WO2007053096A1

WO2007053096A1 - Tobacco product with reduced content of nitrosamines

Info

Publication number: WO2007053096A1
Application number: PCT/SE2006/001257
Authority: WO
Inventors: Thomas Ericsson
Original assignee: Njette Ab
Priority date: 2005-11-07
Filing date: 2006-11-07
Publication date: 2007-05-10
Also published as: WO2007053096B1

Abstract

The invention relates to tobacco products comprising less than 0.1 ug nitrosamines/g dry tobacco as well as methods producing said tobacco products containing said tobacco.

Description

0 7 -11- 2006

TOBACCO PRODUCT

FIELD OF INVENTION

BACKGROUND OF INVENTION

Smoking articles, (e. g., cigarettes, cigars, pipes, etc) and smokeless tobacco products (e. g., chewing tobacco, dry and moist snuff, etc.) are made from natural tobacco, reconstituted tobacco, and blends thereof. Natural tobacco may contain carcinogenic nitrosamines as well as other harmful components, such as Pb, Hg, Cd etc. Such components are formed during the curing of tobacco, e.g., tobacco-specific nitrosamines (TSNAs) and non-tobacco specific nitrosamines. Likewise, reconstituted tobacco formed from natural tobacco by-products may also contain nitrosamines. The nitrosamines may be transferred from tobacco products to man when used in snuff, chewing tobacco or other products made thereof. In addition, the smoke produced by tobacco products containing nitrosamines can also contain nitrosamines, which are either transferred from tobacco or pyro-synthesized. These substances have been shown to be harmful to man, mainly due to their carcinogenic properties. Extensive research has been conducted on the removal of nitrosamines and TSNAs in tobacco products.

Despite such attempts to remove TSNAs from tobacco, a need currently exists for an improved method of reducing the content of nitrosamines (e. g., TSNAs) in tobacco. Smokeless tobacco (SLT) has been associated with oral cancer for many decades. The epidemiologic studies addressing the human risks of SLT use, consists primarily of case-control studies performed over a 40-year period starting in 1957. Some of the compounds may promote cancer development in long-term SLT users.

WO 02/28209 discloses a method in which the content of the nitrosamines are reduced from 0,84 microgram/g dry tobacco to 40 nanograms per gram tobacco in solution, i.e., not dry tobacco (see example 3) or from 4 microgram/g dry tobacco to 0,8 microgram/gram (see example 1). Activated charcoal or zeolites was used to reduce the amount of the nitrosamines.

WO 02/087365 discloses a smokeless product suitable for human consumption in which said product comprises a content of the nitrosamines which are 0,3 ug /g tobacco or less. The tobacco is prepared from tobacco plants having a low content of TSNA, i.e., the variety Virginia flue (see page 6).

Therefore there is a need of developing new method that enables the possibility to produce nicotine with a reduced amount of carcinogenic components and thereby be able to produce new nicotine as well as new tobacco products with less amounts of the carcinogenic components, such as the nitrosamines as well as the heavy metals.

BRIEF DISCLOSURE OF THE INVENTION

The invention relates to a new tobacco product having a reduced level of at least nitrosamines. The tobacco products comprises less than 0.1 ug nitrosamines/g dry tobacco as well as reduced amount of other components such as as Fe (iron), Cr

(chromium), Al (aluminium), Pb (lead), As (arsenic), Ni (nickel), Hg (mercury) and Cd (cadmium), which are harmful for the human being.

The invention also relates to a method how to obtain such a tobacco product, wherein said method comprising the steps of; A method of producing a tobacco product comprising the steps of; providing tobacco material comprising nicotine and fibres, separating said fibres from said tobacco material, extracting nicotine from said tobacco material, obtaining nicotine, wherein said nicotine comprises less than 0.1 ug nitrosamines/mg nicotine and preparing a tobacco product from said nicotine and said fibres. By a unique combination of how to grow the tobacco plants, harvesting of the plant material as well as the purification process it is for the first time possible to provide a new tobacco product having s reduced amount of nitrosamines as well as Cr, Pb, As, Fe, Al, Ni, Hg and Cd, which are harmful for the human being as well as giving the nicotine product a bitter taste and reducing the coloration upon use as a tobacco product, such as Swedish snus. DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the context of the present application and invention, the following definitions apply:

The term " tobacco" refers to any part of a tobacco plant or mixtures of tobacco plants arising from the same or different species as well as being genetically modified (GMO). The tobacco plant belongs to the family Solanaceae and the genus Nicotiana. Examples of tobacco plants are those from Nicotiana tabacum and Nicotiana rustica. Examples of parts are leaves, stems, roots etc, which may be obtained by any method such as being fresh, frozen, deep-frozen, freeze dried or cured.

The term "tobacco product" refers to any product made from or containing tobacco such as nasal snuff, oral moist or dry snuff, chewing tobacco, pipe tobacco, cigar or cigarette tobacco, tablet, capsule, lozenge, chewing gum, mucosal or transdermal patch etc, used in any application where nicotine is to be delivered to the human body. It also refers to any such nicotine-free product.

The term "tobacco specific nitrosamines" (TSNA) refers to a number of nitrosamines, such as N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N'- nitrosoanabasine (NAB), and 4-(N-methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), N'-nitrosonornicotine.

The term "tobacco fibre" is intended to mean any part of the tobacco plant comprising cellulose, cellulose fibres and/or starch.

