Biotechnological strategies for controlled accumulation of flavones in hairy root culture of Scutellaria lateriflora L.

Accumulation of medicinally important flavones and acteoside was evaluated in Scutellaria lateriflora hairy root cultures subjected to different experimental strategies - feeding with precursors of phenolics biosynthesis (phenylalanine, cinnamic acid, and sodium cinnamate), addition of elicitors (chitosan, jasmonic acid) and Amberlite XAD-4 and XAD-7 resins and permeabilization with dimethyl sulfoxide (DMSO) and methanol. The production profile of S. lateriflora cultures changed under the influence of the applied strategies. Hairy roots of S. lateriflora were found to be a rich source of wogonoside or wogonin, depending on the treatment used. The addition of sodium cinnamate (1.0 mg/L) was the most effective approach to provide high production of flavonoids, especially wogonoside (4.41% dry weight /DW/; 566.78 mg/L). Permeabilization with DMSO (2 µg/ml for 12 h) or methanol (30% for 12 h) resulted in high biosynthesis of wogonin (299.77 mg/L and 274.03 mg/L, respectively). The obtained results provide new insight into the selection of the optimal growth conditions for the production of in vitro biomass with a significant level of flavone accumulation. The data may be valuable for designing large-scale cultivation systems of hairy roots of S. lateriflora with high productivity of bioactive compounds - wogonin or wogonoside.

The Impact of Nigella sativa Essential Oil on T Cells in Women with Hashimoto's Thyroiditis.

BACKGROUND: Hashimoto's thyroiditis (HT) is an autoimmune disease mediated by T cells. It is characterized by the presence of thyroid autoantibodies in the serum, such as anti-thyroid peroxidase antibodies (TPO-Ab) and anti-thyroglobulin antibodies (TG-Ab). The essential oil extracted from Nigella sativa seeds is rich in bioactive substances, such as thymoquinone and cymene. METHODS: Therefore, we examined the effect of essential oil from Nigella sativa (NSEO) on T cells from HT patients, especially their proliferation capacity, ability to produce cytokines, and susceptibility to apoptosis. RESULTS: The lowest ethanol (EtOH) dilution (1:10) of NSEO significantly inhibited the proliferation of CD4+ and CD8+ T cells from HT patients and healthy women by affecting the percentage of dividing cells and the number of cell divisions. In addition, 1:10 and 1:50 NSEO dilutions induced cell death. Different dilutions of NSEO also reduced the concentration of IL-17A and IL-10. In healthy women, the level of IL-4 and IL-2 significantly increased in the presence of 1:10 and 1:50 NSEO dilutions. NSEO did not influence the concentration of IL-6 and IFN-γ. CONCLUSIONS: Our study demonstrates that NSEO has a strong immunomodulatory effect on the lymphocytes of HT patients.

Evaluation of the yield, chemical composition and biological properties of essential oil from bioreactor-grown cultures of Salvia apiana microshoots.

Microshoot cultures of the North American endemic Salvia apiana were established for the first time and evaluated for essential oil production. Stationary cultures, grown on Schenk-Hildebrandt (SH) medium, supplemented with 0.22 mg/L thidiazuron (TDZ), 2.0 mg/L 6-benzylaminopurine and 3.0% (w/v) sucrose, accumulated 1.27% (v/m dry weight) essential oil, consisting mostly of 1,8-cineole, ß-pinene, α-pinene, ß-myrcene and camphor. The microshoots were adapted to agitated culture, showing biomass yields up to ca. 19 g/L. Scale-up studies demonstrated that S. spiana microshoots grow well in temporary immersion systems (TIS). In the RITA bioreactor, up to 19.27 g/L dry biomass was obtained, containing 1.1% oil with up to ca. 42% cineole content. The other systems employed, i.e. Plantform (TIS) and a custom made spray bioreactor (SGB), yielded ca. 18 and 19 g/L dry weight, respectively. The essential oil content of Plantform and SGB-grown microshoots was comparable to RITA bioreactor, however, the content of cineole was substantially higher (ca. 55%). Oil samples isolated from in vitro material proved to be active in acetylcholinesterase (up to 60.0% inhibition recorded for Plantform-grown microshoots), as well as hyaluronidase and tyrosinase-inhibitory assays (up to 45.8 and 64.5% inhibition observed in the case of the SGB culture).

