U.S. patent application number 13/276670 was filed with the patent office on 2012-03-22 for activator including biosurfactant as active ingredient, mannosyl erythritol lipid, and production method thereof.
This patent application is currently assigned to National Institute of Advanced Industrial Science and Technology. Invention is credited to Tokuma Fukuoka, Tomohiro Imura, Masaru Kitagawa, Dai Kitamoto, Tomotake Morita, Atsushi Sogabe, Michiko Suzuki, Shuhei Yamamoto.
Application Number | 20120070396 13/276670 |
Document ID | / |
Family ID | 39032978 |
Filed Date | 2012-03-22 |
United States Patent
Application |
20120070396 |
Kind Code |
A1 |
Suzuki; Michiko ; et
al. |
March 22, 2012 |
ACTIVATOR INCLUDING BIOSURFACTANT AS ACTIVE INGREDIENT, MANNOSYL
ERYTHRITOL LIPID, AND PRODUCTION METHOD THEREOF
Abstract
The present invention includes as an active ingredient at least
one biosurfactant, in particular mannosyl alditol lipid (such as
MEL and MML) or triacylated mannosyl alditol lipid. This allows
providing an activator and anti-aging agent that is excellent in
activating and anti-aging effects on cells and that is safe enough
to be used for a long time, and also providing cosmetics,
quasi-drugs, drugs, and drinks and foods including the activator
and the anti-aging agent as active ingredients. Further, the
present invention provides MEL whose mannosyl erythritol skeleton
in a molecular structure is
1-O-.beta.-D-mannopyranosyl-meso-erythritol and a method for
producing the MEL with use of a microorganism.
Inventors: |
Suzuki; Michiko; (Otsu-shi,
JP) ; Kitagawa; Masaru; (Osaka, JP) ;
Yamamoto; Shuhei; (Tsuruga-shi, JP) ; Sogabe;
Atsushi; (Osaka, JP) ; Kitamoto; Dai;
(Tsukuba-shi, JP) ; Morita; Tomotake;
(Tsukuba-shi, JP) ; Fukuoka; Tokuma; (Tsukuba-shi,
JP) ; Imura; Tomohiro; (Tsukuba-shi, JP) |
Assignee: |
National Institute of Advanced
Industrial Science and Technology
Tokyo
JP
TOYO BOSEKI KABUSHIKI KAISHA
Osaka
JP
|
Family ID: |
39032978 |
Appl. No.: |
13/276670 |
Filed: |
October 19, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12376805 |
Feb 9, 2009 |
|
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PCT/JP2007/065427 |
Aug 7, 2007 |
|
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13276670 |
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Current U.S.
Class: |
424/64 ; 424/63;
514/25 |
Current CPC
Class: |
A61Q 19/08 20130101;
A61K 8/60 20130101; A61K 36/06 20130101; C07H 15/06 20130101; A61P
3/00 20180101; C11D 1/662 20130101; A61P 17/14 20180101; C07H 15/04
20130101; A61K 31/7032 20130101; A23L 33/10 20160801; A61P 43/00
20180101 |
Class at
Publication: |
424/64 ; 514/25;
424/63 |
International
Class: |
A61K 8/60 20060101
A61K008/60; A61Q 1/10 20060101 A61Q001/10; A61Q 1/08 20060101
A61Q001/08; A61Q 1/06 20060101 A61Q001/06; A61Q 19/08 20060101
A61Q019/08; A61Q 19/00 20060101 A61Q019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 11, 2006 |
JP |
2006-219170 |
Jul 9, 2007 |
JP |
2007-179892 |
Claims
1. A method for activating a skin cell, comprising contacting a
cell in a skin on a subject that is other than a scalp skin with a
composition containing an effective amount of a mannosylerythritol
lipid or a mannosylmannitol lipid.
2. The method of claim 1, wherein the skin is on the face, lip,
neck, body from the neck down, hands, arms, or legs of the
subject.
3. The method of claim 1, wherein the composition is a skin lotion,
an ointment, a cream, a facial wash, a skin cleansing agent, a
foundation, a face powder, a ceruse, a lipstick, a blusher, an
eyeshadow, an eyeliner, a perfume, or a bath agent.
4. The method of claim 1, wherein the mannosylerythritol lipid
comprises a mannosylerythritol lipid-A, a mannosylerythritol
lipid-B, a mannosylerythritol lipid-C, a mannosylerythritol
lipid-D, or a triacylmannosylerythritol lipid.
5. The method of claim 1, wherein the mannosylmannitol lipid
comprises a triacylmannosylmannitol lipid.
6. A method of alleviating skin aging in a subject in need thereof,
comprising applying a composition containing an effective amount of
a mannosylerythritol lipid or a mannosylmannitol lipid to a skin on
the subject that is other than a scalp skin.
7. The method of claim 6, wherein the skin is on the face, lip,
neck, body from the neck down, hands, arms, or legs of the
subject.
8. The method of claim 6, wherein alleviating skin aging comprises
alleviating a wrinkle or alleviating a sag of a skin.
9. The method of claim 6, wherein the composition is a skin lotion,
an ointment, a cream, a facial wash, a skin cleansing agent, a
foundation, a face powder, a ceruse, a lipstick, a blusher, an
eyeshadow, an eyeliner, a perfume, or a bath agent.
10. The method of claim 6, wherein the mannosylerythritol lipid
comprises a mannosylerythritol lipid-A, a mannosylerythritol
lipid-B, a mannosylerythritol lipid-C, a mannosylerythritol
lipid-D, or a triacylmannosylerythritol lipid.
11. The method of claim 6, wherein the mannosylmannitol lipid
comprises a triacylmannosylmannitol lipid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 12/376,805, filed Feb. 9, 2009, which is a
National Stage entry of PCT/JP2007/065427 filed Aug. 7, 2007, which
claims priority to Japanese Application Serial Nos. 2007-179892
filed Jul. 9, 2007 and 2006-219170 filed Aug. 11, 2006. The
contents of each of the foregoing applications are incorporated by
reference herein.
TECHNICAL FIELD
[0002] The present invention relates to an activator including a
biosurfactant as an active ingredient. In particular, the present
invention relates to cosmetics, quasi-drugs, drugs, drinks and
foods, each including a biosurfactant which activates various cells
and is effective for anti-aging, hair growth, and prevention of
loss of hair.
[0003] Further, the present invention relates to new
mannosylerythritol lipid (which may be hereinafter referred to as
MEL) and a production method thereof. To be specific, the present
invention relates to: an MEL which is one of glycolipids produced
by microorganism and whose mannosylerythritol skeleton in a
molecular structure is 1-O-.beta.-D-mannopyranosyl-meso-erythritol;
and a production method of the MEL by microorganism.
BACKGROUND ART
[0004] Aging of individuals and various diseases due to the aging
are greatly involved with aging of all dividing cells (drop of
dividing speed, drop of cell function). For example, skin includes
epidermic cells, fibroblasts, and extracellular matrixes for
supporting skin structures other than these cells, such as elastion
and collagen. In young skin, interactivities between these skin
tissues maintain homeostasis, which keeps moisture, flexibility,
and resiliency. Consequently, skin appears to have tension and
smoothness, and is kept fresh. However, aging, ultra-violet ray,
dryness, stress etc. decrease functions of extracellular matrixes
and fibroblasts in particular. Consequently, flexibility of skin
and moisture-keeping function of skin drop, skin loses tension and
smoothness, and senile symptoms such as chaps, wrinkles, and
somberness appear.
[0005] In order to stop or prevent aging in cell level, activators
and anti-aging agents have been searched. Known examples of
activators derived from animals include hydrolysis of connective
tissue (Patent Document 1), water-soluble protein derived from
thymus gland and spleen (Patent Document 2), and essence of bovine
placenta (Patent Document 3). Known examples of activators derived
from plants include sesame, Chinese yam, pepper, angelica
acutiloba, houttuynia, mondo grass (Patent Document 4), almond,
taraxacum officinale, elder, Cnidium officinale, swertia japonica,
morus ihou, inner core of seed of peach, ginseng, hop, althaea, and
Job's tears. A part of these is used as an activator and an
anti-aging agent in quail-drugs and cosmetics. However, an
activator and an anti-aging agent that show satisfactory working
effects are not yet obtained.
[0006] Mannosylerythritol lipid (MEL) is a natural surfactant
produced by yeast, and it is reported that MEL has various
physiological functions (Non-patent Document 1). Further, recently,
mannosylmannitol lipid (MML) in which erythritol is replaced with
mannitol has been found (Patent Document 9). As for usage as
external medicines and cosmetics, effectiveness as an
anti-inflammatory agent and an anti-allergy agent (Patent Document
10) and baldness remedy and hair growth (Patent Document 11), an
anti-bacterial effect (Patent Document 12), and
surface-tension-reduction function (Patent Document 13) are
known.
[0007] However, activating function of MEL for cells has been
completely unknown. Further, hair growth function described in
Patent Document 11 was confirmed through animal experiments, and it
has not been reported that MEL activates human head hair-papilla
cells.
[0008] As described above, it is reported that a biosurfactant such
as glycolipid has environment-friendly features such as high
biodegradability and low toxicity, and has new physiological
functions. In view of these features, widely applying a
biosurfactant to food industry, cosmetic industry, medicine
industry, chemical industry, and field of environment allows
attaining a sustainable society and providing high-function
products, and therefore very significant.
[0009] One of representative glycolipid biosurfactants is MEL. MEL
is a material found from Ustilago nuda and Shizonella melanogramma
(see Non-patent Documents 2 and 3). Later, it is reported that MEL
can be produced by yeasts such as Candida yeast that is a mutated
strain producing itaconic acid (see Patent Document 14 and
Non-patent Document 4), Candida antarctica (currently called as
Pseudozyma antarctica) (see Non-patent Documents 5 and 6), and
Kurtzmanomyces yeast (see Non-patent Document 7). Nowadays,
long-time continuous cultivation and production allows producing
300 g/L or more of MEL.
[0010] [Patent Document 1] [0011] Japanese Unexamined Patent
Publication No. Tokukaisho 62-84024
[0012] [Patent Document 2] [0013] Japanese Unexamined Patent
Publication No. Tokukaisho 63-188697
[0014] [Patent Document 3] [0015] Japanese Unexamined Patent
Publication No. Tokukaihei 03-141299
[0016] [Patent Document 4] [0017] Japanese Unexamined Patent
Publication No. Tokukaihei 10-45615
[0018] [Patent Document 5] [0019] Japanese Unexamined Patent
Publication No. Tokukaihei 10-036279
[0020] [Patent Document 6] [0021] Japanese Unexamined Patent
Publication No. Tokukaihei 10-36279
[0022] [Patent Document 7] [0023] Japanese Unexamined Patent
Publication No. Tokukai 2004-75632
[0024] [Patent Document 8] [0025] Japanese Unexamined Patent
Publication No. Tokukai 2005-89375
[0026] [Patent Document 9] [0027] Japanese Unexamined Patent
Publication No. Tokukai 2005-104837
[0028] [Patent Document 10] [0029] Japanese Unexamined Patent
Publication No. Tokukai 2005-68015
[0030] [Patent Document 11] [0031] Japanese Unexamined Patent
Publication No. Tokukai 2003-261424
[0032] [Patent Document 12] [0033] Japanese Unexamined Patent
Publication No. Tokukaisho 57-145896
[0034] [Patent Document 13] [0035] Japanese Unexamined Patent
Publication No. Tokukaisho 61-205450
[0036] [Patent Document 14] [0037] Japanese Unexamined Patent
Publication No. Tokukosho 57-145896
[0038] [Non-patent Document 1] [0039] Journal of bioscience and
bioengineering, 94, 187 (2002)
[0040] [Non-patent Document 2] [0041] R. H. Haskins, J. A. Thorn,
B. Boothroyd, Can. J. Microbiol., Vol 1, p'749-'756, 1955.
[0042] [Non-patent Document 3] [0043] G. Deml, T. Anke, F.
Oberwinkler, B. M. Giannetti, W. Steglich, Phytochemistry, Vol 19,
p 83-87, 1980.
[0044] [Non-patent Document 4] [0045] T. Nakahara, H. Kawasaki, T.
Sugisawa, Y. Takamori, T. Tabuchi, J. Ferment.Technol. Vol 61, p
19-23, 1983.
[0046] [Non-patent Document 5] [0047] D. Kitamoto, S. Akiba, C.
Hioki, T. Tabuch, Agric. Biol. Chem., Vol 54. P 31-36, 1990.
[0048] [Non-patent Document 6] [0049] H.-S. Kim, B.-D. Yoon, D.-H.
Choung, H.-M. Oh, T. Katsuragi, Y. Tani, Appl. Microbiol.
Biotechnol., Springer-Verlag, Vol 52, p 713-721, 1999.
[0050] [Non-patent Document 7] [0051] K. kakukawa, M. Tamai, K.
Imamura, K. Miyamoto, S. Miyoshi, Y. Morinaga, O, Suzuki, T.
Miyakawa, Biosci. Biotechnol. Biochem., Vol 66, p 188-191,
2002.
[0052] [Non-patent Document 8] [0053] D. Crich, M. A. Mora, R.
Cruz, Tetrahedron, Elsevier, Vol 58, p 35-44, 2002.
[0054] [Non-patent Document 9] [0055] Dai Kitamoto, Oleoscience
(Japan), Japan Oil Chemists' Society, Vol. 3, p 663-672 (2003).
[0056] [Non-patent Document 10] [0057] T. Imura, N. Ohta, K. Inoue,
N. Yagi, H. Negishi, H. Yanagishita, D. Kitamoto, Chem. Eur. J,
Wiley, Vol 12, p 2434-2440, 2006.
DISCLOSURE OF INVENTION
[0058] It is an extremely important object to carry out activation
and anti-aging of mammals, in particular, humans. Although various
activating materials and anti-aging agents derived from animals and
plants have been discovered, there has not been yielded an effect
that is so sufficient and stable as to allow industrial use of the
activating materials and the anti-aging agents, and new activating
materials and anti-aging agents have been searched.
[0059] Therefore, an object of the present invention is to provide
an activator and anti-aging agent that are excellent in an
activating effect and an anti-aging effect on cells and are safe
enough to be used for a long time. The other object of the present
invention is to provide cosmetics, quasi-drugs, drugs, and drinks
and foods each including the activator and the anti-aging agent as
active ingredients.
[0060] Further, as described above, in order that a biosurfactant
that has environment-friendly features such as high
biodegradability and low toxicity and that has new physiological
functions is used in food industry, cosmetic industry, medicine
industry, chemical industry, field of environment etc., it is
necessary to increase production efficiency of the biosurfactant
and to widen the variety of a structure and a function of the
biosurfactant. In particular, MEL is excellent not only in
productivity and surface properties but also in specific
self-assembling property and bioactivity, and various applications
of MEL have been developed by taking advantage of the specific
self-assembling property and bioactivity.
[0061] However, microorganism-derived MEL having been reported so
far has a sugar skeleton that is
4-O-.beta.-D-mannopyranosyl-meso-erythritol structure. Therefore,
widening the variety of a structure and a function of MEL has been
strongly requested.
[0062] In particular, chirality of molecule of an organic compound
having bioactivities is very important point. It has been reported
that MEL has various bioactivities such as antibacterial activity,
anti-tumor property, and glycoprotein binding ability (Non-patent
Document 9). Further, MEL shows a very unique self-assembling
property, and application of MEL to a liposome material and a
liquid crystal technique is tried by taking advantage of the
self-assembling property. It is reported that a slight difference
in a molecular structure has a great influence on formation of a
self-assembling body (Non-patent Documents 9 and 10).
