WO2023075491A1 - Composition for preventing, alleviating, or treating muscular dystrophy - Google Patents

Abstract

The present invention relates to a composition comprising a peony extract for preventing, alleviating, or treating muscular dystrophy. The composition comprising a peony extract can be used to reduce muscle damage and HMGB1 levels, inhibit the HMGB1-TLR4-NFkB signaling, or lower cytokine and chemokine levels, thereby effectively preventing, alleviating, or treating muscular dystrophy.

Description

Composition for preventing, improving or treating muscular dystrophy

The present invention relates to a composition for preventing, improving or treating muscular dystrophy.

Muscular dystrophy refers to the progression of skeletal muscle degeneration independent of the central nervous system and the peripheral nervous system, resulting in muscle weakness, contracture, and deformation, and pseudohypertrophy or progressive symmetrical muscle atrophy in specific muscles. Muscular dystrophy is classified into several types according to the degree of muscle weakness and genetic pattern, such as Duchenne type, Becker type, limb-girdle muscular dystrophy, and facial scapulohueral dystrophy. ) and the like.

Duchenne muscular dystrophy (DMD) is an X chromosome-linked degenerative muscle disease that affects about 1 in 3,500 men and is characterized by severe inflammation of the muscle and progressive loss of muscle function.

Duchenne muscular dystrophy is mainly caused by mutations in the dystrophin gene, and patients with these mutations mediate the interaction between actin filaments and the extracellular matrix to transmit physical force and organize the muscle cytoskeleton. this will be lacking A lack of dystrophin protein weakens the membranes of muscle cells, making them vulnerable to muscle contraction-induced damage, and repetitive contraction-induced damage leads to chronic muscle inflammation.

Early symptoms of Duchenne muscular dystrophy include difficulty climbing stairs, abnormal gait, and frequent falls. Most patients with Duchenne muscular dystrophy lose the ability to walk between the ages of 6 and 12 and require assisted ventilation by the age of 20. With proper treatment, people can survive to the age of 40, but most patients die between the ages of 20 and 40 from heart failure or respiratory failure.

Duchenne muscular dystrophy is characterized by persistent muscle inflammation. Inflammatory cytokines and chemokines act as major causes of muscle damage in Duchenne muscular dystrophy. These cytokines and chemokines activate NF-kB, a major inflammatory transcription factor, and further up-regulate the expression of cytokines and chemokines to induce muscle inflammation, resulting in loss of muscle fibers. Therefore, controlling muscle inflammation is an important goal in maintaining muscle function in patients with Duchenne muscular dystrophy.

Currently, there is no effective treatment for Duchenne muscular dystrophy, but corticosteroids such as prednisone and prednisolone are used to slow the progression of the disease. Currently, corticosteroids such as prednisolone are the only drugs clinically recommended for Duchenne muscular dystrophy. The most recent guideline for the treatment of Duchenne muscular dystrophy is the continuous use of corticosteroids in patients throughout their life, beginning at the age of 4–5 years. There is evidence that corticosteroids maintain muscle strength and motor function for a period of time and slow the progression of loss of walking function and loss of lung function.

Since the aforementioned corticosteroids are not direct therapeutic agents, they only slow down the progression of the disease, such as the amount of time the patient becomes dependent on a wheelchair. In addition, the average delay in disease progression in patients who take daily corticosteroids is only 2 years. In addition, chronic use of corticosteroids causes various side effects such as high blood pressure, fluid retention, weight gain, and skin atrophy. Therefore, there is an urgent need to develop an effective and safe treatment for muscular dystrophy.

On the other hand, Paeoniaceae Peony ( Paeonia lactiflora Pall.) belonging to the family is used as a treatment for various inflammatory diseases such as rheumatoid arthritis, hepatitis, and systemic lupus erythematosus in many Asian countries such as China, Korea, Japan, Taiwan, and Thailand. However, no study has been conducted on the efficacy of peony in the treatment of muscular dystrophy.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating muscular dystrophy.

In addition, an object of the present invention is to provide a food composition for preventing or improving muscular dystrophy.

A pharmaceutical composition containing a peony extract according to one embodiment improves muscle function, reduces muscle damage, reduces HMGB1 level, inhibits signaling of HMGB1-TLR4-NFkB, or reduces the level of cytokines and chemokines to prevent or improve muscular dystrophy. Or it can be usefully used for therapeutic purposes.

1A and B show that oral administration of water, 30% ethanol, and 70% ethanol extract of peony improved muscle function in mdx mice. The result of normalizing the grip strength measured for each mouse (n=8) to the body weight (A) and the result of measuring the motor coordination ability through the rotarod test (B) are shown. Subsequent experiments were conducted using 30% ethanol extract (PL) of peony.

1C and D show that oral administration of peony 30% ethanol extract (PL) in mdx mice improved muscle function. The result of normalizing the grip strength measured for each mouse (n=8) to the body weight (C) and the result of measuring the motor coordination ability through the rotarod test (D) are shown.

