DESCRIPTION
TITLE OF THE INVENTION: SKIN WHITENING COMPOSITION
TECHNICAL FIELD
[0001]
The present invention relates to a skin whitening composition.
More specifically, the present invention relates to a skin whitening
composition which contains a proaporphine or aporphine alkaloid as an active
ingredient.
BACKGROUND ART
[0002]
Nelumbo nucifera is a large-size perennial shallow-water plant
classified in the genus Nelumbo in the family Nelumbonaceae of dicotyledon.
It is native to regions in India and China, and is distributed in
south-eastern Europe, Iran, India, China, Japan, Australia and North America .
It grows in ponds, rice paddies, moats, and shallow swamps and marshy areas.
[0003]
In Japan, Nelumbo nucifera is cultivated in rice paddies, moats and
marshy areas as ritual flowers in the Buddhist scriptures and for decorative
purposes, or as rhizomes (lotus root) for edible use.
Nearly all the portions of Nelumbo nucifera are used for medicinal
purposes. Rhizomes, leaves, leafstalks, stamens, fruits, seeds, and fruit
receptacles of Nelumbo nucifera are used as crude drugs.
[0004]
The rhizomes, leaves and stamens of Nelumbo nucifera having a
hemostatic action are used for the treatment of gastric ulcer and
metrorrhagia in combination with other crude drugs. The seeds, a
revitalizer, are used for chronic diarrhea and heart disease in combination
with other crude drugs (Non-patent Document 1).
[0005]
Moreover, the leaves having antipyretic and diuretic actions are
used' to treat hemorrhoid and diarrhea, and the stamens are used as a
revitalizer and a hemostatic agent. Furthermore, the seeds are used for
revitalizing, diuresis, sedation, and as an emmenagogue.
[0006]
Furthermore, the leaves, rhizomes, seeds and flower parts of Nelumbo
nucifera are used for the treatment of pharyngeal disease, chest pain,
spermatorrhea, leucoderma, smallpox, diarrhea, cough, epistaxis,
hemoptysis, metrorrhagia, hyperlipidemia, pyrexia, cholera, hepatic
dysfunction and severe dry mouth as traditional medicine . In Ayurveda, they
are used for diuresis, anthelmintic, and the treatment of painful urination
disorders, emesis, Hansen's disease, skin disease, and neurasthenia. They
are used as general medicine to treat diseases such as skin diseases and
cancer (Non-patent Document 2).
[0007]
It is known that leaves, fruits and seeds of Nelumbo nucifera contain
alkaloids (Non-patent Document 1) and its rhizomes contain starch, catechin
and the like.
[0008]
Patent Document 1 discloses a reactive oxygen species scavenger
containing at least one plant extract selected from several plant extracts
including Nelumbo nucifera and a skin cosmetic composition wherein the
reactive oxygen species scavenger is formulated, describing that the
intended composition in,the patent significantly scavenges and suppresses
the reactive oxygen species as well as improves chalk mark and adds glow
and firmness to the skin. However, it does not disclose that the reactive
oxygen species scavenger and the composition thereof have a skin whitening
effect.
PRIOR ART DOCUMENTS
PATENT DOCUMENT
[0009]
Patent Document 1: Japanese Unexamined Patent Application
Publication No. 11-279069
NON-PATENT DOCUMENTS
[0010]
Non-patent Document 1: Useful Plants of the World, p. 637, Heibonsha
Limited, Publishers
Non-patent Document 2 : Pulok K. Mukher jee et al., Journal of Pharmacy
and Pharmacology, 2009, 61, 407-422.
SUMMARY OF THE INVENTION
PROBLEM TO BE SOLVED BY THE INVENTION
[0011]
The present invention aims to develop a novel method of use of an
extract of flower parts of Nelumbo nucifera.
MEANS FOR SOLVING THE PROBLEM
[0012]
As the result of intensive effort and investigation, the present
inventor has found that an extract from flower parts of Nelumbo nucifera
extracted with a lower alcohol, particularly an alkaloid-containing
fraction obtained from the extract and alkaloids in the alkaloid-containing
fraction have surprisingly an inhibitory effect on melanin production, and
attained the present invention.
[0013]
Therefore, according to the present invention, there is provided
a skin whitening composition containing an active ingredient represented
by general formula (I):
wherein Rlf R2 and R3 each represent a hydrogen atom or a methyl group;
R4, R5 and R6 each represent a hydrogen atom or a methyl group, or
alternatively R3 and R4 as well as R5 and R6 combine together and respectively
represent a bonding hand;
[0014]
two R7s each represent a hydrogen atom, or alternatively the two R7s
combine together and represent an oxo group;
n represents an integer of 0 or 1;
when n is 0, two R8s combine together with carbon atoms to which the
two R8s are bonded and form a 4-oxo-cyclohexadiene ring; and
when n is 1, two R6s and R9 combine together with carbon atoms to
which the two R8s and R9 are bonded and form a benzene ring, wherein
the active ingredient is a proaporphine or aporphine alkaloid.
[0015]
According to the present invention, there is also provided the
composition wherein the alkaloid is at least one proaporphine or aporphine
alkaloid selected from the compounds represented by the following chemical
formulae:
[0016]
According to the present invention, there is also provided the
composition wherein the alkaloid is contained in an extract prepared from
flower parts of Nelumbo nucifera belonging to the genus Nelumbo in the family
Nelumbonaceae.
[0017]
Furthermore, according to the present invention, there is provided
the composition wherein the alkaloid is contained in an organic
solvent-soluble fraction obtained by extracting flower parts of Nelumbo
nucifera belonging to the genus Nelumbo in the family Nelumbonaceae with
a lower alcohol to give an extraction liquid, and further partitioning the
extraction liquid with ethyl acetate/water and n-butanol/water when needed;
or an alkaloid-containing fraction obtained by partitioning the extraction
liquid with diethyl ether/weak-acidic water to give an aqueous layer,
extracting the aqueous layer with diethyl ether after being weakly basified
by an ammonia solution, and washing the ether layer with diluted aqueous
sodium hydroxide solution.
ADVANTAGEOUS EFFECT OF INVENTION
[0018]
A skin whitening composition according to the present invention
contains an extract prepared by the extraction of flower parts of Nelumbo
nucifera with a lower alcohol, or a concentrate thereof or a purified
compound thereof, particularly an alkaloid-containing fraction as an active
ingredient, and is used safely mainly for the purpose of preventing sunburn.
MODES FOR CARRYING OUT OF THE INVENTION
[0019]
Productive regions of the flower parts of Nelumbo nucifera used in
the present invention are not particularly limited.
The "flower part" used in the present invention is intended to mean
every bud before blossoming, petal in full bloom, and the whole flower.
[0020]
Flower parts, however, are used right after flower-picking as they
are, or used after desiccation.
Flower parts are used that are picked in the summer months (blooming
season) , namely at any time between June and September, although it is not
necessarily limited.
[0021]
The term "lower alcohol" as used in the present invention for
extraction of flower parts of Nelumbo nucifera is intended to mean alcohols
having 1 to 4 carbon atom(s), particularly methanol, ethanol, n-propanol,
isopropanol, n-butanol, isobutanol and t-butanol.
[0022]
The lower alcohols may be used alone, or in the mixture, or as an
aqueous alcohol that contains water up to about 50% by volume.
Among the lower alcohols, methanol or ethanol is preferred from the
viewpoint of extraction efficiency and ease of evaporation of a solvent
after the extraction.
[0023]
These extraction solvents are used by a factor of about 1 to 50 by
volume, preferably by a factor of about 2 to 10 by volume relative to the
material for extraction.
[0024]
The extraction temperature can be arbitrarily set between room
temperature and the boiling point of a solvent, and preferably a material
for extraction is soaked in the extraction solvent and extracted, e.g.,
at a temperature between 50°C and the boiling point of the extraction solvent
under the condition of shaking, non-shaking, or refluxing.
In the case where the material for extraction is soaked under the
condition of shaking, a period of 30 minutes to 10 hours is suitable, in
the case where the material for extraction is soaked under the condition
of non-shaking, a period of 1 hour to 20 days is suitable.
[0025]
Furthermore, in the case where the material for extraction is
extracted under the condition of refluxing, the materials are preferably
refluxed under heating for the period of 30 minutes to several hours.
Soaking and extraction of materials at a temperature under 50°C or
room temperature may be feasible, however, in such a case, a longer soaking
time than mentioned above is preferred.
[0026]
The procedure of extraction may be carried out only once for the
same material, but it is preferably repeated more than once, e.g., about
2 to 5 times, from the viewpoint of extraction efficiency.
An extraction liquid obtained by eliminating solid matters from the
extract mixture is concentrated under reduced pressure by a common method
to give an extract essence and an extract, and the solvent recovered during
the concentration under reduced pressure can be reused for further
extraction.
[0027]
The concentration may be done under reduced pressure until an extract
essence is dried.
Therefore, an extract in the present invention is intended to mean
both an extract essence from which the solvent has been eliminated thoroughly
and an extract obtained by evaporating a solvent from the extract essence
under reduced pressure until the extract essence is dried, and the extract
and the extract essence are synonymous.
[0028]
The extract essence can be used as it is to prepare the composition
of the present invention, however, it may be used after solidification such
as in a powdered form or a lyophilized product. Methods known to those of
skill in the related art can be adopted for solidification.
