Journal of

Clinical Medicine

Article
Response to Treatment with Melatonin and Clonazepam versus
Placebo in Patients with Burning Mouth Syndrome
Candela Castillo-Felipe 1 , Asta Tvarijonaviciute 2 , Marina López-Arjona 2 , Luis Pardo-Marin 2 ,
Eduardo Pons-Fuster 3 and Pia López-Jornet 4, *

1 Colaborate Department Stomatology School of Medicine, Oral Medicine University of Murcia,

30100 Murcia, Spain; [email protected]
2 Interdisciplinary Laboratory of Clinical Analysis, Interlab-UMU, Regional Campus of International Excellence
‘Campus Mare Nostrum’, University of Murcia, 30100 Murcia, Spain; [email protected] (A.T.);
[email protected] (M.L.-A.); [email protected] (L.P.-M.)
3 Faculty of Medicine, Department Anatomy Psicobiology Regional Campus of International Excellence
‘Campus Mare Nostrum’, University of Murcia, 30100 Murcia, Spain; [email protected]
4 Department of Oral Medicine, Faculty of Medicine, Regional Campus of International Excellence ‘Campus
Mare Nostrum’, IMIB Instituto Murciano de Investigación Biosanitaria-Arrixaca University of Murcia,
30100 Murcia, Spain
* Correspondence: [email protected]

Abstract: Objective: to evaluate the efficacy of melatonin and clonazepam versus placebo in patients
with burning mouth syndrome (BMS). Methods: a prospective double-blind study was carried out
in patients with BMS and randomized to three groups: melatonin (1 mg once a day), clonazepam
(0.5 mg/twice a day), or a placebo once a day, for 8 weeks. The clinical changes were evaluated,
including xerostomia, the Oral Health Impact Profile 14 (OHIP-14) score, Pittsburg Sleep Quality
Citation: Castillo-Felipe, C.; Index, and the Hospital Anxiety and Depression Scale (HADS). Oxygen saturation and heart rate
Tvarijonaviciute, A.; López-Arjona, were recorded, with an analysis of salivary biomarkers in the forms of oxytocin, ferritin, adenosine
M.; Pardo-Marin, L.; Pons-Fuster, E.; deaminase (ADA), total proteins, and alpha-amylase. Results: a total of 64 patients were analyzed.
López-Jornet, P. Response to
A significant decrease in burning sensation was recorded with melatonin (7.8 ± 1.54 pre-treatment,
Treatment with Melatonin and
5.78 ± 2.54 post-treatment; p < 0.001) and clonazepam (8.75 ± 1.2 pre-treatment, 5.5 ± 3.6 post-
Clonazepam versus Placebo in
treatment (p < 0.01). With regard to quality of life (OHIP-14), significant improvements were observed
Patients with Burning Mouth
before and after the administration of melatonin (p < 0.001) and clonazepam (p = 0.001). On the other
Syndrome. J. Clin. Med. 2022, 11,
2516. https://doi.org/10.3390/
hand, with regard to the changes in salivary biomarkers following treatment, negative correlations
jcm11092516 were found between oxytocin and drainage (r = −0.410; p = 0.009) and between the HADS-D score
and ferritin (r = −0.312; p = 0.05). While salivary amylase showed positive correlation with heart
Academic Editor: Cheng-Chia Yu
rate (r = 0.346; p = 0.029) and oxygen saturation (r = 0.419; p = 0.007). Conclusions: melatonin and
Received: 31 March 2022 clonazepam were shown to be effective at reducing the burning sensation and improving quality of
Accepted: 27 April 2022 life. Both drugs were found to be safe, with no major adverse effects in patients with BMS. Melatonin
Published: 29 April 2022 may be regarded as an alternative treatment for patients with BMS, though further studies are needed
Publisher’s Note: MDPI stays neutral
to confirm its effectiveness.
with regard to jurisdictional claims in
published maps and institutional affil- Keywords: burning mouth syndrome; pain; xerostomia; sleep; melatonin; clonazepam; placebo
iations.

