Passali et al. Multidisciplinary Respiratory Medicine (2017) 12:29
DOI 10.1186/s40248-017-0110-7
RESEARCH ARTICLE
Open Access
Nasal Muco-ciliary transport time alteration:
efficacy of 18 B Glycyrrhetinic acid
Desiderio Passali1*, Chiara Cappello1, Giulio Cesare Passali2, Cemal Cingi3, Codrut Sarafoleanu4
and Luisa Maria Bellussi1
Abstract
Background: Mucociliary clearance is the main self-clearing system of the nasal cavity and paranasal sinuses.
This is a very important means of non specific defence against continuous organic and inorganic contamination
conveyed by air. It works by trapping particles and microorganisms in the mucus and then by transporting the
mucous film to the pharynx where it is eliminated with a cough or swallowed. Its congenital or acquired
abnormalities are involved in the occurrence of widespread infectious and often severe nose and paranasal sinuses
diseases; generally concerning the rhinopharyngealtubal district.
Restoring mucociliary clearance of the nasal epithelium when altered thus represents a key therapeutic tool against
rhinosinus chronic diseases.
This study evaluates the clinical efficacy of the inhalation of a natural compound (Narivent® nasal spray) in chronic
vasomotor rhinitis.
Methods: The study involved 79 patients suffering from chronic vasomotor rhinitis presenting an increased
mucociliary clearance time. Patients were randomized into 2 groups: a first group of 49 subjects and a second
group of 30 subjects.
The first group was treated with a nasal spray (Narivent® nasal spray) (2 sprays per nostril twice a day) for 30 days.
The second group was treated with a nasal spray containing isotonic solution in the same way and for the same
period of the first group.
Nasal Mucociliary transport time was measured in the patients of both groups before treatment, after 15 days of
treatment, and at the end of the 30 days treatment.
Results: The study shows how one of the treatments carried out determines a major objective reduction of the
mucociliary clearance time in the patients under examination, using the method which involves the use of an
insoluble coloured tracer (vegetable carbon), bringing the values back within normal range. At the end of the study
we objectivated an increase in the rate of mucociliary transport in 97.9% percentage of patients we enrolled.
Conclusions: This study shows the effectiveness of treatment with natural extracts with nasal mucosa restoring
function in the treatment of chronic vasomotor rhinitis, a nasal inflammatory disease characterized by
morphological and functional alteration of the normal nasal mucosa.
Keywords: MCT, Glycyrrhetinic acid, HMGB1, Sirt 6, Nasal inflammation
* Correspondence: d.passali@virgilio.it
1
ENT Clinic, Policlinico S. M. alle Scotte, Siena, Italy
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
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Passali et al. Multidisciplinary Respiratory Medicine (2017) 12:29
Background
The mucosa of the nose and paranasal sinuses is formed
by a respiratory cylindrical motile epithelium, completely
analogous to the one of the trachea and bronchi.
Among the cells with cilia which, with their undulating
movement, sweep nasal secretions from the outside towards the inside, there are numerous serummucous
glands that produce nasal mucus, which has the function
of retaining the dust particles inhaled and releasing
moisture and heat to the breathed in air.
Nasal mucus is rich in glycoproteins and contains numerous molecules (IgA, lysozyme, defensins etc.) which contribute to a joint specific and nonspecific defence of the nasal
epithelium. Its rheological properties influence the smooth
operation of that function called mucociliary clearance.
This clearance particularly takes advantage of the mucociliary transport (MCT) system which allows all secretions
to be pushed by the ciliated cells towards the back of the
nasal passages to then descend into the pharynx.
The absence or deficiency of this self-clearing system
of the nasal cavity and paranasal sinuses can constitute
evidence or be the consequence of many diseases.
The present study was conducted to evaluate the clinical efficacy of nasal inhalation of a medical device (nasal
spray) in selected patients with chronic vasomotor rhinitis, a nasal pathology which is characterised by an increased mucociliary transport time.
Narivent® is a natural compound with antioedema and
anti-inflammatory osmotic properties mainly consisting
of Mannitol, Glycerol and Dipotassium Glycyrrhizate.
The treatment effectiveness was evaluated following
objective methods, with the measurement of nasal MCT
speed before, during and after treatment.
During the recruitment and follow up visits, samples
of nasal mucosa cells were taken by scraping, subsequently examined by optical microscope.
