Thin-layer Chromatographic Technique Research Articles

In Vitro Cytotoxic Study for Purified Resveratrol Extracted from Grape Skin Fruit Vitis vinifera


 This study was conducted with the aim to extract and purify a polyphenolic compound “ Resveratrol” from the skin of black grapes Vitis vinifera cultivated in Iraq. The purified resveratrol is obtained after ethanolic extraction with 80% v/v solution for fresh grape skin, followed by acid hydrolysis with 10% HCl solution then the aglycon moiety was taken with organic solvent
 ( chloroform). Using silica gel G60 packed glass column chromatography with mobile phase benzene: methanol: acetic acid 20:4:1 a partial purified resveratrol was obtained then preperative thin layer chromatography technique yielded pure crystals identified as resveratrol (mixture of two isomers cis and trans) in relation to resveratrol standard (35 mg resveratrol crystals / 0.5 kg fresh grape skin was obtained as a result of these processes ). The study was also employed an in vitro evaluation the cytotoxic effect of pure resveratrol on some cell line including : the murine mammary adenocarcinoma AMN-3 cell line, the human laryngeal carcinoma (Hep-2) cell line and the Rat embryo fibroblast (Ref) cell line, at different concentrations and different expousure time. The cytotoxic effect of the pure resveratrol was studied in comparison with trans- resveratrol standard in concentrations of (12.5, 25, 50 and 100) µg/ml for both purified resveratrol and the standard, also the comparism included methotrexate drug in concentrations (0.05 , 0.1 , 0.2 and 0.4) µg/ml toward the growth effects of the three types of cell lines and at three exposure times (24, 48 and 72) hours. The cytotoxic inhibition effect for the purified extracted resveratrol revealed that the highest significant effect (P<0.01) was achieved after 24 hr of exposure on both AMN-3 and Ref cell lines. Hep-2 cell line responded to extracted resveratrol in different manners.
 Keywords: Resveratrol , grape skin , polyphenols, cytotoxic study

Technetium-99m-Labeled Sulfadiazine: a Targeting Radiopharmaceutical for Scintigraphic Imaging of Infectious Foci Due To Escherichia coli in Mouse and Rabbit Models.

Bacterial infection is one of the vital reasons of morbidity and mortality, especially in developing countries. It appears silently without bothering the geological borders and imposes a grave threat to humanity. Nuclear medicine technique has an important role in helping early diagnosis of deep-seated infections. The aim of this study was to develop a new radiopharmaceutical 99mTc-labeling sulfadiazine as an infection imaging agent. Radiolabeling of sulfadiazine with technetium-99m (99mTc) was carried out using stannous tartrate as a reducing agent in the presence of gentistic acid at pH=5. The quality control tests revealed ~98% labeling efficiency. Paper chromatographic (PC) and instant thin-layer chromatographic (ITLC) techniques were used to analyze radiochemical yield. Biodistribution and infection specificity of the radiotracer were performed with Escherichia coli (E. coli) infection-induced rats. Scintigraphy and glomerular filtration rate (GFR) study was performed in E. coli-infected rabbits. Scintigraphy indicated E. coli infection targeting potential of 99mTc-SDZ, while biodistribution study showed minimal uptake of 99mTc-SDZ in non-targeted tissues. The uptake in the kidneys was found 2.56±0.06, 2.09±0.10, and 1.68±0.09% at 30min, 1h, and 4h, respectively. The infected muscle (target) to non-infected muscle (non-target) ratio (T/NT) was found 4.49±0.04, 6.78±0.07, and 5.59±0.08 at 30min, 1h, and 4h, respectively.

