Article Chemical Defense of Yacón ( sonchifolius) Leaves against Phytophagous Insects: Insect Antifeedants from Yacón Leaf Trichomes

Kaisei Tsunaki and Masanori Morimoto *

Department of Applied Biological Chemistry, Faculty of Agriculture, Kindai University, Nara 6318505, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-742-43-7162; Fax: +81-743-43-1445

 Received: 26 May 2020; Accepted: 4 July 2020; Published: 6 July 2020 

Abstract: Yacón is a perennial crop with high insect resistance. Its leaves have many glandular trichomes, which may be related to pest resistance. In order to collect the constituents of glandular trichomes, leaves were rinsed using dichloromethane (DCM) to obtain the rinsate, and the residues were subsequently extracted by DCM to obtain a DCM extract containing the internal constituents of yacón leaves. Biologic evaluations revealed that insect antifeedant activity was stronger for the rinsate than for the DCM extract against the common cutworm. The major constituents of rinsate were isolated by silica gel flash chromatography and were identified as sesquiterpene lactones (SLs), uvedalin (1) and enhydrin (2) and uvedalin aldehyde (3), collectively known as melampolides. Although SLs 1 and 2 exhibited remarkably strong insect antifeedant activity, SL 3 and reduced corresponding derivatives (4 and 5) of 1 and 2 exhibited moderate insect antifeedant activity. Additionally, the two analogs, parthenolide (6) and erioflorin (7) showed moderate insect antifeedant activity. The results indicate that the substituent patterns of SLs may be related to the insect antifeedant activities. The insect antifeedant activities of SLs 1 and 2 were similar to that of the positive control azadirachtin A (8), and thus these natural products may function in chemical defense against herbivores.

Keywords: ; Smallanthus sonchifolius (Poepp. & Endl.) H. Rob.; sesquiterpene lactones; uvedalin; enhydrin; insect antifeedants; Spodoptera litura F.

1. Introduction The is a subfamily of the Asteraceae and includes the genera , , and . Tanacetuem cinerariifolium (Trevir.) Sch. Bip. syn. Pyrethrum cinerariifolium Trevir. produces sesquiterpene lactones (SLs) as well as pyrethroids [1,2]. It is well known that most of the Asteroideae produce SLs [3]. In general, SLs show a remarkable biologic activity toward various insect species [4–7]. Yacón (Smallanthus sonchifolius (Poepp. & Endl.) H. Rob) is a perennial crop plant of the Asteroideae, and its enlarged roots are used as a foodstuff (Figure1). Since this plant exhibits high resistance to pests, it is easy to cultivate using small amounts of pesticides. Yacón has a large number of glandular trichomes on the abaxial surface of its leaves and this was presumed to function as one of its insect-resistance factors (Figure2). However, yac ón produces a large amounts of SLs and polyphenols, the aerial parts are sometimes used as functional foods [8–10].

Plants 2020, 9, 848; doi:10.3390/plants9070848 www.mdpi.com/journal/plants Plants 2020, 9, x FOR PEER REVIEW 2 of 9

a b

Figure 1. Yacón (S. sonchifolius (Poepp. & Endl.), H. Rob): (a) aerial parts and (b) root parts.

Most trichomes are the aerial epidermal protuberances of plants, where they play a critical role as contributing to avoiding feeding damage by herbivores [11–13]. Trichomes are classified into hairy trichome or glandular trichome. Glandular trichomes consist of several cells and synthesizes biologic active compound. Moreover, then they store various chemicals, and various kinds of biologic active compounds in the glandular trichomes have been reported from various plant species [14]. In a function of glandular trichomes, a tomato cultivar without hairy trichomes produced large amounts of terpenoids compared with its cultivar with a hairy trichomes, and these terpenoids contributePlants 2020 , to9, 848 the herbivore resistance in tomato plants [15]. Additionally, plant trichomes can2 of 9 physicalPlants 2020ly, 9 ,interfere x FOR PEER with REVIEW insect locomotion over the leaf surface and as a consequence avoid feeding2 of 9 damage from the herbivores [16]. In particular, most glandular trichomes synthesize and store various biologica active compounds with herbivore repellebnt and antimicrobial activities and they may provide a function as a chemical defense system for them. In our previous studies, the Asteraceae weed Heterotheca subaxillaris (Lam.) Britt. & Rusby was disclosed to produce and store sesquiterpene carboxylates in glandular trichomes on the leaf surface. These sesquiterpenes was found to exhibit phytotoxicity and insect antifeedant activity [16,17]. The Orobanchaceae weeds yellow bartsia (Parentucellia viscosa (L.) Caruel., syn. Bartsia viscosa L.) and Mediterranean linseed (Bellardia trixago (L.) All., syn. Bartsia trixago L.) also produce large amounts of exudates consisting of diterpenes for tiny insect entrapment on the trichomes of their leaf surface. The trichome density and insect antifeedant diterpene, kolavenic acid in glandular trichomes were both important for herbivore resistance of yellow bartsia [16]. The aim of present study was to unravel the resistant factors in yacón. In this study, we report the functionalFigureFigure properties 1. Yacón 1. Yac ó(S. nand (sonchifoliusS. sonchifoliusSLs in glandular (Poepp.(Poepp. & Endl.),trichomes & Endl.), H. H.Rob), from Rob):: (a )chemical (aeriala) aerial parts partsecological and and (b) (rootb points) root parts. parts. of view.

