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Chlorophylls: Chemistry and Biological Functions

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Chlorophylls: Chemistry and Biological Functions Sunil Pareek,1∗ Narashans Alok Sagar,1 Sunil Sharma,1 Vinay Kumar,1 Tripti Agarwal,1 Gustavo A. González-Aguilar,2 and Elhadi M. Yahia3

1Department of Agriculture and Environmental Sciences, National Institute of Food Technology Entrepreneurship and Management (NIFTEM), Ministry of Food Processing Industries, Kundli, Sonepat, Haryana, India 2Technology of Food of Vegetable Origin, Research Center for Food and Development, Hermosillo, Sonora, Mexico 3Faculty of Natural Sciences, Autonomous University of Querétaro, Avenida de las Ciencias s/n, Juriquilla, Querétaro, Mexico

14.1 Introduction bacteria. Although there are different photosynthetic pig­ ments such as carotenoids and phycobilins which entrap Chlorophylls are unique pigments with green color and are solar radiation, chlorophyll is the most important of these found in diverse plants, algae, and cyanobacteria (Inanc, molecules. It converts solar energy into chemical energy 2011). The term chlorophyll is derived from the Greek that is used to build essential carbohydrate molecules chloros meaning “green” and phyllon meaning “leaf.” Iso­ (glucose) which are used as food source for the whole plant lation and naming of the chlorophyll was first carried out (Hynninen and Leppakases, 2002). The process can be by Joseph Bienaimé Caventou (French pharmacist) and described by the equation shown in Figure 14.1. Pierre-Joseph Pelletier (French chemist) in 1817 (Gopi Broadly, the conversion process of solar energy into et al., 2014). Chlorophyll is made up of carbon and nitrogen chemical energy is called photosynthesis. Chlorophyll atoms along with a magnesium ion in central position. pigment absorbs blue light and red light of solar radiation Chlorophyll is found in almost every green part of plants, at 430 nm and 660 nm, respectively, and it reflects the i.e. leaves and stem, within the chloroplast, the main green spectrum (Inanc, 2011) (Figure 14.2). organelle which contains the highest amount. Chloroplasts are found in the mesophyll layer, in the middle of plant leaves. Chloroplasts possess thylakoid membranes which 14.1.2 Types and Distribution of Chlorophylls contain green chlorophyll pigment. Chloroplast can be referred to as the “food factory” of the plant cell because it The numbers of naturally occurring chlorophylls may not produces energy and glucose for the whole plant in asso­ yet be fully known. Chlorophyll a and chlorophyll b are ciation with CO2, water, and sunlight. the main components of photosystems in photosynthetic The name “chlorophyll” was first given to the chloro­ organisms. The types of different chlorophyll pigments plast of higher plants only, but later it was extended to all present in nature in decreasing order of importance are photosynthetic porphyrin pigments (Vernon and Seely, given in Table 14.1. 1966). It comes under the special class of compounds Initially, chlorophyll was classified into four – chlorophyll called tetrapyrroles because it contains four pyrrole rings a, chlorophyll b,chlorophyllc, and chlorophyll d (Vernon joined together with a covalent bond, as are vitamin B12 and Seely, 1966) – but later a new type of chlorophyll was and the heme molecule (Willows, 2004). discovered within stromatolite (a hard rock structure made by cyanobacteria) in western Australia, which was named chlorophyll f. Thus eventually chlorophyll was divided into 14.1.1 Importance five classes, a, b, c, d,andf.

The main source of life on earth is the solar energy that is captured by green plants, algae, and various photosynthetic 14.1.2.1 Chlorophyll a This type of chlorophyll is found in almost all photo­ ∗ Corresponding author. synthetic organisms, i.e. plants, algae, cyanobacteria, and

Fruit and Vegetable Phytochemicals: Chemistry and Human Health, Volume I, Second Edition. Edited by Elhadi M. Yahia. © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd. 270 Fruit and Vegetable Phytochemicals

Table 14.1 Types of different chlorophylls in nature, in order of abundance from most (1) to least (7).

Figure 14.1 Photosynthetic reaction. Abundance Chlorophyll types

1 Chlorophyll a aquatic species (Jordan et al., 2001; Nakamura et al., 2003). 2 Chlorophyll b Previously it had been called chlorophyll α. It is found in all 3 Chlorophyll c light harvesting complexes (LHCs) and both reaction centers (RCs) in organisms, photosystem I (PS I) and 4 Chlorophyll d photosystem II (PS II). It absorbs mainly red light from 5 Chlorophyll f the solar spectrum; the absorption peak is captured at 6 Protochlorophyll 420 nm and 660 nm in organic solvents and at 453 nm and 7 Bacteriochlorophyll 670–480 nm in photosynthetic cells (in vivo). It works as primary donor in the RCs of PS I and PS II (Scheer, 1991). 8 Chlorobium chlorophyll Subsequently, two types of chlorophyll a, Ca 670 and Ca 680, were identified which are responsible for absorption as an accessory pigment. It has three subclasses, named of different wavelengths from the light spectrum (French chlorophyll c1, c2,andc3, which have been found in et al., 1972). This observation was confirmed by curve various algae (Beale, 1999). This pigment is widely assim­ analysis of the absorption spectra of many plants and ilated in different marine organisms like diatoms, brown algae. Various experiments were conducted, and the algae, and other marine algae (Smith and Benitez, 1955; results reported were that Ca 670 possessed greater Strain et al., 1963). The absorption peak of chlorophyll c half-width than Ca 680 in the fraction in PS I, and Ca for the photosynthetic spectrum was obtained at 445 nm 680 possessed greater half-width than Ca 670 in PS II and 625 nm in organic solvent and 645 nm in vivo. (French et al., 1969a, 1969b; French 1970).

