Effect of glycyrrhizin on inflammatory factors and fibrotic factors in high glucose-induced glomerular mesangial cells / 解放军医学杂志

Objective To investigate the effect of glycyrrhizic acid(GA)on the inflammatory and fibrotic factors in high glucose-induced glomerular mesangial cells(SV40 MES13).Methods Cultured mouse SV40 MES13 were divided into normal group(NG,5.6 mmol/L glucose),high glucose group(30 mmol/L glucose)and HG+GA group(30 mmol/L glucose+200 μmol/L GA).The expression levels of inflammatory cytokines interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),IL-6,IL-8 and α-smooth muscle actin(α-SMA)in different groups were detected by Western blotting.The fluorescence intensity of IL-1β,TNF-α and α-SMA in different groups were detected by immunofluorescence.The levels of IL-1β,TNF-α,IL-6 and IL-8 in the culture supernatant of different populations were detected by enzyme-linked immunosorbent assay(ELISA).Results The protein expressions of IL-1β,TNF-α,IL-6,IL-8 and α-SMA in HG group were significantly higher than those in NG group(P<0.01);Compared with HG group,the protein expression levels of IL-1β,TNF-α,IL-6,IL-8 and α-SMA decreased significantly in HG+GA group(P<0.05).The fluorescence intensity of inflammatory cytokines IL-1β,TNF-α and α-SMA increased in HG group than those in NG group(P<0.05);While compared with the HG group,the fluorescence intensity of IL-1β,TNF-α and α-SMA in HG+GA group decreased markedly(P<0.05).The experimental results of ELISA showed that compared with NG group,the levels of IL-1β,IL-6,TNF-α and IL-8 in cell supernatent increased in HG group(P<0.01);while the levels of IL-1β,TNF-α,IL-6,IL-8 in HG+GA group significantly lower than those in HG group(P<0.05).Conclusion Glycyrrhizic acid has certain inhibitory effect on high glucose-induced inflammatory factors and fibrotic factors in glomerular mesangial cells,which may play an important role in prevention of diabetic nephropathy.

Optimization of Preparation Technology of Xinqingduyin Granules Based on Characteristic Chromato-grams and Index Components / 中药新药与临床药理

Objective To establish high performance liquid chromatography(HPLC)characteristic chromatograms of Xinqingduyin Granules(composed of Taraxaci Herba,Lonicerae Japonicae Flos,Chrysanthemi Indici Flos,etc.)and content determination of chicory acid and glycyrrhizic acid,and to optimize the preparation process of Xinqingduyin Granules.Methods Using the characteristic chromatograms of Xinqingduyin and the retention rate of chicory acid and glycyrrhizic acid as indexes,we carried out orthogonal experiment to optimize the extraction process of Xinqingduyin,and studied the concentration process.The molding process of Xinqingduyin Granules was conducted by screening the types and dosage of auxiliary materials,then three batches of pilot experiments were carried out.Results HPLC characteristic chromatograms of Xinqingshuyin Granules and the determination methods of chicory acid and glycyrrhizic acid were established.The optimal preparation technology was as follows:8 times amount of water was added,the drug was decocted for 3 times,with 1 hour per time.After the extract was concentrated under reduced pressure at 80℃,the appropriate amount of steviol glycoside and lactose was added into the extract and mixed.One-step granulation and packaging were adopted.The retention rates of chicoric acid and glycyrrhizic acid in the 3 batches of Xinqingduyin Granules,which were prepared on the pilot scale,were(54.56±1.63)%and(54.96±1.08)%,and the rate of finished product was(87.47±0.49)%,respectively.The quality is uniform,and the characteristic map of Xinqingduyin Granules showed high similarity with that of decoction prepared from the same batch of slices.Conclusion The optimized preparation technology is reasonable,feasible and reproducible.This preparation can be used to obtain the granule with similar materials of Xinqingduyin decoction.

