Evaluation of the anticandidal activity of clotrimazole using Lactobacillus caseie ghosts as biological drug carrier.

BACKGROUND: Candida albicans cause oral and vaginal mucosa infections as well as bloodstream and deep-tissue infections. Commonly, clotrimazole as a broad-spectrum antimitotic drug applied for treatment of Candida albicans infections. Bacterial ghosts are dead cells that have the broad potential to target the various body tissues and cells as drug vector. OBJECTIVES: We hope to conquest this resistance by using clotrimazole loaded on bacterial ghosts. METHODS: Lactobacillus ghosts were produced by using tween 80 and lactic acid according to the protocol and the amount of the DNA and protein in supernatant was measured by Nano-drop spectrophotometry. Ghost's morphological characteristics were detected by using light microscopy, SEM and AFM. Antifungal activities of the synthesized ghosts were measured by plate methods. Three independent vertical Franz cells were used to evaluate drug release profile. BG-clotrimazole was added into cream base and was examined for dispensability as well as uniformity of the formulation on the skin. RESULTS: Results of the Nano-drop analysis showed that protein and DNA was seen in supernatant of treatment compared to control groups. AFM results showed well-dispersed and rod-shaped L. casei ghost cells. Lysis pores formation in the L. casei ghosts was indicated by SEM micrographs. BGs represent an excellent drug delivery system because of the high loading capability. Nearly, 50% of clotrimazole was released from BGs during 90 min. Highest anticandidal activity occurred using 100 mg/l clotrimazole-BG, while toxic effects were also seen with 10 mg/l clotrimazole. IC50 clotrimazole-BG was found at 0.001 mg/l. Chemical stability results showed that about 90% of clotrimazole remained in the formulation. CONCLUSION: It could be concluded that the bacterial ghosts are very talented to high loading capability, keeping and releasing drug during six months, therefore these could act as an excellent drug delivery system.

Ferulago angulata as a Good Radioprotector Against Genotoxicity.

INTRODUCTION: Natural products can be used as radioprotector agents because of containing phenolic compounds and several flavonoids with antioxidant properties. When the normal cells are exposed to ionizing radiation, they generate free radicals and reactive oxygen species that can cause damage in DNA, which leads to cellular dysfunction or even cell death. However, it is necessary to identify new radioprotective agents to protect normal cells. Ferulago angulata (F. angulata), a medicinal plant, can be used as a new radioprotective agent. PURPOSE: In this study, the radioprotective effect of F. angulata was evaluated against genotoxicity and oxidative stress induced by ionizing radiation in human blood lymphocytes. METHODS: The antioxidant activity of F. angulata was assayed using FRAP and DPPH methods. Then, the human blood samples were incubated with F. angulata at different concentrations (25, 50, 100, and 200 µM) and subsequently exposed to IR at a dose of 2Gy. The radioprotective effect of F. angulata on the exposed cells was assessed by the micronucleus (MN) method. Also, biomarkers of oxidative stress in the exposed cells were evaluated by malondialdehyde (MDA) and superoxide dismutase (SOD) methods. RESULTS: Our findings showed that F. angulata reduced the frequency of MN induced by IR in exposed cells. At a 200 µM concentration of F. angulata, the maximum reduction in the frequency of MN (63.11%) was observed that demonstrated a high degree of radioprotection. Afterward, pretreatment at 200 µM concentration of F. angulata inhibited oxidative stress in irradiated lymphocytes, leading to a reduction in MN frequency and MDA levels while SOD activity was enhanced in the exposed cells. CONCLUSION: F. angulata as a natural radioprotective agent can protect normal cells against reactive oxygen species and genetic damage induced by IR.

High-Level Resistance to Erythromycin and Tetracycline and Dissemination of Resistance Determinants among Clinical Enterococci in Iran.