14.1.2.4 Chlorophyll d 14.1.2.2 Chlorophyll b Chlorophyll d is the minor chlorophyll that was identified Previously, this was called chlorophyll β. Chlorophyll b in red algae (Rhodophyta) by Strain (1958). It captures the has been confirmed to be found in green algae and also in extreme red end of the spectrum of sunlight. The absorp­ higher plants. It assists chlorophyll a in the photo­ tion spectra were obtained for chlorophyll d at 450 nm synthesis process. This pigment has yellow color in its and 690 nm in vitro conditions and up to 740 nm on red natural state but absorbs blue light from the whole solar band in vivo. It can be prepared in the laboratory with the spectrum. For chlorophyll b the characteristic absorption help of permanganate by the oxidation of chlorophyll a peak was observed at 453 nm and 625 nm in vitro and at (Holt and Morley, 1959; Holt, 1961). 480 nm and 650 nm in vivo (Strain et al., 1963).

14.1.2.5 Chlorophyll f 14.1.2.3 Chlorophyll c Chlorophyll f was the last main chlorophyll to be dis­ Chlorophyll c is a brownish-golden colored pigment that covered. It was revealed in cyanobacteria from the deep accompanies chlorophyll a in the photosynthesis process stromatolites in the western region of Australia by Min

Figure 14.2 Light absorption of different photosynthetic pigments. 14 Chlorophylls: Chemistry and Biological Functions 271

Chen and his colleagues. Among all known types of Table 14.2 Distribution of different chlorophylls in nature. chlorophyll, it is chlorophyll f that can utilize the lowest solar light from the extreme end of the infrared spec­ Types of chlorophylls Presence and occurrence trum for photosynthesis. The in vitro absorption and fluorescence of chlorophyll f was obtained at 706 nm Chlorophyll a All photosynthetic plants (excluding and 722 nm, respectively (Chen et al., 2010). Min Chen bacteria) highlighted the need for a revised view of the dynamic Chlorophyll b Higher plants and various green algae role of chlorophyll, as “discovering this new chlorophyll Chlorophyll c Brown algae and diatoms has completely overturned the traditional notion that Chlorophyll d Various red algae, reported in Rhodophyta photosynthesis needs high energy light.” However, all Chlorophyll e Golden-yellow algae (Vaucheria hamata) pigments are often considered accessory pigments except chlorophyll a. Chlorophyll f Cyanobacteria In addition to the chlorophyll types described above, Protochlorophyll Found in yellow leaves of seedlings some other chlorophylls are found in nature, as discussed (grown in dark) and seed coat of pumpkin in the following sections. Bacteriochlorophyll a Purple and green bacteria Bacteriochlorophyll b Purple bacterium (Rhodopseudomonas) Bacteriochlorophyll c Reported in Chloroflexaceae, 14.1.2.6 Chlorophyll e Chlorobiaceae Chlorophyll e is a rare type, which was reported in golden- Bacteriochlorophyll d Reported in Chloroflexaceae, yellow algae named Vaucheria hamata and Tribonema Chlorobiaceae bombycinum (Eugene and Govindjee, 1969). Its proper Bacteriochlorophyll e Reported in Chloroflexaceae, working mechanism has not been revealed clearly. Chlorobiaceae Bacteriochlorophyll g Discovered in Heliobacterium chlorum Chlorobium Green bacteria 14.1.2.7 Protochlorophyll chlorophylls Protochlorophyll occurs in very small amounts along with pheoporphyrin a and protochlorophyllide. It is found in pumpkin seeds at the inner coat and in the dark-grown yellow leaves of seedlings (Madsen, 1963). 14.2 Chemistry of Chlorophylls

The porphyrin unit has a very crucial role in nature 14.1.2.8 Bacteriochlorophyll because it participates in the fundamental skeleton of Bacteriochlorophyll is the main chlorophyll of various chlorophyll (Eugene and Govindjee, 1969). Many photosynthetic bacteria (French, 1969b; Sistrom and researchers have performed various experiments, and Clayton, 1964; Jensen et al., 1964). Several forms of both analyzed the chemical properties and elucidated bacteriochlorophylls have been reported, namely a, b, c, the structure of chlorophyll molecules (Aronoff, 1966; d, e, and g (Eimhjellen et al., 1963; Scheer, 1991). 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 14.1.2.9 Chlorobium Chlorophylls creates an isocyclic ring with the help of CH bridges. These are abundantly found in Chlorobacteriaceae Chemically, chlorophylls possess a magnesium ion in the (green-sulfur bacteria). Chlorobium chlorophylls some­ central position which is found connected with the tetra­ times work in association with bacteriochlorophylls pyrrole ring (Scheer, 1991). Moreover, chlorophylls are (Kaplan and Silberman, 1959; Moshentseva and Kondra­ hydrophobic molecules because they contain phytol, an fi teva, 1962; Mathewson et al., 1963). esteri ed isoprenoid C20 alcohol. The phytol (C20H30OH) possesses a double bond in the trans configuration (Gross, 1991).

