U.S. patent application number 10/767268 was filed with the patent office on 2005-02-17 for spray and/or soil treatment of tobacco to reduce tsnas.
This patent application is currently assigned to PHILIP MORRIS USA INC.. Invention is credited to Hempfling, Walter P., Krauss, Marc R., Li, Qinglin.
Application Number | 20050034365 10/767268 |
Document ID | / |
Family ID | 32850803 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050034365 |
Kind Code |
A1 |
Li, Qinglin ; et
al. |
February 17, 2005 |
Spray and/or soil treatment of tobacco to reduce TSNAs
Abstract
Methods for increasing the concentration of antioxidants during
senescence and curing of tobacco leaves include application of a
chemical solution. The methods include spraying the solution onto
the tobacco plant prior to harvest and/or applying the chemical
solution to the soil surrounding roots of growing tobacco plants.
The application of the chemical solution preferably occurs between
topping and harvest, and is optimized to stimulate the production
of antioxidants and interfere with the formation of TSNAs during
curing. The tobacco can be burley tobacco subjected to air
curing.
Inventors: |
Li, Qinglin; (Richmond,
VA) ; Hempfling, Walter P.; (Mechanicsville, VA)
; Krauss, Marc R.; (Midlothian, VA) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
PHILIP MORRIS USA INC.
|
Family ID: |
32850803 |
Appl. No.: |
10/767268 |
Filed: |
January 30, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60443848 |
Jan 31, 2003 |
|
|
|
Current U.S.
Class: |
47/58.1FV |
Current CPC
Class: |
A24B 15/28 20130101;
A01G 7/06 20130101; A24B 15/30 20130101 |
Class at
Publication: |
047/058.1FV |
International
Class: |
A01G 007/06 |
Claims
What is claimed is:
1. A method of reducing tobacco-specific nitrosamines in cured
tobacco, comprising raising the levels of antioxidants in tobacco
leaves by spraying a chemical solution onto leaves of a growing
tobacco plant at least one time prior to harvesting, the
antioxidants being raised at least 25% compared to harvested
tobacco plants grown without being sprayed with the chemical
solution.
2. The method of claim 1, wherein the chemical solution contains at
least abscicic acid or analog thereof.
3. The method of claim 1, wherein the chemical solution contains at
least salicylic acid or analog thereof.
4. The method of claim 1, wherein the chemical solution contains at
least jasmonic acid.
5. The method of claim 1, wherein the chemical solution contains at
least methyl viologen (MV) or analog thereof.
6. The method of claim 1, wherein the chemical solution contains
hydrogen peroxide, sodium chloride or sulfur dioxide.
7. The method of claim 1, wherein the chemical solution contains a
herbicide, a plant activator, plant growth hormone and/or stress
inducing agent.
8. The method of claim 7, wherein the herbicide, activator and/or
growth hormone has an elevated concentration in the chemical
solution compared to conventional tobacco plant treating solutions
of the same ingredients.
9. The method of claim 1, wherein the chemical solution effects
reduction in available carbon dioxide used in photosynthesis.
10. The method of claim 1, further comprising preparing cured
tobacco by harvesting the tobacco plants and subjecting at least
some of the tobacco leaves of the tobacco plants to a curing
process, the level of antioxidants in the tobacco leaves being
sufficient to reduce nitrosation during the yellowing and browning
phases of the curing process.
11. The method of claim 1, wherein the chemical solution contains
at least one chemical compound which can affect chloroplast
activity, the chemical compound being sprayed onto the tobacco
leaves in an amount sufficient to stimulate formation of reactive
oxygen species in the tobacco leaves.
12. The method of claim 1, wherein the chemical solution is sprayed
onto the tobacco plants at different times prior to harvest.
13. The method of claim 1, wherein the tobacco is burley tobacco,
the method further comprising air curing the burley tobacco after
harvesting the tobacco plants.
14. The method of claim 1, further comprising subjecting the
growing tobacco plant to mechanical stress sufficient to raise the
level of antioxidants in the tobacco leaves.
15. The method of claim 1, wherein the tobacco plants are sprayed
only once with the chemical solution at least about one week prior
to harvesting of the tobacco plants.
16. The method of claim 1, wherein the tobacco plants are sprayed
with the chemical solution between topping to remove the flowers
from the tobacco plants and harvesting of the tobacco plants.
17. The method of claim 1, further comprising root pruning or xylem
cutting of the tobacco plants.
18. A method of reducing tobacco-specific nitrosamines in cured
tobacco, comprising raising the levels of antioxidants in tobacco
leaves by spraying a chemical solution onto leaves of a growing
tobacco plant at least one time prior to harvesting, the
antioxidants being raised at least 25% compared to harvested
tobacco plants grown without being sprayed with the chemical
solution, the chemical solution comprising an aqueous solution
which effects reduction in available carbon dioxide used in
photosynthesis.
19. A method of reducing tobacco-specific nitrosamines in cured
tobacco, comprising raising the levels of antioxidants in tobacco
leaves by spraying a chemical solution onto leaves of a growing
tobacco plant at least one time prior to harvesting, the
antioxidants being raised at least 25% compared to harvested
tobacco plants grown without being sprayed with the chemical
solution, the chemical solution comprising at least one chemical
compound selected from abscicic acid, salicylic acid, harpin,
methyl viologen, acifluorfen, acifluorfen sodium, jasmonic acid,
hydrogen peroxide, sodium chloride, and sulfur dioxide, the
chemical compound being sprayed onto the tobacco leaves in an
amount sufficient to stimulate formation of reactive oxygen species
in the tobacco leaves.
