antibiotics

Article Examining Safety of Biocolourants from Fungal and Plant Sources-Examples from Cortinarius and Tapinella, Salix and Tanacetum spp. and Dyed Woollen Fabrics

Riikka Räisänen 1,*, Anja Primetta 1,2, Sari Nikunen 1, Ulla Honkalampi 3, Heli Nygren 4, Juha-Matti Pihlava 5, Ina Vanden Berghe 6 and Atte von Wright 3

1 Craft Studies, University of Helsinki, P.O. Box 8, 00014 Helsinki, Finland; anja.primetta@helsinki.fi (A.P.); [email protected] (S.N.) 2 Department of Environmental and Biological Sciences, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland 3 Department of Pharmacology and Toxicology, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland; u.honkalampi@innolact.fi (U.H.); atte.vonwright@uef.fi (A.v.W.) 4 VTT Technical Research Centre of Finland Ltd., P.O. Box 1000, 02044 Espoo, Finland; heli.nygren@vtt.fi 5 Natural Resources Institute Finland (Luke), Tietotie 4, 31600 Jokioinen, Finland; juha-matti.pihlava@luke.fi 6 Royal Institute for Cultural Heritage (IRPA/KIK), Parc du Cinquantenaire 1, 1000 Brussels, Belgium; [email protected] * Correspondence: riikka.raisanen@helsinki.fi; Tel.: +358-503183973



Received: 31 March 2020; Accepted: 18 May 2020; Published: 20 May 2020


Abstract: Biocolourants have been investigated as alternatives to synthetic dyes. However, natural origin per se is not a label of harmlessness and research is needed to obtain safe dyes. We studied the cytotoxicity of the extracts from fungal (Cortinarius semisanguineus, Tapinella atrotomentosa) and plant (Tanacetum vulgare, Salix phylicifolia) sources and the woollen fabrics dyed with the extracts. Cytotoxicity in vitro using hepa-1 mouse hepatoma cells for 24 h and 72 h exposure was observed as the highest tolerated dose. All biocolourants produced intensive colour on fabrics with fastness properties from moderate to good. The Salix and Cortinarius samples did not show any cytotoxic effects, whereas the Tanacetum and Tapinella samples had slightly higher test values but were not interpreted as being significantly toxic. Higher than zero values of the undyed fabrics showed the importance of examining their toxicity as well. It was found that the cytotoxicity of the samples dyed with the biocolourants did not differ significantly from the undyed wool fabric. The concentrations of dyes used in the assays were very low, imitating the dose of the user. In addition to colouring properties, natural dyes may have pharmaceutical and antibacterial properties which would enhance the interest in using them in products for added value.

Keywords: natural dye; biocolourant; secondary metabolite; in vitro; cytotoxicity; mouse hepatoma cell; highest tolerated dose; HPLC-UV/Vis-MS

1. Introduction

1.1. Background The environmental disadvantages of synthetic dyes are well known. The textile industry discharges large amounts of highly coloured effluent wastewater, the dyes of which severely affect photosynthesis and aquatic life. Effluents may also contain toxic amounts of metal ions and chlorine [1]. In textile workers and end-users, synthetic colourants may cause adverse reactions, such as allergies, urticaria

Antibiotics 2020, 9, 266; doi:10.3390/antibiotics9050266 www.mdpi.com/journal/antibiotics Antibiotics 2020, 9, 266 2 of 24 and dermatitis, or respiratory difficulties like asthma, in addition to cytotoxic, genotoxic, carcinogenic and mutagenic effects [2–5]. Alternative colourants among secondary metabolites from plant and microbial sources such as fungi, yeast and algae have been explored. The natural origin does not automatically mean non-toxicity. Also, the different stages of the textile production process have an impact on the final product, its safety to the consumer, and the environmental load. The degradability of biocolourants is a positive feature in circular material production, whereas in the textile dyeing process, the requirements of colourants are set in high affinity to fibres and stability under end-use conditions. Thus, the utilisation of bio-based colourants requires careful consideration of dye technical and toxicological properties as well as economic feasibility, in which the multi-functionality through antimicrobial and UV-protective properties can create interesting prospects. Our aim in this article is to examine the information about the safety of natural colourants and the results from the preliminary tests of a few dyes of fungal and plant origins. In this study, fungi surprise webcap [Cortinarius semisanguineus (Fr.) Gillet] and velvet roll-rim [Tapinella atrotomentosa (Batsch.) Šutara] and plants tansy (Tanacetum vulgare L.) and willow bark (Salix phylicifolia L.) were used as sources of biocolourants for dyeing of woollen fabrics.

1.2. Safety and Medicinal Use Related to Fungal and Plant Colourants Correct identification and characterisation of the biocolourant’s source is crucial when assessing potential toxicity, as toxic compounds are concentrated in certain taxa [6,7]. Different taxa, whether fungi or plants, also have their own typical colourants [8–11]. For Rubia and Cortinarius genera, the anthraquinones provide a variety of red hues, whereas yellow-orange flavonoids and carotenoids can be obtained from Tanacetum species, and bluish-violet terphenyl quinones from Tapinella fungi [12,13]. Plant parts such as stems, leaves, flowers, roots, bark, berries, and cones vary in the composition and contents of biocolourants according to the developmental stage and harvesting time [6,10,14–17]. The contents may also vary depending on the geographical area, cultivation practices and storage conditions [18–22]. The pre-handling steps such as drying and grinding and subsequent extraction conditions (e.g., solvent) affect the content of biocolourants [13,23–25]. However, after drying at 45 ◦C for 1 week, milling to a powder and storage for more than a year, at least two native enzymes in madder roots (Rubia tinctorum L.) were still highly active upon addition of water, and depending on the conditions, caused conversion of molecules into forms, some of which were toxic [26]. Natural dyes are complex mixtures of dye components, setting challenges for industrial utilisation. Repeatable colour can be achieved by using standardised extraction and quality analyses of the dye components in the mixtures or by producing single component purified dyes [13,25]. The improved repeatability of colour components will also minimise variation of other secondary metabolites and risks of toxic properties. By understanding reaction mechanisms and how the reactions can be controlled [26], the safety risks can be minimised. Those reactions may also affect the dyeing potential, as the products of hydrolysis, i.e., aglycone structures, differ in their properties and affinities to various materials compared to their derivatives [9,13]. The historical background does not necessarily give any guarantee of safeness as even widely-used dye sources can contain toxic secondary metabolites: madder root extracts (R. tinctorum) having alizarin (1) as the main anthraquinone aglycone, also contained toxic lucidin (2), rubiadin (3) and xanthopurpurin (4) (Table1)[ 26–30], of which lucidin and xanthopurpurin were hydrolysis and decarboxylation products of lucidin primeveroside and munjistin. Furthermore, it has been proposed that lucidin is converted into other compounds, such as nordamnacanthal or quinone methide [26,28,31] or degrade to xanthopurpurin. Antibiotics 2020, 9, 266 3 of 25 Antibiotics 2020, 9, 266 3 of 25 Antibiotics 2020, 9, 266 3 of 25 Antibiotics 2020, 9, 266 3 of 25 Previously, Yasui and Takeda [29] had found that the madder root extracts (EtOAc) were mutagenic Previously, Yasui and Takeda [29] had found that the madder root extracts (EtOAc) were mutagenic forPreviously, both strains Yasui of and S. typhimuriumTakeda [29] had(TA100 found and that TA98). the madder They concludedroot extracts that (EtOAc) all mutagenic were mutagenic activity Previously,for both strains Yasui of and S. typhimuriumTakeda [29] had(TA100 found and that TA98). the madder They concludedroot extracts that (EtOAc) all mutagenic were mutagenic activity foundfor both in strainsthe EtOAc of S. extracts typhimurium was due (TA100 to lucidin. and TA98). However, They Kawasaki concluded et thatal. [32] all mutagenicsuggested thatactivity the forfound both in strainsthe EtOAc of S. extracts typhimurium was due (TA100 to lucidin. and TA98). However, They Kawasaki concluded et thatal. [32] all mutagenicsuggested thatactivity the mutagenicityfound in the EtOAcof the extractsextracts ofwas R. tinctorumdue to lucidin. was not However, exclusively Kawasaki due to etlucidin al. [32] but suggested also a result that andthe foundmutagenicity in the EtOAcof the extractsextracts ofwas R. duetinctorum to lucidin. was not However, exclusively Kawasaki due to etlucidin al. [32] but suggested also a result that andthe contributionmutagenicity of of other the extracts mutagenic of R. compounds. tinctorum was not exclusively due to lucidin but also a result and mutagenicitycontribution of of other the extracts mutagenic of R. c ompounds.tinctorum was not exclusively due to lucidin but also a result and contribution of other mutagenic compounds. Antibioticscontribution2020, 9 ,of 266 other mutagenic compounds. 3 of 24 Table 1. Chemical structures of some natural anthraquinones. Table 1. Chemical structures of some natural anthraquinones. Table 1. Chemical structures of some natural anthraquinones. Table 1. Chemical structures of some natural anthraquinones. Chemical Compound Structure TableChemical 1. Chemical Compound structures of some naturalStructure anthraquinones. Chemical Compound Structure Chemical Compound Structure Chemical Compound Structure Alizarin (1) Alizarin (1) AlizarinC14H8O (41 ) AlizarinC14H8O (41 ) M = 240.21Alizarin g mol (1) −1 C14H8O4 −1 M = 240.21CC14HH8 OgO4 mol M = 240.2114 g8 mol4 −1 MM == 240.21240.21 g g mol mol−1 1 −

Lucidin (2) Lucidin (2) LucidinCLucidin15H10O (25 ()2 ) LucidinC15H10O (25 ) M = 270.24 g mol−1 CC1515HH1010OO5 5 −1 M = 270.24C15H10 Og 5mol 1 MM == 270.24270.24 g gmol mol−1− M = 270.24 g mol−1

Rubiadin (3) RubiadinRubiadin (3 ()3 ) RubiadinC15H10O (4 3) RubiadinCC15HH10O O(43 ) M = 254.2415 10g mol4 −1 C15H10O4 −1 1 MM == 254.24C254.2415H10 Og g 4mol mol− M = 254.24 g mol−1 M = 254.24 g mol−1

Xanthopurpurin/Xanthopurpurin / Xanthopurpurin/ PurpurPurpuroxanthineXanthopurpurin/oxanthine (4 ()4 ) PurpurXanthopurpurin/oxanthine (4 ) PurpurCCoxanthine1414HH8O8O4 4 (4) PurpurCoxanthine14H8O4 (4)1 MM == 240.21240.21 g gmol mol−1− C14H8O4 −1 M = 240.21C14H8 Og4 mol M = 240.21 g mol−1 M = 240.21 g mol−1

