Anti-leishmanial effect of the hydroalcoholic extract of the leaves, roots and seeds of Arctium lappa

Abstract

Objective: Zoonotic cutaneous leishmaniasis is a common including endemic infectious disease in many parts of the world and Iran. Due to Arctium lappa wide therapeutic applications, the anti-leishmanial effect of the hydroalcoholic extract of its leaves (L), roots (R) and seeds (S) has been investigated in this research.

Materials and methods: The leaves, seeds and roots of the greater burdock plant were extracted. In the in vitro phase, its cytotoxic and anti-leishmanial effects on promastigote and amastigote forms of Leishmania major(L.major) were investigated. In the in vivo stage, the leishmaniasis mouse model was dosed with concentrations of 50, 100, and 200 mg/kg and the liver and spleen parasite burden was checked.

Results: The results of this research in the in vitro phase showed that the antileishmanial effect of the hydroalcoholic extract of leaves, roots and seeds on the promastigote and amastigote forms of L. major has a significant relationship with the increase in the concentration of the extract (for all p≤0.001). Also, exposure time and interaction effect of concentration and exposure time were significant. In the in vivo phase the significant effect of the increase in concentration (L: p≤0.001, R: p=0.02, S: p=0.03), exposure time (L: p≤0.001, R: p≤0.001, S: p≤0.01) and the interaction effect of these two factors (L: p=0.002, R: p≤0.001, S: p≤0.001) on reducing the size of the wound was shown.

Conclusion: The investigation established that hydroalcoholic extract of the leaves, roots, and seeds of the greater burdock in high concentration exhibited beneficial inhibitory effects on the leishmanial lesions.

Keywords: Anti-leishmanial; Arctium Lappa; BALB/c mice; Glucantime; MTT.

Figure 1

The viability% of promastigotes after exposure to different concentrations of leaves, roots and seeds of Arctium lappa after 24 and 48 hours using MTT method. L,leaves; R, roots; S,seeds. Data analysis with two-way ANOVA method showed the significant effect of the concentration of each extract (L: p≤0.001 R: p≤0.001 (S: p≤0.001), exposure time (L: p=0.002, R: p=0.004, S: p≤0.001(and the interaction effect of these two factors (L: p≤0.001, R: p≤0.001, S: p≤0.001) on the viability of promastigote form. The results illustrated that the highest inhibitory effect on promastigotes of L. majar is respectively related to the seed (IC50 = 480 and 420 µg/ml) then the root ((IC50 = 983 and 771 µg/ml) and finally the leaf (IC50 = 1025 and 920 µg/ml). Positive control (Positive ctrl), Negative control (Negative ctrl).

Figure 2

The viability (%) of macrophage cells after exposure to different concentrations of leaves, roots and seeds of Arctium lappa after 24 and 48 hours. L,leaves; R, roots; S,seeds. The results of the data analysis by two-way ANOVA statistical tests showed the significant effect of the concentration of the extracts (L: p≤0.001, R: p≤0.001, S: p≤0.001), the exposure time (L: p≤0.001, R: p=0.004, S: p≤0.001) and the interaction effect of these two factors (L: p=0.002, R: p=0.019, S: p=0.024) on the viability of macrophages. Also, by performing Tukey's post hoc test, it was found that there is a significant relationship between the different concentrations of the hydroalcoholic extract of the leaves, roots and seeds of the Arctium lappa with each other and with the negative control (p<0.05). In the period of 48 hours, there was a significant difference between high concentrations (100, 200, and 400 µg/ml) and the positive control (p<0.05). Positive control (Positive ctrl), Negative control (Negative ctrl).

Figure 3

Viability % of amastigotes after exposure to different concentrations of the three types of extracts in 24 and 48 hr. L,leaves; R, roots; S,seeds. Data analysis with two-way ANOVA method showed the significant effect of the increasing concentration (L: p≤0.001, R: p≤0.001, S: p≤0.001), increasing contact time (L: p≤0.001, R: p≤0.001, S: p≤0.01) and the interaction effect of concentration and exposure time (L: p=0.002, R: p≤0.001, S: p≤0.001) on reducing the intracellular form of the parasite. Also, by performing Tukey's post-hoc test, it was found that there was a significant relationship between different concentrations of hydroalcoholic extract of leaves, roots and seeds of Arctium lappa and the positive and negative controls (p<0.05). Positive control (Positive ctrl), Negative control (Negative ctrl).

Figure 4

percentage of wound reduction in the groups treated with hydroalcoholic extracts of leaves, roots, and seeds of the Arctium lappa. L,leaves; R, roots; S,seeds. By using the two-way ANOVA statistical method, the significant effect of the increase in concentration (L: p≤0.001, R: p=0.02, S: p=0.03), the increase in exposure time to the extract (L: p≤0.001, R: p≤0.001, S: p≤0.01) and the interaction effect of these two factors (L: p=0.002, R: p≤0.001, S: p≤0.001) on reducing the size of the wound was determined. According to the obtained results and the comparison of the average lesions, it could be suggested that greatest effect is related to Glucantime, then the concentration of 200 mg/kg of leaves, roots and seeds respectively. Also, the statistical analysis of Variance test showed that in each group of L, R and S extracts, the effect of the extract increases with the increase of concentration and exposure time (p≤0.001). Positive control (Positive ctrl), Negative control (Negative ctrl).

Figure 5

The effect of Arctium lappa leaves, roots and seeds extract (the concentration of 200 mg/kg) on the CL mouse model before (A (and after (B (treatment compared to the negative control. CL, Cutaneous Leishmaniasis.