High-performance Thin-layer Chromatography Chromatogram Research Articles

Quality control of marketed herbal products of Asparagus racemosus Willd. through high performance thin layer chromatography (HPTLC) analysis

Asparagus racemosus Willd. is a valuable medicinal plant which is used all over the world. There are several marketed products of A. racemosus. The high demand for this herb has increased the risk of adulteration in its commercial products. The adulterated herbal products might pose serious ill effects on health. Therefore, it is necessary to check the quality of marketed products in terms of the presence of their major bioactive compounds. The present study aimed to carry out the qualitative and quantitative analysis of Shatavarin IV in marketed products of A. racemosus through a validated high performance thin layer chromatography (HPTLC) method. Ten marketed products were analysed and all of them had shown the presence of Shatavarin IV which was quantified. The identification and quantification were done by taking a standard Shatavarin IV as reference. The Shatavarin IV was detected at Rf 0.4±0.05 and showed maximum absorption at 425 nm. The Shatavarin IV was quantified using a 6-point calibration curve having a standard deviation of 3.89 % with an R2 value of 0.9968. The amount of Shatavarin IV varied between 1.47±0.25 to 2.69±0.51 mg/g on a dry weight basis which is a normal range in the raw plant materials. Thus, the present findings would be a simple, reliable and cost-effective method for the quality determination of herbal products of A. racemosus. The developed HPTLC chromatograms would serve as a reference for the quality assessment of commercial products of A. racemosus in future.

HPTLC Fingerprinting Profile of Vacha (Acorus calamus Linn.)

Introduction: Vacha (Acorus calamus Linn.) commonly called Sweet flag is a significant herb with well-recognized therapeutic potential. Choorna (powder) of its rhizome is the commonly used dosage form according to Ayurveda. High-performance thin-layer chromatography (HPTLC) fingerprinting of rhizome powder of Vacha was carried out to estimate the active constituents in the dosage form choorna. ß-asarone, which is the major phytoconstituent of rhizome, was used as standard.Materials and Methods: HPTLC plate 5 × 10 cm, pre-coated with silica gel 60 F254 TLC plates, was used as stationary phase. Toluene: ethyl acetate: acetic acid (90:10:2) was used as mobile phase. Visualization was done under ultraviolet light 254 nn and 366 nm.Results and Discussion: HPTLC chromatogram showed the presence of 3 comparable peaks at 254 nm and 2 comparable peaks at 366 nm, in methanolic extract of rhizome powder and standard.Conclusion: This approach creates profile analysis of the plant rhizome and can be helpful for quality control and further researches

HPTLC fingerprinting reveals leaf and roots phytochemical variability in developmental stages of Withania somnifera

Background: Withania somnifera has been the foundation of numerous contemporary medicines today for treatment of various diseases. However, phytochemical variability profile is lacking in the root and leaf organs. Aim: This study was aimed to elucidate the phytochemical profile in seedling, vegetative and reproductive stages of leaf and roots in W. somnifera using High performance thin layer chromatography (HPTLC) fingerprinting. Methods: The HPTLC with dual wavelength UV (254/366 nm) was used for the analysis of phytochemical variation in leaf and root methanolic extracts of W. somnifera. Results: HPTLC chromatograms in 366 nm, 254 nm, and normal day light, seedling, vegetative and reproductive stages leaf and root exhibited bands variability. At 450 nm leaf HPTLC fingerprints in seedling stage showed five peaks with Rf values in the range of 0.42 to 0.90, vegetative stage with nine peaks of Rf values in range of 0.10 to 0.89, and reproductive stage with six peaks of Rf values in range of 0.30 to 0.92. In root showed six peaks with Rf values in range of 0.00 to 0.82 in seedling stage, ten peaks with Rf values in range of 0.00 to 0.96 in vegetative stage, seven peaks with Rf values in range of 0.01 to 0.91 in reproductive stage. In roots, higher peak areas were found at the reproductive stage (3.33%) compared to vegetative stage (1.65%). Conclusion: The comparative HPTLC fingerprints of leaves and roots in W. somnifera presented phytochemical variability in leaf and root that varied with developmental stages.

HPTLC-based metabolomics for the investigation of metabolic changes during plant development: The case study of Artemisia annua

The application of high performance thin layer chromatography (HPTLC)-based non-targeted metabolomics as a holistic approach to compare fingerprints of metabolite changes during Artemisia annua development is described. Images of HPTLC chromatograms obtained after derivatization with anisaldehyde-sulphuric acid reagent were used as a dataset for multivariate analysis. Principal component analysis and orthogonal partial least squares discriminant analysis confirmed the differentiation of samples belonging to vegetative phase, flowering stage, and seed formation stage of the plant development. The obtained results showed that the HPTLC-based metabolomics approach can be a very reliable technique for the investigation of metabolic changes during plant development, complementary to gas chromatography?mass spectrometry and nuclear magnetic resonance-based metabolomics.