[0063] In view of the above, it is expected that producing a large
amount of optical isomers of conventionally known MEL and comparing
properties of the optical isomers and evaluating functions of the
optical isomers would greatly contribute to development of
applications of MEL.
[0064] Non-patent Document 8 describes that MEL having
1-O-.beta.-D-mannopyranosyl-meso-erythritol structure was
chemically synthesized. According to the description, only one MEL
is synthesized through a very complex process and therefore the
synthesis lacks versatility and is difficult to use.
[0065] In another aspect, the present invention was made in view of
the foregoing problem, and an object of the present invention is to
provide: MEL having 1-O-.beta.-D-mannopyranosyl-meso-erythritol
structure, that is an optical isomer of conventional MEL having
4-O-.beta.-D-mannopyranosyl-meso-erythritol structure; and a
production method thereof.
[0066] The inventors of the present invention have diligently
studied in order to achieve the foregoing objects, and found that
MEL and triacyl MEL are effective for activating cells, and thus
completed the present invention. Further, the inventors of the
present invention have diligently studied in order to achieve the
foregoing objects, and found microorganism that produces MEL having
1-O-.beta.-D-mannopyranosyl-meso-erythritol structure (which may
hereinafter referred to as "MEL of the present invention" or
"1-O-MEL"), which is an optical isomer of conventional MEL having
4-O-.beta.-D-mannopyranosyl-meso-erythritol structure (which may be
hereinafter referred to as "conventional MEL" or "4-O-MEL"), and
thus completed the present invention. That is, the present
invention includes the following subject matters.
(1) An activator, including a biosurfactant as an active
ingredient. (2) The activator as set forth in (1), wherein the
biosurfactant is mannosylalditol lipid or a triacyl derivative of
mannosylalditol lipid. (3) The activator as set forth in (2),
wherein the mannosyl alditol lipid is mannosylerythritol lipid
(MEL) or mannosylmannitol lipid (MML). (4) An anti-aging agent,
including as an active ingredient an activator as set forth in any
one of (1) to (3). (5) A hair growth agent, including as an active
ingredient an activator as set forth in any one of (1) to (3). (6)
An external agent, including as an active ingredient one of an
activator as set forth in any one of (1) to (3), an anti-aging
agent as set forth in (4), and a hair growth agent as set forth in
(5). (7) A cosmetic, including as an active ingredient one of an
activator as set forth in any one of (1) to (3), an anti-aging
agent as set forth in (4), and a hair growth agent as set forth
(5). (8) A quasi-drug, including as an active ingredient one of an
activator as set forth in any one of (1) to (3), an anti-aging
agent as set forth in (4), and a hair growth agent as set forth in
(5). (9) A drug, including as an active ingredient one of an
activator as set forth in any one of (1) to (3), an anti-aging
agent as set forth in (4), and a hair growth agent as set forth in
(5). (10) Food and drink, including as an active ingredient one of
an activator as set forth in any one of (1) to (3), an anti-aging
agent as set forth in (4), and a hair growth agent as set forth in
(5). (11) Mannosylerythritol lipid, including a structure
represented by formula (1)
##STR00001##
wherein substituents R.sup.1 may be a same as each other or
different from each other and represent fatty series acyl groups
having 4-24 carbon atoms, substituents R.sup.2 may be a same as
each other or different from each other and represent hydrogen or
acetyl groups, and a substituent R.sup.3 represents hydrogen or a
fatty series acyl group having 12 carbon atoms, excluding a
structure wherein the substituents R.sup.1 are fatty series acyl
groups having 12 carbon atoms, the substituents R.sup.2 are acetyl
groups, and the substituent R.sup.3 is hydrogen. (12) The
mannosylerythritol lipid as set forth in (11), wherein in the
formula (1), one of the substituents R.sup.2 is an acetyl group and
the other of the substituents R.sup.2 is hydrogen. (13) The
mannosylerythritol lipid as set forth in (11) or (12), wherein in
the formula (1), the substituent R.sup.3 is a fatty series acyl
group having 2-24 carbon atoms. (14) The mannosylerythritol lipid
as set forth in any one of (11)-(13), the mannosylerythritol lipid
being produced by microorganism. (15) A method for producing
mannosylerythritol lipid, comprising the steps of culturing
microorganism that belongs to Pseudozyma genus and that is capable
of producing mannosylerythritol lipid, so as to produce
mannosylerythritol lipid including a structure represented by
formula (1)
##STR00002##
wherein substituents R.sup.1 may be a same as each other or
different from each other and represent fatty series acyl groups
having 4-24 carbon atoms, substituents R.sup.2 may be a same as
each other or different from each other and represent hydrogen or
acetyl groups, and a substituent R.sup.3 represents hydrogen or a
fatty series acyl group having 2-24 carbon atoms. (16) The method
as set forth in (15), wherein the microorganism is one of
Pseudozyma tsukubaensis and Pseudozyma crassa.
[0067] The biosurfactant used as an active ingredient in the
activator of the present invention has a notable activating
function on various cells. Therefore, the activator of the present
invention yields an excellent effect as an anti-aging agent and a
hair-growth agent. further, the biosurfactant is a natural
surfactant derived from living organism and therefore has highly
safe, which yields an effect that the activator of the present
invention can be sufficiently used for a long time. Further, the
biosurfactant can be produced by culturing microorganism.
Therefore, the costs for the raw material of the biosurfactant is
low and a large number of the biosurfactant can be produced. This
yields an effect that the activator of the present invention can be
provided in a low price.
[0068] Further, with the production method of MEL of the present
invention, it is possible to easily produce a large number of an
optical isomer of conventionally known MEL.
[0069] Additional objects, features, and strengths of the present
invention will be made clear by the description below. Further, the
advantages of the present invention will be evident from the
following explanation in reference to the drawings.
BRIEF DESCRIPTION OF DRAWINGS
[0070] FIG. 1 is a graph showing the result of examining a
cell-activating function of MEL (MEL-A produced from soybean oil)
on normal human skin fibroblasts.
[0071] FIG. 2 is a graph showing the result of examining a
cell-activating function of MEL (MEL-A produced from soybean oil)
on human head hair papilla cells.
[0072] FIG. 3 is a graph showing the result of examining a
cell-activating function of triacyl MEL (triacyl MEL-A obtained by
adding oleinic acid to a hydroxide group of an erythritol part of
MEL-A produced from soybean oil) on normal human skin
fibroblasts.
[0073] FIG. 4 is a drawing showing the result of thin layer
chromatography on a culture of Pseudozyma tsukubaensis JCM 10324
strain.
[0074] FIG. 5 is a drawing showing the result of analyzing
high-performance liquid chromatography on a culture of Pseudozyma
tsukubaensis JCM 10324 strain.
[0075] FIG. 6 is a drawing showing the result of thin layer
chromatography on a culture of Pseudozyma crassa CBS 9959
strain.
[0076] FIG. 7 is an enlarged drawing (3.4-5.7 ppm) of a sugar
skeleton part in .sup.1H-NMR spectrum of MEL produced by Pseudozyma
tsukubaensis JCM 10324 strain and Pseudozyma antarctica KM-34
(FERMP-20730) strain.
[0077] FIG. 8 is an enlarged drawing (3.3-4.2 ppm) of a sugar
skeleton part in .sup.1H-NMR spectrum of mannosylerythritol
synthesized from a starting material that is MEL produced by
Pseudozyma tsukubaensis JCM 10324 strain and Pseudozyma antarctica
KM-34 (FERMP-20730) strain.
[0078] FIG. 9 is an enlarged drawing (3.4-5.7 ppm) of a sugar
skeleton part in .sup.1H-NMR spectrum of MEL produced by Pseudozyma
crassa CBS 9959 strain.
[0079] FIG. 10 is a drawing showing the result of polarization
microscope observation of liquid crystal forming ability of MEL
produced by Pseudozyma tsukubaensis JCM 10324 strain and Pseudozyma
antarctica KM-34 (FERMP-20730) strain, the liquid crystal forming
ability being evaluated by a water-invading method.
[0080] FIG. 11 is a drawing showing the result of polarization
microscope observation of liquid crystal forming ability of MEL
produced by Pseudozyma tsukubaensis JCM 10324 strain and Pseudozyma
antarctica KM-34 (FERMP-20730) strain, the liquid crystal forming
ability being evaluated by a water-invading method.
[0081] FIG. 12 shows graphs that illustrate .sup.1H-NMR spectrum of
triacyl MEL produced by Pseudozyma tsukubaensis JCM 10324 strain
and an enlarged drawing (3.4-5.7 ppm) of a sugar skeleton part in
the .sup.1H-NMR spectrum, respectively.
[0082] FIG. 13 shows graphs that illustrate .sup.1H-NMR spectrum of
triacyl MEL produced by Pseudozyma hubeiensis and an enlarged
drawing (3.0-5.7 ppm) of a sugar skeleton part in the .sup.1H-NMR
spectrum, respectively.
[0083] FIG. 14 is a drawing illustrating the result of lipid domain
analysis by HPLC(ODS)-MS analysis of MEL produced by Pseudozyma
tsukubaensis JCM 10324 strain.
[0084] FIG. 15 is a drawing illustrating the result of lipid domain
analysis by HPLC(ODS)-MS analysis of triacyl MEL produced by
Pseudozyma hubeiensis.
BEST MODE FOR CARRYING OUT THE INVENTION
Embodiment 1
<1. Activator>
[0085] "Activating" in the present specification indicates
maintaining or prompting cell functions or cell activities.
Consequently, it is possible to prevent the drops of the cell
functions or cell activities, i.e. it is possible to prevent aging
of cells. Therefore, "activator" is synonymous to "cell activator",
and is effective as "anti-aging agent".
[0086] For example, "activating" and "anti-aging" in skin cells
indicate reducing drop of functions of the skin cells due to
accumulation of structural change of basement membrane derived from
aging and photoaging, thereby preventing or improving wrinkles,
sags, hardening etc. of skins, so as to maintain resilient, young,
and healthy skins. Further, in a case of hair-papilla cells or hair
matrix cells, "activating" and "anti-aging" indicates reducing drop
of functions of hair-papilla cells or hair matrix cells due to
aging, stress, and hormone balance, thereby keeping hair cycle so
as to prevent loss of hair.
[0087] "Biosurfactant" is a general term for a substance that is
produced by living organisms and that has a surfactant property and
an emulsification property. The biosurfactant not only includes an
excellent surfactant property and a high emulsification property,
but also includes various physiological functions, which may attain
behaviors and functions other than those of a synthesized
surfactant. The biosurfactant can be mass-produced through
cultivation of microorganism, and may be used as a premixed
product.
[0088] "Premixed product" is a product obtained by adding a
dispersing agent to a functional material or by diluting the
functional material with use of a solvent so as to be usable when
producing cosmetics.
[0089] Examples of the biosurfactant include mannosylerythritol
lipid (MEL), mannosylmannitol lipid (MML), trehaloselipid,
rhamnolipid, sophorose lipid, surfactin, spiculisporic acid, and
emulsan. These examples can be used in the activator of the present
invention. Among them, it is preferable to use a biosurfactant
having a lamellar structure, and it is particularly preferable to
use MEL and MML.
[0090] Four kinds of MEL are known according to whether an acetyl
group at 4- and 6-positions of mannose exists or not. The four
kinds include MEL-A, MEL-B, MEL-C, and MEL-D. Chemical formula (2)
shows a structure of MEL-A. In chemical formula (2), R1 and R2
indicate carbon hydride groups. That is, MEL-A includes alkanoyl
groups having 5-19 carbon atoms at 2- and 3-positions of mannose
and acetyl groups at 4- and 6-positions of mannose in chemical
formula (2). MEL-B includes H instead of an acetyl group
(CH.sub.3CO) at the 4-position of mannose in chemical formula (2).
MEL-C includes H instead of an acetyl group (CH.sub.3CO) at the
6-position of mannose in chemical formula (2). MEL-D includes H
instead of acetyl groups (CH.sub.3CO) at the 4- and 6-positions of
mannose in chemical formula (2).
##STR00003##
[0091] Chemical formula (3) indicates a structure of MML. In
chemical formula (3), R1 and R2 indicate carbon hydride groups.
Further, in chemical formula (3), at least one of or both of acetyl
groups (CH.sub.3CO) at the 4- and 6-positions of mannose may be
replaced with H.
##STR00004##
[0092] The biosurfactant used in the activator of the present
invention may be a tryacyl derivative of MEL or a tryacyl
derivative of MML. The tryacyl derivative of biosurfactant is a
biosurfactant having a new structure with higher hydrophobicity
than that of MEL or MML. For example, in a case of obtaining a
large amount of the biosurfactant from other than a culture
solution of MEL-producing bacteria, it is possible to produce the
biosurfactant by reacting MEL with various plant oils with use of
enzymes.
[0093] Triacyl derivative of MEL, i.e. triacylmannosylerythritol
lipid (which may be referred to as triacyl MEL) includes a
structure shown by chemical formula (4).
##STR00005##
[0094] In chemical formula (4), R1, R2, and R3 independently
indicate a carbon hydride group or a carbon hydride group including
an oxygen atom. At least one of or both of hydroxyl groups at the
4- and 6-positions of mannose may be replaced with an acetyl group.
A carbon hydride group may include only a saturation bond or may
include an unsaturation bond. When including an unsaturation bond,
the unsaturation bond may include a plurality of double bonds. A
carbon chain may be a straight chain or a branched chain. Further,
in a case of the carbon hydride group including an oxygen atom, the
number and the position of an oxygen atom included in the carbon
hydride group are not limited.
[0095] In chemical formula (4), it is preferable that R1 and R2
include 6-20 carbon atoms. R1 and R2 make, as fatty series acyl
groups (RCO--), ester bonds with hydroxyl groups at 2- and
3-positions of mannose. An acetyl group may make ester bond with
other hydroxyl group. It is preferable that R3 has 6-20 carbon
atoms. R3 makes, as a fatty series acyl group (RCO--), ester bond
with a primary hydroxyl group of erythritol.
[0096] A triacyl derivative of MEL has a structure to which fatty
acid ester is added and has high hydrophobicity. Therefore, the
tryacyl derivative of MEL is excellent as emollient since it is
more familiar with various oil components, compared with
conventional MEL.
[0097] These biosurfactants may be used singularly or two or more
of the biosurfactants may be used in combination.
[0098] A method for producing the biosurfactant is not particularly
limited. Any one of fermentation methods with use of well known
biosurfactant-producing-microorganism may be selected. For example,
cultural production of MEL can be carried out by culturing
Pseudozyma antarctica NBRC 10736 through common procedures.
Examples of MEL-producing-microorganism include Candida antarctica,
Candida sp., etc. in addition to the above. It is well known to a
person skilled in the art that cultivation of the microorganism
easily provides MEL. The biosurfactant-producing-microorganism is
not particularly limited and may be suitably selected according to
purposes.
[0099] A fermentation medium for producing biosurfactants may be a
medium with a general composition, made of N source such as yeast
essence and peptone, C source such as glucose and fructose, and
inorganic salts such as sodium nitrate, dibasic potassium
phosphate, and magnesium heptahydrate. Fats and oils such as olive
oil, soybean oil, sunflower oil, corn oil, canola oil, and coconut
oil, and water-unsoluble bases of carbon hydride such as liquid
paraffin and tetradecan may be added singularly to the medium or
two or more of them may be added in combination.
