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Different types of microshoot cultures (agar, stationary liquid, agitated, and bioreactors) of

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.

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). The influence of bioreactor type on the accumulation of phenolic acids and flavonoids in microshoot cultures of Schisandra chinensis was proven and optimized.

Rhododendron tomentosum (marsh tea, previously Ledum palustre), a fragrant shrub with characteristic evergreen leaves and white flowers, grows in Europe, Asia, and North America. It has been used for centuries in folk medicine to treat rheumatic diseases, lung problems, and infections as well as due to its repellent properties. In North America, the tonic beverage known as “Labrador tea”, derived from the indigenous tradition and made from R. tomentosum, R. groenlandicum and R. neoglandulosum leaves, is prepared until now. The modern biological research confirm anti-inflammatory, analgesic, antimicrobial and insecticidal effect of the discussed plant material, indicating an important role of the essential oil as an active ingredient. However, obtaining the volatile fraction from R. tomentosum ground material for pharmacological studies is difficult because marsh tea is the endangered species in some countries. Moreover, as many as ten chemotypes of R. tomentosum on the Eurasian continent have been distinguished, due to the chemical composition of the essential oil. Such heterogeneity of the plant material is problematic, assuming its use for medical purposes. Therefore, the shoot in vitro culture was initiated for the first time for receiving the R. tomentosum biomass, being the complex source of biologically active volatile compounds, regardless of environmental conditions. The microshoots were subsequently adapted for large laboratory scale cultivation in commercial and prototype bioreactors. The RITA® temporary immersion system, containing SH medium with 24.60 μM 2-isopentenyladenine and 592.02 μM adenine, provided the highest growth parameters of biomass (Gi = 280%) and the intensified biosynthesis of the essential oil (500 μl 100 g−1 dry weight), surpassing the productivity of the aged shoots of the mother plant (300 μl 100 g−1 dry weight). The main terpenes of the obtained volatile fraction were ledene oxide (II) (13%), shyobunone (8%), p-cymene (7%), and alloaromadendrene (6%). In order to increase the essential oil content in the R. tomentosum microshoots, elicitation strategy was applied, using methyl jasmonate and the selected abiotic and biotic elicitors. In response to stress caused by the aphid extract and Pectobacterium carotovorum lysate, the accumulation of the volatile fraction increased by 14%. In addition, the full protocol for micropropagation of marsh tea was developed, including initiation, multiplication, elongation, rooting, hardening, and adaptation of the seedlings to in vivo conditions, for ex situ protection of the discussed endangered species. This article reviews the importance of R. tomentosum from a medical point of view as well as the biotechnological approach obtaining an alternative source of this valuable biomass.

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.

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&mdash, 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&mdash, 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&mdash, 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.

Rhododendron tomentosum Harmaja, previously Ledum palustre L., is a fragrant plant growing in swampy areas, which is characterized by well-known repellent properties and the anti-arthritic potential. In the present study, R. tomentosum microshoots cultivated in RITA® bioreactor, providing the alternative, environment-independent source of the valuable essential oil, were treated for 7 days with copper and nickel salts, methyl jasmonate, chitosan, ergosterol, lysates of: Candida albicans, Escherichia coli, Enterobacter sakazaki, Pectobacterium carotovorum and Dickeya dadantii, to stimulate the production of secondary metabolites. Moreover, for the first time, the aphid ethanol extract was tested as an elicitor of insect origin. Suspensions of all pathogens added to in vitro culture reduced significantly the biomass growth parameters. The application of the aphid extract and lysate of P. carotovorum increased the volatile fraction content by 14%, while Ni2+ and ergosterol by 8%. The influence of the studied elicitors on the chemical composition of R. tomentosum essential oils was presented and discussed thoroughly, taking into account a percentage of 22 main terpenoid compounds, determined by GC/MS (i.a. p-cymene, geranyl acetate, δ-cadinene, shyobunone, methyl everninate, ledene oxide (II) and alloaromadendrene). The biosynthesis of monoterpenes was primarily proven to be affected by the elicitation. The conducted experiments suggest that R. tomentosum is rather resistant to elicitation strategies, both abiotic and biotic as well as methyl jasmonate.