Radical Scavenging and Antimicrobial Properties of Polyphenol Rich Waste Wood Extracts.

The main focus of this study is to assess radical scavenging and antimicrobial activities of the 11 wood extracts: oak (Quercus petraea (Matt.) Liebl., Q. robur L., and Q. cerris L.), mulberry (Morus alba L.), myrobalan plum (Prunus cerasifera Ehrh.), black locust (Robinia pseudoacacia L.), and wild cherry (Prunus avium L.). High-performance thin-layer chromatography (HPTLC) provided initial phenolic screening and revealed different chemical patterns among investigated wood extracts. To identify individual compounds with radical scavenging activity DPPH-HPTLC, assay was applied. Gallic acid, ferulic and/or caffeic acids were identified as the compounds with the highest contribution of total radical scavenging activity. Principal component analysis was applied on the data set obtained from HPTLC chromatogram to classify samples based on chemical fingerprints: Quercus spp. formed separate clusters from the other wood samples. The wood extracts were evaluated for their antimicrobial activity against eight representative human and opportunistic pathogens. The lowest minimum inhibitory concentration (MIC) was recorded against Staphylococcus aureus for black locust, cherry and mulberry wood extracts. This work provided simple, low-cost and high-throughput screening of phenolic compounds and assessments of the radical scavenging properties of selected individual metabolites from natural matrix that contributed to scavenge free radicals.

Chemometric evaluation of alfalfa sprouting impact on its metabolic profile using HPTLC fingerprint-efficacy relationship analysis modelled with partial least squares regression

Sprouting is a commonly applied food processing practice specially in Western countries. Tracking the impact of sprouting of Medicago sativa L. (alfalfa) seeds on their phytochemical composition and curative efficacy was implemented in the current study. Sprouting of alfalfa seeds under controlled conditions for eleven days was performed in a biochemical incubator and three samples were randomly taken each day. A total of thirty-six samples (three ungerminated seeds and thirty-three sprouts samples) were collected, extracted and their cytotoxic, antioxidant and antimicrobial activities against five pathogenic microbial strains were measured. Samples were subjected to High performance thin layer chromatography (HPTLC) as a pattern-oriented strategy for metabolite fingerprinting to discover the fluctuations occurring during the sprouting process accompanied by multivariate chemometric analysis. Unsupervised pattern recognition was carried out using Principal Component Analysis (PCA) after extracting the chromatographic fingerprints from HPTLC chromatograms using ImageJ® software. PCA- loading plots demonstrated that luteolin-7-O-glucoside, ferulic acid and P-coumaric acid were the metabolically significant markers. Thus, simultaneous quantification of these crucial three markers in different aged alfalfa seeds/ sprouts extracts was performed using a newly developed and validated HPTLC-image analysis method. The results of the biological activities together with the quantitative data were further subjected to a Partial Least Squares Regression (PLSR) model for implementing HPTLC fingerprint-efficacy relationship analysis. The results obtained from metabolic pool profiling revealed that sprouting can cause remarkable changes in the phytochemical, nutritional and efficacy characteristics of alfalfa seeds.

Altitudinal variation in gallic acid content in fruits of Phyllanthus emblica L. and its correlation with antioxidant and antimicrobial activity

Gallic acid is one of the most important organic compounds in fruits of Phyllanthus emblica. Therefore, the objective of the present study is to find out the effect of altitude on gallic acid content in fruits of P. emblica and its correlation with antioxidant and antimicrobial activity. Phytochemicals such as phenolics, tannins, flavonoids, carbohydrates, glycosides, phytosteroids, alkaloids and saponins were detected in fruit extracts of P. emblica collected from different regions of Himachal Pradesh. Fruits extract from Mandi (239.74 ± 39.28 mg/g gallic acid equivalents, GAE) district showed a higher amount of total phenolic content (TPC), whereas, total flavonoid content (TFC) was higher from Kangra (356 ± 27.63 mg/g rutin equivalents, RE) district. Methanolic extracts showed inhibition to the growth of both Gram-positive (B. subtilis, S. aureus), and Gram-negative bacteria (E. coli, K. pneumoniae). The methanolic extract of fruits of Bilaspur district showed the highest antibacterial activity against B. subtilis (19.5 ± 0.71 mm), S. aureus (21.0 ± 1.41 mm), E. coli (17.5 ± 0.71) and K. pneumoniae (21.5 ± 2.12) as compared to other regions and amoxyclav. High-performance thin-layer chromatography (HPTLC) method was used for the quantification of gallic acid in the extracts of fruits of P. emblica. HPTLC chromatogram showed the highest content of gallic acid in methanolic extracts of fruits from Kangra followed by Bilaspur, Mandi, and Una. However, antioxidant and antibacterial activity was higher in fruits extracts of high altitude (Bilaspur region). In summary, Bilaspur region of Himachal Pradesh could be used for mass cultivation of fruits of P. emblica because of their high antioxidant and antimicrobial potential under these geographical conditions.

