Methyl jasmonate- and salicylic acid-induced <Emphasis Type="SmallCaps">d</Emphasis>-chiro-inositol production in suspension cultures of buckwheat (<Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>)

The aim of this work is to investigate the effects of methyl jasmonate (MeJ) and salicylic acid (SA) on d-chiro-inositol (DCI) production in buckwheat (Fagopyrum esculentum) suspension cultures. In this study, adding optimal concentrations of MeJ and SA at an appropriate time markedly increased DCI production (yield 6.141 and 5.521 mg/g DW, respectively). In addition, treatment of buckwheat cultures with a combination of 0.2 mM MeJ and 0.6 mM SA on days 0 and 9 increased the DCI yield to 7.579 mg/g DW, which was 3.726 times higher than that in the control; furthermore, the former yield was higher than that achieved by the addition of either elicitor alone. Moreover, unlike MeJ, SA did not exert a negative effect on cell growth.     

Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs

Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.     

Induction of phenolic compounds in Hypericum perforatum L. cells by Colletotrichum gloeosporioides elicitation

Changes in phenolic metabolism after elicitation with Colletotrichum gloeosporioides (CG) has been studied in Hypericum perforatum L. (HP) cell suspension cultures. Soluble phenolics were analysed by HPLC-DAD and HPLC-DAD-MS/MS. HP cultures elicited with the CG elicitor showed a significant increase in xanthone accumulation. Xanthone accumulation increased twelve fold when the cells were primed with methyl-jasmonate (MeJ) or salicylic acid (SA), before elicitation. HP cultures exposed only to MeJ produced a set of flavonoids, the flavones which represent a substantial part (approx. 40%) of the total flavonoids accumulated in these cells. The possible importance of xanthones as a component of defence mechanism of HP against biotic stress is discussed.     

Medium salt strength induced changes in growth, physiology and secondary metabolite content in adventitious roots of Morinda citrifolia: the role of antioxidant enzymes and phenylalanine ammonia lyase

In an attempt to improve growth and secondary metabolite production, and to understand the possible mechanism involved in relation to the changes in physiology and activities of antioxidant enzymes, we cultured Morinda citrifolia adventitious roots in different strength (0.25, 0.50, 0.75, 1.0, 1.5 and 2.0) of Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ indole butyric acid and 30 g l⁻¹ sucrose. Quarter-strength MS medium was proven suitable for the production of both root biomass and secondary metabolites [anthraquinone (AQ), phenolics and flavonoids]. With the increasing salt strength, root growth and AQ accumulation decreased significantly. Higher (1.5 and 2 MS) salt strength provoked osmotic stress resulted in more than twofold free proline accumulation than lower salt strength treated roots and induced free radical scavenging activity. Phenylalanine ammonia lyase activity showed a positive correlation in relation to salt strength that leads to an increase in phenol biosynthesis in expense of AQ formation. The elevated catalase (CAT), guaiacol peroxidase (G-POD) and superoxide dismutase activities and decreased ascorbate peroxidase (APX) activities were observed in roots treated with 2.0 MS. On the other hand, APX activity was strongly activated along with considerable increase in CAT activity at 0.25 MS treated culture. However, the joint functions of CAT, G-POD and APX at 0.25 MS treated cultures were efficient to eliminate the potential danger of hydrogen peroxide (H₂O₂) as evidenced from low H₂O₂ accumulation and low level of lipid peroxidation.     

Silicone-stimulated anthraquinone production and release by Morinda citrifolia in a two-liquid-phase system

Summary A 2.5-fold increase in the release of intracellular anthraquinones was obtained by adding 1 ml L^-1 silicone A to suspension cultures of Morinda citrifolia. Cell growth and secondary metabolite production were not affected even at high silicone A concentrations. Performance of the silicone treatment in a two-liquid-phase system (5 ml n-hexadecane/50 ml medium) resulted in a 150% increase of the overall secondary metabolite productivity.     

