U.S. patent application number 14/021409 was filed with the patent office on 2015-03-12 for smokeless tobacco composition incorporating a botanical material.
This patent application is currently assigned to R.J. Reynolds Tobacco Company. The applicant listed for this patent is R.J. Reynolds Tobacco Company. Invention is credited to Dwayne William Beeson, Daniel Verdin Cantrell, Jerry Wayne Marshall, Serban C. Moldoveanu, Wayne Allen Scott.
Application Number | 20150068544 14/021409 |
Document ID | / |
Family ID | 52624305 |
Filed Date | 2015-03-12 |
United States Patent
Application |
20150068544 |
Kind Code |
A1 |
Moldoveanu; Serban C. ; et
al. |
March 12, 2015 |
SMOKELESS TOBACCO COMPOSITION INCORPORATING A BOTANICAL
MATERIAL
Abstract
A smokeless tobacco product configured for insertion into the
mouth of a user of the product is provided, the smokeless tobacco
product including a tobacco material mixed with at least one
botanical material, wherein the botanical material comprises at
least about 0.1% of the total dry weight of the smokeless tobacco
product. One exemplary product is in the form of a snus product
contained within a water-permeable pouch, which includes at least
about 50% of a pasteurized and fermented particulate tobacco
material, based on the total dry weight of the smokeless tobacco
product, and a botanical material in particulate form. The
botanical material and the smokeless tobacco product can be
characterized based on antioxidant content using the ORAC index or
the FRAP index. An exemplary product has an ORAC index value of at
least about 50 (.mu.mol TE)/g or a FRAP index value of at least
about 80 (.mu.mol/Fe.sup.2+)/g.
Inventors: |
Moldoveanu; Serban C.;
(Winston-Salem, NC) ; Marshall; Jerry Wayne;
(Stokesdale, NC) ; Scott; Wayne Allen;
(Lewisville, NC) ; Beeson; Dwayne William;
(Kernersville, NC) ; Cantrell; Daniel Verdin;
(Lewisville, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
R.J. Reynolds Tobacco Company |
Winston-Salem |
NC |
US |
|
|
Assignee: |
R.J. Reynolds Tobacco
Company
Winston-Salem
NC
|
Family ID: |
52624305 |
Appl. No.: |
14/021409 |
Filed: |
September 9, 2013 |
Current U.S.
Class: |
131/352 |
Current CPC
Class: |
A24B 13/00 20130101;
A24B 15/10 20130101 |
Class at
Publication: |
131/352 |
International
Class: |
A24B 15/10 20060101
A24B015/10 |
Claims
1. A smokeless tobacco product configured for insertion into the
mouth of a user of the product, the smokeless tobacco product
comprising a tobacco material and at least one botanical material,
wherein the botanical material comprises at least about 0.1% of the
total dry weight of the smokeless tobacco product.
2. The smokeless tobacco product of claim 1, wherein the tobacco
formulation is contained within a water-permeable pouch.
3. The smokeless tobacco product of claim 1, wherein the botanical
material has an ORAC index value of about 250 (.mu.mol TE)/g or
greater.
4. The smokeless tobacco product of claim 1, wherein the botanical
material has an ORAC index value of about 500 (.mu.mol TE)/g or
greater.
5. The smokeless tobacco product of claim 1, wherein the botanical
material has an ORAC index value of about 1000 (.mu.mol TE)/g or
greater.
6. The smokeless tobacco product of claim 1, wherein the botanical
material has an FRAP index value of about 250 (.mu.mol/Fe.sup.2+)/g
or greater.
7. The smokeless tobacco product of claim 1, wherein the botanical
material has an FRAP index value of about 500 (.mu.mol/Fe.sup.2+)/g
or greater.
8. The smokeless tobacco product of claim 1, wherein the botanical
material has an FRAP index value of about 1000
(.mu.mol/Fe.sup.2+)/g or greater.
9. The smokeless tobacco product of claim 1, wherein the smokeless
tobacco product has an ORAC index value of at least about 50
(.mu.mol TE)/g or an FRAP index value of at least about 80
(.mu.mol/Fe.sup.2+)/g.
10. The smokeless tobacco product of claim 1, wherein the smokeless
tobacco product has an ORAC index value of at least about 75
(.mu.mol TE)/g or an FRAP index value of at least about 100
(.mu.mol/Fe.sup.2+)/g.
11. The smokeless tobacco product of claim 1, wherein the botanical
material is selected from the group consisting of rosemary,
oregano, sage, hibiscus, clove, rose hip, yerba mate, cocoa,
turmeric, guayusa, honeybush, green tea, black tea, rooibos, yerba
santa, bacopa monniera, gingko biloba, withania somnifera, and
combinations thereof.
12. The smokeless tobacco product of claim 1, wherein the botanical
material is a tea or a tisane material.
13. The smokeless tobacco product of claim 1, wherein both the
tobacco material and the botanical material are in particulate
form.
14. The smokeless tobacco product of claim 1, wherein at least one
of the tobacco material and the botanical material are in the form
of an oil or aqueous extract.
15. The smokeless tobacco product of claim 1, wherein the botanical
material comprises at least about 1% of the total dry weight of the
smokeless tobacco product.
16. The smokeless tobacco product of claim 1, wherein the botanical
material comprises at least about 5% of the total dry weight of the
smokeless tobacco product.
17. The smokeless tobacco product of claim 1, further comprising
one or more additional components selected from the group
consisting of flavorants, fillers, binders, pH adjusters, buffering
agents, salts, sweeteners, colorants, disintegration aids,
humectants, and preservatives.
18. The smokeless tobacco product of claim 1, wherein the product
is in the form of a snus product contained within a water-permeable
pouch and comprises at least about 50% of a pasteurized and
fermented particulate tobacco material, based on the total dry
weight of the smokeless tobacco product, and wherein the botanical
material is in particulate form.
19. The smokeless tobacco product of claim 18, wherein the
botanical material is selected from the group consisting of
rosemary, oregano, sage, hibiscus, clove, rose hip, yerba mate,
cocoa, turmeric, guayusa, honeybush, green tea, black tea, rooibos,
yerba santa, bacopa monniera, gingko biloba, withania somnifera,
and combinations thereof.
20. The smokeless tobacco product of claim 18, wherein the
smokeless tobacco product has an ORAC index value of at least about
50 (.mu.mol TE)/g or an FRAP index value of at least about 80
(.mu.mol/Fe.sup.2+)/g.
21. The smokeless tobacco product of claim 1, wherein the botanical
material is either (i) a shredded or particulate botanical material
present in an amount of at least about 1% of the total dry weight
of the smokeless tobacco product; or (ii) a botanical material
extract present in any amount of at least about 0.1% of the total
dry weight of the smokeless tobacco product.
22. The smokeless tobacco product of claim 1, wherein the botanical
material is selected from the group consisting of honeybush,
rooibos, yerba mate, and combinations thereof.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to products made or derived
from tobacco, or that otherwise incorporate tobacco, and are
intended for human consumption.
BACKGROUND OF THE INVENTION
[0002] Cigarettes, cigars and pipes are popular smoking articles
that employ tobacco in various forms. Such smoking articles are
used by heating or burning tobacco, and aerosol (e.g., smoke) is
inhaled by the smoker. Tobacco also may be enjoyed in a so-called
"smokeless" form. Particularly popular smokeless tobacco products
are employed by inserting some form of processed tobacco or
tobacco-containing formulation into the mouth of the user.
[0003] See for example, the types of smokeless tobacco
formulations, ingredients, and processing methodologies set forth
in U.S. Pat. No. 1,376,586 to Schwartz; U.S. Pat. No. 3,696,917 to
Levi; U.S. Pat. No. 4,513,756 to Pittman et al.; U.S. Pat. No.
4,528,993 to Sensabaugh, Jr. et al.; U.S. Pat. No. 4,624,269 to
Story et al.; U.S. Pat. No. 4,991,599 to Tibbetts; U.S. Pat. No.
4,987,907 to Townsend; U.S. Pat. No. 5,092,352 to Sprinkle, III et
al.; U.S. Pat. No. 5,387,416 to White et al.; U.S. Pat. No.
6,668,839 to Williams; U.S. Pat. No. 6,834,654 to Williams; U.S.
Pat. No. 6,953,040 to Atchley et al.; U.S. Pat. No. 7,032,601 to
Atchley et al.; and U.S. Pat. No. 7,694,686 to Atchley et al.; US
Pat. Pub. Nos. 2004/0020503 to Williams; 2005/0115580 to Quinter et
al.; 2006/0191548 to Strickland et al.; 2007/0062549 to Holton, Jr.
et al.; 2007/0186941 to Holton, Jr. et al.; 2007/0186942 to
Strickland et al.; 2008/0029110 to Dube et al.; 2008/0029116 to
Robinson et al.; 2008/0173317 to Robinson et al.; 2008/0196730 to
Engstrom et al.; 2008/0209586 to Neilsen et al.; 2008/0305216 to
Crawford et al.; 2009/0065013 to Essen et al.; 2009/0293889 to
Kumar et al.; 2010/0291245 to Gao et al; and 2011/0139164 to Mua et
al.; PCT WO 04/095959 to Arnarp et al. and WO 2010/132444 A2 to
Atchley, each of which is incorporated herein by reference.
Exemplary smokeless tobacco products that have been marketed
include those referred to as CAMEL Snus, CAMEL Orbs, CAMEL Strips
and CAMEL Sticks by R. J. Reynolds Tobacco Company; GRIZZLY moist
tobacco, KODIAK moist tobacco, LEVI GARRETT loose tobacco and
TAYLOR'S PRIDE loose tobacco by American Snuff Company, LLC; KAYAK
moist snuff and CHATTANOOGA CHEW chewing tobacco by Swisher
International, Inc.; REDMAN chewing tobacco by Pinkerton Tobacco
Co. LP; COPENHAGEN moist tobacco, COPENHAGEN Pouches, SKOAL
Bandits, SKOAL Pouches, RED SEAL long cut and REVEL Mint Tobacco
Packs by U.S. Smokeless Tobacco Company; and MARLBORO Snus and
Taboka by Philip Morris USA.
[0004] Through the years, various treatment methods and additives
have been proposed for altering the overall character or nature of
tobacco materials utilized in tobacco compositions. For example,
additives or treatment processes are sometimes utilized in order to
alter the chemistry or sensory properties of the tobacco material,
or in the case of smokable tobacco materials, to alter the
chemistry or sensory properties of mainstream smoke generated by
smoking articles including the tobacco material.
[0005] It would be desirable in the art to provide compositions
that alter the character and nature of tobacco (and tobacco
compositions and formulations) useful in smoking articles or
smokeless tobacco products.
SUMMARY OF THE INVENTION
[0006] The present invention relates to a smokeless tobacco product
comprising a tobacco material and a separate botanical component.
The addition of a botanical material to a smokeless tobacco product
can offer numerous advantages such as enhancement of antioxidant
character, altered sensory characteristics, and possible addition
of health or medicinal benefits. Although the focus of the
application is on smokeless tobacco products, the tobacco/botanical
compositions set forth herein could also be incorporated into
smoking articles, such as cigarettes or electronic cigarettes.
[0007] In various embodiments of the present invention, a smokeless
tobacco product configured for insertion into the mouth of a user
of the product is provided, the smokeless tobacco product
comprising a tobacco material mixed with at least one botanical
material, wherein the botanical material comprises at least about
0.1% of the total dry weight of the smokeless tobacco product. The
tobacco formulation can be contained within a water-permeable
pouch, for example.
[0008] In various embodiments of the smokeless tobacco product
described herein, the botanical material can have an ORAC index
value of about 250 (.mu.mol TE)/g or greater, about 500 (.mu.mol
TE)/g or greater, or about 1000 (.mu.mol TE)/g or greater. In some
embodiments, the botanical material can have an FRAP index value of
about 250 (.mu.mol/Fe.sup.2+)/g or greater, or about 500
(.mu.mol/Fe.sup.2+)/g or greater, or about 1000
(.mu.mol/Fe.sup.2+)/g or greater.
[0009] In various embodiments of the smokeless tobacco product
described herein, the smokeless tobacco product can have an ORAC
index value of at least about 50 (.mu.mol TE)/g or an FRAP index
value of at least about 80 (.mu.mol/Fe.sup.2+)/g. In some
embodiments, the smokeless tobacco product can have an ORAC index
value of at least about 75 (.mu.mol TE)/g or an FRAP index value of
at least about 100 (.mu.mol/Fe.sup.2+)/g.
[0010] Various types of botanical materials can be included in a
smokeless tobacco product. For example, the botanical material can
be selected from the group consisting of rosemary, oregano, sage,
hibiscus, clove, rose hip, yerba mate, cocoa, turmeric, guayusa,
honeybush, green tea, black tea, rooibos, yerba santa, bacopa
monniera, gingko biloba, withania somnifera, and combinations
thereof. In some embodiments, a smokeless tobacco product can
incorporate honeybush, rooibos, yerba mate or a combination
thereof. In some embodiments of a smokeless tobacco product the
botanical material is a tea or a tisane material. In addition,
botanical materials can be provided in various forms. In various
embodiments, both the tobacco material and the botanical material
can be in particulate form. In some embodiments of a smokeless
tobacco material, at least one of the tobacco material and the
botanical material are in the form of an oil or aqueous extract or
an ethanol or other alcohol extract.
[0011] In various embodiments of a smokeless tobacco product, the
botanical material can comprise at least about 1%, or at least
about 5% of the total dry weight of the smokeless tobacco product.
