U.S. patent application number 14/781863 was filed with the patent office on 2016-02-25 for therapeutic compositions and uses thereof.
This patent application is currently assigned to Manuka Health New Zealand Limited. The applicant listed for this patent is MANUKA HEALTH NEW ZEALAND LIMITED. Invention is credited to Owen CATCHPOLE, Darina LAZAROVA, Kerry PAUL.
Application Number | 20160051594 14/781863 |
Document ID | / |
Family ID | 51658696 |
Filed Date | 2016-02-25 |
United States Patent
Application |
20160051594 |
Kind Code |
A1 |
PAUL; Kerry ; et
al. |
February 25, 2016 |
Therapeutic Compositions and Uses Thereof
Abstract
The invention provides pharmaceutical compositions, including
anti-gastrointestinal cancer compositions, containing propolis and
cyclodextrin. Methods of using such compositions, in particular in
the treatment or prevention of gastrointestinal cancers, and in the
resenitisation of gastrointestinal cancers to therapy, are also
provided.
Inventors: |
PAUL; Kerry; (Auckland,
NZ) ; CATCHPOLE; Owen; (Auckland, NZ) ;
LAZAROVA; Darina; (Scranton, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MANUKA HEALTH NEW ZEALAND LIMITED |
Auckland |
|
NZ |
|
|
Assignee: |
Manuka Health New Zealand
Limited
Auckland
NZ
|
Family ID: |
51658696 |
Appl. No.: |
14/781863 |
Filed: |
April 7, 2014 |
PCT Filed: |
April 7, 2014 |
PCT NO: |
PCT/NZ2014/000060 |
371 Date: |
October 1, 2015 |
Current U.S.
Class: |
424/493 ;
424/537; 435/375; 536/103 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 31/192 20130101; A61K 31/216 20130101; A61K 47/40 20130101;
A61P 1/00 20180101; A61K 35/644 20130101; A61P 43/00 20180101; A61K
9/5161 20130101; A61K 31/352 20130101; A61K 9/19 20130101; A61P
35/00 20180101; A61K 47/6951 20170801; A61K 9/1652 20130101; A61K
31/353 20130101; A61K 35/63 20150115 |
International
Class: |
A61K 35/644 20060101
A61K035/644; A61K 31/353 20060101 A61K031/353; A61K 31/216 20060101
A61K031/216; A61K 47/48 20060101 A61K047/48 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 5, 2013 |
NZ |
609074 |
Claims
1. A method of treating or preventing a gastrointestinal cancer in
a subject or of inducing apoptosis of one or more neoplastic
gastrointestinal cells in a subject, the method comprising
administering an effective amount of a composition comprising,
consisting essentially or, or consisting of propolis and
cyclodextrin, to a subject in need thereof.
2. A method of inhibiting gastrointestinal tumour formation,
inhibiting gastrointestinal tumour growth, inhibiting
gastrointestinal tumour metastasis, or treating or preventing a
gastrointestinal cancer in a subject, the method comprising
separate, simultaneous or sequential administration of an effective
amount of a composition comprising, consisting essentially or, or
consisting of propolis and cyclodextrin, to a subject in need
thereof.
3. A method of increasing the responsiveness of a subject to a
gastrointestinal cancer therapy or of increasing the sensitivity of
a gastrointestinal tumour in a subject to a gastrointestinal cancer
therapy, the method comprising administration to the subject of a
composition comprising, consisting essentially or, or consisting of
propolis and cyclodextrin.
4. A method of resensitising one or more gastrointestinal cancer
cells that are resistant to treatment, the method comprising
administering an effective amount of a composition comprising,
consisting essentially or, or consisting of propolis and
cyclodextrin to the one or more cancer cells.
5. A method of at least partially reversing the resistance of a
neoplastic cell in a subject suffering from gastrointestinal cancer
to a gastrointestinal cancer therapy, or of reversing, wholly or in
part, the resistance of a gastrointestinal cancer-burdened patient
to a gastrointestinal cancer therapy, or of re-sensitising one or
more tumours of a gastrointestinal cancer-burdened patient which
are, or are predicted to either be or become, resistant to
treatment with a gastrointestinal cancer therapy, the method
comprising administration to the subject of a composition
comprising, consisting essentially or, or consisting of propolis
and cyclodextrin.
6. A method of improving gut health, the method comprising
administering to a subject in need thereof a composition
comprising, consisting essentially of, or consisting of propolis
and cyclodextrin; and in particular, propolis and
gamma-cyclodextrin, for example European propolis and
gamma-cyclodextrin.
7. The method according to any one of claims 1 to 6, wherein the
composition consists of propolis and cyclodextrin.
8. The use of propolis and cyclodextrin in the manufacture of a
composition for use in inhibiting gastrointestinal tumour
formation, inhibiting gastrointestinal tumour growth, inhibiting
gastrointestinal tumour metastasis or treating or preventing
gastrointestinal cancer in a subject; inducing apoptosis of one or
more neoplastic gastrointestinal cells in a subject; increasing the
responsiveness of a subject to a gastrointestinal cancer therapy;
increasing the sensitivity of a gastrointestinal tumour in a
subject to a gastrointestinal cancer therapy; resensitising one or
more gastrointestinal cancer cells in a subject that are resistant
to treatment; at least partially reversing the resistance of a
neoplastic cell in a subject suffering from gastrointestinal cancer
to a gastrointestinal cancer therapy; reversing, wholly or in part,
the resistance of a gastrointestinal cancer-burdened patient to a
gastrointestinal cancer therapy; or re-sensitising one or more
tumours of a gastrointestinal cancer-burdened patient which are, or
are predicted to either be or become, resistant to treatment with a
gastrointestinal cancer therapy to treatment with a
gastrointestinal cancer therapy.
9. The use according to claim 8 wherein the cyclodextrin is
gamma-cyclodextrin.
10. The use according to claim 8 or 9 wherein the composition
comprises at least one additional therapeutic agent.
11. An anti-gastrointestinal cancer composition comprising propolis
and cyclodextrin.
12. The method of any one of claims 1 to 7, the use of any one of
claims 8 to 10, or the composition of claim 11, wherein the
cyclodextrin is gamma-cyclodextrin, or the cyclodextrin is present
as a combination of cyclodextrins comprising
gamma-cyclodextrin.
13. The method of any one of claim 1 to 7 or 12, the use of any one
of claim 8 to 10 or 12, or the composition of claim 11 or 12,
wherein the cyclodextrin is chemically-modified cyclodextrin.
14. The method of any one of claim 1 to 7, 12 or 13, the use of any
one of claim 8 to 10, 12, or 13, or the composition of any one of
claims 11 to 13, wherein the propolis is a propolis extract or
fraction.
15. The method, use, or composition of claim 14 wherein the
propolis is free of wax.
16. The method of any one of claims 1 to 7, or 12 to 14, the use of
any one of claim 8 to 10, or 12 to 14, or the composition of any
one of claims 11 to 14, wherein the propolis is European or Poplar
propolis.
17. The method of any one of claims 1 to 7, or 12 to 14, the use of
any one of claim 8 to 10, or 12 to 14, or the composition of any
one of claims 11 to 14, wherein the propolis is Brazilian Green
propolis.
18. The method, use, or composition of claim 17 wherein the
Brazilian Green propolis is at least in part obtained by bees from
leaf exudates of the tree Baccharis dracanculifolia.
19. The method of any one of claims 1 to 7, or 12 to 18, the use of
any one of claim 8 to 10, or 12 to 18, or the composition of any
one of claims 11 to 18, wherein the composition comprises from
about 1.0% wt to about 99% wt propolis.
20. The method of any one of claims 1 to 7, or 12 to 18, the use of
any one of claim 8 to 10, or 12 to 18, or the composition of any
one of claims 11 to 18, wherein the composition comprises from
about 1.0% wt to about 99% wt propolis resin.
21. The method, use, or composition of claim 19 wherein the
composition comprises from about 1% wt to about 99% wt propolis,
from about 1% wt to about 25% wt propolis, from about 1% wt to
about 30% wt propolis, from about 5% wt to about 25% wt propolis,
from about 5% wt to about 30% wt propolis, from about 5% wt to
about 99% wt propolis, from about 10% wt to about 25% wt propolis,
from about 10% wt to about 30% wt propolis, from about 10% wt to
about 99% wt propolis, from about 15% wt to about 25% wt, from
about 15% wt to about 30% wt, from about 15% wt to about 99% wt,
from about 20% wt to about 25% wt, from about 20% wt to about 30%
wt, from about 20% wt to about 99% wt, or about 25% wt propolis, or
about 30% wt propolis.
22. The method, use, or composition of claim 20 wherein the
composition comprises from about 1% wt to about 99% wt propolis
resin, from about 1% wt to about 25% wt propolis resin, from about
1% wt to about 30% wt propolis resin, from about 5% wt to about 25%
wt propolis resin, from about 5% wt to about 30% wt propolis resin,
from about 5% wt to about 99% wt propolis resin, from about 10% wt
to about 25% wt propolis resin, from about 10% wt to about 30% wt
propolis resin, from about 10% wt to about 99% wt propolis resin,
from about 15% wt to about 25% wt, from about 15% wt to about 30%
wt, from about 15% wt to about 99% wt, from about 20% wt to about
25% wt, from about 20% wt to about 30% wt, from about 20% wt to
about 99% wt, or about 25% wt propolis resin, or about 30% wt
propolis resin.
23. The method of any one of claims 1 to 7, or 12 to 22, the use of
any one of claims 8 to 10, or 12 to 22, or the composition of any
one of claims 11 to 22, wherein the propolis in the composition is
entirely encapsulated within the cyclodextrin.
24. The method of any one of claims 1 to 7, or 12 to 23, the use of
any one of claims 8 to 10, or 12 to 23, or the composition of any
one of claims 11 to 23, wherein the molar ratio of propolis to
cyclodextrin in the composition is no greater than about 1:1.
25. A composition comprising, consisting essentially of or
consisting of propolis and gamma-cyclodextrin.
26. A composition of any one of the preceeding claims for use in
inhibiting gastrointestinal tumour formation, inhibiting
gastrointestinal tumour growth, inhibiting gastrointestinal tumour
metastasis or treating or preventing gastrointestinal cancer in a
subject; inducing apoptosis of one or more neoplastic
gastrointestinal cells in a subject; increasing the responsiveness
of a subject to a gastrointestinal cancer therapy; increasing the
sensitivity of a gastrointestinal tumour in a subject to a
gastrointestinal cancer therapy; resensitising one or more
gastrointestinal cancer cells in a subject that are resistant to
treatment; at least partially reversing the resistance of a
neoplastic cell in a subject suffering from gastrointestinal cancer
to a gastrointestinal cancer therapy; reversing, wholly or in part,
the resistance of a gastrointestinal cancer-burdened patient to a
gastrointestinal cancer therapy; or re-sensitising one or more
tumours of a gastrointestinal cancer-burdened patient which are, or
are predicted to either be or become, resistant to treatment with a
gastrointestinal cancer therapy to treatment with a
gastrointestinal cancer therapy.
27. The composition of claim 26 for use in the treatment or
prevention of gastrointestinal cancer.
28. An anti-gastrointestinal cancer composition comprising
cyclodextrin.
29. The method, use, or composition as claimed in claim 16, wherein
the European propolis has a caffeic acid phenylether ester
concentration of greater than about 1 mg/g.
30. The method, use, or composition as claimed in claim 16 or claim
29, wherein the European propolis has a pinocembrin concentration
of greater than about 10 mg/g.
31. The method, use, or composition as claimed in claim 16, 29 or
30, wherein the European propolis has a galangin concentration of
greater than about 5 mg/g.
32. The method, use, or composition as claimed in claim 16 or any
one of claims 29 to 31, wherein the European propolis has chrysin
concentration of greater than about 3.5 mg/g.
33. The method, use, or composition as claimed in any one of claims
1 to 32, wherein the composition comprises two or more of caffeic
acid phenylether ester (CAPE), caffeic acid, pinocembrin, benzyl
caffeate, chrysin, galangin, and pinobanksin.
34. The method, use, or composition as claimed in any one of claims
1 to 33, wherein the composition is one to which has been added one
or more of CAPE, caffeic acid, pinocembrin, benzyl caffeate,
chrysin, galangin, and pinobanksin.
35. The method, use, or composition as claimed in any one of claims
1 to 34, wherein the composition has a CAPE concentration of
greater than about 1 mg/g.
36. The method, use, or composition as claimed in any one of claims
1 to 35, wherein composition has a pinocembrin concentration of
greater than about 1 mg/g.
37. The method, use, or composition as claimed in any one of claims
1 to 36, wherein the composition has a galangin concentration of
greater than about 1 mg/g.
38. The method, use, or composition as claimed in any one of claims
1 to 37, wherein the composition has a chrysin concentration of
greater than about 1 mg/g.
39. The method, use, or composition as claimed in any one of claims
1 to 38, wherein the composition has a pinobanksin concentration of
greater than about greater than about 1 mg/g.
40. The method, use, or composition as claimed in any one of claims
1 to 39, wherein the composition has a caffeic acid concentration
of greater than about greater than about 1 mg/g.
Description
FIELD OF INVENTION
[0001] This invention relates to compositions for the treatment and
prevention of gastrointestinal cancers. In particular, this
invention relates to anti-gastrointestinal cancer compositions
containing propolis and cyclodextrin, including
anti-gastrointestinal cancer compositions comprising one or more
propolis extracts. Particularly contemplated are
anti-gastrointestinal cancer compositions comprising propolis and
.gamma.-cyclodextrin, for example European propolis and
.gamma.-cyclodextrin, and the use of such compositions in the
treatment or prevention of gastrointestinal cancers such as
colorectal, gastric and throat cancers.
BACKGROUND OF THE INVENTION
[0002] Crude propolis is a resinous substance produced by bees from
the resin collected from botanical sources, such as buds and sap,
which is admixed with beeswax. The colour of crude propolis can
vary from yellow, through browns to almost black depending on the
botanical source. Beeswax is normally separated from the propolis
by extraction using ethanol, in which the wax is insoluble but the
resinous compounds are highly soluble. There are a number of types
of propolis, which are based on the botanical sources of resin
compounds, and the geographical region. The most well-known types
of propolis are "European" propolis, where the resin compounds are
obtained by bees mostly from leaf and bud exudates of poplars, and
to a lesser extent birches and willows; and "Brazilian Green"
propolis, which is mainly obtained by bees from leaf exudates of
the tree Baccharis dracanculifolia. Propolis produced in New
Zealand can be categorized as "European" as its composition broadly
matches other European propolis (Markham et al, 1996. HPLC and
GC-MS identification of the major organic constituents in New
Zealand propolis. Phytochemistry, 42(1): 205-211). Crude propolis
and extracts derived from propolis have been reported to have
antibacterial, antifungal, and antiviral activities. Its use in the
treatment of several types of cancer, in particular breast cancer,
has also been investigated.
[0003] Identification and verification of the anti-cancer
constituent(s) present in propolis resin has been challenging
because of the complex and multicomponent nature of the resin. In
Brazilian Green propolis, the anti-cancer activity is mainly
attributed to artepillan C, while in European propolis resin the
anti-cancer activity is mainly attributed to caffeic acid phenethyl
ester (CAPE), a dihydroxy cinnamic acid ester; and chrysin, an
aglycone flavonoid, see for example, Sawicka et al (2012) "The
anticancer activity of propolis", Folia Histochemica et
Cytobiologica, 50 (1), 25-37.
[0004] Colorectal cancer is reportedly the second and third most
common cancer in women and men, respectively, from developed
countries. Colorectal cancer is more prevalent in developed
countries--the US, Australia, Europe, and New Zealand having the
highest rates--with incidence being as much as 10 times greater
than in developing countries. While surgery can be effective, early
detection is critical to positive surgical outcomes. Other
therapies are largely directed at life extension and palliative
care, as the efficacy of current chemotherapies and radiotherapies
in treating primary tumours, or metastases outside the lymph nodes
is debated.
[0005] Throat cancer, also referred to as oesophaegeal cancer,
pharyngeal cancer, or laryngeal cancer, encompasses tumours that
develop in the tissues of the pharynx, nasopharynx, oropharynx,
hypopharynx, larynx (voice box) or tonsils. Therapies for throat
cancer include surgery, radiotherapy or chemotherapy. Treatment for
throat cancer can damage the throat and may cause changes to the
way a patient eats, breathes and sleeps.
[0006] Gastric or stomach cancer is the second most common cause of
cancer-related death in the world. Diagnosis is often delayed
because symptoms may not occur in the early stages of the disease.
Surgery to remove the stomach (gastrectomy) is the only treatment
that can cure gastric cancers. Chemotherapy and radiation therapy
after surgery may improve the chance of a cure.
[0007] Accordingly, there is a need for anti-gastrointestinal
cancer compositions, including those suitable for use in the
treatment or prevention of colorectal cancer, gastric cancer and
throat cancer and those which are able to support the maintenance
of anti-gastrointestinal cancer activity or augment
anti-gastrointestinal cancer activity.
[0008] It is an object of the present invention to provide
anti-gastrointestinal cancer compositions, including stable
anti-gastrointestinal cancer compositions for use in the treatment
or prevention of colorectal cancer, gastric cancer and throat
cancer, or to at least provide the public with a useful choice.
SUMMARY OF THE INVENTION
[0009] Accordingly, in a first aspect the invention relates to an
anti-gastrointestinal cancer composition comprising propolis and
cyclodextrin, and in particular, gamma cyclodextrin.
[0010] In one embodiment the anti-gastrointestinal composition is
an anti-colorectal cancer composition. In another embodiment the
anti-gastrointestinal composition is an anti-gastric cancer
composition. In a further embodiment the anti-gastrointestinal
composition is an anti-throat cancer composition.
[0011] In another aspect, the present invention relates to a
pharmaceutical composition comprising, consisting essentially of,
or consisting of propolis and gamma-cyclodextrin. In one
embodiment, the composition is for maintaining or improving gut
health. Accordingly, in one embodiment the invention relates to a
pharmaceutical composition for maintaining or improving gut health,
the composition comprising, consisting essentially of, or
consisting of propolis and cyclodextrin.
[0012] In one embodiment the propolis is propolis resin. In one
embodiment the resin is propolis resin and the cyclodextrin is
gamma-cyclodextrin.
[0013] In another aspect the invention relates to a method of
treating or preventing a gastrointestinal cancer in a subject, the
method comprising administering an effective amount of a
composition comprising, consisting essentially of, or consisting of
propolis and cyclodextrin, to a subject in need thereof.
[0014] In another aspect the invention relates to a method of
inhibiting gastrointestinal tumour formation, inhibiting
gastrointestinal tumour growth, inhibiting gastrointestinal tumour
metastasis or treating or preventing a gastrointestinal cancer in a
subject, the method comprising separate, simultaneous or sequential
administration of an effective amount of a composition comprising,
consisting essentially of, or consisting of propolis and
cyclodextrin, to a subject in need thereof.
