U.S. patent application number 10/053237 was filed with the patent office on 2002-10-24 for process for extracting compounds from plants.
Invention is credited to Krasutsky, Pavel A., Nesterenko, Vitaliy V..
Application Number | 20020155177 10/053237 |
Document ID | / |
Family ID | 26929917 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155177 |
Kind Code |
A1 |
Krasutsky, Pavel A. ; et
al. |
October 24, 2002 |
Process for extracting compounds from plants
Abstract
The present invention provides a method for selectively
extracting acidic and/or non-acidic compounds from natural material
such as plant tissue.
Inventors: |
Krasutsky, Pavel A.;
(Duluth, MN) ; Nesterenko, Vitaliy V.; (Duluth,
MN) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Family ID: |
26929917 |
Appl. No.: |
10/053237 |
Filed: |
January 17, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10053237 |
Jan 17, 2002 |
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09969130 |
Oct 1, 2001 |
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60236579 |
Sep 29, 2000 |
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Current U.S.
Class: |
424/725 |
Current CPC
Class: |
B01D 11/0219 20130101;
B01D 11/0288 20130101; C11B 1/10 20130101; B01D 11/0284 20130101;
B01D 11/0203 20130101 |
Class at
Publication: |
424/725 |
International
Class: |
A61K 035/78 |
Claims
What is claimed is:
1. A method for selectively extracting one or more non-acidic
compounds from plant tissue in the presence of one or more acidic
compounds, the method comprising: (a) contacting a mixture of a
basic component and a first solvent with the plant tissue to
immobilize the acidic compound as a salt on the plant tissue; and
(b) contacting the plant tissue with a second solvent suitable to
remove the one or more non-acidic compounds; thereby effectively
providing a solution comprising the one or more non-acidic
compounds.
2. The method of claim 1, wherein the basic component comprises an
alcoholate.
3. The method of claim 2, wherein the alcoholate is an aluminum
alcoholate.
4. The method of claim 3, wherein the aluminum alcoholate is
aluminum iso-propoxide.
5. The method of claim 3 wherein the aluminum alcoholate is
aluminum ethoxide or aluminum methoxide.
6. The method of claim 1 wherein the basic component comprises an
amine of formula NR.sub.3, wherein each R is independently
hydrogen, (C.sub.1-C.sub.12)alkyl, aryl (e.g., phenyl), or
arylalkyl (e.g., benzyl), wherein each alkyl, aryl (e.g., phenyl),
or arylalkyl (e.g., benzyl) can be optionally substituted on carbon
with one or more hydroxy, halo, or --N(R.sub.b).sub.2; wherein
R.sub.b is H, (C.sub.1-C.sub.6)alkyl, aryl (e.g., phenyl), or
arylalkyl (e.g., benzyl).
7. The method of claim 1 wherein the basic component comprises a
heterocycle.
8. The method of claim 7 wherein the heterocycle is pyridine,
morpholine, piperidine, pyrrole, or pyrrolidine; each optionally
subtituted on any suitable carbon with oxo, hydroxy, sulfo,
(C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)hydroxyalkyl, or
--N(R.sub.b).sub.2, wherein R.sub.b is H or (C.sub.1-C.sub.4)alkyl,
or on nitrogen with (C.sub.1-C.sub.4)alkyl or
(C.sub.1-C.sub.4)hydroxyalkyl.
9. The method of claim 1 wherein the basic component comprises an
alkaline earth metal hydroxide.
10. The method of claim 9 wherein the basic component is NaOH, KOH,
LiOH, Mg(OH).sub.2, Ca(OH).sub.2, or a mixture thereof.
11. The method of claim 1 wherein the basic component comprises an
alkaline earth metal oxide.
12. The method of claim 1 wherein the basic component comprises an
alkaline earth metal carbonate or an alkaline earth metal
bicarbonate.
13. The method of claim 12 wherein the basic component is
Na.sub.2CO.sub.3, K.sub.2CO.sub.3, KNaCO.sub.3, Li.sub.2CO.sub.3,
CaCO.sub.3, MgCO.sub.3, or a mixture thereof.
14. The method of claim 1 wherein the basic component comprises an
alkaline earth metal sulfite or alkaline earth metal sulfide.
15. The method of claim 1, wherein the first solvent and the second
solvent are each independently an optionally substituted aromatic
compound, an optionally substituted heterocyclic compound, an
optionally substituted cyclic compound, an optionally substituted
linear or branched compound, or combination thereof, wherein
suitable substituents include (C.sub.1-C.sub.6)alkyl, hydroxyl,
halo, cyano, nitro, oxo, thioxo, amino, carboxyl, or combinations
thereof.
16. The method of claim 1, wherein the first solvent and the second
solvent are each independently isopropanol, ethanol, methanol,
methylene chloride, toluene, o-xylene, m-xylene, p-xylene, carbon
dioxide, Xe, Freon-23, ethane, N.sub.2O, SF.sub.6, propane,
ammonia, n-C.sub.4H.sub.10, (C.sub.2H.sub.5).sub.2O, or a
combination thereof.
17. The method of claim 1, wherein the first solvent, the second
solvent, or a combination thereof, comprises an additive.
18. The method of claim 17 wherein the additive is methanol;
ethanol; 1-propanol; 2-propanol; 1-hexanol; 2-methoxy ethanol;
tetrahydrofuran; 1,4-dioxane; acetonitrile; dichloromethane;
ammonia; chloroform; propylene carbonate; N,N-dimethylacetamide;
dimethyl sulfoxide; formic acid; water; carbon disulfide; acetone;
propane; toluene; hexanes; pentanes; o-xylene; m-xylene; p-xylene;
toluene; or a combination thereof.
19. The method of claim 1 wherein the plant tissue comprises bark,
roots, leaves, flowers, needles, bulbs, berries, rhizomes,
rootstocks, stems, seeds, or any combination thereof.
20. The method of claim 1 wherein the plant tissue comprises Taxus
yunnanesis bark, yew tree needles, Echinacea spp. root, Ginkgo
biloba root bark, Ginkgo biloba leaves; Allium sativum bulbs,
Valeriana officinalis root, Panax ginseng root, Aloe vera leaves,
Vaccinium macrocarpon berries, Eleutherococcus senticosus root,
Eleutherococcus senticosus rhizome, Eleutherococcus senticosus
stems, Eleutherococcus senticosus leaves, Piper methysticum
rootstock, dill seeds, kola nut seeds, cinchona red bark, chinchona
yellow bark, or a combination thereof.
21. The method of claim 1 wherein the one or more non-acidic
compounds comprises lupeol, betulin, taxol, echinacea extract,
valerian root extract, ginkgolide A, ginkgolide B, ginkgolide C,
bilobalide, garlic extract, ginseng extract, aloe gel, barbaloin,
cranberry extract, eleutheroside A, eleutheroside B, eleutheroside
C, eleutheroside D, eleutheroside E, eleutheroside G, kava extract,
dill seed oil, kola extract, quinoline alkoloids, or a combination
thereof.
22. The method of claim 1 wherein the one or more non-acidic
compounds obtained in the selective extraction comprises less than
about 5 wt. % acidic compounds.
23. The method of claim 1, further comprising contacting the plant
tissue with an acid in a third solvent, to neutralize the salt and
to remove the one or more acidic compounds from the plant
tissue.
24. The method of claim 23 wherein the acid comprises hydrochloric
acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic
acid, formic acid, or a combination thereof.
25. The method of claim 23, wherein the third solvent comprises an
optionally substituted aromatic compound, an optionally substituted
heterocyclic compound, an optionally substituted cyclic compound,
an optionally substituted linear or branched compound, or
combination thereof, wherein suitable substituents include
(C.sub.1-C.sub.6)alkyl, hydroxyl, halo, cyano, nitro, oxo, thioxo,
amino, carboxyl, or combinations thereof.
26. The method of claim 23, wherein the third solvent comprises
isopropanol, ethanol, methanol, methylene chloride, toluene,
o-xylene, m-xylene, p-xylene, carbon dioxide, Xe, Freon-23, ethane,
N.sub.2O, SF.sub.6, propane, ammonia, n-C.sub.4H.sub.10,
(C.sub.2H.sub.5).sub.2O, or a combination thereof.
27. The method of claim 23, wherein the third solvent comprises an
additive.
28. The method of claim 27 wherein the additive is methanol;
ethanol; 1-propanol; 2-propanol; 1-hexanol; 2-methoxy ethanol;
tetrahydrofuran; 1,4-dioxane; acetonitrile; dichloromethane;
ammonia; chloroform; propylene carbonate; N,N-dimethylacetamide;
dimethyl sulfoxide; formic acid; water; carbon disulfide; acetone;
propane; toluene; hexanes; pentanes; o-xylene; m-xylene; p-xylene;
toluene; or a combination thereof.
29. The method of claim 1, wherein the one or more acidic compounds
comprises betulin acid, betulin-3-caffeate, tannin, phenol, caffeic
acid, cichoric acid, valerenic acid, isovaleric acid, flavonoid,
quercetin, kaempferol, catechin, lignin, shikimic acid, succinic
acid, amino acid, nicotinic acid, pantothenic acid, anthraquinone,
acidic galactan, benzoic acid, quinic acid, malic acid, citric
acid, hippuric acid, phenolic acid, ferulic acid, chlorogenic acid,
norsolorinic acid, fatty acids, tartaric acid, dicaffeate, cinnamic
acid, or a combination thereof.
30. The method of claim 29 wherein the one or more acidic compounds
obtained from the selective extraction comprises less than about 5
wt. % non-acidic compounds.
31. A method for selectively extracting one or more non-acidic
compounds from plant tissue in the presence of one or more acidic
compounds, the method comprising: (a) contacting the plant tissue
with an aluminum alkoxide in a first solvent to immobilize the
acidic compound as a salt on the plant tissue; and (b) contacting
the plant tissue with a second solvent suitable to remove the one
or more non-acidic compounds; thereby effectively providing a
solution comprising the one or more non-acidic compounds; wherein
the one or more non-acidic compounds comprises lupeol, betulin,
taxol, paclitaxel, echinacea extract, valerian root extract,
ginkgolide A, ginkgolide B, ginkgolide C, bilobalide, garlic
extract, ginseng extract, aloe gel, barbaloin, cranberry extract,
eleutheroside A, eleutheroside B, eleutheroside C, eleutheroside D,
eleutheroside E, eleutheroside G, kava extract, dill seed oil, kola
extract, quinoline alkoloids, or a combination thereof; and wherein
the one or more acidic compounds comprises betulin acid,
betulin-3-caffeate, tannin, phenol, caffeic acid, cichoric acid,
valerenic acid, isovaleric acid, flavonoid, quercetin, kaempferol,
catechin, lignin, shikimic acid, succinic acid, an amino acid,
nicotinic acid, pantothenic acid, anthraquinone, acidic galactan,
benzoic acid, quinic acid, malic acid, citric acid, hippuric acid,
phenolic acid, ferulic acid, chlorogenic acid, norsolorinic acid, a
fatty acid, tartaric acid, dicaffeate, cinnamic acid, or a
combination thereof.
32. The method of claim 31, further comprising treating the plant
tissue with an acid in a third solvent to provide a solution
comprising the one or more acidic compounds.
