Plant Extracts As Modulators of Genotoxic Effects

THE BOTANICAL REVIEW
VOL. 62 OCTOBER-DECEMBER
1996 NO. 4
Plant Extracts as Modulators of Genotoxic Effects
DEBISRI SARKAR, ARCHANA SHARMA
Centre of Advanced Study in Cell and Chromosome Research,

Department of Botany,
University of Calcutta,
35, Ballygunge Circular Road
Calcutta 700 019,
India
AND
GEETA TALUKDER
Vivekananda Institute of Medical Sciences,

Calcutta 700 020,
India
I. Abstract/Zusammenforschung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
II. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
III. Plant Extracts as Inhibitors of Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
A. Extracts from Vegetables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
B. Extracts from Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
C. Extracts from Underground Storage Organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
D. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
IV. Mechanism of Antimutagenic Activity of Plant Extracts . . . . . . . . . . . . . . . . . . . . . . 283
A. Certain Specific Factors Observed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
B. Steps Involved in Protecting against Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . 285
V. Plant Extracts as Inhibitors of Clastogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
VI. Mechanism of Anticlastogenic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
VII. Plant Extracts as Genotoxic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
A. As Mutagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
B. As Promutagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
C. As Cytotoxic and Clastogenic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
D. Synergistic and Antagonistic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
Copies of this issue [62(4)] may be purchased from the Scientific

Publications Department, The New York Botanical Garden, Bronx,
NY 10458-5125 USA. Please inquire as to prices.
TheBotanicalReview62(4): 275-300, October-December1996

9 1996The New YorkBotanicalGarden 275
276 THE BOTANICALREVIEW
VIII. Plant Products as Modulators of Mutagenesis in Traditional Systems of Medicine. 292

A. Indian. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
B. Chinese. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
IX. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
X. Literature Cited . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
I. A b s t r a c t
Higher plants used extensively in traditional medicines are increasingly being screened
for their role in modulating the activity of environmental genotoxicants. The property of
preventing carcinogenesis has been reported in many plant extracts. The observation of a
close association between carcinogenesis and mutagenesis has extended the survey to
include plant extracts and plant products able to modify the process of mutagenesis, which
involves alteration in the genetic material. Natural plant products may, apart from inducing
mutations, modify the action of other known mutagens on the living organisms by 1)
activating the existing mutagens within the cell, 2) inhibiting the production of mutagens
in the cell, 3) synergising the activity of existing mutagens, or 4) activating the promu-
tagens within the cell into mutagens. This review deals with data obtained in the course
of research on the modulatory effects of plant extracts on mutagenesis and clastogenesis,
two genotoxic phenomena associated with carcinogenesis.
In screening for antimutagenic effects, extracts of different plant parts have been used,
ranging from leafy vegetables, fruits, and underground storage organs to whole plants.
The extracts were prepared mainly in water or organic solvents. Several of these assays
have indicated the involvement of certain factors that are intrinsic components of the
extracts, ranging from specific compounds like ascorbic acid to vegetable fibres which
could act as nonspecific redox agents, free radical scavengers, or ligands for binding
metals or toxic principles. The possible ways in which inhibitors of mutagenesis can act
include the inhibition of interaction between genes and biochemically reactive mutagens
and the inhibition of metabolic activation of indirectly acting mutagens.
The effects of toxicants can be observed at the level of chromosomes (clastogenesis)
through alterations in chromosome structure (chromosomal aberrations) and number
(aneuploidy, polyploidy). A wide range of short-term and long-term screening procedures
is available. The most common ones use higher plants or rodents in vivo as test systems
for monitoring chromosomal aberrations. Experiments with a number of crude vegetable
and fruit extracts have demonstrated their anticlastogenic activities against known cyto-
toxic agents. The individual components of the extractsme.g., sulfhydryl and flavonoid
compounds, gallic acid, ellagic acid, mucic acid, citric acid, reducing sugars, tannin--are
observed to have an additive interaction with the major constituents chlorophyll and
ascorbic acid, when modulating the effects of the clastogens.
Under certain conditions, plant products may induce mutagenic effects, due to the
presence of multiple biological properties. Some inhibitors can stimulate simultaneously
both enhancing and detoxifying mechanisms, e.g., inducers of coordinated enzyme
activities. Many oxidants can, depending on the redox potential, either accept or donate
electrons, rendering them protective or harmful. Plants also play an active role in the
accumulation, metabolism, and environmental distribution of xenobiotics. The property
of plants to activate promutagens that may enter the food chain is of great significance in
view of the large number and types of chemicals to which the plants are exposed. A
promutagen is a chemical that is not mutagenic itself but that can be biologically
PLANT EXTRACTS 277
transformed by a plant system into a mutagen. Several methods for studying promutagens
from plants were developed both in vivo and in vitro, including plant cell-free systems.
Both mutagens and antimutagens can be extracted from the same plant extract depending
on the nature of solvents used for extraction. Interaction between inhibitors may lead to
synergistic effects. Such combined action may take place through the different inhibitors
acting at different levels or being localised at different cellular areas. The greater protection
afforded by crude plant extract as compared with an equivalent amount of the purified or
synthetic ingredients, as observed with Phyllanthus emblica L. and Beta vulgaris L. var.
benghalensis Hort., may be related to this phenomenon.
Specific biological action of a drug is due to its specific binding to a functional
molecular receptor. In complex plant extracts, the variable observed effects can be
attributed to the many chemically reactive species that are formed during the processing
and ingestion of the extract, which could act as non-specific redox agents, scavengers of
free radicals, and ligands for binding to toxicants. The final effects are obviously the
outcome of interactions between the components and their individual and collective
interaction with the toxicant. The specificity and efficacy of such responses will be
influenced also by the physiological factors influencing the plants and the process of
administration of the extract.
In utilizing pharmacologically active herbs, both beneficial and potential adverse
effects must be taken into account. The actual dose and form of the plant also need to be
worked out.
Zusammenforschung
Die Pflanze den hoeher Gruppen, die so lange in traditionale Medizine bentitzen waren,
sind jetzt, fuer ihre Rolle in der Veraenderung der Aktivitaet der environmental
Genotoxicants, ausprobiert. Es ist schok berichtet dass viele Pflanze-extrakt einiqe
Eigenschaften haben damit die Carcinogenesis verhuettet ist. Es ist bemerkt dass eine
tiefe verbinding Zwieschen Carcinogenesis und Mutagenesis existrert und diese
Bemerkung fuehrt zu einer weileren Forschung zur Pflanze-extrakt und pflanze-produkte,
die in dem Modifikation-prozess von Mutagenesis helfen Dieser Modifikation prozess its
moeglich mit Aenderung den Geneticmaterial. Die Naturpflanzprodukte, zusaetzlich zur
Mutation induktion koenner auch die Effekten den andere Mutagens den existierten
organismus, mit folgenden Methoden veraendern. 1) Die existierene Mutagens zwischen
die Cell sind leblos gemacht. 2) Die weitere Produktion von mutagens zwischen die Cell
sind verboten. 3) Die Aktivitat von den existierene mutagens sind erhoehen. 4) Die
Promutagens inzwischen die Cell sind effective gemacht.
Diese Untersuchung handelt sich un die Information, die durch die aktuella Experiment
notieren, iiber Veraenderung effekter von Pflanze extraktion auf Mutagenisis and
Clastogenesis. Z. 13. Zwei Genotoxic phenomenon zusammen mit Carcinogenesis.
In dem Ausprobierenprozess fuer Antimutagenic Effekte, sind verschiedene Pflanze-
extrakten Z. 13. belaubte Gemuesse, fruechte, unterirdische gelagerte Pflanz organs
benutzt worden. Die Extrakte waren meisten mit Wasser oder Organicsolvent dargestellt.
Meherere von dieser Untersuchung haben bezeichnet dass die innerwirkliche Teilen von
dieser Extrakten eine Rolle in dieser Aussprobieren prozess gespielt haben. Dieser
Extrakten sind die spezifische Gemische Z. 13. Ascorbic acids und Gemtisse fibers. Dieser
spezifisch Gemische koennen als ein Non-spezifisch Redox Agents, frei Radical
skavangers oder als eine typische Metals. die eine klebrige Eigenschaft haber. Oder als
ein Toxic Prinziple Moeglicherweise koennen weitere Mutagens Produktion mit

