U.S. patent application number 17/427168 was filed with the patent office on 2022-03-31 for pharmaceutical composition comprising a fraction of melissa officinalis leaf extract.
The applicant listed for this patent is ANGIOLAB, INC.. Invention is credited to Hyun Jeung KIM, Jeong Eun KIM, Min-Young KIM, Hee Suk LEE, Byung Young PARK, Hyung Soo YUH.
Application Number | 20220096586 17/427168 |
Document ID | / |
Family ID | 1000006037347 |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220096586 |
Kind Code |
A1 |
KIM; Min-Young ; et
al. |
March 31, 2022 |
PHARMACEUTICAL COMPOSITION COMPRISING A FRACTION OF MELISSA
OFFICINALIS LEAF EXTRACT
Abstract
The present invention relates to a pharmaceutical composition
for preventing or treating otitis media with effusion comprising a
fraction of Melissa officinalis leaf extract as an effective
ingredient.
Inventors: |
KIM; Min-Young; (Daejeon,
KR) ; PARK; Byung Young; (Daejeon, KR) ; LEE;
Hee Suk; (Daejeon, KR) ; YUH; Hyung Soo;
(Sejong-Si, KR) ; KIM; Jeong Eun; (Daejeon,
KR) ; KIM; Hyun Jeung; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ANGIOLAB, INC. |
Daejeon |
|
KR |
|
|
Family ID: |
1000006037347 |
Appl. No.: |
17/427168 |
Filed: |
May 21, 2020 |
PCT Filed: |
May 21, 2020 |
PCT NO: |
PCT/KR2020/006669 |
371 Date: |
July 30, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 27/16 20180101;
A61K 2236/333 20130101; A61K 2236/39 20130101; A61K 36/53 20130101;
A61K 2236/51 20130101 |
International
Class: |
A61K 36/53 20060101
A61K036/53; A61P 27/16 20060101 A61P027/16 |
Foreign Application Data
Date |
Code |
Application Number |
May 22, 2019 |
KR |
10-2019-0060305 |
Claims
1. A pharmaceutical composition for preventing or treating otitis
media with effusion comprising a fraction of Melissa officinalis
leaf extract.
2. The pharmaceutical composition according to claim 1, wherein the
fraction of the extract is obtained by extracting Melissa
officinalis leaves with 50 to 100% alcohol, concentrating,
fractionating with ethyl acetate, and drying.
3. The pharmaceutical composition according to claim 1, wherein the
fraction of Melissa officinalis leaf extract is an ethyl acetate
fraction of ethanol extract of Melissa officinalis leaves.
4. A method for preventing or treating otitis media with effusion,
the method comprising: administering to a subject in need thereof a
therapeutically effective amount of the pharmaceutical composition
comprising the fraction of Melissa officinalis leaf extract
according to claim 1.
5-6. (canceled)
7. The method composition according to claim 4, wherein the
fraction of the extract is obtained by extracting Melissa
officinalis leaves with 50 to 100% alcohol, concentrating,
fractionating with ethyl acetate, and drying.
8. The method according to claim 4, wherein the fraction of Melissa
officinalis leaf extract is an ethyl acetate fraction of ethanol
extract of Melissa officinalis leaves.
Description
TECHNICAL FIELD
[0001] The present invention relates to a pharmaceutical
composition comprising a fraction of Melissa officinalis leaf
extract. More specifically, the present invention relates to a
pharmaceutical composition for preventing or treating otitis media
with effusion, comprising a fraction of Melissa officinalis leaf
extract of the present invention as an effective ingredient.
BACKGROUND ART
[0002] Melissa officinalis is an herbaceous perennial plant, which
belongs to the Labiatae family, and is also nicknamed Lemon
Balm.
[0003] A Melissa officinalis leaf extract contains flavonoid,
triterpene acids, volatile oils, glycosides of alcohol, phenol
compounds and the like, as well as caffeic acid derivatives. In
particular, the flavonoids contained in Melissa officinalis leaves
are cynaroside, cosmosin, rhamnocitrin, isoquercitrin, etc., and
ursolic acid as triterpene acid. The Melissa officinalis leaf
extract contains hydroxycinnamic acid derivatives such as
rosmarinic acid, which is one of non-volatile components drawing
much attention these days, and also contains geraniol, neral,
citronellal and eugenol as volatile oils.
[0004] An inflammation, which occurs in tympanic cavity, is called
otitis media. Otitis media is roughly divided into acute otitis
media and otitis media with effusion, of which about 40 to 50% is
otitis media with effusion.