TO ASSESS THE POTENTIAL OF CURCUMIN AGAINST GUT MICROBIOTA-INDUCED ALTERATION IN CHOLINE METABOLISM IN C57BL/6J MICE

Objective: The present study was focused on assessing the antimicrobial activity of curcumin against Providencia rettgeri, a gut microbe, which was further corelated to the effect of curcumin on choline metabolism and its role in preventing atherosclerosis and on the evaluation of potential of curcumin on the inhibition of conversion of choline to trimethylamine (TMA), and further its conversion to trimethylamine-N-oxide (TMAO) in liver.Methods: The minimum inhibitory concentration of curcumin was determined using Cup-plate method. The estimation of trimethylamine was performed using high performance thin layer chromatography (HP-TLC) technique. Estimation of blood serum parameters were performed using kits supplied by Span-diagnostics for estimation of triglycerides, total cholesterol and high density lipoprotein (HDL) cholesterol. All surgical procedures on animals including blood withdrawal and isolation of livers were carried out in accordance with CPCSEA guidelines.Results: The minimum inhibitory concentration of curcumin was found to be 10 µg/ml. There was a significant increase (p<0.001) in the mean triglyceride, total cholesterol, low-density and very low-density lipoprotein, along with a significant decrease (p<0.001) in high-density lipoprotein in choline-fed mice. Also, there was significant decrease (p<0.001) in the mean triglyceride, total cholesterol, low-density and very low-density lipoprotein, along with a significant increase (p<0.001) in high-density lipoprotein cholesterol in choline-fed mice treated with curcumin. Further, there was a decrease in the Flavin mono-oxygenase (FMO3) activity in the mice livers treated with curcumin by 21.57%.Conclusion: It was concluded that curcumin has the potential to inhibit gut microbiota thus prevention the conversion of choline to trimethylamine, has serum lipid lowering effect in female C57BL/6J mice and has an inhibitory effect on hepatic FMO3 thus preventing the conversion of trimethylamine to trimethylamine-N-oxide.

Structural analysis of phosphatidylcholine using a thin layer chromatography‐based method

In this study, a novel technique for positional analysis of phosphatidylcholine (PC) was developed and investigated. This technique is based on a well‐defined connection between two‐dimensional chromatography and in situ enzymatic hydrolysis. Characterizations of PC composition mainly occurred on a thin layer chromatography (TLC) plate. This method could reveal the relative percentage of each fatty acyl residue on the sn‐1 and sn‐2 ester linkages of PC, respectively. PC was isolated from total lipids by TLC, and then its purity was confirmed by high performance liquid chromatography (HPLC). Without further extraction from the TLC plate, PC was directly hydrolyzed by phospholipases on the plate. A PLA1 enzyme loading of 0.12 mL appeared to be optimum for the in situ enzymatic hydrolysis of PC, with an 87.7% PC conversion; while for a PLA2 enzyme loading of 0.30 mL, the PC conversion was 84.8%. From HPLC, no acyl migration was observed above the HPLC detection limit in both in situ enzymatic hydrolysis reactions. Free fatty acids (FFAs) released from different positions of the PC were isolated from the product mixture by two‐dimensional chromatography. The FFAs bands were chemically trans‐esterified into fatty acids methyl ester (FAMEs) and analyzed by GC‐MS to uncover the PC positional structure.Practical applications: This study developed a new method to directly perform enzymatic hydrolysis reaction on a TLC plate. With a connection between two‐dimensional chromatography and in situ enzymatic hydrolysis, a TLC plate can play multiple roles, including a separation tool, an enzymatic reaction carrier, and an identification tool. Thus, TLC technique has high potential and this study helped extend its applications.Step 1: Total lipids were applied to a thin layer chromatography (TLC) plate, and then phosphatidylcholine (PC) is separated from other lipids. Step 2: Enzyme solution and wetting agent are applied to the PC band. Step 3: After in situ enzymatic reaction, PC is partly hydrolyzed into lysophosphatidylcholine (LPC) and free fatty acids (FFAs), and the hydrolysis rate are 87.7% (with 0.12 mL PLA1 enzyme) and 84.8% (with 0.30 mL PLA2 enzyme), respectively. Step 4: The TLC plate is rotated by 90°. Step 5: The FFAs released from PC are isolated from products mixtures by a second‐dimensional chromatography.

Estimation of Exopolysaccharides (EPS) Producing Ability of Cr (VI) Resistant Bacterial Strains from Tannery Effluent

Chromium is a known heavy metal and recognized as a carcinogen to the biological systems. Previously isolated Cr (VI) resistant Exiguobacterium UE1 and UE4 were used in this study. These strains were analysed for exopolysaccharides (EPS) production for the remediation of Cr (VI) contaminated soils. Both the strains could tolerate about 250µg/ml of Cr (VI) stress. Strain UE1 showed 100% Cr (VI) removal whereas UE4 reduced 99.2% at an initial concentration of K2CrO4 100µgml-1. Optimum growth was observed at 37ºC and pH 7 for both strains. Strains exhibited significant EPS production under Cr (VI) stress and non-stress conditions. However, UE1 showed increased production of released as well as loosely bound EPS (0.36g/100ml and 0.152g/100ml respectively) under Cr (VI) supplemented condition. Thin Layer Chromatography (TLC) technique confirmed the presence of sugars in EPS samples after hydrolysis. Fourier Transforms Infrared Spectroscopy (FTIR) analysis showed the involvement of various functional groups such as hydroxyl group and aromatic compounds in the binding of Cr (VI) ions to the EPS. These findings suggest that strains UE1 and UE4 isolated from local tanneries of Pakistan can be used for remediation of Cr (VI) pollutes soils.

