Composition for treating rheumatic bone pain, preparation method and application thereof

Abstract

The present invention provides a composition for treating rheumatic bone pain, a preparation method and an application thereof, and belongs to the field of medical technology. Ibuprofen / diclofenac sodium cocrystal is prepared, the surface is modified by tannic acid, volatile oils of asarum and frankincense are extracted by supercritical fluid, and the volatile oils are mixed with permeation-enhancing molecules to obtain a permeation-enhancing agent composition, the remaining solid is mixed with Zanthoxylum bungeanum, Gentiana macrophylla, Cinnamomum cassia, and Epimedium ephamii leaves for mixed extraction to obtain a water extract, and fermentation extraction is performed to obtain a fermentation extract, and the modified ibuprofen / diclofenac sodium cocrystal, the permeation-enhancing agent composition, the water extract and the fermentation extract are mixed and added to a thermosensitive hydrogel to obtain a composition for treating rheumatic bone pain, which has good percutaneous absorption effect and sustained release effect, prolongs the action time, improves the drug effect, has the effects of antipyretic, analgesic, blood circulation, anti-inflammatory, and dehumidification, can significantly improve the symptoms of rheumatic bone pain, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a composition for treating rheumatic bone pain, a preparation method thereof and an application thereof. Background Art

[0002] Rheumatic diseases are a serious threat to human health, which can affect joints and surrounding tissues. With the aging of society, the prevalence of osteoarthritis has reached 10% of the population, which is equivalent to hypertension. At present, such diseases are often treated clinically with pure Western medicine or pure Chinese medicine theory system methods, which have certain effects but each has certain shortcomings.

[0003] The difference between Chinese medicine and Western medicine lies mainly in the differences in theoretical systems and application methods. Chinese medicine refers to drugs with therapeutic effects that are used under the guidance of the basic theories of traditional Chinese medicine. If the Chinese medicine theory is used to analyze the medicinal properties of Western medicine and use them dialectically, the scope of Chinese medicine can be expanded and the application of Western medicine can be made more precise and individualized.

[0004] Traditional Chinese medicine believes that rheumatic bone pain is caused by the combination of wind, cold and dampness, and is a disease caused by the combination of various pathogenic factors. Wind, cold and dampness are retained in the joints, blocking the meridians, resulting in joint pain, numbness, swelling, stiffness, and even paralysis, which brings great pain to patients. Currently, Chinese medicines used to treat rheumatic bone diseases include oral medicines and external medicines. Oral medicines generally cannot directly reach the lesion site, so that the efficacy of the medicine cannot be efficiently utilized, which easily affects the treatment effect. External medicines are external medicines that can be directly applied to the painful area, but if the drug power cannot reliably penetrate from the body surface into the lesion site in the body, it cannot achieve the desired therapeutic effect and can only be used to alleviate symptoms.

[0005] Chinese invention patent CN107802791B discloses a Chinese medicine for treating rheumatic bone pain, wherein the raw material composition and weight proportion of the effective ingredients of the Chinese medicine are: 3.5-4.5 weight parts of gold foil, 400-600 weight parts of rosin, 500-600 weight parts of Strychnos nux vomica, 300-400 weight parts of oleander leaves, 300-400 weight parts of Atractylodes macrocephala, 300-400 weight parts of Tripterygium wilfordii, 300-500 weight parts of Peppergrass, 300-400 weight parts of Beehive, 200-300 weight parts of safflower seed oil, 100-200 weight parts of yellow ochre, and 10-18 weight parts of toad venom. It has no toxic side effects and has a significant effect on the treatment of rheumatic bone pain. After it is made into a plaster, it is applied externally to the part of the patient suffering from rheumatic bone pain. The drug penetrates directly into the lesion, removes wind, cold, and moisture, eliminates inflammation, and clears the meridians. However, the efficacy of the Chinese medicine composition is relatively slow, so patients need to endure long-term treatment, which is costly, and at the same time, it does not have a good effect in relieving pain. Summary of the invention

[0006] The purpose of the present invention is to provide a composition for treating rheumatic bone pain, a preparation method thereof and an application thereof, which has good percutaneous absorption effect and sustained release effect, prolongs the action time, improves the drug effect, has the effects of antipyretic, analgesic, blood circulation, anti-inflammatory, and dehumidifying, can significantly improve the symptoms of rheumatic bone pain, and has broad application prospects.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The invention provides a preparation method of a composition for treating rheumatic bone pain, comprising the steps of preparing an ibuprofen / diclofenac sodium cocrystal, modifying the surface of the cocrystal with tannic acid, extracting volatile oils of asarum and frankincense with supercritical fluid, mixing the volatile oils with permeation-enhancing molecules to obtain a permeation-enhancing agent composition, mixing the remaining solids with Zanthoxylum bungeana, Gentiana macrophylla, Cinnamomum cassia and Epimedium leaves to obtain a water extract, performing fermentation extraction to obtain a fermentation extract, mixing the modified ibuprofen / diclofenac sodium cocrystal, the permeation-enhancing agent composition, the water extract and the fermentation extract and adding them into a thermosensitive hydrogel to obtain a composition for treating rheumatic bone pain;

[0009] Wherein, the structural formula of the penetration-enhancing molecule is shown in Formula I:

[0010]

[0011] As a further improvement of the present invention, the following steps are included:

[0012] S1. Preparation of ibuprofen / diclofenac sodium cocrystal: dissolving ibuprofen in acetone and diclofenac sodium in water, mixing the two evenly, leaving the mixture open at room temperature to volatilize the solvent, filtering, and crushing the solid to obtain ibuprofen / diclofenac sodium cocrystal;

[0013] S2. Modification: adding the ibuprofen / diclofenac sodium eutectic obtained in step S1 to water, adding tannic acid and a catalyst, heating and stirring the reaction, cooling to room temperature, filtering, washing, and drying to obtain a modified ibuprofen / diclofenac sodium eutectic;

