DOI:
10.1039/C5RA16071E
(Paper)
RSC Adv., 2015,
5, 89503-89514
Nimbolide from Azadirachta indica and its derivatives plus first-generation cephalosporin antibiotics: a novel drug combination for wound-infecting pathogens†
Received
10th August 2015
, Accepted 2nd October 2015
First published on 6th October 2015
Abstract
The shortage of effective drugs against wound-infecting pathogens poses a serious public health threat. Combination treatment may represent a good choice for treating infections caused by these pathogens. The aim of the present study is to evaluate the in vitro efficacy of nimbolide, desacetylnimbin isolated from Azadirachta indica, and the amide derivatives of nimbolide in combination with first-generation cephalosporin antibiotics against major wound-associated bacterial pathogens. The antibacterial activities of the compounds and antibiotics were studied by calculating minimum inhibitory concentrations and minimum bactericidal concentrations. The checkerboard and time–kill assay were used to evaluate the interactions between the compounds and antibiotics. Nimbolide recorded the highest antimicrobial activity against all tested strains followed by desacetylnimbin, but the amide derivatives of nimbolide were found to be less active. The MICs of the tested compounds ranged from 64 to 2000 μg mL−1. In the checkerboard test, nimbolide and its derivatives markedly reduced the MIC values of the antibiotics. A significant synergistic effect was recorded with nimbolide as well as desacetylnimbin in combination with antibiotics, and this combination recorded significant reduction in the number of colony forming units (CFUs) in the time kill assay, and the maximum reduction was recorded between 4 and 12 h. The above combination was also found to be effective against methicillin-resistant Staphylococcus aureus (MRSA), an important drug-resistant bacterium. The cytotoxicities of the compounds were tested against H9c2 and they recorded no toxicity up to 200 μM. In summary, the combination of nimbolide/desacetylnimbin and antibiotics demonstrated synergistic activity against the major wound-associated bacteria tested in this study. Furthermore, these compounds may potentially widen the therapeutic window of antibiotics, suggesting that these combinations could be used clinically to control infections caused by wound pathogens after in vivo experiments.
1. Introduction
Chronic wounds are a global health problem, independent of socioeconomic and geographic boundaries. The occurrence and prevalence of chronic wounds are set to increase given the disease pathophysiology and ageing populations.1 Enhanced wound microorganisms, and an increase in bacterial pathogenicity and virulence, has a significant influence on increasing the possibility of a wound becoming more fatal.2 Accordingly, when human wound infections occur, the colonising bacteria often grow in a biofilm and show resistance to antibiotics, thus making our ability to control these infections much more difficult.1 In these circumstances, sepsis in wound patients becomes a major concern.2 Moreover, wound infections are one of the most common surgical complications associated with hospital patients, leading to significantly high levels of mortality and morbidity worldwide. Surgical and burn infections are reported to be the most common form of nosocomial infections for surgical patients, and are reported in both the UK and the USA to occur in more than 40% of patients.3
Pathogenic microorganisms that are routinely isolated from wounds include Staphylococcus aureus, Corynebacterium sp., Candida albicans and Pseudomonas aeruginosa.4 However, chronic wounds are often colonized with diverse microorganisms known to affect healing rates and also to increase the risk of infection developing. The microorganisms in chronic wounds exist both in the planktonic and biofilm phenotypic states.5 Consequently, antimicrobials used for the management of chronic wound infections or the prevention of an infection must therefore demonstrate activity against microorganisms in both phenotypic states, as tolerance to antimicrobials is significantly different. In particular, the efficacy of antimicrobials on wound biofilms is of paramount importance because biofilms are now considered to delay wound healing.2 Therefore, disruption and control of biofilms in chronic wounds represents a fundamental requirement of a ‘wound management strategy’.6
To combat wound infection and wound-associated biofilms, a number of strategies have been employed. Combined antibiotic treatment is commonly practised in the clinic to control wound infections, and such treatments can result in synergy, in order to provide increased efficiency and a reduction in the amount of each antibiotic used, which can reduce the risk of side-effects and possible treatment costs.7 Moreover, combined use of antibiotics and natural products with different modes of action reduces the risk of antibiotic resistance arising during therapy.8,9 This is particularly important for chronic wounds where antibiotic therapy is often long-term.
Several studies have revealed that natural compounds, especially from medicinal plants, in combination with clinically used antibiotics offers a novel strategy for developing therapies for controlling infections caused by pathogenic bacterial species.10–12 One such plant that finds extensive application in traditional systems of medicine is Azadirachta indica. It is commonly known in India as ‘neem’ (Family: Meliaceae) and has universally been accepted as a ‘wonder tree’ because of its varied usefulness.13 Almost all parts of the tree are used in traditional systems of medicine for the treatment of various ailments.14 Indigenous people in India used neem leaves for the treatment of gastrointestinal disorders.15–17 The aqueous extract of A. indica leaves significantly reduces blood sugar levels and decreases hyperglycemia.18 The methanolic extract of neem leaves shows chemopreventive, antibacterial and antisecretory activity against Vibrio cholerae.19,20 Even though neem leaf extracts have showed promising antibacterial activity, the active constituents of the neem leaf have not been evaluated in detail. Neem is a rich source of bioactive limonoids and the major compound isolated from neem leaves is the limonoid nimbolide. In this study, we demonstrate a potent synergistic effect between the two limonoids, viz. nimbolide and desacetylnimbin, isolated from Azadirachta indica, and the amide derivatives of nimbolide in combination with commercially available first-generation cephalosporin antibiotics on major wound-associated pathogens. This study highlights the potential of the combined use of neem limonoids and the amide derivatives of nimbolide in combination with cephalosporin antibiotics to develop novel therapies for chronic wounds and severe skin infections, which in turn improve efficacy and reduce the risk of antibiotic resistance.
2. Experimental
2.1. Materials and methods
IR spectra were recorded on a Bruker Alpha FT-IR spectrometer.1H NMR spectra were recorded at 500 MHz and 13C NMR at 125 MHz using deuterated chloroform (CDCl3) as the solvent on a Bruker AV 500 spectrometer. Tertramethylsilane (TMS) was used as the internal standard and chemical shift values are expressed in δ-scale in units of parts per million (ppm) and coupling constants (J) in Hz. Mass spectra were recorded using a Thermo Scientific Exactive mass spectrometer under ESI/HRMS at 61
800 resolution. Analytical thin layer chromatography was performed on Merck silica gel 60 F254 aluminium sheets and column chromatography using Merck silica gel (100–200 mesh). Chromatography was carried out using varying polarities of hexane–ethyl acetate mixtures as the solvent. Amines used were purchased from Sigma-Aldrich.
