Glycyrrhizic Acid Protects Glomerular Podocytes Induced by High Glucose by Modulating SNARK/AMPK Signaling Pathway

Objective:Diabetic nephropathy is one of the most important microvascular complications of diabetes,which mainly refers to glomerular capillary sclerosis.Podocytes are an important part of glomerular capillaries.Previous clinical and basic studies have shown that fibrosis is the main factor of diabetic nephropathy.This study aimed to assess the protective mechanism of glycyrrhizic acid(GA)on glomerular podocytes induced by high glucose as we hypothesized that GA may have antifibrotic and anti-inflammatory effects on podocytes through regulation of the adenosine 5'-monophosphate-activated protein kinase(AMPK)/sucrose nonfermenting AMPK-related kinase(SNARK)signaling pathway.Methods:SNARK siRNA was used to transfect podocytes.Real-time quantitative polymerase chain reaction and immunofluorescence staining assays were used for molecular and pathological analysis.The expression levels of key pathway proteins(including TGF-β1,α-SMA,SITR1,AMPKα,LKB1,PGC-1α,NF-κB,IL-6,and TNF-α)were verified by Western blotting.The expression of inflammatory factors in podocytes was detected by ELISA.Results:We demonstrated that GA decreased the expression of podocyte fibrosis signaling pathway-related factors by upregulating the AMPK pathway and its related factors.However,after transfection of podocytes with SNARK siRNA,there was an increased expression of fibrosis-related factors and inflammation-related factors.Conclusion:GA can protect podocytes and alleviate fibrosis and inflammation induced by high glucose,which is related to the AMPK signaling pathway.Meanwhile,knockdown of SNARK protein can inhibit the AMPK signaling pathway,aggravate fibrosis,and increase inflammation.

The Potential Efficacy of Glycyrrhizic Acid and Its Nanostructure Against Brown Rot of Peach fruits

Production of peaches(Prunus persica(L.)Batsch)for both local market and export is increasing each year in Egypt.Brown rot disease,caused by Monilinia laxa and Monilinia fructigena,is considered one of the most important postharvest rots affecting peaches in Egypt and economic losses are increasing.Antifungal activity of glycyrrhizic acid nanoparticles(GA-NPs)and glycyrrhizic acid(GA)at 0.2 and 0.4 mmol/L was investigated as a control for both these brown rot pathogens on peach fruits in both in vitro and in vivo studies.In the in vitro studies,GA-NPs were the most effective as shown by the ability to decrease linear growth of both brown rot pathogens in potato dextrose agar(PDA)amended with 0.4 mmol/L GA-NPs.Micrographs of M.fructigena exposed to 0.4 mmol/LGA showed mycelial deformations,nodule formation,detachment of the cell wall,shrinkage and inhomogeneous cytoplasmic materials with large vacuoles.Mycelium of M.laxa exposed to 0.4 mmol/LGA-NPs resulted in thinner and distorted hyphae,nodule formation,cell wall thinning,and swellings.The GANPs and GA treatments improved fruit quality by maintaining firmness and total soluble solids(TSS).GA-NPs were more effective in decreasing decay incidence than their bulk material.The 0.4 mmol/L GA-NPs completely inhibited the disease on naturally infected peach fruits for both seasons of 2018 and 2019.Furthermore,0.4 mmol/L GA-NPs reduced the disease incidence in inoculated fruits by 95(M.laxa)and 88%(M.fructigena)in 2018 season and 96(M.laxa)and 85%(M.fructigena)in 2019 season.In conclusion,GA-NPs could enhance the resistance of peaches against brown rot caused by M.laxa and M.fructigena.

Intervention effects and related mechanisms of glycyrrhizic acid on zebrafish with Hirschsprung-associated enterocolitis

BACKGROUND The prevention and treatment of Hirschsprung-associated enterocolitis(HAEC)is a serious challenge in pediatric surgery.Exploring the mechanism of HAEC is conducive to the prevention of this disease.AIM To explore the possible mechanism of glycyrrhizic acid(GA)and its therapeutic effect on HAEC.METHODS We developed a model of enteritis induced by trinitrobenzenesulfonic acid(TNBS)in zebrafish,and treated it with different concentrations of GA.We analyzed the effect of GA on the phenotype and inflammation of zebrafish.RESULTS After treatment with TNBS,the area of the intestinal lumen in zebrafish was significantly increased,but the number of goblet cells in the intestinal lumen was significantly reduced,but these did not increase the mortality of zebrafish,indicating that the zebrafish enteritis model was successfully developed.Different concentrations of GA protected zebrafish with enteritis.In particular,high concentrations of GA were important for the prevention and control of HAEC because it significantly reduced the intestinal luminal area,increased the number of goblet cells in the intestinal lumen,and reduced the levels of interleukin(IL)-1βand IL-8.CONCLUSION GA significantly reduced the intestinal luminal area,increased the number of intestinal goblet cells,and decreased IL-1βand IL-8 in zebrafish,and is important for prevention and control of HAEC.