The influence of Nigella sativa essential oil on proliferation, activation, and apoptosis of human T lymphocytes in vitro.

In previous work, we tested the immunomodulatory effect of Nigella sativa (NS) fatty oil. Our results demonstrated that unrefined, obtained by cold pressing black cumin seed oil inhibited lymphocytes' proliferation and induced their apoptosis in a dose-dependent manner. In this study, we examined the immunomodulatory properties of essential oil (EO) obtained from the NS seeds by hydrodistillation and its two main constituents: thymoquinone (TQ) and p-cymene. We analyzed the proliferation, activation phenotype, and apoptosis rates of human T lymphocytes stimulated with an immobilized monoclonal anti-CD3 antibody in the presence of serial ethanol dilutions of tested oil or serial distilled water dilutions of tested compounds with flow cytometry. Our results showed that NSEO significantly inhibited the proliferation of CD4+ and CD8+ T lymphocytes, induced cell death in a dose-dependent manner, and reduced the expression of CD28 and CD25 antigens essential for lymphocyte activation. TQ inhibited the proliferation of T lymphocytes and induced cell death, particularly in high concentrations. Meanwhile, p-cymene did not influence lymphocyte proliferation. However, its high concentration induced cell necrosis. These results show that the essential oil from Nigella sativa has powerful immunomodulatory properties, which, at least partially, are related to the TQ component.

Phenylpropanoid Glycoside and Phenolic Acid Profiles and Biological Activities of Biomass Extracts from Different Types of Verbena officinalis Microshoot Cultures and Soil-Grown Plant.

Different types of microshoot cultures (agar, stationary liquid, agitated, and bioreactors) of Verbena officinalis were optimized for biomass growth and the production of phenylpropanoid glycosides and phenolic acids. Using ultra-high performance liquid chromatography with high-resolution time-of-flight mass spectrometry, the presence of verbascoside, isoverbascoside, leucoseptoside A/isomers, and cistanoside D/isomer was confirmed in the methanolic extracts obtained from all types of in vitro cultures. The compound's content was determined by ultra-high-performance liquid chromatography. The main metabolites in biomass extracts were verbascoside and isoverbascoside (maximum 4881.61 and 451.80 mg/100 g dry weight (DW)). In the soil-grown plant extract, verbascoside was also dominated (1728.97 mg/100 g DW). The content of phenolic acids in the analyzed extracts was below 24 mg/100 g DW. The highest radical scavenging activity was found in the biomass extract from agitated cultures, the most effective reducing power in agar culture extract, and the highest chelating activity in extract from bioreactor cultures. The extracts showed significantly stronger bacteriostatic and bactericidal activity against Gram-positive bacteria (minimum inhibitory concentration (MIC) of 0.3-2.2 mg/mL and minimum bactericidal concentration (MBC) of 0.6-9 mg/mL) than against Gram-negative bacteria (MIC 0.6-9 mg/mL, MBC of 0.6-18 mg/mL). The biomass extract from liquid stationary culture showed the strongest antibacterial activity, while the extract from soil-grown herb had the lowest.

White Sage (Salvia apiana)-a Ritual and Medicinal Plant of the Chaparral: Plant Characteristics in Comparison with Other Salvia Species.

Salvia apiana, commonly known as white sage, is an aromatic evergreen subshrub of the chaparral, commonly found in coastal plains in California and Baja California. It has been traditionally used by the Chumash people as a ritual and medicinal plant and used as a calmative, a diuretic, and a remedy for the common cold. However, until recently, relatively little has been known about the composition and biological activity of white sage. Phytochemical studies on S. apiana revealed the presence of substantial amounts of essential oil, accompanied by a variety of triterpenes, C23 terpenoids, diterpenes, and flavonoids. Extracts of the plant have been shown to exhibit antioxidative, antimicrobial, and cytotoxic effects. The influence of white sage constituents on the nervous system, including GABA, opioid, and cannabinoid receptors, has also been documented. The review aimed to compile information on the taxonomy, botany, chemical composition, and biological activities of S. apiana. White sage was compared with other representatives of the genus in terms of chemical composition. The differences and similarities between S. apiana and other sage species were noted and discussed in the context of their therapeutic applications. Reports on ethnomedicinal uses of white sage were confronted with reports on chemistry, bioactivity, and bioavailability of S. apiana constituents. Finally, a critical assessment of the available data was made and perspectives for the use of white sage preparations in modern phytomedicine were discussed.

