Phytochemicals Identification and Bioactive Compounds Estimation of Artemisia Species Grown in Saudia Arabia.

Artemisia species are very important medicinal plants, particularly in the Middle East and in developing countries. Their products have been used in traditional and medicine contemporary for the treating of infectious ulcers, gangrenous ulcers, inflammations, and malaria. Artemisinin derived from Artemisia species has been used as a drug in many countries for malaria disease treatment. Hence, this study aimed to identify and evaluate the bioactive compounds of three species of Artemisia (Artemisia judaica, Artemisia monosperma, and Artemisia sieberi) growing in Saudi Arabia. Therefore, several analytical techniques, such as gas chromatography-mass spectrometry (GC-MS), UV-Visible spectrophotometry (UV-Vis), and high-performance liquid chromatography (HPLC), with reference standards, were used. The GC-MS analysis of the artemisia species revealed many bioactive constituents associated with plant secondary metabolites; some of these identified phytochemical components have biological activity. A. Judaica showed the highest number of bioactive compounds, followed by A. sieberi and A. monosperma. Further, the total phenol, total flavonoid, total tannin, terpenoids, and TCA were estimated. Furthermore, biomolecules such gallic acid, tannin acid, quercetin, and artemisinin in different artemisia species were quantified using HPLC with the reference standard. The amount of artemisinin in the leaf extract of these species (A. sieberi, A. Judaica, and A. monosperma) was found to be about 3.01, 2.5, and 1.9 mg/g DW, respectively. Moreover, the antioxidant activity of the samples was estimated. The obtained results have shown that these species possessed high antioxidant activity, and the scavenging of the DPPH radical and hydrogen peroxide were found to be raised with the increase in the plant extract concentration. This reflects the number of bioactive compounds in these species. The findings of this research support and justify the utilization of Artemisia species in folk medicine in the Middle East.

Callus-Mediated High-Frequency Plant Regeneration, Phytochemical Profiling, Antioxidant Activity and Genetic Stability in Ruta chalepensis L.

Efficient methods for callus induction and the high-frequency plant regeneration of Ruta chalepensis L. were established, and the phytochemical potential and antioxidant activity of a donor plant, ex-vitro-established micropropagated plants, and callus were also studied. Yellowish-green callus was induced with a frequency of 97.8% from internode shoot segments of the donor plant growing in soil in the botanical garden cultured on Murashige and Skoog (MS) medium containing 10 µM 2,4-D (2,4-dichlorophenoxyacetic acid) and 1 µM BA (6-benzyladenine). Adventitious shoots were regenerated from the yellowish-green callus on MS medium containing 5.0 µM (BA) and 1.0 µM 1-naphthaleneacetic acid (NAA), with a regeneration frequency of 98.4% and a maximum of 54.6 shoots with an average length of 4.5 cm after 8 weeks. The regenerated shoots were rooted in a medium containing 1.0 µM IBA (indole-3-butyric acid) and successfully transferred to ex vitro conditions in pots containing normal garden soil, with a 95% survival rate. The amounts of alkaloids, phenolics, flavonoids, tannins, and antioxidant activity of the ex-vitro-established micropropagated plants were higher than in the donor plant and callus. The highest contents of hesperidin and rutin (93.3 and 55.9 µg/mg, respectively) were found in the ex-vitro-established micropropagated plants compared to those obtained from the donor plant (91.4 and 31.0 µg/mg, respectively) and callus (59.1 and 21.6 µg/mg, respectively). The genetic uniformity of the ex-vitro-established micropropagated plants was appraised by the ISSR markers and compared with the donor plant. This is the first report describing the callus-mediated plant regeneration, as well as the production of phenolic compounds and antioxidant activities in R. chalepensis, which might be a potential alternative technique for the mass propagation and synthesis of bioactive compounds such as hesperidin and rutin.

High-Frequency Plant Regeneration, Genetic Uniformity, and Flow Cytometric Analysis of Regenerants in Rutachalepensis L.

