Thin-layer Chromatographic Technique Research Articles

Invasive diatom Didymosphenia geminata as a source of polysaccharides with antioxidant and immunomodulatory effects on macrophage cell lines

This study addresses for the first time the chemistry and biological activity of polysaccharides isolated from Didymosphenia geminata. Fourier-transform infrared spectroscopy (FTIR) and high-performance thin-layer chromatography (HPTLC) techniques were used to characterize the crude and acidic polysaccharides of D. geminata; galactose and xylose were found to be the main monomer components. The antioxidant activity as well as the effect on the expression of IL-6 and TNF-α in RAW 264.7 murine cell lines were determined for both types of polysaccharides. The antioxidant activity of crude polysaccharides proved to be stronger than acidic polysaccharides by means of quenching experiments of ABTS·+ and Trolox equivalent antioxidant capacity (TEAC) (p < 0.05). Crude polysaccharides induced an increase in both IL-6 and TNF-α contents. Acidic polysaccharides produced a similar effect on these cytokines, but to a lesser extent. These results suggest that polysaccharides from D. geminata could be explored as natural antioxidant and immunomodulatory agents in medicine and the nutraceutical industry. This investigation suggests an opportunity to turn the invasive mats of D. geminata, which currently cause environmental problems, into a source of polysaccharides with nutraceutical application.

Analysis of tetrahydrocannabinol derivative from cannabis‐infused chocolate by QuEChERS‐thin layer chromatography‐desorption electrospray ionization mass spectrometry

Recently in Canada and some states of the United States, marijuana (cannabis) has become fully legalized and regulated, for both medical and recreational purposes. This fact is going to make cannabis products such as edibles even more popular than ever before. Therefore, it is assumed that there will be a high demand for analytical methods, which are accurate and sensitive enough to be used in different forensic and pharmaceutical cannabis-related applications. Cannabis derivatives have an extreme range and number of constituents with possible interactions with one another. Thus, this characteristic leads to their vast and highly complex chemistry, which requires robust analytical tools to be able to precisely and accurately quantify and qualify them. We developed and validated an analytical method using desorption electrospray ionization (DESI)-mass spectrometry (MS) to accurately detect, characterize, and quantify cannabinoids and also offer an easy, cost-effective, and reliable technique, which can be performed in a short time for infused edibles in complex matrices such as chocolate. We evaluated a quantitative analysis of tetrahydrocannabinol (THC) in cannabis-infused chocolate with thin-layer chromatography (TLC)-DESI-MS and QuEChERS extraction method. Both techniques of TLC and QuEChERS are cost-effective and can be run in short time.

Synthesis, Characterization and study biological activity of some Schiff bases derivatives from 4-amino antipyrine as a starting material.

This study includes formation Schiff bases compounds [A1- A3] were synthesis from raction4-aminoantipyrine with many aromatic aldehydes [4- chloro benzaldehyde, 4-bromo benzaldehyde and 2-hydroxy1-naphthaldehyde] in absolute ethanol. These compounds [A1-A3] has been characterized by melting points, [FT.IR] spectroscopy, thin layer chromatography technique (TLC) has been used for reactions steps and sequence by using solvent (ethyl acetate:toluene, 1:4). Antibacterial activity test was done according to agar well diffusion method against two types of multi-drug resistance of bacteria isolated from patients with infection of urinary tract. Three concentrations were prepared for each compound (50, 100 and 150) mg/ml. The results proved that derivative compound A3150 mg/ml provided the best antibacterial activity against K.pneumoniae with inhibition zone diameter 21± 0.25mm and against C.freundii 22± 0.5mm as compared with other derivatives compounds.

Simultaneous High-Performance Thin-Layer Chromatographic Quantification of Withaferin A and Withanolide A in Solanum nigrum L. “Black Nightshade”

A simple, selective, sensitive, and precise high-performance thin-layer chromatographic technique was followed to determine the diversity within accessions of Solanum nigrum. Fruit, stem, leaf, and root samples extracted in methanol were used to quantify withanolide A and withaferin A. Significant difference has been detected among the different plant parts. The maximum amount of withaferin A has been found to be present in the leaf sample (71.65 ± 2.86 mg g−1 DW), followed by the stem sample, while the least amount of withaferin A has been detected in the mature fruit sample (5.97 ± 1.91 mg g−1 DW).