Figure 2 shows that oral administration of PL in mdx mice reduced muscle damage. Figure 2A shows the results of observing degenerative fibers (arrows) by staining the gastrocnemius muscle with hematoxylin and eosin, and Figure 2B shows the results of quantifying plasma creatine kinase activity with a kit.

Figure 3a is a result showing 100 DEPs (differentially expressed proteins) obtained by comparing the gastrocnemius of a wildtype control group (wildtype) and mdx mice administered with distilled water as a volcano plot (volcano plot). One up-regulated DEPs is marked with a red dot, and 99 down-regulated DEPs are marked with a green dot, and a gray dot indicates that DEPs were not selected.

Figure 3b is a result showing nine DEPs obtained by comparing gastrocnemius muscles of mdx mice orally administered with PL and mdx mice administered with distilled water in a Volcano plot. Eight up-regulated DEPs are marked with a red dot, one down-regulated DEPs are marked with a green dot, and a gray dot indicates that DEPs were not selected.

Figure 3c shows the result of identifying 100 DEPs as a heat map by comparing the gastrocnemius of mdx mice with wildtype controls (wildtype) to which distilled water was administered. Up-regulated DEPs are marked in red, and down-regulated DEPs are marked in blue.

Figure 3d shows the result of identifying 9 DEPs as a heat map by comparing gastrocnemius muscles of mdx mice to which PL was orally administered and mdx mice to which distilled water was administered. Up-regulated DEPs are marked in red, and down-regulated DEPs are marked in blue.

Figure 3e is a Venn diagram of DEPs between wild-type control and mdx mice administered with distilled water, and DEPs between mdx mice orally administered PL and mdx mice administered with distilled water. Up-regulated DEPs are marked in red, and down-regulated DEPs are marked in green.

Figure 3f shows the results of analyzing the interaction of HMGB1 using the STRING database.

Figure 4a is a result of examining major proteins of the HMGB1-TLR4-NF-kB signaling pathway in skeletal muscle of mdx mice.

4b and 4c show that the oral administration of PL in mdx mice significantly reduced the protein level (b) and TLR4 level (c) of HMGB1.

4d to 4f show that p-IRAK (d), p-IKK (e), and p-IkB (f) showed higher levels in the muscles of mdx mice than wild-type mice, and oral administration of PL compared to the distilled water administration group. indicates a significant decrease by

Figure 4g shows the level of IkB, an inhibitor of F-kB, decreased in muscle of mdx mice than wild-type mice, but increased by oral administration of PL.

Figure 4h (left) and Figure 4i show that the level of cytoplasmic NF-kB decreased in mdx mice compared to wild-type controls, but increased with oral administration of PL.

Figure 4h (right) and Figure 4j show that the level of nuclear NF-kB increased in mdx mice compared to wild-type controls, but decreased with oral administration of PL.

Figure 4k shows that NF-kB binding to the target DNA sequence was increased in mdx mice compared to wild-type controls, but decreased by oral administration of PL.

5a to 5i show the mRNA levels of cytokines such as IL-6, IL-1β, IL-1α, IFN-γ, and TNF-α and chemokines such as CCL2, CCL3, CCL4, and CCL5 in the gastrocnemius muscle of the wild-type control group. It was increased in mdx mice compared to , but decreased by oral administration of PL.

6a to 6i show the mRNA levels of cytokines such as IL-6, IL-1β, IL-1α, IFN-γ, and TNF-α and chemokines such as CCL2, CCL3, CCL4, and CCL5 in the diaphragm compared to wild-type controls. It was increased in mdx mice compared to , but decreased by oral administration of PL.

7a and 7b show that the levels of TNF-α and IL-6 protein in the gastrocnemius muscle of mdx mice were significantly higher than those of the wild-type control group, but were reduced by oral administration of PL.

7c and 7d show that plasma protein levels of TNF-α and IL-6 in mdx mice were significantly higher than those in wild-type controls, but were reduced by oral administration of PL.

[Correction under Rule 91 29.12.2022]
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8 shows a possible mechanism by which oral administration of PL increases muscle function in mdx mice. Pathological changes in mdx mice compared to wild-type controls are indicated by red arrows, and changes caused by oral administration of PL in mdx mice are indicated by blue arrows.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating muscular dystrophy comprising a peony extract as an active ingredient.