[0015] Another aspect of the invention relates to a method of
inducing apoptosis of one or more neoplastic gastrointestinal cells
in a subject, the method comprising administration of an effective
amount of a composition comprising, consisting essentially of, or
consisting of propolis and cyclodextrin, to a subject in need
thereof.
[0016] In one embodiment the apoptosis is of gastrointestinal
tumour cells, such as colorectal, gastric or throat tumour
cells.
[0017] Another aspect of the invention relates to a method of
modulating proliferation of one or more neoplastic gastrointestinal
cells in a subject, the method comprising administration of an
effective amount of a composition comprising, consisting
essentially of, or consisting of propolis and cyclodextrin, to a
subject in need thereof.
[0018] For example in one embodiment the modulation is reduction.
Accordingly the invention relates to a method of reducing
proliferation of one or more neoplastic gastrointestinal cells in a
subject, the method comprising administration of an effective
amount of a composition comprising, consisting essentially of, or
consisting of propolis and cyclodextrin, to a subject in need
thereof.
[0019] In one embodiment the proliferation is of gastrointestinal
tumour cells, such as colorectal, gastric or throat tumour
cells.
[0020] Another aspect of the invention relates to a method of
increasing the responsiveness of a subject to a gastrointestinal
cancer therapy comprising administration to the subject of a
composition comprising, consisting essentially of, or consisting of
propolis and cyclodextrin.
[0021] Another aspect of the invention relates to a method of
increasing the sensitivity of a gastrointestinal tumour in a
subject to a gastrointestinal cancer therapy comprising
administration to the subject of a composition comprising,
consisting essentially of, or consisting of propolis and
cyclodextrin.
[0022] In a further aspect, the invention relates to a method of
resensitising one or more gastrointestinal cancer cells that are
resistant to treatment, the method comprising administering an
effective amount of a composition comprising, consisting
essentially of, or consisting of propolis and cyclodextrin to the
one or more gastrointestinal cancer cells.
[0023] In one embodiment, the gastrointestinal cancer cells
comprise a tumour present in a subject. In one embodiment the
gastrointestinal cancer cells are colorectal cancer cells. In
another embodiment the gastrointestinal cancer cells are gastric
cancer cells. In a further embodiment the gastrointestinal cancer
cells are throat cancer cells.
[0024] The invention also relates to a method of at least partially
reversing the resistance of a neoplastic cell in a subject
suffering from a gastrointestinal cancer to a gastrointestinal
cancer therapy, the method comprising administration to the subject
of a composition comprising, consisting essentially or, or
consisting of propolis and cyclodextrin.
[0025] The present invention further relates to a method of
reversing, wholly or in part, the resistance of a gastrointestinal
cancer-burdened patient to a gastrointestinal cancer therapy, the
method comprising the step of administering to said patient a
composition comprising, consisting essentially of, or consisting of
propolis and cyclodextrin.
[0026] In another aspect, the invention provides a method of
re-sensitising one or more tumours of a gastrointestinal
cancer-burdened patient which are, or are predicted to either be or
become, resistant to treatment with a gastrointestinal cancer
therapy, said method comprising the step of administering to said
patient a composition comprising, consisting essentially or, or
consisting of propolis and cyclodextrin.
[0027] In one embodiment, the one or more tumours are or are
predicted to be or to become resistant to a gastrointestinal cancer
therapy due increased activation of one or more pro-cancer cell
survival signaling pathways within the one or more tumours or
within the patient, including increased activation of one or more
of the AKT, JNK or JAK/STAT signaling pathways, for example within
a sample from the patient, such as a tissue sample, a tumour
biopsy, or a blood or plasma sample.
[0028] In one embodiment, the invention provides a method of
inactivating or suppressing one or more pro-cancer cell survival
signalling pathways within the one or more tumours or within the
patient. For example, the invention relates to a method of
inactivating or suppressing one or more of the AKT, JNK or JAK/STAT
signaling pathways within the one or more tumours.
[0029] In one embodiment, the one or more tumours are or are
predicted to be or to become resistant to a gastrointestinal cancer
therapy due to increased activation of one or more of the AKT, JNK
or JAK/STAT signaling pathways within the tumour(s).
[0030] In one embodiment, the invention provides a method of
preventing tumours becoming resistant to a primary gastrointestinal
cancer therapy, wherein the resistance is at least in part mediated
by increased activation of one or more of the AKT, JNK or JAK/STAT
signaling pathways, for example within the tumour(s).
[0031] In one embodiment, the tumours are resistant to treatment
with a chemotherapeutic.
[0032] In one embodiment the gastrointestinal cancer is colorectal
cancer. In another embodiment the gastrointestinal cancer is
gastric cancer. In a further embodiment the gastrointestinal cancer
is throat cancer.
[0033] In still a further aspect, the present invention relates to
a method of improving gut health, the method comprising
administering to a subject in need thereof a composition
comprising, consisting essentially of, or consisting of propolis
and cyclodextrin; and in particular, propolis and
gamma-cyclodextrin, for example European propolis and
gamma-cyclodextrin.
[0034] In a further aspect, the invention provides a synergistic
composition comprising propolis and gamma-cyclodextrin. In one
embodiment, the composition is a synergistic therapeutic
composition. In one embodiment, the composition provides a
synergistic therapeutic effect.
[0035] In one embodiment the propolis and gamma-cyclodextrin
provide a synergistic therapeutic effect that is greater than the
effect of either one alone or greater than the additive effects of
either one alone. For example, there is a greater effect on
induction of apoptosis, on gastrointestinal cancer cell survival or
proliferation, on resensitisation to therapy, on treatment or
prevention of gastrointestinal cancer, or the responsiveness of a
subject or a tumour to the treatment method. In one embodiment, the
propolis and gamma-cyclodextrin allow the administration of a
co-administered or sequentially administered gastrointestinal
cancer therapy to be reduced or increased in dose or in length of
administration, as appropriate.
[0036] Another aspect of the invention relates to use of propolis
and cyclodextrin in the manufacture of a composition for a purpose
as herein described.
[0037] Another aspect of the invention relates to use of propolis
and gamma-cyclodextrin with at least one additional therapeutic
agent in the manufacture of a composition for a purpose as herein
described.
[0038] Another aspect of the invention relates to use of a complex
comprising propolis and gamma-cyclodextrin, with at least one
additional therapeutic agent in the manufacture of a composition
for a purpose as herein described, wherein the composition is
formulated to provide separate, simultaneous or sequential
administration of the propolis and gamma-cyclodextrin complex and
the at least one additional therapeutic agent.
[0039] Another aspect of the invention relates to a composition
comprising, consisting essentially of or consisting of propolis and
gamma-cyclodextrin.
[0040] Another aspect of the invention relates to a product
comprising, consisting essentially of or consisting of propolis and
gamma-cyclodextrin, optionally with one or more, two or more or
three or more additional therapeutic agents as a combined
preparation for simultaneous, separate or sequential use for a
purpose as described herein.
[0041] Another aspect of the invention relates to a composition of
the invention for use in the treatment or prevention of a
gastrointestinal cancer.
[0042] The following embodiments may relate to any of the above
aspects.
[0043] In various embodiments, the cyclodextrin is
gamma-cyclodextrin, or the cyclodextrin is present as a combination
of cyclodextrins comprising gamma-cyclodextrin.
[0044] In one embodiment, the cyclodextrin is chemically-modified
cyclodextrin.
[0045] In one embodiment, the propolis is present in the
anti-gastrointestinal cancer composition as a propolis extract or
fraction.
[0046] In one embodiment, the propolis present in the
anti-gastrointestinal cancer composition is free of wax. For
example, the propolis has been dewaxed using extraction processes
known in the art.
[0047] In one embodiment, the propolis is "European" or "Poplar"
propolis. For example, the "European" propolis is at least in part
derived from the bud and leaf exudates of one or more species of
poplars, birches, larches or willows. In another embodiment, the
propolis is "Brazilian Green" propolis. For example, the
"Brazilian" propolis is at least in part obtained by bees from leaf
exudates of the tree Baccharis dracanculifolia.
[0048] In one embodiment, the composition comprises from about 1.0%
wt to about 99% wt propolis. In an alternative embodiment the
composition comprises from about 1.0% wt to about 99% wt propolis
resin.
[0049] In various embodiments, the composition comprises from about
1% wt to about 99% wt propolis, from about 1% wt to about 25% wt
propolis, from about 1% wt to about 30% wt propolis, from about 5%
wt to about 25% wt propolis, from about 5% wt to about 30% wt
propolis, from about 5% wt to about 99% wt propolis, from about 10%
wt to about 25% wt propolis, from about 10% wt to about 30% wt
propolis, from about 10% wt to about 99% wt propolis, from about
15% wt to about 25% wt, from about 15% wt to about 30% wt, from
about 15% wt to about 99% wt, from about 20% wt to about 25% wt,
from about 20% wt to about 30% wt, from about 20% wt to about 99%
wt, or about 25% wt propolis, or about 30% wt propolis.
[0050] In various embodiments, the composition comprises from about
1% wt to about 99% wt propolis resin, from about 1% wt to about 25%
wt propolis resin, from about 1% wt to about 30% wt propolis resin,
from about 5% wt to about 25% wt propolis resin, from about 5% wt
to about 30% wt propolis resin, from about 5% wt to about 99% wt
propolis resin, from about 10% wt to about 25% wt propolis resin,
from about 10% wt to about 30% wt propolis resin, from about 10% wt
to about 99% wt propolis resin, from about 15% wt to about 25% wt,
from about 15% wt to about 30% wt, from about 15% wt to about 99%
wt, from about 20% wt to about 25% wt, from about 20% wt to about
30% wt, from about 20% wt to about 99% wt, or about 25% wt propolis
resin, or about 30% wt propolis resin.
[0051] In one embodiment the propolis in the composition is
entirely encapsulated within the cyclodextrin.
[0052] In one embodiment the molar ratio of propolis to
cyclodextrin in the composition is no greater than about 1:1.
[0053] In one embodiment, the European propolis comprises caffeic
acid phenylether ester (CAPE). In one embodiment the European
propolis comprises chrysin. In one embodiment the European propolis
comprises caffeic acid. In one embodiment the European propolis
comprises benzyl caffeate. In one embodiment the European propolis
comprises pinobanksin. In one embodiment the European propolis
comprises pinocembrin. In one embodiment the European propolis
comprises galangin.
[0054] In various embodiments, the European propolis comprises any
combination of two or more of CAPE, chrysin, galangin, pinocembrin,
pinobanksin, benzyl caffeate, and caffeic acid.
[0055] In one embodiment, the European propolis has a CAPE
concentration of greater than about 1 mg/g, than about 1.5 mg/g,
than about 2 mg/g, about 2.5 mg/g, about 3 mg/g, about 3.5 mg/g,
about 4 mg/g, about 4.5 mg/g, about 5 mg/g, about 5.5 mg/g, about 6
mg/g, about 7.5 mg/g, about 10 mg/g, about 15 mg/g, about 20 mg/g,
about 25 mg/g, about 30 mg/g, about 40 mg/g, about 50 mg/g, about
75 mg/g, about 100 mg/g, about 125 mg/g, about 150 mg/g, about 175
mg/g, about 200 mg/g, 250 mg/g, about 300 mg/g, about 350 mg/g,
about 400 mg/g, about 450 mg/g, about 500 g/g, about 550 mg/g,
about 600 mg/g, about 650 mg/g, about 700 mg/g, about 750 mg/g,
about 800 mg/g, about 850 mg/g, about 900 mg/g, about 950 mg/g, up
to about 1000 mg/g.
[0056] In one embodiment, the European propolis has a pinocembrin
concentration of greater than about 1 mg/g, than about 1.5 mg/g,
than about 2 mg/g, about 2.5 mg/g, about 3 mg/g, about 3.5 mg/g,
about 4 mg/g, about 4.5 mg/g, about 5 mg/g, about 5.5 mg/g, about 6
mg/g, about 7.5 mg/g, about 10 mg/g, about 15 mg/g, about 20 mg/g,
about 25 mg/g, about 30 mg/g, about 40 mg/g, about 50 mg/g, about
75 mg/g, about 100 mg/g, about 125 mg/g, about 150 mg/g, about 175
mg/g, about 200 mg/g, 250 mg/g, about 300 mg/g, about 350 mg/g,
about 400 mg/g, about 450 mg/g, about 500 mg/g, about 550 mg/g,
about 600 mg/g, about 650 mg/g, about 700 mg/g, about 750 mg/g,
about 800 mg/g, about 850 mg/g, about 900 mg/g, about 950 mg/g, up
to about 1000 mg/g.
[0057] In one embodiment, the European propolis has a galangin
concentration of greater than about 1 mg/g, than about 1.5 mg/g,
than about 2 mg/g, about 2.5 mg/g, about 3 mg/g, about 3.5 mg/g,
about 4 mg/g, about 4.5 mg/g, about 5 mg/g, about 5.5 mg/g, about 6
mg/g, about 7.5 mg/g, about 10 mg/g, about 15 mg/g, about 20 mg/g,
about 25 mg/g, about 30 mg/g, about 40 mg/g, about 50 mg/g, about
75 mg/g, about 100 mg/g, about 125 mg/g, about 150 mg/g, about 175
mg/g, about 200 mg/g, 250 mg/g, about 300 mg/g, about 350 mg/g,
about 400 mg/g, about 450 mg/g, about 500 mg/g, about 550 mg/g,
about 600 mg/g, about 650 mg/g, about 700 mg/g, about 750 mg/g,
about 800 mg/g, about 850 mg/g, about 900 mg/g, about 950 mg/g, up
to about 1000 mg/g.
[0058] In one embodiment, the European propolis has chrysin
concentration of greater than about 1 mg/g, than about 1.5 mg/g,
than about 2 mg/g, about 2.5 mg/g, about 3 mg/g, about 3.5 mg/g,
about 4 mg/g, about 4.5 mg/g, about 5 mg/g, about 5.5 mg/g, about 6
mg/g, about 7.5 mg/g, about 10 mg/g, about 15 mg/g, about 20 mg/g,
about 25 mg/g, about 30 mg/g, about 40 mg/g, about 50 mg/g, about
75 mg/g, about 100 mg/g, about 125 mg/g, about 150 mg/g, about 175
mg/g, about 200 mg/g, 250 mg/g, about 300 mg/g, about 350 mg/g,
about 400 mg/g, about 450 mg/g, about 500 mg/g, about 550 mg/g,
about 600 mg/g, about 650 mg/g, about 700 mg/g, about 750 mg/g,
about 800 mg/g, about 850 mg/g, about 900 mg/g, about 950 mg/g, up
to about 1000 mg/g.
[0059] In one embodiment, the European propolis has benzyl caffeate
concentration of greater than about 1 mg/g, than about 1.5 mg/g,
than about 2 mg/g, about 2.5 mg/g, about 3 mg/g, about 3.5 mg/g,
about 4 mg/g, about 4.5 mg/g, about 5 mg/g, about 5.5 mg/g, about 6
mg/g, about 7.5 mg/g, about 10 mg/g, about 15 mg/g, about 20 mg/g,
about 25 mg/g, about 30 mg/g, about 40 mg/g, about 50 mg/g, about
75 mg/g, about 100 mg/g, about 125 mg/g, about 150 mg/g, about 175
mg/g, about 200 mg/g, 250 mg/g, about 300 mg/g, about 350 mg/g,
about 400 mg/g, about 450 mg/g, about 500 mg/g, about 550 mg/g,
about 600 mg/g, about 650 mg/g, about 700 mg/g, about 750 mg/g,
about 800 mg/g, about 850 mg/g, about 900 mg/g, about 950 mg/g, up
to about 1000 mg/g.
[0060] In various embodiments, the composition comprises one or
more of caffeic acid phenylether ester (CAPE), caffeic acid,
pinocembrin, benzyl caffeate, chrysin, galangin, and
pinobanksin.
[0061] In exemplary embodiments, the composition is one to which
has been added one or more of CAPE, caffeic acid, pinocembrin,
benzyl caffeate, chrysin, galangin, and pinobanksin.
[0062] In one embodiment, the composition has a CAPE concentration
of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350,
400, 450, 500, 600, 700, 800, 900 or 999 mg/g and useful ranges may
be selected between any of these values (for example, about 1 to
about 5, about 1 to about 10, about 2 to about 20, about 5 to about
20, about 5 to about 25, about 10 to about 25, about 10 to about
40, about 15 to about 100, or about 20 to about 999 mg/g).
[0063] In one embodiment, the composition has a pinocembrin
concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250,
300, 350, 400, 450, 500, 600, 700, 800, 900 or 999 mg/g and useful
ranges may be selected between any of these values (for example,
about 1 to about 5, about 1 to about 10, about 2 to about 20, about
5 to about 20, about 5 to about 25, about 10 to about 25, about 10
to about 40, about 15 to about 100, or about 20 to about 999
mg/g).
[0064] In one embodiment, the composition has a galangin
concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250,
300, 350, 400, 450, 500, 600, 700, 800, 900 or 999 mg/g and useful
ranges may be selected between any of these values (for example,
about 1 to about 5, about 1 to about 10, about 2 to about 20, about
5 to about 20, about 5 to about 25, about 10 to about 25, about 10
to about 40, about 15 to about 100, or about 20 to about 999
mg/g).
[0065] In one embodiment, the composition has a chrysin
concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250,
300, 350, 400, 450, 500, 600, 700, 800, 900 or 999 mg/g and useful
ranges may be selected between any of these values (for example,
about 1 to about 5, about 1 to about 10, about 2 to about 20, about
5 to about 20, about 5 to about 25, about 10 to about 25, about 10
to about 40, about 15 to about 100, or about 20 to about 999
mg/g).
[0066] In one embodiment, the composition has a pinobanksin
concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250,
300, 350, 400, 450, 500, 600, 700, 800, 900 or 999 mg/g and useful
ranges may be selected between any of these values (for example,
about 1 to about 5, about 1 to about 10, about 2 to about 20, about
5 to about 20, about 5 to about 25, about 10 to about 25, about 10
to about 40, about 15 to about 100, or about 20 to about 999
mg/g).
[0067] In one embodiment, the composition has a caffeic acid
concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200, 250,
300, 350, 400, 450, 500, 600, 700, 800, 900 or 999 mg/g and useful
ranges may be selected between any of these values (for example,
about 1 to about 5, about 1 to about 10, about 2 to about 20, about
5 to about 20, about 5 to about 25, about 10 to about 25, about 10
to about 40, about 15 to about 100, or about 20 to about 999
mg/g).
[0068] In various embodiments, the composition comprises or is
administered separately, simultaneously or sequentially with at
least one additional therapeutic agent, preferably the at least one
additional therapeutic agent is an anti-tumour agent, preferably
the anti-tumour agent is selected from an anti-tumour food factor,
a chemotherapeutic agent, or an immunotherapeutic agent.
