm and 625 nm in vitro and at
480 nm and 650 nm in vivo (Strain et al., 1963).
14.1.2.3 Chlorophyll c
Chlorophyll cis a brownish-golden colored pigment that
accompanies chlorophyll ain the photosynthesis process
as an accessory pigment. It has three subclasses, named
chlorophyll c
1
,c
2
,andc
3
, which have been found in
various algae (Beale, 1999). This pigment is widely assim-
ilated in different marine organisms like diatoms, brown
algae, and other marine algae (Smith and Benitez, 1955;
Strain et al., 1963). The absorption peak of chlorophyll c
for the photosynthetic spectrum was obtained at 445 nm
and 625 nm in organic solvent and 645 nm in vivo.
14.1.2.4 Chlorophyll d
Chlorophyll dis the minor chlorophyll that was identified
in red algae (Rhodophyta) by Strain (1958). It captures the
extreme red end of the spectrum of sunlight. The absorp-
tion spectra were obtained for chlorophyll dat 450 nm
and 690 nm in vitro conditions and up to 740 nm on red
band in vivo. It can be prepared in the laboratory with the
help of permanganate by the oxidation of chlorophyll a
(Holt and Morley, 1959; Holt, 1961).
14.1.2.5 Chlorophyll f
Chlorophyll fwas the last main chlorophyll to be dis-
covered. It was revealed in cyanobacteria from the deep
stromatolites in the western region of Australia by Min
Table 14.1 Types of different chlorophylls in nature, in order of
abundance from most (1) to least (7)
Abundance Chlorophyll types
1 Chlorophyll a
2 Chlorophyll b
3 Chlorophyll c
4 Chlorophyll d
5 Protochlorophyll
6 Bacteriochlorophyll
7 Chlorobium chlorophyll
Figure 14.2 Light absorption of different photosynthetic
pigments
Sunlight
CO2+ Water O2+ Glucose + Energy
Chloro
p
h
y
ll
Figure 14.1 Photosynthetic reaction
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Chen and his colleagues. Among all known types of
chlorophyll, it is chlorophyll fthat can utilize the lowest
solar light from the extreme end of the infrared spec-
trum for photosynthesis. The in vitro absorption and
fluorescence of chlorophyll fwas obtained at 706 nm
and 722 nm, respectively (Chen et al., 2010). Min Chen
highlighted the need for a revised view of the dynamic
role of chlorophyll, as “discovering this new chlorophyll
has completely overturned the traditional notion that
photosynthesis needs high energy light.”However, all
pigments are often considered accessory pigments
except chlorophyll a.
In addition to the chlorophyll types described above,
some other chlorophylls are found in nature, as discussed
in the following sections.
14.1.2.6 Chlorophyll e
Chlorophyll eis a rare type, which was reported in golden-
yellow algae named Vaucheria hamata and Tribonema
bombycinum (Eugene and Govindjee, 1969). Its proper
working mechanism has not been revealed clearly.
14.1.2.7 Protochlorophyll
Protochlorophyll occurs in very small amounts along with
pheoporphyrin aand protochlorophyllide. It is found in
pumpkin seeds at the inner coat and in the dark-grown
yellow leaves of seedlings (Madsen, 1963).
14.1.2.8 Bacteriochlorophyll
Bacteriochlorophyll is the main chlorophyll of various
photosynthetic bacteria (French, 1969b; Sistrom and
Clayton, 1964; Jensen et al., 1964). Several forms of
bacteriochlorophylls have been reported, namely a,b,c,
d,e, and g(Eimhjellen et al., 1963; Scheer, 1991).
14.1.2.9 Chlorobium Chlorophylls
These are abundantly found in Chlorobacteriaceae
(green-sulfur bacteria). Chlorobium chlorophylls some-
times work in association with bacteriochlorophylls
(Kaplan and Silberman, 1959; Moshentseva and Kondra-
teva, 1962; Mathewson et al., 1963).
14.1.3 Distribution of Chlorophylls among
Photosynthetic Organisms
The distribution of chlorophylls in their natural state is
summarized in Table 14.2 (Scheer, 1991; Chen et al.,
2010).
14.2 Chemistry of Chlorophylls
The porphyrin unit has a very crucial role in nature
because it participates in the fundamental skeleton of
chlorophyll (Eugene and Govindjee, 1969). Many
researchers have performed various experiments, and
both analyzed the chemical properties and elucidated
the structure of chlorophyll molecules (Aronoff, 1966;
Seely, 1966). Research has revealed that the chlorophylls
are tetrapyrroles, a cyclic form of porphyrin and chlorin
(the parent molecule of all chlorophylls). This cyclic form
creates an isocyclic ring with the help of CH bridges.
Chemically, chlorophylls possess a magnesium ion in the
central position which is found connected with the tetra-
pyrrole ring (Scheer, 1991). Moreover, chlorophylls are
hydrophobic molecules because they contain phytol, an
esterified isoprenoid C
20
alcohol. The phytol (C
20
H
30
OH)
possesses a double bond in the trans configuration (Gross,
1991).
