Thin Layer Chromatography Fingerprinting Research Articles

English

Fresh, shade-dried and powdered samples of leaf, stem and root of Chitrak (Plumbago zeylanica L) were subjected to fractional distillation in a Soxhlet apparatus using four organic solvents. Chloroform, acetone and ethanol extracts from root of Plumbago showed higher zone of inhibition of 22.66±1.52, 21.5±1.29 and 16.5±1.29 mm and minimum inhibitory concentration (MIC) of 1.0, 10.0 and 10.0 μg/ml, respectively against Escherichia coli. Root and leaf extracts by ethanol, chloroform and acetone showed higher antibacterial activity against E. coli as compared to standard Kanamycin (MIC-100 μg/ml). The high performance thin layer chromatography (HPTLC) fingerprints were used for the quantitation of two bioactive markers: gibberellic acid and quinol R in the plant powder of different organs. Maximum content of gibberellic acid was found in acetone extract of the root (59.74%, Rf 0.79) followed by methanol root extract (53.01%). HPTLC provides a chromatographic fingerprint of phytochemicals and is suitable for confirming the identity and purity of medicinal plant raw materials. Key words: Plumbago zeylanica, antimicrobial property, bioactive compound, kanamycin, Escherichia coli, high performance thin layer chromatography (HPTLC), phytochemical fingerprint.

Effect of whole plant of Rostellularia diffusa Willd. on experimental stress in mice.

Background:Rostellularia diffusa is an unexplored medicinal plant used as brain tonic in traditional medicine system.Objective:This study was designed to investigate the antioxidant and anti-stress potential of R. diffusa by experimental animal models.Materials and Methods:The extracts of R. diffusa were subjected to preliminary phytochemical screening and high performance thin layer chromatography (HPTLC) finger printing analysis. The antioxidant potential of the extracts was found by different in vitro models. The anti-stress activity was investigated by using acetic acid induced writhing test, swimming endurance test, and restraint stress in experimental mice. Serum parameters such as glucose, triglyceride and cholesterol, oxidative stress parameter thiobarbituric acid reactive substance, antioxidant parameters such as reduced glutathione, superoxide dismutase and catalase and organ weights were evaluated after restraint stress in mice. Diazepam was used as reference standard to compare the anti-stress activity of plant extract.Results:High performance thin layer chromatography finger printing analysis revealed the presence of flavone compounds in both extracts. The extracts also showed good antioxidant property in different in vitro antioxidant models. Administration of extracts of R. diffusa decreased the number of wriths and immobility time when compared with control group in acetic acid-induced writhing test and swimming endurance test respectively in experimental mice. They also suppressed the restraint stress-induced alterations in serum parameters, oxidative stress, and antioxidant parameters in brain and also restored the organ weights in normal level.Conclusion:From these results, it has been concluded that the potential anti-stress activity of R. diffusa is through its adaptogenic and antioxidant properties.

Comparative pharmacognosy of Pashanbhed.

Background:Pashanbhed is a commercially available diuretic and lithotropic drug, used to treat renal problems. It is a controversial name as it is assigned to various plants such as Bergenia ligulata, Kalanchoe pinnata, Coleus aromaticus and Rotula aquatica.Objective:To perform the comparative preliminary phytochemical screening, diuretic activity, and thin layer chromatography (TLC) finger printing profile of three plants (B. ligulata, C. aromaticus, and K. pinnata), most commonly used as Pashanbhed.Materials and Methods:Diuretic potential of methanolic extract (ME) of three plants were evaluated at two dose levels (500 and 1,000 mg/kg p.o.), using normal Wistar rats (Lipschitz method). Furosemide (20 mg/kg p.o.) was used as a standard drug. The effect on urine output and electrolyte changes were measured for 24 h and compared. All MEs were screened preliminarily for their constituents and their TLC finger printing profiles were prepared. One-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test. P < 0.05 was considered statistically significant.Results:The MEs of all three plants have shown diuresis in normal rats. However, in intercomparison of the ME C. aromaticus (1,000 mg/kg p.o.) produced more significant diuresis (P < 0.05) and electrolyte excretion compared to other test groups, the effect was at par with furosemide. The ME of these plants showed presence of alkaloids, glycosides, steroids, terpenoids, saponins, flavonoids, etc.Conclusion:The ME of C. aromaticus (1,000 mg/kg p.o.) has showed highest diuretic action (4.2) among the tested extracts. This suggests the use of C. aromaticus leaves as “Pashanbhed”; the most effective diuretic drug.