OBJECTIVES: The purpose of this study was to investigate the distribution pattern of genes responsible for erythromycin and tetracycline resistance and their association with resistance phenotypes in enterococcus isolates. MATERIALS AND METHODS: Eighty-six Enterococcus faecalis and 26 E. faecium isolates were collected from 2 hospitals in Kerman, Iran. Minimum inhibitory concentration of erythromycin and tetra-cycline was determined and then genes encoding resistance to erythromycin - erm (A-C), mef, and msr - and tetracycline - tet (M), tet (O), tet (S), tet (K), and tet (L) - were investigated. RESULTS: In all resistant isolates (n = 72, 64%), high-level resistance to both tested antibiotics was found. The most prevalent erm gene was erm (B) (77.7%), followed by erm (A) (15.2%) and erm (C) (8.3%). Genes mediating erythromycin efflux were detected in 70.8% (mef) and 9.7% (msr) of resistant isolates. Regarding tetracycline, tet (M) was detected at the highest rate (50%), followed by tet (O) (31%) and tet (S) (11%). Export of tetracycline was found in 31% (tet (K)) and 12% (tet (L)) of isolates. CONCLUSION: A high prevalence of high-level resistance to both erythromycin and tetracycline was documented. Alterations at the ribosomal level was more frequently detected in erythromycin and tetracycline resistance than efflux systems. Concurrent resistance mechanisms were more involved in resistance to erythromycin than tetracycline.

The art of suicidal molecular seduction for targeting drug resistance.

The development of drug resistance is one of the most significant challenges of the current century in the pharmaceutical industry. Superinfections, cancer chemoresistance, and resistance observed in many non-infectious diseases are nullifying the efforts and monetary supplies, put in the advent of new drug molecules. Millions of people die because of this drug resistance developed gradually through extensive use of the drugs. Inherently, some drugs are less prone to become ineffective by drug resistance than others. Covalent inhibitors bind to their targets via a biologically permanent bound with their cognate receptor and therefore display more potent inhibiting characteristics. Suicide inhibitors or mechanism-based inhibitors are one of the covalent inhibitors, which require a pre-activation step by their targeting enzyme. This step accrues their selectivity and specificity with respect to other covalent inhibitors. After that pre-activation step, they produce an analogue of the transition state of the catalytic enzyme, which is practically incapable of dissociating from the enzyme. Suicide inhibitors, due to their high intrinsic affinity toward the related enzyme, are resistant to many mechanisms involved in the development of drug resistance and can be regarded as one of the enemies of this scientific hurdle. These inhibitors compete even with monoclonal antibodies in terms of their cost-effectiveness and efficacy.

Novel 5-(nitrothiophene-2-yl)-1,3,4-Thiadiazole Derivatives: Synthesis and Antileishmanial Activity against promastigote stage of Leishmania major.

In this study, a series of novel compounds based on 5-(5-nitrothiophene-2-yl)-1,3,4-thiadiazole possessing (het) aryl thio pendant at C-2 position of thiadiazole ring is developed and evaluated as antileishmanial agents using MTT colorimetric assay. 10 New compounds containing aryl and heteroaryl derivatives, started from thiophene-2-carbaldehyde in five steps, were synthesized in good to excellent yields and characterized by 1H-NMR, 13C-NMR, and IR spectroscopy. Through the compounds 6a-j, methylimidazole containing derivative 6e was recognized as the most active compound against L. major promastigotes exhibiting IC50 values of 11.2µg/mL and 7.1µg/mL after 24 and 48 h, respectively. This compound is > 4 fold more effective than Glucantime as a standard drug (IC50 = 50 µg/mL after 24 h and 25 µg/mL after 48 h).

N-substituted piperazinyl sarafloxacin derivatives: synthesis and in vitro antibacterial evaluation.

