Bone fracture liniment, preparation method thereof and application of bone fracture liniment in preparation of medicine for treating acute soft tissue injury

Abstract

The invention provides a liniment for bone fracture, a preparation method thereof and application of the liniment in preparation of a medicine for treating acute soft tissue injury, and relates to the technical field of medicines. The liniment for bone fracture is prepared from seven medicinal materials, namely safflower, circium japonicum, folium isatidis, herba violae, radix rubiae, folium artemisiae argyi and rumex madder, the dosage of each medicinal material is equal, and each 100ml of liniment for bone fracture is equivalent to 6g of each of the seven medicinal materials. The preparation method comprises the following steps: adding safflower, circium japonicum, folium isatidis, herba violae, rubia cordifolia, folium artemisiae argyi and rumex madder into an ethanol aqueous solution, heating, refluxing and extracting to obtain an extract, then adding the ethanol aqueous solution for dissolving, uniformly mixing and subpackaging to obtain the traditional Chinese medicine composition. The traditional Chinese medicine composition has the effects of promoting blood circulation to remove blood stasis, easing pain, resisting inflammation and resisting oxidative stress, can be used for treating acute soft tissue injury, and has a better treatment effect than common medicines such as bonesetting water and blood-activating and pain-relieving ointment. The liniment for bone fracture developed by the invention can provide a new drug choice for clinical treatment of acute soft tissue injury.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to an orthopedic liniment, a preparation method thereof, and an application thereof in the preparation of a drug for treating acute soft tissue injuries. Background Art

[0002] Acute soft tissue injury is an injury to tissues such as muscles, tendons, joint ligaments, and bursae caused by a large external violent damage to the body, mainly manifested as no wound on the body surface, tissue swelling, pain, and limited movement. According to statistics, 70% of the sports system injuries in military training belong to acute soft tissue injuries, and this type of injury is also the most common injury type among the general public in daily sports. In military training or daily sports, even a slight change in the soft tissues such as the ankle joint, knee joint, and the surrounding ligaments and tendon sheaths of the lower limbs will cause symptoms of acute joint and soft tissue injuries such as pain and limping. Acute soft tissue injury is a common disease type in the emergency department and orthopedic outpatient clinic. After its occurrence, it often restricts the joint and limb activities of patients and is accompanied by a certain degree of physiological pain, seriously affecting the daily work, study, and life of patients. Preventing and treating military training injuries and sports injuries has become an important issue that urgently needs to be studied and solved in the fields of military medicine and training medicine. Early diagnosis and timely treatment of acute soft tissue injuries help to promote the health recovery of patients and effectively improve the prognosis of patients.

[0003] After the occurrence of acute soft tissue injury, modern medicine generally uses antipyretic and analgesic drugs, non-steroidal anti-inflammatory drugs, and hormonal drugs for treatment in clinical practice. For those with severe dysfunction, a fixed frame is also used for auxiliary treatment. Although chemically synthesized drugs take effect quickly, they are prone to produce toxic and side effects and adverse reactions during use, and even cause organ damage. The fixed frame helps to relieve the clinical symptoms of patients, but it is inconvenient to use and is prone to induce infection and produce a series of complications.

[0004] Traditional Chinese medicine has a unique basic theoretical system and clinical practice efficacy in the treatment of orthopedic diseases. Traditional Chinese medicine theory believes that orthopedic diseases such as acute soft tissue injuries belong to the category of "muscle injuries". The causes of "muscle injuries" include external violence, violent impacts, heavy object crushing, accidental falls, and strong torsion, etc.; the pathological mechanism is that after human body injuries, blood stasis will be formed, resulting in qi stagnation and blood stasis, and then the collaterals will be blocked; the main clinical manifestations are local swelling, pain, limited movement and other symptoms. From the perspective of traditional Chinese medicine treatment, the occurrence of bone injuries is due to the influence on the qi and blood of bones and joints, resulting in poor qi and blood circulation and limb movement disorders. Therefore, the treatment of orthopedic diseases not only needs to improve the patient's trauma surface, but also pays more attention to dredging the meridians, promoting qi and activating blood circulation, and regulating the patient's body functions from the inside. Traditional Chinese medicine for the treatment of orthopedic diseases mainly uses drugs for promoting blood circulation to remove blood stasis, promoting qi to relieve pain, clearing heat and cooling blood. Although it takes a long time, the treatment effect is good and the incidence of adverse reactions is low.

[0005] Based on the above, there is an urgent need to develop a traditional Chinese medicine compound drug that has a definite therapeutic effect on acute soft tissue injuries and has no serious adverse reactions. Summary of the Invention

[0006] In order to solve the technical problems existing in the above background technology, the research team of this application, after in-depth research and through a large number of scientific experiments, proposed an orthopedic liniment, its preparation method, and its application in the preparation of drugs for the treatment of acute soft tissue injuries. At present, there are no relevant literature reports on the preparation, quality control of orthopedic liniment and its therapeutic effect on acute soft tissue injuries.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides an orthopedic liniment, which is prepared from seven medicinal materials including safflower, thistle, folium isatidis, viola yedoensis, madder, folium artemisiae argyi and rumex obtusifolius.

[0009] As a further illustration of the present invention, the dosages of each of the seven medicinal materials are equal, and every 100 ml of the orthopedic liniment is equivalent to 6 g of each of the seven medicinal materials.

[0010] As a further illustration of the present invention, the medical auxiliary material used in the preparation is an ethanol aqueous solution.

[0011] As a further illustration of the present invention, the relative density of the orthopedic liniment is 0.89 - 0.93, the pH value is 5.7 - 6.0, and the ethanol content is not less than 65%; the content of hydroxysafflor yellow A is not less than 141 μg / ml, and the content of aesculetin is not less than 110 μg / ml.

[0012] The second aspect of the present invention provides a preparation method of an orthopedic liniment, which includes the following steps:

[0013] Step 1: Add the medicinal materials of Carthamus tinctorius, Cirsium japonicum, Isatis indigotica, Viola philippica, Rubia cordifolia, Artemisia argyi, and Rumex madaio Makino to the first medical adjuvant, and perform heating and reflux extraction to obtain an extract.

[0014] Step 2: Add the extract obtained in Step 1 to the second medical adjuvant for dissolution, mix well, and divide into portions to obtain the bone injury liniment.

[0015] As a further description of the present invention, Step 1 specifically includes the following process: Take the medicinal materials of Carthamus tinctorius, Cirsium japonicum, Isatis indigotica, Viola philippica, Rubia cordifolia, Artemisia argyi, and Rumex madaio Makino with the same mass, crush them into coarse powder, mix well, place them in an ethanol aqueous solution for soaking, perform multiple heating and reflux extractions, combine the extraction solutions, concentrate under reduced pressure and evaporate to dryness to obtain the extract.

[0016] As a further description of the present invention, Step 2 specifically includes the following process: Add the extract obtained in Step 1 to an ethanol aqueous solution for dissolution, mix well, and divide into portions to obtain the bone injury liniment.

[0017] As a further description of the present invention, the first medical adjuvant used in Step 1 is an 80% ethanol aqueous solution; the second medical adjuvant used in Step 2 is a 70% ethanol aqueous solution.

[0018] The third aspect of the present invention provides the application of the bone injury liniment described in any one of the above or the bone injury liniment prepared by the preparation method of the bone injury liniment described in any one of the above in the preparation of drugs for treating acute soft tissue injuries.

[0019] As a further description of the present invention, the bone injury liniment can be used to treat acute soft tissue injuries, and can effectively control the tissue swelling and congestion symptoms in the injury area, accelerate the soft tissue regeneration and repair process, and promote the recovery of motor function in the injury area by improving the microcirculation disorder in rats with acute soft tissue injuries, increasing the pain threshold, inhibiting local inflammatory reactions and oxidative stress injury mechanisms.

[0020] As a further description of the present invention, the formula analysis of the bone injury liniment: In the formula, Carthamus tinctorius promotes blood circulation to remove stasis and benefits the blood vessels, and Cirsium japonicum cools the blood to stop bleeding and disperse stasis and reduce swelling, and they are the monarch drugs together; Isatis indigotica cooperates with Viola philippica and Rubia cordifolia to enter the blood aspect, clear heat and cool the blood to eliminate macules, and they are the minister drugs together; Artemisia argyi dispels wind-cold-dampness arthralgia, so that arthralgia and stasis are resolved together, and it is the assistant drug; Rumex madaio Makino is bitter in taste and cool in nature, enters the lung and large intestine meridians, cools the blood to stop bleeding, and dispels stasis and reduces swelling. Traumatic injuries often cause qi stagnation and blood stasis to cause pain, and combined with the invasion of wind-cold-damp pathogens to form arthralgia, arthralgia and stasis are intertwined, and the pain is even more severe. The above various drugs are combined, and have the effects of promoting blood circulation to remove stasis, dispersing stasis and dredging meridians, relaxing tendons and activating collaterals, reducing swelling and relieving pain, and can make the stasis disperse and be resolved, and the pathogenic factors be dispelled and the disease be cured.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects:

[0022] The bone injury liniment provided by the present invention is a traditional Chinese medicine compound containing seven medicinal materials, which is prepared by heating and refluxing extraction with an ethanol aqueous solution using traditional techniques, and the operation is simple; it is a liniment dosage form containing ethanol, with strong drug permeability and convenient use; it has clear quality standards such as character, physical and chemical identification, thin-layer chromatography identification, relative density inspection, pH value inspection, ethanol content inspection, filling quantity inspection, microbial limit inspection, determination of the content of main components, etc., and the quality is controllable; it has the effects of promoting blood circulation to remove blood stasis, relieving pain, anti-inflammatory and anti-oxidative stress, and can be used to treat acute soft tissue injuries, and the treatment effect is better than common drugs such as Zheng Gu Shui and Huoxue Zhitong Plaster, and the curative effect is definite.

[0023] Other features and advantages of this technical solution will be described in the subsequent specification, and, in part, will be obvious from the specification, or will be understood by implementing this technical solution. The purpose and other advantages of this technical solution can be achieved and obtained through the structures specifically pointed out in the written specification and the drawings.