Some Observations on Thin Layer Chromatography Technique

The present study is to report the various problems faced during TLC methodology. Although used regularly some technical aspects must be kept in mind to get better and uniform results. During our experiments with TLC methods we came across some problems and here these aspects of TLC methodology are being highlighted. It is suggested that the solvent use as mobile phase should also be used for extraction of any particular phytochemical. TLC plates should be 3 to 4 mm thick, to be dried for at least 72 hrs. It is also suggested that potassium permanganate solution gives better clarity in visualizing the spots.

Novel and Rapid Methods for Histamine Quantification in Wine and Culture Medium

Fermented beverages are frequently contaminated with histamine produced by lactic acid bacteria exhibiting histidine decarboxylase activity. In wine, the presence of histamine is associated with a lack of hygiene during the winemaking process. Determination of histamine is important because it can be toxic to humans. Therefore, we developed two rapid, inexpensive, and simple methods—a colorimetric technique and a thin-layer chromatography technique—to determine histamine concentration in wine and culture medium without the need for complicated extraction procedures. The new colorimetric method showed a good correlation between absorbance and histamine concentration in the range of 1.0 to 100 mg/L of histamine in wine and Moeller culture broth samples (r2 = 0.9981 and 0.9941, respectively). Thin-layer chromatography with a new visualization reagent also demonstrated a good correlation between histamine concentrations of 5.0 to 100 mg/L and absorbance (r2 = 0.9882 and 0.9839 for wine and Moeller culture broth samples, respectively). Both methods showed a high correlation with ELISA (colorimetric method: r2 = 0.9972; thin-layer chromatography: 0.9968) and exhibited good histamine recovery (higher than 87%). The detection limits of the new methods were comparable to those of existing analytical methods. Both methods seem suitable for routine analysis to monitor histamine in any research or quality-control laboratory.

Effect-directed screening of Bacillus lipopeptide extracts via hyphenated high-performance thin-layer chromatography

Bacillus species produce a wide array of biologically active metabolites, including nonribosomaly synthesized lipopeptides (LPs). The high-performance thin-layer chromatography (HPTLC) technique hyphenated with different bioassays and mass spectrometry was demonstrated as a valuable tool for effect-directed analysis of iturins, surfactins, fengycins and kurstakins homologues from complex mixtures of LPs. As proof of this straightforward strategy, the found surfactin and iturin A homologues were characterized and compared with reference substances. This study considered two different extraction methods for LPs produced by five Bacillus strains. The ethyl acetate extraction (Ex-1), and the acidic precipitation followed by methanol extraction (Ex-2) were investigated. Diverse enzyme inhibitions and antimicrobial potentials of LPs were analyzed, and in parallel, high-resolution mass spectra (HRMS) were online recorded from the HPTLC zones of interest. No antimicrobial effect against Gram-positive B. subtilis was evident for iturin, whereas a response was detected for surfactin. The nonpolar kurstakin compounds showed a pronounced B. subtilis antimicrobial activity in Ex-1 of almost all strains, whereas the fengycin homologues were detected in Ex-2 of SS-10.7 and SS-27.2. Iturin had also no activity against Gram-negative Aliivibrio fischeri, while again surfactin showed an enhancing luminescent activity. Contrary, kurstakin compounds caused a decrease in the luminescence in Ex-1 of all strains, particularly for SS-13.1. Both, iturin and surfactin showed a strong acetylcholinesterase (AChE) and α-glucosidase inhibition, but surfactin caused a much stronger inhibition. This was evident in all bacterial strains, except for SS-13.1 in Ex-1 and for SS-38.4 in Ex-2. Although, iturin and surfactin exhibited no DPPH˙ scavenging activity, Ex-1 of all strains contained more intense DPPH˙ scavenging compounds compared to Ex-2, and surfactin methyl esters showed a pronounced DPPH˙ activity, particularly in SS-12.6 in Ex-1. This study pointed to active metabolites of strains that can be used as therapeutics and biocontrol agents with beneficial effects on human health. The straightforward HPTLC profiling served as an excellent bioanalytical tool to control the formed bioactive metabolites. As the fermentation process is very sensitive to external influences, it could be a helpful control tool for standardization of the biotechnological processing.

