U.S. patent application number 15/452376 was filed with the patent office on 2017-07-06 for chlorophyll c containing degranulation suppressor.
This patent application is currently assigned to MARUHA NICHIRO CORPORATION. The applicant listed for this patent is MARUHA NICHIRO CORPORATION. Invention is credited to Akira KAMATA, Tatsuya KONISHI, Hiroshi ODA, Tadakazu TAMAI, Hisashi YOSHIOKA.
Application Number | 20170189377 15/452376 |
Document ID | / |
Family ID | 48697613 |
Filed Date | 2017-07-06 |
United States Patent
Application |
20170189377 |
Kind Code |
A1 |
YOSHIOKA; Hisashi ; et
al. |
July 6, 2017 |
CHLOROPHYLL C CONTAINING DEGRANULATION SUPPRESSOR
Abstract
Degranulation suppressors are provided that are effective
against allergic disease and osteoarthritis. The degranulation
suppressors contain chlorophyll c.
Inventors: |
YOSHIOKA; Hisashi;
(Kumamoto, JP) ; KAMATA; Akira; (Ibaraki, JP)
; KONISHI; Tatsuya; (Ibaraki, JP) ; ODA;
Hiroshi; (Ibaraki, JP) ; TAMAI; Tadakazu;
(Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MARUHA NICHIRO CORPORATION |
Tokyo |
|
JP |
|
|
Assignee: |
MARUHA NICHIRO CORPORATION
Tokyo
JP
|
Family ID: |
48697613 |
Appl. No.: |
15/452376 |
Filed: |
March 7, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14367638 |
Jun 20, 2014 |
9636326 |
|
|
PCT/JP2012/084114 |
Dec 28, 2012 |
|
|
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15452376 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 33/105 20160801;
A61K 31/409 20130101; A23V 2002/00 20130101; A61K 36/03 20130101;
A61P 43/00 20180101; A61K 31/40 20130101; A61P 19/02 20180101; A23V
2200/304 20130101; A23V 2002/00 20130101; A61K 31/737 20130101;
A61K 31/7008 20130101; A61P 37/08 20180101; A23V 2250/21 20130101;
A23L 33/10 20160801; A23L 17/60 20160801 |
International
Class: |
A61K 31/409 20060101
A61K031/409; A61K 31/7008 20060101 A61K031/7008; A61K 31/737
20060101 A61K031/737; A61K 36/03 20060101 A61K036/03 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 28, 2011 |
JP |
2011-288085 |
Claims
1. A method for alleviating or treating osteoarthritis in a
subject, comprising: administering to the subject a degranulation
suppressor composition comprising an effective amount of
chlorophyll c and a pharmaceutically acceptable additive.
2. The method of claim 1, wherein the chlorophyll c has been
extracted from a brown alga.
3. The method of claim 2, wherein the brown alga is selected from
the group consisting of hormeri (Turner) C. Agardh, cava Kjellman,
kurome Okamura, and kelp (Laminariaceae Bory).
4. The method of claim 1, wherein the amount of chlorophyll c
ranges from about 0.1 to 100 mg by dry weight in the total
composition.
5. The method of claim 1, wherein the degranulation suppressor
composition further contains glucosamine and/or chondroitin
sulfate.
6. The method of claim 4, wherein the degranulation suppressor
composition further contains glucosamine and/or chondroitin
sulfate.
7. The method of claim 1, wherein the composition comprises an
algal powder or an algal extract, said algal powder or algal
extract having said chlorophyll c.
8. The method of claim 7, wherein the chlorophyll c has been
extracted from a brown alga.
9. The method of claim 8, wherein the brown alga is selected from
the group consisting of hormeri (Turner) C. Agardh, cava Kjellman,
kurome Okamura, and kelp (Laminariaceae Bory).
10. The method of claim 1, wherein the amount of chlorophyll c
ranges from about 0.1 to 10 wt % in the total composition.
11. The method of claim 7, wherein the degranulation suppressor
composition further contains glucosamine and/or chondroitin
sulfate.
Description
[0001] This application is a Division of U.S. patent application
Ser. No. 14/367,638 filed on Jun. 20, 2014, which is the National
Phase of PCT/JP2012/084114 filed Dec. 28, 2012, which claims
priority under 35 U.S.C. .sctn.119(a) to Patent Application No.
2011-288085 filed in Japan on Dec. 28, 2011, all of which are
hereby expressly incorporated by reference into the present
application.
TECHNICAL FIELD
[0002] The present invention relates to degranulation suppressors
containing chlorophyll c.
BACKGROUND ART
[0003] In recent years, as the eating habit of Japanese people has
changed and as air pollution and infections have decreased, the
incidences of various allergic diseases typified by pollinosis,
food allergy, atopic dermatitis, bronchial asthma and contact
hypersensitivity are increasing rapidly, and so are the numbers of
patients suffering from osteoarthritis and other bone/joint related
diseases as aging accelerates in the Japanese society.
[0004] According to the 2003 Survey of Health and Welfare compiled
by the Ministry of Health, Labor and Welfare (MHLW), more than
forty million people accounting for about a third (35.9%) of
Japan's population, irrespective of sex and age, present with
allergic symptoms in either the skin, respiratory organs, or the
eye and nose.
[0005] Data by disease can be found in the 2010 Report of Akazawa
Group in Scientific Research under MHLW grant; in the 2005-2008
period, the prevalence of infantile bronchial asthma was 13.8% at
ages 6-7, 9.5% at ages 13-14, and 8.3% at ages 16.about.18 whereas
the prevalence of bronchial asthma in adults (aged 20-44) was 5.4%.
As regards allergic rhinitis and pollinosis, the 2009 Clinical
Guideline for Nasal Allergy reports that in the 1998-2008 period,
the overall prevalence of allergic rhinitis increased from 29.8% to
39.4% and that of Japanese cedar pollinosis increased from 16.2% to
26.5%. Speaking of atopic dermatitis, its prevalence was 13.2% at
age 3, 10.6% in sixth graders at elementary school, 9.4% in the
twenties, 8.3% in the thirties, 4.8% in the forties, and 2.5% in
the fifties to sixties (the 2008 Clinical Guideline for Atopic
Dermatitis).
[0006] Allergic diseases occurring in the daily life of Japanese
people are one of the major factors that lower their quality of
life (QOL). According to the 2003 MHLW Survey, idem, approximately
a half (53.4%) of patients complaining of allergic symptoms in
respiratory organs responded that they "could not sleep well" and
approximately 40% (37.2%) of patients suffering allergic symptoms
in the eye and nose responded that they "could not concentrate on
work, housekeeping, or schoolwork."
[0007] Conventional treatments of allergic diseases have involved
medication of drugs such as antihistamines and steroids as part of
symptomatic therapy but a problem with these drugs is that they
cause serious side effects if administered for an extended period
of time. It has, therefore, been desired to develop an ingredient
that can be manufactured in a safe and easy way and which is
capable of alleviating, suppressing or preventing allergic
symptoms, with the additional feature that it can be integrated
into the daily dietary life.
[0008] As aging accelerates in the Japanese society, bone/joint
diseases such as osteoarthritis and osteoporosis are also
increasing in number year by year. According to the 2007 National
Livelihood Survey compiled by MHLW, two major causes for needed
care were joint disease and fracture/falling which accounted for
20.2% (ranking first) and 12.5% (ranking fourth), respectively, of
the persons in need of support, whereas they accounted for 9.4%
(ranking fourth) and 8.4% (ranking fifth), respectively, of the
persons in need of nursing care; thus, bone/joint diseases are one
of the major factors that degrade the activities of daily living
(ADL) and QOL of Japanese people. In 2007, the Japanese Orthopaedic
Association proposed a concept called "locomotive syndrome" which
refers to either a state that requires nursing care or one
presenting a high risk for the need of nursing care, both due to a
locomotor disability and in 2009, Yoshimura et al. at the
University of Tokyo Hospital conducted a large-scale resident's
cohort study called Research on Osteoarthritis/Osteoporosis Against
Disability (ROAD) and reported that as many as forty-seven million
people (aged 40 and above) were found to have a locomotive
syndrome. Obviously, there exists an urgent need to cope with
allergic disease and bone/joint disease because, for one thing,
they are quite serious diseases that can potentially degrade ADL
and QOL as well as shortening the health span and, for another
thing, the ever-growing medical costs must be reduced.
[0009] Under these circumstances, active efforts are being made to
develop pharmaceutical drugs and food ingredients that can
alleviate, suppress or prevent allergic disease and
osteroarthritis. Food ingredients so far reported to have an
allergy suppressing action include, for example, Lactobacillus
(Patent Document 1), royal jelly (Patent Document 2), fucoidan
which is a water-soluble polysaccharide component in seaweeds
(Patent Documents 3 and 4), and alginic acid (Patent Document
5).
[0010] It is known that the degranulation reaction of mast cells or
basophiles is critical to the onset of various allergic diseases
including pollinosis, atopic dermatitis, allergic rhinitis, and
allergic conjunctivitis. When an allergen invades the body, the
production of an allergen-specific antibody is induced and the
allergen-specific antibody produced binds to the surface of a cell
such as a mast cell or a basophile, whereupon sensitization is
established. When the body is invaded again by an allergen, it
binds the allergen-specific antibody on the surface of the mast
cell or basophile, whereupon a granule containing a chemical
mediator is released out of the cell, triggering an allergic
reaction such as enhancement of vascular permeability or
constriction of bronchial smooth muscle. Hence, one effective way
to suppress various allergic diseases including pollinosis, atopic
dermatitis, allergic rhinitis, and allergic conjunctivitis is by
suppressing this degranulation reaction.
[0011] Food ingredients so far reported to have the degranulation
suppressing action include, for example, a plant (galena) extract
(Patent Document 6) and phycoerythrin found in red algae
(Non-Patent Document 1).
