U.S. patent application number 10/303643 was filed with the patent office on 2003-08-14 for echinacea supplement and method of manufacture.
This patent application is currently assigned to Factors R & D Technologies Ltd.. Invention is credited to Barton, Richard E., Chang, Chuck C., Gahler, Roland J., Slama, Jan V..
Application Number | 20030152653 10/303643 |
Document ID | / |
Family ID | 22562708 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030152653 |
Kind Code |
A1 |
Gahler, Roland J. ; et
al. |
August 14, 2003 |
Echinacea supplement and method of manufacture
Abstract
In one aspect, the present invention provides Echinacea
compositions including standardized amounts of at least two
Echinacea components. More preferably the Echinacea compositions of
the present invention include standardized amounts of at least
three Echinacea components. Presently preferred Echinacea
components are cichoric acid, Echinacea alkylamides and Echinacea
polysaccharides. In another aspect, the present invention provides
methods for preparing Echinacea compositions, the methods including
the step of combining amounts of at least two Echinacea components,
preferably three Echinacea components, sufficient to yield an
Echinacea composition including standardized amounts of each of the
combined Echinacea components. In yet another aspect, the present
invention provides Echinacea extracts, and methods for preparing
Echinacea extracts. Presently preferred Echinacea extracts are
enriched in one or more members of the group consisting of
polysaccharides, alkylamides and cichoric acid. In yet another
aspect, the present invention provides methods for enhancing immune
system activity in a mammal.
Inventors: |
Gahler, Roland J.; (North
Vancouver, CA) ; Barton, Richard E.; (Richmond,
CA) ; Slama, Jan V.; (West Bank, CA) ; Chang,
Chuck C.; (Vancouver, CA) |
Correspondence
Address: |
CHRISTENSEN, O'CONNOR, JOHNSON, KINDNESS, PLLC
1420 FIFTH AVENUE
SUITE 2800
SEATTLE
WA
98101-2347
US
|
Assignee: |
Factors R & D Technologies
Ltd.
|
Family ID: |
22562708 |
Appl. No.: |
10/303643 |
Filed: |
November 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10303643 |
Nov 22, 2002 |
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09672729 |
Sep 28, 2000 |
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6511683 |
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09672729 |
Sep 28, 2000 |
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PCT/CA00/00931 |
Aug 11, 2000 |
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60157194 |
Sep 30, 1999 |
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Current U.S.
Class: |
424/737 |
Current CPC
Class: |
A61P 37/02 20180101;
A61K 31/222 20130101; A23L 33/105 20160801; A61K 36/28 20130101;
A61P 43/00 20180101; A61K 31/16 20130101; A61P 37/04 20180101; A61K
31/222 20130101; A61K 31/715 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 31/715 20130101; A61K 31/16 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/737 |
International
Class: |
A61K 035/78 |
Claims
The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. An Echinacea composition comprising standardized amounts of a
first Echinacea component and a second Echinacea component.
2. The Echinacea composition of claim 1 wherein each of said first
and said second Echinacea components are independently selected
from the group consisting of essential oils, alkamides,
alkylamides, cichoric acid, polysaccharides, polyalkynes and
polyalkenes.
3. The Echinacea composition of claim 2 wherein each of said first
and said second Echinacea components are independently selected
from the group consisting of alkylamides, cichoric acid and
polysaccharides.
4. The Echinacea composition of claim 3 wherein said standardized
amount of cichoric acid is from about 0.2 mg/ml to about 500 mg/ml,
said standardized amount of Echinacea alkylamides is from about
0.02 mg/ml to about 50 mg/ml and said standardized amount of
Echinacea polysaccharides is from about 10 mg/ml to about 800
mg/ml.
5. The Echinacea composition of claim 4 wherein said standardized
amount of cichoric acid is from about 0.3 mg/ml to about 30 mg/ml,
said standardized amount of Echinacea alkylamides is from about
0.05 mg/ml to about 50 mg/ml and said standardized amount of
Echinacea polysaccharides is from about 20 mg/ml to about 800
mg/ml.
6. The Echinacea composition of claim 5 wherein said standardized
amount of cichoric acid is from about 5.0 mg/ml to about 30 mg/ml,
said standardized amount of Echinacea alkylamides is from about 0.8
mg/ml to about 50 mg/ml and said standardized amount of Echinacea
polysaccharides is from about 50 mg/ml to about 800 mg/ml.
7. The Echinacea composition of claim 3 wherein said standardized
amount of cichoric acid is from about 0.01 mg/g to about 500 mg/g,
said standardized amount of Echinacea alkylamides is from about
0.005 mg/g to about 100 mg/g and said standardized amount of
Echinacea polysaccharides is from about 5 mg/g to about 900
mg/g.
8. The Echinacea composition of claim 7 wherein said standardized
amount of cichoric acid is from about 0.05 mg/g to about 500 mg/g,
said standardized amount of Echinacea alkylamides is from about 0.1
mg/g to about 100 mg/g and said standardized amount of Echinacea
polysaccharides is from about 10 mg/g to about 900 mg/g.
9. The Echinacea composition of claim 8 wherein said standardized
amount of cichoric acid is from about 15 mg/g to about 500 mg/g,
said standardized amount of Echinacea alkylamides is from about 5
mg/g to about 100 mg/g and said standardized amount of Echinacea
polysaccharides is from about 200 mg/g to about 900 mg/g.
10. An Echinacea composition comprising standardized amounts of
cichoric acid, Echinacea alkylamides and Echinacea
polysaccharides.
11. The Echinacea composition of claim 10 wherein said standardized
amount of cichoric acid is from about 0.2 mg/ml to about 500 mg/ml,
said standardized amount of Echinacea alkylamides is from about
0.02 mg/ml to about 50 mg/ml and said standardized amount of
Echinacea polysaccharides is from about 10 mg/ml to about 800
mg/ml.
12. The Echinacea composition of claim 11 wherein said standardized
amount of cichoric acid is from about 0.3 mg/ml to about 30 mg/ml,
said standardized amount of Echinacea alkylamides is from about
0.05 mg/ml to about 50 mg/ml and said standardized amount of
Echinacea polysaccharides is from about 20 mg/ml to about 800
mg/ml.
13. The Echinacea composition of claim 12 wherein said standardized
amount of cichoric acid is from about 5.0 mg/ml to about 30 mg/ml,
said standardized amount of Echinacea alkylamides is from about 0.8
mg/ml to about 50 mg/ml and said standardized amount of Echinacea
polysaccharides is from about 50 mg/ml to about 800 mg/ml.
14. The Echinacea composition of claim 10 wherein said standardized
amount of cichoric acid is from about 0.01 mg/g to about 500 mg/g,
said standardized amount of Echinacea alkylamides is from about
0.005 mg/g to about 100 mg/g and said standardized amount of
Echinacea polysaccharides is from about 5 mg/g to about 900
mg/g.
15. The Echinacea composition of claim 10 wherein said standardized
amount of cichoric acid is from about 0.05 mg/g to about 500 mg/g,
said standardized amount of Echinacea alkylamides is from about 0.1
mg/g to about 100 mg/g and said standardized amount of Echinacea
polysaccharides is from about 10 mg/g to about 900 mg/g.
16. The Echinacea composition of claim 10 wherein said standardized
amount of cichoric acid is from about 15 mg/g to about 500 mg/g,
said standardized amount of Echinacea alkylamides is from about 5
mg/g to about 100 mg/g and said standardized amount of Echinacea
polysaccharides is from about 200 mg/g to about 900 mg/g.
17. The Echinacea composition of claim 1 wherein said composition
is in a form selected from the group consisting of a solid, a gel
and a liquid.
18. A method for preparing an Echinacea composition comprising
combining an amount of a first Echinacea component and an amount of
a second Echinacea component sufficient to yield an Echinacea
composition including a standardized amount of said first Echinacea
component and a standardized amount of said second Echinacea
component.
19. The method of claim 18 wherein said first Echinacea component
and said second Echinacea component are each independently selected
from the group consisting of cichoric acid, Echinacea alkylamides
and Echinacea polysaccharides.
20. A method for preparing an Echinacea extract comprising:
selecting an Echinacea plant at a developmental stage having an
enhanced amount of a Echinacea component; excising a portion of the
selected plant that is enriched in the desired Echinacea component;
and contacting the excised plant portion with an amount of a
solvent, selected from the group consisting of ethanol and water,
effective to extract the desired Echinacea component from the plant
portion.
21. The method of claim 20 further comprising the step of
concentrating the extracted Echinacea component.
22. The method of claim 20 wherein said excised plant portion is
contacted with solvent in a ratio by weight of no more than about
two parts plant material to about one part solvent.
23. The method of claim 22 wherein said excised plant portion is
contacted with solvent in a ratio by weight of about one part plant
material to about four parts solvent.
24. The method of claim 21 wherein said Echinacea component is
Echinacea polysaccharide.
25. The method of claim 24 wherein: said selected Echinacea plant
is from about one to about four years old; said portion of the
selected plant that is enriched in polysaccharide is selected from
the group consisting of flowers, leaves, stems and roots; said
excised plant material is contacted with a mixture of ethanol and
water comprising an amount of ethanol that is from about 0.1% (w/w)
to about 50% (w/w); said excised plant material is contacted with
said solvent for a period of from about 48 hours to about 14 days
at a temperature of from about 20.degree. C. to about 80.degree.
C.; and said extract is concentrated to a polysaccharide
concentration of from about 1 mg/ml to about 900 mg/ml.
26. The method of claim 25 wherein the excised plant material is
contacted with a mixture of ethanol and water comprising an amount
of ethanol that is from about 5% (w/w) to about 20% (w/w).
27. The method of claim 25 wherein said excised plant material is
contacted with said solvent for a period of from about three days
to about 12 days.
28. The method of claim 27 wherein said excised plant material is
contacted with said solvent for a period of from about four days to
about eight days.
29. The method of claim 25 wherein said excised plant material is
contacted with said solvent at a temperature of from about
30.degree. C. to about 70.degree. C.
30. The method of claim 25 wherein said excised plant material is
contacted with said solvent at a temperature of from about
30.degree. C. to about 50.degree. C.
31. The method of claim 25 wherein said extract is concentrated to
a polysaccharide concentration of from about 60 mg/mL to about 900
mg/mL.
32. The method of claim 25 wherein said extract is concentrated to
a polysaccharide concentration of from about 100 mg/mL to about 900
mg/mL.
33. The method of claim 21 wherein said Echinacea component is a
alkylamide.
34. The method of claim 33 wherein: said selected Echinacea plant
is from about two to about four years old; said portion of the
selected plant that is enriched in alkylamide is selected from the
group consisting of flowers and roots; said excised plant material
is contacted with a mixture of ethanol and water comprising an
amount of ethanol that is from about 50% (w/w) to about 95% (w/w);
said excised plant material being contacted with said solvent for a
period of from about 24 hours to about 7 days at a temperature of
from about 4.degree. C. to about 85.degree. C.; and said extract
being concentrated to an alkylamide concentration of from about 0.1
mg/mL to about 300 mg/mL.
35. The method of claim 34 wherein the excised plant material is
contacted with a mixture of ethanol and water comprising an amount
of ethanol that is from about 65% (w/w) to about 85% (w/w).
36. The method of claim 34 wherein the excised plant material is
contacted with a mixture of ethanol and water comprising an amount
of ethanol that is from about 70% (w/w) to about 80% (w/w).
37. The method of claim 34 wherein said excised plant material is
contacted with said solvent for a period of from about one day to
about four days.
38. The method of claim 34 wherein said excised plant material is
contacted with said solvent for a period of from about two days to
about three days.
39. The method of claim 34 wherein said excised plant material is
contacted with said solvent at a temperature of from about
30.degree. C. to about 70.degree. C.
40. The method of claim 34 wherein said excised plant material is
contacted with said solvent at a temperature of from about
40.degree. C. to about 60.degree. C.
41. The method of claim 34 wherein said extract is concentrated to
an alkylamide concentration of from about 1 mg/mL to about 300
mg/mL.
42. The method of claim 34 wherein said extract is concentrated to
an alkylamide concentration of from about 20 mg/mL to about 300
mg/mL.
43. The method of claim 21 wherein said Echinacea extract is a
cichoric acid extract.
44. The method of claim 43 wherein: said selected Echinacea plant
is from about one to about three years old; said portion of the
selected plant that is enriched in cichoric acid is selected from
the group consisting of whole, young Echinacea plants, leaves and
immature flowers; said excised plant material is contacted with a
mixture of ethanol and water comprising an amount of ethanol that
is from about 40% (w/w) to about 95% (w/w); said excised plant
material is contacted with said solvent for a period of from about
24 hours to about 3 days at a temperature of from about 4.degree.
C. to about 45.degree. C.; and said extract is concentrated to a
cichoric acid concentration of from about 1 mg/mL to about 700
mg/mL.
