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Huperzia selago sporophyte in its natural habitat in the Babia Góra National Park, Poland. Side-view of the H. selago shoots presenting distribution of bulbils (bl) and sporangia (sp).
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This study presents a protocol for a fast and effective in vitro axenic culture of Huperzia selago (Huperziaceae Rothm.) sporophytes, a club moss which is a source of huperzine A, an alkaloid of a considerable therapeutic potential extensively investigated for its uses as treatment for some neurodegenerative diseases. The proposed procedure allowed...
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... we established H. selago sporophyte cultures using shoot fragments obtained from sporophytes growing in natural stations [12,29]. That required large amounts of plant mate- rial, which had to undergo time-consuming and complicated disinfection. On the other hand, when bulbils are procured from the wild, even small fragments of shoots of these rare and protected plants are not cut. Bulbils (Fig. 1, Fig. 2) are formed spontaneously in the apical shoot parts of H. selago sporophytes. They are best collected in the period of their complete develop- ment, preferably in spring and the collection does not cause any mechanical damage to the mother sporophytes. Both surface and internal disinfection is easier with bulbils than with shoots. In the present study, with the disinfection methods described, up to 90% of explants were free from ...
Context 2
... selago sporophyte in vitro cultures were initiated using vegetative propagules (bulbils; Fig. 1, Fig. 2) procured from different local populations in the Babia Góra National Park (The Carpathians, Poland). They were harvested from the mother sporophytes and protected from drying during transport by placing on blotting paper moistened with water in tightly closed containers. The plants were collected in spring, from April to June, and in autumn, in September and October. In the laboratory, the plants were cleaned by manually remov- ing litter, soil and leaves of other plants, and then rinsed in a stream of running tap water for 1 hour. Voucher specimens, including sporophytes with bulbils, were taken and deposited in the herbarium at Department of Biology and Pharmaceutical Botany, Medical University of Warsaw. ...
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During the present study an effort has been made to propagate (in-vitro) the endangered and endemic Indian liverwort Stephensoniella brevipedunculata Kashyap using different culture media under controlled Laboratory conditions. Axenic cultures of the taxon have been established using tubers as explants. Seven combinations of media with Full Strengt...
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... H. selago adult plants were grown on their original soil (Russian accession) or on Arabidopsis potting soil (Spanish and French accessions) under long-day conditions (16-h light/8-h dark) at 22°C and covered with a transparent plastic lid. Bulbils were sterilized as previously described (42) and grown on modified Moore's medium (43). Despite our attempts to propagate H. selago through propagules (bulbils), we observed growth arrest in 2-cm plants grown on regular soil or young plants after radicle emergence when grown in vitro. ...
Jasmonates are phytohormones that regulate defense and developmental processes in land plants. Despite the chemical diversity of jasmonate ligands in different plant lineages, they are all perceived by COI1/JAZ co-receptor complexes, in which the hormone acts as a molecular glue between the COI1 F-box and a JAZ repressor. It has been shown that COI1 determines ligand specificity based on the receptor crystal structure and the identification of a single COI1 residue, which is responsible for the evolutionary switch in ligand binding. In this work, we show that JAZ proteins contribute to ligand specificity together with COI1. We propose that specific features of JAZ proteins, which are conserved in bryophytes and lycophytes, enable perception of dn-OPDA ligands regardless the size of the COI1 binding pocket. In vascular plant lineages beyond lycophytes, JAZ evolved to limit binding to JA-Ile, thus impeding dn-OPDA recognition by COI1.
... Till date, procedures for establishing and maintaining in vitro cultures of Huperzia selago sporophytes ( Figure 2A) and gametophytes ( Figure 2B) have been developed using various starting materials, including shoot fragments, vegetative propagules, somatic embryos, and spores (Szypuła & Pietrosiuk, 2021;Szypuła et al., 2005Szypuła et al., , 2013Szypuła et al., , 2020. e first studies on HupA biosynthesis were conducted by Szypuła et al. (2005Szypuła et al. ( , 2011Szypuła et al. ( , 2013. ...
... Till date, procedures for establishing and maintaining in vitro cultures of Huperzia selago sporophytes ( Figure 2A) and gametophytes ( Figure 2B) have been developed using various starting materials, including shoot fragments, vegetative propagules, somatic embryos, and spores (Szypuła & Pietrosiuk, 2021;Szypuła et al., 2005Szypuła et al., , 2013Szypuła et al., , 2020. e first studies on HupA biosynthesis were conducted by Szypuła et al. (2005Szypuła et al. ( , 2011Szypuła et al. ( , 2013. e establishment of H. selago sporophyte in vitro cultures initiated from shoot explants and vegetative propagules (bulbils) allowed approximately tenfold reduced developmental stages with the omission of the gametophyte stage, thus increasing the sporophyte mass tenfold within 6 months. ...
