molecules

Article Anti-Inflammatory Activity of the Wild Mushroom, tinctorium, in RAW264.7 Macrophage Cells and Mouse Microcirculation

Sumreen Javed 1, Wai Ming Li 1, Mehreen Zeb 1, Almas Yaqoob 1, Linda E. Tackaberry 2, Hugues B. Massicotte 2 , Keith N. Egger 2 , Peter C.K. Cheung 3 , Geoffrey W. Payne 4 and Chow H. Lee 1,*

1 Chemistry and Biochemistry Program, University of Northern British Columbia, Prince George, BC V2N 4Z9, Canada; [email protected] (S.J.); [email protected] (W.M.L.); [email protected] (M.Z.); [email protected] (A.Y.) 2 Ecosystem Science and Management Program, University of Northern British Columbia, Prince George, BC V2N 4Z9, Canada; [email protected] (L.E.T.); [email protected] (H.B.M.); [email protected] (K.N.E.) 3 Food and Nutritional Sciences Program, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; [email protected] 4 Northern Medical Program, University of Northern British Columbia, Prince George, BC V2N 4Z9, Canada; Geoff[email protected] * Correspondence: [email protected]; Tel.: +1-250-960-5413; Fax: +250-960-5170

Academic Editor: Maria da Graça Costa G. Miguel   Received: 9 September 2019; Accepted: 24 September 2019; Published: 27 September 2019

Abstract: The aim of this study was to investigate the anti-inflammatory activity of a previously un-studied wild mushroom, , collected from the forests of north-central British Columbia. The lipopolysaccharide (LPS)-induced RAW264.7 macrophage model was used to study the in vitro anti-inflammatory activity. The crude alkaline extract demonstrated potent anti-inflammatory activity, and was further purified using a “bio-activity-guided-purification” approach. The size-exclusion and ion-exchange chromatography yielded a water-soluble anti-inflammatory polysaccharide (AIPetinc). AIPetinc has an average molecular weight of 5 kDa, and is a heteroglucan composed of mainly glucose (88.6%) with a small amount of galactose (4.0%), mannose (4.4%), fucose (0.7%), and xylose (2.3%). In in vivo settings, AIPetinc restored the histamine-induced inflammatory event in mouse gluteus maximus muscle, thus confirming its anti-inflammatory activity in an animal model. This study constitutes the first report on the bioactivity of Echinodontium tinctorium, and highlights the potential medicinal benefits of fungi from the wild forests of northern British Columbia. Furthermore, it also reiterates the need to explore natural resources for alternative treatment to modern world diseases.

Keywords: Echinodontium tinctorium; polysaccharide; anti-inflammation; mushroom; British Columbia

1. Introduction Fungi represent a major and largely untapped source of potentially powerful new pharmaceutical natural products. Despite this, there have been relatively few studies directed at finding medicinal compounds from forest fungi in British Columbia (BC), Canada. To this end, we have begun to explore medicinal properties of BC native fungi, focusing first on three bioassays that are relevant to cancer: growth-inhibitory, immuno-stimulatory, and anti-inflammatory activities [1,2].

Molecules 2019, 24, 3509; doi:10.3390/molecules24193509 www.mdpi.com/journal/molecules Molecules 2019, 24, 3509 2 of 15

The selected for the present study was Echinodontium tinctorium (Ellis & Everh.) Ellis & Everh. It is also known as Indian paint fungus due to its distinctive red brick color, and its primary use by Indigenous peoples was in preparing red paint pigments [3]. Echinodontium tinctorium is a white rot, able to decay both lignin and cellulose. It produces a woody, large hydnaceous or conk [4,5] that typically grows beneath dead branch stubs on live tree trunks [6]. Most studies pertaining to E. tinctorium relate to its , disease cycle, and management [4,5,7–9], but this conk also has a history of use as an anti-bacterial agent by Indigenous peoples [10]. A recent comprehensive study on the DNA sequences spanning ITS1-5.8S-ITS2 and the D1-D2 domains of 28S rDNA showed that the Echinodontium is represented by E. tinctorium, E. ryvardenii, and E. tsugicola, while a new genus, Echinodontiellum, was proposed to accommodate E. japonicum [8]. To date, there has only been one compound, echinodol, a triterpene acetate, isolated from E. tinctorium [11]. However, the bioactivity of this triterpene acetate was not studied, nor has there been any other study on extracts isolated from this fungus. On the other hand, a number of sesquiterpenes have been isolated from E. tsugicola, but only two show weak anti-bacterial activity [12–14]. Two sesquiterpenoids were also isolated from E. japonicum, but they proved to have no anti-bacterial activity [15]. Inflammation is a component of the body’s own immune response that combats the invasive pathogens, irritants, and cell debris. At the system level, inflammation causes fever, swelling, and pain. At the cellular level, it causes vasodilation and compromises vascular permeability. While the initial intention of an inflammatory event is to support the human body, a prolonged and excessive inflammation is correlated with various clinical presentations like arthritis, cardiovascular complications, and tumor progression [16]. Non-steroidal anti-inflammatory drugs (NSAIDS) are used as first line therapy against inflammation, but their long-term use is associated with gastrointestinal bleeding and other complications, including peptic ulcers [16,17]. This suggests a need to explore alternative medicines, especially natural products that could provide potentially safer options to treat inflammation [16]. The aim of this study was to explore the medicinal bioactivity of E. tinctorium collected from north-central BC. Dried and pulverized mushroom fruiting bodies were sequentially extracted using 80% ethanol, 50% methanol, water, and 5% NaOH, a modified approach used to extract anti-tumor polysaccharide and described previously [18]. The extracts were studied for immuno-stimulatory and anti-inflammatory activities, based on their ability to either stimulate TNF-α production or inhibit lipopolysaccharide-induced TNF-α production, respectively, in RAW264.7 mouse macrophage cells. The potent extract for anti-inflammatory activity was purified using a two-step “bioactivity-guided-purification” approach with the help of size-exclusion and ion-exchange chromatography. Gas chromatography (GC) with a flame ionization detector (FID) was employed to analyze the monosaccharide composition. Intravital microscopy coupled with conducted vasodilation measurements in mice gluteus maximus muscle (second order arterioles) were used to confirm its anti-inflammatory activity in vivo. Thus, the main purpose of this study was to investigate the anti-inflammatory activity of E. tinctorium in both RAW264.7 cells and mice microcirculation.

2. Results and Discussion

2.1. Identification of Echinodontium tinctorium Based on morphological characteristics, the collected fungi were tentatively identified as Echinodontium tinctorium. DNA sequencing analysis of the ITS2 region and the 50 end of the D1-D2 region of the 28S gene using the consensus sequence of the ITS3 and NLB4 primers, followed by a BLAST search of GenBank, further confirmed this finding. The DNA sequence of collections CL37 and CL103 were identical, 481 bp in length, and had highest sequence similarity to collections of E. tinctorium from the USA (GenBank KF996509.1: 100% identity/62% coverage) and Canada (GenBank Molecules 2019, 24, 3509 3 of 15 Molecules 2019, 24, x 3 of 15

AY854088.1:AY854088.1: 99% 99% identity identity/66%/66% coverage);coverage); neither Ge GenBanknBank entry entry extended extended into into the the 28S 28S D1/D2 D1/D2 region, region, hencehence the the lower lower percent percent coverage. coverage.

