Composition for preventing or treating cachexia and muscle loss containing paeoniflorin

Abstract

The present invention relates to a pharmaceutical composition containing paeoniflorin for preventing or treating cachexia and muscle loss. The pharmaceutical composition containing paeoniflorin can be used to effectively prevent or treat cachexia and muscle loss.

Description

[Technical Field]

[0001] The present invention relates to compositions containing paeoniflorin. [Background technology]

[0002] Cachexia is a common symptom of chronic diseases such as cancer, tuberculosis, AIDS, and chronic obstructive pulmonary disease. It is characterized by persistent loss of appetite and weight loss, resulting in malnutrition, metabolic imbalance, and a catabolic state of the body's metabolism accompanied by loss of muscle and fat. However, unlike other chronic illnesses, cancer patients are prone to not only cachexia but also to side effects of various anticancer treatments used to treat cancer (Fearon K. et al., Lancet Oncol 2011;12(5):489-495).

[0003] Cachexia occurs in 50-80% of cancer patients, including those with gastrointestinal and lung cancer, and its mortality rate reaches 20-30%. Cachexia in cancer patients is characterized by muscle loss and weight loss due to an increased catabolic response caused by inflammatory responses and metabolic changes triggered by various cytokines. These changes reduce the response rate to anti-cancer chemotherapy and radiation therapy, hinder the progress of effective anti-cancer treatment, and reduce the patient's quality of life.

[0004] The pathogenesis of cachexia is known to involve altered neuroendocrine activity, the secretion of various inflammatory cytokines (TNF-α, IL-6, IL-1β), GDF-15, and cancer-specific cachexia factors, resulting in decreased food intake and metabolic changes. Cytokines are proteins secreted directly by tumors or produced by immune cells in response to tumors. They play an important role in immune regulation and contribute to the development of cachexia by causing inflammation, anorexia, muscle loss, and weight loss (Argiles JM et al., Curr. Opin. Clin. Nutr. Metab. Care 1998;1:245-251). Inflammatory cytokines such as TNF-α, IL-6, and IL-1β are known to alter appetite center and gastrointestinal function, resulting in decreased appetite and weight loss (Durham WJ et al., Curr. Opin. Clin. Nutr. Metab. Care 2009;12:72-77).

[0005] In addition, it has recently been discovered that GDF-15 (growth differentiation factor 15), a type of cytokine, is responsible for the loss of appetite and weight loss caused by chemotherapy and tumors, and that inhibiting GDF-15 significantly improves appetite and weight loss (Breen DM et al., Cell Metabolism. 2020;32:938-950).

[0006] Muscle loss is one of the most prominent features of cachexia and is known to be caused by increased protein catabolism and decreased protein synthesis due to excessive activity of various cytokines. Cachexia is a condition that includes muscle loss (sarcopenia), and there is considerable overlap between the two. While the majority of patients with cachexia are exposed to muscle loss (sarcopenia), not all patients who experience muscle loss exhibit cachectic symptoms. Clinically, muscle loss (sarcopenia) can be considered a precursor to cachexia. Among the causes of weight loss in surgical patients, sarcopenia and cachexia are common in elderly patients, and both phenomena are associated with a loss of muscle mass (Ryu, Seung-wan, J. Clin. Nutr. 2017;9:2-6).

[0007] Currently, progesterone preparations (megestrol acetate; megace) and steroids (dexamethasone, prednisone) are used to treat cachexia. However, steroids are only prescribed for short periods of time due to their short-term therapeutic effects and the potential for side effects such as fluid retention, insulin resistance, and adrenal insufficiency when used long-term. Progesterone preparations (megestrol acetate) are approved by the U.S. FDA for use in AIDS patients with anorexia and weight loss, and are currently the most commonly prescribed drug for cancer cachexia patients. They were used as a comparative drug in the present invention. However, they increase adipose tissue rather than muscle tissue, and are associated with side effects such as thromboembolism, edema, erectile dysfunction, uterine bleeding, hyperglycemia, and adrenal insufficiency.

[0008] Patients with cachexia respond poorly to anticancer treatment and experience severe side effects. Anticancer treatments for cancer patients generally include surgery, chemotherapy, and radiotherapy. Surgery is a treatment method that removes the lesion (i.e., cancer). While early-stage cancer can be cured by surgery alone, it is not possible to cure cancer with surgery alone in the mid-stage or later stages. Chemotherapy, radiotherapy, and concurrent therapy commonly eliminate cancer cells through cytotoxic effects. However, due to their low specificity, they also damage normal cells that divide and proliferate rapidly, such as hematopoietic cells and immune cells. Therefore, these treatments can cause side effects such as vomiting, loss of appetite, stomatitis, diarrhea, constipation, fever, infections, leukopenia, thrombocytopenia, anemia, abdominal pain, nephrotoxicity, hepatotoxicity, cardiotoxicity, peripheral neurotoxicity, central neurotoxicity, muscle pain, bone pain, and bone marrow suppression. Cachexia and muscle loss, which occur frequently in cancer patients, reduce the response rate to anti-cancer treatment, complicate the progression of effective anti-cancer treatment, and reduce the quality of life of patients.

[0009] Paeoniflorin has been proven to have anti-inflammatory and antioxidant properties (Tao Y et al., Experimental and Therapeutic Medicine, 2016;11:263-268), preventive or therapeutic effects against infertility (Korean Patent Publication No. 10-2016-0121023), memory-enhancing effects (Korean Patent Publication No. 10-2016-0089930), and fat-reducing effects (Korean Patent Publication No. 10-2011-0138322), but its preventive, ameliorative, or therapeutic effects against cachexia and muscle loss have not been clarified.