Antibacterial Activity of Three Medicinal Lasianthus (Rubiaceae) Extracts on Human Resistant Pathogenic Bacteria

Aim: To evaluate the activity of medicinal Lasianthus extracts on bacteria and to determine the chemical characters among three medicinal Lasianthus (Rubiaceae) species. Materials and Methods: The phytochemical investigation of L. cyanocarpus, L. hirsutus and L. lucidus were performed using thin layer chromatography and high performance liquid chromatography (HPLC) techniques. Four antibacterial resistant strains including Staphylococcus aureus ATCC 43300 (MRSA), Klebsiella pneumoniae ATCC BAA 1705 (carbapenemase; KPC-producing strain) and Pseudomonas aeruginosa ATCC 27853 (AmpC β-lactamase producing strain) were used. The phytochemical investigation of L. cyanocarpus, L. hirsutus and L. lucidus were performed using thin layer chromatography and high performance liquid chromatography (HPLC) techniques. We also detected the effects of extracts on bacteria by using a scanning electron microscope. Results: The lipophilic extracts from the three plants revealed the terpenoids, coumarin and iridoid. HPLC showed similarities in the chemical profiles of both leaf and stem bark extracts. L. lucidus lipophilic extracts revealed the greatest effect against S. aureus and P. aeruginosa at 400 and 100 μg/mL respectively, whereas L. cyanocarpus extracts prominently affected K. pneumoniae at 400 μg/mL. Cell lysis and leakage of bacteria treated with extracts were observed. Conclusion: Our findings surprisingly showed the potential of antibacterial effect among resistant pathogenic bacteria. We also revealed the comparable signals of the chemical characters from the three Lasianthus. These findings support the traditional use related infectious diseases and it might be possible to further develop the antibiotic agents.

New Multi-Walled Carbon Nanotubes Functionalized with Dendrimer Stabilized Palladium Nanoparticles Catalyst for C-C Cross Coupling Reactions

Two types of multi-walled carbon nanotubes are (MWCNTs) functionalized with dendrimer/palladium nanoparticle catalysts viz., MWCNTs-poly(propylene imine) generation 2 (G2)-PdNPs (MWCNTs-PPI(G2)-PdNPs) and MWCNTs-poly(propylene imine) generation 3 (G3)-PdNPs (MWCNTs-PPI(G3)-PdNPs) were prepared. The newly developed catalysts were characterized by the spectroscopic/microscopic techniques. The catalytic activity of these catalysts were investigated by performing C-C cross coupling reactions viz., (i) Suzuki-Miyaura, (ii) Heck and (iii) Sonogashira as model reactions. The formation of product in each reaction was identified by thin layer chromatographic (TLC) technique (2:8 percentages of ethyl acetate and hexane) and then purified by column chromatography.The resulting products were characterized by 1H and 13CNMR spectroscopy and the percentage of yield was calculated by gravimetrical method. The study of reusability of the superior catalyst viz.,MWCNT-PPI(G3)-PdNPs was examined so as to know the stability of the catalyst for the same reactions.

Extraction, Purification and Analysis of Sweet Compounds in Stevia rebaudiana Bertoni using Chromatographic Techniques

Stevioside and rebaudioside-A are two commercially important diterpenoid glycoside natural sweeteners present in Stevia rebaudiana Bertoni. Stevioside is the major component about 300 times sweeter than sugar, while rebaudioside-A is 1.6 to 1.8 times sweeter than stevioside. Some of the modern extraction and purification techniques developed have been found to be complex and expensive. Therefore, an attempt was made to extract and purify stevioside and rebaudioside-A using simple, easy and inexpensive thin-layer chromatography and column chromatography techniques. Dried leaves were extracted with methanol followed by purification through column chromatography and further analysis was done through high performance liquid chromatography. This extraction method provided a simple and cost effective route to obtain almost pure stevioside and rebaudioside-A.

