Anti-MRSA Constituents from Ruta chalepensis (Rutaceae) Grown in Iraq, and In Silico Studies on Two of Most Active Compounds, Chalepensin and 6-Hydroxy-rutin 3',7-Dimethyl ether.

Ruta chalepensis L. (Rutaceae), a perennial herb with wild and cultivated habitats, is well known for its traditional uses as an anti-inflammatory, analgesic, antipyretic agent, and in the treatment of rheumatism, nerve diseases, neuralgia, dropsy, convulsions and mental disorders. The antimicrobial activities of the crude extracts from the fruits, leaves, stem and roots of R. chalepensis were initially evaluated against two Gram-positive and two Gram-negative bacterial strains and a strain of the fungus Candida albicans. Phytochemical investigation afforded 19 compounds, including alkaloids, coumarins, flavonoid glycosides, a cinnamic acid derivative and a long-chain alkane. These compounds were tested against a panel of methicillin-resistant Staphylococcus aureus (MRSA) strains, i.e., ATCC 25923, SA-1199B, XU212, MRSA-274819 and EMRSA-15. The MIC values of the active compounds, chalepin (9), chalepensin (10), rutamarin (11), rutin 3'-methyl ether (14), rutin 7,4'-dimethyl ether (15), 6-hydroxy-rutin 3',7-dimethyl ether (16) and arborinine (18) were in the range of 32-128 µg/mL against the tested MRSA strains. Compounds 10 and 16 were the most active compounds from R. chalepensis, and were active against four out of six tested MRSA strains, and in silico studies were performed on these compounds. The anti-MRSA activity of compound 16 was comparable to that of the positive control norfloxacin (MICs 32 vs 16 µg/mL, respectively) against the MRSA strain XU212, which is a Kuwaiti hospital isolate that possesses the TetK tetracycline efflux pump. This is the first report on the anti-MRSA property of compounds isolated from R. chalepensis and relevant in silico studies on the most active compounds.

Chemical Composition, Antibacterial and Phytotoxic Activities of Peganum harmala Seed Essential Oils from Five Different Localities in Northern Africa.

Peganum harmala L., also known as Syrian rue or Pègano, is a herbaceous plant belonging to the Zygohpyllaceae family, and is widely used in traditional medicine. The chemical composition of essential oils of P. harmala seeds from five different regions of Northern Africa (Algeria, Egypt, Libya, Morocco and Tunisia) was studied by GC and GC-MS analyses. A total of 105 compounds were identified, the main components being oxygenated monoterpenes and oxygenated sesquiterpenes. Eugenol is the main component in all oils. The antimicrobial activity of the essential oils was assayed against some bacterial strains: Staphylococcus aureus (DSM 25693), Bacillus cereus (DSM 4313), Bacillus cereus (DSM4384), Escherichia coli (DMS 857) and Pseudomonas aeruginosa (ATCC 50071). All the oils showed different inhibitory activity. In the twentieth century this is an important result; we need possible new botanical drugs because the problem of resistance to antimicrobial drugs has become apparent. Moreover, the essential oils were evaluated for their possible in vitro phytotoxic activity against germination and initial radicle growth of Raphanus sativus L., Lepidium sativum L., and Ruta graveolens L. The results showed that both germination and radical elongation were sensitive to the oils.

Chemical composition and antimicrobial activity of the essential oils from four Ruta species growing in Algeria.

Antimicrobial properties of plants essential oils have been investigated in order to suggest them as potential tools to overcome the microbial drug resistance and the increasing incidence of food borne diseases problems. The aim of this research is to study the antibacterial and antifungal effects of four traditional plants essential oils, Ruta angustifolia, Ruta chalepensis, Ruta graveolens and Ruta tuberculata, against standard bacterial and fungal strains. The chemical compounds of the oils were examined by GC/MS. Results revealed a powerful antifungal activity against filamentous fungi. Aspergillus fumigatus and Cladosporium herbarum are the most sensitive strains to these oils with MIC values less than 3.5 µg ml(-1) for certain oils, reaching 7.8 µg ml(-1) for other. GC/MS essay exhibited ketones as the most abundant constituent of these oils except for R. tuberculata essential oil which has a completely different composition, monoterpenes alcohols being the most abundant. These compositions explain their potential antifungal activity.

Floral ontogeny of Ruteae (Rutaceae) and its systematic implications.

Floral development was investigated in Ruta graveolens and Psilopeganum sinense, representing two genera in the tribe Ruteae. Special attention was paid to the sequence of initiation of organ whorls in the androecium and gynoecium. The antepetalous stamens arise at the same level as the antesepalous stamens in both species. The carpels are antepetalous in both taxa, indicating the androecium in both genera is obdiplostemonous. Compared with floral ontogeny of the ancestral genus Phellodendron (Toddalioideae), the obdiplostemonous androecium is a derived condition. The floral apex in P. sinense is quadrangular before initiation of the two carpels. Additionally, there are four dorsal and four ventral traces in the ovary. Integrated morphological and anatomical evidence indicates that the bicarpellate gynoecium in Psilopeganum most likely evolved from a tetracarpellate ancestor. Considering the similarities in morphological, geographical and chromosomal features, the ancestor may be Ruta-like. Further molecular phylogenetic and genetic studies are needed to verify this assumption.

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.

Agrobacterium-mediated transformation of Ruta graveolens L.

Agrobacterium tumefaciens is used to develop a genetic transformation method for a medicinal plant Ruta graveolens. The direct plant regeneration strategy is preferred to callus line establishment. In vitro seedlings, 2- -to 3-wk-old, are used to excise hypocotyls and co-cultivated for 3 d with A. tumefaciens strain C58C1Rif containing plasmid pTDE4 harbouring neomycin phosphotransferase (npt II, kanamycin resistance) and beta-glucuronidase encoding genes. The Southern blot analysis has shown that 78% kanamycin resistant plants contain gene encoding beta-glucuronidase. The GUS histochemical assay shows that 67% transgenic plants exhibit the corresponding enzymatic activity. Routine transformation efficiency of R. graveolens L. is 11% and could reach up to 22%. Transgenic plants are grown in the greenhouse within 4 months after the initial seedlings.

Novel technique for scaling up of micropropagated Ruta graveolens shoots using liquid culture systems: a step towards commercialization.

Wide applications of Ruta graveolens L. in pharmaceutical industry has led to increased interest in large-scale plant production, with emphasis on use of in vitro cultures. Earlier reports describe use of in vitro germinated seedlings for raising shoot cultures and not regeneration. There is only a single regeneration protocol of R. graveolens; however, it employs conventional labour intensive techniques deterring automation. The aim of present investigation was to establish a cost effective protocol for large-scale plant production. We report for the first time a one-step protocol with improved regeneration efficiency for multiple shoots induction employing liquid culture systems. Effect of polyamines (putrescine and spermine) on growth and furanocoumarin was studied. Addition of spermine enhanced the number of multiple shoots formed (2.5-fold) and reduced the time taken by half. Spermine addition resulted in 1.47-fold in furanocoumarin production. The selected shoot line, RS2 was successfully scaled up to 5L in culture vessels, with 1.53-fold increase in biomass without affecting the productivity of these cultures. This proves to be a commercially feasible alternative to bioreactors for large-scale biomass and furanocoumarin production.

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 agitated cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams.

This study was designed to investigate the dynamics of accumulation of linear furanocoumarins (psoralen, bergapten, xanthotoxin, isopimpinellin, imperatorin) and their biogenetic precursor, umbelliferone, in agitated cultures of Ruta graveolens L. and Ruta graveolens ssp. divaricata (Tenore) Gams during 6-week growth cycles. The metabolites under study were almost exclusively accumulated in the cultured biomass. The total content of all metabolites increased 4.8- and 2.0-fold, in R. graveolens and R. graveolens ssp. divaricata cultures, respectively. Xanthotoxin and bergapten, which are the most important therapeutic compounds, were the dominating metabolites in cultures of both plants. The maximum content of xanthotoxin (25.0 mg/100 g dry wt.) and bergapten (18.4 mg/100 g dry wt) and the maximum content of all metabolites (64.0 mg/100 g dry wt) in R. graveolens ssp. divaricata callus obtained on the 35th culture day were relatively low. However, maximum contents of xanthotoxin (136.8 mg/100 g dry wt), bergapten (210.4 mg/100 g dry wt.) and isopimpinellin (96.7 mg/100 g dry wt), and total content of all metabolites in R. graveolens shoots (520.8 mg/100 g dry wt) obtained on the 42nd culture day are interesting from a 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.