Search

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).

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.

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).