Antileishmanial effect of podophyllotoxin and podophyllin on Leishmania major in vitro and in vivo

Abstract

Therapeutic research is very important in the prevention and treatment of leishmaniasis due to problems such as drug resistance, scarring and disease recurrence. The aim of this study was to determine how Leishmania major responds to the anti-leishmaniasis properties of podophyllotoxin and podophyllin. Cultured Leishmania promastigotes were exposed to different concentrations of podophyllotoxin and podophyllin for 24 and 48 h. Then, during the animal phase, Balb/c mice were experimentally injected with Leishmania promastigotes. After wounding, the effects of 0.5% podophyllotoxin and 25% podophyllin on reducing wound diameter and the number of amastigotes in the wound were evaluated. Podophyllotoxin and podophyllin were 83% and 59% lethal to Leishmania major promastigotes at the highest concentrations (200 µg/ml) and time (48 h). In the in vivo study, the mean lesion diameter at the end of treatment in the negative control group was 15.10 mm compared to 14.21 mm and 11.55 mm in the 25% podophyllin and 0.5% podophyllotoxin groups, respectively. Although both agents reduced the size of mice wounds and the number of amastigotes in the wounds, podophyllotoxin was more effective in this regard. Based on the results, podophyllotoxin and podophyllin can be used as leishmaniasis drugs after further research.

Graphical abstract

Introduction

Leishmaniasis is a zoonotic disease transmitted by vectors with a diverse epidemiology and clinical presentation. 350 million people are at risk of contracting leishmaniases, and there are 12 million infected cases in more than 98 countries (Torres-Guerrero et al. 2017; Sabzevari et al. 2021; WHO 2022). The most prevalent type of leishmaniasis is cutaneous leishmaniasis (CL), which is mostly brought on by Leishmania major and Leishmania tropica. More than half of Iran’s states are endemic for the CL, the most prevalent form of leishmaniasis (Hajjaran et al. 2014; Sabzevari et al.. 2021). Leishmania tropica and Leishmania major, which stand for the two main types of CL, anthroponotic CL and zoonotic CL, respectively, are the two parasites that cause it (Sarkari et al. 2016; Sabzevari et al. 2021).

Currently available therapies are still not sufficient (Zulfiqar and Avery 2022). In considering the rising prevalence of CL, the general awareness of this infection, and potential aesthetic side effects, there is an urgent need for significant global research to investigate the anti-leishmanial efficacy of novel medications and herbal-based derivatives. The building of the mitosis spindle, cell form, cilia and flagellar motion, and intracellular transport can all be affected by microtubules, making them appealing targets for antiparasitic and antifungal drug development (Chatterji et al. 2011). Several medications that have modest toxicity toward their mammalian relatives have been utilized to target trypanosomatid microtubules by blocking or increasing the polymerization of tubulin (Fennell et al. 2008; Jiang et al. 2020). Podophyllotoxin, an antineoplastic and antiviral destructive cyclolignan obtained from Podophyllum spp. (Berberidaceae) and multiple different species of other genera and families have been observed to indicate determined biological actions, in particular antineoplastic effects, by blocking the microtubule formation (Gordaliza et al. 2004; Umesha et al. 2015) and was considered as the precursor of important anti-cancer drugs such as Etoposide and Teniposide (Cragg and Newman 2005). Additionally, by contact at the colchicine-binding site, podophyllotoxin and its derivatives suppress tubulin polymerization (Ravelli et al. 2004). The capacity of podophyllotoxin to suppress steroidogenesis, reduce cytokine levels, and directly inhibit tumor necrosis factor alpha may help to explain its anti-inflammatory properties in addition to its antimitotic activity (Carlstrom et al. 2000). Recently, the antiparasitic activities of podophyllotoxin have been investigated including: Trypanosoma brucei, Giardia and leishmania (Uddin et al. 2010; Hamidzadeh et al.2011; Kazemzadeh et al. 2014; Gutiérrez-Gutiérrez et al. 2017; Alvarez et al. 2017).

Podophyllotoxin has been investigated in numerous randomized studies and is currently offered as a solution or cream. An herbal extract with 4 to 8% podophyllotoxin, also known as podophyllum resin or podophyllin, is frequently used as a treatment for plantar warts and genital warts. While podophyllotoxin is stable and pure, it is non-standardized and comprises a number of active ingredients (Gilson et al. 2020). With little adverse effects and ease of patient application at home, podophyllotoxin is the first-line treatment for condylomata acuminate (Johnson et al. 2018).In order to better manage CL, the current study examines the effects of podophyllotoxin and podophyllin in vivo and in vitro.