[0029]
Furthermore, the extraction liquid may be subjected to a purification
treatment prior to or after concentration.
Adsorption chromatography, ion-exchange chromatography, and
partitioning extraction by solvents can be adopted alone or in combination
thereof as a step of purification.
[0030]
For example, either one or combination of chromatography techniques
such as column chromatography using a normal-phase or reversed-phase carrier,
or ion exchange resins, thin-layer chromatography, high-performance liquid
chromatography, and centrifugal liquid chromatography can be applied to
fractionate an alkaloid-containing fraction.
In this case, the purification conditions such as a carrier and an
eluting solvent can be suitably selected in accordance with various
chromatography techniques.
[0031]
Among them, it is also possible to concentrate the extraction liquid
under reduced pressure to obtain an extract essence and further partition
the extract essence with water and a non-hydrophilic organic solvent to
give an organic solvent-soluble fraction.
[0032]
Examples of the non-hydrophilic organic solvent include ethyl
acetate, n-butanol, diethyl ether, hexane, and chloroform. Above all,
particularly partitioning with n-butanol/water is preferred against water
after the partitioning with ethyl acetate-water to fractionate a methanol
extract of the flower parts of Nelumbo nucifera.
[0033]
More specifically, the resulting methanol extract essence or the
extract obtained by concentration under reduced pressure can be dissolved
in water, partitioned with ethyl acetate, and separated as an ethyl
acetate-soluble fraction. The residual aqueous layer can be partitioned
with n-butanol to isolate an n-butanol-soluble fraction.
[0034]
The combination of diethyl ether and a weak acid aqueous solution,
e.g., diethyl ether-1% aqueous acetic acid solution is preferably used to
fractionate a methanol extract of flower parts of Nelumbo nucifera.
More specifically, the obtained methanol extract essence or the
extract obtained by concentration under reduced pressure is dissolved in
a 1% aqueous acetic acid solution and is partitioned with diethyl ether.
A separated aqueous layer is again partitioned with diethyl ether after
being acidified by adding an ammonia solution to obtain a diethyl
ether-soluble fraction. A diluted aqueous sodium hydroxide solution is
added to the fraction and is partitioned to obtain a diethyl ether-soluble
fraction as an alkaloid-containing fraction.
[0035]
Furthermore, the separated ethyl acetate-soluble fraction, the
n-butanol-soluble fraction, and the diethyl ether-soluble fraction (the
alkaloid-containing fraction) were further repeatedly separated and
purified by normal-phase silica gel or reversed-phase ODS column
chromatography and HPLC, and five known aporphine alkaloids and one
proaporphine alkaloid were isolated.
[0036]
Partitioning extraction can be performed according to conventional
methods commonly applied in the relevant art such as agitation, a shake-flask
method, and droplet countercurrent distribution. For example, under the
condition of room temperature and shaking or non-shaking, the partitioning
extraction by adding ethyl acetate, n-butanol or diethyl ether by a factor
of about 1/5 to 5 by volume (1 : 0.2 to 1 : 5) relative to the aqueous solution
of the extract essence may be suitable.
[0037]
Moreover, the lower alcohol extract or the ethyl acetate-soluble
fraction, or a vacuum-concentrate of n-butanol-soluble fraction or diethyl
ether-soluble fraction can be further purified by the purification treatment.
More specifically, normal-phase chromatography or normal-phase
high-performance liquid chromatography, or reversed-phase chromatography
or reversed-phase high-performance liquid chromatography can be used alone
or in combination for the purification.
Preferably, the presence or absence of an alkaloid is checked by
the detection of an alkaloid with Dragendorff reagent in each step of the
purification and in each fraction.
[0038]
The inventor of the present invention obtained a MeOH-extract essence
of dried flower parts of Nelumbo nucifera originating in Thailand after
the extraction with MeOH under heating at 80°C followed by the evaporation
of the solvent under reduced pressure.
The resulting MeOH extract essence was partition-extracted with
AcOEt and H20 followed by partitioning extraction of the H20 layer with n-BuOH
to obtain an AcOEt layer, an n-BuOH layer, and an H20 layer.
[0039]
The resulting AcOEt layer and the n-BuOH layer were repeatedly
separated and purified by normal-phase silica gel column chromatography,
reversed-phase ODS column chromatography, and HPLC to obtain four known
aporphine alkaloids i.e., nuciferine, (O)-nornuciferine,
N-methylasimilobine, and lysicamine, and one known proaporphine alkaloid,
i.e., pronuciferine.
[0040]
Furthermore, two known flavonoid glycosides i.e.,
kaempferol 3-O-p-D-glucopyranoside and isorhamnetin 3-O-p-Dglucopyranoside,
one kind of sterol i.e., p-sitosterol, and one kind of
fatty acid i.e., palmitic acid were isolated.
[0041]
On the other hand, dried flower parts of Nelumbo nucifera originating
in Thailand were extracted with MeOH at room temperature for the purpose
of isolation of alkaloid components. Afterward, the solvent was removed
under reduced pressure to obtain a MeOH extract essence. The resulting MeOH
extract essence was partition-extracted with Et20 and 1% AcOH, and then the
1% AcOH layer was extracted with Et20 after being basified by an ammonia
solution.
Next, the Et20 layer was further washed with a 10% aqueous NaOH
solution to obtain an Et20 elution as an alkaloid-rich fraction.
[0042]
The resulting alkaloid-rich fraction was repeatedly separated and
purified by normal-phase silica gel column chromatography, reversed-phase
ODS column chromatography and HPLC to obtain five known aporphine alkaloids
i.e., nuciferine, (0)-nornuciferine, lirinidine, N-methylasimilobine, and
lysicamine and one known proaporphine alkaloid i.e., pronuciferine.
Furthermore, one kind of sterol i.e., p-sitosterol was obtained.
[0043]
Compounds isolated from dried flower parts of Nelumbo nucifera are
shown below.
[0044]
Among the compounds containing the dried flower parts of Nelumbo
nucifera, the inventor of the present invention has found that the aporphine
alkaloids i.e., nuciferine, (0)-nornuciferine, lirinidine,
N-methylasimilobine, and lysicamine, and the proaporphine alkaloids i.e.,
pronuciferine and an alkaloid-containing fractions containing these
alkaloids have an inhibitory action on melanin production and have a skin
whitening effect, therefore the inventor of the present invention has
attained the present invention.
[0045]
According to the present invention, the proaporphine or aporphine
alkaloid alone or in combination, or the alkaloid-containing fraction
containing the alkaloids can be used as a skin whitening composition.
In this case, the used amount can be, for example in products
containing other known additives or vehicles used in cosmetics, e.g., 0.001
to 5% by weight.
[0046]
Therefore, appropriate embodiments of the skin whitening
composition according to the present invention include a skin cosmetic for
a topical or whole-body usage; a pharmaceutical and/or cosmetic preparation
being applicable for scalp and head hair; a bath agent to mix with bath
water; and a skin patch and a cosmetic sheet.
[0047]
Specifically, examples of the embodiments include basic skin care
cosmetics e.g., a skin lotion, a milk lotion, a cream, an ointment, a gel,
a lotion, an oil, a facial mask, a mist and sheets for facial cosmetic; '
head hair cosmetics e.g., a shampoo, a rinse, and a hair conditioner; and
makeup cosmetics e.g., a foundation and a rouge.
[0048]
The various cosmetics can be formulated by methods known to persons
having ordinary skill in the art (for example, "Cosmetics", Tetsusaku Ikeda,
Nanzando, 1979) by adding, in addition to the active ingredients, the
following various components and additives to the skin whitening composition
according to the present invention when needed, as long as they do not damage
the advantageous effects of the invention.
[0049]
Oils and fats
Avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange
oil, orange roughy oil, sesame oil, cocoa butter, chamomile oil, carrot
oil, cucumber oil, tallowate, kukui nut oil, safflower oil, shea butter,
liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil,
apricot kernel oil, castor oil, cotton seed oil, peanut oil, turtle oil,
mink oil, egg-yolk oil, palm oil, palm kernel oil, Japan wax, coconut oil,
tallow, lard, squalene, squalane, and pristine or hydrogenated compounds
thereof (e.g., hydrogenated oil).
[0050]
Waxes
Beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced
lanolin, lanolin wax, candelilla wax, montan wax, shellac wax, and rice
wax.
[0051]
Mineral oils
Liquid paraffin, vaseline, paraffin, ozokerite, ceresin, and
microcrystalline wax.
[0052]
Fatty acids
Natural fatty acids such as lauric acid, myristic acid, palmitic
acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid,
docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid,
undecylenic acid, tall oil, and lanolin fatty acid; and synthetic fatty
acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid,
isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and
isopentanoic acid.
[0053]
Alcohols
Natural alcohols such as ethanol, isopropanol, lauryl alcohol,
cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol,
phytosterol, and phenoxyethanol; and synthetic alcohols such as
2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol.
[0054]
Polyhydric alcohols
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, pentyl glycol, glycerin, pentaerythritol,
threitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol,
lactitol, and maltitol.
[0055]
Esters
Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl
laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl
myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate,
diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol
monostearate, propylene glycol monostearate, and propylene glycol
dioleate.
