CN109310660B - Therapeutic compositions and uses thereof - Google Patents

Therapeutic compositions and uses thereof Download PDF

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CN109310660B
CN109310660B CN201780027190.8A CN201780027190A CN109310660B CN 109310660 B CN109310660 B CN 109310660B CN 201780027190 A CN201780027190 A CN 201780027190A CN 109310660 B CN109310660 B CN 109310660B
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propolis
glycerol
diacetoxy
composition
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CN109310660A (en
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O·J·卡奇普尔
S·J·布卢尔
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Manuka Health New Zealand Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/76—Salicaceae (Willow family), e.g. poplar
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The present invention provides medicaments, including anti-epithelial cancer compositions, comprising glyceride compounds and compositions containing one or more of these compounds. The invention also provides methods of making and using these compositions, particularly for treating or preventing epithelial cancers, such as skin cancer, gastrointestinal cancer, and skin disorders.

Description

Therapeutic compositions and uses thereof
Technical Field
The present invention relates to compositions for skin and intestinal health care and for the treatment and prevention of epithelial cancers, including skin cancers and gastrointestinal cancers. In particular, the invention relates to anti-epithelial cancer compositions comprising one or more glycerides containing dihydroxyfatty acids. Skin and intestinal tract health and anti-epithelial cancer compositions comprising one or more dihydroxyfatty acid compounds are particularly contemplated, as well as the use of these compositions in the treatment or prevention of skin disorders, digestive tract disorders, mucosal surface disorders, and epithelial cancers, including gastrointestinal cancers, such as colorectal, laryngeal, buccal and gastric cancers, and skin cancers, such as basal cell carcinoma, squamous cell carcinoma, and melanoma, among others.
Background
Epithelial cancers include some of the most frequent cancers worldwide. Colorectal cancer is reported to be the second and third most common cancer in women and men in developed countries, respectively. Colorectal cancer is more frequent in developed countries-the highest incidence in the united states, australia, europe and new zealand-10 times higher than in developing countries. While surgery may be effective, early findings are critical to positive surgical outcomes. Other therapies are directed primarily to life-prolonging and palliative treatment, as the efficacy of chemotherapy and radiation therapy in treating primary tumors or extra-lymph node metastases is currently controversial.
Laryngeal cancer, also known as esophageal, pharyngeal, or throat cancer, includes tumors that occur in the pharynx, nasopharynx, oropharynx, hypopharynx, larynx (larynx), or tonsil tissue. Treatment of laryngeal cancer includes surgery, radiation therapy or chemotherapy. Treatment of laryngeal cancer can damage the throat and can also alter the patient's diet, respiration, and sleep patterns. Currently, similar problems exist with cheek cancer treatments.
Among the deaths associated with cancer worldwide, gastric cancer is secondary. Since gastric cancer is not symptomatic in early stages, diagnosis is always delayed. The only way to treat gastric cancer is by surgical resection of the stomach (gastrectomy). Post-operative chemotherapy and radiation therapy may increase the chances of healing.
The incidence of skin cancer is also quite high, with more than 300 thousands of cases diagnosed annually in the united states alone. The incidence of skin cancer is particularly high in australia and new zealand, 4 times higher than in the united states. While surgery may be effective, early findings are critical to positive surgical outcomes. Other therapies are primarily directed at low risk diseases, as the efficacy of chemotherapy and radiation therapy in the treatment of primary tumors, particularly malignant melanoma or extra-lymph node metastasis, is currently controversial.
Thus, there is a need for anti-epithelial cancer compositions, including compositions suitable for treating or preventing skin cancer and/or gastrointestinal cancer, as well as compositions capable of supporting maintenance of anti-epithelial cancer activity or enhancing anti-epithelial cancer activity.
It is an object of the present invention to provide anti-epithelial cancer compositions for the treatment or prophylaxis of epithelial cancers (e.g. skin cancers, including basal cell carcinomas, squamous cell carcinomas and melanomas) and/or gastrointestinal cancers (e.g. colorectal cancers, laryngeal cancers, buccal cancers and gastric cancers), or at least to provide the public with a useful choice.
Disclosure of Invention
Accordingly, in a first aspect, the present invention relates to a method of treating or preventing epithelial cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I):
Figure GDA0004178698530000021
or a pharmaceutically acceptable salt or solvate thereof, wherein R 2 、R 3 、R 4 And R is 5 Is H or acetyl (CH) 3 CO-)，
R 6 Is H or CH 3 ，R 7 Is CH 3 Or C2-C6 saturated or unsaturated hydrocarbon, and
x and y are integers of 3 to 14 independently of each other,
provided that when R 6 When H, x+y is greater than 10 and less than or equal to 18, when R 6 Is CH 3 When x+y is greater than 9 and less than or equal to 17. In one embodiment, the invention is directed to a method of treating or preventing epithelial cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound selected from any one or more of the following compounds
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In one embodiment, the method comprises administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of an effective amount of at least one compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In one embodiment, the present invention is directed to a method of treating or preventing skin cancer in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising, consisting essentially of, or consisting of at least one compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following groups
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In another aspect, the present invention relates to a method of inhibiting epithelial neoplasia, epithelial tumor growth or epithelial tumor metastasis in a subject, comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In one embodiment, the present invention relates to a method of inhibiting neoplasia, growth or metastasis in a subject, comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
Or a pharmaceutically acceptable salt or solvate of any one of a) to u).
Another aspect relates to a method of inducing apoptosis in one or more tumor epithelial cells in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one selected compound selected from any one or more of the following
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In one embodiment, the method is a method of inducing apoptosis in one or more neoplastic skin cells in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following groups
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In one embodiment, apoptosis refers to apoptosis of basal carcinoma cells. In another embodiment, the apoptosis refers to apoptosis of squamous cancer cells. In yet another embodiment, the apoptosis refers to apoptosis of melanoma cells.
In another embodiment, the method is a method of inducing apoptosis of one or more tumor gastrointestinal cancer cells in an individual.
For example, apoptosis refers to apoptosis of colorectal, laryngeal, buccal or gastric cancer cells.
Another aspect relates to a method of modulating proliferation of one or more tumor epithelial cells in a subject, comprising administering to a subject in need thereof an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In one embodiment, the invention relates to a method of modulating proliferation of one or more neoplastic skin cells in an individual.
For example, in one embodiment, the adjustment fingers are reduced. Accordingly, the present invention relates to a method of reducing proliferation of one or more neoplastic skin cells in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound selected from any one or more of the following: 3, 8-dihydroxyeicosanoic acid, 1- (3, 8-dihydroxyeicosanoyl) glycerol, 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol, 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol, 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol, 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol, 3, 8-dihydroxyeicosanoyl methyl ester, 3, 8-dihydroxyheneicosanoic acid, 1- (3, 8-dihydroxyheneicosanoyl) glycerol, 1- (3, 8-dihydroxyheneicosanoyl) 2-acetoxyglycerol, 1- (3, 8-dihydroxyheneicosanoyl) 3-acetoxyglycerol, 1- (3, 8-dihydroxyheneicosanoyl) 2, 3-diacetoxy glycerol, 1- (3, 8-diacetoxy) 2, 3-diacetoxy glycerol, 3-dihydroxyhenicosanoyl) 2, 3-dihydroxyhenoyl 2, 8-dihydroxyhenicosanoyl 2, 8-dihydroxydodecanoyl 2, 3-dihydroxydodecanol, 3-dihydroxydodecanoyl 2, 8-dihydroxydodecanoyl glycerol 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol and methyl 3, 8-dihydroxybehenate.
In one embodiment, the proliferation refers to proliferation of basal cancer cells. In another embodiment, the proliferation refers to proliferation of squamous carcinoma cells. In yet another embodiment, the proliferation refers to proliferation of melanoma cells.
In one embodiment, the method is a method of modulating proliferation of one or more tumor gastrointestinal cancer cells in an individual. For example, the proliferation refers to proliferation of colorectal cancer cells, laryngeal cancer cells, buccal cancer cells, or gastric cancer cells.
Accordingly, the present invention relates to a method of reducing proliferation of one or more tumor gastrointestinal cancer cells in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound selected from any one or more of the following: 3, 8-dihydroxyeicosanoic acid, 1- (3, 8-dihydroxyeicosanoyl) glycerol, 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol, 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol, 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol, 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol, 3, 8-dihydroxyeicosanoyl methyl ester, 3, 8-dihydroxyheneicosanoic acid, 1- (3, 8-dihydroxyheneicosanoyl) glycerol, 1- (3, 8-dihydroxyheneicosanoyl) 2-acetoxyglycerol, 1- (3, 8-dihydroxyheneicosanoyl) 3-acetoxyglycerol, 1- (3, 8-dihydroxyheneicosanoyl) 2, 3-diacetoxy glycerol, 1- (3, 8-diacetoxy) 2, 3-diacetoxy glycerol, 3-dihydroxyhenicosanoyl) 2, 3-dihydroxyhenoyl 2, 8-dihydroxyhenicosanoyl 2, 8-dihydroxydodecanoyl 2, 3-dihydroxydodecanol, 3-dihydroxydodecanoyl 2, 8-dihydroxydodecanoyl glycerol 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol and methyl 3, 8-dihydroxybehenate.
Another aspect relates to a method of inducing apoptosis or modulating proliferation of one or more tumor epithelial cells, e.g., in vitro or in vitro, comprising contacting one or more cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In another aspect, the present invention relates to a composition for treating or preventing epithelial cancer in an individual, said composition comprising, consisting essentially of, or consisting of a therapeutically effective amount of a compound of formula (I):
Figure GDA0004178698530000111
or a pharmaceutically acceptable salt or solvate thereof, wherein R 2 、R 3 、R 4 And R is 5 Is H or acetyl (CH) 3 CO-)，
R 6 Is H or CH 3 ，R 7 Is CH 3 Or C2-C6 saturated or unsaturated hydrocarbon, and
x and y are integers of 3 to 14 independently of each other,
provided that when R 6 When H, x+y is greater than 10 and less than or equal to 18, when R 6 Is CH 3 When x+y is greater than 9 and less than or equal to 17.
In one embodiment, x=4, r6=h and y=9, 10, 11, 12 or 13.
For example, x=4, r 4 ，R 5 ；R 6 =h; y=10, 11 or 12.
Thus, in a further aspect, the present invention relates to an anti-epithelial cancer composition comprising, consisting essentially of, or consisting of at least one compound as defined herein, including a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following groups
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In a further aspect, the present invention relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of at least one compound as defined herein, including a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following groups
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In one embodiment, the composition is for use in treating or preventing epithelial cancer.
In one embodiment, the composition is for maintaining or improving skin health.
In one embodiment, the composition is for maintaining or improving intestinal health.
In another aspect, the present invention relates to a method for inhibiting epithelial neoplasia, preferably skin neoplasia, inhibiting epithelial tumor growth, preferably skin tumor growth, inhibiting epithelial tumor metastasis, preferably skin tumor metastasis, or treating or preventing epithelial cancer, preferably skin cancer, in a subject, the method comprising administering separately, simultaneously or consecutively an effective amount of a composition comprising, consisting essentially of, or consisting of a therapeutically effective amount of at least one compound as defined in the present invention, the at least one compound comprising a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
Another aspect relates to a method of increasing responsiveness of a subject to treatment of epithelial cancer comprising administering to the subject an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I), or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or a composition of the present invention.
In one embodiment, the epithelial cancer treatment refers to gastrointestinal cancer treatment. For example, gastrointestinal cancer treatment refers to colorectal cancer treatment, laryngeal cancer treatment, cheek cancer treatment, or gastric cancer treatment.
In one embodiment, the invention relates to a method of increasing responsiveness of an individual to treatment of skin cancer comprising administering to the individual a composition of the invention.
In one embodiment, the skin cancer treatment refers to basal cell carcinoma treatment. In another embodiment, the skin cancer treatment refers to squamous cell carcinoma treatment. In yet another embodiment, the skin cancer treatment refers to melanoma treatment.
Another aspect relates to a method of increasing the sensitivity of a subject to treatment of epithelial cancer comprising administering to the subject an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I), or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) of the invention, or a pharmaceutically acceptable salt or solvate thereof, or a composition of the invention.
Another aspect relates to a method of re-sensitizing one or more epithelial cancer cells that are resistant to treatment comprising administering an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I), or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) of the invention, or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the epithelial tumor refers to a tumor of the gastrointestinal tract. For example, a tumor of the gastrointestinal tract refers to a colorectal tumor, a throat tumor, an oral tumor, or a gastric tumor.
In one embodiment, the invention relates to a method of increasing the susceptibility of a skin tumor of an individual to cancer treatment comprising administering to the individual a composition of the invention.
In one embodiment, the skin cancer is basal cell carcinoma. In one embodiment, the skin cancer is squamous cell carcinoma. In yet another embodiment, the skin tumor is melanoma.
In another aspect, the invention relates to a method of re-sensitizing one or more epithelial cancer cells that are resistant to treatment comprising administering to the one or more epithelial cancer cells an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof or a composition according to the invention.
In one embodiment, the epithelial cancer cell comprises a tumor in the individual.
In one embodiment, the present invention relates to a method of re-sensitizing one or more epithelial cancer cells, preferably skin cancer cells that are resistant to treatment, comprising administering to the one or more epithelial cancer cells, preferably skin cancer cells, an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a composition according to the invention.
In one embodiment, the skin cancer cells comprise a tumor in an individual.
The present invention also relates to a method of at least partially reversing the resistance of tumor cells of a subject suffering from an epithelial cancer to the treatment of an epithelial cancer, the method comprising administering to the subject an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) of the present invention, or a pharmaceutically acceptable salt or solvate thereof, or a composition of the present invention.
In one embodiment, the method is a method of at least partially reversing the resistance of tumor cells of an individual having skin cancer to a cancer treatment, the method comprising administering to the individual an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, or a composition according to the invention.
The present invention also relates to a method for reversing, in whole or in part, the resistance of a patient with an epithelial cancer, preferably a patient with a skin cancer, to an epithelial cancer treatment, preferably a skin cancer treatment, comprising the step of administering to said patient an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) of the invention, or a pharmaceutically acceptable salt or solvate thereof, or a composition of the invention.
In another aspect, the invention provides a method of re-sensitizing one or more tumors in a patient with an epithelial cancer, the one or more tumors being resistant to, or predicted to be resistant to, an epithelial cancer treatment. The method comprises the step of administering to said patient an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) of the present invention, or a pharmaceutically acceptable salt or solvate thereof.
In one embodiment, the method is a method of re-sensitizing one or more tumors of a patient with skin cancer that are resistant to, or are expected to be resistant to, skin cancer treatment. The method comprises the step of administering to said patient an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a composition according to the invention.
In one embodiment, the tumor is resistant to chemotherapy.
In one embodiment, the individual is a human. In a further aspect, the present invention relates to a method of improving skin health or intestinal health comprising administering to a subject in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof or a composition according to the invention.
In one embodiment, the composition is a synergistic therapeutic composition. In one embodiment, the composition has a synergistic therapeutic effect.
In one embodiment, the composition comprises a compound of the invention and at least one additional therapeutic agent that produces a synergistic therapeutic effect that is greater than the effect of one alone or greater than the additive effect of one alone. For example, there is a greater impact on induction of apoptosis, survival or proliferation of epithelial cancer cells, such as survival or proliferation of skin cancer cells, re-sensitization to treatment, treatment or prevention of epithelial cancers, such as skin cancer, or responsiveness of an individual or tumor to a method of treatment. Without being bound by theory, the inventors believe that this enhanced effect may be due to the bioavailability of the composition, for example, by improving water dispersibility. In one embodiment, the compound and at least one other therapeutic agent may be administered in combination or sequentially for epithelial cancer treatment, e.g., skin cancer treatment, with the dosage or time of administration being reduced or increased as appropriate.
In one embodiment, the composition, e.g., a synergistic therapeutic composition, includes at least one other compound or extract of poplar propolis. For example, the composition further comprises at least one compound selected from pinosylvin, CAPE, chrysin, pinosylvin chalcone, homocurcumin, benzyl caffeic acid, benzyl ferulic acid, or caffeic acid.
Another aspect relates to at least one compound according to the invention, including a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the following
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
For the preparation of a medicament or composition for the purposes of the present invention.
In one embodiment, the pharmaceutical or composition of the invention for the purposes of the present invention is prepared with at least one other therapeutic agent.
Another aspect relates to compounds of the present invention, including compounds of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or compounds selected from any one or more of the following
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
For use in the manufacture of a medicament or composition for use with at least one other therapeutic agent for the purposes of the present invention, wherein the medicament or composition is formulated for separate, simultaneous or sequential administration of the at least one compound and the at least one other therapeutic agent.
In one embodiment, the drug or composition comprises cyclodextrin. In one embodiment, the medicament or composition comprises one or more dihydroxyfatty acid compounds described herein, such as a compound of formula (I) complexed with cyclodextrin.
In another embodiment, the compound is dissolved in a solvent or solvent mixture (e.g., ethanol/water, propanol/water, isopropanol/water, ethyl acetate, hydrocarbon solvents, edible oils, or propylene glycol).
In one embodiment, the medicament or composition is a medicament or composition to which has been added at least one compound selected from any one or more of the following groups
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt or solvate of any one of a) to u).
Another aspect relates to a composition for inhibiting epithelial neoplasia, epithelial tumor growth, epithelial tumor metastasis or treating or preventing epithelial cancer in a human subject; inducing apoptosis of one or more tumor epithelial cells in a human subject; increasing responsiveness of a human individual to treatment of epithelial cancer; increasing the sensitivity of an epithelial tumor in a human individual to treatment of an epithelial cancer; re-sensitizing one or more epithelial cancer cells in a human individual that are resistant to the treatment; at least partially reversing the resistance of tumor cells to treatment of an epithelial cancer in a human subject having the epithelial cancer; reversing, in whole or in part, resistance of an epithelial cancer patient to treatment of epithelial cancer; or re-sensitising one or more tumors of a human patient with an epithelial cancer that are resistant to, or are expected to be resistant to, an epithelial cancer treatment.
In one embodiment, the composition is for inhibiting skin tumor formation, skin tumor growth, skin tumor metastasis, or treating or preventing skin cancer in a human subject; inducing apoptosis of one or more tumor skin cells in a human subject; increasing responsiveness of a human individual to skin cancer treatment; increasing the sensitivity of a human individual skin tumor to skin cancer treatment; re-sensitizing one or more skin cancer cells of a human subject that are resistant to the treatment; at least partially reversing the resistance of tumor cells in a human subject suffering from skin cancer to treatment of skin cancer; reversing, in whole or in part, the resistance of a patient with skin cancer to treatment with skin cancer; or re-sensitising one or more tumors of a human patient with skin cancer that are resistant to, or are expected to be resistant to, skin cancer treatment.
In one embodiment, the invention relates to a composition for treating or preventing skin cancer.
In one embodiment, the composition further comprises cyclodextrin.
In one embodiment, the composition further comprises propolis, such as poplar-type propolis.
In one embodiment, the composition further comprises poplar extract, such as poplar leaf or bud exudates as described in embodiments of the present invention.
In various embodiments, the composition comprises propolis, such as poplar propolis, including propolis extract or component, propolis resin extract and/or poplar extract, wherein propolis, propolis resin extract and/or poplar extract is enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the composition further comprises propolis, propolis resin or propolis resin extract, and cyclodextrin.
In one embodiment, the cyclodextrin is α -cyclodextrin.
In one embodiment, the cyclodextrin is β -cyclodextrin.
In one embodiment, the cyclodextrin is gamma-cyclodextrin.
In one embodiment, the cyclodextrin is hydroxypropyl β -cyclodextrin.
In one embodiment, the cyclodextrin is hydroxypropyl gamma cyclodextrin.
In one embodiment, the cyclodextrin is a mixture of cyclodextrins.
In one embodiment, the anti-skin cancer composition is an anti-basal cell carcinoma composition. In another embodiment, the anti-skin cancer composition is an anti-squamous cell carcinoma composition. In yet another embodiment, the anti-skin cancer composition is an anti-melanoma composition.
In another aspect, the present invention relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the composition comprises propolis, propolis resin or propolis resin extract and one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, for example, the composition comprises propolis, propolis resin or propolis resin extract, to which one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof are added. In another embodiment, the composition comprises propolis and/or poplar bud or leaf exudates, propolis resin or propolis resin extract component enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the propolis is poplar type propolis.
In one embodiment, the composition further comprises cyclodextrin.
In one embodiment, the composition is for maintaining or improving skin health. Accordingly, in one embodiment, the present invention relates to a pharmaceutical composition for maintaining or improving skin health, the composition comprising or consisting essentially of propolis, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the propolis enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof is a propolis resin, such as a poplar type propolis resin. In one embodiment, the resin is a propolis resin, such as a poplar type propolis resin; the cyclodextrin is alpha, beta or gamma cyclodextrin.
In another aspect, the invention relates to a method of treating or preventing skin cancer in an individual, a method of inhibiting skin tumor formation, inhibiting skin tumor growth, or inhibiting skin tumor metastasis, a method of inducing apoptosis of one or more neoplastic skin cells in an individual, a method of modulating proliferation of one or more neoplastic skin cells in an individual, a method of increasing responsiveness of an individual to skin cancer treatment, a method of increasing sensitivity of a skin tumor in an individual to skin cancer treatment, a method of re-sensitizing one or more skin cancer cells resistant to treatment; the method comprises administering to a subject in need thereof or to the one or more cells an effective amount of a composition comprising, consisting essentially of, or consisting of propolis, a propolis resin or a propolis resin extract and cyclodextrin, enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the apoptosis refers to apoptosis of skin tumor cells, such as basal carcinoma, squamous carcinoma, or melanoma cells.
In one embodiment, the adjustment fingers are reduced. Accordingly, the present invention relates to a method of reducing proliferation of one or more neoplastic skin cells in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising or consisting essentially of propolis, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the proliferation refers to proliferation of skin tumor cells, such as basal carcinoma, squamous carcinoma, or melanoma cells.