Rhododendron tomentosum (Ledum palustre) is a bog shrub used in traditional medicine for treatment of respiratory and rheumatic diseases. Due to the large variability of the chemical composition of its essential oil, depending on the habitat, the in vitro cultures were established as the alternative source of the volatile fraction. There is a need to monitor a quality of the field grown as well as in vitro plant material, especially if potentially toxic aromadendrane derivatives are concerned. In this study the HPTLC method was developed and validated for quantification of ledol and alloaromadendrene in R. tomentosum essential oils obtained from the plants collected in various locations as well as in vitro cultures. For qualitative analysis, chromatograms were developed on HPTLC Si 60 plates at distance of 50 mm without preconditioning using hexane: ethyl acetate (9:1) and visualized with p-anisaldehyde reagent. For quantitative analysis, chromatograms were developed on HPTLC Si 60 plates at distance of 40 mm with 20 min preconditioning using hexane: ethyl acetate (9:1), visualized with vanillin/phosphoric acid reagent and subjected to densitometric detection (560 nm). The content of ledol and alloaromadendrene in different samples was determined using the validated HPTLC method (12–280 mg and 2–60 mg 100 g−1 dried plant material, respectively) and was compared with GC/MS results. The bioautographic antioxidant HPTLC assays in DPPH and in riboflavin-light-NBT systems as well as the test for xanthine oxidase inhibition were employed for screening of the biological activity of the R. tomentosum essential oils.

Anthocyanins, flavonols, and phenolic acids were estimated in methanolic extracts from the fruits and leaves of three chokeberries—Aronia melanocarpa, Aronia arbutifolia, and Aronia ×prunifolia. The fruits contained significant amounts of cyanidin glycosides (0.3–323.2 mg/100 g DW) and two phenolic acids: chlorogenic acid (16.3–273.5 mg/100 g DW) and neochlorogenic acid (92.3–212.6 mg/100 g DW). The leaf extracts contained high amounts of flavonols: quercetin, quercitrin, and rutin (62.1–367.0 mg/100 g DW), as well as chlorogenic acid, neochlorogenic acid, and rosmarinic acid (max. 724.2, 482.7, 154.7 mg/100 g DW, respectively). Of the examined materials, A. arbutifolia leaves were characterized by the highest total phenolics content (9148.2 mg gal. ac. Eq./100 g DW) and showed the highest antioxidant activity in DPPH and FRAP assays. The results demonstrate that fruits of A. arbutifolia and A. ×prunifolia are a rich source of antioxidants and can be used as plant raw materials, alternatively to A. melanocarpa berries. Leaves of the investigated species are of potential therapeutic and dietary interest because of their high flavonol and phenolic acid content.

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.

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 (

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. Electronic supplementary material The online version of this article (10.1007/s00253-018-8981-x) contains supplementary material, which is available to authorized users.

In the presented work, plant growth regulators (PGRs) were tested for their influence on biomass growth and accumulation of therapeutically-relevant indolizidine alkaloids in Securinega suffruticosa (Phyllanthaceae) callus cultures. The study included 9 auxins [2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid, indole-3-acetic acid (IAA), 4-chlorophenoxyacetic acid, indole-3-butyric acid, 4-amino-3,5,6-trichloropicolinic acid, β-naphthoxyacetic acid, 1-naphthaleneacetic acid, indole-3-propionic acid, one polar auxin transport inhibitor (2,3,5-triiodobenzoic acid) and 7 cytokinins [2-isopentenyladenine, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea, N-(2-chloro-4-pyridinyl)-N′-phenylurea, diphenylurea, zeatin, kinetin (KIN), 6-benzylaminopurine]. The respective PGRs were applied at 0.5 and 5.0 mg l−1, together with 5.0 or 0.5 mg l−1 KIN or 2,4-D (for auxins and cytokinins, respectively). The calli subjected to different phytohormone combinations (39 modifications in total) were evaluated for growth and alkaloid content, and the results tested for statistical differences. The highest concentrations of securinine (1.73 mg g−1 DW) and allosecurinine (3.11 mg g−1 DW) were recorded in the callus grown in the presence of 0.5 mg L−1 IAA and 5.0 mg L−1 KIN. To the best of the authors’ knowledge, this is the first such comprehensive report concerning the influence of PGRs on alkaloid accumulation in plant in vitro cultures.