Materials and methods
Patients
The study involved 79 subjects suffering from chronic
vasomotor rhinitis with increased mucociliary clearance
time from a minimum of 24 min to over 30 min, between the ages of 16 and 61 years, of both genders. The
Page 2 of 6
subjects were randomised into two groups of 49 (first
group) and 30 (second group) subjects.
The main recruitment criterion was the presence of
chronic vasomotor rhinitis not associated with nasal
polyposis but associated with a deficiency of mucociliary
clearance. No other drugs were allowed during the treatment and in the 30 days before the beginning of the study.
The study was conducted by administering two sprays
per nostril twice a day for 30 days the drug1 in the first
group and an isotonic solution in the second group.
At the time of the recruitment visit, all personal data
and clinical history were collected and ENT examination
with a fiberoptic examination of the nasal cavity and
mucociliary transport test (condensed results are reported
in Table 1) were performed.
Patients of both groups were assessed before and after
treatment by measuring MCT time.
A nasal scraping was performed before and after treatment on some consenting subjects (12 cases).
All patients recruited in the study were previously informed and gave their consent to participate in this study.
Mucociliary clearance
Patients with rhinosinus pathologies often exhibit a decreased mucociliary clearance, evaluated as an increase
in the mucociliary transport time, expressed in minutes.
The MCT time is calculated in our clinic by placing a
tracking powder (vegetal charcoal powder) to the head of
the inferior turbinate [1]; through direct pharyngoscopy it
is then possible to detect the transit of the tracer from the
posterior pharyngeal wall. The test requires quite some collaboration by the patient, who must keep a seated position
without either blowing or sniffing. The charcoal powder
used as a tracer, as it is insoluble and clearly visible in a
pink environment such as that of the nasopharyngeal area,
behaves as an inert material, adhering to the mucosa and
transported passively by the movement of mucus and cilia.
Our experience shows that this method for the measurement of MCT, compared to others at our provision such as
isotopic measurement with iodine 131 / technitium 99, is
reliable, low invasive and not expensive [1].
Normally, the tracer is found in the pharynx after about
10–15 min [2], but there are intersubjective and subjective
variations, and only values higher than 25 min are
Table 1 Patients data
Mean MCT
Group 1 (49 Patients)
Group 2 (30 Patients)
Responders
Normalization of MCT
σ
σ2
Before treatment
28.7
–
–
1787
3194
After 15 days of treatment
19.1
85.7% (42)
75.5% (37)
6542
42,802
After 30 days of treatment
15.3
97.9% (48)
97.9% (48)
4234
17,934
Before treatment
27.9
–
–
1905
3632
After 15 days of treatment
26
56.66% (17)
13.3% (4)
4262
18,165
After 30 days of treatment
24.3
83.33% (25)
30% (9)
4453
19,832
Passali et al. Multidisciplinary Respiratory Medicine (2017) 12:29
considered pathological, and for values exceeding 30 min
the mucociliary clearance is considered “blocked”.
The MCT time is calculated for each patient as the
average of the values obtained in both nasal cavities.
Analysis of the samples
Nasal scraping was carried out at the same time as the
MCT test in 12 cases.
Page 3 of 6
Table 2 Results: ANOVA test in both groups and final
comparative T-Test
First group ANOVA test
Second group ANOVA test
f-ratio = 108,02377
f-ratio = 6,78,346
p <0,00001
p = 0.001829 (<0.05)
Comparison between groups
t = 8,91,363
p <0.00001
Collection and analysis of cytology specimens
In this study, the collection of cytology specimens was
performed noninvasively, by nasal scraping. After having
the patient blown his/her nose in order to eliminate any
excess secretions, the top layer of the mucous membrane at
the level of the middle third of the inferior turbinate was removed bilaterally. An appropriate plastic curette was used.
A sample was collected both from the right and from the
left nostril for each patient.
The cellular material was then transferred and laid,
swiping it as uniformly as possible, on a slide.
Staining
We used the May Grunwald Giemsa rapid staining procedure (quick stain).
The test was carried out as follows: after drying the
smear at room temperature, the slide is immersed 5
times for 1 s in the three solutions, one after the other,
washing it under running water.
With this staining procedure the nuclei became purple,
cytoplasmic filaments pink, eosinophil granules orange.
Microscopic observation
Examination was carried out with a Nikon optical
microscope at 100× magnification.
Each slide was evaluated dividing it into 6 equal fields
in order to count the amount of each cell type per
microscopic field. A comparison between the cells
amount before and after treatment was then carried on.
