Modulated effect of Ammi majus L. on Psoriasis-Like Skin Inflammation Caused by Imiquimod in Vivo

Ammar A Ramadhan^1* and Shihab H Mutlag²

^*Correspondence: Ammar A Ramadhan, Center for Allergy and Asthma, Al Zahraa Consulting, Ministry of Health and Environment, Iraq, Email:

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Introduction

Psoriasis is an autoimmune disorder characterized by changes in the components of the skin, such as infiltration of white blood cells and an increase in blood vessels in the dermis, resulting in localized inflammation with enlargement of the epidermis. They generally appear as undesirable local symptoms [1-4]. To date, the pathogenicity is not entirely clear [5], although extensive research continues to explore the underlying mechanisms involved. Imiquimod (IMQ) is an immune response adaptor that was firstly approved by the U.S Food and Drug Administration [6], it is used for the topical treatment of genital and perianal warts caused by human papillomavirus. Additionally, for the treatment of other virus-related skin abnormalities [7,8]. Animal models have been recruited to mimic skin conditions such as human psoriasis for many years [9,10]. In a mouse model, topical application of IMQ has been confirmed to cause psoriasislike skin inflammation [11]. Whereas, these laboratory animals are considered a model that can allow the explain the basic mechanisms and the estimate of proposed new treatments for psoriasis [12]. Humans have used plants as medicines for thousands of years, and there are many medicinal plants that can prevent diseases and reduce their complications as much as possible, because they are highly effective with fewer side effects [13,14]. Ammi majus L. (AM) is belongs to the Apiaceae plants, native to northern Africa, southern Europe, western Asia and India [15,16]. In Iraq, it is found in fields and gardens in several areas especially in Baghdad [17]. Several studies have evaluated the efficacy of Ammi majus in curing some skin disorders in vivo, so the aim of the study was to evaluate the efficacy of A. majus seed extract against psoriasis-like skin inflammation caused by Imiquimod in a mouse model.

Materials and Methods

Drugs

Imiquimod )IMQ) was obtained commercially from Aldara TM 5% Cream. Clobetasol propionate 0.05% (DermovateTM Cream) was punched from a private pharmacy.

Preparation of plant extract

The seeds of the medicinal herb Ammi majus were purchased from Erbil, northern Iraq. The plant was then kindly cleaned to remove dirt and dried in the laboratory at room temperature for a few days. Then, the dry plant material was crushed followed by sieving to get a tough grind. The plant matter powder was then extracted with petroleum ether (40-60°C) by re-uX using a sohxlet for 10 h, and the resulting crude extraction solution was evaporated by a rotary vacuum evaporator and concentrated to obtain the fractional petroleum ether A. majus extract. The resulting crude extract was stored at 4 °C before formulation.

Preparation of a topical emulsion (water in oil)

Wool fat (70 g) was melted in a water bath at 75°C until it became a liquid. A. majus fraction extract (5 g) was added to a laboratory beaker containing up to 30 ml of distilled water and stirred until solutions were obtained. Then the last solutions were added to the melted wool fat baker and the final mixture was cooled with stirring until it was congeal .

Animals and study design

In this experiment, (40) adult male mice weighing (22-42 g), and their ages ranged between (6-7) weeks, were used. They were obtained from the animal centers of the University of Baghdad/Iraq after the study protocol was approved by the Iraqi Review Board (IBR) for the College of Pharmacy. The animals were housed in polypropylene cages and in environmentally controlled conditions in terms of humidity, temperature and appropriate lighting, with free access to water and diet. Laboratory mice were separated into 5 groups (8 animals in each group) and divided as shown in Table 1.

Table 1: Experimental groups and dose treatments

Ear and skin thickness measurement

Ear thickness (mm) was measured for each rat using a digital micrometer, on days 2, 4, 6, 8 and 14 of the experiment. The change in ear thickness was used to demonstrate the gravity of inflammation. Also, the skin thickness of the dorsal region of each mouse (mm) was measured at the end of the experiment (day 14) to compare the thickness measured with that of the other groups.

