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Antibacterial oral sprays from Kaffir lime ( DC.) fruit peel oil and leaf
Citrus hystrix
oil and their activities against respiratory tract pathogens
Somporn Srifuengfung, Nuntavan Bunyapraphatsara, Veena Satitpatipan, Chanwit
Tribuddharat, Varaporn Buraphacheep Junyaprasert, Walla Tungrugsasut, Vimol Srisukh
PII: S2225-4110(18)31036-8
DOI: https://doi.org/10.1016/j.jtcme.2019.09.003
Reference: JTCME 357
To appear in:
Journal of Traditional and Complementary Medicine
Received Date: 07 March 2019
Accepted Date: 12 September 2019
Please cite this article as: Somporn Srifuengfung, Nuntavan Bunyapraphatsara, Veena
Satitpatipan, Chanwit Tribuddharat, Varaporn Buraphacheep Junyaprasert, Walla Tungrugsasut,
Vimol Srisukh, Antibacterial oral sprays from Kaffir lime ( DC.) fruit peel oil and leaf oil
Citrus hystrix
and their activities against respiratory tract pathogens,
Journal of Traditional and Complementary
(2019),
Medicine
https://doi.org/10.1016/j.jtcme.2019.09.003
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© 2019 Published by Elsevier.
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Antibacterial oral sprays from Kaffir lime (Citrus hystrix DC.) fruit peel oil and leaf oil and their
activities against respiratory tract pathogens
Somporn Srifuengfunga, Nuntavan Bunyapraphatsarab, Veena Satitpatipanb, Chanwit
Tribuddharata, Varaporn Buraphacheep Junyaprasertc, Walla Tungrugsasutd, Vimol Srisukhd*
aFaculty of Medicine, Siam University, Phet Kasem Road, Phasi Charoen District,
Bangkok 10160, Thailand.
bDepartment of Pharmacognosy, Faculty of Pharmacy, Mahidol University,
447, Sri-Ayutthaya Road, Bangkok 10400, Thailand.
cDepartment of Pharmacy, Faculty of Pharmacy, Mahidol University,
447, Sri-Ayutthaya Road, Bangkok 10400, Thailand.
dDepartment of Food Chemistry, Faculty of Pharmacy, Mahidol University,
447, Sri-Ayutthaya Road, Bangkok 10400, Thailand.
*Corresponding author.
E-mail address: vimol.sri@mahidol.ac.th (V. Srisukh).
Conflict of Interest Statement
Authors declare that there is no conflict of interest regarding the publication of this paper.
List of Abbreviations
KLO = Kaffir lime oil; KLLO = Kaffir lime leaf oil; MIC = Minimal inhibitory
concentration; MBC = Minimal bactericidal concentration; ˚C = Degree Celsius; mg =
milligram; g = gram; ml = milliliter; μl = microliter; %v/v = percent in volume by volume; μm =
micrometer; mm = millimeter; ppm = part per million; min = minute.
Keywords:
Essential oil; Alpha-terpineol; Terpinene-4-ol; Citronellal; Pharyngitis; Sore throat
Highlights of the findings and novelties:
Clear antibacterial oral sprays were prepared.
Active major compounds were alpha-terpineol, terpinene-4-ol, and citronellal.
The sprays showed strongest activities against Haemophilus influenzae ATCC 49247.
The antibacterial activity was shown throughout 4-month storage.
Type of Article:
Short Communication
Length of the Manuscript
Title: 127 characters; Abstract: 220 words; Text: 2981 words; References: 15; Figures
and Tables: 3; Supplementary files: none.
Section:
1. Natural Products
Taxonomy (classification by EVISE): Respiratory System Disease; Analytical Chemistry;
Infectious Disease; Disease
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Antibacterial oral sprays from Kaffir lime (Citrus hystrix DC.) fruit peel oil and leaf oil
and their activities against respiratory tract pathogens
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ABSTRACT
Background and Aim: Kaffir lime fruit peel oil and Kaffir lime leaf oil have been
reported for their activities against respiratory tract pathogens. The purpose of the study
was to develop clear oral sprays to be used as a first-defense oral spray.