[0005] Otitis media with effusion frequently occurs to young
children and old people. When eustachian tube function that connect
the middle ear to the nasopharynx is deteriorated, a pressure
control of inside and outside the eardrum becomes poor, and thus
the middle ear comes to have a negative pressure, such that liquid
is soaked out of a mucous membrane and stagnates. Thus, there occur
symptoms such as difficulty in hearing, feeling of ear occlusion,
etc. If acute upper respiratory inflammation, chronic sinusitis,
allergic rhinitis, adenoidal hypertrophy and the like are present
in a patient's background, an inflammation in a nose or a throat
affects a middle ear through the eustachian tube all the time, and
thus becomes chronic and prone to intractable. If otitis media with
effusion becomes intractable, it may cause an after-effect such as
difficulty in hearing, etc., thereby requiring a surgery. Thus,
early treatment is considered important. In case of young children,
it is difficult to frequently carry out a surgical procedure such
as myringotomy, tympanostomy tube placement or the like. Thus,
there is a need for a medicament to minimize a surgical
procedure.
[0006] In general, the therapeutic agent used for otitis media with
effusion mainly includes expectorants (for example, carbocysteine,
etc.), macrolide antibiotics (for example, erythromycin
ethylsuccinate, clarithromycin, etc.) or the like, but have failed
to achieve a satisfactory effect. Thus, there is an urgent need for
developing an oral agent with high efficacy.
PRIOR ART REFERENCES
Non-Patent Documents
[0007] (Non-Patent Document 1) Human Reproduction Vol. 18, No. 12
pp. 2668-2671, 2003
DISCLOSURE OF INVENTION
Technical Problem
[0008] An objective of the present invention is to provide a
pharmaceutical composition for preventing or treating otitis media
with effusion, comprising a fraction of Melissa officinalis leaf
extract.
[0009] Another objective of the present invention is to provide a
method for preventing or treating otitis media with effusion by
administering a therapeutically effective amount of a fraction of
Melissa officinalis leaf extract to the subject in need
thereof.
[0010] Still another objective of the present invention is to
provide a use of a pharmaceutical composition comprising a fraction
of Melissa officinalis leaf extract, in the manufacture of a
medicament for preventing or treating otitis media with
effusion.
Solution to Problem
[0011] The present inventors have recognized a need for study on
prevention or treatment of otitis media with effusion and have
achieved the objectives of the study on natural substances capable
of preventing or treating this disease.
[0012] Specifically, the present inventors have found that the case
of containing a fraction of Melissa officinalis leaf extract as an
effective ingredient has an effect on prevention and treatment of
otitis media with effusion, thereby completing the present
invention.
[0013] The present invention provides a pharmaceutical composition
for preventing or treating otitis media with effusion comprising a
fraction of Melissa officinalis leaf extract.
[0014] It has been identified that the pharmaceutical composition
comprising a fraction of Melissa officinalis leaf extract according
to the present invention as an effective ingredient may prevent or
treat otitis media with effusion in such a way that a middle ear
effusion (MEE) is ameliorated, the MEE is reabsorbed and thus not
observed, a thickness of middle ear mucosa is remarkably decreased,
and IL-23 and TNF-.alpha. genes are significantly decreased when
examining the expression of IL-23 and TNF-.alpha. genes.
[0015] Hereinafter, the present invention will be described in more
detail.
[0016] The pharmaceutical composition for preventing or treating
otitis media with effusion according to the present invention
contains a fraction of Melissa officinalis leaf extract as an
effective ingredient, which is obtained in such a way that Melissa
officinalis leaves is extracted with 50 to 100% alcohol (v/v),
concentrated, fractionated with ethyl acetate, and then dried to
give a fraction of Melissa officinalis leaf extract.
[0017] Specifically, the fraction of Melissa officinalis leaf
extract is obtained in such a way that Melissa officinalis leaves
is extracted with 50 to 100% alcohol (v/v) and dried, and the
alcohol extract is suspended with water, fractionated with ethyl
acetate, and then dried, and the ethyl acetate fraction is
resuspended with water and dried.
[0018] In the present specification, the term "pharmaceutical
composition" is defined as a chemical or biological compound or
substances, or a mixture or combination of at least two compounds
or substances, which are intended to be used for medically
diagnosing, dealing with, treating or preventing diseases or
pathologies.
[0019] When preparing the fraction of Melissa officinalis leaf
extract of the present invention, dried Melissa officinalis leaves,
non-dried Melissa officinalis leaves or a mixture thereof may be
used. For an effective extraction, the Melissa officinalis leaves
may be used after being cut into small pieces.
[0020] In the present specification, the term "alcohol" means a
compound in which a hydroxyl group is bound to a carbon atom of an
alkyl or substituted alkyl group, the term "alkyl" means a linear
saturated hydrocarbon group or a branched saturated hydrocarbon
group, and the term "substituted alkyl group" means the one in
which a substituent bonded to the carbon of an alkyl group is
substituted with hydroxy, cyano, halo, etc.
[0021] In the present specification, the term "extract" includes an
extract liquid itself and all dosage forms of extract which may be
formed by using the extract liquid, such as the extract liquid
obtained by carrying out an extraction on the Melissa officinalis
leaves, a diluent or concentrate of the extract liquid, a dried
matter obtained by drying the extract liquid, a crude purified
product or a purified product of the extract liquid, a mixture
thereof, or the like.