Plant cell and organ cultures are potential sources of valuable secondary metabolites that can be used as food additives, nutraceuticals, cosmeceuticals, and pharmaceuticals. Phytochemical biosynthesis in various in vitro plant cultures, in contrast to that in planta, is independent of environmental conditions and free from quality fluctuations.
Pharmaceutical application of plant biotechnology is of interest to almost all departments of the Faculty of Pharmacy and Institute of Pharmacology in Poland with a botanical profile (Pharmaceutical Botany, Pharmacognosy, and Pharmacology).
This study discusses the advances in plant biotechnology for the production of known metabolites and/or biosynthesis of novel compounds in plant cell and organ in vitro cultures in several scientific centers in Poland.
... Currently, HupA is isolated from Huperzia serrata, occurring commonly in Asia. However, the uncontrolled harvesting of this species has reduced its original range and depleted its natural resources [10,25]. Therefore, the aim of our work was to search for alternative sources of HupA and new potent AChE inhibitors from three species commonly encountered in the Euro-Asia region, belonging to the Lycopodiaceae family: Lycopodium clavatum L., Lycopodium annotinum L. and Huperzia selago (L.) Bernh. ...
... Based on these studies, it was found that the highest percentage of Lycopodium alkaloids were isolated under high pressure and temperature (100 bar, 80 • C) from methanolic extracts, so these conditions were used in this approach. Due to the fact that Huperzia species are endangered in some regions, the amount of plant materials was limited [10]. Extracts of 100 mL each were prepared: 4 × 5 g from L. clavatum, 3 × 5 g from L. annotinum and 2 × 5 g from H. selago, which were further processed. ...
In view of the abundant evidence that Lycopodiaceae alkaloids, including the well-known huperzine A (HupA), are among the potent acetylcholinesterase (AChE) inhibitors, an attempt was made to search for new compounds responsible for this property. For this purpose, three plant species belonging to the Lycopodiaceae family, commonly found in the Euro-Asia region, were subjected to the isolation of bioactive compounds, their identification and subsequent evaluation of their anticholinesterase and cytotoxic activities. Methanolic extracts of two Lycopodium and one Hupezia species were obtained via optimized pressurized liquid extraction (PLE) and then re-purified using innovative gradient vacuum liquid chromatography (gVLC). For the first time, three sorbents of different porosity packed in polypropylene cartridges and mobile phase systems of different polarity were used to elute the target compounds. This technique proved to be a rapid tool for the obtainment of alkaloid fractions and allowed one to select the appropriate process conditions to yield potent AChE inhibitors in each of the species studied. More than 100 collected fractions were analyzed via HPLC/ESI-QTOF-MS, which enabled one to detect more than 50 compounds, including several new ones previously unreported. Some of them were present in high purity fractions (60–90% of the established purity). TLC bioautography assays proved that the analyzed species are rich sources of AChE inhibitors, but H. selago showed the highest anti-AChE activity. Additionally, the modified silanized silica gel sorbent used allowed one to isolate L. clavatum alkaloids more efficiently using an aqueous reversed-phase solvent system. Furthermore, the tested extracts from the three plant extracts were found to be safe, as they did not exhibit cytotoxicity to skin fibroblasts.
... They were the first to observe apogamous development of sporophytes in the gametophyte cultures of H. selago. In recent years, procedures for establishing and maintaining in vitro cultures of H. selago sporophytes and gametophytes have been developed which use a variety of starting material: shoot fragments, vegetative propagules, somatic embryos, and spores (Szypuła et al. 2005(Szypuła et al. , 2013(Szypuła et al. , 2020Szypuła and Pietrosiuk 2021) (Fig. 20.9). Establishing sporophyte cultures using vegetative propagules as the initiating material is a fast and effective method and vegetative propagules seem superior to shoot fragments in this respect (Szypuła et al. 2005). ...
... Obtaining propagules from plants growing in the wild does not demand cutting off parts of rare plants often protected by the law as propagules are formed spontaneously in the apical shoot parts of H. selago sporophytes. When the decontamination and disinfection protocol proposed by Szypuła et al. (2013) is followed, up to 90% of explants are free from contaminants. Sporophytes developed from the propagules and grew roots. ...
