U.S. patent application number 16/759364 was filed with the patent office on 2020-12-03 for immunity-boosting agent, immuno-therapeutic anti-cancer agent, and anti-cancer therapy adverse effect mitigating agent containing anthocyanin-fucoidan complex as active ingredient.
This patent application is currently assigned to JBKLAB CO., LTD.. The applicant listed for this patent is JBKLAB CO., LTD.. Invention is credited to Bong Keun JANG, Young-Um JO, Joo Young LEE, Kun NA, Jeong Deok SEO.
Application Number | 20200376066 16/759364 |
Document ID | / |
Family ID | 1000005060777 |
Filed Date | 2020-12-03 |
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United States Patent
Application |
20200376066 |
Kind Code |
A1 |
NA; Kun ; et al. |
December 3, 2020 |
IMMUNITY-BOOSTING AGENT, IMMUNO-THERAPEUTIC ANTI-CANCER AGENT, AND
ANTI-CANCER THERAPY ADVERSE EFFECT MITIGATING AGENT CONTAINING
ANTHOCYANIN-FUCOIDAN COMPLEX AS ACTIVE INGREDIENT
Abstract
The present invention relates to a composition including an
anthocyanin-fucoidan complex formed by ionic bond between
anthocyanin and fucoidan as an active ingredient, and more
particularly, it is confirmed that the anthocyanin-fucoidan complex
in which anion of the fucoidan, which is a natural extract having
high biocompatibility and biodegradability, is ionically bonded
with a cation of the anthocyanin improves the stability and the
solubility of the anthocyanin even in acidic conditions in vivo,
thereby increasing the immune activity in vivo and therefore it is
intended to provide the anthocyanin-fucoidan complex as an immune
enhancer, an immune-cancer agent and an anticancer adjuvant
composition for alleviating side effects of anti-cancer agents.
Inventors: |
NA; Kun; (Bucheon-si,
Gyeonggi-do, KR) ; JANG; Bong Keun; (Yongin-si,
Gyeonggi-do, KR) ; JO; Young-Um; (Seoul, KR) ;
SEO; Jeong Deok; (Chungcheongnam-do, KR) ; LEE; Joo
Young; (Ansan-si, Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JBKLAB CO., LTD. |
Seongnam-si, Gyeonggi-do |
|
KR |
|
|
Assignee: |
JBKLAB CO., LTD.
Seongnam-si, Gyeonggi-do
KR
|
Family ID: |
1000005060777 |
Appl. No.: |
16/759364 |
Filed: |
October 26, 2018 |
PCT Filed: |
October 26, 2018 |
PCT NO: |
PCT/KR2018/012849 |
371 Date: |
August 13, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/737 20130101;
A61K 47/61 20170801; A61K 36/73 20130101; A61P 35/00 20180101 |
International
Class: |
A61K 36/73 20060101
A61K036/73; A61K 31/737 20060101 A61K031/737; A61K 47/61 20060101
A61K047/61; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 27, 2017 |
KR |
10-2017-0141184 |
Oct 26, 2018 |
KR |
10-2018-0128919 |
Claims
1-3. (canceled)
4. An immune enhancer comprising an aronia extract-fucoidan complex
composed of an aronia extract and a fucoidan, wherein an ionic bond
is formed between a cation of a cyanidine of the aronia extract and
an anion of the fucoidan, and .pi.-.pi. bonds are formed between
the cyanidine molecules, as an active ingredient.
5. The immune enhancer of claim 4, wherein the aronia
extract-fucoidan complex induces an expression of immune factors to
increase an activity of immune cells.
6. A method of inhibiting carcinogenesis in a subject in need
thereof, comprising: providing a pharmaceutical composition
comprising an aronia extract-fucoidan complex composed of an aronia
extract and a fucoidan, wherein an ionic bond is formed between a
cation of a cyanidine of the aronia extract and an anion of the
fucoidan, and .pi.-.pi. bonds are formed between the cyanidine
molecules, as an active ingredient; and administering the
pharmaceutical composition to the subject, wherein the
carcinogenesis is inhibited.
7. The method of claim 6, wherein the aronia extract-fucoidan
complex induces an expression of immune factors to increase an
activity of immune cells and kill cancer cells.
8. The method of claim 7, wherein the cancer cells are cancer cells
of solid cancers.
9. (canceled)
10. An anticancer adjuvant comprising an aronia extract-fucoidan
complex composed of an aronia extract and a fucoidan, wherein an
ionic bond is formed between a cation of a cyanidine of the aronia
extract and an anion of the fucoidan, and .pi.-.pi. bonds are
formed between the cyanidine molecules, as an active
ingredient.
11. The anticancer adjuvant of claim 10, wherein the anticancer
adjuvant relieves side effects caused by administration of an
anticancer agent.
12. The anticancer adjuvant of claim 11, wherein the side effects
caused by administration of the anticancer agent are selected from
the group consisting of weight loss, immune factor reduction and
cachexia.
Description
TECHNICAL FIELD
[0001] The present invention relates to an immune enhancer and an
anticancer adjuvant composition for relieving side effects of
anticancer drugs comprising anthocyanin-fucoidan complex as an
active ingredient, which is formed by ionic bonding between the
anthocyanin and the fucoidan.
BACKGROUND ART
[0002] With the goal of conquering cancer, extensive research
worldwide is being conducted for patient-specific precision
medicine based on various factors such as next-generation
sequencing (NGS)-based cancer genome analysis and the environment,
but cancer disease is recognized as a fearful disease that is still
difficult to treat and can lead to death. Even if it is treated
with anticancer drugs, it was reported that the recurrence rate for
gastric cancer is 55%, colon cancer is 20-50%, lung cancer is 20
-50%, uterine cancer is 5-20% and breast cancer is 10-15% (National
Cancer Center).
