U.S. patent application number 10/235636 was filed with the patent office on 2003-03-27 for method for the reduction of tobacco specific nitrosamines by increasing antioxidants in tobacco.
Invention is credited to Hempfling, Walter P., Krauss, Marc R., Li, Qinglin.
Application Number | 20030056801 10/235636 |
Document ID | / |
Family ID | 23233873 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030056801 |
Kind Code |
A1 |
Krauss, Marc R. ; et
al. |
March 27, 2003 |
Method for the reduction of tobacco specific nitrosamines by
increasing antioxidants in tobacco
Abstract
The present invention is drawn to methods for reducing
tobacco-specific nitrosamine (TSNA) content in cured tobacco by
increasing the levels of antioxidants in the tobacco prior to
harvesting. Methods to be used in the present invention include
root pruning of the tobacco plant prior to harvesting; severing the
xylem tissue of the tobacco plant prior to harvesting; and
administering antioxidants and/or chemicals which increase
antioxidants to the tobacco plant after harvesting.
Inventors: |
Krauss, Marc R.;
(Midlothian, VA) ; Li, Qinglin; (Richmond, VA)
; Hempfling, Walter P.; (Mechanicsville, VA) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
23233873 |
Appl. No.: |
10/235636 |
Filed: |
September 6, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60317487 |
Sep 7, 2001 |
|
|
|
Current U.S.
Class: |
131/290 |
Current CPC
Class: |
A24B 15/28 20130101;
A24B 15/245 20130101 |
Class at
Publication: |
131/290 |
International
Class: |
A24B 003/10 |
Claims
1. A method for reducing tobacco-specific nitrosamines in cured
tobacco, said method comprising raising the levels of antioxidants
in a tobacco plant or leaf prior to being cured.
2. The method of claim 1, wherein the levels of antioxidants are
raised by root pruning the tobacco plant prior to harvest.
3. The method of claim 1, wherein the levels of antioxidants are
raised by severing xylem tissue of the tobacco plant prior to
harvest.
4. The method of claim 1, wherein the levels of antioxidants are
raised by inserting a pill containing (i) one or more antioxidants
and/or (ii) one or more chemicals which increase production of
antioxidants into the stalk of the tobacco plant after
harvesting.
5. The method of claim 1, wherein the levels of antioxidants are
raised by inserting the stalk of the tobacco plant after harvesting
into a solution comprising one or more antioxidants and/or one or
more chemicals which increases production of antioxidants.
6. The method of claim 1, wherein the levels of antioxidants are
raised by cutting the top of the tobacco plant after harvesting and
inserting a treatment substance containing (i) one or more
antioxidants and/or (ii) one or more chemicals which increase
production of antioxidants, therein.
7. The method of claim 1, wherein the levels of antioxidants are
raised by root pruning the tobacco plant about 1-3 weeks prior to
harvest.
8. The method of claim 1, wherein the levels of antioxidants are
raised by severing xylem tissue of the tobacco plant about 1-3
weeks prior to harvest.
9. The method of claim 2, wherein the tobacco is Burley tobacco and
the method further includes air curing the Burley tobacco.
10. The method of claim 3, wherein the tobacco is Burley tobacco
and the method further includes air curing the Burley tobacco.
11. The method of claim 4, wherein the tobacco is Burley tobacco
and the method further includes air curing the Burley tobacco.
12. The method of claim 5, wherein the tobacco is Burley tobacco
and the method further includes air curing the Burley tobacco.
13. The method of claim 6, wherein the tobacco is Burley tobacco
and the method further includes air curing the Burley tobacco.
14. The method of claim 2, wherein the tobacco plant is growing in
normal or dry soil conditions.
15. The method of claim 3, wherein the tobacco plant is growing in
wet soil conditions.
16. The method of claim 3, wherein the tobacco plant is cut in two
or more locations on the stalk of the tobacco plant such that the
stalk's water transport capacity is diminished.
17. The method of claim 16, wherein the water transport capacity is
reduced by at least 25%.