[0014] S3. Preparation of a penetration enhancer composition: extracting asarum and frankincense through a supercritical fluid to obtain a volatile oil, and retaining the solid; mixing the volatile oil and the penetration enhancer molecule uniformly to obtain a penetration enhancer composition;

[0015] S4 drug extraction: the solid in step S2, two needles, Qinjiao, cinnamon, Yinyanghuo leaves mixed, crushed to obtain drug powder, added to water, heated to boil extraction, filtered, the residue retained, the filtrate was dried to obtain an aqueous extract;

[0016] S5. Fermentation: adding the filter residue in step S3 to water, sterilizing, inoculating Bacillus, fermenting, filtering, and drying the filtrate to obtain a fermentation extract;

[0017] S6. Preparation of thermosensitive hydrogel: dissolving chitosan in an acid solution, cooling, adding sodium β-glycerophosphate, stirring and mixing, adjusting the pH value of the solution, and preparing a thermosensitive hydrogel;

[0018] S7. Preparation of a composition for treating rheumatic bone pain: add the modified ibuprofen / diclofenac sodium cocrystal prepared in step S2, the permeation enhancer composition prepared in step S3, the water extract prepared in step S4, and the fermentation extract prepared in step S5 to the thermosensitive hydrogel prepared in step S6, stir and mix well, and prepare a composition for treating rheumatic bone pain.

[0019] As a further improvement of the present invention, the mass ratio of ibuprofen to diclofenac sodium in step S1 is 5-7:3-5, and the time for volatilizing the solvent is 2-4 days; the mass ratio of ibuprofen / diclofenac sodium cocrystal, tannic acid and catalyst in step S2 is 10:3-5:0.5-1, the catalyst is a Tris-HCl solution with a pH of 8.5-9, and the temperature of the heating and stirring reaction is 45-55° C., and the time is 1-3 h.

[0020] As a further improvement of the present invention, the conditions of the supercritical fluid extraction in step S3 are: extraction pressure: 18-27MPa, extraction temperature: 35-45°C, CO2 flow rate: 7-12L / h, extraction time: 1-3h, the mass ratio of asarum to frankincense is 7-10:2-4, and the mass ratio of volatile oil to penetration-enhancing molecule is 10-20:3-5; the synthesis method of the penetration-enhancing molecule is as follows:

[0021] T1. After activation, salicylic acid reacts with lysine to obtain an intermediate with the following structure:

[0022]

[0023] T2. After lauric acid is activated, it reacts with the intermediate to obtain the product.

[0024] As a further improvement of the present invention, the activation method is to add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, activate at 0-4°C for 20-30 minutes, the molar ratio of salicylic acid to lysine is 1.05-1.1:1; the molar ratio of lauric acid to the intermediate is 1.05-1.1:1.

[0025] As a further improvement of the present invention, the mass ratio of the solid, Zanthoxylum bungeanum, Gentiana macrophylla, cinnamon twig, and Epimedium brevicornum leaf in step S4 is 10:2-4:1-3:3-5:3-5, the solid-liquid ratio of the drug powder and water is 1:5-10 g / mL, and the heating and boiling extraction time is 2-4 h; the mass ratio of the filter residue and water in step S5 is 15-20:300, the Bacillus includes Bacillus subtilis and Bacillus licheniformis seed solution, and the bacterial content of the seed solution is 10 8 -10 9 cfu / mL, the inoculation amounts were 1-2v / v% and 0.5-1.2v / v%, respectively, and the fermentation culture conditions were 45-50°C, 100-150r / min, and the fermentation culture was 36-48h.

[0026] As a further improvement of the present invention, the acid solution in step S6 is a 1-3wt% acetic acid or lactic acid solution, the mass ratio of the chitosan, β-sodium glycerophosphate, and the acid solution is 10-15:3-5:120-150, the temperature is lowered to 0-4°C, and the pH value of the solution is adjusted to 7.2-7.6; the mass ratio of the modified ibuprofen / diclofenac sodium cocrystal, the permeation enhancer composition, the water extract, the fermentation extract, and the thermosensitive hydrogel in step S7 is 2-3:0.2-0.3:5-7:4-6:100.

[0027] As a further improvement of the present invention, the present invention specifically comprises the following steps:

[0028] S1. Preparation of ibuprofen / diclofenac sodium cocrystal: dissolve 5-7 parts by weight of ibuprofen in 100 parts by weight of acetone, dissolve 3-5 parts by weight of diclofenac sodium in 100 parts by weight of water, mix the two evenly, leave them open to evaporate the solvent at room temperature for 2-4 days, filter, and crush the solid to obtain ibuprofen / diclofenac sodium cocrystal;

[0029] S2. Modification: 10 parts by weight of the ibuprofen / diclofenac sodium eutectic prepared in step S1 was added to 100 parts by weight of water, 3-5 parts by weight of tannic acid and 0.5-1 parts by weight of a catalyst were added, heated to 45-55 ° C, stirred for 1-3h, cooled to room temperature, filtered, washed, and dried to obtain a modified ibuprofen / diclofenac sodium eutectic;

[0030] The catalyst is a Tris-HCl solution with a pH of 8.5-9;

[0031] S3. Preparation of a penetration enhancer composition: 7-10 parts by weight of asarum and 2-4 parts by weight of frankincense are subjected to supercritical fluid extraction to obtain volatile oil, and the solid is retained; 10-20 parts by weight of volatile oil and 3-5 parts by weight of penetration enhancer molecules are mixed uniformly to obtain a penetration enhancer composition;

[0032] The supercritical fluid extraction conditions are: extraction pressure: 18-27 MPa, extraction temperature: 35-45° C., CO2 flow rate: 7-12 L / h, extraction time: 1-3 h;

