CN117946203A - Glycyrrhetinic acid-gold complex and preparation method and application thereof - Google Patents
Glycyrrhetinic acid-gold complex and preparation method and application thereof Download PDFInfo
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- CN117946203A CN117946203A CN202410100603.2A CN202410100603A CN117946203A CN 117946203 A CN117946203 A CN 117946203A CN 202410100603 A CN202410100603 A CN 202410100603A CN 117946203 A CN117946203 A CN 117946203A
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- compound
- glycyrrhetinic acid
- gold complex
- preparation
- gold
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Abstract
The invention discloses a glycyrrhetinic acid-gold complex, a preparation method and application thereof, wherein the gold complex is used for treating tendinosis (Tendinopathy). According to the invention, the anti-oxidative stress and tendinosis treatment activity of the gold complex are evaluated through in-vitro and in-vivo experiments, and the result shows that the glycyrrhetinic acid-gold complex not only has the effects of resisting oxidative stress and inflammation of tendon stem cells in vitro, but also inhibits the collagen arrangement disorder caused in the tendon injury process in vivo, reduces the expression of lipid peroxide MDA, inflammatory factors IL-1 beta and HMGB1, and plays a good role in resisting tendinosis. The research innovatively discovers that the glycyrrhetinic acid and the metal elements are covalently combined and then exert the anti-tendinosis effect and mechanism through anti-inflammatory and anti-oxidation, and provides theoretical basis and new direction for developing medicaments for treating tendinosis.
Description
Technical Field
The invention belongs to the field of small molecular compound medicines, and particularly relates to a glycyrrhetinic acid-gold complex, and a preparation method and application thereof.
Background
Tendinopathy (tendinopathy) is a series of pathological changes occurring in injured or diseased tendons, and clinical symptoms are mainly manifested by pain and stiffness, hypofunction and decreased exercise tolerance. As one of the more common diseases of the current rehabilitation medical science and orthopedics clinic, the incidence of tendinosis has a rising trend year by year on the global scale, and is particularly frequently sent to athletes. Nowadays, tendinosis treatment includes drug or injection treatment, physical treatment based on exercise strategies, shock wave therapy, tendon load management, and the like, and is mostly focused on the aim of alleviating symptoms, and meanwhile, poor prognosis effect is often caused by being limited by poor self-repairing ability of tendons, so that an insufficient physical and psychological burden and economic stress are brought to patients and families thereof.
It is generally considered that the inflammatory reaction is a core link of the occurrence of tendinosis, and mechanical injury and other multiple factors together cause tendon inflammation states, so that the steady state of the normal physiological system of tendons is destroyed, and finally, the collagen disorder and extracellular matrix deposition of tendon tissues are caused, thereby seriously affecting the tendon functions, and the tendinitis is regarded as simplification of tendinosis classification in histopathology. The existing research shows that the damage related molecular pattern (DAMP) theory has applicability to tendinosis, which is a non-exogenous infectious inflammation, and is believed to release endogenous danger signals, called siren, after cell damage, activate innate immune responses, and further directly or indirectly activate adaptive immune responses, causing inflammation. High mobility group box B1 (HMGB 1) is one of the key siren, which is distributed in the nucleus in physiological state, and released to extracellular stimulus immune cells to secrete inflammatory mediators after cell damage, producing inflammatory response. HMGB1 has been found in tenocytes and has been found to be involved in the development and progression of tenopathy inflammation.
The licorice has mild taste and has the effects of regulating the middle warmer, regulating the functions of the medicines, and has long administration history. The main chemical components of the liquorice have multiple pharmacological activities, can play roles of enhancing macrophage functions, regulating cellular immunity and resisting viruses, and are widely applied to clinical treatment of liver diseases. Notably, recent studies have found that both are natural inhibitors of HMGB1, and that direct binding to HMGB1 inhibits its expression and thus inhibits inflammation. At present, the related report of glycyrrhetinic acid for treating tendinosis is not yet seen.
The exploration of metal ions and bioactive materials thereof in bone tissue engineering is reported in numerous studies, which predicts a considerable prospect for treating bone diseases. Auranofin is a classical oral medicine for treating rheumatoid arthritis, shows excellent anti-inflammatory effect of gold complex, and also suggests possibility of applying gold element to the field of orthopedic diseases. At present, no report on the treatment of tendinosis by gold complexes is available.
