Use of bletilla striata glycoside compound, composition containing same, and preparation method therefor

Abstract

Disclosed in the present invention are use of a Bletilla striata glycoside compound, a composition containing same, and a preparation method therefor. The present invention provides use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating osteoarthritis and / or pain. It has been discovered for the first time that a Bletilla striata extract or a Bletilla striata glycoside can significantly increase the mechanical pain threshold of arthritis pain model animals and inhibit inflammatory responses, etc., and with re-administration, the improvement effect on the mechanical pain threshold is enhanced again. Moreover, the bipedal weight-bearing difference between the hind limbs of the model animals can also be significantly reduced, showing that the Bletilla striata extract or the Bletilla striata glycoside has the effect of preventing and / or treating osteoarthritis and / or pain, especially having a good therapeutic effect on the inflammatory responses and / or pain in the bone joints caused by bone joint injury.

Description

Application of bletillae rhizoma glycosides, composition containing the same and preparation method thereof

[0001] This application claims priority to Chinese patent application No. 2024107863956, filed on June 18, 2024, and Chinese patent application No. 2024118261828, filed on December 12, 2024. This application incorporates the entire contents of the above-mentioned Chinese patent applications. TECHNICAL FIELD

[0002] The present application relates to the technical field of natural active drugs, in particular to the application of bletillae rhizoma glycosides, composition containing the same and preparation method thereof. BACKGROUND

[0003] Osteoarthritis (OA) is a degenerative joint disease that seriously affects the quality of life of patients, can cause joint pain, deformity and dysfunction, and can also lead to the risk of deep venous thromboembolism, hip fracture and all-cause mortality. It is reported that there are more than 300 million OA patients worldwide, and the overall prevalence of primary OA among people over 40 years old in China has reached as high as 46.3%. Moreover, with the increasing degree of population aging in China, the prevalence of OA is gradually increasing. Knee osteoarthritis (KOA) is the most common site of OA. Pain is a major associated factor of KOA disability, and can seriously affect the quality of life of patients, including functional status and mental health, and many chronic pain patients often have anxiety and depression. The pathogenic principle and pathogenesis of OA are not clear, and there is no specific drug to reverse joint degeneration. Under such circumstances, how to reduce joint pain and improve the function of the joint of patients has become the main goal of various methods for treating OA.

[0004] In recent years, traditional Chinese medicine has made some progress in the treatment of OA. Many clinical studies have shown that traditional Chinese medicine can improve the symptoms of limited knee joint activity or pain in patients, reduce adverse reactions in patients, and thus improve the quality of life of patients. However, there is still no traditional Chinese medicine preparation with clear efficacy that has successfully entered the clinic or been marketed, providing patients with more drug options.

[0005] Bletilla striata (Thunb.) Reichb. f., the dry tuber of which is recorded in Chinese Pharmacopoeia 2020 Edition Part I. It has the effect of "astringing and stopping bleeding, reducing swelling and promoting tissue regeneration", and is used for treating hemoptysis, hematemesis, trauma bleeding, sore and swelling, and skin chapping. However, there is no report on bletillae rhizoma glycosides and their compositions in the treatment of osteoarthritis. SUMMARY

[0006] The technical problem to be solved by the present application is that the application field of the bletilla striata glycosides in the prior art is relatively single, therefore, the present application provides an application of the bletilla striata glycosides, a composition containing the same and a preparation method thereof. It is found for the first time that the bletilla striata extract or the bletilla striata effective part or the bletilla striata glycosides can obviously improve the mechanical pain threshold of an arthritic pain model animal and inhibit inflammatory response, and the improvement of the mechanical pain threshold is enhanced again with re-dosing. Meanwhile, the bletilla striata extract or the bletilla striata effective part or the bletilla striata glycosides can obviously reduce the weight-bearing difference of the hind limbs of the model animal, which shows that the bletilla striata extract or the bletilla striata effective part or the bletilla striata glycosides have the effects of preventing and / or treating osteoarthritis or pain, and especially have good therapeutic effects on the inflammatory response and / or pain of the osteoarthritis caused by the osteoarticular injury (for example, meniscus injury, ligament injury).

[0007] The present application provides an application of a compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing and / or treating osteoarthritis and / or pain, wherein the compound is a compound as shown in formula I or handanenoside I.

[0008] In the compound as shown in formula I:

[0009] represents a single bond or a double bond; X is CR 1 when the double bond is present, R 1 is H or C 1-6 alkyl;

[0010] X is C*R 2 when the single bond is present, R 3 , R 2 is H or C 1-6 alkyl; R 3 is H, C 1-6 alkyl, hydroxyl or R 4 is hydroxyl or -C(O)-C 1-6 alkyl;

[0011] The carbon atom marked with "*" is a chiral carbon atom, and the configuration thereof is R configuration, S configuration or a mixture of R configuration and S configuration;

[0012] L is a single bond or wherein the O atom is connected to M;

[0013] M and K are independently a single bond or C 1-6 alkylene;

[0014] R 5 and R 6 are independently H or C 1-6 alkoxy.

[0015] In certain embodiments of the compound of Formula I, or a pharmaceutically acceptable salt thereof, certain groups are defined as follows, and the definitions of groups not mentioned are as described in any of the embodiments of the application (hereinafter in this paragraph, "in certain embodiments").

[0016] In certain embodiments, R 1 , R 2 , R 3 and -C(O)-C 1-6 In certain embodiments, the C 1-6 alkyl is independently methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, or t-butyl.

[0017] In certain embodiments, in M and K, the C 1-6 alkylene is independently a straight chain alkylene, for example methylene, ethylene, for example methylene.

[0018] In certain embodiments, R 5 and R 6 , the C 1-6 alkoxy is independently methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, or t-butoxy, for example methoxy.

[0019] In certain embodiments, R 1 is H.

[0020] In certain embodiments, R 2 is C 1-6 alkyl, for example

[0021] In certain embodiments, in -C(O)-C 1-6 alkyl, the C 1-6 alkyl is methyl.

[0022] In certain embodiments, is

[0023] In certain embodiments, R 3 is hydroxyl or

[0024] In certain embodiments, the carbon atom marked "*" is a chiral carbon atom, and its configuration is the R configuration.

[0025] In certain embodiments, is a double bond, M and L are single bonds.

[0026] In certain embodiments, M is C when is a single bond 1-6 alkylene, for example methylene; L is wherein the O atom is attached to M.

[0027] In one embodiment, R is M is C when is a single bond

[0028] In one embodiment, R is 5 M is C when is a single bond 1-6 alkoxy, for example methoxy.

[0029] In one embodiment, K is methylene;

[0030] In one embodiment, R is 6 M is C when is a single bond

[0031] In one embodiment, the compound of Formula I is any one of the following:

[0032] In one embodiment, the pain is pain associated with osteoarthritis.

[0033] In one embodiment, the osteoarthritis in the osteoarthritis and the pain associated with osteoarthritis is independently traumatic osteoarthritis or inflammatory osteoarthritis.

[0034] In one embodiment, the osteoarthritis is knee osteoarthritis.

[0035] In one embodiment, the traumatic osteoarthritis is traumatic osteoarthritis associated with meniscal injury and / or ligament injury, for example traumatic osteoarthritis associated with meniscal injury and ligament injury.

[0036] In one embodiment, the ligament injury is a ligament rupture, for example an anterior cruciate ligament transection.

[0037] In one embodiment, the meniscal injury is a meniscectomy.

[0038] In one embodiment, the inflammatory osteoarthritis is inflammatory osteoarthritis mediated by IL-1β.

[0039] In one embodiment, the medicament, the compound or pharmaceutically acceptable salt thereof is the sole active ingredient.

[0040] The medicament can be administered by any suitable route of administration in the art; in one embodiment, the medicament is administered orally.

[0041] The drug can be administered according to the body weight of the subject, in a certain embodiment, the single dose of the drug is 30-80 mg / kg, for example 40-60 mg / kg, again for example 50 mg / kg, calculated as the dose of the active ingredient.

[0042] In a certain embodiment, the mass concentration of the compound or its pharmaceutically acceptable salt in the drug is 1-30 mg / mL; for example 1-10 mg / mL, again for example 5 mg / mL.