Biosynthesis stimulation of nor‐secotriterpene anxiolytics in cell suspension cultures of Galphimia glauca Cav

Galphimia glauca produces compounds denominated galphimines (galphimine‐A, galphimine‐B and galphimine‐E). Due to their important anxiolytic activity, we initiated in vitro cultures of the species with the purpose of developing a biotechnological process for obtaining galphimines. In this work, we stimulated the biosynthesis and excretion of galphimines with two‐phase batch‐type cell suspension cultures of G. glauca. The effect of nutritional variation and the 2,4‐dichlorophenoxy acetic acid added to Murashige & Skoog(MS) culture medium was evaluated. Later, we evaluated the effect of the stimulation with calcium and methyl jasmonate (MeJ). The greatest production of galphimine‐B (3.39 × 10^?5 g/L day^?1) was obtained on day 40 of kinetics, and induced by a treatment containing concentrations of nitrates and phosphate that are double of those normally used in MS medium, without sucrose but with added 2,4‐dichlorophenoxy acetic acid (4 mg/L). Time of galphimine‐B biosynthesis diminished due to the effect of MeJ in combination with calcium, and induced the excretion (100%) of galphimine‐B (6.35 × 10^?5 g/L day^?1) into the culture medium. Thus, the use of calcium and MeJ comprises a viable alternative to stimulate the production and excretion of galphimine‐B and galphimine‐A in batch‐type cultures of G. glauca in modified MS medium. Once optimized, the production of the anxiolytic compounds can be scaled up to the industrial level.     

Stimulation of the proline cycle and anthraquinone accumulation in <Emphasis Type="Italic">Rubia tinctorum</Emphasis> cell suspension cultures in the presence of glutamate and two proline analogs

Anthraquinone biosynthesis in Rubia tinctorum L. involves different metabolic routes. Chorismic acid, the end-product of the shikimate pathway, becomes the branch point between primary and secondary metabolism. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP⁺ generated by proline reduction from glutamate could act as a cofactor of the first enzymes of the PPP. This pathway generates erythrose-4-phosphate, the substrate of the shikimate pathway. The aim of the present work was to study the effect of the addition of glutamate and two proline analogs, azetidine-2-carboxylic acid and thiazolidine-4-carboxylic acid (T4C), on the PPP, the proline cycle, and anthraquinone production in R. tinctorum cell suspension cultures. The addition of 5 mM of glutamate enhanced both anthraquinone (up to 30%) and total phenolic content (12%), which correlated well with proline accumulation. Only the addition of 200 μM of T4C resulted in an increase in anthraquinone production, which was accompanied by a rise in the proline content. Neither the addition of glutamate nor proline analogs resulted in the induction of PPP, so this route was not a limiting factor as a carbon donor to the shikimate pathway.     

Immobilization of Rubia tinctorum L. suspension cultures and its effects on alizarin and purpurin accumulation and biomass production

The present study is investigating the immobilization of Rubia tinctorum L. suspension cultures. The effects of three inoculation volumes and three immobilization materials (loofa, sisal and jute) on fresh and dry weights of biomass as well as on alizarin and purpurin production were determined in this study. Two grams of four-week old callus tissue were transferred to liquid medium to establish suspension cultures. After four weeks, suspension cultures of R. tinctorum at concentration of 8?×?10⁵?living cells/ml were immobilized with lignocellulosic materials and the cells were attached to all immobilization materials at the end of the first week and started to form aggregates on them. At the fourth week of these batch systems, biomass was measured approximately three times higher than the starting suspension cultures. The highest fresh weight was obtained (339.40?g/l) from sisal with ? inoculation ratio. Immobilization materials and inoculation volumes had an effect on dry weights, and accordingly, the most effective combinations were jute with ? (J3) and ? (J1) inoculation volumes with 7.86 and 7.82?g/l dry weights, respectively. Alizarin and purpurin contents of immobilized cells, analyzed with U-HPLC method, were 6.05 and 22.91 times higher than inoculated cells. All immobilization materials used in this study had no negative effect on to cells and biomass accumulation was enhanced. Concomitantly with rapid biomass increase, alizarin and purpurin production was ascended.     