[0100] Fermentation conditions such as pH and temperature and
culture time etc. may be freely set. A culture solution after the
fermentation may be used as the biosurfactant of the present
invention. Further, the culture solution after the fermentation may
be subjected to any operation such as filtering, centrifugal
separation, extraction, purification, and sterilization. The
obtained essence may be diluted, condensed, and dried.
[0101] The fat and oil used as a raw material is preferably plant
fat and oil. The plant fat and oil is not particularly limited and
may be suitably selected according to necessity. Examples of the
plant fat and oil include soybean oil, colza oil, corn oil, peanut
oil, cotton seed oil, safflower oil, sesame oil, olive oil, and
palm oil. Among them, soybean oil is particularly preferable since
it allows increasing production efficiency (production amount,
production speed, and yield) of a biosurfactant (MEL in
particular). These may be used singularly or two or more of them
may be used in combination.
[0102] An inorganic nitrogen source is not particularly limited and
may be suitably selected according to purposes. Examples of the
inorganic nitrogen source include ammonium nitride, urea, sodium
nitride, ammonium chloride, and ammonium sulfate.
[0103] Recovery and purification of a biosurfactant are not
particularly limited and may be suitably selected according to
purposes. For example, a culture solution is subjected to
centrifugal separation so as to recover oil, and a biosurfactant is
recovered by extraction and condensation with use of an organic
solvent such as acetic ether.
[0104] As an extraction solvent, water, alcohols (lower alcohol
such as methanol, absolute ethanol, and ethanol, or polyvalent
alcohol such as propylene glycol and 1,3-btyleneglycol), ketones
such as acetone, esters such as diethyl ether, dioxane,
acetonitrile, and acetic ether, and organic solvents such as
xylene, benzene, and chloroform may be used singularly or two or
more of them may be used in any combination. Further, various
extracts by solvent may be used in combination
[0105] A method for extracting a biosurfactant is not particularly
limited. In general, extraction is carried out in a range from a
room temperature to a boiling point of a solvent at a normal
pressure. After the extraction, the biosurfactant is filtered or
absorbed, decolorized, and purified with use of ion exchange resin
so that the biosurfactant is in the shape of a solution, paste,
gel, or powder. In many cases, the biosurfactant may be used as it
is. If necessary, the biosurfactant may be subjected to a further
purification process such as deodorization and decolorization in a
range that does not influence the effect of the biosurfactant.
Activated carbon column etc. may be used as purification process
means for deodorization and decolorization. Normal means generally
applied according to an extracted substance may be freely selected.
If the biosurfactant is purified with use of a silica gel column
according to necessity, it is possible to obtain a biosurfactant
with higher purity.
[0106] A method for obtaining a triacyl derivative of a
biosurfactant is explained below using a method for producing a
triacyl derivative of MEL. The triacyl derivative of the
biosurfactant used in the present invention is not limited to
triacyl MEL.
[0107] For example, the triacyl MEL may be obtained by purifying a
fraction of the triacyl MEL from a culture solution produced by
fermenting a microorganism as described above. Further, in order to
obtain a large amount of the triacyl MEL, MEL is dissolved in an
organic solvent, a fatty acid derivative of plant oil etc. is
added, and esterification reaction or ester exchange reaction is
carried out in the presence of hydrolytic enzyme.
[0108] Fatty acid introduced to an erythritol part of MEL may be
univalent carboxylic acid of a long-chain carbon hydride. Further,
the fatty acid may be saturated fatty acid or unsaturated fatty
acid. The unsaturated fatty acid may include a plurality of double
bonds. A carbon chain may be straight chain or a branched chain.
Further, a fatty acid derivative that is a derivative of fatty acid
may be used in the present invention, and a mixture of fatty acid
and fatty acid derivative may be used in the present invention. It
is preferable that fatty acid or fatty acid derivative introduced
into the erythritol part of MEL is derived from oils, higher fatty
acid, or synthesized ester.
[0109] Examples of the oils include plant oil, animal oil, mineral
oil, and hardened oil thereof. Specific examples of the oils
include animal/plant oils such as avocado oil, olive oil, sesame
oil, camellia oil, evening primrose oil, turtle oil, macadamia nut
oil, corn oil, mink oil, colza oil, yolk oil, persic oil, wheat
germ oil, sasanqua oil, castor oil, linseed oil, safflower oil,
cotton seed oil, perilla oil, soybean oil, peanut oil, tea oil,
Japanese torreya seed oil, rice oil, tung oil, jojoba oil, cacao
oil, coconut oil, horse oil, palm oil, palm kernel oil, beef
tallow, sheep tallow, pig tallow, lanoline, whale wax, beeswax,
carnauba wax, Japan wax, candelilla wax, and squalan, and hardened
oils thereof; mineral oils such as liquid paraffin and Vaseline,
and synthesized triglycerin such as tripalmitin acid glycerin.
Preferable examples of the oils include avocado oil, olive oil,
sesame oil, camellia oil, evening primrose oil, turtle oil,
macadamia nut oil, corn oil, mink oil, colza oil, yolk oil, persic
oil, wheat germ oil, sasanqua oil, castor oil, linseed oil,
safflower oil, cotton seed oil, perilla oil, soybean oil, peanut
oil, tea oil, Japanese torreya seed oil, rice oil. Further
preferable examples of the oils include olive oil and soybean
oil.
[0110] Examples of the higher fatty acid include caproic acid,
caprylic acid, capric acid, lauric acid, myristic acid, palmitic
acid, oleic acid, linoleic acid, linolenic acid, stearic acid,
behenic acid, 12-hydroxy stearic acid, isostearic acid, undecynoic
acid, tall acid, eicosapentaenoic acid, and docosahexaenoic acid.
Preferable examples of the higher fatty acid include lauric acid,
myristic acid, palmitic acid, oleic acid, linoleic acid, linolenic
acid, stearic acid, and undecylenic acid. Further preferable
examples of the higher fatty acid include oleic acid, linoleic
acid, and undecylenic acid.
[0111] Examples of the synthesized ester include methyl caproate,
methyl caprylate, methyl caprate, methyl laurate, methyl myristate,
methyl palmitate, methyl oleate, methyl linoleate, methyl
linolenate, methyl stearate, methyl undecynoate, ethyl caproate,
ethyl caprylate, ethyl caprate, ethyl laurate, ethyl myristate,
ethyl palmitate, ethyl oleate, ethyl linoleate, ethyl linolenate,
ethyl stearate, ethyl undecynoate, vinyl caproate, vinyl caprylate,
vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate,
vinyl oleate, vinyl linoleate, vinyl linolenate, vinyl stearate,
vinyl undecynoate, cetyl octanoate, octyldodecyl myristate,
isopropyl myristate, myristyl myristate, isopropyl palmitate, butyl
stearate, hexyl laurate, decyl oleate, dimethyl octanoic acid,
cetyl lactate, and myristyl lactate. Preferable examples of the
synthesized ester include methyl laurate, methyl myristate, methyl
palmitate, methyl oleate, methyl linoleate, methyl linolenate,
methyl stearate, and methyl undecylenate. Further preferable
examples of the synthesized ester include methyl oleate, methyl
linoleate, and methyl undecylenate.
[0112] The triacyl MEL can be produced by dissolving MEL in an
organic solvent and reacting the MEL. The organic solvent is not
particularly limited as long as it can solubilize MEL. The organic
solvent only has to solubilize a part of MEL and does not
necessarily have to solubilize the whole part of MEL. The organic
solvent may be a mixture of a plurality of organic solvents.
Specific examples of the organic solvent include methanol, ethanol,
propanol, butanol, acetone, propanone, butanone, pentane-2-on,
1,2-ethanediol, 2,3-butanediol, dioxane, acetonitrile,
2-methyl-butane-2-ol, tertiary butanol, 2-methylpropanol,
4-hydroxy-2-methylpentanone, tetrahydrofuran, hexane, DMF, DMSO,
pyridine, methyl ethyl ketone. Preferable examples of the organic
solvent include acetone, tetrahydrofuran, tertiary butanol,
acetonitrile, and dioxane. Further preferable example of the
organic solvent is acetone.
[0113] Examples of the hydrolytic enzyme include lipase, protease,
and esterase. It is preferable to use at least one selected from
them. A plurality of the hydrolytic enzymes may be used. Lipase and
esterase are preferable, and lipase is further preferable.
[0114] Specifically, MEL purified from a culture solution of
MEL-producing-microorganism is dissolved in an organic solvent
(e.g. acetone) and a commercially available lipase (e.g. novozyme
435 (manufactured by Novozymes)) and plant fat and oil are added to
the organic solvent.
[0115] In this method, the mixture is stirred for 1-7 days at a
reaction temperature of 10-100.degree. C., preferably 20-50.degree.
C., and more preferably 25-40.degree. C. Further, molecular sieves
may be added to the reaction solution. This method allows MEL added
as a material to be a triacyl derivative substantially
quantitatively.
[0116] Purification of the triacyl MEL may be carried out in
accordance with the above purification of MEL.
[0117] The biosurfactant obtained as described above may be used as
an activator as it is. However, it is preferable to use the
biosurfactant in such a manner that the biosurfactant is mixed in
cosmetics, quasi-drugs, drugs, and drinks and foods. The
concentration with which the biosurfactant is mixed is suitably
determined according to the degree of absorption, the degree of
operation, the form of a product, the frequency of usage etc., and
is not particularly limited. The mixture concentration may be
determined in a range that does not impair an operation for
activating cells. In general, the mixture concentration is
preferably 0.001-50% by mass, more preferably 0.1-20% by mass, and
further preferably 1-15% by mass, and particularly preferably 3-10%
by mass, with respect to the whole weight of the activator.
[0118] Here, the biosurfactant to be mixed in the activator may be
used in any form. For example, the biosurfactant may be used as an
extract from a culture solution, may be purified and presented as a
highly purified product, may be used after being suspended in
water, or may be used after being dissolved in an organic solvent
such as ethanol.
[0119] Although the method of the present invention for producing
an activator including a biosurfactant is not particularly
limited,
[0120] Although the method of the present invention for producing
an activator including a biosurfactant is not particularly limited,
it is preferable to use the biosurfactant in such a manner that the
biosurfactant is dissolved in a non-ionic surfactant, lower
alcohol, polyvalent alcohol, or natural fat and oil such as olive
oil, squalan, and fatty acid, since the biosurfactant is highly
hydrophobic.
[0121] Examples of the non-ionic surfactant include sorbitan fatty
acid esters (e.g. sorbitan monooleate, sorbitan monoisostearate,
sorbitan monolaurate, sorbitan monopalmitate, sorbitan
monostearate, sorbitan sesquioleate, sorbitan trioleate,
penta-2-ethyl hexyl acid diglycerol sorbitan, tetra-2-ethyl hexyl
acid diglycerol sorbitan etc.); glycerin polyglycerin fatty acids
(e.g. mono cotton seed oil fatty acid glycerin, glycerin
monoerucate, glycerin sesquioleate, glycerin monostearate, .alpha.,
.alpha.'-oleic acid pyroglutamic acid glycerin, glycerin
monostearate malate etc.); propylene glycol fatty acid esters (e.g.
propylene glycol monostearate); hardened castor oil derivative;
glycerin alkyl ester etc.
[0122] Examples of POE hydrophilic non-ionic surfactant include
POE-sorbitan fatty acid esters (e.g. POE-sorbitan monomonooleate,
POE-sorbitan monostearate, POE-sorbitan monooleate, POE-sorbitan
tetraoleate etc.); POE sorbit fatty acid esters (e.g. POE-sorbit
monolaurate, POE-sorbit monooleate, POE-sorbit pentaoleate,
POE-sorbit monostearate etc.); POE-glycerin fatty acid esters (e.g.
POE-monooleate etc. such as POE-glycerin monostearate, POE-gycerin
monoisostearate, POE-glycerin triisostearate); POE-fatty acid
esters (e.g. POE-distearate, POE-monodioleate, ethylene glycol
distearate etc.); POE-alkylethers (e.g. POE-lauryl ether, POE-oleyl
ether, POE-stearyl ether, POE-behenyl ether,
POE-2-octyldodecylether, POE-cholestanolether etc.); Pluronic type
(such as Pluronic); POE.cndot.POP-alkylethers (e.g.
POE.cndot.POP-cetyl ether, POE.cndot.POP-2-decyltetradecylether,
POE.cndot.POP-monobutylether, POE.cndot.POP hydrogenated lanolin,
POE.cndot.POP-glycerin ether etc.); tetra POE.cndot.tetra
POP-ethylene diamine condensates (e.g. Tetronic); POE-castor oil
hardened castor oil derivative (e.g. POE-castor oil, POE-hardened
castor oil, POE-hardened castor oil monoisostearate, POE-hardened
castor oil triisostearate, POE-hardened castor oil
mono-pyroglutamic acid mono-isostearic acid diester, POE-hardened
castor oil maleic acid); POE-beeswax lanolin derivative (e.g.
POE-sorbit beeswax etc.); alkanolamide (e.g. palm oil fatty acid
diethanolamide, lauric acid monoethernol amide, fatty acid
isopropanol amide etc.); POE-propylene glycol fatty acid ester;
POE-alkylamine; POE-fatty acid amide; simple sugar fatty acid
ester; alkylethoxydimethylamineoxide; trioleyl phosphoric acid
etc.
[0123] Examples of lower alcohol include ethanol, propanol,
isopropanol, isobutylalcohol, t-butylalcohol etc.
[0124] Examples of the polyvalent alcohol include bivalent alcohols
(such as ethylene glycol, propylene glycol, trimethylene glycol,
1,2-butylene glycol, 1,3-butylene glycol, tetramethylene glycol,
2,3-butyleneglycol, pentamethyleneglycol, 2-butene-1,4-diol,
hexylene glycol, octylene glycol); trivalent alcohols (such as
glycerin and trimethylolpropane); quadrivalent alcohols (e.g.
pentaerythritol etc. such as 1,2,6-hexane triol); pentavalent
alcohols (such as xylitol); hexavalent alcohols (such as sorbitol
and mannitol); polyvalent alcohol polymers (such as diethylene
glycol, dipropylene glycol, triethylene glycol, polypropylene
glycol, tetraethylene glycol, diglycerin, polyethylene glycol,
triglycerin, tetraglycerin, and polygycerin); bivalent alcohol
alkyl ethers (such as ethylene glycol monomethyl ether, ethylene
glycol monoethyl ether, ethylene glycol monobutyl ether, ethylene
glycol monophenyl ether, ethylene glycol monohexyl ether, ethylene
glycol mono 2-methylhexyl ether, ethylene glycol isoamyl ether,
ethylene glycol benzyl ether, ethylene glycol isopropyl ether,
ethylene glycol dimethyl ether, ethylene glycol diethyl ether, and
ethylene glycol dibutyl ether); bivalent alcohol alkyl ethers (such
as diethylene glycol monomethyl ether, diethylene glycol monoethyl
ether, diethylene glycol monobutylether, diethyleneglycol dimethyl
ether, diethylene glycol diethyl ether, diethylene glycol butyl
ether, diethylene glycol methyl ethyl ether, triethylene glycol
monomethyl ether, triethylene glycol monoethyl ether, propylene
glycol monomethyl ether, propylene glycol monoethyl ether,
propylene glycol monobutyl ether, propylene glycol isopropyl ether,
dipropylene glycol methyl ether, dipropylene glycol ethyl ether and
dipropylene glycol butyl ether); bivalent alcohol ether ester (such
as ethylene glycol monomethyl ether acetate, ethylene glycol
monoethyl ether acetate, ethylene glycol monobutyl ether acetate,
ethylene glycol monophenyl ether acetate, ethylene glycol
diadipate, ethylene glycol disuccinate, diethylene glycol monoethyl
ether acetate, diethylene glycol monobutyl ether acetate, propylene
glycol monomethyl ether acetate, propylene glycol monoethyl ether
acetate, propylene glycol monopropyl ether acetate, propylene
glycol monophenyl ether acetate); glycerin monoalkyl ether (such as
chimyl alcohol, selachyl alcohol, and batyl alcohol); sugar alcohol
(such as sorbitol, maltitol, maltotriose, mannitol, simple sugar,
erythritol, glucose, fructose, amylolytic sugar, maltose, xylitose,
and amylolytic sugar reducing alcohol); glysolid;
tetrahydrofurfuryl alcohol; POE-tetrahydrofurfuryl alcohol;
POP-butyl ether; POP.cndot.POE-butyl ether; tripolyoxypropylene
glycerin ether; POP-glycerin ether; POP-glycerin ether phosphoric
acid; POP.cndot.POE-pentaerythritol ether, and polyglycerin.