Phytochemical Analysis and Antimicrobial Activity of Bersama abyssinica Fresen against Multidrug-Resistant Bacterial Uropathogens: Picolinyl Hydrazide Is a Major Compound

ABSTRACTThe phytochemical constituent and antimicrobial activities of Bersama abyssinica leaf extracts against multidrug-resistant bacterial uropathogens were evaluated. Seven organic solvents were used in the extraction of the leaves of B. abyssinica. Agar well diffusion assay, minimum inhibitory and minimum bactericidal concentration analysis, bioassay-guided fractionation, and spectral and elemental analysis were used to identify the principal phytochemical ingredients. Confirmation of detected compound was done by thin layer chromatography, elemental and spectroscopic (1H-NMR, 13C NMR, and IR) techniques. Ethyl acetate-based extract demonstrated the highest antimicrobial activity against the tested bacteria. The highest zone of inhibition (395.3 ± 26.7 mm2) was observed by the ethyl acetate extract against multidrug-resistant uropathogenic Staphylococcus aureus. Totally, 11 main compounds were identified from the active fraction of which picolinyl hydrazide was the principal compound. Antimicrobial activities of B. abyssinica varied with the extraction solvents, concentration of extracts, and types of test microorganisms. Gram-positive bacteria were more susceptible to the plant extracts, and ethyl acetate was the best extraction solvent.

FINGER PRINTING ANALYSIS OF THE PHENOLS FROM PERGULARIA DAEMIA FORSK PLANT LEAVES AND STEM USING HPTLC

The research study was carried out to identify the phenols from methanol extracts (MPD) of medicinally and efficiently useful leaves and stem of Pergularia daemia (Forsk) plant using High Performance Thin Layer Chromatography (HPTLC) technique. Preliminary phytochemical screening was done and HPTLC studies were carried out. Densitometry scanning was performed in the reflectance absorbance mode at 540 nm and operated by Win CATS software with the help of tungsten lamp. Preliminary phytochemical screening of methanol extract of P. daemia shows the presence of alkaloids, glycosides, flavonoids, phenols and carbohydrates. HPTLC finger printing of phenols of methanolic extract of leaf and stem revealed eleven polyvalent phytoconstituents (13 and 10 peaks) and corresponding ascending order of Rf values in the range from 0.08 to 0.65 in leaf and 0.02 to 0.66 in the stem. From the result of preliminary phytochemical analysis and above Rf values from HPTLC, we have concluded the presence of phenols in methanol extracts.

Antimicrobial and Antioxidant Properties of kaempferol-3-O-glucoside and 1-(4-Hydroxyphenyl)-3-phenylpropan-1-one Isolated from the Leaves of Annona muricata (Linn.)

Aim: To evaluate the antimicrobial and antioxidant activities of bioactive compounds isolated from Annona muricata (Linn.) leaf extract.&#x0D; Study Design: In vitro antimicrobial assay of bioactive compounds isolated from solvent fractions of plant leaf extract against selected clinical bacterial and fungal isolates. Antioxidant assay of plant leaf extract.&#x0D; Place and Duration of Study: All the work was carried out in the Departments of Chemistry and Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria between March, 2015 and January, 2016.&#x0D; Methodology: Isolation of bioactive compounds was by column and thin layer chromatographic techniques. Isolated compounds were characterized by nuclear magnetic resonance spectroscopic analysis. Antimicrobial activities were evaluated by disc diffusion and broth microdilution methods while antioxidant activity was investigated using the 2,2-dipheny-1-picrylhydrazyl (DPPH) radical-scavenging assay.&#x0D; Results: Two compounds kaempferol-3-O-glucoside (1) and 1-(4-Hydroxyphenyl)-3-Phenylpropan-1-one (2) were isolated from the ethyl acetate fraction of leaf extract of A. muricata. The two compounds showed broad spectrum antimicrobial activities with zones of inhibition ranging from 26.00 ± 1.73 to 31 ± 1.00 mm and 17.33 ± 1.15 to 31.33 ± 1.15 mm respectively, for compounds 1 and 2 for the test bacteria species and 15.33 ± 1.15 to 31.33 ± 1.15 mm and 17.67 ± 0.58 to 29.67 ±1.53 mm respectively, for compounds 1 and 2 for the test fungi. Minimum inhibitory concentrations ranged between 0.625-5.00 µg/mL and 1.25-5.00 µg/ml respectively, for compounds 1 and 2. Minimum bactericidal concentrations ranged between 2.5-10.00 µg/mL for both compounds which compared favourably with the reference drugs used. DPPH radical-scavenging activities were IC50 = 13.41 ± 0.64 µg/mL and 7.42 ± 0.90 µg/mL for compounds 1 and 2 respectively, compared with IC50 = 51.99 ± 1.44 µg/ml obtained for the standard ascorbic acid. The results show that both isolated compounds from A. muricata leaf possess in vitro antimicrobial and antioxidant properties and they may be useful as active ingredients in antimicrobial drug formulations and as agents for the control of free radical-related pathological disorders.

