U.S. patent application number 09/741215 was filed with the patent office on 2001-05-03 for method for the prevention and treatment of chronic venous insufficiency.
Invention is credited to Farrow, Thomas M., Jia, Qi, Mahiou, Belaid, Nissanka, Ajith, Qiu, Zhihua.
Application Number | 20010000731 09/741215 |
Document ID | / |
Family ID | 23287270 |
Filed Date | 2001-05-03 |
United States Patent
Application |
20010000731 |
Kind Code |
A1 |
Jia, Qi ; et al. |
May 3, 2001 |
Method for the prevention and treatment of chronic venous
insufficiency
Abstract
The present invention provides a method for the prevention and
treatment of chronic venous insufficiency. Specifically, the
present invention provides a method for the prevention and
treatment of varicose veins, particularly hemorrhoids by
application of an effective amount of an isoquinoline alkaloid.
Included in this invention is an improved method for isolating and
purifying alkaloids, particularly isoquinoline alkaloids from
plants.
Inventors: |
Jia, Qi; (Arvada, CO)
; Qiu, Zhihua; (Westminster, CO) ; Nissanka,
Ajith; (Boulder, CO) ; Mahiou, Belaid;
(Westminster, CO) ; Farrow, Thomas M.; (Denver,
CO) |
Correspondence
Address: |
Swanson & Bratschun, L.L.C.
Suite 330
1745 Shea Center Drive
Highlands Ranch
CO
80129
US
|
Family ID: |
23287270 |
Appl. No.: |
09/741215 |
Filed: |
December 19, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09741215 |
Dec 19, 2000 |
|
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09329848 |
Jun 11, 1999 |
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Current U.S.
Class: |
424/725 ;
514/277 |
Current CPC
Class: |
A61K 31/485 20130101;
A61K 31/473 20130101 |
Class at
Publication: |
424/725 ;
514/277 |
International
Class: |
D21C 001/00 |
Claims
What is claimed is:
1. A method of prophylaxis and treatment of varicose veins and
hemorrhoids comprised of the administration to a patient in need
thereof an effective amount of a composition containing an
isoquinoline alkaloid or analog thereof.
2. The method of claim 1 wherein said isoquinoline alkaloid is
selected from an aporphine alkaloid or a benzophenanthridine
alkaloid.
3. The method of claim 2 wherein said aporphine alkaloid is
selected from the group of compounds having the following
structure: 6wherein R.sub.1 and R.sub.2 are independently selected
from the group consisting of H, alkyl, substituted alkyl,
cycloalkyl, substituted cycloalkyl, alkenyl or substituted alkenyl;
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are
independently selected from the group consisting of H, hydroxy,
thiol, methoxy, methyl sulfide, methylenedioxy, alkoxy, alkyl
sulfide; and the pharmaceutically acceptable acid addition salts,
selected from the group consisting of chloride, iodide, fluoride,
sulfate, phosphate, acetate or carbonate and a pharmaceutically
acceptable carrier thereof.
4. The method of claim 2 wherein said aporphine alkaloid is
selected from Magnoflorine or Laurifoline.
5. The method of claim 2 wherein said benzophenanthridine alkaloid
is selected from the group of compounds having the following
structure: 7wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are
independently selected from the group consisting of H, hydroxy,
alkoxy, methoxy, methylenedioxy, thiol, methyl sulfide and alkyl
sulfide; and R.sub.5 is selected from the group consisting of H,
alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,
alkenyl and substituted alkenyl; and the pharmaceutically
acceptable acid addition salts selected from the group consisting
of chloride, iodide, fluoride, sulfate, phosphate, acetate and
carbonate and a pharmaceutically acceptable carrier thereof.
6. The method of claim 2 wherein said benzophenanthridine alkaloid
is Chelerythrine.
7. The method of claim 1 wherein said isoquinoline alkaloid is
obtained by organic synthesis.
8. The method of claim 1 wherein said isoquinoline is isolated from
a plant.
9. The method of claim 1 wherein said plant is selected from the
group consisting of the following genera: Zanthoxylum, Tinospora,
Mahonia, Phellodendron, Aristolochia, Magnolia, Thalictrum, Coptis,
Epimedium, Ranunculus, Sinomenium, Nandina, Manodora, Berberis,
Fumaria, Chelidonium, Pachygone, Dioscoreophyllum, Glaucium,
Clematis, Aconitum or Cocculus, Xanthoxylium, Toddalia, Papaver,
Hypecoum, Hylomecon, Prantl, Argemone, Eschscholtxia, Sanguinaria,
Corydalis, Dicentra, Fagara, Symphoricarpos, Bocconia, Xylocarpus,
and Mocleaya.
10. The method of claim 8 wherein the isoquinoline alkaloid is
obtained from the stem and root bark or whole plant.
11. The method of claim 1 wherein the isoquinoline alkaloid is
formulated as a component in an herb powder, a crude herb extract,
as a mixture containing enriched isoquinoline alkaloids from the
plant source, or as a substantially purified compound.
12. The method of claim 1 wherein the composition is
pharmaceutically formulated for enteral administration, parenteral
administration and topical application at a dose selected from 0.01
to 50 mg/kg of body weight.
13. A method for the isolation and purification of a isoquinoline
alkaloids from a plant comprising: (a) extraction of the ground
biomass of a plant containing isoquinoline alkaloids with a
solvent; and (b) neutralization and concentration of the
neutralized extract; and.
14. The method of claim 13 further comprising: (c) purification of
said extract by a chromatographic method.
15. The method of claim 13 wherein the plant is selected from the
group consisting Araceae, Aristolochiaceae, Berberidaceae,
Caprifoliaceae, Euphorbiaceae, Fumariaceae, Helleboraceae,
Lauraceae, Magnoliaceae, Menispermaceae, Mrliaceae, Papaveraceae,
Ranunculaceae, Rharnaceae and Rutaceae.
16. The method of claim 13 wherein the plant is selected from the
group consisting of: Zanthoxylum, Tinospora, Mahonia,
Phellodendron, Aristolochia, Magnolia, Thalictrum, Coptis,
Epimedium, Ranunculus, Sinomenium, Nandina, Manodora, Berberis,
Fumaria, Chelidonium, Pachygone, Dioscoreophyllum, Glaucium,
Clematis, Aconitum or Cocculus, Xanthoxylium, Toddalia, Papaver,
Hypecoum, Hylomecon, Prantl, Argemone, Eschscholtxia, Sanguinaria,
Corydalis, Dicentra, Fagara, Symphoricarpos, Bocconia, Xylocarpus,
and Mocleaya.