45. The method of claim 44 wherein the excised plant material is
contacted with a mixture of ethanol and water comprising an amount
of ethanol that is from about 60% (w/w) to about 95% (w/w).
46. The method of claim 44 wherein the excised plant material is
contacted with a mixture of ethanol and water comprising an amount
of ethanol that is from about 75% (w/w) to about 95% (w/w).
47. The method of claim 44 wherein said excised plant material is
contacted with said solvent for a period of from about 24 hours to
about 40 hours.
48. The method of claim 44 wherein said excised plant material is
contacted with said solvent for a period of from about 24 hours to
about 36 hours.
49. The method of claim 44 wherein said excised plant material is
contacted with said solvent at a temperature of from about
4.degree. C. to about 30.degree. C.
50. The method of claim 44 wherein said extract is concentrated to
a cichoric acid concentration of from about 5 mg/mL to about 700
mg/mL.
51. The method of claim 44 wherein said extract is concentrated to
acichoric acid concentration of from about 30 mg/mL to about 700
mg/mL.
52. An Echinacea cichoric acid extract comprising cichoric acid at
a concentration of greater than about 0.005% (w/w).
53. An Echinacea cichoric acid extract of claim 52 comprising
cichoric acid at a concentration of greater than about 0.05%
(w/w).
54. An Echinacea cichoric acid extract of claim 52 comprising
cichoric acid at a concentration of greater than about 1.5%
(w/w).
55. An Echinacea polysaccharide extract comprising polysaccharide
at a concentration of greater than about 0.01% (w/w).
56. An Echinacea polysaccharide extract of claim 55 comprising
polysaccharide at a concentration of greater than about 0.05%
(w/w).
57. An Echinacea polysaccharide extract of claim 55 comprising
polysaccharide at a concentration of greater than about 20.0%
(w/w).
58. An Echinacea alkylamides extract comprising alkylamides at a
concentration of greater than about 0.001% (w/w).
59. An Echinacea alkylamides extract of claim 58 comprising
alkylamides at a concentration of greater than about 0.01%
(w/w).
60. An Echinacea alkylamides extract of claim 58 comprising
alkylamides at a concentration of greater than about 0.5%
(w/w).
61. An Echinacea cichoric acid extract comprising cichoric acid at
a concentration of greater than about 0.25% (w/v).
62. An Echinacea cichoric acid extract of claim 61 comprising
cichoric acid at a concentration of greater than about 0.5%
(w/v).
63. An Echinacea cichoric acid extract of claim 61 comprising
cichoric acid at a concentration of greater than about 3.0%
(w/v).
64. An Echinacea polysaccharide extract comprising polysaccharide
at a concentration of greater than about 1.0% (w/v).
65. An Echinacea polysaccharide extract of claim 64 comprising
polysaccharide at a concentration of greater than about 5.0%
(w/v).
66. An Echinacea polysaccharide extract of claim 64 comprising
polysaccharide at a concentration of greater than about 10.0%
(w/v).
67. An Echinacea alkylamides extract comprising alkylamides at a
concentration of greater than about 0.1% (w/v).
68. An Echinacea alkylamides extract of claim 67 comprising
alkylamides at a concentration of greater than about 1.0%
(w/v).
69. An Echinacea alkylamides extract of claim 67 comprising
alkylamides at a concentration of greater than about 3.0%
(w/v).
70. An Echinacea cichoric acid extract prepared according to the
method of claim 44.
71. An Echinacea polysaccharide extract prepared according to the
method of claim 25.
72. An Echinacea alkylamides extract prepared according to the
method of claim 34.
73. A method for enhancing immune system activity in a mammal
comprising administering to said mammal an effective daily dosage
of an Echinacea composition comprising standardized amounts of
Echinacea cichoric acid, Echinacea alkylamides and Echinacea
polysaccharides.
74. The method of claim 73 wherein said standardized amount of
Echinacea cichoric acid is from 5 .mu.g/Kg body weight to 2000
.mu.g/Kg body weight, said standardized amount of Echinacea
alkylamides is from 0.5 .mu.g/Kg body weight to 200 .mu.g/Kg body
weight, and said standardized amount of Echinacea polysaccharides
is from 125 .mu.g/Kg body weight to 50 mg/Kg body weight.
75. The method of claim 74 wherein said standardized amount of
Echinacea cichoric acid is from 120 .mu.g/Kg body weight to 1000
.mu.g/Kg body weight, said standardized amount of Echinacea
alkylamides is from 12 .mu.g/Kg body weight to 100 .mu.g/Kg body
weight, and said standardized amount of Echinacea polysaccharides
is from 3 mg/Kg body weight to 30 mg/Kg body weight.
76. The method of claim 75 wherein said standardized amount of
Echinacea cichoric acid is 800 .mu.g/Kg body weight, said
standardized amount of Echinacea alkylamides is 80 .mu.g/Kg body
weight, and said standardized amount of Echinacea polysaccharides
is 20 mg/Kg body weight.
77. The method of any one of claims 73, 74, 75 or 76 wherein said
method enhances macrophage phagocytic activity.
78. The method of, any one of claims 73, 74, 75 or 76 wherein said
method enhances nitric oxide production by macrophages.
79. The method of any one of claims 73, 74, 75 or 76 wherein said
method enhances TNF-.alpha. production by macrophages.
80. The method of any one of claims 73, 74, 75 or 76 wherein said
method enhances IFN-.gamma. production by splenocytes.
81. The method of any one of claims 73, 74, 75 or 76 wherein said
method enhances TNF-.alpha. production by splenocytes.
82. A method for enhancing immune system activity in a mammal
comprising administering to said mammal an effective daily dosage
of an Echinacea extract selected from the group consisting of
Echinacea alkylamides and Echinacea polysaccharides.
83. The method of claim 82 comprising administering to said mammal
an effective daily dosage of Echinacea alkylamides, wherein said
dosage is from 0.5 .mu.g/Kg body weight to 200 .mu./Kg body
weight.
84. The method of claim 82 comprising administering to said mammal
an effective daily dosage of Echinacea alkylamides, wherein said
dosage is from 12 .mu.g/Kg body weight to 100 .mu.g/Kg body
weight.
85. The method of claim 82 comprising administering to said mammal
an effective daily dosage of Echinacea alkylamides, wherein said
dosage is 80 .mu.g/Kg body weight.
86. The method of any one of claims 82, 83, 84 or 85 wherein said
method enhances macrophage phagocytic activity.
87. The method of any one of claims 82, 83, 84 or 85 wherein said
method enhances nitric oxide production by macrophages.
88. The method of any one of claims 82, 83, 84 or 85 wherein said
method enhances TNF-.alpha. production by macrophages.
89. The method of claim 82 comprising administering to said mammal
an effective daily dosage of Echinacea polysaccharides, wherein
said dosage is from 125 .mu.g/Kg body weight to 50 mg/Kg body
weight.
90. The method of claim 82 comprising administering to said mammal
an effective daily dosage of Echinacea polysaccharides, wherein
said dosage is from 3 mg/Kg body weight to 30 mg/Kg body
weight.
91. The method of any one of claims 89 and 90 wherein said method
enhances TNF-.alpha. production by macrophages.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation of co-pending
International Patent Application Number PCT/CA00/00931, filed Aug.
11, 2000, which relates to and claims the benefit of priority from
U.S. Provisional Patent Application No. 60/157,194, filed Sep. 30,
1999, the benefit of which is hereby claimed under 35 U.S.C.
.sctn..sctn. 119 and 120.
FIELD OF THE INVENTION
[0002] The present invention relates to nutritional supplements
prepared from plants of the genus Echinacea.
BACKGROUND OF THE INVENTION
[0003] Numerous studies attest to the health-promoting properties
of extracts derived from plants of the genus Echinacea. See, e.g.,
A. Awang & D. Kindack, Canadian Pharmaceutical Journal, 124:
512-516 (1991). There is a strong commercial market for Echinacea
compositions containing biologically active components that are
believed to promote good health. Further, it is desirable to
formulate Echinacea compositions to contain standardized amounts of
biologically active components derived from Echinacea plants. Such
standardized, Echinacea compositions provide the consumer with a
consistent, effective dose of one or more, biologically active,
Echinacea components.
[0004] In particular, there is a strong commercial market for
Echinacea extracts containing a high concentration of one or more,
biologically active, Echinacea components believed to promote good
health. Such highly enriched extracts can be used directly as
dietary supplements, or can be blended with other Echinacea
extracts to prepare dietary supplements containing standardized
amounts of biologically active, Echinacea components.
[0005] Scientific studies indicate that Echinacea-derived
polysaccharides, alkylamides and cichoric acid (a caffeic acid
derivative also known as chicoric acid, 2,3-o-di-caffeoyl-tartaric
acid) each possess health-promoting properties. For example,
alkylamides from Echinacea have been shown to stimulate
phagocytosis in mice granulocytes at concentrations of about 0.1
parts per million (ppm). Bauer, R. et al., Arzneim.-Forsch./Drug
Research, 38: 276-281 (1988). Similarly, cichoric acid has been
shown to increase phagocytosis in granulocytes, and may stimulate
the immune system at concentrations as low as 0.01 ppm. See e.g, A.
Awang et al., supra. Echinacea polysaccharides have been shown to
inhibit hyaluronidase, increase phagocytosis, induce the release of
interferon-6, and enhance resistance to C. albicans infection in
mice. See, e.g., A. Awang et al., supra; Wagner, H, et al.
Arzneim.-Forsch./Drug Research, 35: 1069-1075 (1985).
[0006] Numerous factors must be considered and optimized in order
to produce Echinacea extracts having a high concentration of
polysaccharides, alkylamides and/or cichoric acid. For example, the
amounts of polysaccharides, alkylamides and cichoric acid in
Echinacea plants are influenced by the species of the plant, the
age of the plant and the plant growth conditions. Additionally, the
solvents and process parameters, such as temperature and length of
extraction period, utilized to extract polysaccharides, alkylamides
and cichoric acid from Echinacea plants can greatly affect the
yield of these components.
[0007] Thus, there is a need for methods for efficiently extracting
polysaccharides, alkylamides and cichoric acid from Echinacea
plants, and for Echinacea extracts containing a high concentration
of polysaccharides, alkylamides and/or cichoric acid. Further,
there is a need for standardized Echinacea compositions containing
a predetermined, desired amount of Echinacea extracts, including
polysaccharide, alkylamide and/or cichoric acid extracts.
SUMMARY OF THE INVENTION
[0008] In one aspect, the present invention provides Echinacea
compositions including standardized amounts of at least two
Echinacea components. More preferably the Echinacea compositions of
the present invention include standardized amounts of at least
three Echinacea components. In this context the term "standardized
amount", or grammatical equivalents thereof, means a desired,
quantified amount of an Echinacea component. Presently preferred
Echinacea components are Echinacea polysaccharides, Echinacea
alkylamides and cichoric acid. Presently preferred Echinacea
extracts useful for preparing the Echinacea compositions of the
present invention are extracts enriched in cichoric acid (most
preferably cichoric acid prepared in accordance with the present
invention), Echinacea alkylamides (most preferably Echinacea
alkylamides prepared in accordance with the present invention) or
Echinacea polysaccharides (most preferably Echinacea
polysaccharides prepared in accordance with the present invention).
The Echinacea compositions of the present invention can be in
liquid form, gel form or solid form, such as a powder, or tablets,
or capsules, and are preferably adapted for administration (more
preferably oral administration) to a human being.
[0009] In another aspect, the present invention provides methods
for preparing Echinacea compositions, the methods including the
step of combining amounts of at least two Echinacea components,
preferably three Echinacea components, sufficient to yield an
Echinacea composition including standardized amounts of each of the
combined Echinacea components. In this context the term
"standardized amount", or grammatical equivalents thereof, means a
desired, quantified amount of an Echinacea component. Presently
preferred Echinacea components are Echinacea polysaccharides,
Echinacea alkylamides and cichoric acid. Presently preferred
Echinacea extracts useful for preparing the Echinacea compositions
of the present invention are extracts enriched in cichoric acid
(most preferably cichoric acid prepared in accordance with the
present invention), Echinacea alkylamides (most preferably
Echinacea alkylamides prepared in accordance with the present
invention) or Echinacea polysaccharides (most preferably Echinacea
polysaccharides prepared in accordance with the present
invention).
[0010] In another aspect, the present invention provides methods
for preparing Echinacea extracts enriched in at least one Echinacea
component. The methods of the present invention for preparing
enriched Echinacea extracts include the steps of selecting an
Echinacea plant at a developmental stage having an enhanced amount
of a desired component (such as cichoric acid, alkylamides or
polysaccharides); selecting a portion of the plant that is enriched
in the desired component, and contacting the selected portion with
an amount of a solvent, selected from the group consisting of
ethanol and water, effective to extract the desired component from
the plant portion. Preferably the Echinacea extract is then
concentrated. Any species of Echinacea plant can be utilized in the
practice of the present invention, but the presently preferred
species is E. purpurea. Extraction of Echinacea plant material
utilizes a ratio (by weight) of plant material to solvent of no
more than about two parts plant material to about one part solvent.