BACKGROUND: Fluoroquinolones (FQs) are compounds of major interest with broad antimicrobial activities against community and hospital-acquired infections such as respiratory tract infections (nosocomial pneumonia, chronic bronchitis and tuberculosis), skin and soft tissue infections, bone and joint infections, intra-abdominal infections and sexually transmitted diseases. This broad range of activities along with favorable pharmacokinetic and low toxicity introduced this class of compounds as important antimicrobial chemotherapy agents. The rapid increase in prevalence of FQs resistant microbes in environment motivated medicinal chemists to discover new quinolone-based compounds with potent activities against Gram-positive bacteria. METHODS: The designed compounds were prepared through the two-component reaction between aromatic α-haloketones or α-halooximes and sarafloxacin in the presence of NaHCO3 in DMF, affording the corresponding N-[2-(aryl-3-yl) ethyl] piperazinyl quinolone derivatives in good yields. All synthesized compounds were evaluated for antibacterial activities against Gram-positive [Staphylococcus aureus ATCC 6538p, Micrococcus luteus, ATCC 1110, Staphylococcus epidermidis ATCC 12228 and Bacillus subtilis ATCC 6633] and Gram-negative [Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 10031 Pseudomonas aeruginosa ATCC 9027 and Serratia marcescens PTCC 1111] bacteria. RESULTS: The antibacterial activities of 24 new compounds were reported as MIC values in comparison to sarafloxacin. The most active compound, 4 g, exhibited similar inhibitory activity against Gram-positive bacteria including S. aureus, S. epidermidis and B. subtilis compared to positive control. Furthermore, benzyloxime incorporated derivatives (4 s-4x) showed poor activity against all tested strains, except 4x. CONCLUSION: The obtained results indicated that the synthesized compounds containing substituted piperazine moiety at the C-7 position displayed same or weak inhibitory activities compared to sarafloxacin. Graphical abstract ᅟ.

The Effects of Chlorhexidine and Persica Mouthwashes on Colonization of Streptococcus mutans on Fixed Orthodontics O-rings.

STATEMENT OF THE PROBLEM: Fixed orthodontic appliances predispose patients to dental caries. Use of mouthrinses has been introduced as the effective way for reducing dental plaque accumulation. PURPOSE: The aim of this study was to compare the effects of Persica mouthwash and Chlorhexidine (CHX) on colonization of Streptococcus mutans (S. mutans) on fixed orthodontic O-rings. MATERIALS AND METHOD: Thirty patients with fixed orthodontic appliances and proper oral hygiene were randomly provided by CHX and Persica and trained to use these mouthwashes according to the manufacturer's instruction. Sampling was carried out right before and 4 weeks after mouthrinsing treatment. The mean amounts of S. mutans colonies in these groups were compared. RESULTS: Comparison of S. mutans colonization within each group revealed both mouthrinses to be efficient. However, this difference was found to be significant only in CHX group. CONCLUSION: Persica cannot be a good alternative mouthwash and patients on orthodontic treatment are still recommended to use CHX.

Synthesis and cytotoxic activity of novel poly-substituted imidazo[2,1-c][1,2,4]triazin-6-amines.

A novel series of 3,4-diphenyl-7-(hetero)arylimidazo[2,1-c][1,2,4]triazin-6-amine derivatives were synthesized via three-component reaction of 5,6-diphenyl-1,2,4-triazin-3-amine, various aromatic aldehydes, and cyclohexyl isocyanide. All synthesized compounds were tested against HL60 (human promyelocytic leukemia), MOLT-4 (human T lymphoblastic leukemia), and MCF-7 (human breast adenocarcinoma) cell lines, as cytotoxic agents. The structure-activity relationships study revealed that the introduction of hydroxyl and methoxy groups on the 7-phenyl ring can modulate the cytotoxic activity of these compounds. Among the 7-aryl derivatives, 3-hydroxyphenyl and 3-hydroxy-4-methoxyphenyl derivatives (6h and 6o) were the most potent compounds against HL60 and MCF-7 cells (IC(50s) = 9.8 - 20.4 µM). However, the replacement of the 7-aryl moiety with pyridyl or furan-2-yl resulted in compounds 6p or 6r with more promising cytotoxicity against MOLT-4 cell line (IC50 values 12.1 and 13.0 µM, respectively). Also, the acridine orange/ethidium bromide staining assay in MCF-7 cells suggested that the cytotoxic activity of compound 6r occurs via apoptosis.

Determination of antimicrobial resistance profile and inducible clindamycin resistance of coagulase negative staphylococci in pediatric patients: the first report from Iran.