33. A method for selectively extracting lupeol, betulin, or a
combination thereof from birch bark in the presence of betulinic
acid, betulin-3-caffeate, or a combination thereof, the method
comprising: (a) contacting the birch bark with an aluminum alkoxide
in a first solvent comprising xylene thereby effectively
immobilizing the betulinic acid, betulin-3-caffeate, or combination
thereof as a salt on the birch bark; and (b) contacting the birch
bark with a second solvent suitable to remove the lupeol, betulin,
or combination thereof.
34. The method of claim 33, further comprising treating the birch
bark with an acid in a third solvent to provide the betulinic acid,
betulin-3-caffeate, or combination thereof.
35. A composition of matter comprising the one or more non-acidic
compounds as prepared in any one of claims 1-34.
36. A composition of matter comprising the one or more acidic
compounds as prepared in any one of claim 23, 32, or 34.
37. Taxol obtained by the process of contacting a mixture of a
basic component and a first solvent with Taxus yunnanesis bark to
immobilize one or more acidic compounds on the Taxus yunnanesis
bark and contacting the Taxus yunnanesis bark with a second solvent
suitable to remove the taxol, to effectively provide a solution
comprising taxol comprising less than about 5 wt. % of tannins,
fatty acids, phenols, or a combination thereof.
38. Betulin obtained by the process of contacting a mixture of a
basic component and a first solvent with birch bark to immobilize
one or more acidic compounds on the birch bark and contacting the
birch bark with a second solvent suitable to remove the betulin, to
provide a solution comprising betulin comprising less than about 5
wt. % of betulinic acid, betulin-3-caffeate, or a combination
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a Continuation In Part application
claiming benefit under 35 U.S.C. .sctn.120 of U.S. application Ser.
No. 09/969130, filed Oct. 1, 2001, which in turn claimed benefit
under 35 U.S.C. 119(e) of U.S. Provisional Application Serial No.
60/236,579 filed Sep. 29, 2000, both of which are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Many plants accumulate organic substances in quantities
sufficient to be economically useful as chemical feedstocks or raw
materials for various scientific, technological, and commercial
applications. Economically useful organic substances serve as
sources of industrial oils, resins, tannins, saponins, natural
rubber, gums, waxes, dyes, pharmaceuticals, and many specialty
products.
[0003] Plant chemicals are often classified as either primary or
secondary metabolites. Primary plant metabolites are substances
widely distributed in nature, occurring in one form or another in
virtually all organisms. Secondary plant metabolites are compounds
biosynthetically derived from primary metabolites and are more
limited in distribution in the plant kingdom. Secondary metabolites
are frequently accumulated by plants in smaller quantities than are
primary metabolites.
[0004] Secondary plant metabolites present a broad range of
medicinal properties. Many folk remedies are based on the isolation
and purification of secondary metabolites from trees, shrubs, and
flowers. Recently, some plant secondary metabolites have been found
to exhibit cancer-inhibiting activity, or other activity related to
inhibiting diseases. For example, camptothecin, colchicine,
docetaxel, etopside, paclitaxel, podophyllotoxin,
tetrahydrocannabinol, topotecan, vinblastine, vincristine,
vindesine, betulinc acid, as well as others, have been found to
have anticancer activity.
[0005] The use of secondary metabolites to treat diseases such as
cancer or human immunovirus (HIV) has been impeded, in part, by the
difficulty associated with synthesizing secondary plant
metabolites, using conventionally industrial chemical techniques.
Because secondary plant metabolites often have highly complex
structures with many chiral centers that may impart biological
activity, such complex compounds cannot by synthesized
economically. As a result, there is a need for an inexpensive,
efficient, bulk method for selectively extracting secondary
metabolites from plants.
SUMMARY OF THE INVENTION
[0006] The present invention provides a method for selectively
extracting compounds from plants in commercial (e.g., kg)
quantities. The method includes contacting a mixture of a basic
component and a first solvent with plant tissue, wherein the plant
tissue is optionally contained in an extraction vessel. This
contacting provides for the formation of salts with the acidic part
of the plant tissue. This contacting between plant tissue and basic
component can be mild enough to not cause any structural changes
(by processes such as hydrolysis, oxidation, or isomerization) of
the chemical sought to be extracted, or of other compounds present
in plant tissue, other than removal of one or more protons from
acidic compounds or protonated basic compounds. The contacting
should result in ionic salts between the basic pretreatment
component and acidic compounds of plant tissue. A second solvent
can be contacted with the plant tissue to remove non-acidic
compounds. A mixture of an acidic component and a third solvent can
then be contacted with the plant tissue to remove acidic compounds,
thereby providing the acidic compounds. The contact between the
mixture of the third solvent and acidic component with the plant
tissue can also be mild enough to not cause any structural change
of the acidic compound sought to be extracted, or of the compounds
present in plant tissue, other than to protonate the basic
compounds and the salts of acidic compounds.
[0007] The present invention provides for a method for selectively
extracting one or more non-acidic compounds from plant tissue in
the presence of one or more acidic compounds, comprising: (a)
contacting a solution of a basic component and a first solvent with
the plant tissue to immobilize the acidic compound on the plant
tissue; and (b) contacting the plant tissue with a second solvent
suitable to remove the non-acidic compounds to provide a solution
comprising the non-acidic compounds. The resulting plant tissue can
optionally be contacted with a solution of an acidic component and
a third solvent to remove the acidic compounds from the plant
tissue.
[0008] The present invention also provides for a method for
selectively extracting one or more non-acidic compounds from plant
tissue in the presence of one or more acidic compounds comprising:
(a) contacting plant tissue with a solution of an aluminum alkoxide
in a first solvent comprising xylene, thereby effectively
immobilizing the one or more acidic compounds on the plant tissue;
and (b) contacting the plant tissue with a second solvent suitable
to remove the one or more non-acidic compounds, wherein the one or
more non-acidic compounds comprises lupeol, betulin, taxol,
paclitaxel, echinacea extract, valerian root extract, ginkgolide A,
ginkgolide B, ginkgolide C, bilobalide, garlic extract, ginseng
extract, aloe gel, barbaloin, cranberry extract, eleutheroside A,
eleutheroside B, eleutheroside C, eleutheroside D, eleutheroside E,
eleutheroside G, kava extract, dill seed oil, kola extract,
quinoline alkoloids, or a combination thereof. The method can
optionally include contacting the plant tissue with a solution of
acetic acid and a third solvent comprising xylene, isopropanol, or
a combination thereof, to effectively remove the one or more acidic
compounds, wherein the one or more acidic compounds comprises
betulin acid, betulin-3-caffeate, tannin, lipid, phenol, caffeic
acid, cichoric acid, valerenic acid, isovaleric acid, flavonoid,
quercetin, kaempferol, catechin, lignin, shikimic acid, succinic
acid, amino acid, nicotinic acid, pantothenic acid, anthraquinone,
acidic galactan, benzoic acid, quinic acid, malic acid, citric
acid, hippuric acid, phenolic acid, ferulic acid, chlorogenic acid,
norsolorinic acid, or a combination thereof.
[0009] The present invention also provides for a method for
selectively extracting lupeol, betulin, or a combination thereof
from birch bark in the presence of a mixture of acidic compounds
comprising: (a) contacting the birch bark with a solution of an
aluminum alkoxide in a first solvent comprising xylene, thereby
effectively immobilizing the one or more acidic compounds on the
birch bark; and (b) contacting the birch bark with a second solvent
suitable to remove the lupeol, betulin, or a combination thereof.
The method can optionally include contacting the plant tissue with
a solution of acetic acid and a third solvent comprising xylene,
isopropanol, or a combination thereof, to effectively remove the
one or more acidic compounds, wherein the one or more acidic
compounds comprises betulin acid, betulin-3-caffeate, or a
combination thereof.
[0010] The present invention also provides for the one or more
non-acidic compounds as described herein, as prepared by any one of
the methods disclosed herein.
[0011] The present invention also provides for the one or more
acidic compounds as described herein, as prepared by any one of the
methods disclosed herein.
[0012] The present invention also provides for a composition of
matter comprising the one or more acidic compounds described
herein, as prepared by any one of the methods disclosed herein.
[0013] The present invention also provides for Taxol obtained by
the process of contacting a solution of a basic component and a
first solvent with Taxus yunnanesis bark to immobilize one or more
acidic compounds on the Taxus yunnanesis bark and contacting the
Taxus yunnanesis bark with a second solvent suitable to remove the
taxol, thereby providing taxol comprising less than about 5 wt. %
of tannins, fatty acids, phenols, or a combination thereof.
[0014] The present invention also provides for betulin, lupeol, or
a combination thereof, obtained by the process of contacting a
mixture of a basic component and a first solvent with birch bark to
immobilize one or more acidic compounds on the birch bark; and
contacting the birch bark with a second solvent suitable to remove
the betulin, lupeol, or the combination thereof, thereby providing
betulin, lupeol, or the combination thereof that comprises less
than about 5 wt. % of betulinic acid, betulin-3-caffeate, or a
combination thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 depicts an exemplary apparatus for the use of
selectively extracting compounds from natural materials, such as
plant tissue.
DETAILED DESCRIPTION OF THE INVENTION
[0016] All plant tissue includes both acidic and non-acidic
compounds. This complicates extraction processes employed to
isolate non-acidic compounds from acidic compounds of plant tissue.
Therefore, the present extraction method can be employed for the
selective extraction of a wide-range of plant materials. The
present extraction procedure is therefore advantageous for many
commercial industries, including, e.g., pharmaceutical, cosmetic,
and nutraceutical industries. For example, undesirable acidic
components may be present in natural extracts along with desirable
non-acidic compounds. These acidic components may not only have
little or no therapeutic utility, but many mammals (e.g., humans)
may have adverse reactions to these undesirable acidic components.
The acidic compounds, such as betulinic acid, may also be very
desirable. The method of the present invention can be used to
selectively extract non-acidic components from plant tissue,
wherein the non-acidic compounds are essentially free of acidic
compounds. The method of the present invention can also be used to
selectively extract acidic compounds from plant tissue, wherein an
acidic compounds are essentially free from the non-acidic
compounds.
[0017] Plant Tissue
[0018] As used herein, "plant tissue" refers to the tissue of any
organism of the plant kingdom, as opposed to one of the animal
kingdom or of the kingdoms of Fungi, Protista, or Monera. The plant
tissue can be any portion or portions of the plant (e.g., bark,
roots, leaves, flowers, needles, bulbs, berries, rhizomes,
rootstocks, stems, and seeds), as well as the entire plant. The
tissues of a plant ("plant tissue") generally fall into three main
categories: dermal tissue, ground tissue, and vascular tissue.
Dermal tissue refers to the "skin" layer of all plant organs and is
responsible for environmental interaction (light passage, gas
exchange, pathogen recognition and protection, color display,
etc.). Dermal tissue is composed of epidermal cells, closely packed
cells that secrete a waxy cuticle that aids in the prevention of
water loss. Ground tissue lies between dermal tissue and vascular
tissue. The ground tissue comprises the bulk of the primary plant
body. Parenchyma, collenchyma, and sclerenchyma cells are common in
the ground tissue. In roots, the ground tissue may store sugars or
starches to fuel the spring sap flow; in leaves, the ground tissue
is the layer responsible for photosynthesis (the mesophyll).
Vascular tissue transports food, water, hormones and minerals
within the plant. Vascular tissue includes xylem, phloem,
parenchyma, and cambium cells.
[0019] As used herein, "bark" refers to the dry, dead outer
covering of woody branches, stems and roots of plants that is very
distinct and separable from the wood itself. It includes all tissue
outside the cambium (growth layer between bark and wood).