Different in vitro culture systems of the East-Asian origin medicinal plant species − Schisandra chinensis , were tested in order to investigate their potential for the accumulation of two groups of phenolic compounds. In vitro cultures were maintained on the Murashige and Skoog (MS) medium supplemented with 3 mg/l BA and 1 mg/l NAA in an agar system (30- and 60-day growth cycles), and also in two different liquid systems: stationary and agitated. Stationary liquid cultures were grown in batch (30- and 60-day growth cycles) and fed-batch modes. Of the twenty compounds, seven free phenolic acids and of the eleven compounds, five flavonoids were quantified in methanolic extracts from lyophilized biomass and in the growth media using the RP-HPLC-DAD method. For comparison purposes, phytochemical analyses of leaf and fruit extracts from the parent plant were also conducted. The estimated compounds were not detected in the growth media. The highest total amounts of phenolic acids (71.48 mg/100 g DW) and flavonoids (29.36 mg/100 g DW) were found in extracts from the biomass of agar cultures harvested after 30 days of cultivation. The main metabolites in all the tested systems were: protocatechuic acid (max. 35.69 mg/100 g DW), chlorogenic acid (max. 13.05 mg/100 g DW), and quercitrin (max. 27.43 mg/100 g DW).

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia and progressive joint destruction. Despite aggressive treatment with anti-rheumatic drugs, progressive destruction of joints continues to occur in RA patients, who subsequently require joint surgery. A lot of evidence suggest that fibroblast-like synovial cells (FLS) play crucial role in joint degradation and the propagation of inflammation in RA. The expansion of fibroblast populations in the joint results primarily from the inhibition of pro-apoptotic pathways, rather than large scale proliferation. Because multiple factors, which contribute to fibroblast activation and enhance their destructive potential, are under control of transcription factor NF-κB, this pathway presents an interesting target for RA therapy. However, due to the lack of specificity, NF-κB inhibitors may exert severe side effects. Given the above, there has recently been more interest in natural substances of plant origin which are regarded as a safe alternatives for synthetic drugs. Mangiferin, the naturally occurring polyphenol with excellent anti-inflammatory and antioxidative properties, exhibits strong pro-apoptotic effect toward synoviocytes isolated from human synovia. Moreover, it shows no cytotoxicity toward cultivated chondrocytes and reduces the levels of matrix metalloproteinases. Considering that mangiferin is a natural constituent of foods and traditional herbal medicines, showing fewer adverse effects and low toxicity, we hypothesize that it may prove effective in the treatment of RA and prevention against joint destruction.

The influence of plant growth regulators on biomass growth and the accumulation of medicinally-relevant isoflavone phytoestrogens, derivatives of genistein and daidzein (8 compounds including aglycones, glucosides and glucoside esters) in callus cultures of Genista tinctoria (Fabaceae) was examined. The experiments included 10 auxins [2,4-dichlorophenoxyacetic acid (2,4-D), p-chlorophenoxyacetic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, 1-naphthaleneacetic acid, β-naphthoxyacetic acid, picloram, 2,3,5-triiodobenzoic acid (TIBA), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)] and 7 cytokinins [6-benzylaminopurine, forchlorfenuron, 1,3-diphenylurea, 2-isopentenyladenine, kinetin (KIN), thidiazuron, zeatin] applied at 0.5 and 5.0 mg l−1, jointly with 5.0 or 0.5 mg l−1 KIN or 2,4-D (for auxins and cytokinins, respectively—36 phytohormone combinations in total). Statistical analysis of the relationships between callus growth [expressed as growth index (Gi)] and the accumulation of isoflavones showed positive correlation in the cytokinin group (rxy values from 0.13 to 0.61) and negative correlation within auxins (rxy values from −0.31 to −0.39). Among the cytokinins tested, the highest isoflavone content (6,436.26 mg/100 g dry weight) and the fastest biomass growth (Gi = 892.46 %) were obtained for 0.5 mg l−1 KIN used jointly with 5.0 mg l−1 2,4-D. In the group of auxins, the combination of 0.5 mg l−1 TIBA and 5.0 mg l−1 KIN provided the fastest culture growth (Gi = 983.07 %) and the isoflavone concentration of 10,474.23 mg/100 g dry weight, which is so far the highest amount of these metabolites achieved in callus cultures of higher plants.

