THE EFFECT OF CHIA CONSUMPTION ON

DEPRESSION AND SLEEP QUALITY

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

In

Agriculture

By

Emily N. Tai

2020 SIGNATURE PAGE

THESIS: THE EFFECT OF CHIA CONSUMPTION ON DEPRESSION AND SLEEP QUALITY

AUTHOR: Emily N. Tai

DATE SUBMITTED Spring 2020

Department of Nutrition & Food Science

Erik Froyen, Ph.D. Thesis Committee Chair Nutrition and Food Science ______

David Edens, Ph.D. Nutrition and Food Science ______

Emily Kiresich, Ph.D. Nutrition and Food Science ______

ii ABSTRACT

In recent years, Major Depressive Disorder (MDD) has been documented with both higher rates of incidence and earlier age of onset. Though treatments for MDD currently exist, they are limited by inherent obstacles, such as anergia and decreased motivation, or by external obstacles such as lack of knowledge or social stigma.

Complications such as these could potentially be improved upon through dietary intervention, with dietary changes allowing for a mitigation of depressive symptoms that can compliment traditional antidepressant treatment. Chia, as a food product high in omega-3 fatty acids, tryptophan, and antioxidants, could be beneficial for the management of MDD. In addition, because chia can be easily incorporated into different foods, it can be easily introduced to the diets of individuals who suffer decreased motivation or energy due to depression.

To study the potential effects, participants from the California State Polytechnic

University, Pomona campus were recruited for an 8-week study period, taking either water or chia mixed with water 30 minutes prior to each meal. Depressive symptoms, sleep quality, and urinary serotonin were measured through the Major Depression

Inventory, the Pittsburg Sleep Quality Index, and urinary serotonin ELISA results taken before, during, and after the treatment period. Though chia supplementation was not confirmed to reduce depressive symptoms, improve sleep quality, or increase urinary serotonin in this study, further examination into alternative methods of processing chia could still prove beneficial.

Keywords: Chia, Tryptophan, Omega-3, Serotonin, Melatonin, Antioxidants

iii TABLE OF CONTENTS

Signature Page ...... ii

Abstract ...... iii

Table of Contents ...... iv

List of Tables ...... vi

List of Figures ...... vii

CHAPTER 1: INTRODUCTION...... 1

Statement of the Problem...... 1

Purpose of Study...... 4

Statement of the Hypotheses...... 4

Study Significance ...... 5

Definitions...... 6

CHAPTER 2: LITERATURE REVIEW ...... 8

Depression and Sleep Disruptions ...... 8

Chia Nutrient Composition ...... 9

Consumption of Chia + Foods with Similar Properties ...... 10

Nutrients Associated with Depression/Sleep Quality ...... 11

CHAPTER 3: METHODS...... 20

Participants...... 20

Experimental Design...... 21

Dietary Intervention ...... 21

Data Collection ...... 22

iv Data Analysis ...... 23

CHAPTER 4: RESULTS...... 25

Hypothesis 1...... 25

Hypothesis 2...... 26

Hypothesis 3...... 28

CHAPTER 5: DISCUSSION...... 30

Depressive Symptoms...... 30

Sleep Quality...... 31

Chia Nutrient Bioavailability...... 32

Limitations ...... 34

CHAPTER 6: CONCLUSION ...... 37

Implications of the Study ...... 37

References...... 38

Appendices...... 44

Appendix A. MDI Form ...... 44

Appendix B. PSQI Form...... 45

Appendix C. Raw Data – MDI Full ...... 46

Appendix D. Wilcoxon Analysis (within group analysis) - MDI...... 49

Appendix E. Raw Data, PSQI Full ...... 51

Appendix F. ELISA Scores...... 54

Appendix G. ELISA Standards...... 55

v LIST OF TABLES

Table 1. Essential Amino Acids in Chia...... 12

Table 2. Ratio of L-TRP:CAA...... 13

Table 3. Participant Demographics...... 20

Table 4. Wilcoxon Signed Rank Test Results for Major Depression Inventory ...... 26

Table 5. Wilcoxon Signed Ranks Test for Pittsburg Sleep Quality Index Scores ...... 27

Table 6. Mann-Whitney Test for Pittsburg Sleep Quality Index scores ...... 27

Table 7. Wilcoxon Signed Ranks Test for Urinary Serotonin ...... 28

Table 8. Mann-Whitley Test for Urinary Serotonin ...... 29

vi LIST OF FIGURES

Figure 1. MDI Score Comparison Between Control and Treatment Groups ...... 25

Figure 2. PSQI Score Comparisons Between Control and Treatment Groups ...... 26

Figure 3. ELISA Score Mean Comparisons between Control and Treatment Groups ..... 29

Figure 4. ELISA Serotonin Standard Curve ...... 55

vii CHAPTER 1: INTRODUCTION

Statement of the Problem

Major Depressive Disorder (MDD), which affects over 264 million people world- wide, is known by the World Health Organization to be a leading cause of disability.

While the age of onset is known to rage from mid-to-late adolescence to the early 40’s with peak incidence noted to be around the mid 20’s, MDD has recently been documented with higher overall rates and increasingly earlier age of onset (Santos et al.,

2017).

Though MDD is associated with almost 200 known genes, it can be difficult to research and treat due to the wide variety of symptoms and potential etiologies (Santos et al., 2017; Rao et al, 2016). Furthermore, while known and effective treatments for MDD currently exist, proper utilization of these treatments may be limited by income level, incorrect diagnosis, or limited access to health coverage resulting from the social stigma around addressing mental health. MDD is additionally known to be closely related to sleep disruptions, with depression potentially resulting in disturbed sleep or with sleep disturbances contributing to depressive symptoms (Depression & Sleep, n.d.)

Common medical treatments for depression, such as selective serotonin reuptake inhibitors (SSRIs), can improve mood by increasing the availability of extracellular serotonin in the brain, but also cause significant side effects relating to other serotonergic functions (Ferguson 2001). In many cases, the detrimental side effects from antidepressants can be severe enough to reduce patient adherence to their prescription, thus limiting their efficacy in treatment. Furthermore, due to the challenges in treating

MDD, pharmaceutical treatments may fail to target the correct mechanisms for a patient’s

1 depression, resulting in a treatment with negative side effects and few benefits. Over a third of MDD patients may also have treatment-resistant depression (TRD) that responds inadequately to both pharmacological and psychological therapy (Xu et al. 2016; Yang et al., 2018).

Considering these obstacles to managing depression, it is important to consider alternative avenues of treatment. For example, mild or moderate depression can be treated non-pharmacologically with cognitive and behavioral psychotherapies or through exercise. However, since some of the primary symptoms of depression include anergia, a persistent lack of energy that’s not inherently linked to exertion, and anhedonia, a deficit in hedonic function that impacts desire, effort, and motivation, these kinds of treatment can be difficult to initiate and/or maintain. Dietary intervention, on the other hand, can potentially be implemented even with low energy or low motivation, thus making it appealing as an initial or complimentary treatment for depression.

Chia

Salvia hispanica L., which is known colloquially as chia, is an annually herbaceous plant originating from Southern Mexico and Northern Guatemala. Chia has been used for thousands of years in the preparation of folk medicines, foods, and cosmetics, as well as for religious rituals. In the past it has been used as whole seeds, seed flour, and seed oil for a significant part of the Latin American diet, as documented by both natives and historians. Because it is nutrient-dense with a high concentration of healthy fatty acids, a near-complete amino acid profile, and a variety of different phytochemical and antioxidant compounds, chia has served as an important staple for the

2 people of alongside other crops such as and (Hrnčič et al.,

2019) (Valdivia-Lopez et al., 2015).

Previous studies have found that incorporating chia into beef burgers had a beneficial effect on both the omega-6/omega-3 ratio and antioxidant bioavailability

(Antonini et al., 2019). These benefits, along with chia’s high tryptophan concentration, could potentially be a beneficial addition to one’s diet, reducing depressive symptoms and sleep disruptions by improving brain health and serotonin production.