Glycyrrhizic Acid-Loaded Poly-ɛ-Caprolactone Nanoparticles Decrease PRRSV Infection in MARC-145 Cells

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating disease with worldwide distribution caused by Betaarterivirus suid (PRRSV). The virion has great genetic and antigenic variability with a marked increase in virulence. Vaccines tested to date have been of little use in controlling the problems caused by PRRSV, so the present study was conceived to evaluate the antiviral effect of polymeric nanoparticles (PNPs) made with glycyrrhizic acid (GA). Recent work has proven that this nanoparticle system is stable. These nanoparticles have good GA carrying capacity, a size < 250 nm, a spherical morphology, and a wide safety range. The integrity of cell morphology can be maintained for up to 72 h. The antiviral effect of this nanoparticle system was tested in cultures of MARC-145 cells in pre- and coinfection assays with PRRSV to evaluate changes in cell morphology and effects on cell viability. The use of PNPsGA with the real-time quantitative polymerase chain reaction (RT-qPCR) decreased viral infection by 38% in 3 amplification cycles. These results suggest that this system has an antiviral effect against PRRSV under the study conditions established.

MICROWAVE-ASSISTED EXTRACTION OF GLYCYRRHIZIC ACID FROM LICORICE ROOT-EFFECT OF THE PROPERTY OF SOLUTION ON EXTRACTION OF GA

The property of extraction solution is an important factor influencing the extraction efficiency. In the present work, the effect of the property of solution on extraction of GA was studied, which including the concentration of ethanol, ammonia and cation (M+), pH of extraction solution, different kinds of organic solvent etc. The results show that 50%-60%(v/v) ethanol can reach high percentage extraction of GA. If 1% (v/v) ammonia solution was added into 60%(v/v) ethanol, the percentage extraction can be increased from 2.0% to 2.31%. Without ammonia, 50mmol/L [M+] (M+ = K+, NH4+) was added into 60%(v/v) ethanol, percentage extraction of GA can reach about 2.26%. If pH of solution (60% ethanol) was adjust to pH=4.0, it can reach high percentage extraction. If pH of solution (60% ethanol + 50mmol [M+], pH=6.1) was adjust tO PH=4.0, especially M+ is K+ or NH4+, it can reach almost same extraction efficiency as that of 1% ammonia solution + 60% ethanol, and the operation environment can be greatly improved.

Glycyrrhizic acid promotes sciatic nerves recovery in type 1 diabetic rats and protects Schwann cells from high glucose-induced cytotoxicity

The present study aims to investigate the therapeutic effect and mechanism of glycyrrhizic acid(GA)in diabetic peripheral neuropathy(DPN).GA significantly mitigated nerve conduction velocity(NCV)deficit and morphological abnormality and reduced high-mobility group box-1(HMGB1)expression in the sciatic nerves of diabetic rats independent of blood glucose and body weight.Notably,GA alleviated the increase of HMGB1 and the decrease of cell viability in high glucose-stimulated RSC96 cells.Furthermore,GA obviously reduced the concentration of inflammatory cytokines in the sciatic nerves of diabetic rats and supernatants of high glucose-exposed RSC96 cells,then restored the decreased expression levels of nerve growth factor(NGF)and neuritin-1,and the increased expression levels of cleaved caspase-3 and neuron-specific enolase.Additionally,GA markedly inhibited receptor for advanced glycation end products(RAGE)expression,p38MAPK phosphorylation,and the nuclear translocation of NF-κBp65 in diabetic rats and high glucose-exposed RSC96 cells.The promotional effect of high glucose in RSC96 cells was diminished following Hmgb1 siRNA treatment.Our findings indicate that GA may exert neuroprotection on DPN by suppressing HMGB1,which lead to extenuation of inflammation response,balance of NGF,neuritin-1 and caspase-3,as well as inactivation of RAGE/p38MAPK/NF-κBp65 signaling pathway.