In one embodiment, the skin cancer cells comprise a tumor in an individual. In one embodiment, the skin cancer cell is a basal cancer cell. In another embodiment, the skin cancer cell is a squamous cancer cell. In yet another embodiment, the skin cancer cell is a melanoma cell.
The present invention also relates to a method of at least partially reversing the resistance of tumor cells in a subject suffering from skin cancer to treatment of skin cancer, the method comprising administering to the subject a composition comprising, consisting essentially of, or consisting of propolis, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
The present invention also relates to a method of reversing, in whole or in part, the resistance of a patient with skin cancer to treatment with skin cancer, the method comprising the step of administering to said patient a composition comprising or consisting essentially of propolis, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In another aspect, the present invention provides a method of re-sensitizing one or more tumors in a patient suffering from skin cancer, said tumors being resistant to, or predicted to be resistant to, skin cancer treatment, said method comprising the step of administering to said patient a composition comprising, consisting essentially of, or consisting of propolis, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the one or more tumors are resistant to, or are expected to be resistant to, or develop resistance to, skin cancer treatment due to an increase in the activity of one or more pre-cancerous cell survival signaling pathways, including an increase in the activity of one or more AKT, JNK, or JAK/STAT signaling pathways, e.g., in a sample from the patient (e.g., a tissue sample, a tumor biopsy, or a blood or plasma sample).
In one embodiment, the invention provides a method of inactivating or inhibiting one or more pre-cancerous cell survival signaling pathways within the one or more tumors or within a patient. For example, the invention relates to a method of inactivating or inhibiting one or more AKT, JNK or JAK/STAT signaling pathways within said one or more tumors.
In one embodiment, the one or more tumors are resistant to, or are predicted to be resistant to, skin cancer treatment due to an enhancement in the activity of one or more of the AKT, JNK, or JAK/STAT signaling pathways within the tumor.
In one embodiment, the invention provides a method of preventing a tumor from developing resistance to a primary skin cancer treatment, wherein the resistance is mediated at least in part by, for example, an enhancement of the activity of one or more of the AKT, JNK or JAK/STAT signaling pathways within the tumor.
In one embodiment, the tumor is resistant to chemotherapy.
In one embodiment, the skin cancer refers to basal cell carcinoma. In another embodiment, the skin cancer is squamous cell carcinoma. In yet another embodiment, the skin cancer is melanoma.
In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, alpha-cyclodextrin (e.g., poplar propolis, propolis resin or propolis resin extract), and cyclodextrin (e.g., poplar propolis and alpha-cyclodextrin).
In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, beta-cyclodextrin (e.g., poplar propolis, propolis resin or propolis resin extract), and cyclodextrin (e.g., poplar propolis, propolis resin or propolis resin extract and beta-cyclodextrin).
In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, gamma-cyclodextrin (e.g., poplar propolis, propolis resin or propolis resin extract), and cyclodextrin (e.g., poplar propolis, propolis resin or propolis resin extract and gamma-cyclodextrin).
In another aspect, the present invention provides a synergistic composition comprising propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin. In one embodiment, the composition is a synergistic therapeutic composition. In one embodiment, the composition has a synergistic therapeutic effect.
In one embodiment, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof have a synergistic therapeutic effect, greater than the effect of one alone or greater than the additive effect of one alone. For example, to induce apoptosis, survival or proliferation of skin cancer cells, re-sensitization of treatment, treatment or prevention of skin cancer, or responsiveness of an individual or tumor to a method of treatment. In one embodiment, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof may be administered, co-administered or administered continuously for skin cancer treatment, with reduced or increased dosages or times as appropriate.
In another aspect, it relates to propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin, which can be used to prepare a composition for the purposes of the present invention.
In another aspect, it relates to propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin, which can be used with at least one other therapeutic agent for the preparation of a composition for the purposes of the present invention. In one embodiment, concentrated propolis, propolis resin or propolis resin extract and alpha-cyclodextrin are used. In one embodiment, concentrated propolis, propolis resin or propolis resin extract and beta-cyclodextrin are used. In one embodiment, concentrated propolis and gamma cyclodextrin are used.
Another aspect of the invention relates to a complex comprising propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin, said complex being used together with at least one other therapeutic agent for the preparation of a composition for the purposes of the invention, wherein said composition is formulated for separate, simultaneous or sequential administration with propolis, propolis resin or propolis resin extract, cyclodextrin complex and at least one other therapeutic agent.
Another aspect relates to a composition for improving skin health comprising, consisting essentially of, or consisting of propolis, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin or propolis resin extract and alpha-cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin or propolis resin extract and gamma-cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In another aspect, the present invention relates to a composition for treating or preventing skin cancer in an individual, the composition comprising or consisting essentially of propolis, propolis resin or propolis resin extract and cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin or propolis resin extract and alpha-cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin or propolis resin extract and gamma-cyclodextrin enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
Another aspect relates to a product comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof enriched in cyclodextrin and, optionally, one or more, two or more or three or more other therapeutic agents for simultaneous, separate or sequential administration as a combined preparation according to the objects of the invention.
Another aspect relates to a composition for treating or preventing skin cancer.
One aspect relates to a process for the isolation or purification of a compound or a mixture of compounds of formula (I) from propolis or poplar, propolis resin or extracts or exudates thereof, comprising the steps of
Providing poplar, propolis resin or extract or exudate thereof, and
separating or purifying a compound or mixture of compounds from poplar, propolis resin or an extract or exudate thereof.
In various embodiments, the method includes one or more of the following non-limiting steps:
a) Fractionating poplar, propolis resin or an extract or exudate thereof by chromatography (e.g. column chromatography, reverse phase chromatography, normal phase chromatography or supercritical fluid chromatography, and/or solvent separation and/or supercritical extraction) to produce one or more components comprising one or more compounds of formula (I),
b) Fractionation of poplar, propolis resin or extracts or exudates thereof or one or more components by preparative HPLC and/or polymeric resin separation to produce one or more components comprising one or more compounds of formula (I), and optionally,
c) Further purifying the one or more compounds of formula (I) by one or more components of step b) and/or step c).
In another embodiment, the poplar, propolis resin or extract or exudate may be subjected to hydrolysis, methylation or acetylation reactions, followed by one or more of steps a), b) or c) to isolate and purify one or more compounds of formula (I).
Another aspect relates to markers of anti-epithelial cancer efficacy comprising compounds of formula (I).
Another aspect relates to a method of evaluating the efficacy of a composition or product in treating epithelial cancer comprising
a) Providing a sample of said composition or product, and
b) Or (b)
i) Determining the presence or absence of one or more compounds of formula (I) in a sample, or
ii) measuring the amount and/or concentration of one or more compounds of formula (I) in the sample and determining whether the amount and/or concentration of one or more compounds in the sample is equal to or greater than a reference amount of a known compound having anti-epithelial cancer efficacy, and optionally,
iii) The biological activity of a compound and/or a component containing the compound is determined using in vitro or in vivo bioassays.
Another aspect relates to a method of evaluating the efficacy of a composition or product in treating epithelial cancer comprising
a) Receiving information about the presence and/or amount of one or more compounds of formula (I) in a sample of the composition or product, and
b) Determining whether the amount of each compound in the sample is equal to or greater than a reference amount of a known compound having anti-epithelial cancer efficacy.
Another aspect relates to a method of evaluating the efficacy of a composition or product in treating epithelial cancer comprising
a) Determining the presence and/or amount of one or more compounds of formula (I) in a composition or product sample using gas, liquid or supercritical fluid chromatography, and
b) Determining whether the amount of each compound in the sample is equal to or greater than a reference amount of a known compound having anti-epithelial cancer efficacy.
In various embodiments, the method of evaluating the anti-epithelial cancer efficacy of a composition or product comprises any one or more of the following steps:
a) Fractionating the sample by solvent separation and/or supercritical extraction and/or chromatography (e.g., reverse phase chromatography) to produce one or more components enriched in one or more compounds of formula (I),
b) Further fractionating the one or more components enriched in the one or more compounds of formula (I) by one or more solvents or supercritical extraction or chromatography to produce one or more components enriched in the one or more compounds of formula (I),
c) The highly concentrated component is analyzed, for example, by one or more gas, liquid or supercritical chromatography, to determine whether one or more compounds of formula (I) and/or the amounts thereof are present in the sample.
In various embodiments, the method of evaluating the anti-epithelial cancer efficacy of a composition or product comprises any one or more of the following steps:
a. fractionating the sample by chromatography (e.g., reverse phase chromatography) to produce one or more chromatographic components,
b. fractionating the sample or chromatographic component by preparative HPLC to produce one or more HPLC components,
c. analyzing trace amounts produced by preparative HPLC to determine the presence and/or amount of a compound in a sample, and/or
d. Mass spectrometry is performed on the sample, one or more reversed phase chromatographic components, or one or more HPLC components to determine whether one or more compounds of formula (I) and/or the amounts thereof are present in the sample.
In one embodiment, the presence and/or amount of one or more compounds of formula (I) in a sample is determined by mass spectrometry, such as liquid chromatography mass spectrometry (LC-MS).
In one aspect, the invention relates to a composition or product comprising one or more compounds of formula (I), wherein the amount and/or concentration of the one or more compounds of formula (I) is specified by an indication, such as, but not limited to, a product label, an assay certificate, a website, promotional material associated with the composition or product.
In various embodiments, the compound of formula (I) is selected from the group consisting of
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate.
In one embodiment, the composition or product is or includes propolis, propolis resin or propolis resin extract, for example propolis, propolis resin or propolis resin extract that includes or is enriched in one or more compounds of formula (I).
In one embodiment, the indication associated with the composition or product refers to a label or package insert.
In one embodiment, the indication associated with the composition or product refers to a certificate of assay (COA).
In one embodiment, the indication associated with the composition or product refers to a website advertising the product.
In one embodiment, the indication associated with the composition or product refers to a booklet advertising the product.
The following embodiments may relate to any of the above aspects.
In various embodiments, the epithelial cancer refers to an epidermoid cancer or epidermoid carcinoma.
In various embodiments, the epithelial cancer refers to a gastrointestinal cancer, such as colorectal cancer, laryngeal cancer, esophageal cancer, cheek cancer, or gastric cancer. For example, in one embodiment, the colorectal cancer refers to colon adenocarcinoma. In another embodiment, the esophageal cancer refers to esophageal squamous carcinoma. In another embodiment, the gastric cancer refers to a gastric tumor.
In other embodiments, the epithelial cancer refers to skin cancer, such as basal cell carcinoma, squamous cell carcinoma, or melanoma.
In various embodiments, the composition further comprises one or more of Caffeic Acid Phenethyl Ester (CAPE), caffeic acid, pinocembrin, benzyl caffeic acid, benzyl ferulic acid, chrysin, cinnamic acid, pinocembrin chalcone, homocurcumin, and pinocembrin alcohol.
In exemplary embodiments, the composition incorporates one or more of CAPE, caffeic acid, pinocembrin, benzyl caffeic acid, benzyl ferulic acid, isophthalic ferulic acid, cinnamic acid, pinocembrin chalcone, chrysin, homohaemagglutinin, and pinocembrin alcohol-3-acetate.
In one embodiment, the CAPE concentration of the composition is 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, or 600mg/g, and an effective range may be selected from any of these values (e.g., from about 1 to about 5, from about 1 to about 10, from about 2 to about 20, from about 5 to about 25, from about 10 to about 40, from about 15 to about 100, or from about 20 to about 600 mg/g).
In one embodiment, the composition has a pinosylvin concentration 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, or 600mg/g, and an effective range may be selected from any of these values (e.g., about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In one embodiment, the composition has a high good curcumin 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, or 600mg/g, and an effective range may be selected from any of these values (e.g., about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In one embodiment, the chrysin concentration of the composition is 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, or 600mg/g, and the effective range may be selected from any of these values (e.g., about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In one embodiment, the pinosylvin alcohol concentration of the composition is 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, or 600mg/g, and the effective range may be selected from any of these values (e.g., about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In one embodiment, the composition has a caffeic acid concentration 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, or 600mg/g, an effective range being selectable from any of these values (e.g., about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In one embodiment, the composition has a concentration of benzyl caffeic acid 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, or 600mg/g, an effective range being selectable from any of these values (e.g., about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In one embodiment, the composition has a benzyl ferulic 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, or 600mg/g, and an effective range may be selected from any of these values (e.g., about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In one embodiment, the composition has a cinnamic 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, or 600mg/g, and an effective range may be selected from any of these values (e.g., from about 1 to about 5, from about 1 to about 10, from about 2 to about 20, from about 5 to about 25, from about 10 to about 40, from about 15 to about 100, or from about 20 to about 600 mg/g).
In one embodiment, the composition has a cinnamic ferulic 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, or 600mg/g, and an effective range can be selected from any of these values (e.g., about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In various embodiments, the cyclodextrin is an alpha-cyclodextrin, or the cyclodextrin is present as a combination of cyclodextrins including an alpha-cyclodextrin.
In various embodiments, the cyclodextrin is a β -cyclodextrin, or the cyclodextrin is present as a combination of cyclodextrins including β -cyclodextrin.
In various embodiments, the cyclodextrin is a gamma-cyclodextrin, or the cyclodextrin is present as a combination of cyclodextrins including gamma-cyclodextrin.
In one embodiment, the cyclodextrin refers to a modified cyclodextrin, examples of which are shown in stilla He, toxicology pathology, 2008, 36 th edition, pages 30-42.
In one embodiment, the modified cyclodextrin is hydroxypropyl beta cyclodextrin or hydroxypropyl gamma cyclodextrin.
In one embodiment, the propolis is present in the anti-epithelial cancer composition as a propolis extract or component.
In one embodiment, the propolis contained in the anti-epithelial cancer composition is free of wax. For example, propolis has been dewaxed using extraction methods known in the art. Dewaxed propolis is generally known or referred to as "propolis resin".
In one embodiment, the propolis is a "poplar" or "poplar" propolis. For example, "poplar" propolis is derived at least in part from the bud and leaf exudates of one or more of poplar, birch, larch or willow.
In one embodiment, the composition comprises from about 1.0% wt to about 99% wt propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I). In one embodiment, the composition comprises 1.0% wt to about 99% wt propolis resin enriched in one or more compounds of formula (I).
In various embodiments, the composition comprises from about 1% wt to about 99% wt propolis, a propolis resin or a propolis resin extract enriched in one or more compounds of formula (I), from about 1% wt to about 25% wt propolis, a propolis resin or a propolis resin extract enriched in one or more compounds of formula (I), from about 1% wt to about 30% wt propolis, a propolis resin or a propolis resin extract enriched in one or more compounds of formula (I), from about 1% wt to about 40% wt propolis, a propolis resin or a propolis resin extract enriched in one or more compounds of formula (I), from about 1% wt to about 50% wt propolis, from about 5% wt to about 25% wt propolis, from about 5% wt to about 25% propolis resin extract enriched in one or more compounds of formula (I), from about 5% to about 5% wt to about 50% propolis resin extract, from about 5% wt to about 5% propolis resin extract, from about 5% to about 50% propolis resin extract, from about 5% to about 5% wt to about 5% propolis resin extract, from about 50% to about 5% propolis resin extract from about 5% to about 5% propolis resin extract from about 50% to about 5% by weight of one or more compounds of formula (I), about 10% to about 30% by weight of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I), about 10% to about 40% by weight of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I), about 10% to about 50% by weight of propolis, propolis resin or propolis resin extract enriched in about 10% to about 99% by weight of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I), about 15% to about 25% by weight of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I), about 15% to about 40% by weight of propolis enriched in one or more compounds of formula (I), about 15% to about 50% by weight of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I), about 15% to about 99% by weight of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I), about 20% to about 25% by weight of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I), about 20% to about 30% by weight of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I), about 20% to about 40% by weight of propolis, or propolis extract enriched in one or more compounds of formula (I), propolis resin or propolis resin extract, is enriched in about 20% wt to about 50% wt of propolis, propolis resin or propolis resin extract of one or more compounds of formula (I), is enriched in about 20% wt to about 99% wt of propolis, propolis resin or propolis resin extract of one or more compounds of formula (I), is enriched in about 25% wt of propolis, propolis resin or propolis resin extract of one or more compounds of formula (I), or is enriched in about 30% wt of propolis, propolis resin or propolis resin extract of one or more compounds of formula (I).
In various embodiments, the composition comprises from about 1% to about 99% by weight of the propolis resin enriched in the one or more compounds of formula (I), from about 1% to about 25% by weight of the propolis resin enriched in the one or more compounds of formula (I), from about 1% to about 30% by weight of the propolis resin enriched in the one or more compounds of formula (I), from about 1% to about 40% by weight of the propolis resin enriched in the one or more compounds of formula (I), from about 1% to about 50% by weight of the propolis resin enriched in the one or more compounds of formula (I), from about 5% to about 30% by weight of the propolis resin enriched in the one or more compounds of formula (I), from about 5% to about 40% by weight of the propolis resin enriched in the one or more compounds of formula (I), from about 10% to about 10% by weight of the propolis resin enriched in the one or more compounds of formula (I), from about 5% to about 50% by weight of the propolis resin enriched in the one or more compounds of formula (I) to about 25% by weight of the propolis resin enriched in the one or more compounds of formula (I), about 10% wt to about 99% wt of the propolis resin enriched in the one or more compounds of formula (I), about 15% wt to about 25% wt of the propolis resin enriched in the one or more compounds of formula (I), about 15% wt to about 30% wt of the propolis resin enriched in the one or more compounds of formula (I), about 15% wt to about 40% wt of the propolis resin enriched in the one or more compounds of formula (I), about 15% wt to about 50% wt of the propolis resin enriched in the one or more compounds of formula (I), about 15% wt to about 99% wt of the propolis resin enriched in the one or more compounds of formula (I), about 20% wt to about 25% wt of the propolis resin enriched in the one or more compounds of formula (I), about 20% wt to about 30% wt of the propolis resin enriched in the one or more compounds of formula (I), about 40% wt to about 20% wt to about 40% wt of the propolis resin enriched in the one or more compounds of formula (I), about 20% to about 20% wt to about 30% wt of the propolis resin enriched in the one or more compounds of formula (I).
In one embodiment, the propolis, propolis resin or propolis resin extract in the composition is completely contained within cyclodextrin.
In one embodiment, the molar ratio of propolis, propolis resin or propolis resin extract to cyclodextrin in the composition is no greater than about 1:1.
In one embodiment, the propolis, propolis resin or propolis resin extract is poplar type propolis, propolis resin or propolis resin extract.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract comprises and/or is enriched in 3, 8-dihydroxyeicosanoic acid. In one embodiment of the present invention, in one embodiment, the poplar type propolis, propolis resin or propolis resin extract comprises and/or is rich in 1- (3, 8-dihydroxyeicosanoyl) glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is rich in methyl 3, 8-dihydroxyeicosanoate. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 3, 8-dihydroxyundecanoic acid. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxydi-undecanoyl) glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxyglycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxydi-undecanoyl) 2, 3-diacetoxy glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is rich in methyl 3, 8-dihydroxyundecanoate. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 3, 8-dihydroxybehenic acid. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxybehenoyl) glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is enriched in 1- (3, 8-diacetoxy-behenyl) 2, 3-diacetoxy-glycerol. In one embodiment, the poplar-type propolis, propolis resin or propolis resin extract comprises and/or is rich in methyl 3, 8-dihydroxybehenate.
In one embodiment, the poplar-type propolis has any one or more of compounds a) to u) of the present invention at a concentration of greater than about 1mg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 3.5mg/kg, about 4mg/kg, about 4.5mg/kg, about 5mg/kg, about 5.5mg/kg, about 6mg/kg, about 7.5mg/kg, about 10mg/kg, about 15mg/kg, about 20mg/kg, about 25mg/kg, about 30mg/kg, about 40mg/kg, about 50mg/kg, about 75mg/kg, about 100mg/kg, about 125mg/kg, about 150mg/kg, about 175mg/kg, about 200mg/kg, 250mg/kg, about 300mg/kg, about 350mg/kg, about 400mg/kg, about 450mg/kg, about 500mg/kg, about 600mg/kg, or about 600 mg.
In some embodiments, the poplar propolis, propolis resin or propolis resin extract has any two or more, three or more, four or more, of compounds a) to u) of the present invention of at least about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 7.5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000mg/g; the effective range may be selected from any of these values (e.g., about 1 to about 400, about 1 to about 100, about 5 to about 1000, about 5 to about 500, about 5 to about 300, about 5 to about 100). For example, in some embodiments, the poplar propolis has 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol and 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol, or 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol, 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol and 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol, or 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol, and a concentration of about 1mg/g to 1000mg/g of any one or more of compounds a) to c), e) to j) or l) to u).
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg, about 550mg, about 600mg or about 600mg of dihydroxyalkane.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg, about 550mg/g, about 550mg, about 600mg or about 1-to about 7.5mg of dihydroxypropane.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg, about 500mg of acetyl triol, or about 2-2 mg of hydroxyl groups.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg, about 3-to about 500mg of acetyl triol, or about 1-2 mg.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg, about 3-2 mg of acetyl glycerol, or about 2-hydroxy.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg of diacetyl triol, or about 2.8 mg of acetyl.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg, about 550mg, about 600mg or about 8 mg of the dihydroxymethane ester.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg or about 8 mg of dihydroxyalkane.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 500mg, about 8 mg of dihydroxyglycerol or about 1-8 mg.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 500mg of diacetyl triol, or about 2 mg-1.8 mg of dihydroxyl.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 3-undecyl glycerol, or about 3-3 mg.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 3-undecyl glycerol, 2-1, 2mg or about 3-2 mg.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg of diacetyl triol, 2-acetyl, 3-2 mg or about 3-acetyl triol.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg or about 8 mg of the dihydroxymethyl ester.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg, about 550mg, about 600mg or about 600mg of dihydroxyalkane.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg, about 550mg, about 600mg of dihydroxyglycerol or about 1.8 mg.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg of diacetyl triol, or about 2 mg.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg of diacetyl triol, or about 3-2 mg.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg, about 2mg of diacetyl triol, or about 3-2 mg.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg of diacetyl triol, 2-2 mg, or about 2mg of diacetyl.