Statistical analysis
A clinical study in vivo was carried out making on a comparison between pre and after treatment phase with a mid
therapy course assessment, based on objective clinical
criteria.
The statistical significance of the results was assessed
by a one way ANOVA analysis and two tailed T-Test.
Results
The analysis of the results was conducted by analysing
only those subjects who had completed the treatment
and had carried out the recruitment and control visits.
The condensed data collected in this study are listed
in Table 1.
The analysis of the results is shown in Table 2.
In both groups of patients included in this study the
average mucociliary transport time (MCT) detected
during/before treatment was around 28 min. (28.8 min.
in the first group and 28 min. in the second group) with
a minimum value of 24 min.
31 out of the 49 cases included in the first group
of study (63.3%) and 11 out of the 30 subjects included
in the second group (36.7%) had a blocked MCT
(> 30 min.).
At the end of the treatment no adverse reactions were
reported.
In the first group the results were the following.
A MCT mean time of 19.2 min. with a minimum value
of 8 min. was found at the first follow up exam, performed after 15 days of continuous therapy, thus a mean
decrease of 9.6 min. representing a 33.3% decrease from
the starting value. The percentage of responder patients
was equal to 85.7%. In 75.5% of the examined cases we
witnessed a normalisation of the MCT time. In 5 cases,
MCT time remained blocked.
The results of the final follow up exam, carried out
after 30 days, at the end of therapy, show an average
MCT time of 15.3 min. with a minimum value of
10 min., a final average decrease of 13.4 min. representing a 46.7% decrease from the starting value and of 20%
compared with the previous follow up. The percentage
of patients where there was an increase in the rate of
mucociliary clearance was 97.9%. Only in one case we
observed persisted in having a MCT time > 30 min.
Therefore, the treatment was successful in 97.9% of
cases, bringing the MCT time within the threshold of normality in 48 out of 49 cases. Almost all patients answering
to a simple questionnaire referred a favourable opinion on
the treatment and reported an improvement in their nasal
symptoms and especially on nasal obstruction almost accordingly with the MCT time improvement.
At the end of the observation period there was only
one non responder patient documented.
The data analysis performed using the ANOVA
method was statistically significant (p <0.00001).
In the second group the results were the following.
A MCT mean time of 26 min. with a minimum value
of 10 min. was found at the first follow up exam, performed after 15 days of continuous therapy, thus a mean
Passali et al. Multidisciplinary Respiratory Medicine (2017) 12:29
decrease of 1.9 min. representing a 6.9% decrease from
the starting value. The percentage of responder patients
was equal to 56.6%. In 13.3% of the examined cases we
witnessed a normalisation of the MCT time. Only in one
case MCT time remained blocked.
The results of the final follow up exam, carried out after
30 days, at the end of therapy, show an average MCT time
of 24.4 min. with a minimum value of 10 min., a final average decrease of 3.6 min. representing a 12.9% decrease from
the starting value and of 13.8% compared with the previous
follow up. The percentage of patients where there was an increase in the rate of mucociliary clearance was 83.3%.
The treatment was completely successful in 30% of
cases, bringing the MCT time within the threshold of
normality in 9 out of 30 cases.
At the end of the observation period there were four
non responder patients documented.
The data analysis performed using the ANOVA
method was statistically significant (p <0.05).
Discussion
Some studies report how the effects of the most
common formulations of nasal sprays and solutions used
in clinical practice for treating patients suffering from
nose/sinus disorders (isotonic or hypertonic saline solution, Ringer’s lactate and cortisone formulations) result
in an improvement in the symptoms and quality of life,
but not in a significant decrease in mucociliary transport
time [3, 4].
A 2008 systematic review published in the Cochrane Library analysed more than 2000 articles, including 64 clinical
trials, on the efficacy of nasal washes to treat the symptoms
of chronic rhinosinusitis. The conclusions highlighted that
the use of hypertonic saline solution promotes mucociliary
clearance, but there is no evidence of a greater effectiveness
compared with the isotonic solution or with other agents in
relieving rhinosinusitis symptoms [5].
In this regard, a trial on human nasal mucosa models
carried out by Kim and colleagues shows that given the
lack of a direct correlation between ciliary movement frequency in vitro and mucociliary clearance in vivo and the
presence of contradictory results in the literature on the
Page 4 of 6
effects of irrigation with various concentrations of saline
solutions on ciliary activity, the most important factors
which determine mucociliary clearance are the quantity
and the rheological properties of the secreted mucus [6].