1. Introduction
Copyright: © 2022 by the authors.
Burning mouth syndrome (BMS) is characterized by chronic pain of the oral cavity,
Licensee MDPI, Basel, Switzerland.
and is estimated to affect 0.1–3.9% of the population [1–3]. It is defined as a recurrent daily
This article is an open access article
oral burning sensation or dysesthesia lasting over two hours a day for a period of over
distributed under the terms and
three months, in the absence of clinical lesions, capable of explaining the symptoms [4].
conditions of the Creative Commons
Attribution (CC BY) license (https://
The precise etiology of BMS remains unclear [5–7]. This complicates patient management,
creativecommons.org/licenses/by/
and no definitive treatment for the disorder has been established to date [8]. Many stud-
4.0/).
ies have evidenced that in addition to neuropathic alterations, psychological factors are

J. Clin. Med. 2022, 11, 2516. https://doi.org/10.3390/jcm11092516 https://www.mdpi.com/journal/jcm

J. Clin. Med. 2022, 11, 2516 2 of 12

implicated in this disease [9–11]. In this regard, anxiety and depression are the most com-
mon psychopathological disorders observed in these patients [10]. Likewise, BMS seems
to be related to endocrine and metabolic changes that can give rise to alterations of the
hypothalamic–pituitary–adrenal axis, with an impact on salivary composition [12]. Ade-
quate management of the pain requires due evaluation of its presence and severity [13,14].
In this regard, patient descriptions of the pain experience remain the gold standard for
the evaluation of pain, and this is conditioned to adequate communication capacity. It is
therefore of interest to establish biomarkers capable of contributing to the assessment of
different aspects of BMS [14–17]. Saliva can offer noninvasive fluid samples of great diag-
nostic potential that may contribute to identify and monitor the disease. In this respect, the
salivary alpha-amylase concentrations increase in situations of stress and activation of the
sympathetic nervous system. On the other hand, adenosine deaminase (ADA) participates
in different processes related to the immune system. In turn, oxytocin is a neuropeptide
that plays a key role in health and has been implicated in the attenuation of pain [17–24]. It
is important to note that increased salivary ferritin can change the properties of saliva and
also has the potential to bring about changes in the oral environment. Ferritin, being acidic,
can reduce the salivary pH and the buffering capacity of saliva; this can in turn lead to an
increased incidence of dental caries [25].
Despite the advances in the knowledge and treatment of BMS, the disorder remains
a challenge [8]. Very diverse therapeutic strategies have been evaluated, with controversial
results [26–29] In this regard, the treatments used have included antiseizure drugs, ben-
zodiazepines, antidepressants, analgesics, laser therapy, topical agents, physical barriers
such as tongue protectors, psychotherapy, and nutritional supplements [8,25–28]. The best
therapeutic outcomes have been recorded with clonazepam, with a significant decrease in
pain following administration of the drug, though not all patients show a good response
to this treatment. In 2016, Arduino et al. studied the efficacy of photobiomodulation
and topical clonazepam therapy in BMS; there were indications that photobiomodulation
is capable of reducing the symptoms in patients with BMS, with patients experiencing
constant and long-lasting effects after the end of the first applications [29].
Melatonin (N-acetyl−5-methoxytryptamine) is best known for its participation in
the regulation of circadian rhythms. In addition, melatonin has neuroprotective, anti-
inflammatory, and immune-modulating actions [30–35]. On the other hand, melatonin is
reported to have analgesic effects in application to neuropathic disorders, hyperalgesia,
and allodynia [36]. The location and activity of its receptors have been associated with
the main pain-regulating centers, including the trigeminal pathway and the trigeminal
nucleus, which are intimately related to orofacial pain [36]. Likewise, melatonin binding to
its receptors has been intimately related to the dopaminergic and GABAergic pathways.
Thus, some studies have suggested that the application of melatonin acts in a way similar
to certain benzodiazepines, such as clonazepam [30,34,36,37].
Therefore, we have hypothesized that melatonin-based treatment could be beneficial
for people suffering from BMS. The present study evaluates the response to treatment with
melatonin and clonazepam versus placebo in patients with BMS via assessment of clinical
variables (such as burning sensation score, xerostomia, quality of life among others) and
salivary biomarkers related to stress (salivary alpha-amylase, oxytocin) and the immune
system (ADA, ferritin).