Chemotaxonomic differentiation of Clerodendrum species based on high-performance thin-layer chromatographic fingerprinting of key secondary metabolites and chemometric data analysis

Clerodendrum viscosum leaves are used in indigenous systems of medicines of mainland and maritime Southeast Asian countries for the treatment of fever, pain, dysentery, colic, and removal of Ascarids. The Clerodendrum species under study exhibit various phytochemical and morphological similarities. Therefore, it is very challenging to distinguish raw powdered materials used for therapeutic purposes. A validated high-performance thin-layer chromatography (HPTLC) method with 4 key markers, viz., 24β-ethylcholesta-5,22E,25-triene-3β-O-D-glucoside, clerodinin-A, 24β-ethylcholesta-5,22E,25-triene-3β-ol, and lupeol coupled with a chemometric analysis was used to distinguish 3 closely related Clerodendrum species, viz., C. inerme, C. multiforum, and C. viscosum. PRISMA approach was applied for effective HPTLC fingerprint development. The HPTLC—densitometry method was validated following the current International Conference on Harmonisation (ICH) guidelines. Taxonomic differentiation was established by fingerprint-based similarity analysis, a chemotaxonomic study using hierarchical clustering analysis (HCA), and principal component analysis (PCA) was done. HPTLC chromatogram similarity was calculated as correlation coefficient and congruence coefficient values, demonstrating poor similarities (0.26–0.86). However, PCA has resulted in 2 principal component (PC) loadings. PC1 separated C. multiforum, explaining 85.48% of variance mainly due to distribution of 2 triterpenoids. The present HPTLC method is coupled to marker-based quality determination of raw plants as well as discrimination of Clerodendrum species. Chemometric analysis based on 4 metabolites clearly establishes a practical identification of Clerodendrum species intended for therapeutic use.

Effect-directed profiling of aqueous, fermented plant preparations via high-performance thin-layer chromatography combined with in situ assays and high-resolution mass spectrometry

Aqueous, fermented preparations from a vast range of medical plants are among the active pharmaceutical ingredients of homoeopathic medicinal products. In this study aqueous, fermented plant preparations from Chamomilla recutita L. (C.r.), Allium cepa L. (A.c.), Equisetum arvense L. (E.a.) and Hamamelis virginiana L. (H.v.) of different years of harvest were screened using a generic high-performance thin-layer chromatography (HPTLC) method hyphenated to effect-directed analysis (EDA) and high-resolution mass spectrometry (HRMS). Using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•), Gram-positive Bacillus subtilis and α-/β-glucosidase assays, followed by recording of elution head-based HPTLC-ESI-HRMS spectra, a fast assignment of multi-potent compounds was possible. This was exemplified for herniarin identified in C.r. and gallic acid in H.v. preparations. Strong radical scavenging and β-glucosidase inhibiting effects were observed for E.a. and H.v. preparations, whereas the most pronounced antibiotic activity against Gram-positive bacteria was found in C.r. and E.a. preparations. Besides the successful screening for radical-scavenging, antibiotic and anti-diabetic components, HPTLC-UV/Vis/FLD-EDA-HRMS provided profound information on aqueous, fermented plant preparations. Batch-to-batch variations of the plant preparations were not necessarily obvious from their HPTLC chromatograms, but could be substantiated based on their respective (bio)autograms. It became clear that effect-directed fingerprints allow the visualization and control of the fermentation processes in a meaningful sense.

