Stability and Photoisomerization of Stilbenes Isolated from the Bark of Norway Spruce Roots

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Article Stability and Photoisomerization of Stilbenes Isolated from the Bark of Norway Spruce Roots

Harri Latva-Mäenpää 1,2,* , Riziwanguli Wufu 1 , Daniel Mulat 1, Tytti Sarjala 3, Pekka Saranpää 3,* and Kristiina Wähälä 1,4,*

1 Department of Chemistry, University of Helsinki, P.O. Box 55, FI-00014 Helsinki, Finland; riziwanguli.wufu@helsinki.fi (R.W.); [email protected] (D.M.) 2 Foodwest, Kärryväylä 4, FI-60100 Seinäjoki, Finland 3 Natural Resources Institute Finland, Tietotie 2, FI-02150 Espoo, Finland; tytti.sarjala@luke.fi 4 Department of Biochemistry and Developmental Biology, University of Helsinki, P.O. Box 63, FI-00014 Helsinki, Finland * Correspondence: harri.latva-maenpaa@foodwest.fi (H.L.-M.); pekka.saranpaa@luke.fi (P.S.); kristiina.wahala@helsinki.fi (K.W.); Tel.: +358-50-4487502 (H.L.-M. & P.S. & K.W.)

Abstract: Stilbenes or stilbenoids, major polyphenolic compounds of the bark of Norway spruce (Picea abies L. Karst), have potential future applications as drugs, preservatives and other functional ingredients due to their antioxidative, antibacterial and antifungal properties. Stilbenes are photosensitive and UV and fluorescent light induce trans to cis isomerisation via intramolecular cyclization. So far, the characterizations of possible new compounds derived from trans-stilbenes under UV light exposure have been mainly tentative based only on UV or MS spectra without utilizing more detailed structural spectroscopy techniques such as NMR. The objective of this work was to study the stability Citation: Latva-Mäenpää, H.; Wufu, of biologically interesting and readily available stilbenes such as astringin and isorhapontin and R.; Mulat, D.; Sarjala, T.; Saranpää, P.; their aglucones piceatannol and isorhapontigenin, which have not been studied previously. The Wähälä, K. Stability and effects of fluorescent and UV light and storage on the stability of trans stilbenes were assessed and the Photoisomerization of Stilbenes identification and characterisation of new compounds formed during our experiments were carried Isolated from the Bark of Norway out by chromatographic (HPLC, GC) and spectroscopic techniques (UV, MS, NMR). The stilbenes Spruce Roots. Molecules 2021, 26, 1036. https://doi.org/10.3390/ undergo a trans to cis isomerisation under extended UV irradiation by intramolecular cyclisation (by molecules26041036 the formation of a new C-C bond and the loss of two hydrogens) to phenanthrene structures. The characterised compounds are novel and not described previously. Academic Editors: Paola Di Donato and Brigida Silvestri Keywords: stilbenes; spruce bark; UV-light; fluorescent light; photoisomerization; trans to cis isomerization; phenanthrenes; astringin; piceid; isorhapontin; piceatannol; isorhapontigenin Received: 27 December 2020 Accepted: 5 February 2021 Published: 16 February 2021 1. Introduction Publisher’s Note: MDPI stays neutral Stilbenes or stilbenoids are expected to show many future applications as drugs and with regard to jurisdictional claims in functional ingredients or preservatives in food products or improving material properties. published maps and institutional affil- The best known stilbenoid compound is resveratrol, but there are also other interesting iations. stilbenoids such as astringin and isorhapontin, which are derived from forest biomass and are potential starting materials for new products. The bark of Norway spruce (Picea abies L. Karst) contains the stilbenoid glucosides astringin and isorhapontin as the major polyphenolic compounds that are even found in roots [1]. Stilbenes are secondary metabolites and Copyright: © 2021 by the authors. play an important role in the tree defence mechanism [2]. They provide protection against Licensee MDPI, Basel, Switzerland. ultraviolet (UV) light and attacks by fungal pathogens and other microorganisms [3–5] This article is an open access article and they also show potential to prevent pathologies associated with oxidative stress [6]. distributed under the terms and Stilbenoid glucosides are mainly localized to the inner part of the bark in the functional conditions of the Creative Commons phloem [7] and they can be converted to their aglucones by enzymatic hydrolysis [8]. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ Stilbenes may exist as the trans and cis stereoisomeric forms, but naturally they 4.0/). overwhelmingly occur as the trans isomers due to their higher thermodynamical stability

Molecules 2021, 26, 1036. https://doi.org/10.3390/molecules26041036 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1036 2 of 15

compared to the cis isomers [9]. However, trans-stilbenes may be isomerized to the cis form under certain conditions such as irradiation with light. Recently, Välimaa and co- authors [10] showed that UVA-induced modification of the stilbene-rich inner bark extracts increased their radical scavenging activity. The results even indicated a slightly increased antimicrobial activity after UVA-modification. The authors concluded that stilbenes from spruce bark may have potential as preservatives. The conflicting discoveries regarding the stability of stilbenes has raised questions on the future use of stilbenes [11–13]. According to Piñeiro et al. [11], the utilization of the beneficial effects of resveratrol is limited because it is an easily oxidizable and extremely photosensitive compound. In contrast, Prokop et al. [12] showed that piceid and its aglycone resveratrol are stable polyphenols. In solution, trans-resveratrol is converted to cis-resveratrol when exposed to daylight [14]. The same photoisomerization reaction takes place when the solution is exposed to UV light [15]. According to the authors trans- resveratrol was fully isomerized to cis-resveratrol when exposed to UV light at 366 nm. Montsko et al. [16] used HPLC-MS and -MS/MS together with UV spectroscopy and semiempirical quantum chemical calculations to examine the UV isomerization reaction of trans-resveratrol in solution under 365 nm UV light irradiation. Besides the formation of cis- resveratrol, they claimed the formation of an oxidized derivative of trans-resveratrol with a triple bond at the centre of the molecule or the formation of a phenanthrene structure. The formation of a trihydroxy phenanthrene was supported also by Tˇríska et al. [17]. Yang et al. [18] showed that the UV irradiation of trans-resveratrol leads to the formation of a highly fluorescent compound, resveratrone, which was properly characterized by UV, MS and NMR. Computational exploration by Rodríguez et al. [19] uncovered the feasibility of singlet oxygen addition to cis or trans-resveratrol to furnish a dioxetane intermediate which spontaneously cycloreverts to benzaldehydes. Recently, Francioso et al. [20] showed that upon UV-exposure resveratrol in red wine can isomerise from the trans to the cis-isomer, which may further cyclise to 2,4,6-trihydroxy-phenanthrene. The identifications of possible new compounds derived from trans stilbenes have been mainly tentative and based only on UV or MS spectra without utilizing more detailed structural spectroscopy such as NMR. In summary, the findings about the chemical stability of trans-resveratrol and trans- piceid in solution are not fully in agreement with each other. Although the cis-isomer was formed in every case when exposed to UV or fluorescent light, the formation of additional oxidized product(s) was not observed by all investigators. A clear conclusion about the stability of trans-piceid and trans-resveratrol in solution cannot be made. The stability studies of stilbenes have given contradictory results and been concen- trated mainly on resveratrol and its glucoside piceid. Thus, the objective of this work was to study the stability of other biologically interesting and readily available stilbenes such as astringin and isorhapontin and their aglucones piceatannol and isorhapontigenin, which have not been studied in detail previously. To resolve this issue, we assessed the effects of light and storage on the stability of trans stilbenes and put more effort on the identification and characterization of new compounds formed during our experiments. The isolation and analysis of the compounds derived from the trans stilbenes studied were carried out by chromatographic (HPLC, GC) and spectroscopic techniques (UV, MS, NMR).

2. Results and Discussion 2.1. The Stability of Stilbenes in Solution The stability of trans isomers of the isolated isorhapontin, astringin, piceid, piceatannol, resveratrol and isorhapontigenin in methanol as well as that of crude spruce bark extract dissolved in methanol was monitored for 2 weeks under two different conditions: (1) light protected: solutions of isolated stilbenes were stored in a freezer (−20 ◦C) with protection from light and (2) light unprotected: solutions of isolated stilbenes were placed in a laboratory hood and exposed to continuous ﬂuorescent light. Similar conditions were applied to monitor the stability of stilbene constituents in crude extract prior to isolation. Trans isomers of all the isolated stilbenes were stable for the duration of the test (2 weeks, 366 h) Molecules 2021, 26, 1036 3 of 15

at −20 ◦C if protected from light. The recovery [21] of trans isomers of five isolated stilbenes (astringin, isorhapontin, piceid, resveratrol and iso-rhapontigenin) varied in the range of 97–101% and that of trans-piceatannol 73–106%. These compounds as well as the crude extract were stable for at least two weeks in darkness at −20 ◦C. Isolated stilbenes were unstable when exposed to fluorescent light. HPLC–UV analysis of trans-resveratrol revealed that irradiation with fluorescent light reduced the trans-resveratrol peak and a new one appeared with a longer retention time [21]. HPLC-DAD, HPLC-DAD/MS and HPLC-DAD/MS/MS analyses were used for the tentative identification of the new peaks from photoisomerized isorhapontin, astringin, piceid, piceatannol, resveratrol and isorhapontigenin. UV absorption maxima of the new products at ~220 and ~280 nm are characteristic for the cis-stilbene chromophore [22]. The determined m/z of [M − H]− ion indicated molecular weights similar to the starting material. NMR analysis confirmed that trans-stilbenes (isorhapontin, isorhapontigenin, piceid and resveratrol) gave new products as cis isomers (Table1). Light exposure has been shown to cause the isomerization of trans-piceid and trans-resveratrol to cis-piceid and cis-resveratrol respectively [15,23] which is in line with our observations.