Experimental procedure: Clear antibacterial oral sprays were prepared and analyzed for
their respective active major compounds, using GC-MS. The sprays were tested against a
Gr. A streptococcal clinical isolate and 3 standard respiratory tract pathogens, using
Broth microdilution method. A 4-month stability test was carried out as well.
Results and Conclusion: Six clear oral sprays, three formulae composed of Kaffir lime
fruit peel oil (6, 10, 13%v/v KLO) and the other three formulae containing Kaffir lime
leaf oil (4, 8, 12%v/v KLLO), were developed. The active compounds in KLO were α-
terpineol and terpinene-4-ol whereas that in KLLO was citronellal. All oral sprays
exhibited antibacterial activity against one Group A streptococcal clinical isolate and
three respiratory pathogenic pathogens, Staphylococcus aureus ATCC 29213,
Streptococcus pneumoniae ATCC 49619, and Haemophilus influenzae ATCC 49247,
among which the strongest activity was against H. influenzae ATCC 49247. The
antibacterial activity of all oral sprays remained unchanged in an accelerated stability test,
at 4, 30, and 45°C under 75% relative humidity, throughout the 4-month storage.
Keywords:
Essential oil; Alpha-terpineol; Terpinene-4-ol; Citronellal; Pharyngitis; Sore throat
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Graphical abstract
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1.Introduction
Kaffir lime is a member of the genus Citrus, family Rutaceae.1 The scientific
name is Citrus hystrix DC. Common names of Citrus hystrix DC are kaffir lime, leech
lime and makrut (Thai).2 Kaffir lime leaves have been used in Southeast Asian recipes
since they provide unique and strong aroma2.
Analyses by using gas chromatography-mass spectrometry (GC-MS), with
headspace SPME-GC-MS techniques showed that the volatile compounds in Kaffir lime
belonged to the terpenoids group. Related biological and pharmacological effects
included cardioprotective, hepatoprotective,3 anticholinesterase activities,4 antibacterial
effect,5etc.
In a previous study, Kaffir lime fruit peel oil, a volatile oil from fruit peel of C.
hystrix DC., was analyzed for its constituents, using GC-MS. The major constituents
were l-limonene, -terpineol, 2--pinene, terpinene-4-ol, -terpinene, -terpinene, and
–terpinolene. The minimal inhibitory concentration (MIC) against Bacillus subtilis
ATCC 6633, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and
Salmonella typhimurium ATCC 13311 were 0.1, 0.3, 0.4, and 0.6% v/v, respectively.6 In
another study, two essential oils from C. hystrix DC., Kaffir lime fruit peel oil and Kaffir
lime leaf oil were reported to exhibit the antibacterial activities against 411 isolates of
groups A, B, C, F, G streptococci, S. pneumoniae, H. influenzae, S. aureus (methicillin-
resistant and methicillin-sensitive S. aureus) and Acinetobacter baumannii, obtained from
patients with respiratory tract infections, with MIC ranges of 0.03-17.40 and 0.06-68
mg/ml, respectively. The major compounds in Kaffir lime fruit peel oil were α-terpineol,
terpinene-4-ol, and l-limonene whereas Kaffir lime leaf oil contained citronellal as the
major compound.7
Sore throat (pharyngitis) has an infectious or non-infectious etiology.11 Most cases
of sore throat are infectious.9 In a mild to moderate stages which do not require
antibiotics prescription, the patients are often prescribed a mild antiseptic/anti-
inflammatory spray to reduce the pain and the mild infection. This study aimed to
develop antibacterial oral sprays from Kaffir lime fruit peel oil and Kaffir lime leaf oil
and to test the activity of these sprays against the bacteria that caused sore throat.