Glycyrrhizic acid alleviates cisplatin-induced ototoxicity by inhibiting in-flammation of cochlear in C57BL/6J mice / 中国病理生理杂志

AIM:To study whether glycyrrhizic acid(GL)can resist the ototoxicity of cisplatin(CDDP)in mice and its molecular mechanism.METHODS:Male C57BL/6J mice were divided into 5 groups:control group,DMSO(5%)group,CDDP(4 mg/kg)group,CDDP+low-dose(50 mg/kg)GL group,and CDDP+high-dose(100 mg/kg)GL group(n=14).Auditory brainstem response(ABR)was used to detect hearing changes of mice.HE staining was used to observe the morphological change of cochlear stria vascular in mice.Evans blue(EB)staining was used to observe the per-meability change of the blood-labyrinth barrier(BLB).Immunohistochemical technique was used to detect the expression and distribution of adhesion protein VE-cadherin and tight junction protein ZO-1 on the cochlear stria.ELISA assay and immunofluorescence technology were employed to detect the expression of tumor necrosis factor-α(TNF-α)and interleu-kin-1β(1L-1β).RESULTS:In CDDP group,ABR waveforms of all frequencies were disturbed,the hearing threshold was significantly increased,and I wave latency was prolonged(P＜0.05).In CDDP+GL group,ABR waveforms of various frequencies were well differentiated,the hearing threshold was significantly decreased,and the latency of I-wave was shortened(P＜0.01).The disordered morphology and more vacuoles in the stria vascularis were observed by HE staining in CDDP group.The GL alleviated CDDP-induced damage in the stria vascularis.In EB staining,CDDP caused an increase in per-meability of BLB(P＜0.01),which was improved by GL treatment(P＜0.01).Immunohistochemical results showed that the expression of VE-cadherin and ZO-1 in CDDP group were decreased(P＜0.01),which was restored in CDDP+GL group(P＜0.01).The ELISA and immunofluorescence results showed that the expression of IL-1β and TNF-α was in-creased after CDDP treatment(P＜0.01),which was restored in CDDP+GL group(P＜0.01).CONCLUSION:The GL alleviates CDDP-induced hearing loss in mice by inhibiting CDDP-induced inflammation and reducing the permeability of BLB.

Glycyrrhizic Acid Showed Therapeutic Effects on Severe Pulmonary Damages in Mice Induced by Pneumonia Virus of Mice Infection / 实验动物与比较医学

Objective In this study, inbred BALB/c mice infected with the pneumonia virus of mice (PVM) were used to establish an animal model of viral pneumonia, and the changes in the pro-inflammatory alarmin molecule, high mobility group box 1 protein (HMGB1), during PVM infection were observed, as well as the in vivo intervention effects of the HMGB1 inhibitor, glycyrrhizic acid (GA), on PVM-induced lung injury. Methods Three-week-old female BALB/c mice were randomly divided into three groups, each consisting of 6 mice. One group, uninfected by PVM, served as the control group (Control). The other two groups were inoculated intranasally with PVM at a dose of 1×104 50% tissue culture infective dose (TCID50)/25 μL, and subsequently treated with GA saline solution (GA group) or plain saline solution (normal saline, NS group) via gavage for 15 consecutive days. During this period, changes in body weight and appearance were monitored in each group. At the end of the experiment, lung tissue samples were collected from all groups. The distribution of PVM and HMGB1 proteins in the lung tissues was analyzed using hematoxylin-eosin staining and immunohistochemistry. The expression levels of HMGB1 and its Toll-like receptor 4 (TLR-4), advanced glycosylation end-product-specific receptor (AGER), and inflammatory cytokines such as interleukin (IL)-1β, IL-2, and tumor necrosis factor-α (TNF-α) in lung tissues of mice were measured using real time fluorescence quantitative PCR. Results Compared with the Control group, the NS group showed a significant weight loss after 6 days (P<0.05). Histopathological tests revealed pronounced inflammatory lesions in their lungs. Immunohistochemistry results showed that HMGB1 was released from the nucleus to the cytoplasm, and real time fluorescence quantitative PCR results indicated that the expression levels of HMGB1, IL-1β, and IL-2 were significantly upregulated (P<0.05). In the GA group, there was no significant change in the clinical symptoms or body weight. However, compared with the NS group, the pathological damages of lung tissues in the GA group were significantly reduced, and the expression levels of HMGB1, IL-1β, IL-2, and interferon-γ (IFN-γ) in lung tissues were also significantly decreased (P<0.05), although the expression level of AGER was significantly increased (P<0.05).ConclusionPVM infection can cause significant inflammatory pathological lung damages in mice, and GA can effectively alleviate the damages. Its therapeutic effect may be related to the activation of HMGB1 signaling pathway.