14.1.3 Distribution of Chlorophylls among Photosynthetic Organisms 14.2.1 Structures of Different Chlorophylls

The distribution of chlorophylls in their natural state is Chemically, the basic skeleton of chlorophyll is composed summarized in Table 14.2 (Scheer, 1991; Chen et al., of a cyclic tetrapyrrole ring, which is a large planar 2010). structure of a symmetric arrangement in which the 272 Fruit and Vegetable Phytochemicals

four pyrrole rings are joined together by methine (CH=) present in algae and bacteria. The structures of different bridges, and four nitrogen atoms are coordinated with a chlorophylls are described below: central metal atom. In addition, they have a phytol group that confers a hydrophobic characteristic; the metal bound to the chlorophylls is magnesium. Therefore, 14.2.1.1 Chlorophyll a this structure contains a chromophore of several conju­ The molecular formula of chlorophyll a is gated double bonds responsible for absorbing light in the C55H72MgN4O5. It contains a chlorin ring in which a visible region, i.e. red (peak at 670–680 nm) and blue magnesium ion is surrounded centrally by four nitrogen (peak at 435–455 nm). The reflection and/or transmission atoms (Taiz et al., 2006) (the structures of the different of the non-absorbed green light (intermediate wave­ chlorophylls are shown in Figure 14.3). The side chains of length) give the characteristic green color to plants and various chlorophyll molecules determine the characters chlorophyll solutions. Higher plants, ferns, mosses, green of other chlorophyll types and create changes in the algae, and the prokaryotic organism prochloron only have absorption spectrum of solar light (Niedzwiedzki and two chlorophylls (a and b); the remaining chlorophylls are Blankenship, 2010). Chlorophyll a possesses a long

Figure 14.3 Structures of different chlorophylls. 14 Chlorophylls: Chemistry and Biological Functions 273 hydrophobic tail that anchors the molecule to alternative other tetrapyrrole pigments because this pathway pro­ hydrophobic proteins within the thylakoid membrane of duces key precursors and the intermediate C-5 com­ the plastid (Raven et al., 2005). pound δ-aminolevulinic acid (ALA) (Wettstein et al., 1995; Tanaka and Tanaka, 2007). Chlorophyll bio­ synthesis is depicted in Figure 14.4. 14.2.1.2 Chlorophyll b The whole biosynthetic pathway of chlorophyll can be The chemical formula of chlorophyll b is C55H70MgN4O6. elaborated in the steps described in the following sections. There is only a minor difference between the structures of chlorophyll a and chlorophyll b: in the latter, a CHO group is found in place of CH3 at the C-7 position 14.2.2.1 Forming of 5-aminolevulinic acid (ALA) (Figure 14.3). The first step in the C-5 pathway is glutamate activation by t-RNAGlu. A similar step is found in protein synthesis for glutamate incorporation (Hori and Kumar, 1996; Kumar 14.2.1.3 Chlorophyll c et al., 1996). The second step is the formation of glutamyl-1­ Chlorophyll c was confirmed as a mixture of magnesium semialdehyde by the reduction of glutamyl t-RNAGlu, cata­ Glu tetradehydro- and hexadehydropheoporphyrin a3 mono- lyzed by glutamyl t-RNA reductase. In this step, t-RNA- methyl ester (Ia, Ib) (Dougherty et al., 1970). It has three Glu is released. It has been revealed that illumination Glu sub classes, c1, c2, and c3. Their respective formulas are increases the activity of glutamyl t-RNA reductase in C35H30MgN4O5, C35H28MgN4O5, and C36H28MgN4O7. seedling cotyledons of green cucumber (Masuda et al., The c1 is considered the most common form of chloro­ 1996). The final step is the conversion of glutamate-1­ phyll c; its structure differs from c2 in that it contains an semialdehyde into ALA by the transamination process. ethyl group in place of a vinyl group at the C-8 position The C-5 pathway occurs in the plastids and is the only (Figure 14.3). 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 14.2.1.4 Chlorophyll d pathway is found in mitochondria (Drechsler-Thielmann The molecular formula of chlorophyll d is C54H70MgN4O6. et al., 1993; Weinstein et al., 1993). The structure of chlorophyll d was elucidated by Manning and Strain (1943). They revealed that it differs from chlo­ rophyll a in that it has a formyl group in place of divinyl 14.2.2.2 Formation of Protoporphyrin from ALA group in ring A of porphyrin (Larkum and Kuhl, 2005) In this process, two ALA molecules create the monopyrrole (Figure 14.3). porphobilinogen via the enzyme ALA dehydratase. The next step consists of the formation of hydroxymethylbilane, a tetrapyrrole from porphobilinogen. Four molecules of 14.2.1.5 Chlorophyll f porphobilinogen are converted by the enzyme porphobili­ The molecular structure of chlorophyll f is similar to that nogen deaminase (hydroxymethylbilane synthase). The of chlorophyll b, namely C55H70MgN4O6. However, after condensation reaction takes place in this conversion analysis using nuclear magnetic resonance (NMR), mass (Jordan, 1991). The structure of the hydroxymethylbilane spectroscopy (MS), and high-performance liquid chro­ synthase enzyme was elucidated first among all the enzymes matography (HPLC), a major difference was revealed: that of tetrapyrrole biosynthesis (Louie et al., 1992). is, the existence of CHO at the C-2 position of the In the next step, uroporphyrinogen III is formed from porphyrin ring (Figure 14.3) (Willows et al., 2013). hydroxymethylbilane; the reaction is catalyzed by uropor­ phyrinogen III synthase. Uroporphyrinogen III is the first cyclic compound of the chlorophyll biosynthesis path­ 14.2.2 Synthesis of Chlorophyll way (Leeper, 1994). The subsequent steps involve the decarboxylation of acetate chains of uroporphyrinogen III Chlorophyll synthesis is a very crucial mechanism for the which gives coproporphyrinogen III, catalyzed by uropor­ existence of photosynthetic organisms as well as of other phyrinogen decarboxylase. Protoporphyrinogen IX forma­ creatures that are dependent on them. The chlorophyll tion takes place from coproporphyrinogen III by an oxidative biosynthesis process is highly complicated because of the decarboxylation reaction. Coproporphyrinogen oxidase is complex combination of different enzymes and the many the catalytic enzyme for this decarboxylation reaction. resulting compounds. The last step, proporphyrinogen formation, is catalyzed The C-5 pathway, a complex biosynthetic pathway, is by protoporphyrinogen oxidase, which has two features. responsible for naturally synthesizing the chlorophyll and First, this is the only enzyme of plant porphyrin 274 Fruit and Vegetable Phytochemicals