2.2.2.2. Chemical Chemical Extraction Extractionand and AssessmentAssessment of Crude Extracts Extracts of of E. E. tinctorium tinctorium for for Bio-Activities Bio-Activities PowderedPowderedE. E. tinctoriumtinctorium waswas sequentially sequentially extracted extracted with with 80% 80% ethanol, ethanol, 50% methanol, 50% methanol, water, water,2% 2%ammonium ammonium oxalate oxalate and and5% NaOH, 5% NaOH, and assessed and assessed for immuno-stimulatory for immuno-stimulatory activity as shown activity in as Figure shown in1a. Figure At 1 1mg/mL,a. At 1the mg water/mL, extracts the water showed extracts potent showed activity potentthat was activity stronger that than was the strongerpositive control thanthe positivelipopolysaccharide control lipopolysaccharide (LPS), while the ammonium (LPS), while oxalate the extract ammonium showed oxalate modest extract immuno-stimulatory showed modest immuno-stimulatoryactivity. The 80% ethanol, activity. 50% The methanol, 80% ethanol, and 50%5% NaOH methanol, extracts and 5%showed NaOH little extracts to no showed immuno- little tostimulatory no immuno-stimulatory effects and were eff furtherects and assessed were furtherfor anti-inflammatory assessed for anti-inflammatory activity in vitro (Figure activity 1b). Bothin vitro (Figurethe 80%1b). ethanol Both theand 80% 50% ethanolmethanol and extracts 50% methanolshowed potent extracts activity showed in inhibiting potent activityLPS-induced in inhibiting TNF- LPS-inducedα production, TNF- however,α production, the 5% however,NaOH extract the 5% was NaOH the strongest extract was among the strongest the three among extracts the. We three subsequently omitted the ammonium oxalate extraction step with the new collection of E. tinctorium extracts. We subsequently omitted the ammonium oxalate extraction step with the new collection and proceeded with 5% NaOH extraction after water extraction [1,2]. As shown in Figure 1c, the new of E. tinctorium and proceeded with 5% NaOH extraction after water extraction [1,2]. As shown in 5% NaOH extraction method (without ammonium oxalate extraction) has potent anti-inflammatory Figure1c, the new 5% NaOH extraction method (without ammonium oxalate extraction) has potent activity that is similar to the old 5% NaOH extract. anti-inflammatory activity that is similar to the old 5% NaOH extract.

* 3000 *

2000 (pg/ml) α * TNF- 1000

0 l H ter nol no O EM a LPS a ater M W t h W m. Ox Na D Et ha e m M A 5% Extracts (a)

3000 Without LPS 6000 With LPS Withou t LPS

2000 4000 With LPS (pg/ml) (pg/ml) α ** α

TNF- 2000 1000 TNF- * * 0 * 0 * * r l l r ) ) M e B o o H M te B ld w E t M n n O E a M o e M a P a a a M P ( (n D W t h th N D W H E e % O H M 5 a aO N N (b) (c) FigureFigure 1. 1.Immuno-modulatory Immuno-modulatory activities activities of of crude crude extracts extracts obtained obtained from from EchinodontiumEchinodontium tinctorium. tinctorium. (a) (a)At At 1 1mg/mL, mg/mL, crude crude extracts extracts (80% (80% ethanol, ethanol, 50% 50% me methanol,thanol, water, water, 2% 2% ammonium ammonium oxalate, oxalate, and and 5% 5% NaOH)NaOH) were were assessed assessed for for theirtheir abilityability to induce TNF- TNF-αα productionproduction in in RAW264.7 RAW264.7 macrophage macrophage cells cells as as anan indicator indicator of of immuno-stimulation. immuno-stimulation. LipopolysaccharLipopolysaccharideide (LPS) (LPS) was was used used asas positive positive control, control, whereas whereas mediamedia and and water water were were used used asas negativenegative controls. ( (bb) )Crude Crude extracts extracts (1 (1 mg/mL) mg/mL) that that were were inactive inactive for for immuno-stimulationimmuno-stimulation (80% (80% ethanol, ethanol, 50% 50% methanol, methanol, and and 5% 5% NaOH) NaOH) were were tested tested for theirfor their ability ability to inhibit to LPS-inducedinhibit LPS-induced TNF-α production TNF-α production as an indicator as an indicator of anti-inflammatory of anti-inflammatory activity. activity. Polymyxin Polymyxin B (PMB; B an inhibitor(PMB; an of inhibitor LPS) was of used LPS) as was a positive used as a control, positive whereas control, mediawhereas and media water and treated water with treated LPS with were LPS used aswere negative used controls.as negative (c )controls. Comparison (c) Comparison of the anti-inflammatory of the anti-inflammato activityry ofactivity 5% NaOH of 5% extractNaOH extract from two differentfrom two collections different ofcollectionsE. tinctorium. of E.Due tinctorium. to the largeDue to quantity the large of aquantity new collection, of a new the collection, new NaOH the new extract

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NaOH extract was used for further purification studies and in vivo experiments. Data shown were was used for further purification studies and in vivo experiments. Data shown were pooled from three pooled from three biological replicates (n = 3). Error bars are S.D., and one-way ANOVA was biological replicates (n = 3). Error bars are S.D., and one-way ANOVA was performed as statistical performed as statistical analysis. * denotes p < 0.05 as compared to the water control using Sidak’s analysis. * denotes p < 0.05 as compared to the water control using Sidak’s multiple comparison test. multiple comparison test. 2.3. Purification of Anti-Inflammatory Polysaccharide from E. tinctorium 2.3. Purification of Anti-Inflammatory Polysaccharide from E. tinctorium The 5% NaOH extract was selected for further purification and characterization of the anti-inflammatoryThe 5% NaOH compound. extract was A selected summary for further of the schemepurification used and to purifycharacterization the anti-inflammatory of the anti- compoundinflammatory AIPetinc compound. is shown A summary in Figure of the2a. scheme We first used subjected to purify the the anti-inflammatory NaOH extractto compound Sephadex LH-20AIPetinc chromatography is shown in and,Figure as shown2a. We infirst Figure subjected2b, fractions the NaOH 10–22 extract and 26–30 to Sephadex possessed LH-20 potent chromatography and, as shown in Figure 2b, fractions 10–22 and 26–30 possessed potent anti- anti-inflammatory activity. The first anti-inflammatory activity peak (fractions 10–22) correlated inflammatory activity. The first anti-inflammatory activity peak (fractions 10–22) correlated well with well with the maximal carbohydrate (Figure2c) and protein (Figure2d) content, suggesting that the the maximal carbohydrate (Figure 2c) and protein (Figure 2d) content, suggesting that the bioactive bioactive compound may contain polysaccharide as well as protein. The second anti-inflammatory compound may contain polysaccharide as well as protein. The second anti-inflammatory activity activity peak (fractions 26–30) correlated well with the maximal carbohydrate (Figure2c), but not peak (fractions 26–30) correlated well with the maximal carbohydrate (Figure 2c), but not the protein thecontent protein (Figure content 2d), (Figure suggesting2d), suggesting that the that bioa thective bioactive compound compound is composed is composed predominantly predominantly of ofpolysaccharide. polysaccharide.