Most trichomes are the aerial epidermal protuberances of plants, where they play a critical role as contributinga to avoiding feeding damage by herbivoreb s [11–13]. Trichomes are classified into hairy trichome or glandular trichome. Glandular trichomes consist of several cells and synthesizes biologic active compound. Moreover, then they store various chemicals, and various kinds of biologic active compounds in the glandular trichomes have been reported from various plant species [14]. In a function of glandular trichomes, a tomato cultivar without hairy trichomes produced large amounts of terpenoids compared with its cultivar with a hairy trichomes, and these terpenoids contribute to the herbivore resistance in tomato plants [15]. Additionally, plant trichomes can physically interfere with insect locomotion over the leaf surface and as a consequence avoid feeding damage from the herbivores [16]. In particular, most glandular trichomes synthesize and store various biologic active compounds with herbivore repellent and antimicrobial activities and they FigureFigure 2. L 2.owLow-vacuum-vacuum scanning scanning electron electron microscope microscope (LVSEM)(LVSEM) images images of of hairy hairy (black (black triangle) triangle) and may provide a function as a chemical defense system for them. In our previous studies, the andglandular glandular (white (white triangle) triangle trichomes) trichomes on theon yactheó yacónn leaf surfaceleaf surface ( 160). (×160 (a)) adaxial;. (a) adaxial (b) abaxial; (b) abaxial (bar: 100 (bar:µm) . × Asteraceae100 µm) weed. Heterotheca subaxillaris (Lam.) Britt. & Rusby was disclosed to produce and store sesquiterpeneMost trichomes carboxylates are the in aerial glandular epidermal trichomes protuberances on the leaf ofplants, surface. where These they sesquiterpenes play a critical was role as foundcontributing to exhibit to phytotoxicity avoiding feeding and damage insect antifeedant by herbivores activity [11– 13[16]., Trichomes17]. The Orobanchaceae are classified into weeds hairy yellowtrichome bartsia or glandular (Parentucellia trichome. viscosa Glandular (L.) Caruel., trichomes syn. Bartsia consist viscosa of several L.) and cells Mediterranean and synthesizes linseed biologic (Bellardia trixago (L.) All., syn. Bartsia trixago L.) also produce large amounts of exudates consisting of active compound. Moreover, then they store various chemicals, and various kinds of biologic active diterpenescompounds for tiny in the insect glandular entrapment trichomes on the havetrichomes been of reported their leaf from surface various. The planttrichome species density [14]. and In a insectfunction antifeedant of glandular diterpene, trichomes, kolavenic a tomato acid cultivar in glandular without hairy trichomes trichomes were produced both important large amounts for herbivoreof terpenoids resistance compared of yellow with bartsia its cultivar [16]. with a hairy trichomes, and these terpenoids contribute to theThe herbivore aim of present resistance study in tomato was to plants unravel [15 the]. Additionally, resistant factors plant in trichomesyacón. In canthis physicallystudy, we report interfere thewith functional insect locomotionproperties and over SLs the in leaf glandular surface trichomes and as a, consequence from chemical avoid ecological feeding points damage of view from. the herbivores [16]. In particular, most glandular trichomes synthesize and store various biologic active compoundsa with herbivore repellent and antimicrobialb activities and they may provide a function as a chemical defense system for them. In our previous studies, the Asteraceae weed Heterotheca subaxillaris (Lam.) Britt. & Rusby was disclosed to produce and store sesquiterpene carboxylates in glandular trichomes on the leaf surface. These sesquiterpenes was found to exhibit phytotoxicity and insect antifeedant activity [16,17]. The Orobanchaceae weeds yellow bartsia (Parentucellia viscosa (L.) Caruel., syn. Bartsia viscosa L.) and Mediterranean linseed (Bellardia trixago (L.) All., syn. Bartsia trixago L.) also produce large amounts of exudates consisting of diterpenes for tiny insect entrapment on the trichomes of their leaf surface. The trichome density and insect antifeedant diterpene, kolavenic acid in glandular trichomes were both important for herbivore resistance of yellow bartsia [16]. The aim of present study was to unravel the resistant factors in yacón. In this study, we report the functionalFigure 2. propertiesLow-vacuum and scanning SLs in glandular electron trichomes,microscope from (LVSEM) chemical images ecological of hairy (black points triangle of view.) and glandular (white triangle) trichomes on the yacón leaf surface (×160). (a) adaxial; (b) abaxial (bar: 100 µm).

PlantsPlants 20202020,, 99,, 848x FOR PEER REVIEW 33 ofof 99

2. Results and Discussion 2. Results and Discussion The antifeedant activity evaluation showed that the rinsate had stronger insect antifeedant The antifeedant activity evaluation showed that the rinsate had stronger insect antifeedant activity activity than dichloromethane (DCM) extract against common cutworms (Spodoptera litura F.) than dichloromethane (DCM) extract against common cutworms (Spodoptera litura F.) (Figure3). (Figure 3).

