Study The Efficacy of Polyherbal Formulations (Vetricare Integumentary) Against DNCB Induced Atopic Dermatitis in Mice

Atopic dermatitis (AD) presents a significant challenge in veterinary dermatology due to its persistent nature and the wide variety of clinical manifestations it can produce. This inflammatory skin condition affects many animals and is marked by chronic symptoms, recurrent flare-ups, and a diverse range of issues, including itching, redness, swelling, and skin lesions. The causes of AD are complex, involving a combination of genetic factors, dysregulated immune responses, environmental triggers, and compromised skin barrier functions.  Therapeutic options for atopic dermatitis include emollients, topical and systemic corticosteroids, antimicrobials, and immunomodulating agents (Leung et al., 2007). Corticosteroids exert potent actions by terminating cytokine production, suppressing the expression of adhesion molecules, and inhibiting leukocyte chemotaxis. However, they are also associated with adverse effects such as abnormal metabolism, growth suppression, and increased susceptibility to infections (Hon et al., 2011). Although topical steroids and oral antihistamines are commonly used to treat atopic dermatitis, their use can be accompanied by side effects. Consequently, research efforts have focused on identifying safer and more effective plant-derived compounds that can modulate the pathological mechanisms of atopic dermatitis, including antihistamine effects and inhibiting Th2 responses and IgE production (Abraham et al., 2019). Despite advancements in treatment options, such as topical corticosteroids and immunomodulatory agents, many animals suffering from AD continue to experience persistent symptoms and frequent flare-ups. This ongoing struggle emphasizes the urgent need for new and effective treatment approaches that can address the intricate nature of this disorder. Vetricare Integumentary, developed by Vetrina Healthcare Pvt Ltd in Pune, India, claims to have antiatopic activity in dogs. Consequently, this study was designed to assess the in-vivo efficacy of Vetricare Integumentary in managing atopic dermatitis in mice. Recently, nutraceuticals have been proposed for the treatment of various conditions (Innes, 2004; Colitti et al., 2012; Henrotin et al., 2005). Additionally, some herbal medicinal products have demonstrated the ability to interact with mediators of inflammation, which may make them useful in the management of osteoarthritis (Cameron et al., 2009). These products can also act as antioxidants through various mechanisms. However, so far, there have been few clinical trials conducted to verify the efficacy of these herbal medicinal products.  In light of this situation and the increasing acceptance of traditional herbal preparations, Vetrina Healthcare Pvt Ltd (Pune, India) developed a Poly-herbal formulation called Vetricare Integumentary. Vetricare Integumentary claims to provide supportive therapy for chronic dermatitis and atopic dermatitis. It aims to reduce trans epidermal water loss, thereby helping to maintain skin hydration. This syrup is designed to enhance skin health from within and protect against pathogens, significantly aiding the healing process. Consequently, this study evaluated the in-vivo efficacy of Vetricare Integumentary in managing atopic dermatitis in mice.

MATERIAL AND METHODS:

The experiments was carried out in the strict accordance with the guidelines set by the committee for the purpose of Control and Supervision of Experiments on Animals (CCSEA), New Delhi. The procedures used in this study were reviewed and approved by the Institutional Animal ethics committee (MVC/IAEC/02/51/2023)

Product Detail: Vetricare Integumentary is a proprietary polyherbal formulation developed by Vetrina Healthcare Pvt Ltd., Pune. It contains Pantothenic acid, Inositol, Nicotinamide, Choline, Histidine, Zinc, Biotin, Taurine, Ascorbic acid, EPA, DHA, Aloe Indica, Curcuma longa, and Terminalia Chebula.

Experimental Animals

A total of 54 healthy BALB/c mice of uniform age and weight were procured for the study from NIRRH, Mumbai. The mice were kept in cages as per the specifications of CPCSEA and given standard feed (Pelleted rat feed from NCLAS, NIN, Hyderabad) and water. An acclimatization period of 2 weeks was observed before the start of the experiment. Polyherbal formulations Vetricare Integumentary (Pantothenic acid, Inositol, Nicotinamide, Choline, Histidine, Zinc, Biotin, Taurine, Ascorbic acid, EPA, DHA, Aloe vera, Curcuma Longa and Terminalia arjuna) was obtained from Vetrina Healthcare Pvt Ltd Pune as a gift sample for research purposes along with a no objection certificate of the manufacturer.