[0003] Immune response refers to cellular and humoral reactions for
checking self and non-self and removing the non-self, and when
immunity decreases, it cannot perform its original function and
eventually our body is easily infected with foreign substances. In
the case of cancer, immune cells involved in immunity cannot attack
cancer cells due to reduced immune activity, and thus cancer cannot
be removed.
[0004] Anthocyanin is a natural pigment glycoside mainly contained
in plant flowers and fruits, and is contained a lot in plants such
as blackcurrant, blueberry, aronia, cherry, black rice, grape and
red cabbage. High concentrations of anthocyanins are found mainly
in strong sunlight UV rays, harsh cold and high humidity
environments, because intense sunlight UV rays destroy the DNA of
plant cell nuclei and affect plant life, thus anthocyanins, which
are a UV absorber, are produced on the surface or the middle layer
of the plant for the protection which are protected.
[0005] It is known that such anthocyanins are easy to extract from
natural products and thus can be mass-produced, and have excellent
pharmacological activity in aging, immune response, diabetes,
bacterial infections, nervous system diseases, and cancer.
Accordingly, people are increasingly interested in anthocyanins and
the market size of anthocyanin-containing products is gradually
increasing due to the global well-being craze.
[0006] Anthocyanin has the greatest effect when ingested as food,
but it exhibits a low activity of 5% or less in vivo due to a long
residence time in the gastrointestinal tract, low intestinal wall
permeability, low solubility, instability during the process for
commercialization, or temperature, ambient oxygen and light, pH in
the digestive tract and enzymes or other nutrients.
[0007] Accordingly, there is a need for research and development
for anthocyanin that can be used as an effective anticancer drug by
improving the in vivo stability of anthocyanin, a natural product
extracted from a plant such as aronia, to enhance immune
function.
DISCLOSURE
Technical Problem
[0008] In order to solve the above problems, the present invention
provides an anthocyanin complex having improved stability and
solubility by ionic bonding fucoidan, which is a natural extract,
with anthocyanin, as an immune enhancer, an immune-cancer agent and
an anticancer adjuvant composition for alleviating side effects of
anti-cancer agents.
Technical Solution
[0009] The present invention provides an aronia extract-fucoidan
complex comprising an aronia extract and a fucoidan, wherein an
ionic bond is formed between a cation of a cyanidine of the aronia
extract and an anion of the fucoidan, and .pi.-.pi. bonds are
formed between the cyanidine molecules.
[0010] The present invention provides an immune enhancer comprising
an aronia extract-fucoidan complex composed with an aronia extract
and a fucoidan, wherein an ionic bond is formed between a cation of
a cyanidine of the aronia extract and an anion of the fucoidan, and
.pi.-.pi. bonds are formed between the cyanidine molecules, as an
active ingredient.
[0011] The present invention provides an immune anticancer drug
comprising an aronia extract-fucoidan complex composed with an
aronia extract and a fucoidan, wherein an ionic bond is formed
between a cation of a cyanidine of the aronia extract and an anion
of the fucoidan, and .pi.-.pi. bonds are formed between the
cyanidine molecules, as an active ingredient.
[0012] The present invention provides a health food for anticancer
comprising an aronia extract-fucoidan complex composed with an
aronia extract and a fucoidan, wherein an ionic bond is formed
between a cation of a cyanidine of the aronia extract and an anion
of the fucoidan, and .pi.-.pi. bonds are formed between the
cyanidine molecules, as an active ingredient.
[0013] In addition, the present invention provides an anticancer
adjuvant containing an anthocyanin-fucoidan complex comprising an
aronia extract-fucoidan complex composed with an aronia extract and
a fucoidan, wherein an ionic bond is formed between a cation of a
cyanidine of the aronia extract and an anion of the fucoidan, and
.pi.-.pi. bonds are formed between the cyanidine molecules, as an
active ingredient.
Advantageous Effects
[0014] According to the present invention, it is confirmed that the
aronia extract-fucoidan complex in which anion of the fucoidan,
which is a natural extract having high biocompatibility and
biodegradability, is ionically bonded with a cation of the
anthocyanin improves the stability and the solubility of the
anthocyanin even in acidic conditions in vivo, thereby increasing
the immune activity of the tumor animal model and restoring the
body weight reduced by treatment with anticancer drugs and thus
extending the life span, and therefore it is intended to provide
the aronia extract-fucoidan complex as an immune enhancer, an
immune-cancer agent and an anticancer adjuvant composition for
alleviating side effects of anti-cancer agents.
DESCRIPTION OF DRAWINGS
[0015] FIG. 1 shows a structure of a complex by cyanidine-fucoidan
bond (Cyaplex-F8).
[0016] FIG. 2 shows a result of confirming an absorbance and an
optical image of the aronia extract-fucoidan complex.
[0017] FIG. 3 shows DLS result of the aronia extract-fucoidan
complex (left) and a scanning electron microscope analysis result
of the aronia extract-fucoidan complex.
[0018] FIG. 4 shows a result of confirming the difference in the
degree of degradation of aronia extract in an aronia extract and
the aronia extract-fucoidan complex.
[0019] FIG. 5 shows a result of confirming the anticancer efficacy
of the aronia extract-fucoidan complex and the killing rate for
normal cells, the result of confirming the IC.sub.50 according to
the content of aronia extract-fucoidan in (a) HCT-116, (b) Hep-G2,
(c) NIH/3T3 and (d) HUVEC.
