Protective effect of glycyrrhetinic acid against cantharidin-induced hepatotoxicity through reducing oxidative stress in mice

Background:Cantharidin(CTD)is a commonly used natural product with anticancer properties;however,it has significant adverse effects,particularly hepatotoxicity.Glycyrrhetinic acid(GA),the active component of licorice,shows potential hepatoprotective effects.The protective effects and mechanism of GA against CTD-induced hepatotoxicity are still unclear.Objective:This study aims to elucidate the effect and mechanism of GA on CTD-induced hepatotoxicity in mice experiments.Methods:Construction of CTD-induced hepatotoxicity models and oral gavage GA intervention for 14 d.The liver index,ALT,AST and LDH levels in the serum of the mice were examined;HE staining was performed to observe pathological changes in the liver.The MDA level and SOD activities in liver tissue were tested.Western blot was conducted to determine Keap1/Nrf2 signaling pathway-related protein expression.Results:The results showed that GA significantly reduced the levels of ALT,AST,and LDH in the serum,which were increased by CTD.Additionally,it also exerted a substantial inhibitory effect on the reduction of SOD activity and the elevation of malondialdehyde content in liver tissue.Notably,the phenomena of nuclear swelling,necrosis,and inflammatory infiltration of liver tissue were significantly attenuated following oral administration of GA in mice.Subsequent research has demonstrated that GA effectively suppressed the CTD-triggered upregulation of Keap1 while increasing the CTD-induced downregulation of Nrf2,HO-1,and NQO1.Conclusion:These findings suggested that GA may protect against CTD-induced hepatotoxicity in mice by exerting antioxidative stress through the Keap1/Nrf2 signaling pathway.

Exploring the targets and molecular mechanism of glycyrrhetinic acid against diabetic nephropathy based on network pharmacology and molecular docking

BACKGROUND Diabetic nephropathy(DN)stands as the most prevalent chronic microvascular complication of diabetes mellitus.Approximately 50%of DN patients progress to end-stage renal disease,posing a substantial health burden.AIM To employ network pharmacology and molecular docking methods to predict the mechanism by which glycyrrhetinic acid(GA)treats DN,subsequently validating these predictions through experimental means.METHODS The study initially identified GA targets using Pharm Mapper and the TCMSP database.Targets relevant to DN were obtained from the Genecards,OMIM,and TTD databases.The Venny database facilitated the acquisition of intersecting targets between GA and DN.The String database was used to construct a protein interaction network,while DAVID database was used to conducted Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis and Gene Ontology(GO)analysis.Molecular docking experiments were performed using Autodock software with selected proteins.Experimental validation was conducted using renal proximal tubular cells(HK-2)as the study subjects.A hyperglycemic environment was simulated using glucose solution,and the effect of GA on cell viability was assessed through the cell counting kit-8 method.Flow cytometry was employed to detect cell cycle and apoptosis,and protein immunoblot(western blot)was used to measure the expression of proteins of the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway and insulin resistance pathway,including insulin receptor(INSR),PI3K,p-PI3K,AKT,p-AKT,and glycogen synthase kinase-3(GSK3).RESULTS A total of 186 intersecting targets between GA and DN were identified,which were associated with 144 KEGGrelated enrichment pathways,375 GO biological process entries,45 GO cellular component entries,and 112 GO cellular function entries.Molecular docking demonstrated strong binding of GA to mitogen-activated protein kinase(MAPK)-1,SRC,PIK3R1,HSP90AA1,CASPASE9,HARS,KRAS,and MAPK14.In vitro experiments revealed that GA inhibited HK-2 cell viability,induced cell cycle arrest at the G2/M phase,and reduced apoptosis with increasing drug concentration.Western blot analysis showed that GA differentially up-regulated GSK3 protein expression,up-regulated AKT/p-AKT expression,down-regulated INSR,AKT,p-AKT,PI3K,and p-PI3K protein expression,and reduced p-PI3K/PI3K levels under high glucose conditions.CONCLUSION GA may protect renal intrinsic cells by modulating the PI3K/AKT signaling pathway,thereby inhibiting HK-2 cell viability,reducing HK-2 cell apoptosis,and inducing cell cycle arrest at the G0/G1 phase.