The presently preferred ratio (by weight) of plant material to
solvent is about one part plant material to about four parts
solvent.
[0011] In yet another aspect, the present invention provides liquid
Echinacea extracts enriched in one or more member of the group
consisting of Echinacea polysaccharides, Echinacea alkylamides and
cichoric acid. Presently preferred liquid Echinacea extracts
enriched in polysaccharides include polysacchaiides at a
concentration greater than about 1% (w/w), more preferably greater
than about 5% (w/w), most preferably greater than about 10% (w/w).
Presently preferred liquid Echinacea extracts enriched in
alkylamides include alkylamides at a concentration greater than
about 0.1% (w/w), more preferably greater than about 1.0% (w/w),
most preferably greater than about 3.0% (w/w). Presently preferred
liquid Echinacea extracts enriched in cichoric acid include
cichoric acid at a concentration greater than about 0.2% (w/w),
more preferably greater than about 0.5% (w/w), most preferably
greater than about 3.0% (w/w).
[0012] In yet another aspect, the present invention provides solid
Echinacea extracts enriched in one or more member of the group
consisting of Echinacea polysaccharides, Echinacea alkylamides and
cichoric acid. Presently preferred solid Echinacea extracts
enriched in polysaccharides include polysaccharides at a
concentration greater than about 0.01% (w/w), more preferably
greater than about 0.05% (w/w), most preferably greater than about
20% (w/w). Presently preferred solid Echinacea extracts enriched in
alkylamides include alkylamides at a concentration greater than
about 0.001% (w/w), more preferably greater than about 0.01% (w/w),
most preferably greater than about 0.5% (w/w). Presently preferred
solid Echinacea extracts enriched in cichoric acid include cichoric
acid at a concentration greater than about 0.005% (w/w), more
preferably greater than about 0.05% (w/w), most preferably greater
than about 1.5% (w/w).
[0013] The Echinacea extracts of the present invention are useful,
for example, as dietary supplements and as sources of
polysaccharides, alkylamides or cichoric acid for blending to
produce the Echinacea compositions of the present invention. The
Echinacea compositions of the present invention are useful, for
example, as human dietary supplements. By way of non-limiting
example, when administered to a mammal the Echinacea compositions
and extracts of the invention stimulate one or more of the
following aspects of the immune system: macrophage phagocytic
activity; nitric oxide production by macrophages; TNF-.alpha.
production by macrophages; IFN-.gamma. production by splenocytes;
and TNF-.alpha. production by splenocytes.
[0014] In yet another aspect, the present invention provides
methods for enhancing immune system activity in a mammal. In one
embodiment, the methods comprise administering to a mammal an
effective daily dosage of an Echinacea composition comprising
standardized amounts of Echinacea cichoric acid, Echinacea
alkylamides and Echinacea polysaccharides. In another embodiment,
the methods comprise administering to the mammal an effective daily
dosage of an Echinacea extract selected from the group consisting
of Echinacea alkylamides and Echinacea polysaccharides. The term
"effective daily dosage," and grammatical equivalents thereof,
means a daily dosage that is effective to enhance immune system
activity in a mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The foregoing aspects and many of the attendant advantages
of this invention will become more readily appreciated as the same
become better understood by reference to the following detailed
description, when taken in conjunction with the accompanying
drawings, wherein:
[0016] FIG. 1 shows the effect of different dosages of an Echinacea
composition of the invention on various immune system parameters in
rat. The Echinacea composition includes Echinacea cichoric acid,
alkylamide and polysaccharide extracts as set forth in Example 8
herein. The dosage levels are set forth in Table 1 herein.
Abbreviations are: IL2 SP: interleukin-2 production in splenocytes;
TNF-.alpha. SP: tumor necrosis factor alpha production in
splenocytes; INF-.gamma. SP: interferon gamma production in
splenocytes; TNF-.alpha. MA: tumor necrosis factor alpha in
alveolar macrophages; NO MA: nitric oxide production in alveolar
macrophages; PI MA: phagocytic index in alveolar macrophages; PA
MA: phagocytic activity in alveolar macrophages. Results are
normalized to control values.
[0017] FIG. 2 shows the effect on the phagocytic index of rat
alveolar macrophages of different dosages of an Echinacea
composition of the invention. The Echinacea composition includes
Echinacea cichoric acid, alkylamide and polysaccharide extracts as
set forth in Example 8 herein. The dosage levels are set forth in
Table 1 herein.
[0018] FIG. 3 shows the effect of an Echinacea cichoric acid
extract of the invention on various immune system parameters in
rat. The Echinacea cichoric acid extract was prepared as described
in Example 2 herein. Abbreviations are: IL2 SP: interleukin-2
production in splenocytes; TNF-.alpha. SP: tumor necrosis factor
alpha production in splenocytes; INF-.gamma. SP: interferon gamma
production in splenocytes; TNF-.alpha. MA: tumor necrosis factor
alpha in alveolar macrophages; NO MA: nitric oxide production in
alveolar macrophages; PI MA: phagocytic index in alveolar
macrophages; PA MA: phagocytic activity in alveolar macrophages.
Results are normalized to control values.
[0019] FIG. 4 shows the effect of an Echinacea polysaccharide
extract of the invention on various immune parameters in rat. The
Echinacea polysaccharide extract was prepared as described in
Example 3 herein. Abbreviations are: IL2 SP: interleukin-2
production in splenocytes; TNF-.alpha. SP: tumor necrosis factor
alpha production in splenocytes; INF-.gamma. SP: interferon gamma
production in splenocytes; TNF-.alpha. MA: tumor necrosis factor
alpha in alveolar macrophages; NO MA: nitric oxide production in
alveolar macrophages; PI MA: phagocytic index in alveolar
macrophages; PA MA: phagocytic activity in alveolar macrophages.
Results are normalized to control values.
[0020] FIG. 5 shows the effect of an Echinacea alkylamide extract
of the invention on various immune parameters in rat. The Echinacea
alkylamide extract was prepared as described in Example 1 herein.
Abbreviations are: IL2 SP: interleukin-2 production in splenocytes;
TNF-.alpha. SP: tumor necrosis factor alpha production in
splenocytes; INF-.gamma. SP: interferon gamma production in
splenocytes; TNF-.alpha. MA: tumor necrosis factor alpha in
alveolar macrophages; NO MA: nitric oxide production in alveolar
macrophages; PI MA: phagocytic index in alveolar macrophages; PA
MA: phagocytic activity in alveolar macrophages. Results are
normalized to control values.
[0021] FIG. 6 shows the effect of an Echinacea composition of the
invention, and the effect of each of its component cichoric acid,
alkylamide and polysaccharide extracts, on the phagocytic index of
rat alveolar macrophages. The Echinacea composition includes
Echinacea cichoric acid, alkylamide and polysaccharide extracts as
set forth in Example 8 herein. The dosage of the Echinacea
composition was Level 4 as set forth in Table 1 herein, i.e., the
Echinacea composition provided a daily dosage of Echinacea cichoric
acid at 800 .mu.g/kg, Echinacea polysaccharides at 20 mg/kg, and
Echinacea alkylamides at 80 .mu.g/kg. Echinacea cichoric acid was
separately administered at a daily dosage of 800 .mu.g/kg,
Echinacea polysaccharides were separately administered at a daily
dosage of 20 mg/kg, and Echinacea alkylamides were separately
administered at a daily dosage of 80 .mu.g/kg. A mixture of water
and ethanol was administered to control rats.
[0022] FIG. 7 shows the effect on TNF-.alpha. production in rat
alveolar macrophages of different dosages of an Echinacea
composition of the invention. The Echinacea composition includes
Echinacea cichoric acid, alkylamide and polysaccharide extracts as
set forth in Example 8 herein. TNF-.alpha. MA: tumor necrosis
factor alpha in alveolar macrophages. Relative concentrations were
obtained by normalizing each of the dose concentrations in Table 1
to the concentrations at dose level 2 set forth in Table 1.
[0023] FIG. 8 shows the effect of different dosages of an Echinacea
composition of the invention on rat splenocyte production of
Interleukin-2. The Echinacea composition includes Echinacea
cichoric acid, alkylamide and polysaccharide extracts as set forth
in Example 8 herein. The Echinacea composition was administered at
the dosage levels set forth in Table 1 herein. IL2 SP: Splenocyte
production of interleukin-2. Results are normalized to the Control
values. Relative concentrations were obtained by normalizing each
of the dose concentrations in Table 1 to the concentrations at dose
level 2 set forth in Table 1.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0024] Echinacea can be propagated from seed, crown division and by
planting root sections, preferably 10-12 cm in length. The seeds
can be sown outdoors immediately after ripening in the autumn and
germinate the following spring, but the rate of emergence is
affected by the degree of seed dormancy, which varies with species.
Seed germination is affected by several factors. Long-term seed
storage at room temperature for three years or more will reduce
germination rate. Seed size and inflorescence position do not
affect seed germination, however seeds harvested at physiological
maturity, but prior to senescence, have a higher germination rate
than seeds harvested after desiccation. In the experience of the
present inventors, seed source (i.e., the seed supplier) accounts
for the majority of variation of E. purpurea germination rate. In
particular, the presence of Echinacea hybrid seeds in
commercially-purchased batches of seed is undesirable. A presently
preferred method of identifying Echinacea hybrid seeds is as
follows. The seeds to be tested are germinated in planters with
daily watering. Seedlings are allowed to grow for one week after
germination. The seedlings are then removed and washed thoroughly
with water and dried. The clean seedlings are next-processed with a
kitchen blender until they are finely blended. The plant material
is removed by filtering through 5 .mu.m filters. The remaining
liquid is concentrated by evaporation under vacuum to half of its
original volume and then filtered through a 0.45 .mu.m filter for
HPLC analysis. HPLC analysis, using the cichoric acid assay method
described in Example 6 herein, will show a distinct cichoric acid
peak for non-hybridized seeds, whereas the cichoric acid peak is
missing in hybridized seeds.
[0025] Seedling emergence from directly-seeded fields may not be as
uniform as germinating indoors after stratification. One presently
preferred method for seed stratification is to mix seeds with clean
sand (in a ratio of 1:1, v/v) in a plastic bag and maintain
moisture at about ten percent throughout a four to six week period
at a temperature of from about 1.degree. C. to about -4.degree. C.
The stratified seeds are then separated from the sand and can be
sown on the soil surface or 0.5 cm deep in flats or small pots
filled with a mixture of peat and perlite, (in a ratio of 1:1,
v/v). Seeds planted in flats maintained at about 18-20.degree. C.
typically start to emerge within seven to ten days after planting.
Seedlings are preferably grown in a green house or indoors with
supplementary light until transplanting to the outdoors. Typically
there are about 257,000 seeds/kg for E purpurea and up to about
319,000 seeds/kg for E. angustifolia. About 500 g of seed is
required to provide transplants for a 0.5 hectare (ha) field.
[0026] Asexual propagation through cuttings and cell cultures is
also possible. Asexual propagation can produce a large population
of genetically identical plants which can be improved through
selective breeding or genetic engineering for higher yields of
active compounds.
[0027] In the wild, Echinacea typically grows in poor, rocky soil
under full sun light. However, it also thrives under cultivation in
moderately rich and well-drained loam or sandy loam soil, which is
neutral to slightly acid (pH 6-7). Echinacea is a root crop, rocky
or heavy soil should therefore be avoided. Although Echinacea is
drought tolerant, water is important for its growth, and regular
irrigation is recommended.
[0028] Intensive fertilization produces a high herb yield but low
root yield. A balanced fertilizer, low in nitrogen and including an
adequate amount of phosphorus and potassium, is usually sufficient.
Bone meal or phosphatic rock (14.5-20 kg/ha) and wood ash (45-51.5
kg/ha) applied before sowing, and cow or horse manure applied in 3
applications after planting, is a presently preferred combination
of organic fertilizer for growing Echinacea. Fish fertilizer
(including N-P.sub.2O.sub.5-K.sub.2O in a ratio of 5-2-2,
respectively) applied at a working concentration of 4 mL/L promotes
the growth of E. purpurea. Besides manure and decomposed municipal
waste, a green cover crop such as quinoa, stinging nettle or red
clover is also beneficial. Chemical fertilizer can also be used for
non-organic cultivation. Nitrogen fertilizer is used for general
plant health and more specifically, for producing a leafy, dark
green plant. Phosphate fertilizers will increase flowering.
[0029] Additionally, plant spacing affects yield. Preferably,
Echinacea should be planted 30 cm apart in 120 cm wide beds,
allowing five rows per bed and giving approximately 74,000
plants/ha. This is a maximum density that may be too dense if a
vigorous Echinacea species is used, or if the plants are grown for
longer than four years.