Studies on the Polyphenolic Composition and the Antioxidant Properties of the Leaves of Poplar (Populus spp.) Various Species and Hybrids.

The chemical composition in terms of flavonoid and salicylic compounds of leaves from 6 species and 3 hybrids of poplars (Populus) was identified with the use of TLC and HPLC-DAD/ESI-MS methods. Chromatographic analyses were carried out with 21 standard compounds including salicylic compounds (2), phenolic acids (3) and flavonoids (16). Moreover, on the basis of the obtained chromatographic data from the HPLC-DAD/ESI-MS and TLC separations, the presence of salicortin, tremulacin and chlorogenic acid was confirmed, depending on the analyzed poplar species or hybrid. The content of salicylic compounds was determined by HPLC-UV method and expressed on salicin as free and total fraction. Total flavonoid content was determined by spectroscopic method as quercetin equivalent. Significant qualitative and quantitative differences in the chemical composition of the analyzed leaves were demonstrated. The highest concentration of flavonoids (8.02 mg/g) was found in the leaves of Populus nigra, while the highest content of salicylic compounds (47.14 mg/g) was found in the leaves of P.×berolinensis. The antioxidant and xanthine oxidase inhibition properties of extracts from poplar leaves were investigated by TLC bioautography. It has been shown that the richest set of compounds with antioxidant properties are present in the leaves of P. alba, P.×candicans and P. nigra.

Production of Verbascoside, Isoverbascoside and Phenolic Acids in Callus, Suspension, and Bioreactor Cultures of Verbena officinalis and Biological Properties of Biomass Extracts.

Callus, suspension and bioreactor cultures of Verbena officinalis were established, and optimized for biomass growth and production of phenylpropanoid glycosides, phenolic acids, flavonoids and iridoids. All types of cultures were maintained on/in the Murashige and Skoog (MS) media with 1 mg/L BAP and 1 mg/L NAA. The inoculum sizes were optimized in callus and suspension cultures. Moreover, the growth of the culture in two different types of bioreactors-a balloon bioreactor (BB) and a stirred-tank bioreactor (STB) was tested. In methanolic extracts from biomass of all types of in vitro cultures the presence of the same metabolites-verbascoside, isoverbascoside, and six phenolic acids: protocatechuic, chlorogenic, vanillic, caffeic, ferulic and rosmarinic acids was confirmed and quantified by the HPLC-DAD method. In the extracts from lyophilized culture media, no metabolites were found. The main metabolites in biomass extracts were verbascoside and isoverbascoside. Their maximum amounts in g/100 g DW (dry weight) in the tested types of cultures were as follow: 7.25 and 0.61 (callus), 7.06 and 0.48 (suspension), 7.69 and 0.31 (BB), 9.18 and 0.34 (STB). The amounts of phenolic acids were many times lower, max. total content reached of 26.90, 50.72, 19.88, and 36.78 mg/100 g DW, respectively. The highest content of verbascoside and also a high content of isoverbascoside obtained in STB (stirred-tank bioreactor) were 5.3 and 7.8 times higher than in extracts from overground parts of the parent plant. In the extracts from parent plant two iridoids-verbenalin and hastatoside, were also abundant. All investigated biomass extracts and the extracts from parent plant showed the antiproliferative, antioxidant and antibacterial activities. The strongest activities were documented for the cultures maintained in STB. We propose extracts from in vitro cultured biomass of vervain, especially from STB, as a rich source of bioactive metabolites with antiproliferative, antioxidant and antibacterial properties.

Verbena officinalis (Common Vervain) - A Review on the Investigations of This Medicinally Important Plant Species.

