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Journal of Functional Foods 85 (2021) 104671
Available online 5 August 2021
1756-4646/Crown Copyright © 2021 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Effects of a Bacopa monnieri extract (Bacognize®) on stress, fatigue, quality
of life and sleep in adults with self-reported poor sleep: A randomised,
double-blind, placebo-controlled study
Adrian L. Lopresti
a
,
b
,
*
, Stephen J Smith
a
,
b
, Sinan Ali
c
, Alexandra P. Metse
d
, John Kalns
e
,
Peter D. Drummond
b
a
Clinical Research Australia, Perth, Western Australia 6023, Australia
b
Healthy Ageing Research Centre, Murdoch University, Perth, Western Australia 6150, Australia
c
Salpath Functional Pathology, Sydney, New South Wales 2016, Australia
d
School of Health and Behavioural Sciences, University of the Sunshine Coast, Sippy Downs, Queensland 4556, Australia
e
Hyperion Biotechnology, San Antonio, Texas 78216, USA
ARTICLE INFO
Keywords:
Bacopa monnieri
Sleep
Stress
Cortisol
Herbal
Clinical trial
ABSTRACT
In this 28-day, randomised, double-blind, placebo-controlled trial, 100 adults with self-reported poor sleep
received either a placebo or a standardised Bacopa monnieri extract (150 mg twice daily). Outcome measures
included the Bergen Insomnia Scale (primary outcome measure), Functional Outcomes of Sleep Questionnaire,
Pittsburgh Sleep Diary, Short Form-36 Health Survey, and the Depression, Anxiety, and Stress Scale. Changes in
salivary concentrations of cortisol, dehydroepiandrosterone sulfate, immunoglobulin A (sIgA),
α
-amylase (sAA),
C-reactive protein, melatonin, and the fatigue biomarker index were also assessed. Based on the Bergen Insomnia
Scale, Bacopa monnieri did not improve sleep patterns more than the placebo; however, it was associated with
greater improvements in emotional wellbeing, general health, and pain-related symptoms. Bacopa monnieri was
also associated with greater reductions in sIgA and sAA compared to the placebo. Future clinical trials using
varying doses, treatment periods, and objective outcome measures will be important to validate these ndings.
1. Introduction
Common treatments for insomnia include pharmacological in-
terventions, and behavioural and psychological interventions such as
cognitive behavioural therapy for insomnia, stimulus control, sleep re-
striction therapy, relaxation training, and sleep hygiene recommenda-
tions (Edinger et al., 2021; Palagini et al., 2020). However, despite their
efcacy, a signicant portion of individuals obtain no benet or
continue to experience residual insomnia-related symptoms (Davidson,
Dickson, & Han, 2019). Pharmacological treatments for insomnia
include controlled-release melatonin, tricyclic antidepressants, benzo-
diazepines, antihistamines, antiepileptics, and atypical antipsychotics
(Matheson & Hainer, 2017). These medications have moderate efcacy
but are associated with several adverse effects including sedation,
weight gain, dizziness, headaches, and gastrointestinal complaints
(Fitzgerald & Vietri, 2015; Lie, Tu, Shen, & Wong, 2015). Many sleep-
promoting medications, particularly some benzodiazepines, are also
associated with problematic withdrawal effects including rebound
insomnia (Hintze & Edinger, 2018).
The pathophysiology of insomnia is not fully understood but is
believed to be multifaceted. These include genetic variations and dis-
turbances in the hypothalamus-pituitary-adrenal (HPA) axis activity,
melatonergic activity, neurotransmitter action (e.g., gamma-
aminobutyric acid, noradrenaline, and serotonin), and neural circuitry
Abbreviations: BIS, Bergen Insomnia Scale; BMI, body mass index; CAR, cortisol awakening response; COX-2, cyclooxygenase-2; CRP, C-reactive protein; DASS-21,
Depression, Anxiety, and Stress Scale-21; DHEA-S, dehydroepiandrosterone sulfate; ELISA, enzyme-linked immune sorbent assays; FBI, Fatigue biomarker index;
FOSQ-10, Functional Outcomes of Sleep Questionnaire; HPA, hypothalamus-pituitaryadrenal; HRP, horse radish peroxidase; IL, interleukin; MOA, monoamine
oxidase; OD, optical density; OF, oral uid; PSD, Pittsburgh Sleep Diary; sAA, salivary
α
-amylase; SF-36, Short Form-36 Health Survey; sIgA, salivary immunoglobulin
A; TMB, tetramethyl benzidine; TNF-
α
, Tumor necrosis factor-
α
.
* Corresponding author at: A: 38 Arnisdale Rd Duncraig, WA 6023, Australia.
E-mail address: adrian@clinicalresearch.com.au (A.L. Lopresti).
Contents lists available at ScienceDirect
Journal of Functional Foods
journal homepage: www.elsevier.com/locate/jff
https://doi.org/10.1016/j.jff.2021.104671
Received 24 March 2021; Received in revised form 1 July 2021; Accepted 2 August 2021
Journal of Functional Foods 85 (2021) 104671
2
(Levenson, Kay, & Buysse, 2015; Pigeon & Cribbet, 2012). There is also
increasing research to suggest a bi-directional relationship between
inammation, oxidative stress, and sleep quality (Gulec et al., 2012;
Villafuerte et al., 2015). For example, in a representative sample of 319
Swedish women, higher C-reactive protein (CRP) concentrations were
positively associated with self-reported sleep disturbances, specically
sleep maintenance and early morning awakenings (Ghilotti et al., 2021).
In a study on adults with primary insomnia, the mean blood concen-
tration of the antioxidant enzyme glutathione peroxidase was lower, and
concentrations of malondialdehyde, a marker of lipid peroxidation,
were higher compared to matched healthy volunteers (Gulec et al.,
2012).
Plants, herbs, spices, and their extracts (henceforth referred to as
herbs) contain multiple constituents that have been demonstrated in in
vitro, animal, and human trials to have anti-inammatory, antioxidant,
adaptogenic, analgesic, and neuroprotective effects (Liu et al., 2013;
Wink, 2015). Bacopa monnieri (also known as Brahmi, water hyssop, and
Herpestis monniera) is a creeping perennial plant that has been shown to
have anticonvulsant, antidepressant, analgesic, anti-inammatory,
anxiolytic, adaptogenic, and neuroprotective effects (Aguiar & Bor-
owski, 2013; P. S. Saha et al., 2020; Sukumaran, Amalraj, & Gopi, 2019).
Bacopa monnieri also has an important role in cellular homeostasis by
modulating apoptosis through autophagy (Das et al., 2016; S. Saha et al.,
2020; Smith et al., 2018). As a functional food, Bacopa monnieri is often
referred to as a nootropic agent that improves memory and mental
acuity (Brimson et al., 2021; Sukumaran et al., 2019). In India, it has
been used as a component in drinks, biscuits, syrups, jellies, and
breakfast cereals (Devendra et al., 2018). The main bioactive constitu-
ents of Bacopa monnieri believed to be associated with its cognitive
effects are saponins called bacosides, with bacosides A and B the most
studied constituents (Sukumaran et al., 2019). Specically relating to
the pathophysiological processes associated with sleep disturbances,
Bacopa monnieri has been shown to inuence HPA-axis activity (S.