20. A cigarette comprising the cured tobacco of claim 10.
21. A method of reducing tobacco-specific nitrosamines in cured
tobacco, comprising raising the levels of antioxidants in tobacco
leaves by a soil treatment wherein a chemical solution is applied
to the soil surrounding the roots of a growing tobacco plant at
least one time prior to harvesting, the antioxidants being raised
at least 25% compared to harvested tobacco plants grown without the
soil treatment.
22. The method of claim 21, wherein the chemical solution contains
at least abscicic acid or analog thereof.
23. The method of claim 21, wherein the chemical solution contains
at least salicylic acid or analog thereof.
24. The method of claim 21, wherein the chemical solution contains
at least jasmonic acid.
25. The method of claim 21, wherein the chemical solution contains
at least methyl viologen (MV) or analog thereof.
26. The method of claim 21, wherein the chemical solution contains
hydrogen peroxide, sodium chloride or sulfur dioxide.
27. The method of claim 21, wherein the chemical solution contains
a herbicide, a plant activator, plant growth hormone and/or stress
inducing agent.
28. The method of claim 27, wherein the herbicide, activator or
growth hormone has an elevated concentration in the chemical
solution compared to conventional tobacco plant treating solutions
of the same ingredients.
29. The method of claim 21, wherein the chemical solution effects
reduction in available carbon dioxide used in photosynthesis.
30. The method of claim 21, further comprising preparing cured
tobacco by harvesting the tobacco plants and subjecting at least
some of the tobacco leaves of the tobacco plants to a curing
process, the level of antioxidants in the tobacco leaves being
sufficient to reduce nitrosation during the yellowing and browning
phases of the curing process.
31. The method of claim 21, wherein the chemical solution contains
at least one chemical compound which can affect chloroplast
activity, the chemical compound being applied to the soil in an
amount sufficient to stimulate formation of reactive oxygen species
in the tobacco leaves.
32. The method of claim 21, wherein the chemical solution is
applied to the soil at different times prior to harvest.
33. The method of claim 21, wherein the tobacco is burley tobacco,
the method further comprising air curing the burley tobacco after
harvesting the tobacco plants.
34. The method of claim 21, further comprising subjecting the
growing tobacco plant to mechanical stress sufficient to raise the
level of antioxidants in the tobacco leaves.
35. The method of claim 21, wherein the soil is treated only once
with the chemical solution at least about one week prior to
harvesting of the tobacco plants.
36. The method of claim 21, wherein the soil is treated with the
chemical solution between topping to remove the flowers from the
tobacco plants and harvesting of the tobacco plants.
37. The method of claim 21, further comprising root pruning or
xylem cutting of the tobacco plants.
38. A method of reducing tobacco-specific nitrosamines in cured
tobacco, comprising raising the levels of antioxidants in tobacco
leaves by a soil treatment wherein a chemical solution is applied
to the soil surrounding roots of a growing tobacco plant at least
one time prior to harvesting, the antioxidants being raised at
least 25% compared to harvested tobacco plants grown without the
soil treatment, the chemical solution comprising an aqueous
solution which effects reduction in available carbon dioxide used
in photosynthesis.
39. A method of reducing tobacco-specific nitrosamines in cured
tobacco, comprising raising the levels of antioxidants in tobacco
leaves by a soil treatment wherein a chemical solution is applied
to the soil surrounding the roots of a growing tobacco plant at
least one time prior to harvesting, the antioxidants being raised
at least 25% compared to harvested tobacco plants grown without the
soil treatment, the chemical solution comprising at least one
chemical compound selected from abscicic acid, saliclic acid,
acifluorfen, acifluorfen sodium, harpin, methyl viologen, jasmonic
acid, hydrogen peroxide, sodium chloride, and sulfur dioxide, the
chemical compound being applied to the soil in an amount sufficient
to stimulate formation of reactive oxygen species in the tobacco
leaves.
40. A cigarette comprising the cured tobacco of claim 30.
41. The method of claim 4, wherein the chemical solution further
comprises salicylic acid or an analog thereof.
42. The method of claim 4, wherein the chemical solution further
comprises abscicic acid, methyl viologen or an analog of methyl
viologen, hydrogen peroxide, sodium chloride or sulfur dioxide.
43. The method of claim 4, wherein the chemical solution further
comprises a herbicide, a plant growth hormone, or a stress inducing
agent.
44. The method of claim 4, wherein the chemical solution further
comprises a herbicide, a plant growth hormone or a stress inducing
agent.
45. The method of claim 44, wherein the chemical solution further
comprises a second plant activator.
46. The method of claim 4, wherein the chemical solution further
comprises a second plant activator.
47. The method of claim 1 further comprising pruning roots or
cutting xylem of the tobacco plant.
48. The method of claim 4, further comprising pruning roots or
cutting xylem of the tobacco plant.
49. The method of claim 1, wherein the chemical solution contains
at least harpin or an analog thereof.
50. The method of claim 21, wherein the chemical solution contains
at least harpin or an analog thereof.
51. The method of claim 1, wherein the chemical solution contains
at least acifluorfen or acifluorfen sodium.
52. The method of claim 21, wherein the chemical solution contains
at least acifluorfen or acifluorfen sodium.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Ser. No.
60/443,848 filed on Jan. 31, 2003, which is herein incorporated by
reference in its entirety for all purposes.
FIELD OF INVENTION
[0002] The invention relates to methods for increasing the
concentration of advantageous antioxidants in tobacco leaves by
spraying at least one chemical solution onto a growing tobacco
plant and/or soil treatment of growing tobacco plants. The
application of the chemical solution preferably occurs between
topping and harvest, and can be optimized to stimulate the
production of antioxidants in tobacco leaves which interfere with
the formation of TSNAs during curing of the tobacco leaves.
