Latest ArticlesTopically applied traditional Chinese medicines (TCM) generally show low transdermal rates and doses, leading to weak therapeutic efficacy. Here, cataplasm of white mustard seed varnish was prepared. The effects of iontophoresis and sonophoresis on the transdermal delivery of this TCM and its anti-asthma effect were evaluated. Active components of white mustard seed, rhizoma corydalis and Radix Kansui were obtained after alcohol extraction, respectively. The volatile oil in Asarum Heterotropoides were obtained with volatile oil extraction equipment. The drug loading ratio of cataplasms was 17% (w/w). Franz cell diffusion method was used to evaluate the accumulated permeation amount and the steady-state transdermal absorption rate of sodium thiocyanate. The improvement of sonophoresis on transdermal absorption was higher than that of iontophoresis.Asthma model of guinea pigs were established. Cataplasms of white mustard seed varnish were applied on the back of guinea pigs. Iontophoresis and sonophoresis obviously promoted the transdermal absorption and enhanced anti-asthma efficacy of white mustard seed varnish cataplasms. Pathological analysis showed that iontophoresis and sonophoresis significantly alleviated inflammation. Compared with the model group, IL-4 and IgE levels in the cataplasm group, cataplasm/iontophoresis group, cataplasm/sonophoresis group decreased obviously, although the IFN-γ levels markedly increased. Moreover, the therapeutic efficacy of cataplasm/sonophoresis group was the best in all the groups, even leading to levels similar to that of healthy animals. Iontophoresis and sonophoresis are effective methods to promote transdermal absorption of cataplasms. Moreover, the effect of sonophoresis is higher than that of iontophoresis. Physical penetration improvement techniques provide a novel insight for the wide application of transdermal TCM.
UDP-glycosyltransferase PgUGT74AE2 from Panax ginseng can transfer a glucose moiety to the free C-3 hydroxyl of protopanaxadiol (PPD) to produce ginsenoside Rh2. However, no report demonstrates that PgUGT74AE2 can transfer a glucose moiety to the free C-3 hydroxyl of protopanaxatriol (PPT) to produce a PPT-type ginsenoside. In this study, the expression plasmid pET-32a-PgUGT74AE2 was constructed for expression of the recombinant protein and transferred into Escherichia coli Transetta (DE3) to generate the recombinant strain Transetta-PgUGT74AE2. The recombinant enzyme PgUGT74AE2 was expressed by induction of isopropyl-β-D-thiogalactoside (IPTG). An in vitro enzymatic reaction system was established with the recombinant enzyme PgUGT74AE2 and the substrate PPT. PgUGT74AE2 catalyzed the glycosylation of the free C-3 hydroxyl of PPT to produce 3-O-β-D-glucopyranosyl-dammar-24-ene-3β, 6α, 12β, 20S-tetraol, a new PPT-type ginsenoside. This study provides an efficient approach for the biosynthesis of a new PPT-type ginsenoside through in vitro enzymatic reaction, which may pave a way to produce promising lead in drug discovery.
Farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep. (AoFPPS) is considered as one of the important rate-limiting enzymes in the biosynthetic pathway of protostane triterpenes. In order to investigate the expression and function of AoFPPS, the gene (accession No. HQ724508) was cloned into a bacterial expression vector pCzn1, then the combined plasmid pCzn1-AoFPPS was transformed into Escherichia coli BL21, and a fusion protein was obtained after induction. The fusion protein was purified by Ni resin, and the function was verified through in vitro enzymatic reaction. High performance liquid chromatography (HPLC) analysis revealed that the products were able to catalyze the synthesis of farnesyl pyrophosphate (FPP). High purity recombinant protein was used to immunize New Zealand rabbits to generate a polyclonal antibody. The titer of the antibody was determined by enzyme linked immunosorbent assay (ELISA), and Western blot results demonstrated that the antibody could specifically recognize the AoFPPS protein in A. orientale (Sam.) Juzep. So, the method of rapid immunoassay to detect AoFPPS was established. This study lays the foundation for further study of the AoFPPS gene expression, regulation and mechanism of action in A. orientale (Sam.) Juzep., and it also provides a scientific basis on improving the quality of Alismatis Rhizoma using the plant genetic engineering.