Study design and Experimental details

For the experimental design, the mice were divided into Five groups, each consisting of six mice. This grouping allows for proper allocation and comparison of the studied treatments or conditions. Groups are as follows:

Experimental design:

At the end of the study on the 30^th day, all animals were sacrificed, and ear and skin were collected for histopathological examination, score, and Cytokine estimation. Blood was collected on day 0 and day 30, from the retro-orbital plexus using a capillary tube, following the method outlined by Sorg and Buckner (1964), preceding the animals' sacrifice. The collected blood was preserved in K3-EDTA anticoagulant vials for subsequent hematological analysis, with processing completed within 8 hours of collection. Additionally, a thin blood smear was prepared to facilitate the differential leukocyte count.

Induction of Atopic Dermatitis by DNCB:

The 2,4-dinitrochlorobenzene (DNCB) solutions, prepared by dissolving the compound in a 1:3 mixture of acetone/olive oil to achieve concentrations of 1% (w/v) and 0.5% (w/v), were administered for the induction of atopic dermatitis. The sensitization phase involved cleaning and shaving the dorsal skin using a depilatory cream. For three consecutive days, mice, except Group A, received daily administration of a 200 µL solution containing DNCB 0.5% (3:1, v/v) on a designated 1 cm×1 cm area. Subsequently, on days 14, 17, 20, 23, 26, and 29, re-exposure was administered, involving the application of 20 µL DNCB 1% (3:1, v/v) on both ears and 100 µL DNCB 1% on the dorsal skin, excluding Group A from this re-exposure regimen (Suryawati et al., 2022).

Evaluation of study parameters:

Clinical signs: All the animals were monitored thrice daily (morning, afternoon, and evening) for clinical signs, abnormal behavior, and ill health.

Body weight: The body weight of all the mice was monitored twice a week.

Ear thickness: The thickness of the skin on the right ear of all the mice was assessed three times per week utilizing a digital vernier caliper.

Evaluation of Dermatitis Score The severity of dermatitis was evaluated using the Eczema Area and Severity Index (EASI), which considers four clinical signs:

i. Redness (erythema, inflammation)

ii. Thickness (induration, papulation, swelling – acute eczema)

iii. Scratching (excoriation)

iv. Lichenification (lined skin, furrowing, nodules – chronic eczema)

The average intensity of each sign in each body region was assessed on a scale ranging from none (0), mild (1), moderate (2), to severe (3).

Hematological examination: A volume of 0.5 ml of blood was gathered in EDTA vials to conduct a complete blood count (CBC), encompassing assessments such as white blood cell (WBC) count, WBC differential, red blood cell (RBC) count, and platelet count. These analyses were performed utilizing a Hematology Auto-analyzer. Additionally, the Differential Leukocyte count was manually conducted on a blood smear using Leishman’s stain, following the methodology outlined by Benjamin (2001).

Evaluation of Cytokine: IL-4 and INF-?: Serum samples were harvested from blood samples collected at the end of the experiment and the levels of IL-4 and IFN-ϒ were analysed using ELISA kits following the manufacturer’s instructions.

Histopathology: Skin and ear tissue samples were collected and preserved in 10% neutral buffered formalin (NBF), with subsequent processing using the routine paraffin embedding method outlined by Aziz and Zeman (2022). The resulting tissue sections, measuring 4 microns, were then stained following the standard Hematoxylin (H) and Eosin (E) protocol as recommended by Cardiff et. al.,1995.

Statistical analysis

The data generated during the experiment were statistically analyzed by using a One-way analysis of variance (ANOVA). The comparisons between the groups and between the days were tested by the Tukey test. Values are represented as mean, standard error mean and (P < 0.05) considered as significant. Mean values and standard error of mean were calculated and all the values were expressed as mean ± SEM (Graph Pad Prism 5 Software, 2007).

RESULT AND DICUSSION:

The results of the study were presented in the table and graphically represented in graphs and tables. The mean body weight of groups B (DNCB alone), and C (Dexamethasone @3mg), was similar and lowest among all groups. There was an improvement in body weight gain in groups D (Vetricare Integumentary 0.1ml), E (Vetricare Integumentary 0.4ml), and F (Vetricare Integumentary 1.0 ml) and G (Vetricare Integumentary @ 2ml) had the highest weight among the treatment groups on the 30^th day of treatment. There was a significant difference in body weight (P≤0.001) between the treatment and control group. The Group H and I group have similar values like normal values. The mean body weight of groups B (DNCB alone), and C (Dexamethasone @3mg), was similar and lowest among all groups. There was an improvement in body weight gain in groups D (Vetricare Integumentary 0.1ml), E (Vetricare Integumentary 0.4ml), and F (Vetricare Integumentary 1.0 ml) and G (Vetricare Integumentary @ 2ml) had the highest weight among the treatment groups on the 30^th day of treatment. There was a significant difference in body weight (P≤0.001) between the treatment and control group. The Group H and I group have similar values like normal values.