Optimization of preparation technology and in vitro release of baicalin-glycyrrhizic acid solid nanocrystals / 中国药房

OBJECTIVE To optimize the preparation technology of baicalin （BCN）-glycyrrhizic acid （GA） solid nanocrystals （BCN-GA-SN）， to characterize them and investigate their in vitro release characteristics. METHODS According to the compatibility ratio of classic couplet medicinals “Scutellaria baicalensis-Glycyrrhiza uralensis”， the compatibility ratio of BCN and GA was determined as 6∶1 （m/m）； BCN-GA nanosuspension was prepared by precipitation method combined with high-pressure homogenization method. The preparation technology of BCN-GA nanosuspension was optimized by using mean particle size and polydispersity index （PDI） as indexes and with types and dosage of stabilizers， stirring speed and time， high-pressure homogenization pressure and frequency as factors. The freeze-dried consolidation process of BCN-GA nanosuspension was optimized to prepare BCN-GA-SN using average particle size， PDI and redispersibility index （RDI） as indicators， with the type and dosage of freeze-dried protective agents as factors； then， the physicochemical properties and in vitro release of BCN-GA-SN were investigated. RESULTS The optimal preparation technology of BCN-GA-SN was as follows： BCN-GA nanosuspension was prepared by using 15% sodium dodecyl sulfate as a stabilizer， stirring at 1 000 r/min for 15 minutes， and homogenizing at 100 MPa for 20 times； then， BCN-GA nanosuspension was freeze-dried and solidified with 5% mannitol （corresponding to the dosage of BCN）. The average particle size of prepared BCN-GA-SN was （442.2±5.7） nm with PDI of 0.225±0.015 and RDI of 1.055± 0.013. The prepared BCN-GA-SN presented as the irregularly spherical shape with more uniform size； the drug-loading amount of BCN in the nanocrystal was （62.5±0.7）%， and that of GA was （9.4±0.2）%； the in vitro release results showed that the cumulative dissolution of BCN-GA-SN was higher than that of the physical mixture of BCN and GA. CONCLUSIONS BCN-GA-SN is prepared successfully in this study with uniform particle size and even distribution， which can effectively improve the dissolution of BCN.

Effect of HMGB1/Caspase-1/GSDMD signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury / 器官移植

Objective To evaluate the effect of high mobility group box 1 (HMGB1)/ cysteinyl aspartate specific proteinase (Caspase)-1/Gasdermin D (GSDMD) signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury (IRI). Methods C57BL/6 mice were randomly divided into the sham operation group (Sham group), IRI 2 h group, IRI 6 h group, IRI 12 h group, glycyrrhizic acid (GA)+Sham group and GA+IRI 12 h group (n=8 in each group). AML12 cells were evenly divided into the Sham group, IRI 12 h group, GA+Sham group and GA+IRI 12 h group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1β and IL-6 in each group were detected by enzyme-linked immune absorbent assay(ELISA). The messenger ribonucleic acid (mRNA) levels of IL-1β and IL-6 were detected by reverse transcription polymerase chain reaction(RT-PCR). The pathological score of liver ischemia and cell apoptosis were compared among all groups. The expression level of HMGB1 in the liver tissues of each group was determined by immunohistochemistry. The expression levels of HMGB1, Caspase-1 and GSDMD proteins in the mouse liver tissues and AML12 cells were measured by Western blot. Results Compared with the Sham group, the serum levels of ALT, AST, IL-1β and IL-6 and the relative expression levels of IL-1β and IL-6 mRNA in the liver tissues were all significantly up-regulated after IRI in each group (all P < 0.05), and showed significant time-dependent pattern along with the prolongation of reperfusion time. Compared with the Sham group, the pathological score of hepatic ischemia and the apoptosis rate of hepatocytes were significantly increased after IRI in each group (all P < 0.05). Immunohistochemical results showed that the expression level of HMGB1 in the liver tissues was significantly up-regulated after IRI, which showed an increasing trend along with the prolongation within the period of 2-12 h. Western blot showed that compared with the Sham group, the relative expression levels of HMGB1, Caspase-1 and GSDMD proteins in vivo and in vitro were up-regulated in the IRI 12 h group. The relative expression level of HMGB1 protein was significantly up-regulated, whereas those of Caspase-1 and GSDMD proteins were significantly down-regulated in the GA+IRI 12 h group compared with those in the IRI 12 h group (all P < 0.05). Conclusions Hepatocytes probably activate the Caspase-1/GSDMD signaling pathway by releasing HMGB1, thereby triggering hepatocyte pyroptosis and leading to liver IRI. Inhibition of extracellular release of HMGB1 by GA may mitigate liver IRI.