Phytochemical Analysis of Two Cytotypes (2x and 4x) of Physalis angulata an Important Medicinal Plant, Collected from Rajasthan

A simple, selective, sensitive and precise high performance thin layer chromatographic technique was followed to determine the diversity within two cytotypes of P. angulata. Fruit, stem, leaf and root samples extracted in n-hexane were used to quantify phenols and phytosterols and methanolic extracts for triterpinoids. The amounts of phytochemical differ in both the cytotypes of the same species.

Characterization of the Phenolic Compound, Gallic Acid from Sansevieria roxburghiana Schult and Schult. f. Rhizomes and Antioxidant and Cytotoxic Activities Evaluation.

Background:Sansevieria roxburghiana Schult. and Schult. f. (Asparagaceae) grows in India, Indonesia, Sri Lanka, and tropical Africa. Even though the plant has been traditionally used for the treatment of many ailments, the antioxidant and antiproliferative activities of S. roxburghiana methanol extract and its fractions have not yet been explored.Materials and Methods:Quantitative estimation of phenols and different antioxidant assays were performed using standard methods. Anti-proliferative effect of the extract and fractions were evaluated in HCT-116, HeLa, MCF-7, HepG2, and A-549 cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine B (SRB) assay methods. High-performance liquid chromatography (HPLC) and high-performance thin layer chromatography (HPTLC) fingerprint profiling were carried out for extract and different fractions.Results:Significant antioxidant and anti-proliferate activity were detected in ethyl acetate fraction. Ethyl acetate fraction showed prominent scavenging activity in 1,1-diphenyl-2-picrylhydrazyl, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, and nitric oxide antioxidant assays with an concentration yielding 50% inhibition (IC50) 15.33 ± 1.45, 45.3 ± 1.93 and 48.43 ± 0.46 mg/ml, respectively. Cytotoxicity of ethyl acetate fraction was the highest among other fractions against HCT-116, HeLa, and MCF-7cancer cell lines with IC50 values 16.55 ± 1.28, 12.38 ± 1.36, and 8.03 ± 1.9 μg/ml, respectively, by MTT assay and 15.57 ± 0.70, 13.19 ± 0.49, and 10.34 ± 0.9 μg/ml, respectively, by SRB assay. The presence of gallic acid in the ethyl acetate fraction of S. roxburghiana rhizomes was confirmed by HPLC and HPTLC analysis.Conclusion:Results suggested that ethyl acetate fraction exhibited effective antioxidant and antiproliferative activities. The phenolic compounds identified in ethyl acetate fraction could be responsible for the activities.SUMMARY Sansevieria roxburghiana has been selected for in vitro antioxidant and cytotoxicity screeningEthyl acetate fraction of methanol extract of S. roxburghiana exhibited effective antioxidant and antiproliferative activitiesThe activity of ethyl acetate fraction may be due to the presence of phenolic compound which is identified by high-performance liquid chromatography and high-performance thin layer chromatography techniques. Abbreviations used: %: Percent, ºC: Celsius, mg: Microgram, ml-Microlitre, ANOVA: Analysis of variance, DMSO: Dimethyl sulfoxide, g: Grams, IC50: Concentration yielding 50% inhibition, Kg: Kilogram, mg: Milligram, min: Minutes, ml: Milliliter, HPLC: High-performance liquid chromatography, HPTLC: High-performance thin layer chromatography, DPPH: 1,1-diphenyl-2-picrylhydrazyl, ABTS: 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, GAE: Gallic acid equivalents, SRME: Methanol extract of S. roxburghiana, ROS: Reactive oxygen species, SRPE: Petroleum ether fraction of S. roxburghiana, SREA: Ethyl acetate fraction of S. roxburghiana, SRAQ: Aqueous fraction of S. roxburghiana, DMEM: Dulbecco's Minimum Essential Medium, FBS: Fetal bovine serum, OD: Optical density, TPC: Total phenolic content, SRBU: Butanol fraction of S. roxburghiana.

