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Cancer Patent Abstract
Vegetable sources of cancer chemoprotective agents have been identified
which are extraordinarily rich in glucosinolates, metabolic precursors
of isothiocyanates. The vegetable sources are used to provide a
dietary means of reducing the level of carcinogens in mammals.
Cancer Patent Claims
What is claimed is:
1. A method of extracting glucosinolates and isothiocyanates from
plant tissue comprising homogenizing said plant tissue in an excess
of a mixture of dimethyl sulfoxide, acetonitrile and dimethylformamide
at a temperature sufficient to inactivate myrosinase enzyme activity.
2. The method of claim 1, wherein the ratio of dimethyl sulfoxide:acetonitrile:dimethylformamide
is 1:1:1.
3. The method of claim 1, wherein said temperature is between 0.degree.
C. and the freezing temperature of the extraction mixture.
4. The method of claim 1, wherein said temperature is between -50.degree.
C. and the freezing temperature of the extraction mixture.
5. The method of claim 1, wherein said plant tissue is selected
from the group consisting of cruciferous sprouts measured after
3 days of growth, cruciferous seeds, plants or plant parts.
6. The method of claim 5, wherein said sprouts, seeds, plants or
plant parts have at least 200,000 units per gram fresh weight of
Phase 2 enzyme-inducing potential.
7. The method of claim 5, wherein said sprouts, seeds, plants or
plant parts have at least 300,000 units per gram fresh weight of
Phase 2 enzyme-inducing potential.
8. The method of claim 5, wherein said sprouts, seeds, plants or
plant parts have at least 400,000 units per gram fresh weight of
Phase 2 enzyme-inducing potential.
9. The method of claim 5, wherein said sprouts, seeds, plants or
plant parts have at least 500,000 units per gram fresh weight of
Phase 2 enzyme-inducing potential.
10. A method of making a food product comprising extracting glucosinolates
and isothiocyanates from cruciferous plant tissue having at least
200,000 units per gram fresh weight of Phase 2 enzyme-inducing potential,
recovering said glucosinolates and isothiocyanates and adding said
glucosinolates and isothiocyanates to food; wherein said extracting
comprises contacting said plant tissue with a non-toxic solvent
at a temperature sufficient to inactivate myrosinase enzyme activity.
11. The method according to claim 10, wherein said solvent is water.
12. The method of claim 11, wherein said water is at 100.degree.
C.
13. The method according to claim 10, wherein said solvent is liquid
carbon dioxide.
14. The method according to claim 10, wherein said solvent is ethanol.
15. The method of claim 10, wherein said plant tissue is selected
from the group consisting of cruciferous sprouts measured after
3 days of growth, cruciferous seeds, plants and plant parts.
16. The method of claim 15, wherein said sprouts, seeds, plants
or plant parts have at least 300,000 units per gram fresh weight
of Phase 2 enzyme-inducing potential.
17. The method of claim 15, wherein said sprouts, seeds, plants
or plant parts have at least 400,000 units per gram fresh weight
of Phase 2 enzyme-inducing potential.
18. The method of claim 15, wherein said sprouts, seeds, plants
or plant parts have at least 500,000 units per gram fresh weight
of Phase 2 enzyme-inducing potential.
19. The method of claim 10 wherein said food product is selected
from the group consisting of a bread, a drink, a soup, a salad,
a sandwich and a cereal.
20. The method of claim 19 wherein said drink is a tea.
21. The method of claim 10 wherein said extracting further comprises
homogenizing said plant tissue with said non-toxic solvent.
22. The method of claim 15 wherein said sprouts, seeds, plants
or plant parts have at least 250,000 units per gram fresh weight
of Phase 2 enzyme-inducing potential.
23. The method of claim 15, wherein said plants are broccoli.
24. The method of claim 15, wherein said plant parts are from broccoli.
25. The method of claim 15, wherein said cruciferous sprouts are
broccoli sprouts.
26. The method of claim 15, wherein said cruciferous seeds are
broccoli seeds.
Cancer Patent Description
BACKGROUND OF THE INVENTION
I. Field of Invention
This invention relates to a dietary approach to reducing the level
of carcinogens in animals and their cells and thereby reducing the
risk of developing cancer. In particular, this invention relates
to the production and consumption of foods which are rich in cancer
chemoprotective compounds. More specifically, this invention relates
to chemoprotective compounds that modulate mammalian enzymes which
are involved in metabolism of carcinogens. This invention relates
to food sources which are extremely rich in compounds that induce
the activity of Phase 2 enzymes, without inducing biologically significant
activities of those Phase 1 enzymes that activate carcinogens.
II. Background
It is widely recognized that diet plays a large role in controlling
the risk of developing cancers and that increased consumption of
fruits and vegetables reduces cancer incidence in humans. It is
believed that a major mechanism of protection depends on the presence
of chemical components in plants that, when delivered to mammalian
cells, elevate levels of Phase 2 enzymes that detoxify carcinogens.
Early studies on the mechanism of chemoprotection by certain chemicals
assumed that these chemoprotectors induced activities of monooxygenases,
also known as Phase 1 enzymes or cytochromes P-450. However, Talalay
et al., [reviewed in "Chemical Protection Against Cancer by
Induction of Electrophile Detoxication (Phase II) Enzymes"
In: CELLULAR AND MOLECULAR TARGETS OF CHEMOPREVENTION, L. Wattenberg
et al., CRC Press, Boca Raton, Fla., pp 469-478 (1992)] determined
that administration of the known chemoprotector butylated hydoxyanisole
(BHA) to rodents resulted in little change in cytochromes P-450
(Phase 1 enzyme) activities, but profoundly elevated Phase 2 enzymes.
Phase 2 enzymes such as glutathione transferases, NAD(P)H:quinone
reductase (QR) and glucuronosyltransferases, detoxify DNA-damaging
electrophilic forms of ultimate carcinogens. Selective inducers
of Phase 2 enzymes are designated monofunctional inducers. Prochaska
& Talalay, Cancer Res. 48: 4776-4782 (1988). The monofunctional
inducers are nearly all electrophiles and belong to 8 distinct chemical
classes including (1) diphenols, phenylenediamines and quinones;
(2) Michael reaction acceptors containing olefins or acetylenes
conjugated to electron-withdrawing groups; (3) isothiocyanates;
(4) 1,2-dithiole-3-thiones; (5) hydroperoxides; (6) trivalent inorganic
and organic arsenic derivatives; (7) heavy metals with potencies
related to their affinities for thiol groups including Hg.sup.2+,
and Cd.sup.2+; and (8) vicinal dimercaptans. Prestera et al., Proc.
Natl. Acad. Sci. USA 90: 2963-2969 (1993). The only apparent common
property shared by all of these inducers is their ability to react
with thiol groups.
Chemoprotective agents can be used to reduce the susceptibility
of mammals to the toxic and neoplastic effects of carcinogens. These
chemoprotectors can be of plant origin or synthetic compounds. Synthetic
analogs of naturally occurring inducers have also been generated
and shown to block chemical carcinogenesis in animals. Posner et
al., J. Med. Chem. 37: 170-176 (1994); Zhang et al., Proc. Natl.
Acad. Sci. USA 91: 3147-3150 (1994); Zhang et al., Cancer Res. (Suppl)
54: 1976s-1981s (1994).
Highly efficient methods have been developed for measuring the
potency of plant extracts to increase or induce the activities of
Phase 2 enzymes. Prochaska & Santamaria, Anal. Biochem. 169:
328-336 (1988) and Prochaska et al., Proc. Natl. Acad. Sci. USA
89: 2394-2398 (1992). In addition, these methods have been employed
for isolating the compounds responsible for the inducer activities
in plants and for evaluating the anticarcinogenic activities of
these compounds and their synthetic analogs. Zhang et al., Proc.
Natl. Acad. Sci. USA 89: 2399-2403 (1992) and Posner et al., J.
Med. Chem. 17: 170-176 (1994).
Although inducer activity has been found in many different families
of edible plants, the amounts are highly variable, depending on
family, genus, species, variety, or cultivar of the plant selection
and on growth and harvesting conditions. Thus, there is a need in
the art to identify particular edible plants and methods of growing
and preparing them that yield high levels of Phase 2 enzyme-inducer
activity for chemoprotection. There is also a need for methods of
growing and preparing edible plants that produce a known spectrum
of specific inducers of Phase 2 enzyme activity in order to increase
the efficiency with which specific carcinogens, or classes of carcinogens,
are targeted for inactivation. In addition, there is a need for
methods of plant breeding and selection to increase the level of
Phase 2 inducer activity and to manipulate the spectrum of inducers
produced in particular cultivars.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide food products
and food additives that are rich in cancer chemoprotective compounds.