BACKGROUND OF THE INVENTION
[0003] Tobacco-specific nitrosamines (TSNAs), such as
N-nitrosonornicotine (NNN) and
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), can be found
in smokeless tobacco; mainstream smoke; and side stream smoke of
cigarettes. It has been reported that air-cured and flue-cured
tobacco contain tobacco-specific nitrosamines. See, "Effect of
Air-Curing on the Chemical Composition of Tobacco", Anna Wiernik et
al., Recent Adv. Tob. Sci, (1995), 21: 39-80. According to Wiernik
et al., TSNAs are not present in significant quantities in growing
tobacco plants or fresh cut tobacco (green tobacco), but are formed
during the curing process. Bacterial populations which reside on
the tobacco leaves are stated to largely cause the formation of
nitrites from nitrate during curing and possibly effect the direct
catalysis of the nitrosation of secondary amines at physiological
pH values. The affected secondary amines include tobacco alkaloids,
which form TSNAs when nitrosated.
[0004] Because curing of tobacco leaves is normally performed by
the farmer who grows the tobacco, a simple, economical and
non-labor-intensive method of reducing TSNA levels in the cured
tobacco leaves is desirable.
SUMMARY OF THE INVENTION
[0005] According to one embodiment, a method of reducing
tobacco-specific nitrosamines in cured tobacco, comprises raising
the levels of antioxidants in tobacco prior to being cured, wherein
the levels of antioxidants are raised by spraying a chemical
solution onto an aerial part (i.e., that part of the plant growing
above ground) of a tobacco plant prior to harvesting and wherein
the chemical solution effects an increase in the level of
antioxidants in the tobacco plant. The chemical solution preferably
includes a plant growth hormone such as abscicic acid (ABA) or
analog thereof, a plant activator such as salicylic acid (SA)
(commercially available as ACTIGARD.RTM.) or analog thereof, a
plant activator such as a harpin protein containing product
commercially available as MESSENGER.RTM., a herbicide such as
methyl viologen (MV) or analog thereof, a stress inducing agent
such as hydrogen peroxide, sodium chloride (NaCl), or sulfur
dioxide, or combinations thereof. Preferably, the analog of MV is
Paraquat (a quaternary nitrogen herbicide widely used for weed
control). Preferably, the tobacco plants are sprayed with the
chemical solution in a single application or multiple applications
about 1-3 weeks prior to harvest. Preferably, the tobacco is burley
tobacco and the method further includes air curing the burley
tobacco. The tobacco is preferably incorporated in a cigarette.
[0006] According to another embodiment, a method of reducing
tobacco-specific nitrosamines in cured tobacco, comprises raising
the levels of antioxidants in tobacco prior to being cured, wherein
the levels of antioxidants are raised by soil treatment of tobacco
plants prior to harvesting and wherein the soil treatment effects
an increase in the level of antioxidants in the tobacco plant. The
soil can be treated with a chemical solution which preferably
includes a plant growth hormone such as abscicic acid (ABA) or
analog thereof, a plant activator such as salicylic acid (SA)
(commercially available as ACTIGARD.RTM.) or analog thereof, a
plant activator such as a harpin protein containing product
commercially available as MESSENGER.RTM., a herbicide such as
methyl viologen (MV) or analog thereof, a stress inducing agent
such as hydrogen peroxide, sodium chloride, or sulfur dioxide, or
combinations thereof. Preferably, the analog of MV is Paraquat (a
quaternary nitrogen herbicide widely used for weed control).
Preferably, the soil is treated in a single application or multiple
applications about 1-3 weeks prior to harvest. Preferably, the
tobacco is burley tobacco and the method further includes air
curing the burley tobacco. The tobacco is preferably incorporated
in a cigarette.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 depicts a graph of antioxidant capacity of green and
cured Oriental, bright and burley tobacco grown without a spray
treatment to raise the antioxidant capacity.
[0008] FIG. 2 depicts a mechanism by which antioxidants in tobacco
inhibit TSNA formation.
[0009] FIG. 3 depicts a graph of the effects on antioxidant
capacity of spraying salicylic acid and methyl viologen onto
greenhouse tobacco.
DETAILED DESCRIPTION OF PREFERRED
[0010] EMBODIMENTS OF THE INVENTION
[0011] The amount of tobacco specific nitrosamines (TSNAs) in cured
tobacco leaves can be reduced by spraying growing tobacco plants
with a chemical solution and/or treating the soil surrounding roots
of growing tobacco plants with a chemical solution. In particular,
TSNAs in cured tobacco leaves may be reduced by raising the levels
of antioxidants in the tobacco leaves prior to harvesting the
plants.
[0012] The antioxidant levels in tobacco leaves sprayed with the
chemical solution are preferably raised at least 25%, preferably at
least 30%, and more preferably at least 50% compared to tobacco
leaves of untreated plants grown without the spray treatment.
Likewise, the antioxidant levels in tobacco leaves of tobacco
plants grown in soil treated with the chemical solution are
preferably raised at least 25%, preferably at least 30%, and more
preferably at least 50% compared to tobacco leaves of untreated
plants which without the soil treatment. FIG. 1 shows an example of
the antioxidant capacity of green and cured Oriental, bright and
burley tobacco obtained from untreated plants.
[0013] It is believed that TSNAs are formed predominantly during
the curing process. While not wishing to be bound by theory, it is
believed that the amount of TSNAs in cured tobacco leaves results
from the reaction of a reactive nitrosating species with the
tobacco alkaloids.
