Glycyrrhizin Production in Licorice Hairy Roots Based on Metabolic Redirection of Triterpenoid Biosynthetic Pathway by Genome Editing.

Glycyrrhizin, a type of the triterpenoid saponin, is a major active ingredient contained in the roots of the medicinal plant licorice (Glycyrrhiza uralensis, G. glabra and G. inflata), and is used worldwide in diverse applications, such as herbal medicines and sweeteners. The growing demand for licorice threatens wild resources and therefore a sustainable method of supplying glycyrrhizin is required. With the goal of establishing an alternative glycyrrhizin supply method not dependent on wild plants, we attempted to produce glycyrrhizin using hairy root culture. We tried to promote glycyrrhizin production by blocking competing pathways using CRISPR/Cas9-based gene editing. CYP93E3 CYP72A566 double-knockout (KO) and CYP93E3 CYP72A566 CYP716A179 LUS1 quadruple-KO variants were generated, and a substantial amount of glycyrrhizin accumulation was confirmed in both types of hairy root. Furthermore, we evaluated the potential for promoting further glycyrrhizin production by simultaneous CYP93E3 CYP72A566 double-KO and CYP88D6-overexpression. This strategy resulted in a 3-fold increase (∼1.4 mg/g) in glycyrrhizin accumulation in double-KO/CYP88D6-overexpression hairy roots, on average, compared with that of double-KO hairy roots. These findings demonstrate that the combination of blocking competing pathways and overexpression of the biosynthetic gene is important for enhancing glycyrrhizin production in G. uralensis hairy roots. Our findings provide the foundation for sustainable glycyrrhizin production using hairy root culture. Given the widespread use of genome editing technology in hairy roots, this combined with gene knockout and overexpression could be widely applied to the production of valuable substances contained in various plant roots.

Glycyrrhizic acid improves tacrolimus-induced renal injury by regulating autophagy.

Tacrolimus (TAC)-induced renal injury is detrimental to long-term kidney function, but a treatment medication is not available. Glycyrrhizic acid (GA) is an active ingredient in licorice widely used to treat kidney disease. Thus, this study explored the mechanisms of renoprotection by GA on TAC-induced renal injury. C57BL/6 mice were subjected daily to TAC or a combination of TAC and GA for 4 weeks, and then renal function, histopathology, and autophagy were assessed to examine the effect of GA on a renal injury. Next, Human kidney proximal tubular epithelial (HK-2) cells were pretreated with GA for 2 h and then treated with TAC for 24 h. The effect of GA on TAC-induced HK-2 cell injury was assessed by measuring cell viability, apoptosis, autophagy, and lysosomes. Mice exposed to TAC and treated with GA had significantly greater improvements in renal function and tubulointerstitial fibrosis in comparison to mice not treated with GA. In addition, fibrosis-related protein expression, including α-smooth muscle actin and fibronectin, decreased after GA treatment. GA treatment also relieved autophagic clearance in TAC-induced renal injury. Several in vitro studies found that TAC inhibited cell viability, autophagy, lysosomal acidification, and promoted apoptosis. However, these results were less pronounced with GA pretreatment. In addition, bafilomycin A1 (which inhibits lysosomal function) reduced the protective effect of GA, indicating that lysosomal function plays an important role in this effect. Our data suggest that GA improves lysosomal function and regulates autophagy to protect against TAC-induced renal injury.

Glycyrrhizin regulates antioxidation through Nrf2 signaling pathway in rat temporomandibular joint osteoarthritis.

BACKGROUND: Regulation of redox homeostasis could reduce osteoarthritis severity and limit disease progression, while glycyrrhizin (GL) shows great antioxidant and anti-inflammatory capacity. OBJECTIVE: The aim of this study was to investigate the role of GL on oxidative stress and the potential regulatory mechanism in rat temporomandibular joint (TMJ) chondrocytes under oxidative stress, and investigate the effect of GL in the rat temporomandibular joint osteoarthritis (TMJOA) model. METHODS: Rat TMJ chondrocytes were cultured in oxidative stress with different doses of GL. The effect of glycyrrhizin on the nuclear factor-erythroid 2-related factor 2 (Nrf2) in oxidative stress was evaluated by western blot and immunofluorescence staining. A rat model of TMJOA was treated with GL. Micro-computed tomography, histological and immunohistochemical analysis were used to assess the pathological change of TMJOA. RESULTS: The expression of superoxide dismutase 1 (SOD1), heme oxygenase-1 (HO-1), and peroxiredoxin 6 (PRDX6) were decreased, and intracellular Nrf2 signaling pathway was activated in chondrocytes in oxidative stress. GL upregulates the expression of antioxidants, especially PRDX6, as well as increases Nrf2 expression and nuclear translocation in rat condylar chondrocytes. Administration of GL attenuates condylar bone destruction, cartilage degeneration, and synovitis in rats TMJOA. Meanwhile, GL alleviated oxidative stress and enhanced the antioxidant capacity of TMJOA cartilage. CONCLUSION: This study suggested that GL alleviates rat TMJOA by regulating oxidative stress in condylar cartilage.

