The article is published in Ukrainian in the journal.
The English text is given in the author's version.
UDC :615.281.9:616.5-001/-002:615.262.1
EXPERIMENTAL STUDY ANTI-INFLAMMATORY ACTIVITY
OF COMBINED GEL WITH SILVER NANOPARTICLES AND GLUCOSAMINE
L. A. Buliga, Y. A. Butko
National University of Pharmacy
Keywords: treatment of dermatitis, inflammation, wounds, silver
nanoparticles, glucosamine hydrochloride.
The search for new drugs for the local treatment of dermatitis that would
combines the anti-inflammatory, reparative, antimicrobial effect and minimum
side effects is extremely important problem for the modern pharmaceutical
science. This study investigates the anti-inflammatory properties of a new
topical agent in gel form, which comprises silver nanoparticles and glucosamine
on the model of subchronic inflammation of skin in rats induced turpentine. It is
found that the investigated gel reduces visual signs of inflammation on 66.7%.
Reducing the thickness of skin rolls of damaged tissue held by 15.5% in the
group which applied gel (p <0,005), 13.0% - in the comparator group (p
<0,005) and 4.9% - in the control group compared with the start of treatment.
According to the dynamics of biochemical parameters in the treatment of
animals with dermatitis suggests the presence of a pronounced antiinflammatory,membrane-stabilizing and antioxidant effects study drug that does
not deny the existing literature data on the mechanism of antiinflammatory of
action nanoparticles of silver and glucosamine hydrochloride. The expression of
anti-inflammatory activity of the test drug is superior to the comparison gel
1
cream "Dermazin", which makes it a promising use for the treatment of
inflammatory skin diseases.
The high incidence of dermatitis among the population of Ukraine, the
tendency to increase the number of severe clinical forms, and reducing the period of remission and low rate of recovery made problem of dermatitis one of the
most important in modern dermatology [1, 11]. The dominant element of the
pathogenesis of dermatitis is inflammation - typical pathological process that occurs as a result of various pathogenic factors exogenous and endogenous nature
and characterized by a complex vascular and tissue changes [1, 8, 10, 11]. One
of the most common complications are joining dermatitis fungal and bacterial
infections due to violation of skin integrity and reduce local immunity. For the
treatment of severe chronic dermatitis, especially in the domestic environment,
using local medicines in the form of ointments, gels, creams, lotions, which
preferably comprise glucocorticosteroids (ACS) and their combination with antimicrobial agents. Despite the high anti-inflammatory effect of corticosteroids
have some significant drawbacks: violation of trophic skin atrophy, delayed recovery, reducing resistance to external infection [7, 9, 11]. Thus, the search for
new drugs for the local treatment of dermatitis that would combines the antiinflammatory, reparative, antimicrobial effect and minimum side effects is extremely important for the modern pharmaceutical science
One of the achievements of modern medicine is the use of a wide range of
pharmacological actions of derivatives of D - (+) - glucosamine: antioxidant,
membrane stabilization (as glycolipids and glycoproteins is a structural component of cytoplasmic membranes), antiplatelet, anti-inflammatory, immunomodulatory. Furthermore there is evidence that glucosamine has its antimicrobial activity and potentiates the effect of some other antimicrobial agents [5]. Now
popular is the use of nanoparticles of heavy metals for making new methods and
tools for the treatment of inflammatory skin diseases. Thus, the benefits of silver
2
nanoparticles are multilevel an antibacterial effect, slow development of resistance, low toxicity, anti-inflammatory and reparative properties. [8]
Work objective was to study the anti-inflammatory properties of the new
wound healing drug in gel form with nanoparticles of silver and glucosamine.
Materials and methods. The gel containing 0.1% of silver nanoparticles
stabilized PVP, and 1% glucosamine hydrochloride (Gel NPS + G) developed
the department of industrial technology of NPHaU. Synergy mechanism of action of glucosamine hydrochloride and silver nanoparticles enhances the antiinflammatory activity of the drug. It is composed of silver nanoparticles stabilized polyvinylpyrrolidone (PVP) (PVP nanocomposite / Ag - 0,164%, corresponding to 0,1% Ag, glucosamine (1.0%) and PVP (2.0%). The size of nanocomposite PVP / Ag is 2 microns. As the comparator used cream «Dermazin»
(manufacturer Salyutas Pharma GmbH, Germany, Series SR0680) containing
1% silver sulfadiazine (corresponding to 0.301% pure ionized silver (Ag +) and
is indicated for the treatment of purulent wounds, burns, inflammatory infectious
skin diseases the composition and indications are well matched with gel investigated [3, 4].