14.2.1 Structures of Different Chlorophylls
Chemically, the basic skeleton of chlorophyll is composed
of a cyclic tetrapyrrole ring, which is a large planar
structure of a symmetric arrangement in which the
Table 14.2 Distribution of different chlorophylls in nature
Types of chlorophylls Presence and occurrence
Chlorophyll aAll photosynthetic plants (excluding
bacteria)
Chlorophyll bHigher plants and various green algae
Chlorophyll cBrown algae and diatoms
Chlorophyll dVarious red algae, reported in Rhodophyta
Chlorophyll eGolden-yellow algae (Vaucheria hamata)
Chlorophyll fCyanobacteria
Protochlorophyll Found in yellow leaves of seedlings
(grown in dark) and seed coat of pumpkin
Bacteriochlorophyll aPurple and green bacteria
Bacteriochlorophyll bPurple bacterium (Rhodopseudomonas)
Bacteriochlorophyll cReported in Chloroflexaceae,
Chlorobiaceae
Bacteriochlorophyll dReported in Chloroflexaceae,
Chlorobiaceae
Bacteriochlorophyll eReported in Chloroflexaceae,
Chlorobiaceae
Bacteriochlorophyll gDiscovered in Heliobacterium chlorum
Chlorobium
chlorophylls
Green bacteria
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four pyrrole rings are joined together by methine (
CH=)
bridges, and four nitrogen atoms are coordinated with a
central metal atom. In addition, they have a phytol group
that confers a hydrophobic characteristic; the metal
bound to the chlorophylls is magnesium. Therefore,
this structure contains a chromophore of several conju-
gated double bonds responsible for absorbing light in the
visible region, i.e. red (peak at 670–680 nm) and blue
(peak at 435–455 nm). The reflection and/or transmission
of the non-absorbed green light (intermediate wave-
length) give the characteristic green color to plants and
chlorophyll solutions. Higher plants, ferns, mosses, green
algae, and the prokaryotic organism prochloron only have
two chlorophylls (aand b); the remaining chlorophylls are
present in algae and bacteria. The structures of different
chlorophylls are described below:
14.2.1.1 Chlorophyll a
The molecular formula of chlorophyll ais
C
55
H
72
MgN
4
O
5
. It contains a chlorin ring in which a
magnesium ion is surrounded centrally by four nitrogen
atoms (Taiz et al., 2006) (the structures of the difference
chlorophylls are shown in Figure 14.3). The side chains of
various chlorophyll molecules determine the characters
of other chlorophyll types and create changes in the
absorption spectrum of solar light (Niedzwiedzki and
Blankenship, 2010). Chlorophyll apossesses a long
CH2CH3CH3
CH3
CH3CH3CH3
CH3
CH3
CH3CH3CH3CH3
CH3
CH3
CH2
CH3
H3C
H3C
H3C
H3C
H3C
H3CNN
N
N
N
N
NMg
Mg
NN
O
O
O
OO
OCH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OCH3
H3C
H3C
O
O
O
O
2
Chloro
p
h
y
ll
f
Chlorophyll dChlorophyll c3
Chlorophyll c1
Chlorophyll c2
Chlorophyll aChlorophyll b
OCH3
CH3
CH2
NN
NN
Mgll
CH3
CH3
CH2
CH3
CH3
CH3
CH3
CH3
H3C
H3C
H3C
H3C
H3C
H3C
H3C
H3C
H3C
H3C
H2C
H3C
H3C
H2C
H2C
N
NN
NN
N
N
N
NN
N
N
N
-
N
-
N
N
Mg
Mg
Mg
Mg 2+
O
O
O
O
O
OH
HO
O
O
O
O
O
OHO
OO
O
O
O
O
OO
HO
O
O
O
OO
Figure 14.3 Structures of different chlorophylls
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hydrophobic tail that anchors the molecule to alternative
hydrophobic proteins within the thylakoid membrane of
the plastid (Raven et al., 2005).
14.2.1.2 Chlorophyll b
The chemical formula of chlorophyll bis C
55
H
70
MgN
4
O
6.
There is only a minor difference between the structures of
chlorophyll aand chlorophyll b: in the latter, a CHO
group is found in place of CH
3
at the C-7 position
(Figure 14.3).
14.2.1.3 Chlorophyll c
Chlorophyll cwas confirmed as a mixture of magnesium
tetradehydro- and hexadehydropheoporphyrin a
3
mono-
methyl ester (Ia, Ib) (Dougherty et al., 1970). It has three
sub classes, c
1
,c
2
, and c
3
. Their respective formulas are
C
35
H
30
MgN
4
O
5,
C
35
H
28
MgN
4
O
5
, and C
36
H
28
MgN
4
O
7
.
The c
1
is considered the most common form of chloro-
phyll c; its structure differs from c
2
in that it contains an
ethyl group in place of a vinyl group at the C-8 position
(Figure 14.3).
14.2.1.4 Chlorophyll d
The molecular formula of chlorophyll dis C
54
H
70
MgN
4
O
6
.
The structure of chlorophyll dwas elucidated by Manning
and Strain (1943). They revealed that it differs from chlo-
rophyll ain that it has a formyl group in place of divinyl
group in ring A of porphyrin (Larkum and Kuhl, 2005)
(Figure 14.3).
14.2.1.5 Chlorophyll f
The molecular structure of chlorophyll fis similar to that
of chlorophyll b, namely C
55
H
70
MgN
4
O
6
. However, after
analysis using nuclear magnetic resonance (NMR), mass
spectroscopy (MS), and high-performance liquid chro-
matography (HPLC), a major difference was revealed: that
is, the existence of CHO at the C-2 position of the
porphyrin ring (Figure 14.3) (Willows et al., 2013).
14.2.2 Synthesis of Chlorophyll
Chlorophyll synthesis is a very crucial mechanism for the
existence of photosynthetic organisms as well as of other
creatures that are dependent on them. The chlorophyll
biosynthesis process is highly complicated because of the
complex combination of different enzymes and the many
resulting compounds.