Glycyrrhizin, an inhibitor of HMGB1 induces autolysosomal degradation function and inhibits Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori is a key agent for causing gastric complications linked with gastric disorders. In response to infection, host cells stimulate autophagy to maintain cellular homeostasis. However, H. pylori have evolved the ability to usurp the host's autophagic machinery. High mobility group box1 (HMGB1), an alarmin molecule is a regulator of autophagy and its expression is augmented during infection and gastric cancer. Therefore, this study aims to explore the role of glycyrrhizin (a known inhibitor of HMGB1) in autophagy during H. pylori infection. MAIN METHODS: Human gastric cancer (AGS) cells were infected with the H. pylori SS1 strain and further treatment was done with glycyrrhizin. Western blot was used to examine the expression of autophagy proteins. Autophagy and lysosomal activity were monitored by fluorescence assays. A knockdown of HMGB1 was performed to verify the effect of glycyrrhizin. H. pylori infection in in vivo mice model was established and the effect of glycyrrhizin treatment was studied. RESULTS: The autophagy-lysosomal pathway was impaired due to an increase in lysosomal membrane permeabilization during H. pylori infection in AGS cells. Subsequently, glycyrrhizin treatment restored the lysosomal membrane integrity. The recovered lysosomal function enhanced autolysosome formation and concomitantly attenuated the intracellular H. pylori growth by eliminating the pathogenic niche. Additionally, glycyrrhizin treatment inhibited inflammation and improved gastric tissue damage in mice. CONCLUSION: This study showed that inhibiting HMGB1 restored lysosomal activity to ameliorate H. pylori infection. It also demonstrated the potential of glycyrrhizin as an antibacterial agent to address the problem of antimicrobial resistance.

Site-specific crosslinking and assembly of tetrameric ß-glucuronidase improve glycyrrhizin hydrolysis.

In this study, eight nonconserved residues with exposed surfaces and flexible conformations of the homotetrameric PGUS (ß-glucuronidase from Aspergillus oryzae Li-3) were identified. Single-point mutation into cysteine enabled the thiol-maleimide reaction and site-specific protein assembly using a two-arm polyethylene glycol (PEG)-maleimide crosslinker (Mal2 ). The Mal2 (1k) (with 1 kDa PEG spacer)-crosslinked PGUS assemblies showed low crosslinking efficiency and unimproved thermostability except for G194C-Mal2 (1k). To improve the crosslinking efficiency, a lengthened crosslinker Mal2 (2k) (with 2 kDa PEG spacer) was used to produce PGUS assembly and a highly improved thermostability was achieved with a half-life of 47.2-169.2 min at 70°C, which is 1.04-3.74 times that of wild type PGUS. It is found that the thermostability of PGUS assembly was closely associated with the formation of inter-tetramer assembly and intratetramer crosslinking, rather than the PEGylation of the enzyme. Therefore, the four-arm PEG-maleimide crosslinker Mal4 (2k) (with 2 kDa PEG spacer) was employed to simultaneously increase the inter-tetramer assembly and intratetramer crosslinking, and the resulting PGUS assemblies showed further improved thermostabilities compared with Mal2 (2k)-crosslinked assemblies. Finally, the application of PGUS assemblies with significantly improved thermostability to the bioconversion of GL proved that the PGUS assembly is a strong catalyst for glycyrrhizin (GL) hydrolysis in industrial applications.

Disruption of a licorice cellulose synthase-derived glycosyltransferase gene demonstrates its in planta role in soyasaponin biosynthesis.