A larger amount of botanical material can be used to achieve a
higher antioxidant character of the product. In certain embodiments
where a botanical extract is used, the botanical material can
comprise about 1% or less, or about 0.5% or less of the total dry
weight of the smokeless tobacco product. In one embodiment, the
botanical material is either (i) a shredded or particulate
botanical material present in an amount of at least about 1% of the
total dry weight (or at least about 5%) of the smokeless tobacco
product; or (ii) a botanical material extract present in any amount
of at least about 0.1% of the total dry weight (or at least about
0.5%) of the smokeless tobacco product.
[0012] Various embodiments of a smokeless tobacco product can
further comprise one or more additional components selected from
the group consisting of flavorants, fillers, binders, pH adjusters,
buffering agents, salts, sweeteners, colorants, disintegration
aids, other antioxidants, humectants, and preservatives.
[0013] In an embodiment of a smokeless tobacco product described
herein, the product can be in the form of a snus product contained
within a water-permeable pouch and can comprise at least about 50%
of a pasteurized and fermented particulate tobacco material, based
on the total dry weight of the smokeless tobacco product, and the
botanical material can be in particulate form. Furthermore, the
botanical material can be selected from the group consisting of
rosemary, oregano, sage, hibiscus, clove, rose hip, yerba mate,
cocoa, turmeric, guayusa, honeybush, green tea, black tea, rooibos,
and combinations thereof. Also, the smokeless tobacco product can
have an ORAC index value of at least about 50 (.mu.mol TE)/g or an
FRAP index value of at least about 80 (.mu.mol/Fe.sup.2+)/g.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In order to provide an understanding of embodiments of the
invention, reference is made to the appended drawings, which are
not necessarily drawn to scale, and in which reference numerals
refer to components of described exemplary embodiments of the
invention. The drawings are exemplary only, and should not be
construed as limiting the invention.
[0015] FIG. 1 is a top view of a smokeless tobacco product
embodiment, taken across the width of the product, showing an outer
pouch filled with a combination of a tobacco material and a
botanical material; and
[0016] FIG. 2 is a cross-sectional view of a smokeless tobacco
product embodiment, taken across the width of the product, showing
an outer pouch filled with a combination of a tobacco material and
a botanical material and optional microcapsules disposed
therein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawing. The
inventions may be embodied in many different forms and should not
be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout. As used in this specification and the claims,
the singular forms "a," "an," and "the" include plural referents
unless the context clearly dictates otherwise. Reference to "dry
weight percent" or "dry weight basis" refers to weight on the basis
of dry ingredients (i.e., all ingredients except water).
[0018] The invention provides a tobacco composition incorporating a
separate botanical material (e.g., leaf or extract), smokeless
tobacco products incorporating such tobacco compositions, and
methods for preparing a tobacco composition that includes a
botanical material and for incorporating such compositions within
smokeless tobacco products. The addition of a botanical material to
a smokeless tobacco product can offer numerous advantages that will
vary with selection of the specific botanical material and the
nature and type of the smokeless tobacco product. Exemplary
advantages include enhancement of antioxidant character of the
smokeless tobacco product, altered sensory characteristics of the
smokeless tobacco product, and possible addition of health or
medicinal benefits associated with the smokeless tobacco product.
In certain embodiments of the invention including a component
derived or isolated from a botanical or herbal source, the
botanical or herbal component can add advantageous biological
functions to the product, such as immune system boosting effects,
antioxidant effects, and the like. See, e.g., U.S. Pat. No.
4,696,315 to Summers; U.S. App. Pub. Nos. 2011/0061666 to Dube et
al. and 2012/0272976 to Byrd et al., each of which is herein
incorporated by reference.
[0019] As used herein, the term "botanical material" refers to any
plant material, including plant material in its natural form and
plant material derived from natural plant materials, such as
extracts or isolates from plant materials or treated plant
materials (e.g., plant materials subjected to heat treatment,
fermentation, or other treatment processes capable of altering the
chemical nature of the material). For the purposes of the present
disclosure, a "botanical material" includes but is not limited to
"herbal materials," which refer to seed-producing plants that do
not develop persistent woody tissue and are often valued for their
medicinal or sensory characteristics (e.g., teas or tisanes).
Reference to botanical material is not intended to include tobacco
materials (i.e., does not include any Nicotiana species). The
botanical materials used in the present invention may comprise,
without limitation, any of the compounds and sources set forth
herein, including mixtures thereof. Certain botanical materials of
this type are sometimes referred to as dietary supplements,
nutraceuticals, "phytochemicals" or "functional foods."
[0020] Exemplary botanical materials, many of which are associated
with antioxidant characteristics, include without limitation acai
berry, alfalfa, allspice, annatto seed, apricot oil, basil, bee
balm, wild bergamot, black pepper, blueberries, borage seed oil,
bugleweed, cacao, calamus root, catnip, catuaba, cayenne pepper,
chaga mushroom, chervil, cinnamon, dark chocolate, potato peel,
grape seed, ginseng, gingko biloba, Saint John's Wort, saw
palmetto, green tea, black tea, black cohosh, cayenne, chamomile,
cloves, cocoa powder, cranberry, dandelion, grapefruit, honeybush,
echinacea, garlic, evening primrose, feverfew, ginger, goldenseal,
hawthorn, hibiscus flower, jiaogulan, kava, lavender, licorice,
marjoram, milk thistle, mints (menthe), oolong tea, beet root,
orange, oregano, papaya, pennyroyal, peppermint, red clover,
rooibos (red or green), rosehip, rosemary, sage, clary sage,
savory, spearmint, spirulina, slippery elm bark, sorghum bran
hi-tannin, sorghum grain hi-tannin, sumac bran, comfrey leaf and
root, goji berries, gutu kola, thyme, turmeric, uva ursi, valerian,
wild yam root, wintergreen, yacon root, yellow dock, yerba mate,
yerba santa, bacopa monniera, withania somnifera, and silybum
marianum.
[0021] Botanical materials often include compounds from various
classes known to provide certain bioactive effects, such as
minerals, vitamins such a ascorbic acid, isoflavones,
phytoesterols, allyl sulfides, dithiolthiones, isothiocyanates,
indoles, lignans, flavonoids, polyphenols, plant phenolics,
tocopherols, ubiquinone, benzodioxoles, carotenoids, etc. More
specifically, typical antioxidants from botanicals can be
classified in the following groups: monoterpenoid phenols; alcohols
such as thymol, carvacol, menthol; p-cymene; diterpene phenols such
as carnosic acid, carnosol, rosmanol; hydroxycinnamic type
compounds such as caffeic acid, chlorogenic acid, rosmarinic acid,
p-coumaric acid, resveratrol, curcumin, eugenol, cinnamaladehyde;
hydroxybenzoic acids and derivatives such as gallic acid,
protocatechuic acid, propyl gallate; 2-benzopryrones such as
scopoletin, coumarin; 4-benzopyrones such as quercetin, genistein,
naringenin, diosmin, rutin; dihydrochalcones such as aspalathin,
notophagin; flavanols such as epicatechin, epigallocatechin,
epicatechin gallate, epigallocatechin gallate; anthocyanins and
anthocyanidins; triterpenes such as ursolic acid, oleanolic acid,
betulinic acid, betulonic acid; tocopherols such as .alpha.,
.beta., .gamma., .delta.-tocopherols; tocotrienols; carotenoids
such as .beta.-carotene or lutein; ubiquinone, CoQ10; ascorbyl
palmitate; benzodioxoles such as myristicin, piperine, safrole; and
other compounds such as gambogic acid, gingerol, and the like.
Beyond antioxidant properties, certain compounds noted above can
also have properties such as distinctive flavor, color, antiseptic
properties, anti-carcinogenic effects, etc.
[0022] Exemplary compounds found in botanical materials include,
but are not limited to, propylene glycol, lactic acid, glycolic
acid, alanine, camphor, pyruvic acid, aspalathin, borneol, menthol,
phosphate, glycerin, proline, succinic acid, thymol, glyceric acid,
2-butenedioic acid, 3-hydroxyglutaric acid, malic acid,
5-oxoproline (pyroglutamic acid), aspartic acid, trihydroxybutanoic
acid, glutamine, asparagine, levoglucosan, xylitol, ribitol,
2-keto-L-gluconic acid, fructose, caffeine, citric acid,
glucosamine, neophytadiene, altrose, quinic acid, xylulose,
glucose, inositol, 2-amino-2-deoxyglucose, glucitol, ascorbic acid,
glucose, gallic acid, gluconic acid, galactaric acid, hexadecanoic
acid, 3,4-dihydroxyphenyl-2-hydroxypropionic acid, glucuronic acid,
myoinositol, caffeic acid, tryptophan, linolenic acid, octadecanoic
acid, galacturonic acid, rosmaricin, carnosic acid, melibiose,
carnosol, phitosterol, sucrose, rosmanol, 2,5-deoxyfructosazine,
2,6-deoxyfructosazine, fructosazine, maltitol, epicatechin,
nothofagin, orientin, catechin, epigallocatechin, coumaroyl quinic
acid, tocoferol, chloro genic acid, stigmasterol, rosmarinic acid,
betulinic acid, oleanolic acid, ursolic acid, glyderinine,
epicatechin gallate, catechin gallate, epigallocatechin gallate,
gallocatechin gallate, solanesol, and the like. For additional
exemplary compounds, see, e.g., Santhosh et al., Phytomedicine, 12
(2005) 216-220, which is incorporated herein by reference.
[0023] The botanical material can be used in the smokeless tobacco
compositions and products of the invention in a variety of forms.
Exemplary forms include plant materials in shredded or particulate
(e.g., a milled or ground product in a form characterized as
granular or powder) form. Exemplary forms also include isolated
components of plant materials in forms such as oils, aqueous
extracts, or alcohol (e.g., ethanol) extracts, which can be
optionally used in solid form (e.g., freeze-dried or spray-dried
form).
[0024] Botanical materials can be evaluated for their antioxidant
character and for other potential benefits. The evaluation of a
botanical material or evaluation of a tobacco product incorporating
a botanical material can be done, for example, based on the
antioxidant character as described by two indices, ORAC (Oxygen
Radical Absorbance Capacity) and FRAP (Ferric Reducing Ability
Potential).
[0025] The ORAC assay provides an index which describes the degree
and length of time in which a substance (antioxidant) is able to
inhibit the action of an oxidizing agent acting on a fluorescent
substrate. The assay is based on the measurement of the kinetics of
the oxidation reaction. The antioxidant activity (or index) can be
characterized by the number of moles of free radicals scavenged by
an antioxidant. The antioxidant mechanisms include hydrogen atom
transfer (HAT), single electron transfer followed by proton
transfer (SET or ET-PT), and the sequential proton loss electron
transfer (SPLET). Each of these mechanisms involves different
kinetics. For the HAT mechanism, the reactions can be summarized as
follows:
ROO.+AH.fwdarw.ROOH+A.
ROO.+A..fwdarw.ROOA
For the SET mechanism, the reactions can be summarized as
follows:
ROO..sub.-+AH.fwdarw.ROO..sub.-.sup.-+AH.sup.+
ROO..sub.-+AH.sup.+.fwdarw.ROO.sup.-AH..sup.+
ROO.sup.-AH..sup.+.fwdarw.ROOH+A.
ROO.+A..fwdarw.ROOA
For the SPLET mechanism, the reactions can be summarized as
follows:
ROO..sub.-+AH.fwdarw.ROO..sub.-.sup.-+AH.sup.+
ROO.sup.-AH..sup.+.fwdarw.ROOH+A.
ROO.+A..fwdarw.ROOA
The end result of the three mechanisms is essentially the same: the
transformation of a peroxide radical into a much less reactive
species.
[0026] Antioxidant measurements methods are typically based on the
measurement of the rate of reaction of a free radical generator
with a compound that has color or fluorescence, in the presence of
a standard antioxidant, or the presence of an antioxidant substance
to be evaluated. For example, in various embodiments of the
invention, the radical used in the ORAC assay can be a peroxyl
radical produced by 2,2-azobis(2-amidinopropane) dihydrochloride
(AAPH), for example. This compound decomposes and generates N.sub.2
and two radicals. These radicals react with molecular oxygen to
produce peroxyl radicals. The peroxyl radicals can either react
with the antioxidant by removing a hydrogen atom from it or by
damaging a specific fluorescent reagent resulting in a loss of
fluorescence. For example, a fluorescent protein (R-PE or
R-phycoerythrin) can be used as the fluorescent material.
Fluorescein can also be used as a fluorescent probe material. Upon
reaction with the peroxyl radicals, the fluorescent material yields
a non-fluorescent product and the loss of fluorescence can be
measured with a fluorimeter. The degree of an antioxidant
protection is quantified by comparison with a standard antioxidant
(e.g., 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, also
known as Trolox which is a vitamin E analogue) and expressed in TEs
(trolox equivalents). Different concentrations of Trolox can be
used to make a standard curve. The measurement of ORAC index can be
performed for example, following a procedure described in Ou B.,
Hampsch-Woodill M., Prior R.L. Development and validation of an
improved oxygen radical absorbance capacity assay using fluorescein
as the fluorescent probe, J. Agric. Food Chem., 49 (2001) 4619-4626
and Ou B., Hampsch-Woodill M., Flanagan J., Deemer E. K., Prior R.
L., Huang D. J., Novel fluorometric assay for hydroxyl radical
prevention capacity using fluorescein as the probe, J. Agric. Food.
Chem. 50 (2002) 2772-2777, each of which is herein incorporated by
reference.
[0027] In general, ground botanical material can be extracted with
a solution containing approximately 50% acetone and 50% water.
