Comparative Molecular Mechanisms of Biosynthesis of Naringenin and Related Chalcones in Actinobacteria and Plants: Relevance for the Obtention of Potent Bioactive Metabolites

Naringenin and its glycosylated derivative naringin are flavonoids that are synthesized by the phenylpropanoid pathway in plants. We found that naringenin is also formed by the actinobacterium Streptomyces clavuligerus, a well-known microorganism used to industrially produce clavulanic acid. The production of naringenin in S. clavuligerus involves a chalcone synthase that uses p-coumaric as a starter unit and a P450 monoxygenase, encoded by two adjacent genes (ncs-ncyP). The p-coumaric acid starter unit is formed by a tyrosine ammonia lyase encoded by an unlinked, tal, gene. Deletion and complementation studies demonstrate that these three genes are required for biosynthesis of naringenin in S. clavuligerus. Other actinobacteria chalcone synthases use caffeic acid, ferulic acid, sinapic acid or benzoic acid as starter units in the formation of different antibiotics and antitumor agents. The biosynthesis of naringenin is restricted to a few Streptomycess species and the encoding gene cluster is present also in some Saccharotrix and Kitasatospora species. Phylogenetic comparison of S. clavuligerus naringenin chalcone synthase with homologous proteins of other actinobacteria reveal that this protein is closely related to chalcone synthases that use malonyl-CoA as a starter unit for the formation of red-brown pigment. The function of the core enzymes in the pathway, such as the chalcone synthase and the tyrosine ammonia lyase, is conserved in plants and actinobacteria. However, S. clavuligerus use a P450 monooxygenase proposed to complete the cyclization step of the naringenin chalcone, whereas this reaction in plants is performed by a chalcone isomerase. Comparison of the plant and S. clavuligerus chalcone synthases indicates that they have not been transmitted between these organisms by a recent horizontal gene transfer phenomenon. We provide a comprehensive view of the molecular genetics and biochemistry of chalcone synthases and their impact on the development of antibacterial and antitumor compounds. These advances allow new bioactive compounds to be obtained using combinatorial strategies. In addition, processes of heterologous expression and bioconversion for the production of naringenin and naringenin-derived compounds in yeasts are described.

Appraisal of Anti-Arthritic Potential of Quercus Leucotrichophora Methanolic Extract in Complete Freund’s Adjuvant Induced Arthritic Rats; a Mechanistic Study

This research work was conducted to validate the folkloric use and therapeutic potential of Quercus leucotrichophora (QL) leaf methanolic and aqueous extracts against inflammation and arthritis and to determine the chemical composition by HPLC. The in-vitro anti-oxidant and anti-inflammatory activities were carried out along with in-vivo assays such as carrageenan induced paw edema, xylene induced ear edema and Complete Freund’s Adjuvant induced arthritis in Wistar rats. The CFA (0.1 ml) was inoculated to the left hind paw at day 1 to induce arthritis and oral dosing with QLME at 150, 300 and 600 mg/kg was begun at 8th day till the 28th day in all groups while methotrexate was given as standard treatment. There was a noteworthy (p<0.05-0.0001) restoration in body weight, paw edema, arthritic index, altered blood parameters and oxidative stress biomarkers in treated rats as compared to diseased group. Moreover, QLME considerably (p<0.0001) downregulated TNF-α, IL-6, IL-1β, COX-2, and NF-κB, while significantly (p<0.0001) upregulated IL-10, I-κB, IL-4 in relation to diseased group. The QLME exhibited no mortality in acute toxicity study. It was concluded that QLME possessed substantial anti-inflammatory and anti-arthritic potential at all dosage levels, mainly at 600 mg/kg might be due to presence of quercetin, sinapic acid and ferulic acid.

Effect of Issatchenkia terricola WJL-G4 on Deacidification Characteristics and Antioxidant Activities of Red Raspberry Wine Processing

Our previous study isolated a novel Issatchenkia terricola WJL-G4, which exhibited a potent capability of reducing citric acid. In the current study, I. terricola WJL-G4 was applied to decrease the content of citric acid in red raspberry juice, followed by the red raspberry wine preparation by Saccharomyces cerevisiae fermentation, aiming to investigate the influence of I. terricola WJL-G4 on the physicochemical properties, organic acids, phenolic compounds and antioxidant activities during red raspberry wine processing. The results showed that after being treated with I. terricola WJL-G4, the citric acid contents in red raspberry juice decreased from 19.14 ± 0.09 to 6.62 ± 0.14 g/L, which was further declined to 5.59 ± 0.22 g/L after S. cerevisiae fermentation. Parameters related to CIELab color space, including L*, a*, b*, h°, and ∆E* exhibited the highest levels in samples after I. terricola WJL-G4 fermentation. Compared to the red raspberry wine pretreated without deacidification (RJO-SC), wine pretreated by I. terricola WJL-G4 (RJIT-SC) exhibited significantly decreased contents of gallic acid, cryptochlorogenic acid, and arbutin, while significantly increased contents of caffeic acid, sinapic acid, raspberry ketone, quercitrin, quercetin, baicalein, and rutin. Furthermore, the antioxidant activities including DPPH· and ABTS+· radical scavenging were enhanced in RJIT-SC group as compared to RJO-SC. This work revealed that I. terricola WJL-G4 had a great potential in red raspberry wine fermentation.

