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Title:
Method for treating allergic diseases
United States Patent: 7,935,684
Issued: May 3, 2011
Inventors: Wu; Rong-Tsun
(Taipei, TW)
Assignee: National
Yang-Ming University (Taipei, TW)
Appl. No.: 12/177,728
Filed: July 22, 2008
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Outsourcing Guide
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Abstract
The present invention relates to use of
polysaccharides obtained from Dendrobium for treating an allergic disease
or for reducing airway-remodeling caused by inflammation.
Description of the
Invention
TECHNICAL FIELD
This invention relates to treating allergic diseases with polysaccharides
isolated from Dendrobium.
BACKGROUND
Dendrobium is a genus of tropical orchids widely distributed in Asia,
Europe, and Australia. Some of its species have a long history of use in
traditional Chinese medicine for improving skin quality and vision. It has
also been reported that Dendrobium exhibits antioxidation,
anti-inflammation, and immune-regulation activities. See Satoh et al.,
Biochem. Pharmacol. 51: 339-343, 1996; Yamaguchi et al., Cordyceps
sinensis. Phytoher. Res. 14: 647-649, 2000; Zhao et al., J. Biochem.
Molecular Bio. 40(5): 670-677, 2007; and US 20060251739.
SUMMARY OF THE INVENTION
This invention is based on the unexpected discoveries that polysaccharides
prepared from Dendrobii Herba (i.e., Dendrobium stem) are effective in
treating allergic diseases such as asthma, pollen allergy, and atopic
dermatitis.
Accordingly, the present invention provides a method of treating an
allergic disease (e.g., asthma, pollen allergy, allergic conjunctivitis,
or atopic determitis) by administering (e.g., orally) to a subject an
effective amount of polysaccharides prepared from Dendrobii Herba.
The term "treating" as used herein refers to the application or
administration of a composition including one or more active agents to a
subject, who has an allergic disease, a symptom of the allergic disease,
or a predisposition toward the allergic disease, with the purpose to cure,
heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disease, the symptoms of the disease, or the predisposition toward the
disease. "An effective amount" as used herein refers to the amount of each
active agent required to confer therapeutic effect on the subject, either
alone or in combination with one or more other active agents. Effective
amounts vary, as recognized by those skilled in the art, depending on
route of administration, excipient usage, and co-usage with other active
agents.
In one example, the polysaccharides used to practice the method of this
invention are prepared by a process including at least the following five
steps: i. soaking a first Dendrobii Herba preparation in a first alcohol
(e.g., methanol, ethanol, or a mixture thereof), ii. removing the first
alcohol to produce a second Dendrobii Herba preparation; iii. extracting
the second Dendrobii Herba preparation with water (i.e., pure water or a
suitable water-containing solvent) to obtain an aqueous solution; (iv)
mixing the aqueous solution with a second alcohol (e.g., 50% ethanol by
volume) to allow precipitation of polysaccharides, and (v) collecting the
precipitated polysaccharides.
The present invention further provides a method of reducing
airway-remodeling resulted from inflammation by administrating to a
subject in need thereof an effective amount of the polysaccharides
described above.
Also within the scope of this invention is use of the polysaccharides
described herein for treating an allergic disease or reducing
inflammation-induced airway-remodeling, or for the manufacture of a
medicament for these treatments.
DETAILED DESCRIPTION OF THE INVENTION
Described herein is use of Dendrobii Herba polysaccharides for treating an
allergic disease. "Polysaccharides" are polymers each consisting of a
number of monosaccharides joined by glycosidic bonds. A polysaccharide
molecule includes more than 10 monosaccharides of any types, e.g.,
glucose, fructose, galactose, xylose, or ribose. It can have a linear or a
branched structure.
