Meniscus repair agent

Abstract

The present invention aims to provide a formulation capable of repairing the meniscus. [Solution] A formulation containing paeoniflorin, glycyrrhizic acid, and / or liquiritin can repair meniscus damage.

Description

Technical Field

[0001] The present invention relates to a meniscus repair agent.

Background Art

[0002] The meniscus is a tissue shaped like a crescent (C-shaped) between the femur and tibia of the knee joint and plays an important role in facilitating smooth movement of the joint. There are an inner meniscus and an outer meniscus, and blood vessels exist only in the outer edge portion that occupies about 1 / 3 of them, and there are no defects in the remaining portions. When damaged in the portion where blood vessels exist, it may be repaired, but when damaged in the portion where blood vessels do not exist, repair becomes difficult.

[0003] When the meniscus is damaged due to a crack or defect, pain and swelling occur in the knee, and the range of motion is restricted (meniscus injury). Meniscus injury occurs not only due to injuries to sports players but also due to aging. Also, if meniscus injury is left untreated or degeneration or wear occurs in the meniscus due to further aging, the synovium becomes inflamed and pain occurs (osteoarthritis of the knee). Although both meniscus injury and osteoarthritis of the knee are diseases that cause pain in the knee, since the causes, diagnostic methods, and treatment methods are different, if an incorrect treatment method is applied, the condition will deteriorate further (Non-Patent Document 1).

[0004] A typical treatment for meniscus injury is the RICE treatment. In this treatment, based on rest, cooling, compression, and elevation of the affected part are performed.

[0005] For osteoarthritis of the knee, oral administration, external application, injection, and surgery are recognized as useful treatment methods. Among these treatment methods, in the osteoarthritis of the knee treatment guidelines, for oral administration, there is a recommended statement that non-steroidal anti-inflammatory drugs (NSAIDs) are useful because they have short-term analgesic and function-improving effects, and for external application, there is a recommended statement that non-steroidal anti-inflammatory drugs (NSAIDs) have analgesic and function-improving effects (Non-Patent Document 2).

[0006] Glucosamine and chondroitin are used as supplements for osteoarthritis. However, major studies on glucosamine for osteoarthritis of the knee have reported conflicting results, and studies on chondroitin have not shown any usefulness for the pain of osteoarthritis of the knee. Experts have ruled out the possibility that glucosamine and chondroitin are useful for osteoarthritis of the knee, and the American College of Rheumatology (ACR) does not recommend the use of glucosamine and chondroitin in people with osteoarthritis of the knee (Non-Patent Literature 3). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Differences between osteoarthritis of the knee and meniscus tear: symptoms and treatment methods, [online], November 6, 2024, [Retrieved November 24, 2024], Internet<URL:https: / / fuelcells.org / topics / 15271 / > [Non-Patent Document 2] Guidelines for the Treatment of Osteoarthritis of the Knee 2023, supervised by the Japanese Orthopaedic Association, May 2023. [Non-Patent Document 3] What is osteoarthritis?, [online], October 2023, National Center for Complementary and Integrative Health [Retrieved November 24, 2024], Internet <URL:https: / / www.nccih.nih.gov / health / glucosamine-and-chondroitin-for-osteoarthritis-what-you-need-to-know> [Overview of the project] [Problems that the invention aims to solve]

[0008] Nonsteroidal anti-inflammatory drugs (NSAIDs), which are considered effective for osteoarthritis of the knee, have a relatively high frequency of gastrointestinal side effects. Therefore, there is a need for alternative effective medications for osteoarthritis of the knee. Furthermore, oral or topical treatments for osteoarthritis of the knee only suppress inflammation and do not repair worn-down menisci. While the treatments for meniscal tears and osteoarthritis are recognized as different, considering that both involve structural defects in the meniscus, it is thought that a medication capable of meniscal repair could be effectively applied to both conditions. However, to date, no active ingredients capable of meniscal repair have been identified.

[0009] The purpose of this disclosure is to provide a formulation that can repair the meniscus. [Means for solving the problem]

[0010] The inventors of the present invention conducted diligent research to solve the aforementioned problems and discovered that paeoniflorin, glycyrrhizic acid, and liquiritin each have the effect of repairing the meniscus. This disclosure was completed by further research based on this finding.

