Menthol Acts As a Positive Allosteric Modulator on Nematode Levamisole

Entomology Publications Entomology

12-30-2018 Menthol acts as a positive allosteric modulator on nematode levamisole sensitive nicotinic acetylcholine receptors Shivani Choudhary Iowa State University, [email protected]

Djordje S. Marjianović University of Belgrade

Colin R. Wong Iowa State University, [email protected]

Xiaoyu Zhang Iowa State University

Melanie Abongwa IFoowlalo Swta tthie Usn iaverndsit ay,dd maitboniongalwa@i workastast ae.te:duhttps://lib.dr.iastate.edu/ent_pubs Part of the Entomology Commons, Natural Products Chemistry and Pharmacognosy Commons, See next page for additional authors and the Plant Sciences Commons The ompc lete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ ent_pubs/495. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

This Article is brought to you for free and open access by the Entomology at Iowa State University Digital Repository. It has been accepted for inclusion in Entomology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Menthol acts as a positive allosteric modulator on nematode levamisole sensitive nicotinic acetylcholine receptors

Abstract The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100 μM the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1 μM and 10 μM menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1 μM menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics.

Keywords Menthol, allosteric modulation, nAChR, monoterpenoid, nematode

Disciplines Entomology | Natural Products Chemistry and Pharmacognosy | Plant Sciences

Comments This is a manuscript of an article published as Choudhary, Shivani, Djordje S. Marjianović, Colin R. Wong, Xiaoyu Zhang, Melanie Abongwa, Joel R. Coats, Saša M. Trailović, Richard J. Martin, and Alan P. Robertson. "Menthol acts as a positive allosteric modulator on nematode levamisole sensitive nicotinic acetylcholine receptors." International Journal for Parasitology: Drugs and Drug Resistance (2018). doi: 10.1016/ j.ijpddr.2018.12.005.

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Authors Shivani Choudhary, Djordje S. Marjianović, Colin R. Wong, Xiaoyu Zhang, Melanie Abongwa, Joel R. Coats, Saša M. Trailović, Richard J. Martin, and Alan P. Robertson

This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/ent_pubs/495 Accepted Manuscript

Menthol acts as a positive allosteric modulator on nematode levamisole sensitive nicotinic acetylcholine receptors

Shivani Choudhary, Djordje S. Marjianović, Colin R. Wong, Xiaoyu Zhang, Melanie Abongwa, Joel R. Coats, Saša M. Trailović, Richard J. Martin, Alan P. Robertson

PII: S2211-3207(18)30134-9 DOI: https://doi.org/10.1016/j.ijpddr.2018.12.005 Reference: IJPDDR 284

To appear in: International Journal for Parasitology: Drugs and Drug Resistance

Received Date: 7 September 2018 Revised Date: 5 December 2018 Accepted Date: 29 December 2018

Please cite this article as: Choudhary, S., Marjianović, D.S., Wong, C.R., Zhang, X., Abongwa, M., Coats, J.R., Trailović, Saš.M., Martin, R.J., Robertson, A.P., Menthol acts as a positive allosteric modulator on nematode levamisole sensitive nicotinic acetylcholine receptors, International Journal for Parasitology: Drugs and Drug Resistance (2019), doi: https://doi.org/10.1016/j.ijpddr.2018.12.005.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT

7 **** ****

6 50 pEC 5 MANUSCRIPT

4 AChACh+ 0.1+ µM 0.1 + Mentholμ M10 +µM 10 MentholμM menthol menthol ACCEPTED ACCEPTED MANUSCRIPT 1 Menthol acts as a positive allosteric modulator on nematode levamisole sensitive

2 nicotinic acetylcholine receptors

3 Shivani Choudhary a, Djordje S. Marjianovi ćb, Colin R. Wong c, Xiaoyu Zhang a, Melanie

4 Abongwa a, Joel R. Coatsc, Saša M. Trailovi ćb, Richard J. Martin a & Alan P. Robertson a*

5 aDepartment of Biomedical Sciences, College of Veterinary Medicine, Iowa State

6 University, Ames, IA 50011, USA.

7 bDepartment of Pharmacology and Toxicology, College of Veterinary Medicine, University

8 of Belgrade, Belgrade, Serbia.

9 cDepartment of Entomology, Iowa State University, Ames, IA 50011, USA.

10 *Corresponding Author: [email protected]

12 Abstract

13 The ongoing and widespread emergence of resistance to the existing anti-nematodal 14 pharmacopeia has made it imperative to developMANUSCRIPT new anthelminthic agents. Historically, 15 plants have been important sources of therapeutic compounds and offer an alternative to

16 synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce

17 acute toxic effects in insects and nematodes. Previous studies have shown nicotinic

18 acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant

19 metabolites such as carvacrol and carveol. In this study we examined the effects of

20 monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum

21 dentatum and a nicotine sensitive nAChR from Ascaris suum . We expressed the receptors 22 in Xenopus laevis ACCEPTED oocytes and used two-electrode voltage-clamp to characterize the effect 23 of various compounds on these cys-loop receptors. At 100 µM the majority of these

24 compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1

25 µM and 10 µM menthol potentiated acetylcholine and levamisole responses in the

26 levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated 1

ACCEPTED MANUSCRIPT 27 the effects of 0.1 µM menthol on the contractility of A. suum somatic muscle strips.

28 Menthol produced significant potentiation of peak contractions at each concentration of

29 acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in

30 vitro experiments suggests menthol as a promising candidate for combination therapy with

31 cholinergic anthelmintics.

33 Keywords

34 Menthol, allosteric modulation, nAChR, monoterpenoid, nematode

36 Abbreviations

37 Asu , Ascaris suum ; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N ′,N ′-tetraacetic acid

38 tetrakis (acetoxymethyl ester); cRNA, complementary RNA; DMSO, dimethyl sulfoxide;

39 Hco, Haemonchus contortus ; nAChR, nicotinic acetylcholine receptor; Ode , 40 Oesophagustomum dentatum ; PAM, positive allostericMANUSCRIPT modulator; STHs, soil transmitted 41 helminths.

ACCEPTED

ACCEPTED MANUSCRIPT 43 1. Introduction:

44 Parasitic nematode infections are a threat to health and have significant socio-

45 economic consequences. Among the different classes of nematodes, soil transmitted

46 helminths (STHs) are major contributors to the global parasite burden. According to WHO

47 (2018) , more than 1.5 billion people, or 24% of the world’s population, are infected with at

48 least one species of soil transmitted helminth, which mainly include Ascaris lumbricoides ,

49 Trichuris trichiura and hookworms ( Necator americanus and Ancylostoma duodenale ).

50 These infections are most commonly found in tropical and sub-tropical countries due to

51 suboptimal sanitation, lack of access to clean water and favorable climatic conditions for

52 the parasites (Bethony et al., 2006; Brooker et al., 2006; Hotez et al., 2008; Savioli and

53 Albonico, 2004). Although mortality is rare, high morbidity is a common feature of intestinal

54 helminthiasis. The disease manifestations include weight loss, anemia, diarrhea,

55 abdominal pain, malnutrition, general malaise, weakness, as well as impaired growth and 56 physical development (Bethony et al., 2006). ChildrMANUSCRIPTen bear the highest burden of these 57 infections which profoundly affect their physical growth, cognitive development and cause

58 educational deficits (Alum et al., 2010; Brooker, 2010; Stephenson et al., 2000). STH

59 infections such as Ascariasis have also been reported to exacerbate other diseases such

60 as malaria, tuberculosis and HIV/AIDS (Fincham et al., 2003; Le Hesran et al., 2004). In

61 addition, production losses caused by intestinal nematodes of livestock have adverse

62 effects on nutritional status and the food economy especially in poverty-stricken regions

63 (Morgan et al., 2013). 64 ACCEPTED 65 Effective vaccines are the most desirable approach but even after almost 30 years of

66 dedicated research, a licensed vaccine against human parasites remains elusive. Current

67 control strategies for treatment and prevention of helminth infections rely heavily on

68 chemotherapy. Unfortunately, there are a limited number of classes of anthelmintic agents,

ACCEPTED MANUSCRIPT 69 which include benzimidazoles, macrocyclic lactones and nicotinic agonists (Abongwa et

70 al., 2017; Martin, 1997; Robertson and Martin, 2007). Moreover, widespread and

71 indiscriminate administration combined with genetic factors has led to the development of

72 resistance in animals (Albonico et al., 2003; Taman and Azab, 2014). There have also

73 been reports of reduced therapeutic efficacy to anti-nematodal drugs in humans. De

74 Clercq et al. (1997) and Flohr et al. (2007) reported poor efficacy of benzimidazoles

75 against human hookworm infections. There is a need to focus research on drug discovery

76 and development to overcome the issue of increasing resistance and reduced efficacy

77 against existing drugs.