Charge/polarity-dependent 2D separation: a new chromatography for monitoring biomolecules

Thin-layer chromatography is a fast and easy tool for analyzing biomolecules, but opportunities for its use have up to now been limited due to its low resolution of separation. To overcome this problem, we have designed a new two-dimensional biomolecule analysis tool, in which analytes are first subjected to isoelectric focusing in a charge-specific-separation step, which is then followed by thin-layer chromatography in a polarity-specific-separation step. This new technique enables the separation of mixed large proteins as well as that of mixed synthetic oligopeptides that have similar physical properties. We propose here a newly developed thin-layer chromatographic technique that can be used to analyze various combinations of biomolecules, such as proteins, amino acids, and oligopeptides, within a single chip. The entire system is designed to be as rapid and easily applied as classic thin-layer chromatography to permit its use for high-throughput testing.

Методика тонкослойной хроматографии в анализе субстанции 8-бром-3-метилксантина.

Методика тонкослойной хроматографии в анализе субстанции 8-бром-3-метилксантина.

A comparative estimation of the morphine content of the ayurvedic formulation ʻKamini Vidrawan Rasʼ using validated high-performance thin-layer chromatography and high-performance liquid chromatography techniques

The separation and estimation of natural products by chromatographic techniques such as high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) are wide...

Antiangiogenic Human Monoclonal Antibody Ramucirumab Radiolabelling: In Vitro Evaluation on VEGFR2-positive Cell Lines.

Background/Aim: Radiolabelling of monoclonal antibodies (mAbs) could be beneficial in cancer diagnosis and therapy, however it may cause structural changes and consequently deteriorate their immunoreactivity. Materials and Methods: The therapeutic mAb ramucirumab (RAM) was technetium-99m labelled using either a direct or an indirect method with the use of two bifunctional chelating agents (HYNIC, DTPA). The radiochemical purity was assessed using instant thin-layer chromatography (ITLC) and high-performance liquid chromatography (HPLC) technique. The affinity of radiolabelled RAM was tested on human cancer cell lines. Results: The radiolabelling provided the following stable compounds: [ 99m Tc]RAM, [ 99m Tc]HYNIC-RAM and [ 99m Tc]DTPA-RAM. Their radiochemical purity was over 95%. All prepared radiopharmaceuticals showed moderate affinity to the targeted receptor, in vitro. However, their affinity was one order lower compared to that of the natural mAb. Moreover, directly and DTPA-radiolabelled RAM demonstrated less favourable binding kinetics. Conclusion: Radiolabelling negatively affected the affinity of RAM to its targeted receptor.

Chenopodium ambrosioides induces an endothelium-dependent relaxation of rat isolated aorta

ObjectiveThis study aims to evaluate the vasodilatory effect of Chenopodium ambrosioides on the isolated rat aorta, and to explore its mechanism of action. MethodsThe vasorelaxant effect and the mode of action of various extracts from the leaves of C. ambrosioides were evaluated on thoracic aortic rings isolated from Wistar rats. In addition, ethyl acetate and methanol fractions were analyzed, using thin-layer chromatography and high-performance liquid chromatography techniques, for their polyphenolic content. ResultsThe various active extracts of C. ambrosioides at four concentrations (10−3, 10−2, 10−1 and 1 mg/mL) relaxed the contraction elicited by phenylephrine, in a concentration-dependent manner. This effect seems to be endothelium-dependent, since the vasodilatory effect was entirely absent in denuded aortic rings. The vasorelaxant effect of the methanol fraction (MF) of C. ambrosioides at 1 mg/mL was also inhibited by atropine and tetraethylammonium. This effect remained unchanged by Nω-nitro-l-arginine methyl ester hydrochloride and glibenclamide. The preliminary phytochemical analysis showed that the leaves of C. ambrosioides are rich in phenolic and flavonoid derivatives. ConclusionThese results suggest that the MF of C. ambrosioides produces an endothelium-dependent relaxation of the isolated rat aorta, which is thought to be mediated mainly through stimulation of the muscarinic receptors, and probably involving the opening of Ca2+-activated potassium channels.