[0033] S4 drug extraction: 10 parts by weight of the solid in step S2, 2-4 parts by weight of Zanthoxylum bungeanum, 1-3 parts by weight of Gentiana macrophylla, 3-5 parts by weight of cinnamon twig, 3-5 parts by weight of Epimedium erythrorhizon leaves were mixed and crushed to obtain a drug powder, which was added to water, wherein the solid-liquid ratio of the drug powder and water was 1: 5-10 g / mL, heated to boiling extraction for 2-4h, filtered, the residue was retained, and the filtrate was dried to obtain an aqueous extract;

[0034] S5 fermentation: 15-20 parts by weight of the filter residue in step S3 was added to 300 parts by weight of water, sterilized, inoculated with a bacterial content of 10 8 -10 9 cfu / mL of Bacillus subtilis and Bacillus licheniformis seed liquid, inoculated at 1-2 v / v% and 0.5-1.2 v / v%, respectively, at 45-50°C, 100-150 r / min, fermented for 36-48 hours, filtered, and dried the filtrate to obtain a fermentation extract;

[0035] S6. Preparation of thermosensitive hydrogel: 10-15 parts by weight of chitosan was dissolved in 120-150 parts by weight of 1-3wt% acetic acid or lactic acid solution, cooled to 0-4 ° C, 3-5 parts by weight of sodium β-glycerophosphate was added, stirred and mixed, and the pH value of the solution was adjusted to 7.2-7.6 to obtain a thermosensitive hydrogel;

[0036] S7. Preparation of a composition for treating rheumatic bone pain: add 2-3 parts by weight of the modified ibuprofen / diclofenac sodium cocrystal prepared in step S2, 0.2-0.3 parts by weight of the penetration enhancer composition prepared in step S3, 5-7 parts by weight of the water extract prepared in step S4, and 4-6 parts by weight of the fermentation extract prepared in step S5 to 100 parts by weight of the thermosensitive hydrogel prepared in step S6, stir and mix well to prepare a composition for treating rheumatic bone pain.

[0037] The present invention further protects a composition for treating rheumatic bone pain prepared by the above preparation method.

[0038] The present invention further protects the use of the above-mentioned composition for treating rheumatic bone pain in the preparation of a composition for treating rheumatic lumbar disc herniation bone pain and rheumatic joint bone pain.

[0039] The present invention has the following beneficial effects:

[0040] Diclofenac sodium preparation belongs to non-steroidal anti-inflammatory drugs, has obvious analgesic effect, can also play anti-inflammatory, antipyretic effect, can relieve mild to moderate and chronic pain. However, this drug has a relatively large stimulation to the gastrointestinal tract, and its absorption and utilization are low when applied externally, which often limits its application. Ibuprofen belongs to non-steroidal anti-inflammatory drugs, has good antipyretic and analgesic effects, and is widely used in the relief of osteoarthritis, rheumatoid arthritis, multiple fevers and various pain symptoms, but ibuprofen is difficult to dissolve in water, so that its bioavailability is relatively low. Therefore, the present invention prepares a kind of ibuprofen / diclofenac sodium eutectic, greatly improves the water solubility of ibuprofen, thereby improving its bioavailability, and at the same time, also avoids diclofenac sodium to stimulate the stomach and intestines, and improves the absorption and utilization of the drug through external administration. After surface modification by tannic acid, the eutectic surface has a rich hydroxyl structure, which can interact with the chitosan molecules in the hydrogel, thereby playing the effect of uniform dispersion and sustained-release drugs.

[0041] The invention selects volatile oil obtained by supercritical fluid extraction of asarum and frankincense, wherein the asarum volatile oil has surface anesthesia and infiltration anesthesia, and plays a good analgesic effect, and the frankincense volatile oil has the effects and functions of promoting blood circulation and removing blood stasis, reducing swelling and relieving pain, and antibacterial and anti-inflammatory. The obtained mixed volatile oil can play a good analgesic and blood circulation effect on the one hand, and can also promote the percutaneous absorption effect of the drug on the other hand, and improve the effect of promoting penetration.

[0042] The invention prepares a penetration-enhancing molecule, and uses salicylic acid as a raw material to prepare an amide structure compound, thereby avoiding the large skin irritation of salicylic acid when used alone, and at the same time, also playing a good anti-inflammatory and analgesic effect. In addition, using lauric acid as a raw material, a long-chain alkyl chain can move the phospholipid bilayer, thereby improving the effect of drug transdermal absorption. At the same time, the penetration-enhancing molecule is safe and non-toxic, can be used for a long time, and can be mixed with volatile oil for use, thereby greatly improving the effect of drug transdermal absorption, and having a synergistic effect.

[0043] The pharmaceutical composition of the present invention comprises asarum, frankincense, gentiana macrophylla, cassia twig and epimedium leaf, wherein the gentiana macrophylla can dispel rheumatism, clear away heat from deficiency, and stop arthralgia. The main component is gentianine A, which has anti-inflammatory, analgesic, antiallergic, sedative, antipyretic and other effects, and is an important anti-rheumatic drug; cassia twig has antipyretic, analgesic, vasodilatory, sweat-promoting, anti-inflammatory, antiallergic and antibacterial effects; asarum has sedative, analgesic and anti-inflammatory effects; epimedium leaf can enhance the nonspecific immune function and cellular immune function of the body, regulate the humoral immune function, promote bone growth, has a "bone-tonifying" effect, has a good preventive and therapeutic effect on osteoporosis, and enhances hematopoietic function. Olibanum has the effects of promoting blood circulation and relieving pain, reducing swelling and promoting tissue regeneration, and has multiple pharmacological effects such as anti-platelet adhesion, anti-inflammatory, anti-ulcer, analgesic, and promoting wound healing. The gentiana macrophylla can disperse blood stasis and reduce swelling, dispel wind and dredge collaterals, and has the effects of promoting blood circulation and dispersing blood stasis, and dispersing and reducing swelling.