Potential therapeutic targets for the prevention of diabetic nephropathy:Glycyrrhetinic acid

Uncontrolled hyperglycemia or poorly managed disease increases the propensityfor a number of diabetes-related complications targeting major organs includingthe heart, eyes, and kidney. Although the mechanisms by which diabetes inducescardiovascular diseases include oxidative stress and inflammation, when insulinresistance remains the key to the pathogenesis, as implicated in the two reviews inthis issue. This editorial mainly comments on the potential preventive applicationof glycyrrhetinic acid (or 18β-GA) in relation to diabetic nephropathy. The therapeuticor preventive effects of 18β-GA, as a hydrolytic product of glycy-rrhizicacid that is a component of licorice, have been appreciated in other disorders, buthave received much less attention in relation to diabetic complications. A study inthis issue has identified 18β-GA as a therapeutic for preventing diabeticnephropathy and provides evidence to support efficacy in cultured human renaltubule cells in vitro. Although it represents a pilot study, the observations supporta new therapeutic approach that warrants further ex-ploration.

Effect of glycyrrhetinic acid on Th1/Th2 balance in cough variant asthma mice

Objective:To evaluate the therapeutic effect of Glycyrrhetinic Acid on cough variant asthma(CVA)mice and to investigate the possible mechanism in reducing lung inflammation.Methods:48 young female Balb/c mice were divided into Control,CVA,Prednisone Acetate,Glycyrrhetinic Acid high-dose,Glycyrrhetinic Acid middle-dose and Glycyrrhetinic Acid lowdose groups randomly,with 8 mice in each group.The CVA mice model was established by ovalbumin(OVA)sensitization and OVA challenge,the animal asthma behavior was observed after drug administration,and the index of the lung of mice were recorded.The level of OVAsIgE in the bronchoalveolar lavage fluid(BALF)was tested by ELISA.The pathological changes of the lung tissue were observed by Hematoxylin and Eosin(H&E)staining.The protein expressions of T-bet,IFN-γ,Gata3,IL-4 and IL-13 in the lung tissue were determined by Western blot.Results:Compared with the CVA group,the index of lung of mice,the OVA-sIgE level in BALF and expression levels of Th2-related factor in the lung tissue of mice in Prednisone Acetate and Glycyrrhetinic Acid groups were significantly decreased(P<0.05 or P<0.01),the infiltration of inflammatory cells in the lung tissue was reduced,while expressions of Th1-related factor in the lung tissue was significantly increased(P<0.05 or P<0.01).Conclusion:Glycyrrhetinic acid has therapeutic effect on CVA mice,the underlying mechanism of Glycyrrhetinic acid alleviating lung impairment and airway inflammation may be associated with mediating the Th1/Th2 imbalance in the lung tissue.

Effects of glycyrrhetinic acid on collagen metabolism of hepatic stellate cells at different stages of liver fibrosis in rats

INTRODUCTIONLiver fibrosis is a dynamic course leading tocirrhosis from a various chronic liver diseases. Thepathological basis of fibrosis is the disturbance ofproduction and degradation of the extracellularmatrix (ECM), which causes accumulation of ECMin the liver[1,2].

Regulating effect of glycyrrhetinic acid on bronchial asthma smooth muscle proliferation and apoptosis as well as inflammatory factor expression through ERK1/2 signaling pathway