[0022] In the present specification, the term "fraction" means a
product obtained by performing a fractionation to separate a
specific component or a specific component group from a mixture
comprising many different components.
[0023] In the present invention, a fractionation method of
obtaining the fraction of extract is not specifically limited, and
may be performed according to a method conventionally used in the
art. Specifically, there are a solvent fractionation method
performed by treating various solvents, a fractionation method
using ultrafiltration performed by passing through an
ultrafiltration membrane having a certain molecular weight cut-off
value, a chromatography fractionation method performed with various
chromatography types (manufactured for separation according to
sizes, electric charges, hydrophobicity or affinity), a combination
thereof, and the like. Specifically, there may be a method in which
an extraction is performed on the Melissa officinalis leaves of the
present invention, after which an obtained extract is treated with
a pre-determined solvent so as to obtain a fraction from the
extract.
[0024] In the present invention, the type of fractionation solvent
used for obtaining the fraction are not specifically limited, and
any solvents generally known in the art may be used. Specifically,
it may be ethyl acetate, which is a non-polar solvent.
[0025] In the present specification, the term "effective
ingredient" means including a main component as a substances or a
substance group (including herb medicine, etc., of which a
pharmacologically active component, etc., has not been identified
yet) expected to directly or indirectly exhibit the efficacy and
effect of a composition thereof by an intrinsic pharmacological
action.
[0026] To prepare an alcohol extract of Melissa officinalis leaves,
an extraction may be performed according to a conventional
extraction method by using 50 to 100% alcohol (v/v), preferably 70
to 80% alcohol (v/v) in an amount of 5 to 15 times (v/w) of the
Melissa officinalis leaves.
[0027] The alcohol refers to C1-C6 alcohol including ethanol,
methanol, etc., preferably may be ethanol or methanol.
[0028] In the present specification, the term "C1-C6" means a
functional group or a main chain having one or more and six or less
carbon atoms.
[0029] In one embodiment of the present invention, a fraction of
Melissa officinalis leaf extract was prepared by suspending 75%
ethanol extract of the Melissa officinalis leaves in water, then
fractionating with ethyl acetate and drying.
[0030] The fraction of Melissa officinalis leaf extract may be an
ethyl acetate fraction of ethanol extract of Melissa officinalis
leaves.
[0031] The method has an effect of effectively extracting a
water-soluble material and a water-insoluble material and
extracting the water-insoluble material having a low solubility to
water, but having a high solubility to ethyl acetate, by using 50
to 100% alcohol (v/v).
[0032] Ethyl acetate is selected as a secondary extraction solvent
of Melissa officinalis leaves by considering a yield, toxicity of a
residual solvent, a relative content of a marker compound, etc.
[0033] For mass production of the fraction of Melissa officinalis
leaf extract, the fraction of Melissa officinalis leaf extract may
be prepared in large quantities according to a method of suspending
50 to 100% alcohol extract of Melissa officinalis leaves in water,
then fractionating with ethyl acetate, and then freeze-drying or
hot-air drying.
[0034] The fraction of Melissa officinalis leaf extract has a
content of 20% to 80% by weight with regard to the total weight of
the composition comprising the fraction of Melissa officinalis leaf
extract.
[0035] The pharmaceutical composition of the present invention may
contain the fraction of Melissa officinalis leaf extract as an
effective ingredient and further comprises pharmaceutically
acceptable carriers, and may be formulated to an oral dosage form
such as powder, granule, tablet, capsule, suspension, emulsion,
syrup, aerosol, etc., as well as a form of an external preparation
and a sterile solution for injection according to a conventional
method.
[0036] In the present specification, the term "pharmaceutically
acceptable" refers to a composition which is physiologically
acceptable and does not conventionally cause an allergic response
such as gastrointestinal disturbance, dizziness, etc., or other
responses similar thereto, when administered to humans.
[0037] In the present specification, the term "pharmaceutically
acceptable carrier" typically includes the liquid or non-liquid
basis of a pharmaceutical composition. If the pharmaceutical
composition is provided in liquid form, the carrier will typically
be pyrogen-free water, isotonic saline or buffer (aqueous)
solutions, for example, phosphate, citrate, etc. The injection
buffer may be hypertonic, isotonic or hypotonic with reference to
the specific reference media. In other words, the buffer may have a
higher, identical or lower salt content with reference to the
specific reference media. Preferably, it is possible to use the
concentration of the salt mentioned above, which do not lead to
damage of cells due to osmosis or other concentration effects. The
reference media are for example, liquid occurring in "in vivo"
methods, such as blood, lymph, cytosolic liquid or other body
liquids, or for example, liquids, which may be used as reference
media in "in vitro" methods, such as common buffers or liquids.
Such common buffers or liquids are known to a skilled person.