79 The use of phytotherapeutics for parasitic infections has emerged as an attractive

80 alternative. Plants have dominated the human pharmacopoeia for thousands of years

81 (Balick and Cox, 1996; Newman and Cragg, 2016; Raskin and Ripoll, 2004). Many 82 therapeutic products, including codeine, quinine,MANUSCRIPT artemisinin, digitoxin, morphine, 83 paclitaxel, galantamine, currently used in modern pharmacotherapy are of plant origin

84 (Butler, 2004; Heinrich and Lee Teoh, 2004; Newman and Cragg, 2016; Newman et al.,

85 2000; Petrovska, 2012; Pirttila et al., 2004; Samuelsson, 2004). Plant based therapeutic

86 compounds are environment-friendly and exhibit a wide array of medicinal properties

87 (Balunas and Kinghorn, 2005; Coats, 1994; Dias et al., 2012; Harvey et al., 2015; Hu and

88 Coats, 2008). Interestingly, an estimated four billion people living in the developing world

89 rely on medicinal plants for their primary pharmaceutical care (WHO, 2005). Herbal 90 remedies in the ACCEPTEDform of complementary and alternative medicine (CAM) are also being 91 widely embraced in many developed countries (Anquez-Traxler, 2011; Calapai, 2008;

92 Committee on the Use of Complementary, 2005). Among phytochemicals, essential oils

93 and their active principles have been well documented for their broad range of functions

94 including insecticidal and nematocidal activities (Camurca-Vasconcelos et al., 2007; Cetin

ACCEPTED MANUSCRIPT 95 et al., 2009; Coskun et al., 2008; Enan, 2005; Macedo et al., 2009; Panella et al., 2005).

96 Historically, thymol (found in oil of thyme), santonin (derived from buds of the local

97 Artemisia spp.) and ascaridol (isolated from Chenopodium oil) were used in the late

98 nineteenth and early twentieth century for the treatment of ascarids and hookworms in

99 humans (Ferrell, 1914; Kaplan et al., 2014; Lamson and Ward, 1932). Terpene and

100 terpenoid biomolecules, particularly monoterpenes and sesquiterpenes, found in essential

101 oils have been identified as promising anthelmintic compounds and are of great

102 pharmaceutical interest (Jaradat et al., 2016; Katiki et al., 2011; Lei et al., 2010; Pessoa et

103 al., 2002; Squires et al., 2010). Lei et al. (2010) reported in vitro nematocidal activity of

104 thymol and carvacrol (major components of thyme and oregano essential oils)

105 against Caenorhabditis elegans and Ascaris suum . Trailovi ć et al. (2015) also

106 demonstrated the potential anthelmintic effects of carvacrol in A. suum .

107 108 In the present study, we tested the activity of monMANUSCRIPToterpenoid compounds on two different 109 nematode nAChR types: levamisole sensitive (L-type) nAChRs from Oesophagostomum

110 dentatum and a nicotine sensitive (N-type) nAChR (ACR-16) from A. suum . The receptors

111 were expressed in Xenopus laevis oocytes and we used two-electrode voltage-clamp to

112 study the pharmacological effects of the compounds. We also investigated the effects of

113 menthol on the contractility of A. suum somatic muscle strips. The focus of this study was

114 to evaluate the effects produced by monoterpenoid compounds to identify potential

115 alternatives to current chemotherapeutic agents. 116 ACCEPTED 117 2. Materials and Methods

118 2.1 cRNA preparation and X. laevis oocyte expression

119 All nAChR subunit and ancillary factor cRNAs from O. dentatum (Ode-unc-29, Ode-unc-

120 38, Ode-unc-68 and Ode-acr-8), A. suum (Asu-acr-16 and Asu-ric-3) and Haemonchus

ACCEPTED MANUSCRIPT 121 contortus (Hco-ric-3, Hco-unc-50 and Hco-unc-74 ) were prepared as previously described

122 (Abongwa et al., 2016b; Buxton et al., 2014). The receptors were expressed in

123 defolliculated X. laevis oocytes purchased from Ecocyte Bioscience (Austin, Texas, USA).

124 N-type (nicotine sensitive) nAChRs from A. suum were expressed by co-injecting 25 ng

125 of Asu -acr-16 with 5 ng of Asu-ric-3 in a total volume of 50 nL in nuclease-free

126 water. Heteromeric (levamisole sensitive) nAChRs from O. dentatum were expressed by

127 co-injecting 1.8 ng of each subunit cRNA ( Ode-unc-29 , Ode-unc-38 , Ode-unc-68 and Ode-

128 acr-8) with 1.8 ng of each H. contortus ancillary factor ( Hco-ric-3, Hco-unc-50 and Hco-

129 unc-74 ) in a total volume of 36 nL in nuclease-free water. The injections were made in the

130 animal pole of the oocytes using a nanoject II microinjector (Drummond Scientific,

131 Broomall, PA, USA). The injected oocytes were separated into 96-well plates containing

132 200 µL incubation solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2.2H 20, 1 mM

−1 133 MgCl 2.6H 20, 5 mM HEPES, 2.5 mM Na pyruvate, 100 U·mL penicillin and 134 100 µg·mL −1 streptomycin, pH 7.5); each well MANUSCRIPT contained one oocyte. The solution was 135 changed daily to maintain oocytes in optimal condit ions. The oocytes were incubated at 19

136 oC for at 5-7 days to allow functional expression of the receptor.

137

138 2.2 Two-electrode voltage-clamp (TEVC) electrophysiology

139 We used two-electrode voltage-clamp electrophysiology to record the inward current

140 generated by the activation of expressed receptors in X. laevis oocytes. The oocytes were

141 incubated with 100 µM BAPTA-AM (an intracellular calcium chelator) for ∼3 hours prior to 142 recordings to preventACCEPTED activation of endogenous calcium-activated chloride currents. The 143 oocytes were clamped at -60mV for all the experiments with an Axoclamp 2B amplifier; all

144 data were acquired on a desktop computer with Clampex 10.2 (Molecular Devices Inc.,

145 CA, USA). The microelectrodes used to impale oocytes were pulled using a

146 Flaming/Brown horizontal micropipette puller (model P-97, Sutter Instruments Co., USA)

ACCEPTED MANUSCRIPT 147 and filled with 3 M KCl. The tips of the microelectrodes were carefully broken with a tissue

148 paper to achieve a resistance of 2-5 M Ω in recording solution (100 mM NaCl, 2.5 mM KCl,

149 1 mM CaCl 2·2H 2O and 5 mM HEPES, pH 7.3). The low resistance allowed passage of

150 large currents required to maintain adequate voltage-clamp of the oocyte. Oocytes were

151 placed into a tiny groove of the narrow oocyte-recording chamber. The Digidata 1322A

152 (Molecular Devices, CA, USA) was used to control the switches that controlled the

153 perfusion of the chamber at a speed of ~6 mL/min. Un-injected oocytes served as the

154 negative control.

155

156 2.3 A. suum muscle flap contraction measurements

157 The adult female A. suum worms were collected from the slaughterhouse at Surčin,

158 Belgrade, Serbia and maintained at 32 °C in Locke’s solution (NaCl 155 mM, KCl 5 mM,

159 CaCl 2 2 mM, NaHCO 3 1.5 mM and glucose 5 mM). The Locke’s solution was changed 160 twice daily. Each batch of worms was used withinMANUSCRIPT 4 days of collection. For contraction 161 studies, A. suum muscle flaps were prepared as described in Trailovi ć et al. (2015).

162 Briefly, an anterior part of the worm, 2-3 cm caudal to the head was dissected to prepare a

163 1 cm muscle flap and the lateral line was removed from the edge of the flaps. Each muscle

164 flap was attached to a force transducer in an experimental bath maintained at 37 °C,

165 containing 20 mL Ascaris Perienteric Fluid Ringer (23 mM NaCl, 110 mM Na acetate,

166 24 mM KCl, 6 mM CaCl 2, 5 mM MgCl 2, 11 mM glucose, 5 mM HEPES, pH 7.6) and

167 bubbled with room air. The isometric contractions were monitored on a PC computer using

168 a BioSmart interfaceACCEPTED with eLAB software (EIUnit, Belgrade). The preparations were

169 allowed to equilibrate for 15 min under an initial tension of 0.5 g following

170 which acetylcholine (1–100 µM) in the absence and presence of menthol (0.1 µM) was

171 applied to the preparation. The responses for each concentration were expressed in grams

172 (g).

ACCEPTED MANUSCRIPT 173

174 2.4 Drug Applications

175 Acetylcholine chloride, L-menthol, (-)-menthone, eugenol, (S)-(-)-β-Citronellol, (+)-

176 limonene oxide, carvacrol, ((R)-(-))-carvone, menthyl acetate, phenethyl propionate, trans -

177 cinnamaldehyde and BAPTA-AM were purchased from Sigma-Aldrich (St Louis, MO,

178 USA). (+)-Pulegone was purchased from Kodak (Rochester, NY, USA) and geraniol from

179 Berje (Carteret, NJ, USA). Acetylcholine was dissolved in recording solution while

180 monoterpenoid compounds (Figure 1) were dissolved in DMSO such that the final working

181 concentration did not exceed 0.1%.