In one embodiment, the poplar propolis, propolis resin or propolis resin extract has a concentration of at least about 0.1mg/g, about 0.5mg/g, about 1mg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about 10mg/g, about 15mg/g, about 20mg/g, about 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about 100mg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g,250mg/g, about 300mg/g, about 350mg/g, about 400mg, about 450mg, about 550mg, about 600mg, or about 600mg of dihydroxymethyl ester.
In various embodiments, the poplar propolis, propolis resin or propolis resin extract further comprises any combination of two or more of CAPE, chrysin, homocurcumin, pinocembrin alcohol, benzyl caffeic acid, benzyl ferulic acid, benzyl isoferulic acid, cinnamic caffeic acid, cinnamic ferulic acid, pinocembrin chalcone, and caffeic acid.
In various embodiments, the composition comprises at least one additional therapeutic agent, preferably an anti-tumor agent, preferably selected from an anti-tumor food factor, a chemotherapeutic agent or an immunotherapeutic agent, or is administered alone, simultaneously or sequentially with at least one additional therapeutic agent.
In various embodiments, apoptosis is effectively induced by a skin cancer treatment, therapeutic agent, or anti-neoplastic agent, e.g., induction of apoptosis of one or more skin cancer cells or one or more tumor cells.
In one embodiment, the composition is a consumable.
In one embodiment, the composition is a composition for topical administration.
In one embodiment, the topical composition includes one or more of a soaking agent, a photoprotectant, a uv protectant, a vitamin, a humectant, an oil, a hydrophilic or lipophilic gelling agent, a hydrophilic or lipophilic active agent, a preservative, an antioxidant, a solvent, a fragrance, a filler, a pigment, a deodorizing agent, or a dye.
In one embodiment, the composition is a medicament or drug.
In various embodiments, the compositions are formulated for oral, topical, or parenteral administration.
In one embodiment, a composition formulated for oral administration includes gamma cyclodextrin.
In one embodiment, a composition formulated for topical administration includes an α -cyclodextrin.
In one embodiment, a composition formulated for topical administration includes beta-cyclodextrin.
In one embodiment, the composition includes one or more additional anti-epithelial cancer agents.
In one embodiment, the composition is a pharmaceutical composition.
In various embodiments, the chemotherapeutic agent is selected from the group consisting of: mitotic inhibitors, such as vinca alkaloids, including vincristine, vinblastine, vinorelbine, vindesine, vinflunine, podophyllotoxin, paclitaxel (including docetaxel, ralostazol, ostazol, paclitaxel, and tesetaxel), and epothilones, such as ixabepilone; topoisomerase I inhibitors such as topotecan, irinotecan, camptothecin, lubitecan and belotecan, topoisomerase II inhibitors including amsacrine, etoposide phosphate and teniposide, anthracyclines such as aclarubicin, daunorubicin, doxorubicin, epirubicin, huang Dansu, amrubicin, pirarubicin, valrubicin and zorubicin, and anthracenedione drugs such as mitoxantrone and pitaxron; antimetabolites including dihydrofolate reductase inhibitors such as aminopterin, methotrexate, pemetrexed, thymidylate synthase inhibitors such as raltitrexed and pemetrexed, adenosine deaminase inhibitors including pentososides, halo or ribonucleotide reductase inhibitors such as cladribine, clofarabine and fludarabine, thiopurines including thioguanine and mercaptopurine, thymidylate synthase inhibitors including fluorouracil, capecitabine, tegafur, carmofur and fluorouridine, DNA polymerase inhibitors such as cytarabine, ribonucleotide reductase inhibitors such as gemcitabine, hypomethylation drugs including azacytidine and decitabine, and ribonucleotide reductase inhibitors such as hydroxyurea; cell cycle non-specific antineoplastic agents including alkylating agents such as nitrogen mustard including dichloromethyldiethylamine, cyclophosphamide, ifosfamide, chlorocyclophosphamide, chlorambucil, phenylalanine, prednisone mustard, bendamustine, uracil mustard, estramustine, nitrosoureas including carmustine, lomustine, semustine, fotemustine, nimustine, ramustine and streptozotocin, alkyl sulfonates including busulfan, mannosulfan and troxosul, aziridines including carboquinone, thiotepa, triamcinolone and trotaamine, alkylating agents including platinoids such as cisplatin, carboplatin, oxaliplatin, nedaplatin, trinuclear platinum 4 nitrate, satraplatin, hydrazines such as methylbenzyl hydrazine, triazenes such as dacarbazine, temozolomide, hexamethyl and dibromomannitol, and streptomycin such as dactinomycin, lymycin, nuhomomycin and mitomycin; photosensitizers including aminolevulinic acid, methylaminoketovalerate, etoricoxil, and porphyrin derivatives such as porphin sodium, talaporfin, temoporfin, and verteporfin; enzyme inhibitors, including farnesyl transferase inhibitors such as tepirfenib, cyclin dependent kinase inhibitors such as alvocidib and selicillib, proteasome inhibitors such as bortezomib, phosphodiesterase inhibitors such as anagrelide, IMP dehydrogenase inhibitors such as thifluzaine, lipoxygenase inhibitors such as masoprocol, and PARP inhibitors such as olapanib; receptor antagonists, such as endothelin receptor antagonists, including atrasentan, retinoid X receptor antagonists, such as besartan and testicle lactone; and other chemotherapeutic agents including amsacrine, trabectedin, retinoids such as aliskiric acid and tretinoin, arsenic trioxide, asparagine consuming agents such as asparaginase or pergolidase, celecoxib, dimecoxin, illitemo, elsamitrucin, etodolac and lonidamine.
All references to a range of numbers (e.g., 1 to 10) disclosed herein are intended to be incorporated by reference to all rational numbers (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) within that range, as well as any range of rational numbers (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7) within that range; accordingly, all subranges from all ranges explicitly disclosed herein are also explicitly disclosed. These are only examples of what is specifically intended, and all possible combinations of numerical values between the minimum and maximum values listed are to be considered as being expressly stated in this application in a similar manner.
Some patent specifications, other external documents or other sources of information are also incorporated into this specification, primarily by reference in the context of 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 sources of information, in any jurisdiction, are prior art, or are part of the common general knowledge in the art.
The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the invention, individually or collectively, and any or all combinations of two or more of said parts, elements and features, and if specific integers are mentioned herein which have known equivalents in the art to which the invention relates, then such known equivalents are incorporated herein as if individually set forth.
Detailed Description
The present invention relates to fatty acid compounds and compositions comprising these dihydroxyfatty acid glycerides having anti-epithelial cancer activity. Pharmaceutical compositions, such as anti-epithelial cancer compositions, have and/or may enhance the efficacy against epithelial cancer, and in certain embodiments may enhance the activity and physicochemical properties of such compositions including such compounds, such as propolis and poplar extracts. Nutritional compositions, such as skin and intestinal health compositions, have health care value. In certain embodiments, the activity and physicochemical properties of these compositions are enhanced, for example, in the presence of propolis or propolis extract or component or poplar extract or component.
In one aspect, the present invention relates to a method of treating or preventing epithelial cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I):
Figure GDA0004178698530000351
or a pharmaceutically acceptable salt or solvate thereof, wherein R 2 、R 3 、R 4 And R is 5 Is H or acetyl (CH) 3 CO-)，
R 6 Is H or CH 3 ，R 7 Is CH 3 Or C2-C6 saturated or unsaturated hydrocarbon, and
x and y are integers of 3 to 14 independently of each other,
provided that when R 6 When H, x+y is greater than 10 and less than or equal to 18, when R 6 Is CH 3 When x+y is greater than 9 and less than or equal to 17.
In one embodiment, x is greater than or equal to 4. In one embodiment, the invention is directed to a method of treating or preventing epithelial cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a therapeutically effective amount of at least one compound selected from any one or more of the group,
a) 3, 8-dihydroxyeicosanoic acid,
Figure GDA0004178698530000352
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol
Figure GDA0004178698530000353
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
Figure GDA0004178698530000361
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
Figure GDA0004178698530000362
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
Figure GDA0004178698530000363
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
Figure GDA0004178698530000364
g) Methyl 3, 8-dihydroxyeicosanoate,
Figure GDA0004178698530000365
h) 3, 8-dihydroxydi-undecanoic acid,
Figure GDA0004178698530000366
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol
Figure GDA0004178698530000367
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
Figure GDA0004178698530000368
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
Figure GDA0004178698530000371
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
Figure GDA0004178698530000372
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
Figure GDA0004178698530000373
n) methyl 3, 8-dihydroxyundecanoate,
Figure GDA0004178698530000374
o) 3, 8-dihydroxybehenic acid,
Figure GDA0004178698530000375
p) 1- (3, 8-dihydroxybehenoyl) glycerol
Figure GDA0004178698530000376
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
Figure GDA0004178698530000377
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
Figure GDA0004178698530000381
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
Figure GDA0004178698530000382
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol
Figure GDA0004178698530000383
u) methyl 3, 8-dihydroxybehenate,
Figure GDA0004178698530000384
a salt or solvate thereof.
The compounds and pharmaceutical compositions, e.g., anticancer compositions, are used in some embodiments to treat or prevent epithelial cancer, or in other embodiments, e.g., in the presence of propolis, propolis extract or component, propolis resin or propolis resin extract or poplar extract or component, to improve skin health, and/or to enhance the activity and physicochemical properties of propolis, propolis extract or component, propolis resin or propolis resin extract or propolis-containing material.
Thus, anti-epithelial cancer compositions may be used to prepare anti-epithelial cancer pharmaceutical compositions and medicaments so long as they are formulated for administration to a mammalian subject, e.g., such compositions are comprised of materials that are safe to the human body.
Furthermore, since the anti-epithelial cancer activity may be maintained for a period of time in embodiments of the compositions of the present invention, the dosage or frequency of administration of the composition may be reduced, or better efficacy may be produced, or both.
The phrase "anti-epithelial cancer composition" or "composition having anti-epithelial cancer activity" of the present invention (used interchangeably herein) contemplates any kind of composition. Examples include anti-epithelial cancer compositions consisting of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin, or anti-epithelial cancer compositions containing propolis, propolis resin or propolis resin extract and a cyclodextrin. Particularly synergistic compositions, including compositions that enhance any anti-epithelial cancer activity (observed only in propolis or cyclodextrin). In certain embodiments, the anti-epithelial cancer composition may be an anti-basal cell carcinoma, anti-squamous cell carcinoma, or anti-melanoma composition. In other embodiments, the anti-epithelial cancer composition is an anti-gastrointestinal cancer composition, such as an anti-colorectal cancer, anti-gastric cancer, or anti-laryngeal cancer composition.
The term "and/or" may mean "and" or ".
The terms "cancer" and "cancerous" refer to a physiological condition of a mammal that is typically characterized by abnormal or unregulated cell proliferation, cell survival, cell movement, neoplasia and/or tumorigenicity. Cancers and cancer pathologies may be associated with, for example, metastasis, interfering with the normal function of adjacent cells, abnormal release of cellular agents or other exudates, inhibiting or exacerbating inflammation or immune response, neoplasia, precancerous lesions, malignant tumors, invasion of surrounding or distant tissues or organs, such as lymph nodes, and the like. Specifically included are basal cell carcinoma, squamous cell carcinoma, melanoma, and precancerous conditions, which may include dermal tumors, epithelial tumors, oral tumors, such as oral squamous cell carcinoma, carcinoma in situ, and invasive basal cell carcinoma, squamous cell carcinoma, or melanoma cancer, as well as secondary tumors derived therefrom. Also specifically included are gastrointestinal cancers, such as colorectal cancer and precancerous conditions, which may include epithelial tumors, non-epithelial tumors, carcinomas, such as carcinoma in situ, and invasive colorectal cancer. Also included are gastric cancer and precancerous conditions, including epithelial tumors, adenocarcinoma, gastric lymphoma, carcinoid tumors, and stromal tumors. Also included are laryngeal and precancerous conditions, including epithelial tumors, squamous cell carcinoma, and adenocarcinoma. Likewise, mucosal tissue cancers are particularly contemplated. The cancer may be, for example, carcinoma in situ, as well as invasive carcinoma.
The term "comprising" as used in this specification means "consisting at least in part of". In understanding the statements containing this term in this specification, the features starting with the term in each statement need to be present, but other features may also be present. The relative verb variants of the term are understood in the same way.
An "effective amount" is that amount required to achieve a therapeutic effect. Freireich et al (1966) describe the relationship of animal and human dosages (in milligrams per square meter of body surface area). The body surface area may be approximately determined based on the height and weight of the individual. See, e.g., scientific forms, 1970, 537 of Geigy pharmaceutical company, ardley, new york. As will be appreciated by those skilled in the art, the effective dosage will vary depending upon the route of administration, excipient usage, and the like.
The poplar "extract" or "component" as used herein is suitable for use herein if it retains at least one or more anti-epithelial cancer activities or comprises one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. Exemplary extracts or components of poplar as used in the present invention are enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. Examples of such functional extracts include the anti-epithelial cancer formulations described in the examples. It will be appreciated that the poplar extract or component enriched in one or more compounds of formula (I) may be enriched or reduced in preparation, e.g. at least part of the isolated or purified compound of formula (I) may be added to the composition, e.g. the poplar or poplar extract or component, or removed from the poplar or poplar extract or part of the compound other than the one or more compounds of formula (I).
Propolis "extracts" or "fractions" as used in the present invention are suitable for use in the present invention if at least one or more anti-epithelial cancer activities or effects possessed by propolis are retained and/or one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof are included. Exemplary extracts or components of propolis useful in the present invention are enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. Examples of such functional extracts include the anti-epithelial cancer tinctures described in the examples. It will be appreciated that the propolis extract or fraction enriched in one or more compounds of formula (I) may be enriched in the preparation whereby at least part of the isolated or purified compound of formula (I) may be added to the composition, for example propolis or propolis extract or fraction, or removed from propolis or propolis extract or fraction of the compound other than the one or more compounds of formula (I).
Chromatography as used herein refers to a separation process in which a compound dissolved or dispersed or otherwise transferred in a solvent phase is contacted with a solid phase, thereby allowing the solid phase to selectively delay one or more compounds depending on the other compounds present in the solvent, thereby enabling separation thereof. Examples of various types of chromatography include column chromatography, liquid chromatography, gas chromatography, supercritical chromatography, size exclusion chromatography, preparative chromatography, and solid phase extraction. Chromatography may be used to determine the concentration of one or more compounds in a mixture, identify unknown compounds, and/or isolate one or more compounds, e.g., to obtain detailed structural analysis data, for use as an analytical standard and/or for use in a bioassay.
Methods and assays for determining one or more biological effects produced by one or more compounds or compositions of the present invention (e.g., compositions comprising one or more compounds of formula (I), and optionally, propolis or poplar extract) are well known in the art, and related examples are also described herein; and such methods and assays can be used to identify or verify efficacy, e.g., efficacy of one or more compounds or compositions described herein, including compositions comprising one or more functional extracts or functional components of propolis, e.g., propolis extract, poplar extract, or propolis or poplar components enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. For example, to determine the ability of one or more compounds or compositions of the invention to increase one or more oncogene characteristics in a cell, as described in the examples of the invention, the efficacy of one or more compounds or compositions of the invention, or one or more functional extracts or functional components of propolis or poplar, is verified.
As used herein, "propolis" includes propolis obtained and produced by bees from any plant source. In one embodiment, the propolis is "poplar" propolis. "Poplar" propolis is also known by different names, such as "Poplar" propolis. For example, the propolis is derived primarily from the bud and leaf exudates of one or more poplar varieties, followed by white birch, larch or willow. Plant sources have enabled the presence of characteristic compounds, and propolis has been classified into seven major classes according to plant sources (Sforcin and Bankova, 2011. Propolis: has new drug development potential. These classes are European, north American, south American, new Zealand "poplar", respectively, which have extremely high levels of flavonoid aglycones, such as chrysin and high-grade curcumin; "Brazil green" poplar, containing isopentenyl p-coumaric acid, such as actipine C; russia "birch", also rich in flavonoid aglycones such as apigenin, cascara sagittifolia He Shannai; "Red propolis", derived from Kluyveromyces species of Gouba, brazil, mexico, venezuela, containing polyisopentenyl benzophenones, including nemoroson and gambogic alcohol; "Mediterranean" poplar from Greek, sicily island, criter island, maranta, is rich in diterpenes derived from conifer; and "Pacific" poplar from Okinawa, taiwan, indonesia, which contains "propolis".
Because of the complex and multicomponent nature of propolis resins, it presents challenges in identifying and verifying the presence of anticancer components in propolis resins. "aspen" propolis resins have been shown to contain a variety of compounds including sugars, glycerol, simple fatty acids, volatile oils and phenolic acid glycerides. The present invention describes a group of compounds in propolis which have not been previously reported and whose anticancer activity has heretofore been unknown. These compounds are long chain fatty acids and their equivalent glycerides. In addition, these dihydroxyl free fatty acids and glycerides may be partially acetylated until fully acetylated. These compounds have the structure of formula (I):
Figure GDA0004178698530000401
wherein R is 2 、R 3 、R 4 And R is 5 Is H or acetyl (CH) 3 CO-)，
R 6 Is H or CH 3 ，R 7 Is CH 3 Or C2-C6 saturated or unsaturated hydrocarbon, and
x and y are integers of 3 to 14 independently of each other,
provided that when R 6 When H is the number, x+y is greater than 10 and less than or equal to 18, when R 6 Is CH 3 When x+y is greater than 9 and less than or equal to 17.
It will be appreciated by those skilled in the art that the identity of propolis, and in some cases its suitability for a particular use, may be determined by analysis of the components of propolis, including use in the present invention. By the presence and amount of a particular compound (including the compounds discussed herein), i.e., a "marker compound", it is possible to determine whether a particular propolis source, such as poplar propolis, is suitable for a particular use as compared to Brazil propolis. In certain embodiments of the invention, the presence or amount of one or more marker compounds may be determined or analyzed as a preliminary fractionation step prior to formulating the composition. In other embodiments, the presence or amount of one or more marker compounds is determined and reported, for example in an indication, such as an indication of efficacy or activity, associated with the compositions of the present invention.
The "indication" may take any form, including, but not limited to, a label as part of the product, such as a label etched or printed on the surface of the product or composition, an indication printed or etched on a capsule or tablet, a package or label associated with the product, such as a label attached to or incorporated into a package containing the product or composition, a material provided with the product or composition but separate from the product or composition, such as an assay certificate; websites, brochures, or display materials related to the sale or marketing of products or combinations.
The compounds of formula (I) may be isolated and/or purified from poplar propolis using methods known in the art. The present invention provides an exemplary method. Likewise, the compounds of formula (I) may also be isolated and/or purified from plant sources such as poplar and poplar extracts or exudates using methods known in the art. The present invention also provides an exemplary method. These methods are also suitable for determining the concentration or amount of one or more compounds of formula (I) or a pharmaceutically acceptable salt or solvate thereof present in the compositions of the invention, for example in order to report such concentration or amount.
Alternatively, the compounds of formula (I) may be prepared synthetically using methods well known in the art. For example, in the synthesis of the compounds of formula (I), particular consideration is given to the synthesis by the Grignard reaction (Smith, michael B.; march, jery (2007); higher organic chemistry: reaction, mechanism and Structure (6 th edition), new York: wiley-Interscience publication, ISBN 0-471-72091-7). For example, consider the preparation of the compound of formula (I) using the starting materials 3, 8-dihydroxyoctanoic acid and a Grignard reagent selected from the group consisting of magnesium dodecyl bromide, magnesium tridecyl bromide, and magnesium tetradecyl bromide. These grignard reagents may be prepared using the corresponding long chain fatty alcohols dodecane-1-ol (lauryl alcohol), 1-tridecyl alcohol or 1-tetradecyl alcohol (myristyl alcohol). Selective oxidation of primary alcohols of 3, 8-dihydroxyoctanoic acid to aldehydes is also contemplated to effect grignard coupling of the starting materials. The resulting 3, 8-dihydroxyl fatty acid can then be esterified using a suitable lipase, or by standard chemical esterification with glycerol, glycerol-2-acetate or glycerol-2, 3-acetate, to give the main compound of formula (I).
The phrase "enhanced activity" or "enhanced anti-epithelial cancer activity" and grammatical equivalents and derivatives thereof, when used in connection with a composition of the present invention or components of such a composition, are intended to mean that the anti-epithelial cancer agent has enhanced anti-epithelial cancer activity when present in the composition, compared to the agent (e.g., the isolated agent) that is not present in the composition, and/or that the stability of the composition is increased relative to the individual components and/or that the bioavailability of the composition is increased relative to the individual components. For example, 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 an effective range (e.g., about 105% to about 200%, about 120% to about 200%, about 140% to about 200%, about 150% to about 200%, about 180% to about 200%, and about 190% to about 200%) can be selected between any of these values. In certain embodiments, the composition may have enhanced anti-epithelial cancer activity, i.e., 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-epithelial cancer activity of propolis alone or cyclodextrin alone. Also, by using the methods of the present invention, preferably the composition is capable of maintaining enhanced anti-epithelial cancer activity, it is also understood that enhanced anti-epithelial cancer activity is maintained. Without being bound by theory, the enhanced activity (including maintaining the enhanced activity) is due to a synergistic effect between the various components of the composition, or due to, for example, improved water dispersibility, making the composition more stable or improved bioavailability.
The term "oral" includes buccal administration, enteral administration and gastric administration.
The term "parenteral administration" includes, but is not limited to, topical administration (including administration via any skin, epithelial or mucosal surface), subcutaneous administration, intravenous administration, intraperitoneal administration, intramuscular administration, and intratumoral administration (including any direct administration to a tumor).
The term "pharmaceutically acceptable carrier" is intended to mean a carrier, including but not limited to excipients, diluents or adjuvants that can be administered to an individual as a component of a composition. Preferably, the carrier does not reduce the activity of the composition and is not toxic when administered in a dosage sufficient to release an effective amount of propolis or an extract thereof, or when another anti-epithelial cancer agent is administered.
"s" following a noun refers to either the singular or plural form, or both.