The findings of this study were consistent with those of Lee et al, (2020), Kim et al,. (2022), and Zhang et al, (2022), Although there was no significant change in body weight for the control and experimental mice initially, there was a noticeable and statistically significant (P≤0.001) decrease in body weight for mice with DNCB-induced atopic dermatitis and those treated with Dexamethasone by the end of the 30^th day of the study. Zhao et al., (2020) observed comparable outcomes, linking the loss of bone density to the adverse effects of dexamethasone. Furthermore, the reduced body weight gain following glucocorticoid treatment may be attributed to diminished lean mass, as previously noted by Macedo et al., (2016) and Bönisch et al., (2016).

Table 1.  Effect of Vetricare Integumentary on body weight in DNCB-induced atopic dermatitis on different groups

The impact of various treatments including Normal control, DNCB, DNCB+ Dexamethasone, on body weight was assessed. Data are expressed as mean ± standard error of the mean (SEM). The DNCB and dexamethasone groups exhibited a significant decrease in body weight compared to the control (P ≤ 0.001). Conversely, the DNCB Vetricare Integumentary showed a higher body weight gain compared to the DNCB group. Administration of Vetricare Integumentary showed a significant effect in comparison to DNCB control and Normal control group. A significant decrease in ear thickness was observed in all three groups. Presence of polyphenolic compound Curcumin in Curcuma longa L. and chebulic acid, gallic acid, corilagin, chebulanin, choragic acid, ellagic acid, and chebulinic acid in Terminalia chebula Retz. responsible for the ear's thickness due to anti-inflammatory effects. The Vetricare integumentary at different doses showed significant effect in comparison to control and DNCB groups.  Significant increases in ear thickness were observed in groups B (DNCB alone) and I (Aloe vera), while comparatively lesser increments were noted in groups D (Vetricare Integumentary 0.1ml), E (Vetricare Integumentary 0.4ml), F (Vetricare Integumentary @ 1.0ml) and G (Vetricare Integumentary @ 2ml). The least increment in ear thickness was observed in groups C (Prednisolone 3mg), E (Vetricare Integumentary @ 0.4ml)), and F (Vetricare Integumentary @ 1.0ml) compared to the normal group. There was a statistically significant difference among the groups (P ≤ 0.001). Administration of Vetricare Integumentary at different doses showed a significant effect in comparison to DNCB control and DNCB + Prednisolone group. A significant decrease in ear thickness was observed in all four groups of Vetricare Integumentary (@ 0.1ml, 0.4ml and 1.0ml and 2.0ml). Presence of polyphenolic compound Curcumin in Curcuma longa L. and chebulic acid, gallic acid, corilagin, chebulanin, choragic acid, ellagic acid, and chebulinic acid in Terminalia chebula Retz. Similar observations were observed by Sharma et al., (2019) conducted a study and found that curcumin led to considerable clinical improvements in patients with AD-like skin lesions. These improvements included a reduction in the thickness of the epidermis and the infiltration of inflammatory cells in the dermis. Inflammatory cells in AD skin affect the redox balance and worsen the condition by promoting Th2 polarization. According to Jami et al., (2014), the fruit of Terminalia chebula Retz contains hydrolyzable tannins and polyphenols which can inhibit the synthesis of arachidonic metabolites resulting in anti-inflammatory and anti-analgesics effects, leading to a decrease in skin thickness. Cronin (2003) reported that Curcuma longa reduces swelling by inhibiting the production of inflammatory prostaglandins from arachidonic acid, as well as regulating neutrophil function.