Verbena officinalis (common vervain) is a medicinal plant species widely distributed in the world and commonly used in folk medicine of different countries, including traditional Chinese medicine. Monographs on "Verbenae herba" have been included in the European Pharmacopoeia since 2008, and in the Chinese Pharmacopoeia since 1995. This work presents botanical characteristics of this species. It reviews the current knowledge of its chemical composition, which is a rich source mostly of iridoids, phenylpropanoid glycosides, phenolic acids, flavonoids, terpenoids, and essential oil. A large part of this article summarizes traditional medicinal uses and professional pharmacological in vitro and in vivo studies that prove new important applications, e.g., antioxidant, antimicrobial, anti-inflammatory, neuroprotective anticancer, analgesic, or anticonvulsant of verbena herb extracts and individual metabolites. Moreover, emphasis is put on the use of V. officinalis in the food and cosmetics industries, especially due to its antioxidant, antibacterial, and anti-inflammatory properties, and the presence of essential oil with an attractive fragrance composition. This paper also presents the state of biotechnological studies of this species.

Chemical variability of Rhododendron tomentosum (Ledum palustre) essential oils and their pro-apoptotic effect on lymphocytes and rheumatoid arthritis synoviocytes.

Rhododendron tomentosum (Ledum palustre) is an aromatic plant traditionally used for alleviating rheumatic complaints which makes it a potential candidate for a natural drug in rheumatoid arthritis (RA) treatment. However, the effects of plants' volatiles on apoptosis of synovial fibroblasts and infiltrating leucocytes of RA synovia, have not been reported. Volatile fraction of R. tomentosum is chemically variable and chemotypes of the plants need to be defined if the oil is to be used for therapeutic purposes. In the presented work, cluster analysis of literature data enabled to define 10 chemotypes of the plant. The volatile fractions of known composition were then tested for bioactivity using a RA-specific in vitro models. Essential oils of two wild types (γ-terpineol and palustrol/ledol type) and one in vitro chemotype (ledene oxide type) were obtained by hydrodistillation and their bioactivity was tested in two in vitro models: I - peripheral blood lymphocytes of healthy volunteers and II - synoviocytes and immune cells isolated from synovia of RA patients. The influence of oils on blood lymphocytes' proliferation and apoptosis rates of synovia-derived cells was determined by flow cytometry. Dose-dependent inhibitory effect of the serial dilutions of R. tomentosum oils on proliferation rates of blood lymphocytes was found. At 1:400 dilutions, all the tested oils increased the number of necrotic cells in synovial fibroblasts from RA synovia. Additionally, increased proportions of late apoptotic cells were observed in leucocyte populations subjected to oils at 1:400 dilution.

Phenolic acid and flavonoid production in agar, agitated and bioreactor-grown microshoot cultures of Schisandra chinensis cv. Sadova No. 1 - a valuable medicinal plant.

In vitro cultures and raw materials (fruits and leaves) of the valuable medicinal plant species - Schisandra chinensis cultivar Sadova No. 1 (SchS) - were evaluated for the production of two groups of phenolic compounds, phenolic acids and flavonoids, and their antioxidant potential. A series of experiments was conducted, aimed at optimizing culture conditions for maximum growth and phenolic production in SchS microshoots. Different concentrations of plant growth regulators (6-benzyladenine - BA and 1-naphthaleneacetic acid - NAA, from 0 to 3 mg/l) in Murashige-Skoog (MS) medium were tested in several cultivation systems (agar, agitated, bioreactor) over various growth periods (10, 20, 30, 40, 50 and 60 days). Furthermore, an elicitation experiment was conducted in which the bioreactor-grown microshoots were exposed to yeast extract. HPLC-DAD analyses confirmed the presence of eight phenolic acids - chlorogenic, cryptochlorogenic, gallic, neochlorogenic, protocatechuic, salicylic, syringic and vanillic, and two flavonoids: kaempferol and quercitrin, in the in vitro biomasses. The highest total phenolic acid (357.93 mg/100 g DW) and flavonoid (105.07 mg/100 g DW) contents were obtained in agar culture extracts cultivated for 30 days on MS medium containing 2 mg/l BA and 0.5 mg/l NAA and for 50 days on MS medium containing 0.1 mg/l BA and 2 mg/l NAA, respectively. These amounts were 1.59- and 5.95-fold lower than in parent plant leaf extracts (569.66 mg/100 g DW), and 4.30- and 1.25-fold higher than in fruit extracts (83.17 mg/100 g DW), respectively. Microshoots grown in a Plantform bioreactor also proved to be a good source of phenolic compounds, however, the elicitor treatment had no noticeable effect on their accumulation. Antioxidant capacity assessed by the Folin-Ciocalteu, FRAP, DPPH and CUPRAC assays revealed significantly higher potential in extracts from in vitro biomass and leaves of the parent plant, as compared to the parent plant fruit extracts.