STANDARDIZED SUPERCRITICAL CO2 EXTRACT OF ACANTHUS ILICIFOLIUS (LINN.) LEAVES INHIBITS THE PRO-INFLAMMATORY CYTOKINE TUMOR NECROSIS FACTOR-Α IN LIPOPOLYSACCHARIDE-ACTIVATED MURINE RAW 264.7 MACROPHAGE CELLS

Objective: Acanthus ilicifolius Linn. (Acanthaceae) is a medicinal mangrove plant used in the treatment of inflammation. Previous phytochemical studies have identified 2-benzoxazolinone (BOA) from the leaves of A. ilicifolius. In the present study, we attempted to standardize the supercritical CO2 leaf extract of A. ilicifolius (SCFE-AI) for BOA content and investigate the tumor necrosis factor-α (TNF-α) inhibitory effect of SCFE-AI and BOA on the lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. The acute oral toxicity of SCFE-AI and BOA was also established. Methods: SCFE-AI was standardized for BOA content using high-performance thin-layer chromatography (HPTLC) method. The cytotoxicity of SCFEAI and BOA was evaluated using MTS colorimetric method. The in vitro anti-inflammatory effect of SCFE-AI and BOA on TNF-α production in LPSactivated RAW 264.7 cells was quantified using ELISA method. Acute oral toxicity studies were performed following the Organization for Economic Co-operation and Development test guideline No. 423. Results: The amount of BOA was found 0.8% w/w of SCFE-AI. The RAW 264.7 cell viability was unaffected by SCFE-AI and BOA treatments within a concentration range <1000 mg/ml after 24 h incubation. SCFE-AI decreased the production of TNF-α in a dose-dependent manner compared to BOA. The LD50 value for SCFE-AI was found to be >2000 mg/kg and ranges from 300 to 2000 mg/kg with BOA. Conclusion: The HPTLC chromatogram could serve as an analytical tool for authentication and quantification of BOA content. The anti-inflammatory mechanism of A. ilicifolius might be through the inhibition of TNF-α production.

Effect of different extraction techniques on total phenolic and flavonoid contents, and antioxidant activity of betelvine and quantification of its phenolic constituents by validated HPTLC method.

The objectiveof the study is to analyze the effect of different extracting methods on the polyphenolic content and antioxidant activities in Piper betle leaves. In the present research, P. betle leaf extract was prepared by sonication, Soxhlet and maceration methods using acetone (100%, v/v). The efficiency of the extraction methods was estimated by quantifying the total phenolic content (TPC) by the Folin-Ciocalteu method and total flavonoid content (TFC) by AlCl3 colorometric methods, and antioxidant power of the various extracts was determined by DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging assay. Thin-layer chromatography (TLC) bioautography was carried out to identify antioxidants, and their amount was determined by the newly developed high-performance thin-layer chromatography (HPTLC) method. DPPH free radical scavenging capacity of the different extracts from strongest to weakest was as follows: ascorbic acid (4.27µg/mL) > sonication (5.35µg/mL) >, maceration (5.53µg/mL)>, soxhlet extraction (5.83µg/mL). Same trend was also observed for the ABTS radical scavenging capacity. Similarly, findings of this study also showed that sonication extract possessed highest phenolic and flavonoid contents followed by maceration and Soxhlet extraction. In addition, important bioactive phenolic constituents which contribute largely towards antioxidant potential such as eugenol and eugenol acetate were quantified using HPTLC (high-performance thin-layer chromatography) method. The average percent recovery of eugenol and eugenol acetate was found to be 97.28% and 98.04%, respectively. The LOD (limit of detection) and LOQ (limit of quantification) for eugenol were 5 and 15ng/spot, whereas that of eugenol acetate were 10 and 30ng/spot. The HPTLC densitometric determination also supported the results of antioxidant assays by revealing the presence of higher amount of identified antioxidants in sonication followed by maceration and Soxhlet extraction. The developed HPTLC chromatogram profile may be used as a reference for the standardization of P. betle leaf extracts.

Design and development of microparticulate delivery system for Curcumin

Background: Curcumin has been associated with remarkable beneficial effects; despite having a broad spectrum of activities, curcumin is characterized by poor water solubility and low bioavailability. Objective: The objective of this study is to enhance the solubility of curcumin using polyethylene glycol 400 (PEG400) and establish a microparticulate drug delivery system in a sodium alginate polymer matrix (microspheres containing curcumin triturated with PEG400 [Cur-PEGMS]) to improve the bioavailability of curcumin. Materials and Methods: The microspheres were formulated using the ionic gelation technique. Nine batches (F1 to F9) were prepared using 32 factorial design. The amount of sodium alginate and calcium chloride was selected as a formulation variable. The prepared Cur-PEGMS were characterized by Fourier-transform infrared spectroscopy, scanning electron microscope, differential scanning calorimeter, ultraviolet spectrophotometer, and high-performance thin-layer chromatography (HPTLC). The pharmacokinetics of curcumin was characterized in rats using high-performance liquid chromatography, and calculations were performed using WinNonlin standard edition version 1.1 software. Results: We found that solubility was affected by the use of cosolvent. All F1–F9 batches were investigated for entrapment efficiency, and batch F5 was found to be optimum batch which was further evaluated. HPTLC chromatogram of Cur-PEGMS shows peak retention for curcumin. The particle size of microspheres was found to be in the range of 384–468 μm. Remarkable improvement in maximum plasma concentration and bioavailability was observed. Maximum the time after administration of a drug when maximum plasma concentration is reached (Tmax) was found to be 0.5 h, and AUC0–24was found to be 374.75 ng/ml and 622 ng/ml for curcumin suspension and curcumin microspheres, respectively. Conclusion: The developed formulation enhances the bioavailability of the drug. This enhanced oral absorption of Cur-PEGMS may provide a practical formulation to conduct a correlative pharmacodynamic study.