2. Material and Methods

A randomized prospective study was carried out on the efficacy of melatonin and
clonazepam versus placebo in patients with BMS. Double-blinding was applied to the
administration of melatonin and the placebo but not to clonazepam. The study was carried
out at the Dental Clinic of Morales Meseguer University Hospital (University of Murcia,
Spain). Randomization was performed by an external agent. All patients gave written
informed consent to participate in the study, which involved a two-month treatment
duration. The study protocol abided with the principles of the Declaration of Helsinki
J. Clin. Med. 2022, 11, 2516 3 of 12

and was approved by the Bioethics Committee of the University of Murcia (reference:
2203/2018). The study is registered at ClinicalTrials.gov (accessed on 28 December 2018)
with the identifier: NCT03788733. Melatonin and placebo randomization were conducted
by a professional unrelated to the investigation and the randomization codes were kept in
a sealed envelope until all the patients had completed the study.
We included 80 consecutive patients diagnosed with BMS according to the criteria
of the Scala [2]. The patients were assigned in a 1:1:1 proportion to one of the treatments
(melatonin, clonazepam, or placebo) using a computer-generated randomization algorithm.
Individuals allergic to melatonin or clonazepam were excluded from the study, in the
same way as patients working night shifts, pregnant or nursing women, subjects receiving
estrogen or hormone replacement therapy, and individuals using vitamins or nutritional
supplements in the month prior to participation in the study.

2.1. Study Protocol: Clinical Variables and Data Compilation

The patients were divided into three groups: melatonin, clonazepam, and placebo.
Following confirmation of the inclusion criteria, data compilation was carried out by
a single investigator (CCF) (Figure 1). Patient age and gender were recorded, as well as
smoking habits, alcohol intake, and oral hygiene. A buccodental exploration was carried
out, with assessment of the pain symptoms and location of the burning sensation. Then,
heart rate and arterial oxygen saturation (SatO2) were recorded by pulsioximetry. The
following questionnaires were applied:
• Burning sensation visual analog scale (VAS pain): the patient scores burning sen-
sation on a VAS from 0 to 10, where 0 = no burning sensation and 10 = maximum
burning sensation [38].
• Xerostomia score (VAS xeros): the patient scores xerostomia (dry mouth) on a VAS
from 0 to 10, where 0 = no xerostomia and 10 = maximum xerostomia.
• Oral Health Impact Profile 14 (OHIP-14) (Spanish version): this questionnaire is
composed of 7 dimensions: functional limitation, physical discomfort, psychological
discomfort, physical disability, psychological disability, social disability, and handicaps.
Each dimension comprises two questions (with a total of 14 questions), and the answers
are scored from 0 to 4 (0 = lowest level and 4 = highest level). The higher the score, the
poorer the oral quality of life of the patient [39].
• Hospital Anxiety and Depression Scale (HADS): this instrument consists of two
subscales that respectively assess anxiety state (HADS-A) and depressive state (HADS-
D). Each subscale has 7 items scored from 0 to 3, where a total score of over 10 reflects
the presence of anxiety or depression, scores between 8 and 10 are borderline, and
a score of under 7 indicates the absence of anxiety or depression [40].
• Pittsburg Sleep Quality Index: this instrument consists of 19 questions divided into
7 sections, with each section addressing a specific characteristic of the patient sleep
pattern: subjective quality of sleep, latency of sleep, duration of sleep, usual efficiency
of sleep, alterations of sleep, use of medication to sleep, and daytime dysfunction. Each
section receives a score of 0–3, where 0 = no problems and 3 = great problems. The
final score corresponds to the sum of the scores of the 7 sections, yielding a maximum
score of 21 points. A score of 5 or less is indicative of satisfactory quality of sleep,
while scores of over 5 are indicative of sleep disorders [41].
• Epworth daytime sleepiness scale: this scale comprises 8 questions that simulate situ-
ations in which the patient is asked to score the probability of experiencing sleepiness.
The result of the questionnaire is the sum of the individual scores of the 8 questions,
and higher scores are indicative of a greater probability of daytime sleepiness [42].
J.J. Clin.
Clin. Med.
Med. 2022,
2022, 11,
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x FOR PEER REVIEW 44 of 12
of 12

Figure 1.
Figure 1. Flow
Flow Diagram.
Diagram.