folgenden Methoden verhindert werden. The inhibition of interaction between genes and
biochemically reactive mutagens and the inhibition of metabolic activation of indirectly
acting mutagens.
Durch veraenderung in Chromosome structure und Chromosome Nummer ist die
Effekt von Toxicants auf die Chromosome Quantitat ist notiert.
Es gibt ein klein-Zeitdauer und der Verchiedenen Chemisch in der. Umbegung wo die
Pflanzen sind. Die Pflanzen eine besundere Eigenschaften haben, danif die "Promutagens",
die in der Speise eintreten koennen, angespornen sind. Promutagen ist chemish die nicht
eigentlich mutagenic ist aber das Pflanzesystem diese Promutagen zu Mutagen biolo-
gisch umformen kann. Viele Methode, denif die Promutagens chalakteur, von Pflanze
studieren koennen sind schon esturckelt.
Solvent charakteur, der fuer den. Extraktionprozess besmotzt ist, bestimmt, ob
Extraktion von beide Mutagens und Antimutagens von gleichem Pflanze extrakt moeglich
irb.
Weehselceirkung zurischen Inhibitors kann zu Anspornung eftekb resultieren. Diese
Vereinigtro periation koennen, durech verschiedene Inhibitors operieren nerschieder teif
oder oertlich in verschiederen cells, stattfinden. Als Aequivalent quantitaet den raffinierenen
Bestandteil, koennen die rohe Pflanze extrakt lesser Beschmustung leirten. Observetion mit
Phyllanthus emblica und Beta vulgaris vat. benghalensis Hort. hab ein Verhaeltnis mit
diesem Phaenomen.
Einer spezifischer Einband zu einern Functional Molecular Auffang bestimut die
spezifische biologische operation von einer lang Zeitdamer Ausprobieren prozess. Der
normeler Ausprobieren prozess benutzt hoeher gruppe Pflanze oder Rodents in vivo als.
Test systems um die Chromosome Veraenderung zu notieren. Viele Erprobungen mit. rohe
Gemuesse und Frucht Extrakt haben, ihre Anticlastogenic Eigenschaften gezeigt
Waehrend die veraenderung auf die Effekte von Clastogens notiert ist, ist es beobachtet
dass die induviduelle Teile Z. B. Sulfahydryl und Flavonoid Compounds, Dehydroascorbic
Acid, Gallic Acid, Ellagic Acid, Mucic Acid, Citric Acid, Reducing sugars, Tannin U.S.W.
eine positive wechselwirkung mit meiste Bestandteil haben, wesen der Anwesenheit der
multiple biologische Eigenschaften koennen die pflanzeprodukten unter bestimmten
zustend mutagenic effekt produzteren. Einige Vorbeuger koennengleichzeitig erhoehen
und detoxifying mechanismus anspornen, Z. B "Inducers of coordinated enzyme activi-
ties".
Viebe Oxydants koennen Elektron akzeptieren oder Elektron schenken. Das haengbaber
yon den Redoxpotential ab. Auf diese weise machem sie ihnen schuetzend oder schaedlich.
Die Pflanze spielt auch eine grosse Rolle in der Anhaeufung, Metabobsm und in der
Verteilung von Xenobotics in der umgebung. Weger der Anwesenheisb medizine. Die
veraenderliche Effecter notierb in complex Pflanze extrakt sind wegen viele chemisch
reagierete gertung die waehrend des Prozess periation yon den Extrakten produziert.
Diese Extrakten Operieron als "Redox Agents, scavengers of free radicals und ligands for
binding to toxicants. Die enabliche Effekten sind Sicher das Ergebnis von Wechselwirkung
zwischer die Teiler und ihre individuell und gesannelte Wechselwirkung mit Toxicants
Die physiologische Fektoren, die Pflanzen und der Einspritzungs prozess den Extrakten
eincoirken, wirkb auch die spezialitat und die.
Mann muss beide vorteile und Nachteile danken wenn diese Aktive. Herbs in der
Medizine benuzt sind. Die genane Dose und Pflanze typ muessen auch calculieret
werden.
PLANT EXTRACTS 279
II. Introduction
Traditional medicines based on plants have been useful in discovering new drugs
from natural products, in helping to identify new biochemical loci for drug action, and
in developing new classes of bioactive molecules. At present, about 75% of the human
population depends on plant extracts as tools of traditional medicine. There are about
121 clinically useful prescription drugs derived from indigenous medicine. In 1985, of
a total of 3500 new chemical structures discovered, 2619 were isolated from higher
plants (see Abelson, 1990; Kinghorn & Balandrin, 1993). The potential for future uses
is vast, since of an estimated total of 250,000 to 300,000 species of higher living plants,
only about 5000 have been studied extensively for possible medical application (see
Cordell, 1977).
Interest has intensified in plant-based pharmaceuticals with the development of new
methods of screening for anticarcinogenic drugs (Hartwell, 1967). One of the earliest
reports is the use of the extract of Colchicum autumnale bulb (Liliaceae) in reducing
uncontrolled cell division. The property of preventing carcinogenesis has been reported
in many plant extracts, from cruciferous vegetables against alimentary cancers (Bjelke,
1974; Graham et al., 1978; Haenszel et al., 1972, 1976) to degradation of carcinogenic
nitrosamine formation factors by extracts of onion, pepper, and ginger (Kim et al.,
1987). Protective activity of total vegetable or fruit extract as dietary supplement against
epithelial, ovarian, oral, and esophageal cancers has also been reported (Barone et al.,
1992; Shu et al., 1989). Anticancer activity has been observed for extracts of bamboo
leaf (Kuboyama et al., 1981), Chinese tea (Han & Yong, 1990), and betel leaf (Azuine
et al., 1991; Bhide et al., 1991; Padma et al., 1989); for Cupressus semipervirens L.,
Anchusa strigosa L., Myrtus communis L., and Crataegus monogyna (Alwan et al.,
1990); for Trifolium pratense L. (Cassady et al., 1988); for root, stem, and leaf of
Ervatamia heynaena (Chitnis et al., 1971); and for rhizomes of turmeric (Kuttan et al.,
1985).
The observation of a close association between carcinogenesis and mutagenesis has
extended the survey to include plant extracts and plant products able to modify the process
of mutagenesis, alteration in the genetic material.
Natural plant products may, apart from inducing mutations, modify the action of other
known mutagens on the living organisms by 1) inactivating the existing mutagens within
the cell (desmutagens), 2) inhibiting the production of mutagens in the cell (bioantimu-
tagens), 3) synergising the activity of existing mutagens (comutagens), or 4) activating
the promutagens within the cell into mutagens. Such activities, particularly the first two,
often overlap. Some plant extracts can also act differently under different experimental
conditions.
A vast amount of literature, both scientific and folkloric, is available on plants and
cancer. In this review, the scope has been confined to actual data obtained from research
on the modulatory effects of plant extracts on mutagenesis and clastogenesis--two
genotoxic phenomena associated with carcinogenesis.
III. Plant Extracts as Inhibitors of Mutagenesis
In screening for antimutagenic effects, various test systems have been used, principally
bacterial ones such as Salmonella typhimurium and Escherichia coli (Ames et al., 1973).
Both mutagens and the plant products were administered in culture.
280 TIlE BOTANICALREVIEW
The term "antimutagen" was originally used to denote an agent that reduced the
apparent yield of mutations--spontaneous or induced. These included both desmutagens
and bioantimutagens. The former cause chemical and biochemical modification of mu-
tagens before DNA damage, and the latter reduce the apparent frequencies of mutations
interfering with cellular processes of mutation fixation. Environmental toxicants entering
the living systems are metabolised in two stages: biotransformation (phase 1) and
conjugation (phase 2). In the detoxication of toxic chemicals, the oxidative reactions of
phase 1 result in the formation of metabolites that subsequently undergo conjugation in
phase 2, through the action of epoxide hydrolase, glutathione transferase, UDP-glucoronyl
transferase, etc., to form conjugates. The latter are eliminated from the cell and finally
from the organism. In contrast, activation by oxidation results in the formation of
proximate carcinogens or reactive intermediates. The latter are usually poor substrates for
the conjugating enzymes. Therefore, a non-enzymatic interaction of these reactive inter-
mediates takes place with intracellular constituents, including proteins, RNA, and DNA,
leading to covalent binding and formation of neo-antigens, mutations, cancer, and cell-
death. These two alternative routes of oxidative biotransformation, leading to detoxication
or to activation, indicate two different modes of oxygenation and the probable existence
of two families of enzymes, separately responsible for the alternative pathways. The
cytochromes P-450 result mainly in detoxication, while the cytochromes P-448, flavopro-
tein mono-oxygenase, and non-enzymatic free radical hydroxylations are responsible for
oxidative activation.
A. EXTRACTSFROM VEGETABLES
A large amount of data is available on the antimutagenic activity of plant extracts from
different plant groups on bacterial test systems. These observations deal with extracts
from leafy vegetables, from fruits, from underground storage organs, and from whole
plants (Table I). The extracts were prepared mainly in water or organic solvents. Vegeta-
bles such as cabbage, spinach, celery, and sprouts were recorded to suppress the mu-
tagenicity of pyrolysate mutagens derived from tryptophan (Kada et al., 1978; Morita et
al., 1978). Lai et al. (1980) reported that addition of acetone extract of vegetables to the
Salmonella mutagenicity assay mixture of benzo(a)pyrene (BaP) and 3-methylcholan-
threne, reduced the number of revertant colonies. Some vegetables have also been found
to have desmutagenic effects against products obtained from boiled fish (Yoshikawa et
al., 1981). Juices of cauliflower, spinach, and lettuce have been reported to inhibit the
mutagenicity of nitrite combined with nitrosable compounds in mice in vivo (Barale et
al., 1983). Aqueous dialysates of 16 kinds of vegetables and fruits such as burdock,
eggplant, spinach, and apple were found to be antimutagenic against a number of known
mutagens in Salmonella typhimurium TA 100 strain (Shinohara et al., 1988). Investigation
of the urinary mutagenicity of 3 nonsmoking healthy men using Ames Salmonella/mi-
crosome assay, showed a reduction in the number of revertants in the urine of the subjects
who consumed fried salmon and parsley leaves simultaneously (Ohyama et al., 1987).
Among six vegetable juices tested, parsley juice was most effective in suppressing
mutagenicity (88.5%) of roasted beef extract (Nakashima, 1989). Extracts of lettuce and
chard leaves reduced the mutagenicity of BaP in urine samples of Balb/c mice (Perez &
Gago, 1991). Vegetables belonging to the families Compositae, Labiatae, Cruciferae,
and Umbelliferae showed antimutagenic activities against Trp-P-1 in an assay using
Salmonella typhimurium TA 98 strain. Sixty mushroom samples were also tested in the
PLANT EXTRACTS 281
Table I
S o m e plants s h o w n to have a n t i m u t a g e n i c p r o p e r t i e s
Plant parts
used Taxa Antimutagenic against Test systems Authors
Leafy Apium graveolens, Tryptophan pyrolysate Salmonella Kada et ai,, 1978;
vegetables Brassica oleracea var. products typhimurium Morita et al., 1978
capitata and gemnifera
Apium graveolens, Benzo(a)pyrene (BaP) S. typhimurium Lai et al., 1980
Brassica oleracea and 3-methyl
vat. botrytis L. and cholanthrene
capitata, Cucumis
sativus, Lactuca sativa,
Spinacea oleracea
Brassica oleracea var. Nitrite combined with Mice Barale et a1.,1983
botrytis, Lactuca sativa, nitrosable compounds
Spinacea oleracea
Anthriscus sylvestris Fried salmon Ames Ohyama et al.,
Salmonella/ 1987
mierosome
assay
Solanum melongena Trp-P-1, BaP, aflatoxin S. typhimurium Shinohara et al.,
Spinacea oleracea Bl, N-methyl-N'-nitro- 1988
N-nitrosoquanidine
(MNNG)
Anthriscus sylvestris Roasted beef extract S. typhimurium Nakashima, 1989
Lactuca sativa BaP Balb/c mice Perez & Gago,
1991
Fruits Citrus fruits N-nitro-O-phenylene S. typhimurium Bala & Grover,