[0069] In various embodiments, the gastrointestinal cancer therapy,
the therapeutic agent, or the anti-tumour agent is effective to
induce apoptosis, for example, induce apoptosis in one or more
gastrointestinal cancer cells or in one or more neoplastic
cells.
[0070] In one embodiment, the gastrointestinal cancer therapy, the
therapeutic agent, or the anti-tumour agent is butyrate or a source
of butyrate.
[0071] In a further embodiment, the butyrate is generated by the
digestion of cyclodextrin by intestinal or colonic microflora.
[0072] In one embodiment, the composition is a consumer good.
[0073] In one embodiment the composition is a food, drink, food
additive, drink additive, dietary supplement, nutritional product,
medical food, nutraceutical, medicament or pharmaceutical.
[0074] In various embodiments, the composition may be formulated
for oral, topical, or parenteral administration.
[0075] In one embodiment, the composition comprises one or more
additional anti-gastrointestinal cancer agents.
[0076] In one embodiment, the composition is a pharmaceutical
composition.
[0077] In various embodiments, the chemotherapeutic agent is
selected from the group comprising mitotic inhibitors, such as
vinca alkaloids, including vincristine, vinblastine, vinorelbine,
vindesine, vinflunine, podophyllotoxin, taxanes, including
docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel, and
epothilones, such as ixabepilone; topoisomerase I inhibitors, such
as topotecan, irinotecan, camptothecin, rubitecan, and belotecan,
topoisomerase type II inhibitors, including amsacrine, etoposide,
etoposide phosphate, and teniposide, anthracyclines, such as
aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin,
amrubicin, pirarubicin, valrubicin, and zorubicin, and
anthracenediones, such mitoxantrone and pixantrone;
antimetabolites, including dihydrofolate reductase inhibitors, such
as aminopterin, methotrexate, pemetrexed, thymidylate synthase
inhibitors, such as raltitrexed and pemetrexed, adenosine deaminase
inhibitors, including pentostatin, halogenated or ribonucleotide
reductase inhibitors, such as cladribine, clofarabine, and
fludarabine, thiopurines, including thioguanine and mercaptopurine,
thymidylate synthase inhibitors, including fluorouracil,
capecitabine, tegafur, carmofur, and floxuridine, DNA polymerase
inhibitors, such as cytarabine, ribonucleotide reductase
inhibitors, such as gemcitabine, hypomethylating agents, including
azacitidine, and decitabine, and ribonucleotide reductase
inhibitors, such as hydroxyurea; cell-cycle nonspecific
antineoplastic agents, including alkylating agents such as nitrogen
mustards, including mechlorethamine, cyclophosphamide, ifosfamide,
trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine,
uramustine, estramustine, nitrosoureas, including carmustine,
lomustine, semustine, fotemustine, nimustine, ranimustine, and
streptozocin, alkyl sulfonates, including busulfan, mannosulfan,
and treosulfan, aziridines, including carboquone, thioTEPA,
triaziquone, and triethylenemelamine, alkylating-like agents,
including platinum agents such as cisplatin, carboplatin,
oxaliplatin, nedaplatin, triplatin tetranitrate, satraplatin,
hydrazines, such as procarbazine, triazenes, such as dacarbazine,
temozolomide, altretamine, and mitobronitol, and streptomycins,
such as actinomycin, bleomycin, daunomycin, mitomycin, and
plicamycin; photosensitizers, including aminolevulinic acid, methyl
aminolevulinate, efaproxiral, and porphyrin derivatives, such as
porfimer sodium, talaporfin, temoporfin, and verteporfin; enzyme
inhibitors, including farnesyltransferase inhibitors such as
tipifarnib, cyclin-dependent kinase inhibitors, such as alvocidib
and seliciclib, proteasome inhibitors, such as bortezomib,
phosphodiesterase inhibitors, such as anagrelide, IMP dehydrogenase
inhibitors, such as tiazofurine, lipoxygenase inhibitors, such as
masoprocol, and PARP inhibitors, such as olaparib; receptor
antagonists, such as endothelin receptor antagonists including
atrasentan, retinoid X receptor antagonists, such as bexarotene,
and testolactone; and other chemotherapeutics, including amsacrine,
trabectedin, retinoids such as alitretinoin and tretinoin, arsenic
trioxide, asparagine depleters such as asparaginase or
pegaspargase, celecoxib, demecolcine, elesciomol, elsamitrucin,
etoglucid, and lonidamine.
[0078] It is intended that reference to a range of numbers
disclosed herein (for example, 1 to 10) also incorporates reference
to all rational numbers within that range (for example, 1, 1.1, 2,
3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of
rational numbers within that range (for example, 2 to 8, 1.5 to 5.5
and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges
expressly disclosed herein are hereby expressly disclosed. These
are only examples of what is specifically intended and all possible
combinations of numerical values between the lowest value and the
highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.
[0079] In this specification where reference has been made to
patent specifications, other external documents, or other sources
of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless
specifically stated otherwise, reference to such external documents
is not to be construed as an admission that such documents, or such
sources of information, in any jurisdiction, are prior art, or form
part of the common general knowledge in the art.
[0080] The invention may also be said broadly to consist in the
parts, elements and features referred to or indicated in the
specification of the application, individually or collectively, in
any or all combinations of two or more of said parts, elements or
features, and where specific integers are mentioned herein that
have known equivalents in the art to which the invention relates,
such known equivalents are deemed to be incorporated herein as if
individually set forth.
BRIEF DESCRIPTION OF THE FIGURES
[0081] FIG. 1. Graphic showing summarised data for the DLD-1 assay
using European propolis-cyclodextrin complex, CAPE-enriched
European propolis-cyclodextrin, European propolis tincture and
unmodified cyclodextrin as described in Example 1. 1A In this
graphic, taller bars represent higher activity of the fraction in
the assay (% inhibition), and vice versa. 1B This graph presents %
Efficacy for samples comprising propolis (wherein % Efficacy=%
Inhibition/propolis concentration), and % Inhibition for the
identified samples, as described in Example 1.
[0082] FIG. 2. A comparison of the efficacy of CAPE in compositions
of the invention versus pure CAPE. Efficacy is presented as the
relative inhibition (% inhibition/effective concentration in the
test medium) versus effective concentration. A high value equates
to a high efficacy.
[0083] FIG. 3. Effect of CAPE, European propolis cyclodextrin
complex, butyrate and mixtures thereof on apoptosis of HCT-116 and
HCT-116-R cells relative to a negative control (cells only).
DETAILED DESCRIPTION
[0084] The present invention is based on the finding that
compositions comprising propolis and cyclodextrin; and particularly
gamma cyclodextrin, for example compositions comprising European
propolis and gamma-cyclodextrin, have anti-gastrointestinal cancer
efficacy, including enhanced efficacy, and preventative activity.
The pharmaceutical compositions of the invention, for example the
anti-gastrointestinal cancer compositions of the invention, enhance
the activity and physicochemical properties of propolis. The
nutraceutical compositions of the invention, for example the gut
health compositions of the invention also enhance the activity and
physicochemical properties of the propolis present in the
composition.
[0085] Accordingly, provided that the anti-gastrointestinal cancer
compositions are formulated so as to be suitable for administration
to a mammalian subject, for example they consist of materials that
are safe to the human body, they can be used for manufacturing
anti-gastrointestinal cancer pharmaceutical compositions and drugs,
as well as nutraceutical compositions, consumer goods, such as
beverages, foods, and the like.
[0086] Furthermore, as the anti-gastrointestinal cancer activity of
embodiments of the compositions of the invention is maintained for
a sustained period, the dosage or frequency of administration of
the composition can be reduced, or higher efficacy is provided, or
both.
[0087] The phrases "anti-gastrointestinal cancer compositions" or
"compositions having anti-gastrointestinal cancer activity" (used
interchangeably herein) of this invention contemplate any kind of
compositions. Examples include anti-gastrointestinal cancer
compositions containing propolis and cyclodextrin or
anti-gastrointestinal cancer compositions containing materials with
propolis contained and cyclodextrin. Synergistic compositions which
enhance any anti-gastrointestinal cancer activity observed in
either propolis or in cyclodextrin alone are particularly
contemplated. The anti-gastrointestinal cancer compositions may be
anti-colorectal cancer, anti-gastric cancer or anti-throat cancer
compositions.
[0088] The term "and/or" can mean "and" or "or".
[0089] The terms "cancer" and "cancerous" refer to a physiological
condition in mammals that is typically characterized by abnormal or
unregulated cell proliferation, cell survival, cell motility,
neoplasticity, and/or oncogenicity. Cancer and cancer pathology can
be associated, for example, with metastasis, interference with the
normal functioning of neighbouring cells, release of cytokines or
other secretory products at abnormal levels, suppression or
aggravation of inflammatory or immunological response, neoplasia,
premalignancy, malignancy, invasion of surrounding or distant
tissues or organs, such as lymph nodes, etc. Specifically included
are colorectal cancers, gastric cancers, throat cancers and
precancerous conditions, which can include epithelial tumours,
nonepithelial tumours, carcinomas, adenocarcinomas, gastric
lymphomas, carcinoid tumours, stromal tumours or squamous cell
carcinomas for example, carcinomas in situ, as well as invasive
colorectal, gastric or throat cancers.
[0090] The term "comprising" as used in this specification means
"consisting at least in part of". When interpreting statements in
this specification that include that term, the features, prefaced
by that term in each statement, all need to be present but other
features can also be present. Related terms such as "comprise" and
"comprised" are to be interpreted in the same manner.
[0091] An "effective amount" is the amount required to confer
therapeutic effect. The interrelationship of dosages for animals
and humans (based on milligrams per meter squared of body surface)
is described by Freireich, et al. (1966). Body surface area can be
approximately determined from height and weight of the subject.
See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y.,
1970, 537. Effective doses also vary, as recognized by those
skilled in the art, dependent on route of administration, excipient
usage, and the like.
[0092] As used herein, an "extract" or a "fraction" of propolis are
suitable for use in the present invention provided they at least
retain one or more anti-gastrointestinal cancer activity exhibited
by propolis. Such functional extracts or functional fractions may
have greater or lesser activity than the crude propolis. In one
example, one or more of the biological activities of the crude
propolis possessed by the functional extract or functional fraction
may be present to a greater or lesser degree in the functional
extract or functional fraction than is found in the crude propolis.
In another example, each of the biological activities of the crude
propolis possessed by the functional extract or functional fraction
is present to a greater or lesser degree in the functional extract
or functional fraction than is found in the crude propolis. In
still a further example, it may be desirable to provide a
functional extract or functional fraction in which one or more of
the biological activities of the crude propolis is maintained or is
present to a greater degree than is found in the crude propolis,
but one or more other biological activities of the crude propolis
is not present or is present to a lesser degree than is found in
the crude propolis. Examples of such functional extracts include
the anti-gastrointestinal cancer tincture described herein in the
Examples.
[0093] Methods and assays to determine one or more biological
effects elicited by propolis are well known in the art and examples
are described herein, and such methods and assays can be used to
identify or verify one or more functional extracts or functional
fractions of propolis. For example, an assay of the ability of
propolis to increase one or more oncogenic traits in a cell, such
as those described herein in the Examples, is amenable to
identifying one or more functional extracts or functional fractions
of propolis.
[0094] As used herein, "propolis" contemplates propolis produced by
bees from any botantical source. In one embodiment, the propolis is
"European" propolis. "European" propolis is also known under
different names, such as "Poplar" propolis. For example, the
propolis is derived principally from the bud and leaf exudates of
one or more species of poplars, and to a lesser extent birches,
larches or willows. Propolis has been classified into seven major
classes based on plant source (Sforcin and Bankova, 2011. Propolis:
is there a potential for the development of new drugs? J.
Ethnopharmacology, 133: 253-260.). These classes are "Poplar" from
Europe, North America, Southern South America, New Zealand;
"Brazilian green", which contains artepillan C; "Birch" from
Russia; "Red propolis" from Cuba, Brazil, Mexico; "Mediterranean"
from Greece, Sicily, Crete, Malta rich in diterpenes that are
sourced from conifers; "Clusia" from Cuba and Venezuela; and
"Pacific" from Okinawa, Taiwan, Indonesia, which contain
`propolins`.
[0095] Exemplary compounds and concentrations of the compounds
reported in European type propolis from various countries are
presented in Table 1 below, along with a comparative example of
Brazilian green propolis. These compounds are useful, for example
in identifying the source of propolis as being a European-type
propolis, or in characterizing and identifying propolis suitable
for use in the present invention. It has been reported that
Brazilain green propolis contains none of the flavoinoid and
caffeic acid ester compounds characteristic of European-type
propolis
TABLE-US-00001 TABLE 1 Compositional data for ethanol soluble
extract of propolis from various geographic regions (adapted from
Kumazawa et al., 2004 Identification of metabolites in plasma and
urine of Uruguayan propolis-treated rats. Journal of Agricultural
and Food Chemistry 52(10): 3083-3088.) Arg Aus Bra Bul Chil Chi a
Chi b Chi c Hun SA Ukr Uru USA Uzb NZ MHNZ1 MHNZ2 Caffeic acid 0.7
1.7 1.6 7.2 0.4 3.3 2.8 2.4 3.1 0.2 0.8 0.7 0.8 1.5 2.8 9.4 7.2
p-Coumaric 1.8 3.6 27.4 3.5 1.9 4 4 3 3.7 1.5 8.9 8.4 19.4 0.9 3.1
3.4 2.6 3,4-Diemethoxy 2.2 8.6 4 1.8 10.1 7.4 7.9 5.2 0.8 1.1 2 2.6
9.2 NQbp NQbp cinnamic acid Quercetin 2.2 4.8 4.7 1.5 4.4 3.8 .8
4.4 2.5 3.8 0.8 1.2 Pinobanksin-5- 15 23.8 19.7 18.8 19.8 26.2 21.1
21.8 5.9 7.5 51 23.8 10.8 20 methyl ether Apigenin 12 18.4 13.4
14.2 17.1 17.1 14.3 9 3.9 14.8 0.6 8.1 78.3 Kaemferol 2.3 3.9 5 1.4
2.1 2.6 2.5 4.8 1 10.9 2.5 10.3 3.8 3.7 Pinobanksin 22.5 32.1 84.8
21.4 36.1 35 22.5 21.3 31.4 6.6 36.5 23.2 29.4 36.3 35.2 25
Cinnamylidene-a.sup.1 30.4 14.6 6.3 31.2 11.2 12.7 10.5 7.8 13.7
5.2 2.9 18.1 NQbp NQbp Chrysin 68.5 139 120 66.3 127.3 138 138 82.9
11.2 12.9 77.3 39.4 87 102 32.9 24.9 Pinocembrin 68.7 58.7 94.4
86.2 54.8 61.5 46.9 51.2 69.8 9.2 75 46.7 44.5 100 119 95.7
Galangin 32.5 42.5 45.6 37.7 39.6 33.5 32.6 44.2 18.9 13.4 48.8
21.5 41.4 58.2 50.6 37.3 Pinobanksin-3- 56.3 79.7 41.2 63.4 52.5
64.2 51.2 59.9 7.7 14.7 80 27.6 58.4 66.2 71.3 65.5 acetate CAPE
8.6 10.4 5.6 7.4 29.2 24.5 19.3 15.4 2.6 12.4 7.2 18.6 12 9.4 6.1
Cinnamyl caffeate 6.6 16.6 0.7 6.1 16.3 20.3 14.4 13.8 2.1 8.9 7.5
2.2 12.7 NQbp NQbp Tectochrysin 31.4 58.2 96.9 33.1 62 45.4 35.5
39.0 7.7 12.4 23.8 36.1 7.3 62.2 3.5 3.3 Artepillin-c 43.9 All
values are mg/g propolis resin. NQbp = Not quantified but present
.sup.1Cinnamylideneacetic acid (or phenyl petadienoic acid). Arg =
Argentina, Aus = Australia, Bra = Brazilian green? propolis, Bul =
Bulgaria, Chil = Chile, Chi = Chinese (a = Hebei, b = Hubei and c =
Zheijiang province), Hun = Hungary, NZ = New Zealand, SA = South
Africa, Ukr = Ukraine, Uru = Uruguay, Uzb = Uzbekistan, MHNZ 1 =
New Zealand propolis (Manuka Health NZ Ltd), MHNZ 2 = New Zealand
propolis (Manuka Health NZ Ltd).
[0096] As will be appreciated by those skilled in the art, the
identity, and in certain cases the suitability for particular uses,
including use in the present invention, of propolis may be
determined by analysis of the composition of the propolis. The
presence and amount of specific compounds, (including the compounds
discussed herein)--frequently referred to as "marker compounds",
allows a determination of the suitability of a particular source of
propolis, for example European propolis compared to Brazilian
propolis, for a particular use. In certain embodiments of the
present invention, the presence or amount of one or more marker
compounds is assayed, as a preliminary grading step, prior to
formulation of the composition of the invention.
[0097] When used in respect of an agent having
anti-gastrointestinal cancer activity, such as a composition of the
invention or a component of a composition of the invention, the
phrase "retaining anti-gastrointestinal cancer activity" and
grammatical equivalents and derivatives thereof is intended to mean
that the agent still has useful anti-gastrointestinal cancer
activity. Preferably, the retained activity is at least about 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% of the
original activity, and useful ranges may be selected between any of
these values (for example, from about 35 to about 100%, from about
50 to about 100%, from about 60 to about 100%, from about 70 to
about 100%, from about 80 to about 100%, and from about 90 to about
100%). Exemplary compositions of the invention are capable of
supporting the maintenance of useful anti-gastrointestinal cancer
activity of the anti-gastrointestinal cancer agent (s) they
comprise, and can be said to retain anti-gastrointestinal cancer
activity, ideally until utilized in the methods contemplated
herein.
[0098] When used in respect of a composition of the invention or a
component of a composition of the invention, the phrase "enhancing
anti-gastrointestinal cancer activity" and grammatical equivalents
and derivatives thereof is intended to mean that when present in
the composition, an equivalent amount or concentration of the
anti-gastrointestinal cancer agent has increased
anti-gastrointestinal cancer activity compared to that of the agent
in the absence of the composition (such as the isolated agent).
Preferably, the enhanced activity is at least about 105, 110, 115,
120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,
185, 190, 195, 200%, or more of the original activity, and useful
ranges may be selected between any of these values (for example,
from about 105 to about 200%, from about 120 to about 200%, from
about 140 to about 200%, from about 150 to about 200%, from about
180 to about 200%, and from about 190 to about 200%). In certain
embodiments, compositions of the invention may exhibit enhanced
anti-gastrointestinal cancer activity, that is, exhibit at least
about 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160,
165, 170, 175, 180, 185, 190, 195, 200%, or more of the
anti-gastrointestinal cancer activity of propolis alone, or of
cyclodextrin alone. Similarly, preferred compositions of the
invention are capable of supporting the maintenance of enhanced
anti-gastrointestinal cancer activity, and can be said to retain
enhanced anti-gastrointestinal cancer activity, ideally until
utilised using the methods contemplated herein. The enhanced
activity (including enhanced maintenance of activity) is believed,
without wishing to be bound by any theory, to result from synergy
amongst the various components of the compositions of the
invention.
