Amphotericin B Resistant Strain Development
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In vitro activity of Malia azedarach Linn. Against amphotericn B resistant Leishmania tropica strain
Cutaneous leishmaniasis is still a big health problem around the world and drug resistance has emerged as a major problem in treatment. The purpose of the study was to generate Amphotericin B resistant strain and evaluate the crude extract of Melia azedarach against wild and lab generated resistant strain of Leishmania tropica. The result obtained revealed that the continuous increase of drug pressure for 60 days cause resistance in L. tropica by 09 fold compared to wild type. The LC50 value recorded for wild and resistant type strain was 0.024 and 0.224µg/ml, respectively against Amp B drug. The antileishmanial activity of crude extracts against wild and resistant type strain was 0.57 and 123.3 µg/ml for green fruit and 1220.8 and 4010.1µg/ml for ripe fruit, respectively. A significant difference (P<0.05) in activities was observed between green and ripe fruit against wild and resistant type of strain. Based on the results obtained, it can be concluded that green fruit show promising results against resistant type of L. tropica and could be a novel candidate for anti-protozoal activity.
Keywords: Leishmania tropica, Melia azedarach, Resistance.
Infections associated with protozoa are a global health problem, predominantly in the Third World countries (Kondrashin, Baranova et al. 2010, Tengku and Norhayati 2011, Waldron, Ferrari et al. 2011, Al Musayeib, Mothana et al. 2013), and roughly 14% of the world population are at risk. Protozoal infections are considered as a neglected tropical disease and therefore, immense concern has been shown by WHO (Organization 2010). These deadly tropical diseases have negative impacts on the socioeconomic status of the patients (Meshnick 1997). There is a study so far conducted on protozoan infections, including charges, malaria, sleep sickness and leishmaniasis. These diseases considered a major killing factor due to different difficulties are connected with controlling the parasite, high cost, resistant, poor safety, low efficacy and toxicity to the drug (Nwaka and Ridley 2003). Among these protozoan infections leishmaniasis got a special interest in various developing countries due to the increase of resistance to the drug (Pérez-Victoria, Pérez-Victoria et al. 2001).
Leishmania is a fatal disease caused by parasite Leishmania and transmitted through female phlebotomines sand fly bite. Leishmaniasis present in three different clinical forms, i.e. cutaneous leishmaniasis, visceral leishmaniasis and muco-cutaneous leishmaniasis. It is present in tropical and subtropical areas of 88 countries of the world and approximately 10 million population of the world is suffering from cutaneous leishmaniasis (Organization 2007).
Toxicity, inconsistent efficacy and resistant between species or strains of the drug mostly used or for some of them, the long lasting parenteral administration is the need of the day led the researcher for novel antileishmanial drug, most particularly from plant source used in traditional medicine, as a foundation of new leads with new mechanisms of actions (Hoet, Stévigny et al. 2004).
The present study focused on the in vitro antileishmanial activity of crude extract of M. azedarach against wild and lab produced resistant type L. tropica strain.
Material and methods
Collection of Plant Materials
Fresh and ripe fruit of the plant was collected from the vicinity of Quaid-i-Azam University, Islamabad. The plant materials were washed with distilled water and kept in the shade till process. The plant materials were authenticated by the Department of Plant Sciences, Quaid-i-Azam University. A voucher specimen was then submitted to the herbarium.
Fruit (1 Kg) was macerated using mortar and pestle. The powdered obtained were soaked in 5 liter plastic beaker containing distilled water for 6 days with occasional shaking. The materials were filtered through muslin cloth and dried under reduce pressure using a rotary evaporator (Heidolph Laborta4000 efficient). The paste like materials obtained were dried at 40oC in water bath and stored at 4OC for further use.
Production of resistant strain
Resistant strain of L. tropica was developed according to the protocol described by Seifert et al. with slight modification (Seifert, Matu et al. 2003). The wild type culture was maintained in M199 medium with 10% heat inactivated Fetal Bovine Serum. Briefly, drug pressure was generated in step wise manner for 60 days and the sensitivity of strain to Amp B was investigated. The culture showed resistance was maintained in drug pressure for further use at 24oC.