TA 97a &
diamine (NPD), sodium 1989
azide TA 100
Emblica officinalis Sodium azide S. typhimurium Grover & Kanr,
1987
Terminalia chebula MNNG & UV-radiation E. coli WP2 & Jain et al., 1987
S. typhimurium
TA 98.
Cnidium monnieri Known mutagens Ames system Liu et al., 1988
Terminalia chebula
Psidium guajava NPD and sodium azide Salmonella Grover & Bala,
tester strains 1992, 1993
Underground Curcuma longa BaP, dimethyl S. typhimurium Nagabhushan &

parts benzanthracene (DMBA) Bhide, 1987
Daucus carrota Metabolites of S. typhimurium Darroudi et al.,
cyelophosphamide 1988
Allium sativum Ionizing radiation, S. typhimurium Knasmueller et al.,
peroxides, adriamycin, 1989
and MNNG
AUium sativum 4-nitroquinoline-1- E. coli Zhang et al., 1989
oxide (4 NQO)
282 THE BOTANICAL REVIEW
Table I (continued)
Some plants shown to have antimutagenic properties
Plant parts
used Taxa Antimutagenic against Test systems Authors
Others Acacia arabica, BaP, Trp-P-2, UV- E. coil & S. Jain et al., 1987
Aegilops spp., Camellia irradiation typhimurium
sinensis, Cassiafistula,
Eucalyptus spp.,
Psidium guajava,
Terminalia chebula,
T. arjuna, Zizyphus
jujuba
Camellia sinensis MNNG E. coli WP2 Jain et al., 1989
Triticum vulgare BaP S. typhimurium Perytet al., 1988,
1992
Leersiajaponica, BaP, 2-nitrofluorene,2- S. typhimurium Fujimoto et al.,
Polygonum hydropiper, aminofluorene 1987
Potamogeton crispus
Potamogeton oxyphylus BaP, AF-2, 2- S. typhimurium Satoet al., 1984,
nitrofluorene TA 98 & TA 1990
100
Craterellus Aflatoxin B1, BaP, S. typhimurium Grater et al., 1990
cornucopioides acridine half-mustard TM 677
ICR-191 and
2-nitrofluorene
same assay system but were found to be relatively weak in reducing mutagenicity as
compared to the vegetables (Ueda et al., 1991).
B. EXTRACTS FROM FRUITS