This study also states that "the isotonic saline solution is
the most physiologic irrigation solution in terms of cell
morphology and mucin secretion from airway epithelial
cells". Washes of distilled water or hypotonic solutions, in
fact, harm the integrity of the cell junctions creating disruptions in the mucosal barrier, promoting the secretion
of large amounts of mucus and decreasing the total number of ciliated cells, while the action in vitro of hypertonic
saline solutions promotes the secretion of mucins, albeit
without damaging the nasal mucosa significantly from a
morphological point of view.
The optical microscope analysis of the nasal mucosa
samples obtained from 12 subject by scraping highlights
the normalization of the after treatment nasal cytology
(Fig. 1a-1b and 2a-2b).
The therapy cycle was well tolerated, with no side effects reported by patients.
This study allows underlining how the administration
of the medical device under consideration improves an
important nasal functionality parameter, such as mucociliary clearance is and should be regarded. In addition,
it shows that this improvement is statistically significant
in comparison with the improvement achieved in the
second group (p <0.05).
The reason for this beneficial effect is justified by our
present knowledge on the importance of HMGB1 (High
Mobility Group Box 1) during an inflammatory process and
on the inhibiting action of glycyrrhetinic acid on it [7–14].
HMGB1 is a non histonic nuclear protein that is primarly expressed in the nucleus of normal nasal mucosa
cells. Higher extracellular levels and lower nuclear levels
of HMGB1 are normally found in chronic inflammatory
conditions [15].
It has been recently observed that HMGB1 extracellular release is related to the nuclear concentration of Sirt
6, a protein member of Sirtuines family, NAD+
dependent class III histone deacetylases closely correlated to inflammatory conditions [15–17].
Fig. 1 a Neutrophil rhinopathy before treatment. b After treatment normalization of rhinocytology: ciliate cells of nasal epithelium
Passali et al. Multidisciplinary Respiratory Medicine (2017) 12:29
Page 5 of 6
Fig. 2 a Before treatment muciparous rhinopathy. b After treatment: restoration of physiological rhinocytology with normal ciliate cells
Sirt 6 is found in the nucleus of normal nasal mucosa
cells. It promotes epithelialization of nasal mucosa. A depletion of this protein induces a dramatic translocation of
HMGB1 from nucleus to cytoplasm and leads to the suppression of the number of human nasal epithelial cell cilia
[15]. It has recently been observed [15] that glycyrrhetinic
acid increases also Sirt6 protein levels and activity.
This study was approved by CEAVSE Ethic Committee (Comitato Etico di Area
Vasta Sud Est dell’Azienda Ospedaliere Universitaria Senese), on April 22, 2014
with protocol number n°3.
Conclusions
This study shows the effectiveness of a natural extract in
the treatment of chronic vasomotor rhinitis. Glycyrrhetinic acid is a specific medical compound that may be isolated from the licorice plant that binds selectively to
HMGB1 protein released extracellularly and so inhibits
its cytokine activities through a scavenger mechanism
on the protein accumulation [7, 15]. Glycyrrhetinic acid
increases also Sirt6 protein levels and activity, decreasing
HMGB1 release and promoting nasal epithelial cells differentiation stimulating ri-epithelialization of nasal mucosa and increasing the number of cells cilia.
Publisher’s Note
Endnotes
1
Narivent® nasal spray
Abbreviations
HMGB1: High Mobility Group Box 1; MCT: Muco-Ciliary Transport
Acknowledgements
Not applicable.
Funding
The authors declare that they had no founding.
Availability of data and materials
All data generated or analysed during this study are included in this
published article.
Authors’ contributions
DP: General managing and writing of the manuscript. CC2: Statistical analysis
and writing of the manuscript. GCP: Interpretation of patients’ data and
discussion. CC4: Clinical data and collection of cytology specimens collection.
CS: Clinical data and collection of cytology specimens collection.
LMB: Statistical analysis and interpretation of patients’ data. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
All patients recruited in the study were previously informed and gave their
consent to participate in this study.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
ENT Clinic, Policlinico S. M. alle Scotte, Siena, Italy. 2ENT Department, Sacred
Heart University, Rome, Italy. 3ENT Department, Faculty of Medicine, Eskisehir
Osmangazi University, Eskisehir, Turkey. 4ENT Dept, Santa Maria Hospital
University of Medicine and Pharmacy, Bucharest, Romania.
Received: 30 August 2017 Accepted: 23 October 2017
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