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Article Activity of Yeast Particle-Encapsulated Terpenes

Zeynep Mirza 1, Ernesto R. Soto 1 , Yan Hu 1,2, Thanh-Thanh Nguyen 1, David Koch 1, Raffi V. Aroian 1 and Gary R. Ostroff 1,* 1 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA; [email protected] (Z.M.); [email protected] (E.R.S.); [email protected] (Y.H.); [email protected] (T.-T.N.); [email protected] (D.K.); raffi[email protected] (R.V.A.) 2 Department of Biology, Worcester State University, Worcester, MA 01602, USA * Correspondence: gary.ostroff@umassmed.edu; Tel.: 508-856-1930

 Academic Editor: Vaclav Vetvicka  Received: 2 June 2020; Accepted: 24 June 2020; Published: 27 June 2020

Abstract: Soil-transmitted (STN) infect 1–2 billion of the poorest people worldwide. Only are currently used in mass administration, with many instances of reduced activity. Terpenes are a class of compounds with anthelmintic activity. Thymol, a natural monoterpene , was used to help eradicate in the U.S. South circa 1910. However, the use of terpenes as was discontinued because of adverse side effects associated with high doses and premature absorption. Furthermore, the dose–response activity of specific terpenes against STNs has been understudied. Here we used hollow, porous yeast particles (YPs) to efficiently encapsulate (>95%) high levels of terpenes (52% w/w) and evaluated their anthelmintic activity on hookworms ( ceylanicum), a rodent parasite (Nippostrongylus brasiliensis), and whipworm (Trichuris muris). We identified YP–terpenes that were effective against all three parasites. Further, YP–terpenes overcame -resistant Caenorhabditis elegans. These results demonstrate that terpenes are broad-acting anthelmintics. Terpenes are predicted to be extremely difficult for parasites to resist, and YP encapsulation provides water-suspendable terpene materials without surfactants and sustained terpene release that could lead to the development of formulations for oral delivery that overcome fast absorption in the stomach, thus reducing dosage and toxic side effects.

Keywords: terpenes; essential oils; yeast particles; anthelmintic; ; whipworm

1. Introduction Soil-transmitted helminths or nematodes (STHs or STNs) are parasites of the (hookworm and ascarids) and large intestine (whipworm) that pose enormous health problems for humans. Approximately 1–2 billion people are infected [1]. The effects of STN are most pronounced on children, as the infections can result in physical growth stunting, delayed intellectual development, cognitive impairment, , malnutrition/lower nutritional status, school absenteeism, decreased future earnings, and immune defects that can lead to increased susceptibility to/virulence of malaria, tuberculosis, HIV/AIDS, and to failure [2–5]. Infections are also associated with an increased risk of maternal death, infant death, and low birth-weight babies in pregnant women [6] and with decreased adult worker health and productivity [7]. Thus, STN infections currently contribute significantly to trapping large populations in poverty worldwide. Because of the scale of the problem, the treatment of STN infections in humans depends upon mass drug administration (MDA). Only four , belonging to the class of compounds, are approved by the World Health Organization (WHO) for human STN therapy, and of these, only

Molecules 2020, 25, 2958; doi:10.3390/molecules25132958 www.mdpi.com/journal/molecules Molecules 2020, 25, 2958 2 of 13 one drug, albendazole, has adequate efficacy for MDA [8,9]. Albendazole has good efficacy against Ascaris, moderate and variable efficacy against hookworms [10], and poor efficacy against whipworms. Recent data from MDAs have revealed instances of the unusually low efficacy or reduced efficacy of albendazole over time against hookworms, Ascaris, and whipworms [10–15], raising the specter of parasite resistance (already rampant in veterinary medicine [16]). Furthermore, after repeated annual rounds of MDA, the transmission indices can reach plateau levels, indicating that the complete elimination of STN parasites from the community cannot be achieved with the limited anthelmintics in hand [14,17,18]. New classes of anthelmintics with broad specificity, and that are more difficult to resist than current therapies, are urgently needed. Terpenes and terpenoids are naturally occurring compounds that constitute the primary component of essential oils obtained from plants, and have the potential to be used as anthelmintics. Terpenes (e.g., cymene, limonene, pinene) are hydrocarbons consisting of isoprene repeating units. Terpenoids (e.g., terpineol, carvacrol, thymol) are modified terpenes containing additional functional groups (usually oxygen-containing groups). In this paper, the term terpene is used to include both terpenes and terpenoids. These compounds have been long recognized for a wide range of functional properties, such as their insecticidal, , and antibacterial activity [19,20], and many terpenes are safe and approved for applications in food, cosmetics, and in the pharmaceutical industry. The use of terpenes has played an important role in traditional medicine worldwide, for example, such as Asian wormwood and American wormseed contain terpenes that are thought to confer their reported anthelmintic properties [21–23]. Thymol was successfully used to help eradicate hookworm infections in the United States in the early 1900s [19,24]. Although effective, the use of thymol as an anthelmintic has long been discontinued. Large doses are required, as >90% of orally administered thymol is rapidly absorbed in the stomach and proximal intestine, resulting in less than 10% reaching the target sites where STNs reside. These large doses are also associated with toxic side effects, including mucosal irritation and nausea [24,25]. This limitation of thymol and other terpene-based therapies might be overcome by using encapsulation techniques and enteric coated formulations to target effective terpene doses directly at the site of STN infections, thus minimizing adverse side effects. Additionally, despite the success of thymol as a cure for hookworms, little systematic work has been done to study the efficacy of specific terpenes against STNs. A potential benefit of terpenes as an anthelmintic is that, given their generalized and multiple mechanisms of action evolved over the millennia as plant defense molecules, terpenes are extremely difficult to be resisted by target pathogens [26]. Screens for terpene-resistant variants have yet to uncover any significant resistance. For instance, in a forward genetic transposon screen for E. coli resistant to thymol, the best mutant barely increased the E. coli minimum inhibitory concentration (MIC) of thymol from 0.41 mM (wild type) to 0.53 mM (mutant) [27]. Another significant advantage of terpenes is their putative modes of action, worked out in their use as fungicides and bactericides. Terpenes have general membrane-disrupting capabilities, resulting in chemo-osmotic stress and the generalized death of their targets [28,29]. We have developed methods using yeast particles (YPs) to efficiently encapsulate high levels of terpenes. YPs are 3–5 µm hollow and porous microspheres, a byproduct of the food grade yeast (Saccharomyces cerevisae) extract manufacturing process. We have used yeast particles for the encapsulation of a broad range of molecules for drug delivery and agricultural applications [30–36]. In this article, we systematically studied the dose–response impact of terpenes on STN parasites. We screened 17 YP-encapsulated terpenes and three YP-encapsulated essential oil samples for in vitro activity against three parasitic species: the human hookworm parasite (a major human health problem in [10,37]), the murine whipworm Trichuris muris (a good model for human whipworm, Trichuris trichuria [38]), and the rat parasite Nippostrongylus brasiliensis, which is closely related to human hookworms. The results showed that terpenes vary in efficacy and can be categorized into five classes: (1) fast-acting and potent at moderate–high doses, (2) fast-acting at high doses, (3) slow-acting and potent at low doses, (4) slow-acting at moderate–high doses, and Molecules 2020, 25, x FOR PEER REVIEW 3 of 13

Molecules 2020, 25, 2958 3 of 13 terpenes vary in efficacy and can be categorized into five classes: 1) fast-acting and potent at moderate–high doses, 2) fast-acting at high doses, 3) slow-acting and potent at low doses, 4) slow-acting(5) non-effective at moderate–high at the doses tested. doses, Importantly, and 5) non-e activeffective terpenes at thewere doses equally tested. e ffImportantly,ective against active wild terpenestype and were an albendazole-resistant equally effective againstCaenorhabditis wild type elegans and anstrain, albendazole-resistant suggesting that terpenes Caenorhabditis could improve elegans strain,treatment suggesting against that drug-resistant terpenes could nematodes. improve treatment against drug-resistant nematodes.

