Accumulation of furanocoumarins in Ruta graveolens L. shoot culture

Ruta graveolens L. shoots cultured in stationary liquid phase produced furanocoumarins: psoralen, bergapten, xanthotoxin, isopimpinellin and imperatorin at the amount totalling almost 1 g/100 g dry wt of the shoots. The dominating metabolites were therapeutically important compounds: xanthotoxin – 0.33 g/100 g dry wt and bergapten – 0.32 g/100 g dry wt. Maximum contents of the majority of the compounds were observed on 28th day of culture.     

Enhanced in vitro production of Ruta graveolens L. coumarins and rutin by mannitol and ventilation

Ruta graveolens shoot cultures were maintained on static medium supplemented with 0, 1, 2 and 3?% mannitol. The cultures were grown in vessels that ensured a ventilation rate of 7.44, 10.82 or 62.83 air exchanges per day (V1, V2 or V3, respectively). The growth index and fresh weight were significantly increased at 1?% mannitol and decreased with increasing mannitol concentrations, whereas the dry weight (DW) and DW?% increased at higher concentrations of mannitol. Improving the culture ventilation significantly increased all of these parameters. A higher concentration of mannitol resulted in a higher proline content and percentage of coumarins and rutin, but the final accumulation of these bioactive molecules decreased. The coumarins, calculated as xanthotoxin, were increased from 8.15 to 13.60?mg?g^?1 DW using (V1 and mannitol-free medium) and (V2 with medium enriched with 2?% mannitol), respectively. Rutin was linearly increased by raising the mannitol concentrations, achieving the highest content of 54.87?mg?g^?1 DW using V2 and medium supplemented with 3?% mannitol. The lowest accumulation of coumarins and rutin (32, 144?mg vessel^?1, respectively) were found on mannitol-free medium using V1, whereas the highest rutin contents were found on medium with 1?% mannitol using V3. A GC analysis revealed the presence of five main compounds in all of the cultures, coumarin, 7-hydroxucoumarin, scopoletin, xanthotoxin and bergapten, whereas pasoralen was not detected when the cultures were maintained on mannitol-free medium, regardless of the type of vessel. Moreover, the concentrations of these compounds varied according to the mannitol concentration and ventilation.     

Improved furanocoumarin production in Ruta graveolens L. regenerated via in vitro stem internode cultures

A rapid and efficient in vitro propagation protocol by enhanced multiple shoot proliferation from internode cultures of Ruta graveolens was established. Mean shoot number was maximum (55.83) in Murashige and Skoog (MS) basal medium fortified with 1.0 mg L^?1 benzyl amino purine and 0.25 mg L^?1 indole-3-acetic acid. The elongated shoots rooted within 10–12 days in 1/2-strength MS medium supplemented with 2.0 mg L^?1 indole 3-butyric acid. About 80 % of the rooted plantlets survived acclimatization and transfer to the field. Phytochemical analysis revealed that micropropagated plants produced linear furanocoumarins, characteristic of the species, in greater quantities as compared to the in vivo-grown plants. The results will facilitate the conservation of this valuable medicinal plant and to obtain plants with improved phytochemical constituents.     

Furanocoumarins: Novel topoisomerase I inhibitors from Ruta graveolens L.

Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA–topoisomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxation of plasmid in enzyme–substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme–extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA–enzyme complex was observed after enzyme–extract preincubation. The activity could be assigned to furanocoumarins—psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors.     

Production of biologically active phenolic acids in Aronia melanocarpa (Michx.) Elliott in vitro cultures cultivated on different variants of the Murashige and Skoog medium

Phenolic acids, both benzoic and cinnamic acid derivatives, are plant metabolites with high therapeutic and cosmetic values. Methanolic extracts from the biomass of shoot and callus cultures of Aronia melanocarpa growing on seven variants of the Murashige and Skoog (MS) medium with different concentrations of plant growth regulators, BA and NAA, ranging from 0.1 to 3.0 mg l^?1, were examined for the production of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. The extracts from the shoot and callus cultures were confirmed to contain five of the twelve compounds tested for: caffeic, p-coumaric, p-hydroxybenzoic, syringic and vanillic acids. The shoot extracts contained additionally salicylic acid. Both the total amounts and the amounts of individual compounds in either the shoot or callus extracts were dependent on the concentration of cytokinin and auxin in the MS medium variants. The total amounts in the shoot and callus cultures were in the range from 93.52 to 217.00 mg 100 g^?1 DW and from 47.11 to 83.83 mg 100 g^?1 DW, respectively. The amounts of individual compounds showed wide variation, from 1.31 to 91.86 mg 100 g^?1 DW in the shoot extracts, and from 2.58 to 40.16 mg 100 g^?1 DW in the callus extracts. Salicylic acid (max. 91.86 mg 100 g^?1 DW), p-coumaric acid (max. 62.39 mg 100 g^?1 DW) and p-hydroxybenzoic acid (max. 50.66 mg 100 g^?1 DW) dominated in the shoot extracts, while syringic acid (max. 40.16 mg 100 g^?1 DW) and p-hydroxybenzoic acid (max. 23.59 mg 100 g^?1 DW) were the main metabolites in the callus extracts. This is the first report on the quantitative analysis of benzoic and cinnamic acid derivatives in shoot and callus cultures of A. melanocarpa growing on MS-based media with different concentrations of selected plant growth regulators—BA and NAA. The obtained maximum amounts of some metabolites are of interest from a practical perspective.     

Influence of Acid and Salt Sprays on Furanocoumarin Concentrations on the Ruta graveolens Leaf Surface

Ruta graveotens subshrubs, known to contain a large fractionof their furanocoumarins on the leaf surface, were used forinvestigation of salt and acid rain effects on the plant surfaceand substances occurring there. Groups of shoots on each plantcovered by plastic bags were sprayed with either sulphuric acid,pII 2.5, saturated sodium chloride solution, or a distilledwater control. Uncovered shoots, an additional control, hadthe highest concentrations of furanocoumarins both on the surfaceand in the whole leaf. The upper leaves contained less furanocoumarinsin absolute amounts but were similar to the lower leaves ona percentage basis. Spraying caused a decrease in total furanocoumarinconcentrations, slight in the case of distilled water and greaterfor acid and salt, but the percentage on the surface after sprayingincreased. The upper and lower leaves reacted differently tosalt than to sulphuric acid and water: with salt, the percentagesof furanocoumarins on the surface of both kinds of leaves showedsimilar increases; with the other two sprays, upper leaves showeda similar increase, but the lower leaves had a much higher percentageon the surface owing to a high rate of extrusion. Simulated acid rain, salt sprays, plant surface, furanocoumarins, Ruta graveolens     

Accumulation of hydroxybenzoic acids and other biologically active phenolic acids in shoot and callus cultures of Aronia melanocarpa (Michx.) Elliott (black chokeberry)

Phenolic acids are plant metabolites important in phytotherapy and also in cosmetology. In this study, proliferating shoot and callus cultures of Aronia melanocarpa were established and maintained on Linsmaier and Skoog (L-S) medium containing different levels of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), ranging from 0.1 to 3.0 mg l^?1. Methanolic extracts from the biomass of these cultures and from the fruits of soil-grown plants were used to determine the amounts of free phenolic acids and cinnamic acid using the high-performance liquid chromatography (HPLC) method. Out of a total of twelve analyzed compounds, all of the extracts contained four of them: caffeic acid, p-hydroxybenzoic acid, syringic acid, and vanillic acid. Moreover, shoot extracts also contained salicylic acid (o-hydroxybenzoic acid), while callus extracts contained p-coumaric acid. On the other hand, fruit extracts also contained both salicylic acid and p-coumaric acid. The total amount of the analyzed compounds in extracts from both shoot and callus cultures depended on the L-S medium used, and varied between 103.05 and 150.95 mg 100 g^?1 dry weight (DW), and between 50.23 and 81.56 mg 100 g^?1 DW, respectively. Both types of culture contained higher levels of phenolic acids than the fruit extracts (32.43 mg 100 g^?1 DW). In shoot cultures, p-hydroxybenzoic acid and salicylic acid were the predominant metabolites (reaching 55.14 and 78.25 mg 100 g^?1 DW, respectively), while in callus cultures, p-hydroxybenzoic acid (25.60 mg 100 g^?1 DW) and syringic acid (41.20 mg 100 g^?1 DW) were the main compounds. In fruit extracts, salicylic acid (15.60 mg 100 g^?1 DW) and p-hydroxybenzoic acid (5.29 mg 100 g^?1 DW) were predominant.     

Seasonal variation of chemical profile of Ruta graveolens extracts and biological activity against Fusarium oxysporum,Fusarium proliferatum and Stemphylium vesicarium

Ruta graveolens L. is a plant of agricultural interest due to its biological activity against phytopathogenic microorganism. This activity has been attributed to the presence of alkaloids, furanocoumarins, terpenes and fatty acids. The accumulation of these compounds is affected by environmental factors. The aim of this work was to analyze the seasonal variation the chemical profile of rue and to evaluate the biological activity against phytopathogenic fungi. Extracts were obtained in January, April, June and September 2017, chemically characterized by GC-MS. The antifungal activity was determined by absorbance, four concentrations were evaluated: 4, 8, 16 and 32 mg/mL. In extracts from the aerial parts, fatty acids were the most abundant group in the January sample (29.7%), and alkaloids in the April and June (27.7%) samples. In extracts from the roots, alkaloids were the most abundant group in the four collections. The highest percentage of growth inhibition on Fusarium and Stemphylium was obtained with the extracts of the January sample. Ruta graveolens can be considered an alternative for the management of phytopathogenic fungi of crops of economic importance.     

Light and temperature conditions affect bioflavonoid accumulation in callus cultures of Cyclopia subternata Vogel (honeybush)

Callus cultures of the endemic South-African legume Cyclopia subternata were cultivated under varying light and temperature conditions to determine their influence on biomass growth and bioflavonoids accumulation. Experimental modifications of light included complete darkness, light of different spectral quality (white, red, blue and yellow) and ultraviolet C (UVC) irradiation. The calli were also subjected to elevated temperature or cold stress. Among the tested light regimes, cultivation under blue light resulted in the highest levels of hesperidin (H)—118.00 mg 100 g^?1 dry weight (DW) on 28 days of experiment, as well as isoflavones: 7-O-β-glucosides of calycosin (CG), pseudobaptigenin (PG) and formononetin (FG)—28.74, 19.26 and 10.32 mg 100 g^?1 DW, respectively, in 14-days old calli. UVC irradiation applied on 20 days stimulated the accumulation of H (204.14 mg 100 g^?1 DW), CG (31.84 mg 100 g^?1 DW) and PG (18.09 mg 100 g^?1 DW) in 28 days culture by 140, 46 and 165 %, respectively, without negatively influencing callus growth. Low temperature (13 °C) increased CG content by over 1,500 % (235.29 mg 100 g^?1 DW) when applied during the whole 28-days growth cycle, at the same time causing 95 % decrease in culture growth in comparison to reference calli maintained at 24 °C. On the contrary, elevated temperature (29 °C) applied during the second half of the culture period resulted in over 300 and 500 % increase in CG and PG content (61.76 and 58.89 mg 100 g^?1, respectively) while maintaining relatively high biomass yield.     

Hairy root cultures of Rehmannia glutinosa and production of iridoid and phenylethanoid glycosides

Hairy root lines through the infection of Agrobacterium rhizogenes strain (A4) were established from shoot tips and leaves of Rehmannia glutinosa Libosch. Ten lines of hairy roots were selected on the basis of biomass increase in half-strength Gamborg medium (¹/₂ B5). Transgenic status of the roots was confirmed by polymerase chain reaction using rolB and rolC specific primers. Iridoid glycosides (catalposide, loganin, aucubin and catalpol) and phenylethanoid glycosides (verbascoside and isoverbascoside) identified using HPLC?CESI?CMS, and their contents were compared with untransformed root culture and roots of 1-year-old field-grown plants of R. glutinosa by RP-HPLC. The growth and production of secondary metabolites in ten hairy root lines varied considerably as to the media. Woody plant (WP) medium displayed higher growth in terms of fresh (FW) and dry weights (DW) compared to ¹/₂ B5 medium. High-yielding hairy root lines produced higher amounts of loganin, catalposide, verbascoside and isoverbascoside in comparison to the untransformed root culture and roots of 1-year-old field-grown plants. The highest amounts of catalposide and loganin in transformed roots were 4.45?mg?g^?1 DW (RS-2 hairy root line) and 4.66?mg?g^?1 DW (RS-1 hairy root line), respectively. Aucubin and catalpol were detected in some lines in trace amounts. The highest amounts of verbascoside (16.9?mg?g^?1 DW) and isoverbascoside (3.46?mg?g^?1 DW) were achieved in RS-2 root line. The contents of catalposide, verbascoside and isoverbascoside in high-producing lines were several times higher than in untransformed root culture and roots of R. glutinosa plants grown in soil. Loganin and aucubin could not be detected in roots of field-grown plants. However, the levels of catalpol were much lower in the in vitro roots.     

Formation of edulinine and furoquinoline alkaloids from quinoline derivatives by cell suspension cultures of Ruta graveolens

The formation of furoquinoline alkaloids and of edulinine, elaborated by cell suspension cultures of Ruta graveolens, was found to occur by way of 4-hydroxy-2-quinolone. Other substrates transformed to furoquinolines included 4-hydroxy- and 4-methoxy-3-(3-methyl-2-butenyl)-2-quinolone, known earlier as natural precursors in studies with whole plants. Involvement of dictamnine as a natural precursor of 8-methoxydictamnine (γ-fagarine) and skimmianine was proved using ¹⁴C-labelled compounds. Edulinine in the cell suspensions was formed from such substrates as 4-hydroxy-N-methyl-2-quinolone, 4-hydroxy-3-(3-methyl-2-butenyl)-N-methyl-2-quinolone and its 4- methyl ether; this is probably the natural biosynthetic sequence. Changes in alkaloid yields were noted upon prolonged subculturing.     

Furanocoumarin biosynthesis in Ruta graveolens cell cultures

The biosynthetic routes to four linear furanocoumarins—psoralen, xanthotoxin, bergapten. isopimpinellin-co-occurring in Ruta graveolens cell cultures have been investigated with six ¹⁴C-labelled compounds. Mevalonic acid was only poorly incorporated, in contrast to umbelliferone. In support of previous suggestions, 7-demethylsuberosin and (±)-marmesin were very good precursors of the linear furanocoumarins. 7-O-Prenylumbelliferone also was fairly well utilized, but this was probably owing to a prior ether cleavage yielding umbelliferone. Psoralen was well incorporated into bergapten and xanthotoxin, but not into the dimethoxylated isopimpinellin. Differences exist between the organized plant and its cell culture in terms of metabolic products and, by implication, precursor utilization. S(+)-Marmesin was obtained in small quantity from an acid-hydrolysable conjugate present in the culture medium. Syntheses of [2-¹⁴C]7-demethylsuberosin, [2-¹⁴C]osthenol, [2-¹⁴C]7-O-prenylumbelliferone, [3-¹⁴C] (±)-marmesin, and [3-¹⁴C]psoralen are described, as well as an improved method for separation of furanocoumarin mixtures by TLC and GLC.     

Optimized culture conditions for the production of furanocoumarins by micropropagated shoots of Ruta graveolens

Ruta graveolens in vitro cultures are a potential source of secondary metabolites (furanocoumarins) of significant medical interest. Experiments led in our laboratory showed that micropropagated shoots were richer in furanocoumarins than any other plant material. In order to optimize the molecule production by such cultivation systems, several factors related to the culture medium were studied. Effects of medium composition on biomass growth and furanocoumarin content were analysed and optimal conditions were determined for phosphate (300 mg l⁻¹ of NaH₂PO₄), nitrate (2527 mg l⁻¹ of KNO₃), carbon source (10 g l⁻¹ of sucrose) and phytohormones (2,4-dichlorophenoxyacetic acid (2,4-D) 50 μM and benzylaminopurine (BAP) 50 μM). Ruta shoot growth and furanocoumarin production were compared for optimized and standard culture conditions. Specific medium gave better results in terms of growth (t_D equal to 6.9 days against 8.6 for standard conditions) but no significant differences appeared concerning metabolite concentrations. However, the present study opens the way to scale-up studies with bioreactor cultivation systems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Elicitor induced secondary metabolism in Ruta graveolens L.

In vitro cultures of Ruta graveolens L. respond with rapid accumulation of acridone epoxides, furoquinolines and furanocoumarins, when challenged with autoclaved homogenate of the yeast Rhodotorula rubra. A transient increase of several enzymes of the respective biosynthetic pathways was measured but we still look for the key regulatory enzymes. We investigated whether the branch point enzymes of the shikimic acid pathway anthranilate synthase (AS) and chorismate mutase (CM) possibly play such a role. The two enzymes compete for chorismate. AS forms anthranilate, the precursor amino acid of acridone and furoquinoline alkaloids. CM channels chorismate into phenylalanine, tyrosine and phenylpropanoid biosynthesis. Elicitation resulted in a transient increase of the activity of both enzymes. Relative induction rates were 2–4 fold for AS and about 1.5 fold for CM. Constitutive CM activity, however, is about 1000 fold higher than AS activity. As in other plants 2 isoforms of CM are expected to be present in R. graveolens. A differential determination of the activity of the isoforms via the tryptophan activation rate proved to be ambiguous. Some evidence for the specific induction of a plastidic form of CM was obtained by inhibition of translation. The time courses of CM induction show CM not to be a key enzyme in elicitor induction of furanocoumarin accumulation. In comparison to other enzyme activities induction of anthranilate synthase activity corresponds closest to inducible acridone epoxide accumulation indicating a key role in its regulation. Induction of AS and CM was inhibited by actinomycin D and chloramphenicol while cycloheximid inhibited AS induction only.Abbreviations ACT actinomycin D - AS anthranilate synthase - CAP chloramphenicol - CHX cycloheximid - 4-CL 4-coumarate CoA ligase - CM chorismate mutase - DTT dithiothreitol - NMT S-adenosyl-L-methionine:anthranilic acid N-methyltransferase - PAL phenylalanine ammonia lyase - XOMT S-adenosylmethionine: xanthotoxol-O-methyltransferase     

Ruta graveolens L. Induces Death of Glioblastoma Cells and Neural Progenitors,but Not of Neurons,via ERK 1/2 and AKT Activation

Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens’ effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue’s noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.     

Bioreactor type affects the accumulation of phenolic acids and flavonoids in microshoot cultures of Schisandra chinensis (Turcz.) Baill.

Microshoots of the East Asian medicinal plant species Schisandra chinensis (Chinese magnolia vine) were grown in bioreactors characterized by different construction and cultivation mode. The tested systems included two continuous immersion systems—a cone-type bioreactor (CNB) and a cylindric tube bioreactor (CTB), a nutrient sprinkle bioreactor (NSB), and two temporary immersion systems (TIS)—RITA® and Plantform. Microshoots were grown for 30 and 60 days in the MS medium enriched with 1 mg l^?1 NAA and 3 mg l^?1 BA. The accumulation of two groups of phenolic compounds: phenolic acids and flavonoids in the bioreactor-grown S. chinensis biomass, was evaluated for the first time. In the microshoot extracts, seven phenolic acids: chlorogenic, gallic, p–hydroxybenzoic, protocatechuic, syringic, salicylic and vanillic, and three flavonoids: kaempferol, quercitrin and rutoside, were identified. The highest total amount of phenolic acids (46.68 mg 100 g^?1 DW) was recorded in the biomass maintained in the CNB for 30 days. The highest total content of flavonoids (29.02 mg 100 g^?1 DW) was found in the microshoots maintained in the NSB for 30 days. The predominant metabolites in all the tested systems were: gallic acid (up to 10.01 mg 100 g^?1 DW), protocatechuic acid (maximal concentration 16.30 mg 100 g^?1 DW), and quercitrin (highest content 21.00 mg 100 g^?1 DW).

     

Micropropagation of Anethum graveolens L Through Axillary Shoot Proliferation

A protocol is described for rapid and large-scale in vitro propagation of Anethum graveolens by enhanced axillary shoot induction that was dependent on BAP supply. The synergistic combination of 0.5 mg l^?1 BAP and 0.1 mg l^?1 IBA induced 100% shoot formation as well as shoot number (6.6 ± 0.48 per explant). Subculturing of shoot tips of in vitro plants on multiplication medium enabled continuous production of healthy shoots with similar frequency. Rooting of shoots was achieved on a medium with 1mg l^?1 IBA and 0.5 mg l^?1 Kn. Micropropagated plants established in garden were uniform and identical to the donor plant with respect to morphological and cytological characteristics.     

Purification and characterization of chorismate mutase isoenzymes from ruta graveolens l.

Two isoenzymes of chorismate mutase (EC 5.4.99.5), designated as CM-1 and CM-2, were isolated and partially purified from suspension-cultured cells of Ruta gravelens by DEAE-sephacel chromatography and gel filtration. 60–72% of the total activity measured after DEAE-sephacel chromatography were obtained as CM-1 and 28–40% were CM-2 activity. CM-1 was inhibited by phenylalanine (K₁ = 4 · 10^?6 M) and tyrosine (K₁ = 8. 10^?6M) and activated by tryptophan. In contrast, CM-2 was not influenced by these three amino acids. The molecular weights estimated by gel filtration on SEPHADEX G-150 were 56000 for CM-1 and 45000 for CM-2, respectively. Both isoenzymes were stable at ?20°C, but exhibited different behaviour during thermal inactivation and different optima of reaction temperature. CM-1 catalysed the reaction at a pH optimum of pH 7.8 and CM-2 showed a broad optimum between 6–10. The K_m-values for chorismic acid were determined to be 1.1 mM for CM-1 and 0.5 mM for CM-2. The isoenzymes showed different behaviour to inhibitors of sulfhydryl groups. There were no differences in all parameters of chorismate mutase examined for two various cell lines of Ruta graveolens.     

Biosynthesis of rutacridone in tissue cultures of Ruta graveolens L.

The dihydrofuroacridone, rutacridone, is the main alkaloid in tissue cultures of Ruta graveolens L. strain R-19. The biosynthesis of this particular acridone alkaloid was investigated by using calluses and suspension cultures of strain R-19. Anthranilic acid is specifically incorporated into ring A of rutacridone. Some further evidence was provided that acetate via a polyketide is involved in acridone biosynthesis. Mevalonic acid gave a poor incorporation into rutacridone. Thus the origin of the isopropyldihydrofuran moiety of the investigated alkaloid is still obscure.