Another object of the present invention is to provide food products
which contain substantial quantities of Phase 2 enzyme-inducers
and are essentially free of Phase 1 enzyme-inducers.
It is a further object of the present invention to provide food
products which contain substantial quantities of Phase 2 enzyme-inducing
potential and non-toxic levels of indole glucosinolates and their
breakdown products and goitrogenic hydroxybutenyl glucosinolates.
These objects, and others, are achieved by providing cruciferous
sprouts, with the exception of cabbage, cress, mustard and radish
sprouts, harvested prior to the 2-leaf stage. The cruciferous sprouts
include Brassica oleracea varieties acephala, alboglabra, botrytis,
costata, gemmifera, gongylodes, italica, medullosa, palmifolia,
ramosa, sabauda, sabellica, and selensia.
Another embodiment of the present invention provides cruciferous
sprouts, with the exception of cabbage, cress, mustard and radish
sprouts, harvested prior to the 2-leaf stage, wherein the sprouts
are substantially free of Phase 1 enzyme-inducing potential.
Yet another embodiment of the present invention provides a non-toxic
solvent extract of cruciferous sprouts, with the exception of cabbage,
cress, mustard and radish sprouts, harvested prior to the 2-leaf
stage. The non-toxic solvent extract can be a water extract. In
addition, the water extract can comprise a cruciferous vegetable,
such as a cruciferous vegetable of the genus Raphanus, comprising
an active myrosinase enzyme.
Another embodiment of the present invention provides a food product
comprising cruciferous sprouts, with the exception of cabbage, cress,
mustard and radish sprouts, harvested prior to the 2-leaf stage;
extracts of the sprouts or cruciferous seeds; or any combination
of the sprouts or extracts.
A further embodiment of the present invention provides a method
of increasing the chemoprotective amount of Phase 2 enzymes in a
mammal, comprising the step of administering an effective quantity
of cruciferous sprouts, with the exception of cabbage, cress, mustard
and radish sprouts, harvested prior to the 2-leaf stage.
Yet another embodiment of the present invention provides a method
of increasing the chemoprotective amount of Phase 2 enzymes in a
mammal, comprising the step of administering an effective quantity
of a food product comprising cruciferous sprouts, with the exception
of cabbage, cress, mustard and radish sprouts, harvested prior to
the 2-leaf stage.
Another embodiment of the present invention provides cruciferous
sprouts harvested prior to the 2-leaf stage, wherein the sprouts
have at least 200,000 units per gram fresh weight of Phase 2 enzyme-inducing
potential when measured after 3 days of growth from seeds that produce
said sprouts and contain non-toxic levels of indole glucosinolates
and their breakdown products and goitrogenic hydroxybutenyl glucosinolates.
The cruciferous sprouts include Brassica oleracea varieties acephala,
alboglabra, botrytis, costata, gemmifera, gongylodes, italica, medullosa,
palmifolia, ramosa, sabauda, sabellica, and selensia.
A further embodiment of the present invention provides a food product
comprising sprouts harvested prior to the 2-leaf stage, wherein
the sprouts have at least 200,000 units per gram fresh weight of
Phase 2 enzyme-inducing potential when measured after 3 days from
growth of seeds that produce the sprouts and contain non-toxic levels
of indole glucosinolates and their breakdown products and goitrogenic
hydroxybutenyl glucosinolates; extracts of the sprouts or cruciferous
seeds; or any combination of the sprouts or extracts.
Yet another embodiment of the present invention provides cruciferous
sprouts harvested prior to the 2-leaf stage, wherein the sprouts
have at least 200,000 units per gram fresh weight of Phase 2 enzyme-inducing
potential when measured after 3 days of growth from seeds that produce
the sprouts and contain non-toxic levels of indole glucosinolates
and their breakdown products and goitrogenic hydroxybutenyl glucosinolates
and are substantially free of Phase 1 enzyme-inducing potential.
Another embodiment of the present invention provides a non-toxic
solvent extract of cruciferous sprouts harvested prior to the 2-leaf
stage, wherein the sprouts have at least 200,000 units per gram
fresh weight of Phase 2 enzyme-inducing potential when measured
after 3 days of growth from seeds that produce the sprouts and contain
non-toxic levels of indole glucosinolates and their breakdown products
and goitrogenic hydroxybutenyl glucosinolates. The non-toxic solvent
extract can be a water extract. In addition, the water extract can
comprise a cruciferous vegetable, such as a cruciferous vegetable
of the genus Raphanus, comprising an active myrosinase enzyme.
Yet another embodiment of the present invention provides a method
of increasing the chemoprotective amount of Phase 2 enzymes in a
mammal, comprising the step of administering an effective quantity
of cruciferous sprouts harvested prior to the 2-leaf stage, wherein
the sprouts have at least 200,000 units per gram fresh weight of
Phase 2 enzyme-inducing potential when measured after 3 days of
growth from seeds that produce the sprouts and contain non-toxic
levels of indole glucosinolates and their breakdown products and
goitrogenic hydroxybutenyl glucosinolates.
Yet another embodiment of the present invention provides a method
of increasing the chemoprotective amount of Phase 2 enzymes in a
mammal, comprising the step of administering an effective quantity
of a food product comprising sprouts harvested prior to the 2-leaf
stage, wherein the sprouts have at least 200,000 units per gram
fresh weight of Phase 2 enzyme-inducing potential when measured
after 3 days of growth from seeds that produce the sprouts and contain
non-toxic levels of indole glucosinolates and their breakdown products
and goitrogenic hydroxybutenyl glucosinolates.
A further embodiment of the present invention provides a method
of preparing a food product rich in glucosinolates, comprising germinating
cruciferous seeds, with the exception of cabbage, cress, mustard
and radish seeds, and harvesting sprouts prior to the 2-leaf stage
to form a food product comprising a plurality of sprouts. The cruciferous
sprouts include Brassica oleracea varieties acephala, alboglabra,
botrytis, costata, gemmifera, gongylodes, italica, medullosa, palmifolia,
ramosa, sabauda, sabellica, and selensia and contain non-toxic levels
of indole glucosinolates and their breakdown products and goitrogenic
hydroxybutenyl glucosinolates.
Yet another embodiment of the present invention provides a food
product rich in glucosinolates made by germinating cruciferous seeds,
with the exception of cabbage, cress, mustard and radish seeds,
and harvesting sprouts prior to the 2-leaf stage to form a food
product comprising a plurality of sprouts.
Yet another embodiment of the present invention provides a method
of preparing a food product comprising extracting glucosinolates
and isothiocyanates from cruciferous sprouts, with the exception
of cabbage, cress, mustard and radish sprouts, harvested prior to
the 2-leaf stage, with a non-toxic solvent and recovering the extracted
glucosinolates and isothiocyanates. Myrosinase enzyme, or a vegetable,
such as Raphanus species, containing the enzyme is mixed with the
cruciferous sprouts, the extract, or both the sprouts and the extract.
An embodiment of the present invention provides a method of preparing
a food product rich in glucosinolates, comprising germinating cruciferous
seeds having at least 200,000 units per gram fresh weight of Phase
2 enzyme-inducing potential when measured after 3 days of growth
from seeds that produce the sprouts and which contain non-toxic
levels of indole glucosinolates and their breakdown products and
goitrogenic hydroxybutenyl glucosinolates, and harvesting sprouts
prior to the 2-leaf stage to form a food product comprising a plurality
of sprouts. The seeds may be Brassica oleracea, including the varieties
acephala, albogabra, botrytis, costata, gemnifera, gongylodes, italica,
medullosa, palmifolia, ramosa, sabauda, sabellica, and selensia.
Yet another embodiment of the present invention provides a food
product rich in glucosinolates made by germinating cruciferous seeds
having at least 200,000 units per gram fresh weight of Phase 2 enzyme-inducing
potential when measured after 3 days of growth from seeds that produce
the sprouts and which contain non-toxic levels of indole glucosinolates
and their breakdown products and goitrogenic hydroxybutenyl glucosinolates,
and either harvesting sprouts at the 2-leaf stage to form a food
product comprising a plurality of sprouts. The nutritional product
contains non-toxic levels of indole glucosinolates and their breakdown
products and goitrogenic hydroxybutenyl glucosinolates.
A further embodiment of the present invention provides a method
of preparing a food product comprising extracting glucosinolates
and isothiocyanates with a solvent from cruciferous seeds, sprouts,
plants or plant parts, wherein seeds that produce the sprouts, plants
or plant parts producing sprouts having at least 200,000 units per
gram fresh weight of Phase 2 enzyme-inducing potential when measured
after 3 days of growth and wherein the seeds, sprouts, plants or
plant parts have non-toxic levels of indole glucosinolates and their
breakdown products and goitrogenic hydroxybutenyl glucosinolates,
and recovering the extracted glucosinolates and isothiocyanates.