The polysaccharides used to practice the method of this invention can be
prepared from Dendrobii Herba via any conventional methods. One example
follows. Stems of a Dendrobium plant (e.g., Dendrobium loddigesii,
Dendrobium fimbriatum var. oculatum, Dendrobium chrysanthum, Dendrobium
candidum ex Lindl, or Dendrobium nobil) are collected, dried, and soaked
in an alcohol (e.g., methanol, ethanol, or a mixture thereof) for a
suitable period of time (e.g., 1-12 hours). After removing the alcohol
via, e.g., evaporation, the resultant Dendrobium stems are soaked in pure
water or a suitable water-containing solvent for a sufficient period of
time (e.g., 12-16 hours). The water-containing solvent can be an aqueous
salt (e.g., NaCl or KCl) solution, or a mixture containing >70% (e.g.,
80%, 90%, or 95%) by volume water and <30% by volume a water miscible
solvent (e.g., methanol or ethanol). Insoluble substances are removed via
centrifugation or filtration, resulting in a soluble fraction. This
fraction is then mixed with an alcohol (e.g., 30-70% ethanol) to
precipitate polysaccharides. Optionally, before being mixed with the
alcohol, the water-soluble fraction is diluted in warm water and kept at
50-70.degree. C. for 20-60 minutes. The precipitates are collected by,
e.g., centrifugation, and preferably, dried, to obtained the
polysaccharides to be used in the method of this invention.
The Dendrobii Herba polysaccharides described above can be mixed with a
pharmaceutically acceptable carrier, and optionally with another
therapeutically active agent, to form a pharmaceutical composition. The
carrier in the pharmaceutical composition must be "acceptable" in the
sense that it is compatible with the active ingredient of the composition
(and preferably, capable of stabilizing the active ingredient) and not
deleterious to the subject to be treated. One or more solubilizing agents
can be utilized as pharmaceutical excipients for delivery of the
polysaccharide-containing pharmaceutical composition. Examples of other
carriers include dextrine, silicon oxide, magnesium stearate, cellulose,
sodium lauryl sulfate, D&C Yellow #10, microcrystalline cellulose,
mannitol, glucose, defatted milk powder, polyvinylpyrrolidone, and starch,
or a combination thereof This pharmaceutical composition can then be
presented in a variety of forms, such as tablet, capsule, powder, gel, or
liquid.
The pharmaceutical composition is administered to a subject via suitable
routes, e.g., oral administration, once or multiple times per day or
administered once every several days. A solid formulation for oral
administration can contain suitable carriers or excipients, such as corn
starch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose,
kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium chloride,
or alginic acid. Disintegrators that can be used include, without
limitation, microglycolate, and alginic acid. Tablet binders that can be
used include acacia, methylcellulose, sodium carboxymethylcellulose,
polyvinylpyrrolidone (Povidone3), hydroxypropyl methylcellulose, sucrose,
starch, and ethylcellulose. Lubricants that can be used include magnesium
stearates, stearic acid, silicone fluid, talc, waxes, oils, and colloidal
silica.
This solid formulation can be designed such that the composition is
released in the intestine. For example, the composition is confined in a
solid sub-unit or a capsule compartment that have respectively a matrix or
a wall or a closure comprising an enteric polymer which dissolves or
disperses at the pH of the small or large intestine to release the drug
substance in the intestine.
In another example, the polysaccharides described above is mixed with one
or more edible carriers to form a food product (e.g., yogurt, milk, or soy
milk), or a food supplement (e.g., a nutrient supply or an herbal
product). Examples of an edible carrier include starch, cyclodextrin,
maltodextrin, methylcellulose, carbonmethoxy cellulose, xanthan gum, and
aqueous solutions thereof Such food products can be prepared by methods
well known in the food industry. As used herein, the term "food" broadly
refers to any kinds of liquid and solid/semi-solid materials that are used
for nourishing humans and animals, for sustaining normal or accelerated
growth, or for maintaining stamina or alertness.
The polysaccharides described herein can be used to treat allergic
diseases, e.g., allergic diseases associated with an increased level of
eosinophil, IgE, a chemokine, or a Th2 cytokine. It is well known that an
allergen triggers a cascade of immune responses, including T cell
differentiation to Th2 effector cells; release of Th2 cytokine (e.g.,
IL-4, IL-5, IL-6, and IL-13), production of IgE; and activation of
neutrophils such as eosinophils or mast cells. The immune responses
triggered by an allergen result in various allergic diseases, such as
asthma, pollen allergy, allergic conjunctivitis, and atopic dermatitis.