After extraction, the liquid and solid can be separated by
filtration. A sample from the extract can then be diluted with
diluent. The ORAC assay can measure both hydrophilic materials and
lipophilic materials. For example, in a first version of the
procedure, the dilution can be done with a phosphate buffer
solution at approximately pH of 7.2. This returns hydrophilic ORAC
values. A second version of the procedure can use a diluent of
approximately 50% acetone and 50% water, which returns lipophilic
ORAC values. The hydrophilic and lypophilic ORAC values tend to be
in the same general range for a given sample. Therefore, for the
purposes of the discussion herein, reference to an ORAC value can
include both hydrophilic or lypophilic ORAC measurements.
[0028] In some embodiments of the present invention, botanical
materials that can be useful in a tobacco product have an ORAC
index value of about 250 (.mu.mol/TE)/g or greater, about 350
(.mu.mol/TE)/g or greater, about 500 (.mu.mol/TE)/g or greater, or
about 1000 (.mu.mol/TE)/g or greater.
[0029] The FRAP analysis is based on the reduction of a Fe.sup.3+
salt to a Fe.sup.2+ salt, and the reaction of Fe.sup.2+ with
2,4,6-tripyridyl-s-triazine which generates a colored complex. The
color of the complex (absorbance) can be measured at a specified
wavelength using a spectrophotometer. The measurement of the FRAP
index can be performed, for example, following a procedure
described in Benzie, I. F. F., Strain, J. J., The ferric reduction
ability of plasma (FRAP) as a measure of "antioxidant power": the
FRAP assay, Anal. Biochem., 239 (1996) 70-76, herein incorporated
by reference. Since both the ORAC index and the FRAP index measure
the antioxidant property of a material, the correlation between the
two indices is relatively strong. The FRAP index values are
expressed in (.mu.mol/Fe.sup.2+)/g values. In some embodiments of
the present invention, botanical materials useful in tobacco
products described herein can have an FRAP index value of
approximately 250 (.mu.mol/Fe.sup.2+)/g or greater, about 350
(.mu.mol/Fe.sup.2+)/g or greater, about 500 (.mu.mol/Fe.sup.2+)/g
or greater, about 1000 (.mu.mol/Fe.sup.2+)/g, or about 1500
(.mu.mol/Fe.sup.2+)/g or greater.
[0030] The selection of the plant from the Nicotiana species (i.e.,
tobacco material) utilized in the products and processes of the
invention can vary; and in particular, the types of tobacco or
tobaccos may vary. Tobaccos that can be employed include flue-cured
or Virginia (e.g., K326), burley, sun-cured (e.g., Indian Kurnool
and Oriental tobaccos, including Katerini, Prelip, Komotini, Xanthi
and Yambol tobaccos), Maryland, dark, dark-fired, dark air cured
(e.g., Passanda, Cubano, Jatin and Bezuki tobaccos), light air
cured (e.g., North Wisconsin and Galpao tobaccos), Indian air
cured, Red Russian and Rustica tobaccos, as well as various other
rare or specialty tobaccos. Descriptions of various types of
tobaccos, growing practices and harvesting practices are set forth
in Tobacco Production, Chemistry and Technology, Davis et al.
(Eds.) (1999), which is incorporated herein by reference. Various
representative types of plants from the Nicotiana species are set
forth in Goodspeed, The Genus Nicotiana, (Chonica Botanica) (1954);
U.S. Pat. No. 4,660,577 to Sensabaugh, Jr. et al.; U.S. Pat. No.
5,387,416 to White et al.; U.S. Pat. No. 7,025,066 to Lawson et
al.; and U.S. Pat. No. 7,798,153 to Lawrence, Jr.; and US Patent
Appl. Pub. No. 2008/0245377 to Marshall et al.; each of which is
incorporated herein by reference. Tobacco compositions including
dark air cured tobacco are set forth in US Patent Appl. Pub. No.
2008/0245377 to Marshall et al., which is incorporated herein by
reference. See also, types of tobacco as set forth, for example, in
US Patent Appl. Pub. No. 2011/0247640 to Beeson et al., which is
incorporated herein by reference.
[0031] Exemplary Nicotiana species include N. tabacum, N. rustica,
N. alata, N. arentsii, N. excelsior, N. forgetiana, N. glauca, N.
glutinosa, N. gossei, N. kawakamii, N. knightiana, N. langsdorffi,
N. otophora, N. setchelli, N. sylvestris, N. tomentosa, N.
tomentosiformis, N. undulata, N. .times.sanderae, N. africana, N.
amplexicaulis, N. benavidesii, N. bonariensis, N. debneyi, N.
longiflora, N. maritina, N. megalosiphon, N. occidentalis, N.
paniculata, N. plumbaginifolia, N. raimondii, N. rosulata, N.
simulans, N. stocktonii, N. suaveolens, N. umbratica, N. velutina,
N. wigandioides, N. acaulis, N. acuminata, N. attenuata, N.
benthamiana, N. cavicola, N. clevelandii, N. cordifolia, N.
corymbosa, N. fragrans, N. goodspeedii, N. linearis, N. miersii, N.
nudicaulis, N. obtusifolia, N. occidentalis subsp. Hersperis, N.
pauciflora, N. petunioides, N. quadrivalvis, N. repanda, N.
rotundifolia, N. solanifolia and N. spegazzinii.
[0032] Nicotiana species can be derived using genetic-modification
or crossbreeding techniques (e.g., tobacco plants can be
genetically engineered or crossbred to increase or decrease
production of components, characteristics or attributes). See, for
example, the types of genetic modifications of plants set forth in
U.S. Pat. No. 5,539,093 to Fitzmaurice et al.; U.S. Pat. No.
5,668,295 to Wahab et al.; U.S. Pat. No. 5,705,624 to Fitzmaurice
et al.; U.S. Pat. No. 5,844,119 to Weigl; U.S. Pat. No. 6,730,832
to Dominguez et al.; U.S. Pat. No. 7,173,170 to Liu et al.; U.S.
Pat. No. 7,208,659 to Colliver et al. and U.S. Pat. No. 7,230,160
to Benning et al.; US Patent Appl. Pub. No. 2006/0236434 to
Conkling et al.; and PCT WO 2008/103935 to Nielsen et al. See,
also, the types of tobaccos that are set forth in U.S. Pat. No.
4,660,577 to Sensabaugh, Jr. et al.; U.S. Pat. No. 5,387,416 to
White et al.; and U.S. Pat. No. 6,730,832 to Dominguez et al., each
of which is incorporated herein by reference.
[0033] For the preparation of smokeless tobacco products, it is
typical for harvested plants of the Nicotiana species to be
subjected to a curing process. Descriptions of various types of
curing processes for various types of tobaccos are set forth in
Tobacco Production, Chemistry and Technology, Davis et al. (Eds.)
(1999). Especially preferred techniques and conditions for curing
flue-cured tobacco are set forth in Nestor et al., Beitrage
Tabakforsch. Int., 20 (2003) 467-475 and U.S. Pat. No. 6,895,974 to
Peele, which are incorporated herein by reference. Representative
techniques and conditions for air curing tobacco are set forth in
Roton et al., Beitrage Tabakforsch. Int., 21 (2005) 305-320 and
Staaf et al., Beitrage Tabakforsch. Int., 21 (2005) 321-330, which
are incorporated herein by reference. Certain types of tobaccos can
be subjected to alternative types of curing processes, such as fire
curing or sun curing. Typically, harvested tobaccos that are cured
are then aged.
[0034] Various parts or portions of the plant of the Nicotiana
species can be employed. For example, virtually all of the plant
(e.g., the whole plant) can be harvested, and employed as such.
Alternatively, various parts or pieces of the plant can be
harvested or separated for further use after harvest. For example,
the flower, leaves, stem, stalk, roots, seeds, and various
combinations thereof, can be isolated for further use or
treatment.
[0035] The post-harvest processing of the tobacco plant or portion
thereof can vary. After harvest, the plant, or portion thereof, can
be used in a green form (e.g., the plant or portion thereof can be
used without being subjected to any curing process). For example,
the plant or portion thereof can be used without being subjected to
significant storage, handling or processing conditions. A tobacco
plant or portion thereof can be refrigerated or frozen for later
use, freeze-dried, subjected to irradiation, yellowed, dried,
cured, cooked (e.g., roasted, fried or boiled), or otherwise
subjected to storage or treatment for later use.
[0036] The harvested tobacco plant or portion thereof can be
physically processed. The plant or portion thereof can be separated
into individual parts or pieces (e.g., the leaves can be removed
from the stems, and/or the stems and leaves can be removed from the
stalk). The harvested plant or individual parts or pieces can be
further subdivided into parts or pieces (e.g., the leaves can be
shredded, cut, comminuted, pulverized, milled or ground into pieces
or parts that can be characterized as filler-type pieces, granules,
particulates or fine powders). The plant, or parts thereof, can be
subjected to external forces or pressure (e.g., by being pressed or
subjected to roll treatment). When carrying out such processing
conditions, the plant or portion thereof can have a moisture
content that approximates its natural moisture content (e.g., its
moisture content immediately upon harvest), a moisture content
achieved by adding moisture to the plant or portion thereof, or a
moisture content that results from the drying of the plant or
portion thereof. For example, powdered, pulverized, ground or
milled pieces of plants or portions thereof can have moisture
contents of less than about 25 weight percent, often less than
about 20 weight percent, and frequently less than about 15 weight
percent.
[0037] The tobacco material can be used in the smokeless tobacco
compositions and products of the invention in a variety of forms.
Exemplary forms include tobacco materials in shredded or
particulate (e.g., a milled or ground product in a form
characterized as granular or powder) form. Exemplary forms also
include isolated components of tobacco materials in forms such as
oils or aqueous extracts, which can be optionally used in solid
form (e.g., freeze-dried or spray-dried form). In certain
embodiments, the tobacco is employed in the form of parts or pieces
that have an average particle size less than that of the parts or
pieces of shredded tobacco used in so-called "fine cut" tobacco
products. Typically, the very finely divided tobacco particles or
pieces are sized to pass through a screen of about 18 or 16 Tyler
mesh, generally are sized to pass a screen of about 20 Tyler mesh,
often are sized to pass through a screen of about 50 Tyler mesh,
frequently are sized to pass through a screen of about 60 Tyler
mesh, may even be sized to pass through a screen of 100 Tyler mesh,
and further may be sized so as to pass through a screen of 200
Tyler mesh. If desired, air classification equipment may be used to
ensure that small sized tobacco particles of the desired sizes, or
range of sizes, may be collected. In one embodiment, the tobacco
material is in particulate form sized to pass through an 18 or 16
Tyler mesh, but not through a 60 Tyler mesh. If desired,
differently sized pieces of granulated tobacco may be mixed
together.
[0038] Typically, the very finely divided tobacco particles or
pieces suitable for snus products have a particle size greater than
-8 Tyler mesh, often -8 to +100 Tyler mesh, frequently -16 to +60
Tyler mesh. In certain embodiments, the tobacco is provided with an
average particle size of about 0.3 to about 2 mm, more often about
0.5 to about 1.5 mm, and most often about 0.75 to about 1.25 mm
(e.g., about 1 mm). The milled tobacco material is typically
employed in the form of parts or pieces that have an average
particle size less than about 50 microns. In one embodiment, the
average particle size of the tobacco particles may be less than or
equal to about 25 microns.
[0039] The manner by which the tobacco is provided in a finely
divided or powder type of form may vary. Preferably, tobacco parts
or pieces are comminuted, ground or pulverized into a powder type
of form using equipment and techniques for grinding, milling, or
the like. Most preferably, the tobacco is relatively dry in form
during grinding or milling, using equipment such as hammer mills,
cutter heads, air control mills, or the like. For example, tobacco
parts or pieces may be ground or milled when the moisture content
thereof is less than about 15 weight percent to less than about 5
weight percent.
[0040] The plant of the Nicotiana species or portions thereof can
be subjected to other types of processing conditions. For example,
components can be separated from one another, or otherwise
fractionated into chemical classes or mixtures of individual
compounds. Typical separation processes can include one or more
process steps such as solvent extraction using polar solvents,
organic solvents, or supercritical fluids, chromatography,
distillation, filtration, recrystallization, and/or solvent-solvent
partitioning. Exemplary extraction and separation solvents or
carriers include water, alcohols (e.g., methanol or ethanol),
hydrocarbons (e.g., heptane and hexane), diethyl ether methylene
chloride and supercritical carbon dioxide. Various extraction
techniques of tobacco materials can be used to provide a tobacco
extract and tobacco pulp. See, for example, the extraction
processes described in US Pat. Appl. Pub. No. 2011/0247640 to
Beeson et al., which is incorporated herein by reference. Other
exemplary techniques for extracting components of tobacco are
described in U.S. Pat. No. 4,144,895 to Fiore; U.S. Pat. No.
4,150,677 to Osborne, Jr. et al.; U.S. Pat. No. 4,267,847 to Reid;
U.S. Pat. No. 4,289,147 to Wildman et al.; U.S. Pat. No. 4,351,346
to Brummer et al.; U.S. Pat. No. 4,359,059 to Brummer et al.; U.S.
Pat. No. 4,506,682 to Muller; U.S. Pat. No. 4,589,428 to Keritsis;
U.S. Pat. No. 4,605,016 to Soga et al.; U.S. Pat. No. 4,716,911 to
Poulose et al.; U.S. Pat. No. 4,727,889 to Niven, Jr. et al.; U.S.
Pat. No. 4,887,618 to Bernasek et al.; U.S. Pat. No. 4,941,484 to
Clapp et al.; U.S. Pat. No. 4,967,771 to Fagg et al.; U.S. Pat. No.