Disclosure of Invention
The invention aims to provide a glycyrrhetinic acid-gold complex, a preparation method and application thereof, wherein the glycyrrhetinic acid-gold complex can effectively inhibit the expression of HMGB 1in vivo and in vitro, regulate and control inflammatory response and relieve the tendinosis process, so as to solve the problems of multiple, long and complex treatment course, inaccurate curative effect and the like of the existing tendinosis treatment scheme.
The aim of the invention is achieved by the following technical scheme:
the invention provides a glycyrrhetinic acid-gold complex, the structure of which is shown as I (product 10)
The invention provides a preparation method of the glycyrrhetinic acid-gold complex, which comprises the following steps:
Firstly, a key intermediate compound 3 (3- (5-bromopentyl) -1- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1H-imidazole-3-onium bromide) is synthesized by taking a compound 1 (4, 5-bis (4-methoxyphenyl) -1H-imidazole) as a starting raw material through two-step reaction, then the compound 3 is respectively esterified with glycyrrhetinic acid (glycyrrhizinic acid, GA) and BMS-1 (compound 8, (S) -1- (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzyl) piperidine-2-formic acid to prepare the compound 4 (3- (5- (((1S, 4R) -3- ((4 bS,7S,10 aR) -7-hydroxy-1, 4b,8, 10 a-hexamethyl-4-oxo-1, 4a,4b,5,6,7, 8a,9,10 a-dodecahydro-phenanthren-2-yl) -1, (4-dimethylcyclohexane-1-formyloxy) pentyl) -1- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1H-imidazol-3-ium bromide) and the compound 9 ((S) -3- (5- ((1- (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzyl) piperidine-2-carbonyl) oxy) pentyl) -1- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1H-imidazol-3-ium bromide). Finally, the compound 9 is subjected to silver transfer method to prepare the N-heterocyclic carbene gold bromide, and the N-heterocyclic carbene gold bromide is reacted with the compound 4 after no treatment or simple treatment to generate a final product 10 (18 beta-glycyrrhetinic acid gold complex, the chemical name of the compound is (1- (5- (((S) -1- (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzyl) piperidine-2-carbonyl) oxy) pentanyl) -3- (5-hydroxyphenyl) -4, 5-di (4-methoxyphenyl) -1, 3-dihydro-2H-imidazole-2-subunit) (1- (5- ((-); 2S,4aS, 6bR,10S,12 aS) -10-hydroxy-2, 4a, 6b,9, 12 a-heptamethyl-13-oxo-1, 2,3, 4a,5, 6a,6b,7, 8a,9,10,11,12 a,12b,13,14 b-didodecylpyridine-2-carbonyl) oxy) pentanyl) -3- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1, 3-dihydro-2H-imidazol-2-ylidene) gold (i)).
Further, the preparation method of the key intermediate compound 3 comprises the following steps:
(1) Reflux reaction is carried out on the compound 1 and 5-bromopentanol serving as raw materials, and after the reaction is finished, the intermediate compound 2 (5- (4, 5-bis (4-methoxyphenyl) -1H-imidazol-1-yl) pentan-1-ol) is obtained through extraction, reduced pressure rotary evaporation, column chromatography purification, concentration and drying;
(2) And (3) dissolving the compound 2 in acetonitrile, carrying out reflux reaction with 1, 5-dibromopentane, and carrying out reduced pressure rotary evaporation, separation and purification, concentration and drying after the reaction is finished to obtain the compound 3.
Further, the preparation method of the compound 8 comprises the following steps:
(1) Dissolving a compound 5 (3- (chloromethyl) -2-methyl-1, 1 '-biphenyl) and a compound 6 (4-hydroxy-2, 6-dimethoxy benzaldehyde) into anhydrous tetrahydrofuran under the protection of nitrogen for reaction, and performing reduced pressure rotary evaporation, separation, purification, concentration and drying after the reaction is finished to obtain an intermediate compound 7 (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzaldehyde;
(2) Mixing the compound 7 with D- (+) -2-piperidoic acid and DMF, adding sodium borohydride acetate in batches, reacting with glacial acetic acid as a catalyst, extracting, performing rotary evaporation under reduced pressure, separating, purifying, concentrating and drying to obtain a compound 8.