Improved anthraquinone accumulation in cell cultures of Cinchona `Robusta' by feeding of biosynthetic precursors and inhibitors

The effects of feeding of biosynthetic precursors and pathway specific inhibitors on anthraquinone (AQ) accumulation in fungal elicited cell cultures of Cinchona`Robusta' were studied. Addition of glyceraldehyde (1 mM), the initial precursor in the methyl-d-erythritol 4-phosphate (MEP) pathway, did not increase AQ accumulation, suggesting that the endogenous level of this precursor is not a limiting factor of AQ flux. It is proposed that AQs in Cinchona might be derived from the phenylpropanoid pathway, e.g. from caffeic acid. Addition of ferulic acid (1 mM) did not stimulate AQ accumulation, while addition of caffeic acid increased AQ accumulation by 48% compared to the control. The stimulating effect of feeding caffeic acid on AQ accumulation might be due to activation of other pathways. Addition of tectoquinone (2-methyl-anthraquinone) did not change the AQ patterns nor the shifts between AQs in control and tectoquinone-treated cell cultures. Addition of lovastatin, a specific inhibitor of the mevalonic acid pathway, did not inhibit the AQ accumulation. Clomazone, an inhibitor in the MEP pathway, inhibited the AQ accumulation, however. The simultaneous addition of lovastatin and clomazone inhibited both cell growth and AQ accumulation. These results further support the finding that isopentenyl diphosphate, which constitutes ring C of AQs in Cinchona `Robusta', is derived from the MEP pathway, and not from the mevalonic acid pathway.     

Aeration volume and photosynthetic photon flux affect cell growth and secondary metabolite contents in bioreactor cultures ofMorinda citrifolia

To improve their growth and secondary metabolite production, we culturedMorinda citrifolia leaf cells for 3 weeks in bioreactors with different aeration volumes (0.05, 0.1, 0.2, or 0.3 vvm; or 0.05/0.1/0.2/0.3 vvm, as increased at 5-d interval), and photosynthetic photon fluxes (PPF; 0, 15, 30, or 45 μ,moL m^-2 s^-1). Cell growth was greatest (15.6 g L^-1 dry weight) at 0.3 vvm whereas the accumulation of secondary metabolites (total anthraquinones, phenolics, and flavonoids) was maximized at 0.1 vvm. A PPF of 15 μmoLm^-2 s^-1 accelerated the accumulation of both cell biomass and metabolites. Dark-culturing suppressed cell growth, while a high PPF (45 μmoLm^-2 s^-1) inhibited metabolite biosynthesis. Further studies are required to understand the reason for differences in the effect of light on cell growth and secondary metabolite contents inM. citrifolia cell cultures.     

Elicitation with methyl-jasmonate stimulates peruvoside production in cell suspension cultures of <Emphasis Type="Italic">Thevetia peruviana</Emphasis>

Thevetia peruviana is a small tree that produces several compounds with pharmaceutical application, among which peruvoside could be highlighted. However, these compounds are produced in low concentration in the plant, making it important to develop strategies such as plant cell culture and elicitation to obtain higher quantities of the desired product. In this work, cell suspension cultures of T. peruviana were established in four different culture media: Murashige–Skoog (MS), half Murashige–Skoog (half MS), Schenk–Hildebrandt (SH), and Gamborg (B5) to study their effect on cell growth. Cell growth kinetics were studied in SH medium, and the extracellular peruvoside production during the culture time was determined. The best culture medium for the establishment of cell suspension cultures was MS with a growth index of 3.17 ± 0.2 g g⁻¹ inoculum. The cell growth kinetics showed the four characteristic growth phases of a cell culture (lag, exponential, stationary, and death), and during none of these phases was it possible to observe peruvoside production. The elicitor effect of methyl-jasmonate (MeJ) was studied in cell suspension cultures established in SH medium. The effect of MeJ concentration and the time in which it should be applied were determined. The best results were obtained at a concentration of 100 mg l⁻¹ of MeJ applied at the beginning of the culture, which induced a peruvoside production of 8.93 mg l⁻¹ medium. The current results are the first report of an in vitro peruvoside production system.     