[0011] In other words, this disclosure provides inventions in the following embodiments. Item 1. A meniscus repair agent comprising paeoniflorin, glycyrrhizic acid, glycyrrhizinate, and / or liquiritin. Item 2. A meniscus repair agent containing peony and / or licorice. Item 3. A meniscal repair agent comprising Keishikajutsuto, Sokeikakketsuto, Keishikoshikuto, Keishibakoshijukuichito, Keishibakoshikukuichito Kashutsu, Shakuyakukanzoto, Reikyojukanto, and / or Shakuyakukanzofushito. Item 4. A meniscal repair agent as described in any of items 1 to 3, applicable to meniscal injuries. Item 5. A meniscal repair agent as described in any of items 1 to 3, applicable to osteoarthritis of the knee. Item 6. A meniscus repair agent described in any of items 1 to 5, used for repairing the portion of the meniscus in which blood vessels are absent. Item 7. A method for meniscus repair, comprising administering an effective amount of paeoniflorin, glycyrrhizic acid, and / or liquiritin to a subject requiring meniscus repair. Item 8. Use of paeoniflorin, glycyrrhizic acid, and / or liquiritin for the manufacture of meniscus repair agents. Item 9. Paeoniflorin, glycyrrhizic acid, and / or liquiritin for use in meniscus repair. [Effects of the Invention]

[0012] According to this disclosure, a formulation capable of repairing the meniscus is provided. [Brief explanation of the drawing]

[0013] [Figure 1] This report shows the results of cartilage matrix production induced by differentiation induction into chondrocytes using test samples (10 μM each) in three-dimensional culture of human bone marrow-derived mesenchymal stem cells, as indicated by the safranin-0 staining ratio. The safranin-0 staining ratio is shown as the mean value for N=2 and error bars (standard deviation). An asterisk (*) indicates p<0.05 compared to water (control). [Figure 2] This report shows the results of cartilage matrix production induced by differentiation into chondrocytes using test samples (10 μg / mL each) in three-dimensional culture of human bone marrow-derived mesenchymal stem cells, as indicated by the safranin-0 staining ratio. The safranin-0 staining ratio is shown as the mean value for N=2 and error bars (standard deviation). An asterisk (*) indicates p<0.05 compared to water (control). [Figure 3] This report shows the results of cartilage matrix production induced by differentiation into chondrocytes using test samples (10 μg / mL each) in three-dimensional culture of human bone marrow-derived mesenchymal stem cells, as indicated by the safranin-0 staining ratio. The safranin-0 staining ratio is shown as the mean value for N=2 and error bars (standard deviation). An asterisk (*) indicates p<0.05 compared to water (control). [Figure 4]The meniscus degeneration scores of a knee joint degeneration mouse model induced by forced running, with each of 9 types of herbal extracts administered, are shown. The meniscus degeneration scores are shown as the mean value and error bars (standard deviation). The * mark indicates p < 0.05 compared to water (control). [Figure 5] The measurement results of the width of the meniscus of a knee joint degeneration mouse model induced by forced running, with each of 9 types of herbal extracts administered, are shown. The measured values are shown as the mean value and error bars (standard deviation). The * mark indicates p < 0.05 compared to water (control). [Figure 6] The measurement results of the amount of blood pain marker TNFα in a knee joint degeneration mouse model induced by forced running, with each of 9 types of herbal extracts administered, are shown. The measured values are shown as the mean value and error bars (standard deviation). The * mark indicates p < 0.05 compared to water (control), and the ** mark indicates 0.05 < p < 0.1 compared to water (control). [Figure 7] The measurement results of the amount of blood inflammation marker CRP in a knee joint degeneration mouse model induced by forced running, with each of 9 types of herbal extracts administered, are shown. The measured values are shown as the mean value and error bars (standard deviation). The * mark indicates p < 0.05 compared to water (control). [Figure 8] The measurement results of the amount of blood cartilage synthesis marker PIICP in a knee joint degeneration mouse model induced by forced running, with each of 9 types of herbal extracts administered, are shown. The measured values are shown as the mean value and error bars (standard deviation). The * mark indicates p < 0.05 compared to water (control), and the ** mark indicates 0.05 < p < 0.1 compared to water (control). [Figure 9] The measurement results of the amount of blood cartilage degradation marker CTXII in a knee joint degeneration mouse model induced by forced running, with each of 9 types of herbal extracts administered, are shown. The measured values are shown as the mean value and error bars (standard deviation). The * mark indicates p < 0.05 compared to water (control).

Mode for Carrying Out the Invention

[0014] The meniscus repair agent of the present disclosure is characterized by containing paeoniflorin, glycyrrhizic acid, and / or liquiritin as active ingredients. Hereinafter, the meniscus repair agent of the present disclosure will be described in detail. In this specification, a numerical range indicated by two numerical values and "~" shall include those two numerical values as the lower limit value and the upper limit value. For example, the notation of 2~15% by weight means 2% by weight or more and 15% by weight or less.