The structures of the above compounds 1 to 9 are shown as II to X
The synthetic route of the glycyrrhetinic acid-gold complex is shown as follows:
The glycyrrhetinic acid-gold complex has remarkable inhibiting effect on HMGB1, has effective anti-action on acute and early inflammation of tendinosis, can delay and even reverse tendon injury, has a considerable tendon function recovery effect, and can be hopefully used for preparing medicines for treating tendinosis.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of glycyrrhetinic acid-gold complex (product 10);
FIG. 2 is a nuclear magnetic resonance carbon spectrum of glycyrrhetinic acid-gold complex (product 10);
FIG. 3 is a mass spectrum of glycyrrhetinic acid-gold complex (product 10);
FIG. 4 is a high performance liquid chromatography purity analysis chart of glycyrrhetinic acid-gold complex (product 10);
FIG. 5 shows the effect of glycyrrhetinic acid-gold complexes on tendon stem cell viability, wherein: a is the effect result of glycyrrhetinic acid-gold complex administration with different concentration gradients on the survival rate of tendon stem cells; b is the effect result of glycyrrhetinic acid-gold complex administration with different concentration gradients on the survival rate of tendon stem cells under the condition of oxidative stress;
FIG. 6 shows the inhibition of HMGB1 expression by glycyrrhetinic acid-gold complexes, wherein: a is immunofluorescence result of tendon stem cell HMGB1 expression and nuclear mass distribution condition of glycyrrhetinic acid-gold complex administration under oxidative stress condition; b is the detection result of ELISA kit of tendon stem cell HMGB1 secretion condition under oxidative stress condition when glycyrrhetinic acid-gold complex is administered.
Figure 7 shows the effect of glycyrrhetinic acid-gold complex administration on lipid peroxidation and inflammation levels in tendon stem cells under oxidative stress conditions, wherein: A-C are evaluation results of oxidation resistance of tendon stem cells under oxidative stress condition by administration of glycyrrhetinic acid-gold complex; d is the Western blotting detection result of tendon stem cell inflammatory factor IL-1 beta and IL-6 under oxidative stress condition of glycyrrhetinic acid-gold complex administration.
FIG. 8 shows the tendinosis treatment effect of glycyrrhetinic acid-gold complexes on rats, wherein: a is a graph of H & E staining and Masson staining results of tendon tissues separated from rats in each group after anesthesia; B-C is the detection result of the GSH and MDA kit of the serum of each group of rats; d is the detection result of the IL-1 beta level of inflammatory factors in serum of each group of rats; e is a graph of immunohistochemical results of patellar tendon tissue isolated after anesthesia in each group of rats.
Detailed Description
The preparation of the Chinese herbal compound preparation and the efficacy of treating liver fibrosis are further described below by examples.
Correlation definition: the invention discloses a glycyrrhetinic acid-gold complex, gA-Au, a product 10, an 18 beta-glycyrrhetinic acid gold complex "(1- (5- (((S) -1- (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzyl) piperidine-2-carbonyl) oxy) pentanyl) -3- (5-hydroxyphenyl) -4, 5-bis (4-methoxyphenyl) -1, 3-dihydro-2H-imidazol-2-ylidene) (1- (5- (((2S, 4aS, 6bR,10S,12 aS)) and method of preparing the same 10-hydroxy-2, 4a, 6b,9, 12 a-heptamethyl-13-oxo-1, 2,3, 4a,5, 6a,6b,7, 8a,9,10,11,12 a,12b,13,14 b-docosa-dinehydropyridine-2-carbonyl) oxo Pentyl) -3- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1, 3-dihydro-2H-imidazol-2-ylidene" gold (I) "may be used interchangeably.
The glycyrrhetinic acid-gold complex has a structure shown in I:
The invention discloses a preparation method of the complex and application of the complex in medicines for inhibiting HMGB1 to play an anti-inflammatory role and treating tendinosis.
According to the invention, the activity of the glycyrrhetinic acid-gold complex GA-Au against tendinosis is evaluated through in vitro and in vivo experiments, and the preliminary exploration of the action mechanism is carried out, so that the GA-Au pretreatment administration can relieve the oxidative damage and the inflammation level of TSCs, recover the cell viability of the TSCs and inhibit the expression of HMGB1 in vitro; in rats of the tendinosis model, gA-Au administration inhibited the expression level of HMGB1 in rat tendon tissue and the expression of inflammatory factors and lipid peroxides in serum, and immunohistochemical results also showed that the collagen arrangement of rat tendon tissue was restored.