Figure 14.4 Chlorophyll biosynthesis.

biosynthesis that is found in two organelles, mitochondria capsulatus which possessed inter alia the genes respon­ and plastid (Jacobs et al., 1982; Jacobs and Jacobs, 1984). sible for the production of all the enzymes related to Mg Second, protoporphyrinogen is a colorless compound, as branch formation in the biosynthetic pathway. are previously formed intermediate compounds; proto­ The insertion of the magnesium ion is catalyzed by the porphyrinogen oxidase produces the first pigment, pro­ Mg chelatase enzyme which needs ATP for activation. Mg toporphyrin, of the chlorophyll biosynthesis process. chelatase is a significant bottleneck enzyme that specifi­ cally binds to Mg ions only (Castelfranco et al., 1979).

14.2.2.3 Addition of Magnesium Ion The identification and analysis of photosynthetic gene 14.2.2.4 Conversion of Magnesium Protoporphyrin into cluster, a 46 kbp gene sequence, was the real discovery in Protochlorophyllide the field of chlorophyll and bacteriophyll biosynthetic This step involves esterification at position 6 of the pathways. This gene cluster was identified in Rhodobacter propionic acid side chain to produce Mg protoporphyrin 14 Chlorophylls: Chemistry and Biological Functions 275

IX monoethyl ester; the reaction is catalyzed by S-adeno- Table 14.3 Different conditions of chlorophyll degradation in plants along with the parts affected. syl-L-methionine:Mg-protoporphyrin-O-methyltransfer­ ase. The catalytic mechanism of this enzyme was confirmed as a pingpong mechanism in wheat (Richards Condition and factors Part affected (chlorophyll breakdown) et al., 1987) and as an ordered mechanism in Rhodobacter sphaeroides (Bollivar et al., 1994). Various investigations At the point of life cycle initiation have demonstrated that Mg protoporphyrin IX works as a substrate for the methylation step. After that, an isocyclic 1 Germination of seed Cotyledons loss ring is formed that constitutes divinyl protochlorophyl­ 2 Initiation of first leaves lide. The divinyl protochlorophyllide (Mg-2,4-divinyl­ 3 Maturation of inflorescence Bracts loss β pheoporphyrin a5) (MgDVP) is created by -oxidation 4 Fruit ripening Green color loss of fruit and cyclization at position 6 in ring C. This reaction is During the life cycle catalyzed by Mg protoporphyrin IX oxidative cyclase or MPE oxidative cyclase. Turnover (continuous synthesis) Degradation of chloroplast parts Premature death 14.2.2.5 Reduction of Protochlorophyllide into Biotic (disease, harvesting, Whole plant Chlorophyllide grazing, etc.) The photoreduction process occurs at this step, which Climatic (UV rays, temperature, Whole plant converts protochlorophyllide into chlorophyllide a, and darkness) the macrocycle (the dihydroporphyrin or chlorin) is Edaphic (deficiency of water and Whole plant developed. NADPH protochlorophyllide oxidoreductase minerals) is the enzyme responsible for photoreduction. A chro­ Source: Hendry et al., 1987. mophore is found in the macrocycle that provides the green color to chlorophyll a. Prothylakoids of etioplasts contain this enzyme. biological phenomena that degrade chlorophylls such as flowering, fruit ripening, germination, bleaching, etc. 14.2.2.6 Esterification of Chlorophyllide a These mechanisms are considered to be part of the aging This is the last step in the process of chlorophyll a processes. However, the chlorophyll degradation process biosynthesis. Here, phytol, a C-20 isoprenoid alcohol, is not fixed or the same in all cases (Gossauer and Engel, reacts with 7-propionic acid residue by an esterification 1996). Table 14.3 demonstrates some natural and biolog­ process. Chlorophyll dihydrogeranylgeranyl ester and ical mechanisms in which chlorophyll breakdown occurs tetrahydrogeranylgeranyl ester are formed as intermedi­ (Hendry et al., 1987). ates when the geranyl geraniol is esterified by chlorophyll Two main steps are involved in the chlorophyll degra­ a, which is transformed into phytol by three gradual steps. 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 14.2.2.7 Biosynthesis of Chlorophyll b via Chlorophyll a (Amir-Shapira et al., 1987). Chlorophyllase is believed to As discussed previously, the only difference between be present in thylakoid membrane of plastids but it has chlorophyll a and chlorophyll b is the presence of an also been detected near the light harvesting complex aldehyde (formyl) group at ring B in place of a 3-methyl (Schellenberg and Matile, 1995; Brandis et al., 1996). group. In the conversion process of chlorophyll b, trans- The phytol residue is esterified with acetic acid, since it formation occurs (Espineda et al., 1999). Earlier experi­ is not stored as free alcohol (Bortlik et al., 1990). ments suggested that chlorophyll a was the precursor of The opening of the chlorophyll macrocycle by an oxida­ chlorophyll b (Rudiger, 1997). 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 14.2.3 Degradation or Catabolism of Chlorophyll Kräutler, 1996; Engel et al., 1996). The enzyme which has the key role here is called pheophorbide oxygenase (Curty The global degradation of chlorophyll is found to be et al., 1995), which is localized in gerontoplast (senescent always equal to the global synthesis of chlorophyll (Hen­ plastids) membrane (Matile and Schellenberg, 1996). dry et al., 1987). Senescence of plant leaves involves a Many structures of chlorophyll catabolites have been chlorophyll degradation process, and there are various reported in algae and higher plants (Mühlecker and 276 Fruit and Vegetable Phytochemicals