BACKGROUND: Currently, coagulase negative staphylococci (CoNS) have got much attention as a serious health problem especially in neonates and children. High incidence of antibiotic resistance, in particular methicillin resistance, has complicated the treatment of these organisms. The aim of this study is to determine the susceptibility to different antimicrobial agents and the prevalence of macrolideslincosamides-streptogramins B (MLSB) resistance in CoNS isolates obtained from pediatric patients. METHODS: Totally 157 CoNS isolates from various clinical samples were examined for antibiotic resistance using disk diffusion and E-test methods. Double-disk test was applied to detect constitutive and inducible MLSB resistance (cMLSB and iMLSB) phenotypes. RESULTS: Resistance to methicillin was seen in 98 (62.4%) isolates. All isolates were susceptible to vancomycin and linezolid. The prevalence of resistance to antibiotics tested was as follows: fusidic acid (n=58, 36.9%), gentamicin (n=73, 46.5%), ciprofloxacin (n=81, 51.6%), clindamycin (n=112, 71.3%), erythromycin (n=129, 82.2%) and trimethoprim/sulfamethoxazole (n=133, 84.7%). iMLSB phenotype was seen in 14 (8.9%) isolates, and 18 (11.5%) and 98 (62.4%) isolates showed MS and cMLSB phenotypes, respectively. We observed that high overall antibiotic resistance rates were associated significantly with methicillin resistance. Conversely, iMLSB phenotype was correlated neither with methicillin resistance nor with invasiveness. CONCLUSION: Given the similarity observed between the prevalence of iMLSB and MS phenotypes, the performance of disk diffusion induction test is strongly recommended in our region.

Design, synthesis, docking study and cytotoxic activity evaluation of some novel letrozole analogs.

BACKGROUND: Breast cancer is the most common type of female cancer. One class of hormonal therapy for breast cancer drugs -non steroidal aromatase inhibitors- are triazole analogues. In this work, some derivatives of these drugs was designed and synthesized. All synthesized compounds were evaluated for their cytotoxic activities on breast cancer cell lines (MDA-MB-231, T47D and MCF-7). METHODS: Our synthetic route for designed compounds started from 4-bromotolunitrile which was reacted with 1H-1,2,4-triazole to afford 4-(4-cyanobenzyl)-1,2,4-triazole. The reaction of later compound with aromatic aldehydes led to formation of the designed compounds. Eleven novel derivatives 1a-k were tested for their cytotoxic activities on three human breast cancer cell lines. RESULTS: Among the synthesized compound, 4-[2-(3-chlorophenyl)-1-(1H-1,2,4-triazol-1-yl)ethenyl]benzonitrile (1c) showed the highest activity against MCF-7 and MDA-MB-231 cell lines and 4-[2-(4-methoxyphenyl)-1-(1H-1,2,4-triazol-1-yl)ethenyl]benzonitrile (1 h) exhibited highest activity against T47D cell line. According to cytotoxic activities results, compound 4-[2-(4-dimethylamino)-1-(1H-1,2,4-triazol-1-yl)ethenyl]benzonitrile (1 k) showed comparative activity against T47D and MDA-MB-231 cell lines with compound (1 h) and our reference drug Etoposide. CONCLUSION: In the process of anti-cancer drug discovery, to find new potential anti-breast cancer agents, we designed and synthesized a novel series of letrozole analogs. Cytotoxicity evaluation revealed that compounds (1c) and (1 k) were the most potent compounds with comparative activity with Etoposide. The results revealed that π-π interactions are responsible for the enzyme inhibitions of compounds (1 c) and (1 k).

The prevalence of aminoglycoside-modifying enzymes among coagulase negative staphylococci in Iranian pediatric patients.

In spite of widespread emergence of aminoglycoside resistance, these drugs are still used in the treatment of staphylococcal infections. This study aimed to investigate the distribution of aminoglycoside resistance and genes encoding aminoglycoside - modifying enzymes (AMEs) as well as Staphylococcal Cassette Chromosome mec (SCCmec) type in coagulase negative staphylococci (CoNS) in pediatric patients. Totally, 93 CoNS isolates were examined for susceptibility to aminoglycosides using disk diffusion and/or E-test methods. AMEs genes and SCCmec types were detected using multiplex PCR. Strain typing was performed using repetitive extragenic palindromic (REP) - PCR assay. The non-susceptibility rates to kanamycin, tobramycin, gentamicin, amikacin and netilmicin were 73%, 59%, 49.5%, 16% and 7.5%, respectively. aac(6')-Ie-aph(2″)-Ia, ant(4')-Ia and aph(3')-IIIa were encountered in 56 (60.2%), 38 (40.8%) and 18 (19.3%) isolates, respectively. In aac(6')-Ie-aph(2″)-Ia- positive isolates, the non- susceptibility rates to kanamycin, gentamicin, tobramycin, amikacin and netilmicin were 83%, 74%, 73%, 49% and 43%, respectively. SCCmec types included type IV (n = 31), I (n = 17), II (n = 5), III (n = 4), and V (n = 2). Three isolates had two types; I + III (n = 2) and III + IV (n = 1) whereas 11 isolates were non-typeable. AMEs genes carriers were distributed frequently into type IV. We found diverse fingerprint patterns among our isolates. In conclusion, there was a strong correlation between alarming rate of aminoglycoside resistance and methicillin resistance. Discordances between phenotypic and genotypic detection of aminoglycoside resistance were discernible. AMEs genes might be related to SCCmec types.