Table No. 2: Effect of Vetricare Integumentary on ear thickness in DNCB-induced atopic dermatitis in different groups

The impact of various treatments including Normal control, DNCB, DNCB+ Prednisolone, DNCB + (Vetricare Integumentary(0.1ml), DNCB + Vetricare Integumentary (0.4), DNCB + Vetricare Integumentary (1.0ml) on ear thickness was assessed. Data are expressed as mean ± standard error of the mean (SEM). The DNCB group exhibited a significant increase in ear thickness compared to the control (P ≤ 0.001). Conversely, the DNCB + Prednisolone, DNCB + Vetricare Integumentary(0.1ml) , DNCB + Vetricare Integumentary (0.4ml), and DNCB + Vetricare Integumentary (1.0ml) groups showed a decrease in ear thickness compared to the DNCB group. The mean thickness of the dorsal skin (means ± S.E.) of animals on day 30^th for groups A, B, C, D, E, F, G, H, and I were 0.595±0.008, 1.248±0.08, 0.62±0.014, 0.777±0.01, 0.788±0.018, 0.777±0.011, 0.665±0.021, 0.635±0.014, and 1.133±0.0415 respectively depicted in table 3 and Figure 3. 

Table No. 3: Effect of Vetricare Integumentary on skin thickness in DNCB-induced atopic dermatitis in different groups

The impact of various treatments including Normal control, DNCB, DNCB+ Prednisolone, DNCB + Vetricare Integumentary @ 0.1ml, DNCB+ Vetricare Integumentary @ 0.4ml DNCB + Vetricare Integumentary @ 1.0ml on dorsal skin thickness was assessed. Data are expressed as mean ± standard error of the mean (SEM). The DNCB group exhibited a significant increase in dorsal skin thickness compared to the control (P ≤ 0.001). Conversely, the DNCB + Prednisolone, and DNCB + Vetricare Integumentary  groups (0.1ml, 0.4ml and 1.0ml) showed a decrease in dorsal skin thickness compared to the DNCB group. The mean dermatitis score of all mice from different groups was calculated and shown in Table 4 and the pattern of change in the score is depicted in Figure 4.4. The mean were calculated, and statistical analysis for the treatment-wise comparison was performed. On day 0, all animals in groups A, B, C, D, E, F, G, H and I had a mean dermatitis score of 0, which means no dermatitis symptoms were observed among them. Statistical analysis showed no significant difference (P≥0.05) between the groups. On day 7, the mean dermatitis score (mean ± S.E.) for groups A, B, C, D, E, F, G, H, and I were 0, 4.83 ± 0.307, 4.5 ± 0.223, 4.5 ± 0.562, 4.66 ± 0.494, 4.67 ± 0.21, 4.33 ± 0.494, 4.83 ± 0.307, and 4.83 ± 0.307, respectively. It's worth noting that except for group A, all other groups exhibited a nearly uniform increase in dermatitis scores. These differences were found to be statistically significant (P≤0.001). On day 21, the mean dermatitis score (mean ± S.E.) for groups A, B, C, D, E, F, G, H, and I were 0, 7.66 ± 0.558, 4.5 ± 0.341, 5.66 ± 0.210, 5.83 ± 0.401, 6.33 ± 0.21, 4.83 ± 0.542, 4.33 ± 0.494 and 7.16 ± 0.6 respectively. There was a significant increase in the mean dermatitis score of groups B and I which was found to be statistically significant (P≤0.001). On day 30, the mean dermatitis score (mean ± S.E.) for groups A, B, C, D, E, F, G, H, and I were 0, 11.66 ± 0.333, 1.33 ± 0.494, 4.5 ± 0.223, 4.66 ± 0.614, 4.66 ± 0.421, 2.16 ± 0.477, 1.5 ± 0.428 and 11 ± 0.365 respectively. There was a significant increase in the mean dermatitis score of groups B (DNCB induced group), while the scores for groups D (Vetricare Integumentary @ 0.1ml) reduced considerably, followed by groups E (Vetricare Integumentary @ 0.4ml), F (Vetricare Integumentary @ 1.0ml), and G (Vetricare Integumentary @ 2ml). These differences were found to be statistically significant (P≤0.001). The findings of present study align with Wang et al. (2022) observations that mice with atopic dermatitis experience severe symptoms such as edema, exfoliation, erythema, scaling, and itching. However, treating the mice with bisdemethoxycurcumin significantly improved these dermatitis symptoms and reduced itching. Scratching behavior, which is an indicator of itching, was significantly reduced in mice treated with bisdemethoxycurcumin compared to the atopic dermatitis group. Kim et al. (2022) also reported similar findings, demonstrating that administering Terminalia chebula Retz reduced dermatitis scores compared to control mice. They attributed this effect to the inhibition of NF-κB, STAT1, and STAT3 nuclear translocation, along with a decrease in inflammatory cytokine expression at the mRNA level. Also, Suryawati et al. (2022) observed outcomes consistent with the present study, showing that repairing skin barrier damage with a moisturizing nanoemulgel containing turmeric rhizome extract reduced dermatitis scores.