Overexpression of (3S)-linalool synthase gene (LIS) regulates the glycyrrhizic acid biosynthesis in Glycyrrhiza uralensis hairy roots / 药学学报

(3S)-Linalool synthase (LIS) is a key enzyme involved in the monoterpene biosynthetic pathway. Based on our previous transcriptome study, the expression level of LIS gene was exceedingly related to glycyrrhizic acid (GA) biosynthesis. Therefore, we used hairy root culturing to further investigate the effect of LIS on the GA biosynthesis. A LIS gene (GenBank accession number: MZ169552) was cloned from Glycyrrhiza uralensis. The plant binary overexpression vector pCA-LIS was constructed by gene fusion. G. uralensis hairy roots overexpressing LIS were induced by the Agrobacterium rhizogenes ATCC15834. The expression levels of LIS were analyzed by real-time quantitative PCR (RT-qPCR) and the contents of GA in hairy root lines were determined by UPLC. It was found that in the hairy root lines overexpressing LIS, the expression levels of LIS were significantly higher than that in the wild type, while the contents of GA were remarkably lower than those in the wild type and negative control. These findings indicate that the expression level of LIS is negatively correlated with the accumulation of GA. In this study, LIS was cloned from G. uralensis for the first time and the negative regulatory effect of LIS on GA biosynthesis was confirmed by reverse genetics. This work provides support for further improvement of the molecular regulatory network of GA biosynthesis in G. uralensis.

Anti-pseudo-allergic components in licorice extract inhibit mast cell degranulation and calcium influx / 中国天然药物

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-α 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.

Simultaneous determination of five ingredients in Taohe-Chengqi Decoction by UPLC-MS/MS / 国际中医中药杂志

Objective:To develop the UPLC-MS/MS method for the determination of amygdalin, cinnamic acid, rhein, emodin and glycyrrhizic acid in Taohe-Chengqi Decoction simutaneously. Methods:The separation was performed on Supelco Discovery C18, and isocratic elution was carried out with mobile phase consisting of acetonitrile - 4 mmol/L ammonium formate. The mass spectrometer was operated in the positive and negative ionization electrospray (ESI) mode using multiple monitoring (MRM) to analize of five ingredients. The precursor to product ion transitions monitored for amygdalin, cinnamic acid, rhein, emodin and glycyrrhizic acid were m/z 458.2→296.0, 146.8→103.1, 283.7→239.9, 269.7→226.1 and 821.4→350.9, respectively. Results:Amygdalin, cinnamic acid, rhein, emodin and glycyrrhizic acid were analyzed, the linear ranges were 0.001 6-0.102 4, 0.001 6-0.102 4, 0.001 6-0.102 4, 0.000 8-0.051 2 and 0.000 4-0.256 0 ng, respectively. The r were 0.998 7, 0.999 1, 0.999 5, 0.998 9 and 0.998 6, respectively. The recovery of five analytes ranges from 97.33% to 105.33% and the Relative Standard Deviations were all below 2.69%. Conclusion:This UPLC-MS/MS method is exclusive, rapid and sensitivewhich could be applied for the determination of amygdalin, cinnamic acid, rhein, emodin and glycyrrhizic acid in Taohe-Chengqi Decoction.