The term "individual" is intended to mean an animal, preferably a mammal, more preferably a mammalian companion animal or a human. Preferred companion animals include cats, dogs and horses. Other mammalian subjects involve farm animals including horses, pigs, sheep, goats, cattle, deer or poultry, or laboratory animals including monkeys, rats or mice.
The term "treatment" and its derivatives should be construed in as broad a context as possible. The term should not be construed to imply that the individual is treated until complete recovery. Thus, "treating" broadly includes maintaining a disease progression or symptoms in an individual at a substantially quiescent level, increasing the rate of recovery in an individual, preventing the occurrence of symptoms, and/or improving the severity of a particular disorder, or extending the life of a patient. The term "treatment" also broadly includes maintaining the physical health of a sensitive individual and enhancing the resistance to disease.
Exemplary uses of the invention
The methods and compositions of the invention are useful for treating or preventing epithelial cancers, neoplastic diseases associated with epithelial cells, and symptoms of such cancers, including symptoms of cancer treatment and related diseases.
In one example, the methods and compositions of the present invention can be used to treat or prevent skin cancers, such as melanoma, tumor diseases associated with melanoma, and symptoms of melanoma, melanoma treatment, and related diseases. Melanoma (also known as malignant melanoma) is a neoplastic disease that affects melanocytes.
Melanoma is the least common, but most invasive and threatening skin cancer. Where possible, the preferred treatment is total surgical excision, which is curable if no metastasis has occurred.
The methods and compositions of the invention are useful for treating or preventing basal cell carcinoma, neoplastic diseases associated with basal cell carcinoma, and symptoms of basal cell carcinoma, basal cell carcinoma treatment, and related diseases. Basal cell carcinoma is a neoplastic disease affecting dermal basal cells.
Basal cell carcinoma occurs at the lowest layer of the epidermis. Where possible, the preferred treatment is total excision by surgery, which is curable.
In certain embodiments, the methods and compositions are useful for treating or preventing squamous cell carcinoma, neoplastic diseases associated with squamous cell carcinoma, and symptoms of squamous cell carcinoma, squamous cell carcinoma treatment, and related diseases.
Squamous cell carcinoma is a neoplastic disease that occurs in the middle layer of cells in the epidermis. Although squamous cell carcinoma is less common than basal cell carcinoma, the probability of metastasis is higher and can be fatal if left untreated.
The present invention provides methods and compositions for inhibiting skin tumor formation, skin tumor growth, skin tumor metastasis, or treating or preventing skin cancer in an individual in need thereof. Without being bound by theory, applicants believe that at least partial inhibition may be achieved by, for example, preventing ultraviolet-induced DNA damage or preventing or reducing the formation of reactive oxygen species.
In certain embodiments, the invention also relates to a method of at least partially reversing the resistance of tumor cells in an individual having skin cancer to a skin cancer treatment, or to a method of completely or partially reversing the resistance of a patient having skin cancer to a skin cancer treatment, a method of re-sensitizing one or more tumors in an epithelial cancer patient, said one or more tumors being resistant to an epithelial cancer treatment, or being predicted to be resistant to an epithelial cancer treatment, said method comprising the step of administering to said patient a composition comprising, consisting essentially of, or consisting of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I).
In one embodiment, the one or more tumors are resistant to, or are expected to be resistant to, or develop resistance to, skin cancer treatment due to an increase in the activity of one or more pre-cancerous cell survival signaling pathways, including an increase in the activity of one or more AKT, JNK, or JAK/STAT signaling pathways, e.g., in a sample from the patient (e.g., a tissue sample, a tumor biopsy, or a blood or plasma sample).
The pathways for pre-cancerous cell survival signaling associated with the occurrence and progression of skin cancer are known in the art.
The methods and compositions are useful for treating or preventing melanoma, neoplastic diseases associated with melanoma cells, and symptoms of melanoma, melanoma treatment, and related diseases.
The most common treatments are surgical removal of the tumor and radiation therapy. Chemotherapeutic agents may be used in combination with surgery and/or radiation therapy.
The methods and compositions may also be used to maintain or improve skin health.
Including the treatment or prevention of diseases associated with poor skin health, low immunity and skin inflammation. For example, the methods and compositions are useful for treating or preventing skin aging, sunburn, dermatitis, eczema, psoriasis, ichthyosis, and related inflammatory diseases, and treating or preventing skin redness, inflammation, dryness, cracking, or itching.
Propolis and propolis material
Propolis is a generally resinous, tacky solid in New Zealand and elsewhere. Propolis may be obtained from beehives and stored in a warehouse, for example, for use in assessing the content of propolis. Propolis or its extract is generally processed into fine granule or concentrated tincture. Various methods for preparing active propolis or its extract into granule or concentrated tincture are known. Most commonly, natural propolis is extracted with ethanol or an ethanol/water mixture to make a thin tincture. Waxes associated with natural propolis are the least soluble in solvents and therefore do not substantially extract. Any extracted wax may be removed by cooling the diluted tincture and then settling, filtration or centrifugation. The solvent can then be evaporated completely by partial concentration of the tincture to give a concentrated tincture, optionally followed by freeze-drying to give a powder. Or spray drying tincture to obtain powder. The components may be prepared using methods known in the art, such as chromatography (e.g., HPLC) using a size exclusion matrix or a reverse phase matrix, or supercritical separation. A typical solvent for such chromatography is ethanol or another water miscible alcohol.
In one embodiment, the propolis or concentrated propolis tincture is combined with other compounds that enhance the properties of propolis, such as compounds that enhance formulation or ease of administration, or compounds that enhance anti-epithelial cancer activity, or compounds that enhance stability of one or more anti-epithelial cancer activities present in propolis. Examples of other compounds are those that enhance the therapeutic effect of propolis. Specifically contemplated are exemplary compositions wherein one or more compounds are added to propolis, particularly to poplar-type propolis, including bioactive compounds such as CAPE, caffeic acid, pinocembrin, benzyl caffeic acid, cinnamic acid, benzyl ferulic acid, cinnamic acid, chrysin, homocurcumin, pinocembrin alcohol, pinocembrin chalcone, and pinocembrin alcohol-3-acetate. In other examples, other compounds are included that improve or maintain the physiological effects of the composition, such as mannitol may be added to enhance the diuretic effect of the resulting composition. Or other compounds, such as excipients and/or propellants, may be added to improve the dosage, manufacturability, or delivery of the composition.
In a particularly contemplated embodiment, the dewaxed propolis resin, optionally with one or more other compounds, is included with cyclodextrin, and the mixture is dried. The mixture is further processed, for example, to obtain a particle size distribution that can be mixed with the other components of the composition, easily tableted, or easily administered to an individual.
In typical embodiments, the propolis or propolis resin is sterilized, such as by heating to kill bacteria, protozoa, yeasts, fungi and other organisms naturally present in the propolis.
Poplar extract
As described in the examples of the present invention, poplar (populus) extracts, especially exemplary compounds of formula (I) from shoot, leaf and shoot exudates, were identified. Without being bound by theory, applicants believe that the presence of the compounds of the present invention or the compounds of the present invention in poplar propolis and propolis resins are associated with poplar propolis and propolis resins. It will be appreciated by those skilled in the art that for use in the present invention, the poplar or propolis extract may be processed into a form suitable for further processing and encapsulation, for example containing cyclodextrin, or extraction of the poplar or propolis extract with a solvent (e.g. alcohol) while retaining the bioactive ingredient. The poplar or propolis extract is generally processed into fine particles or concentrated tinctures.
Cyclodextrin and cyclodextrin material
Cyclodextrins are cyclic molecules consisting of glucopyranose ring units forming a cyclic structure. The cyclodextrin molecule is water-soluble because the cyclodextrin molecule has hydrophobic inside and hydrophilic outside. The solubility of cyclodextrin can be altered by substitution of the external hydroxyl groups. Also, the hydrophobicity of the interior can be altered by substitution, although the hydrophobicity of the interior generally allows for the accommodation of relatively hydrophobic objects within the cavity. The presence of one molecule within another is known as complexation and the resulting product is known as an inclusion complex. Cyclodextrins are generally identified with reference to the number of monomer units comprising the molecule, wherein the α -cyclodextrin comprises six monomer units, the β -cyclodextrin comprises seven monomer units, and the γ -cyclodextrin comprises eight monomer units. Larger cyclodextrin molecules have been described, including well-characterized cyclodextrins containing 32 1, 4-anhydroglucopyranose units.
Cyclodextrin molecules can be conveniently derivatized, for example by modification, to alter one or more of their physicochemical properties. Examples of cyclodextrin derivatives include methylated cyclodextrin, sulfobutylcyclodextrin, maltosyl cyclodextrin, hydroxypropyl cyclodextrin, such as β -hydroxypropyl cyclodextrin and γ -hydroxypropyl cyclodextrin, and salts thereof. Those skilled in the art will appreciate that various derivatives of cyclodextrin may be suitable for a particular purpose, e.g., certain derivatives of cyclodextrin may not be useful for human administration, but are suitable for industrial use.
Cyclodextrins comprising the anti-epithelial cancer compositions of the present invention are commercially available or may be prepared separately by methods well known to those skilled in the art. It will be apparent to those skilled in the art that cyclodextrins in anti-epithelial cancer compositions for administration to individuals, such as those used in the manufacture of beverages, foods or pharmaceuticals, should be safe to the human body, and preferably are pharmaceutically acceptable cyclodextrins.
In particularly contemplated embodiments, alpha-cyclodextrin, gamma-cyclodextrin, or a combination comprising alpha-cyclodextrin, gamma-cyclodextrin, or both, is used. In these examples, as described in the examples of the present invention, the anti-epithelial cancer activity was significantly enhanced. These compositions comprising alpha-cyclodextrin and/or gamma-cyclodextrin can be formulated to provide a better mouthfeel or palatability, e.g., compositions comprising alpha-cyclodextrin and/or gamma-cyclodextrin and propolis cover any unpleasant taste in propolis.
Cyclodextrins suitable for use in the present invention may be obtained from commercial sources or prepared separately by methods well known in the art, such as by enzymatic conversion from starch. In certain embodiments, a CAVAMAX W6, W7 or W8 food product, α, β or γ cyclodextrin sold by Wacker AG, is used.
In certain embodiments, such as those directed to topical administration of the compositions, non-food grade cyclodextrins are used, e.g., substituted cyclodextrins within the scope of cavalsol are also contemplated.
Cancer of epithelium
Epithelial cancers suitable for treatment using the compositions of the present invention include squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, large cell carcinoma, small cell carcinoma, and transitional cell carcinoma. It is understood that cancer is a term commonly used for malignant epithelial tumors. There are two major types of cancer, classified by the type of epithelium from which they originate. Squamous cell carcinoma derived from squamous epithelium and adenocarcinoma derived from glandular epithelium, respectively.
In various embodiments, the epithelial cancer is selected from the group consisting of gastric cancer (intestinal type), gastric cancer (diffuse/mucinous), mid-differentiated colon cancer, low-differentiated colon cancer (mucinous), hepatocellular cancer (including low-differentiated hepatocellular cancer), renal cell cancer (glaviz's tumor), endometrioid cancer, breast cancer (including invasive breast cancer), metastatic cancer (lymph node), colorectal cancer, oral cancer (including esophageal cancer, pharyngeal cancer or laryngeal cancer), skin cancer (including basal cell skin cancer, squamous cell skin cancer and melanoma), and ovarian cancer.
The methods and compositions described herein are particularly contemplated for treating oral cancer or laryngeal cancer, also known as esophageal cancer, pharyngeal cancer or laryngeal cancer, including tumors arising in the pharynx, nasopharynx, oropharynx, hypopharynx, larynx (larynx) or tonsillar tissue. Also, the treatment of gastric cancer, basal cell skin cancer, squamous cell skin cancer and melanoma is particularly contemplated.
Composition and method for producing the same
Exemplary anti-epithelial cancer compositions include one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and a pharmaceutically acceptable carrier.
Compositions suitable for administration to an individual may be formulated as medical supplies, medical devices, medicaments or medicaments. Those skilled in the art can refer to the techniques and teachings of this specification to prepare suitable formulations.
Compositions useful in the present invention may be formulated for administration to an individual by a selected route, including but not limited to oral or parenteral (including topical, subcutaneous, intramuscular, and intravenous). It will be appreciated by those skilled in the art that the route of administration of the composition will generally take into account the purpose of administration of the composition-for example, in order to improve skin health or to treat or prevent skin cancer, the route of administration of the pharmaceutical composition will generally take into account health considerations or the nature of the skin cancer.
In general, the pharmaceutical compositions useful in the present invention can be formulated by one skilled in the art according to known formulation techniques when administered orally. In certain embodiments, the composition formulated for oral administration comprises gamma cyclodextrin. In certain embodiments, the composition formulated for oral administration comprises α -cyclodextrin.
Thus, pharmaceutical compositions useful according to the present invention may be formulated with suitable pharmaceutically acceptable carriers (including excipients, diluents, adjuvants, and combinations thereof) selected according to the intended route of administration and standard pharmaceutical practice. See, for example, ramington pharmaceutical science, 16 th edition, osol, a. Mack publishing Co., ltd., 1980.
While certain embodiments are suitable for administration, e.g., including administration of one or more compounds of formula (I) orally, it will be appreciated that any administration is suitable for any composition, including administration by multiple routes, the administration routes of the different agents being different. Thus, nasal mucosal administration (nasal or buccal inhalation) and vaginal and rectal administration of any composition are also contemplated. Intramedullary, epidural, intra-articular and intrapleural administration of any composition is also contemplated. Also contemplated are compositions administered via a first route of administration and a second route of administration, optionally including at least one additional anti-epithelial cancer factor, wherein the first route of administration is adapted for the separate, simultaneous or sequential administration of one or more additional agents, including one or more additional anti-epithelial cancer agents; the second route of administration is oral, with the topical administration of at least one other anti-epithelial cancer agent.
The composition may also be formulated into dosage forms. The dosage forms useful in the present invention may be orally administered in the form of a powder, liquid, tablet or capsule. Suitable dosage forms may contain additives including emulsifiers, antioxidants, flavoring or coloring agents, or have an enteric coating, as desired. Suitable enteric coatings are known. Enteric coatings encapsulate the active ingredient and prevent release of the active ingredient in the stomach, but allow the dosage form to release after exiting the stomach. The dosage forms useful in the present invention may be adapted for immediate release, sustained release, modified release, sustained release, pulsed or controlled release of the active ingredient. Suitable formulations may contain additives including emulsifiers, antioxidants, flavouring or colouring agents as required.
The capsule may comprise any standard pharmaceutically acceptable material, such as gelatin or cellulose. Tablets may be formulated in accordance with conventional procedures by compressing a mixture of the active ingredient with a solid carrier and a lubricant. Examples of solid carriers include starch and sugar bentonite. The active ingredient may also be administered in the form of hard shell tablets or capsules containing binders such as lactose or mannitol, conventional fillers and compressed tablets. The pharmaceutical compositions may also be administered by parenteral route. Examples of injectable pharmaceutical forms include aqueous solutions of the active agent, isotonic saline or 5% glucose, or other well known pharmaceutically acceptable excipients. Solubilizers well known to those skilled in the art may be used as pharmaceutical excipients for the release of anti-epithelial cancer drugs.
The injectable dosage forms may be formulated as liquid solutions or suspensions. Solid forms suitable for dissolution in a liquid or suspension in a liquid prior to injection may also be prepared. The dosage form may also be emulsified. The one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof may be admixed with a carrier, such as water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof.
Can be prepared into sustained release preparation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and optionally, propolis and/or poplar extract and/or cyclodextrin, and also containing at least one other anti-epithelial cancer drug, if any. The matrix can be formed byIn the form of shaped articles, such as films or microcapsules. Examples of slow release matrices include polyesters, hydrogels (e.g. poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (see U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, nondegradable ethylene vinyl acetate and degradable lactic acid-glycolic acid copolymers, e.g. LUPRON DEPOT TM (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprorelin acetate).
In particular, topical formulations comprising the composition are contemplated, such as formulations comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, or concentrated propolis, propolis resin or propolis resin extract and cyclodextrin, and/or formulations when at least one other anti-epithelial cancer agent is present. Topical formulations may be formulated as lotions, creams, ointments, pastes or salves using known carriers. In certain embodiments, the compositions formulated for topical administration comprise alpha, beta, or gamma cyclodextrin.
In certain embodiments, the topical formulation includes one or more osmotic agents, such as one or more alkyl lactate esters, one or more antioxidants, such as vitamin E (α -tocopherol) or another naturally occurring antioxidant, including polyphenol antioxidants, such as procyanidins and chlorogenic acid, quinic acid, and ferulic acid, one or more photoprotectants or uv protectants, such as titanium dioxide or carnosic acid, one or more lipids, collagen, keratin, or other proteins.
In certain embodiments, the topical composition includes one or more carriers common in cosmetics, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preservatives, antioxidants, solvents, fragrances, fillers, uv protectors, pigments, deodorants, and dyes. Typically, the composition will comprise amounts conventionally used in the art, for example from 0.01% to 20% relative to the total weight of the composition. These carriers are added to the lipid phase, the aqueous phase or one or more phases, vesicles, e.g., one or more lipid vesicles, microparticles or other components of a topical formulation, depending on the nature and specific example.
In certain embodiments, particularly where the composition is an emulsion, the proportion of lipid/fatty phase may be from 5% to 80% by weight, for example from 5% to 50% by weight, based on the total weight of the composition. The oils, emulsifiers and co-emulsifiers that may be used in the composition in emulsion form are selected from the group consisting of oils, emulsifiers and co-emulsifiers conventionally used in the art. The emulsifiers and co-emulsifiers, when present in the composition, are present in a proportion of from 0.3% to 30% by weight, for example from 0.5 to 20% by weight, based on the total composition.
In certain embodiments, the composition comprises one or more oils, such as one or more of mineral oil (liquid petrolatum), vegetable oil (avocado oil or soybean oil), animal oil (lanolin), synthetic oil (perhydro squalene), silicone oil (cyclomethicone), and fluoro oil, including perfluoropolyethers. Fatty alcohols, such as cetyl alcohol, fatty acids and waxes (carnauba or ozokerite) are also used as fatty substances in certain embodiments.
In certain embodiments, the emulsifier and co-emulsifier is a fatty acid ester of polyethylene glycol (e.g., PEG stearate) or a fatty acid ester of glycerol (e.g., glycerol stearate), or a mixture of both.
Hydrophilic gelling agents are contemplated for use in certain formulations, where such agents include carbopol (e.g., carbomers), acrylic acid copolymers (e.g., acrylate/alkyl acrylate copolymers), polyacrylamides, polysaccharides, natural gums and clays, and lipophilic gelling agents (including modified clays such as bentonite), fatty acid metal salts, hydrophobic silica and polyethylene.
In certain embodiments, the moisturizing creams Dermabase, unibase and vaniteam are representative examples of commercially available base creams that are used as pharmaceutically acceptable carriers.
In certain embodiments, the topical formulation further comprises a humectant, a depigmenting or coloring agent, an antimicrobial agent, or a free radical scavenger.
In certain embodiments, the topical composition is formulated as an aqueous formulation, such as a water-soluble skin cream (water-in-oil or oil-in-water emulsion).
Such formulations may be used directly, e.g., directly on a wound, sprayed onto a surgical site, etc., or may be used indirectly, e.g., by dipping into a bandage or dressing or spraying onto surgical equipment, dressing, etc.
Also provided are parenteral unit dosage forms comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, optionally containing at least one other therapeutic agent.
In one example, the anti-epithelial cancer composition is a powder obtained by mixing a propolis tincture, e.g., a propolis tincture enriched in one or more compounds of formula (I) or pharmaceutically acceptable salts or solvates thereof, with cyclodextrin and one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, then adding water and homogenizing the composition, followed by spray drying or freeze drying. Other exemplary anti-epithelial cancer compositions of the invention include solutions, for example, including solutions in which a propolis tincture or component thereof is mixed with cyclodextrin and then dispersed in water; propolis or propolis-containing material and cyclodextrin are dissolved or dispersed in water separately and then mixed (e.g., kneaded); and first dissolving the propolis powder or resin in another organic solvent, or an organic solvent-water mixture in which the propolis powder or resin is soluble, such as ethanol, propylene glycol, ethyl acetate, isopropyl alcohol, and mixtures thereof with water, and mixing the resulting solution with cyclodextrin, adding to water and further mixing (e.g., kneading), and then drying by means known in the art (e.g., spray drying or freeze drying). Alternatively, the water and cyclodextrin may be mixed under conditions where the solubility is equal to or lower than the solubility of cyclodextrin in water, then the propolis tincture is mixed therein, then water is added and mixed, and then the resulting dispersion is dried by methods known in the art, such as spray or freeze drying. In certain embodiments, for example, the above-described anti-epithelial cancer composition as a powder may be preferred because the above-described anti-epithelial cancer composition as a powder may retain stronger anti-epithelial cancer activity, or may retain longer anti-epithelial cancer activity, than a solution of the above-described anti-epithelial cancer composition.
The propolis, propolis resin or propolis resin extract enriched in at least one compound of formula (I) and cyclodextrin of the present invention may be in any amount as long as the desired anti-epithelial cancer activity is achieved. Also, the compound of formula (I), propolis resin or propolis resin extract and cyclodextrin of the present invention may be in any amount as long as the desired anti-epithelial cancer activity is achieved.
Without being bound by theory, applicants believe that when the molar ratio of propolis, propolis resin or propolis resin extract to cyclodextrin is no greater than 1:1, the propolis, propolis resin or propolis resin extract enriched in at least one compound of formula (I) in the composition will be completely encapsulated.
In some embodiments, the molar ratio of propolis, propolis resin or propolis resin extract enriched in at least one compound of formula (I) to cyclodextrin in the composition may be greater than 1:1. In these compositions, the excess propolis, propolis resin or propolis resin extract is not included by cyclodextrin.
Other well-known anti-epithelial cancer substances may be combined with the anti-epithelial cancer composition depending on the use of the composition.