The C-5 pathway, a complex biosynthetic pathway, is
responsible for naturally synthesizing the chlorophyll and
other tetrapyrrole pigments because this pathway pro-
duces key precursors and the intermediate C-5 com-
pound δ-aminolevulinic acid (ALA) (Wettstein et al.,
1995; Tanaka and Tanaka, 2007). Chlorophyll bio-
synthesis is depicted in Figure 14.4.
The whole biosynthetic pathway of chlorophyll can be
elaborated in the steps described in the following sections.
14.2.2.1 Forming of 5-aminolevulinic acid (ALA)
The first step in the C-5 pathway is glutamate activation by
t-RNA
Glu
. A similar step is found in protein synthesis for
glutamate incorporation (Hori and Kumar, 1996; Kumar
et al., 1996). The second step is the formation of glutamyl-1-
semialdehyde by the reduction of glutamyl t-RNA
Glu
, cata-
lyzed by glutamyl t-RNA
Glu
reductase. In this step, t-RNA-
Glu
is released. It has been revealed that illumination
increases the activity of glutamyl t-RNA
Glu
reductase in
seedling cotyledons of green cucumber (Masuda et al.,
1996). The final step is the conversion of glutamate-1-
semialdehyde into ALA by the transamination process.
The C-5 pathway occurs in the plastids and is the only
process that has been reported in plants. Apart from plants,
ALA synthesis also takes place in animals and fungi, but the
pathway is different and is known as the C-4 pathway. This
pathway is found in mitochondria (Drechsler-Thielmann
et al., 1993; Weinstein et al., 1993).
14.2.2.2 Formation of Protoporphyrin from ALA
In this process, two ALA molecules create the monopyrrole
porphobilinogen via the enzyme ALA dehydratase. The
next step consists of the formation of hydroxymethylbilane,
a tetrapyrrole from porphobilinogen. Four molecules of
porphobilinogen are converted by the enzyme porphobili-
nogen deaminase (hydroxymethylbilane synthase). The
condensation reaction takes place in this conversion
(Jordan, 1991). The structure of the hydroxymethylbilane
synthase enzyme was elucidated first among all the enzymes
of tetrapyrrole biosynthesis (Louie et al., 1992).
In the next step, uroporphyrinogen III is formed from
hydroxymethylbilane; the reaction is catalyzed by uropor-
phyrinogen III synthase. Uroporphyrinogen III is the
first cyclic compound of the chlorophyll biosynthesis path-
way (Leeper, 1994). The subsequent steps involve the
decarboxylation of acetate chains of uroporphyrinogen III
which gives coproporphyrinogen III, catalyzed by uropor-
phyrinogen decarboxylase. Protoporphyrinogen IX forma-
tion takes place from coproporphyrinogen III by an oxidative
decarboxylation reaction. Coproporphyrinogen oxidase is
the catalytic enzyme for this decarboxylation reaction.
The last step, proporphyrinogen formation, is catalyzed
by protoporphyrinogen oxidase, which has two features.
First, this is the only enzyme of plant porphyrin
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biosynthesis that is found in two organelles, mitochondria
and plastid (Jacobs et al., 1982; Jacobs and Jacobs, 1984).
Second, protoporphyrinogen is a colorless compound, as
are previously formed intermediate compounds; proto-
porphyrinogen oxidase produces the first pigment, pro-
toporphyrin, of the chlorophyll biosynthesis process.
14.2.2.3 Addition of Magnesium Ion
The identification and analysis of photosynthetic gene
cluster, a 46 kbp gene sequence, was the real discovery in
the field of chlorophyll and bacteriophyll biosynthetic
pathways. This gene cluster was identified in Rhodobacter
capsulatus which possessed inter alia the genes respon-
sible for the production of all the enzymes related to Mg
branch formation in the biosynthetic pathway.
The insertion of the magnesium ion is catalyzed by the
Mg chelatase enzyme which needs ATP for activation. Mg
chelatase is a significant bottleneck enzyme that specifi-
cally binds to Mg ions only (Castelfranco et al., 1979).
14.2.2.4 Conversion of Magnesium Protoporphyrin into
Protochlorophyllide
This step involves esterification at position 6 of the
propionic acid side chain to produce Mg protoporphyrin
ALA-synthase
Glutamyl-tRNA
Glutomyl-tRN A synthetase
Glutomyl-tRNA red uctase
aminotransterase
Glutamate 1-semialdehyde
ALA dehydratase
Glycine +
Glutamate + tRNAGiu
5-Aminolaevulinic acid
Glutamate 1-semialdehyde
Succinyl-CoA
Hydroxymethylbilane synthase
Porphobilinogen
Uroporphyrinogen III synthase
Hydroxymethylbilane
Uroporphyrinogen Ill decarboxylase
Uroporphyrinogen III
Coproporphyrinogen oxidase
Coproporphyrinogen III
Protoporphyrin ogen IX oxidase
Protoporphyrinogen IX
Protoporphyri n IX
Mg-protopo rphyrin IX
methyltransferase
Mg-protoporphyrin IX
Mg-chelatose
Mg-protoporphyrin IX monomethyl ester
Cyclase
Protochlorophyllide a
Divinyl protochlorophyllide
Vinyl-reductase
Protochlorophyllide
oxidoreductase
Chlorophyll syntha se
Chlorophyllide a
Chlorophyllide b
Chlorophyll s ynthase
Chloro
p
h
y
ll a
Chlorophyll
Figure 14.4 Chlorophyll biosynthesis
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IX monoethyl ester; the reaction is catalyzed by S-adeno-
syl-L-methionine:Mg-protoporphyrin-O-methyltransfer-
ase. The catalytic mechanism of this enzyme was
confirmed as a pingpong mechanism in wheat (Richards
et al., 1987) and as an ordered mechanism in Rhodobacter
sphaeroides (Bollivar et al., 1994). Various investigations
have demonstrated that Mg protoporphyrin IX works as a
substrate for the methylation step. After that, an isocyclic
ring is formed that constitutes divinyl protochlorophyl-
lide. The divinyl protochlorophyllide (Mg-2,4-divinyl-
pheoporphyrin a5) (MgDVP) is created by β-oxidation
and cyclization at position 6 in ring C. This reaction is
catalyzed by Mg protoporphyrin IX oxidative cyclase or
MPE oxidative cyclase.
