Journal of Chemical Ecology (2018) 44:510–524 https://doi.org/10.1007/s10886-018-0956-y

REVIEW ARTICLE

Yellow-Cedar, Callitropsis (Chamaecyparis) nootkatensis, Secondary Metabolites, Biological Activities, and Chemical Ecology

Joseph J. Karchesy1 & Rick G. Kelsey2 & M. P. González-Hernández3

Received: 22 December 2017 /Revised: 26 March 2018 /Accepted: 28 March 2018 /Published online: 14 April 2018 # This is a U.S. government work and its text is not subject to copyright protection in the United States; however, its text may be subject to foreign copyright protection 2018

Abstract Yellow-cedar, Callitropsis nootkatensis, is prevalent in coastal forests of southeast Alaska, western Canada, and inland forests along the Cascades to northern California, USA. These trees have few microbial or animal pests, attributable in part to the distinct groups of biologically active secondary metabolites their tissues store for chemical defense. Here we summarize the new yellow-cedar compounds identified and their biological activities, plus new or expanded activities for tissues, extracts, essential oils and previously known compounds since the last review more than 40 years ago. Monoterpene hydrocarbons are the most abundant compounds in foliage, while heartwood contains substantial quantities of oxygenated monoterpenes and oxygenated sesquiterpenes, with one or more tropolones. Diterpenes occur in foliage and bark, whereas condensed tannins have been isolated from inner bark. Biological activities expressed by one or more compounds in these groups include fungicide, bactericide, sporicide, acaricide, insecticide, general cytotoxicity, antioxidant and human anticancer. The diversity of organisms impacted by whole tissues, essential oils, extracts, or individual compounds now encompasses ticks, fleas, termites, ants, mos- quitoes, bacteria, a water mold, fungi and browsing animals. Nootkatone, is a heartwood component with sufficient activity against arthropods to warrant research focused toward potential development as a commercial repellent and biopesticide for ticks, mosquitoes and possibly other arthropods that vector human and animal pathogens.

Keywords Nootkatone . Chemical defense . Repellents . Biopesticides . Monoterpenes . Sesquiterpenes . Tropolones . Tannins

Introduction coastal forests of southeast Alaska and British Columbia, with a southern extension primarily in the Cascade Range through Yellow-cedar, Callitropsis nootkatensis (D.Don)Oerst.exD.P. WashingtonandOregontonorthernCalifornia,whereittypi- Little, also known as Alaska cedar, Alaska yellow-cedar or cally grows above 600 m (Harris 1990;Hennonetal.2016). Nootka cypress, is an ecologically and economically important Investigations of yellow-cedar secondary metabolites have conifer in the Cupressaceae family that indigenous people have been ongoing for nearly a century, beginning with the isolation valued and used for centuries as a material resource (Hennon of α-andβ-pinene (Fig. 1) from foliage essential oil with other et al. 2016;Stewart1984). It is naturally most abundant in the monoterpenes not fully characterized (Clark and Lucas 1926). Subsequent investigations of the leaf essential oil by gas-liquid chromatography (GC) found (−)-α-pinene, (+)-δ-3-carene and * Rick G. Kelsey (+)-limonene to be the major components (Fig. 1) with several [email protected] additional monoterpenes in lower quantities (Andersen and Syrdal 1970; Cheng and von Rudloff 1970a). Sesquiterpenes, 1 Wood Science and Engineering, Oregon State University, diterpenes, n-alkanes, alkanals, and esters of short chain (C4, Corvallis, OR 97331, USA C5) acids also occur in foliage oil, but usually in low abundance 2 USDA Forest Service, Pacific Northwest Research Station, (trace-0.5%, Cheng and von Rudloff 1970a, b). Initial studies of Corvallis, OR 97331, USA heartwood essential oils identified five monoterpenes (Fig. 1,), 3 Department of Crop Production and Projects of Engineering, carvacrol, methyl carvacrol, chamic acid, isochamic acid and Santiago de Compostela University, 27002 Lugo, Spain chaminic acid (Carlsson et al. 1952; Duff and Erdtman 1953; J Chem Ecol (2018) 44:510–524 511

Fig. 1 Monoterpenes in tissues of yellow-cedar. Compounds in the top two rows occur only in foliage, except α-terpineol and terpinen-4-ol that sometimes are found in heartwood. Compounds in the last two rows have been reported only in heartwood

Erdtman et al. 1956;Norin1964a), two tropolones (Fig. 2), Fungal bioassays with some of these compounds began as nootkatin and chanootin (Carlsson et al. 1952; Duff et al. sufficient quantities were isolated and purified. Nootkatin con- 1954,Norin1964b), and two sesquiterpenes, nootkatene and centrations of 0.001–0.002% inhibited eight out of 12 fungal nootkatone (Erdtman and Topliss 1957; Erdtman and Hirose species growing on agar, and it was fungicidal to seven of the 1962;MacLeod1965). taxa at 0.001–0.005% (Rennerfelt and Nacht 1955). In the 512 J Chem Ecol (2018) 44:510–524

1993), however further details about their biochemistry and storage of monoterpenes relative to leaf morphology, age, etc. as observed in western redcedar (Foster et al. 2016)remainto be determined.