KEY MESSAGE: CRISPR-Cas9-mediated disruption of a licorice cellulose synthase-derived glycosyltransferase gene, GuCSyGT, demonstrated the in planta role of GuCSyGT as the enzyme catalyzing 3-O-glucuronosylation of triterpenoid aglycones in soyasaponin biosynthesis. Triterpenoid glycosides (saponins) are a large, structurally diverse group of specialized metabolites in plants, including the sweet saponin glycyrrhizin produced by licorice (Glycyrrhiza uralensis) and soyasaponins that occur widely in legumes, with various bioactivities. The triterpenoid saponin biosynthetic pathway involves the glycosylation of triterpenoid sapogenins (the non-sugar part of triterpenoid saponins) by glycosyltransferases (GTs), leading to diverse saponin structures. Previously, we identified a cellulose synthase-derived GT (CSyGT), as a newly discovered class of triterpenoid GT from G. uralensis. GuCSyGT expressed in yeast, which could transfer the sugar glucuronic acid to the C3 position of glycyrrhetinic acid and soyasapogenol B, which are the sapogenins of glycyrrhizin and soyasaponin I, respectively. This suggested that GuCSyGT is involved in the biosynthesis of glycyrrhizin and soyasaponin I. However, the in planta role of GuCSyGT in saponin biosynthesis remains unclear. In this study, we generated GuCSyGT-disrupted licorice hairy roots using CRISPR-Cas9-mediated genome editing and analyzed the saponin content. This revealed that soyasaponin I was completely absent in GuCSyGT-disrupted lines, demonstrating the in planta role of GuCSyGT in saponin biosynthesis.

Incorporation of glycyrrhizic acid and polyene phosphatidylcholine in lipid nanoparticles ameliorates acute liver injury via delivering p65 siRNA.

Liver injury caused by hepatitis is the pathological basis of varied hepatic diseases with high morbidity and mortality. Although siRNA appears promising in therapeutics of hepatitis, efficient and safe delivery remains a challenge. In this study, we developed a new strategy of incorporating glycyrrhizic acid (GA) and polyene phosphatidylcholine (PPC) into lipid nanoparticles (GA/PPC-modified LNPs), which was capable of promoting cellular uptake, enhancing gene-silencing, reducing cytotoxicity and improving siRNA stability. GA/PPC-modified LNP and siRNA lipoplex targeting NF-κB, a key mediator of inflammation, mitigates acute liver injury, as assessed by liver histology, hematological and pro-inflammatory cytokine analysis. Furthermore, GA/PPC-modified LNPs reveal efficiently intracellular delivery of antisense oligonucleotides (ASOs) and mRNA inhibiting viral infection. In conclusion, GA/PPC-modified LNPs could be used as a promising delivery system for nucleic acid-based therapy.

Multi-Omics Elucidates Difference in Accumulation of Bioactive Constituents in Licorice (Glycyrrhiza uralensis) under Drought Stress.

Licorice is a frequently applied herb with potential edible and medicinal value based on various flavonoids and triterpenes. However, studies on detailed flavonoid and triterpene metabolism and the molecular basis of their biosynthesis in licorice are very limited, especially under drought conditions. In the present study, we carried out transcriptome, proteome, and metabolome experiments. To ultimately combine three omics for analysis, we performed a bioinformatics comparison, integrating transcriptome data and proteome data through a Cloud platform, along with a simplified biosynthesis of primary flavonoids and triterpenoids in the KEGG pathway based on metabolomic results. The biosynthesis pathways of triterpenes and flavonoids are enriched at both gene and protein levels. Key flavonoid-related genes (PAL, 4CL, CHS, CHI, CYP93C, HIDH, HI4OMT, and CYP81E1_7) and representative proteins (HIDH, CYP81E1_7, CYP93C, and VR) were obtained, which all showed high levels after drought treatment. Notably, one R2R3-MYB transcription factor (Glyur000237s00014382.1), a critical regulator of flavonoid biosynthesis, achieved a significant upregulated expression as well. In the biosynthesis of glycyrrhizin, both gene and protein levels of bAS and CYP88D6 have been found with upregulated expression under drought conditions. Most of the differentially expressed genes (DEGs) and proteins (DEPs) showed similar expression patterns and positively related to metabolic profiles of flavonoid and saponin. We believe that suitable drought stress may contribute to the accumulation of bioactive constituents in licorice, and our research provides an insight into the genetic study and quality breeding in this plant.