2. Materials and methods
2.1. Chemical analysis of the constituents of Kaffir lime fruit peel oil (KLO) and Kaffir
lime leaf oil (KLLO)
Kaffir lime fruit peel oil (KLO) (Batch no. 5209234/2009; density 0.87 g/ml) and
Kaffir lime leaf oil (KLLO) (Batch no. 5209234-1/2009) were purchased from Thai
China Flavours and Fragrances Co. Ltd. and stored at 4°C. The products were prepared
by steam distillation. The diluted oils (100 ppm) were analyzed by gas chromatography-
mass spectrometry (GC-MS)10, using a Hewlett-Packard HP 6890 Series GC System and
Hewlett-Packard HP 5973 Mass selective detector, as stated in details in our previous
study7. The standard compounds in analytical grade, citronellal (density = 0.86 g/ml),
limonene (density = 0.84 g/ml), terpinene-4-ol (density = 0.93 g/ml), α-terpineol (density
= 0.94 g/ml) were purchased from Sigma Chemical Co, USA.
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2.2. Preparation of antibacterial clear oral sprays
KLO was formulated into 6, 10, and 13% v/v clear oral sprays, using a
solubilizing agent and a co-solvent; with the addition of flavoring agents and coloring
agents. KLLO was also formulated into 4, 8, 12% v/v clear sprays, in the same manner.
2.3. Chemical analysis of KLO oral sprays and KLLO oral sprays
Three KLO oral sprays and three KLLO oral sprays were extracted, using hexane,
at the proportion of 1:4 (oral spray: hexane) in separatory funnels for 5 times. The hexane
extracts were combined and analyzed with GC-MS as in 2.1. The analysis was carried
out in triplicate, comparing with the corresponding standard curves, α-terpineol (0.5, 1, 3,
5, 8, and 10 ppm) and citronellal (5, 10, 20, 40, 60, and 80 ppm), for KLO and KLLO,
respectively.
2.4. Antibacterial activity of KLO oral sprays and KLLO oral sprays
All six oral sprays were diluted and tested for their antibacterial activity, using
broth microdilution method according to the standard microbial techniques11,12.. One
Group A streptococcal clinical isolate was obtained from respiratory tract specimens of
the patients with respiratory symptoms at Siriraj Hospital, a tertiary care center in
Bangkok, as previously described7. The specimens were discarded samples from the
hospital’s routine laboratory identification use with no links to the source/names and thus
exempted from the ethical committee approval. Three standard strains7, Streptococcus
pneumoniae ATCC 49619, Staphylococcus aureus ATCC 29213, and Haemophilus
influenzae ATCC 49247 were also used. MIC and MBC of all samples were recorded.
2.5. Stability test
2.5.1. Antibacterial activity of KLO oral sprays and KLLO oral sprays during 4-month
storage
All six oral sprays were transferred into 10-ml injection bottles, crimped, and
stored at 4, 30 and 45°C, under 75% relative humidity in closed containers. Samples were
collected at 1, 2, 3, and 4 months for the antibacterial testing, using broth microdilution
method according to standard microbiological techniques11,12. The bacteria used were the
same as in 2.4. MIC and MBC of the oral sprays were recorded.
2.5.2. Correlation between active compounds and their antibacterial activity of selected
KLO oral sprays and KLLO oral sprays during a 4-month accelerated stability test
Selected formulae (10%v/v KLO and 12%v/v KLLO) were collected at 0, 1, 2, 3,
and 4 months, extracted and determined by comparing with the standard curves of the
corresponding markers (α-terpineol and citronellal, respectively), using GC-MS, under
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the condition as previously described. Contents of the markers were compared with the
antibacterial activity results of both oral sprays at 0, 1, 2, 3, and 4 months.
3. Result and discussion
3.1. Chemical constituents of KLO and KLLO
From GC-MS analysis of KLO, the essential oil consisted of 3 major constituents,
l-limonene (40.65%), terpinene-4-ol (13.71%), α-terpineol (13.20%), and other
constituents (Table 1). In our previous study,7 α-terpineol showed the strongest
antibacterial activity (MIC range = 0.07-2.40 mg/ml) and was chosen as a marker for the
subsequent GC-MS analysis of KLO oral sprays.