Figure 3. Insect antifeedant activity of rinsate and dichloromethane (DCM) extract from aerial parts Figure 3. Insect antifeedant activity of rinsate and dichloromethane (DCM) extract from aerial parts of yacón. of yacón. This plant species has many glandular trichomes on abaxial leaf surfaces (Figure2). The rinsate containedThis plant the extract species from has their many glandular glandular trichomes, trichomes therefore on abaxial the glandularleaf surface trichomess (Figure may 2). T playhe rinsate a role incontained the defense the system extract against from their phytophagous glandular insecttrichomes, pests intherefore this plant. the The glandular SLs uvedalin trichomes (1) and may enhydrin play a (role2) were in the found defense to exist system in glandular against trichomesphytophagous on the insect abaxial pests leaf in surfaces this plant of yac. Theón usingSLs uvedalin matrix-assisted (1) and laserenhydrin desorption (2) were/ionization found to mass exist spectrometry in glandular (MALDI-MS) trichomes on imaging the abaxial [18]. Similarleaf surface resultss of were yacón reported using formatrix another-assisted member laser of desorption/ionization the Asteroideae, Mexican mass sunflower spectrometry ( (MALDI diversifolia-MS) imaging(Hermsl.) [18] A.. Similar Gray), whichresultsalso were has reported glandular for another trichomes member that produce of the Asteroideae, SLs—the rinsate Mexican prepared sunflower from ( thisTithonia plant diversifolia showed insect(Hermsl antifeedant.) A. Gray), activity which againstalso has a glandular lepidopteran trichomes insect that [19]. produce Camphorweed SLs—the (H. rinsate subaxillaris prepared), a weed from fromthis plant the Asteraceae, showed insect had antifeedant a huge amount activity of glandular against a trichomes lepidopteran on the insect leaf [ surface19]. Camphorweed in our previous (H. studies.subaxillaris The), rinsatea weed preparedfrom the from Asteraceae, this plant ha alsod a huge showed amount strong of insect glandular antifeedant trichomes activity on the against leaf commonsurface in cutworms, our previous and several studies calamenene-type. The rinsate prepared sesquiterpene from this carboxylates plant also were showed identified strong as insect antifeedantsantifeedant [ activity16]. against common cutworms, and several calamenene-type sesquiterpene carboxylatesThe major were constituents identified as of insect the rinsate antifeedant prepareds [16 from]. aerial parts of yacón were identified as uvedalinThe ( major1) and constituents its oxidative ofcongener the rinsate enhydrin prepared (2) and from uvedalin aerial aldehyde parts of yacón (3), collectively were identified known as as melampolidesuvedalin (1) and [20 its] (Figure oxidative4). It congener was presumed enhydrin that (2 these) and SLs uvedalin prevent aldehyde feeding damage(3), collectively by herbivores, known butas melampolides these natural products [20] (Figure did not 4). kill It herbivores was presumed in the that field these (personal SLs observation prevent feeding by M. damage Morimoto). by Actually,herbivores, the but speed these of natural common products cutworms did eating not kill yac herbivoón leavesres wasin the very field low (personal in the field. observation Based on by this M. fieldMorimoto) observation,. Actually, these the compounds speed of common were evaluated cutworms for eating insect yacón antifeedant leaves was activity very against low in commonthe field. cutworms.Based on this The field SLs 1 observation,and 2 exhibited these remarkably compounds strong were insect evaluated antifeedant for insect activity antifeedant and, excepting activity SL 3against, showed common similar cutworms. insect antifeedant The SLs 1 activity and 2 exhibited to the positive remarkably control strong azadirachtin insect antifeedant A (8). Thus, activity these melampolidesand, excepting maySL 3, functionshowed similar in theyac insectón chemicalantifeedant defense activity system to the againstpositive herbivores. control azadirachtin As seen inA other(8). Thus, structure–activity these melampolides relationship may (SAR) function information in the onyacón these chemical melampolides, defense hydrogenation system against of theherbivores. exomethylene As seen on their in g-lactone other structure ring slightly–activity decreased relationship their biologic (SAR) activity information (Figure 4, on Table these1). Amelampolides similar case, was hydrogenation reported concerning of the exomethylene exomethylene on onthe their g g-lactone-lactone ring ring slightly being critical decreased for insect their antifeedantbiologic activity activity (Figure of eudesmanolide 4, Table 1). A from similarEncelia casespp., was also reported of the concerning Asteraceae [exomethylene21]. In general, on since the ang-lactone exomethylene ring being on thecritical g-lactone for insect ring antifeedant will combine activity with an of SH eudesmanolide group of protein, from this Encelia chemical spp., moietyalso of the Asteraceae [21]. In general, since an exomethylene on the g-lactone ring will combine with an SH

Plants 2020, 9, 848 4 of 9

Plants 2020, 9, x FOR PEER REVIEW 4 of 9 is the important to appear biologic activity. For the herbicidal guaianolide, its glutathione adduct (dehydrozaluzaningroup of protein, this C) chemical was also moiety reported is [the22]. important to appear biologic activity. For the herbicidal guaianolide, its glutathione adduct (dehydrozaluzanin C) was also reported [22].