Introduction Hyoscyamine and scopolamine, anti-cholinergic agents widely used in medicine, are typically obtained from plants grown under natural conditions. Since field cultivation entails certain difficulties (changeable weather, pests, etc.), attempts have been made to develop a plant in vitro culture system as an alternative source for the production of these compounds. During experiments to locate the limiting steps in the biotechnological procedure, it is important to monitor not only the levels of the final products but also the changes in the concentration of their precursors. Objective To develop a HPTLC method for the separation and quantitation of the main tropane alkaloids hyoscyamine and scopolamine, their respective direct precursors littorine and anisodamine, and cuscohygrine, a product of a parallel biosynthetic pathway that shares a common precursor (N-methyl-∆1-pyrrolium cation) with tropane alkaloids. Methods Using alkaloid extracts from Atropa baetica hairy roots, different TLC chromatographic systems and developing procedures were investigated. Results Full separation of all compounds was obtained on HPTLC Si60 F254 plates preconditioned with mobile phase vapours (chloroform:methanol:acetone:25% ammonia ratios of 75:15:10:1.8, v/v/v/v). The chromatograms were developed twice (at distances of 4.0 and 3.0 cm) in a Camag twin trough chamber and visualised with Dragendorff's reagent. Densitometric detection (λ = 190 and 520 nm) was used for quantitative analyses of the different plant samples. Conclusion This method can be recommended for quantitation of hyoscyamine, scopolamine, anisodamine, littorine and cuscohygrine in different plant material (field grown vs. in vitro cultures). Copyright © 2013 John Wiley & Sons, Ltd.

Thin layer chromatographic methods for quantitative determination of nightshade-specific tropane (l-hyoscyamine, scopolamine) and steroidal alkaloids (α-solanine, α-chaconine) in goji berries (L. barbarum L., Solanaceae) were developed. The analysis of tropane derivatives included separation on silica gel-coated HPTLC plates using mobile phase consisted of chloroform:methanol:acetone:25% ammonium hydroxide (75:15:10:1.6 v/v/v/v), derivatization with Dragendorff reagent and scanning densitometry at λ - 520nm in reflectance/absorption mode. Steroidal alkaloid analysis employed silica gel-coated TLC plates, mobile phase composed of chloroform:methanol:water:25% ammonium hydroxide (70:30:4:2 v/v/v/v), derivatization with Carr-Price reagent and video densitometry under white light in the reflectance mode. Both methods were validated with respect to linearity, precision, accuracy and sensitivity, enabling reliable detection of tropane derivatives and α-solanine at 1.20 and 7.84mgper100g fresh material, respectively. None of the analyzed compounds were detected in fruits, leaves, stems and roots of three L. barbarum varieties ('No. 1', 'New Big' and 'Amber Sweet Goji').

The use of celery (Apium graveolens, Apiaceae family) as a vegetable dates back to sixteenth century Italy when the first cultivated varieties appeared. At present, it is cultivated worldwide as a spice and food crop. Celery owes its distinctive aroma to the presence of essential oil, of which the most characteristic compounds are phthalides including 3-n-butylphthalide and sedanolide. Celery volatile oil is obtained mostly from specialized seed crops and used as a flavoring agent in food and perfume industries. The essential oil obtained from celery also gained some interest as a potential antispoilage agent devoid of the drawbacks of synthetic preservatives. This review summarizes the production, chemical composition, and selected properties (antibacterial, antifungal, and antioxidant) of celery volatiles. Their possible utilization in food preservation is also discussed.

In the presented work, an insight was made into the polyphenolic composition of intact plant material and in vitro cultures of indigenous South African plant Cyclopia subternata Vogel (honeybush). Ethyl acetate fractions of methanol extracts were separated by means of gravity column chromatography and/or semipreparative HPLC on two serially connected monolithic RP-18 columns. The structures of the isolated compounds were determined by means of 1D and 2D NMR techniques and additionally confirmed by LC-DAD-ESI-MS. Apart from the previously described honeybush components, that is mangiferin ( 1 ), scolymoside ( 2 ), hesperidin ( 3 ) and narirutin ( 4 ), three additional compounds: iriflophenone 3- C - β -glucoside (benzophenone) ( 5 ), phloretin 3′,5′-di- C - β -glucoside (dihydrochalcone) ( 6 ), and isorhoifolin (flavone) ( 7 ) were identified for the first time in the herb of C. subternata . Additionally, three isoflavone glucosides, namely calycosin 7- O - β -glucoside ( 8 ), rothindin ( 9 ) and ononin ( 10 ), which had not been previously reported in Cyclopia plants, were identified in the callus of the above species. As far as the authors are concerned, this is the first report on the presence of benzophenone and dihydrochalcone derivatives in Cyclopia genus.