Asthma is a chronic illness involving the respiratory system. The cardinal
features in humans are airway inflammation, hypersecretion of highly
viscous mucus, and bronchoconstriction. This airway narrowing causes
symptoms such as wheezing, shortness of breath, chest tightness, and
coughing. These features may be triggered by such things as exposure to an
environmental stimulant (or allergen), cold air, warm air, moist air, or
emotional stress. In addition, allergic sensitization is also defined by
production of IgE against environmental antigens, recruitment of
lymphocyte and eosinophil into the lungs and in serum, and inducing the
production of Th2 cytokines or chemokines. Public attention in the
developed world has recently focused on asthma because of its rapidly
increasing prevalence, affecting up to one in four urban children. See
Craig M. Lilly, Journal of Allergy and Clinical Immunology, Vol. 115,
Issue 4, Supplement 1, April 2005, Pages S526-S531.
The same as asthma, allergic conjunctivitis and atopic dermatitis both
result from hypersensitive to allergen, such as pollen; however, the
inflamed region and symptoms are varied. Similarly, these allergic
diseases are also related to excess of Th2 cytokines or chemokines, and
proportion to the number of IgE level and eosinophils in the inflamed
region.
The conjunctiva is a thin membrane that covers the eye. When an allergen
irritates the conjunctiva, common symptoms that occur in the eye include:
ocular itching, eye lid swelling, tearing, photophobia, watery discharge,
and foreign body sensation. The allergen will trigger a typical allergic
progress aforementioned. The study showed that patients limited certain
activities such as going outdoors, reading, sleeping, and driving.
Therefore, treating patients with allergic conjunctivitis can improve
their everyday quality of life.
Atopy, or atopic syndrome, is an allergic hypersensitivity affecting parts
of the body not in direct contact with the allergen. It may involve eczema
(atopic dermatitis), allergic conjunctivitis, allergic rhinitis and
asthma. There appears to be a strong hereditary component. Atopic syndrome
can be fatal for those who experience serious allergic reactions such as
anaphylaxis, brought on by reactions to food or environment. Although
atopy has various definitions, most consistently it is defined by the
presence of elevated levels of total and allergen-specific IgE in the
serum of patient, leading to positive skin-prick tests to common
allergens. Since the twentieth century, many mucosal inflammatory
disorders have become dramatically more common; atopy is a classic example
of such a disease. It now affects 10-20% of children and 1-3% of adults in
industrialized countries, and its prevalence there has more than doubled
in the past thirty years.
The Dendrobii Herba polysaccharides described herein can also be used to
increase the population of T regulatory cells in the intestinal lamina
propria site via, e.g., oral administration. The term "T regulatory cell"
used herein refers to a specialized subpopulation of T cells that act to
suppress activation of immune system and thereby maintain immune system
homeostasis and tolerance to self antigens. Sometimes it also called
suppressor T cells. T regulatory cells come in many flavors, including
those that express the CD8 transmembrane glycoprotein (CD8+ T cells),
those that express CD4, CD25 and Foxp3 (CD4+CD25+ regulatory T cells or "Tregs")
and other T cell types that have suppressive function. In one embodiment,
the T regulatory cells refer to the population which expressed CD4 and
CD25.
The term "intestinal lamina propria" used herein refers to a thin layer of
loose connective tissue which lies beneath the epithelium and together
with the epithelium constitutes the mucosa. In this region, there are many
immune cells retained here, for example, dendritic cells, macrophages, T
cells, B cells or mast cells etc.
In another example, the polysaccharides also can be administrated to a
subject to reduce the subject's IgE levels in serum and BALF. IgE plays a
key role in the pathogenesis of allergy. Like other immunoglobulins, IgE
is produced by B-lymphocytes following exposure of a foreign antigen from
environment. When two or more molecules of IgE bound to the surface of a
cell are simultaneously linked to its specific allergen, the cell will
immediately release preformed inflammatory cytokines including histamine,
which induce the immediate affects or early phase of allergy. These
cytokines recruit eosinophils, macrophages and more basophils to the area.
