Nutrition Research 27 (2007) 1 – 12

www.elsevier.com/locate/nutres
Review Article
Digestion, absorption, and cancer preventative activity of
dietary chlorophyll derivatives
Mario G. Ferruzzia,4, Joshua Blakesleeb
a
Department of Food Science, Purdue University, 745 Agriculture Mall Dr, West Lafayette, IN 47907
b
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, J Walter Wilson Laboratory, Providence, RI 02912
Received 8 June 2006; revised 11 December 2006; accepted 14 December 2006

Abstract

The growing body of epidemiological and experimental evidence associating diets rich in fruits
and vegetables with prevention of chronic diseases such as cancer has stimulated interest in plant
food phytochemicals as physiologically active dietary components. Chlorophyll and its various
derivatives are believed to be among the family of phytochemical compounds that are potentially
responsible for such associations. Dietary chlorophyll is predominantly composed of lipophilic
derivatives including chlorophyll a and b (fresh fruits and vegetables), metal-free pheophytins and
pyropheophytins (thermally processed fruits and vegetables), as well as Zn-pheophytins and
Zn-pyropheophytins (thermally processed green vegetables). Water-soluble derivatives including
chlorophyllides, pheophorbides, as well as a commercial-grade derivative known as sodium copper
chlorophyllin (SCC) also contribute to the diversity of dietary chlorophyll derivatives. Although the
use of chlorophyll derivatives, especially SCC, in traditional medical applications is well
documented, it is perhaps the potential of chlorophyll as a cancer preventative agent that has drawn
significant attention recently. Biological activities attributed to chlorophyll derivatives consistent
with cancer prevention include antioxidant and antimutagenic activity, mutagen trapping, modulation
of xenobiotic metabolism, and induction of apoptosis. Although most research has focused on
commercial-grade SCC, the extent to which natural chlorophyll derivatives modulate biomarkers of
cancer risk is also being explored. Recent research efforts have also included investigation of the
impact of digestive factors on chlorophyll structure and bioaccessibility as a means to better
understand the extent to which these pigments may be bioavailable in humans and therefore have
more systemic impact in the prevention of cancer.
D 2007 Elsevier Inc. All rights reserved.
Keywords: Chlorophyll; Chlorophyllin; Cancer prevention; Antimutagenic; Antioxidant; Xenobiotic metabolism; Apoptosis;
Bioavailability

1. Introduction natural pigments, reaching levels that can exceed 1000 to

Increased fruit and vegetable consumption has been
associated with a decreased cancer risk [1-4], intensifying
interest in plant food phytochemicals as potential physiolog-
ically active agents. Chlorophyll is the most ubiquitous of all
4 Corresponding author. Tel.: +1 765 494 0625; fax: +1 765 494 7953.
E-mail address: mferruzz@purdue.edu (M.G. Ferruzzi).
0271-5317/$ – see front matter D 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.nutres.2006.12.003
2000 ppm wet weight in some species [5], and is responsible
for the color of all green plants [6]. Considering the primary
role of chlorophyll in photosynthesis and its close association
with yellow/orange carotenoid pigments well known for their
bioactivity, these blue-green pigments have often been
overlooked with regard to their potential physiological
impact and role in the prevention of chronic disease.
Chlorophyll and its various derivatives have a long-
established history of use in traditional medicine and for
2 M.G. Ferruzzi, J. Blakeslee / Nutrition Research 27 (2007) 1–12
therapeutic purposes [7]. Both natural chlorophyll and
commercial-grade derivatives such as sodium copper
chlorophyllin (SCC) have been widely investigated for a
range of beneficial biological activities including wound
healing [8], anti-inflammatory properties [9,10], control of
calcium oxalate crystals [11], and internal deodorization
[12]. Furthermore, the ability of both natural and commer-
cial chlorophyll derivatives to act as photosensitizers have
enabled their utilization as effective agents in photodynamic
therapy of cancer [13-15].
Although these applications illustrate the potential
usefulness of chlorophyll derivatives in medical applica-
tions, it is perhaps the potential role of these pigments in
the prevention of human cancers that has drawn more
recent attention. Both SCC and natural chlorophyll deri-
vatives have demonstrated significant biological activities
in vitro and in vivo consistent with the prevention of cancer
including antioxidant activity, antimutagenic activity, mod-
ulation of xenobiotic metabolizing enzymes, and induction
of apoptotic events in cancer cell lines. These encouraging
results have led to the investigation of chemopreventative
effects in humans [16,17] and further stimulated interest in
the absorption and potential tissue distribution of these
pigments in humans. Although traditionally assumed to be
unabsorbable by humans, limited research efforts have
begun to offer evidence supporting the notion that
chlorophyll derivatives may in fact be bioavailable. These
efforts remain critically important to better understand the
mechanism by which dietary chlorophyll derivatives may
prevent cancer.