Combined multivariate data analysis of high-performance thin-layer chromatography fingerprints and direct analysis in real time mass spectra for profiling of natural products like propolis

Sophisticated statistical tools are required to extract the full analytical power from high-performance thin-layer chromatography (HPTLC). Especially, the combination of HPTLC fingerprints (image) with chemometrics is rarely used so far. Also, the newly developed, instantaneous direct analysis in real time mass spectrometry (DART-MS) method is perspective for sample characterization and differentiation by multivariate data analysis. This is a first novel study on the differentiation of natural products using a combination of fast fingerprint techniques, like HPTLC and DART-MS, for multivariate data analysis. The results obtained by the chemometric evaluation of HPTLC and DART-MS data provided complementary information. The complexity, expense, and analysis time were significantly reduced due to the use of statistical tools for evaluation of fingerprints. The approach allowed categorizing 91 propolis samples from Germany and other locations based on their phenolic compound profile. A high level of confidence was obtained when combining orthogonal approaches (HPTLC and DART-MS) for ultrafast sample characterization. HPTLC with selective post-chromatographic derivatization provided information on polarity, functional groups and spectral properties of marker compounds, while information on possible elemental formulae of principal components (phenolic markers) was obtained by DART-MS.

Two-dimensional thin-layer chromatographic fingerprint ofHelleborus thibetanusFranch. Using a polyamide plate with nonaqueous and reversed micellar mobile phases

A two-dimensional thin-layer chromatographic fingerprint has been developed on a polyamide plate for the quality control of Helleborus thibetanus Franch. The investigated sample was separated by chloroform-ethyl acetate-methanol (3.0:8.0:4.4, v/v) in the first dimension and isooctane-n-propyl alcohol-water (10:2.5:1.0, v/v) adding 0.28 mol L-1 sodium dodecyl sulfate, a reversed micelle, in the second dimension. The plate was dried in the air at room temperature and examined in ultraviolet (UV) light at lambda = 365 nm after development. The two developments were carried out over a distance of 70 mm. The two-dimensional thin-layer chromatographic method was validated in terms of repeatability, stability, and robustness. For fingerprint analysis, nine spots were identified as common spots. Statistical method (canonical correlation analysis) was first used to calculate the degree of similarity between the two-dimensional chromatograms. The proposed method was novel and accurate for the identification and quality evaluation of H. thibetanus Franch.

HPTLC determination of chemical composition variability in raw materials used in botanicals

Besides the chemotaxonomic value, nowadays determination of biodiversity and chemical variability has a commercial impact. The exact identity of raw material and constituents of botanical products, such as food supplements or herbal remedies, is a very important argument, being the real prerequisite for quality control and traceability, followed by the determination of active components. However, the analytical approach must consider the natural great variability in secondary metabolites and product form, such as in extracts. Against the reductive approach, on the basis of single chemical standards, so far dominant in Pharmacopoeias monographs, we report applications and utility of the high-performance thin-layer chromatography fingerprint in determination of species of the same genus, of populations of the same species and of different drugs of the same plant.

Standardization and validated high-performance thin-layer chromatographic fingerprint method for quantitative determination of plumbagin in a traditional Indian formulation

Standardization and validated high-performance thin-layer chromatographic fingerprint method for quantitative determination of plumbagin in a traditional Indian formulation