FigureFigure 2. Purification2. Purification of theof anti-inflammatorythe anti-inflammatory polysaccharide polysaccharide from Echinodontium from Echinodontium tinctorium tinctorium(AIPetinc). (a)(AIPetinc).Summary of(a)the Summary purification of the scheme purification used; (schemeb) Profile used; of anti-inflammatory (b) Profile of anti-inflammatory activity of the NaOH activity extract of afterthe elutionNaOH fromextract Sephadex after elution™ LH-20 from column. Sephadex™ The assaysLH-20 column. were performed The assays in triplicatewere performed and the in data showntriplicate is a representativeand the data shown from three is a representa biological replicatestive from (threen = 3). biological Error bars replicates represent (nstandard = 3). Error deviation. bars One-wayrepresent ANOVA standard was deviation. performed One-way as described ANOVA in Materials was performed and Methods. as described * denotes pin< Materials0.05 as compared and Methods. * denotes p < 0.05 as compared to DMEM using Sidak’s multiple comparison test. Fractions to DMEM using Sidak’s multiple comparison test. Fractions which contained anti-inflammatory activity which contained anti-inflammatory activity (expressed as % inhibition) were also assessed for

Molecules 2019, 24, x 5 of 15 Molecules 2019, 24, 3509 5 of 15 carbohydrate (c) and protein (d) contents. The data shown in (c) and (d) also result from three biological replicates (n = 3). (expressed as % inhibition) were also assessed for carbohydrate (c) and protein (d) contents. The data shownSeveral in large-scale (c) and (d) alsoSephadex result from LH-20 three runs biological were pe replicatesrformed ( nand= 3). fractions which contained the first anti-inflammatorySeveral large-scale activity Sephadex peak LH-20were runspooled, were lyophilized, performed andand fractions dose-dependently which contained assessed the firstfor anti-inflammatoryactivity. When assessed activity at peak 0.1–1 were μg/ pooled,μL, the lyophilized, Sephadex andLH-20 dose-dependently purified sample assessed had higher for activity. anti- Wheninflammatory assessed activity at 0.1–1 thanµg/µ theL, thecrude Sephadex 5% NaOH LH-20 E4 purifiedextracts sample(data not had shown), higher anti-inflammatorydemonstrating the activityefficiency than of theSephadex crude 5%LH-20 NaOH in purifying E4 extracts the (data anti-inflammatory not shown), demonstrating compound. We the then effi ciencyassessed of Sephadexseveral buffers, LH-20 each in purifying with different the anti-inflammatory pH, for possible use compound. with DEAE We Sephadex then assessed in purifying several the bu ffanti-ers, eachinflammatory with diff erentcompound. pH, for Amongst possible the use five with buffers DEAE (N Sephadex-methyl piperazine in purifying pH the4.5, anti-inflammatoryPiperazine pH 5.5, compound.L-Histidine pH Amongst 6.1, Tris the pH five 7.7, bu ffanders (diethanolaN-methylmine piperazine pH 8.7) pH tested, 4.5, Piperazine we determined pH 5.5, thatl-Histidine 20 mM pHPiperazine 6.1, Tris pH 7.7,5.5 andwas diethanolamine the most effective pH 8.7) buff tested,er for we purification determined thatof the 20 mManti-inflammatory Piperazine pH 5.5polysaccharide was the most in e ffthatective the buboundffer for bioactive purification polysacch of thearide anti-inflammatory could be eluted polysaccharide with 1 M NaCl in (data that thenot boundshown). bioactive Finally, polysaccharide we subjected the could bioactive be eluted poly withsaccharide 1 M NaCl eluted (data notwith shown). 1 M NaCl Finally, from we the subjected DEAE theSephadex bioactive column polysaccharide to a higher resolution eluted with size-exclusion 1 M NaCl from chromatography, the DEAE Sephadex Superdex column 200. Results to a higher of the resolutionanalysis of size-exclusion fractions collected chromatography, from Superdex Superdex 200 are 200. shown Results in of Figure the analysis 3. The of anti-inflammatory fractions collected fromactivity Superdex peak obtained 200 are shownfrom fractions in Figure 12–153. The is anti-inflammatory estimated to have activity a size of peak 5 kDa obtained based fromon the fractions dextran 12–15standards is estimated (Figure to 3a). have The a size activity of 5 kDa peak based was on foun thed dextran to correlate standards well (Figure with carbohydrate3a). The activity content peak wasanalysis found (Figure to correlate 3b), but well not with with carbohydrate protein content content analysis analysis (Figure 3 3c).b), but These not withresults protein suggest content that analysisAIPetinc (Figure is a polysaccharide3c). These results and suggestthat the thatpurificat AIPetincion steps is apolysaccharide following Sephadex and that LH-20 the purificationwere able to stepsfurther following purify the Sephadex anti-inflammatory LH-20 were compound. able to further purify the anti-inflammatory compound.

FigureFigure 3.3.Purification Purification of theof anti-inflammatorythe anti-inflammatory polysaccharide polysaccharide from Echinodontium from Echinodontium tinctorium (AIPetinc)tinctorium using(AIPetinc) Superdex using 200.Superdex Fractions 200. collectedFractions werecollected assessed were forassessed anti-inflammatory for anti-inflammatory activity whereactivity percent where inhibitionpercent inhibition represents represents the inhibition the inhibition of LPS-induced of LPS-induced TNF-α plotted TNF-α against plotted (a )against dextran (a standards,) dextran (standards,b) carbohydrate (b) carbohydrate contents, and contents, (c) protein and ( contents.c) protein Resultscontents. are Results representative are representative from two from separate two experimentsseparate experiments (n = 2). (n = 2).

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2.4.2.4. AIPetinc InhibitedInhibited LPS-InducedLPS-Induced NONO ProductionProduction and and Histamine-Induced Histamine-Induced TNF- TNF-ααProduction Production in in RAW264.7 MacrophageRAW264.7 Macrophage Cells Cells ToTo understandunderstand the the mechanism mechanism of of the the anti-inflammatory anti-inflammatory activity activity AIPetinc, AIPetinc, we first we performedfirst performed MTT assaysMTT assays to test to for test potential for potential toxicity toxicity of the of polysaccharide the polysaccharide on RAW264.7 on RAW264.7 cells. Ascells. shown As shown in Figure in Figure4a, at a4a, cell at densitya cell density of 10,000 of 10,000 cells/well cells/well and incubated and incubated for six for hours, six hours, the normal the normal incubation incubation period period to assess to anti-inflammatoryassess anti-inflammatory response, response, cell viability cell viability was similar was amongstsimilar amongst all groups all (Figuregroups4 (Figurea). At a 4a). cell At density a cell normallydensity normally used in theused anti-inflammatory in the anti-inflammatory assay (100,000 assay (100,000 cells/well), cells/well), there was there a slight was a decrease slight decrease in cell viabilityin cell viability for the for crude the extractcrude extract (E4), and (E4), the and semi-purified the semi-purified fractions fractions post LH-20 post LH-20 and post-LH20-DEAE and post-LH20- thatDEAE contained that contained AIPetinc AIPetinc (Figure (Fig4b).ure The 4b). small The reduction small reduction in cell viabilityin cell viability (7–16%) (7–16%) obtained obtained for these for fractionsthese fractions cannot cannot account account for the substantialfor the substantial inhibition inhibition of inflammatory of inflammatory response (> response90%) observed (> 90%) in Figureobserved3. As in such,Figure the 3. As inhibition such, th ofe inhibition LPS-stimulated of LPS-stimulated TNF- α response TNF-α from response RAW264.7 from RAW264.7 cells cannot cells be attributedcannot be toattributed the mild to cytotoxic the mild e cytotoxicffects of AIPetinc. effects of AIPetinc.

