Note: Descriptions are shown in the official language in which they were submitted.
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Tobacco product, preparation and uses thereof
Field of the Invention
The invention relates generally to smokeless tobacco, tobacco derivative or
tobacco
substitute products incorporating cyclodextrins or cyclodextrin derivatives.
More
specifically, the invention relates to smokeless tobacco, tobacco derivatives,
or
tobacco substitute products comprising cyclodextrins or cyclodextrin
derivatives
which are incorporated in or bound to a cellulosic material.
>0 Background of the Invention
Cyclodextrins are cyclic oligosaccharides consisting of 1,4-a-glucoside
monomers.
They exist naturally as a-, P-, and y-cyclodextrins consisting of 6, 7, and 8
glucose
monomers in a ring, respectively. Their respective internal diameters are
0.57, 0.78,
and 0.95 nm; each has a torus depth of about 0.78 nm. The toriod shape offers
unequal opening sizes for the inner cavity. The smaller opening exposes a
primary
hydroxyl group to the surrounding solvent while the larger opening exposes a
secondary hydroxyl group. The presence of these hydroxyl groups results in the
exterior surface of the toroidal shaped molecule having a hydrophilic
character,
while the interior of the toroid is less hydrophilic and may be considered as
a
hydrophobic cavity.
Consequently, the interior of the toroid can host hydrophobic molecules, or
hydrophobic chemical moieties, while the exterior allows the complex to be
solubilised in an aqueous environment. In the liquid phase hydrophobic
molecules
or moieties can be used to form strong inclusion complexes with cyclodextrins
or
cyclodextrin derivatives. For example it has been shown that the aqueous
solubility
of vanillin is much higher in the presence of (3-cyclodextrin, present at a
molecular
ratio of 1:1, compared to when E3-cyclodextrin is absent from the solution
(Karathanos et al. Food Chemistry 101, pp 652-658). Furthermore, from
analytical
measurements it may be concluded that the molecule of vanillin inside the
cyclodextrin cavity is protected from oxidation.
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This ability to form inclusion complexes yet remain water soluble has helped
make
cyclodextrins a subject of considerable interest. Complexes can be
administered to
an area of interest, be it a body organ or a component of a product, whereupon
the
molecules inside the cyclodextrin are released by various factors such as
heat,
enzymatic activity, and pH change. Cyclodextrins are also relied upon to
scavenge
or filter.out certain molecules, often toxic molecules. Environmental clean-up
operations can utilize the ability of cyclodextrins to complex with heavy
metals such
as cadmium, removing these materials from a contaminated area.
Cyclodextrin derivatives are also widely used. For example, 2-hydroxypropyl-(3-
cyclodextrin is used to formulate drug complexes which facilitate the aqueous
solubility of certain pharmaceutically-active compounds. One application for
cyclodextrin derivates is to complex them with nicotine then incorporate that
complex into chewing gum, providing a smoking cessation aid as described in WO
91/09599. A chewing gum could alternatively offer flavour complexed in the
cyclodextrin. Another use of cyclodextrins and derivatives thereof complexed
with
flavour particles is their application to smoking products. Flavourant use is
common
in the tobacco field, a wide variety of flavourants are known and flavours are
continually being developed.
Flavourants are traditionally added to whole tobacco, reconstituted tobacco,
wrapping paper, cigarette filters, or packaging, often to affect the flavour
of the
product in use but also to improve the aroma of the unused product or that
experienced by persons in the vicinity of the product during use. One
manufacturing and packaging challenge presented by many known flavourants is
that they tend to be highly volatile and easily sublime. One solution has been
to
complex the flavourant with a cyclodextrin. For example, US 5,144,964 proposes
a
water-soluble molecular inclusion complex of a(3-cyclodextrin derivative and a
lipophilic organic flavourant compound which can be incorporated in the
wrapping
paper for a cigarette.
Despite the known uses of cyclodextrins, there remains an unmet need in the
art to
provide further, advanced uses for these powerful compounds.
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Summary of the Invention
It is therefore an object of the present invention to provide a new product
incorporating cyclodextrins or cyclodextrin derivatives, namely, smokeless
oral
tobacco products.
It is a further object of the present invention to provide a new product
incorporating cyclodextrins or cyclodextrin derivatives complexed with
flavourants
and provided in a smokeless oral tobacco product.
According to an embodiment of the invention, a smokeless oral tobacco product
is
provided, which comprises at least one of tobacco, a tobacco derivative, or a
tobacco substitute and a cellulosic material. The cellulosic material
comprises at
least one cyclodextrin or cyclodextrin derivative. The tobacco can be
encapsulated
in a cellulosic wrapper. The cellulosic material may be, for example, fibrous,
a
woven web, or a non-woven web.
The cyclodextrin or cyclodextrin derivative may be chemically attached to the
cellulosic material. A spacer group may be located between the cellulosic
material
and the cyclodextrin or cyclodextrin derivative. A complex agent may be
complexed
with the cyclodextrin or cyclodextrin derivative, such complex agent being any
agent
which can be located in the cavity of the cyclodextrin or cyclodextrin
derivative or
otherwise provided in or on such as by chemical bonding. Examples of complex
agent include flavourants, aroma modifiers, and antimicrobial agents.
Flavourants can comprise monoterpene flavourant, monoaromatic flavourant, or
polyaromatic flavourant, and examples include (+), (-)-limonene,
cinnamaldehyde,
cinnamonitrile, euginol, cis-isoeuginol, trans-isoeuginol, eugenyl acetate,
eugenol
methyl/ethyl esters, trans-anethole, cis-anethole, menthol, isomenthol,
neomenthol,
(+)-menthone, (-)-menthone, (+)-citronellal, S(+)-carvone, R(-)-carvone, trans-
methyl cinnamate, cis-methyl cinnamate, vanillin, capsaicin, phenylpropanoids,
aspartame, chocolate, coffee, pyrazines, salt, y-Glu-Tyr, y-Glu-Phe, orinithyl
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containing peptides, aromatic aldehydes, aromatic aldehydes derivatized as
acetals,
and lactones.