[0056]
Metallic soaps
Aluminum stearate, magnesium stearate, zinc stearate, calcium
stearate, zinc palmitate, magnesium myristate, zinc laurate, and zinc
undecylenate.
[0057]
Gum substances, sugars or water-soluble macromolecular compounds
Gum arabic, gum benzoin, partially hydrogenated rosin, gum guaiac,
Irish moss, Karaya gum, gum tragacanth, carob gum, quince seed, agar, casein,
lactose, fructose, sucrose or esters thereof, trehalose or derivatives
thereof, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl
chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan added with
alkylene (C2-C4) oxide such as ethylene oxide, low-molecular-weight chitin
or chitosan, chitosan salts, sulfated chitin or chitosan, phosphated chitin
or chitosan, alginic acid or salts thereof, hyaluronic acid or salts thereof,
chondroitin sulfate or salts thereof, heparin, ethyl cellulose, methyl
cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium
carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose,
nitrocellulose, crystalline cellulose, polyvinyl alcohol, poly(vinyl
methyl ether), polyvinylpyrrolidone, poly(vinyl methacrylate),
poly(acrylic acid) salts, polyalkylene oxides such as polyethylene oxide
and polypropylene oxide or cross-linked polymers thereof, carboxy vinyl
polymer, and polyethylenimine.
[0058]
Surfactants
Anionic surfactants: alkyl carboxylate salts, alkyl sulfonate salts,
alkyl sulfate ester salts, alkyl phosphate ester salts; cationic
surfactants: alkyl amine salts, alkyl quaternary ammonium salts; amphoteric
surfactants; carboxylic acid type amphoteric surfactants: amino-type,
betain-type; sulfate type amphoteric surfactants; sulfonic acid type
amphoteric surfactants; phosphate type amphoteric surfactants; nonionic
surfactants: ether-type nonionic surfactants, ether ester-type nonionic
surfactants, ester-type nonionic surfactants, block polymer-type nonionic
surfactants, nitrogen-containing-type nonionic surfactants; other
surfactants: natural surfactants, protein hydrolysates and derivatives
thereof, polymeric surfactants, surfactants containing titanium/silicon,
and fluorocarbon-based surfactants.
[0059]
Various vitamins
Vitamin A group: retinol, retinal (vitamin Al), dehydro retinal,
(vitamin A2), carotene, lycopene (provitamin A) , vitamin B group: thiamine
hydrochloride, thiamine sulfate (vitamin Bl), riboflavin (vitamin B2),
pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids,
nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin
C group: ascorbic acid or derivatives thereof, vitamin D group:
ergocalciferol (vitamin D2), cholecalciferol (vitamin D3),
dihydrotachysterol, vitamin E group: vitamin E or derivatives thereof,
ubiquinones, vitamin K group: phytonadione (vitamin Kl), menaquinone
(vitamin K2), menadione (vitamin K3), menadiol (vitamin K4), others,
essential fatty acid (vitamin F), carnitine, ferulic acid, y-oryzanol,
orotic acid, vitamin Ps (rutin, eriocitrin, hesperidin), and vitamin U.
[0060]
Various amino acids
Valine, leucine, isoleucine, threonine, methionine, phenylalanine,
tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine,
cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid,
glutamic acid, hydroxylysine, arginine, ornithine, histidine and the like,
and sulfates thereof, phosphates thereof, nitrates thereof, citrates
thereof, or amino acid derivatives such as pyrrolidonecarboxylic acid.
[0061]
Various additives from plant or animal-origin raw materials
Such agents may be suitably selected and used from various materials
to be added in response to product types and formulation types, after common
fabrications (e.g., arbitrarily selected from grinding, milling, cleansing,
hydrolysis, fermentation, purification, compression, extraction,
fractionation, filtration, desiccation, powderization, granulation,
dissolution, sterilization, pH adjustment, deodorization, decolorization,
and a combination of them) are performed.
[0062]
An extraction solvent may be selected with due consideration of
intended purposes and types of products or fabrication treatments in the
following steps and the like, however, under normal circumstances, at least
one mixture is preferably used wherein the mixture is selected from water,
lower alcohols or aqueous lower alcohols e.g., methanol, ethanol, propyl
alcohol, isopropyl alcohol, butanol, and isobutanol, polyhydric alcohols
or aqueous polyhydric alcohols e.g., propylene glycol, 1, 3-butylene glycol,
and glycerin, and various organic solvents e.g., acetone and ethyl acetate.
However, in the case where the presence of an organic solvent is not preferred
for the intended purpose, only water may be used or ethanol which is easy
to eliminate after the extraction may be used alone or in an arbitrary mixture
with water. Solvents extracted may also be used.
[0063]
In the case of using additives originating in plant or animal raw
materials for drugs for external usage or cosmetics for whole-body or topical
applications, cosmetic effects including protection of skin or head hair,
moisture retention, improvement of feeling/tone, addition of flexibility,
alleviation of stimulation, alleviation of stress by fragrance, cellular
stimulation (prevention of cellular senescence), suppression of
inflammation, improvement of skin tone and hair type, prevention of chalk
mark and improvement thereof, hair growth, hair restoration, prevention
of hair loss, additional hair shine, cleaning effect, relaxation from
fatigue, improvement of blood flow, and warm-bath effects can be expected
as well as other effects e.g. , added fragrance, odor elimination, thickening,
preservation, and buffering.
Crude drugs or plant-origin materials known to those skilled in the
relevant art are used as raw material plants (crude drugs).
[0064]
Algae
Examples of algae include green algae, brown algae, red algae,
blue-green algae, charales, and golden algae (Chrysophytes).
[0065]
Materials from animal-origin raw materials
Materials from animal-origin raw materials include collagen
derivatives including collagen hydrolysates (e.g., by an acid, an alkali,
or an enzyme) of cow, pig, chicken or fish, a water-soluble collagen and
an acylated collagen, elastin or elastin hydrolysates (e.g., by an acid,
an alkali, or an enzyme) of cow or pig, or water-soluble elastin derivatives,
keratin and degradation products thereof or derivatives thereof, silk
protein and degradation products thereof or derivatives thereof, milk,
casein and degradation products thereof or derivatives thereof, powdered
skim milk and degradation products thereof or derivatives thereof,
lactoferrin or degradation products thereof, chicken egg components, fish
meat degradation products, and nucleic acid-related compounds (ribonucleic
acid and deoxyribonucleic acid).
[0066]-
Maritime components
Examples include marine water such as deep ocean water, for example,
sea salts, dry substances from marine water, mineral salts (e.g., sodium
chloride, magnesium chloride, and potassium chloride) obtained from the
Dead Sea, the Atlantic Ocean, or the Pacific Ocean, sea mud or mud (fangos) ,
for example, sea mud or mud (components contained: silicon dioxide, titanium
dioxide, aluminum oxide, iron oxide, manganese oxide, sodium oxide,
potassium oxide, magnesium oxide, calcium oxide, strontium oxide, sodium,
potassium, magnesium, calcium, chromium, iron, copper, nickel, zinc, lead,
manganese, arsenic, and water) from various regions such as Italian fango,
Germany's fango, Eifel fango, and Freiburg fango, Shotoku-seki (stone
particularly mined in the subsurface layer of Miyagi prefecture in Japan),
and obsidian.
[0067]
Metabolites from cell culture of microorganisms
Examples include yeast metabolites, an extract essence from yeast
fungus, bacterial metabolites, an extract essence from bacteria, fungus
or Actinomyces metabolites, an extract essence from fungus or Actinomyces,
Bacillus subtilis var. natto metabolites, an extract essence from natto,
an extract from fermented rice, an extract from fermented rice bran (red
rice bran, white rice bran), an Euglena extract or degraded substances
thereof or water-soluble derivatives thereof, trehalose or derivatives
thereof, products of lactic acid fermentation of raw milk or powdered skim
milk, products of lactic acid fermentation of leguminous plants, and
products of lactic acid fermentation of genus Cocos plant.
[0068]
Inorganic pigments
Examples include anhydrous silicic acid, magnesium silicate, talc,
kaolin, bentonite, mica, titanated 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.
[0069]
Ultraviolet absorbers/blockers
Examples of such agents include benzophenone derivatives (e.g.,
2-hydroxy-4-methoxybenzophenone,
2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, sodium
2-hydroxy-4-methoxybenzophenone-5-sulfonate, dihydroxy
dimethoxybenzophenone, sodium dihydroxy dimethoxybenzophenone sulfonate,
2,4-dihydroxy benzophenone, and tetrahydroxy benzophenone),
p-aminobenzoic acid derivatives (e.g., p-aminobenzoic acid, ethyl
p-aminobenzoate, glyceryl p-aminobenzoate, amyl p-dimethylaminobenzoate,
and octyl p-dimethylaminobenzoate), methoxycinnamic acid derivatives (e.g.,
p-methoxycinnamic acid ethyl ester, p-methoxycinnamic acid isopropyl ester,
p-methoxycinnamic acid octyl ester, p-methoxycinnamic acid 2-ethoxyethyl
ester, p-methoxycinnamic acid sodium salt, p-methoxycinnamic acid
potassium salt, and di-p-methoxycinnamic acid mono-2-glyceryl
ethyhexanoate) , salicylic acid derivatives (e.g., octyl salicylate, phenyl
salicylate, homomenthyl salicylate, dipropylene glycol salicylate,
ethylene glycol salicylate, myristyl salicylate, and methyl salicylate),
anthranilic acid derivatives (e.g., methyl anthranilate), urocanic acid
derivatives (e.g., urocanic acid and ethyl urocanate) , coumarin derivatives,
amino acid-based compounds, benzotriazole derivatives, tetrazole
derivatives, imidazoline derivatives, pyrimidine derivatives, dioxane
derivatives, camphor derivatives, furan derivatives, pyrone derivative,
nucleic acid derivatives, allantoin derivatives, nicotinic acid
derivatives, vitamin B6 derivatives, umbelliferone, esculin, benzyl
cinnamate, cinoxate, oxybenzone, dioxybenzone, octabenzone, sulisobenzone,
benzoresorcinol, arbutin, guaiazulene, shikonin, baicalin, baicalein,
berberine, neo heliopan, escalol, zinc oxide, talc, and kaolin.