Foliage Monoterpenes dominating yellow-cedar foliage have been studied further. Leaf essential oil from trees growing in their native range contain primarily limonene (35.4–42.2%), and lesser quantities of δ-3-carene (11.5–23.4%), α-pinene (8.7–16.3%), and sabinene (0.2–10.5%), with 0.4–0.9% ses- quiterpenes and 5.0–7.4% diterpenes (Adams et al. 2007). Outside its native environment in Spain the essential oil from young stems and leaves contain higher quantities of limonene (53.2%), similar proportions of δ-3-carene (21.0%), α-pinene (12.2%), and myrcene (6.1%), but considerably lower levels of sesquiterpenes (0.1%) and diterpenes (0.3%, Palá-Paúl et al. 2009). None of the major foliage components occur in other tissues, but there are five less abundant foliage com- pounds also in heartwood, two monoterpenes (terpinen-4-ol, and α-terpineol) and three sesquiterpenes (α-cadinol, δ- cadinene, and β-bisabolene). When present in foliage their ≤ Fig. 2 Tropolones that may occur in yellow-cedar heartwood concentrations are typically 0.7% (Adams et al. 2007; Andersen and Syrdal 1970; Cheng and von Rudloff 1970a; Palá-Paúl et al. 2009), and when present in heartwood oils or same study, chamic acid inhibited growth for six fungal spe- extracts they are usually ≥1.0% (Addesso et al. 2017; Kelsey cies at 0.01–0.02% and was fungicidal for two species at the et al. 2015; Khasawneh et al. 2011;Manteretal.2007). same concentrations. Wood infused with 1.1–1.2% nootkatin or carvacrol, or 1.0% carvacrol was protected from decay with Heartwood Several new compounds have been isolated from varying levels of efficacy, depending on the fungal species yellow-cedar heartwood since Barton’s(1976) review, includ- (Scheffer and Cowling 1966). In addition, yellow-cedar heart- ing two new oxygenated monoterpenes (1S)-2-oxo-3-p- wood blocks containing a natural mix of these compounds at menthenol and (4R)-4-hydroxy-4-isopropylcyclohex-1- their constitutive concentrations resisted decay from several enecarboxylic acid (Fig. 1) obtained from a methanol extract fungal species (Smith 1970; Smith and Cserjesi 1970). (Khasawneh et al. 2011). Four new sesquiterpenes have been The focus of this synthesis is on biological activities of new identified, although all were previously known in the litera- yellow-cedar compounds, and new or expanded activities for ture. Nootkatol, and valencene-13-ol were isolated from steam nootkatone and other previously known compounds, extracts, distilled essential oil (Khasawneh et al. 2011), while essential oils, and tissues reported since Barton’s(1976)re- valencene-11, 12-diol (syn tedonodiol) and kudtdiol (Fig. 3) view. Emphasis is placed on chemical studies linked closely to were isolated from a methanol extract (Khasawneh and yellow-cedar. It is not an exhaustive assessment of all biolog- Karchesy 2011). The absolute stereochemistry for nootkatol ical activities tested for each compound, since many occur in was resolved (Khasawneh et al. 2011), and both nootkatol and other plant species, are commercially available, or synthesized epi-nootkatol were detected in heartwood ethyl acetate ex- by the investigators. tracts by GC analysis (Kelsey et al. 2015). Heartwood essential oil analyzed by GC contained carva- crol (35.4%) as the major component, followed by four ses- Tissue Chemical Composition quiterpenes (Fig. 3), nootkatene (20.1%), nootkatone (17.4%), valencene-13-ol (6.4%) and nootkatol (5.2%) and the Conifer tissues often store constitutive defense chemicals in tropolone, nootkatin (3.5%, Khasawneh et al. 2011). specialized ducts or glands to limit their interference with life Heartwood ethyl acetate extracts analyzed by GC contained sustaining metabolic functions (Foster et al. 2016; Franceschi valencene-11, 12-diol (16.8%), nootkatol (10.6%), et al. 2005; Zulak and Bohlmann 2010). Yellow-cedar outer nootkatone (8.2%), valencene-13-ol (7.3%), carvacrol bark and phloem have resin ducts (Hennon et al. 2016), but the (7.1%) and nootkatin (6.4%, Kelsey et al. 2015). The same sapwood and heartwood do not (Wheeler and Arnette 1994). study reported additional tropolones, procerin (5.7%), a pos- The scale-like leaves are occasionally glandular (Michener sible procerin isomer (0.7%), and hinokitiol (β-thujaplicin, J Chem Ecol (2018) 44:510–524 513

Fig. 3 Sesquiterpenes in yellow-cedar heartwood. The bottom three compounds have also been reported in foliage at low concentrations

0.9%), but they were not isolated further (Fig. 2,Kelseyetal. additional hydroxyl (Zavarin et al. 1967). Carlsson et al. 2015). Compositional differences between essential oils and (1952) noted the loss of nootkatin from heartwood oil if dis- extracts noted above may result from environmental and tree tilled in an iron or copper still because of metal complex genetic differences at the geographic sites sampled. Also, dur- formation. ing essential oil distillation the hot steam and presence of any acidic constituents can facilitate structural conversions of sev- Bark Several diterpenes have been isolated by bioassay- eral compounds, especially the sesquiterpene diols that were guided fractionations (Fig. 4), including (+)-totarol from the found in extracts, but not in oil. Nootkatol can be converted to outer bark (Constantine et al. 2001), and five others from a nootkatene, and nootkatin can yield hydronootkatinol (Bicho mixed sample of stemwood/bark (Pettit et al. 2004). et al. 1963) that may go undetected with typical chromato- Nootkastatins 1 and 2 were totally new compounds, whereas graphic procedures because of its greater polarity from the 6, 7-dehydrototarol, 7α-hydroxytotarol, and 7α- 514 J Chem Ecol (2018) 44:510–524