The purpose of this review is to summarize informa-
tion from relevant scientific studies covering chlorophyll
structure; the impact of food processing and digestive
conditions; the potential for intestinal uptake and bioavail-
ability; and cancer preventative activities including antiox-
idant and antimutagenic activity, modulation of xenobiotic
metabolism, and induction of apoptosis. Furthermore,
an effort is made to further elaborate the differences
between research on natural chlorophylls and studies
Fig. 1. Structure of chlorophyll derivatives found in higher plants including
chlorophyll a (R = CH3) and chlorophyll b (R = CHO).
Table 1
Representative chlorophyll and carotenoid content of common green
vegetables
Fruit/Vegetable Total chlorophyll content Total carotenoid content
tissue (lg/g fresh tissue) (lg/g fresh tissue)
Green beans 52 8.6
Broccoli 79 42
Brussel sprouts 68 49
Kale 1870 776
Peas 134 34
Spinach 1250 364
Data compiled from Lopez-Hernandez et al [124], Khachik et al [125], and
Edelenbos et al [126].
focused on commercially derived semisynthetic derivatives
such as SCC.
2. Chlorophyll structure, stability, and derivatives
Structurally, chlorophyll is a substituted tetrapyrrole with
a centrally bound magnesium atom (Fig. 1). The porphyrin
macrocycle is further esterified to a diterpene alcohol, phytol,
to form chlorophyll. In nature, chlorophyll a and b pre-
dominate in higher plants, whereas chlorophyll c, d, and e
derivatives are found throughout various photosynthetic
algae and diatomic species including brown, red, and
yellow-green algae [6]. Furthermore, several additional
classes of bacteriochlorophylls, not covered in this review,
have additionally been isolated in photosynthetic bacteria
[6,18].
Widely distributed in green fruits and vegetables as
primary photosynthetic pigments, chlorophyll a is typically
found in higher amounts than chlorophyll b by a 3 to
1 margin. The distribution and content of chlorophyll in
fruits and vegetables are dependent on a number of factors
including species, agroclimactic conditions, pre- and post-
harvest treatment, and type and degree of food processing
[19]. Although difficult to generalize across the diversity of
plant tissue, the chlorophyll content of commonly consumed
green vegetables typically exceeds the levels of other
bioactive pigments, such as carotenoids, by up to a 5-fold
margin (Table 1). This relatively high concentration makes
chlorophyll a significant contributor to the total dietary
phytochemical pool.
The sensitivity of natural chlorophylls to extremes in pH
and temperature allows for the formation of several distinct
derivatives through processing of vegetable tissue and
human digestion. The impact of food processing operations
on chlorophylls in green fruits and vegetables has been
thoroughly studied and is the subject of several reviews
[20-22]. The main degradative reactions are summarized in
Fig. 2. Thermal processing and/or acidification results in a
perceivable discoloration of vegetable tissue from green to
brown known as pheophytinization. This color loss is a
result of the conversion of natural chlorophylls to Mg2+-free
derivatives such as pheophytins and pyropheophytins
[23-25]. Chlorophyllide derivatives can also be formed
 M.G. Ferruzzi, J. Blakeslee / Nutrition Research 27 (2007) 1–12 3

Fig. 2. Major chlorophyll degradation and derivatization reactions occurring during food processing operations. Exposure to severe heat and/or acidic
conditions, as is common in canning operations, results in loss of the central Mg2+ metal generating olive brown pheophytin and pyropheophytin pigments.
Inclusion of divalent metals such as Zn2+ results in bregreeningQ by generation of Zn-pheophytin and Zn-pyropheophytins from respective metal-free
derivatives. Chlorophyllase activity, induced by blanching operations, results in the formation of water-soluble chlorophyllides that further degrade through
thermal processing and/or acidification to pheophorbide pigments. Commercial-grade SCC is a mixture of Cu-chlorin derivatives prepared by saponification of
chlorophyll with NaOH followed by replacement of the Mg2+ atom by Cu2+.

through mild thermal processing because an increase in
endogenous chlorophyllase enzyme activity occurs from the
mild heating of plant tissues [26]. In a process known as
regreening, metallochlorophyll complexes of pheophytin a
and pyropheophytin a are rapidly formed by the addition of
divalent metal salts of zinc (Zn2+) and copper (Cu2+) to
thermally processed vegetables before thermal treatment
[27-31]. Both Cu2+ and Zn2+ complexes of chlorophylls
have been shown to be significantly more stable to food
processing conditions than their native counterparts [32] and
have been used commercially both in the canning of green
beans to improve the color of the finished product and in the
generation of green color additives [27,33].
Commercial-grade water-soluble chlorophyll derivatives
known as chlorins are used as food-grade colorants.
