Antibacterial and Oxidative Stress-Protective Effects of Five Monoterpenes from Softwood.

Volatile organic compounds (VOC) affect the quality of indoor air. Terpenes and especially monoterpenes are the main molecules emitted from softwood material (coniferous species), which is widely used in construction. The corneal epithelium is one of the first human membranes to encounter VOCs in the air. Moreover, the industrial use of pleasant-scented monoterpenes in cosmetics, food, and detergents exposes people to monoterpenes in their daily lives. In the present study, the health effective properties of five monoterpenes from softwood were tested; cytotoxicity and oxidative stress-protective effects of α- and ß-pinenes, R- and S-limonene, and 3-carene were tested in a human corneal epithelial (HCE) cell model system and with two additional in vitro antioxidant tests: oxygen radical absorbance capacity (ORAC) and hydrogen peroxide (H2O2) scavenging. Antibacterial efficacies were tested with two bioluminescent bacterial biosensor strains (Escherichia coli K12+pcGLS11 and Staphylococcus aureus RN4220+pAT19) and with minimum inhibitory concentration (MIC) test against Escherichia coli. Only very high concentrations of monoterpenes (0.3-0.5 mg/mL) demonstrated cytotoxicity against HCE cells. Contrary to the original hypothesis, monoterpenes did not exhibit strong antioxidant properties in tested concentrations. However, biosensors and MIC tests indicated clear antibacterial activities for all tested monoterpenes.

Field-Grown and In Vitro Propagated Round-Leaved Sundew (Drosera rotundifolia L.) Show Differences in Metabolic Profiles and Biological Activities.

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.

Bioactive Properties of the Aqueous Extracts of Endophytic Fungi Associated with Scots Pine (Pinus sylvestris) Roots.

Despite the continuing interest in various plant and natural products, only a small portion of the biologically active compounds from nature has been discovered and exploited. In this study, antioxidant and antibacterial properties of aqueous fractions of three endophytic fungi isolated from the roots of 8-year-old Scots pines (Pinus sylvestris) growing on a drained peatland were investigated. The endophytic fungi species were Acephala applanata, Phialocephala fortinii, and Humicolopsis cephalosporioides/Coniochaeta mutabilis. The bioactivities were examined using hydrogen peroxide scavenging and oxygen radical absorbance capacity tests as well as sensitive Escherichia coli-based biosensors, which produce a luminescent signal in the presence of substances with oxidative or genotoxic properties. In addition, cell models for Parkinson's disease, age-related macular degeneration, and osteoarthritis were used to evaluate the potential for pharmaceutical applications. The aqueous extracts of fungi and 19 out of 42 fractions were found to be active in one or more of the tests used. However, no activity was found in the age-related macular degeneration and osteoarthritis cell model tests. Additionally, bioactivity data was connected with metabolites putatively annotated, and out of 330 metabolites, 177 were interesting in view of the bioactivities investigated. A majority of these were peptides and all three fungal species shared a highly similar metabolome. We propose that Scots pine endophytic fungi are a rich source of interesting metabolites, and synergistic effects may cause the bioactivities, as they were found to vary after the fractionation process.

Correction: Tienaho, J., et al. Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC-Orbitrap-MS. Molecules 2019, 24, 2330.

The authors wish to make the following corrections to this paper published in Molecules[...].

Metabolic Profiling of Water-Soluble Compounds from the Extracts of Dark Septate Endophytic Fungi (DSE) Isolated from Scots Pine (Pinus sylvestris L.) Seedlings Using UPLC-Orbitrap-MS.

Endophytes are microorganisms living inside plant hosts and are known to be beneficial for the host plant vitality. In this study, we isolated three endophytic fungus species from the roots of Scots pine seedlings growing on Finnish drained peatland setting. The isolated fungi belonged to dark septate endophytes (DSE). The metabolic profiles of the hot water extracts of the fungi were investigated using Ultrahigh Performance Liquid Chromatography with Diode Array Detection and Electron Spray Ionization source Mass Spectrometry with Orbitrap analyzer (UPLC-DAD-ESI-MS-Orbitrap). Out of 318 metabolites, we were able to identify 220, of which a majority was amino acids and peptides. Additionally, opine amino acids, amino acid quinones, Amadori compounds, cholines, nucleobases, nucleosides, nucleotides, siderophores, sugars, sugar alcohols and disaccharides were found, as well as other previously reported metabolites from plants or endophytes. Some differences of the metabolic profiles, regarding the amount and identity of the found metabolites, were observed even though the fungi were isolated from the same host. Many of the discovered metabolites have been described possessing biological activities and properties, which may make a favorable contribution to the host plant nutrient availability or abiotic and biotic stress tolerance.

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.

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.

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.

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.

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.

A panel of bioluminescent whole-cell bacterial biosensors for the screening for new antibacterial substances from natural extracts.

Whole-cell bacterial biosensors can be applied for the screening of antibacterial properties of extracts. We constructed a biosensor panel consisting of four different bacterial biosensor strains: Escherichia, Staphylococcus, Acinetobacter and Pseudomonas for expanded screening potential. The functionality of the panel was first evaluated with known antibacterial compounds: ethanol, naphthoquinones (juglone, lawsone, plumbagin) and a flavonoid (quercetin). Natural extracts comprise a vast source of potential new antibacterials for diverse functional purposes. To demonstrate the utilization of the panel for screening of a demanding sample material, round-leaved sundew (Drosera rotundifolia) extracts were used as an example. Differences between field- and laboratory originating sundew extracts could be detected. This demonstrates the efficiency of the developed biosensor panel in the rapid screening of the antibacterial properties of plant extracts.

A Bioscreening Technique for Ultraviolet Irradiation Protective Natural Substances.

Ultraviolet radiation (UV-R) causes genotoxic and aging effects on skin, and sunscreens are used to alleviate the damage. However, sunscreens contain synthetic shielding agents that can cause harmful effects in the environment. Nature-derived substances may have potential as replacement materials for the harmful sunscreen chemicals. However, screening of a broad range of samples is tedious, and often requires a separate genotoxicity assessment. We describe a simple microplate technique for the screening of UV protective substances using a recombinant Escherichia coli biosensor. Both absorbance-based and bioactivity-based shields can be detected with simultaneous information about the sample genotoxicity. With this technique, a controversial sunscreen compound, oxybenzone offers physical or absorbance-based shield but appears genotoxic at higher concentrations (3.3 mg/mL). We also demonstrate that pine needle extract (PiNe ) shields the biosensor from UV-R in a dose-dependent manner without showing genotoxicity. The physical shield of 5 mg/mL PiNe was similar to that of one of the most common UV-shielding compound TiO2 concentration 0.80 mg/mL. The bioactivity-based shield of PiNe also reaches the extent of the physical shield with the highest concentration (3.3 mg/mL). We conclude that our technique is suitable in detecting the UV-shielding potential of natural substances, and gives simultaneous information on genotoxicity.

Method with high-throughput screening potential for antioxidative substances using Escherichia coli biosensor katG'::lux.

A new method is described for the rapid real-time screening of antioxidative properties using a recombinant Escherichia coli DPD2511 biosensor. This microplate technique, without time-consuming pre-incubations and handling, has potential for a high-throughput search of bioactive compounds. Special emphasis was given to obtaining highly reliable and repeatable results.