The non-toxic extraction solvent can be water. Myrosinase enzyme,
or a vegetable, such as Raphanus species, containing the enzyme
is mixed with the cruciferous sprouts, seeds, plants, plant parts
or extract, or any combination thereof.
A further embodiment of the present invention provides a method
of reducing the level of carcinogens in mammals, comprising administering
cruciferous sprouts, with the exception of cabbage, cress, mustard
and radish sprouts.
Yet another embodiment of the present invention provides a method
of reducing the level of carcinogens in mammals, comprising administering
cruciferous sprouts having at least 200,000 units per gram fresh
weight of Phase 2 enzyme-inducing potential when measured after
3 days of growth from seeds that produce the sprouts and non-toxic
levels of indole glucosinolates and their breakdown products and
goitrogenic hydroxybutenyl glucosinolates.
Another embodiment of the present invention provides a method of
preparing a food product by introducing cruciferous seeds, having
at least 200,000 units per gram fresh weight of Phase 2 enzyme-inducing
potential when measured after 3 days of growth from seeds that produce
the sprouts and non-toxic levels of indole glucosinolates and goitrogenic
hydroxybutenyl glucosinolates, into an edible ingredient.
A further embodiment of the present invention provides a method
of extracting glucosinolates and isothiocyanates from plant tissue
which comprises homogenizing the plant tissue in an excess of a
mixture of dimethyl sulfoxide, acetonitrile, and dimethylformamide
(DMF/ACN/DMSO) at a temperature that prevents myrosinase activity.
Another embodiment of the present invention provides cruciferous
sprouts harvested prior to the 2-leaf stage, wherein the ratio of
monofunctional to bifunctional inducers is at least 20 to 1.
Another object of the present invention is to provide a food product
supplemented with a purified or partially purified glucosinolate.
Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
the specific examples, while indicating preferred embodiments of
the invention, are given by way of illustration only, since various
changes and modifications within the spirit and scope of the invention
will become apparent to those skilled in the art from this detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the total inducing potential of organic solvent extracts
of broccoli and daikon cultivars as a function of age.
FIG. 2 shows the high resolution NMR spectra of isolated glucosinolates
obtained from hot aqueous extracts of 3-day old Saga broccoli sprouts.
DETAILED DESCRIPTION
I. Definitions
In the description that follows, a number of terms are used extensively.
The following definitions are provided to facilitate understanding
of the invention.
A bifunctional inducer is a molecule which increases activities
of both Phase 1 enzymes such as cytochromes P-450 and Phase 2 enzymes
and requires the participation of Aryl hydrocarbon (Ah) receptor
and its cognate Xenobiotic Response Element (XRE). Examples include
flat planar aromatics such as polycyclic hydrocarbons, azo dyes
or 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD).
A chemoprotector or chemoprotectant is a synthetic or naturally
occurring chemical agent that reduces susceptibility in a mammal
to the toxic and neoplastic effects of carcinogens.
A food product is any ingestible preparation containing the sprouts
of the instant invention, or extracts or preparations made from
these sprouts, which are capable of delivering Phase 2 inducers
to the mammal ingesting the food product. The food product can be
freshly prepared such as salads, drinks or sandwiches containing
sprouts of the instant invention. Alternatively, the food product
containing sprouts of the instant invention can be dried, cooked,
boiled, lyophilized or baked. Breads, teas, soups, cereals, pills
and tablets, are among the vast number of different food products
contemplated.
Inducer activity or Phase 2 enzyme-inducing activity is a measure
of the ability of a compound(s) to induce Phase 2 enzyme activity.
In the present invention, inducer activity is measured by means
of the murine hepatoma cell bioassay of QR activity in vitro. Inducer
activity is defined herein as QR inducing activity in Hepa 1c1c7
cells (murine hepatoma cells) incubated with extracts of sprouts,
seeds or other plant parts untreated with myrosinase. Inducer activity
is measured in Hepa 1c1c7 murine hepatoma cells grown in 96-well
microtiter plates. Typically 10,000 Hepa 1c1c7 cells are introduced
into each well. Hepatoma cells are grown for 24 hours and a plant
extract containing microgram quantities of fresh plant tissue is
serially diluted across the microtiter plates into fresh culture
medium containing 0.15 ml .alpha.MEM culture medium amended with
10% Fetal Calf Serum (FCS) and streptomycin and penicillin. The
cells are further incubated for 48 hours. QR activity (based on
the formation of the blue-brown reduced tetrazolium dye) is measured
with an optical microtiter plate scanner in cell lysates prepared
in one plate, and related to its protein concentration. Quantitative
information on specific activity of QR is obtained by computer analysis
of the absorbances. One unit of inducer activity is the amount that
when added to a single microtiter well doubles the QR activity.
(See Prochaska and Santamaria, Anal. Biochem. 169: 328-336 (1988)
and Prochaska et al., Proc. Natl. Acad. Sci. USA 89: 2394-2398 (1992)).
Inducer potential or Phase 2 enzyme-inducing potential is a measure
of the combined amounts of inducer activity in plant tissue provided
by isothiocyanates, plus glucosinolates that can be converted by
myrosinase to isothiocyanates. Glucosinolates are not themselves
inducers of mammalian Phase 2 enzymes, whereas isothiocyanates are
inducers. Inducer potential therefore is defined herein as QR activity
in murine 1c1c7 hepatoma cells incubated with myrosinase-treated
extracts of the sprouts, seeds or other plant parts. In the present
invention therefore inducer potential is measured by means of the
murine hepatoma cell bioassay of QR activity in vitro as described
above. Inducer potential is measured in Hepa 1c1c7 murine hepatoma
cells grown in 96-well microtiter plates. Typically, 10,000 Hepa
1c1c7 cells are introduced into each well. Hepatoma cells are grown
for 24 hours and a plant extract containing microgram quantities
of fresh plant tissue is serially diluted across the microtiter
plates into fresh culture medium containing 0.15 ml .alpha.MEM culture
medium amended with 10% Fetal Calf Serum (FCS) and streptomycin
and penicillin. Myrosinase (6 units/ml plant extract) is added to
the plant extract. Myrosinase is purified by modification of the
technique of Palmieri et al., Anal. Biochem. 35: 320-324 (1982)
from 7 day old Daikon sprouts grown on agar support containing no
added nutrients. Following 234-fold purification, the myrosinase
had a specific activity of 64 units/mg protein [unit=amount of enzyme
required to hydrolyze 1 .mu.mol sinigrin/min]. Plant extract is
diluted 200-fold into the initial wells of the microtiter plate
followed by 7 serial dilutions. The cells are further incubated
for 48 hours. QR activity (based on the formation of the blue-brown
reduced tetrazolium dye) is measured with an optical microtiter
plate scanner in cell lysates prepared in one plate, and related
to its protein concentration. Quantitative information on specific
activity of QR is obtained by computer analysis of absorbances.
One unit of inducer potential is the amount that when added to a
single microtiter well doubles the QR activity. (See Prochaska and
Santamaria, Anal. Biochem. 169: 328-336 (1988) and Prochaska et
al., Proc. Natl. Acad. Sci. USA 89: 2394-2398 (1992)).
A monofunctional inducer increases the activity of Phase 2 enzymes
selectively without significantly altering Phase 1 enzyme activities.
Monofunctional inducers do not depend on a functional Ah receptor
but enhance transcription-of Phase 2 enzymes by means of an Antioxidant
Responsive Element (ARE).
A cruciferous sprout is a plant or seedling that is at an early
stage of development following seed germination. Cruciferous seeds
are placed in an environment in which they germinate and grow. The
cruciferous sprouts of the instant invention are harvested following
seed germination through and including the 2-leaf stage. The cruciferous
sprouts of instant invention have at least 200,000 units per gram
fresh weight of Phase 2 enzyme-inducing potential at 3-days following
incubation under conditions in which cruciferous seeds germinate
and grow.
II. Description
A major mechanism of protection provided by fruits and vegetables
in reducing the cancer incidence in humans depends on minor chemical
components which, when delivered to mammalian cells, elevate levels
of Phase 2 enzymes that detoxify carcinogens. It has now been discovered
that the anticarcinogenic activity of certain edible plants can
be increased. Plants such as Brassica oleracea variety italica (broccoli)
are normally not harvested until they form heads. By growing these
plants only to the seedling or sprout stage, that is between the
onset of germination and the 2-leaf stage, the levels of inducers
of enzymes that detoxify carcinogens and protect against cancer
can be increased at least five-fold over those found in commercial
stage vegetables of the same cultivars. Often increases of between
10 and 1000-fold have been observed.
