Anti Leishmanial Effect of Plantago psyllium (Ovate) and White Vinegar on Leishmania major Lesion in BALB/c Mice

Abstract

Background:

Leishmania major is the etiologic agent of zoonotic cutaneous leishmaniasis in Iran, and glucantime injection is currently used for its treatment. This study aimed to evaluate the anti-leishmanial effect of topical Plantago psyllium and white vinegar in L. major infected BALB/c mice.

Methods:

Thirty infected mice were divided into five groups as follows: Group 1: treated with the combination of ovata powder and white vinegar, Group 2: treated with glucantime, Group 3: treated with white vinegar, Group 4: treated with the combination of ovata powder and water, and Group 5: without any treatment. All the groups were treated for 18 days. Lesion size was measured, and final smears were prepared for microscopic examination.

Results:

The findings indicated that the difference in the mean areas of the ulcers in all the groups before and after treatment was not significant, except for the second (glucantime) and third (vinegar) groups. Also, the results showed that in the first, second, third, and fourth group, 6 (60%), 4 (80%), 3 (60%), and 2 (40%) mice were healed, respectively. However, ulcers remained in all the five mice of the control group.

Conclusion:

The combination of ovata powder and white vinegar has been traditionally used to treat leishmanial lesions in Iran. It seems the most anti-leishmanial effect is related to vinegar and supported by Plantago. The route of treatment with this combination is very simple and painless in comparison with injection. Thus, further studies on this issue could help to design more effective and easy-to-use drugs.

Introduction

Leishmaniasis is one of the most important zoonotic diseases caused by more than 20 Leishmania species. This disease has a worldwide distribution and is transmitted by sand-flies between animals and human hosts (1, 2). According to the World Health Organization (WHO) reports, leishmaniasis has a high mortality rate and is a health problem in endemic areas, especially in the developing countries (3, 4). Cutaneous leishmaniasis (CL) can be observed in the two forms of rural and urban in 97 different countries, and approximately 1.5 million new cases are reported throughout the world annually, with over 80% of the cases occurring in the developing countries (5, 6). Brazil, Iran, Afghanistan, and Sudan are the most infected countries (7).

Leishmania major is the main etiologic agent of zoonotic cutaneous leishmaniasis in some parts of Iran, and it has caused economic and health problems in these areas (8). For a long time, pentavalent antimonial drugs have been used as the first drugs of choice in the treatment of leishmaniasis. Currently, glucan-time is used in many endemic countries because of its production as a generic drug. In some regions, mainly where resistance has developed, miltefosine, paramycin, and liposomal amphotericin B are gradually replacing the antimonials (9, 10). Nowadays, due to some factors such as side effects, drug resistance, excessive and economic costs of medical services, lack of easy access to these medications in some areas, researchers are looking for effective alternative drugs to reduce the side effects of CL treatment (11, 12, 13). One way to do so is the use of safe plant extracts in the treatment of the disease. The utilization of herbal extracts for the treatment of cutaneous leishmaniasis in many cities of Iran has a long history based on Avecina traditional medicine. According to the studies conducted in Iran (Refer to the review studies of Moghaddas et al. 2017, Oryan et al. 2015, and Soosaraei et al. 2017) in this field, more than fifty different local plants and extracts, oil, and powder of them (such as hydroalcoholic, dichloromethane, ethanolic, methanolic, and aqueous extract) have been used to treat cutaneous leishmaniasis, the findings of the mentioned studies showed that some of them like Achillea millefolium (Persian name: bumadaran), Tanacetum parthenium (Persian name: babouneh), Nigella sativa (Persian name: siah daneh), and Satureja khuzestanica (Persian name: Marzeh khuzestani) were exhibited anti-Leishmania effects activity (14, 15, 16). In the present study, Plantago psyllium (ovate) and white vinegar was used in the treatment on Leishmania major lesion in BALB/c mice, which was not previously reported in the anti-leishmania experimental studies.