[0020] As used here the terms "leaf" or "leaves" refer to those
parts of a plant which grow along the sides of branches or stems or
at the bases of plants. Most are green and contain chlorophyll,
though they vary in their shapes and sizes. Leaves are the part of
the plant that ordinarily performs photosynthesis (the process that
converts sunlight and carbon dioxide into energy).
[0021] As used herein, "needle" generally refers to a narrow stiff
leaf, such as those of conifers (e.g., pine trees).
[0022] As used herein, "root" refers to the part of a plant,
normally underground, that absorbs nutrients and anchors the plant
into the ground.
[0023] As used herein, "bulb" refers to a spheroidal body growing
from a plant either above or below the ground (usually below),
which is usually a bud, consisting of a cluster of partially
developed leaves, and producing, as it grows, a stem above, and
roots below, (e.g., the onion or tulip bulb). A true bulb is a
complete package containing next year's plant (flower) already
forming inside. The contents of the bulb are often enclosed in
protective, fleshy scales, which are held together by a small basal
plate. The scales are modified leaves that contain enough nutrients
to sustain the plant through dormancy and early growth. They may be
loose and open like those of a lily, or tightly closed like those
of a hyacinth. In many bulbs, a paper-thin tunic protects the
scales (lilies don't have a tunic). Roots will grow from the bulb's
basal plate.
[0024] As used herein, "berry" refers to any small fruit that is
pulpy or succulent throughout, having seeds loosely imbedded in the
pulp, such as the currant, grape, or blueberry. Berry can be
further defined as an indehiscent fruit derived from a single ovary
and having the whole wall fleshy, such as the grape or tomato.
Furthermore, berries come in various structures including simple,
such grape; blueberry, cranberry, or aggregate, such as blackberry;
raspberry, strawberry mulberry.
[0025] As used herein, "rhizome" refers to a horizontal, usually
underground stem that often sends out roots and shoots from its
nodes (also called rootstalk or rootstock).
[0026] As used herein, "rootstock" refers to a robust plant that
provides the root system in grafting, also known as a stock. Scions
and buds are grafted and budded to a rootstock or stock. Rootstock
also refers to the elongated and often thick rhizomes of certain
perennial herbaceous plants such as the Iris, Aspidistra and
Solomon's Seal.
[0027] As used herein, "stem" refers to the main (usually aerial)
axis (sometimes referred to as the trunk or stalk) of a tree,
shrub, or plant. "Stem" also refers to the part of the plant that
supports the leaves, flowers or fruits of a plant, such as the
peduncle of a fruit or the pedicel of a flower.
[0028] As used herein, "seed" refers to a ripened ovule, consisting
of an embryo with one or more integuments, or coverings, such as an
apple seed, a currant seed, dill seed, or kola nut seed. By
germination, most seeds produces a new plant. "Seed" also refers to
any small seedlike fruit, though it may consist of a pericarp, or
even a calyx, as well as the seed proper, such as a parsnip seed or
thistle seed. The seed proper has an outer and an inner coat, and
within these the kernel or nucleus. The kernel is either the embryo
alone, or the embryo enclosed in the albumen, which is the material
for the nourishment of the developing embryo. The scar on a seed,
left where the stem parted from it, is called the hilum, and the
closed orifice of the ovule, the micropyle.
[0029] Plant
[0030] The plant can be a bryophyte or vascular plant. More
specifically, the plant can be grass, flower or a tree and the
plant tissue can be any part of the grass, flower or tree. Specific
plants, flowers, and trees include, e.g., Moss (e.g., Club Moss),
Horsetail, Fern, Conifer, Cycad, Ginkgo biloba (Ginkgo), Taxus
yunnanesis (yew tree), Echinacea spp., Valeriana officinalis,
Allium sativum (garlic), Panax ginseng, aloe vera, Vaccinium
macrocarpon, Eleutherococcus senticosus, Piper methysticum, dill,
kola nut, and cinchona.
[0031] Another specific plant is the birch tree, wherein the
suitable plant tissue can be the bark of the birch tree. As used
herein, "birch" or "birch tree" refers to any of the several
deciduous tress of the genus Betula. The birches comprise the
family Betulaceae in the order Fagales. Birch trees include, for
example, white birch, B. alba; sweet, black or cherry birch, B.
lenta; monarch birch, B. maximowicziana; dwarf or arctic birch; B.
nana; Japanese white birch, B. platphylajaponica; smooth-bark
birch, B. pubescens; yellow birch, B. alleghaniensis; paper, white
or canoe birch, B. papyrifera; gray birch, B. populifolia; river
birch, B. nigra; and the European birches, B. pubescens; B. alba
and B. pendula. Specifically, birch can be B. alba, B. lenta, B.
maximowicziana, B. nana, B. platyphyla japonica, B. pubescens, B.
alleghaniensis, B. papyrifera, B. populifolia, B. nigra or B.
pendula. A specific birch for use in the processes of the present
invention is B. papyrifera.
[0032] As used herein, "Taxus" or "yew" refers to plants belonging
to Taxaceae Gymnopemnae. There are 11 species and five sub-species
of Taxus in the world, mainly found in East Asia, North America,
and Europe;
[0033] "Echinacea spp." refers to a perennial native to North
American which resembles a black-eyed Susan and is called
echinacea, purple coneflower or snake root;
[0034] "Valeriana officinalis" or "valerian" refers to the plant
Valeriana officinalis of the valerianaceae family, which is also
known as valerian, phu, all-heal, great wild valerian, amantilla,
setwall, setewale, capon's tail;
[0035] "Allium sativum" refers to garlic;
[0036] "Panax ginseng" refers to ginseng, commonly called Korean
ginseng, Chinese ginseng or American ginseng. Asian ginseng is a
member of the Araliaceae family, which also includes the closely
related American ginseng, Panax quinquefolius, and less similar
Siberian ginseng;
[0037] "Eleutherococcus senticosus," refers to "eleuthero" (which
contains eleutheroside A, eleutheroside B (syringin), eleutheroside
C, eleutheroside D, eleutheroside E (syringaresinol
di-O-.beta.-D-glucoside, liriodendrin), and eleutheroside G, among
other constituents);
[0038] "Aloe" refers to any of the over 500 different species of
Aloe. Aloe Vera is a member of the Lily family and is very-cactus
like in its characteristics. This unique plant also belongs to a
larger plant family called "Xeroids". Of the 500+ species of Aloe,
Aloe barbadensis miller (Aloe Vera species) is preferred;
[0039] "Vaccinium macrocarpon" refers to cranberry;
[0040] "Piper methysticum," a member of the pepper family, refers
to a plant native to the South Sea Islands of Micronesia, Melanesia
and Polynesia;
[0041] "Kola vera," of the family N.O. Sterculiaceae, also known as
"Kola nut" refers to the tree that grows about 40 feet high and has
yellow flowers spotted with purple; and
[0042] "Cinchona," belongs to the family N.O. Rubiaceae and refers
to Peruvian bark (Cinchona succirubra) which is an evergreen tree
that grows 15 to 45 feet in height.
[0043] Plant Components (Non-Acidic Compounds and Acidic
Compounds)
[0044] The specific non-acidic compounds and acidic compounds that
can be isolated from the plant tissue will depend, in part, upon
the specific plant tissue that is being extracted. For example, the
bark of Taxus yunnanesis can be extracted employing the methods
described herein to provide taxol (paclitaxel) as the non-acidic
compound and tannin, fatty acids, and phenols as the acidic
compounds;
[0045] the needles of the Yew tree can be extracted employing the
methods described herein to provide taxol (paclitaxel) as the
non-acidic compound, and tannin, fatty acids, and phenols as the
acidic compounds;
[0046] the root of the Echinacea spp. can be extracted employing
the methods described herein to provide Echinacea extract as the
non-acidic compound and tannin, caffeic acid, and cichoric acid as
the acidic compounds;
[0047] the root of the Valeriana officinalis can be extracted
employing the methods described herein to provide Valerian root
extract as the non-acidic compound and valerenic acid, isovaleric
acid, and tannins as the acidic compounds;
[0048] the roots, bark, leaves, or any combination thereof of the
Ginkgo biloba can be extracted employing the methods described
herein to provide Ginkgolide A, Ginkgolide B, Ginkgolide C, and
bilobalide as the non-acidic compounds and tannins, flavonoids
(e.g., quercetin, kaempferol, catechin), lignins, shikimic, and
succinic acids as the acidic compounds;
[0049] the bulb of the Allium sativum can be extracted employing
the methods described herein to provide garlic extract as the
non-acidic compound and fatty acids and amino acids as the acidic
compounds;
[0050] the root of the Panax ginseng can be extracted employing the
methods described herein to provide Ginseng extract as the acidic
compound and tannin, fatty acids, nicotinic acid and pantothenic
acid as the acidic compound;
[0051] the leaves of the Aloe Vera can be extracted employing the
methods described herein to provide aloe gel and barbaloin as the
non-acidic compounds and fatty acids, anthraquinones, acidic
gelactan, and amino acids as the acidic compounds;
[0052] the berries of the Vaccinium macrocarpon can be extracted
employing the methods described herein to provide cranberry extract
as the non-acidic compounds and benzoic acid, quinic acid, malic
acid, citric acid, and hippuric acid as the acidic compounds;
[0053] the roots, rhizomes, stems, leaves, or combination thereof
of the Eleutherococcus senticosus can be extracted employing the
methods described herein to provide Eleutherosides A-G as the non
acidic compounds and tannin, fatty acids, and caffeic acid as the
acidic compounds;
[0054] the rootstock of the Piper methysticum can be extracted
employing the methods described herein to provide Kava extract as
the non-acidic compounds and tannin, fatty acids, and amino acids
as the acidic compounds;
[0055] the seeds of the Dill can be extracted employing the methods
described herein to provide seed oil as the non-acidic compound and
phenolic acids (caffeic acid, ferulic acid, and chlorogenic acid)
as the acidic compounds;
[0056] the seeds of the Kola nut can be extracted employing the
methods described herein to provide kola extract as the non-acidic
compounds and tannin and catechins as the acidic compounds; and
[0057] the bark of the cinchona (yellow or red) can be extracted
employing the methods described herein to provide quinolone
alkaloids as the non-acidic compounds and norsoloric acid, tannins,
and quinic acid as the acidic compounds.
1TABLE 1 Non-acidic compounds and acidic compounds that can be
isolated from specific plant tissue. Components of Interest Acidic
Plant Tissue (non-acidic) Components Taxus yunnanesis Bark Taxol
(paclitaxel) Tannin, fatty acids, phenols Yew tree Needles Taxol
(paclitaxel) Tannin, fatty acids, phenols Echinacea spp. Root
Echinacea extract Tannin, caffeic, cichoric acid, tartaric acid
& dicaffeate Valeriana Root Valerian Roots Valerenic acid,
officinalis extract Isovaleric acid, tannins Ginkgo biloba Root
bark Ginkgolide A, B Tannins, and leaves and C, bilobalide
flavonoids (quercetin, kaempferol, catechin), lignins, shikimic and
succinic acids Allium sativum Bulb Garlic extract Fatty acids,
(garlic) amino acids Panax ginseng Root Ginseng extract Tannin,
fatty acids, nicotinic acid, pantothenic acid Aloe Vera Leaves Aloe
gel, Fatty acids, barbaloin anthraquinones, acidic galactan, amino
acids Vaccinium Berry Cranberry extract benzoic, macrocarpon
quinic, malic, citric and hippuric acid Eleutherococcus Root,
rhizome, Eleutherosides Tannin, fatty senticosus stems, leaves A-G
acids, caffeic acid Piper methysticum Rootstock Kava extract
Tannin, fatty acids, amino acids Dill Seeds Seed oil Phenolic acids
(caffeic, ferulic, chlorogenic) Kola nut Seeds Kola extract Tannin,
catechins Cinchona (red and Bark Quinoline Cinnamic acid, yellow)
alkaloids tannins, quinic acid
[0058] "Paclitaxel" refers to
[2aR-[2a.alpha.,4.beta.,4a.beta.,6.beta.,9.a-
lpha.(.alpha.R*,.beta.S*),-11,.alpha.12.alpha.,12a.alpha.,12b.alpha.]]-.be-
ta.-(Benzoylamino)-.alpha.-hydroxybenzenepropanoic acid
6,12b-bis(acetyloxy)-12-(benzoyloxy)-2a,
3,4,4a,5,6,9,10,11,12,12a,12b-do-
decahydro-4,11,-dihydroxy-4a,8,13,13-tetramethyl-5-5
oxo-7,11-methano-1H-cyclodeca[3,4]benz[1,2-b]oxet-9-yl ester.