[0014] The amount of antioxidants in tobacco leaves during the time
of air-curing is believed to be advantageous to the inhibition of
TSNA formation. An elevated concentration of native antioxidants
during senescence and air-curing of tobacco can be obtained by
spraying of a chemical solution preferably comprising an aqueous
solution containing one or more chemical compounds onto the growing
tobacco plant and/or treating the soil surrounding roots of growing
tobacco plants with the aqueous solution. For example, it is
possible to increase foliar contents of native antioxidants by
stressing the tobacco plant, e.g., using ABA to reduce the
availability of carbon dioxide necessary for photosynthesis.
Preferably the tobacco is burley tobacco. The spraying of the
solution and/or soil treatment preferably occurs between topping
(i.e., removal of the flower from the tobacco plant to stimulate
leaf production) and harvest, and is intended to stimulate the
production of antioxidants and interfere with the formation TSNAs
during curing of the tobacco. Preferably, the level of antioxidants
is increased to an amount sufficient to prevent significant
nitrosation during the yellowing and browning phases of curing. The
chemical spraying and/or soil treatment can be carried out only
once or the tobacco plants can be sprayed and/or subjected to soil
treatment more than once. For example, the plants can be sprayed at
layby (when the plants are about knee-high) and the spray treatment
can be repeated periodically such as every 5 to 15 days until
harvest. Likewise, the soil in which the plants are grown can be
treated at layby and the soil treatment can be repeated
periodically such as every 5 to 15 days until harvest.
[0015] The spray and/or soil treatment can be combined with other
methods of elevating tobacco leaf antioxidants to a level that
inhibits nitrosation. For example, the antioxidants can be raised
by pruning of the tobacco plants (e.g., root pruning and xylem
cutting) prior to harvest. Thus, root pruning and/or xylem cutting
may be used to inhibit TSNA formation during air-curing. Under the
stressful conditions caused by root pruning and xylem cutting, the
plant's capacity to assimilate CO.sub.2 is reduced, and the
photosynthetic electron flux to O.sub.2 will increase resulting in
the increased production of superoxide and hydroxyl radicals. These
molecules induce antioxidants to cope with the oxidative stress.
(See commonly-owned U.S. patent application Ser. No. 10/235,636,
filed on Sep. 6, 2002, which is incorporated by reference in its
entirety herein).
[0016] Disturbances in photosynthetic activity-can be used to cause
the formation of reactive oxygen species. Thus, chemicals that
directly affect chloroplast activity can stimulate processes that
induce formation of reactive oxygen species. Redox-active
herbicides such as the diphenyl ethers (e.g., acifluorfen) can act
through the production of reactive oxygen. Such herbicides can
cause the activity of antioxidants such as ascorbate, glulathione
and glutathione reductase to increase while the plant is actively
defending itself against the herbicide. Bipyridyl herbicides, such
as Paraquat (also known as methyl viologen) and Diquat, can be used
to increase oxidative stress directly by generating reactive oxygen
radicals.
[0017] Growing tobacco plants can be sprayed with a chemical
solution, preferably an aqueous solution, which effects an increase
in antioxidant levels in the tobacco leaves and thus reduced TSNAs
during curing of the tobacco leaves. Likewise, the soil of tobacco
plants can be treated with a chemical solution, preferably an
aqueous solution, which effects an increase in antioxidant levels
in the tobacco leaves and thus reduced TSNAs during curing of the
tobacco leaves. Preferably, a chemical solution containing at least
one chemical compound is sprayed onto growing tobacco plants using
farm equipment suitable for spray application of liquids to tobacco
plants. If desired, spraying of such compounds can be combined with
other nitrosamine-reducing treatments such as root pruning and
xylem cutting, to result in tobacco leaves with exceptionally low
nitrosamine contents during the curing process. Further, the soil
surrounding the roots of tobacco plants can be sprayed and/or
irrigated using a chemical solution which effects the desired
increase in the antioxidant levels in the tobacco leaves.
[0018] 1. Definitions and Abbreviations
[0019] 1.1 Definions
[0020] By the term "herbicide" is meant to include reagents that
increase antioxidant activity. For example, preferred herbicides
include but are not limited to redox-active herbicides and
bipyridyl herbicides. Preferred redox-active herbicides include
diphenyl ethers (e.g., acifluorfen and acifluorfen sodium).
Preferred bipyridyl herbicides include methyl viologen (e.g.,
Paraquat) and Diquat.
[0021] By the term "plant growth hormone" is meant to include those
plant growth hormones which modulate the antioxidant level and
thereby TSNA in the tobacco plant. Preferred plant growth hormones
are abscicic acid (ABA) and jasmonic acid.
[0022] By the term "plant activator" is meant to include reagents
which modulate the antioxidant level and thereby TSNA in tobacco
plants. A preferred plant activator is a salicylic acid, or an
analog of salicylic acid, (e.g., ACTIGARD.RTM.), or a harpin
protein (e.g., MESSENGER.RTM.).
[0023] By the term "stress inducing reagents" is meant to include
reagents which modulate the antioxidant level and thereby TSNA in
tobacco. Preferred stress inducing reagents include sulfur dioxide,
sodium chloride and hydrogen peroxide.
[0024] 1.2 Abbreviations
[0025] ABA abscicic acid
[0026] FRAP Ferric-Reducing Antioxidant Power
[0027] HPLC high performance liquid chromatography
[0028] MV methyl viologen
[0029] NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
[0030] NNN N-nitrosonornicotine
[0031] SA salicylic acid
[0032] TSNA tobacco-specific nitrosamines
[0033] 2. Herbicides
[0034] One embodiment comprises spraying an effective amount of at
least one herbicide such as methyl viologen (MV) or one or more
analogs thereof (e.g., Paraquat), or Diquat (chemical name:
1',1'-ethylene-2,2'-bipyridyl- diylium) and/or other herbicides, to
the aerial part of topped tobacco plants several weeks or days
before harvesting. Also contemplated are diphenyl ethers. Preferred
diphenyl ethers include but are not limited to acifluorfen and
acifluorfen sodium, which respectively have the chemical names of
5-[2-chloro-4-(trifluoro-methyl)phenoxy]-2-nitrobenzoate and sodium
5-[2-chloro-4-(trifluoro-methyl)phenoxy]-2-nitrobenzoate.