Antioxidant monoammonium glycyrrhizinate alleviates damage from oxidative stress in perinatal cows.

This study was conducted to evaluate the antioxidant capability of dietary supplementation with monoammonium glycyrrhizinate (MAG) in perinatal cows. Glycyrrhizic acid has been shown to have strong antioxidant activity and we hypothesised that the aglycone of glycyrrhizin and MAG, could reduce damage from oxidative stress in perinatal cows by enhancing antioxidant capacity. Blood and milk samples were collected from three groups of healthy perinatal cows that were similar in body weight, parity, milk yield in the last milk cycle, etc., receiving dietary MAG supplementation ([Day 0 = parturition]: 0 g/day, [n = 13)] 3 g/day [n = 13] or 6 g/day [n = 11]) from -28 to 56 day (0 day = parturition). Compared with 0 g/day controls (CON), milk fat was significantly decreased in cows fed with MAG, and 3 g/day had the greatest effect. A diet containing 3 g/day MAG decreased the serum alanine aminotransferase (ALT) level compared with CON at -7 day post-partum. ALT was also lower at 5 day post-partum in cows fed with 3 g/day MAG compared to 6 g/day. The administration of 3 g/day and 6 g/day MAG decreased serum aspartate transaminase (AST) at 3 day post-partum. Supplementation of MAG in cows increased total antioxidant capacity (T-AOC) in serum, and cows given 3 g MAG per day had higher T-AOC than controls on post-partum 7 day. At the end of the experiment, we isolated and cultured primary hepatocytes to determine the effect of MAG on oxidative stress caused by incubation with the sodium oleate (SO). SO increased lipid synthesis, but pre-treatment with MAG prevented the fatty buildup. SO treatment increased AST and ALT levels and malondialdehyde concentration, but decreased T-AOC and superoxide dismutase (SOD). Incubation with MAG increased antioxidant capacity and inhibited oxidant damage in bovine hepatocytes. SO stimulated expression of the antioxidant genes, NAD(P)H quinone dehydrogenase 1 (NQO1) and SOD1, in the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, and catalase 1 (CAT1); this increase was accentuated by MAG pre-treatment. The results suggest that MAG can alleviate the damage caused by oxidative stress in perinatal cows by enhancing antioxidant activity.

Multi-omics profiling reveals comprehensive microbe-plant-metabolite regulation patterns for medicinal plant Glycyrrhiza uralensis Fisch.

Glycyrrhiza uralensis Fisch is a medicinal plant widely used to treat multiple diseases in Europe and Asia, and its efficacy largely depends on liquiritin and glycyrrhizic acid. The regulatory pattern responsible for the difference in efficacy between wild and cultivated G. uralensis remains largely undetermined. Here, we collected roots and rhizosphere soils from wild (WT) G. uralensis as well as those farmed for 1 year (C1) and 3 years (C3), generated metabolite and transcript data for roots, microbiota data for rhizospheres and conducted comprehensive multi-omics analyses. We updated gene structures for all 40 091 genes in G. uralensis, and based on 52 differentially expressed genes, we charted the route-map of both liquiritin and glycyrrhizic acid biosynthesis, with genes BAS, CYP72A154 and CYP88D6 critical for glycyrrhizic acid biosynthesis being significantly expressed higher in wild G. uralensis than in cultivated G. uralensis. Additionally, multi-omics network analysis identified that Lysobacter was strongly associated with CYP72A154, which was required for glycyrrhizic acid biosynthesis. Finally, we developed a holistic multi-omics regulation model that confirmed the importance of rhizosphere microbial community structure in liquiritin accumulation. This study thoroughly decoded the key regulatory mechanisms of liquiritin and glycyrrhizic acid, and provided new insights into the interactions of the plant's key metabolites with its transcriptome, rhizosphere microbes and environment, which would guide future cultivation of G. uralensis.

18ß-Glycyrrhetinic acid altered the intestinal permeability in the human Caco-2 monolayer cell model.