17. The method in claim 13 wherein the biomass is extracted in a
dynamic mode.
18. The method of claim 17 wherein said dynamic mode is a vat
extractor.
19. The method of claim 13 wherein the biomass is extracted in a
static mode.
20. The method of claim 19 wherein said static mode is a column
extractor.
21. The method of claim 14 wherein said chromatographic method is
selected from ion exchange chromatography, absorption
chromatography, reverse phase chromatography, size exclusive
chromatography, ultra-filtration or a combination of two or more of
these methods.
Description
FIELD OF INVENTION
1. The present invention relates generally to a method for the
prevention and treatment of chronic venous insufficiency.
Specifically, the present invention relates generally to a method
for the prevention and treatment of varicose veins, particularly
hemorrhoids by administration of an isoquinoline alkaloid. Included
in this invention is an improved method for isolating and purifying
alkaloids, particularly isoquinoline alkaloids from plant
sources.
BACKGROUND OF THE INVENTION
2. Varicose veins are swollen and knotted veins that are both
unsightly and uncomfortable. Up to 20% of the adult population have
varicose veins and experience discomfort as a result. They can
cause swelling of the legs and feet and the skin to itch. Factors
such as prolonged standing or sitting, obesity and pregnancy play a
large part in the development of varicosity. Varicose veins can
occur in almost any part of the body, however they most often occur
in the calf or on the inside of the leg between the groin and the
ankle.
3. Hemorrhoids are a varicose dilation of the veins around the
anus. Hemorrhoids are similar to varicose veins in the legs, in
that the supporting walls of the veins weaken. According to the
National Institutes of Health, about half of the United States
population over age 50 suffer from hemorrhoids. While most
hemorrhoids are not serious, they can have a major effect on one's
quality of life. Prevention and treatment of hemorrhoids can
include dietary changes, stool softeners, sitz baths and
application of conventional topical anti-inflammatory ointments,
such as Hemorid, Hemcure, Hemorr-X, Delicare, Preparation H and
Ultroid. In more extreme cases, destroying the hemorrhoid by
freezing or heating, ligation of the hemorrhoid or even laser
surgery may be warranted. There are also a variety of traditional
remedies available in India, China and other countries.
4. Alkaloids are basic nitrogenous organic compounds of plant
origin. Isoquinoline alkaloids are a class of alkaloids derived
from isoquinoline which has the following structure: 1
5. Aporphine alkaloids, are a class of isoquinoline alkaloids,
which have the following general structure: 2
6. wherein R.sub.1 and R.sub.2 are independently selected from the
group consisting of H, alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, alkenyl or substituted alkenyl, methylene;
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are
independently selected from the group consisting of H, hydroxy,
thiol, methoxy, methyl sulfide, methylenedioxy, alkoxy, alkyl
sulfide or pharmaceutically acceptable acid addition salts,
selected from the group consisting of chloride, iodide, fluoride,
sulfate, phosphate, acetate or carbonate. Aporphine alkaloids have
been isolated from the genera of more than twenty plant families,
including but not limited to, Araceae, Aristolochiaceae,
Berberidaceae, Euphorbiaceae, Helleboraceae, Lauraceae,
Magnoliaceae, Menispermaceae, Papaveraceae, Ranunculaceae,
Rhamnaceae and Rutaceae. Within these plant families aporphine
alkaloids have been isolated from species in numerous genera,
including but not limited to, Aconitum, Aristolochia, Berberis,
Chelidonium, Clematis, Cocculus, Coptis, Dioscoreophyllum,
Epimedium, Fumaria, Glaucium, Magnolia, Mahonia, Manodora, Nandina,
Pachygone, Phellodendron, Ranunculus, Sinomenium, Thalictrum,
Tinospora and Zanthoxylum. Aporphine alkaloids can be isolated from
the whole plant, stems, stem bark, twigs, tubers, flowers, fruits,
roots, root barks, young shoots, seeds, rhizomes and aerial parts.
To date, more than 500 aporphine alkaloids have been synthesized
and/or isolated from more than 90 genera of plants.
7. Aporphine alkaloids have been reported as having diverse
biological activity. Compounds within this class of alkaloids have
been patented for the treatment of duodenal ulcers and epileptic
seizures (U.S. Pat. Nos. 4,543,256 and 4,543,256), cardiac
arrhythmia (U.S. Pat. No. 5,594,033), hypertension (U.S. Pat. No.
4,120,964) and colds and allergies (U.S. Pat. No. 4,461,895).
Compounds within this class of alkaloids have also been reported to
enhance dopaminergic activity by inactivating a dopamine receptor
(U.S. Pat. Nos. 4,353,912 and 4,687,773), improve circulatory
performance (U.S. Pat. Nos. 4,761,417 and 5,153,178) and improve
wound-healing (U.S. Pat. Nos. 5,156,847 and 5,474,782). Finally,
compounds within this class of alkaloids have been reported as an
antidote to counteract the effects of cocaine, as appetite
suppressants (U.S. Pat. No. 5,258,384), as having analgesic and
antitussive effects (U.S. Pat. Nos. 4,358,592, 4,265,912 and
4,315,010) and antipsychotic or sedative effects (U.S. Pat.
No.4,687,773). A number of these compounds have been prepared by
synthetic processes (see, e.g., U.S Pat. Nos. 4,309,542 and
4,202,980).
8. Magnoflorine (see FIG. 1) is a typical aporphine alkaloid, which
is widely distributed in a number of plant families, including but
not limited to, Aristolochiaceae, Berberidaceae, Helleboraceae,
Magnoliaceae, Menispermaceae, Papaveraceae, Ranunculaceae,
Rhamnaceae and Rutaceae. Magnoflorine has been shown to decrease
arterial blood pressure by activation of the nicotinic receptors in
the parasympathetic ganglia and the release of the vasodilator ACh
in a dose-dependent manner, without affecting the heart rate and
the respiration; induce dose-dependent hypothermia resulting from
peripheral vasodilation; induce contractions of isolated rat
uterus; and suppress the induction phase, but not the effector
phase of the cellular immune response. This compound has also been
reported as having antimicrobial activity, significant
cytotoxicity, and to inhibit lipoxygenase. To date there have been
no reports of the use of this compound for the prevention and
treatment of venous insufficiency.