[0024] The technical solution of this technical solution will be further described in detail below through the drawings and examples. Description of the Drawings

[0025] The drawings are used to provide a further understanding of this technical solution, and constitute a part of the specification. Together with the embodiments of this technical solution, they are used to explain this technical solution and do not constitute a limitation to this technical solution. In the drawings:

[0026] Figure 1 It is the thin-layer chromatography identification diagram of the bone injury liniment [A. Cirsium japonicum; B. Isatis indigotica; C. Rubia cordifolia; D. Artemisia argyi (D1. Viewed under an ultraviolet lamp at 365 nm; D2. Sprayed with 5% sulfuric acid ethanol solution, heated and developed, and then viewed under natural light); E. Rumex madaio (E1. Viewed under an ultraviolet lamp at 365 nm; E2. Fumigated with ammonia vapor and then viewed under natural light); G1 - G3. Samples of the bone injury liniment (batch numbers 20221109, 20230106, 20230308); GH. Mixture of the sample of the bone injury liniment (batch number 20230308) and the control medicinal material of Cirsium japonicum; Hc. Control medicinal material of Cirsium japonicum; HN. Mixture of the control medicinal material of Cirsium japonicum and the negative sample without Cirsium japonicum; Nhc. Negative sample without Cirsium japonicum; Fi. Control medicinal material of Isatis indigotica; In. Indirubin reference substance; Nfi. Negative sample without Isatis indigotica; GR. Mixture of the sample of the bone injury liniment (batch number 20230308) and the control medicinal material of Rubia cordifolia; Rru. Control medicinal material of Rubia cordifolia; Nru Negative sample without Rubia cordifolia; GF. Mixture of the sample of the bone injury liniment (batch number 20230308) and the control medicinal material of Artemisia argyi; Fa. Control medicinal material of Artemisia argyi; Nfa. Negative sample without Artemisia argyi; Rrc. Control medicinal material of Rumex madaio; Em. Emodin reference substance; Nrc. Negative sample without Rumex madaio]

[0027] Figure 2HPLC chromatogram of Gushang Liniment [A. Mixed reference solution; B. Test solution (batch number 20230308); C. Negative sample solution without Carthami Flos and Violae Herba; D. Blank solution; 1. Hydroxysafflor yellow A; 2. Aesculetin]

[0028] Figure 3 Standard curve for the determination of the content of Gushang Liniment (A. Hydroxysafflor yellow A; B. Aesculetin; n = 6)

[0029] Figure 4 Comparison of the swelling degree scores at the injury site in rats of each group after treatment [A1. Administered for 1 day ( n = 12); A2. Administered for 3 days ( n = 12); A3. Administered for 5 days ( n = 6); A4. Administered for 7 days ( n = 6); B. Estimated marginal mean of the swelling degree scores after treatment ( n = 6). Note: Compared with the N group, * P < 0.05, ** P < 0.01, **** P < 0.0001; compared with the M group, # P < 0.05]

[0030] Figure 5 Comparison of the congestion degree scores at the injury site in rats of each group after treatment [A1. Administered for 1 day ( n = 12); A2. Administered for 3 days ( n = 12); A3. Administered for 5 days ( n = 6); A4. Administered for 7 days ( n = 6); B. Estimated marginal mean of the congestion degree scores after treatment ( n = 6). Note: Compared with the N group, ** P < 0.01, *** P < 0.001, **** P < 0.0001; compared with the M group, # P < 0.05]

[0031] Figure 6 Comparison of the activity scores in rats of each group after treatment [A1. Administered for 1 day ( n = 12); A2. Administered for 3 days ( n = 12); A3. Administered for 5 days ( n = 6); A4. Administered for 7 days ( n = 6); B. Estimated marginal mean of the activity scores after treatment ( n = 6). Note: Compared with the N group, * P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001]

[0032] Figure 7 Comparison of the pain withdrawal reflex thresholds of rats in each group after treatment [A1. One day after drug administration ( n = 12); A2. Three days after drug administration ( n = 12); A3. Five days after drug administration ( n = 6); A4. Seven days after drug administration ( n = 6); B. Estimated marginal means of PWT values before and after treatment ( n = 6). Note: Compared with the N group, **** P < 0.0001; compared with the M group, # P < 0.05, ## P < 0.01]

[0033] Figure 8 Comparison of whole blood viscosity and plasma viscosity of rats in each group after treatment (A1. Three days after drug administration, WBV-L; A2. Seven days after drug administration, WBV-L; B1. Three days after drug administration, WBV-M; B2. Seven days after drug administration, WBV-M; C1. Three days after drug administration, WBV-H; C2. Seven days after drug administration, WBV-H; D1. Three days after drug administration, plasma viscosity, D2. Seven days after drug administration, plasma viscosity. n = 6. Note: Compared with the N group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; compared with the M group, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001; compared with the ZT group, && P < 0.01, &&&& P < 0.0001; compared with the HP group, @ P < 0.05, @@ P < 0.01)

[0034] Figure 9 Comparison of histopathology of rats in each group after treatment (A. Three days of treatment; B. Seven days of treatment. HE staining, 200×)

[0035] Figure 10 Comparison of tissue inflammatory factor levels of rats in each group after treatment (A1. Three days after drug administration, IL-1β; A2. Seven days after drug administration, IL-1β; B1. Three days after drug administration, IL-6; B2. Seven days after drug administration, IL-6; C1. Three days after drug administration, TNF-α; C2. Seven days after drug administration, TNF-α. n = 6. Note: Compared with group N, *** P < 0.001, **** P < 0.0001; compared with group M, # P < 0.05, ## P < 0.01, #### P < 0.0001; compared with group ZT, & P < 0.05; compared with group HP, @ P < 0.05, @@ P < 0.01, @@@ P < 0.001)

[0036] Figure 11 Comparison of the levels of tissue oxidative stress indexes in rats of each group after treatment (A1. SOD on the 3rd day of drug administration; A2. SOD on the 7th day of drug administration; B1. GSH on the 3rd day of drug administration; B2. GSH on the 7th day of drug administration; C1. MDA on the 3rd day of drug administration; C2. MDA on the 7th day of drug administration. n = 6. Note: Compared with group N, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; compared with group M, # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001; compared with group ZT, & P < 0.05, && P < 0.01; compared with group HP, @ P < 0.05, @@ P < 0.01)

[0037] Figure 12 Comparison of the swelling degree score, congestion degree score and activity score at the injury site of rats after treatment with the bone injury liniment and its formula-deficient preparation (A. Swelling degree score; B. Congestion degree score; C. Activity score. n = 6. Note: Compared with group M, # P < 0.05, ## P < 0.05) Specific embodiments

[0038] The following is a description of the preferred embodiments of the present technical solution with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present technical solution and are not used to limit the present technical solution.

[0039] The following is an illustration with specific embodiments:

[0040] Example 1: Preparation experiment of bone injury liniment

[0041] 1 Materials

[0042] 1.1 Instruments MTS2100 Meilen electronic balance, Shenzhen Meifu Electronics Co., Ltd. HGZF-II-101-1 electric constant temperature blast drying oven, Shanghai Yuejin Medical Instrument Co., Ltd. Q-500B3 Chinese medicine pulverizer, Shanghai Bingdu Electric Appliance Co., Ltd. ZNHW-2000ml temperature control electric heating jacket, Beijing Zhongyi Hongrui Technology Development Co., Ltd. N-1100 rotary evaporator, Japan Eyela Co., Ltd. DLSB-5L / 30 low temperature cooling circulation pump, Shanghai Yike Instrument Co., Ltd. SHZ-D (III) circulating water multi-purpose vacuum pump, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.

[0043] 1.2 Reagent ethanol, pharmaceutical excipient grade, 20kg / barrel, Xinxiang Xianfeng Pharmaceutical New Materials Co., Ltd. Water is purified water, which is made by the hospital preparation room through a secondary reverse osmosis pure water unit.

[0044] 1.3 Test drugs Carthami Flos, batch number 1231226; Cirsii Japonici Herba, batch number 20231001; Isatidis Folium, batch number 1230215; Violae Herba, batch number 2230315; Rubiae Radix et Rhizoma, batch number 1231025; Artemisiae Argyi Folium, batch number 1240106; all purchased from Shaohuatang Chinese Medicine Co., Ltd., Bozhou City, Anhui Province. Rumex Crispus Radix et Rhizoma, batch number 20240407, was purchased from Huixingtang Pharmacy, Bozhou City, Anhui Province. The above medicinal materials were identified by Zhu Bin, deputy chief pharmacist of the hospital's pharmacy department, and are the dried flowers of safflower (Carthamus tinctorius L.) of the Asteraceae family, the dried aerial parts of thistle (Cirsium japonicum Fisch.ex DC.) of the Asteraceae family, the dried whole herb of Viola yedoensis Makino of the Violaceae family, the dried roots and rhizomes of Rubia cordifolia L. of the Rubiaceae family, the dried leaves of Isatis indigotica Fort. of the Cruciferae family, the dried leaves of Artemisia argyi Lévl. et Van. of the Asteraceae family, and the dried roots and rhizomes of Rumex crispus Linn. of the Polygonaceae family.

[0045] 2 Methods and Results

[0046] The prescription of Gushang Liniment includes seven medicinal materials: Flos Carthami, Herba Cirsii Japonici, Folium Isatidis, Herba Violae, Radix Rubiae, Folium Artemisiae Argyi, and Radix Rumicis Acetosellae.

[0047] Take 120 g of Flos Carthami, 120 g of Herba Cirsii Japonici, 120 g of Folium Isatidis, 120 g of Herba Violae, 120 g of Radix Rubiae, 120 g of Folium Artemisiae Argyi, and 120 g of Radix Rumicis Acetosellae according to the proportion of the prescription of Gushang Liniment, crush them into coarse powder, mix them evenly, soak them in 5 times the volume of 80% ethanol aqueous solution for 48 h, then extract by heating under reflux twice, combine the extracts, concentrate under reduced pressure and evaporate to dryness to obtain 232 g of extract, with a yield of 27.6%; add 70% ethanol aqueous solution to dissolve, adjust the total volume of the solution to 2000 ml, mix evenly, and dispense into 100-ml bottles, thus obtaining 20 bottles of Gushang Liniment.

[0048] Example 2: Experiment on the establishment of the quality standard of Gushang Liniment

[0049] 1 Materials

[0050] 1.1 Instruments CPA225D electronic analytical balance, with a maximum load of 220 g and a scale value of 0.01 mg, manufactured by Sartorius, Germany. Nexera-i LC-2040C 3D Plus high-performance liquid chromatography system, including LC-40B XR binary pump, SIL-40C automatic sampler, CTO-40C column oven, and SPD-40V diode array detector, manufactured by Shimadzu, Japan. N-1100 rotary evaporator, manufactured by Eyela, Japan. DLSB-5L / 30 low-temperature cooling circulating pump, manufactured by Shanghai Yike Instrument Co., Ltd. SHZ-D(III) circulating water multi-purpose vacuum pump, manufactured by Shanghai Lichen Bangxi Instrument Technology Co., Ltd. FSJ-A03D1 crusher, manufactured by Little Bear Electric Co., Ltd. ZNHW-2000ml temperature-controlled electric heating mantle, manufactured by Beijing Zhongyi Hongrui Technology Development Co., Ltd. SK7200H numerical control ultrasonic cleaner, manufactured by Shanghai Kedao Ultrasonic Instrument Co., Ltd. ZF-8 four-purpose ultraviolet analyzer in a dark box, manufactured by Shanghai Jihui Scientific Analysis Instrument Co., Ltd. DB-1 digital display stainless steel electric heating plate, manufactured by Changzhou Putian Instrument Manufacturing Co., Ltd. PHS-3C pH meter, manufactured by Shanghai Yidian Scientific Instrument Co., Ltd. MJX-160B-Z mold incubator, manufactured by Shanghai Boxun Industry Co., Ltd., Medical Equipment Factory. YXQ-LS-50SII vertical pressure steam sterilizer, manufactured by Shanghai Sanshen Medical Instrument Co., Ltd. BSC-1304IIA2 biological safety cabinet, manufactured by Suzhou Antai Air Technology Co., Ltd.