2.2. Isolation of nimbolide and desacetylnimbin
Fresh leaves of Azadirachta indica were dried and powdered. 500 g of the powdered leaves was extracted using acetone at room temperature (2.5 L × 3) to obtain 30 g of the crude extract after removal of the solvent. It was then subjected to purification using silica gel (100–200 mesh) column chromatography using gradient mixtures of hexane and ethyl acetate. The nimbolide-rich fraction, obtained by eluting the column with 25% ethyl acetate in hexane, was subjected to crystallization using a petroleum ether–dichloromethane solvent system to yield 450 mg of pale yellow crystals of nimbolide (1) (Fig. 1). Spectral details of the compound matched well with the reported data.21
 |
| Fig. 1 Structures of nimbolide (1) and desacetylnimbin (2). | |
2.2.1. Nimbolide (1). Pale yellow crystals; C27H30O7; FT-IR (KBr, νmax, cm−1): 2978, 1778, 1730, 1672, 1433, 1296, 1238, 1192, 1153, 1069, 951, 827, 750; 1H NMR (500 MHz, CDCl3) δH: 7.32 (t, J = 1.5 Hz, 1H), 7.28 (d, J = 9.5 Hz, 1H), 7.22 (s, 1H), 6.25 (m, 1H), 5.93 (d, J = 10.0 Hz, 1H), 5.53 (m, 1H), 4.62 (dd, J = 3.67 Hz, 12.5 Hz, 1H), 4.27 (d, J = 3.5 Hz, 1H), 3.67 (d, J = 9.0 Hz, 1H), 3.54 (s, 3H), 3.25 (dd, J = 5.0 Hz, 16.25 Hz, 1H), 3.19 (d, J = 12.5 Hz, 1H), 2.73 (t, J = 5.5 Hz, 1H), 2.38 (dd, J = 5.5 Hz, 16.25 Hz, 1H), 2.22 (dd, J = 6.5 Hz, 12.0 Hz, 1H), 2.10 (m, 1H), 1.70 (s, 3H), 1.47 (s, 3H), 1.37 (s, 3H), 1.22 (s, 3H); 13C NMR (125 MHz, CDCl3) δC: 200.8 (CO), 175.0 (COO), 173.0 (COO), 149.6 (CH), 144.8 (C), 143.2 (CH), 138.9 (CH), 136.4 (C), 131.0 (CH), 126.5 (C), 110.3 (CH), 88.5 (CH), 82.9 (CH), 73.4 (CH), 51.8 (OCH3), 50.3 (C), 49.5 (CH), 47.7 (CH), 45.3 (C), 43.7 (C), 41.2 (CH2), 41.1 (CH), 32.1 (CH2), 18.5 (CH3), 17.2 (CH3), 15.2 (CH3), 12.9 (CH3); HR-MS (m/z): 467.20795 [(M + H)+].The supernatant liquid left after the crystallization of nimbolide on flash column chromatographic purification followed by crystallization using petroleum ether–dichloromethane solvent system yielded 20 mg of colourless crystals, which were characterised as desacetylnimbin (2) (Fig. 1) using various spectroscopic techniques and on comparison with the literature reports.22
2.2.2. Desacetylnimbin (2). Colorless crystals; C28H34O8; FT-IR (KBr, νmax, cm−1): 3574, 1771, 1731, 1681, 1547, 1536, 1435, 1397, 1257, 1228; 1H NMR (500 MHz, CDCl3) δH: 7.33 (t, J = 1.5 Hz, 1H), 7.24 (s, 1H), 6.41 (d, J = 10.5 Hz, 1H), 6.33 (d, J = 1.5 Hz, 1H), 5.86 (d, J = 10.5 Hz, 1H), 5.55 (m, 1H), 4.03 (d, J = 3.5 Hz, 1H), 3.93 (m, 1H), 3.71 (s, 3H), 3.68 (m, 1H), 3.66 (s, 3H), 3.40 (d, J = 12.0 Hz, 1H), 2.90 (dd, J = 5.5 Hz, 16.5 Hz, 1H), 2.77 (m, 1H), 2.20 (m, 2H), 2.04 (m, 2H), 1.69 (s, 3H), 1.61 (s, 3H), 1.29 (s, 3H), 1.22 (s, 3H); 13C NMR (125 MHz, CDCl3) δC: 201.6 (CO), 175.6 (COO), 173.7 (COO), 148.1 (CH), 146.8 (C), 143.1 (CH), 139.0 (CH), 134.9 (C), 126.8 (CH), 126.4 (C), 110.4 (CH), 87.4 (CH), 86.9 (CH), 66.2 (CH), 53.0 (OCH3), 51.7 (OCH3), 49.6 (CH), 47.8 (C), 47.5 (C), 47.3 (C), 43.9 (CH), 41.4 (CH2), 39.1 (CH), 34.4 (CH2), 17.5 (CH3), 17.2 (CH3), 16.4 (CH3), 12.9 (CH3); HR-MS (m/z): 499.23434 [(M + H)+].
2.3. Synthesis of amide derivatives of nimbolide (1a–c)
In a typical experiment, nimbolide (100 mg) was dissolved in dry THF and to that 3.5 equivalents of the respective amine was added, and the contents were refluxed under stirring for a period of 24 h. The progress of the reaction was monitored using TLC. After completion of the reaction THF was removed under reduced pressure and the products were isolated in good yields using silica gel column chromatography by eluting the column with mixtures of ethyl acetate and hexane. Three different amide derivatives of nimbolide were synthesized and the structures of the compounds were confirmed using various spectroscopic techniques. A general scheme of the reaction, amines used, and their respective yields are given in Table 1.
Table 1 Scheme for the synthesis of amide derivatives
2.3.1. Compound 1a. Colourless crystals; C33H45NO7; FT-IR (KBr, νmax, cm−1): 3558, 3380, 2952, 1739, 1680, 1640, 1532, 1442, 1371, 1263, 1163, 1062, 1029; 1H NMR (500 MHz, CDCl3) δH: 7.33 (s, 1H), 7.23 (s, 1H), 6.36 (d, J = 10 Hz, 1H), 6.32 (s, 1H), 5.87 (d, J = 10 Hz, 1H), 5.71 (t, J = 5.5 Hz, 1H), 5.54 (t, J = 7 Hz, 1H), 4.01 (d, J = 3 Hz, 1H), 3.94 (m, 1H), 3.67 (brs, 1H), 3.65 (s, 3H), 3.35 (d, J = 11.5 Hz, 1H), 3.25 (m, 2H), 2.88 (dd, J = 5.5 Hz, 16.5 Hz, 1H), 2.78 (t, J = 5 Hz, 1H), 2.48 (d, J = 10.5 Hz, 1H), 2.22 (dd, J = 3.5 Hz, 16.5, 1H), 2.18 (m, 1H), 2.04 (m, 1H), 1.68 (s, 3H), 1.59 (s, 3H), 1.40 (t, J = 8 Hz, 2H), 1.29 (s, 3H), 1.24 (s, 3H) 0.92 (s, 9H); 13C NMR (125 MHz, CDCl3) δC: 202.4 (CO), 175.0 (CONH), 173.6 (COO), 149.2 (CH), 146.9 (C), 143.1 (CH), 139.0 (CH), 134.8 (C), 126.9 (CH), 126.3 (C), 110.4 (CH), 87.7 (CH), 86.8 (CH), 66.4 (CH), 51.7 (OCH3), 49.6 (CH), 48.1 (C), 47.4 (C), 47.3 (C), 43.7 (CH), 43.0 (CH2), 41.4 (CH2), 39.0 (CH), 37.2 (CH2), 34.5 (CH2), 29.9 (C), 29.2 (3 × CH3), 17.5 (CH3), 16.9 (CH3), 16.6 (CH3), 12.9 (CH3); HR-MS (m/z): 568.32440 [(M + H)+].
