The leaves of the L.latifolium was isolated and dried under shade. After grinding the leaves, 100 g of milled leaves was added to 400 ml of pure methanol and mixed. The obtained solution was left at room temperature for 24 h. After filtering the mixture and separating coarser particles, the alcohol was separated in vacuum conditions and finally, the obtained dry powder was placed into sterile containers and stored at -70 °C (Moazeni and Nazer 2010).
In vitro study
After collection of head lice, in vitro tests were started within 30 min. A filter paper contact bioassay (Rassami and Soonwera 2013a; WHO 1981) was used to evaluate the toxicity. For in vitro study, concentrations of 0.12, 0.25, 0.50 and 0.75 ml/cm2 from herbal extract were provided. Pieces of filter papers were soaked in this solution and put on separate plates, and then 10 head lice were placed on the filter paper. The mortalities of head lice were recorded at 5, 15, 30, 45 and 60 min. The test was repeated 3 times for each concentration. Permethrin %1 and distilled water were used as the positive and negative control, respectively.
In vivo study
With respect to the results of in vitro study, the best and most effective concentration and time were selected to be used for in vivo study. The alcoholic extract was used as a shampoo and Permethrin %1 was used as positive control. Patients were under the supervision throughout the treatment period and the incidence of adverse events causing withdrawal from the study. Finally, Statistical analysis was performed using the SPSS software. Four stages with 48 hours interval were considered for medication. In each treatment, 250 ml of the extract was applied on the patients’ hair for 30 minutes and then rinsed. Twenty four hours after treatment, the individuals were examined for infection and the presence of adult lice was regarded as the infection index (Soonwera 2014).
The results are illustrated in Table 1. Furthermore, Permethrin shampoo was used as the positive and distilled water as the negative control. After 30 minutes of exposure to 0.75 ml/cm2 concentration, all lice were dead. The same concentration was used for infected individuals. 24 hours after each treatment, patients were examined for infection and the number of adult parasites was considered as infection index with results presented in Table 2. After 4 stages of treatment in the Permethrin-treated group, one individual was still infected and five adult parasites were removed from the patients’ hair. However, in the group treated with herbal extract, all individuals were completely treated and no adult lice were found in their hairs.
In Iran, head lice infestation is a serious problem affecting primary schoolchildren. The infestation rates were 57.7, 52.3, 44.1, and %36.6 of primary school children in the northeastern, southern, central, and northern parts of Iran, respectively. In our study, the potential of pediculicidal activity of alcoholic extract of L.latifolium was evaluated in comparison with chemical medicine. After in vitro assay, herbal shampoos or lotion based on native plant (L. latifolium) in Iran used for treatment against head lice and compared with a commercial shampoo in order to assess theirs in vivo efficacy. Acaricidal effect of this herb showed in 2015 and thymol is one of its effective ingredients for treatment of arthropods (Razavi et al. 2015).
Since L. latifolium can act against mites, this plant was selected for head lice treatment and Permethrin shampoo as the positive control. Due to the low impact of drugs on parasite eggs, treatments were repeated and patients were examined after each treatment. In each stage of the treatment, individuals were studied in terms of allergies and side effects and any kinds of side effects such as itching, redness of the skin or hair loss excluded patients from the study. Fortunately, no side effects were observed and all patients tolerated the medication well. Considering the results, the herbal extract was evaluated as a better alternative for treating infected individuals.
In addition, in vitro pediculicidal activity has been reported for some plant-based pediculicide such as Annona squamosal, Averrhoabilimbi, Canagaodorata, Citrus limon, Cymbopogonnardus, Cymbopogonwinteratus, Clitoriaternatea, M. alternifolia, M. azedarach, M. fragrans, Piper betle, Piper ribesioides, S. aromaticum, Taccachantrieri, and Zingibercassumunar (Bagavan et al 2011; Carpinella et al 2007; Heukelbach et al 2008a; Intaranongpai et al 2006; Rassami and Soonwera2013a226; Rossini et al 2008; Shrivastava et al. 2010; Soonwera and Rassami2011).
Rassami and Soonwera(222011″2011) also reported herbal shampoo based on %10 of long pepper (Piper retrofactum) fruit extract which was shown to be highly effective against head lice infestations. It was recorded that more than 95.0 % mortality of head lice at 10.0 min after application of these herbal shampoos to infested schoolchildren of Ladkrabang area, Bangkok, Thailand as compared to %0.47 mortality in the control group treated with commercial pediculicide. Moreover, Abdel-Ghaffar and Semmler (222007″2007) and Abdel-Ghaffar et al. (222010b%22b”2010HYPERLINK “%222010b%222010HYPERLINK%20%226%222010b%22b”) proved the shampoo with neem seed extract and grape fruit extract to be highly effective against all stages of head lice of naturally infected human in Egypt.
Finally, it is worth mentioning that head lice must be controlled as a serious disease at an early stage. Also, due to certain kinds of drug resistance, available shampoos may not be effective in this regard. The present study showed that Lepidium latifolium can well controls the disease without side effects. Further studies are needed to be carried out for testing higher doses to decrease medication time and a number of replications.