Tobacco product The invention relates to a new tobacco product having improved properties compared to other tobacco products, such as the content of the nitrosamines being less than 0.1 ug nitrosamines/g dry tobacco, such as less than 0.09, 0,08, 0,07, 0,06, 0,05, 0,04, 0,03, 0,02, 0,01 ug/g dry tobacco or even less than O.Olug nitrosamines/g dry tobacco. Examples are 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, or 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002 or 0.001 ug nitrosamines/g dry tobacco or even lower, i.e., the nicotine may be substantially free from nitrosamines. The nitrosamines may be N'-nitrosonornicotine (NNN)₅ N'-nitrosoanatabine (NAT), N'- nitrosoanabasine (NAB), and 4-(N-methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), and 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone. The amount of the nitrosamines may be determined by the method shown in example 9. Additionally the tobacco product may have a Cd content less than 0.5 ug/g dry tobacco, such as less than 0.4, 0.3, 0.2, 0.1, 0.05 or 0.01 ug/g dry tobacco, a Pb content less than 10 ug/g dry tobacco, such as less than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 or 0.1 ug/g dry tobacco, an As content less than 1 ug/g dry tobacco, such as less than 0.5, 0,4, 0,3, 0,2, 0.1 or 0.01 ug/g dry tobacco, aNi content less than 10 ug/g dry tobacco, such as less than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 or 0.1 ug/g dry tobacco, a Cr content less than 10 ug/g dry tobacco, such as less than 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5 or 0.1 ug/g dry tobacco. The nitrite content may be less than 100 ug/g dry tobacco, such as 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ug/g dry tobacco. The nitrate content may be less than 100 ug/g dry tobacco, such as 9O₅ 80, 70, 60, 50, 40, 30, 2O₅ 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 ug/g dry tobacco. A reduction of the above mentioned compounds will, give rise to a more healthy tobacco product, since the above mentioned compounds are carcinogenic or otherwise harmful to the body and gives the tobacco product a bitter taste as well as discolouration of the teeth. Accordingly, the nitrates as well as the nitrites are reduced. The tobacco product may be a smoking or a non-smoking product. Examples of smoking articles are cigarettes, cigars, fine cut smoking articles and examples of nonsmoking articles are nasal snuff, oral moist or dry snuff, and chewing tobacco.

The tobacco product may also comprise one or more additives selected from the group consisting of natural, natural identical, synthetic and semisynthetic additives such as menthol, bergamot, orange, mandarin, acai berry, eucalyptus and coffee, citrus or other fruit flavour, liquorice, etc.

The tobacco product may have the tobacco fibre material as a core and the nicotine adsorbed onto the tobacco fibre material or absorbed into the tobacco fibre material, such as by spraying the nicotine onto the tobacco fibre material. Additionally, the tobacco and/or the fibres from tobacco may be encapsulated into non- woven materials, well-known for a person skilled in the art.

The tobacco product may be formulated into one or more preparations, such as sachets, pouches, stick-pack, buccal pads or patches or compressed into chewing tablets, lozenges, resoriblettes or tablets for sucking. The resulting product is then packed in airtight packages to prevent losses by oxidization and/or vaporization of the nicotine. The tobacco product may have a release profile in vitro analysed during 35 minutes, with following intervals;

Nicotine released after 5 minutes is 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3,4,5,6,7,8,9, 10 mg.

Nicotine released after 10 minutes is 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3,4,5,6,7,8,9, 10 mg. Nicotine released after 15 minutes is 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2,

3,4,5,6,7,8,9, 10 mg.

Nicotine released after 20 minutes is 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3,4,5,6,7,8,9, 10 mg.

Nicotine released after 25 minutes is 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3,4,5,6,7,8,9, 10 mg.

Nicotine released after 30 minutes is 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3,4,5,6,7,8,9, 10 mg.

Nicotine released after 35 minutes is 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3,4,5,6,7,8,9, 10 mg. The release profile may be designed with respect to the rate and amount of nicotine and unwanted substances released by the formulation. The release of nicotine from the tobacco product may be designed to be released in a sufficient amount and/or a sufficient rate to enable a desired effect and on the other hand be designed to retain substances, which normally give rise to side-effects, such as, e. g., nitrosamines, and/or to release such substances in a substantially low amount and/or at a substantially slow rate.

In another embodiment of the invention the release of the invention, may be delayed compared to the commercially available moist snuff, to mimic the effect of nicotine replacement products such as, e.g., patches. The release profile may be 0.1-10 mg/h. The tobacco products may be designed so that at least about 50% w/w of the total content of nicotine is released within at the most 60 min when subject to the release test described in the European pharmacopoeia latest ed.

The tobacco product according to the invention may be designed so that at least about 50% w/w of the total content of nicotine is released within at the most 120 min when subject to the release test.

Extracted release

Normally release is tested in in- vitro studies. Extracted release is the amount release during use of snuff, snus, or another tobacco product. Mean extracted amount is calculated by subtraction of individual or mean values of used product from the mean content often "unused products".

Whether the nicotine steady state or single plasma levels from a refined tobacco product based on natural tobacco and/or genetically modified and/or a tobacco substitute and/or nicotine according to the invention, the tobacco and/or tobacco substitute may preferably have the calculated extracted amount in humans as typically; extraction (mg) 0,1, 0,2, 0,3, 0,4, 0,5, 0,6, 0,7, 0,8, 0,9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg.