Table No. 4:  Effect of Vetricare Integumentary on dermatitis score in DNCB-induced atopic dermatitis in different groups

The impact of various treatments including Normal control, DNCB, DNCB+ Prednisolone, DNCB + Vetricare Integumentary (0.1ml, 0.4ml and 1.0ml) on dermatitis score was assessed. Data are expressed as mean ± standard error of the mean (SEM). The DNCB group exhibited a significant increase in dermatitis score compared to the control (P ≤ 0.001). Conversely, the DNCB + Prednisolone, and DNCB + Vetricare Integumentary groups (0.1ml, 0.4ml and 1.0ml) showed a decrease in dermatitis score compared to the DNCB group. The mean total leukocyte count (TLC) values (mean ± S.E.) of animals on day 0 for groups A, B, C, D, E, F, G, H, and I were 7.017±0.304, 7.033±0.328, 7.333±0.327, 7.05±0.281, 7.3±0.434, 7.35±0.308, 7.18±0.341 and 7.33±0.210 respectively as show in Table No 5. The mean TLC values of mice in all groups were similar. There was no statistically significant difference (P≥0.05). On day 30, the mean TLC values (mean ± S.E.) of animals for groups A, B, C, D, E, F, G, H, and I were 7.983±0.156, 21.817±1.08, 8.883±0.299, 12.983±0.33, 13.25±0.634, 13.283±0.697, 9.733±0.577, 9.3±0.447 and 21.617±1.565 respectively. The mean TLC values of groups B (DNCB alone), D (Vetricare Integumentary @ 0.1ml), E (Vetricare Integumentary @ 0.4ml), F (Vetricare Integumentary @ 1.0ml), Vetricare Integumentary @ 2ml were significantly higher than those of group A (P≤0.001). In contrast, the TLC values of Group C (Prednisolone 3mg), Group E (Vetricare Integumentary), and Group F (Vetricare Integumentary) were statistically like the normal group.The following study found that applying DNCB to mice led to an increase in the total white blood cell count as well as individual subtypes like neutrophils, basophils, eosinophils, monocytes, and lymphocytes. This suggests that there was an inflammatory reaction in mice following DNCB application, which is consistent with the findings of Yousif & Abu-Raghif (2020) and Hwang et al. (2021). The study also proposes that the anti-inflammatory effects of Curcuma longa L. and Terminalia chebula Retz. may be due to a decrease in total white blood cell counts, as suggested by Mujumdar et al. (2000) and Chandrasekaran et al. (2013), respectively.

Table No.5:  Effect of Vetricare Integumentary on total WBC count in DNCB-induced atopic dermatitis in different groups

The impact of various treatments including Normal control, DNCB, DNCB+ Prednisolone, DNCB+ Vetricare Integumentary @ 0.1ml, DNCB+ Vetricare Integumentary @ 0.4ml, DNCB + Vetricare Integumentary @ 1.0ml on TLC was assessed. Data are expressed as mean ± standard error of the mean (SEM). The DNCB group exhibited a significant increase in TLC value compared to the control (P ≤ 0.001). Conversely, the DNCB+ Prednisolone, DNCB + Vetricare Integumentary groups showed a decrease in TLC value compared to the DNCB group. On day 30, the mean IL-4 concentration values (mean ± S.E.) for groups A, B, C, D, E, F, G, H, and I were 18.365±1.47, 50.175±1.60, 23.428±1.40, 34.508±2.50, 38.015±2.35, 37.235±2.15, 26.908±1.27, 26.107±1.47, and 48.782±1.24, respectively. The mean IL-4 concentration values in group B (DNCB alone) were significantly higher than those in groups D (Vetricare Integumentary@ 0.1ml), E (Vetricare Integumentary @ 0.5ml), F (Vetricare Integumentary @ 1.0ml) and G (Vetricare Integumentary @ 2.0ml). However, there was no significant difference in mean IL-4 concentration values between groups C (Prednisolone), D (Vetricare Integumentary @ 0.1ml), E (Vetricare Integumentary @ 0.4ml) and F (Vetricare Integumentary @ 1.0ml) and G (Vetricare Integumentary @ 2ml. It was observed that the mean IL-4 concentration values in the DNCB-induced atopic dermatitis group were significantly higher than those in the healthy control group. Treatment groups showed a significant decrease in IL-4 values compared to DNCB induced group.   Similar observations were reported by Lee et al., (2016) Sharma et al., (2019), and Wang et al., (2022) reported that Curcumin administration @200mg reduced serum IL-4 levels and IgE. Th2-promoting cytokines (TSLP/IL-33) and Th2 cytokines (IL-4/IL-5/IL-13/IL-31) were markedly suppressed, along with reduced STAT-6 phosphorylation and GATA-3 expression. Development of Atopic Dermatitis (AD) is caused by an imbalance in the Th1/Th2 cell ratio among patients. This results in Th2 dominance, leading to an increase in IgE and IL-4 levels. The researchers identified IL-6 and IL-4 as primary pro-inflammatory cytokines that regulate immune cell differentiation and the production of various inflammatory mediators. These cytokines play a crucial role in the complex immunological milieu associated with AD (Bernard et al 2012). Curcumin interacts with several proteins, suppresses kinase activity, modulates the expression of cytokines, enzymes, and cell survival proteins, and influences the activation of transcription factors (Agrawal et al., 2017).