Quantification and Identification of Bioactive Eugenol in Myristica fragrans Seed Using Validated High Performance Thin Layer Chromatography Technique

A sensitive, selective, precise and stability indicating high-performance thin layer chromatographic method (HPTLC) was developed and validated for analysis of eugenol from methanolic extract of Myristica fragrans seed. The chromatography study was performed on aluminium foil-backed silica gel 60 F-254 HPTLC plates using toluene-ethyl acetate-formic acid (2:5:0.2, v/v/v) as the mobile phase. The densitometric determination was carried out by TLC scanner (CAMAG) at 560 nm in reflectance/absorbance mode. The developed method was validated for different parameters like linearity, precision, recovery, robustness, detection limits, quantification limits and stressed stability study in accordance with ICH guidelines. The developed analytical method were found to be linear in the concentration range of 100-1000 ng band-1 with regression value closer to unity (r2=0.998). The developed system was found to give compact spots for eugenol (Rf 0.55) with the limit of detection and limit of quantification (21 and 63 ng band-1) respectively. Further the study showed recovery of eugenol in the range of 99.3-99.8% with the precision value ≤ 1.85% and ≤ 1.71%. The all validation parameters result showed satisfactory result in comparison to pure eugenol. The stress degradation study of eugenol showed well separated degraded peak from the pure eugenol. This developed stability indicating HPTLC method was found to ideal method to separate the eugenol from its degradation products. Further this method will be successfully used in the analysis and routine quality control of eugenol loaded formulations.

Purification and Characterization on an Antibacterial Agent from (Eugenia Caryophllate) Against of Some Pathogenic Bacteria Isolated From Fishes

Local bacterial cultures could be isolated from 200 Oreochromis niloticus and were caught as random samples from ELAbassa, Abou- Hammad, and Sharkia, Egypt. Fish farms with an average body weight 40±5g. Suffered From signs of septicemia as hemorrhages on several parts of the body surface (mouth) base of fins, abdominal part. opercula , and around the anal opening turbidity of the eyes and slight exophthalmia roughness of scales and sometime scale losses, postmortemally showing, hepatomegaly, splenomegaly, congestion of gills, kidney and accumulation, of bloody fluid in abdominal wall to bacterial strains are Gram negative bacteria were isolated from fish. The bacterial isolate taxonomic classification clarified that the bacterial isolates was likely belonging to Pseudomonas aeruginiosa-I, Aeromonas hydrophila-2 according to its physiological morphological, and biochemical characters. The active extract using bioactivity-guided technique of aqueous and organic extracts of (Eugenia caryophllate). The separation of the active ingredient and its purification was performed using both thin layer chromatography (TLC) and column chromatography techniques. The physico-chemical characteristics of the purified antibacterial agent viz. color, melting point, solubility, elemental analysis and spectroscopic characteristics (GC – mass techniques) have been investigated. This analysis indicates a suggested empirical formula of C10 H12 O2. The biological activities i.e. MICs of the Purified antibacterial agent were also determined.

Determination of dantrolene sodium in the presence of its process-related impurity by high-performance thin-layer chromatography—spectrodensitometry

Dantrolene sodium (DAN) is widely used as a muscle relaxant drug for the treatment of malignant hyperthermia. 5-(4-Nitrophenyl)-2-furaldehyde is reported to be DAN-related compound C (DC), and it is the synthetic precursor of DAN that may be presented as process-related impurity in a pharmaceutical formulation. Also, it is considered to be the acidic and photolytic degradation product of DAN. The synthesis of DC has been performed from p-nitroaniline with good yield and high purity; structure elucidation has been confirmed by infrared (IR), mass spectrometry (MS), and nuclear magnetic resonance (NMR) analyses. In the present study, high-performance thin-layer chromatography (HPTLC) technique was developed and validated for the separation and quantification of DAN along with DC. Separation was carried out on HPTLC silica gel 60 F254 plates using a developing system consisting of chloroform—ethylacetate—acetic acid (10:0.5:0.01, by volume) with ultraviolet (UV) scanning at 380 nm. The method was highly sensitive and could be used for the determination of DAN and DC in the range of 0.1–1.5 μg band−1 and 0.1–2.0 μg band−1, respectively. The method has been validated in compliance with the International Conference on Harmonization (ICH) guidelines and was successfully applied to a pharmaceutical formulation. The method was compared favorably with the reported British Pharmacopoeia high-performance liquid chromatography (BP HPLC) method.