Kumar & Mondal, 2016; Zu et al., 2017), neurotransmitter concentra-
tions of dopamine and serotonin (Rauf et al., 2012; Sheikh et al., 2007),
and antioxidant and inammatory activity (Nemetchek, Stierle, Stierle,
& Lurie, 2017; Stough, Singh, & Zangara, 2015). In experimental
studies, bacoside A inhibited inammatory cytokine production
(Madhu, T, & S, 2019), reduced free radical damage in the liver and
brain (Sekhar, Viswanathan, & Baby, 2019), inhibited inammatory
cytokine production in the brain (Nemetchek et al., 2017), and inhibited
beta-amyloid cytotoxicity (Malishev et al., 2017). Due to the association
between inammation, oxidative stress, HPA-axis activity, and neuro-
transmitter concentrations, Bacopa monnieri has promise as a sleep-
promoting and mood-enhancing agent. In human trials, Bacopa mon-
nieri has been shown to have positive cognitive (Abdul Manap et al.,
2019; Hingorani, Patel, & Ebersole, 2012; N. Kumar et al., 2016; Stough
et al., 2008) and anxiolytic (Benson et al., 2014; Calabrese et al., 2008)
effects; however, there have been no trials examining its effect on sleep.
The aim of this human trial was to examine the effects of a Bacopa
monnieri extract (Bacognize®) on sleep, quality of life, and fatigue in
adults with self-reported poor sleep. Changes in several salivary hor-
mones associated with stress, sleep, fatigue, and inammation were also
evaluated to help elucidate the potential mechanisms of action associ-
ated with Bacopa monnieri supplementation.
Fig. 1. Systematic Illustration of Study Design. BIS =Bergen Insomnia Scale; DASS-21 =Depression, Anxiety, Stress Scale −21; FOSQ-10 =Functional Outcomes of
Sleep Questionnaire; PSD =Pittsburgh Sleep Diary; SF-36 =Short Form-36.
A.L. Lopresti et al.
Journal of Functional Foods 85 (2021) 104671
3
2. Materials and methods
2.1. Study design
This was a two-arm, parallel-group, 28-day, randomised, double-
blind, placebo-controlled trial (Fig. 1). The trial protocol was
approved by the Human Research Ethics Committee at the National
Institute of Integrative Medicine (approval number 0066E_2020) and
was prospectively registered with the Australian New Zealand Clinical
Trials Registry (Trial ID. ACTRN12620000770965). Based on a single
outcome variable, an a priori power analysis was undertaken to estimate
the required sample size. Even though there has been no study exam-
ining the effects of Bacopa monnieri on sleep quality, an effect size of 0.6
was predicted based on a previous trial examining the sleep-enhancing
effects of a herbal ingredient (Lopresti, Smith, Metse, & Drummond,
2020). Assuming a power of 80% and a type one error rate (alpha) of
5%, the number of participants required per group to nd an effect on
the Bergen Insomnia Scale (BIS) was estimated as 36. After allowing for
an approximate 20% drop out rate, we aimed to recruit 50 participants
per group.
2.2. Recruitment and randomisation
Participants were recruited across Perth, Western Australia through
social media advertisements between July and August 2020. Interested
participants were directed to a website page that provided information
about the trial and a link to complete an online screening questionnaire.
This questionnaire screened for mental health symptoms, medication
use, history of medical or psychiatric disorders, alcohol, nicotine, and
other drug use, supplement and vitamin intake, and pregnancy/breast-
feeding status. If assessed as likely eligible, volunteers participated in a
telephone interview comprising a structured series of questions to
further clarify details pertaining to the eligibility criteria and to obtain
further demographic details. Suitable participants were then required to
complete online versions of the Bergen Insomnia Scale (BIS), Depres-
sion, Anxiety, and Stress Scale (DASS-21), and an informed consent
form. Eligible and consenting participants were randomly allocated to
one of two groups (Bacopa monnieri or placebo) using a randomisation
calculator (http://www.randomization.com). The randomisation
calculator ensured sequence concealment. The randomisation structure
comprised 10 randomly permuted blocks, containing 10 participants per
block. The participant identication number was assigned based on the
order of participant enrolment in the study. All capsules were packed in
identical bottles labelled by two intervention codes (held by the study
sponsor until nal data collection). Participants and study investigators
were blind to treatment group allocation until all outcome data were
collected. No nancial compensation was provided to participants for
volunteering in this study, although at the end of the study, participants
allocated to the placebo condition were offered a free 4-week supply of
Bacopa monnieri capsules.
2.3. Participants
Inclusion criteria: physically-healthy, male and female participants
aged 18 to 70 years, with a score of 3 or more on at least one of the rst
four questions on the BIS and a score of 3 or more on at least one of the
last two questions on the BIS were recruited for this trial. All participants
were medication-free for at least 4 weeks apart from the contraceptive
pill and no more than once per week use of pain-relieving medications.
Volunteers had a body mass index (BMI) between 20 and 30 and a usual
bedtime between 9 pm and 12 am. Participants were uent in English
and consented (via an online consent form) to all pertinent aspects of the
trial.
Exclusion criteria: Participants were ineligible to participate in the
study if they were employed in night shift work or rotational shift work.
People experiencing a sleep disorder other than moderate insomnia (e.g,
sleep apnoea, periodic limb movement disorder, restless legs syndrome),
persistent, severe sleep disturbance greater than 1 year, diagnosis of a
mental health disorder (other than mild depressive or anxiety symptoms
as measured by the Depression, Anxiety and Stress Scale-21), coffee
intake greater than 3 cups per day (or equivalent caffeine intake from
other caffeinated drinks e.g., tea, energy drinks), and alcohol con-
sumption greater than 14 standard drinks per week were also ineligible
for the study. Participants were also ineligible for the study if they re-
ported experiencing external factors that may affect their sleep patterns
(e.g., infant/children regularly awakening, excessive noise, snoring
partner); were receiving non-pharmacological treatment for sleep dis-
orders (e.g., cognitive behavioural therapy, relaxation therapy); had a
current or 12-month history of illicit drug use; were taking supplements
that may affect sleep; were taking Bacopa monnieri supplements; or had a
diagnosed medical condition including but not limited to: diabetes,
hyper/hypotension, cardiovascular disease, a gastrointestinal disease
requiring regular use of medications, gallbladder disease/gallstones/
biliary disease, endocrine disease, psychiatric disorder, neurological
disease (Parkinson’s, Alzheimer’s disease, intracranial haemorrhage,
head or brain injury), or acute or chronic pain affecting sleep. Pregnant
women, women who were breastfeeding, or women who intended to fall
pregnant were also ineligible to participate in the study.