PHYTOCHEMICAL INVESTIGATION ON WITHANIA SOMNIFERA ROOT MARC GENERATED IN AYURVEDIC INDUSTRY

Water extraction is a major process in Ayu rvedic medicine manufacturing industry. This process involves water extraction of huge quantity of herbs, which culminates in the generation of large amount of marc or herbal residue. Usually the residue is either incinerated or converted to herbal manure. This paper investigates the marc of Withania somnifera roots obtained from Ayurvedic Industry for presence of valuable and potent bioactive phytochemicals. The chances of the phytochemicals getting altered by the process of prolonged heating is also looke d into and found that no such genesis or degradation of phytochemical s leading to a new compound ha s occurred in the marc. As part of study, physicochemical analysis such as total ash percentage, acid insoluble ash percentage and extractive values are perf ormed. Preliminary phytochemical analysis w as carried out with different extracts, like hexane, ethyl acetate and methanol of both herb and marc which showed the presence of similar compounds. High Performance Thin Layer Chromatography finger printing and densitogram of hexane , ethyl acetate and methanol extracts of marc and herb showed that there was a reduction in the concentration of the actives in marc. The densitogram of ethyl acetate extract showed absence of certain prominent peaks in marc. In the ca se of methanol extract the prominent peaks were present in the marc and the concentration of some major actives were reduced in the hexane extract of marc.

Spectrophotometric and bioassay methods for the estimation of erythromycin formulation

Erythromycin is a naturally occurring chemotherapeutic agent used in the treatment of aerobic gram positive cocci, bacilli and a few gram negative organisms. In view of its resurgent use due to resistance to anti-bacterial chemotherapy, the study sets out to provide simple, sensitive and cost-effective analytical and bioassay techniques involving UV-spectrophotometric and Microbiological methods. Method validation was by means of a precision and accuracy assays. Ten brands of erythromycin formulation comprising, seven tablets and three suspension dosage forms were assayed by UV spectrophotometric at the λmax of 285nm and bioassay methods. Thin Layer Chromatographic (TLC) method was used to establish the identity of the active ingredient in the formulations. The spectrophotometric results, expressed as a percentage of stated amounts of erythromycin were 79 - 120%w/w, which shows that 70% of the test samples conformed to the official stated standard. The bioassay determination gave MICs ranging from 0.5-2µg/ml for all the samples, 0.5μg/ml and 1.0μg/ml for reference erythromycin against S. aureus and B. subtilis , respectively. Thus, 80% of the samples met the bioassay requirement. TLC fingerprint gave Rf values ranging from 0.71 - 0.79 for 80% of the samples while the remaining 20% gave no change in colour, elution nor increase in size of the principal spot. Hence, no single method could be adequate in the determination of the quality of pharmaceutical formulations.

Chemopreventive Effect and HPTLC Fingerprinting Analysis of Jasminum sambac (L.) Ait. Extract Against DLA-Induced Lymphoma in Experimental Animals

The anticancer activity of the ethanolic extract of Jasminum sambac against Dalton's lymphoma ascites-induced lymphatic cancer in Swiss albino mice was investigated. The anticancer activity of J. sambac was studied against lymphoma using lipid profiles, biochemical parameters, and membrane-bound marker enzymes by standard procedures. A high-performance thin-layer chromatography fingerprinting analysis showed the presence of terpenoids and flavonoids. The levels of cholesterol, triglyceride, VLDL cholesterol, and LDL cholesterol were significantly decreased in tumor-induced mice, while HDL cholesterol showed increased levels compared with those profiles. On treatment with J. sambac, the levels were brought back to near normal. The albumin, creatinine, total protein, urea, and uric acid contents were also approaching normal values. There was s significant increase in the levels of ATPase in group II. These levels were brought back to normal upon plant extract treatment of mice. DNA fragmentation occurred in the tumor-induced group of tissue, and treatment with ethanolic extract reduced the DNA damage caused by lymphoma. Expression of lactate dehydrogenase (LDH) isoenzymes shows an increase in the levels of LDH-4 and LDH-5 in cancer-bearing animals which is brought back to near normal. Histopathological investigation showed normal sections of liver tissues in the treatment group. The results found in mice treated with ethanolic extract 100mg kg(-1) body weight quite promising and were comparable with the standard drug 5-fluorouracil. The statistically processed results support the conclusion that the ethanolic extract of J. sambac flower (100mg kg(-1)) possesses a dose-dependent significant anticancer activity against lymphoma.