Without being bound by theory, applicants believe that enhanced anti-epithelial cancer activity is observed in the exemplary compositions of the invention comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin, at least in part due to the synergistic effect between the one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, particularly when present as a propolis resin or concentrated component of a propolis resin and cyclodextrin. In certain embodiments, exemplary synergistic compositions are compositions comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof (e.g., propolis extract or fraction enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, propolis resin extract or poplar extract or exudate) and alpha-cyclodextrin. In another contemplated embodiment, the synergistic composition is a composition comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof (e.g., propolis extract or fraction enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, propolis resin extract or poplar extract or exudate) and gamma-cyclodextrin.
In one embodiment, the present invention relates to propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin (e.g. propolis and α -cyclodextrin and/or γ -cyclodextrin), optionally together with at least one anti-epithelial cancer drug, for the preparation of a medical device, medical article, medicament or medicament.
In one embodiment, exemplary compositions comprising propolis, propolis resin extract, poplar extract, or cyclodextrin are formulated for oral administration. In another embodiment, the composition is formulated for parenteral administration, including topical administration. In certain embodiments, the composition is used to induce apoptosis, treat or prevent skin cancer, maintain or improve skin health, or one or more other uses described above.
In one embodiment, the composition is a powder, tablet, caplet, pill, hard or soft capsule or lozenge.
In one embodiment, the composition is in the form of a pouch, dispensable powder, granules, suspension, elixir, liquid, beverage or any other form that can be added to food or beverage, including water or juice. In one embodiment, the composition is an enteral product, a solid enteral product, or a liquid enteral product.
In one embodiment, the composition may be a cream, ointment, paste, drop (including eye or ear drops), inhalant or inhalable composition, dressing, pad or spray.
In one embodiment, the composition further comprises one or more components (e.g., antioxidants) that prevent or reduce degradation of the composition during storage or after administration.
Of particular concern are compositions that additionally comprise milk or one or more milk products or milk ingredients, such as milk proteins, whey proteins, colostrum, any component of milk fat or milk, or one or more milk products or milk ingredients, such as milk fat components, milk protein components, whey protein components, colostrum components, and the like.
The compositions useful in the present invention also include other factors such as calcium, zinc, magnesium, selenium, vitamin C, vitamin D, vitamin E, vitamin K2, complex carbohydrates, edible oils (including palm oil, olive oil, soybean oil, canola oil, corn oil, sunflower oil, safflower oil, peanut oil, grapeseed oil, sesame oil, walnut oil, almond oil, cashew oil, hazelnut oil, macadamia nut oil, pecan oil, pistachio seed oil, and walnut oil), and other foods (brazil berry, amaranth, apricot, argan nut, artichoke, avocado, babassu, beans, blackcurrant seed, glass-seed, sallowry nut, calabash, buffalo gourd, carob (algaroba), pinus, corium sativum, coriander seed, evening primrose, pseudoflax, hemp, cottonseed, banjo, mustard, sunflower seed, columbum, perilla seed, pine, peach seed, pine nut, pumpkin seed, black melon seed, black sesame seed, wheat germ, or combinations thereof).
In various embodiments, the composition may include at least one other therapeutic agent, wherein the at least one other therapeutic agent is an antibiotic, such as an aminoglycoside antibiotic (amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, or paromomycin); ansamycin antibiotics (geldanamycin or herbimycin); carbacephem antibiotics (lauterol); carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastat Ding Huomei robipenem); first generation cephalosporins (cefadroxil, cefazolin, cefalotin or cefalexin); second generation cephalosporins (cefaclor, cefamandole, cefoxitin, cefprozil or cefuroxime); third generation cephalosporin antibiotics (cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftizoxime or ceftriaxone); fourth generation cephalosporin antibiotics (cefepime); a fifth generation cephalosporin antibiotic (ceftolterone); glycopeptide antibiotics (teicoplanin or vancomycin); macrolide antibiotics (azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, vinegared marcomycin, telithromycin or spectinomycin); monocyclic beta-lactam antibiotics (aztreonam); penicillin antibiotics (amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, meloxicam, nafcillin, oxacillin, penicillin, piperacillin or ticarcillin); a polypeptide antibiotic (bacitracin, colistin or polymyxin b); quinolone antibiotics (ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin or ofloxacin); sulfonamide antibiotics (sulfamilone, azosulfadiazine (old), sulfacetamide, sulfadiazine, sulfamethylthiadiazole, sulfadimidine (old), sulfasalazine, sulfadimidine, trimethoprim or trimethoprim-sulfamethoxazole (compound neonoxazole) (tmp-smx)); tetracyclines (demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline); others (arsenicol, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, dull-line mycin, pyrazinamide, quinine/daplatin, rifampin (U.S. rifampin), thiamphenicol, tinidazole, dapsone, clofazimine); or a cyclic lipopeptide antibiotic (daptomycin); glycylcyclines (tigecycline), or oxazolidinone antibiotics (linezolid).
In other embodiments, the at least one other therapeutic agent is an antifungal agent, such as a polyene antifungal agent (natamycin, spinosyns, filipins, nystatin, amphotericin B, candesamin); imidazole antifungals (miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole or tioconazole); triazole antifungal agents (fluconazole, itraconazole, isaconazole, lifuconazole, posaconazole, voriconazole or terconazole); thiazole antifungals (abafungin); allylamine antifungal agents (terbinafine, amorolfine, naftifine, or butenafine); echinocandin antifungal agents (anidulafungin, caspofungin or micafungin); others (benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine or 5-flucytosine, griseofulvin, haloprogin and sodium bicarbonate); or substitute (garlicin, tea tree oil, citronella oil, iodine, lemon grass, olive leaf, orange oil, rose grass oil, patchouli, lemon bole, neem seed oil, coconut oil, zinc or selenium). Or the drug is selected from any one of the drugs of the invention.
The compositions useful according to the invention can be evaluated for their efficacy in vitro and in vitro. See examples below. Briefly, in one embodiment, a composition may be tested for its ability to, for example, inhibit tumor cell proliferation in vitro. For in vivo studies, the composition may be fed to an animal (e.g., a mouse) or injected or topically administered, and then evaluated for its effect on one or more symptoms of skin cancer cell survival, proliferation, metastasis, or skin cancer or related disease or disorder. From the results, the appropriate dosage range, frequency and route of administration can be determined.
The compositions useful in the present invention may be used either alone or in combination with one or more other anti-epithelial cancer agents or one or more other therapeutic agents. The anti-epithelial cancer drug or other therapeutic agent may be or include a medical device, medical article, medicament or drug. The primary efficacy of an anti-epithelial cancer drug or other therapeutic agent is to be effective in alleviating one or more neoplastic diseases or conditions or one or more symptoms of one or more neoplastic diseases or conditions, or to otherwise benefit the individual. Preferred therapeutic agents include therapeutic food factors, immunogenic or immunostimulating agents, wound healing agents, and the like.
It will be appreciated that other anti-epithelial cancer or therapeutic agents (food-based drugs and medicaments) described above may also be used in the methods of the invention, either alone, simultaneously or sequentially with the compositions useful in the invention.
It will be appreciated that the dosage of the composition, the time of administration and the general dosage regimen will vary from individual to individual depending on various variables such as the severity of the individual's symptoms, the type of disease being treated, the mode of administration selected, and the age, sex and/or general health of the individual. However, as can be seen by way of conventional example, for compositions useful in the present invention, the dosage is from about 1mg to about 5000mg/kg, from 1mg to about 4000mg/kg, from 1mg to about 3000mg/kg, from 1mg to about 2000mg/kg, from 1mg to about 1000mg/kg per kilogram of body weight, preferably from about 5mg to about 100mg/kg per kilogram of body weight, preferably daily. In one embodiment, the compositions useful in the present invention are administered in a dose of about 0.05mg to about 250mg per kilogram of body weight.
In various embodiments, sufficient composition is administered daily to achieve delivery of about 0.001mg to about 50mg, about 0.001mg to about 40mg, about 0.001mg to about 30mg, about 0.001mg to about 20mg, about 0.001mg to about 10mg, about 0.001mg to about 5mg, about 0.001mg to about 1mg, about 0.001mg to about 0.5mg, about 0.001mg to about 0.1mg, or about 0.001mg to about 0.05mg of a compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, per kilogram of body weight.
It will be appreciated that administration may comprise a single dose, for example a single daily dose, or multiple discrete doses as required. It will be appreciated that in view of this technology and this disclosure, one of ordinary skill in the art will be able to determine an effective dosage regimen (including dosage and time of administration) for a given condition without undue experimentation.
The invention also relates to a dietary, nutritional, cosmeceutical or oral pharmaceutical composition comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and optionally, propolis or a material comprising propolis in combination with cyclodextrin; or consist essentially of, or consist of, it. In certain embodiments, the composition consists essentially of about 1-99wt% propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and about 1-80wt% cyclodextrin. For example, the composition consists essentially of about 20-80wt% propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and about 20-80wt% cyclodextrin. In another example, the composition consists essentially of about 20-40wt% propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and about 60-80wt% cyclodextrin.
There is provided a dietary, nutritional, cosmeceutical or oral pharmaceutical composition comprising, consisting essentially of, or consisting of propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, or a material comprising propolis, propolis resin or propolis resin extract, which is entrapped by cyclodextrin. In certain embodiments, the composition consists essentially of about 1-30wt% propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and about 70-99wt% cyclodextrin. For example, the composition consists essentially of about 10-25wt% propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and about 75-90wt% cyclodextrin. In another example, the composition consists essentially of about 20-30wt% propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and about 70-80wt% cyclodextrin.
In one embodiment, the composition comprises propolis, propolis resin or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, or a propolis component enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. 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 99wt% propolis, propolis resin or propolis resin extract or propolis component, the effective range being selectable from any of these values (e.g., about 1-25wt%, 1-30wt%, 5-30wt%, 15-30wt%, 20-30wt%, 25-30wt%, 10-50wt%, 15-50wt%, 40-99wt%, 45-99wt%, 50-99wt%, 55-99wt%, 60-99wt%, 65-99wt%, 70-99wt%, 75-99wt%, 80-99wt%, 85-99wt%, 90-99wt%, or 95-99 wt%).
In one embodiment, the composition comprises a cyclodextrin, such as an alpha-cyclodextrin and/or a 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, or 99wt% cyclodextrin, the effective range being selectable from any of these values (e.g., about 1-99wt%, about 5-99wt%, about 10-99wt%, about 15-99wt%, about 20-99wt%, about 25-99wt%, about 30-99wt%, about 35-99wt%, about 40-99wt%, about 45-99wt%, about 50-99wt%, about 55-99wt%, about 60-99wt%, about 65-99wt%, about 70-99wt%, about 75-99wt%, about 80-99wt%, about 85-99wt%, about 90-99wt%, or about 95-99 wt%).
When used in combination with another anti-epithelial cancer drug or therapeutic agent, the compositions useful in the present invention and the other anti-epithelial cancer drug or therapeutic agent may be administered simultaneously or sequentially. Simultaneous administration includes administration of a single dosage form (all ingredients) or administration of separate dosage forms at substantially the same time. Continuous administration includes administration according to different schedules, preferably such that there is overlap in the times at which the compositions and other therapeutic agents useful in the present invention are provided.
Furthermore, it is contemplated that the compositions of the present invention may be formulated with other active ingredients that are beneficial to the individual in particular circumstances. For example, therapeutic agents directed to the same or different aspects of the disease process may be used.
Exemplary anti-epithelial cancer compositions and methods of making such compositions will now be described with reference to the following examples.
Example
Example 1: initial bioassay guided fractionation of propolis tincture
This example describes the evaluation of the anti-gastrointestinal cancer activity of propolis components produced by preparative chromatography. The proliferation assay was performed on the human colon adenocarcinoma cell line DLD-1.
Column chromatography for preparing propolis tincture
Separation was performed using a glass chromatography column packed with Merck Lichroprep CI8 reversed phase stationary phase (16 x4 cm), wherein the stationary phase was washed with methanol (MeOH) (200 ml) and equilibrated with 20% aqueous ethanol (EtOH) (500 ml). A dry solid of propolis tincture (5.446 g) dissolved in EtOH (5 ml) was loaded onto the top of the column with a piston pump. Elution was performed with a graded gradient of 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% aqueous ethanol (250 ml), followed by two 100% EtOH gradient solutions, followed by 2-propanol (IPA), ethyl acetate (EtOAc), acetone and chloroform (CHCl 3). The solvents in the different components were removed in vacuo on a rotary evaporator and then freeze-dried overnight. Two 100% etoh fractions and the remaining four less polar fractions (IPA, etOAc, acetone and CHCl 3) were combined for bioassay work due to the relatively low mass. The components are shown in table 1 as percentages of ethanol (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% aqueous EtOH and 100% EtOH) used in the elution step from the column.
TABLE 1 propolis fractionation quality and sample number
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Anti-gastrointestinal cancer, anti-proliferation detection materials and methods for DLD-1 human colon adenocarcinoma cells, KYSE-30 human esophageal squamous carcinoma and NCI-N87 human gastric cancer
Samples obtained by dry solid isolation of propolis tincture as shown in table 1 above were evaluated for their ability to modulate the activity and proliferation of human colorectal adenocarcinoma cells (DLD-1) according to the MTT assay. The bioassays also require dry solid raw materials of propolis tincture. In addition to the non-supplemented cell control (negative control), the study included the positive control 5-fluorouracil (5-FU). In the examples that follow, the ability of the samples to modulate survival and proliferation of human esophageal cancer cells (KYSE-30) and human gastric cancer cells (NCI-N87) was also assessed by MTT assay.
Description of test materials and test methods
Human gastrointestinal cancer cell lines DLD-1, KYSE-30 and NCI-N87 were thawed and cultured with the addition of the sample and the reference sample. The cell culture conditions should meet the requirements of the cell suppliers (ATCC). The cultures were then subjected to MTT assay to determine the effect of the sample on cell proliferation.
The method is based on the following reported steps:
smolka, AJ, goldering, JR, gupta, S and Charles E Hammond, CE. Pyrrolizine derivatives ML 3000 inhibit gastric H, K-ATPase activity and gastric epithelial IL-8 secretion (2004) 4:4 of BMC gastrointestinal liver disease.
Establishment of human gastric epithelial (HE) cell lines with barrier function, progenitor cells and proenzyme characteristics Chailler, P and Menard, D (2005) & journal of cytophysiology 202:263-274.
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 properties of human colorectal cancer cell lines and oncogene expression (J.International J.cancer 41:287-296).
Features of the newly established esophageal cancer cell line of Shimada, Y, imamura, M, wagata, T, yamaguchi, N, tobe, T. (1992) 21 strain (cancer) 69:277-284.
Sutter, AP, hopfner, M, huetherer, A, maaser, K, scherubi, H. (2006) erlotinib (Tarceva) TM ) Treatment of esophageal cancer by targeting Epithelial Growth Factor Receptor (EGFR) J.International cancer 118:1814-1822
Heterogeneity of the tumor cell populations in response to differentiation inducers and cytostatics, P lyi, i. (1989) 184:11-17.
The single copy mutant p33 gene of Fritzsche, C, zeller, G, knaup, KX, reemer, k. (2004) has no anti-apoptotic effect in drug-treated tumor cells anticancer drugs 15:679-688.
Yang, Y, zhou, Z, he, S, fan, T, et al (2012) treated prostate cancer with (Galectin-3) -targeted HPMA copolymer- (G3-C12) -5-fluorouracil conjugate (biological Material. 33:2260-2271).
Characteristic mechanism of Nakagawa, y., iinuma, m., naoe, t., nozawa, y. and Akao, y. (2007) a-mangostin to induce cell death: apoptosis, unrelated to caspase, increased expression of miR-143 in mitochondrial release endonuclease-G and human colorectal cancer DLD-1 cells was reported in Bioorganic chemistry and medicinal chemistry 15:5620-5628.
Minegaki, T., takara, K., hamaguchi, R., tsujimoto, M.and Nishiguchi, K. (2013) factors affecting the sensitivity of human esophageal cancer cell lines to 5-fluorouracil and cisplatin are reported in tumor flash 5:427-434.
Nakamura, A., nakajima, G., okuyama, R., kuramoch, H., kondoh, Y., kanemura, T., takechi, T., yamamoto, M.and Hayashi, K. (2014) 5-fluorouracil-induced cytotoxicity of calcium folinate against 5-fluorouracil-resistant gastric cancer cells is enhanced following upregulation of thymidylate synthase expression (17:188-195).
Tankiewicz-kwedo, a., pawlak, d., domaniewski, t., and Buczko, w. (2010). Effects of erythropoietin, 5-fluorouracil, and SN-38 on DLD-1 cell growth, pharmacological report 62:926-937.
Sample preparation
The test components were dissolved in 15% Ethanol (ETOH)/HBSS to a concentration of 2mg/ml solids to prepare a working solution.
Experimental procedure
Characteristics of the test System
1. Human colon adenocarcinoma cells were obtained from ATCC (CCl-221, DLD-1).
2. Human gastric cancer cells were obtained from ATCC (CRL-5822, NCI-N87).
3. Human esophageal squamous carcinoma cells were obtained from Sigma Aldrich (ECACC, KYSE-30).
4. The DLD-1 cell culture medium obtained from GIBCO was Dulbecco's Modified Eagle's Medium (DMEM) to which 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and 100mg/ml streptomycin were added. The medium was stored at 4 ℃.
5. NCI-N87 cell culture medium obtained from Sigma was RPMI-1640 medium modified to contain 2mM L-glutamine, 10mM HEPES, 1mM sodium pyruvate, 4500mg/L glucose and 1500mg/L sodium bicarbonate. The medium was stored at 4 ℃.
6. KYSE-30 cell culture medium obtained from Sigma was Ham's F12:RPMI-1640 (50:50) medium, which was modified to contain 2mm L-glutamine. 100U/ml penicillin and 2.5. Mu.g/ml gentamicin were added to the complete medium, 10% FBS. The medium was stored at 4 ℃.
7. Penicillin-streptomycin solution obtained from Sigma (#P-0781) included 10000 units/mL penicillin in 0.9% NaCl, 10mg/mL streptomycin, stored at-20 ℃.
8. The trypsin-EDTA solution obtained from Invitrogen (15400054) included 0.25% trypsin/EDTA.
9. Phosphate Buffered Saline (PBS) was prepared internally.
10. Hanks Balanced Salt Solution (HBSS) was obtained from GIBCO (14185-052) and stored at 4 ℃.
11. Fetal bovine serum was obtained from GIBCO (10091-148) and stored at-20 ℃.
12. 100 mg/vial MTT reagent was obtained from SIGMA (M-2128), dissolved at 10mg/ml in PBS and stored at-20 ℃. A5 mg/ml MTT solution was prepared in PBS and stored as a working solution at 4 ℃.
13. Preparation of MTT lysis buffer consisting of 10% Sodium Dodecyl Sulfate (SDS)/45% dimethylformamide: 20g SDS was dissolved in 100mL Double Distilled Water (DDW), 90mL dimethylformamide was added, and the pH was adjusted to 4.7 with glacial acetic acid, and then diluted with DDW to a final volume of 200mL.
14. 5-fluorouracil (5-FU) was obtained from Sigma (F-6627). Working solutions of 15% etoh/HBSS were prepared as required and then diluted to the desired concentration prior to bioassay.
Preparation of the culture Medium
Proliferation medium for each cell line was as described above. Each medium was prepared according to the manufacturer's instructions and supplemented with penicillin-streptomycin solution (10 ml per liter). 10% FBS was added before use.
Cell culture
1. Each cell line was thawed.
2. After initial proliferation using the above medium (see medium preparation), the culture was re-cultured using trypsin-EDTA as described below. The medium was removed, 5ml of trypsin-EDTA solution was added and incubated at 37℃for 5 minutes or until all cells were isolated. Trypsin was neutralized by adding an equal amount of DMEM medium and the culture was centrifuged at 300g (1200 rpm) for 5 minutes at 4 ℃.
3. The supernatant was decanted and the cell pellet was resuspended in medium DMEM, FBS (10%), penicillin (100 units/ml), streptomycin (100. Mu.g/ml). At 37℃at 5% CO 2 Cells were cultured in air at/95%.
4. After reaching the confluence point, the cells were isolated using trypsin-EDTA and centrifuged as described in step 2.
5. The supernatant was discarded as described in step 3, 1.0x10 4 Cells/ml cells were resuspended in DMEM and supplements.
6. 180 μl of cells (1800 cells/well) or medium was added to each well of the three 96-well plates. The plates were exposed to 5% CO at 37 ℃ 2 Culture in 95% air for 48 hours was sufficient to allow cell adhesion.
7. To each well 20 μl of sample or positive control was added. For the "medium" or "cell only" control, 20 μl of 15% ETOH/HBSS was added to each well. Each sample was evaluated 3-6 times in duplicate, while the control on each of the three microtiter plates was evaluated 3-9 times (in triplicate). The final concentration of the sample in each well was 200. Mu.g/ml unless otherwise noted.
8. The total volume of each well was 200. Mu.l.
9. The plates were incubated at 37℃with 5% CO 2 Culture in 95% air for 19h.
Cell proliferation assay
1. After the completion of the culture, all wells were added20 μl MTT working solution (5 mg/ml) and at 37℃at 5% CO 2 Culturing in 95% air for 3-4 hr.
2. During incubation, it was noted that high color development unexpectedly occurred in wells of some samples, including cell sample wells and sample medium blank wells. After the end of the incubation period, the supernatant from each well was removed, each well was gently washed twice with HBSS to remove purple, and then MTT lysis buffer was added as described in step 3 below.
3. Then 100. Mu.l MTT lysis buffer was added at 37℃at 5% CO 2 Plates were incubated overnight in 95% air. The plates were centrifuged at 1200rpm for 10 minutes to pellet any remaining insoluble material. 200 μl aliquots were transferred from each well into fresh 96-well plates. Plates were read at 570nm using a Versamax microplate reader.
4. Results are expressed as the percentage proliferation of cells cultured in the sample compared to cells cultured in the control sample alone. Blank readings were subtracted from all wells as background readings.
