In-Vivo Effectiveness of 5 % Azadirachta indica Oil Cream as Anti-Scabies

Scabies is an infectious skin disease caused by mite Sarcoptes scabiei. Neem tree (Azadirachta indica) has the potential to be used as an anti-parasite due to the presence of azadirachtin compound that is commonly found in the seeds. The aim of this study was to evaluate in-vivo effectiveness of neem oil as an anti-scabies. This study used an experimental method. The effectiveness of the cream as an anti-scabies was tested on New Zealand white rabbits which were infected with scabies. Permethrin cream was used as a positive control and cream base was used as a negative control. Cream was applied once daily and left for 8 hours. The data were analyzed using Kruskal Wallis and Mann Whitney. Dermal acute irritation test was performed by applying 0.5 g cream on the rabbit dorsal. We found that 5% neem oil cream was effective as an anti-scabies with 20-21 days recovery time. The recovery time is longer than permethrin cream (7-8 days), but shorter compared to negative control with recovery time over 30 days. Primary irritation index for 5% neem oil creams was 0, indicating negligible irritation category. In conclusion, A. indica cream was effective for the treatment of scabies although its recovery time is shorter than permethrin cream.


Introduction
Scabies is a contagious skin disease caused by mites Sarcoptes scabiei.The infection cause erythema, papules, and vesicles which can lead to skin damage.Itching usually occurs at night.S. scabiei is an obligate parasite whose breeding requires a host.Scabies affects many children.Transmission can occur through direct contact with people with scabies or through indirect contact, such as the use of same clothing, towels, etc. Dense population with poor hygiene level can facilitate transmission of scabies.Indonesia is one of the five countries with the highest prevalence of scabies. 1,2Scabies can be treated through systemic and topical therapy.However, systemic treatmet is only given for severe scabies.Thus, topical therapy is the main treatment choice for scabies. 3,4em tree (A.indica) is a medicinal plant originating from India.Neem tree can grow well in tropical and subtropical areas.Every part of this plant has its own function that can be used for empirical treatment.This plant has many biological activities, e.g., as anti-histamines, anti-dermatitis, anti-fungi, anti-inflammatory, anti-scabies, diuretics, insecticides, anti-protozoa, spermicidal, and other biological activities.Previous study found that there was no difference in the effectiveness of the cream containing 10% of neem oil and 5% of permethrin cream as positive control for scabies. 5,6here is an urgent need to further evaluate the effectiveness of neem oil at different concentration with in-vivo method.In-vivo evaluation needs to be conducted to ensure the safety and effectiveness of the cream before being clinically tested in humans.Skin irritation potential also should be investigated to obtain complete safety profile of the cream. 7Therefore, we conducted this study to evaluate in-vivo safety and effectiveness of neem oil as an antiscabies.

Methods
This study used an experimental method.

Cream formulation
Certificated neem oil which was obtained from Happy Green Co, Jakarta, Indonesia.
The cream was formulated: 5% neem oil, 1 g adeps lanae, 14.2 g stearic acid, 10 g glycerin, 0.25 g sodium tetraborate, 1 g triethanolamine, 75 g distilled water, and 0.1% nipagine.The oil phase (adeps lanae and stearic acid) was placed in a vaporizer dish and heated over a waterbath at 70 0 C. The water phase (glycerin, sodium tetraborate, triethanololamine and distilled water) was placed in the evaporator cup and heated under the same conditions.
The oil and water masses were mixed at the same temperature until cream was formed.Nipagin was dissolved in distilled water and then mixed into the cream.Neem oil was added to the base cream.The cream was then mixed until homogenous. 9,10

Acclimatization of the animals
The experimental animals used were New Zealand albino male rabbits (6-8 weeks) with the weight of 1.5-2 kilograms.The animals were normally fed and maintained in a room with good air circulation, temperature, and lighting.The animals were acclimatized for 5 days.The body weight of the animals were observed every day.Acute irritation test A dermal acute irritation test was performed on healthy skin.Animal hair was shaved on the back area (2 x 3 cm).An irritation test was performed by applying 0.5 g of cream on the dorsal.It was then covered with gauze and plaster.The animals were divided into 2 group, i.e., control formula (base) and 5% neem oil cream.Each group has 3 animals.The degree of irritation was assessed after 1 hour, 24, 48, and 72 hours after exposure. 11rimary irritation index was calculated with the following formula: Primary Irritation Index = (A-B) C Information: A: The number of erythema and udema in tested observation area after 24, 48 and 72 hours divided by the number of observations.B: The number of erythema and udema in control observation area after 24, 48 and 72 hours divided by the number of observations.C: Number of animals