18. The method of claim 6, wherein the treatment substance is in
the form of at least one pill, capsule, caplet, paste, emulsion,
foam, powder or combination thereof.
19. The method of claim 6, wherein the treatment substance
comprises 0.5 to 12 grams of the antioxidants and/or chemicals.
Description
[0001] The invention relates generally to methods for reducing
tobacco specific nitrosamines (TSNAs) comprising increasing the
levels of antioxidants in the harvested tobacco. The invention also
relates generally to methods for increasing advantageous
antioxidants in any vegetable or fruit.
BACKGROUND OF THE INVENTION
[0002] 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, pp. 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.
[0003] Star Tobacco and Pharmaceutical Co., Inc., has reported that
it treats tobacco leaves before or during flue-curing by
microwaving for purposes of reducing tobacco-specific nitrosamines.
See WO 98/58555. The microwaving adds significant cost to the
tobacco farmer, including the costs of excess handling and breakage
of tobacco leaves, the microwave process, the microwave facility
and the extra labor and time necessitated by the microwaving
process. A further drawback to this method of reducing TSNAs is
that microwaving of the tobacco leaves has a thermal effect upon
the tobacco tissue resulting in heating of the tobacco leaves which
may affect the taste and aroma of the smoke from the tobacco.
[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] The present inventors unexpectedly found that by root
pruning (i.e. partially removing the root structure) growing
tobacco plants about 1-3 weeks (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) prior
to harvesting, or, alternatively, 1-3 weeks after topping (i.e.
cutting of the apex of the plant), significant reductions of TSNAs
during curing (i.e., air-curing, flue-curing, fire-curing,
sun-curing, and any other means of curing known or contemplated by
one of skill in the art) of the tobacco is obtained. The present
inventors also found that a similar effect can be obtained by
cutting the xylem tissue about 1-3 weeks prior to harvest. Finally,
by administering antioxidants into tobacco plant upon harvesting
significant reductions of TSNAs can be obtained.
[0006] The methods disclosed herein for increasing antioxidant
content in tobacco can also be used for increasing antioxidant
content in other plants, vegetables or fruit. Such methods would be
advantageous since they would provide an important antioxidant
source to those eating the plants, vegetables or fruit.
[0007] Thus, in a first embodiment of the present invention a
method for reducing tobacco-specific nitrosamines in cured tobacco
is provided, wherein the method comprises raising the levels of
antioxidants in tobacco leaves prior to curing the tobacco
leaves.
[0008] In a first preferred embodiment of the present invention,
the levels of antioxidants in the tobacco are raised by root
pruning a tobacco plant about 1-3 weeks before harvesting and
curing.
[0009] In another preferred embodiment of the present invention,
the levels of antioxidants in the tobacco are raised by severing
the xylem tissue of the tobacco plant about 1-3 weeks before
harvesting and curing.
[0010] In a further preferred embodiment of the present invention,
the levels of antioxidants in the tobacco are raised by inserting a
pill containing (i) one or more antioxidants and/or (ii) one or
more chemicals which increase production of antioxidants into the
stalk of the tobacco plant after harvesting.
[0011] Yet another preferred embodiment of the present invention
involves increasing the levels of antioxidants in the tobacco by
cutting the top of the tobacco plant after harvesting and inserting
a pill containing (i) one or more antioxidants and/or (ii) one ore
more chemicals which increase production of antioxidants,
therein.
[0012] Another preferred embodiment of the present invention
involves increasing the levels of antioxidants in the tobacco by
dipping the stalk of the tobacco plant after harvesting into a
solution comprising one or more antioxidants and/or one or more
chemicals which increase production of antioxidants.
[0013] Another embodiment of the present invention involves
increasing the levels of antioxidants in any plant, vegetable or
fruit using any of the methods described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a graph of antioxidant capacity of green and cured
Oriental, bright and Burley tobacco.
[0015] FIG. 2 is a graph correlating the amount of polyphenols
present in tobacco extract with the antioxidant capacity of the
tobacco extract.
[0016] FIG. 3 depicts a mechanism by which antioxidants in tobacco
inhibit TSNA formation.