2.2. Results Results

2.1.2.1. Yeast Yeast Particle Particle Encapsulation Encapsulation of of Terpenes Terpenes YeastYeast particles (YPs) (YPs) are are hollow, hollow, porous microparticles (3–5 (3–5 μµm)m) derived derived from from baker’s baker’s yeast. yeast. TheThe porous cell wall structurestructure makesmakes thesethese particlesparticles excellentexcellent absorbentabsorbent materials.materials. Terpene Terpene encapsulationencapsulation in in YPs YPs is is based based on on the the loading loading of ofterpenes terpenes inside inside the thehydrophobic hydrophobic YP cavity YP cavity by the by passivethe passive diffusion diffusion of terpene of terpene through through the porous the porous cell walls cell in walls an aqueous in an aqueous suspension suspension of YPs (Figure of YPs 1).(Figure We selected1). We selected17 low-cost, 17 low-cost, commerc commerciallyially available available terpenes terpenes(chemical (chemical structures structures depicted in depicted Figure S1in and Figure key S1 properties and key properties for YP loading for YP and loading characterization and characterization are listed in are Table listed S1) in and Table three S1) andessential three oilsessential (lavender, oils (lavender, tea tree, and tea tree,peppermint and peppermint oil) that contain oil) that several contain terpenes several terpenesfor encapsulation. for encapsulation. A high loadingA high loadingcapacity capacity (1.1:1 w/w (1.1:1 payload:YP)w/w payload:YP) was achieved was achieved with all with tw allenty twenty target target materials. materials. Nile red Nile dye red wasdye wasused used to stain to stain terpenes terpenes to qualitatively to qualitatively assess assess loading. loading. The The pictures pictures in in Figure Figure 11 provide visual evidenceevidence of of empty empty YPs YPs and and YPs fully loaded with a terpene (citral). Selected YP–terpene materials materials werewere extracted extracted in in 90% 90% methanol–10% methanol–10% water water to to quantify quantify terpene terpene loading loading by by HPLC. HPLC. The The HPLC HPLC results results showedshowed that that terpenes terpenes were were encapsulated encapsulated with with > 98% eefficiencyfficiency (Table S2).S2).

YP Terpene

Terpene loading in YP

10 μm

FigureFigure 1. 1. SchematicSchematic of diffusion-controlled diffusion-controlled terpeneterpene loadingloading in in yeast yeast particles particles (YPs) (YPs) and and micrographs micrographs of ofsamples samples stained stained with with Nile Nile red, red, showing showing empty empt YPsy YPs and and YPs YPs loaded loaded with with terpene terpene (citral). (citral). 2.2. Anthelmintic Activity of YP–Terpenes In Vitro 2.2. Anthelmintic Activity of YP–Terpenes In Vitro The anthelmintic activity of YP–terpenes was evaluated in vitro against four species of The anthelmintic activity of YP–terpenes was evaluated in vitro against four species of worms in the adult stage: the human hookworm parasite A. ceylanicum, the murine whipworm T. muris, in the adult stage: the human hookworm parasite A. ceylanicum, the murine whipworm T. muris, the the rat parasite N. brasiliensis, and albendazole-resistant and wild-type C. elegans. All studies were rat parasite N. brasiliensis, and albendazole-resistant and wild-type C. elegans. All studies were carried out at terpene concentrations from 0 to 333 µg/mL. Free terpenes and empty YPs were also carried out at terpene concentrations from 0 to 333 µg/mL. Free terpenes and empty YPs were also evaluated as controls. Empty YPs do not show toxicity on worms and active free terpenes require high evaluated as controls. Empty YPs do not show toxicity on worms and active free terpenes require concentrations (1 mg/mL) for effective toxicity on worms. All in vitro results below used YP–terpenes. high concentrations (1 mg/mL) for effective toxicity on worms. All in vitro results below used YP– YP–terpene activity against A. ceylanicum: The hookworm screen results showed that the efficacy terpenes. of YP–terpenes varies, and terpenes can be classified into five groups based on their dose–response YP–terpene activity against A. ceylanicum: The hookworm screen results showed that the curves (Figure2). The first class of terpenes, which consists of thymol, carvacrol, eugenol, geraniol, efficacy of YP–terpenes varies, and terpenes can be classified into five groups based on their dose– cinnamic aldehyde, and citronellol, was very effective at inactivating hookworms at moderate–high response curves (Figure 2). The first class of terpenes, which consists of thymol, carvacrol, eugenol, (200–333 µg/mL) doses in one to three hours. Yet, 100 µg/mL doses of these terpenes did not show geraniol, cinnamic aldehyde, and citronellol, was very effective at inactivating hookworms at activity in the same timeframe of 1 to 3 h. A second group of terpenes, consisting of , cymene, moderate–high (200–333 µg/mL) doses in one to three hours. Yet, 100 µg/mL doses of these terpenes limonene, and L-carvone, were effective only at high doses (333 µg/mL) at the three-hour time point. did not show activity in the same timeframe of 1 to 3 h. A second group of terpenes, consisting of anethole, cymene, limonene, and L-carvone, were effective only at high doses (333 µg/mL) at the three-hour time point.

Molecules 2020, 25, x FOR PEER REVIEW 4 of 13

The results for the other groups are as follows: The third group of terpenes, which consists of farnesol and nerolidol, were not effective in one to three hours; however, these terpenes killed the worms at lower doses (66 µg/mL) in 24 h. The fourth group of terpenes, which consists of alpha pinene, citral, alpha terpineol, linalool, and peppermint oil, showed activity only at the 24-h time Molecules 2020, 25, 2958 4 of 13 point at moderate–high doses. Finally, linalyl acetate, , and lavender oil did not show any activity against hookworms at the doses tested.

a. Fast-acting terpenes at moderate–high doses b. Fast-acting terpenes at high doses

Thymol Carvacrol Eugenol Anethole Cymene

Geraniol Cinnamic aldehyde Citronellol L-carvone Limonene

c. Slow-acting terpenes at low doses d. Slow-acting terpenes at moderate–high doses Farnesol Nerolidol Alpha-terpineol Alpha-pinene Citral

e. Not effective at doses tested Peppermint oil Linalool Lavender oil Tea tree oil Linalyl acetate

Figure 2. Dose–response of YP–terpenes on adult Ancylostoma ceylanicum in vitro, as shown for (a)Figure fast-acting 2. Dose–response terpenes at moderate–high of YP–terpenes doses, on adult (b) Ancylostoma fast-acting terpenes ceylanicum at highin vitro, doses, as shown (c) slow-acting for (a) terpenesfast-acting at lower terpenes doses, at (moderate–highd) slow-acting terpenesdoses, (b at) fast-acting moderate–high terpenes doses, at andhigh ( edoses,) terpenes (c) slow-acting not effective atterpenes the doses at tested. lower Terpenesdoses, (d were) slow-acting evaluated terpenes at concentrations at moderate–high from 0 to doses, 333 µ gand/mL. (e Parasitic) terpenes worms not effective at the doses tested. Terpenes were evaluated at concentrations from 0 to 333 µg/mL. were scored at the times indicated for motility, where 3 is actively moving, 2 is slow moving, 1 is Parasitic worms were scored at the times indicated for motility, where 3 is actively moving, 2 is slow not moving unless touched, and 0 is not moving even when touched (likely dead). N = eight adult moving, 1 is not moving unless touched, and 0 is not moving even when touched (likely dead). N = worms/condition. eight adult worms/condition. The results for the other groups are as follows: The third group of terpenes, which consists of YP–terpene activity against T. muris: The same YP–terpene samples were screened against T. farnesol and nerolidol, were not effective in one to three hours; however, these terpenes killed the muris (Figure 3). Overall, T. muris was less susceptible to terpenes than A. ceylanicum (minimum worms at lower doses (66 µg/mL) in 24 h. The fourth group of terpenes, which consists of alpha pinene, effective terpene doses against each worm are summarized in Table 1). The fast-acting terpenes at citral, alpha terpineol, linalool, and peppermint oil, showed activity only at the 24-h time point at moderate–high doses against A. ceylanicum, with the exception of eugenol, also immobilized T. muris moderate–high doses. Finally, linalyl acetate, tea tree oil, and lavender oil did not show any activity within between one and three hours. Eugenol, cymene, limonene, L-carvone, alpha pinene, citral, against hookworms at the doses tested. and linalyl acetate were effective at moderate–high doses at the 24-h time point. As seen with A. ceylanicumYP–terpene, farnesol activity and against nerolidolT. muris: actedThe slowly same against YP–terpene T. muris samples but required were screened low doses. against Lastly,T. muriswe (Figure3). Overall, T. muris was less susceptible to terpenes than A. ceylanicum (minimum effective terpene doses against each worm are summarized in Table1). The fast-acting terpenes at moderate–high doses against A. ceylanicum, with the exception of eugenol, also immobilized T. muris within between one and three hours. Eugenol, cymene, limonene, L-carvone, alpha pinene, citral, and linalyl acetate were effective at moderate–high doses at the 24-h time point. As seen with A. ceylanicum, farnesol and nerolidol acted slowly against T. muris but required low doses. Lastly, we did not observe any Molecules 2020, 25, 2958 5 of 13

