Studies on the Accumulation of Secondary Metabolites and Evaluation of Biological Activity of In Vitro Cultures of Ruta montana L. in Temporary Immersion Bioreactors.

The present work focuses on in vitro cultures of Ruta montana L. in temporary immersion PlantformTM bioreactors. The main aim of the study was to evaluate the effects of cultivation time (5 and 6 weeks) and different concentrations (0.1-1.0 mg/L) of plant growth and development regulators (NAA and BAP) on the increase in biomass and the accumulation of secondary metabolites. Consequently, the antioxidant, antibacterial, and antibiofilm potentials of methanol extracts obtained from the in vitro-cultured biomass of R. montana were evaluated. High-performance liquid chromatography analysis was performed to characterize furanocoumarins, furoquinoline alkaloids, phenolic acids, and catechins. The major secondary metabolites in R. montana cultures were coumarins (maximum total content of 1824.3 mg/100 g DM), and the dominant compounds among them were xanthotoxin and bergapten. The maximum content of alkaloids was 561.7 mg/100 g DM. Concerning the antioxidant activity, the extract obtained from the biomass grown on the 0.1/0.1 LS medium variant, with an IC50 0.90 ± 0.03 mg/mL, showed the best chelating ability among the extracts, while the 0.1/0.1 and 0.5/1.0 LS media variants showed the best antibacterial (MIC range 125-500 µg/mL) and antibiofilm activity against resistant Staphylococcus aureus strains.

QM/MM Modeling of the Flavin Functionalization in the RutA Monooxygenase.

Oxygenase activity of the flavin-dependent enzyme RutA is commonly associated with the formation of flavin-oxygen adducts in the enzyme active site. We report the results of quantum mechanics/molecular mechanics (QM/MM) modeling of possible reaction pathways initiated by various triplet state complexes of the molecular oxygen with the reduced flavin mononucleotide (FMN) formed in the protein cavities. According to the calculation results, these triplet-state flavin-oxygen complexes can be located at both re-side and si-side of the isoalloxazine ring of flavin. In both cases, the dioxygen moiety is activated by electron transfer from FMN, stimulating the attack of the arising reactive oxygen species at the C4a, N5, C6, and C8 positions in the isoalloxazine ring after the switch to the singlet state potential energy surface. The reaction pathways lead to the C(4a)-peroxide, N(5)-oxide, or C(6)-hydroperoxide covalent adducts or directly to the oxidized flavin, depending on the initial position of the oxygen molecule in the protein cavities.

The role of iron nanoparticles on morpho-physiological traits and genes expression (IRT1 and CAT) in rue (Ruta graveolens).

The iron nanoparticles with different physic-chemical properties induce inconsistent effects on various studied plant species. Thus, the effect of ferric oxide (Fe2O3) nanoparticles was compared with Fe2O3 microparticles and FeSO4complexes of EDTA for major physiological and gene expression in Rue (Ruta graveolens). Iron root content increased as Fe-MPs + EDTA ˂˂ Fe-NPs + EDTA˂ FeSO4 + EDTA. The shoot's iron remained unchanged or slightly increased under most of FeSO4 and Fe-MPs + EDTA treatments. Under Fe-NPs + EDTA treatment, 50 and 250 µM concentration decreased on shoot iron by 23.2% and 19.4% compared to control, respectively. But the shoot iron at 500 µM NPs was 28.2% higher than that of the control. A 46-58 fold lower Fe translocation was observed under Fe-NPs + EDTA than Fe-MPs + EDTA. The effect of Fe-NPs + EDTA was more significant on plant fresh and dry mass than the control. All treatments showed an increase in anthocyanin by 19-84% in leaves compared to the control. The Fe-NPs + EDTA and MPs + EDTA induced similar effects on enhanced growth parameters, total chlorophyll, catalase enzyme activity, gene, and reduced chlorophyll a/b and oxidants. Catalase enzyme activity in FeSO4 and MPs + EDTA was similar, and in Fe-NPs + EDTA treatments were influenced by coarse and fine regulation mechanisms, respectively. Iron MPs + EDTA had a more negative effect on IRT1 relative gene expression in roots as compared to other iron forms. The IRT1 relative gene expression in shoots was positively affected by 31-81% under all treatment types (except control and 250 µM Fe-NPs + EDTA, and 250 µM MPs + EDTA). These results could reveal the potential mechanism of plant response to nanoparticles.