2

(a) (b)

Figure 4. MTT assay demonstrating mild toxicity of AIPetinc on RAW264.7 cells. RAW264.7 cells plated Figure 4. MTT assay demonstrating mild toxicity of AIPetinc on RAW264.7 cells. RAW264.7 cells at 10,000 cells/well (a) and at 100,000 cells/well (b) were incubated with crude (E4) and semi-purified extractsplated at (post-LH20 10,000 cells/well and post-LH20-DEAE) (a) and at 100,000 ofcells/wellE. tinctorium (b) werefor sixincubated hours before with crude the MTT (E4) assayand semi- was performedpurified extracts to determine (post-LH20 cell and viability. post-LH20-DEAE) Error bars are of E. S.D., tinctorium obtained for fromsix hours two before biological the MTT replicates assay (wasn = 2).performed to determine cell viability. Error bars are S.D., obtained from two biological replicates (n = 2). To further examine the anti-inflammatory response of AIPetinc in RAW264.7 cells, we performed an experiment,To further where examine RAW264.7 the anti-inflammatory cells were pre-incubated response with of theAIPetinc extracts in before RAW264.7 exposure cells, to LPS.we Theperformed rationale an was experiment, to test whether where AIPetinc RAW264.7 is similar cells to polymyxinwere pre-incubated B (PMB) that with binds the to LPS.extracts As shownbefore inexposure Figure5 toa, theLPS. TNF- The αrationaleresponse was indeed to test was whethe not changedr AIPetinc when is similar the cells to werepolymyxin pre-incubated B (PMB) with that PMB,binds washed,to LPS. As and shown then stimulatedin Figure 5a, with the LPS. TNF- However,α response when indeed PMB was was not co-incubated changed when with the LPS, cells a substantialwere pre-incubated decrease with in TNF- PMB,α responsewashed, and was then noted stimulated (Figure5 b).with This LPS. is However, because PMB when is PMB required was toco- bindincubated to LPS with extracellularly LPS, a substantial to prevent decrease LPS in binding TNF-α to response the Toll-Like was noted Receptor (Figure 4. Similarly, 5b). This is the because crude extractPMB is (E4) required and semi-purified to bind to LPS fraction extracellularly (post-LH20) to containing prevent LPS AIPetinc binding also to had the no Toll-Like effect on Receptor the TNF- α4. responseSimilarly, to the LPS, crude when extract they (E4) were and pre-incubated semi-purified with fraction the RAW264.7 (post-LH20) cells contai (Figurening5a), AIPetinc suggesting also that had theno actioneffect on of AIPetincthe TNF- alsoα response acts like to PMB LPS, extracellularly when they were to inhibit pre-incubated LPS binding with to itsthe receptor. RAW264.7 Since cells it can(Figure be viewed 5a), suggesting that all polysaccharides that the action haveof AIPetinc the same also potential acts like to PMB neutralize extracellularly LPS extracellularly, to inhibit LPS we includedbinding to another its receptor. polysaccharide Since it fromcan be a diviewedfferent mushroomthat all polysaccharides species, Phaeolepiota have the aurea same, for comparison.potential to Asneutralize shown inLPS Figure extracellularly,5b, ISPaurea, we which included is a polysaccharide another polysaccharide with glucose from as a the different major component mushroom similarspecies, to Phaeolepiota AIPetinc (see aurea below),, for does comparison. not inhibitLPS-stimulated As shown in TNF- Figureα response. 5b, ISPaurea, Thus, although which manyis a polysaccharide with glucose as the major component similar to AIPetinc (see below), does not inhibit LPS-stimulated TNF-α response. Thus, although many polysaccharides have been reported to have

Molecules 2019, 24, 3509 7 of 15 Molecules 2019, 24, x 7 of 15 polysaccharidesanti-inflammatory have activity, been reported their action to have cannot anti-inflammatory be simply attributed activity, to their the actiongeneral cannot structure be simply of the attributedpolysaccharide to the as general a β-glucan. structure of the polysaccharide as a β-glucan.

(a) (b)

Figure 5. The inhibitory effect of AIPetinc on LPS-stimulated TNF-α response requires co-incubation Figure 5. The inhibitory effect of AIPetinc on LPS-stimulated TNF-α response requires co-incubation with LPS. (a) LPS-stimulated TNF-α production in RAW264.7 cells with pre-incubation of mushroom extractswith LPS. isolated (a) LPS-stimulated from P. aurea TNF-(ISPaurea)α production or E. tinctoriumin RAW264.7crude cells extract with pre-incubation (E4) and the semi-purified of mushroom fractionextracts post-LH20.isolated from After P. aurea incubation (ISPaurea) for six or hours E. tinctorium with the crude indicated extract extracts (E4) andand controls,the semi-purified the cells werefraction washed post-LH20. two times After before incubati LPSon was for added six hours for a furtherwith the six indicate hours incubation.d extracts and TNF- controls,α production the cells in thewere medium washed was two then times measured. before LPS (b) LPS-stimulatedwas added for TNF-a furtherα production six hours inincubation. RAW264.7 TNF- cellsα co-incubated production within the mushroom medium was extracts. then Errormeasured. bars are(b) S.D.,LPS-stimulated obtained from TNF- threeα production biological replicatesin RAW264.7 (n = cells3), and co- incubated with mushroom extracts. Error bars are S.D., obtained from three biological replicates (n = one-way ANOVA was performed as statistical analysis. * denotes p < 0.05 as compared to H2O control using3), and Sidak’s one-way multiple ANOVA comparison was performed test. as statistical analysis. * denotes p < 0.05 as compared to H2O control using Sidak’s multiple comparison test. To further assess the anti-inflammatory potential of AIPetinc, we determined its effect on nitric oxideTo (NO) further production. assess the NO, anti-inflammatory a critical biomarker potential for of inflammation, AIPetinc, we playsdetermined a major its role effect in on chronic nitric inflammationoxide (NO) production. and carcinogenesis NO, a critical [16,19 biomarker]. It is produced for inflammation, by inducible plays nitric a oxidemajor synthaserole in chronic upon inductioninflammation by the and transcription carcinogenesis factor [16,19]. NF-κβ Itthat is isproduced a downstream by inducible target of nitric LPS and oxide TNF- synthaseα. As shown upon ininduction Figure6 a,by 1 the mg transcription/mL of AIPetinc factor significantly NF-κβ that inhibited is a downstream NO production target of in LPS response and TNF- to LPS,α. As further shown confirmingin Figure 6a, the 1 anti-inflammatorymg/mL of AIPetinc activity significantly of AIPetinc. inhibited NO production in response to LPS, further confirmingHistamine, the anti-inflammatory a commonly usedinflammation activity of AIPetinc. inducer, was dose-dependently assessed for its ability to induceHistamine, TNF-α aproduction commonly in used RAW264.7 inflammation cells. At indu 1 µM,cer, histamine was dose-dependently was found to stronglyassessed inducefor its TNF-abilityα productionto induce TNF- (dataα not production shown).Figure in RAW264.76b shows cells. that indeed,At 1 μM, at 1histamine mg /mL, thewas 5% found NaOH to AIPetincstrongly extractinduce couldTNF-α significantly production (data inhibit not histamine-induced shown). Figure 6b TNF-showsα thatproduction. indeed, at The 1 mg/mL,in vitro theresults 5% NaOH using RAW264.7AIPetinc extract cells suggestcould significantly that AIPetinc inhibit behaves histamine-induced similarly to PMB, TNF- anα production. antibiotic that The binds in vitro to LPSresults to neutralizeusing RAW264.7 it from cells stimulating suggest anthat innate AIPetinc immune behaves response. similarly It remainsto PMB, toan be antibiotic tested whether that binds AIPetinc to LPS hasto neutralize bactericidal it from effects stimulating by binding an to innate LPS on immune bacterial response. cell walls. It remains to be tested whether AIPetinc has bactericidal effects by binding to LPS on bacterial cell walls.