9. Benzophenanthridine alkaloids, are another class of isoquinoline
alkaloids having the following structure: 3
10. wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently
selected from the group consisting of H, hydroxy, alkoxy, methoxy,
methylenedioxy, thiol, methyl sulfide and alkyl sulfide; and
R.sub.5 is selected from the group consisting of H, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl and
substituted alkenyl. Benzophenanthridine alkaloids have been
isolated from the genera of numerous plant families, including but
not limited to, Rutaceae, Fumariaceae, Papaveraceae,
Caprifoliaceae, Mrliaceae. Within these plant families these
alkaloids can be isolated from numerous genera including, but not
limited to, Zanthoxylum, Xanthoxylium, Toddalia, Chelidonium,
Papaver, Hypecoum, Hylomecon, Prantl, Argemone, Eschscholtxia,
Sanguinaria, Corydalis, Dicentra, Fumaria, Fagara, Symphoricarpos,
Bocconia, Xylocarpus, and Mocleaya. Benzophenanthridine alkaloids
have also been reported as having diverse biological activity.
Chelerythrine (see FIG. 1) has been reported for use as an
anti-hypertensive and as an inhibitor of platelet aggregration.
(U.S. Pat. No. 5,137,912, issued 1992).
11. Zanthoxylum (also referred to in the literature as
Xanthoxylum), a member of the Yellow Wood family (Rutaceae), is a
widely distributed genus of plants which has more than thirty
species. Two species of Zanthoxylum are indigenous to the mainland
of the United States: Z. americanum Mill. (Northern prickly Ash)
and Z. clava-herculis L. (Southern prickly Ash), these two species
are referred to herein collectively as Prickly Ash. Prickly Ash,
commonly known as the toothache tree, is a shrub or small tree,
5-10 feet in height, which has had a long history as a botanical
remedy. It is a traditional native North American remedy for
toothaches. This plant has also been used as an internal treatment
for ulcers, skin sores, diarrhea, indigestion and circulatory
problems; and as an external remedy for chronic joint pain and
rheumatism. The plant parts used are the bark and fruit. The stem
bark of Zanthoxylum is a rich source of alkaloids and lignans.
Chemical investigations on the bark of Prickly Ash have led to the
isolation of a number of lignans and alkaloids, including both
benzophenanthridine and aporphine alkaloids. Northern Prickly Ash
is native to southern Canada and northern, central and western
parts of the United States. The bark is harvested in spring and
fall and the berries are collected in summer. Dry prickly ash bark
powders are available in the botanical raw materials market.
Southern Prickly Ash is native to central and southern United
States.
12. There have been numerous reports of Prickly Ash and its active
ingredients having diverse biological activity, including high
protein kinase C inhibition activity (Chelerythrine),
anti-hypertensive activity (Asarinin), antiplatelet activity (total
alkaloids and coumarins), anti-malarial activity (crude extract),
neuromuscular effects (crude extract), anti-sickling activity,
(benzoic acid derivatives), cytotoxic activity (alkaloids), and
elevated cytochrome P450 (essential oil).
13. Terry et al. have disclosed a method of treating vascular
disorders by administration of Xanthoxylum (Zanthoxylum). (Terry et
al. U.S. patent application Ser. No. 5,562,906, issued Oct. 8,
1996). Terry et al. did not disclose any specific compounds from
the Zanthoxylum that could be used for this purpose. Other reported
uses of extracts from the Zanthoxylum genus include use in herbal
compositions (Japanese Patent No. 07324039JP, 1995); use in herbal
powders for the treatment of addictive diseases (U.S. Pat. No.
5,198,230, 1993); use as a remedy for anemia and arthritis (U.S.
Pat. No. 4,767,626, 1988); use in a cream to enhance male sexual
function (Japan Patent No. 06211678JP, 1994); and use as a hair
tonic (Japan Patent No.05201833JP, 1976).
14. Current products containing Prickly Ash bark as an ingredient
include Multi nutrition Fruit Drink (minor ingredient);
Pro-Essence(detoxic) herbal formula--aqueous extract (major
ingredient); Circulatory tonic--aqueous extract (major ingredient);
and Self defense (bark powder).
SUMMARY OF THE INVENTION
15. The present invention includes a method for the prevention and
treatment of chronic venous insufficiency. Specifically, the
present invention includes a method for the prevention and
treatment of varicose veins, particularly hemorrhoids by
administration to a patient in need thereof an effective amount of
an isoquinoline alkaloid. In one embodiment of the present
invention the isoquinoline alkaloid is selected from the group
consisting of aporphine alkaloids and/or benzophenanthridine
alkaloids. In a preferred embodiment the isoquinoline alkaloid is
selected from the group consisting of Magnoflorine, Laurifoline and
Chelerythrine. The isoquinoline alkaloid may be obtained by organic
synthesis or it may be isolated from a plant containing said
alkaloid.
16. This invention also includes an improved method for isolating
and purifying isoquinoline alkaloids from plants containing these
compounds. The improved method of the present invention comprises:
extraction of the ground biomass of a plant containing a
isoquinoline alkaloid; neutralization and concentration of the
neutralized extract; and purification of said extract by
chromatography. The present invention provides a commercially
viable process for the isolation and purification of isoquinoline
alkaloids having desirable physiological activity.
17. The advantages of using isoquinoline alkaloids, particularly
Magnoflorine, Laurifoline and Chelerythrine, for the prevention and
treatment of varicose veins, particularly hemorrhoids is that they
are a natural alternative to conventional ointments, can be
administered in convenient oral dosage with a short course of
treatment, and they exhibit a remarkable curative effect against
hemorrhoids. Additionally, these compounds are safe.
18. It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE FIGURES
19. FIG. 1 depicts the chemical compounds isolated and identified
from Prickly Ash bark as described in Examples 2 and 3.
20. FIG. 2 illustrates graphically the results of the hemorrhoid in
vivo assay using Prickly Ash extract, as described in Example
5.
21. FIG. 3 illustrates graphically the results of the hemorrhoid in
vivo assay using three of the isoquinoline alkaloids isolated from
Prickly Ash, as described in Example 6.
22. FIG. 4 illustrates graphically the dose dependent reduction of
the swelling of the recto-anus induced by croton oil by Prickly Ash
Extract as described in Example 7.
DETAILED DESCRIPTION OF THE INVENTION
23. The present invention includes a method for the prevention and
treatment of chronic venous insufficiency. Specifically, the
present invention includes a method for the prevention and
treatment of varicose veins, particularly hemorrhoids by
administration to a patient in need thereof an effective amount of
an isoquinoline alkaloid.
