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Funding Information: This work was supported by Business Finland corona-co-creation funding for the project Antiviral Fibers?pilot with extracts from Finnish forests (grant: 40699/31/2020). This study was also funded by the Natural Resources Institute Finland?s strategic research funding to the projects ?More, faster, higher quality: potential of short-rotation aspen and willow biomass for novel products in bioeconomy? (AspenWill) and ?Added value potential of new and under-utilized fibre sources in Finnish value networks of green bioeconomy: prefeasibility, prototyping, and market acceptance? (VALUEPOT). In addition, Academy of Finland has supported this study via the project ?Antivirals from Forest Biomasses: Structure, Function, and Applicability? (grant: 342250). Work was also funded by Jane and Aatos Erkko Foundation: Novel probes for discovering antivirals. Publisher Copyright: Copyright © 2021 Tienaho, Reshamwala, Sarjala, Kilpeläinen, Liimatainen, Dou, Viherä-Aarnio, Linnakoski, Marjomäki and Jyske. Earlier studies have shown that the bark of Salix L. species (Salicaceae family) is rich in extractives, such as diverse bioactive phenolic compounds. However, we lack knowledge on the bioactive properties of the bark of willow species and clones adapted to the harsh climate conditions of the cool temperate zone. Therefore, the present study aimed to obtain information on the functional profiles of northern willow clones for the use of value-added bioactive solutions. Of the 16 willow clones studied here, 12 were examples of widely distributed native Finnish willow species, including dark-leaved willow (S. myrsinifolia Salisb.) and tea-leaved willow (S. phylicifolia L.) (3 + 4 clones, respectively) and their natural and artificial hybrids (3 + 2 clones, respectively). The four remaining clones were commercial willow varieties from the Swedish willow breeding program. Hot water extraction of bark under mild conditions was carried out. Bioactivity assays were used to screen antiviral, antibacterial, antifungal,yeasticidal, and antioxidant activities, as well as the total phenolic content of the extracts. Additionally, we introduce a fast and less labor-intensive steam-debarking method for Salix spp. feedstocks. Clonal variation was observed in the antioxidant properties of the bark extracts of the 16 Salix spp. clones. High antiviral activity against a non-enveloped enterovirus, coxsackievirus A9, was found, with no marked differences in efficacy between the native clones. All the clones also showed antibacterial activity against Staphylococcus aureus and Escherichia coli, whereas no antifungal (Aspergillus brasiliensis) or yeasticidal (Candida albicans) efficacy was detected. When grouping the clone extract results into Salix myrsinifolia, Salix phylicifolia, native hybrid, artificial hybrid, and commercial clones, there was a significant difference in the activities between S. phylicifolia clone extracts and commercial clone extracts in the favor of S. phylicifolia in the antibacterial and antioxidant tests. In some antioxidant tests, S. phylicifolia clone extracts were also significantly more active than artificial clone extracts. Additionally, S. myrsinifolia clone extracts showed significantly higher activities in some antioxidant tests than commercial clone extracts and artificial clone extracts. Nevertheless, the bark extracts of native Finnish willow clones showed high bioactivity. The obtained knowledge paves the way towards developing high value-added biochemicals and other functional solutions based on willow biorefinery approaches.

