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TREATMENT OF ATOPIC DERMATITIS IN CHILDREN WITH BIOAPIFIT® ANTI-
INFLAMATORY HERBAL OINTMENT - A PRELIMINARY STUDY
Višnja Oreščanin
OREŠČANIN Ltd., Laboratory for herbal drugs development, A. Jakšića 30, 10000
Zagreb, Croatia,
Correspondence to: prof. dr. sc. Višnja Oreščanin, senior scientist;
OREŠČANIN Ltd, Laboratory for herbal drugs development, Ante Jaksica 30, 10000
Zagreb, Croatia, Tel. +385914377905; vorescan@gmail.com
Abstract
Objectives: The purpose of this work was development, formulation and testing of new herbal
ointment for the treatment of mild to severe atopic dermatitis in infants and children (4 to 56 months
old) as possible alternative therapy to standard corticosteroids and immunomodulants.
Patients and methods: 50 patients were treated 8 weeks (every four hours) with the ointment
containing the following ingredients: Avena sativa, Nigella sativa, Argania spinosa, Prunus amygdalus,
Daucus carota, Helichrysum italicum, Calendula officinalis, Matricaria chamomilla, Bellis perennis,
Lavandula officinalis, Achillea millefolium,Thymus serpyllum, Salvia officinalis, Symphytum
officinale, Plantago major, Olea europaea, Melaleuca alternifolia, Cera alba, honey, and glycerol. The
severity of the disease prior and after the therapy was assessed by SCORAD index.
Results: The total score before the therapy ranged from 23.1 to 99.4 (66.50±23.43). Among the tested
patients 76%, 20% and 4% had severe, moderate and mild symptoms, respectively. Following the eight
weeks treatment the extent, intensity and subjective symptoms decreased significantly. The total
score decreased more than 20 times compared to the initial values and ranged from 0 to 14
(13.18±4.29). The intensity parameters (swelling, oozing/crusting and scratch marks) as well as
subjective parameters (itch and sleeplessness) disappeared completely following two months therapy.
Conclusion: Two months of the topical treatment with Bioapifit® herbal anti-inflammatory ointment
resulted in complete remission of the symptoms in 56% of the patients while in another 44% of them
only mild symptoms persisted. Such excellent results could be atributed to the ointment’s formulation
containing emollients, strong anti-inflammatory, immunomodulating, wound healing and antimicrobial
agents of herbal origin that simultaneously targeted the multiple mechanisms involved in AD
pathogenesis. This ointment could be used as alternative therapy to the topical corticosteroids and
immunomodulants.
Key words- atopic dermatitis, children, Bioapifit® herbal ointment, SCORAD index
1. INTRODUCTION
Atopic dermatitis (AD) is a chronic, relapsing, inflammatory skin disease which starts early in
life (Bieber, 2010). The main symptom of AD is constant itching throughout the day which usually
worsens during the night, causing insomnia, irritability, exhaustion that impairs the quality of life both
the patient and his family significantly. It is often associated with other atopic diseases such as food
allergies, asthma and allergic rhinitis. The symptoms of AD may vary from mild to moderate to severe
forms of the disease depending of the age of the patient. It usually starts in early infancy between 2nd
and 6th month of life. It was found that in 45% of children disease begins during the first six months of
life, in 60% of them during the first year, and in 85% the symptoms appeared before five years of age
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(Bieber, 2010). AD has a tendency of disappearance with age. In 50% of the children the symptoms
disappeared before seven years of age, and in 70% before adolescence. If AD lasts until adolescence, it
is very likely that the patient will have difficulties in adulthood (Bieber, 2010). The clinical feature
may differ with age and severity of the disease. The acute phase is characterized by oozing, crusted,
eroded vesicles or papules on erythematous plaques while in sub-acute phase thick and excoriated
plaques are usually formed and the chronic phase is characterized by lichenified, slightly pigmented,
excoriated plaques (Bieber, 2010).
The prevalence of AD in the last 30 years has increased up to three times (Pustišek et al.,
2016). It is estimated that in the industrialized countries 15-30% of children and 2-10% of adults are
affected by AD (Bieber, 2008; Bieber, 2010). The highest incidence was obtained in northern Europe
(ISAAC). It was confirmed that the current prevalence of AD in developed countries is up to 20%
(Deckers et al., 2012). The prevalence of AD also increased in developing countries, especially in Africa
and East Asia (Flohr and Mann, 2014).
AD could be associated with elevated levels of immunoglobulin E (IgE). It was confirmed
(Spergel, 2010; Carlsten et al., 2012; Dharmage et al., 2014) that AD is the first disease to appear in a
series of allergic diseases such as food allergy, asthma, and allergic rhinitis, leading to the “atopic
march”. Early or severe AD and cutaneous sensitization to environmental allergens may lead to
subsequent allergic disease at other epithelial barriers.
Almost 30% of the children suffering from AD will develop asthma later in life, and almost 70%
of them will develop allergic sensitization and the symptoms of allergic rhinitis (Dharmage et al., 2014;
Gustafsson et al., 2000). High correlation between food allergy and AD was also reported (Allen and
Dharmage, 2010).
Genetic and environmental factors represent the main risk factors for the development of AD
(Carson, 2013). 37.9% and 50% of the children under age of four will develop AD if one and both parents
have atopic disease compared to 27.1% of the atopic children with no family history of atopy (Bohme et
al., 2003). Although, heredity factors are clearly linked with increased risk of AD development, the
influence of environmental factors onto AD gave contradictory results (Carson, 2013).