Toxicity studies of colour providing secondary metabolites, e.g., those of anthraquinones, The chemical structures of secondary metabolites and the ir functional groups, for example, have givenThe chemical somewhat structures contradictory of secondary results. metabolites Jäger et al. [and27] detected their functional lucidin ( groups,2), rubiadin for example (3) and , hydroxyl,The chemical alkyl, alkoxy structures or chlorine, of secondary attached metabolites at specific and positions, their functional can confer groups, different for biological example, xanthopurpurinhydroxyl,The chemical alkyl, ( 4 alkoxy) being structures ormutagenic, chlorine, of secondary but attached not metabolites alizarin at specific (1) (Tableand positions, the1),ir in functional madder can confer root groups, different extracts for (EtOAc). biological example, activitieshydroxyl, andalkyl, ge alkoxynotoxicity or chlorine, profiles attached [33–35]. at Lucidin specific positions,(2) and rubiadin can confer (3 different), which biological are 1,3- However,hydroxyl,activities all andalkyl, the madderge alkoxynotoxicity dyedor chlorine, profileswool samples attached [33–35]. had at Lucidin mutagenic specific positions,(2) e ff andects in rubiadin can Ames confer assays (3 different), which (strain biological TA100). are 1,3- dihydroxyanthraquinonesactivities and genotoxicity possessing profiles a [33methyl–35]. or Lucidin hydroxymethyl (2) and group rubiadin at carbon (3), 2, which showed are strong 1,3- Previously,activitiesdihydroxyanthraquinones Yasui and and genotoxicity Takeda possessing [29 profiles] had found a [33methyl that–35]. the or Lucidin hydroxymethyl madder ( root2) and extracts group rubiadin (EtOAc) at carbon (3 were), 2, which mutagenicshowed are strong for 1,3- mutagenicity,dihydroxyanthraquinones whereas alizarin possessing (1), 1,2 a-dihydroxyanthraquinone methyl or hydroxymethyl without group atother carbon substituents 2, showed, had strong no bothdihydroxyanthraquinonesmutagenicity, strains of S. whereas typhimurium alizarin possessing(TA100 (1), 1,2 and a-dihydroxyanthraquinone methyl TA98). or They hydroxymethyl concluded that without group all mutagenic at other carbon substituents activity2, showed found, hadstrongin no mutagenicity.mutagenicity, whereasFurther, 4,5alizarin-dihydroxylation (1), 1,2-dihydroxyanthraquinone of 2-methyl anthraquinones without did other not substituents have any appa, hadrent no themutagenicity,mutagenicity. EtOAc extracts whereasFurther, was due 4,5alizarin- todihydroxylation lucidin. (1), 1,2 However,-dihydroxyanthraquinone of 2 Kawasaki-methyl anthraquinones et al. [32 without] suggested did other not that substituents have the mutagenicityany appa, hadrent no effectmutagenicity. on the mutagenicity.Further, 4,5-dihydroxylation The results suggested of 2-methyl that anthraquinonesboth the number did of not OH have-groups any andappa theirrent ofmutagenicity.effect the extracts on the of mutagenicity.Further,R. tinctorum 4,5-dihydroxylation was The not results exclusively suggested of 2- duemethyl that to lucidin anthraquinonesboth the but number also a did result of not OH and have-groups contribution any andappa theirrent of positionseffect on are the relevant mutagenicity. for the Tmutagenicityhe results suggested [32]. that both the number of OH-groups and their othereffectpositions mutagenic on are the relevant mutagenicity. compounds. for the Tmutagenicityhe results suggested [32]. that both the number of OH-groups and their positionsMany are st relevantudies have for the found mutagenicity antimicrobial [32]. properties in biocolourants [36–38]. When testing positionsTheMany chemical are st relevantudies structures have for the found ofmutagenicity secondary antimicrobial [32]. metabolites properties and in their biocolourants functional [36groups,–38]. forWhen example, testing toxicityMany in vitro studies, it is important have found to analyse antimicrobial the conditions properties and inobserved biocolourants changes [36in detail–38]. Whento distinguish testing hydroxyl,toxicityMany in alkyl, vitro studies alkoxy, it is important have or chlorine, found to attachedanalyse antimicrobial the at specific conditions pro positions,perties and inobserved can biocolourants confer changes different [36 in biologicaldetail–38]. Whento distinguish activities testing betweentoxicity in toxic vitro and, it is non important-toxic antimicrobial to analyse the activity. conditions Antimicrobial and observed ac changestivity can in bedetail considered to distinguish toxic andtoxicitybetween genotoxicity in toxic vitro and, profiles it is nonimportant [-33toxic–35 ].antimicrobial to Lucidin analyse (2 the) and activity. conditions rubiadin Antimicrobial and (3), whichobserved are ac changes 1,3-dihydroxyanthraquinonestivity can in detailbe considered to distinguish toxic (i.e.between, cytotoxic) toxic andif it inducesnon-toxic changes antimicrobial at the level activity. of cell Antimicrobial structure [39]. ac tivity can be considered toxic possessingbetween(i.e., cytotoxic) toxic a methyl andif it inducesnon or hydroxymethyl-toxic changes antimicrobial at the group level activity. at of carboncell Antimicrobial structure 2, showed [39]. ac strongtivity mutagenicity,can be considered whereas toxic (i.e., cytotoxic) if it induces changes at the level of cell structure [39]. alizarin(i.e., cytotoxic) (1), 1,2-dihydroxyanthraquinone if it induces changes at the without level of othercell structure substituents, [39]. had no mutagenicity. Further, 1.3. Flavonoids and Carotenoids in Tanacetum Vulgare 4,5-dihydroxylation1.3. Flavonoids and C ofarotenoids 2-methyl in anthraquinones Tanacetum Vulgare did not have any apparent effect on the mutagenicity. 1.3. Flavonoids and Carotenoids in Tanacetum Vulgare The1.3. results FlavonoidsT. vulgare suggested L.,and commonly Carotenoids that both called in the Tanacetum tansy number (Table V ofulgare OH-groups2), is a flowering and theirherbaceous positions plant are widely relevant distribut for theed T. vulgare L., commonly called tansy (Table 2), is a flowering herbaceous plant widely distributed mutagenicityin theT. Northern vulgare [32 L.,]. H commonlyemisphere calledand known tansy (Tablefor its curing2), is a floweringproperties herbaceous for colds and plant fevers widely [39]. distribut ed in theT. Northern vulgare L., H commonlyemisphere calledand known tansy (Tablefor its curing2), is a floweringproperties herbaceous for colds and plant fevers widely [39]. distribut ed in theManyTansy Northern studies has H been haveemisphere foundreported and antimicrobial forknown its varietyfor properties its curing of chemotypes in properties biocolourants forbased colds [36 on–38 and ].the When fevers most testing [39]. predominant toxicity in theTansy Northern has H beenemisphere reported and forknown its varietyfor its curing of chemotypes properties forbased colds on and the fevers most [39]. predominant inconstituent vitroTansy, it is importantin has the been essential to reported analyse oil, which the for conditions its may variety change and of observed according chemotypes changes to itsbased growth in detail on stagthe to distinguishe most[16]. In predominant addition between to constituentTansy in has the been essential reported oil, which for its may variety change of according chemotypes to itsbased growth on stagthee most[16]. In predominant addition to toxictypicalconstituent and monoterpenes, non-toxic in the antimicrobialessential less oil volatile, which activity. sesquiterpenes, may Antimicrobial change according sesquiterpene activity to can its be growth lactones, considered stag none toxic [16].-volatile (i.e., In addition cytotoxic) terpenes, to constituenttypical monoterpenes, in the essential less oil volatile, which sesquiterpenes, may change according sesquiterpene to its growth lactones, stag none [16].-volatile In addition terpenes, to ifflavonoidstypical it induces monoterpenes,, changes and phenolic at the less acids level volatile ofare cell also sesquiterpenes, structure present [[15,4039]. – sesquiterpene45]. Flavonoids lactones, and carotenoids non-volatile are the terpenes, major typicalflavonoids monoterpenes,, and phenolic less acids volatile are also sesquiterpenes, present [15,40 – sesquiterpene45]. Flavonoids lactones, and carotenoids non-volatile are the terpenes, major colouringflavonoids constituents, and phenolic in T.acids vulg areare also. For presentdyeing [15,40purposes,–45]. the Flavonoids whole plant and can carotenoids be used, areresulting the major in a flavonoidscolouring constituents, and phenolic in acidsT. vulg areare also. For present dyeing [15,40purposes,–45]. the Flavonoids whole plant and cancarotenoids be used, areresulting the major in a 1.3.greenishcolouring Flavonoids-yellow constituents and colour, Carotenoids in while T. vulg in flower Tanacetumare. For heads dyeing Vulgare produce purposes, bright the yellow whole [13,46]. plant can be used, resulting in a colouringgreenish-yellow constituents colour, in while T. vulg flowerare. For heads dyeing produce purposes, bright the yellow whole [13,46]. plant can be used, resulting in a greenish-yellow colour, while flower heads produce bright yellow [13,46]. greenishT. vulgare-yellowL., commonlycolour, while called flower tansy heads (Table produce 2), is a bright flowering yellow herbaceous [13,46]. plant widely distributed in the Northern Hemisphere and known for its curing properties for colds and fevers [39]. Tansy has been reported for its variety of chemotypes based on the most predominant constituent in the essential oil, which may change according to its growth stage [16]. In addition to typical monoterpenes, less volatile sesquiterpenes, sesquiterpene lactones, non-volatile terpenes, flavonoids, and phenolic acids are also present [15,40–45]. Flavonoids and carotenoids are the major colouring Antibiotics 2020, 9, 266 4 of 24 constituents in T. vulgare. For dyeing purposes, the whole plant can be used, resulting in a greenish-yellow colour, while flower heads produce bright yellow [13,46]. In the flower heads, luteolin (5), quercetin (6), violaxanthin (7), β-carotene (8) (Table 2) and their derivatives are dominating colourant components [12,13], whereas caffeic, ferulic and neochlorogenic acid and their derivatives occur in all plant parts [12,37,47–49]. The amounts of potentially harmful alkaloids and terpenes (e.g., sesquiterpene lactones) depend largely on the chemotype. Exo-methylene group in the lactone is regarded to be essential for the cytotoxicity, but also the presence of the O=C-C=CH2 system has been shown to be responsible [40,42,50]. Devrnja et al. [37] identified neochlorogenic acid, 3,5-O-dicaffeoylquinic acid, dicaffeoylquinic acid and quercetin-3-O-glucoside as the main secondary metabolites from the MeOH extracts of tansy flower heads. The extracts exhibited a strong antiproliferative effect on human cervical adenocarcinoma (HeLa) cells causing cell shrinkage and detachment but did not lead to a significant cell detachment of human non-tumour cells (MRC-5) under the same conditions. The tansy was categorised as trans-chrysanthenyl acetate chemotype with low proportions (< 10%) of generally harmful monoterpenes trans- and cis-thujone [37]. The water extract of tansy leaves possessed a strong diuretic action but no renal toxicity or other detrimental effects in tests on rats [51–54].

1.4. Tannins in Salix spp. Salix species (Table 2) are deciduous shrubs, trees or subshrubs occurring mainly in the temperate and arctic areas of the Northern Hemisphere. Leaves and bark are rich in secondary metabolites such as condensed tannins, constituting approx. 2–3% of dry weight (dw) [10,23,55,56] but even 10% (dw) in twigs (Salix pyrolifolia Ledeb.) [10]. Tannins bind to proteins and carbohydrates and accumulate alkaloids and metals (e.g., Al, Fe, Cu). Traditionally, willows (Salix spp.) have been considered safe, as they have been used as a medicine to reduce fever, inflammation and pain. Adverse effects of bark extracts appear to be minimal. The primary risk may relate to allergic reactions in salicylate-sensitive individuals [57,58]. The composition of secondary metabolites varies highly among Salix species, between genotypes and hybrids [10,14,56,59,60]. The condensed tannins in willow bark are procyanidins, which consist of chains with varying numbers of flavan-3-ol monomers linked to each other. The bark contains higher amounts of phenolics and higher diversity of glycosides than the leaves [14,59,61,62]. In dyeing, tannins produce browns and can be used as biomordants [13]. Salix bark contains simple phenolic glucosides like picein (9), triandrin (10), catechin (11) and salireposide (12) as main colourants reported by Dou et al. [63]. Salix spp. also contain other phenolics, such as phenolic acids, flavones and chalcones and yellow providing flavonols, especially quercetin (6) derivatives.