Results
The effect of controls and samples on DLD-1 cell proliferation of propolis fractions s#1-s#10 and comparative propolis dry solids s#11 is summarized in table 2, where nc=negative control (cells only), pc=positive control (5-fluorouracil, tested at 7.50 ng/ml). In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value that makes the measurement statistically significant by student's t-test, here a value <0.05 (ns=nonsensical); the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
TABLE 2 Effect of propolis fraction samples on DLD-1 cell proliferation
Figure GDA0004178698530000551
Figure GDA0004178698530000561
The most active propolis fractions were fractions S#1, S#2, S#5 and S#8. Propolis components S#1, S#2 and S#5 contain known propolis phenols and flavonoids, so no further investigation is made here. Other studies were also performed to determine the presence of compounds in propolis component s#8, HPLC showed not to contain any previously identified phenolic compounds. These studies are described in example 2.
Example 2: initial bioassay guided fractionation without phenolic components
This example describes further bioassay guided fractionation of propolis component s#8 prior to compound identification. Four additional subfractions, numbered s#28 to s#31, were generated. The study performed proliferation assays for colon cancer adenocarcinoma cell line DLD-1 by MTT assay as described in example 1.
Generation of subfractions S#28 to S#31 by preparative HPLC
The 90% aqueous ethanol eluate fraction (propolis fraction s#8) produced in example 1 was further fractionated by preparative HPLC. Propolis component S#8 was dissolved in pure ethanol and then chromatographed on a Phenomenex Synergi C-12C-12 column (4 μ, -RP Max 80A 250x30 mm) using a Gilson 321 preparation pump and an Agilent 1100 series diode array detector by preparative HPLC. The sample injection amount is 0.5-1.5ml. The flow rate was 20ml/min. The solvent was 80% aqueous methanol (0.1% TFA) and EtOAc/methanol (4:1 vol/vol). The initial eluent composition consisted of 80% aqueous methanol. The solvent composition was held for 5 minutes under initial conditions before the EtOAc/MeOH solvent concentration increased linearly to 100% after 35 minutes. Chromatography was performed at room temperature (18-20 ℃). Fractions were collected manually and tested on-line and Evaporative Light Scattering (ELSD) at 210nm, 268nm and 327nm, respectively. The main component of this fraction is a weakly polar component, which is then eluted in the chromatogram, and the minimum ultraviolet absorption occurs at the wavelengths commonly used for analysis of other phenolic-containing propolis fractions, namely 268nm and 327nm. ELSDs have shown that the mixing of ingredients is relatively complex. Due to the destructive nature of ELSD, the preparative HPLC fractions were collected manually and tested on-line at 210 nm. Because chromatographic analysis did not produce significant isolated peaks, but rather a large rise and fall in baseline, four components were collected during the test, all of which were ready for bioassays.
Materials and methods for DLD-1 colon cancer antiproliferative assays
The samples shown in Table 3 were evaluated for their ability to modulate the activity and proliferation of human colorectal adenocarcinoma cells (DLD-1) according to the MTT assay. In addition to the non-supplemented cell control (NC, negative control), the study included the positive control 5-fluorouracil (5-FU). The concentration of 5-FU was increased compared to example 1 to induce a stronger antiproliferative response. The DLD-1 antiproliferation assay was performed as described in example 1.
TABLE 3 sample
Sample numbering Sample ID and concentration Sample numbering Sample and concentration
S#28 90％F1,200μg/ml NC-1 Cell only
S#29 90％F2,200μg/ml PC-1 Cell +5-FU 0.65. Mu.g/ml
S#30 90％F3,200μg/ml PC-2 Cell +5-FU 1.95. Mu.g/ml
S#31 90％F4,200μg/ml
The sample was dissolved as a 2mg/ml working solution in HBSS containing 15% ethanol. The final concentration of the sample was 200. Mu.g/ml and the final EtOH concentration was 1.5% when measured.
Results and discussion
Table 4 summarizes the effect of positive control and test samples on cell proliferation after 24 hours of incubation, where nc=negative control (cells only), pc=positive control (5-fluorouracil, tested at 0.65 and 1.95 μg/ml, respectively). In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the measured average optical density value; p is a probability value that makes the measurement statistically significant by student's t-test, here a value <0.05 (ns=nonsensical); the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
TABLE 4 Effect of propolis subfraction samples on DLD-1 cell proliferation
Figure GDA0004178698530000571
All propolis subfraction samples tested inhibited proliferation of DLD-1 colon cancer cells. The most active component is s#30 90% f3, which completely inhibits proliferation but is cytotoxic at the concentrations tested. Further work was carried out in example 3 to identify the nature of the bioactive compounds of the samples used in examples 1 (propolis fraction S#8) and 2 (propolis subcomponent S#30).
Example 3: identification of glycerides in propolis as active Compounds
This example describes the isolation and identification of novel dihydroxyfatty glyceride from NZ propolis resin components equivalent to examples 1 (propolis component s#8) and 2 (propolis subcomponent s#30). A40% propolis tincture containing 40% propolis resin (400 mg dry solids) was fractionated on a silica gel column with a stepwise solvent gradient to give 14 components, and the weight distribution is shown in Table 5. The components were analyzed by UHPLC using UV (phenol detection at 268 nm) and ELSD detector (for detection of all compounds). Propolis resin and selected components were also analyzed by LC-MS for structural resolution. The purified subfractions were also analyzed by NMR. The column was washed sequentially with hexane, hexane/diethyl ether and diethyl ether eluting most of the phenolic materials, including flavonoids, caffeates and ferulates, from the silica (components 1-2;3-7; 8-9). The brown component remained on the silica. When ethyl acetate (fractions 11-12) was used, the elution of brown color began. The majority of the remaining dark color was then eluted with acetone and methanol (fractions 13, 14). The mass distribution showed that most of the propolis mass was present in the pale yellow diethyl ether fraction, but a significant portion of the total mass was eluted in the fractions eluted with ethyl acetate (fractions 11, 12). The water-soluble material is recovered in the components 13, 14 or remains on the column.
TABLE 5 weight and solvent gradient for silica gel fractionation of propolis
Component (A) Eluent Volume (ml) Weight (mg) Percentage of total elution volume (%)
1 Hexane 10 1 0.3
2 Hexane 10 <1 <0.3
3 20% diethyl ether/hexane 10 1 0.3
4 40% diethyl ether/hexane 10 2 0.6
5 40% diethyl ether/hexane 10 8 2.3
6 60% diethyl ether/hexane 10 10 2.8
7 60% diethyl ether/hexane 10 62 17.5
8 Diethyl ether 10 70 19.8
9 Diethyl ether 10 51 14.4
10 Dichloromethane (dichloromethane) 20 53 15.0
11 Acetic acid ethyl ester 20 47 13.3
12 Acetic acid ethyl ester 20 38 10.7
13 Acetone/methanol 11 3.1
14 Methanol 3 0.8
Results and discussion
These novel compounds peak in the 34-44 minute region of the ELSD chromatogram of components 12, 13. The sharp and well resolved late peaks observed in ELSD can be correlated with a set of peaks seen in LC-MS analysis of native propolis and enriched fractions 12 and 13. These peaks have no uv absorption associated therewith. Mass Spectrometry (MS) showed that the ideal peaks appear in the positive ion mode, with m+1 and m+na peaks observed. The main components of this group of compounds have molecular weights of 460, 474, 488 and 502, corresponding to a same family, which components differ by methylene. All mass spectra exhibited common peaks at 159 and 117 atomic mass units (amu). In the negative ion mode, the compound exhibits an excimer ion peak at m+45 due to the formate adduct. High resolution MS gives 460 molecular weight compounds of formula C25H48O7, as well as other methylene groups of the other compounds of the series. The subfractions of the natural components 12, 13 are then fully acetylated to aid in the separation of related compounds and provide more defined spectral data. Mass spectra of the peracetylated mixtures showed that these compounds formed essentially triacetates, the indicated molecular weight increased by 3x42 atomic mass units.
The initial chromatographic separation described above was repeated several times to produce more enriched components 11-13. Related compounds are also easily separated from these enriched components by silica gel chromatography to yield sufficient NMR analytical material. The spectra showed the presence of four acetate groups, including a single acetate found in some natural compounds. Substituted glycerols are present and acids attached to the glycerols are also hydroxylated at position 3. The remaining molecule consists of a fatty acid chain, which also has a second hydroxyl group. Fatty acids are the primary compounds of dihydroxy C20, C21C 22 or C23. Thus, these compounds are glycerides.
GC-MS analysis of TMS-treated glyceride-enriched fractions from reverse phase column chromatography showed that the natural mixture of hydroxylated fatty acid lipids in propolis was very complex. The component contains the common fatty acids (palmitic, oleic and stearic) known to exist in propolis and monoglycerides. The glycerin and acetyl groups are removed by alkaline water to obtain four main and trace dihydroxyl fatty acids, thereby reducing the complexity. This corresponds to the above structural type, but the specific position of the acetyl group is not determined. In combination with the fatty acid and the position/stereoisomer range of the hydroxyl group, a large number of analogues are produced. The primary compounds identified were monoacylglycerides of dihydroxyc 20, C21C 22 or C23 fatty acids (esterification of the C1 position of glycerol) by chromatographic separation followed by structural analysis using MS and NMR. The fatty acids in monoglycerides are predominantly linear C20-C22. Hydroxylation of fatty acids at the C3 position, the second hydroxyl group being separated from the first hydroxyl group by at least one methylene group; and not on the final methyl group of the fatty acid. The glycerol moiety may be acetylated at one or both of the hydroxyl groups at the C2 and C3 positions. Further work was performed to determine the position of the acetyl and second hydroxyl groups at the fatty acid in example 4.
Example 4: preparation of glyceride concentrate and isolation and purification of glycerides
In this example, the position of the second hydroxyl group on the fatty acid is determined, as well as the position of the acetyl group. Monoglyceride compounds were also isolated. A large amount of extract was prepared using 1.1kg of dewaxed propolis resin with ethyl acetate as a solvent. After the propolis resin is coarsely crushed, the propolis resin is soaked in ethyl acetate for 2 hours at room temperature, and mechanical stirring is used in the soaking process. The solvent containing the dissolved extract was filtered, but not concentrated. First, the extract was adsorbed onto a portion of silica gel and ethyl acetate was evaporated using a rotary evaporator; thereafter, about 20% of the extract solution was subjected to chromatography using silica gel. Next, the previously adsorbed solid was smeared on top of a large silica gel column, and the column eluted with 1:1 hexane/diethyl ether and 8×150ml fractions were collected, of which 4 150ml fractions were eluted again with diethyl ether and finally with ethyl acetate. The 4 ethyl acetate fractions (fractions #13- # 16) collected were much darker in color than the previous fraction. The final fraction is collected after elution with acetone (# 17). Progress of the purification process was followed using silica TLC (phosphomolybdic acid developed in ethanol by heating) and LC-MS. The glyceride-rich fractions from the silica gel chromatography are then combined and subjected to reversed-phase C18 column chromatography using a mixture of water and ethanol free of acid to give the final glyceride-rich fraction (GLY-conc). The process was repeated with more ethyl acetate extract as required.
Analysis of all the fractions by LC-MS showed that ethyl acetate fraction #13- #16 and acetone fraction #17 contained the main glyceride compounds. These components are also rich in brown materials, consistent with example 3. Components #13 and #14 and components #15 and #16 are combined, respectively. The combined fractions #13/#14 were used for structural identification as follows. About 100mg of the #13/#14 mixture was mixed with 4ml of THF: meOH: H2O and 55mg of LiOH. Stirred at ice temperature for 30 minutes and then at room temperature for 48 hours. The reaction mixture was acidified with acetic acid and extracted with chloroform. The dry extract was then esterified with diazomethane and chromatographed on silica gel (ethyl acetate/hexane). The fractions containing one major spot (Rf about 0.3, wherein EtOAc: hexane 1:1) were collected and phosphomolybdic acid was developed in ethanol by heating. After BSTFA treatment, the purified samples were analyzed by GC-MS. The 9 peaks (numbered 4-12) of free fatty acids generated by glyceride hydrolysis in the mass spectrum are identical. Main peak 4 has a characteristic peak at 383 (corresponding to the loss of methyl and tmssoh), while the following main peaks 5 (397), 7 (411), 10 (425), 11 (439) and 12 (453) are C19 to C24 dihydroxyfatty acids (methyl ester TMS ether). Another feature is that there are fragment ions at m/z 301 and 247 on all peaks. These fragments, in particular fragment 301, are characteristic of 3, 8-dihydroxy acid TMS ether and are obtained by saponification, methylation and sialylation of 3-acetoxy, 8-hydroxy fatty acid glycerides esterified at the C2 position on glycerol [ Asai, t., hara, n., kobayashi, s., kohshima, s., fujimoto, y. (2009). Acyl glycerol (=glyceride) in the leaf gland hair exudate of paulownia tomentosa. Helvetica Chimica acta.92:1473-1494]. Other possible dihydroxy acids 3,6, 3,7 and 3,9 have different cleavage patterns. The dihydroxy acid in the glyceride component is thus a C19 to C24 3, 8-dihydroxy fatty acid. The relative peak sizes indicate that the C20 and C22 chain lengths are the predominant compounds. Minor peaks 6, 8, 9, 13 were also observed to have nearly the same MS fragmentation pattern as the larger peaks. Peak 9 is very similar to peak 10, while peak 12 is very similar to peak 13. These minor peaks are the same versions of C22 and C24 acids, i.e., methyl branches at the fatty acid terminus. This is consistent with NMR evidence for peracetylated compounds, which indicates that there is only a small amount of methyl branching.
For the combined fractions #15/16, preparative HPLC was performed followed by Sephadex LH20 size exclusion chromatography to give two monoacetate compounds GLY-1Ac-1 and GLY-1Ac-2 (C20 and C21 dihydroxyfatty acid monoacetate, respectively). For component #17, preparative HPLC was performed followed by Sephadex LH20 size exclusion chromatography to give three non-acetic compounds GLY-0Ac-1, GLY-0Ac-2 and GLY-0Ac-3 (C20, C21 and C22 dihydroxyfatty acid monoglycerides, respectively). The isolation process was followed by HPLC in combination with ELSD, and then the isolated individual compounds were evaluated by HPLC, LC-MS and NMR for purity >95%.
Example 5: preparation of glyceride concentrate, free fatty acids, methyl esters, diacetate, peracetate and cyclodextrin complexes
The glyceride concentrate samples obtained in example 4 were hydrolyzed as described in Reis et al to produce free fatty acids (FFA-con) (Reis, m.g., de Faria, a.d., do Amaral, m.d., c.e., marsaioli, a.j. (2003) Oncodinol-a novel diglyceride from Ornithophora radicans Barb. About 100mg of glyceride concentrate was mixed with 4ml THF: meOH: H2O and 55mg LiOH. Stirred at ice temperature for 30 minutes and then at room temperature for 48 hours. The reaction mixture was acidified with acetic acid and extracted with chloroform. The free fatty acid mixture was purified using silica gel chromatography (ethyl acetate/hexane).
A sample of the purified free fatty acid mixture is methylated to produce dihydroxyfatty acid methyl ester (ME-cont). Methylation was performed by adding a solution of diazomethane (Aldrich, trimethylsilylated diazomethane, 2.0M in hexane) to 10mg of the free fatty acid mixture. After 1 hour of reaction at room temperature, the reaction was quenched with acetic acid and the solution was evaporated to dryness under N2. The methyl ester mixture was purified using silica gel chromatography (ethyl acetate/hexane). The fractions containing one major spot (Rf about 0.3, wherein EtOAc: hexane 1:1) were collected and phosphomolybdic acid was developed in ethanol by heating.
Dihydroxyfatty acid glyceride diacetate (GLY-2 Ac-1 mixture) was prepared by partial acetylation of a glyceride concentrate (15 mg was placed in THF containing a catalytic amount of dimethylaminopyridine, and stirred with 1 equivalent of acetic anhydride overnight at room temperature). The reaction mixture was purified to give a spot on TLC.
The partially purified glyceride fraction (about 150 mg) was dissolved in acetic anhydride (20 mL) and a small amount of dimethylaminopyridine was added, thereby peracetylating the glyceride-enriched fraction (PerAc-cont). After stirring overnight at room temperature, methanol and toluene (about 20mL each) were added and evaporated to dryness using a rotary evaporator to strengthen the reaction. The residue was chromatographed on silica gel using a hexane/ethyl acetate mixture, followed by obtaining a purified peracetate mixture (about 80 mg).
The cyclodextrin complexes of the glyceride concentrates obtained in example 4 were prepared as follows. About 400mg of glyceride concentrate was accurately weighed and placed in a flask. Then, 1.2g of ethanol was added to the vial, and stirred to dissolve the solid, to obtain a 25% glyceride ethanol solution (by mass). The glycerides were rapidly dissolved in ethanol. Approximately 300mg of alpha, beta and gamma cyclodextrin, respectively, were accurately weighed and placed in a mortar. About 400mg of 25% glyceride tincture was then added to each mortar containing one cyclodextrin using a calibrated syringe. The materials in each mortar were then mixed with a pestle to form a uniform paste. Then, an excessive amount of water was added to each mortar, and the substances therein were mixed until the complex was uniformly dispersed in water and was pale yellow. The contents of each mortar were added to a weighed round bottom flask. The contents of the flask were frozen by rotating the flask in a dry ice/acetone mixture. The contents of the flask were then freeze-dried. 370mg of gamma-cyclodextrin glyceride complex (g-CD glyceride concentrate), 390mg of beta-cyclodextrin glyceride complex (beta-CD glyceride concentrate), and 360mg of alpha-cyclodextrin glyceride complex (alpha-CD glyceride concentrate) were recovered from the flask.
Example 6: dihydroxyfatty acid glycerides versus human skin cancer cell lines: antiproliferative activity of melanoma cell line A-2058
This example shows that many glyceride compounds in NZ propolis have novel anti-skin cancer activity against the human melanoma cell line a-2508. This example also describes a general in vitro bioassay method for determining the antiproliferative activity of purified components and highly purified glycerides on human skin cancer cell lines and the activity of these test compounds on human melanoma cell line a-2058. Three human skin cancer cell lines are:
1) Human melanoma cell line (A-2058).
2) Human epidermoid (squamous) carcinoma cell line (A-431).
3) Human basal cell carcinoma cell line (TE 354. T).
After thawing these cell lines, the samples and reference samples were added for culture. The cultures were then subjected to MTT analysis to determine the effect of the samples on cell activity and proliferation. Evaluating the standard error percentage of the average value; if SEM% >15, the extreme outliers are removed. When α.ltoreq.0.05, the preliminary significance differences (whether outliers were present or not) were assessed using independent student t-test. The method is based on the following reported steps:
metabolic markers of mitogen-activated protein kinase in Ahn, NG, campbell JS (1993) A431 cells demonstrated phosphorylation of serine and threonine residues (Proc. Natl. Acad. Sci. USA 90:5143-5147).
Galan-Cobo, A et al (2014). Inhibition of aquaporin-3 function by gold-based compounds results in a blockage of cell proliferation journal of cytophysiology 229:1787-1801.
Effect of Roomi, MW, kalinovsky, J et al (2013) nutrient Medium on matrix metalloproteinase-9 dimer in various human cancer cell lines (J.International journal of oncology 44:936-942).
The evaluation of the cytotoxicity of cardiotonic steroids and bufadienolide in the American bells and of the bufadienolide in Huang, HC, lin, MK et al (2013) Planta medical 79:1362-1369.
Tilli, CMLJ, stavasct-Kooy, AJW et al (2003) garlic-derived organosulfur fraction ajoene reduced basal cell carcinoma tumor size by induction of apoptosis. Skin disease research archive. 295:117-123.
Sample preparation
Unless otherwise indicated, samples were dissolved in 15% ethanol (EtOH)/HBSS to make working solutions, and then diluted 10-fold to give a final concentration in cells of 50g/ml.
Experimental procedure
Characteristics of the test System
1. A human melanoma cell line (A-2058,ATCC CRL11147) was obtained from ATCC of Besseda, malyland, U.S.A.
2. Human squamous (epidermoid) carcinoma cell lines were obtained from ATCC of bezidas, maryland, U.S. Pat. (a-431, ATCC crl 1555).
3. Human basal cell carcinoma cell line (TE 354.T,ATCC CRL7762) was obtained from ATCC of bescens, maryland, usa.
4. Dulbecco's Modified Eagle's Medium (DMEM) was obtained from GIBCO (12100-038). 10% Fetal Bovine Serum (FBS), 100U/ml penicillin and 100mg/ml streptomycin were added prior to use.
5. Penicillin-streptomycin solution obtained from Sigma (#P-0781) included 10000 units/mL penicillin in 0.9% NaCl, 10mg/mL streptomycin, stored at-20 ℃.
6. The trypsin-EDTA solution obtained from Invitrogen (15400054) included 0.25% trypsin/EDTA.
7. Phosphate Buffered Saline (PBS) was prepared internally.
8. Hanks Balanced Salt Solution (HBSS) was obtained from GIBCO (# 14185-052) and stored at 4 ℃.
9. Fetal bovine serum was obtained from GIBCO (# 10091-148) and stored at-20 ℃.
10. 100 mg/vial MTT reagent was obtained from Sigma (M-2128), dissolved at 10mg/ml in PBS and stored at-20 ℃. A5 mg/ml MTT solution was prepared in PBS and stored as a working solution at 4 ℃.
11. Preparation of MTT lysis buffer consisting of 10% Sodium Dodecyl Sulfate (SDS)/45% dimethylformamide: 20g of SDS was dissolved in 100mL of Double Distilled Water (DDW), and then 90mL of dimethylformamide was added to the solution. The pH was adjusted to 4.7 with glacial acetic acid and then diluted with DDW to a final volume of 200mL.
12. 5-fluorouracil (5-FU) was obtained from Sigma (F-6627). Three working solutions of 19.5. Mu.g/ml, 6.5. Mu.g/ml and 1.95. Mu.g/ml were prepared, respectively, and dissolved in 15% EtOH/HBSS. The final concentrations were 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), respectively.
Preparation of the culture Medium
The culture conditions of the cells were as described by the cell suppliers (ATCC). The proliferation medium for each cell line was DMEM as described above. The medium was prepared according to the manufacturer's instructions and supplemented with penicillin-streptomycin solution (10 ml per liter). 10% FBS was added before use.