The development of inflammation that leads to AD was connected either by primary immune
dysfunction resulting in IgE sensitization or by epithelial-barrier disturbance. However, the disturbance
of the epithelial barrier will lead to the immunologic dysregulation leading to the development of
inflammation.
The imbalance in the T-cell subsets, especially Th2 cells will lead to the production of Th2
cell‒associated pro-inflammatory, pro-allergic cytokines, primarily IL–4, IL-5, and IL-13, causing higher
expression of the adhesion molecules on endothelial cells, inhibition of apoptosis of eosinophils and
increased level of IgE and consequently, decreased interferon-gamma levels (Bieber, 2008, Bieber,
2010; Peng and Novak, 2015). During the chronic phase of AD, the Th0 and Th1 cells predominate
resulting with elevated levels of the IFNG-γ, IL-12, IL-5, and granulocyte-macrophage colony
stimulating factor (GM-CSF) as well as IL-18, IL-11, IL-17 TGF-β1 (Bieber, 2008, Bieber, 2010). Besides,
Th17 cells could be also elevated in patients with AD. However, more research was needed to
understand the precise contributions of Th1 and Th17 cell to the pathogenesis of AD (Peng and Novak,
2015; Koga et al., 2008).
Moreover, eosinophils and mast cells have also significant role in the pathogenesis of AD
(Molfino et al., 2011; Hershko et al., 2011). In the last few years, basophils and group 2 innate
lymphoid cells (ILC2s) are connected to the pathogenesis of AD (Kim et al., 2013; Kim et al., 2014;
Roediger et al., 2013; Imai et al., 2013; Salimi et al., 2013) since they are important sources of IL-4, IL-
5, and IL-13 pro-allergic cytokines (Kim et al., 2013; Kim et al., 2014). Those cells were regulated by a
family of epithelial cell‒derived cytokines directly released from the damaged keratinocytes, including
thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 (Kim, 2015).
The epidermal barrier dysfunction is connected to AD development due to the entrance of
antigens through the damaged skin which will lead to the production of inflammatory cytokines. It was
found that mutations in the gene encoding filaggrin, a key protein that facilitates terminal
differentiation of the epidermis and formation of the skin barrier is the strongest known genetic risk
factors for the development of AD (Osawa et al., 2011; Smith et al., 2006; Palmer et al., 2006).
Filaggrin dysfunction could lead to the release of the epithelial cell‒derived cytokines,
including TSLP, IL-25, and IL-33 and consequently, development of inflammation that will lead to AD
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(Hvid et al., 2011; Savinko et al., 2012; Soumelis et al., 2002; Brandt et al., 2011). Aside from FLG
mutations it was found that genetic variants of TSLP interact with mutations in filaggrin to influence
AD disease persistence in patients. Among the patients with FLG- loss of function mutation, those with
TSLP variation were more likely to have less persistent disease (Margolis et al., 2014).
Taking all together it was obvious that loss of filaggrin function leads to increased trans-
epidermal penetration of environmental allergens, increasing inflammation and sensitivity and
potentially leading to the atopic march (Kubo et al., 2012).
Although, there is no cure for AD warious treatment approaches were developed so far in order
to alleviate the symptoms. The data were summarized in the work of Leung and Hon, 2015. In short,
aside from continues usage of emollients and wet bandages in order to hydrate the skin and form the
protective barrier, the therapy include either topical or systemic anti-allergic or immunomodulating
drugs. Topical treatment include topical corticosteroids of different potency depending on the severity
of the symptoms, topical immunomodulants (topical calcineurin inhibitors), topical sodium
cromoglicate (anti-allergic, anti-itch, and anti-inflammatory drug). In the case of Staphylococcus
aureus infection oral antibiotics were also included while antihistamines were sometimes prescribed in
order to reliefs the itching regardless of the lack of evidence of their efficiency as anti-pruritics. In the
case of chronic severe AD in children and adults that not responding to the topical therapy systemic
immunomodulating agents are used. Among them, cyclosporin A, azathioprine and methotrexate are
most commonly prescribed. Other approaches include phototherapy which is mostly used for the
treatment of chronic lichenified forms of moderate-to-severe AD. Dietary interventions, allergen-
specific immunotherapy, Chinese herbal medicine, psychological and educational interventions could
be also of some help. However, more research is needed to confirm their efficiency.
Since there was no reliable biomarker for the diagnosis of AD, it is predominantly based on a
constellation of clinical feature (Leung and Hon, 2015). Various tools were developed so far to
assessing AD. However, the Severity Scoring of Atopic Dermatitis Index (SCORAD) is best validated and
most commonly used instrument to assess the clinical signs of AD (Schmitt et al., 2013). The SCORAD
index (Oranje, 2011; Oranje et al., 2007) assessing the extent (Score A), the intensity (Score B) and
subjective symptoms (Score C). The total score is calculated according to the formula A/5 + 7B/2 + C
with maximum value of 103. For the extant the rule of nines is applied on a front/back drawing of the
patient's inflammatory lesions (Head and neck 9%, Upper limb 9% each, Lower limb 18% each, Anterior
trunk 18%, Back 18% and genitals 1%). The total score A may range from 0 to 100. Six parameters (signs)
were included for the assessment of the intensity (Redness, Swelling, Oozing / crusting, Scratch marks,
Skin thickening (lichenification) and Dryness). Each of these signs is assessed as none (0), mild (1),
moderate (2) or severe (3). B score was calculating by summing the individual results for each sign. The
maximum value for score B is 18. Subjective symptoms i.e., itch and sleeplessness, are each scored by
the patient or relative using a visual analogue scale (VAS) where 0 is no itch (or no sleeplessness) and
10 is the worst imaginable itch (or sleeplessness). These scores are summed to obtain Score C
(maximum 20).