2.2.
2.2. Sialometry
Sialometry
Resting
Restingsaliva
salivaoutput
outputwas
wasmeasured
measuredfor 5 min,
for 5 min,using thethe
using drainage
drainage technique.
technique.Measure-
Meas-
ments were always performed in the morning, taking into account
urements were always performed in the morning, taking into account the circadian the circadian rhythm,
and following
rhythm, a fasting aperiod
and following fastingofperiod
at leastoftwo hours.
at least twoThe saliva
hours. The was collected
saliva in graded
was collected in
polyethylene tubes, which were visually inspected, and any samples
graded polyethylene tubes, which were visually inspected, and any samples containingcontaining blood were
discarded.
blood wereThe samplesThe
discarded. were then immediately
samples centrifuged (3000
were then immediately rpm for(3000
centrifuged 10 minrpm 4 ◦ C),
at for 10
and ◦
−80 stored
min the
at 4supernatant
°C), and thewas stored at was
supernatant C until
at analysis.
−80 °C until analysis.
With
With regard
regard toto both
both melatonin
melatonintreatment
treatmentand andthe
the administration
administration of placebo,
of placebo, the the
pa-
patients received edible cellulose and starch strips allowing instant
tients received edible cellulose and starch strips allowing instant release and rapid release and rapid
ab-
absorption
sorption of ofthethe active
active ingredient
ingredient (1 mg(1 of
mgmelatonin
of melatonin per strip).
per strip). Eachwas
Each strip strip was sup-
supplied in
plied in aaluminum
sealed aluminum × 32 mm 2
a sealed sachet forsachet for both melatonin
both melatonin and placebo and placebo
(size 22 x 32(size
mm222, thickness < 1,
thickness
mm). Neither< 1 mm). Neither thenor
the investigator investigator
the patientnor wasthe patient
aware was
of the aware ofadministered.
treatment the treatment
administered.
The clonazepam group received Rivotril®® 2.5 mg/mL oral solution, administering 5
drops (0.5clonazepam
The group received
mg of clonazepam) Rivotril and
in the morning 2.5 5mg/mL
drops atoral solution,
night administering
in the form of a rinse
5 drops (0.5 mg of clonazepam) in the morning and 5 drops at night in the form of a rinse
retained in the mouth for 1.5 min [43,44]. The patients were instructed not to swallow the
retained in the mouth for 1.5 min [43,44]. The patients were instructed not to swallow
drug.
the drug.
The duration of the treatments was two months, after which all the clinical variables
were again recorded, together with patient satisfaction with the treatment and its
J. Clin. Med. 2022, 11, 2516 5 of 12

The duration of the treatments was two months, after which all the clinical vari-
ables were again recorded, together with patient satisfaction with the treatment and its
organoleptic properties. We likewise recorded the possible presence of side effects during
the two-month treatment period.

2.3. Biochemical Analysis

The salivary alpha-amylase activity was measured by a commercial kit using the
International Federation of Clinical Chemistry and Laboratory Medicine method [18,20,23].
ADA was measured using a commercially available spectrophotometric assay (Adenosine
Deaminase assay kit, Diazyme Laboratories, Poway, California, USA) adapted to an auto-
mated analyzer (Olympus AU400 automated biochemical analyzer, Olympus Diagnostica
GmbH, Ennis, Ireland) [18]. Ferritin was measured by a commercial immunoturbidimetric
assay that uses polyclonal anti-human ferritin antibodies (Tina-quant ferritin, Roche Diag-
nostics, Indianapolis, United States) in an automated analyzer (Olympus AU400 automated
biochemical analyzer, Olympus Diagnostica GmbH, Ennis, Ireland) [23].
Salivary total protein content was determined using a colorimetric assay (protein in
urine and CSF, Spinreact, Spain) adapted for its use in automatic analyzers (Olympus
UA600 automated biochemical analyzer, Olympus Diagnostica GmbH, Ennis, Ireland)
following the manufacturer’s instructions.

2.4. Statistical Analysis

Data validation was performed by assessing normal distribution (Shapiro–Wilk test),
homoscedasticity (Levene’s test), and sphericity. In cases lacking any of these properties,
and following log transformation, nonsignificant values were obtained in contrast testing,
with all the data being considered valid for analysis. Statistical significance was considered
for p < 0.05. The SPSS version 25.0 statistical package (SPSS Inc., New York, NY, USA) was
used throughout.

3. Results
A total of 64 patients with a mean age of 57.8 years (range 39–83) were divided into
three groups (melatonin, placebo, clonazepam). No statistically differences were detected
between groups in terms of patient age, sex, oral hygiene, or smoking habits, with exception
of alcohol consumption (Table 1).

Table 1. Description and comparison of the demographic data and habits of the study subjects.