[0030] Mulch serves the purpose of maintaining soil moisture and
temperature, and more importantly controls weeds. Seedlings thrive
under black plastic mulch or bark mulch. If seeding outdoors, a
mulch of clean straw (2-3 cm) over the seed is considered
essential. Echinacea is not tolerant of weeds, thus weed control is
an important factor. In the experience of the present inventors,
with respect to organically grown Echinacea, black plastic or bark
mulch is the best weed control measure, especially for newly
established plants. Plastic mulch can decrease the labor cost for
weed control by seventy to eighty percent. Bark mulching should be
done immediately after planting and should be coarse (i.e.,
utilizing pieces of bark mulch having dimensions of greater than
about 2 cm by 2 cm) to enable the surface to dry out and create
conditions unfavorable for weed germination. Land preparation, at
least one year before planting, is another way to get rid of
perennial weeds and reduce the annual weed population. A preferred
method of land preparation is to turn the soil over in Spring when
the weeds have grown up, but before planting the Echinacea plants.
In this way, the weeds are destroyed by exposing their roots.
[0031] Thorough soil cultivation is essential to optimize growth
and yield of Echinacea. Normally the roots do not reach desirable
size until 3-4 years after sowing. With carefully regulated growing
conditions, the yield from E. angustifolia can reach up to 2.5
tons/ha. Yields for E. purpurea are typically higher than that for
E. angustifolia.
[0032] The Echinacea compositions of the present invention include
standardized amounts of at least two Echinacea components. More
preferably the Echinacea compositions of the present invention
include standardized amounts of at least three Echinacea
components. In this context the term "standardized amount", or
grammatical equivalents thereof, means a desired, quantified amount
of an Echinacea component. It is desirable to formulate Echinacea
compositions to contain standardized amounts of Echinacea
components because such standardized, Echinacea compositions
provide the consumer with a consistent (i.e., does not vary between
batches), effective dose of the biologically active
component(s).
[0033] Presently preferred Echinacea extracts useful for preparing
the Echinacea compositions of the present invention are enriched in
at least one Echinacea component that is capable of inducing a
biological response, such as stimulation of immune function, when
introduced into a living organism. By way of non-limiting example,
Echinacea components useful for preparing the Echinacea
compositions of the present invention include: essential oils,
alkamides (including alkylamides), cichoric acid, polysaccharides,
polyalkynes and polyalkenes. Representative examples of Echinacea
essential oils include, borneol, bornylacetate,
pentadeca-8-en-2-one, germacrene D, caryophyllene, caryophyllene
epoxide and palmitic acid. Echinacea essential oils are described,
for example, in A. Awang and D. Kindack, Canadian Pharmaceutical
Journal 124: 512-516 (1991), incorporated herein by reference, and
in Bauer, R., "Echinacea Species as Potential Immunostimulatory
Drugs", Economic and Medicinal Plant Research, vol. 5, p. 261-267
(1991). Presently preferred Echinacea components useful for
preparing the Echinacea compositions of the present invention are
cichoric acid (most preferably cichoric acid prepared in accordance
with the present invention), Echinacea alkylamides (most preferably
Echinacea alkylamides prepared in accordance with the present
invention) and Echinacea polysaccharides (most preferably Echinacea
polysaccharides prepared in accordance with the present
invention).
[0034] In one presently preferred embodiment, the present invention
provides Echinacea compositions including a standardized amount of
a first Echinacea component and a standardized amount of a second
Echinacea component, wherein the first and second Echinacea
components are each independently selected from the group
consisting of cichoric acid (most preferably cichoric acid prepared
in accordance with the present invention), Echinacea alkylamides
(most preferably Echinacea alkylamides prepared in accordance with
the present invention) and Echinacea polysaccharides (most
preferably Echinacea polysaccharides prepared in accordance with
the present invention), and wherein the standardized amount of
cichoric acid is from about 0.2 mg/ml to about 500 mg/ml, more
preferably from about 0.3 mg/ml to about 30 mg/ml, most preferably
from about 5 mg/ml to about 30 mg/ml; the standardized amount of
Echinacea alkylamides is from about 0.02 mg/ml to about 50 mg/ml,
more preferably from about 0.05 mg/ml to about 50 mg/ml, most
preferably from about 0.8 mg/ml to about 50 mg/ml; and the
standardized amount of Echinacea polysaccharides is from about 10
mg/ml to about 800 mg/ml, more preferably from about 20 mg/ml to
about 800 mg/ml, most preferably from about 50 mg/ml to about 800
mg/ml.
[0035] In a second presently preferred embodiment, the present
invention provides Echinacea compositions including a standardized
amount of cichoric acid (most preferably cichoric acid prepared in
accordance with the present invention), Echinacea alkylamides (most
preferably Echinacea alkylamides prepared in accordance with the
present invention) and Echinacea polysaccharides (most preferably
Echinacea polysaccharides prepared in accordance with the present
invention), and wherein the standardized amount of cichoric acid is
from about 0.2 mg/ml to about 500 mg/ml, more preferably from about
0.3 mg/ml to about 30 mg/ml, most preferably from about 5 mg/ml to
about 30 mg/ml; the standardized amount of Echinacea alkylamides is
from about 0.02 mg/ml to about 50 mg/ml, more preferably from about
0.05 mg/ml to about 50 mg/ml, most preferably from about 0.8 mg/ml
to about 50 mg/ml; and the standardized amount of Echinacea
polysaccharides is from about 10 mg/ml to about 800 mg/ml, more
preferably from about 20 mg/ml to about 800 mg/ml, most preferably
from about 50 mg/ml to about 800 mg/ml.
[0036] In a third presently preferred embodiment, the present
invention provides Echinacea compositions including a standardized
amount of a first Echinacea component and a standardized amount of
a second Echinacea component, wherein the first and second
Echinacea components are each independently selected from the group
consisting of cichoric acid (most preferably cichoric acid prepared
in accordance with the present invention), Echinacea alkylamides
(most preferably Echinacea alkylamides prepared in accordance with
the present invention) and Echinacea polysaccharides (most
preferably Echinacea polysaccharides prepared in accordance with
the present invention), and wherein the standardized amount of
cichoric acid is from about 0.01 mg/g to about 500 mg/g, more
preferably from about 0.05 mg/g to about 500 mg/g, most preferably
from about 15 mg/g to about 500 mg/g; the standardized amount of
Echinacea alkylamides is from about 0.005 mg/g to about 100 mg/g,
more preferably from about 0.1 mg/g to about 100 mg/g, most
preferably from about 5 mg/g to about 100 mg/g; and the
standardized amount of Echinacea polysaccharides is from about 5
mg/g to about 900 mg/g, more preferably from about 10 mg/g to about
900 mg/g, most preferably from about 200 mg/g to about 900
mg/g.
[0037] In a fourth presently preferred embodiment, the present
invention provides Echinacea compositions including a standardized
amount of cichoric acid (most preferably cichoric acid prepared in
accordance with the present invention), Echinacea alkylamides (most
preferably Echinacea alkylamides prepared in accordance with the
present invention) and Echinacea polysaccharides (most preferably
Echinacea polysaccharides prepared in accordance with the present
invention), and wherein the standardized amount of cichoric acid is
from about 0.01 mg/g to about 500 mg/g, more preferably from about
0.05 mg/g to about 500 mg/g, most preferably from about 15 mg/g to
about 500 mg/g; the standardized amount of Echinacea alkylamides is
from about 0.005 mg/g to about 100 mg/g, more preferably from about
0.1 mg/g to about 100 mg/g, most preferably from about 5 mg/g to
about 100 mg/g; and the standardized amount of Echinacea
polysaccharides is from about 5 mg/g to about 900 mg/g, more
preferably from about 10 mg/g to about 900 mg/g, most preferably
from about 200 mg/g to about 900 mg/g.
[0038] In another aspect, the present invention provides methods
for preparing Echinacea compositions, the methods including the
step of combining amounts of at least two Echinacea components,
preferably three Echinacea components, sufficient to yield an
Echinacea composition including standardized amounts of each of the
combined Echinacea components. In this context the term
"standardized amount", or grammatical equivalents thereof, means a
desired, quantified amount of an Echinacea component. Presently
preferred Echinacea components useful for preparing Echinacea
compositions, in accordance with the present invention, are capable
of inducing a biological response, such as stimulation of immune
function, when introduced into a living organism. By way of
non-limiting example, Echinacea components useful for preparing
Echinacea compositions, in accordance with the methods of the
present invention, include: essential oils, alkamides (including
alkylamides), cichoric acid, polysaccharides, polyalkynes and
polyalkenes. Presently preferred Echinacea components useful for
preparing Echinacea compositions, in accordance with the methods of
the present invention, are cichoric acid (most preferably cichoric
acid prepared in accordance with the present invention), Echinacea
alkylamides (most preferably Echinacea alkylamides prepared in
accordance with the present invention) and Echinacea
polysaccharides (most preferably Echinacea polysaccharides prepared
in accordance with the present invention). The Echinacea
compositions prepared in accordance with the present invention can
be in liquid form, gel form or solid form, such as a powder or
capsules, and are preferably adapted for administration (more
preferably oral administration) to a human being.
[0039] In one presently preferred embodiment, the present invention
provides methods for preparing Echinacea compositions including the
step of combining an amount of a first Echinacea component and an
amount of a second Echinacea component, each of said first and
second Echinacea components being independently selected from the
group consisting of cichoric acid (most preferably cichoric acid
prepared in accordance with the present invention), Echinacea
alkylamides (most preferably Echinacea alkylamides prepared in
accordance with the present invention) and Echinacea
polysaccharides (most preferably Echinacea polysaccharides prepared
in accordance with the present invention), said amount of cichoric
acid being an amount sufficient to yield an Echinacea composition
including a cichoric acid standardized amount of from about 0.2
mg/ml to about 500 mg/ml, more preferably from about 0.3 mg/ml to
about 30 mg/ml, most preferably from about 5 mg/ml to about 30
mg/ml; said amount of Echinacea alkylamides being an amount
sufficient to yield an Echinacea composition including an
alkylamides standardized amount of from about 0.02 mg/ml to about
50 mg/ml, more preferably from about 0.05 mg/ml to about 50 mg/ml,
most preferably from about 0.8 mg/ml to about 50 mg/ml; and said
amount of Echinacea polysaccharides being an amount sufficient to
yield an Echinacea composition including an Echinacea
polysaccharides standardized amount of from about 10 mg/ml to about
800 mg/ml, more preferably from about 20 mg/ml to about 800 mg/ml,
most preferably from about 50 mg/ml to about 800 mg/ml.
[0040] In a second presently preferred embodiment, the present
invention provides methods for preparing Echinacea compositions
including the step of combining an amount of cichoric acid (most
preferably cichoric acid prepared in accordance with the present
invention), an amount of Echinacea alkylamides (most preferably
Echinacea alkylamides prepared in accordance with the present
invention) and an amount of Echinacea polysaccharides (most
preferably Echinacea polysaccharides prepared in accordance with
the present invention), said amount of cichoric acid being an
amount sufficient to yield an Echinacea composition including a
cichoric acid standardized amount of from about 0.2 mg/ml to about
500 mg/ml, more preferably from about 0.3 mg/ml to about 30 mg/ml,
most preferably from about 5 mg/ml to about 30 mg/ml; said amount
of Echinacea alkylamides being an amount sufficient to yield an
Echinacea composition including an alkylamides standardized amount
of from about 0.02 mg/ml to about 50 mg/ml, more preferably from
about 0.05 mg/ml to about 50 mg/ml, most preferably from about 0.8
mg/ml to about 50 mg/ml; and said amount of Echinacea
polysaccharides being an amount sufficient to yield an Echinacea
composition including an Echinacea polysaccharides standardized
amount of from about 10 mg/ml to about 800 mg/ml, more preferably
from about 20 mg/ml to about 800 mg/ml, most preferably from about
50 mg/ml to about 800 mg/ml.
[0041] In a third presently preferred embodiment, the present
invention provides methods for preparing Echinacea compositions
including the step of combining an amount of a first Echinacea
component and an amount of a second Echinacea component, each of
said first and second Echinacea extracts being independently
selected from the group consisting of cichoric acid (most
preferably cichoric acid prepared in accordance with the present
invention), Echinacea alkylamides (most preferably Echinacea
alkylamides prepared in accordance with the present invention) and
Echinacea polysaccharides (most preferably Echinacea
polysaccharides prepared in accordance with the present invention),
said amount of cichoric acid being an amount sufficient to yield an
Echinacea composition including a cichoric acid standardized amount
of from about 0.01 mg/g to about 500 mg/g, more preferably from
about 0.05 mg/g to about 500 mg/g, most preferably from about 15
mg/g to about 500 mg/g; said amount of Echinacea alkylamides being
an amount sufficient to yield an Echinacea composition including an
alkylamides standardized amount of from about 0.005 mg/g to about
100 mg/g, more preferably from about 0.1 mg/g to about 100 mg/g,
most preferably from about 5 mg/g to about 100 mg/g; and said
amount of Echinacea polysaccharides being an amount sufficient to
yield an Echinacea composition including an Echinacea
polysaccharides standardized amount of from about 5 mg/g to about
900 mg/g, more preferably from about 10 mg/g to about 900 mg/g,
most preferably from about 200 mg/g to about 900 mg/g.