2.4. Interventions
Placebo and Bacopa monnieri capsules were identical in appearance,
being matched for colour coating, shape, and size. The active treatment,
supplied by Verdure Sciences Inc. (Noblesville, Indiana, USA), contained
150 mg of a standardised Bacopa monnieri extract (Bacognize®). This
dose was chosen as previous studies using this identical extract and dose
have demonstrated cognitive-enhancing effects in both younger and
older adults (Hingorani et al., 2012; N. Kumar et al., 2016; Stough et al.,
2008). Bacognize® is a standardised hydroalcoholic extract of the whole
herb Bacopa monnieri (L.) Wettst. It is standardised by the United States
Pharmacopeial Convention method (using High-performance liquid
chromatography) to 12% total Bacopa glycosides (Bacopaside I, Baco-
side A3, Bacopaside II, Jujubogenin isomer of Bacopasaponin C, Baco-
pasaponin C). Each capsule contained 150 mg of Bacognize® extract.
The placebo capsules (microcrystalline cellulose powder) contained the
same excipients as the active capsules. All participants were instructed
to take one capsule, twice daily (morning and evening), with or without
food for 27 days. Medication adherence was measured by participants
providing a capsule count every 7 days. Efcacy of participant treatment
blinding was examined by asking participants to predict group alloca-
tion (placebo, bacopa, or uncertain) at the completion of the study.
Bacopa monnieri and placebo capsules were mailed to participants with
directions for use provided on capsule bottles. Participants were also
provided with an information sheet about capsule intake and what to do
if they missed a dose. This information was also verbally conveyed to
participants during their initial telephone interview.
2.5. Outcome measures
2.5.1. Primary outcome measure:
Bergen Insomnia Scale (BIS): The BIS is a validated six-item ques-
tionnaire that assesses difculties with sleep initiation, sleep mainte-
nance, early morning awakening, nonrestorative sleep, daytime
impairment, and satisfaction with sleep. The BIS correlates highly with
other validated sleep questionnaires such as the Pittsburgh Sleep Quality
Index (Pallesen et al., 2008). For each question, respondents indicate
how many days per week (0 to 7 days) they experienced each sleep-
related problem. A total composite score is calculated by adding
together the scores for each item, yielding a total score with a possible
range of 0 to 42. The BIS was completed at baseline (from days −5 to
−3), day 7, 14, 21, and 27.
A.L. Lopresti et al.
Journal of Functional Foods 85 (2021) 104671
4
2.5.2. Secondary outcome Measures: Questionnaires and diaries
Functional Outcomes of Sleep Questionnaire (FOSQ-10): The FOSQ-10
comprises 10 questions evaluating the respondent’s quality of life as it
relates to disorders of excessive sleepiness. A total score is calculated
plus subscale scores for the ve domains of day-to-day life comprising
(1) general productivity, (2) activity levels, (3) vigilance, (4) social
outcomes, and (5) intimacy and sexual relationships. The FOSQ-10 has
good psychometric properties and similar reliability and validity to the
longer version of the original FOSQ (Chasens, Ratcliffe, & Weaver,
2009). The FOSQ-10 was completed on day −2, 7, 14, 21, and 27.
Pittsburgh Sleep Diary (PSD): the PSD is a 14-item sleep diary that
respondents complete upon awakening. The PSD shows good retest
reliability over a mean inter-test interval of 22 months. Scores also
correlate with circadian type, subjective sleep quality, and objective
actigraphy measurements (Monk et al., 1994). Scores are calculated for
total sleep time (hours), sleep latency (minutes), number of awakenings
after sleep onset, sleep quality rating [5-point Likert rating ranging from
very bad (1) to very good (5)], mood rating at nal awakening [5-point
Likert rating ranging from very calm (1) to very tense (5)], and alertness
rating at nal awakening [5-point Likert rating ranging from very sleepy
(1) to very alert (5)]. The PSD was completed on day −2, day −1, 3, 7,
14, 21, 27 and 28.
Short Form-36 Health Survey (SF-36): The SF-36 is a self-report
measure assessing quality of life. It consists of eight scaled scores
measuring (1) vitality, (2) physical functioning, (3) bodily pain, (4)
general health perceptions, (5) physical role functioning, (6) emotional
role functioning, (7) social role functioning, and (8) emotional well-
being. The SF-36 is a commonly-used outcome measure of quality of life
with strong psychometric properties (McHorney, Ware, & Raczek, 1993;
Ware & Sherbourne, 1992). Scoring for the SF-36 was based on the al-
gorithm developed by RAND Health Care where high scores indicate a
more favourable health state (Hays, Sherbourne, & Mazel, 1993). Based
on previous factor analyses, the SF-36 has been identied as having two
factors: a mental component consisting of the social functioning,
emotional wellbeing, role limitations due to emotional problems, and
vitality subscale scores; and a physical component consisting of the
physical function, general health, pain, and role limitations due to
physical health subscale scores (Ware et al., 1995). The SF-36 was
completed on days −1 and day 27.
Depression, Anxiety, and Stress Scale – 21 (DASS-21): The DASS-21 is a
validated self-report measure assessing symptoms of stress, anxiety, and
depression (Brown, Chorpita, Korotitsch, & Barlow, 1997). Twenty-one
questions are rated on a 4-point scale (0–3), ranging from never to
almost always (lower scores indicate a reduction in symptoms). Subscale
scores for depression, anxiety, and stress are calculated. The DASS-21
was completed at baseline (days −5 to −3) and on day 27.
2.5.3. Secondary outcome Measures: Salivary hormones
Salivary cortisol (morning and evening). Cortisol provides a measure of
HPA axis activity. Concentrations are altered during times of stress, and
differences (increased and decreased concentrations) have been
observed in adults with insomnia compared to adults with healthy sleep
(Nicolaides, Vgontzas, Kritikou, & Chrousos, 2000).
Dehydroepiandrosterone sulfate (DHEA-S). DHEA-S is an endogenous
steroid that is produced by the adrenal cortex. Concentrations of DHEA-
S are altered during times of stress (Walker, Pngst, Carnevali, Sgoifo, &
Nalivaiko, 2017) and are higher in adults with post-traumatic stress
disorder and trauma-exposed adults (van Zuiden et al., 2017).
Salivary immunoglobulin A (sIgA). sIgA has important immunological
functions and is altered after exposure to various psychosocial stressors
(Brandtzaeg, 2013; Tsujita & Morimoto, 1999; Valdimarsdottir & Stone,
1997).
Salivary
α
-amylase (sAA). sAA is a marker of stress and autonomic
nervous system activity. Concentrations are lowered after mind–body
interventions such as stress-reduction programs, self-compassion
training, and mindfulness-based interventions (Ali & Nater, 2020;
Arch et al., 2014; Duchemin, Steinberg, Marks, Vanover, & Klatt, 2015;
Limm et al., 2011).
Salivary C-reactive protein (CRP). CRP is an acute-phase inammatory
protein that increases in response to injury, infection, and inammation
(Sproston & Ashworth, 2018). CRP is typically measured in blood
although salivary concentrations have been shown to correlate modestly
with serum concentrations (Out, Hall, Granger, Page, & Woods, 2012;
Pay & Shaw, 2019). Concentrations of CRP are higher in people with
insomnia and sleep disturbances (Ghilotti et al., 2021; Meier-Ewert
et al., 2004).
Salivary melatonin. Melatonin is a hormone primarily released by the
pineal gland and has an important role in the regulation of the sleep-
wake cycle (Low, Choo, & Tan, 2020). Salivary melatonin concentra-
tions are altered during times of stress and have been found, albeit
inconsistently, to be associated with sleep quality (Ito et al., 2013;
Kennaway, 2020).