Materials and Methods

In this experimental study, after disinfecting the leishmanial ulcer surface in mice, which were previously infected by the standard strain of Leishmania major (acquired from Shiraz School of Medicine with MHOM/64/IR/ER75 code), some of the ulcer margins were collected by a scalpel and dissolved in 6mL of normal saline. Then, by an insulin syringe, 0.2mL of the prepared solution (containing L. major amastigote) was injected into the tail of 30 male BALB/c mice aged 10 weeks old. This strain of mice was used because it is the most sensitive to L. major; the mice were purchased from Razi Institute in Shiraz in April 2016.

After infecting the mice, they were kept on special shelves under the same conditions. After three weeks, an ulcer appeared in the tail of all the mice. Then, to confirm the presence of the parasite (amastigote or Leishman body) in the lesions, Giemsa-stained slides were observed at 100× magnification. Afterwards, all of the 30 infected BALB/c mice were randomly divided into five groups.

The first group was treated with the combination of Plantago psyllium powder and white vinegar (10 mice). For this purpose, Plantago psyllium seeds were powdered by grinding. Then, 6.2g of P. psyllium powder was mixed in 25mL of white vinegar and a dough was prepared. A 4mm thick layer of the dough was used for treating the ulcers. The second group (as positive control), which consisted of five mice, was treated with 0.2mL of glucantime by intraperitoneal injection (According to the treatment method in many articles such as Nilforoushzadeh et al. 2008 and Taran et al. 2010). For the treatment of the third group (5 mice), a cotton ball soaked in 5ml of vinegar was used and placed on the wound for 2 minutes. Also, P. psyllium powder and water combination (4mm thick layer) were used for treatment in the fourth group (5 mice). Group five (5 mice) as the negative control did not receive any treatment. Before and after treatment (once every 72 hours), the ulcers’ diameters were measured and recorded in all the groups by a caliper (ulcer size was calculated by measuring two diameters perpendicular to each other). All the groups were treated for 18 days, and at the end of the treatment period, smears were prepared for microscopic examination. The data obtained from microscopic observations, diameter changes, and lesions’ healing were analyzed by SPSS version 20 using paired samples t-test. A P-value of less than 0.05 was considered significant.

Results

The findings of paired samples t-test indicated that the mean areas of the ulcers in all the groups were not significantly different before and after treatment, except for the second (glucantime) and third (vinegar) groups, (Table 1). The mean difference of ulcer area in all the groups was evaluated before and after treatment, and each group was compared separately with the second group (glucantime), which revealed a significant difference between the third, fourth, and fifth groups and the glucan-time group (P< 0.05, Table 2). Also, in the first, second, third, and fourth groups, 6 (60 %), 4 (80%), 3 (60%), and 2 (40%) mice were healed, respectively. On the other hand, ulcers remained in all the five mice of the control group. The results showed a significantly (P< 0.05) smaller lesion size in the treated groups, especially in the vinegar and P. psyllium with water groups, compared to the control group. Anti-leishmanial effect of vinegar and glucan-time were more than P. psyllium on under treatment mice.

Table 1.

Comparison of the mean diameter (± SD) of lesions area in Balb/c mice due to L. major, before and after treatment in cases and control group

Group Name	Numbers	X±SD Before	X±SD After	Sig/NS
P. psyllium with Vinegar	10	53.7±5.60	42.4±1.99	NS T= 0.36, DF= 9, p= 0.72
Glucantime	5	69±6.44	1.4±0.62	Sig T= 3.39, DF= 4, p= 0.028
Vinegar	5	23.5±4.13	9.2±5.8	Sig T= 3.3, DF= 3, p= 0.045
P. psyllium with Water	5	38.7±2.47	34.5±9.47	NS T= 0.17, DF= 4, p= 0.87
Control	5	56.67±5.42	81.6±10.3	NS T= 0.64, DF= 4, p= 0.55

SD= Standard deviation, X= Mean ulcer area, DF= Degrees of freedom, NS= Non-significant, Sig= Significant, p= p value, and T= the t-value of the paired sample t-test

Table 2.