[0012] Furthermore, a recent study reported that a glucosamine
hydrochloride having an osteroarthritis suppressing action exerted
a degranulation suppressing action and also reported was an
osteroarthritis suppressing effect mediated by the degranulation
suppressing action (Non-Patent Document 2). These results suggest
that by investigating the degranulation suppressing action, not
only the effect for suppressing allergic disease but also the
effect for suppressing osteroarthritis can be verified.
[0013] Aside from the glucosamine hydrochloride, various other
ingredients have been reported to have the osteroarthritis
suppressing action and they include a chondroitin sulfate
(Non-Patent Documents 3, 4, 5, 6, 7 and 8) and plant extracts such
as those of a plant of the family Compositae (Patent Document No.
7) and spices (Patent Document 8).
[0014] Chlorophyll which is a photosynthesis pigment typically
contained in plants may be defined as a metal complex of cyclic
tetrapyrrole coordinated with Mg or Zn and while it is primarily
used as a colorant and food additives that take advantage of its
nature as pigment, chlorophyll's recent medical application in
photodynamic therapy (PDT) is worth mention (Non-Patent Document
9). In this connection, chlorophyll of high purity is extracted
from plants, algae, etc. using organic solvents, etc. and is
subsequently separated and purified using an absorption photometer,
high-performance liquid chromatography, etc. (Non-Patent Documents
10, 11, and 12). Concerning chlorophyll's functionality, a report
was made of a chlorophyllin copper complex as a chlorophyll analog
having an antioxidization-based antimicrobial, anticancer,
antiatherosclerotic, antipsoriatic and other beneficiary actions
(Non-Patent Document 13); however, no report has yet been published
about the degranulation suppressing effect of chlorophyll that
contributes to allergy suppression as by suppressing vascular
permeability or to osteroarthritis suppression.
CITATION LIST
Patent Literature
[0015] Patent Document 1: Japanese Patent No. 4212838
[0016] Patent Document 2: WO2004/019971 A1
[0017] Patent Document 3: JP Hei 11-21247 A
[0018] Patent Document 4: JP Hei 10-72362 A
[0019] Patent Document 5: Japanese Patent No. 4012977
[0020] Patent Document 6: JP Hei 2010-70531 A
[0021] Patent Document 7: WO2008/010335
[0022] Patent Document 8: JP Hei 2006-22002 A
Non-Patent Literature
[0023] Non-Patent Document 1: Food science and technology research.
Vol. 17, No. 2, 171-177 (2011)
[0024] Non-Patent Document 2: Life science. 86(9-10), 337-343
(2010)
[0025] Non-Patent Document 3: The journal of Rheumatology. 27,
205-211 (2000)
[0026] Non-Patent Document 4: The journal of Rheumatology. 23,
1385-1391 (1996)
[0027] Non-Patent Document 5: The journal of Rheumatology. 28,
173-181 (2001)
[0028] Non-Patent Document 6: The new ENGLAND journal of medicine.
354, No. 8, 795-808 (2006)
[0029] Non-Patent Document 7: Journal of the American pharmacists
association. Vol. 46, No. 1, 14-24 (2006)
[0030] Non-Patent Document 8: Drug aging. 24(7), 573-580 (2007)
[0031] Non-Patent Document 9: Chlorophyll--Structures, Reactions,
Functions--; pp. 69-70 (2011)
[0032] Non-Patent Document 10: Researches in Organic Geochemistry.
22, pp. 43-55 (2007)
[0033] Non-Patent Document 11: PNAS. Vol. 102, pp. 850-855
(2005)
[0034] Non-Patent Document 12: Organic & Biomolecular
Chemistry. Vol. 7, No. 10, pp. 1989-2224 (2009)
[0035] Non-Patent Document 13: New jersey medicine. 85(8), 669-673
(1988)
SUMMARY OF INVENTION
Technical Problem
[0036] The problem the present invention addresses is the recent
rapid increase in the number of patients with allergic disease and
bone/joint disease about which something must be done as soon as
possible; to reduce the incidence of these diseases, the present
invention provides degranulation suppressing agents that contain
chlorophyll c as an active ingredient that shows a degranulation
suppressing action.
Solution to Problem
[0037] As a result of intensive studies, the present inventors
found that while there are a lot of chlorophylls available,
chlorophyll c or an algal powder or an algal extract that contain
chlorophyll c as an ingredient involved in activity has a
degranulation suppressing action, and this finding has led to the
accomplishment of the present invention. Briefly, the present
invention has as its objective providing chlorophyll c or a
composition for alleviating, treating or preventing allergic
disease or osteoarthritis by suppressing the degranulation reaction
of mast cells, basophiles, etc., said composition being
characterized by comprising chlorophyll c as an active
ingredient.
[0038] Briefly, the present invention relates to a degranulation
suppressing composition comprising chlorophyll c. This
degranulation suppressing composition may be used as an
osteroarthritis suppressing agent or an allergy suppressing agent.
It may also be used as a food in which chlorophyll c has been
incorporated. Specifically, it may be used as a degranulation
suppressing, functional food comprising chlorophyll c. It may also
be used as a feed or pet food in which chlorophyll c has been
incorporated. Specifically, it may be used as a degranulation
suppressing feed or pet food in which chlorophyll c has been
incorporated.
Advantageous Effects of Invention
[0039] According to the present invention, by using chlorophyll c
or an extract of a particular kind of seaweeds that contain
chlorophyll c, there can be obtained a degranulation suppressor
that can be taken orally and which comprises chlorophyll c as an
active ingredient; use can be made of the suppressor as a
pharmaceutical, a food or beverage, a feed or a pet food. By orally
taking such pharmaceutical, food or beverage, feed or pet food, the
degranulation reaction of mast cells or basophiles can be
suppressed to enable alleviation, suppression or prevention of
allergic disease or osteoarthritis.
BRIEF DESCRIPTION OF DRAWINGS
[0040] FIG. 1 is a graph showing in terms of percent degranulation
the result of a test conducted to evaluate the activity of
chlorophyll species a to d for suppressing degranulation of rat
basophile derived cell line RBL-2H3.
[0041] FIG. 2 is a graph showing in terms of percent cell survival
the result of a test conducted to evaluate the cytotoxicity of
chlorophyll c2 on rat basophile derived cell line RBL-2H3.
[0042] FIG. 3 is a graph showing in terms of percent degranulation
the result of a test conducted to evaluate how effectively
chlorophyll c as extracted from hormeri (Turner) C. Agardh with
water, or hydrous ethanol at 20%, 40%, 60% or 80%, or 100% ethanol
were able to suppress degranulation of rat basophile derived cell
line RBL-2H3.
[0043] FIG. 4 is a graph showing the percent degranulation of each
of the fractions as obtained by HPLC on an extract of hormeri
(Turner) C. Agardh with an aqueous ethanol solution.
[0044] FIG. 5 is a photograph showing a spot of an active fraction
(Fr. J in FIG. 4) as observed in thin-layer chromatographic
analysis.
[0045] FIG. 6 is a set of charts showing the absorbances at
absorption wavelength 450 nm of an active fraction (Fr. J in FIG.
4) as obtained by HPLC and an authentic sample of chlorophyll
c2.
[0046] FIG. 7 is a set of charts showing the absorption spectra of
an active fraction (Fr. J in FIG. 4) as obtained by HPLC and an
authentic sample of chlorophyll c2.
[0047] FIG. 8 is a graph showing in terms of percent degranulation
the result of a test conducted to evaluate the activity of
chlorophyll c1 or c3 for suppressing degranulation of rat basophile
derived cell line RBL-2H3.
[0048] FIG. 9 is a graph showing in terms of percent cell survival
the result of a test conducted to evaluate the cytotoxicity of
chlorophyll c1 or c3 on rat basophile derived cell line
RBL-2H3.
[0049] FIG. 10 is a graph showing the percent vascular permeability
of SD rat groups that were subjected to a test for evaluating the
vascular permeation suppressing activity of a feed supplemented
with chlorophyll c as extracted from hormeri (Turner) C.
Agardh.
[0050] FIG. 11 is a graph showing the number of nose scratches per
hour by C57BL/6 mice that were subjected to a test for evaluating
the pollinosis suppressing activity of a feed supplemented with
chlorophyll c as extracted from hormeri (Turner) C. Agardh.
[0051] FIG. 12 is a graph showing the result of a test performed on
DA rats to evaluate the osteoarthritis suppressing activity after
they were given a feed supplemented with chlorophyll c as extracted
from hormeri (Turner) C. Agardh, a feed supplemented with
glucosamine, a feed supplemented with SCP (NB), a feed supplemented
with a chlorophyll c extract and glucosamine, and a feed
supplemented with a chlorophyll c extract and SCP (NB); the data
represent the sum of arthritis scores from the day of onset of the
disease (day 0) up to the 15.sup.th day of onset.
DESCRIPTION OF EMBODIMENTS
[0052] As noted above, the present invention relates to
degranulation suppressor compositions comprising chlorophyll c and
it will be described below in greater details.
[0053] Chlorophyll c is a porphyrin compound contained in certain
algae whose names are mentioned below; it has the structural
formula:
##STR00001##
[0054] and consists of three species c1, c2 and c3. The structures
of chlorophylls c1, c2 and c3 are, unlike molecules having the
chlorin .pi. skeleton such as chlorophylls a, b and d which are
incapable of suppressing the degranulation reaction, characterized
by having the porphyrin .pi. skeleton with a C17-C18 double bond as
depicted in the above structural formula. Their molecular weights
are close to each other--c1 (610), c2 (608) and c3 (653)--and so
are the spectra for absorbance maxima in diethyl ether--c1 (444.4
nm), c2 (447.8 nm) and c3 (450.8 nm). Either form of chlorophyll c
starts to be biosynthesized in algae from divinyl
protochlorophyllide a and exhibits a photosynthetic action and an
antioxidizing action.