[0015] Active ingredients Paeoniflorin is a known component with known effects such as sedation, analgesia, anti-inflammation, blood pressure lowering, vasodilation, and smooth muscle relaxation.

[0016] Glycyrrhizic acid is a known component with known effects such as anti-inflammatory effect. The salts of glycyrrhizic acid are not particularly limited as long as they are pharmaceutically acceptable. Specifically, alkali metal salts such as sodium salt and potassium salt; ammonium salts and the like can be mentioned. These salts may be used alone or in combination of two or more.

[0017] Liquiritin is a known component with known effects such as anti-allergy and anti-inflammatory effect.

[0018] In the meniscus repair agent of the present disclosure, purified products of these active ingredients may be used for the formulation of paeoniflorin, glycyrrhizic acid, and / or liquiritin, or natural products containing the active ingredients may also be used.

[0019] When the meniscus repair agent of the present disclosure contains a natural product containing paeoniflorin, glycyrrhizic acid, and / or liquiritin, the natural product is not particularly limited. For example, natural products containing paeoniflorin include peony, natural products containing glycyrrhizic acid include licorice, and natural products containing liquiritin include licorice. Therefore, the meniscus repair agent of the present disclosure may contain peony and / or licorice. <​​In the meniscus repair agent of this disclosure, specific embodiments of peony include peony powder and peony extract. Specific embodiments of licorice include licorice powder and licorice extract. Specific embodiments of peony extract include a single extract of peony and an extract of a crude drug formulation containing peony and other crude drugs. Specific embodiments of licorice extract include a single extract of licorice and an extract of a crude drug formulation containing licorice and other crude drugs.

[0021] If the meniscus repair agent of this disclosure contains peony and / or licorice, one may be selected from peony powder, a single extract of peony, an extract of a crude drug preparation containing peony and other crude drugs, licorice powder, a single extract of licorice, and an extract of a crude drug preparation containing licorice and other crude drugs, or two or more may be used in combination.

[0022] Furthermore, as the extract of the herbal medicine preparation containing peony and other crude drugs, one type may be selected and used from the following: an extract of an herbal medicine preparation consisting of peony and licorice; an extract of an herbal medicine preparation containing peony, licorice and other crude drugs; or an extract of an herbal medicine preparation containing peony, peony and other crude drugs; or two or more types may be used in combination. In addition, as the extract of the herbal medicine preparation containing licorice and other crude drugs, one type may be selected and used from the following: an extract of an herbal medicine preparation consisting of licorice and peony; an extract of an herbal medicine preparation containing licorice, peony and other crude drugs; or an extract of an herbal medicine preparation containing licorice, licorice and other crude drugs; or two or more types may be used in combination.

[0023] Peony (Paeonia lactiflora Pallas) is the root of the Paeoniaceae family, or other closely related plants, and is used as a crude drug (Japanese Pharmacopoeia) mainly as an analgesic and antispasmodic (gastrointestinal medicine), women's medicine, medicine for cold sensitivity, and cold medicine. Peony is both the name of the crude drug (Japanese Pharmacopoeia) and the name of the plant.

[0024] The single-ingredient extract of peony is listed in the Japanese Standards for Quasi-Drug Ingredients as peony extract and is publicly known. Specifically, the single-ingredient extract of peony can be obtained by extracting the roots of peony or other closely related plants using an extraction solvent. Furthermore, extracts of herbal medicine preparations containing peony and other crude drugs can be obtained by extracting the herbal medicine preparations containing peony and other crude drugs using an extraction solvent. Examples of extraction solvents used in the extraction process include water; lower alcohols such as ethanol; polyhydric alcohols such as 1,3-butylene glycol; and polar solvents such as mixtures thereof. Preferably, the solvents are water, ethanol, 1,3-butylene glycol, or mixtures thereof.

[0025] Licorice (Glycyrrhiza uralensis Fischer) or Glycyrrhiza glabra Linne are roots and stolons of the legume family Glycyrrhiza, sometimes with the periderm removed (peeled licorice). As a crude drug (Japanese Pharmacopoeia), it is mainly used as an expectorant and for treating stomach ulcers.