... Sporophytes developed from the propagules and grew roots. The optimal results were achieved using Moore medium without plant growth regulators or supplemented with 0.05 μM IBA and 1.4 μM kinetin (Szypuła et al. 2013) which ensured both viability of the propagules and their further development ( Fig. 19.9a, c). The biomass growth index for H. selago sporophytes grown from propagules, determined at 3 months of culture (1 passage) on Moore medium ensuring optimal growth was 650% and at 6 months the biomass growth index increased to 1114%. ...
Pteridophytes found in Europe have been used for centuries for a variety of ailments but compared to angiosperms they constitute a relatively small group of medicinal plants. The term pteridophytes refer to a polyphyletic group of taxa that consists of club mosses, horsetails, psylophytes and ferns. Now, according to the Peridophyte Phylogeny Group they are members of the monophyletic class of Lycopodiopsida comprising some 1388 species and of the Polypodiopsida class which includes most of all pteridophytes (some 10,597 species). This review presents historical and updated information on pteridophyte taxonomy, secondary metabolites isolated from species screened for medicinal properties, their pharmacological activities, and the use of in vitro plant tissue culture techniques for the conservation of pteridophyte biodiversity and for the biosynthesis of their secondary metabolites. The analysis is based on a comprehensive review of the literature with the relevant papers retrieved from online databases (PubMed, Web of Science, Wiley, Science Direct, Elsevier’s Scopus, Google Scholar) and print sources (ACS Publications, SpringerLink and Elsevier journals). The analysis demonstrates that numerous pteridophyte species are a source of popular and valued herbal medicines used for the treatment of a variety of health problems and diseases. The major secondary metabolites isolated from pteridophytes and reported for their medicinal properties include alkaloids, polyphenols and flavonoids, although secondary metabolites belonging to other compound classes also may have a therapeutic value or they may modify the pharmacological activity of the major secondary metabolites. Numerous studies have demonstrated that pteridophyte extracts and their isolated secondary metabolites have a broad spectrum of pharmacological activity including anti-inflammatory, anticancer, cytotoxic, antibacterial, antiparasitic, antifungal, antiviral, and acetyl- and butyrylcholinesterase inhibitory effects. Importantly, of the 205 European pteridophyte species, only some 40 species have been investigated phytochemically and/or biologically. For some, protocols for in vitro micropropagation have been developed to aid ex-situ species conservation. Further studies are necessary to determine the composition of secondary metabolites and investigate biologically and pharmacologically the remaining 80% of the European pteridophyte flora.
... trideca-2(7),3,10-trien-5-one), can be isolated from a traditional Chinese medicinal (TCM) plant Huperzia serrata [1,2]. H. serrata belongs to Huperzia genus, in the Huperziaceae family, which comprises two genera and 150-450 species [3,4], distributed in Eastern, Southern, and Southeast Asia, Oceania, and Central America [5,6]. The dried whole grass has been extensively used as a traditional Chinese medicinal (TCM) herb, named Qian Ceng Ta, to cure fever, contusions, strains, hematuria, schizophrenia, and snakebite for several hundreds of years in China [7][8][9][10][11][12]. ...
As the population ages globally, there seem to be more people with Alzheimer’s disease. Unfortunately, there is currently no specific treatment for the disease. At present, Huperzine A (HupA) is one of the best drugs used for the treatment of Alzheimer’s disease and has been used in clinical trials for several years in China. HupA was first separated from Huperzia serrata, a traditional medicinal herb that is used to cure fever, contusions, strains, hematuria, schizophrenia, and snakebite for several hundreds of years in China, and has been confirmed to have acetylcholinesterase inhibitory activity. With the very slow growth of H. serrata, resources are becoming too scarce to meet the need for clinical treatment. Some endophytic fungal strains that produce HupA were isolated from H. serrate in previous studies. In this article, the diversity of the endophytic fungal community within H. serrata was observed and the relevance to the production of HupA by the host plant was further analyzed. A total of 1167 strains were obtained from the leaves of H. serrata followed by the stems (1045) and roots (824). The richness as well as diversity of endophytic fungi within the leaf and stem were higher than in the root. The endophytic fungal community was similar within stems as well as in leaves at all taxonomic levels. The 11 genera (Derxomyces, Lophiostoma, Cyphellophora, Devriesia, Serendipita, Kurtzmanomyces, Mycosphaerella, Conoideocrella, Brevicellicium, Piskurozyma, and Trichomerium) were positively correlated with HupA content. The correlation index of Derxomyces with HupA contents displayed the highest value (CI = 0.92), whereas Trichomerium showed the lowest value (CI = 0.02). Through electrospray ionization mass spectrometry (ESI-MS), it was confirmed that the HS7-1 strain could produce HupA and the total alkaloid concentration was 3.7 ug/g. This study will enable us to screen and isolate the strain that can produce HupA and to figure out the correlation between endophytic fungal diversity with HupA content in different plant organs. This can provide new insights into the screening of strains that can produce HupA more effectively.