Pharmacognostic and high performance thin layer chromatography finger printing of Pyrus malus Linn

Background: Pyrus malus L. Syn. Malus sylvestris Mill., commonly known as 'Apple' is a well known tree belonging to the family Rosaceae. Leaves and young aerial parts are used as medicine in homoeopathy. Objective: The Pharmacognostic and physico-chemical studies are carried out to facilitate use of correct species and standard raw materials. Material and Methods: Pharmacognostic examinations of aerial parts (leaf, petiole and stem) of authentic sample of Pyrus malus L., have been carried out. Physico-chemical parameters of raw drug viz., extractive value, ash values, formulation besides weight per mL, total solids, alcohol content along with high performance thin layer chromatography (HPTLC) and UV-visible studies have been worked out for mother tincture. Results: The leaves are ovate to oblong, margin serrate, acute, glabrous above and tomentose beneath with a short petiole. The leaf is hypostomatic with anomocytic stomata. Uniseriate macroform flagellate conical and unicellular filiform cylindrical hairs occur on the leaf. In transverse section (TS) the midvein is conspicuously ridged on abaxial side. Epidermis is 1-layered with larger cells on abaxial. Cells over midvein are papillated. Palisade is 2-layered. The vascular bundle at midvein is single, large and arc shaped. The petiole in T.S. is shield like with an adaxial groove. The cortex is aerenchymatous towards adaxial. The vascular tissue consists of a single, large, arcuate bundle and two small adaxial bundles. The young stem is oval to spherical. The vascular tissue is in the form of a continuous cylinder. 1-3 seriate rays are present in the xylem. The xylem towards the centre possess secretory canals arranged in a ring. Conclusion: The powder microscopic features and organoleptic characters along with the anatomical and physico-chemical studies are diagnostic to establish the standards for the drug.

Phytochemical analysis of ethanolic extract of Dichrostachys Cinerea W and Arn leaves by a thin layer chromatography, high performance thin layer chromatography and column chromatography.

Background:The leaves of Dichrostachys cinerea are used as laxative, diuretic, painkiller. It is also used in the treatment of gonorrhoea, boils, oedema, gout, veneral diseases and nasopharyngeal affections, etc.Materials and Methods:The Phytochemical investigation of ethanolic extract of D. cinerea leaves were performed by standard chemical tests, thin layer chromatography (TLC) by using various solvent systems, and by high performance liquid chromatography (HPTLC). Two compounds were isolated by column chromatography and one of the compounds was identified by various spectral studies.Result:Preliminary phytochemical screening of ethanolic extract of D. cinerea leaves showed the presence of Carbohydrates, proteins, Glycosides, Saponins, Tannins, Aminoacids and Terpenoids. The TLC and HPTLC fingerprint of ethanolic extract were studied and various fractions were isolated by column chromatography and one of the fraction contain β-amyrin glucoside which was confirmed by Infra Red[IR] Spectroscopy, 1H-Nuclear Magnetic Resonance (NMR), C-13 NMR and Mass spectroscopic (MS) studies.

Wound Healing Potential of Formulated Extract from Hibiscus Sabdariffa Calyx

Wound healing agents support the natural healing process, reduce trauma and likelihood of secondary infections and hasten wound closure. The wound healing activities of water in oil cream of the methanol extract of Hibiscus sabdariffa L. (Malvaceae) was evaluated in rats with superficial skin excision wounds. Antibacterial activities against Pseudomonas aeroginosa, Staphylococcus aureus and Echerichia coli were determined. The total flavonoid content, antioxidant properties and thin layer chromatographic fingerprints of the extract were also evaluated. The extract demonstrated antioxidant properties with a total flavonoid content of 12.30±0.09 mg/g. Six reproducible spots were obtained using methanol:water (95:5) as the mobile phase. The extract showed no antimicrobial activity on the selected microorganisms, which are known to infect and retard wound healing. Creams containing H. sabdariffa extract showed significant (P<0.05) and concentration dependent wound healing activities. There was also evidence of synergism with creams containing a combination of gentamicin and H. sabdariffa extract. This study, thus, provides evidence of the wound healing potentials of the formulated extract of the calyces of H. sabdariffa and synergism when co-formulated with gentamicin.