14.2.2.5 Reduction of Protochlorophyllide into
Chlorophyllide
The photoreduction process occurs at this step, which
converts protochlorophyllide into chlorophyllide a, and
the macrocycle (the dihydroporphyrin or chlorin) is
developed. NADPH protochlorophyllide oxidoreductase
is the enzyme responsible for photoreduction. A chro-
mophore is found in the macrocycle that provides the
green color to chlorophyll a. Prothylakoids of etioplasts
contain this enzyme.
14.2.2.6 Esterification of Chlorophyllide a
This is the last step in the process of chlorophyll a
biosynthesis. Here, phytol, a C-20 isoprenoid alcohol,
reacts with 7-propionic acid residue by an esterification
process. Chlorophyll dihydrogeranylgeranyl ester and
tetrahydrogeranylgeranyl ester are formed as intermedi-
ates when the geranyl geraniol is esterified by chlorophyll
a, which is transformed into phytol by three gradual steps.
14.2.2.7 Biosynthesis of Chlorophyll b via Chlorophyll a
As discussed previously, the only difference between
chlorophyll aand chlorophyll bis the presence of an
aldehyde (formyl) group at ring B in place of a 3-methyl
group. In the conversion process of chlorophyll b, trans-
formation occurs (Espineda et al., 1999). Earlier experi-
ments suggested that chlorophyll awas the precursor of
chlorophyll b(Rudiger, 1997).
14.2.3 Degradation or Catabolism of Chlorophyll
The global degradation of chlorophyll is found to be
always equal to the global synthesis of chlorophyll (Hen-
dry et al., 1987). Senescence of plant leaves involves a
chlorophyll degradation process, and there are various
biological phenomena that degrade chlorophylls such as
flowering, fruit ripening, germination, bleaching, etc.
These mechanisms are considered to be part of the aging
processes. However, the chlorophyll degradation process
is not fixed or the same in all cases (Gossauer and Engel,
1996). Table 14.3 demonstrates some natural and biolog-
ical mechanisms in which chlorophyll breakdown occurs
(Hendry et al., 1987).
Two main steps are involved in the chlorophyll degra-
dation process. These are phytol removal by chlorophyl-
lase, and removal of Mg catalyzed by magnesium
dechelatase, but these two steps may be different in plants
(Amir-Shapira et al., 1987). Chlorophyllase is believed to
be present in thylakoid membrane of plastids but it has
also been detected near the light harvesting complex
(Schellenberg and Matile, 1995; Brandis et al., 1996).
The phytol residue is esterified with acetic acid, since it
is not stored as free alcohol (Bortlik et al., 1990).
The opening of the chlorophyll macrocycle by an oxida-
tion process is the key step in chlorophyll degradation,
which produces a different resulting structure (Matile and
Kräutler, 1995; Gossauer and Engel, 1996; Mühlecker and
Kräutler, 1996; Engel et al., 1996). The enzyme which has
the key role here is called pheophorbide oxygenase (Curty
et al., 1995), which is localized in gerontoplast (senescent
plastids) membrane (Matile and Schellenberg, 1996).
Many structures of chlorophyll catabolites have been
reported in algae and higher plants (Mühlecker and
Table 14.3 Different conditions of chlorophyll degradation in
plants along with the parts affected
Condition and factors Part affected (chlorophyll
breakdown)
At the point of life cycle initiation
1 Germination of seed Cotyledons loss
2 Initiation of first leaves
3 Maturation of inflorescence Bracts loss
4 Fruit ripening Green color loss of fruit
During the life cycle
Turnover (continuous synthesis) Degradation of chloroplast
parts
Premature death
Biotic (disease, harvesting,
grazing, etc.)
Whole plant
Climatic (UV rays, temperature,
darkness)
Whole plant
Edaphic (deficiency of water and
minerals)
Whole plant
Source: Hendry et al., 1987.
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Kräutler, 1996), algae (Engel et al., 1991, 1993, 1996;
Iturraspe et al., 1994). This results in oxygenase catalysis
and formation of a structure having 19-formyl-1
(21H,22H)bilinone derivate.
Catabolic products derived from chlorophyll aand
chlorophyll bhave been reported among algae. The
responsible modification behind this is the esterification
hydrolysis of the methyl ester group at ring E. In higher
plants, catabolites are hydrogenated in a few metabolic
steps, and after ring opening a reductase reaction is
indicated for higher plants (Gossauer and Engel, 1996).
Subsequently, modification includes hydroxylation
reactions, found only at ethyl group of ring B in Brassica
napus and at side chains of ring A and B in Hordeum
vulgare. This change facilitates transport into storage and
vacuole for further formation of malonic acid ester and
β-glucoside in Brassica napus.