Molecules 2020, 25, x FOR PEER REVIEW 5 of 13 anthelmintic activity with alpha-terpineol, linalool, anethole, peppermint oil, lavender oil, or tea tree oildid against not observeT. muris. any anthelmintic activity with alpha-terpineol, linalool, anethole, peppermint oil, lavender oil, or tea tree oil against T. muris. (a) Fast-acting terpenes at moderate–high doses (b) Fast-acting terpenes at high doses Thymol Carvacrol Cinnamic aldehyde No active terpenes against T. muris in this group

(c) Slow-acting terpenes at low doses

Geraniol Citronellol Farnesol Nerolidol

(d) Slow-acting terpenes at moderate–high doses (e) Not effective at the doses tested

Eugenol Alpha-pinene Cymene Lavender oil Tea tree oil

Citral Linalyl acetate Alpha terpineol Linalool

L-carvone Limonene Peppermint oil Anethole

Figure 3. Dose–response of YP–terpenes on adult Trichuris muris in vitro, as shown for (a) fast-acting Figure 3. Dose–response of YP–terpenes on adult Trichuris muris in vitro, as shown for (a) fast-acting b c terpenesterpenes at at moderate–high moderate–high doses,doses, ((b)) fast-actingfast-acting terpenes at at high high doses, doses, (c () )slow-acting slow-acting terpenes terpenes at at lowerlower doses, doses, (d )(d slow-acting) slow-acting terpenes terpenes at moderate–highat moderate–high doses, doses, and and (e) terpenes(e) terpenes not not effective effective at the at dosesthe µ tested.doses Terpenestested. Terpenes were evaluated were evaluated at concentrations at concentr fromations 0 tofrom 333 0 tog/ mL.333 µg/mL. Parasitic Parasitic worms wereworms scored were at thescored times at indicated the times for indicated motility, for where motility, 3 is actively where 3 moving, is actively 2 is moving, slow moving, 2 is slow 1 is notmoving, moving 1 is unless not touched,moving andunless 0 is touched, not moving and even 0 is whennot moving touched even (likely when dead). touched N = eight(likely adult dead). worms N =/ condition.eight adult worms/condition.

Molecules 2020, 25, x FOR PEER REVIEW 6 of 13 MoleculesMolecules 2020Molecules, 25 2020, x 2020 FOR,Molecules 25,, x25PEER FOR, x FOR2020 REVIEWPEER ,PEER 25 REVIEW, x FORREVIEW PEER REVIEW 6 of 136 of6 13of 13 6 of 13 Molecules 2020, 25, x FOR PEER REVIEW 6 of 13

TableTable 1. TableMinimum 1.Table Minimum1. MinimumTable 1.effective Minimum 1.effective Minimum effectivedoses effective dosesand doseseffective classification and doses and classification doses andclassification classificationofand YP–terpenes classificationof YP–terpenesof YP–terpenes of YP–terpenesagainst of YP–terpenesagainst A.against ceylanicum againstA. ceylanicumA. against ceylanicum A.and ceylanicum A.T. and ceylanicum and T. andT. T.and T. Table 1. Minimum effective doses and classification of YP–terpenes against A. ceylanicum and T. muris.muris. muris. muris. muris. muris.

A. ceylanicumA. A.ceylanicum ceylanicumA. ceylanicumA. ceylanicum T. murisT. murisT. murisT. muris T. muris A. ceylanicum T. muris Molecules 2020Terpene, 25,Terpene 2958 TerpeneTerpene Terpene 1 h 1 h 3h1 h 1 3hh 24 3h 1h h 24 3h h 241 h 3hh 24 1 h 3 1 24 hh h 1 3 h h 24 3 1h h 3 24 h h 24 3 h h 24 h 24 h 6 of 13 Terpene 1 h 3h 24 h 1 h 3 h 24 h CarvacrolCarvacrolCarvacrol Carvacrol Carvacrol 200 200 200200 200200 200200 200 200 200 333200 200 200 333 200333 200 333 200 20066 333 200 66 66200 66 66 Carvacrol 200 200 200 333 200 66 CinnamicCinnamicCinnamic aldehydeCinnamic aldehyde Cinnamicaldehyde aldehyde200 aldehyde200 200200 200200 200200 200 200 200 333200 200 200 333 200333 200 333 200 20066 333 200 66 66200 66 66 Table 1. MinimumCinnamic effective aldehyde doses and classification200 200 of YP–terpenes200 333 against 200A. ceylanicum66 and T. muris. ThymolThymol ThymolThymol Thymol200 200 200200 200200 100200 200 200 100>333 100 200 100>333 >333 200 100 >333 200100200 >333 200100 100 200 100 100 Thymol 200 200 100 >333 200 100 GeraniolGeraniol GeraniolGeraniol Geraniol200 200 200200 A.200200 ceylanicum 200200 200 200 200>333 200 200 200>333 >333 200 200 >333 200100200 >333 200100 T. muris100 200 100 100 Geraniol 200 200 200 >333 200 100 CitronellolCitronellolCitronellolTerpene Citronellol Citronellol 200 200 1 h200 200 200200 200200 3h200 200 200>333 200 200 24 h200>333 >333 200 200 >333 1200 h200200 >333 200200 3200 h200 200 24200 h Citronellol 200 200 200 >333 200 200 CarvacrolEugenolEugenol Eugenol 200 200 200 200200200 200 200200200 200 >333 200 >333 333>333 >333 >333 200200 >333 200 20066 EugenolEugenolCinnamic 200 200200 200200 200>333 >333>333 >333200 200 Eugenol 200 200200 200 200 200 333>333 >333200 200 66 AnetholeAnetholealdehydeAnethole Anethole Anethole333 333 333333 333333 200333 333 333 200>333 200 333 200>333 >333>333 200 >333>333 >333>333 >333 >333>333 >333 >333 >333 >333 Anethole 333 333 200 >333 >333 >333 CymeneCymene ThymolCymeneCymene Cymene>333 >333200 >333333 >333333200200333 >333 333200 >333 200 333100 200>333 >333>333 200 >333>>333 333>333333 >333 >333333 200333 >333 333 333100 GeraniolCymene 200 >333200 333 200 200 >333>333 >333200 333 100 LimoneneLimoneneLimonene Limonene Limonene >333 >333>333333 >333333333333 >333 333333 >333 333 333 333>333 >333>333 333 >333>333 >333200 >333 >333200 200 >333 200 200 CitronellolLimonene 200 >333200 333 200 333 >333>333 >333200 200 200 L-carvoneL-carvoneL-carvoneEugenol L-carvone L-carvone >333 >333200>333333 >333333333200333 >333 333333 >333 333 333200 333>333 >333>333 333 >333>>333 333>333333 >333 >333333 >333 >333 333 333200 L-carvone >333 333 333 >333 >333 333 FarnesolFarnesolAnethole FarnesolFarnesol Farnesol>333 >333333 >333>333 >333>333>333333 66 >333 >333 66>333 66>333200 >33366 >333>333 66 >333 >>333333>333 33 >333 >333 33> 333 33>333 33 >33333 CymeneFarnesol >333 >333333 >333 200 66 >333>333 >333>333 33 333 NerolidolNerolidolNerolidol Nerolidol Nerolidol>333 >333>333>333 >333>333>333 66 >333 >333 66>333 66>333 >33366 >333>333 66 >333 >333>333 66 >333 >333 66 66>333 66 66 LimoneneNerolidol >333 >333333 >333 333 66 >333>333 >333>333 66 200 AlphaAlpha pineneAlphaL-carvone pineneAlpha pineneAlpha pinene>333 pinene >333>333>333>333 >333>333>333200333 >333 >333200 >333200 >333333 200>333 >333>333 200 >333>>333 333>333200 >333 >333200 >333200 >333 200 200333 Alpha pinene >333 >333 200 >333 >333 200 Citral CitralFarnesolCitral Citral >333Citral >333> 333>333>333 >333>333>>333333 333 >333 >333333 >333333 >33366 333>333 >333 -333 >333> 333- 200 >333- 200 - >333200 - 200 20033 Nerolidol Citral >333 >333>333 >333 66 333 >333>333 >-333 200 66 AlphaAlpha terpineolAlpha terpineolAlpha terpineol Alpha terpineol >333 terpineol >333 >333>333 >333>333>333333 >333 >333333 >333333 >333 333>333 >333>333 333 >333>333 >333>333 >333 >333>333 >333 >333 >333 >333 AlphaAlpha pinene terpineol>333 >333>333 >333 200 333 >333>333 >333>333 >333200 LinaloolLinalool LinaloolCitralLinalool Linalool>333 >333>333 >333>333 >333>333>>333333 333 >333 >333333 >333333 >333333 333>333 >333>333 333 >333>>333 333>333>333 >333 >333>333 >333 ->333 >333 >333200 Linalool >333 >333 333 >333 >333 >333 PeppermintPeppermintAlphaPeppermint oilPeppermint terpineol oilPeppermint oil>333 oil >333> 333 >333>333oil >333>333>>333333 333 >333 >333333 >333333 >333333 333>333 >333>333 333 >333>>333 333>333>333 >333 >333>333 >>333333 >333 >333 >333>333 LinaloolPeppermint oil>333 >333>333 >333 333 333 >333>333 >333>333 >333> 333 LinalylLinalyl acetateLinalyl acetateLinalyl acetateLinalyl acetate>333 acetate >333 >333>333 >333>333>333>333 >333 >333>333 >333>333 >333 >333>333 >333>333 >333 >333>333 >333333 >333 >333333 333 >333 333 333 PeppermintLinalyl oil acetate>333 >333>333 >333 333 >333 >333>333 >333>333 333> 333 LavenderLavenderLinalylLavender oil Lavender acetate oil Lavenderoil>333 oil >333> 333oil>333>333 >333>333>333>>333 333 >333 >333>333 >333>333 >333>333 >333>333 >333>333 >333 >333>>333 333>333>333 >333 >333>333 >>333333 >333 >333 >333333 Lavender oil >333 >333 >333 >333 >333 >333 Tea treeTeaLavender oilTea tree treeTea oil oil oiltreeTea >333 oil tree >333> oil333>333>333 >333>333>333>>333 333 >333 >333>333 >333>333 >333>333 >333>333 >333>333 >333 >333>>333 333>333>333 >333 >333>333 >>333333 >333 >333 >333>333 Tea treeTea oil tree oil> 333 >333>333 >333> 333>333 >333>333 >333>333 >333> 333