[0099] As used herein, the term "stable" when used in relation to a
composition of the invention means a composition capable of
supporting anti-gastrointestinal cancer activity for preferably
more than two hours, more than three hours, 6 hours, 9 hours, 12
hours, 15 hours, 18 hours, 20 hours, more than one day, preferably
about two, about three, about four, preferably about five, more
preferably about six days, preferably a week, two weeks, three
weeks, a month, or longer. It will be appreciated that in certain
embodiments, stable compositions include those which have
anti-gastrointestinal cancer activity for a period greater than
does the propolis or cyclodextrin alone.
[0100] The term "oral administration" includes oral, buccal,
enteral and intra-gastric administration.
[0101] The term "parenteral administration" includes but is not
limited to topical (including administration to any dermal,
epidermal or mucosal surface), subcutaneous, intravenous,
intraperitoneal, intramuscular and intratumoural (including any
direct administration to a tumour) administration.
[0102] The term "pharmaceutically acceptable carrier" is intended
to refer to a carrier including but not limited to an excipient,
diluent or auxiliary that can be administered to a subject as a
component of a composition of the invention. Preferred carriers do
not reduce the activity of the composition and are not toxic when
administered in doses sufficient to deliver an effective amount of
propolis or extracts thereof, or, when administered, of another
anti-gastrointestinal cancer agent.
[0103] The term "(s)" following a noun contemplates the singular or
plural form, or both.
[0104] The term "subject" is intended to refer to an animal,
preferably a mammal, more preferably a mammalian companion animal
or human. Preferred companion animals include cats, dogs and
horses. Other mammalian subjects include an agricultural animal,
including a horse, a pig, a sheep, a goat, a cow, a deer, or a
fowl, or a laboratory animal, including a monkey, a rat, or a
mouse.
[0105] The term "treat" and its derivatives should be interpreted
in their broadest possible context. The term should not be taken to
imply that a subject is treated until total recovery. Accordingly,
"treat" broadly includes maintaining a subject's disease
progression or symptoms at a substantially static level, increasing
a subject's rate of recovery, amelioration and/or prevention of the
onset of the symptoms or severity of a particular condition, or
extending a patient's quality of life. The term "treat" also
broadly includes the maintenance of good health for sensitive
individuals and building stamina for disease prevention.
Exemplary Uses of the Invention
[0106] The methods and compositions of the invention may be used in
the treatment or prevention of colorectal cancers, neoplastic
disorders associated with colorectal cancer cells, and the symptoms
of colorectal cancer, colorectal cancer treatment, and associated
disorders. Colorectal cancer is a neoplastic condition affecting
the large intestine, particularly the colon and rectum--hence it is
commonly referred to as colon cancer or bowel cancer. Risk factors
include age, diet, in particular high intake of fat, alcohol or red
meat, male gender, obesity, smoking and lack of exercise.
[0107] Colorectal cancer originates from epithelial cells in the
colon or rectum of the gastrointestinal tract, and is most commonly
associated with defects in the Wnt-APC-beta-catenin signaling
pathway. Where possible, the preferred treatment is complete
surgical removal which can be curative. If metastasis has occurred
and the cancer has entered the lymph nodes, the chemotherapeutic
agents fluorouracil or capecitabine have been reported to increase
life expectancy. Other chemotherapeutics considered for use include
fluorouracil, capecitabine, UFT, leucovorin, irinotecan, or
oxaliplatin, and combinations of these agents. However, where the
cancer is more widespread, or when surgery is not possible,
treatment generally focuses on palliative care and extension of
life expectancy. Combinations of radiation and chemotherapy can be
considered for rectal cancer, but radiotherapy is not typically
used in colon cancer due to radiosensitivity of the bowels.
[0108] In certain embodiments, the methods and compositions of the
invention are used in the treatment or prevention of gastric
cancers, neoplastic disorders associated with gastric cancer cells,
and the symptoms of gastric cancer, gastric cancer treatment, and
associated disorders.
[0109] Gastric cancer is a neoplastic condition arising from any
part of the stomach. Prognosis is poor because most patients
present with advanced disease. Risk factors include age, diet, in
particular high intake of smoked foods, salted fish, cured meats
and pickled vegetables, smoking, male gender and a history of
autoimmune atrophic gastritis or intestinal metaplasia.
[0110] Approximately 90% of gastric cancers are adenocarcinomas,
originating in the glandular epithelium of the gastric mucosa.
Other types include lymphomas (MALTomas OR MALT lymphoma), and
carcinoid and stromal tumours. Where possible, the preferred
treatment is complete surgical removal of all or part of the
stomach and surrounding lymph nodes. Gastric cancers are not
particularly sensitive to chemotherapeutic agents, however,
chemotherapeutics including fluorouracil, capecitabine, BCNU
(carmustine), methyl-CCNU (Semustine), and doxorubicin
(Adriamycin), Mitomycin C, cisplatin and taxotere have been used in
palliative care regimes. Combinations of radiation and chemotherapy
are sometimes used for the treatment of gastric cancers.
[0111] In certain embodiments, the invention also relates to
methods of at least partially reversing the resistance of a
neoplastic cell in a subject suffering from a gastrointestinal
cancer to a gastrointestinal cancer therapy, or to a method of
reversing, wholly or in part, the resistance of a gastrointestinal
cancer-burdened patient to a gastrointestinal cancer therapy, or to
a method of re-sensitising one or more tumours of a
gastrointestinal cancer-burdened patient which are, or are
predicted to either be or become, resistant to treatment with a
gastrointestinal cancer therapy, said methods comprising the step
of administering to said patient a composition comprising,
consisting essentially or, or consisting of propolis and
cyclodextrin.
[0112] In one embodiment, the one or more tumours are or are
predicted to be or to become resistant to a gastrointestinal cancer
therapy due increased activation of one or more pro-cancer cell
survival signaling pathways within the one or more tumours or
within the patient, including increased activation of one or more
of the AKT, JNK or JAK/STAT signaling pathways, for example within
a sample from the patient, such as a tissue sample, a tumour
biopsy, or a blood or plasma sample.
[0113] Pro-cancer cell survival signaling pathways implicated in
the onset and development of gastrointestinal cancers are known in
the art.
[0114] The methods and compositions of the invention may be used in
the treatment or prevention of throat cancers, neoplastic disorders
associated with throat cancer cells, and the symptoms of throat
cancer, throat cancer treatment, and associated disorders.
[0115] Throat cancer, also referred to as oesophaegeal cancer,
pharyngeal cancer, or laryngeal cancer, encompasses tumours that
develop in the tissues of the pharynx, nasopharynx, oropharynx,
hypopharynx, larynx (voice box) or tonsils. Approximately 90% of
throat cancers are squamous cell carcinomas originating from the
mucosal lining (epithelium) of these regions. Risk factors include
diet, in particular high intake of alcohol, smoking, use of
smokeless tobacco, betel nut chewing and exposure to environmental
carcinogens including occupational exposures nickel refining,
textile fibres and woodworking.
[0116] The most common therapies are surgical excision of tumours
and radiation therapy. Chemotherapeutic agents may be used in
combination surgery and/or radiation. Agents include paclitaxel,
carboplatin, cetuximab, taxotere and docetaxel.
[0117] The methods and compositions of the invention may also be
used for maintaining or improving gut health.
[0118] Robust gut health is associated with intestinal comfort,
resistance to infectious diseases and the prevention of chronic
gastrointestinal diseases.
[0119] This includes the treatment or prevention of a condition
associated with poor gut health, low immunity and gastrointestinal
tract inflammation. For example, the methods and compositions of
the invention are useful for the treatment or prevention of
inflammatory bowel disease, irritable bowel syndrome, environmental
enteropathy, infectious diarrhoea and for the removal or
alleviation of visceral pain.
[0120] Propolis and Materials Comprising Propolis
[0121] Propolis is available in New Zealand and elsewhere, commonly
as a resinous sticky solid. Propolis may be obtained from bee-hives
with the resulting propolis held in storage, for example to assess
the propolis content. Those skilled in the art will recognise that
for use in the present invention, propolis may be processed to a
form suitable for admixture, for example with cyclodextrin, while
maintaining the bioactive ingredients. Typically the propolis, or
an extract thereof, is processed to a fine particulate form or a
concentrated tincture. Various methods of preparing active
propolis, or an extract thereof, to a particulate form or
concentrated tincture are known. Most commonly, crude propolis is
extracted using ethanol or ethanol/water mixtures to produce a
dilute tincture. Wax associated with the crude propolis is at best
poorly soluble in the solvent and so is mostly not extracted. Any
extracted wax can be removed by cooling the dilute tincture and
then settling, filtration, or centrifugation. The tincture can then
be concentrated by partial to complete evaporation of the solvent
to give a concentrated tincture, optionally followed by freeze
drying to give a powder. Alternatively, the tincture can be spray
dried to give a powder. Fractions can be prepared by using methods
known in the art such as chromatography (such as HPLC) using, for
example, a size exclusion matrix or a reverse phase matrix, or
supercritical fractionation. A typical solvent for use in such a
chromatographic process is ethanol or another water miscible
alcohol.
[0122] In one embodiment propolis or concentrated propolis tincture
is combined with other compounds that enhance the properties of
propolis, for example a compound that enhances the ease of
formulation or administration, or that enhances
anti-gastrointestinal cancer activity, or that enhances the
stability of one or more anti-gastrointestinal cancer activities
present in propolis. Examples of additional compounds are those
that improve the therapeutic benefits of the propolis. Exemplary
compositions in which one or more compounds present in propolis,
and in European propolis in particular, including biologically
active compounds such as CAPE, caffeic acid, pinocembrin, benzyl
caffeate, chrysin, galangin, pinobanksin, and pinobanksin-3-acetate
are added are specifically contemplated. In other examples,
additional compounds are included to improve or maintain the
physiological benefits of the composition, for example mannitol can
be added to enhance the diuretic properties of the resulting
composition. Alternatively or additionally other compounds such as
excipients, and/or propellants could be added to improve the
dosing, manufacturability or delivery properties of the
composition.
[0123] In particularly contemplated embodiments, dewaxed propolis
resin, optionally with one or more additional compounds added, is
admixed with cyclodextrin and the admixture dried. Further
processing of the admixture, for example, to obtain a particle size
distribution that enables ready admixture with the other components
of the composition, ease of tableting, or ease of administration to
a subject, is conducted.
[0124] In typical embodiments, the propolis or propolis resin is
sterilized, for example by heating to kill bacteria, protozoa,
yeast, fungi and other organisms that naturally may be present in
the propolis.
[0125] Cyclodextrins and Materials Comprising Cyclodextrin
[0126] Cyclodextrins are cyclic molecules composed of glucopyranose
ring units which form toroidal structures. The interior of the
cyclodextrin molecule is hydrophobic and the exterior is
hydrophilic, making the cyclodextrin molecule water soluble. The
degree of solubility can be altered through substitution of the
hydroxyl groups on the exterior of the cyclodextrin. Similarly, the
hydrophobicity of the interior can be altered through substitution,
though generally the hydrophobic nature of the interior allows
accommodation of relatively hydrophobic guests within the cavity.
Accommodation of one molecule within another is known as
complexation and the resulting product is referred to as an
inclusion complex. Cyclodextrins are typically identified with
reference to the number of monomeric units that comprise the
molecule, wherein alpha-cyclodextrin (.alpha.-cyclodextrin)
comprises six monomeric units, beta-cyclodextrin
(.beta.-cyclodextrin) comprises seven monomeric units, and
gamma-cyclodextrin (.gamma.-cyclodextrin) comprises eight monomeric
units. Larger cyclodextrin molecules have been described, including
a well-characterised cyclodextrin containing 32
1,4-anhydroglucopyranoside units
[0127] Cyclodextrin molecules may conveniently be derivatised, by
for example chemical modification, for example to alter one or more
of the physicochemical properties thereof. Examples of cyclodextrin
derivatives include methylated cyclodextrins,
sulfobutylcyclodextrin, maltosylcyclodextrin,
hydroxypropylcyclodextrin, and salts thereof. Those skilled in the
art will recognise that various derivates of cyclodextrin may be
suitable for particular purposes, for example, certain derivatives
of cyclodextrin are not be acceptable for administration to human
subjects, but are suitable for industrial uses.
[0128] Cyclodextrins comprising the anti-gastrointestinal cancer
compositions of the present invention may be commercially
available, or may be prepared independently by methods well known
to those skilled in the art. It will be apparent to those skilled
in the art that cyclodextrins used in the anti-gastrointestinal
cancer compositions for administration to a subject, for example a
cyclodextrin for manufacturing a beverage, food, or pharmaceutical
of the invention should be safe to human body, and preferably is a
pharmaceutically acceptable cyclodextrin.
[0129] In particularly contemplated embodiments, gamma-cyclodextrin
or combinations comprising gamma-cyclodextrin are used. In such
embodiments, anti-gastrointestinal cancer activity is substantially
enhanced, as presented herein in the examples. Such compositions
comprising gamma-cyclodextrin can be formulated to provide enhanced
mouth feel or palatability, for example compositions comprising
gamma-cyclodextrin and propolis exhibit a stronger tendency to mask
any distasteful flavours present in the propolis.
[0130] Cyclodextrins suitable for use in the present invention can
be obtained from commercial sources, or can be prepared
independently by methods well known in the art, such as from starch
by enzymatic conversion. In certain embodiments, CAVAMAX W8 FOOD, a
gamma-cyclodextrin commercially supplied by Wacker AG, is used.
[0131] Compositions of the Invention
[0132] Exemplary anti-gastrointestinal cancer compositions of the
present invention include a powder that is obtained after mixing
propolis tincture with cyclodextrin, then adding water and
homogenizing the composition, and then spray-drying or
freeze-drying. Other exemplary anti-gastrointestinal cancer
compositions of the present invention include solutions, including
for example, those in which propolis tincture and cyclodextrin are
mixed and then dispersed in water, those in which propolis or
materials with propolis contained and cyclodextrin are
independently dissolved or dispersed in water, and then admixed,
for example by kneading, and further those in which propolis powder
or resin is firstly dissolved in another organic solvent in which
it is soluble, such as for example propylene glycol, ethyl acetate,
isopropyl alcohol, and the resultant solution is admixed with
cyclodextrin, then added to water, and further mixed, for example
by kneading and then dried by means known in the art, such as spray
or freeze-drying. In certain embodiments, anti-gastrointestinal
cancer compositions prepared as powders as described above may be
preferred, for example because they may maintain stronger
anti-gastrointestinal cancer activity or may maintain
anti-gastrointestinal cancer activity for a longer period than that
of solutions of anti-gastrointestinal cancer compositions prepared
as described above.
[0133] The content of propolis and cyclodextrin of the present
invention can be at any level as long as the expected
anti-gastrointestinal cancer activity is realized.
[0134] Without wishing to be bound by any theory, the applicants
believe that the propolis in the composition will be entirely
encapsulated when the molar ratio of propolis to cyclodextrin is no
greater than 1:1.
[0135] In some embodiments the molar ratio of propolis to
cyclodextrin may exceed 1:1 in the compositions of the invention.
In such compositions the excess propolis will not be encapsulated
by the cyclodextrin.
[0136] Other anti-gastrointestinal cancer substances generally
known can be combined with the anti-gastrointestinal cancer
compositions of this invention, depending upon the application to
which the composition is to be put.
[0137] Without wishing to be bound by any theory, the applicants
believe that the enhanced anti-gastrointestinal cancer activity
observed in exemplary compositions of the present invention may be
due at least in part to a synergy between propolis, particularly
when present as propolis, and gamma-cyclodextrin. Again, without
wishing to be bound by any theory, the applicants acknowledge that
there may be a role of other components of the exemplary
compositions, such as polyphenols present in propolis, in achieving
the observed enhanced anti-gastrointestinal cancer activities.
[0138] Compositions suitable for administration to a subject may be
formulated as a food, drink, food additive, drink additive, dietary
supplement, nutritional product, medical food, nutraceutical,
medical supply, medical device, medicament or pharmaceutical.
Appropriate formulations may be prepared by an art skilled worker
with regard to that skill and the teaching of this
specification.
[0139] In one embodiment the present invention relates to use of
propolis and gamma cyclodextrin, optionally with at least one
anti-gastrointestinal cancer agent, in the manufacture of a food,
drink, food additive, drink additive, dietary supplement,
nutritional product, medical food, nutraceutical, medical device,
medical supply, medicament or pharmaceutical. In one embodiment,
the composition is formulated for oral administration. In another
embodiment, the composition is formulated for parenteral, including
topical, administration. In certain embodiments, the composition is
for inducing apoptosis, treating or preventing a gastrointestinal
cancer, maintaining or improving gut health or one or more other
uses as described above.
[0140] In one embodiment the composition is in the form of a
powder, a tablet, a caplet, a pill, a hard or soft capsule or a
lozenge.
[0141] In one embodiment the composition is in the form of a
sachet, a dispensable powder, granules, a suspension, an elixir, a
liquid, a drink, or any other form that can be added to food or
drink, including for example water or fruit juice. In one
embodiment the composition is an enteral product, a solid enteral
product or a liquid enteral product.
[0142] In one embodiment, the composition is in the form of a
cream, ointment, a paste, a drop solution including eye drops or
ear drops, an inhaler or as an inhalable composition, a dressing, a
pad, or a spray.
[0143] In one embodiment the composition further comprises one or
more constituents (such as antioxidants) which prevent or reduce
degradation of the composition during storage or after
administration.
[0144] In one embodiment, compositions useful herein include any
edible consumer product which is able to carry one or more
cyclodextrins. When the composition comprises a proteinaceous
factor as the at least one additional anti-gastrointestinal cancer
agent, the edible consumer product is one able to carry protein.
Examples of suitable edible consumer products include baked goods,
powders, liquids, confectionery products, reconstituted fruit
products, snack bars, food bars, muesli bars, spreads, sauces,
dips, dairy products including ice creams, yoghurts and cheeses,
drinks including dairy and non-dairy based drinks (such as milk
drinks including milk shakes, and yogurt drinks), milk powders,
sports or nutritional supplements including dairy and non-dairy
based sports or nutritional supplements, food additives such as
protein sprinkles and dietary supplement products including daily
supplement tablets. Within this embodiment, a composition useful
herein may also be an infant formula, in powder or liquid form.
Suitable nutraceutical compositions useful herein may be provided
in similar forms. Particularly contemplated are compositions
additionally comprising milk or one or more milk products or
components of milk, such as milk protein, whey protein, colostrums,
milk fat, or any fractions of milk or one or more milk products or
components of milk, such as a milk fat fraction, a milk protein
fraction, a whey protein fraction, a colostrums fraction, or the
like.
