Production of tropane alkaloids in Hyoscyamus niger (black henbane) hairy roots grown in bubble-column and spray bioreactors

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

Bioreactor type affects the accumulation of phenolic acids and flavonoids in microshoot cultures of Schisandra chinensis (Turcz.) Baill

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 $1^{-1}$ NAA and 3 mg $1^{-1}$ 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)

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

Schisandra chinensis plant in vitro cultures were maintained on Murashige and Skoog (MS) medium supplemented with 3 mg/l 6-benzyladenine (BA) and 1 mg/l 1-naphthaleneacetic acid (NAA) in an agar system and also in two different liquid systems: stationary and agitated. Liquid cultures were grown in batch (30 and 60 days) and fed-batch modes. In the methanolic extracts from lyophilized biomasses and in the media, quantification of fourteen dibenzocyclooctadiene lignans identified based on co-chromatography with authentic standards using high-performance liquid chromatography with diode array detection (HPLC-DAD) and/or liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI-MS) methods. For comparison purposes, phytochemical analyses were performed of lignans in the leaves and fruits of the parent plant. The main lignans detected in the biomass extracts from all the tested systems were schisandrin (max. 65.62 mg/100 g dry weight (DW)), angeloyl-/tigloylgomisin Q (max. 49.73 mg/100 g DW), deoxyschisandrin (max. 43.65 mg/100 g DW), and gomisin A (max. 34.36 mg/100 g DW). The highest total amounts of lignans in the two tested stationary systems were found in extracts from the biomass harvested after 30 days of batch cultivation: 237.86 mg/100 g DW and 274.65 mg/100 g DW, respectively. In the agitated culture, the total content reached a maximum value of 244.80 mg/100 g DW after 60 days of the fed-batch mode of cultivation. The lignans were not detected in the media. This is the first report which documents the potential usefulness of S. chinensis shoot cultures cultivated in liquid systems for practical purposes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-015-7230-9) contains supplementary material, which is available to authorized users

Production of verbascoside, isoverbascoside and phenolic acids in callus, suspension, and bioreactor cultures of Verbena officinalis and biological properties of biomass extracts

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