[0038] The pharmaceutically acceptable carriers may include the
carriers conventionally used in the art, for example, lactose,
dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,
maltitol, starch, acacia rubber, alginate, gelatin, calcium
phosphate, calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl
hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate,
mineral oil and the like, but are not limited thereto.
[0039] Also, the pharmaceutical composition of the present
invention may contain a diluent or an excipient such as filler,
extender, binder, humectant, disintegrant, surfactant, etc., or
other pharmaceutically acceptable additives.
[0040] The pharmaceutical composition of the present invention may
be prepared into forms of liquid, suspension, powder, granule,
tablet, capsule, pill or extract.
[0041] The composition of the present invention may be administered
orally or parenterally (for example, applied or injected
intravenously, subcutaneously or intraperitoneally).
[0042] In the present specification, the term "oral administration"
is a route of administration where the substance for ameliorating a
pathological symptom is taken through the mouth. In the present
specification, the term "parenteral administration" means a route
of administration where the substance is administered
subcutaneously, intramuscularly, intravenously and
intraperitoneally through a tube, excluding administration through
the mouth.
[0043] A solid preparation for oral administration may include
powder, granule, tablet, capsule, soft capsule, pill, etc. A liquid
preparation for oral administration may include a suspension,
liquid for internal use, emulsion, syrup, aerosol, etc., but may
also include various excipients, for example, humectant, sweetening
agent, flavoring agent, preservative, etc. in addition to water and
liquid paraffin, which are commonly used simple diluents.
[0044] A preparation for parenteral administration may be used by
being formulated into a form of external preparation and sterilized
injectable preparation such as sterilized aqueous solution, liquid,
non-aqueous solvent, suspension, emulsion, eye drop, eye ointment,
syrup, suppository, aerosol, etc., according to respective
conventional methods, and may be preferably used by preparing a
pharmaceutical composition of cream, gel, patch, spray, ointment,
plaster, lotion, liniment, eye ointment, eye drop, paste or
cataplasma, but is not limited thereto. A composition for local
administration may be an anhydrous or aqueous form depending on a
clinical prescription. As the non-aqueous solvent and the
suspension, propylene glycol, polyethylene glycol, vegetable oil
such as olive oil, injectable ester such as ethyl oleate, etc., may
be used. As a base of the suppository, witepsol, macrogol, tween
61, cacao butter, laurinum, glycerogelatin, etc., may be used.
[0045] The pharmaceutically acceptable additive according to the
present invention may be contained in an amount of 0.1 to 99.9
parts by weight, specifically in an amount of 0.1 to 50 parts by
weight with regard to the composition, but is not limited
thereto.
[0046] A preferred dosage of the composition of the present
invention may vary depending on in vivo absorption degree of an
active ingredient, a patient's age, sex and degree of obesity, but
may be appropriately selected by a skilled person in the art.
However, in case of oral administration for a preferable effect,
the composition of the present invention may be generally
administered to an adult at a dose of 0.0001 to 40 mg/kg a day,
preferably 0.001 to 30 mg/kg a day.
[0047] The administration may be performed once a day or several
times a day by dividing the preparation. The dosage above does not
limit the scope of the present invention in any aspect.
[0048] Pharmaceutical Composition for Preventing or Treating Otitis
Media with Effusion Comprising a Fraction of Melissa officinalis
Leaf Extract
[0049] The present invention provides a pharmaceutical composition
for preventing or treating otitis media with effusion comprising a
fraction of Melissa officinalis leaf extract.
[0050] In the present invention, the term "otitis media with
effusion (OME)" means a disease in which a middle ear, a space
inside an eardrum of an ear, is filled with liquid called effusion
without any symptoms of earache, fever or the like.
[0051] The pharmaceutical composition for preventing or treating
otitis media with effusion comprising a fraction of Melissa
officinalis leaf extract according to the present invention as an
effective ingredient has an effect of preventing or treating otitis
media with effusion in such a way that middle ear effusion (MEE) is
ameliorated, the effusion is reabsorbed and thus not observed, a
thickness of a middle ear mucosa is remarkably decreased, and IL-23
and TNF-.alpha. genes are significantly decreased when examining
the expression of IL-23 and TNF-.alpha. genes.
[0052] The pharmaceutical composition for preventing or treating
otitis media with effusion comprising a fraction of Melissa
officinalis leaf extract of the present invention as an effective
ingredient may be administered in a conventional manner orally,
rectally, intravenously, intra-arterially, intraperitoneally,
intramuscularly, intrasternally, percutaneously, locally,
intraocularly or through an intradermal route, and specifically may
be orally administered.
[0053] It is preferable that the pharmaceutical composition for
preventing or treating otitis media with effusion comprising a
fraction of Melissa officinalis leaf extract of the present
invention as an effective ingredient may be administered at a dose
of 0.001 to 40 mg/kg when administered once to several times a
day.
[0054] The fraction of Melissa officinalis leaf extract is the same
as described above.