[0055] It is through the suppression of the degranulation from mast
cells or basophiles that chlorophyll c is capable of alleviating,
suppressing and preventing allergic disease or osteoarthritis.
[0056] The starting materials from which the chlorophyll c to be
contained in the composition of the present invention can be
obtained are not particularly limited. Chlorophyll c which is one
of the photosynthetic pigments found in plants is known to be
contained in oxygen-producing photosynthetic organisms,
particularly of the Division Dinoflagellata, the Division
Cryptophyta, the Division Heterokontophyta including the Class
Chrysophyceae, the Class Raphidophyceae, the Class Phaeophyceae,
the Class Bacillariophyceae (Diatomea) and the Class
Eustigmatophyceae, as well as the Division Haptophyta. Preferred
examples are brown algae of: the order ECTOCARPALES Setchell et
Gardner, 1922, the family Ectocarpaceae C. Agardh, 1828, the genus
Acinetospora Bomet, 1892 [Ectocarpus filamentosus, Ectocarpus
ugoensis], the genus Asteronema Delepine et Asensi, 1975 nom. cons.
[breviarticulatum (J. Agardh) Ouriques et Bouzon, Ectocarpus
breviarticulatus, Feldmannia breviarticulata, Hincksia
breviarticulata], the genus Ectocarpus Lyngbye, 1819 nom. cons.
[arctus Kuetzing, confervoides, fusiformis Nagai, laurenciae
Yamada, siliculosus (Dillwyn) Lyngbye, socialis Setchell et
Gardner, yezoensis Yamada et Tanaka], the genus Feldmannia Hamel,
1939 [formosana (Yamada) Itono, Ectocarpus formosanus, irregularis
(Kuetzing) Hamel, Ectocarpus irregularis, Ectocarpus izuensis], the
genus Hincksia J. E. Gray, 1864 [granulosa (J. E. Smith) Silva,
Ectocarpus granulosus, Giffordia granulosa, indica (Sonder) J.
Tanaka, Ectocarpus indicus, Feldmannia indica, Giffordia indica,
mitchellae (Harvey) Silva, Ectocarpus mitchellae, Giffordia
mitchellae, Ectocarpus ovatus, Giffordia ovata, Ectocarpus
sandrianus, Giffordia sandriana], the genus Laminariocolax Kylin,
1947 [aecidioides (Rosenvinge) Peters, Gononema aecidioides,
Streblonema aecidioides], the genus Laminarionema Kawai et
Tokuyama, the genus Spongonema Kuetzing, 1849 [tomentosum (Hudson)
Kuetzing], the genus Streblonema Derbes et Solier in Castagne, 1851
[evagatum Setchell et Gardner, fasciculatum Thuret], the family
Pylaiellaceae Pedersen, 1984, the genus Bachelotia (Bornet) Kuckuck
ex Hamel, 1939 [antillarum (Grunow) Gerloff], the genus Pylaiella
Bory, 1823 orth. cons. [littoralis (Linnaeus) Kjellman], the family
Sorocarpaceae Pedersen, 1977, the genus Botrytella Bory, 1822
[parva (Takamatsu) Kim, micromora, Sorocarpus uvaeformis], the
genus Polytretus Sauvageau, 1900 [reinboldii (Reinke) Sauvageau,
Ectocarpus intricatus, Ectocarpus iwadatensis, Ectocarpus
recurvatus]; the order RALFSIALES Nakamura, 1972 emend. Lin et
Kawai (61), the family Lithodermataceae Hauck,1883, the genus
Pseudolithoderma Svedelius in Engler et Prantl, 1911 [subextensum
(Waern) S. Lund], the family Ralfsciaceae Fardlow, 1881, the genus
Analipus Kjellman, 1889 [filiformis (Ruprecht) Papenfuss, gunjii
(Yendo) Kogame et Yoshida, Chordaria gunjii, japonicus (Harvey)
Wynne, Heterochordaria abietina], the genus Diplura Hollenberg,
1969 [simplex J. Tanaka et Chilhara], the genus Endoplura
Hollenberg, 1969 [aurea Hollenberg],_the genus Hapalospongidion
Saunders, 1899 [schmidtii (Weber-van Bosse) Silva, Mesospora
schmidtii], the genus Heteroralfsia Kawai, 1989 [saxicola (Okamura
et Yamada) Kawai, Gobia saxicola, Saundersella saxicola], the genus
Ralfsia Berkeley in Smith et Sowerby, 1843 [fungiformis (Gunnerus)
Setchell et Gardner, verrucosa (Areschoug) Areschoug]; the order
SYRINGODERMATALES Henry, 1984, the family Synringodermataceae
Henry, 1084, the genus Syringoderma Levring, 1940 [abyssicola
(Setchell et Gardner) Levring]; the order SPHACELARIALES Migula
1909, the family Sphacelariaceae Decaisne, 1842, the genus
Sphacelaria Lyngbye in Hornemann, 1818 [californica Sauvageau,
cornuta Sauvageau, divaricata Montagne, furcigera sensu Reinke,
hizikiae, prostrata, radicans sensu Yendo, tenuis, nipponica
Kitayama, plumigera Holmes ex Hauck, rigidula Kuetzing, apicalis,
caespitosa, expansa, furcigera, iridaeophytica, iwagasakensis,
linearis, sessilis, subfusca, variabilis, solitaria (Pringsheim)
Kylin, divaricata, f. japonica, shiiyaensis, viridis, tribuloides
Meneghini, yamadae Segawa, pyriformis, radiata], the family
Stypocaulaceae Oltmanns, 1922, the genus Halopteris Kuetzing, 1843
[filicina (Grateloup) Kuetzing], the genus Stypocaulon Kuetzing,
1843 [durum (Ruprecht) Okamura]; the order DICTYOTALES Kjellman in
Engler et Prantl, 1896, the family Dictyotaceae Lamouroux ex
Dumortier, 1822, the genus Dictyopteris Lamouroux, 1809 nom. cons.
[divaricata (Okamura) Okamura, fucoides Tanaka, latiuscula
(Okamura) Okamura, papenfussii Tanaka, plagiogramma (Montagne)
Vickers, polypodioides (De Candolle) Lamouroux, membranacea,
prolifera (Okamura) Okamura, punctata Noda, repens (Okamura)
Boergesen, undulata Holmes], the genus Dictyota Lamouroux, 1809
nom. cons. [bartayresii Lamouroux, dentata Lamouroux, dichotoma
(Hudson) Lamouroux, dilatata Yamada, divaricata Lamouroux, friabdis
Setchell, linearis (C. Agardh) Greville, maxima Zanardini, patens
J. Agardh, spathulata Yamada, spinulosa Harvey], the genus
Distromium Levring, 1940 [decumbens (Okamura) Levring,
Chlanidophora repens, Chlanidote decumbens], the genus
Homoeostrichus J. Agardh, 1894 [flabellatus Okamura], the genus
Lobophora J. Agardh, 1894 [variegata (Lamouroux) Womersley ex
Oliveira, Gymnosorus collaris, Pocockiella variegata], the genus
Pachydictyon J. Agardh, 1984 [coriaceum (Holmes) Okamura], the
genus Padina Adanson, 1763 nom. cons. [arborescens Holmes,
australis Hauck, boryana Thivy, crassa Yamada, fasciata Ni-Ni-Win,
Arai et Kawai, japonica Yamada, melemele Abbot et Magruder, minor
Yamada, moffittiana Abbot et Huisman, okinwaensis Ni-Ni-Win, Arai
et Kawai, ryukyuana Y. P. Lee et Kamura, stipitata Tanaka et
Nozawa, terricolor Ni-Ni-Win, Arai et Kawai, undulata Ni-Ni-Win,
Arai et Kawai], the genus Ruglopteryx DeClerk et Coppjans 2009
[okamurae (Dawson) Hwang, Lee et Kim in Hwang et al., Dilophus
okamurae, Dilophus marginatus], the genus Spatoglossum Kuetzing,
1843 [crassum J. Tanaka, variabile sensu Yendo, latum J. Tanaka,
pacificum Yendo, cornigerum sensu Yendo, solieri sensu Yendo], the
genus Stypopodium Kuetzing, 1843 [zonale (Lamouroux) Papenfuss,
lobatum], the genus Zonaria C. Agardh, 1817 nom. cons. [diesingiana
J. Agardh, stipitata Tanaka et K. Nozawa]; the order CHORDARIALES
Setchell et Gardner, 1925, the family Acrotrichaceae Kuckuck, 1929,
the genus Acrothrix Kylin, 1907 [gracilis Kylin, pacifica Okamura
et Yamada], the family Chordariaceae Greville, 1830, the genus
Chordaria C. Agardh, 1817 nom. cons. [chordaeformis (Kjellman)
Kawai et Kim, flagelliformis (O. F. Mueller) C. Agardh, gracilis
Setchell et Gardner], the genus Cladosiphon Kuetzing, 1843
[Okamuranus Tokida, Eudesme virescens sensu Okamura, umezakii
Ajisaka], the genus Eudesme J. Agardh, 1882 [virescens (Carmichael
ex Berkeley) J. Agardh], the genus Heterosaundersella Tokida, 1942
[hattoriana Tokida], the genus Myriogloea Kuckuck ex Oltamanns,
1922 [simplex (Segawa et Ohta) Inagaki], the genus Papenfussiella
Kylin, 1940 [kuromo (Yendo) Inagaki, Myriocladia kuromo], the genus
Saundersella Kylin, 1940 [simplex (Saunders) Kylin, Gobia simplex],
the genus Sauvageaugloia Hamel ex Kylin, 1940 [ikomae (Narita)
Inagaki], the genus Sphaerotrichia Kylin, 1940 [divaricata (C.