182

183 Oocytes were perfused with drugs by an 8-channel system (VC-8 valve controller) (Warner

184 Instruments, USA) at a flow rate of ~6 mL/min. For the agonist experiments, all the

185 essential oils were used at a concentration of 100 µM. All the responses were normalized 186 to the control (100 µM acetylcholine) response.MANUSCRIPT Each drug was applied for 10s and the 187 wash off time between each drug was 2 minutes. The application sequence of agonists for

188 determining the potency series was random and not predetermined. The concentration-

189 response relationship for acetylcholine was generated by applying increasing

190 concentrations of acetylcholine (0.1-300 µM) for 10 s.

191

192 For positive allosteric modulator experiments, we tested the effects of menthol (0.1 and 10

193 µM) on acetylcholine and levamisole concentration-response relationship. Control 194 concentration-responsesACCEPTED were obtained by applying ascending concentrations of the 195 agonist for 10 s. Concentration-response relationships in the presence of 0.1 µM and 10

196 µM menthol were generated by first applying a control 100 µM acetylcholine for 10 s,

197 followed by a 2-min application of menthol, and 10 s applications of increasing

198 concentrations of the agonist in the continued presence of menthol. The responses were

ACCEPTED MANUSCRIPT 199 normalized to the control 100 µM acetylcholine current. Between each drug application a

200 2 min wash off time was allowed, and the responses were normalized to the control

201 100 µM acetylcholine current.

202

203 In order to characterize the antagonist properties of the phytocompounds, a control

204 concentration of 100 µM ACh was first applied for 30 s followed by a 2 min wash with

205 recording solution. Thereafter, 100 µM acetylcholine was applied for 10 s, immediately

206 followed by 10 s application of the antagonist (100 µM) in the continued presence of 100

207 µM ACh and then a final 10 s application of 100 µM ACh. At least 2 min drug wash off

208 interval was allowed between applications in order to minimize desensitization. Note that,

209 the potency for each antagonist is likely to be slightly underestimated due to the short time

210 of drug application with this approach (Abongwa et al., 2016a). Acetylcholine

211 concentration-response relationships in the presence of 100 µM limonene oxide and 100 212 µM carvacrol were generated by first applyingMANUSCRIPT a control 100 µM acetylcholine for 10 s, 213 followed by a 2-min application of the antagonist, and 10 s applications of 3, 10, 30, 100

214 and 300 µM acetylcholine in the continued presence of antagonist. The responses were

215 normalized to the control 100 µM acetylcholine current.

216

217 2.5 Data and statistical analysis

218 Data acquired from electrophysiological recordings were analyzed with Clampfit 10.3

219 (Molecular Devices, Sunnyvale, CA, USA) and GraphPad Prism 7.0 (Graphpad Software 220 Inc., La Jolla, CA,ACCEPTED USA). The peak current responses in oocytes to applied drugs were 221 normalized to the 100 µM ACh control current (unless otherwise indicated) and expressed

222 as mean ± SEM. All completed drug application sequences on the oocytes were used for

223 analysis without exclusion. If the recording became unstable, indicated by a change in the

224 baseline holding current, all of that recording was rejected for analysis. The mean %

ACCEPTED MANUSCRIPT 225 inhibition produced by monoterpenoid compounds on currents elicited by 100 µM

226 acetylcholine were calculated as previously described (Zheng et al., 2016). For A. suum

227 contraction experiments, mean contraction responses in grams (g) to each concentration

228 of acetylcholine in the absence and presence of menthol was determined. The sigmoidal

229 concentration-responses for the oocytes experiments and isometric contraction studies

230 were described using the following equation:

231 % response= 1/1+[EC 50 /Xa] nH

232 where EC 50 is the concentration of agonist (Xa) producing 50 % of the maximum

233 response and nH is the Hill coefficient (slope). Differences in pEC 50 and Imax were

234 assessed using extra sum of squares F-test. Two-way ANOVA and paired t-tests were

235 used to test differences between control contractions and test contractions on the same

236 muscle flap. The difference was considered significant if P<0.05.

237 238 3. RESULTS: MANUSCRIPT 239 3.1 Pharmacology of monoterpenoids on Ode (29-38-63-8) receptor

240 3.1.1 Agonist pharmacology

241 The agonist properties of 12 monoterpenoid compounds were investigated on the

242 levamisole sensitive Ode -unc-29∶Ode -unc-38 ∶Ode -unc-63 ∶Ode -acr-8 nAChR (Figure 2).

243 11 of the 12 monoterpenoids we tested showed no significant agonistic effect (<1.0% of

244 control acetylcholine current; Figure 2A) on the expressed levamisole sensitive channel.

245 Menthol was the only compound tested that produced an agonist effect, showing only ≈

246 6.5% of the controlACCEPTED acetylcholine currents.

247

248 3.1.2 Antagonist pharmacology

249 We tested potential antagonistic effects of the same 12 monoterpenoids on the levamisole

250 sensitive receptor (Figure 3). Among all the phytocompounds tested limonene oxide, 10

ACCEPTED MANUSCRIPT 251 citronellol, carvone, carvacrol, pulegone and eugenol reduced the acetylcholine response.

252 Limonene oxide was the most potent inhibitor (mean inhibition% = 36.0 ± 3.2%). The rank

253 order potency of inhibition was: limonene oxide > citronellol > carvone > carvacrol =

254 pulegone = eugenol (Figure 3A). Interestingly, monoterpenoids including menthol,

255 menthone, menthyl acetate and geraniol increased the amplitude of currents produced by

256 acetylcholine instead of inhibiting them (data not shown). Menthol produced the most

257 potent positive allosteric modulation and increased the response by 3.1 ± 0.7% (mean ±

258 SEM) with a maximum potentiation of 6.3%.

259

260 3.1.3 Antagonistic effects of limonene oxide and carvacrol on acetylcholine concentration-

261 response relationship

262 The concentration-response relationship for acetylcholine was examined by applying

263 increasing concentrations of acetylcholine 0.3–300 µM to oocytes expressing the

264 levamisole sensitive nAChR (Figure 3B & C). The sigmoidal concentration-response fit MANUSCRIPT 265 gave pEC 50 = 5.3 ± 0.0 (EC 50 = 5.3 µM), a hillslope ( nH) of 2.0 ± 0.2 and Imax value of 104.6

266 ± 1.6%. We investigated the effects of limonene oxide (100 µM) and carvacrol (100 µM) on

267 the acetylcholine concentration-response relationship. Imax for acetylcholine in the

268 presence of limonene oxide and carvacrol were 62.8 ± 7.0% and 74.1 ± 3.2%,

269 respectively. Both the monoterpenoid compounds produced a statistically significant

270 reduction in the maximum response ( P<0.001 for limonene oxide; P<0.0001 for carvacrol;

271 Figure 3B & C). Neither carvacrol, pEC 50 = 5.7 ± 0.1 (EC 50 = 2.0 µM) nor limonene oxide,

272 pEC 50 = 5.4 ± 0.2ACCEPTED (EC 50 = 4.0 µM) increased the EC 50 values c.f. control. This indicates 273 limonene oxide and carvacrol both produced non-competitive inhibition and are not binding

274 at the agonist-binding site to produce their effect.

275

276 3.1.4 Menthol as positive allosteric modulator

ACCEPTED MANUSCRIPT 277 Menthol was a weak agonist on the expressed levamisole sensitive receptor. However, it

278 appeared to potentiate acetylcholine responses in our antagonist experiments (data not

279 shown). To investigate positive allosteric modulation further, we analyzed the effect of

280 menthol on acetylcholine and levamisole concentration-response relationships (Figure 4

281 and Figure 5). We observed a left shift in the sigmoidal concentration-response curves for

282 both levamisole and acetylcholine in the presence of menthol. Sensitivity of the receptor

283 was increased significantly for acetylcholine, pEC 50 = 5.3 ± 0.0 (EC 50 = 5.0 µM); in the

284 presence of 0.1 µM menthol, pEC 50 = 6.4 ± 0.1 (EC 50 = 0.4 µM) and with 10 µM menthol,

285 pEC 50 = 6.5 ± 0.1 (EC 50 = 0.3 µM).

286 For levamisole alone, pEC 50 and EC 50 values were 6.3 ± 0.1 and 0.5 µM respectively. In

287 the presence of 0.1 µM menthol pEC 50 and EC 50 values were 6.5 ± 0.1 and 0.3 µM

288 respectively. In the presence of 10 µM menthol the pEC 50 and EC 50 values were 6.8 ± 0.2

289 and 0.2 µM. Only 10 µM menthol produced a significant left shift in the levamisole 290 concentration-response curve (P<0.05; FigureMANUSCRIPT 5C). The positive allosteric modulation 291 produced by menthol was more pronounced with acetylcholine as an agonist in

292 comparison to levamisole. In conclusion, menthol not only acts as an agonist on the

293 levamisole sensitive receptor but also displays significant PAM activity on this nAChR.

