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Title: In vitro cultures of Schisandra henryi C.B. Clarke as a potential source of bioactive lignans and polyphenolic compounds
Abstract:
The subject of this doctoral thesis was a species of vine, endemic to Yunnan Province (China), known for its potential medicinal properties in traditional Chinese medicine (TCM) - Schisandra henryi C.B. Clarke. S. henryi belongs to the genus Schisandra (family Schisandraceae), just like the recognized pharmacopoeial species - Schisandra chinensis Turcz. Baill. (Chinese magnolia vine). S. chinensis fruit extracts show, among others: antioxidant, anti-inflammatory, anticancer, hepatoprotective and neuroprotective effects. Species of the Schisandra genus are distinguished by their specific chemical composition, dominated by dibenzocyclooctadiene lignans. As part of this work, interdisciplinary research were carried out for the species S. henryi in the field of plant biotechnology, phytochemistry and biological activity. The main goal of the work was the initiation and cultivation of various types of in vitro cultures and the optimization of culture conditions in terms of the qualitative and quantitative selection of plant growth regulators (PGRs) in the medium acc. to Murashige-Skoog (MS) and the duration of breeding cycles, in order to obtain high endogenous production of secondary metabolites (lignans, triterpenoid compounds and polyphenolic compounds), and to study the biological activity of extracts obtained from the biomass of S. henryi in vitro cultures. As part of the resea ; rch, the cultivation of microshoot cultures - agar, agitated and multiplied in temporary immersion systems (TIS) - PlantForm bioreactors, as well as undifferentiated cultures - callus and suspension cultures, was optimized. The qualitative and quantitative selection of PGRs in the culture medium (MS) and the duration of culture cultivation influenced the appearance of biomass and the production of metabolites. Qualitative analysis using the UHPLC-DAD-MS/MS method of methanol extracts from biomass of microshoot cultures confirmed the presence of compounds from three groups of lignans: dibenzocyclooctadiene lignans (schisandrin, gomisin G, schisantherin A and B, deoxyschisandrin and schisandrin C), dibenzylbutane lignans (hernicin B) and aryltetralin lignans (wulignan A1 and A2, epiwulignan A1, ensicine, epiensicine and dimethylwulignan A1), in addition, two triterpenoid compounds were identified - kadsuric acid and schisanhenric acid. The analysis of polyphenolic compounds using the UHPLC-DAD-ESI-MS3 method showed, in extracts from in vitro cultures, the presence of, among others: phenolic acids and their derivatives (neochlorogenic acid, protocatechuic acid O-hexoside, coumaroylquinic acid and its isomers), flavan-3-ols (catechin) and procyanidin derivatives (procyanidin trimer type C isomers, procyanidin dimer type A and B isomers, tetramer and pentamer procyanidin isomers). M ; icroshoot cultures grown in PlantForm bioreactors on MS medium containing 2 mg/L IBA (3-indolylbutyric acid) were selected as optimal conditions for conducting in vitro cultures of S. henryi, favoring the production of lignans (max. 543.99 mg/100g DW (dry weight)) and 0.5 mg/L BA (6-benzyladenine). Agar microshoot cultures grown on MS medium containing 2 mg/L IBA and 0.5 mg/L BA were selected as the optimal conditions for conducting in vitro cultures of S. henryi, favoring the production of polyphenolic compounds (max. 112.56 mg/100g DW). Among the lignans, schisantherin A and schisantherin B dominated in in vitro cultures (73.16 mg/100g DW and 390.16 mg/100g DW, respectively), similarly to the leaves of the parent plant (61.65 mg/100g DW and 361.24, respectively). mg/100g DW). The phytochemical profile of extracts from in vitro cultures partially differed from the profile of the extract from the leaves of the parent plant. Extracts from in vitro cultures contained schisandrin (0.35 mg/100 g dry DW), which was not detected in leaf extracts. Similarly, the presence of rubriflorin A (0.02 mg/100g DW) was found in leaf extracts, which was not found in microshoot extracts. Quantitative analysis of polyphenolic compounds in extracts from various types of in vitro cultures of S. henryi using the UHPLC-DAD-ESI-MS3 and HPLC-DAD methods showed the highest production of these compounds i ; n agar microshoot cultures (neochlorogenic acid - max. 210.13 mg/100g DW, catechin - max. 112.56 mg/100g DW) and in microshoot cultures grown in PlantForm bioreactors (catechin - max. 124.25 mg/100g DW). As part of the work, the biological activity of extracts from S. henryi in vitro cultures and from the leaves of the parent plant, was tested for the first time. Determinations of antioxidant potential (DPPH, FRAP and FIC (chelating capacity) tests) and anti-inflammatory activity of extracts (15-LOX (15-lipoxygenase), COX-1 and 2 (cyclooxygenase-1 and 2), sPLA2 (secretory phospholipase A2) inhibition tests), showed higher results for extracts from microshoot cultures grown in PlantForm bioreactors than for leaf extracts. Antiproliferative activity was determined (against the following lines: cervical adenocarcinoma HeLa, colon adenocarcinoma HT-29, breast adenocarcinoma MCF-7 and Jurkat T-cell-like lymphoblasts), and the cytotoxic activity (against the normal human cell line HEK-293) of extracts from microshoot cultures grown in PlantForm bioreactors and from the leaves of the parent plant was assessed. A stronger anti-proliferative effect, in particular against the HT-29 cell line, was found for leaf extracts. Activity tests were carried out antimicrobial - antibacterial (against Gram positive bacteria: Staphylococcus aureus ATCC 25923, S. aureus ATCC 43300, S. epidermidis ATC ; C 12228 and Gram negative bacteria: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Helicobacter pylori ATCC 43504) and antifungal (against strains: Candida albicans ATCC 10231, C. parapsilosis ATCC 22019, C. glabrata ATCC 90030 and Aspergilus niger ATCC 16404). Similar antimicrobial activity of S. henryi extracts from the biomass of in vitro cultures and leaves was found. The strongest antibacterial activity was found against H. pylori strain, and the strongest fungicidal activity was confirmed for C. albicans and C. parapsilosis. The results of carried out experimental works under this doctoral thesis were published in three experimental publications in international journals. The studies outcomes were presented at foreign conferences in the form of posters (3x), and at national conferences in the form of oral presentations (4x) and posters (8x). Moreover, in order to systematize and expand knowledge regarding the medicinal, dietary and cosmetic applications of a little-known species - S. henryi and compounds from the lignan group, as well as to present the results of own research, two English-language and one Polish-language review publications were published. To sum up, this doctoral thesis proved that S. henryi cultures can be a potential, rich source of obtaining biologically active metabolites. Noteworthy is the innovative scope of research on in vitro cul ; tures of the species S. henryi and the research conducted on extracts from the leaves of the parent plant. The obtained results are not only cognitive, but also of application nature.
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Rada Dyscypliny Nauki farmaceutyczne
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Feb 27, 2025
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Feb 27, 2025
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http://dl.cm-uj.krakow.pl:8080/publication/5227
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| Edition name | Date |
|---|---|
| ZB-142193 | Feb 27, 2025 |
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