Elucidating the Absorption and Metabolism of Linalool to Understand Its Potential Health Benefits

ELUCIDATING THE ABSORPTION AND METABOLISM OF LINALOOL TO UNDERSTAND ITS POTENTIAL HEALTH BENEFITS

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Angela Nicole Kerns

Graduate Program in Food Science and Technology

The Ohio State University

2017

Master's Examination Committee:

Christopher T. Simons Ph.D., Co-Advisor

Ken Lee Ph.D., Co-Advisor

Luis Rodriguez-Saona Ph.D.

Copyrighted by

Angela Kerns

2017

Abstract

Linalool, the impact aroma compound present in lavender, is a volatile compound that is prevalent in various foods, flavorings, beverages and perfumes. Lavender essential oil has been used in aromatherapy for centuries, and is known for its sedative and anxiety-reducing effects. There is a proposed pharmacological hypothesis for the beneficial effects of essential oils, which posits that volatile compounds, such as linalool, enter the bloodstream via the nasal or lung mucosa. Following entrance in the blood stream, the active constituent is able to interact and directly affect the central nervous system, resulting in observed changes in mood and physiology. A few studies document the appearance of linalool in blood following ingestion, inhalation and dermal application, supporting our hypothesis that the health benefits of linalool are mediated by the absorption of the compound. However, little is known about the metabolism of linalool following ingestion and inhalation, and it is unknown whether linalool metabolites are responsible for the biological and pharmacological action observed following linalool exposure.

Our research aims to better understand linalool absorption and metabolism following ingestion of a linalool beverage or inhalation of volatilized linalool. A small pilot clinical study was conducted wherein participants (n=6) received three different treatments: ingestion of a linalool beverage over 10 minutes, inhalation of linalool orthonasally for 10 minutes and inhalation of linalool retronasally for 10 minutes. Blood

ii samples were taken at various time points (t= 0, 15, 30, 45, 60, 90, 120 minutes), and urine was collected for 24 hours after each treatment. Standards of expected linalool metabolites were synthesized (8-OH-linalool and linalool glucuronide) or purchased, and

LC-MS/MS methods were developed to profile the metabolites in blood and urine.

Pharmacokinetic parameters for linalool glucuronide in plasma were determined for each of the treatments, and urine analysis revealed linalool glucuronide as the only metabolite detected with our methodology, which may have potential for pharmacological action.

Orthonasal and retronasal inhalation treatments led to significantly earlier metabolism of linalool, which was evidenced by an earlier Tmax at 20 minutes for linalool glucuronide in both inhalation treatments compared to Tmax at 37 minutes for ingestion of the linalool beverage. Future work with linalool-containing functional foods may generate insight on mechanisms of health promotion.

iii

Acknowledgments

I am very grateful for the opportunity to have completed my Master’s in Food

Science at OSU, and have many people to thank who played an important role in the success of my project. First off, special thanks to Dr. Christopher Simons, for being an incredible advisor, and guiding me through every step of the way. I could not have asked for a better mentor—he really made this an enjoyable experience and I feel so lucky to have studied under him for the past two years.

Many thanks to Dr. Steve Schwartz, for providing me with this opportunity to work in his lab under many brilliant scientists and for opening doors for a lot of collaboration in my project. To Dr. Jessica Cooperstone, who was like another advisor to me and went above and beyond in her role. I could not have achieved as much as I did without her assistance through each part of my project. Thanks to Dr. Ken Lee for stepping up as my co-advisor and to Dr. Luis Rodriguez-Saona, for being part of my committee. To Ken Riedl for being an integral part of method development and providing me with a plethora of knowledge along the way.

Many thanks to Dr. Curley—for being so patient in teaching a biologist like myself how to get into an organic chemistry lab and synthesize metabolites. Thanks for teaching me the real meaning of patience and how to jam out to Eric Clapton along the way.

Thanks to Dr. Bruno for allowing us to use his clinical space and for providing help from his lab as well--- to Josh McDonald for his phelobtomy help and resilience iv with a few unexpected events, to Julie Chitchumroonchokchai for lighting up a room every time help was needed and to Bryan Olmstead and Meredith Moller for their assistance during the clinical trial.

To the Simons’ lab & Schwartz’ lab both past and present for their amazing support and advice throughout this journey. Special thanks to Alex Pierce for being a huge help in the clinical trial and for making everyone laugh during those early stressful mornings. Thanks to my friends and officemates for making my time at OSU so memorable and fun along the way.

Finally, many thanks to Lisa & Dan Wampler, for providing part of my funding that made this research possible. I am very grateful for the opportunities their funding provided.

Last but certainly not least, to my family for their endless love and support.

Thanks for their constant encouragement—I would not be where I am today without them.

Vita

May 2011 ...... Lawrence North High School, Indpls., IN

May 2015 ...... B.S. Biology, Indiana University

August 2015 to present ...... Graduate Research Associate, Department

of Food Science and Technology, The Ohio

State University, Columbus, OH

March 2017 to present ...... Lisa and Dan Wampler Endowed Fellow for

Food and Health Research, Department of

Food Science and Technology, The Ohio

State University, Columbus, OH

Fields of Study

Major Field: Food Science and Technology

Table of Contents

Abstract ...... ii

Acknowledgments...... iv

Vita ...... vi

List of Tables ...... xii

List of Figures ...... xiii

CHAPTER 1- Literature Review ...... 1

1.1 Lavender Essential Oil- Why linalool? ...... 1

1.1.1 Linalool properties ...... 2

1.2.1 Mouse studies ...... 3

1.2.2 Human Clinical Trials ...... 4

1.3 Potential mechanisms ...... 5

1.4 Essential oil routes of delivery ...... 6

1.4.1 Inhalation ...... 6

1.4.2 Airflow pattern- Orthonasal Olfaction ...... 7

1.4.3 Airflow pattern- Retronasal Olfaction ...... 8

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1.4.4 Oral ...... 10

1.5 Pharmacokinetics of linalool ...... 11

1.5.1 Absorption ...... 12

1.5.2 Distribution ...... 13

1.5.3 Metabolism ...... 13

1.5.4 Excretion ...... 15

1.6 HPLC-MS/MS Analysis ...... 15

1.7 Overall objective: Better understand linalool absorption and metabolism in blood

and urine following inhalation (orthonasal & retronasal delivery) and ingestion of a

linalool beverage...... 16

CHAPTER 2: Introduction ...... 19

CHAPTER 3: Materials and Methods ...... 23

3.1 Materials ...... 23

3.2 General Methods ...... 23

3.2.1 Nuclear Magnetic Resonance (NMR) Spectroscopy ...... 24

3.3 Synthesizing 8-OH linalool by the methods of Curley & Ticoras and Bhalerao &

Rapoport51,52 ...... 24

3.4 Synthesizing linalool glucuronide by the methods of Lucas, Alcantara & Morales53

...... 26

3.5 Subjects ...... 28 viii

3.6 Study Design ...... 28

3.6.1 Aroma Apparatus ...... 31

3.6.2 Linalool Beverage ...... 31

3.7 Sample preparation ...... 31

3.7.1 Plasma extraction ...... 31

3.7.2 Urine dilution ...... 32

3.8 Ultra High Performance Liquid Chromatography- Tandem Mass Spectrometry

(UHPLC-MS/MS) ...... 32

3.9 Statistical Analysis ...... 35

CHAPTER 4: Results ...... 37

4.1 Plasma analysis ...... 37

4.1.1 Orthonasal inhalation pharmacokinetics ...... 39

4.1.2 Retronasal inhalation pharmacokinetics ...... 40

4.1.3 Beverage ingestion pharmacokinetics ...... 42

4.1.4 Comparing Tmax between all three treatments ...... 43

4.1.5 Comparing orthonasal and retronasal parameters (AUC, Cmax) ...... 45

4.2 Urine analysis ...... 48

4.2.1 Orthonasal and retronasal linalool glucuronide excretion ...... 49

4.2.2 Beverage linalool glucuronide excretion ...... 53

CHAPTER 5: Discussion ...... 55

5.1 Method Development...... 55

5.1.1 Confirming synthesized linalool metabolite standards with UHPLC-QTOF-MS

...... 55

5.2 Linalool glucuronide metabolism in plasma ...... 58

5.2.1 Linalool metabolism- primary metabolite ...... 59

5.2.2 Comparison of inhalation treatments to beverage treatment ...... 60

5.2.3 Orthonasal and retronasal inhalation differences ...... 61

5.3 Excretion of linalool glucuronide in urine ...... 62

CHAPTER 6: Conclusion ...... 64

Appendix A: Consent Form ...... 71

Appendix B: Subjective Stress Assessment ...... 80

Appendix C: Aroma Delivery Apparatus ...... 85

1.1 General Synthesis ...... 88

1.2 Synthesizing 8-hydroxylinalool ...... 88

1.2.1 First Approach based on method by Elsharif et al.1 ...... 88

1.2.2 Second approach based the methods of Curley & Ticoras and Bhalerao &

Rapoport51,52 ...... 89

1.2.3 Third & final approach based on the methods of Curley & Ticoras and

Bhalerao & Rapoport51,52 ...... 90

1.3 Linalool glucuronide synthesis ...... 91

1.3.1 First approach based on methods by Alonen et al60 ...... 91

1.3.2 Second approach based on methods by Lucas, Alcantara & Morales53 ...... 97

1.4 8-carboxylinalool synthesis ...... 98

1.4.1 First approach based on methods by Paquette & Heidelbaugh61 ...... 98

1.4.2 Second approach based on methods by Corey & Posner62 ...... 99

List of Tables

Table 1: MS/MS parameters for linalool and metabolites...... 34

Table 2: Analysis of linalool glucuronide in urine following orthonasal and retronasal inhalation treatments...... 52

Table 3: Analysis of linalool glucuronide in urine following beverage treatment...... 54

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List of Figures

Figure 1: Structure of linalool...... 1

Figure 2: Orthonasal and retronasal olfaction pathways (modified illustration from

Patrick L. Lynch, medical illustrator27)...... 7

Figure 3: Proposed linalool metabolic pathway in rats (Taken from Jager 201038)...... 14

Figure 4: Structures of linalool and metabolites (1=linalool, 2=8-hydroxylinalool, 3=8- carboxylinalool, 4=linalool glucuronide, 5=dihydrolinalool, 6=tetrahydrolinalool)...... 17

Figure 5: Allylic oxidation of linalool (1) to give 8-hydroxylinalool (2)...... 25

Figure 6: Synthesis of linalool glucuronide (4) from linalool (1)...... 27

Figure 7: Study design for linalool clinical trial...... 30

Figure 8: Chromatogram of linalool glucuronide in Subject 2 plasma after ingestion of linalool beverage. Linalool glucuronide eluted at 2.2 minutes, which coincides with peak of standard that can be found in Figure 17...... 38

(P<0.05), where average peak area of linalool glucuronide was significantly greater than

xiii time zero at 15 minutes, 30 minutes, 45 minutes, 60 minutes and 90 minutes. Mean average peak area was plotted ± standard error of mean (SEM)...... 40

Dunnett’s post-hoc test (P<0.05), where average peak area of linalool glucuronide was significantly greater than time zero at 15 minutes, 30 minutes, 45 minutes and 60 minutes points. Mean average peak area was plotted ± standard error of mean (SEM)...... 41

Figure 11: Relative concentration of linalool glucuronide in plasma following ingestion of a linalool beverage. Here, linalool glucuronide peaked on average across subjects at 30 minutes and decreased by 120 minutes, however was still significantly different from zero by the 120-minute time point. Note that the ordinate ranges from 0-20, which is two orders of magnitude greater than orthonasal inhalation (Figure 9) and retronasal inhalation (Figure 10), whose ordinates range from 0-0.20. * timepoints are significantly different by Dunnett’s post-hoc test (P<0.05), where average peak area of linalool glucuronide was significantly greater than time zero at all time points. Error bars are

SEM...... 43

Figure 12: Time to maximum peak intensity (Tmax) for all three treatments (orthonasal inhalation, retronasal inhalation, ingestion of beverage). Linalool glucuronide concentration peaked significantly later for the beverage compared to both orthonasal and retronasal inhalation conditions. Different letters denote significant differences using

xiv

ANOVA, modeling treatment, subject and treatment:subject, with Tukey’s post-hoc test

(P<0.05)...... 44

Figure 13: Relative concentration of linalool glucuronide in plasma following orthonasal and retronasal treatments. Average peak area across all subjects was plotted ± SEM. * denotes significance level P<0.05, wherein the concentration of linalool glucuronide was significantly greater following retronasal inhalation compared to orthonasal inhalation at

15 minutes, 45 minutes and 120 minutes, in addition to marginal significance at 30 minutes (p=0.052)...... 46

Figure 14: Calculated Area Under the Curve (AUC) of linalool glucuronide pharmacokinetic curve for orthonasal and retronasal inhalation. Error bars are SEM.