Materials and methods

Chemicals Podophyllotoxin and podophyllin were purchased from Santa Cruz Biotechnology Company (United States).

Experimental animals A total of 40 pathogen-free BALB/c mice, approximately 6–7 weeks, have been bought from the Pasteur Institute in Karaj, Iran. The animals were kept in typical settings with complete access to both water and food (temperature 22 °C, lights on from 6:00 to 18:00). Iranian strains of L. major promastigotes (MRHO/IR/75/ER) in the amount of 2 × 10⁶ stationary stages were subcutaneously injected into mice at the base above the tail.

In vitro study Leishmania major promastigotes were procured from the Pasteur Institute in Karaj, Iran. Promastigotes were cultivated in RPMI1640 media enriched with 10% fetal bovine serum, 100 g/ml of streptomycin and 100 IU/ml of penicillin (Mohammadi et al. 2021). One million parasites were added to each well of the 48-well plate, and after the addition of podophyllotoxin and podophyllin the number of parasites per well was calculated after 24, 48, and 72 h.

In vivo study Forty mice were randomly assigned to four groups, each with ten members. These groups included a negative control group that received Eucerin treatment, a positive control group that received Glucantime treatment, and two experimental groups that received podophyllotoxin 0.5% and podophyllin 25% treatment. When local ulcers emerged, a thin layer of podophyllotoxin was applied to the skin with a sterile tip once every other day for four weeks. The same method was used to administer podophyllin once each week for four weeks. The treated area was then left uncovered and undressed. Ulcers were observed for changes in color, and the diameter of the lesions was measured both before and after therapy. Two diameters [small (d) and big (D)] of lesions were measured by a caliper before and weekly after treatment for 30 days. Then, their sizes were calculated using the formula D + d/2 (Batool et al. 2021).

Direct smears were made from the lesions in all groups at the conclusion of the treatment, and counting the number of amastigotes allowed for an assessment of the parasite load.The Ethical Committee of Kashan University of Medical Sciences (IR.KAUMS.MEDNT.REC.1399.009), Kashan, Iran, gave its approval to this project.

Results

In vitro study The growth inhibitory effect of four concentrations (80, 120, 160, and 200μg/ml) of podophyllotoxin on the promastigotes was evaluated at 24 and 48 h. Direct counting was used to assess this medication’s antileishmanial activity, which was then contrasted with a control group. Finally, the fatality percentage was calculated (Fig. 1). After 48 h, 59% of Leishmania major promastigotes were killed when exposed to a concentration of 200μg/ml of podophyllotoxin.

Fig. 1.

Fatality percentage of Podophylotoxin on L.major promastigotes

After 48 h, 200 µg/ml of podophyllin showed 83% lethality for promastigotes (Fig. 2).

Fig. 2.

Fatality percentage of podophyllin on L.major promastigotes

In vivo study The three groups were compared weekly based on the diameters of the lesions, indurations, and local side effects at similar sessions. There were no statistically significant variations in lesion diameter between the groups (P > 0.05), and neither infection recovery nor spontaneous healing were seen. The mean lesion diameter was 15.10 mm in the negative control group, 14.21 mm in the podophyllin 25% group, and 11.55 mm in the podophyllotoxin 0.5% group at the end of the treatment. The lesion diameter significantly continued to progress in the podophyllin group, especially after the second week of the therapy, which lead to a significantly greater lesion size at the end of 4 weeks compared to the podophyllotoxin group(Fig. 3). Local side effects, including erhythema, irritation, and desquamation, were visible in the podopyllin and podopyllotoxin groups, which were more severe in the podophyllin group. Although systemic complications such as weight loss and shedding were observed in the podophyllin group, mice in the podophyllotoxin group were normal and did not show any of these complications. Finally, the topical application of podophyllotoxin 0.5% had antileishmanial effects and reduced the progression of lesion diameters and induration, compared negative control group. It was more effective than podopyllin and had fewer local or systemic complications (Table 1, 2).

Fig. 3.

Picture of lesion after treatment with 1. Podophyllin 25%, 2. Podophyllotoxin 0.5%, 3. Negative control

Table 1.