As used herein, the term "muscular dystrophy" refers to degeneration of skeletal muscles that is not related to the central nervous system and the peripheral nervous system, showing muscle weakness, contracture, and deformation, and symmetrical muscular atrophy that comes to specific muscles in pseudohypertrophy or progressively. means to appear

According to one embodiment of the present invention, the peony extract is red peony ( Paeonia lactiflora Pall.), Cham peony ( Paeonia lactiflora var. trichocarpa (Bunge) Stern), mincham peony ( Paeonia lactiflora f. nuda Nakai), ho peony ( Paeonia lactiflora f. pilosella Nakai), white peony ( Paeonia japonica (Makino) Miyabe & Takeda), hairy white peony ( Paeonia japonica var. pillosa Nakai), mountain peony ( Paeonia obovata Maxim), peony ( Paeonia veitchii Lynch) and other closely related plants It may be derived from one or more species selected from the group consisting of (Peonyaceae and Paeoniaceae), and preferably may be derived from dried roots of red peony.

According to one embodiment of the present invention, the red peony extract can be prepared according to a conventional method known in the art. For example, a peony extract may be prepared by pulverizing the peony, adding a solvent commonly used for extraction, and extracting at an appropriate temperature and pressure.

According to one embodiment of the present invention, the peony extract may be extracted with one or more solvents selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixed solvents thereof, preferably 10 to 90, 15 to 85, It may be extracted with 20 to 80, 25 to 75, or 30 to 70%, or 30 or 70% ethanol.

On the other hand, the peony extract includes not only the extract by the above-described solvent extraction method, but also an extract that has undergone a conventional purification process. For example, fractions obtained through various additional purification methods, such as separation using an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. It may also be included in the peony extract of the present invention. In addition, the peony extract may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying.

As used herein, the term "active ingredient" means a component that exhibits the desired activity alone or that can exhibit activity in combination with a carrier that is not active itself.

Muscular dystrophy includes types such as Duchenne type, Becker type, limb-girdle muscular dystrophy, facial scapulohueral dystrophy, dystonia, and Emery-Dreifes.

According to one embodiment of the present invention, the muscular dystrophy is Duchenne muscular dystrophy, Becker muscular dystrophy, Limb-girdle muscular dystrophy, Facioscpulohueral dystrophy, muscular dystrophy It may be one or more types selected from the group consisting of myotonic dystrophy and Emery-Dreifuss muscular dystrophy.

Duchenne type is the most common type, and it occurs most often in boys between the ages of 2 and 4 due to hemi-recessive inheritance. Usually, the signs of Duchenne type include obvious muscle weakness around the age of 5, and pseudohypertrophy (adipose connective tissue, not actual muscle), in which the gastrocnemius muscle is enlarged due to disability-compensatory hypertrophy. Early in the disease, children show Gower's sign, which occurs when rising from a prone position, usually by placing one hand on the thigh and then pushing the hand. It is mainly caused by a mutation of the dystrophin gene, and a significant number of patients have an abnormality on the X chromosome.

Becker's onset usually occurs between the ages of 5 and 20, a little later than Duchenne's, and the progression of the disease is slow. Compared to the Duchenne type, who has myocardial disorders and usually dies in their teens, the Becker type survives into their 20s and does not have myocardial disorders.

Limb-macrodystrophy usually occurs after the age of 20 and corresponds to autosomal recessive muscular dystrophy.

Facial scapulohumeral muscular dystrophy usually begins before the age of 20 and is an autosomal dominant muscular dystrophy with asymmetrical defects and atrophy of the muscles around the eyes, mouth, shoulders, abdominal muscles, upper arms, and lower legs.

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of muscular dystrophy by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities that improve or beneficially change the symptoms of muscular dystrophy by administration of the pharmaceutical composition according to the present invention.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the peony extract described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of a tablet or capsule, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, or water. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc. can be administered.

A suitable dosage of the pharmaceutical composition of the present invention may be determined by factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. .

A typical daily dosage of peony raw materials for humans may be 3 g when the standard human weight is 60 kg, and the dosage of ethanol extract of peony is 50 to 10000 mg/day, 100 to 8000 mg/day, 200 to 6000 mg /day, 300 to 4000 mg/day, 400 to 2000 mg/day, 450 to 1000 mg/day, 500 to 800 mg/day, preferably 600 to 700 mg/day. The dose of the 30% ethanol extract (yield, 22.2%) of peony according to an embodiment of the present invention may be 666mg/day.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container.

According to one embodiment of the present invention, the pharmaceutical composition comprising the peony extract as an active ingredient improves muscle function, reduces muscle damage, reduces HMGB1 level, inhibits HMGB1-TLR4-NFkB signaling or cytokine and chemokine levels By reducing, muscular dystrophy can be prevented or treated.

Another aspect of the present invention provides a method for preventing or treating muscular dystrophy, comprising administering to a subject a pharmaceutical composition containing a peony extract as an active ingredient.

Another aspect of the present invention provides a preventive or therapeutic use of a composition containing a peony extract as an active ingredient, muscular dystrophy.

Another aspect of the present invention provides a use of a composition containing a peony extract as an active ingredient for preparing a medicament for preventing or treating muscular dystrophy, or a food for preventing or improving muscular dystrophy.