Drosera rotundifolia L. is a carnivorous plant used in traditional medicine for its therapeutic properties. Because of its small size, its collection in nature is laborious and different cultivation methods have been studied to ensure availability. However, only a few studies exist where the lab-grown sundew tissue and field-grown sundew would have been compared in their functionality or metabolic profiles. In this study, the antioxidant and antiviral activities of lab-grown and field-grown sundew extracts and their metabolic profiles are examined. The effect of drying methods on the chromatographic profile of the extracts is also shown. Antioxidant activity was significantly higher (5–6 times) in field-grown sundew but antiviral activity against enterovirus strains coxsackievirus A9 and B3 was similar in higher extract concentrations (cell viability ca. 90%). Metabolic profiles showed that the majority of the identified compounds were the same but field-grown sundew contained higher numbers and amounts of secondary metabolites. Freeze-drying, herbal dryer, and oven or room temperature drying of the extract significantly decreased the metabolite content from −72% up to −100%. Freezing was the best option to preserve the metabolic composition of the sundew extract. In conclusion, when accurately handled, the lab-grown sundew possesses promising antiviral properties, but the secondary metabolite content needs to be higher for it to be considered as a good alternative for the field-grown sundew. peerReviewed

Luonnonmateriaalien sisältämien biologisesti aktiivisten komponenttien seulontaan tarvitaan uusia tehokkaita menetelmiä. Tässä tutkimuksessa metsästä saatavien uutteiden bioaktiivisuuden määrittämiseen käytettiin kokosolubiosensoreita. Tätä vaihtoehtoista seulontamenetelmää on tutkittu tarkoituksessa vain vähän, mutta herkkyytensä vuoksi se on varteenotettava. Tutkimuksessa aktiivisuushavaintoja täydentämään käytettiin myös perinteisempiä menetelmiä kuten antioksidanttikuoppalevytestejä. Lisäksi aineenvaihduntatuotteet määritettiin bioaktiivisiksi havaituista endofyyttisieniuutteista nestekromatografia-massaspektrometriaa (LC-MS) käyttäen. Tutkimuksen osana kehitettiin kaksi biosensorimenetelmää Escherichia coli kokosolubiosensoreille. Ensimmäinen sovellus parantaa aiemmin kuvattua antioksidanttisuuden mittaamisen menetelmää sen seulonnan tehopotentiaalia lisäämällä. Toinen esittelee menetelmän UV-säteilysuojan mittaamiseksi määrittäen sekä biologista että absorbanssipohjaista suojauskykyä. Menetelmällä voidaan saada samalla tietoa myös näytteen genotoksisuudesta. Kehitettyjen menetelmien toimivuus vahvistettiin malliyhdisteitä käyttäen, ja ne todettiin toimiviksi myös luonnonaineuutteilla joillakin rajoituksilla. Bakteeribiosensoreita voidaan käyttää etenkin konsentraatioltaan pienien näytteiden ja fraktioiden aktiivisuus- tai inaktiivisuusseulonnoissa, jolloin minimoidaan rajoitukset, kuten näytematriisin signaalin peittovaikutus ja induktioviive. Endofyyttisienet elävät kasvien sisällä aiheuttamatta oireilua isäntäkasvissa, ja ne voivat lisätä esimerkiksi isäntien kasvua ja stressinsietokykyä. Endofyyttisienet sisältävät suuren määrän toistaiseksi hyödyntämättömiä bioaktiivisia yhdisteitä, joita voidaan käyttää erilaisiin tarkoituksiin. Tutkimuksessa männyn taimien juurista eristettiin Acephala applanata, Philocephala fortinii ja Humicolopsis cephalosporioides/ Coniochaeta mutabilis -endofyyttisienilajit, ja niistä löydettiin suuri määrä yhdisteitä LC-MS: lla (yht. 318 kpl). Yhdisteistä tunnistettiin eri varmuusasteilla 220 kpl. Sienilajien uutteiden ja fraktioiden antioksidantti- ja antibakteerisuuspotentiaali seulottiin käyttäen E. coli -biosensorimenetelmiä sekä täydentäviä menetelmiä ja havaittiin useita aktiivisuuksia. Johtopäätöksenä endofyyttisienet sisältävät valtavan määrän mahdollisesti toiminnallisia aineenvaihduntatuotteita, ja kokosolubiosensorimenetelmät soveltuvat erilaisten luontopohjaisten näytteiden tutkimiseen antaen samanaikaisesti tietoa sekä näytteen bioaktiivisuudesta että biosaatavuudesta, mikäli myös niiden heikkoudet huomioidaan mittausasettelussa. Efficient methods are needed in the search for novel biologically active components from natural sources. Bacterial whole-cell biosensors offer a less-examined alternative with high sensitivity. In this thesis, forest-derived extracts were screened for their bioactivity with bacterial whole-cell biosensors and complementary measures, such as antioxidant microplate tests. The main metabolites were also identified from endophytic fungi extracts using liquid chromatography-mass spectrometry (LC-MS). Two novel applications of biosensors