PURPOSE: Glycyrrhizin (GL) and its metabolites 18α-glycyrrhetinic acid (18α-GA) and 18ß-glycyrrhetinic acid (18ß-GA) are used as traditional medicine and food sweeteners. As the major rout of their administration is oral way, therefore their impact on intestinal epithelial cells are investigated. METHODS: The effects of GL and its metabolites on cell viability using MTT assay, on cytotoxicity using LDH release, on integrity of intestinal epithelial cells by measuring the transepithelial electrical resistance (TEER) and Luciferase permeability tests, on the expression of tight junction proteins at mRNA and protein level by qPCR and western blot techniques, and ultimately on the rate of test compounds absorption via Caco-2 cells monolayer were investigated. RESULTS: MTT assay showed a concentration- and time-dependent decrease in metabolic activity of Caco-2 cells induced by GL, 18α-GA, and 18ß-GA, while only 18ß-GA increased the LDH leakage. The monolayer integrity of Caco-2 cells in TEER assay only was affected by 18ß-GA. The permeability of paracellular transport marker was increased by 18α-GA and 18ß-GA and not GL. In transport studies, only metabolites were able to cross from Caco-2 cells monolayer. qPCR analyses revealed that 18ß-GA upregulated the expression of claudin-1 and -4, occludin, junctional adhesion molecules and zonula occludens-1, while 18α-GA upregulated only claudin-4. The expression of claudin-4 at protein level was downregulated non-significantly at 50 µM concentration of 18ß-GA. CONCLUSION: Our results suggest that 18ß-GA may cause cellular damages at higher concentrations on gastrointestinal cells and requires a remarkable attention of the nutraceutical and pharmaceutical industries.

Glycyrrhizic Acid Derivatives Bearing Amino Acid Residues in the Carbohydrate Part as Dengue Virus E Protein Inhibitors: Synthesis and Antiviral Activity.

Dengue virus (DENV) is one of the most geographically distributed mosquito-borne flaviviruses, like Japanese encephalitis virus (JEV), and Zika virus (ZIKV). In this study, a library of the known and novel Glycyrrhizic acid (GL) derivatives bearing amino acid residues or their methyl/ethyl esters in the carbohydrate part were synthesized and studied as DENV inhibitors in vitro using the cytopathic effect (CPE), viral infectivity and virus yield assays with DENV1 and DENV-2 in Vero E6 and A549 cells. Among the GL conjugates tested, compound hits GL-D-ValOMe 3, GL-TyrOMe 6, GL-PheOEt 11, and GL-LysOMe 21 were discovered to have better antiviral activity than GL, with IC50 values ranging from <0.1 to 5.98 µM on the in vitro infectivity of DENV1 and DENV2 in Vero E6 and A549 cells. Compound hits 3, 6, 11, and 21 had a concentration-dependent inhibition on the virus yield in Vero E6, in which GL-D-ValOMe 3 and GL-PheOEt 11 were the most active inhibitors of DENV2 yield. Meanwhile, the time-of-addition assay indicated that conjugates GL-D-ValOMe 3 and GL-PheOEt 11 exhibited a substantial decrease in the DENV2 attachment stage. Subsequently, chimeric single-round infectious particles (SRIPs) of DENV2 C-prM-E protein/JEV replicon and DENV2 prM-E/ZIKV replicon were utilized for the DENV envelope I protein-mediated attachment assay. GL conjugates 3 and 11 significantly reduced the attachment of chimeric DENV2 C-prM-E/JEV and DENV2 prM-E/ZIKV SRIPs onto Vero E6 cells in a concentration-dependent manner but did not impede the attachment of wild-type JEV CprME/JEV and ZIKV prM-E/ZIKV SRIPs, indicating the inhibition of Compounds 3 and 11 on DENV2 E-mediated attachment. Molecular docking data revealed that Compounds 3 and 11 have hydrophobic interactions within a hydrophobic pocket among the interfaces of Domains I, II, and the stem region of the DENV2 envelope (E) protein. These results displayed that Compounds 3 and 11 were the lead compounds targeting the DENV E protein. Altogether, our findings provide new insights into the structure−activity relationship of GL derivatives conjugated with amino acid residues and can be the new fundamental basis for the search and development of novel flavivirus inhibitors based on natural compounds.

Physiology-Based Pharmacokinetic Study on 18ß-Glycyrrhetic Acid Mono-Glucuronide (GAMG) Prior to Glycyrrhizin in Rats.