[0017] FIG. 4 is a table listing antioxidant capacity of various
phenolic compounds.
[0018] FIG. 5 is a graph of HPLC chromatograms of phenolic
compounds from cured Oriental tobacco.
[0019] FIG. 6 is a graph of antioxidant capacity of phenolic
fractions of phenolic compounds from tobacco extracts and a graph
of the HPLC chromatograms of the phenolic compounds.
[0020] FIG. 7 shows graphs of the approximate percent of
antioxidant capacity for chlorogenic acid, rutin and scopletin in
Oriental and bright tobacco.
[0021] FIG. 8 shows graphs of antioxidant capacity of green Burley
tobacco and cured Burley tobacco.
[0022] FIG. 9 is a graph of antioxidant capacity of Burley tobacco
compared to TSNA and nitrite capacity over a two plus week curing
process.
[0023] FIG. 10 is a graph of TSNA content in cured Burley tobacco,
with and without root pruning.
[0024] FIG. 11 is a graph of antioxidant levels in cured Burley
tobacco, with and without root pruning.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention provides a method for reducing
tobacco-specific nitrosamines, or TSNAs, which are generated during
the curing of tobacco leaves, wherein said method comprises root
pruning the tobacco plant immediately prior to harvesting. The
invention also provides a method for reducing tobacco-specific
nitrosamines wherein said method comprises administering
antioxidants into tobacco plants upon harvesting. A further method
of the present invention for reducing tobacco-specific nitrosamines
comprises cutting the xylem tissue of the tobacco plant about 1-3
weeks prior to harvesting.
[0026] The present inventors have found that methanolic extract of
cured Oriental, bright and Burley tobaccos had high, intermediate,
and low antioxidant activities, respectively (see FIG. 1). This
extract proved to be rich in polyphenols, which were well
correlated with the extracts antioxidant capacity (see FIG. 2). The
present inventors separated phenolic compounds in tobaccos and
characterized the compounds by reversed-phase HPLC and then studied
the effect of curing processes on the phenolic composition. It was
determined that different means of curing resulted in different
phenolic contents.
[0027] Furthermore, the inventors found that different treatments
to the tobacco stalk, either before harvesting or after harvesting,
can result in higher levels of antioxidant activities, and thus
polyphenols.
[0028] For example, partial removal of the root structure (i.e.
root pruning) of Burley tobacco in the field about 1-3 weeks before
harvesting results in a significant reduction of TSNAs during
air-curing. The inventors found that this reduction of TSNAs was
accompanied by an increase in total antioxidant activity, as
measured by the Ferric-reducing Antioxidant Potential assay
("FRAP"), that persists during the curing process. This effect
appears to depend largely upon the imposition of rapid
water-deficiency stress upon the plant owing to the partial removal
of the organ of water uptake. It is also possible that a wounding
response is manifested, with one outcome being the augmentation of
antioxidants.
[0029] The expected rise of antioxidants and the attending
reduction of curing-dependent nitrosation does not occur when
excess water is available in the soil surrounding the roots (the
"rhizosphere"). In water-deficient or dry soil conditions,
root-pruned tobacco plants undergo extensive
transpiration-dependent wilting soon after pruning occurs, but then
recover turgor as water is taken up through the residual root
structure and distributed throughout the plant. Such root
pruning-dependent wilting is not apparent in the presence of excess
water or wet soil conditions, indicating that the remaining intact
root is capable of supplying water to the shoot sufficient to
retain turgor despite transpiration. Therefore, when there is
excess water present in the soil, it is preferred that rather than
root pruning the tobacco plants, the xylem is severed, as disclosed
therein. When the xylem is severed, as in the method of the present
invention, antioxidant capacity is raised regardless of the
availability of water in the soil. When there are moderate amounts
of water in the soil (normal soil conditions), the method of the
present invention wherein the tobacco plants are root pruned may be
used.
[0030] It should be noted that in the presence of a great excess of
water (swamp-like conditions), antioxidants also rise and TSNAs are
diminished in the cured leaf. This is likely due to stress induced
by anoxia in the rhizosphere. However, in the presence of excess
water, the quality and yield of the tobacco suffers and makes the
notion of flooding the field not practical for the ultimate goal of
reducing TSNAs while maintaining tobacco yield.