[0020] FIG. 6 shows a result of confirming the level of immune
induction through the secretion of immune factors of the aronia
extract-fucoidan complex.
[0021] FIG. 7 shows a result of confirming whether or not the tumor
was generated in laboratory animals treated with a carcinogen and a
tumor accelerator after daily oral administration of PBS, fucoidan
(Fu), aronia bio-active fractions (ABF) and aronia extract-fucoidan
complex (Cyaplex-F8) for 22 weeks.
[0022] FIG. 8 shows results of confirming the tumor growth
inhibitory effect in 5 weeks, 9 weeks, 12 weeks, 16 weeks, 18
weeks, 20 weeks and 22 weeks, in laboratory animals treated with a
carcinogen and a tumor accelerator after daily oral administration
of PBS, fucoidan (Fu), aronia (ABF) and aronia extract-fucoidan
complex (Cyaplex-F8) for 22 weeks.
[0023] FIG. 9 shows results of confirming the tumor growth
inhibitory effect in 9 weeks, 9 weeks, 11 weeks, 14 weeks, 16
weeks, 18 weeks and 22 weeks, in laboratory animals treated with a
carcinogen and a tumor accelerator after daily oral administration
of PBS, fucoidan (Fu), aronia extract (ABF) and aronia
extract-fucoidan complex (Cyaplex-F8) for 22 weeks.
[0024] FIG. 10 shows a result of confirming the body weight and the
survival rate in laboratory animals treated with a carcinogen and a
tumor accelerator after oral administration of PBS, fucoidan (Fu),
aronia extract (ABF) and aronia extract-fucoidan complex
(Cyaplex-F8)
[0025] FIG. 11 shows a result of confirming the increase rate of
the tumor determination number (Nodule counts) and the tumor area
in laboratory animals treated with a carcinogen and a tumor
accelerator after oral administration of PBS, fucoidan (Fu), aronia
extract (ABF) and aronia extract-fucoidan complex (Cyaplex-F8).
[0026] FIG. 12 shows a result of hemotoxin & eosin staining
tissues of major organs of heart, lung, liver, kidney and spleen of
laboratory animals treated with a carcinogen and a tumor
accelerator after oral administration of PBS, fucoidan (Fu), aronia
extract (ABF) and aronia extract-fucoidan complex (Cyaplex-F8).
[0027] FIG. 13 shows a result of hemotoxin & eosin staining
tumor tissues of laboratory animals treated with a carcinogen and a
tumor accelerator after oral administration of PBS, fucoidan (Fu),
aronia extract (ABF) and aronia extract-fucoidan complex
(Cyaplex-F8).
[0028] FIG. 14 shows a result of confirming the IFN-.gamma. level
in plasma of laboratory animals treated with a carcinogen and a
tumor accelerator after oral administration of PBS, fucoidan (Fu),
aronia extract (ABF) and aronia extract-fucoidan complex
(Cyaplex-F8).
[0029] FIG. 15 shows a result of confirming the tumor growth
inhibitory effect of a tumor-induced experimental animal by cancer
cell transplantation after intravenous injection of doxorubicin
followed by oral administration of PBS, fucoidan (Fu), aronia
extract (ABF) and aronia extract-fucoidan complex (Cyaplex-F8).
[0030] FIG. 16 shows the tumor growth inhibitory effect (A), weight
change (B) and survival rate (C) of a tumor-induced experimental
animal by cancer cell transplantation after intravenous injection
of doxorubicin followed by oral administration of PBS, fucoidan
(Fu), aronia extract (ABF) and aronia extract-fucoidan complex
(Cyaplex-F8).
[0031] FIG. 17 shows a result of confirming the level of CD8+T
cells and NK cells in the blood on Day 5 and Day 14 of the
experiment after intravenous injection of doxorubicin in a mouse
animal model on the 1st and 5th day of the experiment (2 times in
total) followed by oral administration of PBS, fucoidan (Fu),
aronia extract (ABF) and aronia extract-fucoidan complex
(Cyaplex-F8) for 14 days.
[0032] FIG. 18 shows a result of confirming the spleen weight in a
mouse animal model after intravenous injection of doxorubicin in
the mouse animal model on the 1st and 5th day of the experiment (2
times in total) followed by oral administration of PBS, fucoidan
(Fu), aronia extract (ABF) and aronia extract-fucoidan complex
(Cyaplex-F8) for 14 days and extracting the spleen of the animal
model on the 14th day of the last day of the experiment.
BEST MODE
[0033] Hereinafter, the present invention will be described in more
detail.
[0034] The present inventors have confirmed that a complex can be
produced by ionically binding an anion of fucoidan having
biocompatibility with a cation of cyanidin, an anthocyanin derived
from aronia to structurally stabilize the cyanidine in the stomach,
small intestine, and blood, and to improve immune activity under
conditions similar to the body and to provide as a complex having
excellent bioavailability and physiological activity and thus they
have completed the present invention.
[0035] The present invention may provide an aronia extract-fucoidan
complex comprising an aronia extract and a fucoidan, wherein an
ionic bond is formed between a cation of a cyanidine of the aronia
extract and an anion of the fucoidan, and .pi.-.pi. bonds are
formed between the cyanidine molecules.
[0036] More specifically, the cyanidine may be
cyanidine-3-glucoside (C3G) separated and purified from an aronia
extract, but it is not limited thereto.