Harvesting plants at an early seedling or sprout stage, or otherwise
arresting their growth, leads to the greatest inducer potential
and yields a food product of a type to which consumers are already
accustomed. The Phase 2 enzyme-inducing potential of such sprouts
may be as much as several hundred times higher than that observed
in adult, market stage vegetables obtained from the same seeds.
Thus it is possible that humans can consume the same quantities
of inducer potential by eating relatively small quantities of sprouts,
rather than large quantities of market-stage vegetables.
It has now been found that most of the inducer potential of crucifer
plants is due to their content of isothiocyanates and their biogenic
precursors, glucosinolates. Glucosinolates are converted to isothiocyanates
by the enzyme myrosinase which is a thioglucosidase. Normally myrosinase
and glucosinolates are separated in the cell and if the cell is
damaged, with loss of compartmentalization, myrosinase comes into
contact with glucosinolates, which are then converted to isothiocyanates.
In order to screen large numbers of edible plants and to evaluate
the effects of environmental perturbation on Phase 2 enzyme-inducer
potential in those vegetables, it was necessary to improve upon
the previously described techniques for homogenization and extraction
of those vegetables. Techniques initially described for the extraction
of Phase 2 inducers from vegetables involved homogenization of the
vegetables in cold water, lyophilization, extraction of the resultant
powder with acetonitrile, filtration and evaporative concentration,
Prochaska et al., Proc. Natl. Acad. Sci. USA 89: 2394-2398 (1992).
Following identification of sulforaphane as the principal Phase
2 inducer from broccoli, comparative extractions were performed
into hot 80% methanol, yielding similar inducer activity as the
aforementioned acetonitrile extracts. When myrosinase was added
to these hot methanol extracts in which glucosinolates are freely
soluble, there was a dramatic enhancement of the Phase 2 inducer
activity of these extracts (data summarized in Table 1). The deliberate
conversion of these glucosinolates to isothiocyanates using exogenous
myrosinase thus gave a better index of the inducers for Phase 2
enzymes of the vegetables tested. It was thus clear that the majority
of the potential Phase 2 inducers in crucifers was usually present
in whole plants as the glucosinolate precursors of isothiocyanates.
The preponderance of glucosinolates and the rapidity with which,
upon wounding of cruciferous plant tissue, glucosinolates are converted
to isothiocyanates, led to the development of an improved extraction
procedure. By manipulation of solvent mixtures and of the water
activity of fresh vegetable/solvent homogenates, a procedure was
developed that permits both glucosinolate and isothiocyanate quantification
from the same, non-concentrated sample. In addition to being the
rate-limiting step in an extraction protocol, evaporative concentration
allows volatile inducers to escape detection. The improved procedure
is both simple and efficient, requiring only that the plant sample
be completely homogenized in solvent. Using this technique, the
present inventors have thus been able to demonstrate dramatic increases
in the recovery of inducer activity and inducer potential from cruciferous
vegetables over previously described techniques.
If fresh-picked vegetables are promptly and gently harvested, directly
into organic solvents comprising a mixture of DMF/ACN/DMSO and a
temperature that prevents myrosinase activity, both glucosinolates
and isothiocyanates are efficiently extracted into the organic solvent
mixture. Preferably, the DMF, ACN and DMSO are mixed in equal volumes.
However, the volumes of the three solvents in the mixture can be
varied to optimize extraction of specific glucosinolates and isothiocyanates
from any plant tissue. The temperature of the extraction mixture
is preferably less than 0.degree. C., and most preferably less than
-50.degree. C. The temperature of the extraction solvent must be
kept above freezing. At the same time the enzyme myrosinase, which
invariably accompanies these constituents in the plants and rapidly
converts glucosinolates into isothiocyanates, is inactive. Such
extracts typically contain high quantities of glucosinolates and
negligible quantities of isothiocyanates. The in planta myrosinase
activity varies between different plant species.
Glucosinolates are not themselves inducers of mammalian Phase 2
enzymes, whereas isothiocyanates are monofunctional inducers in
the murine hepatoma cell bioassay of QR activity. The inducer potential,
as distinct from inducer activity, of plant extracts can be measured
by adding purified myrosinase, obtained from the same, or other
plant sources, to the assay system.
Glucosinolates are converted at least partially to isothiocyanates
in humans. If, however, it is desirable to accelerate this conversion,
broccoli or other vegetable sprouts, high in glucosinolates, can
be mixed with myrosinase. The mixture can be in water, or some other
non-toxic solvent that does not inactivate myrosinase. The myrosinase
can be from a partially purified or purified preparation. Alternatively,
the myrosinase can be present in plant tissue, such as a small quantity
of crucifer sprouts rich in myrosinase, including Raphanus sativus
or daikon. Such a preparation can be used to produce a "soup"
for ingestion that is high in isothiocyanates and low in glucosinolates.
Inducer potential can be measured using a multiwell plate screen
with murine hepatoma cells for in vitro measurement of QR specific
activity as described above.
The ratio of monofunctional to bifunctional inducer activity of
plant tissue is measured by bioassaying plant extracts, as described
above, not only in wild-type Hepa 1c1c7 cells, but also, in mutants
designated c1 and BP.sup.rc1 that have either defective Ah receptors
or defective cytochrome P.sub.1-450 genes, respectively. Prochaska
and Talalay, Cancer Research 48: 4776-4782 (1988).
A harvested sprout according to the present invention can be incorporated
immediately into food products such as fresh salads, sandwiches
or drinks. Alternatively, the growth of the harvested sprout can
be arrested by some active human intervention, for example by refrigeration,
at a stage of growth prior to the 2-leaf stage, typically between
1 and 14 days after germination of seeds. Growth arrest can also
be accomplished by removing a sprout from its substrate and/or water
source. Freezing, drying, baking, cooking, lyophilizing and boiling
are among the many treatments that can be used to arrest growth.
These may also be useful for either preserving myrosinase activity
in the sprout (e.g., lyophilizing) or for inactivating myrosinase
activity in the sprout (e.g., boiling), as is desired in a particular
application.
The harvested sprout can also be allowed to mature further, under
different growing conditions, prior to incorporation into a food
product. For example, the sprout can be harvested at a very young
age of development, such as 1 to 2 days after seed imbibition. The
sprout can then be allowed to mature under different growing conditions,
such as increased or decreased light intensity, temperature or humidity;
exposure to ultraviolet light or other stresses; or addition of
exogenous nutrients or plant growth regulators (hormones). The sprout
is then immediately incorporated into a food product, such as for
fresh consumption in salads. Alternatively, the growth of the sprout
is arrested and/or further treated by means of lyophilization, drying,
extracting with water or other solvents, freezing, baking, cooking,
or boiling, among others.
A sprout is suitable for human consumption if it does not have
non-edible substrate such as soil attached or clinging to it. Typically
the sprouts are grown on a non-nutritive solid support, such as
agar, paper towel, blotting paper, Vermiculite, Perlite, etc., with
water and light supplied. Thus, if a sprout is not grown in soil,
but on a solid support, it does not need to be washed to remove
non-edible soil. If a sprout is grown in a particulate solid support,
such as soil, Vermiculite, or Perlite, washing may be required to
achieve a sprout suitable for human consumption.
Sprouts can be grown in containers which are suitable for shipping
and marketing. Typically such containers are plastic boxes or jars
which contain a wetted pad at the bottom. The containers allow light
to penetrate while providing a mechanically protective barrier.
Numerous methods for the cultivation of sprouts are known, as exemplified
by U.S. Pat. Nos. 3,733,745, 3,643,376, 3,945,148, 4,130,964, 4,292,760
or 4,086,725. Food products containing the sprouts of the instant
invention can be stored and shipped in diverse types of containers
such as jars, bags and boxes, among many others.
Sprouts suitable as sources of cancer chemoprotectants are generally
cruciferous sprouts, with the exception of cabbage (Brassica oleracea
capitata), cress (Lepidiumsativum), mustard (Sinapis alba and S.
niger) and radish (Raphanus sativus) sprouts. The selected sprouts
are typically from the family Cruciferae, of the tribe Brassiceae,
and of the subtribe Brassicinae. Preferably the sprouts are Brassica
oleracea selected from the group of varieties consisting of acephala
(kale, collards, wild cabbage, curly kale), medullosa (marrowstem
kale), ramosa (thousand head kale), alboglabra (Chinese kale), botrytis
(cauliflower, sprouting broccoli), costata (Portuguese kale), gemmifera
(Brussels sprouts), gongylodes (kohlrabi), italica (broccoli), palmifolia
(Jersey kale), sabauda (savoy cabbage), sabellica (collards), and
selensia (borecole), among others.