[0145] Compositions useful herein may further include other factors
such as calcium, zinc, magnesium, selenium, vitamin C, vitamin D,
vitamin E, vitamin K2, complex carbohydrates, edible or cooking
oils including palm, olive, soybean, canola, corn, sunflower,
safflower, peanut, grape seed, sesame, nut, almond, cashew,
hazelnut, macadamia, pecan, pistachio, and walnut, and other
edibles include acai, amaranth, apricot, argan, artichoke, avocado,
babassu, ben, blackcurrant seed, borage seed, borneo tallow nut,
bottle gourd, buffalo gourd, carob pod (algaroba), cohune,
coriander seed, evening primrose, false flax, hemp, kapok seed,
lallemantia, meadowfoam seed, mustard, okra seed (hibiscus seed),
perilla seed, pequi, pine nut, poppyseed, prune kernel, pumpkin
seed, quinoa, ramtil, rice bran, tea (camellia), thistle,
watermelon seed, or wheat germ oil, or a combination thereof.
[0146] The compositions useful herein may be formulated to allow
for administration to a subject by any chosen route, including but
not limited to oral or parenteral (including topical, subcutaneous,
intramuscular and intravenous) administration. Those skilled in the
art will appreciate that the route of administration to a subject
will typically take into account the purpose for which the
composition is being administered--for example, where a
pharmaceutical composition of the invention is being administered
to treat a microbial disease or disorder, the route of
administration will typically be chosen taking into account the
nature of the microbial disease or disorder.
[0147] In general, for oral administration a dietary (a food, food
additive or food supplement for example), nutraceutical or
pharmaceutical composition useful herein may be formulated by a
skilled worker according to known formulation techniques.
[0148] Thus, a pharmaceutical composition useful according to the
invention may be formulated with an appropriate pharmaceutically
acceptable carrier (including excipients, diluents, auxiliaries,
and combinations thereof) selected with regard to the intended
route of administration and standard pharmaceutical practice. See
for example, Remington's Pharmaceutical Sciences, 16th edition,
Osol, A. Ed., Mack Publishing Co., 1980.
[0149] While the preferred route of administration is oral, it
should be understood that any mode of administration may be
suitable for any composition of the invention, including
administration by multiple routes, including different routes for
different agents. Therefore, inhalation (nasal or buccal
inhalation) and vaginal and rectal administration of any
composition of the invention is also contemplated. Intramedullar,
epidural, intra-articular, and intra-pleural administration of any
composition of the invention is also contemplated. Administration
of a composition of the invention, optionally with at least one
additional anti-gastrointestinal cancer factor, by a first
administration route accompanied by separate, simultaneous or
sequential administration of one or more other agents, including
one or more other anti-gastrointestinal cancer agents, by a second
administration route is also contemplated; for example, oral
administration of a composition of the invention accompanied by
topical administration of the at least one additional
anti-gastrointestinal cancer agent.
[0150] The compositions of the invention may also be formulated as
a dosage form. A dosage form useful herein may be administered
orally as a powder, liquid, tablet or capsule. Suitable dosage
forms may contain additional agents as required, including
emulsifying, antioxidant, flavouring or colouring agents, or have
an enteric coating. Suitable enteric coatings are known. Enteric
coatings surrounding the active ingredients and prevent the release
of the active ingredients in the stomach but allow release after
the dosage form has left the stomach. Dosage forms useful herein
may be adapted for immediate, delayed, modified, sustained, pulsed
or controlled release of the active components. Suitable
formulations may contain additional agents as required, including
emulsifying, antioxidant, flavouring or colouring agents.
[0151] Capsules can contain any standard pharmaceutically
acceptable materials such as gelatin or cellulose. Tablets can be
formulated in accordance with conventional procedures by
compressing mixtures of the active ingredients with a solid carrier
and a lubricant. Examples of solid carriers include starch and
sugar bentonite. Active ingredients can also be administered in a
form of a hard shell tablet or a capsule containing a binder, e.g.,
lactose or mannitol, a conventional filler, and a tabletting agent.
Pharmaceutical compositions can also be administered via the
parenteral route. Examples of parenteral dosage forms include
aqueous solutions, isotonic saline or 5% glucose of the active
agent, or other well-known pharmaceutically acceptable excipient.
Solubilising agents well-known to those familiar with the art, can
be utilized as pharmaceutical excipients for delivery of the
anti-gastrointestinal cancer agent.
[0152] Injectable dosage forms may be formulated as liquid
solutions or suspensions. Solid forms suitable for solution in, or
suspension in, liquid prior to injection may also be prepared. The
dosage form may also be emulsified. Propolis, or a material
comprising propolis, and cyclodextrin or a material comprising
cyclodextrin, and when present the at least one additional
anti-gastrointestinal cancer factor may be mixed with carriers such
as, for example, water, saline, dextrose, glycerol, ethanol, or the
like and combinations thereof.
[0153] Sustained-release preparations may be prepared incorporating
propolis and cyclodextrin. Suitable examples of sustained-release
preparations include semi-permeable matrices of solid hydrophobic
polymers containing propolis and cyclodextrin, and when present the
at least one additional anti-gastrointestinal cancer agent. The
matrices may be in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (see U.S. Pat. No. 3,773,919), copolymers of
L-glutamic acid and ethyl-L-glutamate, non-degradable
ethylene-vinyl acetate, and degradable lactic acid-glycolic acid
copolymers such as the LUPRON DEPOT.TM. (injectable microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide
acetate).
[0154] Topical formulations comprising propolis and cyclodextrin,
and when present the at least one additional anti-gastrointestinal
cancer agent, may be prepared as lotions, creams, ointments, pastes
or salves using known carriers for such applications. Such
formulations may be administered directly, for example, applied
directly on to a wound, sprayed onto a surgical site, etc, or may
be applied indirectly, such as by impregnation into a bandage or
dressing or sprayed onto surgical equipment, dressings and the
like.
[0155] The present invention also relates to a parenteral unit
dosage form comprising propolis and cyclodextrin, optionally with
at least one additional therapeutic agent.
[0156] In various embodiments, the at least one additional
therapeutic agent is an antibiotic, such as an aminoglycoside, such
as amikacin, gentamicin, kanamycin, neomycin, netilmicin,
streptomycin, tobramicin, or paromomycin; an ansamycin, such as
geldanamycin, or herbimycin; a carbacephem, such as loracarbef;
carbapenems, such as, ertapenem, doripenem, imipenem/cilastatin, or
meropenem; cephalosporins (first generation), such as cefadroxil,
cefazolin, cefalotin or cefalothin, or cefalexin; cephalosporins
(second generation), such as cefaclor, cefamandole, cefoxitin,
cefprozil, or cefuroxime; cephalosporins (third generation), such
as cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime,
cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, or ceftriaxone;
cephalosporins (fourth generation), such as cefepime;
cephalosporins (fifth generation), such as ceftobiprole;
glycopeptides, such as teicoplanin, or vancomycin; macrolides, such
as azithromycin, clarithromycin, dirithromycin, erythromycin,
roxithromycin, troleandomycin, telithromycin, or spectinomycin;
monobactams, such as aztreonam; penicillins, such as amoxicillin,
ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin,
flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin,
penicillin, piperacillin, or ticarcillin; polypeptides, such as
bacitracin, colistin, or polymyxin b; quinolones, such as
ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,
moxifloxacin, norfloxacin, or ofloxacin; sulfonamides, such as
mafenide, sulfonamidochrysoidine (archaic), sulfacetamide,
sulfadiazine, sulfamethizole, sulfanilimide (archaic),
sulfasalazine, sulfisoxazole, trimethoprim, or
trimethoprim-sulfamethoxazole (co-trimoxazole) (tmp-smx);
tetracyclines, such as demeclocycline, doxycycline, minocycline,
oxytetracycline, tetracycline; others such as arsphenamine,
chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin,
fusidic acid, furazolidone, isoniazid, linezolid, metronidazole,
mupirocin, nitrofurantoin, platensimycin, pyrazinamide,
quinupristin/dalfopristin, rifampicin (rifampin in US),
thiamphenicol, tinidazole, dapsone, clofazimine; or a cyclic
lipopeptides, such as daptomycin, a glycylcycline, such as
tigecycline, or an oxazolidinones, such as linezolid.
[0157] In other embodiments, the at least one additional
therapeutic agent is an antifungal, such as a polyene antifungal,
such as natamycin, rimocidin, filipin, nystatin, amphotericin B,
candicin; imidazoles, such as miconazole, ketoconazole,
clotrimazole, econazole, bifonazole, butoconazole, fenticonazole,
isoconazole, oxiconazole, sertaconazole, sulconazole, or
tioconazole; triazoles, such as fluconazole, itraconazole,
isavuconazole, ravuconazole, posaconazole, voriconazole, or
terconazole; thiazoles such as abafungin; allylamines, such as
terbinafine, amorolfine, naftifine, or butenafine; echinocandins,
such as anidulafungin, caspofungin, or micafungin; others such as
benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine
or 5-fluorocytosine, griseofulvin, haloprogin, and sodium
bicarbonate; or alternatives such as allicin, tea tree oil,
citronella oil, iodine, lemon grass, olive leaf, orange oil,
palmarosa oil, patchouli, lemon myrtle, neem seed oil, coconut oil,
zinc, or selenium
[0158] Alternatively the agent is selected from any of those
described herein.
[0159] The efficacy of a composition useful according to the
invention can be evaluated both in vitro and in vivo. See, e.g.,
the examples below. Briefly, in one embodiment the composition can
be tested for its ability, to for example, inhibit neoplastic cell
proliferation in vitro. For in vivo studies, the composition can be
fed to or injected into an animal (e.g., a mouse) and its effects
on gastrointestinal cancer cell survival, proliferation,
metastasis, or one or more symptoms of a gastrointestinal cancer or
associated disease or disorder are then assessed. Based on the
results, an appropriate dosage range, frequency, and administration
route can be determined.
[0160] The compositions useful herein may be used alone or in
combination with one or more other anti-gastrointestinal cancer
agents, or one or more additional therapeutic agents. The
anti-gastrointestinal cancer agent or additional therapeutic agent
may be or comprise a food, drink, food additive, drink additive,
food component, drink component, dietary supplement, nutritional
product, medical food, nutraceutical, medical device, medical
supply, medicament or pharmaceutical. The anti-gastrointestinal
cancer agent or additional therapeutic agent is preferably
effective to attenuate one or more neoplastic diseases or disorders
or one or more of the symptoms of one or more neoplastic diseases
or disorders, or otherwise confer a benefit on the subject to whom
it is administered. Preferred therapeutic agents include
therapeutic food factors, immunogenic or immunostimulatory agents,
wound healing agents, and the like.
[0161] It should be understood that the additional
anti-gastrointestinal cancer or therapeutic agents listed above
(both food based and pharmaceutical agents) may also be employed in
a method according to the invention where they are administered
separately, simultaneously or sequentially with a composition
useful herein.
[0162] As will be appreciated, the dose of the composition
administered, the period of administration, and the general
administration regime may differ between subjects depending on such
variables as the severity of symptoms of a subject, the type of
disorder to be treated, the mode of administration chosen, and the
age, sex and/or general health of a subject. However, by way of
general example, from about 1 mg to about 5000 mg per kg body
weight of a composition useful herein is administered, 1 mg to
about 4000 mg per kg body weight of a composition useful herein is
administered, 1 mg to about 3000 mg per kg body weight of a
composition useful herein is administered, 1 mg to about 2000 mg
per kg body weight of a composition useful herein is administered,
1 mg to about 1000 mg per kg body weight of a composition useful
herein is administered, per administration or per day, preferably
about 50 to about 1000 mg per kg, preferably per day. In one
embodiment, the administration is of from about 0.05 mg to about
250 mg per kg body weight of a composition useful herein.
[0163] In various embodiments, sufficient composition is
administered to deliver from about 0.001 mg to about 50 mg of
propolis per kg body weight, from about 0.001 mg to about 40 mg of
propolis per kg body weight, from about 0.001 mg to about 30 mg of
propolis per kg body weight, from about 0.001 mg to about 20 mg of
propolis per kg body weight, from about 0.001 mg to about 10 mg of
propolis per kg body weight, from about 0.001 mg to about 5 mg of
propolis per kg body weight, from about 0.001 mg to about 1 mg of
propolis per kg body weight, from about 0.001 mg to about 0.5 mg of
propolis per kg body weight, from about 0.001 mg to about 0.1 mg of
propolis per kg body weight, or from about 0.001 mg to about 0.05
mg of propolis per kg body weight, per administration or per
day.
[0164] It should be appreciated that administration may include a
single dose, such as a single daily dose, or administration of a
number of discrete divided doses as may be appropriate. It should
be understood that a person of ordinary skill in the art will be
able without undue experimentation, having regard to that skill and
this disclosure, to determine an effective dosage regime (including
dose and timing of administration) for a given condition.
[0165] The present invention also relates to a dietary,
nutraceutical or oral pharmaceutical composition comprising,
consisting essentially of or consisting of propolis or a material
comprising propolis in combination with cyclodextrin. In certain
embodiments the composition consists essentially of about 1 to 99
wt % propolis or a material comprising propolis and about 1 to 99
wt % cyclodextrin. For example, the composition consists
essentially of about 10 to 80 wt % propolis or a material
comprising propolis and about 20 to 90 wt % cyclodextrin. In
another example, the composition consists essentially of about 20
to 40 wt % propolis and about 60 to 80 wt % cyclodextrin.
[0166] The present invention also relates to a dietary,
nutraceutical or oral pharmaceutical composition comprising,
consisting essentially of or consisting of propolis or a material
comprising propolis that is encapsulated by cyclodextrin. In
certain embodiments the composition consists essentially of about 1
to 30 wt % propolis or a material comprising propolis and about 70
to 99 wt % cyclodextrin. For example, the composition consists
essentially of about 10 to 25 wt % propolis or a material
comprising propolis and about 75 to 90 wt % cyclodextrin. In
another example, the composition consists essentially of about 20
to 30 wt % propolis and about 70 to 80 wt % cyclodextrin.
[0167] In one embodiment a composition of the invention comprises
propolis or a propolis fraction. In one embodiment the composition
comprises at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
60, 70, 80, 90, or 99% by weight propolis or a propolis fraction,
and useful ranges may be selected from any of these values (for
example, from about 1 to about 25% by weight, from about 1 to about
30% by weight, from about 5 to about 30% by weight, from about 15
to about 30% by weight, from about 20 to about 30% by weight, from
about 25 to about 30% by weight, from about 10 to about 50% by
weight, from about 15 to about 50% by weight, from about 40 to
about 99% by weight, from about 45 to about 99% by weight, from
about 50 to about 99% by weight, from about 55 to about 99% by
weight, from about 60 to about 99% by weight, from about 65 to
about 99% by weight, from about 70 to about 99% by weight, from
about 75 to about 99% by weight, from about 80 to about 99% by
weight, from about 85 to about 99% by weight, from about 90 to
about 99% by weight, or from about 95 to about 99% by weight).
[0168] In one embodiment a composition of the invention comprises
cyclodextrin, preferably gamma-cyclodextrin. In one embodiment the
composition comprises at least about 1, 5, 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% by weight
cyclodextrin, and useful ranges may be selected from any of these
values (for example, from about 1 to about 99% by weight, from
about 5 to about 99% by weight, from about 10 to about 99% by
weight, from about 15 to about 99% by weight, from about 20 to
about 99% by weight, from about 25 to about 99% by weight, from
about 30 to about 99% by weight, from about 35 to about 99% by
weight, from about 40 to about 99% by weight, from about 45 to
about 99% by weight, from about 50 to about 99% by weight, from
about 55 to about 99% by weight, from about 60 to about 99% by
weight, from about 65 to about 99% by weight, from about 70 to
about 99% by weight, from about 75 to about 99% by weight, from
about 80 to about 99% by weight, from about 85 to about 99% by
weight, from about 90 to about 99% by weight, or from about 95 to
about 99% by weight).
[0169] When used in combination with another anti-gastrointestinal
cancer agent or therapeutic agent, the administration of a
composition useful herein and the other anti-gastrointestinal
cancer agent or therapeutic agent may be simultaneous or
sequential. Simultaneous administration includes the administration
of a single dosage form that comprises all components or the
administration of separate dosage forms at substantially the same
time. Sequential administration includes administration according
to different schedules, preferably so that there is an overlap in
the periods during which the composition useful herein and other
therapeutic agent are provided.
[0170] Additionally, it is contemplated that a composition in
accordance with the invention may be formulated with additional
active ingredients which may be of benefit to a subject in
particular instances. For example, therapeutic agents that target
the same or different facets of the disease process may be
used.
[0171] Accordingly, "foods and beverages comprising
anti-gastrointestinal cancer compositions" of this invention can be
used for general foods and health food. Since the
anti-gastrointestinal cancer compositions of the present invention
mask the taste of propolis, they can be eaten as they are or in the
form of powder. They can be used as an ingredient or raw material
for cake, biscuit, cookie, chocolate, sweets and other
confectionary, including drops or chewing gum. The compositions of
the invention may be added to water as a drink, can be used as
sweetener for beverages such as milk, tea, coffee, hot chocolate,
etc., and as an ingredient or raw material for fruit juice
beverages, sports drink, etc.
[0172] Exemplary anti-gastrointestinal cancer compositions of the
invention, and methods for preparing such compositions will now be
described with reference to the following examples.
EXAMPLES
Example 1
[0173] This example describes the preparation and characterization
of compositions of the invention comprising propolis and
cyclodextrin.
Complex CD1
[0174] A propolis-.gamma. cyclodextrin complex comprising 27% by
mass propolis solids was made using an ethanolic tincture of
propolis comprising 23.1% by weight propolis solids (wax free,
supplied by Manuka Health). 6.16 kg of .gamma. cyclodextrin
(CAVAMAX W8, supplied by Wacker AG) was mixed with 9.50 kg of
propolis tincture in a stainless steel tank by hand until a
homogenous, pasty brown liquid was obtained. 12.3 kg of water was
then added in 4 steps and then the resultant mixture was
homogenized at 6000 rpm for 1 hour. A further 30.43 kg of water was
then added in 4 steps with continuous stirring. This solution was
then spray dried to give a fine yellow powder. The flavonoid and
hydroxycinnamic acid content of the complex CD1 is shown in Table
2.
Complex CD2
[0175] The same procedure for complex CD1 was used to manufacture a
propolis-.gamma. cyclodextrin complex with added CAPE, but with the
following modification. 12 g of solid CAPE (supplied by Sigma
Aldrich at >97% purity) was mixed with 9.5 kg of propolis
containing 23.1.degree. Jo by weight solids until fully dissolved.