4,986,286 to Roberts et al.; U.S. Pat. No. 5,005,593 to Fagg et
al.; U.S. Pat. No. 5,018,540 to Grubbs et al.; U.S. Pat. No.
5,060,669 to White et al.; U.S. Pat. No. 5,065,775 to Fagg; U.S.
Pat. No. 5,074,319 to White et al.; U.S. Pat. No. 5,099,862 to
White et al.; U.S. Pat. No. 5,121,757 to White et al.; U.S. Pat.
No. 5,131,414 to Fagg; U.S. Pat. No. 5,131,415 to Munoz et al.;
U.S. Pat. No. 5,148,819 to Fagg; U.S. Pat. No. 5,197,494 to Kramer;
U.S. Pat. No. 5,230,354 to Smith et al.; U.S. Pat. No. 5,234,008 to
Fagg; U.S. Pat. No. 5,243,999 to Smith; U.S. Pat. No. 5,301,694 to
Raymond et al.; U.S. Pat. No. 5,318,050 to Gonzalez-Parra et al.;
U.S. Pat. No. 5,343,879 to Teague; U.S. Pat. No. 5,360,022 to
Newton; U.S. Pat. No. 5,435,325 to Clapp et al.; U.S. Pat. No.
5,445,169 to Brinkley et al.; U.S. Pat. No. 6,131,584 to
Lauterbach; U.S. Pat. No. 6,298,859 to Kierulff et al.; U.S. Pat.
No. 6,772,767 to Mua et al.; and U.S. Pat. No. 7,337,782 to
Thompson, all of which are incorporated by reference herein.
[0041] In certain embodiments, a solvent is added to the tobacco
material and the material is soaked for a given period of time
(e.g., about 1 h). Following the extraction process, the tobacco
pulp is generally isolated from the tobacco extract, for example,
by filtration or centrifugation, although these methods are not
intended to be limiting. Alternatively, in some embodiments, the
tobacco pulp can be isolated from the extract by means of
distillation (e.g., steam distillation) of the tobacco mixture. See
also, the types of separation techniques set forth in Brandt et
al., LC-GC Europe, p. 2-5 (March, 2002) and Wellings, A Practical
Handbook of Preparative HPLC (2006), which are incorporated herein
by reference. In addition, the plant or portions thereof can be
subjected to the types of treatments set forth in Ishikawa et al.,
Chem. Pharm. Bull., 50, 501-507 (2002); Tienpont et al., Anal.
Bioanal. Chem., 373, 46-55 (2002); Ochiai, Gerstel Solutions
Worldwide, 6, 17-19 (2006); Coleman, III, et al., J. Sci. Food and
Agric., 84, 1223-1228 (2004); Coleman, III et al., J. Sci. Food and
Agric., 85, 2645-2654 (2005); Pawliszyn, ed., Applications of Solid
Phase Microextraction, RSC Chromatography Monographs, (Royal
Society of Chemistry, UK) (1999); Sahraoui et al., J. Chrom., 1210,
229-233 (2008); and U.S. Pat. No. 5,301,694 to Raymond et al.,
which are all incorporated herein by reference.
[0042] The tobacco extract can also be subjected to further
treatment steps. In some embodiments, the extract is brought into
contact with an imprinted polymer or non-imprinted polymer such as
described, for example, in US Pat. Pub. Nos. 2007/0186940 to
Bhattacharyya et al; 2011/0041859 to Rees et al.; 2011/0159160 to
Jonsson et al; and 2012/0291793 to Byrd et al., all of which are
incorporated herein by reference. In some embodiments, the extract
is clarified and/or concentrated by reverse osmosis, Still further,
the tobacco extract could be treated with ion exchange materials
having amine functionality, which can remove certain aldehydes and
other compounds. See, for example, U.S. Pat. No. 4,033,361 to
Horsewell et al and U.S. Pat. No. 6,779,529 to Figlar et al., which
are incorporated by reference herein.
[0043] Tobacco pulp that has been provided and isolated following
the extraction step can be whitened in certain embodiments
according to any means known in the art. For example, whitening
methods using various bleaching or oxidizing agents and oxidation
catalysts can be used. Exemplary oxidizing agents include peroxides
(e.g., hydrogen peroxide), chlorite salts, chlorate salts,
perchlorate salts, hypochlorite salts, ozone, ammonia, and
combinations thereof. Exemplary oxidation catalysts are titanium
dioxide, manganese dioxide, and combinations thereof.
[0044] If desired, the tobacco materials can be irradiated,
pasteurized, or otherwise subjected to controlled heat treatment.
Such treatment processes are detailed, for example, in US Pat. Pub.
No. 2009/0025738 to Mua et al., which is incorporated herein by
reference. The tobacco material could also be treated with enzymes
and/or probiotics to inhibit acrylamide formation or otherwise
chemically alter the tobacco material as discussed in US Pat. Pub.
Nos. 2010/0300463, 2011/0048434, and 2012/0060854 to Chen et al.,
U.S. patent application Ser. No. 13/444,272 to Marshall et al.,
filed on Apr. 11, 2012 and U.S. patent application Ser. No.
13/553,222 to Moldoveanu, filed on Jul. 19, 2012, which are
incorporated herein by reference. Exemplary enzymes that can be
used to treat the tobacco extract include amylases (which catalyze
the breakdown of starch into sugars) or proteases (which catalyze
the hydrolysis of peptide bonds of proteins) or a combination
thereof. Certain types of probiotics and compositional ingredients
that can be used to treat tobacco include examples set forth in
U.S. Pat. No. 8,097,245 to Harel et al.; U.S. Pat. No. 8,097,281 to
Heim et al.; U.S. Pat. No. 8,101,167 to Gueniche; and U.S. Pat. No.
8,101,170 to Plail et al., which are all incorporated herein by
reference.
[0045] The relative amount of tobacco material within the smokeless
tobacco products of the invention may vary. Typically, the amount
of tobacco material within the smokeless tobacco product is at
least about 10%, at least about 25%, or at least about 50%, and in
some cases, at least about 60%, at least about 70%, at least about
80%, or at least about 90% on a dry weight basis of the product. A
typical range of tobacco material within the product is about 10 to
about 99%, more often about 25 to about 75% by weight on a dry
basis.
[0046] The amount of botanical material incorporated into a
smokeless tobacco product will vary, depending in part on the
desired functional or sensory characteristics of the product, the
type and form of the botanical material, the type and form of the
smokeless tobacco product, and the like. A representative amount of
botanical material in the smokeless tobacco products of the
invention is at least about 0.1%, at least about 1%, at least about
5%, at least about 10%, or at least about 15%, and in some cases,
at least about 20%, at least about 30%, at least about 40%, or at
least about 50% on a dry weight basis of the product. A typical
range of botanical material within the product is about 0.1 to
about 60%, more often about 5 to about 40% by weight on a dry
basis.
[0047] A larger amount of certain botanical materials can be used
to achieve a higher antioxidant character of the product. Where an
extract is used as the botanical material, lesser amounts may be
needed to achieve the desired product characteristics. In certain
embodiments where a botanical extract is used, the botanical
material can comprise about 1% or less, or about 0.5% or less of
the total dry weight of the smokeless tobacco product. In one
embodiment, the botanical material is either (i) a shredded or
particulate botanical material present in an amount of at least
about 1% of the total dry weight (or at least about 5%) of the
smokeless tobacco product; or (ii) a botanical material extract
present in any amount of at least about 0.1% of the total dry
weight (or at least about 0.5%) of the smokeless tobacco
product.
[0048] The relative amount of tobacco material and botanical
material can also be expressed in terms of weight ratio of tobacco
to botanical within the smokeless tobacco product, with exemplary
ratios including about 99:1 to about 40:60, such as about 80:20 to
about 50:50.
[0049] The components of the smokeless tobacco composition,
including the tobacco material, the botanical material, and any
additional ingredients described herein, can be brought together in
admixture using any mixing technique or equipment known in the art.
The various components can be admixed together in multiple steps or
in a single step. Any mixing method that brings the tobacco
composition ingredients into intimate contact can be used. A mixing
apparatus featuring an impeller or other structure capable of
agitation is typically used. Exemplary mixing equipment includes
casing drums, conditioning cylinders or drums, liquid spray
apparatus, conical-type blenders, ribbon blenders, mixers available
as FKM130, FKM600, FKM1200, FKM2000 and FKM3000 from Littleford
Day, Inc., Plough Share types of mixer cylinders, and the like. As
such, the overall mixture of various components with the tobacco
material may be relatively uniform in nature. See also, for
example, the types of methodologies set forth in U.S. Pat. No.
4,148,325 to Solomon et al.; U.S. Pat. No. 6,510,855 to Korte et
al.; and U.S. Pat. No. 6,834,654 to Williams, each of which is
incorporated herein by reference.
[0050] The overall smokeless tobacco composition or product can
also be characterized based on ORAC index value or FRAP index
value. For example, exemplary smokeless tobacco products (including
snus type products) have an ORAC index value of at least about 50
(.mu.mol/TE)/g, at least about 75 (.mu.mol/TE)/g, or at least about
100 (.mu.mol/TE)/g, or in certain embodiments, at least about 120
(.mu.mol/TE)/g, at least about 140 (.mu.mol/TE)/g, or at least
about 160 (.mu.mol/TE)/g. Exemplary smokeless tobacco products
(including snus type products) have an FRAP index value of at least
about 80 (.mu.mol/Fe.sup.2+)/g, at least about 100
(.mu.mol/Fe.sup.2+)/g, at least about 140 (.mu.mol/Fe.sup.2+)/g, at
least about 200 (.mu.mol/Fe.sup.2+)/g, or in certain embodiments,
at least about 220 (.mu.mol/Fe.sup.2+)/g, at least about 260
(.mu.mol/Fe.sup.2+)/g, at least about 300
(.mu.mol/Fe.sup.2+)/g.
[0051] The form of the smokeless tobacco product of the invention
can vary. In one particular embodiment, the product is in the form
of a snus-type product containing a particulate tobacco material
and a particulate botanical material. Manners and methods for
formulating snus-type tobacco formulations will be apparent to
those skilled in the art of snus tobacco product production.
[0052] Referring to FIG. 1, there is shown a first embodiment of a
smokeless tobacco product 10. The tobacco product 10 includes a
moisture-permeable container in the form of a pouch 12, which
contains a snus-type particulate mixture 14 comprising a mixture of
a tobacco material and a botanical material. As shown in FIG. 2,
for example, the smokeless tobacco product also may optionally
comprise a plurality of microcapsules 16 dispersed within the
particulate material 14, the microcapsules containing an additive
(e.g., a flavorant) such as described in greater detail below.
[0053] The tobacco product 10 is typically used by placing one
pouch containing the tobacco formulation in the mouth of a human
subject/user. During use, saliva in the mouth of the user causes
some of the components of the product to pass through the
water-permeable pouch and into the mouth of the user. The pouch
preferably is not chewed or swallowed. The user is provided with
tobacco flavor and satisfaction, and is not required to spit out
any portion of the product. After about 10 minutes to about 60
minutes, typically about 15 minutes to about 45 minutes, of
use/enjoyment, substantial amounts of the product and have been
ingested by the human subject, and the pouch may be removed from
the mouth of the human subject for disposal.
[0054] The moisture content of the smokeless tobacco product prior
to use by a consumer of the formulation may vary. For example, a
representative snus-type product may exhibit a moisture content of
about 20 weight percent to about 50 weight percent, preferably
about 20 weight percent to about 40 weight percent. The manner by
which the moisture content of the formulation is controlled may
vary. For example, the formulation may be subjected to thermal or
convection heating.
[0055] Most preferably, moist tobacco formulations, such as the
types of tobacco formulations employed within snus types of
products, are subjected to pasteurization or fermentation.
Techniques for pasteurizing/heat treating and/or fermenting snus
types of tobacco products will be apparent to those skilled in the
art of snus product design and manufacture.
[0056] As noted above, the smokeless tobacco composition can be
housed within a moisture-permeable packet or pouch that acts as a
container for use of the tobacco. The composition/construction of
such packets or pouches, such as the container pouch 12 in the
embodiment illustrated in FIG. 1, may be varied. Suitable packets,
pouches or containers of the type used for the manufacture of
smokeless tobacco products are available under the tradenames
CatchDry, Ettan, General, Granit, Goteborgs Rape, Grovsnus White,
Metropol Kaktus, Mocca Anis, Mocca Mint, Mocca Wintergreen, Kicks,
Probe, Prince, Skruf and TreAnkrare. The smokeless tobacco
formulation may be contained in pouches and packaged, in a manner
and using the types of components used for the manufacture of
conventional snus types of products. The pouch provides a
liquid-permeable container of a type that may be considered to be
similar in character to the mesh-like type of material that is used
for the construction of a tea bag. Components of the loosely
arranged smokeless tobacco formulation readily diffuse through the
pouch and into the mouth of the user.
[0057] An exemplary pouch may be manufactured from materials, and
in such a manner, such that during use by the user, the pouch
undergoes a controlled dispersion or dissolution. Such pouch
materials may have the form of a mesh, screen, perforated paper,
permeable fabric, or the like. For example, pouch material
manufactured from a mesh-like form of rice paper, or perforated
rice paper, may dissolve in the mouth of the user. As a result, the
pouch and tobacco formulation each may undergo complete dispersion
within the mouth of the user during normal conditions of use, and
hence the pouch and tobacco formulation both may be ingested by the
user. Other exemplary pouch materials may be manufactured using
water dispersible film forming materials (e.g., binding agents such
as alginates, carboxymethylcellulose, xanthan gum, pullulan, and
the like), as well as those materials in combination with materials
such as ground cellulosics (e.g., fine particle size wood pulp).