− Table 1. HPLC-DAD-MS characteristics (tR (min), UV proﬁle and [M − H] , m/z) of the new products after light exposure of stilbene solutions.

− Sample Name Products tR(min) UV Proﬁle [M − H] , m/z trans-astringin 11.8 trans-stilbene 405 trans-astringin new peak 16 λ max~260 403 trans-piceid 16.3 trans-stilbene 389 trans-piceid new peak 27.5 cis-stilbene 389 trans-isorhapontin 17.5 trans-stilbene 419 trans-isorhapontin new peak 28.3 cis-stilbene 419 trans-piceatannol 18.6 trans-stilbene 243 trans-piceatannol no peak detected trans-resveratrol 25 trans-stilbene 227 trans-resveratrol new peak 32.3 cis-stilbene 227 trans-isorhapontigenin 26.4 trans-stilbene 257 trans-isorhapontigenin new peak 33.1 cis-stilbene 257

trans-Astringin exposed to fluorescent light suffers a loss of two hydrogen atoms from the trans-astringin molecule yielding a new product (Table1) with maximum absorption at ~260 nm and molecular mass 404 Da as can be deduced from [M − H]− = 403 m/z. Trans-piceid, trans-resveratrol and trans-astringin showed the highest instability under fluorescent light exposure whereas trans-isorhapontigenin and trans-isorhapontin were more stable (Figure1). The steric hindrance due to the methoxy substituent in the structure of isorhapontin and its aglucone discourages the trans to cis isomerization. The effect of fluorescent light on the stability of isolated stilbenes can also be seen from the yield of new products (Figure2). Maximum conversion resulted from the isomerization of the trans forms of resveratrol and piceid to their corresponding cis isomers after 2 week fluorescent light exposure at room temperature. As shown in Figure2, the conversion rate is faster for the initial 36 h and becomes slower thereafter. Crude spruce bark extract solutions were not significantly destablized by exposure to fluorescent light. It is known that in addition to stilbenes, spruce bark contains phenolic compounds such as lignans, flavonoids and tannins [1,7,10] which may influence the action of light on the stilbenes. Molecules 2021, 26, x FOR PEER REVIEW 4 of 16

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100 90 100100100 100 80 909090 90 70 808080 80 60 707070 70 50 606060 60 40 505050 50 Recovery (%) 30 404040 40 Recovery (%) Recovery (%) Recovery (%) 20 303030 Recovery (%) 30 10 202020 20 0 101010 10 0 50 100 150 200 250 300 350 400 000 0 000 50 50 50 100 100 100 150 150 150 200 2000 200 250 50 250 250 300100 300 300 350150 350 350 400200 400 400 250 300 350 400 Exposure time (h) ExposureExposureExposure time time time (h) (h) (h) Exposure time (h) Figure 1. Effect of fluorescent light exposure on the stability of trans-stilbenes isolated in MeOH FigureFigureFigure 1. 1. 1.Effect Effect Effect of of offluorescent fluorescent fluorescent light light light exposure exposure exposureFigure on on on the the1. the Effectstability stability stability of offluorescent of oftrans trans trans-stilbenes-stilbenes-stilbenes light exposureisolated isolated isolated in onin inMeOH MeOHthe MeOH stability solution. of trans -stilbenes trans -astringin,isolated in MeOHtrans -piceid, trans-isorhapontin, trans- Molecules 2021, 26, x FOR PEER REVIEW 4 of 16 solution.solution.solution. trans trans trans-astringin,-astringin,-astringin, transtranstranssolution.-piceid,-piceid,-piceid, trans transtrans trans-isorhapontin,-astringin,-isorhapontin,-isorhapontin, trans -piceid,trans trans trans- - - resveratrol trans-isorhapontin, and trans-isorhapontigenin. trans- resveratrolresveratrolresveratrol and and and trans trans trans-isorhapontigenin.-isorhapontigenin.-isorhapontigenin. resveratrol and trans-isorhapontigenin. The effect of fluorescent light on the stability of isolated stilbenes can also be seen from the yield of new products (Figure 2). Maximum conversion resulted from the isom- TheTheThe effect effect effect of of offluorescent fluorescent fluorescent100 light light light on on on the the the Thestabil stabil stabil effectityityity of of of ofisolated isolated fluorescentisolated stilbenes stilbenes stilbenes light can oncan can alsothe also also stabil be be be seen seen ityseen of isolated stilbenes can also be seen fromfromfrom the the the yield yield yield of of ofnew new new products products products (Figure (Figure (Figure 2). 2). 2). Maximum Maximum Maximum conversion conversion conversion resulted resulted resulted from from from the the the isom- isom- isom-erization of the trans forms of resveratrol and piceid to their corresponding cis isomers 90 from the yield of new products (Figure 2). Maximum conversion resulted from the isom- erizationerizationerization of of ofthe the the trans trans trans forms forms forms of of ofresveratrol resveratrol resveratrol and and and piceid piceid piceid to to totheir their their corresponding corresponding corresponding cis cis cis isomers isomers isomersafter 2 week fluorescent light exposure at room temperature. As shown in Figure 2, the 80 erization of the trans forms of resveratrol and piceidconversion to their ratecorresponding is faster for cisthe isomers initial 36 h and becomes slower thereafter. afterafterafter 2 2 week2 week week fluorescent fluorescent fluorescent light light light exposure exposure exposureafter at at at room2 room roomweek temperature. temperature. temperature.fluorescent Aslight As As shown shown shownexposure in in inFigure Figure atFigure room 2, 2, 2,the temperature.the the As shown in Figure 2, the conversionconversionconversionMolecules Moleculesrate rate rate2021 is is ,isfaster 262021faster faster, x, FOR26 for ,for 1036for thePEER 70the the init init REVIEWinitialialial 36conversion 36 36 h h andh and and becomes becomes becomesrate is fasterslower slower slower for thereafter. thereafter. thereafter.the initial 36 h and becomes slower thereafter. 4 of 154 of 16

60 100 100100100 10050 90 909090 40 80 10090 8080 Recovery (%) 30 80 8090 70 20 707070 7080 60 10 606060 6070 50

0 ield (%) 505050 5060 Y 40 ield (%) ield (%) ield (%) 0 50 100 150 200 250 300 350 400 ield (%) Y Y Y 404040 50 30 Y 40 Exposure time (h) 40 303030 30 20

Recovery (%) 30 202020 20 10 20 Figure 1. Effect of fluorescent light exposure on the stability of trans-stilbenes isolated in MeOH 101010 10 0 10 solution. trans-astringin, trans-piceid,0 50 trans-isorhapontin, 100 150 200 trans- 250 300 350 400 000 0 0 resveratrol and trans-isorhapontigenin. 000 50 50 50 100 100 100 150 150 1500 200 200 200 50 250 250 250 100 300 300 300 150 350 350 350 200 400 400 400 250 300 350 400 Exposure time (h) 0 50 100 150 200 250 300 350 400 ExposureExposureExposure time time time (h) (h) (h) The effect of fluorescentExposure light time on (h) the stability of isolated stilbenes can also be seen Exposure time (h) Figure 2. The yield of cis stilbenes and a new compound (derived from trans-astringin) formed by from the yield of new products (Figure 2). Maximum conversion resulted from the isom- Figure 2. The yield of cis stilbenes and a new compound (derived from trans-astringin) formed bythe exposure of corresponding trans stilbenes to fluorescent light in MeOH solution. peak FigureFigure 2. 2. The The yield yield of of cis cis stilbenes stilbenes and and a anew newerizationFigure compound compound 2. The of yield (derivedthe (derived oftrans cis from fromstilbenes forms trans trans -astringin)andof-astringin) resveratrol a new formedcompound formed and by by (derived piceid fromto their trans -astringin)corresponding formed bycis isomers the exposure of corresponding trans stilbenes to fluorescent light in MeOH solution. peakderived from cis-astringin, cis-piceid, cis-isorhapontin, cis-resveratrol and thethe exposure exposure of of corresponding correspondingFigure 1. Effect trans trans of stilbenes ﬂuorescentstilbenesFigureafterthe exposureto to fluorescent2 lightfluorescent1. week Effect exposure of correspondingfluorescent of light light fluorescenton in in theMeOH MeOH stabilitylight trans lightsolution. solution. stilbenesexposure ofexposuretrans to-stilbenes fluorescent aton peak peak roomthe isolatedstability temperature.light in of MeOH trans-stilbenes solution. solution.As shown isolated in trans peakFigure in- MeOH 2, the astringin, trans-piceid, trans-isorhapontin, trans-resveratrol and trans-isorhapontigenin. derivedderivedderived from from from cis cis cis-astringin,-astringin,-astringin, cisciscis-piceid,-piceid,-piceid,solution.conversionderived fromcis cis cis-isorhapontin,-isorhapontin, -isorhapontin,cisrate-astringin, trans is faster-astringin, for ciscis thecis -piceid,cis-resveratrol-resveratrol-resveratrol inittransial 36-piceid, and and hand cis and -isorhapontin, becomes cis trans-isorhapontigenin. slower-isorhapontin, cis -resveratrolthereafter. and trans -