14.3 Presence and Distribution in Fruits
and Vegetables
Chlorophyll is mostly composed of a lipophilic derivative
which includes chlorophyll aand band is found in fresh
fruits and green vegetables (Kimura and Rodriguez-
Amaya, 2002). It is frequently found in the range 1000
to 2000 ppm in some species (Khachik et al., 1986). Chlo-
rophyll is a widely distributed plant pigment (Table 14.4) in
all green fruits and vegetables, and it is the most abundant
pigment on earth. Chlorophylls are registered as food
additives used to give color and other properties to a
variety of foods and green beverages.
Generally, chlorophyll comprises 0.6% to 1.6% of plants
on a dry weight basis; however, wide variation has been
noted. In nature, six classes of chlorophyll exist in plants
and photosynthetic organisms: a,b,c,d,e,andf. However,
only aand bare predominant in angiospermic plants,
while c,d,e, and fare found in different photosynthetic
algal and diatomic species. Chlorophyll ais predominant
over chlorophyll bby a 3:1 margin. Lichtenthaler (1968)
surveyed the molar ratio of chlorophyll ato bin higher
plants in 24 species, and the range was from 2.56 to 3.45.
The a:bratio was approximately 3 depending upon spe-
cies. But the ratio can vary due to different environmental
factors and growth conditions, and particularly with high
exposure to sunlight.
The concentration of chlorophyll correlates with the
photosynthetic capacity of plants, which gives some indi-
cation of the physiological status of the plant system
(Gamon and Surfus, 1999).
14.4 Biological Functions of
Chlorophyll
Chlorophyll is an extremely important biomolecule found
in green plants. Plants utilize chlorophyll and sunlight to
make food materials. Chlorophyll is important not only as
a color pigment and for its physiological role in plants, but
also for its health benefits (Liu et al., 2007).
Chlorophylls have been the subject of extensive
research efforts because of their eminent role in plant
physiology and their role as derivatives in the food sector.
They have a major role in interrupting diverse diseases
such as cancer, cardiovascular, and other chronic diseases
(Sangeetha and Baskaran, 2010). Chlorophyll is used for
bad breath, and it reduces colostomy odor. Chlorophylls
have been used for a long time as a traditional medicine
for therapeutic functions.
Humans have no potential to synthesize chlorophyll
pigment, but they are able to deposit dietary chlorophyll
as absorbed or with minor alteration to its structure
(Larsen and Christensen, 2005). Healthcare providers
use chlorophyll intravenously for the pain and swelling
(inflammation) with pancreas problems (pancreatitis).
Common sources of chlorophyll used for medicine
are alfalfa (Medicago sativa) and silkworm droppings.
Early research suggests that chlorophyll not only is
responsible for green color but also can prevent lung
and skin cancer by intravenous injection along with the
drug talaporfin, followed by laser therapy, in early stages
of cancer.
Both natural and artificial commercial grade deriva-
tives such as copper chlorophyll have been extensively
used for their beneficial biological activities, which
include wound healing, control of calcium oxalate crys-
tal (Tawashi et al., 1980), and anti-inflammatory prop-
erties (Bowers, 1947; Larato and Pfau, 1970). Some
reports do provide sufficient evidence for the beneficial
effects of chlorophyll. However, many researchers have
made claims regarding the healing properties of
chlorophyll, but most have been disproved by the com-
panies that are marketing them. Quackwatch (www
.quackwatch.org) claims that no deodorant effect can
possibly occur from chlorophyll added to products such
as gum, foot powder, and cough drops (Kephart, 1955).
More evidence is essential to rate the effectiveness of
chlorophyll for different biological uses.
Table 14.4 Distribution of chlorophylls in living organisms
Chlorophyll
type
Occurrence Chlorophyll
type
Occurrence
Chlorophyll aUniversal Chlorophyll dVarious algae
Chlorophyll bMostly plants Chlorophyll eCyanobacteria
Chlorophyll cVarious algae Chlorophyll fCyanobacteria
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14.5 Changes in Chlorophyll during
Processing of Fruits and Vegetables
Chlorophyll degradation is a unique phenomenon that
happens during the processing of fruits and vegetables.
Color is one of the important sensory characteristics and
plays a vital role in the acceptability of food items in the
food industry. Chlorophyll aimparts blue-green color
whereas chlorophyll bimparts yellow-green color. They
also possess different thermal stabilities (Steet and Tong,
1996). Chlorophyll bwas reported to be thermally more
stable, whereas chlorophyll awas found to be thermally
unstable (Schwartz and Elbe, 1983; Canjura et al., 1991;
Schwartz and Lorenzo, 1991). In the temperature range
70–100 °C, green peas showed degradation of chlorophyll
aand bfollowing a first-order kinetics model. The deg-
radation of chlorophyll awas reported to be 12–18 times
faster than that of chlorophyll b, and was found to be
primarily dependent upon the temperature regime during
the processing; at the same time, this indicated the higher
susceptibility of chlorophyll ain a thermal environment
(Erge et al., 2008). The higher thermal stability of chloro-
phyll bhas been attributed to the electron-withdrawing
effect of its C-3 formyl group (Belitz and Grosch, 1987).
Chlorophylls are highly susceptible to degradation dur-
ing processing conditions, resulting in food color changes
(Schwartz and Elbe, 1983). The simple reaction mecha-
nism that occurs with the chlorophyll molecule during
degradation is shown in Figure 14.5. The chlorophyll
degradation in foods may occur via chemical and bio-
chemical reactions. Chemical reactions involve the forma-
tion of pheophytin from the chlorophyll by the
replacement of magnesium ions from the porphyrin
ring. The magnesium replacement can occur by acidic
substitution, by heat treatment, or after the action of Mg
chelatase. Decarbomethoxylation may occur during strong
heat treatment, leading to the conversion of pheophytin to
pyropheophytin (Schwartz et al., 1981; Diop et al., 2011).