Cell culture
1. Various cell lines obtained from the American type culture Collection were thawed.
2. After initial propagation using DMEM medium as described above, the culture was again cultured with trypsin-EDTA. The medium was removed, 5ml of trypsin-EDTA solution was added and incubated at 37℃for 5 minutes or until all cells were isolated. Trypsin was neutralized by adding an equal amount of DMEM medium and the suspension was centrifuged at 300g (1200 rpm) for 5 min at 4 ℃.
3. The supernatant was decanted and the cell pellet was resuspended in DMEM containing FBS (10%), penicillin (100 units/ml), streptomycin (100. Mu.g/ml).
4. After reaching the confluence point, the cells were isolated using trypsin-EDTA and centrifuged as described in step 2.
5. The supernatant was discarded and the cells were resuspended in DMEM and supplements as described in step 3, concentrated per ml of cellsThe degrees are different. Cell concentration of A-431 cultures from 1X10 4 Cells/ml increased to 5x10 4 The concentration of cells/ml, the other two cell lines (A2058 and TE 354) increased to 2X10 4 Cells/ml.
6. For three cell lines, 180 μl of cells or medium was added to each well of a 96-well plate. Using 5% CO 2 Air/95% air plates were incubated at 37℃for various times to allow cell adhesion. The preculture time was 3 hours for the A-431 cultures and 24 hours for the other two cell lines (A2058 and TE 354. T).
7. To each well 20. Mu.l of test compound or 5-FU was added. To wells labeled "medium" or "cell only" 20. Mu.l 15% EtOH/HBSS were added. The number of evaluations for each sample or control is recorded in the example table.
8. The total volume of each well was 200. Mu.l.
9. The plates were incubated at 37℃with 5% CO 2 Culturing in 95% air for 24 hr.
Cell proliferation assay
1. After the completion of the incubation, 20. Mu.l of MTT working solution (5 mg/ml) was added to all wells, and the plates were incubated at 37℃in 5% CO2/95% air for 3-4 hours. Plates were monitored every 30-60 minutes, and lysis buffer was added as step 2 if a few cells showed crystals.
2. Mu.l of MTT lysis buffer was then added and the plates incubated overnight at 37℃in 5% CO2/95% air. The flat wells were centrifuged at 300g (1200 rpm) for 10 minutes to pellet any remaining insoluble material. 200 μl aliquots were transferred from each well into fresh 96-well plates. Plates were read at 550nm using a Versamax microplate reader.
3. The results are expressed as the percentage of adsorption of cells cultured in the sample to cells cultured in the control sample alone. Blank readings were subtracted from all wells as background readings.
Glyceride samples GLY-1Ac-1 and GLY-1Ac-2 (dihydroxyfatty acid glycerides acetylated at the C3 position of glycerol, C20 and C21 fatty acids, respectively) and GLY-0Ac-1, GLY-0Ac-2 and GLY-0Ac-3 (dihydroxyfatty acid glycerides each free of acetate, C20, C21 and C22 fatty acids) were tested at final concentrations of 50. Mu.g/ml and compared to negative control NC-1 (cell only), positive control PC-1, PC-2, PC-3 (consisting of 5-FU, test concentrations of 0.195, 0.65 and 1.95. Mu.g/ml, respectively) and PC-4 (consisting of Monopalmitin (MGP)) tested at concentrations of 50. Mu.g/ml.
Results and discussion
This example shows that many glyceride compounds in NZ propolis have novel anti-skin cancer activity against the human melanoma cell line a-2508. The bioassay results are shown in Table 6. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value which makes the measurement statistically significant by student t test, where the value is <0.05; the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
TABLE 6 antiproliferative Activity of dihydroxyfatty acid glycerides on human melanoma cell line A-2508
Sample number and repetition number, n Average value (OD 570 nm) SEM P value [ (]<0.05) Inhibition ratio (%)
NC-1 cells only, n=6 0.1260 0.0046 1 0.00
PC-1 cells +5-FU,0.195 μg/ml, n=6 0.1339 0.0067 NS 0
PC-2 cells +5-FU,0.65 μg/ml, n=6 0.1187 0.0043 NS 5.78
PC-3 cells+5-FU, 1.95 μg/ml, n=6 0.1383 0.0045 NS 0
PC-4 cells+MGP, 50 μg/ml, n=3 0.0548 0.0016 1.7E-05 56.5
S#12 cells+GLY-1 Ac-1, 50. Mu.g/ml, n=2 0.0249 0.0037 2.4E-05 80.2
S#13 cells+GLY-1 Ac-2, 50. Mu.g/ml, n=2 0.0236 0.0006 1.4E-06 81.3
S#14 cells+GLY-0 Ac-1, 50. Mu.g/ml, n=2 0.0157 0.0001 1.3E-05 87.5
S#15 cells+GLY-0 Ac-2, 50. Mu.g/ml, n=2 0.0106 0.0001 9.7E-06 91.6
S#16 cells+GLY-0 Ac-3, 50. Mu.g/ml, n=2 0.0358 0.0055 5.2E-05 71.6
All highly purified compounds inhibited proliferation of 72-92% of human melanoma skin cancer cell lines when tested at 50 μg/ml. Most effective of these compounds is the non-acetylated dihydroxyfatty glyceride GLY-0Ac-2 having a fatty acid chain length of 22 carbons. The antiproliferative activity of these dihydroxyfatty acid glycerides was superior to the positive control monoglyceride palmitate (MGP) also when tested at 50 μg/ml. The activity of 5-fluorouracil was low and meaningless when tested at three concentrations of 0.195, 0.65 and 1.95. Mu.g/ml, respectively.
Example 7: antiproliferative activity of dihydroxyfatty acid glycerides on HER A-431
This example shows that many glyceride compounds in NZ propolis have novel anti-skin cancer activity against human epidermoid carcinoma cell line a-431. The antiproliferative activity of glyceride samples GLY-1Ac-1 (C20 dihydroxyfatty acid glyceride acetylated at the C3 position of glycerol) and GLY-0Ac-1 (C20 dihydroxyfatty acid glyceride without acetate on glycerol) on HER cell line A-431 was tested at a final concentration of 50. Mu.g/ml. Cell proliferation assays were performed as described in example 6. The samples were compared with negative controls NC-1 (cells only) and positive controls PC-1, PC-2, PC-3 (consisting of 5-FU, test concentrations of 0.195, 0.65 and 1.95. Mu.g/ml respectively) and PC-4 (consisting of glycerol Monopalmitate (MGP), test concentrations of 50. Mu.g/ml).
The bioassay results are shown in Table 7. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value that makes the measurement statistically significant by student's t-test, where the value is <0.05 (n=nonsensical); the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
Table 7: antiproliferative activity of dihydroxyfatty acid glycerides on HER cell line A-431
Sample number and repetition number, n Average value (OD 570 nm) SEM P value [ (]<0.05) Inhibition ratio (%)
NC-1 cells only, n=6 0.2621 0.0158 NS 0.00
PC-1 cells +5-FU,0.195 μg/ml, n=6 0.2364 0.0064 NS 9.8
PC-2 cells +5-FU,0.65 μg/ml, n=6 0.2416 0.0111 NS 7.8
PC-3 cells+5-FU, 1.95 μg/ml, n=6 0.2561 0.0086 NS 2.3
PC-4 cells+MGP, 50 μg/ml, n=3 0.0360 0.0034 2.5E-05 86.3
S#12 cells+GLY-1 Ac-1, 50. Mu.g/ml, n=3 0.0507 0.0037 3.3E-04 80.6
S#14 fineCell +GLY-0Ac-1,50 μg/ml, n=3 0.0484 0.0025 3.6E-05 81.5
The proliferation inhibition levels of the two monoglyceride compounds GLY-1Ac-1 and GLY-0Ac-1 tested were nearly identical, 80.6% and 81.5%, respectively, when tested at 50. Mu.g/ml. This example shows that the amount of acetate in glycerol is not significant in terms of activity. The antiproliferative activity of these dihydroxyfatty acid glycerides, also tested at 50 μg/ml, was similar to that of the positive control monoglyceryl palmitate (MGP). The activity of 5-fluorouracil was low and meaningless when tested at three concentrations of 0.195, 0.65 and 1.95. Mu.g/ml, respectively.
Example 8: antiproliferative activity of dihydroxyfatty acid glycerides on human basal cell carcinoma TE 354.T
The antiproliferative activity of glyceride samples GLY-1Ac-1 (C20 dihydroxyfatty acid glyceride acetylated at the C3 position of glycerol) and GLY-0Ac-1 (C20 dihydroxyfatty acid glyceride without acetate on glycerol) on human basal cell carcinoma cell line TE 345 was tested at a final concentration of 50. Mu.g/ml. Cell proliferation assays were performed as described in example 6. The samples were compared with negative controls NC-1 (cells only) and positive controls PC-1, PC-2, PC-3 (consisting of 5-FU, test concentrations of 0.195, 0.65 and 1.95. Mu.g/ml respectively) and PC-4 (consisting of glycerol monopalmitate, test concentrations of 50. Mu.g/ml).
This example shows that many glyceride compounds in NZ propolis have novel anti-skin cancer activity against human basal carcinoma cell line TE 354. The bioassay results are set forth in Table 8. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value that makes the measurement statistically significant by student's t-test, here a value <0.05 (ns=nonsensical); the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
Table 8: antiproliferative activity of dihydroxyfatty acid glycerides on human basal cell carcinoma cell line TE 354
Sample number and repetition number, n Average value (OD 570 nm) SEM P value [ (]<0.05) Inhibition ratio (%)
NC-1 cells only, n=6 0.2696 0.0096 NS 0.00
PC-1 cells +5-FU,0.195 μg/ml, n=6 0.2548 0.0051 NS 5.49
PC-2 cells +5-FU,0.65 μg/ml, n=6 0.2656 0.0061 NS 1.49
PC-3 cells +5-FU,1.95μg/ml,n＝6 0.2553 0.0070 NS 5.31
PC-4 cells+MGP, 50 μg/ml, n=3 0.2277 0.0065 0.0247 15.6
S#12 cells+GLY-1 Ac-1, 50. Mu.g/ml, n=3 0.1959 0.0037 1.3E-03 27.3
S#14 cells+GLY-0 Ac-1, 50. Mu.g/ml, n=3 0.1672 0.0236 1.7E-03 38.0
The two monoglyceride compounds GLY-1Ac-1 and GLY-0Ac-1 tested had similar proliferation inhibition levels of 27.3% and 38.0%, respectively, when tested at 50 μg/ml. This example shows that the absence of acetate on glycerol can enhance the biological activity of this skin cancer cell line. The antiproliferative activity of these dihydroxyfatty acid glycerides was also positive over the positive control monoglyceryl palmitate (MGP) when tested at 50 μg/ml. The activity of 5-fluorouracil was low and meaningless when tested at three concentrations of 0.195, 0.65 and 1.95. Mu.g/ml, respectively.
Example 9: antiproliferative activity of dihydroxyfatty acid glycerides and derivatives thereof on human colon adenocarcinoma cell line DLD-1
In this example, the antiproliferative activity of monoglycerides GLY-1Ac-1 and GLY-1Ac-2 (acetylated dihydroxyfatty acid glycerides at the C3 position of glycerol, C20 and C21 fatty acid chain lengths) and GLY-0Ac-1 and GLY-0Ac-2 (non-acetylated dihydroxyfatty acid glycerides, C20 and C21 fatty acid chain lengths) against the colon adenocarcinoma cell line DLD-1 was tested at a final concentration of 50. Mu.g/ml. GLY-1Ac-1 was retested at 50 and 25 μg/ml, respectively, at the second assay to provide an indicative dose response. Cell proliferation assays were performed as described in examples 1 and 2. The samples were compared with negative control NC-1 (cells only) and positive controls PC-1, PC-2 (consisting of 5-FU, test concentrations of 0.65 and 1.95. Mu.g/ml, respectively) and PC-3 (consisting of glycerol Monopalmitate (MGP), test concentrations of 50. Mu.g/ml). Also included in the table are the cell data NC-1 and NC-2 for the first and second bioassay experiments.
This example shows that many glyceride compounds in NZ propolis have novel anti-gastrointestinal cancer activity against human colon adenocarcinoma cell line DLD-1. The bioassay results are shown in Table 9. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value that makes the measurement statistically significant by student's t-test, here a value <0.05 (ns=nonsensical); the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
Table 9: antiproliferative activity of dihydroxyfatty acid glycerides on human adenocarcinoma cell line DLD-1
Figure GDA0004178698530000681
The proliferation inhibition of all monoglycerides with or without acetate attached to glycerol was between 68-91% when tested at 50 μg/ml. The most effective of these compounds in the first assay was monoacetylated dihydroxyfatty acid glyceride GLY-1Ac-2. The antiproliferative activity of these dihydroxyfatty acid glycerides, also tested at 50 μg/ml, was similar to or better than the positive control monoglyceryl palmitate (MGP). GLY-1Ac-1 was retested at 50 and 25g/ml, respectively. The antiproliferative activity was 94% at a concentration of 50. Mu.g/ml; the antiproliferative activity at a concentration of 25. Mu.g/ml was only 13%; the LD50 of this compound is therefore between 25 and 50. Mu.g/ml. The activity of 5-fluorouracil was low and meaningless when tested at two concentrations of 0.65 and 1.95. Mu.g/ml, respectively.
Example 10: antiproliferative activity of dihydroxyfatty acid glyceride mixtures, derivatives thereof and cyclodextrin complexes on human adenocarcinoma cell line DLD-1
This example shows the antiproliferative activity of purified glyceride components GLY-conc, derivatives of purified glycerides, including free fatty acids (FFA-conc), methyl esters (ME-conc) and peracetylated glycerides (PerAc-conc), and cyclodextrin complexes of glycerides g-CD GLY-conc (gamma-cyclodextrin inclusion glyceride concentrate) and alpha-CD GLY-conc (alpha-cyclodextrin inclusion glyceride concentrate) on human colon cancer cell line DLD-1. These components and samples were prepared as described in examples 4 and 5 and the antiproliferative activity of glyceride concentrates on the colon adenocarcinoma cell line DLD-1 was tested at a final effective concentration of 50. Mu.g/ml. At a concentration of 200. Mu.g/ml (corresponding to 50. Mu.g/ml glyceride) a cyclodextrin-coated glyceride sample containing 25% glyceride concentrate (by mass) was tested. The samples were compared with negative controls NC-1 (cells only) and positive controls PC-1, PC-2, PC-3 (consisting of 5-FU, test concentrations of 6.5, 19.5 and 58.5. Mu.g/ml respectively) and PC-4 (consisting of monopalmitate of glycerol, test concentrations of 50. Mu.g/ml). The bioassay results are shown in Table 10. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value that makes the measurement statistically significant by student's t-test, here a value <0.05 (ns=nonsensical); the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
Table 10: antiproliferative activity of dihydroxyfatty glyceride concentrates, derivatives and complexes thereof on human colon adenocarcinoma cell line DLD-1
Figure GDA0004178698530000691
This example shows that isolated glyceride mixtures, glyceride derivatives, including free fatty acids, alkyl esters and peracetic esters, and cyclodextrin-coated glycerides in NZ propolis have novel anti-gastrointestinal cancer activity against human colon adenocarcinoma cell line DLD-1. The most effective test substance is a monoglyceride concentrate with a proliferation inhibition of 82%. The most effective derivative and complex is methyl ester with an inhibition of 47% and then the alpha and gamma cyclodextrin complexes with inhibition of 33% and 28%, respectively. The antiproliferative activity of peracetate and free fatty acid was the lowest, 19% and 10%, respectively. The test concentrations of these derivatives may be too low. The activity of 5-fluorouracil was low and meaningless when tested at three concentrations of 6.5, 19.5 and 58.5. Mu.g/ml, respectively.
Example 11: antiproliferative activity of dihydroxyfatty acid glycerides on human gastric cancer cell line NCI-N87
In this example, the antiproliferative activity of monoglycerides GLY-1Ac-1 and GLY-1Ac-2 (dihydroxyfatty acid glycerides acetylated at the C3 position of glycerol, C20, C21, C22 fatty acid chain lengths), GLY-0Ac-1, GLY-0Ac-2, GLY-0Ac-3 (non-acetylated dihydroxyfatty acid glycerides, C20, C21, C22 fatty acid chain lengths) and GLY-2Ac-1 mixtures (mixtures of dihydroxyfatty acid glycerides acetylated at the C2 and C3 positions, dihydroxyfatty acid chain lengths) on human gastric cancer cell line NCI-N87 were tested at final concentrations of 50 μg/ml. Meanwhile, GLY-1Ac-1 was also tested at concentrations of 25 and 10 μg/ml, respectively, to provide an indicative dose response. Cell proliferation assays were performed as described in examples 1 and 2. The samples were compared with negative controls NC-1 (cells only) and positive controls PC-1, PC-2, PC-3 (consisting of 5-FU, test concentrations of 6.5, 19.5 and 58.5. Mu.g/ml, respectively) and PC-4 (consisting of glycerol Monopalmitate (MCP), test concentrations of 50. Mu.g/ml). The bioassay results are shown in Table 11. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value which makes the measurement statistically significant by student t test, where the value is <0.05; the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
Table 11: antiproliferative activity of dihydroxyfatty acid glycerides on human gastric cancer cell line NCI-N87
Sample number and repetition number, n Average value (OD 570 nm) SEM P value [ (]<0.05) Inhibition ratio (%)
NC-1 cells only, n=6 0.0864 0.0128 1.00 0.00
PC-1 cells +5-FU,6.5 μg/ml, n=6 0.0693 0.0062 NS 19.83
PC-1 cells +5-FU,19.5 μg/ml, n=6 0.0792 0.0067 NS 8.32
PC-1 cells +5-FU,58.5 μg/ml, n=6 0.0793 0.0023 NS 8.22
PC-4 cells + MGP,50 μg/ml, n=6 0.0000 0.0000 N/A 100
S#1 cells+GLY-0 Ac-1, 50. Mu.g/ml, n=6 0.0000 0.0000 N/A 100.00
S#2 cells+GLY-0 Ac-2, 50. Mu.g/ml, n=6 0.0000 0.0000 N/A 100.00
S#3 cells+GLY-0 Ac-3, 50. Mu.g/ml, n=6 0.0000 0.0000 5.0E-05 99.97
S#4a cells+GLY-1 Ac-1, 50. Mu.g/ml, n=6 0.0000 0.0000 N/A 100.00
S#4b cells+GLY-1 Ac-1,25 μg/ml, n=2 0.0118 0.0016 1.9E-02 86.38
S#4c cells+GLY-1 Ac-1,10 μg/ml, n=6 0.0530 0.0041 3.2E-02 38.61
S#7 cells+GLY-1 Ac-2, 50. Mu.g/ml, n=6 0.0000 0.0000 N/A 100.00
S#8 cells+GLY-2 Ac-1mix, 50. Mu.g/ml, n=4 0.0414 0.0042 2.5E-02 52.08
This example shows that a large number of glyceride compounds in NZ propolis have novel and very potent anti-gastrointestinal cancer activity against the human gastric cancer cell line NCI-N87. All isolated acetate-free and monoacetate compounds were cytotoxic (100% inhibition of proliferation) when tested at 50 μg/ml. The glyceryl diacetate compound mixture, GLY-2Ac-1 mixture, had very strong antiproliferative effect at 52% inhibition, but no cytotoxicity. When the test concentration of monoacetate compound GLY-1Ac-1 was reduced from 50. Mu.g/ml to 25. Mu.g/ml, the antiproliferative activity was also reduced to 86% (still very potent). The test concentration was further reduced from 25. Mu.g/ml to 10. Mu.g/ml and the antiproliferative activity to 39%. Thus, LD50 is between 25 μg/ml and 10 μg/ml. The activity of 5-fluorouracil was low and meaningless when tested at three concentrations of 6.5, 19.5 and 58.5 μg/ml, respectively; the activity was highest when tested at 6.5. Mu.g/ml. Glycerol Monopalmitate (MGP) was also cytotoxic at the assay concentration of 50. Mu.g/ml.
Example 12: antiproliferative activity of dihydroxyfatty acid glyceride mixtures, derivatives and cyclodextrin complexes on human gastric cancer cell line NCI-N87
This example shows the antiproliferative activity of purified glyceride components GLY-conc, derivatives of purified glycerides, including free fatty acids (FFA-conc), methyl esters (ME-conc) and peracetylated glycerides (PerAc-conc), and cyclodextrin complexes of glycerides g-CD GLY-conc (gamma-cyclodextrin inclusion glyceride concentrate) and alpha-CD GLY-conc (alpha-cyclodextrin inclusion glyceride concentrate) on human gastric cancer cell line NCI-N87. These fractions and samples were prepared as described in examples 4 and 5 and the antiproliferative activity of glyceride concentrates on gastric cancer cell line NCI-N87 was tested at a final effective concentration of 50 μg/ml. At a concentration of 200. Mu.g/ml (corresponding to 50. Mu.g/ml glyceride) a cyclodextrin-coated glyceride sample containing 25% glyceride concentrate (by mass) was tested. The samples were compared with negative controls NC-1 (cells only) and positive controls PC-1, PC-2, PC-3 (consisting of 5-FU, test concentrations of 6.5, 19.5 and 58.5. Mu.g/ml, respectively) and PC-4 (consisting of glycerol Monopalmitate (MCP), test concentrations of 50. Mu.g/ml). The bioassay results are shown in Table 12. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value that makes the measurement statistically significant by student's t-test, here a value <0.05 (ns=nonsensical); the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
Table 12: antiproliferative activity of dihydroxyfatty glyceride concentrates, derivatives and complexes on the human gastric cancer cell line NCI-N87
Figure GDA0004178698530000711
This example shows that isolated glyceride mixtures, derivatives including free fatty acids, alkyl esters and peracetic esters, and cyclodextrin-coated glycerides in NZ propolis have novel anti-gastrointestinal cancer activity against human gastric cancer cell line NCI-N87. The most effective test substance is a monoglyceride concentrate with a proliferation inhibition of 82%. The most effective derivatives and complexes are methyl ester (inhibition of 70%), beta-cyclodextrin complex (inhibition of 78%), alpha-cyclodextrin complex (inhibition of 68%) and gamma-cyclodextrin complex (inhibition of 65%), respectively. The antiproliferative activity of peracetate and free fatty acids was relatively low, 60% and 55%, respectively. The activity of 5-fluorouracil was low and meaningless when tested at three concentrations of 6.5, 19.5 and 58.5 μg/ml, respectively; the activity was highest when tested at 6.5. Mu.g/ml. Glycerol Monopalmitate (MGP) was cytotoxic at the assay concentration of 50. Mu.g/ml.
