CLIAMS:A solvent system to reduce tobacco specific nitrosamine (TSNA) and polycyclic aromatic hydrocarbon (PaH) compounds in tobacco comprising;
atleast one polar solvent; and atleast one non-polar solvent.
2. A solvent system as claimed in claim 1, wherein the solvent system maintains the nicotine content in the tobacco.
3. A solvent system as claimed in claim 1, wherein said polar solvent is selected from ethanol, dichloromethane (DCM) or a combination thereof.
4. A solvent system as claimed in claim 1, wherein said non-polar solvent is n-hexane.
5. A solvent system as claimed in claim 1, wherein the ratio of n-hexane:dichloromethane is 80:20.
6. A solvent system as claimed in claim 1, wherein the ratio of n-hexane:ethyl alcohol is 90:10.
7. A solvent system as claimed in claim 1, wherein the ratio of n-hexane:ethyl alcohol and DCM is 85:10:5.
8. A solvent system as claimed in claim 1, wherein the reduction in B(a)P is about 80%.
9. A solvent system as claimed in claim 1 claimed in claim 1, wherein the reduction in TSNA is about 45%.
10. A solvent system as claimed in claims 1 and 2, wherein the loss in nicotine content is not greater than 15%.
11. A method to reduce nitrosamine and PaH compounds employing the solvent system of claim 1 comprising the steps of:
(i) soaking tobacco in said solvent system for about 20 to48 hours;
(ii) removing the solvent; and
(iii) further soaking of tobacco of step (ii) in freshly prepared solvent system for about 12 to 48 hours.
12. A combustible or smokeable product comprising tobacco washed in solvent system of claim 1.
13. The combustible or smokeable products as claimed in claim 12 can be selected from cigarette, cigar, cheroot, heat-not-burn formats and the like.
,TagSPECI:Field of invention

The present invention relates to a solvent system to treat tobacco leaves so as to reduce the levels of tobacco specific nitrosamines (TSNAs), polynuclear aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP), without substantially altering the nicotine content. It also relates to a method of employing said solvent system and a combustible product comprising such treated tobacco.

Background and prior art of the invention

Tobacco materials such as natural tobacco leaves, cut tobacco, midribs, stems and roots contain various components including nicotine, nitrosamines, hydrocarbons and proteins. Several chemicals such as tobacco specific nitrosamines like tobacco specific nitrosamines (TSNAs), polynuclear aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BaP), carbonyls, carbazole, phenol, catechol are undesirable components of tobacco. PAHs are a group of chemicals where the constituent atoms of carbon and hydrogen are linked by chemical bonds in such a way as to form two or more rings, or “cyclic” arrangements. For this reason, these are also referred to as polycyclic hydrocarbons or polynuclear aromatics. Examples of such chemical arrangements are anthracene (3 rings), pyrene (4 rings), benzo(a)pyrene (5 rings), and similar polycyclic compounds. TSNAs are formed by reactions involving free nitrate during processing and storage of tobacco, and by combustion of tobacco containing nicotine and nornicotine in a nitrate rich environment. It is also known that fresh-cut, green tobacco contains virtually no tobacco specific nitrosamines. It is widely accepted that such nitrosamines are formed post-harvest, during the conventional curing process, and in the combustion process. Generally, in a 750 mg of tobacco which forms a cigarette, the total TSNA constitutes in the range of 0.4-8 µg/g, depending upon the type of tobacco. Whereas, when the smoke is analyzed, the total TSNA attributes to 150-400 ppb. Similarly, the total B(a)P amounts to 0.12-0.18 µg/g.

It is thus desirable that some of these natural tobacco material components be reduced in amount or removed while retaining the others at certain amounts to maintain smoking flavor. This process of reducing or removing the undesirable components is often referred to as tobacco washing.

However, as the structure of TSNAs is similar to nicotine, which is a desirable component, it is very difficult and complicated to remove TSNAs selectively without reducing the content of nicotine. Many attempts have been made to reduce the amounts of TSNA and B(a)P in tobacco, which apart from being undesirable, impacts the smoke and sensory profile of tobacco. For instance US 2002/134394 discloses a method for reducing nitrosamine content in tobacco by extraction with an aqueous solvent and activated charcoal or a resin and regenerating said tobacco. WO 02/28209 discloses a method in which an extract solution obtained by extracting tobacco with a solvent is brought into contact with an adsorbent such as activated carbon or zeolite in order to reduce tobacco-specific nitrosamines (TSNAs). US 5,810,020 discloses that nitrosamines are removed by a liquid-liquid extraction treatment using a great amount of crown ether. WO 01/65954 discloses that tobacco is treated with supercritical carbon dioxide to extract nitrosamines and the extract is subjected to a nitrosamine removing process. US20120125354 and WO 2013142483 disclose a process for preparing a smokeless tobacco composition containing a cured tobacco material and a reduced benzo(a)pyrene concentration. These disclose the method of extracting B(a)P from a tobacco material involves treating the material with methanol or a relatively non-polar solvent such as hexane, cyclohexane or methylene chloride.

