Chalcone isomerases (CHIs) play an essential role in the biosynthesis of flavonoids important in plant self-defense. Based on the transcriptome data of Aquilaria sinensis Calli, a full-length cDNA sequence of CHI1 (termed as AsCHI1) was cloned by reverse transcription PCR. AsCHI1 contains a complete open frame (ORF) of 654 bp. The deduced protein is composed of 217 amino acids, with a predicted molecular weight of 23.11 kDa. The sequence alignment and phylogenetic analysis revealed that AsCHI1 has conserved most of the active site residues in type I CHIs, indicating a close relationship with the CHI from Gossypium hirsutum. The recombinant AsCHI1 protein was obtained by heterologous expression of AsCHI1 in E. coli BL21(DE3). The purified AsCHI1 protein exhibited CHI activity by catalyzing the production of naringenin from naringenin chalcone. Remarkably, AsCHI1 expression in A. sinensis Calli treated with various abiotic stresses including salt, mannitol, cold, and heavy metals could be markedly increased, and plant hormones such as abscisic acid (ABA), gibberellin (GA3), and salicylic acid (SA) could also increase the expression of AsCHI1, suggesting that AsCHI1 might play an important role in plant self-defense. The results expand our understanding of the biosynthesis of flavonoids in A. sinensis and give further insight into the defensive responses of A. sinensis to abiotic and biotic stresses.

Transporters have a great influence on the transportation and distribution of drugs in the body. On the one hand, solute carrier transporters could transport drugs into tissues and organs, which may improve the oral bioavailability or change the tissue-distribution of the drugs. On the other hand, the ATP-binding cassette could pump some drugs out of the cell, which decreases the intracellular drug concentrations and leads to drug resistance.This paper summarizes the distribution, substrate characteristics and drug design strategies of several important drug transporters, such as improving bioavailability by prodrug design, introducing acid group to improve hepatic selectivity and adjusting the polarity of compounds to decrease efflux ratio.

Network pharmacological approaches were used to predict the components, targets and pathways of Erhuang decoction (EhD) in the treatment of acute lung injury (ALI). The SwissTargetPrediction platform, DisGeNET, Therapeutic Target Database (TTD), GeneCards and Online Mendelian Inheritance in Man (OMIM) databases were used to predict potential targets of EhD and were integrated with the predicted targets for the treatment of ALI. A protein-protein interaction network model was constructed by using String database and Cytoscape software; the DAVID platform was used for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. A network of drug components-targets-pathways was constructed by Cytoscape software and the SwissDock platform was used to dock the molecules of EhD found in blood with the key disease targets. An ALI model was established in mice and inflammatory factor detection and Western blot protein expression experiments with lung tissue sections were carried out to verify the effect of EhD in the treatment of ALI. Animal experiment ethical requirements were approved by the Ethical Committee Experimental Animal Center of Shandong University (Grant Number: 2016020). We identified 148 potential targets including signal transducer and activator of transcription 3 (STAT3), vascular endothelial cell growth factor A (VEGFA), RAC-alpha serine/threonine-protein kinase (AKT1), and nuclear factor-kappa B/p65 (RELA). The potential targets are largely associated with the biological processes of inflammation, oxidative stress, and apoptosis. Additional pathways relate to cancer, VEGF signaling, mitogen-activated protein kinase (MAPK) signaling, and Toll-like receptors (TLRs) signaling, along with other signaling pathways. Pharmacodynamic experiments showed that EhD could significantly reduce the content of inflammatory factors and the degree of lung injury of ALI mice. Western blot revealed that EhD could significantly decrease the expression of NF-κB/p65 and upregulate the expression of NF-kappa-B inhibitor alpha(IκBα). From the perspective of network pharmacology, the mechanisms of EhD in the treatment of ALI is consistent with the characteristics of multiple ingredients, multiple targets and multiple pathways. This research provides a reference for further study of the mechanism of this traditional Chinese medicine.

The quality markers(Q-markers) of traditional Chinese medicine(TCM) have become a topic of interest in TCM research in recent years. Nonetheless, there is still no consensus on how to scientifically characterize TCM Q-markers. Our study establishes an identification method for TCM Q-markers based on the analytical hierarchy process(AHP) and the entropy weight comprehensive method. By constructing an evaluation system encompassing the target layer, the factor layer and the control layer, AHP can be used to analyze the weight of three core TCM quality attributes, including effectiveness, testability and specificity. Following that, the entropy weight method is employed to analyze the specific indicators for each attribute based on the literature and experimental data. Finally, the comprehensive weight of each index is obtained by combining the two weights, and the comprehensive weight and the specific value of each component is multiplied and summed to obtain the integrated score ranking, and thereby identify the TCM Q-markers. Taking Shaoyao Gancao decoction as an example, the analysis revealed that the top 8 components are as follows: paeoniflorin > quercetin > albiflorin > glycyrrhizic acid > naringenin > liquiritin > oxypaeoniflorin > benzoylpaeoniflorin, and can be identified as Q-markers of Shaoyao Gancao decoction. This study not only provides support for the establishment of quality standards and process quality control of TCM formulae, but also provides innovative ideas and methods for quantitative evaluation and accurate identification of TCM Q-markers.