Fast acting Fast Fast acting at Fastactingmoderate-high at acting Fast moderate-high at moderate-high acting at moderate-high doses at moderate-high doses doses doses Fast doses acting Fast Fast acting at Fastactinghigh at acting Fastdoses high at high acting dosesat high doses at doseshigh Slow doses acting Slow Slow acting at Slow actinglow at Slowdosesacting low at low acting doses at doseslow at doses low doses FastFast acting acting at at moderate-high moderate-high dosesdoses Fast Fast acting acting at high at high doses doses Slow acting Slow at acting low doses at low doses Slow acting Slow Slow acting at Slow actingmoderate-high at Slowacting moderate-high at moderate-high acting at moderate-high doses at moderate-high doses doses doses Not doses effective Not Not effective effectiveat Not doses effective Notat doses testedat effective doses at tested doses tested at doses tested tested Slow acting Slow at moderate-high acting at moderate-high doses Not doses effective at doses Not tested. effective at doses tested

YP–terpeneYP–terpeneYP–terpene activity activity againstactivity against N.against brasiliensis: N. brasiliensis: N. brasiliensis: After After testing After testing the testing terpenesthe terpenesthe terpeneson A.on ceylanicum A. on ceylanicum A. ceylanicum and andT. T.and T. YP–terpeneYP–terpene activityYP–terpene activity against activityagainstactivity N. brasiliensis: againstN.against brasiliensis:N. N. After brasiliensis brasiliensis: After testing: testing After Afterthe testingterpenes the testing terpenes the onthe terpenes A. terpeneson ceylanicum A. on ceylanicumA. on ceylanicum A.and ceylanicum T.and and T. T. and muris T., murismuris, wemuris ,screened we screened, we screenedselected selected YP–terpenesselected YP–terpenes YP–terpenes on N. on brasiliensis N. on brasiliensis N. brasiliensis (Figure (Figure 4) (Figure. The 4). Thedose–response 4). dose–responseThe dose–response curves curves were curves were were murismuris, we screened, wemuriswe screened screened ,selected we screened selected selected YP–terpenes YP–terpenesselected YP–terpenes on YP–terpenes N. on brasiliensis on N.N. brasiliensis brasiliensis on (FigureN. brasiliensis (Figure(Figure 4). The 4) (Figure4 .).dose–response The The dose–response dose–response4). The dose–response curves curves curves were were werecurves similar were similarsimilar tosimilar those to those toobtained those obtained obtainedwith with A. ceylanicum withA. ceylanicum A. ceylanicum and andT. muris T.and muris . T.While muris. While carvacrol,. While carvacrol, carvacrol, thymol, thymol, andthymol, andcinnamic cinnamicand cinnamic similarsimilar to those tosimilarto those thoseobtained toobtained obtained those with obtained withA. with ceylanicum A. ceylanicum with A.and ceylanicum T.and muris T.T. murismuris. andWhile. .T. WhileWhile carvacrol,muris carvacrol,carvacrol,. While thymol, carvacrol, thymol, thymol, and and cinnamicthymol,and cinnamic cinnamic and aldehydecinnamic aldehydealdehydealdehyde immobilized immobilized immobilized N. brasiliensis N. brasiliensis N. brasiliensis worms worms within worms within one within onehour hourone at 333 hour at 333and at and200333 200µg/mLand µg/mL200 doses, µg/mL doses, geraniol doses, geraniol geraniol aldehydealdehyde immobilizedaldehydeimmobilized immobilized N.immobilized brasiliensisN. N. brasiliensis brasiliensis N.worms brasiliensisworms worms within within within worms one one hour one within hour hourat at333 one 333at and 333hour and 200and 200at µg/mL 333200µg /andmLµg/mL doses, doses,200 doses, µg/mLgeraniol geraniol geraniol doses, and geraniol eugenol and andeugenol eugenoland wereeugenol were less were lesseffective. effective.less effective. Farnesol Farnesol Farnesolwas wasstill stilleffectivewas effectivestill effectiveat low at lowdoses at doseslow at thedoses at 24-hthe at 24-h timethe 24-htime point. timepoint. point. and eugenoland eugenol wereandwere eugenolwereless less effective. elessffective. were effective. lessFarnesol Farnesol effective. Farnesol was was still Farnesolwas still effective estillffective waseffective atstill at low low effectiveat doses doseslow doses at at the thelow at 24-h 24-h dosesthe time24-h time at point.thetime point. 24-h point. time point.

FigureFigure 4.Figure Effect 4.Figure Effect of4. selectEffectFigure 4. of Effect selectYP-terpenesof 4. select Effect of YP-terpenes select YP-terpenes of selecton YP-terpenes N. onbrasiliensis.YP-terpenes N.on brasiliensis.N. on brasiliensis. N. N on brasiliensis.= eightN. N brasiliensis. = N adulteight = eightN adultworms/condition.= eight adultN =worms/condition. eightadult worms/condition. adultworms/condition. worms/condition. Figure 4. EffectEffect of select YP-terpenes on N. brasiliensis. N = eighteight adult adult worms/condition. worms/condition. YP-terpeneYP-terpeneYP-terpene activity activity against activity against C. against elegans: C. elegans: C. Terpenes elegans: Terpenes Terpenes classified classified classified as fast-a as fast-acting as fast-acting at moderate–highcting at moderate–high at moderate–high doses doses doses YP-terpeneYP-terpene activityYP-terpene activity against againstactivity activity C. elegans: C. against elegans: Terpenes C. Terpenes elegans: classified TerpenesTerpenes classified as fast-a classified classified as fast-acting ctingasat moderate–highfast-a fast-acting at moderate–highcting at moderate–high doses doses doses in thein hookwormthein hookworm the hookworm and andwhipworm whipwormand whipworm screening screening screening were were evaluated were evaluated ev onaluated wild-type on wild-type on wild-type and andalbendazole-resistant albendazole-resistantand albendazole-resistant in thein hookworm the hookworminin the the and hookworm whipworm and whipworm and and screening whipworm screening were screening screening wereevaluated ev aluatedwere on wild-typeev evaluated onaluated wild-type andon wild-type albendazole-resistant and albendazole-resistant and albendazole-resistant C. elegans to assess whether these terpenes can overcome albendazole ben-1(e1880) resistance. The results

showed that LD50 values were very similar for each terpene on wild-type and ben-1(e1880) worms (Figure5), indicating that the mechanism of action of these terpenes is di fferent than albendazole. Molecules 2020, 25, x FOR PEER REVIEW 7 of 13

C. elegans to assess whether these terpenes can overcome albendazole ben-1(e1880) resistance. The results showed that LD50 values were very similar for each terpene on wild-type and ben-1(e1880) wormsMolecules (Figure2020, 25, x FOR5), indicatingPEER REVIEW that the mechanism of action of these terpenes is different7 of 13than albendazole. C. elegans to assess whether these terpenes can overcome albendazole ben-1(e1880) resistance. The

results showed that LD50 values were very similar for each terpene on wild-type and ben-1(e1880) Moleculesworms2020 (Figure, 25, 2958 5), indicating that the mechanism of action of these terpenes is different than7 of 13 albendazole.