[0037] The anthocyanin of the present invention is a combination of
the Greek words Anthos meaning flower and cyanos meaning blue, and
anthocyanin pigment, a type of phytochemical is produced in the
fruits, flowers, and stems of berry to protect themselves from
external stimuli. About 600 species of these anthocyanins exist in
nature, and among these, cyanidin-3-glucoside (C3G) has the best
activity and particularly, anti-aging, antioxidant, anti-cancer,
and anti-metabolism thereof are excellent.
[0038] The complex may be formed in a weight ratio of 0.1:10 to
10:0.1 of the aronia extract and the fucoidan.
[0039] The complex may be a nanocomposite having an average
diameter in a range of 50 nm to 500 nm.
[0040] The present invention may provide an immune enhancer
comprising an aronia extract-fucoidan complex composed with an
aronia extract and a fucoidan, wherein an ionic bond is formed
between a cation of a cyanidine of the aronia extract and an anion
of the fucoidan, and .pi.-.pi. bonds are formed between the
cyanidine molecules, as an active ingredient.
[0041] The aronia extract-fucoidan complex may induce an expression
of immune factors to increase an activity of immune cells.
[0042] According to an example of the present invention, an aronia
extract (ABF) solution containing 50 .mu.g/ml of an aronia extract
(ABF) or a fucoidan solution containing 500 .mu.g/ml of a fucoidan
and aronia extract-fucoidan complex containing 50 .mu.g/ml of an
aronia extract (ABF) and 500 .mu.g/ml of fucoidan was treated to
HCT-116 (human colon cells), SKBR-3 (human breast cancer cells) and
Hep-G2 (human liver cells), respectively and as a result, as shown
in FIG. 6, it was confirmed that IL-6 was secreted higher when the
aronia extract-fucoidan complex was treated than when the aronia
extract (ABF) and fucoidan were treated alone, and in the cells
treated with the aronia extract (ABF) and fucoidan, and the amount
of IL-6 secreted from the cells treated with the aronia
extract-fucoidan complex was higher than the sum of the amount of
IL-6 secreted from the cells treated with the aronia extract (ABF)
and the fucoidan.
[0043] From the results, it was confirmed that the aronia
extract-fucoidan complex can induce the expression of immune
factors in vivo, thereby increasing the activity of immune
cells.
[0044] Accordingly, the present invention may provide an immune
anticancer drug comprising an aronia extract-fucoidan complex
composed with an aronia extract and a fucoidan, wherein an ionic
bond is formed between a cation of cyanidine of the aronia extract
and an anion of the fucoidan, and .pi.-.pi. bonds are formed
between the cyanidine molecules, as an active ingredient.
[0045] The aronia extract-fucoidan complex may induce an expression
of immune factors to increase an activity of immune cells and to
kill cancer cells.
[0046] The cancer cells may be cancer cells of solid cancers, and
more specifically, the solid cancer may be selected from the group
consisting of colon cancer, breast cancer, lung cancer, stomach
cancer, epithelial ovarian cancer, brain cancer, skin cancer and
liver cancer, but it is not limited thereto.
[0047] According to another example of the present invention, in
order to confirm the anticancer effect against cancer cells and
toxicity to normal tissue cells of complex with anthocyanins, each
eight samples were prepared by the serial dilution method to dilute
1/2 from initial concentration using an aronia extract (ABF)
solution containing 50 .mu.g/ml of an aronia extract (ABF) or a
fucoidan solution containing 200 .mu.g/ml of fucoidan and a aronia
extract-fucoidan complex containing 50 .mu.g/ml of aronia extract
(ABF) and a 200 .mu.g/ml of fucoidan and they were treated to
HCT-116 (human colon cells), NIH/3T3 (rat embryonic fibroblasts),
HUVEC (endothelial cells; ATCC) and Hep-G2 (human liver). Cells) to
confirm the cancer cell killing level of the sample, respectively,
as shown in FIG. 5, it was confirmed that the IC.sub.50 values of
the aronia extract (ABF) solution for the HCT-116 and Hep-G2 were
27 .mu.g/ml and 17 .mu.g/ml, respectively and the IC.sub.50 values
of the aronia extract-fucoidan complex were 12 .mu.g/ml and 5.2
.mu.g/ml. In addition, in normal tissue cells NIH/3T3 and HUVEC,
IC.sub.50 values for the aronia extract (ABF) solutions were 41
.mu.g/ml and 53 .mu.g/ml, respectively, and IC.sub.50 values for
the aronia extract-fucoidan complex were 38 .mu.g/ml and 38
.mu.g/ml.
[0048] From the above results, as the IC.sub.50 value of the aronia
extract-fucoidan complex for cancer cells was lower than that of
the aronia extract (ABF) solution, it was confirmed that the aronia
extract-fucoidan complex effectively killed cancer cells in a
smaller amount than the aronia extract (ABF). As the IC.sub.50
value of the aronia extract-fucoidan complex for normal tissue
cells was higher than that of cancer cells, it was confirmed that
the aronia extract-fucoidan complex could effectively kill cancer
cells without cytotoxicity to normal cells.
[0049] In one embodiment of the present invention, the
pharmaceutical composition for preventing or treating cancer
disease comprising the aronia extract-fucoidan complex as an active
ingredient can be used as any one formulation selected from the
group consisting of injection, granule, powder, tablet, pill,
capsule, suppository, gel, suspensions, emulsions, drops or liquids
according to a conventional method.