[0059] "Echinacea extract" is believed to include essential oil,
polysaccharides, such as inulin, polyacetylenes, betain, glycoside,
sesquiterpenes and caryophylene. Echinacea extract is also believed
to contain copper, iron, tannins, protein, fatty acids, fat-soluble
alkylamides, caffeic acid glycoside (echinacoside), and vitamins A,
C, and E.
[0060] "Valeriana officinalis extract" is a very effective sedative
and is used most often to help insomnia, especially due to stress.
It has an advantage over prescription sedatives in that it is not
habit forming. Valerian has many actions besides its well-known
sedative effects. It strengthens the heart and in some cases lower
blood pressure. It promotes wound healing and has some antibiotic
activity and may be used externally to relieve muscle cramps. It
has some expectorant activity and may help a tickly cough. It may
actually balance the nervous system helping to calm agitated states
and stimulate cases of extreme fatigue. There are several species
of valerian, which vary in potency and can be used similarly,
although V. officinalis is the preferred plant. Other constituents
are a volatile oil, which includes isovalerianic acid and borneol;
choline; flavonoids; sterols and several alkaloids, including
actinidine, valerianine, valerine, and chatinine. Valepotriates are
not water-soluble, but valeric acid is and may be the constituent
most likely to produce valerian's sedative effect, especially when
used as it was traditionally in water extracts (teas) or
water/alcohol extracts (tinctures).
[0061] Many studies have provided clinical evidence that ginkgo
prevents many problems throughout the entire body. Ginkgo is
gaining recognition as a brain tonic that enhances memory because
of its positive effects on the vascular system, especially in the
cerebellum. It is also used as a treatment for vertigo, tinnitus
(ringing in the ears) and a variety of neurological disorders and
circulation problems. Ginkgo may help to counteract the effects of
aging, including mental fatigue and lack of energy. Ginkgo has two
groups of active substances, flavonoids (a three-ringed molecule
with hydroxyl (OH) groups attached) and terpene lactones, including
ginkgolides A, B, and C, bilobalide (a sesquiterpene), quercetin (a
flavonoid), and kaempferol (a flavonoid). The constituents of
gingko include terpenoids (bilobalide), diterpenoids (ginkgolide A,
ginkgolide B, ginkgolide C, ginkgolide J, ginkgolide M),
flavonoids: flavones (luteolin, tricetin, 2-hydroxyluteolin),
biflavones (amentoflavone, ginkgetin, isoginkgetin, sciadoptysin,
5-methoxybilobetin, bilobetin), flavonols (caempherol, quercetin,
isorhammetin), flavone glycosides, flavone acyl glycosides,
catechins, and steroids (sitosterol, sitosterol glucoside). The
ginkgolides have been shown to control allergic inflammation,
anaphylactic shock and asthma. Ginkgo extract is generally derived
from dried ginkgo leaves, but also may be derived from gingko root
or bark.
[0062] "Garlic" contains compounds that are antibacterial,
antifungal and reduce blood clotting. In order for the active
ingredient that gives garlic its characteristic odor and its
therapeutic effects to be released, the garlic clove (or bulb) must
be cut or crushed. This releases an enzyme that causes the
formation of allicin, the component responsible for garlic's odor
and medicinal activity. Active constituents present in garlic
include the sulphur compound allicin, produced by crushing or
chewing fresh garlic, which in turn produces other sulphur
compounds: ajoene, allyl sulfides, and vinyldithiins.
[0063] "Ginseng" is believed to increase energy, counter the
effects of stress, and enhance intellectual and physical
performance. Thirteen ginsenosides have been identified in ginseng,
including ginsenosides Rg1 and Rb1. Other constituents include the
panaxans, which are believed to help lower blood sugar, and the
polysaccharides (complex sugar molecules), which are believed to
support immune function. Also, long-term intake may be linked to a
reduced risk of cancer.
[0064] Applied to wounds, "aloe" is a mild anesthetic, relieving
itching, swelling, and pain: it also is antibacterial and
antifungal, increases blood flow to wounded areas, and stimulates
fibroblasts, the skin cells responsible for wound healing.
[0065] "Cranberry" has astringent applications for the urinary
tract and is a traditional remedy for bladder infections and
kidney-related disorders. Two components of cranberry juice have
been shown to inhibit the adherence of E. coli to uroepithelial
cells. The first is fructose. The second is proanthocyanidin, the
chemical structure of which has been elucidated. Fructose inhibits
the adherence of type-1 fimbriated E. coli and proanthocyanidin
inhibits the adherence of P-fimbriated E. coli to uroepithelial
cells. Cranberry is also a natural diuretic and urinary antiseptic
agent.
[0066] Although "kava" has undergone much research as to its active
ingredients, there is still no definite conclusion. It consists of
an oleoresin from which kavalactones originate, starch, sugars,
proteins, vitamins B1, B2, B3, B6, folic acid and E, potassium,
manganese, biotin, choline, inositol, fat, glycyrrhizin, lecithin,
pantothenic acid, para-aminobenzoic acid, pentacyclic terpenes,
phosphorous, and a yellow dye. Kavalactones are considered the most
active constituents in the plant. The main use for kava today is in
the treatment of anxiety. It is also an excellent muscle relaxant
and has diuretic and urinary antiseptic properties, so it may be
useful in urinary cystitis and prostatitis. Kava also shows
pain-relieving properties.
[0067] "Kola vera" or "Cola vera" seeds are said to contain a
glucoside, Kolanin (this substance may be a mixture of Kola red and
caffeine). The seeds also contain starch, fatty matter, sugar, and
a fat decomposing enzyme acting on various oils.
[0068] "Dill seed" is an herbal medicine that is used to reduce
gas, upset stomach, and colic pains. It is also used to promote the
flow of milk in breastfeeding mothers, and to help control bad
breath and hiccups. Other names for Dill Seed include: Anethum
Graveolens, Dill, and Dillweed.
[0069] As used herein, "tannin" refers to tannic acid or
gallotannic acid. Tannin varies somewhat in composition, depending
on the source, having the approximate empirical formula
C.sub.76H.sub.52O.sub.46. Tannic acid is a colorless to pale yellow
solid; it is believed to be a glucoside in which each of the five
hydroxyl groups of the glucose molecule is esterified with a
molecule of digallic acid. Tannin is used in tanning animal skins
to make leather; it transforms certain proteins of animal tissue
into compounds that resist decomposition. It is also used in
manufacturing inks, as a mordant in dyeing, and in medicine as an
astringent and for treatment of bums.
[0070] As used herein, "fatty acids" refers to a long-chain of
carboxylic acids that may either be saturated (without double bond)
or non-saturated (with double bond). It refers to any acid derived
from fats by hydrolysis (e.g., oleic acid, palmitic acid, or
stearic acid); any long-chain monobasic organic acid.
[0071] As used herein, "phenols" refers to compounds that include a
C.sub.6H.sub.5OH backbone. They are aromatic alcohols that are
optionally substituted by one or more substituents. Phenols
exhibits weak acidic properties and are sometimes called carbolic
acids, especially when in water solution.
[0072] As used herein, "caffeic acid" refers to
3-(3,4-Dihydroxyphenyl)-2-- propenoic acid.
[0073] As used herein, "valeric acid" refers to pentanoic acid;
valerianic acid; and propylacetic acid.
[0074] As used herein, "isovaleric acid" refers to 3-Methylbutanoic
acid and isovalerianic.
[0075] As used herein, "flavonoid" refers to polyphenols that have
a carbon skeleton. They have an acidic nature due to the phenol
groups.
[0076] As used herein, quercetin refers to
2-(3,4-Dihydroxyphenol)-3,5,7-t-
rihydroxy-4H-1-benzopyran-4-one.
[0077] As used herein, "kaempferol" refers to
3,5,7-Trihydroxy-2-(4-hydrox- yphenyl)-4H-1-benzopyran-4-one.
[0078] As used herein, "catechin" refers to
(2R-trans)-2-(3,4-dihydroxyphe-
nyl)-3,-4-dihydro-2H-1-benzopyran-3,5,7-triol.
[0079] As used herein, "lignin" refers to a highly polymerized and
complex chemical compound especially common in woody plants. The
cellulose walls of the wood become impregnated with lignin, a
process called lignification, which greatly increases the strength
and hardness of the cell and gives the necessary rigidity to the
tree. It is essential to woody plants for them to stand erect.
[0080] As used herein, "amino acids" refers to any one of a class
of simple organic compounds containing carbon, hydrogen, oxygen,
nitrogen, and in certain cases sulfur. These compounds are the
building blocks of proteins. They are characterized by the presence
of a carboxyl group (COOH) and an amino group (NH.sub.2). The 20
amino acids commonly found in animals are alanine, arginine,
asparagine, aspartic acid, cysteine, glutamic acid, glutamine,
glycine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
and valine. More than 100 less common amino acids also occur in
biological systems, particularly in plants. Every amino acid except
glycine can occur as either of two optically active stereoisomers,
D or L; the more common isomer in nature is the L-form. When the
carboxyl carbon atom of one amino acid covalently binds to the
amino nitrogen atom of another amino acid with the release of a
water molecule, a peptide bond is formed.
[0081] As used herein, "shikimic acid" refers to
[3R-(3.alpha.,4.alpha.,5.-
beta.]-3,4,5-Trihydrooxy-1-cyclohexene-1-carboxylic acid.
[0082] As used herein, "succinic acid" refers to butanedoic acid
(HOOCCH.sub.2CH.sub.2COOH).
[0083] As used herein, "nicotinic acid" refers to
3-Pyridinecarboxylic acid.
[0084] As used herein, "pantothenic acid" refers to
"(R)-N-(2,4-Dihydroxy-3,3-dimethyl-1-oxobutyl)-.beta.-alanine.
[0085] As used herein, "anthraquinone" refers to
9,10-anthracenedione.
[0086] As used herein, "acidic galactan" refers to a poly sugar
with attached carboxylic groups.
[0087] As used herein, "benzoic acid" refers to benzoic acid,
C.sub.6H.sub.5CO.sub.2H. It is the simplest aromatic carboxylic
acid. In addition to being synthesized from a variety of organic
compounds (e.g., benzyl alcohol, benzaldehyde, toluene, and
phthalic acid), it may be obtained from resins, notably gum
benzoin. It is used largely for making its salts and esters, most
notably sodium benzoate, which is widely used as a preservative in
foods and beverages and as a mild antiseptic in mouthwashes and
toothpastes.