... Today, H. serrata, the Chinese Huperzia species, is the main source of HupA for the pharmaceutical industry. Uncontrolled harvesting observed in recent years has considerably depleted the natural resources of the club moss [175]. According to Ishiuchi et al. [176], HupA content in sporophytes of different species varies from 0.0 to 1.766 mg g À1 dry weight (DW) with the highest content in the sporophytes of H. pinifolia (an average content of 1.766 mg g À1 DW). ...
... Importantly, H. serrata is widely distributed throughout the boreal and temperate regions of Europe, Asia, and North America. It is also found in Australia, Tasmania, and New Zealand [18,175]. In Europe, it is distributed from the Arctic archipelago of Svalbard, Iceland, and the Scandinavian Peninsula to the mountain ranges of the Northern Mediterranean. ...
... Studies by Szypuła et al. [168,169] confirm that H. selago is a rich source of HupA, but procurement of the raw material from plants growing in the wild is difficult if not impossible. In many countries (e.g., Poland, Luxembourg, or the USA), H. selago is listed as a protected species or has an endangered or threatened species status [175]. Its natural populations are shrinking, and the habitats are unique. ...
This review provides a comprehensive overview of the cholinesterase-inhibiting compound production in in vitro culture of club mosses (Lycopodiaceae sensu lato). Some hallmarks in a rich history of studies on club mosses, their complex systematics and phylogeny, and secondary metabolites with potential medicinal uses are presented. The review summarizes the published literature, from historical times to the present, including reports on the latest developments in the biosynthesis of alkaloids using different methods. Currently, over 30 compounds which are AChE inhibitors are being assessed in various phases of preclinical and clinical trials, and a few have been approved for use. Huperzine A (HupA, selagine), an alkaloid isolated from some club mosses, has been chosen as a promising drug candidate for Alzheimer’s disease. Although the procedure for the total HupA synthesis has been developed, the pharmaceutical industry uses mainly Huperzia serrata sporophytes collected in its natural habitat. Currently, a total of approximately 350 different Lycopodium alkaloids are known, and some of them demonstrate very strong acetylcholinesterase inhibitory or butyrylcholinesterase-inhibiting activity. Since the 1950s, a number of approaches to Lycopod regeneration have been described, and some appeared to be potentially useful for large-scale propagation of the plant material, including the production of secondary metabolites. Some other established cultures which partially succeeded used Lycopod gametophytes or sporophytes. Some of those studies dealt specifically with the biosynthesis of alkaloids and obtaining the biomass for their isolation. These values clearly demonstrate that the highest HupA content in the plant material from in vitro tissue cultures exceeds by approximately 26-fold or by 13-fold the mean HupA level in the whole plant of H. serrata or by 10-fold the highest HupA content in the H. serrata sporophytes with the most efficient biosynthesis of the alkaloid. Importantly, the in vitro methods successfully shorten the life cycle of the plants which under natural conditions can take many years to develop.
... Importantly, H. selago is a rich source of HupA, since its sporophytes contain much more of the alkaloid than found in Huperzia serrata native to Asia [17,20,23]. The preliminary studies of HupA production in in vitro culture of H. selago gametophytes indicate that this is a more efficient method of obtaining secondary metabolites than sporophyte culture [17,23,25]. So far, there have been no studies on the biosynthesis of alkaloids or any other metabolites using in vitro culture of gametophytes (prothallus). ...
... Up to now, club moss gametophytes from natural populations have been only infrequently described and in very rare cases they were obtained in vitro, but they have not been effectively used for the biosynthesis of secondary metabolites [23]. The first studies on the biosynthesis of HupA were conducted by Szypuła et al. [17,20,25] and Ma and Gang [24]. Szypuła et al. [17] presented a protocol for the establishment and maintenance of in vitro culture of Huperzia selago sporophytes from shoot explants (Figure 1). ...