24. In one embodiment the isoquinoline alkaloid is an aporphine
alkaloid having the following general structure: 4
25. wherein R.sub.1 and R.sub.2 are independently selected from the
group consisting of H, alkyl, substituted alkyl, cycloalkyl,
substituted cycloalkyl, alkenyl or substituted alkenyl, methylene;
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are
independently selected from the group consisting of H, hydroxy,
thiol, methoxy, methyl sulfide, methylenedioxy, alkoxy, alkyl
sulfide or pharmaceutically acceptable acid addition salts,
selected from the group consisting of chloride, iodide, fluoride,
sulfate, phosphate, acetate or carbonate. In a preferred embodiment
the aporphine alkaloid is selected from Magnoflorine, wherein
R.sub.1 and R.sub.2=CH.sub.3; R.sub.3 and R.sub.8 =H; R.sub.4 and
R.sub.7=OCH.sub.3 and R.sub.5 and R.sub.6=OH or Laurifoline,
wherein R.sub.1 R.sub.2=CH.sub.3; R.sub.3 and R.sub.6=OH; R.sub.4
and R.sub.7=OCH.sub.3 and R.sub.5 and R.sub.8=H. (see FIG. 1).
26. The aporphine alkaloids of this invention may be obtained by
synthetic methods or may be isolated from a plant source. The
aporphine alkaloids of this invention can be isolated from the
genera of numerous plant families, including but not limited to,
Araceae, Aristolochiaceae, Berberidaceae, Euphorbiaceae,
Helleboraceae, Lauraceae, Magnoliaceae, Menispernaceae,
Papaveraceae, Ranunculaceae, Rhamnaceae and Rutaceae. Within these
plant families these alkaloids can be isolated from Zanthoxylum,
Tinospora, Mahonia, Phellodendron, Aristolochia, Magnolia,
Thalictrum, Coptis, Magnolia, Epimedium, Ranunculus, Sinomenium,
Nandina, Manodora, Berberis, Fumaria, Chelidonium, Pachygone,
Dioscoreophyllum, Glaucium, Clematis, Aconitum and Cocculus. In a
preferred embodiment the plant is selected from the Zanthoxylum
genus of plants, a member of the Rutaceae family. In the most
preferred embodiment the plant is selected from two species of
Zanthoxylum genus: Z. americanum Mill. (Northern prickly Ash) and
Z. clava-herculis L. (Southern prickly Ash).
27. In a second embodiment the isoquinoline alkaloid is a
benzophenanthridine alkaloid having the following structure: 5
28. wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are independently
selected from the group consisting of H, hydroxy, alkoxy, methoxy,
methylenedioxy, thiol, methyl sulfide and alkyl sulfide; and
R.sub.5 is selected from the group consisting of H, alkyl,
substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl and
substituted alkenyl. In a preferred embodiment the
benzophenanthridine alkaloid is Chelerythrine, wherein R.sub.1 and
R.sub.2=OCH.sub.3; R.sub.3 and R.sub.4 together=CH.sub.2;
R.sub.5=CH.sub.3.
29. The benzophenanthridine alkaloids of this invention may be
obtained by synthetic methods or may be isolated from a plant
source. The benzophenanthridine alkaloids of this invention can be
isolated from the genera of numerous plant families, including but
not limited to Rutaceae, Fumariaceae, Papaveraceae, Caprifoliaceae,
Mrliaceae. Within these plant families these alkaloids can be
isolated from Zanthoxylum, Xanthoxylium, Toddalia, Chelidonium,
Papaver, Hypecoum, Hylomecon, Prantl, Argemone, Eschscholtxia,
Sanguinaria, Corydalis, Dicentra, Fumaria, Fagara, Symphoricarpos,
Bocconia, Xylocarpus, and Mocleaya. In a preferred embodiment the
plant is selected from the Zanthoxylum genus of plants, a member of
the Rutaceae family. In the most preferred embodiment the plant is
selected from two species of Zanthoxylum: Z. americanum Mill.
(Northern prickly Ash) and Z clava-herculis L. (Southern prickly
Ash).
30. The isoquinoline alkaloids of this invention can be
administered by any method known to one of ordinary skill in the
art. The modes of administration include, but are not limited to,
enteral (oral) administration, parenteral (intravenous,
subcutaneous, and intramuscular) administration and topical
application. The method of treatment according to this invention
comprises administering internally or topically to a patient in
need thereof an effective amount of the isoquinoline alkaloid.
Doses of the isoquinoline alkaloids and pharmaceutical compositions
containing same are an efficacious, nontoxic quantity generally
selected from the range of 0.01 to 50 mg/kg of body weight. Persons
skilled in the art using routine clinical testing are able to
determine optimum doses for the particular ailment being
treated.
31. The preparation of products for administration in
pharmaceutical preparations may be performed by a variety of
methods well known to those skilled in the art. The isoquinoline
alkaloid may be formulated in an herb powder, a crude herb extract,
as a mixture containing enriched isoquinoline alkaloids from the
plant source or as the substantially purified compound, isolated
from a plant source or prepared by synthetic methods.
32. This invention also includes an improved method for isolating
and purifying isoquinoline alkaloids from plants. This improved
method comprises: extraction of the ground biomass of a plant
containing a isoquinoline alkaloid; neutralization and
concentration of the neutralized extract; and purification of said
extract by chromatography. As provided above, these alkaloids,
particularly aporphine alkaloids have been isolated from the genera
of more than twenty plant families. The method of this invention
can be extended to the isolation of these compounds from any plant
source containing these compounds. In one embodiment the plant used
is selected from the family of plants including, but not limited
to, Araceae, Aristolochiaceae, Berberidaceae, Caprifoliaceae,
Euphorbiaceae, Fumariaceae, Helleboraceae, Lauraceae, Magnoliaceae,
Menispermaceae, Mrliaceae, Papaveraceae, Ranunculaceae, Rhamnaceae
and Rutaceae. Within these plant families the plant used to isolate
aporphine alkaloids is selected from a number of genera including,
but not limited to, Zanthoxylum, Tinospora, Mahonia, Phellodendron,
Aristolochia, Magnolia, Thalictrum, Coptis, Magnolia, Epimedium,
Ranunculus, Sinomenium, Nandina, Manodora, Berberis, Fumaria,
Chelidonium, Pachygone, Dioscoreophyllum, Glaucium, Clematis,
Aconitum and Cocculus. For the isolation of benzophenanthridine
alkaloids the plant is selected from a number of genera including,
but not limited to Zanthoxylum, Xanthoxylium, Toddalia,
Chelidonium, Papaver, Hypecoum, Hylomecon, Prantl, Argemone,
Eschscholtxia, Sanguinaria, Corydalis, Dicentra, Fumaria, Fagara,
Symphoricarpos, Bocconia, Xylocarpus, and Mocleaya. In a preferred
embodiment the plant is selected from the Zanthoxylum genus of
plants, a member of the Rutaceae family. In the most preferred
embodiment the plant is selected from two species of Zanthoxylum
genus: Z. americanum Mill. (Northern prickly Ash) and Z.
clava-herculis L. (Southern prickly Ash).