Agardh) Kylin, japonica, sadoensis, Castagnea divaricata, Chordaria
cladosiphon, firma (E. Gepp) Zinova (20), Chordaria firma], the
genus Tinocladia Kylin, 1940 [crassa (Suringar) Kylin, Eudesme
crassa], the family Elachistaceae Kjellman, 1890, the genus
Elachista Duby, 1830 nom. et orth. cons. [coccophorae Takamatsu,
orbicularis, Gonodia orbicularis, Okamura Yoshida, fucicola sensu
Okamura, globosa Takamatsu, taeniaeformis Yamada, crassa, flaccida
auct. Japon., sadoensis, tenuis Yamada, vellosa Takamatsu], the
genus Halothrix Reinke,1888 [ambigua Yamada, lumbricalis sensu
Takamatsu, tortuosa], the genus Leptonematella Silva, 1959
[fasciculata (Reinke) Silva], the family Leathesiaceae Farlow,
1881, the genus Kurogiella Kawai, 1993 [saxicola Kawai], the genus
Leathesia S. F. Gray, 1821 [crassipilosa Takamatsu, difformis
(Linnaeus) Areschoug, monilicellulata Takamatsu, primaria
Takamatsu, pulvinata Takamatsu, sadoensis Inagaki, saxicola
Takamatsu, granulosa], the genus Phaeophysema Tanaka, Uwai et Kawai
2010 [pulvinata (Takamatsu) Tanaka, Uwai et Kawai, sphaerocephala
(Yamada) Tanaka, Uwai et Kawai], the genus Vimineolaethesia Tanaka,
Uwai et Kawai [japonicaa (Inagaki) Tanaka, Uwai et Kawai], the
genus Myriactula Kuntze, 1898 [sargassi (Yendo) Feldmann,
saromaensis Yamada et Iwamoto], the family Petrospongiaceae Recault
in Recault et al. 2009, the genus Petrospongium Naegeli ex
Kuetzing, 1858 [rugosum (Okamura) Setchell et Gardner,
Cylindrocarpus rugosus], the family Myrionemataceae Naegeli, 1847,
the genus Compsonema Kuckuck, 1899 [nummuloides Setchell et
Gardner], the genus Hecatonema Sauvageau [maculans (Collins)
Sauvageau, terminale (Kuetzing) Kylin], the genus Microspongium
Reinke, 1888 [Myrionema globosum], the genus Myrionema Greville,
1827 [corunnae Sauvageau], the genus Protectocarpus Kuckuck et
Kommann, 1955 [speciosus (Boergesen) Kuckuck ex Kommann, Compsonema
ramulosum sensu Noda], the family Spermatochnaceae Kjellman, 1890,
the genus Nemacystus Derbes et Solier, 1850 [decipiens (Suringar)
Kuckuck], the genus Stilophora J. Agardh, 1841 nom. cons. [tenella
(Esper) Silva, rhizodes]; the order DICTYOSIPHONALES Setchell et
Gardner, 1925, the family Asperococcaceae Farlow, 1881, the genus
Asperococcus Lamouroux, 1813 [bullosus Lamouroux, turneri], the
genus Melanosiphon Wynne, 1969 [intestinalis (Saunders) Wynne,
Myelophycus intestinalis], the family Coilodesmaceae Setchell et
Gardner, 1925, the genus Coilodesme Stroemfelt, 1886 [cystoseirae
(Ruprecht) Setchell et Gardner, japonica Yamada, cystoseirae sensu
Yendo], the family Delamareaceae A. D. Zinova, 1953, the genus
Delamarea Hariot, 1889 [attenuata (Kjellman) Rosenvinge], the genus
Stschapovia A. D. Zinova, 1954, the family Dictyosiphonaceae
Kuetzving, 1849, the genus Dictyosiphon Greville, 1830 nom. cons.
[chordaria Areschoug, foeniculaceus (Hudson) Greville,
hippuroides], the family Punctariaceae (Thuret) Kjellman, 1880, the
genus Pogotrichum Reinke, 1892 [yezoense (Yamada et Nakamura) Sakai
et Saga, Litosiphon yezoense], the genus Punctaria Greville, 1830
[flaccida Nagai, kinoshitae Yamada et Iwamoto, tenuis, latifolia
Greville, mageshimensis Tanaka, occidentalis Setchell et Gardner,
chartacea sensu Umezaki, conglomerata, pilosa Umezaki, plantaginea
(Roth) Greville, rubescens sensu Yendo, projecta Yamada], the genus
Trachynema Pedersen, 1985 [groenlandicum (Lund) Pedersen], the
family Striariaceae Kjellman, 1890, the genus Coelocladia
Rosenvinge, 1893 [arctica Rosenvinge], the genus Stictyosiphon
Kuetzing, 1843 [soriferus (Reinke) Rosenvinge, Kjellmania
arasakii], the genus Striaria Greville, 1828 [attenuata (Greville)
Greville]; the order SCYTOSIPHONALES J. Feldmann, 1949, the family
Chnoosporaceae Setchell et Gardner, 1925, the genus Chnoospora J.
Agardh,1847 [implexa J. Agardh, minima (Hering) Papenfuss,
pacifica], the family Scytosiphonaceae Farlow, 1881, the genus
Colpomenia (Endlicher) Derbes et Solier in Castagne, 1851 [bullosa
(Saunders) Yamada, sinuosa f. deformans, peregrina (Sauvageau)
Hamel, phaeodactyla Wynne et J. N. Norris, sinuosa (Mertens ex
Roth) Derbes et Solier], the genus Hydroclathrus Bory, 1825
[clathratus (C. Agardh) Howe, tenuis Tseng et Lu], the genus
Myelophycus Kjellman in Engler et Prantl, 1893 [cavus J. Tanaka et
Chihara, simplex (Harvey) Papenfuss, caespitosus], the genus
Petalonia Derbes et Solier, 1850 nom. cons. [binghamiae (J. Agardh)
Vinogradova, Endarachne binghamiae, fascia (O. F. Mueller) Kuntze,
Ilea fascia, zosterifolia (Reinke) Kuntze], the genus Rosenvingea
Boergesen, 1914 [intricata (J. Agardh) Boergesen], the genus
Scytosiphon C. Agardh, 1820 nom. cons. [canaliculatus (Setchell et
Gardner) Kogame, gracilis Kogame, lomentaria (Lyngbye) Link,
tenellus Kogame]; the order CUTLERIALES Oltmanns, 1922, the family
Cutleriaceae Hauck, 1883, the genus Cutleria Greville, 1830
[adspersa (Roth) De Notaris, cylindrica Okamura, multifida (Turner)
Greville]; the order SPOROCHNALES Sauvageau, 1926, the family
Sporochanaceae Greville, 1830, the genus Carpomitra Kuetzing, 1843
nom. cons. [costata (Stackhouse) Batters, cabrerae, the genus
Nereia Zanardini, 1846 [intricata Yamada], the genus Sporochnus C.
Agardh, 1817 [radiciformis (R. Brown ex Turner) C. Agardh,
scoparius]; the order DESMARESTIALES Setchell et Gardner, 1925, the
family Desmarestiaceae (Thuret) Kjellman, 1880, the genus
Desmarestia Lamouroux, 1813 nom. cons. [ligulata (Stackhouse)
Lamouroux, tabacoides Okamura, viridis (Mueller) Lamouroux]; the
order LAMINARIALES Kylin, 1917, the family Akkesiphycaceae Kawai et
Sasaki, 2000, the genus Akkesiphycus Yamada et Tanaka, 1944
[lubricus Yamada et Tanaka], the family Alariaceae Setchell et
Gardner, 1925, the genus Alaria Greville, 1830 nom. cons. [angusta
Kjellman, crassifolia Kjellman, paradisea (Miyabe et Nagai)
Widdowson, Pleuropterum paradiseum, praelonga Kjellman, taeniata
Kjellman], the genus Druehlia Lane et Saunders, 2007 [fistulosa
(Postels et Ruprecht) Lane et Saunders], the genus Undaria
Suringar, 1873 [peterseniana (Kjellman) Okamura, pinnatifida
(Harvey) Suringar, undarioides (Yendo) Okamura], the family
Chordaceae Dumortier, 1822, the genus Chorda Stackhouse, 1797
[asiatica Sasaki et Kawai, Chora filum auct. japon.], the genus
Agarum Dumortier, 1822 nom. cons. [clathratum Dumortier, cribrosum,
oharaense Yamada], the genus Costaria Greville, 1830 [costata (C.
Agardh) Saunders], the family Laminariaceae Bory, 1827, the genus
Ecklonia Hornemann, 1828 [cava Kjellman, kurome Okamura,
stolonifera Okamura], the genus Eckloniopsis Okamura, 1927
[radicosa (Kjellman) Okamura], the genus Eisenia Areschoug, 1876
[arborea Areschoug, bicyclis (Kjellman) Setchell], the genus
Arthrothamnus Ruprecht, 1848 [bifidus (Gmelin) Ruprecht], the genus
Cymathaere[Cymathaere japonica, crassifolia sensu Miyabe et Nagai],
the genus Laminaria Lamouroux, 1813 nom. cons. [yezoensis Miyame],
the genus Saccharina Stackhouse 1809 [angustata (Kjellman) Lane,
Mayes, Druehl et Saunders, Laminaria angustata, cichorioides
(Miyabe) Lane, Mayes, Druehl et Saunders, Laminaria cichorioides,
coriacea (Miyabe) Lane, Mayes, Druehl et Saunders, Laminaria
coriacea, diabolica (Miyabe) Lane, Mayes, Druehl et Saunders,
Laminaria diabolica, gyrata (Kjellman) Lane, Mayes, Druehl et
Saunders, Laminaria gyrata, Kjellmaniella gyrata, japonica
(Areschoug) Lane, Mayes, Druehl et Saunders, Laminaria japonica,
kurilensis Lane, Mayes, Druehl et Saunders, Cymathaere japonica
Miyabe et Nagai, latissima (Linneaus) Lane, Mayes, Druehl et
Saunders, Laminaria saccharina, longipedalis (Okamura) Lane, Mayes,
Druehl et Saunders, Laminaria longipepedalis, longissima (Miyabe)
Lane, Mayes, Druehl et Saunders,
Laminaria longissima, ochotensis (Miyabe) Lane, Mayes, Druehl et
Saunders, Laminaria ochotensis, religiosa (Miyabe) Lane, Mayes,
Druehl et Saunders, Laminaria religiosa, Sculpera Lane, Mayes,
Druehl et Saunders, Kjellmaniella crassifolia, Saccharina
crassifolia, yendoana (Miyabe) Lane, Mayes, Druehl et Saunders,
Laminaria yendoana], the genus Streptophyllopsis Kajimura, 1981
[kuroshioensis (Segawa) Kajimura, Hedophyllum kuroshioense], the
family Pseudochordaceae Kawai et Kurogi, 1985, the genus
Pseudochorda Yamada, Tokida et Inagaki in Inagaki, 1958 [gracilis
Kawai et Nabata, nagaii (Tokida) Inagaki, Chordaria nagaii]; the
order ISHIGEALES Cho et Boo in Cho et al., 2004, the family
Ishigeaceae Okamura in Segawa, 1935, the genus Ishige Yendo, 1907
[okamurae Yendo, sinicola (Setchell et Gardner) Chihara, foliacea];
the order FUCALES Kylin, 1917, the family Fucaceae Adanson, 1763,
the genus Fucus Linnaeus, 1753 [distichus Linnaeus subsp.