However none of the prior art teaches an effective manner of removal of TSNA, B(a)P which is simple, cost effective and does not require complex technicalities or manpower yet retaining the desirable nicotine content.

Therefore, there exists a need to develop a method for effective removal of TSNA, B(a)P from tobacco yet retaining the desired amount of nicotine.

Accordingly, the present invention provides a solvent system which is simple, convenient and cost effective for washing tobacco and a method thereof by rendering the use of high throughput machines or technical complexities unnecessary.

Objects of the Invention

It is an object of the present invention to overcome the disadvantages of the prior arts.
It is another object of the present invention to provide a simple, convenient and cost effective system for washing of tobacco without employing high throughput machines or technical complexities.

It is another object of the present invention to provide a simple, convenient and cost effective system for efficient removal of TSNA, B(a)P washing of tobacco.

It is another object of the present invention to provide a simple, convenient and cost effective system for efficient removal of TSNA, B(a)P washing of tobacco yet retaining the desired amount of nicotine.

It is yet another object of the present invention is to provide a solvent system comprising at least one polar and one non-polar solvent, at a specific concentration to effectively reduce TSNA, B(a)P without substantially altering the nicotine content.

It is yet another object of the present invention is to provide a method employing a solvent system comprising atleast one polar and one non-polar solvent, at a specific concentration to effectively reduce TSNA, B(a)P without substantially altering the nicotine content.

SUMMARY OF THE INVENTION

In one aspect the present invention provides a solvent system comprising at least one polar and one non-polar solvent, at a specific concentration to effectively reduce TSNA, B(a)P without substantially altering the nicotine content.

In one other aspect the present invention provides a method employing said solvent system to effectively reduce the amount of TSNA, B(a)P without substantially altering the nicotine content.

In another aspect the present invention provides a combustible product comprising the tobacco obtained after being washed or treated according to the said method employing said solvent system.

BRIEF DESCRIPTIONS OF ACCOMPANYING DRAWINGS

Figure 1 graphically represents a comparison among various concentrations of the polar and non-polar solvents in the solvent mixture. It can be seen that solvent mixtures having various ratios effect on TSNA, B(a)P and nicotine. Hex in the graph indicates Hexane; DCM indicates Dichloromethane; EtOH indicates Ethyl alcohol; and AA indicates Acetic acid)

Figure 2 represents the reduction in TSNA, B(a)P and nicotine when a solvent system comprising Hexane:Dichloromethane at a ratio of 80:20 is employed to treat or wash tobacco.

Figure 3 represents the reduction in TSNA, B(a)P and nicotine when a solvent system comprising Hexane:Ethanol at a ratio of 90:10 ratio is employed to treat or wash tobacco.

DETAILED DESCRIPTION OF THE INVENTION

As briefly discussed in the background section, it is known that tobacco smoke contains carcinogenic compounds that are the cause of morbidity and mortality to smokers. Such compounds include PAHs, TSNAs, carbazole, phenol, and catechol. PAHs are a group of chemicals where constituent atoms of carbon and hydrogen are linked by chemical bonds in such a way as to form two or more rings, or “cyclic” arrangements and their carcinogenic potential is widely known. B(a)P is one such compound. Likewise, TSNAs are electrophilic alkylating agents and are potent carcinogens. They are formed by reactions involving free nitrate during processing and storage of tobacco, and by combustion of tobacco containing nicotine and nornicotine in a nitrate rich environment. As mentioned above, it is widely accepted that such nitrosamines are formed post-harvest, during the conventional curing process, and in the combustion process. Commonly known TSNAs are N-nitrosonornicotine (NNN), 4-methyl-N-nitrosamino-1-(3-pyridyl)-1-butanone (NNK), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB).TSNA levels are particularly high in chewing tobaccos and snuff. The partially anaerobic processes that occur during fermentation promote the formation of TSNAs from tobacco alkaloids by promoting increased nitrite levels; in particular, over-fermentation can increase TSNA levels in snuff by its effects on nitrate levels and microbial enzymatic activity.

Since the nitrate level of tobacco is important for nitrosamine formation in cigarette smoke, a significant reduction of nitrosamines in smoke negatively affects the smokability or the taste of the tobacco.