are described for Escherichia coli whole-cell biosensors. The first application provides an improved antioxidant method via increased high-throughput screening potential and the other describes a method for measuring the UV protection capacity for both the biological and absorbance-based shield. There is also an opportunity to obtain simultaneous information of the genotoxicity of the sample. The methodologies were validated using reference compounds and were found to be usable in the field of forest-derived extracts with certain limitations. The bacterial biosensors were used in an activity/inactivity type of screening. By using small concentrations of forest-derived extracts or fractionated samples, the limitations, such as possible sample matrix masking effect and induction delay phenomenon, can be reduced. Endophytic fungi live asymptomatically inside a plant host and are often beneficial for the host plant growth and stress resistance. They comprise a large untapped source of potential bioactive compounds to be utilized for different purposes. Many metabolites (318) were discovered using an LC-MS methodology from Acephala applanata, Phialocephala fortinii and Humicolopsis cephalosporioides/ Coniochaeta mutabilis isolated from the roots of Scots pine seedlings. Out of the metabolites, 220 were identified with varying degree of certainty. The antioxidant and antimicrobial potential of the extracts and fractions were screened with the biosensors and complementary methods and various bioactivities were discovered. In conclusion, endophytic fungi are a rich source of potential functional metabolites and whole-cell biosensor methodologies are usable for nature-based samples and provide valuable insights into both the bioactivity and bioavailability of the material, if their limitations are also considered.

Natural extracts have been used in traditional medicine as remedies to treat for example inflammations. During the era of modern medicine, there has been a shift towards usage of synthetic substances. However, many bacteria have already developed resistances against antibiotics or other antibacterial substances. These resistant bacteria cause serious health issues, especially in the hospital environment. The natural extracts used in traditional medicine are new sources for antibacterial sub-stances. One of these extracts is round-leaved sundew extract, which has been used for example as cough remedy. The compounds of sundew extract, such as naphthoquinones and flavonoids, have proven health effects. The extract of round leaved sundew includes naphthoquinones, such as 7-methyljuglone and plumbagin in small concentrations, as well as flavonoid quercetin. New measurement methods, which can measure bioavailability and toxicity at the same time, can be useful in the screening of natural extracts. The natural extracts are usually screened as crude extracts, and therefore the concentrations of the active substances can be low, and there can be plenty of different samples to screen. Thus, the methods have to be sensitive, fast and easy to perform and easily adoptable into different laboratories. This diploma thesis was done as a part of collaboration between Tampere University of Technology and the Natural Resources Institute of Finland (Luke). In this thesis, the an-tibacterial effects of ethanol extracted Drosera rotundifolia from pure tissue culture and plants gathered from two different peatlands were studied using genetically modified bi-oluminescent bacterial cells. Luke Parkano research station provided the round leaved sundew extracts as well as the standard substances, which were quercetin, plumbagin, lawsone and juglone. Recombinant biosensor cells are bacterial cells, which have been genetically modified to include reporter genes from another organism. In this work, four different biosensor strains were used, and they all included a bioluminescent genetic re-porter. The strains were Staphylococcus aureus RH4220, Escherichia coli K12 + pcGLS11, Acinetobacter baylyi ADP1 + pBAV1K-T5-LUX and Pseudomonas putida. The last one was genetically modified during the work by transforming a bioluminescence plasmid pBAV1K-T5-LUX into the P. putida cells using electroporation. The sundew extracts and the standards showed antibacterial effects on all of the strains. However, there were dissimilarities in the effects between the strains. Therefore, in further studies, it is important to consider the sensitivities of different bacteria. These variances might be caused for example by the gram positive bacteria having dissimilar reactions towards the compounds extracted from the round-leaved sundew than the gram negative.