Aroma modifiers can comprise, for example, maltol, ethyl maltol, cis-jasmone,
methyl jasmonate, geraniol, nerol, geranyl esters, neryl esters, (+)-
citronellol, (-)-
citronellol, citral, (+)-limonene, (-)-limonene, and monoterpenes.
An example of an antimicrobial agent is hinokitol.
According to another embodiment, a cyclodextrin or cyclodextrin derivative is
used
in the preparation of a smokeless oral tobacco product, wherein the product
comprises tobacco. Alternatively, a cyclodextrin or cyclodextrin derivative
complexed with at least one flavourant can be used in the preparation of a
smokeless oral tobacco product, wherein the product comprises tobacco. The
product can further comprise a cellulosic material, in which case the
cyclodextrin or
cyclodextrin derivative is complexed to the cellulosic material such as by
chemical
attachment.
According to another embodiment, a method for complexing at least one
cyclodextrin or cyclodextrin derivative with a material in or formed by a
smokeless
oral tobacco product is provided which comprises the steps of providing a
smokeless oral tobacco product comprising at least one cyclodextrin or
cyclodextrin
derivative, contacting the product with moisture to moisten the product, and
allowing the material to complex with the cyclodextrin or cyclodextrin
derivative.
Where the cyclodextrin or cyclodextrin derivative comprises at least one
flavourant
the method can further comprise the step of allowing the flavourant to be
released
from the cyclodextrin or cyclodextrin derivative after the contacting step.
Examples
of materials complexed according to this embodiment include benzo(a)pyrene and
cadmium.
As used herein, the term "cellulosic materiaP" means a material consisting
wholly or
in part of natural or synthetic cellulose (C6H,0O5),,, a long chain polymeric
polysaccharide carbohydrate of (3-glucose, for example lignocellulose, flax
fibres,
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hardwood pulp, softwood pulp, heinp fibres, esparto fibres, kenaf fibres, jute
fibres,
and sisal fibres. It also means a material consisting wholly or in part of
cellulose
derivates, wherein the hydroxyl groups of cellulose have been partially or
fully
reacted with any appropriate entity. Examples of derivatives include cellulose
esters
such as cellulose acetate or cellulose triacetate. The term also encompasses
mixtures
of more than one type of cellulosic material as well as materials which have
undergone treatments to improve performance or appearance, such as bleaching.
"Cyclodextrin(s)" "cyclodextrin derivative" or "CD" means any molecule from
the
class of naturally-occurring or synthetic cyclodextrins. It further
encompasses
molecules or portions thereof derived from cyclodextrins or synthesized to
resemble cyclodextrins in whole or in part. Such cyclodextrins or cyclodextrin
derivatives are toroidal, chiral structures having a hydrophilic, water-
soluble outer
surface comprising hydroxyl groups and a less hydrophilic or hydrophobic
internal
cavity. A cyclodextrin as denoted herein could refer to an a, (3 or y
cyclodextrin, a
methyl substituted cyclodextrin, an ethyl substituted cyclodextrin , an alkyl
substituted cyclodextrin with straight chain or branch chain alkyl groups, a
hydroxyalkyl substituted cyclodextrin (e.g. 2-hydroxypropyl cyclodextrin , an
anionic
cyclodextrin, a cationic cyclodextrin, a quaternary ammonium cyclodextrin, an
amphoteric cyclodextrin, or mixture of the same).
"Flavour" or "Flavourant" refers to any compound or chemical entity which may
stimulate a taste sensation when consumed or when placed in the oral cavity of
a
user. Stimulation may be in the form of actual taste stimulation or perceived
taste
stimulation (simulation), such as that provided by scented materials. Examples
of
flavours include but are not limited to citrus ((+), (-)-limonene), fruit
(benzaldehyde) aromatic and spice (cinnamon could be cinnamaldehyde, green
leafy
odour could be cis-3-hexenal, clove could be euginol, other monoterpene
flavourants), mint (mentha flavours such as menthol, isomenthol, neomenthol),
pepper (S(+)-carvone), dill (R(-)-carvone), ginger (phenylpropanoids), vanilla
(vanillin and other aromatic aldehydes), sweet (sweeteners such as aspartame),
chocolate and coffee (pyrazines), liquorice, salt (y-Glu-Tyr, y-Glu-Phe,
orinithyl
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containing peptides). Flavourants which can achieve desired flavours can be
natural
or synthetic and include but are not limited to the above examples.
To achieve a flavour sensation or in addition to flavourant, aroma modifier
compounds may be complexed to cyclodextrins according to the invention.
Examples of aroma modifiers include but are not limited to maltol and ethyl
maltol,
cis-jasmone, methyl jasmonate, geraniol, nerol, geranyl and neryl esters, (+)-
citronellol, (-)-citronellol, citral, (+)-limonene, (-)-limonene, monoterpene
aroma
compounds.
By using flavourants it can be possible to provide not only a product with
improved
flavour, but also one which has additional benefits over existing products.
For
example, flavourants which mimic the taste of salt can be used to provide a
reduced
salt product, and artificial sweeteners can be used to provide a reduced
calorie
product.