[0010] Therefore, there is a need to develop materials that are superior to existing therapeutic agents and safe for the human body in order to treat cachexia and muscle loss that occur in cancer and other diseases. Summary of the Invention [Problem to be solved by the invention]

[0011] One embodiment provides a pharmaceutical composition for preventing or treating cachexia and muscle loss, comprising paeoniflorin as an active ingredient.

[0012] Another embodiment provides a food composition for preventing or ameliorating cachexia and muscle loss, comprising paeoniflorin.

[0013] Yet another embodiment provides an anti-cancer adjunct for preventing or treating cachexia and muscle loss, comprising paeoniflorin. [Means for solving the problem]

[0014] One embodiment provides a pharmaceutical composition for preventing or treating cachexia and muscle loss, comprising paeoniflorin as an active ingredient.

[0015] According to one embodiment, said cachexia may be cancer-induced.

[0016] According to one embodiment, the cachexia may be induced by an anti-cancer drug.

[0017] In one embodiment, the composition may improve a symptom selected from the group consisting of weight loss, muscle mass loss, muscle strength loss, endurance loss, decreased muscle coordination, and increased inflammatory cytokines and GDF-15.

[0018] According to one specific example, the prevention or treatment of cachexia and muscle loss may be achieved by suppressing the increase of any one selected from the group consisting of TNF-α, IL-6, IL-1β, and GDF-15.

[0019] According to one specific example, the paeoniflorin may be derived from any one plant species selected from the group consisting of peony (Paeonia suffruticosa Andrews), red peony (Paeonia lactiflora Pall.), ginseng peony (Paeonia lactiflora var. trichocarpa (Bunge) Stern), naked ginseng peony (Paeonia lactiflora f. nuda Nakai), Chinese 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), thousand peony (Paeonia veitchii Lynch), and other closely related plants of the same genus (Paeoniaceae).

[0020] Another embodiment provides a food composition for preventing or ameliorating cachexia and muscle loss, comprising paeoniflorin.

[0021] Yet another embodiment provides an anti-cancer adjunct for preventing or treating cachexia and muscle loss, comprising paeoniflorin. [Effects of the Invention]

[0022] According to one embodiment, a pharmaceutical composition containing paeoniflorin improves weight loss, muscle mass loss, muscle strength loss, endurance loss, and muscle coordination loss, and inhibits increases in inflammatory cytokines and GDF-15, and is therefore useful for preventing or treating cachexia and muscle loss. [Brief explanation of the drawings]

[0023] [Figure 1] Paeoniflorin was administered to C57BL / 6 mice in which cachexia was induced with cisplatin, and the weights of (A) body weight, (B) gastrocnemius muscle, and (C) quadriceps muscle were measured. [Figure 2] Paeoniflorin was administered to Balb / c mice in which cachexia was induced with cisplatin, and the following data were obtained: (A) body weight, (B) gastrocnemius muscle weight, and (C) quadriceps muscle weight. [Figure 3] Paeoniflorin was administered to C57BL / 6 mice with cisplatin-induced cachexia, and skeletal muscle function was tested. (A) Muscle strength, (B) endurance, and (C) muscle coordination were measured. [Figure 4] Balb / c mice with cachexia induced by cisplatin were administered paeoniflorin and then their skeletal muscle function was tested. (A) Muscle strength and (B) endurance were measured. [Figure 5] These are data obtained by measuring the mRNA levels of MuRF1 and MAFbx in the gastrocnemius muscle by real-time PCR after paeoniflorin was administered to C57BL / 6 mice in which cachexia was induced with cisplatin. [Figure 6] Western blotting data show that the protein levels of (A) MuRF1, (B) MAFbx, (C) MyHC, (D) MyoD, and (E) β-actin were measured in the gastrocnemius muscle of C57BL / 6 mice with cisplatin-induced cachexia after paeoniflorin administration. [Figure 7]After paeoniflorin administration to C57BL / 6 mice with cisplatin-induced cachexia, the intensities of (A) MyHC and (B) MyoD protein bands observed by Western blotting in the gastrocnemius muscle were quantified using ImageJ software (NIH, Bethesda, MD, USA). [Figure 8] Paeoniflorin was administered to C57B / 6 mice with cachexia induced by cisplatin, and the mRNA expression levels of (A) TNF-α, (B) IL-6, (C) IL-1β, (D) IL-1α, (E) CCL2, and (F) CCL5 in the gastrocnemius muscle were measured and compared. [Figure 9] This is the data comparing the protein concentrations of (A) TNF-α, (B) IL-6, and (C) IL-1β in the gastrocnemius muscle of Balb / c mice in which cachexia was induced with cisplatin after paeoniflorin administration. [Figure 10] After paeoniflorin administration to C57B / 6 mice with cisplatin-induced cachexia, (A) p-IKK (phospho-IKK) and (B) p-IκB (phospho-IκB) levels in the gastrocnemius muscle were quantified using ImageJ software analysis of Western blot protein bands. [Figure 11] After paeoniflorin administration to C57B / 6 mice with cisplatin-induced cachexia, IκB levels in the gastrocnemius muscle were quantified using ImageJ software analysis of Western blotting protein bands. [Figure 12] After paeoniflorin administration to C57B / 6 mice with cisplatin-induced cachexia, (A) cytoplasmic NF-κB levels and (B) nuclear NF-κB levels in the gastrocnemius muscle were quantified using ImageJ software analysis of Western blotting protein bands. (C) NF-κB target DNA binding activity in the gastrocnemius muscle was also quantified. [Figure 13]This figure shows the results of measuring plasma concentrations of (A) TNF-α and (B) GDF-15 by ELISA after paeoniflorin administration to C57B / 6 mice with cachexia induced by cisplatin. [Figure 14] Paeoniflorin was administered to C57BL / 6 mice in which cachexia was induced with Lewis lung carcinoma, and the weights of (A), (B) gastrocnemius muscle, and (C) quadriceps muscle were measured. [Figure 15] Balb / c mice with cachexia induced by C26 colon carcinoma were administered paeoniflorin, and then the weights of (A), (B) gastrocnemius muscle, and (C) quadriceps muscle were measured. [Figure 16] The graph shows the results of measuring (A) muscle strength and (B) endurance after administering paeoniflorin to C57BL / 6 mice in which cachexia was induced with Lewis lung carcinoma. [Figure 17] Balb / c mice with cachexia induced by C26 colon carcinoma (Colon-26 carcinoma) were administered paeoniflorin, and then (A) muscle strength and (B) endurance were measured. [Figure 18] 1 shows the results of measuring plasma GDF-15 by ELISA after administration of paeoniflorin to mice in which cachexia was induced with Lewis lung carcinoma. [Figure 19] This is a photographic observation of the effect of paeoniflorin on promoting myotube differentiation of C2C12 myoblasts. [Figure 20] The effect of paeoniflorin on promoting myofiber (myotube) differentiation of C2C12 myoblasts was quantitatively analyzed using the ImageJ program. DETAILED DESCRIPTION OF THE INVENTION