[0070]
skin whitening agents
Examples of such agents include p-aminobenzoic acid derivatives,
salicylic acid derivatives, anthranilic acid derivatives, coumarin
derivatives, amino acid-based compounds, benzotriazole derivatives,
tetrazole derivatives, imidazoline derivatives, pyrimidine derivatives,
dioxane derivatives, camphor derivatives, furan derivatives, pyrone
derivatives, nucleic acid derivatives, allantoin derivatives, nicotinic
acid derivatives, vitamin C or derivatives thereof (e.g., vitamin C
phosphate magnesium salt and vitamin C-glucoside) , vitamin E or derivatives
thereof, kojic acid or derivatives thereof, oxybenzone, benzophenone,
arbutin, guaiazulene, shikonin, baicalin, baicalein, berberine, placenta
extract, ellagic acid, and rucinol.
[0071]
Tyrosinase activity inhibitors
Examples of such agents include vitamin C or derivatives thereof
(e.g., vitamin C-phosphate magnesium salt and vitamin C-glucoside),
hydroquinone or derivatives thereof (e.g., hydroquinone benzyl ether),
kojic acid or derivatives thereof, vitamin E or derivatives thereof,
N-acetyl tyrosine or derivatives thereof, glutathione, hydrogen peroxide,
zinc peroxide, placenta extract, ellagic acid, arbutin, rucinol, a silk
extract, and plant extracts (e.g., Chamomilla recutita, Morus alba, Gardenia
florida, Angelica acutiloba, Sanguisorba officinalis, Sophora angustifolia,
Artemisia princeps, Lonicera japonica, Phellodendron amurense, Houttuynia
cordata, Poria cocos, Coix lacryma-jobi var ma-yuen, Lamium album, Humulus
lupulus, Crataegus cuneata, Eucalyptus globulus, Achillea millefolium,
Althea officinalis, cinnamon bark, Vitex rotundifolia L. fruit, Hamamelis
virginiana, Morus alba L. or Morus bombycis, Isodon japonicus, Platycodon
grandiflorum, Cuscuta australis seed, Euphorbia lathyris L. seed, Iris
japonica, Ephedra sinica Stapf (orE, equisetina Bunge) , Cnidium of ficinale
Makino, Angelica pubescens Maxim root, Bupleurum falcatum L., Ledebouriella
seseloides, Glehnia littoralis, Scutellaria baicalensis, Paeonia moutan
Sims bark, Paeonia albiflora, Geranium thunbergii, Pueraria lobata root,
Glycyrrhiza glabra L. (or Glycyrrhiza uralensis) root, Rhus javanica L.
gullnut, Aloe ferox (or Aloe africana) , Zingiber officinale, Carthamus
tinctorius L., Camellia sinensis (green tea), Camellia sinensis (black tea),
and Uncaria gambir Roxb.).
[0072]
Melanin pigment-reducing/-degrading substances
Examples of such substances include phenylmercuric hexachlorophene,
mercuric oxide, mercurous chloride, hydrogen peroxide, zinc peroxide, and
hydroquinone and derivatives thereof (hydroquinone benzyl ether).
[0073]
Substances to augment cell activity
Substances which accelerate cell turnover/cellular augmentation
include hydroquinone, an extract from lactobacilli, a placenta extract,
a reishi mushroom extract, vitamin A, vitamin E, allantoin, a spleen extract,
a thymus extract, a yeast extract, a fermented milk extract, and plant
extracts (e.g., Aloe ferox (or Aloe africana), Scutellaria baicalensis,
Equisetum arvense, Gentiana lutea, Arctium lappa, Lithospermum
erythrorhizon root, Daucus carota sativa, Hamamelis virginiana, Humulus
lupulus, Coix lacryma-jobi ma-yuen seed, Lamium album, Swertia japonica,
Angelica acutiloba root, Calendula officinalis L., Hydrangea macrophylla
var. thunbergii, Hypericum erectum, Cucumis sativus, Thymus vulgaris,
Rosmarinus officinalis L., and Petroselinum crispum.).
[0074]
Astringent agents
Examples of such agents include succinic acid, allantoin, zinc
chloride, zinc sulfate, zinc oxide, calamine, zinc p-phenolsulfonate,
alminum potassium sulfate, resorcin, ferric chloride, and tannic acid
(including catechin compounds).
[0075]
Reactive oxygen species scavengers
Examples of such agents include SOD, catalase, and glutathion
peroxidase.
[0076]
Antioxidants
Examples of such agents include vitamin C or salts thereof, stearates,
vitamin E or derivatives thereof, nordihydroguaiaretic acid, butyl hydroxy
toluene (BHT), butyl hydroxy anisole (BHA), hydroxytyrosol,
p-hydroxyanisol, propyl gallate, sesamol, sesamolin, gossypol, and
propolis.
[0077]
Inhibitors on lipid peroxide production
Examples of such agents include P-carotene, and plant extracts (e.g.,
sesame cultured cells, Hydrangea macrophylla var. thunbergii, Hypericum
erectum, Hamamelis virginiana, Eugenia caryophillus flower powder, Melissa
officinalis, Isodon japonicus, Betula alba, Salvia officinalis, Rosmarinus
officinalis L., Nandina domestica, Rosa multiflora fruit, Ginkgo biloba,
and Camellia sinensis (green tea)) .
[0078]
Anti-inflammatory drugs
Examples of such drugs include ichthammol, indometacin, kaolin,
salicylic acid, sodium salicylate, methyl salicylate, acetylsalicylic acid,
diphenhydramine hydrochloride, d-camphor, dl-camphor, hydrocortisone,
guaiazulene, chamazulene, chlorpheniramine maleate, glycyrrhizic acid or
salts thereof, glycyrrhetinic acid or salts thereof, Glycyrrhiza glabra
extract, Lithospermum erythrorhizon root extract, Rosa multiflora fruit
extract, and propolis.
[0079]
Antibacterial and bactericidal agents and disinfectants
Examples of such agents include acrinol, sulfur, calcium gluconate,
chlorhexidine gluconate, sulfamic acid, mercurochrome, lactoferrin or
hydrolysates thereof, alkyl diaminoethyl glycine hydrochloride solution,
triclosan, sodium hypochlorite, chloramine T, bleaching powder, iodine
compounds, iodoform, sorbic acid or salts thereof, propionic acid or salts
thereof, salicylic acid, dehydroacetic acid, p-hydroxybenzoates,
undecylenic acid, thiamine lauryl sulfate, thiamine lauryl nitrate, phenol,
cresol, p-chlorophenol, p-chloro-m-xylenol, p-chloro-m-cresol, thymol,
phenethyl alcohol, O-phenylphenol, Irgasan CH3565, halocarban,
hexachlorophene, chlorhexidine, ethanol, methanol, isopropyl alcohol,
benzyl alcohol, ethylene glycol, propylene glycol, 2-phenoxyethanol,
1,2-pentanediol, zinc pyrithione, chlorobutanol, isopropyl methylphenol,
nonionic surfactants (e.g., polyoxyethylene lauryl ether, polyoxyethylene
nonylphenyl ether, and polyoxyethylene octyl phenyl ether), amphoteric
surfactants, anionic surfactants (e.g., sodium lauryl sulfate and lauroyl
sarcosine potassium salt), cationic surfactants (e.g.,
cetyltrimethylammonium bromide, benzalkonium chloride, benzethonium
chloride, and methylrosanilinium chloride), formaldehyde, hexamine,
brilliant green, malachite green, crystal violet, germall, photosensitive
pigment 101, photosensitive pigment 201, photosensitive pigment 401,
N-long-chain acyl basic amino acid derivatives and acid addition salts
thereof, zinc oxide, hinokitiol, Sophora flavescens ait root, and propolis.