Different letters denote significant differences using a paired t-test (P<0.05). Retronasal

AUC was found to be significantly greater than orthonasal AUC (p=0.003)...... 47

Figure 15: Average relative maximum linalool glucuronide concentration absorbed in plasma (Cmax) following orthonasal and retronasal inhalation. Different letters denote significance using a paired t-test (P<0.05). Statistical analysis revealed Cmax was significantly greater following retronasal inhalation, compared to orthonasal inhalation, which was a similar trend noted in Figure 14. Error bars are SEM...... 48

Figure 16: Chromatogram of linalool glucuronide in urine of Subject 2 after ingestion of linalool beverage. Linalool glucuronide eluted at 2.2 minutes, which coincides with peak of standard that can be found in Figure 18...... 49

Figure 17: Arbitrary units of linalool glucuronide excreted in urine following orthonasal inhalation and retronasal inhalation. Total units excreted were significantly greater

(p=0.029, significance level α<0.05) following orthonasal inhalation compared to retronasal inhalation, which provides further evidence for this trend that was previously noted in plasma (Figure 13 & Figure 14)...... 50

Figure 18: Chromatogram of linalool glucuronide, 8-hydroxylinalool and linalool where each eluted at 2.2 min, 2.7 min, and 2.9 min, respectively. Metabolites were measured in ion abundance, however units were omitted given that the units remained arbitrary due to the unknown purity of the synthesized metabolites...... 57

(p=0.009), 45 minutes (p=0.009), 60 minutes (p=0.009) and 90 minutes (p=0.02) were all significantly lower than initial rating (2.5 ± 2.26), however no significance was found for time 120 minutes (p=0.166). For retronasal inhalation, post-hoc Dunnett’s t-test demonstrated significance for each time point compared to the initial rating of 3.33 ±

1.03 (15 minutes- p=0.002; 30 minutes- p=0.008; 45 minutes, 60 minutes, 90 minutes &

120 minutes- p<0.0001)...... 82

xvi minutes (p<0.0001). Interestingly, the stress ratings did not become significantly lower from the initial rating until 45 minutes and onward, which was the first blood collection time point after Tmax (30 minutes) in plasma for linalool glucuronide following the beverage treatment...... 83

Figure 21 Aroma delivery apparatus shematic. Breathing grade air was filtered, flowed at

6 liters per minute (for both inhalation conditions), humidified through water, bubbled through a 12% linalool in miglyol solution, flowed through an air trap to prevent any overflow from being inhaled by subjects, and finally was split to flow to orthonasal nosepiece or retronasal mouthpiece. If subjects were doing the orthonasal treatment, the tubing that connected the retronasal mouthpiece was clamped off, and vice versa...... 85

Figure 22: Orthonasal nosepiece...... 86

Figure 23: Retronasal mouthpiece...... 87

Figure 24: First attempt at 8-hydroxylinalool (2) synthesis where 8-oxolinalool predominated over 8-hydroxylinalool (2) in the resulting product...... 89

Figure 25: Second attempt at 8-hydroxylinalool (2) synthesis where 2 equivalents of selenium dioxide were used to make 8-oxolinalool followed by sodium borohydride reduction to 8-hydroxylinalool (2)...... 90

Figure 26: Third & final approach at 8-hydroxylinalool synthesis...... 91

Figure 27: Step one in approach one for linalool glucuronide synthesis where the protected glucuronide was linked with bromide to give glucuronosyl bromide...... 92

Figure 28: Step two in approach one of glucuronide synthesis where linalool and glucuronosyl bromide were combined to give protected linalool glucuronide...... 93

xvii

Figure 29: Mechanism of potential formation of glucuronide orthoester in step two of approach one for linalool glucuronide synthesis...... 94

Figure 30: Saponifaction of glucuronic acid protecting groups to give glucuronic acid conjugate...... 96

Figure 31: Second and final approach to synthesize linalool glucuronide...... 98

Figure 32: First approach to synthesize 8-carboxylinalool where 8-oxolinalool was first synthesized from 8-hydroxylinalool and then oxidized in air to give 8-carboxylinalool. 99

Figure 33: Second approach to synthesize 8-carboxylinalool where 8-carboxymethyl ester was made from 8-oxolinalool and then saponified to give 8-carboxylinalool...... 100

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CHAPTER 1- Literature Review

1.1 Lavender Essential Oil- Why linalool?

Essential oils have been used in aromatherapy for centuries as an alternative medicinal approach to relieve pain, reduce anxiety, enhance energy and improve mood

(Elsharif, Banerjee, & Buettner, 2015). Lavender essential oil in particular, is known for its floral fragrance and has traditionally been used to aid relaxation and sleep and also for its anti-bacterial and anti-inflammatory properties (Cavanagh & Wilkinson, 2002)(A.

Cavanagh & Wilkinson, 2005). Lavender is thought to be comprised of 26 main constituents, of which linalool and linalyl acetate predominate in the mixture (26% each).

Despite the approximate equal presence of linalool and linalyl acetate in lavender oil, linalool (Figure 1) was found to be an active constituent in lavender that led to its anxiolytic effects and consequently became the focus of this study (Umezu et al., 2006).

Figure 1: Structure of linalool.

1.1.1 Linalool properties

Linalool is an acylic monterpene alcohol and also the proposed compound responsible for the anxiolytic and sedative properties of lavender(Umezu et al., 2006). It is an oil at room temperature, however it is still an appreciable water-soluble compound, with a solubility of 850-1590 mg/L in water (UNEP Publications, 2002). Its fragrance is described as having a “floral, spicy, wood odor”, and is one of the most commonly used fragrant ingredients in cosmetic and beauty products, appearing in 70-90% of body lotions, shampoos, anti-perspirants, hairsprays and soaps (Burdock & Fenaroli, 2005; Cal

& Krzyzaniak, 2006). It is additionally added as a flavor or aroma to foods and beverages. Linalool is naturally found in a variety of citrus fruits, grapes, wine, spices and herbs. Consequently, people are exposed to linalool regularly, with an estimated oral exposure of 0.0438 mg/kg per day from food products in both the US and Europe (UNEP

Publications, 2002). Linalool is also synthesized and added to products to impart a lavender-like aroma.

1.2 Anxiolytic properties of linalool

Linalool has a variety of biological properties, including anxiolytic, sedative, anti- inflammatory, anticonvulsant and analgesic effects, however of primary interest in regard to this study is the anxiolytic property of linalool (Aprotosoaie, Hǎncianu, Costache, &

Miron, 2014).

2 1.2.1 Mouse studies

A variety of animal studies have demonstrated the anxiolytic and sedative effects of lavender and linalool following oral administration or inhalation utilizing pharmacological behavioral tests such as the elevated plus maze (B. F. Bradley, Starkey,

Brown, & Lea, 2007; Buchbauer, Jirovetz, Jäger, Dietrich, & Plank, 2015; Buchbauer,

Jirovetz, Jäger, Plank, & Dietrich, 1993; Linck et al., 2010; Souto-Maior et al., 2011). It was even found that lavender increased social interaction and decreased aggressive behavior in mice (Linck et al., 2010).

Furthermore, linalool has well-documented effects on the nervous system.

Linalool had a dose-dependent inhibitory effect on glutamate binding in rat cortex, which is the main excitatory neurotransmitter in the central nervous system (CNS)(Elisabetsky,

Marschner, & Onofre Souza, 1995). This suggests that the sedative effects of linalool could be mediated through inhibition of glutamatergic transmission. Additionally, in vitro linalool caused a rise in cyclic adenosine monophosphate (cAMP) levels in guinea pig ileum smooth muscle preparation, of which a rise in such levels normally also happens during stress (Lis-Balchin & Hart, 1999). Consequently, there is a proposed dichotomy between peripheral action in guinea pigs and overall relaxant effect in humans (Lis-

Balchin & Hart, 1999). A study conducted by Schuwald et al (2013) administered nanomolar range doses of Silexan™, the lavender oil capsule used in clinical trials, to mice that correlated to the dose administered to humans (80 mg/day) in the Silexan clinical trials (Schuwald et al., 2013). In contrast to other studies that administered supraphysiologically high doses of lavender oil, this study demonstrated that even at low concentrations (1-10 mg/kg body weight), Silexan still demonstrated anxiolytic effects in 3 the behavioral pharmacological tests (elevated plus maze)(Schuwald et al., 2013). In anxiety and stress disorders, it is proposed that there is an enhanced influx of Ca2+ that occurs through N and P/Q type voltage operated calcium channels (VOCCs), which subsequently causes a release of glutamate and norepinephrine neurotransmitters. An established anxiolytic, pregabalin, selectively binds at the P/Q type calcium channels, thus reducing the Ca2+ influx. Interestingly, Silexan was found to unselectively inhibit several VOCCs, which suggests a potential mechanism of linalool-evoked anxiolytic action. This unselective inhibition of VOCCs thereby is speculated to inhibit release of neurotransmitters glutamate and norepinephrine (Schuwald et al., 2013). Linalool additionally has an inhibitory effect on potassium-stimulated glutamate release in adult albino mice (Silva Brum, Emanuelli, Souza, & Elisabetsky, 2001).

1.2.2 Human Clinical Trials

There have been a few clinical studies conducted on the patented Silexan formula, which is 80 mg encapsulated lavender essential oil made from Lavandula angustifolia flowers with elevated levels of linalool and linalyl acetate. This Silexan formula is the active substance in the dietary supplement Lasea®, available in Germany (Uehleke,

Schaper, Dienel, Schlaefke, & Stange, 2012). A multi-center, double blind, randomized trial demonstrated anxiolytic efficacy of Silexan compared to a placebo. Subjects with

‘subsyndromal’ anxiety disorder received the Silexan preparation (n=107) or placebo

(n=109) for 10 weeks and anxiety was assessed with Hamilton Anxiety Rating Score

(HAMA) every two weeks. A significant reduction in HAMA total score was seen from week 4 and the remaining 6 weeks between both treatments, with final HAMA score 4 reduction of 59.3% in the Silexan group and 35.4% in the placebo group (Kasper et al.,

2010). Additionally, another multi-center, double blind, randomized clinical trial evaluated anxiolytic efficacy of Silexan however they compared it to a benzodiazepine,

Lorazepam. Subjects with Generalized Anxiety Disorder (GAD) were treated daily with either Silexan (80 mg capsule; n=40) or Lorazepam (standard dosage of 0.5 mg capsule; n=37) for 6 weeks and anxiety was again assessed with HAMA scale. HAMA scores decreased similarly between the two groups over the 6-week period and ultimately were reduced 45% in the Silexan group and 46% in the Lorazepam group (Woelk & Schlafke,

2010). Furthermore, a recent study on Silexan have also found an 80mg dose and 160mg dose to be significantly better at reducing HAMA scores in GAD and at minimum just as effective as paroxetine, another benzodiazepine also regularly used to treat GAD (Kasper et al., 2014). These clinical trials demonstrate the strong anxiolytic properties of lavender.

1.3 Potential mechanisms

Aromatherapy and lavender essential oil use have been around for centuries, however it has only been in the last 50 years that studies have looked into biological activity of lavender and more specifically linalool. There has been a bit of skepticism looking at aromatherapy and its efficacy, due to the strength of the placebo effect and studies lacking proper controls. There certainly is potential for a placebo effect, where the aroma can cause therapeutic effects by means of an expectation bias and associative conditioning through emotional learning/olfactory cognition. Regarding the latter effect, aromas can have an impact on mood due to the proximity of the olfactory system and the limbic system, where there can be an association with certain moods and memories from 5 the initial aroma exposure (Köteles & Babulka, 2014). However, as mentioned previously, my interest lies in the biological activity of linalool.

There is a proposed pharmacological hypothesis for the beneficial effects of essential oils, which posits that volatile compounds, such as linalool, enter the bloodstream via the nasal or lung mucosa. Following entrance in the blood stream, the active constituent is able to interact and directly affect the CNS, resulting in observed changes in mood and physiology. This may result in the observed change in mood, physiology and behavior as observed when individuals are exposed to aromatherapy, or in the case of linalool, an anxiolytic or sedative effect (Herz, 2009).

1.4 Essential oil routes of delivery

The main routes of delivery for essential oils are dermal, inhalation and oral.

However, for the purpose of this study, I am interested solely in inhalation (orthonasal and retronasal) and ingestion delivery routes given that the pharmacokinetics of the ingestion route and retronasal inhalation has yet to be elucidated.

1.4.1 Inhalation

olfactory epithelium

ORTHONASAL

RETRONASAL pharynx

Figure 2: Orthonasal and retronasal olfaction pathways (modified illustration from Patrick L. Lynch, medical illustrator(Lynch, 2006)).

1.4.2 Airflow pattern- Orthonasal Olfaction

One function of the nose is to warm, humidify and filter the inspired air. In orthonasal olfaction, as air flows through the nose, some volatile molecules are 7 transported through the ciliated olfactory epithelium, where the olfactory receptors are located, consequently resulting in odorant perception. Due to the protected structure of the olfactory epithelium, only an estimated 10% of the inhaled air reaches the olfactory region during normal breathing (Zhao, Scherer, Hajiloo, & Dalton, 2004). Airflow primarily occurs on the nasal floor, with the lowest air flow occurring in the superior region of the nasal cavity, which also contributes to a small percentage of volatiles that actually make it into the olfactory epithelium (Hahn, Scherer, & Mozell, 1993; Keyhani,

Scherer, & Mozell, 1995). Another factor affecting absorption into the olfactory epithelium is the polarity of the inhaled volatile compound, which can affect aroma partitioning (Ployon, Morzel, & Canon, 2017). Once absorbed in the mucosa, the compounds make their way to the capillaries where they can then be in circulation throughout the body and exert pharmacological effects. The remaining air continues through the bronchioles until the alveoli are reached, which are lined with many capillaries and thus facilitate transportation of the volatiles from the gaseous lungs into circulation (Rhind, 2012).

1.4.3 Airflow pattern- Retronasal Olfaction

In retronasal olfaction, volatile compounds travel through the pharynx, over the soft palate and ultimately reach the olfactory epithelium. This occurs during mastication and swallowing of foods and beverages, where the soft palate controls the volatiles that reach olfactory epithelium (Aylor, 2002). Volatile compounds that are inhaled retronasally go through a different environment than through the orthonasal route, due, in part, to the presence of saliva in the mouth. Saliva is primarily made up of water (~98%) 8 and contains over a thousand proteins, of which many are enzymes that catalyze chemical reactions, which can subsequently affect aroma release (Denny et al., 2008). Such enzymes include α-amylase, carbonic anhydrase IV and lysozyme, which also are responsible for antimicrobial activity and regulating the oral microbiome (Ployon et al.,

2017).

A predominate protein in saliva are mucins, and they are responsible primarily for the thick viscosity of saliva and maintaining lubrication in the mouth (Denny et al.,

2008). Mucins are large glycoproteins that are negatively charged and have been found to be the key component in flavor release where hydrophobic binding with volatile compounds can subsequently affect the concentration of the volatile in the headspace

(Friel & Taylor, 2001). α-Amylase was also found to decrease aroma release in saliva due to hydrophobic effects as well, but there was not a cumulative effect noted on aroma release when the two proteins were observed in conjunction. Therefore, it is possible that protein-protein interactions between mucin and α-amylase can decrease the available binding sites for volatile compounds (Pagès-Hélary, Andriot, Guichard, & Canon, 2014).