Wherein, the animal experiments are all carried out according to guidelines and protocols approved by the ethical committee of the animal experiment center of the university of Nanjing traditional Chinese medicine (approval number: 202211A 011).
The experimental procedure and experimental results of the present invention are specifically described below.
Example 1: preparation method of glycyrrhetinic acid-gold complex
1. Experimental method
(1) Preparation of compound 2: compound 1 was dissolved in anhydrous tetrahydrofuran, cooled to 0 ℃ in an ice salt bath, sodium hydride with 60% purity was added in portions, and reacted at room temperature for 30min. 5-bromopentanol was added thereto, and the mixture was heated to reflux for 2 hours. Cooling to room temperature, slowly dripping the reaction solution into ice water to quench the reaction, sequentially extracting with ethyl acetate, washing with saturated saline, drying with anhydrous sodium sulfate, removing the organic solvent by rotary evaporation under reduced pressure, purifying by column chromatography, concentrating, drying to obtain white solid, and obtaining the yield: 92.7%.
(2) Preparation of compound 3: compound 2 was dissolved in acetonitrile, 1, 5-dibromopentane was added, and the mixture was heated under reflux for 5 days. Naturally cooling to room temperature, removing the organic solvent by rotary evaporation under reduced pressure, purifying by column chromatography, concentrating, and oven drying to obtain white solid with yield: 74.5%.
(3) Preparation of Compound 4: compound 3 was dissolved in DMF, followed by addition of potassium carbonate and 18 β -glycyrrhetinic acid, and stirred overnight at room temperature. Extracting with water and ethyl acetate, washing with saturated saline, drying with anhydrous sodium sulfate, removing solvent by rotary evaporation under reduced pressure, purifying by column chromatography, concentrating, and oven drying to obtain white solid, yield: 76.5%.
(4) Preparation of compound 7: compound 5, compound 6, triphenylphosphine were added sequentially to anhydrous tetrahydrofuran under nitrogen atmosphere, the ice salt bath was cooled to about 0 ℃, DIAD was dissolved in anhydrous tetrahydrofuran and added dropwise to the reaction solution, and the reaction was carried out overnight at room temperature. After the reaction is completed, removing most of the solvent by rotary evaporation under reduced pressure, adding diethyl ether, cooling for about 1h in an ice bath, removing the solid by suction filtration, rinsing the solid with a small amount of diethyl ether, concentrating the diethyl ether solution (which cannot be evaporated), purifying by column chromatography, concentrating to remove the solvent, drying to obtain a white solid, and obtaining the yield: 83.6%.
(5) Preparation of Compound 8: compound 7, D- (+) -2-pipecolic acid and DMF were added sequentially in a 100ml single neck round bottom flask and stirred at room temperature for 1h. Sodium borohydride acetate was added in portions, glacial acetic acid was added as a catalyst, and stirred at room temperature overnight. TLC monitoring, adding saturated ammonium chloride aqueous solution after the reaction is completed, stirring at room temperature for 10min, extracting with ethyl acetate, mixing organic phases, washing with saturated saline, drying with anhydrous sodium sulfate, removing the solvent by rotary evaporation under reduced pressure, purifying by column chromatography, concentrating, and drying to obtain light yellow solid, and obtaining the yield: 70.7%.
(6) Preparation of Compound 9: compound 3 was dissolved in DMF, followed by addition of potassium carbonate and compound 8, and stirred at room temperature overnight. Extracting with water and ethyl acetate, washing with saturated saline, drying with anhydrous sodium sulfate, removing solvent by rotary evaporation under reduced pressure, purifying by column chromatography, concentrating, and oven drying to obtain white solid, yield: 80.0%.