In addition to or instead of flavourants other complex agents can be provided
with
the cyclodextrins of the present invention. For example, use of an anti-
microbial
agent such as hinokitol can increase shelf-life and/or provide a product which
can
be suitably stored under less stringent conditions. Complex agents such as
anti-
refrigerants or preservative agents could be used to improve product stability
and/or minimize undesired limitations on storage and transport conditions. The
complex agents used with the present invention thus give the blender greater
latitude in the use of flavourants, buffers, additives and moisture when
preparing
the tobacco or tobacco substitute.
Smokeless oral tobacco products are a class of tobacco products which are
intended
for oral administration. Such products are inserted in the mouth of the user
and
retained for a period of time, often for between 5 and 60 minutes. The product
may
be actively chewed or allowed to remain in the mouth without mastication. The
user
may expectorate saliva during use or not. The tobacco portion may comprise
loose
tobacco leaves or leaves which are chopped, shredded, or pulverized,
alternatively,
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the tobacco may be compressed into a plug. Chopped, shredded or pulverized
tobacco may be provided in a pouch, which pouch is inserted orally.
Thus "smokeless oral tobacco product" is used herein to denote any tobacco
product which is not intended for combustion but instead designed to be placed
in
the oral cavity of a user for a limited period of time, during which there is
contact
between the user's saliva and the product. The term encompasses conventional
products such as Swedish-style snus and American-style chewing tobacco, as
well as
less conventional and forthcoming products.
"Spacer group" or "SG" is used herein to describe any of a class of molecules
which
can be used to create a physical or chemical span between a cyclodextrin and
cellulosic material. Spacer entities are well known in the art; see, for
example, WO
06/53628. A spacer group located between a cyclodextrin and cellulosic
material
thus could be represented CD-SG-cellulosic material wherein SG could be an
alkylene with up to 10 carbon atoms which may be unsubstituted, mono or poly-
substituted by halogens, alternatively one or more non-adjacent CH2 groups
could
be replaced, in each case independently from one another, with 0, S, NH-NR,
CO,
COO, OCO, OCO-O, S, CO, CO-S, CH=CH, or C=C in such a manner that 0
and/or S atoms are not linked directly to each other. Examples of spacer
entities
include (CH2)p and (CH2CH2O)q where p is an integer from 2 to 6 and q is an
integer from 1 to 3. In some embodiments the spacer group could be an
imidazolidone moiety or (CH2)r or (CH2O)I where r is an integer between 1 and
5.
"Tobacco, tobacco derivative, or tobacco substitute" as used herein includes
pure
tobacco and reconstituted tobacco. It includes derivatives of tobacco such as
specific compounds, e.g., nicotine, whether extracted from actual tobacco or
otherwise produced, as well as structural derivatives such as the fibrous
portion of a
tobacco leaf. Tobacco substitutes can comprise individual chemical entities as
well
as complex chemical solutions which, when appropriately prepared, physically
resemble actual tobacco.
Detailed Description
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Unless specifically noted, all materials and equipment are standard,
commercially
available products.
As noted previously herein, the invention is directed to smokeless oral
tobacco
products which incorporate at least one cyclodextrin or cyclodextrin
derivative.
Smokeless oral tobacco products, particularly those comprising natural
tobacco,
contain compounds or release compounds in use that may present a risk of
injury to
the user after extensive and extended use of the product. Technologies for
identifying and extracting such compounds from the natural tobacco have
progressed; however, it can be preferable, perhaps particularly so for newly-
identified compounds, to provide a way to remove the compounds during use of
the
product so as to minimize the complex steps related to preparing the tobacco
for
inclusion in a product.
The cyclodextrins of the present invention thus provide a novel means for
complexing with potentially harmful compounds, before the compounds can be
carried away with the user's saliva and move freely about the oral cavity. It
is
therefore advantageous to provide the cyclodextrins in a manner so that they
are
bound to one or more substrates that will be expectorated after use of the
product,
thereby removing the cyclodextrin-compound complex from the mouth of the user.
According to an embodiment of the invention cyclodextrins are attached, for
example by chemical bonds, to cellulosic material. That material is then
incorporated into the smokeless oral tobacco product, e.g., a filler material
or as a
wrapper for a pouch-like product. Attachment of cyclodextrins is a routine
matter,
technology related to their applicability in chromatography provide numerous
examples of the process.
Where chemical bonds are used, so that the cyclodextrin is not subject to
interference from the cellulosic material to which it is attached, a spacer
group can
be provided. This ensures the cavity of the cyclodextrin is free to interact
with the
surrounding solution either to release a complexed compound or to remove a
target
molecule from free solution.
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Cyclodextrins are known in the art, as are methods and materials for selecting
particular cyclodextrins based on the molecule to be contained within the
cavity.
For example, in use it can be expected that certain smokeless oral tobacco
products
release polyaromatic hydrocarbons such as benzo(a)pyrene, a molecule which is
suspected of potentially causing harm for long-term heavy users.
Benzo(a)pyrene
has been calculated to be 0.88 nm across its wide axis. It is thought that
benzo(a)pyrene cannot form an inclusion complex with a-cyclodextrin, but that
it
forms a stable 1:1 host-guest complex with (3- cyclodextrin and a 1:2 host-
guest
complex with y- cyclodextrin (Fielden and Packham, J.Chromatog. 516(1990) 355-
364).
To remove benzo(a)pyrene from a product during use it can be preferred to take
advantage of its high propensity to complex within the y-cyclodextrin cavity.
Once
complexed, benzo(a)pyrene is unlikely to return to free solution, i.e., the
user's
saliva, as it is an extremely hydrophobic molecule. By providing the
cyclodextrins in
or attached to cellulosic material one ensures that both the cyclodextrins and
the
molecules they have complexed with are expectorated after use of the product.