Rapid Identification of 3,6′-Disinapoyl Sucrose Metabolites in Alzheimer’s Disease Model Mice Using UHPLC–Orbitrap Mass Spectrometry

Alzheimer’s disease (AD) is a degenerative disease of the central nervous system characterized by the progressive impairment of neural activity. Studies have shown that 3,6′-disinapoyl sucrose (DISS) can alleviate the pathological symptoms of AD through the activation of the cAMP/CREB/BDNF signaling pathway. However, the exact biochemical mechanisms of action of DISS are not clear. This study explores metabolism of DISS in an AD mouse model, induced by the microinjection of a lentiviral expression plasmid of the APPswe695 gene into CA1 of the hippocampus. After gavage administration of DISS (200 mg/kg), the kidneys, livers, brains, plasma, urine, and feces were collected for UHPLC–Orbitrap mass spectrometry analysis. Twenty metabolites, including the prototype drug of DISS, were positively or tentatively identified based on accurate mass measurements, characteristic fragmentation behaviors, and retention times. Thus, the metabolic pathways of DISS in AD mice were preliminarily elucidated through the identification of metabolites, such as ester bond cleavage, demethoxylation, demethylation, and sinapic acid-related products. Furthermore, differences in the in vivo distribution of several metabolites were observed between the model and sham control groups. These findings can provide a valuable reference for the pharmacological mechanisms and biosafety of DISS.

Research on Crystal Structure and Fungicidal Activity of the Amide Derivatives Based on the Natural Products Sinapic Acid and Mycophenolic Acid

Structural optimization based on natural products is an important and effective way to discover new green pesticides. Here, two series of amide derivatives based on sinapic acid and mycophenolic acid were designed in combination with the fungicidal natural product piperlongumine and synthesized by preparing the carboxylic acid into acyl chloride and then reacting with the corresponding aromatic amines, respectively. The resulting structures were successively characterized by 1H NMR, 13 C NMR, and HRMS. The crystal structures of molecules I-4 and II-5 were analyzed for structure validation. The in vitro inhibitory activity indicated that most of the target products exhibited fungicidal activity equivalent to or even better than fluopyram against Physalospora piricola. The in vivo fungicidal activity demonstrated that the compounds I-5 and II-4 displayed almost the same preventative activity as carbendazim and fluopyram at 200 μg mL−1. The TEM observation revealed that the fungicidal activity of the target molecules against Physalospora piricola may be due to the influence on the mitochondria in the cell structure. These results will provide valuable theoretical guidance for developing the new green fungicides.

Synthesis of hydroxycinnamoyl shikimates and their role in monolignol biosynthesis

Hydroxycinnamoyl shikimates were reported in 2005 to be intermediates in monolignol biosynthesis. 3-Hydroxylation of p-coumarate, originally thought to occur via coumarate 3-hydroxylase (C3H) from p-coumaric acid or its CoA thioester, was revealed to be via the action of coumaroyl shikimate 3′-hydroxylase (C3′H) utilizing p-coumaroyl shikimate as the substrate, itself derived from p-coumaroyl-CoA via hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase (HCT). The same HCT was conjectured to convert the product, caffeoyl shikimate, to caffeoyl-CoA to continue on the pathway starting with its 3-O-methylation. At least in some plants, however, a more recently discovered caffeoyl shikimate esterase (CSE) enzyme hydrolyzes caffeoyl shikimate to caffeic acid from which it must again produce its CoA thioester to continue on the monolignol biosynthetic pathway. HCT and CSE are therefore monolignol biosynthetic pathway enzymes that have provided new opportunities to misregulate lignification. To facilitate studies into the action and substrate specificity of C3H/C3′H, HCT, and CSE enzymes, as well as for metabolite authentication and for enzyme characterization, including kinetics, a source of authentic substrates and products was required. A synthetic scheme starting from commercially available shikimic acid and the four key hydroxycinnamic acids (p-coumaric, caffeic, ferulic, and sinapic acid) has been developed to provide this set of hydroxycinnamoyl shikimates for researchers.