Phenolics of the Inner Bark of Silver Birch: Characterization and Intraspecific Variation

This thesis describes work related to the in-depth characterization of the phenolic compounds of silver birch (Betula pendula) inner bark. Phenolic compounds are the most ubiquitous class of plant secondary compounds. The unifying feature of this structurally diverse group is an aromatic ring containing at least one hydroxyl group. Due to the structural diversity, phenolics have various roles in the plant defense against biotic and abiotic stresses. In addition, they can confer several health-promoting properties to humans. Furthermore, the structural diversity of this class of compounds causes challenges for their analysis. The study species in the present work, silver birch, is economically the most important hard wood species in northern Europe. Its inner bark contains a high level of phenolic compounds and it has shown one of the strongest antioxidant activities among 92 Finnish plant materials. The literature review surveys the diversity and organ specific distribution of phenolic compounds in silver birch as well as the proposed ecological functions of phenolic compounds in nature. In addition, the basis for the characterization of phenolics by mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and circular dichroism spectroscopy (CD) are reviewed. The objective of the experimental work was to extract, purify, characterize, and quantify the inner bark phenolic compounds. Overall 36 compounds were characterized by MS and ultraviolet spectroscopy (UV). 24 compounds were isolated and their structures confirmed by NMR and CD spectroscopy. Five novel natural compounds were identified. Special emphasis was placed on the establishment of a method for the characterization of proanthocyanidins (PAs). Hydrophilic interaction liquid chromatography (HILIC) was utilized because of its high resolution power and predictable elution order of oligomeric and polymeric PAs according to an increasing degree of polymerization. The combination of HILIC and high-resolution MS detection allowed the identification of procyanidin (PC) polymers up to the degree of polymerization of 22. In addition, a series of oligomeric and polymeric PC monoxylosides were observed for the first time in nature. Season and genotype influenced the quantities of the main inner bark phenolics, yet qualitative differences were not observed. However, manual wounding of the inner bark induced the production of ellagitannins (ETs) in the wounded tissues, i.e. callus. Since ETs were not detected in the intact inner bark, this finding may reflect the capacity of silver birch to exploit ellagitannins in its defense.Siirretty Doriast

Untargeted Metabolic Fingerprinting Reveals Impact of Growth Stage and Location on Composition of Sea Buckthorn (Hippophaë Rhamnoides) Leaves

Sea buckthorn (Hippophaë rhamnoides) is increasingly cultivated to produce raw materials for food and nutraceuticals. There is little knowledge on composition of sea buckthorn leaves (SBLs) and the key factors influencing the composition. This research aims to unravel the metabolic profile of SBLs and the effects of cultivar, location and stage of growth, and climatic conditions on the metabolic profile of SBLs. Leaves of two sea buckthorn cultivars grown in the south and north of Finland during two consecutive growth seasons were studied using untargeted nuclear magnetic resonance (NMR) metabolomics. The highest variance in the metabolic profile was linked to the growth stage, wherein leaves from the first 7 weeks of harvest were characterized with higher abundance of polyphenols, while relatively higher abundance of carbohydrates and sugars was observed in the later weeks. The growth location attributed for the second highest variation, wherein the north–south comparison identified fatty acids and sugars as discriminatory metabolites, and the potential association of metabolome to natural abiotic stressors was revealed. An inverse correlation between carbohydrate/sugar content as well as fatty acids of higher carbon chain length with the temperature variables was evident. The supervised chemometric models with high sensitivity and specificity classified and predicted the samples based on growth stage and location, and cultivar. Nontargeted NMR-metabolomics revealed the metabolic profile of SBLs and their variation associated with various biotic and abiotic factors. Cultivar and growth stage are key factors to consider when harvesting SBLs for use in food and nutraceuticals.Practical ApplicationGlobally, sea buckthorn cultivation has been rapidly increasing due to the known health-promoting benefits of the berries and leaves of the plant. The current research obtained new comprehensive information on the compositional profile of sea buckthorn leaves as well as the impact of major contributory factors, such as cultivars, the advancement of growth stage, geographical location, and weather parameters. The findings of this research provide new knowledge and guidance for plant breeding, cultivation and commercial utilization of sea buckthorn leaves as raw materials for food, feed, and nutraceuticals.</p