Picrolemma sprucei quassinosides inhibits breast and prostate cell growth and impairs behavioral phenotype in mice

Background: It is estimated that in Brazil 59,700 new cases of breast and 68,220 new cases of prostate cancer will occur, in 2018, in Brazil. For that reason, there is a need for introducing new antitumor drugs in therapy. Objective: The objective of the study is to bioguide fractionate the organic/aqueous extracts from the aerial organs of Picrolemma sprucei (EB1151 and EB1152) aiming the identification of antitumor compounds and to evaluate the influence of EB1151 on mice behavioral phenotype. Materials and Methods: The breast (MCF-7) and prostate (PC-3) cancer cell lines cytotoxicity assay were assessed using the sulforhodamine B method. Fractionation was carried out by thin layer and column chromatographic techniques. The influence of the intraperitoneal administration of nonlethal dose (NLD, 39.1 mg/kg) of EB1151 to Balb-c male mice was assessed in open cage and open-field (OF) apparatuses. Results: FCHCl3partition phases from EB1151 and EB1152 showed cytotoxic activity against both cancer cells, as did fractions UNIP343, which contains sergeolide and isobruceine B, and UNIP344, which contains sergeolide and 15-deacetylsergeolide. Higher doses of EB1151 showed significant alterations in response to touch, hindquarter fall, irritability, auricular and corneal reflexes, ataxia, micturition, ptosis, piloerection, hypothermia, and cyanosis, and NLD showed alterations in general activity and response to touch, but recovery was obtained up to the end of the experiment. Conclusions: Quassinosides from the aerial organs of P. sprucei showed significant cytotoxicity against MCF-7 and PC-3 cell lines. Extract EB1151 has impaired general activity and activity in the OF apparatus, but recovery was observed. Abbreviations used: IBAMA/CGen/MMA: Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renovaveis/Conselho de Gestao do Patrimonio Genetico/Ministerio do Meio Ambiente; UNIP: Universidade Paulista; g: Gram; h: Hour; mL: Milliliters; FCHCl3: Fraction chloroform; FBuOH: Fraction butanol; FH2O: Fraction water; FHEX: Fraction hexane; FDCM: Fraction dichloromethane; FMeOH: Fraction methanol; FACN15: Fraction acetonitrile 15%; FACN50: Fraction acetonitrile 50%; FAcEt: Fraction ethyl acetate; CDCl3: Deuterated chloroform; NH4OH: Amonium hydroxide; DOXO: Doxorubicin; M: Molar; TCA: Trichloroacetic acid; μg: Microgram; IC50: 50% inhibitory concentration; CEUA/ICS/UNIP: Comissao de Etica no Uso de Animais/Instituto de Ciencias da Saude/Universidade Paulista; kg: cm3: Cubic centimeters; Kilogram; BP: Behavioral phenotype; OF: Open-field apparatus; NLD: Nonlethal dose; mL: Microliters; ANOVA: Analysis of variance; FAkl: Fraction alkaloids; mM: Millimolar; DNA: Desoxyribonucleic acid.

Preliminary Phytochemical Screening and HPTLC Fingerprinting of Extracts of Thuja occidentalis

Objective: To establish the fingerprint profile of Thuja occidentalis using high performance thin layer chromatography (HPTLC) technique. Methods: Preliminary phytochemical screening was done and HPTLC studies were carried out. CAMAG HPTLC system equipped with Linomat V applicator, TLC scanner 3 and visionCATS software were used. Results: Preliminary phytochemical screening of the extract showed the presence of alkaloids, triterpenes, tannins, saponins, glycosides, phenolic compounds and flavonoids. HPTLC finger printing of methanol extract of plant with Rf values at 0.5. Conclusions: It can be concluded that HPTLC fingerprint analysis of extract of Thuja occidentalis can be used as a diagnostic tool for the correct identification of the plant and it is useful as a phytochemical marker.