... Importantly, spore disinfection according the protocol developed specifically for the present study allowed tissue cultures free from bacterial and fungal contamination to be established and maintained. Achieving axenic cultures of the sporophytes of H. selago and other club mosses is the most challenging step [17,23,25]. Club mosses are mycorrhizal plants and their aboveground tissues are colonized by endophytic bacteria and fungi. ...
This is the first report of an efficient and effective procedure to optimize the biosynthesis of huperzine A (HupA) and huperzine B (HupB) in vitro from Huperzia selago gametophytes. Axenic tissue cultures were established using spores collected from the sporophytes growing in the wild. The prothalia were obtained after 7–18 months. Approximately 90 up to 100% of the gametophytes were viable and grew rapidly after each transfer on to a fresh medium every 3 months. The best biomass growth index for prothallus calculated on a fresh (FW) and dry weight (DW) basis, at 24 weeks of culture, was 2500% (FW) and 2200% (DW), respectively. The huperzine A content in the gametophytes was very high and ranged from 0.74 mg/g to 4.73 mg/g DW. The highest yield HupA biosynthesis at >4 mg/g DW was observed on W/S medium without growth regulators at 8 to 24 weeks of culture. The highest HupB content ranged from 0.10 mg/g to 0.52 mg/g DW and was obtained on the same medium. The results demonstrate the superiority of H. selago gametophyte cultures, with the level of HupA biosynthesis approximately 42% higher compared to sporophyte cultures and 35-fold higher than when the alkaloid was isolated from H. serrata, its current source for the pharmaceutical industry. Moreover, the biosynthesis of HupB was several-fold more efficient than in H. selago sporophytes growing in the wild. HPLC-HR-MS analyses of the extracts identified eight new alkaloids previously unreported in H. selago: deacetylfawcettine, fawcettimine, 16-hydroxyhuperzine B, deacetyllycoclavine, annopodine, lycopecurine, des-N-methylfastigiatine and flabelline.
... Today, H. serrata, the Chinese Huperzia species, is the main source of HupA for the pharmaceutical industry. Uncontrolled harvesting observed in recent years has considerably depleted the natural resources of the club moss [175]. According to Ishiuchi et al. [176], HupA content in sporophytes of different species varies from 0.0 to 1.766 mg g À1 dry weight (DW) with the highest content in the sporophytes of H. pinifolia (an average content of 1.766 mg g À1 DW). ...
... Importantly, H. serrata is widely distributed throughout the boreal and temperate regions of Europe, Asia, and North America. It is also found in Australia, Tasmania, and New Zealand [18,175]. In Europe, it is distributed from the Arctic archipelago of Svalbard, Iceland, and the Scandinavian Peninsula to the mountain ranges of the Northern Mediterranean. ...
... Studies by Szypuła et al. [168,169] confirm that H. selago is a rich source of HupA, but procurement of the raw material from plants growing in the wild is difficult if not impossible. In many countries (e.g., Poland, Luxembourg, or the USA), H. selago is listed as a protected species or has an endangered or threatened species status [175]. Its natural populations are shrinking, and the habitats are unique. ...
This review provides a comprehensive overview of the cholinesterase-inhibiting compound production in in vitro culture of club mosses (Lycopodiaceae sensu lato). Some hallmarks in a rich history of studies on club mosses, their complex systematics and phylogeny, and secondary metabolites with potential medicinal uses are presented. The review summarizes the published literature, from historical times to the present, including reports on the latest developments in the biosynthesis of alkaloids using different methods. Currently, over 30 compounds which are AChE inhibitors are being assessed in various phases of preclinical and clinical trials, and a few have been approved for use. Huperzine A (HupA, selagine), an alkaloid isolated from some club mosses, has been chosen as a
promising drug candidate for Alzheimer’s disease. Although the procedure for the total HupA synthesis has been developed, the pharmaceutical industry uses mainly Huperzia serrata sporophytes collected in its natural habitat. Currently, a total of approximately 350 different Lycopodium alkaloids are known, and some of them demonstrate very strong acetylcholinesterase inhibitory or butyrylcholinesterase-inhibiting activity. Since the 1950s, a number of
approaches to Lycopod regeneration have been described, and some appeared to be potentially useful for large-scale propagation of the plant material, including the production of secondary metabolites. Some other established cultures which partially succeeded used Lycopod gametophytes or sporophytes. Some of those studies dealt specifically with the biosynthesis of alkaloids and obtaining the biomass for their isolation. These values clearly demonstrate that the highest HupA content in the plant material from in vitro tissue cultures exceeds by approximately 26-fold or by 13-fold the mean HupA level in the whole plant of H. serrata or by 10-fold the highest HupA content in the H. serrata sporophytes with the most efficient biosynthesis of the alkaloid. Importantly, the in vitro methods successfully shorten the life cycle of the plants which under natural conditions can take many years to develop.
