Anti-Inflammatory Properties of Extract of the Hairy Root of Native Portulaca Oleracea L. and Its Effect on Some Inflammatory Genes Expression in Rat Microglial Cells.

Background: Most herbs play significant roles in the treatment of various diseases. Because dopamine functions in the anti-inflammatory process and the presence of this substance in Portulaca Oleracea L. native plant, investigating this plant's anti-inflammatory properties in treating neurological diseases is interesting. Objectives: The objective of this study was to estimate the NO production and the expression level of inflammatory genes in lipopolysaccharide (LPS)-treated microglial cells affected by P. oleracea L. extraction. Materials and Methods: P. oleracea L. hairy root extract was isolated, and the primary microglial cell of the rat was isolated from glial cells and confirmed by immunocytochemistry analysis. Microglial cells were pretreated with different concentrations of P. oleracea L. extract and then treated with 1 µg.mL-1 LPS. The control group did not receive any treatment. The NO level in culture supernatants was measured by the Griess method. The mRNA expression levels of iNOS (inducible Nitric oxide synthase) and TNF-α (tumor necrosis factor-alpha) in LPS-treated microglial cells were evaluated using Real-Time PCR. Results: The present study determined that 0.1 mg. mL-1 of the P. oleracea L. extract decreased the NO production in rat microglial cells. Different concentrations of the P. oleracea L. extract had no prominent effects on LPS-treated cell viability. The results of real-time PCR indicated that P. oleracea L extracts suppressed the mRNA expression levels of iNOS and TNF-α in LPS-treated cells. MTT assay determined that P. oleracea L. extract was not cytotoxic, and the anti-inflammatory P. oleracea L. extract effects observed were not because of cell death. Conclusion: P. oleracea L. extract might be helpful as an anti-inflammatory agent in treating inflammatory diseases.

A novel spray bioreactor for the proliferation of plant callus; Hyoscyamus niger and Arnebia pulchra.

OBJECTIVE: Unlike plant cell suspension culture, the proliferation of callus in bioreactors has received inadequate attention. The magnificent potential of plant callus becomes more appreciated as the research unfolds and promises interesting applications including the production of valuable metabolites, therapeutic antibodies, bioactive extracts with regenerating effects, and the generation of genetically improved plants. Issues such as the lack of 3D-access of the cells to the nutrients, using an interfering gelling substance as the support matrix, and the changes in the medium formulation during the growth phase were discouraging factors for extending research on this topic. Considering the existing drawbacks, a novel open-flow spray bioreactor (OFSB) was configured to circumvent the associated problems with the solid cell culture and promote the applicability of plant callus culture via improving the feeding strategy. METHODS: Applying similar subculture conditions, the proliferation of Arnebia pulchra and Hyoscyamus niger calli as the examples of two important plant families (Boraginaceae and Solanaceae) was studied in the OFSB in comparison with similar calli that grew in Petri dishes and jars. RESULTS: A. pulchra and H. niger calli obtained the weight gains of (%87.3 and %106.7) in the Petri dishes, (%208.7 and %226) in the jars, and (%288.6 and %320.0) in OFSB, respectively, while no significant changes were observed in the productivity indices of the examined calli. CONCLUSION: The simple design of OFSB bypasses most of the notorious problems associated with solid plant callus culture. OFSB technical features allow the bioreactor to be used for growth optimization of various types of plant calli in a cost-effective manner.

Ethyl Acetate Extract of Licorice Root (Glycyrrhiza glabra) Enhances Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Glycyrrhiza glabra (G. glabra) has been used as a flavoring and sweetener agent, in addition to its therapeutic properties. It is rich in phytoestrogen and may prevent osteoporosis caused by estrogen deficiency; however, there is no evidence for its effects on proliferation and osteogenesis in mesenchymal stem cells. So, we were encouraged to investigate whether the ethyl acetate extract of licorice root as a source of phytoestrogen can act similar to estrogen in cell culture. Furthermore, the analysis of the licorice extract (LE) based on HPLC-DAD-ESI-MS indicated that LE comprises phytoestrogen compounds, such as glabridin and glabrene. In this study, the effects of LE on proliferation of human bone-marrow mesenchymal stem cells (hBM-MSCs) were investigated using MTT assay. In addition, its effects on the osteogenesis were evaluated using alkaline phosphatase activity (ALP), calcium deposition, and bone specific gene expression such as ALP, osteocalcin, Runx2, and BMP-2. The quantitative gene expression was studied by real-time RT-PCR. Our results showed a significant increase in proliferation in presence of LE in concentration 10-50 µg/mL. The differentiation of hBM-MSCs increased in doses of LE (10-25 µg/mL) compared to the control group. The effects of LE were similar to those of 17ß-estradiol (E2) (10-8 M) and were abolished by ICI 182,780 an antagonist of estrogen receptor (ER) (10-7), indicating that the stimulatory effects of LE occur through estrogen receptor-mediated mechanism . Taking these into account, LE may be a potential candidate for prevention of osteoporosis in menopausal women.