[0051] 1.2 Reagents HSG thin layer chromatography silica gel G plate, specification 5cm×20cm, batch number 20140610, Yantai Jiangyou Silica Gel Development Co., Ltd. Thin layer chromatography polyamide film, specification 5cm×10cm, batch number 20230208, Taizhou Luqiao Sijia Biochemical Plastic Factory, Zhejiang Province. Microporous filter membrane, 0.45μm, batch number 20210318, Changde Beekman Biotechnology Co., Ltd. Acetonitrile is chromatographic grade, Merck, Germany. The rest of the reagents are analytical grade, Sinopharm Chemical Reagent Co., Ltd. Water is purified water, which is made by the secondary reverse osmosis pure water unit in the hospital preparation room.

[0052] 1.3 Test bone injury liniment, self-made by the preparation room of the hospital pharmacy department, specification 100ml / bottle, batch number 20221109, 20230106, 20230308. In addition, according to the prescription and its preparation method, negative samples lacking Cirsium japonicum, negative samples lacking Folium Isatidis, negative samples lacking Rubia cordifolia, negative samples lacking Artemisia argyi, negative samples lacking Rheum officinale, negative samples lacking Carthamus tinctorius and negative samples lacking Viola yedoensis were prepared respectively.

[0053] Cirsii Japonici Herba (Cirsii Japonici Herba) reference medicinal materials, batch number 121411-201903; Isatidis Folium (Isatidis Folium) reference medicinal materials, batch number 121367-202204; Rubiae Radix et Rhizoma (Rubiae Radix et Rhizoma) reference medicinal materials, batch number 121049-201906; Artemisiae Argyi Folium (Artemisiae Argyi Folium) reference medicinal materials, batch number 121345-201804; Rumex Crispus Radix et Rhizoma) reference material, batch number 121291-201803; indirubin reference substance, batch number 110717-202106, purity 99.1%; emodin reference substance, batch number 110756-201913, purity 96.0%; hydroxysafflor yellow A reference substance, batch number 11637-202111, purity 96.8%; aesculetin reference substance, batch number 110741-202109, purity 96.0%, were all purchased from China Food and Drug Inspection Institutes.

[0054] 2 Methods and Results

[0055] The quality standards of bone injury ointment include: properties, physical and chemical identification, thin layer chromatography identification, relative density inspection, pH value inspection, ethanol content inspection, filling volume inspection, microbial limit inspection, and main component content determination.

[0056] 2.1 Properties The Bone Injury Liniment is a dark brown liquid preparation, which turns yellowish brown after dilution. It has a special aroma and a slight alcohol irritation.

[0057] 2.2 Physical and Chemical Identification Take 5 ml of the Bone Injury Liniment, add 45 ml of warm water, take 1 ml in a test tube, add 1 drop of ferric trichloride test solution, the solution turns dark green, and then add 2 drops of ammonia water to produce a large amount of reddish brown flocculent precipitate.

[0058] 2.3 Thin Layer Chromatography Identification

[0059] 2.3.1 Cirsium japonicum DC. Take 10 ml of the Bone Injury Liniment, extract it three times with 30 ml of petroleum ether (60℃ - 90℃) respectively, combine the extraction solutions, evaporate to dryness under reduced pressure, dissolve the residue in 10 ml of 70% ethanol solution, ultrasonically treat it (240W, 45Hz) for 30 min, filter, and concentrate the filtrate to 2 ml under reduced pressure as the test solution. Take 10 ml of the negative sample without Cirsium japonicum DC. and prepare the negative sample solution in the same way. Additionally, take 1 g of the Cirsium japonicum DC. reference medicinal material, add 10 ml of methanol, ultrasonically treat it (240W, 45Hz) for 30 min, filter, evaporate the filtrate to dryness under reduced pressure, dissolve the residue in 2 ml of methanol as the reference medicinal material solution. Conduct the experiment according to the thin layer chromatography method (Appendix 0502, Volume IV of Chinese Pharmacopoeia 2020 Edition). Absorb 5 μl of each of the above three solutions and spot them on the same polyamide film respectively. Use acetylacetone - butanone - ethanol - water (1∶3∶3∶17) as the developing agent, develop, take out, and dry. Spray with aluminum trichloride test solution, dry, and examine under an ultraviolet lamp (365 nm). At the corresponding position of the reference medicinal material chromatogram, the test solution chromatogram shows spots of the same color; the negative sample chromatogram has no interference. See Figure 1 A.

[0060] 2.3.2 Isatis indigotica Fortune Take 20 ml of the Bone Injury Liniment, evaporate to dryness under reduced pressure, dissolve the residue in 20 ml of chloroform, filter, and concentrate the filtrate to 2 ml under reduced pressure as the test solution. Take 20 ml of the negative sample without Isatis indigotica Fortune and prepare the negative sample solution in the same way. Additionally, take 0.5 g of the Isatis indigotica Fortune reference medicinal material, add 20 ml of water, heat under reflux for 30 min, filter, evaporate the filtrate to dryness under reduced pressure, dissolve the residue in 20 ml of chloroform, filter, and concentrate the filtrate to 2 ml under reduced pressure as the reference medicinal material solution. Then take an appropriate amount of indirubin reference substance and make a reference substance solution of 1 mg / ml with chloroform. Conduct the experiment according to the thin layer chromatography method (Appendix 0502, Volume IV of Chinese Pharmacopoeia 2020 Edition). Absorb 5 μl of each of the above four solutions and spot them on the same silica gel G thin layer plate respectively. Use cyclohexane - chloroform - acetone (5∶4∶2) as the developing agent, develop, take out, and dry. Examine under natural light. At the corresponding positions of the reference medicinal material chromatogram and the reference substance chromatogram, the test solution chromatogram shows the same purplish red spots; the negative sample chromatogram has no interference. See Figure 1 B.

[0061] 2.3.3 For 10 ml of Rubia cordifolia L. fracture injury liniment, it was evaporated to dryness under reduced pressure, 10 ml of methanol was added, ultrasonicated for 30 min, filtered, and the filtrate was concentrated to 1 ml under reduced pressure to obtain the test solution. Separately, 0.5 g of the reference crude drug of Rubia cordifolia L. and 10 ml of the negative sample without Rubia cordifolia L. were used, and the reference crude drug solution and the negative sample solution were prepared in the same method. The experiment was carried out according to the thin-layer chromatography method (Part IV of the Chinese Pharmacopoeia 2020 Edition, General Rule 0502). 5 μl of each of the above three solutions was respectively spotted on the same silica gel G thin-layer plate, and petroleum ether (60 °C - 90 °C)-acetone (2:1) was used as the developing agent. After development, it was taken out and dried. Sprayed with 5% sulfuric acid ethanol solution, heated at 120 °C until the spots were clearly visible, and examined under daylight. At the position corresponding to the reference crude drug chromatogram, the test solution chromatogram showed spots of the same color; the negative sample chromatogram had no interference. See Figure 1 C.

[0062] 2.3.4 For 20 ml of Artemisia argyi Levl. et Vant. fracture injury liniment, it was extracted three times with 20 ml of petroleum ether (60 °C - 90 °C) each time, and the extraction solutions were combined and evaporated to dryness under reduced pressure. The residue was dissolved in 1 ml of n-hexane to obtain the test solution. 20 ml of the negative control sample without Artemisia argyi Levl. et Vant. was used, and the negative sample solution was prepared in the same method. Separately, 1 g of the reference crude drug of Artemisia argyi Levl. et Vant. was added with 25 ml of petroleum ether (60 °C - 90 °C), heated under reflux for 30 min, filtered, and the filtrate was evaporated to dryness under reduced pressure. The residue was dissolved in 1 ml of n-hexane to obtain the reference crude drug solution. The experiment was carried out according to the thin-layer chromatography method (Part IV of the Chinese Pharmacopoeia 2020 Edition, General Rule 0502). 5 μl of each of the above three solutions was respectively spotted on the same silica gel G thin-layer plate, and cyclohexane-ethyl acetate (6:1) was used as the developing agent. After development, it was taken out and dried. First, it was examined under an ultraviolet lamp (365 nm). At the position corresponding to the reference crude drug chromatogram, the test solution chromatogram showed fluorescent spots of the same color; then it was sprayed with 5% sulfuric acid ethanol solution, heated at 120 °C until the spots were clearly visible, and examined under daylight. At the position corresponding to the reference crude drug chromatogram, the test solution chromatogram showed spots of the same color; the negative sample chromatogram had no interference. See Figure 1 D.

[0063] 2.3.5 Take 10 ml of bone injury liniment from Rhubarb, evaporate to dryness under reduced pressure, add 10 ml of methanol to the residue to dissolve, ultrasonically treat (240W, 45Hz) for 30 min, filter, evaporate the filtrate to dryness, add 10 ml of water to the residue to dissolve, add 1 ml of hydrochloric acid, ultrasonically treat (240W, 45Hz) for 30 min, cool and extract by shaking with cyclohexane twice (20 ml each time), combine the cyclohexane solution, evaporate to dryness under reduced pressure, and add 1 ml of chloroform to the residue to dissolve as the test solution. Take 0.5 g of Rhubarb control medicinal material and 10 ml of Rhubarb negative sample, and prepare control medicinal material solution and negative sample solution in the same way. Take an appropriate amount of Rhubarb reference substance and add methanol to prepare a reference substance solution of 1 mg / ml. Perform the experiment according to the thin layer chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV, General Principle 0502). Take 5μl of each of the above four solutions and spot them on the same silica gel G thin layer plate. Use the upper layer solution of petroleum ether (30℃~60℃)-ethyl acetate-formic acid (15:5:1) as the developing agent, develop, take out and dry. First place it under ultraviolet light (365nm) for inspection. At the corresponding position of the reference medicinal material chromatogram and the reference substance chromatogram, the same color yellow fluorescent spots will appear in the chromatogram of the test sample; then place it in ammonia vapor for fumigation, and place it under sunlight for inspection. The spots of the same color will turn orange-red; there is no interference in the chromatogram of the negative sample. Figure 1 E.

[0064] 2.4 Relative density inspection The relative density determination method (Chinese Pharmacopoeia 2020 Edition Part IV, General Chapter 0601) was used to inspect the relative density of the three batches of bone injury ointment, and the relative density was measured to be 0.9093±0.0054. The results are shown in Table 1.

[0065] Table 1 Results of relative density test of bone injury liniment

[0066]

[0067] 2.5 pH value inspection According to the pH value determination method (Chinese Pharmacopoeia 2020 Edition Part IV, General Chapter 0631), the pH value of the three batches of bone injury ointment was measured to be 5.87±0.04. The results are shown in Table 2.

[0068] Table 2 Results of pH value test of bone injury liniment

[0069]

[0070] 2.6 Ethanol content inspection According to the ethanol content determination method "the ethanol content of the second distillation method is higher than 30%" (Chinese Pharmacopoeia 2020 Edition Part IV, General Rule 0711), the ethanol content of the three batches of bone injury liniment was measured to be (68.53±1.00)%. The results are shown in Table 3.