3 Purpose of Study

The purpose of this study is to determine whether chia seed consumption can alleviate depressive symptoms and poor sleep quality.

Specifically, chia seed consumption will be examined for its impact on depressive symptoms, sleep quality, and urinary serotonin concentration to determine whether chia seed consumption has a beneficial effect on these factors.

Statement of the Hypotheses

1. H0 - Major Depressive Inventory score index will show no significant difference

between the chia-supplementation and control populations by the end of the treatment

period.

H1 - Major Depressive Inventory score index will be significantly statistically

different in the chia-supplementation population compared to the control population

by the end of the treatment period.

2. H0 - PSQI score index will show no significant difference between the chia-

supplementation and control populations by the end of the treatment period.

H1 - PSQI score index will be significantly statistically different in the chia-

supplementation population compared to the control population by the end of the

treatment period.

3. H0 - Urinary serotonin will show no significant difference between the chia-

supplementation and control populations by the end of the treatment period.

H1 – Urinary serotonin will be significantly statistically different in the chia-

supplementation population compared to the control population by the end of the

treatment period.

4 Study Significance

Though there are currently effective treatments for depression, there also exist obstacles, both inherent to depression and related to public understanding of it, that can obstruct it. Anergia, anhedonia, and low mood can make it difficult for patients to seek out or adhere to medical treatment, while social stigma or a lack of understanding about depression can make it difficult for people to seek out treatment. Furthermore, because depression has a variable and potentially unknown etiology, it can be difficult to identify or treat when it arises. In light of these complications, it would be beneficial to further research complimentary or alternative methods to alleviating depressive symptoms, in order to improve both patient treatment and quality of life.

Since nourishment is an immediate and essential element for life, it is likely to be addressed by patients with anhedonia or anergia. Therefore, finding a function food that can be easily incorporated into a person’s diet could help improve depression treatments, as a first step towards mitigating depressive symptoms, providing a synergistic compliment to current antidepressant treatments and potentially minimizing its significant side effects.

Because it can be easily incorporated into the diet, and because it has high tryptophan, omega-3, and antioxidant concentrations, chia has potential as a beneficial addition to the diet, as an unobtrusive and high impact way of alleviating depressive symptoms and improving quality of life.

5 Definitions

Anergia - Anergia is a lack of energy that, while conceptually similar to fatigue, is more persistent and not specifically post-exertional (Rizvi et al., 2016).

Anhedonia - Anhedonia a deficit in hedonic function, which can affect desire, effort/motivation, anticipation, and consummatory pleasure (Rizvi et al., 2016).

Antioxidant - Antioxidants are molecules that stop the formation of free radicals, thus reducing oxidative stress that can result in cell damage or death (Wahlqvist, 2013)

Major Depressive Disorder (MDD)/Clinical Depression (CD) - MDD/CD refers to a mood condition where depressive symptoms, such as low mood, anhedonia, and/or anergia, persist for two weeks or greater. Further complications resulting from MDD/CD include increased school failure, lower marriage probability, higher divorce probability, teen childbearing, negative parenting, lower income, work absenteeism, and increased risk of early death (Santos et al., 2017, Rao et al., 2016). MDD is also associated with increased risk for comorbid disorders, such as , that result from the impaired quality of life, loss of daily functionality, and loss of productivity (Parekh et al.,

2017).

Melatonin - Melatonin is a hormone synthesized from serotonin in the pineal gland which entrains circadian rhythms and regulates the sleep-wake cycle. It is also known to act as an antioxidant, protect mitochondrial and neuronal function, and tends to decrease cell senescence (Jiang et al., 2016).

Polyunsaturated fatty acids (PUFA) - PUFAs are fatty acids with multiple double bonds in their backbones (Ander et al, 2003)

6 Serotonin - Serotonin a monoamine neurotransmitter that modulates cognition, reward, learning, memory, and physiological processes such as vomiting and vasoconstriction

(Young, 2007)

Treatment-Resistant Depression (TRD) - TRD refers to a subset of MDD in which the patient responds insufficiently to initial and subsequent antidepressant treatment, including combined pharmacotherapy and psychotherapy treatments. This affects over one-third of MDD patients and accounts for a significant amount of the overall costs of

MDD treatment.

Tryptophan (TRP) - TRP is an essential amino acid that functions as a precursor to the hormones serotonin and melatonin

7 CHAPTER 2: LITERATURE REVIEW

Depression and Sleep Disruptions

Major depressive disorder (MDD) is defined as a mood condition with persistent depressive symptoms such as low mood, anhedonia, and/or anergia that last for two weeks or greater, with genetic, neurological, hormonal, immunological, and neuroendocrinological mechanisms that can play a role in its development. Many of these factors appear to center around stressor responses and emotional information processing, with stressful experiences such as acutely stressful life events, chronic stress, and childhood exposure to adversity triggering depression in those with a biological or psychological susceptibility to it (National Academies Press, 2009).

While there is no clear etiology for it, there is a strong association between depression and impaired processing of serotonin, a neurotransmitter known to modulate both cognitive and physiological processes. For example, a Hong Kong study found that two missense mutations in the SLC6A4 coding region, known for serotonin transport, were found in Chinese patients with both clinical depression and suicide attempts. By increasing the SLC6A4 ’s affinity to serotonin, these mutations increased clearance of serotonin from the synaptic cleft, thus terminating its effect in the brain (Rao et al., 2016). Further studies of untreated depressed patients have suggested that serotonin function is decreased with depression, while antidepressant treatments tend to act on serotonin and/or noradrenaline transmission (Garrido et al., 2012).

In addition, over one-third of MDD patients have treatment resistant depression

(TRD), which means they do not respond sufficiently to initial or subsequent antidepressant treatments. Recent research suggests that this may be a result of

8 inflammatory processes and oxidative stress in the brain, which cause damage resulting in structural or functional abnormalities that impair brain processes. Further studies suggest that, by reducing inflammation and oxidative stressors in the brain, anti- inflammatory treatment for these patients can minimize or reverse damage, thus providing a beneficial effect against depressive symptoms (Yang et al., 2018).

Disruptions of the sleep/wake cycle, which can occur as a result of decreased melatonin levels, are known to be associated with higher degrees of pain, depression, and disease activity that resulting in a lower mood and quality of life (Bravo et al., 2012, Lee et al., 2016). Sleep cycles in the body are managed through circadian rhythms, entrained by melatonin (Jiang et al. 2016) Because of this melatonin, which is produced from serotonin, is used as an indicator of sleep quality. Clinically, melatonin is used by doctors and researchers to improve sleep quality, treat insomnia and depression, and to reduce jet lag (Fukushige et al., 2016).

Chia Nutrient Composition

Salvia hispanica L., also known as chia, has been historically been used in ancient

Mesoamerican cultures in the preparation of for medicine and food, alongside staples such as corn, beans, and amaranth. While Salvia hispanica L. produces seeds that vary from black, grey, spotted, or white, it has been noted that there is a marginal enough difference between white and black seed varieties that they are considered to be equal, though the chemical composition of chia seeds can vary by the environment of cultivation (Hrnčič et al., 2019).

Chia is composed of 30-33% , 26-42% , 18-30% , and 15-25% protein, along with a variety of , minerals, and antioxidants.

9 Tryptophan (TRP), which is the least common essential amino acid (EAA) in the human diet, is present at 440 mg per 100 g of chia, while omega-3 fatty acids can make up 68% of chia seed oil (Kulczyński et al., 2019). In addition, chia is about 8.8% various phenolic compounds, such as antioxidants (Hrnčič et al., 2019).

The current recommended chia consumption is no more than 48 g/day, equivalent to 1/4 cup, providing 211.2 mg of tryptophan total. The recommended daily allowance

(RDA) of tryptophan is estimated to be 250-425 mg/day (Richard et al., 2009).