Whether the nicotine steady state or single plasma levels from a refined tobacco product based on natural tobacco and/or genetically modified and/or a tobacco substitute and/or nicotine according to the invention, the tobacco and/or tobacco substitute may preferably have the biological parameters in humans as typically;

Cmax (ng/ml) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23,

24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more up to 50 ng/ml.

AUC (hours x ng/ml) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more up to 50 hours x ng/ml.

Whether the nicotine pH levels from a nicotine or a tobacco product according to the invention may have the pH parameters as typically; pH 7,0, 7,1, 7,2, 7,3, 7,4, 7,5, 7,6 7,8, 7,9, 8,0, 8,1, 8,2, 8,3, 8,4, 8,5, 8,6, 8,8, 8,9, 9,0, 9,1 to 10. The tobacco product may be produced by any method such as one of those described in the examples. A method of producing said tobacco product

The invention also relates to a method of producing nicotine and a tobacco product as described above. Tobacco cuttings as well as seeds may be planted in the fields. For example, the soil in the field may have a low content of heavy metals to secure that heavy metals will not be accumulated in the tobacco plants and later on be present in the nicotine after the nicotine has been purified from the tobacco plants. The nicotine is extracted from tobacco, such as from Tobacco tabacwn. The tobacco plant may be a genetic modified plant, such as a genetic modified plant in which the content of the nitrosamines have been reduced. The tobacco plant material may be any part of the plant such as roots, stems or leaves such as lamina of the leaf. The tobacco plants may be grown on farmland or in greenhouse, with or without fertilizers or addition of other growth stimulating components. The choice of fertilizer depends on the quality of the soil in the field and well-known for a person skilled in the art. Examples of fertilizers are organic fertilizers or NPK or UREA. In one embodiment the plant material is lamina, which is harvested and directly transferred into a temperature being below 0°C, such as being frozen, by freezing in liquid nitrogen and/or dry ice and/or deep freezing containers and/or any other technique, which halt all the ongoing processes within the plant and thereby reduce and/eliminate the possibility that nitrosamines will be expressed to elevated amounts within the plant material. Alternatively the tobacco plant material is green material without any visible yellow parts. The harvested plant material may also be treated in other ways as long as the production of nitrosamines is inhibited. By securing that the plant material is not stressed the levels of the nitrosamines will be kept lower as compared to when the plants are under any pressure. The harvested plant material may then be crushed, cut, milled or sliced and mixed with a water solution to which a base is added such as sodium hydroxide (NaOH) or calcium carbonate (CaCCβ, limestone) or calcium hydroxide (slaked lime) 10 % by weight and 25 % moisture. The plant mixture may then be packed into a vessel, such as a percolator or a reactor vessel and water may then be passed through the percolator to enable the nicotine to be extracted from the plant material. The fibres are then removed from the nicotinized broth and the nicotinized broth is further processed to purify the nicotine therein.

The nicotinized broth may be added to a cationic exchange resin, such as Amberlite™, such as Amberlite™ IPR 64, or Sepharose Fast Flow, (both may be obtained from Sigma- Aldrich) or Indion 464 supplied by Ion Exchange (India) Ltd. passed through the column whereby the nicotine will be bound to the column (cationic exchange resin). The nicotine may then be released from the column by the use of an acid such as by the use of acetic or phosphoric acid. After such method the nicotine will be released/extracted from the plant fibres. By the use of a cationic column the nicotine that will be obtained after passing said column, will be purer as compared to not using a cationic column.

The method gives rise to the opportunity to separate the nicotine and the fibre material and removing and/or minimising the amount of certain compounds within the nicotine as well as within the fibre material, such as carcinogenic as well as bitter tasting components. The plant material may be exposed to a subset of washing steps such as both alkaline and acid washing steps to remove unsuitable components on or within the material. Examples of washing steps are washing with NaOH and water or purified water.

The washing steps may be performed one or several times depending on the starting material. Samples may be taken after every washing step to follow the washing effect on the unwanted components, which are reduced and/or eliminated by the washing steps. The numbers of washing steps are dependent on the tobacco plant material and the number of washing steps may vary from time to time. However, a person skilled in the art will know how many washing steps that are needed to obtain the invented tobacco product by using the different tests that are shown in the examples. Finally, a tobacco product is obtained in which the nitrosamines are reduced as well as other components, such as Fe, Al, Ni, Hg and particularly nitrates and nitrites and the heavy metals Pb, Cd and As.

To obtain nicotine as well as fibres from the tobacco plant material, with minor amounts of impurities several factors are of importance. The quality of the chemicals used during the process is of importance, including the invented method in which the chemicals are used. Additionally, the growth of the tobacco plant in the field is also of importance and finally the harvest is also of importance. All these factors influence the quality of the obtained nicotine and tobacco products-

The nicotine obtained by the invented method having the same characteristics as the above defined nicotine. Following examples are intended to illustrate, but not to limit, the invention in any manner, shape, or form, either explicitly or implicitly.

EXAMPLES

EXAMPLE l

PURIFICATION OF NICOTINE FROM DEEP FROZEN TOBACCO LEAVES USING A CATION EXCHANGE RESIN.

Stage 1 Harvesting and deep freezing Fresh tobacco leaves from tobacco plants (="green tobacco") are collected and deep frozen (-2O⁰C) within less than 3 hours.

Stage II Mixing and Percolation

The deep frozen tobacco leaves and calcium carbonate (CaCO3, limestone) or calcium hydroxide (slaked lime) 10 % by weight are mixed in a Mixing machine

(ribbon) of Machine Steel Water is added to bring the moisture of the mixture to 25 % humidity. The slightly humid raw tobacco mixture is packed into a Machine Steel percolator, i.e. a cylindrical extractor. Purified water is percolated through the mixture to get a nicotinized broth.