[0044] The present invention breaks the cell walls in plant cells through water extraction and fermentation extraction, dissolves most of the active components, and greatly improves the efficacy of the medicine. After fermentation treatment with Bacillus subtilis and Bacillus licheniformis, the polyene compounds and lipopeptide compounds produced, including a variety of antimicrobial peptides such as polymyxin, can also play an antibacterial role and inhibit the growth of harmful bacteria, thereby reducing the impact of viral arthritis.

[0045] The present invention prepares a thermosensitive hydrogel. At the temperature of the skin body temperature, the hydrogel solution gels and adheres to the skin surface, prolonging the action time of the drug and playing a good drug sustained release effect. At the same time, under the action of the penetration-promoting composition, the drug effect is greatly improved and the drug effect time is prolonged.

[0046] The composition for treating rheumatic bone pain prepared by the present invention has good percutaneous absorption effect and sustained release effect, prolongs the action time, improves the drug effect, has the effects of antipyretic, analgesic, blood circulation, anti-inflammatory, and dampness removal, can significantly improve the symptoms of rheumatic bone pain, and has broad application prospects. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Bacillus subtilis, ATCC9372, 100 billion cfu / g, Bacillus licheniformis, ATCC11946, 100 billion cfu / g, were purchased from Shanxi Xinyu Biotechnology Co., Ltd. Chitosan, content>99%, was purchased from Henan Dongyi Chemical Co., Ltd.

[0049] The preparation method of the bacterial seed solution is as follows: the bacterial strain is inoculated into the slant culture medium, and the activation culture is carried out at 50°C, 100 r / min, and the activation culture is carried out for 24 hours to obtain a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution.

[0050] Preparation Example 1 Synthesis of penetration-enhancing molecules

[0051] Synthesis route:

[0052]

[0053] Here’s how:

[0054] T1. Add 0.0105 mol salicylic acid to 100 mL dichloromethane, add 0.02 mol 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.02 mol N-hydroxysuccinimide, activate at 0°C for 30 min, slowly add 0.01 mol lysine, stir at 35°C for 5 h, remove the solvent under reduced pressure, separate by column chromatography (ethyl acetate: petroleum ether volume ratio = 1:1), and obtain the intermediate; ESI-MS calculated value: C 13 H 19 N2O4(M+H) + 267.13, found: 267.1, yield: 81.2%.

[0055] NMR results: 1 H NMR (400MHz, CDCl3) δ11.05 (br, 1H), 8.05 (br, 1H), 7.75 (d, J = 6.2Hz, 1H), 7.31 (m, 1H), 6.95-6.97 (m , 2H), 5.00(br, 1H), 3.45(t, 1H), 3.22(t, 2H), 2.07(br, 2H), 1.75(m, 2H), 1.56(m, 2H), 1.28(m, 2H).

[0056] T2. Add 0.0105 mol of lauric acid to 100 mL of dichloromethane, add 0.02 mol of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.02 mol of N-hydroxysuccinimide, activate at 0°C for 30 min, add 0.01 mol of the intermediate dropwise, heat under reflux for 4 h, remove the solvent under reduced pressure, separate by column chromatography (ethyl acetate: petroleum ether volume ratio = 2:1), and obtain the product. ESI-MS calculated value: C 25 H 41 N2O5(M+H) + 449.29, found value: 440.3, yield: 83.5%.

[0057] NMR results: 1 H NMR (400MHz, CDCl3) δ11.00 (br, 1H), 8.04 (br, 2H), 7.78 (d, J = 6.6Hz, 1H), 7.32 (m, 1H), 7.02 (m, 1H), 6.94 (d, J = 5.7Hz, 1H ), 5.02 (br, 1H), 4.45 (t, 1H), 3.21 (t, 2H), 2.15 (t, 2H), 1.75 (m, 2H), 1.55-1.57 (m, 4H), 1.27-1.30 (m, 18H), 0.94 (t, 3H).

[0058] Example 1

[0059] This embodiment provides a method for preparing a composition for treating rheumatic bone pain, which specifically comprises the following steps:

[0060] S1. Preparation of ibuprofen / diclofenac sodium cocrystal: 5 parts by weight of ibuprofen were dissolved in 100 parts by weight of acetone, and 3 parts by weight of diclofenac sodium were dissolved in 100 parts by weight of water, the two were mixed, stirred for 15 minutes, left open at room temperature for 2 days to evaporate the solvent, filtered, and the solid was crushed to obtain ibuprofen / diclofenac sodium cocrystal;

[0061] S2. Modification: 10 parts by weight of the ibuprofen / diclofenac sodium eutectic prepared in step S1 was added to 100 parts by weight of water, 3 parts by weight of tannic acid and 0.5 parts by weight of a catalyst were added, heated to 45 ° C, stirred for 1 h, cooled to room temperature, filtered, washed, and dried to obtain a modified ibuprofen / diclofenac sodium eutectic;

[0062] The catalyst is a Tris-HCl solution with a pH of 8.5;

[0063] S3. Preparation of a penetration enhancer composition: 7 parts by weight of asarum and 2 parts by weight of frankincense were subjected to supercritical fluid extraction to obtain volatile oil, and the solid was retained; 10 parts by weight of volatile oil and 3 parts by weight of the penetration enhancer molecule prepared in Preparation Example 1 were mixed for 20 minutes to obtain a penetration enhancer composition;

[0064] The supercritical fluid extraction conditions are: extraction pressure: 18 MPa, extraction temperature: 35°C, CO2 flow rate: 7 L / h, extraction time: 1 h;

[0065] S4. Extraction of the drug: 10 parts by weight of the solid in step S2, 2 parts by weight of Zanthoxylum bungeanum, 1 part by weight of Gentiana macrophylla, 3 parts by weight of cinnamon twig, 3 parts by weight of Epimedium brevicornum leaf were mixed and crushed to obtain a drug powder, which was added to water, wherein the solid-liquid ratio of the drug powder to water was 1: 5 g / mL, and the extraction was heated to boiling for 2 h, filtered, the residue was retained, and the filtrate was dried to obtain an aqueous extract;