Ruta chalepensis L., an evergreen shrub in the citrus family, is well-known around the world for its essential oils and variety of bioactivities, indicating its potential medicinal applications. In this study, we investigated the effect of different culture conditions, including plant growth regulators, media types, pH of the medium, and carbon sources, on in vitro regeneration from nodal explants of R. chalepensis. Following 8 weeks of culture, the highest percentage of regeneration (96.3%) and maximum number of shoots (40.3 shoot/explant) with a length of 4.8 cm were obtained with Murashige and Skoog (MS) medium at pH 5.8, supplemented with 3.0% sucrose and 5.0 µM 6-Benzyladenine (BA) in combination with 1.0 µM 1-naphthaleneacetic acid (NAA). For rooting, individually harvested shootlets were transferred on ½ MS (half-strength) supplemented with IAA (indole-3-acetic acid), IBA (indole 3-butyric acid), or NAA, and the best response in terms of root induction (91.6%), number of roots (5.3), and root mean length (4.9 cm) was achieved with 0.5 µM IBA after 6 weeks. An average of 95.2 percent of healthy, in vitro regenerated plantlets survived after being transplanted into potting soil, indicating that they were effectively hardened. DNA assays (PCR-based markers) such as random amplification of polymorphic DNA (RAPD) and directed amplification of minisatellite-region (DAMD) were employed to assess in vitro cultivated R. chalepensis plantlets that produced a monomorphic banding pattern confirming the genetic stability. Additionally, no changes in the flow cytometric profile of ploidy between regenerated plantlets and donor plants were detected. Regeneration of this valuable medicinal plant in vitro will open up new avenues in pharmaceutical biotechnology by providing an unconventional steadfast system for mass multiplication and might be effectively used in genetic manipulation for enhanced bioactive constituents.

Tissue-specific analysis of Coffea arabica L. transcriptome revealed potential regulatory roles of lncRNAs.

Long non-coding RNAs (lncRNAs) play pivot roles in regulating mRNA expression in eukaryotic organisms without coding any proteins. In the current study, a comprehensive analysis of 260 published RNA-Seq datasets collected from different tissues (fruits, leaves, stems, and roots) of Coffea arabica L. was performed to discover potential lncRNAs. A total of 10,564 unique lncRNAs were identified. Our results showed that 77.14% of the lncRNAs were intergenic and 60.39% of them are located within 5 Kbp from the partner gene. In general, all the identified lncRNAs showed shorter lengths, fewer number of exons, and lower expression levels as compared to mRNAs in different studied tissues. Several lncRNAs were determined as differentially expressed (DE) in fruits as compared to leaves, stems, or roots. The functional characterization of the DE lncRNAs revealed their roles in regulating significant biological processes in different tissues of C. arabica. The current study provides a comprehensive analysis and dataset of lncRNAs in C. arabica that could be utilized in further studies concerning the roles of these molecules in plant cells.

Inhibitory Effect of Multimodal Nanoassemblies against Glycative and Oxidative Stress in Cancer and Glycation Animal Models.

In recent years, there has been a progress in the study of glycation reaction which is one the possible reason for multiple metabolic disorders. Glycation is a nonenzymatic reaction between nucleic acids, lipids, and proteins resulting into the formation of early glycation products that may further lead to the accumulation of advanced glycation end products (AGEs). The precipitation of AGEs in various cells, tissues, and organs is one of the factors for the initiation and progression of various metabolic derangements including the cancer. The AGE interaction with its receptor "RAGE" activates the inflammatory pathway; yet, the downregulation of RAGE and its role in these pathways are not clear. We explore the effect of anticancer novel nanoassemblies on AGEs to determine its role in the regulation of the expression of RAGE, NFƙB, TNF-α, and IFN-γ. This paper is based on the in vivo and in vitro study in glycation and lung cancer model systems. Upon the treatment of nanoassemblies in both the model systems, we observed a protective effect of nanoassemblies over the inhibition of glycative and oxidative stress via mRNA expression analysis. The mRNA expression results corroborated with the reactive oxygen species (ROS), carboxy-methyl-lysine (CML), and fluorescence studies. In this study, we found that the presence of common factors for glycation and lung cancer is oxidative and glycative stress. This oxidation and glycation might be responsible for the initiation of inflammation which may further lead to uncontrolled growth of cells leading to cancer. This can be a strong association between lung cancer and glycation reaction. The intervention of the anticancer and antiglycation effects of multimodal nanoassemblies throughout the study promises a new pathway for cancer research.

Methylglyoxal mediated glycation leads to neo-epitopes generation in fibrinogen: Role in the induction of adaptive immune response.

In diabetes mellitus, hyperglycemia mediated non-enzymatic glycosylation of proteins results in the pathogenesis of diabetes-associated secondary complications via the generation of advanced glycation end products (AGEs). The focus of this study is to reveal the immunological aspects of methylglyoxal (MG) mediated glycation of fibrinogen protein. The induced immunogenicity of modified fibrinogen is analyzed by direct binding and inhibition ELISA. Direct binding ELISA confirmed that MG glycated fibrinogen (MG-Fib) is highly immunogenic and induces a high titer of antibodies in comparison to its native analog. Cross-reactivity and antigen-binding specificity of induced antibodies were confirmed by inhibition ELISA. The enhanced affinity of immunoglobulin G (IgG) from immunized rabbits' sera and MG glycated fibrinogen is probably the aftermath of neo-epitopes generation in the native structure of protein upon modification. Thus, we deduce that under the glycative stress, MG-mediated structural alterations in fibrinogen could induce the generation of antibodies which might serve as a potential biomarker in diabetes mellitus and its associated secondary disorders.