[0024] To achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating cachexia and muscle loss, comprising paeoniflorin.

[0025] As used herein, the term "cachexia" refers to a severe generalized debilitating symptom seen in the terminal stages of cancer, tuberculosis, diabetes, acquired immunodeficiency syndrome (AIDS), and other conditions. It is frequently observed in patients with gastrointestinal cancers, such as stomach cancer, esophageal cancer, and colon cancer, as well as lung cancer. Symptoms include loss of appetite, weight and strength loss, muscle loss, anemia, lethargy, and indigestion. In particular, cachexia can cause difficulty in maintaining normal dietary intake due to a loss of appetite, or weight and muscle loss despite normal dietary intake. Cachexia can lead to poor responses to anticancer chemotherapy and radiation therapy, resulting in a decline in patients' quality of life and a shortened life expectancy, and is responsible for the deaths of 20-30% of all cancer patients.

[0026] As used herein, the term "muscle loss" refers to a loss of muscle tissue within the body, such as loss of muscle tissue due to disuse, loss of muscle tissue due to muscle disease, or loss of muscle tissue due to damage to the nerves that control the muscles, and such muscle loss can occur, for example, due to cachexia.

[0027] According to one embodiment of the present invention, the "paeoniflorin" may be a compound represented by the following chemical formula 1: [ka]

[0028] According to one embodiment of the present invention, the cachexia is induced by cancer, or by an anticancer drug used in cancer treatment, such as, but not limited to, cisplatin, doxorubicin, irinotecan, paclitaxel, daunorubicin, docetaxel, and 5-fluorouracil.

[0029] The paeoniflorin used as the active ingredient in this composition was extracted from the peony (Paeoniae Radix). Paeoniae Radix corresponds to the specifications of the Korean Pharmacopoeia and is made from the root of the peony (Paeonia lactiflora P. all.), a plant in the Paeoniaceae family. It is known to have antibacterial properties, inhibiting the growth of Staphylococcus aureus and other bacteria, and to be effective against abdominal pain and diarrhea, but its effects in inhibiting muscle loss and improving cachexia are unknown.

[0030] According to one embodiment, the paeoniflorin may be isolated from the aqueous or ethanol extract of Paeonia lactiflora Pall.

[0031] Paeoniflorin is derived from tree peony (Paeonia suffruticosa Andrews), red peony (Paeonia lactiflora Pall.), Chinese peony (Paeonia lactiflora var. trichocarpa (Bunge) Stern), naked peony (Paeonia lactiflora f. nuda Nakai), Chinese peony (Paeonia lactiflora f. pilosella Nakai), white peony (Paeonia japonica (Makino) Miyabe & Takeda), white haired peony (Paeonia japonica var. pillosa Nakai), mountain peony (Paeonia obovata Maxim), and thousand peony (Paeonia veitchii). Lynch), and other closely related plants of the same genus (Family Paeoniaceae), or preferably, isolated from an extract of red peony.

[0032] According to one embodiment of the present invention, the red peony extract can be prepared by a conventional method known in the art, for example, by crushing the peony, adding a solvent commonly used for extraction, and extracting at an appropriate temperature and pressure to prepare the peony extract.

[0033] According to one embodiment of the present invention, the solvent is selected from the group consisting of distilled water, C1-C4 lower alcohols, hexane, ethyl acetate, chloroform, diethyl ether, dichloromethane, acetone, and mixtures thereof.

[0034] The peony extract of the present invention includes not only extracts obtained by the above-mentioned solvent extraction method but also extracts that have undergone conventional purification processes. For example, fractions obtained by various additional purification methods, such as separation using an ultrafiltration membrane with a certain molecular weight cutoff value or various chromatographic methods (designed for separation by size, charge, hydrophobicity, or affinity), are also included in the peony extract of the present invention. The peony extract may also be prepared in powder form by additional processes such as vacuum distillation and freeze-drying or spray-drying.

[0035] According to one embodiment of the present invention, paeoniflorin can improve symptoms selected from the group consisting of weight loss, muscle mass loss, muscle strength loss, inhibition of muscle fiber differentiation, decreased stamina, decreased muscle coordination ability, and increased levels of inflammatory cytokines and GDF-15, and can therefore be useful for preventing, improving, or treating cachexia and muscle loss. Furthermore, paeoniflorin can significantly improve appetite and body weight by inhibiting the increase of GDF-15, a cytokine recently shown to be responsible for anorexia and weight loss caused by chemotherapy and tumors, and can also promote muscle fiber differentiation, making it useful for preventing, improving, or treating cachexia and muscle loss.