[0026] The single-ingredient extract of licorice is listed in the Japanese Standards for Quasi-Drug Ingredients as licorice extract and is publicly known. Specifically, the single-ingredient extract of licorice can be obtained by extracting the roots or stolons of Glycyrrhiza uralensis Fischer or Glycyrrhiza glabra Linne using an extraction solvent. Furthermore, extracts of herbal medicine preparations containing licorice and other crude drugs can be obtained by extracting the herbal medicine preparations containing licorice and other crude drugs using an extraction solvent. Examples of extraction solvents used in the extraction process include water; lower alcohols such as ethanol; polyhydric alcohols such as 1,3-butylene glycol; and polar solvents such as mixtures thereof. Preferably, the solvents are water, ethanol, 1,3-butylene glycol, or mixtures thereof.

[0027] If the meniscus repair agent of this disclosure contains at least one of the following: a mixture of herbal powders containing peony powder, licorice powder, and other herbal powders; a mixture of herbal powders containing peony powder and other herbal powders; a mixture of herbal powders containing licorice powder and other herbal powders; an extract of an herbal preparation containing peony, licorice, and other herbals; an extract of an herbal preparation containing peony, peony, and other herbals; and an extract of an herbal preparation containing licorice and other herbals, the formulation of such herbal powder mixtures or herbal preparations can be selected from known Kampo formulations. In other words, the meniscus repair agent of this disclosure may contain a Kampo medicine containing peony and / or licorice or an extract thereof (Kampo extract).

[0028] There are no particular restrictions on such herbal medicines, but examples include those shown in Tables 1 and 2.

[0029] [Table 1]

[0030] [Table 2]

[0031] These herbal medicines may be selected individually or in combination of two or more. Among these herbal medicines, preferred examples include Keishi-ka-jutsu-bu-to, Sokkei-kakketsu-to, Keishi-etsubi-to, Keishi-ni-etsubi-ichi-to, Keishi-ni-etsubi-ichi-to-ka-jutsu-bu, Shakuyaku-kanzo-to, Ryokyo-jutsu-kan-to, and Shakuyaku-kanzo-bushi-to.

[0032] Extracts of Kampo medicine containing peony and / or licorice can be obtained using the same methods as for ordinary Kampo extracts. For example, an extraction method can be used in which approximately 10 to 20 times the weight of the crude drug preparation is added to the above extraction solvent, preferably water, and the mixture is stirred at approximately 80 to 100°C for approximately 1 to 3 hours. After extraction, the solids are removed by solid-liquid separation such as centrifugation and filtration, and if necessary, the mixture is subjected to concentration and / or drying to obtain an extract of Kampo medicine containing peony and / or licorice.

[0033] When obtaining the above-mentioned extracts as extract powders, the extract, from which the solid components have been removed, can be concentrated as needed and then subjected to drying treatments such as spray drying, vacuum concentration drying, or freeze-drying. Furthermore, when subjecting the extract to drying (especially spray drying), excipients such as dextrin may be added as needed. Adding excipients in this way makes it possible to shorten the drying time. The type and amount of excipients added are the same as those used when manufacturing general extract powders.

[0034] Furthermore, to obtain the above-mentioned various extracts as soft extracts, the extract from which the solid components have been removed can be concentrated by vacuum concentration or the like. Alternatively, an appropriate adsorbent (e.g., anhydrous silicic acid, starch, etc.) may be added to the soft extract to form an adsorbent powder.

[0035] If the meniscus repair agent of this disclosure contains at least one of the above-mentioned extracts, the extract may be either an extract powder or a soft extract.

[0036] Other ingredients The meniscus repair agent of this disclosure may consist solely of the above-mentioned active ingredient, or it may contain additives and / or bases depending on the formulation. Such additives and bases, whether present or absent, are not particularly limited to those that are pharmaceutically acceptable, but examples include excipients, binders, disintegrants, lubricants, isotonic agents, plasticizers, dispersants, emulsifiers, solubilizers, wetting agents, stabilizers, suspending agents, adhesives, coating agents, glossing agents, water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, water-soluble polymers, surfactants, metal soaps, lower alcohols, polyhydric alcohols, pH adjusters, buffers, antioxidants, UV inhibitors, preservatives, flavoring agents, fragrances, powders, thickeners, dyes, chelating agents, etc. These additives may be used individually or in combination of two or more. The content of these additives and bases is appropriately determined depending on the type of additive and base used, as well as the formulation of the meniscus repair agent.