Stage III Extraction

This nicotinized broth, i.e. the nicotine containing aqueous mother-liquor is transferred to a moderate-strong acid cation exchange resin, i.e., Amberlite™ IPR64 (Rohm and Haas/Ion Exchange Resins, Philadelphia, PA, USA or Indion 464 supplied by Ion Exchange (India) Ltd Tiecicon House, Dr. E. Moses Road,

Mahalaxmi, Mumbai-400 011, India) packed in tall columns of Machine Steel or Stainless Steel. The nicotine - nicotine cation exchange resin complex is washed with purified water to remove impurities.

Stage IV Separation The nicotine is eluted from the cation exchange resin column by IM sulphuric acid. The Nicotine salt is precipitated and collected from the bottom of the vessel.

Stage V Filtration and purification

The Nicotine salt is filtrated and purified by passing it through a stainless steel super centrifuge (8000 rpm for at least 6 minutes) to remove traces of water.

Stage VI Testing

After the filtration and purification of Nicotine Alkaloid, it was then tested for nitrosamine content (TSNA, i. e. NNN, NNK, NAT, and NAB). The result is shown in the table below batch 001 T. The total content, when tested, was at least less than 0.1 ug/g dry tobacco.

Thus, as indicated, the total level of nitrosamines in the tobacco can be selectively reduced.

Stage VII Packing

The purified nicotine is packed in epoxycoated drums under an inert gas such as nitrogen and stored in cool and dry place.

EXAMPLE 2 EXTRACTION AND PURIFICATION OF NICOTINE FROM FRESH LEAVES

USING AN ORGANIC SOLVENT AND SUBSEQUENT SEPARATION USING A CATION EXCHANGE RESIN.

Stage 1 Mixing Fresh tobacco leaves from tobacco plants (="green tobacco") and calcium carbonate (CaCO3, limestone) or calcium hydroxide (slaked lime) 10 % by weight are mixed in a Mixing machine (ribbon) of Machine Steel. Water is added to bring the moisture of the mixture to 25 % humidity.

Stage II Percolation The slightly humid raw tobacco mixture is packed into a Machine Steel percolator, i.e. a cylindrical extractor. Water is percolated through the mixture to get a nicotinized broth.

Stage III Extraction This nicotinized broth, i.e. the nicotine containing aqueous mother-liquor is subjected to liquid - liquid extraction with a water-immiscible non-polar solvent in tall columns of Machine Steel or Stainless Steel for liquid-liquid extraction. Several solvents have been used including ethyl-acetate, hexane, cyclohexane and heptane. Nicotine is thereby transferred to the organic solvent. The liquor leaving the column by the bottom is essentially free of nicotine alkaloid. Liquid-liquid extraction not only separates and isolates the alkaloid rapidly, but it also contributes to its purification, since some impurities are insoluble in the organic solvent.

Stage IV Separation The nicotine concentration in an organic water-immiscible non-polar solvent is estimated by titration. Ten (10) ml of the solvent is mixed with 10 ml of IOnormal H2SO4 in a beaker and titrated against IOnormal NaOH solution using Phenolpthalin as indicator. The count will determine the quantity of Nicotine in the solvent. The greater the count the greater the quantity of Nicotine dissolved in the solvent and a calculated quantity of dilute aqueous Sulphuric acid is added under agitation in a Reactor vessel Machine Steel or Stainless Steel to form Nicotine Sulphate 40 %. Nicotine Sulphate 40 % being insoluble and heavier than the organic water-immiscible non-polar solvent is collected from the bottom of the vessel. The Nicotine Sulphate 40 % is subsequently dissolved in purified water and transferred to a moderate-strong acid cation exchange resin, i.e., Amberlite™ IPR64 packed in tall columns of Machine Steel or Stainless Steel. The nicotine - nicotine cation exchange resin complex is washed with purified water to remove impurities. The nicotine is eluted from the cation exchange resin column by IM sulphuric acid. The Nicotine salt is precipitated and collected from the bottom of the vessel.

Stage V Filtration and purification

Stage VI Packing The purified Nicotine Sulphate is packed in epoxycoated drums and stored in cool and dry place.

Stage VII Alkalization

Nicotine sulphate 40 % is taken into a Stainless Steel reactor and Sodium Hydroxide is added under slow agitation. After alkalization is complete, Nicotine 95 % separates out on top and is decanted.

Stage VIII Purification of Nicotine Alkaloid

Nicotine Alkaloid is washed again with strong alkali (NaOH) water solution and separated by Super centrifugation to give pure Nicotine Alkaloid 98 %.

Stage IX Distillation of Nicotine Alkaloid

Nicotine Alkaloid of 97 % to 99,9 % purity is taken in the distillation equipment (preferably of glass or glass lined). Under high vacuum (almost 760mm), the impurity (water) starts distilling at approx 70 degrees centigrade. After the distilling slows down (after approx 4 % of the quantity taken), the receiver flask is emptied (preferable changed) for the collection of Nicotine Alkaloid. With the vacuum kept high, Nicotine starts distilling at approx 140-160 degrees centigrade. The distilled alkaloid is then stored in either glass or stainless steel vessel under inert atmosphere. Stage X Testing.