Kräutler and Matile (1999) and Matile et al. (1999)
described the role of chlorophyllase, Mg dechelatase,
and pheophorbide aoxygenase in chlorophyll degradation.
Among these enzymes, chlorophyllase catalyzes the first
step in chlorophyll catabolism and removes the phytol
chain from the porphyrin ring to form chlorophyllide.
Lipoxygenase is widely distributed in vegetables and is
involved in off-flavor development and color loss. The
latter is due to hydroperoxide and radical formation by
oxidation of lipids, which can destroy chlorophyll and
carotenoids during frozen storage (Vamos-Vigyazo and
Haard, 1981; Adams, 1991). Lopez-Ayerra et al. (1998)
concluded that lipid oxidation was related to chlorophyll
degradation in spinach. Peroxidases are assumed to play an
important role in chlorophyll degradation, a process
accompanying ripening and senescence in most fruits
and vegetables. Peroxidase is responsible for off-flavor
development during storage of canned products, especially
of non-acidic vegetables which often exhibit high levels of
activity. They impair not only the sensory properties and,
hence, the marketability of the product, but also its nutri-
tive value (Vamos-Vigyazo and Haard, 1981). Martinez
et al. (2001) reported that chlorophyll degrading peroxi-
dase activity in strawberry and canola seeds decreased with
chlorophyll content in their ripening stage, while the
reverse occurred in mustard.
Kato and Shimizu (1985) reported that a phenolic
peroxidase H
2
O
2
system was involved in in vitro bleaching
of chlorophyll. The phenolics active in the system were
derivatives of monophenols having an electron-attracting
group in the para position. Yamauchi et al. (2004) con-
cluded that in peroxidase-mediated chlorophyll degrada-
tion, peroxidase oxidizes the phenolic compounds with
hydrogen peroxide and forms the phenoxy radical. The
phenoxy radical then oxidizes chlorophyll to colorless low
molecular weight compounds through the formation of
C-13-hydroxychlorophyll-aand a bilirubin-like com-
pound as an intermediate.
It is very important to consider the factors affecting
chlorophyll degradation during the processing of fruits
and vegetables. Diverse factors are responsible for degra-
dation, including exposure to dilute acids, heat, and
oxygen (Tonucci and Von Elbe, 1992). Under thermal
processing conditions, chemical and structural variations
Chlorophyll
Heat, acid
Heat, acid
Methyl chlorophyllide
Heat, acid
Pheo
p
horbide
Pheophytins
Phytol ChlorophyllaseMg2+
Mg2+, phytol
Figure 14.5 Reactions of chlorophylls
14 Chlorophylls: Chemistry and Biological Functions 277

C14 04/28/2017 1:18:25 Page 278
take place in the chlorophyll which in turn lead to changes
in color (Canjura et al., 1991; Heaton et al., 1996). In green
cellular tissues subjected to various processing condi-
tions, chlorophyll undergoes distinct types of degradation
reactions. The changes in the chlorophyll molecule dur-
ing heating have been discussed by Gauthier-Jaques et al.
(2001). Demetalation and epimerization were observed
during heat treatment, and prolonged heating leads to
additional demethoxycarbonylation of the molecule.
Demethoxycarbonylation also occurs during the canning
of vegetables. Dephytylation of chlorophyll is achieved
enzymatically during fermentation and storage and is
often observed together with demetalation.
In food products, chlorophyll degradation studies
revealed that chlorophyll degrades to pheophorbide via
pheophytins or chlorophyllide. Shwartz and Lorenzo
(1990) demonstrated that chlorophyll degradation termi-
nates at pheophorbide. However, studies by Matile et al.
(1992) and Ginsburg et al. (1994) have shown that chlo-
rophyll degradation continues beyond pheophorbide to
colorless compounds. Heaton et al. (1996) observed that
chlorophyll degradation of cabbage heads in cold storage
did not lead to pheophorbide accumulation, leaving the
degradation of pheophorbide to colorless byproducts as
the only explanation.
Chlorophyll degradation has been shown to follow
different pathways depending on the commodity (Heaton
et al., 1996). However, the mechanisms and kinetics of
those reactions have been only partially characterized.
Heaton and Marangoni (1996) summarized the accepted
mechanism of chlorophyll degradation in fruits and veg-
etables and extended it to include the degradation of
pheophorbides into colorless compounds. Takamiya
et al. (2000), in a study on the mechanism of chlorophyll
degradation using gene cloning, determined that after
successful removal of phytol and Mg
2+
from the chloro-
phyll molecule by the use of chlorophyllase and Mg
dechelatase, pheophorbide is cleaved and reduced to yield
a colorless, open tetrapyrrole intermediate.
Variation in pH is one of the important factors that
influence the variations in the color of chlorophylls by the
conversion of chlorophylls to pheophytins (Minguez-
Mosquera et al., 1989). During the processing of peas
for puree formation, three chlorophyll-linked degraded
products were reported at high pH under high-tempera-
ture, short-time conditions after the storage of puree at
room temperature (Buckle and Edwards, 1969). Schwartz
and Lorenzo (1990), in a study on continuous aseptic
processing and storage, concluded that oxidation during
storage was not a dominant factor in chlorophyll degra-
dation and color loss. A major problem in thermal proc-
essing of green vegetables is the continuous decrease in
pH values due to acid formation. To avoid this problem,
Ryan-Stoneham and Tong (2000) conducted degradation
studies in a specially designed reactor with an online pH
control capability. The authors concluded that the acti-
vation energy was independent of pH, with a magnitude
varying between 17 and 17.5 kcal/mol for both chloro-
phyll aand b. The degradation rate was found to decrease
linearly as the pH increased.