[0050] In another embodiment of the present invention, a
pharmaceutical composition for preventing or treating cancer
disease comprising aronia extract-fucoidan complex as an active
ingredient may further include one or more suitable additives
selected from the group consisting of carriers, excipients,
disintegrants, sweeteners, coating agents, swelling agents, slip
modifiers, flavoring agents, antioxidants, buffers, bacteriostatic
agents, diluents, dispersants, surfactants, binders and lubricants,
which are commonly used in the manufacture of pharmaceutical
compositions
[0051] Specifically, carriers, excipients or diluents include
lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,
erythritol, maltitol, starch, acacia rubber, alginate, gelatin,
calcium phosphate, calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinylpyrrolidone, water,
methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium
stearate, mineral oil and the like. Solid preparations for oral
administration include tablets, pills, powders, granules, capsules,
etc. and these solid preparations can be prepared by mixing at
least one excipient such as starch, calcium carbonate, sucrose or
lactose, gelatin, etc. in the composition. Further, in addition to
simple excipients, lubricants such as magnesium stearate, talc are
also used. Oral liquid preparations include suspensions, oral
solutions, emulsions, and syrups, and may include various
excipients such as wetting agents, sweeteners, fragrances,
preservatives and the like in addition to commonly used simple
diluents such as water and liquid paraffin. Formulations for
parenteral administration include sterile aqueous solutions,
non-aqueous solvents, suspensions, emulsions, freeze drying agents,
suppositories. As the non-aqueous solvents and suspensions,
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, injectable esters such as ethyl oleate, and the like may be
used. As the base of the suppository, witepsol, macrogol, tween 61,
cacao butter, laurinum, glycerogelatin and the like may be
used.
[0052] According to an example of the present invention, the
pharmaceutical composition can be administered to a subject in a
conventional manner through intravenous, intraarterial,
intraperitoneal, intramuscular, intraperitoneal, intrasternal,
transdermal, intranasal, inhalation, topical, rectal, oral,
intraocular or intradermal routes.
[0053] The preferred dosage of the aronia extract-fucoidan complex
may vary depending on the condition and weight of the subject, the
type and extent of the disease, the drug form, the route and
duration of administration, and may be appropriately selected by
those skilled in the art. According to one example of the present
invention, it is not limited thereto, but the daily dosage may be
0.01 to 200 mg/kg, specifically 0.1 to 200 mg/kg, and more
specifically 0.1 to 100 mg/kg. The administration may be
administered once a day or divided into several administrations,
and the scope of the present invention is not limited thereby.
[0054] In the present invention, the `subject` may be a mammal,
including a human, but it is not limited to these examples.
[0055] In addition, the present invention may provide a health food
for anticancer comprising an aronia extract-fucoidan complex
composed with an aronia extract and a fucoidan, wherein an ionic
bond is formed between a cation of a cyanidine of the aronia
extract and an anion of the fucoidan, and .pi.-.pi. bonds are
formed between the cyanidine molecules, as an active
ingredient.
[0056] The above health food may be used with other foods or food
additives other than the aronia extract-fucoidan complex, and may
be appropriately used according to a conventional method. The
mixing amount of the active ingredient may be appropriately
determined according to its purpose of use, for example,
prevention, health or therapeutic treatment.
[0057] The effective dose of the compound contained in the health
functional food may be used in accordance with the effective dose
of the therapeutic agent. However, in the case of long-term intake
for the health and hygiene or for health control purposes, it may
be used in the above range or lower, and it is clear that the
active ingredient can be used in an amount of at least the above
range because there is no problem in terms of safety.
[0058] There is no particular limitation regarding the kind of the
health functional food. Examples of the health functional food
include meat, sausage, bread, chocolate, candy, snack,
confectionery, pizza, ramen, other noodles, gums, dairy products
including ice cream, various soups, beverages, tea, drinks,
alcoholic beverages and vitamins complex, etc.
[0059] In addition, the present invention may provide an anticancer
adjuvant comprising an aronia extract-fucoidan complex composed
with an aronia extract and a fucoidan, wherein an ionic bond is
formed between a cation of a cyanidine of the aronia extract and an
anion of the fucoidan, and .pi.-.pi. bonds are formed between the
cyanidine molecules, as an active ingredient.
[0060] The anticancer adjuvant may relieve side effects caused by
administration of an anticancer agent, and the side effects caused
by administration of the anticancer agent may be selected from the
group consisting of weight loss, immune factor reduction and
cachexia.
[0061] Hereinafter, examples of the present invention will be
described in detail to understand the present invention. The
present invention may, however, be embodied in many different forms
and should not be limited to the embodiments set forth herein in
order to clearly illustrate the present invention for those skilled
in the art to which the present invention pertains.
EXAMPLE 1
Preparation of Aronia Extract-Fucoidan Complex
[0062] 20 mg of aronia extract (ABF) [JBK Lab.] was dissolved in 20
ml of an aqueous solution of phosphate buffer pH 3 (PB 3) so that
no precipitate was visible and 200 mg of fucoidan (Haewon Biotech)
was dissolved in 20 ml of distilled water so that no precipitate
was visible and then a aronia extract solution was added to the
fucoidan solution and stirred at room temperature for 72 hours to
prepare a aronia extract-fucoidan complex (Cyaplex-F8).
[0063] On the other hand, the aronia extract (ABF) and the fucoidan
were dissolved in the same amount used to prepare the complex and
used as a comparative example.
EXAMPLE 2
Confirmation of Aronia Extract-Fucoidan Complex Properties
[0064] 1. Confirmation of Aronia Extract-Fucoidan Complex
Formation
[0065] From the first day to the sixth day, samples of an aronia
extract (ABF) solution and a aronia extract-fucoidan complex were
obtained, and after diluting the samples to 1/10, the optical
density was measured by a spectrophotometer (UV-1601, Shimadzu,
JAPAN). In addition, an optical photograph was obtained with an
aronia extract (ABF) solution and a aronia extract-fucoidan complex
stock solution.