Of fruit juices, citrus juices are effective antimutagens. Juices from ten citrus varieties
reduced significantly the number of revertant colonies induced by N-nitro-o-phenyle-
nediamine (NPD) in TA 97a and sodium azide in TA 100 strains of Salmonella ty-
phimurium (Bala & Grover, 1989). A similar reduction in the number of his § revertants
was observed with water, acetone, and chloroform extracts of Emblica officinalis Gaertn.
(syn. Phyllanthus emblica L.) (Grover & Kaur, 1989). Extracts of Terminalia chebula
Willd. fruit suppressed the mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine
(MNNG) and UV radiation in E. coli WP-2 and S. typhimurium TA 98 strains (Jain et al.,
1987). Similar action was shown by water extract of Cnidium monnieri (L.) Cuss. fruits,
using Ames test (Liu et al., 1988). Water and chloroform extracts of guava (Psidium
guajava L.) and myroblan (Terminalia chebula Willd.) fruits were tested against two
direct-acting mutagens NPD and sodium azide and the S9-dependent mutagen 2-
aminofluorene (AF-2) in Salmonella tester strains. Only the water extract was effective
in reducing the mutagenic effects. Enhancement of the inhibitory activity of the water
extracts on pre-incubation with the mutagens showed that desmutagens may be present
in the extract itself (Grover & Bala, 1992, 1993).
PLANT EXTRACTS 283
C. EXTRACTSFROM UNDERGROUNDSTORAGEORGANS
Amongst underground parts, aqueous extracts of garlic bulbs (Allium sativum L.) have
been observed to inhibit a number of known mutagens, including ionizing radiations,
peroxides, adriamycin, and MNNG in Salmonella strains and also in eukaryotic Chinese
hamster cells (Knasmueller et al., 1989). In two tester strains of E. coli, this extract
suppressed the mutagenicity of 4-nitroquinoline-l-oxide (4 NQO) but not that induced
by UV (Zhang et al., 1989). Turmeric extract effectively decreased the level of BaP and
7,12-dimethylbenzanthracene (DMBA)-induced mutagenesis in strain TA 98 of S.
typhimurium (Nagabhushan & Bhide, 1987). Carrot juice, rich in vitamin A, has been
found to suppress mutagenic activities of metabolites of cyclophosphamide (Darroudi et
al., 1988). Peony root extract inhibited BaP-induced mutagenicity in S. typhimurium
reversion test (Sakai et al., 1990)
D. OTHERS
Several plant parts were tried against certain common mutagens. Lemon, black and
green tea, extracts of guava, and Cassia fistula L. leaves and Zizyphus jujuba L. bark
suppressed the mutagenicities of BaP and Trp-p-2, while Psidium guajava L., Terminalia
chebula Willd., T. arjuna Willd., Zizyphus jujuba L., Eucalyptus spp., Aegilops spp. and
Acacia arabica Willd. extracts were antimutagenic against UV irradiation (Jain et al.,
1987).
Extracts of green and black tea leaves decreased the mutagenic activity of MNNG in
E. coli WP2 in vitro and in the stomachs of rats as shown by bacterial mutagenicity in
vitro assays. Priming the rats with the extracts proved to be more effective than simulta-
neous administration of extract and mutagen (Jain et al., 1989).
Aqueous fractions of roots and leaves of sprouted wheat selectively inhibited the
mutagenicity of known mutagens requiring metabolic activation, as demonstrated by the
Ames~Salmonella microsome test (Lai, 1979). Later studies on the subfractions of extracts
from wheat sprout also reduced the mutagenic effects of BaP in S. typhimurium tester
strains (Peryt et al., 1988, 1992).
Different aquatic plants have been screened for their antimutagenic activities. Whole
plant extracts of curled pondweed (Potamogeton crispus L.), European cutgrass (Leersia
japonica Makino), and smartweed (Polygonum hydropiper L.) decreased mutagenic
effects of BaP, 2-nitrofluorene, and 2-aminofluorene (Fujimoto et al., 1987). Water
extract of grass wrack pondweed (Potamogeton oxyphyllus Miquel) also reduced reverse
mutations induced by BaP, AF-2, and 2-nitrofluorene in S. typhimurium TA 98 and TA
100 strains (Sato et al., 1984, 1990).
Ethanol extract of the fungus CratereUus cornucopioides completely inhibited the
mutagenic action of aflatoxin Bh BaP, acridine half-mustard ICR-191, and 2-nitrofluorene
in a forward mutation system using Salmonella typhimurium TM 677 (Gruter et al., 1990).
IV. Mechanism of Antimutagenic Activity of Plant Extracts
A. CERTAINSPECIFICFACTORSOBSERVED
Studies on antimutagenic activities of plant extracts have, in some cases, indicated the
involvement of certain factors, which are mainly intrinsic components of the extracts.
These components range from specific compounds such as ascorbic acid to vegetable
fibres, which could act as nonspecific redox agents, free radical scavengers, or ligands
for binding metals or toxic principles.
Morita et al. (1978) identified a desmutagenic factor in cabbage which was sensitive
to heat (100 ~ C) and pronase treatment, suggesting a protein character. Kada et al. (1978)
suggested a similar vegetable factor in cabbage and radish, capable of reducing
tryptophan-induced mutagenesis in Salmonella tester strains. In vegetables such as
burdock, a heat- and enzyme-resistant desmutagenic factor was isolated with molecular
weight higher than 300,000 and showing characteristics of a polyanionic substance, It
reduced the action of mutagens both with and without S9-mix (Morita et al., 1984). Of
the four compounds isolated from dried leaves of cabbage, nonacosane and pheophytin
did not inhibit 2-aminoanthracene (2AA) or N-methyl-N-nitrosourea-induced mutagene-
sis, whereas 15 nonacosane was more effective than 13-sitosterol. In V-79 cell mammalian
mutagenicity assay, all the four fractions were active against mutagenicity of 2 AA
(Lawson et al., 1989). Vegetable juices modified the mutagenic effects of beef extract and
nitrosated beef extract only in the presence of S9-mix as shown by Ames test. It was
therefore suggested that the constituents do not act directly upon the mutagens, but
interact with the metabolic activation system (Muenzner, 1986). The antimutagenic
principle of the green fruits of Momordica charantia L. was found to be an inextractable
mixture of novel acylglucosylsterols. Ingestion of these compounds may result in their
adsorption in the plasma membrane lipid bilayer, which could adversely affect the
membrane permeability toward the known mutagen mitomycin C and disrupt cellular
activity of the latter (Guevera et al., 1990).
The antimutagenic activity of some common vegetables was attributed to their chlo-
rophyll content (Lai et al., 1980). Chlorophyll was suggested to be the major active factor
in wheat sprout extract inhibiting the metabolic activation of carcinogens in vitro (Lai,
1979). However, later studies with wheat sprout gave conflicting results (Peryt et al., 1988,
1992). Two heat-resistant compounds were isolated from such extracts, located within
the cell cytosol, that showed antimutagenic activity--one with low and the other with
high molecular weight. The strong inhibition of BaP mutagenicity with non-chlorophyllic
wheat sprout extract suggested that chlorophyll is not the main factor involved in
antimutagenicity (see Sarkar et al., 1994). Among the aquatic plants, two species of
Potamogeton (grasswrack pond weed and curled pond weed) reduced BaP-induced mu-
tagenesis (Fujimoto et al., 1987; Sato et al., 1984, 1990). The factor involved in both these
plants was water-soluble, heat-resistant, and with a molecular weight above 300,000.
Smartweed (Polygonum sp.) also has similar desmutagenic factors, but the molecular
weights were both above and below 300,000. The active factor in European cutgrass may
not be a desmutagen. There is a possibility that it is a bioantimutagen or an inhibitor of
S9-mix (Fujimoto et al., 1987). Whatever may be the nature of antimutagenic factor, all
these studies have shown that chlorophyll does not play an important role in antimutagene-
sis. On the other hand, plant mucilage present throughout the body of the plants may be
considered to have inhibitory properties and may act by absorbing mutagens (Sato et al.,
1990). Antimutagenic effects of garlic extract have been attributed to molecules with
sulfur moieties which act as scavengers of free radicals (Knasmueller et al., 1989). It has
also been suggested that garlic bulbs reduce markedly the mutagenicity of 4 NQO by
inactivating the electrophilic groups of the mutagen or inhibiting metabolic activation
(Zhang et al., 1989).
Turmerin, a water-soluble 5-KDa peptide from turmeric, has been found to be an
efficient antioxidant/DNA protectant/antimutagen (Srinivas et al., 1992). The antimu-
PLANT EXTRACTS 285
tagenic action of the ethanol extract of mushroom Craterellus cornucopioides may be due
to direct chemical interaction with the mutagen and/or inhibition of the activation process
of promutagen (Gruter et al., 1990).
Inhibitory activity of extracts of the fruits of Emblica officinalis Gaertn., Psidium
guajava L., and Terminalia chebula Willd. was enhanced when the extracts were
pre-incubated with the mutagen at 37 ~ C for 30 minutes prior to plating, suggesting a
desmutagenic action (Bala & Grover, 1989; Grover & Bala, 1992, 1993). The antimu-
tagenic activity of citrus fruits had been attributed to the principal components, ascorbic
and citric acids. However, in general, the effects of the crude extract are more than that
of an equivalent amount of any single component, indicating that the different compo-
nents, both major and minor, are involved in the process.
Vegetable fibres have been shown to be able to suppress the action of pyrolysates by
absorbing them (Kada et al., 1984). Such fibres may be responsible for the activity of
most crude plant extracts in eliminating possible mutagens from the system.
B. STEPS INVOLVEDIN PROTECTINGAGAINSTMUTAGENESIS