Figure 4. Melampolides and derivatives 1–5 from yacón leaf trichomes. Figure 4. Melampolides and derivatives 1–5 from yacón leaf trichomes. Table 1. Insect antifeedant activity of tested sesquiterpene lactones and azadirachtin A against common cutwormTo determine (S. litura the) third-instar critical structural larvae. factor(s), the analog SLs 6 and 7 with different functional groups were evaluated for insect antifeedant activity. These SLs drastically decreased the biologic activity. These findings suggested thatAntifeedant the g-lactone Activity ring against moietyS. litura wasLarvae not essential for insect 2 2 2 antifeedant activityCompounds in the testedED50 (mgSLs/.cm The) common ED50 (µmol structural/cm ) 95% features CI (mg / werecm ) hemiterpene, methylester and acetoxy1 groups0.0080, among the strong 0.018 insect antifeedant SLs 0.0062–0.012 1 and 2 and azadirachtin A (8) used as a positive 2control. Additionally,0.0042 the exomethylene 0.0090 moiety on 0.0026–0.0086 the g-lactone ring may have increased the biologic3 activity compared0.030 with hydrogenated 0.072 derivatives 0.022–0.043 (Table 1). As seen in other 4 0.040 0.058 0.015–0.070 SAR information on these melampolides, the substituent patterns of SLs may be related to the 5 0.027 0.088 0.0076–0.053 appearance of the insect6 antifeedant0.25 activities (Figure 7.3 5, Table 1). Additionally, 0.15–0.69 the two analogs, parthenolide (6) from7 T. parthenum0.20 L. and erioflorin 7.9 (7) from Helianthus 0.18–0.21 strumosus L., showed moderate insect antifeedant8 (Aza) activity.0.00023 Parthenolide 0.00032 (6) has no functional 0.00021–0.00027 groups other than the g-lactone ring and epoxide and erioflorin (7) also has few substituents in addition to methacrylate esterTo and determine hydroxyl the group. critical The structural SLs 6 and factor(s), 7 showed the analog moderate SLs insect6 and antifeedant7 with diff erent activity functional against groupscommon were cutworms. evaluated Thus for, the insect substituent antifeedant patterns activity. of melampolide These SLs drasticallys may be decreasedrelated to thepresence biologic of activity.these insect These antifeedant findings suggested activities. that Interestingly, the g-lactone the ring g-lactone moiety stereochemistry was not essential of for SLs insect was antifeedantalso found activityto be related in the to tested herbivore SLs. Theresistance common in structuralthe field for features Xanthium were strumarium hemiterpene, L. methylester(Asteraceae), and which acetoxy has groups,glandular among trichome thes strong contain insecting the antifeedant SL xanthanolide SLs 1 and [232] andisomers azadirachtin of cis/trans A-fused (8) used lactone as a positivediffered control.in herbivore Additionally, resistance the potential, exomethylene with moietythe trans on-fused the g-lactone form showing ring may stronger have increased resistance the than biologic the activitycis-fused compared form [24]. with The hydrogenated insect antifeedant derivatives SLs 1– (Table3, from1). glandular As seen in trichomes other SAR of information yacón leaves on, thesewere melampolides,also in trans-fused the substituentform (Figure patterns 4). of SLs may be related to the appearance of the insect antifeedant activities (Figure5, Table1). Additionally, the two analogs, parthenolide ( 6) from T. parthenum L. and erioflorin (7) from Helianthus strumosus L., showed moderate insect antifeedant activity. Parthenolide (6) has no functional groups other than the g-lactone ring and epoxide and erioflorin (7) also has few substituents in addition to methacrylate ester and hydroxyl group. The SLs 6 and 7 showed moderate insect antifeedant activity against common cutworms. Thus, the substituent patterns of melampolides may be related to presence of these insect antifeedant activities. Interestingly, the

Plants 2020, 9, 848 5 of 9 g-lactone stereochemistry of SLs was also found to be related to herbivore resistance in the field for Xanthium strumarium L. (Asteraceae), which has glandular trichomes containing the SL xanthanolide [23] isomers of cis/trans-fused lactone differed in herbivore resistance potential, with the trans-fused form showing stronger resistance than the cis-fused form [24]. The insect antifeedant SLs 1–3, from glandular trichomesPlants 2020, 9 of, x yacFORó PEERn leaves, REVIEW were also in trans-fused form (Figure4). 5 of 9

Figure 5. Melampolide analogs 6, 7 and azadirachtin A (8) used for the evaluation of insect antifeedantFigure 5. Mela activity.mpolide analogs 6, 7 and azadirachtin A (8) used for the evaluation of insect antifeedant activity. Consequently, SLs 1–3 from glandular trichomes of yacón leaves were presumed to play a role in chemicalConsequently, defense. SLs The 1–3 termfrom “allelopathy”glandular trichomes is defined of yacón an interaction leaves were between presumed diff erentto play species. a role Anyin chemical chemical defense acting. The as an term allelochemical “allelopathy should” is defined directly an ainteractionffect the species between concerned, different whichspecies in. Any this studychemical were acting yacó asn andan allelochemical common cutworms. should In directly the case affect of plant–plant the species chemical concerned, interactions, which in this when study the constituentswere yacón producedand common by the cutworms. donor plant In a ff theect the case receiver of plant plant–plant it is usuallychemical via interactions, soil and environmental when the media.constituents In such produced cases, identifying by the donor and demonstrating plant affect anthe allelochemical receiver plant is occasionally it is usually di ffiviacult. soil In and our study,environmental it was easy media. to determine In such case thats, theidentif SLsying were and allelochemicals demonstrating because an allelochemical they provided is occasionally yacón with herbivoredifficult. In resistance. our study, it was easy to determine that the SLs were allelochemicals because they provideFinally,d yacón we evaluatedwith herbivore the relationship resistance. between the insect antifeedant potential of SLs and herbivore resistance of tested plants using intact plant leaves. In this dual-choice type test for preference by tested phytophagousTable 1. Insect insects, antifeedant the herbivore activity commonof tested sesquiterpene cutworm consumed lactones manyand azadirachtin more leaf disksA against prepared from freshcommonH. strumosus cutwormleaves (S. litura compared) third- withinstar those larvae prepared. from fresh yacón leaves (Figure6). The result seemed to be related to the insect antifeedantAntifeedant potential Activity of constituentsagainst S. litura and L herbivorearvae resistance of tested plants—it was similar to the stronger insect antifeedant activity of SLs 1 and 2 extracted from Compounds ED50 (mg/cm2) ED50 (µmol/cm2) 95% CI (mg/cm2) yacón compared to SL 7 extracted from H. strumosus (Table1). 1 0.0080 0.018 0.0062–0.012 2 0.0042 0.0090 0.0026–0.0086 3 0.030 0.072 0.022–0.043 4 0.040 0.058 0.015–0.070 5 0.027 0.088 0.0076–0.053 6 0.25 7.3 0.15–0.69 7 0.20 7.9 0.18–0.21 8 (Aza) 0.00023 0.00032 0.00021–0.00027