Development and Validation of a Robust RP-HPLC Method for Analysis of Calcipotriol in Pharmaceutical Dosage Form

Calcipotriol is a very efficient drug used in the treatment of psoriasis. In present work a novel, hasty, effortless and cost-effective Reversed -Phase High-Performance Thin-Layer Chromatography (RP-HPTLC) technique has been developed and subsequently validated for quantitative assessment of calcipotriol in bulk and in ointment. Optimized chromatographic condition was employed to accomplish separation on a Phenomenex Luna C18 column (250mm X 4.6 mm in diameter) with typical particle size of 5µ column employing Shimadzu HPLC system. Mobile phase constituting of methanol: water (80:20, v/v) was pumped during the chromatographic separation at 1 mL/min flow rate and detection was made by ultraviolet-visible detector at 264 nm. The method was validated for linearity, precision, robustness, accuracy, limit of detection and limit of quantification as per guidelines of International conference on Harmonization (ICH). The method resulted into sharp and well resolved peak for Calcipotriol at 8.2 min retention time. Method was also found to be linear (regression coefficient: 0.999), accurate (average recovery: 100%), precise and robust. Thus the developed analytical method is acceptable for identification and quantitative estimation of calcipotriol in bulk formulation and ointment formulation.

Identification and quantification of fucoxanthin in selected carotenoid-producing marine microalgae and evaluation for their chemotherapeutic potential

Background: Although there are about 18,500 compounds have been isolated and reported from marine resources, the prominence of drug discovery research on marine microalgae is still very less while comparing to other natural resources. Hence, this investigation was designed, especially on some carotenoid-producing marine microalgae to evaluate their chemotherapeutic efficacies including antibacterial, antifungal, antioxidant, hemolytic, and anthelmintic activities. Objective: The objective of this research is to evaluate the suitability of the selected marine microalgae for biological activities and to perform the identification and quantification of fucoxanthin in their methanol extracts using high-performance liquid chromatography (HPLC)–diode-array detector technique. Materials and Methods: The methanolic extracts of all 10 marine microalgae were screened for antibacterial, antifungal, antioxidant, hemolytic, and anthelmintic activities. The fucoxanthin was identified and quantified by thin-layer chromatography and HPLC techniques, respectively. Results: Among the test microalgae, Isochrysis galbana (IG) showed the presence of the highest concentration of fucoxanthin (5.93 mg/g dry weight) and also exhibited notable antioxidant activities (86%) at 80 mg/mL. In antimicrobial activities, Dunaliella salina (DS) demonstrated the promising antimicrobial activities (minimum inhibitory concentration [MIC]: 40 mg/mL) against Gram-negative bacteria and fungi while Thalassiosira species showed activity (MIC: 40 mg/mL) against Staphylococcus aureus and fungi. It was also noted that all test extracts were resistant to Escherichia coli. In anthelmintic activity against Pheretima posthuma, there are two microalgae, namely IG and Chaetoceros gracilis, exhibited considerable anthelmintic potential (with P

Application of High-Performance Thin-Layer Chromatography and Bioautography Techniques for Determination of Antibiotic Residues in Mastitic Cow Milk Following Treatment

A method to identify and quantify multiple antibiotic residues like ceftriaxone and enrofloxacin in mastitic cow’s milk by high-performance thin-layer chromatography coupled with bioautography was developed. The antibiotic residues were extracted from antibiotic treated cow’s milk suffering from mastitis with the help of acetonitrile by eliminating fat using petroleum ether and finally isolated with dichloromethane. The chromatogram of peaks of antibiotic residue confirmed the presence of antibiotics in the milk samples with presence of 0.108±0.011mg/ml (ceftriaxone) and 0.173±0.015mg/ml (enrofloxacin) in the milk sample, collected 10 days post-administration of drug with initial dose of 15g of ceftriaxone and 9g of enrofloxacin, respectively. In the present study, the presence of antibiotic residue beyond 10 days is contradictory from the withdrawal period given by the pharmaceutical company for 5 days. Bioautography confirmed the presence of active antibiotic residue in milk sample-1, 2, 3, 4, 5 and 6 with zone of inhibition of 6.5±0.407mm, 6.0±0.495mm, 5.0±0.666mm, 5.2±0.4mm, 5.1±0.432mm and 6.0±0.401mm, respectively. The test microorganism used for bioautography was Streptococcus spp.