Example 13: antiproliferative activity of dihydroxyfatty acid glycerides on human esophageal cancer cell line KYSE-30
In this example, the antiproliferative activity of monoglycerides GLY-1Ac-1 and GLY-1Ac-2 (dihydroxyfatty acid glycerides acetylated at the C3 position, C20, C21 fatty acid chain lengths), GLY-0Ac-1, GLY-0Ac-2, GLY-0Ac-3 (non-acetylated dihydroxyfatty acid glycerides, C20, C21, C22 fatty acid chain lengths) and GLY-2Ac-1 mixtures (mixtures of dihydroxyfatty acid glycerides acetylated at the C2 and C3 positions, fatty acid chain lengths) on human esophageal cancer cell lines KYSE-30 were tested at final concentrations of 50 μg/ml. Meanwhile, GLY-1Ac-1 was also tested at concentrations of 25 μg/ml and 10 μg/ml to provide an indicative dose response. Cell proliferation assays were performed as described in examples 1 and 2. The samples were compared with negative controls NC-1 (cells only) and positive controls PC-1, PC-2, PC-3 (consisting of 5-FU, test concentrations of 6.5, 19.5 and 58.5. Mu.g/ml, respectively) and PC-4 (consisting of glycerol Monopalmitate (MCP), test concentrations of 50. Mu.g/ml). The bioassay results are shown in Table 13. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density (ns=nonsensical); p is a probability value which makes the measurement statistically significant by student t test, where the value is <0.05; the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
Table 13: antiproliferative activity of dihydroxyfatty acid glycerides on human esophageal cancer cell line KYSE-30
Figure GDA0004178698530000721
This example shows that many glyceride compounds in NZ propolis have novel and potent anti-gastrointestinal cancer activity against the human esophageal cancer cell line KYSE-30. All isolated non-acetate and monoacetate compounds had moderate to strong antiproliferative effects (34-86%) when tested at 50 μg/ml, with GLY-1Ac-1 being the most effective. The glyceryl diacetate compound mixture GLY-2Ac-1 mixture had a moderate antiproliferative effect at an inhibition of 25%. When the test concentration of monoacetate compound GLY-1Ac-1 was reduced from 50. Mu.g/ml to 25. Mu.g/ml, the antiproliferative activity was also reduced to 50% (still very potent). The test concentration was further reduced from 25. Mu.g/ml to 10. Mu.g/ml, and the antiproliferative activity reached a statistically significant level of 9%. Thus, the LD50 is about 25 μg/ml. At a test concentration of 58.5. Mu.g/ml, 5-fluorouracil has a low but significant activity of 17%; at concentrations as low as 6.5 and 19.5. Mu.g/ml, activity was meaningless. At a test concentration of 50 μg/ml, glycerol Monopalmitate (MGP) has a strong antiproliferative activity of 75%.
Example 14: antiproliferative activity of dihydroxyfatty acid glyceride mixtures and derivatives thereof on human esophageal squamous carcinoma cell line KYSE-30
This example shows the antiproliferative activity of purified glyceride components GLY-coc, purified glyceride derivatives, including free fatty acids (FFA-coc), methyl esters (ME-coc) and peracetylated glycerides (PerAc-coc) on human esophageal squamous carcinoma cell line KYSE-30. These components and samples were prepared as described in examples 4 and 5 and the anti-proliferative activity of glyceride concentrates on the esophageal squamous carcinoma cell line KYSE-30 was tested at a final effective concentration of 50 μg/ml. The samples were compared with negative controls NC-1 (cells only) and positive controls PC-1, PC-2, PC-3 (consisting of 5-FU, test concentrations of 6.5, 19.5 and 58.5. Mu.g/ml, respectively) and PC-4 (consisting of glycerol Monopalmitate (MCP), test concentrations of 50. Mu.g/ml). The bioassay results are shown in Table 14. In the table, OD is the optical density measured at 570 nm; SEM is the standard error associated with the average value of the measured optical density; p is a probability value that makes the measurement statistically significant by student's t-test, here a value <0.05 (ns=nonsensical); the% inhibition is the percentage of proliferation reduction compared to the negative control and a large number of data indicate that the test compounds have anti-cancer proliferation potential.
Table 14: antiproliferative activity of dihydroxyfatty glyceride concentrate and its derivatives on human esophageal squamous cell carcinoma line KYSE-30
Sample number and repetition number, n Average value (OD 570 nm) SEM P value [ (]<0.05) Inhibition ratio (%)
NC-1 cells only, n=6 0.1519 0.0072 1.000 0.00
PC-1 cells +5-FU,6.5 μg/ml, n=6 0.1336 0.0083 NS 12.02
PC-2 cells +5-FU,19.5 μg/ml, n=6 0.1407 0.0053 NS 7.34
PC-3 cells +5-FU,58.5 μg/ml, n=6 0.1263 0.0064 2.4E-02 16.85
PC-4 cells + MGP,50 μg/ml, n=6 0.0385 0.0047 1.2E-07 74.68
S#9 cells+GLY-coc, 50. Mu.g/ml, n=6 0.1137 0.0033 7.2E-04 44.03
S#10 cells+ffa-cont, 50 μg/ml, n=6 0.0850 0.0039 1.0E-05 21.47
S#11 cells+me-cont, 50 μg/ml, n=6 0.1193 0.0028 1.8E-03 27.59
S#12 cells+perac-coc, 50 μg/ml, n=6 0.1100 0.0028 2.9E-04 32.03
This example shows that the isolated glyceride mixtures and their derivatives, including free fatty acids, alkyl esters and peracetic esters, in NZ propolis have novel anti-gastrointestinal cancer activity against human esophageal squamous carcinoma cell line KYSE-30. The most effective test substance is a monoglyceride concentrate with a proliferation inhibition of 44%. The most effective derivative is peracetate with an inhibition of 32%. The antiproliferative activity of methyl esters and free fatty acids was low, 28% and 21%, respectively. At a test concentration of 58.5. Mu.g/ml, 5-fluorouracil has a low but significant activity of 16.8%; at concentrations as low as 6.5 and 19.5. Mu.g/ml, activity was meaningless. At a test concentration of 50 μg/ml, glycerol Monopalmitate (MGP) has a strong antiproliferative activity of 75%.
Example 15: isolation of dihydroxyl fatty acid glycerides from poplar
This example shows that certain poplar varieties or hybrids are the source of dihydroxyfatty acid glycerides found in New Zealand propolis and are therefore alternative plant sources for these glycerides in propolis or chemical synthesis.
Poplar shoot, shoot and leaf samples were collected from the plant and food research poplar collection station of aokauere (Manawatu, new zealand), the wheatstone regional committee seedling farm of Akura protection center (Masterton, new zealand) from 9 months to 2 months next year, respectively. Most samples were taken from short stem trees because they are more readily available.
The samples were weighed and extracted with absolute ethanol at room temperature for about 2 hours. Samples were extracted as received, without maceration or slicing. The extracts were dried and weighed, and samples of each extract were prepared for LC-MS analysis by formulating an ethanol solution at a concentration of 5 mg/ml. Leaves and patterns were collected from the leaves and buds of two plants of paulownia tomentosa and Hutt valley mature willow for comparative analysis. In extracting the material, the plant material is immersed in ethyl acetate and only the waxy outer layer of the material is extracted.
All samples were analyzed as described in example 4 using the LC-MS method developed for propolis glycerides. This assay allows the detection of very low levels of novel dihydroxyfatty acid glycerides and other glycerides, if any.
Results
The qualitative content of dihydroxyfatty acid glycerides in the shoots, leaves or buds of each poplar species is shown in Table 15.
Table 15: qualitative content of glycerides in New Zealand poplar varieties
Figure GDA0004178698530000741
np=no glycerides present, ++content of glycerides, higher+++ content is used for the preparation of the glyceride, nd=undetermined.
Analysis showed that dihydroxyfatty acid glycerides found in propolis were also present in many new zealand poplar varieties that were more commonly cultivated for analysis. The presence of glycerides in the leaves, shoots and buds of many poplars appears to be generally associated with the resin at these sites.
The fatty acid composition of the glycerides was substantially the same as that in propolis, indicating that poplar is the source of propolis glycerides. It is contemplated that monoglycerides may be separated from poplar exudates in a similar manner as used in example 4. Populus varieties such as 'Tasman' and 'Fraser' are typical examples of widely planted populus deltoidea (also known as Canadian poplar and Carolina poplar), and populus delwyn et al are also common in New Zealand. In other varieties such as the Liaoyang x black Yang Moxing line and the Chinese aspen variety 'Yunan', no dihydroxyfatty acid glycerides are generally present. No glycerides were found in the hybrid varieties that did not cross the populus tremuloides. There are also no such glycerides in the samples of the leaves and shoots, but small amounts of such glycerides are present in paulownia, indicating that these plant species are not a source of propolis glycerides nor are they suitable for isolation of the glyceride compounds of the present invention.
Example 16: chiral determination of novel glycerides
This example shows that the naturally occurring novel dihydroxyfatty acid glycerides have 3R,8R stereochemical configuration. Chiral analysis of the alcohol groups of organic molecules is typically performed by using chiral derivatizing reagents. These agents are in both R and S forms, and typically contain an aromatic group; when attached to an alcohol group, the aromatic group can affect the 1H NMR chemical shift of the adjacent group through shielding. The change in adjacent group displacement of the R and S derivatives prepared separately indicates the original alcohol chirality. Of most interest for the novel glycerides is the stereochemical configuration of the alcohol group of 3, 8-dihydroxyfatty acids. This work used natural glyceride mixtures since the length of the fatty acid chains had no effect on the chemical shift of the protons at the C-2, -3 and-8 positions.
Preparation of R, S diester and 1H-NMR analysis
The natural glyceride mixtures were hydrolyzed using LiOH as described in example 4. Free fatty acid methyl esters were then prepared using diazomethane with the hydrolyzed glyceride mixtures described in example 4. R-and S-alpha-methoxyphenylacetic acid (MPA), N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) and 4-Dimethylaminopyridine (DMAP) were purchased from Sigma. The chiral ester derivatization method used, freire et al, was described in 2005. About 0.8mL CH in a 5mL reaction flask was dissolved 10mg of the dihydroxyfree fatty acid methyl ester mixture 2 Cl 2 (dry). The solution was stirred at room temperature using a magnetic stirrer and 23m was addedg R-alpha-methoxyphenylacetic acid, 10. Mu.L of EDC, and a catalytic amount of DMAP. The reaction solution was washed with water, 1M HCl, sodium bicarbonate and water to complete the reaction. The organic layer was dried in vacuo and purified by a small silica gel column to give the diester product (about 8 mg). S-diester was obtained in a similar manner. 1H NMR samples were dissolved in CDCl 3 In (3) at 500 hz. The product was then checked by 1H NMR.
Results and discussion
The signals of H-2, H-3 and H-8 were separated sufficiently for detailed analysis. It was first observed that both products became single species by NMR. This will only occur if the original alcohol is a chiral alcohol, i.e. the C-3 and C-8 hydroxyl groups have stereospecificity, R or S each other. The shift changes of these derivatives Δδ (s—r) are shown in table 16. Based on comparison with similar compounds in the literature (tables 17 and 18), the results show that all glycerides have a stereochemical configuration of 3R,8R.
Table 16: 1H NMR chemical Displacement difference between R and S MPA esters of methyl 3,8 dihydroxyfatty acid esters
Hydrogen gas Delta dihydroxyfatty acid methyl ester Delta Di R fatty acid methyl ester Delta Di S fatty acid methyl ester Δδ(S-R)
H a -C(2) 2.5 2.6 2.43 -0.17
H b -C(2) 2.41 2.5 2.35 -0.15
H-3 4.0 5.22 5.15 -0.07
H-8 3.6 4.8 4.9 +0.1
Confirmation of stereochemical configuration by comparison with literature
Then, stereochemistry was confirmed by comparing delta (S-R) displacement changes with the relevant examples in the literature. Two examples in the literature are effectively directly compared and similar derivatizing agents are 3, 7-diacetoxy docosahexaenoic acid, also known as byrsonic acid (Reis et al, 2007), and 1-acetyl-2- (3, 7-diacetoxy-eicosanoyl) -glycerol, also known as oncondinol (Reis et al, 2003), respectively, are used. Hydrolysis of the byrsonic acid removes the acetoxy group to produce the 3,7 dihydroxyl fatty acid. The 3,7 diester was prepared with the chiral derivatizing agent Mosher acid (methoxy { trifluoromethyl } phenylacetic acid) and then detected by 1H NMR. The change in Δδ (S-R) is shown in table 17, whereby Reis et al (2007) named this acid as (3R, 7R) -3, 7-dihydroxybehenic acid.
Table 17: 1H NMR chemical Displacement difference between R and S Mosher esters of methyl 3,7 dihydroxyfatty acid esters derived from Byrsonic acid
Hydrogen gas Δδ(S-R)
H a -C(2) -0.11
H b -C(2) -0.13
H-3 -0.08
H-7 +0.07
Similarly, reis et al (2003) also produced 3,7 dihydroxyfatty acids by hydrolyzing encidinol to remove glycerol and acetoxy groups; the acid was derivatized to the diester using Mosher acid and the 3,7 diester was detected by 1H NMR. As shown in Table 18, the acid was designated as 3R,7R dihydroxy acid due to the change in Δδ (S-R).
Table 18: 1H NMR chemical Displacement difference between R and S Mosher esters of methyl 3,7 dihydroxyfatty acid esters derived from encidinol
Hydrogen gas Δδ(S-R)
H a -C(2) -0.11
H b -C(2) -0.12
H-3 -0.06
H-7 +0.08
The results for byrsonic acid and oncinidol were very similar to those of the new glycerides in table 16. Delta (S-R) values are negative for H-2 and H-3 and positive for the distal hydroxyl position H8, demonstrating that the stereochemical configuration of all glycerides is 3R,8R.
Freire F,Seco JM,
Figure GDA0004178698530000761
E, riguera r. Absolute stereochemical configuration of primary and secondary 1, 2-diols was predicted by 1H NMR spectroscopy: principle and application chemistry 2005 19;11 (19):5509-22).
Reis, M.G., de Faria, A.D., dos Santos, I.A., amaral, M.D.C.E., marsaioli, A.J., byrsonic acid-a floral clue to oil flowers and bees for oil production J.Chemie ecology, 2007,33 (7), pp.1421-1429
Reis, M.G., de Faria, A.D., do Amaral, M.D.C.E., marsaioli, A.J. Oncodinol-a novel diglyceride of flower oils from Ornithophora radicans Barb. Rodr (Orchidaceae) 2003 tetrahedral communication 44 (46), pp.8519-8523
Industrial applicability
The anti-epithelial cancer composition of the present invention, comprising propolis or an extract or component thereof enriched in the compound of formula (I) and cyclodextrin, and further comprising an isolated or purified compound derived from propolis or a component thereof, is useful in medical and medical fields such as medical devices, medical supplies and medicines, and compositions. Methods of treating epithelial cancers, skin cancers, and symptoms thereof using these compositions have been used in the medical field.

Claims (23)

1. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound selected from any one or more of the following:
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt of any one of a) to f) and h) to u).
2. The pharmaceutical composition of claim 1, wherein the composition is formulated for oral administration.
3. The pharmaceutical composition of claim 2, wherein the composition is in the form of a powder, liquid, tablet, caplet, hard or soft capsule or lozenge.
4. The pharmaceutical composition of claim 1, wherein the composition is formulated for topical administration.
5. The pharmaceutical composition of claim 4, wherein the composition is in the form of a lotion, cream, paste or salve.
6. The pharmaceutical composition of claim 4, wherein the composition is in the form of an ointment.
7. At least one compound selected from any one or more of the group consisting of
a) 3, 8-dihydroxyeicosanoic acid,
b) 1- (3, 8-dihydroxyeicosanoyl) glycerol,
c) 1- (3, 8-dihydroxyeicosanoyl) 2-acetoxyglycerol,
d) 1- (3, 8-dihydroxyeicosanoyl) 3-acetoxyglycerol,
e) 1- (3, 8-dihydroxyeicosanoyl) 2, 3-diacetoxy glycerol,
f) 1- (3, 8-diacetoxy eicosanoyl) 2, 3-diacetoxy glycerol,
g) Methyl 3, 8-dihydroxyeicosanoate,
h) 3, 8-dihydroxydi-undecanoic acid,
i) 1- (3, 8-dihydroxydi-undecanoyl) glycerol,
j) 1- (3, 8-dihydroxydi-undecanoyl) 2-acetoxy glycerol,
k) 1- (3, 8-dihydroxydi-undecanoyl) 3-acetoxyglycerol,
l) 1- (3, 8-dihydroxyundecanoyl) 2, 3-diacetoxy glycerol,
m) 1- (3, 8-diacetoxy-di-undecanoyl) 2, 3-diacetoxy-glycerol,
n) methyl 3, 8-dihydroxyundecanoate,
o) 3, 8-dihydroxybehenic acid,
p) 1- (3, 8-dihydroxybehenoyl) glycerol,
q) 1- (3, 8-dihydroxybehenoyl) 2-acetoxyglycerol,
r) 1- (3, 8-dihydroxybehenoyl) 3-acetoxyglycerol,
s) 1- (3, 8-dihydroxybehenoyl) 2, 3-diacetoxy glycerol,
t) 1- (3, 8-diacetoxy-behenoyl) 2, 3-diacetoxy-glycerol, and
u) methyl 3, 8-dihydroxybehenate,
or a pharmaceutically acceptable salt of any one of a) to u)
Use for preparing a composition having the following uses: inhibiting epithelial neoplasia, epithelial tumor growth, epithelial tumor metastasis or treating or preventing epithelial cancer in a subject; inducing apoptosis of one or more tumor epithelial cells in an individual; increasing responsiveness of the individual to treatment of the epithelial cancer; increasing sensitivity of an individual's epithelial tumor to treatment of epithelial cancer; re-sensitizing one or more epithelial cancer cells in the individual that are resistant to the treatment; at least partially reversing the resistance of tumor cells in an individual having an epithelial cancer to treatment of the epithelial cancer; reversing, in whole or in part, resistance of an epithelial cancer patient to treatment of epithelial cancer; or re-sensitize one or more tumors of the epithelial cancer patient that are resistant to, or are expected to be resistant to, the epithelial cancer treatment.
8. The use of claim 7, wherein the at least one compound is used with at least one other therapeutic agent to prepare the composition.
9. The use of claim 7, wherein the subject is a human subject.
10. The use of any one of claims 7-9, wherein the composition is formulated for oral administration.
11. The use of claim 10, wherein the composition is in the form of a powder, liquid, tablet, caplet, hard capsule or soft capsule or lozenge.
12. The use of any one of claims 7-9, wherein the composition is formulated for topical administration.
13. The use according to claim 12, wherein the composition is in the form of a lotion, cream, paste or salve.
14. The use according to claim 12, wherein the composition is in the form of an ointment.
15. Use of a pharmaceutical composition according to any one of claims 1-6 in the manufacture of a medicament for: inhibiting epithelial neoplasia, epithelial tumor growth, epithelial tumor metastasis or treating or preventing epithelial cancer in a human subject; inducing apoptosis of one or more tumor epithelial cells in a human subject; increasing responsiveness of a human individual to treatment of epithelial cancer; increasing the sensitivity of an epithelial tumor in a human individual to treatment of an epithelial cancer; re-sensitizing one or more epithelial cancer cells in a human individual that are resistant to the treatment; at least partially reversing the resistance of tumor cells to treatment of an epithelial cancer in a human subject having the epithelial cancer; reversing, in whole or in part, resistance of an epithelial cancer patient to treatment of epithelial cancer; or re-sensitize one or more tumors of the epithelial cancer patient that are resistant to, or are expected to be resistant to, the epithelial cancer treatment.
16. The use of claim 15, wherein the medicament is for treating or preventing epithelial cancer.
17. A process for the isolation or purification of at least one compound or a mixture of compounds as defined in any one of claims 1 to 6 from propolis or poplar or an extract or exudate thereof, comprising the steps of
a. Providing poplar extract, poplar propolis or extract or exudate thereof, and
b. separating or purifying compounds from poplar, propolis or extracts or exudates thereof,
wherein the poplar extract, poplar propolis or extract or exudate thereof is from populus nigra or populus tremulosa.
18. The method of claim 17, wherein the populus deltoidea comprises a variety or interspecific hybrid thereof.
19. The method of claim 18, wherein the populus euphratica is a species selected from the group consisting of: selwyn, tasman, luisa Avanzo and Fraser.
20. A process for the isolation or purification of at least one compound or a mixture of compounds as defined in any one of claims 1 to 6 from propolis or poplar or an extract or exudate thereof, comprising the steps of
a. Providing poplar extract, poplar propolis or extract or exudate thereof, and
b. Separating or purifying compounds from poplar, propolis or extracts or exudates thereof,
wherein the poplar extract, poplar propolis or extract or exudate thereof is from a hybrid of populus americana.
21. The method of claim 20, wherein the hybrid of populus jaborandi comprises populus jaborandi x populus jaborandi.
22. A process for the isolation or purification of at least one compound or a mixture of compounds as defined in any one of claims 1 to 6 from propolis or poplar or an extract or exudate thereof, comprising the steps of
a. Providing poplar extract, poplar propolis or extract or exudate thereof, and
b. separating or purifying compounds from poplar, propolis or extracts or exudates thereof,
wherein the poplar extract, poplar propolis or extract or exudate thereof is derived from a hybrid of Europe and America poplar.
23. A pharmaceutical composition comprising one or more compounds as defined in any one of claims 1 to 6, wherein the amount and/or concentration of the one or more compounds is specified by an indication associated with the pharmaceutical composition.
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