Preferred pouch materials, though water dispersible or dissolvable,
may be designed and manufactured such that under conditions of
normal use, a significant amount of the tobacco formulation
contents permeate through the pouch material prior to the time that
the pouch undergoes loss of its physical integrity. If desired,
flavoring ingredients, disintegration aids, and other desired
components, may be incorporated within, or applied to, the pouch
material.
[0058] The amount of material contained within each pouch may vary.
In smaller embodiments, the dry weight of the material within each
pouch is at least about 50 mg to about 150 mg. For a larger
embodiment, the dry weight of the material within each pouch
preferably does not exceed about 300 mg to about 500 mg. In some
embodiments, each pouch/container may have disposed therein a
flavor agent member, as described in greater detail in U.S. Pat.
No. 7,861,728 to Holton, Jr. et al., which is incorporated herein
by reference. If desired, other components can be contained within
each pouch. For example, at least one flavored strip, piece or
sheet of flavored water dispersible or water soluble material
(e.g., a breath-freshening edible film type of material) may be
disposed within each pouch along with or without at least one
capsule. Such strips or sheets may be folded or crumpled in order
to be readily incorporated within the pouch. See, for example, the
types of materials and technologies set forth in U.S. Pat. No.
6,887,307 to Scott et al. and U.S. Pat. No. 6,923,981 to Leung et
al.; and The EFSA Journal (2004) 85, 1-32; which are incorporated
herein by reference.
[0059] Descriptions of various components of snus types of products
and components thereof also are set forth in US Pat. App. Pub. No.
2004/0118422 to Lundin et al., which is incorporated herein by
reference. See, also, for example, U.S. Pat. No. 4,607,479 to
Linden; U.S. Pat. No. 4,631,899 to Nielsen; U.S. Pat. No. 5,346,734
to Wydick et al.; and U.S. Pat. No. 6,162,516 to Den, and US Pat.
Pub. No. 2005/0061339 to Hansson et al.; each of which is
incorporated herein by reference. See, also, the types of pouches
set forth in U.S. Pat. No. 5,167,244 to Kjerstad, which is
incorporated herein by reference. Snus types of products can be
manufactured using equipment such as that available as SB 51-1/T,
SBL 50 and SB 53-2/T from Merz Verpackungmaschinen GmBH. Snus
pouches can be provided as individual pouches, or a plurality of
pouches (e.g., 2, 4, 5, 10, 12, 15, 20, 25 or 30 pouches) can
connected or linked together (e.g., in an end-to-end manner) such
that a single pouch or individual portion can be readily removed
for use from a one-piece strand or matrix of pouches.
[0060] Products of the present invention may be packaged and stored
in much the same manner that conventional types of smokeless
tobacco products are packaged and stored. For example, a plurality
of packets or pouches may be contained in a cylindrical container.
If desired, moist tobacco products (e.g., products having moisture
contents of more than about 20 weight percent) may be refrigerated
(e.g., at a temperature of less than about 10.degree. C., often
less than about 8.degree. C., and sometimes less than about
5.degree. C.). Alternatively, relatively dry tobacco products
(e.g., products having moisture contents of less than about 15
weight percent) often may be stored under a relatively wide range
of temperatures.
[0061] The invention is not limited to snus-type smokeless tobacco
products. For example, the mixture of tobacco material and
botanical material can also be incorporated into various smokeless
tobacco forms such as loose moist snuff, loose dry snuff, chewing
tobacco, pelletized tobacco pieces, extruded tobacco strips or
pieces, finely divided or milled agglomerates of powdered pieces
and components, flake-like pieces (e.g., that can be formed by
agglomerating tobacco formulation components in a fluidized bed),
molded tobacco pieces (e.g., formed in the general shape of a coin,
cylinder, bean, cube, or the like), pieces of tobacco-containing
gum, products incorporating mixtures of edible material combined
with tobacco pieces and/or tobacco extract, products incorporating
tobacco (e.g., in the form of tobacco extract) carried by a solid
inedible substrate, and the like. For example, the smokeless
tobacco product can have the form of compressed tobacco pellets,
multi-layered extruded pieces, extruded or formed rods or sticks,
compositions having one type of tobacco formulation surrounded by a
different type of tobacco formulation, rolls of tape-like films,
readily water-dissolvable or water-dispersible films or strips
(see, for example, US Pat. Appl. Pub. No. 2006/0198873 to Chan et
al.), or capsule-like materials possessing an outer shell (e.g., a
pliable or hard outer shell that can be clear, colorless,
translucent or highly colored in nature) and an inner region
possessing tobacco or tobacco flavor (e.g., a Newtoniam fluid or a
thixotropic fluid incorporating tobacco of some form).
[0062] In some embodiments, smokeless tobacco products of the
invention can have the form of a lozenge, tablet, microtab, or
other tablet-type product. See, for example, the types of lozenge
formulations and techniques for formulating or manufacturing
lozenges set forth in U.S. Pat. No. 4,967,773 to Shaw; U.S. Pat.
No. 5,110,605 to Acharya; U.S. Pat. No. 5,733,574 to Dam; U.S. Pat.
No. 6,280,761 to Santus; U.S. Pat. No. 6,676,959 to Andersson et
al.; U.S. Pat. No. 6,248,760 to Wilhelmsen; and U.S. Pat. No.
7,374,779; US Pat. Pub. Nos. 2001/0016593 to Wilhelmsen;
2004/0101543 to Liu et al.; 2006/0120974 to Mcneight; 2008/0020050
to Chau et al.; 2009/0081291 to Gin et al.; and 2010/0004294 to
Axelsson et al.; which are incorporated herein by reference.
[0063] Depending on the type of smokeless tobacco product being
processed, the tobacco product can include one or more additional
components in addition to the tobacco material and the botanical
material as described above. For example, the tobacco material and
botanical material can be processed, blended, formulated, combined
and/or mixed with other materials or ingredients, such as other
tobacco materials or flavorants, fillers, binders, pH adjusters,
buffering agents, salts, sweeteners, colorants, disintegration
aids, humectants, and preservatives (any of which may be an
encapsulated ingredient as shown in FIG. 2). See, for example,
those representative components, combination of components,
relative amounts of those components and ingredients relative to
tobacco, and manners and methods for employing those components,
set forth in US Pat. Pub. Nos. 2011/0315154 to Mua et al. and
2007/0062549 to Holton, Jr. et al. and U.S. Pat. No. 7,861,728 to
Holton, Jr. et al., each of which is incorporated herein by
reference.
[0064] Exemplary flavorants that can be used are components, or
suitable combinations of those components, that act to alter the
bitterness, sweetness, sourness, or saltiness of the smokeless
tobacco product, enhance the perceived dryness or moistness of the
formulation, or the degree of tobacco taste exhibited by the
formulation. Flavorants may be natural or synthetic, and the
character of the flavors imparted thereby may be described, without
limitation, as fresh, sweet, herbal, confectionery, floral, fruity,
or spicy. Specific types of flavors include, but are not limited
to, vanilla, coffee, chocolate/cocoa, cream, mint, spearmint,
menthol, peppermint, wintergreen, eucalyptus, lavender, cardamon,
nutmeg, cinnamon, clove, cascarilla, sandalwood, honey, jasmine,
ginger, anise, sage, licorice, lemon, orange, apple, peach, lime,
cherry, strawberry, and any combinations thereof. See also,
Leffingwell et al., Tobacco Flavoring for Smoking Products, R. J.
Reynolds Tobacco Company (1972), which is incorporated herein by
reference. Flavorings also may include components that are
considered moistening, cooling or smoothening agents, such as
eucalyptus. These flavors may be provided neat (i.e., alone) or in
a composite (e.g., spearmint and menthol, or orange and cinnamon).
Representative types of components also are set forth in U.S. Pat.
No. 5,387,416 to White et al.; US Pat. App. Pub. No. 2005/0244521
to Strickland et al.; and PCT Application Pub. No. WO 05/041699 to
Quinter et al., each of which is incorporated herein by reference.
Types of flavorants include salts (e.g., sodium chloride, potassium
chloride, sodium citrate, potassium citrate, sodium acetate,
potassium acetate, and the like), natural sweeteners (e.g.,
fructose, sucrose, glucose, maltose, mannose, galactose, lactose,
and the like), artificial sweeteners (e.g., sucralose, saccharin,
aspartame, acesulfame K, neotame, and the like); and mixtures
thereof. The amount of flavorants utilized in the tobacco
composition can vary, but is typically up to about 10 dry weight
percent, and certain embodiments are characterized by a flavorant
content of at least about 1 dry weight percent, such as about 1 to
about 10 dry weight percent. Combinations of flavorants are often
used, such as about 0.1 to about 2 dry weight percent of an
artificial sweetener, about 0.5 to about 8 dry weight percent of a
salt such as sodium chloride and about 1 to about 5 dry weight
percent of an additional flavoring.
[0065] Exemplary filler materials include vegetable fiber materials
such as sugar beet fiber materials (e.g., FIBREX.RTM. brand filler
available from International Fiber Corporation), oats or other
cereal grain (including processed or puffed grains), bran fibers,
starch, or other modified or natural cellulosic materials such as
microcrystalline cellulose. Additional specific examples include
corn starch, maltodextrin, dextrose, calcium carbonate, calcium
phosphate, lactose, manitol, xylitol, and sorbitol. The amount of
filler, where utilized in the tobacco composition, can vary, but is
typically up to about 20 dry weight percent, and certain
embodiments are characterized by a filler content of up to about 10
dry weight percent, up to about 5 dry weight percent or up to about
1 dry weight percent. Combinations of fillers can also be used.
[0066] Typical binders can be organic or inorganic, or a
combination thereof. Representative binders include povidone,
sodium carboxymethylcellulose and other modified cellulosic
materials, sodium alginate, xanthan gum, starch-based binders, gum
arabic, pectin, carrageenan, pullulan, zein, and the like. The
amount of binder utilized in the tobacco composition can vary, but
is typically up to about 30 dry weight percent, and certain
embodiments are characterized by a binder content of at least about
5 dry weight percent, such as about 5 to about 30 dry weight
percent.
[0067] Preferred pH adjusters or buffering agents provide and/or
buffer within a pH range of about 6 to about 10, and exemplary
agents include metal hydroxides, metal carbonates, metal
bicarbonates, and mixtures thereof. Specific exemplary materials
include citric acid, sodium hydroxide, potassium hydroxide,
potassium carbonate, sodium carbonate, and sodium bicarbonate. The
amount of pH adjuster or buffering material utilized in the tobacco
composition can vary, but is typically up to about 5 dry weight
percent, and certain embodiments can be characterized by a pH
adjuster/buffer content of less than about 0.5 dry weight percent,
such as about 0.05 to about 0.2 dry weight percent.
[0068] A colorant may be employed in amounts sufficient to provide
the desired physical attributes to the tobacco formulation.
Exemplary colorants include various dyes and pigments, such as
caramel coloring, titanium dioxide, and curcumin. The amount of
colorant utilized in the tobacco composition can vary, but is
typically up to about 3 dry weight percent, and certain embodiments
are characterized by a colorant content of at least about 0.1 dry
weight percent, such as about 0.5 to about 3 dry weight
percent.
[0069] Exemplary humectants include glycerin and propylene glycol.
The amount of humectant utilized in the tobacco composition can
vary, but is typically up to about 5 dry weight percent, and
certain embodiments can be characterized by a humectant content of
at least about 1 dry weight percent, such as about 2 to about 5 dry
weight percent.
[0070] Other ingredients such as preservatives (e.g., potassium
sorbate) or disintegration aids (e.g., microcrystalline cellulose,
croscarmellose sodium, crospovidone, sodium starch glycolate,
pregelatinized corn starch, and the like) are typically used in
amounts of up to about 10 dry weight percent and usually at least
about 0.1 dry weight percent, such as about 0.5 to about 10 dry
weight percent. A disintegration aid is generally employed in an
amount sufficient to provide control of desired physical attributes
of the tobacco formulation such as, for example, by providing loss
of physical integrity and dispersion of the various component
materials upon contact of the formulation with water (e.g., by
undergoing swelling upon contact with water).
[0071] In certain embodiments, one or more substances are added to
the smokeless tobacco composition for the purpose of soothing
throat irritation that may develop during use of the product. The
throat irritation mitigant can include any substance capable of
mitigating or soothing irritation caused by the smokeless tobacco
product, and expressly includes counter-irritants, anesthetics, and
demulcents. Examples of throat irritation mitigants include sodium
citrate, honey, ginger, pectin, capsaicin, camphor,
dextromethorphan, echinacea, zinc gluconate, peppermint oil,
spearmint oil, eucalyptus oil, glycerin, organic acids (e.g.,
citric acid, lactic acid, levulinic acid, or succinic acid), and
combinations or extracts thereof (e.g., oleoresin ginger). Organic
acids function by shifting the pH of the product into the acidic
range, such as the pH range of about 3 to about 6.5. Typically, the
organic acid is an organic acid containing at least one carboxylic
acid group (e.g., carboxylic acids, dicarboxylic acids, and
tricarboxlic acids). The amount of throat irritation mitigant used
in the product can vary, but will be an amount sufficient to
provide some degree of relief for throat irritation (e.g., a
decrease in scratchy, lumpy sensations in the throat, a decrease in
pain upon swallowing, a decrease in hoarseness while speaking, or a
decrease in coughing). In exemplary embodiments, the throat
irritation mitigant is present in an amount of at least about 1
weight percent, or at least about 2 weight percent, or at least
about 3 weight percent (e.g., about 1 to about 10 weight
percent).
