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.

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.

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.

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.

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.

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.

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.

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.

Preparation of evodiamine-glycyrrhizic acid micelles with glycyrrhizic acid as carrier and their anti-hepatic fibrosis activity / 中国中药杂志

This study aimed to prepare evodiamine-glycyrrhizic acid(EVO-GL) micelles to enhance the anti-hepatic fibrosis activity of evodiamine. Firstly, EVO-GL micelles were prepared with use of thin film dispersion method. With particle size, encapsulation efficiency, loading capacity of micelles and the solubility of evodiamine as the indexes, the effect of different factors on micelles was observed to screen the optimal preparation methods and process. Then the pharmaceutical properties and the therapeutic effects of EVO-GL micelles prepared by optimal process were evaluated on CCl_4-induced hepatic fibrosis. The results showed that the micelles prepared by the thin film dispersion method had an even size, with an average particle size of(130.80±12.40)nm, Zeta potential of(-41.61±3.12) mV, encapsulation efficiency of 91.23%±1.22%, drug loading of 8.42%±0.71%, high storage stability at 4 ℃ in 3 months, and slow in vitro release. Experimental results in the treatment of CCl_4-induced hepatic fibrosis in rats showed that EVO-GL micelles had a synergistic anti-hepatic fibrosis effect, which significantly reduced the liver function index of hepatic fibrosis rats. In conclusion, the EVO-GL micelles prepared with glycyrrhizic acid as a carrier would have a potential application prospect for the treatment of hepatic fibrosis.

Near-infrared spectroscopy in rapid determination of glycyrrhizic acid and liquiritin in Glycyrrhizae Radix et Rhizoma Yinpian / 第二军医大学学报

Objective To establish a rapid determination model based on near-infrared spectroscopy (NIRS) for glycyrrhizic acid and liquiritin in Glycyrrhizae Radix et Rhizoma Yinpian. Methods The contents of glycyrrhizic acid and liquiritin in Glycyrrhizae Radix et Rhizoma Yinpian from different places of origin were determined by high performance liquid chromatography (HPLC) as reference values. At the same time 2 200-2 049, 1 750-1 450, 1 151-1 001 nm and 1 795-1 475, 1 395-1 293, 1 125-1 030 nm wavelength ranges of near-infrared spectra were selected to establish the rapid determination model by combining partial least squares (PLS) regression analysis with cross validation method. Results The correlation coefficient and root-mean-squares error of cross validation of the established content calibration model were 0.980 and 0.184 for glycyrrhizic acid, and 0.919 and 0.144 for liquiritin, respectively. Conclusion The NIRS-PLS method is convenient, rapid and nondestructive for the content determination of glycyrrhizic acid and liquiritin for large number of Glycyrrhizae Radix et Rhizoma Yinpian, which provides a new and feasible method for the rapid quality evaluation of Glycyrrhizae Radix et Rhizoma Yinpian.

Total saponins of Glycyrrhiza inflata and its decoction on intestinal flora in rats with liver injury / 中草药

Objective: To explore the effect of total saponins of Glycyrrhiza inflata and its decoction on intestinal flora in rats with liver injury. Methods: Model of rats with liver injury was induced by intraperitoneal injection of carbon tetrachloride, which were randomly divided into four groups, including total saponins group (Sap group), water decoction group (Dec group), bifendate group (Bif group) and physiological saline group (NS group), then they were given drugs by oral admission. At the same time, a blank control was used (Control group), and the rats were given physiological saline by oral admission. Finally, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of each group of rats were measured; The rat feces were collected for 16 S rRNA sequencing of intestinal flora. Results: Compared with the control group, the levels of ALT and AST in the serum of the model group were significantly increased (P < 0.05, P < 0.01). Compared with the model group, the levels of ALT and AST in the serum of the rats in each administration group were significantly reduced (P < 0.05, P < 0.01). Compared with the NS group, the intestinal flora in Bif group did not change significantly, while the Sap group and the Dec group showed different community composition. Total saponins significantly increased the relative abundance of Lactobacillus and Bacteroidales_S24-7 compared with water decoction. However, the intestinal flora of the Sap group and the Dec group were still different from the intestinal flora of the control group, and did not completely return to the normal state. Conclusion: Total saponins of G. inflata and its decoction may play different roles in regulating the composition of intestinal flora in rats with liver injury, and then improve the pathological condition of liver injury.