[0043] The present application provides a pharmaceutical composition comprising 1%-70% of Bletilla Striata Glycosides and 0.5%-20% of 2-O-glucosyl Bletilla Striata Glycosides (Dactylorhin A), the % being the percentage of the mass of each component accounting for the total mass of the pharmaceutical composition.

[0044] In a certain embodiment, the mass percentage of the Bletilla Striata Glycosides is 1%-30%, for example 10%-15%, again for example 12.76%.

[0045] In a certain embodiment, the mass percentage of the Bletilla Striata Glycosides is 10%-70%, for example 20%-50%, again for example 40.24%.

[0046] In a certain embodiment, the mass percentage of the Bletilla Striata Glycosides is 10%-50%, for example 12%-41%.

[0047] In a certain embodiment, the mass percentage of the 2-O-glucosyl Bletilla Striata Glycosides is 0.5%-10%, for example 1%-6%, again for example 3.76%.

[0048] In a certain embodiment, the mass percentage of the 2-O-glucosyl Bletilla Striata Glycosides is 3%-20%, for example 5%-15%, again for example 10.51%.

[0049] In a certain embodiment, the mass percentage of the 2-O-glucosyl Bletilla Striata Glycosides is 1%-15%, for example 3%-11%.

[0050] In a certain embodiment, the pharmaceutical composition further comprises a substance C, the substance C comprising one or more components selected from 2-O-glucosyl-2-isobutyl malic acid (Dactylorhin C), 2-isobutyl malic acid (α-isobutylmalic acid), Densiflorin I, Bletilloside A, Handianoside I and Handianoside III.

[0051] In a certain embodiment, the substance C is selected from any of the following schemes:

[0052] Scheme 1 : the material C consists of 2-O-glucosyl-2-isobutylmalic acid, 2- isobutylmalic acid, anoectochilins I, picroside A, handeliumside I and handeliumside III;

[0053] Scheme 2: the material C consists of anoectochilins I, picroside A and handeliumside III;

[0054] Scheme 3, the material C consists of 2-O-glucosyl-2-isobutylmalic acid, 2- isobutylmalic acid and anoectochilins I;

[0055] Scheme 4, the material C is 2-O-glucosyl-2-isobutylmalic acid.

[0056] Scheme 5, the material C consists of picroside A, handeliumside I and handeliumside III.

[0057] In a certain embodiment, the active ingredient of the pharmaceutical composition is selected from one or more of picroside, 2-O-glucosylpicroside and the material C, for example the active ingredient of the pharmaceutical composition is picroside, 2-O- glucosylpicroside, consists of picroside and 2-O-glucosylpicroside or consists of picroside, 2-O-glucosylpicroside and the material C.

[0058] In a certain embodiment, the active ingredient of the pharmaceutical composition is selected from the following schemes:

[0059] Scheme 1 : the active ingredient of the pharmaceutical composition consists of 10-50% picroside and 1-15% 2-O-glucosylpicroside;

[0060] Scheme 2: the active ingredient of the pharmaceutical composition consists of 10-50% picroside, 1-15% 2-O-glucosylpicroside and the material C, the material C consists of 2-O- glucosyl-2-isobutylmalic acid, 2-isobutylmalic acid, anoectochilins I, picroside A, handeliumside I and handeliumside III; preferably, the mass percentage of the picroside is 10%-15%, the mass percentage of the 2-O-glucosylpicroside is 1%-6%; or the mass percentage of the picroside is 20%-50%, the mass percentage of the 2-O- glucosylpicroside is 5%-15%;

[0061] Scheme 3: the active ingredients of the pharmaceutical composition are composed of 10-50% of bletilla striata glycosides, 1-15% of 2-O-glucosyl bletilla striata glycosides and substance C, which is composed of 2-O-glucosyl-2-isobutyl malic acid, 2-isobutyl malic acid and panacrin I; preferably, the mass percentage of the bletilla striata glycosides is 10%-15%, the mass percentage of the 2-O-glucosyl bletilla striata glycosides is 1%-6%; or the mass percentage of the bletilla striata glycosides is 20%-50%, the mass percentage of the 2-O-glucosyl bletilla striata glycosides is 5%-15%;

[0062] Scheme 4: the active ingredients of the pharmaceutical composition are composed of 10-50% of bletilla striata glycosides, 1-15% of 2-O-glucosyl bletilla striata glycosides and substance C, which is composed of bletilla striata glycoside A, handan glycoside I and handan glycoside III; preferably, the mass percentage of the bletilla striata glycosides is 10%-15%, the mass percentage of the 2-O-glucosyl bletilla striata glycosides is 1%-6%; or the mass percentage of the bletilla striata glycosides is 20%-50%, the mass percentage of the 2-O-glucosyl bletilla striata glycosides is 5%-15%.

[0063] In an embodiment, the active ingredients of the pharmaceutical composition are selected from the following schemes:

[0064] Scheme 1: the active ingredients of the pharmaceutical composition are composed of 12.76% of bletilla striata glycosides, 3.76% of 2-O-glucosyl bletilla striata glycosides and substance C, which is composed of 2-O-glucosyl-2-isobutyl malic acid, 2-isobutyl malic acid, panacrin I, bletilla striata glycoside A, handan glycoside I and handan glycoside III.

[0065] Scheme 2: the active ingredients of the pharmaceutical composition are composed of 40.24% of bletilla striata glycosides, 10.51% of 2-O-glucosyl bletilla striata glycosides and substance C, which is composed of 2-O-glucosyl-2-isobutyl malic acid, 2-isobutyl malic acid, panacrin I, bletilla striata glycoside A, handan glycoside I and handan glycoside III.

[0066] In an embodiment, the pharmaceutical composition is prepared by a preparation method selected from the following method 1 or method 2:

[0067] Method 1 comprises the following steps: mixing and extracting bletilla striata with an aqueous alcohol solvent to obtain the pharmaceutical composition;

[0068] Method 2 comprises the following steps: eluting the crude extract of bletilla striata in a chromatographic column to obtain the pharmaceutical composition; the filler of the chromatographic column is a styrene type macroporous adsorption resin.

[0069] In an embodiment, in method 1, the bletilla striata is bletilla striata tuber, such as tuber powder.

[0070] In a certain embodiment, in method 1, the alcoholic solvent is methanol and / or ethanol, for example ethanol.

[0071] In a certain embodiment, in method 1, in the alcoholic solvent aqueous solution, the volume percentage of the alcoholic solvent is 30-90%, for example 50-70%, further for example 58%-62%, more for example 60%, the volume percentage being the percentage of the volume of the alcoholic solvent / the volume of the alcoholic solvent aqueous solution.

[0072] In a certain embodiment, in method 1, the ratio of the mass of the Bletilla striata to the volume of the alcoholic solvent aqueous solution is (0.05-0.15) g:1 mL, for example (0.08-0.13) g:1 mL, further for example 0.1 g:1 mL.

[0073] In a certain embodiment, in method 1, the extraction is performed by ultrasonic extraction (for example the ultrasonic power is 20-25 KHz) or solvent reflux method, for example solvent reflux method.

[0074] In a certain embodiment, in method 1, the extraction is performed at a temperature of 70-100°C, for example 85-100°C.

[0075] In a certain embodiment, in method 1, the extraction is performed for a time of 15 min-80 min, for example 40-80 min, for example 60 min.

[0076] In a certain embodiment, in method 1, the extraction is performed for ≥1 time, for example 1-3 times, further for example 2 times.

[0077] Method 1 further comprises post-treatment methods that are conventional in the art, for example filtering the extract obtained after the extraction, removing the solvent and drying; the method of removing the solvent is conventional in the art, for example reduced pressure distillation.

[0078] In a certain embodiment, in method 2, the styrene type macroporous adsorption resin is AB-8 macroporous resin.

[0079] In a certain embodiment, in method 2, the elution solvent is an alcoholic solvent aqueous solution, and the alcoholic solvent is methanol and / or ethanol, for example ethanol.