[0125] Examples of the oils include animal and plant oils such as
avocado oil, olive oil, sesame oil, camellia oil, evening primrose
oil, turtle oil, macadamia nut oil, corn oil, mink oil, colza oil,
yolk oil, persic oil, wheat germ oil, sasanqua oil, castor oil,
linseed oil, safflower oil, cotton seed oil, Perilla oil, soybean
oil, peanut oil, tea oil, Japanese torreya seed oil, rice oil, tung
oil, jojoba oil, cacao oil, coco oil, horse oil, palm oil, palm
kernel oil, beef tallow, lard, lanoline, whale tallow, beeswax,
carnauba wax, Japan wax, candellila wax, squalan and hardened oil
thereof, mineral oils such as liquid paraffin and Vaseline, and
synthesized triglycerin such as tripalmtin acid glycerin.
[0126] Examples of the higher fatty acid include lauric acid,
myristic acid, palmitic acid, oleic acid, linoleic acid, linolenic
acid, stearic acid, behenic acid, 12-hydroxy stearic acid,
isostearic acid, undecynoic acid, tall acid, eicosapentaenoic acid,
and docosahexaenoic acid. Examples of the higher alcohols include
lauric alcohol, cetyl alcohol, stearyl alcohol, behenyl alcohol,
myristyl alcohol, oleyl alcohol, cetostearyl alcohol, jojoba
alcohol, lanoline alcohol, batyl alcohol, 2-decyltetratececynol,
cholesterol, phytosterol, and isostearyl alcohol. Examples of the
synthesized ester include cetyl octanoate, octyl dodecyl myristate,
isopropyl myristate, myristyl myristate, isopropyl palmitate, butyl
stearate, hexyl laurate, decyl oleate, dimethyl octanoic acid,
cetyl lactate, and myristyl lactate. Examples of the silicone
include chain polysiloxane such as dimethyl polysiloxane and methyl
phenyl polysiloxane, cyclic polysiloxane such as
decamethylcyclopolysiloxane, and a 3-D matrix structure such as
silicone resin.
[0127] As described above, it is preferable to embody the activator
of the present invention as a composition by mixing a biosurfactant
that is an active ingredient with cosmetics, quasi-drugs, drugs,
and foods.
[0128] In a case of embodying the activator of the present
invention in the form of cosmetics, quasi-drugs, and drugs, it is
preferable that the activator is for external use. However, since
the biosurfactant can be also taken orally, the activator is not
limited to external use and may be used for internal use and for
foods and drinks.
[0129] In a case of using the activator of the present invention as
a drug, since it is verified that the biosurfactant has a function
for activating human head hair papilla cells, it is possible to use
the activator as drugs for promoting hair-growth and preventing
progression of loss of hair.
[0130] The dosage form of the activator is not limited and may be
various forms such as ample, capsule, powder, granulated powder,
pill, tablet, a solid agent, a liquid agent, gell, foam, emulsion,
cream, ointment, sheet, mousse, and bath agent.
[0131] Specifically, examples of the cosmetics, quasi-drugs, and
drugs include: drug products for internal and external uses; basic
skin care such as skin lotion, emulsion, cream, ointment, lotion,
oil, pack; facial wash and skin cleansing agent, hair cosmetics
such as shampoo, rinse, hair treatment, hair cream, pomade, hair
spray, hair dressing, permanent reagent, hair tonic, hairdye, and
hair growth drug and baldness remedy; makeup cosmetics such as
foundation, face powder, ceruse, lipstick, blusher, eyeshadow,
eyeliner, mascara, eyebrow pencil, and eyelash; make-up cosmetics
such as manicure; perfumes; bath agents; tooth pastes; mouth
deodorant; mouth wash; hircismus preventing agent/deodorant;
sanitary goods; sanitary cottons; and wet tissues.
[0132] Specifically, examples of the foods and drinks include:
drinks such as soft drink, carbonated drink, energy drink, juice,
and lactic acid drink; frozen desserts such as ice cream, ice
sherbet, and shaved ice; noodles such as soba, udon, bean-starch
vermicelli, crust of potsticker, crust of dumpling, Chinese noodle,
and instant noodle; sweets such as lozenge, candy, gum, chocolate,
tablet candy, munch, biscuit, jelly, jam, cream, baked cake, and
bread; marine products such as crab, salmon, clam, tuna, sardine,
shrimp, bonito, mackerel, whale, oyster, saury, squid, ark shell,
scallop, ear shell, sea urchin, salmon caviar, and sulculus
diversicolor supertexta; marine and animal processed foods such as
boiled fish paste, ham, and sausage; dairy products such as
processed milk and fermented milk; fats and oils and fat and oil
processed foods such as salad oil, frying oil, margarine,
mayonnaise, shortening, whip cream, and dressing; seasonings such
as source and baste; retort pouch foods such as curry, stew,
chicken and egg bowl, rice gruel, zosui, Chinese bowl, pork cutlet
bowl, tempura bowl, spicy broiled eel bowl, hashed rice, oden, mapo
doufu, beef bowl, meat source, egg soup, omelet rice, potsticker,
dumpling, hamburg steak, and meat ball; health and nutriceutical
supplements in various forms; functional foods; tablets; capsules;
health drinks; and troches.
[0133] The activator of the present invention is preferably
applicable to human, but may be applied to animals other than
human.
[0134] The activator of the present invention may include not only
the biosurfactant that is an active ingredient but also, if
necessary, components and additives that are used in cosmetics,
quasi-drugs, drugs, and foods and drinks in a range that does not
reduce the effect of the present invention.
[0135] Examples of the fats and oils include avocado oil, almond
oil, fennel oil, perilla oil, olive oil, orange oil, orange roughy
oil, sesame oil, cacao oil, chamomile oil, carrot oil, cucumber
oil, beef tallow, beef tallow fatty acid, kukui nut oil, safflower
oil, soybean oil, camellia oil, corn oil, colza oil, persic oil,
castor oil, cotton seed oil, peanut oil, turtle oil, mink oil, yolk
oil, cacao oil, palm oil, palm kernel oil, Japan wax, coco oil,
beef tallow, lard, hardened oil, and hardened castor oil.
[0136] Examples of tallow include beeswax, carnauba wax, whale wax,
lanoline, liquid lanoline, reduced lanoline, hardened lanoline,
candellila wax, montan wax, and shellac wax.
[0137] Examples of the mineral oil include liquid paraffin,
Vaseline, paraffin, ozokerite, ceresin, microcrystalline wax,
polyethylene powder, squalene, squalan, and pristane.
[0138] Examples of the fatty acids include: natural fatty acids
such as lauric acid, myristic acid, palmitic acid, stearic acid,
behenic acid, oleic acid, 12-hydroxy stearic acid, undecynoic acid,
tall oil, and lanoline fatty acid; and synthesized fatty acids such
as isononanoic acid, caproic acid, 2-ethylbutane acid, isopentane
acid, 2-methylpentane acid, 2-ethylhexane acid, and isopentane
acid.
[0139] Examples of the alcohols include: natural alcohols such as
ethanol, isopropanol, lauric alcohol, cetanol, stearyl alcohol,
oleyl alcohol, lanoline alcohol, cholesterol, and phytosterol;
synthesized alcohols such as 2-hexyldecanol, isostearyl alcohol,
and 2-octyldodecanol; polyvalent alcohols such as ethylene oxide,
ethylene glycol, diethylene glycol, triethylene glycol, ethylene
glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene
glycol monomethyl ether, diethylene glycol monoethyl ether,
polyethylene glycol, propylene oxide, propylene glycol,
polypropylene glycol, 1,3-butylene glycol, glycerin, batyl alcohol,
pentaerythritol, sorbitol, mannitol, glucose, and simple sugar.
[0140] Examples of the esters include isopropyl myristate,
isopropyl palmitate, butyl stearate, hexyl laurate, myristyl
myristate, oleyl oleate, decyl oleate, octyl dodecyl myristate,
hexyl decile dimethyloctanoate, cetyl lactate, myristyl lactate,
diethyl phthalate, dibutyl phthalate, lanoline acetate,
ethyleneglycol monostearate, propylene glycol monostearate,
propylene glycol dioleate.
[0141] Examples of the metal soap include aluminum stearate,
magnesium stearate, zinc stearate, calcium stearate, zinc
palmitate, magnesium myristate, zinc laurate, and zinc
undecylenate.
[0142] Examples of gummy and water-soluble macromolecular
compositions include gum Arabic, benzoin gum, dammar gum, guaiac,
Irish moth, karaya gum, tragacanth gum, carob gum, quince seed,
agar, casein, dextrin, gelatin, pectin, starch, carrageenan,
carboxy alkyl chitin, chitosan, hydroxy alkyl chitin, low molecular
chitosan, chitosan salt, sulfated chitin, phosphorylated chitin,
alginic acid and salt thereof, hyaluronic acid and salt thereof,
chondroitin sulfate, heparin, ethylcellulose, methylcellulose,
carboxy methylcellulose, carboxy ethylcellulose, carboxyethyl
cellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose,
nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl
methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate,
polyacrylic acid salt, polyalkylene oxide such as polyethylene
oxide and polypropylene oxide and crosslinking polymer thereof,
carboxy vinyl polymer, polyethylene imine.
[0143] Examples of the surfactants include anionic surfactant (such
as carboxylate, sulfonate, sulfate ester salt, and phosphoric ester
salt), cationic surfactant (such as amine salt and quaternary
ammonium salt), ampholytic surfactant (carboxylic acid ampholytic
surfactant, sulfate ester ampholytic surfactant, sulfonic acid
ampholytic surfactant, and phosphate ester ampholytic surfactant),
non-ionic surfactant (such as ether non-ionic surfactant, ether
ester non-ionic surfactant, ester non-ionic surfactant, block
polymer non-ionic surfactant, and nitrogen-containing non-ionic
surfactant), and other surfactant (such as natural surfactant,
derivative of protein hydrolysate, macromolecular surfactant,
surfactant including titan and silicon, and carbon fluoride
surfactant.
[0144] Examples of vitamins include retinol, retinal (vitamin A1),
dehydroretinal (vitamin A2), carotin, and lycopene (protovitamine
A) in vitamin A group; thiamine hydrochloride, thiamine
hydrosulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine
(vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic
acids, pantothenic acids, biotins, choline, inositols in vitamin B
group; ascorbic acid and derivative thereof in vitamin C group;
ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), and
dihydrotachysterol in vitamin D group; tocopherol and derivative
thereof and ubiquinone in vitamin E group; phytonadione (vitamin
K.sub.1), menaquinone (vitamin K.sub.2), menadione (vitamin
K.sub.3), and menadiol (vitamin K.sub.4) in vitamin K group.
[0145] Examples of amino acids include valine, leucine, isoleucine,
threonine, methionine, phenylalanine, tryptophan, lysine, glycine,
alanine, asparagine, glutamine, serin, cysteine, cystine, tylosine,
proline, hydroxyproline, asparaginic acid, glutamic acid,
hydroxylysine, arginine, ornithine, histidine, hydrosulfates
thereof, phosphates thereof, nitrates thereof, citrates thereof,
and amino acid derivatives such as pyrrolidone carboxylic acid.
[0146] Examples of whitening agents include ascorbic acid and
derivative thereof, sulfur, hydrolysate of placenta, ellagic acid
and derivative thereof, kojic acid and derivative thereof,
glucosamine and derivative thereof, arbutin and derivative thereof,
hydroxycinnamic acid and derivative thereof, glutathione, arnica
essence, essence of root of scultellaria baicalensis, essence of
root bark of morus ihou, essence of root of bupleurum
scorzonerifoliumL, essence of root of saposhnikovia seseloides,
culture of mycelium of Ganoderma lucidum and extract thereof,
essence of linden, essence of peach leaves, essence of fruit of
rosa multiflora, essence of root of sophora flavescens, essence of
Sanguisorba officinalis, essence of angelica acutiloba, essence of
seed of Job's tears, essence of persimmon leaves, essence of
pieplant, essence of root bark of peony, essence of hamamelis,
essence of marronnier, essence of hypericum erectum, essence of
oil-soluble licorice.
[0147] Examples of moisture retention agents include hyaluronic
acid, polyglutamine acid, serin, glycine, threonine, alanine,
collagen, hydrolyzed collagen, hydronectin, fibronectin, keratin,
elastin, royal jelly, chondroitin heparin sulfate,
glycerophospholipid, glyceroglycolipid, sphingophospholipid,
sphingoglycolipid, linoleic acid and esters thereof,
eicosapentaenoic acid and esters thereof, pectine, bifidobacteria
fermentation product, lactic acid fermentation product, yeast
extract, culture of mycelium of Ganoderma lucidum and extract
thereof, wheat germ oil, avocado oil, rice oil, jojoba oil, soybean
phospholipid, .gamma.-oryzanol, essence of Althaea officinalis,
essence of seed of Job's tears, essence of root of Rehmannia
glutinosa, essence of fruit of jujube, essence of seaweed, essence
of aloe arborescens, essence of burdock, essence of rosemary,
essence of arnica, and wheat bran.
[0148] Examples of the hair growth drug include pentadecanic acid
glyceride, essence of coleus, essence of gentiana lutea, essence of
conifer cone, essence of royal jelly, essence of sasa veitchii,
t-flavanone, 6-benzyl amino purine, essence of swertia japonica,
carpronium chloride, minoxidil, finasteride, adenosine, nicotinic
acid amide, essence of mulberry roots, essence of rehmannia
glutinosa, and 5-aminolevulinic acid.