In the treatment method and use, the peony, extract, muscular dystrophy, prevention, treatment, improvement, pharmaceutical composition and food are as described above or below.

Another aspect of the present invention provides a food composition for preventing or improving muscular dystrophy comprising a peony extract.

Descriptions common to the foregoing here are omitted in order to avoid excessive complexity.

As used herein, the term "improvement" refers to all actions that at least reduce the parameters related to the condition to be treated, for example, the severity of symptoms. In this case, the food composition may be used before or after the onset of the disease to prevent or improve muscular dystrophy, simultaneously with or separately from a drug for treatment.

According to one embodiment of the present invention, the peony extract is red peony ( Paeonia lactiflora Pall.), Cham peony ( Paeonia lactiflora var. trichocarpa (Bunge) Stern), mincham peony ( Paeonia lactiflora f. nuda Nakai), ho peony ( Paeonia lactiflora f.pilosella Nakai), white peony ( Paeonia japonica (Makino) Miyabe & Takeda), hairy white peony ( Paeonia japonica var. pillosa Nakai), mountain peony ( Paeonia obovata Maxim), peony ( Paeonia veitchii Lynch) and other closely related plants It may be derived from one or more species selected from the group consisting of (Peonyaceae and Paeoniaceae), and preferably may be derived from dried roots of red peony.

According to one embodiment of the present invention, the peony extract may be extracted with one or more solvents selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixed solvents thereof, preferably 10 to 90, 15 to 85, It may be extracted with 20 to 80, 25 to 75, or 30 to 70%, or 30 or 70% ethanol.

According to one embodiment of the present invention, the muscular dystrophy consists of Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy and facial scapulohueral dystrophy. It may be one or more selected from the group.

The food composition according to the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, chewing gum, ice cream, vitamin complexes, and health supplements. .

In addition to the peony extract, the food composition of the present invention may include ingredients commonly added during food preparation, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink or beverage, in addition to the peony extract of the present invention, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included. there is.

According to one embodiment of the present invention, the food composition containing the peony extract improves muscle function, reduces muscle damage, reduces HMGB1 level, inhibits signaling of HMGB1-TLR4-NFkB or reduces the level of cytokines and chemokines to treat muscular dystrophy can be prevented or ameliorated.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of peony extract

Dried roots of Paeonia lactiflora Pall. were used. Red peony dried roots were grown and harvested in Uiseong, Korea, and were purchased from Sunil Oriental Medicine Company (Hongcheon, Korea) (lot # SQ-17053-2-2), and identified and standardized according to the Korean Pharmacopoeia. The dried roots of red peony were put in 30% and 70% ethanol, which are 10 times the volume thereof, and extracted at 65 ° C. for 4 hours. The extract was filtered through a 5 μm filter and concentrated by vacuum evaporation at 45 °C. The concentrated extract was sterilized at 85 °C for 1 hour, and spray-dried to prepare an ethanol extract (PL) of peony. The yield of PL was 22.2%. The main compounds included in PL are paeoniflorin and albiflorin, and information on them is shown in Table 1 below.

information	Peony Florin	albiflorin
molecular formula	C 23 H 28 O 11	C 23 H 28 O 11
Molecular Weight	480.46 g/mol	480.46 g/mol
CAS registration number	23180-57-6	39011-90-0
chemical structure
Content in PL (w/w)	8.9-13.5%	0.8-1.1%

Preparation Example 1. Preparation of other reagents and drugs

Anti-HMGB1 (high mobility group box 1) (cat# 3935), anti-TLR4 (cat# 14358), anti-IRAK1 (cat# 4504), anti-IKKβ (cat# 8943), anti-IKKβ (cat# 8943) used as primary antibodies -p-IKKα/β (Ser176/180) (cat# 2694), anti-IkB (cat# 9242), anti-p-IkB (Ser 32/36) (cat# 9246) and anti-NF-kB p65 ( cat# 8242) and anti-mouse antibody (cat# 7076) and anti-rabbit antibody (cat# 7074) used as secondary antibodies were purchased from Cell Signaling Technology (USA). Anti-p-IRAK1 (Thr209) (cat# ABP54915) antibody was purchased from Abbkine (China). Anti-β-actin (cat# 47778) and anti-TATA box-binding protein (TBP, cat# 204) antibodies were purchased from Santa Cruz Biotechnology, Inc. USA. Prednisolone was purchased from Tokyo Chemical Industry Co., Japan.

Preparation Example 2. Preparation of experimental animals

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Chung-Ang University (approval number 202000095). 4-week-old male mdx mice (C57BL/10ScSn-Dmd ^mdx /J) and wild-type controls (C57BL/10ScSnJ) of the same strain were all purchased from Jackson Laboratory (USA). The mdx mouse is widely used as an animal model for Duchenne muscular dystrophy (DMD), which is characterized by severe decline in muscle function. Prior to the experiment, they went through a 7-day acclimatization period at the Chung-Ang University breeding farm, and were housed in a specific pathogen-free breeding room, four per cage. Light and dark were maintained in a 12-hour cycle. During the adaptation period, solid food and water were freely consumed.