Ferulate 5-hydroxylase gene (F5H) regulation of glycyrrhizic acid biosynthesis determined by gene overexpression and knockout / 药学学报

Ferulate 5-hydroxylase (F5H) is a key enzyme involved in the phenylpropane metabolism pathway. Based on our previous transcriptome sequencing study, F5H played a negative regulatory role in glycyrrhizic acid (GA) biosynthesis. Therefore, in this study we cloned the F5H gene and investigated its regulatory effect on GA accumulation through gene overexpression and knockout. F5H was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MK882511). A plant binary expression vector pCA-F5H was constructed by inserting F5H into pCAMBIA1305.1 at Spe I and Bgl II sites. The sgRNA sequences were designed based on the first exon of F5H. The CRISPR/Cas9 gene editing vector pHSE-F5H was constructed by inserting F5H sgRNA into pHSE401 at two Bsa Ⅰ sites. PCA-F5H and pHSE-F5H were transfected into Agrobacterium tumefaciens ATCC15834, which was used to induce hairy root overexpressing or knocking out F5H with licorice hypocotyl as explants. At the same time, wild type and negative control hairy roots were also generated. UPLC was used to assay the GA content in different hairy root lines, and results showed that the GA content in hairy root lines knocking out F5H was significantly higher, whereas in hairy root lines overexpressing F5H GA content was lower than that in the wild-type and negative control. In this work, through a reverse genetics strategy, the negative regulatory effect of F5H on GA biosynthesis was confirmed through gene overexpression and knockout. This work will lay a foundation for further elucidation of the molecular regulatory network of GA biosynthesis.

The mechanism of toxicity enhancement with the combination of Flos Genkwa and Radix et Rhizoma Glycyrrhizae based on the solubilization of glycyrrhizic acid / 药学学报

In order to study the contraindications of the compatibility of Flos Genkwa-Radix et Rhizoma Glycyrrhizae, in this study, the solubilizing and poisoning essence were explored. In this experiment, chromatographic assay, field emission scanning electron microscopy, MTT cytotoxicity evaluation, and other methods were used to study the main chemical components, morphology and toxicity of the ethyl acetate part of Flos Genkwa and its co-decoction with glycyrrhizic acid, in order to clarify Flos Genkwa-Radix et Rhizoma Glycyrrhizae incompatibility provides a new idea for the research on incompatibility of Flos Genkwa-Radix et Rhizoma Glycyrrhizae. The results showed that after co-decoction of the ethyl acetate part of Flos Genkwa with glycyrrhizic acid, high performance liquid chromatography (HPLC) detected the dissolution of the toxic component yuanhuacine of 54.8%, while yuanhuacine chromatographic peak was not detected in the Flos Genkwa ethyl acetate part of the single decoction. The increase of co-decoction dissolution rate was observed by scanning electron microscopy, and it was found that glycyrrhizic acid uniformly dispersed the fat-soluble components of Flos Genkwa into nano-scale particles, which improved the solubility and stability in the solution. Furthermore, the results of cytotoxicity evaluation showed that the survival rate of cells decreased after co-decoction, 4',6-diamidino-2-phenylindole (DAPI) staining also gave the same results. In summary, the co-decoction of the ethyl acetate part of Flos Genkwa with glycyrrhizic acid promotes the dissolution of the toxic component yuanhuacine, and makes the part form uniformly distributed nanoparticles, which is conducive to the absorption of the ingredient and increases the toxicity.