33. Isoquinoline alkaloids can be isolated from various parts of
the plant including, but not limited to, the whole plant, stems,
stem bark, twigs, tubers, flowers, fruit, roots, root barks, young
shoots, seeds, rhizomes and aerial parts. In a preferred embodiment
the alkaloids are isolated from the stem and stem bark or the whole
plant.
34. The solvent used for extraction of the ground biomass of the
plant includes, but is not limited to water, acidified water, an
acidified water miscible hydroxylated organic solvent including,
but not limited to, methanol or ethanol and an acidified mixture of
alcohol or other water miscible hydroxylated organic solvent and
water. In one embodiment the acidic extract is neutralized to pH of
4.5-5.5 with aqueous base. The neutralized extract is then
concentrated and dried to yield a powder. The alkaloids can then be
purified by various chromatographic methods including, but not
limited to, ion exchange chromatography, absorption chromatography,
reverse phase chromatography, size exclusive chromatography,
ultra-filtration or a combination of two or more of these
chromatographic methods.
35. Example 1 describes the isolation and identification of the
chemical components of prickly ash bark. Using the method of
Example 1, five alkaloids were isolated and identified from the
bark of Southern Prickly Ash: Asarinin, a lignan, Chelerythrine,
Liriodenine, Magnoflorine and Laurifoline (see FIG. 1).
36. Example 2 describes the determination of the alkaloid content
in different species of Prickly Ash using different methods of
extraction. The results of this example are set forth in Table 1.
These studies demonstrate that isoquinoline alkaloids can be
extracted from plants with deionized (DI) water, acidic aqueous
solutions or high polarity solvents, such as ethanol, methanol,
butanol with or without water. Acidic aqueous solutions are the
best solvents, as they generate high extractable solids and
reasonable amounts of isoquinoline alkaloids. The preferred solvent
was determined to be 1% HCl in MeOH.
37. Example 3 describes a commercial process for the isolation of
isoquinoline alkaloids from Prickly Ash bark. The preferred
commercial extraction process is using a column extractor,
percolating the ground biomass with 0.3% H.sub.2SO.sub.4 at
60.degree. C. The crude extract can be concentrated using a solvent
evaporator, ultra-filtration or column chromatography to reach the
optimum concentration of isoquinoline alkaloids.
38. Example 4 describes the separation of isoquinoline alkaloids
from prickly ash aqueous extract using ion exchange
chromatography.
39. Example 5 illustrates the reduction of the swelling, induced by
croton oil, of the recto-anus in rats by prickly ash extract. The
results are depicted in FIG. 2, which shows that the prickly ash
extract, administered orally or topically, completely inhibited the
swelling of the recto-anus induced by croton oil at both
concentrations studied.
40. Example 6 illustrates the reduction of the swelling, induced by
croton oil, of the recto-anus in rats by individual isoquinoline
alkaloids isolated from Prickly Ash. The results are depicted in
FIG. 3, which shows that Magnoflorine completely inhibited the
swelling of the recto-anus induced by croton oil. Under the same
conditions, Laurifoline and Chelerythrine partially reduced the
swelling.
41. Example 7 illustrates the dose dependent reduction of the
swelling, induced by croton oil, of the recto-anus in rats by
Prickly Ash Extract. As depicted in FIG. 4, the Prickly Ash
extract, administered orally, reduced the swelling of the
recto-anus by 79.5%, 67.5% and 42.1%, at dosage levels of 6.0
mg/kg, 2.0 mg/kg, and 0.67 mg/kg, respectively.
42. The following examples are provided for illustrative purposes
only and are not intended to limit the scope of the invention.
EXAMPLES
Example 1
Isolation and Identification of the Chemical Components of Prickly
Ash Bark
43. Prickly Ash (Zanthoxylum clava-herculis) bark, dried powdered
stem bark (800 g), was exhaustively extracted using hexane followed
by a mixture of methanol and dichloromethane (1:1). The hexane
extract was concentrated to give a brownish oil (105 g) and the
dichloromethane/methanol extract was concentrated to give a brown
solid (102 g). The concentrated hexane extract (20 g) was purified
by column chromatography (38 cm.times.5.0 cm column, silica gel),
eluting with a step-wise gradient of ethyl acetate in hexane (0%-
100%). A solid (1.57 g) was obtained from the fraction eluted with
30% EtOAc/hexane, which was further purified by column
chromatography (38 cm.times.5.0 cm column, silica gel), eluting
with 30% EtOAc/hexane to yield a white solid (1.0 g). The white
solid (1 g) was recrystallized by dissolving it into a minimum
amount of dichloromethane and by precipitating with hexane to give
needle-like crystals (0.17 g). The compound isolated was identified
by spectral analysis as Asarinin, a lignan (FIG. 1). UV
.lambda..sub.max (MeOH) nm: 207, 237, 286, 320; .sup.1H NMR
(CDCl.sub.3) .delta.: 2.82 (q, 1H), 3.28 (m, 2H), 3.82 (m, 2H),
4.07 (d, 4.37 (d, 1H), 4.81 (d, 1H), 5.93 (s, 2H), 5.93 (s, 2H),
6.78 (m, 5H), 6.85 (s, 1H).
44. TLC analysis (2% MeOH in dichloromethane with 1 % ammonium
hydroxide) of the dichloromethane extract revealed the presence of
two major alkaloids. The dichloromethane extract (3.2 g) was
chromatographed on a medium pressure liquid chromatography column
(MPLC), eluting with 6% methanol in dichloromethane. Five fractions
were collected. The first fraction (0.54 g) was further purified by
silica gel preparative thin layer chromatography (TLC) using 2%
methanol in chloroform, to yield a greenish yellow solid (6 mg).