Consumption of Chia + Foods with Similar Properties

Previous experiments, using chia seeds and/or goji puree as a supplement for beef burger patties, found that incorporation of chia seeds increased the total content in the beef patties increased PUFA content up to 10.5% with the addition of 5 g chia per 100 g beef patty. The PUFA/SFA and omega-6/omega-3 ratios of these beef patties were also improved, and they were noted to have improved polyphenol content, increasing from the control at 20.9 mg/100g to 25 mg/100g with 2.5g chia and 29 mg/100g with 5g chia respectively. This indicates that chia, as incorporated into food products, has a beneficial effect on PUFA concentrations and polyphenol intake (Antonini et al, 2020).

Furthermore, studies on tryptophan-rich food products have found a correlation between the ingestion of tryptophan-rich food products and mood. For example, one study found that consumption of Jerte Valley cherries, with high tryptophan, serotonin, and melatonin content due to the soil, was connected to a significant improvement in mood for healthy young, middle aged, and elderly participants, while also significantly decreasing anxiety scores for middle aged and elderly participants and urinary cortisol levels in young and elderly participants (Garrido et al, 2012).

10 Nutrients Associated with Depression/Sleep Quality

Tryptophan

Tryptophan, which serves as the precursor for both serotonin and melatonin, is significant in the regulation of mood and sleep quality. It is also the amino acid found in the lowest concentration among most with low tissue storage, thus making it the amino acid with the lowest concentration in the human body. Furthermore, tryptophan competes with other large neutral amino acids (LNAAs) for transport across the blood- brain barrier. consumption can increase tryptophan availability through insulin activation, which reduces the concentration of competing amino acids in the blood. Conversely, ingestion of most proteins decreases tryptophan concentration through the blood-brain barrier by either increasing protein anabolism and drawing from tryptophan stores, or by increasing the competition of tryptophan absorption with other amino acids (Richard et al., 2009).

Chia, as indicated in Tables 1 and 2, contains 440 mg tryptophan per 100 g serving and has a tryptophan to competing amino acid (CAA) ratio of 0.093. This ratio, larger than most other high-tryptophan food products, indicates that the tryptophan in chia may be more readily absorbed through the blood-brain barrier due to increased competitive power relative to other CAAs.

11 Table 1. Essential Amino Acids in Chia

Content (mg) per 100 g Amino Acids per 48 g per tbsp per tsp (USDA) Essential Amino Acids Arginine 2140.0 1027.2 256.8 85.6 Histidine 530.0 254.4 63.6 21.2 Isoleucine 800.0 384.0 96.0 32.0 Leucine 1370.0 657.6 164.4 54.8 Lysine 970.0 465.6 116.4 38.8 Methionine 590.0 283.2 70.8 23.6 Phenylalanine 1020.0 489.6 122.4 40.8 Threonine 710.0 340.8 85.2 28.4 Tryptophan 440.0 211.2 52.8 17.6 Valine 950.0 456.0 114.0 38.0 Nonessential Amino Acids Cystine 410.0 196.8 49.2 16.4 Tyrosine 560.0 268.8 67.2 22.4 Alanine 1040.0 499.2 124.8 41.6 Aspartic acid 1690.0 811.2 202.8 67.6 Glutamic acid 3500.0 1680.0 420.0 140.0 Glycine 940.0 451.2 112.8 37.6 Proline 780.0 374.4 93.6 31.2 Serine 1050.0 504.0 126.0 42.0 *Summarized from Kulczyński et al, 2019

12 Table 2. Ratio of L-TRP:CAA

L-TRP Product CAA (mg) Ratio (mg) Chia (100 g) 440 4700 0.093

Turkey (light meat, per lb, raw) 410 9,525 0.043

Turkey (dark meat per lb, raw) 303 7,036 0.043

Chicken (light meat per lb, raw) 238 5,122 0.046

Chicken (dark meat per lb, raw) 256 5,492 0.047

Whole (per qt) 732 8,989 0.081

2% milk (per qt) 551 12,516 0.044 *CAA = Isoleucine, Leucine, Phenylalanine, Tyrosine, Valine (Richard et al, 2009)

Intake of pure tryptophan has been shown to significantly increase plasma tryptophan levels, and the ratio of tryptophan to other LNAAs, around 60 minutes after administration, with peak levels reached around 2 hours after intake. These elevated TRP levels are maintained for 7-12 hours after intake. Previous studies have shown that tryptophan administration can reverse changes in circulating levels of serotonin and melatonin for mammals and ring doves (Steenbergen et al., 2016)

Sleep and depression have both been associated with serotonergic functions in the brain, with depression connecting with either poor or excessive sleep (Li et al., 2017,

Lieberman et al., 2016). Conversion of brain tryptophan to 5-hydroxytryptophan with tryptophan hydroxylase is the first and rate-limiting step to serotonin synthesis. Since tryptophan hydroxylase is typically 50% saturated with tryptophan, a change in brain tryptophan availability can alter serotonin synthesis (Richard et al., 2009)

13 Acute tryptophan depletion can produce depressive symptoms in patients, associated with disturbances in circadian rhythm or increases in anxiety (Bravo et al.,

2012), while decreased tryptophan levels are associated with decreased serotonin synthesis and turnover that results in a subsequent negative bias in automatic information processing, along with a promotion in negative social behaviors such as aggression

(Steenbergen et al., 2016).

Conversely, increased plasma tryptophan availability is associated with enhanced positive mood and dampened cortisol response after an acute environmental stress, likely due to the serotonin mechanisms involved in stress adaptation. A Jerte Valley cherry study determined that cherries from the area, with high tryptophan, serotonin, and melatonin content, had a beneficial effect on mood and presented antidepressant-like actions when compared to the placebo (Garrido et al., 2012).

Though pure tryptophan supplementation is limited by regulatory restrictions, alpha-lactalbumin (ALAC), which is a whey-derived tryptophan-rich protein, has been used as a dietary method for increasing brain serotonin levels. One study found that consumption of ALAC resulted in a 50-130% increase in the plasma TRP:LNAA ratio, subsequently increasing tryptophan absorption through the blood-brain barrier and improving brain serotonin function as related to mood and stress behaviors (Markus et al.

2008).

A later study on depressive moods in subjects with high or low chronic stress vulnerabilities found that morning consumption of tryptophan-rich egg protein hydrolysate improved depressive mood. For subjects who had low chronic stress vulnerabilities, the egg protein hydrolysate was also found to improve perceptual-motor

14 and vigilance performance. Though plasma tryptophan availability is known to increase through both the consumption of pure tryptophan and the consumption of carbohydrates, this study found that the high-tryptophan egg protein hydrolysate increased the plasma

TRP:LNAA ratio even further (Markus et al. 2010).

Melatonin, produced from serotonin derived from tryptophan in the pituitary gland, participates in sleep management through the regulation of circadian rhythms, thus giving it an effect on mood as well. Though tryptophan intake has been associated with an increase in self-reported sleep duration in a study by Lieberman in 2016, analysis of the data for men and women separate did not show an association with sleep duration.

However, after adjusting for total protein intake, a small positive association between tryptophan intake and sleep duration was found, suggesting an independent effect of tryptophan on sleep quality. In relatively high doses, tryptophan administration increased self-reported ratings of sleepiness and decreased sleep latency, with a stronger association for those with mild insomnia or a longer baseline sleep latency. For the range of tryptophan ingested by the US population, higher doses may correlate with lower levels of depression and longer sleep.