To understand that 18ß-Glycyrrhetic acid 3-O-mono-ß-D-glucuronide (GAMG) showed better pharmacological activity and drug-like properties than 18ß-Glycyrrhizin (GL); a rapid and sensitive HPLC-MS/MS method was established for the simultaneous determination of GAMG and its metabolite 18ß-Glycyrrhetinic acid (GA) in rat plasma and tissues after oral administration of GAMG or GL. This analytical method was validated by linearity, LLOQ, specificity, recovery rate, matrix effect, etc. After oral administration, GAMG exhibited excellent Cmax (2377.57 ng/mL), Tmax (5 min) and AUC0-T (6625.54 mg/L*h), which was much higher than the Cmax (346.03 ng/mL), Tmax (2.00 h) and AUC0-T (459.32 mg/L*h) of GL. Moreover, GAMG had wider and higher tissue distribution in the kidney, spleen, live, lung, brain, etc. These results indicated that oral GAMG can be rapidly and efficiently absorbed and be widely distributed in tissues to exert stronger and multiple pharmacological activities. This provided a physiological basis for guiding the pharmacodynamic study and clinical applications of GAMG.

Allylic Hydroxylation Activity Is a Source of Saponin Chemodiversity in the Genus Glycyrrhiza.

Licorice (Glycyrrhiza) produces glycyrrhizin, a valuable triterpenoid saponin, which exhibits persistent sweetness and broad pharmacological activities. In the genus Glycyrrhiza, three species, Glycyrrhiza uralensis, Glycyrrhiza glabra and Glycyrrhiza inflata, produce glycyrrhizin as their main triterpenoid saponin, which has a ketone group at C-11. Other Glycyrrhiza species produce mainly oleanane-type saponins, which harbor homoannular or heteroannular diene structures that lack the C-11 ketone. Although the glycyrrhizin biosynthetic pathway has been fully elucidated, the pathway involving saponins with diene structures remains unclear. CYP88D6 from G. uralensis is a key enzyme in glycyrrhizin biosynthesis, catalyzing the sequential two-step oxidation of ß-amyrin at position C-11 to produce 11-oxo-ß-amyrin. In this study, we evaluated the functions of CYP88D6 homologs from the glycyrrhizin-producing species G. glabra and G. inflata and from the non-glycyrrhizin-producing species Glycyrrhiza pallidiflora and Glycyrrhiza macedonica, using yeast engineered to supply ß-amyrin as a substrate. Yeast expressing CYP88D6 homologs from glycyrrhizin-producing species produced 11-oxo-ß-amyrin. However, yeast expressing CYP88D6 homologs (such as CYP88D15) from the non-glycyrrhizin-producing Glycyrrhiza species accumulated oleana-9(11),12-dien-3ß-ol and oleana-11,13(18)-dien-3ß-ol; these diene compounds are non-enzymatic or yeast endogenous enzymatic dehydration derivatives of 11α-hydroxy-ß-amyrin, a direct reaction product of CYP88D15. These results suggest that the activities of CYP88D6 homologs, particularly their ability to catalyze the second oxidation, could influence glycyrrhizin productivity and diversify the chemical structures of saponins in Glycyrrhiza plants. A synthetic biological approach to engineer CYP88D15 could enable the production of pharmacologically active saponins with diene structures, such as saikosaponins, whose biosynthetic pathways have yet to be fully characterized.

ARPI, ß-AS, and UGE regulate glycyrrhizin biosynthesis in Glycyrrhiza uralensis hairy roots.

KEY MESSAGE: ARPI, ß-AS, and UGE were cloned from G. uralensis and their regulatory effects on glycyrrhizin biosynthesis were investigated. ß-AS and UGE but not ARPI positively regulate the biosynthesis of glycyrrhizin. Glycyrrhiza uralensis Fisch. has been used to treat respiratory, gastric, and liver diseases since ancient China. The most important and widely studied active component in G. uralensis is glycyrrhizin (GC). Our pervious RNA-Seq study shows that GC biosynthesis is regulated by multiple biosynthetic pathways. In this study, three target genes, ARPI, ß-AS, and UGE from different pathways were selected and their regulatory effects on GC biosynthesis were investigated using G. uralensis hairy roots. Our data show that hairy roots knocking out ARPI or UGE died soon after induction, indicating that the genes are essential for the growth of G. uralensis hairy roots. Hairy roots with ß-AS knocked out grew healthily. However, they failed to produce GC, suggesting that ß-AS is required for triterpenoid skeleton formation. Conversely, overexpression of UGE or ß-AS significantly increased the GC content, whereas overexpression of ARPI had no obvious effects on GC accumulation in G. uralensis hairy roots. Our findings demonstrate that ß-AS and UGE positively regulate the biosynthesis of GC.

