Synergic Antibacterial Effect of Curcumin with Ampicillin ; Free Drug Solutions in Comparison with SLN Dispersions

2016 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2016, 6(3), 461-465 doi: 10.15171/apb.2016.060 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
3] Ampicillin as a β-lactam could inhibit bacteria cell wall synthesis to kill the bacteria. 4eta-lactam antibiotics are the most preferred antibiotic group in the treatment of infections due to their wide spectrum and minimal side effects but bacteria go to be resistance against them. 5Some studies have previously demonstrated that curcumin potentiates the effects of chemical antibiotics against bacteria strains. 6ur previous studies showed that loading of antibiotics on SLNs could cause to decrease MIC and MBC against S.aureus, Ecoli and P.aeroginosa significantly. 7,8he aim of this study was to investigate the synergistic effect of curcumin and ampicillin against P.aeroginosa, S.aureus, Ecoli, C.diphtheria, B.Subtilis and MRSA as well as to determine the antibacterial activity of their combination when loaded on SLNs.

Preparation of Solutions Solution A
A stock solution of free ampicillin was prepared in sterile water that was further diluted in Muller-Hinton broth to reach a concentration of 0.125 μg/mL.

Solution B
A stock solution of free curcumin was prepared in sterile water that was further diluted in Muller-Hinton broth to reach a concentration of 0.125 μg/mL.

Solution C
Then a stock solution of free ampicillin and curcumin was prepared by mixing of 50-50% of the two above solutions.

Preparation of SLNs
Curcumin and ampicillin loaded SLNs were prepared separately using high pressure homogenization method based on the Varshosaz et al method in which hot oily phase was added to aqueous phase under homogenization. 9Table 1 shows the formulations.

E
Mixture of formulation A and B in 50-50% ratio, formulation E was designed to compare that is it better that active ingredients load together on SLNs or each of them load separately and then prepared drug loaded SLNs mix in 50-50% ratio (comparison of formulation D and E).

Determination of particle size of SLNs
All SLNs, were investigated to determine particle size distribution by Malvern instrument.

Morphology studies
Morphology of the nanoparticles was characterized by scanning electron microscopy.

MIC and MBC Detection
The conventional broth macrodilution tube method was used to determine MIC and MBC of all above solutions and formulations with respect to P.aueroginosa, S.aureus, Ecoli, C.diphtheria, B.subtilis and MRSA in vitro. 7,10lling contact time Determination In this procedure, 0.1 ml of bacteria suspension equivalent to 0.5 McFarland standards were added to tubes contain 9.9 ml Muller-Hilton broth with MIC concentration of formulations A, B, C, D and E Muller-Hilton broth as control, and incubation was done at 37°C.Then samples were taken from growing bacteria cultures in desired times and spread on Muller-Hilton agar and colonies were count after incuation.

Results and Discussion
Particle size of SLNs Results (Z-average) showed that the particle size of formulations A,B,C and D were 159, 149, 163 and 145 nm respectively.Poly Dispersity Indexes were less than 0.5 for all formulations.

Morphology studies
All desired SLN formulations were spherical.

Antibacterial studies Determination of inhibition zone
Table 2 shows the result of inhibition zone of formulation A-D.Results show that although MRSA and C.diphtheria are resistant against curcumin SLNs (formulation A) but these SLNs could increase inhibition zone when is combine with ampicillin on SLNs (formulation C), so a synergy could be seen.There are not significant differences between formulation D and E for none of bacteria strains except S.aureus, MRSA and P.aueroginosa in which seems that it will be better if drugs load together in formulation process (formulation D) instead of preparing of each formulation (A and B) and then mix them in 50-50% ratio (formulation E).

MIC and MBC studies
Results were shown in Tables 3 and 4. The enhancement of antibacterial effect when curcumin used with ampicillin was obtained as reported in Table 3 for free drugs solutions especially for MIC regarding to B.subtilis, C.diphteria and MRSA.Comparisons of data series of Table 3 and 4, show that MIC and MBC of ampicillin and curcumin which were loaded together on SLNs (formulation C) were much more decreased than solution C in which free ampicillin and curcumin was mixed together.About B.subtilis MIC of formulation C is practically the same as solution C but the resistancy of bacteria to free drugs in MBC was broken by using SLNs.P.aueroginosa is resistant against free drug mixture of ampicillin and curcumin but formulation C is effective on this strain and MBC is two times larger than MIC in this situation.For C.diphteria, no significant changes were observed in MIC of Solution C and formulation C but MBC decreased 4 times when drugs loaded on SLNs in comparison with free drugs.About MRSA MIC decreased 2 times with using SLNs and the resitancy of MRSA about MBC was broken.The increasing of antibacterial effect of ampicillin SLNs in comparison with free ampicillin against Ecoli, S.aureus and P.aueroginosa was reported by Alihoseyni et al. 8 Previously the efficacy of SLNs to decrease MIC and MBC of Curcumin was demonstrated. 11lling contact time Table 5 shows killing contact time results.Formulation C after 10 hours contact with S.aureus, Ecoli, P.aueroginosa, C.diphtheria, B.subtilis show the highest killing percentage in comparison with formulations A, B, D and E. The efficacy of all formulations against all bacteria strains is time dependent and the most bacteria killing percentage was reported after 10 hours, may be due to sustained drug release profiles.Presented results demonstrated the probability of benefits of using nanotechnology to decrease the dosage of antibiotics and decrease risk of having antibiotic resistance bacteria strains.

Table 3 .
MIC and MBC results for desired solutions against six bacteria strains

Table 4 .
MIC and MBC results for desired formulations against six bacteria strains

Table 5 .
Results of killing contact time studies for SLNs