(7) Preparation of product 10: under the protection of nitrogen, sequentially adding the compound 9, ag 2 O and anhydrous DCM into a 50ml single-neck round bottom flask, keeping out of the sun with tinfoil paper, reacting for 12 hours at room temperature, adding Me 2 SAuCl and NaBr solid, and continuing reacting for 12 hours. After the reaction is completed, the solid is removed by suction filtration through diatomite, a small amount of anhydrous DCM is used for rinsing the solid, the solvent is removed by rotary evaporation under reduced pressure, the solvent is dissolved in a small amount of DCM and is added into a proper amount of n-hexane in a dropwise manner, white solid is separated out, and the white solid is obtained through suction filtration and drying and is directly put into the next step without further purification. Dissolving the solid in anhydrous acetone, sequentially adding the compound 4 and K 2CO3 under the protection of nitrogen, keeping out of the sun, stirring at room temperature for 12h by tinfoil paper, finally adding KPF 6, and continuously stirring at room temperature for 48h. TLC monitoring, directly removing solvent by rotary evaporation under reduced pressure after reaction, purifying by column chromatography, concentrating, dissolving appropriate amount of anhydrous dichloromethane, slowly dripping into appropriate amount of anhydrous n-hexane, separating out solid, removing solvent, oven drying to obtain white solid, namely the 18 beta-glycyrrhetinic acid gold complex ((1- (5- (((S) -1- (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzyl) piperidine-2-carbonyl) oxy) pentanyl) -3- (5-hydroxyphenyl) -4, 5-di (4-methoxyphenyl) -1, 3-dihydro-2H-imidazole-2-subunit) of the invention 1- (5- (((2S, 4as, 6br,10S,12 as) -10-hydroxy-2, 4a, 6b,9, 12 a-heptamethyl-13-oxo-1, 2,3, 4a,5, 6a,6b,7, 8a,9,10,11,12 a,12b,13,14 b-didodecylpyridine-2-carbonyl) oxy) pentanyl) -3- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1, 3-dihydro-2H-imidazol-2-ylidene) gold (i)), yield: 65%.
2. Results
The structure and purity of the resulting glycyrrhetinic acid-gold complex (product 10) were identified.
(1) Nuclear magnetic resonance hydrogen spectrum
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of the glycyrrhetinic acid-gold complex (product 10) .1H NMR(500MHz,CDCl3)δ7.43(d,J＝5.9Hz,3H),7.36(dd,J＝23.1,7.0Hz,3H),7.19(d,J＝6.5Hz,8H),6.90(d,J＝6.6Hz,10H),6.28(d,J＝9.8Hz,2H),5.58(s,1H),5.13(s,2H),4.41(s,2H),4.24(d,J＝5.9Hz,10H),4.16(s,2H),3.98(d,J＝18.6Hz,2H),3.88(s,1H),3.82(d,J＝14.3Hz,20H),3.56(s,4H),3.45(s,1H),3.24(d,J＝6.1Hz,1H),2.94(d,J＝38.7Hz,2H),2.76(d,J＝12.6Hz,1H),2.35(s,2H),2.29(s,3H),2.14(s,1H),2.02(s,2H),1.94(d,J＝10.3Hz,2H),1.86(s,1H),1.83(s,1H),1.81(s,1H),1.79(s,2H),1.77(s,2H),1.76(s,2H),1.75–1.74(m,2H),1.73–1.72(m,1H),1.67(s,1H),1.64(s,2H),1.62(s,1H),1.61(s,2H),1.60–1.58(m,1H),1.54(s,1H),1.53(s,1H),1.52–1.51(m,1H),1.48(s,1H),1.46(s,2H),1.46–1.45(m,1H),1.44–1.42(m,2H),1.37(s,6H),1.34(s,3H),1.32(s,2H),1.20(d,J＝14.1Hz,2H),1.14–1.10(m,9H),1.03(s,1H),1.02(s,3H),0.99(s,1H),0.82(s,3H),0.78(s,3H),0.71(d,J＝11.5Hz,1H).
(2) Nuclear magnetic resonance carbon spectrum
FIG. 2 is a nuclear magnetic resonance carbon spectrum of the glycyrrhetinic acid-gold complex (product 10) .13C NMR(126MHz,CDCl3)δ200.58,182.48,176.47,160.40,160.18,143.09,141.85,134.65,134.56,131.96,131.76,130.49,129.38,128.47,128.27,128.11,126.90,125.68,119.54,114.38,91.35,78.68,69.53,62.21,62.04,61.86,55.95,55.29,54.89,48.84,48.51,45.46,43.97,43.28,41.05,39.14,37.69,37.09,32.73,31.93,31.84,31.35,31.15,31.02,28.59,28.33,28.09,27.27,26.46,26.35,23.38,23.02,22.91,18.68,17.47,16.39,16.27,15.59.