[0042] In a fourth presently preferred embodiment, the present
invention provides methods for preparing Echinacea compositions
including the step of combining an amount of cichoric acid (most
preferably cichoric acid prepared in accordance with the present
invention), an amount of Echinacea alkylamides (most preferably
Echinacea alkylamides prepared in accordance with the present
invention) and an amount of Echinacea polysaccharides (most
preferably Echinacea polysaccharides prepared in accordance with
the present invention), said amount of cichoric acid being an
amount sufficient to yield an Echinacea composition including a
cichoric acid standardized amount of from about 0.01 mg/g to about
500 mg/g, more preferably from about 0.05 mg/g to about 500 mg/g,
most preferably from about 15 mg/g to about 500 mg/g; said amount
of Echinacea alkylamides being an amount sufficient to yield an
Echinacea composition including an alkylamides standardized amount
of from about 0.005 mg/g to about 100 mg/g, more preferably from
about 0.1 mg/g to about 100 mg/g, most preferably from about 5 mg/g
to about 100 mg/g; and said amount of Echinacea polysaccharides
being an amount sufficient to yield an Echinacea composition
including an Echinacea polysaccharides standardized amount of from
about 5 mg/g to about 900 mg/g, more preferably from about 10 mg/g
to about 900 mg/g, most preferably from about 200 mg/g to about 900
mg/g.
[0043] Echinacea compositions prepared in accordance with the
present invention can be in liquid form, gel form or solid form,
such as a powder or capsules, and are preferably adapted to be
administered (more preferably orally administered) to a human
being. Typically, prior to blending to form Echinacea compositions
of the present invention, aliquots of Echinacea extracts are tested
for potency. Extracts are adjusted to the desired concentration(s)
by concentration or dilution with appropriate fillers or solvents
(including water, alcohol, soy oil or other oils, lecithin,
glycerin, and so on). Vessels for blending can be purged with
nitrogen gas and the blending can be carried out under nitrogen to
prevent oxidation. The Echinacea compositions, or the individual
Echinacea extracts, can be formulated in an alcohol or glycerin
base. Powdered Echinacea extracts or compositions can be
manufactured through spray-drying or freeze-drying. Liquid
Echinacea extracts or compositions can also be microencapsulated
into a free-flowing powder form. Liquid Echinacea compositions or
extracts can be used directly as ingredients in soft-gelatin liquid
capsules and hard-gelatin capsules, and tablets can be made from
the powdered material.
[0044] In general, the Echinacea extracts and compositions prepared
in accordance with the present invention can include one or more
excipients. Suitable pharmaceutically acceptable excipients include
processing agents and drug delivery modifiers and enhancers, such
as, for example, vegetable oil powder, croscarmellose sodium,
acacia gum and guar gum. Other suitable pharmaceutically acceptable
excipients are described in "Remington's Pharmaceutical Sciences,"
Mack Pub. Co., New Jersey (1991), incorporated herein by
reference.
[0045] In another aspect, the present invention provides methods
for preparing enriched Echinacea extracts. The presently preferred
Echinacea extracts of the invention are enriched in Echinacea
alkylamides, Echinacea polysaccharides or cichoric acid. The
methods of the present invention include the steps of selecting an
Echinacea plant at a developmental stage having an enhanced amount
of a desired component (such as Echinacea alkylamides, Echinacea
polysaccharides or cichoric acid); selecting a portion of the plant
that is enriched in the desired component, and contacting the
selected portion with an amount of a solvent, selected from the
group consisting of ethanol and water, effective to extract the
desired component from the plant portion. Preferably the Echinacea
extract is then concentrated. Any species of Echinacea plant can be
utilized in the practice of the present invention, but the
presently preferred species is E purpurea. Presently preferred
components extracted from Echinacea plants, in accordance with the
present invention, are polysaccharides, alkylamides and cichoric
acid. When the methods of the present invention are used to extract
polysaccharides, the extracted Echinacea plants are preferably from
about one to about four years old. Preferably polysaccharides are
extracted from the flowers, leaves, stems and/or roots of Echinacea
plants. When the methods of the present invention are used to
extract alkylamides, the extracted Echinacea plants are preferably
from about two to about four years old. Preferably alkylamides are
extracted from mature flowers and roots of Echinacea plants. When
the methods of the present invention are used to extract cichoric
acid, the extracted Echinacea plants are preferably from about one
to about three years old. Preferably cichoric acid is extracted
from young Echinacea plants, from leaves of Echinacea plants, and
from immature flowers. Extraction of Echinacea plant material
utilizes a ratio (by weight) of plant material to solvent of no
more than about two parts plant material to about one part solvent.
The presently preferred ratio (by weight) of plant material to
solvent is about one part plant material to about four parts
solvent.
[0046] In accordance with one aspect of the present invention,
polysaccharides may be extracted from selected Echinacea plant
material by contacting the plant material with a mixture of ethanol
and water containing from about 0.1% ethanol to about 50% ethanol
(w/w), preferably from about 5% ethanol to about 20% ethanol.
[0047] Polysaccharides are preferably extracted from the flowers,
leaves, stems and/or roots of Echinacea plants. The aerial parts of
the plant typically contain higher concentrations of
polysaccharides than the roots, and mature flowers typically
contain higher concentrations than immature flowers. The preferred
extraction temperature for extracting polysaccharides is from about
20.degree. C. to about 80.degree. C., more preferably from about
30.degree. C. to about 70.degree. C., most preferably from about
30.degree. C. to about 50.degree. C. The duration of polysaccharide
extraction is preferably from about 48 hours to about 14 days, more
preferably from about three days to about twelve days, most
preferably from about four days to about eight days. Extended
extraction times at elevated temperatures ensure that
polysaccharides are extracted as completely as possible, although
some hydrolysis of polysaccharides occurs at higher temperatures,
but cold temperatures slow the extraction process significantly.
Polysaccharide extracts containing low concentrations of ethanol
must be monitored closely to prevent bacterial, yeast, or fungal
growth and consequent spoilage.
[0048] Polysaccharide extracts prepared in accordance with the
present invention typically contain polysaccharide at a
concentration of from about 0.5 mg/mL to about 20.0 mg/mL. In the
practice of the present invention, polysaccharide extracts are
preferably concentrated to a final polysaccharide concentration of
from about 1 mg/mL to about 900 mg/mL, more preferably to a
polysaccharide concentration of from about 60 mg/mL to about 900
mg/mL, most preferably to a polysaccharide concentration of from
about 100 mg/mL to about 900 mg/mL. The presently preferred method
for concentrating polysaccharide extracts is evaporation.
Evaporation is preferably performed at a temperature of from about
20.degree. C. to about 85.degree. C. for a period of from about 5
hours to about 6 days. The vacuum applied is typically from about 5
millibars (mbar) to about 320 mbar absolute vacuum.
[0049] In accordance with one aspect of the present invention,
alkylamides may be extracted from selected Echinacea plant material
by contacting the plant material with a mixture of ethanol and
water containing from about 50% ethanol to about 95% ethanol (w/w),
preferably from about 65% ethanol to about 85% ethanol, more
preferably from about 70% ethanol to about 80% ethanol. Alkylamides
are found in all Echinacea species and the total alkylamide
concentration (and the complexity of the alkylamide fraction) is
greater in roots than in leaves. Isobutylamides are the major
component of the alkylamide fraction in the roots of E.
angustifolia and E. purpurea, although they are a minor component
of the alkylamide fraction in the roots of Epallida. Isobutylamides
are present in the aerial parts of E. angustifolia, E. purpurea and
E. pallida. Mature flowers and roots of Echinacea plants are
preferably used as starting materials to prepare alkylamide
extracts since these plant materials have the highest amount of
alkylamides. Alkylamides are preferably extracted from Echinacea
plants that are greater than about two years old.
[0050] The preferred extraction temperature for extracting
Echinacea alkylamides is from about 4.degree. C. to about
85.degree. C., more preferably from about 30.degree. C. to about
70.degree. C., most preferably from about 40.degree. C. to about
60.degree. C. The duration of alkylamide extraction is preferably
from about 24 hours to about 7 days, more preferably from about one
day to about four days, most preferably from about two days to
about three days. The chosen alkylamide extraction temperature
depends, in part, on the content of other compounds in the
starting, raw plant material. In the case of plant material that
contains a large proportion of alkylamides and little cichoric
acid, extractions can be carried out at 85.degree. C.; however,
when a significant amount of cichoric acid is also present in the
starting plant material, and it is desirable to preserve the
cichoric acid in the final extract, the extraction temperature
should be lowered to less than 40.degree. C. to avoid degradation
of the chicoric acid.
[0051] Alkylamide extracts prepared in accordance with the present
invention typically contain alkylamides at a concentration of from
about 0.1 mg/mL to about 3.0 mg/mL. In the practice of the present
invention, alkylamide extracts are preferably concentrated to a
final alkylamide concentration of from about 0.1 mg/mL to about 300
mg/mL, more preferably to an alkylamide concentration of from about
1 mg/mL to about 300 mg/mL, most preferably to an alkylamide
concentration of from about 20 mg/mL to about 300 mg/mL. The
presently preferred method for concentrating alkylamide extracts is
evaporation. Evaporation is typically performed at a temperature of
from about 10.degree. C. to about 90.degree. C. for a period of
from about 3 hours to about 3 days. The vacuum applied is typically
from about 5 millibars (mbar) to about 320 mbar absolute
vacuum.
[0052] In accordance with the present invention, cichoric acid may
be extracted from selected Echinacea plant material by contacting
the plant material with a mixture of ethanol and water containing
from about 40% ethanol (w/w) to about 95% ethanol (w/w), preferably
from about 40% ethanol (w/w) to about 85% ethanol (w/w), more
preferably from about 50% ethanol (w/w) to about 70% ethanol (w/w).
The parts of the Echinacea plant containing the highest
concentrations of cichoric acid are preferably extracted in order
to yield extracts having a high concentration of cichoric acid. In
E. purpurea, the highest concentrations of cichoric acid are found
in the roots, flowers and leaves, whereas in E angustifolia
cichoric acid is only found in the roots. In general, cichoric acid
is preferably extracted from young plants, leaves, and immature
flowers. In this aspect of the invention, Echinacea plants are
selected that are preferably less than one year old.
[0053] The preferred extraction temperature for extracting cichoric
acid is from about 4.degree. C. to about 45.degree. C., more
preferably from about 4.degree. C. to about 30.degree. C. The
duration of cichoric acid extraction is preferably from about 24
hours to about 3 days, more preferably from about 24 hours to about
40 hours, most preferably from about 24 hours to about 36 hours.
Cichoric acid degradation becomes significant at extraction
temperatures higher than about 40.degree. C. As much as 95% of the
cichoric acid initially present in the plant material can be lost
if the extraction is carried out at greater than 40.degree. C. for
more than 48 hours. More extended extractions can give extracts
completely devoid of cichoric acid. Cichoric acid extracts prepared
in accordance with the present invention typically contain cichoric
acid at a concentration of from about 0.2 mg/mL to about 2.0 mg/mL.
In the practice of the present invention, cichoric acid extracts
are preferably concentrated to a final cichoric acid concentration
of from about 1 mg/mL to about 700 mg/mL, more preferably to a
cichoric acid concentration of from about 5 mg/mL to about 700
mg/mL, most preferably to a cichoric acid concentration of from
about 30 mg/mL to about 700 mg/mL. The presently preferred method
for concentrating cichoric acid extracts is evaporation.
Evaporation is preferably performed at a temperature of from about
10.degree. C. to about 45.degree. C. for a period of from about 5
hours to about 2.5 days. The vacuum applied is typically from about
5 millibars (mbar) to about 320 mbar absolute vacuum.
[0054] In the methods of the present invention for preparing
Echinacea extracts, the Echinacea extraction conditions preferably
minimize degradation of polysaccharides, alkylamides and cichoric
acid. Many biologically active compounds in Echinacea are sensitive
to degradation and each compound or compound group is sensitive to
different conditions which may cause degradation. For example,
cichoric acid is not stable in solution. Degradation of cichoric
acid can occur quickly by phenol oxidase-mediated oxidation, by
hydrolysis, and by chemical degradation. Heat can also cause
degradation of cichoric acid. Similarly, endogenous plant enzymes
(such as glycoside hydrolases and amylases) can degrade
polysaccharides by hydrolysis. In addition, polysaccharide extracts
are easily spoiled by mold, yeast, and fungal growth. Alkylamides
degrade quickly once dried or purified.