Fatigue biomarker index (FBI). The FBI is described as an objective
fatigue measure based on the ratio in concentrations of two salivary
peptide fragments. The FBI has been shown to correlate with evaluations
of perceived exertion in male cyclists (Michael, Daugherty, Santos,
Ruby, & Kalns, 2012), predicted success or failure in military training
candidates (Kalns et al., 2011), and was altered after 48 hrs of sleep
deprivation (Michael, Valle, Cox, Kalns, & Fogt, 2013). Lower FBI re-
ects greater fatigue.
Adverse events: Tolerability and safety of supplement intake by par-
ticipants were assessed every 7 days via an online question querying
adverse effects that were believed to be associated with supplement
intake. Participants were also requested to contact the researchers
immediately if any adverse effects were experienced.
2.5.4. Data collection procedures
Initial screening questionnaires comprising the BIS and DASS-21
were completed online. A response booklet containing copies of the
required questionnaires and sleep diaries was then mailed to all par-
ticipants. The dates for completion of each questionnaire, diary, and
saliva collection were recorded in the booklet. Participants were also
advised to keep their response booklet near their bed and to complete it
within 30 min after awakening.
To measure salivary hormones, participants were provided with
small collection tubes and the whole saliva was collected by unstimu-
lated passive drool. These samples were collected in participants’ homes
on days −2, −1, 26 and 27. There were exactly 28 days between saliva
collections to ensure pre- and post-saliva collections occurred on the
same day of the week. Salivary testing procedures used in this study are
detailed in supplementary le 1 and saliva collection details are as
follows:
(1) On days −2 and 26 (morning collection) before brushing their
teeth and consuming any food or drink, participants were
instructed to collect approximately 5 mls of saliva 30 min after
waking. To ensure good saliva ow, participants were permitted
to drink a glass of water no sooner than 15 min prior to their
saliva collection. This sample was used to measure cortisol,
DHEA-S, sIgA, sAA, and C-reactive protein (CRP).
(2) On days −2 and 26 (evening collection) participants were
instructed to collect approximately 5 mls of saliva at 10 pm. They
were requested to not consume any food or drink at least 15 min
before collecting this sample. If participants went to bed earlier
than this time, they were requested to collect the sample before
going to bed. However, they were requested to collect the days
−2 and 26 evening saliva samples at the same time. This sample
was used to measure cortisol and melatonin.
(3) On days −1 and 27, participants were requested to collect a saliva
sample three times throughout the day, upon waking, at midday,
and in the evening. Participants were instructed to collect this
sample at least 15 min away from consuming any food or drink.
A.L. Lopresti et al.
Journal of Functional Foods 85 (2021) 104671
5
Samples were collected by chewing on a cotton sponge for 2 min.
This sample was used to measure the FBI.
2.6. Statistical analysis
An independent samples T-test or Mann-Whitney U test was used to
compare demographic variables across the two treatment groups for
continuous variables, and Pearson’s Chi-square was used to compare
categorical data. To assess for the effects of Bacopa monnieri, compared
to placebo, on primary and secondary outcomes (BIS and FOSQ-10; PSD;
SF-36 physical component; SF-36 mental component; and salivary hor-
mones), multivariate, repeated-measures analyses of variance (ANOVA)
were computed. If the time ×group (Bacopa monnieri versus placebo)
interaction was signicant, further univariate repeated measures
ANOVA analyses were conducted to examine within-group changes over
time and group ×time interaction effects for relevant subscale scores
and hormonal measurements. Normality of data was assessed by the
visual inspection of Q-Q plots and an analysis of skewness and kurtosis.
This indicated that the questionnaire and diary data were mostly nor-
mally distributed. However, salivary hormone concentrations were not
normally distributed so logarithmic transformations were conducted,
which resulted in normalisation of data. FBI is a log-transformed mea-
sure and was thus evaluated without transformative steps. Where
necessary, degrees of freedom in univariate analyses were adjusted
using the Greenhouse-Geisser approach to correct for violations of the
sphericity assumption. The BIS total score was analysed for changes
from baseline (day −5 to −3), days 7, 14, 21, and 27; the FOSQ-10 total
score from days −2, 7, 14, 21, and 27; the DASS-21 from baseline (day
−5 to −3) to day 27; the PSD scores from mean baseline (days −2 and
−1) to mean follow-up (mean score across days 3, 7, 14, 21, 26, and 27);
salivary hormone concentrations from baseline to day 26; and mean FBI
concentrations (morning, midday, and evening collections) from base-
line to day 27. Data from all participants who returned their response
booklets were included in analyses. All data were analysed using SPSS
(version 26; IBM, Armonk, NY). The critical p-value was set at p ≤0.05
for all analyses.
3. Results
3.1. Study population
3.1.1. Baseline questionnaire and demographic information
As detailed in Fig. 1, from 289 people who completed the initial
online screening questionnaire, 189 individuals did not complete the
initial baseline questionnaires (n =28) or were ineligible (n =161).
One-hundred volunteers participated in the study and questionnaire/
diary data from 89 participants who returned questionnaire booklets
were used for statistical analyses. Pre and post salivary hormone samples
(cortisol, DHEA-S, melatonin, CRP, sIgA, and sAA) were obtained from
87 participants. Full pre and post-FBI samples (morning, midday, and
evening) were obtained from 78 participants. Details of participant de-
mographic information and baseline scores of the total recruited sample
are detailed in Tables 1 and 2. Eleven participants withdrew or did not
return response booklets. There were no signicant differences in
dropout rates across groups. Reasons for withdrawal included exacer-
bation of a pre-existing medical condition (n =4), social/family
stressors (n =3) no reason given (n =2), and skin irritation (n =1).
3.2. Outcome measures
3.2.1. BIS and FOSQ-10
Change in the BIS and FOSQ-10 scores across the two treatment
groups and repeated measures ANOVA signicance levels are detailed in
Table 3. BIS and FOSQ-10 scores decreased over time (F
8,80
=20.47, p <
.001), comprising a reduction from baseline to day 27 in BIS scores of
50% and 47% in the Bacopa monnieri and placebo groups, respectively
Table 1
Baseline Demographic Details of Participants.