Several approaches to reduce the amount of said carcinogens including TSNAs, B(a)Ps are known, however, they mostly involve multi-step extraction. Consequently, these processes require use of high throughput machines thus making them expensive and complicated.

Unexpectedly, the present inventors have found a simple yet effective solvent system comprising at least one polar and one non-polar solvent, at a specific concentration to effectively reduce TSNA, B(a)P without altering the nicotine content. Consequently, having no adverse affect on smokability and taste of the tobacco.

The present invention also relates to a method of tobacco washing employing said solvent system. As stated before, said system and method are economically significant as it avoids any high throughput machines or technical complexities.

In one embodiment suitable polar solvent include ethanol and dichloromethane and the non-polar solvent is n-hexane.

In an embodiment of the present invention the solvent system comprises n-hexane and dichloromethane in a ratio of 8:2. The solvent system preferably comprises 60-80% of n-hexane and 20 to 40% of dichloromethane.

In another embodiment the solvent system comprises n-hexane and ethanol in a ratio of 7:3 or 9:1. The solvent system preferably comprises 70 to 98% of n-hexane and 2 to 30% of ethanol.

In yet another embodiment the solvent system comprises n-hexane, ethanol and dichloromethane in a ratio of 8.5:1:0.5. The solvent system preferably comprises 80 to 85% of n-hexane, 5-10% of ethanol and 5-10% of dichloromethane.

The novel solvent system at the said ratios was unexpectedly found to effectively reduce the levels of TSNA up to 45% and B(a)P up to 79.4%. Most importantly, the novel system has minimal effect on nicotine level wherein the loss in nicotine is not greater than 15%.

In another embodiment the present invention provides a method to reduce such toxicants including nitrosamine and/or PaH compounds present in tobacco with minimal loss on nicotine which involves soak the tobacco with the said solvent system for a suitable time period followed by removing the solvent. Fresh solvent is added and there is a further soaking for a certain time period. After the steps of soaking the solvent is removed and the levels of the carcinogens are analyzed in the tobacco. For effective removal, the soaking in the first step is for about 32 to 44 hours and followed by another 24 to 48 hours of soaking in the freshly added solvent.

The present invention further relates to a combustible product comprising the tobacco washed or treated according to said method employing said solvent system. The combustible product can be a cigarette, cigar, cheroot, heat-not-burn formats and the like.

The present inventors found that at the specific ratios of the concentrations of hexane:dichloromethane and hexane: ethanol are 80:20 and 90:10 respectively, an optimum reduction in the levels of TSNA and B(a)P was observed. As observed in figures 2, the combination of hexane:dichloromethane at a ratio of 80:20 significantly reduces TSNA to about 45% and B(a)P to about 79.4% while retaining the nicotine to the levels of 87%.

Similarly the solvent system comprising of hexane and ethanol at a ratio of 90:10 provides significant reduction in TSNA and B(a)P amounting to 35% and 66% respectively and retains nicotine at the levels atleast 92%.

In accordance with the present invention, TSNA is reduced to the extent of 48%, B(a)P at 64% and remarkably, nicotine loss is not more than 11%.

Further as demonstrated in figure 3 the amount of TSNA and B(a)P present in the extracted solvent after treatment with the solvent system.

The present invention is described by way of non-limiting illustrative examples.

Determination and analysis of nicotine in tobacco: 0.25 gm of ground tobacco was dissolved in 25 ml water, extraction was done using orbitek shaker for 30 mins. Resulting solution was filtered on whatman 40 filter paper. Water layer was analyzed for Nicotine content using Flow Analyzer.

Nicotine analysis in smoke: The Cambridge filter pad post smoking was extracted using Isopropanol along with reference standard (n-heptadecane). Resulting solution was analyzed for Nicotine using Gas Chromatography.

Example 1 (Comparative example)

1 kilogram of tobacco was added to a solvent system comprising varying ratios of of n-hexane,DMC and ethanol. The total volume of the solvent system was about 15 liters. The tobacco was soaked for at least 20 hours (first wash). After soaking, it was filtered and fresh solvent was added amounting to 10 liters, and further soaked for at least 12 hours (second wash). This was followed by filtration and drying the tobacco at room temperature for about 12 hrs. The ratios of the solvents used in the system are mentioned in the graph represented in Figure 1.