[0071] Table 3 Results of the test on the amount of ethanol in bone injury liniment

[0072]

[0073] 2.7 The filling quantity inspection was carried out according to the "volumetric method" in the Minimum Filling Quantity Inspection Method (Volume IV of Chinese Pharmacopoeia 2020 Edition, General Rule 0942). Three bottles of Gushang Liniment of each of the 3 batches were taken, and the average filling quantity of the Gushang Liniment of the 3 batches was not less than the labeled filling quantity, and the filling quantity of each bottle was not less than 97% of the labeled filling quantity, meeting the requirements of relevant regulations. The results are shown in Table 4.

[0074] Table 4 Results of the filling quantity inspection of Gushang Liniment

[0075]

[0076] 2.8 Microbiological limit inspection

[0077] 2.8.1 Culture media and diluents Tryptose soya liquid (TSL) medium, batch number 2201262; Tryptose soya agar (TSA) medium, batch number 2204122; Sabouraud dextrose liquid (SDL) medium, batch number 220815; Sabouraud dextrose agar (SDA) medium, batch number 2201092; Mannitol salt agar (MSA) medium, batch number 2202192; Cetrimide agar (CA) medium, batch number 221012; pH 7.0 Sodium chloride peptone (SCP) buffer solution, batch number 220521, all purchased from Beijing Sanyao Science and Technology Development Company.

[0078] 2.8.2 Bacterial strains Staphylococcus aureus (SA strain), CMCC(B)26003; Pseudomonas aeruginosa (PA strain), CMCC(B)10104; Candida albicans (CA strain), CMCC(F)98001; Bacillus subtilis (BS strain), CMCC(B)63501; Aspergillus niger (AN strain), CMCC(F)98003, all purchased from the National Institutes for Food and Drug Control, and each bacterial strain was the 3rd generation.

[0079] 2.8.3 Verification method

[0080] The microbial limit test was carried out according to the microbial counting method (Part IV of Chinese Pharmacopoeia 2020 Edition, General Rule 1105) and the test for controlled bacteria (Part IV of Chinese Pharmacopoeia 2020 Edition, General Rule 1106).

[0081] 2.8.3.1 Preparation method of bacterial suspension. SA, BS, and PA bacterial cultures (fresh) were inoculated into TSL medium (5 ml), placed in a bacterial incubator, and cultured for 24 h (at 32.5 °C). 1 ml of each culture was taken and added to 9 ml of SCP buffer to be diluted to 10 -5 , and a bacterial suspension was prepared (the number of bacteria ≤ 10000 cfu / ml).

[0082] CA bacterial culture (fresh) was inoculated into SDL medium (5 ml), placed in a bacterial incubator, and cultured for 2 d (at 25 °C). 1 ml of the culture was taken and added to 9 ml of SCP buffer to be diluted to 10 -5 , and a bacterial suspension was prepared (the number of bacteria ≤ 10000 cfu / ml).

[0083] AN bacterial culture (fresh) was inoculated onto SDA slant medium, placed in a bacterial incubator, and cultured for 7 d (at 25 °C) to form a large number of spores. 5 ml of SCP buffer [containing 0.05% polysorbate 80 (v / v)] was used to elute the spores, which were filtered into a sterile test tube. 1 ml of the spore solution was taken and added to 9 ml of SCP buffer [containing 0.05% polysorbate 80 (v / v)] to be diluted to 10 -5 , and a bacterial suspension was prepared (the number of bacteria ≤ 10000 cfu / ml).

[0084] 2.8.3.2 Preparation method of test solution. 10 ml was taken from 2 bottles of Gushang Liniment, and SCP buffer was slowly added to make up to 100 ml. After mixing and standing still, it was used as the test solution (1:10). The same operation was carried out for 3 batches of Gushang Liniment samples.

[0085] 2.8.3.3 Membrane filtration method was used to verify the counting methods for the total number of aerobic bacteria, the total number of molds and yeasts

[0086] ① Counting the total number of aerobic bacteria in the test group. 9.9 ml of the test solution under item "2.8.3.2" was taken respectively, and 0.1 ml of the SA, BS, PA, CA, and AN bacterial suspensions under item "2.8.3.1" was added respectively. After mixing, the test solution (the bacterial content ≤ 100 cfu) was respectively injected into 100 ml of SCP buffer, filtered, and rinsed with 5 bottles of SCP buffer (100 ml / bottle). After filtration, the filter membrane was taken out, with the bacterial surface facing up, and was respectively pasted on the prepared TSA medium plates, cultured for 3 d (at 32.5 °C), and counted.

[0087] ②Total count of molds and yeasts in the test group. Respectively take 9.9 ml of the test solution under item "2.8.3.2", add 0.1 ml of the CA bacteria and AN bacteria suspensions under item "2.8.3.1", mix well, so that the test solution (bacterial content ≤ 100 cfu), respectively inject into 100 ml of SCP buffer solution, filter, and rinse with 5 bottles of SCP buffer solution (100 ml / bottle), after filtration, take out the filter membrane, make the bacterial surface face up, respectively stick it on the prepared SDA culture medium plate, culture for 5 d (at 25 °C), and count.

[0088] ③Test article control group. Respectively take 9.9 ml of the test solution under item "2.8.3.2", add the dilution solution (substituting the bacterial solution) and mix well, and operate in the same way as in "①Total count of aerobic bacteria in the test group" and "②Total count of molds and yeasts in the test group", and count.

[0089] ④Bacterial solution control group. Substitute the dilution solution for the test solution under item "2.8.3.2", add the test bacteria, and operate in the same way as in "①Total count of aerobic bacteria in the test group" and "②Total count of molds and yeasts in the test group", and count.

[0090] ⑤Verification results. According to the formula "Recovery ratio of bacteria in the test group = (Average colony count in the test group - Average colony count in the test article control group) / Average colony count in the bacterial solution control group", calculate the recovery ratios of the five bacteria, and the results are shown in Table 5. The recovery ratios of the five test bacteria in 3 batches of orthopedic liniment were all within the range of 0.5 - 2, meeting the requirements of the microbial limit standard for non-sterile drugs (Part IV of the Chinese Pharmacopoeia 2020 Edition, General Rule 1107). It shows that the membrane filtration method can be used for the total count of aerobic bacteria, molds and yeasts in orthopedic liniment.

[0091] Table 5 Verification results of the microbial counting method for orthopedic liniment

[0092]

[0093]

[0094] 2.8.3.4 Verification of the membrane filtration method for the determination of Staphylococcus aureus and Pseudomonas aeruginosa

[0095] ① Test group. Staphylococcus aureus: Take 10 ml of the test solution under item "2.8.3.2" and 1 ml of SA bacteria under item "2.8.3.1", mix well, inject into 100 ml of SCP buffer solution, filter, and rinse with 5 bottles of SCP buffer solution (100 ml / bottle). After filtration, take out the filter membrane, place it in TSB culture medium (100 ml), for 24 h (at 35 °C), streak-inoculate the above TSB culture on MSA culture medium plate for 72 h (at 35 °C). Pseudomonas aeruginosa: Take 10 ml of the test solution under item "2.8.3.2" and 1 ml of PA bacteria under item "2.8.3.1", mix well, inject into 100 ml of SCP buffer solution, filter, and rinse with 5 bottles of SCP buffer solution (100 ml / bottle). After filtration, take out the filter membrane, place it in TSB culture medium (100 ml), for 24 h (at 35 °C), streak-inoculate the above TSB culture on CA culture medium plate for 72 h (at 35 °C).

[0096] ② Negative control group. Replace the test solution under item "2.8.3.2" with diluent, without adding SA bacteria and PA bacteria, and operate according to the method under "① Test group".

[0097] ③ Verification results The verification results of the inspection methods for Staphylococcus aureus and Pseudomonas aeruginosa are shown in Table 6. Staphylococcus aureus and Pseudomonas aeruginosa in the test group were both detected normally, and none were detected in the negative control group, meeting the requirements of the microbial limit standard for non-sterile drugs (Part IV of the Chinese Pharmacopoeia 2020 Edition, General Rule 1107). It shows that the membrane filtration method can be used for the determination of Staphylococcus aureus and Pseudomonas aeruginosa in the traumatic injury liniment.

[0098] Table 6 Verification results of the inspection methods for controlled bacteria in the traumatic injury liniment

[0099]

[0100] Note: "+" represents bacterial growth, and "-" represents no bacterial growth.

[0101] 2.9 Determination of main component content

[0102] 2.9.1 Chromatographic conditions The chromatographic column is Shimadzu C of Shimadzu Corporation 18Column (4.6 mm × 250 mm, 5 μm); The mobile phase was a mixed solution of acetonitrile (A) - 1% aqueous acetic acid solution (B), gradient elution (v / v): 0 min to 10 min, 5% A → 21% A; 10 min to 13 min, 21% A → 26% A; 13 min to 16 min, 26% A → 95% A; 16 min to 19 min, 95% A → 5% A. The flow rate was 1.0 ml / min. The column temperature was 30 °C. The injection volume was 10 μl. Detection wavelength: For hydroxysafflor yellow A was 403 nm, and for aesculetin was 344 nm.

[0103] 2.9.2 Preparation of reference substance solution Weigh accurately about 15 mg of hydroxysafflor yellow A reference substance, place it in a 5 ml volumetric flask, dissolve it with methanol and make up the volume to the mark, shake well to obtain a 3.08 mg / ml hydroxysafflor yellow A reference substance stock solution; Weigh accurately about 10 mg of aesculetin reference substance, place it in a 5 ml volumetric flask, dissolve it with methanol and make up the volume to the mark, shake well to obtain a 2.02 mg / ml aesculetin reference substance stock solution. Store it in a -20 °C refrigerator for later use. Accurately pipette 2 ml each of the hydroxysafflor yellow A reference substance stock solution and the aesculetin reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with 50% methanol aqueous solution, mix well, and make up the volume to prepare a mixed reference substance solution with 616.0 μg / ml of hydroxysafflor yellow A and 404.0 μg / ml of aesculetin.

[0104] 2.9.3 Preparation of test sample solution Accurately pipette 10 ml of the traumatic injury liniment, place it in a 25 ml volumetric flask, dilute to 25 ml with 50% methanol aqueous solution, make up the volume to the mark, tightly stopper it, weigh accurately, ultrasonically treat it (240 W, 45 kHz) for 30 min, let it cool and then weigh accurately again, add 50% methanol aqueous solution to make up for the lost mass, shake well; Take this solution, centrifuge it at 1500 rmp for 15 min in a centrifuge, accurately pipette 20 ml of the supernatant, concentrate it under reduced pressure to dryness, dissolve the residue with 50% methanol aqueous solution, transfer it to a 10 ml volumetric flask, make up the volume to the mark, shake well to prepare the test sample solution. Before detection, filter the test sample solution through a 0.45 μm microporous membrane, take the subsequent filtrate for injection.

[0105] 2.9.4 Preparation of negative sample solution Take the negative sample lacking safflower and viola yedoensis, and prepare the negative sample solution lacking safflower and viola yedoensis according to the above preparation method of the test sample solution.