It was demonstrated that chlorophylls have higher sta-
bility at alkaline pH conditions (Clydesda and Francis,
1968). Gupte and Francis (1964) reported that the use of
magnesium carbonate in combination with high-tempera-
ture, short-time processing (at 150 °C) initially improved
chlorophyll retention in pureed spinach. However, the
effect was not stable during storage. Magnesium com-
pounds resulted in the formation of hard white crystals
of magnesium-ammonium-phosphate (Eheart and Odubi,
1973). Ahmed et al. (2002) studied the color degradation
kinetics of coriander leaf puree; however, they worked on
puree at unmodified pH. The effect of pH (3–8) and
microbial growth on green color degradation of blanched
broccoli during storage was investigated by Gunawan and
Barringer (2000). Color degradation accelerated when
broccoli was submerged in McIlvaine’sbufferatdecreasing
pH. Pheophytinization followed first-order reaction kinet-
ics. The logarithmic values of reaction rate constants were
linearly correlated with the ambient pH up to pH 7. Acids
containing a benzene ring were found to cause a faster color
change than acids with simple chains due to their hydro-
phobicity. There is synthesis of olive-brown pheophytins
under the acidic conditions, which interchanged the mag-
nesium in the chlorophyll with the two hydrogen ions
(Mangos and Berger, 1997; Van Boekel, 1999, 2000).
When the process temperatures are high and the pH of
the tissue is low, pheophytins are formed in the processed
vegetables (La and Von Elbe, 1990). The excessive heating
leads to the formation of pyropheophytins as a result of
removal of the carbomethoxy group from the pheophytins
(Schwartz and Elbe, 1983; Canjura et al., 1991; Mangos and
Berger, 1997). The treatment of broccoli juice at tempera-
tures more than 60 °C leads to the degradation of chloro-
phyll aand bto pheophytin aand b(Weemaes et al., 1999a,
1999b). Pheophorbides are formed by the loss of magne-
sium from the chlorophyllides under the acidic environ-
ment (Heaton et al., 1996). The hydrolysis of the phytol
chain in pheophytins produced olive-brown colored pheo-
phorbides (Heaton et al., 1996). Higher temperature sen-
sitivity was found in mustard leaves than in spinach in
relation to color (Ahmed et al., 2004).
The thermal degradation of chlorophyll pigment in
thermally processed green vegetables is an aspect of vital
importance for the food processing sector. The color
degradation of green peas occurred when green peas
were subjected to temperatures of 70, 80, 90, and 100
°C to analyze the effect of heat treatment (Erge et al.,
2008). It was reported that chlorophyll aand chlorophyll b
278 Fruit and Vegetable Phytochemicals

C14 04/28/2017 1:18:25 Page 279
followed a kinetics model of the first order during degra-
dation, which was recorded using a tristimulus colorime-
ter (Baardseth and Von Elbe, 1989). The blanching of
green peas was studied under temperatures of 70, 80, 90,
and 100 °C in solutions of buffers with pH of 5.5, 6.5, and
7.5. The pH effect on the degradation of chlorophyll was
noted, and the results showed the rise in degradation rate
of chlorophyll aand chlorophyll bwith the decrease in pH
(Koca et al., 2007). The constant degradation rate of
chlorophyll aand chlorophyll bwere highly related to
the changes in the green pea CIE a∗color values, when the
products were at a pH of 5.5 or 6.5. The a∗parameter is
related to the greenish color of the food; thus the degra-
dation of chlorophylls was followed by a reduction in the
CIE LAB parameter. Moyano et al. (2008) reported a
correlation between the chlorophyll content of a broad
range of virgin olive oils and their lightness.
The degradation of green color of broccoli (Brassica
oleracea) by blanching at a storage temperature of 7 °C
was carried out; the broccoli was then submerged in McIl-
vaine’s buffer at a pH of 3–8, and it was observed that
chlorophyll degradation increased as the pH decreased
(Gunawan and Barringer, 2000). The thermal degradation
of chlorophyll and chlorophyllides in a puree of spinach at
temperatures of 100 to 145 °C(2–25 min) and 80 to 115 °C
(2.5–39 min), respectively, led to the formation of deriva-
tives pheophorbides, pyropheophorbides, pheophytins, and
pyropheophytins (Canjura et al., 1991). Ahmed et al. (2000)
studied the effect of particle size on chlorophyll content and
total color of green chili puree. Both chlorophyll content and
green color varied significantly with processing temperature
and mesh size. Chlorophyll content increased with
increased mesh fineness, but decreased linearly with proc-
essing temperature. Up to a 60% loss in chlorophyll was
noted after heating at 100 °Cfor15minutes.
Chlorophyll degradation and yellowing of peel in lime
(Citrus latifolia Tan.) is one of the dominant problems
worldwide. Application of UV-B treatment at 8.8 kJ/m
2
led to effective delay in the reduction of chlorophyll
content. The pheophorbide content decreased, followed
by an increase in the pheophytin acontent during later
stages of storage. Therefore, the study concluded that
there was mitigation of degradation of chlorophyll by the
application of UV-B irradiation (Srilaong et al., 2011).