After the distillation of Nicotine Alkaloid, it was then tested for nitrosamine content (TSNA, i. e. NNN, NNK, NAT, and NAB). The result is shown in the table below batch 002 T. The total content, when tested, was at least less than 0.1 ug/g dry tobacco.

Stage XI Packing The purified nicotine is packed in epoxycoated drums under an inert gas such as nitrogen and stored in cool and dry place.

EXAMPLE 3

EXTRACTION AND PURIFICATION OF NICOTINE FROM DEEP FROZEN LEAVES USING AN ORGANIC SOLVENT, CATION EXCHANGE RESIN AND SUBSEQUENT DISTILLATION.

Stage 1 Mixing

Tobacco leaves from tobacco plants (="green tobacco") are collected and deep frozen (-2O⁰C) within less than 3 hours.

Stage II Mixing and Percolation

The deep frozen tobacco leaves and calcium carbonate (CaCO3, limestone) or calcium hydroxide (slaked lime) 10 % by weight are mixed in a Mixing machine (ribbon) of Machine Steel Water is added to bring the moisture of the mixture to 25 % humidity. The slightly humid raw tobacco mixture is packed into a Machine Steel percolator, i.e. a cylindrical extractor. Purified water is percolated through the mixture to get a nicotinized broth.

Stage III Extraction This nicotinized broth, i.e. the nicotine containing aqueous mother-liquor is subjected to liquid - liquid extraction with an organic water-immiscible non-polar solvent, i.e., ethyl-acetate or another solvent as mentioned above, in tall columns of Machine Steel or Stainless Steel for liquid-liquid extraction. Nicotine is thereby transferred to the organic solvent. The mother-liquor leaving the column by the bottom is essentially free of nicotine alkaloid. Liquid-liquid extraction not only separates and isolates the alkaloid rapidly, but it also contributes to its purification, since some impurities are insoluble in the organic solvent.

Stage IV Separation

The nicotine concentration in the organic solvent is estimated by titration and a calculated quantity of dilute aqueous Sulphuric acid is added under agitation in a Reactor vessel Machine Steel or stainless steel to form Nicotine Sulphate 40 %. Nicotine Sulphate 40 % being insoluble and heavier than the organic solvent, is collected from the bottom of the vessel.

The Nicotine Sulphate 40 % is subsequently dissolved in purified water and transferred to a moderate-strong acid cation exchange resin i.e., Amberlite™ IPR64 (Rohm and Haas/Ion Exchange Resins, Philadephia, PA, USA) packed in tall columns of Machine Steel or Stainless Steel. The nicotine - nicotine cation exchange resin complex is washed with purified water to remove impurities. The nicotine is eluted from the cation exchange resin column by IM sulphuric acid. The Nicotine salt is precipitated and collected from the bottom of the vessel.

Stage V Filtration and purification The Nicotine salt is filtrated and purified by passing it through a stainless steel super centrifuge (8000 rpm for at least 6 minutes) to remove traces of water.

Stage VI Packing

The purified Nicotine Sulphate is packed in epoxycoated drums and stored in cool and dry place. Stage VII Alkalization

The purified Nicotine Sulphate is taken into a Stainless Steel reactor and Sodium Hydroxide is added under slow agitation. After alkalization is complete, Nicotine 95 % separates out on top and is decanted.

Stage VIII Purification of Nicotine Alkaloid

Stage IX Distillation of Nicotine Alkaloid

Nicotine Alkaloid of 97% to 99,9% purity is taken in the distillation equipment (preferably of glass or glass lined). Under high vacuum (almost 760mm), the impurity (water) starts distilling at approx 70 degrees Centigrade. After the distilling slows down (after approx 4% of the quantity taken), the receiver flask is emptied (preferable changed) for the collection of Nicotine Alkaloid. With the vacuum kept high, Nicotine starts distilling at approx 140-160 degrees centigrade. The distilled alkaloid is then stored in either glass or stainless steel vessel under inert atmosphere.

Stage X Testing. After the distillation of Nicotine Alkaloid, it was then tested for nitrosamine content (TSNA, i. e. NNN, NNK, NAT, and NAB). The result is shown in the table below batch 003 T. The total content, when tested, was at least less than 0.1 ug/g dry tobacco.

Stage XI Packing

The purified nicotine is packed in epoxycoated drums under an inert gas such as nitrogen and stored in cool and dry place. EXAMPLE 4

EXTRACTION AND PURIFICATION OF NICOTINE FROM TOBACCO DUST USING AN ORGANIC WATER-IMMISCIBLE NON-POLAR SOLVENT, CATION EXCHANGE RESIN AND SUBSEQUENT DISTILLATION.

Stage 1 Mixing

Tobacco dust (stalks, top leaves, stem, scraps and dusts are tobacco wastes) and calcium carbonate (CaCO3, limestone) or calcium hydroxide (slaked lime) 10 % by weight are mixed in a Mixing machine (ribbon type) of Machine Steel. Water is added to bring the moisture of the mixture to 25 % humidity.

Stage II Percolation

The slightly humid raw tobacco mixture is packed into a Machine Steel percolator, i.e. a cylindrical extractor. Water is percolated through the mixture to get a nicotinized broth.

Stage III Extraction

This nicotinized broth, i.e. the nicotine containing aqueous mother-liquor is subjected to liquid - liquid extraction with an organic water-immiscible non-polar solvent, ethyl acetate, in tall columns of Machine Steel or Stainless Steel for liquid- liquid extraction. Nicotine is thereby transferred to an organic water-immiscible non- polar solvent. The liquor leaving the column by the bottom is essentially free of nicotine alkaloid. Liquid-liquid extraction not only separates and isolates the alkaloid rapidly, but it also contributes to its purification, since some impurities are insoluble in the organic solvent.