[0066] As a result, as shown in FIG. 2, the aronia extract-fucoidan
complex was formed by ionic bond formed by cationic property of the
cyanidine of an aronia extract and anionic property of the
fucoidan, and it was also found that .pi.-.pi. interactions between
cyanidine molecules also contribute to the formation of the
complex. In addition, it was confirmed that the maximum absorbance
of cyanidine possessed by .pi.-.pi. interaction while aronia
extract-fucoidan formed a complex is red-shifted as shown in FIG.
2, and it was confirmed from the optical photograph that the
complex-formed sample had a purple color than the aronia extract
(ABF) solution.
[0067] 2. Confirmation of Aronia Extract-Fucoidan Complex Size
[0068] After placing 2 ml of the completed complex into a
polystyrene cuvette (DTS0012), the size of the complex was
confirmed using a zeta potential & nanoparticle size analyzer
(Zetasizer nano ZS, Malvern Instruments Ltd., England). In
addition, 10 .mu.l of the completed complex was placed on a cover
glass and dried in an oven at 60.degree. C. overnight to obtain an
image with a scanning electron microscope (S-4800, HITACHI, Ltd.,
U.S.A).
[0069] Referring to the photo on the left of FIG. 3, which is a
result of the zeta potential & nanoparticle size analyzer
analysis, the complex was confirmed to be about 380 nm in nano
size. In addition, the size of the scanning electron microscope
image, which is a picture on the right in FIG. 3, was confirmed to
be about 85 nm.
[0070] 3. Confirmation of Structural Stability Improving Effect of
Complex Under Various pH Conditions
[0071] After centrifuging 40 ml of the complex obtained by the
method for preparing the aronia extract-fucoidan complex for 13000
rpm for 30 minutes, supernatant was removed and the remaining
precipitate was re-dispersed with PB 3 (pH 3) and phosphate buffer
saline (pH 7) and the effect of improving structural stability was
confirmed by measuring with anthocyanin solution with a
spectrophotometer over time.
[0072] As a result, as shown in FIG. 4, the aronia extract (ABF) is
stable in a low pH environment (pH 3), and decomposition occurs as
the pH increases.
[0073] In more detail, if the aronia extract (ABF) is treated in
human body, it will be exposed to pH 7.4 of the human body, and
eventually decomposition may be expected and indeed, as shown in
FIG. 4, anthocyanin was decomposed with time, but it was confirmed
that the aronia extract-fucoidan complex maintains the absorbance
value to a certain degree over time.
[0074] From the above results, it was confirmed that the aronia
extract-fucoidan complex exhibits excellent stability even in
acidic conditions in vivo.
EXAMPLE 3
Confirmation of In Vitro Cytotoxicity and Cytokine Secretion of
Aronia Extract-Fucoidan Complex
[0075] 1. Confirmation of In Vitro Cytotoxicity of Aronia
Extract-Fucoidan Nanoparticles
[0076] The anticancer effect of complex of aronia extract and
fucoidan against cancer cells and toxicity to normal tissue cells
were confirmed.
[0077] First, an aronia extract (ABF) solution containing 50
.mu.g/ml aronia extract (ABF) or a fucoidan solution containing 200
.mu.g/ml fucoidan and sample of aronia extract-fucoidan complex
containing 50 .mu.g/ml aronia extract (ABF) and 200 .mu.g/ml
fucoidan were prepared, and each 8 samples for the remaining
samples were prepared using a serial dilution method diluting to
1/2 from the aforementioned initial concentration.
[0078] Experimental cells HCT-116 (human colon cell carcinoma;
Korea Cell Line Bank) and HUVEC (endothelial cells; ATCC) were
cultured in RPMI 1640 (Wellgene) medium containing penicillin and
streptomycin and 10% FBS, and Hep-G2 (Human liver cell carcinoma;
Korean cell line bank) and NIH/3T3 (rat embryonic fibroblasts;
Korean cell line bank) were cultured in DMEM medium containing
penicillin and streptomycin.
[0079] The cell lines were seeded at 5.times.10.sup.4 cells per
well on 24-well plates and cultured overnight. Thereafter, the
cells were incubated with DMEM- and FBS-free RPMI and samples of
various concentrations at 37.degree. C. for 24 hours, followed by
exposing to FBS-added DMEM, RPMI medium and 10% cell counting kit-8
(CCK-8) solution (CCK-8 kit, Enzo Life Sciences, Inc., KOREA) and
incubating at 37.degree. C. for 4 hours to confirm cytotoxicity at
an optical density of 450 nm.
[0080] As a result, as shown in FIG. 5, IC.sub.50 values for aronia
extract (ABF) solutions of the HCT-116 and Hep-G2 were 27 .mu.g/ml
and 17 .mu.g/ml, respectively, and IC.sub.50 values for the aronia
extract-fucoidan complex was confirmed to be 12 .mu.g/ml and 5.2
.mu.g/ml. In addition, in normal tissue cells NIH/3T3 and HUVEC,
IC.sub.50 values for aronia extract (ABF) solutions were 41
.mu.g/ml and 53 .mu.g/ml, respectively, and IC.sub.50 values for
aronia extract-fucoidan complex were 38 .mu.g/ml and 38
.mu.g/ml.