Melatonin Clonazepam Placebo p

Gender (%) 19 (82.61) 15 (92) 23 (92)
p > 0.05
FemaleMale 4 (17.39) 1 (6.25) 2 (8)
Age (mean;SD) 57.68 (10.32) 63.81 (10.69) 60.40 (13.8) p > 0.05
Smoking n (%)
6 (26.1) 3 (18.8) 4 (16)
Smoking
13 (56.5) 13 (81.3) 17 (68) p > 0.05
No Smoking
4 (17.4) 0 (0) 4 (16)
Ex Smoking
Alcohol n (%)
11 (47.8) 15 (93.7) 20 (80)
less than once a week
6 (26.1) 0 (0) 2 (8) p < 0.05
Once a day
6 (26.1) 1 (6.3) 3 (12)
weekends
Oral hygiene (brushing)
n (%) 0 (0) 1 (6.3) 1(4)
No 13 (59.9) 7 (43.8) 10 (40)
p > 0.05
Once a day 0(0) 1 (6.3) 1 (4)
Twice a day 9 (39.1) 7 (43.8) 13 (52)
Three times a day
J. Clin. Med. 2022, 11, 2516 6 of 12

The VAS pain for the intensity of the burning sensation as a dependent variable in the
three treatment groups, before and after treatment, evidenced a decrease in the mean score
in all three groups at the end of treatment. In the melatonin group, the mean (±standard
deviation (SD)) score decreased from 7.8 ± 1.5 before treatment to 5.8 ± 2.54 after treatment
(p < 0.001). In the clonazepam group, the score decreased from 8.8 ± 1.2 to 5.5 ± 3.6
(p < 0.01), and in the placebo group, the score decreased from 8.1 ± 1.8 to 7.7 ± 1.9, though
in this case, the difference failed to reach statistical significance (p > 0.05).
With regard to the differences in decreased burning sensations between groups, signif-
icant differences were observed between the administration of melatonin versus placebo
(p = 0.049), but not between clonazepam and the placebo (p = 0.590) or between the admin-
istration of clonazepam and melatonin (p = 0.290).
Likewise, with regard to xerostomia, a decrease was observed in the mean VAS xeros
score in all three groups at the end of treatment, although without reaching statistical
significance in any of them. In the melatonin group, the mean (± standard deviation [SD])
score decreased from 3.73 ± 3.7 before treatment to 3.26 ± 3.12 after treatment. In the
clonazepam group, the score decreased from 4.68 ± 3.53 to 3.56 ± 2.80, and in the placebo
group, the score decreased from 5.06 ± 3.63 to 4.93 ± 3.52.
The saliva drainage test was the dependent variable in all three treatment groups, and
the results were evaluated before and after administration of each of the drugs. An increase
was recorded in the melatonin group (from 3.47 ± 2.46 to 4.13 ± 2.56), with a slight decrease
in the clonazepam group (from 2.65 ± 1.98 to 2.47 ± 1.69), and no changes in the placebo
group (2.85 ± 1.63 before treatment versus 2.85 ± 2.08 after treatment). The difference
proved statistically significant in the melatonin group (p = 0.04). No significant differences
were observed in the comparison of the changes in salivary drainage between the groups
(p > 0.05).
The Oral Health Impact Profile 14 (OHIP-14) corresponding to perceived oral quality
of life evidenced a decrease in the mean score in all three study groups. The largest decrease
was recorded in the clonazepam group (36 ± 10.00 before treatment versus 28.13 ± 8.82
after treatment), followed by the melatonin group (31 ± 7.80 before treatment versus
27.7 ± 8.11 after treatment) and the placebo group (35.5 ± 10.95 before treatment versus
34.48 ± 11.46 after treatment). The decrease proved significant for melatonin (p < 0.001) and
clonazepam (p = 0.001), but not for the placebo (p = 0.133). In turn, significant differences
were recorded between melatonin and the placebo (p = 0.036), but not in the rest of the
comparisons between groups.
The anxiety levels were measured with the Hospital Anxiety and Depression Scale
(HADS). Regarding the items referred to anxiety, the highest scores corresponded to the
clonazepam group, which showed the greatest reductions of anxiety (11.25 ± 4.59 before
treatment versus 9.81 ± 4.32 after treatment), followed by melatonin (9.13 ± 3.84 ver-
sus 8.78 ± 4.17), while the placebo group showed a slight increase (10.24 ± 4.48 versus
10.40 ± 4.32). Despite the variations in mean scores, there were no significant differences
between the pre- and post-treatment scores in any of the groups, or in the comparisons
between groups (p > 0.05 in all cases).
With regard to depression, the melatonin group showed a slight increase in score
(4.17 ± 3.12 before treatment versus 4.22 ± 3.33 after treatment), while the clonazepam
group showed a slight decrease (6.25 ± 4.48 versus 5.81 ± 4.43). A slight increase in the
depression score was recorded in the placebo group (4.60 ± 5.33 before treatment versus
4.76 ± 5.31 after treatment). There were no significant differences between the pre- and
post-treatment scores in any of the groups, or in the comparisons between groups (p > 0.05
in all cases).
The quality of sleep was assessed using the Pittsburg Sleep Quality Index. There
were no significant differences between the first and second visits in any group, though
the clonazepam group showed values very close to statistical significance (p = 0.055). In
turn, the Epworth Daytime Sleepiness Scale showed no significant differences between
J. Clin. Med. 2022, 11, 2516 7 of 12