2.3.2. Compound 1b. Colourless crystals; C32H43NO7; FT-IR (KBr, νmax, cm−1): 3561, 3377, 2954, 1739, 1680, 1642, 1531, 1442, 1375, 1262, 1162, 1198, 1062, 1029; 1H NMR (500 MHz, CDCl3) δH: 7.33 (s, 1H), 7.23 (s, 1H), 6.36 (d, J = 10 Hz, 1H), 6.32 (s, 1H), 5.86 (d, J = 10 Hz, 1H), 5.77 (m, 1H), 5.53 (t, J = 7 Hz, 1H), 4.01 (d, J = 3 Hz, 1H), 3.93 (t, J = 4 Hz, 1H), 3.67 (brs, 1H), 3.65 (s, 3H), 3.35 (d, J = 11.5 Hz, 1H), 3.25 (m, 2H), 2.89 (dd, J = 5.5 Hz, 17.5 Hz, 1H), 2.77 (t, J = 5.0 Hz, 1H), 2.46 (d, J = 10 Hz, 1H), 2.23 (dd, J = 4 Hz, 16.5 Hz, 1H), 2.17 (m, 1H), 2.04 (m, 1H), 1.69 (s, 3H), 1.60 (m, 1H), 1.59 (s, 3H), 1.39 (m, 2H), 1.29 (s, 3H), 1.24 (s, 3H), 0.91 (d, J = 6.5 Hz, 6H); 13C NMR (125 MHz, CDCl3) δC: 202.4 (CO), 175.0 (CONH), 173.6 (COO), 149.3 (CH), 146.9 (C), 143.0 (CH), 139.0 (CH), 134.8 (C), 126.9 (CH), 126.3 (C), 110.5 (CH), 87.7 (CH), 86.8 (CH), 66.4 (CH), 51.6 (OCH3), 49.6 (CH), 48.1 (C), 47.5 (C), 47.3 (C), 43.7 (CH), 41.4 (CH2), 39.0 (CH), 38.7 (CH2), 38.1 (CH2), 34.5 (CH2), 25.9 (CH), 22.5 (CH3), 22.4 (CH3), 17.5 (CH3), 16.9 (CH3), 16.6 (CH3), 12.9 (CH3); HR-MS (m/z): 576.29425 [(M + Na)+].
2.3.3. Compound 1c. Colourless crystals; C35H41NO7; FT-IR (KBr, νmax, cm−1): 3547, 3379, 2929, 1735, 1678, 1648, 1529, 1444, 1374, 1262, 1163, 1062, 1026; 1H NMR (500 MHz, CDCl3) δH: 7.31 (m, 3H), 7.21 (m, 4H), 6.33 (m, 1H), 6.24 (d, J = 10 Hz, 1H), 5.86 (t, J = 5 Hz, 1H), 5.80 (d, J = 10 Hz, 1H), 5.53 (m, 1H), 4.00 (d, J = 3.5 Hz, 1H), 3.93 (dd, J = 3 Hz, 12 Hz, 1H) 3.67 (s, 1H), 3.64 (s, 3H), 3.56 (m, 1H), 3.45 (m, 1H), 3.34 (d, J = 11.5 Hz, 1H), 2.88 (dd, J = 5.5 Hz, 15 Hz, 1H), 2.82 (m, 2H), 2.21 (m, 3H), 2.05 (m, 2H), 1.51 (s, 3H), 1.28 (s, 3H), 1.26 (s, 3H), 1.21 (s, 3H); 13C NMR (125 MHz, CDCl3) δC: 202.4 (CO), 175.1 (CONH), 173.6 (COO), 149.2 (CH), 146.9 (C), 143.1 (CH), 139.0 (CH), 138.9 (C), 134.8 (C), 128.9 (2 × CH), 128.7 (2 × CH), 126.9 (CH), 126.6 (CH), 126.2 (CH), 110.5 (CH), 87.7 (CH), 86.8 (CH), 66.4 (CH), 51.6 (OCH3), 49.6 (CH), 48.1 (C), 47.5 (C), 47.4 (C), 43.6 (CH), 41.5 (CH2), 41.4 (CH2), 39.0 (CH), 35.4 (CH2), 34.5 (CH2), 17.5 (CH3), 16.8 (CH3), 16.5 (CH3), 12.9 (CH3); HR-MS (m/z): 588.29303 [(M + H)+].
2.4. Antibacterial activity of the compounds
2.4.1. Test microorganisms. The following four major wound-associated medically important bacteria were used in the present study: Staphylococcus aureus, Staphylococcus epidermis MTCC 435, Klebsiella pneumoniae MTCC 109 and Pseudomonas aeruginosa MTCC 2642. The study was also carried out on the methicillin-resistant Staphylococcus aureus (MR-S. aureus) ATCC 43300. All the strains were cultured at 37 °C on nutrient agar (NA) medium and stored at 4 °C. All the test microorganisms except MR-S. aureus were purchased from Microbial Type Culture collection Centre, IMTECH, Chandigarh, India. MR-S. aureus was gifted by Dr Pratap Chandran, Associate Professor, SDV College of Arts and Applied Science, Alappuzha, Kerala, India.