1.5. Anthraquinones in Cortinarius Semisanguineus Cortinarius spp. are ectomycorrhizal fungi growing mainly in coniferous forests. Of the secondary metabolites, fungi Cortinarius, subgroup Dermocybe, are rich in monomeric anthraquinones also containing dimeric pre-anthraquinones of flavomannin type [13,64–68]. The fruiting bodies of C. semisanguineus (Table 2) are relatively small (< 10 cm, cap 3–9 cm), ∅ but are produced in large numbers by the mycelia. Among the more abundant compounds are emodin (13), emodin-1-glucoside, dermocybin (14), dermocybin-1-glucoside and dermorubin (15). Also, physcion (16), dermolutein, 5-chlorodermolutein, 5-chlorodermorubin, dermoglaucin, endocrocin, and erythroglaucin have been found [68]. Anthraquinones from Cortinarius species have inhibited the growth of Staphylococcus aureus and P. auerugosa [69], and many anthraquinones have bioactive properties [70]. Macromycetes have mostly been studied for their oral toxicity in humans [71]. Highly nephrotoxic bipyridine orellanine, which is a good aluminium ion chelator, has been found in Cortinarius orellanus from Europe and Cortinarius armillatus native to North America [67,72,73]. Because Cortinarius fungi Antibiotics 2020, 9, 266 5 of 24 are endemic, it should not be assumed that toxins in European species are similarly present or absent in species of other regions, even if they appear morphologically similar [72,73]. Orellanine has not been found in the European species of C. armillatus, nor in Cortinarius sanguineus (syn. Dermocybe sanguinea)[74] or C. semisanguineus [75], which all contain anthraquinones and have been used for dyeing [13]. Toxicity tests for anthraquinones [33,37] show that the structural requirements for their mutagenicity in Salmonella typhimurium are not so clear. It has been suggested that the presence of alkyl or alkoxy groups in the anthraquinone structure, e.g., of chrysophanol, emodin (13), icelandicin, lucidin-2-ethyl ether, dermoglaucin, catenarin, and cynodontin, results in changes in cell tests [33]. Emodin (13) and dermoglaucin were found to be non-toxic for Bacillus subtilis in rec-assay and E. coli in pol A assay. However, dermocybin (14) showed very slight mutagenicity for B. subtilis, but no toxicity for E. coli [33]. There are an increasing number of reports regarding adverse effects of emodin (13). It has been reported to induce genotoxicity, reproductive toxicity, and also hepatotoxicity and nephrotoxicity, presumably connected to high doses and long-term use [76,77]. Von Wright et al. [78] studied the genotoxicity of the crude fractions from the extracts of C. sanguineus using an in vitro bacterial repair assay (E. coli; strains WP2 and CM871), a sister-chromatid exchange (SCE) test and a mouse hepatoma cell assay (Hepa-1c1c7). Emodin (13) was the most abundant (44%) compound in Fraction I. Analysis showed that standard emodin was not toxic to either of the E. coli strains. However, it induced more SCEs than Fractions I and IV. Fraction I was clearly positive in both SCE- and Hepa-1 tests and in E. coli assays, indicating that other emodin-related but yet unidentified compounds were responsible for the observed cytotoxic and clastogenic effects. It was concluded that the different test results had somewhat contrary results and discrepancies existed between SCEs and E. coli assay tests results.

1.6. Terphenyl Quinones in Tapinella Atrotomentosa T. atrotomentosa (syn. Paxillus atrotomentosus) (Table2) is a sturdy dark brown-capped wood rotting fungus, with a dark brown, velvety stalk, and rim of the unsymmetrical cap. It is common on moss-covered pine stumps in boreal forests, pine moors and forest edges in Europe, North America and Asia [13,38]. The easily harvestable pale flesh fruiting body can grow to a relatively large size (cap 20 cm) and is rich in colourants. T. atrotomentosa has not proved to be toxic but no dietary use ∅ ≥ has been recommended [36,79]. The colourants isolated from its fresh fruiting bodies typically have a terphenyl quinone structure [80]. The p-terphenyl core (Table2, 17) is essentially restricted to fungi and lichens and the structure was found to possess notable antiproliferative, antibacterial, antioxidant, and anti-inflammatory activities [38]. Atromentin (17) isolated from T. atrotomentosa is responsible for the brown colour. It occurs as colourless precursors, leucomentins 2 and 3 (18, 19) in the flesh [38,80], and its content may be ca. 2% of the fungal mass (dw), those of leucomentins about 5% [80]. Orange-yellow flavomentins and violet spiromentins (20)[81,82], osmundalactones and ergostane-type ecdysteroids (e.g., paxillosterone and atrotosterones A–C) have also been found [38,82]. T. atrotomentosa has a broad-spectrum of antimicrobial activity against Gram+ and Gram- pathogens, and a significant inhibitory activity against resistant bacterial strains, e.g., MRSA [38]. A recent investigation showed that violet colour providing spiromentins B and C (spiromentin core (20), Table2) have strong antibacterial activity against multiresistant Acinetobacter baumannii and E. coli [38]. AntibioticsAntibioticsAntibioticsAntibiotics 2020 2020 2020, 9 ,,2020 9266,, 9266, ,266 9 , 266 7 of7 of 725 of 25 7 25 of 25 Antibiotics 2020, 9, 266 6 of 24

AntibioticsAntibioticsAntibiotics 2020Antibiotics 2020, 9, 2662020, 9, 2020266, 9, 266, 9, 266 7 of 725 of 725 of 7 25 of 25 TableTableTable Table2. 2.S 2.ources S ourcesS 2.ources Sources used used used in used in this in this this inresearch, research,this research, research, their their their main their main main colouring main colouring colouring colouring compounds compounds compounds compounds as as aglycones as aglycones aglyconesas aglycones and and and dyeing dyeingand dyeing dyeing result result results result ons ons woolon swool onwool as wool as CIELab as CIELab CIELabas CIELab coordinates coordinates coordinates coordinates L, L, a*, L, a*, b* a*,L, b*. a*,(Photos:b*. (Photos:. b*(Photos:. (Photos: Riikka Riikka Riikka Riikka Räisän Räisän Räisän Räisänen,en, en, en, exceptexceptexceptTableexcept Salix Salix Salix 2.phylicifolia Salix phylicifoliaSources phylicifolia phylicifolia usedby by Joukoby Jouko in byJouko this JoukoRikkinen Rikkinen research, Rikkinen Rikkinen [74] [74] their [74]). ). [74] ). main ). colouring compounds as aglycones and dyeing results on wool as CIELab coordinates L, a*, b*. (Photos: Riikka Räisänen, except Salix phylicifolia by Jouko Rikkinen [74]). TableTable 2.Table Sources 2.Table Sources 2. Sources 2.used Sources used in used this in used this research,in this inresearch, this research, their research, their main their main theircolouring main colouring main colouring compoundscolouring compounds compounds compounds as aglycones as aglycones as aglyconesas aglyconesand and dyeing and dyeing and dyeingresults dyeing results onresults woolresults on woolon as woolon CIELab as wool CIELab as CIELab ascoordinates CIELab coordinates coordinates coordinates L, a*, L, b*. a*, L, (Photos: b*.a*, L, (Photos:a*,b*. b*.(Photos: Riikka (Photos: Riikka Räisänen,Riikka RiikkaRäisänen, Räisänen, Räisänen, CortinariusCortinariusCortinariusCortinarius semisanguineus semisanguineus semisanguineus semisanguineus (Fr.) (Fr.) (Fr.) (Fr.) TapinellaTapinellaTapinellaTapinella atrotomentosa atrotomentosa atrotomentosa atrotomentosa ScientificScientificScientificScientific Nexcept ameN Nameexceptame NSalixexcept ame except Salix phylicifolia Salix phylicifolia Salix phylicifolia phylicifoliabyTanacetum Jouko TanacetumbyTanacetum JoukobyTanacetum Rikkinen Joukoby RikkinenJouko vulgare Rikkinenvulgare vulgare[74] Rikkinen vulgare[74]). L. [74]L.). L. [74]). L.). SalixSalixSalix phylicifoliaSalix phylicifolia phylicifolia phylicifolia L. L. L. L. Cortinarius semisanguineusGillet Tapinella(Batsch.) atrotomentosa Šutara Scientific Name Tanacetum vulgare L. Salix phylicifolia L. GilletGillet Gillet (Batsch.)(Batsch.)(Batsch.) Šutara Šutara Šutara CortinariusCortinariusCortinariusCortinarius semisanguineus semisanguineus(Fr.) semisanguineus semisanguineus Gillet (Fr.) (Fr.) (Fr.) (Fr.)Tapinella TapinellaTapinella atrotomentosaTapinella atrotomentosa(Batsch.) atrotomentosa atrotomentosa Šutara CommonCommonCommonCommon names namesScientific names Scientificnames Scientific ScientificN ame Name Name NTancy,ame Tancy,Tancy, Tancy, goldenTanacetum golden goldenTanacetum golden Tanacetumbuttons buttonsTanacetum buttonsvulgare buttonsvulgare vulgare L. vulgare L. L. L. Willow,Willow,Willow,SalixWillow, Salixtea phylicifoliateaSalix -tealeaved phylicifolia-Salixleaved tea-leaved phylicifolia- leavedphylicifolia willow L. willow willow L. willow L. L. SurpriseSurpriseSurpriseSurprise webcap webcap webcap webcap VelvetVelvetVelvetVelvet roll roll roll-rim- rimroll-rim -rim GilletGillet Gillet Gillet (Batsch.)(Batsch.)(Batsch.) Šutara(Batsch.) Šutara Šutara Šutara Common names Tancy, golden buttons Willow, tea-leaved willow Surprise webcap Velvet roll-rim CommonCommonCommon namesCommon names names names

AppearanceAppearanceAppearanceAppearance AppearanceAppearanceAppearanceAppearanceAppearance