The mean lesions diameter (± SD) of area in Balb/c mice due to L. major, in cases and control group compared with glucantime group, before and after treatment.

Group Name	Mean	SD	Mean	SD	Sig/NS
P. Psyllium with vinegar	−30.11	44.98	−45.68	15.45	NS T= 1.21, DF= 9, p= 0.12
Vinegar	−60.20	41.12	−45.68	15.45	Sig T= −2.3, DF= 7, p= 0.038
P. Psyllium with water	−4.23	23.54	−45.68	15.45	Sig T= −2.3 DF= 8, p= 0.038
Control	96.24	86.08	−45.68	15.45	Sig T= −2.14, DF= 8, p= 0.032

SD= Standard deviation, X= Mean ulcer area, DF= Degrees of freedom, NS= Non-significant, Sig= Significant, p= p value, and T=the t-value of the paired sample t-test

Discussion

Cutaneous leishmaniasis is a great health problem in Iran. At the present time, no effective drug and vaccine for the inhibition of pathogenic leishmania spp and chemicals for eradication of its vectors is provided. According to the published research in the world, efforts are still ongoing to discover an effective treatment to cure cutaneous leishmaniasis with negligible side effects and low price. The current and standard medicine against cutaneous leishmaniasis is Glucantime, but it has many side effects. Therefore, traditional treatment of CL is a common habit of natives in many endemic areas in Iran. More than fifty different local plants are used to traditional treatment of cutaneous leishmaniasis lesions in Iran (14, 15, 16). Most of these herbal and traditional medicines (same as the findings of present study) showed anti-leishmanial effect, and also, they can play an effective role in healing wounds and finding an effective way to reduce injection pain and treatment costs. On the other hand, production of ointments containing herbal extract or essential oil are noninvasive method for treatment of cutaneous leishmaniasis (17, 18, 19, 20).

The results indicated that there was a significant difference between the third, fourth, and fifth groups, and no significant difference between the glucantime group and the P. psyllium and vinegar groups. The findings of Hejazi et al. showed a significant difference in the means of lesion diameter before and after treatment in the control, yarrow, and thyme groups (P< 0.05). Also, paired t-test showed no significant difference in mean lesion diameter after treatment between treatment and glucantime groups (P> 0.05) (21). According to paired t-test, there was no significant difference in lesions’ healing among different groups after treatment. Furthermore, the difference in the mean lesion areas before and after treatment in each group was compared with a positive control group (Group 2: treated with the glucantime) separately. Only the study by Westerhof et al. investigated the effect of P. psyllium on the treatment of cutaneous leishmaniasis. That study was aimed at investigating the effect of mucopolysaccharides on wound healing. The results of that study indicated that mucopolysaccharides limited the formation of scars (22). Other studies have also evaluated on the effects of herbal extracts on the treatment of CL. For instance, Ahmadi et al. examined the effect of garlic extract on CL, and their findings showed that the diameters of lesions were reduced by the hydrous extract of garlic over 30 days of treatment. However, the maximum reduction was achieved when mice were exposed to 10 days of vitamin A ointment, then treated for 45 days with garlic extract (23). Doroodgar et al. showed that Artemisia sieberi extract had no effect on the treatment of Leishmania major ulcers in BALB/c mice (24). Most studies on the treatment of CL using herbal extracts and natural products with anti-leishmanial activity so far indicate that none of these herbal extracts had a 100% effect on wound healing. Although some of these herbal extracts have been somewhat effective in the wound healing process and reduced the period of the disease (25, 26, 27).

Conclusion

The combination of Plantago p. powder and white vinegar has been traditionally used to treat leishmanial lesions in Iran. It seems the most anti-leishmanial effect is related to vinegar and supported by Plantago. The route of treatment with this combination is very simple and painless in comparison with injection. Thus, further studies on this issue could help to design more effective and easy-to-use drugs.