[0037] Furthermore, the meniscus repair agent of this disclosure may or may not contain, in addition to the above-mentioned active ingredients, nutritional components and / or other pharmacological components as needed. Such optional nutritional and pharmacological components are not particularly limited to those that are pharmaceutically acceptable, but examples include antacids, stomachic agents, digestive agents, other intestinal regulators, antispasmodics, mucosal repair agents, anti-inflammatory agents, astringents, antiemetics, antitussives, expectorants, anti-inflammatory enzymes, sedatives, antihistamines, caffeines, cardiotonic and diuretic agents, antibacterial agents, vasoconstrictors, vasodilators, local anesthetics, other herbal medicines, other herbal extracts, vitamins, menthols, etc. These nutritional and pharmacological components may be used individually or in combination of two or more. The content of these components will be appropriately determined depending on the type of component used and the formulation of the meniscus repair agent.

[0038] Dosage form The meniscus repair agent of this disclosure may be an oral preparation, a topical preparation, or an injectable preparation. Examples of oral preparations include solid preparations such as powders, granules, granules (including dry syrups), tablets, pills, and capsules (soft capsules and hard capsules); semi-solid preparations such as jellies; and liquid preparations such as solutions, suspensions, and syrups. Examples of topical preparations include solutions (including lotions, sprays, aerosols, and emulsions), foams, ointments, creams, gels, and patches.

[0039] To prepare the meniscus repair agent of this disclosure into the dosage form, the active ingredient and, if necessary, additives, bases, nutritional components and / or other pharmacological components may be used to formulate the agent according to conventional formulation methods used in the pharmaceutical field.

[0040] Purpose The meniscus repair agent of this disclosure is used to repair the meniscus of the knee. Through its meniscus repair action, the meniscus repair agent can be used to support the meniscus's inherently poor self-repair capacity. Because the meniscus repair agent of this disclosure exerts its effect through its meniscus repair action, it can be applied to both damaged meniscuses (specifically, cracks, defects, etc.) and meniscuses that have degenerated or worn down, and more specifically, to both meniscal injuries and osteoarthritis of the knee. Furthermore, the meniscus repair agent of this disclosure can be applied not only to meniscal injuries caused by injury, but also to meniscal injuries due to aging. Moreover, because the meniscus repair agent of this disclosure has excellent knee meniscus repair capabilities, it can effectively repair even the portion of the meniscus that is inherently difficult to repair, located inside the outer edge where blood vessels are present, where blood vessels are absent. Therefore, the meniscus repair agent of this disclosure is preferably used to repair the portion of the meniscus where blood vessels are absent.

[0041] The meniscus repair agent of this disclosure can be administered orally, topically, or by injection. The dosage of the meniscus repair agent of this disclosure should be appropriately determined according to the method of use, the age, sex, and constitution of the recipient. For example, when the meniscus repair agent of this disclosure is taken orally, the daily dose may be, for example, 0.012 to 0.384 g of paeoniflorin, for example, 0.02 to 0.32 g of the total amount of glycyrrhizic acid and glycyrrhizinate, and for example, 0.012 to 0.192 g of liquiritin. The total amount of paeoniflorin, glycyrrhizic acid, glycyrrhizinate, and liquiritin may be, for example, 0.012 to 0.896 g, preferably 0.024 to 0.448 g. Furthermore, if the meniscus repair agent of this disclosure is taken orally and contains peony and / or licorice, the amount of peony in crude drug equivalent is 0.5 to 16 g, the amount of licorice in crude drug equivalent is 0.5 to 8 g, and the total amount of peony and licorice in crude drug equivalent is, for example, 0.5 to 24 g, preferably 1 to 12 g. [Examples]

[0042] The present disclosure will be explained in more detail below with reference to examples, but the present disclosure is not limited to these examples.

[0043] 1. Test sample 1-1. Paeoniflorin, Glycyrrhizic Acid, Liquiritin, Glucosamine Hydrochloride Paeoniflorin, glycyrrhizic acid, and liquiritin were prepared as 10 mM aqueous solutions using special grade reagents from Fujifilm Wako Pure Chemical Industries, respectively. For glucosamine hydrochloride (comparative example), Sigma-Aldrich's D-(+)-glucosamine hydrochloride was used and prepared as a 10 mg / mL aqueous solution.

[0044] 1-2. Peony extract, licorice extract Dried peony and dried licorice were extracted with hot water, the residue was removed, and the extracts were freeze-dried to obtain single-ingredient extract powders of peony and licorice. Each of the obtained single-ingredient extracts was prepared as a 10 mg / mL aqueous solution.

[0045] 1-3. Herbal extracts Each of the crude drug formulations shown in Table 3 was extracted with hot water, the residue was removed, and the extract was freeze-dried to obtain the herbal extract powders (1) to (9). Of these herbal extracts (1) to (9), extracts (1) to (8) contain paeoniflorin, glycyrrhizic acid, and / or liquiritin because the crude drug formulations contain peony and / or licorice. Each of the obtained herbal extracts was prepared as a 10 mg / mL aqueous solution.