The Aronia genus (Rosaceae family, Maloideae subfamily) includes two species of native North American shrubs: Aronia melanocarpa (Michx.) Ell. (black chokeberry) and Aronia arbutifolia (L.) Pers. (red chokeberry). The fruits of A. melanocarpa have been traditionally used by Potawatomi Native Americans to cure colds. In the first half of the 20(th) century, cultivars of black chokeberry were introduced to the Soviet Union and other European countries, providing fruits used by food industry. At present, it is used mainly for juice, jam, and wine production, as well as an ornamental plant. Among other substances, the berries of A. melanocarpa contain anthocyanins and procyanidins, possessing strong antioxidative potential. Numerous health-promoting activities-namely, antioxidative, antimutagenic, anticancer, cardioprotective, hepatoprotective, gastroprotective, antidiabetic, anti-inflammatory, antibacterial, antiviral, radioprotective, and immunomodulatory-have been demonstrated for black chokeberry extracts by both in vitro and in in vivo studies. The presented review summarizes the information concerning botany, cultivation, chemical composition, and pharmacological activities of Aronia plants.

The chemical composition of hydrodistilled essential oils obtained from aerial parts and roots of selected Caryopteris (‘bluebeard’) species (C. incana, C. mongolica, Caryopteris × clandonensis), as well as the newly established in vitro shoot and adventitious root cultures of the above plants, was analyzed by gas chromatography–mass spectrometry. Essential oil content and composition differed significantly depending on the type of plant material analyzed. Adventitious roots were characterized by the highest essential oil yield, reaching 1.8 % V/DW in Caryopteris × clandonensis. Limonene and cedrol were the main components of the essential oil derived from aerial parts of the intact plants (11.9–16.0 and 10.7–10.9 %, respectively), whereas the volatile fractions of the in vivo roots of all species contained large amounts of 3,5-bis(1,1-dimethyl)-phenol (12.9–26.2 %). 1,8-cineole, absent in the intact plant materials, was the dominating volatile constituent of the essential oils obtained from in vitro shoots (24.8–34.2 %). The volatile oil derived from adventitious root cultures consisted primarily of 1-octen-3-ol (19.7–31.5 %) and medicinally relevant diterpenoids: abietatriene and trans-totarol, which were accumulated in considerable quantities, especially in the adventitious roots of C. clandonensis (21.6 and 29.2 %, respectively).

Labrador tea is a name for the dried leaves of Rhododendron groenlandicum, R. tomentosum or R. neoglandulosum (family Ericaceae, previously genus Ledum) as well as for the beverage native to North America, which is made from them. The above species are rich in the essential oil, which gives a conifer aroma to the tisane. Labrador tea is a valuable source of ascorbic acid, with tonic, improving digestion and relaxing activity. However, this beverage should not be drunk more than once daily because of the ledol and grayanotoxin toxicity. The common recipe for making Labrador tea is to add one teaspoonful of dried leaves to one cup of boiling water and to brew for 5 min. It is often sweetened or enriched with other flavors. Additionally, Labrador tea dried leaves are used to spice meat, soups, sauces, salads, beer, cakes and other dishes. In agriculture, its insecticidal properties can be useful for controlling pests. © 2014 Society of Chemical Industry

From the flowers of meadowsweet and inflorescence of hawthorn the whole set of phenolic acids and flavonoids was analysed by TLC . Phenolic compounds were determined both as free ones and those liberated by hydrolysis. Moreover, ethyl ether and ethyl acetate extracts obtained from the analysed plants before and after alkaline and enzymatic hydrolyses were evaporated under reduced pressure and residues were analysed for their antioxidative properties. The weakest antioxidative activity was observed with the remaining residue after evaporation of ethyl ether extract obtained from enzymatically (β-glucosidase) hydrolysed hawthorn inflorescence water extract. The strongest antioxidative activity was noticed with the remaining residues after evaporation of ethyl ether extracts obtained from non-hydrolysed and hydrolysed in alkaline conditions of meadowsweet flower water extracts. The residues from meadowsweet flowers exhibited stronger antioxidative properties than residues obtained from hawthorn inflorescence and can be recommended as margarine preservatives.