High Performance Thin Layer Chromatography: A Mini Review

High Performance Thin layer Chromatography (HPTLC) technique is a sophisticated and automated form of the thin-layer chromatography (TLC) with better and advanced separation efficiency and detection limits, and is often an excellent alternative to GC and HPLC. Applications of HPTLC include phytochemical and biomedical analysis, herbal drug quantification, active ingredient quantification, fingerprinting of formulations, and check for adulterants in the formulations. HPTLC is useful in detecting chemicals of forensic concern. Various advance techniques in reference to HPTLC like hyphenations in HPTLC-MS, HPTLC-FTIR and HPTLC-Scanning Diode Laser have made HPTLC a power analytical tool in the field of analysis. Experts are of the opinion that HPTLC future to combinatorial approach and the utilization of instrumental HPTLC toward the analysis of drug formulations, bulk drugs, and natural products will increase in the future

English

Control of leishmaniasis remains a challenge due the high toxicity of the chemotherapeutic drugs presently available. The ongoing search for better leishmanicidal compounds has brought herbal drugs into the limelight as safe and effective substitutes to conventional therapies which have various drawbacks. The current study was designed to evaluate the antileishmanial effect of Quassia amara against Leishmania amazonensis and Leishmania infantum. Different fractions (Hexane (Q1), dichloromethane (Q2), ethyl acetate (Q3) and butanol (Q4)) obtained from the liquid-liquid partition of the crude Q. amara methanol extract were tested against Leishmania promastigote forms. After 120 h of treatment with Q. amara fractions (1 to 500 µg/ml), the minimal inhibitory concentration (MIC) was determined using resazurin. The most active fraction (Q2) was analyzed using thin layer chromatography (TLC) and high performance liquid chromatography-diode array detector (HPLC-DAD) techniques. The effects of Q2 on parasite ultrastructure were investigated by transmission electron microscopy. In addition, Leishmania-infected macrophages were treated with Q2 in order to evaluate the anti-amastigote effect. Among the fractions tested, Q2 showed the highest activity against L. amazonensis and L. infantum promastigotes MIC values of 62.5 and 31.25 µg/ml, respectively. There were ultrastructural alterations, such as nuclear chromatin condensation, intense vacuolization and autophagic and myelin-like figures in parasites treated with Q2 (62.5 µg/ml). Macrophages previously infected with L. amazonensis and L. infantum promastigotes showed a drastic reduction in the number of parasites recovered in the supernatant after Q2 treatment at MIC values. The TLC and HPLC fingerprints of Q2 showed that alkaloids were the main chemical constituents in this fraction. The results presented herein showed that the alkaloid-rich fraction Q2 is a promising source of antileishmanial agents. Further investigation will be necessary in order to isolate and test the substance(s) responsible for the bioactivity. Key words: Antileishmanial activity, Quassia genus, alkaloids, macrophage infection, nitric oxide.

NEW INDOLIZIDINE ALKALOIDS FROM PSYCHROPHILIC YEAST Glyciozyma antarctica PI12

Two alkaloid compounds: namely 3-(1-aminoethyl)tetrahydro-1H-pyrrolo[1,2-d][1,2,4]oxadiazine-1,4(3H)-dione (1) and tetrahydro-1H-pyrrolo[2,1-c][1,4]oxazine-1,3(4H)-dione (2) was isolated from Glyciozyma antarctica (PI12), a psychrophilic yeast. The isolated compounds were isolated by Radial Chromatography and Thin Layer Chromatography techniques. The chemical structures were elucidated by Ultraviolet-visible, Infrared, Nuclear Magnetic Resonance and Mass Spectroscopy.

Stability-indicating high-performance thin-layer chromatographic method for simultaneous estimation of the active pharmaceutical ingredients metolazone and spironolactone

This work represents the validation of a stability-indicating thin-layer chromatographic technique for the simultaneous estimation of metolazone (METO) and spironolactone (SPIRO) from marketed formulation (tablets). Thin-layer chromatography was performed using precoated silica gel plate 60 F254 using ethyl acetate—chloroform—GAA (5:5:0.1 v/v) as the mobile phase for the separation of METO and SPIRO. The stability study forms an integral part of the formulation development process, and its use is also encouraged by various guidelines. Stress study was performed on active pharmaceutical ingredients (APIs) as well as on formulation for establishing a stability-indicating thin-layer chromatographic method for both drugs. The APIs were subjected to change under various environmental conditions such as pH, temperature, oxidation, etc. to determine their effect on the stability of drugs. The developed method was able to resolve drugs and their degradation products formed under the aforementioned conditions. The...

From Traditional Resource to Global Commodities:-A Comparison of Rhodiola Species Using NMR Spectroscopy-Metabolomics and HPTLC.

