Paweł Kubica’s research while affiliated with Jagiellonian University and other places

Plant biotechnology creates opportunities for the cultivation of plants regardless of their natural habitats, which are often protected or difficult to access. Maintaining suspension cell cultures in bioreactors is an advanced part of biotechnological research that provides possibilities for obtaining plant tissue on a large scale. In this study, the suspension culture cultivation of a Chinese endemic plant, Schisandra henryi, in a stirred tank bioreactor was elaborated for the first time. The phytochemical profile of the tissue extracts was determined with UHPLC-MS/MS for the lignans (fifteen dibenzocyclooctadiene lignans, one aryltetralin lignan, and two neolignans) and UHPLC-DAD-ESI-MS3 for the phenolic compounds (procyanidins and their derivatives and catechin). The maximum total lignan content of 1289 µg/100 g DW was detected for the extracts from suspensions cultured in a bioreactor for over 10 days. For the phenolic compounds, catechin was the dominant compound (390.44 mg/100 g DW). The biological activity of the extracts was tested too. To determine antioxidant potential we used DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), Molybdenum reduction, and β-carotene bleaching tests. The inhibition activity of the S. henryi extract on the enzymes responsible for skin aging, hyaluronidase and tyrosinase, was assessed with spectrophotometry. The cytotoxic activity of the extracts was estimated on human ovarian SKOV-3, cervical HeLa, and gastric AGS cancer cells and non-cancer, normal fibroblasts by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results showed the great potential of the obtained cell biomass extracts. The results of the antioxidant tests indicated their strong ability to reduce the level of free radicals, similarly to that of ascorbic acid, as well as the weak capacity to protect lipids from oxidation. Moreover, anticancer potential, particularly on the cervical and gastric cancer cells, was confirmed too.

A precursor feeding strategy was used for the first time in agitated microshoot cultures of Aronia × prunifolia. This strategy involved the addition of biogenetic precursors of simple phenolic acids (phenylalanine, cinnamic acid, and benzoic acid) and depsides (caffeic acid) into the culture media, with an assessment of its effect on the production of these bioactive compounds. The in vitro cultures were maintained in Murashige–Skoog medium (1 mg/L BAP and 1 mg/L NAA). Precursors at five concentrations (0.1, 0.5, 1.0, 5.0, and 10.0 mmol/L) were fed into the medium at the time of culture initiation (point “0”) and independently on the 10th day of growth cycles. The contents of 23 compounds were determined in methanolic extracts of biomass collected after 20 days of growth cycles using an HPLC method. All extracts contained the same four depsides (chlorogenic, neochlorogenic, rosmarinic, and cryptochlorogenic acids) and the same four simple phenolic acids (protocatechuic, vanillic, caffeic, and syringic acids). Chlorogenic and neochlorogenic acids were the predominant compounds in all extracts (max. 388.39 and 263.54 mg/100 g d.w.). The maximal total contents of all compounds were confirmed after feeding with cinnamic acid (5 mmol/L, point “0”) and caffeic acid (10 mmol/L, point “0”), which caused a 2.68-fold and 2.49-fold increase in the contents of the estimated compounds vs. control cultures (603.03 and 558.48 mg/100 g d.w., respectively). The obtained results documented the efficacy of the precursor feeding strategy in enhancing the production of bioactive compounds in agitated cultures of A. × prunifolia and suggest a potential practical application value.

Sundews (Drosera sp.) are the source of biologically active secondary metabolites: phenolic acids, flavonoids, and 1,4-naphtoquinones. Because obtaining them from the natural environment is impossible (rare and endangered species), in this study modifications of traditional tissue cultures grown in solid medium (SM), such as agitated cultures (ACs) (cultures in liquid medium with rotary shaking) and temporary immersion bioreactors PlantformTM (TIB), were used for multiplication of four sundew species: Drosera peltata, Drosera indica, Drosera regia, and Drosera binata, with simultaneously effective synthesis of biologically active phenolic compounds. Each species cultivated on SM, AC, and TIB was tested for biomass accumulation, the content of total phenols and selected phenolic derivative concentrations (DAD-HPLC), the productivity on of phenolic compounds, as well as its antibacterial activity against two human pathogens: Staphylococcus aureus and Escherichia coli. The results showed that the type of culture should be selected for each species separately. Phytochemical analyses showed that the synthesis of secondary metabolites from the groups of phenolic acids, flavonoids, and 1,4-naphthoquinones can be increased by modifying the cultivation conditions. D. regia turned out to be the richest in phenolic compounds, including 1,4-naphtoquinones: plumbagin and ramentaceone. Extracts from D. indica and D. regia tissue showed strong antibacterial activity against both pathogens. It has also been shown that the growth conditions of sundews can modify the level of secondary metabolites, and thus, their biological activity.

Reynoutria japonica Houtt. (Japanese knotweed) is an invasive plant belonging to the Polygonaceae family. However, being native to East Asia, it has been used in natural medicine for ages because of its broad range of biological activity. Although R. japonica is known as a rich source of phenolic compounds, plant biomass collected from the field may be contaminated with toxic elements like heavy metals, and the level of metabolite accumulation depends on environmental conditions. Therefore, the aim of this study was to derive Japanese knotweed tissue cultures and investigate biomass production and phenolic compound synthesis in in vitro conditions. Plants were cultivated in a traditional agar-solidified medium, in a liquid medium with rotary shaking (agitated culture), and in a temporary immersion bioreactors Plantform™, as well as in soil (ex vitro conditions). Analyses of the growth index and dry weight accumulation were performed on the collected material. In the extracts obtained from examined plants, qualitative and quantitative analysis of phenolic derivatives using DAD-HPLC was conducted to determine the sum of phenolic compounds, as well as the quantity of selected phenolic acids, catechins, and other flavonoids. Results have shown that agitated cultures and temporary immersion bioreactors increased biomass accumulation compared to solid medium cultures. Tissue cultures of R. japonica had increased synthesis of phenolic compounds compared to plants from ex vitro conditions. Shoots and roots from agitated cultures were 2.8- and 3.3-fold richer in catechins, respectively, compared to plants cultivated in soil. Based on the obtained results it can be concluded that agitated and bioreactor cultures are the best source of Japanese knotweed biomass rich in valuable secondary metabolites.