Additionally, when human saliva was compared to artificial saliva composed of mucins and α-amylase, there was an increase in hydrolysis of esters to their acid forms in human saliva, demonstrating the presence of esterase activity and the potential for enzymatic degradation of aroma compounds in saliva (Pagès-Hélary et al., 2014).

In addition to enzymes and proteins, saliva also contains the oral microbiome, which can also affect the composition of aroma compounds in retronasal inhalation. The oral microbiome is very diverse, containing a total of 707 bacterial species (Mark Welch,

Rossetti, Rieken, Dewhirst, & Borisy, 2016) . A study on odorless grape glycosidic 9 aroma precursors demonstrated the ability of typical oral bacteria and human whole oral microbiota to hydrolyze these glycosidic precursors to form aromatic aglycones including: terpenes, benzenic compounds and lipid derivatives (Muñoz-González, Cueva,

Ángeles Pozo-Bayón, & Moreno-Arribas, 2015). In this study, linalool was one of the terpenes monitored over time (specifically incubation time of glycosidic grape precursor with whole saliva) and an inter-individual variability was noted over time between the three subjects when comparing linalool produced in aerobic conditions and anaerobic conditions. Therefore, the type of bacteria in the saliva had a significant effect on hydrolytic activity of the oral microbiota, which affected the observed type of aglycone compounds (Muñoz-González et al., 2015).

1.4.4 Oral

Another potential route of delivery could be through ingestion, as there are now oral supplements available for lavender such as Lasea®, which is made of the Silexan formula (Kasper et al., 2010). In this case, the active constituent (linalool in this case) would be absorbed through the gastrointestinal tract and ultimately circulate in blood and have potential to interact with the central nervous system (Jager, 2010; Rhind, 2012).

Digestion begins in the oral cavity, where the capsule or beverage is mixed with saliva and when swallowed, is propelled through the pharynx (throat) and esophagus until it reaches the stomach. In the stomach, the beverage or capsule is mixed with gastric juices that contain hydrochloric acid, enzymes (pepsin, gastric lipase and α-amylase) and mucus, which facilitate breakdown of food. Only a small amount of absorption occurs in the stomach, including water, alcohol and some fat-soluble drugs. From the stomach, 10 digestion continues through the duodenum followed by the jejunum and ileum (all making up the small intestine) where the majority of nutrient absorption occurs.

Absorption occurs in the enterocytes that line the lumen of the gastrointestinal tract through diffusion, facilitated diffusion, active transport or pinocytosis or endocytosis.

Water and small lipid molecules are absorbed into enterocytes via diffusion, where the molecules move freely across a membrane from higher concentration to lower concentration. Once in the enterocytes, the compound or molecule can diffuse into the surrounding interstitial fluid and then capillaries via osmotic pressure and is subsequently filtered through the kidneys to prepare for excretion in urine. The remaining unabsorbed material traverse the colon and are excreted in feces (Gropper & Smith, 2013).

To my knowledge, this route of delivery has only been investigated with the use of encapsulated lavender (Silexan) and pharmacokinetics have not been determined following consumption of linalool in a beverage. Therefore this is a novel route for delivery for an essential oil constituent. Additionally, it should be noted that beverage consumption contains a small amount of retronasal olfaction that occurs when the beverage is swallowed (Figure 2) and contributes to the perceived flavor of the beverage

(Rozin, 1982).

1.5 Pharmacokinetics of linalool

It is plausible that a metabolite, or metabolites of linalool are responsible for its observed biological efficacy. For this reason, I am additionally interested in understanding linalool metabolism in humans. Linalool metabolism has been elucidated

11 in rats however a comprehensive overview of all linalool metabolites has yet to be completed in humans.

1.5.1 Absorption

Linalool was found to be absorbed rapidly from the gut following oral and gavage dosing (500 mg/kg bw) of 14C-labelled linalool to rats, with 10% of the dose excreted in urine after around 7 hours following dose administration (Parke, Rahman, & Walker,

1974). In humans, a few studies document the appearance of linalool in blood following dermal application, inhalation and ingestion, supporting my hypothesis that the volatile compound enters the blood through absorption through nasal or lung mucosa. After application of a massage oil containing lavender oil and peanut oil (2:28 w/w) to a defined area of skin (376 cm2), linalool appeared in the bloodstream within 5 minutes of finishing the massage (total massage time was 10 minutes) and the peak concentration was observed at 19 minutes (Buchbauer & Jirovetz, 1992). Additionally, a separate study assessed the pharmacokinetics of linalool in blood following either inhalation of linalool for 45 minutes (10%, w/w solution of (-)-linalool in propylene glycol) or dermal application of (-)-linalool in peanut oil over a 200 cm2 area of skin. In the inhalation group, linalool appeared in blood at 25 minutes and decreased around 40-45 minutes, while linalool was detected in blood around 30 minutes following the massage in the dermal group (Friedl et al., 2010). Lastly, linalool appeared in blood of subjects at around

10-15 minutes following oral ingestion of 200 μl lavender essential oil, with peak concentrations observed at 30 minutes (Belinda F Bradley, Brown, Chu, & Lea, 2009).

12 1.5.2 Distribution

The study conducted by Parke et al. mentioned previously found that 72 hours following the 500 mg/kg bw 14C-labeled linalool administration to rats, 0.5% of the dose was found in liver, 0.6% in gut, 0.8% in skin and 1.2% in skeletal muscle, accounting for

3% of original dose (Parke, Rahman, et al., 1974).

1.5.3 Metabolism

Once ingested or inhaled, linalool is metabolized to a more polar and water- soluble compound to facilitate excretion, which is deemed xenobiotic metabolism. In general, despite most organs being capable of metabolic biotransformation, the liver is the primary site of metabolism. There are two phases of biotransformation, including phase I and phase II reactions. Phase I reactions are mostly catalyzed by the cytochrome

P450 enzyme, and the compound is oxidized, reduced or hydrolyzed. Subsequently, in phase II reactions, the phase I metabolites are conjugated to highly water-soluble endogenous entities including glucuronic acids and sulfates to facilitate excretion further(Jager, 2010).

In rats, the predominant metabolite detected in urine was the glucuronic acid conjugate following an oral dose of 500 mg/kg 14C-linalool (Figure 3)(Parke, Rahman, et al., 1974). Furthermore, in a separate study this same linalool dose (without the radioactivity) was administered to rats by intragastric intubation daily for a prolonged period of 64 days and a 17% increase in 4-methylumbelliferone glucuronyltransferase activity was seen by day 3. There was also a delayed induction of cytochrome P-450 enzymes (Parke, Quddusur Rahman, & Walker, 1974). Other reduced metabolites 13 dihydrolinalool and tetrahydrolinalool were detected in rat urine in a separate study following single oral dose of linalool (Figure 3)(Rahman, 1974). Additionally, when rats were dosed orally with 800 mg/kg body weight linalool daily for 20 days, the oxidized linalool metabolites 8-hydroxylinalool and 8-carboxylinalool were detected in urine

(Figure 3). Treatment with linalool also resulted in a ~50% induction of liver microsomal cytochrome P-450 (Chadha & Madyastha, 1984).

Figure 3: Proposed linalool metabolic pathway in rats (Taken from Jager 2010(Jager, 2010)).

1.5.4 Excretion

In rats orally administered 500 mg/kg body weight 14C-linalool, the primary route of excretion was via urine as the glucuronic acid conjugate (55% of original dose).

14 Additionally, 23% was excreted as expired C labeled CO2 within a few hours of administration, and 15% excreted in feces between 36 and 48 hours after dosing. The delay in excretion via expiration and fecal route suggests intermediary metabolism and presence of biliary excretion. Moreover, 25% of the original dose appeared in bile as glucuronic acid conjuguate within 4 hours of dosing thus confirming enterohepatic circulation (Parke, Rahman, et al., 1974).

1.6 HPLC-MS/MS Analysis

Previous studies that quantified linalool in biological fluids in humans utilized gas chromatography- mass spectrometry (GC-MS) due to the volatile nature of linalool(Buchbauer & Jirovetz, 1992; Friedl et al., 2010). Furthermore, most studies that analyzed linalool in plant samples also used GC-MS techniques(Qin, Lu, & Chen, 1999;

Shang, Yaoming, Deng, & Hu, 2002). However, there was a study that developed a reversed-phase high performance- liquid chromatography (HPLC) method to determine the distribution of linalool in the Michelia alba plant. In this method, a C18 analytical column (4.6 mm x 250 mm, 5 μm) was used with a 210 nm detection wavelength and linalool eluted isocratically at 8.5 minutes in a mobile phase of 55:45 acetonitrile:water

(Xia et al., 2010a). Because of the lack of a chromophore in linalool, this method was reasonably non-specific. To my knowledge, this is the only publication of an HPLC 15 method for linalool. Therefore, an HPLC method with tandem mass spectrometry would be of benefit to this area of literature, specifically with the mass spectrometry aspect, as it would provide additional information to confirm the presence of linalool and its metabolites.

1.7 Overall objective: Better understand linalool absorption and metabolism in blood and urine following inhalation (orthonasal & retronasal delivery) and ingestion of a linalool beverage.

Linalool has been documented in blood/plasma following inhalation and dermal application (Buchbauer & Jirovetz, 1992; Friedl et al., 2010). However, linalool metabolites found in Figure 4, have only been documented in rat urine after different oral doses (Chadha & Madyastha, 2009; Parke, Rahman, et al., 1974; Rahman, 1974).

Therefore, what is missing is a comprehensive overview of linalool and metabolites in human plasma and urine.

I hypothesize that linalool will appear around the same time in orthonasal and retronasal inhalation conditions and will lead to the same metabolites in blood and urine.

Additionally, I hypothesize that linalool will appear later in blood following ingestion conditions compared to both inhalation conditions and that it will lead to different metabolites

The overall objective was accomplished by the following:

A) Synthesize linalool metabolites that could not be purchased.

Three of the metabolites of interest were synthesized because they could not be purchased. This included: 8-hydroxylinalool (2), 8-carboxylinalool (3) and linalool 16 glucuronide (4) (Figure 4). These metabolites were needed to serve as standards for identification of linalool and metabolites in biological samples.

Figure 4: Structures of linalool and metabolites (1=linalool, 2=8-hydroxylinalool, 3=8- carboxylinalool, 4=linalool glucuronide, 5=dihydrolinalool, 6=tetrahydrolinalool).

B) Specific Aim 2:Develop HPLC-MS/MS methods for linalool and metabolites.

High performance liquid chromatography with tandem mass spectrometry

(HPLC-MS/MS) methods were developed for linalool (1), 8-hydroxylinalool (2), 8- carboxylinalool (3), linalool glucuronide (4), dihydrolinalool (5) and tetrahydrolinalool

(6) to ultimately be able to profile linalool and metabolites in plasma and urine.

C) Quantify and compare linalool and metabolites in blood and urine following inhalation (orthonasal & retronasal delivery) and ingestion of a linalool beverage.

HPLC-MS/MS methods were then used to quantify linalool (1) and metabolites

(8-hydroxylinalool (2), 8-carboxylinalool (3), linalool glucuronide (4), dihydrolinalool

(5) and tetrahydrolinalool (6)) in plasma and urine and subsequently compare any differences between treatments.

CHAPTER 2: Introduction

Lavender essential oil is known for its floral fragrance and has traditionally been used to aid relaxation and sleep and also for its anti-bacterial and anti-inflammatory properties (Cavanagh & Wilkinson, 2002)(A. Cavanagh & Wilkinson, 2005). Lavender is comprised of 26 main constituents, of which linalool and linalyl acetate predominate in the mixture (26.12% and 26.32%, respectively). Despite the approximate equal presence of linalool and linalyl acetate in lavender oil, linalool (Figure 1) was found to be an active constituent in lavender that led to its anxiolytic effects and consequently became the focus of this study (Umezu et al., 2006).

(850-1590 mg/l)(UNEP Publications, 2002). Its fragrance is described as “floral, spicy, wood odor”, and is one of the most commonly used fragrant ingredients in cosmetic and beauty products, appearing in 70-90% of body lotions, shampoos, anti-perspirants, hairsprays and soaps (Burdock & Fenaroli, 2005; Cal & Krzyzaniak, 2006). It is additionally added as a flavor or aroma to foods and beverages and also naturally found in a variety of citrus fruits, grapes, wine, spices and herbs.

19 Linalool has a variety of biological properties, including anxiolytic, sedative, anti- inflammatory, anticonvulsant and analgesic effects, however of primary interest in regard to this study is the anxiolytic property of linalool(Aprotosoaie et al., 2014).

The main routes of delivery for essential oils are dermal application, inhalation and ingestion. However, for the purpose of this study, I am interested solely in inhalation

(orthonasal and retronasal) and ingestion delivery routes given that the pharmacokinetics of the ingestion route and retronasal inhalation has yet to be elucidated.

There are two different inhalation pathways, orthonasal and retronasal inhalation, the former of which is more commonly known and the other latter less so. Orthonasal inhalation is associated with the detection of odorants arising from the outward environment, while retronasal inhalation refers to volatile compounds associated inwardly with foods (Aylor, 2002). When odorants are inhaled orthonasally or retronasally, they ultimately reach the same olfactory epithelium, however despite this, there is a proposed duality of smells hypothesis which posits that the two routes lead to different perceptions (Rozin, 1982). I am interested in better understanding pharmacological differences between the two inhalation routes, since much of the previous research has focused on perceptual differences(Hummel et al., 2006). Another potential route of delivery could be through ingestion, as there are now oral supplements available for lavender such as LASEA®, which is made of the Silexan formula (Kasper et al., 2010). To my knowledge, this route of delivery has only been investigated with the use of encapsulated lavender (Silexan) and pharmacokinetics have not been determined following a beverage. Therefore this is a novel route for delivery for an essential oil constituent. 20

There is a proposed pharmacological hypothesis for the beneficial effects of essential oils, which posits that volatile compounds, such as linalool, enter the bloodstream via the nasal or lung mucosa. Following entrance in the blood stream, the active constituent is able to interact and directly affect the central nervous system, resulting in observed changes in mood and physiology. In humans, a few studies document the appearance of linalool in blood following dermal application, inhalation and ingestion, supporting our hypothesis that the volatile compound enters the blood through absorption of nasal or lung mucosa (Belinda F Bradley et al., 2009; Buchbauer &

Jirovetz, 1992; Friedl et al., 2010).