cis cis cis-isorhapontigenin.-isorhapontigenin.-isorhapontigenin. resveratrol cis-isorhapontigenin. and trans-isorhapontigenin. Crude spruce bark extract solutions were not significantly destablized by exposure 100 CrudeCrudeCrude spruce spruce spruce bark bark bark extract extract extract solutions solutions solutions were were were not not not significantly significantly significantly destablized destablized destablized by by by exposure exposure exposureto fluorescent light. It is known that in addition to stilbenes, spruce bark contains phenolic CrudeThe effect spruce of barkfluorescent extract solutions light on were the stabilnot significantlyity of isolated destablized stilbenes by canexposure also be seen toto tofluorescent fluorescent fluorescent light. light. light. It It isIt is isknown known known90 that that that in in inaddition addition addition to to tostilbenes, stilbenes, stilbenes, spruce spruce spruce bark bark bark contains contains contains phenolic phenolic phenolic fromto fluorescent the yield light. of new It is knownproducts that (Figure in addition 2). Maximumto stilbenes, conversionspruce bark containsresulted phenolic from the isom- 80 erization of the trans forms of resveratrol and piceid to their corresponding cis isomers 70 after 2 week fluorescent light exposure at room temperature. As shown in Figure 2, the conversion rate is faster for the initial 36 h and becomes slower thereafter. 60

10050 ield (%)

Y 40 90 8030 20 70 6010 500 0 50 100 150 200 250 300 350 400 ield (%)

Y 40 Exposure time (h) 30 20 Figure 2. The yield of Figurecis stilbenes 2. The and yield a new of cis compound stilbenes (derivedand a new from compoundtrans-astringin) (derived formed from by trans the-astringin) exposure of formed by corresponding10trans stilbenesthe exposure to ﬂuorescent of corresponding light in MeOH trans solution. stilbenes peakto fluorescent derived from lightcis -astringin,in MeOH solution.cis-piceid, peak cis-isorhapontin, cis-resveratrol and cis-isorhapontigenin. 0 derived from cis-astringin, cis-piceid, cis-isorhapontin, cis-resveratrol and

0 50 100 150 200 250 300 350 400 2.2. cis Stability-isorhapontigenin. Assessment of Stilbenes in Solid Crude Extract Stilbene glucosidesExposure (trans-astringin, time (h)trans-isorhapontin and trans-piceid) in the solid crudeCrude extract spruce showed bark 100% extract recovery solutions when assessedwere not for significantly their stability destablized under three differentby exposure to fluorescentconditions: (1) light. Light It protected:is knownsolid that extract in addition in a capped to stilbenes, glass vial spruce stored bark in freezer contains (−20 phenolic◦C) Figure 2. The yield of cis stilbenes and a new compound (derived from trans-astringin) formed by with protection from light; (2) Light unprotected capped glass vial: solid extract in a capped the glassexposure vial exposedof corresponding to continuous trans stilbenes ﬂuorescent to fluorescent light and (3) light Light in unprotectedMeOH solution. uncapped glass peak derivedvial: solidfrom extractcis-astringin, in glass vial leftcis-piceid, uncapped and placedcis-isorhapontin, on laboratory cabinet cis-resveratrol to expose itand to cis-isorhapontigenin.

Crude spruce bark extract solutions were not significantly destablized by exposure to fluorescent light. It is known that in addition to stilbenes, spruce bark contains phenolic

Molecules 2021, 26, x FOR PEER REVIEW 5 of 16

Molecules 2021, 26, x FOR PEER REVIEW 4 of 16 compounds such as lignans, flavonoids and tannins [1,7,10] which may influence the ac- MoleculesMoleculesMolecules 2021 2021 2021, 26, 26, ,26 x, x,FOR xFOR FOR PEER PEER PEER REVIEW REVIEW REVIEW tion of light on the stilbenes. 4 4of 4of of16 16 16 Molecules 2021, 26, x FOR PEER REVIEW 4 of 16

2.2. Stability Assessment of Stilbenes100 in Solid Crude Extract Stilbene glucosides (trans90-astringin, trans-isorhapontin and trans-piceid) in the solid 100100100 100 crude extract showed 100% recovery80 when assessed for their stability under three differ- 909090 90 ent conditions: (1) Light protected:70 solid extract in a capped glass vial stored in freezer (−20 808080 80 °C) with protection from light;60 (2) Light unprotected capped glass vial: solid extract in a 707070 70 capped glass vial exposed to 50continuous fluorescent light and (3) Light unprotected un- 606060 60 capped glass vial: solid extract in40 glass vial left uncapped and placed on laboratory cabinet 505050 50 to expose it to continuous fluorescentRecovery (%) 30 light [21]. Even if in contact with air in uncapped 404040 40

Recovery (%) vials, the oxidation or isomerization of the evaluated stilbenes were not observed. In other Recovery (%) Recovery (%) 20 303030 Recovery (%) 30 studies, solid resveratrol and 10piceid in glass vials protected from light were stable [12], 202020 20 and furthermore the solid trans0 -resveratrol and trans-piceid are not very sensitive to 101010 10 UV/fluorescent light, elevated temperature0 50 or humidity 100 or 150atmospheric 200 oxidants 250 at ambi- 300 350 400 000 0 000 50 50 50 100 100 100 150 150 150 200 200 200 250 250 250ent 300conditions 300 300 350 350 350 [21,23]. 400 400 400 Exposure time (h) Molecules 2021, 260, 1036 50 100 150 200 250 300 350 400 5 of 15 ExposureExposureExposure time time time (h) (h) (h) 2.3. UV StabilityExposure of Stilbenes time (h) Figure 1. Effect of fluorescent light exposure on the stability of trans-stilbenes isolated in MeOH The isomerization of crude trans stilbenes extract, hydrolysed (containing stilbene FigureFigure 1. 1. Effect Effect of of fluorescent fluorescent light light exposure exposure on on the the stability stability of of trans trans-stilbenes-stilbenes isolated isolated in in MeOH MeOH solution. trans-astringin, trans-piceid, trans-isorhapontin, trans- Figure 1. Effect of fluorescent light exposureFigure on the 1. stabilityEffectaglucones)continuous of offluorescent trans and-stilbenes ﬂuorescent lightnon-hydrolysed exposureisolated light [in 21on MeOH]. the Even (containingstability if in contactof trans stilbene-stilbenes with air inglucosides),isolated uncapped in MeOH vials, to thessss the oxidation cis forms solution.solution.solution. trans trans trans-astringin,-astringin,-astringin, transtranstranssolution.-piceid,-piceid,-piceid, started or trans isomerizationtranstrans transimmediately-isorhapontin,-astringin,-isorhapontin,-isorhapontin, of after the trans evaluated exposure trans -piceid,trans trans- - - stilbenes toresveratrol UV trans light were-isorhapontin, and (366 not nm) observed. trans and-isorhapontigenin. transmost In other- of the studies, trans solidstructures resveratrol and piceid in glass vials protected from light were stable [12], and furthermore resveratrolresveratrolresveratrol and and and trans trans trans-isorhapontigenin.-isorhapontigenin.-isorhapontigenin.resveratrolhad and disappeared trans-isorhapontigenin. after 10–30 min (Figure 3). trans-Astringin and trans-isorhapontin the solid trans-resveratrol and trans-piceid areThe not effect very of sensitive fluorescent to UV/ﬂuorescent light on the stabil light,ity of isolated stilbenes can also be seen showed the lowest rate of disappearance of the trans form. elevated temperature or humidity or atmosphericfrom the yield oxidants of new at products ambient (Figure conditions 2). Maximum [21,23]. conversion resulted from the isom- TheTheThe effect effect effect of of offluorescent fluorescent fluorescent light light light on on on the the the stabilThe stabil stabil effectityityity of Theof ofisolated isolated isolatedfluorescentexposure stilbenes stilbenes stilbenes oflight the can can on canisolated also thealso also stabilbe be be stilbenesseen seen ityseen of isolated in methanol stilbenes solution, can also hydrolysedbe seen (containing fromfromfrom the the the yield yield yield of of ofnew new new products products products (Figure (Figure (Figure 2). 2). 2). Maximum Maximum Maximum conversion conversion conversion resulted resulted resulted from from from the the the isom- isom- isom-erization of the trans forms of resveratrol and piceid to their corresponding cis isomers from the stilbeneyield2.3. of UV newaglucones) Stability products of Stilbenesand (Figure non-hydrolysed 2). Maximumafter conversion (containing2 week fluorescent resulted stilbene from light glucosides), the exposure isom- at crude room temperature.extract As shown in Figure 2, the erizationerizationerization of of ofthe the the trans trans trans forms forms forms of of ofresveratrol resveratrol resveratrolerization and and and piceid ofpiceid piceid the totrans to totheir their their forms corresponding corresponding corresponding of resveratrol cis cis cis andisomers isomers isomers piceid to their corresponding cis isomers under UVThe isomerizationirradiation lamp of crude (366trans nm)conversionstilbenes caused extract,the rate isomerization is hydrolysedfaster for the (containingof init transial 36 stilbenesh and stilbene becomes to cis slower thereafter. afterafterafter 2 2 week2 week week fluorescent fluorescent fluorescent light light light exposure exposure exposureafter at at at room 2room roomweek temperature. temperature. temperature.fluorescent As lightAs As shown shown shownexposure in in inFigure Figure Figureat room 2, 2, 2,the thetemperature. the As shown in Figure 2, the forms.aglucones) After and10–30 non-hydrolysed min of UV exposure (containing most stilbene of the glucosides), trans structures to thessss werecis forms absent started (Figure conversionconversionconversion rate rate rate is is isfaster faster faster for for for the the the init init initialialial 36conversion 36 36 h h andh and and becomes becomes becomes rate is slowerfaster slower slower for thereafter. thereafter. thereafter.the initial 36 h and becomes slower thereafter. 3). immediatelyThe proportion after of exposure cis stilbenes100 to UV increased light (366 10–60 nm) and min most after of starting the trans thestructures exposure had (Figure 4). disappearedAfter that the after proportion 10–30 min (Figureof cis 3structures). trans-Astringin decreased and transdue -isorhapontinto transformation showed of the cis stil- 100100100 100 90 beneslowest to other rate of products. disappearance of the trans form. 909090 90 80 808080 70 80 100 707070 70 90 60 606060 80 50