These newly synthesized substances and the cells they recruit are key
players in the late phase of the allergic response. The close association
between IgE, allergy, and asthma has long been recognized (Platts-Mills T
A E, Am J Respir Crit Care Med, 164:S1-S5, 2001; Milgrom H, et al., N Eng
J Med., 341:1966-73, 1999). In one embodiment, mice fed with the
polysaccharides extracted from Dendrobii Herba had lower level of IgE in
serum and in BALF. Also these mice had fewer eosinophils and cytokines or
chemokines which attracted the immune cells and stimulate airway
epithelium. Therefore, the polysaccharides extracted from Dendrobii Herba
could treat asthma.
In yet another example, the polysaccharides are used to reduce
airway-remodeling resulted from inflammation by orally administrating the
subject with polysaccharides extracted from Dendrobii Herba.
The term "airway remodeling" used herein refers to structural changes that
occur in conjunction with, or because of, chronic airway inflammation.
Airway remodeling results in alterations in the airway epithelium, lamina
propria, and submucosa, leading to thickening of the airway wall.
Consequences of airway remodeling in asthma include incompletely
reversible airway narrowing, bronchial hyper-responsiveness, airway edema,
and mucus hypersecretion.
Without further elaboration, it is believed that one skilled in the art
can, based on the above description, utilize the present invention to its
fullest extent. The following specific embodiments are, therefore, to be
construed as merely illustrative, and not limitative of the remainder of
the disclosure in any way whatsoever. All publications cited herein are
incorporated by reference.
Example 1
Preparation of Polysaccharides from Dendrobii Herba
2 kg of fresh Dendrobii Herba was dried to form the dry material, and then
the dry material was immersed and treated with 20 L of methanol to obtain
a treated plant. The treated plant was treated with a de-methanol process,
and then immersed in water overnight to obtain an overnight water
solution. The overnight water solution was extracted with 24 L warm water
at the temperature ranged 60.degree. C. for 30 min, and then centrifuged
by the centrifuge (ER-RC13 C-124, HITACH) at 5,000 rpm and 10.degree. C.
After the centrifugation, the supernatant was filtered through 6 .mu.m
filter paper. The filtrate was precipitated with 50% (w/v) ethanol to
obtain the polysaccharides.
Example 2
The Effect of Polysaccharides Extracted from Dendrobii Herba on Asthma
Set Up an Animal Model of Asthma
BALB/c male mice were randomly grouped into normal group (n=6, healthy
mice as negative control), the control group (n=10, mice sensitized with
OVA but without the treatment of the polysaccharides extracted from
Dendrobii Herba), the treatment groups sensitized with OVA fed with a dose
of 10 mg/kg/day (n=8), 30 mg/kg/day (n=8) and 90 mg/kg/day (n=8). Except
the normal group, all the mice were immunized with an intraperitoneal
injection of 20 and 50 .mu.g/ml OVA (Sigma, USA) in phosphate-buffered
saline (PBS) combined with the 2 mg and 5 mg Al(OH).sub.3 as adjuvant on
Day 1 and 14, respectively. Negative control mice were given
intraperitoneal injection of 1.times.PBS on each immunization. All mice
were exposed to 1% OVA in PBS for 20 min by ultrasonic nebulizer (Buxco)
on Day 28, 29, 30. On Day 31, all mice were subjected to AHR (Airway
hyperresponsiveness) test by various concentration of methacholine, and
sacrificed the next day (the method was referred to Charles Perkins et
al., J. Allergy Clin. Immunol., 118(2):410-419). On Day 0, 21 and after
AHR test, bloods of the mice were collected from the retro-orbital venous
plexus. The blood samples were tested for OVA-specific antibodies.