Stage IV Separation

The nicotine concentration in an organic water-immiscible non-polar solvent is estimated by titration. Ten (10)ml of the solvent is mixed with 10 ml of IOnormal H2SO4 in a beaker and titrated against IOnormal NaOH solution using Phenolpthalin as indicator. The count will determine the quantity of Nicotine in the solvent. The greater the count the greater the quantity of Nicotine dissolved in the solvent and a calculated quantity of dilute aqueous Sulphuric acid is added under agitation in a Reactor vessel Machine Steel or Stainless Steel to form Nicotine Sulphate 40 %. Nicotine Sulphate 40 % being insoluble and heavier than the organic water-immiscible non-polar solvent is collected from the bottom of the vessel.

The Nicotine Sulphate 40 % is subsequently dissolved in purified water and transferred to a moderate-strong acid cation exchange resin i.e., Amberlite™ IPR64 (Rohm and Haas/Ion Exchange Resins, Philadephia, PA, USA) packed in tall columns of Machine Steel or Stainless Steel. The nicotine - nicotine cation exchange resin complex is washed with purified water to remove impurities. The nicotine is eluted from the cation exchange resin column by IM sulphuric acid. The Nicotine salt, 95%, is precipitated and collected from the bottom of the vessel.

Stage V Filtration and purification Nicotine Sulphate 95 % is filtrated and purified by passing it through a Stainless

Steel super centrifuge (8000 rpm for at least 6 minutes) to separate suspended particles and remove traces of the organic water-immiscible non-polar solvent.

Stage VI Packing The purified Nicotine Sulphate is packed in epoxycoated drums under an inert gas such as nitrogen and stored in cool and dry place.

Stage VII Alkalization

The purified Nicotine Sulphate is taken into a Stainless Steel reactor and Sodium Hydroxide is added under slow agitation. After alkalization is complete, Nicotine

95-97 % separates out on top and is decanted.

Stage VIII Purification of Nicotine Alkaloid

Nicotine Alkaloid is washed again with strong alkali (NaOH) water solution and separated by Super centrifugation to give pure Nicotine Alkaloid 97-98 %. Stage IX Distillation of Nicotine Alkaloid

Nicotine Alkaloid of 97 % to 99,9 % purity is taken in the distillation equipment (preferably of glass or glass lined). Under high vacuum (almost 760mm), the impurity (water) starts distilling at approx 70 degrees Centigrade. After the distilling slows down (after approx 4% of the quantity taken), the receiver flask is emptied (preferable changed) for the collection of Nicotine Alkaloid. With the vacuum kept high, Nicotine starts distilling at approx 140-160 degrees centigrade. The distilled alkaloid is then stored in either glass or stainless steel vessel under inert atmosphere.

Stage X Testing.

After the distillation of Nicotine Alkaloid, it was then tested for nitrosamine content (TSNA, i. e. NNN, NNK, NAT, and NAB). The result is shown in the table below batch 004 T. The total content, when tested, was at least less than 0.1 ug/g dry tobacco. Thus, as indicated, the total level of nitrosamines in the tobacco can be selectively reduced.

Stage XI Packing

EXAMPLE 5

REFINED TOBACCO CONTAINING PURIFIED NICOTINE

Stage I Washings of the insoluble fraction After extraction of tobacco dust (stalks, top leaves, stem, scraps and dusts are tobacco wastes), fresh or deep frozen tobacco leaves according to the examples 1-4 above the insoluble fraction is refined by washing 5 times with pure water (acidic, alkaline and neutral) and finally with ethyl alcohol for 30 minutes each. Stage II Centrifugation and drying of insoluble fraction

The insoluble fraction is centrifuged for at least 6 minutes at 8000 revolutions per minute to remove the major part of the water.

Stage III Centrifugation and drying of insoluble fraction

After centrifugation the refined fibers are dried by Fluidized Bed Drying until having less than 1% humidity.

Stage IV Reconstitution of the tobacco by impregnation After centrifugation and Fluidized Bed Drying the refined fibers (the web), are impregnated with the high purity nicotine solution according to examples 1 -4 so that a purified finished tobacco is obtained that has nicotine content of 0.5-10% by weight.

Stage V Testing of the tobacco After the purification and impregnation the tobacco was then tested for nitrosamine content (TSNA, i. e. NNN, NNK, NAT, and NAB). The total content, was at least less than 0.1 ug/g dry tobacco. Thus, as indicated, the total level of nitrosamines in the tobacco can be selectively reduced without substantially reducing other levels of components in the tobacco, such as carbohydrates, starch and cellulose. See table 1.

Stage VI Pacldng

The tobacco product may be formulated into one or more preparations, such as sachets, pouches, stick-pack, buccal pads or patches or compressed into chewing tablets, lozenges, resoriblettes or tablets for sucking. The resulting product is the packed in air tight packages to prevent losses by oxidization and/or vaporization of the nicotine.

During storage pending packaging in consumer packs the purified tobacco as well as the formulated products derived from it are packed in epoxycoated drums under an inert gas such as nitrogen and stored in cool and dry place. Table 1. Total content of some impurities in different batches of tobacco produced by the different method in the examples above as well as some natural sources. Tobacco 1-5 represent natural sources.

a/ Tobacco purchased from different suppliers. b/ Results from farmed, harvested, treated and/or processed. c/ Results from farmed, harvested and treated.