Fig. 4 Diterpenes from yellow- cedar bark or foliage

methoxytotarol were previously known, but new to yellow- removed by steam distillation, or its solvent extracts. The di- cedar tissues (Pettit et al. 2004). versity of organisms impacted by the tissues or individual Condensed tannins were isolated and purified from fresh compounds has expanded to include ticks, fleas, termites, inner bark of yellow-cedar. Structural analysis by C-13 NMR ants, mosquitoes, bacteria, a water mold, and more fungi, and MALDI-TOF MS showed they were homogeneous and Table 1. Many flea, tick, and mosquito species targeted in heterogeneous oligomers of procyanidin (catechin/epicate- these bioassays are important to public health as common chin) and prodelphinidin (gallocatechin/epigallocatechin) vectors of human and animal pathogens causing disease flavan-3-ol monomer units (Rosales-Castro et al. 2014). The around the world (Dietrich et al. 2006; Panella et al. 2005; oligomers ranged in size from trimers to heptamers with Table 1). interflavan C8 → C4, or C6 → C4 linkages of the B-type dominant, as it is most common in proanthocyanidins (Fig. 5). This gave a condensed tannin profile including size and monomer composition of each individual oligomer chain. Such structural information is important when considering chemical and biological functions and activity.

Biological Activity

Tissues, Essential Oils, and Extracts Growth inhibition of wood decay fungi, and fungicide attributes were the most thorough- ly documented activity for yellow-cedar tissues and isolated compounds in Barton’s(1976) review. Since then, additional studies have expanded the activity categories to include bac- tericide, sporicide, acaricide, insecticide, general cytotoxicity, and anticancer activity (Table 1). Much of this work has been directed toward the heartwood, its volatile essential oils Fig. 5 Tannins in yellow-cedar inner bark J Chem Ecol (2018) 44:510–524 515

Table 1 Bioactivity of yellow-cedar tissues, essential oils, extracts, or isolated compounds

Bioactivity Plant part source Material tested Bioassay organismsa Reference

Arthropods (insects, arachnids, brine shrimp) Acaricide, Heartwood Sawdust, wood chips Ixodes scapularis Piesman 2006 or repellent Essential oil I. scapularis Panella et al. 1997 3-Carene, carvacrol, methyl carvacrol, I. scapularis, Panella et al. 2005; nootkatene, nootkatin, nootkatol, Dietrich et al. 2006 nootkatone, nootkatone 1 → 10 epoxide, nootkatone 11 → 12 epoxide, nootkatone diepoxide, terpinen-4-ol, valencene, valencene-13-aldehyde, valencene-13-ol, valencene-11, 12- diol Commercial Nootkatone I. scapularis, Behle et al. 2011 chemicals Bharadwaj et al. 2012 Amblyomma americanum, Dermacentor Flor-Weiler et al. 2011 variablis, I. scapularis, Rhipicephalus sanguineus Nootkatone, carvacrol Amblyomma americanum, Dolan et al. 2009; I. scapularis Jordan et al. 2011, 2012; Schulze et al. 2011 Insecticide Heartwood Essential oil Solenopsis invicta × S. richteri hybrid Addesso et al. 2017 Commercial Wood, heartwood, heartwood-sapwood mix, Coptotermes formosanus Morales-Ramos and trees, wood, or extracted heartwood Rojas 2001, lumber Morales-Ramos et al. 2003; Cornelius et al. 2004; Taylor et al. 2006 Reticulitermes flavipes Kirker et al. 2013 Isolated compounds and derivatives listed A. aegypti, X. cheopis Panella et al. 2005 above for Panella et al. 2005 Commercial Nootkatone, carvacrol Anopheles gambiae McAllister and Adams chemicals 2010 Cytotoxic Foliage, Extracts Artemia salina Karchesy et al. 2016 heartwood, inner bark, outer bark, sapwood

Microorganisms Bactericide Young stems/ Essential oil Bacillus subtilis (8) Palá-Paúl et al. 2009 leaves Branches/leaves, Nootkastatin 1, nootkastatin 2, Enterococcus faecalis, Micrococcus luteus, Pettit et al. 2004 stemwood/- 6, 7-dehyrototarol, 7α-hydroxytotarol, Neisseria gonorrhoeae, Rhodococcus bark 7α-methoxytotarol spp., Staphylococcus aureus, S. epidermidis, S. pneumoniae, S. pyogenes (11) Heartwood Essential oil Actinomyces bovis, Clostridium Johnston et al. 2001 perfringens, Fusobacterium necrophorum Outer bark Totarol Mycobacterium tuberculosis Constantine et al. 2001 Fungicide Young stems/ Essential oil Candida albicans Palá-Paúl et al. 2009 leaves Branches/leaves, Nootkastatin 1, nootkastatin 2, Cryptococcus neoformans (3) Pettit et al. 2004 stemwood/- 6, 7-dehyrototarol, 7α-hydroxytotarol, bark 7α-methoxytotarol Heartwood Essential oil, nootkatin C. albicans Johnston et al. 2001 Extracted wood Postia placenta Taylor et al. 2006 516 J Chem Ecol (2018) 44:510–524

Table 1 (continued)

Bioactivity Plant part source Material tested Bioassay organismsa Reference

Extract Gloeophyllum trabeum, Trametes Du et al. 2011 versicolor Wood blocks G. trabeum (3) DeGroot et al. 2000 Wood stakes Soil microbes (Alaska, Mississippi) Hennon et al. 2007 Wood, lumber Heartwood-sapwood mix Marasmiellus troyanus Cornelius et al. 2004 Wood, extracted wood G. trabeum, Irpex lacteus, P. placenta, T. Kirker et al. 2013 versicolor, Tyromyces palustris Extract infused wood G. trabeum, Kirker et al. 2016 I. lacteus, T. versicolor (4) Sporicide, Heartwood Wood shavings, essential oil, extract, Phytophthora ramorum Manter et al. 2006, 2007 hyphal nootkatin, carvacrol, methyl carvacrol, growth nootkatone, valencene, nootkatol, inhibitor valencene-13-ol, valencene-11, 12-diol