Particularly useful broccoli cultivars to be used in the claimed
method are Saga, DeCicco, Everest, Emerald City, Packman, Corvet,
Dandy Early, Emperor, Mariner, Green Comet, Green Valiant, Arcadia,
Calabrese Caravel, Chancellor, Citation, Cruiser, Early Purple Sprouting
Red Arrow, Eureka, Excelsior, Galleon, Ginga, Goliath, Green Duke,
Greenbelt, Italian Sprouting, Late Purple Sprouting, Late Winter
Sprouting White Star, Legend, Leprechaun, Marathon, Mariner, Minaret
(Romanesco), Paragon, Patriot, Premium Crop, Rapine (Spring Raab),
Rosalind, Salade (Fall Raab), Samurai, Shogun, Sprinter, Sultan,
Taiko, and Trixie. However, many other broccoli cultivars are suitable.
Particularly useful cauliflower cultivars are Alverda, Amazing,
Andes, Burgundy Queen, Candid Charm, Cashmere, Christmas White,
Dominant, Elby, Extra Early Snowball, Fremont, Incline, Milkyway
Minuteman, Rushmore, S-207, Serrano, Sierra Nevada, Siria, Snow
Crown, Snow Flake, Snow Grace, Snowbred, Solide, Taipan, Violet
Queen, White Baron, White Bishop, White Contessa, White Corona,
White Dove, White Flash, White Fox, White Knight, White Light, White
Queen, White Rock, White Sails, White Summer, White Top, Yukon.
However, many other cauliflower cultivars are suitable.
Suitable sprouts will have at least 200,000 units per gram of fresh
weight of Phase 2 enzyme-inducing potential following 3-days incubation
of seeds under conditions in which the seeds germinate and grow.
Preferably the sprouts will have at least 250,000 units of inducer
potential per gram of fresh weight, or even 300,000 units, 350,000
units, 400,000 units, or 450,000 units. Some samples have been found
to contain greater than 500,000 units per gram of fresh weight at
3-days of growth from seeds.
The level of inducing activity and inducing potential has been
found to vary among crucifers and even among cultivars. Most preferably,
the sprouts are substantially free of indole glucosinolates and
their breakdown products which have Phase 1 enzyme-inducing potential
in mammalian cells, and substantially free of toxic levels of goitrogenic
nitriles and glucosinolates such as hydroxybutenyl glucosinolates,
which upon hydrolysis yield oxazolidonethiones which are goitrogenic.
Mature Brussels sprouts and rapeseed are rich in these undesirable
glucosinolates.
Non-toxic solvent extracts according to the invention are useful
as healthful infusions or soups. Non-toxic or easily removable solvents
useful for extraction according to the present invention include
water, liquid carbon dioxide or ethanol, among others. The sprouts
can be extracted with cold, warm, or preferably hot or boiling water
which denature or inactivate myrosinase. The residue of the sprouts,
post-extraction, may or may not be removed from the extract. The
extraction procedure may be used to inactivate myrosinase present
in the sprouts. This may contribute to the stability of the inducer
potential. The extract can be ingested directly, or can be further
treated. It can, for example, be evaporated to yield a dried extracted
product. It can be cooled, frozen, or freeze-dried. It can be mixed
with a crucifer vegetable which contains an active myrosinase enzyme.
This will accomplish a rapid conversion of the glucosinolates to
isothiocyanates, prior to ingestion. Suitable vegetables that contain
active myrosinase are of the genus Raphanus, especially daikon,
a type of radish.
Seeds, as well as sprouts have been found to be extremely rich
in inducer potential. Thus it is within the scope of the invention
to use crucifer seeds in food products. Suitable crucifer seeds
may be ground into a flour or meal for use as a food or drink supplement.
The flour or meal is incorporated into breads, other baked goods,
or health drinks or shakes. Alternatively, the seeds may be extracted
with a non-toxic solvent such as water, liquid carbon dioxide or
ethanol to prepare soups, teas or other drinks and infusions. The
seeds can also be incorporated into a food product without grinding.
The seeds can be used in many different foods such as salads, granolas,
breads and other baked goods, among others.
Food products of the instant invention may include sprouts, seeds
or extracts of sprouts or seeds taken from one or more different
crucifer genera, species, varieties, subvarieties or cultivars.
It has been found that genetically distinct crucifers produce chemically
distinct Phase 2 enzyme-inducers. Different Phase 2 enzyme-inducers
detoxify chemically distinct carcinogens at different rates. Accordingly,
food products composed of genetically distinct crucifer sprouts
or seeds, or extracts or preparations made from these sprouts or
seeds, will detoxify a broader range of carcinogens.
Glucosinolates and/or isothiocyanates can be purified from seed
or plant extracts by methods well known in the art. See Fenwick
et al., CRC Crit. Rez. Food Sci. Nutr. 123-201 (1983) and Zhang
et al., Pro. Natl Acad. Sci. USA 89: 2399-2403 (1992). Purified
or partially purified glucosinolate(s) or isothiocyanate(s) can
be added to food products as a supplement. The dose of glucosinolate
and/or isothiocyanate added to the food product preferably is in
the range of 1 .mu.mol to 1,000 .mu.mols. However, the dose of glucosinolate
and/or isothiocyanate supplementing the food product can be higher.
The selection of plants having high Phase 2 enzyme-inducer potential
in sprouts, seeds or other plant parts can be incorporated into
Cruciferae breeding programs. In addition, these same breeding programs
can include the identification and selection of cultivars that produce
specific Phase 2 enzyme-inducers, or a particular spectrum of Phase
2 enzyme-inducers. Strategies for the crossing, selection and breeding
of new cultivars of Cruciferae are well known to the skilled artisan
in this field. Brassica Crops and Wild Allies: Biology & Breeding;
S. Tsunoda et al. (eds), Japan Scientific Societies Press, Tokyo
pp. 354 (1980). Progeny plants are screened for Phase 2 inducer
activity or the chemical identity of specific Phase 2 enzyme-inducers
produced at specific plant developmental stages. Plants carrying
the trait of interest are identified and the characteristic intensified
or combined with other important agronomic characteristics using
breeding techniques well known in the art of plant breeding.
EXAMPLE 1
Comparison of Cruciferous Sprout Inducing Potential
Sprouts were prepared by first surface sterilizing seeds of different
species from the cruciferae family with a 1 min treatment in 70%
ethanol, followed by 15 min in 1.3% sodium hypochlorite containing
approximately 0.001% Alconox detergent. Seeds were grown in sterile
plastic containers at a density of approximately 8 seeds/cm.sup.2
for from 1 to 9 days on a 0.7% agar support that did not contain
added nutrients. The environment was carefully controlled with broad
spectrum fluorescent lighting, humidity and temperature control.
The seeds and sprouts were incubated under a daily cycle of 16 hours
light at 25.degree. C. and 8 hours dark at 20.degree. C.
Sprouts were harvested following 3-days of incubation and immediately
plunged into 10 volumes of a mixture of equal volumes of DMF/ACN/DMSO
at -50.degree. C. This solvent mixture has a freezing point of approximately
-33.degree. C., but when admixed with 10% water, as found in plant
material, the freezing point is depressed to below -64.degree. C.
The actual freezing point depression is even greater with plant
material.
Homogenization was accomplished either by manually grinding the
samples in a glass-on-glass homogenizer in the presence of a small
amount of the total solvent used, then gradually adding more solvent
or homogenizing the sample in 10 volumes of solvent using a Brinkman
Polytron Homogenizer for 1 min at half-maximum power. The homogenate
was then centrifuged to remove remaining particulates and stored
at -20.degree. C. until assayed.
Inducer potential of plant extracts prepared as described above,
was determined by the microtiter plate bioassay method as described
in the Definitions section above.
Broccoli and cauliflower sprouts harvested and assayed at 3-days
after incubation of seeds under growth conditions have Phase 2 enzyme-inducer
potential greater than 200,000 units/g fresh weight. On the other
hand, cabbage, radish, mustard and cress have Phase 2 enzyme-inducer
potential of less than 200,000 units/g fresh weight when assayed
at the same time point.
EXAMPLE 2
Variation in Inducer Potential Among Differ Broccoli Cultivars
There is variation in inducer potential among different broccoli
cultivars. In addition, most of the inducer potential in crucifers
is present as precursor glucosinolates. The inducer activity and
inducer potential of market stage broccoli heads was determined
following DMF/ACN/DMSO extractions and assay of QR activity as described
above.
Bioassay of homogenates of such market stage broccoli heads, with
and without the addition of purified plant myrosinase, showed that
the amount of QR activity found in the absence of myrosinase was
less than 5% of that observed with added myrosinase. These observations
confirmed previous suggestions (see Matile et al., Biochem. Physiol.