CIELabCIELabCIELabCIELab coordinates coordinates coordinatesCIELab coordinatesCIELabCIELab coordinates CIELab coordinates coordinates coordinates L L70.74; L 70.74; 70.74;L a*70.74; a* 2.54; a* 2.54; 2.54;a* b* 2.54;b* 29.60 b* 29.60 29.60b* 29.60 L L56.39; L56.39; 56.39;L a*56.39; a* 13.43; a* 13.43; 13.43;a* b*13.43; b* 24.98 b* 24.98 24.98b* 24.98 L L49.90; L49.90; 49.90;L a*49.90; a* 21.22; a* 21.22; 21.22;a* b*21.22; b* 20.14 b* 20.14 20.14b* 20.14 L L38.64; L38.64; 38.64;L a*38.64; a* 4.07; a* 4.07; 4.07;a* b* 4.07;b* 18.27 b* 18.27 18.27b* 18.27 CIELab coordinates L 70.74; a* 2.54; b* 29.60 L 56.39; a* 13.43; b* 24.98 L 49.90; a* 21.22; b* 20.14 L 38.64; a* 4.07; b* 18.27 DirectDirectDirect Directdye dye dye, NM, Direct dyeNM, NM Direct, NMdyeDirect Directdye, NM dye, NM dye, NM , NM Direct dye, NM L L68.66; L68.66; 68.66;L La*68.66; 68.66;a* 3.30; La* 3.30;68.66; 3.30;L a*a* 68.66;b* L 3.30;b* a*68.66;49.18 b* 3.30;49.18 a* b*49.18b* 3.30; 49.18a* b*49.18 3.30; 49.18 b* 49.18 b* 49.18 L L57.09;L L 57.09; 57.09;LL 57.09; a*57.09;L a* a*57.09; 12.58; L12.58;a* a*12.58;57.09; 12.58;a*12.58; a* b*b*12.58; 12.58;a* b*2 2 b*6.5412.58;b*6.54 2 2 6.54 b*26.54b* 6.54 2 b*26.54 6.54 2 6.54 L 43.65;L 43.65;L43.65;L L43.65;a* 43.65; 43.65; L34.49;L a*43.65; a*43.65; 34.49; a* 34.49; b*a* 34.49;34.49;a* 30.05 34.49; b*a*34.49; 30.05 b*34.49;b* b* 30.05b*b* 30.05 30.05 30.05b* 30.05 L 33.68; L 33.68;L a* 33.68; L3.42; a*L33.68; L3.42;33.68; a* b*33.68; L 3.42;16.50a* b*a*33.68; 3.42; a*16.50 3.42;b* a* 3.42; 16.50 b* 3.42;a* b*16.50 3.42; b* 16.50 b* 16.50 16.50b* 16.50 KAl(SOKAl(SOKAl(SO4)2 KAl(SO4)2 4)2 4)2 L 68.66; a* 3.30; b* 49.18 L 57.09; a* 12.58; b* 26.54 L 43.65; a* 34.49; b* 30.05 L 33.68; a* 3.42; b* 16.50 KAl(SOKAl(SOKAl(SOKAl(SO4)24 )24 )2 4)2 KAl(SO4)2 L 35.07;L 35.07;L a* 35.07; L0.45; a*35.07; 0.45; a*b* 0.45;13.64a* b* 0.45; 13.64 b* 13.64b* 13.64 L 43.81;L 43.81;L a* 43.81; L5.06; a*43.81; 5.06; a*b* 5.06;15.58a* b* 5.06; 15.58 b* 15.58b* 15.58 L 35.62;L 35.62;L a* 35.62; L8.84; a*35.62; 8.84; a*b* 8.84;10.63a* b* 8.84; 10.63 b* 10.63b* 10.63 L 23.63;L 23.63;L a* 23.63; L- 0.24; a*23.63; - 0.24;a* b* -a* 0.24;10.66 b* -0.24; 10.66 b* 10.66b* 10.66 L L35.07; L35.07; 35.07; a* a* 0.45; a* 0.45; 0.45; b* b* 13.64 b* 13.64 13.64 L L43.81; L43.81; 43.81; a* a* 5.06; a* 5.06; 5.06; b* b* 15.58 b* 15.58 15.58 L L35.62; L35.62; 35.62; a* a* 8.84; a* 8.84; 8.84; b* b* 10.63 b* 10.63 10.63 L L23.63; L23.63; 23.63; a* a* - 0.24;a* -0.24; -0.24; b* b* 10.66 b* 10.66 10.66 FeSOFeSO4 FeSO4 FeSO4 4 L 35.07; a* 0.45; b* 13.64 L 43.81;L 43.81; a* 5.06;a* 5.06; b* 15.58b* 15.58 L 35.62;L 35.62; a* 8.84; a* 8.84; b* 10.63 b* 10.63 L 23.63;L a*23.63; -0.24; a* b*-0.24; 10.66 b* 10.66 FeSO4 4 4 4 FeSOFeSOMain FeSO Main colourantsMain4 colourantsMain colourants colourants [ref] [ref] [ref] [ref] [12,13] [12,13] [12,13] [12,13] [6 3] [6 3][6 3][6 3] [64,66,[64,68[64,66,] 68[64, 66,] 6866,] 68 ] [79,80][79,80] [79,80] [79,80] MainMainMain colourantsMain colourants colourants colourants [ref] [ref] [ref] [ref] [12,13][12,13][12,13]Lu[12,13] teolinLu teolinLu (5 teolinLu) (teolin5) (5) (5) [6[63][63 ]3 [6] 3] [64,[64,[64,66,66,[64,6866,68] 6866,] ]68 ] AtromentinAtromentinAtromentinAtromentin (17) ( [79,80]17[79,80] )( 17[79,80] )( 17[79,80] ) Main colourants [ref] [12,13][63][EmodinEmodinEmodin (6413Emodin) ,( 6613, )68( 13 ][)( 13) 79,80] LuLuteolinLuteolinteolinLuLuteolin (teolin5 ()5 ()5 ) (5) AtromentinAtromentinAtromentinAtromentin (17 (17 )( 17) ()17 ) PiceinPicein (Picein9) Picein(9) (9) (9) EmodinEmodinEmodinEmodin (13 (1313 )( 13)) ()13 ) PiceinPiceinPicein Picein(9 ()9 ()9 ) (9)

−1 −1 −1 C14HC1814OHC718 14MOHC 714=18 M HO298.29 187= MO 298.297 =M g·mol298.29 = 298.29g·mol g·mol−1 g·mol 15 10 515 1510 105 5 −1 −1 −1 −1 C HC15OHC10 MOHC 5= M HO270.24 = MO 270.24 =M g·mol270.24 = 270.24g·mol g·mol g·mol −1 −1 −1 −1 −1 −1 −1 C18H1218OC612 18MHC 618=12 HO324.29126 MO6 =M g·mol324.29 = 324.29 g·mol−1 g·mol C15HC1015OHC610 15MOHC 615=10 M HO286.24 106= MO 286.246 =M g·mol286.24 = 286.24g·mol g·mol g·mol −1 −1 −1 C H O M = 324.29 g·mol 14 14C1814H1814718O7 187 M7 = 298.29 g·mol 1 −1 CCCH14HHOC18O HOM M7 =OM 298.29= =298.29M298.29 = 298.29g·mol g·mol g mol g·mol − · −1 −1 −1 C15CH15CH1015OH1015O510 MO5 M105 = M 270.24=5 =270.24 270.24 g·mol g·mol g·mol 1 −1 −1 −1 −1 C15H10CO5HMO= 270.24M = 270.24 g mol g·mol− −11−1 −1 −1 C15CH15CH1015COCH1015O610H HMO6 M106 = OOM 286.24=6 =286.24M 286.24 = 286.24286.24g·mol g·mol g·mol g·mol g mol −1 1 · CC1818CHH18C12H1218OOCH12618O612 MHO6 M126 = OM 324.29=324.296 =324.29M 324.29 = 324.29g·mol g g·molmol g·mol −g·mol 15 10 6 · − · Antibiotics 2020, 9, 266 7 of 24

Table 2. Cont. AntibioticsAntibioticsAntibiotics 2020 2020 2020, 9,, 9266, 9266, 266 8 of8 of 825 of25 25 AntibioticsAntibioticsAntibiotics 2020 2020, , 2020 9,,, , 9 266266,, 266,266 9 , 266 Cortinarius semisanguineus Tapinella atrotomentosa 8 of8 of 258 25 of 25 AntibioticsScientific 2020,, 9,, 266266 Name Tanacetum vulgare L. Salix phylicifolia L. 8 of 25 (Fr.) Gillet (Batsch.) Šutara Antibiotics 2020, 9, 266 LeucomentinLeucomentinLeucomentin 28 2(of18 ( 22518 )( 18) ) LeucomentinLeucomentinLeucomentin 2 2(18 (18 2) )( 18 ) Antibiotics 2020, 9, 266 LeucomentinLeucomentinLeucomentin 2 28 (of2 (1818 (2518)) ) QuercetinQuercetinQuercetin (6 ()6 )( 6) DermocybinDermocybinDermocybin (14 (14 )( 14) ) QuercetinQuercetinQuercetinQuercetin (6 () 6( 6) )( 6) DermocybinDermocybin (14 (14) ) Quercetin (6) DermocybinDermocybinDermocybin ( (1414 (14)) ()14 ) Leucomentin 2 (18) TriandrinTriandrinTriandrin (10 (10 )( 10) ) Leucomentin 2 (18) Quercetin (6) TriandrinTriandrin (10 (10) ) TriandrinTriandrinTriandrinTriandrin ( 10( ( 1010) )() 10 ) Dermocybin (14) Quercetin (6) Dermocybin (14) Triandrin (10) Triandrin (10) −1 1515 201520 7207 7 −1 −1 CCHCHOHO MO M = M =312.31 312.31= 312.31·g·g mo ·gmo lmol −1l −1 CC15H1515H20O20O7 M7 M = =312.31 312.31·g·g mo mol−1−1l −1 1 −1 CC15H1515HC2015O20HO7 M207 MOM =7 = =312.31M 312.31312.31 = 312.31·g·g·g mo mo mo·gll mo−1l l CC15HH20O7 MM == 312.31312.31·gg mo moll − −1 −1 −1 −1−1 −1 −1 CC16H16CH1216O12HO712 M7O M 7= M =316.27 316.27= 316.27 g·mol g·mol g·mol CC15H15CH1015O10HO710 M7O M 7= M =302.2 302.2= 302.24 4g·mol g·mol4 g·mol C15H20O7 M = 312.31·g mo· l −1−1−1 −1−1−1 CCC1616H16H12O12O7 M7 M = 316.27=316.27 316.27 g·molg·mol g·mol−1−1 1 −1 CC15H15CH1510HO10O107 OM7 M7 =M =302.2 =302.2 302.24 4g·mol4 g·mol g·mol−1 −1 −11 15 20 7 −1 CCC1616HHC1216OHO7 M12 OM =7 316.27=316.27M 316.27 = 316.27 g·molg·mol g·mol g·mol CC15HCHC151015HOHO107 OM10 OM7 =M7 =302.2M =302.2 302.2= 302.24 4g·mol4 g·mol g·mol4 g·mol C H O M = 312.31·g mol C16H12O7 M = 316.27−1 g mol− C15H10O7 M = 302.24 g mol−1− 16 12 7 −1−1 −1 C15H10O7 M = 302.24 g·mol C H O M = 316.27 g·mol · CC30H30CH2830O28HO1028 10OM M10 = M =54 54=8 .54548.548 g·mol.54 g·mol g·mol · −1 −1 −1−1 −1 C16H12O7 M = 316.27 g·mol CC30C30HH3028HO2830O2810O 10M 2810M =M 5410= =548 54.548.548 g·mol.54 g·mol g·mol−1 1−1 −1 C15H10O7 M = 302.24 g·mol C30CC30HC30H28H28OHCO2810O10HO 10M M O =M 54=548.54 =54M8 54.54−18 =.548 54g·mol.54 g 8g·molmol .54g·mol −g·mol (+)(+) Cat(+) Cat eCatcehcinehcin h(11in (11 )( and11) and) and epicat epicat epicatechechinechin in C30H28O10 M = 548.54 g·mol · (+)(+)(+) Cat CatCatecehcinhin (11 ((11) and)) andand epicat epicatepicatechechechininin LeucomentinLeucomentinLeucomentin−1 3 3(19 (319 )( 19) ) (+)(+)(+)(+) Cat (+) CatCatCate (Catc+ehc)hinh Catechininein c( h(11 (11(in11)) and)( 11andand) (epicat 11epicatand epicatepicat) and epicatechechechechinin inechin in C30H28O10 M Leucomentin=Leucomentin 54Leucomentin8.54 g·mol 3 (319 3(19 )( 19) ) differentiatedifferentiatedifferentiate in in stereochemistry in stereochemistry stereochemistry DermorubinDermorubinDermorubin (15 (15 )( 15) ) LeucomentinLeucomentinLeucomentinLeucomentinLeucomentin 3Leucomentin (19) 3 (3 (1919 3(19) )( 193) ()19 ) differentiatedifferentiate(+)differentiate epicatechinCatdifferentiateechin (in11 inin )stereochemistry di and stereochemistrystereochemistry infferentiate stereochemistryepicatechin in DermorubinDermorubin (( 1515 (15)) ) differentiatedifferentiateofof C2of C2 in inOHC2 OH stereochemistrystereochemistry OH-gr-gr-gr DermorubinDermorubinDermorubinDermorubin (15) ((1515 (15)) ()15 ) Leucomentin 3 (19) ViolaxanthinViolaxanthinViolaxanthinViolaxanthin (7 ()7 )( ( 77)) differentiatestereochemistryofof C2 C2in OHstereochemistry OHOH- ofgr--grgr C2 OH-gr ViolaxanthinViolaxanthinViolaxanthinViolaxanthinViolaxanthin (7 () 7( 7) )( 7)) ofofof C2 C2 C2of OHOH OHC2OH-- grgrOH--grgr-gr Dermorubin (15) Violaxanthin (7) of C2 OH-gr