In vitro Antileishmanial Assay
Antileishmanial assay was performed according to the procedure described by Khan et al. 2014. Promastigotes of L. tropica strain were cultured in M199 media (Invitrogen, USA) with 10% Fetal Bovine Serum (PAA, GmbH). Briefly, stock solution of the test samples and standard drug Amphotericin B drug (10,000 ppm) were prepared in distilled water. Promastigotes were harvested at 1x106 cells/ml and inoculated in 96 well plate. The assay was performed at different concentration (1000-0.001 ppm). For negative control, sterilized distilled water was used. The 96 well plate was incubated for 48 h at 24OC. The live promastigotes were counted using improved Neubauer chamber under light microscope (40x magnification). The results obtained were statistically analyzed using SPSS Version 21 software (Khan, Yasinzai1 et al. 2014).
All the experiment was carried out in triplicate. For comparison Post Hoc Multiple comparison test was applied using SPSS. Ver. 21 software. All the data were given mean ± standard deviation (M±SD).
Resistance in L. tropica against Amp B was developed by a step wise increase of drug pressure. The sensitivity of Amp B against wild and resistant type given in Table 1. A significant different (P<0.05) in LC50 value was observed for wild and resistant type 0.024 and 0.224 µg/ml, respectively. An increase of 09 fold was observed for resistant type compared to wild type.
The antileishmanial activity of green fruit (LC50 value 0.57µg/ml) significant different (P<0.05) when compared to ripe fruit (LC50 value 1220.8µg/ml). The LC50 values for green and ripe fruit against wild type strain are depicted in Table 2. At concentration 0.05µg/ml, the percent survival for green fruit is 95±2.22%, whereas 75.0±3.51% for ripe fruit is recorded at 500µg/ml concentration (Figure 1).
A significant difference (P<0.05) in activity was also observed between green and ripe fruit against resistant type strain. The LC50 value established for green and ripe fruit is 123.3µg/ml and 4010.1µg/ml, respectively as shown in Table 2. The percent survival of the parasite against green and ripe fruit is depicted in Figure 2. The results indicated that at concentration 50µg/ml, the percent survival of the green and ripe fruit is 85.0±3.5% and 100%, respectively.
Parasitic protozoan infection constitutes a major health problem in developing countries. Resistance shown by the parasite and limited knowledge on the mechanism(s) on which these parasites acquire resistance cause additional burden in the development of practical public health policies for parasite control (Grögl, Oduola et al. 1989). The term “resistance” describes the irresponsiveness of a microorganism to an antimicrobial drug as investigated in vitro and compared with other isolates of the same species. While, clinical failure of a drug for appropriate treatment may include antimicrobial resistance, impaired immune function, accelerated metabolism and poor availability of the given drug. Primary resistance is the resistance that occurs in an organism never before to that specific drug of choice, while secondary resistance, also known as acquired resistance occurs after exposure to the drug (Rex, Pfaller et al. 1997). The resistance of the Leishmania to the Amp B was studied. Amp B a polyene antibiotic is the second line treatment for leishmaniasis, binds specifically to ergosterol, the main sterol present of Leishmania, trypanosome cruzi and fungi (Davidson, Scott et al. 1991). Amp B resistant promastigotes lines were selected by step by step increase in drug pressure, as previously shown by other antileishmanial drugs (Arana, Pérez–Victoria et al. 1998). A resistant type of parasite was generated at 0.1 µg/ml in M199 medium as parasite found dead at 0.2 µg/ml of concentration. In this context, plant originated drug showed promising results to overcome resistance. We have previously reported the potential application of green and ripe fruit of M. azedarach against wild type L. tropica strain and the results indicated that green fruit significantly (P<0.05) reduced the parasites than ripe fruit (Khan, Yasinzai1 et al. 2014), which is parallel to our findings. The effect of green fruit on resistant type L. tropica strain was significantly higher than ripe fruit. The elevated activity of the green fruit may be attributed to the high amount of lemonoids and azadirachtin present or may be due to some novel compounds (Khan, Yasinzai1 et al. 2014).
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