This compound was identified as Chelerythrine (see FIG. 1) by
comparison of its TLC and spectral data with a standard solution of
Chelerythrine. Chelerythrine: UV .lambda..sub.max (MeOH) nm: 236,
282, 319; IR .gamma.(KBr): 2916, 2849, 1737, 1598, 1494, 1365,
1283, 1253, 930, 876, 821.
45. The third fraction (3.22 g) was purified by column
chromatography (38 cm.times.5.0 cm column, silica gel), eluting
with 3% methanol in chloroform to yield 72 mg of a greenish yellow
solid, which was identified as Liriodenine (see FIG. 1). UV
.lambda..sub.max (MeOH) nm: 201.6, 222.2, 271.6, 300.60 (sh),
328.1, 385.6; .sup.1H NMR (CDCl.sub.3) .delta.: 6.28 (s, 2H), 7.05
(s, 1H), 7.47 (t, 1H), 7.64 (m, 2H), 8.41 (d, 1H), 8.48 (d, 1H),
8.70 (d, 1H).
46. A freeze dried sample of Prickly Ash, aqueous extract (Z.
clava-herculis, 20 g) was dissolved in water and chromatographed by
MPLC (CG-161 resin), eluting with a step-wise gradient of methanol
in water (0%, 20%, 40%, 60%, 80% and 100%). Three major fractions
were collected: 4.07 g eluted with 40% methanol, 2.02 g eluted with
60% methanol and 1.5 g eluted with 80% methanol, respectively.
Orange red color spots obtained with Dragendorff s reagent
confirmed the presence of alkaloids in these three fractions. The
fraction eluted with 40% methanol (190 mg) was further purified by
reverse phase preparative HPLC (C18 column; 41.1 mm.times.250 mm),
eluting with a mixture of solvents (water (0.05 M TEA at pH 3.6
with phosphoric acid) and 1% THF in acetonitrile (85:15)) to give
89 mg of a yellow solid, which was further purified on an LH-20
column (2.0 cm.times.40 cm), eluting with DI water to yield 7.9 mg
of a yellow solid, identified as Magnoflorine (see FIG. 1).
(C.sub.20H.sub.24NO.sub.4).sup.+molecular weight 342.50; mass
spectra: ESI+ =342, ESI-=340, FAB=342; UV .lambda..sub.max (MeOH)
nm: 234, 279, 325; IR .gamma.(KBr): 3400, 2932, 2912, 1598 1515,
1462, 1378; .sup.1H NMR (D.sub.2O) .delta.: 2.13 (t,1H), 2.72 (s,
3H), 2.63-3.28 (m, 5H), 3.15(s, 3H), 3.55 (m,1H), 3.77 (s, 3H),
3.83 (s, 3H) 6.77 (m, 2H), 6.87 (s, 1H); .sup.13C NMR: 23.24,
29.61, 42.86, 53.62, 55.98, 60.99, 68.63, 110.41, 111.57, 115.56,
119.03, 120.19, 120.47, 121.21, 125.38, 140.33, 140.63 147.99,
149.04.
47. The fraction eluted with 60% methanol (179 mg) was further
purified by reverse phase preparative HPLC (C18 column: 41.1
mm.times.250 mm), eluting with a mixture of solvents (water (0.05 M
TEA at pH 3.6 with phosphoric acid) and 1% THF in acetonitrile
(90:10)) to yield 42.9 mg of a solid, which was further purified on
an LH-20 column, eluting with water to yield a yellow solid (6.8
mg), which was identified as Laurifoline (see FIG. 1). LC-MS: a
single peak at m/z 342, having a different retention time than
Magnoflorine; (C.sub.20H.sub.24NO.sub.4).su- p.+molecular weight
342.41; mass spectra ESI+m/z=342; UV .lambda..sub.max (EtOH) nm:
230, 282, 306; IR .gamma.(KBr): 3400, 2932, 2912, 1598, 1515, 1462,
1378, 1249, 1044; .sup.1H NMR (D.sub.2O) .delta.: 1.83 (t, 1H),
2.66 (s, 3H), 2.58-3.50 (m, 5H), 3.10 ( 3.67 (s, 3H), 3.72 (s, 3H)
6.46 (s,1H), 6.53 (s, 1H), 7.52 (s, 1H); .sup.13C NMR: 23.96,
28.40, 43.44, 54.08, 56.49, 56.61, 62.04, 69.45, 109.78, 112.58,
115.52, 118.88, 119.55, 120.37, 123.64, 125.92, 141.90, 144.99,
146.59, 148.65.
Example 2
Determination of Alkaloid Content in Different Species of Prickly
Ash Using Different Methods of Extraction
48. Southern Prickly Ash bark powder (Z. clava-herculis, 100 g )
was extracted exhaustively with hot methanol (Soxhlet extraction)
for 48 hours. The methanol extract was evaporated at 40.degree. C.
using a rotary evaporator at low pressure to yield a brown sticky
solid (24.47 g). The solid was analyzed for alkaloid content and
the results are set forth in Table 1. The alkaloid content in the
extract was determined to be 9.1 % and the total alkaloid content
in the bark was determined to be 2.23%.
49. Prickly Ash bark from three different species of Zanthoxylum
(Z. americanum, Z. frazineum and Z. clava-herculis) was ground to a
powder using a mechanical grinder. The three samples (60 g, 60 g,
and 35 g, respectively) were mixed with 600 mL of distilled water
and shaken at room temperature for two hours. The solution was
decanted, filtered and the filtrate was freeze dried to give three
brown colored powders, which were analyzed for alkaloid content.
The results are set forth in Table 1. As can be seen in Table 1,
the aqueous extracts contained a high alkaloid content with low
extractable solids.
50. Southern prickly ash bark (Z. clava herculis), three samples,
60 g each, was extracted with 600 mL of three different acidic
aqueous solutions (5% tartaric acid, 0.6% sulfuric acid, and 1%
HCl, respectively) at 60.degree. C. for two hours. The solution was
filtered through a Buchner funnel, neutralized with NaOH to pH=7
and the solvent was evaporated with a freeze-drier. Each of the
extracts was analyzed for alkaloid content. The results are set
forth in Table 1. As can be seen in Table 1, extraction with acidic
water solutions increases the amount of extractable solids. The
alkaloid concentration in the extracts, however, is decreased.