The purpose of this work was development, formulation and testing of new herbal ointment for
the treatment of mild to severe atopic dermatitis in infants and children as possible alternative
therapy to standard corticosteroids and immunomodulants.
2. PATIENTS AND METHODS
2.1. Study Design
50 patients (31 females and 19 males) in the age range from 4 to 56 months diagnosed from mild to
severe atopic dermatitis whose parents rejected corticosteroid therapy were included. All the parents
signed informed consent and completed the questioner including general questions about child and
the parents, the onset of the disease, family history of atopy. The diagnosis and the severity of the
disease were based on the SCORAD index. The patients were treated 8 weeks with the herbal
ointment. The ointment was applied on affected area every four hours during the whole course of the
study. It was also applied on whole body after each bathing or at least twice a day in order to
moisturize the skin. The parents were advised to make daily notes about the changes of the
symptoms. The parents were also advised to use soft cotton clothing next to the child’s skin, maintain
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mild temperatures particularly at night, use humidifier, use mild detergent, with no bleach or fabric
softener for clothes washing, provide non-smoking environment, avoid contact with animals, avoid
hipper-allergenic food in the case of concomitant food allergy. Two follow-ups (after four and eight
weeks) were carried out in order to assess the efficacy of the therapy.
2.2 Preparation of the Ointment
The process of the production of macerate and ointment was described in details in our previous
papers (Orescanin et al., 2015 a,b; Orescanin and Findri, 2016). The final product consists of: 15% of
colloidal oat (Avena sativa), 5% of black cumin oil (Nigella sativa), 5% of argan oil (Argania spinosa),
5% of almond oil (Prunus amygdalus), 2% of carrot oil (Daucus carota), 5% of immortelle flowers
(Helichrysum italicum), 10% of marigold flowers (Calendula officinalis), 5% of chamomile flowers
(Matricaria chamomilla), 2% of daisy flowers (Bellis perennis), 2% of lavender flowers (Lavandula
officinalis), 5% of aerial part of yarrow (Achillea millefolium), 2% of aerial part of wild thyme (Thymus
serpyllum), 2% of aerial part of sage (Salvia officinalis), 5% of comfrey leaves (Symphytum officinale),
7% of plantain leaves (Plantago major), 4% of olive leaves (Olea europaea), 0.2% of tea tree
(Melaleuca alternifolia) essential oil, 8% of bee wax (Cera alba), 5% of honey, 4.8% of glycerol.
2.3. Statistical Analysis
For statistical evaluation Statistica 11.0 software package was employed. The description of the
treated population was done by basic statistics and frequency tables. Statistical significance was set
to p<0.05 in all the tests performed. The differences in the mean values of each parameter prior and
after the therapy were assessed by Kruskal-Wallis ANOVA and Multiple Comparisons test. The influence
of the predictor variables on the values of SCORAD index was tested by Multiple regression method
and General regression model (Oreččanin et al., 2015a).
3. RESULTS AND DISCUSSION
3.1 Description of the Population
The study included the infants and children in the age range from 4 to 56 months (18.48±16.54
months). The onset of the disease was from 2 to 6 months (4.24±1.93) which was in accordance with
previously published data (Bieber, 2010; Pustisek, 2016). Most of the children (82%) came from the
middle to upper class, mostly from high educated (76%) parents. Similar results were obtained by
Pustisek, 2016. When we looked at the extent of AD (Figure 1) the head and neck was the most
affected area of the body (92%), followed by upper limbs (56%), lower limbs (36%), genitals (32%),
anterior trunk (24%) and back (8%). The mean values and standard deviations for the extent (Score A),
intensity (Score B), subjective symptoms (Score C) and total score were presented in Fig. 2. Score A
ranged from 9 to 82 (37.40±16.95), score B from 5 to 18 (12.92±.4.40), score C from 2 to 20
(13.80±5.55) and total score from 23.1 to 99.4 (66.50±23.43)
The following mean values and standard deviations were obtained for the intensity parameters
(Fig. 3): 2.60±0.56 for redness, 2.24±0.78 for swelling, 1.64±0.99 for oozing/crusting, 2.28±0.84 for
scratch marks, 2.04±0.79 for lichenification and 2.12±0.78 in the case of dryness. In the case of
subjective factors the mean value and standard deviation for itch was 6.88±2.74 and sleeplessness
6.92±2.78.
According to the total score 76% of the patients developed severe symptoms, 20% of them had
moderate symptoms while only 4% them had mild symptoms (Fig. 4).
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Figure 1. Percentage of the patients affected by atopic dermatitis for each part of the body assessed
by SCORAD index before and following four and eight weeks of the therapy with Bioapifit anti-
inflammatory ointment
Family history of atopic diseases was obtained in 48% of the patients. Bohme et al., 2003 found
significantly higher percentage of children with AD if one or both parents have atopic disease compared
to those without family history of atopy. Pustisek et al., 2016 reported that among 134 participants,
41% of them had family history of atopy. In the present study 40% of the patients had also some other
allergic disease. Among them food allergies prevailed (24%), followed by allergic rhinitis (8%) and
asthma (8%). Atopic dermatitis was the first allergic disease that occurred in the examined children.