[0106] 2.9.5 Specificity test Take the mixed reference substance solution (after dilution), the test sample solution, the negative sample solution lacking safflower and viola yedoensis, and the blank reagent solution, and perform the determination according to the above chromatographic conditions respectively, record the chromatogram, see Figure 2The retention times of hydroxysafflor yellow A and aesculin were 10.297 min and 13.082 min respectively, and the resolution with the adjacent chromatographic peaks was greater than 1.5, and it was completely separated from other components; the theoretical plate numbers were 40870 and 62236 respectively, both greater than 10000. There were no interfering peaks in the chromatograms of the negative sample solution and the blank solution at the peak positions of the reference substances. It shows that the specificity of this determination method is good.

[0107] 2.9.6 Investigation of linear relationship Accurately measure an appropriate amount of the above mixed reference substance solution, dilute it in multiples, and prepare a series of mixed reference substance solutions with 6 different concentration gradients. Among them, the mass concentrations of hydroxysafflor yellow A were (616.0, 246.4, 123.2, 61.6, 24.6, 12.3) μg / ml respectively; the mass concentrations of aesculin were (404.0, 161.6, 80.8, 40.4, 16.2, 8.1) μg / ml respectively. Carry out the determination according to the above chromatographic conditions respectively, and perform linear regression with the reference substance concentration (μg / ml) as the abscissa and the peak area as the ordinate. The linear regression equations, correlation coefficients, and linear ranges of the reference substances are shown in Table 7; the standard curves are shown in Figure 3 The regression equation of hydroxysafflor yellow A was y = 31163x - 171296, r = 0.9998; the regression equation of aesculin was y = 39508x - 156876, r = 0.9998, indicating that hydroxysafflor yellow A showed a good linear relationship in the concentration range of 12.3 μg / ml - 616.0 μg / ml, and aesculin showed a good linear relationship in the concentration range of 8.1 μg / ml - 404.0 μg / ml.

[0108] Table 7 Linear regression equations, correlation coefficients, and linear ranges of hydroxysafflor yellow A and aesculin reference substances

[0109]

[0110] 2.9.7 Precision experiment Take the mixed reference substance solution with the mass concentrations of hydroxysafflor yellow A and aesculin being 61.6 μg / ml and 40.4 μg / ml respectively, and continuously determine it 6 times on the same day according to the above chromatographic conditions, record the peak area, and the within-day RSDs of hydroxysafflor yellow A and aesculin were 0.17% and 0.12% respectively. For three consecutive days, measure it once a day according to the above chromatographic conditions, and the between-day RSDs of hydroxysafflor yellow A and aesculin were 0.40% and 0.09% respectively. The results of the within-day precision test are shown in Table 8, and the results of the between-day precision test are shown in Table 9. It shows that the precision of this detection method is good.

[0111] Table 8 Results of within-day precision experiment (n = 6)

[0112]

[0113] Table 9 Results of daytime precision experiment (n = 3)

[0114]

[0115]

[0116] 2.9.8 Stability experiment Take the Bone Injury Liniment (batch number 20230308), prepare the test solution according to the above-mentioned preparation method of the test solution, and place it at room temperature for 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h, and then measure it respectively according to the above-mentioned chromatographic conditions. The measured mass concentrations of hydroxysafflor yellow A and aesculin are (178.97 ± 0.53) μg / ml and (132.87 ± 2.04) μg / ml respectively, and the RSDs are 0.30% and 1.54% respectively. The results are shown in Table 10. It shows that the test solution has good stability within 10 h.

[0117] Table 10 Results of stability experiment (n = 6)

[0118]

[0119] 2.9.9 Repeatability experiment Take the Bone Injury Liniment (batch number 20230308), prepare 6 portions of the test solution according to the above-mentioned preparation method of the test solution, and measure them respectively according to the above-mentioned chromatographic conditions. The measured mass concentrations of hydroxysafflor yellow A and aesculin are (178.90 ± 0.57) μg / ml and (132.91 ± 2.22) μg / ml respectively, and the RSDs are 0.32% and 1.67% respectively. The results are shown in Table 11. It shows that the detection method has good repeatability.

[0120] Table 11 Results of repeatability experiment (n = 6)

[0121]

[0122] 2.9.10 Spike recovery experiment Accurately measure 6 portions of 1 ml of the Bone Injury Liniment with known mass concentration (batch number 20230308), add 1 ml of the mixed reference solution containing 200.4 μg / ml of hydroxysafflor yellow A reference substance and 150.6 μg / ml of aesculin reference substance to each portion respectively, prepare the test solution according to the above-mentioned preparation method of the test solution, and then measure it respectively according to the above-mentioned chromatographic conditions. Calculate the spike recovery rate. The average spike recovery rates of hydroxysafflor yellow A and aesculin are 98.88% and 95.44% respectively, and the RSDs are 1.85% and 2.41% respectively. The results are shown in Table 12. It shows that the detection method meets the requirements of spike recovery rate.

[0123] Table 12 Results of spike recovery experiment (n = 6)

[0124]

[0125] 2.9.11 Content determination results The contents of hydroxysafflor yellow A and aesculin in 3 batches of GuShang liniment were determined respectively according to the above method, and the results are shown in Table 13.

[0126] Table 13 Determination results of hydroxysafflor yellow A and aesculin in GuShang liniment (n = 3)

[0127]

[0128] Example 3: Study on the treatment of acute soft tissue injury in rats with GuShang liniment

[0129] 1 Materials and methods

[0130] 1.1 Instruments SQP Quintix 124-1CN electronic analytical balance, with a maximum load of 120 g and a division value of 0.1 mg, manufactured by Sartorius, Germany. MP3002 electronic balance, with a maximum load of 300 g and a division value of 10 mg, manufactured by Shanghai Shunyu Hengping Scientific Instrument Co., Ltd. NC12775 Touch-Test tactile threshold tester and Von Frey cilia needle filament kit, manufactured by NorthCoast Stoelting, USA. LBY-N6C full-automatic blood rheometer, manufactured by Beijing Prisun Instrument Co., Ltd. Tissuelyser-24 multi-sample tissue grinder, manufactured by Shanghai Jingxin Industrial Development Co., Ltd. Microfuge 20R high-speed refrigerated centrifuge, manufactured by Beckman Coulter, Germany. DM6 B upright optical biological microscope, manufactured by Leica, Germany. RT-6100 microplate reader, manufactured by Shenzhen Rayto Life Science Co., Ltd.

[0131] 1.2 Reagents Rat interleukin-1β (IL-1β) enzyme-linked immunosorbent assay (ELISA) kit, batch number F3822-A (96T); Rat interleukin-6 (IL-6) ELISA kit, batch number F2874-A (96T); Rat serum tumor necrosis factor-α (TNF-α) ELISA kit, batch number F8751-A (96T), Shanghai Kexing Trading Co., Ltd. Rat superoxide dismutase (SOD) ELISA kit, batch number 20240721 (96T); Rat malonaldehyde (MDA) ELISA kit, batch number 20240617 (96T); Rat reduced glutathione (GSH) ELISA kit, batch number 20240828 (96T); All were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. The rest of the reagents were of analytical grade, from Sinopharm Chemical Reagent Co., Ltd. The water was purified water.

[0132] 1.3 Medicinal Agents Bone Injury Liniment, specification 100 ml / bottle, batch number 20230308, prepared by the hospital pharmacy preparation room. Zheng Gu Shui, specification 30 ml / bottle, batch number 2312283, purchased from Guangxi Yulin Pharmaceutical Group Co., Ltd. Huoxue Zhitong Plaster, specification 7 cm × 10 cm, batch number 20240130, purchased from Anhui Anke Yuliangqing Pharmaceutical Co., Ltd.

[0133] 1.4 Experimental Animals Sprague-Dawley rats, 84 in number, SPF grade, half male and half female, body weight 200 g - 250 g, purchased from Changzhou Cavens Experimental Animal Co., Ltd., production license number of experimental animals SCXK (Su) 2021-0013, quality certificate number 320730240100300375. They were fed with standard rat food in the animal house, with free access to water and activities, room temperature 20°C - 25°C, relative humidity 20% - 35%, and continuous 12-hour alternating light and dark fluorescent lighting. The experiment started after 7 days of adaptive feeding. This study was approved by the hospital ethics committee, animal welfare ethics approval number LL-2024DW01.

[0134] 1.5 Animal Experiment Methods

[0135] 1.5.1 Animal grouping and model preparation After the rats were fasted for 12 h, they were randomly divided into 7 groups according to the random number table method, namely the normal control group (Group N), the model control group (Group M), the positive drug Zheng Gu Shui group (Group ZT), the positive drug Huoxue Zhitong Plaster group (Group HP), the low-dose group of Gushang Liniment (Group GL1), the medium-dose group of Gushang Liniment (Group GL2), and the high-dose group of Gushang Liniment (Group GL3), with 12 rats in each group. One day before modeling, the hair on the right hind limb thigh of the rats in each group was removed with 10% sodium sulfide; on the day of modeling, except for the rats in Group N, the rats in the remaining groups were fixed on the soft tissue impactor, the soft tissue at the mid-lateral part of the right hind limb thigh was marked for positioning, and a 100 g impact hammer was pulled to a height of 10 cm and the soft tissue at the calibrated position was impacted 10 times. After the impact, it was obvious that the impacted area was red, swollen and congested, indicating successful modeling [Ye Yuehua, Wei Wei, Wu Fanjing. Experimental study on the treatment of acute closed soft tissue injury, anti-inflammatory and analgesic effects of Wuwei Xiaotong Tincture. Journal of Emergency in Traditional Chinese Medicine, 2021, 30(11): 1929-1931, 1954.].

[0136] 1.5.2 Treatment method According to "Methodology for the Research on Traditional Chinese Medicine Pharmacology", the equivalent dose ratio between humans and animals was calculated according to the body surface area to calculate the drug dosage for rats ([Chen Qi. Methodology for the Research on Traditional Chinese Medicine Pharmacology. Beijing: People's Medical Publishing House, 1993: 1103-1104.]). For the rats in Group GL1, Group GL2, and Group GL3, the gauze was soaked with the liquid medicine of Gushang Liniment and wrapped around the entire right hind limb for wet compress. The liquid medicine dosage was 0.4 ml / cm 2 (Reference [Liu Hongbo. Study on the effect of Zheng's New Injury Lotion on the content of NO in tissues and histomorphology after acute blunt contusion in rats. Chengdu: Master's thesis of Chengdu Sport University, 2016.]), and plastic food wrap was used to wrap it to prevent biting. The wet compress time for the three groups of rats was 0.5 h, 1 h, and 2 h in turn each time, 3 times a day; for the rats in Group ZT, the liquid medicine of Zheng Gu Shui was used for wet compress in the same way with the same liquid medicine dosage, 1 h each time, 3 times a day; for the rats in Group HP, the Huoxue Zhitong Plaster was cut into a suitable shape and pasted on the affected area, with a dosage of 4 cm 2 / rat (Reference [Xu Rong, Sun Jingying, Shen Hong, et al. Effect of Huoxue Tongluo Plaster on nerve root pain in rats. Chinese Journal of Experimental Traditional Medical Formulae, 2011, 17(2): 181-183.]), and it was removed after 8 h and changed once a day; for the rats in Group N and Group M, normal temperature physiological saline was used for wet compress in the same way, 1 h each time, 3 times a day. Each group started to administer drugs 6 h after modeling and administered drugs continuously for 7 days.