[0046] [Table 3]

[0047] 2. In vitro study: Induction of chondrocyte differentiation by human mesenchymal stem cells (preliminary study) 2-1. Human mesenchymal stem cells Human mesenchymal stem cells (hMSC-BM) were purchased from PromoCell and used. These cells were cultured and proliferated using PromoCell's Mesenchymal Stem Cell Growth Medium 2 (Ready-to-use). After harvesting, an equal volume of cell cryoprotection solution CP-1 (Kyokuto Pharmaceutical Co., Ltd.) was added to the harvested cells, and they were cryopreserved.

[0048] 2-2. Examination Schedule and Examination Methods After thawing, cryopreserved human bone marrow-derived mesenchymal stem cells were grown to 2.5 x 10⁻¹⁰. 5Each cell was dispensed into a 15 mL tube (TPP). These were centrifuged at 1,000 rpm for 3 minutes at room temperature to form cell pellets. The supernatant was removed, and the culture medium was replaced with 0.5 mL of chondrocyte differentiation induction medium according to PLoS ONE, (US), 2014, 9(12), e112291. DOI:10.1371 / journal.pone.0112291. This medium alone was used as the negative control, a medium supplemented with TGFβ3 to a concentration of 1 ng / mL was used as the control, and a medium supplemented with TGFβ3 and BMP-7 (Bone morphogenetic protein-7) to concentrations of 1 ng / mL and 0.1 μg / mL, respectively, was used as the positive control. At the same time as the addition of the positive controls, the test substances were added to the medium to a final concentration of 10 μg / mL each. Every two days, the culture medium was changed with 0.5 mL of chondrocyte differentiation induction medium containing the positive control and test substance at the same concentrations as above. After 21 days of culture, the cells were fixed in 4% paraformaldehyde-phosphate buffer for 12 hours and then dehydrated with 70% ethanol. Subsequently, the cells were embedded in paraffin and specimens were prepared. The prepared specimens were stained with safranin O for proteoglycan staining of the cartilage matrix, and the morphology of differentiated chondrocytes was observed under a light microscope (magnification 10x). Using the image analysis software ImageJ, the area showing safranin O proteoglycan staining (red) was derived as the relative area (safranin O staining ratio) with the control area set to 1. The results are shown in Figures 1-3. A staining ratio greater than 1 indicates the ability to induce chondrocyte differentiation.

[0049] 2-3.Results As shown in Figures 1-3, when paeoniflorin, glycyrrhizic acid, liquiritin, single extracts of peony, single extract of licorice, and the herbal extracts (1)-(8) were used as test samples, the ability to induce chondrocyte differentiation was observed.

[0050] 3. In vivo study: Meniscus repair study in a mouse model of accelerated knee joint degeneration. 3-1. Mouse model for accelerating knee joint degeneration Seventy 12-week-old female C57BL / 6 mice (SLC Japan) were prepared and acclimatized for one week. Following the procedures described in Osteoarthritis Cartilage, 2012, 20(8), 887-895. DOI:10.1016 / j.joca.2012.04.012 and Kinki University Medical Journal, 2012, Vol. 37, No. 1-2, pp. 11-19, the acclimatized mice were forced to run up a 15-degree incline on a treadmill (belt conveyor, manufactured by Muromachi Machinery) at a rate of 20 m / min for 20 minutes / day for two weeks to promote meniscal degeneration. This created a non-surgical animal model that reflects age-related osteoarthritis of the knee in humans, i.e., meniscal wear and tear.

[0051] 3-2. Exam Schedule The mice were forced to run for two weeks, and the following day, the herbal extracts (1) to (9) were orally administered at the following doses for eight weeks (N=6 in each group). A control group was also prepared, consisting of mice administered water ("Water (Control)"; N=6). In addition, a group of mice that were not forced to run for two weeks but were dissected immediately afterward ("2W No Forced Running"; N=3), a group of mice that were forced to run for two weeks but were dissected immediately afterward ("2W Forced Running"; N=3), and a group of mice that were not forced to run for two weeks but were orally administered water for eight weeks (without forced running during the eight weeks of water administration), and then dissected immediately afterward ("10W No Forced Running"; N=4).

[0052] The dosage of the test sample administered to mice was set to be equivalent to the full dose (g / day) of the herbal extract administered to humans, as shown in Table 4.

[0053] [Table 4]

[0054] The test subjects were orally administered the above dose for 8 weeks, and after the completion of administration, autopsy and blood collection were performed.