Effects of different softening methods on quality of Glycyrrhizae Radix et Rhizoma / 中草药

Objective: A method was established to obtain fingerprint and determination of six components in Glycyrrhizae Radix et Rhizoma Pieces (GRRP) based on HPLC-PDA, and samples with four kinds of softening methods (showering moistening, steaming, 70 ℃ decompression steaming, 85 ℃ decompression steaming) were analyzed. Methods: The content of total flavonoids and total saponins was determined by ultraviolet spectrophotometry with liquiritin and glycyrrhizic acid as reference materials. Simultaneous determination of six components of liquiritin, ononin, isoliquiritin, glycyrrhizin, echinatin, glycyrrhizic acid was performed based on HPLC. Changes of the components content in the samples which treated by different softening methods were compared. The similarity evaluation of samples with different softening methods was carried out by the chromatographic fingerprint similarity evaluation system of traditional Chinese medicine, and cluster analysis was also carried out. Results: The results showed that the content of total flavonoids and total saponins in untreated samples was the highest, and the content of total flavonoids and total saponins in samples treated by showering moistening was the lowest. The three treatment methods of atmospheric pressure steaming, steaming decompression at 70 ℃ and steaming decompression at 85 ℃ had little effect on the samples. The content determination showed that the content of isoliquiritin was decreased significantly after softening treatment. The difference among the different softening treatment groups was not significant. The samples with different softening methods of the three batches of samples were grouped together with their raw products. Different softening methods had no significant difference in the composition of the medicinal herbs. Conclusion: The established method can quickly and accurately determine the six components, and in particular, the content of isoglycyrrhizin should be monitored. Combining production efficiency, production cost and quality evaluation, steaming is the most feasible in the production process. This study provided theoretical guidance for the large-scale production of softening, which was conducive to further standardizing the production process of GRRP.

Preparation of silymarin nanosuspension with glycyrrhizic acid as stabilizer and study of its stabilization mechanism / 中草药

Objective: To prepare silymarin nanosuspension (SM-NS) with glycyrrhizic acid as stabilizer, and investigate the in vitro release characteristics and charge stabilization mechanism. Methods: SM-NS was prepared by high-speed shear-high pressure homogenization method. SM-NS lyophilized powder were prepared by freeze-drying method and characterized by physical and chemical characterization and in vitro release. The stability mechanism of SM-NS was studied from the ionic strength and pH value. Results: The dosage of glycyrrhizic acid (GA) was 0.15%. The preparation process was shear rate of 19 000 r/min, shear time of 4 min, homogenization pressure of 100 MPa, homogenization times of 12 times, and lyoprotectant was mannitol 3%, the average particle size of SM-NS lyophilized powder was (516.4 ± 10.4) nm, PDI was (0.260 ± 0.046); The in vitro release results showed that the dissolution rate and solubility of SM-NS lyophilized powder were significantly higher than the physical mixture; The study of charge stability mechanism showed that licorice acid can provide good charge stabilization and strong resistance to environmental impact. Conclusion: SM-NS is a potential and new nano-drug with high safety, which is formed by the charge stability of GA to significantly improve the solubility and stability of silymarin.