Determination of Airway Hyperresponsiveness to Methacholine
Mice were placed, unrestrained, in cylindrical plexiglass plethysmograph
chambers that were connected to a Buxco nebulized control aerosol delivery
system and a Buxco Max II apparatus for analyzing barometric
plethysmography (Buxco Electronics, Sharon, Conn.). Baseline measurements
of enhanced pause (Penh) were made over a 5-minute period. Penh, referring
to a unit-less measure of airflow obstruction, reflects changes in
pulmonary function related to quantitative differences in time and extent
between inspiration and expiration. Specifically, Penh is considered a
parameter that reflects changes in waveform of the measured box pressure
signal that are a consequence of bronchoconstriction and is an index of
airway obstruction. The severity of asthma can be evaluated by the level
of Penh (Zhang et al., J. Allergy Clin. Immunol., February; 113(2):320-6,
2007).
Mice were then challenged for 3 minutes by means of inhalation of
aerosolized, .beta.-methacholine in PBS produced with a nebulizer (Buxco
Electronics, Sharon, Conn.), starting at a methacholine concentration of
6.25 mg/mL. Penh measurements were starting 3 minutes after completion of
exposure to the aerosolized methacholine, and average Penh values for the
3-minute period were calculated. Subsequently, the procedure was then
serially repeated and the concentration of methacholine was changed to
12.5 mg/mL and 25 mg/mL. All the recorded data were normalized to the
baseline measurements, and the results of each group were showing in FIG.
1 (see Original Patent).
AHR contributes significantly to airway obstruction and the cardinal
symptoms in asthma, such as breathlessness, chest tightness, coughing and
difficultly speaking. Accordingly, inhibition of AHR can ease the severity
of asthma. As shown in FIG. 1, all of the three treatment groups exhibited
significantly decreased Penh responses to aerosolized 25 mg/mL
methacholine compared to the control group. It was indicated that the
polysaccharides extracted from Dendrobii Herba could inhibit AHR,
therefore it could treat asthma.
Brochoalveolar Lavage Fluid (BALF) Analysis
"Brocho-alveolar lavage fluid" is the fluid obtained by washout of the
alveolar compartment of the lung. It is used to determine biochemical and
inflammatory changes in the interstitial lung tissue, thereby assessing
the efficacy of the method of this invention.
After cervical dislocation, the trachea were exposed and incubated with a
polyethylene catheter. BALF was collected from the mice by lavaging with 1
mL of PBS through the trachea. After the wash was centrifuged, the
supernatant was used for antibody detection and a total cell count was
obtained from the pellet. Cytospin slides were prepared and stained by
Liu's staining, a modification of Giemsa staining (Sevens, M. L.
Fundamentals of clinical hematology., W.B. Saunders company, 1997).
The cells of BALF from the mice were first stained by addition of 0.5 mL
Liu's A solution (prepared by dissolving 0.18 g Eosin Y and 0.05 g
Methylene blue in 100 mL methanol, followed by filtering the resultant
solution through a No. 3 filter paper) for 30 seconds. Thereafter, 1 mL
Liu's B solution (prepared by dissolving 0.12 g Methylene blue, 0.14 g
Azure B, 2.52 g Na.sub.2HPO.sub.4 and 1.26 g KH.sub.2HPO.sub.4 in 100 mL
H.sub.2O, followed by filtering the resultant solution through a No. 3
filter paper) was added to mix with the Liu's A stain immediately. After a
reaction time of 60 seconds, the slide was washed with water to clean out
the stain solution. The slide were air-dried and then examined under an
optical phase microscope, and the differentials were obtained after
counting 300 cells. The results were showed in FIG. 2 and FIG. 3 (see Original Patent).
As shown in FIG. 2, the number of total cells in 90 mg/kg/day group
compared to the control group had significant decreased. Furthermore, the
percentages of each cell type (eosinophils, neutrophils, monocytes and
lymphocytes) are shown in FIG. 3. Eosinophil and lymphocyte percentages
were significant reduced in the group orally treated with 90 mg/kg/day
Dendrobii Herba polysaccharides compared to the control group.
Without being bound by theory, the polysaccharides obtained from Dendrobii
Herba may be effective in treating asthma through the following mechanism.
