Hip joint cartilage repair agent

Abstract

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

Description

Technical Field

[0001] The present invention relates to a hip joint cartilage repair agent.

Background Art

[0002] The hip joint is formed by the acetabulum of the pelvis and the femoral head of the femur. The surfaces of the acetabulum of the broad acetabulum and the surface of the femoral head are covered with cartilage, and these hip joint cartilages have the role of cushioning to absorb impact and the function of smoothing the movement of the foot.

[0003] The morbidity rate of osteoarthritis of the hip joint by X-ray diagnosis in Japan has been reported to be 1.0 to 4.3% (Non-Patent Document 1). Compared with the reported morbidity rate of radiographic osteoarthritis of the knee joint in Japan being about 55% (Non-Patent Document 2), the frequency of osteoarthritis of the hip joint is low. However, since the hip joint supports the body weight as the largest joint in the human body and is responsible for various movements such as twisting (rotation) of the foot, walking, standing, and squatting, the onset of osteoarthritis of the hip joint significantly reduces the quality of life. In view of the long life expectancy expected in today's aging society, an effective countermeasure against osteoarthritis of the hip joint is desired.

[0004] As useful treatment methods for osteoarthritis of the hip joint, oral administration, injection, and surgery are recognized. Among these treatment methods, in the osteoarthritis of the hip joint treatment guidelines, for oral administration, a recommendation statement that non-steroidal anti-inflammatory drugs (NSAIDs) are useful for pain relief in the short term is published (Non-Patent Document 1).

[0005] Glucosamine and chondroitin are used as supplements for osteoarthritis. However, the guidelines for the treatment of hip osteoarthritis include a recommendation stating that there is no consensus on the therapeutic effects of glucosamine and chondroitin for hip osteoarthritis (Non-Patent Literature 1). The American Academy of Orthopaedic Surgeons has also published clinical practice guidelines for the management of hip osteoarthritis, concluding that it does not support the use of glucosamine sulfate for hip osteoarthritis. This conclusion is based on reliable studies showing that glucosamine is not superior to placebo in terms of effects on pain, joint function, or joint structure (joint space narrowing) after two years of treatment with either glucosamine sulfate or placebo (Non-Patent Literature 3). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Guidelines for the Treatment of Osteoarthritis of the Hip, 2016 Revised 2nd Edition, supervised by the Japanese Orthopaedic Association and the Japanese Hip Society, July 20, 2018. [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]

[0007] Nonsteroidal anti-inflammatory drugs (NSAIDs), which are considered effective for osteoarthritis of the hip, are medications with a relatively high frequency of gastrointestinal side effects. Therefore, there is a need for alternative effective medications for osteoarthritis of the hip. Furthermore, oral medications for osteoarthritis of the hip only suppress inflammation and do not repair worn-down hip cartilage. However, to date, no active ingredients capable of repairing hip cartilage have been identified.

[0008] The purpose of this disclosure is to provide a formulation that can repair hip joint cartilage. [Means for solving the problem]

[0009] The inventors of the present invention conducted diligent research to solve the aforementioned problems and found that paeoniflorin, glycyrrhizic acid, and liquiritin each have the effect of repairing hip joint cartilage. This disclosure was completed by further research based on this finding.

[0010] In other words, this disclosure provides inventions in the following embodiments. Item 1. A hip joint cartilage repair agent comprising paeoniflorin, glycyrrhizic acid, glycyrrhizinate, and / or liquiritin. Item 2. A hip joint cartilage repair agent containing peony and / or licorice. Item 3. A hip joint cartilage repair agent containing Keishikajutsuto, Sokeikakketsuto, Keishikotsukuto, Keishibakoshijyuichito, Keishibakoshijyuichito Kashutsu, Shakuyakukanzoto, Reikyojukanto, and / or Shakuyakukanzofushito. Item 4. A method for hip joint cartilage repair, comprising administering an effective amount of paeoniflorin, glycyrrhizic acid, and / or liquiritin to a subject requiring hip joint cartilage repair. Item 5. Use of paeoniflorin, glycyrrhizic acid, and / or liquiritin for the manufacture of hip joint cartilage repair agents. Item 6. Paeoniflorin, glycyrrhizic acid, and / or liquiritin for use in hip joint cartilage repair. [Effects of the Invention]

[0011] According to the present disclosure, a preparation capable of repairing hip joint cartilage is provided.