[0080]
Moisturizers
Examples of such agents include glycerin, propylene glycol,
1,3-butylene glycol, polyethylene glycol, glyceryl
tricaprylate/tricaprate, glycolic acid (a-hydroxy acid), hyaluronic acid
or salts thereof, chondroitin sulfate or salts thereof, water-soluble chitin
or derivatives thereof or chitosan derivatives, pyrrolidonecarboxylic acid
or salts thereof, sodium lactate, urea, sorbitol, amino acids or derivatives
thereof (e.g., valine, leucine, isoleucine, threonine, methionine,
phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine,
serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic
acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine and
sulfates thereof, phosphates thereof, nitrates thereof, citrates thereof,
or pyrrolidonecarboxylic acid), fats and oils (e.g., avocado oil, almond
oil, fennel oil, perilla oil, olive oil, orange oil, orange roughy oil,
sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, tallowate,
quince seed oil, kukui nut oil, safflower oil, shea butter, liquid shea
butter, soybean oil, camellia oil, corn oil, rapeseed oil, jojoba oil,
apricot kernel oil, castor oil, sunflower oil, cottonseed oil, peanut oil,
turtle oil, tea tree oil, mink oil, egg-yolk oil, palm oil, palm kernel
oil, Japan wax, coconut oil, eucalyptus oil, tallow, lard, squalene,
squalane, pristane or hydrogenates thereof (hydrogenated oil)), waxes
(beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced
lanolin, lanolin wax, candelilla wax, montan wax, shellac wax, and rice
wax), mineral oils (e.g., liquid paraffin, vaseline, paraffin, ozokerite,
ceresin, and microcrystalline wax), metabolites from Bacillus subtilis var
natto, a natto extract essence, a silk fiber extract, and plant extracts
(e.g., Pinus densiflora bark extract, Aloe ferox (or Aloe africana),
Pueraria lobata root, Chamomilla recutita, Glycyrrhiza glabra, Cucumis
sativus, Glycyrrhiza glabra, rice or rice bran, Lithospermum erythrorhizon
root, Betula alba or Betula alba juice, Swertia japonica, Morus alba bark,
Eriobotrya japonica leaf, Luffa cylindrical, Paeonia moutan Sims bark, and
Artemisia princeps).
[0081]
Fragrances
Examples include natural animal-origin fragrances such as musk,
civet, castoreum, and ambergris, plant-origin fragrances such as anise
essential oil, angelica essential oil, ylang ylang essential oil, iris
essential oil, fennel seed essential oil, orange essential oil, kananga
essential oil, caraway essential oil, cardamom essential oil, guaiacwood
essential oil, cumin essential oil, Lindera umbellata essential oil,
cinnamon bark essential oil, cinnamon essential oil, geranium essential
oil, copaiba balsam essential oil, coriander essential oil, perilla
essential oil, cedarwood essential oil, citronella essential oil, jasmine
essential oil, ginger glass essential oil, cedar essential oil, spearmint
essential oil, peppermint oil, star anise 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 essential oil, Japanese cypress essential oil, yellow-cedar
essential oil, sandalwood essential oil, petitgrain essential oil, bay
essential oil, vetiver essential oil, bergamot essential oil, Peru balsam
essential oil, bois de rose essential oil, Ho-Leaf essential oil, mandarin
orange essential oil, eucalyptus essential oil, lime essential oil, lavender
essential oil, linaloe essential oil, lemongrass essential oil, lemon
essential oil, rosemary essential oil, and Japanese mint essential oil,
and other synthetic fragrances.
[0082]
Colorants
Examples include colorants such as red cabbage color, red rice color,
madder color, annatto extract pigment, sepia color, turmeric oleoresin
pigment, enju color, krill color, Japanese persimmon color, caramel, gold,
silver, gardenia color, maize morado color, onion color, tamarind color,
spirulina color, buckwheat extract color, cherry color, laver color,
hibiscus color, grape juice color, marigold color, purple sweet potato color,
purple yam color, lac color, and rutin.
[0083]
Other additives
Examples of other additives include a hormone, a sequestering agent,
a pH adjuster, a chelator, a preservative, an anti-fungal agent, a freshener,
a stabilizer, an emulsifier, a protein of animal/plant origin and
degradation products thereof, a polysaccharide of animal/plant origin and
degradation products thereof, a glycoprotein of animal/plant origin and
degradation products thereof, a blood flow improvement agent, an
anti-inflammatory drug, an antiphlogistic drug, an antiallergic drug, a
wound healing drug, a foaming agent, a thickener, an enzyme, purified water
(including electron ionized water, small clustered water, and the like),
an odor eliminating agent, and a deodorizer.
[0084]
By the concomitant use of these components and the additives, the
enhancement of the intended effect of the present invention or other cosmetic
and esthetic effects can be expected additively or synergistically.
Hereinafter, preparation examples, validation tests, and prescription
examples will be described in more detail. However, the present invention
is not limited to them.
[0085]
Since the proaporphine or aporphine alkaloids having a skin whitening
function are known substances, the alkaloid-containing fraction containing
the alkaloids is not particularly limited to flower parts of Nelumbo nucifera.
Any plants containing the proaporphine or aporphine alkaloids can be used
as raw materials in the present invention.
EXAMPLES
[0086]
Optical rotation was measured with Horiba high sensitive SEPA-300
digital polarimeter (I = 0.5) (HORIBA,.Ltd.).
High-resolution mass spectrometry (High resolution FAB-MS and
EI-MS) and mass analysis (FAB-MS and EI-MS) were performed by using JEOL
JMS-SX 102 and JMS-GCMATE mass spectroscope (manufactured by JEOL Ltd.).
[0087]
Hydrogen NMR spectrum ^H-NMR) and Carbon 13 nuclear magnetic
resonance spectrum (13C-NMR) were measured by using JEOL JNM-EX270 (270 MHz) ,
JNM-LA500 (500 MHz) , and JNM-ECA600 (600 MHz) (manufactured by JEOL Ltd. ) ,
and tetramethylsilane (TMS) was used as an internal standard.
[0088]
For High-performance liquid chromatography (HPLC), Shimadzu LC-6AD
(manufactured by Shimadzu Corporation) was used as a pump and Shimadzu
RID-10A (manufactured by Shimadzu Corporation), HITACHI L-2130, or HITACHI
L-2490 (manufactured by Hitachi, Ltd.) was used as a refractive index
detector.
[0089]
As the adsorbent for column chromatography, silica gel BW-200
(manufactured by Fuji Silysia Chemical Ltd., 150 to 350 mesh) was used for
a normal-phase type support and Chromatorex ODS DM1020T (manufactured by
Fuji Silysia Chemical Ltd., 100 to 200 mesh) was used for a reversed-phase
type support.
[0090]
To perform thin-layer chromatography (TLC), silica gel 60F254
(manufactured by Merck, normal-phase), RP-18 60F254 (manufactured by Merck,
reversed-phase), silica gel 60F254 HPTLC (manufactured by Merck,
normal-phase), and RP-18 WF254 HPTLC (manufactured by Merck,
reversed-phase) were used, and the detection of spots on the TLC was
performed by the observation of the color reaction under UV (254 nm) after
spraying a 1% Ce(SO4)2/10% H2S04 aqueous solution on heating.
[0091]
Unless specifically described otherwise, all the reagents (special
grade) used to perform the present invention were from Wako Pure Chemical
Industries, Ltd.
The values resulting from experiments were expressed as the mean
± standard error, data were analyzed using the Dunnett method to validate
significant differences over the mean values from the control group, and
data were considered significant when their p-values were 0.05 or less.
[0092]
Cells were cultured in MCO-18AIC (manufactured by SANYO Electric
Co., Ltd.) C02 incubator in the presence of 5%C02 at 37°C under saturated
humidity.
[0093]
Evaluation of inhibition on melanin production of mouse melanoma
cells
Mouse B16 melanoma 4A5 cells (RCB0557) given by the RIKEN bioresource
center cell bank were incubated in a DMEM (4500 mg/L glucose) culture medium
(Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS), 100 unit/mL
penicillin, and 100 (ig/mL streptomycin in the presence of 5%C02 at 37°C.
[0094]
Next, cells were seeded at 2. 0 x 104 cells/400 nL/well into a 24-well
multiplate (manufactured by SUMITOMO Bakelite Co., Ltd.) and were
preincubated for a period of 24 hours, and a substance of interest and
theophylline (1 jiM at a final concentration) were added. After 72 hours
of incubation, cells were recovered by stripping by trypsin treatment and
were washed with PBS and were dissolved in a NaOH solution (120 |LtL/well,
80°C, 30 min) . The cell lysates were plated (100 fiL/well) into a 96-well
microplate (manufactured by SUMITOMO Bakelite Co. , Ltd.) and the absorbance
of the produced melanin was measured by a microplate reader (model 550,
manufactured by BiO-RAD) (measurement wavelength: 405 nm, reference
wavelength: 655 nm) . A substance of interest was dissolved in DMSO and added
to a culture medium (0.1% at a final concentration of DMSO).
[0095]
The inhibitory ratio was calculated by the equation shown below:
Inhibitory ratio (%) = [(A-B)/A]/(C/100) x 100
wherein A represents the absorbance of a control (without a substance
of interest), B represents the absorbance in the presence of a substance
of interest, and C represents the cell viability.
[0096]
Evaluation of inhibition of cell proliferation and cytotoxicity on
mouse melanoma cells by WST-8 assay
B16 melanoma 4A5 cells were incubated in a DMEM (4500 mg/L glucose)
culture medium (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) ,
100 unit/mL penicillin and 100 |ag/mL streptomycin in the presence of 5%
C02 at 37°C.
[0097]
Next, cells (5.0 x 103 cells/100 |aL/well) were seeded into a 96-well
microplate (manufactured by SUMITOMO Bakelite Co., Ltd.) and were
preincubated for a period of 24 hours. After the preincubation, a substance
of interest and theophylline (at a final concentration of 1 mM) were added.