−1−1 −1 CC40H40CH5640O5640HO456 M456O40 M 4= 4M 56 =600.88 600.88=4 600.88 g·mol g·mol g·mol −1 −1 40C40H56C56O4H4 MO =M 600.88 = 600.88 g·mol g·mol−1−1−1 −1 1 CC40H40CH5640OH56HO4 MO564 OM =M4 =600.88M =600.88 600.88= 600.88 g·mol g·mol g·mol g−1mol −1 − CC40HHC5640OCHO44056 MH OM56 =O4 =600.88M4 M600.88 = = 600.88 600.88 g·mol g·mol g·molg·mol −1 −1 −1 −1 · CC17C17HH171217COH12178O12H12 MO812 M=8O M344.28 8= M =344.28 344.28= g·mol344.28 g·mol g·mol g·mol−1 C17H12O8 M = 344.28 g·mol−1−1 1−1 CCC17H17HH12O12O8 M8 M = =344.28 344.28 −1g·molg·mol g·mol−1 −1 −1 −1 −1 C17CHC1712C17HOH128HC 12MO17O H=8O8 344.28M12 OM =8 =344.28M344.28 g·mol344.28 = 344.28 g·mol g g·molmol g·mol− C 36H 34OC1236 HM36 34 36=O34 658.661234 12M12 =g·mol 658.66 g·mol−1 −1 −1 15 14 6 −1−1 −1 −1−1 CC36HCH34HOO12 OM M = M =658.66 658.66= 658.66 g·mol g·mol g·mol−1 −11 −1 CC15H15CCH141515OH14O61414 OM6O M6 6 M= MM =290.27 = = 290.27=290.27 290.27 290.27 g·mol g·molg·mol g·mol g·mol −1 · C C36HC36H3634OH34O3412O 12M12 =M 658.66658.66 = 658.66−1 g·mol g mol g·mol−1 −1 −1 C15H14O6 M = 290.27 g·mol−1 −1−1 1 C36H34O3612C MC3634H 36= HC34658.661236O34HO12 34 12M Og·mol M 12= =658.66M 658.66 = 658.66 g·mol g·mol −g·mol CCC15C15H1515HH14141415O14OO6 6M146 M MM ==6 =290.27= 290.27290.27 g·mol g·mol g·mol g mol −1 − −1 · C15HC14OH6 MO = 290.27M = 290.27 g·mol· g·mol SpiromentinSpiromentinSpiromentinSpiromentinSpiromentin (20) ( (2020 (20) ) ( 20) ) Salireposide (12) Physcion (16) SpiromentinSpiromentinSpiromentinSpiromentin (20Spiromentin) (20 (20 )( 20) )( 20 ) SalireposideSalireposideSalireposideSalireposide (12 ( 12( )12 ( 12) ) ) PhyscionPhyscionPhyscion ( 16(( 1616 )( )16) ) Spiromentin (20) β-Carotene (8) SalireposideSalireposideSalireposide ((12 12 ((12)12 ) )) PhyscionPhyscionPhyscion (16) ((1616 (16)) ) β-βCarotene-βCaroteneβ-Carotene-βCaroteneβ-Carotene-Carotene (8 ()8 )( (88) ) ( 88)) SalireposideSalireposideSalireposide (12 (12) ()12 ) PhyscionPhyscionPhyscion (16 (16) ()16 ) β-βCarotene-Caroteneβ-Carotene (8 ()8 ) (8)

C40H56 M = 536.89 g·mol−1 −1 −1 C40H56 M = 536.89 g·mol−1−1 1 C20H22O9 M = 406.37 g·mol C16H12O5 M = 284.27 g·mol 40 56C40H56 M = 536.89 g·mol−1−1 −1−1 CCH40CHC40C 56H40M40 H56MH =56 M 56 =536.89M 536.89=M = 536.89 =536.89 536.89g·mol g·mol g·mol g·mol g mol −1 − 20 22 9 −1 16 12 5 −1 4040 5656 −1 −1 −1 C20H 22O 9 M = 406.37 g·mol−1 −1 C H 16O 12M =5 284.27 g·mol −1 −1 22 14 7 −1 CC40H40H5640 56M M 56= =536.89 536.89 g·mol g·mol· 20 C2220H922O9 M = 406.37 g·mol −1 −1−1 1 1C6 16H12 12O5 5 M = 284.27 g·mol −1−1 1−1C H O M = 390.35 g·mol C HC MH = 536.89M = 536.89 g·mol g·mol CCCH20C20H20HOH2222O22 MO9O M9 9 = MM =40 =40=6.37 40406.37406.376.376.37 g·mol g·mol g·mol g·mol g mol −1 − CCCCH1C6HHH16O12HOO12 M5O M 5= M =284.2 284.2=284.27 284.27 77g·mol g·molg·mol7 g g·molmol −1 −1 2020 2222 9 9 −1 −1 −1 16161612121255 5 −1−−1C22−1H14O7 M = 390.35 g·mol CC20H20HC2220O22HO9 M229 MO =9 =40M 406.37 =6.37 40 g·mol6.37 g·mol· g·mol CC16H16HC121O126HO5 M125 MO =5 =284.2M 284.2 = 284.27 7g·mol g·mol· 7 g·mol 22 14 7 −1 C H O M = 406.37 g·mol C H O M = 284.27 g·mol C2222H1414O77 M = 390.35 g·mol−1−1 −11 −1 CCCCHH22HCH22O14HOO14 M7O M 7 = M =390.35390.35 390.35= 390.35 g·mol g g·molmol g·mol −1 22 C221422H14714O7 7 M = 390.35 g·mol−−1 −1 −1 CC22H22HC1422O14HO7 M147 OM =7 =390.35M 390.35 = 390.35 g·mol· g·mol g·mol

Antibiotics 2020, 9, 266 8 of 24

1.7. Objectives Our aim with this paper was to discuss the toxicity of the examined biocolourants, their sources and the dyed textile samples. We evaluated the results of cytotoxicity tests when two plant species, T. vulgare and S. phylicifolia, and two fungal species, C. semisanguineus and T. atrotomentosa, were used as natural dye sources. The extracts were obtained directly from plant and fungal material and the wool fabrics dyed with them. Preliminary toxicity screening was performed using Hepa-1 mouse hepatoma cell tests in vitro and examining the highest tolerated dose (HTD) after exposures of 24 and 72 h. The data obtained were analysed by statistical means. Common natural dye sources T. vulgare, S. phylicifolia, C. semisanguineus, and T. atrotomentosa were chosen due to their high colouring and pharmacological potential, and the information we already had gained of their characteristics and dyeing properties [13,18,46,63,68,83,84]. The characteristic colourants of the studied plants and fungi are described in Table2.

2. Results

2.1. Colourants and Colourfastness The main colourants of the fungal and plant species under study were tentatively identified and the results can be seen in the HPLC-UV/Vis-MS spectra in Figure1 and Table3. Our analysis is in accordance with the previously published works as described in Introduction. The tentative identification of flavonoids (T. vulgare) from the MS data revealed the presence of several flavonol and flavone glycosides such as the derivatives of quercetin, apigenin and luteolin. Furthermore, caffeoylquinic acid isomers and dicaffeoylquinic acids were tentatively identified, which is in accordance with the literature [37].

Table 3. The results of the HPLC-UV/Vis-MS data of Tanacetum vulgare, Cortinarius semisanguineus and Tapinella atrotomentosa. The chemical structures and the calculated MS of the numbered compounds are found in Table2. The MS data for C. semisanguineus refers to our earlier data published in [68] and [85].

MS Data: m/z UV/Vis Ret. Time [M H]/[M + H]+ Molecular Formula Compound λ [nm] max and Main− Fragment Tanacetum vulgare, Figure1, chromatogram (370 nm) and spectra A

13.61 218, 244, 298 sh ‡, 328 353.0864; 191.0535 C16H18O9 Caffeoylquinic acid isomer I 25.42 204, 256, 366 463.0878; 301.0331 C21H20O12 Quercetin (6) derivative 27.10 218, 244, 298 sh, 328 515.1186; 353.0859 C25H24O12 Caffeoylquinic acid isomer II 29.75 200, 266, 336 445.0759; 269.0428 C21H18O11 Apigenin derivative 30.13 218, 244, 300 sh, 328 515.1191; 353.0863 C25H24O12 Caffeoylquinic acid isomer III 31.55 204, 254, 266, 346 639.3185; 285.0378 - Luteolin (5) derivative 40.09 206, 266, 336 269.0427 C15H10O5 Apigenin 41.13 204, 254, 266, 346 285.0388 C15H10O6 Luteolin (5) Cortinarius semisanguineus, Figure1, chromatogram (440 nm) and spectra B

15.91 224, 286, 438 327; 283 C17H12O7 Dermolutein 16.64 250, 285, 434 - C17H11ClO7 5-Cl-Demolutein 18.16 224, 288, 439 684 C21H20O10 Emodin-1-glucoside (13)* 18.86 231, 278, 482 343; 299 C17H12O8 Dermorubin (15) 20.23 229, 285, 452 303; 239 C15H9ClO5 7-Cl-Emodin 23.03 211, 283, 431 283; 240 C16H12O5 Physcion (16) # 26.28 263, 484 770 C22H22O12 Dermocybin-1-glucoside (14) Tapinella atrotomentosa, Figure1, chromatogram (370 nm) and spectra C

07.40 263, 371 325.07; 307.06 C18H12O6 Atromentin (17) # ‡ shoulder, * Emodin-1-glucoside heksaacetate, Dermocybin-1-glucoside heptaacetate. Antibiotics 2020, 9, 266 9 of 25

2. Results

2.1. Colourants and Colourfastness The main colourants of the fungal and plant species under study were tentatively identified and the results can be seen in the HPLC-UV/Vis-MS spectra in Figure 1 and Table 3. Our analysis is in accordance with the previously published works as described in Introduction. The tentative identification of flavonoids (T. vulgare) from the MS data revealed the presence of several flavonol and flavone glycosides such as the derivatives of quercetin, apigenin and luteolin. Furthermore, caffeoylquinic acid isomers and dicaffeoylquinic acids were tentatively identified, which is in accordance with the literature [37]. In C. semisanguineus dermolutein, physcion, dermocybin-1-glucoside, and dermorubin were identified together with three other antraquinones and the results follow our previous findings [68]. Antibiotics 2020, 9, 266 9 of 24 In HPLC, T. atrotomentosa showed one major peak which was identified as atromentin with the UV/Vis and mass spectrometric data (Table 3).

FigureFigure 1. 1. HPLCHPLC chromatogram, chromatogram, UV/Vis UV/Vis and and MS MS spectra spectra of of the the main main compounds compounds of of A (A. Tanacetum) Tanacetum vulgarevulgare ((HPLCHPLC detection at 370370 nm),nm), (BB.) CortinariusCortinarius semisanguineussemisanguineus(HPLC (HPLC detection detection at 440at 440 nm) nm) and and (C ) CTapinella. Tapinella atrotomentosa atrotomentosa(HPLC (HPLC detection detection at 370 at nm) 370 extracts. nm) extracts Detailed. Detailed data isdata in Table is in 3Table. 3. In C. semisanguineus dermolutein, physcion, dermocybin-1-glucoside, and dermorubin were identified together with three other antraquinones and the results follow our previous findings [68]. In HPLC, T. atrotomentosa showed one major peak which was identified as atromentin with the UV/Vis and mass spectrometric data (Table3). The colours of the dyed woollen samples are shown in Table2 and the colour fastness results in Table4. The results presented in Table4 show that mordant increases the stability of the dye structure, which can be seen from the higher light fastness values compared to the non-mordanted samples. The use of iron mordant gives slightly higher values, which can be explained by its ability to form stabilising coordination complexes [86]. Washing fastness tests reveal that staining is quite low, but highly basic (pH ~10) washing liquor results in colour changes that are particularly visible in yellow and red hues. In all cases, colour after washing was stronger than before, and its hue was different. This is due to bathochromic change of the dye molecule’s absorption maxima as a result Antibiotics 2020, 9, 266 10 of 24 from the ionisation of the free OH-groups. Metal complexes formed by Al3+ and Fe3+ ions stabilise the structure and decrease colour change as seen from their higher values.

Table 4. Colour fastnesses to light (scale 1 8) and washing (scale 1 5). LF = light fastness (200 h − − without side change), Cc = colour change, s = staining, NM = no mordant.