(3) Mass spectrometry
FIG. 3 is a mass spectrum of the resulting glycyrrhetinic acid-gold complex (product 10): ESI-MS (+) [ m/z ]:2011.06[ M-PF 6+H]+.
(4) High Performance Liquid Chromatography (HPLC)
The purity of the resulting glycyrrhetinic acid-gold complex (product 10) was checked using HPLC. High performance liquid chromatography was performed using Waters ACQUITY HPLC systems (Massachusetts, U.S.A.), equipped with RP-C18 columns (Inertex-C18, 250 mm. Times.4.6 mm, particle size 5 μm), flow rate 0.5mL/min, mobile phase acetonitrile/water. The proportion of mobile phase in the gradient elution over time is shown in the following table:
as shown in FIG. 4, the purity of the glycyrrhetinic acid-gold complex (product 10) was 96.25% by HPLC.
Example 2: in vitro evaluation of Glycyrrhetinic acid-gold Complex anti-inflammatory and antioxidant stress
1. Experimental procedure
(1) Cell culture
Rat tendon stem cells (Tendon STEM CELLS, TSCs) were cultured in a 5% CO 2 incubator at 37℃until the cells grew to 80% -90% density for passaging. To ensure the characteristics of stem cells, 2-6 passages of cells were used for experimental investigation. Growth medium: DMEM/F-12 medium with 10% fetal bovine serum (Fetal bovine serum, FBS) +1% penicillin/streptomycin mix.
(2) Cell viability assay
TSCs were seeded in 96-well plates (100 μl per well) at a cell density of 1×10 5/ml, dosed in groups according to experimental design, and cell viability was determined using MTT. Mu.l MTT was added to each well and the wells were incubated at 37℃for 4h. Absorbance was measured at 490nm for each well using a microplate reader.
(3) Western blot detection
The glycyrrhetinic acid-gold complex of the present invention prepared in example 1 was treated and then washed with pre-chilled PBS for 3 times. Then cell lysates (RIPA: PMSF: protease inhibition=100:1:1) were added, and after sufficient grinding, they were lysed on ice for 30min. Centrifuging, and taking the supernatant to measure the protein concentration. Protein loading buffer (5 XSDS-PAGE) was added and the mixture was boiled in a metal bath at 95℃for 15min to denature the protein. The extracted proteins were electrophoretically separated on a polyacrylamide gel and then transferred using a 0.22 mu mPVDF membrane. Then closing for 2 hours, applying the primary antibody overnight, and incubating the secondary antibody for 2 hours. Protein expression was detected on a chemiluminescent gel imager by ECL chemiluminescent development.
(4) Reactive oxygen species level detection
Cell-loaded slide was placed in 24-well plates and TSCs were seeded into the plates at a density of 2 x 10 4 per well. After the grouping dosing treatment, 1ml of DCFH-DA probe diluted 1000 times by serum-free medium was added to each well, and the mixture was placed in an incubator to incubate for 20min in the absence of light, and observed and photographed under a fluorescence microscope.
(5) Malondialdehyde (MDA) and reduced Glutathione (GSH) were measured using Malondialdehyde (MDA) detection kit and reduced Glutathione (GSH) detection kit, and absorbance was measured at a prescribed wavelength according to the instructions.
(6) ELISA kit for detecting HMGB1 level in cell supernatant
After cells were dosed in groups, cell supernatants were collected and treated according to the instructions of the enzyme-linked immunosorbent assay kit.
2. Results
FIG. 5A is a graph showing the effect of varying concentration gradients of the administration of the glycyrrhetinic acid-gold complex GA-Au of the present invention on the viability of TSCs. The results show that with increasing GA-Au concentration gradient, the cell viability of each group of TSCs decreased, but without significant differences. FIG. 5B shows that pretreatment of TSCs with GA-Au at the same concentration gradient for 4H followed by H 2O2 (100. Mu.M) stimulation for 24H, gA-Au pretreatment at 1, 2. Mu.M promotes cell survival, indicating that GA-Au concentrations below 2. Mu.M can enhance the survival of TSCs under oxidative stress conditions.