Kräutler, 1996), algae (Engel et al., 1991, 1993, 1996; factors and growth conditions, and particularly with high Iturraspe et al., 1994). This results in oxygenase catalysis exposure to sunlight. and formation of a structure having 19-formyl-1 The concentration of chlorophyll correlates with the (21H,22H)bilinone derivate. photosynthetic capacity of plants, which gives some indi­ Catabolic products derived from chlorophyll a and cation of the physiological status of the plant system chlorophyll b have been reported among algae. The (Gamon and Surfus, 1999). 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 14.4 Biological Functions of steps, and after ring opening a reductase reaction is Chlorophyll indicated for higher plants (Gossauer and Engel, 1996). Subsequently, modification includes hydroxylation Chlorophyll is an extremely important biomolecule found reactions, found only at ethyl group of ring B in Brassica in green plants. Plants utilize chlorophyll and sunlight to napus and at side chains of ring A and B in Hordeum make food materials. Chlorophyll is important not only as vulgare. This change facilitates transport into storage and a color pigment and for its physiological role in plants, but vacuole for further formation of malonic acid ester and also for its health benefits (Liu et al., 2007). β-glucoside in Brassica napus. 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. 14.3 Presence and Distribution in Fruits They have a major role in interrupting diverse diseases and Vegetables such as cancer, cardiovascular, and other chronic diseases (Sangeetha and Baskaran, 2010). Chlorophyll is used for Chlorophyll is mostly composed of a lipophilic derivative bad breath, and it reduces colostomy odor. Chlorophylls which includes chlorophyll a and b and is found in fresh have been used for a long time as a traditional medicine fruits and green vegetables (Kimura and Rodriguez- for therapeutic functions. Amaya, 2002). It is frequently found in the range 1000 Humans have no potential to synthesize chlorophyll to 2000 ppm in some species (Khachik et al., 1986). Chlo­ pigment, but they are able to deposit dietary chlorophyll rophyll is a widely distributed plant pigment (Table 14.4) in as absorbed or with minor alteration to its structure all green fruits and vegetables, and it is the most abundant (Larsen and Christensen, 2005). Healthcare providers pigment on earth. Chlorophylls are registered as food use chlorophyll intravenously for the pain and swelling fl additives used to give color and other properties to a (in ammation) with pancreas problems (pancreatitis). variety of foods and green beverages. Common sources of chlorophyll used for medicine Generally, chlorophyll comprises 0.6% to 1.6% of plants are alfalfa (Medicago sativa) and silkworm droppings. on a dry weight basis; however, wide variation has been Early research suggests that chlorophyll not only is noted. In nature, six classes of chlorophyll exist in plants responsible for green color but also can prevent lung and photosynthetic organisms: a, b, c, d, e,andf. However, and skin cancer by intravenous injection along with the fi only a and b are predominant in angiospermic plants, drug talapor n, followed by laser therapy, in early stages while c, d, e, and f are found in different photosynthetic of cancer. fi algal and diatomic species. Chlorophyll a is predominant Both natural and arti cial commercial grade deriva­ over chlorophyll b by a 3:1 margin. Lichtenthaler (1968) tives such as copper chlorophyll have been extensively fi surveyed the molar ratio of chlorophyll a to b in higher used for their bene cial biological activities, which plants in 24 species, and the range was from 2.56 to 3.45. include wound healing, control of calcium oxalate crys­ fl The a:b ratio was approximately 3 depending upon spe­ tal (Tawashi et al., 1980), and anti-in ammatory prop­ cies. But the ratio can vary due to different environmental erties (Bowers, 1947; Larato and Pfau, 1970). Some reports do provide sufficient evidence for the beneficial Table 14.4 Distribution of chlorophylls in living organisms. effects of chlorophyll. However, many researchers have made claims regarding the healing properties of Chlorophyll Occurrence Chlorophyll Occurrence chlorophyll, but most have been disproved by the com­ type type panies that are marketing them. Quackwatch (www .quackwatch.org) claims that no deodorant effect can Chlorophyll a Universal Chlorophyll d Various algae possibly occur from chlorophyll added to products such Chlorophyll b Mostly plants Chlorophyll e Cyanobacteria as gum, foot powder, and cough drops (Kephart, 1955). Chlorophyll c Various algae Chlorophyll f Cyanobacteria More evidence is essential to rate the effectiveness of chlorophyll for different biological uses. 14 Chlorophylls: Chemistry and Biological Functions 277