The model of allergic contact dermatitis, which is caused turpentine, reproduced on 35 white laboratory rats weighing 200-240 g by the standard method [2]. Animals were divided into 5 groups (n = 7): 1 - intact control (IC), 2 control pathology on day 10 of the experiment (before treatment - CP10), 3 - control pathology on the 15th day of the experiment (CP15), 4 - animals which
treated with the experimental gel, 5 - a group that was treated comparator. To
assess the severity of edema in animals studied thickness of skin convolution
(mm), which is measured using calipers. Anti-inflammatory activity (AIA%)
products determined by the formula: AIA = 100% - 100 x Ie / IKP15 where Ie - intensity of skin lesions in the experimental group, balls; IKP15 - the intensity of
skin damage in group control pathology on the 15th day of the experiment,
balls.
3
To assess changes in systemic manifestations of inflammation in the body
for treatment of animal blood were determined laboratory (the rate of subsidence
erythrocytes (ESR), the level of white blood cells and hemoglobin) and biochemical indicators (total protein, g / l, the intensity lipid peroxidation (TBA-R,
mcmol / l), antioxidant status (middle molecules SH-groups mcmol / l), the level
of circulating immune complexes (CIC, c. un.). Selection of blood for analysis
performed on the 10-day application of turpentine (before treatment) and after
treatment (15 day experiment.) All interventions and euthanasia of animals was
performed under general anesthesia followed "Procedure for Conducting research institutions experiments, experiments on animals" (Ukraine, 2012) harmonized with the "European Convention for the Protection of vertebrate animals
used for experimental and other scientific purposes "(Strasbourg, 1985). Compliance with these requirements shown to the committee on bioethics pharmacy
(Minutes № 3 of 03.20.2013).
Results processed using Statistica 6.0 t St'udenta the criterion in the case
of normal distribution, the criterion Manna-Whitney U - in his absence. Intragroup differences were analyzed by paired Wilcoxon criterion [6].
Results and discussion. Simulation subchronic inflammation of the
skin, caused turpentine, led to the emergence in animals characteristic signs of
inflammation: redness, edema (thickening of skin convolution), complete disappearance of the derivatives of skin (fur) in the affected zone. There were observed exfoliate the epidermis, bleeding, sores with crusts in the some animals
(Figure 1 a, b).
а) A intensity lesion, balls
*/
4,00
20,00
3,00
15,00
2,00
*
**/
***
**
0,00
*
* ***/ ***
# #
5,00
0,00
The initial values
10th day
*
10,00
**
1,00
*/
в)A thickness of skin convolution **/ **/
15th day
The control patology
10th day
Gel NPS+G
15th day
cream Dermasine
4
Fig. 1. Study of anti-inflammatory action gel with silver nanoparticles
and glucosamine on model subchroniс skin inflammation in rats induced by
turpentine
Notes. Value are probable comparatively: to the source data – * (p <
0,05); to the indicators on the 10th day of the experiment – ** (p < 0,05); ); to
the indicators on the 15th day of the experiment – *** (p < 0,05), # (p <
0,005); (n = 7); gel NPS+G – gel with silver nanoparticles and glucosamine
The intensity of skin lesions before treatment averaged in groups 3,083,33 balls. Increased thickness of the skin convolution in groups of animals after
drawing turpentine held by 16%.
After treatment, the animals, observed a gradual normalization of condition of the skin. The intensity of skin lesions in all groups was lower than at the
beginning of treatment (p <0,05). However, compared with the group CP15 skin
condition indicators differed significantly only in the group which applied experimental gel (0,50 ± 0,11 balls). Anti-inflammatory activity was for gel NPS+G –
n n66.67% and cream "Dermazin" – 38.68%. In the affected zones of redness
disappeared and decreased swelling, skin was smooth, whereas in the group CP15
in some animals remained the flaking phenomenon. In general, thinning the
skin convolution of damaged tissues held by 15.5% in the group which applied
gel NPS + D (p <0,005), 13.0% –
in the comparator group (p <0,005) and
4.9% – CP15 group compared to the beginning of treatment (Fig. 1 d).