Preferably, the spraying is performed one or more times in the
field about 1-3 weeks before harvesting (i.e., 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25
days before harvesting). It has been found that such spraying
results in a significant reduction of TSNAs during air-curing of
burley tobacco. The reduction of TSNAs during curing may be
accompanied by an increase in total antioxidant activity, as
measured by the Ferric-Reducing Antioxidant Power assay
("FRAP").
[0035] Another embodiment comprises applying an effective amount of
at least one herbicide such as methyl viologen (MV) or one or more
analogs thereof, such as Paraquat (chemical name:
1,1'-dimethyl-4,4'-bipyridinium- ), and/or other herbicides, to the
soil surrounding roots of topped tobacco plants several weeks or
days before harvesting. Preferably, the soil treatment is performed
one or more times in the field about 1-3 weeks before harvesting
(i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 days before harvesting).
[0036] The herbicide(s) can be applied in a concentration of about
0.01 mM to about 0.3 mM, more preferably 0.05 to about 0.15 mM and
most preferably in an amount of about 0.08 to about 0.11 mM (e.g.,
about 0.1 mM Paraquat) (and every 0.01 mM unit inbetween these
ranges). Typically about 50 mL to about 500 mL of the solution is
sprayed per plant, more preferably an amount of about 100 mL to
about 300 mL and most preferably about 150 mL to about 250 mL of
solution is sprayed onto the leaves or the soil of each plant.
[0037] 3. Plant Activators
[0038] Another embodiment comprises spraying an effective amount of
a plant activator, such as salicylic acid (SA), or one or more
analogs thereof, preferably ACTIGARD.RTM. (active ingredient
chemical name: 1,2,3-benzothiadiazole-7-thiocarboxylic
acid-5-methyl-ester), or a harpin protein such as MESSENGER.RTM.
(harpin, HrpN from Erwinia amylovora; GenBank Accession No:
AAC31644), to, for example, the aerial part of topped tobacco
plants several weeks or days before harvesting. ACTIGARD.RTM. is
manufactured by Syngenta Crop Protection, Greensboro, N.C.
MESSENGER.TM. is available from Eden Bioscience Corporation,
Bothell, Wash. Preferably, the spraying is performed one or more
times in the field about 1-3 weeks before harvesting (i.e., 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 days before harvesting). For example, ACTIGARD.RTM. can
be sprayed onto the tobacco plants in a single application at a
concentration of about 1 ounce (oz) per acre about 1 week prior to
harvest or in a series of applications at the same or different
concentrations, e.g., 1 oz/acre, 0.5 oz/acre and/or 0.25 oz/acre.
MESSENGER.RTM. can be sprayed onto the tobacco plants in a single
application at a concentration of 9 ounces per acre or in a series
of applications at the same or different concentrations.
ACTIGARD.RTM. is believed to disable enzyme activity thereby
creating oxidative stress to which the tobacco plant responds by
production of antioxidants. MESSENGER.RTM. is believed to create a
plant response known as systemic acquired resistance that manifests
a production of antioxidants. Due to the increase in antioxidant
level in the treated tobacco leaves, a significant reduction of
TSNAs can be obtained during curing of the tobacco leaves.
[0039] A further embodiment comprises applying an effective amount
of salicylic acid (SA), or one or more analogs thereof, preferably
ACTIGARD.RTM., or a harpin protein such as MESSENGER.RTM., to the
soil surrounding roots of topped plants several weeks or days
before harvesting. Preferably, the soil treatment is performed one
or more times in the field about 1-3 weeks before harvesting (i.e.,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, or 25 days before harvesting). Salicylic acid and its
analogs such as ACTIGARD.RTM. can be used to treat plants in
concentrations of about 0.1 mM to about 10 mM, or more preferably
about 0.5 mM to about 2 mM and most preferably in an amount of
about 0.8 mM to about 1.2 mM. Typically about 50 mL to about 500 mL
of the solution is sprayed per plant, more preferably an amount of
about 100 mL to about 300 mL and most preferably about 150 mL to
about 250 mL of solution is sprayed onto the leaves of each plant
or administered onto the soil surrounding the roots of each
plant.
[0040] A harpin protein such as MESSENGER.RTM. can be used to treat
the leaves or roots of the plants in an amount of about 4.5 oz/acre
to about 9 oz/acre (9 oz/acre is the FDA approved amount).
[0041] 4. Plant Growth Hormones
[0042] Another embodiment of the present invention comprises
spraying an effective amount of at least one plant growth hormone
such as abscicic acid (ABA), or one or more of its more stable
analogs, or jasmonic acid or one or more analogs thereof preferably
onto the aerial part of topped tobacco plants several weeks or days
before harvesting. Preferably, the spraying is performed one or
more times in the field about 1-3 weeks before harvesting (i.e., 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25 days before harvesting). Due to the increase in
antioxidant level in the treated tobacco leaves, a significant
reduction of TSNAs can be obtained during curing of the tobacco
leaves.
[0043] ABA is a major controller of stomatal closing, and naturally
increases in concentration in tobacco leaves as a response to
water-deficit stress. See FIG. 2. The CO.sub.2 supply for
photosynthesis can be interrupted leading to enhancement of the
native leaf antioxidants. A solution of ABA, or a suitable analog,
can be sprayed onto the tobacco plant such that stomata remain at
least partially closed for a short time period, but then open again
to support photosynthesis, akin to the action of root pruning, but
without loss of turgor due to water deficit. The effects of
spraying ABA on growing tobacco plants as compared to MV is shown
in FIG. 3. When administering ABA onto the leaves of the plant or
on the soil surrounding the roots of the tobacco plant, preferably
an amount of about 0.05 mM to about 1 mM ABA is used, more
preferably about 0.1 to about 0.5 mM ABA and most preferably about
0.2 mM to about 0.4 mM ABA.
[0044] Jasmonic acid is preferably administered to the leaves or
the soil surrounding the roots of the tobacco plants in an amount
of about 0.01 mM to about 2.0 mM and more preferably in an amount
of about 0.1 mM to about 1 mM.
[0045] A further embodiment of the present invention comprises
applying an effective amount of at least one plant growth hormone
such as abscicic acid (ABA), or one or more of its more stable
analogs or jasmonic acid or one or more analogs thereof onto the
soil surrounding roots of topped tobacco plants several weeks or
days before harvesting. Preferably, the soil treatment is performed
one or more times in the field about 1-3 weeks before harvesting
(i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 days before harvesting).
[0046] Typically about 50 mL to about 500 mL of the solution is
sprayed per plant, more preferably an amount of about 100 mL to
about 300 mL and most preferably about 150 mL to about 250 mL of
solution is sprayed onto each plant (e.g., the leaves or the soil
surrounding the roots).
[0047] 5. Stress Inducing Agent
[0048] Another embodiment of the present invention comprises
spraying a solution containing an effective amount of at least one
stress inducing agent such as hydrogen peroxide, NaCl (salt) or
sulfur dioxide onto the aerial part (the leaves) of topped tobacco
plants several weeks or days before harvesting. Preferably, the
spraying is performed one or more times in the field about 1-3
weeks before harvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before
harvesting).
[0049] A further embodiment of the present invention comprises
applying a solution containing an effective amount of at least one
stress inducing agent such as hydrogen peroxide, a salt (e.g.,
NaCl) or sulfur dioxide onto the soil surrounding the roots of
topped tobacco plants several weeks or days before harvesting.
Preferably, the soil treatment is performed one or more times in
the field about 1-3 weeks before harvesting (i.e., 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or
25 days before harvesting).
[0050] The foregoing methods of soil treatment and spraying one or
more chemical compounds onto the leaves of the tobacco plants
before harvesting can be combined with other methods of increasing
antioxidants, to reduce nitrosamines in tobacco plants during the
curing process. For example, partial removal of the root structure
(root pruning) of burley tobacco in the field, about a week before
harvesting, results in significant reduction of tobacco-specific
nitrosamines during air-curing, probably due to the resulting
increase in total antioxidant capacity (measured by the
Ferric-Reducing Antioxidant Power assay) that persists during the
curing process.
[0051] Hydrogen peroxide treatment is preferably applied as a
solution containing hydrogen peroxide in a range of about 1% to
about 5%, more preferably in a range of about 2% to about 4%, and
most preferably in a range of about 2.5% to about 3.5% being
sprayed on the leaves and/or onto the soil surrounding the
roots.
[0052] NaCl is administered to the plant by spraying on the leaves
or onto the soil surrounding the roots a solution of sodium
chloride comprising about 0.8% to 0.1% sodium chloride, more
preferably from about 0.6% to about 0.2% NaCl, and most preferably
0.5% to about 0.3% (e.g., about 0.4%) NaCl.
[0053] The sulfur dioxide is administered to the plant in an amount
of about 50 nL/L to about 500 nL/L, more preferably in an amount of
about 100 nL/L to about 250 nL/L and most preferably in an amount
of about 150 nL/L to about 200 nL/L.
[0054] Typically about 50 to about 500 mL of a solution with one or
more of these reagents is sprayed per plant, more preferably an
amount of about 100 to about 300 mL and most preferably about 150
to about 250 mL of solution is sprayed onto each plant.
[0055] 6. Combinations of Reagents
[0056] Also contemplated herein are combinations of reagents. For
example, one or more herbicides, stress inducing reagents, plant
growth hormones, and plant activators can be combined either in an
admixture or applied separately. The combination of reagents can be
used to treat the tobacco such that the tobacco has increased
antioxidant activity and/or production of antioxidants, which
correspondingly reduces TSNAs. Combinations of such reagents
include a plant growth hormone in combination with a plant
activator (e.g., a salicylic acid combined with jasmonic acid).
Additional combinations include jasmonic acid in combination with
one or more stress reducing reagents, one or more herbicides, one
or more plant activators, or with a different plant growth hormone.
When used in combinations, preferably the combinations of reagents
may require less of each reagent to be administered to the tobacco
plant than if the reagent was administered alone (i.e., not in
combination with other antioxidant inducing reagents).
[0057] Such combinations can be administered onto the aerial part
of a tobacco plant (topped or prior to being topped). The
combination of reagents can be administered several weeks or days
before harvesting, or both. Preferably, the combination of reagents
are applied by spraying. Such spraying can be performed one or more
times in the field. Preferably, the spraying of the combination of
reagents occurs about 1-3 weeks before harvesting (i.e., 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 days before harvesting). The combination of reagents can
also be administered to the plant, or portions of the plant (e.g.,
leaves, stems), after the plant has been harvested.
