J Arthropod-Borne Dis, March 2022, 16(1): 45–50 A Moshfe et al.: Anti Leishmanial Effect of … 45 http://jad.tums.ac.ir Published Online: March 31, 2022 Original Article Anti Leishmanial Effect of Plantago psyllium (Ovate) and White Vinegar on Leishmania major Lesion in BALB/c Mice Abdolali Moshfe1,2, Keianoush Karami1, Maryam Bahmani1, Mohsen Naghmachi1, Shahrbanoo Askarian1, Abbas Rezaei1, Roohallah Zare1, *Ali Jamshidi3 1Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran 2Department of Microbiology, School of Medicine, Yasuj University of Medical Sciences, Yasuj, Iran 3Behbahan Faculty of Medical Sciences, Behbahan, Iran *Corresponding author: Dr Ali Jamshidi, Email: ajamshidi@behums.ac.ir (Received 13 Mar 2019; accepted 18 Jan 2022) Abstract Background: Leishmania major is the etiologic agent of zoonotic cutaneous leishmaniasis in Iran, and glucantime in- jection 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, respective- ly. 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. Keywords: Leishmaniasis; Plantago psyllium; Ulcer; Mice Introduction Leishmaniasis is one of the most important zoonotic diseases caused by more than 20 Leishmania species. This disease has a world- wide 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 mor- tality 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 econom- ic 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 be- cause of its production as a generic drug. In Copyright © 2022 The Authors. Published by Tehran University of Medical Sciences. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International license (https://creativecommons.org/licenses/by- nc/4.0/). Non-commercial uses of the work are permitted, provided the original work is properly cited. http://jad.tums.ac.ir/ https://creativecommons.org/licenses/by-nc/4.0/ https://creativecommons.org/licenses/by-nc/4.0/ J Arthropod-Borne Dis, March 2022, 16(1): 45–50 A Moshfe et al.: Anti Leishmanial Effect of … 46 http://jad.tums.ac.ir Published Online: March 31, 2022 some regions, mainly where resistance has de- veloped, miltefosine, paramycin, and liposomal amphotericin B are gradually replacing the an- timonials (9, 10). Nowadays, due to some fac- tors such as side effects, drug resistance, ex- cessive and economic costs of medical ser- vices, lack of easy access to these medications in some areas, researchers are looking for ef- fective alternative drugs to reduce the side ef- fects of CL treatment (11-13). One way to do so is the use of safe plant extracts in the treat- ment of the disease. The utilization of herbal extracts for the treatment of cutaneous leish- maniasis in many cities of Iran has a long his- tory 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 (Per- sian name: bumadaran), Tanacetum partheni- um (Persian name: babouneh), Nigella sativa (Persian name: siah daneh), and Satureja khuzestanica (Persian name: Marzeh khuzestani) were exhibited anti-Leishmania effects activ- ity (14-16). 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 disinfect- ing 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 collect- ed by a scalpel and dissolved in 6mL of nor- mal 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 pur- chased 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 pres- ence 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 ran- domly divided into five groups. The first group was treated with the com- bination 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 treat- ment (once every 72 hours), the ulcers’ diam- eters 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 exami- nation. The data obtained from microscopic http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 45–50 A Moshfe et al.: Anti Leishmanial Effect of … 47 http://jad.tums.ac.ir Published Online: March 31, 2022 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 in- dicated that the mean areas of the ulcers in all the groups were not significantly different be- fore 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 treat- ment, and each group was compared separate- ly 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 treat- ment 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= Signifi- cant, 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 com- pared with glucantime group, before and after treatment. Group Name Sig/NS Mean SD Mean SD 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= Signifi- cant, p= p value, and T=the t-value of the paired sample t-test http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 45–50 A Moshfe et al.: Anti Leishmanial Effect of … 48 http://jad.tums.ac.ir Published Online: March 31, 2022 Discussion Cutaneous leishmaniasis is a great health problem in Iran. At the present time, no effec- tive drug and vaccine for the inhibition of path- ogenic leishmania spp and chemicals for erad- ication of its vectors is provided. According to the published research in the world, efforts are still ongoing to discover an effective treat- ment to cure cutaneous leishmaniasis with neg- ligible side effects and low price. The current and standard medicine against cutaneous leish- maniasis is Glucantime, but it has many side effects. Therefore, traditional treatment of CL is a common habit of natives in many endem- ic areas in Iran. More than fifty different local plants are used to traditional treatment of cu- taneous leishmaniasis lesions in Iran (14-16). 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 find- ing an effective way to reduce injection pain and treatment costs. On the other hand, pro- duction of ointments containing herbal extract or essential oil are noninvasive method for treatment of cutaneous leishmaniasis (17-20). The results indicated that there was a sig- nificant difference between the third, fourth, and fifth groups, and no significant difference between the glucantime group and the P. psyl- lium and vinegar groups. The findings of He- jazi et al. showed a significant difference in the means of lesion diameter before and after treat- ment in the control, yarrow, and thyme groups (P< 0.05). Also, paired t-test showed no signif- icant 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 le- sions’ 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 glu- cantime) separately. Only the study by Wester- hof et al. investigated the effect of P. psyllium on the treatment of cutaneous leishmaniasis. That study was aimed at investigating the ef- fect of mucopolysaccharides on wound heal- ing. The results of that study indicated that mu- copolysaccharides 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 find- ings 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 ex- posed 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 Leish- mania major ulcers in BALB/c mice (24). Most studies on the treatment of CL using herbal ex- tracts and natural products with anti-leishma- nial activity so far indicate that none of these herbal extracts had a 100% effect on wound healing. Although some of these herbal ex- tracts have been somewhat effective in the wound healing process and reduced the period of the disease (25-27). 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 sim- ple and painless in comparison with injection. Thus, further studies on this issue could help to design more effective and easy-to-use drugs. Acknowledgements This article was financially supported by Yasuj University of Medical Sciences and ap- http://jad.tums.ac.ir/ J Arthropod-Borne Dis, March 2022, 16(1): 45–50 A Moshfe et al.: Anti Leishmanial Effect of … 49 http://jad.tums.ac.ir Published Online: March 31, 2022 proved by the Ethics committee of Yasuj Uni- versity of Medical Sciences (IR.YUMS.REC. 1397.023). 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