[0088] As used herein, "quinic acid" refers to
[1R-(1.alpha.,3.alpha.,4.al-
pha.,5.beta.]-1,3,4,5-Tetrahydroxycyclohexanecarboxylic acid.
[0089] As used herein, "malic acid" refers to hydroxybutanedioic
acid.
[0090] As used herein, "citric acid" refers to citric acid or
2-hydroxy-1,2,3-propanetricarboxylic acid,
HO.sub.2CCH.sub.2C(OH)(CO.sub.- 2H)CH.sub.2CO.sub.2H, an organic
carboxylic acid containing three carboxyl groups. It is responsible
for the tart taste of various fruits in which it occurs, e.g.,
lemons, limes, oranges, pineapples, and gooseberries.
[0091] As used herein, "hippuric acid" refers to
N-Benzoylglycine.
[0092] As used herein, "ferulic acid" refers to
3-(4-Hydroxy-3-methoxyphen- yl)-2-propenoic acid.
[0093] As used herein, "chlorogenic acid" refers to
[1S-(1.alpha.,3.beta.,4.alpha.,5.alpha.]-3-[[3-(3,4-Dihydroxyphenyl)-1-ox-
o-2-propenyl]oxy]-1,4,5,trihydroxycyclohexanecarboxylic acid.
[0094] As used herein, "cinnamic acid" refers to
3-phenyl-2-propenoic acid.
[0095] Extraction Procedures
[0096] As used herein, "extraction" refers to a technique for
separating a mixture of chemical components from plant tissue,
wherein the components that are separated have different
solubilities and adsorption strengths. A "solvent extraction" is a
type of extraction wherein a mixture of components adsorbed on
plant tissue are separated utilizing the differences in the
solubilities and adsorption strengths of the components that are
separated. Suitable extraction techniques are disclosed, e.g., in
"Experiments in Organic Chemistry: From Microscale to Macroscale,"
Jonathan S. Nimitz (New York: Prentice Hall, 1990).
[0097] As used herein. "selective extraction" refers to the process
of extracting a class of one or more compounds (e.g., one or more
non-acidic compounds) from another class of one or more compounds
(e.g., one or more acidic compounds).
[0098] As used herein, "acidic compound" in plant tissue refers to
any compound naturally found in plant tissue that is acidic enough
to form salts with the basic components upon treatment with the
basic components. For instance, plant phenols, flavonoids,
flavones, flavolonoles, and tannins are acidic enough to form salts
that are immobilized on plant tissue by treatment with the basic
components.
[0099] As used herein, an "acid" refers to any compound or mixture
of compounds, in any suitable and effective amount, that can
effectively lower the pH of a neutral solution to below 7.0. The
acid will act as a proton donor and can neutralize a basic
component or solution of basic components, thereby forming a salt
and water. Any suitable acid can be employed, provided the acid
effectively neutralizes the one or more salts, which are formed
after the plant tissue immobilization. The suitable acid may be an
inorganic acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric
acid, phosphoric acid, or a combination thereof); an organic acid
(e.g., acetic acid, formic acid, or a combination thereof); or a
combination of an inorganic acid and an organic acid.
[0100] As used herein, a "basic component" refers to any compound
or mixture of compounds, in any suitable and effective amount, that
can effectively form non-soluble salts with the one or more acidic
compounds and effectively immobilizes the one or more acidic
compounds on the plant tissue. The basic component will act as a
proton acceptor. Any suitable basic component can be employed,
provided the basic component effectively forms non-soluble salts
with the one or more acidic compounds and effectively immobilizes
the one or more acidic compounds on the plant tissue. One suitable
class of basic components that effectively forms non-soluble salts
with the one or more acidic compounds and effectively immobilizes
the one or more acidic compounds on the plant tissue are the
alcoholates.
[0101] As used herein, an "alcoholate" or "alkoxide" refers to a
base formed from an alcohol in which the hydroxyl hydrogen atom has
been replaced by a metal atom (e.g., sodium, lithium, potassium,
calcium, or aluminum). One suitable alcoholate includes the
aluminum alcoholates.
[0102] As used herein, an "aluminum alcoholate" refers to an
alcoholate or alkoxide in which the metal atom is aluminum.
Suitable aluminum alcoholates include compounds of the formula
Al(OR).sub.3, wherein each R is independently
(C.sub.1-C.sub.12)alkyl, aryl (e.g., phenyl), or arylalkyl (e.g.,
benzyl), wherein each alkyl, aryl, or arylalkyl can be optionally
substituted on carbon with one or more hydroxy, halo, or
--N(R.sub.b).sub.2. Each R.sub.b can idependently be H,
(C.sub.1-C.sub.6)alkyl, aryl (e.g., phenyl), or arylalkyl (e.g.,
benzyl). Suitable specific aluminum alcoholates include, e.g.,
aluminum isopropoxide [Al(i-OPr).sub.3], aluminum ethoxide
[Al(OEt).sub.3], and aluminum methoxide [Al(OMe).sub.3].
[0103] As used herein, "alkyl" can be straight-chain or
branched.
[0104] Other suitable alcoholates include sodium alcoholates
(NaOR), lithium alcoholates (LiOR), potassium alcoholates (KOR),
magnesium alcoholates [Mg(OR).sub.2], calcium alcoholates
[Ca(OR).sub.2], and germanium alcoholates [Ge(OR).sub.3]; wherein
each R is independently (C.sub.1-C.sub.12)alkyl, aryl (e.g.,
phenyl), or arylalkyl (e.g., benzyl), wherein each alkyl, aryl, or
arylalkyl can be optionally substituted on carbon with one or more
hydroxy, halo, or --N(R.sub.b).sub.2. R.sub.b is H,
(C.sub.1-C.sub.6)alkyl, aryl (e.g., phenyl), or arylalkyl (e.g.,
benzyl). Specific examples of alcoholates include sodium methoxide,
sodium ethoxide, potassium ethoxide, potassium tert-butoxide, and
dimethoxymagnesium.
[0105] Another suitable class of basic components includes amines.
As used herein, "amines" includes ammonia, as well as primary
(NH.sub.2R), secondary (NHR.sub.2), and tertiary (NR.sub.3) amines.
Each R can independently be (C.sub.1-C.sub.12)alkyl, aryl (e.g.,
phenyl), or arylalkyl (e.g., benzyl); wherein each alkyl, aryl, or
arylalkyl can be optionally substituted on carbon with one or more
hydroxy, halo, or --N(R.sub.b).sub.2. Each R.sub.b can idependently
be H, (C.sub.1-C.sub.6)alkyl, aryl, or arylalkyl. Specific examples
of amines are ammonia, triethylamine, trimethylamine,
N(CH.sub.2CH.sub.2OH).sub.3, and (HOCH.sub.2).sub.3CNH.sub.2.
[0106] Another suitable class of basic components includes
heterocycles. As used herein, "heterocycle" refers to an aromatic
or non-aromatic compound that contains in the ring at least one
basic nitrogen atom. A heterocyclic ring system can be simple,
ortho-fused, or bicyclic. The ring system can optionally comprise
one or more non-peroxide oxygen or sulfur. Examples of heterocycles
include pyridine, morpholine, piperidine, N-methylpiperidine,
pyrrole, pyrrolidine, azabicyclo[2.2.2]octane, and
diazabicyclo[2.2.2]octane. The heterocyle ring system can
optionally be subtituted on carbon with one or more oxo, hydroxy,
amino, sulfo, (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)hydroxyal-
kyl, or --N(R.sub.b).sub.2, wherein R.sub.b is H or
(C.sub.1-C.sub.4)alkyl; or on nitrogen with one or more
(C.sub.1-C.sub.4)alkyl or (C.sub.1-C.sub.4)hydroxyalkyl.
[0107] As used herein, "hydroxyalkyl" can be straight-chain or
branched, and the hydroxy group can be on any suitable carbon
atom.
[0108] Another suitable class of basic components includes alkaline
earth metal hydroxides. These comprise an alkaline earth cation and
one or more hydroxide ions. Examples of alkaline earth metal
hydroxides include NaOH, KOH, LiOH, Mg(OH).sub.2, and
Ca(OH).sub.2.
[0109] Another suitable class of basic components includes alkaline
earth metal oxides. These compounds consist of one or more alkaline
earth metals and oxygen. Examples of basic oxides include K.sub.2O,
Na.sub.2O, Li.sub.2O, KNaO, CaO, and MgO.
[0110] Another suitable class of basic components includes alkaline
earth metal carbonates and bicarbonates. The compounds consist of
CO.sub.3.sup.2- or HCO.sub.3.sup.- and alkaline earth metal
cations. Examples of alkaline earth metal carbonates and
bicarbonates include Na.sub.2CO.sub.3, NaHCO.sub.3,
K.sub.2CO.sub.3, KHCO.sub.3, KNaCO.sub.3, Li.sub.2CO.sub.3,
LiHCO.sub.3, CaCO.sub.3, and MgCO.sub.3.
[0111] Another suitable class of basic components includes alkaline
earth metal sulfites. These compounds consist of sulfite anion and
alkaline earth metal cations. Examples of alkaline earth metal
sulfites include Na.sub.2SO.sub.3, K.sub.2SO.sub.3, KNaSO.sub.3,
Li.sub.2SO.sub.3, CaSO.sub.3, and MgSO.sub.3.
[0112] Another suitable class of basic components includes alkaline
earth metal sulfides. These compound consist of S.sup.2- and
alkaline earth metal cations. Examples of alkaline earth metal
sulfides include Li.sub.2S, K.sub.2S, and Na.sub.2S.
[0113] Another suitable class of basic components includes alkaline
earth metal hydrogen sulfides. These compounds consist of HS.sup.-
and an alkaline earth metal cation. Examples of alkaline earth
metal hydrogen sulfides include LiHS, KHS, and NaHS.
[0114] The basic component can also be generated in a mixture from
compounds that generate a basic component. Examples of compounds
that can generate basic components would be elemental sodium,
elemental magnesium, elemental potassium, and elemental calcium.
Each of these compounds in contact with water or alcohols will
generate the corresponding alkline earth metal hydroxide or
alkaline earth metal alcoholate.
[0115] Suitable basic components are commercially available from,
e.g., Aldrich (Milwaukee, Wis.)
[0116] The first solvent can effectively dissolve the basic
component but will not effectively dissolve the salts that are
formed in the process of the neutralization of acidic compounds.
Any suitable solvent can be employed as the first solvent, provided
the solvent effectively dissolves the basic component but does not
effectively dissolve the salts that are formed in the process of
the neutralization of acidic compounds. The first solvent can
include any suitable: (1) optionally substituted aromatic compound,
(2) optionally substituted heterocyclic compound, (3) optionally
substituted cyclic compound, (4) optionally substituted linear or
branched compound, (5) or any combination thereof Suitable
substituents include, e.g., (C.sub.1-C.sub.6)alkyl, hydroxyl, halo,
trihalo(C.sub.1-C.sub.6)alkyl, cyano, nitro, oxo, thioxo, amino,
carboxyl, and combinations thereof. Compounds suitable as a first
solvent are disclosed and commercially available from, e.g., 2001
Aldrich Catalogue (Milwaukee, Wis.). Specific compounds suitable as
a first solvent include isopropanol, ethanol, methanol, methylene
chloride, toluene, xylene (e.g., o-xylene, m-xylene, or p-xylene),
carbon dioxide, or combinations thereof. Other compounds suitable
as a first solvent include Xe, Freon-23, ethane, N.sub.2O,
SF.sub.6, propane, ammonia, n-C.sub.4H.sub.10,
(C.sub.2H.sub.5).sub.2O, and combinations thereof. The first
solvent can include a single compound or can include a mixture of
compounds. In addition, the first solvent can optionally include an
additive.