Brief Description of the Drawings

[0012] [Figure 1] The results of cartilage matrix production induced by test samples (each 10 μM) in the three-dimensional culture of human bone marrow-derived mesenchymal stem cells into chondrocytes are shown by the safranin O staining ratio. The safranin O staining ratio is shown by the average value of N = 2 and the error bar (standard deviation). The * mark indicates p < 0.05 compared to water (control). [Figure 2] The results of cartilage matrix production induced by test samples (each 10 μg / mL) in the three-dimensional culture of human bone marrow-derived mesenchymal stem cells into chondrocytes are shown by the safranin O staining ratio. The safranin O staining ratio is shown by the average value of N = 2 and the error bar (standard deviation). The * mark indicates p < 0.05 compared to water (control). [Figure 3] The results of cartilage matrix production induced by test samples (each 10 μg / mL) in the three-dimensional culture of human bone marrow-derived mesenchymal stem cells into chondrocytes are shown by the safranin O staining ratio. The safranin O staining ratio is shown by the average value of N = 2 and the error bar (standard deviation). The * mark indicates p < 0.05 compared to water (control). [Figure 4] The hip joint cartilage degeneration scores of a hip joint degeneration mouse model by forced running, each administered with 9 kinds of herbal extracts, are shown. The hip joint cartilage degeneration score is shown by the average value and the error bar (standard deviation). The * mark indicates p < 0.05 compared to water (control). [Figure 5] The measurement results of the amount of blood pain marker TNFα in a hip joint degeneration mouse model by forced running, each administered with 9 kinds of herbal extracts, are shown. The measured values are shown by the average value and the error bar (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 6]The measurement results of the amount of blood inflammatory marker CRP in a femoral joint degeneration mouse model caused by forced running, after administration of each of 9 types of herbal extracts, 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 7] The measurement results of the amount of blood cartilage synthesis marker PIICP in a femoral joint degeneration mouse model caused by forced running, after administration of each of 9 types of herbal extracts, 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 8] The measurement results of the amount of blood cartilage degradation marker CTXII in a femoral joint degeneration mouse model caused by forced running, after administration of each of 9 types of herbal extracts, 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

[0013] The femoral joint cartilage repair agent of the present disclosure is characterized by containing paeoniflorin, glycyrrhizic acid, and / or liquiritin as active ingredients. Hereinafter, the femoral joint cartilage repair agent of the present disclosure will be described in detail. In this specification, a numerical range indicated by two numerical values and "~" includes 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.

[0014] Active ingredients Paeoniflorin is a known component with known effects such as sedation, analgesia, anti-inflammation, blood pressure lowering, vasodilation, and smooth muscle relaxation.

[0015] Glycyrrhizic acid is a known component with anti-inflammatory effects and the like. The salts of glycyrrhizic acid are not particularly limited as long as they are pharmaceutically acceptable. Specifically, examples include alkali metal salts such as sodium salt and potassium salt; ammonium salts and the like. These salts may be used alone or in combination of two or more.

[0016] Licorice is a known component with anti-allergic and anti-inflammatory effects and the like.

[0017] In the femoral joint cartilage repair agent of the present disclosure, purified products of these active ingredients may be used for the formulation of paeoniflorin, glycyrrhizic acid, and / or licorice, or natural products containing the active ingredients may be used.

[0018] When the femoral joint cartilage repair agent of the present disclosure contains a natural product containing paeoniflorin, glycyrrhizic acid, and / or licorice, the natural product is not particularly limited. For example, as the natural product containing paeoniflorin, peony can be mentioned, as the natural product containing glycyrrhizic acid, licorice can be mentioned, and as the natural product containing licorice, licorice can be mentioned. Therefore, the femoral joint cartilage repair agent of the present disclosure may contain peony and / or licorice.