[0080] In a certain embodiment, in method 2, in the alcoholic solvent aqueous solution, the volume percentage of the alcoholic solvent is 1%-95%, for example 1%-20% or 80%-95%, further for example 10% or 90%; the volume percentage being the percentage of the volume of the alcoholic solvent / the volume of the alcoholic solvent aqueous solution.

[0081] In certain embodiments, in Method 2, the elution is divided into a first elution and a second elution.

[0082] In certain embodiments, in Method 2, the volume percentage of the alcoholic solvent in the aqueous alcoholic solvent of the first elution is 1%-20%, for example, 10%.

[0083] In certain embodiments, in Method 2, the volume percentage of the alcoholic solvent in the aqueous alcoholic solvent of the second elution is 80%-95%, for example, 90%.

[0084] In certain embodiments, in Method 2, in the first elution and the second elution, the volume of the elution solvent is (2-5) times the column volume, for example, 3 times the column volume.

[0085] In Method 2, the loading mode of the crude bletilla striata extract is a loading mode conventional in the art, for example, loading in the form of a crude bletilla striata extract solution, the solvent of the crude bletilla striata extract solution being an aqueous alcoholic solvent, the volume percentage of the alcoholic solvent being 1%-20%, for example, 10%.

[0086] In certain embodiments, in Method 2, the ratio of the mass of the crude bletilla striata extract to the volume of the solvent in the crude bletilla striata extract solution is (40-60) g:1 L, for example, 50 g:1 L.

[0087] In Method 2, the preparation method of the pharmaceutical composition further comprises a post-treatment method conventional in the art, for example, removing the solvent in the eluate of the second elution and drying, the removal of the solvent in the eluate of the second elution being conventional in the art, for example, distillation under reduced pressure.

[0088] In certain embodiments, in Method 2, the crude bletilla striata extract is prepared by using Method 1.

[0089] The present application provides a preparation method of a pharmaceutical composition as described above, the preparation method being selected from the following Method 1 or Method 2:

[0090] Method 1 comprises the following steps: mixing bletilla striata with an aqueous ethanol solution and extracting to obtain the pharmaceutical composition;

[0091] Method 2 comprises the following steps: eluting a crude bletilla striata extract in a chromatographic column to obtain the pharmaceutical composition; the filler of the chromatographic column is a styrene type macroporous adsorption resin.

[0092] The operations and conditions of the Method 1 and Method 2 can also be as described in any one of the present application.

[0093] The present application provides a pharmaceutical composition prepared by a preparation method selected from the following method 1 or method 2:

[0094] Method 1 comprises the following steps: mixing and extracting Bletilla striata with an aqueous ethanol solution to obtain the pharmaceutical composition;

[0095] Method 2 comprises the following steps: eluting the crude extract of Bletilla striata in a chromatographic column to obtain the pharmaceutical composition; the packing material of the chromatographic column is a styrene macroporous adsorption resin;

[0096] Preferably, the pharmaceutical composition is as described in any one of the present application; the operation and conditions of the method 1 and method 2 can also be as described in any one of the present application.

[0097] The present application provides a pharmaceutical preparation comprising the pharmaceutical composition as described above and a pharmaceutical excipient.

[0098] The present application provides the use of the pharmaceutical composition or pharmaceutical preparation as described above in the preparation of a drug for preventing and / or treating osteoarthritis and / or pain.

[0099] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, thereby obtaining each preferred example of the present application.

[0100] The reagents and raw materials used in the present application are commercially available.

[0101] The positive progress effect of the present application is that the present application provides the use of Bletilla striata extract or Bletilla striata active site or Bletilla striata glycoside, a main component of Bletilla striata, in the preparation of a drug for preventing and / or treating osteoarthritis and / or pain, which enriches the optional types of drugs for treating osteoarthritis and pain and has a good therapeutic effect. Specifically, the present application finds that Bletilla striata extract or Bletilla striata active site or Bletilla striata glycoside can significantly improve the mechanical pain threshold of an arthritic pain model animal and inhibit inflammatory response, etc., and the improvement of the mechanical pain threshold is enhanced again with re-dosing. At the same time, it can also significantly reduce the difference in the weight bearing of the hind limbs of the model animal, which shows that Bletilla striata extract or Bletilla striata active site or Bletilla striata glycoside, a main component of Bletilla striata, has a preventive and therapeutic effect on osteoarthritis or pain, and especially has a good therapeutic effect on the inflammatory response and / or pain of the osteoarticular injury (such as meniscus injury, ligament injury). DETAILED DESCRIPTION

[0102] The present application will be further described by way of examples below, but the present application is not limited to the scope of the examples. The experimental methods in the following examples, for which no specific conditions are indicated, are selected according to conventional methods and conditions, or according to the instructions of the commercial products.

[0103] The structural formula of each component of the Bletilla striata extract is shown in the following table:

[0104] Example 1:

[0105] This example investigates the extraction method of the Bletilla striata extract.

[0106] I. Experimental instruments and materials

[0107] A. Name: Intelligent digital display PID control electric heating jacket, manufacturer: Gongyi Yuhua Instrument Co., Ltd., model: ZNHW-II.

[0108] B. Name: Electronic balance, manufacturer: Mettler-Toledo Instruments (Shanghai) Co., Ltd., model: ML3002T.

[0109] C. Name: Rotary evaporator, manufacturer: Heidolph Instruments and Equipment (Shanghai) Co., Ltd., model: HEIDOLPH.

[0110] D. Name: Low-temperature cooling pump, manufacturer: Zhengzhou Great Wall Science and Technology Co., Ltd., model: DLSB-5.

[0111] E. Name: Circulating water type multi-purpose vacuum pump, manufacturer: Zhengzhou Great Wall Science and Technology Co., Ltd., model: SHB-III.

[0112] II. Preparation method:

[0113] Take 1 kg of dry tuber powder of Bletilla striata (Thunb.) Reichb. f. purchased from Shanghai Kangqiao Chinese Herbal Medicine Slice Co., Ltd., produced in Guizhou, batch number: 220110, add 10 L of 60% volume percentage ethanol aqueous solution, heat reflux extraction twice at 85-100°, heat reflux extraction for 1 hour, filter using 300 mesh filter cloth, a total of 2 times, combine the filtrate, recover the solvent, dry at 50°C under reduced pressure, to obtain the Bletilla striata extract (Bletilla striata 60% ethanol extract), a total of 140.96 g.

[0114] Example 2:

[0115] This example investigates the preparation method of the Bletilla striata effective fraction.

[0116] I. Experimental instruments and materials

[0117] A. Name: Electronic balance, manufacturer: Mettler-Toledo Instruments (Shanghai) Co., Ltd., model: ML3002T.

[0118] B. Name: Rotary Evaporator; Manufacturer: HEIDOLPH Instruments (Shanghai) Co., Ltd.; Model: HEIDOLPH.

[0119] C. Name: Cryogenic Cooling Pump, Manufacturer: Zhengzhou Great Wall Science & Industry Trade Co., Ltd., Model: DLSB-5.

[0120] D. Name: Circulating water type multi-purpose vacuum pump, Manufacturer: Zhengzhou Great Wall Science & Industry Trade Co., Ltd., Model: SHB-Ⅲ.

[0121] E. Name: AB-8 macroporous resin, Manufacturer: Shanghai Titan Technology Co., Ltd., Model: AB-8, Item No.: 013763538.

[0122] II. Preparation method:

[0123] 100g of Bletilla striata 60% ethanol extract was dissolved in 2L of 10% ethanol and loaded onto 2L of AB-8 macroporous resin. The sample was eluted sequentially with 10% ethanol and 90% ethanol, with each elution being three column volumes. The eluents of 90% ethanol were combined, the solvent was recovered, and the solution was dried to obtain the effective fraction of Bletilla striata (90% ethanol effective fraction), totaling 31.53g.

[0124] Example 3:

[0125] This embodiment characterizes the extract of Bletilla striata and the main components of the effective parts of Bletilla striata.