English

Microorganisms play an important role in the development of periodontal diseases in human that lead to serious complications. Since ages, medicinal plants, singly or in combinations are used for the treatment of various dental disorders. However, their scientific validation and characterization is scarce. Therefore, the goal of this study was to develop knowledge based poly-herbal formulation as a remedy for oral hygiene. After validations of antimicrobial potential, extracts of 11 plants were combined in different ratios, and finally one poly-herbal formulation was selected for further characterization. Antimicrobial activity was determined through disc and well diffusion assays. Further level of tumour necrosis factor (TNF-&alpha;) for assessing anti-inflammatory activity and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for safety assessment was determined. High performance thin layer chromatography (HPTLC) fingerprint profile was developed using thin layer chromatography (TLC) scanner and densitometer. The poly-herbal formulation, PHF 12-05 showed antimicrobial activity against both pathogenic bacteria and fungi with zone of inhibition ranging from 7 to 13 mm. PHF 12-05&nbsp; was found to possess anti-inflammatory activity by significantly (P&lt;0.05) reducing the level of tumour necrosis factor (TNF-&alpha;). HPTLC characterized PHF 12-05 was found to be quite stable and safe. The observations clearly indicate that PHF 12-05 holds promise in improving oral hygiene and hence help in preventing oral problems. &nbsp; Key words:&nbsp;&nbsp;Poly-herbal formulation, medicinal plants, antimicrobial, anti-inflammatory, oral hygiene, High performance thin layer chromatography (HPTLC) fingerprinting.

Extracts of Artemisia annua leaves and seeds mediate programmed cell death in Leishmania donovani

Leishmaniasis is one of the major tropical parasitic diseases, and the condition ranges in severity from self-healing cutaneous lesions to fatal visceral manifestations. There is no vaccine available against visceral leishmaniasis (VL) (also known as kala-azar in India), and current antileishmanial drugs face major drawbacks, including drug resistance, variable efficacy, toxicity and parenteral administration. We report here that n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) possess significant antileishmanial activity against Leishmania donovani promastigotes, with GI(50) of 14.4 and 14.6 µg ml(-1), respectively, and the IC(50) against intracellular amastigotes was found to be 6.6 and 5.05 µg ml(-1), respectively. Changes in the morphology of promastigotes and growth reversibility analysis following treatment confirmed the leishmanicidal effect of the active fractions, which presented no cytotoxic effect on mammalian cells. The antileishmanial activity was mediated via apoptosis, as evidenced by externalization of phosphatidylserine, in situ labelling of DNA fragments by terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G(0)/G(1) phase. High-performance thin-layer chromatography (HPTLC) fingerprinting showed that the content of artemisinin in crude bioactive extracts (~1.4 µg per 100 µg n-hexane fraction) was too low to account for the observed antileishmanial activity. Characterization of the active constituents by GC-MS showed that α-amyrinyl acetate, β-amyrine and derivatives of artemisinin were the major constituents in AAL and cetin, EINECS 211-126-2 and artemisinin derivatives in AAS. Our findings indicate the presence of antileishmanial compounds besides artemisinin in the n-hexane fractions of A. annua leaves and seeds.

HPTLC method Development & Validation for quantification of Markers of Dhatrinisha churna

Introduction: Dhatrinisha churna has been traditionally used in the Ayurvedic system of medicine and by traditional medical practices of India to treat hyperlipidemia. A sensitive, reliable, selective, precise and accurate densitometric High Performance Thin Layer Chromatography (HPTLC) fingerprinting method has been developed for the simultaneous determination and quantification of Curcumin and Ellagic acid in Dhatrinisha churna. Method: Quantification of Curcumin and Ellagic acid were done by developed densitometric HPTLC method. Validation of method performs in order to demonstrate its selectivity, accuracy, precision, repeatability and recovery study. Result: All calibration curves showed good linear correlation coefficients (r2>0.997) within the tested ranges. The chromatogram of Dhatrinisha churna was quantified with respect to Curcumin (1.072% w/w) and Ellagic acid (0.867% w/w). Intra-and inter-day RSDs of retention times and peak areas were less than 1.92%. The recoveries were between 96.60 and 101.40%. Conclusion: A method has been developed for the simultaneous quantification of two markers in Dhatrinisha churna. The proposed HPTLC method was found to be simple, précised and accurate and can be used for the quality control of the raw materials as well as formulations.