FIG. 6A shows immunofluorescence results of HMGB1 expression and nuclear mass distribution. The results showed that HMGB1 expression was increased after H 2O2 stimulation and the amount of expression in the cytoplasm was increased, whereas the fluorescence intensity of HMGB1 expression was decreased after GA-Au administration. FIG. 6B shows the detection of HMGB1 content in cell supernatants by ELISA kit, and the secretion of HMGB1 by cells after H 2O2 stimulation compared with control group, was inhibited by GA-Au.
FIG. 7 is the effect of GA-Au pretreatment on lipid peroxidation and inflammation levels in TSCs under oxidative stress conditions. The results of FIGS. 7A-C demonstrate that GA-Au pretreatment, compared to the H 2O2 building block, relieves GSH depletion of TSCs caused by oxidative stress and accumulation of lipid peroxidation product MDA, enhances the antioxidant capacity of cells, and also suppresses intracellular ROS levels. The WB results of FIG. 7D show that the GA-Au pretreatment group inhibited protein expression of the TSCs inflammatory factors IL-1 beta, IL-6 due to H 2O2 modeling.
Example 3: in vivo evaluation of the tendinosis treating effect of glycyrrhetinic acid-gold complexes
1. Experimental procedure
(1) Animal model building and processing
15 Wistar rats weighing 90-110 g are selected, and after 1 week of adaptive feeding, the rats are randomly divided into 3 groups: the control group, tendinosis model group, administration group, model group and administration group were intragastric with ciprofloxacin hydrochloride (0.5% CMC-Na solubilizing aid) at a dose of 800mg/kg, and the blank control group was intragastric with 0.5% CMC-Na once daily for two weeks. Two weeks later, a drug treatment was given in which the administration group was given by intraperitoneal injection of the glycyrrhetinic acid-gold complex GA-Au (5 mg/kg) of the present invention prepared in the above example 1, once a day, and the blank control group and the model group were given by intraperitoneal injection of 0.5% CMC-Na, and after two weeks of treatment, rats were anesthetized, and then they were subjected to abdominal aortic blood collection and rat tendon tissue collection.
(2) Immunohistochemical detection
Rat patellar tendon tissue is taken, fixed by 4% paraformaldehyde, gradually dehydrated by ethanol, then subjected to xylene transparentization, paraffin embedding, slicing and wax removal. Antigen retrieval treatment is then performed, followed by blocking. And adding corresponding primary antibodies and secondary antibodies, and finally performing DAB color development treatment. After completion, the image was observed and photographed using a microscope.
(3) Kit for detecting expression level of GSH and MDA in rat serum
After the rat is obtained, the plasma is kept at normal temperature for 4 hours, and the supernatant is obtained after centrifugation (3000 rmp, 15 min) and is treated according to the corresponding operation steps of the kit.
2. Results
The results in FIG. 8A show that the tendon tissue structure of the rats in the control group is complete and the collagen arrangement is compact; the collagen fibers of the model group are arranged in disorder, cavitation occurs, and the cell nucleus is circular; compared with the model group, the GA-Au administration group has more compact collagen arrangement, and is loose but has no fracture deformation compared with the blank group. The GSH and MDA kit results of fig. 8B-C show that the serum GSH of the model rats is consumed and the lipid peroxidation product MDA accumulates, which is alleviated after administration, indicating that GA-Au inhibits oxidative stress in the progression of tendinopathy. The results in FIG. 8D show that inflammatory factor IL-1β levels were also inhibited in the serum of rats in the dosing group compared to the modeling group. The immunohistochemical results of fig. 8E show high expression of HMGB1 in the building block and inhibition of HMGB1 expression after administration. The above results demonstrate that GA-Au inhibits the expression of HMGB1 and improves the progression of tendinosis in rats.