The propolis tincture with added CAPE was then mixed with .gamma.
cyclodextrin, and then water, and then spray dried as above. The
flavonoid and hydroxycinnamic acid content of the complex CD2 is
shown in Table 2. This composition comprises twice the
concentration of CAPE as Complex CD1.
Complex CD3
[0176] A propolis-.gamma. cyclodextrin complex comprising 24% by
mass propolis solids was made using an ethanolic tincture of
propolis comprising 25.0% by weight propolis solids (wax free,
supplied by Manuka Health). 300 g of .gamma. cyclodextrin (CAVAMAX
W8, supplied by Wacker AG) was mixed with 375 g of propolis
tincture in a glass vessel by hand until a homogenous, pasty brown
liquid was obtained. 1350 g of water was then added in 4 steps, and
then the resultant mixture was homogenized at 6000 rpm for 1 hour.
The solution was then added to a freeze drier tray, and placed in
front of a forced fan until 600 g of mass was lost by evaporation.
The residual solution was then frozen overnight before
freeze-drying to give a light yellow, easily crushable powder. The
flavonoid and hydroxycinnamic acid content of the complex CD3 is
shown in Table 2.
Complex CD4
[0177] A propolis-.gamma. cyclodextrin complex comprising 19% by
mass propolis solids was made using a propylene glycol solution of
propolis comprising 19% by weight propolis solids (wax free,
supplied by Manuka Health). 300 g of .gamma. cyclodextrin (CAVAMAX
W8, supplied by Wacker AG) was mixed with 1350 g of water at
60.degree. C. until fully dissolved. 375 g of propylene glycol
solution of propolis was gradually added to the mixture with
continuous stirring. The mixture was stirred for a further hour at
60.degree. C. at high speed after all solution was added. The
solution was then poured onto a freeze-drier tray, air cooled to
room temperature, and then frozen overnight before being
freeze-dried to give a yellow, easily crushable powder. The
flavonoid and hydroxycinnamic acid content of the complex CD4 is
shown in Table 2.
Complex CD5:
[0178] A propolis-.gamma. cyclodextrin complex comprising 29% by
mass propolis solids was made using an ethanolic tincture of
propolis comprising 50.0% by weight propolis solids (wax free,
supplied by Manuka Health). 7 kg of concentrated propolis tincture
was mixed in steps in a stainless steel bowl with 9.5 kg of .gamma.
cyclodextrin (CAVAMAX W8, supplied by Wacker AG). When all the
tincture had been added to the cyclodextrin, the resultant paste
was mixed for a further hour using a whisk attachment on a
planetary mixer. 50.1 kg of water was then added in 4 steps to give
a yellow coloured solution. When all the water was added the
solution was then stirred for a further hour. The solution was then
weighed onto freeze drier trays, and then placed in a blast
freezer. When the tray contents were fully frozen, the trays were
transferred to a freeze drier and then freeze-dried to give a
yellow, easily crushable powder. The flavonoid and hydroxycinnamic
acid content of the complex CD5 is shown in Table 2.
Complex CD6:
[0179] A propolis-.gamma. cyclodextrin complex containing 21% by
mass propolis solids was made using an ethanolic tincture of
propolis containing 40.3% by weight propolis solids (wax free,
supplied by Manuka Health). 6.35 kg of concentrated propolis
tincture was slowly and continuously added to a stainless steel
bowl containing 8.13 kg of .gamma. cyclodextrin (CAVAMAX W8,
supplied by Wacker AG) whilst stirring with a K-type mixer attached
to a planetary mixer. When all the tincture had been added to the
cyclodextrin, the resultant paste/powder was mixed for a further
1/2 hour. The paste was then removed from the bowl, the stirrer
changed to a whisk, and then 60.0 kg of water was added to the
bowl. Mixing was restarted, and the paste/powder was
semi-continuously added. When all the paste/powder was added the
solution was then stirred for a further hour. The resultant
dispersion was transferred into trays, which were then heated
overnight at 40.degree. C. to drive off some water and to also
induce settling of the encapsulated propolis as a pasty
precipitate. The dilute solution was then carefully poured off the
top of the trays, the paste was then weighed onto freeze drier
trays, and these trays were then placed in a blast freezer. When
the tray contents were fully frozen, the trays were transferred to
a freeze drier and then freeze-dried to give a yellow, easily
crushable powder. The flavonoid and hydroxycinnamic acid content of
the complex CD6 is shown in Table 2.
Complex CD7:
[0180] A propolis-.alpha. cyclodextrin complex containing around
22% by mass NZ propolis solids was made using an ethanolic tincture
of propolis containing 40.3% by weight propolis solids (wax free,
supplied by Manuka Health). 10.91 g of concentrated propolis
tincture was mixed in steps by hand in a stainless steel bowl with
12.92 g of a cyclodextrin (CAVAMAX W6, supplied by Wacker AG). When
all the tincture had been added to the cyclodextrin, the resultant
paste was mixed for a further 1/2 hour. 96.3 g of water was then
added 4 steps by mixing the contents of the stainless steel bowl
using a whisk attachment on a lab scale food mixer. When all the
water was added the solution was then stirred for a further hour.
The bulk of the well mixed solution was then added to a glass
round-bottom flask, which was then manually rotated in an
acetone-dry ice bath until the flask contents were fully frozen.
The flask was then transferred to a lab scale freeze drier and then
freeze-dried to give a light yellow, easily crushable powder. The
flavonoid and hydroxycinnamic acid content of the complex CD7 is
shown in Table 2.
Complex CD8:
[0181] A propolis-.beta. cyclodextrin complex containing around 22%
by mass NZ propolis solids was made using an ethanolic tincture of
propolis containing 40.3% by weight propolis solids (wax free,
supplied by Manuka Health). 10.06 g of concentrated propolis
tincture was mixed in steps by hand in a stainless steel bowl with
12.81 g of a cyclodextrin (supplied by Sigma Chemicals). When all
the tincture had been added to the cyclodextrin, the resultant
paste was mixed for a further 1/2 hour. 98.4 g of water was then
added 4 steps by mixing the contents of the stainless steel bowl
using a whisk attachment on a lab scale food processor. When all
the water was added the solution was then stirred for a further
hour. The bulk of the well mixed solution was then added to a glass
round-bottom flask, which was then manually rotated in an
acetone-dry ice bath until the flask contents were fully frozen.
The flask was then transferred to a lab scale freeze drier and then
freeze-dried to give a light yellow, easily crushable powder. The
flavonoid and hydroxycinnamic acid content of the complex CD8 is
shown in Table 2.
Complex CD9:
[0182] A propolis-.gamma. cyclodextrin complex containing around
20% by mass Brazilian green propolis solids was made using a
propylene glycol solution of propolis containing 40.0% by weight
propolis solids (wax free, supplied by Polenectar, Brazil). 8.13 g
of .gamma. cyclodextrin (CAVAMAX W8, supplied by Wacker AG) was
dissolved into 60.0 g of water in a stainless steel bowl of a lab
scale food processor. When the cyclodextrin was fully dissolved,
6.35 g of the propylene glycol solution of Brazilian green propolis
was added drop wise to the solution with stirring using a whisk
attachment on the lab scale food processor. When all the propolis
solution was added the solution was then stirred for a further 1/2
hour. The bulk of the well mixed solution was then added to a glass
round-bottom flask, which was then manually rotated in an
acetone-dry ice bath until the flask contents were fully frozen.
The flask was then transferred to a lab scale freeze drier and then
freeze-dried to give a light green, easily crushable powder. The
flavonoid and hydroxycinnamic acid content of the complex CD9 is
shown in Table 2.
TABLE-US-00002 TABLE 2 CAPE and flavonoid composition of
cyclodextrin complexes, mg/g of complex pinobanksin- Complex CAPE
pinobanksin 3-acetate pinocembrin chrysin galangin CD1 2.0 5.2 14.6
19.5 5.4 8.1 CD2 4.9 5.1 14.4 19.1 5.3 7.9 CD3 1.6 6.4 17.5 25.4
6.6 10.1 CD4 1.1 4.0 10.0 14.6 4.3 6.0 CD5 1.7 6.6 17.2 24.4 8.5
10.1 CD6 1.2 5.0 9.1 17.7 5.7 8.1 CD7 1.5 5.7 10.0 21.0 6.5 9.2 CD8
1.4 5.3 9.6 20.2 6.3 8.9
Example 2
[0183] This example describes an assessment of the anti-colon
cancer efficacy of compositions of the present invention. This
study was performed using proliferation assays in the colon cancer
adenocarcinoma cell line, DLD-1.
Materials and Methods
[0184] Test samples shown below in Table 3 were assessed for their
ability to modulate the viability and proliferation of human
colorectal adenocarcinoma cells (DLD-1) as assessed by the MTT
assay. A positive control, 5-Fluorouracil (5-FU) was included in
addition to an unsupplemented cell control (negative control) in
the study. All samples were normalized to 200 .mu.g/ml total
solids.
[0185] Thus, DLD-1 colon cancer cells were exposed to: [0186] a
tincture originally containing 25% solids by mass propolis which
then diluted in the test solution to give 200 .mu.g/ml total
solids. The tincture was used to manufacture the
cyclodextrin-encapsulated propolis samples. [0187] Cyclodextrin
propolis (.gamma.-cyclodextrin encapsulated with 25% propolis
tincture), CD1 from Table 2 in Example 1 [0188] Cyclodextrin
propolis+CAPE (.gamma.-cyclodextrin encapsulated 25% propolis
tincture with added CAPE to double the concentration of CAPE
relative to the tincture), CD2 from Table 2 in Example 1 [0189]
.gamma.-cyclodextrin alone (CAVAMAX W8 food gamma cyclodextrin)
[0190] 5-flurorouracil at 7.50 ng/ml as a positive control
[0191] All cyclodextrin-encapsulated samples and
.gamma.-cyclodextrin alone were assayed on a per weight of sample,
and so contain at most 50 .mu.g/ml total solids as propolis.
TABLE-US-00003 TABLE 3 Test Samples Sample No Test Sample ID #2 CD1
Cyclodextrin Propolis Complex #3 CD2 Cyclodextrin propolis + added
CAPE Complex #4 25% Propolis Tincture #5 CAVAMAX W8 Food
gamma-cyclodextrin
Description of Test Materials and Test Methods
[0192] Human colorectal adenocarcinoma epithelial cell lines were
revived from cryostorage and cultured in the presence of the test
and reference samples. The culture conditions for the cells were
those described by the supplier of the cells (ATCC). An MTT assay
was then performed on the cultures to determine the effect of the
samples on the cell proliferation.
[0193] The methodology was based on the procedures reported by:
[0194] Smolka, A J, Goldenring, J R, Gupta, S and Charles E
Hammond, C E. Inhibition of gastric H,K-ATPase activity and gastric
epithelial cell IL-8 secretion by the pyrrolizine derivative ML
3000. (2004). BMC Gastroenterology. 4: 4. [0195] Chailler, P and
Menard, D (2005). Establishment of Human Gastric Epithelial (HGE)
Cell Lines Exhibiting Barrier Function, Progenitor, and
Prezymogenic Characteristics. Journal of Cellular Physiology 202:
263-274. [0196] Trainer, D. L., Kline, T., McCabe, F. L., Faucette,
L. F., Field, J., Chaikin, M., Anzano, M., Rieman, D., Hoffstein,
S., Li, D-J., Gennaro, D., Buscarino, C., Lynch, M., Poste, G. And
Greig, R. (1988). Biological characterization and oncogene
expression in human colorectal carcinoma cells lines. International
Journal of Cancer 41: 287-296.
Sample Preparation
[0197] The test items were dissolved in 15% ethanol (ETOH)/Hanks
Balanced Salt Solution (HBSS) at concentrations given in the
following table.
TABLE-US-00004 TABLE 4 Concentrations of the test samples used in
this project Sample Preparation Stock Working Solution in No Sample
Concentrations 15% ETOH/HBSS Final Conc. in Well #2 CD1
Cyclodextrin Propolis Complex powder 2 mg/ml solids 200 .mu.g/ml
solids #3 CD2 Cyclodextrin + CAPE powder 2 mg/ml solids 200
.mu.g/ml solids #4 25% Propolis Tincture 250 mg/ml solids 2 mg/ml
solids 200 .mu.g/ml solids #5 CAVAMAX W8 Food gamma powder 2 mg/ml
solids 200 .mu.g/ml solids Cyclodextrin
[0198] Sample #4 was diluted 50-fold to give a working stock
solution of 2 mg/mL solids.
[0199] Samples #2, #3 and #5 were powders. Working stock solutions
were prepared by weighing 2 mg and dissolving in 1 ml of 15%
ETOH/HBSS.
Experimental Procedures
Characterisation of the Test System
[0200] 1. Human colorectal adenocarcinoma cells (ATCC CCl-221,
DLD-1). [0201] 2. Penicillin-streptomycin solution: 10000 units/ml
penicillin, 10 mg/ml streptomycin in 0.9% NaCl (Sigma Cat #P-0781).
Stored at -20.degree. C. [0202] 3. DMEM culture medium (Invitrogen
Cat #12100-046) Stored at -20.degree. C. [0203] 4. Trypsin-EDTA
solution: 0.25% Trypsin/EDTA, Invitrogen Cat#15400054 (.times.10 in
stock). [0204] 5. Phosphate buffered saline (PBS) (Prepared by
TBL). [0205] 6. Hanks Balanced Salt Solution (HBSS). (GIBCO Cat No.
14185-052). Stored at 4.degree. C. [0206] 7. Foetal Bovine Serum
(GIBCO Cat #10091-148). Stored at -20.degree. C. [0207] 8. MTT
Reagent: 100 mg/vial, (SIGMA Cat. No. M-2128) dissolved in PBS at
10 mg/ml and stored at -20.degree. C. 5 mg/ml MTT solution were
prepared in PBS and stored at 4.degree. C. as working solution.
[0208] 9. MTT lysis buffer: 10% sodium dodecyl sulphate (SDS)/45%
Dimethyl Formamide (20 g SDS was dissolved in 100 ml of
double-distilled water (DDW), and 90 ml of Dimethyl Formamide added
to the SDS solution). The pH was adjusted to 4.7 by glacial acetic
acid, and DDW added up to a final volume of 200 ml. [0209] 10.
5-Fluorouracil (5-FU), (Sigma Cat. No. F-6627). Two working
solutions were prepared at 150 ng/ml and 75 ng/ml, dissolved in 15%
Ethanol/HBSS. Final concentrations were 7.50 ng/ml and 3.75
ng/ml.
Medium Preparation
[0210] The medium for the propagation of the colorectal
adenocarcinoma (DLD-1) cells was DMEM, supplemented with
penicillin-streptomycin solution (10 ml per litre). FBS was added
just before use to give 10% w/v.
Culturing of Cells
[0211] 1. The human colorectal adenocarcinoma cells (DLD-1)
obtained from the American Type Culture Collection USA were revived
from cryostorage. [0212] 2. Following initial propagation using the
media described above (see Medium Preparation), the culture was
subcultured using the trypsin-EDTA as follows. The media was
removed and 5 ml of the trypsin-EDTA solution added and incubated
at 37.degree. C. for 5 min or until all the cells had detached. The
trypsin was neutralised by adding an equal volume of DMEM medium
and the culture centrifuged at 300 g (1200 rpm) for 5 min at
4.degree. C. [0213] 3. The supernatant was decanted and the cell
pellets resuspended in medium DMEM, FBS (10%), penicillin (100
units/ml), streptomycin (100 .mu.g/ml). The cells were cultured at
37.degree. C. in 5% CO.sub.2/95% air. [0214] 4. After reaching
confluence, the cells were detached using trypsin-EDTA and
centrifuged as described in 2 above. [0215] 5. The supernatants
were discarded and the cells resuspended in DMEM and supplements as
described in 3 above at 7.5.times.10.sup.4 cells per ml. A total
volume of 23 mls of the cell suspension was required. [0216] 6.
Into each well of three 96 well plates, 180 .mu.l of the cells
(13,550 cells/well) or medium was added as indicated in the plate
layout below. The plates were incubated at 5% CO.sub.2/95% air at
37.degree. C. for 8 h which was sufficient to allow the cells to
strongly adhere. [0217] 7. To each well, 20 .mu.l of each of the
test samples or positive control was added as indicated in the
diagrams above. To the wells labelled `medium` or `cells only`, 20
.mu.l of 15% ETOH/HBSS was added. Each sample was assessed in
replicates of 6, while the controls on all three plates were
assessed in replicates of 9 (combined triplicates) [0218] 8. The
total volume in each well was 200 .mu.l. [0219] 9. The plates were
incubated at 37.degree. C. in 5% CO.sub.2/95% air for 19 h.
Cell Proliferation Assay
[0219] [0220] 1. On completion of the incubation, 20 .mu.l of MTT
working solution (5 mg/ml) was added to all wells and incubated for
2 hr at 37.degree. C. in 5% CO.sub.2/95% air. [0221] 2. 100 .mu.l
of MTT lysis buffer was then added and the plates incubated for 3
hr at 37.degree. C. on a shaker in 5% CO.sub.2/95% air, followed by
pipetting to break up and dissolve the crystals until fully
solubilized. The plates were centrifuged at 1200 rpm for 10 minutes
to pelletize any remaining insoluble material. From each well 200
.mu.l aliquots were transferred to fresh 96 well plates. The plates
were read by a VersaMax microplate reader at 570 nm. [0222] 3.
Results were expressed as the percentage proliferation of cells
cultured in the presence of the sample in comparison to the cells
only control. The blank reading was subtracted from all wells as a
background reading.
Results and Discussion
[0223] A summary of the effects of controls and test samples on the
proliferation of the cells is presented in Table 5 and in FIG.
1.
TABLE-US-00005 TABLE 5 Effects of the test samples on the
proliferation of the DLD-1 cells. Viability of Human Colon Cancer
Cells (DLD-1) as Measured by the MTT Assay. Mean (OD p Values %
Sample ID 570 nm) SEM (<0.05) Inhibition Cells only (n = 9)
0.7047 0.0252 1.00000000 0.00 Cells + 5-FU (7.50 ng/ml). (n = 9)
0.6575 0.0172 NS 6.69 Cells + S#2 CD1 Cyclodextrin Propolis 0.5363
0.0161 0.00024874 23.90 Complex (200 .mu.g/ml). (n = 6) Cells + S#3
CD2 Cyclodextrin & CAPE 0.5057 0.0155 0.00004986 28.24 (200
.mu.g/ml). (n = 6) Cells + S#4 25% Propolis 0.5295 0.0339
0.00097212 24.86 Tincture (200 .mu.g/ml). (n = 6) Cells + S#5
CAVAMAX W8 Food gamma 0.6405 0.0365 NS 9.11 Cyclodextrin (200
.mu.g/ml). (n = 6) NS = Not Significant.