Phytochemicaland proximate analyses and thin layer chromatography fingerprinting of the aerial part of Chenopodium ambrosioides Linn. (Chenopodiaceae)

The aerial part of Chenopodium ambrosioides L., reputable for the treatment of malaria and diabetes in Nigeria, was qualitatively screened for the presence of secondary metabolites using standard methods. Some proximate parameters were also determined. The result of the phytochemical screening revealed the presence of alkaloids, tannins, saponins, flavonoids, terpenes, sterols, cardenolide aglycone, volatile oils and carbohydrates. The proximate analysis revealed moisture content of 10.90%, total ash value of 14.65%, acid-insoluble ash value of 3.05%, water-soluble ash of 6.25%, water-soluble extractive value of 3.18% and alcohol-soluble extractive value of 13.20%. The thin layer chromatography fingerprinting and phytochemical screening revealed several chemical components, which could be isolated from the plant. This study shows that C. ambrosioides is a potential drug plant considering the rich phytochemical and proximate pharmacognostic profile of the aerial part, hence its folkloric uses. The result of this study is the first of its kind on the Nigerian species of this plant drug, and is informative for standardization and monograph development of this herbal plant. Key words: Chenopodium ambrosioides, secondary metabolites, thin layer chromatography, pharmacognostic analysis, antimalaria, Mexican tea, ash value.

Secondary metabolite credentials of Evolvulus alsinoides by high performance thin layer chromatography (HPTLC)

Plants and plant-based products are the bases of many modern pharmaceuticals that are current in use today for various diseases. The aim of the study was to investigate the biochemical constituents and high performance thin layer chromatography (HPTLC) finger printing of the ethanolic extract of Evolvulus alsinoides. Phytochemi-cal screening was done by standard procedures and HPTLC method was also established to analyze alkaloids, fla-vonoids and phenolic compounds from the ethanolic extract of Evolvulus alsinoides. Preliminary phytochemical screening showed that ethanol extracted more secondary metabolites than other solvents. HPTLC fingerprinting analysis showed the presence of various alkaloids, flavonoids and phenols (quercetin) in the ethanolic extract. It can be concluded that Evolvulus alsinoides may serve as a source of potent antioxidants that may be used in the prevention of various diseases such as cancer, diabetes and cardiovascular diseases due to the presence of phenolic compounds. HPTLC finger print of Evolvulus alsinoides may be useful in the differentiation of the species from adulterants and act as a biochemical marker for this medicinally important plant in the pharmaceutical industry and plant systematic studies.

Optimum Ultrasound Assisted Extraction Conditions of Some Flavonoids from Green Tea Leaves. Control Quality of Green Tea Product by TLC Fingerprinting

The optimization of the green tea flavonoid extraction conditions was investigated. The experiments were carried out with two extraction methods: ultrasound assisted extraction (UAE) and reflux extraction (RE). The parameters that were varied in this study were: the extraction solvent system composition, the type of organic modifier of the extraction mixture, temperature, and time. The highest efficiency was obtained with an extraction mixture of ethanol: water, 80:20, v/v. An extraction performed at temperature of 45°C in 50–60 minutes led to optimum results. Moreover, a new fingerprinting procedure based on thin layer chromatography (TLC) image analysis was employed in order to compare the chemical composition of green tea in comparison with white and black tea.

Biological Fingerprinting of Herbal Samples by Means of Liquid Chromatography

Biological chromatographic fingerprinting is a relatively new concept in the quality control of herbal samples. Originally it has been developed with the application of HPLC, and recently herbal samples' biological profiles have been obtained by means of thin-layer chromatography (TLC). This paper summarizes the application of liquid chromatographic techniques for the purpose of biological fingerprint analysis (BFA) of complex herbal samples. In case of biological TLC fingerprint, which is a relatively novel solution, perspectives of its further development are outlined in more detail. Apart from already published data, some novel results are also shown and briefly discussed. The paper aims at drawing scientists' attention to the unique solutions offered by biological fingerprint construction.