An application of QSRR approach and multiple linear regression method for lipophilicity assessment of flavonoids.

The analysis of quantitative structure-retention relationships (QSRR) is useful tool for assessment of compound's lipophilicity/hydrophobicity due to similarity between its retention in chromatographic system and ability to permeation through biological membranes. The main goal of this study was to compare usefulness of two reversed-phase chromatographic columns (Synergy POLAR and Synergy-FUSION) for lipophilicity assessment of 30 structurally diverse flavonoids using the QSRR approach and multiple linear regression method. The developed MLR models included the mechanistically interpretable geometrical descriptors: 3D Molecule Representation of Structure based on Electron diffraction (3D-MoRSE) and Radial Distribution Function (RDF). Both models were evaluated by the internal and external validation and selected descriptors were further interpreted. According to obtained results the FUSION-RP column can be recommended to log kw prediction of flavonoids. The comprehensive interpretation of molecular descriptors was used to present the molecular mechanisms and structural features governing the chromatographic retention of tested compounds.

Phytochemical and biotechnological studies on Schisandra chinensis cultivar Sadova No. 1-a high utility medicinal plant.

In the presented work, raw materials (fruits and leaves) and in vitro biomass of a highly productive Schisandra chinensis Sadova No. 1 cultivar (SchS) were evaluated for the production of therapeutically useful schisandra lignans (SL). In vitro cultures of SchS were initiated, followed by extensive optimization studies focused on maximizing secondary metabolite production, with the aim of establishing a sustainable source of SL. Different cultivation systems (agar, agitated, bioreactor), experiment times (10, 20, 30, 40, 50 and 60 days) and plant growth regulators (6-benzyladenine-BA and 1-naphthaleneacetic acid-NAA, from 0 to 3 mg/l) in Murashige-Skoog (MS) medium were tested. Moreover, an elicitation procedure was applied to bioreactor-grown microshoots in order to increase SL production. Validated HPLC-DAD protocol enabled to detect fourteen SL in the extracts from in vitro and in vivo materials. The main compounds in the in vitro cultures were as follows: schisandrin (max. 176.3 mg/100 g DW), angeloylgomisin Q (max. 85.1 mg/100 g DW), gomisin A (max. 71.4 mg/100 g DW) and angeloylgomisin H (max. 67.0 mg/100 g DW). The highest total SL content (490.3 mg/100 g DW) was obtained in extracts from the biomass of agar cultures cultivated for 30 days on the MS medium variant containing 3 mg/l BA and 1 mg/l NAA. This amount was 1.32 times lower than in fruit extracts (646.0 mg/100 g DW) and 2.04 times higher than in leaf extracts (240.7 mg/100 g DW). The study demonstrated that SchS is a rich source of SL, thus proving its value for medical, cosmetic and food industry.

Improved production of dibenzocyclooctadiene lignans in the elicited microshoot cultures of Schisandra chinensis (Chinese magnolia vine).