As observed in Figure 1, the combination of dichloromethane and hexane at ratio of 70:30 and 60:40 fails to reduce TSNA and B(a)P levels within the acceptable limits.
Likewise solvents such as ethyl acetate, acetic acid show the highest percent reduction of B(a)P to the extent of 82.6, 78.2% respectively but fails to reduce the TSNA content without compromising the nicotine levels.
With hexane and ethanol at various ratios such as 70:30, 80:20, 95:5 and 97.5:2.5, there is variation in the reduction of TSNA, B(a)P and nicotine levels, which is again unacceptable.
A combination of three solvents as mixture such as hexane, dichloromethane and ethanol yet again is unacceptable as it results in heavy reduction of nicotine to the extent of 16.3%.

Example 1 (Working)

1 kilogram of tobacco was added to a solvent system of n-hexane:DMC 80:20 of 15 liters, and soaked for 20 hours (first wash). After soaking, it was filtered and fresh solvent was added amounting to 10 liters, and further soaked for 24 hours (second wash). This was followed by filtration and drying the tobacco at room temperature for about 12 hrs. The amounts of TSNA, B(a)P and nicotine was measured according to the method mentioned hereinbefore.

Non polar solvent Polar solvent TSNA reduction BaPreduction Nicotine reduction
Hexane Dichloro methane (DCM)
60-80% 20-40% 30.18% – 45% 70-80% 10.2-13.5%

Example 2 (Working)
1 kilogram of tobacco was added to a solvent system of n-hexane:ethanol in a ratio of 90:10 of 15 liters, and soaked for 48 hours (first wash). After soaking, it was filtered and fresh solvent was added amounting to 10 liters, and further soaked for 24 hours (second wash). This was followed by filtration and drying the tobacco at room temperature for about 12 hrs. The amounts of TSNA, B(a)P and nicotine was measured according to the method mentioned hereinbefore.

(I wash: 48 hrs; II wash: 24 hrs)
Non polar solvent Polar solvent TSNA reduction BaP reduction Nicotine reduction
Hexane Ethanol (EtOH)
70-98% 2-30% 15-35% 64.6 - 75% 2.1-15.6%

Example 3 (working)
1 kilogram of tobacco was added to a solvent system of n-hexane:DMC:ethanol in a ratio of 85:5:10 of 15 liters, and soaked for 24 hours (first wash). After soaking, it was filtered and fresh solvent was added amounting to 10 liters, and further soaked for 12 hours (second wash). This was followed by filtration and drying the tobacco at room temperature for about 12 hrs. The amounts of TSNA, B(a)P and nicotine was measured according to the method mentioned hereinbefore.

Non polar solvent Polar solvents combined TSNA reduction BaP reduction Nicotine reduction
Hexane DCM Ethanol (EtOH)
80-85% 5-10% 0-10% 27.4-35.3% 68.8-78.2% 11.56-16.3%

Example 4 (working)
1 kilogram of tobacco was added to a solvent systems of n-hexane:DMC, hexane:ethanol and hexane:DCM:ethanol in a ratio of 80:20, 90:10 and 85:5:10 ratio of 15 liters, and soaked for 44 hours (first wash). After soaking, it was filtered and fresh solvent was added amounting to 10 liters, and further soaked for 48 hours (second wash). This was followed by filtration and drying the tobacco at room temperature for about 12 hrs. The amounts of TSNA, B(a)P and nicotine was measured according to the method mentioned hereinbefore.
Non-polar solvent Polar solvents TSNA reduction B(a)P reduction Nicotine reduction
Hexane 80% DCM-20% 45% 80% 13.5%
Hexane 90% EtOH-10% 35% 66% 8.5%
Hexane 85% DCM 5%, EtOH 10% 27.4% 78.2% 11.56%

Example 5 (Working)
20 gram of tobacco (source: commercial cigarette samples) was added to a solvent system of n-hexane:ethanol 90:10 of 200 ml, and soaked for 22 hours. After soaking, it was filtered and fresh solvent was added amounting to 150 ml, and further soaked for 12 hours (second wash). This was followed by filtration and drying the tobacco at room temperature for about 12 hrs. The amounts of TSNA and B(a)P and nicotine was measured according to method discussed hereinbefore.

Non-polar solvent Polar solvent TSNA reduction B(a)P reduction Nicotine reduction
Hexane-90% EtOH-10% 48.5% 64.4% 10.7%

Smoke analysis of a cigarette containing tobacco washed using invention solvent system
Cigarettes were made from the sample washed with Hex: MDC (80:20) and Hex: EtOH (90:10). Smoke analysis showed reduction in TSNA and B(a)P levels as tabulated.
Solvent system TSNA reduction B(a)P reduction % Ni reduction
Hex : MDC (80:20) 28.9% 17.64% 6%
Hex : EtOH (90:10) 38.76% 30.5% 11.3%