Search

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.

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.

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).

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.

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.

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).

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.

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.

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.

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

An efficient micropropagation protocol of Cyclopia genistoides (L.) Vent., an indigenous South African shrub of economic importance, was established. In vitro shoot cultures were obtained from shoot tip fragments of sterile seedlings cultured on solid Schenk and Hildebrandt (SH) medium supplemented with 9.84 microM 6-(gamma,gamma-dimethylallylamino)purine (2iP) and 1.0 microM thidiazuron (TDZ). Maximum shoot multiplication rate [(8.2 +/- 1.3) microshoots/explant)] was observed on this medium composition. Prior to rooting, the multiplied shoots were elongated for 60 days (two 30-days passages) on SH medium with one-half sucrose concentration, supplemented with 4.92 microM indole-3-butyric acid (IBA). The rooting of explants was only possible in the case of the elongated shoots. The highest root induction rate (54.8%) was achieved on solid SH medium with one-half sucrose and one-half potassium nitrate and ammonium nitrate concentration, respectively, supplemented with 28.54 microM indole-3-acetic acid (IAA) and 260.25 microM citric acid. The plantlets were acclimatized for 30 days in the glasshouse, with the use of peat/gravel/perlite substrate (1:1:1). The highest acclimatization rate (80%) was obtained for explants rooted with the use of IAA-supplemented medium. The phytochemical profile of the regenerated plants was similar to that of the reference intact plant material. HPLC analyses showed that C. genistoides plantlets obtained by the micropropagation procedure kept the ability to produce xanthones (mangiferin and isomangiferin) and the flavanone hesperidin, characteristic of wild-growing shrubs.