Tryptophan intake at breakfast has been associated with higher melatonin secretion and easier sleep when coupled with exposure to morning light (Fukushige et al.,

2016), while tryptophan enriched cereals have been found to improve sleep quality and reduce sleep problems. Because tryptophan intake was directly related to increased brain serotonin and melatonin availability, it can affect sleep latency and sleep quality. The positive effect of tryptophan administration on melatonin, serotonin, and sleep quality is well documented. (Bravo et al., 2012)

15 Tryptophan rich diets were also able to consolidate sleep for newborns and improve sleep and antioxidant capacity levels for young, middle-aged, and elderly individuals. The consumption of tryptophan-rich cereals was able to produce beneficial effects in the sleep/wake cycle (Bravo et al., 2012)

Omega-3/Antioxidants

Patients with increased inflammatory activity are more susceptible to treatment- resistant depression (TRD), which affects about one-third of all MDD patients. In these cases, patients do not respond sufficiently to antidepressant treatments, including pharmacotherapy and psychotherapy combined treatments. Because inflammatory processes and oxidative stress are also biological effects that can stem from depression, it is likely that neuro-inflammation, associated with structural and functional abnormalities in the brain, plays a role in treatment-resistant depression. Supporting this, anti- inflammatory drugs have been found to show benefit for MDD treatment, with positive effects being higher for patients with increased inflammatory activity (Yang et al., 2018).

Increases in macrophage activity and peripheral pro-inflammatory cytokines, such as IL-1β and IL-6, have been recognized in MDD patients, resulting glucocorticoid stimulation in the hypothalamic–pituitary–adrenal axis (HPA axis) that increases stress response and impairs neurotransmission and causes behavior similar to those in depressed patients. In addition, microglia in the brains of depressed patients can be activated, thus secreting inflammatory mediators in the brain and potentially inducing neuro- inflammation, depressive symptoms, and neuronal apoptosis. By contrast, some effective antidepressant treatments are known to suppress inflammatory cytokines and prevent cytokine-induced depression (Song et al., 2016). Research has also shown that patients

16 with anxiety or depression have significantly lower levels of blood A, C, and E when compared to a healthy population, and that supplementation with these vitamins results in a significant drop in depression and anxiety scores when compared to a placebo

(Gautam et al, 2012).

Consequently, it is likely that MDD can be linked to cortisol-induced neuronal atrophy, neuro-inflammation due to activated microglia in the brain, reduced neurotrophins and receptors, and/or increased endogenous neurotoxins (Song et al.,

2016).

Omega-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are essential components of cell membranes, increasing membrane fluidity and permeability as well as modulating the activity of membrane-bound proteins. EPA is known to have a pronounced effect on myelinogenesis, by increasing the expression of proteolipid proteins (PLP), myelin basic protein and myelin oligodendrocyte protein to help improve the speed of neuronal transmission. Meanwhile, synapse (the primary unit of a neuronal circuit) are known to have DHA-rich membranes. Poly-unsaturated fatty acids (PUFAs) also have beneficial effects on white matter structural integrity and grey matter volume for the frontal, temporal, parietal, and limbic areas of the left hemisphere.

Conversely, omega-3 deficiencies have been linked with deteriorated serotonergic

(mediating mood), noradrenalergic (mediating stress cooldown), and dopaminergic

(mediating reward behavior) neurotransmission (Song et al., 2016).

In addition to these inherent properties, EPA and DHA are known to suppress the neuro-immune network, decreasing inflammation in the brain by working against oxidative stress. While the production of reactive oxygen species (ROS) is known to

17 facilitate neuronal damage, clearance of ROS, which can be achieved with omega-3 fatty acids and other antioxidants, help to prevent inflammation (Song et al., 2016).

Deficiencies in EPA and DHA, as well as higher omega-6:omega-3 ratios, are associated with MDD. In cases of chronic or severe MDD, decline in cognitive function is also associated with lower EPA concentrations. Meanwhile, magnetic resonance imaging (MRI studies) show that patients with lower blood DHA also have lower total brain volume along with a greater white matter hyper intensity. Lower plasma EPA and

DHA concentrations are also correlated with reduced visual working memory and executive function, making it more difficult for depressed patients to self-motivate for the tasks they need or want to accomplish (Song et al., 2016).

Conversely, higher intake of fish or omega-3 PUFAs is associated with decreased risk of depressive disorders as well as fewer depressive symptoms, with evidence that

MDD responds to EPA and DHA synergistically. A 2-week pretreatment with 3.5 g

EPA/day was found to significantly decrease IFN-alpha induced depression, and MDD treatment with selective serotonin reuptake inhibitors (SSRIs) is known be more effective when combined with EPA supplementation than when taken alone (Song et al., 2016).

Omega-3 consumption has a favorable impact on sleep-related issues and on neurologic functions that can affect cognition, emotion, and behavior. It has been found to have a positive effect on subjective sleep, which is more closely related to psychological function than objective sleep, and is associated with longer sleep duration and favorable inflammatory markers for women during gestation. In addition, omega-3 consumption has been found to decrease sleep disturbances, anxiety sensitivity, and uncertainty intolerance to a greater degree than a placebo, which also being associated

18 with higher emotional regulation, perception, and acknowledgement. (Janghard et al.,

2018)

19 CHAPTER 3: METHODS

This study was conducted in conjunction with two other chia studies following the same pattern, with one analyzing chia seed consumption as related to food displacement, and the other analyzing the effect of chia seed consumption on blood sugar. As a result, methodology and recruitment for those studies were utilized for this study as well.

Participants

Participants (n = 16, age = 22.56±3.86, BMI = 31.71±5.07) selected for the study, to examine the effect of chia seed consumption on depressive symptoms, sleep quality, and urinary serotonin in overweight or obese women, were students of California State

Polytechnic University, Pomona. Recruitment was done through fliers and emails, with consent forms utilized in screening the participants of the study to determine eligibility.

Table 3. Participant Demographics

Demographic Information Pre Mid Pst Age 21.25 ± 2.03 Control Height (cm) 164.88 ± 6.53 (n=8) Weight (kg) 81.53 ± 11.94 81.06 ± 11.40 81.23 ± 11.08 BMI 29.88 ± 2.95 29.7 ± 2.74 29.83 ± 2.56 Age 24.00 ± 4.81 Treatment Height (cm) 161.38 ± 7.39 (n=8) Weight (kg) 87.1 ± 15.46 87.25 ± 15.72 86.90 ± 15.37 BMI 33.56 ± 6.22 33.55 ± 6.18 33.44 ± 6.08

20 The exclusion criteria were as follows: younger than 18 or older than 45, averse to chia consumption, adherence to a diet program, pregnancy or plans to become pregnant, and irregular sleep schedule.

Inclusion criteria were as follows: between ages 18 and 45, no aversion to chia, no current adherence to a specific diet plan, not pregnant or planning to become pregnant, and regular sleep schedule.

Participant records were stored in folders labeled with only participant numerical

ID, in a locked cabinet in the on-campus laboratory.

Experimental Design

16 participants, between the ages of 18-45, were included in the study. The study was a randomized parallel design over the course of 8 weeks, with the dietary interventions being 1) chia-free control and 2) chia consumption.

Dietary Intervention

Participants were asked to maintain normal diet and exercise for the duration of the study, except for the consumption of either 10 oz water or 10 oz water + chia 30 minutes before each meal, depending on their dietary intervention. While most participants reported eating three meals a day, with occasional snacks in between, one participant in the treatment group consistently ate two meals per day, rather than three.

All participants were asked to refrain from chia consumption outside of the allocation provided to them. While the control group was provided with no chia, the treatment groups were provided with chia to the equivalent of 10% of their daily caloric intake, as calculated by their estimated energy expenditure. Compliance for the study was

21 determined by dietary logs indicating the time and amount of chia consumed and by participants returning each week with any chia that was left unused.

Data Collection

The Chia study procedures included anthropometric data collection, urinary sample collection, and survey questionnaires for depressive symptoms and sleep quality.

Participant data was collected during an on-site morning meeting after an 8-hour fasting period. Participants also filled out a dietary log for two weekdays and a weekend during the pretest, midtest, and posttest portions of the study, with participants in the treatment group additionally logging the amount of chia they had prior to each meal.

Depressive Symptoms

Major Depression Inventory questionnaires were filled in on paper copies at weeks 0, 4, and 8, when participants came in for an on-person meeting.

Sleep Quality

Pittsburg Sleep Quality Index questionnaires were completed at weeks 0, 4, and 8, when participants came in for an on-person meeting.