Similar results were obtained by Pustisek, 2016 where 44% of the patients in association with AD had
some other allergic disease and the highest percentage was associated with food allergies (33.6%)
followed by more than two allergic diseases. Two of our participants had also diagnosis of ulcerative
colitis and one of them Crohn disease. All those results support the “atopic march” theory where early
or severe AD and cutaneous sensitization to environmental allergens may lead to subsequent allergic
disease at other epithelial barrier surfaces (Spergel, 2010; Carlsten et al., 2012; Dharmage et al.,
2014).
Figure 2. Mean values and standard deviations of the results of the severity scoring of atopic dermatitis
(SCORAD) index assessing the percentage of the affected area of the body (Score A), intensity of the
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symptoms (Score B), subjective symptoms (Score C) and total score before (0) and after 4 and 8 weeks
of the treatment with Bioapifit anti-inflammatory ointment.
Figure 3. Intensity of atopic dermatitis symptoms assessed by SCORAD index before and following four
and eight weeks of the treatment with Bioapifit anti-inflammatory ointment.
Based on the results of multiple regression analysis there was a strong, statistically significant
correlation (R = 0.89; p< 0.00000) between the total score and predictor variables (age, family history,
other allergic diseases). The severity of the disease was negatively correlated with age (β = -0.44;
p<0.000630). Indeed, the highest SCORAD index was obtained in the youngest patients < 6 months old.
Statistically significant, positive correlation was obtained between SCORAD index and family history of
atopic disease (β = 0.57; p<0.000597) as well as SCORAD index and the presence of other allergic
diseases (β = 0.32; p<0.037002). According to the beta coefficients and their significance level the
influence of the predictor variables on the result of SCORAD index increased in the following order:
other allergic diseases<age<family history.
The results of general regression model expressed as Pareto charts of t-values were completely
in agreement with the results of multiple regression analysis (Figure 5). All three predictor variables
showed statistically significant influence in the results of SCORAD index with family history as most
important predictor variable. As we mentioned previously, in our study 48% of the patients have family
history of atopic diseases. Among the family members, atopic disease was most commonly found
between siblings followed their mothers.
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Figure 4. Percentage of the patients affected by atopic dermatitis depending on the degree of severity
assessed by SCORAD index before and following four and eight weeks of the treatment with Bioapifit
anti-inflammatory ointment.
Figure 5. Pareto charts of t-values testing for the influence of predictor variables (family history, age,
other allergic diseases) on the results of SCORAD index
3.2 The Response to the Therapy
Following the 30 days of the treatment with Bioapifit® anti-inflammatory ointment the mean value of
the total score of SCORAD index was decreased more than three times compared to the results prior to
the therapy (20.68±11.26). The mean values and standard deviations for the extent (24.72±14.58),
intensity (3.96±2.32) and subjective symptoms (0.96±1.34) also decreased significantly (Fig. 2). Among
the intensity parameters, the highest decrease compared to the results prior to the therapy (Fig. 3)
was obtained for scratch marks (9.5 times), oozing/crusting (8.2 times) and swelling (5.1 times) while
other three parameters decreased from 1.9 to 2.5 times.
According to the total score following the 30 days therapy, 40% of the patients had moderate
and 60% of them had mild symptoms of AD. Itching as most important symptoms of AD disappeared
completely in 60% of the patients while 40% of them graded the severity of the symptom from 1 to 3 of
10 on the VAS scale (0.92±1.29).
Prolonged treatment for another 30 days resulted in further decrease of all considered
parameters of SCORAD index (Figs. 1-3) and the degree of the severity (Fig. 4). Mean value and
standard deviation of the total score was 3.18±4.29 (range from 0 to 14.1) which was decreased more
than 20 times compared to the period before the therapy. Only mild symptoms of AD persisted in 44%
of the patients (Fig. 4).
The intensity parameters swelling, oozing/crusting and scratch marks disappeared completely
while redness, lichenification and dryness decreased for 6.5, 25.5 and 17.7 times compared to the
period prior to the treatment. The subjective parameters itch and sleeplessness disappeared
completely following two months of the therapy.
According to the results of Kruskal-Wallis ANOVA and Multiple Comparisons test the total score
as well as all SCORAD index parameters showed statisticaly significant diference amnong the initial
values (before the treatment) and the values obtained after 30 and 60 days of the therapy.
Two months of the topical treatment with Bioapifit® herbal ointment resulted in complete
remission of the symptoms in 56% of the patients while in another 44% of them only mild symptoms are
recorded. Itching, as the basic symptom of AD disappeared in all 50 patients. Such excellent results
could be ascribed to the ointment formulation containing emollients, strong anti-inflammatory,
immunomodulating, wound healing and antimicrobial agents of herbal origin.
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As mentioned earlier the disturbance of epidermal barrier is highly associated with the
pathogenesis of AD since it allows the entrance of the allergens through the damaged skin resulting in
the production of inflammatory cytokines and progression of AD (Osawa et al., 2011; Smith et al.,
2006; Palmer et al., 2006). Besides, trans-epidermal water loss is also increased in AD due to skin
barrier dysfunction (Kelleheretal et al., 2015). So, skin hydration and restoration of the normal
function of epidermal barrier is essential in the management of AD.