Objective: To study the influence of glycyrrhetinic acid(GA) on bronchial asthma(BA)smooth muscle proliferation and apoptosis as well as inflammatory factor expression and its molecular mechanism.Methods: Male SD guinea pigs were selected and made into asthma models, bronchial asthma smooth muscle cells were cultured and divided into BA group, GA group and GA + LM group that were treated with serum-free RPMI1640 culture medium, serumfree RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid, serum-free RPMI1640 culture medium containing 50 ng/mL glycyrrhetinic acid and 100 ng/mL LM22B-10 respectively; normal guinea pigs were collected and bronchial smooth muscle cells were cultured as control group. The cell proliferation activity as well as the expression of proliferation and apoptosis genes, inflammatory factors and p-ERK1/2 was determined.Results: Proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6,YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in BA group were significantly higher than those of control group while m RNA expression levels of Bax,caspase-9 as well as caspase-3 were significantly lower than that of control group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40, protein expression of p-ERK1/2 of airway smooth muscle cell in GA group were significantly lower than those of BA group(P < 0.05) while the m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly higher than those of BA group(P < 0.05); proliferation activity value and m RNA expression of Bcl-2, TNF-α, IL-4, IL-6, YKL-40 of airway smooth muscle cell in GA + LM group were significantly higher than those of GA group(P < 0.05) while m RNA expression levels of Bax, caspase-9 as well as caspase-3 were significantly lower that of GA group(P < 0.05).Conclusion: GA can inhibit the proliferation of bronchial smooth muscle cells and reduce the expression of inflammatory factors by inhibiting the phosphorylation of ERK1/2.

18beta-glycyrrhetinic acid induces ROS-mediated apoptosis to ameliorate hepatic fibrosis by targeting PRDX1/2 in activated HSCs

Hepatic stellate cells(HSCs)are essential drivers of fibrogenesis.Inducing activated-HSC apoptosis is a promising strategy for treating hepatic fibrosis.18beta-glycyrrhetinic acid(18b-GA)is a natural compound that exists widely in herbal medicines,such as Glycyrrhiza uralensis Fisch,which is used for treating multiple liver diseases,especially in Asia.In the present study,we demonstrated that 18b-GA decreased hepatic fibrosis by inducing the apoptosis in activated HSCs.18b-GA inhibited the expression of a-smooth muscle actin and collagen type Ⅰ alpha-1.Using a chemoproteomic approach derived from activity-based protein profiling,together with cellular thermal shift assay and surface plasmon resonance,we found that 18b-GA covalently targeted peroxiredoxin 1(PRDX1)and peroxiredoxin 2(PRDX2)proteins via binding to active cysteine residues and thereby inhibited their enzymatic activities.18b-GA induced the elevation of reactive oxygen species(ROS),resulting in the apoptosis of activated HSCs.PRDX1 knockdown also led to ROS-mediated apoptosis in activated HSCs.Collectively,our findings revealed the target proteins and molecular mechanisms of 18b-GA in ameliorating hepatic fibrosis,highlighting the future development of 18b-GA as a novel therapeutic drug for hepatic fibrosis.

18β-Glycyrrhetinic Acid Improves Cardiac Diastolic Function by Attenuating Intracellular Calcium Overload

Summary:Ranolazine,a late sodium current inhibitor,has been demonstrated to be effective on heart failure.18B-glycyrrhetinic acid(18β-GA)has the similar inhibitory effect on late sodium currents.However,its effect on diastolic function is still unknown.This study aimed to determine whether 18β-GA can improve the diastolic function and to explore the underlying mechanisms.Eighty male Sprague Dawley(SD)rats of Langendorff model were randomly divided into the following groups:group A,normal cardiac perfusion group;group B,ischemia-reperfusion group;group C,ischemia-reperfusion with anemoniasulcata toxinⅡ(ATX-Ⅱ);group D,ranolazine group;and group E,18β-GA group with four different concentrations.Furthermore,a pressure-overloaded rat model induced by trans-aortic constriction(TAC)was established.Echocardiography and hemodynamics were used to evaluate diastolic function at 14th day after TAC.Changes of free intracellular calcium(Ca27)concentration was indirectly detected by laser scanning confocal microscope to confirm the inhibition of late sodium currents.With the intervention of ATX-Ⅱon ischemia reperfusion injury group,5 umol/L ranolazine,and 5,10,20,40μmol/L 18β-GA could improve ATX-I-induced cardiac diastolic dysfunction.630 mg/kg glycyrrhizin tablets could improve cardiac diastolic function in the pressure-overloaded rats.18B-GA and ranolazine had similar effects on reducing the free calcium in cardiomyocytes.The study demonstrates that 18B-GA and glycyrrhizin could improve diastolic dysfunction induced by ischemia-reperfusion injury in Langendorff-perfused rat hearts and pressure-overloaded rats.The mechanism may be attributed to the inhibition of enhanced late sodium currents.