The role of licorice DXS knockout and overexpression in glycyrrhizic acid biosynthesis / 药学学报

1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme involved in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for terpenoid precursor biosynthesis. DXS plays an essential role in glycyrrhizic acid (GA) biosynthesis. Based on our previous transcriptome study, there was a negative correlation between DXS expression and GA content. Therefore, we explored the regulatory role of DXS in GA biosynthesis using both gene overexpression and gene knockout in a hairy root culture system. DXS was cloned from Glycyrrhiza glabra L. (GenBank Accession No. MN158121). A plant binary expression vector pCA-DXS was constructed by a gene fusion method. The sgRNA sequence was designed based on the first exon of DXS to construct the gene editing vector pHSE-DXS. Hairy roots overexpressing or knocking out DXS were generated through an Agrobacterium-mediated method with licorice hypocotyls as explants. Wild-type hairy roots and negative control hairy roots containing empty plasmids were also evaluated. UPLC was used to determine the GA content in each licorice hairy root line. Results showed that the content of GA in the hairy root group knocking out DXS was significantly higher than that in the wild-type and negative control groups, while in the hairy root group overexpressing DXS was significantly lower, suggesting that DXS plays a negative role in GA biosynthesis. This study provides a foundation for determining the function of DXS in terpenoid metabolism and for further establishment of a molecular regulatory network of GA biosynthesis.

Glycyrrhizic acid: a potential drug against COVID-19 / 药学学报

Pneumonia caused by SARS-CoV-2 has seriously threatened human life and health worldwide and caused a large number of deaths. Viral infection and acute inflammation are important causes of death, so it is particularly important to combine antiviral therapy with anti-inflammatory therapy. Glycyrrhizic acid, the main component of the glycyrrhizic root extract, has a wide range of pharmacological effects as well as high efficiency and low toxicity, its preparation has been widely used in the treatment of chronic hepatitis and other diseases. Glycyrrhizic acid can regulate the expression and release of a variety of cytokines and play a significant anti-inflammatory effect. At the same time, glycyrrhizic acid also showed significant inhibition towards a variety types of viruses. Therefore, the potential application of glycyrrhizic acid as COVID-19 treatment should be explored.

Metabolomic investigation of secondary metabolites of prepared slices of Glycyrrhiza uralensis with different degrees of honey processing / 药学学报

A metabolomics method was used to search for chemical markers in prepared slices of Glycyrrhiza uralensis with different degrees of honey processing. Coupled with these metabolomics analytical methods, ultra-performance liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) was used to generate global chemical profiles of the raw material of Glycyrrhiza uralensis and the prepared slices. The samples were collected in Shanxi, Hebei Zhangjiakou and Inner Mongolia. A total of 57 chemical components were identified in Glycyrrhiza uralensis by using the UNIFI theoretical database combined with the library of reference samples. Among them, 37 compounds were identified in positive ion mode and 56 compounds were identified in negative ion mode. Unsupervised principal component analysis (PCA) showed that the chemical ingredients differed considerably depending on the extent of processing. Supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was used to differentiate the moderate processing group and the raw group, and partial least squares discriminant analysis (PLS-DA) was used to differentiate the less, the moderate, and the excessive processing groups. The results showed that the contents of glycyrrhizic acid, licoricesaponin G2, and licoricesaponin E2 varied with the extent of processing. The content of these components increased after processing, and reached the highest level when the extent of processing was moderate (P < 0.05). Glycyrrhizic acid, licoricesaponin G2 and licoricesaponin E2 can be regarded as the chemical markers to differentiate the samples with different degrees of processing. These three compounds can be used to monitor the processing of Glycyrrhiza uralensis.

Effects of dipotassium glycyrrhizinate on wound healing

ABSTRACT Purpose: Dipotassium glycyrrhizinate (DPG) has anti-inflammatory properties, besides promoting the regeneration of skeletal muscle. However, it has not been reported on skin wound healing/regeneration. This research aimed to characterize the effects of DPG in the treatment of excisional wounds by second intention. Methods: Male adults (n=10) and elderly (n=10) Wistar rats were used. Two circular wounds were excised on the dorsal skin. The excised normal skins were considered adult (GAN) and elderly (GIN) naïve. For seven days, 2% DPG was applied on the proximal excision: treated adult (GADPG) and elderly (GIDPG), whereas distal excisions were untreated adult (GANT) and elderly (GINT). Wound healing areas were daily measured and removed for morphological analyses after the 14th and the 21st postoperative day. Slides were stained with hematoxylin-eosin, Masson's trichrome, and picrosirius red. Results: Histological analysis revealed intact (GAN/GIN) and regenerated(GANT/GINT/GADPG/GIDPG) skins. No differences of wounds' size were found among treated groups. Epidermis was thicker after 14 days and thinner after 21 days of DPG administration. Higher collagen I density was found in GIDPG (14th day) and GADPG (21st day). Conclusions: DPG induced woundhealing/skin regeneration, with collagen I, being more effective in the first 14 days after injury.