[0055] 3-3. Test Method 3-3-1. Organizational Structure Evaluation During the dissection, knee joint tissue from the left leg was excised. The excised knee joint tissue was fixed in 10% formalin buffer for 48 hours, dehydrated with 70% ethanol, and then embedded in paraffin to prepare a specimen. The prepared specimen was stained with safranin O for proteoglycan staining. A cross-sectional specimen of the medial meniscus (medial meniscus), sandwiched between the articular cartilage of the femur and tibia, was observed under a light microscope (10x magnification), and scored on a 4-point scale from 0 to 3 for each of the following based on the meniscus degeneration evaluation method described in J Orthop Res. 2017 Jun;35(6):1274-1282. doi: 10.1002 / jor.23211.: (1) surface including the lamellar layer on the femoral side, (2) surface including the lamellar layer on the tibia side, (3) cellularity of chondrocytes, (4) collagen tissue / arrangement and fibrous tissue, and (5) staining ability of safranin O (red). The total score is 0 for a normal state with no cartilage degeneration, and 15 for a diseased state where cartilage has completely disappeared. The results are shown in Figure 4.

[0056] 3-3-2. Evaluation of meniscus width Furthermore, the width of the meniscus was also measured in the above cross-sectional specimen. The results are shown in Figure 5.

[0057] 3-3-3. Measurement of blood markers The collected blood was centrifuged to prepare serum, and blood markers such as the pain marker (TNFα), anti-inflammatory marker (CRP), cartilage synthesis marker (PIICP), and cartilage degradation marker (CTXII) were measured. The results are shown in Figures 6-9.

[0058] 3-4.Results 3-4-1.Organizational form As shown in Figure 4, the group of mice subjected to forced running for two weeks had a higher meniscal degeneration score compared to the group not subjected to forced running for two weeks, suggesting that forced running caused meniscal degeneration. This meniscal degeneration score in mice subjected to forced running was almost the same in the group of mice given water for eight weeks afterward (control group). Among the groups of mice subjected to forced running for two weeks and then administered herbal extracts (1) to (9) for eight weeks, the group administered herbal extracts (1) to (8), which contain peony and / or licorice in their herbal formulations (i.e., paeoniflorin, glycyrrhizic acid, and / or liquiritin), had a lower meniscal degeneration score compared to the group given water for eight weeks (control group) (the herbal extracts marked with an asterisk in Figure 4 showed a statistically significant difference of p<0.05). In other words, meniscal tissue repair was observed with the administration of preparations containing paeoniflorin, glycyrrhizic acid, and / or liquiritin.

[0059] 3-4-2. Width of the meniscus In the group without forced running for 2 weeks and the group that received water for 8 weeks without forced running, the meniscus width could not be measured because the meniscus was not torn, and no degeneration of meniscal tissue occurred. As shown in Figure 5, in the group with forced running for 2 weeks and the group that received water for 8 weeks with forced running (control group), the meniscus width was approximately 400-500 μm. Among the groups in which mice that underwent forced running for 2 weeks were administered herbal extracts (1)-(9) for 8 weeks, the group that received herbal extracts (1)-(8), which contain peony and / or licorice in their crude drug formulations (i.e., paeoniflorin, glycyrrhizic acid, and / or liquiritin), for 8 weeks showed a larger meniscus width compared to the group that received water for 8 weeks (control group) (the herbal extracts marked with * in Figure 5 showed a statistically significant difference of p<0.05). In other words, meniscal tissue repair was observed with the administration of preparations containing paeoniflorin, glycyrrhizic acid, and / or liquiritin. In particular, tissue repair was observed in the area of ​​the meniscus that is inherently difficult to repair, located inside the outer edge where blood vessels are present, and where blood vessels are absent.

[0060] 3-4-3. Blood Markers As shown in Fig. 6, in the group of mice with 2 weeks of forced running, the amount of TNFα in the blood increased significantly compared to the group without forced running. This suggests that forced running induces pain, that is, knee joint pain. The increase in the amount of TNFα in the blood due to this forced running in mice was maintained even in the group of mice given water for 8 weeks later (control group). In all groups where the 9 kinds of Kampo extracts (1) to (9) were administered to the mice that had been forced to run for 2 weeks for 8 weeks, a decrease in the amount of TNFα in the blood was confirmed compared to the group given water for 8 weeks (control group) (*-marked Kampo extracts in Fig. 6 have a significant difference with p < 0.05, and **-marked Kampo extracts have a significant difference with 0.05 < p < 0.1). That is, an analgesic effect was recognized in all 9 kinds of Kampo extracts. In particular, the administration of (2) Keishi-Ninjin-Yoei-To showed a significant analgesic effect because the amount of TNFα decreased compared to mice without forced running.