[0055] Prevention and Treatment Method
[0056] Another objective of the present invention is to provide a
method for preventing or treating otitis media with effusion by
administering a therapeutically effective amount of a fraction of
Melissa officinalis leaf extract to the subject in need
thereof.
[0057] In the present specification, the term "subject in need for
treatment" means mammals such as monkey, cow, horse, dog, cat,
rabbit, rat, mouse, etc., and in particular includes humans. The
term "administration" means providing a desired substance to a
patient through any appropriate method.
[0058] In the treatment method of the present invention, the
pharmaceutical composition for preventing or treating otitis media
with effusion comprising a fraction of Melissa officinalis leaf
extract of the present invention as an effective ingredient may be
administered in a common manner through orally, rectally,
intravenously, intra-arterially, intraperitoneally,
intramuscularly, intrasternally, percutaneously, locally,
intraocularly or intradermal route, and specifically may be orally
administered.
[0059] In the present specification, the term "therapeutically
effective amount" means an amount of an effective ingredient or a
pharmaceutical composition which induces a biological or medical
response in a tissue system, animal or human being considered by a
researcher, veterinarian, physician or other clinician, and which
includes an amount that induces a relief of the symptoms of disease
or disorder to be treated. It is apparent to those skilled in the
art that the therapeutically effective dosage and the number of
administration for effective ingredient of the present invention
may vary depending on a desired effect. Thus, an optimal dosage to
be administered may be easily determined by those skilled in the
art, and may be adjusted depending on various factors including a
type of disease, severity of disease, contents of effective
ingredient and other component contained in a composition, a type
of dosage form, a patient's age, body weight, general health
condition, sex and diet, an administration time, an administration
route, a secretion rate of the composition, a treatment period and
concomitant drugs therewith. In the treatment method of the present
invention, it is preferable for adults that the pharmaceutical
composition for preventing or treating otitis media with effusion
comprising a fraction of Melissa officinalis leaf extract of the
present invention as an effective ingredient may be administered at
a dose of 0.001 to 40 mg/kg when administered once to several times
a day.
[0060] In the present specification, the term "prevention" refers
to any act of inhibiting or delaying otitis media with effusion by
administering the pharmaceutical composition according to the
present invention.
[0061] In the present specification, the term "treatment" refers to
any act of improving or beneficially altering otitis media with
effusion by administering the pharmaceutical composition according
to the present invention.
[0062] Use of Pharmaceutical Composition
[0063] An objective of the present invention is to provide a use of
a pharmaceutical composition comprising a fraction of Melissa
officinalis leaf extract, in the manufacture of a medicament for
preventing or treating otitis media with effusion.
[0064] In one embodiment of the present invention, the
pharmaceutical composition comprising a fraction of Melissa
officinalis leaf extract in the manufacture of a medicament may be
mixed with an acceptable carrier, etc. and further contain other
agents.
[0065] The matters mentioned in the pharmaceutical composition,
treatment method and use of the present invention are applied
equally unless contradictory to each other.
Advantageous Effects of Invention
[0066] The pharmaceutical composition comprising a fraction of
Melissa officinalis leaf extract according to the present invention
as an effective ingredient may prevent or treat otitis media with
effusion in such a way that a middle ear effusion (MEE) is
ameliorated, the MEE is reabsorbed and thus not observed, a
thickness of middle ear mucosa is remarkably decreased, and IL-23
and TNF-.alpha. genes are significantly decreased when examining
the expression of IL-23 and TNF-.alpha. genes.
BRIEF DESCRIPTION OF DRAWINGS
[0067] FIG. 1 is a graph showing the results of an experiment on
the inhibitory effects on NO production in LPS-induced inflammation
in RAW 264.7 cells when treated with an experimental group
(fraction of Melissa officinalis leaf extract according to Example
1) and a control group.
[0068] FIG. 2 is a graph showing the experiment results of
inhibitory effects on IL-6 protein expression level in LPS-induced
inflammation in RAW 264.7 cells when treated with an experimental
group (fraction of Melissa officinalis leaf extract according to
Example 1) and a control group.
[0069] FIG. 3 is a picture showing otitis media with effusion in an
experimental model of SD white rat, which was induced by blocking a
eustachian tube, according to one embodiment of the present
invention.
[0070] FIG. 4 is a picture showing a therapeutic effect (Grade I)
on otitis media with effusion in an experimental model of SD white
rat, which was induced by blocking a eustachian tube, when
administered with an experimental group (fraction of Melissa
officinalis leaf extract according to Example 1), according to one
embodiment of the present invention.
[0071] FIG. 5 is a picture showing a therapeutic effect (Grade 0)
on otitis media with effusion in an experimental model of SD white
rat, which was induced by blocking a eustachian tube, when
administered with an experimental group (fraction of Melissa
officinalis leaf extract according to Example 1), according to one
embodiment of the present invention.