Fabrication of an immunosensor for early and ultrasensitive determination of human tissue plasminogen activator (tPA) in myocardial infraction and breast cancer patients.

Sensitive detection of biomarkers will mean accurate and early diagnosis of diseases. A tissue plasminogen activator (tPA) has a crucial role in many cardiovascular diseases and it is related to many processes such as angiogenesis in cancer cells. Therefore, sensitive determination of tPA is important in diagnosis and clinical research. tPA monoclonal antibody was covalently attached onto single-wall carbon nanotubes (SWCNTs) using diimide-activated imidation coupling. Functionalized SWCNTs were immobilized onto a glassy carbon electrode and the modification process was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), SEM, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Cyclic voltammograms (CVs) in a scan rate of 100 mVs-1 was studied and comparisons were made between the modified glassy carbon electrodes (immobilized with antibodies) as a working electrode before and after the formation of tPA-antibody complex. Results of the SDS-PAGE demonstrated that the antibody was covalently and site directly attached to the SWCNTs. The fabricated biosensor provided a good linear response range from 0.1 to 1.0 ng mL-1 with a low detection limit of 0.026 ng mL-1. The immunosensor showed selectivity, reproducibility, good sensitivity, and acceptable stability. Satisfactory results were observed for early and sensitive determination of tPA in human serum samples. For the first time, such specific biosensor is currently being fabricated for tPA in our laboratories and successfully could determine tPA in myocardial infraction and breast cancer patients. Graphical abstract Fabricated biosensor for determination of tPA.

Expression of active chimeric-tissue plasminogen activator in tobacco hairy roots, identification of a DNA aptamer and purification by aptamer functionalized-MWCNTs chromatography.

Tissue-type plasminogen activator (tPA) is a serine protease that plays a crucial role in the fibrinolytic system. We increased the activity of tPA by splicing the active site of dodder-cuscutain gene to human tPA. The chimeric cDNA of tPA was constructed by Splicing by Overlap Extension Polymerase Chain Reaction (SOEing-PCR) method and transferred to the hairy roots of tobacco using different strains of Agrobacterium rhizogenes. Chimeric-tPA was purified by lysine-sepharose chromatography and specific aptamers were designed using SELEX method. Multi wall carbon nanotubes were functionalized with selected aptamers, packed in a column, and used for purification. The results demonstrated that selected aptamer having KD values of 0.320 nM and IC50 of 28.9 nM possessed good affinity to tPA, and the chimeric-tPA was properly purified by aptamer-chromatography. Hairy roots expressing chimeric-tPA and normal-tPA produced 900 and 450 ngmg-1 of total protein, respectively. The activities of chimeric-tPA and normal-tPA were 90 and 60 IUml-1, respectively. Compared to the normal-tPA, chimeric-tPA showed more activity.

FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna.

A simple, sensitive, selective, and rapid optical nanobiosensor based on FRET was designed to detect tropane alkaloids as anti-cholinergic agents in natural and transgenic hairy roots extracts of Atropa belladonna. To achieve that, conjugation of tioglycolyic acid capped cadmium telluride quantum Dots, M2 muscarinic receptor (Cd/Te QDs-M2R) and conjugation of scopolamine-rhodamine123 (Sc-Rho123) were performed. More specifically, proportional amounts of M2 muscarinic receptor and quantum dots (QDs) were conjugated while scopolamine (as a tropane alkaloid) and rhodamine123 were also combined and these moieties functioned as donor and acceptor pairs, respectively. The system response was linear over the range of 0.01-4µmolL-1 of scopolamine hydrochloride concentration with a detection limit of 0.001µmolL-1. The developed nanobiosensor was successfully used for in vitro recognition of scopolamine as an anti-cholinergic agent in the investigated plant extracts. In addition, Agrobacterium rhizogenesis mediated gene transfer technique was employed to generate hairy roots and to enhance the production of tropane alkaloids in the studied medicinal plant.