[0036] The pharmaceutical composition may contain, in addition to the paeoniflorin described above, one or more additional pharmaceutically acceptable carriers for administration, and may be formulated to be suitable for pharmaceutical compositions.

[0037] The pharmaceutical composition may be formulated in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, infusions, or injectable solutions. For example, to formulate tablets or capsules, the active ingredient can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, or the like. If desired or necessary, suitable binders, lubricants, disintegrating agents, 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, tragacanth, or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like.

[0038] Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, and, if necessary, other common additives such as antioxidants, buffers, bacteriostats, etc. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0039] The pharmaceutical composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

[0040] The suitable dosage of the pharmaceutical composition of the present invention can be determined depending on factors such as the formulation method, administration method, age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate and reaction sensitivity of the patient.

[0041] According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention may be 1 to 10,000 mg / kg, 1 to 1,000 mg / kg, or 1 to 100 mg / kg, preferably 10 or 20 mg / kg.The daily dosage for humans may be multiplied by the human equivalence dose factor (HED) of 0.081 to obtain 0.081 to 810 mg / kg, 0.081 to 81 mg / kg, or 0.081 to 8.1 mg / kg, preferably 0.81 to 1.62 mg / kg.

[0042] The pharmaceutical compositions of the present invention may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients in a manner readily practiced by those skilled in the art to which the invention pertains.

[0043] Another aspect of the present invention provides a food composition for preventing or ameliorating cachexia and muscle loss, comprising paeoniflorin as an active ingredient.

[0044] The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food, or can be added to various foods, such as beverages, confectioneries, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, chewing gum, ice cream, vitamin complexes, and dietary supplements.

[0045] In addition to paeoniflorin as an active ingredient, the food composition of the present invention may contain ingredients commonly added during food production, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings. Examples of the carbohydrates mentioned above include common sugars such as monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., dextrin, cyclodextrin, etc.), as well as sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavorings (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is made into a drinkable preparation, in addition to the paeoniflorin of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts can be added.

[0046] Yet another aspect of the present invention provides an anti-cancer adjuvant comprising paeoniflorin as an active ingredient.

[0047] The term "anticancer adjuvant" as used herein means an adjuvant that is effective in ameliorating side effects of anticancer drugs during anticancer treatment, including hematopoietic toxicity, loss of appetite, weight loss, etc.

[0048] The anti-cancer adjuvant contains paeoniflorin as an active ingredient and may further contain one or more active ingredients exhibiting the same or similar functions. The anti-cancer adjuvant can be administered orally or parenterally in clinical practice. When administered parenterally, it may be administered by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, intracerebrovascular injection, or intrathoracic injection, and can be used in the form of a general pharmaceutical formulation.

[0049] The anti-cancer adjuvant agents can be used alone or in combination with surgery, radiation therapy, hormonal therapy, chemotherapy and methods using biological response modifiers.

[0050] The daily dose of the anticancer adjuvant may be about 0.0001 to 1000 mg / kg, 0.001 to 100 mg / kg, 0.01 to 100 mg / kg, 0.1 to 100 mg / kg, or 1 to 100 mg / kg, and is preferably 10 or 20 mg / kg.

[0051] The human daily dose is calculated by multiplying this by the human equivalence dose factor (HED) of 0.081, or 8.1 × 10 -6 ~81 mg / kg, 8.1 × 10 -5 ~8.1 mg / kg, or 8.1 × 10 -4 The dose may be up to 8.1 mg / kg, and is preferably 0.81 to 1.62 mg / kg.

[0052] The dosage range is preferably one to several times daily, depending on the patient's weight, age, sex, health condition, dietary requirements, administration time, administration method, excretion rate, and disease severity. The anticancer adjuvant of the present invention can be administered in various parenteral dosage forms during clinical practice. Conventional diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, are used for formulation. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases include witepsol, macrogol, Tween 61, cocoa butter, lauric butter, and glycerol gelatin.

[0053] Yet another aspect of the present invention provides a method for preventing or treating cachexia and muscle loss by administering a pharmaceutical composition comprising paeoniflorin.

[0054] The pharmaceutical composition containing paeoniflorin of the present invention can improve weight loss, muscle loss and fatigue, and can therefore be useful in preventing or treating cachexia and muscle loss.

[0055] The composition containing paeoniflorin of the present invention suppresses an increase in any one selected from the group consisting of TNF-α, IL-6, IL-1β and GDF-15, and can therefore be useful in preventing or treating cachexia and muscle loss.

[0056] Example Hereinafter, one or more embodiments will be described in more detail through the following examples, but these examples are intended to illustrate one or more embodiments and are not intended to limit the scope of the present invention.

[0057] Example 1. Preparation of Materials - Preparation of Paeoniflorin 1-1. Manufacturing of Peony Extract After crushing the roots of red peony (Paeonia lactiflora Pall.), an extraction solvent consisting of 75% ethanol and water (5 times the volume of the crushed material) was added. Extraction was performed three times, for three hours each time, and the extract was then concentrated. After removing the residual ethanol, the concentrate was dissolved by adding an equal volume of water and heating. The solution was fractionated three times by adding an equal volume of ethyl ether to obtain the aqueous layer, which was then concentrated. The concentrate was loaded onto a 200-300 mesh silica gel column, separated using chloroform:acetone (4:1) as the mobile phase, concentrated, and dried.

[0058] 1-2. Isolation of Paeoniflorin The dried material was resuspended in water and injected into a 10 mm diameter x 250 mm long column packed with 4 μm octadecylsilylated silica gel. The mobile phase was a 75:25 volumetric mixture of 0.5% TFA (trifluoroacetic acid) and methanol. Paeoniflorin was detected using a UV detector (230 nm). The mobile phase was run at a rate of 2.5 mL / min, and the main peak detected between 18 and 20 minutes was repeatedly separated. The resulting fractions were distilled under reduced pressure to obtain paeoniflorin. The mass of the resulting paeoniflorin was analyzed using an LCQ mass spectrometer (Finnigan, USA) and qualitative analysis was performed using NMR (Bruker).