60 % , ield (%)

70 Y 505050 50 40 ield (%) ield (%) ield (%) 60 ield (%) Y Y Y 404040 30 Y 40 50 stilbenes 303030 30 40 20 20 2020 20 trans 30 10 101010 10 20 0 10 0 50 100 150 200 250 300 350 400 000 0 000 50 50 50 100 100 100 150 150 150 200 200 2000 250 250 250 300 300 300 350 350 350 400 400 400 Exposure time (h)

The loss of loss The 0 50 100 150 200 250 300 350 400 0 1030601203001440 ExposureExposureExposure time time time (h) (h) (h) Exposure time (h) Exposure time (min) Figure 2. The yield of cis stilbenes and a new compound (derived from trans-astringin) formed by the exposure of corresponding trans stilbenes to fluorescent light in MeOH solution. peak FigureFigureFigure 2. 2. 2.The The The yield yield yield of of ofcis cis cis stilbenes stilbenes stilbenes and and and a a newa new Figurenew compound compound compound 2. The yield(derived (derived (derived of cis from fromstilbenes from trans trans trans-astringin) and-astringin)-astringin) a new formed compoundformed formed by by by (derived from trans-astringin) formed by thethethe exposure exposure exposure of of ofcorresponding corresponding correspondingFigure trans trans trans 3. stilbenes Effectstilbenes stilbenesthe of toexposure UVto tofluorescent fluorescent fluorescent lightFigure of exposure corresponding light3. light lightEffect in onin inMeOH MeOHtheof MeOH UVstability trans solution. solution.light solution. stilbenes ofexposure isolated to fluorescent peak transon peak peak the -stilbenesderived stability light from in in ofMeOH MeOH cis isolated-astringin, solution. solution. trans -stilbenescis-piceid, trans peak in-astringin, MeOH cissolution.-isorhapontin, cis-resveratrol and trans-piceid, trans-isorhapontin, trans-resveratrol and trans-isorhapontigenin. derivedderivedderived from from from cis cis cis-astringin,-astringin,-astringin, cisciscis-piceid,-piceid,-piceid,derived fromcis cis cis-isorhapontin,-isorhapontin,-isorhapontin, cis-astringin, trans-astringin, cis cis cis-resveratrol-piceid,-resveratrol-resveratroltrans -piceid, and and and cis -isorhapontin, cis trans-isorhapontigenin. -isorhapontin, cis-resveratrol and trans -resveratrol and

cis cis cis-isorhapontigenin.-isorhapontigenin.-isorhapontigenin. cis-isorhapontigenin. transThe-isorhapontigenin. exposure of the isolated stilbenesCrude in methanol spruce solution, bark extract hydrolysed solutions (containing were not significantly destablized by exposure stilbene aglucones) and non-hydrolysed (containing stilbene glucosides), crude extract Crude spruce bark extract solutions were not significantly destablized by exposureto fluorescent light. It is known that in addition to stilbenes, spruce bark contains phenolic CrudeCrude spruce spruce bark bark extract extract solutions solutions were wereCrude not not underspruce significantly significantly UV bark irradiation extract destablized destablized solutions lamp by (366by exposurewere exposure nm) not caused significantly the isomerization destablized of trans by exposurestilbenes to cis forms. to fluorescent light. It is known that in addition to stilbenes, spruce bark contains phenolic toto fluorescent fluorescent light. light. It It is is known known that that in in additionto addition fluorescent to to stilbenes,After stilbenes, light. 10–30 It spruce is spruce known min bark of bark UVthat contains containsexposure in addition phenolic phenolic most to stilbenes, of the trans sprucestructures bark contains were absent phenolic (Figure 3). The proportion of cis stilbenes increased 10–60 min after starting the exposure (Figure4). After that the proportion of cis structures decreased due to transformation of cis stilbenes to other

products. The GC-MS information of the trans stilbenes isolated and the compounds derived from those during UV irradiation (366 nm) is presented in Table2. In general, trans-to-cis- isomerisation took place rapidly and after that all the compounds lost two hydrogens from their structures presumably by oxidation. In the case of piceatannol, the cis form was not detected by GC-MS. Molecules 2021, 26, x FOR PEER REVIEW 4 of 16

MoleculesMoleculesMolecules 2021 2021 2021, 26, 26, ,26 x, x,FOR xFOR FOR PEER PEER PEER REVIEW REVIEW REVIEW 4 4of 4of 16of 16 16 Molecules 2021, 26, x FOR PEER REVIEW 4 of 16

100 90 100100100 100 80 909090 90 70 808080 80 60 707070 70 50 606060 60 40 505050 50 Recovery (%) 30 404040 40 Recovery (%) Recovery (%) Recovery (%) 20 303030 Recovery (%) 30 10 202020 20 0 101010 10 0 50 100 150 200 250 300 350 400 000 0 000 50 50 50 100 100 100 150 150 150 200 200 2000 250 250 250 50 300 300 300 100 350 350 350 150 400 400 400 200 250 300 350Exposure 400 time (h) ExposureExposureExposure time time time (h) (h) (h) Exposure time (h) Figure 1. Effect of fluorescent light exposure on the stability of trans-stilbenes isolated in MeOH FigureFigureFigure 1. 1. Effect1. Effect Effect of of offluorescent fluorescent fluorescent light light light exposure exposure exposure on on Figureon the the the stability stability stability1. Effect of of oftrans trans transfluorescent-stilbenes-stilbenes-stilbenes lightisolated isolated isolated exposure in in inMeOH MeOH MeOH onsolution. the stability of trans-astringin,-stilbenes isolated trans in MeOH-piceid, trans-isorhapontin, trans- solution.solution.solution. trans trans trans-astringin,-astringin,-astringin, transtranstrans-piceid,-piceid,-piceid,solution. trans trans trans -isorhapontin,trans-isorhapontin,-isorhapontin,-astringin, transtrans trans trans--piceid,- - resveratrol and trans -isorhapontin, trans-isorhapontigenin. trans- resveratrolresveratrolresveratrol and and and trans trans trans-isorhapontigenin.-isorhapontigenin.-isorhapontigenin. resveratrol and trans-isorhapontigenin. The effect of fluorescent light on the stability of isolated stilbenes can also be seen The effectMolecules of 2021fluorescent, 26, 1036 light on the stability of isolated stilbenes can also be fromseen the yield of new products (Figure 2).6 of Maximum 15 conversion resulted from the isom- TheThe Moleculeseffect effect of of 2021 fluorescent fluorescent, 26, x FOR PEERlight light REVIEW on on the the stabil stabilTheityity effect of of isolated isolated of fluorescent stilbenes stilbenes light can can also onalso the be be stabilseen seen ity of isolated stilbenes can also 6be of seen16 erization of the trans forms of resveratrol and piceid to their corresponding cis isomers fromfromfrom the the the yield yield yield of of ofnew new new products products products (Figure (Figure (Figure 2). 2). 2). fromMaximum Maximum Maximum the yield conversion conversion conversion of new productsresulted resulted resulted from(Figure from from the the the2). isom- isom- Maximumisom- conversion resulted from the isom- erizationerizationerization of of ofthe the the trans trans trans forms forms forms of of ofresveratrol resveratrol resveratrol and and and piceid piceid piceid to to totheir their their corresponding corresponding corresponding cis cis cis isomers isomers isomersafter 2 week fluorescent light exposure at room temperature. As shown in Figure 2, the erization of the trans forms of resveratrolconversion and piceid rate to theiris faster corresponding for the initial cis 36 isomers h and becomes slower thereafter. afterafterafter 2 2 week2 week week fluorescent fluorescent fluorescent light light light exposure exposure exposure at at atafterroom room room 2 temperature. weektemperature. temperature. fluorescent As As As shown shown lightshown exposurein in inFigure Figure Figure at2, 2, room2,the the the temperature. As shown in Figure 2, the conversionconversionconversion rate rate rate is is fasteris faster faster100 for for for the the the init init initialialial 36 36 36 h h conversionandh and and becomes becomes becomes rate slower slower sloweris faster thereafter. thereafter. thereafter. for the init ial 36 h and becomes slower thereafter. 100 90 100100100 100 90 80 909090 90 80 808080 70 80 70 707070 60 60 70 606060 60 50 isomer, expressed as % as expressed isomer, 50 ield (%) 505050 50 Y 40 cis ield (%) ield (%) ield (%) ield (%) Y