Figure 5. Dose–response curves and LD50 values of fast-acting YP-terpenes on (a) wild-type (WT) and (b) albendazole ben-1(e1880)-resistant C. elegans. N = 20 L4–adult worms per condition scored at 6 days. FigureFigure 5. 5.Dose–response Dose–response curvescurves and and LD50 LD50 values values of of fast-acting fast-acting YP-terpenes YP-terpenes on on ( a()a wild-type) wild-type (WT) (WT) and 2.3. Kinetics(band) albendazole (b) ofalbendazole Terpeneben-1(e1880 Release ben-1(e1880)-resistant from)-resistant YPsC. elegans C. elegans.N =. N20 = L4–adult 20 L4–adult worms worms per per condition condition scored scored at 6at days. 6 days. 2.3. KineticsTerpene of release Terpene from Release YPs from is YPsbased on passive diffusion out of the particles and is a function of terpene2.3. TerpeneKinetics water of release Terpenesolubility. from Release YP–terpene YPs from is basedYPs samples on passive were di ffdilutedusion out at ofdifferent the particles terpene and concentrations is a function ofin water, and the terpene release from particles into the supernatant was quantified at different terpeneTerpene water solubility.release from YP–terpene YPs is based samples on passive were diluteddiffusion at out diff erentof the terpeneparticles concentrations and is a function in water, of andtimepoints the terpene by HPLC. release fromThe graphs particles of into cumulative the supernatant terpene was release, quantified as a function at different of timepointstime at three by terpene water solubility. YP–terpene samples were μdiluted at different terpene concentrations in HPLC.different The dilutions graphs ofin cumulative water (66, terpene 200, and release, 333 asg/mL), a function are ofshown time atin threeFigure diff erent6 for dilutionsterpenes representativewater, and the of terpene each grouprelease classified from particles based inonto anthelminticthe supernatant activity. was quantified Carvacrol, at a different fast-acting intimepoints water (66, by 200, HPLC. and 333Theµ ggraphs/mL), areof cumulative shown in Figure terpene6 for release, terpenes as a representative function of time of each at three group terpene with a maximum solubility in water of 1.3 mg/mL, shows a fast release (> 90% at 1 h) from classifieddifferent baseddilutions on anthelmintic in water (66, activity. 200, Carvacrol,and 333 μ ag/mL), fast-acting are terpeneshown within Figure a maximum 6 for solubilityterpenes in YPs at all concentrations. L-carvone has a similar solubility in water as carvacrol, but it shows a waterrepresentative of 1.3 mg /mL,of each shows group a fast classified release ( >based90% aton 1 h)anthelmintic from YPs at activity. all concentrations. Carvacrol, L-carvonea fast-acting has a slower rate of release from YPs (40–60% at 3 h) and almost complete terpene release at 24 h. The lipid similarterpene solubility with a maximum in water assolubility carvacrol, in water but it of shows 1.3 mg/mL, a slower shows rate ofa fast release release from (> YPs90% (40–60%at 1 h) from at 3 h) content in YPs is likely to impact the release of terpenes upon dilution in water, as shown in the andYPs almost at all concentrations. complete terpene L-carvone release athas 24 a h. similar The lipid solubility content in in water YPs is as likely carvacrol, to impact but theit shows release a of different release patterns of two terpenes (carvacrol and L-carvone) with similar solubility in water. terpenesslower rate upon of release dilution from in water, YPs (40–60% as shown at 3 in h) the and di almostfferent complete release patterns terpene ofrelease two terpenesat 24 h. The (carvacrol lipid andThiscontentl -carvone)reduction in YPs withisin likelyterpene similar to impactrelease solubility thecorrelates inrelease water. ofwith Thisterpenes the reduction experimental upon indilution terpene observation in releasewater, correlatesas that shown L-carvone in with the the is fast-acting on nematodes only at high concentrations. Terpenes that are slow-acting at high experimentaldifferent release observation patterns of that two L-carvone terpenes (carvacrol is fast-acting and L on-carvone) nematodes with onlysimilar at solubility high concentrations. in water. TerpenesconcentrationsThis reduction that are (e.g.,in slow-acting terpene citral) releaseshow at high acorrelates slow concentrations release with profile the (e.g., experimental from citral) YPs show (30–40% observation a slow at release t that= 3h profile L-carvoneat 200 from and is YPs333 μ (30–40%fast-actingg/mL). Terpenes at ton= 3hnematodes atthat 200 do and not only 333show atµ ganthelmintichigh/mL). concentrations. Terpenes acti thatvity also doTerpenes not do shownot thatshow anthelmintic are significant slow-acting activity terpene at alsohigh release do notfromconcentrations show YPssignificant (e.g., (e.g.,linalyl terpenecitral) acetate). show release The a slow fromterpenes release YPs in (e.g., profile this linalyl group from acetate). haveYPs (30–40% very The low terpenes at solubilityt = 3h in at this 200in group waterand 333 have(< 10 µg/mL) and remain trapped in the hydrophobic YPs. Finally, two highly water insoluble terpenes veryμg/mL). low Terpenes solubility that in waterdo not (

Figure 6. Cumulative terpene release from YP-terpene samples diluted in water at terpene concentrationsFigure 6. Cumulative of 66, 200, terpene and 333 µreleaseg/mL. from YP-terpene samples diluted in water at terpene concentrations of 66, 200, and 333 μg/mL. 2.4. DiFigurefference 6. in Cumulative Uptake of YP–Terpenes terpene release by Hookwormsfrom YP-terpe andne Whipworms. samples diluted in water at terpene concentrations of 66, 200, and 333 μg/mL. Hookworms and whipworms showed differential susceptibility to YP–terpenes. We hypothesized that the difference in terpene activity between these two parasite species might be explained by YP–terpenes acting with two different mechanisms: (1) terpene release from YP–terpenes to the medium, followed by terpene absorption by the worms and (2) YP–terpene by worms and subsequent terpene release inside the worms. Rhodamine-labeled YPs (rYPs) were used to evaluate YP uptake by worms and the results after a 24 h incubation show that A. ceylanicum avidly Molecules 2020, 25, x FOR PEER REVIEW 8 of 13

2.4. Difference in Uptake of YP–Terpenes by Hookworms and Whipworms. Hookworms and whipworms showed differential susceptibility to YP–terpenes. We hypothesized that the difference in terpene activity between these two parasite species might be explained by YP–terpenes acting with two different mechanisms: 1) terpene release from YP– terpenesMolecules 2020 to ,the25, 2958medium, followed by terpene absorption by the worms and 2) YP–terpene ingestion8 of 13 by worms and subsequent terpene release inside the worms. Rhodamine-labeled YPs (rYPs) were usedeats rYPsto evaluate (Figure YP7). uptake These resultsby worms support and the that results the YP–terpene after a 24 h e incubationffect on A. show ceylanicum that A.proceeds ceylanicum by avidlytwo mechanisms, eats rYPs but(Figure YP–terpene 7). These activity results on supportT. muris thatis limited the YP–terpene to only terpene effect release on A. from ceylanicum YPs to proceedsthe medium. by two mechanisms, but YP–terpene activity on T. muris is limited to only terpene release from YPs to the medium.

Figure 7. MicroscopyMicroscopy picturespictures showing showing the the ingestion ingestion of of YPs YPs by hookworms,by hookworms, but notbut whipworms,not whipworms, after aftera 24 ha incubation24 h incubation with with rhodamine-labeled rhodamine-labeled YPs. YPs. 3. Discussion 3. Discussion Although terpenes have been used in humans as anthelmintics for more than 100 years, there is a Although terpenes have been used in humans as anthelmintics for more than 100 years, there is surprising dearth of careful research in this area. Here, we systematically look at the dose–response a surprising dearth of careful research in this area. Here, we systematically look at the dose–response efficacy of a wide range of terpenes against a phylogenetically diverse range of STNs. The potential efficacy of a wide range of terpenes against a phylogenetically diverse range of STNs. The potential major advantages of terpenes are: (1) they are broad acting, (2) they are predicted to be extremely major advantages of terpenes are: 1) they are broad acting, 2) they are predicted to be extremely difficult for parasites to resist, and (3) their pharmacodynamics may be vastly improved by modern difficult for parasites to resist, and 3) their pharmacodynamics may be vastly improved by modern formulation strategies (e.g., encapsulation and enteric coated capsules to protect premature release and formulation strategies (e.g., encapsulation and enteric coated capsules to protect premature release absorption in the stomach) that are predicted to overcome their traditional limitations with regards to and absorption in the stomach) that are predicted to overcome their traditional limitations with unpleasant taste and toxicity. regards to unpleasant taste and toxicity. The entrapment of hydrophobic terpenes in YPs occurs by passive diffusion through the YP shell The entrapment of hydrophobic terpenes in YPs occurs by passive diffusion through the YP into its hydrophobic interior, forming a terpene oil droplet filling the hollow cavity inside the YP shell into its hydrophobic interior, forming a terpene oil droplet filling the hollow cavity inside the (Figure1). Terpene encapsulation in YPs o ffers several major advantages for in vitro testing: (1) high YP (Figure 1). Terpene encapsulation in YPs offers several major advantages for in vitro testing: 1) terpene load levels (50% terpene w/w), (2) water-suspendable terpene formulations without surfactants, high terpene load levels (50% terpene w/w), 2) water-suspendable terpene formulations without (3) high terpene bioavailability, and (4) sustained terpene release. surfactants, 3) high terpene bioavailability, and 4) sustained terpene release. The dose–response screening of YP–terpenes in vitro against the hookworm A. ceylanicum The dose–response screening of YP–terpenes in vitro against the hookworm A. ceylanicum (Figure2) and the whipworm T. muris (Figure3) show that the terpenes may be grouped into (Figure 2) and the whipworm T. muris (Figure 3) show that the terpenes may be grouped into five five pharmacodynamic classes: (1) fast-acting terpenes at moderate–high doses (100–333 µg/mL), pharmacodynamic classes: 1) fast-acting terpenes at moderate–high doses (100–333 μg/mL), 2) (2) fast-acting at high doses (333 µg/mL), (3) slow-acting at lower doses (33–66 µg/mL), (4) slow-acting fast-acting at high doses (333 μg/mL), 3) slow-acting at lower doses (33–66 μg/mL), 4) slow-acting at at moderate–high doses (100–333 µg/mL), and (5) not effective at the doses tested. moderate–high doses (100–333 μg/mL), and 5) not effective at the doses tested. Most fast-acting terpenes have a solubility in water higher than the dose tested, allowing for rapid Most fast-acting terpenes have a solubility in water higher than the dose tested, allowing for terpene release from YPs in 1–3 h (e.g., carvacrol, Figure6). Terpenes like L-carvone also have a high rapid terpene release from YPs in 1–3 h (e.g., carvacrol, Figure 6). Terpenes like L-carvone also have solubility but the kinetics of release are slow, likely due to terpene–lipid interaction in YPs, and as a high solubility but the kinetics of release are slow, likely due to terpene–lipid interaction in YPs, result of the slower rate of terpene release, L-carvone is fast-acting only at high doses. Terpenes in the and as result of the slower rate of terpene release, L-carvone is fast-acting only at high doses. slow-acting at a moderate–high dose group have a low water solubility, requiring a longer incubation Terpenes in the slow-acting at a moderate–high dose group have a low water solubility, requiring a time ( 24 h) to achieve an effective terpene dose to release from YPs. Interestingly, terpenes in the longer≥ incubation time (≥ 24 h) to achieve an effective terpene dose to release from YPs. Interestingly, slow-acting at a low dose group are extremely water insoluble, but we found out that terpenes in this group (farnesol, nerolidol) self-emulsify in YP–terpene aqueous suspensions, releasing the terpene at rates 20–30 times higher than its maximum solubility in water. Overall, T. muris was less susceptible to terpenes than A. ceylanicum. For example, the fast-acting terpenes at moderate–high doses showed an effect on A. ceylanicum in 1 h at 200 µg/mL, but it took Molecules 2020, 25, 2958 9 of 13 a slightly longer time (3 h) for these terpenes to be active on T. muris at the same concentration. Additionally, we did not identify any terpenes in the second group (fast-acting at a high dose) against T. muris. Relative to A. ceylanicum, more terpenes were slow-acting at moderate–high doses against whipworm, or not even effective at all. The screening results of selected YP–terpenes on N. brasiliensis (Figure4) show that this hookworm species is susceptible to all the selected terpenes with carvacrol, thymol, and cinnamic aldehyde, showing complete worm incapacitation in 1 h at 200 µg/mL. This rat parasite is closely related to human hookworms, making it a convenient intestinal nematode/rodent laboratory model that we will use in future studies to test the efficacy of orally administered YP–terpenes. To determine if terpenes are able to overcome anthelmintic resistance, we treated the free-living laboratory nematode Caenorhabditis elegans, both wild-type and albendazole-resistant genotypes, with YP–terpenes (Figure5). Both strains are fully susceptible to all terpenes tested. Our results demonstrate the possibility of using YP–terpenes as a high efficacy pan-anthelmintic therapeutic with the potential to overcome resistance. Future work depends upon marrying traditional plant-based medicines that were effective, but have been long out of favor, with modern formulation and therapeutic technologies that have the potential to overcome the drawbacks of these “old-school” therapies (e.g., bad taste, premature absorption in the stomach, and mucosal irritancy) that historically limited their use. Surprisingly, purified terpenes, as a broad class, have not been probed in great breadth or depth against STNs. To our knowledge, there has also been no systematic attempt at using the very best and modern formulation technologies to allow terpenes to reach their full, and likely vast, therapeutic potential against STNs.

4. Materials and Methods YPs were purchased from Biorigin (Louisville, KY, USA). Terpenes and HPLC solvents were obtained from Thermo Fisher Scientific (Waltham, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA). Essential oils were obtained from the following suppliers: tea tree oil (Melaleuca alternifolia) from Vitacost (Lexington, NC, USA), peppermint oil (Mentha piperita L.) from Laboratoire I.R.I.S. (Eygluy-Escoulin, France), and lavender oil (Lavandula angustifolia) from Custom Essence (Somerset, NJ, USA). Reagents for worm culture medium were purchased from Gibco (Gaithersburg, MD, USA).

4.1. YP Loading of Terpenes Dry YPs were mixed with water (180 g YP/L) and the slurry was passed through an Emulsiflex®-C3 high-pressure homogenizer (Avestin, Ottawa, ON, USA) to obtain a uniform single YP suspension. Samples of homogenized YPs (0.9 g dry YPs) were mixed with terpene (990 mg) and incubated at room temperature with the exception of YP–limonene (40 ◦C) and YP–thymol (60 ◦C). Samples were incubated for a minimum of 24 h to allow for complete terpene loading. Samples were stained with Nile red to assess loading by the fluorescence microscopy of the encapsulated terpene–Nile red complex. YP–terpene samples (100 mg) were centrifuged to collect excess liquid (free terpene) and the pellet fraction was resuspended in 10 mL of 90% methanol–10% water to extract the encapsulated terpene. Both free and encapsulated terpene fractions were quantified by HPLC operated with 32 KaratTM software version 7.0 (Beckman Coulter, Inc, Brea, CA, USA), using a Waters Symmetry® C18 column (3.5 µm, 4.6 150 mm) with acetonitrile:water 70:30 as a mobile phase, flow rate at 1 mL/min, injection × volume of 10 µL, and terpene detection at 254 nm. This isocratic HPLC method allows the detection of single terpene samples with terpene retention times varying from 2.7 to 18.5 min. The quantification of terpenes was done by measuring the peak area and interpolating the concentration using a calibration curve obtained with terpene standards. For the analysis of the three essential oils, the peak of the main terpene in the oil was used for quantification. All samples were run in triplicate. Molecules 2020, 25, 2958 10 of 13

4.2. Terpene Release from YP–Terpenes YP–terpene samples were resuspended in water and incubated at room temperature. Aliquots were collected at predetermined times, centrifuged, and the supernatant collected to measure the terpene released from the particles by HPLC.