It has been reported that eosinophils play the key role in asthma
development. See Walsh G. M., Curr Opin Hematol., 8(1):28-33, 2001; and
Menzies-Gow A et al., J Asthma., 38(8):605-13, 2001. As shown above, the
Dendrobium polysaccharides reduced numbers of eosinophils in the BALF,
thereby alleviating asthmatic symptoms.
Effect of the Polysaccharides Extracted From Dendrobii Herba on
Eosinophilia and Airway Remodeling in Lungs
After BALF was obtained, the lung tissue was fixed in 10% neutral buffered
formalin for 24 h. The lung tissue was embedded in paraffin, and then cut
into 5 .mu.m thickness sections. The slices were stained with H&E solution
(hematoxylin; Sigma MHS-16 and eosin, sigma HT110-1-32). The results were
photographed and showed in FIG. 4 (see Original Patent).
Asthma is characterized by a specific pattern of inflammation in the
airway mucosa, and involves the infiltration of eosinophils, increased
number of T.sub.H2 cells relative to T.sub.H1 cells. In addition, there
are characteristic structure changes to the airway, termed remodeling,
some of which might even precede the development of the disease. These
structural changes seen in asthmatic patients can include thickening of
the airway wall reticular basement membrane, formation of an abnormal
elastic fiber network, alterations in airway cartilage structure,
angiogenesis, and increase in airway smooth muscle mass.
In the present invention, control group obviously displayed the thickening
of the airway epithelium basement and the narrowness in bronchial tubes.
As the dose of Dendrobii Herba polysaccharides treatment went up, so the
features of airway remodeling reduced and the diameter of bronchial tubes
came to nature. Combination with the results in FIG. 2, the present
invention features a method to decrease the number of eosinophil and
lymphocyte, also relief the symptom of airway remodeling.
Oral Treatment of the Polysaccharides Extracted From Dendrobii Herba Can
Increase the Percentage of Treg in Intestinal Lamina Propria
Mice were killed by cervical dislocation. The small intestine was
immediately removed, and flushed in HBSS (with HEPES, Ca.sup.2+ and
Mg.sup.2+-free). Peyer's patchs were carefully dissected out and cut into
segments. To remove the epithelium cells, the segments were shaken with 1
mM dithiothreitol solution (DTT, Amresco, USA) and 1 mM
ethylenediaminetetraacetic acid (EDTA, Sigma, USA) at 37.degree. C. for
one hour. After the incubation, the segments were minced into 1-mm pieces
and digested in RPMI 1640 (Gaithersburg, USA) contained with 30 unit/mL
Collagenase Type I (Sigma, USA) and 10 unit/mL Collagenase Type II (Sigma,
USA) as well as 10% fetal calf serum at 37.degree. C. for one hour. The
digested tissue were passed through a nylon mesh (Small Parts, USA) to
obtain the cells. After thorough washing, the mononuclear cells were
obtained by the centrifugation on Percoll.
The mononuclear cells were adjusted to 1.times.10.sup.6 cells/mL, and
added to the tube to incubated with FITC conjugated CD4 antibody (BD
Biosciences, USA) and PE conjugated CD25 antibody (BD Biosciences, USA)
for 30 min at 4.degree. C. in the dark. The samples were washed twice in
cold PBS and analyzed by two-color cytometry performed with a FACScan
(Becton Dickinson Bioscience, USA). The number of CD4+CD25+ cells divided
by the number of CD4+ cell was the percentage of Treg. The results were
shown in FIG. 5 (see Original Patent). Mice fed with 90 mg/kg/day
Dendrobii Herba polysaccharides had more Treg in the intestinal lamina
propria.
Effects of the Polysaccharides Extracted From Dendrobii Herba on the Serum
and BALF Levels of OVA-specific IgE in the Mice Sensitized With OVA
The serum collected on Day 1, 14 and the day before sacrificed and BALF
were used to detect the OVA-specific IgE by ELISA. The assay was performed
according to the manufacture's direction. Serum samples in duplicate were
diluted to 1:50 and BALF was not diluted. The antibody used here was AP
conjugated goat-anti-mouse IgE bought from Southern BioTech (USA). As
shown in FIG. 6, serum IgE significant decreased in 90 mg/kg/day Dendrobii
Herba polysaccharides group. However, the IgE level of BALF was also on
the downward trend in the same group.