[0126] I. Experimental Instruments and Materials

[0127] Waters Acquity I-CLASS TM UPLC (Ultra-High Performance Liquid Chromatography) system (Waters Corporation, USA); Waters Xevo G2-XS quadrupole tandem time-of-flight mass spectrometer (Waters Corporation, UK). Mettler Toledo MS105 electronic analytical balance (Mettler Toledo, Switzerland); Milli-Q Advantange A10 pure water system (Millipore, USA); KQ-250DE ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.). Acetonitrile, methanol (LC-MS grade, Fisher Scientific, USA); methanol (chromatographic grade, Fisher Scientific, USA); formic acid, acetic acid (LC-MS grade, Fisher Scientific, USA).

[0128] II. Preparation of the test solution

[0129] Bletilla striata 60% ethanol extract 30 mg, Bletilla striata 90% ethanol extract 10 mg, respectively, into a 2 mL volumetric flask, ultrasonic methanol for 30 minutes, then add water to the mark, as the test solution.

[0130] Respectively, precision weighing of Densiflorin I (DSTDP003901), Bletilloside A (CFN91183), Chiratin I (DST231010-096), Dactylorhin A (DSTDD023301), Bletilla striata glycoside (DSTDD023301), Chiratin III (CFN95033) standard configuration into 0.02 mg / ml, 0.02 mg / ml, 0.03 mg / ml, 0.07 mg / ml, 0.03 mg / ml, 0.04 mg / ml, 0.02 mg / ml, 0.02 mg / ml of standard solution. Among them, Bletilloside A, Chiratin III purchased from Wuhan Tianzhi Biological Technology Co., Ltd., Densiflorin I, Chiratin I, Dactylorhin A and Bletilla striata glycoside purchased from Chengdu Lemaitian Pharmaceutical Technology Co., Ltd.

[0131] III. Chromatography and mass spectrometry conditions

[0132] The positive and negative ion modes were used, and the following chromatographic conditions and mass parameters were detected.

[0133] Chromatographic column: Waters ACQUITY HSS T3 chromatographic column (2.1 x 100 mm, 1.7 μm);

[0134] Column temperature: 30℃;

[0135] Flow rate: 0.30 mL / min;

[0136] Injection volume: 2 μL

[0137] Detection wavelength: 190-410 nm

[0138] Mobile phase: A phase: 0.5% formic acid-methanol (percentage of formic acid / methanol volume percentage): acetonitrile (1:1), B phase: 0.1% acetic acid-water (percentage of acetic acid / water volume percentage)

[0139] Elution gradient:

[0140] Mass spectrometry parameters: electrospray ion source (ESI), positive and negative ion mode, Desolvation Gas flow rate of 800 L / h, Desolvation temperature of 600℃, Source temperature 120℃, Capillary voltage 3.0 kV. Sampling Cone 40 V, Source Offset 80 V. MSE scan mode detection, low energy scan transmission collision energy of 6 eV, high energy scan transmission collision energy of 40-60 eV, nebulizer pressure of 6.5 x 105 Pa, Cone gas flows of 50 L / h, scan range m / z 50-1500. Leucine-enkephalin (m / z 554.2615 [M-H]-) and (m / z 556.2771 [M+H]+) as external standard (Lock Spray™) for mass real-time correction, volume flow set to 5 μL / min.

[0141] Four, chemical composition analysis

[0142] Through mass spectrometry fragmentation fragment analysis combined with standard comparison, 8 chemical components were identified in Bletilla striata 60% ethanol extract and Bletilla striata 90% ethanol effective fraction, as shown in Table 1.

[0143] Table 1. Identification results of chemical components of Bletilla striata 60% ethanol extract and 90% ethanol effective fraction * indicates consistent comparison with the control.

[0144] Example 4

[0145] This example investigates the content of the main components Bletilla striata glycoside and Dactylorhin A in Bletilla striata extract and Bletilla striata effective fraction.

[0146] I. Experimental instruments and materials

[0147] A. Name: ultra-high performance liquid chromatograph, manufacturer: Agilent Technologies, USA, model: Agilent 1290 Infinity II.

[0148] B. Name: electronic balance, manufacturer: Mettler-Toledo Instruments (Shanghai) Co., Ltd., model: MS204S.

[0149] C. Name: medical digital ultrasonic cleaner, manufacturer: Kunshan Ultrasonic Instruments Co., Ltd., model: KQ-250DE.

[0150] II. Preparation of test solution

[0151] The white and 60% ethanol extract and white 90% ethanol effective site powder were dissolved in methanol, and then diluted with methanol to prepare 21.80 mg / ml and 7.99 mg / ml solutions, respectively. The solutions were filtered through a 0.22 μm filter to obtain the sample solution for UPLC analysis.

[0152] III. Chromatographic conditions

[0153] Chromatographic column: Waters ACQUITY HSS T3 chromatographic column (2.1 x 100 mm, 1.7 μm);

[0154] Column temperature: 30°C;

[0155] Flow rate: 0.30 mL / min;

[0156] Injection volume: 2 μL

[0157] Detection wavelength: 221 nm

[0158] Mobile phase: A phase: methanol: acetonitrile (1:1), B phase: water

[0159] Elution gradient:

[0160] IV. Content determination results

[0161] The content determination results are shown in Table 2. At the same time, the Dactylorhin A and white glycoside contents were increased after the white 60% ethanol extract was enriched by a macroporous resin.

[0162] Table 2. Content determination results

[0163] The content percentage is the percentage of Dactylorhin A or white glycoside in the total mass of the extract.

[0164] As shown in the above table, the content of small molecule components in the 90% ethanol fraction was significantly increased compared with the 60% ethanol extract.

[0165] Example Five

[0166] This example investigated the therapeutic effect of white extract and white effective site on the pain and bone joint damage of SD rats caused by partial meniscectomy combined with anterior cruciate ligament transection (pMMx+ACLT) osteoarthritis.

[0167] I. Test substances

[0168] Test substances: white (Bletilla striata (Thunb.) Reichb. f.) 60% ethanol extract, white 90% ethanol effective site.

[0169] II. Experimental instruments and materials

[0170] A. Name: Von Frey filaments, manufacturer: DanMic Global, model: Aesthesio.

[0171] B. Name: Two-foot balance pain tester, manufacturer: Nanjing Calvin Biological Technology Co., Ltd., model: KW-11A.

[0172] C. Name: Respiratory anesthesia machine, manufacturer: Shanghai Yuren Scientific Instrument Co., Ltd., model: ABS-4A.

[0173] D. Name: Rat NO ELISA Kit detection kit, Rat IL-1β ELISA Kit detection kit, manufacturer: Shanghai Junrun Biological Technology Co., Ltd.

[0174] III. Experimental animals and feeding

[0175] 1. Animals

[0176] Species and strain: SD rats.

[0177] Level: SPF level.

[0178] Gender: male.

[0179] Source: Shanghai Slike Experimental Animals Co., Ltd.

[0180] Experimental animal quality certificate number: 20230009003824 / 20230009003825.

[0181] Experimental animal production license number: SCXK (Shanghai) 2023-0009.

[0182] Number of animals: 40.

[0183] The weight of the animals at the beginning of the experiment: 200-250g.

[0184] Adaptation time: 7 days.

[0185] Animal number method: Each mouse cage is hung with an identity card with information such as project number, experimental group, experimenter's name, animal species, etc. Experimental rats are labeled with tail root lines.

[0186] 2. Environment

[0187] The environment of the animal room is kept at a temperature of 23±2℃, humidity of 40-70%, and 12 hours of light and dark alternation. Animals are housed in cages with 4 animals each, and the bedding (corn cob bedding, Suzhou Gule Laboratory Equipment Co., Ltd.) is changed twice a week.

[0188] 3. Food and drinking water

[0189] SPF size mouse growth and reproduction feed Co during the experiment 60 Sterilization, purchased from Suzhou Glee Laboratory Equipment Co., Ltd. The experimental animals used water with high pressure sterilization filter.

[0190] 4. Animal selection and fasting

[0191] The animals used for the experiment will maintain a healthy condition, and the animals will be free to eat and drink during the experiment.

[0192] Four, experimental methods

[0193] 1. Animal grouping

[0194] One week after the operation modeling, the animals were randomly divided into 5 groups, 8 in each group.

[0195] Table 3. Animal grouping and drug administration scheme

[0196] Note: The sham operation group and the model group were given solvent normal saline, Qd*2w means once a day, continuous two weeks of drug administration. Duloxetine group, white and 60% ethanol extract group and white and 90% ethanol effective part group were dissolved in deionized water respectively.