Finally, we evaluated the relationship between the insect antifeedant potential of SLs and herbivore resistance of tested plants using intact plant leaves. In this dual-choice type test for preference by tested phytophagous insects, the herbivore common cutworm consumed many more leaf disks prepared from fresh H. strumosus leaves compared with those prepared from fresh yacón leaves (Figure 6). The result seemed to be related to the insect antifeedant potential of constituents and herbivore resistance of tested plants—it was similar to the stronger insect antifeedant activity of SLs 1 and 2 extracted from yacón compared to SL 7 extracted from H. strumosus (Table 1).

Plants 2020, 9, 848 6 of 9 Plants 2020, 9, x FOR PEER REVIEW 6 of 9

Figure 6. Comparison of herbivore resistance between yacón leaf and H. tuberosus or H. strumosus leaf. Figure 6. Comparison of herbivore resistance between yacón leaf and H. tuberosus or H. strumosus 3. Materialsleaf. and Methods

3.1.3. Materials General and Methods The 1D- and 2D-NMR (COSY, HSQC and HMBC) spectra were obtained using an Avance 3.1. General 400 instrument (Bruker Co. Ltd., Bremen, Germany) with solvent signal as an internal reference. The EI-MSThe 1D spectra- and 2D were-NMR measured (COSY, usingHSQC a and JMS-K9 HMBC) (JEOL spectra Co. Ltd.,were Tokyo,obtained Japan). using Aan low-vacuum Avance 400 scanninginstrument electron (Bruker microscope Co. Ltd., (LVSEM)Bremen, wasGermany) used for with observation solvent signal of leaf as surfaces an internal by SU-1510 reference. (Hitachi The High-Technologies,EI-MS spectra were Tokyo, measured Japan). using Optical a JMS rotations-K9 (JEOL were Co. measured Ltd., Tokyo, with a Japan). high-sensitivity A low-vacuum SEPA 300scanning polarimeter electron (Horiba). microscope Flash (LVSEM) column was chromatography used for observation was performed of leaf surface on ans Isolera by SU- One1510 (Biotage,(Hitachi Uppsala,High-Technologies, Sweden). Tokyo, Japan). Optical rotations were measured with a high-sensitivity SEPA 300 polarimeter (Horiba). Flash column chromatography was performed on an Isolera One (Biotage, 3.2.Uppsala, Chemicals Sweden). and Insects All naturally occurring SLs (1–3) were isolated from DCM rinsate prepared from the aerial parts 3.2. Chemicals and Insects of fresh yacón leaves cultivated and collected in Ikoma City, Nara Pref., Japan. The corresponding derivativesAll naturally (4, 5) were occurring prepared SLs by (1– organic3) were synthesis isolated from [25]. ErioflorinDCM rinsate (7) wasprepared isolated from from the DCM aerial rinsate parts preparedof fresh yacón from theleaves aerial cultivated parts of andH. strumosus collectedcultivated in Ikoma andCity collected, Nara Pref. in, Ikoma Japan. City. The corresponding derivativesThe authentic (4, 5) were reference prepared compounds, by organic parthenolide synthesis (> [25]97%). Erioflorin (6) and azadirachtin (7) was isolated A (Aza) from (>95%) DCM (8), wererinsate purchased prepared from from Tokyo the aerial Kasei parts Kogyo of Co.H. strumosus Ltd., Japan cultivated and Sigma-Aldrich and collected Co., in LLC, Ikoma UK, City respectively.. CommonThe authentic cutworms reference (S. compoun litura F.,ds, Lepidoptera: parthenolide Noctuidae)(>97%) (6) and were azadirachtin purchased A from(Aza) Sumika (>95%) Technoservice(8), were purchased (Takarazuka from Tokyo City, Japan). Kasei Kogyo The insects Co. Ltd., were Japan reared and onSigma an artificial-Aldrich diet Co., (InsectLLC, UK LF,, Nihonrespectively Nosan. Kogyo Co. Ltd., Japan) in a controlled environment at 26.5 ◦C and 60% humidity. Common cutworms (S. litura F., Lepidoptera: Noctuidae) were purchased from Sumika 3.3.Technoservice Plant Materials (Takarazuka City, Japan). The insects were reared on an artificial diet (Insect LF, NihonBoth Nosan yacó Kogyon (S. sonchifolius Co. Ltd., )Japan) and H. in strumosus a controlledwere environment cultivated and at 26.5 harvested °C and in 60% Ikoma humidity. City during 2017–2019. The H. tuberosus was collected from the riverside of Tomio River in Nara City in 2019. These3.3. Plant mature Materials plants were cultivated at Nov. in these years. Both yacón (S. sonchifolius) and H. strumosus were cultivated and harvested in Ikoma City 3.4. LVSEM Observation during 2017–2019. The H. tuberosus was collected from the riverside of Tomio River in Nara City in 2019.The These plant mature specimens plants werewere preparedcultivated as at a Nov. small in piece these from years. fresh yacón leaf. The specimens were mounted using double-sided carbon tape. For LVSEM, SU-1510 (Hitachi High-Technologies, Tokyo, Japan)3.4. LVSEM was used Observation with the vacuum condition and accelerating voltage set at 30 Pa and 15 kV, respectively. The eTheffect plant of DCM specimen rinseds withinwere prepared a few seconds as a small was measuredpiece from both fresh for yacón intact leaf. (a) and The treated specimen (b)s leaves were (Figuremounted7). using double-sided carbon tape. For LVSEM, SU-1510 (Hitachi High-Technologies, Tokyo, Japan) was used with the vacuum condition and accelerating voltage set at 30 Pa and 15 kV,