After 70 hours of incubation, WST-8
[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2
H-tetrazolium monosodium salt) (Dojindo Laboratories) was added in 10 (ig/mL
installments. After 2 hours of incubation, the absorbance of the resulting
WST-8 formazan was measured by a microplate reader (model 550, manufactured
by BiO-RAD) (measurement wavelength: 450 nm, reference wavelength: 655 nm) .
The substance of interest was dissolved in DMSO and added to a culture medium
(0.1% at a final concentration of DMSO).
[0098]
Inhibitory ratio (%) = [(A-B)/A] x 100
wherein A represents the absorbance of control (without a substance
of interest) , and B represents the absorbance in the presence of a substance
of interest.
[0099]
Tyrosinase inhibitory activity assay
To a 96-well microplate (manufactured by SUMITOMO Bakelite Co., Ltd.),
70 ^/well of a 2.5 mM L-DOPA phosphate buffer was loaded and 20 (XL of the
adjusted test solution (DMSO solution) was added. In the next step, 120
HL of tyrosinase (46 unit/mL at a final concentration) in a phosphate buffer
was added to initiate an enzyme reaction at room temperature for a period
of 5 minutes. A phosphate buffer was used as a blank. The absorbance of
the produced melanin was measured by a microplate reader (model 550,
manufactured by BiO-RAD) (measurement wavelength: 4 50 nm, reference
wavelength: 655 nm).
[0100]
Inhibitory ratio (%) = [(A-B)/A] x 100
wherein A represents the absorbance of the control (without a
substance of interest), and B represents the absorbance in the presence
of a substance of interest.
[0101]
Example 1
Inhibitory action of methanol extract essences extracted from each
portion of Nelumbo nucifera on melanin production
Melanin-producing melanocytes are found throughout the basal layer
of the epidermis, and melanocytes produce more pigments when the skin is
exposed to the rays of the sun, resulting in a brownish yellow or dark brown
skin color. Melanin is a pigment polymer which is synthesized in
specialized cells such as melanocytes or melanoma, and plays an important
role in the protection from the exposure to UV light and in the absorption
of toxic compounds.
[0102]
On the other hand, there have recently been searched components that
have an inhibitory effect on melanin production for the purpose of a skin
whitening effect.
[0103]
In the present invention, the inhibitory action of a MeOH extract
essence (hereinafter, also referred to as an extract) prepared by extraction
of flower parts, stamens, seeds and leaves of Nelumbo nucifera on heating
(80°C) on melanin production of mouse B16 melanoma 4A5 cells was examined
and the result is shown in Table 1.
[0104]
[0105]
As a result, the activity was recognized in flower parts, stamen,
and leaves. The inhibitory ratio of melanin production was calculated in
view of cell viability.
[0106]
As described in Table 1 above, it was found that the extract essence
from the flower parts had the significant inhibitory effect on melanin
production.
[0107]
Moreover, arbutin is known to inhibit melanin synthesis due to direct
action on tyrosinase which is associated with melanin synthesis, and is
believed to have a skin whitening effect; therefore, it has been widely
used in cosmetics. Thus, arbutin was used as a comparative existing
substance having a skin whitening effect.
The result is shown in Table 2 below.
[0108]
[0109]
From the results shown in Tables 1 and 2 above, the extract essence
prepared from the dried flower parts of Nelumbo nucifera has an inhibitory
effect on melanin production more potent than arbutin which has been widely
used as a skin whitening component in cosmetics.
[0110]
Furthermore, the inhibitory action of the MeOH extract essence
prepared from the flower parts, stamens, seeds and leaves of Nelumbo nucifera
on B16 melanoma 4A5 cell growth was investigated. However, these extracts
did not show the cytotoxicity as shown in Table 3 below.
[0111]
[Table 3]
[0112]
Example 2
Inhibitory action of methanol extract essence of flower parts of
Nelumbo nucifera and each fraction on melanin production
Since the MeOH extract essence extracted from the flower parts of
Nelumbo nucifera on heating (80°C) was confirmed to have the significant
inhibitory action on melanin production, the MeOH extract essence prepared
from the flower parts of Nelumbo nucifera was fractionated with AcOEt, n-BuOH
and H20 as shown in Scheme 1 below. The inhibitory action of each resulting
fraction on melanin production of B16 melanoma 4A5 cells was investigated.
[0113]
[0114]
As a result, the activity was recognized in the AcOEt fraction and
the n-BuOH fraction.
Meanwhile, the inhibitory action of the MeOH extract essence prepared
from the flower parts of Nelumbo nucifera at room temperature, and the Et20
fraction, the H20 fraction, and the alkaloid-rich fraction obtained by
fractionating the extract as shown in Scheme 2 below on melanin production
was also investigated.
[0115]
[0116]
As a result, the potent inhibitory activity of cell growth was found,
particularly in the alkaloid-containing fraction. Moreover, as shown in
Table 5, the cytotoxicity was recognized in the n-BuOH fraction and the
alkaloid-containing fraction at a high dose (100 ng/mL).
[0117]
Example 3
Isolation and purification method A of component contained in flower
parts of Nelumbo nucifera (hot extraction method)
[0118]
Dried flower parts of Nelumbo nucifera (1.0 kg) originating in
Thailand were extracted with MeOH on heating (80°C) and then the solvent
was removed under reduced pressure to obtain a MeOH extract essence (155.0
g, yield 15.5%) .
145.0 g out of the resulting MeOH extract essence was
partition-extracted with AcOEt and H20. The H20 layer was further
partition-extracted with n-BuOH to obtain an AcOEt layer (31.2 g, 3.1%),
an n-BuOH layer (41.0 g, 4.1%), and an H20 layer (72.8 g, 7.3%) (Scheme 1).
[0119]
The resulting AcOEt layer (31.2 g) and the n-BuOH layer (40.0 g)
were repeatedly separated and purified by using normal-phase silica gel
column chromatography, reversed-phase ODS column chromatography and HPLC
to isolate four known aporphine alkaloids i.e., nuciferine (299.2 mg,
0.0299%), (0)-nornuciferine (11.2 mg, 0.0011%), N-methylasimilobine (6.6
mg, 0.0007%) (all are described in Helene G., Michel L., Andre C , Lloydia,
38, 275-338 (1975)) and lysicamine (141.1 mg, 0.0141%) (described in Zhang
Z., ElSohly N. H., Jacob R. M., Pasco S. D., Walker A. L., Clark M. A.,
J. Nat. Prod., 65, 856-859 (2002)) and one known proaporphine alkaloid i. e.,
pronuciferine (56.0 mg, 0.0056%) (described in Fajardo V., ArayaM., Cuadra
P., Oyarzun A. , Gallardo A. , Cueto M., J. Nat. Prod., 72, 1355-1356 (2009)) .
[0120]
Furthermore, two known flavonoid glycosides i.e.,
kaempferol 3-O-p-D-glucopyranoside (6.1 mg, 0.0006%) (described in Markham
R.K., TernalB., Stanly R., GeigerH., MabryJ.T., Tetrahedron, 34, 1389-1397
(1977)) and isorhamnetin 3-O-p-D-glucopyranoside (81.0 mg, 0.0081%)
(described in Lee S. Y., Lee S., Lee S. H., Kim B., Ohuchi K., Shin H. K.,
Biol. Pharm. Bull., 28, 916-91-8), p-sitosterol (a type of sterol) (140.6
mg, 0.0141%) (described in Kovganko V. N., Kashkan N. Zh., Borisov V. E.,
Batura V. E., Chemistry of Naturul Compounds, 35, 646-649 (1999)), and
palmitic acid (a type of fatty acid) (96.3 mg, 0.0096%) were isolated
(above-mentioned Scheme 1).
[0121]
These known compounds were identified by comparing the obtained
spectrum data with the document values of 1H-NMR, 13C-NMR, MS spectrum data
and optical rotation except the identification of palmitic acid. Palmitic
acid was identified by comparing the obtained spectrum data with the standard
compound (commercialized product) on 1H-NMR, 13C-NMR, and MS spectrum data.
The yields of these isolated components are the isolation yields
from the flower parts of Nelumbo nucifera.
[0122]
More specifically, the dried flower parts of Nelumbo nucifera (1.0
kg) originating in Thailand were extracted with MeOH on heating. The
extraction liquid was filtered and MeOH was added to the residue, and the
same extraction procedure was repeated 3 times. The MeOH extraction liquids
were combined and the solvent was evaporated under reduced pressure to obtain
a MeOH extract essence (155.0 g, yield 15.5%). After 145.0 g out of the
resulting MeOH extract essence was partition-extracted with AcOEt and H20,
the resulting H20 layer was further partition-extracted with n-BuOH. The
solvent in each layer was evaporated under reduced pressure to obtain an
AcOEt layer (31.2 g, 3.1%), an n-BuOH layer (41.0 g, 4.1%), and an H20 layer
(72.8 g, 7.3%) .