It is plausible that a metabolite, or metabolites of linalool are responsible for its observed biological efficacy. For this reason, I am interested in understanding linalool metabolism in humans. Linalool metabolism has been elucidated in rats however a comprehensive overview of all linalool metabolites has yet to be completed in humans. In rats, the predominant metabolite detected in urine was the glucuronic acid conjugate following an oral dose of 500 mg/kg 14C-linalool. Other reduced metabolites dihydrolinalool and tetrahydrolinal were detected in rat urine in a separate study following single oral dose of linalool. Additionally, when rats were dosed orally with 800 mg/kg body weight linalool daily for 20 days, the oxidized metabolites 8-hydroxylinalool and 8-carboxylinalool were detected in urine.

Previous studies that quantified linalool in biological fluids in humans utilized gas chromatography- mass spectrometry analytical methods (GC-MS) due to the volatile nature of linalool (Buchbauer & Jirovetz, 1992; Friedl et al., 2010). However, there was a 21 study that developed a reversed-phase high performance- liquid chromatography (HPLC) method to determine the distribution of linalool in the Michelia alba plant. To my knowledge, this is the only publication of an HPLC method for linalool.

The first objective was to develop high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) methods for linalool (1), 8-hydroxylinalool

(2), 8-carboxylinalool (3), linalool glucuronide (4), dihydrolinalool (5) and tetrahydrolinalool (6) to ultimately profile linalool and metabolites in plasma and urine.

Linalool, dihydrolinalool and tetrahydrolinalool standards were purchased, and linalool glucuronide and 8-hydroxylinalool standards were synthesized since they could not be purchased. The second objective was to then quantify and compare linalool and metabolites (linalool glucuronide, 8-hydroxylinalool, dihydrolinalool, tetrahydrolinalool) in blood and urine following inhalation (orthonasal and retronasal delivery) and ingestion of a linalool beverage.

CHAPTER 3: Materials and Methods

3.1 Materials

Linalool (1), selenium dioxide, methyl-2,3,4-tri-O-acetyl-α-D-glucuronic acid trichloroacetimidate, and boron trifluoride diethyl etherate were purchased from Sigma-

Aldrich (St. Louis, MO). Dihydrolinalool (5) and tetrahydrolinalool (6) were purchased from TCI chemicals (Portland, OR). LCMS optima grade water, acetonitrile and 99% formic acid were obtained from Fisher Scientific (Waltham, MA). Other reagents used were purchased from Sigma Aldrich.

3.2 General Methods

All synthesis reactions were carried out in anhydrous conditions, and dry glassware was used. Thin Layer Chromatography (TLC) was conducted on 1 x 5 cm silica gel F254 plates and preparative TLC was performed on 20 cm x 20 cm plates with

1000 μm silica gel F254 layers from Analtech (Newark, DE). TLC plates were visualized through phosphomolybdic acid staining for all reactions except for the derivitization reaction, which was visualized through fluorescence quenching. Column chromatography purifications were performed on silica gel 60Å packed columns.

23 3.2.1 Nuclear Magnetic Resonance (NMR) Spectroscopy

1H and 13C-NMR spectra were recorded at the appropriate digital resolution in

CDCl3 at 400 and 100 MHz frequencies respectively on a Bruker DRX400 instrument

(Billerica, MA). Chemical shifts are reported in parts per million (ppm) and signal multiplicities are indicated by dd (doublet of doublets), m (multiplet), and s (singlet).

3.3 Synthesizing 8-OH linalool by the methods of Bhalerao & Rapoport, 1971; Curley Jr

& Ticoras, 1986

Linalool (1) (4.85 g; 31.4 mmol), 1.75 g selenium dioxide (SeO2)(15.8 mmol) and

25 mL ethanol were refluxed with stirring for 3 hours (Figure 5). Solvent was then evaporated under reduced pressure and the residue dissolved in ethyl acetate. This organic layer was extracted with saturated sodium bicarbonate solution, washed with saturated NaCl solution, dried (MgSO4) and concentrated to dryness. The product was purified twice by silica gel column chromatography (53.5 cm x 3 cm) with a mobile phase of 10% then 20% ethyl acetate in hexanes for the first purification and 10%, 15%,

25%, 30% and 50% ethyl acetate in hexanes for the second purification. Fractions were monitored by TLC and fractions that contained the same compound were pooled and evaporated under reduced pressure to provide 630 mg of oil. NMR spectroscopy showed the presence of some remaining impurities.

Figure 5: Allylic oxidation of linalool (1) to give 8-hydroxylinalool (2).

To further purify the product (Figure 5) the mixture was derivatized as the p- nitrobenzoate ester to facilitate purification. The 8-hydroxylinalool (2) (400 mg), 1.5 g 4- nitrobenzoyl chloride, 5 mL anhydrous acetonitrile (ACN), 1 mL pyridine and a catalytic amount of DMAP were combined and stirred for 4 days. ACN was then evaporated with a stream of air and subsequently re-dissolved in ethyl acetate. This organic layer was extracted with 1M HCl, washed with water, extracted with sodium bicarbonate, washed with saturated NaCl solution, dried (MgSO4) and then concentrated to dryness. The residue was then re-dissolved in acetone and purified with preparative TLC (solvent system: 15% ethyl acetate in hexanes). Two fractions were collected and 1H-NMR spectroscopy revealed at least two products in each fraction. Consequently, both fractions were again re-dissolved in acetone and re-purified twice more with preparative TLC

(solvent system: 15% ethyl acetate in hexanes). Finally, both fractions were re-dissolved in ethanol and saponified separately (combined with 5M KOH and stirred for 4 days).

Ethanol was evaporated, and each residue was extracted with ethyl acetate, washed with brine, dried (sodium sulfate) and concentrated to dryness. 1H-NMR spectra of both 25 fractions were compared against previously published spectra of 8-hydroxylinalool (2)

(Elsharif et al., 2015) and it was determined which fraction was the desired 8- hydroxylinalool, which provided 12.3mg of oil. Final yield was 0.007% and NMR

1 characteristics, reported in parts per million (ppm): H NMR (CDCl3) δ 5.79 (dd, 1H, J =

9.5, 17.7 Hz), 5.41 (m, 1H), 5.14 (m, 2H), 4.00 (s, 2H), 2.06 (m, 2H), 1.67 (s, 3H), 1.58

13 (m, 2H), 1.26 (s, 3H); C NMR (CDCl3) δ 142.9, 134.2, 125.9, 113.8, 76.5, 68.6, 38.8,

21.9, 21.5, 13.2. An explanation of all approaches taken to synthesize 8hydroxylinalool can be found in Appendix D.

3.4 Synthesizing linalool glucuronide by the methods of Lucas, Alcantara, & Morales,

2009

Linalool (300ul, 1 mmol), 1.0 g Methyl-2,3,4-tri-O-acetyl-α-D-glucuronic acid trichloroacetimidate (2.1 mmol), 50ul BF3·H2O (0.53mmol) and excess dichloromethane were combined over ice and the solution was left to stir at room temperature for 2 hours

(Figure 6). The organic layer was extracted with sodium bicarbonate, washed with saturated NaCl solution, dried (Na2SO4) and concentrated to dryness. The residue was then saponified (combined with ~5mL 5M KOH and ethanol for 4 days) and subsequently concentrated to dryness (Figure 6). The residue was re-dissolved in water, ether and ethanol then concentrated to dryness. The aqueous layer was extracted with ether, then HCl was added dropwise over an ice bath until the pH lowered to ~3. The organic layer was subsequently extracted with ethyl acetate, the organic layer washed with saturated NaCl solution, dried (Na2SO4) and concentrated to dryness. The product was purified by silica gel column chromatography (50 cm x 3 cm) with a stepwise 26 gradient mobile phase of 10%, 20%, 40%, 80% ethyl acetate in hexanes and finally 20%

MeOH in ethyl acetate to successfully produce a small amount of the glucuronic acid conjugate which was confirmed by tandem mass spectrometry.

Figure 6: Synthesis of linalool glucuronide (4) from linalool (1).

An explanation of all approaches taken to synthesize linalool glucuronide can be found in

Appendix D.

27 3.5 Subjects

Nine female subjects were recruited for this study via an email sent to The Ohio

State University Department of Food Science and Technology listserv, and six of those subjects (ranging from 21 to 32 years of age, average BMI 22.05 ± 1.98) completed the whole study. Two subjects dropped out before any data were collected and one subjected completed the first visit however their data was not used in analyses. Each subject went through an informed consent process where the goals, procedures, risks and benefits of the study were discussed with them (Appendix A). Subjects had to meet the following inclusion criteria: age 18-70 years, female, BMI 18.5-30.0kg/m2, not lactating, pregnant or plan to be pregnant during study, no tobacco use (cigarettes and chewing tobacco), no metabolic disease, no malabsorption disorders, no history of cancer, esophageal or intestinal ulcers, no history of livery or kidney insufficiency or failure, no chemosensory deficits (no anosmics), no pulmonary problems (e.g. asthma), no cardiac problems, no use of psychoactive drugs, no fragrance allergies, no blood donation within the last 8 weeks, must weigh above 110 pounds and no antibiotic use in the last six months.

Subjects were compensated $10 after the information session, and $30 on day 3, day 10, and day 17 when they returned their 24-hour urine collection, totaling to $100 for completion of all aspects of this entire study. This study was approved by The Ohio State

University’s (OSU) Institutional Review Board (IRB protocol # 2016H0138).

3.6 Study Design

The study design was a three-way crossover wherein subjects were randomized to order, with three treatments: orthonasal linalool delivery, retronasal linalool delivery and 28 ingestion of an 8 ounce linalool beverage Figure 7. Each subject went through three total clinical visits (at Campbell Hall), each of which was 2.5 hours long and separated by one week from one another. Forty-eight hours prior to each visit, subjects were asked to avoid any products that would contain linalool, including: fragrant lotions, essential oils, and perfumes. They were additionally asked to fast 12 hours prior to each visit, however they were encouraged to stay hydrated. Upon arrival to Campbell Hall, a spot urine was collected, blood pressure and heart rate were recorded and subjects were provided a standardized breakfast of a banana and granola bar. A trained phlebotomist then inserted a flexible venous catheter and a baseline blood draw (10 mL, 0 min) was collected in a glass sodium heparin BD Vacutainer® Blood Collection Tube (Franklin Lakes, NJ). The catheter was then flushed with saline to prevent coagulation. Following this was the 10- minute treatment (either orthonasal inhalation, retronasal inhalation or ingestion of a beverage) then subsequent blood draws were taken at 15, 30, 45, 60, 90 and 120 minutes from the start of each treatment (time zero). Following each blood draw, subjects filled out a subjective stress assessment (Appendix B). Subjects were additionally asked to collect their urine for 24 hours following the start of each visit, in 3 different urine containers: 0-8 hours, 8-16 hours and 16-24 hours. Blood samples were immediately put on ice after each blood draw and subsequently centrifuged for 10 minutes at max speed

(Eppendorf™ 5810 Centrifuge; ThermoScientific; Waltham, MA). The plasma was then aliquoted and flash frozen in liquid nitrogen prior to storing at -80°C freezer. Plasma samples were extracted in two batches one day prior to HPLC-MS/MS analysis.

29 Recruit & screen healthy women (Consent Visit) Randomize to treatments

48-hr linalool avoidance n=6, all females

Linalool Orthonasal Retronasal beverage delivery delivery n=2 n=2 n=2 C R O S S O V E R Retronasal Linalool Orthonasal delivery beverage delivery n=2 n=2 n=2 C R O S S O V E R Orthonasal Retronasal Linalool delivery delivery beverage n=2 n=2 n=2

Figure 7: Study design for linalool clinical trial.

30 3.6.1 Aroma Apparatus

Breathing grade air was filtered, humidified then bubbled through a 12% linalool in miglyol solution at 6 liters per minute in both orthonasal and retronasal inhalation conditions (Appendix C). For orthonasal delivery, a glass nosepiece cupped the subject’s nose and they were instructed to breathe in through their nose and out through their mouth. For retronasal delivery, polyethylene tubing was connected to a glass manifold inserted into a silicone mouthpiece. Here, subjects were instructed to breathe in through their mouth and out through their nose. Each inhalation treatment was 10 minutes long.

3.6.2 Linalool Beverage

The linalool beverage was comprised of water (8 oz) and linalool (80 mg) so as to minimize confounding variables in the absorption/metabolism of linalool. The amount of linalool added to the beverage (80 mg) was based on the amount added to a dietary supplement, Silexan (Woelk & Schlafke, 2010). A large stock of the beverage was prepared for the entirety of the clinical trial to ensure consistency in the beverage delivered to each subject and was stored at refrigeration temperature (4°C). The stock was set out a half hour prior to each clinical day to ensure consistency in beverage temperature across subjects.

3.7 Sample preparation

3.7.1 Plasma extraction

Plasma was thawed on ice for one hour prior to extraction. Once thawed, 500μl plasma was combined with 1000μl acetonitrile and then vortexed for 10 seconds to 31 ensure complete mixing. Samples were then centrifuged at max speed (21,130 x g) for 5 minutes (Eppendorf™ 5424 Microcentrifuge; ThermoScientific; Waltham, MA) and the supernatant removed. Then the pellet was re-extracted with another 1000μl acetonitrile, vortexed (Vortex-Genie 2; Scientific Industries, Inc.; Bohemia, NY) and centrifuged for 5 minutes and the supernatant of both extractions were combined. Subsequently, the samples were dried in a speed vacuum (Savant™ SPD131DDA SpeedVac™

Concentrator; ThermoScientific; Waltham, MA) and placed in a -80°C freezer overnight.

The following day, the extracted samples were re-dissolved in 200μl MeOH and sonicated (Fisher Scientific FS30 Ultrasonic Cleaner; Waltham, MA) for 10 seconds.

They were then centrifuged at 21,130 x g for 2 min and the supernatant analyzed by

UHPLC-MS/MS.