Bacopa Placebo p-
value
n =49 n =51
Age Mean 49.02 51.02 0.359
a
SE 1.56 1.50
Sex Female (n) 39 38 0.546
a
Male (n) 10 13
BMI Mean 25.56 26.03 0.508
a
SE 0.49 0.51
Marital status Single (n) 17 11 .144
b
Married/
defacto (n)
32 40
Educational level Secondary (n) 24 17 .137
b
Tertiary (n) 13 23
Post-graduate
(n)
12 11
Exercise level (n) Never/rarely (n) 4 11 .180
b
1 to 2 times a
week (n)
3 3
3 to 5 times a
week (n)
19 12
6 +times a week
(n)
23 25
Duration of sleep problems <6 months (n) 5 3 .472
b
6 to 12 months
(n)
10 6
1 to 2 years (n) 7 8
2 to 5 years (n) 10 10
5 to 10 years (n) 3 9
10 or more years
(n)
14 15
BIS Mean 27.94 28.80 0.576
a
SE 1.14 1.04
FOSQ-10 Mean 30.70 30.11 0.573
a
SE 0.82 0.66
DASS Depression Mean 4.65 5.49 0.416
a
SE 0.64 0.80
DASS Anxiety Mean 3.43 4.35 0.313
a
SE 0.54 0.73
DASS Stress Mean 11.55 9.80 0.237
a
SE 1.10 0.98
DASS Total Mean 19.63 19.65 0.996
a
SE 1.96 2.16
n =44 n =45
SF-36 Physical Functioning Mean 88.98 89.44 0.872
a
SE 2.26 1.82
SF-36 Role limitations due to
physical health
Mean 82.23 84.64 0.509
a
SE 2.45 2.69
SF-36 Role limitations due to
emotional problems
Mean 82.75 82.04 0.860
a
SE 2.72 2.90
SF-36 Vitality Mean 49.77 51.56 0.604
a
SE 2.40 2.44
SF-36 Emotional well-being Mean 69.32 71.78 0.437
a
SE 2.20 2.25
SF-36 Social functioning Mean 78.55 77.69 0.837
a
SE 2.63 3.20
SF-36 Pain Mean 72.61 75.38 0.459
a
SE 2.45 2.79
SF-36 General Health Mean 67.61 69.33 0.609
a
SE 2.52 2.22
PSD Time to sleep onset (min) Mean 30.32 30.92 0.926
a
SE 4.41 4.71
PSD Total sleep time (min) Mean 419.39 414.96 0.798
a
SE 11.95 12.48
PSD Number of waking Mean 3.13 3.68 0.651
a
SE 0.34 1.16
PSD Sleep Quality Rating Mean 2.86 2.91 0.743
a
SE 0.10 0.10
PSD Mood rating after waking Mean 2.81 2.64 0.329
a
SE 0.11 0.12
PSD Alertness rating after
waking
Mean 2.72 2.74 0.869
a
SE 0.12 0.13
a =Independent samples t-test; b =Chi-square Test; BIS =Bergen Insomnia
Scale; BMI =Body Mass Index; SE =standard error; DASS-21 =Depression,
Anxiety, and Stress Scale −21; FOSQ-10 =Functional Outcomes of Sleep
Questionnaire; PSD =Pittsburgh Sleep Diary; SF-36 =Short-Form-36.
A.L. Lopresti et al.
Journal of Functional Foods 85 (2021) 104671
6
(Fig. 2), and an increase in FOSQ-10 scores by 5 and 4% in the Bacopa
monnieri and placebo groups, respectively. However, changes were
similar in both groups; specically, the between-group main effect for
BIS and FOSQ-10 scores (F
2,86
=0.023, p =.978) and the group ×time
interaction (F
8,80
=0.539, p =.823) were not signicant.
3.2.2. Psd
Changes in the PSD scores across the two treatment groups and
repeated measures ANOVA signicance levels are detailed in Table 4.
There was an overall signicant change in PSD scores over time (F
6,82
=
5.15, p <.001) in both groups. However, changes were similar in both
groups; specically, the between-group main effect for PSD scores (F
2,82
=0.573, p =.751) and the group ×time interaction (F
6,82
=0.958, p =
.459) were not signicant. Graphical illustrations of changes in PSD
scores are detailed in supplementary le 1, gure S1.
3.2.3. Sf-36
Changes in the SF-36 scores across the two treatment groups and
repeated measures ANOVA signicance levels are detailed in Table 5.
The multivariate analysis revealed there was an overall signicant in-
crease in both physical (F
4,84
=8.25, p <.001) and mental component
(F
4,84
=13.21, p <.001) scores over time in both groups. There was no
signicant between-group main effect for SF-36 physical (F
4,84
=0.481,
p =.750) or mental component scores (F
4,84
=0.607, p =.659); how-
ever, there were statistically-signicant group ×time interactions for
both physical (F
4,84
=3.10, p =.020) and mental component scores
(F
4,84
=2.59, p =.043). On the mental component subscale scores,
univariate analyses revealed that there was a statistically-signicant
group ×time interaction for the emotional well-being score (F
1,87
=
5.99, p =.016). On the physical component subscale scores, univariate
analyses revealed there were statistically-signicant group ×time in-
teractions for pain (F
1,87
=6.69, p =.011) and general health (F
1,87
=
5.07, p =.027) subscores. Compared to baseline, supplementation with
Bacopa monnieri was associated with a 14% improvement in emotional
well-being (F
1,43
=22.34, p =<0.001), 12% improvement in general -
health (F
1,43
=16.85, p <.001), and 16% reduction in pain (F
1,43
=
20.76, p <.001). In contrast, the placebo was associated with a 6%
improvement in emotional well-being (F
1,44
=8.10, p =.007), 4%
improvement in general health (F
1,44
=4.52, p =.039), and a non-
signicant 3% reduction in pain (F
1,44
=0.52, p =.474). Graphical il-
lustrations of changes in PSD scores are detailed in supplementary le 1,
gure S2.
3.2.4. Dass-21
Changes in the DASS-21 scores across the two treatment groups and
repeated measures ANOVA signicance levels are detailed in Table 3.
There was no overall signicant change in DASS-21 scores over time
Table 2
Baseline Salivary Hormone Concentrations of Participants.
Bacopa (n =
42)
Placebo (n =
45)
p-value
*
CRP (pg/mL) Mean 218.92 138.17 0.143
SE 52.67 28.65
Cortisol, morning (ug/
dL)
Mean 0.40 0.46 0.111
SE 0.04 0.04
Cortisol, evening (ug/
dL)
Mean 0.07 0.13 0.849
SE 0.00 0.05
sIgA (ug/mL) Mean 302.42 215.35 0.065
SE 59.10 77.51
sAA (U/mL) Mean 75.53 62.32 0.282
SE 7.02 4.12
DHEA-S (pg/mL) Mean 4588.20 3275.91 0.745
SE 705.99 276.31
Melatonin (pg/mL) Mean 7.87 7.12 0.550
SE 1.10 1.10
FBI (morning) Mean −1.46 −1.61 0.136
SE 0.076 0.061
n 43 44
FBI (midday) Mean −1.95 −2.04 0.200
SE 0.053 0.044
n 42 43
FBI (evening) Mean −2.00 −2.04 0.604
SE 0.061 0.062
N 43 45
*
Independent-samples t-test (log10 scores)
Table 3
Change in Questionnaire Outcome Measures.
Bacopa (n =44) Placebo (n =45) Multivariate analysis
Time
effects*
Group
Main
effects*
Time ×group
interaction*
Baseline Day 7 Day
14
Day
21
Day 27 Baseline Day 7 Day
14
Day
21
Day 27
BIS Mean 27.77 18 16.25 15.48 13.89 28.18 18.82 16.16 15.48 14.98 <0.001 0.978 0.823
SE 1.20 1.35 1.34 1.54 1.41 1.12 1.26 1.23 1.27 1.36
FOSQ-10 Mean 30.70 30.82 31.02 31.45 32.14 30.11 30.47 31.29 31.64 32.18
SE 0.82 0.95 0.89 0.87 0.88 0.66 0.70 0.68 0.69 0.72
DASS-21
Depression
Mean 4.64 5.05 5.69 6.04 0.394 0.041 0.885
SE 0.646 0.782 0.885 0.891
DASS-21
Anxiety
Mean 3.50 4.05 4.31 4.36
SE 0.582 0.787 0.804 0.863
DASS-21
Stress
Mean 11.77 10.73 9.82 9.38
SE 1.138 1.434 1.063 1.199
*
p-values, repeated measures ANOVA multivariate analysis
Fig. 2. Change in BIS Scores.