[0072] The acidity or alkalinity of the smokeless tobacco product,
which is often characterized in terms of pH, can vary. Typically,
the pH of that formulation is at least about 6.5, and preferably at
least about 7.5. Typically, the pH of that formulation will not
exceed about 9, and often will not exceed about 8.5. A
representative tobacco formulation exhibits a pH of about 6.8 to
about 8.2 (e.g., about 7.8). A representative technique for
determining the pH of a tobacco formulation involves dispersing 5 g
of that formulation in 100 ml of high performance liquid
chromatography water, and measuring the pH of the resulting
suspension/solution (e.g., with a pH meter).
[0073] Certain products of the present invention also can have
outer coatings (e.g., an outer coating can be composed of
ingredients such as carnauba wax and/or pharmaceutically acceptable
forms of shellacs, glazing compositions and surface polish agents).
Application of a coating can be accomplished using techniques such
as airless spraying, fluidized bed coating, use of a coating pan,
or the like. Materials for use as a coating can be polymeric in
nature, such as cellulosic material (e.g., cellulose butyrate
phthalate, hydroxypropyl methylcellulose phthalate, and
carboxymethyl ethylcellulose), and polymers and copolymers of
acrylic acid, methacrylic acid, and esters thereof.
[0074] The smokeless tobacco product can be packaged within any
suitable inner packaging material and/or outer container. See also,
for example, the various types of containers for smokeless types of
products that are set forth in U.S. Pat. No. 7,014,039 to Henson et
al.; U.S. Pat. No. 7,537,110 to Kutsch et al.; U.S. Pat. No.
7,584,843 to Kutsch et al.; D592,956 to Thiellier; D594,154 to
Patel et al.; and D625,178 to Bailey et al.; US Pat. Pub. Nos.
2008/0173317 to Robinson et al.; 2009/0014343 to Clark et al.;
2009/0014450 to Bjorkholm; 2009/0250360 to Bellamah et al.;
2009/0266837 to Gelardi et al.; 2009/0223989 to Gelardi;
2009/0230003 to Thiellier; 2010/0084424 to Gelardi; and
2010/0133140 to Bailey et al; 2010/0264157 to Bailey et al.;
2011/0168712 to Bailey et al.; and 2011/0204074 to Gelardi et al.,
which are incorporated herein by reference.
[0075] Although the focus of the invention is on combinations of
tobacco material with a separate botanical material, any of the
botanical materials set forth herein could also be incorporated
into various oral products without a tobacco component, such as in
non-tobacco pouched products, lozenges, films, tablets, gels,
sticks, and the like.
[0076] The following examples are provided to illustrate further
the present invention, but should not be construed as limiting the
scope thereof. Unless otherwise noted, all parts and percentages
are by weight.
EXPERIMENTAL
[0077] The present invention is more fully illustrated by the
following examples, which are set forth to illustrate the present
invention and are not to be construed as limiting thereof. In the
following examples, g means gram, L means liter, mL means
milliliter, and Da means daltons. All weight percentages are
expressed on a dry basis, meaning excluding water content, unless
otherwise indicated.
Example 1
[0078] Ten botanicals known for their antioxidant character and for
their reported potential health benefits are evaluated. The
botanicals evaluated are: green tea (Camellia sinensis), rosemary
(Rosmarinus officinalis), oregano (Origanum vulgare), sage (Salvia
officinalis), hibiscus flower (Hibiscus rosa), cloves (Syzygium
aromaticum), turmeric (Curcuma longa), rosehip (Rosa canina), yerba
mate (Ilex paraguariensis), and cocoa powder (Theobroma cacao). To
this list, a typical flue-cured tobacco blend (Nicotiana tabacum)
and a burley blend are added and analyzed by the same procedures as
the botanicals.
[0079] The ORAC and FRAP indices are measured for each of the
botanicals and the tobacco blends. The ORAC assay provides an index
which describes the degree and length of time in which each
botanical material evaluated is able to inhibit the action of an
oxidizing agent acting on a fluorescent substrate (expressed as TEs
or trolox equivalents). The FRAP index is based on the measurement
of reduction of Fe.sup.3+ to Fe.sup.2+ by the antioxidants
(expressed as Fe.sup.2+ concentration). These indices provide
guidance as to which botanicals upon addition to tobacco products
will have a higher potential to increase the antioxidant
characteristics of the product.
[0080] The ORAC assay is based on the measurement of the kinetics
of the oxidation reaction, as described above. The reagents used
for the assay are obtained as a kit from Cell Biolabs, Inc. (San
Diego, Calif., 92126, USA), but the same solutions can be made
using separately purchased chemicals. The kit contains 0.5 mL
fluorescein solution (100.times.), a sample of solid free radical
indicator, 0.1 mL of a 5 mM solution of Trolox, and 50 mL solution
of diluent (4.times.) at pH=7.25. The working solutions are about
20 mL of diluent solution 1:4 with deionized water; about 150 .mu.L
per sample evaluated of fluorescein solution made 1:100
fluorescein+diluent (this solution may be slightly adjusted to lead
to a 1000 LU fluorescence reading); 0.2 mM stock Trolox solution
made by 1/25 dilution of solution from the kit, wherein 7 dilutions
standards are prepared from this diluted Trolox stock solution; and
80 mg/mL solution of AAPH in phosphate buffer solution at pH=8.1
(about 250 .mu.L solution is made with approximately 20 mg
AAPH).
[0081] The calibration curve is generated as follows. 25 .mu.L of
Trolox calibration solution is placed in a well from a 96-well
clear bottom black Microtiter plate. To this solution 150 .mu.L of
fluorescein solution is added. The plate with several solutions in
cells is placed in the fluorescence measuring instrument. The
fluorescence measuring is performed with a Gemini XPS Fluorescence
Microplate Reader (Molecular Devices, Sunnyvale, Calif. 94089, USA)
with an extraction wavelength at 480 nm, and emission at 520 nm.
The software of the instrument is SoftMax Pro, which allows
multiple readings and measurement of areas under the curves. The
temperature of the solutions is set at 37.degree. C. The readings
for all plates are performed at intervals of 30 seconds for 20
mins. The readings of all standards should be around 1000 LU. After
20 mins, the plate is removed from the reading instrument and
allowed to cool at room temperature for about 5-10 mins. Following
cooling, about 25 .mu.L of AAPH solution is placed in every cell
with Trolox+fluorescein. Immediately, the 96 well plate is placed
in the Gemini XPS instrument, shook for 5 seconds. Upon reaction
with the peroxyl radicals produced by AAPH, the fluorescent
material yields a non-fluorescent product and the loss of
fluorescence is measured with the fluorimeter. The fluorescence
measurement is performed every 30 seconds for a period of 30
minutes. A graph is generated showing readings for a set of 7
standards showing the kinetics of the oxidation reaction. With the
areas from the standards, a calibration curve is generated which
plots area under the fluorescence curve versus Trolox
concentration. Due to the variability of the kinetic reaction, a
duplicate of standards is run together with the samples
evaluated.
[0082] ORAC values for each evaluated sample are measured by two
versions of the procedure described above, one indicated as
hydrophilic ORAC and the other as lipophilic ORAC. For the
hydrophilic version, the dilution is done with a phosphate buffer
solution at pH=7.2, while for the lipophilic versions the dilution
is performed with 50% acetone and 50% water (v/v). The phosphate
buffer solution with pH=7.2 and at a concentration 7.5 10.sup.-2
mol/L is prepared by mixing 64.2 mL Na.sub.2HPO.sub.4 solution with
35.8 mL solution KH.sub.2PO.sub.4. The solution of
Na.sub.2HPO.sub.4 is prepared by dissolving 13.363 g
Na.sub.2HPO.sub.4 2H.sub.2O in 1 L water, and the solution of
KH.sub.2HPO.sub.4 is prepared by dissolving 10.217 g
KH.sub.2HPO.sub.4 in 1 L water. The solution of fluorescein is
prepared at a concentration of 10.sup.-6 mol/L using double
dilution from solid fluorescein, and the stock trolox solution is
prepared at a concentration of 5 mmol/L from trolox. The
2,2-azobis(2-amidinopropane) dihydrochloride (AAPH) used is solid.
For the calibration of the hydrophilic ORAC, the stock solution of
Trolox is diluted with the phosphate buffer solution at seven
different levels. For the lipophilic ORAC the calibration
concentrations are identical with that for hydrophilic test, but
the dilution is performed using 50% acetone and 50% water
(v/v).
[0083] The preparation of samples for measurement of ORAC values is
performed as follows. First, 500 mg fine ground plant material is
extracted with 20 mL of a solution containing 50% acetone and 50%
water (v/v). The extraction is performed for 30 min on a wrist
action shaker (Burrell Co., Pittsburgh, Pa., USA). After
extraction, the liquid and solid are separated by filtration
through a 0.45 .mu.m pore PVDF Watman Autovial (Watman, Clifton,
N.J.). From the extract, 40 .mu.L are further diluted to 1.0 mL
with diluent. This solution is further diluted 20 times, by taking
10 .mu.L into 190 .mu.L diluent to generate 200 .mu.L, and thereby
bring responses within the calibration range. From this diluted
sample, 25 .mu.L are placed in a well on the 96 well plate, to
which 150 .mu.L of fluorescein is added and then incubated for
about 30 mins. To this solution, 25 .mu.L of 80 mg/mL AAPH solution
is added. Each sample is measured in duplicate. The ORAC value is
measured using the same procedure described above for the standard
samples.
[0084] The ORAC index values for each of the tested materials are
indicated below in Table 1. The ORAC index is expressed in TEs
(trolox equivalent mol/g botanical). The ORAC indices shown below
represent values obtained by the direct duplicate measurements
according to the procedure outlined above, and the relative
standard deviation between the measurements was in the range of
0.5% to 10%. As shown in Table 1, the ORAC hydro and ORAC lipo
numbers are similar for most evaluated botanicals, since the
initial extraction of the sample was performed for both cases in
acetone/water (50/50 v/v), and further dilution did not affect
solubility.
TABLE-US-00001 TABLE 1 ORAC values for botanicals and two tobacco
samples ORAC hydro ORAC lipo Material (.mu.mol TE)/g (.mu.mol TE)/g
Green tea dry leaf 1003 911 Rosemary 602 610 Oregano 637 716 Sage
rubbed 705 636 Hibiscus flower 312 319 Cloves 2616 2999 Turmeric
powder 410 590 Rose hip dry 1246 1498 Yerba mate 1043 1097 Cocoa
430 572 Flue-cured tobacco 471 419 Burley tobacco 134 146
[0085] The FRAP analysis is based on the reduction of a Fe.sup.3+
salt to a Fe.sup.2+ salt, and the reaction of Fe.sup.2+ with
2,4,6-tripyridyl-s-triazine which generates a colored complex. The
color of the complex (absorbance) is measured at 620 nm using a
Microplate Reader spectrophotometer SpectraMax 340 PC.sup.384
(Molecular Devices, Sunnyvale, Calif. 94089, USA). For the
analysis, the following reagents are prepared: 1) 300 mmol/L
acetate buffer at pH 3.6 made from 3.1 g
CH.sub.3COONa.times.3H.sub.2O and 16 mL CH.sub.3COOH in 1.0 L
water, 2) 10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine) and 40
mmol/L HCl, made from 312.33 mg TPTZ and 146 mg HCl for 100 mL
solution, 3) 20 mmol/L FeCl.sub.3 6H.sub.2O made from 540.59 mg
FeCl.sub.3 6H.sub.2O for 100 mL solution. A working FRAP reagent is
prepared by mixing 10 mL acetate buffer with 1 mL TPTZ solution and
1 mL FeCl.sub.3 solution. The working FRAP reagent is made daily
(for utilization). The calibration of FRAP measurement is made
using a set of standards made from a stock solution of FeSO.sub.4
7H.sub.2O in the range 100 to 2000 .mu.mol/L. The stock solution in
this study is made from 56.82 mg FeSO.sub.4 7H.sub.2O in 100 mL
solution in water, and corresponded to 2044 .mu.mol/L Fe.sup.2+.
From this solution, standards containing 2044 .mu.mol/L, 1533
.mu.mol/L, 1022 .mu.mol/L, 511 .mu.mol/L, 255.5 .mu.mol/L, and
127.8 .mu.mol/L are prepared by dilution with water. For the
measurement, 200 .mu.L of reagent is added to each sample cell of a
96 well plate. The plate is placed in the SpectraMax 340 PC.sup.384
instrument, is shaken for 10 seconds, and allowed to equilibrate at
37.degree. C. for 10 minutes. After the equilibration, a 10 .mu.L
sample (or standard) is added to each cell and the absorbance is
measured every 30 seconds for a period of 10 minutes at 620 nm.
[0086] The FRAP index values for each of the analyzed materials are
indicated in Table 2. The FRAP index is expressed in .mu.mol
Fe.sup.2+/g.
TABLE-US-00002 TABLE 2 FRAP values for botanicals and two tobacco
samples FRAP average Material (.mu.mol Fe.sup.2+)/g SD (.mu.mol
Fe.sup.2+)/g) Green tea dry leaf 1799 28.3 Rosemary dry 788 61.1
Oregano leaf 862 45.9 Sage rubbed 710 212.9 Hibiscus flower 367 1.5
Cloves 4647 151.8 Turmeric powder Not analyzed* -- Rose hip dry
1545 54.8 Yerba mate 1113 61.1 Cocoa powder 670 25.3 Flue-cured
tobacco 377 5.1 Burley tobacco 90 5.5 *The yellow color of turmeric
interferes with the analysis
[0087] The ten botanicals known for their antioxidant character and
for their potential health benefits are also evaluated using a
GC/MS scanning technique. For comparison with the evaluated
botanicals, two tobaccos are included and evaluated regarding the
same parameters as the other botanicals. The types of antioxidant
compounds predominantly found in each botanical and tobacco blend
is described below.