3.7.2 Urine dilution

Urine was thawed for 15 minutes in warm water. Urine tubes were then inverted 5 times and 50 μL urine was combined with 450 μL water with 0.1% formic acid to make a

1/10th dilution. Samples were then centrifuged at max speed for 2 min and 200 μL of the supernatant was pipetted into an LC vial.

3.8 Ultra High Performance Liquid Chromatography- Tandem Mass Spectrometry

(UHPLC-MS/MS)

Linalool and metabolites were analyzed using an Agilent 6495 Triple Quadrupole

LC-MS/MS system (Santa Clara, CA). The analytes were separated on an Agilent

ZORBAX Eclipse Plus C18 column (2.1 x 50mm, 1.8μm; Santa Clara, CA) in a binary 32 mobile phase of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid

(B). Starting conditions were 10% B for 0.5 minutes, and a linear gradient was applied over 5 minutes at a flow rate of 0.600 mL/min to 100% B, then held at 100% B for 1 minute and finally returned back to starting conditions of 10% B for re-equilibration of the column. Eluent from the HPLC then entered the triple quadruple mass spectrometer where electrospray ionization (ESI) was applied in positive or negative mode, depending on the analyte (see Table 1 for details). MS/MS parameters included: source temperature,

250°C; gas flow, 16 l/min; nebulizer, 30 psi; sheath gas temp, 400°C; sheath gas flow, 11

L/min; capillary, 3500 V positive and negative; nozzle voltage, 1500 V positive and negative. Multiple reaction monitoring (MRM) transitions of each analyte can be found in Table 1. Samples were injected 16 μL per run and the injection needle was washed in between runs with acetonitrile with 0.1% formic acid.

Table 1: MS/MS parameters for linalool and metabolites.

Ionization Retention Dwell Analyte mode Time Mass Transitions (m/z) Collision Energies (eV) (min) Time (ms) linalool positive 2.9 137.1 > 95, 81, 67, 57 20, 20, 30, 20 298 linalool glucuronide negative 2.2 329.2 > 113, 89, 85, 75, 71, 59 30, 30, 30, 30, 25, 25 298 8-hydroxylinalool positive 2.7 153.1 > 135, 107, 95, 93, 81, 71, 69 20, 20, 20, 20, 20, 20, 20 298

34 Plasma and urine samples were first analyzed for linalool and metabolites described in Table 1, however, linalool glucuronide was the only metabolite that could be tracked thus a second method was used where only linalool glucuronide and transitions were monitored to optimize chromatogram peaks.

A quantification method was developed on Agilent MassHunter Quantification

Software (Santa Clara, CA) and peaks were integrated based on the glucuronic acid transition 329.2 > 85. Since the purity of linalool glucuronide could not be determined

(not enough product for NMR), a semi-quantitative approach was taken to analyze the relative peak areas of linalool glucuronide at each time point, thus providing a pharmacokinetic curve for each treatment.

3.9 Statistical Analysis

To compare the quantity of linalool glucuronide in blood and urine following orthonasal aroma delivery, retronasal aroma delivery and ingestion of a linalool beverage, the area-under-the-curve (AUC) for linalool glucuronide pharmacokinetic curve was calculated using trapezoidal approximation (Phillips & Taylor, 1996). The AUC data was then analyzed by analysis of variance (ANOVA) modeling the effects of treatment and subject.

Additional pharmacokinetic parameters were determined for each treatment in plasma: Cmax and Tmax. For Cmax, relative linalool glucuronide concentration was plotted over time, and the highest relative concentration was selected for each subject then averaged across subjects per treatment. For Tmax, the time in minutes at which linalool glucuronide concentration peaked was selected for each subject for each treatment then 35 averaged across subjects per treatment. Cmax and Tmax were compared using paired-test for orthonasal and retronasal treatments, since the same concentration of linalool was delivered in both inhalation conditions.

Furthermore, the effects of linalool delivery on the individual appearances of linalool metabolites in the plasma over time were compared within subjects by using repeated-measures ANOVA (Paetau, Chen, Goh, & White, 1997).

CHAPTER 4: Results

4.1 Plasma analysis

Plasma samples for the 6 subjects that completed all three clinical visits were first analyzed for linalool, 8-hydroxylinalool, dihydrolinalool, tetrahydrolinalool and linalool glucuronide. However, the only compound detected in plasma was linalool glucuronide

(Figure 8), therefore pharmacokinetic parameters were determined only for this metabolite. Linalool glucuronide was semi-quantitatively assessed in plasma using LC-

MS/MS methods described previously, thus we were able to track this metabolite based on its relative peak area over time (minutes).

4.1.1 Orthonasal inhalation pharmacokinetics

Following orthonasal inhalation, linalool glucuronide peaked on average at 15 minutes then plateaued (Figure 9). A two-way Analysis of Variance (ANOVA) test revealed significant differences due to subject (p<0.0001) and sampling time (p<0.0001).

Post hoc Dunnett t-test compared the average peak area at each time point against the average peak area at 0 minutes (0.0000 ± 0.0000), which served as the control. The

Dunnett t-test revealed significant differences for 15 minutes (0.0618 ± 0.1962, p<0.0001), 30 minutes (0.0642 ± 0.0361, p<0.0001), 45 minutes (0.0472 ± 0.0272, p=0.001), 60 minutes (0.0465 ± 0.0320, p=0.001) and 90 minutes (0.0275 ± 0.0190, p=0.042), however no significance was found for 120 minutes (0.012 ± 0.0084, p=0.415).

Figure 9: Relative concentration of linalool glucuronide in plasma following orthonasal inhalation. Peak concentration occurred at 15 minutes then plateaued and approached zero by 120 minutes. * timepoints are significantly different by Dunnett’s post-hoc test (P<0.05), where average peak area of linalool glucuronide was significantly greater than time zero at 15 minutes, 30 minutes, 45 minutes, 60 minutes and 90 minutes. Average peak area was plotted ± standard error of mean (SEM).

4.1.2 Retronasal inhalation pharmacokinetics

As seen in Figure 10, linalool glucuronide also peaked on average at 15 minutes following retronasal inhalation. A two-way ANOVA test revealed significant differences between sampling time (p<0.0001) and subject (p=0.001). Post hoc Dunnett t-test compared the average peak area at each time point against the average peak area at 0 minutes (0.0022 ± 0.0053), which served as the control. The Dunnett t-test revealed significant differences for 15 minutes (0.1903 ± 0.0531, p<0.0001), 30 minutes (0.1398 ±

0.0761, p<0.0001), 45 minutes (0.1005 ± 0.0512, p<0.0001) and 60 minutes (0.073 ±

40 0.0313, p=0.005), however no significance was found for 90 minutes (0.0447 ± 0.0156, p=0.106) or 120 minutes (0.033 ± 0.0062, p=0.259).

Figure 10: Relative concentration of linalool glucuronide in plasma following retronasal inhalation. Similar to orthonasal inhalation, linalool glucuronide peaked at 15 minutes and approached zero by 90 and 120 minutes. * timepoints are significantly different by Dunnett’s post-hoc test (P<0.05), where average peak area of linalool glucuronide was significantly greater than time zero at 15 minutes, 30 minutes, 45 minutes and 60 minutes points. Average peak area was plotted ± standard error of mean (SEM).

41 4.1.3 Beverage ingestion pharmacokinetics

Following ingestion of the beverage, linalool glucuronide on average peaked at 30 minutes (Figure 11). In comparison to both inhalation conditions, ingestion of the linalool beverage led to a 100-fold increase of linalool glucuronide in plasma. However, different concentrations of linalool were administered in the inhalation and beverage conditions (12% linalool in miglyol solution bubbled at 6 liters per minute for inhalation conditions and 80 mg linalool in beverage) therefore this was expected.

A two-way ANOVA again demonstrated a significance difference between subject (p<0.0001) and sampling time (p<0.0001). Further Dunnett Post hoc t-test compared the average peak area at each time point against the average peak area at 0 minutes (0.0040 ± 0.0063), which served as the control. The Dunnett t-test revealed significant differences for all time points: 15 minutes (9.6722 ± 4.7280, p<0.0001), 30 minutes (14.8897 ± 5.7190, p<0.0001), 45 minutes (13.4646 ± 4.2442, p<0.0001), 60 minutes (11.0368 ± 3.8226, p<0.0001), 90 minutes (7.1335 ± 2.0652, p<0.0001) and 120 minutes (4.4756 ± 1.5125, p=0.016).

4.1.4 Comparing Tmax between all three treatments

Time to maximum peak intensity (Tmax) was determined for each subject, then averaged across subjects and subsequently analyzed by a three-way ANOVA for all three treatments. The results of the ANOVA demonstrated significance in Tmax with treatment

(p=0.007), and post-hoc Tukey’s HSD test showed significant differences in Tmax between the beverage (37.50 ± 8.216 minutes) and both orthonasal inhalation treatment

(20.00 ± 7.746 minutes; p=0.013) and retronasal inhalation treatment (19.29 ± 7.319

43 minutes; p=0.008). Figure 12 depicts the significant difference in treatments, which supports my hypothesis that the beverage would lead to later absorption of linalool.

44 4.1.5 Comparing orthonasal and retronasal parameters (AUC, Cmax)

Orthonasal and retronasal treatments were compared together since subjects were given the same concentration of linalool in both treatments. Unexpectedly, when directly compared, the average peak area of linalool glucuronide following retronasal inhalation trended above that of the average peak area found after orthonasal inhalation (Figure 13).

A 3-way ANOVA demonstrated significance for time*treatment (p<0.0001), therefore further post-hoc paired t-tests were conducted. The paired t-test demonstrated that the average peak area of linalool glucuronide in plasma was significantly greater following retronasal inhalation than orthonasal inhalation for 15 minutes (p<0.0001), 45 minutes

(p=0.048) and 120 minutes (p=0.001), with marginal significance found for 30 minutes

(p=0.052). Figure 13 below displays the average relative absorption of linalool glucuronide in plasma following both orthonasal and retronasal treatments.

To further investigate differences in orthonasal and retronasal treatments, the area under the curve (AUC) was calculated for the orthonasal pharmacokinetic curve (Figure

9) and retronasal pharmacokinetic curve (Figure 10). A paired t-test was conducted to assess differences in AUC depicted in Figure 14 between the two treatments.

Figure 14: Calculated Area Under the Curve (AUC) of linalool glucuronide pharmacokinetic curve for orthonasal and retronasal inhalation. Error bars are SEM. Different letters denote significant differences using a paired t-test (P<0.05). Retronasal AUC was found to be significantly greater than orthonasal AUC (p=0.003).

The paired t-test demonstrated significantly higher AUC in retronasal treatment

(10.549 ± 2.468; p=0.003) compared to orthonasal treatment (4.856 ± 2.408).

Furthermore, Cmax was also significantly higher following retronasal treatment (0.1935 ±

0.0524; p=0.001) compared to orthonasal treatment (0.0742 ± 0.0281) (Figure 15). Both of these findings were unexpected given that the same concentration of linalool (12% linalool in miglyol, bubbled at 6 liters per minute) was administered in both inhalation treatments.

4.2 Urine analysis

Urine samples for the 6 subjects that completed all three clinical visits were also first analyzed for linalool, 8-hydroxylinalool, dihydrolinalool, tetrahydrolinalool and linalool glucuronide. However, the only compound that appeared in urine was linalool glucuronide (same result as plasma), therefore the pharmacokinetic parameters were determined only for this metabolite (example of chromatogram in Figure 16). Linalool glucuronide was semi-quantitatively assessed in urine using LC-MS/MS methods described previously, thus I was able to track excretion of this metabolite based on its relative peak area over a 24-hour period. 48

Ion Abundance

0 1 2 2.2 3 4 Retention time (minutes)

4.2.1 Orthonasal and retronasal linalool glucuronide excretion

There were no significant differences in time to maximum peak intensity (Tmax)

between orthonasal treatment (8.00 ± 0) and retronasal treatments (9.60 ± 3.57, p=0.374).

In both treatments (with the exception of subject 3 retronasal excretion) total arbitrary

units of linalool glucuronide were excreted during the first 8-hour collection period

(Table 2). Additionally, total arbitrary units excreted following retronasal treatment were

significantly higher (1.44 x 105 ± 7.60 x 104) than orthonasal treatment (3.55 x 104 ± 2.58 x 49 104; p=0.029)(Figure 17), a trend that remained consistent with what was observed with linalool glucuronide absorption in plasma. There was a similar trend for Cmax as well, which approached marginal significance between retronasal inhalation treatment (1.27 x

105 ± 6.0 x 104) and orthonasal inhalation treatment (4.27 x 104 ± 2.12 x 104, p=0.053).

Figure 17: Arbitrary units of linalool glucuronide excreted in urine following orthonasal inhalation and retronasal inhalation. Total units excreted were significantly greater (p=0.029, significance level α<0.05) following orthonasal inhalation compared to retronasal inhalation, which provides further evidence for this trend that was previously noted in plasma (Figure 14 & Figure 15).

50 Table 2 below depicts total arbitrary units excreted, Tmax and Cmax parameters for both orthonasal and retronasal inhalation. Note that subject 10 had no observed linalool glucuronide in urine, despite demonstrating metabolite absorption in plasma during this same treatment. Therefore, I believe she did not comply with urine collection for the complete 24 hours and as a result her data for orthonasal inhalation was not included for statistical analyses.

Table 2: Analysis of linalool glucuronide in urine following orthonasal and retronasal inhalation treatments.

Orthonasal Treatment Retronasal Treatment Subject Total arbitrary Tmax Total arbitrary Tmax Cmax Cmax units excreted (hours) units excreted (hours)

4 5 5

52 2 3.47 x 10 8 34662.16 2.23 x 10 8 2.23 x 10 4 4 5 5 3 1.64 x 10 8 1.64 x 10 2.54 x 10 16 1.17 x 10

4 7.46 x 104 8 7.46 x 104 1.12 x 105 8 1.12 x 105

7 3.96 x 104 8 3.96 x 104 6.25 x 104 8 6.25 x 104

9 4.79 x 104 8 4.79 x 104 9.91 x 104 8 9.91 x 104

10 N/A N/A N/A 1.14 x 105 8 1.14 x 105

4.2.2 Beverage linalool glucuronide excretion

Following ingestion of the linalool beverage, linalool glucuronide peaked at 8 hours, which is consistent with the Tmax of orthonasal and retronasal treatments. Similar to both inhalation treatments, linalool glucuronide was observed only after 8-hours of urine collection, while not excreting significant amounts at 16 and 24 hours (Table 3).