5-Nitroimidazole-based 1,3,4-thiadiazoles: heterocyclic analogs of metronidazole as anti-Helicobacter pylori agents.

A series of 5-nitroimidazole-based 1,3,4-thiadiazoles were prepared and tested for antibacterial activity against Helicobacter pylori. The anti-H. pylori activity of target compounds along with the commercially available antimicrobial metronidazole was evaluated by comparing the inhibition-zone diameters determined by the paper disc diffusion bioassay. From our bioassay results against 20 clinical isolates it is evident that piperazinyl, 4-methylpiperazinyl, 3-methylpiperazinyl, and 3,5-dimethylpiperazinyl analogs (6a, 6b, 6e, and 6f, respectively) and pyrrolidine derivative 7 had strong activity at 0.5 µg/disc (average of inhibition zone > 20 mm) while metronidazole had no activity at this dose. Compound 6f containing the 3,5-dimethylpiperazinyl moiety at the 2-position of the 5-(1-methyl-5-nitro-1H-imidazol-2-yl)-1,3,4-thiadiazole skeleton was the most potent compound tested at low concentrations.

2-Substituted-5-nitroheterocycles: in vitro anti-Helicobacter pylori activity and structure-activity relationship study.

Helicobacter pylori infection is the main cause of gastritis and gastroduodenal ulcer disease, and is associated with gastric cancer. In order to develop new potential anti-Helicobacter pylori candidates, we have investigated the antimicrobial activity of some 2-substituted-5-nitroheterocycles against H. pylori. The anti-Helicobacter pylori activity of selected compounds along with commercially available antibacterial metronidazole was evaluated by comparing the inhibition zone diameters determined using the paper disc diffusion bioassay. The compounds that exhibited strong anti-H. pylori activity at concentration of 8-32 microg/disc (average of inhibition zone >20 mm) were further tested against 20 clinical isolates of H. pylori at lower concentrations. In general, we have identified a series of 5-nitroheterocyles including nitrofurans, nitrothiophenes and nitroimidazoles bearing a carboxaldehyde thiosemicarbazone or 2-substituted-1,3,4-thiadiazole residues in the 2-position of the 5-nitroheteroaryl ring as potent anti- Helicobacter pylori agents. It was found that chloro-/ amino-/ mercapto-substituted 1,3,4-thiadiazole moiety attached to 5-nitroheteroaryl ring served as promising C-2 substituents for 2-substituted-5-nitroheterocycles. The Structure-activity relationship of this series indicates that both the structure of the nitroaryl scaffold and the C-2 attached residue have dramatic impact on anti-Helicobacter pylori activity.

Bioassay screening of the essential oil and various extracts from 4 spices medicinal plants.

Four commonly used spices plants in Iran were evaluated for cytotoxicity effect using Brine Shrimp Lethality (BSL) assay. Essential oils and various extracts of Heracleum persicum, Nigella arvensis, Cinnamomum zeylanicum and Zingiber officinale were assessed by two methods of disk and solution of BSL. Data were processed in probit-analysis program to estimate LC50 values. All of the tested fractions have exhibited more cytotoxicity in the solution method. Essential oils of H. persicum and C. zeylanicum have shown the most cytotoxicity with LC50 values 0.007 and 0.03 microg/ml respectively. None of aqueous extracts showed significant cytotoxicity. The analysis of the essential oil of H. persicum showed the hexyl butyrate and octyl acetate as the main compounds. These results suggest some limitation for using of these spices in diet. Furthermore, these plants could be considered as a source of cytotoxic compounds which might be studied in more details.

Synthesis and antibacterial activity of nitroaryl thiadiazole-gatifloxacin hybrids.