[0088] The scanning technique consists of direct silylation of the
sample followed by a GC/MS analysis of the extract. For the
analysis, duplicate samples of 50 mg were weighed (with 0.1 mg
precision) in GC vials (2 mL screw top vials with septa, Agilent,
Wilmington, Del. 19808). The silylation is done to all the
compounds containing active hydrogens, such as acids, alcohols, or
amines. The result is the formation of various trimethylsilyl (TMS)
derivatives.
[0089] A reagent and a solvent are used for the silylation process.
The reagent is bis(trimethylsilyl)-trifluoroacetamide (BSTFA) with
1% trimethylchlorosilane (TMCS). The solvent is
N,N-dimethylformamide (DMF). The solvent also contains as internal
standard tert-butylhydroquinone. The DMF solution with internal
standards is prepared using 100 mL DMF and 40 mg
tert-butylhydroquinone (all compounds from Aldich/Sigma Saint
Louis, Mo. 63178-9916). The final DMF solution contains 0.4 mg/mL
tert-butylhydroquinone.
[0090] For the analysis, 0.4 mL DMF with internal standards and 0.8
mL BSTFA with 1% TMCS are added to each vial. The vials are kept at
78.degree. C. (in a heating block) for about 30 mins. The vials are
then allowed to cool at room temperature for another 30 mins. After
cooling, the solution from each vial is filtered through a 0.45
.mu.m PTFE filter (VWR Suwanee, Ga. 30024) into a screw top vials
with screw caps with septa and used for the GC/MS analysis. The
analysis is done using a GC/MS instrument (such as Agilent
6890/5973 system from Agilent, Wilmington, Del. 19808). Specific
modifications are implemented to the initial procedure for
extending the analysis to heavier molecules.
[0091] The peaks in the chromatograms are characterized by their
retention time, mass spectrum and peak areas. The retention time
and the mass spectrum are utilized for peak identification. Mass
spectral libraries useful for search include Nist8 and Wiley275. A
few individual compounds are identified based on comparison with
standards. The area count of each peak is measured normalized to
the peak area of an internal standard (tertbutylhydroquinone). This
type of presentation of results does not provide the quantitative
level of different compounds in a sample, but still allows a
quantitative comparison indicating which compound is at a higher or
a lower level in the samples. The chemical compounds targeted as
antioxidants and analyzed in various botanicals by this GC/MS
scanning technique do not include molecules with higher molecular
weight (>500 MW) such as polymeric antioxidants. Such molecules
are analyzed using dedicated HPLC methods.
[0092] The GC/MS scan profile of silylated green tea dry leaf
confirms the presence of the following characteristic antioxidants
expected to be found in green tea:
TABLE-US-00003 Menthol (15.87 min) 3-Chlorogenic acid (68.11 min)
Caffeine (37.79 min) 5-Chlorogenic acid (69.60 min) Gallic acid
(42.23 min) A-Tocoferol (68.05 min) Caffeic acid (47.60 min)
Epicatechin gallate (78.14 min) Epicatechin (63.83 min) Catechin
gallate (78.32 min) Catechin (64.32 min) Epigallocatechin gallate
(78.83 min) Epigallocatechin (65.18 min) Gallocatechin gallate
(79.40 min) Coumaroyl quinic acid (67.41 min)
[0093] The GC/MS scan profile of silylated rosemary confirms the
presence of the following characteristic antioxidants expected to
be found in rosemary:
TABLE-US-00004 Camphor (11.09 min) Rosmanol (60.19 min) Borneol
(14.21 min) Rosmarinic acid (72.69 min) Thymol (18.64 min)
Oleanolic acid (74.02 min) Ascorbic acid (41.71 min) Betulinic acid
(74.35 min) Rosmaricin (55.68 min) Ursolic acid (74.77 min)
Carnosic acid (57.54 min) Betulonic acid (75.23 min) Carnosol
(58.90 min)
[0094] It is noted that trace amounts of gallic acid (42.22 min)
and tocoferol (68.05 min) are also found. Caffeic acid (47.60 min)
is present at higher levels than green tea. Also, when comparing
the chromatograms for green tea and rosemary, there are significant
differences which are in particular related to the antioxidants in
the two botanicals. Specifically, green tea contains catechins
where as rosemary contains more acids with antioxidant properties.
In addition, rosemary contains lighter compounds that contribute to
the strong flavor of rosemary, such as camphor, borneol and
thymol.
[0095] The GC/MS scan profile of silylated oregano confirms the
presence of the following characteristic antioxidants expected to
be found in oregano:
TABLE-US-00005 Borneol (14.19 min) Coumaroyl quinic acid (67.41
min) Menthol (15.87 min) Chlorogenic acid (68.11 min) Thymol (18.64
min) Rosmarinic acid (72.65 min) Caffeine (37.84 min) Oleanolic
acid (73.90 min) Gallic acid (42.22 min) Betulinic acid (74.23 min)
Caffeic acid (47.61 min) Ursolic acid (74.65 min) Carnosic acid
(57.52 min) Betulonic acid (75.31 min) Epicatechin (63.83 min)
Epicatechin gallate (78.04 min) Catechin (64.32 min) Catechin
gallate (78.85 min) Epigallocatechin (65.19 min) Epigallocatechin
gallate (78.85 min)
[0096] Oregano leaf contains a wide range of antioxidants, some of
them at relatively low level. Thymol is in particular elevated in
oregano leaf, which contributes significantly to its flavor.
[0097] The GC/MS scan profile of silylated sage confirms the
presence of the following characteristic antioxidants expected to
be found in sage:
TABLE-US-00006 Camphor (11.06 min) Carnosol (58.94 min) Borneol
(14.19 min) Rosmanol (60.19 min) Thymol (18.64 min) Rosmarinic acid
(72.64 min) Caffeine (37.84 min) Oleanolic acid (73.97 min) Gallic
acid (42.22 min) Betulinic acid (74.23 min) Caffeic acid (47.61
min) Ursolic acid (74.72 min) Rosmaricin (55.67 min) Betulonic acid
(75.13 min) Carnosic acid (57.50 min)
[0098] Sage also contains some sabinol and other volatile compounds
typical for essential oils that impart the specific flavor of
sage.
[0099] The GC/MS scan profile of silylated hibiscus flower (dry)
shows several peaks not common to other botanicals evaluated here.
These peaks belong to several polyfunctional hydroxyl acids. As
reflected in its relatively low ORAC index above, hibiscus flower
does not have a significant level of antioxidants. The dry flowers
contain sugars (fructose, glucose) and some acids such as citric
quinic acid, and other hydroxyl acids. Only a low level of ascorbic
acid and some chlorogenic acid were identified as contributors to
the antioxidant properties of this material. However, the flower
contains some anthocyanidins that are not possible to analyze by
the scanning technique described above. These anthocyanidins
include delphinidin and cyanidin.
[0100] The GC/MS scan profile of silylated cloves confirms the
presence of the following characteristic antioxidants expected to
be found in cloves:
TABLE-US-00007 Caffeine (37.84 min) Rosmarinic acid (72.65 min)
Gallic acid (42.22 min) Oleanolic acid (74.03 min) Caffeic acid
(47.61 min) Betulinic acid (74.23 min) Coumaroyl quinic acid (67.41
min) Ursolic acid (74.65 min) Chlorogenic acid (68.11 min)
Betulonic acid (75.31 min)
[0101] Cloves contain a significant proportion of eugenol (24.76
min), and also have a high level of gallic acid and of betulinic
acid. These compounds together, having a strong antioxidant
character, lead to a high ORAC and FRAP value for cloves.
[0102] The GC/MS scan profile of silylated tymeric (ground) shows
that turmeric does not contain a significant level of antioxidants,
as compared to other botanicals evaluated in this example. A low
level of several flavorful volatile compounds such as camphor and
thymol are present in turmeric and these may contribute some
antioxidant character. Also, a very low level of betulinic acid is
present. Different from other botanicals evaluated here, turmeric
contains some oxalic acid (11.08 min), ar-turmerone (31.82 min),
and curcumin (75.31 min) Tumerone is reported to have some
antioxidant properties. Curcumin is a polyphenol which also has
antioxidant properties and is responsible for the yellow color of
turmeric.
[0103] The GC/MS scan profile of silylated rose hip (dry ground)
confirms the presence of the following characteristic antioxidants
expected to be found in rose hip:
TABLE-US-00008 Ascorbic acid (41.71 min) Coumaroyl quinic acid
(67.41 min) Gallic acid (42.28 min) .alpha.-Tocoferol (68.03 min)
Caffeic acid (trace) (47.61 min) Oleanolic acid (74.03 min)
Epicatechin (trace) (63.83 min) Betulinic acid (74.23 min) Catechin
(64.32 min) Ursolic acid (74.65 min)
[0104] The ORAC and FRAP indices for rose are relatively high,
indicating good antioxidant properties. These high values are
likely generated by the presence of ascorbic acid, oleanolic acid
and betulinic acid, all present in the dry ground material. The
main peaks shown in the chromatogram of silylated rose hip belong
to carbohydrates (glucose, fructose, and sucrose).
[0105] The GC/MS scan profile of silylated yerba mate confirms the
presence of the following characteristic antioxidants expected to
be found in yerba mate:
TABLE-US-00009 Caffeine (37.84 min) Coumaroyl quinic acid (67.41
min) Ascorbic acid (trace) (41.71 min) Chlorogenic acid (68.11 min)
Gallic acid (42.22 min) Chlorogenic acid (2) (73.97 min) Caffeic
acid (47.61 min) Oleanolic acid (73.97 min) Epicatechin (63.83 min)
Betulinic acid (74.23 min) Catechin (64.32 min) Ursolic acid (74.63
min)
[0106] Yerba mate does not have a profile similar to green tea,
which also contains a considerable amount of catechins Yerba mate
does have a considerable level of chlorogenic acid.
[0107] The chromatogram of silylated cocoa powder shows trace
levels of caffeine (37.84 min), gallic acid (42.22 min),
epicatechin (63.80 min), and an additional class of antioxidants
known as leucoanthocyanidins. These compounds elute in the
separation used herein between 80 and 86 min and include
theobromine, theophiline, and leucocyanidin gallate.
Leucoanthocyanidins also form proanthocyanidin polymers in which
different units of the flavan groups are connected by bonds. These
compounds have antioxidant properties; however, the larger
molecules cannot be analyzed by the GC/MS scanning technique
described above.
[0108] For comparison purposes, flue-cured tobacco and burley
tobacco are also analyzed. Flue-cured tobacco contains a higher
lever of antioxidants, including in particular chlorogenic acid
(68.12 min) and some .alpha.-tocopherol. This explains the lower
ORAC and FRAP indices for burley tobacco compared to flue-cured
tobacco. Both tobacco materials have less antioxidant
characteristics than the botanicals evaluated.
Example 2
[0109] The antioxidant profiles of guayusa leaves are evaluated to
determine their potential use in tobacco products. Ilex guayusa is
a botanical with antioxidant properties. The plant is grown
primarily in Ecuador, but it is also found in parts of Peru and
Colombia. Traditionally, guayusa leaves are boiled in water and the
resulting beverage is consumed for its stimulative effects, likely
due to the content in caffeine and theobromine. Some studies have
shown that ingesting guayusa helped reduce hyperglycemia and other
side-effects of Type 1 diabetes in animal models.
[0110] The antioxidant character of the guayusa leaves as described
by ORAC and FRAP indices is evaluated and compared to that of green
tea. The measurement of ORAC index is performed following a
procedure described in Example 1. The ORAC index values for guayusa
and for green tea are indicated below in Table 3. The ORAC index is
expressed in TEs (trolox equivalent .mu.mol/g botanical).
TABLE-US-00010 TABLE 3 ORAC values for Guayusa and for Green Tea
Guayusa Guayusa Green Tea Green Tea (hydrophilic) (lipophilic)
(hydrophilic) (lipophilic) Average 562.20 596.68 925.18 872.06
Standard 0.85 5.08 43.48 87.45 Deviation RSD % 0.152 0.851 4.700
10.028
[0111] The FRAP index is measured according to the procedure
described in Example 1. The FRAP index values for guayusa and green
tea are indicated in Table 4. The FRAP index is expressed in mol
Fe.sup.2+/g botanical.
TABLE-US-00011 TABLE 4 FRAP values for Guayusa and for Green Tea
Guayusa Green Tea Average 603.40 1651.56 Standard Deviation 76.88
116.74 RSD % 12.740 7.068
[0112] In addition, the CG/MS scanning technique, as described in
Example 1, provides information about the nature and relative level
of specific antioxidants found within guayusa leaves. The GC/MS
scan profile of guayusa is very different from that of green tea,
however, the profile of compounds in guayusa was similar to that of
yerba mate, which is also an ilex (Ilex paraguariensis). Among the
main antioxidants in guayusa and also present in yerba mate are the
following:
TABLE-US-00012 Caffeine (37.84 min) 2-Chlorogenic acid (68.11 min)
Ascorbic acid (trace) (41.71 min) 5-Chlorogenic acid (69.60 min)
Gallic acid (42.22 min) Oleanolic acid (trace) (73.97 min) Caffeic
acid (47.55 min) Betulinic acid (trace) (74.23 min) Epicatechin
(trace) (63.83 min) Ursolic acid (74.63 min)
[0113] In summary, similar to yerba mate, guayusa has a significant
level of the two chlorogenic acids (3-Chlorogenic acid and
5-Chlorogenic acid). Theobromine reported to be in guayusa, was not
identified in the chromatogram. The level of caffeine in guayusa
and yerba mate is about the same. Guayusa has less antioxidant
character as compared to green tea. The chromatographic profile of
silylated guayusa is different from that of green tea, indicating
different compounds contributing to the antioxidant character.