Dibenzocyclooctadiene lignans are a specific group of secondary metabolites that occur solely in Schisandra chinensis. The aim of the presented work was to boost the accumulation of lignans in the agitated microshoot cultures of S. chinensis, using different elicitation schemes. The experiments included testing of various concentrations and supplementation times of cadmium chloride (CdCl2), chitosan (Ch), yeast extract (YeE), methyl jasmonate (MeJa), and permeabilizing agent-dimethylsulfoxide (DMSO). After 30 days, the microshoots were harvested and evaluated for growth parameters and lignan content by LC-DAD method. The analyses showed enhanced production of lignans in the elicited S. chinensis microshoots, whereas the respective media samples contained only trace amounts of the examined compounds (< 5 mg/l). Elicitation with CdCl2 caused up to 2-fold increase in the total lignan content (max. ca. 730 mg/100 g DW after the addition of 1000 µM CdCl2 on the tenth day). Experiments with chitosan resulted in up to 1.35-fold increase in lignan concentration (max. ca. 500 mg/100 g DW) after the supplementation with 50 mg/l on the first day and 200 mg/l on the tenth day. High improvement of lignan production was also recorded after YeE elicitation. After the elicitation with 5000 mg/l of YeE on the first day of the growth period, and with 1000 and 3000 mg/l on the 20th day, the lignan production increased to the same degree-about 1.8-fold. The supplementation with 1000 mg/l YeE on the 20th day of the growth cycle was chosen as the optimal elicitation scheme, for the microshoot cultures maintained in Plantform temporary immersion system-the total content of the estimated lignans was equal to 831.6 mg/100 g DW.

Naturally occurring xanthone and benzophenone derivatives exert significant anti-proliferative and proapoptotic effects in vitro on synovial fibroblasts and macrophages from rheumatoid arthritis patients.

There is a need for novel, safer and cheaper drugs for the therapy of rheumatoid arthritis (RA), better targeted against the cellular processes involved in the disease pathogenesis. Using advanced analysis of microscopic images and flow cytometry, we demonstrate that naturally occurring xanthone and benzophenone derivatives exert strong, dose- and O2 concentration-dependent anti-proliferative and pro-apoptotic effects on RA patients' fibroblast-like synoviocytes (FLS) and macrophages. Suspensions containing fibroblasts, macrophages and other infiltrating cells were obtained from inflamed synovial tissue collected from female RA patients. Cells were grown in the presence of xanthone (mangiferin, isomangiferin, neomangiferin, norathyriol) or benzophenone (iriflophenone 3-C-glucoside, maclurin) derivatives for 48h or 7days, at 5% or 21% O2. Proportions of macrophages, FLS and infiltrating T cells undergoing apoptosis (annexin- or annexin and 7-AAD-positive) were determined by flow cytometry. The extent of late apoptosis (DNA degradation) was assessed by fluorescent microscopy and image analysis in cultures where DNA was stained with Hoechst 33342. Majority of tested compounds exert anti-proliferative and pro-apoptotic, O2-dependent effects on T cells, FLS and macrophages. The results indicate that xanthone- and benzophenone-rich plant products provide a basis for the development of dietary strategy for rheumatoid arthritis management.

Schisandra lignans production regulated by different bioreactor type.

Schisandra chinensis (Chinese magnolia vine) is a rich source of therapeutically relevant dibenzocyclooctadiene lignans with anticancer, immunostimulant and hepatoprotective activities. In this work, shoot cultures of S. chinensis were grown in different types of bioreactors with the aim to select a system suitable for the large scale in vitro production of schisandra lignans. The cultures were maintained in Murashige-Skoog (MS) medium supplemented with 3mg/l 6-benzylaminopurine (BA) and 1mg/l 1-naphthaleneacetic acid (NAA). Five bioreactors differing with respect to cultivation mode were tested: two liquid-phase systems (baloon-type bioreactor and bubble-column bioreactor with biomass immobilization), the gas-phase spray bioreactor and two commercially available temporary immersion systems: RITA® and Plantform. The experiments were run for 30 and 60 days in batch mode. The harvested shoots were evaluated for growth and lignan content determined by LC-DAD and LC-DAD-ESI-MS. Of the tested bioreactors, temporary immersion systems provided the best results with respect to biomass production and lignan accumulation: RITA® bioreactor yielded 17.86g/l (dry weight) during 60 day growth period whereas shoots grown for 30 days in Plantform bioreactor contained the highest amount of lignans (546.98mg/100g dry weight), with schisandrin, deoxyschisandrin and gomisin A as the major constituents (118.59, 77.66 and 67.86mg/100g dry weight, respectively).

Accumulation of dibenzocyclooctadiene lignans in agar cultures and in stationary and agitated liquid cultures of Schisandra chinensis (Turcz.) Baill.

