Valorizing Assorted Logging Residues: Response Surface Methodology in the Extraction Optimization of a Green Norway Spruce Needle-Rich Fraction To Obtain Valuable Bioactive Compounds.

During stemwood harvesting, substantial volumes of logging residues are produced as a side stream. Nevertheless, industrially feasible processing methods supporting their use for other than energy generation purposes are scarce. Thus, the present study focuses on biorefinery processing, employing response surface methodology to optimize the pressurized extraction of industrially assorted needle-rich spruce logging residues with four solvents. Eighteen experimental points, including eight center point replicates, were used to optimize the extraction temperature (40-135 °C) and time (10-70 min). The extraction optimization for water, water with Na2CO3 + NaHSO3 addition, and aqueous ethanol was performed using yield, total dissolved solids (TDS), antioxidant activity (FRAP, ORAC), antibacterial properties (E. coli, S. aureus), total phenolic content (TPC), condensed tannin content, and degree of polymerization. For limonene, evaluated responses were yield, TDS, antioxidant activity (CUPRAC, DPPH), and TPC. Desirability surfaces were created using the responses showing a coefficient of determination (R2) > 0.7, statistical significance (p ≤ 0.05), precision > 4, and statistically insignificant lack-of-fit (p > 0.1). The optimal extraction conditions were 125 °C and 68 min for aqueous ethanol, 120 °C and 10 min for water, 111 °C and 49 min for water with Na2CO3 + NaHSO3 addition, and 134 °C and 41 min for limonene. The outcomes contribute insights to industrial logging residue utilization for value-added purposes.

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 (SARS-CoV-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.

Willow (Salix spp.) bark hot water extracts inhibit both enveloped and non-enveloped viruses: study on its anti-coronavirus and anti-enterovirus activities.

Introduction: Recurring viral outbreaks have a significant negative impact on society. This creates a need to develop novel strategies to complement the existing antiviral approaches. There is a need for safe and sustainable antiviral solutions derived from nature. Objective: This study aimed to investigate the antiviral potential of willow (Salix spp.) bark hot water extracts against coronaviruses and enteroviruses. Willow bark has long been recognized for its medicinal properties and has been used in traditional medicines. However, its potential as a broad-spectrum antiviral agent remains relatively unexplored. Methods: Cytopathic effect inhibition assay and virucidal and qPCR-based assays were used to evaluate the antiviral potential of the bark extracts. The mechanism of action was investigated using time-of-addition assay, confocal microscopy, TEM, thermal, and binding assays. Extracts were fractionated and screened for their chemical composition using high-resolution LC-MS. Results: The native Salix samples demonstrated their excellent antiviral potential against the non-enveloped enteroviruses even at room temperature and after 45 s. They were equally effective against the seasonal and pandemic coronaviruses. Confocal microscopy verified the loss of infection capacity by negligible staining of the newly synthesized capsid or spike proteins. Time-of-addition studies demonstrated that Salix bark extract had a direct effect on the virus particles but not through cellular targets. Negative stain TEM and thermal assay showed that antiviral action on enteroviruses was based on the added stability of the virions. In contrast, Salix bark extract caused visible changes in the coronavirus structure, which was demonstrated by the negative stain TEM. However, the binding to the cells was not affected, as verified by the qPCR study. Furthermore, coronavirus accumulated in the cellular endosomes and did not proceed after this stage, based on the confocal studies. None of the tested commercial reference samples, such as salicin, salicylic acid, picein, and triandrin, had any antiviral activity. Fractionation of the extract and subsequent MS analysis revealed that most of the separated fractions were very effective against enteroviruses and contained several different chemical groups such as hydroxycinnamic acid derivatives, flavonoids, and procyanidins. Conclusion: Salix spp. bark extracts contain several virucidal agents that are likely to act synergistically and directly on the viruses.

Inspired by nature: Fiber networks functionalized with tannic acid and condensed tannin-rich extracts of Norway spruce bark show antimicrobial efficacy.