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Figure 6. In vitro anti-inflammatory activity of AIPetinc. (a) At 1 mg/mL, AIPetinc inhibited LPS-induced nitric acid production in RAW264.7 macrophage cells. PMB was used as a positive Figure 6. In vitro anti-inflammatory activity of AIPetinc. (a) At 1 mg/mL, AIPetinc inhibited LPS- control for anti-inflammatory response. Purified sample inhibited 98% of stimulated nitric oxide induced nitric acid production in RAW264.7 macrophage cells. PMB≥ was used as a positive control (NO). (b) At 1 mg/mL, 5% NaOH extract containing AIPetinc inhibited histamine-induced TNF-α for anti-inflammatory response. Purified sample inhibited ≥ 98% of stimulated nitric oxide (NO). (b) production in RAW264.7 macrophage cells. The histamine concentration used is 10 6 M. Error bars are At 1 mg/mL, 5% NaOH extract containing AIPetinc inhibited histamine-induced− TNF-α production S.D., data obtained from three biological replicates (n = 3), and one-way ANOVA was performed as in RAW264.7 macrophage cells. The histamine concentration used is 10−6 M. Error bars are S.D., data statistical analysis. * denotes p < 0.05 as compared to DMEM using Sidak’s multiple comparison test. obtained from three biological replicates (n = 3), and one-way ANOVA was performed as statistical 2.5. AIPetincanalysis. Blocked * denotes Histamine-Induced p < 0.05 as compared Inflammatory to DMEM using Event Sidak’s in Mice multiple Microcirculation comparison test.

2.5. AIPetincInflammation Blocked is aHistamine-Induced multi-step physiological Inflammatory response Event that in begins Mice Microcirculation with the stimulation of the immune system to release cytokines leading to localized changes in the vasculature, including vasodilation and increasedInflammation permeability is a multi-step of the blood physiological vessel [20– 22response]. Results that obtained begins from with RAW264.7 the stimulation cells suggest of the thatimmune AIPetinc system is capable to release of inhibiting cytokines the leading release to of localized cytokine changes from macrophages, in the vasculature, an early including event of anvasodilation inflammatory and response.increased permeabi To furtherlity investigate of the blood the vessel anti-inflammatory [20–22]. Results potential obtained of from AIPetinc, RAW264.7 we usedcells ansuggestin vivo thatintravital AIPetinc microscopy is capable of mouse inhibiting model the systemrelease toof cy studytokine acetylcholine from macrop (ACh)-inducedhages, an early conductedevent of an vasodilation inflammatory in the response. microcirculation To further as a measureinvestigate of vasculaturethe anti-inflammatory integrity, a latepotential event inof inflammation.AIPetinc, we used Previous an in studies vivo intravital have shown micros thatcopy histamine, mouse a model strong inflammationsystem to study mediator acetylcholine in the blood(ACh)-induced vessel, can conducted inhibit ACh-induced vasodilation vasodilation in the microcir at aculation distance as from a measure the stimulation of vasculature site, attributed integrity, toa histamine’slate event in inhibitory inflammation. effect onPrevious cell-to-cell studies coupling have shown through that a mechanism histamine, whicha strong is dependent inflammation on NOmediator production in the [ 22blood]. Figure vessel,7a can shows inhibit that ACh-induced the local diameter vasodilation change at at thea distance site of delivery from the was stimulation almost identicalsite, attributed in the secondto histamine’s order (2A) inhibitory arterioles effect under on cell-to-cell the different coupling treatment through groups, a mechanism whereas Figure which7b is demonstratesdependent on that NO histamine production decreased [22]. Figure the vasomotor 7a shows response that the tolocal ACh diameter at 500 µ mchange upstream at the from site the of sitedelivery of ACh was treatment. almost Thisidentical lack of in change the second at the “local”order site(2A) indicates arterioles the under arterioles the aredifferent intact and treatment viable, implyinggroups, whereas a decrease Figure in cellular 7b demonstrates communication that hist (lossamine of decreased vascular integrity the vasomotor via inflammation) response to ACh along at the500 arterioles. μm upstream The from change the in site diameter of ACh droppedtreatment. from This 10 lack0.3 of µchangem in the at controlthe “local” state site to indicates6 0.06 µ them ± ± inarterioles the inflammatory are intact stateand inducedviable, implying by histamine. a decrease The addition in cellular of AIPetinc communication reversed (loss the responseof vascular to histamine,integrity via suggesting inflammation) cellular along communication the arterioles. was The restoredchange in by diameter AIPetinc. dropped Results from strongly 10 ± 0.3 suggest μm in thatthe AIPetinccontrol state has anti-inflammatoryto 6 ± 0.06 μm in the activity inflammatory in the microcirculation state induced to by restore histamine. vasculature The addition integrity, of possiblyAIPetinc due reversed to the abilitythe response of AIPetinc to histamine, to reduce NOsuggesting production. cellular This, communication however, needs was to be restored confirmed by byAIPetinc. further experimentation.Results strongly Thesuggest study onthat AIPetinc AIPetinc and has our previousanti-inflammatory study on extractsactivity from in thethe fungusmicrocirculationInonotus obliquus to restore[23 ]vasculature demonstrate integrity, that inhibition possibly of due LPS-induced to the ability TNF- ofα AIPetincproduction to reduce in mouse NO macrophageproduction. RAW264.7This, however, cells isneeds a good to be biological confirmed system by further to study experime the anti-inflammatoryntation. The study activity on AIPetincin vitro. Furthermore,and our previous the mouse study microcirculationon extracts from the established fungus Inonotus here could obliquus also be[23] used demonstrate as a model that to studyinhibition the inof vivo LPS-induced action of anti-inflammatoryTNF-α production compoundsin mouse macrophage from medicinal RAW264 fungi.7 cells in the is vasculature.a good biological system to study the anti-inflammatory activity in vitro. Furthermore, the mouse microcirculation established here could also be used as a model to study the in vivo action of anti-inflammatory compounds from medicinal fungi in the vasculature.