Lesion diameter during treatment with Podophyllotoxin 0.5% and Podophyllin 25%

Groups	Lesion diameter(mm)
	First day	1st week	2nd week	3rd week	4th week
Podophyllotoxin	2.20 ± 0.25	4.50 ± 1.39	7.00 ± 2.13	8.20 ± 2.20	11.55 ± 2.04
Podophyllin	2.15 ± 0.24	4.45 ± 1.01	7.72 ± 1.46	9.85 ± 1.18	14.21 ± 1.25
Negative control	2.25 ± 0.26	5.20 ± 1.13	9.50 ± 1.17	11.25 ± 1.98	15.10 ± 1.50

Table 2.

Parasite load in mice lesion after treatment with Podophyllotoxin 0.5% and Podophyllin 25%

Groups	Amastigotes/macrophage
Podophyllotoxin 0.5%	3.1 ± 0.8
Podophyllin 25%	3.8 ± 1
Negative control	4 ± 0.6

Discussion

Although spontaneous cures might take months or even years, CL is a self-treatment disease. The primary goals of therapy are thought to be shortening the course of the disease and avoiding scarring (Sharquie et al. 2015). Pentavalent antimonials, liposomal amphotericin B, fluconazole, and miltefosine are a few of the few medications that have been used to treat CL (Mosimann et al. 2018). The first-line treatment for CL is still injections of meglumine antimony, which patients may stop receiving due to major adverse effects include medication resistance, discomfort, and unpredictable efficacy (McGwire and Satoskar 2014). Finding an effective new therapy for CL treatment is crucial due to the paucity of viable medications without severe side effects. Effective drugs derived from plants or extracts of plant are predicted to become a worthy source of new therapeutic agents due to their great variety and ease of access for people with low purchasing power (Jaffary et al. 2014).

In this study, the findings demonstrated that both agents tested in this study had a lethal effect on Leishmania major promastigotes. The fatality rate at the highest concentration employed for podophyllin and podophyllotoxin was 83% and 59%, respectively, 48 h after cultivation. In general, the effect of two agent on promastigotes had a pattern dependent on time and concentration.

Recent studies on the impact of podophyllotoxin and podophyllic aldehyde derived compounds on promastigotes and splenocytes that become infected with amastigotes of L. infantum have demonstrated that altering the structure of podophyllotoxin can change its antileishmanial activity. In addition, even though the author claimed that tubulin is a great target against leishmaniasis (Escudero-Martínez et al. 2017), there was no evidence of the efficacy of podophyllotoxin on L.major promastigotes before. Therefore, the toxicity of podophyllotoxin on L. major promastigotes was evaluated and compared with data from podophyllin. The current study indicated that podophyllotoxin and podophyllin have antileishmanial activity against L. major. In equal concentrations, the lethality of podophyllin in Leishmania major promastigotes was higher than podophyllotoxin.

Previous studies demonstrated that podophyllin could eradicate the leishmania parasite in vitro by arresting cell mitosis (Hamidzadeh et al. 2011). However, the average dimension of lesions increased in the mice treated with different concentrations of podophyllin. In a seperated, blinded, medicinal study, the effects of a topical 25% podophyllin lotion once a week for 3–6 weeks to treat patients with the CL investigated and reported an 85% cure rate with no significant local or systemic adverse effects. Sharquie et al. concluded that a topical 25% podophyllin solution is a helpful, non-costly, available, and painless treatment for CL (Sharquie et al. 2015, 2017). According to earlier research podophyllotoxin 5% was more effective and caused genital warts to disappear faster than podophyllin 20% (Edwards et al. 1988; Lacey et al. 2003). To the best of the information we have, no research has been done to compare the effectiveness of podophyllotoxin and podophyllin in treating CL. Consequently, in the current investigation, the impacts of podophyllotoxin in vivo and in vitro were compared with podophyllin. In the present study, local treatment with podophyllotoxin 0.5% reduced the progression of lesion diameters and induration with a few local side effects. The results of the podophyllin group were not significant and mice treated with podophyllin 25% once a week for 4 weeks showed severe local side effects and even systemic toxicity, which could be related to other active ingredients in podophyllin. These compounds are available as topical solutions or creams and are commonly used for a maximum of 4 weeks, they can be self-administered at home easily in comparison with an injection of pentavalent antimonials.

Conclusion

Based on the findings of the present study, topical treatment with either podophyllotoxin or podophyllin is an effective drug for managing CL. It should be investigated in more complete studies and the mechanisms of its effect should be determined.

Funding

The Vice Chancellor for Research of Kashan University of Medical Sciences, Kashan, Iran granted this study (project No. 99010).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The local Kashan University of Medical Sciences ethics review committee (IR.KAUMS.MEDNT.REC.1399.009) approved all procedures used in this investigation.