Plants 2020, 9, x FOR PEER REVIEW 7 of 9 respectively. The effect of DCM rinsed within a few seconds was measured both for intact (a) and Plants 2020, 9, 848 7 of 9 treated (b) leaves (Figure 7).

a b

FigureFigure 7. LVSEM 7. LVSEM images images of glandular of glandular trichomes trichomes on the on theyacón yac leafón leafsurface surface (a) before (a) before DCM DCM rinse rinse and and (b)( withinb) within a few a few seconds seconds after after DCM DCM rinse rinse (bar: (bar: 100 100 µm)µm)..

3.5.3.5. Extraction Extraction and and Isolation Isolation DCMDCM rinsates rinsates were were prepared prepared from from the the fresh fresh aerial aerial parts parts of ofyacón yacó (4.7n (4.7 kg) kg) by byrinsing rinsing using using DCM DCM solventsolvent (4 (4L) L)for for ca. ca. 10 10s. The s. The DCM DCM solution solution obtained obtained was was concentrated concentrated under under reduced reduced pressure pressure to to obtainobtain a DCM a DCM rinsate rinsate (9.5 (9.5 g, g,0.20% 0.20% yield). yield). Then Then the the plant plant residues residues of ofthe the aer aerialial parts parts of ofyacón yacó weren were extractedextracted using using DCM DCM for for three three days. days. After After filtration, filtration, the the extract extract was was concentrated concentrated under under reduced reduced pressurepressure to toobtained obtained DCM DCM extract extract ( (12.212.2 g, 0.26%0.26% yield).yield). The The constituents constituents (1–3 (1–)3 were) were isolated isolated from from DCM DCMrinsates rinsates by silicaby silica gel flushgel flush column colu chromatographymn chromatography with hexanewith hexane and ethyl and acetateethyl acetate as eluent as eluent [20], and [20the], and corresponding the corresponding hydrogenated hydrogenated derivatives derivatives (4, 5) were (4 prepared, 5) were using prepared hydrogenation using hydrogenation with Pd–C as withcatalyst Pd–C [ 26as]. catalys DCMt rinsates [26]. DCM were rinsates prepared were from prepared the fresh from aerial the parts fresh of H. aerial strumosus parts (0.75of H. kg)strumosus by rinsed (0.75the kg) plant by usingrinsed DCM the plant solvent using (2 L)DCM for ca.solvent 10 s. (2 The L) DCM for ca solution. 10 s. The obtained DCM solution was concentrated obtained was under concentratedreduced pressure under reduced to obtain pressure a DCM rinsateto obtain (1.0 a g, DCM 0.14% rinsate yield). (1.0 The g, constituent 0.14% yield). (7) The was constituent isolated from (7)DCM was isolatedrinsates byfrom silica DCM gel flush rinsates column by silica chromatography gel flush colu withmn hexanechromatography and ethyl acetate with hexane as eluent and [27 ]. ethyl acetate as eluent [27]. 3.6. Insect Antifeedant Biologic Assay (Dual-choice Type Test) 3.6. InsectTest Antifeedant compounds Biologic and extractsAssay (Dual of the-choice insect Type antifeedant Test) activity were evaluated against common cutwormTest compounds (S. litura F.). and Using extracts leaf of disks the insect of 2-cm antifeedant diameter wereactivity prepared were evaluate using a corkd against borer common from fresh cutwormsweet potato (S. litura (Ipomoea F.). Using batata leafcv. disks Narutokintoki) of 2-cm diameter leaves that were was prepared cultivated using at the a cork farm borer in Nara from campus fresh of sweetKindai potato University (Ipomoea (Nara, batata Japan).cv. Narutokintoki The upper) surface leaves ofthat two was leaf cultivated disks were at the treated farm with in Nara test compoundcampus of inKindai acetone University and two (Nara, other leaf Japan). disks The were upper treated surface with acetoneof two leaf only disks as a control. were treat Aftered withthe acetone test compoundhad completely in acetone evaporated and two other at room leaf temperature. disks were treated These with four acetone leaf disks only were as a placed control. in After alternating the acetonepositions had in completely the same 9.5-cm-diameter evaporated at room glass temperature. Petri dish with The moistenedse four leaf filter disks paper were on placed the bottom. in alternatingThe reference positions dish consistedin the same of only9.5-cm four-diameter control glass leaf disks. Petri Then,dish with 10 larvae moistened (third filter instar) paper were on released the bottom.into the The dish reference and allowed dish consisted to feed. The of dishonly wasfour kept control in an leaf insect disks. rearing Then room, 10 larvae at 26.5 (third◦C in instar) darkness werefor released 5–6 h. Each into experimentthe dish and was allowed terminated to feed. when The dish the consumption was kept in a ofn insect four control rearing leaf room disks at 26.5 in the °C referencein darkness dish for was 5–6 completed. h. Each experiment The consumed was terminated leaf disks werewhen digitized the consumption using a PC of scanner.four control Data leaf were disksanalyzed in the onreference a PC using dish NIH was Image completed. J, U. S. The National consumed Institutes leaf of disks Health, were Bethesda, digitized Maryland, using a PC USA scanner.(http:// rsb.info.nih.gov Data were analyzed/ij/index.html on a PC). For using each NIH experiment, Image J, the U. data S. National for an intact Institutes disk were of Health, measured Bethesda,and compared Maryland, with USA those (http://rsb.info.nih.gov/ij/index.html). of a treated disk. To measure the activities For each of test experiment, compounds, the wedata used for an an antifeedantintact disk were index: measured AFI = (% and of treated compared disks with consumed those of/ a(% treated of treated disk. disks To measure consumed the +activities% of control of disks consumed)) 100. The AFI value was converted to the inhibitory rate (%): inhibitory rate (%) test compounds, we ×used an antifeedant index: AFI = (% of treated disks consumed / (% of treated = (50 AFI) 2. The insect antifeedant potency of test compounds was evaluated in terms of the disks consumed− × + % of control disks consumed)) × 100. The AFI value was converted to the median effective dose (ED50) value for the rate of feeding inhibition calculated from the area of the leaf disk consumed [28]. A straight line was fitted to the points obtained using the bioassay, and the ED50 Plants 2020, 9, 848 8 of 9 calculated as the dose corresponding to the midpoint between complete inhibition and no effect using the probit method [29] using R software (ver. R. 2.12.1, R Foundation for Statistical Computing); the 95% confidence interval (95% CI) was also determined.