[0061] As shown in Fig. 7, in the group of mice with 2 weeks of forced running, the amount of CRP in the blood increased compared to the group without forced running. This suggests that forced running induces inflammation associated with knee joint degeneration. The increase in the amount of CRP in the blood due to this forced running in mice was almost the same as that in the group of mice given water for 8 weeks later (control group). In all groups where the 9 kinds of Kampo extracts (1) to (9) were administered to the mice that had been forced to run for 2 weeks for 8 weeks, a significant decrease in the amount of CRP was confirmed compared to the group given water for 8 weeks (control group) (*-marked Kampo extracts in Fig. 7 have a significant difference with p < 0.05).

[0062] As shown in Fig. 8, in the group of mice with 2 weeks of forced running, the amount of PIICP in the blood was significantly decreased compared to the group without forced running. Thus, it was suggested that type II collagen that constitutes the cartilage tissue of the meniscus was not synthesized by forced running. The decrease in the amount of PIICP in the blood due to this forced running in mice was maintained even in the group of mice given water for 8 weeks later (control group). Among the groups in which the crude drug preparations (1) to (9) were administered to the mice that had been forced to run for 2 weeks for 8 weeks, in the groups administered with the crude drug extracts (1) to (8) containing Paeonia lactiflora and / or Glycyrrhiza glabra in the crude drug preparation (that is, containing paeoniflorin, glycyrrhizic acid, and / or liquiritin), a tendency for the amount of PIICP to increase was observed compared to the group given water for 8 weeks (control group) (*-marked crude drug extracts in Fig. 8 had p < 0.05, and **-marked crude drug extracts had a significant difference of 0.05 < p < 0.1). Since the amount of PIICP in the groups administered with the crude drug extracts (1) to (8) increased compared to the group without forced running, it is considered that administration of the preparation containing paeoniflorin, glycyrrhizic acid, and / or liquiritin resulted in cartilage synthesis action.

[0063] As shown in Fig. 9, in the group of mice with 2 weeks of forced running, the amount of CTXII in the blood was significantly increased compared to the group without forced running. Thus, it was suggested that type II collagen was decomposed due to the degeneration of the knee joint caused by forced running. The increase in the amount of CTXII in the blood due to this forced running in mice also occurred in the group of mice given water for 8 weeks later (control group). In all of the groups in which the crude drug extracts (1) to (9) were administered to the mice that had been forced to run for 2 weeks for 8 weeks, the amount of CTXII was significantly decreased compared to the group given water for 8 weeks (control group) (*-marked crude drug extracts in Fig. 9 had a significant difference of p < 0.05). Since the amount of CTXII in the groups administered with the crude drug extracts was almost the same as that in the group without forced running, it is considered that the crude drug extracts could suppress the cartilage degradation action caused by forced running in mice.

[0064] 3-4-4. Summary Table 5 summarizes the results of administering the herbal extracts (1) to (9). As shown in Table 5, effects related to pain suppression, anti-inflammatory effects, and inhibition of cartilage degradation (reduction in pain markers, reduction in anti-inflammatory markers, reduction in cartilage degradation markers) were observed with all herbal extracts. However, effects related to the repair of knee joint cartilage tissue (induction of chondrocyte differentiation, reduction in knee joint cartilage degeneration score, increase in blood cartilage synthesis markers) were characteristically observed in the herbal extracts (1) to (8), which contain peony and / or licorice in their crude drug formulations (i.e., contain paeoniflorin, glycyrrhizic acid, and / or liquiritin).

[0065] [Table 5]

Claims

1. A meniscus repair agent comprising paeoniflorin, glycyrrhizic acid, glycyrrhizinate, and / or liquiritin.

2. A meniscus repair agent containing peony and / or licorice.

3. A meniscal repair agent comprising keishikashutsuto, sokeikakketsuto, keishikoshikuto, keishibatsukoshikukuichito, keishibatsukoshikutsuto, shakuyakukanzoto, ryokyojukanto, and / or shakuyakukansofushito.

4. A meniscus repair agent according to any one of claims 1 to 3, applicable to meniscus injuries.

5. A meniscus repair agent according to any one of claims 1 to 3, applicable to osteoarthritis of the knee.

6. A meniscus repair agent according to any one of claims 1 to 3, used for repairing the portion of the meniscus in which blood vessels are absent.

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