System network analysis of Rosmarinus officinalis transcriptome and metabolome-Key genes in biosynthesis of secondary metabolites.

Medicinal plants contain valuable compounds that have attracted worldwide interest for their use in the production of natural drugs. The presence of compounds such as rosmarinic acid, carnosic acid, and carnosol in Rosmarinus officinalis has made it a plant with unique therapeutic effects. The identification and regulation of the biosynthetic pathways and genes will enable the large-scale production of these compounds. Hence, we studied the correlation between the genes involved in biosynthesis of the secondary metabolites in R. officinalis using proteomics and metabolomics data by WGCNA. We identified three modules as having the highest potential for the metabolite engineering. Moreover, the hub genes highly connected to particular modules, TFs, PKs, and transporters were identified. The TFs of MYB, C3H, HB, and C2H2 were the most likely candidates associated with the target metabolic pathways. The results indicated that the hub genes including Copalyl diphosphate synthase (CDS), Phenylalanine ammonia lyase (PAL), Cineole synthase (CIN), Rosmarinic acid synthase (RAS), Tyrosine aminotransferase (TAT), Cinnamate 4-hydroxylase (C4H), and MYB58 are responsible for biosynthesis of important secondary metabolites. Thus, we confirmed these results using qRT-PCR after treating R. officinalis seedlings with methyl jasmonate. These candidate genes may be employed for genetic and metabolic engineering research to increase R. officinalis metabolite production.

Discovery of the cyclotide caripe 11 as a ligand of the cholecystokinin-2 receptor.

The cholecystokinin-2 receptor (CCK2R) is a G protein-coupled receptor (GPCR) that is expressed in peripheral tissues and the central nervous system and constitutes a promising target for drug development in several diseases, such as gastrointestinal cancer. The search for ligands of this receptor over the past years mainly resulted in the discovery of a set of distinct synthetic small molecule chemicals. Here, we carried out a pharmacological screening of cyclotide-containing plant extracts using HEK293 cells transiently-expressing mouse CCK2R, and inositol phosphate (IP1) production as a readout. Our data demonstrated that cyclotide-enriched plant extracts from Oldenlandia affinis, Viola tricolor and Carapichea ipecacuanha activate the CCK2R as measured by the production of IP1. These findings prompted the isolation of a representative cyclotide, namely caripe 11 from C. ipecacuanha for detailed pharmacological analysis. Caripe 11 is a partial agonist of the CCK2R (Emax = 71%) with a moderate potency of 8.5 µM, in comparison to the endogenous full agonist cholecystokinin-8 (CCK-8; EC50 = 11.5 nM). The partial agonism of caripe 11 is further characterized by an increase on basal activity (at low concentrations) and a dextral-shift of the potency of CCK-8 (at higher concentrations) following its co-incubation with the cyclotide. Therefore, cyclotides such as caripe 11 may be explored in the future for the design and development of cyclotide-based ligands or imaging probes targeting the CCK2R and related peptide GPCRs.

Experimental, molecular docking and molecular dynamic studies of natural products targeting overexpressed receptors in breast cancer.

Natural compounds are proper tools for inhibiting cancer cell proliferation. Hence, the search for these ligands of overexpressed receptors in breast cancer has been a competitive challenge recently and opens new avenues for drug discovery. In this research, we have investigated molecular interactions between natural products and overexpressed receptors in breast cancer using molecular docking and dynamic simulation approaches followed by extraction of the best ligand from Citrus limetta and developing for nanoscale encapsulation composed of soy lecithin using a sonicator machine. The encapsulation process was confirmed by DLS and TEM analyses. Anticancer activity was also examined using MTT method. Among the investigated natural compounds, hesperidin was found to bind to specific targets with stronger binding energy. The molecular dynamics results indicated that the hesperidin-MCL-1 complex is very stable at 310.15 K for 200 ns. The RP-HPLC analysis revealed that the purity of extracted hesperidin was 98.8% with a yield of 1.72%. The results of DLS and TEM showed a strong interaction between hesperidin and lecithin with an entrapped efficiency of 92.02 ± 1.08%. Finally, the cytotoxicity effect of hesperidin was increased against the MDA-MB-231 cell line with an IC50 value of 62.93 µg/mL after encapsulation, whereas no significant effect against the MCF10A cell line. We showed for the first time that hesperidin is a flexible and strong ligand for the MCL-1 receptor. Also, it has the in vitro ability to kill the MDA-MB-231 cell lines without having a significant effect on the MCF10A cell lines. Therefore, hesperidin could be used as a food ingredient to generate functional foods.

The potential application of the protein hydrolysates of three medicinal plants: cytotoxicity and functional properties.

Functional evaluation of encrypted bioactive peptides in protein structure helps to better understand those for using in pharmacy and food sciences. For this purpose, the total protein was extracted from Matricaria chamomilla, Ziziphora clinopodioides, and Cressa cretica, and partially purified with ammonium sulfate. Protein hydrolysates were obtained from pancreatin hydrolysis for 240 min and the enzyme hydrolysis was confirmed using the determination of hydrolysis degree and Fourier transform infrared (FT-IR) followed by the physicochemical and sensory properties were investigated. The results showed that all hydrolysates had both cytotoxic and antioxidant activities. Specifically, C. cretica hydrolysates represented cytotoxic activity against the MCF-7 cell line with the IC50 of 135.21 µg/mL, while showed no significant growth inhibition effect on the HEK293 cell line. Besides, M. chamomilla hydrolysates showed the lowest bitterness value (1.125 ± 0.52). From the perspective of color investigation, M. chamomilla hydrolysates indicated the highest L* and the lowest a* factors. The highest turbidity and surface tension, and 10-fold more cancer cell killing effect under gastrointestinal digestion conditions were observed for M. chamomilla hydrolysates. Therefore, bioactive peptides might be formulated in designing of novel anticancer drugs or could be used in promising protocols for the production of food products with beneficial health effects.