evanescens (C. Agardh) Powell, evanescens], the genus Silvetia
Serrao, Cho, Boo et Brawley in Serrao et al. 1999 [babingtonii
(Harvey) Serrao et al., Pelvetia babingtonii, Pelvetia wrightii],
the family Sargassaceae Kuetzing, 1843, the genus Coccophora
Greville, 1830 [langsdorfii (Turner) Greville], the genus
Cystoseira C. Agardh, 1820 nom. cons. [crassipes (Mertens ex
Turner) C. Agardh, Cystophyllum crassipes, geminata C. Agardh,
Cystophyllum geminatum, hakodatensis (Yendo) Fensholt, Cystophyllum
hakodatense], the genus Hormophysa Kuetzing, 1843 [cuneiformis
(Gmelin) Silva, triquetra, Cystoseira prolifera], the genus
Myagropsis Kuetzing, 1843 [myagroides (Mertens ex Turner) Fensholt,
yendoi, Cystophyllum caespitosum, Cystophyllum sisymbrioides,
Cystophyllum turneri], the genus Sargassum C. Agardh, 1820 nom.
cons. [alternato-pinnatum Yamada, asymmetricum, ammophilum Yoshida
et T. Konno, araii Yoshida, assimile Harvey, autumnale Yoshida,
boreale Yoshida et Horiguchi, bulbiferum Yoshida, carpophyllum J.
Agardh, angustifolium sensu Yamada, vulgare var. linearifolium
sensu Yendo, confusum C. Agardh, crassifolium J. Agardh,
berberifolium, crispifolium Yamada, cristaefolium C. Agardh,
denticarpum Ajisaka, duplicatum Bory, brevifolium sensu Yendo,
sandei, filicinum Harvey, fulvellum (Turner) C. Agardh, enerve,
fusiforme (Harvey) Setchell, Hizikia fusiformis, giganteifolium
Yamada, glaucescens J. Agardh, hemiphyllum (Turner) C. Agardh,
hornei (Turner) C. Agardh, ilicifolium (Turner) C. Agardh var.
conduplicatum Grunow, incanum Grunow, kashiwajimanum Yendo,
kushimotense Yendo, longifructum Tseng et Lu, macrocarpum C.
Agardh, serratifolium auct. japon., micracanthum (Kuetzing)
Endlicher, microceratium (Mertens ex Turner) C. Agardh, miyabei
Yendo, kjellmanianum, muticum (Yendo) Fensholt, kjellmanianum f.
muticum, myriocystum J. Agardh, opacum, nigrifolium Yendo,
nipponicum Yendo, okamurae Yoshida et T. Konno, pallidum (Turner)
C. Agardh, patens C. Agardh, piluliferum (Turner) C. Agardh,
pinnatifidum Harvey, polycystum C. Agardh, polyporum Monagne, ssp.
ringgoldianum, ssp. coreanum (J. Adardh) Yoshida, ryukyuense
Shimabukuro et Yoshida, sagamianum Yendo, salicifolioides Yamada,
hyugaense, segii Yoshida, racemosum Yamada et Segi, ringgoldianum
f. ellipticum, serratifolium (C. Agardh) C. Agardh, siliquastrum
(Turner) C. Agardh, tortile, siliquosum J. Agardh, spathulophyllum
J. Tanaka, Murakami et Arai, tenuifolium Yamada, thunbergii
(Mertens ex Roth) Kuntze, tosaense Yendo, trichophyllum (Kuetzing)
Kuntze, wakayamaense Yoshida, yamadae Yoshida et T. Konno,
Yamamotoi Yoshida, yendoi Okamura et Yamada, henslowianum var.
condensatum, yezoense (Yamada) Yoshida et T. Konno, sagamianum var.
yezoense], and the genus Turbinaria Lamouroux, 1825 [conoides (J.
Agardh) Kuetzing, ornata (Turner) J. Agardh, turibinata (Linnaeus)
Kuntze, trialata]. Particularly preferred for use as the starting
materials for chlorophyll c are hormeri (Turner) C. Agardh, cava
Kjellman, kurome Okamura, and kelp (Laminariaceae Bory) which are
all brown algae.
[0057] The method for obtaining the chlorophyll c to be used in the
present invention is not particularly limited if the chlorophyll c
is neither decomposed nor denatured. For instance, the starting
material to be subjected to extraction may be used as it is or
after being subjected to desalting, water-washing and drying
processes; alternatively, it may be first reduced to a powder in
order to improve the extraction efficiency. If desired, in order to
further facilitate the extraction of chlorophyll c out of the cell,
a decomposition process may be preliminarily performed by treatment
with an enzyme such as protease which degrades such proteins as the
one forming the cell membrane or the one binding to chlorophyll c
or cellulase which degrades cellulose present in the cell wall and
the like. The extract may be concentrated by any suitable technique
such as vacuum concentration, freeze concentration or membrane
concentration.
[0058] The solvent for extracting the chlorophyll c to be used in
the present invention is not particularly limited if the
chlorophyll c is neither decomposed nor denatured). Examples
include water, salt containing aqueous solutions, and organic
solvents such as alcohols (e.g. methanol, ethanol and butanol),
ethers (e.g. diethyl ether), esters (e.g. ethyl acetate), and
ketones (e.g. acetone); preferred examples include ethanol,
methanol, acetone, and diethyl ether.
[0059] Described below is one specific method for producing
chlorophyll c from a chlorophyll c containing alga: for the purpose
of concentrating chlorophyll c in a convenient and inexpensive way
by removing impurities such as salts, polysaccharides, and other
non-chlorophyll c water-soluble and water-insoluble components, the
alga is washed under stirring with 1-100 volumes of water and after
repeating the same washing operation several times, the alga is
dried under appropriate conditions and ground with a mill to
produce a dry algal powder as a concentrated form of chlorophyll
c.
[0060] For the purpose of concentrating chlorophyll c even further,
the thus produced dry algal powder as a concentrated form of
chlorophyll c may be subjected to extraction with 60%-100%,
preferably 80%, hydrous ethanol under stirring overnight at
ordinary temperatures. After the extraction, suction filtration is
performed to remove the insoluble matter and the resulting extract
is rotary evaporated to dryness, thereby yielding an algal hydrous
ethanol extract as a concentrated form of chlorophyll c.
[0061] For the purpose of concentrating chlorophyll c still
further, the thus produced algal hydrous ethanol extract as a
concentrated form of chlorophyll c may be subjected to phase
separation using n-butanol and water, and the resulting n-butanol
fraction may be subjected to further phase separation using hexane
and hydrous methanol. The resulting hydrous methanol fraction is
fractionated on a silica gel column and an ODS column, eluted on a
gradient using an aqueous acetonitrile solution and pure
acetonitrile, and fractionated by HPLC involving peak
identification based on the absorbance at a wavelength of 254 nm.
The resulting fractions and an authentic sample of chlorophyll c
were subjected to HPLC and measured for the absorbance at 450 nm;
by verifying the occurrence of a peak at the same retention time as
for the authentic sample of chlorophyll c, an even more purified
form of chlorophyll c can be produced from the starting algal
hydrous ethanol extract as a concentrated form of chlorophyll
c.
[0062] The protease that may be used in the present invention to
decompose proteins is not particularly limited. Examples include
Newlase F3G, Newlase A, Morushin F, Sumizyme AP, Denapushin 2P,
Orientase 20A, Tetoraze S, Brewer's Clarex, Protease YP-SS,
Protease A "Amano" G, Protease N "Amano" G, Protease S "Amano" G,
Papain W-40, bromelain F, Puromoddo 223LP, Sumizyme LP, Sumizyme
FP, Sumizyme LPL, PROTIN SD-NY-10, PROTIN SD-PC-10F, Brewer's
Protease, Accelazyme NP50.000, purified Papain, purified Papain for
use in foods, Denachimu AP, PTN, Neutrase, nucleicin, Orientase
10NL, Orientase 90N, Orientase ONS, Papain F, trypsin 4.0T,
COROLASE N, VERON L10, COROLASE L10, COROLASE 7089, Panchidaze
NP-2, Panchidaze P, Aroaze AP-10, Aroaze NP-10, Aroaze NS, Enchiron
NBS, Papain, Protex 7L, Protex 14L, Thermoase PC10F, PROTIN
SD-AC-10F, PROTIN SD-AY-10, Proleather FG-F, Protease P "Amano" 3G,
alkali Protease GL, Protex 6L, Protx 89L, Purafect, Purafect OX,
Puroperaze, Protex OXG, Protex 40L, Sumizyme MP, Delvolase,
Esperase, Savinase, Alcalase, Clear Lens Pro, Everlase, Kan'nase,
Novozyme FM, Biopuraze OP, Biopuraze AL-15FG, Biopuraze 30G,
Biopuraze APL-30, Biopuraze XL-416F, Biopuraze SP-20FG, Biopuraze
SP-4FG, Protease CL-15, Orientase 22BF, Aroaze XA-10, Enchiron SA,
Bakezyme PPU95.000, Bakezyme B500, Polarzyme, Protease M "Amano" G,
Kokulase.cndot.P, Actinase AS, Gurindoamiru PR59, Gurindoamiru
PR43, Flavorzyme, Prometax, Noboran, VERON W, Protease AL, Magnax
MT, Sofutagen M2, Collupulin, Fromase, Meito RENNET, Meito RENNNET
SUPER, and Maxiren.