Simultaneous determination of 12,13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture by high performance liquid chromatography

A reversed phase high performance liquid chromatography （HPLC） method was established for the simultaneous determination of 12, 13-dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture. A Grace Apollo Cl8 column （250 mm × 4.6 mm, 5 μm） was used as the stationary phase and the mobile phase was composed of acetonitrile and aqueous phosphoric acid （0.2%, v/v）. Gradient elution was carried out at the flow rate of 1.0 mL/min and the column temperature was 30 ℃. An ultraviolet （UV） detector was used with a selected wavelength of 240 nm. Calibration curves were linear within the concentration range of 4.6-45.75 μg/mL for 12, 13-dihydroxyeuparin （r〉0.9999） and 106.9-1068.9μg/mL for glycyrrhizic acid （r〉0.9999）, respectively. Recoveries were 102.18% for 12, 13-dihydroxyeuparin and 101.17% for glycyrrhizic acid. The method developed could be applied to the simultaneous determination of 12, 13- dihydroxyeuparin and glycyrrhizic acid in Yanyanfang mixture.

Effect of glycyrrhizic acid and 18β-glycyrrhetinic acid on the differentiation of human umbilical cord-mesenchymal stem cells into hepatocytes

BACKGROUND End-stage liver disease is a global health complication with high prevalence and limited treatment options.Cell-based therapies using mesenchymal stem cells(MSCs)emerged as an alternative approach to support hepatic regeneration.In vitro preconditioning strategies have been employed to strengthen the regenerative and differentiation potential of MSCs towards hepatic lineage.Chemical compounds of the triterpene class;glycyrrhizic acid(GA)and 18β-glycyrrhetinic acid(GT)possess diverse therapeutic properties including hepatoprotection and anti-fibrosis characteristics.They are capable of modulating several signaling pathways that are crucial in hepatic regeneration.Preconditioning with hepato-protective triterpenes may stimulate MSC fate transition towards hepatocytes.AIM To explore the effect of GA and GT on hepatic differentiation of human umbilical cord-MSCs(hUC-MSCs).METHODS hUC-MSCs were isolated and characterized phenotypically by flow cytometry and immunocytochemistry for the expression of MSC-associated surface molecules.Isolated cells were treated with GA,GT,and their combination for 24 h and then analyzed at three time points;day 7,14,and 21.qRT-PCR was performed for the expression of hepatic genes.Expression of hepatic proteins was analyzed by immunocytochemistry at day 21.Periodic acid Schiff staining was performed to determine the functional ability of treated cells.RESULTS The fusiform-shaped morphology of MSCs in the treatment groups in comparison with the untreated control,eventually progressed towards the polygonal morphology of hepatocytes with the passage of time.The temporal transcriptional profile of preconditioned MSCs displayed significant expression of hepatic genes with increasing time of differentiation.Preconditioned cells showed positive expression of hepatocyte-specific proteins.The results were further corroborated by positive periodic acid Schiff staining,indicating the presence of glycogen in their cytoplasm.Moreover,bi-nucleated cells,which is the typical feature of hepatocytes,were also seen in the preconditioned cells.CONCLUSION Preconditioning with glycyrrhizic acid,18β-glycyrrhetinic acid and their combination,successfully differentiates hUC-MSCs into hepatic-like cells.These MSCs may serve as a better therapeutic option for degenerative liver diseases in future.

Glycyrrhizic Acid-Targeted Carbon Nanotube Nanocomposites with Polyethylenimines-Mediated for Cancer Drug Delivery

In order to enhance the efficiency and specificity of anticancer drug delivery and realize intelligently controlled release,a new multi-functional nanoparticle drug carrier was synthesized.The drug carrier was prepared by functionalizing multi-walled carbon nanotubes(MWCNTs) with polyethylenimines(PEI),fluorescein isothiocyanate(FITC) and glycyrrhizic acid(GL).After detailed characterization,doxorubicin(DOX) was loaded onto the obtained MWCNT composites through π-π stacking interactions.The drug loading capacity of the GL-functionalized material was up to 92%,and the release behavior was significantly pH-sensitive.Release at pH = 5.8(typical of the tumor cell microenvironment) was much more rapid and reached a greater extent than release under normal physiological conditions(pH = 7.4).The modified MWCNTs had high biocompatibility with the liver cancer cell line SMMC-7721,but were able to induce cell death after 24 h incubation if loaded with DOX.Tests with shorter incubation time(2 h) were undertaken to investigate the selectivity of the MWCNT composites,showed that the nanocomposites could specifically target cancer cells.The above results suggest that the functionalized carbon nanotubes-based material has potential applications for targeted delivery and controlled release of anticancer drug.