Nowadays, many researches have indicated that IgE is the key molecules in
immediate allergic reactions, because it can form a bridge between
allergens and neutrophils and triggers cellular degranulation, with
liberation of countless preformed mediators and cytokines (Holgate S et
al., J Allergy Clin. Immunol., 115:459-465, 2005; Cooper P J, Parasite
Immunol. 26:455-467, 2004; Milgrom H, et al., ut supra). Therefore,
neutralization or inhibition of IgE synthesis could be a rational option
for the treatment of allergic diseases (Sarinho E and Cruz A A, J Pediatr
(Rio J), 82(5 Suppl):S127-32, 2006; Wagelie-Steffen A L et al., Clin Chest
Med., 27:133-147, 2006; Clark J et al., J Asthma, 43:87-93, 2006). In the
present invention, 90 mg/kg/day Dendrobii Herba polysaccharides could
decrease the level of IgE in serum and in lungs of mice with asthma. In
view of these data, Dendrobii Herba polysaccharides have great potential
to treat allergic disease or asthma.
Effects of the Polysaccharides Extracted From Dendrobii Herba on the Gene
Expression in Lung Tissue From the Mice Sensitized with OVA
The tissue was excision and cut into pieces. Total RNA was then isolated
with rare RNA total RNA isolation reagent (Gsharp Corporation, Taiwan)
according to the manufacture's direction. Reverse transcription used 5 .mu.g
RNA for cDNA synthesis with MMLV reverse transcrptase (Promega, USA) in a
total volume of 40 .mu.l. The portion of the resulting
reverse-transcription product (1 .mu.l) was used to PCR amplification. The
primer sequences used for IL-13, eotaxin-1, IDO, IL-17, and TSLP were
listed as following
-- see Original Patent.
PCR conditions to amplify these genes were 95.degree. C. for 5 min for
initial degeneration and followed by 94.degree. C. for 30 sec, 58.degree.
C. for 30 sec and 72.degree. C. for 1 min. All of the gene expression
level was normalized with .beta.-actin mRNA. As shown in FIG. 7, the
expression level of mRNA of cytokine or chemokine in lungs were lower in
the group fed with 30 mg/kg/day and 90 mg/kg/day polysaccharides extracted
from Dendrobii Herba.
Selective recruitment of eosinophils by some chemokines, such as IL-13,
eotaxin-1, IL-17 and TSLP, into the airways during allergic inflammation
suggests that eosinophil-specific chemoattractant are produced and
released throughout the course of pulmonary inflammation (Rothenberg M E.,
N. Engl. J. Med., 338:1592-1600, 1998; Lacy P et al., Chem. Immunol.,
76:134-155, 1998). Furthermore, it is acknowledged that an increase of IDO,
which was induced by eosinophils, will lead to the apoptosis of Th1 and
promote Th2 polarization. In the result, eosinophil may maintain Th1-Th2
imbalance seen in allergic asthma through expression functionally active
IDO in lymphoid tissue (Odemuyiwa S O et al, J Immunol. Nov. 15, 2004;
173(10):5909-13). Therefore, inhibition of these protein of production is
helpful to block the progression of asthma. In the present invention, mice
with oral treatment of the polysaccharides from Dendrobii Herba had lower
gene expression level mentioned above, which were related to recruit
eosinophils or lymphocytes. Because these two cells played the major role
in the process of allergic disease, especially asthma, reduced the
chemokine could prevent the infiltration in the lungs. Consequently, the
symptoms of allergic disease associated to eosinophil, such as asthma can
be treated.
Claim 1 of 20 Claims
1. A method for treating an allergic
disease, comprising administering to a subject in need thereof an
effective amount of polysaccharides obtained from Dendrobii Herba.
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