Although available chelated to various transition metals
(Zn, Fe, Co, Cu) for distinct color and stability, the most
common form is SCC. SCC is synthesized from a crude
natural chlorophyll extract by treatment with methanolic
sodium hydroxide followed by replacement of the central
Mg atom with Cu [7]. The final product consists of a mixture
of numerous chlorin-type compounds derived from natural
chlorophyll, the primary components of which are referred to
as Cu-chlorin e4 and Cu-chlorin e6 (Fig. 2) and sometimes
minor amounts of Cu-chlorin e4 ethyl ester [34-38]. With
thermal sensitivity similar to that of other water-soluble
chlorophyll derivatives [39], SCC has seen common
utilization as a food-grade colorant in Europe, Asia, and, to
a more limited but growing extent, the United States.
3. Digestive behavior and bioavailability of
chlorophyll derivatives
Bioavailability refers to the fraction of any compound
ingested and made available for utilization, metabolism, and/
or storage by the organism. Information on the bioavailability
of chlorophyll derivatives is limited partially because of the
general assumption that chlorophyll is unabsorbable by
humans. However, considering the natural abundance of
chlorophyll in fruit and vegetable tissues, the diversity of
derivatives formed through food processing and preparation,
and the growing use of commercial-grade chlorophyll
4 M.G. Ferruzzi, J. Blakeslee / Nutrition Research 27 (2007) 1–12

Fig. 3. Schematic of potential digestive behavior and absorptive routes for (A) chlorophyll and (B) SCC derivatives. Chlorophyll derivatives: After release from
the plant food matrix, natural chlorophyll (CHL) derivatives are exposed to the acidity of the gastric digestion resulting in conversion to respective metal-free
pheophytins (PHE). Within the upper small intestine, PHE derivatives partition into bile salt lipid micelles resulting in solubilization of PHE and movement
across the unstirred water layer and ultimately into the enterocyte presumably by passive diffusion [49]. The extent to which natural chlorophyll derivatives are
metabolized and/or secreted into circulation is currently unknown. Chlorophyllin: After digestive release of water-soluble SCC derivatives, significant
degradation of Cu-chlorin e6 derivatives occurs, leaving primarily Cu-chlorin e4 and potentially residual amounts of Cu-chlorin e4 ethyl ester [50]. Uptake of
SCC derivatives by intestinal cells seems to proceed predominantly by a facilitated process, with absorbed SCC derivatives efficiently effluxed back to the
luminal compartment [50] by unidentified efflux system (potentially BCRP/ABCG2 [55-57]). A portion of the internalized SCC derivatives is passed from the
enterocyte into circulation as noted by Egner et al [38]. The extent to which SCC derivatives are metabolized and the extent to which the metabolites may be
transported out of the enterocyte are currently unknown.

derivatives such as SCC, dietary exposure to these pigments
can be significant. In this context, even minimal absorption
of chlorophyll derivatives may prove to be physiologically
significant and thus warrants investigation.
Although information on the absorption of natural
chlorophyll derivatives is limited, data on the absorption
and distribution of water-soluble derivatives such as SCC
are available. Henderson and Long [40] orally administered
natural chlorophyll and SCC to rats and discovered
uncharacterized derivatives dispersed throughout the
liver, lymph nodes, and spleen. Harrison et al [41] followed
the plasma levels of rats fed diets of SCC as high as 3% and
noted plasma levels reaching up to 116 lg SCC per
milliliter of plasma. Rapid distribution of SCC in the heart,
liver, skin, kidneys, and lungs was noted in ICR mice
administered 15 mg/kg body weight by gavage [42].
 M.G. Ferruzzi, J. Blakeslee / Nutrition Research 27 (2007) 1–12
Recently, Egner et al [38] provided concrete evidence
that SCC components are in fact absorbable by humans. As
part of a large chemoprevention trial initiated in 1997 in
the People’s Republic of China [16], subjects consumed
300 mg SCC per day (primarily composed of Cu-chlorin e4,
Cu-chlorin e6, and Cu-chlorin e4 ethyl ester). After 4 months
of consumption, it was determined that patients attained a
steady state of ~2.0 lg/mL total SCC in serum predomi-
nantly as a Cu-chlorin e4 ethyl ester, with only minor
amount of Cu-chlorin e4 and no detectable levels of
Cu-chlorin e6. These results indicate that SCC components
are bioavailable but selectively, as specific SCC components
did not elicit a plasma response.