the pre- and post-treatment sleepiness scores in any of the groups, or in the comparisons
between groups.
The evaluation of the number of locations of burning mouth sensation before and after
each of the treatments showed a decrease in the number of locations in all three groups:
melatonin 3.13 ± 1.45 before treatment versus 2.61 ± 1.46 after treatment; clonazepam
3.60 ± 1.35 versus 2.94 ± 1.84; and placebo 3.92 ± 1.28 versus 3.60 ± 1.44. The difference
was shown to be statistically significant in the melatonin group (p = 0.015). In the compari-
son between treatment groups, significant differences were observed between melatonin
and placebo (p = 0.028).
Eight patients in the global sample (12.5%) presented adverse effects, all of which were
mild: five in the melatonin group and three in the placebo group, with no adverse effects in
the clonazepam group. The recorded adverse effects corresponded to mild gastrointestinal
alterations, and none of them prevented the patients from completing the two months of
the study.
No changes in saliva composition were observed in relation to oxytocin, ferritin,
ADA, amylase, or uric acid before versus after the administration of treatment, though
significantly higher total protein content was recorded in the melatonin group after the
treatment versus pre-treatment (Table 2).

Table 2. Pre vs Post different treatments.

Melatonin Placebo Clonazepam

p p p
Pre Post Pre Post Pre Post
804.4 1581 1417 1627 804.4 1581
Oxytocin 0.339 0.953 0.910
(562–1902) (544.6–2128) (907.6–2491) (768.4–2388) (562–3065) (544.6–2467)
3.1 2.8 3.8 3.7 3.1 2.8
ADA 0.718 0.992 0.945
(0.75–5.7) (0.75–6.575) (1.3–11.1) (1.4–11.5) (0.75–7.525) (0.75–5.65)
5.75 9 8.45 6 5.75
Ferritin 6$(2–13.78) 0.266 0.252 0.219
(3.075–22.18) (4.25–12.33) (2.925–19.7) (2–22.63) (3.075–17.55)
198,760 196,720 181,520 166,960 198,760 196,720
SAA (U/L) (132,680– (111,080– 0.266 (97,400– (81,680– 0.891 (132,680– (111,080– 0.910
368,600) 359,160) 364,520) 232,920) 395,180) 382,800)
121.9 149.6 185.9 130 121.9 149.6
PT 0.043 0.679 0.250
(78.35–208.4) (101.2–222.1) (99.38–309.1) (90.16–332.2) (78.35–183.2) (101.2–217.5)
Note: ADA = adenosine deaminase; SAA, salivary alpha-amylase; Pt Total proteins.

On evaluating the pre- versus post-treatment changes in each clinical variable with
respect to the variations in salivary composition, correlations were observed between
oxytocin and saliva drainage (r = −0.410; p = 0.009), heart rate and salivary amylase
(r = 0.346; p = 0.029), oxygen saturation and salivary amylase (r = 0.419; p = 0.007), and
the HAD-D score and ferritin (r = −0.312; p = 0.05), and ADA and protein total (r = 0.56;
p = 0.001).