4.3. Maintenance of Parasites and C. elegans A. ceylanicum worms were maintained in golden Syrian hamsters, as previously described [39]. T. muris parasites were maintained in STAT6-/-mice [40]. N. brasiliensis were maintained in Wistar rats [41]. C. elegans strains were cultured as per standard protocols [40]. Ethical approval: All experiments were carried out under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Massachusetts Medical School (UMMS protocol number A-2483, A-2484, A-2518). Hamsters, mice, and rats used in this study were housed, handled, fed, and experimentally used following the National Institute of Health (NIH) Guide for the Care and Use of Laboratory in Research. Euthanasia was performed by CO2 asphyxiation, followed by bilateral pneumothorax.

4.4. Adult Worm In Vitro Screening Adult worms were harvested from infected rodents and washed using prewarmed medium (RPMI 1640 with 25 mM HEPES (pH 7.2)) and (100 U/mL penicillin, 100 µg/mL streptomycin, 1 µg/mL fungizone). Eight worms were manually picked into each well of the 24-well screening plate containing 500 µL RPMI per well. As serum is incompatible with the assays with YP–terpenes, we left it out. Hence, this limited the duration of the in vitro motility assays with hookworms. YP–terpene samples were evaluated at concentrations of 0 to 333 µg terpene/mL. Assay plates were incubated at 37 ◦C and 5% CO2. Terpene activity was determined at different timepoints using the standard motility index [42]. A motility index of 3 was given to active worms, 2 for slow-moving worms, 1 for motile after stimulation by touching, and 0 for not moving even when touched (likely dead).

5. Conclusions Yeast particles were used to efficiently encapsulate twenty different terpenes and essential oils, and the dose–response of YP–terpenes was evaluated against the human-infecting zoonotic hookworm, A. ceylanicum, and the murine whipworm T. muris (a good model for human whipworm). This is the most systematic examination of a wide range of terpenes against STNs. The anthelmintic activity of YP–terpenes was evaluated based on the average motility index after 1–3 h and 24 h (hookworm) or 24–72 h (whipworm). Five main groups of terpenes can be observed based on anthelmintic activity, fast-acting terpenes at moderate–high doses, fast-acting at high doses, fast-acting at low doses, slow-acting terpenes at moderate–high doses, slow-acting terpenes at low doses, and finally the terpenes that were not effective at the doses tested. Carvacrol, thymol, and cinnamic aldehyde were consistently fast-acting at moderate–high doses on A. ceylanicum and T. muris. Eugenol was fast-acting against A. ceylanicum but slow-acting against T. muris. Anethole, cymene, limonene, l-carvone, alpha-terpineol, linalool, peppermint oil, and linalyl acetate fell into different categories for A. ceylanicum and T. muris and they were less effective for the latter parasite. Alpha-pinene and citral were consistently slow-acting at moderate–high doses against both parasites. Similarly, farnesol and nerodiol exhibited similar killing kinetics—slow but effective at low doses—on A. ceylanicum and T. muris. The difference in the activity of terpenes on the two worm species can be explained at least partly by YP–terpenes acting on A. ceylanicum hookworms by two mechanisms: terpene release from YPs into the medium followed by absorption by worms and a second mechanism of direct YP–terpene ingestion by hookworms. In contrast, T. muris whipworms do not ingest YPs and the terpene mode of action on this whipworm depends only on terpene release from YPs into the medium. Molecules 2020, 25, 2958 11 of 13

We extended our adult worm in vitro screen with lead candidates to the rat hookworm parasite N. brasiliensis, and albendazole-resistant and wild-type C. elegans. The results show that the selected terpenes (carvacrol, thymol, cinnamic aldehyde, farnesol) have broad anthelmintic activity and have the potential to be developed into anthelmintic drugs. Our next step will be to evaluate this in vivo using the N. brasiliensis/rat model to target lethal terpene doses in the intestine using enteric coated YP–terpenes or using YP-encapsulated acid-resistant biodegradable pro-terpene drugs [35] to avoid the premature release of terpene from YPs.

Supplementary Materials: The following are available online, Figure S1: Chemical structure of terpenes, Table S1: Octanol/water partition coefficient (log P), solubility in water, and HPLC retention time with isocratic method (acetonitrile:water 70:30) of terpenes evaluated for encapsulation in YPs, Table S2: Terpene loading of selected YP–terpene samples. Author Contributions: Conceptualization, R.V.A. and G.R.O.; methodology, Z.M., E.R.S., Y.H., T.-T.N., and D.K.; writing—original draft preparation, Z.M. and E.R.S.; writing—review and editing, R.V.A. and G.R.O.; project administration, G.R.O.; funding acquisition, R.V.A. and G.R.O. All authors have read and agreed to the published version of the manuscript. Funding: This project was supported by (1) the National Institutes of Health/National Institute of Allergy and Infectious Diseases grants R01AI056189 and R01AI50866 to R.V.A., (2) Agriculture and Food Research Initiative Competitive Grant no. 2015–11323 from the USDA National Institute of Food and Agriculture to R.V.A., and (3) a Sponsored Research Agreement from Eden Research plc to G.R.O. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Pullan, R.L.; Smith, J.L.; Jasrasaria, R.; Brooker, S.J. Global numbers of and disease burden of soil transmitted helminth infections in 2010. Parasites Vectors 2014, 7, 37. [CrossRef] 2. Barry, M.A.; Simon, G.G.; Mistry, N.; Hotez, P.J. Global trends in neglected tropical disease control and elimination: Impact on child health. Arch. Dis. Child. 2013, 98, 635–641. [CrossRef][PubMed] 3. Bethony, J.; Brooker, S.; Albonico, M.; Geiger, S.M.; Loukas, A.; Diemert, D.; Hotez, P.J. Soil-transmitted helminth infections: , , and hookworm. Lancet 2006, 367, 1521–1532. [CrossRef] 4. Harris, J.B.; Podolsky, M.J.; Bhuiyan, T.R.; Chowdhury, F.; Khan, A.I.; LaRocque, R.C.; Logvinenko, T.; Kendall, J.; Faruque, A.S.; Nagler, C.R.; et al. Immunologic responses to Vibrio cholerae in patients co-infected with intestinal parasites in Bangladesh. PLoS Negl. Trop. Dis. 2009, 3, e403. [CrossRef][PubMed] 5. Olds, G.R. the world. Trans. Am. Clin. Climatol. Assoc. 2013, 124, 265. 6. Hotez, P. Hookworm and poverty. Ann. N. Y. Acad. Sci. 2008, 1136, 38–44. [CrossRef] 7. Crompton, D.; Nesheim, M. Nutritional impact of intestinal during the human life cycle. Annu. Rev. Nutr. 2002, 22, 35–59. [CrossRef] 8. Keiser, J.; Utzinger, J. Efficacy of Current Drugs Against Soil-Transmitted Helminth Infections. JAMA 2008, 16, 1937–1948. [CrossRef] 9. Keiser, J.; Utzinger, J. The Drugs We Have and the Drugs We Need Against Major Helminth Infections. In Advances in ; Elsevier Ltd: London, UK, 2010; Volume 73, pp. 197–230. 10. Soukhathammavong, P.A.; Sayasone, S.; Phongluxa, K.; Xayaseng, V.; Utzinger, J.; Vounatsou, P.; Hatz, C.; Akkhavong, K.; Keiser, J.; Odermatt, P. Low efficacy of single-dose albendazole and against hookworm and effect on concomitant helminth infection in Lao PDR. PLoS Negl. Trop. Dis. 2012, 6, e1417. [CrossRef] 11. Albonico, M.; Rinaldi, L.; Sciascia, S.; Morgoglione, M.E.; Piemonte, M.; Maurelli, M.P.; Musella, V.; Utzinger, J.; Ali, S.M.; Ame, S.M.; et al. Comparison of three copromicroscopic methods to assess albendazole efficacy against soil-transmitted helminth infections in school-aged children on Pemba Island. Trans. R. Soc. Trop. Med. Hyg. 2013, 107, 493–501. [CrossRef] 12. Humphries, D.; Mosites, E.; Otchere, J.; Twum, W.A.; Woo, L.; Jones-Sanpei, H.; Harrison, L.M.; Bungiro, R.D.; Benham-Pyle, B.; Bimi, L.; et al. Epidemiology of in Kintampo North Municipality, Ghana: Patterns of malaria coinfection, anemia, and albendazole treatment failure. Am. J. Trop. Med. Hyg. 2011, 84, 792–800. [CrossRef] Molecules 2020, 25, 2958 12 of 13