With limited but growing evidence that select chloro-
phyll derivatives may be absorbable in humans, efforts to
elucidate factors impacting intestinal uptake and potential
bioavailability have been initiated. Considering that chloro-
phyll derivatives span a broad range of polarity from
lipophilic (natural chlorophylls) to highly water-soluble
(SCC) and have variable sensitivities to pH, individual
differences in digestive behavior and routes of absorption
are likely. It is likely that the general mechanism of
chlorophyll absorption would follow routes similar to those
taken by other xenobiotic compounds that require consid-
eration of (a) efficient release of the chlorophyll from the
food matrix, (b) stability to gastric and small intestinal
digestive conditions, (c) solubilization of lipophilic deriv-
atives, (d) uptake by small intestinal absorptive epithelial
cells, and (e) secretion into circulation (Fig. 3). Co-
consumption of lipid with lipophilic chlorophyll–rich foods
would likely be a requirement for absorption via formation
and association with mixed micelles as is common for
dietary lipids and lipid-soluble compounds in vivo [43-45].
The solubility of water-soluble chlorophyll derivatives,
including chlorins, chlorophyllides, and pheophorbides,
would likely not require co-consumed lipid for micellariza-
tion or secretion; and absorption of these compounds is thus
limited only by release from food matrix, digestive stability,
and intestinal uptake/transport.
Experimental data to support this general outline of
chlorophyll digestive behavior and absorption have been
focused on in vitro assessments and limited in vivo studies.
The sensitivity of chlorophyll to acidic conditions makes the
gastric environment a logical candidate for significant
chlorophyll modification. Early investigations provided
insight into chlorophyll digestive instability by identifying
decomposition products of both natural and commercial-
grade derivatives in urine and feces. In general, Mg2+-free
derivatives (including pheophytins and pyropheophytins),
attributed to gastric acidity, were isolated as a predominant
end-product in human feces [46,47]. Baxter [48] found little
evidence of subsequent pheophytin hydrolysis to water-
soluble pheophorbide and release of phytol by colonic
microflora in humans fed C14-labeled pheophytin or
spinach, leading to the conclusion that the major digestive
end-products of chlorophyll components were pheophytins.
5
More recent examination of the digestive behavior of
natural chlorophyll in vitro has characterized formation of
pheophytins and pheophytin epimers as the primary small
intestinal derivatives of natural chlorophyll from spinach
purees [49]. In the same study, stability of Zn-pheophytin a
and b to simulated gastric and small intestinal conditions
was observed, demonstrating that specific metallochloro-
phyll derivatives stable to food processing conditions may
also survive the acidity of the gastric environment and be
available for absorption or interaction in the intestinal tract.
In vitro experiments with SCC found that, although
Cu-chlorin e4 demonstrated excellent digestive stability,
Cu-chlorin e6, representing ~10% of the total SCC used in
the study, was digestively labile, with approximately 90%
loss from an aqueous solution of SCC (simulating con-
sumption of a supplemental dose) [50]. The sensitivity of
Cu-chlorin e6 to digestive conditions may be responsible, in
part, for the lack of serum response in humans consuming
SCC [38]. From these data, it may be said that digestive
sensitivity of natural and commercial chlorophyll deriva-
tives allows for the creation of a diverse array of digestive
end-products but more critically may serve to limit
bioavailability of sensitive derivatives common in foods
and dietary supplements.
After exposure and degradation in the digestive environ-
ment, association of lipophilic chlorophyll derivatives
(pheophytins and Zn-pheophytins) with bile salt lipid
micelles has been noted in vitro. Although differences have
been noted between chlorophyll a and b derivatives, the
overall extent of micellarization (10%-40%) [49] was
similar to that of other lipid-soluble phytochemicals
including carotenoids [51-53]. In similar in vitro experi-
ments, more water-soluble SCC derivatives did not require
association with bile salt micelles for solubilization and
subsequent intestinal cell uptake [50].
Solubilization of chlorophyll derivatives by virtue of the
hydrophilicity and/or association with bile salt lipid micelles
is required for subsequent uptake by the intestinal lumen. In
cell culture, uptake of micellarized lipophilic chlorophyll
derivatives by human intestinal Caco-2 cells was moderately
efficient, with 5% to 10% of chlorophyll derivatives from
digested spinach purees absorbed by human intestinal cells.
Although uptake seemed relatively inefficient, resulting
intracellular levels were similar to those of carotenoids
(similarly found in spinach) because of the large excess of
chlorophyll in purees [49]. These data again serve to
illustrate that even a small absorption of chlorophyll
derivatives may result in significant intracellular concen-
trations in target tissues such as the gut. In contrast to
lipophilic chlorophyll derivatives, uptake of water-soluble
SCC derivatives has been shown to be very efficient, with
almost 45% to 60% of the SCC internalized by the intestinal
cells in culture. However, retention of SCC derivatives by
Caco-2 cells was found to be very poor, with efficient apical
efflux of Cu-chlorin e4 and e6 observed [50]. There are
several well-established lines of evidence that pheophorbide
6 M.G. Ferruzzi, J. Blakeslee / Nutrition Research 27 (2007) 1–12
a, pyropheophorbide a, and chlorin e6, components similar
to those in SCC, serve as substrates for ABC transporters
facilitating apical efflux. Specifically, BCRP/ABCG2 trans-
porters have demonstrated the ability to transport chloro-
phyll derivative in multiple cell culture lines [54-56] and an
animal model [57], indicating that these transport proteins
may play a pivotal role in limiting/controlling the bioavail-
ability of chlorophyll derivatives in vivo. ABC-transporter–
mediated efflux of chlorin-type compounds may serve to
control the bioavailability of porphyrin derivatives and thus
limit the potential for phototoxicity associated with these
compounds [57,58]. The extent to which these transport
systems may limit uptake and distribution of absorbable
chlorophyll derivatives, such as SCC, to target tissues is
unknown and warrants further investigation.