[0197] 2. Modeling and methods

[0198] After the experimental animals were adaptively fed, the normal pain threshold of the knee joint of the operation side (right hind limb) of the rats was tested by Von Frey (Up-Down method) and double foot balance tester before modeling. Then the rats were anesthetized with isoflurane, the knee joint was depilated and disinfected. The skin and joint cavity near the right knee were incised, the knee joint was in the maximum flexion state, the joint cavity was exposed, the medial meniscus was separated, the medial 1 / 3 meniscus was removed, and the anterior cruciate ligament was cut. The sham operation group was only incised skin, opened the joint cavity, and then sutured. The modeling day was recorded as D0, and the drug treatment was given by gavage (1 time / day).

[0199] Each group of rats was tested for changes in pain threshold of the knee joint by Von Frey on D7, D14, D21, and the weight bearing of the lower limbs of the rats was detected by double foot balance tester.

[0200] 3. Measurement of mechanical pain threshold

[0201] Measurement by Up-Down method: The rats were placed in a transparent cage cover, the bottom of which was a mesh metal plate, and were allowed to adapt for about 15 minutes until the spontaneous exploration and combing activities stopped. A Von Frey probe was used to vertically stimulate the middle of the plantar of each rat's hind foot, making it slightly s-shaped, for 5 seconds, and the withdrawal reaction of the paw was observed. If the rat immediately produced a rapid withdrawal, leg lifting or foot licking phenomenon after stimulation, it was considered a positive reaction, and immediate withdrawal after the probe was removed was also considered a positive reaction. Each measurement was separated by about 10 seconds, and if there was no reaction after the probe stimulation, it was considered a negative reaction.

[0202] The rat's foot was stimulated with a series of probes (0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, 15 g), and the rat started with a 2 g probe. If a withdrawal reaction occurred, it was recorded as X, and a weaker level of 1.4 g probe was used for testing. If there was no withdrawal reaction, it was recorded as O, and a stronger level of 4 g probe was used for testing. Both of them were continued to be tested in the same way, and each time a continuous sequence of probes was selected, and there could be no skipping. Each response was recorded, and when the first OX or XO occurred, it was tested four more times, so as to obtain a sequence such as OXOXOX. For rats that still did not have a withdrawal reaction using a 15 g probe, the 50% withdrawal threshold was recorded as 15 g; if there was still a withdrawal reaction using a 0.4 g probe, the 50% withdrawal threshold was recorded as 0.4 g. If neither the 15 g nor the 0.4 g probe was used, the following formula was used for calculation: 50% MWT (g) = (10 [Xf+kδ] ) / 10000

[0203] 50% MWT indicates that the rat has about a 50% chance of withdrawal at this intensity of stimulation, Xf = the value of the last Von Frey probe used (in log units). K value: obtained from the table according to the measured sequence. δ is the average difference between the series of stimulations (in log units, here 0.224).

[0204] The pain threshold improvement rate was calculated according to the following formula:

[0205] Pain threshold improvement rate (%) = (50% MWT of the administration group - 50% MWT of the model group) / 50% MWT of the model group x 100%.

[0206] 5. Measurement of bilateral foot balance bearing value

[0207] The animals were placed in a plexiglass cover of a bilateral foot balance pain instrument, and after the animals completed the exploration behavior, the rat's front limbs were placed on the inclined plane of the device, and the hind limbs were placed on the left and right balance plates, respectively. When the animal was basically inactive, the button was pressed, and the average bearing force of the left and right hind limbs within 5 seconds was measured. Each animal was measured repeatedly 3 times, and each measurement was separated by 10 seconds.

[0208] 6. Measurement of inflammation level

[0209] After the last administration, the rats in each group were fasted for 24 h, and 2 ml to 5 mL of blood was taken from each rat into an EP tube, which was then placed at 4°C for 1 h and centrifuged for 10 min (4°C, 4000 r) to obtain the serum. The content of NO and IL-1β in the serum was detected according to the kit instructions.

[0210] V. Results

[0211] 1. General clinical symptoms of rats after administration

[0212] After a single gavage administration of each test substance, all animals showed no visible abnormalities.

[0213] 2. Effects of Bletilla 60% ethanol extract and Bletilla 90% ethanol effective parts on the body weight of rats with inflammatory pain induced by pMMX+ACLT

[0214] The results are shown in Table 4 below, which shows that the body weight of animals in each group increased over time after administration of each test substance. There was no significant difference between groups.

[0215] Table 4. Effects of Bletilla 60% ethanol extract and Bletilla 90% ethanol effective parts on the body weight of animals in each group (unit: g) (Mean ± SD)

[0216] 3. Effects of Bletilla 60% ethanol extract and Bletilla 90% ethanol effective parts on the mechanical pain threshold of rats with knee joint inflammatory pain induced by pMMx+ACLT

[0217] Table 5. Effects of Bletilla 60% ethanol extract and Bletilla 90% ethanol effective parts on the mechanical pain threshold of rats with knee joint inflammatory pain induced by pMMx+ACLT Note: ### P<0.001 vs sham operation group, *P<0.05, **P<0.01, ***P<0.001 vs model group.

[0218] As can be seen from the above table, the pain inhibiting effect of the Bletilla 60% ethanol extract group and the Bletilla 90% ethanol effective parts group was better than that of the duloxetine group after 21 days of administration; although the effect of the Bletilla 90% ethanol effective parts group was worse than that of the Bletilla 60% ethanol extract group after 7 days of administration (i.e. on the 14th day), it was inferred that although the Bletilla 90% ethanol parts did not act as quickly as the Bletilla 60% extract, it could achieve an effect comparable to that of the Bletilla 60% extract after two weeks of administration.

[0219] Table 6. Effects of Bletilla 60% ethanol extract and Bletilla 90% ethanol effective parts on the mechanical pain threshold of rats with knee joint inflammatory pain induced by pMMx+ACLT

[0220] Results showed that compared with the model group, the pain threshold of rats with inflammatory pain was increased by 261.0% and 79.7% on D14 and D21 after administration of 600 mg / kg of the 60% ethanol extract of Bletilla striata; the pain threshold of rats with inflammatory pain was increased by 121.0% and 79.7% on D14 and D21 after administration of 200 mg / kg of the 90% ethanol effective fraction of Bletilla striata. The pain threshold of rats with inflammatory pain was increased by 123.2% and 66.2% on D14 and D21 after administration of 5 mg / kg of the positive drug duloxetine.

[0221] According to the pain threshold-time curve, it was found that the improvement of the mechanical pain threshold by the 60% ethanol extract of Bletilla striata, the 90% ethanol effective fraction of Bletilla striata and the positive drug was enhanced with the increase of the administration times. On D14 and D21, compared with the model group, the 50% paw withdrawal threshold of rats with inflammatory pain caused by pMMX+ACLT was significantly increased in the groups of the 60% ethanol extract of Bletilla striata, the 90% ethanol effective fraction of Bletilla striata and duloxetine, and the trend analysis result showed that the 60% ethanol extract of Bletilla striata was superior to duloxetine in improving the 50% paw withdrawal threshold of rats with inflammatory pain caused by pMMX+ACLT.

[0222] 5. The effect of the 60% ethanol extract of Bletilla striata and the 90% ethanol effective fraction of Bletilla striata on the difference in the weight-bearing of the hind limbs of rats with inflammatory pain caused by pMMX+ACLT is shown in Table 7.

[0223] Table 7. Effect of administration of the 60% ethanol extract of Bletilla striata and the 90% ethanol effective fraction of Bletilla striata on the difference in the weight-bearing of the hind limbs of rats with inflammatory pain of the knee joint caused by pMMX+ACLT Note: ###P<0.001 VS sham operation group; *P<0.05, **P<0.01, ***P<0.001 vs model group.

[0224] 6. The effect of the 60% ethanol extract of Bletilla striata and the 90% ethanol effective fraction of Bletilla striata on the serum inflammatory level of rats with inflammatory pain caused by pMMX+ACLT is shown in Table 8.