For KLLO, citronellal (80.04%) was the major constituent (Table 1). In our
previous study,7 citronellal exhibited a strong antibacterial activity against pathogens,
with the MIC range of 0.3-1.1 mg/ml, and was chosen to be the marker for subsequent
GC-MS analysis of KLLO oral sprays.
Table 1
Constituents of Kaffir lime fruit peel oil and Kaffir lime leaf oil as analyzed by
GC-MS.
Kaffir lime fruit peel oil
Kaffir lime leaf oil
Peak
number
Retention
time(min)
Constituents
Area
(%)
Peak
number
Retention
time(min)
Constituents
Area
(%)
1
7.69
unidentified
3.47
1
8.05
l-limonene
0.52
2
8.06
l-limonene
40.65
2
8.50
trans-beta-ocimene
0.38
3
8.93
gamma-terpinene
7.24
3
8.92
gamma-terpinene
0.20
4
9.31
linalool oxide
1.73
4
9.30
linalool oxide
0.54
5
9.80
alpha-terpinolene
5.80
5
9.80
cis-carane-cis-4-ol
0.31
6
10.19
linalool
0.60
6
10.18
linalool
3.04
7
10.92
D-fenchyl alcohol
0.48
7
11.97
citronellal
80.04
8
11.43
3-terpinen-1-ol
0.20
8
12.25
isopulegol
1.29
9
11.90
isopulegol
4.38
9
12.97
terpinene-4-ol
0.94
10
12.23
isopulegol
1.95
10
13.44
alpha-terpineol
0.29
11
12.69
borneol
0.45
11
14.43
beta-citronellol
4.13
12
12.97
terpinene-4-ol
13.71
12
18.55
citronellol acetate
5.46
13
13.44
alpha-terpineol
13.20
13
18.84
unidentified
0.08
14
14.42
beta-citronellol
0.45
14
19.48
neryl acetate
0.70
15
18.55
citronellyl
propionate
0.55
15
20.95
caryophyllene
0.89
16
19.51
alpha-copaene
1.56
16
22.08
alpha-humulene
0.14
17
19.90
unidentified
0.61
17
23.31
bicyclogermacrene
0.33
18
20.94
caryophyllene
0.69
18
23.96
cadinene
0.23
19
22.07
alpha-humulene
0.22
19
25.21
nerolidol
0.49
20
22.86
germacrene D
0.36
21
23.96
delta-cadinene
1.69
3.2. Preparations and chemical analysis of KLO oral sprays and KLLO oral sprays
All oral sprays were clear. It was shown that 6, 10, 13%v/v KLO contained 3.06,
4.28, and 5.11 ppm of α-terpineol, respectively, whereas 4, 8, 12%v/v KLLO contained
53.90, 88.77, and 148.32 ppm of citronellal, respectively. The attempt was to try to obtain
the highest concentrations of both essential oils with acceptable aroma and also the
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lowest concentration that would effectively work against the pathogens, thus 3 varying
formulae for each essential oil were developed. Strong undesirable flavor and odor of
both oils were completely masked. Future sensory evaluation is needed to confirm the
desirable attributes of the oral sprays.
3.3. Antibacterial activity of KLO oral sprays and KLLO oral sprays
The antibacterial activity of all oral sprays was shown in Table 2. All oral sprays
showed the strongest antibacterial activity against H. influenzae. Upon converting the unit
of MIC and MBC of the three KLO oral sprays into “mg of KLO/ml”, the MIC and MBC
ranges against all tested bacteria were 0.26-13.92 and 0.28-18.10 mg KLO/ml,
respectively. They were compared to those of the essential oil KLO (MIC(MBC) = 0.03-
17.40 mg of KLO/ml) in the previous study7. As for the three KLLO oral sprays, the
converted MIC(MBC) range was 0.26-8.26 mg KLLO/ml. The range was compared to
those of the essential oil KLLO in the previous study7(MIC(MBC) = 0.03-68 mg
KLLO/ml). The sprays showed slightly weaker antibacterial effect than their
corresponding essential oils.
Table 2
Antibacterial activity of three Kaffir lime fruit peel oil oral sprays and three Kaffir lime
leaf oil oral sprays against one Group A streptococcal clinical isolate and 3 standard
strains of respiratory tract pathogenic bacteria, using broth microdilution method.