Enhanced Accumulation of Biologically Active Coumarin and Furanocoumarins in Callus Culture and Field-grown Plants of Ruta chalepensis Through LED Light-treatment.

Ruta chalepensis, a medicinal plant, produces biologically active coumarins (CRs) and furanocoumarins (FCRs). However, their yield is quite low in cultivated plants. In this work, the influence of light-emitting diodes (LEDs) was investigated on the accumulation of CRs and FCRs in the callus cultures and field-grown plants of R. chalepensis. Among the various tested wavelengths of LED lights, maximum accumulation of CR and FCRs was recorded under blue LED treatment in both the callus cultures as well as field-grown plants when compared with respective controls treated with white LED. Metabolite analyses of LED-treated field-grown plants showed that highest concentrations of CR (umbelliferone, 2.8-fold), and FCRs (psoralen, 2.3-fold; xanthotoxin, 3.8-fold and bergapten, 1.16-fold) were accumulated upon blue LED-treatment for 6 days. CR and FCRs contents were also analyzed in the blue LED- and red LED-treated in vitro callus tissue. Upon blue LED-treatment, callus accumulated significantly high levels of umbelliferone (48.6 ± 1.2 µg g-1 DW), psoralen (370.12 ± 10.6 µg g-1 DW) and xanthotoxin (10.16 ± 0.48 µg g-1 DW). These findings imply that blue LED-treatment is a viable option as a noninvasive and low-cost elicitation technology for the enhanced production of biologically active CR and FCRs in field-grown plants and callus cultures of R. chalepensis.

Furanocoumarins from Ruta chalepensis with Amebicide Activity.

Entamoeba histolytica (protozoan; family Endomoebidae) is the cause of amoebiasis, a disease related to high morbidity and mortality. Nowadays, this illness is considered a significant public health issue in developing countries. In addition, parasite resistance to conventional medicinal treatment has increased in recent years. Traditional medicine around the world represents a valuable source of alternative treatment for many parasite diseases. In a previous paper, we communicated about the antiprotozoal activity in vitro of the methanolic (MeOH) extract of Ruta chalepensis (Rutaceae) against E. histolytica. The plant is extensively employed in Mexican traditional medicine. The following workup of the MeOH extract of R. chalepensis afforded the furocoumarins rutamarin (1) and chalepin (2), which showed high antiprotozoal activity on Entamoeba histolytica trophozoites employing in vitro tests (IC50 values of 6.52 and 28.95 µg/mL, respectively). Therefore, we offer a full scientific report about the bioguided isolation and the amebicide activity of chalepin and rutamarin.

Colletotrichum gloesporioides inhibition using chitosan-Ruta graveolens L essential oil coatings: Studies in vitro and in situ on Carica papaya fruit.

In this study we assessed the efficacy of chitosan (CHI) (2%) emulsion added with Ruta graveolens L. essential oil (REO) at different concentrations (0.5%, 1.0% and 1.5%) to control C. gloesporioides grows both "in situ" and "in vitro" in papaya Maradol (Carica papaya L.). In vitro studies showed a decrease on fungal growth (mycelia diameter) with the increase of REO concentration, while 0.5% of REO induce a reduction of 56.42%, REO at 1.0% and 1.5% induced a reduction of 97%. Microscopic analysis showed irreversible deleterious morphological and ultrastructural alterations as well as changes in conidia morphology, and conidia germination inhibition up to 90%. Among the most abundant REO constituents, 2-Nonanol showed strong antifungal activity followed by 2-Undecanone, Benzyl acetate, 2-Nonanone, 2-Tridecanone and 2-Dodecanone. Studies "in situ" on papaya fruit during 12 days at 20 °C, showed a reduction of the C. gloesporioides lesion expansion by 50% using CHI-REO 0.5% emulsions and by 100% with treatments of CHI-REO 1.0 and 1.5%, in addition the emulsions were efficacious to reduce the fruit surface microbiota. On the other hand, physicochemical analysis of the papaya fruits demonstrated that CHI-REO emulsions treatment delayed papaya ripening without affecting the organoleptic characteristics. All these results demonstrated for the first time the application of coatings CHI-REO as a postharvest treatment for the control of anthracnose on papaya fruit.