4. Chlorophyll and cancer prevention
The association of diets rich in phytochemicals with the
prevention of cancer has intensified interest in chlorophyll
as a class of plant pigments with potential chemopreventa-
tive effects. Considering the traditional use of chlorophyll
derivatives in medicine [7] and the low toxicity [41], these
pigments serve as attractive cancer preventative and
potentially therapeutic agents. Research highlighting the
bioactivity of chlorophyll in vitro and in vivo as well as
potential mechanisms of cancer preventative actions is
reviewed in the following sections.
4.1. Cancer preventative activity in vitro
Cancer preventative effects of chlorophyll derivatives
have been extensively studied, with particular emphasis on
their in vitro antimutagenic activity against numerous
dietary and environmental mutagens. A number of these
studies have used bacterial mutagenicity assays focusing on
commercially derived SCC as previously reviewed [59-63].
In general, SCC has demonstrated the ability, in vitro, to
effectively protect against mutagenic activity of both direct-
and indirect-acting dietary and environmental mutagens
including the following: heterocyclic amines 3-amino-1-
methyl-5H-pyrido-[4,3-b]indole and 2-amino-3-methylimi-
dazo[4,5-f]quinoline (IQ) [64], chromium (VI) oxide [65],
aflatoxin B1 [64], benzo[a]pyrene [66-68], benzo[a]pyrene-
7,8-dihydrodiol-9-10-epoxide [69], 3-methylchloroanthra-
cene [37], and 7,12-dimethylbenzo[a]anthracene [70].
Natural chlorophyll derivatives have also been investi-
gated with regard to antimutagenic/antigenotoxic activity.
Lai et al [71] found a positive correlation between the
antimutagenic activity of common vegetable extracts and
their chlorophyll content. Natural chlorophylls have dem-
onstrated antigenotoxic and antimutagenic activity in
bacterial, Drosophila wing spot, and mammalian cell–based
assays [72-75]. Metal-free chlorophyll derivatives such as
pheophytins and pheophorbides have also exhibited signif-
icant suppressive activity in Salmonella typhimurium and
Escherichia coli tester bacteria [76-78]. Although the
presence of a transition metal in the tetrapyrrole macrocycle
has been postulated to alter potential antimutagenic activity
[79], reduction in the mutagenic activity of benzo[a]pyrene
by metal-free chlorophyll derivatives was found to be
similar to that of other metalloporphyrins including Mg2+,
Zn2+, and Cu2+ derivatives [68].
4.2. Cancer preventative activity in vivo
In vitro antimutagenicity data on SCC and, to a more
limited extent, with natural chlorophylls provided strong
justification for subsequent investigation of potential cancer
preventative activities in vivo. As with in vitro results, the
primary focus has been on water-soluble SCC, with more
limited work on natural chlorophyll derivatives. Animal mo-
dels used include mice, rats, and rainbow trout, targeting skin
cancer, liver cancer, and, to a limited extent, colorectal cancer.
Dietary SCC was found to modulate aflatoxin B1–
induced hepatocarcinogenesis in rainbow trout by retarding
aflatoxin-DNA binding [64,80]. SCC consumption signifi-
cantly reduced the average number of liver neoplasms and
proliferation but not the overall liver cell cancer incidents in
a diethylnitroamine-phenobarbital hepatocarcinogenesis
model [81]. Harttig and Bailey [82] reported that natural
chlorophylls from spinach inhibited dibenzo[a,l]pyrene-
DNA adduct formation in rainbow trout liver and that
chlorophyll a protected against DNA adduct formation
when trout embryos were coinjected with both chlorophyll
derivative and tritium-labeled aflatoxin B1 [83]. However,
SCC provided better protection against adduct formation,
suggesting that the potential for better distribution of water-
soluble derivatives may improve efficacy.
Water-soluble chlorophyll derivatives have been moder-
ately effective in animal models of skin cancer. SCC
reduced benzo[a]pyrene- and benzo[a]pyrene-7,8-dihydro-
diol-9,10-epoxide–induced skin carcinogenesis in ICR mice
[42]. Inhibitory effects of pheophorbide a on skin tumor
promotion were further demonstrated in ICR mice [84].