A novel biomimetic nanomedicine system with anti-inflammatory and anti-osteoporosis effects improves the therapy efficacy of steroid-resistant nephrotic syndrome.

Clinically, steroid-resistant nephrotic syndrome (SRNS) is always prolonged and difficult to treat and easily develops into end-stage renal disease, resulting in a low survival rate. Strategies to reverse steroid resistance and reduce the long-term use of high doses of steroid medicines are urgently needed. In this study, a novel nanoparticle drug system (Pm-GCH) with a core-shell structure was designed. Metal-organic frameworks, synthesized by glycyrrhizic acid (G) and calcium ions (Ca2+) loaded with hydrocortisone (H) were the core of the nanoparticles. Platelet membrane vesicles were the shells. The natural platelet membrane endows Pm-GCH with good biocompatibility and the ability to promote immune escape. In addition, under the chemotaxis of inflammatory factors, platelet membranes assist Pm-GCH in nonspecific targeting of the inflammatory sites of the kidney. Under an inflammatory acid environment, GCH slowly degrades and releases glycyrrhizic acid and hydrocortisone. Glycyrrhizic acid inhibits the inactivation of hydrocortisone, jointly inhibits the activity of phospholipase A2 (PLA2) and the classic activation pathway of complement C2, blocks the production of inflammatory factors, plays an anti-inflammatory role, and enhances the efficacy of hydrocortisone in the treatment of SRNS. Moreover, glycyrrhizic acid alleviates osteoporosis induced by long-term use of glucocorticoids. These results indicate that Pm-GCH is a promising treatment strategy for SRNS.

The permeability characteristics and interaction of main components from Si-Ni-San in a MDCK epithelial cell monolayer model.

1. Si-Ni-San (SNS) possesses extensive therapeutic effects, however, the extent to which main components are absorbed and the mechanisms involved are controversial. 2. In this study, MDCK cell model was used to determine the permeability characteristics and interaction between the major components of Si-Ni-San, including saikosaponin a, paeoniflorin, naringin and glycyrrhizic acid. 3. The transport of the major components was concentration-dependent in both directions. Moreover, the transport of paeoniflorin, naringin and glycyrrhizic acid was significantly reduced at 4 °C or in the presence of NaN3. Additionally, the efflux of paeoniflorin and naringin were apparently reduced in the presence of P-gp inhibitor verapamil. The transport of glycyrrhizic acid was clearly inhibited by the inhibitors of MRP2, indicating that MRP2 may be involved in the transport of glycyrrhizic acid. However, the results indicated that saikosaponin a was absorbed mainly by passive diffusion. Furthermore, the combined incubation of four major components had a powerful sorbefacient effect than a single drug used alone which may be regulated by tight junctions. 4. Taken together, our study provides useful information for pharmacological applications of Si-Ni-San and offers new insights into this ancient decoction for further researches, especially in drug synergism.

Glycyrrhizic Acid Inhibits SARS-CoV-2 Infection by Blocking Spike Protein-Mediated Cell Attachment.

Glycyrrhizic acid (GA), also known as glycyrrhizin, is a triterpene glycoside isolated from plants of Glycyrrhiza species (licorice). GA possesses a wide range of pharmacological and antiviral activities against enveloped viruses including severe acute respiratory syndrome (SARS) virus. Since the S protein (S) mediates SARS coronavirus 2 (SARS-CoV-2) cell attachment and cell entry, we assayed the GA effect on SARS-CoV-2 infection using an S protein-pseudotyped lentivirus (Lenti-S). GA treatment dose-dependently blocked Lenti-S infection. We showed that incubation of Lenti-S virus, but not the host cells with GA prior to the infection, reduced Lenti-S infection, indicating that GA targeted the virus for infection. Surface plasmon resonance measurement showed that GA interacted with a recombinant S protein and blocked S protein binding to host cells. Autodocking analysis revealed that the S protein has several GA-binding pockets including one at the interaction interface to the receptor angiotensin-converting enzyme 2 (ACE2) and another at the inner side of the receptor-binding domain (RBD) which might impact the close-to-open conformation change of the S protein required for ACE2 interaction. In addition to identifying GA antiviral activity against SARS-CoV-2, the study linked GA antiviral activity to its effect on virus cell binding.