It was reported that argan oil has positive effect on the skin hydration (Boucetta et al., 2014)
which was demonstrated by significantly higher water content of the epidermis and significant
decrease of trans-epidermal water loss following both oral and topical application. The authors
concluded that argan oil improved the skin hydration by restoring epidermal barrier function and by
maintaining the water-holding capacity. These was connected with the high concentration of linoleic
acid in argan oil (31-35%) since this acid is the predominant constituent of the epidermal cement
ceramides, more precisely, ceramide-1 subclass which plays a key role in the organization of the
stratum corneum lipids. This beneficial effect of linoleic acid, on the improvement of skin hydration is
achieved by the activation of peroxisome proliferator-activated hormone which promotes the
activation of epidermal keratinocyte differentiation leading to the synthesis of the cement epidermal
lipids and finally to the restoration of the normal barrier function. Argan oil based nanostructured lipid
carrier hydrogel preparation exhibited promising skin-moisturizing effect (Tichota et al., 2014).
Positive effect of argan oil on skin elasticity was also proven following the 60 days of the topical
application (Boucetta et al., 2015) which was connected with the presence of high value of vitamin E (a
lipid-soluble nutrient) which in addition to its high antioxidant potential can act on skin elasticity by
down-regulation of TGF-β resulting in a signal inducing fibroblasts to produce more collagen and elastic
fibers necessary for the normal appearance and integrity of the skin. Indeed, significant influence of
vitamin E in the management of AD symptoms was reported by Tsoureli-Nikita et al., 2002. They found
a significant decrease of IgE serum level in AD patients following the 8 months of vitamin E treatment.
Furthermore, a remarkable improvement in facial erythema, lichenification, and skin general
appearance was reported. The researchers concluded that eczematous lesions healed mostly as a result
of decreased pruritus. Following the sixty days of the treatment of AD patients with the combination of
E and D vitamins the mean value of SCORAD index decreased for 64.3% (Javanbakht et al., 2011).
Beneficial effects of the combination of vitamins A, C, and E as well as E and C on the treatment of
skin related diseases and conditions were also reported (Keller and Fenske, 1998; Hayakawa et al.,
1981).
From the above mentioned findings it could be concluded that base oils rich in vitamins A, C, D
and especially E, and polyunsaturated fatty acids, especially linoleic acid have very beneficial effect on
the restoration of the normal epidermal barrier function. Keeping that in mind, 5% of almond oil was
also included in the formulation since this oil has long tradition of use in skin care products
(Centerchem, 2006) due to its excellent emollient, moisturizing and skin softening effect that was
connected with high linoleic acid amount (20-30%) and vitamin E (282 mg/kg). Besides, between 62.0
and 86.0% of oleic acid is found in almond oil which promotes the transportation of polyunsaturated
fatty acids in the skin by softening of the horny layer that promote the production of beneficial
eicosanoids that play an integral part in homeostatic mechanisms related to skin health and structural
integrity (Nicolaou, 2013).
2% of carrot root macerate rich in carotenoids, vitamins B, C, D, E also provide similar benefits
to the impaired skin barrier.
It was found that primary immune dysfunction resulting in IgE sensitization plays the key role in
the pathogenesis of AD (Bieber, 2008, Bieber, 2010; Peng and Novak, 2015). Consequently, Nigella
sativa, the plant with well documented immunomodulatory, anti-inflammatory and antihistaminic
activity was included in the formulation.
The oil and certain active ingredients of N. sativa especially thymoquinine (TQ) exhibited
significant immunomodulatory effect by decreasing the T cell- and natural killer cell-mediated immune
responses (Salem, 2005). Strong anti-inflammatory potential of both oil and thymoquinine were also
confirmed on various inflammation-based models which were mostly based on the suppression of the
prostaglandins and leukotriens inflammatory mediators (Salem, 2005).
Most of the beneficial properties of the Nigella sativa seed oil are attributed to TQ. Among its
various beneficial effects, the suppression of histamine production and/or release from basophils and
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mast cells (Darakhshan et al., 2015) is of high importance in the management of AD. Basic mechanism
of its anti-inflammatory activity was trough down-regulation of the expression of pro-inflammatory
mediators such as COX-2, iNOS, 5-lipoxygenase, TNF-α and inhibition of the activation of transcription
factors NF-κB, Akt and ERK signaling pathways.
Immunomodulatory effects of N. sativa is well documented. Following the for 4 weeks intake of
N. sativa the majority of participants exhibited 55% increase in CD4 to CD8 T cells ratio, and a 30%
increase in natural killer (NK) cell function confirming that N. sativa can enhance immune responses in
humans (Haq et al., 1999). Two times increase in the number of splenic NK cells, and their cytotoxicity
against YAC-1 tumor targets when compared to control NK cells was obtained following one week
intake of N. sativa oil (Abuharfeil et al., 2001). Contrary to its enhancing effect on the T cell-mediated
immune response, N. sativa constituents have shown a tendency to down-regulate B cell-mediated
immunity (Haq et al., 1999). So it could be concluded that N. sativa constituent may enhance cellular
immunity, while suppress humoral immunity (Salem, 2005).
After the treatment of human peripheral blood mononuclear cells (PBMCs) with methanolic
extract of N. Sativa with/without previous phytohemagglutinin (PHA) stimulation an immunostimulating
effect on non-PHA-stimulated proliferation of human PBMCs was obtained, while in PHA-stimulated
cells immunosuppressive activity was found (Alshatwi, 2014).