A.L. Lopresti et al.
Journal of Functional Foods 85 (2021) 104671
7
(F
3,85
=1.00, p =.394) in either group (group ×time interaction F
3,85
=
0.217, p =.885), but there was a signicant between-group main effect
for DASS-21 scores (F
3,85
=2.88, p =.041). However, univariate ana-
lyses revealed that there was no signicant group main effect for the
individual subscale scores of depression (F
1,87
=1.14, p =.288), anxiety
(F
1,87
=0.385, p =.537), or stress (F
1,87
=1.14, p =.289).
3.2.5. Salivary hormones
Changes in the salivary hormone concentrations across the two
treatment groups and repeated measures ANOVA signicance levels
(based on logarithmic transformations) are detailed in supplementary
le 1, table S1. The multivariate analysis revealed there was no overall
signicant change in hormone concentrations over time (F
7,79
=1.35, p
=.238) and no signicant between-group main effect (F
7,79
=0.30, p =
.950); however, there was a statistically-signicant group ×time
interaction (F
7,79
=2.97, p =.008). Univariate analyses revealed that
there were statistically-signicant group ×time interactions for morn-
ing cortisol (F
1,85
=5.58, p =.020), sIgA (F
1,85
=4.99, p =.028), and
sAA (F
1,85
=7.23, p =.009) concentrations. Within-group analysis
revealed that in the Bacopa monnieri group, there were statistically-
signicant decreases in sIgA (F
1,41
=4.34, p =.043) and sAA (F
1,41
=
5.09, p =.030) concentrations over time. In the placebo group, there
was a statistically-signicant decrease in morning cortisol concentration
over time (F
1,44
=6.39, p =.015). Percentage change in hormone
concentrations from baseline to day 26 are illustrated in Fig. 3.
3.2.6. Fatigue biomarker index (FBI)
Changes in the salivary FBI concentrations across the two treatment
groups and repeated measures ANOVA signicance levels are detailed in
supplementary le 1, table S2. The multivariate analysis revealed there
was no overall statistically signicant changes in overall FBI concen-
trations over time (F
3,74
=0.84, p =.474), between-group main effect
Table 4
Change in PSD scores.
Bacopa (n
=44)
Placebo (n
=45)
Multivariate
analysis
Mean
Baseline
Mean Days 3
to 27
p-
value
a
Mean
Baseline
Mean Days 3
to 27
p-
value
a
Time effect
c
Group main
effect
c
Time ×group
interaction
c
Time to sleep onset
(min)
Mean 30.32 30.98 0.857 30.92 28.75 0.520 <0.001 0.751 0.459
SE 4.41 3.87 4.71 3.92
Total sleep time
(min)
Mean 419.39 405.95 0.312 414.96 407.07 0.501
SE 11.95 14.31 12.48 9.26
Number of wakings Mean 3.13 2.30 <0.001 3.68 1.94 0.126
SE 0.34 0.20 1.16 0.15
Sleep Quality
Rating
Mean 2.86 3.11 0.039 2.91 3.15 0.045
SE 0.10 0.10 0.10 0.09
Mood rating after
waking
Mean 2.81 2.76 0.620 2.64 2.59 0.552
SE 0.11 0.12 0.12 0.10
Alertness rating
after waking
Mean 2.72 3.22 <0.001 2.74 2.90 0.202
SE 0.12 0.10 0.13 0.08
a =within-group, repeated measures ANOVA univariate analysis; b =between-group, repeated measures ANOVA univariate analysis; c =repeated measures ANOVA
multivariate analysis
Table 5
Change in SF-36 scores.
Bacopa (n =44) Placebo (n =45) Time ×group
interaction
b
Multivariate analysis
Time
effect
c
Group Main
effect
c
Time ×group
interaction
c
Baseline Day
27
p-
value
a
Baseline Day
27
p-
value
a
Physical Component
SF-36 Physical functioning Mean 88.98 89.77 0.667 89.44 90.67 0.288 0.843 <0.001 0.750 0.020
SE 2.26 2.20 1.82 1.49
SF-36 Role limitations due
to physical health
Mean 82.23 89.05 0.015 84.64 90.36 0.036 0.770
SE 2.45 2.39 2.69 2.25
SF-36 Pain Mean 72.61 83.91 <0.001 75.38 77.29 0.474 0.011
SE 2.45 2.31 2.79 2.53
SF-36 General health Mean 67.61 75.68 <0.001 69.33 72.11 0.039 0.027
SE 2.52 2.48 2.22 2.41
Mental Component
SF-36 Emotional well-being Mean 69.32 79.20 <0.001 71.78 75.67 0.007 0.016 <0.001 0.659 0.043
SE 2.20 1.87 2.25 2.36
SF-36 Role limitations due
to emotional problems
Mean 82.75 90.16 0.004 82.04 89.67 0.001 0.948
SE 2.72 2.18 2.90 2.51
SF-36 Social functioning Mean 78.55 88.30 <0.001 77.69 82.91 0.076 0.235
SE 2.63 2.23 3.20 3.35
SF-36 Vitality Mean 49.77 56.91 0.001 51.56 59.93 <0.001 0.635
SE 2.40 2.87 2.44 2.32
a =within-group, repeated measures ANOVA univariate analysis; b =between-group, repeated measures ANOVA univariate analysis; c =repeated measures ANOVA
multivariate analysis
A.L. Lopresti et al.
Journal of Functional Foods 85 (2021) 104671
8
(F
3,74
=1.39, p =.250), or group ×time interaction (F
3,74
=0.39, p =
.758).
3.2.7. Intake of supplements
On day 7, 14, 21, and 27 participants recorded their quantity of
remaining supplements. On day 28, 97% of participants reported taking
greater than 80% of their capsules.
3.2.8. Efcacy of participant blinding
To evaluate the efcacy of condition concealment over the study,
participants were asked at the completion of the study to predict con-
dition allocation (i.e. placebo, Bacopa monnieri, or uncertain). Efcacy of
group concealment was high as 65% of participants either incorrectly
guessed treatment allocation or were unsure.
3.2.9. Adverse events
The frequency of self-reported adverse effects is detailed in supple-
mentary le 1, table S3. There were no signicant differences in the
reports of adverse effects between the groups and no signicant adverse
events were reported by participants. One participant in the placebo
group withdrew from the study due to reports of increased skin itching.
4. Discussion
In this 28-day, randomised, double-blind, placebo-controlled study,
supplementation with 150 mg, twice daily of a Bacopa monnieri extract
was not associated with greater improvements in sleep patterns
compared to the placebo. Changes in sleep quality in adults with self-
reported sleep disturbances improved signicantly in all participants,
with reductions in the BIS total score (primary outcome measure) of
50% and 47% in the Bacopa monnieri and placebo groups, respectively.