[0117] The concentration of the basic component in the first
solvent can vary depending on the carrying capacity of the solvent
for the basic component. Any suitable solvent can be employed that
allows for efficient reaction between the basic component and
acidic compounds in the plant tissue. Typically, the concentration
can be about 0.1 to 25 percent basic component in the solvent by
weight. Specifically, the concentration can be about 0.5 percent to
10 percent base in the solvent by weight. More specifically, the
concentration can be about 1 percent to 5 percent base in the
solvent by weight.
[0118] By treating the plant tissue with a mixture of a basic
component in a first solvent, acidic compounds in the plant tissue
will form salts. The resulting salts can precipitate on the plant
tissue, or otherwise adhere to the plant tissue, so that non-acidic
compounds can be selectively removed from the plant tissue.
Discharging the mixture of the basic component in the first solvent
from the extraction vessel and introducing a second solvent can
accomplish this. The excess basic component can be adsorbed by any
suitable adsorbent (e.g., silica, alumina, or a combination
thereof).
[0119] The second solvent can effectively dissolve the one or more
non-acidic compounds but will not effectively dissolve the salts of
the one or more acidic compounds that were effectively formed
during the treatment with the basic component. The second solvent
can include any suitable: (1) optionally substituted aromatic
compound, (2) optionally substituted heterocyclic compound, (3)
optionally substituted cyclic compound, (4) optionally substituted
linear or branched compound, (5) or any combination thereof.
Suitable substituents include, e.g., (C.sub.1-C.sub.6)alkyl,
hydroxyl, halo, trihalo(C.sub.1-C.sub.6)alkyl, cyano, nitro, oxo,
thioxo, amino, carboxyl, and combinations thereof. Compounds
suitable as a second solvent are disclosed and commercially
available from, e.g., 2001 Aldrich Catalogue (Milwaukee, Wis.).
Specific compounds suitable as a second solvent include
isopropanol, ethanol, methanol, methylene chloride, toluene, xylene
(e.g., o-xylene, m-xylene, or p-xylene), carbon dioxide, or
combinations thereof. Other compounds suitable as a second solvent
include Xe, Freon-23, ethane, N.sub.2O, SF.sub.6, propane, ammonia,
n-C.sub.4H.sub.10, (C.sub.2H.sub.5).sub.2O, and combinations
thereof.
[0120] The second solvent can include a single compound or can
include a mixture of compounds. In addition, the second solvent can
optionally include an additive. The second solvent can be passed
through the vessel to remove non-acidic compounds from the plant
tissue in one pass or in multiple passes. Optionally, the second
solvent can be recirculated through the vessel using the reservoir
optionally attached to the vessel. Non-acidic compounds can be
extracted from the plant tissue using temperature, pressure, and
time parameters that are sufficient to remove a significant amount
(e.g., more than about fifty percent, more than about seventy
percent, or more than about ninety percent) of non-acidic compounds
can be removed from the plant tissue. An optional additional vessel
can be employed for the adsorption of any excess basic material and
some polymeric non-acidic compounds.
[0121] The third solvent can effectively neutralize the basic salts
that are formed during the treatment of plant tissue with the basic
components. This process releases acidic compounds for further
extraction with the third solvent. The third solvent can include
any suitable: (1) optionally substituted aromatic compound, (2)
optionally substituted heterocyclic compound, (3) optionally
substituted cyclic compound, (4) optionally substituted linear or
branched compound, (5) or any combination thereof. Suitable
substituents include, e.g., (C.sub.1-C.sub.6)alkyl, hydroxyl, halo,
trihalo(C.sub.1-C.sub.6)alkyl, cyano, nitro, oxo, thioxo, amino,
carboxyl, and combinations thereof. Compounds suitable as a third
solvent are disclosed and commercially available from, e.g., 2001
Aldrich Catalogue (Milwaukee, Wis.). Specific compounds suitable as
a third solvent include isopropanol, ethanol, methanol, methylene
chloride, toluene, xylene (e.g., o-xylene, m-xylene, or p-xylene),
carbon dioxide, or combinations thereof. Other compounds suitable
as a third solvent include Xe, Freon-23, ethane, N.sub.2O,
SF.sub.6, propane, ammonia, n-C.sub.4H.sub.10,
(C.sub.2H.sub.5).sub.2O, and combinations thereof. The third
solvent can include a single compound or can include a mixture of
compounds. In addition, the third solvent can optionally include an
additive.
[0122] The solution of the acid in the third solvent can be passed
through the vessel in one pass or in multiple passes using
temperature, pressure, and time parameters that are sufficient to
remove a significant amount (e.g., more than about fifty percent,
more than about seventy percent, or more than ninety percent) of
acidic compounds from the plant tissue.
[0123] As used herein, an "additive" is a compound added to the
solvent in an amount of about 1 wt % to about 20 wt. % based on the
solvent. Specifically, the additive may be present in an amount of
about 1 wt. % to about 15 wt. % or about 1 wt. % to about 10 wt. %.
Upon addition, the additive will modify the physical properties of
the solvent. For example, an additive may be useful to modify the
polarity, critical temperature, critical pressure, etc., of the
solvent system. Suitable additives include lower alcohols (e.g.,
methanol, ethanol, 1-propanol, 2-propanol, 1-hexanol, or 2-methoxy
ethanol); ethers (e.g., tetrahydrofuran or 1,4-dioxane);
substituted hydrocarbons (e.g., acetonitrile, dichloromethane,
ammonia or chloroform) propylene carbonate, N,N-dimethylacetamide;
dimethyl sulfoxide; carboxylic acids (e.g., formic acid); water;
carbon disulfide; lower ketones (e.g., acetone), hydrocarbons
(e.g., propane, toluene, hexanes and pentanes); as well as
optionally substituted aromatic compounds (e.g., o-xylene,
m-xylene, p-xylene, and toluene).
[0124] As used herein, "fragmentation" includes chopping,
crunching, crushing, gnashing or pounding. Such fragmentation of
plant tissue will effectively provide smaller pieces of plant
tissue. The smaller pieces of plant tissue will have, combined, a
larger surface area. The fragmentation can conveniently be carried
out, e.g., by introducing plant tissue into a machine with knives
on a rotating disk (e.g., a chipper or shredder). One chipper
suitable for fragmenting the plant tissue is the YardMan Model
246-648D401 chipper.
[0125] As used herein, "pelletization" refers to the process of
forming plant tissue pellets. Any suitable pelletization method
known to those of skill in the art can be employed. For example,
fragmented plant tissue can be sprayed with a solvent (e.g., water)
through a sprinkler in a horizontal mixer. Pelletization can
routinely be performed using, e.g., a Laboratory Pellet Machine
(California Pellet Mill, Co., Calif.) through a die with holes.
Pelletization increases the density of plant tissue. This increases
the efficiency of the extraction process, decreases the extractors'
volume and decreases the amount of solvents needed for extraction.
In addition, plant tissue pellets are relatively easy to handle.
For example, there are little or no problems associated with dust
or filtration.
[0126] As used herein, "drying" refers to the process of removing a
substantial amount (e.g., up to about 50%, up to about 75%, or up
to about 90%) of liquid or moisture in the plant tissue. In an
alternative embodiment, the drying process can remove up to about
95%, up to about 99%, or up to about 100% of liquid or moisture in
the plant tissue. The liquid or moisture can typically include
water. As such, the drying will effectively remove at least a
portion of water present in the plant tissue. Prior to or
subsequent to fragmentation, plant tissue can be dried. Such drying
may increase the efficiency of the fragmentation, which can
increase the efficiency of the extraction. The plant tissue can be
air-dried or dried at an elevated temperature with or without
reduced pressure (i.e., in vacuo). Typically, the drying
temperature employed is any suitable elevated temperature that will
not lead to degradation or decomposition of the plant tissue or the
components therein. Specifically, plant tissue can be dried in
vacuo at an elevated temperature. Machines capable of drying plant
tissue are known in the art and include an oven, or similar device,
such as a rotating air drum drier. The plant tissue can be dried at
any suitable temperature. For example, the plant tissue can be
dried above about 25.degree. C., above about 50.degree. C., or
above about 100.degree. C. Additionally, the plant tissue can be
dried for any suitable period of time. For example, the plant
tissue can be dried for more than about 10 minutes, for more than
about 1 hour, or for more than about 24 hours. Additionally, any
suitable pressure can be employed that does not lead to
decomposition or degradation of the plant tissue. The drying
pressure typically can be from about 0.1 atmosphere to 2.5
atmospheres. Specifically, the drying pressure can be from about
0.1 atmosphere to 1.0 atmosphere. More specifically, the drying
pressure can be from about 0.1 atmosphere to 0.75 atmosphere.
[0127] After the fragmented plant tissue is optionally dried and
optionally pelletized, it typically can be placed in an extraction
vessel to be extracted. Any suitable extraction vessel can be
employed. The extraction vessel will preferably be equipped with
inlets and outlets that can be opened and closed. The vessel will
optionally be capable of being heated and/or pressurized. For
smaller scale extractions, the vessel can be, e.g., a soxhlet
apparatus. For commercial scale (e.g., kilogram) extractions, the
extraction vessel can be a stainless steel tube or similar chamber,
optionally attached to a solvent reservoir. Additionally,
commercial scale (e.g., kilogram) extractions, the extraction
vessel can be a vessel capable of supercritical fluid extraction
(SFE).
[0128] The plant tissue can be packed to fill the capacity (or some
fraction thereof) of the extraction vessel. The extent to which the
extraction vessel can be filled with the packed, fragmented plant
tissue will vary depending upon the dimensions of the extraction
vessel, the plant tissue, and the density to which the plant tissue
can be packed. The fragmented plant tissue typically can be packed
to any suitable density depending upon the extraction vessel and
the scale of the extraction. Preferably, the plant tissue can be
packed to a density of about 0.1 g/cm.sup.3 to about 1 g/cm.sub.3.
More preferably, the fragmented plant tissue can be packed to a
density of about 0.25 g/cm.sub.3 to about 0.85 g/cm.sub.3 in the
extraction vessel. More preferably, the fragmented plant tissue can
be packed to a density of about 0.5 g/cm.sup.3 to about 0.75
g/cm.sup.3. However, the optimal extent to which the extraction
vessel can be packed with plant tissue and the density to which the
plant tissue can be packed can be easily determined by
practitioners in the art. Additionally, the extraction vessel can
be filled up to about 99% of the volume, up to 90% of the volume,
or up to about 80% of the volume.
[0129] Supercritical Fluid Extraction (SFE)
[0130] The second extraction solvent, the third extraction solvent,
or combination thereof, can employ supercritical fluid
extraction.
[0131] Supercritical fluid extraction is an extraction wherein a
fluid at a temperature and pressure above its critical point is
employed; or a fluid above its critical temperature, regardless of
pressure, is employed. Below the critical point, the fluid can
coexist in both gas and liquid phases, but above the critical point
there is only one phase. Equipment and techniques for carrying out
supercritical fluid extraction are known to those skilled in the
art. See, McHugh, M. And Krukonis, V., Supercritical Fluid
Extraction, 2nd ed, Butterworth-Heinemann, Boston, 1994; Johnston,
K. P., Penninger, J. M. L., Supercritical Fluid Science and
Technology, ACS Symposium Series 406, American Chemical Society,
Washington, D.C.; and Taylor, L.T., Supercritical Fluid Extraction,
John Wiley & Sons, New York, 1996.