018 were tested prior to purification (A) and after purification. EXAMPLE 6

NICOTINE CHEWING GUM PRODUCT

To obtain 1000 g of chewing gum 22 g nicotine obtained in the examples 1-4 was mixed with chewing gum premix, consisting of 600 g Dreyco Gum Base (L. A. Dreyfus Company, 3775 Park Avenue, Edison, New Jersey, USA), or Nicol 8 (Gum Base Co, ViaNerviano, 25, 20020 Lainate, Milan, Italy), 205 g sorbitol, 2Og Xylitol, 55 g talc, 40 g sodium bicarbonate, 20 g sodium carbonate 3 g aspartame, 1 g Acesulfame Potassium, 5 g menthol and flavouring oils. Mixing of nicotine, flavouring, buffer and premelted gum base was made in a gum kneader.

After the following steps of manufacture nicotine chewing gum was obtained: Extrusion, Cooling, Rolling, Scoring, Hardening and Wrapping. The following values for strength and purity were achieved:

Testing of the finished gum product Nicotine assay

Note: Carrier being polacrilex, cyclodextrin, starch, cellulose and cellulose derivatives, chewing gum bases, gelatin, alginate, PVP and tobacco and other fibers. The gum was then tested for nitrosamine content (TSNA, i. e. NNN, NNK, NAT, and NAB). The total content was at least less than 0.1 ug nitrosamines/g dry tobacco.

EXAMPLE 7

A SNUFF (SNUS) TOBACCO PRODUCT

The tobacco from example 5 is prepared into portion snuff (snus) pouches (sachets) of woven or non- woven material. Snuff (snus) was manufactured in different strengths e.g. 1, 3, and 6 mg per pouches. The nicotine amounts of tobacco were different in the formulations and were varied due to assay of the tobacco batches. Typically tobacco was mixed with excipients as fillers (dextrin e.g. polydextrin, maltodextrin, natural fibres, cellulose, starches or other suitable fillers), sweeteners (Xylitol, sorbitol, maltitol, mannitol, saccharin, sodium saccharin, cyclamates, Acesulfan K), adjutants to increase floe properties (Avicel; Macrocrystalline cellulose, talc) and natural and nature identical aromas and flavours. A typical formulation for 3 mg snus portion, Tobacco 10 to 24 %, Fibre 10 to 20 %, Polydextrose 5 to 15 %, Sodium Chloride 8 %, Sodium bicarbonate 7 %, polyvinyl pyrrolidon 3 %, Talc 2 %, Avicel 5 to 15 %, flavours quantum sati.

The tobacco product, such as sachet, pouch, stick-pack, is packed in air tight packages to prevent losses by oxidization and/or vaporization of the nicotine.

EXAMPLE 8

ATOBACCO CHEWING GUM PRODUCT A tobacco chewing gum (100Og) was manufactured by mixing a premix of 15 g glycerine and 15 g finely grinded tobacco (example 5) with the chewing gum premix in example 8, consisting of 600 g Dreyco Gum Base (L. A. Dreyfus Company, 3775 Park Avenue, Edison, New Jersey, USA), or Nicol 8 (Gum Base Co, Via Nerviano, 25, 20020 Lainate, Milan, Italy),, 205 g sorbitol, 2Og Xylitol, 55 g talk, 50 g sodium bicarbonate, 3 g aspartame, 1 g Acesulfame Potassium, 5 g menthol and flavouring oils. Mixing of tobacco, flavouring, buffer and premelted gum base was made in a gum kneader.

After the following steps of manufacture a tobacco chewing gum was obtained: Extrusion, Cooling, Rolling, Scoring, Hardening And Wrapping. Tobacco chewing gum:

Nicotine assay (initial) 2.00-2.20 mg/piece

(after 6 months storage) 1.8 - 2.20 mg/piece

The tobacco chewing gum was then tested for nitrosamine content (TSNA, i. e. NNN, NNK, NAT, and NAB). Nitrosamines were assayed by a gas chromatograph system, LC-MS-MS. The total content was at least less than 0.1 ug nitrosamines/g dry tobacco.

EXAMPLE 9

ANALYSIS OF THE NITROSAMINES Nitrosamines were assayed the method published in J Chromatogr A. 2003 Aug

8; 1008(2):135-43, Jansson et al, Analysis of tobacco-specific N-nitrosamines in snuff by ethyl acetate extraction and liquid chromatography-tandem mass spectrometry.

A rapid, selective and sensitive method for routine analysis of the four tobacco-specific

N-nitrosamines, N'-nitroso-nornicotine, N'-nitrosoanatabine, N'-nitrosoanabasine and 4- (methyl-nitrosamino)-l-(3-pyridyl)-l-butanone in snuff has been developed. The nitrosamines were isolated by ethyl acetate extraction and analysed by LC-MS-MS.

Except for evaporation and filtration, no additional clean-up steps are needed in the proposed method. The detection limits for standard in solvent are between 0.0005 and

0.001 microg/ml (0.005 and 0.01 microg/g).

EXAMPLE 10

MEASURMENT OF COLOURINDEX

Colour index was measured on the supernatant after dispersing and incubating the tobacco for 30 minutes followed by filtration. Measurements were made according to a modified European Pharmacopea method. As a measure of the colour of nicotine product and /or tobacco thereof, the Visual Color Index- VCI is introduced.