[0225] Table 8. Effect of administration of the 60% ethanol extract of Bletilla striata and the 90% ethanol effective fraction of Bletilla striata on the content of serum inflammatory factors of rats with inflammatory pain of the knee joint caused by pMMX+ACLT Note: #P<0.05, ##P<0.01 VS sham operation group; *P<0.05, **P<0.0, vs model group.

[0226] The results show that after the animals are given the Bletilla striata 60% ethanol extract and the Bletilla striata 90% ethanol effective part by gavage, the levels of inflammatory factors in the serum of the rats are significantly improved compared with the model group, and the Bletilla striata 60% ethanol extract and the Bletilla striata 90% ethanol effective part can significantly reduce the content of NO and IL-1β in the serum of the rats.

[0227] VI. CONCLUSION

[0228] In the SD rat model of knee osteoarthritis induced by pMMX (partial meniscectomy) plus ACLT (anterior cruciate ligament transection), after the Bletilla striata 60% ethanol extract and the Bletilla striata 90% ethanol effective part are given, the experimental results show that the Bletilla striata 60% ethanol extract and the Bletilla striata 90% ethanol effective part can significantly improve the mechanical pain threshold of the model animals, and with re-dosing, the improvement of the mechanical pain threshold is enhanced again. At the same time, the Bletilla striata 60% ethanol extract and the Bletilla striata 90% ethanol effective part can significantly reduce the weight-bearing difference of the hind limbs of the model animals. And the Bletilla striata 60% ethanol extract and the Bletilla striata 90% ethanol effective part can reduce the levels of inflammatory factors in the serum of the rats and improve the inflammatory state.

[0229] In summary, this embodiment verifies that the test substances (the Bletilla striata 60% ethanol extract and the Bletilla striata 90% ethanol effective part) have good therapeutic effects on the pain and bone joint damage of SD rats with osteoarthritis induced by pMMx (partial meniscectomy) plus ACLT (anterior cruciate ligament transection) by the way of gavage once a day for two consecutive weeks.

[0230] Example Six

[0231] This embodiment investigates the therapeutic effects of the main component Bletillan in Bletilla striata on the pain and bone joint damage of SD rats with osteoarthritis induced by pMMx (partial meniscectomy) plus ACLT (anterior cruciate ligament transection).

[0232] I. Test Substances

[0233] Test substance: Bletillan.

[0234] Source: Chengdu Lemaitian Pharmaceutical Technology Co., Ltd.

[0235] Characteristics: solid powder.

[0236] Storage condition: stored at 4°C.

[0237] II. Experimental Instruments and Materials

[0238] A. Name: Von Frey filaments, manufacturer: DanMic Global, model: Aesthesio.

[0239] B. Name: double-foot balance pain tester, manufacturer: Nanjing Calvin Biological Technology Co., Ltd., model: KW-11A.

[0240] C. Name: Respiratory anesthesia machine, manufacturer: Shanghai Yuren Scientific Instrument Co., Ltd., model: ABS-4A.

[0241] III. Experimental animals and feeding

[0242] 1. Animals

[0243] Strain and line: SD rats.

[0244] Level: SPF level.

[0245] Gender: male.

[0246] Source: Shanghai Sulei Experimental Animal Co., Ltd.

[0247] Experimental animal quality certificate number: 20230009003824 / 20230009003825.

[0248] Experimental animal production license number: SCXK (Shanghai) 2023-0009.

[0249] Number of animals: 32.

[0250] Animal body weight at the beginning of the experiment: 200-250g.

[0251] Adaptation time: 7 days.

[0252] Animal number method: Each mouse cage is hung with an identity card with information such as project number, experimental group, experimenter's name, animal breed, etc. The experimental rats are labeled with tail root lines.

[0253] 2. Environment

[0254] The environment of the animal room is kept at a temperature of 23±2℃, humidity of 40-70%, and 12 hours of light and dark alternation. Animals are housed in cages with 4 animals each, and the bedding (corn cob bedding, Suzhou Gle Laboratory Equipment Co., Ltd.) is changed twice a week.

[0255] 3. Food and water

[0256] SPF rat growth and reproduction feed Co 60 was sterilized and purchased from Suzhou Gle Laboratory Equipment Co., Ltd. Filtered water was used for experimental animals.

[0257] 4. Animal selection and fasting

[0258] The animals used for the experiment will remain in good health, and the animals will be free to eat and drink during the experiment.

[0259] IV. Experimental methods

[0260] 1. Animal grouping:

[0261] One week after surgery modeling, the animals were randomly divided into 4 groups, 8 in each group.

[0262] Table 9. Animal grouping and drug administration scheme

[0263] Note: The sham operation group and the model group were given solvent normal saline, Qd*3w means once a day, continuous three weeks of drug administration. Duloxetine group and Baiji group were dissolved in deionized water respectively.

[0264] 2. Modeling and method

[0265] After the experimental animals were adaptively fed, the normal pain threshold of the knee joint of the operation side (right hind limb) of the rats was tested by Von Frey (Up-Down method) and double foot balance tester before modeling. Then the rats were anesthetized with isoflurane, the knee joint was depilated and disinfected. The skin and joint cavity near the right knee were incised, the knee joint was in the maximum flexion state, the joint cavity was exposed, the medial meniscus was separated, the medial 1 / 3 meniscus was excised, and the anterior cruciate ligament was cut. The sham operation group was only incised skin, opened the joint cavity, and then sutured. The modeling day was recorded as D0, and the drug treatment was given by gavage (1 time / day).

[0266] Each group of rats was tested for changes in pain threshold of the knee joint by Von Frey on D7, D8, D15, D22, and the weight-bearing condition of the lower limbs of the rats was detected by double foot balance tester on D7, D10, D17, D24.

[0267] 3. Measurement of mechanical pain threshold

[0268] Up-Down method was used for measurement: the rats were placed in a transparent cage cover, the bottom was a sieve-shaped metal plate, and the animals were adapted for about 15 min until the spontaneous exploration and combing activity stopped. The Von Frey probe was vertically stimulated in the middle of the sole of each rat, making it slightly s-shaped, for 5s, and the paw withdrawal reaction was observed. The rat immediately produced rapid withdrawal, leg lifting or licking feet after stimulation was considered as positive reaction, and immediate withdrawal after removing the probe was also considered as positive reaction. Each measurement interval was about 10s, and if there was no reaction after probe stimulation, it was considered as negative reaction.

[0269] The rats were stimulated on the plantar surface with a series of probes (0.4, 0.6, 1, 1.4, 2, 4, 6, 8, 10, 15 g). The rats were started with a 2 g probe, and if a withdrawal response occurred, it was recorded as X, and the next weaker probe, 1.4 g, was used. If no withdrawal response occurred, it was recorded as O, and the next stronger probe, 4 g, was used. The same procedure was followed for each subsequent test, using consecutive probes in the series, and no skipping of probes was allowed. The responses were recorded for each test, and when the first OX or XO occurred, the test was repeated four more times, and a series was obtained, such as OXOXOX. For rats that did not show a withdrawal response using the 15 g probe, the 50% withdrawal threshold was recorded as 15 g. If a withdrawal response occurred using the 0.4 g probe, the 50% withdrawal threshold was recorded as 0.4 g. For rats that did not use the 15 g or 0.4 g probe, the 50% withdrawal threshold was calculated according to the following formula: 50% MWT (g) = (10 [Xf+kδ] ) / 10000

[0270] 50% MWT indicates that the rats had approximately a 50% chance of withdrawal at that intensity of stimulation, Xf = the value of the last Von Frey probe used (in log units). K value: obtained from a table according to the sequence measured. Delta is the average difference between consecutive stimuli (in log units, here 0.224).

[0271] The pain threshold increase was calculated according to the following formula:

[0272] Pain threshold increase (%) = (50% MWT of the treatment group - 50% MWT of the model group) / 50% MWT of the model group x 100%.

[0273] 5. Measurement of bilateral balance weight-bearing value

[0274] The animals were placed in the plexiglass cover of the bilateral balance pain instrument, and after the animals completed the exploration behavior, the forelimbs of the rats were placed on the inclined surface of the device, and the hind limbs were placed on the left and right balance plates, respectively. When the animals were basically inactive, the button was pressed, and the average bearing force of the left and right hind limbs within 5 seconds was measured. Each animal was measured repeatedly for 3 times, and the interval between each measurement was 10 seconds.