Human Anticancer Branches/leaves, Nootkastatin 1, nootkastatin 2, Cancer cells Pettit et al. 2004 stemwood/- 6, 7-dehyrototarol, bark 7α-hydroxytotarol, 7α-methoxytotarol a Species names listed only for those with the greatest response. Number in parenthesis is total number of taxa tested

Isolation of individual bioactive compounds has usually (McAllister and Adams 2010). Its repellent concentration, been guided with bioassays (Constantine et al. 2001; RC50, for nymphal I. scapularis in4hrlaboratorybioassays Dietrich et al. 2006; Karchesy et al. 2016; Panella et al. was 0.112% (wt:vol.) compared to 0.073% for technical 2005;Pettitetal.2004) leading to purification and structural grade DEET (N,N-diethyl-3-methylbenzamide) commonly identification. Retesting the purified compounds validated used in commercial insect repellent products (Dietrich et al. those with the strongest bioactivity, but mixtures of purified 2006).Infieldtrialsaqueous5%carvacrolsprayapplications compounds have seldom been tested to determine their poten- on forest floor litter completely suppressed nymphs of tial for synergistic effects. Yellow-cedar tissues contain mix- I. scapularis and the lone star tick Amblyomma americanum tures of several bioactive compounds and it is unknown to for 2 days with a 77.8% and 90.6% reduction in nymphs what extent this could synergize their activity, if any. through day 28 and 14, respectively (Dolan et al. 2009). In another field trial, a 2% carvacrol formulation applied to Monoterpenes Carvacrol antimicrobial activity has been ex- forest leaf litter provided ˃ 90% control of nymphs for both panded to include moderate inhibition of Phytophthora ramorum species 1 day after application (Jordan et al. 2011), but then zoospore germination and hyphal growth (Manter et al. 2006, declined to ˂ 80% by 14 days, when a second treatment was 2007). This microbe is a water mold causing leaf spot, and twig applied. This reapplication elevated activity toward both dieback on many horticultural plants, and mortality of susceptible species, but was most effective for I. scapularis with ˃ 90% tree species, particularly in the western U.S. and Great Britain control at day 35. (Frankel and Palmieri 2014).Ithasbeenreportedinmanyother Applying carvacrol to clothing is another control strategy European countries, but mainly at nurseries (European and tested with 0.8 mg/cm2 on cloth sheets dragged through a Mediterranean Plan Protection Organization 2017). forested area. This provided 100% repellency for nymphal Carvacrol possesses biocidal activity against ticks, Ixodes I. scapularis through day 3 post-treatment, or 95.2% and scapularis,fleas,Xenopsylla cheopis,andmosquitoes, 93.3% repellency for nymphal A. americanum at day 1 and

Aedes aegypti with LC50 (lethal concentration of 50% mor- 3, respectively (Schulze et al. 2011). Spray application of 2 tality, or EC50 half maximal effective concentration) at about 1 mg carvacrol/cm on clothing worn while walking 0.0068,0.0059,and0.0051%(wt:vol)after24hr,respective- through the forest provided 100% field repellency of ly, and some residual activity toward I. scapularis for up to I. scapularis adults for 1 day post-treatment, but little repellent 1week(Panellaetal.2005). Carvacrol also has insecticidal protection against adult A. americanum (Jordan et al. 2012). activity toward adult Anopheles gambiae mosquitoes, with a Carvacrol is also a major component in essential oil from plant mode of action unlike current commercial products genera used as spices, particularly oregano and thyme. The J Chem Ecol (2018) 44:510–524 517 essential oils from these sources and carvacrol are known to biocide resistance in target species (McAllister and Adams possess an extensive diversity of biological activities reviewed 2010). Antimicrobial activity of nootkatol, and valencene- by Baser (2008). 11, 12-diol was rated moderate at Log10 EC50 1.0–2.0 ppm, whereas valencene-13-ol was rated weak at Log10 EC50 2.0– Sesquiterpenes Nootkatone, nootkatol, and valencene-13-ol 3.0 ppm toward P. ramorum (Manter et al. 2007). Valencene are yellow-cedar heartwood compounds with significant bio- and nootkatone inhibited zoospore germination and hyphal cidal and repellent activity towards the flea, tick, and mosquito growth of P. ramorum in culture at concentrations of 100 to species above (Table 1). Nootkatone, is the most thoroughly 1000 ppm (Manter et al. 2006). studied in field trials with applications to the target species Four sesquiterpene derivatives of nootkatone and habitat at various 0.5% to 5% formulations sprayed on the valencene were synthesized (Fig. 6), nootkatone-1, 10-epox- forest litter layer, or lawn perimeters to control nymphs of ide, nootkatone 11, 12-epoxide, nootkatone-(1, 10–11, 12)- I. scapularis or A. americanum in Lyme disease endemic areas diepoxide and valencene-13-aldehyde (Khasawneh et al. (Bharadwaj et al. 2012; Dolan et al. 2009;Jordanetal.2011). 2011), and tested for biocidal activity toward I. scapularis, Efficacy varied by formulation, method of application and X. cheopis and A. aegypti in the same laboratory experiment target species. For example, 2% emulsified nootkatone ap- as the natural compounds (Panella et al. 2005). Valencene-13- plied by high pressure spray provided 96.2 to 100% suppres- aldehyde LC50 concentrations were similar to nootkatone at sion for nymphs of both species in the core treatment area 24 hr and up to 1 week for all test species. Nootkatone-1, 10- through day 42 post-application, whereas backpack applica- epoxide and nootkatone diepoxide were less effective, with tion of the same concentration was more variable and less LC50 concentrations 2–49 times and 8–22 times greater than effective (Dolan et al. 2009), but was enhanced with dual nootkatone at 24 hr, respectively (Panella et al. 2005). treatments 2 weeks apart, yielding 96.5 and 91.9% control Nootkatone-1-10-epoxide repelled nymphal I. scapularis for for I. scapularis and A. americanum nymphs through 42 and 4hratRC50 concentrations similar to DEET, whereas 35 days, respectively (Jordan et al. 2011). In another study, nootkatone-11, 12-diol and nootkatone-(1, 10–11, 12)- 0.84% emulsified nootkatone applied to lawn perimeters with diepoxide had no repellent activity (Dietrich et al. 2006). a hydraulic sprayer gave 100, 49, and 0% control of I. scapularis nymphs at 1, 2, and 3 weeks, respectively Tropolones Nootkatinwasshowntohavestrongactivity