Chung et al [70] found that orally administered SCC
inhibited both tumor promotion and progression of papil-
lomagenesis in 7,12-dimethylbenz[a]anthracene– and 12-O-
tetradecanoyl-phorbol-13-acetate–induced mouse skin car-
cinogenesis. Higashi-Okai et al [85] further demonstrated
that magnesium-free chlorophyll derivatives pheophytin a
and b significantly suppressed tumor promotion and
progression of skin tumorigenesis in a BALB/c mouse
skin model.
Recently, epidemiological evidence has linked diets high
in chlorophyll with a reduced risk of colon cancer in humans
[4]. However, dietary SCC was found to act as a promoter of
colon carcinogenesis in rats treated with dimethylhydrazine
[86]. Interestingly, SCC was found to have both promotional
and suppressive activity (correlating with low and high
doses, respectively) on postinitiation IQ– and 1,2-dimeth-
ylhydrazine (DMH)–induced colon tumors in rats contain-
ing mutations in b-catenin. In the same report, natural
chlorophyll but not chlorophyllin suppressed, postinitiation,
 M.G. Ferruzzi, J. Blakeslee / Nutrition Research 27 (2007) 1–12
azoxymethane- and IQ-induced aberrant crypt foci in rats
[87]. Similarly, de Vogel et al [88,89] found that dietary
chlorophyll from green vegetables, but not water-soluble
SCC, is able to limit heme-induced hyperproliferation of rat
colonocytes in vivo. Combined, these results highlight
potential differences in activity of natural versus commercial
derivatives of chlorophyll and underscore the importance of
understanding the specific chlorophyll forms and dose used
in the diet as well as those present through the extent of
the gastrointestinal (GI) tract and at specific target tissues.
Positive data from in vitro and animal studies have
prompted an increased interest in the potential usefulness of
chlorophylls as preventative agents for humans at elevated
risk for development of specific cancers. The minimal
toxicity of chlorophyll and chlorophyllin, combined with
the strong and mechanistic evidence of the ability of
chlorophyllin to act as an interceptor molecule reducing
the bioavailability of dietary mutagens (see Section 5), led
to the planning and execution of a randomized, double-
blind, placebo-controlled trial in Qidong, People’s Republic
of China [16]. Residents of Qidong are at an elevated risk
for hepatocarcinoma cancer because of long-term exposure
to aflatoxin B1. In this study, participants consumed
3 supplements containing 100 mg chlorophyllin daily
during a 4-month trial. Results indicated a 55% reduction
in urinary levels of aflatoxin-N7-guanine [17]. These results
support the notion that chlorophyllin intervention may
reduce the bioavailability and/or activity of ingested
aflatoxin B1 in humans. Although encouraging, the long-
term use of supplemental forms may be challenged by issues
of compliance associated with adherence to supplement use
Fig. 4. Schematic of major cancer preventative activities associated with
dietary chlorophyll derivatives.
7
over a lifetime. Extension of such interventions to natural
chlorophyll has the potential for broader dietary application
with real foods. The abundance of natural chlorophyll in
green fruits and vegetables would also provide a diversity of
ways to consume chlorophyll in a manner perhaps
complimentary to supplemental strategies.
5. Potential mechanism of chlorophyll cancer
preventative activity
Several studies have been conducted to elucidate specific
mechanisms responsible for the observed cancer preventa-
tive activity of chlorophyll derivatives. Focused on in vitro
and cell culture–based assays, these efforts have primarily
highlighted antioxidant activity, mutagen trapping, modula-
tion of detoxification pathways, and induction of apoptosis
as modes of actions responsible, in part, for chlorophyll’s
protective effects in vivo (Fig. 4).
5.1. Antioxidant activity
Although chlorophyll derivatives are normally thought of
as pro-oxidants because of their participation in photo-
oxidation of fats and oils [90], both chlorophylls and
pheophytins have demonstrated protective activity against
auto-oxidation of vegetable oils in the dark [91,92]. Endo et
al [93] suggested that chlorophyll derivatives may be acting
as electron donors as evidenced by their ability to reduce
free radicals such as 1,1,diphenyl-2-picrylhydrazyl. Chloro-
phyll a was shown to act synergistically with vitamin E by
quenching tocopherol radicals, thereby enhancing the
observed antioxidant effects of vitamin E [94,95].
Commercial-grade SCC has also demonstrated apprecia-
ble antioxidant activity in vivo and ex vivo. Concentration-
dependent inhibition of lipid peroxidation was noted in rat
liver microsomes [96], whereas intraperitoneal and intrave-
nous administration of SCC protected mitochondria and
microsome lipid peroxidation and carbon tetrachloride–
induced injury in rat livers [97,98]. Cu-chlorin e4 was
subsequently identified as a major antioxidative component
in SCC [99].