294

295 3.2 Effects of menthol and carvacrol on A. suum ACR-16 nAChR

296 Figure 6 shows the concentration-response relationship for acetylcholine on the expressed

297 Asu-ACR-16 nicotine sensitive nAChR. The sigmoidal concentration-response fit gave

298 pEC 50 = 5.0 ± 0.1ACCEPTED (EC 50 = 11.1 µM), Imax = 109.1 ± 6.6% and a hillslope ( nH) of 1.6 ± 0.3. 299 We investigated the effects of menthol (10 µM) and carvacrol (10 µM and 100 µM) on the

300 acetylcholine response. There was no significant change in the sensitivity or efficacy of

301 acetylcholine on the N-type receptor in the presence of menthol (pEC 50 = 5.0 ± 0.1 and I max

302 =101.6 ± 7.5%). The concentration-response fits (Figure 6C) show that 100 µM carvacrol

ACCEPTED MANUSCRIPT 303 decreased the maximum response while 10 µM carvacrol failed to exhibit any significant

304 antagonistic properties. In the presence of 10 µM carvacrol pEC 50, EC 50 and Imax values

305 were 5.1 ± 0.1, 8.1 µM and 94.6 ± 5.9% respectively. Carvacrol produced a significant

306 reduction in Imax (55.8 ± 4.4%; P<0.05) when applied at 100 µM concentration but did not

307 produce a significant right shift in the pEC 50 (5.1 ± 0.1). This indicates carvacrol at 100 µM

308 produced non-competitive inhibition.

309

310 3.3 Effect of menthol on A. suum muscle

311 We examined the effect of menthol on the A. suum muscle flap contractions induced by

312 increasing concentrations of acetylcholine. Figure 7A shows the representative trace for

313 acetylcholine, in the absence and presence of 0.1 µM menthol in the experimental bath.

314 Acetylcholine produced concentration-dependent isometric contractions with a pEC 50 of

315 5.2 ± 0.2 (EC 50 = 7.0 µM) and Rmax = 1.7 ± 0.2 g. We did not observe a significant left shift 316 in pEC 50 (5.2 ± 0.2) or change in Rmax (2.0 ±MANUSCRIPT 0.2 g) in the presence of menthol. As 317 expected, based on two-way ANOVA, acetylcholine concentration had a significant effect

318 on muscle contraction ( P<0.0001). Importantly the presence of menthol also had a

319 significant effect on muscle contraction ( P<0.0001). Further analysis revealed a significant

320 potentiation of contractions at each concentration of acetylcholine in the presence of

321 menthol (paired t-tests, P< 0.05, Figure 7B for examples). This modulatory effect of

322 menthol is similar to our observed effects on the levamisole sensitive receptor.

323 324 4. DISCUSSION:ACCEPTED 325 Monoterpenoids are a group of plant secondary metabolites that have been shown to

326 modulate the function of nicotinic acetylcholine receptors of mammals and insects (Lozon

327 et al., 2016; Park et al., 2003; Park et al., 2001; Tong et al., 2013). We were interested in

328 finding possible agonists, antagonists or modulators for these cys loop receptors of

ACCEPTED MANUSCRIPT 329 parasitic nematodes. In this study, we provide evidence of significant modulatory effects of

330 several monoterpenoid compounds on the levamisole sensitive O. dentatum receptor and

331 nicotine sensitive A. suum nAChR. We chose to use the levamisole sensitive nAChR from

332 O. dentatum due to unreliable expression of the levamisole sensitive nAChR from A. suum

333 in our hands.

334

335 The majority of the phytocompounds tested failed to exhibit agonist properties on the

336 nAChRs but we were able to identify inhibitors of the levamisole sensitive nematode

337 receptor with a rank order:

338 limonene oxide > citronellol > carvone > carvacrol = pulegone = eugenol.

339 Limonene oxide and carvacrol produced significant non-competitive inhibition suggesting

340 neither compound acts at the ligand binding sites. Limonene oxide is a monoterpene found

341 in essential oils of various citrus fruits and is an odorous component of some types of 342 mushrooms (Breheret et al., 1997; Lemes et al.,MANUSCRIPT 2018; Lota et al., 2002; Rapior et al., 343 1997; Vieira et al., 2018); it has been reported to stimulate TRPA1 cation channels

344 (Kaimoto et al., 2016). There are limited data available on the effects of limonene oxide on

345 nAChRs and this is the first example of an antagonistic effect of the compound on a

346 levamisole sensitive nematode nAChR. According to a study done by Kim et al. (2013)

347 limonene oxide was reported to be a safe phytochemical in mammals and thus could be

348 used in combination with other nicotinic inhibitors for anti-nematodal therapy. Carvacrol,

349 found in many plants (thyme, oregano, Alaska yellow cedar; (Bouchra et al., 2003; De 350 Vincenzi et al., 2004)ACCEPTED acted as a non-competitive inhibitor on both levamisole sensitive and 351 nicotine sensitive nematode receptors. Various studies have shown alpha-7 homomeric

352 nAChRs, GABA (gamma amino-butyric acid) and tyramine receptors as target sites for

353 carvacrol (Lozon et al., 2016; Tong and Coats, 2012; Tong et al., 2013). In addition,

354 carvacrol was shown to produce significant inhibitory effects on acetylcholine induced

ACCEPTED MANUSCRIPT 355 muscle contractions in A. suum (Trailovi ć et al., 2015). This polypharmacological effect of

356 carvacrol might make it efficacious as an anthelmintic phytotherapeutic, although it may be

357 best considered as an adjunct to other anti-nematodal compounds to increase efficacy due

358 to toxicity considerations when used alone at high concentrations (Bimczok et al., 2008;

359 Roselli et al., 2007; Stammati et al., 1999).

360

361 The most interesting finding was the positive allosteric modulatory activity of menthol on

362 the levamisole sensitive nematode nAChR. Menthol is a monocyclic terpene alcohol

363 extracted from peppermint plants, Mentha spp . and regulates somatosensory sensations

364 such as warmth, pain and irritation (Cliff and Green, 1994; Eccles, 1994; Farco and

365 Grundmann, 2013). Menthol showed very mild agonist activity but produced statistically

366 significant potentiation of acetylcholine induced inward currents at concentrations as low

367 as 0.1 µM and of levamisole induced currents at 10 µM on the levamisole sensitive cation 368 channel. PAM compounds exhibit potentiation ofMANUSCRIPT the agonist-activation effects by binding 369 to a site distinct from the ligand binding sites. This property is highly desired in drug

370 discovery and development as PAMs can achieve subtype selectivity more easily in

371 comparison to compounds binding to orthosteric sites (Chatzidaki and Millar, 2015;

372 Williams et al., 2011) as all the nAChRs have highly conserved acetylcholine binding sites.

373 In comparison, the conservation for amino acid composition for other sites is less which

374 makes PAMs preferable in terms of selectivity and reduced potential off target effects

375 (Chatzidaki and Millar, 2015; Williams et al., 2011). Menthol modulates the 376 pharmacological ACCEPTEDproperties and expression of vertebrate nicotinic acetylcholine receptors; 377 it inhibits the effect of nicotine on α7-nACh receptors in neural cells in a non-competitive

378 manner (Ashoor et al., 2013; Hans et al., 2012; Ton et al., 2015). However, it is worth

379 noting that these negative allosteric modulatory and inhibitory effects of menthol on α7-

380 nACh receptors were produced by much higher concentrations (30-300 µM) in comparison

ACCEPTED MANUSCRIPT 381 to our study. In addition, menthol at 10 µM had no significant effect on the nematode

382 nicotine sensitive nAChR (the closest ortholog to vertebrate α7-nAChRs in the

383 nematodes). This makes menthol an interesting candidate that can potentiate the effects

384 of cholinergic agonist anthelmintics acting on levamisole sensitive nAChRs. Importantly,

385 0.1 µM menthol produced significant potentiation of contractions produced by

386 acetylcholine on A. suum muscle flaps. The presence of positive allosteric modulation in

387 the somatic muscle flap preparation can be attributed to the presence of levamisole

388 receptors and is important in vivo confirmation of our findings from the in vitro

389 heterologous expression studies.

390

391 5. Conclusion

392 Natural products have formed the basis of sophisticated traditional therapies and they

393 continue to play essential roles in modern healthcare. Phytocompounds, with their 394 complex chemistry and structural diversity, MANUSCRIPT offer the potential for discovery and 395 development of new pharmaceuticals. Botanical anthelmintics may reduce the need for

396 chemical drug treatment and alleviate the pressure on the limited group of anthelmintics

397 available. Carvacrol produced significant antagonism on the acetylcholine induced inward

398 currents on both levamisole sensitive and nicotine sensitive nematode ligand gated ion

399 channels. This illustrates a multifaceted mode of action with involvement of multiple target

400 subtypes that can help ameliorate the issue of drug resistance. It may be possible to

401 combine these compounds with the nAChR antagonist derquantel or perhaps with a 402 macrocyclic lactoneACCEPTED as with Startect® (derquantel + abamectin). 403

404 Our results provide promising evidence of positive allosteric modulation by menthol which

405 could be used in combination therapy with cholinomimetic drugs like pyrantel or levamisole

406 to increase efficacy. For future investigations menthol could be used as the starting point

ACCEPTED MANUSCRIPT 407 in structure activity relationship studies to find new potent PAM compounds suitable for

408 stand-alone use as anthelmintics. In summary, the use of plant derived compounds, either

409 alone or in combination, could facilitate sustainable control of parasitic nematodes. The

410 results of the present study are encouraging and suggest monoterpenoids can be

411 exploited as components of new anthelmintic formulations.