Instead of or in addition to providing the cyclodextrin with an empty cavity,
the
invention further provides complexing the cyclodextrins with one or more
flavourants which will be released by the cyclodextrin during use, after which
the
cyclodextrins present an empty cavity for complexing with a compound of
interest
present or generated in the tobacco portion of the product. This provides the
added
benefit of flavour release while still reaping the benefit of reducing the
release of
certain compounds into the oral cavity of the user.
This aspect of the invention takes advantage of the fact that many flavourants
or
other additives, which are also within the scope of the invention, are small
mono-
aromatic compounds which form transient complexes with cyclodextrins and are
readily released upon contact with moisture such as saliva. y-cyclodextrin
presents a
large cavity from which a small compound would more rapidly disassociate,
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providing an open cavity into which another molecule such as benzo(a)pyrene
could
complex.
According to an embodiment of the invention, (.3-cyclodextrin could be a
preferred
cyclodextrin for the complexation of flavourants and aroma molecules because
it
presents a cavity size which is ideal for a large number of flavourant and
aroma
molecules. The bi-aromatic ring compounds in particular would be held stably
in the
cyclodextrin complex until conditions are such that they solubilize. This
would
allow for a high moisture content in the product which would not interfere
with the
inclusion complex, only upon use where the user's saliva causes a much higher
degree of wetting would sufficient solution be available to release the
flavourant.
The cyclodextrins would then be free to complex with other compounds.
Particularly drawn to the cyclodextrins would be hydrophobic molecules such as
those which might potentially cause harm to the user if allowed to pass into
the
saliva and be carried around the oral cavity freely.
Another feature of the invention is that molecules complexed by cyclodextrins
are
presumably more resistive to decomposition, degradation and alteration. It is
known, for example, that the propensity for the flavourant vanillin to undergo
oxidation is lowered while it is complexed to a cyclodextrin.
According to an embodiment of the invention, oc-cyclodextrins could preferably
be
used where the flavourant of interest is a small molecule, such as a mono-
aromatic
flavour compound containing one phenyl-sized moiety, for example,
phenylacetaldehyde dimethyl acetal. Release from the inclusion complex would
be
slow and sustained over a period corresponding to the average time of product
use.
Approximate ranges which can be employed when practicing the present invention
can be readily determined by a skilled person. For example, benzo(a)pyrene is
known to be present in certain smokeless tobacco products in amounts ranging
from approximately 0.1-10 g/kg (e.g., a product with 1 g/kg could release 4
mmole/kg into the user's mouth). So where a 1:1 complex between benzo(a)pyrene
and a cyclodextrin is assumed, and no other molecule is believed to complex
with
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the cyclodextrin, 0.4-40 mmole/kg cyclodextrin could be provided in an effort
to
complex all benzo(a)pyrene from the product. To compensate for less than
efficient
complexing and the possibility of other molecules forming complexes with
certain
of the cyclodextrins, one may choose to use, for example, ten or twenty times
the
base amount of cyclodextrin calculated, in this example, 4.0-800 mmole/kg.
Because the ratio of cellulosic material to tobacco is eminently variable, the
amount
of cyclodextrin relative to the tobacco and tobacco constituents can easily be
manipulated. The cellulosic material may be provided, e.g., as fragments,
fibres,
leaves or sheets blended in the tobacco or as a moisture-permeable wrapper
encapsulating the tobacco.
Cyclodextrins may be provided interstitially within or bonded to the
cellulosic
material in a random pattern or in a specific pattern such as rows. Where the
cellulosic material forms a wrapper for a tobacco pouch, the cyclodextrins may
be
provided on the side of the pouch facing the user or the side facing the
tobacco
during use, or a mixture of both. Methods of providing the cyclodextrins in
the
cellulosic material as well as densities, locations and the hke can be
optimized
depending on factors such as the molecule the cyclodextrin is designed to
capture
and its relative amounts in the product, how and where that molecule is
released,
the timing of the release, and the like.
Where flavourants or other additives are provided as an inclusion complex with
cyclodextrins, they may be provided in a range such that perhaps about 5% of
the
cyclodextrins are complexed, or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or 100% are complexed. A smokeless oral tobacco product may incorporate
all
of its flavourant or additive in the cyclodextrin complex, or only a portion
of its
overall flavourant or additive may be present in complex with cyclodextrin and
the
remainder provided according to conventional means. For example, cyclodextrin
immobilized on a cellulosic wrapper may include 0.85 mol/mol (+)-limonene or
0.95 mol/mol hinokitiol.
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A variety of conditions can be used to attach (3-, y-hydroxypropyl, and/or y-
cyclodextrins to cellulose powder via a spacer or linker group.
Example 1 Cyclodextrin linked to Cellulose Powder with Epichlorohydrin
Two samples were prepared using aqueous potassium carbonate to attach
epichlorohydrin and cyclodextrin to cellulose.
A mixture of cellulose (50g), epichlorohydrin (5g), potassium carbonate (10g)
and ~i-
cyclodextrin (10g) in water (200m1) were stirred at 50 C for 5 hours. The
mixture
was cooled, filtered, washed exhaustively with water and dried in vacuo to
give the
first sample, PD906 (50.1g). The second sample was prepared by stirring a
mixture
of cellulose (50g), epichlorohydrin (5g), potassium carbonate (10g) and y-
cyclodextrin (10g) in water (200m1) at 75 C for 14 hours. The mixture was
cooled,
filtered, washed exhaustively with water and dried in vacuo to give sample
PD907
(50g).
Two samples were prepared using potassium carbonate and butanone to attach
epichlorohydrin and cyclodextrin to cellulose.
A mixture of cellulose (50g), epichlorohydrin (5g), potassium carbonate (10g)
and (3-
cyclodextrin (10g) in 2-butanone (200m1) were stirred at reflux (80 C) for 6
hours.