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Figure 7. In vivo anti-inflammatory activity of AIPetinc. (a) Local response to conducted vasodilation Figure 7. In vivo anti-inflammatory activity of AIPetinc. (a) Local response to conducted vasodilation in intact arterioles of C57BL6 mice (n = 10). Responses to acetylcholine (Ach) (1psi, 500 ms) were in intact arterioles of C57BL6 mice (n = 10). Responses to acetylcholine (Ach) (1psi, 500 ms) were recorded at the site of stimulation (local: distance = 0). There was no significant difference in the local recorded at the site of stimulation (local: distance = 0). There was no significant difference in the local response in arterioles of C57BL6 mice under different treatment. (b) Responses to ACh (1psi, 500 ms) response in arterioles of C57BL6 mice under different treatment. (b) Responses to ACh (1psi, 500 ms) were recorded upstream (with respect to blood flow) at a distance of 500 µm (conducted response). were recorded upstream (with respect to blood flow) at a distance of 500 μm (conducted response). At each site, change in diameter was calculated as peak response diameter minus resting diameter. At each site, change in diameter was calculated as peak response diameter minus resting diameter. There is a significant difference between conducted response of histamine vs. histamine + AIPetinc There is a significant difference between conducted response of histamine vs. histamine + AIPetinc indicating the anti-inflammatory potential of the molecule. Error bars represents mean S.D. One-way indicating the anti-inflammatory potential of the molecule. Error bars represents mean± ± S.D. One- ANOVA was performed as described in methods. * denotes p < 0.05 as compared to control. way ANOVA was performed as described in methods. * denotes p < 0.05 as compared to control. 2.6. Chemical Analysis of the Anti-Inflammatory Polysaccharide from E. tinctoirum 2.6. Chemical Analysis of the Anti-Inflammatory Polysaccharide from E. tinctoirum The monosaccharide composition of AIPetinc was determined by Gas Chromatography (GC). For comparison,The monosaccharide we also performed composition parallel of AIPetinc experiments was determined on the well-known by Gas Chromatography polysaccharide-protein (GC). complexFor comparison, isolated fromwe alsoTrametes performed versicolor parallel, polysaccharide-K experiments on [24 the]. Table well-known1 shows thatpolysaccharide-protein AIPetinc consisted mainlycomplex of isolated glucose (88.6%).from Trametes Other minor versicolor monosaccharides, polysaccharide-K present [24]. included Table galactose 1 shows (4.0%), that mannoseAIPetinc (4.4%),consisted fucose mainly (0.7%), of glucose and xylose (88.6% (2.3%).). Other This minor is diff monosaccharideserent from polysaccharide-K, present included which galactose has a relatively (4.0%), highermannose amounts (4.4%), of fucose mannose (0.7%), (14.0%) and andxylose galactose (2.3%). (7.1%), This is and different from afrom growth-inhibitory polysaccharide-K, polysaccharide which has recentlya relatively isolated higher from amountsPaxillus of mannose involutus ,(14.0%) which and has galactose a relatively (7.1%), high and amount from ofa growth-inhibitory galactose (20.8%) (Tablepolysaccharide1)[ 25]. A complementaryrecently isolated approach, from Paxillus the β–glucan involutu assays, which kit, washas alsoa relatively used to assess high theamount amount of ofgalactoseα– and (20.8%)β–glucans (Table present 1) [25]. in AIPetinc.A complementary Results, summarized approach, the from β–glucan two separate assay kit, experiments, was also used show to thatassess the the total amount glucan of in α AIPetinc– and β–glucans has 93.42% presentβ–glucan in AIPetinc. and 6.57% Resuα–glucan.lts, summarized Overall, ourfrom results two separate suggest thatexperiments, AIPetinc belongsshow that to thethe family total ofglucanβ–glucans in AIPetinc with anti-inflammatory has 93.42% β–glucan activity and [26 6.57%,27]. α–glucan. Overall, our results suggest that AIPetinc belongs to the family of β–glucans with anti-inflammatory activityTable [26,27]. 1. Monosaccharide composition of purified anti-inflammatory polysaccharide from E. tinctorium (AIPetinc). Table 1. Monosaccharide composition of purified anti-inflammatory polysaccharide from E. Anti-Inflammatory Growth-Inhibitory Monosaccharidetinctorium (AIPetinc). Polysaccharide from E. Polysaccharide from P. Polysaccharide-K [24] Composition (%) Anti-Inflammatorytinctorium involutusGrowth-Inhibitory[25] Monosaccharide Polysaccharide-K Polysaccharide from E. Polysaccharide from P. CompositionGlucose (%) 88.6 65.9 74.4 [24] Galactosetinctorium 4.0 20.8involutus [25] 7.1 GlucoseMannose 88.6 4.4 7.8 65.9 14.0 74.4 GalactoseFucose 4.0 0.7 3.2 20.8 1.6 7.1 MannoseXylose 4.4 2.3 2.3 7.8 2.7 14.0 FucoseRibose 0.7 - - 3.2 0.2 1.6 Xylose 2.3 2.3 2.7 Ribose - - 0.2

Molecules 2019, 24, 3509 10 of 15

3. Materials and Methods

3.1. Materials and Chemicals Echinodontium tinctorium was collected from three separate locations in north-central BC: an old growth forest near McBride (voucher not kept); Twin Falls Recreation Site, Smithers (CL37); and Pine Lake Recreation Site, Terrace (CL103). DEAE Sephadex, dextran standards (T1, T5, T12, T25, T50, T80, T150, T270, T410), and the standard monosaccharides (glucose, mannose, rhamnose, ribose, xylose, arabinose, fucose, galactose, galactosamine, and glucosamine) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum was from Life Technologies Inc. (Waltham, MA, USA), and Dulbecco Modified Eagle Medium was from LONZA (Walkersville, MD, USA). Sephadex LH-20 resin and pre-packed Superdex 200 Increase 10/300 GL were from GE Healthcare (Uppsala, Sweden). Polysaccharide-K was purchased from Kureha Pharmaceuticals (Tokyo, Japan). All other reagents used were of analytical grade.

3.2. Identification of Mushroom Species The mushroom collections were initially identified as Echinodontium tinctorium based on morphological characteristics [3,6]. Two (CL37 and CL103) of the three collections were confirmed by DNA sequencing. Small tissue samples were aseptically collected from fungal sporocarps and dried. DNA was extracted using a PowerSoil DNA isolation kit according to the manufacturer’s protocol. Extracted DNA samples underwent polymerase chain reaction (PCR) amplification as described previously [1,2] using primers ITS3 (50-GCATCGATGAAGAACGCA-30) and NLB4 (50-GGATTCTCACCCTCTATGA-30). DNA sequences of the Internal Transcribed Spacer 2 (ITS2) and the 50 end of the D1-D2 region of the 28S gene were aligned and edited using CLC Main Workbench (Qiagen, Carlsbad, CA, USA), then entered into the Basic Local Alignment Search Tool (BLAST) to determine closest sequence identity in GenBank.