[0063] The cellulase that may be used in the present invention to
decompose celluloses is not particularly limited. Examples include
Cellulase A "Amano" 3, Cellulase T "Amano" 4, .beta.-Glucanase,
Spezyme CP, Multifect, Multifect GC Extra, Optiflow RC, Primafast,
IndiAge NeutraFlex, Accellerase, Optimash, Optimase CX, Puradax HX,
GODO-TCF, GODO-TCL, GODO-TCD-H3, VESSELEX, Softagen.cndot.C-1,
Super-heat-resistant Cellulase, Cellulizer CL, Cellulizer ACE,
Cellulase Nagase, Cellzyme C, Cellulase SS. Cellulase XL-531,
Bakezyme concreate, Citrase CL, Bakezyme X-CELL, Varidase TRL,
Varidase ANC40, CELLSOFT, Carezyme, Celluzyme, Celluclast, Cellic
CTec, Cellulosin AC40, Cellulosin AL, Cellulosin TF, Cellulase
"onozuka" 3S, Cellulase Y-NC, PANCELASE br, CellSEB Ts, Sumizyme
AC, Sumizyme C, Sucrase C, Enchiron MC, Enchiron MCH, BioHIT,
Bio-STAR, Cellulase ES, Finizyme, UltraFlo, Viscozyme, Glucanex,
Filtrase L, Filtrase NLCL, Filtrase Premium L, Sumizyme BGA, and
naringinase.
[0064] The dosage form of the composition of the present invention
is not particularly limited. Examples include dusts, granules,
tablets, syrups, injections, drops, powders, suppositories,
suspensions, ointments, etc. The composition of the present
invention may be administered either orally or parenterally (e.g.
intravenous, intramuscular, subcutaneous, rectal or transdermal).
The composition may contain additives such as excipients,
disintegrants, binders, lubricants, and colorants. Exemplary
excipients include lactose, glucose, corn starch, sorbitol, and
microcrystalline cellulose; exemplary disintegrants include starch,
sodium alginate, gelatin powder, calcium carbonate, calcium
citrate, and dextrin; exemplary binders include dimethylcellulose,
poly(vinyl alcohol), poly(vinyl ether), methylcellulose,
ethylcellulose, gum arabic, gelatin, hydroxypropylcellulose, and
poly(vinyl pyrrolidone); exemplary lubricants include talc,
magnesium stearate, poly(ethylene glycol), and hardened vegetable
oils.
[0065] Depending on its dosage form, the composition of the present
invention contains varying amounts of chlorophyll c which typically
range from about 0.1 to 100 mg by dry weight in the total
composition. In the case of an algal powder or extract that
contains chlorophyll c as an active ingredient, its content
typically ranges from about 0.1 to 10 wt %, preferably from about 1
to 3 wt %, in the total composition.
[0066] The dose of administration is determined as appropriate for
individual cases in consideration of various factors such as the
age, body weight and sex of the patient, the type of disease, and
the severity of symptoms, and the frequency of administration may
be once or more than once a day. For instance, the dose of
administration may be such that chlorophyll c is ingested in a dry
weight ranging from 0.1 to 100 mg; an algal powder or extract that
contains chlorophyll c as an active ingredient may be ingested in a
dry weight ranging from about 0.1 g to 10 g, preferably from about
1 g to 3 g.
[0067] The composition of the present invention may, either on its
own or after being added to a processed food, serve as a functional
food or beverage that has degranulation suppressing activity,
allergy suppressing activity or osteoarthritis suppressing
activity. The type of the food or beverage to be produced is not
particularly limited if it does not interfere with the
degranulation suppressing action, allergy suppressing action or
osteoarthritis suppressing action of the active ingredient
chlorophyll c.
[0068] Examples of the processed food to which the composition of
the present invention may be added include: bakery; cereal flour
and noodles; processed fishery products; processed agricultural and
forestry products; processed animal foods; cow's milk and dairy
products; oils and fats, either unprocessed or processed; liquors;
beverages; seasonings; prepared frozen foods; retorted foods;
instant foods; seasoned dried products such as fish/shell senbei;
salt preserved fish roe and other products; tsukudani (fish and
other foods boiled in soy sauce) such as kanroni, shigureni, and
kakuni; broiled processed products; boiled processed products;
seasoned processed products such as fish/shell miso; kamaboko
(minced and steamed fish) such as mushi (steamed)-kamaboko, yaki
(broiled)-kamoboko, age (fried)-kamaboko, yude (boiled)-kamaboko,
fuumi (flavored)-kamaboko, housou (packaged)-kamaboko, saiku
(crafted)-kamaboko, and kunsei (smoked)-kamaboko; pickles such as
shoyu (soy)-zuke, miso-zuke, kasu (sake lees)-zuke, su
(vinegar)-zuke, and koji (rice malt)-zuke; shiokara (salted fish
guts) such as katsuo (bonito)-shiokara, uni (sea urchin)-shiokara,
and ika (squid)-shiokara; canned products; binned products; fish
sauce; and extract products.
[0069] The food and beverage of the present invention encompass
health foods and beverages, dietary supplements, foods for
specified health uses, foods with nutrient functional claims, etc.
The "foods for specified health uses" refers to those foods and
beverages that are ingested in dietary life for a specified health
purpose and which have a label claiming that the specified health
purpose may be achieved by ingesting them. Such foods and beverages
may have labels attached thereto, claiming that they "alleviate
symptoms of allergy," "alleviate symptoms of pollinosis,"
"alleviate symptoms of atopic dermatitis," "alleviate symptoms of
osteoarthritis," or that they are "foods for persons worried about
allergy" or "foods for persons worried about osteoarthritis."
[0070] The composition of the present invention may, either on its
own or after being added to a feed or pet food, serve as a feed or
pet food that has degranulation suppressing activity, allergy
suppressing activity or osteoarthritis suppressing activity. The
type of the feed or pet food to be produced is not particularly
limited if it does not interfere with the degranulation suppressing
action, allergy suppressing action or osteoarthritis suppressing
action of the active ingredient chlorophyll c.
[0071] The feed or pet food of the present invention is not
particularly limited in scope and they may be feeds for cattle and
poultry such as cows, pigs and chickens, or feeds for farming
crustaceans and fish/shellfish, or pet foods for companion animals
such as dogs, cats, hamsters, and squirrels. The form the feed or
pet food of the present invention can take is not particularly
limited and examples include a pellet type, a crumble type, a flake
type, a bulky type, a dry type, a wet type, a semi-moist type, a
biscuit type, a sausage type, a jerky type, a powder type, a
granule type, a capsule type, etc.
EXAMPLES
[0072] The present invention will be described below more
specifically by reference to Examples. It should, however, be
understood that the following Examples are by no means intended to
limit the scope of the present invention.
Example 1
Test for Evaluating the Degranulation Suppressing Activity of
Chlorophyll c
[0073] Chlorophylls, heretofore unknown to have a degranulation
suppressing action, were subjected to a test as described below for
evaluating their degranulation suppressing activity using the rat
basophile derived cell line RBL-2H3.
[0074] First, the rat basophile derived cell line RBL-2H3 was
diluted with a DMEM medium (product of SIGMA) containing 10% fetal
bovine serum (hereinafter referred to as FBS) to give a density of
2.5.times.10.sup.5 cells/ml, dispensed on a 24-well culture plate
in a volume of 1 ml/well, and cultured overnight at 37.degree. C.
under conditions of 5% CO.sub.2 and 100% humidity. After removing
the culture supernatant, the cell surfaces were washed with 1
ml/well of a phosphate buffer solution (hereinafter referred to as
PBS) and an anti DNP-IgE solution prepared by diluting mouse
monoclonal anti-dinitrophenol (product of SIGMA) with a DMEM medium
to give a concentration of 50 ng/ml was added in a volume of 500
.mu.l/well, followed by incubation for 2 hours at 37.degree. C.
under conditions of 5% CO.sub.2 and 100% humidity. After the end of
incubation, the anti DNP-IgE solution was removed and washing was
done using 1.5 ml/well of a buffer solution containing 137 mM NaCl
(product of nacalai tesque), 2.7 mM KCl (JUNSEI CHEMICAL), 1.8 Mm
CaCl.sub.2.2H.sub.2O (JUNSEI CHEMICAL), 1 mM MgCl.sub.2.6H.sub.2O
(JUNSEI CHEMICAL), 5.6 mM glucose (Wako Pure Chemical Industries),
20 mM HEPES (Wako Pure Chemical Industries) and 0.1% BSA (the
buffer solution being a modified Tyrode buffer which is hereinafter
referred to as "MT buffer"); this procedure was repeated twice.