Y 40 Y 404040 30 Y 40 303030 30 30 20 202020 10 20 20 101010 10 0

The proportion of proportion The 10 0 50 100 150 200 250 300 350 400 000 0 000 50 50 50 100 100 100 150 150 1500 200 200 2000 250 250 250 50 300 300 300 100 350 350 350 150 400 400 400 200 250 300 350Exposure 400 time (h) ExposureExposureExposure 0time time time (h) (h) (h) 1030601203001440Exposure time (h) Exposure time (min) Figure 2. The yield of cis stilbenes and a new compound (derived from trans-astringin) formed by the exposure of corresponding trans stilbenes to fluorescent light in MeOH solution. peak FigureFigureFigure 2. 2. The2. The The yield yield yield of of ofcis cis cisstilbenes stilbenes stilbenes and and and a anew anew new compound compound Figurecompound 2. (derivedThe (derived (derived yield from offrom from cis trans stilbenestrans trans-astringin)-astringin)-astringin) and a formed new formed formed compound by by by (derived from trans-astringin) formed by Figure 4. The proportion of cis stilbenes due to the exposure of trans isomers to UV light in MeOH thethethe exposure exposure exposure of of ofcorresponding Figurecorresponding corresponding 4. The trans proportiontrans trans stilbenes stilbenes stilbenes of to tocisthe tofluorescent fluorescent stilbenes fluorescentexposure light dueof light light corresponding toin in in theMeOH MeOH MeOH exposure solution. solution. solution. trans of stilbenes trans isomers peak to peakderived peak fluorescent to from UV light cislight-astringin, inin MeOHMeOH solution.cis-piceid, peak cis-isorhapontin, cis-resveratrol and cis-astringin, cis-piceid,solution.cis -isorhapontin, cis-astringin, cis-resveratrolcis-piceid, and ciscis-isorhapontin,-isorhapontigenin. cis-resveratrol and derivedderivedderived from from from cis cis -astringin,cis-astringin,-astringin, cisciscis-piceid,-piceid,-piceid, derived cis cis cis-isorhapontin,-isorhapontin, from-isorhapontin, cis-astringin, cis cis cis-resveratrol-resveratrol-resveratrolcis-piceid, and and and cis cis-isorhapontin,-isorhapontigenin. cis-resveratrol and

cis cis cis-isorhapontigenin.-isorhapontigenin.-isorhapontigenin. cis-isorhapontigenin. cis-isorhapontigenin. Crude spruce bark extract solutions were not significantly destablized by exposure Table 2. GC-MS1 characteristics of the stilbenes and new products obtained from the UV light (366 nm) exposure experiment. to fluorescent light. It is known that in addition to stilbenes, spruce bark contains phenolic CrudeCrudeCrude spruce spruce spruce bark bark bark extract extract extract solutions solutions solutions were were wereThe Crudenot not GC-MSnot significantly significantly significantly spruce information bark destablized destablized destablizedextract of the solutions bytrans by by exposure exposure exposurestilbenes were not isolated significantly and the destablized compounds by derived exposure toto tofluorescent fluorescent fluorescent light. light. light. It It isIt is knownis known known that that that in in inaddition addition fromaddition those to to tostilbenes, stilbenes,during stilbenes,Major UV spruce Compoundspruce spruce irradiation bark bark bark contains contains contains(366 nm) phenolic phenolic phenolicis presented in Table 2. In general, trans-to-cis- to fluorescent light.After It UV is known thatGC-MS in addition Retention to stilbenes,GC-MS spruce bark containsHPLC phenolic Sample Productsisomerisation took place rapidly and after that all the compounds lost two hydrogens Irradiation Time (min) (TMSI), m/z UV Proﬁle from their structures(0 h, 2presumably h, 24 h) by oxidation. In the case of piceatannol, the cis form was not detected by GC-MS. trans-astringin 0 h 41.72 532 trans-stilbene In the hydrolysed crude extract, all three trans stilbene aglucones isomerized to the trans-astringin cis-astringin 2 h 32.04 532 cis-stilbene cis forms, which were the major compounds at 2 h. The new peaks with loss of two hy- new peak 24 h 34.48 530 λ max~260 transdrogens-piceid derived from 0 hstilbene aglucones 38.42 were found from 444the hydrolysedtrans crude-stilbene extract trans-piceid cis-piceidafter 24 h UV irradiation 2 h by GC-MS as in 30.30the case of isolated 444stilbene aglucones.cis-stilbene The same newwas peak true for the non-hydrolys 24 hed extracts 31.77containing the trans 442 stilbene glucosides.λ max~260 trans-isorhapontin 0 h 42.45 474 trans-stilbene cis-isorhapontinTable 2. GC-MS1 characteristics 2 h/24 h of the stilbenes 31.62 and new products 474obtained from thecis -stilbeneUV light (366 trans λ -isorhapontin newnm) peak exposure 1 experiment. 24 h 30.43 472 max~260 new peak 2 24 h 34.48 472 λ max~260 new peak 3 24 hMajor Compound 43.54 After UV GC-MS Retention 472 GC-MSλ max ~260HPLC Sample Products trans-piceatannol 0 hIrradiation (0h,2h,24h) 22.76 Time (min) 532 (TMSI),trans m/z-stilbene UV Profile trans-piceatannol new peak 1 2 h 21.57 530 λ max~260trans- trans-astringin 0h 41.72 532 new peak 1 24 h 21.57 530 λ max~260stilbene trans-astringin trans-resveratrolcis-astringin 0 h 2h 20.0032.04 444 532trans -stilbenecis-stilbene trans-resveratrol cis-resveratrolnew peak 2 h 24h 13.8934.48 444 530cis -stilbeneλ max~260 new peak 24 h 18.64 442 λ ~260trans- trans-piceid 0h 38.42 444 max trans- stilbene trans-piceid 0 h 22.55 474 trans-stilbene trans- isorhapontigenin cis-piceid 2h 30.30 444 cis-stilbene isorhapontigenin cis-isorhapontigeninnew peak 2 h 24h 15.6231.77 474 442cis -stilbeneλ max~260 new peaktrans 24- h 16.36 472 λ ~260trans- 0h 42.45 474 max isorhapontin stilbene cis- trans- 2h/24h 31.62 474 cis-stilbene In the hydrolysedisorhapontin crude extract, all three trans stilbene aglucones isomerized to the cis isorhapontin forms, which werenew peak the major 1 compounds24h at 2 h. The new30.43 peaks with loss472 of two hydrogensλ max~260 derived from stilbenenew peak aglucones 2 were24h found from the hydrolysed34.48 crude472 extract afterλ max~260 24h new peak 3 24h 43.54 472 λ max~260

Molecules 2021, 26, x FOR PEER REVIEW 7 of 16 Molecules 2021, 26, x FOR PEER REVIEW 7 of 16