[0031] A vascular plant, a tobacco plant transports water taken up
by the root by means of the xylem system, which comprises tubular
structures extending upward through the stalk connecting the root
with the various components of the shoot. Because the xylem tubules
are located in vascular bundles in the outer portion of the stalk
(see Dickison, W. C., Integrative Plant Anatomy, Harcourt Academic
Press, San Diego (2000), pp. 121-129), they can be severed by
superficial wounding. Sufficient wounding is inflicted to produce
prompt wilting, but not so much as to prevent recovery from
wilting. The inventors also found that severing of the xylem tissue
will result in an increase in antioxidant activity.
EXAMPLE 1
[0032] HPLC chromatography was performed on polyphenol obtained
from cured Oriental tobacco (see FIG. 5). Phenolic compounds were
identified by retention times and UV absorbance spectra
(diode-array) obtained from standards. The first three peaks were
identified as chlorogenic acid and its isomers. The forth peak is
scopletin and the seventh peak is rutin. Fractionation of phenolic
compounds was achieved through methanol elution with prepacked C-18
cartridges. Antioxidant tests (using Ferric Reducing Antioxidant
Potential) and subsequent analysis by HPLC were conducted
respectively for each fraction (see FIG. 6). Using this method and
the previously described determination of antioxidant capacity
(Ferric Reducing Antioxidant Potential), it was found that tobaccos
cured in various ways contained widely differing phenolic contents.
Specifically, tobacco cured by air-curing had a significant
reduction in phenolic compounds, while in tobacco cured by
flue-curing the reduction in phenolic compounds was not
significant.
[0033] There were three major phenolic compounds identified as
chlorogenic acid (and its isomers), rutin and scopoletin in
Oriental and Bright tobacco. In Oriental tobacco, chlorogenic acid
accounts for 30.6% of the total antioxidant capacity; rutin 19.65%;
and scopletine 1.5%. In Bright tobacco, chlorogenic acid accounts
for 28.7% of the total antioxidant capacity and rutin accounts for
11.5% (see FIG. 7). In green Burley tobacco, chlorogenic acid
accounts for 13.4% of the total antioxidant capacity and rutin
accounts for 6.2%. In cured Burley tobacco, no chlorogenic acids
were found (see FIG. 8).
[0034] During curing, polyphenol antioxidant capacity was reduced
by 80% in the first two weeks of curing and significant TSNAs
levels were only found after curing for two weeks (see FIG. 9). At
least half of this decline in antioxidant capacity is due to
decreases in isomers of chlorogenic acid and rutin.
EXAMPLE 2
[0035] Xylem Severing
[0036] The tobacco stalk is wounded about 1-3 weeks before harvest.
This is done by cutting, at several places not at the same level,
the lowermost portion of the stalk below the first leaf position by
means of applying a cutting edge at a right angle to the stalk's
axis to cut into the pith (depth of about 5 mm, length about 5 mm).
Alternatively, several plugs of about 5 mm diameter and 5 mm depth
can be taken transversely at different levels, also near the base
of the stalk. The aim is to reduce the stalk's water transport
capacity to a degree sufficient to impose sufficient water stress
on the plant. Preferably, the stalk's water transport capacity is
reduced by about half by cutting about half of the circumference of
the stalk. The stalk's water transport capacity can also be reduced
by three-quarters, two-thirds, one-third, one-quarter, etc., by
cutting the appropriate amount of the circumference of the stalk.
While transpiration of water occurs through leaves, severed xylem
tubules take up air, creating embolisms that further block upward
water transport (see Sperry, J. S., "Limitations on stem water
transport and their consequences," in Gartner B. L. (Ed) Plant
Stems. Physiology and Functional Morphology, Academic Press, New
York (1995), pp. 105-124). Blocking about one-quarter, one-third,
two-thirds, three-quarters, or preferably half of the water
transport in the tobacco plant will suffice to impose sufficient
water stress on the plant and will produce the desired increase of
laminar antioxidant capacity. Severing of the xylem can be done
alone or together with root-pruning. The use of one technique or
the other can be determined by soil structure, cultural practice,
weather and irrigation.