In general, the ways in which inhibitors of mutagenesis can act include 1) the inhibition
of interaction between genes and biochemically reactive mutagens and 2) the inhibition
of metabolic activation of indirectly acting mutagens. The latter mechanism includes
a) inactivation of metabolizing enzymes and b) interaction with promutagens making
them unavailable for the enzymatic process (see Hayatsu et al., 1988). Inhibition of
effect of mutagens can be outside the cells or inside the cells (Table II.)
The majority of inhibitors that naturally occur in many edible plants are phenols,
aromatic isothiocyanates, coumarins, flavones, diterpenes, retinoids, ascorbic acid,
alphatocopherol, selenium salts and plant sterols. Eighteen active chemical antimutagenic
compounds were identified from 200 diverse plants, one of them being protoanemonia
(Minakata et al., 1983; Ramel et al., 1986).
Deactivation of mutagens in the alimentary tract was by crude juices from various
vegetables, which reduced in vitro the mutagenicity of tryptophan pyrolysis products
(Kada et al., 1978, 1984). Inactivation enzymes possessing peroxidase and NADPH-
oxidase activities were also isolated from cabbage and broccoli.
lntracellular inhibition ofmutagenesis: The extracts of cruciferous plants--e.g.,
brussel sprout, cabbage, cauliflower and broccoli--are capable both of activating enzymes
such as arylhydrocarbon hydroxylase and of detoxifying enzymes such as the cytosolic
GSH S-transferase. The latter effect prevails. The extracts contain phenols, isothiocy-
anates and indole derivatives like indole 3-carbinole, 3,3' diindolylmethane, and indole
3-acetonitrile. The protection afforded has been ascribed to a changed balance in the
enzyme activities involved in the biotransformation of these compounds and to the effect
of conjugating enzyme systems.
Blocking of reactive chemical species: Ellagic acid may protect DNA from the attack
of electrophilic species such as BaP diolepoxide or free radicals by binding to its
nucleophilic sites. It is a naturally occurring polyphenol from coffee, nuts, and grapes.
The flavonoids myrecetin and rutin show similar effects, as also gallic acid and sulphydryl
compounds.
Endogenous N-nitroso compound (NOC) formation through interaction between ni-
trites and nitrosable amines or amides in the stomach has been inhibited by complex
mixtures of plant origin, including tea, coffee, vegetables and fruit juices, soya products,
Table II
Mechanisms for inhibition of mutagenesis by plant products

(from DeFlora & Ramel, 1988)
I. Inhibition of mutagens acting outside the cells (stage 1 inhibitors)

A. Inhibiting the uptake of mutagens or precursors by:
1. hindering their penetration into
the organism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . body shielding, washing
the cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . fatty acids, putrescin, aromatic
amino acids
2. favouring their removal . . . . . . . . . . . . . . . . . . . . fibres
B. Inhibiting endogenous formation of mutagens by:
1. Inhibiting nitrosation . . . . . . . . . . . . . . . . . . . . . . ascorbic acid, tocopherols, phenols
2. Modifying microsomal intestinal flora . . . . . . . . fermented products
C. Reactions deactivating mutagens
1. Physical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . maintenance of physiological pH
2. Chemical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . thiols, antioxidants
3. Enzymatic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vegetables with peroxidase activity
II. Inhibition of mutagens acting inside the cells (stage 2 inhibitors)

A. Modulation of metabolism by:
1. Inhibiting cell replication . . . . . . . . . . . . . . . . . . . retinoids
2. Sequestering mutagens in non-target cells . . . . . thiols
3. Inhibiting activation of promutagens . . . . . . . . . extracts of Cruciferae
4. Inducing detoxification . . . . . . . . . . . . . . . . . . . . . phenols, thiols
B. Blocking reactive molecules by:
1. Reacting with electrophiles . . . . . . . . . . . . . . . . . sulphur compounds
2. Scavenging reactive oxygen . . . . . . . . . . . . . . . . . antioxidants
3. Protecting nucleophilic sites of DNA . . . . . . . . . ellagic aid, retinoids
C. Modulation of DNA replication/repair by:
1. Increasing fidelity of DNA replication . . . . . . . . CoC12, NaAsO2
2. Increasing repair of DNA damage . . . . . . . . . . . . cinnamaldehyde coumarins,
umbelliferone, vanillin, thiols
3. Inhibiting error-prone repair . . . . . . . . . . . . . . . . protease inhibitors
and betel nut extracts. This inhibitory action is due to the presence or vitamins A, C, and
E and natural phenols, which have different functions. Caffeic, chlorogenic, ellagic,
ferulic, gallic, and tannic acids and vitamin C inhibit mutagenicity of direct-acting
N-nitroso compounds, and vitamin A inhibits metabolic activation of promutagenic
nitrosamines (Ames, 1982; Gichner & Veleminsky, 1988).
PLANT EXTRACTS 287
Inhibition of uptake ofmutagens: Purified fibres from different vegetables can bind in
vitro and irreversibly adsorb the mutagenic products of proteins and amino acids. Refined
corn bran binds dinitropyrene, thus decreasing its mutagenicity (Takeuchi et al., 1988).
Vitamin E functions as a chain-breaking antioxidant for the lipid phase of biological
systems. It is a mixture of four phenols called tocopherols, of which the alpha form is
most biologically active. It is the most effective natural chain-breaking antioxidant due
to the stereo-electronic properties of its hydroxychroman group.
Diallylsulphide, aUyl methyl disulphide, and diallyl-trisulphide are components of
Allium cepa and A. sativum and are able to suppress BaP-induced neoplasia in the mouse
forestomach. These compounds enhance the level of glutathione-S-transferase in the
forestomaeh, accelerating the detoxication of mutagens.
Protease inhibitors are proteins and peptides found in microbes, animals, and plants,
particularly in the beans. Certain soybean proteins, Edipro A and BBI, suppress turnouts
induced by dimethylhydrazine (DMH). In vitro, they decrease hydrogen peroxide forma-
tion through blockage of active oxygen-mediated processes.
V. Plant Extracts as Inhibitors of Clastogenesis
The effects of toxicants can be observed at the level of chromosomes (clastogenesis)

through alterations in chromosome structure (chromosomal aberrations or CA) and
number (aneuploidy, polyploidy). A wide range of short-term and long-term screening
procedures is available. The most common ones use higher plants (AIlium cepa, Allium
sativum, Viciafaba, Tradescantia virginiana) or rodents (mice, rats) in vivo as test systems
for monitoring chromosomal aberrations (see Hsu, 1982; Kihlman, 1971; Levan, 1949;
Naismith, 1987; Sharma & Sharma, 1989). In vitro studies using leukocytes or cell lines
are relatively rare.
Such experiments with a number of crude vegetable and fruit extracts have demon-
strated the anticlastogenic activities of these extracts against known clastogens (Table III).
The data, however, are not always conclusive.
Ten vegetable juices (both fresh and boiled) were administered to rats exposed to
DMBA and the CA screened in bone marrow cells. Both fresh and boiled juices of onion,
burdock, eggplant, cabbage, and welsh onion reduced the frequency of CA to a significant
level. Fresh pumpkin juice, on the other hand, enhanced the incidence of aberrant
metaphases, while boiled juice reduced it to a significant extent (Ito et al., 1986).
Anticlastogenic effects of carrot and spinach were observed against the chemotherapeutic
drug cyclophosphamide in rat bone marrow cells in vivo following short-term micronu-
cleus tests (Abraham et a1.,1986). Both simultaneous and prior administration of the
vegetable juices reduced the cyclophosphamide-induced micronuclei significantly in the
treated animals.
Priming of Swiss albino mice with fresh spinach leaf extract for 7 days reduced
significantly the frequency of CA induced by chromium compounds (Sarkar et al., 1995,
1996). The frequencies of CA induced by mitomycin C, cyclophosphamide, and sodium
arsenite were reduced in bone marrow cells of mice that had been administered aqueous
garlic extract for different periods as a dietary supplement (Das et al., 1993a, 1993b;
Roychoudhury et al., 1993). The extracts of garlic bulbs and spinach did not themselves
induce appreciable number of chromosome aberrations when administered alone, indicat-
ing that these extracts were non-clastogenic at the doses used.
Green tea extract suppressed chromosome aberrations induced by aflatoxin B l, in rat