[0137] 1.6 Observation indicators

[0138] 1.6.1 Observation and scoring of symptoms and activities at the injury site of rats On the 1st, 3rd, 5th, and 7th days after drug administration respectively, after the drug administration was completed, observe whether there are new erythema, eschar, and aggravated edema at the injury site of rats in each group; at the same time, observe symptoms such as the degree of limb swelling and congestion, as well as the activities of rats, and conduct grading and scoring. Among them, for the degree of swelling, 2 points are counted if the limb at the injury site is significantly swollen, 1 point if it is slightly swollen, and 0 point if there is no swelling; for the degree of congestion, 2 points are counted if the limb at the injury site is dark purple, 1 point if it is dark red, and 0 point if the color is normal; for the activity, 2 points are counted if the injured limb cannot move, 1 point if the movement ability of the injured limb is poor, and 0 point if the movement ability of the injured limb is normal.

[0139] 1.6.2 Detection of pain withdrawal reflex threshold Before drug administration, on the 1st, 3rd, 5th, and 7th days after drug administration respectively, after the drug administration was completed, place the rats in a special plexiglass observation chamber and detect the pain withdrawal reflex threshold (PWT) on the mechanical touch-induced pain metal grid platform. After 30 minutes of adaptation, use Von Frey cilia filaments with different folding forces to stimulate the left hind paw of rats in each group 5 times per minute, and take the obvious paw retraction or foot licking of rats as the positive standard. The intensities of Von Frey cilia filaments are: 0.07 g, 0.16 g, 0.4 g, 0.5 g, 1 g, 1.4 g, 2 g, 4 g, 6 g, 8 g, 10 g, 15 g, 26 g, 60 g, 100 g, etc., and each filament is measured 5 times. Each rat is stimulated starting from the filament with an intensity of 0.07 g, and the minimum filament intensity at which the paw withdrawal reflex occurs more than 3 times is taken as the PWT value of the rat [Hou Gongjin, Bai Zhengping, Zeng Puhua, et al. Effects of Chanlong Analgesic Plaster on pain threshold and serum PEG2, TNF-α, IL-6, β-EP in rats with bone metastatic cancer pain model. New Drugs and Clinical Remedies of Traditional Chinese Medicine, 2019, 30(10): 1222-1227.].

[0140] 1.6.3 Detection of hemorheology indexes After the administration ended on the 3rd day and the 7th day respectively, the body weights of rats in each group were weighed using an electronic balance. Six rats (half male and half female) in each group were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 40 mg / kg, fixed on an animal fixing table, the abdominal cavity was opened, the abdominal aorta was fully exposed, 5 ml of whole blood was drawn and placed in an anticoagulation tube for storage. The whole blood low shear viscosity (10 / s) (whole blood viscosity at low shear rate, WBV-L), whole blood medium shear viscosity (60 / s) (whole blood viscosity at medium shear rate, WBV-M), whole blood high shear viscosity (150 / s) (whole blood viscosity at high shear rate, WBV-H) and plasma viscosity of rats were detected using an automatic hemorheology analyzer.

[0141] 1.6.4 Observation of histopathology at the injury site After the whole blood was drawn, the rats in each group were decapitated and sacrificed. The skin of the injured limb was dissected and removed using surgical scissors, the soft tissue was cut, the surface blood stain was rinsed clean with normal saline, the surface moisture was blotted dry, and it was stored in a -80 °C refrigerator. About 150 mg of tissue specimens from rats in each group were taken, rinsed with normal saline and then fixed in 4% formaldehyde solution, embedded in paraffin, and subjected to routine 5 μm continuous tissue sectioning and hematoxylin and eosin (HE) staining, and the histopathology of the sample tissue was observed under an optical biological microscope.

[0142] 1.6.5 Determination of the levels of inflammatory factors in the tissue at the injury site About 100 mg of the tissue specimens under item "1.64" were taken, weighed precisely, cut into pieces, and tissue homogenate was prepared using an ultra-fine homogenizer in an ice-water bath. The tissue homogenate was placed in a low-temperature high-speed centrifuge and centrifuged at 4 °C and 19060×g for 15 min. The supernatant was collected and stored in a -80 °C refrigerator. The levels of inflammatory factors IL-1β, IL-6, and TNF-α in the supernatant of the tissue homogenate were determined according to the steps of the Elisa kit instructions.

[0143] 1.6.6 Determination of the levels of oxidative stress indexes in the tissue at the injury site The supernatant of the tissue homogenate under item "1.65" was taken, and the levels of oxidative stress indexes SOD, MDA, and GSH in the supernatant of the tissue homogenate were determined according to the steps of the Elisa kit instructions.

[0144] 1.7 Statistical analysis IBM SPSS Statistics 21.0 software was used for statistical analysis of the experimental data. Measurement data were expressed as mean ± standard deviation It was shown that one-way analysis of variance was used to compare the differences in the measurement data of rats in each group, and the LSD method was used for pairwise comparison between groups; two-way repeated measurement was used to compare the differences in the swelling degree score, congestion degree score, activity score and PWT value of rats in each group after administration. A P value < 0.05 was considered statistically significant.

[0145] 2 Results

[0146] 2.1 Effects of Gushang Liniment on tissue swelling, tissue congestion and activity at the injury site of rats After administration, no new erythema, eschar or aggravated edema was observed in the skin at the injury site of rats in each group, indicating that Gushang Liniment has no skin irritation reaction and good safety.

[0147] 2.1.1 Effects of Gushang Liniment on tissue swelling at the injury site of rats On the 1st, 3rd and 5th days after administration, compared with the N group, the swelling degree scores of rats in the M group, ZT group, HP group and GL groups were significantly increased (all P < 0.0001), indicating that acute soft tissue injury can cause tissue swelling at the injury site of rats.

[0148] On the 7th day after administration, compared with the N group, the swelling degree scores of rats in the M group, ZT group, HP group, GL2 group and GL3 group were significantly increased (P < 0.0001, P < 0.01, P < 0.01, P < 0.01, P < 0.01 respectively); compared with the M group, the swelling degree score of the GL3 group was significantly decreased (P < 0.05). It was shown that after treatment with high-dose GL, the tissue swelling at the injury site of rats significantly subsided, and the effect intensity of GL was generally positively correlated with the dose; the inhibitory effects of each dose of GL on tissue swelling at the injury site of rats were equivalent to those of ZT and HP. See Figure 4 A.

[0149] After repeated measurement comparison, the swelling degree scores of rats in the M group and each treatment group gradually decreased with the increase of treatment time (P < 0.0001 for the measurement time item by Greenhouse-Geisser), and the effects of various treatment methods on the swelling degree score did not change with time (P = 0.236 for the interaction item of measurement time and group by Greenhouse-Geisser); there were differences in the swelling degree scores between groups (P < 0.0001), among which, the swelling degree score of the GL3 group was significantly lower than that of the M group (P = 0.030). It was shown that high-dose GL has an obvious inhibitory effect on tissue swelling at the injury site of rats with acute soft tissue injury. The estimated marginal mean plot is shown in Figure 4 B.

[0150] The experimental results showed that Gushang Liniment can effectively inhibit tissue swelling at the injury site of rats with acute soft tissue injury after 7 days of treatment.

[0151] 2.1.2 Effects of Gushang Liniment on tissue congestion at the injury site in rats On the 1st, 3rd, and 5th days of administration, compared with the N group, the congestion degree scores of rats in the M group, ZT group, HP group, and GL groups were significantly increased (all P<0.0001), indicating that acute soft tissue injury can cause tissue congestion at the injury site in rats.

[0152] On the 7th day of administration, compared with the N group, the congestion degree scores of rats in the M group, ZT group, HP group, and GL groups were significantly increased (P<0.0001, P<0.001, P<0.01, P<0.001, P<0.001, P<0.001 respectively); compared with the M group, the congestion degree score of the GL3 group was significantly decreased (P<0.05). It is shown that after treatment with high-dose GL, the tissue congestion at the injury site in rats was significantly alleviated, and the effect intensity of GL was generally positively correlated with the dose; the inhibitory effects of each dose of GL on tissue congestion at the injury site in rats were equivalent to those of ZT and HP. See Figure 5 A.

[0153] After repeated measurement comparison, the congestion degree scores of rats in the M group and each treatment group gradually decreased with the increase of treatment time (P of Greenhouse-Geisser for the measurement time item = 0.002), and the effects of various treatment methods on the congestion degree scores did not change with time (P of Greenhouse-Geisser for the interaction item of measurement time and group = 0.077); there were differences in the congestion degree scores among groups (P<0.0001), among which, the congestion degree score of the GL3 group was significantly lower than that of the M group (P = 0.025). It is shown that high-dose GL has an obvious inhibitory effect on tissue congestion at the injury site in rats with acute soft tissue injury. The estimated marginal mean graph is shown in Figure 5 B.

[0154] The experimental results show that Gushang Liniment can effectively inhibit tissue congestion at the injury site in rats with acute soft tissue injury after 7 days of treatment.

[0155] 2.1.3 Effects of Gushang Liniment on the activity of rats On the 1st, 3rd, and 5th days of administration, compared with the N group, the activity scores of rats in the M group, ZT group, HP group, and GL groups were significantly increased (all P<0.0001), indicating that acute soft tissue injury can cause limited activity in rats.

[0156] After 7 days of administration, compared with the N group, the activity scores of the rats in the M group, ZT group, HP group, GL1 group, and GL2 group were significantly increased (P < 0.001, P < 0.01, P < 0.05, P < 0.05, P < 0.05 respectively). However, there was no significant difference in the activity scores between the N group and the GL3 group (P > 0.05). It was shown that after treatment with high-dose GL, the activity of the rats was significantly improved, and the effect intensity of GL was generally positively correlated with the dose; the improvement effect of each dose of GL on the activity of the rats was equivalent to that of ZT and HP. See Figure 6 A.

[0157] After repeated measurement comparison, the activity scores of the rats in the M group and each treatment group gradually decreased with the increase of treatment time (P of Greenhouse-Geisser for the measurement time item < 0.0001), and the effects of various treatment methods on the activity scores did not change with time (P of Greenhouse-Geisser for the interaction item of measurement time and group = 0.187); there were differences in the activity scores among the groups (P < 0.0001), among which, the activity scores of the GL2 group and the GL3 group were significantly lower than those of the M group (P = 0.023, P = 0.023 respectively). It was shown that medium and high-dose GL had an obvious promoting effect on the activity of rats with soft tissue injury. The estimated marginal mean graph is shown in Figure 6 B.

[0158] The experimental results showed that after 7 days of treatment with the bone injury liniment, it could effectively improve the motor state of rats with acute soft tissue injury and promote the activity of the rats.