Pflanzen 179: 5-12 (1984)) that uninjured plants contain almost
no free isothiocyanates.
TABLE-US-00001 TABLE 1 Effect of Myrosinase on Inducer Activity
of Market-Stage Broccoli Plant Heads Units per gram (wet weight)
Broccoli vegetable cultivar - myrosinase + myrosinase DeCicco 5,882
37,037 Calabrese Corvet 1,250 41,666 Everest * 8,333 Dandy Early
* 20,000 Emperor * 13,333 Saga 5,000 13,333 Emerald City * 12,500
* Below limits of detection (833 units/g).
As can be observed in Table 1, most of the plant inducer potential
is derived from glucosinolates following hydrolysis by myrosinase
to form isothiocyanates. Hence, hydrolysis is required for biological
activity.
EXAMPLE 3
Inducer Potential is Highest in Seeds and Decreases as Sprouts
Mature
Phase 2 enzyme-inducer potential is highest in seeds and decrease
gradually during early growth of seedlings. Plants were prepared
by first surface sterilizing seeds of Brassica oleracea variety
italica cultivars Saga and DeCicco with a 1 min treatment in 70%
ethanol, followed by 15 min in 1.3% sodium hypochlorite containing
approximately 0.001% Alconox detergent. Seeds were grown in sterile
plastic containers at a density of approximately 8 seeds/cm.sup.2
on a 0.7% agar support that did not contain added nutrients. The
environment was carefully controlled with broad spectrum fluorescent
lighting, humidity and temperature control. The seeds and sprouts
were incubated under a daily cycle of 16 hours light at 25.degree.
C. and 8 hours dark at 20.degree. C.
Each day plants were rapidly and gently collected from the surface
of the agar from replicate containers. The plants were harvested
gently to minimize glucosinolate hydrolysis by endogenous myrosinase
released upon plant wounding. Samples containing approximately 40
sprouts were homogenized in 10 volumes of DMF/ACN/DMSO solvent at
-50.degree. C. which dissolves nearly all the non-lignocellulosic
plant material.
Harvested plants were homogenized and QR activity with and without
myrosinase, was determined as described above. As can be seen in
FIG. 1, Phase 2 enzyme-inducer potential per gram of plant is highest
in seeds, but decreases gradually following germination. No detectable
(less than 1000 units/g) QR inducer activity was present in the
absence of added myrosinase.
EXAMPLE 4
Sprouts Have Higher Inducer Potential Than Market Stage Plants
The cruciferous sprouts of the instant invention have higher Phase
2 enzyme-inducer potential than market stage plants. More specifically,
sprouts have at least a 5-fold greater Phase 2 enzyme-inducing potential
than mature vegetables. For example, total inducing potential of
7-day-old broccoli sprouts, extracted with DMF/ACN/DMSO and treated
with myrosinase, as described above, were 238,000 and 91,000 units/g
fresh weight, compared to 25,000 and 20,000 units/g fresh weight
for field-grown heads of broccoli cultivars Saga and DeCicco, respectively.
Sprout extracts of over 40 different members of the Cruciferae
have now been bioassayed and broccoli sprouts remain the most Phase
2 enzyme-inducer-rich plants tested. Total inducing potential of
organic solvent extracts of market stage and sprout stage broccoli
and daikon is shown in Table 2.
TABLE-US-00002 TABLE 2 Comparison of Inducer Potential in Sprouts
and Mature Vegetables Activity (units/g fresh weight) Vegetable
Mature - Fold Cultivar* Vegetable Sprout** Difference DAIKON Miura
625 26,316 42 Tenshun 3,333 33,333 10 Hakkai 1,471 16,667 11 Ohkura
2,857 50,000 18 BROCCOLI Saga 25,000 476,000 19 DeCicco 25,000 625,000
25 Everest 8,333 1,087,000 130 Emerald City 12,500 833,000 67 Packman
20,000 556,000 28 *The commercial portion of each plant was sampled
(e.g. the taproot of Raphanus sativus variety radicola [radish]),
and heads of Brassicsa oleracea variety izalica [broccoli]). Myrosinase
was added to all extracts tested. **Broccoli sprouts were 1-day
old and daikon seedlings were 4-5-days old.
Sprouts of the broccoli cultivar Everest contained 130-fold more
inducer potential (units/g fresh weight) than mature vegetables.
The inducer activity in broccoli was significantly higher than in
daikon.
EXAMPLE 5
Inducer Potential of Broccoli Sprout Extracts
Inducer potential of a series of water extracts of 3-day old broccoli
sprouts of the cultivar Saga were determined. Plants were prepared
by first surface sterilizing seeds of Brassica oleracea variety
italica (broccoli) cultivar Saga by a 1 min treatment in 70% ethanol,
followed by 15 min in 1.3% sodium hypochlorite containing approximately
0.001% Alconox detergent. Seeds were grown in sterile plastic containers
at a density of approximately 8 seeds/cm.sup.2 for 72 hours on a
0.7% agar support that did not contain added nutrients. The environment
was carefully controlled with broad spectrum fluorescent lighting,
humidity and temperature control (16 hours light, 25.degree. C./8
hours dark, 20.degree. C.).
Plants were rapidly and gently collected from the surface of the
agar to minimize glucosinolate hydrolysis by endogenous myrosinase
released upon plant wounding. Sprouts (approximately 25 mg fresh
wt/sprout) were gently harvested and immediately and rapidly plunged
into approximately 3 volumes of boiling water in order to inactivate
endogenous myrosinase as well as to extract glucosinolates and isothiocyanates
from the plant tissue. Water was returned to a boil and maintained
at a rolling boil for 3 min. The sprouts were then either strained
from the boiled infusion [tea, soup] or homogenized in it, and the
residue then removed by filtration or centrifugation.
Data in Table 3. represent both homogenates and infusions. Preparations
were stored at -20.degree. C. until assayed. Inducer potential of
plant extracts, prepared as described above, was determined as described
in Definitions section above.
TABLE-US-00003 TABLE 3 Inducer Potentials of Hot Water Extracts
of 3-Day Saga Broccoli Sprouts EXTRACT NO. units/g fresh weight
1 500,000 2 370,000 3 455,000 4 333,000 5 435,000 6 333,000 7 625,000
8 250,000 9 313,000 10 357,000 11 370,000 12 370,000 13 217,000
14 222,000 15 1,000,000 16 714,000 17 435,000 18 1,250,000 19 263,000
AVERAGE 464,000 .+-. 61,600 S.E.M.
Some variability in the amount of Phase 2 enzyme-inducer potential
was detected. High levels of Phase 2 enzyme-inducer potential, however,
were consistently observed.
EXAMPLE 6
Hot Water Broccoli Extracts Treated with Daikon Myrosinase
QR activity in a hot water broccoli extract increased in the presence
of a vegetable source of myrosinase. An aqueous extraction of 3-day
old sprouts of broccoli cultivar Saga grown on water agar, in which
myrosinase was inactivated by boiling for 3 min, was divided into
6 different 150 ml aliquots. Nine-day old daikon sprouts, a rich
source of the enzyme myrosinase, were added to this cooled infusion
in amounts equivalent to 0, 5, 9, 17, 29 and 40% (w/w) of the broccoli.
QR activity, as determined in the Definition section, of the control
extracts containing 0% daikon was 26,300 units/gram fresh weight
while QR activity of the extracts that had received daikon as a
source of myrosinase ranged from 500,000 to 833,000 units/gram fresh
weight of broccoli. Accordingly, myrosinase present in the daikon
sprouts, increased the QR activity in the broccoli extract greater
than 19-fold.
EXAMPLE 7
Glucoraphanin and Glucoerucin are the Predominant Glucosinolates
in Hot Water Extracts of Broccoli (Cultivar Saga) Sprouts
Paired Ion Chromatography (PIC).
Centrifuged hot water extracts of 3-day-old broccoli (cultivar
Saga) sprouts were subjected to analytical and preparative PIC on
a reverse phase C18 Partisil ODS-2 HPLC column in ACN/H.sub.2O (1/1,
by vol.) with tetraoctylammonium (TOA) bromide as the counter-ion.
Only three well-separated peaks were detected: peak A eluted at
5.5 min, B at 11.5 min, and C at 13 min at a molar ratio [A:B:C]
of ca. 2.5:1.6:1.0 (monitored by UV absorption at 235 nm), and they
disappeared if the initial extracts were first treated with highly
purified myrosinase. Peaks A, B, and C contained no significant
inducer activity, and cyclocondensation assay of myrosinase hydrolysates
showed that only Peaks A and C produced significant quantities of
isothiocyanates, accounting for all the inducer activity. See Zhang
et al., Anal. Biochem. 205: 100-107 (1992). Peak B was not further
characterized. Peaks A and C were eluted from HPLC as TOA salts
but required conversion to ammonium salts for successful mass spectroscopy,
NMR and bioassay. The pure peak materials were dried in a vacuum
centrifuge, redissolved in aqueous 20 mM NH.sub.4Cl, and extracted
with chloroform to remove excess TOA bromide. The ammonium salts
of glucosinolates remained in the aqueous phase, which was then
evaporated.