Nigella sativa reduced significantly the peripheral blood eosinophil count, IgG1 and IgG2a
levels, cytokine profiles and inflammatory cells in lung tissue of conalbumin-treated mice, without
changing IFN-y level (Abbas et al., 2005) confirming its anti-inflammatory and immunoregulatory effect
which may be useful for the treatment of allergic asthma. Pretreatment of the mice with ovalbumin
induced allergic airway inflammation with thymoquinine resulted in significant decrease of Th2
cytokines, lung eosinophilia, and goblet cell hyperplasia together with the inhibition of COX-2 protein
expression and PGD2 production confirming a significant anti-inflammatory and immunomodulatory
effect of TQ (El Mezayen et al., 2006).
Four weeks treatment of hand eczema patients with Nigella sativa oil resulted in significant
improvement of the symptoms which was comparable with the results obtained by Betamethasone
corticosteroid ointment (Yousefi et al., 2013)
Avena sativa represents the main ingredient in Bioapifit® anti-inflammatory ointment. It was
included in the formulation because of its excellent anti-inflammatory, antihistaminic and moisturizing
effect (Pazyar et al., 2012). Avena sativa was used for centuries for topical treatment of various
inflammatory skin diseases. Ilnytska et al., 2016 found that colloidal oatmeal extracts induce the
expression of genes related to epidermal differentiation, tight junctions and lipid regulation in skin,
and provide pH-buffering capacity. It promotes the expression of multiple target genes related to skin
barrier, and resulted in recovery of barrier damage in an in vitro model of atopic dermatitis. In in vivo
study, significant clinical improvements in skin dryness, moisture content, and barrier was obtained
following the treatment with colloidal oat based preparation.
Fowler, 2014 summarized the beneficial effect of avenanthramides (the principle polyphenolic
antioxidants in oats) for alleviation of the symptoms of AD. Avenanthramides decrease the
inflammation in murine models of contact hypersensitivity and neurogenic inflammation as well as
pruritogen-induced scratching in a murine itch model. Another study (Cerio et al., 2010) demonstrated
that avenanthramides are good inhibitors of nuclear factor kappa B activity resulted with the inhibition
of pro-inflammatory cytokines and histamine release from mast cells that play the key role in the
pathophysiology of inflammatory dermatoses. As reported by Sur et al., 2008 avenanthramides at
concentrations as low as 1 µg/kg inhibited the degradation of nuclear factor kappa B-alpha (NFκBα)
inhibitor in keratinocytes which correlated with decreased phosphorylation of p65 subunit of nuclear
factor kappa B (NF-κB) as well as tumor necrosis factor-alpha (TNF-α) induced NF-κB luciferase activity
resulted in decreased production of IL-8 pro-inflammatory cytokine. Furthermore, avenanthramides
also showed anti-itching effect by reducing oxazolone-induced contact hypersensitivity, resiniferatoxin-
induced neurogenic inflammation, and compound 48/80-induced, histamine-mediated itch.
Anti-itching effect of oatmeal preparations was demonstrated by Matheson et al., 2001. Topical
application of liquid paraffin preparation with 5% colloidal oatmeal significantly decreased itching and
need for antihistamine drugs compared to the control group (liquid paraffin only) in the patients with
burn injuries.
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The treatment of skin fragments stimulated by vasoactive intestinal peptide with oatmeal
extract oligomer showed a significant decrease of the mean surface of dilated vessels and edema as
well as decrease of TNF-α confirming excellent anti-inflammatory effect of the oligomer (Boisnic et al.,
2003). The colloidal oatmeal extracts decreased pro-inflammatory cytokines production in vitro while
the application of colloidal oat based skin lotion resulted in significant clinical improvements of skin
dryness, scaling, roughness, and itch intensity (Reynertson et al., 2015).
The application of the oat-based sterile emollient cream twice daily in children with AD
resulted in significant improvement of clinical symptoms and reduction of the number of flare-ups
(Mengeaud et al., 2015).
Criquet et al., 2012 assessed the safety of various oatmeal based personal care products. No
allergies were reported by consumers of 445,820 products sold during a 3-year period confirming that
colloidal oatmeal is a safe and effective ingredient in personal care products.
The studies conducted on the patients in the age ranging from 3 months to 60 years with mild
to moderate atopic dermatitis reviled that the daily use of colloidal oatmeal based moisturizers and/or
cleansers resulted in significant decrease of the symptoms. The products were well tolerated in all age
groups (Fowler et al., 2012).
Moisturizing effect of colloidal oatmeal is attributed with high content of starches and beta-
glucans in oat that are responsible for its protective and water-holding functions (Kurtz and Wallo,
2007).
Bellis perennis was included in the formulation due to its traditional use for the treatment of wounds
and bruises. Karakaş et al., 2012 reported excellent wound healing potential of the extract of B.
perennis flowers on circular excision wound model. Following the 30 days of the treatment with B.
perennis based ointment significantly higher wound closure was observed compared to the control
group. Furthermore, no scars were observed in B. perennis treated rats.
It was reported that oleanane-type triterpene saponins isolated from Bellis perennis flowers
has promoting collagen synthesis in normal human dermal fibroblasts (Morikawa et al., 2015).
Helichrysum italicum was included in the formulation due to its strong anti-inflammatory activity,
antimicrobial and wound healing effect (Antunes Viegas et al., 2014). It was reported that
acetophenones and flavonoids gnaphaliin and pinocembrin isolated from H. italicum could reduce
significantly the production of leukotriene B4 (LTB4) which was resistant to corticosteroid therapy
(Ohnishi et al., 2008). LTB4 is pro-inflammatory mediator with chemoattractant properties which is
rapidly generated from activated innate immune cells such as neutrophils, macrophages, and mast
cells. Elevated levels of leukotriene B4 have been reported in various allergic diseases including AD
(Ohnishi et al., 2008). Besides, acetophenones isolated from H. italicum exibited the COX-1 activity.