[0159] 2.2 Effects of the bone injury liniment on the PWT value of rats On the 1st, 3rd, 5th, and 7th days of administration, compared with the N group, the PWT values of the rats in the M group, ZT group, HP group, and each GL group were significantly decreased (all P < 0.0001), indicating that acute soft tissue injury could cause a significant decrease in the PWT value.

[0160] On the 3rd day of administration, compared with the M group, the PWT value of the GL3 group was significantly increased (P < 0.05); on the 5th day of administration, compared with the M group, the PWT values of the ZT group, HP group, GL2 group, and GL3 group were significantly increased (P < 0.05, P < 0.01, P < 0.05, P < 0.01 respectively); on the 7th day of administration, compared with the M group, the PWT values of the ZT group, HP group, GL2 group, and GL3 group were significantly increased (P < 0.01, P < 0.01, P < 0.05, P < 0.05 respectively). It was shown that after treatment with ZT, HP, and medium and high-dose GL, the PWT values of the rats were significantly increased, and the effect intensity of GL was generally positively correlated with the dose; on the 5th and 7th days of treatment, the improvement effect of each dose of GL on the PWT of the rats was equivalent to that of ZT and HP. See Figure 7 A.

[0161] After repeated measurement and comparison, the PWT values of the rats in group M and each treatment group gradually increased with the increase of treatment time (P of Greenhouse-Geisser for the measurement time item < 0.0001), and the effects of various treatment methods on the PWT values changed with time (P of Greenhouse-Geisser for the interaction item of measurement time and group < 0.0001); there were differences in the PWT values among groups (P < 0.0001). Among them, the PWT values of the ZT group, HP group, GL2 group, and GL3 group were significantly higher than those of group M (P = 0.005, P = 0.019, P = 0.010, P = 0.003, respectively). It showed that ZT, HP, and medium-high dose GL had obvious promoting effects on the PWT values of rats with soft tissue injury. The estimated marginal mean graph is shown in Figure 7 B.

[0162] The experimental results showed that the bone injury liniment could significantly increase the PWT values of rats with acute soft tissue injury after 5 days and 7 days of treatment, and could effectively inhibit the pain of acute soft tissue injury.

[0163] 2.3 Effects of the bone injury liniment on the hemorheological indexes of rats After 3 days of administration, compared with group N, the WBV-L, WBV-M, WBV-H, and plasma viscosity of the rats in group M and each administration group were significantly increased (all P < 0.0001), indicating that there was microcirculation disorder in the rats with acute soft tissue injury model.

[0164] After 3 days of administration, compared with the M group, the WBV-L of rats in the ZT group, HP group, and each GL group was significantly decreased (P < 0.001 or P < 0.0001 respectively); after 7 days of administration, compared with the M group, the WBV-L viscosities of the HP group, GL2 group, and GL3 group were significantly decreased (P < 0.0001, P < 0.05, P < 0.0001 respectively); after 7 days of administration, compared with the ZT group, the WBV-L of rats in the GL3 group was significantly decreased (P < 0.0001). After 3 days of administration, compared with the M group, the WBV-M of rats in each GL group was significantly decreased (P < 0.05, P < 0.05, P < 0.01 respectively); after 7 days of administration, compared with the M group, the WBV-M of rats in the GL3 group was significantly decreased (P < 0.01). After 3 days of administration, compared with the M group, the plasma viscosities of rats in the ZT group, HP group, and each GL group were significantly decreased (P < 0.0001, P < 0.0001, P < 0.01, P < 0.01, P < 0.001 respectively); after 7 days of administration, compared with the M group, the plasma viscosities of rats in the ZT group, HP group, and GL3 group were significantly decreased (P < 0.01, P < 0.001, P < 0.001). It is shown that after treatment with ZT, HP, and GL, the WBV-L, WBV-M, and plasma viscosity decreased significantly, and the effect intensity of GL was generally positively correlated with the dose; after 7 days of treatment, the inhibitory effects of low and medium doses of GL on WBV-L, WBV-M, and plasma viscosity were equivalent to those of ZT, and the inhibitory effect of high-dose GL on WBV-L was better than that of ZT, and the inhibitory effects of high-dose GL on WBV-L, WBV-M, and plasma viscosity were equivalent to those of HP. See Figure 8 A, Figure 8 B, Figure 8 D.

[0165] After 3 days and 7 days of administration, compared with the N group, the WBV-H of rats in the M group and each administration group increased to varying degrees, and there was no statistical difference in the WBV-H of rats in each administration group compared with the M group (P > 0.05), indicating that treatment with ZT, HP, and GL for 7 days had no effect on the WBV-H of rats. See Figure 8 C.

[0166] The experimental results show that after 7 days of treatment with the bone injury liniment, it can significantly reduce the WBV-L, WBV-M, and plasma viscosity of rats with acute soft tissue injury, and can effectively improve the microcirculation disorder of rats.

[0167] 2.4 Effect of bone injury liniment on tissue pathological damage After 3 days of administration, the muscle fibers of rats in group N were neatly arranged, the pathological structure and morphology were intact, there was no obvious abnormality, and the cell nuclei were all located under the basement membrane. The muscle fiber bundles of rats in group M were disordered, the muscle fibers were twisted, deformed, broken, and partially degenerated (or degenerated), the cell nuclei were uneven in size, the intermuscular space was irregularly widened, and edema was visible. The structure and morphology of the muscle fibers of rats in group ZT were improved to varying degrees, some of them were slightly atrophied (or fatty), the cell nuclei were located under the basement membrane, the size was relatively uniform, the intermuscular space was relatively tight (the intermuscular space was slightly widened), and there was no obvious edema. The structure and morphology of the muscle fibers of rats in group HP were improved to varying degrees, the twisting was not obvious, some were slightly atrophied, the cell nuclei were located under the basement membrane, the size was relatively uniform, the intermuscular space was relatively tight, and there was no obvious edema. The tissue lesions of rats in group GL1 were more severe, with edema, widened intermuscular space, atrophy and degeneration of muscle fibers. The rats in group GL2 had pathological conditions such as mild atrophy of some muscle fibers and widened intermuscular space, which were lighter than those in group GL1. The muscle fiber structure and morphology of rats in the GL3 group were basically normal, with some slightly atrophied, the nuclei were located under the basement membrane, the size was relatively uniform, the intermuscular space was relatively tight (the intermuscular space was slightly widened), and there was no obvious edema. Figure 9 A.

[0168] After 7 days of administration, the muscle fibers of the rats in group N were neatly arranged, with intact pathological structure and morphology, without obvious abnormalities, and the cell nuclei were all located under the basement membrane. The muscle fiber bundles of the rats in group M were disordered, the muscle cells were twisted and deformed, the muscle fibers were focally degenerated and necrotic, the intermuscular spaces were irregularly widened, and edema was visible. Some muscle fibers of the rats in group ZT were atrophied, and there was a little edema. The muscle fiber structure and morphology of the rats in group HP were basically normal, the nucleus size was relatively uniform, the intermuscular spaces were relatively tight, and there was no obvious edema. Some muscle fibers of the rats in group GL1 were atrophied, and there was a little edema. Some muscle fibers of the rats in group GL2 were slightly twisted, and the rest of the structure and morphology were basically normal. The structure and morphology of the muscle fibers of the rats in group GL3 were basically normal. See Figure 9 B.

[0169] Experiments have shown that bone injury ointment can reduce edema in damaged tissues and alleviate inflammatory responses, and can promote tissue repair after acute soft tissue injury in rats.

[0170] 2.5 Effect of bone injury ointment on the content of inflammatory factors in the injured tissue of rats. After 3 days and 7 days of administration, compared with the N group, the levels of IL-1β, IL-6, and TNF-α in the tissue homogenate of the soft tissue injury site of the rats in the M group were significantly increased (all P<0.0001), indicating a strong inflammatory response in the rats with acute soft tissue injury model.

[0171] On the 3rd and 7th days of administration, compared with the M group, the levels of tissue IL-1β and IL-6 in the ZT group, HP group, and each GL group were significantly decreased (all P<0.0001 or P<0.01); on the 3rd day of administration, compared with the M group, the level of tissue TNF-α in the GL3 group was significantly decreased (P<0.05); on the 7th day of administration, compared with the M group, the levels of tissue TNF-α in the ZT group, HP group, and each GL group were significantly decreased (all P<0.0001). It is shown that after treatment with ZT, HP, and GL, the levels of tissue IL-1β, IL-6, and TNF-α were significantly decreased, and the effect intensity of GL was generally positively correlated with the dose; on the 7th day of treatment, the inhibitory effect of high-dose GL on IL-1β and the inhibitory effect of each dose of GL on IL-6 were equivalent to those of HP or ZT, and the inhibitory effect of medium-high-dose GL on TNF-α was equivalent to that of ZT or HP. See Figure 10 .

[0172] The experimental results showed that after 7 days of treatment with the traumatic injury liniment, it could significantly inhibit the expression of IL-1β, IL-6, and TNF-α in the injured tissue of rats with acute soft tissue injury, and could effectively improve the inflammatory environment in rats and inhibit tissue inflammatory reactions.

[0173] 2.6 Effects of the traumatic injury liniment on the levels of redox index in the injured part of rats On the 3rd and 7th days of administration, compared with the N group, the levels of SOD and GSH in the tissue homogenate of the soft tissue injury in rats in the M group were significantly decreased (all P<0.0001), and the level of MDA was significantly increased (P<0.0001), indicating a strong oxidative stress response in rats with acute soft tissue injury.

[0174] After 3 days of administration, compared with the M group, the tissue SOD levels in each GL group were significantly increased (P < 0.05, P < 0.01, P < 0.01 respectively); compared with the M group, the tissue GSH levels in the ZT group, HP group, GL2 group, and GL3 group were significantly increased (P < 0.01, P < 0.05, P < 0.05, P < 0.01 respectively); compared with the M group, the tissue MDA levels in the ZT group, HP group, and each GL group were significantly decreased (P < 0.01, P < 0.001, P < 0.01, P < 0.001, P < 0.001 respectively). After 7 days of administration, compared with the M group, the tissue SOD levels in the GL2 group and GL3 group were significantly increased (both P < 0.01); compared with the M group, the tissue GSH levels in the ZT group, HP group, and GL3 group were significantly increased (all P < 0.05); compared with the M group, the tissue MDA levels in the ZT group, HP group, and each GL group were significantly decreased (P < 0.01, P < 0.001, P < 0.01, P < 0.0001, P < 0.0001 respectively); compared with the ZT group and HP group, the tissue SOD levels in the GL3 group were significantly increased (both P < 0.05); compared with the ZT group, the tissue SOD levels in the GL2 group and GL3 group were significantly increased (P < 0.05, P < 0.01 respectively). It is shown that after treatment with ZT, HP, and GL, the tissue SOD and GSH levels were significantly increased, the MDA level was significantly decreased, and the effect intensity of GL was generally positively correlated with the dose; after 7 days of treatment, the promoting effect of each dose of GL on GSH was equivalent to that of ZT or HP, the promoting effect of high-dose GL on SOD was better than that of ZT or HP, and the inhibitory effect of medium-high-dose GL on MDA was better than that of ZT. See Figure 11 。

[0175] The experimental results show that after 7 days of treatment with the orthopedic injury liniment, it can significantly promote the generation of SOD and GSH in the injured tissue of rats with acute soft tissue injury, significantly inhibit the expression of MDA, effectively improve the oxidative stress state in rats, and inhibit tissue oxidative stress injury.