ELISA

Urine samples were collected at weeks 0, 4, and 8 after an 8-hour fast, with samples labeled and stored at -80C until analyzed with the Serotonin Ultra-Sensitive

ELISA assay kit.

22 Data Analysis

Areas of Interest

For the purpose of this study, depressive symptoms, sleep quality, and urinary serotonin were selected as areas of interest for analysis.

Analysis Objective 1: Change in Major Depression Inventory Scores

The Major Depression Inventory (MDI) is a self-reported mood questionnaire developed by the WHO to generate a diagnosis for clinical depression and estimate symptom severity. Scores from the MDI were collected from the participants during the pretest, midtest, and posttest periods to determine changes in depressive symptoms.

Analysis Objective 2: Change in Pittsburg Sleep Quality Index Scores

The Pittsburg Sleep Quality Index (PSQI) is a self-reported questionnaire that assesses sleep quality over a month-long time interval, developed by the University of

Pittsburg to be used in both clinical and research settings. Scores from the Pittsburg Sleep

Quality Index were collected during the pretest, midtest, and posttest periods and analyzed for changes in depressive symptoms.

Analysis Objective 3: Change in Eagle Bioscience Serotonin Ultrasensitive ELISA

Assay readings

Readings for urinary serotonin were collected from urine samples and analyzed with the Eagle Bioscience Serotonin Ultrasensitive ELISA Assay kit.

Statistical Methods

Analysis of depressive symptoms, sleep disturbances, and urinary serotonin were conducted independently. Out of 21 participants selected for the primary study, 13 were included for analysis of MDI scores, 10 were included for analysis of PSQI scores, and

23 14 were included for analysis of urinary serotonin, as determined by total completion of the relevant questionnaires and of the urine sample sequence.

Due to the nature of the study, the Wilcoxon signed rank test was used to determine the significance of the difference within the pre-test, mid-test, and post-test scores of the control and treatment groups independently, while the Mann-Whitney test was used to determine the difference between pre-test, mid-test, and post-test scores between the control and treatment groups. Statistical analysis was conducted using SPSS

24 CHAPTER 4: RESULTS

Primary study results, analyzing the change in participant data between pre, mid, and post-trial periods as well as differences between control and treatment groups, found that differences were insignificant with only a few exceptions.

Hypothesis 1

30

25

20 cores

15 ean S I MD 10 M

5

0 PRE MID PST CONTROL 8.1667 5.1667 4.6667 TREATMENT 14.2857 14.1413 13.4286 Figure 1. MDI Score Comparison Between Control and Treatment Groups Hypothesis 1 states that depressive symptoms, as indicated by Major Depression

Inventory scores, will decrease as a result of chia supplementation when compared to the control. However, within-group analysis of both the control and the treatment groups revealed no significant difference between pre-test (control = 8.19 ± 3.43, treatment =

14.29 ± 8.22), mid-test (control = 5.17 ± 3.13, treatment = 14.14 ± 8.47), and post-test

(control = 4.67 ± 2.34, treatment = 13.43 ± 11.67) scores, as indicated in Figure 1 and

Table 3. This indicates that, for both the control and the treatment groups, there was no significant change in MDI scores over the course of the study. Further analysis of the individual MDI questions (Appendix B) revealed that only question 9 (Have you felt

25 subdued or slowed down?) saw a significant difference between the pretest and the post- test (z=-2.00, p=0.046), with the post-test scores ranking lower than the pretest scores.

Due to the significant difference of the initial MDI scores for the control group and the treatment group, the two groups could not be accurately compared.

Table 4. Wilcoxon Signed Rank Test Results for Major Depression Inventory

MDI Mid-Pre Pst-Mid Pst-Pre Z -1.367 -3.19 -1.581 Water only Asymp. Sig (2-tailed) 0.172 0.75 0.114 Z -0.085 -0.507 -0.954 Water + Chia Asymp. Sig (2-tailed) 0.932 0.612 0.34

Hypothesis 2

14

12

10

coresean S coresean 8

6 SQI M P M SQI 4

2

0 PRE MID POST □ CONTROL 8.3333 6 2.3333 mTRT 5.4286 4.7143 5.2857

Figure 2. PSQI Score Comparisons Between Control and Treatment Groups Hypothesis 2 states that sleep disruptions, as indicated by PSQI test scores, will decrease in the chia supplementation population compared to the control. However,

26 within-group analysis of the control and treatment groups revealed no significant difference between pre-test (control = 8.33 ± 4.93, treatment = 5.42 ± 2.64), mid-test

(control = 6.00 ± 1.73, treatment = 4.71 ± 3.09), and post-test (control = 2.33 ± 1.53, treatment = 5.29 ± 2.81) scores, as indicated in Figure 2 and Table 4. This indicates that, for both the control and the treatment groups, there was no significant change in PSQI scores over the course of the study.

Statistical analysis indicates that for the pre-test, the mid-test, and the post-test, there was no significant difference in PSQI scores between the control group and the treatment group, as indicated in Table 5. However, it should be noted in these cases that the control group sample size is incredibly small (n=3) due to poor questionnaire compliance.

Table 5. Wilcoxon Signed Ranks Test for Pittsburg Sleep Quality Index Scores

PSQI Mid-Pre Pst-Mid Pst-Pre Z -1.342 -1.604 -1.604 Water only Asymp. Sig (2-tailed) 0.180 0.109 0.109 Z -0.974 -0.604 0.000 Water + Chia Asymp. Sig (2-tailed) 0.344 0.546 1.000

Table 6. Mann-Whitney Test for Pittsburg Sleep Quality Index scores

PSQI Pre Mid Post Z -0.575 -0.580 -1.509 Asymp. Sig (2-tailed) 0.565 0.562 0.131 Exact Sig. (2*(1-tailed Sig.) 0.667 0.667 0.183

27 Hypothesis 3

Hypothesis 3 states that Urinary serotonin will be significantly increased in the chia-supplementation population compared to the control population. However, as indicated in Table 6, results showed no difference between the optical densities of serotonin in the urine samples between the pretest and the posttest, for both the control and treatment groups. While the treatment group saw a significant difference between pretest and midtest scores (z=-1.992, p=0.046), indicating a potential decrease in serotonin concentration that then increased back to pretest levels, this could not be confirmed within the study, due to corruption in the data for the standard concentration curve. Since the data for the standard concentration curve was unusable, the observed optical densities obtained from the urine samples were unable to be translated to specific serotonin concentrations.

Table 7. Wilcoxon Signed Ranks Test for Urinary Serotonin

Elisa Mid-Pre Pst-Mid Pst-Pre Z -1.540 -0.700 -0.700 Water only Asymp. Sig (2-tailed) 0.123 0.484 0.484 Z -1.992 -1.782 -0.524 Water + Chia Asymp. Sig (2-tailed) 0.046 0.175 0.600

28 1.4

1.2

nm) 1 054(y

0.8 tiens

0.6 Dlcaipt

0.4 O 0.2

0 PRE MID PST

CTR □ TRT

Figure 3. ELISA Score Mean Comparisons between Control and Treatment Groups Between the control group and the treatment group, there was no significant difference found, as indicated in Table 7 and Figure 5.

Table 8. Mann-Whitley Test for Urinary Serotonin

ELISA Pre Mid Post Z -0.258 -0.258 0.000 Asymp. Sig (2-tailed) 0.796 0.796 1.000 Exact Sig. (2*(1-tailed Sig.) 0.852 0.852 1.000

29 CHAPTER 5: DISCUSSION

The aim of the current study was to examine the effects of chia seed consumption on mood and sleep quality through the analysis of MDI and PSQI scores, as well as through urinary serotonin analysis. Ultimately, the current study found the effect of whole chia seed consumption on these factors to be insignificant.