Diterpene synthases from plants are key enzymes in diterpene biosynthesis, which catalyzed the initial cyclization cascade of (E, E, E,)-geranylgeranyl pyrophosphate, and generated diverse carbon skeletons. Recent research show that the structural and stereo chemical differences lead to diverse natural diterpene compounds of plants. The structures of diterpene synthases play a crucial role in cyclization function. This article mainly reviews the mechanisms, functional characteristics, and structural information of diterpene synthases according to the crystal structures and functions.
Plant flowering regulation is an important mechanism to response to environmental stress. Heat shock protein 70 family is one of the main molecular chaperones to resist stress; miRNA can be used as a negative regulator to participate in post-transcriptional gene in flowering network. In this paper, we obtained an Hsp70 gene from Lonicera japonica transcriptome and combined with Lonicera japonica miRNA library to obtain a novel miRNA that may target Hsp70 gene through bioinformatics method. Bioinformatics and expression during different flowering stages of the obtained Hsp70 gene and miRNA were analyzed. Phylogenetic tree showed that the obtained Hsp70 gene was clustered with Hsp110 subfamily in Oryza sativa and Arabidopis thaliana. The prediction of miRNA secondary structure showed its stable structure and high reliability. The binding site map showed that there were two base mismatches between sequences of miRNA and Hsp70 gene. The expression analysis showed that the expression of Hsp70 and miRNA in different flowering stages had opposite trends, indicating that miRNA might regulate Hsp70 to participate in the flowering stages of Lonicera japonica. This study provided new ideas for Lonicera japonica flowering regulation and response to environmental stress mechanisms.
MCT is an important key enzyme in the terpenoid biosynthesis in MEP pathway. In this study, Gateway technology was used to construct RNAi vector of TwMCT, and a vector fragment with a size of 484 bp was obtained. The TwMCT RNAi vector was transferred into the suspension cells of Tripterygium wilfordii by gene gun. Accumulation of terpenoids was assayed by UPLC, and the result showed that the content of triptolide and celastrol in cells decreased by 23.4% and 42.8%, respectively, compared with the control group pK7GWIWG2D. Moreover, the gene expression of TwMCT and major genes in terpenoid biosynthesis pathway was detected by qRT-PCR, which demonstrated that the expression of TwMCT reduced by 29.2% relative to that of the control group pK7GWIWG2D, and the relative expression of TwDXR, TwGGPS, TwHMGR and TwHMGS diminished by 36.3%, 31.3%, 62.2%, and 29.1%, respectively, but the expression of TwDXS was up-regulated by 114.2%, and there was no significant change in TwFPS. Thus, it was verified in vivo that interference with TwMCT expression significantly inhibited the accumulation of triptolide and celastrol in Tripterygium wilfordii, laying a foundation for further exploring the regulation mechanism of MCT gene on the terpenoid biosynthesis in Tripterygium wilfordii.
The commonly used traditional Chinese medicines Curcumae Rhizome (Ezhu) and Curcumae Radix (Yujin), are representatives of multi-plant sources. The relationship among the original source plant species is intricate. In this study, by using multivariate data analysis, volatile metabolites in rhizomes and radixes of source plants of Curcumae Longae rhizome (Jianghuang), Yujin and Ezhu in traditional Chinese medicine were compared and analyzed. The source plants included Curcuma longa, Curcuma kwangsuensis, Curcuma wenyujing and Curcuma phaeocaulis. The results indicated that:① volatile metabolites were similar in quality but variation in quantity for rhizomes and radixes origin from the same plant species; ② volatile metabolites of C. longa rhizomes showed bigger difference compared with others; ③ although common volatile metabolites were observed in rhizome of C. kwangsuensis, C. wenyujing and C. phaeocaulis, the difference among them were significant; and ④ significant differences were observed for the four kinds of radixes. Results in this study revealed the differences of the four source plants species, and similar metabolites in source plants of Curcumae Rhizome (Ezhu) and Curcumae Radix (Yujin) from the level of volatile metabolites. These results provided a reference for the clinical use of the three kinds of traditional Chinese medicine.