Next, an MT buffer containing 0.5% of a chlorophyll, i.e., an
authentic sample of chlorophyll a (product of DHI), an authentic
sample of chlorophyll b (product of DHI), an authentic sample of
chlorophyll c2 (product of DHI), or an authentic sample of
chlorophyll d (purified from cyanobacteria adhering to surfaces of
red algae, by referring to Non-Patent Documents 10 and 11, for
example) was added in a volume of 490 .mu.l/well and incubation was
conducted for 10 minutes at 37.degree. C. under conditions of 5%
CO.sub.2 and 100% humidity. To a control group that would not
undergo suppression of the degranulation reaction, an MT buffer
containing 0.5% ethanol was added. After 10-min incubation,
Albumin, dinitrophenyl (product of SIGMA) was added to give a
concentration of 200 ng/ml and the mixture was stirred to
homogeneity, followed by 30-min incubation at 37.degree. C. under
conditions of 5% CO.sub.2 and 100% humidity. After the end of
incubation, the mixture was ice-cooled for 10 minutes to quench the
reaction and 500 .mu.l of the supernatant was recovered. To the
cells from which the culture supernatant was recovered, Cell Lysate
buffer (product of SIGMA) was added in a volume of 500 .mu.l/well
and thereafter an ultrasonic cell disrupter was used to form a
homogeneous cell lysate which was recovered in a volume of 500
.mu.l. The culture supernatant and the cell lysate were dispensed
on a 96-well plate, each in a volume of 50 .mu.l/well, and
incubated at 37.degree. C. for 5 minutes. After the end of
incubation, an aqueous solution containing 100 mM citric acid and
3.3 mM p-nitrophenyl-2-acetoamido-2-deoxy-.beta.-D-glucopyranoside
(product of Wako Pure Chemical Industries) (the aqueous solution is
hereinafter referred to as a "substrate solution") was added in a
volume of 100 .mu.l/well and after thorough mixing, incubation was
conducted at 37.degree. C. for 25 minutes. After the end of
incubation, an aqueous solution containing 2 M glycine (the aqueous
solution is hereinafter referred to as a "reaction quencher") was
added in a volume of 100 .mu.l/well and thorough mixing was
conducted to quench the reaction; thereafter, a plate reader was
used to measure the absorbance at 405 nm. Through this sequence of
reactions, degranulation occurred and the activity of
.beta.-hexosaminidase contained in the granules was measured to
calculate the proportion of granules released from the cells to the
culture supernatant. For evaluation, the proportion of granules
released into the culture supernatant from the cells in the control
group to which 0.5% of 100% ethanol had been added instead of the
test sample and which would not undergo suppression of
degranulation was taken as 100% and the proportion of granules
released from the cells in each test sample added group into the
culture supernatant was counted as percent degranulation relative
to this reference value.
[0075] For efficacy evaluation, a Dunnett multiple comparison test
was conducted on those groups that were found to have statistically
significant differences by variance analysis. In a graph showing
the result, the group found to have a significant difference at a
5% significance level was marked by symbol * whereas the group
found to have a significant difference at a 1% significance level
was marked by symbol **. The same test was conducted in Example 2
and subsequent Examples.
[0076] As shown in FIG. 1, the authentic samples of chlorophyll a,
b and d were recognized to have no degranulation suppressing
activity at all; on the other hand, the groups treated with the
authentic sample of chlorophyll c2 were shown to exhibit the
degranulation suppressing activity in a dose-dependent manner which
was already apparent when chlorophyll c2 was added in a very low
concentration which was only about 0.05 .mu.g/ml by dry weight. It
was therefore clear that chlorophyll c2 has a very strong activity
for suppressing degranulation which has heretofore been entirely
unknown in chlorophylls; the use of chlorophyll c2 has thus been
verified to be effective for preventing or treating allergic
symptoms as of pollinosis and osteoarthritis, both involving the
degranulation reaction.
Example 2
Test for Evaluating Percent Cell Survival Using Basophile Derived
Cell Line RBL-2H3
[0077] The rat basophile derived cell line RBL-2H3 diluted to the
same concentration as in Example 1 was dispensed on a 96-well
culture plate in a volume of 0.1 ml/well and cultured overnight at
37.degree. C. under conditions of 5% CO.sub.2 and 100% humidity.
After removing the culture supernatant, the cell surfaces were
washed with 1 ml/well of PBS and then an MT buffer containing an
authentic sample of chlorophyll c2 at the same concentrations by
dry weight as in Example 1 was added in a volume of 100 .mu.l/well
and incubation was conducted for 40 minutes at 37.degree. C. under
conditions of 5% CO.sub.2 and 100% humidity. To a control group
that would not undergo suppression of the degranulation reaction,
an MT buffer containing 0.5% ethanol was added. After 40-min
incubation, WST-1 reagent (product of DOJINDO) was added in a
volume of 10 .mu.g/ml and the mixture was incubated for 30 minutes
at 37.degree. C. under conditions of 5% CO.sub.2 and 100% humidity.
After the end of incubation, the mixture was left to stand at room
temperature for 10 minutes and then the absorbance at 450 nm was
measured to evaluate the percent cell survival.
[0078] The result is shown in FIG. 2; obviously, none of the cell
groups treated with the authentic sample of chlorophyll c2
experienced a drop in percent cell survival and it was verified
that the degranulation suppressing activity of chlorophyll c2 as
demonstrated in Example 1 is not due to cytotoxicity.
Example 3
Preparation of Chlorophyll c
[0079] As noted hereinabove, chlorophyll c is known to be abundant
in algae such as those of the Division Dinoflagellata, the Division
Cryptophyta, the Division Heterokontophyta including the Class
Chrysophyceae, the Class Raphidophyceae, the Class Phaeophyceae,
the Class Bacillariophyceae (Diatomea) and the Class
Eustigmatophyceae, as well as the Division Haptophyta. Hence, from
among chlorophyll c containing algae, hormeri (Turner) C. Agardh
which is readily available and which has been part of people's
dietary life was particularly selected for the purpose of
concentrating chlorophyll c in a convenient and inexpensive way by
removing salts, polysaccharides and other non-chlorophyll c
water-soluble and water-insoluble impurities; the alga was washed
under stirring for 3 minutes using 15 volumes of water and after
repeating the same washing operation three times, the alga was
dried overnight under the condition of 25.degree. C. and ground
with a mill to remove any unwanted matter that was not chlorophyll
c, whereby a dry powder of the brown alga hormeri (Turner) C.
Agardh was obtained as a concentrated form of chlorophyll c.
[0080] For the purpose of concentrating chlorophyll c even further,
the thus produced dry powder of the brown alga hormeri (Turner) C.
Agardh as a concentrated form of chlorophyll c was subjected to
extraction with stirring overnight under the condition of
25.degree. C.; the extraction solvent was water or 20% or 40% or
60% or 80% hydrous ethanol or 100% ethanol, whose volume was 15
times the dry weight of the powder. After the extraction, suction
filtration was performed to remove the insoluble matter and the
resulting extract was rotary evaporated to dryness.
[0081] The extracts thus evaporated to dryness were evaluated for
the degranulation suppressing activity using the rat basophile
derived cell line RBL-2H3 as in Example 1. The results are shown in
FIG. 3; obviously, the extract prepared using water was found to
have no degranulation suppressing activity at all and only the
extracts prepared using 60-80% hydrous ethanol or 100% ethanol were
found to have the activity of interest, which was strongest in the
extract prepared using 80% hydrous ethanol. It was therefore
verified that using 60-80% hydrous ethanol or 100% ethanol is
effective for efficient extraction of chlorophyll c and that
chlorophyll c can be extracted most efficiently when 80% hydrous
ethanol is used; thus, from the dry powder of the brown alga
hormeri (Turner) C. Agardh as a concentrated form of chlorophyll c,
extracts as a highly concentrated form of chlorophyll c were
prepared using hydrous ethanol.
[0082] For the purpose of concentrating chlorophyll c still
further, the 80% hydrous ethanol extract characterized by most
efficient extraction of chlorophyll c from the dry powder of the
brown alga hormeri (Turner) C. Agardh as a concentrated form of
chlorophyll c was subjected to phase separation using n-butanol and
water, and the resulting n-butanol fraction was subjected to
further phase separation using hexane and 90% hydrous methanol. The
resulting 90% hydrous methanol fraction was fractionated on a
silica gel column (product of YAMAZEN CORPORATION) and the same
fraction procedure was repeated three times. The resulting
concentrated fraction of chlorophyll c was loaded on an ODS column
(product of Waters), eluted on a gradient using a 45% aqueous
acetonitrile solution and 100% acetonitrile, and fractionated by
HPLC involving peak identification based on the absorbance at a
wavelength of 254 nm to yield eleven fractions A to K. The
resulting fractions were subjected to a test for evaluating
degranulation suppressing activity using the rat basophile derived
cell line RBL-2H3 as in Example 1; the results are shown in FIG. 4
and only Fr. J was found to have the evaluating degranulation
suppressing activity. The photo of FIG. 5 verified that the active
fraction Fr. J had been purified to a single band; Fr. J and an
authentic sample of chlorophyll c2 (product of DHI) were measured
for the absorbance at 450 nm by HPLC; as FIG. 6 shows, Fr. J and
the authentic sample of chlorophyll c2 were found to have the same
peak at a retention time of about 4.6 minutes; in addition, as FIG.
7 shows, Fr. J and the authentic sample of chlorophyll c2, both
dissolved in an aqueous acetonitrile solution, had an identical
absorption spectrum; with these results taken together, Fr. J was
identified as chlorophyll c2; thus, the 80% hydrous ethanol extract
of chlorophyll c from the dry powder of the brown alga hormeri
(Turner) C. Agardh as a concentrated form of chlorophyll c could be
processed by the sequence of those operations to prepare an even
more purified form of chlorophyll c2.