Reducing the external manifestations of inflammation in animals were
treated was accompanied by normalization of laboratory and biochemical markers of inflammation (Fig. 2). Thus, compared to the beginning of treatment, animals in groups, which applied gel NCHS + G, cream «Dermazin" and the group
CP15 happened reduction ESR in 4.0 times, 2.7 times, 1.6 times and leukocytes
– 1.5 times, 1.4 times, 1.1 times, respectively. It should be noted that animals
treated, took place complete normalization of relevant parameters and their values were lower than CP15 group (p <0,005).
5
A Marker characterizing the state of the body during inflammatory diseases (significant damage to the skin including) the level of total protein (TP). After
treatment, in the blood of animals observed valid normalization of this index
there is. TP level compared to the beginning of treatment increased in groups
that applied both drugs at approximately 1.4 times (p <0,005), and was higher
than the corresponding values in the group CP15 in 1.2 times (p <0,005).
mm/h
10
Erythrocyte sedimentation rate
*
#
8
*/**
#/##
6
*
#
5
0
0
G-SH
3
2
0
*
#
**
##
**
##
0,4
0
*
@
*/**
##
0,6
0,2
80
70
60
50
40
30
20
10
0
*
#
0,8
1
Total protein
g/l
1
mcmol/l
*
#
TBA-R
*/**/***
*/**/***
##
##
@@@
@/@@@
*/**
#/##
Circulating immune complexes
**/*** **/***
##
##
@@@ @@@
0,1
c.un.
mcmol/l
4
**/*** **/***
##/### ##/###
10
2
5
*
#
15
**/***
**/*** ##/###
##/###
4
Leukocytes
109/l
20
*
#
0,05
*/**
#/## **/*** **/***
##/### ##/###
0
IC
KP10
KP15
Gel NPS+G
cream "Dermazin"
Fig. 2. Change haematological and biochemical parameters in blood of
rats with turpentine dermatitis, during the treatment by gel with silver nanoparticles and glucosamine (n = 35)
Notes. Value are probable comparatively: to the intact group (IC) –
(p < 0,05),
@
(p < 0,01),
#
*
(p < 0,005); to the indicators of control pathology
6
group on the 10th day of the experiment (CP10) –
**
(p < 0,05), ## (p < 0,005);
to the indicators of control pathology group on the 15th day of the experiment
(CP15) – *** (p < 0,05), @@@ (p < 0,01), ### (p < 0,005).
After treatment, the animals were also observed normalization of LPO ratio, a marker which is the content of TBA-R and antioxidant defense system
(level middle molecular G-SH-group), which are important indicators of inflammatory diseases, suggesting the presence of antioxidant effect. Thus, compared to the beginning of treatment, marked reduction of TBA-R in the groups
treated with an experimental gel cream "Dermazin", CP15 – in 1.6 times, in 1.4
times, in 1.3 times (p <0,005) and increased G-SH – in 1.4 times, in 1.3 times, in
1,2 (p <0,005) respectively. In addition, the level of G-SH in groups of animals
which applied the study gel was higher than in the group CP15 (p <0,01).
Raising middle molecular CIC is characteristic for chronic allergic and inflammatory processes involving the increase of immunological reactivity. Reduction CIC happened in animals, were treated by the gel NPS + G in 1.8
times, which struck comparator – in 1.7 times, CP15 group – in 1.3 times (p
<0,005). Should be noted this marker was significantly lower in the groups that
were treated compared to the CP15 (p <0,005).
Since there is not significant distinction between the level of ESR, leukocytes, containing TBA-R, TP, CIC in animal blood after treatment and in intact
animals, it can be noted that in the treated groups there was complete normalization of relevant indicators, indicating pronounced anti-inflammatory, membrane
stabilization effect of drugs. As the results of dynamics of hematological and
biochemical parameters on the background treatment subhronical skin inflammation in experimental rats efficacy of gel prevails efficacy of comparator drug,
but likely differences between the values of the relevant parameters have not
been observed.