Dyeing LF Washing Fastness Cc s, WO s, CO Tanacetum NM, direct dye 6 1 5 4/5 Alum mordant 7 3/4 5 4 FeSO4 mordant 7 2/3 5 4 Salix NM, direct dye 5 1 5 5 Alum mordant 6 2/3 5 5 FeSO4 mordant 6 2 5 5 Cortinarius NM, direct dye 3 3/4 4/5 4/5 Alum mordant 4 4 4/5 4/5 FeSO4 mordant 5 4 4/5 4/5 Tapinella NM, direct dye 2 3 5 4/5 Alum mordant 4 3/4 5 4/5 FeSO4 mordant 5 3/4 5 5

2.2. Toxicity

In the HTD tests, the limit of significant toxicity was placed at EC50 [52]. Thus, if over 50% of the exposed cells exhibited changes in cell morphology and viability (i.e., HTD value was 3 or 4), the influence of the sample was interpreted as being toxically significant. HTD value 2 was regarded as the result of a low toxic effect, and subsequently, the value of 1 as a non-toxic, harmless effect. EtOH controls (0.5, 1, 2, 4%) in parallel were first tested on cells. It was found that no cell changes were observed (i.e., HTD values 0) at the EtOH concentrations up to 2% after exposure of 24 h. However, when the EtOH concentration rose to its highest (4%), one of the parallel samples had undergone mild cell changes (< 25%), indicating non-toxic, harmless effect. After longer exposure time (72 h) with similar parallel EtOH concentrations up to 2%, no cell changes were observed. However, with the highest EtOH concentration (4%), the quantity of cell changes rose to the next level (< 25%), similarly than in the shorter time of exposure, but now both of the parallel samples had undergone changes, indicating non-toxic effect. Although the quantity of cell changes (< 25%) with the HTD value 1 had been regarded as harmless by Klemola (2008), excluding any possible toxicity caused by the solvent was wanted, and therefore, EtOH concentrations 0.5, 1 and 2% were selected for further inspections.

2.2.1. The Effect of EtOH Concentration on the Cell Morphology and Viability EtOH extracts were diluted to the concentrations of 0.5, 1 and 2% EtOH with α-MEM. The results showed the stronger the EtOH concentration was, the higher the amount of change in the morphology and the loss of viability% (HTD value) in exposed cells, showing a highly significant correlation (Figure2a). AntibioticsAntibiotics 20202020,, 99,, 266 266 1211 of of 25 24

a

b

FigureFigure 2 2.. TheThe Toxicity Toxicity of of the the Plant Plant and and Fungal Fungal Materials. Materials. a ( a(above).) The eff Theect ofeffect EtOH of concentrationEtOH concentration on the onaverage the average changes changes in the morphologyin the morphology and the and loss the of viability%loss of viability% for the di forfferent the different EtOH dilutions. EtOH dilutions. Samples Samplesof 24 h and of 24 72 h exposureand 72 h exposure times are included.times are included. (rs = 0.863, (rps =< 0.863,0.0005, p n< =0.0005,192). (nb =192).) The e bff ect(below). of time The on effectthe HTD of time values on (changesthe HTD invalues morphology (changes and in mo lossrphology of viability and (%) loss where of viability 1 < 25%, (%) 2 where= –50%, 1 < 3 25%,= –75%, 2 = –450%,> 75% 3 ).= – The75%, ranges 4 > 75%). and averageThe ranges values and are average marked values in the are columns marked and in standardthe columns deviations and standard (SD) at deviationsthe bottom. (SD) The at results the bottom. (n = 195) The include results EtOH(n = 195) concentrations include EtOH of 0.5,concentrations 1 and 2%. of 0.5, 1 and 2%. 2.2.2. The Toxicity of the Plant and Fungal Materials 2.2.2. The Toxicity of the Plant and Fungal Materials None of the dye sources (Tanacetum, Salix, Cortinarius and Tapinella) were significantly toxic when None of the dye sources (Tanacetum, Salix, Cortinarius and Tapinella) were significantly toxic the EtOH concentration was 1% or less. This indicated that 1% EtOH contained extracted compounds when the EtOH concentration was 1% or less. This indicated that 1% EtOH contained extracted in the amounts and composition, which was some kind of threshold value. The changes in the cell compounds in the amounts and composition, which was some kind of threshold value. The changes morphology and the losses of viability, which the diluted 1% EtOH extract induced, were < 50% in the cell morphology and the losses of viability, which the diluted 1% EtOH extract induced, were (i.e., HTD value was 2 at the maximum). < 50% (i.e. HTD value was 2 at the maximum). According to our results, Tanacetum and Tapinella extracts were the most toxic samples. If the According to our results, Tanacetum and Tapinella extracts were the most toxic samples. If the extracts were diluted to 2% EtOH, they caused changes in the cell morphology in the quantity of 75% extracts were diluted to 2% EtOH, they caused changes in the cell morphology in the quantity of 75% (i.e., HTD values were 4), indicating significant toxicity (Figure2a). (i.e. HTD values were 4), indicating significant toxicity (Figure 2a). Time did not have any effect on toxicity i.e., the cell morphological changes were at the same Time did not have any effect on toxicity i.e. the cell morphological changes were at the same quantities (Figure2b) when comparing exposure times of 24 h and 72 h of plant and fungal quantities (Figure 2b) when comparing exposure times of 24 h and 72 h of plant and fungal material. material. However, there were slightly more cell changes with increased exposure time caused However, there were slightly more cell changes with increased exposure time caused by Cortinarius Antibiotics 2020, 9, 266 13 of 25 extracts, but the difference was statistically insignificant compared with the other samples (Mann– AntibioticsWhitney2020 U-test,, 9, 266 p > 0.05, n = 44). 12 of 24

2.2.3. The Toxicity of the Undyed Fabrics by Cortinarius extracts, but the difference was statistically insignificant compared with the other samplesUnmordanted (Mann–Whitney and mordanted U-test, p > undyed0.05, n = fabric44). samples diluted to 0.5% EtOH did not show any changes indicating non-toxicity (i.e. HTD value was 0). However, when these samples were diluted 2.2.3.to 1% Theand Toxicity2% EtOH, of theexposed Undyed cells Fabrics showed changes in morphology and viability in the quantity of < 50%, Unmordantedindicating a low and toxic mordanted effect at the undyed maximum. fabric samples diluted to 0.5% EtOH did not show any changesA longer indicating time non-toxicityof exposure (i.e.,slightly HTD increased value was changes 0). However, in exposed when cells, these but samples the results were dilutedwere still to 1%below and toxicity, 2% EtOH, i.e. exposed average cellsHTD showed values changeswere 1.0 in after morphology exposure andof 24 viability h, and in1.3 the after quantity 72 h (Figure of < 50%, 3). indicatingThe difference a low between toxic eff theect attwo the timespans maximum. was statistically insignificant (Mann–Whitney U-test, p > 0.05, An = longer 24). time of exposure slightly increased changes in exposed cells, but the results were still belowIron toxicity, (FeSO i.e.,4) mordanted average HTD fabric values samples were induced 1.0 after the exposure least cell of changes, 24 h, and as 1.3 HTD after values 72 h (Figure were 23 at). Thethe maximum difference indicating between thefrom two non-toxic timespans to low was toxic statistically effects, but insignificant the differences (Mann–Whitney compared to U-test, alum 2 p(KAl(SO> 0.05,4 n)2=) or24). unmordanted samples were insignificant (Kruskal–Wallis-test, X = 3.37, p > 0.05, n = 24) (Figure 3).

Figure 3. The effect effect of time on the changes in the morphologymorphology and the loss of viability (%) for the mordanted but undyed fabrics, fabrics, wh whereere the the HTD HTD value value of of 1 1 indicated indicated << 25%25% loss loss of of viability viability and and 2 2between between 25 25–50%.–50%. The The results results contain contain the thevalues values of 1 of an 1d and 2% EtOH 2% EtOH dilutions. dilutions. NM NM= no =mordant,no mordant, SD = standardstandard deviation.

2.2.4.Iron The (FeSOToxicity4) mordanted of the Dyed fabric Fabrics samples induced the least cell changes, as HTD values were 2 at the maximum indicating from non-toxic to low toxic effects, but the differences compared to alum The CIELab values of the colours obtained from dyeing are shown in Table 2.2 The dyed fabric (KAl(SO4)2) or unmordanted samples were insignificant (Kruskal–Wallis-test, X = 3.37, p > 0.05, nsamples= 24) (Figure induced3). slightly more changes to exposed cells than undyed fabric samples, but the differences were insignificant (Kruskal–Wallis test, X2(1.120) = 1.339, p > 0.05 (p = 0.247), n = 360) 2.2.4.(Figure The 4). Toxicity of the Dyed Fabrics When the dyed fabrics were diluted to 0.5% or 1% EtOH, their effect on the morphology and The CIELab values of the colours obtained from dyeing are shown in Table2. The dyed fabric viability of the exposed cells was < 25%, i.e. HTD values were 1 at the highest, indicating the diluted samples induced slightly more changes to exposed cells than undyed fabric samples, but the differences dyed fabric samples were non-toxic. were insignificant (Kruskal–Wallis test, X2(1.120) = 1.339, p > 0.05 (p = 0.247), n = 360) (Figure4). All the dyed fabric samples caused changes on exposed cells when they were diluted to 2% When the dyed fabrics were diluted to 0.5% or 1% EtOH, their effect on the morphology and EtOH. In that case, at least one of the two parallel samples induced 25–50% losses of morphology and viability of the exposed cells was < 25%, i.e., HTD values were 1 at the highest, indicating the diluted cell viability, i.e. HTD values were 2, indicating a non-significant but still low toxicity. dyed fabric samples were non-toxic. Iron mordanted dyed samples induced the least changes on exposed cells i.e. low toxic effects at All the dyed fabric samples caused changes on exposed cells when they were diluted to 2% EtOH. the maximum, after exposure of 24 h. However, after exposure of 72 h, the differences between iron In that case, at least one of the two parallel samples induced 25–50% losses of morphology and cell mordanted dyed samples and undyed controls became smaller, and the iron mordanted samples viability, i.e., HTD values were 2, indicating a non-significant but still low toxicity. obtained HTD values similar to all others (Figure 4). Iron mordanted dyed samples induced the least changes on exposed cells i.e., low toxic effects In the case when all the samples were examined, it was observed that the dyed fabrics induced at the maximum, after exposure of 24 h. However, after exposure of 72 h, the differences between slightly more changes to exposed cells than the undyed fabrics. Those changes indicated harmless, iron mordanted dyed samples and undyed controls became smaller, and the iron mordanted samples obtained HTD values similar to all others (Figure4). Antibiotics 2020, 9, 266 14 of 25 non-toxic, or low toxic effects at the maximum. However, the difference between these two groups was statistically insignificant (Kruskal–Wallis test, X2 = 1.34, p > 0.05, n = 192). The length of the exposure time did not increase the difference in inducing cell changes caused by the dyed fabrics compared to the undyed controls. Interestingly, for diluted undyed fabric samples and diluted Tanacetum-, Salix- and Tapinella- dyed fabric samples, effects on exposed cells increased with time, whereas for Cortinarius the situationAntibiotics 2020was, 9 ,contrary: 266 The longer the exposure time, the fewer changes were observed in13 ofthe 24 exposed cells. This was shown by the lower HTD values after the exposure of 72 h.