Changes in sleep as measured by the PSD and FOSQ-10 also revealed
mostly similar improvements over time. However, based on results from
the SF-36, Bacopa monnieri supplementation was associated with greater
improvements in both physical and emotional component scores. Mea-
surements of changes in salivary hormones revealed Bacopa monnieri
was associated with statistically-signicant greater decreases in salivary
concentrations of sIgA and sAA. Moreover, there were signicant
between-group differences in changes in morning cortisol concentra-
tions over the 28 days as demonstrated by decreases in the placebo
group and a trend toward increased concentrations in the Bacopa mon-
nieri group.
These results suggest that Bacopa monnieri supplementation for 28
days at a dose of 150 mg twice daily did not improve sleep patterns more
than the placebo in adults with self-reported sleep problems. However,
whether intake for a longer duration, higher dose, or different dosage
regimen (e.g., once versus twice-daily administration) may result in
greater treatment efcacy requires investigation in future trials. More-
over, identical doses were provided to all participants irrespective of
age, sex, or weight. In future trials, modifying doses based on these
characteristics, particularly weight, will be important to investigate. The
high placebo effect in this trial, as demonstrated by an almost 50%
reduction in the BIS score, requires consideration as such efcacy is not
typical in placebo-controlled trials for insomnia (Perlis, McCall, Jung-
quist, Pigeon, & Matteson, 2005). Reasons for this high placebo effect
could not be determined but may be associated with the easing in work
and social restrictions imposed due to the COVID-19 pandemic in
Australia during the trial.
Even though further investigation is required to validate the sec-
ondary and exploratory outcome ndings, Bacopa monnieri was associ-
ated with greater improvements in emotional wellbeing, pain, and
general health compared to the placebo. Although promising, such
outcomes require conrmation in future trials utilising validated mea-
sures with specically-recruited populations. In this trial, an improve-
ment in emotional wellbeing was identied but there were no changes in
the DASS-21 subscale scores, which is a self-report measure assessing
depressive, anxiety, and stress-related symptoms. However, the lack of
change in DASS-21 scores may be due to oor effects as, at baseline,
participants reported non-clinical levels of depression, anxiety, and
stress. In relation to previous investigations into the mood-enhancing
effects of Bacopa monnieri as an adjunct to an antidepressant medica-
tion, Bacopa monnieri was associated with improvements in mood in
adults with anhedonia (Micheli et al., 2020). Acute, mood-enhancing
effects were also identied in another trial on healthy adults exposed
to a computerised multitasking activity (Benson et al., 2014). Results
from several animal trials have also suggested Bacopa monnieri may have
anxiolytic or antidepressant effects (Hazra, Kumar, Saha, & Mondal,
2017; Zu et al., 2017). Bacopa monnieri contains various metabolites
such as saponins, alkaloids and sterols. The main active constituents of
Bacopa monnieri are saponins known as bacosides with bacoside A the
Fig. 3. Percentage Change in Salivary Hormones.
A.L. Lopresti et al.
Journal of Functional Foods 85 (2021) 104671
9
most studied triterpenoid saponin. Bacoside A is a mixture of four sa-
ponins comprising bacoside A3, bacopaside II, jujubogenin isomer of
bacopasaponin C (bacopaside X), and bacopasaponin C (Sukumaran
et al., 2019). The mood-enhancing effects of Bacopa monnieri may be
associated with its inuence on neurotransmitter availability. For
example, tryptophan hydroxylase, an enzyme involved in serotonin
synthesis was upregulated in the hippocampus of postnatal rats after the
oral treatment of a Bacopa monnieri leaf extract containing bacosides
(Charles, Ambigapathy, Geraldine, Akbarsha, & Rajan, 2011). An in
silico model suggested bacoside A3 may be particularly important in
tryptophan hydroxylase function and thus serotonin synthesis (Rajathei,
Preethi, Singh, & Rajan, 2014). The Bacopa monnieri constituents,
bacopaside I and bacoside A have also been shown to inhibit monoamine
oxidase activity (MAO-A and MAO-B). These are enzymes involved in
the degradation of amine neurotransmitters such as noradrenaline,
adrenaline, serotonin, and dopamine (Singh, Ramakrishna, Bhateria, &
Bhatta, 2014).
The increase in the pain subscale score of the SF-36 (indicating re-
ductions in pain symptoms) is a unique nding which has not been
investigated in any previous human trial. However pain-relieving effects
of Bacopa monnieri have been identied in animal and in vitro trials (Rauf
et al., 2013; Shahid, Subhan, Ahmad, & Ullah, 2017; Taznin, Mukti, &
Rahmatullah, 2015). Bacopa’s analgesic effects may be via its effects on
cyclooxygenase-2 (COX-2) activity and the adrenergic, serotonergic, and
opioidergic systems (Bhaskar & Jagtap, 2011; Rauf et al., 2013).
Bacoside-A also possesses anti-inammatory actions and in an acute and
chronic animal model, downregulated the inammatory cytokines (in-
terleukins-6 and 17a, and tumour necrosis factor-
α
) and the inamma-
tory chemokine CCL-5 (Madhu et al., 2019). In an in vitro model, pre-
treatment with bacoside-A3 before β-amyloid stimulation suppressed
the generation of reactive oxygen species, prostaglandin E2 secretion,
and the over-expression of COX-2 (Bai & Zhao, 2021). Further conr-
mation of the pain-relieving effects of Bacopa monnieri in populations
experiencing pain-related difculties will be required in future trials.
To help understand the physiological mechanisms associated with
Bacopa monnieri administration, salivary hormone (morning and eve-
ning cortisol, evening melatonin, morning sIgA, sAA, DHEA-S) and FBI
concentrations (morning, midday, and evening) were measured.
Compared to the placebo, Bacopa monnieri was associated with greater
reductions in morning sIgA and sAA, and increases in morning cortisol
relative to decreased concentrations in the placebo group. sIgA has
important immunological functions, including assisting in the preven-
tion of bacteria from forming colonies on mucosal surfaces, and neu-
tralising toxins and enzymes produced by bacteria (Brandtzaeg, 2013;
Tsujita & Morimoto, 1999). However, various psychosocial stressors
such as academic examination, daily hassles, negative mood, unfav-
ourable daily events, and work demands can inuence sIgA concentra-
tions (Tsujita & Morimoto, 1999; Valdimarsdottir & Stone, 1997). The
direction of the effects of these stressors on sIgA concentrations is
inconsistent as it is inuenced by the type of stressor, and the intensity
and duration of the stress. For example, sIgA concentrations decreased
when measured several days or weeks after academic stress but
increased immediately after academic stress (Jemmott & Magloire,
1988; Otsuki et al., 2004). Even though ndings are inconsistent,
reduced sIgA concentrations after relaxation and meditative practices
have been generally found (Heckenberg, Hale, Kent, & Wright, 2019;
Tsujita & Morimoto, 1999; Valdimarsdottir & Stone, 1997). The direc-
tion of changes in sIgA may be inuenced by the population examined
and the timing of salivary collections after relaxation and meditation
practice (Hewson-Bower & Drummond, 1996; Taniguchi, Hirokawa,
Tsuchiya, & Kawakami, 2007). It seems that relaxation may have a
modulating effect on sIgA, characterised by increased concentrations in
individuals with low sIgA and reduced concentrations in people with
high sIgA. Whether sIgA concentrations are elevated or depressed in
people with sleep disturbances has not been adequately investigated,
although in a study on pregnant women with pregnancy-induced
hypertension and gestational diabetes, sIgA concentrations increased
from the second and third trimester, and this was inversely correlated
with sleep quality (Hayase, Shimada, & Seki, 2014). In another study,
sleep deprivation for two nights in healthy males increased concentra-
tions of salivary sIgA (Costa et al., 2010). In relation to sAA, there is an
increasing body of evidence conrming it as a valid and reliable marker
of stress and autonomic nervous system activity. Alpha-amylase is a
salivary enzyme involved in the digestion of carbohydrates and starches
but may also reect central noradrenergic activity (Ali & Nater, 2020).