4. Conclusions The isolated SLs from glandular trichomes of yacón showed strong insect antifeedant activity against common cutworms, thus may function as a kind of the resistant factors in this plant. The SLs, uvedalin (1) and its oxidative congener, enhydrin (2), known as melampolide were major constituent and showed remarkable insect antifeedant activity as well as the positive control azadirachtin A (8). Additionally, the two analogs, parthenolide (6) and erioflorin (7) showed moderate insect antifeedant activity against common cutworms. As a result, the substituent patterns of SLs maybe concerned with appearance of insect antifeedant activity. These results suggested that the existence of SLs in glandular trichomes was important to show phytophagous insect resistance of yacón.

Author Contributions: Conceptualization, M.M. and K.T.; methodology, M.M.; validation, M.M.; formal analysis, M.M. and K.T.; investigation, M.M. and K.T.; resources, M.M. and K.T.; writing—original draft preparation, M.M.; writing—review and editing, M.M. and K.T. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflicts of interest.

References

1. Doskotch, R.W.; El-Feraly, F.S.; Hufford, C.D. Sesquiterpene lactones from pyrethrum flowers. Can. J. Chem. 1971, 49, 2103–2110. [CrossRef] 2. Ramirez, A.M.; Saillard, N.; Yang, T.; Franssen, M.C.R.; Bouwmeester, H.J.; Jongsma, M.A. Biosynthesis of sesquiterpene lactones in pyrethrum (Tanacetum cinerariifolium). PLoS ONE 2013, 8, e65030. [CrossRef] [PubMed] 3. Ikezawa, N.; Göpfert, J.C.; Nguyen, D.T.; Kim, S.-U.; O’Maille, P.E.; Spring, O.; Ro, D.-K. Lettuce costunolide synthase (CYP71BL2) and its homolog (CYP71BL1) from sunflower catalyze distinct regio- and stereoselective hydroxylations in sesquiterpene lactone metabolism. J. Biol. Chem. 2011, 286, 21601–21611. [CrossRef] [PubMed] 4. Rees, S.B.; Harborne, J.B. The role of sesquiterpene lactones and phenolics in the chemical defence of the chicory plant. Phytochemistry 1985, 24, 2225–2231. [CrossRef] 5. Arnason, J.T.; Isman, M.B.; Philogène, B.J.R.; Waddell, T.G. Mode of action of the sesquiterpene lactone, tenulin, from amarum against herbivorous insects. J. Nat. Prod. 1987, 50, 690–695. [CrossRef] 6. Rossiter, M.; Gershenzon, J.; Mabry, T.J. Behavioral and growth responses of specialist herbivore, Homoeosoma electellum, to major terpenoid of its host, Helianthus spp. J. Chem. Ecol. 1986, 12, 1505–1521. [CrossRef] 7. Cis, J.; Nowak, G.; Kisiel, W. Antifeedant properties and chemotaxonomic implications of sesquiterpene lactones and syringin from Rhaponticum pulchrum. Biochem. Syst. Ecol. 2006, 34, 862–867. [CrossRef] 8. Khajehei, F.; Hartung, J.; Graeff-Hönninger, S. Total phenolic content and antioxidant activity of yacon (Smallanthus Sonchifolius Poepp. and Endl.) chips: Effect of cultivar, pre-treatment and drying. Agriculture 2018, 8, 183. [CrossRef] 9. Oliveira, R.B.; Chagas-Paula, D.A.; Secatto, A.; Gasparoto, T.H.; Faccioli, L.H.; Campanelli, A.P.; Da Costa, F.B. Topical anti-inflammatory activity of yacon leaf extracts. Rev. Bras. De Farmacogn. 2013, 23, 497–505. [CrossRef] 10. Lachman, J.; Fernández, E.C.; Orsák, M. Yacon [Smallanthus sonchifolia (Poepp. et Endl.) H. Robinson] chemical composition and use—A review. Plant Soil Environ. 2003, 49, 283–290. [CrossRef] 11. Stipanovic, R.D. Function and Chemistry of Plant Trichomes and Glands in Insect Resistance. In Plant Resistance to Insects; Hedin, P.A., Ed.; American Chemical Society: Washington, DC, USA, 1983; Volume 208, pp. 69–100. 12. Sletvold, N.; Huttunen, P.; Handley, R.; Kärkkäinen, K.; Ågren, J. Cost of trichome production and resistance to a specialist insect herbivore in Arabidopsis lyrata. Evol Ecol 2010, 24, 1307–1319. [CrossRef] Plants 2020, 9, 848 9 of 9