FigureFigure 4. 4. TheThe effect effect of of dye dye source, source, mordant mordant and and time time on on the the HTD HTD values values (changes (changes in in morphology morphology and and lossloss of of viability%) viability%) after 2424 hh (above)(above) andand 72 72 h h (below) (below) exposure exposure times. times. EtOH EtOH dilutions dilutions of of 1% 1% and and 2% 2% are areincluded included (n =(n152). = 152). NM NM= no= no mordant, mordant, SD SD= standard= standard deviation. deviation. In the case when all the samples were examined, it was observed that the dyed fabrics induced 2.2.5. The Effect of Mordant on the Toxicity slightly more changes to exposed cells than the undyed fabrics. Those changes indicated harmless, non-toxic,The effect or low of the toxic mordant effects aton the the maximum. colour can However,be seen of thethe diCIELabfference values between in Table these 2. two Iron groups mordant was obviouslystatistically darkened insignificant colours. (Kruskal–Wallis test, X2 = 1.34, p > 0.05, n = 192). TheThe analysis length of showed the exposure that iron time mordant did not increaseinduced thethe di fewestfference changes in inducing to exposed cell changes cells compared caused by tothe other dyed mordants, fabrics compared as shown to by the the undyed lowest HTD controls. values (Figure 4). In all the samples there was slightly more Interestingly,variation (max for and diluted min undyedvalues) fabricobserved samples after and the dilutedexposureTanacetum- time of ,24Salix- h thanand 72Tapinella h, showing-dyed timefabric evening samples, out e fftheects differences. on exposed cells increased with time, whereas for Cortinarius the situation was contrary:Unmordanted The longer but the biocoloured exposure time, fabrics the fewer (Tanacetum changes, Salix were observedand Tapinella in the) exposed did not cells.produce This any was differenceshown by in the the lower HTD HTD values values at either after exposure the exposure times; of nor 72 h.did they produce any difference compared to the unmordanted undyed fabric sample. When examining the HTD values of unmordanted 2.2.5. The Effect of Mordant on the Toxicity The effect of the mordant on the colour can be seen of the CIELab values in Table2. Iron mordant obviously darkened colours. The analysis showed that iron mordant induced the fewest changes to exposed cells compared to other mordants, as shown by the lowest HTD values (Figure4). In all the samples there was slightly Antibiotics 2020, 9, 266 14 of 24 more variation (max and min values) observed after the exposure time of 24 h than 72 h, showing time evening out the differences. Unmordanted but biocoloured fabrics (Tanacetum, Salix and Tapinella) did not produce any difference in the HTD values at either exposure times; nor did they produce any difference compared to the unmordanted undyed fabric sample. When examining the HTD values of unmordanted samples after the exposure of 24 h, it was observed that the dyed fabrics received slightly higher values than the undyed controls, but after 72 h, the differences between dyed and undyed fabrics had disappeared. The only exception was Cortinarius, which received lower HTD values after 72 h than 24 h, with all mordants. The alum mordanted Tanacetum fabric samples differed slightly from other dyed samples and the undyed controls by causing fewer cell changes at both exposure times, the difference being statistically insignificant. The samples that were mordanted with iron and dyed with different biocolourants did not differ significantly from each other. When examining differences between the mordants, it was observed that iron induced the least change in exposed cells, although the differences compared to other mordants were insignificant (Kruskal-Wallis, X2 = 3.33, p > 0.05, n = 120). The most changes were observed after the exposure of 24 h, but the Kruskal–Wallis test revealed that the differences between all the mordants were insignificant (X2 = 5.73, p > 0.05, n = 60).

3. Discussion The sample extracts (99.5% EtOH) were diluted to three EtOH concentrations (0.5, 1.0, 2.0%) and the results showed that the higher the EtOH concentration, the more changes in the morphology and viability of exposed cells were observed. This was likely because of the increase in the concentrations of compounds extracted with EtOH from the plant and fungal material. With the lowest EtOH concentration, no cytotoxicity was observed, indicating that the type and number of toxicity-inducing compounds in the sample was low. However, further studies would be needed to analyse these compounds, their character and behaviour more precisely. Both the plant and fungal extracts and dyed fabrics of Tanacetum and Tapinella showed the most change in the morphology and viability of the exposed cells. If their extracts were diluted to 2% EtOH, the changes were toxically significant. The studied sample of T. vulgare (Finland) was a camphor chemotype (71% camphor), but had no quantifiable thujones [18,41], which are regarded at high concentrations to exhibit toxicity [40]; so, we suppose there are some other components than thujones that are responsible for the observed low to moderate cytotoxicity. Furthermore, the tansy used in this research was collected and dried years ago and long storage time may have affected the composition and content of colourants and other compounds. Our qualitative HPLC-UV/Vis-MS analysis showed that the majority of the secondary compounds had preserved well over the years. According to previous studies that used the same solvent as we did, i.e., EtOH, or similar EtOH:H2O, extracts obtained from the aerial parts of Tanacetum species contained secondary metabolites from the groups of sesquiterpene, non-volatile sesquiterpene lactones, coumarins, phenolic acids, and flavonoids [49,87–89]. Previously, the essential oils of tansy have shown cytotoxicity. The oil distilled from the aerial parts of T. vulgare (Canada) with camphor (31%), borneol (15%) and 1,8-cineole (i.e., eucalyptol) (11%) as the major components were slightly cytotoxic against human healthy cell line WS1 and human keratinocytes (HaCaT). Furthermore, its minor components, α-humulene (0.21%) and caryophyllene oxide (1.13%), possessed a moderate cytotoxicity against both of these [90]. The recent results [44] strongly implied that the toxicity of T. vulgare (β-thujone chemotype) essential oil, obtained from its aerial parts with hydrodistillation, should not be associated exclusively with thujones. The tests using the Artemia salina model indicated that some other (minor) constituents of the oil were responsible for the observed more pronounced acute toxicity. For example, sabinene, Antibiotics 2020, 9, 266 15 of 24 terpinen-4-ol and caryophyllene oxide showed very strong negative correlations (r > 0.8) with the − live nauplii after 48 h, while all other constituents (e.g., linalool, intermedeol, sabinyl acetate) showed strong (r > 0.6) negative correlations [44]. Previously, it has been shown that a linalool rich oil − was more toxic in this brine-shrimp bioassay than the sample that did not contain this monoterpene alcohol [44]. Rosselli and coworkers [42] isolated five sesquiterpene lactones with the eudesmanolides skeleton from the dried flowers of T. vulgare ssp. siculum (Guss). All of them (douglanin, ludovicin B, ludovicin A, 1α-hydroxy-1-deoxoarglanine, 11,13-dehydrosantonin) had high time- and concentration-dependent cytotoxic activity against in vitro cultured cancer and healthy cell lines (V79379A), being more effective against the latter ones. As far as we are aware, there have been no previous cytotoxicity studies concerning T. atrotomentosa, which would have shown significant toxicity when the extract was diluted to 2% EtOH. Despite this, its chemistry holds great potential, as atromentin, the central terphenylquinone intermediate of a large family of homobasidiomycete secondary metabolites, represents the generic precursor molecule for the entire terphenylquinone and pulvinic acid family of colourants. Atromentin may be modified by oxidative ring splitting into atromentic acid (yellow), or by dihydroxylation and symmetric heterocyclisation to thelephoric acid; or by reduction and esterification to produce leucomentins, which can upon saponification, release osmundalactones [91]. The terphenylquinone compounds have gained pharmaceutical attention due to their multiple bioactivities and have been produced under laboratory conditions [91]. The significant cytotoxicity we found in these preliminary studies would need further research. T. atrotomentosa holds great potential as a source of colourants, but also due to its manifold other properties. In recent years, a lot of effort has been focused on the development of cultivation of fungi for colourant production [11,92,93]. For Salix and Cortinarius, the HTD values caused by their extracts and dyed fabrics were quite similar. Both showed low cytotoxicity when diluted to 2% EtOH. The observed toxicity changes did not alter after exposure of 72 h. The compounds and interactions that lie behind the observed toxicity of Cortinarius extracts are probably due to anthraquinones, the chemistry and toxicity of which were found in previous studies, are presented in Section 1.5. Natural anthraquinones are interesting compounds in many applications and they need more detailed studies. The low cytotoxicity of Salix extracts was presented for the first time. Generally, the extracts of Salix bark have been regarded as being safe, although sensitive individuals may get allergic reactions due to salicylates. Studies have also shown that there are other salicin-related compounds that contribute to various effects, which may also concern cytotoxicity [57]. Textile production procedures are complex. During the procedures, many different auxiliary chemicals are used, and extracts may contain remnants of them. In our study, the undyed fabrics were not interpreted as being cytotoxic; however, they caused effects from non-toxic to low toxic, depending on the concentration of EtOH, for both unmordanted and mordanted fabrics, indicating that it is also important to analyse the toxicity of the undyed fabric itself. All unmordanted and alum (KAl(SO4)2) and iron (FeSO4) mordanted fabrics showed slight changes ( 50%) in cell structures when diluted to 1 or 2% EtOH and interpreted toxically insignificant ≤ changes. The study showed that iron mordanted fabric samples induced the least cell changes, but the differences compared to alum (KAl(SO4)2) or unmordanted samples were insignificant. The reason for this is not known, but leucomentins of Tapinella may act as iron chelators [94], and condensed tannins of Salix species form complexes with metal ions [60]. In these cell tests, the concentration of plant/fungal material was approx. 50–800 µg/mL of α-MEM. If organic material contains approx. 4% of biocolourants, a maximum of 2–32 µg pure dye/mL of α-MEM was used in the cell tests. This is considerably less compared with Klemola [52] who used 50–1600 µg pure synthetic dye/mL medium in the studies. In the future studies, higher concentrations of plant/fungal material should be tested. Antibiotics 2020, 9, 266 16 of 24

Using pure dyes in tests would give less ambiguous results. On the other hand, it is difficult to predict the toxicity of a dyed material from just testing the dye, because one chemical itself may be non-toxic but the chemicals and materials together may have a totally different effect [95]. Also, differences exist between a plant material extract which contains all soluble compounds and an extract from a dyeing when only the portion of substances which had been adsorbed during dyeing and released in later extraction are present. Development of biocolourants would be more economic if crude extracts were used instead of fractions or purified compounds. If additional values of biocolourant are to be developed, it is essential to consider whether the product would be purified or a mixture, as in the purification step, activities may be lost or decreased [96]. The cell tests can give information about the potential toxicity of extracts from textile materials. However, the cell type and test method used have an impact on how the analysis results may be interpreted. Only one type of cell, Hepa-1, was used in this research, and conclusions are only directly applicable to this cell type. The cases in which one cell line analysis shows slight cytotoxicity may appear to be harmless to humans. To draw a more reliable picture, several different cell lines should be used, and the total impact evaluated based on all results. Nonetheless, Hepa-1 cells have been observed to predict potential cytotoxicity in humans better than rat cells [52]. Hepa-1 cytotoxicity test can give useful information about the overall toxicity of extracts from textiles and can help in the development of safe textile products [97]. When considering the harmful effects of dyes, discussion is most often focused on environmental effects and the elimination of dyes from textile effluents, instead of the direct influence of textile dyes on human health [5]. The biocolourants are biodegradable, but their production, usage at a larger scale, and safety needs much more research. We tested four extracts of plant/fungal sources and dyed fabrics to answer the question whether it is safe to wear natural dyed textile. The amounts of natural colour compounds in textile after dyeing are low. Washing fastness tests reveal that minimal amounts of colourants were removed during these simulated standardised use phases. This preliminary study showed that risk for harmful effects of natural dyes when wearing textiles are low among the tested dye sources.