Identification of Glucosinolates.
The ammonium salts of Peaks A and C were characterized by mass
spectrometric and NMR techniques: (a) negative ion Fast Atom Bombardment
(FAB) on a thioglyerol matrix; this gave values of 436 (Peak A)
and 420 (Peak C) amu for the negative molecular ions, and (b) high
resolution NMR, as shown in FIG. 2, provided unequivocal identification
of the structure. Peak A is glucoraphanin [4-methylsulfinylbutyl
glucosinolate], and Peak C is the closely related glucoerucin [4-methythiobutyl
glucosinolate]. These identifications and purity are also consistent
with the inducer potencies; Peaks A and C, after myrosinase hydrolysis
had potencies of 36,100 and 4,360 units/.mu.mol, respectively, compared
with reported CD values of 0.2 .mu.M (33,333 units/.mu.mol) for
sulforaphane and 2.3 .mu.M (2,900 units/.mu.mol) for erucin. CD
values are the concentrations of a compound required to double the
QR specific activity in Hepa 1c1c7 murine hepatoma cells. Since
there are no other glucosinolate peaks, and the inducer activity
of peak A and C account for the total inducer activity of the extracts,
it is therefore likely that in this cultivar of broccoli, there
are no significant quantities of other inducers, i.e., no indole
or hydroxyalkenyl glucosinolates. Further, the isolated compounds
are therefore substantially pure.
EXAMPLE 8
Comparison of Aqueous and Organic Solvent Techniques for Extraction
of Inducer Potential
Plants were prepared by first surface sterilizing seeds of Brassica
oleracea variety italica (broccoli) cultivar Saga, with 70% ethanol
followed by 1.3% sodium hypochlorite and 0.001% alconox. The seeds
were grown in sterile plastic containers at a density of approximately
8 seeds/cm.sup.2 for 72 hours on a 0.7% agar support that did not
contain added nutrients. The environment was carefully controlled
with broad spectrum fluorescent lighting, humidity, and temperature
control (16 hours light, 25.degree. C./8 hours dark, 20.degree.
C.).
The plants were rapidly and gently collected from the surface of
the agar to minimize glucosinolate hydrolysis by endogenous myrosinase
released upon plant wounding. A portion of the plants was homogenized
with 10 volumes of the DMF/ACN/DMSO solvent at -50.degree. C., as
described in Example 1, which dissolves nearly all the non-lignocellulosic
plant material. Alternatively, the bulk of the harvested plants
was plunged into 5 volumes of boiling water for 3 min to inactivate
endogenous myrosinase and to extract glucosinolates and isothiocyanates.
The cooled mixture was homogenized, centrifuged, and the supernant
fluid was stored at -20.degree. C.
Inducer potential of plant extracts, prepared by the two methods
described above, was determined by the microtiter plate bioassay
as described above. Typical inducer potentials in-an average of
5 preparations were 702,000 (DMF/ACN/DMSO extracts) and 505,000
(aqueous extracts) units/g fresh weight of sprouts.
Spectrophotometric quantitation of the cyclocondensation product
of the reaction of isothiocyanates with 1,2-benzenedithiole was
carried out as described in Zhang et al., Anal. Biochem. 205: 100-107
(1992). Glucosinolates were rapidly converted to isothiocyanates
after addition of myrosinase. About 6% of the total hot water extractable
material [dissolved solids] consisted of glucosinolates. These results
demonstrate that (a) isothiocyanate levels in the crude plant extracts
are extremely low; (b) myrosinase rapidly converts abundant glucosinolates
to isothiocyanates; (c) hot water extraction releases over 70% of
the inducer activity extractable with a triple solvent mixture permitting
recovery of most of the biological activity in a preparation that
is safe for human consumption; and (d) over 95% of the inducing
potential in the intact plant is present as glucosinolates and therefore
no other inducers are present in biologically significant quantities.
EXAMPLE 9
Development Regulation of Glucosinolate Production
Preliminary experiments in which field grown broccoli (cultivar
DeCicco) was harvested at sequential time points from the same field
indicated that on a fresh weight basis, inducer potential declined
from the early vegetative stage through commercial harvest, but
appeared to increase at late harvest (onset of flowering). These
data suggested that inducer potential might be highest in seeds.
Subsequent studies have shown that when seeds of 8 broccoli cultivars
were surface sterilized and grown under gnotobiotic conditions,
Phase 2 enzyme-inducer potential was highest in seeds and declined
progressively (on a fresh weight basis) over time throughout the
first 14 days of seedling growth.
Expressed on a per plant basis, however, activity remained constant
over this period, suggesting that at this early stage of growth
there was no net synthesis of glucosinolates. However, when the
glucosinolate profiles of market stage broccoli heads and 3 day
old sprouts (cultivar Emperor) were compared, there was a profound
difference in the apparent glucosinolate compositions of these plants.
Sprouts were prepared by first surface sterilizing seeds of Brassica
oleracea variety italica (broccoli) cultivar Emperor with a 1 minute
treatment in 70% ethanol, followed by 15 min in 1.3% sodium hypochlorite
with approximately 0.001% Alconox detergent. Seeds were grown in
sterile plastic containers at a density of approximately 8 seeds/cm.sup.2
for 72 hours on a 0.7% agar support that did not contain added nutrients.
The environment was carefully controlled; broad spectrum fluorescent
lighting, humidity and temperature control (16 hours light, 25.degree.
C./8 hours dark, 20.degree. C.).
Plants were rapidly and gently collected from the surface of the
agar to minimize glucosinolate hydrolysis by endogenous myrosinase
released upon plant wounding. Sprouts [approximately 25 mg fresh
wt/sprout], were gently harvested and immediately and rapidly plunged
into approximately 3 volumes of boiling water in order to inactivate
endogenous myrosinase as well as to extract glucosinolates and isothiocyanates
from the plant tissue. Water was returned to a boil and maintained
at a rolling boil for 3 min. The sprouts were then strained from
the boiled infusion [tea, soup] and the infusion was stored at -20.degree.
C. until assayed.
Market stage heads were obtained by germinating seeds of the same
seedlot in a greenhouse in potting soil, transplanting to an organically
managed field in Garrett County, md and harvested at market stage.
Heads were immediately frozen upon harvest, transported to the laboratory
on ice and extracts were prepared in an identical fashion to those
described above for sprouts except that approximately 3 gram floret
tissue samples were used for extraction.
Inducer potential of plant extracts, prepared as described above,
was determined by the microtiter plate bioassay method as described
in Example 1. Paired ion chromatography revealed two major peaks,
probably glucobrassicin and neo-glucobrassicin, in extracts of market
stage heads with similar retention times to glucobrassicin (indole-3-ylmethyl
glucosinolate) and neo-glucobrassicin (1-methoxyindole-3-ylmethyl
glucosinolate). This observation is consistent with published reports
on the glucosinolate composition of mature broccoli plants. However,
paired ion chromatography under the same conditions of identically
prepared extracts of 3-day-old sprouts showed absence of glucobrassicin
or neo-glucobrassicin. Additionally, 3-day-old sprouts of different
broccoli cultivars produce different mixtures of glucosinolates.
Accordingly, glucosinolate production is developmentally regulated.
EXAMPLE 10
Evaluation of Anticarcinogenic Activities of Broccoli Sprout Preparations
in the Huggins DMBA (9,10 Dimethyl-1,2-Benzanthracene) Mammary Tumor
Model
Sprouts were prepared by first surface sterilizing seeds of Brassica
oleracea variety italica (broccoli) cultivar Saga with a 1 min treatment
in 70% ethanol, followed by 15 min in 1.3% sodium hypochlorite with
approximately 0.001% Alconox detergent. Seeds were grown in sterile
plastic containers at a density of approximately 8 seeds/cm.sup.2
for 72 hours on a 0.7% agar support that did not contain added nutrients.
The environment was carefully controlled with broad spectrum fluorescent
lighting, humidity and temperature control (16 hours light, 25.degree.
C./8 hours dark, 20.degree. C).