bone marrow cells in vivo (Ito et al., 1989). Anticlastogenic effect was also observed with
essential oil of seeds ofApium graveolens against CC14 in mice in vivo (Sobti et al., 1991).
Leaf extract of Apocynum venetum L. is unable itself to induce micronuclei in bone
marrow polychromatic erythrocytes in mice and also prevents the increase of micronuclei
formation induced by cyclophosphamide (Hao et al., 1988).
Extensive work has been done in our laboratory to test the protection afforded by crude
extract of fruits of Phyllanthus emblica L. (Emblica officinalis Gaertn.) and equivalent
amounts of synthetic ascorbic acid (vitamin C) against a number of known clastogens,
including zinc chloride, metanil yellow, and ethyl parathion (Giri & Banerjee, 1986),
nickel and lead (Agarwal et al., 1989), lead and aluminium (Dhir et al., 1990), cesium
chloride (Ghosh et al., 1992), and chlordane (Sarkar, 1992). Separate sets of mice were
administered orally the crude fruit extract and equivalent amount of synthetic ascorbic
acid for periods from 24 hours to 60 days. The toxicants were administered as a single
acute dose or a series of subtoxic doses at intervals for prolonged periods. It was observed
that the crude extract reduced the cytotoxic effects to a greater extent than vitamin C alone.
The protective ability of Emblica officinalis Gaertn. grated fruit extract had been con-
firmed against radiation-induced chromosome damage in Allium sativum root tips (Yadav,
1987).
VI. Mechanism of Anticlastogenic Activity
The mechanisms by which crude vegetable and fruit extracts reduce the cytotoxic
effects of various clastogens may be different for different plants. It has been reported
that intake of diets containing powdered preparations of brussels sprouts, cabbage, coffee
beans, or tea leaves increased the activity of glutathione-S-transferase (GST), which
catalyses the binding of electrophiles to glutathione (GSH) (Sparnins et al., 1982). Thus,
GST activated by vegetable juices may be involved in the anticlastogenic activity of the
vegetables. The oral administration of GSH alone reduced DMBA-induced chromosomal
aberrations (Ito et al., 1984, 1986). The SH-compounds, present abundantly in onion and
welsh onion, have a chemical structure analogous to SH (Whitaker, 1976). These SH
compounds were presumed to be responsible for the CA-suppressing activity of onion
juices.
Many vegetable and fruit juices also contain flavonic compounds such as quer-
cetin and kaempferol. Non-carcinogenic and anti-tumor-promoting activity of quer-
cetin is well documented (Hirono et al., 1981; Morino et al., 1982; Nishino et al.,
1984). Flavone compounds of vegetables may also be involved in the anticlasto-
genic activity.
Crude spinach leaf extract has been found to suppress chromosomal aberrations
induced by cyclophosphamide (Abraham et al., 1986) and chromium compounds (Sarkar
et al., 1995, 1996) in mice bone marrow cells in vivo. It was earlier suggested that
chlorophyll plays an important role in spinach anticlastogenicity (Abraham et al., 1986).
However, other compounds---e.g., ascorbic acid, fibres, other vitamins present in the
extract--are suspected to have an additive interaction with chlorophyll (Barale et al.,
1983).
Chemical constituents of fruits of Phyllanthus emblica L. include ascorbic acid,
dehydroascorbic acid, gallic acid, ellagic acid, mucic acid, citric acid, reducing sugars,
and tannin (Shrivastava & Shrivastava, 1964; Soman & Pillay, 1962). Among these, the
PLANT EXTRACTS 289
Table I I I
Some plants shown to have anticlastogenic effects
Anticlastogenic Test
Plant parts used Taxa against systems Authors
Fresh and Allium cepa, DMBA Mice Ito et al., 1986
boiled juices Solanum melongena,
Brassica oleracea
vat. capitata
Roots and leaves Daucus carrota, Cyclophosphamide Rats Abraham et al.,
Spinacea oleracea 1986
Leaves Apocynum venetum Cyclophosphamide Mice Hao et al., 1988
Leaves Spinacea oleracea Chromium (VI) oxide Mice Sarkar et al., 1994
Bulbs Allium sativum Mitomycin C, Mice Das et al., 1993a,
cyclophosphamide, 1993b;
sodium arsenite Roychoudhury et
al., 1993
Leaves Camellia sinensis-- Aflatoxin B1 Rats Ito et a1.,1989
different varieties
Phyllanthus emblica Zinc chloride, Mice Girl & Banerjee,
metanil yellow, ethyl 1986
parathion
Nickel and lead Mice Agarwal et al., 1989
Fruits Lead and aluminium Mice Dhir et al., 1990
Cesium chloride Mice Ghosh et al., 1991
Chlordane Mice Sarkar, 1992
Emblica officinalis Radiation Allium Yadav,1987
sativum
major component vitamin C, due to its antioxidant and chelating effects, has been already
observed to act as anticarcinogen, antimutagen, and anticlastogen, respectively, in differ-
ent test systems (Mirvish, 1975; Parshad et al., 1978; Gebhart et al., 1985; Ginter et al.,
1989). Ellagic acid may protect DNA from the attack of electrophilic species like BaP
diol epoxide or free radicals by binding to its nucleophilic sites (Wood et al., 1982). Gallic
acid, ellagic acid, tannic acid, and vitamin C inhibit mutagenicity of direct-acting
N-nitroso compounds (Ames, 1982; Takeuchi et al., 1988). Crude plant extracts are
complex mixtures of a number of individual components. Their antimutagenic and
anticlastogenic activity cannot be attributed to any one of the individual components. They
are the sum of interactions between the components and the clastogens or mutagens added
(Sharma, 1990).
VII. Plant Extracts as Genotoxic Agents
Under certain conditions, plant products may induce mutagenic effects, due to the
presence of multiple biological properties. Some inhibitors can stimulate simultaneously
both enhancing and detoxifying mechanisms, as do, e.g., inducers of coordinated enzyme
activities. Many oxidants can, depending on the redox potential, either accept or donate
electrons, rendering them protective or harmful.
A. AS MUTAGENS
Ames (1983) compared mutagenic activities of 16 mutagens of plant origin including
components like coumarins, eugenols, hydrazine and phorbol esters, and plant extracts of
Vicia faba, alfalfa, and others, using bacterial assay. Aqueous extracts of three plant
species (Achyrocline satureoides Gaertn., Baccharis anomala DC., Luchea divarticata
L.) used in Brazilian popular medicine showed positive mutagenic activity in the Ames
test with microsomal activation (Vargas et al., 1991). Chili extract and its pure alkaloid
capsaicin induced mutations in Salmonella typhimurium histidine-deficient tester strains
with metabolic activation. Capsaicin was positive in micronucleus test with V-79 CHO
cells and also inhibited DNA synthesis in tests with Swiss mice (Nagabhushan & Bhide,
1985). Among the six vegetables commonly consumed in the Netherlands, cultivars of
lettuce, paprika, and rhubarb were mutagenic in TA 98 strain of Salmonella typhimurium;
string beans were mutagenic in TA 98 and TA 100. Spinach and brussels sprouts, however,
could not induce any mutation (vander Hoeven et al., 1983). Ingredients of betel quid,
which have been related to high incidence of oral cancers, were examined. Among these,
extract of areca nut was found to enhance the formation of BPV DNA-induced trans-
formed foci, though no such promoting activity was shown by chewing tobacco. A
chemopreventive effect was afforded by the administration of vitamin A to the betel quid
chewers (Stich & Tsang, 1989).
B. AS PROMUTAGENS
Plants play an active role in the accumulation, metabolism, and environmental distri-
bution of xenobiotics. The property of plants to activate promutagens that may enter the
food chain is of great significance in view of the large number and types of chemicals to
which the plants are exposed. A promutagen is a chemical that is not mutagenic itself but
can be biologically transformed by a plant system into a mutagen. Several methods for
studying promutagens from plants were developed both in vivo and in vitro, including
plant cell-free systems (Gentile & Gentile, 1991; Gentile & Plewa, 1988; Plewa et al.,
1988).
Several categories of chemicals have been activated by crude plant extracts and have
induced mutations in Salmonella systems. These range from pesticides, herbicides, and
insecticides to maleic hydrazide and polyaromatic hydrocarbons. The extracts include
those of wheat and corn seedlings, tobacco callus, pea apical bud, potato tuber and tulip
bulb, Tradescantia leaf, and Viciafaba roots.
The extent to which plants can store mutagenic xenobiotics or convert non-mutagenic
chemicals to promutagenic or mutagenic forms has not yet been fully clarified. Since most
carcinogenic chemicals are also mutagenic, the mutagenic properties of xenobiotics and
their metabolities are receiving increasing attention. In plants the mutagenic activation
may be studied at two levels. The mutagenic damage may be caused in the plant itself.
Alternatively, the mutagenic metabolite may be conjugated and stored in the plant until
it is liberated and becomes active upon consumption of the plant by animal or man, e.g.,
after application of pesticides to food crops.
Certain plant microsomal enzymes and peroxidases have been shown to form reactive
intermediates. The best-studied examples are 2-aminofluorene, BaP, and pentachlorophe-
nol. The latter two xenobiotics are converted to quinoid derivatives, which are, in
principle, able to participate in redox cycle and generate active oxygen species. Therefore,
PLANT EXTRACTS 291
covalent binding of reactive intermediates to DNA as well as fragmentation of DNA are