(Bharadwaj et al. 2012). The decline in activity coincided with against P. ramorum at Log10 EC50 1.0 ppm, limiting zoospore nearly complete volatilization of nootkatone after 7 days. germination to 0% at 10 ppm or above, and inhibiting hyphal Nootkatone encapsulated in lignin, or as a Maillard formula- growth by 99.9% at 50 ppm (Manter et al. 2007), but growth tion (heated amino acid, carbohydrate mix) at 0.5 to 0.6% resumed when nootkatin was removed (Manter et al. 2006). concentrations were more persistent and reduced tick numbers Nootkatin was recently tested against Cryptococcus for 1 week or longer in field trials, but the reduction was not neoformans, a human fungal pathogen common in immuno- statistically lower than the controls post-treatment (Bharadwaj suppressed patients (Donlin et al. 2017). The minimum inhib- et al. 2012). itory concentration (MIC80)was18μM and the 50% cytotox- 2 Application directly to clothing was tested with 0.8 mg/cm icity concentration (CC50) was 18.5 μM, but it had a low of nootkatone on cloth sheets dragged through a forested area therapeutic index with limited potential for medicinal use. that provided 100% repellency for nymphs of both tick spe- Hinokitiol, although not very abundant in the heartwood is cies through day 14 post-treatment (Schulze et al. 2011). In a known to possess a variety of bioactivities (Saniewska et al. subsequent experiment, nootkatone applied to clothing at 2007, 2014;Zhao2007). about 1.0 mg/cm2 provided 100% repellency for adults of the same two tick species through day 1 post-treatment while Diterpenes Diterpenes isolated from yellow-cedar (Fig. 4)have walking through the forest (Jordan et al. 2012), plus extended significant antibacterial, antifungal and anticancer activities 100% control on I. scapularis and 96% on A. americanum (Table 1). (+)-Totarol from the outer bark is active against through day 3. After 7 days on clothing, nootkatone was more Mycobacterium tuberculosis, the causative agent of tuberculosis effective against both tick species than Permanone, a (Constantine et al. 2001). This was the first report of its activity permethrin-based commercial clothing repellent, or against this organism, although previously it was shown to im- EcoSMART®, an organic plant-derived essential oil insect pact gram-positive bacteria and other species of Mycobacterium repellent (Jordan et al. 2012). This level of activity is the basis (Constantine et al. 2001). The five related diterpenes, 6, 7- for ongoing develop of nootkatone as a commercial biopesti- dehyrototarol, 7α-hydroxytotarol, 7α-methoxytotarol, and cide discussed below. nootkastatins 1 and 2 from stemwood/bark possess activity to- Insecticidal actions of nootkatone against A. gambiae mos- ward various human cancer cells and several medicinally impor- quitoes, like carvacrol, appears to differ from currently used tant fungi and gram-positive bacteria (Pettit et al. 2004). Given products and has potential for combating the development of the medicinal activities of the diterpenes it is interesting to note 518 J Chem Ecol (2018) 44:510–524

Fig. 6 Semisynthetic derivatives of yellow-cedar sesquiterpenes tested for bioactivity