In conclusion, the 18 beta-glycyrrhetinic acid gold complex can inhibit the expression of HMGB1 in vivo and in vitro, has the functions of resisting oxidative stress and inflammation, and provides a basis for the pharmacological action of the complex in treating tendinosis.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The glycyrrhetinic acid-gold complex is characterized by being 18 beta-glycyrrhetinic acid-gold complex, and the structure of the glycyrrhetinic acid-gold complex is shown as I:
2. the glycyrrhetinic acid-gold complex according to claim 1, wherein the preparation method comprises the steps of:
(1) The key intermediate compound 3 is synthesized by taking the compound 1 as an initial raw material through two-step reaction, wherein the chemical name of the compound 1 is 4, 5-bis (4-methoxyphenyl) -1H-imidazole, and the chemical name of the compound 3 is 3- (5-bromopentyl) -1- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1H-imidazole-3-onium bromide;
(2) The compound 3 is respectively esterified with glycyrrhetinic acid and a compound 8 to prepare a compound 4 and a compound 9, wherein the compound 8 is BMS-1, and the chemical name is (S) -1- (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzyl) piperidine-2-formic acid; compound 4 was chemically named 3- (5- (((1S, 4 r) -3- ((4 bs,7S,10 ar) -7-hydroxy-1, 4b,8, 10 a-hexamethyl-4-oxo-1, 4a,4b,5,6,7, 8a,9,10 a-dodecahydrophenanthren-2-yl) -1, (4-dimethylcyclohexane-1-formyloxy) pentyl) -1- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1H-imidazol-3-ium bromide and compound 9 was chemically named (S) -3- (5- ((1- (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzyl) piperidine-2-formyl) oxy) pentyl) -1- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1H-imidazol-3-ium bromide;
(3) The compound 9 is used for preparing the N-heterocyclic carbene gold bromide by a silver transfer method, and is reacted with the compound 4 to generate a final product 10 after no treatment or simple treatment, the product 10 is 18 beta-glycyrrhetinic acid gold complex, the chemical name of the compound is 1- (5- (((S) -1- (2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzyl) piperidine-2-carbonyl) oxy) pentanyl) -3- (5-hydroxyphenyl) -4, 5-di (4-methoxyphenyl) -1, 3-dihydro-2H-imidazole-2-subunit) (1- (5- (((-); 2S,4aS, 6bR,10S,12 aS) -10-hydroxy-2, 4a, 6b,9, 12 a-heptamethyl-13-oxo-1, 2,3, 4a,5, 6a,6b,7, 8a,9,10,11,12 a,12b,13,14 b-didodecylpyridine-2-carbonyl) oxy) pentanyl) -3- (5-hydroxypentyl) -4, 5-bis (4-methoxyphenyl) -1, 3-dihydro-2H-imidazol-2-ylidene) gold (i).
3. The glycyrrhetinic acid-gold complex according to claim 1, wherein the preparation method of the compound 3 in the preparation method comprises the steps of:
(1) Reflux reaction is carried out by taking a compound 1 and 5-bromopentanol as raw materials, and an intermediate compound 2 is obtained through separation and purification, wherein the chemical name of the compound 2 is 5- (4, 5-bis (4-methoxyphenyl) -1H-imidazol-1-yl) pentan-1-ol;
(2) And (3) dissolving the compound 2 in acetonitrile, carrying out reflux reaction with 1, 5-dibromopentane, and separating and purifying to obtain the compound 3.
4. The glycyrrhetinic acid-gold complex according to claim 1, wherein the method for preparing the compound 8 comprises the steps of:
(1) Dissolving a compound 5 and a compound 6 serving as raw materials into anhydrous tetrahydrofuran for reaction under the protection of nitrogen, and separating and purifying to obtain an intermediate compound 7, wherein the chemical name of the compound 5 is 3- (chloromethyl) -2-methyl-1, 1' -biphenyl; compound 6 is chemically named 4-hydroxy-2, 6-dimethoxy benzaldehyde and compound 7 is chemically named 2, 6-dimethoxy-4- ((2-methyl- [1,1' -biphenyl ] -3-yl) methoxy) benzaldehyde;
(2) And mixing the compound 7 with D- (+) -2-piperidoic acid and DMF, adding sodium borohydride acetate in batches, reacting with glacial acetic acid as a catalyst, and separating and purifying to obtain a compound 8.
5. Use of a glycyrrhetinic acid-gold complex according to claim 1 in the preparation of HMGB1 inhibitor medicaments.
6. Use of a glycyrrhetinic acid-gold complex according to claim 1 for the preparation of a medicament against non-exogenous infectious inflammation.
7. Use of a glycyrrhetinic acid-gold complex according to claim 1 in the preparation of a medicament for the treatment of tendinosis.
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