A cell suspension culture of Tabernaemontana divaricata, that had lost alkaloid production, was still capable of producing a similar pattern of alkaloids as directly after its initiation. When fed with early precursors, such as tryptamine and loganin, 57% of the precursors was converted into indole alkaloids such as strictosidine, vallesamine, O-acetylvallesamine and voaphylline. Apparently most of the cell factory has remained stable during the many years of subculturing. Only an early step of the biosynthesis the flux seems to be diverted to other pathways.

Hairy root cultures of Hyoscyamus niger were cultivated in shake-flasks, a bubble-column bioreactor and a hybrid bubble-column/spray bioreactor and evaluated for alkaloid production. The latter gave the highest anisodamine content (0.67 mg/g dry wt) whereas scopolamine, hyoscyamine and cuscohygrine concentrations were highest in the bubble-column reactor (5.3, 1.6 and 26.5 mg/g dry wt, respectively). Both bioreactors gave similar productivities of scopolamine (1 and 0.98 mg/l day) and cuscohygrine (5 and 5.4 mg/l day), but anisodamine productivity was 3.5-fold higher in the hybrid bioreactor (HB) (0.02 and 0.07 mg/l day, respectively). Elicitation with methyl jasmonate increased scopolamine productivity by 146 % in roots grown in the HB whereas their permeabilization with DMSO caused 4-, 5-, 25- and 28-fold increase in scopolamine, hyoscyamine, anisodamine and cuscohygrine concentrations in the growth medium. In situ extraction with Amberlite XAD-2 doubled scopolamine productivity in the hybrid reactor after 50 days.

Suspension cultures of the endemic South-African plant Cyclopia subternata were established for the first time and evaluated for the presence of isoflavones. The influence of light, as well as medium supplementation strategies with phenylalanine, casein hydrolysate and coconut water on biomass growth and isoflavone production were examined. The highest levels of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (275.57, 125.37 and 147.28 mg/100 g DW, respectively) were recorded for cultures grown in the absence of light, whereas coconut water substantially promoted biomass growth. Cell suspensions were subsequently grown in the 2-l stirred-tank bioreactor. Maximum productivity of 7-O-β-glucosides of calycosin, pseudobaptigenin and formononetin (0.96, 0.44 and 0.22 mg l(-1) day(-1), respectively) in bioreactor-cultivated cells was obtained for biomass grown in the dark and supplemented with coconut water. The results indicate that C. subternata suspension cultures can be utilised for the production of the specified isoflavone derivatives absent in the intact plant.

A fast and efficient method for the isolation of the C-glucosidated xanthones mangiferin and isomangiferin from the South-African plant Cyclopia genistoides was developed for the first time. The procedure involved extraction, liquid–liquid partitioning with ethyl acetate and subsequent precipitation of mangiferin and isomangiferin from methanol and acetonitrile–water fractions, respectively. Additionally, two benzophenone derivatives: 3-C-β-glucosides of maclurin and iriflophenone, were isolated from C. genistoides extracts using semi-preparative HPLC. Apart from the above, the isolation procedure also yielded hesperidin and small amounts of luteolin. The structures of the compounds were determined by 1D and 2D NMR experiments and/or LC-DAD-ESI–MS. The selected Cyclopia constituents were screened for pro-apoptotic activity on TNF-α-stimulated synovial cells isolated from rheumatoid arthritis patients. The strongest effect, measured as percent of apoptotic cells, was recorded for isomangiferin (75%), followed by iriflophenone 3-C-β-glucoside (71%), hesperidin (67%) and mangiferin (65%). The results are encouraging for further studies on the use of the above compounds in the treatment of rheumatoid arthritis.

Honeybush tea is a well-recognized South-African beverage manufactured from the aerial parts of several Cyclopia spp., which are characteristic of the fynbos shrublands of the Cape Floristic Region. The harvested material is traditionally fermented in order to develop the characteristic sweet aroma of the final product, or dried in unfermented state to give the so called ‘green honeybush’. Honeybush infusions are characterized by distinctive honey-like aroma, lack of caffeine and the presence of numerous phenolic compounds, including xanthones, flavanones and flavones. Cyclopi a extracts exhibit substantial antioxidant, antimutagenic and phytoestrogenic effects, which were demonstrated with in vitro and animal models. The growing popularity of honeybush makes it an important export commodity, but has also forced the need to refine the tea production process and standardize procedures in order to obtain a high-quality product. The current review covers botanical issues, tea manufacture, chemical composition and biological activity of Cyclopia extracts.