Pressured Microwave-assisted Hydrolysis of Crude Glycyrrhizic Acid for Preparation of Glycyrrhetinic Acid

A pressured microwave-assisted hydrolysis （PMAH） technique has been developed for hydrolyzing the crude glycyrrhizic acid （GA） extracted from licorice root to prepare glycyrrhetinic acid （GRA）. In order to optimize the efficiency of PMAH, several experimental parameters were investigated, including liquid-solid ratio, hydrolysis time, sulfuric acid concentration and hydrolysis temperature. The optimized hydrolysis conditions were as follows：pressured microwave-assisted hydrolysis of crude GA for 21 min （taking 15 min to reach 150 ℃, and holding it for 6 rain） at 150 ℃ （at a radiation power of 450 W） in 3%-5% sulfuric acid solution with the liquid-solid （ml.g-1 crude GA） ratio of 25 ： 1. As a result of the considerable saving in time and higher product yields （up to 90%）, PMAH was proved more effective than conventional methods.

Synthesis and Anti-tumor Activity of Novel Glycyrrhetinic Acid Derivatives

Twenty-five derivatives of glycyrrhetinic acid（GA） modified on the A-ring,at C30 and C11 positions were synthesized.Their in vitro cytotoxicity against various cancer cell lines[henrietta lacks strain of cancer cells（HeLa）,human hepatocellular liver carcinoma cells（HepG2） and human gastric carcinoma cells（BGC-823）] was evaluated by standard MTT[3-（4,5-dimethyl-2-thiazol-yl）-2,5-diphenyl-2H-tetrazolium bromide] assay.All the tested derivatives were found to have stronger cell growth inhibitory than their parent compound GA.Among them,compounds 3a,5a,and 8d have similar activity on HeLa cell line,and compound 8a has similar activity on HeLa,HepG2 and BGC-823 cell lines as Gefitinib.

Protective mechanisms of hypaconitine and glycyrrhetinic acid compatibility in oxygen and glucose deprivation injury

This study investigated the protective effect of the compatibility of hypaconitine （HA） and glycyrrhetinic acid （GA） on H9c2 cells under oxygen and glucose deprivation （OGD）-induced injury, and the possible mechanisms. We found that HA＋GA significantly improved pathology and morphology of the nucleus and ultrastructure of H9c2 cells under OGD as determined by Hoechst 33342 staining and transmission electron microscopy （TEM） tests. It also reduced the releases of lactate dehydrogenase （LDH）, creatine kinase-myocardial band isoenzyme （CK-MB）, and aspartate transaminase （AST） from the cultured supernatant of H9c2 cells, which were tested by enzyme-linked immune sorbent assay （ELISA） kits. In addition, it lessened the apoptotic rate as determined by a fluorescein isothiocyanate-annexin V/propidium iodide （FITC-AV/PI） double staining assay. It was also found that HA＋GA might regulate the protein expression associated with the phosphatidylinositol 3-kinase （PI3K）/Akt signaling pathway. Overall, the study demonstrated that HA＋GA protected H9c2 cells against OGD-induced injury, and the signaling mechanism might be related to the PI3K/Akt signaling pathway.

Laminaria japonica increases plasma exposure of glycyrrhetinic acid following oral administration of Liquorice extract in rats

The present study was designed to investigate the effects of Laminaria japonica(Laminaria) on pharmacokinetics of glycyrrhetinic acid(GA) following oral administration of Liquorice extract in rats.Following oral administrations of single-dose and multi-dose Liquorice extract and Liquorice-Laminaria extract,respectively,plasma samples were obtained at various times and the concentrations of GA,liquiritigenin,and isoliquiritigenin were measured by LC-MS.The effects of Laminaria extract on pharmacokinetics of GA were also investigated,following single-dose and multidose of glycyrrhizic acid(GL).The effects of Laminaria extract on intestinal absorption of GA and GL were studied using the in situ single-pass intestinal perfusion model.The metabolism of GL to GA in the contents of small and large intestines was also studied.The results showed Liquorice-Laminaria extract markedly increased the plasma concentration of GA,accompanied by a shorter Tmax.Similar alteration was observed following multidose administration.However,pharmacokinetics of neither liquiritigenin nor isoliquiritigenin was affected by Laminaria.Similarly,Laminaria markedly increased concentration and decreased Tmax of GA following oral GL were observed.The data from the intestinal perfusion model showed that Laminaria markedly increased GL absorption in duodenum and jejunum,but did not affect the intestinal absorption of GA.It was found that Laminaria enhanced the metabolism of GL to GA in large intestine.In conclusion,Laminaria increased plasma exposures of GA following oral administration of liquorice or GL,which partly resulted from increased intestinal absorption of GL and metabolism of GL to GA in large intestine.