[0066] S5 fermentation: 15 parts by weight of the filter residue in step S3 was added to 300 parts by weight of water, sterilized, inoculated with Bacillus subtilis and Bacillus licheniformis seed solution, the inoculation amount was 1v / v% and 0.5v / v%, 45 ° C, 100r / min, fermentation culture 36h, filtered, and the filtrate was dried to obtain a fermentation extract;

[0067] S6. Preparation of thermosensitive hydrogel: 10 parts by weight of chitosan was dissolved in 120 parts by weight of 1 wt% acetic acid solution, cooled to 0 ° C, 3 parts by weight of sodium β-glycerophosphate were added, stirred and mixed for 20 min, and the pH value of the solution was adjusted to 7.2 to obtain a thermosensitive hydrogel;

[0068] S7. Preparation of a composition for treating rheumatic bone pain: add 2 parts by weight of the modified ibuprofen / diclofenac sodium cocrystal prepared in step S2, 0.2 parts by weight of the penetration enhancer composition prepared in step S3, 5 parts by weight of the water extract prepared in step S4, and 4 parts by weight of the fermentation extract prepared in step S5 to 100 parts by weight of the thermosensitive hydrogel prepared in step S6, and stir and mix for 30 minutes to prepare a composition for treating rheumatic bone pain.

[0069] Example 2

[0070] This embodiment provides a method for preparing a composition for treating rheumatic bone pain, which specifically comprises the following steps:

[0071] S1. Preparation of ibuprofen / diclofenac sodium cocrystal: 7 parts by weight of ibuprofen were dissolved in 100 parts by weight of acetone, and 5 parts by weight of diclofenac sodium were dissolved in 100 parts by weight of water, the two were mixed, stirred for 15 minutes, left open at room temperature for 4 days to evaporate the solvent, filtered, and the solid was crushed to obtain ibuprofen / diclofenac sodium cocrystal;

[0072] S2. Modification: 10 parts by weight of the ibuprofen / diclofenac sodium eutectic prepared in step S1 was added to 100 parts by weight of water, 5 parts by weight of tannic acid and 1 part by weight of a catalyst were added, heated to 55 ° C, stirred for 3 hours, cooled to room temperature, filtered, washed, and dried to obtain a modified ibuprofen / diclofenac sodium eutectic;

[0073] The catalyst is a Tris-HCl solution with a pH of 9;

[0074] S3. Preparation of a penetration enhancer composition: 10 parts by weight of asarum and 4 parts by weight of frankincense were subjected to supercritical fluid extraction to obtain volatile oil, and the solid was retained; 20 parts by weight of volatile oil and 5 parts by weight of the penetration enhancer molecule prepared in Preparation Example 1 were mixed for 15 minutes to obtain a penetration enhancer composition;

[0075] The supercritical fluid extraction conditions are as follows: extraction pressure: 27 MPa, extraction temperature: 45°C, CO2 flow rate: 12 L / h, extraction time: 3 h;

[0076] S4. Extraction of the drug: 10 parts by weight of the solid in step S2, 4 parts by weight of Zanthoxylum bungeanum, 3 parts by weight of Gentiana macrophylla, 5 parts by weight of cinnamon twig, 5 parts by weight of Epimedium brevicornum leaf were mixed and crushed to obtain a drug powder, which was added to water, wherein the solid-liquid ratio of the drug powder and water was 1: 10 g / mL, and the extraction was heated to boiling for 4 h, filtered, the residue was retained, and the filtrate was dried to obtain an aqueous extract;

[0077] S5 fermentation: 20 parts by weight of the filter residue in step S3 was added to 300 parts by weight of water, sterilized, inoculated with Bacillus subtilis and Bacillus licheniformis seed solution, the inoculation amount was 2v / v% and 1.2v / v%, 50 ° C, 150r / min, fermentation culture 48h, filtered, and the filtrate was dried to obtain a fermentation extract;

[0078] S6. Preparation of thermosensitive hydrogel: 15 parts by weight of chitosan was dissolved in 150 parts by weight of 3 wt% acetic acid solution, cooled to 4 ° C, 5 parts by weight of sodium β-glycerophosphate were added, stirred and mixed for 20 min, and the pH value of the solution was adjusted to 7.6 to obtain a thermosensitive hydrogel;

[0079] S7. Preparation of a composition for treating rheumatic bone pain: add 3 parts by weight of the modified ibuprofen / diclofenac sodium cocrystal prepared in step S2, 0.3 parts by weight of the penetration enhancer composition prepared in step S3, 7 parts by weight of the water extract prepared in step S4, and 6 parts by weight of the fermentation extract prepared in step S5 to 100 parts by weight of the thermosensitive hydrogel prepared in step S6, and stir and mix for 30 minutes to prepare a composition for treating rheumatic bone pain.

[0080] Example 3

[0081] This embodiment provides a method for preparing a composition for treating rheumatic bone pain, which specifically comprises the following steps:

[0082] S1. Preparation of ibuprofen / diclofenac sodium cocrystal: 6 parts by weight of ibuprofen were dissolved in 100 parts by weight of acetone, and 4 parts by weight of diclofenac sodium were dissolved in 100 parts by weight of water, the two were mixed, stirred for 15 minutes, left open at room temperature for 3 days to evaporate the solvent, filtered, and the solid was crushed to obtain ibuprofen / diclofenac sodium cocrystal;

[0083] S2. Modification: 10 parts by weight of the ibuprofen / diclofenac sodium eutectic prepared in step S1 was added to 100 parts by weight of water, 4 parts by weight of tannic acid and 0.7 parts by weight of a catalyst were added, heated to 50 ° C, stirred for 2 h, cooled to room temperature, filtered, washed, and dried to obtain a modified ibuprofen / diclofenac sodium eutectic;

[0084] The catalyst is a Tris-HCl solution with a pH of 8.7;