Functional specialization of UDP-glycosyltransferase 73P12 in licorice to produce a sweet triterpenoid saponin, glycyrrhizin.

Glycyrrhizin, a sweet triterpenoid saponin found in the roots and stolons of Glycyrrhiza species (licorice), is an important active ingredient in traditional herbal medicine. We previously identified two cytochrome P450 monooxygenases, CYP88D6 and CYP72A154, that produce an aglycone of glycyrrhizin, glycyrrhetinic acid, in Glycyrrhiza uralensis. The sugar moiety of glycyrrhizin, which is composed of two glucuronic acids, makes it sweet and reduces its side-effects. Here, we report that UDP-glycosyltransferase (UGT) 73P12 catalyzes the second glucuronosylation as the final step of glycyrrhizin biosynthesis in G. uralensis; the UGT73P12 produced glycyrrhizin by transferring a glucuronosyl moiety of UDP-glucuronic acid to glycyrrhetinic acid 3-O-monoglucuronide. We also obtained a natural variant of UGT73P12 from a glycyrrhizin-deficient (83-555) strain of G. uralensis. The natural variant showed loss of specificity for UDP-glucuronic acid and resulted in the production of an alternative saponin, glucoglycyrrhizin. These results are consistent with the chemical phenotype of the 83-555 strain, and suggest the contribution of UGT73P12 to glycyrrhizin biosynthesis in planta. Furthermore, we identified Arg32 as the essential residue of UGT73P12 that provides high specificity for UDP-glucuronic acid. These results strongly suggest the existence of an electrostatic interaction between the positively charged Arg32 and the negatively charged carboxy group of UDP-glucuronic acid. The functional arginine residue and resultant specificity for UDP-glucuronic acid are unique to UGT73P12 in the UGT73P subfamily. Our findings demonstrate the functional specialization of UGT73P12 for glycyrrhizin biosynthesis during divergent evolution, and provide mechanistic insights into UDP-sugar selectivity for the rational engineering of sweet triterpenoid saponins.

Root-associated endophytic bacterial community composition and structure of three medicinal licorices and their changes with the growing year.

BACKGROUND: The dried roots and rhizomes of medicinal licorices are widely used worldwide as a traditional medicinal herb, which are mainly attributed to a variety of bioactive compounds that can be extracted from licorice root. Endophytes and plants form a symbiotic relationship, which is an important source of host secondary metabolites. RESULTS: In this study, we used high-throughput sequencing technology and high-performance liquid chromatography to explore the composition and structure of the endophytic bacterial community and the content of bioactive compounds (glycyrrhizic acid, liquiritin and total flavonoids) in different species of medicinal licorices (Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata) and in different planting years (1-3 years). Our results showed that the contents of the bioactive compounds in the roots of medicinal licorices were not affected by the species, but were significantly affected by the main effect growing year (1-3) (P < 0.05), and with a trend of stable increase in the contents observed with each growing year. In 27 samples, a total of 1,979,531 effective sequences were obtained after quality control, and 2432 effective operational taxonomic units (OTUs) were obtained at 97% identity. The phylum Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, and the genera unified-Rhizobiaceae, Pseudomonas, Novosphingobium, and Pantoea were significantly dominant in the 27 samples. Distance-based redundancy analysis (db-RDA) showed that the content of total flavonoids explained the differences in composition and distribution of endophytic bacterial communities in roots of cultivated medicinal liquorices to the greatest extent. Total soil salt was the most important factor that significantly affected the endophytic bacterial community in soil factors, followed by ammonium nitrogen and nitrate nitrogen. Among the leaf nutrition factors, leaf water content had the most significant effect on the endophytic bacterial community, followed by total phosphorus and total potassium. CONCLUSIONS: This study not only provides information on the composition and distribution of endophytic bacteria in the roots of medicinal licorices, but also reveals the influence of abiotic factors on the community of endophytic bacteria and bioactive compounds, which provides a reference for improving the quality of licorice.