Rhododendron tomentosum Harmaja (previously: Ledum palustre) is a fragrant evergreen shrub found in peaty soils in northern Europe, Asia and North America, commonly referred to as wild rosemary, marsh tea, marsh rosemary or northern Labrador tea. At least since the eighteenth century it has been used in ethnomedicine for the treatment of various ailments, such as rheumatism, cough, cold and insect bites, as well as a repellent. The essential oil of wild rosemary with the rich polyphenolic fraction possesses analgesic, anti-inflammatory, antimicrobial, antiviral, antifungal and insecticidal potential, demonstrated by in vivo and in vitro studies. In addition, recent scientific research reported the promising antidiabetic, antioxidant and anticancer properties. This review summarizes the information concerning taxonomy, botany, ecology, chemical composition, biological activities, toxicology and traditional and contemporary application of Rhododendron tomentosum plants.

Hyoscyamine and scopolamine, anti-cholinergic agents widely used in medicine, are typically obtained from plants grown under natural conditions. Since field cultivation entails certain difficulties (changeable weather, pests, etc.), attempts have been made to develop a plant in vitro culture system as an alternative source for the production of these compounds. During experiments to locate the limiting steps in the biotechnological procedure, it is important to monitor not only the levels of the final products but also the changes in the concentration of their precursors.To develop a HPTLC method for the separation and quantitation of the main tropane alkaloids hyoscyamine and scopolamine, their respective direct precursors littorine and anisodamine, and cuscohygrine, a product of a parallel biosynthetic pathway that shares a common precursor (N-methyl-∆(1) -pyrrolium cation) with tropane alkaloids.Using alkaloid extracts from Atropa baetica hairy roots, different TLC chromatographic systems and developing procedures were investigated.Full separation of all compounds was obtained on HPTLC Si60 F254 plates preconditioned with mobile phase vapours (chloroform:methanol:acetone:25% ammonia ratios of 75:15:10:1.8, v/v/v/v). The chromatograms were developed twice (at distances of 4.0 and 3.0 cm) in a Camag twin trough chamber and visualised with Dragendorff's reagent. Densitometric detection (λ = 190 and 520 nm) was used for quantitative analyses of the different plant samples.This method can be recommended for quantitation of hyoscyamine, scopolamine, anisodamine, littorine and cuscohygrine in different plant material (field grown vs. in vitro cultures).

The plants from Cyclopia genus (Honeybush) are endemic, South-African shrubs characteristic for the fynbos plant formation of the Cape Floristic Region. Aerial parts of several Cyclopia species, including C. genistoides, are used to manufacture the honeybush herbal tea, recognized by its dinstictive sweet, honey-like aroma, low level of tannins and lack of caffeine [1,2]. Extracts obtained from Cyclopia plants are rich in polyphenols, including xanthones (mangiferin and isomangiferin), flavanones (hesperidin), flavones and isoflavones, and as such exhibit substantial antioxidative and antimutagenic activity [1,2,3]. In vitro propagation protocol for C. genistoides has been established. Solid Schenk-Hildebrandt (SH) medium supplemented with 2.0mg l-1 6-(γ,γ-dimethylallylamino)purine (2iP) and 0.22mg l-1 thidiazuron (TDZ) have been shown to be the best for shoot culture initiation, as well as for the development of high number of microshoots. Shoot elongation procedure involved explant cultivation on the full strength SH medium supplemented with 1.0mg l-1 indole-3-butyric acid (IBA), followed by 30-day long growth on SH medium with reduced sucrose level, supplemented with the same amount of IBA. As a result, elongated shoots representing intact plant morphology were obtained. Root induction was achieved on solid SH medium with reduced sucrose and nitrate concentrations, supplemented with 5.0mg l-1 indole-3-acetic acid (IAA) and 50.0mg l-1 citric acid. The regenerated plants were acclimatized in the glasshouse, with the use of peat/gravel/perlite subtrate (1:1:1). Both regenerated plants and in vitro microshoots of C. genistoides were evaluated for the production of selected polyphenolic compounds (LC-ESI-MS and LC-DAD analysis). Acknowledgements: the study was supported by grant no N N302 041936 from Polish Ministry of Education and Science. References: 1. Joubert E. et al. (2008)J. Ethnopharmacol. 19:376–412. 2. Kokotkiewicz A., Luczkiewicz M. (2009) Fitoterapia 80:3–11. 3. Joubert E. et al. (2008)J. Agric. Food Chem. 56:954–963.