[0072] FIG. 6 is a picture showing results of histopathological
findings on a middle ear cavity removed on a seventh day after
blocking a eustachian tube after treated with an experimental group
(fraction of Melissa officinalis leaf extract according to Example
1) and a control group (0.5% CMC).
[0073] FIG. 7 is a graph showing results of evaluating a thickness
of middle ear mucosa after treated with an experimental group
(fraction of Melissa officinalis leaf extract according to Example
1) and a control group (0.5% CMC).
[0074] FIG. 8 is a graph showing a decrease in IL-23 expression
level when IL-23 genes up-regulated by LPS were treated with an
experimental group (fraction of Melissa officinalis leaf extract
according to Example 1).
[0075] FIG. 9 is a graph showing a decrease in TNF-.alpha. gene
expression level when TNF-.alpha. genes up-regulated by LPS were
treated with an experimental group (fraction of Melissa officinalis
leaf extract according to Example 1).
MODE FOR THE INVENTION
[0076] The terms used herein have been selected from the general
terms that are currently used as widely as possible while
considering functions in the present invention, but this may be
subject to change depending on the intention of those skilled in
the art, precedents, the advent of new technologies, or the like.
In addition, in certain cases, some terms are arbitrarily selected
by the applicant. In this case, their meanings will be described in
detail in the description of the corresponding invention. Thus, the
terms used in the present invention should not be defined simply by
the names of the terms, but should be defined based on the meanings
of the terms and the contents provided throughout the present
invention.
[0077] All the terms used herein including technical or scientific
terms have the same meaning as commonly understood by those
ordinary skilled in the art, to which the present invention
pertains, unless defined otherwise. Such terms as those defined in
a generally used dictionary are to be interpreted to have the
meanings equal to the contextual meanings in the relevant art, and
are not to be interpreted to have ideal or excessively formal
meanings, unless clearly defined in the present application.
[0078] A numerical range includes the numerical value defined in
the above range. All the maximum numerical limits given throughout
the present specification include all the lower numerical limits
thereof as if the lower numerical limits are clearly written. All
the minimum numerical limits given throughout the present
specification include all the higher numerical limits thereof as if
the higher numerical limits are clearly written. All the numerical
limits given throughout the present specification will include all
the narrower numerical ranges thereof within a broader numerical
range, as if the narrower numerical limits are clearly written.
[0079] The examples and the preparation examples are provided for
better understanding of the present invention. The following
examples and preparation examples are provided only for the purpose
of illustrating the present invention, and thus the present
invention is not limited thereto.
EXAMPLE
Example 1. Preparation of a Fraction of Melissa officinalis Leaf
Extract
[0080] 200 kg of dried Melissa officinalis leaves were extracted
with reflux using 75% ethanol (v/v) in an amount of ten times by
weight for 4 hours, then filtered through a cartridge filter, and
then primarily concentrated under reduced pressure. An equal amount
of ethyl acetate was added to the obtained concentrate and the
process of fractionation was repeated twice. Then, an ethyl acetate
layer was collected, and secondarily concentrated under reduced
pressure, and dried, and then pulverized to obtain an ethyl acetate
fraction of ethanol extract of Melissa officinalis leaves (yield:
8.2 kg).
Experimental Example 1. Inhibition of NO Production and Protein
Expression Level
[0081] (1) Experimental Method
[0082] The fraction of Melissa officinalis leaf extract according
to Example 1 was evaluated on the inhibition of NO production and
the protein expression level of interleukin-6 (IL-6) in RAW 264.7
cells, which are murine macrophage cell lines stimulated by
LPS.
[0083] With regard to evaluating the effect of the fraction of
Melissa officinalis leaf extract according to Example 1 on the
inhibition of NO production in RAW 264.7 cells stimulated by LPS,
the RAW 264.7 cells were adjusted to 1.times.10 .sup.6 cells/mL,
inoculated into a 24 well plate, and cultured at 37.degree. C. 5%
CO.sub.2 incubator for 2 hours. After that, the cells were treated
with the fraction of Melissa officinalis leaf extract according to
Example 1 at 0, 25, 50, 75 and 100 ug/mL, then stimulated by LPS at
1 ug/mL in one hour later, and then main cultured for 24 hours. The
obtained supernatant and Griess reagent (1% sulfanilamide+0.1%
naphthylendiamine dihydrochloride, 1:1) were subjected to a
reaction at 1:1 at room temperature for 10 minutes, and the
absorbance was measured at 540 nm by using a microplate reader.