Post-hoc Dunnett t-tests compared each time point against the average spot urine

(0) and demonstrated significance for 8 hours (1.05x107 ± 4.5x106, p<0.0001) but not for

16 hours (1.75x105 ± 3.75x105; p=0.700) or 24 hours (2.04x105 ± 2.20x105; p=0.691).

Based on the raw data, there were two fast metabolizers who excreted all of linalool glucuronide in the first 8 hours, while the rest of the group had varying degrees of metabolizing speed, where linalool glucuronide was still detected at 24 hours. This wide range of metabolism amongst the subjects could be a contributing factor to the high standard deviation reported previously and subsequent lack of significance at time 16 and

24 hours.

7 It is important to note that the average Cmax for the beverage treatment (1.05x10 ) is three and two orders of magnitude higher than orthonasal and retronasal average Cmax, respectively. This again further supports my hypothesis that a greater concentration of linalool was administered during the beverage treatment compared to the inhalation treatments. Table 3 below depicts total arbitrary units of linalool glucuronide excreted following the beverage treatment, as well as Tmax (hours), Cmax and AUC.

Table 3: Analysis of linalool glucuronide in urine following beverage treatment.

Beverage Treatment Subject Total arbitrary T (hours) C AUC units excreted max max 2 2.01E+07 8 1.87E+07 8.04E+07 3 1.13E+07 8 8.22E+06 8.84E+07 4 9.91E+06 8 9.91E+06 7.93E+07 7 4.90E+06 8 4.90E+06 3.92E+07 9 1.12E+07 8 1.08E+07 8.88E+07 10 1.05E+07 8 1.03E+07 8.38E+07

CHAPTER 5: Discussion

5.1 Method Development

The initial method developed for linalool was based upon Xia et al. 2010, thus isocratic conditions were used with a binary mobile phase of 55:45 acetonitrile: water, both with 0.1% formic acid added (Xia et al., 2010b). Linalool eluted at 8 minutes and

MRM transitions were determined and finalized to the four transitions listed in Table 1.

Initially, the column used was a SunFire C18 (4.6 x 150 mm column, 5 um particle size, no split) in electron spray ionization (ESI) mode. However, in order to decrease run time, a smaller column (Agilent ZORBAX Eclipse Plus C18 column (2.1 x 50mm, 1.8μm;

Santa Clara, CA)) was used for the remaining method development and subsequent sample runs. This shortened the runs to 7 minutes, with linalool eluting at 2.9 minutes. A variety of mobile phases, temperature and probe types were used to determine the parameters that yielded the highest peak intensity (Table 1).

5.1.1 Confirming synthesized linalool metabolite standards with UHPLC-QTOF-MS

Linalool glucuronide and 8-hydroxylinalool were both synthesized for qualitative identification in plasma and urine, and a variety of steps were taken to confirm that the correct metabolite was synthesized, mainly by UHPLC-QTOF-MS. This instrument is advantageous to use for confirmation of a compound based on its higher mass accuracy compared to a triple quadruple mass spectrometer. This provides more confidence than a 55 regular mass spectrometer, which only goes out to one decimal point. Linalool glucuronide and 8-hydroxylinalool were confirmed by accurate mass with an Agilent

6550 QTOF mass spectrometer, and chromatograms of both metabolites in addition to linalool can be found in Figure 18.

56 57

57 Linalool glucuronide eluted at 2.2 minutes, which was earlier than linalool (eluted at 2.9 minutes). This was expected given that linalool glucuronide has the added sugar acid, rendering the compound more polar than linalool itself. Moreover, insource fragmentation of linalool glucuronide was noted with the appearance of various linalool peaks (mass 137 amu) surrounding the linalool glucuronide peak. Additionally, one of the in-source linalool fragments also eluted at the same time as linalool glucuronide, which provides further evidence for accurate synthesis of linalool glucuronide.

Interestingly, in both the synthesized linalool glucuronide standard and linalool glucuronide identified in plasma and urine, two peaks consistently appeared together. The second peak was larger and not present in the baseline biological samples, therefore this was used for semi-quantitation of the glucuronide. However, it was interesting to note that the first peak varied in levels between subjects. I hypothesize this could be a potential isomer of the glucuronide, however future studies could look into this further.

5.2 Linalool glucuronide metabolism in plasma

In the present study, I assessed a novel way of delivering linalool through a beverage, in addition to the more traditional route used in aromatherapy through the inhalation pathways (orthonasal and retronasal routes). Furthermore, I looked into linalool metabolism through analysis of plasma and urine following each of the treatments. I hypothesized that linalool would be absorbed and metabolized more quickly in both inhalation routes compared to ingestion of the linalool beverage. I also hypothesized that different metabolites would appear following the ingestion condition compared to the inhalation delivery mode. 58 Consistent with my first hypothesis, linalool was absorbed and metabolized to linalool glucuronide more quickly following both orthonasal inhalation and retronasal inhalation, with linalool glucuronide appearing at 15 minutes in plasma following inhalation, compared to 30 minutes following ingestion of the beverage. However, in contrast to my second hypothesis, the same metabolite, linalool glucuronide, appeared in both inhalation and beverage treatments. I anticipated the oral microbiome and enzymes located in the saliva of the oral cavity and gastrointestinal microbiome would have an impact on such metabolism and lead to different metabolites following ingestion of the linalool beverage. However, it is possible that all of the metabolites of linalool were not detected with this targeted LC-MS/MS approach, therefore this matter would need to be further investigated with different instrumentation such as GC-MS to reach a valid conclusion.

5.2.1 Linalool metabolism- primary metabolite

Linalool glucuronide was the only metabolite detected in plasma and urine following inhalation of linalool and ingestion of a linalool beverage. This is consistent with a previous study that stated linalool glucuronide as a primary metabolite, however, the other three metabolites (dihydrolinalool, tetrahydrolinalool and 8-hydroxylinalool) that were previously documented in rat urine following oral administration of linalool were not detected (Chadha & Madyastha, 2009; Parke, Rahman, et al., 1974; Rahman,

1974). Given that dihydrolinalool, tetrahydrolinalool and 8-hydroxylinalool were previously detected in a rodent model, it is possible those metabolites are not as prominent in human metabolism of linalool. To my knowledge, this is the first human 59 clinical study conducted that investigated linalool metabolism, therefore future studies could further investigate whether these metabolites are detectable. Moreover, it is possible my liquid chromatography-mass spectrometry methods were not able to detect the metabolites; perhaps gas chromatography techniques could be used in future studies to further confirm my findings.

5.2.2 Comparison of inhalation treatments to beverage treatment

As mentioned previously, Tmax of linalool glucuronide was significantly earlier

(20 minutes) in both inhalation treatments compared to the beverage treatment (37 minutes). I suspect the different routes affected the time course. With both inhalation routes, there is systemic delivery of volatiles into circulation within seconds or minutes due to high vascularization and permeability of the nasal mucosa and lung mucosa

(Patton & Byron, 2007; Ugwoke, Verbeke, & Kinget, 2001). In addition, with the inhalation route, first-pass metabolism is bypassed due to the low concentration of drug- metabolizing enzymes, which also accelerates systemic circulation and resulting high bioavailability (Patton & Byron, 2007). On the contrary, with oral delivery route, foreign molecules go through first pass metabolism following absorption into capillary beds of small and large intestine. Therefore, the molecule will not be systemically available until this pass through the liver, which could have contributed to the delayed appearance of linalool glucuronide in blood (Thummel, 1997).

Despite linalool metabolism following beverage ingestion occurring later at 30 minutes, I was still surprised to see how quickly linalool metabolized in all three treatments. It would be interesting to collect plasma at earlier time points to see when 60 initial metabolism occurred, since I only captured the peak of linalool metabolism following inhalation at the first blood collection time point at 15 minutes.

In comparison to both inhalation treatments, the beverage treatment yielded significant differences across all time points. The lack of significance for the 120 minutes in orthonasal treatment and 90 and 120-minute time points in retronasal inhalation treatment could be attributed to the small n of 6 subjects. Both conditions trended towards significance therefore it is possible that if this clinical trial were conducted on a larger scale, significance across all time points would be observed. Furthermore, in the beverage treatment, I hypothesize subjects received a significantly higher concentration of linalool (80 mg), which can be seen in the 100-fold increase in average peak area of linalool glucuronide in plasma following the beverage treatment (Figure 11) compared to both inhalation treatments (Figure 13).

5.2.3 Orthonasal and retronasal inhalation differences

Perhaps one of the most surprising findings from the present study was that linalool glucuronide absorption in plasma was greater following retronasal inhalation compared to orthonasal inhalation. This is evidenced by a significantly greater area under the curve (AUC) and Cmax of linalool glucuronide following retronasal inhalation compared to orthonasal inhalation. This trend was especially surprising since subjects were administered the same concentration of linalool (12% solution of linalool in miglyol).

It is possible that the nature of the mouthpiece and nosepiece (depicted in

Appendix C) had an impact on the difference in linalool glucuronide absorption in 61 plasma following orthonasal and retronasal inhalation. With retronasal inhalation, delivery through the mouthpiece is actively pushing air into the subjects’ mouth whether they are actually inhaling or in-between breaths, prior to inhalation. With orthonasal delivery however, subjects must actively inhale through the nosepiece and, consequently, air is not directly pushed up the nostrils when the subject is not inhaling. Therefore, it is possible linalool volatile compounds are partitioning in the saliva during these stagnant periods found in retronasal inhalation. More specifically, two proteins predominate in saliva, α-amylase and mucins, have both been found to affect the concentration of volatiles in the headspace through hydrophobic bonding (Friel & Taylor, 2001; Pagès-

Hélary et al., 2014). These proteins and enzymes could consequently have an effect on the volatiles absorbed in the oral mucosa, and resulting concentration found in plasma.

Furthermore, retronasal inhalation is not a regular way of breathing, and subjects tended to take greater breaths during this inhalation compared to orthonasal inhalation. As a consequence of both noted differences, it is possible that in retronasal inhalation the subjects are actually absorbing more linalool volatiles in the oral cavity, which could contribute to the larger levels of linalool glucuronide noted in plasma following this delivery mode.

5.3 Excretion of linalool glucuronide in urine

Linalool glucuronide was previously found as the primary linalool metabolite excreted in rat urine (Parke, Rahman, et al., 1974). This was the same case for the present study, where in all 3 treatments linalool glucuronide was excreted within the first 8 hours following initial treatment administration at the clinical visit. In the inhalation treatments, 62 there was no linalool glucuronide detected at 16 and 24 hours. However, interestingly in the beverage treatment there were small levels of the metabolite detected at 16 and 24 hours for four of the subjects. Moreover, there were varying levels of linalool metabolizing efficiency among the subjects, where two of the subjects excreted all of linalool glucuronide in the first 8 hours, while the remaining subjects still excreted the majority of the metabolite at 8 hours, but still had varying levels of linalool glucuronide at 16 and 24 hours. Despite the detectable levels, these values were not found to be significantly different from zero, most likely due to the high variability found within subjects. Furthermore, the small sample size also contributed to the lack of significance at those two time points.

CHAPTER 6: Conclusion

In the present study, LC-MS/MS methods were utilized as a novel way of detecting linalool and metabolites in plasma and urine following different routes of administration: inhalation (orthonasal and retronasal delivery) and ingestion of a linalool beverage. This was the first published human study to screen for linalool metabolites, as all previous studies in the literature detected linalool metabolites in rats.

Analysis of plasma revealed linalool glucuronide as the predominate metabolite for all three treatments. Absorption and metabolism of linalool occurred quickly, with linalool glucuronide appearing in plasma at the earliest blood collection time point (15 minutes) for all three treatments. Moreover, linalool metabolized to linalool glucuronide faster in orthonasal and retronasal inhalation treatments, which is evidenced by an earlier

Tmax at 20 minutes, compared to ingestion Tmax at 37 minutes. Linalool glucuronide was also the predominant metabolite excreted in urine, and was excreted on average within the first 8 hours following each treatment.

I assessed a novel delivery route for linalool, through ingestion of a linalool beverage and demonstrated potential to deliver a larger concentration of linalool, which could potentially be applied as a functional beverage for stress reduction. Future work could assess whether linalool or linalool glucuronide is responsible for biological effects.

Furthermore, a larger clinical study could be conducted and GC-MS/MS methods could be used to track linalool over time. 64

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Appendix A: Consent Form

The Ohio State University Consent to Participate in Research

Study Title: Quantification of Linalool in Biological Fluids Christopher T. Simons, Ph.D. Principal Investigator: None Sponsor:  This is a consent form for research participation. It contains important information about this study and what to expect if you decide to participate. Please consider the information carefully. Feel free to discuss the study with your friends and family and to ask questions before making your decision whether or not to participate.  Your participation is voluntary. You may refuse to participate in this study. If you decide to take part in the study, you may leave the study at any time. No matter what decision you make, there will be no penalty to you and you will not lose any of your usual benefits. Your decision will not affect your future relationship with The Ohio State University. If you are a student or employee at Ohio State, your decision will not affect your grades or employment status.  You may or may not benefit as a result of participating in this study. Also, as explained below, your participation may result in unintended or harmful effects for you that may be minor or may be serious depending on the nature of the research.  You will be provided with any new information that develops during the study that may affect your decision whether or not to continue to participate. If you decide to participate, you will be asked to sign this form and will receive a copy of the form. You are being asked to consider participating in this study for the reasons explained below.

1. Why is this study being done?

Essential oils have been used in aromatherapy for centuries as an alternative medicinal approach to relieve pain, reduce anxiety, enhance energy and improve mood. Lavender oil in particular, is known for reducing stress and anxiety and improving

71 sleep. The active compound in lavender, linalool, provides its floral scent and has shown in a variety of animal and human studies to play a role in stress and anxiety reduction. Linalool has been documented in blood and urine of animals and humans, however the majority of these studies assess when linalool appears in blood and/or urine following putting it on the skin or smelling it through the nose.

