US 20030056801 A1
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.
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.
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 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
FIG. 1 is a graph of antioxidant capacity of green and cured Oriental, bright and Burley tobacco.
FIG. 2 is a graph correlating the amount of polyphenols present in tobacco extract with the antioxidant capacity of the tobacco extract.
FIG. 3 depicts a mechanism by which antioxidants in tobacco inhibit TSNA formation.
FIG. 4 is a table listing antioxidant capacity of various phenolic compounds.
FIG. 5 is a graph of HPLC chromatograms of phenolic compounds from cured Oriental tobacco.
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.
FIG. 7 shows graphs of the approximate percent of antioxidant capacity for chlorogenic acid, rutin and scopletin in Oriental and bright tobacco.
FIG. 8 shows graphs of antioxidant capacity of green Burley tobacco and cured Burley tobacco.
FIG. 9 is a graph of antioxidant capacity of Burley tobacco compared to TSNA and nitrite capacity over a two plus week curing process.
FIG. 10 is a graph of TSNA content in cured Burley tobacco, with and without root pruning.
FIG. 11 is a graph of antioxidant levels in cured Burley tobacco, with and without root pruning.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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.
 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).
 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.
 Xylem Severing
 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.
 Root Pruning
 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.
 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.
 Treatment of Plant Stalks With Antioxidant Treatment Substances Such as Pills and Liquids
 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.
 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.
 In an alternative method, the top of the tobacco plant is cut off and the anchors containing the ascorbic acid powder are inserted therein.
 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.
 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.