4. Discussion
In the present study, melatonin and clonazepam were shown to be effective at reducing
burning mouth sensation and in improving the patient’s quality of life compared with the
placebo. Both melatonin and clonazepam were safe, with no major adverse effects recorded
in the patients with BMS.
Melatonin has been reported to exert beneficial effects upon its target organs, with
anti-inflammatory actions and a reduction of oxidative stress that protects against cell
damage [31,32,45]. Some studies have found melatonin to be able to neutralize free radicals
and protect the mitochondria against oxidative stress, with the restoration of normal
mitochondrial functioning in different inflammatory disease conditions [30,34]. Due to this
range of activities, melatonin has been attributed with neuroprotective, antitumor, and
tissue regeneration properties [30–36].
The only study to date on the application of melatonin in BMS was published by
Varoni et al. [37]. The authors reported no improvement in the intensity of burning sen-
J. Clin. Med. 2022, 11, 2516 8 of 12

sation and no significant differences versus the placebo, despite the administration of
higher doses than in our own study (12 mg/day). This suggests that higher doses do not
necessarily imply improved therapeutic outcomes—though the risk of adverse effects may
increase. In our study, all three treatments (melatonin, clonazepam, and placebo) resulted
in a decrease in the number of sites of burning sensation, though significant differences
were only observed in the melatonin group. In this regard, administration in the form of
soft bands might be a method worth considering.
With regard to saliva drainage, melatonin was associated with a significant increase in
salivary levels following treatment (p = 0.004), coinciding with the observations of other
authors [34]. Melatonin may be useful as a coadjuvant in the treatment of certain disorders
of the oral cavity, improving salivation [34,36]. Since dry mouth is present in many patients
with BMS, melatonin could offer marked improvement of quality of life.
The scientific evidence on the appropriate melatonin dose for the management of sleep
disturbances is limited, with figures ranging between 0.5 and 10 mg [46]. In the present
study, we did not specifically address improvements in the quality of sleep, and the absence
of results of clinical relevance may have been due to the administration of an insufficient
drug dose.
Both anxiety and depression were shown to decrease in the two active drug treatment
groups, while the placebo group exhibited a slight increase in score. The greatest reduction
of anxiety corresponded to clonazepam, though statistical significance was not reached.
In contrast, Varoni et al. [37] documented a significant decrease in anxiety with melatonin
versus a placebo. The differences in results obtained may possibly be explained by the
lower drug dosage used in our study.
The literature offers heterogeneous data regarding the application of clonazepam
in patients with BMS [8,26]. It is one of the drugs of choice for the treatment of sleep
disturbances, although the studies in which clonazepam is used in application to BMS
focus mainly on the reduction of the burning sensation, without addressing the quality
of sleep [43]. The application of clonazepam can afford a rapid analgesic effect that may
prove effective at reducing the burning sensation in BMS through stabilization of the
nerve fiber membrane and cells of the oral mucosa [44–46]. Cui et al. [27], in a review
of the application of clonazepam in BMS, were unable to establish optimum topical or
systemic doses in patients with this syndrome; in this regard, the reported duration, method
of administration and doses of clonazepam used to treat BMS varied. Previous studies
describe dryness, drowsiness, and fatigue as the most common side effects of clonazepam,
and the drug, moreover, may cause dependency [27,44–46]. Adverse effects of melatonin
are not serious and are infrequent; headache and somnolence, nausea, palpitations, and
abdominal pain [47] Circadian clock dysfunction is an emerging area of research that may
underpin many BMS disease manifestations. Pain perception, depression and anxiety, and
sleep disorders are inextricably linked with circadian disturbances. Melatonin may regulate
and restore circadian rhythms [6,30]. Therefore, more investigations into the mechanism of
melatonin in burning mouth syndrome are necessary.
It is important to mention the placebo effect of treatments in BMS. Kuten-Shorrer et al. [48]
published a meta-analysis in which a large percentage of patients (up to 72%) responded
favorably to the application of a placebo. The authors thus suggested that the type of
placebo used in clinical studies on BMS should be standardized, with the conduction of at
least 8 weeks of follow-up, and the inclusion of “no treatment” groups, in order to ensure
greater reliability in evaluating the effectiveness of the studied treatments [48].
Saliva is one of the defense systems of the body and could prove useful as a di-
agnostic fluid. Burning mouth syndrome is a challenge for clinicians; current research
therefore should focus on methods combining physiological parameters, psychological and
behavioral aspects, and the characteristics of saliva [12,18–24,49]. We observed a negative
correlation to drainage and oxytocin when analyzing the changes in the composition of
saliva in relation to the post-treatment clinical parameters. The latter is a neuropeptide
found in the nervous system and has been implicated in pain attenuation. Variations in
J. Clin. Med. 2022, 11, 2516 9 of 12