[0059] Table 1 below shows the results of mass spectrometry, and Table 2 below shows the results of NMR. From the results, paeoniflorin of Chemical Formula 1 (molecular formula C 23 H 28 O 11 ) could be analyzed. [Table 1] [Table 2]

[0060] The structural formula of paeoniflorin is shown in Chemical Formula 1 below. [ka]

[0061] Example 2. Confirmation of the effect of anticancer drugs on improving cachexia and muscle loss and confirmation of the mechanism of action

[0062] 2-1. Experimental method 2-1-1. Preparation of reagents Anti-IκB (cat#9242), anti-phospho-IκB (Ser32 / 36) (cat#9246), anti-phospho-IKK (cat#2694S), IKK (cat#7218), and anti-NF-κB p65 (cat#8242) primary antibodies and anti-mouse (cat#7076S) and anti-rabbit (cat#7074S) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-MuRF1 (cat#172479) and anti-MAFbx (cat#157596) antibodies were purchased from Abcam (Cambridge, UK). Anti-myosin heavy chain (MyHC) antibody (cat# MAB4470) was purchased from R&D Systems (Minneapolis, MN, USA).

[0063] Anti-myoblast determination protein (MyoD) (cat#377460), anti-β-actin (cat#47778), and anti-TATA box-binding protein (TBP, cat#204) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and Megastrol acetate (MA; Megace) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).

[0064] 2-1-2. Preparation for animal experiments Animal experiments were conducted in accordance with internationally accepted principles for the use and care of laboratory animals, and the study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University, Korea (approval number 2021-00022). Four-week-old female C57BL / 6 and Balb / c mice were purchased from Raon Bio (Yongin, Korea) and maintained at the Chung-Ang University Animal Facility on standard laboratory chow and water ad libitum. Each experimental group consisted of 10 mice, as shown in Tables 3 and 4 below. After a week of acclimation, mice were intraperitoneally injected with 3 mg / kg / day of cisplatin suspended in 200 μL of saline three times every other day. Sham-treated control mice were injected with the same volume of saline under the same regimen. To the cisplatin-treated group, paeoniflorin (Pae) ​​or megestrol acetate (MA) suspended in 200 μL of distilled water was orally administered every day until euthanasia.

[0065] Specifically, the cisplatin-treated groups were orally administered paeoniflorin (prepared in Example 1) at 5 (Pae5 group), 10 (Pae10 group), or 20 (Pae20 group) mg / kg once daily for 9 days. The comparative drug treatment group was orally administered 160 mg / kg of megestrol acetate (MA), the most commonly prescribed drug for cancer cachexia patients in clinical practice (800 mg daily human dose / 60 kg average human body weight × 12 (dose equivalent factor of mouse body surface area to human)), once daily for 9 days.

[0066] Cisplatin-treated mice orally administered the same volume of distilled water served as a negative control (vehicle group). After drug treatment, mice in each group were euthanized with 10 mg / kg alphaxalone (Jurox, Rutherford, Australia). Blood was collected in EDTA-containing tubes and centrifuged to separate plasma. Gastrocnemius and quadriceps skeletal muscles were dissected and weighed. Skeletal muscles were quickly frozen in liquid nitrogen for use in RT-qPCR, Western blot, and ELISA.

[0067] [Table 3] [Table 4]

[0068] 2-1-3.Skeletal muscle function test Skeletal muscle function was measured one day before euthanasia. Muscle strength was measured by forelimb grip strength, and endurance was measured by the time the mice could run on a treadmill until exhaustion. Muscle coordination was measured by the time they could stay on a rotating cylinder with a diameter of 3 cm (Castro and Kuang, 2017). Grip strength for each mouse was measured using a grip strength measuring device (Bio-GS3, Bioseb, Vitrolles, France). Running time for each mouse was measured using a treadmill (Exer-3 / 6, Columbus Instruments, Baltimore, MD, USA). The ability of each mouse to stay on the rotating cylinder was measured using a rotarod apparatus (Panlab, Barcelona, ​​Spain).

[0069] 2-1-4. Measurement of mRNA levels in skeletal muscle Total RNA was isolated from skeletal muscle using Qiazol (Qiagen, Genrmany), and the mRNA levels of inflammatory cytokines and muscle-specific ubiquitin E3 ligases (MuRF1 and MAFbx) were measured using real-time PCR.

[0070] 2-1-5. Measurement of protein concentration TNF-α and interleukin (IL)-6 concentrations were measured using the respective ELISA kits (Invitrogen, Waltham, MA, USA), and IL-1β and GDF-15 concentrations were measured using the respective Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA).

[0071] 2-1-6. Western Blot Proteins were extracted from skeletal muscle using a NucleoSpin kit (Macherey-Nagel, Düren, Germany) and quantified using a BCA protein analysis kit (Pierce, Rockford, IL, USA). Anti-β-actin antibodies were used to quantify protein loading. Protein levels in gastrocnemius muscle were measured by Western blot for myosin protein (MyHC, myosin heavy chain), muscle genic factor (MyoD), and NF-κB signaling pathway (p-IKK, IKK, p-IκB, IkB). Band intensity was quantified using ImageJ software (NIH, Bethesda, MD, SUA).