The genus Cyclopia (Fabaceae family) includes a number of shrubs endemic to Cape Fynbos region of South Africa. The most common of these plants is Cyclopia intermedia E. Mey., used together with other Cyclopia species (mainly C. subternata and C. sessiliflora) to manufacture the honeybush herbal tea which has been produced in South Africa roughly since the beginning of the 19th century. Honeybush infusions are gaining popularity due to their characteristic honey-like flavour, low tannin content, absence of caffeine and potential health effects related to their antimutagenic and antioxidant properties. The presented review summarizes information concerning botany, chemistry, biological activity and application of Cyclopia plants.

The UV-honey guides of Rudbeckia hirta were investigated by UV-photography, reflectance spectroscopy, LC-MS analysis and studies of the enzymes involved in the formation of the UV-absorbing flavonols present in the petals. It was shown for the first time that the typical bull's eye pattern is already established at the early stages of flower anthesis on the front side of the petal surface, but is hidden to pollinators until the buds are open and the petals are unfolded. The rear side of the petals remains UV-reflecting during the whole flower anthesis. Studies on the local distribution of 19 flavonols across the petals confirmed that the majority are concentrated in the basal part of the ray flower. However, in contrast to the earlier studies, eupatolitin 3-O-glucoside (6,7-dimethoxyquercetin 3-O-glucoside) was present in both the basal and apical parts of the petals, whereas eupatolin (6,7-dimethoxyquercetin 3-O-rhamnoside) was exclusively found in the apical parts. The enzymes involved in the formation of the flavonols in R. hirta were demonstrated for the first time. These include a rare flavonol 6-hydroxylase, which was identified as cytochrome P450-dependent monooxygenase and did not accept any methylated flavonol as substrate. All enzymes were present in the basal and apical parts of the petals, although some of them clearly showed higher activities in the basal part. This indicates that the local accumulation of flavonols in R. hirta is not achieved by a locally restricted presence of the enzymes involved in flavonol formation.

Callus cultures of Vaccinium corymbosum var. bluecrop were optimized for their isoprene derivatives production by supplementing Schenk-Hildebrandt (SH) medium with constant concentration of kinetin (2.32 μᴍ) and two different amounts of selected auxins. Every auxin, except for IBA, used in 10-time higher concentration (2,4D, NAA, IAA, NOA) stimulated biosynthesis of β-sitosterol and inhibited triterpene synthesis. Quantitative analysis of isoprene derivatives in callus biomass collected on the 25th day of the experiment proved that the analyzed callus of Vaccinium corymbosum var. bluecrop synthesized the highest amount of isoprene derivatives after subculturing on SH medium modified with 22.6 μᴍ of 2,4D and 2.32 μᴍ of kinetin.

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.

In vitro shoots of the commercially important South-African legume Cyclopia genistoides were grown in different liquid culture systems [membrane rafts (MR) and a temporary immersion bioreactor (TIB)] and evaluated for the accumulation of phenolic secondary metabolites. The major constituents of the investigated cultures were medicinally relevant xanthones [mangiferin (M) and isomangiferin (IM)] and benzophenone derivatives [iriflophenone 3-C-β-glucoside (IG)]. The highest concentrations of M, IM and IG in MR-grown shoots were 1,843.59, 712.02 and 594.29 mg 100 g−1 dry wt, respectively. Bioreactor cultivation provided higher peak concentrations of M (2,622.70 mg 100 g−1 dry wt), IM (757.40 mg 100 g−1 dry wt) and IG (648.30 mg 100 g−1 dry wt) which corresponded to the respective productivities of 5.48, 1.58 and 3.04 mg l−1 d−1. The results indicate that TIB cultures of C. genistoides may be utilized as an alternative source of the above constituents, particularly IM and IG, which are relatively expensive and so far hardly available from commercial sources.