transtrans- trans- 0h 22.76 532 transtrans- piceatannol 0h 22.76 532 stilbene trans- piceatannol stilbene piceatannol new peak 1 2h 21.57 530 λ max~260 piceatannol new peak 1 2h 21.57 530 λ max~260 new peak 1 24h 21.57 530 λ max~260 new peak 1 24h 21.57 530 λ max~260 transtrans- trans- 0h 20.00 444 trans- resveratrol 0h 20.00 444 stilbene trans-resveratrol resveratrol stilbene trans-resveratrol cis-resveratrol 2h 13.89 444 cis-stilbene cis-resveratrol 2h 13.89 444 cis-stilbene new peak 24h 18.64 442 λ max~260 new peak 24h 18.64 442 λ max~260 transtrans- trans- isorhapontigen 0h 22.55 474 trans- isorhapontigen 0h 22.55 474 stilbene trans- in stilbene trans- in isorhapontigeni cis- isorhapontigeni cis- Molecules 2021, 26, 1036 n isorhapontigen 2h 15.62 474 7 of 15cis-stilbene n isorhapontigen 2h 15.62 474 cis-stilbene in in new peak 24h 16.36 472 λ max~260 new peak 24h 16.36 472 λ max~260 UV irradiation by GC-MS as in the case of isolated stilbene aglucones. The same was true 2.4. Isolationfor the non-hydrolysed and Identification extracts of the containing New Compounds the trans stilbene Formed glucosides. During UV Irradiation 2.4. Isolation and Identification of the New Compounds Formed During UV Irradiation The new compounds derived from the trans stilbenes during UV light exposure were 2.4.The Isolation new compounds and Identiﬁcation derived of the New from Compounds the trans Formed stilbenes during during UV Irradiation UV light exposure were fractionated by using preparative HPLC and identified by NMR and MS. The stilbenes fractionatedThe new by compoundsusing preparative derived from HPLC the transand stilbenesidentified during by UVNMR light and exposure MS. The were stilbenes showed isomerization from trans to cis form during UV light irradiation followed by the showedfractionated isomerization by using from preparative trans to HPLC cis form and identiﬁed during UV by NMR light and irradiation MS. The stilbenesfollowed by the loss showedof two isomerizationhydrogens ( from−2 H)trans afterto acis longerform during time UVof irra lightdiation. irradiation ESI-TOF-MS followed by was the used to loss of two hydrogens (−2 H) after a longer time of irradiation. ESI-TOF-MS was used to identifyloss ofthe two fractions hydrogens derived (−2H) from after astilbene longer time glucosides, of irradiation. astringin ESI-TOF-MS (Figure was 5) and used isorhapon- to identify the fractions derived from stilbene glucosides, astringin (Figure 5) and isorhapontin (Figureidentify the6) (Table fractions 3) derived and EI-MS from stilbene to identify glucosides, the fractions astringin (Figure derived5) and from isorhapontin hydrolyzed stil- tin (Figure(Figure6 )6) (Table (Table3) and 3) EI-MSand EI-MS to identify to identify the fractions the derivedfractions from derived hydrolyzed from stilbenes hydrolyzed stilbenes piceatannol and isorhapontigenin. benespiceatannol piceatannol and isorhapontigenin.and isorhapontigenin. Datafile Name:F1astringinUV100ul001.lcd SampleDatafile Name:F1astringinUV100ulName:F1astringinUV100ul001.lcd Sample Name:F1astringinUV100ul mAU bar mAU bar 4000 306nm,4nm Pump A Pressure 4000 306nm,4nm Pump A Pressure 0 2 300 0 2 300 3000 250 3000 250 200 2000 200 2000 150 150 1 1000 1 100 1000 100 50 50 0 3 0 3 0 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 min 0 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 min Figure 5. Sample chromatogram of preparative HPLC fractionation of stilbenes derived from UV- Figure 5. Sample chromatogram of preparative HPLC fractionation of stilbenes derived from UV- Figure 5. Sample chromatogramexposed astringin. of preparative HPLC fractionation of stilbenes derived from UV-exposed astringin. exposed astringin. Datafile Name:F3isorhapontinUV100ul001.lcd SampleDatafile Name:F3isorhapontinUVName:F3isorhapontinUV100ul001.lcd Sample Name:F3isorhapontinUV mAU bar mAU bar 4000 306nm,4nm Pump A Pressure 4000 306nm,4nm Pump A Pressure 1 4 300 1 4 300 3000 250 3000 250 200 2000 200 2000 150 150

1000 100 1000 100 50 50 0 0 2 3 5 0 0 2 3 5 0 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 min 0 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 min Figure 6. Sample chromatogram of preparative HPLC fractionation of stilbenes derived from UV-exposed isorhapontin.

Molecules 2021, 26, x FOR PEER REVIEW 8 of 16

Figure 6. Sample chromatogram of preparative HPLC fractionation of stilbenes derived from UV- exposed isorhapontin.

Table 3. Identification of compounds derived from trans-astringin (F1) and trans-isorhapontin (F3) by HR ESI-TOF-MS.

Molecules 2021, 26, 1036 8 of 15 Compound Stilbene [M-](m/z) Calcd. Formula

F1.1 trans-astringin 405.1180 405.1186 C20H21O9 Table 3. Identiﬁcation of compounds derived from trans-astringin (F1) and trans-isorhapontin (F3) by F1.2 new peak 403.1036 403.1029 C20H19O9 HR ESI-TOF-MS. F1.3 cis-astringin 405.1243 405.1186 C20H21O9 CompoundF3.1 Stilbenetrans-isorhapontin [M-]( m/z)419.1479 Calcd. 419.1342 Formula C21H23O9

F1.1F3.2 trans-astringinnew peak 405.1180417.1321 405.1186 417.1186 C20H21O 9 C21H21O9 F1.2F3.3 new peaknew peak 403.1036417.1327 403.1029 417.1186 C20H19O 9 C21H21O9 F1.3 cis-astringin 405.1243 405.1186 C20H21O9 F3.4 cis-isorhapontin 419.1487 419.1342 C21H23O9 F3.1 trans-isorhapontin 419.1479 419.1342 C21H23O9 F3.5 new peak 417.1321 417.1186 C21H21O9 F3.2 new peak 417.1321 417.1186 C21H21O9 F3.3 new peak 417.1327 417.1186 C21H21O9 2.5. TheF3.4 Structural Elucidationcis-isorhapontin of New Peaks 419.1487 Formed From 419.1342 Astringin (and C21H Its23O Aglycone9 F3.5 new peak 417.1321 417.1186 C21H21O9 Piceatannol) and Isorhapontin (and Its Aglycone Isorhapontigenin) [21] 2.5. TheFor Structural the new Elucidation compound of New derived Peaks Formed from fromtrans Astringin-astringin (and (F1.2), Its Aglycone the ESI-MS spectrum showedPiceatannol) 403.1036 and Isorhapontin m/z as the (and parent Its Aglycone peak. Isorhapontigenin) Its molecular mass is two units less than that of trans-For and the cis new-astringin. compound In the derived 1H-NMR from spectrum,trans-astringin there (F1.2), are six the proton ESI-MS signals spectrum between 6.0- 10.0showed ppm. 403.1036 Alkenem/ zHas signals the parent with peak. a larger Its molecular coupling mass constant is two units (ca less 12 than−16 thatHz) ofwere absent trans- and cis-astringin. In the 1H-NMR spectrum, there are six proton signals between from6.0–10.0 the ppm.aromatic Alkene range. H signals The signal with a δ larger 9.008 coupling (1H, s) could constant be (ca an 12 aldehyde−16 Hz) wereproton, but the 13 aldehydeabsent from carbon the aromatic signal range.(around The 200 signal ppm)δ 9.008 however (1H, s) does could not be anappear aldehyde in the proton, C-NMR spectrum.but the This aldehyde proton carbon bound signal to carbon (around (about 200 ppm) 104 however ppm) is does therefore not appear an aromatic in the 13C- proton. The signalsNMR spectrum. 7.435 (1H, This d, protonJ = 9 Hz) bound and to 7.319 carbon (1H, (about d, J 104= 9 ppm)Hz) belong is therefore to a anpair aromatic of ortho-coupled aromaticproton. The protons. signals They 7.435 (1H,do not d, J couple= 9 Hz) with and 7.319 other (1H, aromatic d, J = 9 Hz)protons. belong The to a signals pair of 6.968 (1H, ortho-coupled aromatic protons. They do not couple with other aromatic protons. The d, J = 2.4 Hz) and 6.843 (1H, d, J = 2.7 Hz) had almost the same coupling constants, so they signals 6.968 (1H, d, J = 2.4 Hz) and 6.843 (1H, d, J = 2.7 Hz) had almost the same coupling mayconstants, be from so theythe same may bearomatic from the ring. same 2D aromatic NMR (COSY, ring. 2D HSQC, NMR (COSY, and HMBC) HSQC, also and supported theseHMBC) findings. also supported Based theseon the ﬁndings. above Based evidence on the th abovee new evidence structure the new was structure identified was (F1.2) as 7- Oidentiﬁed-β-D-glucosyl-2,3,5-trihydroxyphenanthrene (F1.2) as 7-O-β-D-glucosyl-2,3,5-trihydroxyphenanthrene (Figure 7). (Figure7).

Figure 7. The proposed structure of F1.2, a phenanthrene molecule derived from the stilbene astringin Figureby a photochemical 7. The proposed cyclization structure and dehydrogenation of F1.2, a phenanthre inducedne by UVmolecule irradiation. derived from the stilbene astringin by a photochemical cyclization and dehydrogenation induced by UV irradiation. For the new compounds derived from trans-isorhapontin, the negative ESI-MS spectrum showed the strongest peak at 417.1321 m/z (for F3.2, F3.3, F3.5) two units less than For the new compounds derived from trans-isorhapontin, the negative ESI-MS spec- trans- and cis-isorhapontin. 1H-NMR and 2D NMR signals of F3.2 were almost the same trum showed the strongest peak at 417.1321 m/z (for F3.2, F3.3, F3.5) two units less than as those in fraction F1.2, except for the OCH3 signal at 3.992. F3.5 showed the shift of transthe methoxy- and cis signal-isorhapontin. to 3.432 ppm, 1H-NMR which and may 2D be dueNMR to thesignals formation of F3.2 of differentwere almost iso- the same asmers those of phenanthrenesin fraction F1.2, since except the positions for the ofOCH hydroxy3 signal and at methoxy 3.992. F3.5 groups showed adjacent the to shift of the methoxyeach other signal have twoto 3.432 alternative ppm, arrangements which may inbe theduecis toisomer the formation from which of is different the start- isomers of phenanthrenesing point of phenanthrene since the positions formation. of F3.3 hydroxy was the and mixture methoxy of the groups fractions adjacent F3.2 and to each other F3.5. The new structures identiﬁed (F3.2 and F3.5) are 7-O-β-D-glucosyl-3,5-dihydroxy- have2-methoxyphenanthrene two alternative arrangements and 7-O-β-D-glucosyl-2,5-dihydroxy-3-methoxyphenanthrene in the cis isomer from which is the starting point of phenanthrene(Figure8). formation. F3.3 was the mixture of the fractions F3.2 and F3.5. The new structures identified (F3.2 and F3.5) are 7-O-β-D-glucosyl-3,5-dihydroxy-2-methoxyphenanthrene and 7-O-β-D-glucosyl-2,5-dihydroxy-3-methoxyphenanthrene (Figure 8).