[0055] One method of reducing degradation of biologically active
compounds during extraction is to utilize an atmosphere rich in
nitrogen or argon, thereby excluding oxygen. Nitrogen can be
applied by thoroughly purging all equipment and containers before
any liquid transfer. Further, nitrogen gas can be bubbled
continuously through Echinacea liquid extracts during storage and
extraction. Strict control of process parameters, such as
temperature and concentration, can also help to reduce oxidation
and hydrolysis. For example, enzymatic hydrolysis of
polysaccharides into monosaccharide residues occurs much more
readily at 74.degree. C. than at 40.degree. C. Chemical hydrolysis
occurs at 75.degree. C. to 120.degree. C. By maintaining lower
temperatures during processing, hydrolysis can be minimized.
Oxidation is also reduced by lower processing temperatures. A
further aid in decreasing endogenous enzymatic activity is to
concentrate the liquid extracts to minimize the water content.
[0056] Since many consumers demand that herb-derived products are
produced with only non-toxic solvents, many solvents routinely used
in the laboratory such as hexane, chloroform, ethers, and ketones
are preferably not used in the practice of the present invention.
The methods of the present invention for preparing Echinacea
extracts utilize water and ethanol as extraction solvents, and
different ethanol and water mixtures are utilized for extracting
different active ingredients from Echinacea plants. Preferably,
plant material is extracted in liquid solvent at a ratio (by
weight) of one part plant material to four parts liquid solvent.
However, extracts can also be made utilizing a plant material to
liquid solvent ratio of as high as 2 parts plant material to one
part liquid solvent. Echinacea extracts have also been prepared in
accordance with the present invention by utilizing a plant material
to liquid solvent ratio of one part plant material to twenty parts
liquid solvent. Extractions with a high solvent to plant material
ratio result in the extraction of a greater amount of active
compounds from the plant in a more dilute form. The dilute extracts
are preferably concentrated to reach the desired concentrations of
polysaccharides, alkylamides and/or cichoric acid.
[0057] Alternatively, supercritical carbon dioxide can be used as a
non-toxic solvent to extract biologically active components from
Echinacea plants. Supercritical fluid is circulated through the
plant material until the extraction has occurred as completely as
desired. Echinacea extracts prepared using supercritical fluid
carbon dioxide contain mostly non-polar compounds (such as oils).
Various additional extraction solvents (such as propane and
acetone) can be added to supercritical fluid carbon dioxide in
order to extract a higher proportion of polar compounds, but only
the addition of water and ethanol is acceptable to the
health-conscious consumer.
[0058] In general, extraction vessels utilized in the methods of
the present invention for preparing Echinacea extracts are large
containers with one or more openings through which plant materials
and extraction solvents can be easily loaded and removed. An
agitation mechanism can be built into the extraction vessel so that
extraction occurs with constant agitation to reduce extraction
time. Extraction vessels as small as 500 mL and as large as 3000 L
have been successfully utilized in the practice of the present
invention. Vessels should be constructed from materials resistant
to degradation by ethanol, preferably polypropylene and stainless
steel.
[0059] In the practice of the methods of the present invention for
preparing Echinacea extracts, Echinacea extracts are preferably
concentrated in order to reduce storage space, to increase storage
stability (i.e., by reducing the amount of water), and to achieve
desired concentrations of polysaccharides, alkylamides and cichoric
acid. The concentration step is preferably optimized to minimize
the loss of biological activity of the polysaccharides, alkylamides
and cichoric acid. Since high temperatures cause rapid degradation
of many compounds, concentration is usually performed at low
temperatures and under reduced pressure. Usually a rotary
evaporator is used. Evaporation reaction flasks typically range in
size from between 20 L and 100 L. Between 5-90% of the solvent used
for the extracts can be recovered.
[0060] In yet another aspect, the present invention provides liquid
Echinacea extracts enriched in one or more member of the group
consisting of Echinacea polysaccharides, Echinacea alkylamides and
cichoric acid. Presently preferred Echinacea extracts enriched in
polysaccharides include polysaccharides at a concentration greater
than about 1% (w/v), more preferably greater than about 5% (w/v),
most preferably greater than about 10% (w/v). Presently preferred
Echinacea extracts enriched in alkylamides include alkylamides at a
concentration greater than about 0.1% (w/v), more preferably
greater than about 1.0% (w/v), most preferably greater than about
3.0% (w/v). Presently preferred Echinacea extracts enriched in
cichoric acid include cichoric acid at a concentration greater than
about 0.2% (w/v), more preferably greater than about 0.5% (w/v),
most preferably greater than about 3.0% (w/v).
[0061] In yet another aspect, the present invention provides solid
Echinacea extracts enriched in one or more member of the group
consisting of Echinacea polysaccharides, Echinacea alkylamides and
cichoric acid. Presently preferred Echinacea extracts enriched in
polysaccharides include polysaccharides at a concentration greater
than about 0.01% (w/w), more preferably greater than about 0.05%
(w/w), most preferably greater than about 20% (w/w). Presently
preferred Echinacea extracts enriched in alkylamides include
alkylamides at a concentration greater than about 0.001% (w/w),
more preferably greater than about 0.01% (w/w), most preferably
greater than about 0.5% (w/w). Presently preferred Echinacea
extracts enriched in cichoric acid include cichoric acid at a
concentration greater than about 0.005% (w/w), more preferably
greater than about 0.05% (w/w), most preferably greater than about
1.5% (w/w).
[0062] A presently preferred Echinacea polysaccharide extract of
the present invention includes at least 60% (by weight) arabinose
and galactose, and the polysaccharide molecules have a molecular
weight greater than about 10,000.
[0063] A presently preferred Echinacea alkylamide extract of the
present invention includes greater than about 70% (w/w)
isobutylamides. A presently more preferred Echinacea alkylamide
extract of the present invention has an isobutylamide concentration
of greater than about 70% (w/w) and includes greater than about 30%
(w/w) 2E,4E,8E,10E/Z tetraene-dodeca-isobutylamide.
[0064] In yet another embodiment, the present invention provides
nutritional compositions formulated to provide a standardized dose
of at least two, preferably three, Echinacea components. The
presently preferred Echinacea components are cichoric acid,
Echinacea polysaccharides and Echinacea alkylamides. The presently
preferred, standardized dose of Echinacea alkylamides is from about
0.01 mg to about 100 mg, more preferably from about 0.01 mg to
about 40 mg, most preferably from about 0.1 mg to about 1 mg; the
presently preferred, standardized dose of cichoric acid is from
about 0.1 mg to about 120 mg, more preferably from about 0.1 mg to
about 15 mg, most preferably from about 0.5 mg to about 3 mg; the
presently preferred, standardized dose of Echinacea polysaccharides
is from about 1 mg to about 500 mg, more preferably from about 5 mg
to about 100 mg, most preferably from about 10 mg to about 50
mg.
[0065] In yet another aspect, the present invention provides
methods for enhancing immune system activity in a mammal. The
methods comprise administering to a mammal an effective daily
dosage of an Echinacea composition comprising standardized amounts
of Echinacea cichoric acid, Echinacea alkylamides and Echinacea
polysaccharides. Representative standardized amounts of Echinacea
cichoric acid are from 5 .mu.g/Kg body weight to 2000 .mu.g/Kg body
weight, such as from 120 .mu.g/Kg body weight to 1000 .mu.g/Kg body
weight (e.g., 800 .mu.g/Kg body weight). Representative
standardized amounts of Echinacea alkylamides are from 0.5 .mu.g/Kg
body weight to 200 .mu.g/Kg body weight, such as from 12 .mu.g/Kg
body weight to 100 .mu.g/Kg body weight (e.g., 80 .mu.g/Kg body
weight). Representative standardized amounts of Echinacea
polysaccharides are from 125 .mu.g/Kg body weight to 50 mg/Kg body
weight, such as from 3 mg/Kg body weight to 30 mg/Kg body weight
(e.g., 20 mg/Kg body weight). By way of non-limiting example, the
methods (that utilize Echinacea compositions as described above)
for enhancing immune system activity in a mammal are useful for
enhancing macrophage phagocytic activity, enhancing nitric oxide
production by macrophages, enhancing TNF-.alpha. production by
macrophages, enhancing IFN-.gamma. production by splenocytes, and
enhancing TNF-.alpha. production by splenocytes.
[0066] In a further aspect, the present invention provides methods
for enhancing immune system activity in a mammal comprising
administering to the mammal an effective daily dosage of an
Echinacea extract selected from the group consisting of Echinacea
alkylamides and Echinacea polysaccharides. Representative effective
daily dosages of Echinacea alkylamides are from 0.5 .mu.g/Kg body
weight to 200 .mu.g/Kg body weight, such as from 12 .mu.g/Kg body
weight to 100 .mu.g/Kg body weight (e.g., 80 .mu.g/Kg body weight).
In accordance with this aspect of the invention, an effective daily
dosage of Echinacea polysaccharides is useful for enhancing
macrophage phagocytic activity, enhancing nitric oxide production
by macrophages, and/or enhancing TNF-.alpha. production by
macrophages.
[0067] Representative effective daily dosages of Echinacea
polysaccharides are from 125 .mu.g/Kg body weight to 50 mg/Kg body
weight, such as from 3 mg/Kg body weight to 30 mg/Kg body weight
(e.g., 20 mg/Kg body weight). In accordance with this aspect of the
invention, an effective daily dosage of Echinacea polysaccharides
is useful for enhancing TNF-.alpha. production by macrophages.
[0068] The following examples merely illustrate the best mode now
contemplated for practicing the invention, but should not be
construed to limit the invention.
EXAMPLE 1
Extraction of Alkylamides from Echinacea Plants
[0069] Echinacea plants are grown about two feet apart and watered
once per week until one week before the flowers are harvested.
Preferably the flowers of two and/or three year old Echinacea
purpurea plants are harvested. Fully mature Echinacea purpurea
flower cones are preferably collected in late August, and
approximately 700 kg of flowers are typically collected from a
one-acre field.
[0070] 500 kg of freshly harvested flowers are macerated with a
hammer mill to particles sizes of about 1.5 cm.sup.3, or smaller,
and then extracted with about 2000 kg of 75% ethanol for 72 hours
at 35.degree. C. Lower ethanol concentrations and temperatures
reduce yield while higher ethanol concentrations and temperatures
increase cost. The liquid and plant material are agitated at 3
revolutions per minute with rotating mechanical paddles spaced
approximately 20 cm apart and radiating from a center axle in the
extraction vessel. At the end of the extraction period, 2300 kg of
the liquid extract is removed from the extracted flowers.
[0071] The liquid extract now contains about 0.6 mg/mL of
alkylamides. Concentration of the liquid extract at 25 mbar and
40.degree. C. for 30 hours yields about 100 kg of alkylamide-rich
liquid concentrate having an alkylamide concentration of about 10
mg/mL. The concentrate is characterized by a dark-green color,
because of the presence of plant chlorophyll, and typically
contains less than about 1 mg/mL of cichoric acid and less than
about 10 mg/mL of polysaccharides.
EXAMPLE 2
Extraction of Cichoric Acid from Echinacea Plants
[0072] The seedlings are germinated from selected, non-hybridized
seeds in late February and early March and transplanted to fields
by middle or late March. Plants are spaced about two feet apart and
watered twice weekly until harvest. Black plastic mulch is used to
reduce growth of weeds and to speed up plant growth. The seeds are
carefully selected since contamination with Echinacea angustifolia
seeds (or seeds from E. angustifolia/E. purpurea hybrids) will
reduce the yield of cichoric acid more than 20-fold because of
increased enzymatic degradation. The plants are harvested as whole
plants in late September, and the stem, leaves and flowers of the
plants are removed for extraction. Typically, one acre of Echinacea
purpurea plants yields approximately 1000 kg of plant material.
Preferably, one year old Echinacea purpurea plants are
harvested.
[0073] 500 kg of the freshly harvested plants are macerated with a
hammer mill to produce particle sizes of about 1.5 cm.sup.2 to
about 4 cm.sup.2. The macerated material is extracted with 2000 kg
of 50% ethanol for 30 hours at 25.degree. C. Lower ethanol
concentration and higher extraction temperature allow for increased
degradation while higher ethanol concentration and lower
temperatures reduce yield. Nitrogen gas is continuously purged
through the extraction liquid to reduce the amount of cichoric acid
degradation. The extraction liquid is circulated through the plant
mash by a rotary pump with a capacity of up to 500 L per hour. At
the end of the extraction period, 2100 kg of liquid extract having
a cichoric acid concentration of about 2.0 mg/ml can be
obtained.
[0074] Concentration of the liquid extract at 10 mbar and
35.degree. C. for 20 hours yields 100 kg of cichoric acid rich
concentrate having a cichoric acid concentration of 30 mg/mL. The
concentrate is characterized by a dark-green color, resulting from
the presence of plant chlorophyll, and contains less than 2 mg/mL
of alkylamides and less than 20 mg/mL of polysaccharides.