Molecular Cloning and Sequence Analysis of GgUGE Gene in Glycyrrhiza glabra / 中国实验方剂学杂志

Objective::To clone the cDNA sequence of UDP-glucose 4-epimerase (UGE) in Glycyrrhiza glabra and analyze its sequence, so as to explore the potential relationship between the UGE gene and the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis. Method::The cDNA sequence of UGE was cloned from the root of G. glabra by reverse transcription polymerase chain reaction (RT-PCR), then sequenced and analyzed by bioinformatics software. Results::A GgUGE cDNA sequence with the full length of 1 121 bp was obtained. The open reading fame (ORF) of GgUGE was 1 053 bp, encoding 350 amino acid residues. The GgUGE cDNA sequence was submitted to GenBank, and the accession No. was MK638908. Sequence analysis showed that GgUGE was an unstable hydrophilic protein, its average relative molecular weight was 39.02 kDa, and isoelectric point was 6.13. It contained no signal peptides or transmembrane domains. Its secondary structure mainly constituted of α-helix and had a conversed domain of UDP-glucose 4-epimerase superfamily. The homologoue analysis showed that the cDNA and amino acid sequences of GgUGE had the closest evolutionary relationship to Leguminosae and relatively distant evolutionary relationship to Salicaceae. Conclusion::In this study, GgUGE cDNA sequence is successfully cloned from G. glabra for the very first time, which will provide reference for studying the function of GgUGE and explaining the molecular regulatory mechanisms of glycyrrhizic acid biosynthesis in G. glabra.

Simultaneous and Rapid Detection of Concentrated Liquid of Compound Dahuang Decoction Based on Near Infrared Spectroscopy / 中国实验方剂学杂志

Objective::Near infrared spectroscopy was used to detect the concentration density (25 ℃), solid-containing content, rhein content and glycyrrhizic acid content of compound Dahuang decoction. Method::The concentrated liquid of compound Dahuang decoction was determined by near infrared optical fiber transmission spectrometry. The contents of rhein and glycyrrhizic acid were determined by HPLC. Fifty-one samples were used for internal cross-validation, and partial least square regression was used to establish correction models between near-infrared spectrum and density, solid-containing content, rhein content and glycyrrhizic acid content, respectively. Ten unknown concentrated liquid samples were collected for external validation and prediction. Result::The external validation complex correlation coefficients between near-infrared spectra and density, solid-containing content, rhein content and glycyrrhizic acid content of the concentrated liquid of compound Dahuang decoction were 0.995 9, 0.999 6, 0.997 0 and 0.992 2, and the root mean square error of prediction (RMSEP) values were 2.50×10-3, 0.17, 7.57 and 67.10, respectively. Conclusion::The near infrared spectroscopy is suitable for the determination of evaluation indexes of the concentrated liquid index of compound Dahuang decoction, and has the characteristics of rapid, simple, stable and reliable.

Cloning and Bioinformatic Analysis of ARPI Gene from Glycyrrhiza glabra / 中国实验方剂学杂志

Objective::To clone the complementary deoxyribonucleic acid (cDNA) of auxin/indole acetic acid protein (Aux/IAA) from Glycyrrhiza glabra (GgARPI) and analyze its sequence by bioinformatics. Method::RNA was extracted from fresh root of G. glabra, the cDNA sequence of GgARPI gene was cloned by reverse transcription polymerase chain reaction (RT-PCR), then sequencing and bioinformatic analysis were performed. Result::The GgARPI cDNA sequence with the full length of 686 bp was obtained from G. glabra. The full open reading frame (ORF) was 585 bp, encoding 194 amino acid residues. The bioinformatic analysis showed that the protein coded by GgARPI was a stable hydrophilic protein, with a relative molecular weight of 21.95 kDa and isoelectric point of 6.85.It contained no signal peptides or transmembrane domain. Its secondary structure mainly consisted of random coil. An Aux/IAA superfamily was included in the conversed domain. Homology analysis indicated that it had a close evolutionary relationship with leguminous plants, and a distant evolutionary relationship with monocotyledon, such as Setaria italica. Conclusion::GgARPI cDNA sequence is successfully cloned from G. glabra for the first time, which will lay a foundation for studying the function of GgARPI and explaining the molecular regulatory mechanism of biosynthesis of glycyrrhizic acid in G. glabra.