[0084] With regard to evaluating the effect of the fraction of
Melissa officinalis leaf extract according to Example 1 on the
protein expression level of interleukin-6 (IL-6) in RAW 264.7 cells
stimulated by LPS, an amount of interleukin-6 (IL-6) secreted into
the cell culture medium of RAW 264.7 cells was measured by using an
ELISA kit (Mouse ELISA set, R&D Systems Inc.). To obtain the
cell culture medium, the RAW 264.7 cells were adjusted to
5.times.10.sup.6 cells/mL, and inoculated into a 24-well plate, and
cultured for 2 hours, and then treated with the fraction of Melissa
officinalis leaf extract according to Example 1 at each
concentration of 0, 25, 50, 75 and 100 ug/mL as well as LPS at 1
ug/mL. After that, a main culture was performed for 24 hours, and
then supernatant was obtained through centrifugation. For the
enzyme-linked immunosorbent assay (ELISA), an anti-mouse IL-6 was
seeded in a microplate as a capture antibody, and subjected to
coating overnight at room temperature, and washed with wash buffer
(#WA126, R&D Systems Inc.), and subjected to blocking with
reagent diluent (#DY995, R&D Systems Inc.), and then washed
with wash buffer. Then, the cell culture supernatant was seeded in
each microplate well and subjected to a reaction at room
temperature for 2 hours. After the reaction, washing was performed
with wash buffer, after which a diluted biotinylated anti-mouse
IL-6 detection antibody and streptavidin-horseradish peroxidase
conjugate were added therein and subjected to a reaction at room
temperature for 20 minutes. After that, washing was performed again
with wash buffer, after which a substrate solution (#DY999, R&D
Systems Inc.) was added and subjected to a dark reaction at room
temperature for 20 minutes. Finally, the reaction was ended with a
stop solution (#DY994, R&D Systems Inc.), after which the
absorbance was measured at 450 nm by using a microplate reader.
[0085] (2) Effect on Inhibition of NO Production
[0086] The effect of the fraction of Melissa officinalis leaf
extract according to Example 1 on the inhibition of NO production
was examined through using LPS-induced inflammation model in RAW
264.7 cells.
TABLE-US-00001 TABLE 1 Absorbance (%) Control 26 LPS 1 ug/mL 100
LPS 1 ug/mL + Experimental group 25 ug/mL 32 LPS 1 ug/mL +
Experimental group 50 ug/mL 31 LPS 1 ug/mL + Experimental group 75
ug/mL 34 LPS 1 ug/mL + Experimental group 100 ug/mL 41
[0087] Referring to FIG. 1, it was identified that an amount of
nitric oxide (NO) was increased in the RAW 264.7 cells induced by
LPS, and the NO production was inhibited in a
concentration-dependent manner when those cells were treated with
the fraction of Melissa officinalis leaf extract according to
Example 1. The fraction of Melissa officinalis leaf extract
according to Example 1 effectively inhibited NO production which
was increased in the RAW 264.7 cells by LPS-induced inflammation
model.
[0088] (3) Effect on IL-6 Protein Expression Level
[0089] The effect of the fraction of Melissa officinalis leaf
extract according to Example 1 on the IL-6 protein expression level
was examined through an experiment on the inhibition of IL-6
protein expression level in RAW 264.7 cells by LPS-induced
inflammation model.
TABLE-US-00002 TABLE 2 IL-6 (%) Control 0.00 LPS 1 ug/mL 100.00 LPS
1 ug/mL + Experimental group 25 ug/mL 58.70 LPS 1 ug/mL +
Experimental group 50 ug/mL 19.12 LPS 1 ug/mL + Experimental group
75 ug/mL 14.85 LPS 1 ug/mL + Experimental group 100 ug/mL 15.61
[0090] Referring to FIG. 2, it was identified that IL-6 protein
expression level which was increased by LPS in RAW 264.7 cells was
is decreased in a concentration-dependent manner when treated with
the fraction of Melissa officinalis leaf extract according to
Example 1. The fraction of Melissa officinalis leaf extract
according to Example 1 effectively decreased the IL-6 protein
expression level increased by LPS-induced inflammation in RAW 264.7
cells.
Experimental Example 2. Identification of Therapeutic Effect on
Otitis Media with Effusion
[0091] (1) Experimental Method
[0092] Experiment 1
[0093] In in vitro test, the effect of the fraction of Melissa
officinalis leaf extract according to Example 1 on the expression
of genes encoding inflammatory cytokines, interleukin (IL)-23 and
tumor necrosis factor alpha (TNF-.alpha.) was examined after an
inflammation was induced in human middle ear epithelial cells
(HMEEC) by lipopolysaccharide (LPS).
[0094] Experiment 2
[0095] After incising a skin of male sprague-dawley (SD) white rat,
a hole was made on a bony part of a eustachian tube (ET) to make a
small pore therein, after which dental cement was injected into the
small pore by using a disposable 1 mL syringe.
[0096] An observation was made on all the SD white rates by using
an otoendoscope so as to identify an occurrence of otitis media
with effusion (OME) (see FIG. 3).
[0097] The fraction of Melissa officinalis leaf extract according
to Example 1 was suspended in 0.5% carboxymethyl cellulose (CMC)
and orally administered, while a control group was dosed with 0.5%
CMC only.