Molecules 2021, 26, x FOR PEER REVIEW 9 of 16

Molecules 2021, 26, x FOR PEER REVIEW 9 of 16 Molecules 2021, 26, 1036 9 of 15

Figure 8. The proposed structures of F3.2 and F3.5, phenanthrene molecules derived from the stilbene isorhapontin due to a photochemical cyclization and dehydrogenation induced by UV irradiation. Figure 8. The proposed structuresFigure of8. F3.2The andproposed F3.5, phenanthrene structures of molecules F3.2 and derived F3.5, ph fromenanthrene the stilbene molecules isorhapontin derived due to afrom the stil- photochemical cyclizationbene and dehydrogenation isorhapontin due induced to a photochemical by UV irradiation. cyclization and dehydrogenation induced by UV irradi- The novel compounds derived from trans-piceatannol and trans-isorhapontigenin ation. show similarThe novel phenanthrene compounds derivedNMR signals. from trans Our-piceatannol NMR data and in transcombination-isorhapontigenin with our UV show similar phenanthrene NMR signals. Our NMR data in combination with our UV and MSThe data novel suggest compounds that the photochemicalderived from transtransformation-piceatannol of andstilbenes trans to-isorhapontigenin phenanthrene structuresand MS datagoes suggest by way that of the an photochemical intramolecular transformation cyclisation offorming stilbenes a tonew phenanthrene C-C bond. The showstructures similar goes phenanthrene by way of an intramolecularNMR signals. cyclisationOur NMR forming data in a combination new C-C bond. with The our UV phenanthrene structures observed in our studies are supported by earlier data based on andphenanthrene MS data suggest structures that observed the photochemical in our studies aretransformation supported by earlier of stilbenes data based to phenanthrene on the thestructuresstudies studies of oftransgoes trans-resveratrol by-resveratrol way of and an itsandintramolecular glucoside, its glucoside, piceid cyclisation [pice16–18id,24 [16–18,24]] andforming by the anda studiesnew by C-Cthe of purestudies bond. Theof purephenanthrenestilbene stilbene molecule molecule structures itself itself [25 ,observed26 [25,26].]. Our study Ourin our ofstudy variousstud ofies various stilbenes are supported stilbenes isolated by fromisolated earlier bark from extractsdata barkbased ex- on tractsthenow studies now establishes establishes of trans their-resveratrol generaltheir general behavior and behaviorits exhibited glucoside, exhibited in thepice photochemicalid in [16–18,24] the photochemical and transformation by the transfor-studies of mationpureto phenanthrene stilbene to phenanthrene molecule structures. itself stru Thectures.[25,26]. suggested TheOur studysuggested electrocyclic of various electrocyclic mechanism stilbenes ofmechanism isolated photochemical from of photo-bark ex- transformation of cis-stilbenes to phenanthrenes is shown in Figure9. chemicaltracts now transformation establishes oftheir cis -stilbenesgeneral behavior to phenanthrene exhibiteds is in shown the photochemical in Figure 9. transformation to phenanthrene structures. The suggested electrocyclic mechanism of photochemical transformation of cis-stilbenes to phenanthrenes is shown in Figure 9.

Figure 9. ProposedFigure mechanism 9. Proposed of photochemicalmechanism of transformation photochemical of transformationcis stilbenes to phenanthrenes. of cis stilbenes to phenanthrenes. Photochemical transformations of trans-astringin and trans-isorhapontin (and of their Photochemical transformations of trans-astringin and trans-isorhapontin (and of their Figureaglycones, 9. Proposedtrans-piceatannol mechanism andof photoctrans-isorhapontigenin)hemical transformation derived of cis from stilbenes Norway to phenanthrenes. spruce aglycones,root bark trans have-piceatannol been reported and and trans described-isorhapontigenin) (Figure 10). These derived molecules from Norway formed by spruce rootphotochemical barkPhotochemical have been transformation reported transformations and of naturallydescribed of trans occurring (Figure-astringin 10). stilbenes and These trans have molecules-isorhapontin not been formed reported (and by of pho- their tochemicalaglycones,earlier in thetransformation trans literature.-piceatannol of naturally and trans occurri-isorhapontigenin)ng stilbenes have derived not beenfrom reported Norway earlier spruce The UVA-induced modiﬁcation of the stilbene-rich inner bark extracts increased the inroot the barkliterature. have been reported and described (Figure 10). These molecules formed by pho- radical scavenging activity [20]. The results also indicate even a slightly increased antimi- tochemicalcrobial activity transformation after UVA-modiﬁcations of naturally [20 occurri]. Thusng stilbenes stilbenes have have commercial not been potential reported as earlier ine.g., thepreservatives. literature.

Molecules 2021, 26, 1036 10 of 15 Molecules 2021, 26, x FOR PEER REVIEW 11 of 16

Figure 10. Proposed photochemicalFigure 10. transformation Proposed photochemical of selected trans transformationstilbenes (astringin of selected and isorhapontin trans stilbenes and (astringin their aglycones and iso- piceatannol and isorhapontigenin)rhapontin derived and fromtheir Norwayaglycones spruce piceatannol root bark. and R is =orhapontigenin) -H or -O-β-D-glucoside derived (Glu).from Norway−2H = loss spruce of root bark. R = -H or -O-β-D-glucoside (Glu). -2H = loss of two hydrogens two hydrogens

Molecules 2021, 26, 1036 11 of 15

3. Materials and Methods 3.1. Bark Material Thick roots of Norway spruce (Picea abies [L.] Karst) were collected from trees growing on mineral soil from Parkano (Lapinneva, Western Finland) in June 2010. The detailed sample information is given in Latva-Mäenpää et al. [1]. The samples were collected from the underground parts of the roots close to the stem. After debarking the bark material of the roots was immediately frozen at −20 ◦C. It was splintered, freeze-dried for 2–3 days, subsequently ground and stored in a freezer (−20 ◦C) prior to analysis.

3.2. Extraction of Bark Material The bark extract of Norway spruce was obtained by sequential extraction of the powdered bark (2 g) ﬁrst with hexane to remove the hydrophobic constituents, followed by 95% V/V ethanol to collect the hydrophilic stilbene constituents using an Accelerated Solvent Extraction (ASE 350, Dionex, Sunnyvale, CA, USA). The ASE sequential extraction method [1,7,21] was: (1) n-hexane extraction: temperature, 90 ◦C; static time, 5 min; two cycles and (2) ethanol:water (95:5 V/V) extraction: temperature, 100 ◦C; static time, 5 min; two cycles for 2 g sample or three cycles for 3 g of sample. The crude extract was diluted 1:10 with MeOH before injecting 5 µL into the HPLC-DAD, HPLC-DAD/ESI-MS and HPLC-DAD/ESI-MS/MS instruments. The stilbene content of the non-hydrolysed samples was enriched in an XAD-7HP polymer resin ﬁlled column, which was previously conditioned with water. Polyphenolic compounds were absorbed in a resin material and water-soluble compounds such as sugars were washed away with water. Stilbene glucosides were collected from ethanol-water eluates. All samples were stored at −20 ◦C with light protection until further processing. For producing and isolating hydrolysed stilbene aglucones the other 95% V/V ethanol extract was evaporated to dryness by rotary evaporation at 40 ◦C and dissolved in 15 mL of sodium acetate buffer (0.1 M, pH 5) solution. 15 mL of β-glucosidase enzyme (3 mg/mL) was suspended in the above solution and incubated in a laboratory oven at 39 ◦C for 3 days. The hydrolysed samples were enriched in a polymer resin as mentioned above and collected from ethyl acetate eluates. The stilbene glucoside and the stilbene aglucone fract e ions were evaporated to dryness by rotary evaporation at 40 ◦C and used for preparativHPLC, HPLC, HPLC-DAD/MS and HPLC-NMR analyses.

3.3. HPLC-DAD Analyses Separation of stilbenes from the extract of the root bark of Norway spruce was carried out by HPLC. A Model 1100 series liquid chromatography system (Hewlett-Packard, Santa Clara, CA, USA) with a quaternary pump, degasser, autosampler, column oven and an Agilent 1100 series diode array detector coupled to a Hewlett-Packard Chem Station was used for analysis. HPLC separation was performed on an Xbridge-C18 column (4.8 × 150 mm, 5 µm, Waters, Milford, MA, USA) using a mobile phase of H2O and MeOH with a flow rate of 0.8 mL/min. The mobile phase gradient program was 90:10 (t) 0 min, 75:25 (t) 3 min, 60:40 (t) 21 min, 50:50 (t) 30 min, 30:70 (t) 33 min, 10:90(t) 35–36 min and 90:10 (t) 37–39 min. The UV profiles of eluates were monitored with DAD in the range of 200–400 nm. Detection was carried out at 286 nm and 306 nm for cis and trans stilbenes, respectively (260 nm was also used for detection in UV experiments). Measurements were carried out in duplicate or in triplicate. The mobile phase (H2O and MeOH) gradient program was further modified for the structural characterisation of the compounds formed after UV irradiation and this separation was performed by Prominence HPLC instrument (Shimadzu, Canby, OR, USA); the modified mobile phase gradient program was 72:28 (t) 0 min, 60:40 (t) 9 min, 50:50 (t) 13.5 min, 30:70 (t) 15 min, 10:90 (t) 17.5 min and 72:28 (t) 18–21.5 min with a flow rate of 1.4 mL/min. Molecules 2021, 26, 1036 12 of 15