[0085] S3. Preparation of a penetration enhancer composition: 8.5 parts by weight of asarum and 3 parts by weight of frankincense were subjected to supercritical fluid extraction to obtain volatile oil, and the solid was retained; 15 parts by weight of volatile oil and 4 parts by weight of the penetration enhancer molecule prepared in Preparation Example 1 were mixed for 20 minutes to obtain a penetration enhancer composition;

[0086] The supercritical fluid extraction conditions are: extraction pressure: 22 MPa, extraction temperature: 40°C, CO2 flow rate: 10 L / h, extraction time: 2 h;

[0087] S4. Extraction of the drug: 10 parts by weight of the solid in step S2, 3 parts by weight of Zanthoxylum bungeanum, 2 parts by weight of Gentiana macrophylla, 4 parts by weight of cinnamon twig, 4 parts by weight of Epimedium brevicornum leaf were mixed and crushed to obtain a drug powder, which was added to water, wherein the solid-liquid ratio of the drug powder to water was 1:7 g / mL, and the extraction was heated to boiling for 3 h, filtered, the residue was retained, and the filtrate was dried to obtain an aqueous extract;

[0088] S5 fermentation: 17 parts by weight of the filter residue in step S3 was added to 300 parts by weight of water, sterilized, inoculated with Bacillus subtilis and Bacillus licheniformis seed solution, the inoculation amount was 1.5v / v% and 0.8v / v%, 47 ° C, 120r / min, fermentation culture 42h, filtered, and the filtrate was dried to obtain a fermentation extract;

[0089] S6. Preparation of thermosensitive hydrogel: 12 parts by weight of chitosan was dissolved in 135 parts by weight of 2 wt% lactic acid solution, cooled to 2 ° C, 4 parts by weight of sodium β-glycerophosphate were added, stirred and mixed for 20 min, and the pH value of the solution was adjusted to 7.4 to obtain a thermosensitive hydrogel;

[0090] S7. Preparation of a composition for treating rheumatic bone pain: add 2.5 parts by weight of the modified ibuprofen / diclofenac sodium cocrystal prepared in step S2, 0.25 parts by weight of the penetration enhancer composition prepared in step S3, 6 parts by weight of the water extract prepared in step S4, and 5 parts by weight of the fermentation extract prepared in step S5 to 100 parts by weight of the thermosensitive hydrogel prepared in step S6, and stir and mix for 30 minutes to prepare a composition for treating rheumatic bone pain.

[0091] Comparative Example 1

[0092] Compared with Example 3, the difference is that step S1 is not performed, and in step S2, ibuprofen and diclofenac sodium are mixed and added in a mass ratio of 6:4.

[0093] Comparative Example 2

[0094] Compared with Example 3, the difference is that step S2 is not performed.

[0095] Comparative Example 3

[0096] Compared with Example 3, the difference is that no penetration-enhancing molecules are added in step S3.

[0097] Comparative Example 4

[0098] Compared with Example 3, the difference is that no volatile oil is added in step S3.

[0099] Comparative Example 5

[0100] Compared with Example 3, the difference is that the Bacillus subtilis seed liquid is not inoculated in step S5.

[0101] Comparative Example 6

[0102] Compared with Example 3, the difference is that the Bacillus licheniformis seed liquid is not inoculated in step S5.

[0103] Comparative Example 7

[0104] Compared with Example 3, the difference is that the modified ibuprofen / diclofenac sodium co-crystal is not added in step S7.

[0105] Comparative Example 8

[0106] Compared with Example 3, the difference is that no penetration enhancer composition is added in step S7.

[0107] Comparative Example 9

[0108] Compared with Example 3, the difference is that no fermentation extract is added in step S7.

[0109] Comparative Example 10

[0110] Compared with Example 3, the difference is that in step S7, the temperature-sensitive hydrogel is replaced by water of equal mass.

[0111] Test Example 1

[0112] Drug preparation: Dissolve 4 mg / mL type II collagen in 0.1 mol / L acetic acid to make a 2 mg / mL solution, mix with pre-cooled Freund's complete adjuvant and make a type II collagen with a mass concentration of 1 mg / mL.

[0113] Grouping and modeling: SPF male SD rats were randomly divided into a blank group, a model group, a positive drug group, Example 1-3 groups, and Comparative Example 1-10 groups, with 10 rats in each group. Except for the blank group, each rat in the other groups was injected with 0.25 mL of the prepared 1 mg / mL type II collagen through the base of the tail, the back, and the right hind foot plantar skin to induce inflammation. After 2 weeks, the immune effect was enhanced by scattered injections at the base of the tail, 0.1 mL per rat.

[0114] Administration: From the day of modeling, the Example 1-3 group and the Comparative Example 1-10 group were sprayed with the prepared composition for treating rheumatic bone pain at the inflamed site at a volume of 0.1 mL per 100 g of body mass, bandaged with transparent adhesive tape, maintained for 3 hours, once a day; the blank group and the model group were replaced with an equal amount of physiological saline, and the positive drug group was externally applied with diclofenac sodium gel, and treated in the same manner as above. The drug was stopped on the 31st day after inflammation, and observation was continued for 7 days.

[0115] 1. Swelling:

[0116] The volume of the (primary) inflamed foot before and 35 days after inflammation was measured by drainage volume method, and the swelling degree and swelling inhibition rate were calculated. Swelling degree = volume after inflammation - volume before inflammation. Swelling inhibition rate (%) = (swelling degree of model group - swelling degree of experimental group) / swelling degree of model group × 100%.

[0117] 2. Arthritis index: The degree of joint lesions in the whole body was recorded from the 7th day after inflammation was induced, and the arthritis index was calculated on the 35th day according to the following scoring criteria: 0 point for no redness and swelling; 1 point for redness and swelling of the little toe joint; 2 points for swelling of the big toe joint and toes; 3 points for swelling of the paw below the ankle joint; 4 points for swelling of all paws including the ankle joint. The accumulated scores of each joint are the arthritis index of each rat.

[0118] The results are shown in Table 1.