PL or prednisolone was suspended in 200 μL of distilled water and orally administered to mice once a day for 4 weeks.

PL was administered based on human titrated doses. A typical daily dose of peony raw material for humans is 3 g (Monograph, Alternative Medicine Review, 2001, volume 6, 495-499), which is equivalent to 666 mg of the 30% ethanol extract (yield, 22.2%) used in this study. Since the standard weight of a human is 60 kg and the human-equivalent dose (HED) constant for a mouse is 12.3 (Nair and Jacob, J. Basic. Clin. Pharm., 2016, volume 7, 27-31), The HED of the mouse was calculated as 666 mg/60 kg Х 12.3 = 136.5 mg/kg. In the mouse dose range experiment, the central dose, the lowest dose, and the highest dose were set at 100 mg/kg, 50 mg/kg, and 200 mg/kg, respectively. Prednisolone (1 mg/kg/day) was used as a comparator.

Control groups (vehicle-treated groups, vehicle) of mdx mice and wild-type mice were orally administered with only 200 μL of distilled water.

Mice were anesthetized with 10 mg/kg alfaxalone (Jurox, Australia) after oral administration of the drug daily for 4 weeks. Blood was collected in EDTA-coated tubes and centrifuged at 2500g for 10 minutes to obtain plasma.

For muscle analysis, the gastrocnemius and diaphragm of the mouse were separated, rapidly frozen in liquid nitrogen, and then stored at -80°C.

Experimental Example 1. Confirmation of muscle function improvement effect of peony extract

1-1. Experiment method - grip strength test and rotarod test

To evaluate the effect of PL on the muscle function of mdx mice, wild-type mice were orally administered with distilled water (wildtype), mdx mice were administered with distilled water (vehicle), prednisolone was administered at 1 mg / kg (predn), The group administered with PL at 50 mg/kg (PL 50), the group administered at 100 mg/kg (PL 100), and the group administered at 200 mg/kg (PL 200), and each drug was administered orally daily for 4 weeks. administered.

The muscle function of the mice was measured by grip strength test and rotarod test. Forelimb grip strength was measured as an index of muscle strength, and a grip dynamometer (Bio-GS3, Bioseb, France) was used. The motor coordination ability was measured by the rotarod test, using the rotarod device (Panlab, Spain) on a rotating rod with a diameter of 3 cm. ) was measured.

Data are expressed as mean ± standard deviation of the results of three or more replicates. Statistical significance was assessed using an unpaired t test. p < 0.05 was considered to indicate statistical significance (* p < 0.05, ** p < 0.01 and *** p < 0.001). Data analysis was performed using SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA). Data analysis for the subsequent experiment was also performed in the same way.

1-2. Experiment result

The forearm grip strength normalized to body weight of the mdx mice and the latencies in the rotarod test were significantly lower than those of the wild-type control group.

However, mdx mice orally administered PL showed significantly higher grip strength and latencies than mdx mice (vehicle) administered with distilled water in a dose-dependent manner. Referring to FIGS. 1C and 1D , it can be seen that the oral administration of PL effectively improved muscle strength and motor coordination ability of mdx mice.

Experimental Example 2. Confirmation of muscle damage reduction effect of peony extract 1

2-1. Experimental method - Histological analysis of muscle

A portion of the gastrocnemius muscle was fixed in formalin and standard procedures for histopathological examination, such as dehydration, paraffin embedding, and sectioning, were performed. Degenerative fibers are stained basophil by hematoxylin due to leukocyte infiltration and nuclear pyknosis. Degenerative fibers of the treated muscle tissue were stained with hematoxylin and eosin and observed under a microscope.

2-2. Experiment result

Histological examination of the gastrocnemius revealed an increased number of degenerative fibers in mdx mice compared to wild-type controls. However, it was confirmed that the number of degenerative fibers in the muscles of mdx mice was reduced due to oral administration of PL (FIG. 2A).

Experimental Example 3. Confirmation of muscle damage reduction effect of peony extract 2

3-1. Experimental Method - Creatine Kinase Activity Assay

Creatine kinase (CK) activity in the blood is a clinically used biomarker for determining muscle damage. The activity of plasma CK was assayed using a kit (Abcam, UK) (cat# ab155901).

3-2. Experiment result

Plasma CK activity was significantly higher in mdx mice than in wild-type controls. However, oral administration of PL resulted in a significant decrease in plasma CK activity in mdx mice in a dose-dependent manner, confirming that PL reduced muscle damage in mdx mice (FIG. 2B).