In this study, we investigated the inhibitory effect of SYT-1, a new compound of tetrahydroisoquinoline, on tumor cell proliferation and underlying mechanisms. Cell counting kit-8(CCK-8) method was used to detect cell proliferation; clone formation experiment was used to detect cell clone formation ability; JC-1 probe was used to detect cell mitochondrial membrane potential; 2', 7'-dichlorodihydrofluorescein diacetate(DCFH-DA)probe was used to detect intracellular reactive oxygen species; Annexin V-FITC/PI(fluorescein isothiocyanate/propidium) counterstaining method was used to detect apoptosis; Western blot assay was used to detect the expression level of related proteins. The experimental results show that SYT-1 has a significant inhibitory effect on the proliferation of six human-derived cancer cells. Among them, the inhibitory effect on breast cancer MCF-7 cells is the strongest, the half maximal inhibitory concentration(IC₅₀) of SYT-1 of 48 h administration on MCF-7 cells is 5.87 μmol·L^-1, which is better than that of cisplatin(8.92 μmol·L^-1). Further studies have shown that SYT-1 can dose-dependently inhibit the monoclonal formation ability of MCF-7 cells, and can cause the mitochondrial membrane potential of the cells to decrease and the level of reactive oxygen species to increase. In addition, SYT-1 can significantly inhibit the activation of PI3 K-Akt(phosphatidylinositol 3-kinase/protein kinase B) signaling pathway and induce apoptosis of MCF-7 cells. The above research results show that, as a new type of tetrahydroisoquinoline compound, SYT-1 has the potential to inhibit tumor cell proliferation.

Xianling Gubao is a common and effective medicine in the treatment of orthopedic diseases. In recent years, it has been reported to be associated with liver injury. However, through the analysis of the adverse drug reaction reports and key hospital cases, we found that there is considerable incomplete information in the reports of Xianling Gubao-related liver injury cases retrieved from the literature. Thus, it is difficult to accurately judge causality between the drug and liver injury. Six cases of liver injury related to Xianling Gubao were identified in key hospitals, two of which achieved the clinical diagnosis according to the assessment of the integrated evidence chain method. We further analyzed the public health data of all residents in Yinzhou. The gross incidence rate of Xianling Gubao-related liver injury was 0.034%, which corresponds to a level of rare incidence. This revealed that Xianling Gubao-related liver injury has significant divergence in individuals and an idiosyncratic nature. The gross incidence of liver injury related to Xianling Gubao was lower than that of other medicines for the treatment of orthopedic diseases. Based on the idiosyncratic drug-induced liver injury model mediated by immune stress, it was found that Epimedii Folium and Psoraleae Fructus were the major components that lead to liver injury, and the liver injury caused by a full prescription was less serious than that encountered with only Epimedii Folium and Psoraleae Fructus. This suggests that the other 4 herbs(Dipsaci Radix, Anemarrhenae Rhizoma, Rehmanniae Radix, Salviae Miltiorrhizae Radix et Rhizoma) can prevent/alleviate the liver injury. Through disassembled prescription analysis, we found that the attenuation efficacy of Salviae Miltiorrhizae Radix et Rhizoma was the most significant.In conclusion, Xianling Gubao may cause idiosyncratic liver injury in a tiny minority of susceptible individuals, but the incidence risk is lower than that of other commonly used drugs for orthopedic disease. Xianling Gubao should be discreetly applied to patients with immune stress. The major components that induced liver injury in Xianling Gubao were Epimedii Folium and Psoraleae Fructus, and Salviae Miltiorrhizae Radix et Rhizoma appears to attenuate this toxicity. This study provides a reference for the rational clinical medication with Xianling Gubao.