Laatua ja laadunhallintaa luonnontuotealalle

Luonnontuotealan vuoden 2019 toimialaraportin mukaan (Honkanen 2019) luonnontuotealan kehityksen edellytyksenä raaka-aineen saatavuuden lisäksi on luonnontuotteiden laatu. Luon-nontuotealalla ei ole vielä kattavia laatunormistoja raaka-aineille ja niiden jalostukselle. Lisäksi etenkin keskeisten yhdisteiden analyysimenetelmiä ja standardeja on kehitettävä. 90 % luon-nontuotealan yrityksistä on mikroyrityksiä (alle 10 hlö), joista suurin osa toimii alkutuotannossa, hyvinvoinnin ja/tai matkailun parissa Lapissa ja Pohjois-Pohjanmaalla. Korkeamman jalostusasteen tuotteita esimerkiksi juoma-, hyvinvointi-, hygienia-, lääketeollisuuden alalla valmistetaan etenkin Uudellamaalla. (Honkanen 2019) Luonnon raaka-aineiden, niin villien kuin viljeltyjen, kirjo on laaja marjoista, sienistä ja mahlasta yrtteihin ja erikoiskasveihin. Lisäksi lainsäädäntö ja suositukset ohjaavat toimintaa raaka-ainekohtaisesti käyttötarkoituksen mukaan. Näin ollen, laatutietouden ja kohdistetun tiedon ohjaaminen yrityksiin on erittäin tärkeää. Tämä opas on suunnattu kerääjille ja luonnontuotealan yrityksille, joissa luonnon raaka-aineita jalostetaan nyt tai tulevaisuudessa korkeamman jalostusarvon tuotteiksi elintarvike-, hyvinvointi- ja kosmetiikka-aloille. Oppaan sisällön on tarkoitus tuoda tietoa jo olemassa olevien omavalvontapohjien ja oppaiden rinnalle laadunhallintaan korkean raaka-aineen laadun ylläpitämiseksi sekä raaka-aineen jalostusarvon nostamiseksi. Laatua ja laadunhallintaa luonnontuotealalle -opas on tuotettu osana Lapissa toteutettua Laatu-sormenjälki arktiselle luonnonraaka-aineelle (Arctic FingerPrint) – EAKR -hanketta. Rahoitus hankkeelle on saatu Euroopan aluekehitysrahoituksesta (EAKR) Kestävää kasvua ja työtä 2014–2020.202

Tannins of Conifer Bark as Nordic Piquancy-Sustainable Preservative and Aroma?

Bark of Norway spruce and Scots pine trees contain large amounts of condensed tannins. Tannins extracted with hot water could be used in different applications as they possess antioxidative and antimicrobial activities. The use of bark tannins as e.g., food preservatives calls for increases in our knowledge of their antioxidative activities when applied in foodstuffs. To assess the ability of bark tannins to prevent lipid oxidation, hot water extracts were evaluated in a liposome model. Isolated tannins were also applied in dry-cured, salty meat snacks either as liquid extracts or in dry-powder form. Consumer acceptance of the snacks was tested by a sensory evaluation panel where outlook, odor, taste, and structure of the snacks were evaluated and compared to a commercial product without tannin ingredients. Our results show that conifer bark tannin-rich extracts have high capacity to prevent lipid oxidation in the liposome model. The efficacies of pine and spruce bark extracts were ten to hundred folds higher, respectively, than those of phenolic berry extracts. The bark extracts did not significantly influence the odor or taste of the meat snacks. The findings indicate that bark extracts may be used as sustainable food ingredients. However, more research is needed to verify their safety.Peer reviewe