[0224] The relatively rapid proliferation rate of the cells
observed during the growth of the stock cell culture allowed the
incubation time of the cells with the test samples to be reduced to
8 hours. This proved to be quite satisfactory as assessed by the
colour intensity of the control culture following reaction with MTT
(Mean OD.sub.570 nm=0.7047). Each sample was assayed in replicates
of six. There was good consistency within the six samples, such
that the SEM. for all replicates was within expected limits and all
six were used in the calculation of the mean absorbance values. For
the controls (both negative and positive) nine replicates were
sufficiently consistent to be used for the statistical
evaluations.
[0225] CD1 Cyclodextrin Propolis Complex
[0226] Cyclodextrin Propolis Complex at 200 .mu.g/ml was a
statistically significant inhibitor of the proliferation of the
colon cancer cells. It reduced proliferation by 23.9%.
[0227] CD2 Cyclodextrin Propolis+CAPE
[0228] The preparation Cyclodextrin propolis+CAPE at 200 .mu.g/ml
was also statistically significant as an inhibitor of the cell
growth. It reduced proliferation by 28.2%.
[0229] 25% Propolis Tincture
[0230] The 200 .mu.g/ml concentration of 25% Propolis Tincture was
a statistically significant inhibitor of colon cancer growth. It
reduced proliferation by 24.9%
[0231] CAVAMAX W8 Food Gamma-Cyclodextrin
[0232] At 200 .mu.g/ml CAVAMAX W8 Food gamma-cyclodextrin caused an
insignificant effect on the proliferation of the cells with a
reduction of only 9.1%.
[0233] In this study the 5-Fluorouacil at 7.5 ng/ml produced an
insignificant 6.7% inhibition of the growth. This was unexpected.
Based on previous studies it was anticipated that, at this
concentration, a statistically significant reduction would be
recorded.
[0234] The assays described above were performed on an equivalent
mass basis of the test material. Thus, for the native propolis
tincture, where the final concentration of test material was 200
.mu.g/mL, the entire 200 .mu.g comprised propolis solids. The
cyclodextrin complexes contained at most 25% propolis, and so only
50 of the 200 .mu.g comprised propolis. As can clearly be seen in
FIG. 1 and as observed above, approximately the same activity for
native propolis (ex-tincture) and the propolis/gamma-cyclodextrin
composition was observed. Accordingly, this data suggests that this
example of a propolis/gamma-cyclodextrin composition of the
invention exhibits approximately four times the activity in this
assay compared to native propolis, since the content of propolis
solids in the complex is only 50 .mu.g/mL (See FIG. 1B).
[0235] Table 6 below and FIG. 1B show the % Efficacy for each
propolis-containing sample, wherein % Efficacy=%
Inhibition/concentration of propolis in the sample.
TABLE-US-00006 TABLE 6 Efficacy of test samples Sample % inhibition
[effective propolis] Efficacy Propolis/CD complex 23.9 50 47.8
Propolis/CD + CAPE 28.2 50 56.4 complex Propolis tincture 24.9 200
12.45 CD 9.1 0 --
[0236] The applicants also note that doubling the amount of CAPE
present in the cyclodextrin complex only marginally increases the
bioactivity, which suggests that CAPE at this concentration is not
having a large effect on proliferation.
[0237] These data support the enhanced efficacy of compositions of
the invention compared to propolis. Without wishing to be bound by
any theory, the applicants suggest this may be due to an unexpected
synergistic effect on colorectal cell proliferation exhibited by
the propolis and cyclodextrin composition.
Example 3
[0238] This example shows the effectiveness of the
cyclodextrin-encapsulated propolis compared to pure standards of
CAPE, chrysin and galangin. All samples were tested against the
same human colorectal adenocarcinoma cell line DLD-1 used in
Example 2.
Materials and Methods
[0239] The test method and experimental procedure are the same as
those used in Example 2. CAPE, galangin and chrysin standards were
obtained at greater than 99% purity from Sigma-Aldrich. The CAPE,
chrysin and galangin concentrations are given in Table 7.
TABLE-US-00007 TABLE 7 Test concentrations of CAPE, chrysin and
galangin Samples tested No Sample CAPE Chrysin Galangin S#2
Cyclodextrin propolis 0.41 .mu.g/ml solids 1.07 .mu.g/ml solids
1.61 .mu.g/ml solids Complex S#3 Cyclodextrin propolis + CAPE 0.98
.mu.g/ml solids 1.06 .mu.g/ml solids 1.58 .mu.g/ml solids S#4 25%
Propolis Tincture, 1.48 .mu.g/ml solids 3.91 .mu.g/ml solids 5.87
.mu.g/ml solids 200 .mu.g/ml solids S#6 CAPE 200 .mu.g/ml solids
S#7 Chrysin 200 .mu.g/ml solids S#8 Galangin 200 .mu.g/ml
solids
[0240] The following samples were tested at a well concentration of
200 .mu.g/ml except as specified in Table 8.
Results and Discussion
TABLE-US-00008 [0241] TABLE 8 Effects of cyclodextrin complexes and
pure compounds on the proliferation of DLD-1 cells Viability of
Human Colon Cancer Cells (DLD-1) as Measured by the MTT Assay. Mean
(OD p Values % Sample ID 570 nm) SEM (<0.05) Inhibition Cells
only (n = 9) 0.3239 0.0135 NS 0.00 Cells + 5-FU (0.65 .mu.g/ml). (n
= 9) 0.3023 0.0154 NS 6.66 Cells + 5-FU (1.95 .mu.g/ml). (n = 9)
0.2689 0.0116 .00534 16.96 Cells + S#2 CD1 Cyclodextrin Propolis
0.5363 0.0161 0.00024874 23.90 Complex (200 .mu.g/ml). (n = 6)
Cells + S#3 CD2 Cyclodextrin & CAPE 0.5057 0.0155 0.00004986
28.24 (200 .mu.g/ml). (n = 6) Cells + S#4 25% Propolis Tincture
0.5295 0.0339 0.00097212 24.86 (200 .mu.g/ml). (n = 6) S#6 CAPE
(200 .mu.g/ml). (n = 5) 0.0777 0.0071 7.43E-8 76.01 S#7 Chrysin
(200 .mu.g/ml). (n = 6) 0.1233 0.0099 3.71E-8 61.92 5#8 Galangin
(200 .mu.g/ml). (n = 6) 0.1411 0.0038 6.74E-8 56.45
[0242] As shown in Table 8, the CAPE, chrysin and galangin
standards were all statistically significant inhibitors of DLD-1
proliferation at a concentration of 200 .mu.g/ml. The proliferation
is also significant when these compounds are present at much lower
concentrations in the cyclodextrin complexes. The relative
inhibition (ratio of % inhibition to concentration) versus the test
substance concentration is shown in FIG. 2.
[0243] The results show that encapsulation of propolis containing
these three compounds in cyclodextrin greatly increases their
activity.
Example 4
[0244] This example demonstrates the ability of compositions of the
invention, cyclodextrin encapsulated propolis to induce apoptosis
in the human colorectal cancer cell line HCT-116, and to augment
the apoptotic effect of butyrate in this human colorectal cancer
cells, and a cancer cell line HCT-116 R made resistant to butyrate.
Butyric acid is generated by intestinal microflora resulting from
the digestion of soluble fibre.
Materials and Methods
Cell Culture and Chemicals
[0245] The human colorectal cell line HCT-116 was obtained from the
American Type Culture Collection (Rockville, Md.). The butyrate
resistant cell line was derived from HCT-116 by culturing the
parental cells in increasing concentrations of butyrate as
previously described in Bordonaro M, Lazarova D L, Sartorelli A C.
The activation of beta-catenin by Wnt signaling mediates the
effects of histone deacetylase inhibitors. Exp Cell Res. 2007;
313:1652-66 (including references therein).
[0246] Colorectal cells were grown in alpha-MEM medium with 10%
fetal bovine serum and antibiotics. The concentration of CAPE in
the propolis-cyclodextrin complex CD1 was 1.79 .mu.g/100 .mu.g
propolis solid encapsulated in the cyclodextrin, or 0.41 .mu.g/100
.mu.g cyclodextrin complex. Stock solutions were prepared in
dimethyl sulfoxide, except for butyrate, which was dissolved in
water at 1 M concentration.
Apoptotic Assays
[0247] Twenty-four hours prior to treatments, colon cancer cells
were plated in 24-well plates at 100,000-120,000 per well. A
negative control of cells only was compared to cells exposed to a
positive control of 5 mM butyrate; 1.2 ug/ml CAPE; 5 mM
butyrate+1,2 ug/ml CAPE, 100 ug/ml propolis-cyclodextrin, or 5 mM
butyrate+100 ug/ml propolis-cyclodextrin for 50 hours. All cells
(floating and attached) were harvested and stained for apoptotic
and necrotic markers using a PE Annexin V Apoptosis Detection Kit I
(BD Biosciences, #559763). Flow cytometry analyses were carried out
with FACS Aria II and DiVa software. Percent apoptosis is the
number or apoptotic cells divided by the number of all analyzed
cells, and multiplied by 100.
Statistics
[0248] All data are presented as mean.+-.standard deviation from at
least three sets of independent experiments. Student T-test
analysis was used to determine the significance of statistical
differences. Differences were considered significant at
P<0.05.
Results
[0249] The apoptotic effect of butyrate on colon cancer cells is
augmented to different extent by CAPE and propolis.
[0250] HCT-116 and HCT-R cells were utilized to compare the
apoptotic effect of butyrate, CAPE and propolis-cyclodextrin
complex, and combinations of butyrate and CAPE or propolis
cyclodextrin. The results are shown in Table 9 and in FIG. 3 after
normalization against the negative control.
TABLE-US-00009 TABLE 9 Effect of pure compounds, propolis gamma
cyclodextrin complex CD1 and their mixtures with butyrate on the
apoptosis of HCT-116 and HCT-116 R cells HCT-116 HCT-116-R % %
Sample I.D. Apoptosis Error Apoptosis Error Negative control, cells
only 22.3 .+-.4.4 12.1 .+-.1.0 Positive control, butyrate (5 mM)
57.5 .+-.1.5 11.2 .+-.2.1 CAPE (1.2 ug/ml) 18.3 .+-.5.8 9.7 .+-.1.2
CAPE (1.2 ug/ml + butyrate 74 .+-.4.3 24.3 .+-.4.3 (5 mM) S#3
propolis cyclodextrin complex 28.7 .+-.4.9 23.4 .+-.4.9 S#3
propolis cyclodextrin 80.5 .+-.1.4 45.5 .+-.1.4 complex +
butyrate
[0251] HCT-116 cells exposed to CAPE had no significant increase in
apoptosis relative to the negative control. HCT-116 cells exposed
to the propolis-cyclodextrin complex CD1 underwent significant
levels of apoptosis at around 8% higher than the negative control
(see FIG. 3), despite the propolis-cyclodextrin complex having only
approximately 1/4 of the CAPE. Butyrate treatment of HCT-116 cells
resulted in 35% apoptosis relative to cells only. The combination
of CAPE or propolis-cyclodextrin and butyrate augmented the
apoptosis of HCT-116 cells achieved with butyrate. The combination
of CAPE and butyrate resulted in 52% apoptosis relative to cells
only (P<0.05 compared to butyrate alone). The combination of
propolis-cyclodextrin and butyrate resulted in 58% apoptosis
relative to cells only (P<0.05 compared to butyrate alone),
despite having only 1/4 of the CAPE. HCT-R cells did not undergo
significant apoptosis when tested with butyrate alone (as expected)
or CAPE alone; however, exposure to the propolis-cyclodextrin
complex resulted in significantly higher levels of apoptosis
relative to the negative control of 11%, P<0.05. The combination
of CAPE or propolis-cyclodextrin and butyrate re-sensitized HCT-R
cells to the apoptotic effect of butyrate. The combination of CAPE
and butyrate resulted in 12% apoptosis relative to the control
(P<0.05 compared to butyrate alone). The combination of
propolis-cyclodextrin complex and butyrate further enhanced
apoptosis to 33% relative to the control, (P<0.05 compared to
butyrate alone).
Discussion
[0252] This example shows that caffeic acid phenethyl ester (CAPE),
and a propolis-cyclodextrin complex containing a 1/4 of the amount
of CAPE tested alone, exert differential effects on apoptosis in
human colon cancer cells. The propolis-cyclodextrin composition
augmented apoptosis of colon cancer cells exposed to butyrate to a
much greater extent than CAPE, which is ineffective by itself at
the concentration used. Furthermore, the propolis-cyclodextrin
complex was more potent than CAPE in resensitizing
butyrate-resistant HCT-R cells to butyrate-induced apoptosis.
[0253] It is expected that a diet containing soluble fibre
digestable by intestinal microflora will generate butyrate in-situ,
and that colon cancer cells could become resistant to butyrate
generated in such a manner, or administered by other means due to
prolonged exposure. This example demonstrates the efficacy of
compositions of the invention for modulating the apoptosis of both
native and butyrate resistant colorectal cancer cells, both without
and with a second apoptotic agent; and that the compositions of the
invention can reverse the resistance of colorectal cells to
apoptosis induced by such agents. These results support the
efficacy of compositions of the invention in the treatment and
prevention of gastrointestinal cancers.
Example 5
[0254] This example demonstrates the ability of compositions of the
invention, .gamma.-cyclodextrin encapsulated European-type propolis
to inhibit proliferation of the human colon cancer adenocarcinoma
cell line, DLD-1; human colon cancer cell line, HCT-116; human
gastric carcinoma cell line, NCI-N87; and human oesophageal
squamous cell carcinoma cell line, KYSE-30. The general method used
for Examples 5-8 is shown below.
Materials and Methods for the Gastro-Intestinal Cancer
Anti-Proliferative Assays
[0255] Sample CD5 .gamma.-cyclodextrin as prepared in Example 1 was
assessed for its ability to modulate the viability and
proliferation of human colorectal adenocarcinoma cells (DLD-1);
human colon cancer cell line (HCT-116); human gastric carcinoma
cell line (NCI-N87); and human oesophageal squamous cell carcinoma
cell line (KYSE-30) as assessed by the MTT assay. The positive
control, 5-fluorouracil (5-FU) was included at three
concentrations; in addition to an unsupplemented cell control as a
negative control in the study. [Do we include test compounds for
comparison?.
Description of Test Materials and Test Methods
[0256] The four human gastrointestinal carcinoma cell lines were
revived from cryostorage and cultured in the presence of the test
and reference samples. An MTT assay was then performed on the
cultures to determine the effect of the samples on the cell
viability and proliferation.
The methodology is based on the procedures outlined in Example 2
above.
Sample Preparation
[0257] The test sample, and all other test samples in Examples 5-8
were dissolved in 15% ethanol/Hanks Balanced Salt Solution
(ETOH)/HBSS to a concentration of 2 mg/mL solids. In the assay the
final concentration of the samples was 200 .mu.g/ml with a final
EtOH concentration of 1.5%.
Experimental Procedures
Characterisation of the Test System
[0258] 1. Human colorectal adenocarcinoma cells (ATCC CCI-221,
DLD-1)(ATCC, Bethesda, Md., USA) [0259] 2. Human gastric carcinoma
cells (ATCC CRL-5822, NCI-N87)(ATCC, Bethesda, Md., USA) [0260] 3.
Human oesophageal squamous cell carcinoma (ECACC, KYSE-30)(Sigma
Aldrich, Auckland, NZ) [0261] 4. Human colon carcinoma cells (ECACC
HCT-116)(Sigma Aldrich, Auckland, NZ) [0262] 5.
Penicillin-streptomycin solution: 10000 units/ml penicillin, 10
mg/ml streptomycin in 0.9% NaCl (Sigma Cat #P-0781). Stored at
-20.degree. C. [0263] 6. For DLD-1 cells: DMEM culture medium
(Invitrogen Cat #12100-046). Stored at 4.degree. C. [0264] 7. For
NCI-N87 cells: RPMI-1640 medium modified to contain 2 mM
L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose,
and 1500 mg/L sodium bicarbonate (Sigma R6504). Stored at 4.degree.
C. [0265] 8. For KYSE-410 cells: RPMI-1640 medium modified to
contain 2 mM L-glutamine (Sigma R6504). Stored at 4.degree. C.
[0266] 9. For HCT-116 cells: McCoy's 5A medium modified to contain
2 mM L-glutamine (Sigma M48792). Stored at 4.degree. C. [0267] 10.
Trypsin-EDTA solution: 0.25% Trypsin/EDTA, Invitrogen Cat#15400054
(.times.10 in stock). [0268] 11. Phosphate buffered saline (PBS)
(Prepared by TBL). [0269] 12. Hanks Balanced Salt Solution (HBSS).
(GIBCO Cat No. 14185-052). Stored at 4.degree. C. [0270] 13. Foetal
Bovine Serum (GIBCO Cat #10091-148). Stored at -20.degree. C.
[0271] 14. MTT Reagent: 100 mg/vial, (SIGMA Cat. No. M-2128)
dissolved in PBS at 10 mg/ml and stored at -20.degree. C. 5 mg/ml
MTT solution will be prepared in PBS and stored at 4.degree. C. as
working solution. [0272] 15. MTT lysis buffer: 10% sodium dodecyl
sulphate (SDS)/45% Dimethyl Formamide (20 g SDS will be dissolved
in 100 ml of double-distilled water (DDW), and 90 ml of Dimethyl
Formamide will be added to the SDS solution). The pH will be
adjusted to 4.7 by glacial acetic acid, and DDW added up to a final
volume of 200 ml. [0273] 16. 5-Fluorouracil (5-FU), (Sigma Cat. No.
F-6627). Three working solutions will be prepared at 19.5 .mu.g/ml.
6.5 .mu.g/ml and 1.95 .mu.g/ml dissolved in 15% Ethanol/HBSS. Final
concentrations will be 1.95 .mu.g/ml (15 .mu.M), 0.65 .mu.g/ml (5
.mu.M) and 0.195 .mu.g/ml (1.5 .mu.M).
Medium Preparation
[0274] The medium for the propagation of the each of the cells
lines is given above. Each medium was prepared following the
ATCC/ECACC instructions and supplemented with
penicillin-streptomycin solution (10 ml per litre). FBS (at 10%)
was added just before use.
Culturing of Cells
[0275] 1. Each of the cell lines obtained from either the American
Type Culture Collection USA, or the European Collection of Cell
Cultures was revived from cryostorage. [0276] 2. Following initial
propagation using the media described above (see Characterisation
of Test System, and Medium Preparation), the culture was
subcultured using trypsin-EDTA. The media was removed and 5 ml of
the trypsin-EDTA solution added and incubated at 37.degree. C. for
5 min or until all the cells have detached. The trypsin was
neutralised by adding an equal volume of the relevant medium and
centrifuging the suspension at 300 g (1200 rpm) for 5 min at
4.degree. C. [0277] 3. The supernatant was decanted and the cell
pellets resuspended in the relevant medium containing FBS (10%),
penicillin (100 units/ml), streptomycin (100 .mu.g/ml). [0278] 4.