3.3. Extraction and Purification of Anti-Inflammatory Polysaccharide The fungal specimens were dried and then crushed into powder form using a Hammer mill. The material was sequentially fractionated into four extracts as previously described [1,2]; this is an extension of a well-established protocol for extraction of anti-tumor polysaccharides [18]. Typically, about 1 g of powdered specimen was extracted with 10 mL of 80% ethanol for 3 h at 65 ◦C. The solution, filtered through Whatman paper No.3, was then referred to as Extract 1, while the residue was subjected to the second step: 50% methanol extraction for 3 h at 65 ◦C. The filtrate (Extract 2) was kept, while the residue was subjected to the third step: water extraction for 6 h at 65 ◦C. The filtrate from the water extraction step was referred to as Extract 3. Finally, this residue was subjected to the last step: 5% sodium hydroxide (NaOH) extraction at 65 ◦C for 6 h. The filtered solution from this step was referred to as Extract 4. Initially, 2% ammonium oxalate extraction was also performed at the fourth step, but it was later omitted because of the incompatibility of oxalate with the medium used for growing RAW264.7 cell lines. In the initial trials, extract 4 represents 2% ammonium oxalate, and extract 5 represents the 5% NaOH fraction. A subsequent collection of Echinodontium tinctorium was extracted without the ammonium oxalate step. All extracts were subjected to roto-evaporation followed by lyophilization. All lyophilized extracts were reconstituted in water at 2 mg/mL and filter-sterilized using a 0.2 µm filter (Sarstedt, QC, Canada) before they were assessed for immuno-stimulatory or anti-inflammatory activities, as described below. Some lyophilized extracts were reconstituted in methanol to solubilize the extract as indicated. All extracts were tested for their ability to stimulate TNF-α in RAW264.7 cells. Extracts negative for the immuno-stimulatory activity were then assessed for their ability to inhibit LPS-induced TNF- α as a measure of their anti-inflammatory potential, as described previously [1,2]. The crude alkaline extract (NaOH extract) with anti-inflammatory activity was suspended in water and centrifuged at 100 g for 5 min to remove any insoluble particulate. Two percent (or 1.12 mL supernatant) was loaded × Molecules 2019, 24, 3509 11 of 15 onto a size exclusion, Sephadex LH-20 column (16 mm 70 cm), and run at a flow rate of 1.0 mL/min. × Thirty-five 2 mL fractions were collected and assessed for anti-inflammatory activity by ELISA, as described in Section 3.6. Carbohydrate and protein contents in the fractions were determined using the phenol-sulfuric acid method [28] and Pierce BCA protein assay kit (Waltham, MA, USA), respectively. Fractions with potent activity were pooled, lyophilized, resuspended in water and loaded onto DEAE Sephadex in XK-50 column (50 mm 100 cm) equilibrated with 20 mM piperazine buffer pH 5.5, and × ran at a flow rate of 1.0 mL/min. After washing the column with the same buffer, the column was eluted with two bed volumes (2 L) containing 1 M NaCl. The collected eluent was lyophilized, dialyzed, re-lyophilized, and reconstituted in water at a concentration of 2 mg/mL before loading 500 µL onto a Superdex 200 larger column (1.0 30 cm), which was run at a flow rate of 1 mL/min. The bioactive × eluent collected in fractions from Superdex 200 was pooled, lyophilized, and designated as AIPetinc.

3.4. Monosaccharide Composition Analysis The monosaccharide composition of AIPetinc was determined by GC analysis. Briefly, samples were subjected to sequential acid hydrolysis (12 M sulfuric acid for 1 h at 35 ◦C, and then 2 M sulfuric acid for 1 h in a boiling water bath). Alditol acetates of the neutral sugars in the acid hydrolysate were prepared according to the previously described method [29] with β-d-allose as the internal standard. Alditol acetates of the monosaccharides were quantified by an HP6890 series II gas chromatography, using an Alltech DB-225 capillary column (15 m 0.25 mm i.d., 0.25 µm film, (Analytical Columns, New × Addington, Croydon, England) with the following oven temperature program: initial temperature, 170 ◦C; temperature rise at 2 ◦C/min to 220 ◦C and final hold for 15 min. The carrier gas was helium, and detection was made by flame ionization.

3.5. Determining the Percentage of α- and β-Glucans in AIPetinc The β–glucan content of AIPetinc was determined using β–Glucan Assay Kit (Megazyme, Ireland) with slight modification. Ten milligrams of AIPetinc and the yeast positive control were first subjected to total glucan determination as according to the manufacturer’s instructions. Two hundred microliters of ice cold 12 M sulfuric acid were added and the mixtures were incubated for 2 h in an ice-water bath with occasional mixing. Four hundred microliters of MilliQ water (Millipore Canada, Etobicoke, ON, Canada) were added and mixed for 10 s, followed by the addition of 600 µL of MilliQ water and mixing and incubation at 100 ◦C for 2 h. After cooling to room temperature, 100 µL of mixtures were transferred to a 10 mL volumetric flask, and the remaining mixtures in the vial were rinsed with 5 mL 200 mM sodium acetate buffer (pH 5) prior to transferring to the 10 mL volumetric flask. Six hundred microliters of 10 M KOH solution were pipetted to the volumetric flask, and the volume was adjusted with 200 mM sodium acetate buffer (pH 5). The contents were mixed and transferred to a 15 mL tube and centrifuged at 1500 g for 10 min. A total of 10 µL of the supernatant (in duplicates) × of the centrifuged extract were transferred to a 1.5 mL tube, followed by the addition of 10 µL of a mixture of exo-1,3-β-glucanase (20 U/mL) plus β-glucosidase (4 U/mL) in 200 mM sodium acetate buffer (pH 5.0). The same procedure was performed with a positive control (glucose) and negative control (sodium acetate buffer), with a total of 10 µL each of the 1 mg/mL Glucose Standard solution or 200 mM sodium acetate buffer (pH 5). The resulting solution was then gently vortexed and incubated at 40 ◦C for 60 min. After one hour, 300 µL of GOPOD reagent was transferred into each tube, followed by incubation at 40 ◦C for 20 min. Upon completion, 100 µL of the solution was transferred into a 96-well UV plate and the absorbance of all solutions (AIPetinc, yeast control, negative control, and glucose control) was measured at 510 nm against the reagent blank using Synergy 2 multi-plate reader (Bio-Tek, Winooski, VT, USA). For determination of the amount of α–glucan in samples, approximately 10 mg of AIPetinc and the yeast positive control were mixed with 200 µL of 2 M KOH solution. The vials were then capped and hand-shaken on an ice-water bath for 20 min, followed by the addition of 800 µL of 1.2 M sodium acetate buffer (pH 3.8) and 20 µL of amyloglucosidase (1.630 U/mL) plus invertase (500 U/mL). Molecules 2019, 24, 3509 12 of 15

The vials were then incubated at 40 ◦C for 30 min with occasional mixing. Subsequently, the contents were transferred into a 1.5 mL tube and centrifuged at 1500 g for 10 min. A total of 10 µL of the × supernatant (in duplicates) was transferred to a separate 1.5 mL tube, followed with the addition of 10 µL of sodium acetate buffer (200 mM, pH 5.0) plus 300 µL of GOPOD reagent and incubated at 40 ◦C for 20 min. The same procedure was performed with a positive control (glucose) and negative control (sodium acetate buffer), with a total of 10 µL each of the 1 mg/mL Glucose Standard solution or 200 mM sodium acetate buffer (pH 5), respectively, mixed with 300 µL of GOPOD reagent. Upon completion, 100 µL of the solution was transferred into a 96-well UV plate and the absorbance of all solutions (AIPetinc, yeast control, negative control, and glucose control) was measured at 510 nm against the reagent blank. The total glucan and α–glucan in % w/w were calculated according to the manufacturer’s guidelines. The amount of β-glucan was determined by the following simple equation: β-glucan (% w/w) = Total glucan (% w/w) α–glucan (% w/w). − 3.6. Cell Line and Assessment for Immuno-Stimulatory Activity RAW264.7 mouse macrophage cell line was purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco Modified Eagle Medium. Cells were grown in media supplemented with 10% fetal bovine serum in a humidified incubator at 37 ◦C supplied with 5% carbon dioxide. To assess immuno-stimulatory activity, we measured TNF-α production in RAW264.7 cells upon treatment with 1 mg/mL fungal extracts for 6 h. Fifty µL of medium was removed and stored at 80 C until TNF-α content determination using enzyme-linked immunosorbent assay (ELISA), as − ◦ previously described [1,2].