Glycyrrhizic Acid Protect IEC-6 Cells from Apoptosis Via HuR Mediated Posttranscription of p21 / 中国实验方剂学杂志

Objective:To investigate the protective effect of glycyrrhizic acid (GA) from tumor necrosis factor-α+actinomycetes ketone （TNF-α+CHX） induced apoptosis in rat small intestine crypt epithelial cell line (IEC-6) via AU rich element mRNA binding protein HuR mediated posttranscription of p21 and the potential mechanism. Method:The cultured IEC-6 cells were observed. The experiment was divided into blank group, GA (60 μmol·L-1) group, TNF-α+CHX group and GA+TNF-α+CHX group. Cytoplasmic and nuclear HuR were measured by Western blot. The interaction of HuR and p21 mRNA was detected by biotin pull down and RNA IP. Luciferase activity was measured after transfection with construct with p21 3'-UTR cloned into downstream of luciferase reporter. Cell apoptosis was detected by real-time dynamic cell analyzer, p21 and cysteine proteinas-3 precursor protein（proCaspase-3） association was analysised by CO-IP. Result:After GA treatment for 48 h, cytoplasmic HuR protein expression increased（P<0.05）,the binding between HuR and p21 mRNA expression up regulated(P<0.05), luciferase activity increased(P<0.01), and p21 mRNA and protein expression also increased(P<0.05), while these results were abolished by HuR silencing with siRNA. GA enhanced p21 and procaspase3 interaction(P<0.05), and attenuated TNF-α+CHX induced apoptosis in IEC-6 cells. Conclusion:GA protected IEC-6 cells from TNF-α/CHX induced apoptosis via HuR mediated p21 posttranscription, which due to GA enhanced HuR binding to endogenous and recombinant p21 mRNA and increased p21 interaction with proCaspase3.

Optimization of puerarin nanoemulsion based on glycyrrhizic acid by central composite design-response surface method and its in vitro release studies / 中草药

Objective: Puerarin nanoemulsion (Pue-NE) was prepared with glycyrrhizic acid as a natural stabilizer, and its release characteristics in vitro were investigated. Methods: Data processing was performed using particle size and polydispersity index (PDI) as independent variables, and using the overall desirability (OD) as the evaluation index. The central composite design-response surface method was used to optimize the prescription, and the physical and chemical properties and release characteristics of Pue-NE prepared by the optimal prescription were investigated. Results: The best prescription for Pue-NE is puerarin at a concentration of 5.0 mg/mL, glycyrrhizic acid at a concentration of 1.75 mg/mL, and caprylic glyceride in an amount of 3.5 mL. The average particle size of the nanoemulsion is (184.5 ± 0.8) nm, the PDI is 0.088 ± 0.002, the zeta potential is (10.56 ± 0.35) mv, the conductivity is (98.3 ± 0.4) μs/cm, pH is 6.750 ± 0.005, solubility (4.970 ± 0.008) mg/mL, drug loading is (99.4 ± 0.2)%, turbidity (24.3 ± 1.0) cm-1 (n = 3). It was identified as O/W emulsion by dyeing method. TEM scanning results show that the droplets are spherical and uniform in size and the stability results showed that Pue-NE has good storage stability at 25 ℃. In vitro release results showed that Pue-NE has the greatest release in phosphate buffered pH 6.8 within 24 hours. Conclusion: The preparation of Pue-NE with glycyrrhizic acid as a natural stabilizer is not only simple and convenient, but also can effectively replace the use of traditional chemical synthetic stabilizers and improve the solubility of puerarin.