After reaching confluence, the cells were detached using
trypsin-EDTA as described in 2 above and centrifuged. [0279] 5. The
supernatants were discarded and the cells resuspended in the
relevant medium and supplements as described in 3 above at
1.0.times.10.sup.4 cells per ml. A total volume of approximately 54
mls of each cell suspension was required. [0280] 6. Into each well
of six 96 well plates, 180 .mu.l of the cells (1,800 cells/well) or
medium was added as indicated in predetermined plate layouts. The
plates were incubated at 5% CO.sub.2/95% air at 37.degree. C. for
.about.48 hrs to allow the cells to adhere. [0281] 7. To each well,
20 .mu.l of each of the test compounds or 5-FU was added as
indicated in the plate layouts. For negative control wells labelled
`medium` or `cells only`, 20 .mu.l of 15% ETOH/HBSS was added. Each
sample or control was assessed in replicates of 3 or 6. [0282] 8.
The total volume in each well was 200 .mu.l. [0283] 9. The plates
were incubated at 37.degree. C. in 5% CO.sub.2/95% air for 24
hr.
Cell Proliferation Assay
[0283] [0284] 1. On completion of the incubation, 20 .mu.l of MTT
working solution (5 mg/ml) was added to all wells and incubated for
3-4 hr at 37.degree. C. in 5% CO.sub.2/95% air. The plates were
monitored every 30-60 minutes and if a few cells showed the
presence of crystals then the lysis buffer was added as in Step 2
below. [0285] 2. 100 .mu.l of MTT lysis buffer was then added and
the plates incubated overnight at 37.degree. C. in 5% CO.sub.2/95%
air. The plates were centrifuged at 1200 rpm for 10 minutes to
pellet any remaining insoluble material. From each well 200 .mu.l
was transferred to fresh 96 well plates. The plates were then read
in a VersaMax microplate reader at 550 nm. [0286] 3. The blank
reading was subtracted from all wells as a background reading.
Results were expressed as the percentage proliferation of cells
cultured in the presence of the sample in comparison to the cells
only control. [0287] 4. A VersaMax 96-well plate reader was used to
colorimetrically (at 550 nm) assess the proliferation of the cells.
[0288] 5. The percentage standard error of the mean was assessed
and extreme outliers will be removed if the SEM %>15.
Preliminary statistical significance will be assessed with an
independent Student t-test at .alpha..ltoreq.0.05 (with and without
outliers).
Observations Regarding the Test Method
[0289] The culturing of the several cancer cells in the presence of
the various test preparations and the 5-FU was conducted for 48
hours as the visual observation of the cell density of the negative
controls of the DLD-1 and NCI-N87 after 24 hours suggested that the
growth was not as rapid as anticipated. For consistency the time
for the other cultures was set as the same.
[0290] The study plan required outliers among the replicates to be
deleted from the calculations if the percentage standard error of
mean (SEM) was greater than 15%. When a sample is a strong
antagonist, the absorbance values are small. Consequently small
changes among these mean that the variance from the mean expressed
as a percentage is relatively large when compared with less
inhibitory samples. This has resulted in some outliers in which
virtually complete inhibition occurred being removed from the
analysis.
Results
[0291] The results of the cell proliferation assay for CD5
.gamma.-cyclodextrin are shown in Table 10, along with results for
the positive control 5-fluorouracil (5-FU) at three concentrations,
and the negative control (cells only with medium and no test
compound or positive control). In the Table, OD is Optical Density
measured at 570 nm; SEM is the Standard Error associated with the
Mean Optical Density value measured; p is the probability value
that the measurement is statistically significant via the Student
t-test, here taken to be <0.05; % stim. is the percentage
stimulation of proliferation compared to the negative control (test
compound inactive); % inhibition is the percentage reduction of
proliferation compared to the negative control, with a large number
indicating the test compound has anticancer proliferation
potential.
TABLE-US-00010 TABLE 10 Antiproliferative activity of CD5
.gamma.-cyclodextrin encapsulated European-type propolis against 4
gastro-intestinal cell lines Mean OD p values % Cell type Sample
(570 nm) SEM (<0.05) % stim. inhibition DLD-1 Cells only (n = 6)
0.4122 0.0104 1 0 0 Cells + 5FU (0.195 .mu.g/ml, n = 6) 0.3029
0.0074 6.36E-06 0 26.53 Cells + 5FU (0.65 .mu.g/ml, n = 6) 0.2584
0.0113 1.59E-06 0 37.31 Cells + 5FU (1.95 .mu.g/ml, n = 6) 0.2409
0.0073 9.50E-08 0 41.56 Cells + CD5 0.1114 0.0029 8.00E-11 0 73
HCT-116 Cells only (n = 6) 0.5084 0.0321 1.00E+00 0 0 Cells + 5FU
(0.195 .mu.g/ml, n = 6) 0.336 0.019 9.50E-04 0 33.92 Cells + 5FU
(0.65 .mu.g/ml, n = 6) 0.234 0.015 1.50E-05 0 53.97 Cells + 5FU
(1.95 .mu.g/ml, n = 6) 0.2169 0.0037 4.10E-06 0 57.34 Cells + CD5
0.1051 0.0071 2.40E-07 0 79.33 NCI-N87 Cells only (n = 6) 0.4417
0.025 1 0 0 Cells + 5FU (0.195 .mu.g/ml, n = 6) 0.4085 0.0081
>0.05 0 7.52 Cells + 5FU (0.65 .mu.g/ml, n = 6) 0.3656 0.0212
0.043 0 17.22 Cells + 5FU (1.95 .mu.g/ml, n = 6) 0.3702 0.0055
0.019 0 16.19 Cells + CD5 0.2973 0.0069 2.40E-04 0 32.68 KYSE-30
Cells only (n = 6) 0.6017 0.0223 1 0 0 Cells + 5FU (0.195 .mu.g/ml,
n = 6) 0.4588 0.0107 1.80E-04 0 23.75 Cells + 5FU (0.65 .mu.g/ml, n
= 6) 0.3647 0.0188 1.00E-05 0 39.38 Cells + 5FU (1.95 .mu.g/ml, n =
6) 0.316 0.0045 1.90E-07 0 47.48 Cells + CD5 0.1773 0.0069 5.30E-09
0 70.54
Discussion
[0292] CD5 .gamma.-cyclodextrin was highly active against three of
the four cell lines resulting in inhibition of proliferation of
human colon adenocarcinoma cell line DLD-1 by 73.0%, human colon
cancer cell line HCT-116 by 79.3%, human gastric carcinoma cell
line NCI-N87 by 32.7% and human oesophageal squamous cell carcinoma
cell line KYSE-30 by 70.5%.
[0293] The degree of inhibition was similar to that achieved using
the known anticancer agent 5-fluororacil (5-FU), and indeed
superior for NCI-N87. The results demonstrate that the
.gamma.-cyclodextrin encapsulated European-type propolis has broad
spectrum anti-proliferative activity for gastrointestinal cancers.
The results also confirm the anti-cancer activity against human
colon adenocarcinoma cell line DLD-1 demonstrated in Examples 2-4,
and anti-cancer activity against human colon cancer cell line
HCT-116 demonstrated in Example 4.
Example 6
[0294] This example demonstrates the ability of compositions of the
invention, .alpha.-cyclodextrin encapsulated European-type propolis
to inhibit proliferation of the human colon cancer adenocarcinoma
cell line, DLD-1; human colon cancer cell line, HCT-116; human
gastric carcinoma cell line, NCI-N87; and human oesophageal
squamous cell carcinoma cell line, KYSE-30. This study was
performed using proliferation assays for DLD-1; HCT-116; NCI-N87;
KYSE-30.
[0295] The .alpha.-cyclodextrin encapsulated European-type propolis
used in the assays at a concentration of 200 .mu.g/ml was
manufactured as per Example 1, test substance CD6 with composition
given in Table 2. The same test procedure given in detail in
Example 5 was also used for Example 6.
Results
[0296] The results of the antiproliferation assays for
.alpha.-cyclodextrin encapsulated European-type propolis are shown
in Table 11. The results for the cells only (negative control), and
for the 5-FU positive controls are the same as for Example 5 and so
are not included in the table.
TABLE-US-00011 TABLE 11 Antiproliferative activity of CD6
.alpha.-cyclodextrin encapsulated European-type propolis against 4
gastro-intestinal cell lines Mean OD P values Cell type (570 nm)
SEM (<0.05) % stim. % inhibition DLD-1 0.158 0.0069 1.80E-09 0
61.67 HCT-116 0.2147 0.0157 9.30E-06 0 57.78 NCI-N87 0.3329 0.0068
1.80E-03 0 24.62 KYSE-30 0.3088 0.0071 1.90E-07 0 48.67
Discussion
[0297] CD6 .alpha.-cyclodextrin was moderately active against two
of the four cell lines, resulting in inhibition of proliferation of
human colon adenocarcinoma cell line DLD-1 by 61.7%, human colon
cancer cell line HCT-116 by 57.8%, human gastric carcinoma cell
line NCI-N87 by 24.6% and human oesophageal squamous cell carcinoma
cell line KYSE-30 by 48.7%.
[0298] The degree of inhibition was similar to that achieved using
the known anticancer agent 5-fluororacil (5-FU). The results
demonstrate that the .alpha.-cyclodextrin encapsulated
European-type propolis has broad spectrum anti-proliferative
activity for gastrointestinal cancers, but the activity is lower
than that of .gamma.-cyclodextrin encapsulated European-type
propolis.
Example 7
[0299] This example demonstrates the ability of compositions of the
invention, .beta.-cyclodextrin encapsulated European-type propolis
to inhibit proliferation of the human colon cancer adenocarcinoma
cell line, DLD-1; human colon cancer cell line, HCT-116; human
gastric carcinoma cell line, NCI-N87; and human oesophageal
squamous cell carcinoma cell line, KYSE-30. This study was
performed using proliferation assays for DLD-1; HCT-116; NCI-N87;
and KYSE-30.
[0300] The .beta.-cyclodextrin encapsulated European-type propolis
used in the assays at a concentration of 200 .mu.g/ml was
manufactured as per Example 1, test substance CD7 with composition
given in Table 2. The same test procedure given in detail in
Example 5 was also used for Example 7.
Results
[0301] The results of the antiproliferation assays for
.alpha.-cyclodextrin encapsulated European-type propolis are shown
in Table 12. The results for the cells only (negative control), and
for the 5-FU positive controls are the same as for Example 5 and so
are not included in the table.
TABLE-US-00012 TABLE 12 Antiproliferative activity of CD7
.beta.-cyclodextrin encapsulated European-type propolis against 4
gastro-intestinal cell lines Mean OD % Cell type (570 nm) SEM P
values (<0.05) % stim. inhibition DLD-1 0.1547 0.005 7.40E-10 0
62.48 HCT-116 0.2563 0.0201 5.60E-05 0 49.6 NCI-N87 0.3451 0.0062
3.70E-03 0 21.85 KYSE-30 0.3438 0.003 4.40E-07 0 42.87
Discussion
[0302] CD7 .beta.-cyclodextrin was moderately active against two of
the four cell lines, resulting in inhibition of proliferation of
human colon adenocarcinoma cell line DLD-1 by 62.5%, human colon
cancer cell line HCT-116 by 49.6%, human gastric carcinoma cell
line NCI-N87 by 21.8% and human oesophageal squamous cell carcinoma
cell line KYSE-30 by 42.9% The degree of inhibition was similar to
that achieved using the known anticancer agent 5-fluororacil
(5-FU).
[0303] The results demonstrate that the .beta.-cyclodextrin
encapsulated European-type propolis has broad spectrum
anti-proliferative activity for gastrointestinal cancers, but the
activity is lower than that of .gamma.-cyclodextrin encapsulated
European-type propolis.
Example 8
[0304] This example demonstrates the ability of compositions of the
invention, .gamma.-cyclodextrin encapsulated Brazilian-type
propolis to inhibit proliferation of the human colon cancer
adenocarcinoma cell line, DLD-1; human colon cancer cell line,
HCT-116; human gastric carcinoma cell line, NCI-N87; and human
oesophageal squamous cell carcinoma cell line, KYSE-30. This study
was performed using proliferation assays for DLD-1; HCT-116;
NCI-N87; and KYSE-30.
[0305] The .gamma.-cyclodextrin encapsulated Brazilian-type
propolis used in the assays at a concentration of 200 .mu.g/ml was
manufactured as per Example 1, test substance CD8. The same test
procedure given in detail in Example 5 was also used for Example
8.
Results
[0306] The results of the antiproliferation assays for
.gamma.-cyclodextrin encapsulated Brazilian green-type propolis are
shown in Table 13. The results for the cells only (negative
control), and for the 5-FU positive controls are the same as for
Example 5 and so are not included in the table.
TABLE-US-00013 TABLE 13 Antiproliferative activity of CD8
.gamma.-cyclodextrin encapsulated Brazilian green-type propolis
against 4 gastro-intestinal cell lines Mean OD % Cell type (570 nm)
SEM P values (<0.05) % stim. inhibition DLD-1 0.3347 0.0128
8.50E-05 0 18.82 HCT-116 0.3847 0.02 8.40E-03 0 24.34 NCI-N87
0.4566 0.0132 >0.05 3.39 0 KYSE-30 0.4733 0.012 4.80E-04 0
21.35
Discussion
[0307] CD8 .gamma.-cyclodextrin was moderately to weakly active
against three of the four cell lines, resulting in inhibition of
proliferation of human colon adenocarcinoma cell line DLD-1 by
18.8%, human colon cancer cell line HCT-116 by 24.3%, and human
oesophageal squamous cell carcinoma cell line KYSE-30 by 21.3% The
proliferation of human gastric carcinoma cell line NCI-N87 was
stimulated by 3.4%, but the stimulation was not significant.
[0308] The degree of inhibition was similar to that achieved using
the known anticancer agent 5-fluororacil (5-FU) except against
NCI-N87, where no effect was seen. The results demonstrate that the
.gamma.-cyclodextrin encapsulated Brazilian green-type propolis has
relatively broad spectrum anti-proliferative activity for
gastrointestinal cancers, but the activity is much lower than that
of .gamma.-cyclodextrin encapsulated European-type propolis.
Example 9
[0309] This example provides a comparative assessment of the
anti-oesophageal cancer activity of European-type NZ cyclodextrin
encapsulated propolis CD8 with Brazilian green
cyclodextrin-encapsulated propolis CD9; and a comparative
assessment of the parent NZ propolis dry solids to Brazilian green
propolis solids; and to gamma-cyclodextrin alone. This study was
performed using proliferation assays for the human oesophageal
squamous cell carcinoma cell line, KYSE-30 using the general method
outlined in Example 5. The NZ propolis was the same as used in
Example 1 to manufacture complex CD8. The Brazilian green propolis
was the same as used in Example 1 to manufacture complex CD9. The
NZ propolis contained 5.81 mg/g CAPE, 20.92 mg/g pinobanksin, 43.84
mg/g pinobanksin 3 acetate, 85.17 mg/g pinocembrin, 27.60 mg/g
chrysin and 39.74 mg/g galangin. The Brazilian propolis contained
8.36 mg/g artepillin-c, 0.26 mg/g caffeic acid, and 4.31 mg/g
para-coumaric acid
Results
[0310] The results of the KYSE-30 cell proliferation assay are
shown in Table 14 for the compounds and propolis tincture samples
tested, along with results for the positive control 5-fluorouracil
(5-FU) at three concentrations, and the negative control (cells
only with medium and no test compound or positive control). In the
Table, n is the number of replicates; OD is Optical Density
measured at 570 nm; SEM is the Standard Error associated with the
Mean Optical Density value measured; p is the probability value
that the measurement is statistically significant via the Student
t-test, here taken to be <0.05 (NS=not significant); %
inhibition is the percentage reduction of proliferation compared to
the negative control, with a large number indicating the test
compound has anticancer proliferation potential.
TABLE-US-00014 TABLE 14 Antiproliferative activity of
cyclodextrin-encapsulated propolis CD8 and CD9 and propolis dry
solids against oesophageal squamous cell carcinoma cell line
KYSE-30 Mean OD P values Compound Concentration (570 nm) SEM
(<0.05) % inhibition Cells only (n = 6) 0.3353 0.0076 1 0 5-FU
(n = 6) 0.195 .mu.g/ml 0.3238 0.0202 NS 3.41 5-FU (n = 6) 0.65
.mu.g/ml 0.2795 0.0100 1.26E-03 16.63 5-FU (n = 6) 1.95 .mu.g/ml
0.2297 0.0052 4.60E-07 31.49 NZ propolis dry solids (n = 6) 200
.mu.g/ml 0.2358 0.0077 3.57E-06 29.67 Brazilian green propolis (n =
6) 50 .mu.g/ml 0.2989 0.0131 3.72E-02 10.86 CD8 Gamma-CD complex NZ
200 .mu.g/ml 0.2307 0.0079 2.55E-06 31.18 propolis (n = 6) CD9
Gamma-CD complex 200 .mu.g/ml 0.3008 0.0138 NS 10.27 Brazilian
propolis (n = 6) Gamma-CD (n = 6) 200 .mu.g/ml 0.3056 0.0219
2.29E-01 8.85
Discussion
[0311] This example shows that gamma-cyclodextrin encapsulated NZ
propolis is more active (31.2% inhibition of proliferation) than
the parent NZ propolis dry solids (29.7% inhibition of
proliferation), despite only containing approximately 1/4 of the
propolis dry solids. The activity is similar to the known
anticancer agent 5-fluororacil (5-FU) at the highest test
concentration. The results are very similar to that found for the
inhibition of proliferation of colon carcinoma cell line DLD-1 in
Example 2 by gamma-cyclodextrin encapsulated NZ propolis and NZ
propolis dry solids. The gamma cyclodextrin alone inhibited
proliferation by 8.8%.
[0312] These data support the enhanced efficacy of compositions of
the invention compared to propolis for anti-gastrointestinal
cancer. Without wishing to be bound by any theory, the applicants
suggest this may be due to an unexpected synergistic effect on
gastrointestinal cancer cell proliferation exhibited by the NZ
propolis and cyclodextrin composition. In contrast, the Brazilian
green gamma-cyclodextrin propolis complex has about the same level
of activity as the equivalent amount of Brazilian green propolis
dry solids, and only slightly higher activity than gamma
cyclodextrin by itself against the proliferation of human
oesophageal squamous cell carcinoma cell line KYSE-30. These data
also show that for this cell line, European-type propolis is the
preferred type of propolis for encapsulation by cyclodextrin.
INDUSTRIAL APPLICABILITY
[0313] Anti-gastrointestinal cancer compositions of this invention
containing propolis or an extract or fraction thereof and
cyclodextrin can be used in consumer goods including foods and
beverages, medical devices, medical supplies, functional foods and
pharmaceuticals. Methods of using such compositions, for example in
the treatment of gastrointestinal cancer and symptoms thereof have
application in the medical field.
* * * * *