Preparation of glycyrrhizic acid-F127/TPGS mixed nanomicelles and its in vivo intestinal absorption in rats / 中草药

Objective: To prepare glycyrrhizic acid (GL)-Pluronic F127 (F127)/polyethylene glycol 1000 vitamin E succinate (TPGS) mixed nanomicelles (MMs) and improve oral absorption of GL. Methods: GL-F127/TPGS-MMs was prepared by thin film dispersion method. The encapsulation efficiency and drug loading of MMs were used as evaluation indexes. The formulation and process, including the ratio of F127 to TPGS, the concentration of polymer and GL, hydration temperature and time, were optimized by the single factor experiment. The morphology of MMs was investigated by transmission electron microscopy. The single-pass perfusion model was established in rats to investigate the intestinal absorption characteristics of GL-F127/TPGS-MMs with absorption rate constant (Ka) and apparent absorption coefficient (Papp) as evaluation indexes. Results: The optimal formulation and process of GL-F127/TPGS-MMs were as follows: TPGS 180 mg, F127 270 mg, GL 70 mg, hydration temperature 50 ℃ and hydration time 3 h. The prepared GL-F127/TPGS-MMs had good clarity and the particle size, polydispersity index, and Zeta potential were (28.20 ± 5.63) nm, 0.20 ± 0.06, and (-5.24 ± 1.55) mV, respectively. The encapsulation efficiency and drug loading were (97.57 ± 5.29) % and (13.13 ± 0.71) %, respectively. The MMs were spherical with distinct vesicle structure. The absorption of GL in the jejunum segment was significantly higher than that in the ileum segment (P < 0.05). Compared with raw GL, GL-F127/TPGS-MMs had a statistically significant higher absorption rate in the intestinal segment (P < 0.05). Conclusion: The prepared GL-F127/TPGS-MMs could significantly improve the absorption of GL in vivo.

Investigating mechanism of Qingfei Dayuan Granules for treatment of COVID-19 based on network pharmacology and molecular docking / 中草药

Objective: To explore the main active components, key targets and signaling pathways of Qingfei Dayuan Granules in treating of COVID-19 based on network pharmacology and molecular docking. Methods: TCMSP, ETCM and YATCM databases were used to search the chemical constituents of Qingfei Dayuan Granules, and the threshold values of OB ≥ 30% and DL ≥0.18 were used to screen the potential active compounds. SIB and STITCH databases were used to query the targets corresponding to the active compounds, and the PPI network and network topology parameters were obtained by using STRING database. Cytoscape 3.6.0 was used to screen the hub targets. The key targets were analyzed by Gene Ontology (GO), the Kyoto Encyclopedia of genes and genomes (KEGG) pathway enrichment and tissue enrichment using DAVID 6.8 software. The molecular docking was performed by AutoDock Tools 1.5.6 software. Results: A total of 251 active compounds and 1 037 targets were obtained, 107 key targets and 185 corresponding compounds were screened. The key targets involved ESR1, AR, EGFR, KDR, MMP2, and 52 genes were coexpressed with ACE2. The results of GO function enrichment analysis showed that Qingfei Dayuan Granules mainly regulated the biological processes of cell surface signaling transduction, molecular function, phosphorylation and transcription. KEGG pathway enrichment mainly involved chemokine signaling pathway, T cell receptor signaling pathway, B cell receptor signaling pathway, natural killer cell mediated cytotoxicity and Toll like receptor signaling pathway. The results of tissue enrichment showed that the key gene expression sites were mainly in lung and epithelial cells, involving a variety of immune cells, such as T cells, B cells, lymphocytes, etc. Molecular docking showed that the compounds with good binding power to SARS-CoV-2-RBD-ACE2 complex in Qingfei Dayuan granules were mainly come from Bupleuri Radix, Codonopsis Radix, Anemarrhenae Rhizoma, and Glycyrrhizae Radix et Rhizoma. Saikosaponin, glycyrrhizic acid, anemarsaponin had good binding power with SARS-CoV-2-S-RBD-ACE2, which may be potential active components against SARS-CoV-2. Conclusion: Qingfei Dayuan Granules has the characteristics of multi-components, multi-targets and multi-pathway regulation. Saikosaponin, glycyrrhizic acid, and anemarsaponin may be the potential active components against SARS-CoV-2. The mechanisms of its treatment against COVID-19 may be related to the regulation of the co-expressed genes with ACE2, inhibition of inflammation and immune related signaling pathways, and the destruction of the complex structure of SARS-CoV-2-S-RBD-ACE2.