This study demonstrated the antibacterial and antiviral potential of condensed tannins and tannic acid when incorporated into fiber networks tested for functional material purposes. Condensed tannins were extracted from industrial bark of Norway spruce by using pressurized hot water extraction (PHWE), followed by purification of extracts by using XADHP7 treatment to obtain sugar-free extract. The chemical composition of the extracts was analyzed by using HPLC, GC‒MS and UHPLC after thiolytic degradation. The test matrices, i.e., lignocellulosic handsheets, were produced and impregnated with tannin-rich extracts, and tannic acid was used as a commercial reference. The antibacterial and antiviral efficacy of the handsheets were analyzed by using bioluminescent bacterial strains (Staphylococcus aureus RN4220+pAT19 and Escherichia coli K12+pCGLS11) and Enterovirus coxsackievirus B3. Potential bonding of the tannin-rich extract and tannic acid within the fiber matrices was studied by using FTIR-ATR spectroscopy. The deposition characteristics (distribution and accumulation patterns) of tannin compounds and extracts within fiber networks were measured and visualized by direct chemical mapping using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and digital microscopy. Our results demonstrated for the first time, how tannin-rich extracts obtained from spruce bark side streams with green chemistry possess antiviral and antibacterial properties when immobilized into fiber matrices to create substitutes for plastic hygienic products, personal protection materials such as surgical face masks, or food packaging materials to prolong the shelf life of foodstuffs and prevent the spread of infections. However, more research is needed to further develop this proof-of-concept to ensure stable chemical bonding in product prototypes with specific chemistry.

Essential Oils and Supercritical CO2 Extracts of Arctic Angelica (Angelica archangelica L.), Marsh Labrador Tea (Rhododendron tomentosum) and Common Tansy (Tanacetum vulgare)-Chemical Compositions and Antimicrobial Activities.

Traditionally, arctic Finnish Angelica (Angelica archangelica L.), marsh Labrador tea (Rhododendron tomentosum, syn. Ledum palustre) and common tansy (Tanacetum vulgare) have been used as medicinal herbs in folklore medicine. However, these underutilised plants are a source of, e.g., oil-based compounds, which could benefit many modern applications implemented by the green chemistry extraction methods, as well. We extracted Angelica, marsh Labrador tea and common tansy by non-toxic and recyclable extraction methods, i.e., hydrodistillation and supercritical carbon dioxide (scCO2) extraction; characterised the essential oils (EOs) and scCO2 extracts by combination of gas chromatography and mass spectrometry (GC-MS), and in addition, analysed the antimicrobial properties. As expected for Angelica root and common tansy inflorescence, the scCO2 extraction method produced less amount of volatile compounds compared to hydrodistillation. On the other hand, more coumarins, alkanes, fatty alcohols and fatty acids were obtained. Additionally, sesquiterpenoids palustrol and ledol were predominant compounds in both marsh Labrador tea EO and scCO2 extract. According to our results, however, all the EOs and scCO2 extracts showed broad spectrum of antimicrobial activities against the selected microbes, but the effects were extract-specific. The strongest and broadest antimicrobial activities were performed by marsh Labrador tea scCO2 extract, which showed extremely strong effect on Staphylococcusaureus subsp. aureus and strong effect on Candida albicans.

Salix spp. Bark Hot Water Extracts Show Antiviral, Antibacterial, and Antioxidant Activities-The Bioactive Properties of 16 Clones.

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.

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.

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 APPLICATION: Globally, 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.

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.

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 aqueous ethanol and analyzed with UPLC-DAD-ESI-MS, HPLC-DAD, and NMR. The total content of phenolics was consistently higher in leaves than in berries (25-7856 vs. 28-711mg/100g fresh weight). Sea buckthorn leaves were richest in phenolic compounds (7856mg/100g f.w.) with ellagitannins as the dominant compound class. Sea buckthorn berries contained mostly isorhamnetin glycosides, whereas quercetin glycosides were typically abundant in most samples investigated. Anthocyanins formed the dominating group of phenolics in most dark-colored berries but phenolic acid derivatives were equally abundant in saskatoon and chokeberry berries. Caffeoylquinic acids constituted 80% of the total phenolic content (1664mg/100g f.w.) in bilberry leaves. B-type procyanidins and caffeoylquinic acids were the major phenolic compounds in hawthorn and rowanberry, respectively. Use of leaves of some species with prunasin, tyramine and ß-p-arbutin, may be limited in food applications.

Online Measurement 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.

Phenolic compounds of the inner bark of Betula pendula: seasonal and genetic variation and induction by wounding.

The contents of individual phenolic compounds in the inner bark of silver birch (Betula pendula Roth) were analyzed by HPLC-DAD. Samples from 21 mature trees originating from three micropropagated parent trees were collected six times over a 1-year period. Significant seasonal variation in the quantities of ten compounds and four chromatographically unresolved compound pairs was found. A majority of the compounds also exhibited significant quantitative variation among birch clones. There were no qualitative differences associated with the season or among the clones. However, wounding of the bark induced the production of new types of bark phenolics: several ellagitannins were detected in the callus tissues of birch for the first time.

Procyanidin xylosides from the bark of Betula pendula.