Example 3
[0114] The antioxidant profile of rooibos (Aspalathus linearis) is
evaluated to determine potential use in tobacco products. Rooibos
grows in the region of the Western Cape province of South Africa.
The leaves are used in a tisane (herbal tea) called bush tea or
simply rooibos. Generally, the leaves are subjected to a
fermentation that produces a reddish-brown color (red rooibos) and
enhanced flavor. Green or unfermented rooibos has a slightly grassy
flavor.
[0115] The antioxidant character of green and red rooibos as
described by ORAC and FRAP indices is evaluated. The measurement of
ORAC index is performed following a procedure described in Example
1. The ORAC index values for green and red rooibos are indicated
below in Table 5. The ORAC index is expressed in TEs (trolox
equivalent .mu.mol/g botanical).
TABLE-US-00013 TABLE 5 ORAC values for Green and Red Rooibos Green
rooibos Green rooibos Red rooibos Red rooibos (hydrophilic)
(lipophilic) (hydrophilic) (lipophilic) Average 1035.51 793.58
629.68 547.85 Standard 59.61 40.89 44.32 26.78 Deviation RSD % 5.76
5.15 7.04 4.89
[0116] The FRAP index is measured according to the procedure
described in Example 1. The FRAP index values for green and red
rooibos are indicated in Table 6. The FRAP index is expressed in
.mu.mol Fe.sup.2+/g botanical.
TABLE-US-00014 TABLE 6 FRAP values for Green and Red Rooibos Green
rooibos Red rooibos Average 1570.37 862.95 Standard Deviation 88.56
170.13 RSD % 5.64 19.71
[0117] As can be seed, green rooibos has larger ORAC values and
FRAP values than red rooibos. The apparent loss of antioxidant
character of red rooibos is analogous to the loss of antioxidant
character of black tea compared to green tea. The ORAC and FRAP
values of rooibos are very similar to green tea.
[0118] In addition, the CG/MS scanning technique, as described in
Example 1, provides information about the nature and relative level
of specific antioxidants and other compounds found within rooibos
leaves. Among the main compounds found in rooibos are the
following: [0119] Malic acid (25.71 min) [0120] I.S. (29.61 min)
[0121] Inositol type (37.90 min) [0122] Inositol (45.85 min) [0123]
Sucrose (59.69 min) [0124] Aspalathin (77.72 min).
Example 4
[0125] The antioxidant profile of honeybush leaves (fermented) is
evaluated to determine their potential use in tobacco products.
Honeybush (Cyclopia) is a genus of flowering plants in the legume
family, and its leaves have antioxidant properties. Several species
of Cyclopia grow in the southwest and southeast of South Africa.
The leaves of honeybush are commonly used to make tisanes or herbal
teas. The plant has many similarities with rooibos and it is
available as green and fermented. The fermentation process is an
enzymatic oxidation caused by the plant's intracellular enzymes,
which reduces in part the ORAC value of the leaves. The honeybush
evaluated here is fermented, but the specific species of plant is
unknown. The ORAC and FRAP values as compared with green tea are
not very high. The antioxidant character of honeybush is produced
by a variety of antioxidant compounds. Unfermented honeybush is
reported to be low in tannin (0.45%), but to contain isofavones,
flavones, cinnamic acids, coumestans, xanthonoids, mangiferin and
isomangiferin. Most of these compounds cannot be evaluated by the
GC/MS method used herein. Therefore, it is not determined which of
these compounds is still active in the fermented honeybush.
[0126] The antioxidant character of the honeybush leaves as
described by ORAC and FRAP indices is evaluated and compared to
that of green tea. The measurement of ORAC index is performed
following a procedure described in Example 1. The ORAC index values
for honeybush and for green tea are indicated below in Table 7. The
ORAC index is expressed in TEs (trolox equivalent .mu.mol/g
botanical).
TABLE-US-00015 TABLE 7 ORAC values for Honeybush and for Green Tea
Honeybush Honeybush Green Tea Green Tea (hydrophilic) (lipophilic)
(hydrophilic) (lipophilic) Average 278.60 300.05 882.29 986.29
Standard 32.42 17.76 51.97 25.38 Deviation RSD % 11.64 5.92 5.89
2.57
[0127] The FRAP index is measured according to the procedure
described in Example 1. The FRAP index values for honeybush and
green tea are indicated in Table 8. The FRAP index is expressed in
.mu.mmol Fe.sup.2+/g botanical.
TABLE-US-00016 TABLE 8 FRAP values for Honeybush and for Green Tea
Honeybush Green Tea Average 340.89 1578.73 Standard Deviation 84.89
392.10 RSD % 24.81 24.84
[0128] In addition, the CG/MS scanning technique, as described in
Example 1, provides information about the nature and relative level
of specific antioxidants and other compounds found within honeybush
leaves. Among the main compounds found in honeybush are the
following:
TABLE-US-00017 Lactic acid (8.05 min) Xylulose (39.97 min) I.S.
(29.60 min) Hexose (43.16 min) Pentose-1 (30.98 min) Glycoside
(58.14 min) Hydrocarbon (33.10 min) Unknown (65.20 min) Inositol
type (37.86 min) Unknown antioxidant glycoside (87.76 min)
[0129] Besides some of the carbohydrates, the compounds seen in the
chromatogram do not match the antioxidants expected to be found in
honeybush, as indicated in the available literature. Furthermore,
the specific species of honeybush plant evaluated herein shows less
antioxidant characteristics as compared to green tea.
Example 5
[0130] The antioxidant properties for an experimental snus with
green tea, a pouched product containing microcrystalline cellulose
(MCC) and green tea, and a snus control are evaluated. The green
tea is a commercial product obtained from the market (Shanghai
Tiantan Intern. Trading Co., Ltd.). The experimental snus with
green tea sample contains about 16.79% dry weight basis milled
green tea. The pouched material with MCC contains about 41.55%
milled green tea. The basic ingredients of the snus control include
milled lamina+milled stem; salt, Na.sub.2CO.sub.3, NaHCO.sub.3;
sucralose and propylene glycol. The experimental snus with green
tea is made by replacing part of the milled lamina and stem in the
control with green tea, without significantly changing the
proportion of other components in the product. The basic
composition of the pouched material is intended to keep the level
of MCC plus green tea at a somewhat equivalent dry weight percent
level with that of tobacco materials plus green tea within the snus
with green tea sample.
[0131] The ORAC values and FRAP values for each sample are measured
using the same procedures described in Example 1 above. The ORAC
values for the samples are given in Table 9 below. The ORAC index
is expressed in TEs (trolox equivalent .mu.mol/g botanical).
TABLE-US-00018 TABLE 9 ORAC values for 3 samples as well as green
tea alone Pouched Suns with Material with Green Tea Green Tea Snus
Control Green Tea Average 178.14 456.33 32.52 1182.67 RSD % 6.38
3.29 6.06 --
[0132] The FRAP index values for samples and green tea are
indicated in Table 10. The FRAP index is expressed in .mu.mol
Fe.sup.2+/g botanical.
TABLE-US-00019 TABLE 10 FRAP values for 3 samples and for green tea
alone Pouched Suns with Material with Green Tea Green Tea Snus
Control Green Tea Average 337.70 1071.17 65.23 2488.54 RSD % 0.64
18.44 10.54 --
[0133] In addition, the GC/MS scanning technique described in
Example 1 above is used for the evaluation of the relative level of
several antioxidant compounds in the three samples and to compare
these levels with that from the green tea "as is." The evaluation
is done based on the normalized area counts of the peaks generated
by the main antioxidant compounds in the chromatographic scans
applied on each material. The normalization is performed using the
area of an internal standard (400 ppm of
tertbutylhydroquinone).
[0134] The results of the normalized area counts in the analyzed
samples show that the proportion of different antioxidant compounds
follows the proportion of green tea in the corresponding product.
In summary, the addition of green tea significantly increases the
antioxidant properties of the sampled products.
Example 6
[0135] The changes in antioxidant properties for an experimental
snus blend upon the addition of a small proportion of two
botanicals (green tea leaf and freeze-dried rosemary) is evaluated.
The ORAC values for snus samples incorporating three levels of
freeze dried rosemary (5%, 10% and 20% dry weight basis), snus
samples incorporating three levels of green tea (5%, 10% and 20%
dry weight basis), and a control snus sample without any botanical
additives were measured. The ORAC values for each sample are
measured using the same procedures described in Example 1
above.
[0136] The ORAC values for the samples are given in Table 11 below.
The ORAC index is expressed in TEs (trolox equivalent .mu.mol/g
botanical).
TABLE-US-00020 TABLE 11 ORAC values for snus tobacco samples mixed
with freeze dried rosemary or with green tea ORAC Average Standard
Deviation (.mu.M TE)/g (.mu.M TE) Freeze Dried Rosemary Proportion
0% 45.9 2.2 5% 73.4 6.0 10% 86.8 6.6 20% 120.6 4.0 Green Tea
Proportion 0% 41.0 3.7 5% 70.8 10.6 10% 91.1 6.3 20% 145.0 4.1
[0137] The addition of green tea as well as the addition of
rosemary to a snus tobacco blend leads to an increase in the ORAC
values. Furthermore, the variation in the ORAC values shows a
linear dependence on the proportion of botanical product added to
the product.
[0138] In addition to the measurement of ORAC values, a GC/MS
screening according to the procedure described in Example 1 above
is performed on the snus samples with either green tea or rosemary
added. The chromatography results for the samples indicate the
presence of typical tobacco compounds such as sugars, tobacco
specific acids, nicotine, phosphate, etc. as well as compounds with
expected antioxidant properties specific to green tea or rosemary.
The results also show that the level of specific compounds present
in the added botanicals can be increased in the composite
material.
Example 7
[0139] Tobacco products may require to be heated at specific
temperatures (often below 100.degree. C.). Also, in order to assure
that the materials added to tobacco products are free from
bacteria, the botanicals may be required to be heated at
temperatures around 100.degree. C. Therefore, the following
non-limiting example determines potential changes in the
antioxidant properties of green tea and of dry rosemary upon
heating for one hour at three different temperatures: 76.degree.
C., 93.degree. C. and 110.degree. C.
[0140] Samples of 2-3 g of green tea and dry rosemary are weighed
in different beakers. Each sample is heated at a different
temperature for one hour. The three temperatures used are
76.degree. C., 93.degree. C. and 110.degree. C. After heating, the
beakers are covered with aluminum foil and allowed to cool to room
temperature. From each sample, the amounts required for each
analysis are precisely weighed in duplicate. The ORAC values and
FRAP values for each sample are measured using the same procedures
described in Example 1 above.
[0141] The ORAC values for the samples are given in Tables 12 and
13 below. The ORAC index is expressed in TEs (trolox equivalent
.mu.mol/g botanical).
TABLE-US-00021 TABLE 12 ORAC values for green tea samples at
different temperatures Room Temperature 76.degree. C. 93.degree. C.
110.degree. C. Average 1182.67 1121.29 1161.24 1241.77 RSD % --
6.91 5.18 1.04
TABLE-US-00022 TABLE 13 ORAC values for rosemary samples at
different temperatures Room Temperature 76.degree. C. 93.degree. C.
110.degree. C. Average 629.92 878.75 899.06 1017.29 RSD % -- 12.54
12.19 5.00
[0142] The FRAP index values for the samples are indicated in
Tables 14 and 15 below. The FRAP index is expressed in .mu.mol
Fe.sup.2+/g botanical.
TABLE-US-00023 TABLE 14 FRAP values for green tea samples at
different temperatures Room Temperature 76.degree. C. 93.degree. C.
110.degree. C. Average 2488.54 2208.51 2382.78 2415.20 RSD % --
1.82 3.85 4.27
TABLE-US-00024 TABLE 15 FRAP values for rosemary samples at
different temperatures Room Temperature 76.degree. C. 93.degree. C.
110.degree. C. Average 998.57 991.82 1185.39 1097.68 RSD % -- 10.35
0.72 8.96
[0143] As seen from Tables 12-15, the ORAC and FRAP values for
either green tea or dry rosemary when heated up to 110.degree. C.
show very little change. In some instances, it even appears that an
increase in the ORAC and FRAP values is seen for the heated
samples. It is possible these increases are a result of variation
in the analytical measurements.
[0144] Levels of the specific antioxidant compounds found in the
room temperature and heated green tea and rosemary samples are
evaluated using the GC/MS scanning technique described above in
Example 1. The evaluation is done based on the normalized area
counts of the peaks generated by the main antioxidant compounds in
the chromatographic scans applied on each material. The
normalization is performed using the area of an internal standard
(400 ppm of tertbutylhydroquinone).
[0145] The results show that heating the samples does not
significantly change the levels of most antioxidant compounds found
within green tea or dry rosemary. Only for rosemary, the level of
carnosic acid seems to decrease upon heating at 110.degree. C. Also
in rosemary, the levels of the compounds rosmaricin and rosmarinic
acid are slightly decreased when the sample is heated at
110.degree. C. for one hour. Overall, the heating of the samples
does not significantly affect the antioxidant properties of the
analyzed botanicals.
[0146] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
* * * * *