Depressive Symptoms

The effect of chia seed consumption on depressive symptoms was analyzed through use of the Major Depression Inventory. Past research indicates that omega-3 fatty acids, antioxidants, and tryptophan, which are all present in high levels in chia, can have beneficial effects on depressive symptoms. (Garrido et al, 2012;Yang et al, 2018; Song et al, 2016)

Results indicate that, overall, MDI scores were not significantly affected by consumption of chia. While the between-groups analysis for control and chia groups was not applicable for this situation, as there was a significant difference between the pre-test medians, within-group analysis indicates that, overall, there was no significant change in

MDI scores before, during, or after the test.

The MDI control group only saw a significant difference between pretest and posttest scores for question 9 (have you felt subdued or slowed down). Conversely, the

Jerte Valley Cherry study, which observed the effects of cherries from the Jerte Valley region on stress and mood due to the high levels of tryptophan, serotonin, and melatonin, saw a significant effect of cherry consumption compared to the placebo (Garrido et al,

2012). However, there were differences between this past study and the current study – potentially, one of the biggest differences was in the preparation of the product. While the

30 cherries for the 2012 study were ground into a powder and mixed with water for consumption by the participants, the chia seeds provided to this study’s participants were whole, mixed in water before consumption. Because of this, it is possible that the nutrients from the Jerte Valley cherries were rendered more bioavailable than the nutrients present in the chia seeds, as a result of the processing accelerating the digestion process.

Other studies suggest that the benefits from chia seeds may be increased by taking the oil, rather than the whole seed. Potentially, to preserve the protein content as well as the healthy fatty acid and antioxidant content, future research into chia seed consumption on mood and sleep could compare the effects of ingesting no chia seeds to whole chia seeds, ground chia seeds, chia seed oil, and dry chia seed matter (Teoh et al, 2020; Enez et al, 2020).

Sleep Quality

The effect of chia seed consumption on sleep quality was analyzed through use of the Pittsburg Sleep Quality Index. Though research on this subject was less in-depth than research on depressive symptoms, there has been research indicating that the omega-3 fatty acids, antioxidants, and tryptophan present at high levels in chia can have beneficial effects on sleep quality. In addition, sleep quality and depressive symptoms are known to be closely interrelated, with depressive symptoms exacerbating poor sleep or poor sleep contributing to depressive symptoms. (Lieberman in 2016; Janghard et al., 2018).

However, in the present study, chia seed consumption was not found to significantly affect sleep quality, as there was no significant difference found between pretest, midtest, and posttest scores for either the control or treatment groups.

31 Chia Nutrient Bioavailability

A meta-analysis on chia supplementation for unbalanced diets, analyzing 17 different animal-based chia studies, found that chia seed supplementation was able to beneficially affect health in regard to lipid profile, glucose metabolism, antioxidant effects, and inflammation. One study, with a chia dosage of 41.7%, was noted to increase antioxidant capacity and decrease inflammatory markers for animals with chia introduced as a whole seed flour. Because of the grinding process, which was not part of the current study, the bioavailability of nutrients and antioxidants already present in the chia was increased.

Therefore, it is possible that this study, as a result of the usage of whole, unground chia seeds, saw reduced nutrient bioavailability that subsequently dampened the potential health effects. The meta-analysis further noted that, for the purposes of their article, they primarily focused on animal studies due to dosage reasons, as acceptable human dosage for chia intake is low compared to those offered to animals. Because of this, they theorized that the amount of chia required for human consumption to improve metabolic parameters may be too high to meet (da Silva et al, 2018; Rosa et al, 2013; Enes et al,

2020). Chia oil may also prove to present greater improvement in inflammation and antioxidant systems than whole chia seeds, though flavonoid bioavailability may be reduced as a result of the oil.

Another meta-analysis, looking at the effect of chia seed consumption on human participants, found that there was no significant difference in inflammation markers, oxidative stress, or cortisol levels between participants who received chia and the control group in the studies they analyzed, with significant clinical effects generally being observed in only one of fourteen studies (Teoh et al, 2020).

32 Considering these factors, it is possible that chia, while possessing nutrients and polyphonic compounds that are beneficial to health, simply cannot convey these benefits in significant enough amounts to affect human health, due to the bioavailability of these compounds and the limited dietary tolerance. Considering this, further studies observing the effect of chia seed consumption on human health should focus on the form of chia consumption and other ways to improve the bioavailability of relevant nutrients. If, accounting for these factors, chia fails to provide significant health benefits to the human population, it may be better relegated to animal diets for the improvement of their health instead.

However, other studies suggest chia can have a beneficial effect on dietary nutrient composition. For example, oil extracted from chia seeds, with a 3:1 ratio of omega-3 to omega-6 fatty acids, was found to significantly decrease adipocyte hypertrophy and foam cell development, indicating successful integration of chia-derived omega-3 fatty acids into the diet and human metabolism. The study also found that inflammation proteins, such as PAI-1, MCP-1, PGE2, TLR-4, and MF-kB were decreased with the use of chia seed oil, as compared to the control (Pandurangan et al, 2020).

Research also indicates that, in the case where whole seeds provide low nutrient bioavailability, further processing can increase its effects. For example, flaxseed fed to chickens has been known to result in low energy utilization and resultant poor growth despite being a rich source of protein, oil and omega-3 fatty acids, due in part to the indigestible increasing the viscosity of chyme in the gastrointestinal tract that decreases the absorption of inherent nutrients. Because of this, study observed the effect that viscosity-reducing enzymes would have on nutrient bioavailability and found that

33 enzyme supplements served to increase fat digestibility. In addition, the study found that digestibility of fat increased with grinding of the flaxseed, compared to the consumption of the whole seed (Jia et al, 2009). Other studies have found that flaxseed oil can double the deposition of omega-3 fatty acids in chicken egg yolk compared to milled flaxseed supplementation, indicating that components in the seed may impede the absorption of nutrients, such as resulting in protein- complexes that bind to relevant nutrients (Ehr et al, 2017).

Considering this, it is possible that the lack of significant difference in the present study was due to reduced bioavailability of the tryptophan, omega-3 fatty acids, and antioxidants in chia, rather than the effect of these nutrients themselves. In this case, future research could benefit from observing the effects of chia on depressive symptoms and sleep quality when presented as whole seeds, milled seeds, and oil extract.

Limitations

While chia has a significant concentration of tryptophan, further research indicates that chia, while having many nutritional benefits, is not a good dietary source of protein due to the low bioavailability. While the bioavailability of protein in chia can be improved through grinding, this study was conducted through the consumption of whole chia seeds, thus limiting the effect that tryptophan would have on the system. Because of this, though there could have been potential effects from the absorption of omega-3 fatty acids, tryptophan likely played a smaller role in this experiment than predicted.

Furthermore, while the current study did not exceed the current recommended limit of 48 g/day, it should still be noted that chia is a food high in fiber. Since a sudden introduction of fiber to the diet can cause unfavorable gastrointestinal symptoms such as bloating,

34 nausea, increased flatulence, or eructation, it is possible that any improvements in sleep disruptions that occurred may have been masked by the subsequent gastrointestinal discomfort, resulting in no net change in sleep quality (National Research Council, 1989).

Another limitation is that, as a result of the corrupted data for the serotonin concentration curve from the ELISA reading, an accurate understanding of the participant’s urinary serotonin concentration could not be obtained. While there is still information to be drawn from the data, with increased optical density correlating with lower serotonin concentration, further analysis was not possible at this time.

Furthermore, there were limitation in data collection for the PSQI. Due to the unclear formatting of some of the questions, many of the forms were not fully completed, thus limiting the testing that could be done with this data, especially in the control group.

In humans, fluid regulation is managed largely through urine excretion and drinking as mediated by the sensation of thirst, signaled by cell shrinkage. For healthy people living in temperate climates, thirst is generally not a factor in day-to-day life.

Rather, people fitting these criteria tend to take fluids in as parts of everyday food (such as soup or milk), in beverages used as mild stimulants (tea or coffee) or simply for pleasure. Mild levels of dehydration have been found to disrupt mood and cognitive functioning, altering concentration, alertness, and short-term memory in young adults.