412

413 Acknowledgments:

414 Research funding was by the NIH National Institute of Allergy and Infectious Diseases

415 grants R21AI121831-01 to APR and R01AI047194-17 to RJM, the College of Veterinary

416 Medicine TA support to SC, the Iowa Agricultural Experiment Station to JRC & CRW, and

417 the E. A. Benbrook Foundation for Pathology and Parasitology to RJM. A. suum muscle

418 flap contraction experiments were supported by the Ministry of Education, Science and

419 Technological Development Republic of Serbia, Grant No. TR31087 to SMT. The funding 420 agencies had no role in the design, execution orMANUSCRIPT publication of this study. The content is 421 solely the responsibility of the authors and does not necessarily represent the official views

422 of any of the funding sources.

423

424 References 425 426 Abongwa, M., Baber, K.E., Martin, R.J., Robertson, A.P., 2016a. The cholinomimetic

427 morantel as an open channel blocker of the Ascaris suum ACR-16 nAChR. Invert Neurosci

428 16, 10. ACCEPTED 429 Abongwa, M., Buxton, S.K., Courtot, E., Charvet, C.L., Neveu, C., McCoy, C.J., Verma, S.,

430 Robertson, A.P., Martin, R.J., 2016b. Pharmacological profile of Ascaris suum ACR-16, a

431 new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues. Br J

432 Pharmacol 173, 2463-2477.

ACCEPTED MANUSCRIPT 433 Abongwa, M., Martin, R.J., Robertson, A.P., 2017. A Brief Review on the Mode of Action of

434 Antinematodal Drugs. Acta Vet (Beogr) 67, 137-152.

435 Albonico, M., Bickle, Q., Ramsan, M., Montresor, A., Savioli, L., Taylor, M., 2003. Efficacy

436 of mebendazole and levamisole alone or in combination against intestinal nematode

437 infections after repeated targeted mebendazole treatment in Zanzibar. Bull World Health

438 Organ 81, 343-352.

439 Alum, A., Rubino, J.R., Ijaz, M.K., 2010. The global war against intestinal parasites--should

440 we use a holistic approach? Int J Infect Dis 14, e732-738.

441 Anquez-Traxler, C., 2011. The Legal and Regulatory Framework of Herbal Medicinal

442 Products in the European Union: A Focus on the Traditional Herbal Medicines Category.

443 Drug Information Journal 45, 15-23. MANUSCRIPT 444 Ashoor, A., Nordman, J.C., Veltri, D., Yang, K.H., Al Kury, L., Shuba, Y., Mahgoub, M.,

445 Howarth, F.C., Sadek, B., Shehu, A., Kabbani, N., Oz, M., 2013. Menthol binding and

446 inhibition of alpha7-nicotinic acetylcholine receptors. PLoS One 8, e67674.

447 Balick, M.J., Cox, P.A., 1996. Plants, people, and culture: the science of ethnobotany/

448 Michael J. Balick, Paul Alan Cox (Scientific American Library series no. 60). New York :

449 Scientific American Library. ACCEPTED

450 Balunas, M.J., Kinghorn, A.D., 2005. Drug discovery from medicinal plants. Life Sci 78,

451 431-441.

ACCEPTED MANUSCRIPT 452 Bethony, J., Brooker, S., Albonico, M., Geiger, S.M., Loukas, A., Diemert, D., Hotez, P.J.,

453 2006. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet

454 367, 1521-1532.

455 Bimczok, D., Rau, H., Sewekow, E., Janczyk, P., Souffrant, W.B., Rothkotter, H.J., 2008.

456 Influence of carvacrol on proliferation and survival of porcine lymphocytes and intestinal

457 epithelial cells in vitro. Toxicol In Vitro 22, 652-658.

458 Bouchra, C., Achouri, M., Idrissi Hassani, L.M., Hmamouchi, M., 2003. Chemical

459 composition and antifungal activity of essential oils of seven Moroccan Labiatae against

460 Botrytis cinerea Pers: Fr. J Ethnopharmacol 89, 165-169.

461 Breheret, S., Talou, T., Rapior, S., Bessière, J.-M., 1997. Monoterpenes in the Aromas of

462 Fresh Wild Mushrooms (Basidiomycetes). Journal of Agricultural and Food Chemistry 45, 463 831-836. MANUSCRIPT

464 Brooker, S., 2010. Estimating the global distribution and disease burden of intestinal

465 nematode infections: adding up the numbers--a review. Int J Parasitol 40, 1137-1144.

466 Brooker, S., Clements, A.C., Bundy, D.A., 2006. Global epidemiology, ecology and control

467 of soil-transmitted helminth infections. Adv Parasitol 62, 221-261.

468 Butler, M.S., 2004.ACCEPTED The role of natural product chemistry in drug discovery. J Nat Prod 67,

469 2141-2153.

470 Buxton, S.K., Charvet, C.L., Neveu, C., Cabaret, J., Cortet, J., Peineau, N., Abongwa, M.,

471 Courtot, E., Robertson, A.P., Martin, R.J., 2014. Investigation of acetylcholine receptor

ACCEPTED MANUSCRIPT 472 diversity in a nematode parasite leads to characterization of tribendimidine- and

473 derquantel-sensitive nAChRs. PLoS Pathog 10, e1003870.

474 Calapai, G., 2008. European legislation on herbal medicines: a look into the future. Drug

475 Saf 31, 428-431.

476 Camurca-Vasconcelos, A.L., Bevilaqua, C.M., Morais, S.M., Maciel, M.V., Costa, C.T.,

477 Macedo, I.T., Oliveira, L.M., Braga, R.R., Silva, R.A., Vieira, L.S., 2007. Anthelmintic

478 activity of Croton zehntneri and Lippia sidoides essential oils. Vet Parasitol 148, 288-294.

479 Cetin, H., Cilek, J.E., Aydin, L., Yanikoglu, A., 2009. Acaricidal effects of the essential oil of

480 Origanum minutiflorum (Lamiaceae) against Rhipicephalus turanicus (Acari: Ixodidae). Vet

481 Parasitol 160, 359-361.

482 Chatzidaki, A., Millar, N.S., 2015. Allosteric modu MANUSCRIPTlation of nicotinic acetylcholine receptors. 483 Biochem Pharmacol 97, 408-417.

484 Cliff, M.A., Green, B.G., 1994. Sensory irritation and coolness produced by menthol:

485 evidence for selective desensitization of irritation. Physiol Behav 56, 1021-1029.

486 Coats, J.R., 1994. Risks from natural versus synthetic insecticides. Annu Rev Entomol 39,

487 489-515. ACCEPTED

488 Committee on the Use of Complementary, a.A.M.b.t.A.P., Board on Health Promotion, and

489 Disease Prevention, Institute of Medicine, 2005. Complementary and Alternative Medicine

490 in the United States. The National Academies Press, Washington, DC.

ACCEPTED MANUSCRIPT 491 Coskun, S., Girisgin, O., Kurkcuoglu, M., Malyer, H., Girisgin, A.O., Kirimer, N., Baser,

492 K.H., 2008. Acaricidal efficacy of Origanum onites L. essential oil against Rhipicephalus

493 turanicus (Ixodidae). Parasitol Res 103, 259-261.

494 De Clercq, D., Sacko, M., Behnke, J., Gilbert, F., Dorny, P., Vercruysse, J., 1997. Failure

495 of mebendazole in treatment of human hookworm infections in the southern region of Mali.

496 Am J Trop Med Hyg 57, 25-30.

497 De Vincenzi, M., Stammati, A., De Vincenzi, A., Silano, M., 2004. Constituents of aromatic

498 plants: carvacrol. Fitoterapia 75, 801-804.

499 Dias, D.A., Urban, S., Roessner, U., 2012. A historical overview of natural products in drug

500 discovery. Metabolites 2, 303-336.

501 Eccles, R., 1994. Menthol and related cooling compoMANUSCRIPTunds. J Pharm Pharmacol 46, 618- 502 630.

503 Enan, E.E., 2005. Molecular and pharmacological analysis of an octopamine receptor from

504 American cockroach and fruit fly in response to plant essential oils. Arch Insect Biochem

505 Physiol 59, 161-171.

506 Farco, J.A., Grundmann, O., 2013. Menthol--pharmacology of an important naturally

507 medicinal "cool". ACCEPTEDMini Rev Med Chem 13, 124-131.