Group A
streptococcal
clinical isolate
S. aureus
ATCC 29213
S. pneumoniae
ATCC 49619
H. influenzae ATCC
49247
Oral sprays
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
6%v/v KLO
4
4
16
16
2
2
0.5
1
10%v/v KLO
4
4
16
16
1
1
0.5
0.5
13%v/v KLO
4
4
8
16
1
1
0.25
0.25
4%v/v KLLO
8
8
8
8
4
4
0.5
0.5
8%v/v KLLO
4
4
8
8
0.5
0.5
0.25
0.5
12%v/v KLLO
4
4
8
8
0.25
0.25
1
0.5
Concentration in µl of oral spray/ 100 µl of broth
3.4. Stability test
3.4.1. Antibacterial activity of KLO oral sprays and KLLO oral sprays during a 4-month
accelerated stability test.
All oral sprays were stored at 4, 30 and 45°C, under 75% relative humidity. The
ranges of MIC and MBC of the six oral sprays, covering all 3 temperatures, were 0.06 –
16 and 0.125 – 32 µl of oral spray/100 µl of broth, respectively. The MIC and MBC
ranges (combining the three temperatures) of the six oral sprays at 1, 2, 3, and 4 months
were compared with those at initial. Except for those against S. aureus which were
slightly weaker than those at initial, the results implied that the antibacterial activities of
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all oral sprays were mostly retained at 4, 30 and 45°C, under 75% relative humidity, for
at least 4 months.
3.4.2. Correlation between active compounds and their antibacterial activity of selected
KLO oral sprays and KLLO oral sprays during a 4-month accelerated stability test
The results were as shown in Table 3.
Ten-percent v/v KLO oral sprays. There was no significant changes (p>0.05) in
the active compound, α-terpineol, during the first two months at 4 and 30°C. By the end
of 4 months, the active compound decreased (p<0.05) to 82.01% and 74.77%, at 4 and
30°C, respectively. Contrastingly, at 45°C, the active compound concentration decreased
drastically. The MIC and MBC of the samples during the 4-month storage were mostly
retained, with some exceptions. Other compounds (Table 1) that might be contributing to
the antibacterial effect included l-limonene7, terpinene-4-ol7,13, ɤ-terpinene14,
isopulegol14, delta-cadinene15, etc.
Twelve-percent v/v KLLO oral spray. The active compound, citronellal, was more
stable at 4°C; there were only slight decreases (p<0.05) of citronellal during the first 3
months. At 30 and 45°C, the concentrations at one month decreased more than at 4°C. By
the end of 4 months, the active compound decreased to 80.06, 60.82, and 33.54%
(p<0.05), at 4, 30 and 45°C, respectively. Similarly, the oral spray still maintained its
antibacterial activity throughout the storage time of 4 months. Other compounds (Table
1) that might be contributing to the antibacterial effect included beta-citronellol13,
linalool13,14, isopulegol14, etc.
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Table 3
Correlation between the active compounds (alpha-terpineol/citronellal) and the antibacterial activity of
Kaffir lime fruit peel oil oral spray (10%v/v KLO) and Kaffir lime leaf oil (12%v/v KLLO) against Gr. A
streptococcal clinical isolate, S. aureus ATCC 29213, S. pneumoniae ATCC 49619, and H. influenza
ATCC 49247, during 4-month storage, at 4, 30, and 45˚C, under 75% relative humidity
Bacterial strains
Gr.A
streptococcal
clinical isolate
S. aureus
ATCC 29213
S. pneumoniae
ATCC 49619
H. influenzae
ATCC 49247
Test
formulae
Storage
temp.