A number of gatifloxacin analogues containing a nitroaryl-1,3,4-thiadiazole moiety attached to the piperazine ring at C-7 position were prepared and evaluated as antibacterial agents against a panel of gram-positive and gram-negative bacteria. Among synthesized compounds, nitrofuran analog 6a exhibited more potent inhibitory activity against gram-positive bacteria including Staphylococcus epidermidis (MIC=0.0078 microg/mL), Bacillus subtilis (MIC=0.0039 microg/mL), Enterococcus faecalis (MIC=0.125 microg/mL) and Micrococcus luteus (MIC=0.125 microg/mL), with respect to other synthesized compounds and reference drug gatifloxacin. The target compounds were also assessed for their cytotoxic activity against normal mouse fibroblast (NIH/3T3) cells using MTT assay. The results indicated that these compounds exhibit antibacterial activity at non-cytotoxic concentrations.

Synthesis and in-vitro antibacterial activity of 5-substituted 1-methyl-4-nitro-1H-imidazoles.

A series of 5-substituted 1-methyl-4-nitro-1H-imidazole derivatives were synthesized and evaluated for in-vitro antibacterial activity against a panel of microorganisms including Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Enterobacter aerogenes, and Helicobacter pylori using conventional agar dilution method. Among the test compounds, 1-methyl-4-nitro-5-(phenylsulfonyl)-1H-imidazole was the most potent against Gram-positive bacteria, with a MIC value of < or =8 microg/mL. All compounds showed no significant activity against Gram-negative bacteria at concentrations < or =64 microg/mL. The MIC values against 15 clinical isolates of H. pylori indicated that compounds 10 and 11 were the most active compounds in this series in terms of inhibiting the growth of H. pylori (MIC = 2 microg/mL). It was also demonstrated that their corresponding activities were four times larger than that of metronidazole.

Synthesis and antibacterial activity of N-[2-[5-(methylthio)thiophen-2-yl]-2-oxoethyl] and N-[2-[5-(methylthio)thiophen-2-yl]-2-(oxyimino)ethyl]piperazinylquinolone derivatives.

A number of N-substituted piperazinylquinolone derivatives were synthesized and evaluated for antibacterial activity against Gram-positive and Gram-negative bacteria. Preliminary results indicated that most compounds tested in this study demonstrated comparable or better activity against Staphylococcus aureus and Staphylococcus epidermidis than their parent piperazinylquinolones as reference drugs. Among these derivatives, ciprofloxacin derivative 5a, containing N-[2-[5-(methylthio)thiophen-2-yl]-2-oxoethyl] residue, showed significant improvement of potency against staphylococci, maintaining Gram-negative coverage.

Synthesis and antibacterial activity of N-(5-benzylthio-1,3,4-thiadiazol-2-yl) and N-(5-benzylsulfonyl-1,3,4-thiadiazol-2-yl)piperazinyl quinolone derivatives.

A series of N-(5-benzylthio-1,3,4-thiadiazol-2-yl) and N-(5-benzylsulfonyl-1,3,4-thiadiazol-2-yl) derivatives of piperazinyl quinolones was synthesized and evaluated for antibacterial activity against Gram-positive and Gram-negative microorganisms. Some of these derivatives exhibit high activity against Gram-positive bacteria; Staphylococcus aureus and Staphylococcus epidermidis, comparable or more potent than their parent N-piperazinyl quinolones norfloxacin and ciprofloxacin as reference drugs. The SAR of this series indicates that both the structure of the benzyl unit and the S or SO(2) linker dramatically impact antibacterial activity.

Synthesis and antibacterial activity of N-[2-(5-bromothiophen-2-yl)-2-oxoethyl] and N-[(2-5-bromothiophen-2-yl)-2-oximinoethyl] derivatives of piperazinyl quinolones.

A series of N-[2-(5-bromothiophen-2-yl)-2-oxoethyl] and N-[2-(5-bromothiophen-2-yl)-2-oximinoethyl]derivatives of piperazinyl quinolones were synthesized and evaluated for antimicrobial activity against Gram-positive and Gram-negative microorganisms. Some of these derivatives exhibit comparable or better activity against Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis, than ciprofloxacin, norfloxacin and enoxacine as reference drugs.