14.5 Changes in Chlorophyll during Among these enzymes, chlorophyllase catalyzes the first Processing of Fruits and Vegetables step in chlorophyll catabolism and removes the phytol chain from the porphyrin ring to form chlorophyllide. Chlorophyll degradation is a unique phenomenon that Lipoxygenase is widely distributed in vegetables and is happens during the processing of fruits and vegetables. involved in off-flavor development and color loss. The Color is one of the important sensory characteristics and latter is due to hydroperoxide and radical formation by plays a vital role in the acceptability of food items in the oxidation of lipids, which can destroy chlorophyll and food industry. Chlorophyll a imparts blue-green color carotenoids during frozen storage (Vamos-Vigyazo and whereas chlorophyll b imparts yellow-green color. They Haard, 1981; Adams, 1991). Lopez-Ayerra et al. (1998) also possess different thermal stabilities (Steet and Tong, concluded that lipid oxidation was related to chlorophyll 1996). Chlorophyll b was reported to be thermally more degradation in spinach. Peroxidases are assumed to play an stable, whereas chlorophyll a was found to be thermally important role in chlorophyll degradation, a process unstable (Schwartz and Elbe, 1983; Canjura et al., 1991; accompanying ripening and senescence in most fruits Schwartz and Lorenzo, 1991). In the temperature range and vegetables. Peroxidase is responsible for off-flavor 70–100 °C, green peas showed degradation of chlorophyll development during storage of canned products, especially a and b following a first-order kinetics model. The deg­ of non-acidic vegetables which often exhibit high levels of radation of chlorophyll a was reported to be 12–18 times activity. They impair not only the sensory properties and, faster than that of chlorophyll b, and was found to be hence, the marketability of the product, but also its nutri­ primarily dependent upon the temperature regime during tive value (Vamos-Vigyazo and Haard, 1981). Martinez the processing; at the same time, this indicated the higher et al. (2001) reported that chlorophyll degrading peroxi­ susceptibility of chlorophyll a in a thermal environment dase activity in strawberry and canola seeds decreased with (Erge et al., 2008). The higher thermal stability of chloro­ chlorophyll content in their ripening stage, while the phyll b has been attributed to the electron-withdrawing reverse occurred in mustard. effect of its C-3 formyl group (Belitz and Grosch, 1987). Kato and Shimizu (1985) reported that a phenolic Chlorophylls are highly susceptible to degradation dur­ peroxidase H2O2 system was involved in in vitro bleaching ing processing conditions, resulting in food color changes of chlorophyll. The phenolics active in the system were (Schwartz and Elbe, 1983). The simple reaction mecha­ derivatives of monophenols having an electron-attracting nism that occurs with the chlorophyll molecule during group in the para position. Yamauchi et al. (2004) con­ degradation is shown in Figure 14.5. The chlorophyll cluded that in peroxidase-mediated chlorophyll degrada­ degradation in foods may occur via chemical and bio­ tion, peroxidase oxidizes the phenolic compounds with chemical reactions. Chemical reactions involve the forma­ hydrogen peroxide and forms the phenoxy radical. The tion of pheophytin from the chlorophyll by the phenoxy radical then oxidizes chlorophyll to colorless low replacement of magnesium ions from the porphyrin molecular weight compounds through the formation of ring. The magnesium replacement can occur by acidic C-13-hydroxychlorophyll-a and a bilirubin-like com­ substitution, by heat treatment, or after the action of Mg pound as an intermediate. chelatase. Decarbomethoxylation may occur during strong It is very important to consider the factors affecting heat treatment, leading to the conversion of pheophytin to chlorophyll degradation during the processing of fruits pyropheophytin (Schwartz et al., 1981; Diop et al., 2011). and vegetables. Diverse factors are responsible for degra­ Kräutler and Matile (1999) and Matile et al. (1999) dation, including exposure to dilute acids, heat, and described the role of chlorophyllase, Mg dechelatase, oxygen (Tonucci and Von Elbe, 1992). Under thermal and pheophorbide a oxygenase in chlorophyll degradation. processing conditions, chemical and structural variations