(°C)
Storage
time
(months)
Active compounds*
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
0
4.28+0.02 (100%) A
4
4
16
16
1
1
0.5
0.5
4
1
4.22+0.07 (98.60%) A
8
8
8
16
2
2
0.25
0.25
2
3.88+0.67 (90.65%)ABC
2
2
8
16
0.5
0.5
1
1
3
3.58+0.54 (83.64%)BCD
2
2
4
8
0.25
0.5
0.25
0.25
4
3.51+0.04 (82.01%) CD
4
4
4
16
0.25
0.5
0.25
0.5
30
1
4.13+0.07 (96.50%) AB
8
8
8
8
1
1
0.125
0.125
2
3.75+0.214 (87.62%) ABCD
2
2
4
16
1
2
0.5
0.5
3
3.56+0.23 (83.18%) BCD
2
2
4
16
0.5
2
0.25
0.25
4
3.20+0.31 (74.77%) D
2
4
4
8
0.5
0.5
0.5
0.5
45
1
2.53+0.41 (59.11%) E
8
8
8
8
2
2
0.25
0.25
2
2.25+0.21 (52.57%) EF
2
2
16
16
0.5
1
0.5
0.5
3
2.04+0.21 (47.66%) EF
8
4
16
16
0.5
0.5
0.5
1
10% v/v
KLO
4
1.75+0.32 (40.89%) F
4
4
8
32
0.5
0.5
0.5
1
0
148.32+1.57 (100%) a
4
4
8
8
0.25
0.25
1
0.5
4
1
144.68+2.69 (97.55%) b
8
8
8
8
1
1
0.25
0.25
2
144.65+1.07 (97.53%) b
2
2
4
8
0.5
0.5
0.06
0.25
3
144.65+1.07 (97.53%) b
2
2
4
16
0.5
0.5
0.125
0.125
4
118.74+1.07 (80.06%) f
2
2
4
4
0.25
0.25
0.25
0.25
30
1
142.03+0.98 (95.76%) c
8
8
8
8
1
1
0.125
0.25
2
98.33+0.99 (66.30%) g
2
2
4
16
1
1
0.25
0.5
3
98.77+0.54 (66.59%) g
2
2
4
16
0.5
1
0.125
0.125
4
90.21+1.03 (60.82%) h
2
2
4
4
0.5
0.5
0.125
0.25
45
1
135.62+1.00 (91.44%) d
ND
ND
ND
ND
ND
ND
ND
ND
2
87.28+0.89 (58.85%) i
2
2
8
8
0.5
0.5
0.25
0.5
3
53.70+0.64 (36.21%) j
2
2
8
8
0.5
0.5
0.25
0.5
12% v/v
KLLO
4
49.74+0.14 (33.54%) k
2
2
4
8
0.25
0.25
0.25
0.25
* α-Terpineol in ppm (in bracket, as %) in 10% v/v KLO oral spray, citronellal in ppm (%) in 12% v/v
KLLO oral spray, triplicate analysis, means not sharing the same alphabets differ significantly (p<0.05);
MIC = Minimal inhibitory concentration, MBC = Minimal bactericidal concentration, in µl of oral spray/
100 µl of broth, ND = not determined
4. Conclusions
Six clear oral sprays, three from Kaffir lime fruit peel oil (6, 10, 13%v/v KLO)
and three from Kaffir lime leaf oil (4, 8, 12%v/v KLLO), were developed. The active
compounds in KLO and KLLO oral sprays were α-terpineol and citronellal, respectively.
All oral sprays exhibited antibacterial activity against a Group A streptococcal clinical
isolate and three respiratory pathogens (S. aureus ATCC 29213, S. pneumoniae ATCC
49619, and H. influenzae ATCC 49247), with the strongest activity against H. influenzae
ATCC 49247. The antibacterial activity of all oral sprays was retained in an accelerated
stability test throughout the 4-month storage.
Conflict of interest
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Authors declare that there is no conflict of interest regarding the publication of
this paper.
Acknowledgments
This work was supported by the Co-ordinating Center for Research and
Development to Increase the Value of the Plants Indigenous to Thailand, Mahidol
University, and The Thailand Research Fund (TRF); and from the Graduate Research
Fund, Faculty of Medicine Siriraj Hospital, Mahidol University.
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Conflict of interest
Authors declare that there is no conflict of interest regarding the publication of this paper.