[0081] From the above results, it was confirmed that the IC.sub.50
value of the aronia extract-fucoidan complex for cancer cells was
lower than that of the aronia extract (ABF) solution, and the
complex effectively killed cancer cells in a smaller amount than
the aronia extract (ABF) solution alone. In addition, it was
confirmed that as the IC.sub.50 value of the aronia
extract-fucoidan complex for normal tissue cells was higher than
that of the cancer cells, the aronia extract-fucoidan complex had a
higher anticancer effect than when the aronia extract (ABF)
solution was used alone and cytotoxicity to normal tissue cells was
low.
[0082] 2. Confirmation of Cytokine Secreted From Cells Killed by
Aronia Extract-Fucoidan Nanoparticles
[0083] An aronia extract (ABF) solution containing 50 .mu.g/ml
aronia extract (ABF) or a fucoidan solution containing 500 .mu.g/ml
fucoidan and a sample of aronia extract-fucoidan complex containing
50 .mu.g/ml aronia extract (ABF) and 500 .mu.g/ml fucoidan were
prepared.
[0084] Experimental cells HCT-116 (human colon cell carcinoma;
Korean cell line bank) and SKBR-3 (human breast cancer cell; Korean
cell line bank) were cultured in RPMI 1640 (Wellgene) medium
containing penicillin and streptomycin and 10% FBS, and Hep-G2
(human liver cell carcinoma; Korea Cell Line Bank) was cultured in
DMEM medium containing penicillin and streptomycin.
[0085] Each cell line cultured on a 6-well plate was seeded at
3.times.10.sup.5 cells per well and cultured overnight. Thereafter,
the cells were incubated for 24 hours at 37.degree. C. in 2 ml of
DMEM- and FBS-free RPMI medium containing various concentrations of
samples and after recovering the culture medium, IL-6, IL-1.beta.
and TNF-.alpha. cytokine levels were confirmed by ELISA (Enzyme
Linked Immunosorbent Assay, Enzo Life Sciences, Inc., KOREA).
[0086] As a result, the cytokines of IL-1.beta. and TNF-.alpha.
were not secreted in all three cell lines, as shown in FIG. 6 and
IL-6 was secreted more when treated with the aronia
extract-fucoidan complex than when the aronia extract (ABF) and
fucoidan were treated alone. In addition, it was confirmed that the
amount of IL-6 secreted from the cells treated with the aronia
extract-fucoidan complex was higher than the sum of the amount of
IL-6 secreted from the cells treated with the aronia extract (ABF)
and fucoidan alone.
EXAMPLE 4
Confirmation of Tumor Inhibition Effect of Aronia Extract-Fucoidan
Complex
[0087] The effect of inhibiting carcinogenesis of the aronia
extract-fucoidan complex was confirmed in experimental animals
treated with a carcinogen and a tumor accelerator.
[0088] After epilating the back of a Balb/c (n=5) mouse, 200 .mu.L
of carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) 200
nmol/acetone 200 .mu.L was applied to the back, and the tumor
accelerator 12-O-tetradecanoylphorbol-13-acetate (TPA) 4
.mu.g/acetone 200 .mu.L was treated twice a week for 22 weeks.
[0089] The experimental animals were divided into PBS, Aronia
Bio-active Fractions (ABF), Fucoidan (Fu) and aronia
extract-fucoidan complex (Cyaplex-F8) experimental groups, and 800
mg/kg of fucoidan (Fu) 200 .mu.l or 100 mg/kg of aronia extract (10
mg/mL) 200 .mu.l was orally administered to each experimental group
suitably and Cyaplex-F8 (Fu 80 mg, ABF 10 mg/mL) 200 .mu.l was
orally administered daily for 22 weeks at Fu 800 mg and ABF 100
mg/kg, and the weight, survival rate and number and area of induced
tumor nodules were checked once a week.
[0090] As a result, as shown in FIG. 7 to FIG. 9, tumor nodules
appeared from the 9.sup.th week in the PBS experimental group,
which was applied with carcinogens and tumor accelerators and
administered drugs and since then, the number and area of tumors
increased, but the effect of inhibiting carcinogenesis was
confirmed in the experimental group administered with Fu or ABF, in
particular, the Cyaplex-F8 experimental group has excellent
carcinogenesis inhibitory efficacy.
[0091] In addition, at week 22 of the last week of the experiment,
heart, lung, liver and spleen tissues of the main organs of each
animal in each experimental group, were extracted and hematoxylin
& eosin staining (H&E staining) was performed.
[0092] As a result, as shown in FIG. 12, no specific difference in
the major organs for each experimental group was confirmed, but in
the case of tumor tissue induced in the back as shown in FIG. 13,
it was confirmed that the tumor was induced in the PBS experimental
group with the largest area, and the Cyaplex-F8 experimental group
had the smallest tumor-induced area.
[0093] On the other hand, from the 11.sup.th week of drug
administration, 200 .mu.l of blood from each experimental animal
was collected by orbital blood-gathering method and then
centrifuged at 13,000 rpm for 10 minutes to separate plasma and
IFN-.gamma. present in plasma was quantitatively analyzed by ELISA
kit (485-MI-100, R&D Systems, Inc, USA).
[0094] As a result, as shown in FIG. 14, it was confirmed that the
homeostasis of immune function in the body was maintained without
significant difference in all the experimental groups from week 11
to week 22.
EXAMPLE 5
Confirmation of Combined Administration Effect of Aronia
Extract-Fucoidan Complex and Anticancer Drug
[0095] The anticancer effect was confirmed according to the
combination administration of a aronia extract-fucoidan complex and
an anticancer agent.