Network pharmacology study of Yinqiao Jiedu Soft Capsules in treatment of COVID-19 / 中草药

Objective: To explore the mechanism of Yinqiao Jiedu Soft Capsules in the treatment of coronavirus disease 2019 (COVID-19). Methods: The interactions between 1 418 compounds of Yinqiao Jiedu Soft Capsules and 48 inflammatory target proteins related to COVID-19 were analyzed by molecule docking. The drug-target network was established to clarify the active compounds and potential targets. Results: The network analysis suggested 50 active compounds of Yinqiao Jiedu Soft Capsules, which were mainly flavonoids and triterpenoids, and 37 potential targets, mainly including MTOR, JAK3, ACE, ACE2, PIK3CA, TNF, AKT2, and MAP2K1. The results of molecular docking exhibited that forsythiaside and vitexin 2″-O-rhamnoside had good affinity with SARS-CoV-2 3CL hydrolase, and glycyrrhizic acid had good affinity with ACE2. Conclusion: The molecular mechanism of Yinqiao Jiedu Soft Capsules for COVID-19 may be involved in interfering SARS-CoV-2 replication and regulating the expression of inflammatory signaling pathway and the secretion of inflammatory cytokines.

Predictive analysis of Q-marker of Glycyrrhizae Radix et Rhizoma based on network pharmacology combined with qualitative and quantitative research / 中草药

Objective: To make a preliminary prediction of the Q-marker of Glycyrrhizae Radix et Rhizoma from the perspective of the effectiveness and measurability of chemical components based on the concept of Q-marker of Chinese materia medica. Methods: Based on literature integration and data analysis, the source range of Glycyrrhizae Radix et Rhizoma Q-marker was screened, and the effectiveness of the ingredients was analyzed through network pharmacology. Qualitative and quantitative analysis of 15 batches of Glycyrrhizae Radix et Rhizoma from four places of origin was performed by HPLC. The pattern recognition method was used to screen out the main marker components that caused the differences between groups, which were combined with network pharmacological results to further determine the Q-marker of Glycyrrhizae Radix et Rhizoma. Results: Literature studies had determined that flavonoids and triterpenoids were the main source of Glycyrrhizae Radix et Rhizoma Q-marker; Network pharmacology results showed that liquiritin, glycyrrhizic acid and other components had high connectivity in the "component-target-pathway" network and were the main active components; The fingerprints of 15 batches of Glycyrrhizae Radix et Rhizoma samples were established, and five components, including liquiritin and liquiritin apioside, were identified as the main marker components by PLS-DA analysis; The content determination results of liquiritin, liquiritin apioside, glycyrrhizic acid and glycyrrhetinic acid showed that there were significant differences in the content of ingredients among different production areas. The qualitative and quantitative research on pharmacology combined with network pharmacology revealed that liquiritin, liquiritin apioside, glycyrrhizic acid and glycyrrhetinic acid can be used as Glycyrrhizae Radix et Rhizoma Q-marker. Conclusion: Taking flavonoids and triterpenoids as the source of Q-marker for Glycyrrhizae Radix et Rhizoma, the qualitative and quantitative (measurability) study of Glycyrrhizae Radix et Rhizoma herbs from multiple producing areas combined with network pharmacology (effectiveness) revealed liquiritin, liquiritin apioside, glycyrrhizic acid and glycyrrhetinic acid as the potential Q-marker of Glycyrrhizae Radix et Rhizoma are scientific and reasonable, which provide reference for quality control of Glycyrrhizae Radix et Rhizoma.