Schisandra chinensis plant in vitro cultures were maintained on Murashige and Skoog (MS) medium supplemented with 3 mg/l 6-benzyladenine (BA) and 1 mg/l 1-naphthaleneacetic acid (NAA) in an agar system and also in two different liquid systems: stationary and agitated. Liquid cultures were grown in batch (30 and 60 days) and fed-batch modes. In the methanolic extracts from lyophilized biomasses and in the media, quantification of fourteen dibenzocyclooctadiene lignans identified based on co-chromatography with authentic standards using high-performance liquid chromatography with diode array detection (HPLC-DAD) and/or liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI-MS) methods. For comparison purposes, phytochemical analyses were performed of lignans in the leaves and fruits of the parent plant. The main lignans detected in the biomass extracts from all the tested systems were schisandrin (max. 65.62 mg/100 g dry weight (DW)), angeloyl-/tigloylgomisin Q (max. 49.73 mg/100 g DW), deoxyschisandrin (max. 43.65 mg/100 g DW), and gomisin A (max. 34.36 mg/100 g DW). The highest total amounts of lignans in the two tested stationary systems were found in extracts from the biomass harvested after 30 days of batch cultivation: 237.86 mg/100 g DW and 274.65 mg/100 g DW, respectively. In the agitated culture, the total content reached a maximum value of 244.80 mg/100 g DW after 60 days of the fed-batch mode of cultivation. The lignans were not detected in the media. This is the first report which documents the potential usefulness of S. chinensis shoot cultures cultivated in liquid systems for practical purposes.

Simultaneous determination of scopolamine, hyoscyamine and anisodamine in in vitro growth media of selected Solanaceae hairy roots by CE method.

An electrophoretic method for fast separation of three tropane alkaloids (hyoscyamine, anisodamine and scopolamine) was presented. The substances were complete resolved in less than one minute due to utilization of relatively short capillary (20.2cm effective length) and high voltage (25kV). Detector probing frequency was found among the parameters that significantly affected the detection sensitivity. The performed experiments showed insufficient available probing frequency of used commercial spectrophotometric detector according to capillary electrophoresis (CE) separation potential. Under the optimized conditions the background electrolyte (BGE) was composed of 20mM Tris, 6mM HCl and 20mM NaCl (pH 8.50). All analyses were carried out in fused silica capillaries of 50µm (inner diameter) and 31.2cm (total capillary length). Samples were injected hydrodynamically (5s; 3.45kPa) without any sample preparation step and separation was performed at 25kV. The elaborated method was applied in plant cultures growth media analysis after incubation with hairy roots of selected Solanaceae species. The performed experiments proved the usefulness of CE in quality control of biotechnological processes.

Production of therapeutically relevant indolizidine alkaloids in Securinega suffruticosa in vitro shoots maintained in liquid culture systems.

Microshoot cultures of the Chinese medicinal plant Securinega suffruticosa (Pall.) Rehd. were established and evaluated for the presence of therapeutically relevant indolizidine alkaloids securinine (S) and allosecurinine (AS). The cultures were maintained in shake flasks (SFs) and a bubble column bioreactor (BCB) using the modified Murashige's shoot multiplication medium supplemented with 1.0 mg l(-1) benzyladenine (BA), 3.0 mg l(-1) 2-isopentenyladenine (2iP), and 0.3 mg l(-1) 1-naphthaleneacetic acid (NAA). The influence of light and medium supplementation strategies with biosynthesis precursor (lysine (LY)) and nutrient formulations (casein hydrolysate (CH) and coconut water (CW)) on biomass growth and alkaloid production were investigated. SF cultures grown in the presence of light yielded up to 6.02 mg g(-1) dry weight (DW) S and 3.70 mg g(-1) DW AS, corresponding to the respective productivities of 98.39 and 60.21 mg l(-1). Among feeding experiments, CW supplementation proved most effective for SF-grown shoots, increasing biomass yield and AS productivity by 52 and 44 %, respectively. Maximum concentrations of securinine (3.25 mg g(-1) DW) and allosecurinine (3.41 mg g(-1) DW) in BCB cultures were achieved in the case of 1.0 g l(-1) LY supplementation. These values corresponded to the productivities of 42.64 and 44.47 mg per bioreactor, respectively.