The mixture was cooled, filtered, washed exhaustively with water and dried in
vacuo
to give sample PD911 (46.2g). For the second sample in this class a mixture of
cellulose (50g), epichlorohydrin (5g), potassium carbonate (10g) and y-
cyclodextrin
(10g) in 2-butanone (200m1) were stirred at reflux (80 C) for 6 hours. The
mixture
was cooled, filtered, washed exhaustively with water and dried in vacuo to
give
sample PD912 (47.1g).
Two samples were prepared using aqueous sodium hydroxide to attach
epichlorohydrin and cyclodextrin to cellulose.
A mixture of cellulose (50g), epichlorohydrin (7g), sodium hydroxide (6g) and
(3-
cyclodextrin (10g) in water (200m1) were stirred at 50 C for 2 hours then at
room
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temperature overnight. The mixture was cooled, filtered, washcd exhaustively
with
water (very slow filtration) and dried in vacuo to yield sample PD913 (49.4g).
The
second sample of this type was created when a mixture of cellulose (50g),
epichlorohydrin (7g), sodium hydroxide (6g) and y-cyclodextrin (10g) in water
(200m1) were stirred at 50 C for 2 hours then at room temperature overnight.
The
mixture was cooled, filtered, washed exhaustively with water (very slow
filtration)
and dried in vacuo to give sample PD914 (50.8g).
On drying, both of samples PD913 and PD914 were very hard to crush in a mortar
and pestle. The samples were thus only semi-crushed.
Where epichlorohydrin is used as the linker group, methods using 2-
butanone/potassium carbonate tend to produce more tanned cellulosic material
whereas methods relying upon aqueous sodium hydroxide reactions generally
afford
the whitest material. In some downstream applications, a whiter or brighter
cellulose material may be favoured.
Example 2 Cyclodextrin linked to Cellulose Powder with Imidasolidone
In these thermal reactions, the general procedure followed was: addition of an
aqueous (deionised water) or aqueous methanolic solution of
CD/imidazolidone/catalytic magnesium chloride/citric acid to cellulose powder
(microcrystalline size) followed by evaporation of the slurry (circa 75 C in
vacuo) and
heat treatment of residue (circa 160 C either in air or under a nitrogen
atmosphere).
The cooled material was exhaustively washed with water and dried at 65-75 C in
vacuo.
The experimental conditions were generally similar to those disclosed in US
Patent
No. 7,109,324. As noted therein, magnesium chloride was added to optimize CD-
cellulose bonding and citric acid was added to keep pH at 5.
A solution of (3-CD (5g), imidazolidone (5g), magnesium chloride hexahydrate
(1g),
citric acid (100mg) in deionised water (50m1) was mixed with cellulose to form
a
paste. This was placed in a glass dish in a 155-180 C oven (temperature
gradient
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from front to back). After 45 minutes, weight check indicated all the water
had
evaporated and the mixture was left for a further 5 minutes. Cooled, washed
exhaustively with water and dried in vacuo at 65-75 C to yield sample PD908
(48g).
A solution of y-CD (5g), imidazolidone (5g), magnesium chloride hexahydrate
(1g),
citric acid (100mg) in deionised water (50ml) was mixed with cellulose to form
a
paste. The mixture was pre-dried in vacuo at 75 C before heating in an oil
bath up to
180 C (oil bath temperature) in a magnetically stirred glass flask until the
temperature of the air inside the flask reached 160 C. Cooled, washed
exhaustively
with water and dried in vacuo at 65-75 C to give sample PD909 (46.6g).
A solution of y-CD (5g), imidazolidone (5g), magnesium chloride hexahydrate
(1g),
citric acid (100mg) in deionised water (50m1) was mixed with cellulose to form
a
paste. The mixture was pre-dried in vacuo at 75 C before heating in an oil
bath to
160-164 C for 5 minutes (temperature of solid -150 C) in a magnetically
stirred
glass flask. Cooled, washed exhaustively with water and dried in vacuo at 65-
75 C to
give sample PD910 (49.2g).
A solution of y-CD (5g), imidazolidone (5g), magnesium chloride hexahydrate
(1g),
citric acid (1 00mg) in methanol (50m1)/water (30m1) was mixed with cellulose
to
form a paste. The mixture was pre-dried in vacuo at 75 C before heating in an
oil
bath up to 180 C (oil bath temperature) in a magnetically stirred glass flask
until the
temperature of the air inside the flask reached 160 C. Cooled, washed
exhaustively
with water and dried in vacuo at 65-75 C. After drying, the material was
placed in a
magnetically stirred glass flask then put in a 175 C oil bath under a stream
of
nitrogen for 15 minutes. The reaction was placed under nitrogen to see if this
would
affect material tanning. The sample produced was PD911 (46.2g).
A solution of y-HP-CD (5g), imidazolidone (5g), magnesium chloride hexahydrate
(1g), citric acid (100mg) in deionised water (50ml) was mixed with cellulose
to form
a paste. The mixture was pre-dried in vacuo at 75 C before heating under
nitrogen in
an oil bath up to 175 C (oil bath temperature) in a magnetically stirred glass
flask
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for 20 minutes. Cooled, washed exhaustively with water and dried in vacuo at
65-
75 C to give sample PD917A (48.5g).
A solution of y-HP-CD (5g), imidazolidone (5g), magnesium chloride hexahydrate
(1g), citric acid (100mg) in deionised water (50m1) was mixed with cellulose
to form
a paste. The mixture was pre-dried in vacuo at 75 C before heating under
nitrogen in
an oil bath up to 175 C (oil bath temperature) in a magnetically stirred glass
flask
for 20 minutes. Cooled, washed exhaustively with water and dried in vacuo at
65-
75 C. The dried paste was slurried in methanol (150m1), dried at 60 C in
vacuo, then
heated under nitrogen in a 175 C oil bath for 20 minutes. The sample produced
was
PD917B (49g).