We aim to assess a different way of delivering linalool (through a linalool beverage) and also through two modes of smelling the linalool aroma (through your nose and through your mouth). We will determine at what time point linalool appears in blood and also whether linalool or metabolites (result of linalool metabolizing, or breaking down in the body) appear in urine and blood as well.

2. How many people will take part in this study? Up to 20 subjects will be screened for the goal of obtaining 9 subjects to complete this study.

3. What will happen if I take part in this study?

To assist you in understanding the procedures described below, please refer to Table 1 and Table 2.

If you decide to take part in this study, we will schedule three morning visits for you at Campbell Hall. Each visit will be one week apart and will last around 2 hours. Before each visit, you will be asked to fast overnight (abstain from eating) for 12 hours prior to your appointment. During your fast you are encouraged to drink water (unflavored) and take any scheduled medications, but no food or other drinks. You will additionally be asked to avoid using fragrant lotions, essential oils or perfumes for 48 hours prior to each visit. During your first visit, you will also fill out a health questionnaire and your weight and height will be measured to calculate your BMI. If your BMI is normal (18.5-30 kg/m2) you will be eligible to participate in this study. Additionally, at each visit we will measure your blood pressure and heart rate. However, if you have certain health conditions that have potential to interfere with the outcomes of the study, you will no longer be eligible to participate in the study.

Upon arrival to Campbell Hall for each visit (3 total visits), we will provide you with a standardized breakfast and will collect a urine sample. You will then be hooked up to instrumentation that will record heart rate, blood pressure and respiration rate throughout each entire visit. A trained phlebotomist will then insert a venous catheter (IV) and take the first blood sample at 0 minutes. The IV is a thin, flexible tube that is inserted into a vein to allow blood to be taken periodically throughout the visit without multiple needle pricks. Each blood sample taken will be approximately 2 teaspoons (10 mL). Following this initial blood draw, will be 10 minutes of linalool delivery (3 different delivery methods). With two of the delivery methods, an aroma delivery device (olfactometer) containing humidified and purified, breathing quality air will be used to

72 deliver flavors to your nose or mouth. The linalool aroma will be delivered directly to your nose by a tube, or delivered into your mouth through tubing inserted into a sterile mouthpiece, which we will ask you to wear. You will be asked to inhale (through your nose or mouth) and exhale (through your nose) normally. The other delivery method will consist of drinking a linalool beverage over a 10 minute long period. More blood draws will be taken at 15, 30, 45, 60, 90 and 120 minutes, with a total of 7 blood draws taken over the course of 2 hours.

During the remaining time following the linalool delivery (1 hour and 50 minutes), we will ask you to answer some questions about how you feel towards the aroma or flavor, including how intense or how pleasant it is.

You will begin collecting your urine in a container after your first urine sample (at time 0 minutes) and continue for 24 hours. This means you will need to finish the urine collection at home and return it to Campbell Hall or Parker Food Science & Technology Building the next day. We ask you to not use fragrant lotions, essential oils and perfumes until after you return the urine container the following day. The overall study schedule is detailed in Table 1 and the blood drawing schematic is outlined in Table 2. Table 1. Study Schedule Study Activity Notes Day Number Initial You learn about details of the study  Learn about Screening and are enrolled and consented. inclusion/exclusion criteria and This can take place either at which products that contain Campbell Hall or at Parker Food high amounts of linalool that Science and Technology Building. will need to be avoided during the study  Informed consent Day 0-1 Avoid using products that contain  Products to avoid: essential linalool. oils, fragrant lotions & perfumes Day 2 First long visit at Campbell Hall. You  Water consumption during (1st visit) should fast overnight prior to fast/during blood draws is arriving at Campbell Hall. encouraged.  You will be at the clinic for ~2 hours, have periodic blood draws and begin collecting your urine after your breakfast. Urine collection will continue for 24 hours from this point. Day 3 Continue to avoid using products  Products to avoid: essential that contain linalool & return 24- oils, fragrant lotions & hour urine collection to Campbell perfumes Hall or Parker Food Science & Technology Building.

73 Day 7-8 Avoid using products that contain  Products to avoid: essential linalool. oils, fragrant lotions & perfumes Day 9 Second long visit at Campbell Hall.  Water consumption during (2nd visit) You should fast overnight prior to fast/during blood draws is arriving at Campbell Hall. encouraged.  You will be at the clinic for ~2 hours, have periodic blood draws and begin collecting your urine after your breakfast. Urine collection will continue for 24 hours from this point. Day 10 Continue to avoid using products  Products to avoid: essential that contain linalool & return 24- oils, fragrant lotions & hour urine collection to Campbell perfumes Hall or Parker Food Science & Technology Building. Day 14- Avoid using products that contain  Products to avoid: essential 15 linalool. oils, fragrant lotions & perfumes

Day 16 Third long visit at Campbell Hall.  Water consumption during (3rd visit) You should fast overnight prior to fast/during blood draws is arriving at Campbell Hall. encouraged.  You will be at the clinic for ~2 hours, have periodic blood draws and begin collecting your urine after your breakfast. Urine collection will continue for 24 hours from this point. Day 17 Continue to avoid using products  Products to avoid: essential that contain linalool & return 24- oils, fragrant lotions & hour urine collection to Campbell perfumes Hall or Parker Food Science & Technology Building.

Table 2. Blood sampling schematic. Study day Quantity of blood taken Purpose Visit 1 (day 2) t = 0 minutes, 10 mL (~2 tsp) Linalool and metabolites t = 15 minutes, 10 mL (~2 tsp) t = 30 minutes, 10 mL (~2 tsp) t = 45 minutes, 10 mL (~2 tsp) t = 60 minutes, 10 mL (~2 tsp) t = 90 minutes, 10 mL (~2 tsp) t = 120 minutes, 10 mL (~2 tsp) Total blood taken = 70 mL, or ~0.30 cups Visit 2 (day 9) t = 0 minutes, 10 mL (~2 tsp) Linalool and metabolites t = 15 minutes, 10 mL (~2 tsp) t = 30 minutes, 10 mL (~2 tsp)

74 t = 45 minutes, 10 mL (~2 tsp) t = 60 minutes, 10 mL (~2 tsp) t = 90 minutes, 10 mL (~2 tsp) t = 120 minutes, 10 mL (~2 tsp) Total blood taken = 70 mL, or ~0.30 cups Visit 3 (day 16) t = 0 minutes, 10 mL (~2 tsp) Linalool and metabolites t = 15 minutes, 10 mL (~2 tsp) t = 30 minutes, 10 mL (~2 tsp) t = 45 minutes, 10 mL (~2 tsp) t = 60 minutes, 10 mL (~2 tsp) t = 90 minutes, 10 mL (~2 tsp) t = 120 minutes, 10 mL (~2 tsp) Total blood taken = 70 mL, or ~0.30 cups Total blood taken 210 mL, or ~0.90 cups (over a minimum of 28 days)

4. How long will I be in the study?

Recruitment and complete description of the study will take place at a screening visit, and will take approx. 30 minutes. Review of the study, enrollment and obtaining informed consent will also take place in person, and will take approx. 10 minutes. The visits at Campbell Hall on day 2, day 9 and day 16 will each take approximately 2 hours. Subjects will drop off 24 hour urine collection on day 3, day 10 and day 17 which will take approximately 10 minutes. For each subject, the total time commitment will be approximately 6.5 hours over at least 17 days.

5. Can I stop being in the study?

You may leave the study at any time. If you decide to stop participating in the study, there will be no penalty to you, and you will not lose any benefits to which you are otherwise entitled. Your decision will not affect your future relationship with The Ohio State University.

6. What risks, side effects or discomforts can I expect from being in the study?

There is minor risk associated with inserting and keeping a plastic, bendable catheter (tube for taking blood) in a patients arm for 2 hours. These will be minimized by having catheters monitored by a trained phlebotomist according to their standards and protocols. Risks of blood draws from a vein include bruising and infection, and more rarely phlebitis (swelling of a vein from a blood clot). A patient may feel lightheaded or may faint after the blood draws, and the catheter used to collect blood during the 2-

75 hour blood draws on day 2, day 9 and day 16 may be uncomfortable. However these are not severe discomforts and there are no long-term consequences. If subjects complain of severe discomfort, the catheter will be removed and the blood draws will stop.

There is also minor risk associated with delivering the aroma through the nose. Even though we humidify the air, it is possible that the nose gets dry. Additionally, with aroma delivery through mouth, subjects could potentially experience discomfort with the mouthpiece. However, we have minimized the aroma delivery to 10 minutes and if the subjects feel any discomfort they are free to take out the nose tubes or mouthpiece and breathe regular air for as long as needed.

7. What benefits can I expect from being in the study?

The most significant benefit from this study is for society. The results of this study will be important in understanding how the body breaks down linalool and how it differs with different delivery routes (drinking a beverage versus aroma delivery through the nose and mouth). Also, linalool has shown to be an anxiety reducing compound in a variety of human and animal studies therefore a stress or anxiety reducing beverage could be made in the future.

8. What other choices do I have if I do not take part in the study?

You may choose not to participate without penalty or loss of benefits to which you are otherwise entitled.

9. Will my study-related information be kept confidential?

Efforts will be made to keep your study-related information confidential. However, there may be circumstances where this information must be released. For example, personal information regarding your participation in this study may be disclosed if required by state law.

Also, your records may be reviewed by the following groups (as applicable to the research):  Office for Human Research Protections or other federal, state, or international regulatory agencies;  U.S. Food and Drug Administration;  The Ohio State University Institutional Review Board or Office of Responsible Research Practices;  The sponsor, if any, or agency supporting the study  Your insurance company (if charges are billed to insurance).

If this study is related to your medical care, your study-related information may be placed in your permanent hospital, clinic, or physician’s office records. Authorized Ohio State University staff not involved in the study may be aware that you are participating in a research study and have access to your information.

10. What are the costs of taking part in this study?

There are no financial costs to you for taking part in this study. The test meals will be provided to you by the research team. The cost of parking, breakfast and lunch during the day-long visits and the blood draws will be covered by the study.

11. Will I be paid for taking part in this study?

If you decide to participate in this study, we will give you parking passes for each study- related visit to the campus. You will be paid $10 for this consent visit, $30 for each visit at Campbell Hall (3 total), with a total of $100 for completion of the entire study. If you choose not to complete the second or third visit, you will still be paid for each completed visit. By law, payments to subjects are considered taxable income.

12. What happens if I am injured because I took part in this study?

If you suffer an injury from participating in this study, you should notify the researcher or study doctor immediately, who will determine if you should obtain medical treatment at The Ohio State University Medical Center.

The cost for this treatment will be billed to you or your medical or hospital insurance. The Ohio State University has no funds set aside for the payment of health care expenses for this study.

13. What are my rights if I take part in this study?

If you choose to participate in the study, you may discontinue participation at any time without penalty or loss of benefits. By signing this form, you do not give up any personal legal rights you may have as a participant in this study.

You will be provided with any new information that develops during the course of the research that may affect your decision whether or not to continue participation in the study.

You may refuse to participate in this study without penalty or loss of benefits to which you are otherwise entitled.

An Institutional Review Board responsible for human subjects research at The Ohio State University reviewed this research project and found it to be acceptable, according to applicable state and federal regulations and University policies designed to protect the rights and welfare of participants in research.

14. Who can answer my questions about the study?

For questions, concerns, or complaints about the study you may contact the study coordinator, Angie Kerns at 614-292-2798 or the Principal Investigator, Christopher Simons, Ph.D. at 614-688-1489 or [email protected].

For questions about your rights as a participant in this study or to discuss other study- related concerns or complaints with someone who is not part of the research team, you may contact Ms. Sandra Meadows in the Office of Responsible Research Practices at 1- 800-678-6251.

If you are injured as a result of participating in this study or for questions about a study- related injury, you may contact the study coordinator, Angie Kerns at 614-292-2798 or the Principal Investigator, Christopher Simons, Ph.D. at 614-688-1489 or [email protected].

Signing the consent form

I have read (or someone has read to me) this form and I am aware that I am being asked to participate in a research study. I have had the opportunity to ask questions and have had them answered to my satisfaction. I voluntarily agree to participate in this study.

I am not giving up any legal rights by signing this form. I will be given a copy of this form.

Printed name of subject Signature of subject

AM/PM

Date and time

Printed name of person authorized to consent for Signature of person authorized to consent for subject subject (when applicable) (when applicable)

AM/PM

Relationship to the subject Date and time

Investigator/Research Staff

I have explained the research to the participant or his/her representative before requesting the signature(s) above. There are no blanks in this document. A copy of this form has been given to the participant or his/her representative.

Printed name of person obtaining consent Signature of person obtaining consent

AM/PM Date and time

Witness(es) - May be left blank if not required by the IRB

Printed name of witness Signature of witness

AM/PM Date and time

Printed name of witness Signature of witness

AM/PM Date and time

Appendix B: Subjective Stress Assessment

The following subjective stress assessment was used at each clinical visit. Subjects were asked to indicate their level of stress (with an X) at each time point after the blood draw was complete.

Please indicate your level of stress (with an X) at the following time points.

Time: 0 minutes (baseline)

0 1 2 3 4 5 6 7 8 9 10 2.8

Time: 15 minutes

0 1 2 3 4 5 6 7 8 9 10 3

Time: 30 minutes

0 1 2 3 4 5 6 7 8 9 10 3.2

Time: 45 minutes 0 1 2 3 4 5 6 7 8 9 10 3.4

Time: 60 minutes

0 1 2 3 4 5 6 7 8 9 10 3.6

Time: 90 minutes

0 1 2 3 4 5 6 7 8 9 10 3.8

Time: 120 minutes

0 1 2 3 4 5 6 7 8 9 10 4

In addition to the categorical stress assessment, subjects were asked to answer the following questions after their last clinical visit:

1) What do you typically do to relax?

2) Do you typically use products that have lavender scents in your home?

3) Do you find that aromas affect the way you feel?

Results

The following figures depict the results from the subjective questionnaire portion for orthonasal & retronasal inhalation (Figure 19) and ingestion of beverage (Figure 20).