Tripterygium wilfordii 3-hydroxy-3-methylglutaryl coenzyme-A reductase (TwHMGR) is an important regulation site in terpenoids metabolic pathway in cytoplasm which is the first speed limit enzyme of MVA pathway. In order to investigate the effects of TwHMGR on the biosynthesis of triptolide and celastrol in Tripterygium wilfordii, the overexpression of TwHMGR (OE-HMGR) was studied in this paper. We cloned the full-length of TwHMGR to construct overexpression vector by Gateway technology then delivered the expression vector into Tripterygium wilfordii suspension cells by gene gun. qRT-PCR was used to detect the expression of TwHMGR:the expression of TwHMGR was increased to 1.75 folds over the control group (empty vector:pH7WG2D) in the overexpression group. The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC, revealing that:the contents of triptolide and celastrol were increased to 163.93% and 190.04% of over the control group in the overexpression group. Based on these findings, the positive effect on the accumulation of active terpenoids, triptolide and celastrol in Tripterygium wilfordii was found and the results laid a foundation of the synthetic biology research on important active terpenoids in Tripterygium wilfordii.
Dammarenediol-Ⅱ is an important precursor in the biosynthesis pathway of ginsenosides which are the main active components of Panax quinquefolius and Panax ginseng. For constructing a dammarenediol- Ⅱ-producing cell factory, the triterpenoid precursors of yeast are improved significantly by the modular pathway engineering strategy on the basis of an MVA optimized strain. The strain overexpressing Salvia miltiorrhiza SmFPS and Arabidopsis thaliana AtSQS2 could yield 67.4 mg·g-1 squalene, accounting for about 6.74% of cell dry weight. In our further work, an Arabidopsis thaliana 2, 3-oxidosqualene synthase AtSQE2 was found to be able to increase the downstream lanosterol yield by 22-fold, reaching 47.9 mg·g-1. Then, regulating dammarenediol-Ⅱ synthase gene expression, using anti-sense RNA technology for regulation of ERG7 in the ergosterol pathway, and optimizing fermentation process were successively performed. Finally, the synthesis flux of triterpenes was increased to 10 g·L-1 for the first time, and we constructed an efficient cell factory that can produce 15 g·L-1 dammarenediol-Ⅱ, which lays a solid foundation of industrial synthesis of dammarane-type ginsenosides.
Selectivity of drug action is a determinant for wide therapeutic window and less adverse response. From the viewpoint of molecular structure the conception and strategy of drug design are mainly embodied in raising selectivity. For the target-based drug discovery it is crucial to precisely obliterate detrimental targets in dimension of time and space, so as to efficaciously translate the in vitro active compounds into in vivo therapeutic medicines. To realize this translation drug molecules must be accurately transported to and destroy the harmful targets. To this end, chemical structures of drugs must be manipulated in multiple dimensions. This article attempts to concisely describe several kinds of bifunctional molecules for raising selectivity from the standpoint of medicinal chemistry. The bifunctionality of antibody-drug conjugates (ADCs) involves in the guidance and carrier of the antibody to guide ADC and reach to target cells, and simultaneously injury quality of the toxin moiety of ADC interacts with and destroys targets. Based upon target 3D structures design of irreversible inhibitors consist in connecting an appropriate electrophilic moiety to a well-defined ligand to endow the molecule with an additional ability to covalently bond to a specific amino acid residue. Hydrophobic tag (HyT), proteosis-targeting chimera (PROTAC), and degradation tag (dTAG) are new developed technologies, which are structurally characterized by bifunctionality, and mechanistically these compounds are capable of recruiting protein of interest (POI), inducing protein-protein interaction (PPI), and cleaving POI. In spite of large molecular size and the bottleneck of pharmacokinetic and physicochemical properties these technologies still have broad development prospect owing to high selectivity and wide adaptations.
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