Glycyrrhizic Acid Attenuates Balloon-Induced Vascular Injury Through Inactivation of RAGE Signaling Pathways

Percutaneous coronary intervention is a well-established technique used to treat coronary artery disease,but the risk of coronary artery in-stent restenosis following percutaneous coronary intervention is still high.Previous studies revealed that high mobility group protein B1(HMGB1)plays a critical role in neointima formation.In this study,we aimed to investigate the role of glycyrrhizic acid(GA),an HMGB1 inhibitor,in the process of neointima formation and the potential mechanisms.We investigated the role of GA in neointima formation through an iliac artery balloon injury model in rabbits.Proliferation,migration,and phenotype transformation of human vascular smooth muscle cells(VSMCs)were observed.Besides,infl ammation and receptor for advanced glycosylation end products(RAGE)signaling pathways were studied.The results indicate that GA attenuated neointima formation and downregulated HMGB1 expression in injured artery in rabbits.HMGB1 promoted proliferation,migration,and phenotype transformation through the activation of RAGE signaling pathways in VSMCs,and blockade of HMGB1 by GA(1,10,and 100μM)could attenuate those processes and reduce proliferation of human VSMCs.In conclusion,the HMGB1 inhibitor GA might be useful to treat proliferative vascular diseases by downregulating RAGE signaling pathways.Our results indicate a new and promising therapeutic agent for restenosis.

POLAROGRAPHIC CATALYTIC WAVE OF GLYCYRRHIZIC ACID

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Study on the Preparation and Characterization of Glycyrrhizic Acid Liposomes

The purpose of this study was to prepare glycyrrhizic acid nanoliposomes and evaluate the encapsulation efficiency and other properties of glycyrrhizic acid nanoliposomes, which would provide a reference for further research on Glycyrrhizic Acid in the treatment of liver diseases. Firstly, the preparation conditions of glycyrrhizic acid liposomes were optimized by the orthogonal design method. Then, glycyrrhizic acid liposomes were prepared by ultrasonic-film dispersion method and the encapsulation efficiency was determined by the HPLC method. Finally, the properties of glycyrrhizic acid nanoliposomes were also studied. The results showed that the optimal preparation conditions of liposomes were as follows: the ratio of drug to lipid was 1:30;Lecithin: cholesterol = 1:1;Hydration medium: pure water;Ultrasonic time: 120 s. The encapsulation efficiency of liposomes was about 90%. The final liposomes were round and uniform in distribution, with an average particle size of about 50 nm and absolute zeta potential of &#8722;28.9 mV. In this study, glycyrrhizic acid liposomes were prepared and the optimal preparation conditions were optimized. The encapsulation efficiency of the liposomes under the optimized conditions was determined. The evaluation of the morphology, size, particle size and stability of the liposomes was completed.

Cloning and characterization of the gene encoding β-amyrin synthase in the glycyrrhizic acid biosynthetic pathway in Glycyrrhiza uralensis

Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic.Based on previous research,these effects are mediated by a number of active ingredients,especially glycyrrhizic acid(GA).In the present study,a gene encodingβ-amyrin synthase(β-AS)involved in GA biosynthesis in G.uralensis has been cloned and expressed in Saccharomyces cerevisiae.The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene intoβ-amyrin.In fact theβ-AS gene is particularly important in the GA biosynthetic pathway in G.uralensis.The complete sequence of the enzyme was determined and a phylogenetic tree based on theβ-AS gene of G.uralensis and 20 other species was created.This showed that Glycyrrhiza glabra had the closest kinship with G.uralensis.The results of this work will be useful in determining how to improve the efficacy of G.uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro.