508 Ferrell, J.A., 1914. The Rural School and Hookworm Disease, 3-8.

ACCEPTED MANUSCRIPT 509 Fincham, J.E., Markus, M.B., Adams, V.J., 2003. Could control of soil-transmitted

510 helminthic infection influence the HIV/AIDS pandemic. Acta Trop 86, 315-333.

511 Flohr, C., Tuyen, L.N., Lewis, S., Minh, T.T., Campbell, J., Britton, J., Williams, H., Hien,

512 T.T., Farrar, J., Quinnell, R.J., 2007. Low efficacy of mebendazole against hookworm in

513 Vietnam: two randomized controlled trials. Am J Trop Med Hyg 76, 732-736.

514 Hans, M., Wilhelm, M., Swandulla, D., 2012. Menthol suppresses nicotinic acetylcholine

515 receptor functioning in sensory neurons via allosteric modulation. Chem Senses 37, 463-

516 469.

517 Harvey, A.L., Edrada-Ebel, R., Quinn, R.J., 2015. The re-emergence of natural products

518 for drug discovery in the genomics era. Nat Rev Drug Discov 14, 111-129.

519 Heinrich, M., Lee Teoh, H., 2004. Galanthamine MANUSCRIPT from snowdrop--the development of a 520 modern drug against Alzheimer's disease from local Caucasian knowledge. J

521 Ethnopharmacol 92, 147-162.

522 Hotez, P.J., Brindley, P.J., Bethony, J.M., King, C.H., Pearce, E.J., Jacobson, J., 2008.

523 Helminth infections: the great neglected tropical diseases. J Clin Invest 118, 1311-1321.

524 Hu, D., Coats, J., 2008. Evaluation of the environmental fate of thymol and phenethyl

525 propionate in the ACCEPTEDlaboratory. Pest Manag Sci 64, 775-779.

526 Jaradat, N., Adwan, L., K'Aibni, S., Shraim, N., Zaid, A.N., 2016. Chemical composition,

527 anthelmintic, antibacterial and antioxidant effects of Thymus bovei essential oil. BMC

528 Complement Altern Med 16, 418.

ACCEPTED MANUSCRIPT 529 Kaimoto, T., Hatakeyama, Y., Takahashi, K., Imagawa, T., Tominaga, M., Ohta, T., 2016.

530 Involvement of transient receptor potential A1 channel in algesic and analgesic actions of

531 the organic compound limonene. Eur J Pain 20, 1155-1165.

532 Kaplan, R.M., Storey, B.E., Vidyashankar, A.N., Bissinger, B.W., Mitchell, S.M., Howell,

533 S.B., Mason, M.E., Lee, M.D., Pedroso, A.A., Akashe, A., Skrypec, D.J., 2014.

534 Antiparasitic efficacy of a novel plant-based functional food using an Ascaris suum model

535 in pigs. Acta Tropica 139, 15-22.

536 Katiki, L.M., Chagas, A.C., Bizzo, H.R., Ferreira, J.F., Amarante, A.F., 2011. Anthelmintic

537 activity of Cymbopogon martinii, Cymbopogon schoenanthus and Mentha piperita

538 essential oils evaluated in four different in vitro tests. Vet Parasitol 183, 103-108.

539 Kim, Y.W., Kim, M.J., Chung, B.Y., Bang du, Y., Lim, S.K., Choi, S.M., Lim, D.S., Cho, 540 M.C., Yoon, K., Kim, H.S., Kim, K.B., Kim, Y.S., MANUSCRIPT Kwack, S.J., Lee, B.M., 2013. Safety 541 evaluation and risk assessment of d-Limonene. J Toxicol Environ Health B Crit Rev 16,

542 17-38.

543 Lamson, P.D., Ward, C.B., 1932. The chemotherapy of helminth infestations. J. Parasitol.

544 18, 173-199.

545 Le Hesran, J.Y., Akiana, J., Ndiaye el, H.M., Dia, M., Senghor, P., Konate, L., 2004.

546 Severe malaria attackACCEPTED is associated with high prevalence of Ascaris lumbricoides infection

547 among children in rural Senegal. Trans R Soc Trop Med Hyg 98, 397-399.

548 Lei, J., Leser, M., Enan, E., 2010. Nematicidal activity of two monoterpenoids and SER-2

549 tyramine receptor of Caenorhabditis elegans. Biochem Pharmacol 79, 1062-1071. 23

ACCEPTED MANUSCRIPT 550 Lemes, R.S., Alves, C.C.F., Estevam, E.B.B., Santiago, M.B., Martins, C.H.G., Santos, T.,

551 Crotti, A.E.M., Miranda, M.L.D., 2018. Chemical composition and antibacterial activity of

552 essential oils from Citrus aurantifolia leaves and fruit peel against oral pathogenic bacteria.

553 An Acad Bras Cienc 90, 1285-1292.

554 Lota, M.L., de Rocca Serra, D., Tomi, F., Jacquemond, C., Casanova, J., 2002. Volatile

555 components of peel and leaf oils of lemon and lime species. J Agric Food Chem 50, 796-

556 805.

557 Lozon, Y., Sultan, A., Lansdell, S.J., Prytkova, T., Sadek, B., Yang, K.H., Howarth, F.C.,

558 Millar, N.S., Oz, M., 2016. Inhibition of human alpha7 nicotinic acetylcholine receptors by

559 cyclic monoterpene carveol. Eur J Pharmacol 776, 44-51.

560 Macedo, I.T., Bevilaqua, C.M., de Oliveira, L.M., Camurca-Vasconcelos, A.L., Vieira Lda, 561 S., Oliveira, F.R., Queiroz-Junior, E.M., Portela, MANUSCRIPT B.G., Barros, R.S., Chagas, A.C., 2009. 562 Ovicidal and larvicidal activity in vitro of Eucalyptus globulus essential oils on Haemonchus

563 contortus. Rev Bras Parasitol Vet 18, 62-66.

564 Martin, R.J., 1997. Modes of action of anthelmintic drugs. Vet J 154, 11-34.

565 Morgan, E., Charlier, J., Hendrickx, G., Biggeri, A., Catalan, D., von Samson-

566 Himmelstjerna, G., Demeler, J., Müller, E., van Dijk, J., Kenyon, F., Skuce, P., Höglund, J.,

567 Kiely, P., van Ranst,ACCEPTED B., de Waal, T., Rinaldi, L., Cringoli, G., Hertzberg, H., Torgerson, P.,

568 Wolstenholme, A., Vercruysse, J., 2013. Global Change and Helminth Infections in

569 Grazing Ruminants in Europe: Impacts, Trends and Sustainable Solutions. Agriculture 3,

570 484.

ACCEPTED MANUSCRIPT 571 Newman, D.J., Cragg, G.M., 2016. Natural Products as Sources of New Drugs from 1981

572 to 2014. J Nat Prod 79, 629-661.

573 Newman, D.J., Cragg, G.M., Snader, K.M., 2000. The influence of natural products upon

574 drug discovery. Nat Prod Rep 17, 215-234.

575 Panella, N.A., Dolan, M.C., Karchesy, J.J., Xiong, Y., Peralta-Cruz, J., Khasawneh, M.,

576 Montenieri, J.A., Maupin, G.O., 2005. Use of novel compounds for pest control: insecticidal

577 and acaricidal activity of essential oil components from heartwood of Alaska yellow cedar.

578 J Med Entomol 42, 352-358.

579 Park, T.J., Park, Y.S., Lee, T.G., Ha, H., Kim, K.T., 2003. Inhibition of acetylcholine-

580 mediated effects by borneol. Biochem Pharmacol 65, 83-90.

581 Park, T.J., Seo, H.K., Kang, B.J., Kim, K.T., 2001. MANUSCRIPT Noncompetitive inhibition by camphor of 582 nicotinic acetylcholine receptors. Biochem Pharmacol 61, 787-793.

583 Pessoa, L.M., Morais, S.M., Bevilaqua, C.M., Luciano, J.H., 2002. Anthelmintic activity of

584 essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus contortus.

585 Vet Parasitol 109, 59-63.

586 Petrovska, B.B., 2012. Historical review of medicinal plants' usage. Pharmacogn Rev 6, 1-

587 5. ACCEPTED

588 Pirttila, T., Wilcock, G., Truyen, L., Damaraju, C.V., 2004. Long-term efficacy and safety of

589 galantamine in patients with mild-to-moderate Alzheimer's disease: multicenter trial. Eur J

590 Neurol 11, 734-741.

ACCEPTED MANUSCRIPT 591 Rapior, S., Marion, C., Pélissier, Y., Bessière, J.-M., 1997. Volatile Composition of

592 Fourteen Species of Fresh Wild Mushrooms ( Boletales ). J. Essent Oil Res. 9, 231-234.

593 Raskin, I., Ripoll, C., 2004. Can an apple a day keep the doctor away? Curr Pharm Des

594 10, 3419-3429.