[0149] Examples of extracts and essences of animals, plants, and
galenicals include Uncaria gambir, Angelica keiskei, acerola,
Althaea, Arnica montana, avocado, Hydrangea macrophylla var.
thunbergii, Aloe, Aloe vera, nettle, Ginkgo, fennel, turmeric,
Asarum sieboldii, ume, Quercus salicina, Arctostaphylos uva-ursi,
Rosa multiflora, Rabdosia japonica, membranous milk-vetch,
Scutellaria baicalensis (dried root of Scutellaria baicalensis),
Prunus jamasakura, Phellodendron amurense, Coptis japonica, Panax
ginseng, Hypericum erectum, Lamium album var. barbatum, Watercress,
orange, Polygala tenuifolia, Prunella vulgaris subsp. asiatica,
Polygonum multiflorum, Pagoda Tree, mugwort, Zedoary, Kudzu,
Valeriana fauriei, chamomile, Trichosanthes kirilowii var.
japonica, Artemisia capillaris Thunb, licorice, Tussilago farfara,
Bramble, kiwifruit, balloon flower, Chrysanthemum, Catalpa ovata,
Rutaceae plant fruit (unripened fruit of Citrus aurantium or Citrus
natsudaidai), Citrus tachibana, cucumber, Aralia cordata, Angelica
pubescens, apricot, Chinese Wolfberry, Sophora flavescens, Camphor
tree, Sasa veitchii, grapefruit, Cinnamon, Schizonepeta tenuifolia,
Senna obtusifolia, Ipomoea purpurea, morning glory, Safflower,
Sumac, Comfrey, Copaiba, gardenia, gentiana, Magnolia obovata (bark
of Magnolia obovata), achyranthes bidentata (root thereof),
tetradium ruticarpum (fruit thereof), burdock, schisandra chinensis
(fruit thereof), rice, rice bran, wheat, bupleurum
schorzonerifolium (root thereof), saffron, Saponaria officinalis,
hawthorn, Japanese pepper, salvia, panax pseudoginseng, Chinese
mushroom, rehmannia glutinosa (root thereof), Quisqualis indica,
lithospermum erythrorhizon (root thereof), perilla, persimmon
(sepal of fruit thereof), peony, plantago asiatica (seed thereof,
whole parts thereof), ginger, iris, glossy privet (fruit thereof),
filipendula multijuga, white birch, Japanese honeysuckle (bud
thereof), hedera helix, achillea millefolium, elder, adzuki bean,
Japanese red elder, malva sylvestris, cnidium officinale makino,
Japanese green gentian, mulberry (root bark thereof, leaves
thereof), jujube, soybean, aralia elata, panax japonicus,
anemarrhena asphodeloides (bulb thereof), sanguisorba officinalis
(root thereof), houttuynia cordata, Caterpillar fungus, pepper,
Chinese lantern plant, thyme, green tea, black tea, clove, citrus
unshiu (exocarp thereof), camellia, contella asiatica, pepper,
angelica acutiloba (root thereof), calendula officinalis, citrus
aurantium (exocarp of fruit thereof), sanguisorba officinalis (root
thereof), corn (style of gynoecium thereof), eucommia ulmoides
(bark thereof, leaves thereof), tomato, nandina domestica (fruit
thereof), garlic, barley (malt), Pictamnus albus (bark of root of
Pictamnus albus), Ophiopogon japonicus (Ophiopogon japonicus
tuber), parsley, batata, mint, hamamelis, rose, leaves of loquat,
Poria cocos, grape or leaves thereof, dishcloth gourd, tilia
miqueliana, peony (root bark thereof), hop, Rosa rugosa, pine
needle, marronnier, rosemary, soapberry, melissa, melilot, Japanese
quince, bean sprout, peach (inner core of seed thereof, leaves
thereof), Belamcanda chinensis, betel palm tree, leonurus
sibiricus, cornflower, saxifraga stolonifera (leaves thereof),
myrica rubura (bark thereof), alnus firma, Job's tears (seed of
Job's tears), Artemisia Mongolia, artemisia montana, lavender,
apple fruit, varnished conk, lemon fruit, Forsythia suspensa (fruit
thereof), Chinese milk vetch, geranium thunbergii (geranium),
scopolia japonica (bulb and root thereof), crest of chicken,
placental extract of cattle and human, extract or resolvent of
stomach, duodenum, or intestines of pig and cattle, water-soluble
collagen, water-soluble collagen derivative, hydrolysis of
collagen, elastin, hydrolysis of elastin, water-soluble elastin
derivative, silk protein, resolvent of silk protein, and
decomposition product of bovine blood cell protein.
[0150] Examples of the microorganism culture metabolites include
yeast essence, zinc-containing yeast essence, germanium-containing
yeast essence, selenium-containing yeast essence,
magnesium-containing yeast essence, fermented rice essence, euglena
extract, lactic fermentation product of skimmed milk.
[0151] Examples of the .alpha.-hydroxy acid include glycol acid,
citric acid, malic acid, tartaric acid, and lactic acid.
[0152] Examples of the inorganic colorings include silicic acid
anhydride, magnesium silicate, talc, kaolin, bentonite, mica,
titanium mica, bismuth oxychloride, zirconium oxide, magnesium
oxide, zinc oxide, titanium oxide, calcium carbonate, magnesium
carbonate, iron oxide yellow, colcothar, iron oxide black,
ultramarine, chromium oxide, chromium hydroxide, carbon black, and
calamine.
[0153] Examples of the ultraviolet ray absorber include p-amino
benzolin acid derivative, salicylic acid derivative, anthranilic
acid derivative, coumarin derivative, amino acid compound,
benzotriazole derivative, tetrazole derivative, imidazoline
derivative, pyrimidine derivative, dioxane derivative, camphor
derivative, furan derivative, pyrone derivative, nuclear acid
derivative, allantoin derivative, nicotinic acid derivative,
vitamin B6 derivative, oxybenzone, benzophenone, guaiazulene,
shikonin, baicalin, baicalein, and berberine.
[0154] Examples of the astringent include laconic acid, tartaric
acid, succinic acid, citric acid, allantoin, zinc chloride, zinc
sulfate, zinc oxide, calamine, p-phenol zinc sulfonate, aluminum
calium sulfonate, resorcin, iron chloride, and tannic acid.
[0155] Examples of the antioxidant include ascorbic acid and salt
thereof, ester stearate, tocopherol and ester derivative thereof,
nordihydroguaceretenic acid, buthylhydroxy toluene (BHT),
buthylhydroxy anisole (BHA), parahydroxy anisole, propyl gallate,
sesamol, sesamolin, and gossypol.
[0156] Examples of the anti-inflammatory agent include ichthammol,
indomethacin, kaolin, salicylic acid, sodium salicylate, methyl
salicylate, acetylsalicylic acid, diphyenhydramine chloride, d- or
d 1-camphor, hydrocortisone, guaiazulene, chamazulene,
chlorpheniramine maleate, glycyrrhizin acid and salt thereof,
glycyrrhetic acid and salt thereof.
[0157] Examples of the fungicide and disinfectant include acrinol,
sulfur, benzalkonium chloride, benzethonium chloride,
methylrosaniline chloride, cresol, calcium gluconate, chlorhexidine
gluconate, sulfamine, mercurochrome, lactoferrin and hydrolysates
thereof.
[0158] Examples of the hair care agent include selenium disulfide,
alkyl isoquinolinium bromide liquid, zinc pyrithione, biphenamine,
thianthol, kasutari tincture, ginger tincture, pepper tincture,
quinine hydrochloride, strong ammonia water, potassium bromate,
sodium bromate, and thioglycolic acid.
[0159] Examples of the aroma chemical include: natural animal aroma
chemical such as musk, civet, castoreum, and ambergris; plant aroma
chemicals such as anis essential oil, angelica essential oil,
ylang-ylang essential oil, iris essential oil, fennel essential
oil, orange essential oil, cananga essential oil, caraway essential
oil, cardamom essential oil, guaiac wood essential oil, cumin
essential oil, lindera essential oil, cassia essential oil,
cinnamon essential oil, geranium essential oil, copaiba balsam
essential oil, coriander essential oil, perrilla essential oil,
cedar wood essential oil, citronella essential oil, jasmine
essential oil, ginger grass essential oil, cedar essential oil,
spearmint essential oil, peppermint essential oil, Ferula gummosa
essential oil, tuberose essential oil, clove essential oil, orange
flower essential oil, wintergreen essential oil, tolu balsam
essential oil, Patchouli essential oil, rose essential oil,
palmarosa oil, Japanese cypress essential oil, Japanese cypress
essential oil, sandal wood oil, petitgrain essential oil, bay
essential oil, vetiver essential oil, bergamot essential oil,
balsam of Peru essential oil, bois de rose essential oil, camphor
tree essential oil, mandarin essential oil, eucalyptus essential
oil, lime essential oil, lavender essential oil, linaloe essential
oil, lemon grass essential oil, lemon essential oil, rosemary
essential oil, and Japanese mint essential oil; and other synthetic
aroma chemicals.
[0160] Examples of the coloring and coloring matter include red
cabbage coloring, red rice coloring, Rubia argyi coloring, annatto
coloring, sepia coloring, turmeric coloring, sophora coloring,
krill coloring, persimmon coloring, caramel, gold, silver, gardenia
coloring, corn coloring, onion coloring, tamarind coloring,
spirulina coloring, buckwheat coloring, cherry coloring, layer
coloring, hibiscus coloring, grape juice coloring, marigold
coloring, purple potato coloring, purple yam coloring, lac
coloring, and rutin.
[0161] Examples of the sweetening include sugar, Hydrangea
macrophylla, fructose, arabinose, galactose, xylose, mannose,
maltlose, honey, glucose, miraculin, and monellin.
[0162] Examples of the nutritional additive include calcinated
shell calcium, cyanocolabamin, yeast, wheat germ, soybean embryo,
yolk powder, hemicellulose, and heme iron.
[0163] Other examples of materials that may be included in the
activator of the present invention include hormones, chelating
agent, pH adjuster, chelating agent, antiseptic, fungicide,
refrigerant, stabilizer, emulsifier, animal/plant proteins and
decomposition products thereof, animal/plant polysaccharides and
decomposition products thereof, animal/plant glycoproteins and
decomposition products thereof, blood flow promoting agent,
anti-inflammatory agent and anti-allergy agent, cell activator,
keratolytic agent, wound healing drug, foam boosting agent,
thickener, agent for oral use, deodorant, bittering agent,
seasoning, and oxygen.
[0164] <2. Anti-Aging Method>
[0165] Usage of the biosurfactant that is an active ingredient of
the activator of the present invention allows providing an
anti-aging method. That is, the present invention encompasses an
anti-aging method with use of the biosurfactant.
[0166] The anti-aging method of the present invention includes the
step (i) of causing the biosurfactant to touch an animal. The
biosurfactant is preferably at least one selected from a group
consisting of mannosyl erythritol lipid (MEL), mannosyl mannitol
lipid (MML), a triacyl derivative of mannosyl erythritol lipid
(MEL), and a triacyl derivative of mannosyl mannitol lipid
(MML).
[0167] The animal is not particularly limited as long as an
anti-aging effect of the biosurfactant can be expected. A
preferable example of the animal is a mammal. A further preferable
example of the animal is a human.
[0168] "Causing the biosurfactant to touch an animal" indicates
causing the biosurfactant to touch a range where an external agent
can be applied, such as skin and mucous membrane of the animal.
[0169] The anti-aging method of the present invention further
includes the step (ii) of activating cells with use of the
biosurfactant. Activating cells with use of the biosurfactant
having a cell-activating function allows yielding an anti-aging
effect such as preventing and improving wrinkles, sags, and
hardening of skin so as to maintain resilient, youthful, and
healthy skin.
Embodiment 2
[0170] The following explains another embodiment of the present
invention.
[0171] <Mannosyl Erythritol Lipid (MEL)>
[0172] In order to aid understanding of MEL of the present
invention, the following outlines conventional MEL.
[0173] The conventional MEL is produced through cultivation of a
MEL-producing microorganism. A representative example of the
chemical structure of the conventional MEL is shown by general
formula (5) below. 4-O-.beta.-D-mannopyranosyl-meso-erythritol as a
basic structure thereof.
##STR00006##
[0174] In the general formula (5), a substituent R is a hydro
carbon group (alkyl group or alkenyl group). Four kinds of the
conventional MEL are known according to whether an acetyl group at
4- and 6-positions of mannose exists or not. The four kinds include
MEL-A, MEL-B, MEL-C, and MEL-D.
[0175] MEL-A is designed such that each of substituents R.sup.1 and
R.sup.2 is an acetyl group in the general formula (5). MEL-B is
designed such that the substituent R.sup.1 is an acetyl group and
the substituent R.sup.2 is hydrogen in the general formula (5).
MEL-C is designed such that the substituent R.sup.1 is hydrogen and
the substituent R.sup.2 is an acetyl group in the general formula
(5). MEL-D is designed such that each of the substituents R.sup.1
and R.sup.2 is hydrogen.
[0176] The number of carbons in the substituent R of the MEL-A,
MEL-B, MEL-C, and MEL-D varies according to the number of carbons
in fatty acid constituting triglyceride in fats and oils included
in an MEL-producing medium and the degree of assimilation of fatty
acid by MEL producing microorganism in use. In a case where the
triglyceride includes an unsaturated fatty acid residue, when the
MEL producing microorganism do not assimilate a double-bonding
section of the unsaturated fatty acid, it is possible for MEL to
include the unsaturated fatty acid residue as the substituent R. As
is evident from the above, each resulting MEL is generally a
mixture of compounds having different fatty acid residues of the
substituents R.
[0177] On the other hand, MEL of the present invention has a
structure represented by the general formula (1) and is an optical
isomer in which erythritol is introduced in a direction opposite to
that of the conventional MEL. In the general formula (1), the
substituents R.sup.1 may be the same or different from each other
and are fatty series acyl groups having 4-24 carbon atoms, and the
substituents R.sup.2 may be the same or different from each other
and represent hydrogen or acetyl groups. Further, the substituent
R.sup.3 represents a fatty series acyl group having 2-24 carbon
atoms. Note that the present invention excludes MEL where both of
the substituents R' are fatty series acyl groups having 12 carbon
atoms, both of the substituents R.sup.2 are acetyl groups, and the
substituent R.sup.3 is hydrogen. This is indented to exclude the
MEL disclosed in Non-patent Document 8 from the present invention,
and is not indented for any other purpose. This exclusion is not a
limitative matter that unduly limits the scope of the present
invention.
[0178] Further, the substituent R.sup.1 in the general formula (1)
may be a fatty series acyl group or an unsaturated fatty series
acyl group, and is not particularly limited. When the substituent
R.sup.1 includes an unsaturated bond, the substituent R.sup.1 may
include a plurality of double bonds. The carbon chain may be a
straight chain or a branched chain. Further, in a case of
hydrocarbon group containing an oxygen atom, the number and the
position of the contained oxygen atom are not limited.
[0179] Further, it is preferable that one of the substituents
R.sup.2 is an acetyl group and the other of the substituents
R.sup.2 is hydrogen. That is, it is preferable that the MEL of the
present invention is a 1-O-MEL and is MEL-B or MEL-C. In
particular, it is further preferable that the MEL of the present
invention has hydrogen at 4-position and an acetyl group at
6-position, i.e. MEL-B.
[0180] For example, compared with MEL-A (having two acetyl groups),
MEL-B or MEL-C (having one acetyl group) has higher polarity and
different in its self-assembly behavior in water. Consequently,
formed liquid crystals have different phases. In MEL-A, a sponge
phase (L.sub.3 phase) etc. is formed in a wide concentration
region, whereas in MEL-B or MEL-C, a lamella phase (L.sub..alpha.
phase) is likely to be formed. The lamella phase is very similar to
a keratin layer of skin, making the MEL have high skin-permeability
and effective as a skincare material. Further, in MEL-B, a
bimolecular film is likely to form a capsuled vesicle (liposome),
which allows the capsule to include a drug therein. Therefore, it
is expected that MEL-B is easily applicable to liposome cosmetics
and drugs (see Non-patent Documents 9 and 10).