Fatigue is a common complication of type 2 diabetes mellitus (T2DM).We examined the relationship between T2DM fatigue and the skeletal muscle 5-hydroxytryptamine (5-HT) system.In animal experiments, a T2DM model was established in mice by feeding a high-fat diet with intraperitoneal injection of streptozotocin.The mice were treated with the 5-HT_2A receptor antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthesis inhibitor carbidopa (CDP)(separately and in combination).In cell culture experiments, C2C12 cells were stimulated with D-glucose, palmitic acid or 5-HT.5-HT_2AR, 5-HT synthesis and 5-HT degradation were inhibited by SH, CDP, or monoamine oxidase A (MAO-A) inhibitor.The animal experiments were in accordance with the regulations of the Animal Ethics Committee of China Pharmaceutical University.The results showed that 5-HT_2AR, 5-HT synthase and MAO-A were expressed in mouse skeletal muscle and C2C12 cells.The expression of these proteins was significantly up-regulated in T2DM mice or when C2C12 cells were exposed to palmitic acid and D-glucose; palmitic acid was a stronger stimulant of their expression than D-glucose.Rotating rod experiments and biochemical index tests have shown that T2DM fatigue is associated with an increase in skeletal muscle 5-HT_2AR, 5-HT synthesis and 5-HT degradation.5HT_2AR mediates the expression of MAO-A and the synthesis of 5-HT, which indirectly regulates the degradation of 5-HT.MAO-A regulates cell inflammation, mitochondrial ROS production and membrane potential depolarization by mediating 5-HT degradation.MAO-A also inhibits the expression of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1), carnitine palmitoyltransferase-1 (CPT1) and ATP synthase-6 (ATP6), thus inhibiting mitochondrial functions such as fatty acid β oxidation and ATP synthesis.SH and CDP can effectively treat T2DM fatigue, and can also reduce blood glucose and blood lipid, and the combination of SH and CDP has a clear synergistic effect.

Rhamnose synthase (RHM) is a key enzyme in the biosynthesis of uridine diphosphate rhamnose (UDP-Rha), reversibly converting uridine diphosphate-glucose (UDP-Glc) into UDP-Rha in the presence of NADH or NADPH. In this research, yeast extract (YE) was used to stimulate Sorbus aucuparia suspension cells. Based on a previous study of the transcriptome database of S. aucuparia suspension cells, two RHMs were cloned from S. aucuparia and named SaRHM1 (GenBank No.: MK213340) and SaRHM2 (GenBank No.: MK213341). The SaRHM1 gene contained a 2 007 bpopen reading frame (ORF) encoding a polypeptide of 668 amino acids with a molecular weight of 75.25 kD, and a theoretical isoelectric point (pI) of 7.24. The SaRHM2 gene contained a 2 040 bpORF encoding a polypeptide of 679 amino acids with a molecular weight of 76.26 kD and pI of 6.41. Bioinformatic analysis indicated that SaRHM1 and SaRHM2 contained two special sequences of GxxGxxG/A and YxxxK. Multiple sequence alignments and phylogenetic trees show that SaRHM1 and SaRHM2 have high sequence similarity with other plant species of RHMs. The results of enzyme activity assays in vitro revealed that both recombinant SaRHM1 and SaRHM2 are able to convert UDP-Glc into UDP-Rha. SaRHMs displayed maximum activity at 40 ℃ and a pH of 8 and 9, respectively. The K_m values of SaRHM1 and SaRHM2 for UDP-Glc were 212.4 ± 56.70 and 361.0 ± 63.74 μmol·L^-1, respectively, with V_max values of 235.5 ± 18.98 and 516.5 ± 22.30 nmol·min^-1·μg^-1, respectively. This study reports the cloning and sequencing of RHMs from S. aucuparia and verifies their function, which likely provide rhamnose donors for the subsequent biosynthesis of rhamnosides.

Ten triterpenoid saponins were isolated and purified from the water extract of Glycyrrhiza glabra by polyamide resin combined with macroporous resin column chromatography, ODS medium pressure column chromatography and semi-preparative RP-HPLC. Their structures were elucidated by physicochemical properties, NMR and MS spectra, and determined as 3β-O-[β-D-glucuronpyranosyl-(1→2)-β-D-glucuronpyranosyl]-30β-O-β-D-glucuronpyranosyl-oleanane-11-oxo-12(13)-ene(1), 3β-O-[β-D-glucuronpyranosyl-(1→2)-β-D-glucuronpyranosyl]-30 β-O-α-L-rhamnopyranosyl-oleanane-11-oxo-12(13)-en-22 β, 30-diol(2), uralsaponin C(3), licorice-saponin A3(4), licorice-saponin P2(5), 22β-acetoxyl-glycyrrhizin(6), macedonoside A(7), 29-hydroxyl-glycyrrhizin(8), licorice-saponin G2(9), glycyrrhizin(10). Compounds 1 and 2 are two new compounds and named as licorice-saponin R3 and licorice-saponin S3.

Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis. Based on our previous studies, we selected the specific CHI haplotype (GenBank Accession No. KY115232) to maximize flavonoid accumulation. We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation. The recombinant A. tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G. uralensis to obtain transgenic hairy roots. A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines. The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5. UPLC results showed that the content of total flavonoids, liquiritin, liquiritigenin, and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples. This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G. uralensis. This work provides a theoretical basis for clarifying the function of CHI. Three transgenic hairy root lines of G. uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.