Table No. 6: The Effect of Vetricare Integumentary on IL-4 concentration in DNCB-induced atopic dermatitis in different groups of mice

The impact of various treatments including Normal control, DNCB, DNCB+ Prednisolone, Vetricare Integumentary on IL-4 concentration was assessed. Data are expressed as mean ± standard error of the mean (SEM). The DNCB group exhibited a significant increase in IL-4 concentration compared to the control (P ≤ 0.001). Conversely, the DNCB+ Prednisolone DNCB + Vetricare Integumentary @ 0.1ml, DNCB + Vetricare Integumentary @ 0.4ml, and DNCB + Vetricare Integumentary @ 1.0ml groups showed a decrease in IL-4 concentration compared to the DNCB group. The mean concentration of IFN-γ (mean ± S.E.) in animals on day 30 was as follows: group A - 69.75±1.52, group B - 308.483±2.53, group C - 81.757±1.95, group D - 154.25±1.88, group E - 160.167±2.11, group F - 157.4±2.38, group G - 97.405±2.06, group H - 89.047±2.41, and group I - 305.85±2.36 represented in table 7. The mean IFN-γ levels in group B was significantly higher than in groups C, D, E, F, and G. However, the mean IFN-γ levels in groups C (Prednisolone 3mg), D (Vetricare Integumentary @ 0.1ml), E (Vetricare Integumentary @ 0.5ml), and F (Vetricare Integumentary @  1.0ml), G (Vetricare Integumentary @ 2ml were lower than in atopic dermatitis group B (DNCB), while in groups D (Vetricare Integumentary), E (Vetricare Integumentary), and F (Vetricare Integumentary), and Vetricare Integumentary @ 2ml the mean concentration of IFN-γ was not significantly different (P≥0.05). The study observed a significant increase in mean IFN-γ levels in the DNCB-induced atopic dermatitis group compared to the healthy control group. Chen et al., (2004) suggested that the onset of atopic dermatitis is primarily driven by Th2 cytokines. They also proposed that the disease process involves the interaction of Th2 and non-Th cytokines, as well as various Th1 proinflammatory cytokines such as IL-12 and IFN-γ.  Similar findings were reported by Kim et al., (2022) with Terminalia chebula extract @ 100mg /kg, they found that inflammatory cytokines IFN?, IL-8, IL-4, and IL-6 significantly decreased after treatment with Terminalia chebula extract. The result might be Terminalia chebula contains chebulagic acid and chebulinic acid which has been reported to exhibit anti-inflammatory effects by reducing the IFN?, IL-8, IL-4, and IL-6. In the line Lee et al., (2016) found that treatment with p-hydroxycinnamic acid (HCA), derived from the roots of Curcuma longa, led to a reduction in the intracellular levels of signature cytokines, such as IFN γ, in both Th1-polarized and Th2-polarized cells. In contrast to the present study result in Zhang et al., (2009); Rubab and Ali (2016); Wang et al., (2022) found an increase in IFN-γ level after DNCB treatment.

Table No. 7: The effect of Vetricare Integumentary on IFN-γ concentration in DNCB-induced atopic dermatitis in different groups of mice