[0176] 3 Conclusion

[0177] The orthopedic injury liniment developed in the present invention has the effects of promoting blood circulation to remove blood stasis, analgesia, anti-inflammation, and anti-oxidative stress. It can effectively control the tissue swelling and congestion symptoms in the injury area, accelerate the soft tissue regeneration and repair process, and promote the recovery of motor function in injured rats by improving microcirculation disorders, increasing pain thresholds, inhibiting local inflammatory reactions, and oxidative stress injuries in rats with acute soft tissue injury.

[0178] This orthopedic injury liniment can be used to treat acute soft tissue injuries, and its therapeutic effect is better than that of the commonly used clinical drugs Zheng Gu Shui and Huoxue Zhitong Plaster.

[0179] Example 4: Comparative experiment on the therapeutic effects of the orthopedic injury liniment and its formula-deficient preparation on acute soft tissue injuries in rats

[0180] 1 Materials and Methods

[0181] 1.1 Instruments and Reagents SQP Quintix 124-1CN electronic analytical balance, with a maximum load of 120 g and a scale value of 0.1 mg, manufactured by Sartorius, Germany. MP3002 electronic balance, with a maximum load of 300 g and a scale value of 10 mg, manufactured by Shanghai Shunyu Hengping Scientific Instrument Co., Ltd. The rest of the reagents are all of analytical grade, produced by Sinopharm Chemical Reagent Co., Ltd. The water used is purified water.

[0182] 1.2 Test Drugs The bone injury liniment, with the same specifications, batch numbers, and preparation methods as in Example 3. Additionally, according to the prescription and its preparation method, the formulations lacking safflower and Cirsium japonicum DC., lacking safflower and Viola philippica Cav., lacking Isatis indigotica Fortune and Rubia cordifolia L., and lacking Artemisia argyi Levl. et Vant. and Rumex obtusifolius L. were prepared respectively.

[0183] 1.3 Experimental Animals Thirty-six Sprague-Dawley rats, SPF grade, with an equal number of males and females, weighing 200 g - 250 g. The purchasing company, experimental animal production license number, and quality certificate number are the same as in Example 3, and the feeding conditions are the same as in Example 3.

[0184] 1.4 Animal Experiment Methods

[0185] 1.4.1 Animal Grouping and Model Preparation After fasting the rats for 12 h, they were randomly divided into 6 groups according to the random number table method, namely the model control group (Group M), the bone injury liniment group (Group GL), the group lacking safflower and Cirsium japonicum DC. (Group Ncc), the group lacking safflower and Viola philippica Cav. (Group Ncv), the group lacking Isatis indigotica Fortune and Rubia cordifolia L. (Group Nir), and the group lacking Artemisia argyi Levl. et Vant. and Rumex obtusifolius L. (Group Nar), with 6 rats in each group. The method for preparing the animal model is the same as in Example 3.

[0186] 1.4.2 Treatment Methods For each group of rats, normal saline at room temperature, bone injury liniment, the formulation lacking safflower and Cirsium japonicum DC., the formulation lacking safflower and Viola philippica Cav., the formulation lacking Isatis indigotica Fortune and Rubia cordifolia L., and the formulation lacking Artemisia argyi Levl. et Vant. and Rumex obtusifolius L. were used to soak the gauze, which was then wrapped around the entire right hind limb for wet compress. The dose of the liquid medicine was 0.4 ml / cm 2 , and plastic food wrap was used to prevent biting. Each wet compress lasted for 2 h, 3 times a day. Each group started administering the drug 6 h after model establishment and continued for 7 consecutive days.

[0187] 1.5 Observation and Scoring of Symptoms and Activity of Rat Injury Sites After the 7th day of drug administration, the swelling degree, congestion degree, and other symptoms of the injured limbs of rats in each group, as well as the activity of the rats, were observed and graded and scored. The grading and scoring criteria are the same as in Example 3.

[0188] 1.6 Statistical analysis: The experimental data were statistically analyzed using IBM SPSS Statistics 21.0 software. Measurement data were expressed as mean ± standard deviation. One-way analysis of variance was used to compare the differences in measurement data among rats in each group, and the LSD method was used for pairwise comparison between groups. A P value < 0.05 was considered statistically significant.

[0189] 2 Results

[0190] 2.1 Effects of Gushang Liniment and its formula-deficient preparations on tissue swelling at the injury site in rats: After 7 days of administration, compared with the M group, the swelling degree scores at the injury site in rats in the GL group, Ncc group, Ncv group, Nir group, and Nar group were significantly decreased (P < 0.01, P < 0.05, P < 0.05, P < 0.05, P < 0.05, respectively). It was shown that after GL treatment, the tissue swelling at the injury site in rats significantly subsided, and the effect was better than that of the formula-deficient preparations lacking safflower and Cirsium japonicum, lacking safflower and Viola yedoensis, lacking Isatis indigotica and Rubia cordifolia, and lacking Artemisia argyi and Rheum palmatum. See Figure 12 A.

[0191] The experimental results showed that after 7 days of treatment with Gushang Liniment, it could effectively inhibit tissue swelling at the injury site in rats with acute soft tissue injury, and its inhibitory effect was better than that of other formula-deficient preparations with the same dose.

[0192] 2.2 Effects of Gushang Liniment and its formula-deficient preparations on tissue congestion at the injury site in rats: After 7 days of administration, compared with the M group, the congestion degree scores at the injury site in rats in the GL group, Nir group, and Nar group were significantly decreased (P < 0.01, P < 0.05, P < 0.05, respectively). It was shown that after GL treatment, the tissue congestion at the injury site in rats significantly alleviated, and its effect was significantly better than that of the formula-deficient preparations lacking safflower and Cirsium japonicum, lacking safflower and Viola yedoensis, better than that of the formula-deficient preparations lacking Isatis indigotica and Rubia cordifolia, and lacking Artemisia argyi and Rheum palmatum. See Figure 12 B.

[0193] The experimental results showed that after 7 days of treatment with Gushang Liniment, it could effectively inhibit tissue congestion at the injury site in rats with acute soft tissue injury, and its inhibitory effect was better than that of other formula-deficient preparations with the same dose.

[0194] 2.3 Effects of Gushang Liniment and its formula-deficient preparations on the activity of rats: After 7 days of administration, compared with the M group, the activity scores of rats in the GL group, Ncv group, Nir group, and Nar group were significantly decreased (P < 0.01, P < 0.05, P < 0.05, P < 0.05, respectively). It was shown that after GL treatment, the activity of rats significantly improved, and the effect was significantly better than that of the formula-deficient preparation lacking safflower and Cirsium japonicum, better than that of the formula-deficient preparations lacking safflower and Viola yedoensis, lacking Isatis indigotica and Rubia cordifolia, and lacking Artemisia argyi and Rheum palmatum. See Figure 12 C.

[0195] The experimental results show that the bone injury liniment can effectively improve the movement state of rats with acute soft tissue injury after 7 days of treatment, and its improvement effect is better than that of other prescriptions lacking certain ingredients with the same dose.

[0196] 3 Conclusions

[0197] The bone injury liniment developed in the present invention can effectively inhibit tissue swelling and tissue congestion at the injury site of rats with acute soft tissue injury, and effectively improve the movement state of rats.

[0198] This bone injury liniment can be used to treat acute soft tissue injury, and its treatment effect is better than that of other prescriptions lacking certain ingredients with the same dose.

[0199] Obviously, those skilled in the art can make various changes and modifications to this technical solution without departing from the spirit and scope of this technical solution. Thus, if these modifications and variations of this technical solution fall within the scope of the claims of this technical solution and its equivalent technologies, then this technical solution is also intended to include these modifications and variations.

Claims

1. A traumatic injury liniment, characterized in that, The bone injury liniment is prepared from seven medicinal materials, namely safflower, thistle, folium isatidis, viola yedoensis, madder, mugwort leaf and rumex obtusifolius.

2. The traumatic injury liniment according to claim 1, wherein The dosages of the seven medicinal materials are equal. Every 100 ml of the bone injury liniment is equivalent to 6 g of each of the seven medicinal materials.

3. The traumatic injury liniment according to claim 1, wherein, The medical adjuvant used in the preparation is an ethanol aqueous solution.

4. The traumatic injury liniment according to claim 1, characterized in that, The relative density of the bone injury liniment is 0.89 - 0.93, the pH value is 5.7 - 6.0, and the ethanol content is not less than 65%; the content of hydroxysafflor yellow A is not less than 141 μg / ml, and the content of aesculin is not less than 110 μg / ml.

5. A preparation method of an orthopedic liniment, characterized in that, It includes the following steps: Step 1: Add the medicinal materials of safflower, thistle, folium isatidis, viola yedoensis, madder, mugwort leaf and rumex obtusifolius to the first medical adjuvant, and extract by heating under reflux to obtain an extract. Step 2: Add the extract obtained in Step 1 to the second medical adjuvant for dissolution, mix well and divide into portions to obtain the bone injury liniment.

6. The preparation method of the bone injury liniment according to claim 5, characterized in that, Step 1 specifically includes the following process: Take the medicinal materials of safflower, thistle, folium isatidis, viola yedoensis, madder, mugwort leaf and rumex obtusifolius with the same mass, crush them into coarse powder, mix well and soak them in an ethanol aqueous solution, conduct multiple heating reflux extractions, combine the extraction solutions, concentrate under reduced pressure and evaporate to dryness to obtain the extract.

7. The preparation method of the traumatic injury liniment according to claim 5, characterized in that, Step 2 specifically includes the following process: Add the extract obtained in Step 1 to an ethanol aqueous solution for dissolution, mix well and divide into portions to obtain the bone injury liniment.

8. The preparation method of the traumatic injury liniment according to claim 5, characterized in that, The first medical adjuvant used in Step 1 is an 80% ethanol aqueous solution; the second medical adjuvant used in Step 2 is a 70% ethanol aqueous solution.

9. Use of the bone injury liniment according to any one of claims 1 - 4 or the bone injury liniment prepared by the preparation method of the bone injury liniment according to any one of claims 5 - 8 in the preparation of a drug for treating acute soft tissue injuries.

10. Use of the traumatic injury liniment according to claim 9 in the preparation of a medicament for treating acute soft tissue injuries, characterized in that, The bone injury liniment can be used to treat acute soft tissue injuries, and can effectively control the tissue swelling and congestion symptoms in the injury area, accelerate the soft tissue regeneration and repair process, and promote the recovery of motor function in the injury site by improving the microcirculation disorder in rats with acute soft tissue injuries, increasing the pain threshold, inhibiting the local inflammatory reaction and oxidative stress injury mechanism.

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