proposed as mechanisms of action of mutagenic plant metabolites (Sandermann, 1988).
Plants also produce mutagen precursors. A nitrosable mutagen precursor, 4-chloro-6-
methoxyindole, was found in fava beans, and a relationship was suggested between the
high incidence of gastric cancers and the intake of fava beans and nitrite in Central and
South American countries. The precursors may be activated by fermentation as well; e.g.,
soybeans do not show mutagenic activity, even when treated with nitrite, unless fermented.
A mutagen precursor isolated from Chinese cabbage was indole-3-acetonitrile (Wakabayashi
et al., 1988).
C. AS CYTOTOXICAND CLASTOGENICAGENTS
Low concentrations of tobacco leaf extract exerted a stimulating effect, whereas
high concentration acted as a mitodepressant, on root-tip cells of Allium sativum L.
(Sopova et al., 1983). Stronger concentrations of extract of immature Solanum nigrum
L. fruits reduced the intensity of mitosis in A. sativum L., whereas weaker concentra.
tions stimulated it. The presence of a cytokinin-like substance in the extract has been
suggested to be responsible (Krivokapic et al., 1970). Extracts of leaves and inflores-
cences of male spinach and aster plants increased the frequency of chromosomal
aberrations and mutations in welsh onion and barley, respectively, whereas the female
plants inhibited the processes (Sidorskii, 1984). Cellular damage including heavy
pycnosis, clumping of chromosomes, fragmentation, and spindle disturbances in
Allium cepa L. root meristem were induced by the leaf extract of Ricinus communis L.
(George & Geethamma, 1990). Abraham and Cherian (1978) investigated the cellular
changes produced by extracts of betel leaves on root tip ceils of onion and demon-
strated the cytotoxicity of such extracts. Chromosome-breaking activity has been
exhibited by aqueous extract of mushroom (Paxillus involutus) in dry and pre-soaked
seeds of Nigella damascena L. (Gilot-delhalle et al., 1991).
Extracts of Viciafaba L. roots and leaves and Zea mays L. leaves were compared for
their ability to induce chromosomaI aberrations and sister-chromatid exchanges in
Chinese hamster ovarian ceils and human lymphocytes. Both the extracts induced CAs
in both systems; however, maize extract was more potent than Vicia extract (Kanaya
et al., 1992). Aqueous extract of Heliotropium curassavicum L., though employed
widely in therapeutics, has been found to induce chromosomal aberrations and
anaphase delay in CHO cell line. This toxic effect was associated with the pyrrolidiz-
ing alkaloids and the N- oxides, which are changed into pyrrolic derivatives through
a process of in vitro metabolism (Carballo et al., 1992).
D. SYNERGISTICAND ANTAGONISTICEFFECTS
Certain plant extracts are observed to induce both mutagenic and antimutagenic effects
in different test systems. Rhizome juice of ginger was found to be antimutagenic against
tryptophan pyrolysate-induced mutagenesis (Kada et a1.,1978; Morita et al., 1978) and
6-gingerol (Nakamura & Yamamoto, 1982). However, when added to known mutagens
such as AF-2 and MNNG, mutagenesis was increased by ginger juice, and the potent
mutagen identified in this case was 6-gingerol (Nakamura & Yamamoto, 1982). It was
presumed that ginger juice contains antimutagenic substances that can suppress the
activity of 6-gingerol and that, in the presence of certain specific mutagens like AF-2 and
MNNG, 6-gingerol is able to express its mutagenicity (Nakamura & Yamamoto, 1982).
Extracts of a desert mushroom, Al-faga (Tirmaniapinoyi), in water and methanol failed
to show any mutagenic activity, but the chloroform extract was mutagenic with and
without metabolic activation. Moreover, the ethanol extract, combined with some known
mutagens, inhibited carcinogen-induced mutagenicity. These results indicated that both
mutagens and antimutagens can be extracted from the same food item using different
solvents (Hannah et al., 1989). Agaricus bisporus has been reported to be carcinogenic
(Toth, 1979) and mutagenic in microbial systems (Sterner et al., 1982). However, no
mutagenicity or genotoxicity of the same fungal extract was detected by Pool-zobel et al.
(1990) in either in vitro or in vivo studies.
Among the chemical constituents of plant extracts, vitamin C inhibits the formation
of some nitrosamines but accelerates the formation of others, which might give rise to
mutagens by transnitrosation. The change from protective to harmful effects may be
related to the dose, the mode of administration, or even the sequence of administration.
The inhibitor may have opposite effects in different tissues. For example, mixed treatment
of rats with BHA and plant antioxidants such as propyl gallate and alpha tocopherol
enhanced or inhibited the induction of hyperplasia at different sites of the forestomach
epithelium (Hirose et al., 1987).
Interactions between inhibitors may lead to synergistic effects, e.g., the potential
preventive effects of vitamins A, C, and E may need a high level of carotene and vice
versa. Similarly, an optimal amount of vitamin E may be essential for protective effects
of vitamin A. These combined actions may be due to the different inhibitors acting at
different levels or being localised at different cellular areas. The higher protection
afforded by crude plant extracts than an equivalent amount of the purified or synthetic
ingredients, as observed with Phyllanthus emblica L., may be related to this phenomenon
(Giri & Banerjee, 1986; Giri et al., 1988).
The administration of aqueous leaf extracts of spinach-beet (Beta vu!garis var.
benghalensis Hort.) to mice as dietary supplements for prolonged periods reduced signifi-
cantly the clastogenic activity of chromium (VI) oxide. Chlorophyll extracted from the
leaf, in equivalent amounts, was itself clastogenic to a lower degree and did not markedly
affect the genotoxicity of chromium. An equivalent amount of chlorophyllin--a synthetic
derivative with Na/Cu replacing the Mg in chlorophyll moleculepwas significantly
anticlastogenic (Sarkar et al., 1993). In protecting against the genotoxic effects, therefore,
the crude extract is significantly more effective than chlorophyll due to the interactive
effects of its ingredients.
VIII. Plant Products as Modulators of Mutagenesis in Traditional