Schisandra henryi is an endemic species of medicinal potential known from traditional Chinese medicine. As part of this study, a complex biotechnological and phytochemical assessment was conducted on S. henryi with a focus on phenolic compounds and antioxidant profiling. The following in vitro cultures were tested: microshoot agar and callus, microshoot agitated, and suspension, along with the microshoot culture in PlantForm bioreactors. Qualitative profiling was performed by ultra-high-performance liquid chromatography with a photodiode array detector coupled with ion-trap mass spectrophotometry with electrospray ionization and then quantitative analysis by high-performance liquid chromatography with a diode array detector using standards. In the extracts, mainly the compounds from procyanidins were identified as well as phenolic acids (neochlorogenic acid, caffeic acid, protocatechuic acid) and catechin. The highest content of phenolic compounds was found for in vitro agar microshoot culture (max. total content 229.87 mg/100 g DW) and agitated culture (max. total content 22.82 mg/100 g DW). The max. TPC measured using the Folin-Ciocalteu assay was equal to 1240.51 mg GAE/100 g DW (agar microshoot culture). The extracts were evaluated for their antioxidant potential by the DPPH, FRAP, and chelate iron ion assays. The highest potential was indicated for agar microshoot culture (90% of inhibition and 59.31 nM/L TEAC, respectively). The research conducted on the polyphenol profiling and antioxidant potential of S. henryi in vitro culture extracts indicates the high therapeutic potential of this species. Key points • Different types of S. henryi in vitro cultures were compared for the first time. • The S. henryi in vitro culture strong antioxidant potential was determined for the first time. • The polyphenol profiling of different types of S. henryi in vitro cultures was shown.

Reynoutria japonica Houtt. (Japanese knotweed) is an invasive plant belonging to the Polygonaceae family. However, being native to east Asia, it has been used in natural medicine since ages because of broad range of biological activity. Although, R. japonica is known as a rich source of phenolic compounds plant biomass collected from the field may be contaminated with toxic elements like heavy metals. Therefore, the aim of this study was to investigate biomass production and phenolic compounds synthesis in Japanese knotweed from in vitro cultures. Plants were cultivated in a traditional agar-solidified medium, in a liquid medium with rotary shaking (agitated culture), and in a temporary immersion bioreactors Plantform™, as well as in soil (ex vitro conditions). Analyses of the growth index and dry weight accumulation were performed on the collected material. In the extracts obtained from examined plants, qualitative and quantitative analysis of phenolic derivatives using DAD-HPLC was conducted to determine the sum of phenolic compounds, as well as the quantity of selected phenolic acids, catechins and other flavonoids. Results have showed that agitated cultures and temporary immersion bioreactors increased biomass accumulation compared to solid medium cultures. Tissue cultures of R. japonica had increased synthesis of phenolic compounds. Shoots and roots from agitated cultures were 2.8- and 3.3-fold richer in catechins, respectively, compared to plants from ex vitro conditions. Based on the obtained results it can be concluded that agitated and bioreactor cultures are the best source of Japanese knotweed biomass rich in valuable secondary metabolites.

This research’s scope encompassed biotechnological, phytochemical, and biological studies of Schisandra henryi, including investigations into its in vitro microshoot culture grown in PlantForm bioreactors (temporary immersion systems, TISs), as well as extracts from leaves of the parent plant, focusing on anti-inflammatory, antioxidant, anticancer, and antimicrobial activities. The phytochemical analysis included the isolation and quantification of 17 compounds from dibenzocyclooctadiene, aryltetralin lignans, and neolignans using centrifugal partition chromatography (CPC), HPLC-DAD, and UHPLC-MS/MS tandem mass spectrometry with triple quadrupole mass filter methods. Higher contents of compounds were found in microshoots extracts (max. 543.99 mg/100 g DW). The major compound was schisantherin B both in the extracts from microshoots and the leaves (390.16 and 361.24 mg/100 g DW, respectively). The results of the anti-inflammatory activity in terms of the inhibition of COX-1, COX-2, sPLA2, and LOX-15 enzymes indicated that PlantForm microshoot extracts showed strong activity against COX-1 and COX-2 (for 177 mg/mL the inhibition percentage was 76% and 66%, respectively). The antioxidant potential assessed using FRAP, CUPRAC, and DPPH assays showed that extracts from microshoot cultures had 5.6, 3.8, and 3.3 times higher power compared to extracts from the leaves of the parent plant, respectively. The total polyphenol content (TPC) was 4.1 times higher in extracts from the in vitro culture compared to the leaves. The antiproliferative activity against T-cell lymphoblast line Jurkat, breast adenocarcinoma cultures (MCF-7), colon adenocarcinoma (HT-29), and cervical adenocarcinoma (HeLa), showed that both extracts have considerable effects on the tested cell lines. The antimicrobial activity tested against strains of Gram-positive and Gram-negative bacteria and fungi showed the highest activity towards H. pylori (MIC and MBC 0.625 mg/mL).