The pouches containing the refined tobacco product was placed in water in a testing tube made of clear glass with or with out stirrer. The Visual Colour Index is regarded as follows

The Visual Colour Index has been tested for the refined tobacco and common Swedish Snus purchased in any ordinary shops, gives the following VCI Index when tested; Results; The sum at each time point is summarised;

The colour Index gives a product that has less risk for colouring the teeth and less smell from the tobacco.

EXAMPLE I l

MEASURMENT OF HEAVY METALS, NITRITES AND NITRATES

Additionally the nicotine product and the tobacco product (example 5) will have a reduced amount of one or more substances such as Pb, As and/or Cd, measured using atomic absorption. The following metals were measured: Fe Al B V Mo Ti Hg Cu

Cr As Bi W Se Co Pb

P S Zn Cd Mn Ni Sr

Na Ms Ca K Nitrates nitrites

The method is a standard method described in Swedish or international standards (SS 028183, ISO). The dried and/or moisture containing tobacco is treated as per the standards and analysed atomic absorption, ICP Optical, ICP-MS, and/or ICP-OES. Results see below in table 2 and 3.

Claims

1. A tobacco product comprising less than 0.1 ug nitrosamines/g dry tobacco.

2. The tobacco product according to claim 1, wherein said tobacco product comprises less than 0.05 ug nitrosamines/g dry tobacco.

3. The tobacco product according to claim 2, wherein said tobacco product comprises less than 0.01 ug nitrosamines/g dry tobacco.

4. The tobacco product according to claim 3, wherein said tobacco product comprises less than 0.005 ug nitrosamines/g dry tobacco.

5. The tobacco product according to any of preceding claims, wherein the nitrosamines is at least one of N'-nitrosonornicotine (NNN), N'-nitrosoanatabine (NAT), N¹- nitrosoanabasine (NAB), and 4-(N-methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK), and 4-(Methylnitrosamino)-l-(3-pyridyl)-l-butanone.

6. The tobacco product according to any of preceding claims, having a Cadmium (Cd) content less than 0.5 ug/g dry tobacco.

7. The tobacco product according to any of preceding claims, having a Lead (Pb) content less than 10 ug/g dry tobacco.

8. The tobacco product according to any of preceding claims, having an arsenic (As) content less than 1 ug/g dry tobacco.

9. The tobacco product according to any of preceding claims, having a Nickel (Ni) content less than 10 ug/g dry tobacco.

10. The tobacco product according to any of preceding claims, having a Chromium content less than 10 ug/g dry tobacco.

11. The tobacco product according to any of preceding claims, having a nitrite content less than 100 ug/g dry tobacco.

12. The tobacco product according to any of preceding claims, having a nitrate content less than 100 ug/g dry tobacco.

13. The tobacco product according to any of preceding claims, comprising tobacco fibres containing less than 0.1 ug nitrosamines/g dry tobacco.

14. The tobacco product according to any of preceding claims, comprising tobacco fibres containing less than 0.05 ug nitrosamines/g dry tobacco.

15. The tobacco product according to any of preceding claims, comprising tobacco fibres containing less than 0.01 ug nitrosamines/g dry tobacco.

16. The tobacco product according to any of preceding claims, comprising tobacco fibres containing less than 0.005 ug nitrosamines/g dry tobacco.

17. The tobacco product according to any of preceding claims, wherein said tobacco product comprises nicotine and tobacco fibres wherein said nicotine and tobacco fibres are encapsulated into a carrier.

18. The tobacco product according to claim 17, wherein said carrier is a non- woven material.

19. The tobacco product according to claims 17-18, wherein said nicotine is absorbed and/or adsorbed into the tobacco fibre material and encapsulated into a carrier.

20. The tobacco product according to any of preceding claims, wherein said tobacco product comprises at least one additive.

21. The tobacco according to claim 20, wherein said additive is selected from the group consisting of menthol, bergamot, eucalyptus, coffee, citrus or other fruit flavour, orange, mandarin, Acai berry and liquorice.

22. The tobacco product according to any of preceding claims, wherein the tobacco product is selected from the group consisting of smoking articles, such as cigarettes, cigars, fine cut smoking articles and non-smoking articles, such as nasal snuff, oral moist or dry snuff, chewing gum, lozenge, capsules and chewing tobacco.

23. The tobacco product according to claim 22, wherein the tobacco product is selected from the group consisting of moist and dry snuff, chewing tobacco and snus.

24. A package containing at least one tobacco product according to any of preceding claims. 25. A method of producing a tobacco product comprising the steps of; a. providing tobacco material comprising nicotine and fibres, b. separating said fibres from said tobacco material, c. extracting nicotine from said tobacco material, d. obtaining nicotine, wherein said nicotine comprises less than 0.1 ug nitrosamines/mg nicotine and e. preparing a tobacco product from said nicotine and said fibres.

25. The method according to claim 24, wherein the tobacco material in step a) is green tobacco plant material.

26. The method according to claim 25, wherein said tobacco material is leaf material.

27. The method according to claims 24-26, wherein the tobacco material in step a) is kept at a temperature below O⁰C.

28. The method according to claim 27, wherein said tobacco material is freezed in liquid nitrogen.

29. The method according to any of claims 24-28, wherein said extraction in step c) is performed by the use of a cationic exchange resin.

30. The method according to claim 29, wherein said cationic exchange resin is Amberlite™ or Sepharose Fast Flow™ or Indion 464 ™

31. The method according to claim 30, wherein said Amberlite™ is Amberlite™ IPR 64.