Systems of Medicine
As mentioned earlier, extensive use is made of plant products in traditional systems of

medicine and as part of life style. A limited screening of some of these products indicates
a combination of effects. Some examples are cited below.
A. INDIAN
Chili and its pure alkaloid capsaicin, and ginger and its phenolics gingerol and shogaol
are mutagenic, Turmeric (Curcuma longa L.) and its pure components are non-mutagenic
and suppress the mutagenicity of chili and capsaicin and also of several mutagens and
PLANT EXTRACTS 293
carcinogens such as tobacco, cigarette, and benzo(a)anthracene. A diet that included 1%

turmeric reduced BaP-and DMBA-induced stomach tumours and spontaneous mammary
tumours in mice (Nagabhushan & Bhide, 1985; Nagabhushan et al., 1987a & 1987b).
The daily intake of turmeric powder by Indian adults is between 3 and 6 g, containing
2-5% curcumin. The exposure of such populations to nitroso-compound precursors
through vegetables, marine foods, and drinking water may be neutralised by the relatively
high consumption of turmeric, which reduces the formation of mutagenic/carcinogenic
nitroso compounds (Nagabhushan et al., 1988).
Long-term studies were made simulating betel-chewing habits in India, with and
without tobacco, on Swiss mice in vivo. Crude aqueous extracts ofAreca catechu L. and
Nicotiana tabacum L. leaf given separately were mitogenic and also increased nuclear
DNA content. Tobacco, in any combination of chewing mixture, induced duration-
dependent clastogenicity. The addition of high lime and betel leaf (Piper betel L.) to the
quid reduced the degree of mitogenicity and induction of aneuploidy but was ineffective
when both tobacco and areca nut were added to the quid (Sen et al., 1987, 1991).
B. CHINESE
Screening of 169 Chinese medicinal herbs used orally as aqueous extracts showed
antimutagenic activity. The following plants reduced the mutations induced by picrolinic
acid: Pteris multifida, Actinidia chinensis Planch., Artemisia vulgaris L., Paris polyphylla
S., and Ampelopsis brevipedunculata Maxim. ex Trautv. Mutagenicity of benzo(a)pyrene
was completely inhibited by Smilax china L., Prunella vulgaris L., and Actinidia chinensis
Planch. and moderately inhibited by Pteris polyphylla S., Ampelopsis brevipedunculata
Maxim. ex Trautv., Gossypium herbaceum L., Lithospermum erythrorhizon Sleb. & Zucc.,
Artemisia lavendulaefolia DC., Selaginella doederleinii H., Dianthus superbus L., Cen-
tipeda minima A. Br. & Aschers., Curcuma zedoaria R., Marsdenia tenacissima Wight &
Am., and Kalopanax septemlobus K. Five of these were antimutagenic against both
mutagens tested (Lee & Lin, 1988).
On the other hand, extracts from Buplei radix, Aurantii nobilis pericarpium, and
Pinelliae tuber increased the mutagenicity of BaP slightly but significantly. Small doses
of Angelicae radix and Cnidii rhizoma extracts enhanced the mutagenicity, but at higher
doses a decreasing effect was noted. The factors isolated contained umbelliferone, pro-
toanemonin, and plant phenols. These medicinal plants containing blocking agents for
mutagenic activity are very important and are used very frequently in Chinese herbal
medicines. In these cases, the effect seems to be unrelated to the part of the plant used or
the family to which it belongs; rather, the substances in each extract combine to produce
the effect on mutagenicity (Sakai et al., 1988).
IX. Conclusions
Specific biological action of a drug is due to its specific binding to a functional

molecular receptor. In complex plant extracts, the variation in effects observed can be
attributed to the many chemically reactive species that are formed during the processing
and ingestion of the extract, which could act as nonspecific redox agents, scavengers of
free radicals, and ligands for binding to toxicants. The final effects are obviously the
outcome of interactions between the components and their individual and collective
interaction with the toxicant. The specificity and efficacy of such responses will be
influenced also by the physiological factors influencing the plants and the process
followed for administration of the extract.
In utilizing pharmacologically active herbs, both beneficial and potential adverse
effects must be taken into account. The actual dose and form of the plant also need to be
worked out (see DeSmet et al., 1992).
The activation of chemicals that have entered the plant system into potential mutagens
by plant enzymes is another field that needs immediate attention, particularly in view of
the large number of agricultural chemicals such as fertilisers, herbicides, and pesticides
that are being continuously added. Since the plants, particularly the crops, that receive
most of these chemicals are at the beginning of the food chain, it is imperative to study
the interaction of plants with environmental agents. The wide diversity of plants and
environmental chemicals make the problem more complicated. The information available
gives the mutagenic/antimutagenicaction of individual components on a limited number
of test systems. But this information is very limited, as is shown by contradictory activity
of the same product under different conditions. The inhibitory action tends to lower the
active dose of genotoxic agents and the accelerating action raises it. The ultimate load of
mutations is the result of interaction between these opposing forces, modified by a large
number of exogenous and endogenous factors. Therefore, a comprehensive overview is
needed before arriving at conclusions regarding the environmental safety of any new
chemical.
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Plant Extracts As Modulators of Genotoxic Effects

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THE BOTANICAL REVIEW

Plant Extracts as Modulators of Genotoxic Effects

DEBISRI SARKAR, ARCHANA SHARMA

Centre of Advanced Study in Cell and Chromosome Research,

Vivekananda Institute of Medical Sciences,

Copies of this issue [62(4)] may be purchased from the Scientific

TheBotanicalReview62(4): 275-300, October-December1996

VIII. Plant Products as Modulators of Mutagenesis in Traditional Systems of Medicine. 292

ein Toxic Prinziple Moeglicherweise koennen weitere Mutagens Produktion mit

III. Plant Extracts as Inhibitors of Mutagenesis

Fruits Citrus fruits N-nitro-O-phenylene S. typhimurium Bala & Grover,

Underground Curcuma longa BaP, dimethyl S. typhimurium Nagabhushan &

B. EXTRACTS FROM FRUITS

IV. Mechanism of Antimutagenic Activity of Plant Extracts

B. STEPS INVOLVEDIN PROTECTINGAGAINSTMUTAGENESIS

Mechanisms for inhibition of mutagenesis by plant products

I. Inhibition of mutagens acting outside the cells (stage 1 inhibitors)

II. Inhibition of mutagens acting inside the cells (stage 2 inhibitors)

V. Plant Extracts as Inhibitors of Clastogenesis

The effects of toxicants can be observed at the level of chromosomes (clastogenesis)

Green tea extract suppressed chromosome aberrations induced by aflatoxin B l, in rat

VI. Mechanism of Anticlastogenic Activity

VII. Plant Extracts as Genotoxic Agents

covalent binding of reactive intermediates to DNA as well as fragmentation of DNA are

VIII. Plant Products as Modulators of Mutagenesis in Traditional

As mentioned earlier, extensive use is made of plant products in traditional systems of

carcinogens such as tobacco, cigarette, and benzo(a)anthracene. A diet that included 1%

Specific biological action of a drug is due to its specific binding to a functional

Abelson, P. 1990. Editorial. Science 247: 513.

--, T. S. Bandopadhyay, A. Mukherjee, G. Talukder & A. Sharma. 1988. Effects of vitamin

, M . Kato, K. Aikawa & S. Kiriyama. 1984. Adsorption of pyrolysate mutagens by vegetable

vander Hoeven, J. C., W. J. Lagerweij, I. M. Bruggeman, F. G. Voragen & J. H. Koeman. 1983.

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