[0096] SK-BR-3 (human breast cancer) was transplanted to Balb/c
(n=5) mice at 1.times.10.sup.5 cells/mice and 1 week after
transplantation, 10 mg/kg of doxorubicin (DOX) was administered
intravenously once a week for 2 weeks and for each experimental
group except for the doxorubicin administration day, 800 mg/kg of
fucoidan (Fu) 200 .mu.l (160 mg/mL) or 100 mg/kg of aronia extract
(ABF; 20 mg/mL) 200 .mu.l was administered orally, respectively and
200 .mu.l (Fu 160 mg, ABF 20 mg/mL) of Cyaplex-F8 was administered
orally for 2 weeks twice a day at Fu 1600 mg and ABF 200 mg/kg.
[0097] As a result, as shown in FIG. 15 and FIG. 16A, it was
confirmed that in the experimental group in which the fucoidan,
aronia extract (ABF), or the aronia extract-fucoidan complex was
administered alone, the tumor size increased, but in the
experimental group in which the doxorubicin and the aronia
extract-fucoidan complex Cyaplex-F8 were treated in combination,
the tumor size was reduced to a level similar to the experimental
group treated with doxorubicin alone.
[0098] In addition, referring to FIG. 16B and FIG. 16C, in the
experimental group treated with doxorubicin alone, a rapid decrease
in body weight of the experimental animals was observed after the
second intravenous administration of doxorubicin, but in the
experimental group treated with doxorubicin and Cyaplex-F8 in
combination, it was found that the weight loss rate was small and
the survival life was extended longer than the doxorubicin-treated
group, compared with the group treated with doxorubicin alone.
[0099] In addition, it was confirmed that in the case of the group
administered with doxorubicin alone, while the leukocyte level was
6.57.times.10.sup.3 cells/.mu.L, as shown in Table 1, the
DOX+Cyaplex-F8 combination administration group increased to
9.54.times.10.sup.3 cells/.mu.L.
TABLE-US-00001 TABLE 1 WBC RBC PLT (X10.sup.3 (X10.sup.6 HGB
(X10.sup.3 cells/ cells/ (g/ HCT MCV MCH MCHC cells/ NEUT LYM MONO
EOS BASO 37 day .mu.L) .mu.L) dL) (%) (fL) (pg) (g/dL) .mu.L) (%)
(%) (%) (%) (%) PBS 20.36 9.8 12.6 42.2 18.2 43 12.8 504 69.7 12.1
6.4 4.2 2.6 Fu 18.41 9.14 10.9 39.8 21.3 43.5 12 1242 67.9 14.3 4.6
6.1 1.5 ABF 14.82 10.81 14.3 39.8 21.3 43.5 12 1740 61.4 20.9 5.4
1.5 1.9 Cyaplex- 17.01 10.47 12.7 47.8 17.3 44.2 13.3 2029 70.2
17.4 2.6 1. 0.5 F8 DOX 6.57 10.99 16.5 52.6 14.8 47.9 15 1954 33.9
53.7 4.3 3 3.2 DOX + 9.54 10.09 14.4 48.1 17.1 47.6 14.3 2600 46.4
36.7 5.1 3.4 2 Cyaplex- F8
EXAMPLE 6
Confirmation of Immune Cell Activity Effect of Aronia
Extract-Fucoidan Complex
[0100] As confirmed in the previous experiment, as the aronia
extract-fucoidan complex was found to increase leukocyte levels,
the effect of the aronia extract-fucoidan complex on immune cells
was confirmed.
[0101] Doxorubicin (DOX) was treated with each 10 mg/kg in Balb/c
(n=5) mice on the 1st and 5th day of the experiment (2 times in
total), and 1,600 mg/kg of Fucoidan (Fu) (160 mg/mL) 500 .mu.l, 200
mg/kg of aronia (ABF) 500 .mu.l (20 mg/mL) and Fu 1,600 mg and ABF
200 mg/kg of aronia extract-fucoidan complex (Cyaplex-F8) 500 .mu.l
(Fu 160 mg, ABF 20 mg/mL) was orally administered daily, excluding
doxorubicin treatment days.
[0102] Before doxorubicin treatment, 200 .mu.l of blood was
collected from all experimental groups, treated with 500 .mu.L of
RBC lysis buffer, reacted for 5 minutes at 4.degree. C., diluted
10-fold with DPBS, centrifuged for 1,500 rpm for 3 minutes, and
then the level of cytotoxic T cells in the blood was checked using
anti-CD3 and anti-CD8, and flow cytometry of NK cells in the blood
was performed using anti-CD49b (DX5).
[0103] In addition, the spleens of the experimental animals were
extracted on the 14.sup.th day, the last day of the experiment to
measure the spleen/body weight.
[0104] As a result, as shown in FIG. 17, after administration of
doxorubicin, immune cells decreased in all experimental groups, but
the experimental group treated with the aronia extract-fucoidan
complex exhibited superior immune cell (CD8 T cell and NK cell)
recovery ability than the experimental group treated with the
aronia extract (ABF) and fucoidan alone and as shown in FIG. 18, it
was confirmed that the reduced body weight was best recovered in
the experimental group in which the doxorubicin and the aronia
extract-fucoidan complex were treated in combination.
[0105] From the above results, it was confirmed that the aronia
extract-fucoidan complex recovers the weight reduced by doxorubicin
treatment through the combination treatment.
[0106] While the present invention has been particularly described
with reference to specific embodiments thereof, it is apparent that
this specific description is only a preferred embodiment and that
the scope of the present invention is not limited thereby to those
skilled in the art. That is, the practical scope of the present
invention is defined by the appended claims and their
equivalents.
* * * * *