Induction of somatic embryogenesis in Brassica juncea L. and analysis of regenerants using ISSR-PCR and flow cytometer.

A new and simple protocol has been developed and standardized for direct somatic embryogenesis and plant regeneration from aseptic seedlings derived from immature Brassica juncea seeds. Depending on the age of immature seeds and nutrient media, in vitro occurrence of embryogenesis and the number of embryos from each seedling have varied greatly. The largest number of somatic embryos, producing 12.7 embryos per seedlings, have been developed by seedlings obtained from immature seeds collected after 21 days of pollination (DAP). Effect of different nutrient media [Gamborg (B5), Murashige and Skoog (MS) and Linsmaier and Skoog (SH)] and carbon sources (fructose, glucose, maltose and sucrose) were assessed to induce somatic embryos and the maximum response were achieved on Nitsch culture medium fortified with sucrose (3% w/v) followed by fructose and maltose. The somatic embryo converted into complete plantlets within 04-weeks of culture on Nitsch medium containing half-strength of micro and macro salts. The regenerated plantlets were successfully established in soil with 90% survival rate. The acclimated plants were subsequently transferred to field condition where they grew normally without any phenotypic differences. Genetic stability of B. juncea plants regenerated from somatic embryos were confirmed by inter-simple sequence repeat (ISSR)-PCR analysis and flow cytometry. No significant difference in ploidy level and ISSR banding pattern were documented between somatic embryo's plants and control plants grown ex vitro.

Genome-wide transcriptome variation landscape in Ruta chalepensis organs revealed potential genes responsible for rutin biosynthesis.

Ruta chalepensis L., most commonly known as 'fringed rue,' is an excellent and valuable bioactive plant that produces a range of complex flavonoids, of which rutin is the major compound present in this plant of great pharmaceutical and medicinal significance. The present study is a pioneering attempt to examine the changes in the transcriptomic landscape of leaf, stem, and root tissues and correlate this with rutin quantity in each tissue in order to identify the candidate genes responsible for rutin biosynthesis and to increase genomic resources in fringed rue. Comparative transcriptome sequencing of leaves, stems and roots were performed using the NovaSeq 6000 platform. The de novo transcriptome assembly generated 254,685 transcripts representing 154,018 genes with GC content of 42.60 % and N50 of 2280 bp. Searching assembled transcripts against UniRef90 and SwissProt databases annotated 79.7 % of them as protein coding. The leaf tissues had the highest rutin content followed by stems and roots. Several differentially expressed genes and transcripts relating to rutin biosynthesis were identified in leaves comparing with roots or stems comparing with roots. All the genes known to be involved in rutin biosynthesis showed up-regulation in leaves as compared with roots. These results were confirmed by gene ontology (GO) and pathway enrichment analyses. Up-regulated genes in leaves as compared with roots enriched GO terms with relation to rutin biosynthesis e.g. action of flavonol synthase, biosynthetic mechanism of malonyl-CoA, and action of monooxygenase. Phylogenetic analysis of the rhamnosyltransferase (RT) gene showed that it was highly homologues with RT sequence from Citrus species and all were located in the same clade. This transcriptomic dataset will serve as an important public resource for future genomics and transcriptomic studies in R. chalepensis and will act as a benchmark for the identification and genetic modification of genes involved in the biosynthesis of secondary metabolites.

Effects of 4-CPPU on in vitro multiplication and sustainable generation of Hibiscus rosa-sinensis L. 'White Butterfly'.

There are more than nine thousand cultivars of Hibiscus rosa-sinensis L., with a series of flowers with shapes, colors and new cultivars continues as generated through both traditional and modern breeding techniques. In this study, advanced biotech methods of in vitro culture have been used to identify a technique for the efficient mass multiplication of H. rosa-sinensis 'White Butterfly', using phenyl urea, N-(2-Chloro-4-pyridyl)-N'-phenylurea (4-CPPU). For the first time, the effects of 4-CPPU for stimulating axillary shoot proliferation and multiple shoot regenerations from nodal explants were evaluated, and the optimal nutrient media deduced. From the diverse concentrations as 0.1, 0.5, 2.5, 5.0 & 10.0 µM of 4-CPPU, the highest frequency of shoots was recorded at 2.5 µM supplied in Murashige and Skoog (MS, pH-5.8) medium. After eight-weeks of culture, on an average of 6.7 shoot were obtained on this media with shoot heights of 4.2 cm from each explant. With the involvement of 0.5 µM-IBA (indole-3-butyric acid) in MS medium the regenerated shoots were rooted and followed by successful acclimation to ex vitro conditions. The ploidy consistency among the micro-plants was analyzed using flow cytometry and compared with ex vitro grown plants. No differences in the ploidy levels were observed among the 4-CPPU induced plants, when compared with the donor plants.