Experimental Example 4. Confirmation of protein expression change by peony extract

4-1. Experimental Method - Intramuscular Proteomic Analysis

Proteomic analysis of gastrocnemius muscles (n = 3) was performed in distilled water-administered wild-type control, distilled-water-administered mdx mice, and PL-administered mdx mice (100 mg/Kg). Gastrocnemius tissue was cryo-pulverized in liquid nitrogen.

The protein of the sample was reduced with tris(2-carboxyethyl)phosphine at 37 °C for 30 minutes, and then reduced with iodoacetic acid at 25 °C in the dark. It was alkylated for 18 hours and treated with trypsin (Promega, US) (trypsin: protein = 1: 50 (w/w)) at 37° C. for 18 hours. The digested peptides were separated using the EkspertTM nanoLC 425 system (Eksigent, USA), and the separated peptides were analyzed using the SCIEX TripleTOF 5600+ mass spectrometry system (Agilent Technologies, USA). Mass spectrometry data were processed using the mouse reference spectrum library MouseRefSWATH (PASS01569). Proteome mass spectrometry data were archived by the ProteomeXchange consortium through the PRIDE database with the dataset identifier PXD028886. Data mining and graphic visualization including volcano plots and heat maps were performed using ExDEGA (Ebiogen Inc., Korea). Interaction network analysis identified known or predictable interactions between genes and/or proteins using the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database.

4-2. Experiment result

A total of 581 proteins were identified in the gastrocnemius muscles of all experimental groups. The fold change distribution and p value for 581 proteins in the experimental group were shown as a Volcano plot (FIGS. 3a and 3b). Proteins showing significant differences (fold change > 2 and p < 0.05) among the experimental groups were selected as differentially expressed proteins (DEPs). 100 DEPs were identified through comparison of gastrocnemius muscles of wild type control (wildtype) and mdx mice (mdx) administered with distilled water. Among the 100 DEPs, peroxiredoxin-2 (PRDX2), which reduces hydrogen peroxide and protects cells from oxidative stress, showed higher levels in the wild-type control group than mdx mice, whereas the remaining 99 proteins were low. level was found (Fig. 3c). Nine DEPs were identified as a result of comparing muscle proteomes of mdx mice orally administered PL with those of mdx mice given distilled water (Fig. 3d). Among these 9 DEPs, HMGB1 (high mobility group box 1) showed a lower level in mdx mice (mdx+PL) orally administered PL than in mdx mice (mdx) administered with distilled water, and the remaining 8 showed higher levels. .

Referring to FIG . 3e, the Venn diagram for the two sets of DEPs shows that HMGB1 is the only protein among 9 proteins whose expression level is different between mdx mice orally administered with PL and mdx mice administered with distilled water. It was found that the difference between In addition, compared to the wild-type control group, mdx mice show that the expression level of HMGB1 is only significantly restored by oral administration of PL among 100 proteins with different expression levels. Thus, HMGB1 may play an important role in muscle injury and PL-induced muscle recovery in mdx mice.

Experimental Example 5. Confirmation of effect of peony extract on HMGB1-TLR4-NF-kB signaling

5-1. Experimental method - Western blot

Protein was extracted from the gastrocnemius muscle using the NucleoSpin kit (Macherey-Nagel, Germany; cat# MN740933). Protein concentration was measured using the BCA protein assay kit (Pierce, USA), then 50 μg protein per sample was separated by 12% polyacrylamide gel electrophoresis and electrophoresis on a nitrocellulose membrane at 150 mA for 1 hour 30 minutes ( electrotransfer). The membrane was blocked for 3 hours with phosphate-buffered saline (PBS) containing 5% skim milk and 0.1% Tween-20 at room temperature (22 to 26 ° C). did Thereafter, after reacting with the primary antibody for 16 hours at 4 ° C., it was reacted with the secondary antibody coupled to horseradish-peroxidase (HRP) for 2 hours at room temperature. Protein bands were detected using an ECL kit (Bio-Rad Laboratories Inc., USA). The intensity of protein bands was quantified using Image J software (NIH, USA).

5-2. Experiment result

Signal transduction of HMGB1-TLR4-NF-kB is known to play a role in progressing inflammation. Considering that HMGB1 interacts with NF-kB and TLR4 (Fig. 3f), Western blot analysis showed that HMGB1- TLR4 -HMGB1-TLR4- The protein expression level of the NF-kB signaling pathway was analyzed (Fig. 4a). Although the HMGB1 protein level was higher in mdx mice compared to wild-type controls, it was found to be significantly reduced by oral administration of PL (FIG. 4b). Although TLR4 protein (HMGB1 receptor) was found to be higher in mdx mice than in wild-type controls, oral administration of PL resulted in a significant decrease in TLR4 levels in mdx mice (Fig. 4c). Compared to the wild-type control group, all signaling proteins from TLR4 to NF-kB activated in the muscles of mdx mice were suppressed by oral administration of PL. That is, p-IRAK, p-IKK, and p-IkB, which showed higher levels in the muscles of mdx mice than wild-type controls, were all significantly reduced by oral administration of PL compared to distilled water treatment (FIGS. 4d to 4f). . Oral administration of PL increased the level of IkB, an inhibitor of NF-kB, in muscle (Fig. 4g).

Experimental Example 6. Confirmation of effects of peony extract on NF-kB nuclear translocation and binding to target DNA

6-1. Experimental method - Evaluation of binding of NF-kB to target DNA

Nuclear lysates were collected from gastrocnemius muscles using NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific; cat# 78833), and protein concentrations were determined using the BCA protein assay kit (Pierce). The binding of NF-kB to the target DNA sequence (5'-GGACTTTCC-3') was evaluated using the TransAM NF-kB ELISA kit (Active Motif, cat# 40096). Briefly, 10 μg of nuclear protein extract was added to a 96-well plate coated with oligonucleotides containing the target DNA sequence. After the incubation and washing steps, the NF-kB antibody included in the kit was added to the wells, and then the HRP-conjugated secondary antibody, peroxidase substrate, and stop solution included in the kit were sequentially added. added.

6-2. Experiment result

Compared to wild-type controls, the cytoplasmic NF-kB level decreased in mdx mice (Figs. 4h and 4i), whereas the nuclear NF-kB level increased (Figs. 4h and 4j), suggesting nuclear translocation of NF-kB in muscular dystrophy. translocation) was confirmed. Oral administration of PL has been shown to inhibit nuclear translocation of NF-kB. NF-kB binding to the target DNA sequence was increased in mdx mice compared to wild-type controls as a result of NF-kB nuclear translocation, and NF-kB binding to the target DNA sequence was significantly decreased by oral administration of PL. appeared (Fig. 4k).

Experimental Example 7. Confirmation of effect of peony extract on cytokine and chemokine levels (gene level)

7-1. Experimental method - RT-qPCR

Total RNA was isolated from gastrocnemius and diaphragm tissues using the RNeasy Kit (Qiagen; cat# 74106). 1 μg of total RNA was reverse transcribed using a cDNA reverse transcription kit (Applied Biosystems Inc., cat# 43-688-13). Quantitative polymerase chain reaction (qPCR) reaction was performed according to a known method (Bae et al. Journal of Ethnopharmacology, 2020, 246, 112222). For RT-qPCR reaction, IL(interleukin)-6 (cat# Mm00446190_m1), IL-1β (cat# Mm00434228_m1), IL-1α (cat# Mm00439620_m1), IFN(interferon)-γ (cat# Mm01168134_m1), TNF( tumor necrosis factor)-α (cat# Mm00443258_m1), CCL (C-C motif chemokine ligand)2 (cat# Mm00441242_m1), CCL3 (cat# Mm00441259_m1), CCL4 (cat# 00443111_m1), CCL5 (cat# Mm01302428_m1), and 18S ribosome RNA (cat# Hs99999901_s1) was ordered and used (Assay-on-Demand Gene Expression Products, Applied Biosystems). For each experimental group, the mRNA level was normalized to the 18S ribosomal RNA level, and the ratio of the normalized mRNA level in each experimental group was compared to the wild-type control group using the comparative Ct method.

7-2. Experiment result

NF-kB is a major inflammatory transcription factor, and is known to bind to target DNA sequences located in the promoters of many cytokine and chemokine genes and activate gene transcription. Since PL significantly reduced the binding of NF-kB to its target DNA (Fig. 4k), we measured the mRNA levels of cytokines and chemokines in muscle. The mRNA levels of inflammatory cytokines, such as IL-6, IL-1β, IL-1α, IFN-γ, and TNF-α, and chemokines, such as CCL2, CCL3, CCL4, and CCL5, were significantly higher in the gastrocnemius muscle of mdx mice compared with wild-type controls. appeared to be high. It was confirmed that the mRNA levels of cytokines and chemokines in the gastrocnemius muscle of mdx mice were significantly lowered due to oral administration of PL (FIG. 5).

In addition, cytokine and chemokine mRNA levels in the diaphragm were measured to confirm the same results as gastrocnemius (Fig. 6), through which oral administration of PL improved the levels of cytokines and chemokines in respiratory muscles as well as skeletal muscles in mdx mice. can

Experimental Example 8. Confirmation of effect of peony extract on cytokine and chemokine levels (protein level)

8-1. Experimental method - ELISA

Levels of TNF-α, IL-6, and HMGB1 were measured using ELISA kits (cat# BMS607-3, cat# BMS603-2 from Invitrogen, USA; and cat# EM0382 from FineTest, China, respectively). In order to measure the levels of TNF-α and IL-6 in muscle, the total protein of gastrocnemius muscle was extracted with NucleoSpin Kit (Macherey-Nagel) and then ELISA was measured.