2.4.2. Antibiotics used. The following two first-generation cephalosporin antibiotics were used in this study: cefalexin (97% pure) and cefazolin (98% pure) (Sigma-Aldrich, USA). The structures of the compounds are given in Fig. 2.
 |
| Fig. 2 Chemical structures of antibiotics used in the present study. | |
2.4.3. Determination of minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The test compounds and antibiotics were screened for antimicrobial activity using the macro broth dilution method in nutrient broth (NB).23 To determine the MIC, the test compounds were dissolved in DMSO to afford a stock concentration of 4000 μg mL−1, while the antibiotics were dissolved in sterile distilled water to afford stock concentrations of 1000 μg mL−1. All stock concentrations of test compounds and antibiotics were filter sterilized using a 0.22 μM syringe filter (Millipore). Two-fold serial dilutions of the test compounds and antibiotics were prepared with NB to afford concentrations ranging from 1 to 1000 μg mL−1. Five hundred microliters of 1 × 106 CFU mL−1 bacterial suspensions was added to the sterile test tubes affording an inoculum of 5 × 105 CFU mL−1. Another 50 μL of antibiotics or test compounds were pipetted into the tubes and incubated at 37 °C for 18 h. The tubes were read visually and spectrophotometrically at 600 nm. The MICs of the extracts that exhibited no turbidity were recorded as the MICs. The control tube did not have any antibiotics or test compounds, but contained the test bacteria, and DMSO was used to dissolve the compounds. DMSO was shown not to affect the growth of the bacteria during the experiments. The MIC was defined as the lowest concentration of the test compound that inhibited the visual growth after incubating the test bacteria for 18 h.For MBC testing, an aliquot of inoculum taken from MIC test tubes that did not show turbidity was serially diluted in saline and plated into nutrient agar plates for each bacterial species. The agar plates were incubated for 24 h at 37 °C. The MBC value was read as the lowest concentration of the compound at which 99.99% or more of the initial inoculum was killed.24 The MIC and MBC experiments were repeated in triplicate.
2.4.4. Antibacterial activity using the disc diffusion method. The antibacterial activity of the test compounds was determined by the disc diffusion method against the test bacteria on Muller-Hinton agar, according to the Clinical and Laboratory Standards Institute (CLSI). The media plates (MHA) were streaked with bacteria 2–3 times by rotating the plate at 60° angles for each streak to ensure the homogeneous distribution of the inocula. After inoculation, discs (6 mm Hi-Media) loaded with 100 μg mL−1 of the test compounds were placed on the bacteria-seeded plates using sterile forceps. The plates were then incubated at 37 °C for 24 h. The inhibition zone around the discs was measured and recorded. Cefalexin and cefazolin (Hi-Media) were used as the positive controls to compare the efficacy of the test samples. DMSO served as the negative control, and assays were carried out in triplicate.
2.4.5. Determination of the in vitro synergistic activity by checkerboard assay. The antimicrobial effects of different combinations of two or more antimicrobial agents were assessed using the checkerboard test.25 Checkerboard testing is one of the most widely used standard methods to determine the synergistic activity of test compound and antibiotic combinations. It is based on microdilution susceptibility testing of antibiotic combinations. The antimicrobial assays were performed with the two natural limonoids and three amide derivatives of nimbolide in combination with antibiotics. The inocula were prepared from colonies that had been grown on Muller Hinton agar (MHA) overnight. The final bacterial concentration after inoculation was 2 × 105 CFU mL−1. The MIC was determined after 24 h incubation at 37 °C. The MIC was defined as the lowest concentration of the test compounds, alone or in combination with antibiotics, visibly inhibiting the growth of bacteria, by measuring the OD at 600 nm using a microplate reader (Bio-rad, USA). Each experiment was repeated thrice. To assess the in vitro interaction of combinations of test compounds (A) and antibiotics (B), the results obtained were further analysed using the fractional inhibitory concentration (FIC) index. The fractional inhibitory concentration and the fractional bactericidal concentration are mathematical expressions of the effect of the combination of antibacterial agents. The interaction of two compounds was regulated as synergistic, indifferent or antagonistic on the basis of the FIC index values. The FIC values were calculated as follows: FIC of test compounds (FICA) = (MIC of A in combination with B)/(MIC of A alone), where A and B were mixed in the ratio 7
:
3; FIC of antibiotics (FICB) = (MIC of B in combination with A)/(MIC of B alone), where A and B were mixed in the ratio 4
:
6.The sum of the fractional inhibitory concentration indices (FICI) of the two compounds (A and B) in the combination was calculated as follows:
A synergistic relationship was defined as FIC index ≤ 0.5, an additive relationship was defined as 0.5 < FIC index ≤ 1.0, an indifferent relationship was defined as 1.0 < FIC index ≤ 4.0, and an antagonistic relationship was defined as FIC index > 4.0.
2.4.6. Killing curve determination. Killing curve determination was carried out in order to confirm the antibacterial and synergistic activities of the two natural limonoids and three amide derivatives of nimbolide when used singly and in combination with antibiotics. The viabilities of drug-resistant bacteria after exposure to these agents alone and in combination at seven distinct times (0, 2, 4, 6, 12, 24 and 48 h) were counted. The assay was followed by the previously described method with some modifications.26 Concisely, inocula (5 × 105 CFU mL−1) were exposed to the test compounds either singly or in combination with antibiotics. Aliquots (0.1 mL) of each exposed time were removed from tubes and diluted in normal saline as needed to enumerate 30–300 colonies. The diluted cultures were plated and spread thoroughly on plates containing MHA. After incubating at 35 °C for 18 h, the growing colonies were counted. The lowest detectable limit for counting is 103 CFU mL−1. The experiment was performed in triplicate; data are shown as mean ± SD.
2.4.7. Determination of cytotoxicity.
2.4.7.1. Cell line maintenance. H9c2 cell lines (rat embryonic cardiomyoblasts) were obtained from ATCC (American Type Culture Collection, USA). For maintenance of cell lines, Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma) containing 10% fetal bovine serum (FBS) (Gibco), antibiotics (100 U mL−1 penicillin and 100 μg mL−1 streptomycin) and amphotericin (0.25 μg mL−1) (Hi-Media) was employed. The cells were maintained in cell culture flasks in CO2 incubators at 37 °C with 5% CO2 in air and 99% humidity. Passaging of cells when confluent was carried out using 0.25% trypsin and 0.02% EDTA (Hi-Media) in phosphate-buffered saline (PBS).
2.4.7.2. MTT assay. Cell viability after incubating the cells with different concentrations of the test substance was determined by methyl thiazolyl tetrazolium (MTT) assay. This is a colorimetric assay based on the ability of live, but not dead, cells to reduce MTT (yellow) to a formazan (purple) product. The cells were spread in 96-well plates at 5 × 103 cells per well. After 36 h of seeding, they were incubated with different concentrations of the test substance individually for 24 h. Subsequently, the cells were exposed to MTT at a concentration of 50 μg per well for 2.5 to 3 h at 37 °C in a CO2 incubator. The working solution of MTT was prepared in Hanks balanced salt solution (HBSS). After viewing formazan crystals under the microscope, the crystals were solubilized by treating the cells with DMSO
:
isopropanol at a ratio of 1
:
1 for 20 min at 37 °C. The plate was read at an absorbance of 570 nm. The relative cell viability in percent was calculated as: (absorbance of treated/absorbance of control) × 100. Control samples used were cells without any treatment. The cell viability of control cells was kept as 100%.