the endogenous oxytocin system are associated to the morphology of the brain regions
implicated in pain processing and modulation. Oxytocin induces analgesia, and different
studies have shown that the production of this neuropeptide contributes to cope with
situations of stress and anxiety, and strengthens affectivity [19,22]. This is crucial in treating
problems of anxiety and stress. In our study, oxytocin in saliva was seen to increase in all
the groups after treatment, with no significant differences in any of the groups.
In recent years, salivary alpha-amylase (AA) has been shown to be a valid and reliable
indicator of the activity of the autonomic nervous system (ANS) in situations of stress. It
is therefore a biomarker worth considering [18,19,49–51]. High salivary alpha-amylase
levels reflect adrenergic activity secondary to biological–psychosocial stress of the adrenal
gland medullary sympathetic system; salivary AA is therefore considered to be a biological
marker of psychological stress. These systems closely interact with the immune system
and are thus implicated in the development and maintenance of pathological conditions.
Nagler et al. [50] indicated that patients with BMS are chronically exposed to stress because
of the persistent oral pain. In our study, we observed a relationship between salivary
AA and certain clinical parameters, such as heart rate (p = 0.029) and oxygen saturation
(p = 0.007).
The measurement of adenosine deaminase (ADA) can yield information on cell me-
diated immunity. This enzyme hydrolyses adenosine and is widely distributed in the
different body tissues. It has been shown to play a role in the function, maturation, and
maintenance of immune responses. In our study, no association was observed between
ADA and the different clinical parameters evaluated. The increase in adenosine levels
during the course of the day contributes to drowsiness [52]. Due to the close relationship
between mood state and sleep behavior, further research in this field may reveal data on
the progression of different psychological disorders, such as chronic anxiety and stress.
Ferritin is an iron storing protein that plays a key role in cellular oxygen metabolism, and
its measurement has been shown to be useful for assessing intracellular iron status [18,23].
Moreover, in recent years, ferritin has been described as a key immune system molecule,
with immunosuppressive properties and proinflammatory effects, and a number of studies
have demonstrated its role as an acute phase reactant. In our study, ferritin was shown to be
correlated to depression—an observation that requires more in-depth investigation.

5. Study Limitations
In the present study, double-blinding was applied to the administration of melatonin
and a placebo but not to clonazepam, which was administered in the form of drops (oral
solution), since it could not be administered in the form of strips, such as melatonin
or a placebo. Despite the noninvasive nature of salivary analysis, this body fluid has
not yet become popular in research. More in-depth research is therefore required of the
different salivary components, due to the broad interindividual ranges involved, with the
recruitment of larger cohorts and longer term assessments of patient responses to treatment.

6. Conclusions
Melatonin and clonazepam were shown to be effective at reducing the burning sensa-
tion and improving quality of life. Both drugs were found to be safe, with no major adverse
effects in patients with BMS. Melatonin may be regarded as an alternative treatment for VAS
pain patients with BMS, although further studies are needed to confirm its effectiveness
and define the optimum dosage.

Author Contributions: Conceptualization, C.C.-F., A.T. and P.L.-J.; Data curation, C.C.-F.; Formal
analysis, A.T., M.L.-A., L.P.-M. and E.P.-F.; Funding acquisition, C.C.-F.; Investigation, C.C.-F., A.T.
and P.L.-J.; Methodology, C.C.-F. A.T. and P.L.-J.; Project administration, A.T. and P.L.-J.; Resources,
L.P.-M., M.L.-A. and E.P.-F.; Supervision, C.C.-F. A.T. and P.L.-J.; Validation, A.T.; Writing—original
draft, E.P.-F. and P.L.-J.; Writing—review & editing, C.C.-F., A.T., M.L.-A. and P.L.-J. All authors have
read and agreed to the published version of the manuscript.
J. Clin. Med. 2022, 11, 2516 10 of 12

Funding: This research received no external funding.

Institutional Review Board Statement: Bioethics Committee of the University of Murcia reference:
2203/2018.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data is contained within the article.
Conflicts of Interest: The authors declare no conflict of interest.

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