... In this family, there are two more interesting genera -Huperzia and Phlegmariurus [16]. Huperzia, comprising 250 species, is a comparatively larger genus [17]. In China, 2 varieties, 2 forma and 29 species of Huperzia and 19 species of Phlegmariurus have been discovered until 2006 [18]. ...
Alzheimer’s disease (AD), which relates to nervous degeneration, is the most popular form of memory loss. The pathogenesis of AD is not fully understood, and there are no therapies for this disorder. Some drugs have been used in clinical applications for preventing and treating AD, but they have significant adverse reactions. Therefore, there is a need to develop treatment for AD. Traditional medicine has used many medicinal plants to alleviate the symptoms of AD. Medicinal plants may reduce neurodegenerative disorders with fewer side effects than chemical drugs, and they are promising drug candidates for AD therapy. This review is the summary of the pathogenesis and treatments of AD and includes information about the chemistry and bioactivities of some medicinal plants from the Huperzia species, such as Huperzia saururus , Huperzia selago , Huperzia phlegmaria , Huperzia fargesii , Huperzia serrata , Huperzia reflexa and Huperzia quadrifariata , that are used for the treatment of AD. We searched literature, including Medline, Embase, Google Scholar and PubMed database, and did a bibliographic review of relevant articles. Key words included Huperzia species, huperzine, huperin, Huperzia and Alzheimer’s disease. We found that the main bioactive compounds of the Huperzia species are alkaloids, which have shown significant effects on preventing the development of AD. They are new promising compounds against AD due to their antioxidant, anti-inflammatory and acetylcholinesterase inhibitory activities in the neural system. Our conclusion from this review is that the Huperzia species are potential source containing various pharmaceutical compounds for the treatment of AD.
... Studies on the pharmacological properties of HupA are being conducted in many centres worldwide, and a comprehensive review of them has been published by Zhang and Tang [60] and Ma et al. [32]. Moreover, studies by Szypuła et al. [46][47][48] have shown that H. selago is a rich source of HupA and other alkaloids, which are much more abundant in this plant than in the Chinese club moss H. serrata. ...
Free radical-induced oxidative damage is implicated in the pathogenesis of neurodegenerative disorders, and antioxidants are presumably of therapeutic value in such diseases. Our previous data indicated that free radicals are strongly associated with brain aging and also play an important role in cytotoxicity of amyloidogenic proteins including -synuclein and amyloid , which accumulate in brains during Parkinson's and Alzheimer's diseases. Disruption of the equilibrium of pro-oxidants and antioxidants results in oxidative stress that leads to the modification of DNA, proteins, carbohydrates, and lipids. It is widely accepted that antioxidants acting as radical scavengers protect the brain against oxidative damage in neurodegenerative diseases. Plant products are rich sources of phytochemicals and have been found to possess a variety of biological activities, including antioxidative potential. The aim of this study was to analyse the antioxidative potential of alkaloid fractions from Huperzia selago and Diphasiastrum complanatum to protect macromolecules against oxidative damage. Thin layer chromatography (TLC) and high-performance liquid chromatography with diode array (HPLC-DAD) and electrospray ionisation mass spectrometric detection (ESI-MS/MS) were used to carry out a comprehensive characterization of alkaloids isolated from the plant material. The effect of the tested compounds on iron/ascorbate-induced lipid peroxidation and carbonyl group formation was analysed in the rat brain homogenate. Direct free radical scavenging (DPPH assay) and the effect on dityrosine formation were measured in cell-free systems. Our results indicated that a number of alkaloid extracts at concentration of 25 µg/ml exhibited antioxidant activity as indicated by DPPH radical scavenging potential (up to 59% inhibition) and inhibition of dityrosine formation. Selected alkaloid fractions provided significant protection against lipid peroxidation and protein oxidation in rat brain tissue homogenate, reducing iron/ascorbate-induced damage by about 20% and 76%, respectively. Overall, the results indicated that selected alkaloids isolated from Huperzia selago effectively protect macromolecules from oxidative stress injury, which will give us an insight into the potential of alkaloids in terms of opening up a new therapeutic approach for oxidative stress-dependent disorders.