[0132] In a supercritical fluid extraction, thermodynamic and
transport properties of supercritical fluid are a function of
density, which depends strongly on the fluid's pressure and
temperature. The density may be adjusted from a gas-like value of
0.1 g/ml to a liquid-like value as high as 1.2 g/ml. Furthermore,
as conditions approach the critical point, the effect of
temperature and pressure on density becomes much more significant.
For example, increasing the density of supercritical carbon dioxide
from 0.2 to 0.5 g/ml requires raising the pressure from 85 atm to
140 atm (8.6 megapascals to 14.2 megapascals) at 158.degree. F.
(70.degree. C.), but at 95.degree. F. (35.degree. C.) the required
change is only from 65 atm to 80 atm (6.61 Mpa to 8.1 Mpa).
[0133] As used herein, "fractional supercritical fluid extraction"
(hereinafter "FSCFE") is a multi-step procedure wherein the
supercritical fluid extraction is carried out at one temperature
and pressure for a given period of time and is then carried out at
one or more other temperatures or pressures.
[0134] The efficiency of supercritical fluid extraction on a
material such as outer birch bark depends in part upon the size of
the outer birch bark pieces. Thus, the smaller the outer birch bark
pieces, the more efficient the supercritical fluid extraction
typically will be. As such, after fragmentation and prior to
extraction, outer birch bark shreds may be further reduced in size
with a Hammermill or suitable means. For example, a 15 horsepower
3B Junior Hammermill made by Jay Bee Manufacturing, Inc can be used
as illustrated in the Examples herein below. The hammermill reduces
large pieces of birch bark by beating the bark with pivoted hammers
until the material is small enough to fall through a mesh.
[0135] For use in the processes of the present invention, the size
of outer birch bark shreds obtained after the Hammermill reduction
is typically less than about 5 mm in diameter. Specifically, the
shreds can be less than about 3 mm in diameter. More specifically,
the shreds can be less than about 1 mm in diameter.
[0136] For use in the processes of the present invention,
supercritical fluid extraction can conveniently be carried out at a
pressure of about 1,000 psi to about 12,000 psi. It is appreciated
that those skilled in the art understand that higher pressures may
enable faster extraction. In this case, it may be necessary to
subsequently separate and purify the product.
[0137] For use in the processes of the present invention,
supercritical fluid extraction can conveniently be carried out at a
pressure of about 750 psi to about 12,000 psi. Specifically, the
pressure may be about 1,000 psi to about 10,000 psi. More
specifically, the pressure may be about 4,000 psi to about 9,000
psi.
[0138] For use in the processes of the present invention, the
temperature of supercritical fluid extraction can conveniently be
about 0.degree. C. to about 150.degree. C. Specifically, the
temperature can be about 25.degree. C. to about 110.degree. C. More
specifically, the temperature can be about 45.degree. C. to about
100.degree. C.
[0139] In one specific embodiment, supercritical fluid extraction
is performed at a temperature of about 40.degree. C. to about
90.degree. C. and a pressure of about 3,000 psi to about 10,000
psi.
[0140] Supercritical fluid extraction employs a solvent which
possesses physical properties suitable as a supercritical fluid.
Suitable solvents include carbon dioxide, Xe, Freon-23, ethane,
N.sub.2O, SF.sub.6, propane, ammonia, n-C.sub.4H.sub.10,
(C.sub.2H.sub.5).sub.2O and the like.
[0141] The physical and environmentally friendly properties of
carbon dioxide make it particularly attractive as a solvent for
supercritical fluid extraction. Carbon dioxide is a major component
of the atmosphere and is therefore relatively safe and abundant. In
addition, carbon dioxide is relatively inexpensive. Compared to
most other suitable solvents, carbon dioxide is environmentally
friendly as it will not harm the atmosphere at the quantities used
in the methods of the invention. Moreover, carbon dioxide is
non-flammable and non-explosive. Further, carbon dioxide leaves no
substantial residue or remnant upon evaporation.
[0142] Carbon dioxide also possesses physical properties which
enable it to change polarity over the temperature range and
pressure range normally employed in supercritical fluid extraction.
As a result, carbon dioxide may act as a nonpolar solvent at one
temperature and pressure but may act as a polar solvent at another
temperature and pressure. By varying the temperature and pressure,
the solvent properties may be modified. This allows for the
isolation of more than one compound using a single solvent
system.
[0143] The solvent employed in supercritical fluid extraction may
be a single compound or may be a mixture of compounds. In addition,
the solvent may include an additive.
[0144] The non-acidic extract (i.e., the non-acidic compounds) will
include relatively little or no acidic compounds therein.
Typically, the non-acidic extract will include less than about 25
wt. % acidic compounds, less than about 10 wt. % acidic compounds,
less than about 5 wt. % acidic compounds, or less than about 1 wt.
% acidic compounds.
[0145] The collected non-acidic extract can be purified by methods
known in the art such as, e.g., crystallization, chromatography,
distillation, or a combination thereof. See in "Experiments in
Organic Chemistry: From Microscale to Macroscale," Jonathan S.
Nimitz (New York: Prentice Hall, 1990).
[0146] An optional adsorption process can be employed in the method
of the present invention. Without an adsorption process, birch bark
can be extracted employing the extraction procedures disclosed
herein to provide a betulin fraction may have a tan appearance. The
color is probably caused by aligomeric tannin admixtures, which are
not acidic enough to be bound by Al(i-OPr).sub.3 in the extraction
vessel. To adsorb the color causing compounds, adsorbents such as
silica, aluminum oxide, calcium carbonate, calcium oxide, molecular
sieves, ionic exchange resins, amberlite, sephadex, sephacryl,
polymeric adsorbents (diaion, ambersorb), cellulose, hydroapatite,
and/or activated charcoal may be employed. Preferably, the
adsorbent for decoloration can be Al.sub.2O.sub.3.
[0147] After the non-acidic compounds are removed from the plant
tissue, the acidic compounds, which underwent reaction with the
basic component to form salts, can optionally be removed.
Discharging the second solvent from the vessel and introducing a
mixture of an acid in a third solvent can accomplish this.
[0148] The acidic extract (i.e., the acidic compounds) will include
relatively little or no non-acidic compounds therein. Typically,
the acidic extract will include less than about 25 wt. % non-acidic
compounds, less than about 10 wt. % non-acidic compounds, less than
about 5 wt. % non-acidic compounds, or less than about 1 wt. %
non-acidic compounds.
[0149] The resulting acidic extract can be purified by methods
known in the art such as, e.g., crystallization, chromatography,
distillation, or a combination thereof See "Experiments in Organic
Chemistry: From Microscale to Macroscale," Jonathan S. Nimitz (New
York: Prentice Hall, 1990).
[0150] The invention is further demonstrated in the following
example. The example is for purposes of illustration and is not
intended to limit the scope of the present invention.
EXAMPLE I
Selective Low-High Pressure Extraction of Pretreated Outer Birch
Bark Pellets in a 3-Liter-Extraction Vessel
[0151] Introduction
[0152] A method for selectively isolating compounds from plant
tissue, such as birch bark, includes the pretreatment of the plant
tissue, e.g., birch bark, selective extraction of the pretreated
tissue, and the isolation and purification of non-acidic and acidic
compounds, such as birch bark triterpenes, which include betulin
(a), lupeol (b), betulinic acid (c) and betulin-3-caffeate (d).
[0153] Birch bark extraction was performed using an apparatus (FIG.
1) purchased from Newport Scientific, Inc. (model: 46-19360-50 Hz).
The apparatus can be used for low and high-pressure operations (up
to 10,000.00 psi or 680 atm). The low-pressure pump (3) (KNF
Flodos, Co., CH-6210 Sursee) and distillation/recycling assembly
(4, 5, 6, 8, 9 and 10) are additional units for the apparatus. The
apparatus may be used with organic solvents, such as i-propanol,
ethanol, methylene chloride, toluene, and o-, p- and m-xylene, at
temperatures from about 50.degree. C. to 200.degree. C., as well as
CO.sub.2 supercritical extraction conditions. For CO.sub.2
supercritical extraction, it is necessary to use the diaphragm type
compressor, which may compress supercritical fluids, gas or liquid,
to 10,000 psi.
[0154] To extract and selectively isolate non-acidic and/or acidic
compounds from birch bark, the bark was first, shredded, ground,
screened and pelletized (cylinder shaped pellets, b.times.h=1
mm.times.1 mm) as described in WO 01 108885. The average density of
the pellets was about 0.5 to 0.6 g/ml. The pellets were dried in a
drying hood at about 100.degree. C. for about 10 hours before
loading into an extraction vessel.
[0155] Step 1: Birch Bark Pellet Pretreatment Process
[0156] Approximately 1.2 kg of dry birch bark pellets were
placed/loaded in a 20 L Rotor Evaporator (Buchi Rotavapor R-153).
Approximately 60 g (1.5%) of Al(O-i-Pr).sub.3 in 4 L of p-xylene
was added to a rotating vessel of the rotor evaporator at room
temperature. The birch bark pellets and Al(O-i-Pr).sub.3 in
p-xylene were rotated in the rotator vessel under normal pressure
at 70.degree. C. for about 2 hours. After 2 hours, the p-xylene was
evaporated at p=30-40 mm at a temperature of about 60-70.degree. C.
Following evaporation, 1 L of p-xylene was added into the rotor
evaporator. This additional solvent was evaporated at p=30-40 mm at
a temperature of about 60-70.degree. C. The additional solvent and
evaporation allowed for the release of birch bark pellets from
undesirable admixtures of i-propanol (see Equation 1 below). As a
result of the pretreatment process, the majority of acidic
compounds present in the birch bark were bound in non-soluble
aluminum salts as demonstrated in Equation 1. Alcohols, such as
betulin and lupeol, are not acidic enough to be bound with
Al(O-i-Pr).sub.3. The birch bark pellets maintained their
cylinder/pellet form (b.times.h=1 mm.times.1 mm) throughout the
pretreatment process. 1 XH + Al ( i - OPr ) 3 X - OAl ( i - OPr ) 2
+ i - PrOH XH -theacidiccomponentofnaturalmaterial Equation 1
[0157] Step 2: The Process of Selective Extraction
[0158] After pretreatment, the pellets, about 1260 g, were loaded
into an extraction vessel for the selection extraction process. The
apparatus, including the parts listed in Table I, were assembled as
shown in FIG. 1. The extraction system consisted of three major
blocks: (1) the extraction vessel assembly; (2) the adsorption
vessel assembly; and (4) the solvent regeneration assembly. The
extraction vessel assembly (1) and adsorption vessel assembly (2)
may maintain the pressure up to 10,000.00 psi (680 atm.). Such
pressure levels may be used for CO.sub.2 supercritical extraction.