The final two thermal reactions were undertaken using similar proportions of
additives to cellulose as described in US Patent No. 7,109,324. The mixture
was
heated to lower temperature to observe the effect on material tanning.
A solution of y-CD (1.75g), imidazolidone (4g), magnesium chloride hexahydrate
(ig) and citric acid (40mg) was made up in deionised water (20m1) and methanol
(100m1). The paste was dried in vacuo at 65 C then heated in a 160-165 C oil
bath
for 15 minutes. Cooled, washed exhaustively with water and dried in vacuo at
65-
75 C to yield sample PD918 (47.8g).
A solution of y-HP-CD (1.75g), imidazolidone (4g), magnesium chloride
hexahydrate (1g) and citric acid (40mg) was made up in deionised water (20m1)
and
methanol (100m1). The paste was dried in vacuo at 65 C then heated in a 160-
165 C
oil bath for 15 minutes. Cooled, washed exhaustively with water and dried in
vacuo at
65-75 C to yield sample PD919 (45.8g).
Tanning was observed for most of the thermal reactions - the least coloured
samples from those described above were PD916 and PD919.
Example 3 Removal of Benzo(a)Pyrene by Derivatised Cellulose
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A feature of the invention was displayed using derivatised cellulose to remove
B(a)P
from an aqueous solution. This also had the effect of confirming the variety
of
cyclodextrin entities described above were successfully bound to cellulose.
Because of the complex nature of tobacco extract, replete with numerous
substances, it can be difficult to quantify molecules such as B(a)P which are
present
at low concentrations in the matrix. This problem is particularly acute with
fluorescence measurements because sometimes the matrix can affect the
measurement; this phenomenon is known as fluorescence quenching. For this
reason fluorescence determinations often require extensive sample clean-up to
minimize the deleterious affects of the matrix.
To avoid problematic fluorescence quenching and clean-up procedures, the
following experiments were conducted without a tobacco matrix. This allowed
the
cyclodextrin - cellulose compounds to be directly assessed for their ability
to
complex B(a)P molecules out of an aqueous environment, thus reducing the
concentration of B(a)P in the sample.
In brief, mini-columns containing a bed of either the cyclodextrin derivatised
cellulose or pure cellulose were prepared. A small quantity of a solution of
B(a)P in
acetonitrile was placed at the head of this column followed by larger amounts
of
water. The water was allowed to flow through the column without assistance
under
gravity and the resultant eluent was collected and analysed for B(a)P.
Preparation of Mini-Columns
Glass Pastuer pipettes 150 mm long (Fisher Scientific, Fisherbrand: FB50251)
were
plugged with a small amount of untreated glass wool (Supelco: 2-0384, lot:
1561-
18). The glass columns were weighed with the glass wool plug, following which
a
quantity of either the cyclodextrin derivatised cellulose or untreated
cellulose
(control, from the same batch as used for the preparation of the CD-
derivatised
cellulose samples) was added into the column. The material was dry packed
without
any compaction; approximately 30 mm of solid material was added into each
column. The column was kept vertical and tapped gently to ensure the bed
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contained no air pockets, and to see that the head of the bed was
approximately
horizontal. The column was weighed after packing to calculate the weight of
the
bed. Details are described below in Table 1.
Table 1
Sample Replicate Weight of Weight of Calculated
Material Number column (g) column + bed weight of bed
material (g) material )
Cellulose 1 2.8451 3.1585 0.3134
2 2.9729 3.266 0.2931
3 2.7864 3.1046 0.3182
4 2.8579 3.1676 0.3097
PD917A 1 2.9121 3.1876 0.2755
2 2.9663 3.2152 0.2489
3 2.8856 3.1082 0.2226
4 2.9948 3.2284 0.2336
PD908 1 3.0032 3.2229 0.2197
2 2.8852 3.1294 0.2442
3 2.9489 3.2839 0.3350
4 2.9338 3.1794 0.2456
PD909 1 2.8405 3.1054 0.2649
2 2.9568 3.236 0.2792
3 2.7631 3.0132 0.2501
4 2.8071 3.1043 0.2972
PD912 1 2.9202 3.1567 0.2365
2 2.9355 3.1815 0.2460
3 2.8662 3.0607 0.1945
4 2.8119 3.0229 0.2110
Application of B(a)P Solution
100 L of a 500 ng/mL (50ng) solution of B(a)P in acetonitrile (Stock Standard
Solution) was carefully pipetted onto the inner surface of the glass column.
This
solution was incorporated into the top 3-5 mm of the absorbent bed.
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Elution of B(a)P from Column
1000 L of water was slow discharged by pipette down the inside of the column.
This addition progressively hydrated the dry material of the bed as well as
mobilized
the B(a)P. Eventually water droplets appeared at the base of the column which
were
collected in a 20 mL glass vials. Approximately 8-11 minutes were required for
the
water to drain to the level of the top of the bed. After this a further 1000
L of
water was slow discharged by pipette down the inside of the column. All of the
water eluent was collected and submitted for B(a)P analysis. Approximately 25-
30
minutes was allowed for liquid to drain out of the column.
Preparation of 100% recovery sample
In order to check the efficacy of the system a 100% recovery sample was also
produced. This sample was not subjected to any column environment; the B(a)P
added would be expected to be conserved. In this sample 100 L of a 500 ng/mL
(50ng) solution of B(a)P in acetonitrile was mixed with 2000 L water in 20mL
vial.