Cahyana et al [100,101] and Hoshina et al [102]
demonstrated a clear structural relationship within porphyr-
ins for inhibition of lipid hydroperoxide formation. Both the
porphyrin structure as well as the presence and nature of the
central metal are considered important to antioxidant
activity of chlorophyll derivatives. Nickel, copper, and
magnesium derivatives suppressed the oxidation of linoleic
acid by superoxide anion, whereas the iron derivatives
enhanced oxidation [103]. Natural chlorophyll a and b were
found to have significantly lower antioxidant activity than
SCC derivatives (Cu-chlorin e4 and Cu-chlorin e6) in radical
scavenging assays. Furthermore, metallochlorophyll deriv-
atives, including Zn- and Cu-pheophytins, exhibited signif-
icantly higher antioxidant capacity than metal-free
derivatives, indicating that the nature of chlorophyll
derivatives present in fresh and processed fruits and
8 M.G. Ferruzzi, J. Blakeslee / Nutrition Research 27 (2007) 1–12
vegetables is important when considering the antioxidant
capacity of these foods [68].
5.2. Mutagen trapping
Sequestration of dietary mutagens and carcinogens by
interceptor molecules is a concept that applies to numerous
compounds including chlorophylls [104]. Although bio-
availability is often considered critical to ultimate physio-
logical relevance, for chlorophyll, it is important to
remember that derivatives that are poorly absorbed may
also be physiologically significant by virtue of their ability
to interact with other compounds in the GI tract. Binding to
dietary mutagens and carcinogens to reduce overall bio-
availability of these toxicants in vivo represents an
alternative mechanism for nonabsorbable chlorophyll deriv-
atives to influence disease risk and outcomes. In vitro
complex formation between SCC and hydrophobic polycy-
clic planar mutagens was first reported by Arimoto et al
[105]. Arimoto-Kobayashi et al [106] further illustrated the
binding of planar mutagens with either 2 or 3 fused rings to
a chlorophyllin-chitosan insoluble complex.
Limiting uptake and transport of several dietary and
environmental mutagens including aflatoxin B1, PhIP, and
dibenzo[a,l]pyrene by chlorophyll in a Caco-2 human
intestinal cells model demonstrated the ability of these
pigments to influence bioaccessibility of carcinogens by
complex formation [107,108]. Animal models have also
demonstrated that formation of chlorophyllin-aflatoxin
complex contributes to a reduction in systemic aflatoxin
bioavailability and thus relative mutagenicity/carcinogenic-
ity [109,110]. Purified chlorophyll derivatives do not seem
to be required because coadministration of Chlorella with
polychlorinated dibenzo-p-dioxin and dibenzofuran retarded
GI absorption and accelerated excretion of these industrial
contaminants in Wistar rats [111].
The nature of mutagen-chlorophyll complexes has been
described as a reversible planar binding of overlapping ring
systems [83] and is dependent on the nature of both porphyrin
structure and mutagen. In the case of SCC complexation, this
may potentially involve both electrostatic attraction between
the porphyrin carboxyl groups with charged moieties such as
exocyclic amines [112] and van der Walls interactions
[113,114], with interactions with specific mutagens such as
aflatoxin B1 being extremely energetically favorable [83].
The presence of a transition metal in the porphyrin macro-
cycle has been postulated to alter potential antimutagenic
activity by alteration of the specific porphyrin:mutagen
complex [79,114]. However, this is likely mutagen-depen-
dent; and the presence of an intact porphyrin macrocycle is
believed to be most essential for effective complex formation
and antimutagenic behavior [68,79].
5.3. Regulation of detoxification pathways
Consideration has also been given to the possibility that
chlorophyll derivatives modulate endogenous xenobiotic
detoxification systems. Singh et al [115,116] demonstrated
significant elevation of glutathione-s-transferase activity
after both topical and gavage administration of SCC to
murine and suckling neonate mice, whereas phase I
cytochrome (CYP) P450 activity remained unchanged.
Nonspecific inhibition of various CYP P450s by chloro-
phyllin has also been observed in human liver microsomes
[117]. Sodium copper chlorophyllin minimized bioactiva-
tion of precarcinogens and accelerated excretion of ultimate
carcinogens by depression of CYP activity and stimulation
of phase II conjugation pathways. More recent mechanistic
studies with chlorophyll, chlorophyll-rich extracts, and SCC
demonstrated significant induction of the phase II enzyme
NAD(P)H:quinone oxidoreductase 1 (NQO1) in murine
hepatoma cells [118,119]. Induction of NQO1 was signif-
icantly higher for SCC derivatives than for chlorophyll
derivatives. Furthermore, the SCC component Cu-chlorin e4
seems to be a bifunctional inducer of NQO1 (requiring
activation by phase I enzymes), whose action is related to
triggering of the gene expression of the antioxidant response
element [119]. Although in vivo evidence has been limited
to date, modest but significant induction of hepatic NQRO1
in vivo (F344 rats) by chlorophyllin (10g/kg body weight)
has been also demonstrated [120].