Both the whole acetone extract and arzanol isolated from H. italicum inhibited NF-κB, while arzanol
inhibited the production of IL-1β, TNF-α, IL-6, IL-8 and PGE2, 5-Lipoxygenase and leukotrienes (Antunes
Viegas et al., 2014).
In addition to its excellent anti-inflammatory effect, it was found that H. italicum and its
constituents posses a strong antimicrobial activity especially against Staphylococcus aureus that was
often found in AD patients. Nostro et al., 2001 reported excellent inhibitory activity against S. aureus
both methicillin-resistant and methicillin-sensitive strains in dose-dependent manner. Inhibitory effect
was also found in the case of coagulase, DNAse, thermonuclease and lipase enzymes which are
considered as virulence factors.
In vivo studies on humans reviled that application of H. italicum oil could reduce post-
operative scars, local inflammation, edema, bruises and hematomas (Antunes Viegas et al., 2014) that
support its traditional use for the treatment of various skin conditions.
Other components are included in the formulations due to its strong wound healing, anti-
inflammatory, and antimicrobial activity. Detailed literature data for each constituent concerning in
vitro and in vivo study on animal models are presented in our previous publications (Oreščanin et al.,
2015a; Oreščanin et al., 2015b). In addition to that available clinical trials on humans concerning the
treatment of various skin conditions will be presented below.
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21 patients with 33 venous ulcers were treated twice a day for three weeks with Calendula
officinalis based ointments. Following the treatment complete epithelialization was achieved in seven
patients while the total surface of all the ulcers were reduced for 41.71% compared to only 14.52% in
the control group treated with saline solution (Đuran et al., 2005). In the study conducted by Pommier
et al., 2004 two nonsteroid topical agents for the prevention of acute dermatitis during adjuvant
radiotherapy for breast carcinoma were compared. 126 patients were treated with calendula
(Pommade au Calendula par Digestion; Boiron Ltd, Levallois-Perret, France) and other 128 patients
with trolamine (Biafine; Genmedix Ltd, France). Significantly lower (P <0.001) occurrence of acute
dermatitis of grade 2 or higher was obtained in calendula (41%) compared to the trolamine group (63%).
Besides, the patients treated with calendula had less frequent interruption of radiotherapy and
significantly reduced radiation-induced pain. The study conducted on 14 male patients (Akhtar et al.,
2011) reviled that topical cream containing 3% calendula extract decreased skin erythema significantly
confirming its anti-inflammatory effect. Besides, the cream showed skin moisturizing effects and
decreased transepidermal water loss.
Nissen et al., 1988 compared 3 different ointments (Kamillosan® ointment, Kamillosan®
ointment base and 0.1% hydrocortisone acetate) for the topical treatment of contact dermatitis. The
results reviled that Kamillosan® ointment is remarkably superior to other reference products with
regard to its soothing effect on human skin. Kamillosan® ointment was found to be about 60% as
effective as 0.25% hydrocortisone cream (Albring et al., 1983). Patzelt-Wenczler et al., 2000 reviled
that Kamillosan® cream may ease discomfort associated with eczema. In a partially double-blind,
randomized study carried out as a half-side comparison, Kamillosan® cream was compared with 0.5%
hydrocortisone cream and a placebo consisting only of vehicle cream in patients suffering from
medium-degree atopic eczema. After 2 weeks of the treatment, Kamillosan® cream showed a slight
superiority over 0.5% hydrocortisone. Glowania et al., 1987 tested the efficacy of the topical use of
chamomile extract to enhance wound healing in a double-blind trial on 14 patients who underwent
dermabrasion of tattoos. The patients treated with chamomile extracts showed significantly faster
wound healing and epithelialization compared to the control. In another randomized, placebo-
controlled trial, radiation-induced skin reactions were less frequent and appeared later in chamomile-
treated areas, but the differences were not statistically significant (Maiche et al., 1991). Merfort et
al., 1994 conducted in vivo skin penetration studies of the chamomile flavones apigenin, luteolin and
apigenin 7-O-beta-glucoside with nine healthy, female volunteers. During seven hours the flavonoids
are not only adsorbed at the skin surface, but penetrate into deeper skin layers. This is important for
their topical use as antiphlogistic agents. In a case study conducted by Carl and Emrich, 1991, 98
cancer patients were used Kamillosan Liquidum® (15 drops in 100 ml of water three times a day),
during head and neck irradiation and/or systemic chemotherapy in order to prevent and or reduce the
intensity of oral mucositis. Of the 66 patients who participated in prophylactic oral care with the
mouthwash, 20 patients underwent radiation therapy and 46 patients received systemic chemotherapy.