The extraction vessel (EV) (1) was equipped with a thermocouple
(19.1), thermocouple temperature controller (20.1), regulating
valves (13.3, 13.6) and heaters (14). The EV (1) was connected to
the adsorption vessel (2) by stainless steel tubing, which was
heated by heating tape (11). The adsorption vessel (2) was also
equipped with a thermocouple (19.2), thermocouple temperature
controller (20.2), regulating valves (13.4, 13.7) and adsorption
vessel heaters (15). The adsorption vessel (2) was connected to the
distilling flask (4) through a valve (13). The distilling flask (4)
was equipped with a Wurtz adapter (8), thermometer (18) and stopper
(7). The horizontal large condenser (6) was attached to adapter (8)
and adapter (9). Adapter (9) was connected to the receiving flask
(5). The pump (3) was attached through a coupling joint and PE
tubing with a receiving flask (5). The pump assembly was equipped
with a back loop system (12). A nitrogen tank was attached to a
regulating valve (13.2).
2TABLE 2 The list of equipment for pretreated birch bark pellets
extraction No. Name of Equipment Quantity 1 Extraction vessel, 3L,
stainless steel 1 2 Adsorption Vessel, 1L, stainless steel 1 3 Low
pressure pump 1 4 Distilling flask, vert, 3N, 45/50, 5L 1 5
Receiving flask, vert, 3N, 45/50, 3L 1 6 Condenser large
horizontal, 45/50, 20" 1 7 Stopper, 45/50 2 8 Wurtz distillation
adapter, 45/50 1 9 Distillation adapter, 45/50 1 10 Condenser small
vertical 1 11 Heating tape 2 12 Back loop system 1 13 Regulating
valve 9 14 EV heater 4 15 AV heater 2 16 DF heater 1 17 RF heater 1
18 Thermometer 1 19 Thermocouples 2 20-24 Flat bottom 4L-flasks
5
[0159] A. Packing of the Extraction 3 L-vessel (1)
[0160] The stainless steel bottom dispersion plate (with 1 mm
diameter holes) was placed at the bottom of extraction vessel (1).
Filter paper, cotton filter and one or more additional filter
papers were placed on the dispersion plate. Approximately 1260 g of
pretreated birch bark pellets were placed in the extraction vessel
through the wide-neck funnel. Filter paper and the dispersion plate
were then placed on top of the pellets. The extraction vessel (1)
was filled with 1500 ml of p-xylene. The extraction vessel was
closed/sealed with a gasket-equipped lid. The thermocouple (19.1)
was inserted and the lid of vessel (1) was closed with eight bolts.
The thermocouple screw was also tightened. The regulating valves
(13.3) and (13.6) were attached to the top and the bottom of the
extraction vessel. The stainless steel tubing was connected with
the out-coming from the pump, to the top-regulating valve (13.1).
The stainless steel tubing, wrapped with heating tape, was also
connected to the bottom-regulating valve (13.9).
[0161] B. 2.2. Packing the Adsorption Vessel (2)
[0162] Filter paper, cotton filter and another filter paper were
placed on the dispersion plate at the bottom of the adsorption
vessel (2). Approximately 300 g of aluminum oxide (active acidic,
activity #1, 7--230 Mesh ASTM) with 0.6 L of p-xylene was poured
carefully and stirred into the adsorption vessel (4). Filter paper
was placed on top of the aluminum oxide, then 400 g of dry
calcinated sand, followed by another filter paper and finally a
dispersion plate. The adsorption vessel (4) was closed with a lid,
the thermocouple (19.2) was inserted and then the lid was tightened
with twelve bolts. After tightening the lid, the thermocouple
(19.2) was tightened with a screw. The regulating valve (13.4) was
attached to the lid of the adsorption vessel. Stainless steel
tubing was connected, out-coming from the extraction vessel, to the
top-regulating valve (13.4). Stainless steel tubing was also
connected from the bottom of the adsorption vessel (2) to the
regulating valve (13.7).
[0163] C. Assembly of the Solvent Distillation System (4)
[0164] The coupling joint was connected to the stainless steel
tubing, out-coming from the regulating valve (13.7). A 5-liter
distilling flask (4) was placed into the distilling flask heater
(16) and the coupling joint inserted. The Wurtz distillation
adapter (8) was placed into the neck of the distilling flask. A
thermometer (18) was installed into the Wurtz adapter (8). A
stopper (7) was placed into the central neck of the distilling
flask. The distillation adapter (9) was attached to the receiving
flask and attached to a large condenser (6). The top of the large
condenser (6) was adjusted to the Wurtz distillation adapter (8).
The drying tube was placed on top of the small condenser (10). The
extraction vessel out-coming tubing was connected to the receiving
flask through the coupling joint.
[0165] D. Extraction Process
[0166] About 3500 ml of p-xylene was added to the distilling flask.
The distilling flask heater was turned on. The solvent was warmed
up to its boiling point. The temperature on the heating tape (11)
and the heaters (14, 15) was set to about 130.degree. C. The
extraction vessel (1), adsorption vessel (2) and tubing had an
average temperature of about 120-130.degree. C. Valves 13.2 and
13.8 were closed, while valves 13.1, 13.3, 13.4, 13.6, 13.7 and
13.9 were open. When the receiving flask (5) was half filled with
freshly distilled p-xylene, the pump was turned on. The pumping
speed was equal to the speed of distillation (about 65-70 ml/min).
The extraction was continued for about 2.5 hours and then the pump
was turned off. All valves were then closed except for valve
(13.5). About 1500 ml of p-xylene was distilled. The distilling
flask heater was turned off. The extract (1.sup.st-fraction) was
transferred from vessel (4) to the 4 L-flat bottom flask (20). The
1.sup.st-fraction, in crystallization flask (20), was placed in a
freezer to enable betulin crystallization at a temperature of about
-5 C. The total volume of the 1.sup.st-fraction extract was
approximately 2300 ml.
[0167] Approximately 1800 ml of p-xylene and 50 ml of acetic acid
was added to the distilling flask (5). About 30 ml of acetic acid
was added to the receiving flask (5). The distilling flask heater
was turned on. The solvent was heated to its boiling point. The
heating tape (15) and heaters (14, 15) were turned on. The
temperature of the vessels and tubing was maintained at
approximately 100.degree. C. Valves 13.2 and 13.8 were closed,
while valves 13.1,13.3, 13.4,13.6, 13.7 and 13.9 were open. Pump
(3) was turned on. The pumping speed was equal to the speed of
distillation (about 65-70 ml/min). The extraction was continued for
2.5 hours and the pump (3) was turned off. All valves, except valve
13.5, were closed. Approximately 1500 ml of solvent was distilled
into the receiving flask (5). The distilling flask heater was
turned off. The contents from the distilling flask (4)
(2.sup.nd-fraction) were transferred into the 3 L-flat bottom flask
(21). Valves 13.1 and 13.8 were closed, while vales 13.2, 13.3,
13.4, 13.6, 13.7, and 13.9 were open. The nitrogen flow valve
(13.2) was opened (200 ml of N.sub.2/min) for 30 minutes and the
residue extract was collected in the distilling flask. The flown
extract was transferred to flask (21). The total volume of the
2.sup.nd-fraction extract was about 2400 ml. The distilled solvent
was transferred from the receiving vessel (5) to the 3 L flat
bottom flask for solvent recycling (22).
[0168] The temperature in the extraction system was maintained
around 100C. 5 L of 95% i-propanol-5% water solvent was prepared
and 1500 ml was loaded in the receiving flask (5) and 3500 ml into
the distilling flask (4). The distilling flask heater was then
turned on. The solvent was heated to its boiling point. Valves 17.2
and 17.8 were closed, while valves 13.1, 13.3, 13.4, 13.6, 13.7 and
13.9 were opened. The pump was then turned on. The pumping speed
was maintained at a level equal to the speed of distillation (about
60-70 ml/min). The extraction continued for 2.5 hours, after which,
the pump was turned off and all valves, except the valve 13.5, were
closed. Approximately 1500 ml of solvent was distilled into the
receiving flask. The distilling flask heater was turned off. The
extract (3.sup.rd-fraction) was transferred from the distilling
flask (4) into the flat bottom flask (23). Valves 13.1 and 13.8
were closed, while valves 13.2, 13.3, 13.4, 13.6, 13.7, and 13.9
were open. The nitrogen flow valve (13.2) was opened (200 ml/min)
for 30 min. The flown extract was collected in vessel (4) and
transferred to flask (23). The total volume of the
3.sup.rd-fraction extract was approximately 2200 ml. The distilled
solvent from the receiving vessel (5) was transferred into the
flask for solvent recycling (24).
[0169] Step 3: Birch Bark Triterpenes Isolation
[0170] A. Betulin Isolation
[0171] The 1.sup.st fraction solution (2.3 L from flask 20) was
cooled down at a temperature of about -5.degree. C. in a freezer
for about 4 hours. The white precipitate was then filtered using a
Buchner funnel 600 ml and Bunzen flask 4 L. The crystals in the
funnel were washed with p-xylene (2.times.100 ml) at a temperature
of approximately 5-10.degree. C. The white crystals were dried in
vacuum at a temperature of about 90.degree. C. up to the constant
weight. About 145 g of 98%.sup.+ pure betulin was obtained (gas
chromatography (GC), high performance liquid chromatography
(HPLC)). The yield of betulin from starting birch bark pellets was
about 12%.
[0172] B. Lupeol Isolation
[0173] The liquid portion (about 2500 ml) remaining after betulin
filtration was evaporated under reduced pressure in a rotor
evaporator. About 32-35 g of dry material was crystallized twice
with dry acetone. The white crystals of lupeol were then dried in
vacuum up to the constant weight. Approximately 14.6 g of 95%.sup.+
pure lupeol was obtained (GC, HPLC). The yield of 95%.sup.+ pure
lupeol from starting birch bark pellets was about 1.2%.
[0174] C. Betulinic Acid Isolation
[0175] The 2.sup.nd-fraction solution (2.4 L from flask 22) was
evaporated under reduced pressure to obtain 27 g of a dark colored
betulinic acid fraction containing about 60% betulinic acid, as
determined by HPLC analysis. The fraction containing approximately
60% of betulinic acid, approximately 27 g, was boiled with 400 ml
of isopropanol and 4.2 g of sodium hydroxide for about 2 hours. The
solution was cooled to a temperature of about 40.degree. C. and the
isopropanol was evaporated under reduced pressure. The remaining
solid material was transferred into the Soxlet apparatus and
extracted with 300 ml of p-xylene for 3 hours. The solid material
was dried under vacuum and transferred to a 1 L beaker to which 700
ml of water was added and stirred at about 1000 rpm for about 1 h.
The precipitate was filtered and washed with about 100 ml of 5%
sodium hydroxide (pH >10). The remaining solid material was
transferred into a 1 L beaker to which 300 ml of water was added
and acidified with approximately 5% hydrochloric acid (pH=5.2-5.5).
The precipitate was filtered and dried on a filter. The dried
precipitate was then crystallized twice from i-propanol.
Approximately 12 g of 95%.sup.+ pure betulinic acid was obtained
(GC, HPLC). The yield of betulinic acid from starting birch bark
pellets was approximately 1.0%.
[0176] C. Betulin 3-Caffeate Isolation
[0177] The 3.sup.rd-fraction solution (2.2 L, flask 23) of the
crystallization flask was evaporated under reduced pressure.
Approximately 47 g of a brown betulin-3-caffeate fraction, which
contains 48% betulin-3-caffeate by HPLC analysis, was obtained.
About 15 g of betulin-3-caffeate was obtained (93%.sup.+ pure,
HPLC, nuclear magnetic resonance (NMR)) by column chromatography on
silica with ether/hexane=6/4. The yield of betulin-3-caffeate from
starting birch bark pellets was about 1.25%.
[0178] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details described
herein may be varied considerably without departing from the basic
principles of the invention.
* * * * *