Psoriasis area and severity index

This index PASI was adopted to assess the psoriasis score, which combines assessment of the severity of lesions and the extent of the affected area into a single indicator.

Inflammatory markers

Blood was taken through a cardiac puncture and serum was separated and stored at -20°C until analysis for determination of IL-23 (pg/ml) and IL-17 (pg/ml) levels.

Statistical analysis

Data were summarized by SPSS software. Using mean ± standard deviation, as well as one-way analysis of variance (ANOVA).

P values ≤ 0.05 were considered statistically significant.

Results

Results for the PASI score, significantly at P ≤ 0.05, the data indicated an increase in the CON-IMQ group compared to the CON group.

Also, in the AM-IMQ group, topical application of A. majus caused a respectable decrease compared to the CON-IMQ group. In the CP-IMQ group, the score was diminished compared to the CON-IMQ.

Whereas in A. majus ointment only (AM) there was no observed change in the score during the study (Table 2).

Table 2: Comparison of psoriasis area score and severity (PASI) among study groups.

Moreover, there was a significant increase in both ear and dorsal skin thicknesses in the CON-IMQ compared to that in the CON group. But in the AM-IMQ group, the measured ear and dorsal skin thicknesses were clearly decreased compared to the CON-IMQ group. While in the CP-IMQ group, there was a significant decrease in the thickness of both ear and back skin as compared to the CON-IMQ as shown in Tables 3 and 4.

Table 3: Comparisons of ear thickness (mm) among study groups.

Table 4 : Comparisons of dorsal skin thickness (mm) among study groups.

As for the results related to interleukins, there was a high increase in both serum levels of IL-23 and IL-17 in the CON-IMQ compared to the corresponding levels in the CON group. In the CP-IMQ, there was a marked decrease in IL-23 and IL-17 serum levels compared to the CONIMQ, and by comparing AM-IMQ with the CON-IMQ group, there was an observed decrease in IL-23 and IL-17 serum levels. Finally, serum IL-23 and IL-17 levels were significantly increased in the AM group compared to the CON-IMQ (Figure 1).

Figure 1: The comparison of mean: (a) IL-17 and IL-23 levels among study groups .* Significantly different when compared with CON mice. # P-value = 0.05 significant in comparison with CON-IMQ group.

Discussion

Based on the evidence of many previous studies, the imiquimod-inducible psoriasis mouse model summarizes signs of inflammatory skin disorders like to human psoriasis. Like the thickness of the epidermal layer, prolongation of retinal-like protrusions and hyperkeratosis next imiquimod induction [18-20]. Physicochemical properties of IMQ can explain the kinetics of penetration and retention of skin layers. The low molecular weight allows easy penetration into the skin but due to its lipophilic the molecule is retained primarily in the stratum corneum [21]. Skin changes appeared from the second day, as the overall result of PASI came to highlight the severity of psoriasis and in particular by noticing the crusting that represents the formation of the scale. The stratum corneum layers are separated thus promoting better IMQ skin penetration [22]. Significant skin thickening was observed on both the back and the treated ear starting from the second day and it increased throughout the study period. Systemic toxicity was also demonstrated synchronous with the appearance of localized psoriasis signs. In skin disorders arising from inflammation such as psoriasis, different types of cytokines such as interleukins are observed to be involved in modulating inflammatory responses [23]. IL-17 has a decisive role in the pathogenicity of psoriasis as its cutaneous expression is tight associated with the up growth of skin lesions, with an increase starting one day after IMQ treatment [24]. A. majus contains four main linear furocoumarins, the most pharmacological of which is xanthotoxin [25]. Previous studies have proven the effectiveness of xanthotoxin in the treatment of psoriasis, and this is consistent with what we have found in this study [26,27]. A. majus has also been shown antiinflammatory activities due to its richness in phytochemicals [28]. For its dermatological role, Shinde and Mohite, have conducted studies on the composition of an herbal gel containing Aloe Vera L and Ammi Majus L for use topically on the skin [29-31].

Conclusion

When Ammi majus L seed extract was used against psoriasis-like inflammation induced by Imiquimod in male mice and compared to clobetasol propionate, modulation of skin thickness changes and inflammation parameters was observed.

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