Anthraquinones production in Rubia tinctorum cell suspension cultures: Down scale of shear effects

The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures.     

Enhanced Dissolution and Stability of Lansoprazole by Cyclodextrin Inclusion Complexation: Preparation, Characterization, and Molecular Modeling

In this study, lansoprazole (LSP)/cyclodextrin (CD) inclusion complexes were prepared using a fluid bed coating technique, with β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (HPCD) as the host molecules, respectively, to simultaneously improve the dissolution and stability of LSP. The dissolution rate and stability of LSP was dramatically enhanced by inclusion complexation regardless of CD type. LSP/HPCD inclusion complex was more stable under illumination than LSP/β-CD inclusion complex. Differential scanning calorimetry and powder X-ray diffractometry proved the absence of crystallinity in both LSP/CD inclusion complexes. Fourier transform infrared spectroscopy together with molecular modeling indicated that the benzimidazole of LSP was included in the cavity of both CDs, while LSP was more deeply included in HPCD than β-CD. The enhanced photostability was due to the inclusion of the sulfinyl moiety into the HPCD cavity. CD inclusion complexation could improve the dissolution and stability of LSP.KEY WORDS: cyclodextrin, dissolution, inclusion complex, lansoprazole, molecular modeling, stability     

Sucrose regulated enhanced induction of anthraquinone,phenolics, flavonoids biosynthesis and activities of antioxidant enzymes in adventitious root suspension cultures of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> (L.)

The effect of initial sucrose concentration was investigated in root suspension cultures of Morinda citrifolia to improve root growth and secondary metabolites production, i.e. anthraquinone, phenolics and flavonoids. Besides, oxidative stress level, antioxidant enzymes activity and membranes damage under different sucrose concentration were estimated. A 5% sucrose supply was shown to be optimal for the production of root dry mass, but higher sucrose concentrations of 7–9% inhibited the accumulation of root dry weight (DW). However, the maximum production of anthraquinone (251.89 g L⁻¹ DW), phenolics (165.14 g L⁻¹ DW) and flavonoids (163.56 g L⁻¹ DW) were achieved at 1% sucrose-treated culture, which may be a source carbon skeletons for secondary metabolism. At the same time was observed low oxidative damage, which could be associated with high levels of secondary metabolites and the increased activity of catalase. Although, catalase (CAT) activity were stimulated at 7–9% sucrose-treated cultures, high accumulation of hydrogen peroxide (H₂O₂) and peroxidation of lipid (MDA) was induced. The observed high activity of CAT and guaiacol peroxidase (G-POD) were not sufficient enough to mitigate the toxic effect of H₂O₂.     

Production of biomass and bioactive compounds by adventitious root suspension cultures of Morinda citrifolia (L.) in a liquid-phase airlift balloon-type bioreactor

To improve root growth and production of bioactive compounds such as anthraquinones (AQ), phenolics, and flavonoids by adventitious root cultures of Morinda citrifolia, the effects of aeration rate, inoculum density, and Murashige and Skoog (MS) medium salt strengths were investigated using a balloon-type bubble bioreactor. The possible mechanisms underlying changes in activities of enzymic (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase) and nonenzymic (vitamin E) antioxidants, phenylalanine ammonia lyase, and stress levels (accumulation of hydrogen peroxide and proline, peroxidation of lipids) were also studied. Low aeration rate (0.05 vvm [air volume/culture volume/min]) accelerated accumulation of root fresh weight and dry weight (DW). High aeration rates (0.1 to 0.3 vvm) stimulated accumulation of AQ, phenolics, and flavonoids and reduced root growth. Low inoculum densities (5 and 10 g l^–1) increased accumulation of those metabolites but inhibited root growth. Culture of adventitious roots with high concentrations of MS salts (1× and 1.5× MS) resulted in induction of oxidative stress that strongly inhibited root growth. Overall, an aeration rate of 0.05 vvm, 15 g l^–1 inoculum density, and half-strength (0.5×) MS medium were optimal for enhancing accumulation of root dry biomass (4.38 g l^–1), AQ (103.08 mg g^–1 DW), phenolics (54.81 mg g^–1 DW), and flavonoids (49.27 mg g^–1 DW).     

Use of non-conventional media inMorinda citrifolia cell cultures

Non-conventional media, containing organic solvents as supplement, were exploited to obtain the non-lethal product release of plant cell secondary metabolites, using suspension cultures ofMorinda citrifolia as model system. The results of our preliminary studies about solvent biocompatibility show that the discrimination between biocompatible and toxic solvents can be achieved by means of two parameters: log P and critical solvent concentration. The last one seems to be a better indicator of solvent toxicity for living cells.Abbreviations log P logarithm of P - P solvent partition coefficient in a standard system n-octanol/water     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

Combined use of six-well polystyrene plates and thin layer chromatography for rapid development of optimal plant cell culture processes: application to taxane production byTaxus sp.

TwoTaxus (T. chinensis andT. baccata) cell suspension cultures were used as a model system to demonstrate the similarities of biomass accumulation and secondary metabolite (taxane) production obtained from cultures in six-well polystyrene plates and glass shake flasks (25 ml and 125 ml). Interference from binding of taxanes in cell-free culture broth to the polystyrene plates was minimal with 85% of the paclitaxel (Taxol®) and 100% of baccatin and 10-deacetyl-7-xylosyl-taxol remaining in the medium after 24 h beyond which no further binding was observed. A simple thin layer chromatography (TLC) procedure with a chloroform: acentonitrile (4:1) solvent system on silica gel was developed to simultaneously test up to 17 cultures for taxane production. The combination of six-well plate technology for experimentation and TLC for rapid taxane analysis can greatly accelerate the establishment of conditions for an optimalTaxus plant-cell culture process for taxane production.Abbreviations TLC Thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - HPLC high pressure liquid chromotography - UV ultraviolet - Rf retention factor     

Growth,secondary metabolite production and antioxidant enzyme response of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> adventitious root as affected by auxin and cytokinin

Morinda citrifolia adventitious roots were cultured in shake flasks using Murashige and Skoog medium with different types and concentrations of auxin and cytokinin. Root (fresh weight and dry weight) accumulation was enhanced at 5 mg l⁻¹ indole butyric acid (IBA) and at 7 and 9 mg l⁻¹ naphthalene acetic acid (NAA). On the other hand, 9 mg l⁻¹ NAA decreased the anthraquinone, phenolic and flavonoid contents more severely than 9 mg l⁻¹ IBA. When adventitious roots were treated with kinetin (0.1, 0.3 and 0.5 mg l⁻¹) and thidiazuron (TDZ; 0.1, 0.3 and 0.5 mg l⁻¹) in combination with 5 mg l⁻¹ IBA, fresh weight and dry weight decreased but secondary metabolite content increased. The secondary metabolite content (including 1,1-diphenyl-2-picrylhydrazyl activity) increased more in TDZ-treated than in kinetin-treated roots. Antioxidative enzymes such as catalase (CAT) and guaiacol peroxidase (G-POD), which play important roles in plant defense, also increased. A strong decrease in ascorbate peroxidase activity resulted in a high accumulation of hydrogen peroxide. This indicates that adventitious roots can grow under stress conditions with induced CAT and G-POD activities and higher accumulations of secondary metabolites. These results suggest that 5 mg l⁻¹ IBA supplementation is useful for growth and secondary metabolite production in adventitious roots of M. citrifolia.