EXAMPLE 3
Extraction of Polysaccharides from Echinacea Plants
[0075] Plants are grown about two feet apart and watered once a
week until one week before the harvest. Fully mature Echinacea
purpurea flower cones are collected in late August. Typically,
approximately 700 kg of flowers are collected from a one-acre field
of Echinacea purpurea plants. Preferably, two and/or three year old
Echinacea purpurea plants are harvested.
[0076] 500 kg of freshly harvested flowers are macerated with a
hammer mill to particle sizes of about 1.5 cm.sup.3, or smaller,
and then extracted with 2000 kg of 20% ethanol for 72 hours at
55.degree. C. Higher ethanol concentrations and lower temperatures
reduce yield while lower ethanol concentrations increase microbial
load of the finished extract, and higher temperatures increase
cost. The liquid and-plant material are agitated at 3 revolutions
per minute with rotating mechanical paddles, spaced approximately
20 cm apart, radiating from a center axle in the extraction vessel.
At the end of the extraction period, 1900 kg of the liquid extract
is removed from the extracted flowers.
[0077] The liquid extract now typically contains about 15 mg/mL of
polysaccharides, which can be concentrated at 50 mbar, 60.degree.
C. for 15 hours to yield about 200 kg of concentrate having a
polysaccharide concentration of about 120 mg/mL. The concentrate is
characterized by a dark brown color and typically contains less
than about 2 mg/mL of alkylamides and less than about 2 mg/mL of
cichoric acid.
EXAMPLE 4
Assay of Polysaccharides Extracted from Echinacea Plants
[0078] The polysaccharide assay method is a modified version of the
method set forth in AOAC Official Methods of Analysis (1995), AOAC
Official Method 988.12, "Dextran in Raw Cane Sugar", 44.1.30, "Part
E--Phenol-H.sub.2SO.sub.4 Test." The polysaccharide assay can be
performed in 13.times.100 mm disposable culture tubes. The final
reaction volume of the assay is 1.5 ml. Distilled water is added to
the polysaccharide sample to yield a water plus polysaccharide
sample volume of 400 .mu.l. 100 .mu.l of a 5% (w/v), reagent-grade
phenol solution is then added to the diluted polysaccharide sample
and mixed. One milliliter of concentrated sulphuric acid (reagent
grade, 95-98% concentrated) is then added and quickly mixed by
vortexing. The tubes containing the samples are then placed in a
boiling water bath for two minutes. The tubes are then allowed to
cool at room temperature for 30 minutes and the absorbency of the
samples at 485 nanometers (nm) are measured against a distilled
water blank using a spectrophotometer. The spectrophotometer should
be able to accurately read A.sub.485 absorbance values over the
range of 0 to 1.5.
[0079] An Echinacea polysaccharides concentration standard is
prepared from a water extraction of Echinacea flowers, with ethanol
precipitations in the following manner. Concentrated Echinacea
polysaccharide material is obtained as described in Example 3
herein. The extract is further concentrated to about 400 mg/mL of
polysaccharides. This concentrate is then precipitated with 4
volumes of 95% ethanol at 4.degree. C. overnight. The precipitate
is removed with centrifugation and redissolved in 35-40.degree. C.
water bath. The material that does not dissolve at this stage is
discarded and the supernatant is precipitated with one volume of
30% (w/v) of trichloroacetic acid. Precipitate is again removed
with centrifugation and the supernatant is precipitated with four
more volumes of ethanol. The precipitated polysaccharides are
dissolved in 2% sodium acetate solutions and precipitated a third
time with ethanol. The supernatant is discarded and the precipitate
is dissolved in water for dialysis with Spectra/Por.RTM. 10,000 MW
cut-off dialysis tubing. Dialysis is carried out over three days
and the remaining material is lyophilized to yield a fluffy solid
material.
[0080] A known weight of the lyophilized polysaccharide is
dissolved in a known volume of water to yield an Echinacea
polysaccharides standard of known concentration. Dilutions of the
Echinacea polysaccharides standard solution are made and the
A.sub.485 of each dilution measured in order to construct a
standard curve of absorbance at 485 nanometers versus
polysaccharide concentration.
EXAMPLE 5
Assay of Alkylamides Extracted from Echinacea Plants
[0081] The presently preferred alkylamides assay method is a
modification of the method published by Bauer in Planta Meidca, 55,
367-371 (1989). The mobile phase of the method was changed from
acetonitrile to methanol due to sample precipitation problems. The
method uses an HPLC (high performance liquid chromatography)
consisting of a gradient pump, an autosampler, and a UV-Visible
detector. Preferably, a Hewlett Packard 1100 series HPLC system and
a Hewlett Packard 1050 series HPLC system are used. A C-18 column,
preferably a 125.times.4.0 mm Hypersil ODS column, with a matching
4.0 mm.times.4.0 mm guard column, is used. The mobile phase
consists of 60% methanol and 40% water (v/v) at the start of the
analysis, and changes in a linear fashion to 95% methanol and 5%
water at 12.5 minutes into the analysis. Total run time is 19.00
minutes with 4 minutes of post-run equilibration. The mobile phase
is pumped at 1.00 mL/minute. 10.mu.L samples are injected and the
sample absorbance at 254 nm is measured. Alkylamides elute the
column at 2.0 minutes to 9.0 minutes. More specifically, dodeca-2E,
4E, 8E, 10E/Z-tetraenoic acid isobutylamide elutes at 5.0 to 8.0
minutes.
[0082] An alkylamide concentration standard is preferably prepared
by extracting 500 ml of alkylamide-rich liquid concentrate,
prepared as described in Example 3 herein, with 500 ml of petroleum
ether at room temperature. The petroleum ether fraction is removed
after one day and evaporated to dryness under vacuum and a dark,
black, oily residue results. The residue is dissolved in a minimum
of methanol (about 20 mL) and centrifuged to removed precipitates.
Octadecyl-functionalized silica is set up in a glass column. A
mobile phase consisting of 70% methanol and 30% water by volume is
used for elution. Material prepared from the liquid concentrate is
applied to the column and 10 mL fractions are collected. The
fractions are analyzed for alkylamide content using the analysis
method described and fractions containing 90% and higher purity of
the alkylamide of interest are pooled. The pooled fractions are
then rotor-evaporated at 45.degree. C. to reduce total volume.
Purified alkylamide fractions from column elution are evaporated to
a clear (yellow colored) liquid under vacuum. Petroleum ether is
added to the residue and warmed in a hot water bath. The residue
and liquid is sonicated to saturate the solution with the purified
alkylamide. The saturated solution is collected and allowed to cool
to room temperature. The solution is then moved to -20.degree. C.
freezer. The crystals form overnight. 500 mL of saturated solution
yields about 0.1-5 g of purified crystals, depending on the
solubility of the specific alkylamide.
[0083] The purified alkylamide standard is dissolved at various
concentrations and passed through an HPLC column as described
herein. A calibration curve is constructed from the resulting peak
areas versus concentrations of the alkylamide standard solutions.
Peak areas from alkylamide samples of unknown concentration are
compared against the calibration curve to determine the
concentration in each sample.
EXAMPLE 6
Assay of Cichoric Acid Extracted from Echinacea Plants
[0084] The cichoric acid analysis method is a modification of the
method published by Bauer in Planta Meidca, 57, 447-449 (1991). The
mobile phase of the method was changed from acetonitrile to
methanol due to sample precipitation problems. The method uses an
HPLC (high performance liquid chromatography) consisting of a
gradient pump, an autosampler, and a UV-Visible detector. In this
case, a Hewlett Packard 1100 series HPLC system and a Hewlett
Packard 1050 series HPLC system are used. A C-18 column, in this
case a 125.times.4.0 mm Hypersil ODS column, with a matching 4.0
mm.times.4.0 mm guard column, is used. The mobile phase consists of
7.0% methanol and 93.0% water with 0.1% phosphoric acid (v/v) at
the start of the analysis and changes in a linear fashion to 32%
methanol and 68% water with 0.1% phosphoric acid (v/v) at 11.6
minutes into the analysis, which then changes to 40.7% methanol and
59.3% water with 0.1% phosphoric acid at 19.1 minutes. Total run
time is 22.00 minutes with 3.50 minutes of post-run equilibration.
The mobile phase is pumped at 1.00 ml/minute. 1011 samples are
injected and the data is collected at 330 nm. The peak area of the
sample is compared against a purified chemical standard for
quantitation. Caffeic acid derivatives elute the column at 7.0
minutes to 18.0 minutes. More specifically, cichoric acid elutes at
14.5 to 17.0 minutes.
[0085] A cichoric acid concentration standard is prepared in the
following manner. Cichoric acid-rich liquid concentrate, prepared
as described in Example 2 herein, is further reduced in volume by
vacuum distillation at 50.degree. C. The polysaccharides are
removed with isopropanol precipitation using 2 parts isopropanol
and 1 part distilled concentrate. The supernatant is removed and
isopropanol is evaporated through vacuum distillation at 37.degree.
C. Distilled water at two times the volume of the concentrate is
added and the pH of the concentrate is adjusted to pH 0-1 with
concentrated hydrochloric acid. Cichoric acid is further purified
by elution through a lipophilic Sephadex LH-20 column with 100%
methanol as mobile phase. Fractions with cichoric acid purity of
greater than 90%, as analyzed by the method described above, are
pooled. The pooled fractions are reduced to dryness under vacuum
and then redissolved in minimum amount of boiling hot water.
Cichoric acid crystallizes out as short, white needles upon
cooling.
[0086] The purified cichoric acid standard is dissolved at various
concentrations and passed through an HPLC column as described
herein. A calibration curve is constructed from the resulting peak
areas versus concentrations of the cichoric acid standard
solutions. Peak areas from cichoric acid samples of unknown
concentration are compared against the calibration curve to
determine the concentration in each sample.
EXAMPLE 7
Exemplary Echinacea Compositions of the Present Invention
[0087] By way of representative example, blended, commercial
Echinacea compositions of the present invention are prepared in the
following manner. Alkylamide-rich concentrate (prepared in
accordance with Example 1, herein), cichoric-acid-rich concentrate
(prepared in accordance with Example 2, herein), and
polysaccharide-rich concentrate (prepared in accordance with
Example 3, herein) are combined in the following volume ratios:
0.4:0.2:0.4, respectively. The concentrates are blended with a
motorized mixer to obtain a homogeneous product. In a presently
preferred embodiment, this combined liquid concentrate contains 5.2
mg/mL of alkylamides, 7.2 mg/mL of cichoric acid, and 56 mg/mL of
polysaccharides. This blended composition is used to prepare
commercial Echinacea compositions as follows:
[0088] (a) An alcohol-free, commercial liquid Echinacea composition
is prepared by evaporating the blended, Echinacea composition
further to remove ethanol, and then diluting the evaporated
composition 10 times by adding 9 times the volume of 70% (w/w)
glycerin. The resulting material is blended thoroughly for a
product containing 0.52 mg/mL of alkylamides, 0.72 mg/mL of
cichoric acid, and 5.6 mg/mL of polysaccharides.
[0089] (b) A commercial, liquid Echinacea composition that contains
alcohol is prepared by diluting the blended, Echinacea composition
10 times by adding 9 times the volume of 40% (w/w) ethanol. The
resulting material is blended thoroughly for a product containing
0.52 mg/mL of alkylamides, 0.72 mg/mL of cichoric acid, and 5.6
mg/mL of polysaccharides.
[0090] (c) Commercial, liquid, Echinacea soft-gelatin capsules are
prepared by diluting the blended, Echinacea composition 10 times by
adding 9 times the volume of a mixture of 30% lecithin and 70% soy
oil. The resulting material is blended thoroughly and then
encapsulated into soft-gelatin capsules at 1.0 mL per capsule for a
product containing 0.52 mg/capsule of alkylamides, 0.72 mg/capsule
of cichoric acid, and 5.6 mg/capsule of polysaccharides.
[0091] (d) Freeze-dried, Echinacea powders are prepared by mixing
the blended, Echinacea composition with 9 times its volume of water
and then lyophilizing with 10% (w/w) tri-calcium phosphate as a
carrier. A dried cake is obtained after 30 hours of lyophilization.
A free-flowing powder is obtained, once the freeze-dried cakes are
milled through a 100 m cone mill, including 2.6 mg/g of
alkylamides, 3.6 mg/g of cichoric acid, and 2.8 mg/g of
polysaccharides.
[0092] (e) Micro-encapsulated Echinacea powders are prepared by
mixing the blended, Echinacea composition with 1% (v/v) of vitamin
E for stability and then micro-encapsulating the mixture with a
cellulose and wax coating at 50%-fill (w/w) with capsules averaging
100 .mu.m in size. The micro-capsules appear as a free-flowing
powder with a composition of 2.6 mg/g of alkylamides, 3.6 mg/g of
cichoric acid, and 2.8 mg/g of polysaccharides.