Analysis of Eluent
Reverse phase HPLC was used with Fluorescence detection. The column used was
125 x 4.60 mm 5 micron (Envirosep PP, Phenomenex), thermostated at 30 C. The
mobile phase was water/acetonitrile proportioned under the gradient regime
detailed in Table 2.
Table 2
Time (minutes) Flow (mL/min) % water % acetonitrile
0.00 2.00 60.0 40.0
2.00 2.00 60.0 40.0
25.00 2.00 0.0 100.0
27.00 2.00 0.0 100.0
30.00 2.00 60.0 40.0
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The wavelength of excitation was 378nm and the wavelength of detection was
405nm. B(a)P elution was after about 20.5 min. Injection volume was 50 L.
The results are summarised in Table 3.
Table 3
Sample Material Replicate B(a)P units Average Standard
B(a)P units Deviation
Cellulose 1 6974 6112.25 1239
2 4814
3 7350
4 5311
PD917A 1 2086 2914 790
2 2702
3 3982
4 2886
PD908 1 3641 2780.25 1091
2 3228
3 3068
4 1184
PD909 1 3671 4181.75 1209
2 3972
3 5924
4 3160
PD912 1 7985 7381.5 1614
2 8311
3 4970
4 8260
Recovery 1 7155 6877.5 1019
sample 2 8108
3 6562
4 5685
As detailed in previous Examples,
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PD917A was a gamma hydroxypropyl cyclodextrin cellulose derivative;
PD908 was a beta cyclodextrin cellulose derivative;
PD909 was a gamma cyclodextrin cellulose derivative; and
PD912 was a beta cyclodextrin cellulose derivative.
The results show that there is a small loss of B(a)P using the pure cellulose
column
compared to the recovery sample. However, in the CD-derivatised cellulose
columns there is a far more dramatic reductions in the amounts of B(a)P
eluting
from the column for samples PD908, PD909 and, in particular, PD917A. In the
case of PD912 no reduction in B(a)P was observed.
The high degree of B(a)P reduction shown by sample PD917A supports the
particularly inventive nature of using gamma cyclodextrin to complex B(a)P. As
shown above, cyclodextrin-cellulose derivatives remove B(a)P in an aqueous
environment.
Examples 1-3 describe methods to provide inventive materials, and the
following
Examples 4-8 demonstrate a few of the ways in which the inventive materials
can be
incorporated in products.
Example 4 Smokeless Oral Tobacco Product with Cellulosic Material
Smokeless tobacco for oral administration is provided. For example, a
commercial
blend of tobaccos can be cut to the suitable size and shape for a loose leaf
chewing
tobacco product. The cut tobacco can then be cased with an aqueous mixture of
flavouring materials and sweetening agents to give a cut and cased tobacco
containing approximately 50 percent moisture. The treated tobacco can be
arranged
on a conveyor belt or loaded onto sheets and moved through or placed in a
dryer.
The dryer may heat the tobacco to about 70 C, briefly, to reduce its moisture
content to about 25 percent. Subsequent processing of the tobacco can be
conventional such as application of a top flavouring solution and temporary
storage
to allow equilibration of the top flavouring additives throughout the tobacco
mass.
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Top flavouring can serve the dual purpose of both adding a top note to the
tobacco
itself and presenting a number of unassociated flavour molecules in the
mixture.
These molecules could be expected to migrate toward cyclodextrins provided in
proximity to the flavoured tobacco and complex with them. Moist conditions
favour
the formation of an equilibration throughout the tobacco mass and nearby
cellulosic
material comprising cyclodextrins.
In addition, or instead of application of a top flavouring solution,
cyclodextrins
previously complexed with flavourant can be provided. Where flavourant-
complexed cyclodextrins are used, it can be found that flavourant migration
occurs
in two directions: some of the complexed flavourant will disassociate and
migrate
toward the tobacco whereas certain of the tobacco-associated flavourant will
complex with the recently-emptied cyclodextrins.
As noted above, either or both of pre-complexed or `empty' cyclodextrins could
be
employed. For example, fibrous cellulosic material is dipped in a water bath
containing 15 mM (3-cyclodextrin and 15 mM imidazolidone. After dipping it is
subjected to oven hearting at 150 C for 1-5 minutes. Where a-cyclodextrin is
used,
one may increase the concentration to about 130 mM and where y-cyclodextrin is
used, to about 170 mM.
The procedure results in cyclodextrin linked with imidazolidone (spacer group)
through the formation of ether (hemiacetal) bonds between the imidazolidone
and
the hydroxyl group (s) on the cyclodextrin. The imidazolidone is attached to
the
cellulose by the formation of ether bonds between it and hydroxyl groups
present
on the cellulose. As such, cellulosic material is provided having a covalently
bonded
spacer group which in turn is linked to a cyclodextrin.
The cyclodextrin-cellulosic material is cut to approximately the same size as
the
tobacco pieces and blended with the tobacco. Moisture, pH, and other
parameters
are adjusted as necessary to provide a stable, useable product. For example,
buffering agents may be used to produce a pH of above about 6.5, or above
about
7.5. The blend is packaged into moisture proof containers.
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When the product is used, moisture liberates molecules. Benzo(a)pyrene and
other
large polyaromatic hydrocarbons in the tobacco will be drawn to the relatively
hydrophobic, suitably-sized inner cavity of the cyclodextrin to form an
inclusion
complex. The hydrocarbons thus bound will be removed from the moist product
and, while in the mouth of a user, the hydrocarbons will not be bioavailable.
Example 5 Smokeless Oral Tobacco Product Comprising Cellulosic Wrapper
This embodiment of the invention comprises a pouch-type product which can be
similar to portioned Swedish-style snus products. That is, as a pouch
containing
tobacco products intended to fit into the gingival fold of the mouth. A non-
woven
fabric can be used as a package material for such pouches.