13. Li, C.-H.; Liu, Y.; Hua, J.; Luo, S.-H.; Li, S.-H. Peltate glandular trichomes of Colquhounia seguinii harbor new defensive clerodane diterpenoids. J. Integr. Plant Biol. 2014, 56, 928–940. [CrossRef][PubMed] 14. Wang, G. Recent progress in secondary metabolism of plant glandular trichomes. Plant Biotechnol. 2014, 31, 353–361. [CrossRef] 15. Tian, D.; Tooker, J.; Peiffer, M.; Chung, S.H.; Felton, G.W. Role of trichomes in defense against herbivores: Comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum lycopersicum). Planta 2012, 236, 1053–1066. [CrossRef] 16. Morimoto, M. Chemical defense against insects in Heterotheca subaxillaris and three Orobanchaceae species using exudates from trichomes. Pest Manag. Sci. 2019, 75, 2474–2481. [CrossRef] 17. Morimoto, M.; Cantrell, C.L.; Libous-Bailey, L.; Duke, S.O. Phytotoxicity of constituents of glandular trichomes and the leaf surface of camphorweed, Heterotheca subaxillaris. Phytochemistry 2009, 70, 69–74. [CrossRef] 18. Lopes, A.A.; Pina, E.S.; Silva, D.B.; Pereira, A.M.S.; da Silva, M.F.D.G.; Da Costa, F.B.; Lopes, N.P.; Pupo, M.T. A biosynthetic pathway of sesquiterpene lactones in Smallanthus sonchifolius and their localization in leaf tissues by MALDI imaging. Chem. Commun. 2013, 49, 9989–9991. [CrossRef] 19. Ambrósio, S.R.; Oki, Y.; Heleno, V.C.G.; Chaves, J.S.; Nascimento, P.G.B.D.; Lichston, J.E.; Constantino, M.G.; Varanda, E.M.; Da Costa, F.B. Constituents of glandular trichomes of Tithonia diversifolia: Relationships to herbivory and antifeedant activity. Phytochemistry 2008, 69, 2052–2060. [CrossRef] 20. Inoue, A.; Tamogami, S.; Kato, H.; Nakazato, Y.; Akiyama, M.; Kodama, O.; Akatsuka, T.; Hashidoko, Y. Antifungal melampolides from leaf extracts of Smallanthus sonchifolius. Phytochemistry 1995, 39, 845–848. [CrossRef] 21. Srivastava, R.P.; Proksch, P.; Wray, V. Toxicity and antifeedant activity of a sesquiterpene lactone from Encelia against Spodoptera littoralis. Phytochemistry 1990, 29, 3445–3448. [CrossRef] 22. Galindo, J.C.G.; Hernández, A.; Dayan, F.E.; Tellez, M.R.; Macías, F.A.; Paul, R.N.; Duke, S.O. Dehydrozaluzanin C, a natural sesquiterpenolide, cause rapid plasma membrane leakage. Phytochemistry 1999, 52, 805–813. [CrossRef] 23. Chen, F.; Hao, F.; Li, C.; Gou, J.; Lu, D.; Gong, F.; Tang, H.; Zhang, Y. Identifying three ecological chemotypes of Xanthium strumarium glandular trichomes using a combined NMR and LC-MS method. PLoS ONE 2013, 8, e76621. [CrossRef][PubMed] 24. Ahern, J.R.; Whitney, K.D. Sesquiterpene lactone stereochemistry influences herbivore resistance and plant fitness in the field. Ann. Bot. 2014, 113, 731–740. [CrossRef] 25. Ali, E.; Dastidar, P.P.G.; Pakrashi, S.C.; Durham, L.J.; Duffield, A.M. Studies on Indian medicinal plants—XXVIII: Sesquiterpene lactones of Enhydra fluctuans Lour. Structures of enhydrin, fluctuanin and fluctuadin. Tetrahedron 1972, 28, 2285–2298. [CrossRef] 26. Herz, W.; Bhat, S.V. Isolation and structure of two new germacranolides from uvedalia. J. Org. Chem. 1970, 35, 2605–2611. [CrossRef] 27. Morimoto, H.; Oshio, H. Isolation of deacetylviguiestenin and erioflorin from Helianthus tuberosus. J. Nat. Prod. 1981, 44, 748–749. [CrossRef] 28. Escoubas, P.; Lajide, L.; Mizutani, J. An improved leaf-disk antifeedant bioassay and its application for the screening of Hokkaido plants. Entomol. Exp. Appl. 1993, 66, 99–107. [CrossRef] 29. Finney, D.J. Probit Analysis, 2nd ed.; Cambridge Univ. Press: London, UK, 1952; p. 318.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).