However, these results are not consistent. In some cases, cognitive performance appears not to be significantly affected in ranges from 2 – 2.6% dehydration, while other studies found dehydration to impair visual perception, short-term memory, and psychomotor ability. Dehydration may result in a mild impairment on cognitive performance, but the most consistent effect of mild dehydration was found to be significant increases in

35 subjective mood scores for fatigue, confusion, anger, and vigor. While little is currently known on the effect of mild dehydration on mental performance, it is possible that, as a psychological stressor, it may compete with other cognitive processes in the brain and reduce cognitive performance or mood as a result (Water, Hydration, and Health).

Because of this, it is possible that, in some cases, participants who were mildly dehydrated would been affected by the study regardless of chia consumption, to the requirement that they consume 10 oz water 30 minutes before each meal.

Finally, it is possible that the texture of the chia, which has a mucilage coating that gels upon contact with water, provided an unfavorable texture to some participants.

Because an aversion to gelatinous food texture was not screened for, it is possible that an aversion might have affected results.

36 CHAPTER 6: CONCLUSION

Depression and disruptions to the sleep cycle prove to be an ever-growing problem in current society that remains complex and challenging to treat. Currently, due to the lack of knowledge on how serotonin and melatonin affect mood, treatments for depression are unrefined, simply targeting serotonin and hoping that it works as desired rather than addressing the problem more specifically. Because of this, SSRIs and other serotonin related treatments show mixed results in the literature. While the exact role of serotonin and melatonin in depression and sleep quality remains unclear, foods that are rich in tryptophan are known to promote both improved mood and sleep quality, due to its role in the synthesis of both serotonin and melatonin. Furthermore, omega-3 fatty acids are also found to contribute to healthy brain function for humans, with low concentrations of DHA, EPA, and omega-3 PUFAs correlating with depression symptoms (Sublette et al., 2013), while antioxidant compounds can help reduce neuroinflammation and increase the efficacy of antidepressant treatment. Because it is a valuable source of tryptophan, omega-3 fatty acids, and antioxidants, chia has great potential to assist in the alleviation of depressive symptoms and sleep cycle disruptions.

Implications of the Study

Chia consumption was not found to be associated with improved mood or sleep quality, though it was associated with a slightly higher urinary serotonin level in the middle of the study. However, greater decreases in depressive symptoms and increase in sleep quality were both observed for the control than for the chia consumption groups, indicating that, more than chia consumption, proper hydration may be pertinent to improving mood and sleep quality.

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43 APPENDICES

Appendix A. MDI Form

Major Depression Inventory Questionaire (0-50 scale, rated as no depression (0-

19), mild (20-24), moderate (25-29), or severe (>30)

44 Appendix B. PSQI Form

45 Appendix C. Raw Data – MDI Full

Participant answers to MDI for Week 0 (PRE) survey for control (CTR) and treatment (TRT) groups

PRE CTR CTR TRT CTR TRT TRT TRT CTR CTR TRT CTR TRT TRT 1 1 1 2 1 1 3 1 1 1 1 1 3 2 2 0 1 2 0 2 3 0 0 1 1 0 2 0 3 2 2 3 0 2 4 0 1 1 2 1 2 0 4 1 1 4 1 2 1 1 0 1 2 1 2 1

46 5 1 0 1 0 1 0 1 1 0 1 0 2 0 6 0 0 0 0 0 1 0 0 0 0 0 1 0 7 0 1 1 0 1 2 0 2 1 3 3 4 0 8 1 1 1 0 2 0 0 0 1 3 0 4 0 9 1 1 4 0 1 1 0 0 1 2 1 4 0 10 0 2 0 1 1 0 0 1 0 1 1 0 0 11 0 0 0 0 0 0 1 2 0 1 3 0 0 12 0 2 4 0 0 3 0 2 0 1 1 0 0 MDI Total PRE 6 12 22 3 13 18 4 10 7 18 12 24 3 Participant answers to MDI for Week 4 (MID) survey for control (CTR) and treatment (TRT) groups

MID CTR CTR TRT CTR TRT TRT TRT CTR CTR TRT CTR TRT TRT 1 1 1 2 0 2 4 0 1 1 1 0 4 1 2 1 0 2 0 1 3 0 1 1 1 0 4 0 3 1 1 1 1 3 2 0 0 1 2 2 4 0 4 2 0 1 0 4 4 0 1 0 2 0 0 0 5 2 0 2 0 1 4 0 0 0 0 0 0 0 47 6 0 0 0 0 0 0 0 0 0 0 0 0 0 7 1 0 2 0 1 1 1 2 1 3 0 4 0 8 1 0 3 0 2 2 0 1 0 3 0 0 0 9 1 0 2 0 1 1 0 0 1 3 1 4 0 10 1 0 0 1 3 0 0 1 0 2 0 0 0 11 0 0 0 0 0 1 2 0 1 2 0 0 0 12 0 0 3 0 1 0 0 0 0 1 1 0 0 MDI Total MID 10 2 18 2 19 22 3 7 6 20 4 20 1 Participant answers to MDI for Week 8 (PST) survey for control (CTR) and treatment (TRT) groups

PST CTR CTR TRT CTR TRT TRT TRT CTR CTR TRT CTR TRT TRT 1 1 0 3 0 1 1 1 1 1 3 0 1 0 2 1 1 3 0 1 0 0 0 1 3 0 1 0 3 1 1 2 0 1 4 0 1 0 3 1 1 0 4 0 0 2 0 2 0 0 0 0 3 1 0 0 5 1 0 3 0 1 1 0 0 0 0 1 0 0

48 6 0 0 0 0 0 0 0 0 0 0 0 0 0 7 1 1 3 0 0 3 1 1 1 4 1 0 0 8 1 0 1 0 1 3 0 1 1 4 1 2 0 9 0 0 1 0 0 2 0 0 0 4 0 0 0 10 1 0 1 1 1 0 0 0 0 3 0 0 0 11 1 1 2 0 0 1 3 0 0 4 0 0 0 12 1 0 1 0 2 0 0 0 1 2 1 0 0 MDI Total PST 9 4 22 1 10 15 5 4 5 33 6 5 0 Pre - 0.816 0.816 2.000 1.134 0.736 0.743 1.732 1.732 1.000 1.732 2.000 1.000 1.000 0.000 1.000 0.414 0.000 1.000 0.046 0.257 0.461 0.458 0.083 0.083 0.317 0.083 0.046 0.000 ------Pst Mid - 0.577 0.577 1.414 1.000 1.000 1.000 0.816 0.577 1.732 1.000 0.317 0.317 0.564 0.157 0.317 0.317 0.000 1.000 0.317 0.414 0.000 1.000 0.000 1.000 0.564 0.000 1.000 0.083 ------Pst Pre - 1.069 1.069 1.414 1.414 1.414 0.577 0.447 0.816 0.816 1.000 0.816 0.414 0.414 0.285 0.157 0.157 0.157 0.564 0.655 0.414 0.000 1.000 0.000 1.000 0.414 0.000 1.000 0.317 ------Mid MDI - Z Z Z Z Z Z Z Z Z Z Z Z tailed) tailed) tailed) tailed) tailed) tailed) tailed) tailed) tailed) tailed) tailed) tailed) tailed) tailed) ------(2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig (2 Asymp. Sig Asymp. Sig (2 (within group (within analysis) Water only 8 9 3 4 5 6 7 1 2 10 11 12 . Wilcoxon Analysis . Wilcoxon D Appendix Appendix 49 Chia Mid-Pre Pst-Mid Pst-Pre Z -0.447 -0.690 -0.849 1 Asymp. Sig (2-tailed) 0.665 0.490 0.396 Z -0.447 -0.736 -0.412 2 Asymp. Sig (2-tailed) 0.655 0.461 0.680 Z -0.378 -0.272 -1.000 3 Asymp. Sig (2-tailed) 0.705 0.785 0.317 Z -0.317 -0.736 -1.730 4 Asymp. Sig (2-tailed) 0.751 0.461 0.084 Z -0.137 -0.447 -0.276 5 Asymp. Sig (2-tailed) 0.891 0.655 0.783 Z 0.000 0.000 0.000