[0181] The MEL synthesized in Non-patent Document 8 is of A type,
and includes two fatty acids each being C12. In contrast thereto,
the present invention allows producing MEL-B or MEL-C, and widely
varying the length of a fatty acid chain. This allows providing MEL
having an ability to form more different liquid crystals.
[0182] Note that the synthesizing method described in Non-patent
Document 8 is limited to a method for synthesizing MEL-A. In order
to synthesize MEL-B and MEL-C, it is necessary to use different
protective groups and to repeat different step. Therefore, the MEL
of the present invention could not have been synthesized based on
Non-patent Document 8.
[0183] In the general formula (1), it is preferable that the
substituent R.sup.3 is a fatty series acyl group having 2-24 carbon
atoms. When both of the substituents R.sup.1 and R.sup.3 are fatty
series acyl groups, the MEL is triacyl MEL that has properties
different from those of diacyl MEL.
[0184] Specifically, a triacyl derivative is a surfactant having
lower HLB (hydrophilic-hydrophobic balance) and higher
lipophilicity than a conventional diacyl derivative. Therefore, the
triacyl derivative is used for purposes different from those of the
diacyl derivative. For example, the triacyl derivative can be used
for W/O emulsion, a dispersing agent etc. Further, as with the
above case, the synthesizing method described in Non-patent
Document 8 is limited to a method for synthesizing a diacyl
derivative of MEL-A, and synthesizing a triacryl derivative would
require entirely different synthesizing route (different protective
groups and different multi-stage reactions). Therefore, the MEL of
the present invention could not have been produced based on
Non-patent Document 8.
[0185] The chemical structure of the MEL of the present invention
can be obtained in the form of a mixture made of compounds that are
different according to the number of carbons in a fatty series acyl
group that is the substituent R.sup.1 in the general formula (1) or
to whether a double bond exists or not. the compounds can be made a
single MEL compound by purifying with use of a preparative
HPLC.
[0186] As with the conventional MEL, the MEL of the present
invention has high surface-activity, and unlike the conventional
MEL, the MEL of the present invention has new physiological
activity and self-assembling property, and therefore can be used as
a surfactant or various catalysts for fine chemicals. Further, the
MEL is very significant since it has high biodegradability and
highly safe. That is, the MEL is a biosurfactant that has high
biodegradability, low toxicity, and is environment-friendly.
[0187] It is reported that the conventional MEL has various
bioactive functions. For example, it is reported that MEL has the
following functions: when MEL is caused to act on strain of human
acute promyelocytic leukemia cellulous HL 60, MEL shows a
promyelocytic cell differentiation inducing function for
differentiating granulocytes; when MEL is caused to act on PC 12
cells derived from rat adrenal medulla melanocytoma, MEL shows
neural system cell differentiation inducing function etc. for
elongating neuritis; and for the first time among glycolipids
produced by a microorganism, MEL can induce apoptosis of melanoma
cells (X. Zhao et. A1, Cancer Research, 59, 482-486 (1999)), and
hence MEL has a function for preventing proliferation of cancer
cells. In consideration of the bioactive functions of the
conventional MEL, it is expected that the MEL of the present
invention also has various bioactive functions and is applicable to
drugs such as an anticancer agent and a new cosmetic material.
[0188] Further, as explained in later-mentioned Examples, the MEL
of the present invention is significantly different in liquid
crystal forming ability from the conventional MEL due to the
difference in chilarity of molecules. Specifically, the MEL of the
present invention has an ability to produce a lamella phase in a
concentration area greatly wider than that of the conventional MEL.
Therefore, the MEL of the present invention is a biosurfactant that
is extremely excellent in the ability to form liquid crystals.
[0189] Evaluation of the ability to form liquid crystals can be
made by a conventional and publicly known method. An example of the
method for easily comparing the ability to form liquid crystals is
a water invading method. In this method, MEL is applied on a slide
glass and distilled water is dropped beside the applied area, and a
liquid crystal phase formed at an interface by dropping of the
distilled water is observed by a microscope. Thus, behavior of
liquid crystal formation can be searched. With the method, it is
possible to easily compare the conventional MEL with the MEL
optical isomer of the present invention in terms of their abilities
to form liquid crystals.
[0190] <2. Method for Producing MEL>
[0191] The method for producing the MFL of the present invention is
characterized by usage of a microorganism capable of producing
1-O-MEL. Specifically, it is a method including the step of
culturing a microorganism that belongs to Pseudozyma genus and is
capable of producing mannosyl erythritol lipid, so as to produce
mannosyl erythritol lipid having the structure represented by the
general formula (1). In the descriptions explaining the method for
producing the MEL of the present invention, in the general formula
(1), the substituents R.sup.1 may be the same or different from
each other and are fatty acid acryl groups having 4-24 carbons, the
substituents R.sup.2 may be the same or different from each other
and represent hydrogen or acetyl groups, and the substituent
R.sup.3 represents hydrogen or fatty series acyl group having 2-24
atoms.
[0192] <2-1. Microorganism in Use>
[0193] Examples of the microorganism useable in the method of the
present invention for producing MEL is not particularly limited as
long as the microorganism belongs to Pseudozyma genus and produces
the MEL optical isomer represented by the general formula (1).
[0194] Examples of the microorganism that produces the MEL
represented by the general formula (1) include microorganism that
belong to Pseudozyma tsukubaensis, Pseudozyma crassa etc. In
particular, the microorganism belonging to Pseudozyma tsukubaensis
is preferable. The microorganism belonging to Pseudozyma
tsukubaensis has high productivity at 25-30.degree. C. for example.
In particular, Pseudozyma tsukubaensis JCM 10324 strain has the
highest productivity at a culture temperature of 30.degree. C.
[0195] <2-2. Culture Medium in Use and Culture Method>
[0196] The culture medium may be a culture medium generally used
for general microorganisms and yeasts, and is not particularly
limited. A culture medium used for yeasts is particularly
preferable. An example of such culture medium is a YPD culture
medium (10 g of yeast extract, 20 g of polypepton, and 100 g of
glucose). It is known that preferable culture temperature for
Pseudozyma tsukubaensis JCM 10324 strain ranges from 27-33.degree.
C. This is because Pseudozyma tsukubaensis JCM 10324 strain has
significantly high productivity of MEL at the temperature.
[0197] The composition of a culture medium suitable for producing
MEL with use of a microorganism usable for the method of producing
the MEL of the present invention, in particular Pseudozyma
tsukubaensis JCM 10324 strain, is as follows. [0198] Yeast essence:
preferably 0.1-2 g/L, and particularly preferably 1 g/L [0199]
Sodium nitrate: preferably 0.1-1 g/L, and particularly preferably
0.3 g/L [0200] Potassium dihydrogen phosphate: preferably 0.1-1
g/L, and particularly preferably 0.3 g/L [0201] Magnesium sulfate:
preferably 0.1-1 g/L, and particularly preferably 0.3 g/L [0202]
Fats and oils: preferably 40 g/L or more, and particularly
preferably 80 g/L
[0203] Further, when culturing the microorganism, it is preferable
that a carbon source is added to a culture medium. The carbon
source includes at least one of, or a mixture of, fats and oils,
fatty acid, fatty acid derivative (fatty acid esters such as fatty
acid triglyceride) and synthesized ester. Other conditions of the
carbon source are not particularly limited and may be determined
suitably in accordance with a technical standard at the time of
usage of the present invention.
[0204] The "fats and oils" may be plant oils, animal oils, mineral
oils, and hardened oils thereof. Specific examples of the fats and
oils include: animal/plant oils such as avocado oil, olive oil,
sesame oil, camellia oil, evening primrose oil, turtle oil,
macadamia nut oil, corn oil, mink oil, colza oil, yolk oil, persic
oil, peanut oil, safflower oil, wheat germ oil, sasanqua oil,
castor oil, linseed oil, safflower oil, cotton seed oil, perilla
oil, soybean oil, arachis oil, tea oil, Japanese torreya seed oil,
rice oil, tung oil, jojoba oil, cacao oil, coconut oil, horse oil,
palm oil, palm kernel oil, beef tallow, sheep tallow, lard,
lanoline, whale wax, beeswax, carnauba wax, Japan wax, candellila
wax, and squalan and hardened oils thereof; mineral oils such as
liquid paraffin and Vaseline; and synthesized triglycerin such as
glycerin tripalmitate. Preferable examples of the fats and oils
include avocado oil, olive oil, sesame oil, camellia oil, evening
primrose oil, turtle oil, macadamian nut oil, corn oil, mink oil,
colza oil, yolk oil, persic oil, wheat germ oil, sasanqua oil,
castor oil, linseed oil, safflower oil, cotton seed oil, perilla
oil, soybean oil, arachis oil, tea oil, Japanese torreya seed oil,
rice oil. Further preferable examples of the fats and oils include
olive oil and soybean oil.
[0205] "Fatty acid" or "fatty acid derivative" preferably derives
from higher fatty acid. Examples of the fatty acid and the fatty
acid derivative include capronic acid, caprylic acid, capric acid,
lauric acid, myristic acid, palmitic acid, oleic acid, linoeic
acid, linolenic acid, stearic acid, behenic acid, 12-hydroxy
stearic acid, isostearic acid, undecynoic acid, tall acid,
eicosapentaenoic acid, and docosahexaenoic acid. Preferable
examples of the fatty acid and the fatty acid derivative include
lauric acid, myristic acid, palmitic acid, oleic acid, linoeic
acid, linolenic acid, stearic acid, and undecynoic acid. Further
preferable examples of the fatty acid and the fatty acid derivative
include oleic acid, linoeic acid, and undecynoic acid.
[0206] Examples of the synthesized ester include methyl caproate,
methyl caprylate, methyl caprate, methyl laurate, methyl myristate,
methyl palmitate, methyl oleate, methyl linoleate, methyl
linolenate, methyl stearate, methyl undecynoate, ethyl caproate,
ethyl caprylate, ethyl caprate, ethyl laurate, ethyl myristate,
ethyl palmitate, ethyl oleate, ethyl linoleate, ethyl linolenate,
ethyl stearate, ethyl undecynoate, vinyl caproate, vinyl caprylate,
vinyl caprate, vinyl laurate, vinyl myristate, vinyl palmitate,
vinyl oleate, vinyl linoleate, vinyl linolenate, vinyl stearate,
vinyl undecynoate, cetyl octanoate, octyldodecyl myristate,
isopropyl myristate, myristyl myristate, isopropyl palmitate, butyl
stearate, hexyl laurate, decyl oleate, dimethyl octanoic acid,
cetyl lactate, and myristyl lactate. Preferable examples of the
synthesized ester include methyl laurate, methyl myristate, methyl
palmitate, methyl oleate, methyl linoleate, methyl linolenate,
methyl stearate, and methyl undecylenate. Further preferable
examples of the synthesized ester include methyl oleate, methyl
linoleate, and methyl undecylenate.
[0207] These may be used singularly or two or more of them may be
used suitably in combination.
[0208] Specific steps of the method for producing MEL of the
present invention are not particularly limited and may be
determined suitably according to purposes. For example, it is
preferable that the steps are scaled up in the order of seed
culture, main culture, and culture for producing MEL. The following
shows culture media and culture conditions for these cultures.
[0209] a) Seed culture; 1 platinum loop is inoculated to a test
tube containing 5 mL of a liquid culture medium including 40 g/L of
glucose, 1 g/L of yeast essence, 0.3 g/L of sodium nitrate, 0.3 g/L
of potassium dihydrogen phosphate, and 0.3 g/L of magnesium
sulfate, and the liquid culture medium is subjected to shaking
culture at 30.degree. C. for 1 day.
[0210] b) Main culture; the culture solution of a) is inoculated to
a Sakaguchi flask containing 100 mL of a liquid culture medium
including a predetermined amount of fat and oil such as plant fat
and oil, 1 g/L of yeast essence, 0.3 g/L of sodium nitrate, 0.3 g/L
of potassium dihydrogen phosphate, and 0.3 g/L of magnesium
sulfate, and the culture solution is cultured at 30.degree. C. for
2 days.
[0211] c) Culture for producing mannosyl erythritol lipid; the
culture solution is inoculated to a jar fermenter containing 1.4 L
of a liquid culture medium including a predetermined amount of fat
and oil such as plant fat and oil, 1 g/L of yeast essence, 0.3 g/L
of sodium nitrate, 0.3 g/L of potassium dihydrogen phosphate, and
0.3 g/L of magnesium sulfate, and cultured at 30.degree. C. with a
stirring speed of 800 rpm. In the culture, it is preferable that
plant fat and oil is flowed into the culture vessel in the course
of the culture so that the concentration of the fat and oil in the
culture medium is kept at 20-200 g/L.
[0212] <2-3. Method for Collecting MEL>
[0213] A method for collecting MEL may be a conventional and
publicly known method and is not particularly limited. For example,
after the culture, a lipid component is extracted with use of ethyl
acetate whose volume is not less than the volume of the lipid
component and not more than four times of the lipid component, and
then ethyl acetate is removed with use of an evaporator so as to
collect the lipid component and glycolipid component. Thereafter,
the lipid component is dissolved in chloroform whose volume is
equal to the volume of the lipid component, and is treated with
silica gel chromatography so that chloroform, chloroform:acetone
(80:20), chloroform:acetone (70:30), chloroform:acetone (60:40),
chloroform:acetone (50:50), chloroform:acetone (30:70), and acetone
are eluted in this order. Each solution is charged to a thin layer
chromatography (TLC) plate, and is developed with a ratio of
chloroform:methanol: ammonia water=65:15:2 (volume ratio). After
the development, whether glycolipid exists or not is confirmed with
use of an anthrone sulfuric acid reagent. An eluate containing
glycolipid is gathered, a solvent is removed, and thus the
glycolipid component can be obtained.
[0214] <2-4. Structural Determination of MEL>
[0215] Structural determination of the MEL obtained by the method
for producing the MEL may be performed by a conventional and
publicly known method and is not particularly limited. For example,
the following explains structural determination of MEL with
reference to a structural determination method of MEL obtained with
use of Pseudozima tsukubaensis JCM 10324 strain.
[0216] The isolated glycolipid component can be determined as
glycolipid component since the glycolipid component shows
blue-green in response to an anthrone sulfuric acid reagent on the
TLC plate. Whether the glycolipid is MEL or not can be easily
confirmed by subjecting the glycolipid to .sup.1H, .sup.13C, and
two-dimensional NMR analyses and comparing the obtained spectrum
with the spectrum of conventional MEL (MEL-A, MEL-B, MEL-C, and
MEL-D) (represented by the general formula (4)) whose structure has
been already known.
[0217] With use of 1) NMR analysis of sugar skeleton and 2)
measurement of optical rotation that are mentioned below, it is
easily confirmed that the MEL of the present invention is an
optical isomer of conventional MEL.
[0218] 1) NMR Analysis of Sugar Skeleton
[0219] In .sup.1H-NMR spectrum measured in chloroform-d, proton of
a sugar chain of MEL is detected near 3.3-5.6 ppm. In particular,
proton at a mannose 1'-position (reducing terminal) that
contributes to glycoside bind and proton at erythritol 4-position
are detected near 4.7 ppm and near 4.0 ppm, respectively. However,
it is reported by D. Crich et al. that when directions in binding
of erythritol are different, the peaks due to the above protons
shift (see Non-patent Document 8). Therefore, it is confirmed
whether the MEL of the present invention shows spectrum patterns
shifted only by the above peaks with respect to the conventional
MEL.