[0019] In the femoral joint cartilage repair agent of the present disclosure, specific forms of peony include peony powder and peony extract. Specific forms of licorice include licorice powder and licorice extract. Specific forms of the peony extract include a simple extract of peony, and an extract of a crude drug preparation containing peony and other crude drugs. Specific forms of the licorice extract include a simple extract of licorice, and an extract of a crude drug preparation containing licorice and other crude drugs.

[0020] If the hip joint cartilage repair agent of this disclosure contains peony and / or licorice, one of the following may be selected and used, or two or more may be used in combination: peony powder, peony extract alone, extract of a crude drug preparation containing peony and other crude drugs, licorice powder, licorice extract alone, and extract of a crude drug preparation containing licorice and other crude drugs.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] If the hip cartilage 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 hip cartilage repair agent of this disclosure may contain a Kampo medicine containing peony and / or licorice or an extract thereof (Kampo extract).

[0027] There are no particular restrictions on such herbal medicines, but examples include those shown in Tables 1 and 2.

[0028] [Table 1]

[0029] [Table 2]

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] If the hip joint cartilage 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.

[0035] Other ingredients The hip cartilage 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. Furthermore, 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 hip cartilage repair agent.

[0036] Furthermore, the hip cartilage 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 hip cartilage repair agent.

[0037] Dosage form The hip joint cartilage repair agent of this disclosure may be either an oral 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.

[0038] To prepare the hip joint cartilage repair agent of this disclosure into the aforementioned 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.

[0039] Purpose The hip cartilage repair agent disclosed herein is used to repair hip cartilage. Through its hip cartilage repair action, the hip cartilage repair agent disclosed herein can be used to support the inherently poor self-repair capacity of hip cartilage.

[0040] The hip cartilage repair agent disclosed herein can be administered orally or by injection. The dosage of the hip cartilage repair agent disclosed herein should be appropriately determined according to the method of use, the age, sex, and constitution of the person taking the drug. For example, when the hip cartilage repair agent disclosed herein 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 hip joint cartilage 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]

[0041] The present disclosure will be explained in more detail below with reference to examples, but the present disclosure is not limited to these examples.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] [Table 3]

[0046] 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.

[0047] 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 g / 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.

[0048] 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.

[0049] 3. In vivo study: Hip cartilage repair study in a mouse model of accelerated hip degeneration. 3-1. Mouse model for accelerating hip joint degeneration Seventy 12-week-old female C57BL / 6 mice (SLC Japan) were prepared and acclimatized for one week. Following the instructions 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 degeneration of the hip cartilage. This created a non-surgical animal model that reflects age-related osteoarthritis of the hip in humans, i.e., the symptoms of hip cartilage wear. Furthermore, this mouse model has been confirmed to exhibit symptoms of meniscus wear in the knee, making it also a model for osteoarthritis of the knee.

[0050] 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).

[0051] 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.

[0052] [Table 4]

[0053] The test subjects were orally administered the above dose for 8 weeks, and after the completion of administration, autopsy and blood collection were performed.

[0054] 3-3. Test Method 3-3-1. Organizational Structure Evaluation During the dissection, femoral head tissue from the right hip joint was excised. The excised femoral head 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. The cross-sectional specimen of the femoral head was observed under a light microscope (10x magnification), and the following method, a modified version of the cartilage degeneration evaluation method described in Osteoarthritis Cartilage. 1996 Jun;4(2):99-110. doi: 10.1016 / s1063-4584(05)80319-x., was adopted. Specifically, the following were scored on a 5-point scale from 0 to 4: (1) staining intensity of safranin O (red), (2) degree of erosion formation, (3) degree of crack formation (roughening), (4) amount of superficial layer loss, (5) degree of irregularity of cell arrangement (columns), (6) amount of chondrocyte loss, and (7) amount of subchondral bone exposure. A total score of 0 indicates a normal state with no cartilage degeneration, while a total score of 28 indicates a diseased state with complete cartilage loss. The results are shown in Figure 4.

[0055] 3-3-2. 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 5-8.

[0056] 3-4.Results 3-4-1.Organizational form As shown in Figure 4, the group of mice subjected to forced running for two weeks showed a significantly higher hip joint cartilage degeneration score compared to the group not subjected to forced running, suggesting that forced running induced cartilage degeneration. This hip joint cartilage degeneration score in mice subjected to forced running was similarly high in the group of mice given water for eight weeks (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), showed a smaller hip joint cartilage 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, the administration of preparations containing paeoniflorin, glycyrrhizic acid, and / or liquiritin resulted in the repair of hip joint cartilage tissue.