8-2. Experiment result

[Correction under Rule 91 29.12.2022]
TNF-α and IL-6 protein levels in the gastrocnemius muscle showed a pattern similar to that of the mRNA levels (FIGS. 7a and 7b). Plasma levels of inflammatory cytokines were also measured to assess systemic inflammation. Plasma levels of TNF-α and IL-6 in mdx mice were significantly higher than those in wild-type controls and were reduced by oral administration of PL (Figs. 7c and 7d). Plasma HMGB1 levels in mdx mice were also significantly higher than wild-type controls in a pattern similar to muscle, and were reduced by oral administration of PL. These data indicate that oral administration of PL significantly reduces muscle and systemic inflammation in mdx mice.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

According to one embodiment, the pharmaceutical composition comprising the peony extract of the present invention improves muscle function, reduces muscle damage, reduces HMGB1 level, inhibits HMGB1-TLR4-NFkB signaling or reduces the level of cytokines and chemokines to treat muscular dystrophy. Since it can be usefully used for preventive, ameliorative or therapeutic purposes, it has industrial applicability.

Claims (12)

1. Containing peony extract as an active ingredient

   A pharmaceutical composition for preventing or treating muscular dystrophy.
2. The method of claim 1,

   The peony extract is red peony ( Paeonia lactiflora Pall.), Cham peony ( Paeonia lactiflora var. trichocarpa (Bunge) Stern), mincham peony ( Paeonia lactiflora f. nuda Nakai), ho peony ( Paeonia lactiflora f. pilosella Nakai), white peony ( Paeonia japonica (Makino) Miyabe & Takeda), hairy peony ( Paeonia japonica var. pillosa Nakai), mountain peony ( Paeonia obovata Maxim), paeonia veitchii Lynch ( Paeonia veitchii Lynch) and other closely related plants (Paeoniaceae and Paeoniaceae). It is derived from one or more selected species,

   A pharmaceutical composition for preventing or treating muscular dystrophy.
3. The method of claim 1,

   The peony extract is extracted with at least one solvent selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixed solvents thereof,

   A pharmaceutical composition for preventing or treating muscular dystrophy.
4. The method of claim 1,

   The muscular dystrophy includes Duchenne muscular dystrophy, Becker muscular dystrophy, Limb-girdle muscular dystrophy, Facioscpulohueral dystrophy, myotonic dystrophy, and Emery - At least one selected from the group consisting of Emery-Dreifuss muscular dystrophy,

   A pharmaceutical composition for preventing or treating muscular dystrophy.
5. According to any one of claims 1 to 5,

   The prevention or treatment of muscular dystrophy is by improving muscle function, reducing muscle damage, reducing HMGB1 level, inhibiting HMGB1-TLR4-NFkB signaling or reducing cytokine and chemokine levels,

   A pharmaceutical composition for preventing or treating muscular dystrophy.
6. Containing peony extract

   A food composition for preventing or improving muscular dystrophy.
7. The method of claim 6,

   The peony extract is red peony ( Paeonia lactiflora Pall.), Cham peony ( Paeonia lactiflora var. trichocarpa (Bunge) Stern), mincham peony ( Paeonia lactiflora f. nuda Nakai), ho peony ( Paeonia lactiflora f. pilosella Nakai), white peony ( Paeonia japonica (Makino) Miyabe & Takeda), hairy peony ( Paeonia japonica var. pillosa Nakai), mountain peony ( Paeonia obovata Maxim), paeonia veitchii Lynch ( Paeonia veitchii Lynch) and other closely related plants (Paeoniaceae and Paeoniaceae). It is derived from one or more selected species,

   A food composition for preventing or improving muscular dystrophy.
8. The method of claim 6,

   The peony extract is extracted with at least one solvent selected from the group consisting of alcohols having 1 to 4 carbon atoms and mixed solvents thereof,

   A food composition for preventing or improving muscular dystrophy.
9. The method of claim 6,

   The muscular dystrophy includes Duchenne muscular dystrophy, Becker muscular dystrophy, Limb-girdle muscular dystrophy, Facioscpulohueral dystrophy, myotonic dystrophy, and Emery-girdle muscular dystrophy. At least one selected from the group consisting of Emery-Dreifuss muscular dystrophy,

   A food composition for preventing or improving muscular dystrophy.
10. The method of claim 6,

    Prevention or improvement of the muscular dystrophy is due to reduction of muscle damage, reduction of HMGB1 level, inhibition of signaling of HMGB1-TLR4-NFkB or reduction of cytokine and chemokine levels,

    A food composition for preventing or improving muscular dystrophy.
11. A method for preventing or treating muscular dystrophy, comprising administering to a subject a pharmaceutical composition containing a peony extract as an active ingredient.
12. Use of a composition comprising a peony extract as an active ingredient for the prevention or treatment of muscular dystrophy.

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