A procyanidin dimer xyloside, catechin-(4α→8)-7-O-ß-xylopyranosyl-catechin, was isolated from the inner bark of Betula pendula and its structure was determined using 1D and 2D NMR, CD and high-resolution ESIMS. Interestingly, the 7-O-ß-xylopyranose unit was found to be present in the lower terminal unit of the dimer. In addition to this procyanidin dimer xyloside, an entire series of oligomeric and polymeric procyanidin xylosides was detected. Their structures were investigated by hydrophilic interaction HPLC-HRESIMS. Procyanidin glycosides are still rarely found in nature.

Birch inner bark procyanidins can be resolved with enhanced sensitivity by hydrophilic interaction HPLC-MS.

A complex mixture of procyanidin aglycones was isolated by Sephadex LH-20 column chromatography from the silver birch inner bark, which is a polyphenol-rich source of natural antioxidants. Procyanidins were studied by using hydrophilic interaction liquid chromatography combined with high-resolution ESI-TOF-MS. A good chromatographic separation was achieved and procyanidins eluted according to their increasing degree of polymerization. Individual procyanidins were detected from dimers up to the degree of polymerization of 22 by their negative-ion mass spectra. The results showed that hydrophilic interaction liquid chromatography can be successfully applied for the analysis of high-molecular-weight procyanidins with enhanced sensitivity in electrospray mass spectrometry.

Two new phenylbutanoids from inner bark of Betula pendula.

Two phenylbutanoids, 7-{3R-[(4-hydroxyphenyl)butyl] beta-glucopyranosid-O-6-yl} 4-O-beta-glucopyranosylvanillin and 3-beta-glucopyranosyloxy-1-(4-hydroxyphenyl)-butanone were isolated from an aqueous methanol extract of the inner bark of Betula pendula. Their structures were determined by NMR spectroscopy and mass spectrometry. The complete assignment of proton and carbon signals was achieved by 1D and 2D NMR experiments: selective 1D TOCSY, HSQC, HMBC and DQF-COSY.

Lignans from the bark extract of Pinus sylvestris L.

Three lignans were isolated from a bioactive fraction of the 70% aqueous acetone extract of the pine (Pinus sylvestris L.) bark. Their structures were identified by high-resolution positive ion EI mass spectrometry and NMR spectroscopy. The complete assignment of proton and carbon signals was achieved by 2D NMR experiments: HSQC, HMBC, DQF-COSY and NOESY.

Characterisation of proanthocyanidin aglycones and glycosides from rose hips by high-performance liquid chromatography-mass spectrometry, and their rapid quantification together with vitamin C.

Fifteen individual proanthocyanidin aglycones and 19 glycosides, together with a complex mixture of chromatographically non-separated tetra- to octameric proanthocyanidin glycosides were detected--the non-separated glycosides being novel natural products--and characterised from dog rose hips using high-performance liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS). Along with these phenolics, a 50% aqueous ethanol extract of rose hips was found to contain high levels of Vitamin C. A simple and rapid HPLC method assisted by diode array detection for the estimation of the total concentration of proanthocyanidin aglycones and glycosides, as well as Vitamin C, in rose hip extracts was developed.

A new dihydroflavonol from Pinus sylvestris L.

A novel dihydroflavonol, C-6,O-7-dimethylaromadendrin, was isolated from a 70% aqueous acetone extract of pine (Pinus sylvestris L.) bark. Its structure was determined by high-resolution negative fast atom bombardment mass spectrometry and NMR spectroscopy. The complete assignment of proton and carbon signals was achieved by 2D NMR experiments: HSQC, HMBC, DQF-COSY and NOESY.

Role of X chromosomal song genes in the evolution of species-specific courtship songs in Drosophila virilis group species.

In the Drosophila virilis group the males of the virilis phylad species produce courtship song consisting of pulse trains with no pauses between successive sound pulses, whereas the males of the montana phylad species produce songs with clear pauses between the sound pulses. We obtained song data for F1 hybrids between D. virilis (representing the virilis phylad) or D. flavomontana (representing the montana phylad) females and the males of several species of the D. virilis group to study the interaction of X chromosomal and autosomal song genes affecting species differences in song. In crosses with D. virilis females, X chromosomal (or maternal) factors masked variation in pulse length despite variation in heterospecific autosomal song genes. To the contrary, in crosses with D. flavomontana females, X chromosomal genes largely determined the pause length and interacted with autosomal genes to determine the pulse length. In the montana phylad species, pulse length showed dominance toward shorter pulses and pause length toward longer pauses. The first-mentioned trait also indicated the epistatic effects of X chromosomal and autosomal components.