The fast developing international trade of products based on traditional knowledge and their value chains has become an important aspect of the ethnopharmacological debate. The structure and diversity of value chains and their impact on the phytochemical composition of herbal medicinal products, as well as the underlying government policies and regulations, have been overlooked in the debate about quality problems in transnational trade. Rhodiola species, including Rhodiola rosea L. and Rhodiola crenulata (Hook. f. & Thomson) H. Ohba, are used as traditional herbal medicines. Faced with resource depletion and environment destruction, R. rosea and R. crenulata are becoming endangered, making them more economically valuable to collectors and middlemen, and also increasing the risk of adulteration and low quality. Rhodiola products have been subject to adulteration and we recently assessed 39 commercial products for their composition and quality. However, the range of Rhodiola species potentially implicated has not been assessed. Also, the ability of selected analytical techniques in differentiating these species is not known yet. Using a strategy previously developed by our group, we compare the phytochemical differences among Rhodiola raw materials available on the market to provide a practical method for the identification of different Rhodiola species from Europe and Asia and the detection of potential adulterants. Nuclear magnetic resonance spectroscopy coupled with multivariate analysis software and high performance thin layer chromatography techniques were used to analyse the samples. Rosavin and rosarin were mainly present in R. rosea but also in Rosea sachalinensis Borris. 30% of the Rhodiola samples purchased from the Chinese market were adulterated by other Rhodiola spp. The utilization of a combined platform based on 1H-NMR and HPTLC methods resulted in an integrated analysis of different Rhodiola species. We identified adulteration at the earliest stage of the value chains, i.e., during collection as a key problem involving several species. This project also highlights the need to further study the links between producers and consumers in national and trans-national trade.

Prevalence of toxigenic fungi in common medicinal herbs and spices in India.

Mycotoxins are unavoidable contaminants of food grains, feeds, medicinal herbs, and spices, posing as health threat to animals and humans. The objective of this study was to screen medicinal herbs and spices for fungi and mycotoxin contamination and evaluate their safety. Sixty-three samples were examined for fungal contamination and fungal load determined using standard microbiological method. Aflatoxin and citrinin were detected using thin layer chromatography and high-performance chromatography technique. Fifty-eight out of the 63 samples were contaminated, while five were free from fungal contamination. Analysis revealed that 47 % of the samples had a fungal load above 1 × 103 cfu/g which is the permissible limit set by World Health Organization. The samples Mesua ferrea-II and Terminalia chebula-III had the highest fungal load, i.e., 5.0 × 104 cfu/g. A total of 187 fungi were isolated, out of which 28 were toxigenic which included 19 aflatoxin-producing Aspergillus flavus and 9 citrinin-producing Penicillium citrinum. The natural contamination with aflatoxin B1 was detected only in one sample, i.e., Arachis hypogaea (groundnut) which was present beyond the permissible limit. Though toxigenic fungi were isolated, mycotoxins were not detected from any of the medicinal herbs and spices. Medicinal herbs and spices are susceptible to toxigenic fungi; however, they also possess intrinsic factors that inhibit mycotoxin contamination. This study provides a basis in assessing the degree of fungal and potential mycotoxin contamination in medicinal herbs and spices.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0476-9) contains supplementary material, which is available to authorized users.

The use of similarity analysis for evaluation of the chromatographic separation of salicylic acid and its selected derivatives studied by adsorption and partition thin-layer chromatography techniques

The aim of the work was the chromatographic separation of salicylic acid and their derivatives, i.e., acetylsalicylic acid, salicylanilide, salicylaldehyde, salicylamide, methyl salicylate, phenyl salicylate, 2,5-dihydroxybenzoic acid, salicylhydroxamic acid, 3,5-dinitrosalicylic acid, 3-aminosalicylic acid, 4-aminosalicylic acid, and 5-aminosalicylic acid by use of adsorption thin-layer chromatography (normal-phase thin-layer chromatography [NPTLC]) and partition thin-layer chromatography (reversed-phase thin-layer chromatography/high-performance thin-layer chromatography [RP-TLC/HPTLC]). Three qualitatively and quantitatively different mobile phases were used for the separation of salicylic acid and its derivatives. Cluster analysis (single linkage method, Euclidean distance) allowed the evaluation of the suitability of the chromatographic conditions used to separate the pairs of tested compounds. The cluster analysis data indicate that the composition of the mobile phase is fundamental in the process o...