[0072] 2-1-7. Measurement of nuclear and cytoplasmic NF-κB levels and target DNA binding activity Nuclear and cytoplasmic lysates were prepared from skeletal muscle using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA). Protein concentrations of the two lysates were measured using a BA protein analysis kit (Pierce). 10 μg of glycoproteins from the lysates were analyzed by Western blotting using anti-NF-κB p65 as the primary antibody. TATA box-binding protein (TBP) and β-actin were used as controls for the nuclear and cytoplasmic lysates, respectively. NF-κB target DNA binding activity was measured using a TransAM NF-κB ELISA kit (Active Motif, USA).

[0073] 2-1-8. Statistical analysis All data were obtained from a minimum of three replicate experiments and are presented as mean ± standard deviation. Statistically significant differences between experimental groups were assessed using unpaired t-tests. A p-value of <0.05 was considered statistically significant. All analyses were performed using SPSS version 18.0 software (SPSS Inc., Chicago, IL, USA).

[0074] 2-2. Confirmation of the effect of paeoniflorin on body weight and muscle mass recovery in cisplatin-treated mice As shown in Figure 1(A), the negative control group (Vehicle) had significantly lower body weight than the control group (Control) in the C57BL / 6 mouse experiment. However, when comparing the results of the Pae10 and Pae20 groups, which were administered paeoniflorin, it was confirmed that cisplatin-induced weight loss was suppressed by paeoniflorin treatment.

[0075] As shown in Figure 1(B) and Figure 1(C), skeletal muscle mass, measured by the weight of the gastrocnemius and quadriceps, was significantly lower in the cisplatin-treated mice than in the control group. However, oral administration of paeoniflorin to the cisplatin-treated mice resulted in recovery of skeletal muscle mass. *P<0.05, **P<0.01, ***P<0.001 (same below).

[0076] Similar results were confirmed in the Balb / c mouse experimental groups. As shown in Figure 2(A), 2(B), and 2(C), the body weight, gastrocnemius muscle, and quadriceps muscle weights of mice in the Pae5, Pae10, and Pae20 groups administered paeoniflorin were increased compared to the negative control group.

[0077] 2-3. Confirmation of the effect of paeoniflorin on skeletal muscle function recovery in cisplatin-treated mice Skeletal muscle function was measured in C57BL / 6 mice by measuring muscle strength, endurance, and coordination through grip strength and treadmill running time and maintenance time on the rotarod.

[0078] As shown in Figure 3, skeletal muscle function was significantly decreased by cisplatin administration. However, oral administration of paeoniflorin significantly restored skeletal muscle function in cisplatin-treated mice.

[0079] Furthermore, as shown in Figure 4, paeoniflorin was also confirmed to restore muscle strength and endurance in Balb / c mice in a similar manner.

[0080] 2-4. Confirmation of the effects of paeoniflorin on muscle-specific ubiquitin E3 ligase inhibition and recovery of skeletal muscle contraction and differentiation-related proteins To measure the mRNA and protein levels of key factors related to muscle loss, muscle contraction, and muscle formation, we measured the mRNA and protein levels in skeletal muscle tissue of C57BL / 6 mice using real-time PCR and Western blotting.

[0081] As shown in Figure 5, the mRNA levels of muscle-specific ubiquitin E3 ligases (MuRF1 and MAFbx) increased in the negative control group (Vehicle) but decreased in the penioflora-treated groups (Pae10 and Pae20). As shown in Figure 6(A) and Figure 6(B), the protein levels of MuRF1 and MAFbx also increased in the negative control group (Vehicle) but decreased in the penioflora-treated groups (Pae10 and Pae20).

[0082] MuRF1 and MAFbx are known to induce the ubiquitination and proteolysis of myosin heavy chain (MyHC), which plays an important role in muscle contraction, and myoblast-determining protein (MyoD), a key transcription factor involved in muscle formation (Bodine & Baehr, Am J Physiol Endocrinol Metab, 307:E469-E484, 2014).

[0083] As shown in the Western blots in Figures 6(C) and 6(D) and in Figure 7, the protein levels of MyHC and MyoD were confirmed to be decreased in skeletal muscle of the negative control group (Vehicle) compared to the control group (Control). In contrast, the protein levels of MyH and MyoD were confirmed to be increased in the paenioflorine-treated groups (Pae10, Pae20) compared to the negative control group (Vehicle).

[0084] The increase in intramuscular levels of MyHC may be directly responsible for the increased muscle function, such as increased grip strength and treadmill running time, caused by paeoniflorin, and the increase in intramuscular levels of MyoD may be directly responsible for the increased muscle mass caused by paeoniflorin.

[0085] The above results suggest that the decrease in MuRF1 and MAFbx and the increase in MyoD and MyHC are involved in the mechanism of the improving effect of paeoniflorin on muscle mass and muscle function.

[0086] 2-5. Confirmation of the effect of paeoniflorin on reducing inflammatory cytokine and chemokine levels in skeletal muscle Real-time PCR was used to measure the mRNA levels of proinflammatory cytokines such as TNF-α, IL-6, IL-1β, and IL-1α, and chemokines such as CCL2 and CCL5 in skeletal muscle.

[0087] As shown in Figure 8, the results of the experiment using C57BL / 6 mice confirmed that the negative control group (Vehicle) had increased intramuscular inflammatory cytokine and chemokine expression levels, while the paeoniflorin-treated groups (Pae10, Pae20) had decreased intramuscular inflammatory cytokine and chemokine expression levels compared to the cisplatin-treated group.

[0088] Furthermore, as shown in FIG. 9, the results of the experiment in Balb / c mice also confirmed that the effects on the expression of inflammatory cytokines were similarly derived.

[0089] 2-6. Confirmation of the inhibitory effect of paeoniflorin on NF-κB signaling in skeletal muscle To confirm the presence of NF-κB signaling, whole extracts and cytoplasmic and nuclear fractions from skeletal muscle of C57BL / 6 mice were analyzed by Western blotting.