3. Results
3.1. Isolation/synthetic modification of limonoids
Nimbolide (1) and desacetylnimbin (2) were isolated from the acetone extract of Azadirachta indica leaves. Three different amide derivatives (1a–c) of nimbolide were synthesized by reacting nimbolide with respective primary amines under reflux conditions using THF as the solvent. The structures of all the compounds were characterised using various spectroscopic techniques.
3.2. MIC and MBC
MIC and MBC ranges of antibiotics, nimbolide, desacetylnimbin and the amide derivatives of nimbolide used alone for wound pathogens are shown in Table 2. The ranges of MIC/MBC for the test pathogens were 64 to 1000 μg mL−1 for S. aureus, 125 to 2000 μg mL−1 for S. epidermis, 32 to 500 μg mL−1 for K. pneumoniae, 125 to 2000 μg mL−1 for P. aeruginosa and 250 to 2000 μg mL−1 for MR-S. aureus. In our study nimbolide recorded the highest activity, followed by desacetylnimbin, but the amide derivatives showed less activity when compared to the natural limonoids.
Table 2 Antibacterial activities of the test compounds and antibiotics against bacteriaa
MIC/MBC (μg mL−1) |
Test compounds |
S. aureus |
S. epidermis |
K. pneumoniae |
P. aeruginosa |
MR-S. aureus |
(—) Recorded no MIC up to 2000 μg mL−1, values represent mean of three replications. |
Nimbolide (1) |
64/125 |
125/125 |
32/64 |
250/500 |
250/250 |
Desacetylnimbin (2) |
125/250 |
250/250 |
125/125 |
250/500 |
500/1000 |
Compound 1a |
125/250 |
125/250 |
250/250 |
500/500 |
1000/2000 |
Compound 1b |
250/500 |
500/1000 |
500/500 |
125/250 |
— |
Compound 1c |
500/1000 |
1000/2000 |
500/500 |
2000/2000 |
— |
Cefalexin |
4/4 |
2/4 |
2/2 |
2/4 |
16/32 |
Cefazolin |
2/4 |
4/4 |
1/2 |
2/4 |
64/64 |
3.3. Antibacterial activity
Quantification of antibacterial activity of the test compounds was confirmed by agar disc diffusion assay and is shown in Table 3. In the agar disc diffusion assay nimbolide recorded the most significant zone of inhibition against the test bacteria, followed by desacetylnimbin. The best zone of inhibition was recorded by nimbolide against K. pneumoniae (29 mm) (Table 3). Photographs of the zones of inhibition by nimbolide against the test bacteria are shown in Fig. 3. The natural limonoid nimbolide was found to be effective against the important drug resistant strain MR-S. aureus as well.
Table 3 Zones of inhibition of the test compounds and antibiotics against bacteriaa
Diameter of zone of inhibition (mm) |
Test compounds |
S. aureus |
S. epidermis |
K. pneumoniae |
P. aeruginosa |
MR-S. aureus |
(—) No zone of inhibition, values represent mean of three replications. |
Nimbolide (1) |
25 ± 0.52 |
21 ± 1.77 |
29 ± 2.52 |
18 ± 1.12 |
17 ± 1.52 |
Desacetylnimbin (2) |
19 ± 0.77 |
15 ± 0.52 |
18 ± 2.1 |
13 ± 0.77 |
10 ± 0.52 |
Compound 1a |
17 ± 1.52 |
19 ± 0.77 |
14 ± 1.52 |
7.0 ± 0.52 |
6.0 ± 1.0 |
Compound 1b |
8.0 ± 1.52 |
— |
6.0 ± 0.52 |
18 ± 1.12 |
— |
Compound 1c |
5.0 ± 1.0 |
— |
6.0 ± 0 |
— |
— |
Cefalexin |
31 ± 0.52 |
28 ± 1.0 |
32 ± 1.12 |
30 ± 1.72 |
21 ± 0 |
Cefazolin |
29 ± 0 |
27 ± 0 |
31 ± 0 |
29 ± 1.0 |
20 ± 0 |
 |
| Fig. 3 Photographs of the zones of inhibition of nimbolide. | |
3.4. Checkerboard assay
The combined activities of the two natural limonoids and amide derivatives of nimbolide with two first-generation cephalosporin antibiotics from their in vitro checkerboard interactions against medically important wound bacteria are summarized in Tables 4 and 5. FICs, FBCs, FIC indices, FBC indices and interpretations for the activities of test compounds and antibiotics against the wound pathogens predominantly showed synergistic interactions. However, some combinations of amide derivatives with antibiotics recorded indifference. Antagonism was not recorded for any of the combinations. During this synergetic combination, the concentrations of the test compounds and antibiotics required to inhibit the pathogens were reduced manyfold when compared with the concentrations of compounds/antibiotics when given individually.
Table 4 Synergistic effects of the test compounds with cefazolin against bacteriad
Test bacteria |
Agent |
MIC/MBC (μg mL−1) |
FIC/FBC |
FICIb/FBCIc |
Outcome |
Alone |
Combinationa |
The MICs and MBCs of the test compounds with cefazolin. The fractional inhibitory concentration index (FIC index). The fractional bactericidal concentration index (FBC index). Significant FICI/FBCI values are shown in bold. |
S. aureus |
Nimbolide |
64/125 |
2/4 |
0.03/0.03 |
0.09/0.09 |
Synergistic/synergistic |
Cefazolin |
2/4 |
0.12/0.25 |
0.06/0.06 |
Desacetylnimbin |
125/250 |
32/64 |
0.26/0.26 |
0.39/0.39 |
Synergistic/synergistic |
Cefazolin |
2/4 |
0.25/0.5 |
0.13/0.13 |
Compound 1a |
125/250 |
64/64 |
0.51/0.26 |
0.10/0.51 |
Indifference/additive |
Cefazolin |
2/4 |
1/1 |
0.50/0.25 |
Compound 1b |
250/500 |
32/32 |
0.13/0.06 |
0.38/0.31 |
Synergistic/synergistic |
Cefazolin |
2/4 |
0.5/1 |
0.25/0.25 |
Compound 1c |
500/1000 |
125/250 |
0.25/0.25 |
0.75/0.50 |
Additive/synergistic |
Cefazolin |
2/4 |
1/1 |
0.50/0.25 |
S. epidermis |
Nimbolide |
125/125 |
16/32 |
0.13/0.26 |
0.26/0.39 |
Synergistic/synergistic |
Cefazolin |
4/4 |
0.5/0.5 |
0.13/0.13 |
Desacetylnimbin |
250/250 |
32/32 |
0.13/0.13 |
0.38/0.38 |
Synergistic/synergistic |
Cefazolin |
4/4 |
1/1 |
0.25/0.25 |
Compound 1a |
125/250 |
32/64 |
0.26/0.26 |
0.51/0.51 |
Additive/additive |
Cefazolin |
4/4 |
1/1 |
0.25/0.25 |