Conclusions.
7
1. Established that the model subchronic skin inflammation in the rat the study
gel helped reduce visual manifestation of inflammation (AIA = 66.67%) and in
accordance with the normalization of hematological and biochemical parameters
in the body.
2. As a result of the impact on the dynamics of biochemical parameters can
suggests the presence of a pronounced anti-inflammatory and antioxidant effects
the study drug that does not deny the existing literature data on the mechanism
of action of anti-inflammatory nanoparticles of silver and glucosamine hydrochloride.
3. In terms of expressiveness anti-inflammatory activity investigational the gel
prevails drug-comparison - cream "Dermazin" that makes perspective its use
for the treatment of inflammatory skin diseases.
References
1. Волкославська, В. М. Стан захворюваності на дерматози в Україні
через 20 років після аварії на ЧАЕС / В. М. Волкославська, О. Л. Гутнєв //
Укр. журн. дерматології, венерології, косметології : Науково-практичне
видання. – 2010. – № 3. – С. 153.
2. Методичні рекомендації «Екпериментальне вивчення нових препаратів
для місцевого лікування ран» / Л.В. Яковлєва, О. В. Ткачова, Я. О. Бутко,
Ю. Б. Лар’яновська. – Харків, 2013. – 52 с.
3. Пат. № 98085 UA, МПК C23C 14/24 (2006.01), C23C 14/28 (2006.01),
C23C 14/30 (2006.01). Пристрій та спосіб електронно-променевого
випарювання й спрямованого осадження парового потоку на підкладку у
вакуумі / Мовчан Б. О., Яковчук К. Ю. – Власник: Міжнародний центр
електронно-променевих технологій інституту електрозварювання ім. Е.
О. Патона Національної академії наук України. – № а201106568; заявл.
25.05.2011; опубл. 10.04.2012, Бюл. № 7.
4. Пат. № 92307 UA, МПК A61K 9/06 (2006.01), A61K 33/38 (2006.01),
A61P 17/02 (2006.01), A61P 31/02 (2006.01), A61P 31/00. Фармацевтична
8
композиція у формі гелю з наночастками срібла для лікування ран та
запальних інфекційних захворювань / Патон Є. Б., Черних В. П., Мовчан Б.
О.
та
ін.
–
Власники:
Патон
Борис
Євгенович,
Національний
фармацевтичний університет. – № u201402559; заявл. 14.03.2014; опубл.
11.08.2014, Бюл. № 15.
5.
Туляков
В.
О.
Фармакологічні
властивості
глюкозаміну:
мембраностабілізуючі, протизапальні, антиоксидантні й імунотропні /
В. О. Туляков, К. О. Зупанець, С. К. Шебеко // Фармакологія та лікарська
токсикологія. – 2009. – Том 9, № 2. – С. 3 – 7.
6. Халафян А. А. STATISTICA 6. Статистический анализ данных
/
А. А. Халафян. – 3-е изд. Учебник. М.: ООО «Бином-Пресс», 2007. – 512 с.
7. Dey V. K. Misuse of topical corticosteroids: A clinical study of adverse effects // Indian Dermatol Online J. – 2014. – Vol. 5, № 4. – Р. 436 – 440.
8. Gunasekaran T. Silver Nanoparticles as Real Topical Bullets for Wound
Healing / Gunasekaran, T. Nigusse, M. D. Dhanaraju // J Am Coll Clin Wound
Spec. – 2011. – Vol. 4, № 3. – P. 82 – 96.
9. Prevalence of self-medication for skin diseases: a systematic review /
M. Corrêa-Fissmer, M. G. Mendonça, A. H. Martins, D. Galato // An Bras
Dermatol. – 2014. – Vol. 4, № 89. – Р. 625 – 630.
10. Reinke J. M. Wound repair and regeneration / J. M. Reinke, H. Sorg //
Eur. Surg. Res. – 2012. – Vol. 1, № 49. – Р. 35 – 43.
11. Zaidi Z. Dermatology in Clinical Practice / Z. Zaidi, S. W. Lanigan. –
L.: Springer-Verland, 2010. – 591 p.
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