Preparation of Glycyrrhetinic Acid Monoglucuronide by Selective Hydrolysis of Glycyrrhizic Acid via Biotransformation

Objective To search for the microorganisms which have the high selectivity of hydrolyzing glycyrrhizic acid(GL) into 18β-glycyrrhetinic acid-3-O-β-D-glucuronide(GAMG) without glycyrrhetinic acid(GA) byproduct.Methods GL was biotransformed by Aspergillus sp.,the products were separated by chromatography on reverse phase C18 column and semi-preparative HPLC,and their structures were elucidated on the basis of HR-ESI-MS,1D NMR(1H-NMR,13C-NMR,and NOESY) and 2D NMR(1H-1H COSY,HSQC,and HMBC) spectral analyses.Results Aspergillus sp.could partially hydrolyze GL into GAMG(3),along with two minor byproducts,3-O-β-D-glucuronopyranosyl-18β-liquiritic acid(1) and 3-O-β-D-glucuronopyranosyl-24-hydroxy-18β-glycyrrhetinic acid(2).Conclusion Aspergillus sp.has the high selectivity of hydrolyzing GL into GAMG without GA byproduct and the yield of GAMG is about 60%.The complete assignments of 1H-NMR and 13C-NMR data for compounds 1 and 2 are reported for the first time.

Allicin and Glycyrrhizic Acid Display Antiviral Activity Against Latent and Lytic Kaposi Sarcoma-associated Herpesvirus

Kaposi sarcoma-associated herpesvirus(KSHV)triggers the development of Kaposi sarcoma,a skin malignancy that is one of the most widespread defining symptoms in acquired immunodeficiency syndrome patients.KSHV manifests in two distinct cycles,a chronic latent cycle and an acute lytic cycle.Current clinical anti-herpesvirus therapeutic agents are predominantly composed of nucleoside analogues that target viral replication in the lytic cycle only,while KSHV latent genes are at the basis tumorigenesis.Currently,there are no effective therapies targeting latent KSHV infections.Therefore,the aim of this study was to identify putative therapeutic compounds with inhibitory activity against latent KSHV.The KSHV-infected primary effusion lymphoma cell line BC-3 was used to study antiviral activity of glycyrrhizic acid(GA),Allicin,and epigallocatechin-3-gallate(EGCG)against latent and lytic KSHV.Activity of GA,Allicin,EGCG,and the established anti-lytic cycle control compound ganciclovir was quantified by real-time polymerase chain reaction of nuclear and virion KSHV DNA yields after treatment compared with the untreated control.GA and Allicin showed antiviral activity against both latent and lytic KSHV,while EGCG displayed activity against lytic KSHV only.Therefore,GA and Allicin are interesting compounds for further development of anti-KSHV therapy against latent cycle infections.

Hypertension Unusual Cause

We expose a case of a woman with hypertension and hypokalemia. The differential diagnosis should include primary hyperaldosteronism, diuretics or lazantes intake, secondary hyperaldosteronism. In this patient, additional tests performed show no cause of hormonal disruption and the whole picture is due to a high intake of licorice. Glycyrrhetinic acid, the active component of licorice, inhibits renal IIbeta-hydroxisteroid dehydrogenase. This allows cortisol to stimulate mineralocorticoid receptors. Licorice ingestion should be considered in the differential diagnosis of hypertension with hypokalemia.

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.

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.

Anti-pseudo-allergic components in licorice extract inhibit mast cell degranulation and calcium influx

Pseudo-allergic reactions(PARs)widely occur upon application of drugs or functional foods.Anti-pseudo-allergic ingredients from natural products have attracted much attention.This study aimed to investigate anti-pseudo-allergic compounds in licorice.The anti-pseudo-allergic effect of licorice extract was evaluated in rat basophilic leukemia 2H3(RBL-2H3)cells.Anti-pseudo-allergic compounds were screened by using RBL-2H3 cell extraction and the effects of target components were verified further in RBL-2H3 cells,mouse peritoneal mast cells(MPMCs)and mice.Molecular docking and human MRGPRX2-expressing HEK293T cells(MRGPRX2-HEK293T cells)extraction were performed to determine the potential ligands of MAS-related G protein-coupled receptor-X2(MRGPRX2),a pivotal target for PARs.Glycyrrhizic acid(GA)and licorice chalcone A(LA)were screened and shown to inhibit Compound48/80-induced degranulation and calcium influx in RBL-2H3 cells.GA and LA also inhibited degranulation in MPMCs and increase of histamine and TNF-a in mice.LA could bind to MRGPRX2,as determined by molecular docking and MRGPRX2-HEK293T cell extraction.Our study provides a strong rationale for using GA and LA as novel treatment options for PARs.LA is a potential ligand of MRGPRX2.