[0057] During the dissection, knee joint tissue from the left leg of the same mouse model was also extracted, and a specimen was prepared in the same manner as above. Proteoglycan staining with safranin O was performed, and a degeneration score (meniscus degeneration score) was derived for the meniscus based on the meniscus degeneration evaluation method described in J Orthop Res. 2017 Jun;35(6):1274-1282. doi: 10.1002 / jor.23211. A reduction in the meniscus deformation score was observed with the administration of the herbal extracts (1) to (8), but the level of reduction in the hip joint cartilage deformation score with the administration of the herbal extracts (1) to (8), as shown in Figure 4, was confirmed to be more significant than the level of reduction in the meniscus deformation score.

[0058] 3-4-2. Blood Markers As shown in Fig. 5, the amount of TNFα in the blood of the group with 2 weeks of forced running in mice was significantly increased compared to the group without forced running, suggesting that forced running caused pain, i.e., hip 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 receiving water administration for 8 weeks (control group) thereafter. In all groups in which the Chinese herbal 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 receiving water administration for 8 weeks (control group) (*-marked Chinese herbal extracts in Fig. 5 had a significant difference at p < 0.05, and **-marked Chinese herbal extracts had a significant difference at 0.05 < p < 0.1). That is, an analgesic effect was recognized in all 9 types of Chinese herbal extracts.

[0059] As shown in Fig. 6, the amount of CRP in the blood of the group with 2 weeks of forced running in mice was increased compared to the group without forced running, suggesting that forced running induced inflammation associated with hip joint degeneration. The increase in the amount of CRP in the blood due to this forced running in mice was approximately the same as that in the group of mice receiving water administration for 8 weeks (control group) thereafter. In all groups in which the Chinese herbal 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 receiving water administration for 8 weeks (control group) (*-marked Chinese herbal extracts in Fig. 6 had a significant difference at p < 0.05).

[0060] As shown in Fig. 7, in the group of mice with 2 weeks of forced running, the amount of PIICP in the blood was significantly decreased compared with the group without forced running. Therefore, it was suggested that type II collagen constituting the cartilage tissue of the hip joint 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 mouse group (control group) with 8 weeks of water administration thereafter. Among the groups in which the crude drug preparations (1) to (9) were administered to the mice with 2 weeks of forced running 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), the amount of PIICP tended to increase compared with the group with 8 weeks of water administration (control group) (*-marked crude drug extracts in Fig. 7 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 with the group without forced running, it is considered that cartilage synthesis was induced by the administration of the preparation containing paeoniflorin, glycyrrhizic acid, and / or liquiritin.

[0061] As shown in Fig. 8, in the group of mice with 2 weeks of forced running, the amount of CTXII in the blood was significantly increased compared with the group without forced running. Therefore, it was suggested that type II collagen was decomposed due to the degeneration of the hip 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 mouse group (control group) with 8 weeks of water administration thereafter. In all of the groups in which the crude drug extracts (1) to (9) were administered to the mice with 2 weeks of forced running for 8 weeks, the amount of CTXII was significantly decreased compared with the group with 8 weeks of water administration (control group) (*-marked crude drug extracts in Fig. 8 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 effect caused by forced running in mice.

[0062] 3-4-3. 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 in all herbal extracts. However, effects related to the repair of hip joint cartilage tissue (induction of chondrocyte differentiation, reduction in hip joint cartilage degeneration score, increase in cartilage synthesis markers in the blood) 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).

[0063] [Table 5]

Claims

1. A hip joint cartilage repair agent comprising paeoniflorin, glycyrrhizic acid, glycyrrhizinate, and / or liquiritin.

2. A hip joint cartilage repair agent containing peony and / or licorice.

3. A hip joint cartilage repair agent comprising keishikashutsuto, sokeikakketsuto, keishikoshikuto, keishibatsukoshikuichito, keishibatsukoshikutsuto, shakuyakukanzoto, reikyojukanto, and / or shakuyakukansofushito.

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