Microbial transformation of glycyrrhetinic acid and potent neural anti-inflammatory activity of the metabolites

The microbial transformation of glycyrrhetinic acid（1） by Cunninghamella blakesleana CGMCC 3.970 led to the production of five new metabolites（2-6）.The structures of the metabolites were determined by extensive spectroscopic（HR-ESIMS,1D and 2D NMR） data analyses.The involved reactions exhibited specific hydroxylations at C-24,C-7,and C-15,and oxidation at C-3.Moreover,compounds 2,5,and 6showed significant neural anti-inflammatory activity by inhibiting lipopolysaccharide-induced NO production in mouse microglia BV2 cells with IC（50） values of 0.76,0.94,and 0.16μmol/L,respectively.

EFFECT OF GLYCYRRHETINIC ACID ON DNA DAMAGE AND UNSCHEDULED DNA SYNTHESIS INDUCED BY BENZO（α）PYRENE

Glyeyrrhetinic acid (GA) is an active component of Glycyrrhiza uraleusis fisch,In this study,GA was found to inhibit ear edema and ornithine decarboxylase (ODC)activity induced by croton oil in mice. GA could also protect rapid DNA damage and decrease the unscheduled DNA synthesis induced by benzo(α)pyrene. The results demonstrate that GA has a potential cancer chemopreventive activity.

Tunable UCST thermoresponsive copolymers based on natural glycyrrhetinic acid

Water-soluble thermoresponsive polymers present either upper critical solution temperature(UCST) or lower critical solution tempe rature(LCST) depending on the location of their miscibility range with water at high temperatures or at low temperatures.Compared with LCST polymers,the water-soluble UCST polymers are still less explored until now.In this work three copolymers of P(AAm-co-GAA) were synthesized by copolymerizing two acrylamide monomers,acrylamide(AAm) and acrylamide functionalized with natural glycyrrhetinic acid(GAA),using reversible addition-fragmentation chain transfer(RAFT) polymerization.These copolymers exhibited the typical UCST thermoresponsive behavior,and their phase transition temperatures could be easily tuned to around 37℃ for potential biological applications.Moreover,the UCST of P(AAm-co-GAA) can be adjusted not only by the content of glycyrrhetinic acid(GA) and polymer concentrations,but also by the host-guest interactions between GA and cyclodextrins(β-and γ-CD).The suitable value of UCST and the biocompatible nature of GA and CDs may endow these copolymers with practical applications in biomedical chemistry.

α-Glucosidase Inhibitors from Glycyrrhiza uralensis Fisch.

Aim To screen for α-glucosidase inhibitor from Glyeyrrhiza uralensis Fisch.. Methods Glycyrrhizic acid, glycyrrhetinic acid, flavonoids of glycyrrhiza, alkaloids of glycyrrhiza, and glycyrrhiza polysaccharides were isolated from the root of Glycyrrhiza uralensis Fisch. respectively. Three compounds were isolated from the flavonoids of glycyrrhiza as guided by the α-glucosidase inhibitory test in vitro. Moreover, the characteristics of inhibitory kinetics of glycyrol and glycyrrhetinic acid were investi- gated. Results The flavonoids of glycyrrhiza and glycyrrhetinic acid had the strongest α-glucosidase inhibitory activity. Glycyrol,β-sitosterol and liquifitin were isolated and identified. Glycyrol was a fast- binding, reversible, noncompetitive α-glucosidase inhibitor, showing IC50 at 0.26 μg·mL^-1 Glycyrrhetinic acid was a fast-binding, irreversible α-glucosidase inhibitor, showing IC50 at 102.4 μg·mL^-1. Conclusion Glycyrol is an effective α-glucosidase inhibitor.