In order to prepare the cyclodextrin-fabric complex, cyclodextrin derivatised
with a
spacer group having a reactive entity at the non-cyclodextrin end of the
spacer
group is obtained (for example, monochlorotriazinyl-(3-cyclodextrin, Wacker
GmbH, Germany). The reactive entity is allowed to interact with the fabric
thus
bonding the spacer group to the fabric.
A unit of pulverized tobacco suitable for an individual dose, for example 0.05-
2.0 g
(dry weight) is placed on the cellulose-bonded fabric and a pouch is formed.
To
increase the amount or to alter the location of the cyclodextrins in the
product,
pieces of cellulosic material incorporating cyclodextrins may be blended with
the
tobacco prior to its addition to the pouch. Conventional snus or tea bag
filling
equipment may be used. The pouch may be sealed shut, for example by heat
sealing.
Where different cellulosic materials are substituted, e.g., long fibre
cellulose
materials, a heat weldable binder may be added to the material to facilitate
heat
sealing. The product is stored in a moisture-proof container, possibly under
refrigeration, prior to use.
By way of example, a mini snus product typically contains 0.5 g tobacco. To
complex the benzo(a)pyrene present in that dose of tobacco, 1.9-19 mg a-
cyclodextrin (MW 972) could be attached to the fabric. Alternatively, 2.3-23
mg (3-
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cyclodextrin (MW 1135) could be attached to the fabric. Alternatively, 2.6-26
mg of
y-cyclodextrin (MW 1297) could be attached to the fabric. A mix of two or more
cyclodextrins could be employed.
Example 6 Smokeless Oral Tobacco Product Comprising Cellulosic Wrapper
In order to prepare a flavourant-cyclodextrin-cellulose complex, cyclodextrin
derivatised with a spacer group having a reactive entity at the non-
cyclodextrin end
of the spacer group is obtained (for example, CAVAMAX W7 CITRAL, Wacker
GmbH, Germany). This reactive entity is designed to react with cellulose
wherein
the spacer group is bonded to the cellulosic material and provides a lemon
flavourant to the resultant product. A sheet of cellulosic material in web
form is
used.
Through extraction processes or chemical synthesis, select components of
tobacco
are provided such as nicotine. These tobacco extracts are applied to
cellulosic or
synthetic tobacco-like material which is then incorporated into a pouch-like
object
formed by folding the sheet of cellulosic web around the cellulosic or tobacco-
like
material.
A pouch so provided does not contain whole, natural tobacco but rather natural
or
synthetic tobacco extracts. During use, moisture permeating the pouch will
encourage the lemon flavourant to disassociate from the cyclodextrin and pass
out
of the pouch and into the surrounding area. Simultaneously, molecules present
in
the extracts or formed by the extract when moistened are transported with the
moisture through the sheet of cellulosic material are attracted to the
relatively
hydrophobic inner cavities of the cyclodextrins, thus prevented from exiting
the
pouch or from remaining in the pouch unbound.
Example 7 Smokeless Oral Tobacco Product Comprising Cellulosic Wrapper
A unit of cellulosic material in web form is provided. 4-40 mg of P-
cyclodextrin is
bound to the cellulosic web. 1.0 g of standard smokeless tobacco is placed on
the
cellulosic web and a sealed pouch is formed.
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As standard smokeless tobacco is expected to comprise about 0.1-0.3 mg/kg
cadmium, the amount of P-cyclodextrins is selected to correspond to this
amount.
Thus during use an amount of cadmium is released from the tobacco product, a
portion of which will be subsequently bound by the (3-cyclodextrins. Cadmium
exposure may be disfavoured; complexation by P-cyclodextrins will reduce the
user's exposure to the substance. The most common oxidation state of cadmium
is
+2, therefore cadmium is at times represented as cadmium(II). Any state and
any
form of cadmium could be encompassed by the present invention.
Example 8 Smokeless Oral Tobacco Product Comprising Cellulosic Wrapper
A sheet of cellulosic material in web form is provided, a spacer group and
subsequently a cyclodextrin derivative are bound to the cellulosic web. The
cellulosic web is treated with a mixture of nicotine and flavourants to
provide
nicotine- and flavourant-cyclodextrin inclusion complexes.
A suitable amount of tobacco in the desired physical form is added to the
sheet,
which is then folded to form a pouch and sealed. In use, moisture permeating
the
pouch will encourage the nicotine and flavourant to disassociate from the
cyclodextrin and pass out of the pouch. Compounds present in or formed by the
tobacco are similarly transported by moisture towards the outside of the
pouch,
however, due to the suitably-sized, relatively hydrophobic cavity offered by
the
bound cyclodextrin they are drawn into an inclusion complex and do not pass
out
of the pouch.
Because small, mono-aromatic compounds typically will not be able to form long-
lasting complexes with cyclodextrins they will be rapidly released from the
inclusion
complexes when moistened, providing nicotine and flavour to the user while
making
the cyclodextrins available to bind with larger molecules of interest. A
mixture of
different cyclodextrins may be used in this configuration, to provide an
extended
release profile for the product.
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As compared with other exemplary products, this configuration may require
stricter
formulation and storage controls to ensure low moisture and appropriate pH
which
would encourage stability of the initial inclusion complexes until use.
The foregoing description and examples have been set forth merely to
illustrate the
invention and are not intended to be limiting. Since modifications of the
described
embodiments incorporating the spirit and substance of the invention may occur
to
persons skilled in the art, the invention should be construed broadly to
include all
variations within the scope of the appended claims and equivalents thereof.