3.7. Anti-Inflammatory Activity In Vitro To assess anti-inflammatory activity, RAW264.7 cells were induced with 250 ng/mL of lipopolysaccharide (LPS) in the presence or absence of 1 mg/mL of fungal extracts or purified samples for 6 h. Polymyxin B (PMB) (100 units, Sigma) was used as a positive control based on its ability to inhibit LPS [1,2,30,31]. The measurement of TNF-α production was determined using ELISA, as described in Section 3.6. Statistical analysis was performed using one-way ANOVA. Sidak’s or Dunnett’s multiple comparisons test was done as post-hoc analysis once the null hypothesis was rejected.

3.8. Nitric Oxide Determination Assay As nitric oxide (NO) is a short-lived chemical in in vitro settings, for the estimation of inhibition of NO, nitrite levels were measured as representative of NO. The assay absolutely requires the use of RAW264.7 macrophage cells that have not been passaged (divided) extensively. Cells were plated at a density of 100,000 cells/well as described previously. After an overnight incubation, cells were treated with: DMEM alone, 1 µg of LPS, 1 µg of LPS + PMB (100 units), 1 µg of LPS + AIPetinc (1 mg). After 24 h incubation, supernatants were collected and assessed for NO content using PromegaTM Griess Reagent System (Promega Corporation, Madison, WI, USA). The protocol was followed as indicated by the supplier with a slight modification. Briefly, NO standards were prepared via serial dilution from stock (0.1 M Sodium Nitrite). One hundred µL of samples were added to each well, followed by 50 µL of Sulphanilamide solution and 50 µL of N-1-napthylethylenediamine dihydrochloride solution. The plates were read at 540 nm, and the amount of NO was quantified by interpolating the serial dilution of nitrite (NaNO2) levels using PRISM software (version 7, GraphPad Software, San Diego, CA, USA).

3.9. Intravital Microscopy and Measurement of Conducted Vasodilation All procedures were approved by the Institutional Animal Care and Use Committee of the University of Northern British Columbia (2016-9 Payne). Male black mice C57BL6 (average weight ~22–30 g and 8–20 weeks of age, n = 10) were purchased from Jackson Laboratories, Bar Harbor, Molecules 2019, 24, 3509 13 of 15

ME, USA. Mice were housed at ~ 24 ◦C on a 12:12-h light dark cycle with free access to food and water. The surgery and gluteus muscle preparation of black C57BL6 mice have been previously described [23]. Briefly, microcirculation in a mouse gluteus maximus muscle was exposed with precise technique. Vessel was equilibrated with physiological saline solution (PSS) to simulate in vivo conditions and vascular integrity was tested prior to the experiment to avoid any experiment-induced inflammatory event. One second order arteriole (2A) with preserved vascular tone was selected as a test vessel. Resting internal diameter was measured under control conditions (PSS). To evaluate conducted vasodilation response under PSS, the vasomotor response to exogenous Acetylcholine (ACh) was measured at the site of delivery (local) and 500 µm upstream from the site of stimulation (conducted). After measuring the local and conducted response under controlled conditions, the 5 preparation was equilibrated with histamine (10− M) for 30 min. The internal diameter was measured as the effect of histamine on local and conducted vasodilation. After recording the effect of histamine, the preparation was super-fused separately with AIPetinc (n = 5) and the added histamine so that the final concentration was 12.5 µg/mL. We have previously established that at 12.5 µg/mL, the reference methanolic extracts of Inonotus obliquus had minimal effects on the vessels [23]. The concentration of the AIPetinc was selected on this same criterion (i.e., the dose at which there was negligible effect on the diameter of 2A arteriole under resting conditions). This mixture (histamine + AIPetinc) was allowed to run on the preparation for 30 min, and the local and conducted response was measured in the same way as described previously while the solution was still running on the muscle preparation. The response to ACh was calculated as the diameter change (i.e., peak response diameters minus the resting diameters). Data was analyzed using One-way ANOVA (PRISM) and is represented as means S.D. Value of n refers to the number of arterioles studied. Differences among groups is statistically ± significant when p < 0.05.

4. Conclusions This is the first description of immuno-modulatory activities of wild fungal extracts of Echinodontium tinctorium collected in north-central BC, Canada. We showed that the 5% NaOH extract from E. tinctorium has potent anti-inflammatory activity. Using Sephadex LH-20, DEAE Sephadex, and Superdex 200, we successfully isolated a 5 kDa anti-inflammatory polysaccharide (AIPetinc). AIPetinc is composed predominantly of β-glucan with small amounts of heteroglycans containing galactose, mannose, fucose, and xylose. The polysaccharide inhibited LPS and histamine-induced TNF-α production, and LPS-induced NO production in RAW264.7 mouse macrophage. It also restored the decreased conducted vasodilation induced by histamine in mouse gluteus maximus muscle, further supporting the anti-inflammatory activity of AIPetinc in vivo. Further mechanistic study is required to identify the mechanism of action and the molecular pathways affected as a result of the anti-inflammatory response.

Author Contributions: Conceptualization, S.J., W.M.L, and C.H.L. Methodology, S.J., W.M.L., A.Y., M.Z., and C.H.L; formal analysis, S.J., M.Z., W.M.L., and C.H.L.; resources, H.B.M., K.N.E., and L.E.T.; data curation, S.J. and W.M.L.; writing–original draft preparation, S.J. and C.H.L.; writing–review and editing, S.J., W.M.L., K.N.E., P.C.K.C., G.W.P., and C.H.L.; supervision, C.H.L., P.C.K.C., and G.W.P.; project administration, C.H.L. funding acquisition, C.H.L. and P.C.K.C. Funding: This work was supported in part by an NSERC Discovery Grant (Grant # 227158), a UNBC Seed Grant, a UNBC Research Project Award, a Canada Foundation for Innovation Grant (# 34711), a BC Knowledge Development Fund Grant and Hong Kong RGC Research Grant General Research Fund (# 463213). S. Javed was recipient of a studentship from the Centre for Blood Research, University of British Columbia. Acknowledgments: We thank Sebastian Mackedenski for performing the Superdex 200 chromatography and Kathy Lewis for pointing out critical reference sources. Conflicts of Interest: The authors declare no conflict of interest. Molecules 2019, 24, 3509 14 of 15

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Sample Availability: Not available.

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