[0220] Further, a sugar chain (mannosyl erythritol; which may
hereinafter abbreviated as ME) obtained by saponifying the
resulting MEL with use of alkali (NaOCH.sub.3) is subjected to NMR
analysis. By comparing a sugar chain of the resulting MEL with a
sugar chain of the conventional MEL in terms of their NMR spectra,
it is possible to confirm that the structure of a sugar chain of
the MEL of the present invention shows a spectrum pattern different
from that of the conventional MEL
(4-O-.beta.-D-mannnopyranosyl-meso-erythritol structure).
[0221] 2) Measurement of Optical Rotation By measuring optical
rotation of MEL or ME, it is possible to compare chirality of
molecules of the conventional MEL with chirality of molecules of
the MEL of the present invention (see Non-patent Document 8).
1-O-(4',6'-di-O-acetyl-2',3'-di-O-dodecyl-.beta.-D-mannno
pyranosyl)-D-erythritol that is reported by D. Crich et al. and
that has the same sugar skeleton as the MEL of the present
invention has specific optical rotation
[.alpha.].sub.D=-25.9.degree. (c=1.5). Comparison of the MEL of the
present invention with the conventional MEL with reference to the
specific optical rotation shows the difference in three-dimensional
structures between the MEL of the present invention and the
conventional MEL.
[0222] The above method allows confirming that the MEL of the
present invention is different from the conventional MEL in terms
of three-dimensional structures of sugar skeletons.
[0223] As described above, with the method for producing MEL of the
present invention, it is possible to selectively produce MEL whose
chirality is different from that of the conventional MEL and which
has not been reported to be produced by a microorganism. As
explained above, difference in chirality of molecules has great
influence on physiological activity and a self-assembling body
forming function. Consequently, although the MEL of the present
invention has the same surface activity as that of the conventional
MEL, the MEL of the present invention is different from the
conventional MEL in terms of other properties. Therefore,
comparison of the MEL of the present invention with the
conventional MEL in terms of their physical properties shows
important factors for evaluation of functions of MEL. Consequently,
storage of data concerning a structure-physical property
relationship such as physiological activity greatly contributes to
development of usage of biosurfactants in various fields such as
drugs, foods, and cosmetics.
[0224] The MEL of the present invention appears to be theoretically
synthesized by a chemical synthesis method. However, the chemical
synthesis of the MEL of the present invention would require an
extremely special synthesis technique and multiple stages of
complicated protection/deprotection reactions. Further, it is
extremely difficult to completely control chirality, and therefore
it is extremely difficult to chemically synthesize the MEL of the
present invention in reality. In contrast thereto, the production
method of the present invention that uses a microorganism includes
an elaborate biosynthesis step and therefore provides a method for
producing MEL with only one step while maintaining a special
structure in which position/three-dimensional structure is
completely controlled. Therefore, the method can be very
effective.
[0225] It is additionally remarked that MEL whose mannosyl
erythritol skeleton in a molecular structure is 1-O-.beta.-D-manno
pyranosyl meso-erythritol, which is described in Embodiment 2, may
be combined with the invention described in Embodiment 1.
[0226] The embodiments and concrete examples of implementation
discussed in the foregoing detailed explanation serve solely to
illustrate the technical details of the present invention, which
should not be narrowly interpreted within the limits of such
embodiments and concrete examples, but rather may be applied in
many variations within the spirit of the present invention,
provided such variations do not exceed the scope of the patent
claims set forth below.
EXAMPLES
[0227] The following explains the present invention further
specifically with reference to Examples. Note that the following is
merely an example and the present invention is not limited to
this.
Example 1
Cell-Activating Function of MEL on Normal Human Skin
Fibroblasts
[0228] Normal human skin fibroblasts were cultured by a common
procedure with use of a normal human skin fibroblast total kit
(CA106K05, manufactured by Cell Applications. Inc. USA, imported
and sold by TOYOBO CO., LTD).
[0229] Normal human skin fibroblasts were inoculated to a
microplate having 48 holes, so that 2.0.times.10.sup.4 cells were
inoculated to each well. An inoculation medium was a Dulbecco's
Modified Eagle's Medium (DMEM) to which 10% of fetal calf serum was
added. The cells were cultured at 37.degree. C. with 5 vol % of
carbon dioxide concentration for 24 hours, and then the cells were
put in a test medium to which MEL-A with final concentration of 1
ng/ml-0.01 mg/ml was added, and the cells were further cultured for
48 hours. MEL-A used in the present Example was obtained by
culturing Pseudozyma antarctica NBRC 10736 in a medium to which
soybean oil was added (3% soybean oil, 0.02% MgSO4.H2O, 0.02%
KH2PO4, 0.1% yeast extract).
[0230] MEL-A was dissolved in ethanol and then diluted stepwise by
ethanol, and was added to each medium so that final concentration
of ethanol was 0.5% in each medium. A solvent control was an
ethanol group (final concentration was 0.5%). Further, in order to
confirm that cytotoxic substance prevents cell proliferation, an
SDS-added group (final concentration was 0.1%) was provided.
Further, the cells were put in a medium containing 100 .mu.g/mL of
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT)
and was cultured for 3 hours, and formazan produced by ring-opening
of a tetrazolium ring was extracted with use of 2-propanol, and
absorption of light of 550 nm was measured by a microplate reader.
At the same time, absorption of light of 650 nm was measured as
turbidity, and cell-activating function was evaluated based on the
difference between the two measurement values.
[0231] The result of the evaluation is shown in FIG. 1 by relative
values with the cell-activating function of an ethanol group
(solvent control group) being 100.
[0232] As is evident from FIG. 1, the MEL-A-added group showed a
higher cell-activating function than the ethanol group with respect
to normal human skin fibroblasts at each concentration. In
particular, in a case of adding MEL-A whose final concentration was
1 ng/ml, a significant cell-activating function that was higher by
65% or more than the ethanol group was observed. This result shows
that MEL has an excellent cell-activating function, which suggests
that application of MEL to skin yields an extremely excellent
anti-aging effect, effectively improving wrinkles, sags etc. of
skin due to aging, exposure to ultra violet ray etc.
Example 2
Cell-Activating Function of MEL on Human Head Hair Papilla
Cells
[0233] (1) How to Culture Human Head Hair Papilla Cells
[0234] Human hair papilla cells were cultured through a normal
procedure with use of human head hair papilla cells (THPC-001)
total kit (HDPC total kit: THPCK-001, manufactured by Cell
Applications. Inc. USA, imported and sold by TOYOBO CO., LTD).
Human head hair papilla cells are widely used for evaluating
medicinal benefits of a hair growth drug (see Japanese Unexamined
Patent Publications No. 2006-83084, No. 2003-81793, and No.
2000-159640).
[0235] Specifically, 10 mL of a PCGM medium for suspending thawed
cells were dispensed in a 15 mL centrifugal tube and cooled by ice.
A vial containing the thawed human head hair papilla cells
(THPC-001) was rapidly melted in a thermostatic chamber at
37.degree. C. The PCGM medium was gradually dropped by
approximately 1 mL into the vial and DMSO was diluted, and then the
total amount was moved to the centrifugal tube containing the PCGM
medium and were suspended. Floating cells were subjected to
centrifugal separation by a cooling slow centrifuge at 4.degree. C.
with 1000 rpm for 5 minutes. Supernatant was sucked while taking
care not to suck precipitated cells, and the supernatant was
suspended again in a 1 mL PCGM medium. The total amount was put in
a T-75 flask coated with a collagen liquid, and the T-75 flask was
put in an incubator under a humidified condition with 5 vol % of
carbon dioxide concentration at 37.degree. C., and the total amount
was subjected to a static culture. One day later, the medium was
replaced. Thereafter, the medium was replaced every two days and a
subculture was carried out.
[0236] The PCGM medium was obtained by adding 2.5 mL of 100-fold
dilution of bovine pituitary extract (BPE), 2.5 mL of 100-fold
dilution of fetal calf serum (FCS), 1.25 mL of 200-fold dilution of
insulin transferrin triiodothyronine solution (ITT), and 1.25 mL of
200-fold dilution of thyroprotein solution (Cyp) to 250 mL of a
PCGM basal medium containing 1% of FBS.
[0237] (2) Evaluation of Human Head Hair Papilla Cell-Activating
Function
[0238] Human head hair papilla cells were inoculated to a
microplate having 48 holes, so that 2.0.times.10.sup.4 cells were
inoculated to each well. An inoculation medium was a Dulbecco's
Modified Eagle's Medium (DMEM) to which 10% of fetal calf serum was
added. The cells were cultured for 24 hours, and then put in a test
medium to which MEL-A with final concentration of 1 ng/ml-0.01
mg/ml was added, and the cells were further cultured for 48 hours.
MEL-A used in the present Example was obtained by culturing
Pseudozyma antarctica NBRC 10736 in a medium to which soybean oil
was added (3% soybean oil, 0.02% MgSO4.H2O, 0.02% KH2PO.sub.4, 0.1%
yeast extract).
[0239] MEL-A was dissolved in ethanol and then diluted stepwise by
ethanol, and was added to each medium so that final concentration
of ethanol was 0.5% in each medium. A solvent control was an
ethanol group (final concentration was 0.5%). Further, the cells
were put in a medium containing 100 .mu.g/mL of
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT)
and was cultured for 3 hours, and formazan produced by ring-opening
of a tetrazolium ring was extracted with use of 2-propanol, and
absorption of light of 550 nm was measured by a microplate reader.
At the same time, absorption of light of 650 nm was measured as
turbidity, and cell-activating function was evaluated based on the
difference between the two measurement values.
[0240] The result of the evaluation is shown in FIG. 2 by relative
values with the cell-activating function of an ethanol group
(solvent control group) being 100.
[0241] As is evident from FIG. 2, the MEL-A-added group showed a
higher cell-activating function than the ethanol group with respect
to human head hair papilla cells in a range of 1 ng/ml-1 .mu.g/ml.
In particular, in a case of adding MEL-A whose final concentration
was 1 ng/ml, a significant cell-activating function that was higher
by 50% or more than the ethanol group was observed. This result
suggests that application of MEL to head skin yields an extremely
excellent effect of activating hair papilla cells, which yields a
hair-growing effect.
Example 3
Cell-Activating Function of Triacyl MEL on Normal Human Skin
Fibroblasts
[0242] Triacyl MEL was OL-MEL (SB) that was obtained by adding
oleic acid to a hydroxyl group of an erythritol portion of MEL-A
cultured in a soybean oil-added medium (3% soybean oil, 0.02%
MgSO.sub.4.H2O, 0.02% KH2PO.sub.4, 0.1% yeast extract).
[0243] Normal human skin fibroblasts were cultured by a common
procedure with use of a normal human skin fibroblast total kit
(CA106K05, manufactured by Cell Applications. Inc. USA, imported
and sold by TOYOBO CO., LTD).
[0244] Normal human skin fibroblasts were inoculated to a
microplate having 48 holes, so that 2.0.times.10.sup.4 cells were
inoculated to each well. An inoculation medium was a Dulbecco's
Modified Eagle's Medium (DMEM) to which 10% of fetal calf serum was
added. The cells were cultured at 37.degree. C. with 5 vol % of
carbon dioxide concentration for 24 hours, and then the cells were
put in a test medium to which the triacyl MEL (OL-MEL (SB)) with
final concentration of 0.01 ng/ml-0.01 mg/ml was added, and the
cells were further cultured for 48 hours. The triacyl MEL was
dissolved in ethanol and then diluted stepwise by ethanol, and was
added to each medium so that final concentration of ethanol was
0.5% in each medium. A solvent control was an ethanol group (final
concentration was 0.5%). Further, in order to confirm that
cytotoxic substance prevents cell proliferation, an SDS-added group
(final concentration was 0.1%) was provided. Further, the cells
were put in a medium containing 100 .mu.g/mL of
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT)
and was cultured for 3 hours, and formazan produced by ring-opening
of a tetrazolium ring was extracted with use of 2-propanol, and
absorption of light of 550 nm was measured by a microplate reader.
At the same time, absorption of light of 650 nm was measured as
turbidity, and cell-activating function was evaluated based on the
difference between the two measurement values.
[0245] The result of the evaluation is shown in FIG. 3 by relative
values with the cell-activating function of an ethanol group
(solvent control group) being 100.
[0246] As is evident from FIG. 3, the triacyl MEL-added group
showed a higher cell-activating function than the ethanol group
with respect to normal human skin fibroblast cells at each
concentration. In particular, in a case of adding the triacyl MEL
whose final concentration was 1 ng/ml, a significant
cell-activating and anti-aging function that was higher by
approximately 50% than the ethanol group was observed. This result
shows that the triacyl MEL has an excellent cell-activating
function as with MEL-A, which suggests that application of the
triacyl MEL to skin yields an extremely excellent anti-aging
effect, effectively improving wrinkles, sags etc. of skin due to
aging, exposure to ultra violet ray etc.
[0247] Examples 4-6 as described below show examples of
prescriptions of various dosage forms of a cell-activator of the
present invention.
Example 4
Essence
[0248] Essence having the following composition was produced by a
common procedure.
TABLE-US-00001 (Composition) (Weight %) Sorbit 4.0 Dipropylene
glycol 6.0 Polyethylene glycol 1500 5.0 POE (20) oleyl alcohol
ether 0.5 Simple sugar fatty acid ester 0.2 Methyl cellulose 0.2
MEL 1.0 Purified water amount that makes the whole amount of
essence 100
Example 5
Emulsion
[0249] An emulsion having the following composition was produced by
a common procedure.
TABLE-US-00002 (Composition) (Weight %) Glyceryl ether 1.5 Simple
sugar fatty acid ester 1.5 Sorbitan monostearate 1.0 Squalan 7.5
Dipropylene glycol 5.0 MEL 1.0 Purified water amount that makes the
whole amount of emulsion 100
Example 6
Cream
[0250] Cream having the following composition was produced through
a common procedure.
TABLE-US-00003 (Composition) (Weight %) Propylene glycol 6.0
Dibutyl phthalate 19.0 Stearic acid 5.0 Glycerin monostearate 5.0
Sorbitan monostearate 12.0 Polyethylene sorbitan monostearate 38.0
Edetate sodium 0.03 MEL 1.0 Purified water amount that makes the
whole amount of cream 100
Example 7
Sensory Evaluation
[0251] Examples 4-6 were subjected to sensory evaluation.
Comparative examples that did not include biosurfactants were also
subjected to the same sensory evaluation. In the sensory
evaluation, a group consisting of six evaluators of 26-48 years
old, conscious about aging symptoms such as wrinkles, used the
Examples and the Comparative Examples twice a day continuously for
3 months, and the evaluators were questionnaired as to the
conditions of their skins after 3 months.
[0252] The result of the sensory evaluation is shown in Table 1 in
which the number of evaluators in individual items is shown. 70% or
more evaluators answered that the Examples made their skins more
resilient and more improved their wrinkles than the Comparative
examples that did not include biosurfactants did. This shows that
the Examples have a significant effect of improving aging symptoms
on skins.