51. Dried stem bark from three different Zanthoxylum species (Z.
americanum, Z. clava-herculis and Z. frazineum) was ground to a
fine powder and extracted with 1% HCL at 60.degree. C. for two
hours. The solution was filtered through a Buchner funnel,
neutralized with NaOH to pH=7 and the solvent was evaporated with a
freeze-drier. The extracts were analyzed for alkaloid content and
the results are set forth in Table 1. As can be seen in Table 1, Z.
frazineum had a higher total alkaloid content than Z. americanum
and Z. clava-herculis. However, the chemical components of Z.
frazineum are different from that of Southern and Northern Prickly
Ash bark.
Example 3
Commercial Process for the Isolation of Isoquinoline Alkaloids from
Prickly Ash Bark
52. Into a 5 L jacketed reactor equipped with a thermocouple
connected to a digital thermometer, a condenser and mixer system
consisting of a variable speed motor and a 316 SS shaft fitted with
an A100 axial flow impeller was placed 2 L of water containing 0.3%
v/v of concentrated sulfuric acid. The water was heated to
60.degree. C. with a recirculating bath, after which 350 g of
coarsely ground bark (average bulk density 2.9 lb/gal) was added
under vigorous stirring. The suspension was vigorously stirred at
60.degree. C. for 54 hours. Aliquots were removed at various time
intervals, vacuum filtered while hot over a hydrophilic non-woven
polypropylene filter cloth and the crude extract was analyzed for
solids and alkaloids. The results after 54 hours of extraction are
summarized in Table 2.
53. The extraction described above was repeated using 3 L of water.
Analysis of aliquots removed at various time intervals established
that the solid and alkaloid content of the extract leveled off
after 24 hours. The solids were removed by vacuum filtration, as
described above and crude extract was analyzed for solids and
alkaloids. The results are set forth in Table 3.
54. Ground Prickly Ash bark was next extracted using a column
extractor. The column extractor used consisted of a flanged
4"id.times.59"ht fiberglass column (rated at 80 psi @ 212.degree.
F.) equipped with 20-mesh stainless steel frit at the bottom. The
bottom outlet of the extractor was equipped with a stainless steel
ball valve. The extractor was charged with 2.831 kg of coarsely
ground bark (average bulk density of 2.91 lb/gal). The extractor
was capped and fitted with a pressure gauge and a stainless steel
ball valve, the extractor was connected to a peristaltic pump using
PHARMED.RTM.24 tubing. Eight liters of room temperature extraction
fluid (water containing 0.3% concentrated sulfuric acid) was pumped
into the extractor and the mixture was allowed to stand at room
temperature for 60 hours. The solvent (first extract) was removed
and replaced with 3 L of virgin extraction solvent that was
re-circulated with the pump for 6 hours. The two extracts were then
analyzed for solids and alkaloids content. The results are shown in
Tables 4 and 5.
55. In a second experiment, the column extractor described above
was charged with 2.803 kg of coarsely ground bark (average bulk
density of 2.91 lb/gal), and 8 L of extraction fluid (water
containing 0.3% concentrated sulfuric acid) at 60.degree. C. was
pumped into the extractor and the mixture was allowed to stand at
room temperature for 60 hours. The solvent (first extract) was then
removed and the bark was extracted a second time with 10 L of the
extraction solvent at of 60.degree. C. The fluid was re-circulated
for 6 hours at 60.degree. C. using a heat exchanger. The solvent
(second extract) was removed and the solids were blown dry with
air. The two extracts were analyzed for solids and alkaloid content
and the results are set forth in Tables 6 and 7.
56. The pH of the combined extract was raised to 5 with aqueous
sodium carbonate, the resulting material was concentrated 6 fold
then freeze-dried to yield 37 g of dried crude extract.
57. Prickly Ash bark was extracted in a 316 stainless steel column
extractor (flanged 10"id.times.12"ht 316 SS column). The bottom
plate was equipped with 20 mesh screen/frit, the outlet was fitted
with a teflon coated SS ball valve and the inlet was fitted with a
pressure gauge and a teflon coated SS ball valve that was connected
to a peristaltic pump using a PHARMED.RTM.LS 24 tubing. The
extractor was charged with 4.23 kg of coarsely ground bark (average
bulk density of 2.91 lb/gal). After capping the extractor, 12 L of
60.degree. C. extraction fluid (water containing 0.3% v/v
concentrated sulfuric acid) was pumped slowly into the reactor,
which was allowed to stand overnight at room temperature. The first
extract was removed and replaced with 13 L of virgin extraction
fluid heated to 60.degree. C. The second extract was re-circulated
for 6 hours while maintaining the temperature at 60.degree. C.
After the second extract was removed, 5 L of fresh hot solvent was
added and the extractor was allowed to stand overnight at room
temperature. The third extract was removed and the marc was
discarded. Each extract was analyzed for solids and alkaloids
content, the results are shown in Tables 8 and 9. The combined
crude extract was treated with aqueous sodium carbonate to bring
the pH to 5, then concentrated 7 fold. The concentrate was
lyophilized to yield 633 g of dried crude extract.
Example 4
Separation of Isoquinoline Alkaloids from Prickly Ash (Aqueous
Extract) Using Ion Exchange Chromatography
58. Ion exchange chromatography is used extensively in the
separation of ionic organic and inorganic molecules. The quaternary
ammonium alkaloids in the aqueous extract of Prickly Ash are ionic
compounds, therefore the use of ion exchange chromatography is a
useful means to separate the ammonium alkaloids from the crude
extract. Analytical grade macroporous cation resin (AG-MP-50, 25-50
mesh hydrogen form) was packed in medium pressure liquid
chromatography column (MPLC, I.D. 5.0 cm, 19.6 mL/cm). The column
bed was washed with 2N HCl (20 column volumes), 1N NaOH (10 column
volumes) and 1N HCl (10 column volumes). The column was then washed
with distilled water until the pH of the medium was neutral. A
sample of Prickly Ash, aqueous extract (20 g) was mixed with water,
decanted and loaded onto the column. The column was washed with
water until all of the unbound components were removed. The column
was then eluted with ammonium hydroxide (4 column volumes). The
ammonium hydroxide eluent was collected and evaporated to yield
1.96 g total alkaloids.
Example 5
Reduction of the Swelling of the recto-anus in Croton Oil-Induced
Hemorrhoid Model in Rats by Prickly Ash Extract
59. Male SD rats (6 weeks old, approximately 140 g) were purchased
from Harlan Sprague Dawley and allowed to acclimate for 1 week. The
rats were maintained in a pathogen-free facility in accordance with
the National Research Council of Laboratory Animal care and use
guidelines. Each experiment was preformed with age-matched rats 7-
8 weeks old. The croton oil-induced hemorrhoid model in rats was
performed according to the method published by Nishiki (Nishiki et
al. (1988) Folia Pharmacology Japan 92:215- 225; Nishiki et al.