Example 4
Test for Evaluating Degranulation Suppressing Activity of
Chlorophyll c1 or c3
[0083] Chlorophyll c1 or c3 which had heretofore been entirely
unknown to have the degranulation suppressing action were evaluated
for the degranulation suppressing activity as in Example 1. The
result is shown in FIG. 8; obviously, the groups respectively
treated with an authentic sample of chlorophyll c1 (prepared from a
brown alga by referring to Non-Patent Document 12, for example) or
an authentic sample of c3 (product of DHI) were found to exhibit
the degranulation suppressing activity as did chlorophyll c2 which
was verified to have the same activity in Example 1. It was
therefore clear that not only chlorophyll c2 which was verified to
be active in Example 1 but also chlorophyll c1 and c3 alike has an
activity for suppressing degranulation which has heretofore been
entirely unknown in chlorophylls; the use of either type of
chlorophyll c was thus verified to be effective for preventing or
treating allergic symptoms as of pollinosis and osteoarthritis,
both involving the degranulation reaction.
Example 5
Test for Evaluating Percent Cell Survival due to Chlorophyll c1 or
c3
[0084] Chlorophyll c1 and c3 were subjected to a test for
evaluating the percent cell survival using the rat basophile
derived cell line RBL-2H3 as in Example 2. The result is shown in
FIG. 9; obviously, neither the cell group treated with the
authentic sample of chlorophyll c1 nor the cell group treated with
the authentic sample of chlorophyll c3 was recognized to experience
a drop in the percent cell survival the groups, verifying that the
degranulation suppressing activity of chlorophyll c1 or c3 which
was demonstrated in Example 4 is not due to cytotoxicity.
Example 6
Test for Evaluating Vascular Permeability Suppressing Activity
Using SD Rat
[0085] To five-week-old SD male rats acclimatized for a week, a
feed consisting of documented formula blend AIN-76 (product of
Oriental Yeast) and 3 wt % or 1 wt % of the dry powder of the brown
alga hormeri (Turner) C. Agardh as a concentrated form of
chlorophyll c which was prepared in Example 3 was given ad libitum
for 28 days. To another group of similarly acclimatized
five-week-old SD male rats, a feed consisting of documented formula
blend AIN-76 and 1.1 wt % of the 80% hydrous ethanol extract of the
brown alga hormeri (Turner) C. Agardh which was also prepared in
Example 3 was given ad libitum for 28 days. To a control group,
only documented formula blend AIN-76 was given ad libitum for 28
days. Each of these SD male rats which were given the respective
feeds ad libitum for 28 days was injected with Evans blue dye into
the tail vein. The animals were then injected intradermally at the
back with Compound 48/80, a drug for invoking
degranulation-mediated vascular permeation. After cervical
dislocation under anesthesia, the dorsal skin of each animal was
peeled and the amount of Evans blue dye penetrating the peeled skin
tissue was quantified by measuring the absorbance at 620 nm with a
plate reader. From the quantified amounts of Evans blue dye, the
percent vascular permeability was calculated as the relative amount
of Evans blue dye penetrating the subcutaneous tissue of each SD
male rat on test feed, with the amount of Evans blue dye
penetrating the subcutaneous tissue of each SD male rat in the
control group being taken as 100%. The results are shown in FIG.
10; obviously, the groups to which the feeds containing 1 wt % or 3
wt % of the dry powder of the brown alga hormeri (Turner) C. Agardh
as a concentrated form of chlorophyll c were given ad libitum for
28 days, as well as the group to which the feed containing 1.1 wt %
of the extract of the brown alga hormeri (Turner) C. Agardh was
given ad libitum for 28 days were suppressed in vascular permeation
as compared with the control group. It was therefore clear that in
the in vivo test, too, oral ingestion of the dry powder of the
brown alga hormeri (Turner) C. Agardh as a concentrated form of
chlorophyll c or the 80% hydrous ethanol extract of the brown alga
hormeri (Turner) C. Agardh, both being prepared in Example 3, were
able to suppress degranulation-mediated vascular permeation, thus
proving effective for preventing or treating allergic diseases such
as pollinosis in which vascular permeation is involved.
Example 7
Test for Evaluating Pollinosis Suppressing Activity Using C57BL/6
Mouse
[0086] Four-week-old C57BL/6 male mice (product of Charles River
Laboratories Japan) were acclimatized for a week. To a group of
these animals, 2 g/kg of the dry powder of the brown alga hormeri
(Turner) C. Agardh as a concentrated form of chlorophyll c which
was prepared in Example 3 was orally administered in a
released-dose and forced manner for 4 weeks as suspended in a 0.5%
CMC solution. To another group of the animals, 0.2 g/kg or 1 g/kg
of the 80% hydrous ethanol extract of an even more concentrated
form of chlorophyll c from the brown alga hormeri (Turner) C.
Agardh which was also prepared in Example 3 was orally administered
in the same manner for 4 weeks as suspended in a 0.5% CMC solution.
To a control group, only a 0.5% CMC solution was orally
administered in the same manner for 4 weeks. To yet another group
of the animals (drug administered group), Olopatadine (product of
SIMGA), a pharmaceutical showing a pollinosis suppressing effect by
the degranulation suppressing action, was orally administered in
the same manner for 4 weeks as suspended in a 0.5% CMC solution.
After the 4-week oral administration in a repeased-dose and forced
manner, a solution having anti-DNP-IgE dissolved in physiological
saline at a concentration of 50 .mu.g/ml was injected into the hip
vein of each mouse for passive sensitization. Five days after the
sensitization, DNP-OVA antigen as dissolved in physiological saline
at a concentration of 50 mg/ml was administered into both nostrils
of each mouse to invoke a rhinitic symptom and the symptom of the
developed pollinosis was evaluated by counting how many times each
rat scratched its nose per hour on account of the pollinosis
symptom. The results are shown in FIG. 11; obviously, the group
that ingested 2 g/kg of the dry powder of the brown alga hormeri
(Turner) C. Agardh as a concentrated form of chlorophyll c which
was prepared in Example 3, as well as the group that ingested 1
g/kg or 0.2 g/kg of the 80% hydrous ethanol extract of an even more
concentrated form of chlorophyll c from the brown alga hormeri
(Turner) C. Agardh which was also prepared in Example 3, scratched
their nose less frequently than the control group, and both the dry
powder and the extract were comparable in potency to the drug
Olopatadine. It was therefore clear that oral ingestion of the dry
powder of the brown alga hormeri (Turner) C. Agardh as a
concentrated form of chlorophyll c or the 80% hydrous ethanol
extract of the brown alga hormeri (Turner) C. Agardh, both being
prepared in Example 3, were able to suppress vascular permeation
which was verified in Example 6 to result from the degranulation
reaction, thereby suppressing the symptom of pollinosis to prove
effective for preventing or treating allergic rhinitis such as
pollinosis.
Example 8
Test for Evaluating Type II Collagen Sensitized Osteoarthritis
Suppressing Activity Using DA Rat
[0087] Six groups of eight-week-old DA/Slc female rats (product of
SANKYO LABO SERVICE CORPORATION) that had been acclimatized for a
week were allowed to ingest ad libitum the following feeds,
respectively, for 4 weeks: a feed consisting of formula blend CRF-1
(product of Oriental Yeast) and 3 wt % of the dry powder of the
brown alga hormeri (Turner) C. Agardh as a concentrated form of
chlorophyll c which was prepared in Example 3; a feed consisting of
formula blend CRF-1 and 2 wt % of glucosamine (product of Yaizu
Suisankagaku Industry); a feed consisting of formula blend CRF-1
and 5 wt % of edible chondroitin sulfate (designated as SCP(NB);
product of Maruha Nichiro Foods); a feed consisting of formula
blend CRF-1 and 3 wt % of the dry powder of the brown alga hormeri
(Turner) C. Agardh as a concentrated form of chlorophyll c which
was prepared in Example 3 plus 2 wt % of glucosamine; a feed
consisting of formula blend CRF-1 and 3 wt % of the dry powder of
the brown alga hormeri (Turner) C. Agardh as a concentrated form of
chlorophyll c which was prepared in Example 3 plus 5 wt % of
SCP(NB); and formula blend CRF-1 only (control group). After the
four-week ingestion ad libitum, bovine type II collagen in solution
(product of Collagen Gijutsu Kenshukai) which was an arthrosis
invoking substance was injected intradermally at four sites of the
lumbar portion of the back at a dose of 0.15 mg to thereby invoke
osteroarthritis. Thereafter, each animal was visually checked for
the state of the footpad of its right and left hind paw and the
developed arthrosis was evaluated by scoring against the criteria
listed in Table 1 below. The results are shown in FIG. 12;
obviously, the rats that were allowed to ingest ad libitum the feed
consisting of formula blend CRF-1 and 3 wt % of the dry powder of
the brown alga hormeri (Turner) C. Agardh as a concentrated form of
chlorophyll c which was prepared in Example 3 had lower arthrosis
scores than the rats of the control group. It was also shown that
the effectiveness of this feed was quite potent and more than
comparable to that observed in the group that was allowed to ingest
ad libitum the feeds consisting of formula blend CRF-1 and
glucosamine or SCP(NB) which are conventionally used to prevent or
treat osteoarthritis. Moreover, the dry powder of the brown alga
hormeri (Turner) C. Agardh as a concentrated form of chlorophyll c
which was prepared in Example 3 was verified to be more potent in
suppressing osteoarthritis when it was ingested together with
glucosamine or SCP(NB) than when the feed consisting of formula
blend CRF-1 and glucosamine or SCP(NB) only was ingested ad
libitum. From these results, it became clear that osteoarthritis
can be suppressed by ingesting the dry powder of the brown alga
hormeri (Turner) C. Agardh as a concentrated form of chlorophyll c
which was prepared in Example 3. It also became clear that a more
potent osteoarthritis suppressing action is exhibited by using the
conventional osteoarthritis suppressor glucosamine or SCP(NB) in
combination with the dry powder of the brown alga hormeri (Turner)
C. Agardh as a concentrated form of chlorophyll c which was
prepared in Example 3.
TABLE-US-00001 TABLE 1 Score State of the footpad of the hind paw 0
Normal 1 Reddened 2 Redness and slight edema on toes 3 Edema spread
from toes to the entire paw 4 Intense edema 5 Joint deformed
* * * * *