|Year : 2016 | Volume
| Issue : 1 | Page : 12-15
Effect of topical gel prepared with hydroalcoholic extract of Echinacea purpurea on treatment of Leishmania major-induced cutaneous leishmaniasis in BALB/C mice
Bahador Sarkari1, Hossein Sattari2, Mahmoud Reza Moein3, Ali Mohammad Tamadon3, Reza Shahriari Rad4, Qasem Asgari5
1 Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
2 School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
3 Department of Pharmacognosy and Medicinal Plants Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
4 Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
5 Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
|Date of Web Publication||19-Feb-2016|
Basic Sciences in Infectious Diseases Research Center, Shiraz University of Medical Sciences, P. O. Box: 71345-1735, Shiraz
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: This study aimed to investigate the effect of alcoholic extract of Echinacea purpurea as a topical gel for the treatment of Leishmania major-induced cutaneous leishmaniasis (CL) in BALB/c mice. Materials and Methods: Leishmania major was inoculated into the tail base of 28 BALB/c mice and the mice were then assigned into four groups, with seven mice in each group. Hydroalcoholic extract of Echinacea purpurea was prepared and 3% carboxymethylcellulose was used for the topical gels. After the development of skin lesions, the gel base without drug and 10% and 20% Echinacea gel, respectively, were applied for the treatment of skin lesions in three groups of mice for 20 days, two times per day. The forth group remained as the control without receiving any treatment. Sizes of the lesions were frequently measured and recorded. The collected data were analyzed by SPSS using the Kruskal-Wallis one-way analysis of variance test. Results: The sizes of the lesions at the tail base of BALB/c mice were found to be increasing in both the treated and untreated mice. Although there were differences in the mean size of the lesions between the control group and those that received various concentration of Echinacea gel, (10% or 20%) the differences were not statistically significant (P > 0.05). Conclusion: The results showed that topical 10% and 20% gel of Echinacea purpurea extract is not considerably effective in the treatment or control of CL.
Keywords: Echinacea purpurea , Leishmania major, Treatment
|How to cite this article:|
Sarkari B, Sattari H, Moein MR, Tamadon AM, Rad RS, Asgari Q. Effect of topical gel prepared with hydroalcoholic extract of Echinacea purpurea on treatment of Leishmania major-induced cutaneous leishmaniasis in BALB/C mice. J Pharm Negative Results 2016;7:12-5
|How to cite this URL:|
Sarkari B, Sattari H, Moein MR, Tamadon AM, Rad RS, Asgari Q. Effect of topical gel prepared with hydroalcoholic extract of Echinacea purpurea on treatment of Leishmania major-induced cutaneous leishmaniasis in BALB/C mice. J Pharm Negative Results [serial online] 2016 [cited 2017 Nov 19];7:12-5. Available from: http://www.pnrjournal.com/text.asp?2016/7/1/12/177054
| Introduction|| |
Leishmaniasis is a tropical disease caused by intracellular protozoan parasites. , This parasite is the causative agent of three different types of leishmaniasis including cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), and visceral leishmaniasis (VL).  It has been estimated that about 12 million cases of CL is present in various regions of the world and about 350 million people worldwide are at risk of infection. 
CL is a common disease in Iran, which is endemically present in several provinces of the country. , The known therapy for this disease is pentavalent antimony derivatives. However, these drugs show several side effects and in about 10-25% of the cases, disease recurrence is observed. Although pentavalent antimony derivatives are still the first line of treatment of CL, drug failure and drug resistance are increasing in various regions of the world. ,
Considering the aforementioned points, it seems necessary to study and evaluate the new drugs for the treatment of leishmaniasis. The use of herbal medicine as a method of therapy has attracted researchers' attention.