595 Robertson, A.P., Martin, R.J., 2007. Ion-channels on parasite muscle: pharmacology and

596 physiology. Invert Neurosci 7, 209-217.

597 Roselli, M., Britti, M.S., Le Huerou-Luron, I., Marfaing, H., Zhu, W.Y., Mengheri, E., 2007.

598 Effect of different plant extracts and natural substances (PENS) against membrane

599 damage induced by enterotoxigenic Escherichia coli K88 in pig intestinal cells. Toxicol In

600 Vitro 21, 224-229.

601 Samuelsson, G., 2004. Drugs of Natural Origin: MANUSCRIPT A Textbook of Pharmacognosy, 5th Edn. 602 Swedish Pharmaceutical Press., Stockholm.

603 Savioli, L., Albonico, M., 2004. Soil-transmitted helminthiasis. Nat Rev Microbiol 2, 618-

604 619.

605 Squires, J.M., Foster, J.G., Lindsay, D.S., Caudell, D.L., Zajac, A.M., 2010. Efficacy of an

606 orange oil emulsion as an anthelmintic against Haemonchus contortus in gerbils (Meriones

607 unguiculatus) andACCEPTED in sheep. Vet Parasitol 172, 95-99.

608 Stammati, A., Bonsi, P., Zucco, F., Moezelaar, R., Alakomi, H.L., von Wright, A., 1999.

609 Toxicity of selected plant volatiles in microbial and mammalian short-term assays. Food

610 Chem Toxicol 37, 813-823.

ACCEPTED MANUSCRIPT 611 Stephenson, L.S., Latham, M.C., Ottesen, E.A., 2000. Malnutrition and parasitic helminth

612 infections. Parasitology 121 Suppl, S23-38.

613 Taman, A., Azab, M., 2014. Present-day anthelmintics and perspectives on future new

614 targets. Parasitol Res 113, 2425-2433.

615 Ton, H.T., Smart, A.E., Aguilar, B.L., Olson, T.T., Kellar, K.J., Ahern, G.P., 2015. Menthol

616 Enhances the Desensitization of Human alpha3beta4 Nicotinic Acetylcholine Receptors.

617 Mol Pharmacol 88, 256-264.

618 Tong, F., Coats, J.R., 2012. Quantitative structure-activity relationships of monoterpenoid

619 binding activities to the housefly GABA receptor. Pest Manag Sci 68, 1122-1129.

620 Tong, F., Gross, A.D., Dolan, M.C., Coats, J.R., 2013. The phenolic monoterpenoid 621 carvacrol inhibits the binding of nicotine to the MANUSCRIPT housefly nicotinic acetylcholine receptor. 622 Pest Manag Sci 69, 775-780.

623 Trailovi ć, S.M., Marjanovic, D.S., Nedeljkovic Trailovic, J., Robertson, A.P., Martin, R.J.,

624 2015. Interaction of carvacrol with the Ascaris suum nicotinic acetylcholine receptors and

625 gamma-aminobutyric acid receptors, potential mechanism of antinematodal action.

626 Parasitol Res 114, 3059-3068.

627 Vieira, A.J., Beserra,ACCEPTED F.P., Souza, M.C., Totti, B.M., Rozza, A.L., 2018. Limonene: Aroma

628 of innovation in health and disease. Chem Biol Interact 283, 97-106.

629 WHO, 2005. National Policy on Traditional Medicine and Regulation of Herbal Medicines-

630 Report of a WHO Global Survey. Geneva, Switzerland: WHO.

ACCEPTED MANUSCRIPT 631 WHO, 2018. Soil-transmitted helminth infections: Fact Sheet. http://www.who.int/en/news-

632 room/fact-sheets/detail/soil-transmitted-helminth-infections [Accessed 20 February 2018].

633 Williams, D.K., Wang, J., Papke, R.L., 2011. Positive allosteric modulators as an approach

634 to nicotinic acetylcholine receptor-targeted therapeutics: advantages and limitations.

635 Biochem Pharmacol 82, 915-930.

636 Zheng, F., Robertson, A.P., Abongwa, M., Yu, E.W., Martin, R.J., 2016. The Ascaris suum

637 nicotinic receptor, ACR-16, as a drug target: Four novel negative allosteric modulators

638 from virtual screening. Int J Parasitol Drugs Drug Resist 6, 60-73.

639

640 Figure Legends:

641 Figure 1: Chemical structures of monoterpenoid compounds used in the present study. 642 MANUSCRIPT 643 Figure 2: Agonist effects of monoterpenoid compounds on levamisole sensitive O. 644 dentatum nAChR. A. Sample traces for the agonist experiment. B. Bar chart (expressed

645 as mean ± SEM%) showing agonistic effects of the monoterpenoid compounds: menthol

646 (6.5 ± 2.4%, n = 6), geraniol (0.9 ± 0.2%, n = 6), cinnamaldehyde (0.6 ± 0.2%, n = 6),

647 citronellol (0.4 ± 0.2%, n = 6), menthyl acetate (0.3 ± 0.1%, n = 6), eugenol (0.3 ± 0.2%, n

648 = 6), phenethyl propionate (0.3 ± 0.1%, n = 6), limonene oxide (0.3 ± 0.2%, n = 6),

649 carvacrol (0.2 ± 0.2%, n = 6), menthone (0.2 ± 0.1%, n = 6), pulegone (0.1 ± 0.1%, n = 6) 650 and carvone (0.0 ACCEPTED± 0.0%, n = 6). 651

652 Figure 3: Effect of monoterpenoid compounds as antagonists on levamisole sensitive O.

653 dentatum receptor acetylcholine mediated responses. A. Bar graph showing the rank order

654 potency of monoterpenoid compounds as antagonists. Results were expressed as %

ACCEPTED MANUSCRIPT 655 mean inhibition ± SEM of currents elicited by 100 µM ACh: limonene oxide (36.0 ± 3.2%, n

656 = 6) > citronellol (18.0 ± 1.6%, n = 6) > carvone (14.0 ± 0.6%, n = 6) > carvacrol (11.0 ±

657 1.6%, n = 6) = pulegone (11.0 ± 2.6%, n = 6) = eugenol (11.0 ± 1.0 %, n = 6). Inset: image

658 showing predicted acetylcholine response in the absence of limonene oxide (dotted line)

659 and inhibition of acetylcholine mediated response in the presence of limonene oxide

660 (highlighted in green). B. Acetylcholine current responses, recorded from the Xenopus

661 oocyte expressing the levamisole sensitive channel, alone and in the presence of 100 µM

662 carvacrol (n=6) & 100 µM limonene oxide (n = 5). C. Concentration-response plots for

663 acetylcholine alone (n = 6, black) and acetylcholine in the presence of 100 µM carvacrol (n

664 = 6, maroon) & 100 µM limonene oxide (n = 5, green). pEC 50 (mean ± SEM), EC 50 (mean,

665 µM), Hill slope (nH) (mean ± SEM) and Imax (mean ± SEM%) values were respectively: 5.3

666 ± 0.0, 5.3 µM, 2.0 ± 0.2 and 104.6 ± 1.6 for acetylcholine alone; 5.4 ± 0.2, 4.0 µM, 2.2 ±

667 1.7 and 62.8 ± 7.0 in the presence of limonene oxide; 5.7 ± 0.1, 2.0 µM, 1.7 ± 1.1 and 74.1 668 ± 3.2 in the presence of carvacrol. Bottom was consMANUSCRIPTtrained to zero for curve fitting. 669

670 Figure 4: Effects of menthol as a PAM on levamisole sensitive O. dentatum nAChR

671 acetylcholine responses. A. Sample traces (above) for two-electrode voltage-clamp

672 recording showing inward currents in response to ascending concentrations of

673 acetylcholine alone, acetylcholine in the presence of 0.1 µM and 10 µM menthol. B.

674 Concentration-response relationships for acetylcholine alone (n = 7, black), acetylcholine

675 in the presence of 0.1 µM (n = 6, blue) and 10 µM menthol (n=7, purple). pEC 50 (mean ±

676 SEM), EC 50 (mean,ACCEPTED µM), Hill slope (nH) (mean ± SEM) and Imax (mean ± SEM%) values 677 were respectively: 5.3 ± 0.0, 5.0 µM, 1.4 ± 0.1 and 107.5 ± 2.6 for acetylcholine alone; 6.4

678 ± 0.1, 0.4 µM, 1.0 ± 0.1 and 109.0 ± 3.6 in the presence of 0.1 µM menthol; 6.5 ± 0.1, 0.3

679 µM, 1.0 ± 0.1 and 110.5 ± 3.7 in the presence of 10 µM menthol. Bottom was constrained

680 to zero for curve fitting. C. Bar chart showing comparison of pEC 50 (expressed as mean ±

ACCEPTED MANUSCRIPT 681 SEM) for acetylcholine in the presence and absence of menthol. ****P<0.0001;

682 significantly different as indicated; extra sum of squares F-test.