Anti-scabies effectiveness test
The S. scabei were injected to the back skin of animal.The transmission was performed indirectly, i.e., by swabing the rabbit dorsal infected with scabies into gauze and then affixed to the back of the test animal using plester.The incubation period of S. scabiei is 14 days from eggs to adult mites.Thus, the rabbit is left 2-3 weeks until the parasites spreads evenly to the surface of the skin.
The effectiveness of anti-scabies cream was calculated using the Federer formula. 11,12ew Zealand male rabbits were divided into 3 groups, i.e, the test group (5% neem oil cream), the positive control (5% permetherin cream), and the negative control group (cream base).The effectiveness of the cream was performed on rabbits infected with scabies by applying the cream to the infected skin once a day and left for 8 hours before it was cleaned.
Observations were conducted every day for 1 month.Examination of wound healing was conducted by observing the wound closure.Score of 1-5 was given in each observation (Table 3).Data analysis was performed using the Kruskal Wallis test and the Mann Whitney test.The Kruskal Wallis test was used to examine value differences in more than two groups.The Mann Whitney test was used to evaluate significant differences between categories.

Results and Discussion
Cream formulation 5% neem oil cream had thick consistency, faded yellow white co, distinctive odor, pH 8, and viscosity around 4195 Cps.Cream was selected as a dosage form since it has good ability to spread on the skin surface and is more convenient for the animals.The pH value was within the recommended

Dermal acute irritation test
The irritation test aimed to evaluate the safety of the neem oil cream by observing the the level of eryhtma and edema.Albino rabbit was selected as it has sensitive skin.Beside, the color of skin is brighter than the other type of rabbits.These characteristics allowed straightforward observation on the skin irritation.The primary irritation index for 5% neem oil cream is 0, indicating very negligible irritation category (a range of 0.0-0.4).Thus, it was safe to be applied to the skin.

Anti-scabies effectiveness
We found that 5% neem oil was effective as an anti-scabies with 20-21 days recovery time (Table 4 and 5).The recovery time is longer than permethrin cream (7-8 days), but shorter compared to negative control with recovery time over 30 days.Statistical analysis showed that there was significant difference between three dose groups.Azadirachtin compound in neem oil acted as an anti-parasites.This compound affects the hormonal cycle of parasites, prevents the growth and development of mites, makes the eggs sterile, and gives an antifeedant effect. 9-14Neem oil does not terminate scabies mites directly, but interfere with the proliferation and reproduction of mites, thus there is a reduction in the population of parasites over time.Therefore, the application of the cream should be conducted daily.Besides, the cream can also relieve itching and moisturize cracked skin. 15,16e 5% permethrin cream was used as the positive control because it is the first line of anti-scabies drugs.Permethrin cream has very low toxicity to mammals but the results of in vivo studies showed children are more sensitive to permethrin. 1,16Besides, it has very little absorption and was metabolized quickly.It was excreted through sweat, sebum, and urine. 17-19Nevertheless, its recovery time is shorther than neem oil.
This study has limitation, particularly in the transmission process of S. scabiei.The administration of parasites on rabbit dorsal

Table 2 . Assessment of Skin Reactions 10 Erythema
Severe edema (the area increases more than 1 mm and widens beyond the exposure area) 4

Table 3 . Assessment of the Effectiveness of the Cream1 1 No Assesment Score 1
The wound closes 100% (the wound closes completely and has no scab) 1 2 The wound closes 75% (the wound closes and has few scabs) 2 3 The wound closes 50% (the wound partially closes and has few scabs) 3 4 The wound closes 25% (the wound slightly closes and has many scabs) 4 5 The wound completely opens and has many scabs 5