EXAMPLE 3
[0037] Root Pruning
[0038] Root pruning can be conducted by any means known in the art.
However, whatever root pruning technique is used, it is desired
that the tobacco plant remain standing after the procedure.
1 Root Pruning - Reduce TSNAs up to 90% Total Antioxidant NNO
Capacity TSNA Nitrogen (.mu.mol/g) (ppb) (ppb) Nicotine % Root
Pruning 29.4 447 81 3.05 Control 19.2 4726 531 4.22
[0039] The table shows the results of experiments using root
pruning to raise Burley native leaf antioxidant capacity in order
to interfere with TSNA production during air curing. Curing
resulted in leaves with nearly 50% antioxidant capacity increases
and reductions of TSNAs of about 90% as compared to untreated
control plants.
EXAMPLE 4
[0040] Treatment of Plant Stalks With Antioxidant Treatment
Substances Such as Pills and Liquids
[0041] Treatments of plants with chemicals typically involve
dusting or spraying the plants directly, or treating the soil and
invoking root uptake systems for systemic delivery. A chemical
delivery system that did not require these traditional methods
needed to be developed for tobacco to preserve the quality and
yield of the leaves. A preferred technique is the use of a
treatment substance in the form of pills, capsules, caplets,
pastes, emulsions, foams or powder to administer customized
mixtures of chemicals to plants. The pills, capsules, caplets,
pastes, emulsions, foams or powder administered to the tobacco
plants in the present invention contain a sufficient amount of
antioxidants or chemicals which will increase the levels of
antioxidants in tobacco. Preferably, the pills, capsules, caplets,
pastes, emulsions, foams or powder comprise 0.5-12 grams of
antioxidants or chemicals which will increase the levels of
antioxidants in tobacco, most preferably about 10 grams. These
pills, capsules, caplets, pastes, emulsions, foams or powder are
injected or placed within the vascular system of the main stem and
rely on dissolution via the intrinsic water and translocation via
the existing vasculature of the plant. Injection can be conducted
by any means known in the art or contemplated by one of skill in
the art. Chemicals to be administered can be compounded
individually, microencapsulated, layered or in concentric layers,
with coatings of varying degrees of solubility, to customize the
rate of administration. Preferred chemicals to be administered are
tobacco antioxidants or chemicals that will elevate the levels of
tobacco antioxidants. During the harvest of Burley tobacco, the
stem is cut at ground level, exposing the main vascular system of
the central stalk. The treatment substance such as a pill
containing anti-oxidants and/or other chemicals (such as sodium
bicarbonate, ascorbic acid, glutathione, selenium), can be easily
inserted into the cut end of the stalk. As the treatment substance
dissolves, the chemicals are dispersed throughout the plant via the
existing vascular network.
2 Plant Pills - Reduce TSNAs up to 28% Total Antioxidant NNO
Capacity TSNA Nitrogen (.mu.mol/g) (ppb) (ppb) Nicotine % Plant
27.5 727 180 3.5 Treatment Substance Control 21.8 1021 334 4.21
[0042] The above results were obtained from experiments conducted
to raise Burley leaf antioxidant levels and interfere with TSNA
production during air curing. Specifically, ascorbic acid powder
was packed into dry wall anchors to form a type of pill. The
anchors containing the ascorbic acid powder were then inserted into
the cut end of the stalk. Curing resulted in leaves with nearly a
28% reduction of TSNAs.
[0043] In an alternative method, the top of the tobacco plant is
cut off and the anchors containing the ascorbic acid powder are
inserted therein.
[0044] In an alternative method of the present invention, after
harvesting, the stalks of the tobacco plants are dipped into a
solution of antioxidants and/or chemicals that will elevate the
levels of tobacco antioxidants, and the vascular system of the
plant is allowed to absorb the solution.
[0045] 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.
* * * * *