683

684 Figure 5: Effects of menthol on levamisole sensitive nAChR levamisole responses. A.

685 Representative current traces for two-electrode voltage-clamp recording showing inward

686 currents in response to ascending application of levamisole alone, levamisole in the

687 presence of 0.1 µM and 10 µM menthol. B. Concentration-response relationships for

688 levamisole alone (n = 7, steel gray) and in the presence of 0.1 µM (n = 6, light green) and

689 10 µM menthol (n=7, dark green). pEC 50 (mean ± SEM), EC 50 (mean, µM), Hill slope (nH)

690 (mean ± SEM) and Imax (mean ± SEM%) values were respectively: 6.3 ± 0.1, 0.5 µM, 1.0 ±

691 0.2 and 96.3 ± 5.2 for levamisole alone; 6.5 ± 0.1, 0.3 µM, 1.1 ± 0.2 and 94.6 ± 5.0 in the

692 presence of 0.1 µM menthol; 6.8 ± 0.2, 0.2 µM, 1.0 ± 0.2 and 89.2 ± 6.0 in the presence of

693 10 µM menthol. Bottom was constrained to zero for curve fitting. C. Bar chart summarizing 694 the results showing comparison of pEC 50 (expressedMANUSCRIPT as mean ± SEM) for levamisole in 695 the presence and absence of menthol. * P<0.05; significantly different as indicated; extra

696 sum of squares F-test.

697

698 Figure 6: Effects of menthol and carvacrol on nicotine sensitive A. suum ACR-16 nAChR

699 acetylcholine responses. A. Representative current traces for two-electrode voltage-clamp

700 recording showing inward currents in response to increasing acetylcholine concentrations

701 in the presence of menthol (10 µM) and carvacrol (10 µM and 100 µM) B. Concentration- 702 response plot forACCEPTED acetylcholine alone (n = 7, black) and acetylcholione in the presence of

703 menthol (n = 6, red). pEC 50 (mean ± SEM) and EC 50 (mean, µM) values were respectively:

704 5.0 ± 0.1 and 11.1 µM for acetylcholine alone, 5.0 ± 0.1 and 9.5 µM in the presence of 10

705 µM menthol. Hill slope (nH) (mean ± SEM) values were: 1.6 ± 0.3 for acetylcholine alone;

706 2.5 ± 1.2 in the presence of 10 µM menthol. Imax (mean ± SEM%) values were: 109.1 ± 6.6

ACCEPTED MANUSCRIPT 707 for acetylcholine alone; 101.6 ± 7.5 in the presence of 10 µM menthol. Bottom was

708 constrained to zero for curve fitting. C. Concentration-response plots for acetylcholine

709 alone (n = 7, black) and in the presence of carvacrol (n=6, blue). pEC 50 (mean ± SEM),

710 EC 50 (mean, µM), Hill slope ( nH) (mean ± SEM) and Imax (mean ± SEM%) values were

711 respectively: 5.1 ± 0.1, 8.1 µM, 2.5 ± 0.8 and 94.6 ± 5.9 in the presence of 10 µM

712 carvacrol, 5.1 ± 0.1, 8.2 µM, 2.0 ± 0.7 and 55.8 ± 4.4 in the presence of 100 µM carvacrol.

713 Bottom was constrained to zero for curve fitting.

714

715 Figure 7: A. Sample trace for the A. suum muscle flap contraction experiments. Inset:

716 concentration-response plots for acetylcholine alone (n = 6, black) and in the presence of

717 0.1 µM menthol (n = 6, red). pEC 50 (mean ± SEM) and EC 50 (mean, µM) values were

718 respectively: 5.2 ± 0.2 and 7.0 µM for acetylcholine alone; 5.2 ± 0.2 and 6.5 µM in the

719 presence of 0.1 µM menthol. Rmax (mean± SEM, g) values were: 1.7 ± 0.2 g for 720 acetylcholine alone and 2.0 ± 0.2 g in the presenceMANUSCRIPT of 0.1 µM menthol. Hill slope (nH) 721 (mean ± SEM) values were: 0.8 ± 0.2 for acetylcholi ne alone and 0.8 ± 0.1 in the presence

722 of 0.1 µM menthol. Bottom was constrained to zero for curve fitting. B. Bar chart

723 summarizing the results showing significant potentiation in amplitude of acetylcholine

724 mediated contractions in the presence of 0.1 µM menthol. *P<0.05, *** P<0.001;

725 significantly different as indicated; paired t-tests.

ACCEPTED

Figure 1 ACCEPTED MANUSCRIPT

carvacrol ((R)-(-))- carvone (+)-limonene oxide

L-menthol (-)-menthone (+)-pulegone

MANUSCRIPT

geraniol (S)-(-)-β-citronellol menthyl acetate

phenethylACCEPTED propionate eugenol

trans-cinnamaldehyde Figure 2 ACCEPTED MANUSCRIPT A 00 nA 5 2 min B 100

20 menthol Normalized current (%) 0 MANUSCRIPT ACh

menthol eugenol geranial carvacolcarvone menthonecitronellol pulegone

limonene oxide menthyl acetatecinnamaldehyde

phenethyl propianate

ACCEPTED Figure 3 A ACCEPTED MANUSCRIPT 50

30 nA 00 2 20 s 20 % Inhibition 10

carvone eugenol citrenellol carvacrolpulegone

limonene oxide B ACh 100 μM 0.3 1 3 10 30 100 300

500 nA 500 2 min ACh 100μM carvacrol MANUSCRIPT 100μM 3 10 30 100 300

500 nA 500 2 min ACh 100μM limonene oxide 100μM 3 10 30 100 300

500 nA 500 2 min

Control carvacrol 100 limonene ACCEPTEDoxide

50 ACh control % Current normalized to 100 µM

0 -6 -5 -4 log[ACh], M Figure 4 ACCEPTED MANUSCRIPT A ACh 100 μM 0.3 1 3 10 30 100 300

500 500 nA 5 min

ACh + 0.1 μM menthol 100 μM 0.1 0.3 1 10 30

ACh + 10 μM menthol

100 μM 0.1 0.3 1 10 30

0 0 50 5 min

ACh

+ 0.1 µM menthol

µ + 10 µM menthol 100

o 50

e MANUSCRIPT

r r

0 -8 -6 -4 log[ACh], M C

7 **** ****

C ACCEPTED

p 5

4 AChACh+ 0.1+ µ 0.1M +Me μ 1M0n thµ+Mo 10 lMe μnMth ol menthol menthol Figure 5 A ACCEPTED MANUSCRIPT ACh lev

100 μM 0.3 1 3 10 30 100 nA

200 200 5 min ACh + 10 μM menthol

100 μM 0.3 1 3 10 30

nA 200 200 5 min ACh + 0.1 μM menthol

100 μM 0.1 0.3 1 3 10 200 nA 200 5 min

Control

M + 0.1 µM menthol

100

0 + 10 µM menthol

t MANUSCRIPT

o 50

0 -8 -7 -6 -5 -4 log[lev], M

C * 7.0 ACCEPTED

6.5

C 6.0

5.5

5.0 levl + 0.1 μl M + 10 μlM ro o o t th th n mentholn mentholn o e e C m m M M µ µ 1 0 . 1 0 + + Figure 6 ACCEPTED MANUSCRIPT A menthol 10 μM Ach 100μM 3 10 30 100 300 nA 00

10 2 min carvacrol 10 μM Ach 100μM 3 10 30 100 300 1000 nA 1000 2 min carvacrol 100 μM Ach 100μM 3 10 30 100 300 1000 nA 1000 2 min B

150 Control +10µM menthol

100 MANUSCRIPT

ACh control 50

% Current normalized to 100 µM 0 -6.0 -5.5 -5.0 -4.5 -4.0 log [ACh], M C

Control +10µM carvacrol +100µM carvacrol 100 ACCEPTED

50 ACh control

% Current normalized to 100 µM 0 -6.0 -5.5 -5.0 -4.5 -4.0 log [ACh], M Figure 7 ACCEPTED MANUSCRIPT A

Control 2.0 + 0.1 µM menthol

)

g 1.5

(

a 1.0

o C 100 μM 0.5

0.0 30 μM -7 -6 -5 -4 log [ACh], M 100 μM

30 μM

10 μM

10 μM 3 μM

3 μM

1 g 1 μM ACh 1 μM ACh

1 min control menthol 0.1 μM MANUSCRIPT B *** 2.0 Control +0.1 µM menthol

1.5 ***

1.0 ACCEPTED*

Contractions (g) 0.5

0.0 1훍M 10 훍M 100훍M ACh ACCEPTED MANUSCRIPT

1 Highlights

2 • We screened 12 monoterpenoid compounds to search for a potential

3 anthelmintic.

4 • Menthol acted as PAM on levamisole sensitive nematode nAChRs in vitro & in

5 vivo .

6 • Carvacrol produced antagonism on both levamisole and nicotine sensitive

7 nAChRs.

8 • Monoterpenoid compounds can be used as an adjunct with cholinergic

9 anthelmintics.

MANUSCRIPT

ACCEPTED