Only one of the 20 patients who had had radiation therapy developed grade 3 mucositis in the final
week of treatment, 65% developed intermediate grade, and 30% developed low-grade mucositis. 36 of
46 patients receiving chemotherapy did not develop clinically significant mucositis. For the 32 patients
with existing mucositis, all noted immediate relief from mouth discomfort and within seven days
almost all patients returned to having no clinical sign of mucositis. In another clinical trial, 161
patients with eczema on their hands, forearms, and lower legs who had been initially treated with 0.1%
difluocortolone valerate were treated with chamomile based Kamillosan® cream, 0.25% hydrocortisone,
0.75% fluocortin butyl ester, or 5% bufexamac. During three to four week maintenance therapy, the
Kamillosan® was as effective as 0.25% hydrocortisone. It was superior to 5% bufexamac and 0.75%
fluocortin butyl ester (Aertgeerts et al., 1985 a,b)
Vakilian et al., 2011 conducted a randomized control trial on 120 primiparous women with
singleton pregnancy who had undergone normal spontaneous vaginal delivery and episiotomy in order
to assess the efficiency of lavender oil in wound healing. Experimental group (60 patients) received
lavender oil and controls received Povidone-iodine. Incision sites were assessed on the 10th day
postpartum. 25 out of 60 women in lavender group and 17 patients in control group had no pain. There
was no significant difference between two groups in surgery site complications. However, redness in
lavender group was significantly less compared to control group (p < 0.001). Similar clinical trial
(Sheikhan et al., 2012) included 60 primiparous women admitted for labor in Kamali Hospital in Karaj,
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Iran. They were randomly categorized into two groups: experimental (using Lavender oil) and control
(using Betadine). Participant’s pain and discomfort were recorded using a Visual Analogue Scale (VAS)
and a Redness, Edema, Ecchymosis, Discharge Scale (REEDA). Pain was evaluated at 4 h, 12 h and 5
days following episiotomy. There was a statistical difference in pain intensity scores between the two
groups after 4 h (p = 0.002, and 5 days (p = 0.000) after episiotomy. The REEDA score was significantly
lower in the experimental group 5 days after episiotomy (p = 0.000). According to these findings, use of
Lavender oil can be effective in reducing perineal discomfort following episiotomy.
Matić et al., 2009 conducted the study on 39 patients with venous leg ulcers in order to test
the ability of the extract of Achillea millefolium to accelerate wound healing. The experimental group
was treated with an ointment containing 7.5% of yarrow extract for three weeks while for the control
group only saline solution dressings were applied to ulcers. Significantly better results were observed in
the experimental group with the reduction of total surface area of all ulcers for 39.64% compared to
only 15.1% reduction in the control group. The parameters like granulation, epithelization and
dermatitis also showed better results in the group treated with yarrow ointment compared to the
control.
Similar study (Đuran et al., 1997) was conducted with the Plantoderm ointment containing the
extracts of four different medicinal herbs (Achillea millefolium, Calendula officinalis, Symphytum
officinale, Salvia officinalis). 40 patients with a total number of 66 venous ulcers were treated for
three weeks which resulted in 58.55% decrease of total ulcer surface while complete epithelization was
recorded in 22 ulcers.
Plantago major leaves extract showed significantly higher antimicrobila potential compared to
standard antibiotics (Samuelsen, 2000). Leaves have also traditionally been used for the treatment of
skin lesions and for bacterial infections (Holetz et al., 2002). Leaves of P. major have been used, and
are still being used as a wound healing remedy in almost all parts of the world in folk medicine. Either
whole or crushed leaves are used to treat for example burns and other kinds of wounds to enhance the
healing process, and to stop bleeding. The leaves of P. major have thus been prescribed for the
treatment of wounds caused by dog bites (Roca-Garcia, 1972). Normally, it is sufficient to apply only
the juice from leaves to heal superficial wounds (Brondegaard, 1987).
Following the treatment of 161 patients with decubitus ulcers with Symphytum officinale based
cream during four weeks resulted in complete healing of the pressure sores in 85.9 % of the patients
and reduction of the total decubitus area for 89.2 % (Stepán et al., 2014). A significant wound healing
potential of the topically applied preparation Traumaplant® containing 10% active ingredient from
medicinal comfrey was confirmed on the patients with fresh abrasions (Barna et al., 2007; Barna et al.,
2012).
4. CONCLUSION
The study included the infants and children with atopic dermatitis in the age range from 4 to 56
months and the onset of the disease between 2 to 6 months. The extent of the affected area of the
body ranged from 9 to 82 (37.40±16.95), intensity of the objective symptoms from 5 to 18
(12.92±.4.40), subjective symptoms from 2 to 20 (13.80±5.55) and total score from 23.1 to 99.4
(66.50±23.43). Among the tested patients 76% of them developed severe symptoms of AD, 20% of them
had moderate and only 4% them had mild symptoms. There was a strong, statistically significant
influence of the predictor variables on the total score (R = 0.89; p< 0.00000) and their influence
increased in the following order: other allergic diseases<age<family history. Following the eight weeks
of the treatment the extent, intensity and subjective symptoms decreased significantly. The total
score decreased more than 20 times compared to the period before the therapy and ranged from 0 to
14 (13.18±4.29). The intensity parameters (swelling, oozing/crusting and scratch marks) as well as
subjective parameters (itch and sleeplessness) disappeared completely following two months of the
therapy. Two months of the topical treatment with Bioapifit® herbal anti-inflammatory ointment
resulted in complete remission of the symptoms in 56% of the patients while in another 44% of them
only mild symptoms are recorded. Such excellent results could be ascribed to the ointment’s
formulation containing emollients, strong anti-inflammatory, immunomodulating, wound healing and
antimicrobial agents of herbal origin that simultaneously targeted the multiple mechanisms involved in
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AD pathogenesis. This ointment could be used as alternative therapy to topical corticosteroids and
immunomodulants.
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