Echinacea consists of nine species, out of which three, namely, as Echinacea purpurea, Echinacea pallida, and Echinacea angustifolia have therapeutic effects. Several studies demonstrated that Echinacea purpurea has an immune stimulatory effect (mainly the stimulation of nonspecific immune responses). The stimulating properties of Echinacea are due to the activation of phagocytosis and fibroblast stimulation, enhancing the respiratory activity and increasing the leukocyte interactions. Several studies on the effects of the polysaccharide purified from Echinacea provide evidence of its immune-stimulating properties. , These properties include the increase of phagocytosis, chemotactic for neutrophil and neutrophil enhancement, and macrophage oxidation. The chemical components and biological activity of Echinacea not only depend on the plant species but also on the part of the plant in use (root or aerial parts), method of extraction, geographical location(s), stage of development, harvesting time, and growth conditions. For instance, hydroextract of Echinacea, unlike the alcoholic form of the extract, consists of polysaccharides that stimulate the nonspecific immune responses.  One of the effects of Echinacea on the immune system is the production of cytokines like tumor necrosis factor-α (TNFα). In leishmaniasis, development of these cytokines can induce the activation of macrophages, resulting in the elimination of the parasite. Since the extract of Echinacea stimulates the immune stimulation and considering the point that immune stimulation and production of proinflammatory cytokines such as TNFα are necessary for killing the parasite(s) in leishmaniasis, it makes sense to postulate that the extract of this plant has antileishmanial effects. , The aim of this study was to evaluate the topical effects of a gel containing Echinacea purpurea, a hydroalcoholic extract, in the treatment of Leishmania major-induced CL.
| Materials And Methods|| |
The current study was conducted at the Department of Parasitology and Mycology of Shiraz University of Medical Science, Shiraz, Fars Province, Iran. The Echinacea plant was provided by Yasuj Medicinal Herbal Garden.
The plant species and herbal numbers were confirmed in Kohgiluyeh va Boyerahmad Agriculture Research Centre. Hydroalcoholic extraction was performed as previously described.  Briefly, the powder was prepared from dried aerial parts of the plant and extraction was done with the help of 80% alcohol.
Carboxymethyl cellulose (CMC), a gel-producing powder, was used in order to facilitate the topical products. Therefore, in this study 3% CMC and 0.2% methylparaben were used for the preparation of the topical gel.
In order to prepare the gel, 50 mL of the extraction (10% or 20%) was placed in a beaker and stirred thoroughly with a homogenizer. Then, 1.5 g of CMC and 0.1 g of methylparaben were slowly added to yield the desired gel. After preparation of the 10% and 20% Echinacea gel, it was placed in a tube and stored in a refrigerator until it was used.
This study was carried out with the prior approval of the Animal Ethics Committee of Shiraz University of Medical Sciences. BALB/c mice were provided by the Comparative and Experimental Medicine Center at Shiraz University of Medical Sciences, Shiraz, Fars Province, Iran. The animals were housed in standard cages under stable condition (humidity 40-50%, temperature 25 ± 3°C, and 12:12-h dark-light cycle). The mice had free access to water ad libitum and were on a standard diet. Leishmania major parasite was taken from the lesions of Leishmania-infected mice and injected into the tail base of 28 of 8-10-week-old male BALB/c mice. The mice were randomly divided into four groups of seven. After the development of scars at the base of the tail of the mice, the three groups received base gel without treatment, 10% Echinacea gel, and 20% Echinacea gel, respectively, twice a day for 20 days. The fourth group was marked as the control group. Skin lesions caused by the Leishmania parasite were measured and noted frequently in all the groups. Measurement of skin lesions at the base of the tail was done in both horizontal and transverse diameters and on the basis of the ellipsoid surface area formula; the area of the skin lesions was measured. Statistical analysis of the findings was carried out by SPSS software (Version 18.0. Chicago: SPSS Inc. IBM Corp.) and evaluation was done with Kruskal-Wallis one-way analysis of variance test.
| Results|| |
The aim of this study was to investigate the effect of Echinacea purpurea extract on the treatment of CL in BALB/c mice. The BALB/c mice were inoculated with Leishmania major and the skin lesions were treated with a topical gel containing 3% CMC and hydroalcoholic extract of Echinacea purpurea. Measurements of the mice skin lesions were done at two different time points. The first was on the 10 th day of the treatment and the second on the 20 th day. It was observed that in the base gel group that received no treatment, area of the scar increased from the time of the first measurement to the next one. This difference provides evidence that the base gel used had no effect.
The mean size of the skin lesions in the group that received 10% Echinacea gel was not different in the first measurement and second measurement in comparison with the control and base gel group, and administration of 10% Echinacea gel did not have any considerable effect on the size of the skin lesions. On the other hand, the mean area of skin lesions in the group of mice that received 20% Echinacea gel in comparison with the control and nontreated group was different but the differences were not statistically significant. [Table 1] represents the mean and standard deviation of the size of skin lesions in the different groups of mice at different period of times throughout the experiments.
|Table 1: The area of scar size (mm) at the tail base of mice caused by Leishmania major in different mice groups |
Click here to view
| Discussion|| |
In this study, the extract of Echinacea purpurea was administered topically to Leishmania major-induced skin lesions in BALB/c mice for 20 days. Although there were decreases in the area of skin lesions in the group of mice that received 20% Echinacea gel, in comparison with the control group, however, these differences were not statistically significant. Therefore, this study did not find a considerable effect for topical administration of gels containing 10% or 20% of Echinacea purpurea extract.
The first lines of treatment for leishmaniasis are tetravalent antimony derivatives like potassium antimony tartrate and pentavalent antimony derivatives. Amphotericin B and pentamidine are used as alternative medications. However, variable side effects, method of administration, long periods of administration, and drug resistances are major problems in the treatment of leishmaniasis by antimonial derivatives.
Lack of effective anti-Leishmania drugs highlighted the need for finding more effective anti-Leishmania drugs with more effective performance and fewer side effects. Therefore, herbal medicine, as a source of chemical components, has received more attention of the researchers.
In endemic areas of leishmaniasis, residents with the help of traditional medicine are using the extract of some plants for the treatment of systemic or cutaneous forms of leishmaniasis.
In herbal medicine, Echinacea purpurea is known for its immune-stimulating properties and is being used recently for the prevention and treatment of common cold, coughs, bronchitis, lung infections, cutaneous disorders, and chronic diseases triggered by the immune system. ,
Echinacea polysaccharides contain immune-stimulating factors and their polyacetylene has anti-inflammatory effects.  Echinacea components increase the number of white blood cells in the circulating system, activate T lymphocytes, increase phagocytosis, and trigger the production of cytokines (interferons, interleukin 1, TNF, and interleukin 6). 
In a study conducted by Hosseini et al., the effect of Echinacea purpurea on promastigote form of Leishmania was evaluated and it was shown that the extraction of the plant, at a concentration of 0.5 mg/mL or higher than that, had anti-Leishmania effects. 
In our study, low doses of the extract might explain the lack of effect of the plant extract in the treatment of CL in the mice and it is tempting to think that higher doses of the extract could have competent effects on the treatment of CL.
Chemical compounds and biological activities of Echinacea not only depend on the species of the plant but also on its part (root, aerial parts), method of extraction, geographical location(s), stage of development, harvesting time, and growth conditions. For instance, hydroextract of Echinacea, unlike the alcoholic form of the extract, consists of polysaccharides that stimulate the nonspecific immune responses. 
In 2007, Zhaiet et al. through their studies provided evidence that the Echinacea plant inhibits the production of NO and TNFα in macrophages. This study showed that alcohol, existing in alcoholic extraction, decreases inflammatory mediators like NO and TNFα.  Considering that these inflammatory mediators play an important role against the Leishmania parasite, it can be postulated that a probable cause of inefficiency of the extract of this plant against Leishmania in this study was due to the alcohol inhibitory effects against NO and TNFα in the extract.
Since the skin lesions induced by Leishmania are a beneficial environment for opportunist bacterial growth, bacillus and other bacteria can play a role in secondary infections of the skin lesions. Secondary infections on the lesion cause an unpleasant appearance and create problem(s) in recovery. The main course of action in the treatment of Leishmania in case of secondary infection is simultaneous treatment of the infections with antibiotics. In this study in order to prevent side effects, no antibiotics were used and the secondary infections could have occurred in the skin lesions. Therefore, topical effects of Echinacea extract might be hindered by these infections.
The properties of CMC gel in inhibiting the release of compounds and active substances of the plant can also explain the gel's lack of effectiveness. Furthermore, deletion and decrease in the amount of gel on the base of the tail of the mice and feeding of the gel in case of other mice are also some reasons that can be accounted for the improper effects of Echinacea purpurea extract in the treatment of CL in the current study.
| Conclusion|| |
Findings of this study showed that topical 10% and 20% gel of Echinacea purpurea extract in the form of CMC gel do not have considerable effects in the treatment of CL in BALB/c mice.
Financial support and sponsorship
The study was financially supported by the office of vice-chancellor for research of Shiraz University of Medical Sciences (Grant No. 89-01-43-2700).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Desjeux P. Leishmaniasis: Current situation and new perspectives. Comp Immunol Microbiol Infect Dis 2004:305-18.
Murray HW, Berman JD, Davies CR, Saravia NG. Advances in leishmaniasis. Lancet 2005;366:1561-77.
Postigo JA. Leishmaniasis in the world health organization eastern mediterranean region. Int J Antimicrob Agents 2010;(Suppl 1):S62-5.
Farahmand M, Nahrevanian H, Shirazi HA, Naeimi S, Farzanehnejad Z. An overview of a diagnostic and epidemiologic reappraisal of Cutaneous leishmaniasis
in Iran. Braz J Infect Dis 2011;15:17-21.
Tiuman TS, Santos AO, Ueda-Nakamura T, Filho BP, Nakamura CV. Recent advances in leishmaniasis treatment. Int J Infect Dis 2011;15:e525-32.
Croft SL, Sundar S, Fairlamb AH. Drug resistancein leishmaniasis. Clin Microbiol Rev 2006;19:111-26.
Wagner H, Stuppner H, Schäfer W, Zenk M. Immunologically active polysaccharides of Echinacea purpurea
cell cultures. Phytochemistry 1988;27:119-26.
Steinmüller C, Roesler J, Gröttrup E, Franke G, Wagner H, Lohmann-Matthes ML. Polysaccharides isolated from plant cell cultures of Echinacea purpurea enhance the resistance of immunosuppressed mice against systemic infections with Candida albicans and Listeria monocytogenes. Int J Immunopharmacol 1993;15:605-14.
Canlas J, Hudson JB, Sharma M, Nandan D. Echinacea and trypanasomatid parasite interactions: Growth-inhibitory and anti-inflammatory effects of Echinacea. Pharm Biol 2010;48:1047-52.
Sadati MS, Sarkari B, Asgari Q, Hatami S, Tavakl A. Effect of Echinacea purpurea
on control of Leishmania major
induced cutaneous leishmaniasis in mice. Armaghan Danesh J Yasouj Univ Med Sci 2011;16:31-40.
Hudson JB. Applications of the phytomedicine Echinacea purpurea
(Purple Coneflower) in infectious diseases. J Biomed Biotechnol 2012;2012:769896.
Zhai Z, Liu Y, Wu L, Senchina DS, Wurtele ES, Murphy PA, et al
. Enhancement of innate and adaptive immune functions by multiple Echinacea species. J Med Food 2007;10:423-34.
Soudi S, Hashemi SM, Hosseini AZ, Ghaemi A, Jafarabadi MA. Antileishmanial effect of Echinacea purpurea
root extract cultivated in Iran. Iran J Pharm Res 2007;6:147-9.
14 Zhai Z, Haney D, Wu L, Salco A, Murphy PA, Wurtele E. Alcohol extracts of Echinacea inhibits production of nitric oxide and tumor necrosis factor-alpha by macrophages in vitro
. Food Agric lmmunol 2007;18:221-36.