[0090] As shown in Figure 10, the levels of p-IKK (phospho-IKK) and p-IκB (phospho-IκB) were significantly increased in the negative control group (Vehicle) compared to the control group (Control), but the levels of p-IKK (phospho-IKK) and p-IκB (phospho-IκB) were again confirmed to be reduced in the paeoniflorin-treated groups (Pae10, Pae20).

[0091] As shown in FIG. 11, it was confirmed that the IκB level was decreased in the negative control group (Vehicle) compared to the control group (Control), and the IκB level was increased in the paeoniflorin-administered groups (Pae10, Pae20).

[0092] Furthermore, as shown in Figures 12(A) and 12(B), the negative control group showed a decrease in cytoplasmic NF-κB levels and an increase in nuclear NF-κB levels compared to the control group. In contrast, the paeoniflorin-treated groups (Pae10, Pae20) showed an increase in cytoplasmic NF-κB levels and a decrease in nuclear NF-κB levels.

[0093] As shown in FIG. 12(C), the target DNA binding activity of NF-κB, which was increased in the negative control group, was also confirmed to be decreased in the paeoniflorin-administered groups (Pae10, Pae20).

[0094] Therefore, these results suggest that cisplatin activates NF-κB signaling, resulting in muscle wasting, and that paeoniflorin significantly attenuates the NF-κB signaling induced by cisplatin.

[0095] 2-7. Paeoniflorin reduces plasma TNF-α The protein concentration of the inflammatory cytokine TNF-α in the mouse plasma was measured using ELISA. As shown in Figure 13(A), plasma TNF-α levels were significantly increased in the cachexia-induced group compared to the control group. In contrast, plasma TNF-α levels were significantly decreased in the cisplatin and paeoniflorin-treated group compared to the cisplatin-treated group within the cachexia-induced group.

[0096] 2-8. Paeoniflorin reduces plasma GDF-15 Plasma GDF-15 protein concentrations were determined using ELISA. As shown in Figure 13(B), plasma GDF-15 levels were significantly increased in the cachexia-induced group compared to the control group. In contrast, plasma GDF-15 levels were significantly decreased in the cisplatin and paeoniflorin-treated group compared to the cisplatin-treated group within the cachexia-induced group.

[0097] Example 3. Confirmation of the effect of improving tumor-induced cachexia and muscle loss 3-1. Experimental animals and experimental methods The experimental animals were 4-week-old female C57BL / 6 mice and Balb / c mice weighing 18-20g. They were supplied by Raon Bio (Yongin, Korea) and were used in the experiment after a week of adaptation in the laboratory animal breeding facility. They were allowed to access solid food and water ad libitum during the experiment, and the temperature of the breeding room was maintained at 20-24°C, the relative humidity at 55-65%, and a 12-hour light / dark cycle.

[0098] The mice were divided into a control group and a cachexia-induced group, and the cachexia-induced group was further divided into a tumor cell injection group, a tumor cell injection and existing drug (Megestrol acetate, MA) administration group, and a tumor cell injection and paeoniflorin (Pae) ​​administration group, with 10 mice per group.

[0099] The control group received a subcutaneous injection of 100 μl of saline, and the cachexia-induced group received a subcutaneous injection of Lewis lung carcinoma cells (C57BL / 6 mice) or C26 colon carcinoma cells (Balb / c mice) at a dose of 106 cells / 100 μl / mouse into the flank of the mice. After two weeks, when significant tumor formation was observed, the tumor cell injection group received distilled water. The tumor cell injection and existing drug administration groups received 160 mg / kg of megestrol acetate (MA), the most commonly prescribed drug for cancer cachexia patients, once daily for nine days. The tumor cell injection and paeoniflorin administration groups received 5 mg / kg (Pae5 group), 10 mg / kg (Pae10 group), and 20 mg / kg (Pae20 group) of paeoniflorin (prepared in Example 1) once daily for nine days. After nine days of administration, the mice's muscle strength was measured using a grip strength testing device (Bioseb, USA), and their endurance was measured using a treadmill device (Columbus Instrument, USA).

[0100] After the experiment, the mice were sacrificed and blood was collected. After removing the tumor, the body weight, gastrocnemius muscle and quadriceps muscle of the hind legs were measured, and GDF-15 in the serum was quantified by ELISA.

[0101] 3-2. Paeoniflorin's effect on improving body weight and muscle mass Paeoniflorin was orally administered to C57BL / 6 mice in which cachexia had been induced with Lewis lung carcinoma, and the mice were then weighed. As a result, as shown in Figure 14(A), the weight of the cachexia-induced group was significantly reduced compared to the control group, and within the cachexia-induced group, the Lewis lung carcinoma-injected and paeoniflorin (Pae)-administered group showed a significant weight increase compared to the Lewis lung carcinoma-injected group.

[0102] Paeoniflorin was orally administered to C57BL / 6 mice with Lewis lung carcinoma-induced cachexia. The mice were then sacrificed and the weights of the gastrocnemius and quadriceps muscles of the hind legs were measured. As shown in Figures 14(B) and 14(C), the weights of the gastrocnemius and quadriceps muscles in the Lewis lung carcinoma-injected and paeoniflorin-treated group were significantly increased compared to the Lewis lung carcinoma-injected group. In this study, megestrol acetate (MA) was used as a positive control drug. Administration of paeoniflorin at 10-20 mg / kg / day demonstrated superior efficacy to administration of megestrol acetate (MA) at 160 mg / kg / day.

[0103] Balb / c mice with cachexia induced by C26 colon carcinoma were orally administered paeoniflorin and weighed. As a result, as shown in Figure 15(A), the weight of the cachexia-induced group was significantly reduced compared to the control group, and within the cachexia-induced group, the C26 colon carcinoma injection and paeoniflorin (Pae) ​​administration group showed a significant increase in weight compared to the C26 colon carcinoma injection group.