[0093] (f) Hard-gelatin Echinacea capsules are prepared by mixing
the freeze-dried powder (described in section (d) of this example)
or the micro-encapsulated powder (described in section (e) of this
example) with five parts (by weight) of microcrystalline cellulose
and 2% (w/w) of magnesium stearate. The resulting powder is
encapsulated in 1000 mg capsules to provide 0.52 mg/capsule of
alkylamides, 0.72 mg/capsule of cichoric acid, and 5.6 mg/capsule
of polysaccharides.
[0094] (g) Echinacea tablets are prepared by mixing the
freeze-dried powder (described in section (d) of this example) or
the micro-encapsulated powder (described in section (e) of this
example) with three parts (by weight) of di-calcium phosphate, two
parts (by weight) of microcrystalline cellulose and 3% (w/w) of
magnesium stearate. The resulting powder is tablet-punched in 1000
mg tablets to provide 0.52 mg/tablet of alkylamides, 0.72 mg/tablet
of cichoric acid, and 5.6 mg/tablet of polysaccharides.
EXAMPLE 8
Effects of Echinacea Compositions and Extracts of the Invention on
Immune Function
[0095] The data set forth in this example shows that Echinacea
extracts and compositions of the invention stimulate various
aspects of the rat immune system.
[0096] Animal Maintenance and Tissue Sampling: Young, adult, male
Sprague-Dawley rats (about 240 g each) were acclimatized in the
laboratory for one week before administering the test material.
One-hundred microliters of the test material of various
concentrations, as specified herein, were administered to each
animal, through a gavage needle, two times a day for four days.
Blood and tissue samples were removed from the animals on the fifth
day for analysis. Each test group included eight animals whose test
results were averaged.
[0097] Test Material. The control sample administered to the
animals was a mixture of ethanol and water. One of the following
test materials was administered to each animal (the dose levels,
expressed as microgrammes (.mu.g) test material per kilogram (kg)
body weight of test animal, are set forth in Table 1): one of four
dose levels of Echinacea alkylamide extract, one of four dose
levels of Echinacea cichoric acid extract, one of four dose levels
of Echinacea polysaccharides extract, or one of four dose levels of
an Echinacea composition that includes Echinacea alkylamide,
cichoric acid, and polysaccharides extracts.
[0098] The Echinacea composition contained the three extracts
listed in Table 1 in the specified amounts for the daily dose. For
example, the level 1 daily dosage of the Echinacea composition
provided 0.5 .mu.g/kg alkylamide, 5.0 .mu.g/kg cichoric acid and
125 .mu.g/kg polysaccharides. The Echinacea composition was
prepared by blending the three Echinacea purpurea extracts
(alkylamides, cichoric acid and polysaccharides) which were
prepared as described in Examples 1-3 herein.
1 TABLE 1 Level 1 Level 2 Level 3 Level 4 Level 5 (.mu.g/kg)
(.mu.g/kg) (.mu.g/kg) (.mu.g/kg) (.mu.g/kg) Alkylamide 0.5 4 12 80
200 Cichoric acid 5 40 120 800 2000 polysaccharides 125 1000 3000
20000 50000
[0099] Processing of blood: Blood was obtained from the test
animals by cardiac puncture and saved in an EDTA vacutainer tube
which was centrifuged at 3000 rpm for 10 minutes (min.) to separate
the plasma. The plasma was stored in a capped plastic tube at
-30.degree. C. until use.
[0100] Preparation of splenocytes. Spleens from the animals were
pressed through a nylon mesh in cold Krebs-Ringer-HEPES solution
(KRH) supplemented with 0.5% bovine serum albumin (BSA) and
collected in 50 mL tubes. The collected cells were then centrifuged
at 4.degree. C. for 10 min. at 2000 rpm and the supernatant was
decanted. One milliliter of lysis buffer ACK (containing aluminum
chloride and potassium) was added to the cells. The cells were then
washed twice with 50 mL of KRH+BSA. Cells were then re-suspended in
1 mL CCM (Complete Culture Medium containing RPMI-1640 and 4% fetal
calf serum. RPMI-1640 is a bicarbonate-based buffer system
developed by Moore and Woods as disclosed in the Tissue Culture
Association Manual, 3: 503-508 (1976)) with penicillin,
streptomycin, amphotericin, glutamine, 2-mercaptoethanol and HEPES,
each of which components were present at a concentration of 1%
(v/v). Cells were diluted to 3.0-5.0.times.10.sup.6/mL and
activated with 20 .mu.L Concanavalin A (ConA) (10 .mu.g/mL) and
incubated for 48 hours. After 48 hours, the cells were centrifuged
and the supernatant was collected and frozen at -30.degree. C. for
cytokine and nitric oxide assays.
[0101] Preparation of Alveolar Macrophages. Alveolar macrophages
were obtained by bronchoalveolar lavaging with 40 mL of phosphate
buffered saline (PBS). The cells were centrifuged at 4.degree. C.
for 20 min at 2000 rpm and the supernatant was discarded. 1 mL ACK
was then added and then washed three times with cold PBS. The cells
were then suspended in 1 mL RPMI supplemented with 10% fetal calf
serum. Cells were diluted to 5-10.times.10.sup.6 cells/mL and 100
.mu.L of the diluted cells were incubated for 2 hours at 37.degree.
C. in V-bottom microtiter plates. The supernatant was discarded and
the microtiter plate was washed 3 times with PBS. 200 .mu.L of LPS
was added to activate the cells, and the cells were then incubated
for 24 hours. Supernatant was collected after incubation and frozen
at -30.degree. C. for cytokine and nitric oxide assays.
[0102] Phagocytosis assay. Macrophages were incubated for 3 hours
at 37.degree. C. for adhesion. They were then washed 3 times with
10% RPMI and then latex beads were added at a concentration of
2.5.times.10.sup.6 beads/200 .mu.L. Cells were incubated for one
more hour and washed 5 times with RPMI. The cells were stained and
phagocytosis was determined by microscopic examination.
[0103] Nitric Oxide assay: a stock solution of 4 mM sodium nitrite
was made and diluted to 1 mM with 4% CCM buffer just before use.
The sodium nitrite stock solution was used at various
concentrations to construct a linear calibration curve. Griess
reagent was prepared by dissolving 0.5 g sulfanilamide and 6 mL 85%
phosphoric acid in 100 mL of water and dissolving 0.05 g
N-(1-naphthyl)ethyl-enediamine in 100 mL of water. Supernatants
from macrophages or splenocytes were added to Gniess reagent and
allowed to stand for 10 minutes. Absorbance was measured at 540 nm
and nitric oxide concentration was calculated from the sodium
nitrite standard curve.
[0104] TNF-.alpha., INF-.gamma., and Interleukin-2 Assays.
Antibodies were obtained from any one of the following suppliers:
Sigma Chemicals, Box 14508 St. Louis, Mo. 63178, U.S.A.; R&D
Systems, or PharMagen, 6300 Kitimat Road, Unit 1, Mississauga,
Ontario L5N 5M1, Canada. The assays were performed using a standard
ELISA protocol.
[0105] Effects of Echinacea Compositions and Extracts of the
Invention on Phagocytic Activity and Phagocytic Index in Alveolar
Macrophages: as shown in FIG. 1, all dosage levels (levels 2-5 as
set forth in Table 1) of the Echinacea composition described in
this Example caused an increase in the phagocytic activity and the
Phagocytic Index of alveolar macrophages. As shown more clearly in
FIG. 2, as the dosage increased, so did the magnitude of the effect
on phagocytic activity and the Phagocytic Index.
[0106] As shown in FIG. 3, an Echinacea cichoric acid extract of
the invention, prepared as described in Example 2 herein and
administered to rats at dosage levels 1-4 as set forth in Table 1,
had no significant effect on the phagocytic activity or the
phagocytic index of rat alveolar macrophages. Similarly, as shown
in FIG. 4, an Echinacea polysaccharide extract of the invention,
prepared as described in Example 3 herein and administered to rats
at dosage levels 1-4 as set forth in Table 1, had no significant
effect on the phagocytic activity or the phagocytic index of rat
alveolar macrophages.
[0107] In contrast, as shown in FIG. 5, an Echinacea alkylamides
extract of the invention, prepared as described in Example 1 herein
and administered to rats at dosage levels 1-4 as set forth in Table
1, enhanced both the phagocytic activity and the phagocytic index
of rat alveolar macrophages compared to control rats treated with a
mixture of ethanol and water. In particular, at dose level 3 the
alkylamide extract significantly increased both the activity and
the phagocytic index of the macrophages, with up to 60% increase in
the activity and 50% increase in the index as compared to the
controls. FIG. 6 shows a comparison of the effect on the phagocytic
index of the Echinacea composition described in this Example, and
the individual Echinacea cichoric acid, alkylamide and
polysaccharide extracts. It is clear from FIG. 6 that the Echinacea
composition showed a statistically significant synergistic activity
compared to the individual compounds.
[0108] Effects of Echinacea Compositions and Extracts of the
Invention on the Production of Nitric Oxide by Alveolar
Macrophages: as shown in FIG. 1, dosage levels 3-5, as set forth in
Table 1, of the Echinacea composition described in this Example
caused an increase in the production of nitric oxide by alveolar
macrophages. As the dosage increased, so did the magnitude of the
level of nitric oxide.
[0109] As shown in FIGS. 3 and 4, Echinacea cichoric acid and
polysaccharide extracts, prepared as described in Examples 2 and 3
herein, did not significantly increase the level of nitric oxide
production by alveolar macrophages. In contrast, as shown in FIG.
5, dosage levels 1-4 (especially dosage levels 3 and 4) of an
Echinacea alkylamides extract, prepared as described in Example 1
herein, caused a significant increase in the level of nitric oxide
produced by alveolar macrophages.
[0110] Effects of Echinacea Compositions and Extracts of the
Invention on the Production of TNF-.alpha. in macrophages: as shown
in FIG. 1, the Echinacea composition described in this Example
caused an increase in the production of TNF-.alpha. by alveolar
macrophages in dosages up to level 4. Initially TNF-.alpha.
production increases with increasing dosage of the Echinacea
composition, then the response decreases. As shown in FIG. 7, the
optimum Echinacea composition dosage occurs between Level 3 and
Level 4.
[0111] As shown in FIG. 3, Echinacea cichoric acid extract,
prepared as described in Example 2 herein, did not significantly
increase the level of TNF-.alpha. production by alveolar
macrophages. In contrast, as shown in FIGS. 4 and 5, Echinacea
polysaccharide extract and Echinacea alkylamide extract, prepared
as described in Examples 3 and 1 herein, both caused a significant
increase in the level of TNF-.alpha. produced by alveolar
macrophages. The stimulatory effect of the alkylamide extract was
especially strong at dosage level 3. However at alkylamide dosage
level 4, the production of TNF-.alpha. returned to that of
controls. This might be a consequence of pronounced increase in
nitric oxide production at this dose.
[0112] Effects of Echinacea Compositions and Extracts of the
Invention on the Production of IFN-.gamma. in splenocytes: as shown
in FIG. 1, all dosage levels (levels 2-5 as set forth in Table 1)
of the Echinacea composition described in this Example caused an
increase in IFN-.gamma. production by splenocytes. The effects were
most pronounced at dosage levels 4 and 5.
[0113] As shown in FIGS. 3-5, the Echinacea cichoric acid,
alkylamide and polysaccharide extracts did not cause a significant
increase in IFN-.gamma. production by the splenocytes.
[0114] Effects of Echinacea Compositions and Extracts of the
Invention on the Production of TNF-.alpha. in splenocytes: as shown
in FIG. 1, dosage level 5 of the Echinacea composition described in
this Example caused a significant increase in TNF-.alpha.
production by splenocytes.
[0115] Effects of Echinacea Compositions and Extracts of the
Invention on the Production of IL-2 in splenocytes: as shown in
FIG. 1, dosage levels 2 and 3 of the Echinacea composition
described in this Example suppressed the production of IL-2 in
splenocytes, while dosage levels 4 and 5 yielded a value close to
that of the controls for the production of IL-2. The suppressive
effect is more clearly observed in FIG. 8 which shows a plot of rat
splenocyte production of IL-2 versus relative concentration of the
Echinacea composition. Suppression of IL-2 results in suppression
of the inflammatory response which can reduce the severity of the
symptoms of inflammatory diseases (and may ameliorate the symptoms
of late-onset diabetes).
[0116] As shown in FIGS. 3-5, the Echinacea cichoric acid,
alkylamide and polysaccharide extracts did not affect the
production of IL-2 in splenocytes.
[0117] While the preferred embodiment of the invention has been
illustrated and described, it will be appreciated that various
changes can be made therein without departing from the spirit and
scope of the invention.
* * * * *