Ruta graveolens Extracts and Metabolites against Spodoptera frugiperda.

The biological activity of Ruta graveolens leaf tissue extracts obtained with different solvents (ethyl acetate, ethanol, and water) and metabolites (psoralen, 2- undecanone and rutin) against Spodoptera frugiperda was evaluated. Metabolites levels in extracts were quantified by HPLC and GC. Ethyl acetate and ethanol extracts showed 94% and 78% mortality, respectively. Additionally, psoralen metabolite showed a high mortality as cypermethrin. Metabolite quantification in extracts shows the presence of 2-undecanone (87.9 µmoles mg(-1) DW), psoralen (3.6 µmoles mg(-1) DW) and rutin (0.001 pmoles mg(-1) DW). We suggest that these concentrations of 2-undecanone and psoralen in R. graveolens leaf tissue extracts could be responsible for S. frugiperda mortality.

Effect of polyamines on shoot multiplication and furanocoumarin production in Ruta graveolens cultures.

The influence of the polyamines putrescine (Put), spermine (Spr) and spermidine (Spd) on growth and furanocoumarin production was investigated by exogenous addition, at different concentrations, to shoot cultures of Ruta graveolens at different phases of growth. Preliminary studies indicated that addition of Put (20 microM) and Spr (80 microM) had a promotive effect on shoot multiplication rate and number of multiple shoots formed. Spd was toxic, even at lower concentrations. The growth-phase of the culture at the time of exogenous addition of polyamines was found to be an important factor. Put was most effective when added at the lag phase, while Spr was most effective when added in the log phase. Time course studies of growth and furanocoumarin content were carried out for each polyamine and phase of addition. It was seen that maximum production of furanocoumarins (256.8 mg/10 g DW) occurred in the second week when Put was added in the lag phase and 260.5 mg/10 g DW in the fourth week when Spr was added in the log phase. Put addition resulted in a 3.10 fold increase in psoralen, 6.12 in xanthotoxin and 1.46 fold in bergapten production. Spr addition resulted in a 1.31 fold increase in psoralen, 4.11 fold in xanthotoxin and 1.49 fold in bergapten production. Results indicate that alteration of growth and furanocoumarin production kinetics is a combined outcome of choice of polyamine and the phase of culture at the time of exogenous addition. Polyamine addition enabled significant enhancement in production of pharmaceutically important bergapten and xanthotoxin in shoot cultures of Ruta graveolens, which could be explored for commercial production.

Bioprocess optimization of furanocoumarin elicitation by medium renewal and re-elicitation: a perfusion-based approach.

Effect of various abiotic (methyl jasmonate, salicylic acid) and biotic (yeast extract, Aspergillus niger) elicitors on furanocoumarin production and in situ product removal was studied using shoot cultures of Ruta graveolens L. Elicitation by yeast extract (1% w/v) on day 15 was most effective. It led to 7.8-fold higher furanocoumarin production that was attained 24 h after elicitation and 43% of the product was released into the medium. Changes in the relative concentration of furanocoumarins produced depend on the elicitor used. Molar ratio of bergapten increased to 93% in response to yeast extract. With the perspective of developing a commercially feasible process, an approach for preserving viability of biomass and its reuse needs to be developed. For this, medium renewal strategy was investigated. Removal of the spent medium 48 h after elicitation allowed in situ product removal and proved effective in revival of cultures, allowing reuse of biomass. A week after medium renewal, the revived biomass was re-elicited and a second furanocoumarin production peak was obtained. A perfusion-based bioprocess optimization approach, employing elicitation coupled with medium renewal with subsequent re-elicitation, as a new strategy for improved furanocoumarin production, has been suggested.

Application of chitin and chitosan as elicitors of coumarins and fluoroquinolone alkaloids in Ruta graveolens L. (common rue).