Identification of potential anti-pneumonia pharmacological components of Glycyrrhizae Radix et Rhizoma after the treatment with Gan An He Ji oral liquid

Glycyrrhizae Radix et Rhizoma,a traditional Chinese medicine also known as Gan Cao(GC),is frequently included in clinical prescriptions for the treatment of pneumonia.However,the pharmacological components of GC for pneumonia treatment are rarely explored.Gan An He Ji oral liquid(GAHJ)has a simple composition and contains GC liquid extracts and paregoric,and has been used clinically for many years.Therefore,GAHJ was selected as a compound preparation for the study of GC in the treatment of pneumonia.We conducted an in vivo study of patients with pneumonia undergoing GAHJ treatments for three days.Using the intelligent mass spectrometry data-processing technologies to analyze the metabolism of GC in vivo,we obtained 168 related components of GC in humans,consisting of 24 prototype components and 144 metabolites,with 135 compounds screened in plasma and 82 in urine.After analysis of the metabolic transformation relationship and relative exposure,six components(liquiritin,liquiritigenin,glycyrrhizin,glycyrrhetinic acid,daidzin,and formononetin)were selected as potential effective components.The experimental results based on two animal pneumonia models and the inflammatory cell model showed that the mixture of these six components was effective in the treatment of pneumonia and lung injury and could effectively downregulate the level of inducible nitric oxide synthase(iNOS).Interestingly,glycyrrhetinic acid exhibited the strongest inhibition on iNOS and the highest exposure in vivo.The following molecular dynamic simulations indicated a strong bond between glycyrrhetinic acid and iNOS.Thus,the current study provides a pharmaceutical basis for GC and reveals the possible corresponding mechanisms in pneumonia treatment.

The antiviral and antimicrobial activities of licorice, a widely-used Chinese herb

Licorice is a common herb which has been used in traditional Chinese medicine for centuries.More than 20 triterpenoids and nearly 300 flavonoids have been isolated from licorice. Recent studies have shown that these metabolites possess many pharmacological activities, such as antiviral,antimicrobial, anti-inflammatory, antitumor and other activities. This paper provides a summary of the antiviral and antimicrobial activities of licorice. The active components and the possible mechanisms for these activities are summarized in detail. This review will be helpful for the further studies of licorice for its potential therapeutic effects as an antiviral or an antimicrobial agent.

Antitumor Activities of Widely-used Chinese Herb—Licorice

Licorice （the roots of Glycyrrhiza uralensis） is widely-used in Chinese herbal compound prescriptions for its functions of nourishing qi, alleviating pain, tonifying spleen and stomach, eliminating phlegm, relieving coughing, and harmonizing prescriptions. It contains more than 20 triterpenoids and approximately 300 flavonoids. In recent years, many studies have reported that it possesses various pharmacological activities, such as antitumor, antimicrobial, and antivirus effects. In this paper, the antitumor activity of licorice is deeply summarized. The antitumor active components and the possible antitumor mechanism are analyzed.

HPLC–MS and HPLC–MS/MS analysis of seven active constituents of Xiao-Xu-Ming decoction and application to a pharmacokinetic study after oral administration to rat

Xiao-xu-ming decoction(XXMD)is a traditional Chinese medicine that has been widely used to treat theoplegia and its sequelae.This paper reports the development of three separate assays based on reversed phase high-performance liquid chromatography–mass spectrometry(HPLC–MS)and HPLC–MS/MS for the determination of seven active constituents of XXMD viz oroxylin A-7-O-glucuronide,wogonoside,liquiritigenin,cimifugin,5-O-methylvisammiol,glycyrrhizic acid and glycyrrhetinic acid in rat plasma.All calibration curves were linear(r >0.99)with lower limits of quantitation(LLOQs)<12.4 ng/mL.Intra-and inter-day precisions(as relative standard deviation)were all <10.7% with recoveries in the range of 88.7–113%.In addition,the seven analytes were shown to be stable in rat plasma samples under relevant storage conditions.The validated methods were successfully applied to a pharmacokinetic study in rat after oral administration of XXMD.