50 6 Asymp. Sig (2-tailed) 1.000 1.000 1.000 Z -0.577 -0.137 -0.431 7 Asymp. Sig (2-tailed) 0.564 0.891 0.666 Z 0.000 -0.276 -0.184 8 Asymp. Sig (2-tailed) 1.000 0.783 0.854 Z -0.447 -0.707 -0.813 9 Asymp. Sig (2-tailed) 0.655 0.480 0.416 Z -1.342 0.000 -1.342 10 Asymp. Sig (2-tailed) 0.180 1.000 0.180 Z -1.732 -1.633 -1.841 11 Asymp. Sig (2-tailed) 0.083 0.102 0.066 Z -0.816 0.000 -0.736 12 Asymp. Sig (2-tailed) 0.414 1.000 0.461 6 5 8 3 7 5 6 8 1 14 PRE PSQITOTAL 1 1 1 3 1 2 1 1 2 0 9 2 1 0 1 0 2 3 2 3 0 8 0 1 0 2 0 0 0 1 3 0 7 0 0 0 0 0 0 0 0 0 0 6 0 1 0 3 0 0 0 0 0 0 5j 0 0 0 0 0 0 0 0 0 0 5i 1 2 0 0 0 0 0 0 0 0 5h 1 1 2 2 0 0 2 2 0 0 5g 3 0 0 0 0 1 1 3 0 0 5f 0 0 0 1 0 0 2 0 0 0 5e 0 0 0 0 0 0 0 0 0 0 5d 0 2 3 3 1 0 0 1 1 0 5c 2 3 3 2 1 1 2 3 1 0 5b questionnaires for control (CTR) and treatment (TRT) groups and (CTR) for control treatment questionnaires 1 0 1 0 0 2 1 1 1 1 5a PSQI 8 7 5 8 9 9 4 10 6.5 6.5 4b 7.67 7.67 7 7 5 7 7 5 4 6.5 6.5 7.5 7.5 4a 7.67 7.67 Week 0 (PRE) 0 (PRE) Week 3 7:15 7:15 0:00 0:00 6:00 6:00 8:00 9:00 7:45 7:00 7:30 7:30 11:00 11:00 10 10 10 30 15 20 20 2 7.5 7.5 7.5 12.5 12.5 11 12 1 2:00 2:00 1:00 1:00 1:30 12:00 12:00 10:15 10:15 11:00 10:00 10:00 PRE CTR TRT CTR TRT TRT TRT CTR TRT TRT TRT Participant responses for Participant Appendix E.Appendix Raw Full Data, PSQI 51 5 7 8 2 5 1 9 7 2 5 PSQITOTAL MID 1 1 1 1 1 0 1 1 1 1 9 1 0 0 0 2 2 2 3 0 1 8 2 0 1 0 0 0 0 3 0 0 7 0 0 0 0 0 0 0 0 0 0 6 1 0 0 0 0 0 0 0 0 0 5j 0 0 0 1 0 0 1 0 0 0 5i 0 0 1 0 0 0 0 0 1 0 5h 2 0 2 0 0 0 2 0 0 0 5g 2 0 0 0 2 1 2 3 0 0 5f 0 0 0 1 0 0 0 0 0 0 5e 0 0 0 0 0 0 0 0 0 0 5d 0 0 3 2 3 0 0 1 1 0 5c 0 0 3 0 2 3 0 2 0 0 5b 0 0 3 0 0 1 1 0 0 0 5a 9 8 9 9 9 6 12 5.2 5.2 6.5 4b 7.25 7.25 5 7 9 5 5 6.5 6.5 1.5 1.5 7.5 7.5 6.5 4a 7.25 7.25 3 7:30 7:30 7:00 7:00 8:00 9:00 8:15 7:00 6:00 8:30 8:30 6:45 6:00 5 5 10 10 30 10 15 12 25 2 12.5 12.5 12 1 1:00 1:00 2:00 12:00 12:00 12:00 12:00 12:00 10:30 10:00 10:30 10:30 10:20 MID CTR TRT TRT CTR CTR TRT TRT TRT TRT TRT Participant responses for Week 4 (MID) PSQI questionnaires for control (CTR) and treatment (TRT) groups and 4 (MID) (CTR) for control responses treatment for PSQIWeek Participant questionnaires

52 2 8 1 3 8 5 8 4 1 4 PST PSQITOTAL 0 2 0 1 2 1 1 0 0 1 9 0 2 0 0 2 0 2 3 0 0 8 1 2 0 0 0 1 1 3 0 0 7 0 0 0 0 1 0 0 0 0 0 6 0 0 0 0 0 2 0 0 0 0 5j 0 0 0 0 0 0 0 0 0 0 5i 1 0 0 0 0 0 0 1 1 0 5h 1 2 0 2 0 0 1 0 0 0 5g 1 0 0 0 0 0 1 3 0 0 5f 0 0 0 0 0 0 0 0 1 0 5e 0 0 1 0 0 0 1 0 0 0 5d 0 1 3 0 3 0 1 1 1 0 5c 1 2 3 0 1 1 1 2 1 0 5b 1 0 2 0 0 1 0 0 0 0 5a 9 5 8 9 3 9 10 10 10 7.5 7.5 4b 7 5 7 9 7 9 3 8 7 7.5 7.5 4a 8 3 8:00 8:00 7:00 7:00 8:00 9:00 8:00 7:00 6:00 6:00 6:00 6:00 5 5 15 10 30 15 10 15 10 2 12.5 12.5 1 1:00 1:00 4:00 4:00 9:00 10:00 10:00 12:00 12:00 11:00 12:30 12:30 12:00 10:20 ST P CTR TRT TRT CTR CTR TRT TRT TRT TRT TRT Participant responses for Week 8 (PST) PSQI questionnaires for control (CTR) and treatment (TRT) groups 8 (PST) PSQI responses and for control (CTR) for treatment Week questionnaires Participant

53 Appendix F. ELISA Scores

Participant readings from ELISA urinary sample analysis, with readings done

twice per sample and averaged (CTR – control, TRT – chia treatment)

PRE AVG MID AVG POST AVG

CTR 0.4795 0.5465 0.384

TRT 0.504 0.4225 0.336

CTR 0.3875 0.473 0.6135

CTR 0.3125 0.5535 0.543

TRT 0.343 0.4885 0.4755

CTR 0.4045 0.4735 0.385

CTR 0.402 0.5 0.4355

TRT 0.5035 1.561 0.4805

CTR 0.515 0.384 0.371

CTR 0.43 0.4935 0.337

TRT 0.3135 0.461 0.5155

CTR 0.4825 0.612 0.782

TRT 0.3845 0.5245 0.3935

TRT 0.444 1.101 0.4605

54 Appendix G. ELISA Standards

Concentration of standards derived from Serotonin Ultrasensitive ELISA Assay Kit for development of serotonin concentration standard curve Serotonin Optical Density Concentration (nm) (pg/sample) Standard 1 1.685 0 Standard 2 1.516 0.67 Standard 3 1.583 2 Standard 4 1.819 6.7 Standard 5 0.89 20 Standard 6 0.679 100 Control 1 1.428 100 Control 2 1.582 100

ELISA Serotonin Standard Curve 1.2 1 0.8 0.6 0.4 0.2

Optical Density (450 nm) (450 Density Optical 0 0 20 40 60 80 100 120 Serotonin Concentration (pg/sample)

Figure 4. ELISA Serotonin Standard Curve Serotonin concentration is calculated by plotting standard concentrations (x-axis)

against OD/ODmax (at 0 pg serotonin, y-axis), as indicated in the Serotonin

Ultrasensitive ELISA Assay Kit manual.

55