[0275] V. Results

[0276] 1. Observation of general clinical symptoms of rats after administration

[0277] After single gavage administration of the test substance, all animals had no visible abnormalities.

[0278] 2. Effect of Bletilla striata glycosides on the mechanical pain threshold of rats with arthritis pain induced by pMMx+ACLT

[0279] Table 10. Effect of Bletilla striata glycosides administration on the mechanical pain threshold of rats with knee arthritis pain induced by pMMx+ACLT Note: ### P<0.001 vs sham group, *P<0.05, **P<0.01, ***P<0.001 vs model group.

[0280] Table 11. Effects of Bailingoside on the improvement rate of pain threshold of rats with inflammatory pain induced by pMMX+ACLT

[0281] The results showed that compared with the model group, the pain threshold of rats with inflammatory pain was increased by 94.9%, 130.4% and 135.7% on D8, D15 and D22 after administration of 50 mg / kg of Bailingoside. The pain threshold of rats with inflammatory pain was increased by 57.8%, 89.5% and 55.9% on D14 and D21 after administration of 5 mg / kg of duloxetine.

[0282] According to the pain threshold-time curve, it was found that the improvement of mechanical pain threshold by Bailingoside and positive drugs was enhanced with the increase of administration times. On D8, D15 and D22, the 50% paw withdrawal threshold of SD rats with inflammatory pain induced by pMMX+ACLT was significantly improved in the Bailingoside and duloxetine groups compared with the model group. Trend analysis showed that Bailingoside was superior to duloxetine in improving the 50% paw withdrawal threshold of SD rats with inflammatory pain induced by pMMX+ACLT.

[0283] 5. Effects of Bailingoside on the difference in weight bearing of both hind limbs of rats with inflammatory pain induced by pMMX+ACLT, and the results are shown in Table 12.

[0284] Table 12. Effects of Bailingoside on the difference in weight bearing of both hind limbs of rats with inflammatory pain of knee joint induced by pMMX+ACLT Note: ### P<0.001 VS sham group, *P<0.05, ***P<0.001 vs model group.

[0285] The results showed that compared with the model group, the difference in weight bearing of both hind limbs of rats with inflammatory pain of knee joint was significantly improved on D10, D17 and D24 after administration of Bailingoside, and Bailingoside could significantly reduce the difference in weight bearing of both hind limbs of model animals.

[0286] Six. Conclusion

[0287] In the model of SD rats with inflammatory pain of knee joint induced by pMMX (partial meniscectomy) plus ACLT (anterior cruciate ligament transection), the results showed that Bailingoside could significantly improve the mechanical pain threshold of model animals, and the improvement of mechanical pain threshold was enhanced again with re-administration. At the same time, Bailingoside could significantly reduce the difference in weight bearing of both hind limbs of model animals.

[0288] In summary, the present example demonstrates that the test substance (albusin) has a good therapeutic effect on osteoarthritis pain and bone and joint damage in SD rats induced by partial meniscectomy combined with anterior cruciate ligament transection (pMMx+ACLT) through once-daily, three-week gavage administration of the test substance.

Claims

1. The use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of osteoarthritis and / or pain, said compound being a compound as shown in Formula I or ginsenoside I; In the compound shown in Formula I: Indicates a single bond or a double bond; When it is a double bond, X is CR. 1 R 1 For H or C 1-6 alkyl; When it is a single bond, X is C*R. 2 R 3 R 2 For H or C 1-6 Alkyl; R 3 For H, C 1-6 alkyl, hydroxy or R 4 It is a hydroxyl group or -C(O)-C 1-6 alkyl; Carbon atoms marked with "*" are chiral carbon atoms, and their configurations are R configuration, S configuration, or a mixture of R and S configurations; L represents a single bond or The O atom is connected to the M atom; M and K are either single bonds or C. 1-6 Alkylene; R 5 and R 6 H or C independently 1-6 Alkyl group.

2. The application as described in claim 1, characterized in that, It meets one or more of the following conditions: (1)R 1 R 2 R 3 and -C(O)-C 1-6 C in alkyl 1-6 The alkyl group is independently methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, or tert-butyl; (2) In M and K, the C 1-6 Alkylenes are independently straight-chain alkylenes, such as methylene, ethylene, etc. (3)R 5 and R 6 In, the C 1-6 The alkoxy group is independently methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, or tert-butoxy; (4) The pain is pain related to osteoarthritis; preferably, the osteoarthritis in the pain related to osteoarthritis is traumatic osteoarthritis or inflammatory osteoarthritis; (5) In the drug, the compound or its pharmaceutically acceptable salt is the sole active ingredient; (6) The drug is administered orally; (7) The single dose of the drug is 30-80 mg / kg, for example 40-60 mg / kg, or even 50 mg / kg, based on the dosage of the active ingredient. (8) In the drug, the mass concentration of the compound or a pharmaceutically acceptable salt thereof is 1-30 mg / mL; for example, 1-10 mg / mL, or even 5 mg / mL; (9) The osteoarthritis mentioned is traumatic osteoarthritis or inflammatory osteoarthritis.

3. The application as described in claim 1, characterized in that, It meets one or more of the following conditions: (1)R 1 For H; (2)R 2 C 1-6 Alkyl, for example (3)-C(O)-C 1-6 C in alkyl 1-6 The alkyl group is methyl; (4) for (5)R 3 hydroxyl or (6) The carbon atom marked with "*" is a chiral carbon atom with an R configuration; (7) When M and L are double bonds, M and L are single bonds; (8) When it is a single bond, M is C. 1-6 Alkylene, such as methylene; L is The O atom is connected to the M atom; (9) for (10)R 5 C 1-6 Alkyl groups, such as methoxy groups; (11) K is methylene; (12)R 6 For H.

4. The application as described in claim 1, characterized in that, The compound shown in Formula I is any of the following compounds:

5. The application as described in claim 2, characterized in that, It meets one or more of the following conditions: (1) The bone joint mentioned is the knee joint; (2) The traumatic osteoarthritis is traumatic osteoarthritis related to meniscus injury and / or ligament injury, such as traumatic osteoarthritis related to meniscus injury and ligament injury; the ligament injury may be a ligament rupture, such as an anterior cruciate ligament transection; the meniscus injury may be a meniscectomy. (3) The inflammatory osteoarthritis mentioned is IL-1β-mediated inflammatory osteoarthritis.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises 1%-70% bletilla striata glycoside and 0.5%-20% 2-O-glucosyl bletilla striata glycoside, wherein the percentage represents the mass of each component as a percentage of the total mass of the pharmaceutical composition.

7. The pharmaceutical composition according to claim 6, characterized in that, It meets one or more of the following conditions, (1) The mass percentage of bletilla striata glycoside is 1%-30%, for example 10%-15%; or, the mass percentage of bletilla striata glycoside is 10%-70%, for example 20%-50%; (2) The mass percentage of the 2-O-glucosyl leucoside is 0.5%-10%, for example 1%-6%; or the mass percentage of the 2-O-glucosyl leucoside is 3%-20%, for example 5%-15%; (3) The pharmaceutical composition further includes substance C, which includes one or more components selected from 2-O-glucosyl-2-isobutylmalic acid, 2-isobutylmalic acid, sucralose I, pamoatein A, ginsenoside I and ginsenoside III.

8. The pharmaceutical composition according to claim 7, characterized in that, It meets one or more of the following conditions, (1) The mass percentage of the bletilla striata glycoside is 10%-50%, for example 12%-41%; (2) The mass percentage of the 2-O-glucosyl leucoside is 1%-15%, for example 3%-11%; (3) The substance C is selected from any of the following schemes: Scheme 1: The substance C is composed of 2-O-glucosyl-2-isobutylmalic acid, 2-isobutylmalic acid, scutellarin I, pyroside A, ginsenoside I and ginsenoside III; Option 2: The substance C is composed of sucrose glycoside I, puerarin A, and ginsenoside III; Scheme 3, wherein substance C is composed of 2-O-glucosyl-2-isobutylmalic acid, 2-isobutylmalic acid and scutellarin I; Option 4, wherein substance C is 2-O-glucosyl-2-isobutylmalic acid; Scheme 5, wherein substance C is composed of senna glycoside A, ginsenoside I and ginsenoside III.