[0098] Right after blocking the eustachian tube (ET), 0.5% CMC
(control group, n=11) and the fraction of Melissa officinalis leaf
extract according to Example 1 (experimental group, n=11) were
administered to SD white rats at a dose of 100 mg/kg/day.
[0099] The therapeutic effect, histopathological findings and a
thickness of middle ear mucosa on otitis media with effusion were
evaluated.
[0100] The therapeutic effect on otitis media with effusion was
defined as Grade II (middle ear cavity fully filled with effusion,
indicated as "++"), Grade I (middle ear cavity partially filled
with effusion, indicated as "+") and Grade 0 (no effusion,
indicated as "O"). The therapeutic effect on otitis media with
effusion was identified three times in total. A first
identification was made on the third day after blocking the
eustachian tube, a second identification was made on the fifth day
after blocking the eustachian tube, and a third identification was
made on the seventh day after blocking the eustachian tube by using
an otoendoscope.
[0101] Histopathological findings were obtained by removing the
middle ear cavity on the seventh day after blocking the eustachian
tube, and observing the extracted middle ear tissue.
[0102] A thickness of middle ear mucosa was observed that a bulla
was removed on the seventh day after blocking the eustachian tube,
and fixed in 10% formalin, and subjected to decalcification and
dehydration by using EDTA, and fixed in paraffin, and then
subjected to hematoxylin and eosin (HE) stain and masson-trichrome
stain. The thickness of subepithelial layer was measured using
Image J software by observing 5 places around the most thickened
mucosa of the middle ear promontory.
[0103] (2) Therapeutic Effect on Otitis Media with Effusion
[0104] The therapeutic effect on otitis media with effusion was
determined in the control group (0.5% CMC) and the experimental
group (the fraction of Melissa officinalis leaf extract according
to Example 1).
[0105] Specifically, referring to Table 3, otitis media with
effusion was occurred as Grade II (++) in the control group and the
experimental group from a third day after blocking the eustachian
tube (first identification).
[0106] In the experimental group (the fraction of Melissa
officinalis leaf extract according to Example 1), a loss of middle
ear effusion (MEE) started to be ameliorated from Grade II (++) to
Grade I (+) from a fifth day after blocking the eustachian tube
(see FIG. 4), and was ameliorated to Grade 0 (90.9%) within seven
days in ten out of eleven SD white rats in total (see FIG. 5).
[0107] In the control group (0.5% CMC), however, only two out of
eleven SD white rats in total were improved to Grade 0 (18.1%), and
seven out of eleven rats maintained otitis media with effusion
without improvement (63.6%).
TABLE-US-00003 TABLE 3 Control Experimental Otoendoscopy group
group Remark 1.sup.st identification grade 0 0 0 (on 3.sup.rd day
after grade I 0 0 blocking the grade II 11 11 eustachian tube)
Total 11 11 2.sup.nd identification grade 0 0 5 (on 5.sup.th day
after grade I 3 5 blocking the grade II 8 0 eustachian tube) Acute
otitis 0 1 Excluding media data Total 11 10 3.sup.rd identification
grade 0 2 10 (7.sup.th day after grade I 4 0 blocking the grade II
3 0 eustachian tube) Acute otitis 2 0 Excluding media data Total 9
10
[0108] (3) Histopathological Findings
[0109] Histopathological findings showed that effusion was present
in a middle ear cavity with inflammatory cells in the control group
(0.5% CMC), while effusion was reabsorbed and thus not observed in
the experimental group (the fraction of Melissa officinalis leaf
extract according to Example 1).
[0110] Specifically, the histopathological findings on the middle
ear cavity removed on a seventh day after blocking the eustachian
tube showed that effusion was not absorbed, but present in the
middle ear cavity in the control group (0.5% CMC), while effusion
was absorbed in the experimental group (the fraction of Melissa
officinalis leaf extract according to Example 1) (see FIG. 6).
[0111] (4) Evaluation of Thickness of Middle Ear Mucosa
[0112] In the experimental group (the fraction of Melissa
officinalis leaf extract according to Example 1), the thickness of
middle ear mucosa was remarkably thinner than that of the control
group (0.5% CMC) (see FIG. 7, p<0.05).
[0113] Specifically, inflammatory cells were present inside the
middle ear cavity of the control group (0.5% CMC), while
inflammatory cells were not seen in the experimental group (the
fraction of Melissa officinalis leaf extract according to Example
1), and the middle ear mucosa became significantly thinner in the
experimental group compared to the control group.
[0114] 5) Evaluation of Inflammatory Factors
[0115] IL-23 genes up-regulated by LPS were significantly decreased
in the experimental group (the fraction of Melissa officinalis leaf
extract according to Example 1) (see FIG. 8), and TNF-.alpha. genes
up-regulated by LPS were significantly decreased in the
experimental group (the fraction of Melissa officinalis leaf
extract according to Example 1) (see FIG. 9).
* * * * *