3.4. Isolation of Stilbenes by Preparative HPLC Preparative isolation was performed using a Waters 600 HPLC instrument on a Waters Xbridge-C18 column (19 × 150 mm, 5 µm) using a mobile phase of water and acetonitrile with a flow rate of 7 mL/min. The mobile phase gradient programs for the non-hydrolysed and hydrolyseSunnd samples were 90:10 (t) 0 min, 85:15 (t) 5 min, 70:30 (t) 25 min, 50:50 (t) 30 min, 10:90 (t) 35 min, 90:10 (t) 37–40 min and 90:10 (t) 0 min, 85:15 (t) 10 min, 70:30 (t) 35 min, 50:50 (t) 38 min, 10:90 (t) 40 min, 90:10 (t) 43–45 min, respectively. The fractions of the hydrolysed extract and the non-hydrolysed extract were collected at 7.5, 11.2, 12.2 min and 13.3, 18.3, 19.5 min. All collected fractions (7 mL each) were evaporated to dryness by a Biotage SB4 evaporator (Uppsala, Sweden) and dissolved in 10 mL of MeOH. The dissolved stilbenes were filtered through a 0.45 µm microporous membrane and stored in freezer at −20 ◦C until analysis. The chemical structures of the stilbenes isolated were confirmed 8 by NMR and MS. The mobile phase (H2O and MeOH) gradient program was further modified for the isolation of stilbenes for the structural characterization of the compounds formed after UV irradiation and this fractionation was performed by Shimadzu Prominence HPLC instrument; the modified mobile phase gradient program was 72:28 (t) 0 min, 60:40 (t) 9 min, 50:50 (t) 13.5 min, 30:70 (t) 15 min, 10:90 (t) 17.5 min and 72:28 (t) 18–21.5 min with a flow rate of 14 mL/min.

3.5. HPLC-DAD/ESI-MS and HPLC-DAD/ESI-MS/MS Analyses For the online HPLC-DAD/ESI-MS and HPLC-DAD/ESI-MS/MS analyses, an Agi- lent 1100 Series liquid chromatography system chromatograph coupled to a Bruker Esquire 3000plus ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) was utilised. The negative ion HPLC-DAD/ESI-MS experiments were conducted using conditions as follows: scan range m/z = 100–1000; dry temperature, 300 ◦C; drying gas, 8.0 L/min; nebulizer, 20 psi; capillary potential, 4 kV; spectra averages, 7; rolling averages 1. The HPLC conditions were the same as the one used for HPLC-DAD analyses. HPLC-DAD/ESI- MS/MS of stilbenes was studied by applying a 5 V supplementary radiofrequency (RF) voltage to the end-caps of the ion trap in order to induce collision with the helium buffer gas present in the ion trap.

3.6. Stability Assessment of Stilbenes in a Solution and Solid Form Stock solutions of reference trans-resveratrol (290 µg/mL), trans-piceatannol (380 µg/mL) and trans-piceid (270 µg/mL) were prepared in MeOH. In addition, stock solutions of isolated stilbenes from our study were prepared in MeOH. The stock solutions of isolated stilbenes were trans-astringin (120 µg/mL), trans-piceid (40 µg/mL), trans- isorhapontin (320 µg/mL), trans-piceatannol (90 µg/mL), trans-resveratrol (14 µg/mL) and trans-isorhapontigenin (190 µg/mL). A 5 mL crude bark extract prior to concentration on a column packed with XAD- 7HP ETAX was directly taken for stability study. The stability of the isolated stilbenes, reference stilbenes and crude extract was tested under various conditions described in Table4. Aliquots of each sample were taken after 0 h (control sample), 24 h, 1 week and 2 weeks for HPLC-DAD, HPLC-DAD/ESI-MS and HPLC-DAD/ESI-MS/MS. The stability of the solid crude extract was tested under three different conditions (the amounts of dried extracts were in the range of 10–11 mg) described in Table4. The samples were taken at intervals of 0 h (control sample), 1 week and 2 weeks for HPLC-DAD analysis. Molecules 2021, 26, 1036 13 of 15

Table 4. Stability assessment of stilbenes.

Stability Conditions in Solution Form Stability CondItions in Solid Form (1) Light protected: The samples were stored in (1) Light protected: The solid extract samples a freezer (−20 ◦C) with protection from light in in capped glass vials were stored in a freezer glass vials capped under ambient air (−20 ◦C) protected from light. conditions (2) Light unprotected: The samples in glass (2) Light unprotected: The solid extract vials (capped under ambient air) were kept on samples in capped glass vials under ambient laboratory bench, exposed to continuous air conditions were kept on laboratory bench, fluorescent light. exposed to continuous fluorescent light. (3) Light unprotected: Solid extracts samples in uncapped glass vials were kept on laboratory bench, exposed to continuous fluorescent light and permanent contact with air.

3.7. UV Stability of Isolated Stilbenes and Crude Extracts The UV stability of the isolated stilbenes (in methanol solution), hydrolysed (containing stilbene aglucones) and non-hydrolysed (containing stilbene glucosides) crude extracts under UV radiation were tested by UV lamp with 366 nm wavelength (UV-A radiation). The stilbenes were placed in vials on the work surface of the UV lamp system and samples were taken at intervals of 0 h (control sample), 10 min, 30 min, 1h, 2 h, 5h and 24 h for HPLC-DAD analysis. A GC-MS method [1] was also applied to analyse samples at 0 h, 2 h and 24 h. Larger amounts of bark-derived stilbenes were isolated by semiprep-HPLC for the structural elucidation of new compounds formed during UV irradiation. The stilbenes isolated were irradiated by UV for 6 days and the compounds formed were isolated by prep-HPLC, dried by vacuum centrifuging and identiﬁed by MS and NMR.

3.8. Mass Spectrometry (MS) and Nuclear Magnetic Resonance Spectroscopy (NMR) The mass spectrometry analyses were conducted for stilbene glucosides by high resolution ESI-TOF-MS with negative ion mode and for stilbene aglucones by EI-MS (high resolution sector instrument). Acetonitrile-water was used as the solvent. The NMR analyses were performed by 500 MHz and 300 MHz spectrometers. The 1H and 13C spectra 1 13 were recorded in CD3OD (300 MHz, 500MHz). H and C chemical shifts were referenced 1 13 to solvent signals of CD3OD, δ ( H) = 3.31 ppm and δ ( C) = 49.86 ppm. The following methods were used to analyse the products formed in UV irradiation studies presented in the chapter concerning the experiments of the UV stability. For the high-resolution mass spectra (HR-ESI-MS) a JMS-SX102mass spectrometer (JEOL, Tokyo, Japan) was used. ESI-TOF MS analysis, a Bruker Daltonics micro-TOF-Q I (Bruker Daltonics), the data was processed by means of DataAnalysis 3.2 (Bruker Daltonics). The positive ion ESI-TOF MS analysis was conducted using source conditions as follows: scan range of 0-3000 m/z; dry temperature, 180 ◦C: Nitrogen was used as the drying gas, 4.0 L/min; nebuliser, 04. bar. Voltages used were: nebulizer end plate 500V, capillary exit 100 V and capillary potential 4.5 kV. NMR analysis: 300 MHz Mercury NMR spectrometer (Varian, Palo Alto, CA, USA) and Avance III 500 NMR spectrometer (Bruker Corporation, Billerica, MA, USA) controlled with TopSpin 3.1 (Bruker, Karlsruhe, Germany). All NMR spectra were acquired at 300 K (for the NMR data see the Supplementary Materials).

4. Conclusions trans-Stilbenes derived from the bark of Norway spruce are stable compounds when stored carefully and protected from light. A trans- to cis-isomerization takes place rapidly upon UV irradiation, but also under ﬂuorescent light after a longer time. Molecules 2021, 26, 1036 14 of 15

In this study, we identified new compounds derived from stilbenes from the bark of Norway spruce roots. The individual stilbene molecules were isolated from the extracts and their photochemical stabilities (under fluorescent and UV light) were studied. Isomerisation was much faster in the presence of UV light. The molecular structures of stilbenes change by intramolecular cyclisation (new C-C bond and loss of two hydrogens) to phenanthrene structures. This is the first time that these new phenanthrene structures are reported to be derived from stilbenes of spruce bark.

Supplementary Materials: The following are available on-line. Figure S1: F3.2 fraction: The phenantrene structure formed in the photoisomerization of trans-isorhapotin. 1H- and 13C-NMR data of F1.2; F1.3; F3.2; F3.4 and F3.5. Author Contributions: Conceptualization, H.L.-M., T.S., P.S., K.W.; methodology H.L.-M., D.M., R.W., T.S., P.S., K.W., analysis H.L.-M., D.M., R.W., supervision K.W., P.S., writing—original draft preparation, H.L.-M. writing—review and editing, H.L.-M., R.W., D.M., T.S., P.S., K.W. All authors have read and agreed to the published version of the manuscript. Acknowledgments: Cooperation between LUKE and University of Helsinki is acknowledged. D.M. is grateful for funding from Erasmus Mundus ASC-master program. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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