In several studies, mind–body interventions such as stress reduction
programs, self-compassion training, and mindfulness-based in-
terventions have been shown to reduce sAA concentrations (Arch et al.,
2014; Duchemin et al., 2015; Limm et al., 2011). The changes in
morning salivary cortisol concentrations (as evidenced by reductions in
morning cortisol in the placebo group) seem to contrast with the nd-
ings of the lowering-effects of Bacopa monnieri on sIgA and sAA. How-
ever, given the signicant diurnal activity of cortisol and ndings of
both hyper- and hypo-cortisol output across different disorders and
stress exposures, this nding needs to be interpreted cautiously. Even
though lowered cortisol is typically viewed as a positive health-related
nding, the research indicates that balance is the key. That is, cortisol
concentrations at both the highest and lowest ends predict future health
and disease outcomes (Turner et al., 2020). Cortisol concentrations are
also signicantly inuenced by the timing of collection, and in this
study, participants were asked to collect saliva samples 30 min after
waking. Morning salivary cortisol concentrations are inuenced by the
cortisol awakening response (CAR) which is typied by an increase of
between 38% and 75% in cortisol concentrations 30 to 45 min after
awakening (Elder, Wetherell, Barclay, & Ellis, 2014; Fries, Dettenborn,
& Kirschbaum, 2009). Delayed or early collections will, therefore, affect
ndings, and despite participants being instructed to collect samples 30
min after waking, this could not be enforced or reliably monitored. For
many participants, waking time was also difcult to determine due to
their poor sleep patterns. The CAR also varies signicantly across con-
ditions with both higher and lower CAR associated with health benets.
For example, in patients with sleep disturbance and treatment-resistant
major depression, a lowered CAR was correlated with more severe
depressive symptoms and worse sleep quality (Santiago et al., 2020). In
a meta-analysis on adults with post-traumatic stress disorder, morning
salivary cortisol concentrations were conrmed to be lower (Meewisse,
Reitsma, de Vries, Gersons, & Olff, 2007). In another meta-analysis, the
CAR was positively associated with job stress and general life stress but
was negatively associated with fatigue, burnout, and exhaustion (Chida
& Steptoe, 2009). Moreover, in a study on adults with chronic fatigue
syndrome, lower salivary morning cortisol concentrations were identi-
ed (Nater et al., 2008). Adverse early life stressors are also associated
with hypocortisolism in adulthood (Juruena, Eror, Cleare, & Young,
2020). These ndings suggest that the relationship between morning
cortisol and mental and physical wellbeing is not simple; therefore,
further research is required to help elucidate the relevance of these
ndings. Due to cortisol’s anti-inammatory effects, it is important to
highlight the nding of reduced pain symptoms in people on Bacopa
monnieri. Further investigation is required but a potential mechanism
associated with bacopa’s analgesic effects may be via its effects on
cortisol (Hannibal & Bishop, 2014). Interestingly, despite the ndings of
reduced pain, and differences in changes in cortisol concentrations in
the placebo and Bacopa monnieri groups, there were no changes in the
inammatory marker, CRP. This may be due to the method of mea-
surement used in this study (saliva versus the more commonly-used
blood sample), CRP being an acute-phase inammatory protein, and/
or the potential of recruiting participants with low baseline concentra-
tions of CRP, thereby increasing the possibility of oor effects (Sproston
& Ashworth, 2018).
A.L. Lopresti et al.
Journal of Functional Foods 85 (2021) 104671
10
5. Conclusions
In conclusion, in this 28-day, randomised, double-blind, placebo-
controlled trial, Bacopa monnieri supplementation did not improve sleep
patterns more than the placebo in adults with self-reported sleep prob-
lems. However, based on ndings from secondary outcomes measures,
Bacopa monnieri was associated with greater improvements in physical
and emotional health as measured by the SF-36. In particular, there were
improvements in emotional wellbeing, general health and bodily pain
subscale scores. An analysis of changes in hormone concentrations over
time also revealed Bacopa monnieri was associated with reductions in
sIgA, sAA, and differences in morning concentrations of salivary cortisol
between the two groups (as evidenced by mostly decreased concentra-
tions in the placebo group). Future clinical trials on specically-
recruited populations will be required to validate these secondary
ndings. To examine the potential effects of Bacopa monnieri on sleep,
future trials using different doses, dosing regimens, treatment durations,
outcome measures (e.g., sleep actigraphy), and adults with varying
sleep-related difculties (e.g., sleep-onset versus sleep-maintenance
insomnia) may be helpful. Bacopa monnieri is traditionally used as a
nootropic herb (Aguiar & Borowski, 2013), and in this study there was
some evidence to suggest an increase in morning alertness as demon-
strated by an 18% increase in alertness ratings, compared to only 6% in
the placebo group (see Supplementary File, Figure S1). This nding will
be important to investigate in future trials. Due to the positive ndings
of Bacopa monnieri on mood and stress-related hormones, further in-
vestigations into the anxiolytic effects of Bacopa monnieri may be war-
ranted using stressed or anxious populations and/or using stress
reactivity models such as the Trier Social Stress Test or Maastricht Acute
Stress Test (Allen et al., 2017; Smeets et al., 2012).
6. Clinical Trial Registration
This study was prospectively registered with the Australian New
Zealand Clinical Trials Registry (Trial ID. ACTRN12620000770965).
https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?
id=379967
Funding
This study was funded by Verdure Sciences Inc. Verdure Sciences was
not involved in the collection and analysis of data, or the writing of the
report.
Declaration of Competing Interest
The authors declare the following nancial interests/personal re-
lationships which may be considered as potential competing interests:
[Dr Lopresti is the managing director of Clinical Research Australia, a
contract research organisation that has received research funding from
nutraceutical companies. Dr Lopresti has also received presentation
honoraria from nutraceutical companies. Mr Smith is an employee of
Clinical Research Australia and declares no other conicts of interest. Dr
Kalns is the Vice-President and Chief Scientic Ofcer of Hyperion
Biotechnology, Inc. who developed the testing methods for the Fatigue
Biomarker Index. No other authors declare any conicts of interest]
Acknowledgements
The authors gratefully acknowledge Verdure Sciences Inc for funding
the project and supplying capsules used for this study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jff.2021.104671.
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