INTRODUCTION:

Psoriasis is a chronic inflammatory autoimmune skin disorder manifesting red, scaly, inflamed skin^1,2. This is a multifactorial disease, which can be exacerbated by genetic, environmental and immune factors³. The underlying mechanism of psoriasis is that it accelerates the life cycle of skin cells, thereby causing a build-up of skin on the surface and is associated with red and painful, inflamed skin. Severity can range from mild localized lesions to severe ones that can cover the whole body. Globally, psoriasis affects 0.09-11.8% of the population. It is mainly associated with HLA locus (HLA-C), particularly with HLA-Cw* 0602 allele. The stimulation of T-cell induces hyper-proliferation of keratinocytes, which results in psoriatic lesions ^4,5. Psoriatic patients are usually treated with medications that block TNF function⁶.

Excessive cell growth and production are observed in psoriasis. This complex pathogenesis of psoriasis has been a challenge to build a standard treatment regimen. Thus, studies are still in progress to come up with better drugs to treat psoriasis. Although the existing treatments are effective, they irritate the skin on prolonged use. Plant source has always been a treasure of medicine since ancient era and many such plants have been used to treat psoriasis as well. Some of the advantages of using drugs of natural origin include, patient compliance, less side effects, easy availability and low costs. Turmeric has been popularly used for its antimicrobial and antiseptic properties in Ayurvedic formulations. These properties of turmeric are due to curcumin; a phyto-constituent of turmeric⁷. It is chemically, diarylheptanoid- a natural phenol, having excellent antioxidant properties⁸. Studies are being conducted on the analogues of curcumin for screening of their various other activities. Antioxidant, antibacterial and anti-inflammatory properties of the structural half analogue of curcumin, dehydrozingerone (DHZ) is reported in scientific literature⁹. There are reports indicating the wound healing and anti-inflammatory properties of DHZ¹⁰. DHZ is also capable of blocking TNF alpha, which is a key cytokine involved in psoriasis¹¹. Therefore, the current study was proposed to evaluate the effectiveness of dehydrozingerone gel to reduce psoriatic like lesions, erythema and scaling using imiquimod-induced psoriatic model in Balb/c strain of mice.

MATERIALS AND METHODS:

Laboratory chemicals used:

Vanillin, acetone, sodium hydroxide, hydrochloric acid, ethanol, carbopol, triethanolamine, methotrexate.

Synthesis of dehydrozingerone:

2.5g of vanillin (4-hydroxy, 3 methoxybenzaldehyde) was taken in a 100mL round bottom flask and was dissolved completely in 10mL of acetone. The mixture was placed in ice bath till a temperature of 5-10⁰C was attained. Then a mixture of 1.4g of sodium hydroxide in 10mL of distilled water (3.5N) was added slowly with continuous stirring using a glass rod which resulted in a bright yellow solution. The magnetic bead was washed with acetone and added to the round bottom flask. The flask was placed on a magnetic stirrer at 300rpm and was continued to stir for 1.5hours on an ice bath. The steps are shown in Figure 1.

Formation of bright yellow precipitate was seen upon continuous stirring that eventually turns to sunset yellow with bright yellow supernatant at the end of 1.5 hours. This mixture was kept open overnight at room temperature for the evaporation of acetone. After acetone completely evaporated, the precipitate turned blood red. The round bottom flask was washed with cold water and the contents were transferred into a 500mL beaker. 300mL of cold water and around 100g crushed ice were also added. Now, the beaker was kept in an ice bath. The solution was further acidified with 10% HCl drop wise and the pH was monitored during the addition. The blood red solution had a pH of 14.0. As the pH decreased to 8.0, the solution turned orange, further turning golden yellow after reaching a pH of 7.0. Acidification was stopped when the pH reached 6.0 giving a turmeric yellow precipitate at the bottom of the beaker. This was filtered using suction and dried in open air for 2 hours. The practical yield was then calculated. The product was recrystallized using 40% ethanol since it is readily soluble in absolute ethanol. Shiny yellow crystals were obtained, and melting point was observed to be 124^ᵒC.