[0123]
The resulting AcOEt layer (31.2 g) was fractionated by using
normal-phase silica gel column chromatography [1.0 kg, hexane (Hex) -.Hex :
AcOEt = (40 : 1) -(15 : 1) -(3 : 1) - AcOEt - CHC13 : MeOH : HzO = (50 :
3 : 1 ) - . ( 3 0 : 3 : 1 ) - ( 1 0 : 3 : 1 ) - ( 7 : 3 : 1 ) - ( 6 : 4 : 1 ) - MeOH]
to obtain a Fr. 1 (0.79 g), a Fr. 2 (0.05 g), a Fr. 3 (3.37 g), a Fr. 4
(0.65 g), a Fr. 5 (7.35 g) , a Fr. 6 (3.32 g) , a Fr. 7 (5.26 g) , a Fr. 8
(4.37 g) , a Fr. 9 (1.27 g) , a Fr. 10 (2.03 g) , and a Fr. 11 (0.30 g)
respectively.
[0124]
The Fr. 7 (5.0 g) was fractionated by using reversed-phase ODS column
chromatography [150.0 g, MeOH : H20 = (50 : 50) - (60 : 40) - (70 : 30) -
(80 : 20) - (90 : 10) -> MeOH] to obtain a Fr. 7-1 (343.5 mg), a Fr. 7-2
(963.2 mg), a Fr. 7-3 (310.1 mg), a Fr. 7-4 (120.7 mg), a Fr. 7-5 (70.6
mg), a Fr. 7-6 (1813.3 mg), and a Fr. 7-7 (1437.8 mg) respectively.
[0125]
The Fr. 7-6 (467.0 mg) was separated and purified by using HPLC
[column: COSMOSIL 5C18-MS-II, eluting solvent: MeOH : H20 = (70 : 30)] to
obtain a Fr. 7-6-1 (38.6 mg), a Fr. 7-6-2 [lysicamine (36.5 mg, 0.0037%)],
and a Fr. 7-6-3 [nuciferine (148.1 mg, 0.0148%)]. The Fr. 7-6-1 (38.6 mg)
was separated and purified by using HPLC [column: COSMOSIL 5C18-MS-II,
eluting solvent: MeOH : H20 = (68 : 32)] to isolate (0)-nornuciferine (11.2
mg, 0.0011%) and lysicamine (2.1 mg, 0.0002%) respectively.
[0126]
The Fr.7-2 (963.2 mg) was separated and purified by using
reversed-phase ODS column chromatography [60.0 g, MeOH : H20 = (45 : 55)
- (60 : 40) - (75 : 25) - MeOH] to obtain a Fr. 7-2-1 (858.0 mg), a Fr.
7-2-2 (17.9 mg), and a Fr. 7-2-3 (45.0 mg) respectively.
[0127]
The Fr. 7-2-1 (858.0 mg) was separated and purified by using HPLC
[column: COSMOSIL 5C18-MS-II, eluting solvent: MeOH : H20 = (35 : 65)] to
isolate kaempferol 3-O-p-D-glucopyranoside (6.1 mg, 0.0006%) and
isorhamnetin 3-O-p-D-glucopyranoside (81.0 mg, 0.0081%).
[0128]
The Fr. 5 (6.6 g) was fractionated by using reversed-phase ODS column
chromatography [200.0 g, MeOH : H20 = (60 : 40) -> (70 : 30) -> (80 : 20) -
(90 : 10) -. MeOH] to obtain a Fr. 5-1 (46.0 mg), a Fr. 5-2 (1117.0 mg) ,
a Fr. 5-3 (844.0 mg), a Fr. 5-4 (751.2 mg), a Fr. 5-5 (2046.5 mg), a Fr.
5-6 (1183.1 mg) , and a Fr. 5-7 (492.0 mg) respectively.
[0129]
The Fr. 5-3 (150.7 mg) was separated and purified by using HPLC
[column: COSMOSIL 5C18-MS-II, eluting solvent: MeOH : H20 = (85 : 15)] to
isolate palmitic acid (96.3 mg, 0.0096%).
The Fr. 5-6 (200.1 mg) was separated and purified by using HPLC
[column: COSMOSIL 5Ci8-MS-II, eluting solvent: MeOH : H20 = (85 : 15)] to
isolate p-sitosterol (140.6 mg, 0.0141%).
[0130]
The resulting BuOH layer (40.0 g) was fractionated by normal-phase
silica gel column chromatography [1.6 kg, CHC13 - CHC13 : MeOH : H20 = (50 :
3 : 1 ) - ( 3 0 : 3 : 1 ) - ( 1 0 : 3 : 1 ) - ( 8 : 3 : 1 ) - ( 6 : 4 : 1 ) - MeOH]
to obtain a Fr. 1 (0.22 g), a Fr. 2 (0.65 g), a Fr. 3 (5.77 g), a Fr. 4
(10.54 g) , aFr. 5 (3.09 g) , aFr. 6 (6.61 g) , and a Fr. 7 (9.87 g) respectively.
[0131]
The Fr. 3 (1.0 g) was separated and purified by using Preparative
TLC [PTLC, silica-gel 60F, CHC13 : MeOH : H20 = 8 : 3 : 1] to obtain a Fr.
3-1 (264.3 mg), a Fr. 3-2 [lysicamine (102.4 mg, 0.0102%)], a Fr. 3-3
[pronuciferine (56.0 mg, 0.0056%)], a Fr. 3-4 (69.4 mg), a Fr. 3-5 (88.8
mg), a Fr. 3-6 (121.2 mg), a Fr. 3-7 (230.8 mg), and a Fr. 3-8 (69.2 mg)
respectively and compounds.
The Fr. 3-4 (69.4 mg) was separated and purified by using HPLC
[column: COSMOSIL 5Ci8-MS-II, eluting solvent: 1% AcOH-containing MeOH :
H20 = (70 : 30)] to isolate N-methylasimilobine (6.6 mg, 0.0007%).
[0132]
Example 4
Isolation and purification method B of component contained in flower
parts of Nelumbo nucifera (extraction at room temperature)
[0133]
Meanwhile, for the purpose of isolation of alkaloid components, dried
flowers of Nelumbo nucifera (900 . 0 g) originating in Thailand were extracted
with MeOH at room temperature. Then, the solvent was removed under reduced
pressure to obtain a MeOH extract essence (60.8 g, yield 6.8%). After the
resulting MeOH extract essence (60.0 g) was partition-extracted with Et20
and 1% AcOH, the 1% AcOH layer was basified by an ammonia solution followed
by extraction with Et20. Next, the Et20 layer was further washed with a
10% aqueous NaOH solution, and the Et20 elution was concentrated to obtain
an alkaloid-containing fraction (4.21 g, 0.47%) (above-mentioned Scheme
2) .
[0134]
The resulting alkaloid-containing fraction (4.20 g, 0.47%) was
repeatedly separated and purified by using normal-phase silica gel column
chromatography, reversed-phase ODS column chromatography and HPLC to obtain
five known aporphine alkaloids i.e., nuciferine (183.1 mg, 0.0203%),
(O)-nornuciferine (121.3 mg, 0.0135%), lirinidine (3.0 mg, 0.0003%),
N-methylasimilobine (36.0 mg, 0.0040%) and lysicamine (38.2m, 0.0042%);
one known proaporphine alkaloid, i.e., pronuciferine (23.0 mg, 0.0026%);
and p-sitosterol (a type of sterol) (1.8 mg, 0.0002%).
[0135] -
These known compounds were identified by comparing the obtained
spectrum data with the document values of 1H-NMR, 13C-NMR, MS spectrum data
and optical rotation.
These yields of the isolated components are the isolation yields
from the dried flower parts of Nelumbo nucifera.
[0136]
The compounds obtained by the isolation and purification methods
A and B are the same as the compounds which are contained in the dried flower
parts of Nelumbo nucifera.
More specifically, the dried flower parts (900.0 g) of Nelumbo
nucifera originating in Thailand were extracted with MeOH at room
temperature for a period of 7 hours.
[0137]
The extraction liquid was filtered and MeOH was added to the residue
and the same extraction process was carried out 3 times. The MeOH extraction
liquids were combined and the solvent was evaporated under reduced pressure
to obtain a MeOH extract essence (60.8 g, yield 6.8%) . After the resulting
MeOH extract essence (60. 0 g) was partition-extracted with Et20 'and 1% AcOH,
the 1% AcOH-acidic aqueous layer was basified with NH4OH and was extracted
with Et20.
[0138]
The Et20 layer was further partition-extracted with a 10% aqueous
NaOH solution to obtain an Et20 elution as an alkaloid-rich fraction (4.21
g, 0.47%).
[0139]
The alkaloid-rich fraction (3.52 g) was fractionated by normal-phase
silica gel column chromatography [120.0 g, hexane : acetone = (8 : 1) —
(5 : 1) -(1 : 2) - CHC13 : MeOH = (10 : 1) -. (5 : 1) - MeOH] to obtain a
Fr.l (48.0 mg), a Fr. 2 (79.7 mg), a Fr. 3 (1337.8 mg), a Fr. 4 (1279.8
mg), a Fr. 5 (76.2 mg) , and a Fr. 6 (79.6 mg) respectively.
[0140]
The Fr. 2 (79.7 mg) was separated and purified by using HPLC [column:
COSMOSIL 5Ci8-MS-II, eluting solvent: MeOH] to obtain a Fr. 2-1 (38.5 mg) ,
a Fr. 2-2 (13.7 mg) , and a Fr. 2-3 [(3-sitosterol (1.8 mg, 0.0002%)].
[0141]
The Fr. 2-1 (38.5 mg) was separated and purified by using HPLC
[column: COSMOSIL 5C18-MS-II, eluting solvent: 1% AcOH-cont'aining MeOH :
H20 = (90 : 10)] to isolate lysicamine (5.0 mg, 0.0006%).
[0142]
The Fr. 3 (450.0 mg) was separated and purified by using HPLC [column:
COSMOSIL 5C18-MS-II, eluting solvent: 1% AcOH-containing MeOH : H20 = (65 :
35)] to obtain a Fr. 3-1 (344.8 mg), a Fr. 3-2 (2.0 mg), and a Fr. 3-3 [=
lysicamine (5, 13.7 mg, 0.0015%)] respectively.
[0143]
The Fr. 3-1 (344.8 mg) was separated and purified by using HPLC
[(column: COSMOSIL 5C18-MS-II, eluting solvent: 1% AcOH-containing MeOH :
H20 = (30 : 70)] to isolatae a Fr. 3-1-1 [N-methylasimilobine (36.0 mg,
0.0040%)], a Fr. 3-1-2 [lirinidine (3.0 mg, 0.0003%)], a Fr. 3-1-3
[nuciferine (183.1 mg, 0.0203%)], and a Fr. 3-1-4 [pronuciferine (23.0 mg,
0.0026%)] respectively, and each alkaloid.
[0144]
The Fr. 4 (420. 0 mg) was separated and purified by using HPLC [column:
COSMOSIL 5C18-MS-II, eluting solvent: 1% AcOH-containing MeOH : H20 = (30 :
70)] to obtain a Fr. 4-1 (15.8 mg), aFr.4-2 (31.3 mg), a Fr. 4-3 (4.0 mg),
and a Fr. 4-4 (189.9 mg) respectively.
[0145]
The Fr. 4-4 (189.9 mg) was separated and purified by using HPLC
[column: COSMOSIL 5Ci8-MS-II, eluting solvent: 1% AcOH-containing MeOH :
H20 = (65 : 35)] to obtain a Fr. 4-4-1 (163.9 mg) andaFr. 4-4-2 [lysicamine
(11.5 mg, 0.0013%)] .
[0146]
The Fr. 4-4-1 (163.9 mg) was separated and purified by using HPLC
[column: COSMOSIL 5C18-MS-II, eluting solvent: 1% AcOH-containing MeOH :
H20 = (75 : 25)] to obtain a Fr. 4-4-1-1 [nornuciferine (121.3 mg, 0.0135%)],
a Fr. 4-4-1-2 (3.0 mg), andaFr. 4-4-1-3 [lysicamine (8.0 mg, 0.0009%)].
[0147]
Example 5
Inhibitory activity of component contained in flower parts of Nelumbo
nucifera on melanin production
Intensive activity was recognized in the six alkaloids isolated from
the AcOEt layer and the n-BuOH layer, and the alkaloid-rich fraction. The
inhibitory action of these six alkaloids on melanin production of B16
melanoma 4A5 cells was studied and the result is shown in Tables 6 and 7.
[0148]
[0149]
[0150]
From the results, it was found that nuciferine, lirinidine, and
N-methylasimilobine had potent inhibitory activity on cell growth.
Meanwhile, lirinidine and lysicamine showed cytotoxicity at a high dose
(100 ng/mL).
[0151]
Example 6
Furthermore, an inhibitory activity of these six alkaloids (1-6)
on mushroom tyrosinase was examined.
[0152]
[0153]
From the above-mentioned result, it was recognized that these
alkaloids has almost no inhibitory activity on tyrosinase activity.
Therefore, the mechanism of inhibition of the alkaloids (or skin whitening
compositions containing the alkaloids) on melanin production in the present
invention is not via the inhibition of tyrosinase activity, but it is
suggested that other mechanisms could exist to exert the inhibitory action
on melanin production.
[0154]
In the following examples, the alkaloids obtained in the procedures
of Examples 3 and 4 could be used in combination of at least one alkaloid
for the formulation of cosmetics, and as examples, the alkaloid-containing
fraction obtained in the process of Example 4 was used to prepare various
cosmetics.
[0155]
Example 7
Preparation of skin lotion
In accordance with the following prescription, 1 kg of skin lotion
was prepared by a common method.
[0156]
Examples 8 to 11
Each 1 kg of skin lotion was prepared in exactly the same manner
as in Example 7 except the alkaloid-containing fraction was used in Example
1 in an amount of 0.01, 0.1, 1 and 5% by weight respectively.
Example 12
Preparation of milk lotion
In accordance with the following prescription, 1 kg of milk lotion
was prepared by a common method.
[0157]
Examples 13 to 16
Each 1 kg of milk lotion was prepared in exactly the same manner
as in Example 12 except that the alkaloid-containing fraction was used in
Example 12 in an amount of 0.01, 0.1, 1 and 5% by weight respectively.
[0158]
Example 17
Preparation of cream
In accordance with the following prescription, 1 kg of cream was
prepared by a common method.
[0159]
Examples 18 to 21
Each 1 kg of cream was prepared in exactly the same manner as in
Example 17 except that the alkaloid-containing fraction in Example 17 was
used in an amount of 0.01, 0.1, 1 and 5% by weight respectively.
[0160]
Example 22
Preparation of sunscreen cream
In accordance with the following prescription, 1 kg of sunscreen
cream was prepared by a common method.
[0161]
Examples 23 to 26
Each 1 kg of sunscreen cream was prepared in exactly the same manner
as in Example 22 except that the alkaloid-containing fraction in Example
22 was used in an amount of 0.01, 0.1, 1 and 5% by weight respectively.
[0162]
Example 27
Preparation of foundation
In accordance with the following prescription, 1 kg of foundation
was prepared by a common method. .
[0163]
Examples 28 to 31
Each 1 kg of foundation was prepared in exactly the same manner as
in Example 27 except that the alkaloid-containing fraction in Example 27
was used in an amount of 0.01, 0.1, 1 and 5% by weight respectively.
[0164]
Example 32
Sunscreen milk lotion for whole-body
In accordance with the following prescription, 1 kg of sunscreen
milk lotion for whole-body was prepared by a common method.
[0165]
Examples 33 to 36
Each 1 kg of sunscreen cream for whole-body was prepared in exactly
the same manner as in Example 32 except that the alkaloid-containing fraction
in Example 32 was used in an amount of 0.01, 0.1, 1 and 5% by weight
respectively.
INDUSTRIAL APPLICABILITY
[0166]
A skin whitening composition prepared in accordance with the present
invention contains an active ingredient, wherein the active ingredient is
an extract prepared by extracting flower parts of Nelumbo nucifera with
a lower alcohol, or a concentrate thereof, or a purified product thereof,
particularly an alkaloid-containing fraction, and the skin whitening
composition according to the present invention can be used safely mainly
for the propose of preventing sunburn.

CLAIMS
1. A skin whitening composition, comprising an active ingredient
represented by general formula ( I ) :
[Chem. 11
whereinR1, R2 andR3 each r e p r e s e n t a hydrogen atomoramethyl group;
R,, R, and R6 each represent a hydrogen atom or a methyl group, or
alternativelyR3 andRq as well as R5 andR,combinetogether and respectively
represent a bonding hand;
two R,s each represent a hydrogen atom or a l t e r n a t i v e l y the two Ris
combine together and represent an 0x0 group;
n represents an integer 0 or 1,
when n is 0, two R8s combine together with carbon atoms t o which the
two R8s are bonded and form a 4-0x0-cyclohexadiene ring; and
when n i s 1, two R8s and R9 combine together with carbon atoms to
which the two R8s and R9 are bonded and form a benzene ring, wherein
the active ingredient is a proaporphine or aporphine alkaloid.
2. The composition of claim 1, wherein the alkaloid is a t l e a s t one
proaporphine or aporphine a l k a l o i d s e l e c t e d fromthe compounds represented
by the following chemical formulae:
[Chem. 21 cH30p ".cH3 =::p; CHso CH3
/ /' / '. '. Nuci f erine ( 0 ) -Nornucif erine uO:y"IFo>eri ne *Lirini dine , cHflp CH30 CH30 .CH,
/ /
\ \ I 1
0
N-Methylasirnilobine Lysicamine Pronuciferine
3. T h e c o m p o s i t i o n o f c l a i m 1 o r 2 , w h e r e i n t h e a l k a l o i d i s comprised
in a lower alcohol e x t r a c t from flower p a r t s of Nelumbo nucifera belonging
t o the genus Nelumbo i n the family Nelumbonaceae.
4 . The composition of anyone of claims 1 t o 3, wherein t h e a l k a l o i d
is comprised in:
an organic solvent-soluble f r a c t i o n obtained by e x t r a c t i n g flower
p a r t s of Nelumbo nucifera belonging to the genus Nelumbo i n the family
Nelumbonaceaewithaloweralcoholto give a n e x t r a c t i o n l i q u i d , and f u r t h e r
p a r t i t i o n i n g t h e e x t r a c t i o n l i q u i d with ethyl acetate/water and
n-butanol/water when needed; or
an alkaloid-containing f r a c t i o n obtained by p a r t i t i o n i n g t h e
extraction l i q u i d with d i e t h y l ether/weak-acidic water t o give an aqueous
layer, extracting the aqueous layer with d i e t h y l ether a f t e r being weakly
basified by an ammonia s o l u t i o n , and washing t h e e t h e r l a y e r with d i l u t e d
aqueous sodium hydroxide s o l u t i o n .