The plants were rapidly and gently collected from the surface of
the agar to minimize glucosinolate hydrolysis by endogenous myrosinase
released upon plant wounding. A large quantity of sprouts was harvested
by immediately and rapidly plunging into approximately 3 volumes
of boiling water in order to inactivate endogenous myrosinase, as
well as extracting glucosinolates and isothiocyanates from the plant
tissue. Water was returned to a boil and maintained at a rolling
boil for 3 min. Sprouts were then strained from the boiled infusion
[tea, soup] and the infusion was lyophilized and stored as a dry
powder at -20.degree. C. [designated Prep A]. Other sprouts, similarly
prepared were extracted with boiling water, cooled to 25.degree.
C. and were amended with a quantity of 7 day old daikon sprouts
equivalent to approximately 0.5% of the original fresh weight of
broccoli sprouts. This mixture was homogenized using a Brinkman
Polytron Homogenizer and incubated at 37.degree. C. for 2 hours
following which it was filtered through a sintered glass filter,
lyophilized as above and stored as a dried powder at -20.degree.
C. [designated Prep B].
QR inducer activity and inducer potential of plant extracts, prepared
as described above, was determined by the microtiter plate bioassay
method as described above. The induction of QR activity in preparation
A is largely due to glucosinolates; predominantly glucoraphanin,
which is the glucosinolate of sulforaphane, but this preparation
also contains some glucoerucin, which is the sulfide analog of glucoraphanin.
The induction QR activity of preparation B is almost exclusively
due to isothiocyanates arising from treatment of glucosinolates
with myrosinase.
Female Sprague-Dawley rats received at 35 days of age were randomized;
4 animals per plastic cage. All animals received 10 mg DMBA, by
gavage in 1 ml sesame oil, at age 50 days. Sprout preparations (A
or B) or vehicle control were given by gavage at 3, 2 & 1 day
prior to DMBA, on the day of DMBA (2 hr prior to the DMBA dose)
and on the day following DMBA dosing. The vehicle used was 50% Emulphor
620P/50% water. Animals were maintained on a semi-purified AIN-76A
diet ad libitum from the time of receipt until termination of the
experiment (167 days of age).
TABLE-US-00004 TABLE 4 ANTICARCINOGENIC ACTIVITIES OF BROCCOLI
SPROUT EXTRACTS IN THE DMBA RAT MAMMARY TUMOR MODEL NUBMER MULTI-
OF PLICITY: ANIMALS NUMBER AT TOTAL OF TERM- TUMOR TUMORS GROUP
TREATMENT INATION NUMBER PER RAT CONTROL DMBA only 19 34 1.79 PREPARATION
324 mg/dose 18 19 1.05 A (100 .mu.mol (Glucosinolate) sulforaphane
equiv.) PREPARATION 424 mg/dose 20 11 0.55 B (100 .mu.mol (Isothiocyanate)
sulforaphane equiv.)
The development of palpable tumors was delayed for as much as 5
weeks by the administration of sprout extracts. Rats treated with
either Preparation A or B had significantly fewer tumors than the
untreated control, and the multiplicity of tumors (tumors per rat)
was significantly lower in the animals receiving Preparations A
or B.
EXAMPLE 11
Metabolism and Clearance of Glucosinolates in Humans
Two male, non-smoking volunteers ages 35 and 40 years, each in
good health, were put on a low vegetable diet in which no green
or yellow vegetables, or condiments, mustard, horseradish, tomatoes
or papayas were consumed. After 24 hours on such a diet, all urine
was collected in 8 hr aliquots. After 24 hours of baseline data,
subjects ingested 100 ml of broccoli sprout soup (prepared as below),
containing 520 .mu.mol of glucosinolates.
The sprouts were prepared by first surface sterilizing seeds of
Brassica oleracea variety italica (broccoli) cultivar Saga with
a 1 min treatment in 70% ethanol, followed by 15 min in 1.3% sodium
hypochlorite with ca. 0.001% Alconox detergent. Seeds were grown
in sterile plastic containers at a density of approximately 8 seeds/cm.sup.2
for 72 hours on a 0.7% agar support that did not contain added nutrients.
The environment was carefully controlled with broad spectrum fluorescent
lighting, humidity and temperature control (16 hours light, 25.degree.
C./8 hours dark, 20.degree. C). The plants were rapidly and gently
collected from the surface of the agar to minimize glucosinolate
hydrolysis by endogenous myrosinase released upon plant wounding.
A large quantity of sprouts was harvested by immediately and rapidly
plunged into approximately 3 volumes of boiling water in order to
inactivate endogenous myrosinase as well as to extract glucosinolates
and isothiocyanates from the plant tissue. Water was returned to
a boil and maintained at a rolling boil for 3 min. Following the
boiling step, sprouts were homogenized directly in their infusion
water for 1 min using a Brinkman Polytron Homogenizer and the preparations
were frozen at -79.degree. C. until use.
Inducer potential of plant extracts, prepared as described above,
was determined by the microtiter plate bioassay method as described
above. Inducer potential is nearly all due to glucosinolates; predominantly
glucoraphanin, which is the glucosinolate of sulforaphane, but some
glucoerucin which is the sulfide analog of glucoraphanin was also
present. When converted to isothiocyanates by the addition of purified
myrosinase, Phase 2 enzyme-inducing potential was 100,000 units/ml
and contained 5.2 .mu.mol of isothiocyanates per ml, as determined
by the cyclocondensation reaction described in Example 7. Thus,
the subjects consumed a total of 520 .mu.mol of glucosinolates.
Collection of 8 hour urine samples was continued for an additional
30 hours. Urinary excretion of isothiocyanate conjugates (dithiocarbamates)
was monitored using the cyclocondensation reaction as described
in Example 7.
TABLE-US-00005 TABLE 5 EXCRETION OF DITHIOCARBAMATES BY TWO SUBJECTS
INGESTING 520 MICROMOLES OF GLUCOSINOLATES EXTRACTED FROM SAGA BROCCOLI
SUBJECT 1 SUBJECT 2 TIME CONDITION .mu.mol Dithiocarbamate Collection
Time per 8 hour urine (hours) collection 8 baseline 1.4 2.7 16 baseline
2.1 0.9 24 baseline 1.7 5.4 32 1st 8 hour 23.2 20.4 post-dose 40
2nd 8 hour 9.9 36.8 post-dose 48 3rd 8 hour 4.4 14.0 post-dose 56
4th 8 hour 4.2 4.1 post-dose Total post-dose minus 39.8 63.2 average
baseline: Total as Percent of dose: 6.7% 12.2%
The two subjects studied metabolically converted a significant
fraction of the ingested glucosinolates to the isothiocyanates which
were converted to cognate dithiocarbamates and measured in the urine.
EXAMPLE 12
Effects of Physical Interventions on Sprout Growth on Production
of Induces of Quinone Reductase
Sprouts were prepared by first surface sterilizing seeds of Raphanus
sativum (daikon) by a 1 minute treatment with 70% ethanol, followed
by a 15 min treatment with 1.3% sodium hypochlorite with approximately
0.001% Alconox detergent. Seeds were grown in sterile plastic containers
at a density of approximately 8 seeds/cm.sup.2 for 7 days on a 0.7%
agar support that did not contain added nutrients. The environment
was carefully controlled with broad spectrum fluorescent lighting,
humidity and temperature control (16 hours light 25.degree. C./8
hours dark, 20.degree. C.).
Treated sprouts were irradiated with germicidal UV light for 0.5
hr on days 5 and 6. Treated sprouts were only half the height of
the untreated controls. Plants were harvested on day 7 by rapidly
and gently collecting the plants from the surface of the agar to
minimize glucosinolate hydrolysis by endogenous myrosinase released
upon plant wounding. Sprouts were harvested by immediate and rapid
plunging into approximately 10 volumes of DMF/ACN/DMSO (1:1:1) at
approximately -50.degree. C. in order to inactivate endogenous myrosinase
as well as to extract glucosinolates and isothiocyanates. Sprouts
were immediately homogenized with a ground glass mortar and pestle
and stored at -20.degree. C.
Inducer potential of plant extracts, prepared as described above,
was determined by the microtiter plate bioassay method as described
above. Inducer potential of the UV-treated sprouts was over three
times that of untreated controls. Treatment of sprouts with ultraviolet
light therefore increased the Phase 2 enzyme-inducer potential of
the plant tissue.
Although the foregoing refers to particular preferred embodiments,
it will be understood that the present invention is not so limited.
It will occur to those of ordinary skill in the art that various
modifications may be made to the disclosed embodiments and that
such modifications are intended to be within the scope of the present
invention, which is defined by the following claims. All publications
and patent applications mentioned in this specification are indicative
of the level of skill of those in the art to which the invention
pertains.
All publications and patent applications are herein incorporated
by reference to the same extent as if each individual publication
or patent application were specifically and individually indicated
to be incorporated by reference in its entirety.
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