Figure 14.5 Reactions of chlorophylls. 278 Fruit and Vegetable Phytochemicals

take place in the chlorophyll which in turn lead to changes studies in a specially designed reactor with an online pH in color (Canjura et al., 1991; Heaton et al., 1996). In green control capability. The authors concluded that the acti­ cellular tissues subjected to various processing condi­ vation energy was independent of pH, with a magnitude tions, chlorophyll undergoes distinct types of degradation varying between 17 and 17.5 kcal/mol for both chloro­ reactions. The changes in the chlorophyll molecule dur­ phyll a and b. The degradation rate was found to decrease ing heating have been discussed by Gauthier-Jaques et al. linearly as the pH increased. (2001). Demetalation and epimerization were observed It was demonstrated that chlorophylls have higher sta­ during heat treatment, and prolonged heating leads to bility at alkaline pH conditions (Clydesda and Francis, additional demethoxycarbonylation of the molecule. 1968). Gupte and Francis (1964) reported that the use of Demethoxycarbonylation also occurs during the canning magnesium carbonate in combination with high-tempera­ of vegetables. Dephytylation of chlorophyll is achieved ture, short-time processing (at 150 °C) initially improved enzymatically during fermentation and storage and is chlorophyll retention in pureed spinach. However, the often observed together with demetalation. effect was not stable during storage. Magnesium com­ In food products, chlorophyll degradation studies pounds resulted in the formation of hard white crystals revealed that chlorophyll degrades to pheophorbide via of magnesium-ammonium-phosphate (Eheart and Odubi, pheophytins or chlorophyllide. Shwartz and Lorenzo 1973). Ahmed et al. (2002) studied the color degradation (1990) demonstrated that chlorophyll degradation termi­ kinetics of coriander leaf puree; however, they worked on nates at pheophorbide. However, studies by Matile et al. puree at unmodified pH. The effect of pH (3–8) and (1992) and Ginsburg et al. (1994) have shown that chlo­ microbial growth on green color degradation of blanched rophyll degradation continues beyond pheophorbide to broccoli during storage was investigated by Gunawan and colorless compounds. Heaton et al. (1996) observed that Barringer (2000). Color degradation accelerated when chlorophyll degradation of cabbage heads in cold storage broccoli was submerged in McIlvaine’sbufferatdecreasing did not lead to pheophorbide accumulation, leaving the pH. Pheophytinization followed first-order reaction kinet­ degradation of pheophorbide to colorless byproducts as ics. The logarithmic values of reaction rate constants were the only explanation. linearly correlated with the ambient pH up to pH 7. Acids Chlorophyll degradation has been shown to follow containing a benzene ring were found to cause a faster color different pathways depending on the commodity (Heaton change than acids with simple chains due to their hydro­ et al., 1996). However, the mechanisms and kinetics of phobicity. There is synthesis of olive-brown pheophytins those reactions have been only partially characterized. under the acidic conditions, which interchanged the mag­ Heaton and Marangoni (1996) summarized the accepted nesium in the chlorophyll with the two hydrogen ions mechanism of chlorophyll degradation in fruits and veg­ (Mangos and Berger, 1997; Van Boekel, 1999, 2000). etables and extended it to include the degradation of When the process temperatures are high and the pH of pheophorbides into colorless compounds. Takamiya the tissue is low, pheophytins are formed in the processed et al. (2000), in a study on the mechanism of chlorophyll vegetables (La and Von Elbe, 1990). The excessive heating degradation using gene cloning, determined that after leads to the formation of pyropheophytins as a result of + successful removal of phytol and Mg2 from the chloro­ removal of the carbomethoxy group from the pheophytins phyll molecule by the use of chlorophyllase and Mg (Schwartz and Elbe, 1983; Canjura et al., 1991; Mangos and dechelatase, pheophorbide is cleaved and reduced to yield Berger, 1997). The treatment of broccoli juice at tempera­ a colorless, open tetrapyrrole intermediate. tures more than 60 °C leads to the degradation of chloro­ Variation in pH is one of the important factors that phyll a and b to pheophytin a and b (Weemaes et al., 1999a, influence the variations in the color of chlorophylls by the 1999b). Pheophorbides are formed by the loss of magne­ conversion of chlorophylls to pheophytins (Minguez- sium from the chlorophyllides under the acidic environ­ Mosquera et al., 1989). During the processing of peas ment (Heaton et al., 1996). The hydrolysis of the phytol for puree formation, three chlorophyll-linked degraded chain in pheophytins produced olive-brown colored pheo­ products were reported at high pH under high-tempera­ phorbides (Heaton et al., 1996). Higher temperature sen­ ture, short-time conditions after the storage of puree at sitivity was found in mustard leaves than in spinach in room temperature (Buckle and Edwards, 1969). Schwartz relation to color (Ahmed et al., 2004). and Lorenzo (1990), in a study on continuous aseptic The thermal degradation of chlorophyll pigment in processing and storage, concluded that oxidation during thermally processed green vegetables is an aspect of vital storage was not a dominant factor in chlorophyll degra­ importance for the food processing sector. The color dation and color loss. A major problem in thermal proc­ degradation of green peas occurred when green peas essing of green vegetables is the continuous decrease in were subjected to temperatures of 70, 80, 90, and 100 pH values due to acid formation. To avoid this problem, °C to analyze the effect of heat treatment (Erge et al., Ryan-Stoneham and Tong (2000) conducted degradation 2008). It was reported that chlorophyll a and chlorophyll b 14 Chlorophylls: Chemistry and Biological Functions 279 followed a kinetics model of the first order during degra­ chlorophyll degradation and maintains quality during dation, which was recorded using a tristimulus colorime­ storage. It is reported that the enzyme activities degrading ter (Baardseth and Von Elbe, 1989). The blanching of chlorophylls, i.e. chlorophyllase, chlorophyll-degrading green peas was studied under temperatures of 70, 80, 90, peroxidase, pheophytinase, and Mg-dechelation activities, and 100 °C in solutions of buffers with pH of 5.5, 6.5, and were mitigated by hot water treatment (Kaewsuksaeng 7.5. The pH effect on the degradation of chlorophyll was et al., 2015). The processing of vegetables like broccoli noted, and the results showed the rise in degradation rate florets, spinach leaves, and green peppers under high of chlorophyll a and chlorophyll b with the decrease in pH pressure does not degrade chlorophylls, whereas high- (Koca et al., 2007). The constant degradation rate of pressure, high-temperature processing leads to the degra­ chlorophyll a and chlorophyll b were highly related to dation of both chlorophylls, and chlorophyll a was found to the changes in the green pea CIE a∗ color values, when the be unstable at 70 °C compared to chlorophyll b. At 117 °C products were at a pH of 5.5 or 6.5. The a∗ parameter is both chlorophylls were degraded (Sanchez et al., 2014). related to the greenish color of the food; thus the degra­ The blanching treatment of vegetables followed by freez­ dation of chlorophylls was followed by a reduction in the ing led to chlorophyll retention in frozen green asparagus, CIE LAB parameter. Moyano et al. (2008) reported a French bean, and peas (Lisiewska et al., 2010). correlation between the chlorophyll content of a broad A previous study related the effect of microwave and range of virgin olive oils and their lightness. conventional cooking on the chlorophyll pigment in peas, The degradation of green color of broccoli (Brassica leek, squash, broccoli, spinach, and green beans. Except in oleracea) by blanching at a storage temperature of 7 °C peas, the reported results showed chlorophyll a to be more was carried out; the broccoli was then submerged in McIl­ resistant than chlorophyll b in five of the six vegetables. vaine’s buffer at a pH of 3–8, and it was observed that There was an increase in the pheophytins of all the chlorophyll degradation increased as the pH decreased vegetables after cooking. Boiled leek showed loss in chlo­ (Gunawan and Barringer, 2000). The thermal degradation rophyll a and chlorophyll b, whereas in the case of boiled of chlorophyll and chlorophyllides in a puree of spinach at and microwaved peas, there was retention of chlorophyll a temperatures of 100 to 145 °C(2–25 min) and 80 to 115 °C and chlorophyll b. The formation of pheophytin a and b (2.5–39 min), respectively, led to the formation of deriva­ was reported at higher levels in boiled squash and boiled tives pheophorbides, pyropheophorbides, pheophytins, and green beans. The maximum amounts of pheophytins were pyropheophytins (Canjura et al., 1991). Ahmed et al. (2000) reported to be formed in boiled vegetables, and the mini­ studied the effect of particle size on chlorophyll content and mum levels in microwaved vegetables (Turkmen et al., total color of green chili puree. Both chlorophyll content and 2006). HPLC (reverse phase gradient) was used to monitor green color varied significantly with processing temperature the effect of freezing (–22 °C) on chlorophyll a and b and mesh size. Chlorophyll content increased with content in Padrón peppers and green beans. In the case increased mesh fineness, but decreased linearly with proc­ of unblanched beans there was considerable reduction in essing temperature. Up to a 60% loss in chlorophyll was the pigments within 1 month, whereas after 11 months the noted after heating at 100 °Cfor15minutes. pigments remained stable; almost similar results were Chlorophyll degradation and yellowing of peel in lime reported in blanched beans. Chlorophyll reductions in (Citrus latifolia Tan.) is one of the dominant problems blanched beans were reported to be induced by blanching. worldwide. Application of UV-B treatment at 8.8 kJ/m2 The reduction of chlorophyll a was also reported in Padrón led to effective delay in the reduction of chlorophyll peppers (Oruna-Concha et al., 1997). content. The pheophorbide content decreased, followed The quantitative determination of chlorophyll a and b by an increase in the pheophytin a content during later and pheophytins a and b under refrigerated storage stages of storage. Therefore, the study concluded that conditions, followed by industrial processing of spinach, there was mitigation of degradation of chlorophyll by the showed that pheophytins a and b were the prominent application of UV-B irradiation (Srilaong et al., 2011). chlorophyll derivatives formed under refrigerated condi­ Pistachio (Pistacia vera L.) kernels are green in color due tions at 8 °C for 3 weeks (Lopez-Ayerra et al., 1998). The to the presence of chlorophyll, which appeals to the food heating of spinach leaves showed degradation of chloro­ industry for its diverse uses. The degradation of the green phylls a and b, and the effect was found more under color in pistachio is one of the unacceptable features. The microwave cooking or blanching than under steaming or heat treatments applied to pistachio during roasting lead to baking. Pheophytins a and b were formed under steamed degradation of chlorophyll a and b to pheophytins and conditions, whereas under the effect of microwave cook­ pyropheophytins, which ultimately impacts the quality and ing pyrochlorophylls a and b were formed (Teng and market value of pistachio kernel (Pumilia et al., 2014). Chen, 1999). In the case of Thai lime (Citrus aurantifolia Swingle The effect of salt on the degradation of visual green ‘Paan’) the postharvest hot water treatment delays color “-a” in spinach puree under a temperature range of 280 Fruit and Vegetable Phytochemicals

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

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