5.4. Induction of apoptosis in cancer cells
More recently, evidence that chlorophyll derivatives may
function to induce apoptosis in transformed and cancerous
cells has provided another mechanism by which the
pigments may exert preventative effects. Sodium copper
chlorophyllin has been found to induce apoptosis in human
colon cancer cells (HCT116) by a mechanism likely
involving the activation of the caspase-8/caspase-3/poly(-
ADP-ribose) polymerase pathway resulting in nuclear
condensation [121]. Chan et al [122] also reported the
induction of apoptosis by water-soluble pheophorbide a in
human hepatocellular carcinoma cells (Hep3B) but rather
through a cytochrome c–mediated pathway involving
activation of caspase-3/caspase-9. Activation of mitogen-
activated protein kinases such as ERK in human breast
cancer cells (MCF-7) was observed to be decreased by SCC
via inhibition of protein activation rather than expression
leading to apoptotic events [123]. Although further research
is required to understand in vivo implications and potential
mechanistic differences between natural and commercial-
grade chlorophyll derivatives, this area represents another
potential avenue for chlorophyll-rich fruits and vegetables to
exert protective activities.
6. Conclusions and future directions
Chlorophylls belong to a larger class of phytochemical
plant pigments implicated as cancer preventative agents.
Investigations highlighting specific derivatives (chloro-
phylls, pheophorbides, pheophytins, and chlorins), prepara-
tions (vegetables, vegetable extracts, and SCC), and
protective actions have been reported in the literature.
 M.G. Ferruzzi, J. Blakeslee / Nutrition Research 27 (2007) 1–12
Following the common assumption that chlorophyll is
unabsorbable by humans and the strong data to indicate
antimutagenic activity in vitro, a central hypothesis has been
formed around the notion that mutagen trapping in the GI
tract is the primary mode of action by which chlorophyll and
its derivatives deliver cancer preventative activity. Acting as
an interceptor molecule that reduces systemic bioavailability
of ingested carcinogens, the commercial-grade chlorophyll
derivative SCC has demonstrated promising results in human
clinical interventions [16,17]. However, after identification
of SCC components in plasma of humans under SCC
supplementation [38] and data suggesting that natural
chlorophyll derivatives are absorbable by human intestinal
cells [49,50], consideration of the extent to which chlorophyll
may deliver more systemic physiological effects consistent
with the prevention of cancer has become important.
Promising data on the ability of chlorophyll to modulate
xenobiotic metabolizing enzymes [115-120] and induce
apoptosis in cancerous cells [121-123] hinge on the ability
of chlorophyll and its derivatives to be present and potentially
absorbable at target tissues including those of the GI tract and
beyond. Further complicating this picture is the complexity of
chlorophyll derivatization induced by food processing,
digestion, and analytical handling (not covered in this
review). Understanding the nature of specific chlorophyll
derivatives present is a challenge that can potentially
confound specific investigation with whole food systems.
Whereas food processing effects are well established, a
more complete understanding of the chlorophyll derivatiza-
tion through digestion, the degree to which chlorophyll
derivatives are absorbable, and the absorption and formation
of any specific chlorophyll metabolites is absolutely required
to further our understanding of the role these pigments may
play in the prevention of chronic disease. Although efforts
have begun, many questions remain, including if natural
chlorophyll derivatives are absorbable intact at all. Informa-
tion such as this is critical and will facilitate the design of
future research on chlorophyll targeting specific forms and
metabolites that represent biologically relevant derivatives.
In this regard, it is important that future studies expand
beyond in vitro and cell culture models to relevant in vivo
models addressing absorption and distribution of chlorophyll
derivatives. With only limited information available from
SCC derivatives, it becomes important to consider that even
low absolute bioavailability of natural chlorophyll deriva-
tives from green vegetables would be significant considering
the high concentration of these pigments in the plant tissues.
Based on recent epidemiological evidence linking
chlorophyll consumption to a decreased risk of colorectal
cancer [4] and the high concentration of these pigments in
the GI tract, the intestinal epithelia should be highlighted as
a logical target tissue to further explore the relationship
between chlorophyll and cancer prevention. Investigation of
chlorophyll uptake and accumulation by these tissues would
serve to expand our understanding of the role these
pigments may play in the prevention of GI tract cancers.
9
Finally, understanding uptake and metabolism of chloro-
phyll by intestinal tissues would also begin to address other
physiological implications of chlorophyll absorption includ-
ing potential interplay with structurally homologous por-
phyrin derivatives such as heme and cytochromes.
Acknowledgment
The authors thank Rodney Green for assistance in
preparation of this manuscript.
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