[0058] A further embodiment of the present invention comprises
applying a solution containing an effective amount of a combination
of reagents onto the soil surrounding the roots of a tobacco plants
(topped or prior to topping). The solution can be applied several
weeks or days before harvesting the plant. Preferably, the soil
treatment is performed one or more times in the field about 1-3
weeks before harvesting (i.e., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 days before
harvesting). The solution can also be administered to the plant
after the plant has been harvested. Typically about 50 to about 500
mL of the solution is sprayed per plant, more preferably an amount
of about 100 to about 300 mL and most preferably about 150 to about
250 mL of solution is sprayed onto each plant.
[0059] 7. Combination of Reagents and Methods for Reducing
TSNAs
[0060] The reagents discussed above can be used alone to reduce
TSNAs or in combination with one another, or in combination with
methods which reduce TSNAs. Thus, the methods of treating the soil
surrounding the roots or the leaves of the plants with one or more
chemical compounds before harvesting can be combined with other
methods of increasing antioxidants, to reduce nitrosamines in
tobacco plants during the curing process. For example, partial
removal of the root structure (root pruning) or xylem cutting of
burley tobacco in the field, about a week before harvesting,
results in significant reduction of tobacco-specific nitrosamines
during air-curing, probably due to the resulting increase in total
antioxidant capacity (measured by the Ferric-Reducing Antioxidant
Power assay) that persists during the curing process.
[0061] The root pruning (or other mechanical stress which produces
a similar result) induces water-deficit stress which is believed to
result in temporary stomatal closure to preserve water in the
plant. Such closure of the stomata also shuts off the supply of
atmospheric carbon dioxide that supports photosynthetic carbon
dioxide fixation and allows the plant to produce more antioxidants.
Other forms of mechanical stress which yield a similar result
(i.e., induce production of antioxidants such as xylem cutting) can
also be used in combination with any one or combination of the
reagents discussed above.
EXAMPLE 1
TSNA Inhibition Using Salicylic Acid or Methyl Viologen
[0062] Topped greenhouse tobacco plants were sprayed with aqueous
solutions of salicylic acid, methyl viologen and a control
substance, in order to measure the total antioxidant capacity
(.mu.mol/g) over 24 hours. Mature burley tobaccos (with about 20-22
leaves per plant) were topped two weeks prior to being sprayed with
either salicylic acid or methyl viologen. An aqueous solution of
salicylic acid (99%; Aldrich Chemical Co.) at 1 M and methyl
viologen (98%; Aldrich Chemical Co.) at 0.1 mM concentration were
sprayed upon the tobacco leaves. Each plant was sprayed with at
least 200 mL of solution with good coverage of each leaf to produce
a more uniform response to the chemical spraying. Green samples
were taken 24 hours after spraying. Leaves were checked after
sampling to make sure all leaves are representative of the plant
conditions. Samples were quickly separated into lamina and midribs
as known in the art. Only the lamina was retained for analysis.
Sampled green lamina was flash-frozen in liquid nitrogen. The
samples can be stored at -80.degree. C. if analysis cannot be
performed immediately.
[0063] Total antioxidant capacity was determined by FRAP assay, and
the values were expressed as .mu.mol/g of fresh weight. The
concentration of chlorogenic acid and rutin, two water soluble
antioxidants, were measured by HPLC and-their contribution to the
total antioxidant capacity (measured by FRAP) was calculated. The
results (Table 1) show that both methyl viologen and salicylic acid
increase antioxidant capacity.
1 TABLE 1 Antioxidant Capacity of Antioxidant Total Antioxidant
Chlorogenic Acid Capacity of Capacity (.mu.mol/g) (.mu.mol/g) Rutin
(.mu.mol/g) Control 40 12 1 Salicylic Acid 50 25 1 Methyl Viologen
55 28 1
[0064] The FRAP assay was performed as follows. The green lamina
were accurately weighed and extracted with 10% methanol and 0.2 M
perchloric acid in water. The mixture was shaken at 4.degree. C. on
an orbital shaker for an hour. The extracts were then centrifuged
at 5,000 rpm for 20 minutes. The supernatant was filtered through
Whatman Autovial 0.45 .mu.m PVDF syringe filters and was ready for
analysis after appropriate dilution with extraction solution (10%
MeOH and 0.2 M perchloric acid in water).
[0065] The procedures for the FRAP assay on tobacco samples were
modified from that previously described by Benzie and Strain, Meth.
Enzymology 299: 15-27 (1999). The assay was done manually on a
spectrophotometer at room temperature. The FRAP reagent was
prepared by combining 300 mM acetate buffer (pH 3.6), 10 mM
2,4,5-tripyridyl-s-triazine in 40 mM HCl and 20 mM FeCl.sub.3 in
the ratio of 10:1:1 (v:v:v). A 100 .mu.L aliquot of the sample
extraction was added to 3 mL of FRAP reagent and mixed. After the
mixture stood at room temperature for 6 min., the absorbance at 593
nm was determined against the FRAP reagent alone. Calibration was
against a standard curve (i.e., 500, 1000 and 2000 .mu.M ferrous
iron) produced by the addition of freshly prepared ferrous sulfate.
FRAP values were calculated as micromolar ferrous ion (ferric
reducing power) from two determinations.
[0066] Chlorogenic acid (i.e., 3-O-caffeoylquinic acid) and rutin
(i.e., quercetin 3-rhamnosidoglucoside) are two antioxidants that
are water soluble and can easily be measured in plants. Chlorogenic
acid and rutin concentrations were determined by high performance
liquid chromatographic (HPLC) analysis. Other polyphenols such as
the isomers of chlorogenic acid (i.e., 4-O-, and 5-O-caffeoylquinic
acid) and scopoletin (i.e., 6-methoxy-7-hydroxycoumarin) were also
separated by reverse phase HPLC using a gradient of methanol with
1% glacial acetic acid. 500 mg of tobacco was extracted with 10%
methanol and 0.2 M perchloric acid in water. The supernatant was
neutralized using K.sub.2CO.sub.3 to a pH of 6.5 before the sample
was injected into the HPLC. Umbelliferone (i.e. 7-hydroxycoumarin)
was used as an internal standard and detection was by UV absorption
at 326 nm.