Compound 1b |
500/1000 |
125/125 |
0.25/0.13 |
0.38/0.38 |
Synergistic/synergistic |
Cefazolin |
4/4 |
0.5/1 |
0.13/0.25 |
Compound 1c |
1000/2000 |
250/250 |
0.25/0.13 |
0.75/0.63 |
Additive/additive |
Cefazolin |
4/4 |
2/2 |
0.50/0.50 |
K. pneumoniae |
Nimbolide |
32/64 |
4/8 |
0.13/0.13 |
0.25/0.26 |
Synergistic/synergistic |
Cefazolin |
1/2 |
0.12/0.25 |
0.12/0.13 |
Desacetylnimbin |
125/125 |
16/16 |
0.13/0.13 |
0.25/0.19 |
Synergistic/synergistic |
Cefazolin |
1/2 |
0.12/0.12 |
0.12/0.06 |
Compound 1a |
250/250 |
32/64 |
0.13/0.26 |
0.38/0.51 |
Synergistic/additive |
Cefazolin |
1/2 |
0.25/0.5 |
0.25/0.25 |
Compound 1b |
500/500 |
64/125 |
0.13/0.25 |
0.38/0.50 |
Synergistic/synergistic |
Cefazolin |
1/2 |
0.25/0.5 |
0.25/0.25 |
Compound 1c |
500/500 |
64/125 |
0.13/0.25 |
0.25/0.38 |
Synergistic/synergistic |
Cefazolin |
1/2 |
0.12/0.25 |
0.12/0.13 |
P. aeruginosa |
Nimbolide |
250/500 |
64/64 |
0.26/0.13 |
0.32/0.19 |
Synergistic/synergistic |
Cefazolin |
2/4 |
0.12/0.25 |
0.06/0.06 |
Desacetylnimbin |
250/500 |
32/64 |
0.13/0.13 |
0.38/0.38 |
Synergistic/synergistic |
Cefazolin |
2/4 |
0.5/1 |
0.25/0.25 |
Compound 1a |
500/500 |
32/32 |
0.06/0.06 |
0.31/0.19 |
Synergistic/synergistic |
Cefazolin |
2/4 |
0.5/0.5 |
0.25/0.13 |
Compound 1b |
125/250 |
16/32 |
0.13/0.13 |
0.63/0.63 |
Additive/additive |
Cefazolin |
2/4 |
1/2 |
0.50/0.50 |
Compound 1c |
2000/2000 |
250/250 |
0.13/0.13 |
0.38/0.38 |
Synergistic/synergistic |
Cefazolin |
2/4 |
0.5/1 |
0.25/0.25 |
MR-S. aureus |
Nimbolide |
125/250 |
16/16 |
0.13/0.06 |
0.38/0.31 |
Synergistic/synergistic |
Cefazolin |
16/32 |
4/8 |
0.25/0.25 |
Desacetylnimbin |
500/1000 |
32/64 |
0.06/0.06 |
0.31/0.31 |
Synergistic/synergistic |
Cefazolin |
16/32 |
4/8 |
0.25/0.25 |
Compound 1a |
1000/2000 |
500/500 |
0.50/0.25 |
1.0/0.75 |
Indifference/additive |
Cefazolin |
16/32 |
8/16 |
0.50/0.50 |
Compound 1b |
4000/4000 |
1000/2000 |
0.25/0.50 |
1.25/1.0 |
Indifference/additive |
Cefazolin |
16/32 |
16/16 |
1.0/0.50 |
Compound 1c |
4000/8000 |
2000/4000 |
0.50/0.50 |
1.0/0.75 |
Indifference/additive |
Cefazolin |
16/32 |
8/8 |
0.50/0.25 |
Table 5 Synergistic effects of the test compounds with cefalexin against bacteriad
Test bacteria |
Agent |
MIC/MBC (μg mL−1) |
FIC/FBC |
FICIb/FBCIc |
Outcome |
Alone |
Combinationa |
The MICs and MBCs of compounds with cefalexin. The fractional inhibitory concentration index (FIC index). The fractional bactericidal concentration index (FBC index). Significant FICI/FBCI values are shown in bold. |
S. aureus |
Nimbolide |
64/125 |
16/32 |
0.25/0.26 |
0.31/0.32 |
Synergistic/synergistic |
Cefalexin |
4/4 |
0.25/0.25 |
0.06/0.06 |
Desacetylnimbin |
125/250 |
16/16 |
0.13/0.06 |
0.38/0.31 |
Synergistic/synergistic |
Cefalexin |
4/4 |
1/1 |
0.25/0.25 |
Compound 1a |
125/250 |
8/8 |
0.06/0.03 |
0.19/0.28 |
Synergistic/synergistic |
Cefalexin |
4/4 |
0.5/1 |
0.13/0.25 |
Compound 1b |
250/500 |
64/125 |
0.26/0.25 |
0.51/0.50 |
Additive/synergistic |
Cefalexin |
4/4 |
1/1 |
0.25/0.25 |
Compound 1c |
500/1000 |
64/64 |
0.13/0.06 |
0.38/0.56 |
Synergistic/indifference |
Cefalexin |
4/4 |
1/2 |
0.25/0.50 |
S. epidermis |
Nimbolide |
125/125 |
8/16 |
0.06/0.13 |
0.38/0.26 |
Synergistic/synergistic |
Cefalexin |
2/4 |
0.5/1 |
0.25/0.25 |
Desacetylnimbin |
250/250 |
16/32 |
0.06/0.13 |
0.31/0.26 |
Synergistic/synergistic |
Cefalexin |
2/4 |
0.5/0.5 |
0.25/0.13 |
Compound 1a |
125/250 |
16/32 |
0.13/0.13 |
0.63/0.38 |
Indifference/synergistic |
Cefalexin |
2/4 |
1/1 |
0.50/0.25 |
Compound 1b |
500/1000 |
32/64 |
0.06/0.06 |
0.31/0.19 |
Synergistic/synergistic |
Cefalexin |
2/4 |
0.5/0.5 |
0.25/0.13 |
Compound 1c |
1000/2000 |
250/500 |
0.25/0.25 |
0.75/0.75 |
Indifference/indifference |
Cefalexin |
2/4 |
1/2 |
0.50/0.50 |
K. pneumoniae |
Nimbolide |
32/64 |
4/8 |
0.13/0.13 |
0.26/0.26 |
Synergistic/synergistic |
Cefalexin |
2/2 |
0.25/0.25 |
0.13/0.13 |
Desacetylnimbin |
125/125 |
16/16 |
0.13/0.13 |
0.19/0.26 |
Synergistic/synergistic |
Cefalexin |
2/2 |
0.12/0.25 |
0.06/0.13 |
Compound 1a |
250/250 |
32/32 |
0.13/0.13 |
0.38/0.38 |
Synergistic/synergistic |
Cefalexin |
2/2 |
0.5/0.5 |
0.25/0.25 |
Compound 1b |
500/500 |
32/64 |
0.06/0.13 |
0.56/0.63 |
Indifference/indifference |
Cefalexin |
2/2 |
1/1 |
0.50/0.50 |
Compound 1c |
500/500 |
64/64 |
0.13/0.13 |
0.63/0.63 |
Indifference/indifference |
Cefalexin |
2/2 |
1/1 |
0.50/0.50 |
P. aeruginosa |
Nimbolide |
250/500 |
64/64 |
0.26/0.13 |
0.39/0.19 |
Synergistic/synergistic |
Cefalexin |
2/4 |
0.25/0.25 |
0.13/0.06 |
Desacetylnimbin |
250/500 |
32/64 |
0.13/0.13 |
0.38/0.26 |
Synergistic/synergistic |
Cefalexin |
2/4 |
0.5/0.5 |
0.25/0.13 |
Compound 1a |
500/500 |
64/64 |
0.13/0.13 |
0.28/0.28 |
Synergistic/synergistic |
Cefalexin |
2/4 |
0.5/1 |
0.25/0.25 |
Compound 1b |
125/250 |
16/32 |
0.13/0.13 |
0.28/0.28 |
Synergistic/synergistic |
Cefalexin |
2/4 |
0.5/1 |
0.25/0.25 |
Compound 1c |
2000/2000 |
125/250 |
0.06/0.13 |
0.56/0.63 |
Indifference/indifference |
Cefalexin |
2/4 |
1/2 |
0.50/0.50 |
MR-S. aureus |
Nimbolide |
125/250 |
16/32 |
0.13/0.13 |
0.26/0.26 |
Synergistic/synergistic |
Cefalexin |
64/64 |
8/8 |
0.13/0.13 |
Desacetylnimbin |
500/1000 |
64/125 |
0.13/0.13 |
0.26/0.38 |
Synergistic/synergistic |
Cefalexin |
64/64 |
8/16 |
0.13/0.25 |
Compound 1a |
1000/2000 |
500/500 |
0.50/0.25 |
0.75/0.75 |
Additive/additive |
Cefalexin |
64/64 |
16/32 |
0.25/0.50 |
Compound 1b |