Figure 1: Steps involved in the synthesis of DHZ

Formulation of gel:

For the preparation of 5g of 3% DHZ gel, the following procedure¹² was adopted. 0.1g carbopol-940 was dissolved in 5mL of water in a 100mL beaker and left overnight for hydration of carbopol. 0.15g of dehydrozingerone was dissolved in 5mL of 60% ethanol and then added to the hydrated carbopol. Stirred well and pHwas adjusted to 7.0 using triethanolamine [2, 2’, 2’’-Nitrilotri (ethan-1-ol)]. The gel was stored at room temperature.

Induction of psoriasis:

Among the various psoriatic animal models, Imiquimod-induced model was shown to be the most developed and validated. Studies have revealed higher correlation between psoriatic gene expression in human psoriatic skin sample to mouse imiquimod (IMQ) induced psoriatic model. A total of 7 trial days with male Balb/c strain of mice (where n=8) that are 8-12 weeks old were used. All the animals were shaved on the dorsal back of their interscapular area using depilatory cream, one day before the commencement of the treatment¹³. The cream was applied on the back or on the ear and were checked for various parameters including erythema, scaling, spleen weight, etc. Animal experimentation was conducted after obtaining the approval from institutional animal ethics committee (IAEC/KMC/118/2018) KMC Manipal. The animal handling, maintenance and care was taken according to the guidelines laid by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)¹⁴. The study included four treatment groups namely normal (N), disease control (IMQ), standard drug (MTX), test drug (DHZ) consisting of 8 mice each. 62.5mg of imiquimod cream was applied on each animal of IMQ group, MTX group and DHZ group and was left for 4-5 hours. After 4-5 hours, carbopol gel base, test drug gel was applied on normal and DHZ groups, respectively. Methotrexate was administered orally at a dose of 300µg/kg body weight. Application was continued in similar pattern for 6 days. Modified PASI scoring was done for scaling and erythema. Once development of psoriatic like lesions were seen, clinical scoring was taken every day on a scale of 4. On day 7, body weight, clinical scoring was measured and spleen and skin samples were isolated.

RESULTS AND DISCUSSION:

Results:

Bright yellow crystals of DHZ was obtained with a melting point of 124⁰C confirming the formation of DHZ with a yield of 82%. Imiquimod cream application caused significant increase in erythema and scaling score from day 2 to 7 of clinical scoring, confirming the induction of psoriasis. Pre-clinical testing of this gel against Imiquimod-induced psoriasis indicated that DHZ gel significantly reduced erythema and scaling score at the end of seventh day of clinical scoring indicating that 3% DHZ Gel can reduce imiquimod induced psoriatic like lesions. 3% Dehydrozingerone gel showed promising results to prevent psoriatic like lesions in imiquimod induced psoriasis (Figure 2, 3 and 4).

Figure 2: Induction of psoriasis (On Day 4)

DISCUSSION:

Generally, psoriasis affects the skin of the elbows, knees, scalp, lumbosacral areas, intergluteal cleft and glans penis^15,16. Typical lesion is a well demarcated, pink to salmon-coloured plaque covered by loosely adherent silver-white scales. Due to the thinning or absence of stratum granulosum, extensive overlying parakeratotic scales are seen. Psoriatic plaques are thinning of portion of the epidermal cell layer that overlies the tips of dermal papillae and dilated, tortuous blood vessels within these papillae. This results in abnormal proximity of vessels within the dermal papillae to the overlying parakeratotic scale and results in characteristic multiple, minute, bleeding points when the scales are lifted from the plaque¹⁷.Most bacterial infections and systemic fungal infections exacerbate psoriasis and often make the treatment difficult¹⁸. Till date, treatment for psoriasis includes topical, systemic, biologics¹⁹ and light therapies. Severe cases of psoriasis require the combination of topical and systemic or light therapy²⁰. Topical corticosteroids diminish inflammation and relieve itching. Long-term usage or the overuse causes thinning of the skin and thus is mainly given in cases of mild psoriasis. Calcipotriene and Calcitriol²¹, a prescription cream of vitamin D analogue, are limitedly used because of their skin irritancy²². Commonly used for short-term application is Anthralin. This aids in removal of scales, thereby resulting in smoother texture of the skin²³. The staining property of anthralin has led to its decreased use. Topical retinoids and Calcineurin inhibitors have anti-inflammatory properties but cannot be given during pregnancy and applied to sensitive areas respectively²⁴. Goeckerman therapy, Psoralen plus ultraviolet A, Excimer laser are all light therapies that uses natural or artificial ultraviolet²⁵. Blistering increased risks of skin cancer, redness, dry and wrinkled skin are a few of the side effects experienced with light therapies ²⁶. Oral or injected medications like methotrexate, cyclosporine and various other biologics suppress the immune system but alongside affect the liver, kidney and red blood cells. The current psoriasis skin models are broadly divided into in-vitro and in-vivo models. But no model is homologous (if the symptoms and course of the disease is same as humans) nor isomorphic (if the symptoms are same but course is different). Direct induction of psoriasis by the application of imiquimod is considered most fitting for this study as it is simple, rapid and a cost-effective method to study induction of psoriasis²⁷. Also, no labour-intensive breeding is necessary and the lesions that are induced are similar to human psoriatic lesions phenotypically and histologically, moreover, are dependent on IL-23 and IL-17. It was observed that the topical application of imiquimod for HPV (Human Papillomavirus Infection), caused psoriasis in patients²⁸. This was the result of the strong activation of the immune system. Imiquimod is a ligand for toll-like receptors TLR7 and TLR8, which are expressed in macrophages, monocytes and dendritic cells, producing cytokines and chemokines²⁹. In this study, the authors tried to explore the effect of DHZ to reduce psoriatic like lesions in imiquimod-induced psoriasis in mice. The effectiveness of drug was tested against standard drug methotrexate³⁰ upon topical and oral administration, respectively. Application was continued in similar fashion for 7 days. Once development of psoriatic like lesions are seen, clinical scoring was taken on a scale of 4. Scoring was done for scaling and erythema. DHZ has shown significant action on the reduction of scaling from day 1 to day 7, in comparison to the disease control. Although there was an increase in erythema scoring on day 3, it had declined by day 7 denoting the effectiveness of the test drug. Furthermore, a study on the TNF alpha estimation is necessary. It was also noticed that DHZ has staining property like that of Anthralin, which can be a limitation.

Authorship and Acknowledgements:

Authorship:

K. Nandakumar has conceptualized the idea, Rekha Shenoy along with K.S.R. Pai has designed the protocol Anusha Prabhu along with Chaitanyamayee Kalakota synthesized and formulated the gel, Gangadhar Hari and Jeslin Johnson carried out the animal study while Kufaila and Gangadhar Hari contributed in the clinical scoring and data interpretation. All the authors provided their valuable inputs and suggestions in writing the paper.

ACKNOWLEDGMENTS:

Study was supported by the Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal. This project would not have been successful without timely guidance and support of Ms. Neesha J Dargad, Mr Manas Kinra, Mr Jayant Singh, Ms. Runali Sankhe, Research Scholars M.Pharm students and Research Scholars of Manipal College of Pharmaceutical Sciences, Manipal.

CONFLICT OF INTERESTS:

There are no conflicts of interest.

FUNDING SOURCE:

This project was funded by Manipal College of Pharmaceutical Sciences, MAHE, Manipal, India.

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Received on 18.05.2020 Modified on 23.07.2021

Research J. Pharm. and Tech 2022; 15(10):4469-4472.

DOI: 10.52711/0974-360X.2022.00749

Development and Pharmacological assessment of dehydrozingerone gel formulation on imiquimod-induced psoriasis in mice

RESULTS AND DISCUSSION:

Results: