Leishmaniasis is an important protozoan disease, which still is a major health problem in endemic countries, such as Iran (1). Despite, recent advances, there is a long way until arriving to the ideal anti-leishmanial agents, with less toxicity, side effects and more potent efficacy. The World Health Organization (WHO) has suggested usage of herbal plants to reach this aim (2). In the Iranian folk medicine, Cordia myxa is an herbal plant, which belongs to the Boraginaceae family and grows in tropical regions, such as Iran (3). Its fruits contain phenolic compounds, which can be a potential therapeutic option for leishmaniasis, similarly to several herbal plants, such as green tea extract (4). The objective of this study was to evaluate the in vitro effects of C. myxa mucilage on Leishmania Major and L. infantum promastigotes. The fruits of C. myxa were collected from a traditional harvesting region for this plant, Ahvaz, Southwest Iran. A total of 50g of C. myxa fruits were boiled slowly, in one liter of hot water, for 30 minutes. The residue was filtrated through Whatman paper No. 33. The mucilage filtrate was freeze-dried. The mucilage extract of C. myxa was initially dissolved in dimethyl sulphoxide (DMSO) (Sigma-Aldrich Corp., St. Louis, MO, USA) and further diluted with the RPMI 1640 medium (GIBCO, Grand Island, NewYork, USA) after a sterilizing the filtrate.
To examine the anti-leishmanial activity of the extract, logarithmic phase promastigotes of L. infantum (MCAN/IR/96/LON49) and L. major (MRHO/IR/75/ER) (1×106 cells/ mL) were seeded in a 96-well microtiter plate, in the presence of the serial concentrations (0, 0.61, 1.22, 2.44, 4.88, 9.75, 19.5, 39, 78, and 156 mg/ mL w/v) and then incubated at 24°C, for 72 hours. Anti-leishmanial activity was assayed by light microscopy and (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) MTT method. Each assay was performed in triplicate, with three independent experiments. The concentration inhibiting parasite growth by 50% (IC50 value) was calculated with a sigmoid dose-response curve. Mucilage extract of C. myxa was active against promastigotes form of L. major and L. infantum, with an IC50 of 26 ± 2.2 mg/mL and an IC50 of 35 ± 2.2 mg/mL, respectively. The survival percentage of L. major and L. infantum promastigotes after 72 hours treatment, with different concentrations of C. myxa mucilage extract, is shown in Table 1.
|Leishmania major, survival||92.1||88.9||85.1||76.8||61.34||49.86||36.82||21.09||17.68|
|L. infantum, survival||88.98||85.44||83.77||76.31||69.44||60.45||32.14||29.76||16.68|
This study demonstrated that C. myxa extract had anti-leishmanial effects against L. infantum and L. major promastigotes, under in vitro conditions. Phytochemical studies have demonstrated C. myxa, as a good source of trace elements (such as selenium, copper, zinc, iron and manganese), phenolic, and flavonoid compounds (robinin, datiscoside, rutin, hesperidin, dihydrorobinetin, caffeic acid and chlorogenic acid) (5). Probably, C. myxa has potential anti-leishmanial activity because of phenolic and flavonoid compounds (rutin and caffeic acid) and several trace element content (selenium). Flavonoides bind to the nucleotide binding domains (NBD) of the ATP binding cassette (ABC) transporters, and their role is well established in most medications resistance phenomena, such as anticancer and anti-leishmaniasis drugs. Finally, these cellular events induced an increase of hydrophobic interactions, which leads to inhibition of multiple drug resistance (6). Several studies have focused on the anti-leishmanial activity of selenium compounds. These findings mention the selenium compounds, as novel anti-leishmanial agents for amastigotes and promastigotes, with more potent activity and lower cytotoxicty than miltefosine and edelfosine (IC50 promastigotes = 0.9 - 17 µM and 0.3 - 9 µM for amastigotes) (7, 8). Other benefits of C. myxa can be attributed to less cytotoxicity and cost, compared to current anti-leishmanial drugs, whose main limitation for their administration, particularly in developing countries (9).
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