4000/4000 |
1000/2000 |
0.25/0.50 |
0.75/1.0 |
Additive/indifference |
Cefalexin |
64/64 |
32/32 |
0.50/0.50 |
Compound 1c |
4000/8000 |
2000/4000 |
0.50/0.50 |
1.0/1.5 |
Indifferent/indifference |
Cefalexin |
64/64 |
32/64 |
0.50/1.0 |
When nimbolide was combined with cefazolin for the inhibition of S. aureus, a significant synergistic effect (FIC = 0.09) was observed and the MIC values of nimbolide and cefazolin were reduced to 6 and 8 times below their individual MIC values, respectively (Table 4). Another prominent synergy was observed (FIC = 0.18) for desacetylnimbin and cefalexin against K. pneumoniae, and the concentration of the desacetylnimbin was reduced to 4 times below its individual MIC value (Table 5). Interestingly the combination of nimbolide and desacetylnimbin with antibiotics recorded a significant synergistic effect against drug resistant S. aureus (MRSA) as well, whereas the combination of amide derivatives of nimbolide with antibiotics did not record any synergistic interaction.
3.5. Time–kill study
The antibacterial effects of nimbolide, desacetylnimbin and the amide derivatives of nimbolide with antibiotics against test bacteria were confirmed by time–kill curve experiments. Time–kill assays for the synergistic combinations on test bacterial strains are shown in Fig. 4. The time–kill assay was conducted to determine the rates of killing of test bacteria when exposed to test compounds with cefalexin or cefazolin (Fig. 4).
 |
| Fig. 4 Time–kill curves of the compounds and antibiotics alone and in combination against bacterial species. control, 1, 2, 1a, 1b, 1c, corresponding antibiotics, 1a plus corresponding antibiotics, 2 plus corresponding antibiotics, 1a plus corresponding antibiotics, 1b plus corresponding antibiotics, and 1c plus corresponding antibiotics. Data are expressed as mean ± standard deviation. The x axis represents time, and the y axis represents logarithmic bacterial survival. | |
When the test compounds with cefalexin or cefazolin were tested against S. aureus (Fig. 4), maximum reduction in the colony count was observed between 6 and 12 h when compared to the control. At 24 to 48 h, an almost complete bactericidal effect was observed with treatments with a combination of nimbolide and cefazolin.
In the case of S. epidermis, maximum reduction was observed between 2 and 6 h (Fig. 4). A significant effect was also noted for the combination of nimbolide and cefazolin. A similar pattern of results was observed for K. pneumoniae (Fig. 4). Regrowth was observed for the compounds and antibiotics alone after 24 h, whereas it was not observed for the combinations even at 48 h (Fig. 4). In the case of P. aeruginosa, maximum reduction was observed between 4 and 6 h. For this organism, complete reduction in the colony count was observed after 24 h. In the case of MR-S. aureus, maximum reduction was observed between 12 and 24 h.
Very interestingly, amide derivatives of nimbolide in combination with antibiotics recorded less activity when compared with nimbolide and desacetylnimbin. Moreover regrowth was observed for the amide derivatives of nimbolide in combination with antibiotics after 24 h.
3.6. Cytotoxicity test
The cytotoxic activity of the compounds was tested against the H9c2 (rat embryonic cardiomyoblasts) cell line by MTT assay. Fig. 5 shows the cytotoxicities of the compounds expressed in bar graphs (control, 10, 25, 50, 100 and 200 μM) and corresponding confocal images of H9c2 cells (control, 100 and 200 μM). The results show that there is no significant cytotoxicity up to 200 μM except in the case of compound 1c (Fig. 5). Compound 1c recorded slight cytotoxicity from 50 μM onwards and at 200 μM concentration approximately 16% of cells were dead, which indicated that this compound may have slight toxicity towards normal cells. Cells were found to be viable in the presence of all other tested compounds. This results of the present study clearly indicate that the compounds may be safe for normal human cells.
 |
| Fig. 5 Cytotoxicities of the compounds against the H9c2 cell line measured by MTT assay. Where A = 1, B = 2, C = 1a, D = 1b, E = 1c. | |
4. Discussion
Medicinal plants are an important source of bioactive compounds and constitute the most common human use of biodiversity.27 They have been used for thousands of years, with more than 75% of the world’s population still depending on traditional medicines to satisfy their healthcare needs.28 The use of medicinal plants, being in existence even before the introduction of antibiotics and other modern drugs,29 led scientists to anticipate that phytochemicals with significant antibacterial properties might be useful for treating various bacterial infections.30 Consequently, many ethnopharmacological studies are being conducted to determine the safety and efficiency, and for the discovery of new bioactive principles associated with plants in recent times.31 Many medicinal plants and plant products with antimicrobial activities have become significant sources of potent and powerful drugs,32,33 and are the main source of new leads for antimicrobial pharmaceutical development.34 In the present study, we have carried out a detailed investigation into the antibacterial activities and synergistic effects of nimbolide, desacetylnimbin and some amide derivatives of nimbolide against major wound-associated bacterial pathogens.