13. Massa, K.; Magnussen, P.; Sheshe, A.; Ntakamulenga, R.; Ndawi, B.; Olsen, A. The combined effect of the Lymphatic Elimination Programme and the and Soil-transmitted Helminthiasis Control Programme on soil-transmitted helminthiasis in schoolchildren in Tanzania. Trans. R. Soc. Trop. Med. Hyg. 2009, 103, 25–30. [CrossRef][PubMed] 14. Speich, B.; Ali, S.M.; Ame, S.M.; Bogoch II, A.R.; Huwyler, J.; Albonico, M.; Hattendorf, J.; Utzinger, J.; Keiser, J. Efficacy and safety of albendazole plus , albendazole plus mebendazole, albendazole plus pamoate, and mebendazole alone against and concomitant soil-transmitted helminth infections: A four-arm, randomised controlled trial. Lancet Infect. Dis. 2015, 15, 277–284. [CrossRef] 15. Stothard, J.R.; Rollinson, D.; Imison, E.; Khamis, I.S. A spot-check of the efficacies of albendazole or , against soil-transmitted helminthiases in young Ungujan children, reveals low frequencies of cure. Ann. Trop. Med. Parasitol. 2009, 103, 357–360. [CrossRef][PubMed] 16. Kaplan, R.M. Drug resistance in nematodes of veterinary importance: A status report. Trends Parasitol. 2004, 20, 477–481. [CrossRef][PubMed] 17. Sunish, I.P.; Rajendra, R.; Munirathinam, A.; Kalimuthu, M.; Kumar, V.A.; Nagaraj, J.; Tyagi, B.K. Impact on prevalence of intestinal helminth infection in school children administered with seven annual rounds of diethyl carbamazine (DEC) with albendazole. Indian J. Med. Res. Suppl. 2015, 141, 330. [CrossRef] 18. Stothard, J.R.; French, M.D.; Khamis, I.S.; Basáñez, M.G.; Rollinson, D. The epidemiology and control of urinary schistosomiasis and soil-transmitted helminthiasis in schoolchildren on Unguja Island, Zanzibar. Trans. R. Soc. Trop. Med. Hyg. 2009, 103, 1031–1044. [CrossRef] 19. Cowan, M.M. Plant products as agents. Clin. Microbiol. Rev. 1999, 12, 564–582. [CrossRef] 20. Rodríguez, A.; Nerín, C.; Batlle, R. New cinnamon-based active paper packaging against Rhizopusstolonifer food spoilage. J. Agric. Food Chem. 2008, 56, 6364–6369. [CrossRef] 21. Bai, C.Q.; Liu, Z.L.; Liu, Q.Z. Nematicidal Constituents from the Essential Oil of Chenopodium Ambrosioides Aerial Parts. E-Journal Chem. 2011, 8.[CrossRef] 22. Squires, J.M.; Ferreira, J.F.S.; Lindsay, D.S.; Zajac, A.M. Effects of and Artemisia extracts on in gerbils (Meriones unguiculatus). Vet. Parasitol. 2011, 175, 103–108. [CrossRef] [PubMed] 23. Zhu, L.; Dai, J.L.; Yang, L.; Qiu, J. In vitro ovicidal and larvicidal activity of the essential oil of Artemisia lancea against Haemonchus contortus (). Vet. Parasitol. 2013, 195, 112–117. [CrossRef][PubMed] 24. Ferrell, J.A. Hookworm disease: Its ravages, prevention and cure. J. Am. Med. Assoc. 1914, 52. [CrossRef] 25. Michiels, J.; Missotten, J.; Dierick, N.; Fremaut, D.; Maene, P.; De Smet, S. “In vitro degradation and in vivo passage kinetics of carvacrol, thymol, eugenol and trans-cinnamaldehyde along the of piglets. J. Sci. Food Agric. 2008, 88, 2371–2381. [CrossRef] 26. Palaniappan, K.; Holley, R.A. Use of natural antimicrobials to increase susceptibility of drug resistant bacteria. Int. J. Food Microbiol. 2010, 140, 164–168. [CrossRef][PubMed] 27. Shapira, R.; Mimran, E. Isolation and characterization of Escherichia coli mutants exhibiting altered response to thymol. Microb. Drug Resist. 2007, 13, 157–165. [CrossRef][PubMed] 28. Gershenzon, J.; Dudareva, N. The function of terpene natural products in the natural world. Nat. Chem. Biol. 2007, 3, 408–414. [CrossRef] 29. Mukherjee, N.; Mukherjee, S.; Saini, P.; Roy, P.; Sinha Babu, S.P. Phenolics and Terpenoids; the Promising New Search for Anthelmintics: A Critical Review. Mini-Rev. Med. Chem. 2016, 16, 1415–1441. [CrossRef] 30. Soto, E.R.; Ostroff, G.R. Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA. Bioconjug. Chem. 2008, 19, 840–848. [CrossRef] 31. Aouadi, M.; Tesz, G.J.; Nicoloro, S.M.; Wang, M.; Chouinard, M.; Soto, E.; Ostroff, G.R.; Czech, M.P. Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation. Nature 2009, 458, 1180–1184. [CrossRef] 32. Soto, E.; Ostroff, G. Glucan particles as carriers of nanoparticles for macrophage-targeted delivery. In Nanomaterials for Biomedicine; American Chemical Society: Washington, DC, USA, 2012; pp. 57–79. [CrossRef] 33. Mirza, Z.; Soto, E.R.; Dikengil, F.; Levitz, S.M.; Ostroff, G.R. Beta-glucan particles as vaccine adjuvant carriers. In for Invasive Fungal Infections; Humana Press: New York, NY, USA, 2017; pp. 143–157. Molecules 2020, 25, 2958 13 of 13

34. Soto, E.R.; O’Connell, O.; Dikengil, F.; Peters, P.J.; Clapham, P.R.; Ostroff, G.R. Targeted Delivery of Glucan Particle Encapsulated Gallium Nanoparticles Inhibits HIV Growth in Human Macrophages. J. Drug Deliv. 2016.[CrossRef][PubMed] 35. Soto, E.; Rus, F.; Ostroff, G.R. Yeast Cell Wall Particle Encapsulation of Pro-Terpene Payloads. Available online: https://pdfs.semanticscholar.org/5229/afbd113d373534ae75bca45e72af023e6b58.pdf (accessed on 17 June 2019). 36. Specht, C.A.; Lee, C.K.; Huang, H.; Hester, M.M.; Liu, J.; Luckie, B.A.; Santana, M.A.; Mirza, Z.; Khoshkenar, P.; Abraham, A.; et al. Vaccination with Recombinant Cryptococcus Proteins in Glucan Particles Protects Mice against Cryptococcosis in a Manner Dependent upon Mouse Strain and Cryptococcal Species. MBio 2017, 8. [CrossRef][PubMed] 37. Inpankaew, T.; Schar, F.; Dalsgaard, A.; Khieu, V.; Chimnoi, W.; Chhoun, C.; Sok, D.; Marti, H.; Muth, S.; Odermatt, P.; et al. High prevalence of Ancylostoma ceylanicum hookworm infections in humans, , 2012. Emerg. Infect. Dis. 2014, 20, 976. [CrossRef][PubMed] 38. Klementowicz, J.E.; Travis, M.A.; Grencis, R.K. Trichuris muris: A model of gastrointestinal parasite infection. Semin. Immunopathol. 2012, 34, 815–828. [CrossRef] 39. Hu, Y.; Zhan, B.; Keegan, B.; Yiu, Y.Y.; Miller, M.M.; Jones, K.; Aorian, R.V. Mechanistic and Single-Dose In Vivo Therapeutic Studies of Cry5B Anthelmintic Action against Hookworms. PLoS Negl. Trop. Dis. 2012, 6, e1900. [CrossRef] 40. Hu, Y.; Xiao, S.H.; Aroian, R.V. The new anthelmintic is an L-type (Levamisole and ) nicotinic acetylcholine receptor agonist. PLoS Negl. Trop. Dis. 2009, 3, e499. [CrossRef] 41. Brombacher, T.M.; De Gouveia, K.S.; Cruywagen, L.; Makena, N.; Booley, F.; Tamgue, O.; Brombacher, F. Nippostrongylus brasiliensis infection leads to impaired reference memory and myeloid cell interference. Sci. Rep. 2018, 8, 1–8. [CrossRef] 42. Hu, Y.; Ellis, B.L.; Yiu, Y.Y.; Miller, M.M.; Urban, J.F.; Shi, L.Z.; Aroian, R.V. An Extensive Comparison of the Effect of Anthelmintic Classes on Diverse Nematodes. PLoS ONE 2013, 8, e70702. [CrossRef]

Sample Availability: Samples of the YP-Terpene formulations are available from the authors.

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