Sephadex LH-20 fractionation and bioactivities of phenolic compounds from extracts of Finnish berry plants

In order to assist developing a natural, safe food-preservative, aqueous ethanolic extracts of leaves and berries of eight Finnish berry plants were fractionated with Sephadex LH-20 column chromatography. For each fraction, phenolic compounds were analyzed with NMR, UPLC-DAD-ESI-MS and HPLC-DAD. The antioxidant activities of the fractions were investigated using oxygen radical absorbance capacity (ORAC) assay, and the antibacterial activities were evaluated against foodborne pathogens Staphylococcus aureus and Escherichia coli. Antioxidant activities of the fractions correlated highly with both the total concentration and structural feature of phenolic compounds, including both flavonoids and non-flavonoid phenolics. ORAC value correlated strongly with the concentration of (+)-catechin, (−)-epicatecin, quercetin glycosides, and anthocyanins. Increase in size and number of sugar moieties may reduce the antioxidative activities of quercetin glycosides. Type of sugar moieties may have a significant role in influencing peroxyl-radicals scavenging ability of quercetin glycosides with monosaccharides as a single sugar moieties. Most of the fractions inhibited the target microbes. S. aureus strains expressed a higher sensitivity to phenolic compounds than E. coli strains.</p

Online measurements of real-time cytotoxic responses induced by multi-component matrices, such as natural products, through electric-cell substrate impedance sensing (ECIS).

Natural products are complex matrices of compounds that are prone to interfere with the label-dependent methods that are typically used for cytotoxicity screenings. Here, we developed a label-free Electric Cell-substrate Impedance Sensing (ECIS)-based cytotoxicity assay that can be applied in the assessment of the cytotoxicity of natural extracts. The conditions to measure the impedance using ECIS were first optimized in mice immortalized hypothalamic neurons GT1-7 cells. The performance of four natural extracts when tested using three conventional cytotoxicity assays in GT1-7 cells, was studied. Betula pendula (silver birch tree) was found to interfere with all of the cytotoxicity assays in which labels were applied. The silver birch extract was also proven to be cytotoxic and, thus, served as a proof-of-concept for the use of ECIS. The extract was fractionated and the ECIS method permitted the distinction of specific kinetic patterns of cytotoxicity on the fractions as well as the extract's pure constituents. This study offers evidence that ECIS is an excellent tool for real-time monitoring of the cytotoxicity of complex extracts that are difficult to work with using conventional (label-based) assays. Altogether, it offers a very suitable cytotoxicity-screening assay making the work with natural products less challenging within the drug discovery workflow.Peer reviewe

Rock Type Effects on Radio Signal Attenuation

This research work is aimed at studying different rock types and the effect of their mineral contents on an active 434 MHz RFID card's radio signal attenuation. This research was done at the ONKALO nuclear waste storage facility using radio frequency identification (RFID) equipment. First, the studied area and research plan, including the used system and equipment, are explained. After this, the researched areas of rock types and their effects on radio signals are presented. This work focused mainly on occupational safety, but it also investigated whether it would be possible to use RFID technology in producing mines as well, especially in the boundary layer of the ore body. This research can help the design of communication frequencies for autonomous devices.publishedVersionPeer reviewe

Phenolic compounds extracted by acidic aqueous ethanol from berries and leaves of different berry plants