Figure 19: Subjective stress assessment on a 1-10 category scale where both orthonasal and retronasal treatments trend downward with the exception of 120 minutes for orthonasal treatment. Post-hoc Dunnett’s t-test compared each rating to the initial rating at time 0 min and determined whether they were significantly lower than this initial rating. For orthonasal inhalation, the ratings at 15 minutes (p=0.02), 30 minutes (p=0.009), 45 minutes (p=0.009), 60 minutes (p=0.009) and 90 minutes (p=0.02) were all significantly lower than initial rating (2.5 ± 2.26), however no significance was found for time 120 minutes (p=0.166). For retronasal inhalation, post-hoc Dunnett’s t-test demonstrated significance for each time point compared to the initial rating of 3.33 ± 1.03 (15 minutes- p=0.002; 30 minutes- p=0.008; 45 minutes, 60 minutes, 90 minutes & 120 minutes- p<0.0001).

Figure 20: Subjective stress ratings over time during beverage treatment. Note the gradual decline compared to both inhalation conditions in Figure 19 where there was an initial steep decline in subjective stress ratings. Post hoc Dunnett’s t-test demonstrated significance from initial rating of 2.67 ± 1.21 for 45 minutes (p=0.004), 60, 90 & 120 minutes (p<0.0001). Interestingly, the stress ratings did not become significantly lower from the initial rating until 45 minutes and onward, which was the first blood collection time point after Tmax (30 minutes) in plasma for linalool glucuronide following the beverage treatment.

A stress questionnaire was also given to subjects on their last visits and the results for each subject can be found in Table 4 that follows.

83 Table 4: Stress questionnaire given to subjects on their last visit.

Question 2: Do you Question 3: Do you find Question 1: What do you typically use products that aromas affect the do to relax? that have lavender scents way you feel? in your home? watch TV, do face masks, yes yes, they make me happy Subject 2 color yes, both in general and my shampoo has lavender walking, watching TV also specifically in this scents in it Subject 3 study likely to be in lotions, but drink tea didn't know that before yes- baking/ cooking Subject 4 study take a nap, watch tv, take yes, I find candles and air freshener Subject 7 a walk sweet scents relaxing exercise, glass of wine with dinner prep, cooking yes many. Hand & body yes, very much! I have dinner in general, nap on soap, body oil very strong scent memory Subject 9 the couch yes, specifically air listen to music, read a fresheners and candles. book, go for a walk, deep no Not so much lavender breaths though. Favors citrus and Subject 10 fruity aromas

Appendix C: Aroma Delivery Apparatus

Figure 21 Aroma delivery apparatus shematic. Breathing grade air was filtered, flowed at 6 liters per minute (for both inhalation conditions), humidified through water, bubbled through a 12% linalool in miglyol solution, flowed through an air trap to prevent any overflow from being inhaled by subjects, and finally was split to flow to orthonasal nosepiece or retronasal mouthpiece. If subjects were doing the orthonasal treatment, the tubing that connected the retronasal mouthpiece was clamped off, and vice versa.

85 The orthonasal nosepiece was made of glass and blown in a shape that fit right over the subject’s nose (Figure 22) The retronasal mouthpiece was constructed from a silicone scuba mouthpiece in which a glass manifold was inserted and then connected to the aroma delivery apparatus with polyethylene tubing (Figure 23).

Figure 22: Orthonasal nosepiece.

Figure 23: Retronasal mouthpiece.

Appendix D: Synthesis Approaches

1.1 General Synthesis

It is worth noting that a common by-product in synthesizing 8-hydroxylinalool, 8- carboxylinaloool and linalool glucuronide was a hydrocarbon elimination product. It consistently appeared at the top of the normal phase TLC plate and came off a similar column first during column chromatography.

1.2 Synthesizing 8-hydroxylinalool

1.2.1 First Approach based on method by Elsharif et al., 2015

A previous study conducted by Elsharif et al., 2015 elucidated methods for synthesizing 8-hydroxylinalool and 8-carboxylinalool. Therefore, this method was utilized as the starting point to begin synthesis of 8-OH linalool. 1.2 equivalents dried

SeO2 were combined with linalool, dioxane and ethanol and were refluxed overnight.

However, 1H-NMR spectroscopy revealed a complex mixture of 8-oxolinalool and numerous isomers of 8-hydroxylinalool (Figure 24).

Figure 24: First attempt at 8-hydroxylinalool (2) synthesis where 8-oxolinalool predominated over 8-hydroxylinalool (2) in the resulting product.

1.2.2 Second approach based the methods of (Bhalerao & Rapoport, 1971; Curley Jr &

Ticoras, 1986

Based on the complex mixture of 8-hydroxylinalool isomers that resulted in the first attempt, a different approach was taken going forward. In the second attempt, 2 equivalents of selenium dioxide were used to drive the reaction more towards 8- oxolinalool. This occurs because in this reaction selenium dioxide makes the alcohol first and then it is oxidized to the aldehyde. Therefore, as equivalents of selenium dioxide are increased, more of the alcohol can be converted to aldehyde. By selecting more for 8- oxolinalool, the aim was to minimize 8-OH isomers and reduce the complexity of the product. However, 1H-NMR analysis of 8-oxolinalool product revealed 4 different aldehydes. Despite this result, half of the product was used for reduction to the alcohol and the other half was used to make 8-carboxymethyl ester, with the intention of ultimately chromatographing each product to eliminate unwanted cis isomers (Figure

25).

Figure 25: Second attempt at 8-hydroxylinalool (2) synthesis where 2 equivalents of selenium dioxide were used to make 8-oxolinalool followed by sodium borohydride reduction to 8-hydroxylinalool (2).

Following the sodium borohydride reduction, the resulting 8-hydroxylinalool product was purified utilizing column chromatography. However despite purification, 8- hydroxylinalool was still a complicated mixture comprised of cis and trans isomers, as well as other side products. The 8-carboxymethyl ester reaction (Figure 25) will be explained in section 1.4.2.

1.2.3 Third & final approach based on the methods of Bhalerao & Rapoport, 1971;

Curley Jr & Ticoras, 1986

Based on the complexity of the mixture that still resulted in the second attempt, another approach was used for the final and successful attempt. Instead of increasing the equivalents of selenium dioxide, a smaller amount (1/2 equivalent) of selenium dioxide was used to stop the reaction at the alcohol and thus select more for 8-hydroxylinalool

90 (Figure 26). This reduced formation of the isomers that were seen with the previous two attempts and consequently selected better for the desired trans 8-hydroxylinalool isomer.

Figure 26: Third & final approach at 8-hydroxylinalool synthesis.

Linalool was combined with 0.5 equivalents SeO2 and refluxed in 95% ethanol to make

8-hydroxylinalool. To further purify the product, 8-OH linalool was derivatized as the p- nitrobenzoate ester to facilitate purification via preparative TLC. This allowed for visualization via fluorescence quenching since the derivatized product absorbed UV light at 254 nm while 8-hydroxylinalool by itself did not. Once purified, the product was saponified to produce the purified 8-hydroxylinalool product.

1.3 Linalool glucuronide synthesis

1.3.1 First approach based on methods by Alonen et al., 2009

In the first approach, glucuronic acid with protecting groups was combined with

HBr and acetic acid until dissolved and then the solution was then refrigerated (Figure

27). The product was evaporated under reduced pressure and toluene was added once the

91 acetic acid was almost completely evaporated to help enhance evaporation. The product was then dissolved in chloroform, extracted with sodium bicarbonate, washed with saturated NaCl solution and then filtered through MgSO4. Ethanol was then added with heat, and crystals formed 3-4 hours later. Crystals were air dried then put under vacuum before storing in the refrigerator. 1H-NMR spectra of the product revealed only minor impurities.

Figure 27: Step one in approach one for linalool glucuronide synthesis where the protected glucuronide was linked with bromide to give glucuronosyl bromide.

From there, the protected glucuronosyl bromide was added drop-wise to a solution containing linalool, Ag2CO3, 4Å molecular sieves and toluene (Figure 28). The solution was refluxed with heat for 6 hours then was left with stirring at room temperature for 5 days.

Figure 28: Step two in approach one of glucuronide synthesis where linalool and glucuronosyl bromide were combined to give protected linalool glucuronide.

Since the solution had a high concentration of linalool relative to the small amount of glucuronosyl bromide in toluene dripping, we were hoping to speed up the reaction and reduce rearrangements (particularly formation of orthoester—seen in Figure 29).

Figure 29: Mechanism of potential formation of glucuronide orthoester in step two of approach one for linalool glucuronide synthesis.

A small amount of solvent remained on top of the thicker oil (all a purple/fuchsia color). TLC revealed this was in fact the elimination hydrocarbon product and was the highest spot on the TLC plate indicating it is most non-polar. It is possible the

94 hydrocarbon elimination product is non-polar enough to be immiscible with the polar glucuronic acid conjugate. We then refrigerated the product to help solidify the oil more and then pipetted off as much as possible the top layer in attempts to speed up the column chromatography. The solution was then filtered with toluene into a separatory funnel and then extracted with HCl, dried with magnesium sulfate and subsequently evaporated under reduced pressure and concentrated to dryness. We did the extraction with HCl because the orthoester is easily destroyed by acid. This helped break up the orthoester into linalool and glucuronic acid since we were only interested in the linalool glucuronide conjugate. The product was finally purified by column chromatography and we were able to separate the product into 3 fractions: linalool, desired protected glucuronic acid conjugate, and glucuronic acid.

Next, the protecting groups were saponified, which consists of two steps: the first being a tranesterification and the second being the saponification (Figure 30).

Figure 30: Saponifaction of glucuronic acid protecting groups to give glucuronic acid conjugate.

In the first step, the samples were re-dissolved in methanol and 4 equivalents of sodium methoxide. The result was ionization of the oxygen on the O-acyl groups.

However, the methyl ester stayed the same because methoxide replaced it with the same functional group. For this reason 4 equivalents were used to ensure that the three acetate functional groups were actually switched to O-. This part is considered the transesterification. The solution was left to stir at room temperature for 2 hours and then

NaOH was added and left to stir for 2 days. This part of the reaction is considered the actual saponification. Here, the O- groups were protonated immediately because these groups have a pKa of 16 so a pH below 7 will immediately be protonated. OH- then attacked the carbon of the methyl ester, thus leaving it as a carboxylic acid.

When we went to work up the saponification we decided to TLC first to see if the reaction had proceeded as anticipated. The product was supposed to be polar enough to not move from the baseline since the protecting groups were removed. However, TLC revealed it was still further up from the baseline, indicating the reaction did not proceed as we would have liked.

1.3.2 Second approach based on methods by Lucas et al., 2009

For the second approach, a different protected glucuronic acid was used. Here, linalool, Methyl-2,3,4-tri-O-acetyl-α-D-glucuronic acid trichloroacetimidate, BF3·H2O and excess dichloromethane were combined over ice and the solution was left to stir at room temperature for 2 hours (Figure 31). The organic layer was extracted with sodium bicarbonate, washed with saturated NaCl solution, dried (Na2SO4) and concentrated to dryness. The residue was then saponified (combined with KOH and ethanol for 4 days) and subsequently concentrated to dryness (Figure 31). The residue was re-dissolved in water, ether and ethanol then concentrated to dryness. The aqueous layer was extracted with ether, then HCl was added dropwise over an ice bath until the pH lowered to ~3.

The organic layer was subsequently extracted with ethyl acetate, the organic layer washed with saturated NaCl solution, dried (Na2SO4) and concentrated to dryness. The product was purified by column chromatography to successfully produce a small amount of the glucuronic acid conjugate, which was confirmed by tandem mass spectrometry.

Figure 31: Second and final approach to synthesize linalool glucuronide.

1.4 8-carboxylinalool synthesis

Two different approaches were taken to synthesize 8-carboxylinalool. However, analysis of products from both attempts by NMR and by Quadrupole Time Of Flight

Mass Spectrometry (QTOF-MS; Agilent 6550 QTOF-LC/MS, Santa Clara, CA) revealed that the correct metabolite was not synthesized. As a result, biological samples obtained from the clinical trial were not analyzed for 8-carboxylinalool.

1.4.1 First approach based on methods by Paquette, Heidelbaugh, Paquette, &

Heidelbaugh, 2003

In the first approach, a small amount of 8-hydroxylinalool was combined with manganese dioxide (MnO2) and put into solution with petroleum ether (Figure 32) to

98 give 8-oxolinalool. 1H-NMR spectra revealed presence of main aldehyde at 9.3 ppm and lack of peak at 4 ppm demonstrating no remaining starting material. A stream of air was consistently put on the product for one month in hopes to oxidize the aldehyde. However, very few crystals formed and it was not sufficient to test the crystal identity with 1H-

NMR.

Figure 32: First approach to synthesize 8-carboxylinalool where 8-oxolinalool was first synthesized from 8-hydroxylinalool and then oxidized in air to give 8-carboxylinalool.

1.4.2 Second approach based on methods by Corey, Gilman, & Ganem, 1968

As a second approach, the idea was to make 8-carboxymethyl ester from 8- oxolinalool, to then cleave the ester and ultimately make 8-carboxylinalool. To begin, 8- oxolinalool product synthesized in the first approach was combined with potassium

- cyanide (KCN ), MnO2, methanol and acetic acid and stirred for 3 days. This residue was then saponified with KOH and ethanol to give 8-carboxylinalool (Figure 33).

Figure 33: Second approach to synthesize 8-carboxylinalool where 8-carboxymethyl ester was made from 8-oxolinalool and then saponified to give 8-carboxylinalool.

TLC revealed typical carboxylic acid behavior (long & narrow smeared spot up the plate) because it travels through the plate as COO- and COOH. It also appeared that a second carboxylic acid traveled further up the plate. There appeared to be some potential crystals starting to form but it was hard to tell, therefore the solution was refrigerated to see if more end up forming. We were expecting the 8-carboxylinalool to be a fine white powder so crystallization was expected, however if there are in fact two products then that could have inhibited crystal formation. Analysis of the product by QTOF-MS revealed an impure sample containing possibly more than one carboxylic acid, as predicted from the preliminary TLC work.

100