[0104] Balb / c mice with cachexia induced by C26 colon carcinoma were orally administered paeoniflorin, and then sacrificed to measure the weights of the gastrocnemius and quadriceps muscles of their hind legs. As a result, as shown in Figure 15(B) and Figure 15(C), it was confirmed that the weights of the gastrocnemius and quadriceps muscles in the C26 colon carcinoma-injected and paeoniflorin-administered group were significantly increased compared to the C26 colon carcinoma-injected group.

[0105] 3-3. Paeoniflorin improves muscle function Paeoniflorin was orally administered for nine days to C57BL / 6 mice injected with Lewis lung carcinoma, and muscle function was measured in terms of strength and endurance. As shown in Figures 16(A) and 16(B), muscle strength and endurance were significantly reduced in the Lewis lung carcinoma-injected group compared to the control group. In contrast, muscle strength and endurance were significantly increased in the Lewis lung carcinoma-injected and paeoniflorin-treated group. Megestrol acetate (MA) was used as the positive control in this study, and administration of paeoniflorin at 10-20 mg / kg / day demonstrated superior efficacy compared to administration of 160 mg / kg / day of megestrol acetate (MA).

[0106] Balb / c mice injected with C26 colon carcinoma were orally administered paeoniflorin for 9 days, and muscle function was measured for strength and endurance. As shown in Figures 17(A) and 17(B), muscle strength and endurance were significantly reduced in the C26 colon carcinoma-injected group compared to the control group. In contrast, muscle strength and endurance were significantly increased in the C26 colon carcinoma-injected and paeoniflorin-administered group.

[0107] 3-4. Paeoniflorin reduces plasma GDF-15 Paeoniflorin was orally administered to tumor-induced mice for 9 days, and blood samples were collected from the mice to measure plasma GDF-15 protein levels using ELISA. As a result, as shown in Figure 18, plasma GDF-15 levels were significantly increased in the Lewis lung carcinoma-injected group compared to the control group. In contrast, serum GDF-15 levels were significantly decreased in the Lewis lung carcinoma-injected and paeoniflorin-administered groups.

[0108] Example 4. Confirmation of the effect of promoting myoblast differentiation into muscle fibers (myotubes) 4-1. Cells and experimental methods C2C12 myoblasts were purchased from ATCC (Manassas, Virginia, USA) and cultured in DMEM medium containing 10% fetal bovine serum. After the cells reached 70% confluence, myofiber differentiation was induced by replacing the medium with DMEM containing 2% horse serum every two days.

[0109] As a negative control, cells were treated with 100 μg / ml of TNF-α, which inhibits myofiber differentiation. The degree of myofiber differentiation was observed by treating cells with paeoniflorin at concentrations of 50 to 200 μM together with TNF-α, and the length of differentiated muscle fibers was measured using the Image J program.

[0110] 4-2. Paeoniflorin promotes muscle fiber differentiation As shown in Figure 19, muscle fiber differentiation was clearly suppressed in the negative control group (TNF-α + Pae 0 μM) compared to the control group (Control). In contrast, treatment with paeoniflorin at concentrations of 50-200 μM together with TNF-α promoted muscle fiber differentiation.

[0111] As shown in Figure 20, paeoniflorin treatment significantly increased muscle fiber length compared to the negative control group (TNF-α + Pae 0 μM), confirming that paeoniflorin promotes muscle fiber differentiation.

[0112] The present invention has been described above with a focus on preferred embodiments. Those skilled in the art will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is defined by the claims, not the above description, and all variations within the scope of the claims should be construed as being within the scope of the present invention.

Claims

1. A pharmaceutical composition for preventing or treating cachexia and muscle loss, comprising paeoniflorin represented by the following formula 1 as an active ingredient: 【Chemistry 1】

2. the cachexia is cancer-induced; The pharmaceutical composition for preventing or treating cachexia and muscle loss according to claim 1.

3. The cachexia is induced by an anticancer drug. The pharmaceutical composition for preventing or treating cachexia and muscle loss according to claim 1.

4. The composition improves a symptom selected from the group consisting of weight loss, muscle mass loss, muscle strength loss, endurance loss, decreased muscle coordination, and increased inflammatory cytokines. The pharmaceutical composition for preventing or treating cachexia and muscle loss according to claim 1.

5. The prevention or treatment of cachexia and muscle loss is achieved by suppressing the increase of any one selected from the group consisting of TNF-α, IL-6, IL-1β, and GDF-15. The pharmaceutical composition for preventing or treating cachexia and muscle loss according to claim 1.

6. The paeoniflorin is a compound of Paeonia suffruticosa Andrews, red peony (Paeonia lactiflora Pall.), ginseng peony (Paeonia lactiflora var. trichocarpa (Bunge) Stern), naked ginseng peony (Paeonia lactiflora f. nuda Nakai), Chinese peony (Paeonia lactiflora f. pilosella Nakai), white peony (Paeonia the active ingredient is derived from any one plant species selected from the group consisting of Paeonia japonica (Makino) Miyabe & Takeda), Paeonia japonica var. pilrosa Nakai, Paeonia obovata Maxim, Paeonia veitchii Lynch, and other closely related plants of the same genus (Peonyaceae family); The pharmaceutical composition for preventing or treating cachexia and muscle loss according to claim 1.

7. The daily dosage of the pharmaceutical composition is 0.81 to 1.62 mg / kg; The pharmaceutical composition for preventing or treating cachexia and muscle loss according to claim 1.

8. A food composition for preventing or improving cachexia and muscle loss, comprising paeoniflorin represented by the following chemical formula 1 as an active ingredient: 【Chemistry 1】

9. An anti-cancer adjuvant containing paeoniflorin represented by the following chemical formula 1 as an active ingredient. 【Chemistry 1】

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