[0119] Table 1

[0120] Group Swelling degree (mL) Swelling inhibition rate (%) Arthritis Index Model Group 0.94±0.14 / 8.03±0.92 Positive drug group 0.45±0.07* 52.13 6.47±0.44* Example 1 0.37±0.03* 60.64 6.11±0.23* Example 2 0.35±0.02* 62.77 6.08±0.19* Example 3 0.34±0.04* 63.83 6.02±0.25* Comparative Example 1 0.47±0.05 50.00 6.54±0.33 Comparative Example 2 0.42±0.08 55.32 6.49±0.43 Comparative Example 3 0.45±0.07 52.13 6.52±0.40 Comparative Example 4 0.40±0.11 57.45 6.45±0.47 Comparative Example 5 0.43±0.06# 54.26 6.51±0.31# Comparative Example 6 0.45±0.05# 52.13 6.54±0.29# Comparative Example 7 0.51±0.09 45.74 6.72±0.32 Comparative Example 8 0.49±0.08 47.87 6.61±0.36 Comparative Example 9 0.57±0.06# 39.36 6.83±0.27# Comparative Example 10 0.50±0.07 46.81 6.69±0.29

[0121] Note: * compared with the model group, P < 0.05, # compared with the Example 3 group, P < 0.05.

[0122] It can be seen from the above table that the compositions for treating rheumatic bone pain prepared in Examples 1-3 of the present invention can significantly reduce inflammation, reduce swelling, and relieve arthritis.

[0123] 3. Contents of PGE2, TNF-α, and IL-6 in serum and inflammatory tissue fluid

[0124] 3 days after drug withdrawal, fasting but not water for 12 hours, anesthetized rats, and blood was collected from the abdominal aorta after the righting reaction disappeared, and serum was separated for later use. The inflamed part was cut 0.5 cm above the ankle joint on the inflamed side, cut longitudinally, soaked in 5 mL of 0.9% sodium chloride solution at 4°C for 2 hours, centrifuged, and the supernatant was taken to obtain inflammatory tissue fluid for later use. The contents of PGE2, TNF-α, and IL-6 in serum and inflammatory tissue fluid were determined according to the instructions of the kit. The results are shown in Table 2.

[0125] Table 2

[0126]

[0127] Note: * compared with the blank group, P < 0.05; # compared with the model group, P < 0.05; a compared with the Example 3 group, P < 0.05.

[0128] PGE2 stimulates synovial cells and chondrocytes to produce collagenase and other neutral proteases, releases osteocalcin to destroy osteocytes, affects the synthesis and degradation of glycoproteins, and causes the destruction of bone and soft tissue. TNF-α participates in a variety of rheumatoid arthritis pathogenic mechanisms, including activating endothelial cells, inducing cytokines, activating osteoclasts, gathering leukocytes, destroying bone quality, etc. IL-6 does not directly stimulate synovial fibroblasts and chondrocytes to produce PGE2, but by promoting the synthesis of acute phase proteins in the liver, and synergistically enhancing the inflammatory effect with TNF-α, accelerating the inflammatory process, and aggravating arthritis lesions. As shown in the above table, the composition for the treatment of rheumatic bone pain obtained by Examples 1-3 of the present invention can significantly reduce the content of PGE2, TNF-α, and IL-6 in rat serum and inflammatory tissue fluid.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a composition for treating rheumatic bone pain, characterized in that: The following steps are involved: S1. Preparation of ibuprofen / diclofenac sodium cocrystal: dissolve ibuprofen in acetone, dissolve diclofenac sodium in water, mix the two evenly, expose to room temperature to volatilize the solvent, filter, and crush the solid to obtain ibuprofen / diclofenac sodium cocrystal; the mass ratio of ibuprofen to diclofenac sodium is 5-7:3-5; S2. Modification: adding the ibuprofen / diclofenac sodium eutectic obtained in step S1 to water, adding tannic acid and a catalyst, heating and stirring to react, cooling to room temperature, filtering, washing, and drying to obtain a modified ibuprofen / diclofenac sodium eutectic; the mass ratio of the ibuprofen / diclofenac sodium eutectic, tannic acid, and catalyst is 10:3-5:0.5-1; the catalyst is a Tris-HCl solution with a pH of 8.5-9; S3. Preparation of a penetration enhancer composition: extracting asarum and frankincense through supercritical fluid to obtain volatile oil, and retaining the solid; mixing the volatile oil and the penetration enhancer molecule uniformly to obtain a penetration enhancer composition; the mass ratio of asarum and frankincense is 7-10:2-4, and the mass ratio of the volatile oil to the penetration enhancer molecule is 10-20:3-5; S4. Extraction of the drug: The solid in step S3, Zanthoxylum bungeana, Gentiana macrophylla, cinnamon twig, and Epimedium pubescens leaves are mixed and crushed to obtain a drug powder, added to water, heated to boiling for extraction, filtered, the residue is retained, and the filtrate is dried to obtain an aqueous extract; the mass ratio of the solid, Zanthoxylum bungeana, Gentiana macrophylla, cinnamon twig, and Epimedium pubescens leaves is 10:2-4:1-3:3-5:3-5; S5. Fermentation: adding the filter residue in step S4 to water, sterilizing, inoculating Bacillus, fermenting, filtering, and drying the filtrate to obtain a fermentation extract; the mass ratio of the filter residue to water is 15-20:300; the Bacillus includes Bacillus subtilis and Bacillus licheniformis seed solution, the bacterial content of the seed solution is 10 8 -10 9 cfu / mL, and the inoculum concentrations were 1-2v / v% and 0.5-1.2v / v% respectively; S6. Preparation of thermosensitive hydrogel: dissolving chitosan in an acid solution, cooling, adding β-sodium glycerophosphate, stirring and mixing, adjusting the pH value of the solution to obtain a thermosensitive hydrogel; the mass ratio of chitosan, β-sodium glycerophosphate, and acid solution is 10-15:3-5:120-150; S7. Preparation of a composition for treating rheumatic bone pain: adding the modified ibuprofen / diclofenac sodium cocrystal prepared in step S2, the permeation enhancer composition prepared in step S3, the water extract prepared in step S4, and the fermentation extract prepared in step S5 to the thermosensitive hydrogel prepared in step S6, stirring and mixing evenly to prepare a composition for treating rheumatic bone pain; the mass ratio of the modified ibuprofen / diclofenac sodium cocrystal, the permeation enhancer composition, the water extract, the fermentation extract and the thermosensitive hydrogel is 2-3:0.2-0.3:5-7:4-6:100; Wherein, the structural formula of the penetration-enhancing molecule is shown in Formula I:

2. The preparation method according to claim 1, characterized in that: The time for evaporating the solvent in step S1 is 2-4 days; the temperature for heating and stirring the reaction in step S2 is 45-55° C. and the time is 1-3 hours.

3. The preparation method according to claim 1, characterized in that: The conditions of supercritical fluid extraction in step S3 are: extraction pressure: 18-27 MPa, extraction temperature: 35-45° C., CO2 flow rate: 7-12 L / h, extraction time: 1-3 h. The synthesis method of the permeation-enhancing molecule is as follows: T1. After activation, salicylic acid reacts with lysine to obtain an intermediate with the following structure: T2. After lauric acid is activated, it reacts with the intermediate to obtain the product.

4. The preparation method according to claim 3, characterized in that: The activation method comprises adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide, activating at 0-4°C for 20-30 minutes, wherein the molar ratio of salicylic acid to lysine is 1.05-1.1:1; and the molar ratio of lauric acid to the intermediate is 1.05-1.1:

1.

5. The preparation method according to claim 1, characterized in that: The solid-liquid ratio of the drug powder and water in step S4 is 1:5-10 g / mL, and the heating boiling extraction time is 2-4 hours; the fermentation culture conditions in step S5 are 45-50°C, 100-150 r / min, and the fermentation culture is 36-48 hours.

6. The preparation method according to claim 1, characterized in that: In step S6, the acid solution is a 1-3 wt % acetic acid or lactic acid solution, the temperature is lowered to 0-4° C., and the pH value of the solution is adjusted to 7.2-7.

6.

7. The preparation method according to claim 1, characterized in that: The specific steps include: S1. Preparation of ibuprofen / diclofenac sodium cocrystal: dissolve 5-7 parts by weight of ibuprofen in 100 parts by weight of acetone, dissolve 3-5 parts by weight of diclofenac sodium in 100 parts by weight of water, mix the two evenly, leave them open to evaporate the solvent at room temperature for 2-4 days, filter, and crush the solid to obtain ibuprofen / diclofenac sodium cocrystal; S2. Modification: 10 parts by weight of the ibuprofen / diclofenac sodium eutectic prepared in step S1 was added to 100 parts by weight of water, 3-5 parts by weight of tannic acid and 0.5-1 parts by weight of a catalyst were added, heated to 45-55 ° C, stirred for 1-3h, cooled to room temperature, filtered, washed, and dried to obtain a modified ibuprofen / diclofenac sodium eutectic; The catalyst is a Tris-HCl solution with a pH of 8.5-9; S3. Preparation of a penetration enhancer composition: 7-10 parts by weight of asarum and 2-4 parts by weight of frankincense are subjected to supercritical fluid extraction to obtain volatile oil, and the solid is retained; 10-20 parts by weight of volatile oil and 3-5 parts by weight of penetration enhancer molecules are mixed uniformly to obtain a penetration enhancer composition; The conditions of the supercritical fluid extraction are: extraction pressure: 18-27 MPa, extraction temperature: 35-45° C., CO2 flow rate: 7-12 L / h, extraction time: 1-3 h; S4 drug extraction: 10 parts by weight of the solid in step S3, 2-4 parts by weight of Zanthoxylum bungeanum, 1-3 parts by weight of Gentiana macrophylla, 3-5 parts by weight of cinnamon twig, 3-5 parts by weight of Epimedium erythrorhizon leaves were mixed and crushed to obtain a drug powder, which was added to water, wherein the solid-liquid ratio of the drug powder and water was 1: 5-10 g / mL, heated to boiling and extracted for 2-4h, filtered, the residue was retained, and the filtrate was dried to obtain an aqueous extract; S5 fermentation: 15-20 parts by weight of the filter residue in step S4 was added to 300 parts by weight of water, sterilized, inoculated with a bacterial content of 10 8 -10 9 cfu / mL of Bacillus subtilis and Bacillus licheniformis seed liquid, inoculated at 1-2 v / v% and 0.5-1.2 v / v%, respectively, at 45-50°C, 100-150 r / min, fermented for 36-48 hours, filtered, and dried the filtrate to obtain a fermentation extract; S6. Preparation of thermosensitive hydrogel: 10-15 parts by weight of chitosan was dissolved in 120-150 parts by weight of 1-3wt% acetic acid or lactic acid solution, cooled to 0-4 ° C, 3-5 parts by weight of sodium β-glycerophosphate was added, stirred and mixed, and the pH value of the solution was adjusted to 7.2-7.6 to obtain a thermosensitive hydrogel; S7. Preparation of a composition for treating rheumatic bone pain: add 2-3 parts by weight of the modified ibuprofen / diclofenac sodium cocrystal prepared in step S2, 0.2-0.3 parts by weight of the penetration enhancer composition prepared in step S3, 5-7 parts by weight of the water extract prepared in step S4, and 4-6 parts by weight of the fermentation extract prepared in step S5 to 100 parts by weight of the thermosensitive hydrogel prepared in step S6, stir and mix well to prepare a composition for treating rheumatic bone pain.

8. A composition for treating rheumatic bone pain obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the composition for treating rheumatic bone pain as claimed in claim 8 in the preparation of a composition for treating rheumatic lumbar disc herniation bone pain and rheumatic joint bone pain.

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