Common rue (Ruta graveolens L.) accumulates various types of secondary metabolites, such as coumarins furanocoumarins, acridone and quinolone alkaloids and flavonoids. Elicitation is a tool extensively used for enhancing secondary-metabolite yields. Chitin and chitosan are examples of elicitors inducing phytoalexin accumulation in plant tissue. The present paper describes the application of chitin and chitosan as potential elicitors of secondary-metabolite accumulation in R. graveolens shoots cultivated in vitro. The simple coumarins, linear furanocoumarins, dihydrofuranocoumarins and fluoroquinolone alkaloids biosynthesized in the presence of chitin and chitosan were isolated, separated and identified. There was a significant increase in the growth rate of R. graveolens shoots in the presence of either chitin or chitosan. Moreover, the results of the elicitation of coumarins and alkaloids accumulated by R. graveolens shoots in the presence of chitin and chitosan show that both compounds induced a significant increase in the concentrations of nearly all the metabolites. Adding 0.01% chitin caused the increase in the quantity (microg/g dry weight) of coumarins (pinnarin up to 116.7, rutacultin up to 287.0, bergapten up to 904.3, isopimpinelin up to 490.0, psoralen up to 522.2, xanhotoxin up to 1531.5 and rutamarin up to 133.7). The higher concentration of chitosan (0.1%) induced production of simple coumarins (pinnarin up to 116.7 and rutacultin up to 287.0), furanocoumarins (bergapten up to 904.3, isopimpinelin up to 490.0, psoralen up to 522.2, xanhotoxin up to 1531.5) and dihydrofuranocoumarins (chalepin up to 18 and rutamarin up to 133.7). Such a dramatic increase in the production of nearly all metabolites suggests that these compounds may be participating in the natural resistance mechanisms of R. graveolens. The application of chitin- and chitosan-containing media may be considered a promising prospect in the biotechnological production of xanthotoxin, isopimpinelin, psoralen, chalepin or methoxylated dictamnine derivatives.

Identification of Ruta graveolens L. metabolites accumulated in the presence of abiotic elicitors.

The study aimed to elucidate the effects of benzothiadiazole (BTH) and saccharin on the biosynthesis of simple coumarins, linear furanocoumarins, dihydrofuranocoumarins, and furoquinolone alkaloids in shoots of R. graveolens cultivated in vitro. The biosynthesized metabolites were analyzed and identified by GC-MS and by comparison of Kovats indices. Eight coumarin metabolites were identified: bergapten, chalepin, isopimpinelin, pinnarin, psoralen, rutacultin, rutamarin, and xanthotoxin, and also four alkaloids: dictamnine, gamma-fagarine, skimmianine, and kokusaginine. Each of the tested BTH concentrations induced a significant production of furanocoumarins and furoquinolone alkaloids. The use of saccharin also increased the production of bergapten, isopimpinelin, pinnarin, psoralen, and xanthotoxin several times.

Effective biotic elicitation of Ruta graveolens L. shoot cultures by lysates from Pectobacterium atrosepticum and Bacillus sp.

Growth of Ruta graveolens shoots was induced when Bacillus sp. cell lysates were added to the culture medium. Elicitation of coumarin by this lysate was also very effective; the concentrations of isopimpinelin, xanthotoxin and bergapten increased to 610, 2120 and 1460 microg g(-1) dry wt, respectively. It also had a significant effect on the production of psoralen and rutamarin (680 and 380 microg g(-1) dry wt) and induced the biosynthesis of chalepin, which was not detected in the control sample, up to 47 microg g(-1) dry wt With lysates of the Pectobacterium atrosepticum, their effect on growth was not so significant and had no effect on the induction of coumarin accumulation. But elicitation with this lysate was much more effective for inducing the production of furoquinolone alkaloids; the concentrations of gamma-fagarine, skimmianine, dictamnine and kokusaginine rose to 99, 680, 172 and 480 microg g(-1) dry wt, respectively.

Accumulation of biologically active furanocoumarins in Ruta graveolens ssp. divaricata (Tenore) Gams in vitro culture.

The study was designed to investigate dynamics of accumulation of five linear furanocoumarins and umbelliferone in stationary liquid cultures of Ruta graveolens ssp. divaricata (Tenore) Gams during 6-week growth cycles. The contents of individual metabolites in biomass increased 1.8-3.5 times while their total content rose 2.3 times. Maximum contents of xanthotoxin, bergapten and isopimpinellin (112.3, 76.2 and 84.0mg/100g d.w., respectively) and maximum total content of all metabolites (283.4 mg/100 g d.w.), obtained on 35th culture day, are interesting from practical point of view.

Specific accumulation and revised structures of acridone alkaloid glucosides in the tips of transformed roots of Ruta graveolens.

The root tips of Ruta graveolens (common rue) show strong autofluorescence of acridone alkaloids, which are characteristic secondary metabolites of this plant. To study the specific distribution and accumulation of acridone alkaloids in various root segments of Ruta graveolens, root material was harvested from genetically transformed root cultures and extracts were investigated by chromatographic techniques and HPLC-(1)H NMR spectroscopy. The cells of the elongation and differentiation zones contained acridone glucosides and large amounts of acridone alkaloids, mainly rutacridone. Gravacridondiol glucoside was identified as the dominant secondary compound of the root tips and its structure revised by means of spectroscopic methods. In addition, minor acridones, including the structurally revised gravacridontriol glucoside and unknown natural products, were found in the root tip.

Cinnamic acid 4-hydroxylase mechanism-based inactivation by psoralen derivatives: cloning and characterization of a C4H from a psoralen producing plant-Ruta graveolens-exhibiting low sensitivity to psoralen inactivation.

Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) complete cDNA was cloned from the leaves of Ruta graveolens, a psoralen producing plant. The recombinant enzyme (classified CYP73A32) was expressed in Saccharomyces cerevisiae. Mechanism-based inactivation was investigated using various psoralen derivatives. Only psoralen and 8-methoxypsoralen were found to inactivate C4H. The inactivation was dependent on the presence of NADPH, time of pre-incubation, and inhibitor concentration. Inactivation stoichiometry was 0.9 (+/-0.2) for CYP73A1 and 1.1 (+/-0.2) for CYP73A32. SDS-PAGE analysis demonstrated that [3H]psoralen was irreversibly bound to the C4H apoprotein. K(i) and k(inact) for psoralen and 8-methoxypsoralen inactivation on the two C4H revealed a lower sensitivity for CYP73A32 compared to CYP73A1. Inactivation kinetics were also determined for CYP73A10, a C4H from another furocoumarin-producing plant, Petroselinum crispum. This enzyme was found to behave like CYP73A32, with a weak sensitivity to psoralen and 8-MOP inactivation. Cinnamic acid hydroxylation is a key step in the biosynthesis of phenylpropanoid compounds, psoralen derivatives included. Our results suggest a possible evolution of R. graveolens and P. crispum C4H that might tolerate substantial levels of psoralen derivatives in the cytoplasmic compartment without a depletive effect on C4H and the general phenylpropanoid metabolism.

Ruta 6 selectively induces cell death in brain cancer cells but proliferation in normal peripheral blood lymphocytes: A novel treatment for human brain cancer.

Although conventional chemotherapies are used to treat patients with malignancies, damage to normal cells is problematic. Blood-forming bone marrow cells are the most adversely affected. It is therefore necessary to find alternative agents that can kill cancer cells but have minimal effects on normal cells. We investigated the brain cancer cell-killing activity of a homeopathic medicine, Ruta, isolated from a plant, Ruta graveolens. We treated human brain cancer and HL-60 leukemia cells, normal B-lymphoid cells, and murine melanoma cells in vitro with different concentrations of Ruta in combination with Ca3(PO4)2. Fifteen patients diagnosed with intracranial tumors were treated with Ruta 6 and Ca3(PO4)2. Of these 15 patients, 6 of the 7 glioma patients showed complete regression of tumors. Normal human blood lymphocytes, B-lymphoid cells, and brain cancer cells treated with Ruta in vitro were examined for telomere dynamics, mitotic catastrophe, and apoptosis to understand the possible mechanism of cell-killing, using conventional and molecular cytogenetic techniques. Both in vivo and in vitro results showed induction of survival-signaling pathways in normal lymphocytes and induction of death-signaling pathways in brain cancer cells. Cancer cell death was initiated by telomere erosion and completed through mitotic catastrophe events. We propose that Ruta in combination with Ca3(PO4)2 could be used for effective treatment of brain cancers, particularly glioma.