TABLE-US-00004 TABLE 1 Examples Comparative Examples (with
biosurfactant) (without biosurfactant) 4 5 6 4 5 6 Resiliency
Improved 2 4 3 0 0 0 of skin Rather 2 0 1 0 0 0 improved No change
2 2 2 6 5 4 Worsened 0 0 0 0 1 2 Improvement Improved 3 2 2 0 0 0
of wrinkles Rather 1 1 2 1 0 1 improved No change 2 3 2 5 6 4
worsened 0 0 0 0 0 1
Example 8
Culture of Pseudozyma Tsukubaensis JCM 10324 Strain
[0253] a) Pseudozyma tsukubaensis JCM 10324 strain preserved in a
preservation medium (3 g/L of malt essence, 3 g/L of yeast essence,
5 g/L of peptone, 10 g/L of glucose, and 30 g/L of agar) was
inoculated by one platinum loop into a test tube containing 2 mL of
a liquid medium including 20 g/L of glucose, 1 g/L of yeast
essence, 0.3 g/L of sodium nitrate, 0.3 g/L of potassium dihydrogen
phosphate, and 0.3 g/L of magnesium sulfate, and the Pseudozyma
tsukubaensis JCM 10324 strain was subjected to shaking culture at
30.degree. C. Then, b) 1 mL of a resulting bacterial culture
solution was inoculated into a Sakaguchi flask containing 20 mL of
a liquid culture including a predetermined amount of soybean oil, 1
g/L of yeast essence, 0.3 g/L of sodium nitrate, 0.3 g/L of
potassium dihydrogen phosphate, and 0.3 g/L of magnesium sulfate,
and was subjected to shaking culture at 30.degree. C.
[0254] The bacterial culture solution obtained in the cultures a)
and b) was subjected to the following test.
Example 9
Confirmation of Ability of Pseudozyma Tsukubaensis JCM 10324 Strain
to Produce Glycolipid
[0255] The culture a) was carried out for 1 day and then the
culture b) was carried out for 7 days, and a culture solution was
sampled. Using the culture solution, production of a biosurfactant
by Pseudozyma tsukubaensis JCM 10324 strain was confirmed by thin
layer chromatography. A developing solvent included chloroform,
methanol, 7N ammonia water in the ratio of 65:15:2, respectively.
An indicator was an anthrone sulfuric acid reagent that colors
glycolipid in blue-green. Standard MEL was a purified authentic
sample obtained by culturing Pseudozyma antarctica KM-34
(FERMP-20730) strain in a soybean oil-added medium and removing
impurities such as raw material fats and oils etc. MEL-A, MEL-B,
MEL-C, and MEL-D indicate compounds represented by the general
formula (5) in the standard MEL.
[0256] The result is shown in FIG. 4. FIG. 4 shows that Pseudozyma
tsukubaensis JCM 10324 strain produced glycolipid that seems to be
MEL-B.
Example 10
Production of MEL in a Medium for Producing MEL
[0257] Using Pseudozyma tsukubaensis JCM 10324 strain, the culture
a) was carried out for 1 day, and then the culture b) was carried
out for 7 days. Thereafter, the culture solution was sampled, a
component that was soluble in ethyl acetate was purified from the
culture solution, and then the produced MEL was detected by high
performance liquid chromatography. In Comparative Example, a
culture solution obtained by culturing Pseudozyma antarctica KM-34
(FERMP-20730) strain with use of soybean oil as a carbon source was
detected by high performance liquid chromatography.
[0258] The results of the detections are shown in FIG. 5. FIG. 5
shows that by culturing Pseudozyma tsukubaensis JCM 10324 strain,
it is possible to obtain glycolipid whose peak was seen at the same
retention time as MEL-B.
Example 11
Culture of Pseudozyma Crassa CBS 9959 Strain and Confirmation of
Ability to Produce MEL
[0259] Pseudozyma crassa CBS 9959 strain was subjected to the
method in Example 8 except that the temperature was 25.degree. C.
The culture a) was carried for 1 day and then the culture b) was
carried for 7 days, and a culture solution was sampled. Using the
culture solution, production of a biosurfactant by Pseudozyma
crassa CBS 9959 strain was confirmed by thin layer chromatography,
as in the method in Example 9.
[0260] The result of the confirmation is shown in FIG. 6. FIG. 6
shows that Pseudozyma crassa CBS 9959 strain produced glycolipid
whose Rf value was a little lower than already known MEL (MEL-A,
MEL-B, and MEL-C).
Example 12
Structure Elucidation of MEL Produced by Pseudozyma Tsukubaensis
JCM 10324 Strain
[0261] Initially, NMR analysis of a sugar skeleton was carried out.
Glycolipid obtained in Example 9 was isolated and purified by a
known separation method using silica gel column chromatography, and
.sup.1H-NMR analysis was carried out using deuterated chloroform
(CDCl.sub.3). As a comparative object, conventional MEL-B that was
produced by culturing Pseudozyma antarctica KM-34 (FERMP-20730)
strain and then isolated and purified was measured in the same
manner.
[0262] The results are shown in FIG. 7. FIG. 7 shows that
glycolipid produced by Pseudozyma tsukubaensis JCM 10324 strain was
MEL-B. Further, it was confirmed that 1'-position of mannose (H-1'
in the drawing) was shifted toward a lower magnetic field, i.e.
from 4.73 ppm to 4.76 ppm, and proton at 4-position of erythritol
that were largely separated into two parts (3.8 ppm, 4.0 ppm) were
greatly shifted to be one (3.9 ppm). This result is completely in
accordance with the descriptions of Non-patent Document 8 reported
by D. Crich et al., which demonstrates that erythritol derived from
MEL produced by Pseudozyma tsukubaensis JCM 10324 strain was bonded
in a manner reverse to the manner of conventional MEL.
[0263] Further, in order to make more detailed comparison of the
structure of sugar skeleton, ester bond in the MEL was subjected to
alkali hydrolysis with use of sodium methoxide in methanol. A
product obtained after the reaction was precipitated again in ethyl
acetate to collect, and was subjected to recrystallizing operation
in a 90% ethanol aqueous solution. Thus, crystals of sugar chains
(manno erythritol; ME) was obtained.
[0264] Sugar chains were collected from conventional MEL in the
similar manner, and the obtained ME was subjected to .sup.1H,
.sup.13C--, and various two-dimensional NMR analyses using heavy
water (D.sub.2O) as a solvent.
[0265] Consequently, as illustrated in FIG. 8, only proton at
4-position of erythritol shifted its peak (H-4a: 3.85
ppm.fwdarw.*3.89 ppm, H-4b: 4.12 ppm.fwdarw.>4.0 ppm). M. Kurz
et al. carried out detailed structure elucidation of
4-O-.beta.-mannnopyranosyl-D-erythritol (described as
1-O-.beta.mannnopyranosyl-L-erythritol in the Document) that was
prepared from usuchi lipid having the same structure as that of
conventional MEL (J. Antibiot., 56, 91-101 (2003)), and described
that chemical shift of proton at 4-position of conventional ME was
such that H-4a was 3.76 ppm and H-4b was 4.09 ppm. The known
document describes that in the conventional ME (and MEL), proton at
4-position of erythritol was largely separated into two parts. This
shows that MEL of the present invention is new MEL that is an
optical isomer of conventional MEL and that includes as a sugar
skeleton structure 1-O-.beta.-mannnopyranosyl-D-erythritol where
erythritol was bonded in a reverse manner.
[0266] Subsequently, optical rotation of the ME was measured. ME
was synthesized through alkali hydrolysis in the above manner from
MEL obtained by culturing Pseudozyma antarctica KM-34 (FERMP-20730)
strain and Pseudozyma tsukubaensis JCM 10324 strain, and dissolved
in distilled water to prepare a 1% aqueous solution. Optical
rotation of each aqueous solution was measured with use of a
polarimeter (digital polarimeter DIP 370 type manufactured by JASCO
Corporation) so as to obtain specific optical rotation of each
ME.
[0267] Consequently, specific optical rotation of ME derived from
Pseudozyma antarctica KM-34 (FERMP-20730) was
[.alpha.].sub.D=-35.2.degree. and specific optical rotation of ME
derived from Pseudozyma tsukubaensis JCM 10324 strain was
[.alpha.].sub.D=-39.6.degree.. This shows that chiralities of sugar
skeletons (ME) of MEL produced from respective strains are
different, which demonstrates that MEL produced by Pseudozyma
tsukubaensis JCM 10324 strain is an optical isomer whose
3-dimensional structure of sugar skeleton is different from that of
conventional MEL.
[0268] Further, ME derived from Pseudozyma antarctica KM-34
(FERMP-20730) was obtained as white powder through the above
collecting operation and had a melting point of 156.9.degree. C.,
whereas ME derived from Pseudozyma tsukubaensis JCM 10324 strain
was obtained as a transparent, colorless, and oily compound, and a
melting point of the ME could not be measured. This shows that the
two ME have different molecular 3-dimensional structures and have
different crystallinity.
[0269] It was confirmed from the above result that MEL produced by
Pseudozyma tsukubaensis JCM 10324 strain obtained in Example 9 is
MEL-B and is
1-(6'-acetyl-2',3'-di-O-alka(ke)noyl-.beta.-D-mannnopyranosyl-)
meso-erythritol that is an optical isomer of conventional
MEL-B.
Example 13
Structure Elucidation of MEL Produced by Pseudozyma Crassa CBS 9959
Strain
[0270] Glycolipids produced by Pseudozyma crassa CBS 9959 strain
obtained in Example 11 were isolated and purified as in Example 12.
Three kinds of glycolipids were subjected to .sup.1H-NMR analysis
and were compared with conventional MEL-A, MEL-B, and MEL-C.
[0271] Consequently, as illustrated in FIG. 9, it was confirmed
that the three kinds of glycolipids produced by Pseudozyma crassa
CBS 9959 strain correspond to MEL-A, MEL-B, and MEL-C,
respectively, and proton at 4-position of erythritol would show two
peaks in the conventional MEL, whereas proton at 4-position of
erythritol shows one peak in MEL of the present invention.
Example 14
Comparison of Ability to Form Liquid Crystal
[0272] MEL produced by Pseudozyma tsukubaensis JCM 10324 strain and
conventional MEL produced by Pseudozyma antarctica KM-34
(FERMP-20730) strain were compared with each other by a
water-invading method in terms of their abilities to form liquid
crystal. The result of the comparison shows that MEL derived from
Pseudozyma tsukubaensis JCM 10324 strain has an ability to form
lamella phase in a very wider concentration range than conventional
MEL, and is a biosurfactant that is excellent in the ability to
form liquid crystal, as illustrated in FIGS. 10 and 11.
Example 15
Production of Triacyl MEL by Culturing Pseudozyma Tsukubaensis JCM
10324 Strain
[0273] Frozen stock of 0.2 mL of P. tsukubaensis was planted in a
500 ml Sakaguchi flask containing 20 ml of a YM seed medium and
cultured at 26.degree. C. at 180 rpm for 1 night to be preinoculum.
0.2 ml of the preinoculum was planted in a 500 ml Sakaguchi flask
containing 20 ml of a YM seed medium and cultured at 26.degree. C.
at 180 rpm for 1 night to be inoculum. 20 ml of the inoculum was
planted in 5 L jar containing 2 L of a YM medium and cultured at
26.degree. C. at 300 rpm (1/4VVM, 0.5 L air/min) for 8 days. The
culture solution was centrifuged at 7,900 rpm for 60 min at
4.degree. C., so that the culture solution was separated into
strain (including MEL-B) and supernatant. 80 ml of ethyl acetate
was added to strain fractions, and stirred upward and downward so
that the strain was suspended sufficiently, and then centrifuged at
7,900 rpm for 60 min at 4.degree. C. To the obtained supernatant
was added the same amount of a saturated saline solution, and the
resultant was stirred to obtain an etyl acetate layer. A suitable
amount of sulfuric anhydride Na was added to the etyl acetate
layer, and left at rest for 30 minutes and then evaporated to
obtain glycolipid.
Example 16
NMR Analysis of Triacyl MEL Produced by Pseudozyma Tsukubaensis JCM
10324
[0274] Glycolipid obtained in Example 15 was isolated and purified
through a known separation method with use of silica gel column
chromatography to obtain 50 g of MEL-B and 1.5 g of triacyl MEL-B.
Triacyl MEL-B fractions were subjected to .sup.1H-NMR analysis with
use of deuterated dimethylsulfoxide (DMSO-d.sub.6) as a solvent and
analyzed in the same manner as that of Example 13. The result is
shown in FIG. 12. As shown in FIG. 12, it was confirmed that the
triacyl MEL-B produced by P. tsukubaensis JCM 10324 strain had
erythritol that was bonded in a manner reverse to the manner of
conventional MEL.
[0275] For comparison, Pseudozyma hubeiensis was cultured and
produced, and 45 g of MEL-C and 1.3 g of triacyl MEL-C were
isolated and purified with use of silica gel column chromatography
in the same manner as above. The triacyl MEL-C was subjected to
.sup.1H-NMR analysis with use of deuterated dimethylsulfoxide
(DMSO-d.sub.6) as a solvent. Consequently, as shown in FIG. 13, it
was confirmed that MEL-C produced by Pseudozyma hubeiensis had
erythritol bonded in the same direction as conventional MEL.
Example 18
Lipid Domain Analysis of MEL-B Produced by Pseudozyma Tsukubaensis
JCM 10324 Strain
[0276] MEL-B produced by Pseudozyma tsukubaensis JCM 10324 strain
was separated by high performance liquid chromatography using
reverse phase column, and then subjected to mass spectrometry
(LC-MS analysis), and a fatty acid structure of lipid domain was
confirmed. Consequently, as shown in FIG. 14, it was confirmed that
fatty acid having 6 carbon atoms was mainly attached to one
hydroxide group of mannose and fatty acid having 10-14 carbon atoms
was attached to the other hydroxide group.
[0277] Analysis conditions of HPLC are as follows. HPLC device:
Agilent 100, column: Imtakt Cadenza CD-C18 2.times.150 mm,
mobile-phase: A 0.1% formic acid, B acetonitrile, 0 min (50% B)-20
min (98% B)-30 min (98% B), flow rate: 0.2 ml/min, column
temperature: 40.degree. C., injection rate: 3 .mu.l. MS conditions
are as follows. MS device: BRUKER DALTONICS esquire 3000 Plus,
ionization method: ESI positive.
[0278] The embodiments and concrete examples of implementation
discussed in the foregoing detailed explanation serve solely to
illustrate the technical details of the present invention, which
should not be narrowly interpreted within the limits of such
embodiments and concrete examples, but rather may be applied in
many variations within the spirit of the present invention,
provided such variations do not exceed the scope of the patent
claims set forth below.
INDUSTRIAL APPLICABILITY
[0279] The present invention provides cosmetics, quasi-drugs
(external agent for skin, bath agent, hair growth agent etc.),
drinks and foods, and drugs for which highly safe cell-activating
function and anti-aging function derived from a biosurfactant can
be expected, and which include a cell-activating component and an
anti-aging component as active ingredients. Therefore, the present
invention is expected to greatly contribute to industries.
[0280] Further, the MEL of the present invention has a structure in
which erythritol is ether-bonded to mannose in the reverse manner
as that of conventional MEL, which makes the MEL of the present
invention have an entirely different chilarity, greatly different
liquid crystal forming behavior, and a greatly different
self-assembling property, from those of the conventional MEL.
Because of these differences in the properties, the MEL of the
present invention is expected to show new physiological activities
that are not seen in the conventional MEL. Therefore, the MEL of
the present invention is expected to be widely used in the field of
cleaning agents, food industries, chemical industries,
environmental fields etc. as the conventional MEL, and in
particular greatly contribute to wider application of MEL in the
fields such as medicine and cosmetic industries.
* * * * *