(1988) Folia Pharmacol. Japan 92:227-240). Briefly, a cotton swab
with a diameter of 4 mm soaked with 0.16 mL of inducer (deionized
water: pyridine : ethyl ether: 6% croton oil/ethyl ether (1:4:5:10)
was applied to the rat's anus for 12 seconds. The final
concentration of croton oil was 3%. The edema developed linearly
until 7-8 hours after application and the severity of the edema was
sustained for more than 24 hours. Twenty-four hours later,
recto-anus tissue (approx. 10 mm long) was isolated after the rats
were euthanized. The weights of rat body and recto-anus were
measured. The recto-anus coefficient (RAC) was calculated using the
formula: weight of recto-anus (mg)/body weight (g).
60. The average RAC of croton oil-treated rats was 2.10, which was
38.2% higher than that the rats treated with vehicle only
(RAC=1.52). Vehicle alone did cause significant swelling of the
recto-anus as compared with the rats without any treatment
(RAC=0.98). Twenty-four hours after hemorrhoid induction with
croton oil followed by treatment orally with prickly ash extract at
either 6 mg/kg or 30 mg/kg and by treatment topically with prickly
ash extract at 6 mg/mL in 1:1 aquaphor, the RAC of rats was
determined. The results are depicted in FIG. 2, which shows that
the prickly ash extract completely inhibited the swelling of the
recto-anus induced by croton oil at both concentrations and by both
routes of administration.
Example 6
Reduction of the Swelling of the Recto-Anus in Croton Oil-Induced
Hemorrhoid Model in Rats by Alkaloids Isolated from Prickly Ash
61. This experiment was performed as described in Example 5. The
average RAC of croton oil-treated rats was 1.92, which was 20%
higher than that the rats treated with vehicle only (RAC=1.60) in
this experiment. Vehicle alone did cause significant swelling of
the recto-anus as compared with the rats without any treatment
(RAC=0.98). Twenty-four hours after hemorrhoid induction with
croton oil followed by treatment orally with the pure alkaloids
isolated from prickly ash extract at 1 mg/kg, the RAC of rats was
determined. As shown in FIG. 3, Magnoflorine completely inhibited
the swelling of the recto-anus induced by croton oil. Under the
same conditions, Laurifoline and Chelerythrine partially reduced
the swelling.
Example 7
Dose Dependent Reduction of the Swelling of the Recto-Anus in
Croton Oil-Induced Hemorrhoid Model in rats by Prickly Ash
Extract
62. This experiment was performed as described in Example 5. The
average RAC of croton oil-treated rats was 1.66 which was 40.7%
higher than that of the rats treated with vehicle only (RAC=1.18).
Twenty-four hours after hemorrhoid induction with croton oil,
followed by oral treatment with Prickly Ash extract at doses of 6.0
mg/kg, 2.0 mg/kg, and 0.67 mg/kg, the RAC of rats was determined.
As shown in FIG. 4, the Prickly Ash extract reduced the swelling of
the recto-anus by 79.5%, 67.5% and 42.1%, respectively.
1TABLE 1 Total Extract- Alka- Plant Sample Solids able loid Latin
Name part Weight Solvent (g) Solids content Zanthoxylum Stem 100 g
MeOH 24.47 24.47% 9.1% clava-herculis bark (reflux) Zanthoxylum
Stem 60 g H.sub.2O 2.65 4.4% 35.3% americanum bark Zanthoxylum Stem
60 g H.sub.2O 6.6 11% 22.4% frazineum bark Zanthoxylum Stem 35 g
H.sub.2O 3.58 10.2% 25.4% clava-herculis bark Zanthoxylum Stem 60 g
H.sub.2O 8.6 14.3% 2.0% clava-herculis bark (5% tartaric acid)
Zanthoxylum Stem 60 g H.sub.2O 7.56 12.6% 6.5% clava-herculis bark
(0.6% H.sub.2SO.sub.4) Zanthoxylum Stem 60 g MeOH 16.51 27.5% 4.7%
clava-herculis bark (1% HCl) Zanthoxylum Stem 60 g MeOH 13.69 22.8%
2.2% americanum bark (1% HCl) Zanthoxylum Stem 60 g MeOH 18.57 31%
9.9% frazineum bark (1% HCl)
63.
2TABLE 2 Parameter Value Yield Liquid Extract 750 mL 37.5% Solids
28.44 g 8.13% Alkaloids 0.96 g 0.27% Purity of Alkaloids 3.27%
64.
3TABLE 3 Parameter Value Yield Liquid Extract 1900 mL 63.3% Solids
52.82 g 16.1% Alkaloids 3.82 g 1.16% Purity of Alkaloids 7.23%
65.
4TABLE 4 Value Yield Value Yield Liquid Extract 2.75 L 34.4% 2.9 L
93.6% Solids 114.3 g 4.0% 87.3 g 3.1% Alkaloids 13.1 g 0.46% 10.6 g
0.37% Purity of Alkaloids 11.5% 12.1%
66.
5TABLE 5 Parameter Value Yield Liquid Extract 5.65 L 50.1% Solids
201.6 g 7.1% Alkaloids 23.7 g 0.82% Purity of Alkaloids 11.7%
67.
6TABLE 6 Value Yield Value Yield Liquid Extract 2.7 L 33.75% 9.5 L
95.0% Solids 135.3 g 4.8% 199.8 g 7.13% Alkaloids 16.2 g 0.58%
29.35 g 1.05% Purity of Alkaloids 11.97% 14.7%
68.
7TABLE 7 Parameter Value Yield Liquid Extract 12.2 L 67.8% Solids
335.1 g 11.96% Alkaloids 45.55 g 1.63% Purity of Alkaloids
13.59%
69.
8TABLE 8 Value Yield Value Yield Value Yield Liquid Extract 5.0 L
41.67% 11.0 L 84.62% 5.0 L 100.0% Solids 103.5 g 2.45% 409.2 g
9.67% 95.5 g 2.26% Alkaloids 11.15 g 0.26% 34.1 g 0.81% 7.65 g
0.18% Purity of 10.8% 8.33% 8.01% Alkaloids
70.
9TABLE 9 Parameter Value Yield Liquid Extract 21.0 L 70% Solids
608.2 g 14.38% Alkaloids 52.9 g 1.25% Purity of Alkaloids 8.70%
* * * * *