the uses of yellow-cedar bark, branches and foliage for tradition- with a double bond (Figs 1, 2, 3, 4 and 5). Carvacrol antimi- al medicines by native peoples. Boughs were used in sweat baths crobial activity is maximized by the aromatic ring and free for arthritis or rheumatism, and infusions to wash sores and hydroxyl group (Ultee et al. 2002; Veldhuizen et al. 2006), swellings (Turner and Bell 1973). with moderate activity against P. ramorum at Log10 EC50 1.8 ppm concentration (Manter et al. 2007), and LC50 concen- Polyphenols Yellow-cedar inner bark tannins (Fig. 5) were trations toward arthropods X. cheopis, I. scapularis,and tested for antioxidant activities using 1,1′-diphenyl-2- A. aegypti ranging from 0.0051 to 0.0068% (wt:vol) after picrylhydrazyl (DPPH), 2,2-azino-bis-3-ethylbenzothiazo- 24 hr exposure. Methyl carvacrol with a blocked hydroxyl line-6-sulfonic acid (ABTS) radical scavenging, and ferric was not active toward P. ramorum or the arthropods above reducing/antioxidant power (FRAP) that all involve electron (Manter et al. 2007; Panella et al. 2005). Interestingly, the transfer mechanisms, plus the β-carotene-linoleic acid model dominant foliage monoterpenes all lack an oxygen atom, system (β-CLAMS) assay based on hydrogen atom transfer and are not aromatic (Fig 1). reactions (Rosales-Castro et al. 2014). The condensed tannins The sesquiterpenes oxygen functionality can vary and still showed significant antioxidant activity with a median inhibi- have activity against arthropods. Nootkatone, valencene-13-al- tion capacity, IC50, comparable to the catechin control re- dehyde, and valencene-13-ol LC50 concentrations for sponse, and they have a high antioxidant capacity based on X. cheopis, I. scapularis,andA. aegypti all ranged from both the electron transfer mechanisms and hydrogen atom 0.0024 to 0.0085% (wt:vol) after 24 hr exposure (Panella transfer reactions. Proanthocyanidins are of great interest as et al. 2005). The two analog hydrocarbons, valencene and materials and medicines for humans, as well as their ecologi- nootkatene (Fig. 3) were also active toward these species, but cal functions for plants producing them (Dixon et al. 2005). less effective, with most LC50 concentrations between 0.011 to 0.041% (Panella et al. 2005). Nootkatone is repellent toward Coptotermes formosanus termites with a 12.5 μg/dish repellen- Structure and Functional Group Influence cy threshold. This activity is reduced by hydrogenating the on Bioactivity ketone to nootkatol, shifting the threshold level to 100 μg/dish, and hydrogenating the conjugated 1, 10 double bond in Bioactive compounds produced in yellow-cedar tissues exist nootkatone enhances this activity, dropping the threshold level in a variety of structures, including three terpenoid classes, to 1.56 μg/dish (Zhu et al. 2010). Other structural modifications tropolones, and tannins, yet there are some general common- also increase or decrease efficacy relative to nootkatone. alities across these groups. The most active compounds all Tropolones, especially hinokitiol have been extensively in- have one or more ring systems, at least one unsaturation, vestigated for their structure-activity relationships and associ- and one or more oxygen atoms as a ketone, alcohol, aldehyde, ated modes of action (see reviews by Meck et al. 2014; or epoxide usually attached to an aromatic ring, or conjugated Saniewska et al. 2007, 2014;Zhao2007). The seven J Chem Ecol (2018) 44:510–524 519 membered aromatic ring, and oxygen atoms are key structural Live Tree and Snag Longevity Yellow-cedar trees can live in components for their activities, modulated by the composition excess of 1000 years (Jozsa 1991), but as the twentieth century and structure of attachments to the ring. Formation of metal began, trees started dying in low elevation forests along the complexes by hinokitiol and other tropolones can inhibit coasts of southeast Alaska and Canada, initiating a decline metalloenzymes requiring the bound metal as a cofactor for (Hennon et al. 2016). This occurs when the roots freeze, as a activity. Proanthocyanidins can also bind metals (Dixon et al. result of complex interactions between adequate snow levels 2005), but this mode of action is not associated with the var- available to insulate roots from freezing that is influenced by ious terpenoids in yellow-cedar. Tropolones can also interfere climate change, and soil drainage, which impacts how deep roots with other enzymes, or protein receptors embedded in mem- can penetrate into soil for protection against freezing (Hennon branes, and microbial membranes may be lysed (Zhao 2007). et al. 2016). Projections suggest up to 50% of the North Pacific Nootkatin was observed bursting the membranes surrounding coast yellow-cedar area is at risk of not receiving adequate snow P. ramorum zoospores at concentrations from 1 to 10 ppm protection during winter in the next century, increasing exposure (Manter et al. 2006). Disruption of microbial membranes is a to potential mortality (Buma et al. 2017). The dead trees can mechanism shared also by the other terpene classes in yellow- stand as snags for 80 to 100 years while they shed foliage, bark, cedar tissues. Carvacrol penetrates microbial cell membranes sapwood, and branches leaving a tapered cylinder of heartwood causing structural changes that disrupt their normal functions until it is weakened by decay and falls to the ground. Five visu- (Chavan and Tupe 2014;Limaetal.2013; Ultee et al. 2002). ally recognizable standing snag classes corresponding with time The antibacterial activity of totarol and other diterpenes in- since death can be identified (Hennon et al. 1990). Snag long- volves penetrating host membranes, and is dependent on the term durability, and the longevity of live trees, are likely influ- structure having a hydrophobic decalin skeleton and a hydro- enced by the rich mixture of heartwood chemicals. philic fragment with one hydrogen-bond-donor group (Urzúa Concentrations of carvacrol, nootkatol, nootkatone, nootkatin et al. 2008). The hydrophobic portion inserts into the mem- and a total extractives (representing the summed quantities for branes lipophilic region, and the hydrophilic portion interacts 16 components) began to show decline in the class 3 snags with hydrogen-bond-acceptor groups within the lipid bilayer. (average of 26 years since death), as the sapwood weathered Heartwood accumulation of oxygenated compounds away after losing the protective bark (Kelsey et al. 2005). without resin ducts could provide additional benefits for Aged heartwood of the oldest class 5 snags (81 average age since defense against microbes by allowing the production of death) had the lowest quantities of these bioactive components. compounds with stronger action to disrupt microbial mem- In a separate study, wood blocks from class 5 snags had lost branes since they are stored in dead tissues. A comparable much of their chemical defense against Gloeophyllum trabeum level of activity might not be attainable if stored in resin compared to heartwood from live trees (DeGroot et al. 2000). ducts or glands because of potential damage to their mem- However, the bioactive chemicals did not protect heartwood of branes, similar to microbes discussed above. Absence of live trees, or class 3 and 5 snags from decay by two other fungi, sequestered storage in glands or ducts likely allows these Postia placenta and Serpula hemitioides (DeGroot et al. 2000). compound to be more uniformly deposited throughout the Environmental influences on decay resistance were detected in tissue, making them more difficult for invading microbes to field trials. Yellow-cedar heartwood stakes in contact with soil avoid. This dispersal has been documented for the phenolic deteriorated more rapidly in Mississippi than at Alaska test sites, antifungal pinosylvins in Scots pine, Pinus sylvestris, heart- likely the result of warmer temperatures over a longer period, wood cell walls, middle lamella, and lumina of tracheids and a different community of soil microbes and insects in (Belt et al. 2017). Similar dispersion occurs in Japanese Mississippi (Hennon et al. 2007). cedar, Cryptomeria japonica, heartwood where the diter- Limits to yellow-cedar heartwood chemical defenses were pene phenol, ferruginol, is uniformly distributed in cell detected in trees infected with unidentified fungi associated walls of tracheids, axial parenchyma cells, ray parenchyma with black-stained wood (Smith 1970; Smith and Cserjesi cells, and also inside these parenchyma cells (Imai et al. 1970). Most of the fungal strains were capable of moderate to 2005;Kurodaetal.2014). heavy growth at 0.5 mg nootkatin per 100 ml agar, and slight to moderate growth at 1.0 mg per 100 ml agar (Smith and Cserjesi 1970). When heartwood cubes were infected with these same fungal strains the nootkatin concentrations dropped to 2.7– Bioactive Constituents and Ecological 22.1% of the control 3 months after inoculation, clearly dem- Relationships onstrating the microbe’s abilities to tolerate and degrade high nootkatin concentrations. In addition to low levels of nootkatin, The bioactive compounds in various yellow-cedar tissues do fully stained heartwood may retain no carvacrol and only about influence the tree’s relationships with other organisms in their 24% of the nootkatone present in unstained tissues (Morales- natural environment, thus contributing to its autecology. Ramos et al. 2003). Thus, stained heartwood is less resistant to 520 J Chem Ecol (2018) 44:510–524 decay than unstained heartwood, but sufficient quantities of for commercial marketing as a repellent and biopesticide bioactive constituents may still remain in the black-stained against mosquitoes and ticks, and possibly other arthropod zones to provide some antifungal defense (Smith 1970). vectors of human and animal pathogens causing disease Reduction in heartwood defensive chemicals from colonization (Federal Laboratory Consortium for Technology Transfer by black stain fungi, or age-related changes in chemical com- 2018). Successful development of these products will require position may allow decay fungi to infect heartwood and even- a large, sustainable supply of nootkatone. tually reach the stem pith causing butt rot, or concentric rings of (+)-Nootkatone was initially isolated from yellow-cedar rot known as ring shake, that older trees commonly experience heartwood (Erdtman and Hirose 1962) and 2 years later from (Hennon et al. 2016). grapefruit, Citrus paradisi, peel oil (up to 0.3%), grapefruit juice, and other citrus fruit (< 0.01%, MacLeod and Buigues Animal Interactions Herbivory by Sitka black-tailed deer is the 1964). Subsequent studies reported it in roots of vetiver grass, primary cause of animal damage to yellow-cedar in southeast Vetiveria zizanioides (Andersen 1970; Zhu et al. 2001), fruit of Alaska, especially for seedlings and young saplings (Hennon Chinese black cardamom, Alpinia oxyphylla (Liang and et al. 2016). The foliar terpenoids appear to play a role in their Zheng 1992), rhizomes of the sedge Cyperus rotundus defense, as unbrowsed trees contained about 0.8 to 4% (dry (Tsoyi et al. 2011), and heartwood of Leyland cypress, matter basis) monoterpene, 0.03 to 0.2% sesquiterpenes, and xHesperotropsis leylandii, a hybrid between yellow-cedar approximately 2 to 3% diterpenes (Vourc’hetal.2002). The and Monterey cypress, Hesperocyparis macrocarpa (Kelsey heavily-browsed trees contained lower quantities of each group, et al. 2015). It was also detected at trace to 0.56% of the with about 0 to 0.35% monoterpenes, 0 to trace sesquiterpenes, essential oil obtained in 0.01% yield from the aerial parts of and 0 to 0.63% diterpenes. Which class of compounds had the six Teucrium species (Velasco-Negueruela and Pérez-Alonso most influence was not determined, although in the same study 1990). Because of limited plant materials, low concentrations, browsed western redcedar trees contained lower total monoter- or challenges and costs of purification from complex mix- pene concentrations than unbrowsed trees. The influence of tures, grapefruit has been the dominant plant source of natural redcedar monoterpenes on deer browsing has been further dem- nootkatone for commercial use as a grapefruit-like flavor and onstrated with a breeding program producing seedlings and cut- fragrance (Zviely 2009). It is also synthesized commercially tings with varying levels of monoterpene concentrations by oxidizing valencene, an abundant orange industry by-prod- impacting the browse resistance of individual trees, with higher uct, using various catalysts and reaction conditions concentrations increasing resistance (Kimball et al. 2012; (Leonheardt and Berger 2015; Zviely 2009). This nootkatone Russell and Ferguson 2010; Russell and Kimball 2010). is less expensive than the natural material but it cannot be Similar improvements may be possible for enhancing the pro- labeled Bnatural^ for use in food, flavor, fragrance, cosmetics tection of yellow-cedar from deer. In addition, yellow-cedar trees and pharmaceutical products (Fraatz et al. 2009; Leonheardt have fewer insect pests than many other conifers (Hennon 1991), and Berger 2015;Zviely2009). attributable in large part to the complex mixtures of compounds Desire for a less expensive, sustainable supply of Bnatural^ with activity toward arthropods, as discussed above. Yellow- nootkatone has focused research on molecular techniques, ge- cedar heartwood is resistant to attack by termites (Cornelius netic engineering, and biotechnological processes that include et al. 2004; Grace and Yamamoto 1994;Kirkeretal.2013; plant catalysts, cell cultures, or biotransformation by bacteria Morales-Ramos and Rojas 2001, Morales-Ramos et al. 2003; and fungi as reviewed by others (Fraatz et al. 2009;Leonhardt Taylor et al. 2006). This defense can be weakened by removing and Berger 2015; Wriessnegger et al. 2014). Yellow-cedar the extractives with solvents (Kirker et al. 2013;Tayloretal. heartwood has contributed toward this goal by providing a 2006), wood aging (Morales-Ramos and Rojas 2001), or mi- gene expressing a valencene synthase enzyme that produces crobe metabolism. Fully black-stained heartwood contains no substantial quantities of enzyme and (+)-valencene when carvacrol, about a quarter of the nootkatone in healthy tissues, expressed in select microorganisms (Beekwilder et al. 2014). and likely minimal nootkatin concentrations (Morales-Ramos A yellow-cedar cytochrome P450 valencene oxidase enzyme et al. 2003; Smith and Cserjesi 1970). Termites consumed has also been identified that catalyzes the oxidation of (+)- black-stained heartwood 3.1 times faster than unstained wood valencene to (+) nootkatone and trans-nootkatol when co- (Morales-Ramos et al. 2003). expressed with yellow-cedar valencene synthase in yeast (Cankar et al. 2014). A whole-cell yeast system with intracel- lular production of (+)-valencene by co-expression of Nootkatone Supply, Demand, and Human valencene synthase from yellow-cedar has shown potential Protection for high-level production of (+)-nootkatone in bioreactors (Wriessnegger et al. 2014). BNatural^ valencene and (+)-Nootkatone is undergoing research and development to nootkatone are now being commercially produced by fermen- support US Environmental Protection Agency registration tation of sugar using bioengineered bacteria or yeast for the J Chem Ecol (2018) 44:510–524 521 flavor and fragrance industries (Davies 2016). If a worldwide numerous chemicals with strong biological activities, espe- market is established for nootkatone as a repellent and biopes- cially in the heartwood. Several of these compounds are active ticide, it remains to be determined what proportion will be against wood decay fungi and a variety of arthropod species, supplied by these bioprocesses. providing an effective tree chemical defense that helps protect them for hundreds of years in their native environment. Nootkatone is one of the compounds with sufficient bioactiv- Research Opportunities ity toward arthropods to stimulate its potential development into commercial products for protecting humans and animals Promising yellow-cedar research topics involving its second- from pathogens vectored by ticks, mosquitoes and possibly ary metabolic products include breeding programs, or trans- other arthropods. Demand for an abundant, less costly genic and molecular manipulations to enhance tissue chemical Bnatural^ source of nootkatone for use in the food, fragrance, defenses that benefit tree management and wood products cosmetic, pharmaceutical, and possibly biopesticide industry durability. For example, deer browsing of young juvenile fo- has driven research toward producing it by enzymatic or mi- liage on seedlings and saplings adversely impacts yellow- crobial bioprocesses because sustainable supplies from plant cedar regeneration and is considered an urgent need for future sources, such as yellow-cedar, have limitations. management (Hennon et al. 2016). Deer resistance may be enhanced by increasing the concentrations of all, or just a few specific terpenes in the foliage, or incorporating genes from another tree, such as red cedar (Kimball et al. 2012; Russell and Ferguson 2010; Russell and Kimball 2010), to References substantially alter the chemical arsenal. Although natural du- rability of yellow-cedar heartwood is rated resistant to moder- Adams RP, Thomas P, Rushforth K (2007) The leaf essential oils of the new conifer genus, Xanthocyparis: Xanthocyparis vietnamensis and ately resistant from decay (Scheffer and Morrell 1998), its X. nootkatensis. J Essent Oil Res 19:30–33. https://doi.org/10.1080/ chemical defense is not active against all microbes. As a con- 10412905.2007.9699223 sequence, heartwood products perform best above ground Addesso KM, Oliver JB, O’Neal PA, Youssef N (2017) Efficacy of noot- with intermittent wetting, and are less desirable for products ka oil as a biopesticide for management of imported fire ants – in direct, long-term contact with soil (DeGroot et al. 2000; (Hymenoptera: Formicidae). J Econ Entomol 110:1547 1555. https://doi.org/10.1093/jee/tox114 Hennon et al. 2007). Modifying the heartwood chemical arse- Andersen NH (1970) Biogenetic implications of the antipodal sesquiter- nal could enhance durability of the final wood products. penes of vetiver oil. Phytochemistry 9:145–151. https://doi.org/10. 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Curr Pharm Des 14:3106–3120. sensitive target organisms with mixtures of two or more active https://doi.org/10.2174/138161208786404227 heartwood compounds will fill a gap in understanding oppor- Beekwilder J, van Houwelingen A, Cankar K, van Dijk ADJ, de Jong RM, tunities for synergistic enhancements of bioactivities. There is Stoopen G, Bouwmeester H, Achkar J, Sonke T, Bosch D (2014) also need for better insight into the relationships between Valencene synthase from the heartwood of Nootka cypress (Callitropsis chemical structure and function, plus the modes of action for nootkatensis) for biotechnological production of valencene. Plant Biotechnol J 12:174–182. https://doi.org/10.1111/pbi.12124 these compounds since there is some evidence they may work Behle RW, Flor-Weiler LB, Bharadwaj A, Stafford KC III (2011) A differently than existing biopesticide products (McAllister and formulation to encapsulate nootkatone for tick control. J Med Adams 2010). 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