The antibacterial activity of A. indica leaf extract against wound-associated pathogens has been reported previously,35,36 but the antimicrobial activity of isolated pure compounds from A. indica leaves against wound pathogens has not been studied. One of the major compounds isolated from A. indica leaves is the limonoid nimbolide, first isolated from fresh leaves by Ekong et al.37 and for which an optimized isolation procedure from dry leaves is also reported by Nair et al.38 Nimbolide has been found to have a very good antiproliferative effect on human cancer cell lines and is emerging as a promising drug candidate for the treatment of cancer.39–42 In 2006 Sastry et al.43 reported the synthesis of amide derivatives of nimbolide and studied their in vitro anticancer activities. It has been found that some of the amide derivatives possessed better activity than nimbolide. Even though nimbolide and its amide derivatives were explored for their anticancer activity, they have not been studied in detail for their antimicrobial activity. Only few reports are available on the antimicrobial activity of nimbolide as well. Previous studies have shown that nimbolide possesses antimalarial activity44 and shows antibacterial activity against Staphylococcus aureus and S. coagulase.45 In the present study the natural limonoids nimbolide and desacetylnimbin recorded significant activity against wound pathogens. Synthetic derivatives recorded less activity when compared to natural ones. The antibacterial activities of nimbolide, desacetylnimbin and the amide derivatives of nimbolide against major wound-associated bacterial pathogens are reported here for the first time.
Chronic wounds are an increasingly urgent health problem and bacterial infection plays a significant role in the inability of these wounds to heal.7 Treatment of such infections often involves combinations of antibiotics in an effort to increase efficacy and stem antibiotic resistance. To date, no studies have evaluated the use of neem limonoids in combination with antibiotics. Therefore it is not known if these compounds might have synergistic or antagonistic effects when co-administered with antibiotics.
Synergy between natural compounds and antimicrobial agents is a thrust area of phytomedicinal research, and has created a new way of looking at phytopharmaceuticals. The synergy between plant-derived compounds and antimicrobial drugs has been evaluated previously against many human pathogenic microorganisms. This approach is not exclusive for extract combinations, since effective combinations of single natural products, essential oils or extracts with chemosynthetics or antibiotics have been described in the literature.46,47 Even then, there are only limited reports available on the synergistic effects of individual phytochemicals with antibiotics (examples include those of lupulone, xanthohumol,48 eugenol,49 baicalein50etc.). In the current study we have investigated the antimicrobial and synergistic activity of nimbolide as well as desacetylnimbin, isolated from Azadirachta indica and the amide derivatives of nimbolide, along with first-generation cephalosporin antibiotics (cefalexin and cefazolin) against bacteria associated with wound infection. In addition to achieving these synergistic effects, the combination of two or more compounds is essential for the following reasons: (1) to prevent or suppress the emergence of resistant strains, (2) to decrease dose-related toxicity, and (3) to attain a broad spectrum of activity.46 Moreover, the process for developing a new drug is very expensive and is not possible in most developing countries, and the use of a new drug also has risks of unknown side-effects. Here in the current study, the neem limonoids plus two first-generation cephalosporin antibiotics recorded significant synergistic effects. Therefore, using well-known drugs in combination with herbal substances is quite an elegant alternative for combating infectious diseases.
In this study, the growth inhibitory effects of nimbolide, desacetylnimbin isolated from Azadirachta indica, and the amide derivatives of nimbolide on wound bacterial strains were observed for their combinations with first-generation cephalosporin antibiotics. In the present study the natural limonoids recorded predominantly synergistic interactions when combined with first-generation cephalosporin antibiotics. Herein, in combination, the MIC values of antibiotics have been reduced four- to five-fold, especially when combined with nimbolide and desacetylnimbin. The above combinations have also been found to be effective against the important drug-resistant bacteria MR-S. aureus. The combination of neem limonoid and first-generation cephalosporin antibiotics may help to reduce the amount of antimicrobial agent used and deliver a medicine with similar or greater potency than the antimicrobial alone. More importantly, these phytochemicals are structurally different from antimicrobial drugs and often have different mechanisms of action, which may provide new means of studying the mechanisms of bacterial control at a molecular level. With the increase in the prevalence of multidrug-resistant wound pathogens, synergy testing using various combinations of plant compounds and their corresponding synthetic derivatives with antimicrobial drugs could be a powerful tool in helping to select appropriate antimicrobial therapy to control pathogens.51–53
The indiscriminate use of various antibiotics in the treatment of bacterial infections, especially against wound pathogens, has led to the emergence and spread of resistant bacterial strains, and this in turn has resulted in a great loss of clinical efficacy of previously effective first-generation antibiotics, resulting in shifting of the antimicrobial treatment regimen to second-generation or third-generation antibiotics that are often more expensive, with many unwanted side-effects to human beings.54 In fact, studies have shown that crude extracts of plants possess the ability to enhance the activity of antimicrobial agents without causing any unwanted side-effects.55,56 In the present study the neem compounds also recorded no toxicity towards H9c2 cell lines.
5. Conclusion
Resistance to antibiotics is a ubiquitous and relentless clinical problem compounded by a dearth of novel therapeutic agents. The retreat of the pharmaceutical industries from research and development of new antibiotics has exacerbated the challenge of widespread resistance and signals a critical need for innovation. Although antimicrobial combinations are commonly used in medicine to broaden the antimicrobial spectrum and generate synergy, this should be promoted and encouraged as a strategy for reducing the emergence of antibiotic-resistant strains. In the present study the antibacterial effect of nimbolide, desacetylnimbin from Azadirachta indica, and the amide derivatives of nimbolide, and their synergistic effect when in combination with antimicrobial agents were recorded. Nimbolide showed the highest synergistic effect with the tested antibiotics, followed by desacetylnimbin. Amide derivatives were found to be less potent when compared to the two natural limonoids. Interestingly, natural limonoids combined with antibiotics recorded significant synergy against MR-S. aureus, an important drug-resistant bacteria. These results may be applied in the near future in alternative therapies for diseases caused by wound-associated pathogenic strains. In addition, more studies, including structural molecular modification together with toxicological studies, should be conducted on these compounds before their therapeutic application in humans.
Acknowledgements
SRD thanks the CSIR for the research fellowship. SNK thanks KSCSTE for the post-doctoral fellowship. The authors thank the CSIR 12th 5-year plan project “NaPAHA” for financial support. Ms Saumini Mathew and Ms S. Viji of CSIR-NIIST are acknowledged for recording the NMR and mass spectra.
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Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra16071e |
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