Pistachio (Pistacia vera L.) kernels are green in color due
to the presence of chlorophyll, which appeals to the food
industry for its diverse uses. The degradation of the green
color in pistachio is one of the unacceptable features. The
heat treatments applied to pistachio during roasting lead to
degradation of chlorophyll aand bto pheophytins and
pyropheophytins, which ultimately impacts the quality and
market value of pistachio kernel (Pumilia et al., 2014).
In the case of Thai lime (Citrus aurantifolia Swingle
‘Paan’) the postharvest hot water treatment delays
chlorophyll degradation and maintains quality during
storage. It is reported that the enzyme activities degrading
chlorophylls, i.e. chlorophyllase, chlorophyll-degrading
peroxidase, pheophytinase, and Mg-dechelation activities,
were mitigated by hot water treatment (Kaewsuksaeng
et al., 2015). The processing of vegetables like broccoli
florets, spinach leaves, and green peppers under high
pressure does not degrade chlorophylls, whereas high-
pressure, high-temperature processing leads to the degra-
dation of both chlorophylls, and chlorophyll awas found to
be unstable at 70 °C compared to chlorophyll b. At 117 °C
both chlorophylls were degraded (Sanchez et al., 2014).
The blanching treatment of vegetables followed by freez-
ing led to chlorophyll retention in frozen green asparagus,
French bean, and peas (Lisiewska et al., 2010).
A previous study related the effect of microwave and
conventional cooking on the chlorophyll pigment in peas,
leek, squash, broccoli, spinach, and green beans. Except in
peas, the reported results showed chlorophyll ato be more
resistant than chlorophyll bin five of the six vegetables.
There was an increase in the pheophytins of all the
vegetables after cooking. Boiled leek showed loss in chlo-
rophyll aand chlorophyll b, whereas in the case of boiled
and microwaved peas, there was retention of chlorophyll a
and chlorophyll b. The formation of pheophytin aand b
was reported at higher levels in boiled squash and boiled
green beans. The maximum amounts of pheophytins were
reported to be formed in boiled vegetables, and the mini-
mum levels in microwaved vegetables (Turkmen et al.,
2006). HPLC (reversephase gradient) was used to monitor
the effect of freezing (–22 °C) on chlorophyll aand b
content in Padrón peppers and green beans. In the case
of unblanched beans there was considerable reduction in
the pigments within 1 month, whereas after 11 months the
pigments remained stable; almost similar results were
reported in blanched beans. Chlorophyll reductions in
blanched beans were reported to be induced by blanching.
The reduction of chlorophyll awas also reported in Padrón
peppers (Oruna-Concha et al., 1997).
The quantitative determination of chlorophyll aand b
and pheophytins aand bunder refrigerated storage
conditions, followed by industrial processing of spinach,
showed that pheophytins aand bwere the prominent
chlorophyll derivatives formed under refrigerated condi-
tions at 8 °C for 3 weeks (Lopez-Ayerra et al., 1998). The
heating of spinach leaves showed degradation of chloro-
phylls aand b, and the effect was found more under
microwave cooking or blanching than under steaming or
baking. Pheophytins aand bwere formed under steamed
conditions, whereas under the effect of microwave cook-
ing pyrochlorophylls aand bwere formed (Teng and
Chen, 1999).
The effect of salt on the degradation of visual green
color “-a”in spinach puree under a temperature range of
14 Chlorophylls: Chemistry and Biological Functions 279

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50–120 °C, open pan cooking, and pressure cooking, was
studied. Salt showed a protective action against the deg-
radation of the green color (Nisha et al., 2004). In canned
slices of kiwi fruit, chlorophyll was found to degrade when
heated at 100 °C for 5 minutes (Robertson, 1985).
Therefore, in conclusion, chlorophyll pigments in horti-
cultural crops require very specific conditions to avoid
degradation under various processing environments. The
congenial temperature conditions during thermal and non-
thermal processing should be optimized to mitigate the
degradation of chlorophyll pigments in different vegetables
and fruits. However, under other processing steps like
blanching, the pH plays an important role in relation to
the degradation of chlorophyll pigments. The susceptibility
of degradation of chlorophyll during processing of different
vegetables is largely dependent on the different processes
employed with a particular horticultural crop. The process-
ing methodology along with its intensity during processing
of vegetables or fruits is also a crucial factor which affects the
degradation of chlorophylls in horticultural crops.
14.6 Conclusions and Research
Needs
Chlorophyll plays a vital role in the photosynthetic
processesinplantsuniversally. Its biological role for
human health facilitates its importance for nurturing
good health. It acts as potent antioxidant, enhances
blood clotting activity, assists with hormonal balancing,
has importance for detoxification, and improves diges-
tion. The presence of chlorophyll in diverse food items
emphasizes its importance; it is a natural colorant which
appeals to customers in the items in which the presence
of high chlorophyll content is acceptable. The majority
of raw vegetables contain chlorophyll, and therefore
chlorophylls act as a potent biomarker to check the
freshness of vegetables. It is commonly observed in
fruits that chlorophyll is degraded during the ripening
process and converted to other pigments like lycopene,
anthocyanin, etc. The emerging food processing sector
worldwide should develop the engineering technology
for the processing of chlorophyllous fruits and vegeta-
bles by stabilizing the chlorophyll pigment, and making
it bioavailable. The processes in which the maximum
amount of chlorophyll pigments could be retained can
be optimized to the utmost levels for keeping the
stability of chlorophyll pigments under consideration
while processing fresh green vegetables. Novel method-
ologies should be developed for the processing of chlo-
rophyll-rich vegetables and fruits. There is an ample
need to apply biotechnological interventions to facili-
tate the long-term preservation of chlorophylls by
enhancing shelf life.
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