Phenolic compounds of berries and leaves of thirteen various plant species were extracted with aqueousethanol and analyzed with UPLC-DAD-ESI-MS, HPLC-DAD, and NMR. The total content of phenolics wasconsistently higher in leaves than in berries (25–7856 vs. 28–711 mg/100 g fresh weight). Sea buckthornleaves were richest in phenolic compounds (7856 mg/100 g f.w.) with ellagitannins as the dominant compoundclass. Sea buckthorn berries contained mostly isorhamnetin glycosides, whereas quercetin glycosideswere typically abundant in most samples investigated. Anthocyanins formed the dominating groupof phenolics in most dark-colored berries but phenolic acid derivatives were equally abundant in saskatoonand chokeberry berries. Caffeoylquinic acids constituted 80% of the total phenolic content(1664 mg/100 g f.w.) in bilberry leaves. B-type procyanidins and caffeoylquinic acids were the major phenoliccompounds in hawthorn and rowanberry, respectively. Use of leaves of some species with prunasin,tyramine and b-p-arbutin, may be limited in food applications.</p

Antiviral functionalization of cellulose using tannic acid and tannin-rich extracts

Due to seasonally appearing viruses and several outbreaks and present pandemic, we are surrounded by viruses in our everyday life. In order to reduce viral transmission, functionalized surfaces that inactivate viruses are in large demand. Here the endeavor was to functionalize cellulose-based materials with tannic acid (TA) and tannin-rich extracts by using different binding polymers to prevent viral infectivity of both non-enveloped coxsackievirus B3 (CVB3) and enveloped human coronavirus OC43 (HCoV-OC43). Direct antiviral efficacy of TA and spruce bark extract in solution was measured: EC50 for CVB3 was 0.12 and 8.41  μg/ml and for HCoV-OC43, 78.16 and 95.49  μg/ml, respectively. TA also led to an excellent 5.8- to 7-log reduction of severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) virus infectivity. TA functionalized materials reduced infectivity already after 5-min treatment at room temperature. All the tested methods to bind TA showed efficacy on paperboard with 0.1 to 1% (w/v) TA concentrations against CVB3 whereas material hydrophobicity decreased activities. Specific signatures for TA and HCoV-OC43 were discovered by Raman spectroscopy and showed clear co-localization on the material. qPCR study suggested efficient binding of CVB3 to the TA functionalized cellulose whereas HCoV-OC43 was flushed out from the surfaces more readily. In conclusion, the produced TA-materials showed efficient and broadly acting antiviral efficacy. Additionally, the co-localization of TA and HCoV-OC43 and strong binding of CVB3 to the functionalized cellulose demonstrates an interaction with the surfaces. The produced antiviral surfaces thus show promise for future use to increase biosafety and biosecurity by reducing pathogen persistence

Chemical composition and bioactivity of hemp, reed canary grass and common reed grown on boreal marginal lands

Underutilised agricultural land and former peat production areas in northern Europe are potentially suitable for growing lignocellulosic biomass that could be used in various non-food applications. In this study, the biorefining process of Phalaris arundinacea (reed canary grass), Phragmites australis (common reed), and Cannabis sativa (oil and fibre hemp cultivars) was assessed based on their chemical composition and biological activity using various analytical techniques. Two-stage accelerated solvent extraction was used first with hexane, followed by EtOH/H2O (95/5, v/v) to extract the lipophilic and hydrophilic fractions of the samples collected during and after the growing season. Later, pressurised hot water extraction (PHWE) and two-stage extraction were performed to examine the biorefinery potential of aqueous extracts focusing on extraction efficiency, quality, and chemical composition of the plant materials. Combining two-stage and elevated extraction temperatures with PHWE resulted in high levels of total dissolved solids (TDS), carbohydrates, phenolics, and bioactivities. Data showed that TDS yielded over 400 mg g−1 for summer oil hemp and approximately 300 mg g−1 for reed canary grass and common reed. Summer-harvested plants had carbohydrate yields of 110–155 mg g−1, while autumn yields were 40–60 mg g−1 for hemp and 120–170 mg g−1 for reed canary grass and common reed, respectively. The findings suggest that aboveground biomass from marginal lands holds potential as a valuable source of bioactive compounds for biorefinery feedstocks, thereby presenting new opportunities for sustainable biomass-based valorisation and future optimisation of two-stage extraction methods targeting hemicellulose-rich fractions