9. The pharmaceutical composition according to claim 7, characterized in that: The active ingredient of the pharmaceutical composition is selected from one or more of bletilla striata glycoside, 2-O-glucosyl bletilla striata glycoside, and substance C. Preferably, the active ingredient of the pharmaceutical composition is bletilla striata glycoside, 2-O-glucosyl bletilla striata glycoside, a combination of bletilla striata glycoside and 2-O-glucosyl bletilla striata glycoside, or a combination of bletilla striata glycoside, 2-O-glucosyl bletilla striata glycoside and substance C. More preferably, the active ingredient of the pharmaceutical composition is selected from the following: Option 1: The active ingredient of the pharmaceutical composition consists of 10-50% bletilla striata glycoside and 1-15% 2-O-glucosyl bletilla striata glycoside; Option 2: The active ingredient of the pharmaceutical composition comprises 10-50% bletilla striata glycoside, 1-15% 2-O-glucosyl bletilla striata glycoside, and substance C, wherein substance C comprises 2-O-glucosyl-2-isobutylmalic acid, 2-isobutylmalic acid, scutellarin I, pamoatechin A, ginsenoside I, and ginsenoside III; preferably, the mass percentage of bletilla striata glycoside is 10%-15%, and the mass percentage of 2-O-glucosyl bletilla striata glycoside is 1%-6%; or the mass percentage of bletilla striata glycoside is 20%-50%, and the mass percentage of 2-O-glucosyl bletilla striata glycoside is 5%-15%. Option 3: The active ingredient of the pharmaceutical composition comprises 10-50% bletilla striata glycoside, 1-15% 2-O-glucosyl bletilla striata glycoside, and substance C, wherein substance C comprises 2-O-glucosyl-2-isobutylmalic acid, 2-isobutylmalic acid, and scutellarin I; preferably, the mass percentage of bletilla striata glycoside is 10%-15%, and the mass percentage of 2-O-glucosyl bletilla striata glycoside is 1%-6%; or the mass percentage of bletilla striata glycoside is 20%-50%, and the mass percentage of 2-O-glucosyl bletilla striata glycoside is 5%-15%. Option 4: The active ingredient of the pharmaceutical composition comprises 10-50% bletilla striata glycoside, 1-15% 2-O-glucosylbletilla striata glycoside, and substance C, wherein substance C comprises scutellarin A, ginsenoside I, and ginsenoside III; preferably, the mass percentage of bletilla striata glycoside is 10%-15%, and the mass percentage of 2-O-glucosylbletilla striata glycoside is 1%-6%; or the mass percentage of bletilla striata glycoside is 20%-50%, and the mass percentage of 2-O-glucosylbletilla striata glycoside is 5%-15%. More preferably, the active ingredient of the pharmaceutical composition is selected from the following: Option 1: The active ingredient of the pharmaceutical composition consists of 12.76% bletilla striata glycoside, 3.76% 2-O-glucosyl bletilla striata glycoside and substance C, wherein substance C includes 2-O-glucosyl-2-isobutylmalic acid, 2-isobutylmalic acid, scutellarin I, pulsatilla striata glycoside A, ginsenoside I and ginsenoside III. Option 2: The active ingredient of the pharmaceutical composition consists of 40.24% bletilla striata glycoside, 10.51% 2-O-glucosyl bletilla striata glycoside, and substance C, wherein substance C includes 2-O-glucosyl-2-isobutylmalic acid, 2-isobutylmalic acid, sennain I, pamoatein A, ginsenoside I, and ginsenoside III.

10. The pharmaceutical composition according to any one of claims 6-9, characterized in that, The pharmaceutical composition is prepared by the following method, wherein the preparation method is selected from method 1 or method 2: Method 1 includes the following steps: mixing and extracting Bletilla striata with an aqueous alcohol solvent to obtain the pharmaceutical composition; Method 2 includes the following steps: eluting crude Bletilla striata extract in a chromatographic column to obtain the pharmaceutical composition; the packing material of the chromatographic column is a styrene-type macroporous adsorption resin.

11. The pharmaceutical composition of claim 10, characterized in that, It meets one or more of the following conditions: (1) In method 1, the Bletilla striata is the tuber of Bletilla striata, for example, tuber powder; (2) In method 1, the alcohol solvent is methanol and / or ethanol, for example, ethanol; (3) In method 1, the volume percentage of the alcohol solvent in the aqueous solution is 30-90%, for example 50-70%, or even 58%-62%, and the volume percentage is the percentage of the volume of the alcohol solvent / the volume of the aqueous solution of the alcohol solvent. (4) In method 1, the mass ratio of Bletilla striata to the volume ratio of the aqueous alcohol solvent is (0.05-0.15) g: 1 mL, for example (0.08-0.13) g: 1 mL; (5) In method 1, the extraction method is ultrasonic extraction or solvent reflux method, such as solvent reflux method; (6) In method 1, the extraction temperature is 70-100℃, for example 85-100℃; (7) In method 1, the extraction time is 15 min-80 min, for example 40-80 min; (8) In method 1, the number of extractions is ≥1, for example, 1-3 times; (9) Method 1 also includes a post-processing method, such as filtering, removing solvent and drying the extract obtained after extraction; (10) In Method 2, the styrene-type macroporous adsorption resin is AB-8 macroporous resin; (11) In method 2, the elution solvent is an aqueous solution of an alcohol solvent, and the alcohol solvent is methanol and / or ethanol, for example, ethanol; (12) In method 2, the volume percentage of the alcohol solvent in the aqueous solution is 1%-95%, for example 1%-20% or 80%-95%; the volume percentage is the percentage of the volume of the alcohol solvent / the volume of the aqueous solution of the alcohol solvent. (13) In method 2, the elution is divided into a first elution and a second elution; the volume percentage of the alcohol solvent in the aqueous solution of the alcohol solvent in the first elution can be 1%-20%; the volume percentage of the alcohol solvent in the aqueous solution of the alcohol solvent in the second elution can be 80%-95%; in the first elution and the second elution, the volume of the eluting solvent can be (2-5) times the column volume; (14) In method 2, the crude extract of Bletilla striata is loaded in the form of a crude extract solution. The solvent of the crude extract solution is an aqueous solution of an alcohol solvent, and the volume percentage of the alcohol solvent can be 1%-20%. (15) In Method 2, the ratio of the mass of crude Bletilla striata extract to the volume of solvent in the crude Bletilla striata extract solution is (40-60) g: 1 L. (16) In method 2, the preparation method of the pharmaceutical composition further includes a post-processing method, such as removing the solvent from the eluent of the second elution and drying; Preferably, in method 2, the crude product of the Bletilla striata extract is obtained by method 1.

12. A method for preparing a pharmaceutical composition according to any one of claims 6-9, characterized in that, The preparation method is selected from either method 1 or method 2: Method 1 includes the following steps: mixing and extracting Bletilla striata with an ethanol-water solution to obtain the pharmaceutical composition; Method 2 includes the following steps: eluting crude Bletilla striata extract in a chromatographic column to obtain the pharmaceutical composition; the packing material of the chromatographic column is a styrene-type macroporous adsorption resin; The operation and conditions of methods 1 and 2 may also be as described in claim 11.

13. A pharmaceutical composition, characterized in that, The pharmaceutical composition is prepared by the following preparation method, wherein the preparation method is method 1 or method 2 as claimed in claim 12; preferably, the pharmaceutical composition is the pharmaceutical composition as claimed in any one of claims 6-9.

14. A pharmaceutical formulation comprising a pharmaceutical composition and pharmaceutical excipients as described in any one of claims 6-9 and 13.

15. Use of a pharmaceutical composition according to any one of claims 6-9 and 13 or a pharmaceutical preparation according to claim 11 in the preparation of a medicament for the prevention and / or treatment of osteoarthritis and / or pain; wherein the definitions of osteoarthritis and pain may also be as described in claim 2 or 5.

Images