4. Materials and Methods

4.1. Plant and Fungal Material Tansy flower heads (Tanacetum vulgare L.), obtained from Natural Resources Institute Finland (Jokioinen, Finland), were harvested in 2000. The plant previously studied by Keskitalo [18] was classified as camphor chemotype. Flowers were dried and stored in the dark at room temperature (RT) until used in June 2010. A 1:1 mass ratio of dried flower heads to textile material was used for the dye liquor. Willow (Salix phylicifolia L.) was gathered from Sipoo, Finland, in May 2008. Bark was separated and left to dry in a dark place at RT until used in June 2010. To prepare the dye liquor, a 5:1 mass ratio of dried bark to textile material was used. The bark was soaked in water for 2 days before heated extraction. Fruiting bodies of surprise webcap [Cortinarius semisanguineus (Fr.) Gillet] were collected from Jyväskylä, Finland, in 2005. They were dried at 60 ◦C and stored in a dark place at RT until used in June 2010. To prepare the dye liquor, a 1:1 mass ratio of dried fungi to textile material was used. Velvet roll-rims [Tapinella atrotomentosa (Batsch.) Šutara] were gathered from Espoo, Finland, in September 2009 and deep-frozen until used in June 2010. To prepare the dye liquor, a 10:1 mass ratio of defrosted fungi to textile material was used. The preparation of dye liquors followed the traditional dyeing recipes in which 1:1 dried plant/fungi for textile material and 10:1 fresh fungi/textile material were used. For Salix, a five times greater amount of material was used to obtain an identically deep colouration of textile [13]. Antibiotics 2020, 9, 266 17 of 24

4.2. Methods for Compound Identification Qualitative analysis of the main colourants of each studied species was performed using HPLC combined with UV/Vis and MS detections. The phenolic compunds in Tanacetum flowers were analysed by HPLC-DAD and UHPLC-QTOF MS/MS analytical equipments and methods described in Pihlava et al. [98]. Briefly, 300 mg of ground sample was extracted with 10 mL of MeOH for 1 h in an ultrasonic water bath. An aliquot of extract was filtered through a 0.20 mm membrane filter into an autosampler vial for the analysis. Anthraquinones of Cortinarius were analysed with methods described earlier by Vanden Berghe et al. [99]. The ground fungal material of Tapinella (100 mg) was extracted with 1 mL of MeOH using ultrasonication for 15 min where after centrifuged and the liquid phase was transferred to another tube. The material was re-extracted twice with 1 mL of MeOH; extracts were combined and evaporated to dryness. The samples were reconstituted in 0.2 mL of 50% aqueous MeOH. Analysis was performed on an Acquity UHPLC system (Waters, Milford, MA, USA) equipped with a Waters Synapt G2-S MS system and Waters Photodiode Array (PDA) Detector. The analytical column ACQUITY UPLC BEH HSS T3 (1.8 µm 2.1 100 mm) was kept at 30 C. The experiment was carried out at a flow rate × ◦ of 0.4 mL/min with mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in MeOH). The gradient elution started at 20% B (0–0.2 min), was increased linearly to 100% B within 28 min and after this returned within 0.1 min to initial percentage where it was maintained for 2 min. The injected sample amount was 2 µL. Mass spectrometry was carried out using electrospray ionization (ESI) in positive polarity, and leucine enkephalin was used as the lock spray reference compound. The capillary voltage was 2.0 kV, cone voltage 40 kV, source temperature 150 ◦C, and desolvation temperature 500 ◦C. The cone and desolvation gas flow were set at 60 L/h (nitrogen) and 1000 L/h (nitrogen), respectively. The data was collected at a mass range of m/z 50–800 with a scan duration of 0.2 sec. The PDA detection wavelength range was 200–500 nm.

4.3. Dyeing of Fabric Samples and the Colour Fastness Testing

Technical grade alum (KAl(SO4)2) and FeSO4 (TetriDesign, Helsinki, Finland) and tap water (pH ~7.8; Helsinki Region Environmental Services Authority HSY, Helsinki, Finland) were used for dyeing. Pre-mordanting and dyeing were performed in an Original Hanau Linitest machine (Hanau, Germany). The colour was measured as CIE L*, a* and b* values [100] using a Konica Minolta (Tokyo, Japan) CM-2600d spectrophotometer (D65, 10◦). Unbleached 100% wool fabric (plain weave, warp and weft densities 6.5 yarns/cm, 84 g/m2) (HAMK/Wetterhof, Hämeenlinna, Finland) was made of the SportLoden yarn (Nm 28/2, with Öko-Tex 100 and Bluesign labels) (Schoeller, Germany). To prepare the dye extracts, the amount of dried or defrosted plant or fungal material, as in Section 4.1, was weighed, chopped or crushed, and boiled in water covering the organic material. After boiling (80 90 C, 60 min), the extract was filtered. Organic material was discarded and the filtrate − ◦ used as a dye liquor. The fabric samples (á 20 g) were pre-mordanted with 10% on the weight of the fibre alum or 2% FeSO4 in a 1:40 material to liquor ratio. The mordant was diluted in water, wetted fabric was added and the vessel closed. The temperature was increased to 80 ◦C and held there for 60 min, continuously agitating the liquid. The samples were squeezed, left to dry at RT and stored in the dark until used. The pH for alum mordant liquor was 3.3–3.9 and for FeSO4 4.5–5.3. The samples without mordant were treated similarly as the pre-mordanted samples. The volume of the prepared dye extract was measured, and water was added to meet the 1:20 ratio for dyeing. Pre-mordanted samples were dyed at 90 ◦C for 60 min and then rinsed until no colour was observed in the water. The samples were left to dry at RT. The colour of each sample was measured as CIELab coordinates (L*, a*, b*) (Table2). L* obtains values from zero for black to 100 for white; a* > 0 describes the redness, a* < 0 the greenness, b* > 0 the yellowness, and b* < 0 the Antibiotics 2020, 9, 266 18 of 24 blueness of the colour [100]. Colour fastness to light and laundering were studied according to the ISO 105-B02/A1-1994 and ISO 105-C06-1997 standards, respectively (Table4).

4.4. Preparation of Plant, Fungal and Fabric Extracts for Toxicity Testing Plant or fungal material was ground in a mortar (Tanacetum and Cortinarius) or cut into small pieces (Salix and Tapinella) and 1.5 g was placed in a 100 mL flask; 37.5 mL EtOH (99.5%, Altia, Helsinki, Finland) was added and the materials were extracted for 24 h at RT. Thereafter, the extract was filtered and refrigerated until use. The colour of the Tanacetum extract was light green, Salix bright green, Cortinarius orange, and Tapinella black. The dyed woollen fabrics and undyed controls were cut into 1 cm 1 cm pieces and treated × similarly to the plant/fungal samples.

4.5. Cytotoxicity Assays The cell tests were performed as described earlier [53,101]. The effects of each sample extract on the morphology and viability of the cells were evaluated using phase contrast microscopy by observing possible alterations in cell shape, e.g., cell rounding, swelling, cell shrinkage, vacuolization, growth inhibition, attachment, cell lysis, and cell death. Hepa-1 cells containing cytochrome P4501A (Department of Physiology, University of Kuopio, Kuopio, Finland) from mouse liver were used in HTD tests. Cell culture reagents were α-MEM (Minimum Essential Medium) without glutamine, endotoxin-free FBS (Fetal Bovine Serum), penicillin-streptomycin solution 100 , l-glutamine 100 (200 mM), × × PBS (Phosphate-Buffered Saline, pH 7.2) 10 without Ca2+ and Mg2+, and trypsin 0.05%/EDTA × (ethylenediaminetetraacetic acid) 0.02% in PBS (EuroClone, Milan, Italy). EtOH was used as a solvent control in concentrations of 0.5, 1 and 2% and α-MEM as a negative control and solvent medium. Hepa-1 cells were grown in α-MEM supplemented with 1% l-glutamine, 10% endotoxin-free FBS and 1% penicillin-streptomycin solution. A short-term cytotoxicity test was carried out in 96-well microplates. Hepa-1 cells were left to grow for 2–3 days in cell culture flasks. Erythrosin B (Sigma, Darmstadt, Germany) was used to release the cells from the flasks: a 1:4 dilution was prepared using a 20 µL volume of cells and that of 80 µL of Erythrosin B, which were adjusted to the concentration of 5 104 cells/mL. A volume of 200 µL from × this dilution was pipetted into each well, resulting in 10,000 cells/well. A Bürker counting chamber measured the number of cells and cell density in each well. Cells were exposed to the extracts prepared as explained in Section 4.4. EtOH extracts were diluted to the concentrations of 0.5, 1 and 2% with α-MEM and a sample (200 µL) was pipetted in a well. Non-exposed cells with α-MEM were used as negative controls. DNP (2,4(α)-dinitrophenol, pH 2.8 4.7, Merck, Kenilworth, NJ, USA) was used as a positive control and was diluted in DMSO − (dimethylsulphoxide, pH 2.8 4.7, Riedel-de Haën, Seelze, Germany). A stock solution of DNP − (100 mg/mL) was diluted with α-MEM to the concentrations of 0.5, 0.05 and 0.005 mg/mL. All results were compared with the controls. After exposures of 24 and 72 h, the cells were washed twice with PBS-buffer. The possible alterations of cells were observed with phase-contrast microscopy and compared to the control cells, which were without exposure. The alterations were described and the quantity of changes was evaluated with a five- step-scale: (0) no changes, (1) < 25%, (2) 25 50%, (3) 50 75%, and (4) > 75% of − − cells had changed. Each plant, fungal and fabric sample was studied using 0.5, 1 and 2% concentrations of EtOH extracts diluted with α-MEM and incubation times of 24 h and 72 h as two parallel samples in different places in the 96-well microplates. Four parallel wells were studied from each sample. The results of parallel wells were reported as a single value. These values (1 4) were the basis for the statistical − analysis. For a few samples, two values were reported (e.g., 0/1); in these cases, the mean value (0.5) was used for statistical calculations. Antibiotics 2020, 9, 266 19 of 24

4.6. Statistical Analyses The SPSS program (IBM SPSS Statistics for Windows, Version 24.0, Armonk, NY, USA) was used for quantitative data analysis. Statistical significance was placed at p < 0.05. The differences were evaluated using non-parametric Kruskal–Wallis one-way analysis of variance and the Mann–Whitney U-test.

5. Conclusions The dilution concentration was shown to be of high importance concerning the toxicity issue. The highest HTD values were observed when the samples were diluted to 2% EtOH concentration, which is due to higher concentrations of compounds including biocolourants. EtOH was used because it dissolves polar and somewhat lipophilic biocolourants, as well as those of potential toxins. Pure 2% EtOH was observed as being harmless. The most cytotoxic effects were induced by the extracts of Tanacetum and Tapinella. With 2% EtOH, the HTD values were at the highest 4, which means that over 75% of the tested cells had changes in morphology and loss of viability, indicating significant toxicity; the extracts of Salix and Cortinarius induced barely perceptible cytotoxicity when diluted to the same EtOH concentration. When examining all the data, Salix induced the least toxic effects of the tested biocolourant sources. Tanacetum showed some toxicity, but the effect declined when the time of exposure increased from 24 h to 72 h. Interpretation of the results could be more feasible if there were standardised methods for biocolourant production, extraction and in vitro testing. In this research, Salix and Cortinarius semisanguineus were at most mildly harmful. Tapinella atrotomentosa and Tanacetum vulgare induced the most changes. At the highest non-toxic EtOH concentrations (2%), all dyed fabrics were interpreted as being not significantly toxic. It seems that biocolourants may be a less harmful alternative to dyes. In all, the cytotoxicity of samples dyed with natural dyes did not differ from untreated wool. When developing the larger scale utilisation of biocolourants, profound bioassays and chemical analyses are necessary from single purified compounds to track the possible hazardous pathways of dyes in the human metabolism and environment.

Author Contributions: Conceptualization, R.R. and A.P.; methodology, R.R., A.P. and U.H.; software, S.N., R.R., H.N., J.-M.P. and I.V.B.; validation, R.R., A.P. and S.N.; investigation, R.R., U.H., A.P., S.N., H.N., J.-M.P. and I.V.B.; resources, R.R. and A.v.W.; writing—original draft preparation, R.R. and A.P.; writing—review and editing, A.v.W.; visualization, R.R.; supervision, R.R. and A.v.W.; project administration, R.R.; funding acquisition, R.R. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Aino-Koti Foundation (2010) and the Strategic Research Council at the Academy of Finland [funding decision no. 327178, 2019]. Acknowledgments: We express our gratitude to the laboratory technician Riitta Venäläinen for skilful assistance in the different parts of this study. We also want to thank Leena Loukojärvi and Saara Hynninen for their work in the dyeing experiments. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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