Removal of Phenol by A. belladonna L. Hairy Root.

Phenolic compounds that present in the several industries are harmful and dangerous for human health. In this study we have studied the potential of Atropa belladonna hairy roots in phenol removal of wastewater. The optimal conditions for the removal process were evaluated using different phenol (10-500 mg.1(-1)) and H2O2 (1-15 Mm) concentrations. In the presence of H2O2, Roots were able to remove phenol concentrations up to 500 mg.1(-1). in the wide range of pH (4-9), reaching high removal efficiency. When roots were re-used for five consecutive cycles, phenol removal efficiency decreased from 98-62%, in the last cycle. After the removal process, the solutions were obtained from the experiment were estimated for their toxicity using a test with Lactaca sativa L. seeds. Results showed that the treated solution was less toxic than the parent solution.

Increasing Scopolamine content in Hairy Roots of Atropa belladonna using Bioreactor

The aim of this study was to use the, hairy root system for increasing the scopolamine content in Atropa belladonna. Agrobacterium rhizogenes ATCC15834 was utilized to produce hairy roots. The culture was carried out in a 1.5-l bioreactor using the inoculum size of 0.5 g fr. wt of 10-day-old hairy roots and various parameters, including agitation, aeration, conductivity and the consumption of sucrose were evaluated. Results revealed that the highest amount of scopolamine production (1.59 mg/g-1 dry wt) occurred in the bioreactor with aeration and agitation 1.25 vvm (volume per volume per minute) and 70 rpm, respectively. Study of conductivity and the consumption of sucrose showed that the highest amount of sucrose consumption and the highest amount of minerals consumption also was at 1.25 vvm and 70 rpm. Transgenic hairy root lines were confirmed by polymerase chain reaction (PCR).

The effect of drought stress on the expression of key genes involved in the biosynthesis of triterpenoid saponins in liquorice (Glycyrrhiza glabra).

Glycyrrhiza glabra is an important medicinal plant throughout the world. Glycyrrhizin is a triterpenoid that is among the most important secondary metabolites produced by liquorice. Drought stress is proposed to enhance the levels of secondary metabolites. In this study, the effect of drought stress on the expression of important genes involved in the glycyrrhizin biosynthetic pathway was examined. Drought stress at the seedling stage was applied to 8-day-old plants using polyethylene glycol. Subsequently, the samples were collected 0, 4, 8 or 24 h post-treatment. At the adult plant stage, 10-month-old plants were subjected to drought stress by discontinuing irrigation. Subsequently, samples were collected at 2, 16 and 28 days after drought imposition (S(2d), S(16d) and S(28d), respectively). We performed semi-quantitative RT-PCR assays to evaluate the gene expression levels of sequalene synthase (SQS), ß-amyrin synthase (bAS), lupeol synthase (LUS) and cycloartenol synthase (CAS) during stress. Finally, the glycyrrhizin content of stolons was determined via HPLC. The results revealed that due to osmotic stress, the gene expression levels of SQS and bAS were increased, whereas those of CAS were relatively unchanged at the seedling stage. At the adult plant stage, the expression levels of SQS and bAS were increased under drought stress conditions, whereas the gene expression level of CAS remained relatively constant. The glycyrrhizin content in stolons was increased only under severe drought stress conditions (S(28d)). Our results indicate that application of controlled drought stress up-regulates the expression of key genes involved in the biosynthesis of triterpenoid saponins and directly enhances the production of secondary metabolites, including glycyrrhizin, in liquorice plants.

Phytochemical composition and in vitro antimicrobial and antioxidant activities of some medicinal plants.

Different parts of three plants (Primula auriculata, Fumaria vaillantii and Falcaria vulgaris) were extracted with three different solvents to yield 72 crude extracts. The phytochemical analysis (chemical screening, GC-MS) of three plants was investigated for their antioxidant and antibacterial activity using nine Gram-positive and Gram-negative bacteria. The principal antioxidant and antimicrobial components were determined using HPLC with UV detection. All extracts possessed antibacterial activity especially methanolic extracts from flowers of P. auriculata. The DPPH-radical scavenging assay exhibited high antioxidant activities in three plants (more than 80% at 50µg). The F. vulgaris showed high content of carvacrol (29.8%) as main component. The contents of carvacrol and fumaric acid in the methanolic-water extracts were 1119 and 1966mg/l respectively. Our results indicate that these plants would be able to promise sources of natural products with potential antibacterial and antioxidant activity.