EXAMPLE 2
[0067] The following table (Table 2) shows the results of
experiments using an aqueous chemical spray to raise burley native
leaf antioxidant capacity in order to interfere with TSNA
production during air curing. Burley tobacco plants were grown
using standard agronomic practices. ACTIGARD.RTM. and
MESSENGER.RTM. were sprayed on the plants after layby in two-week
intervals with a total of five (5) applications of the reagents
until the plants were harvested. Treatment with the ACTIGARD.RTM.
or MESSENGER.RTM. at this time in plant growth is not suitable for
optimum results for the manufacturer recommended uses of those
reagents as plant activators.
[0068] At harvest, care was taken to ensure clean tobacco by
minimizing contact with the soil. Plants were stalk cut and five
stalks were speared on each stick for standard curing in
conventional air-curing barns. All treated and control tobaccos
were hung with even spacing of sticks. The treated (non-control)
tobaccos were surrounded by clean filler tobacco to help to create
normal curing conditions. At least three replicate samples were
taken at each sampling point from five different plants with 3
leaves from the top one-third of the plant (excluding the top 4
leaves). Each plant was sampled only once. Midveins were separated
from the lamina. Both lamina and midveins were weighed and placed
in sample bags stored at -80.degree. C. After freeze-drying,
samples were re-weighed to determine the moisture content. Samples
were ground to pass through a 40-mesh screen and stored at
-40.degree. C. until analysis.
[0069] FRAP analysis was performed as follows. Freeze-dried samples
were accurately weighed and extracted with 10% methanol and 0.2 M
perchloric acid in water. The mixture was shaken at 4.degree. C. on
an orbital shaker for an hour. The extracts were then centrifuged
at 5,000 rpm for 20 minutes. The centrifuged supernatants were then
filtered through Whatman Autovial 0.45 .mu.m PVDF Syringe filters.
The filtered material was then diluted as discussed in Example 1
above. The FRAP assay was conducted as described by Benzie and
Strain (1999) with the following modifications. The FRAP reagent
was prepared daily by combining 300 mM acetate buffer (pH 3.6), 10
mM 2,4,5-tripyridyl-s-triazine in 40 mM HCl and 20 mM FeCl.sub.3 in
a ratio of 10:1:1 (v:v:v). A 100 .mu.L aliquot of the sample
extraction was added to 3,000 .mu.L (3 mL) of FRAP reagent and
mixed. After the mixture stood at room temperature for 6 minutes,
the absorbance at 593 nm was determined against the FRAP reagent.
Calibration was against a standard curve (i.e., 500, 1000 and 2000
.mu.M ferrous ion) produced by the addition of freshly prepared
ferrous sulfate. FRAP values were calculated as micromolar ferrous
ion (ferric reducing power) from two determinations. The total
antioxidant capacity values are expressed in .mu.mol/g of fresh
weight.
[0070] The treated tobaccos resulted in cured leaves with nearly
30% antioxidant capacity increases and reduction of TSNA of about
65-70% as compared to untreated control samples.
2 TABLE 2 ACTIGARD .RTM. Control MESSENGER .RTM. 0.5 oz/ No Spray 9
oz/acre 0.25 oz/acre acre Total Antioxidant 55.4 70.9 71.9 59.1
Capacity (.mu.mol/g) TSNA Lamina 3040 1443 1787 1037 TSNA Mid-rib
5110 1057 1624 1627
EXAMPLE 3
NaCl Induced Inhibition of TSNAs
[0071] In tobacco plants, antioxidants interfere with the
nitrosation of secondary alkaloids. The amount of antioxidants
present at the time of air-curing the tobacco plants is believed to
be effective in inhibiting TSNA formation. The objective is to
increase the foliar concentration of native antioxidants during
senescence and air-curing of burley tobaccos by judicious
application of reagents which inhibit TSNAs. For burley tobaccos,
application preferably occurs between topping of the tobacco plant
and harvest.
[0072] High concentrations of sodium chloride (NaCl) cause ionic,
osmotic and associated secondary stresses to plants. Plant response
to those primary and secondary stresses are complex, but can be
grouped into three general categories: homeostasis, detoxification
and growth control.
[0073] Green house grown burley tobacco plants were sprayed with
either 0.7% NaCl, 0.35% NaCl, 0.18% NaCl, or control after topping
one week before harvest. Once harvested, the leaves were examined
to make sure that the leaves were representative of the plant's
condition, as discussed above for Example 1. Samples were rapidly
separated into the lamina and midribs. The lamina containing
portions were rapidly frozen in liquid nitrogen and either stored
at -80.degree. C. or analyzed. Total antioxidant capacity was
determined by FRAP assay as discussed above for Example 1 and the
values were expressed as .mu.mol/g of fresh weight.
3 TABLE 3 Control 0.7% NaCl 0.35% NaCl 0.18% NaCl Total 148.4 206
229 175 antioxidant capacity (.mu.mol/g)
[0074] While the invention has been described with reference to
preferred embodiments, it is to be understood that variations and
modifications may be resorted to as will be apparent to those
skilled in the art. Such variations and modifications are to be
considered within the purview and scope of the invention as defined
by the claims appended hereto.
* * * * *