Morphological, Anatomical, Genetical and High Performance Thin Layer Chromatography Profiling of Buddleia indica (Scrophulariaceae)

Buddleia indica Lam is an evergreen African shrub, native to Madagascar. Botanical investigations were done on the entire fresh plant leaves, old and young roots and stems, their histological sections as well as air-dried finely powdered samples. Genetic profiling and DNA fingerprinting were accomplished using RAPD-PCR technique (Random Amplified Polymorphic DNR- Poymerase Chain Reaction). Preliminary metabolic screening was performed using high performance thin layer chromatography (HPTLC) analysis. Botanical study of Buddleia indica revealed the presence of non –glandular 4-armed stellate trichomes, cruciferous stomata in the leaves, and the existence of U- shaped median vascular strand accompanied by small accessory bundles in the wings of its petiole all showed its close similarity to the Buddleioideae. Additionally, the cork in the stem that arouses from the pericycle (pericylic cork) and the pericycle which forms a loose ring of fibers surrounding the collateral vascular bundles are common characteristic among the members of Buddleioideae On the genetic level, it can be concluded that the most discriminative and relevant fragment resulting from the successful combination of template and primer was that produced by two primers OPA-09 and OPA-06 followed by OPA-07 RAPD primers attributing to the generation of 14, 13 and 12 amplified DNA fragments. Examination of HPTLC chromatograms of the leaves methanol extract showed ten major spots existing at different percentages that give bluish green color upon spraying with ferric chloride solution indicating its richness by phenolic compounds. Buddleia indica is rich in phenolic compounds and it belongs to Buddleioideae owing to the botanical resemblance.

STANDARDIZED SUPERCRITICAL CO2 EXTRACT OF ACANTHUS ILICIFOLIUS (LINN.) LEAVES INHIBITS THE PRO-INFLAMMATORY CYTOKINE TUMOR NECROSIS FACTOR-Α IN LIPOPOLYSACCHARIDE-ACTIVATED MURINE RAW 264.7 MACROPHAGE CELLS

Objective: Acanthus ilicifolius Linn. (Acanthaceae) is a medicinal mangrove plant used in the treatment of inflammation. Previous phytochemical studies have identified 2-benzoxazolinone (BOA) from the leaves of A. ilicifolius. In the present study, we attempted to standardize the supercritical CO2 leaf extract of A. ilicifolius (SCFE-AI) for BOA content and investigate the tumor necrosis factor-α (TNF-α) inhibitory effect of SCFE-AI and BOA on the lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. The acute oral toxicity of SCFE-AI and BOA was also established.Methods: SCFE-AI was standardized for BOA content using high-performance thin-layer chromatography (HPTLC) method. The cytotoxicity of SCFE-AI and BOA was evaluated using MTS colorimetric method. The in vitro anti-inflammatory effect of SCFE-AI and BOA on TNF-α production in LPS-activated RAW 264.7 cells was quantified using ELISA method. Acute oral toxicity studies were performed following the Organization for Economic Co-operation and Development test guideline No. 423.Results: The amount of BOA was found 0.8% w/w of SCFE-AI. The RAW 264.7 cell viability was unaffected by SCFE-AI and BOA treatments within a concentration range <1000 mg/ml after 24 h incubation. SCFE-AI decreased the production of TNF-α in a dose-dependent manner compared to BOA. The LD50 value for SCFE-AI was found to be >2000 mg/kg and ranges from 300 to 2000 mg/kg with BOA.Conclusion: The HPTLC chromatogram could serve as an analytical tool for authentication and quantification of BOA content. The anti-inflammatory mechanism of A. ilicifolius might be through the inhibition of TNF-α production.

MICROBICIDAL POTENTIALITY OF PURIFIED ANTHOCYANIN FROM IN VITRO CULTURE OF CLERODENDRON INFORTUNATUM L. AGAINST SELECTED PATHOGENS

Objective: Clerodendron infortunatum L. is a widely used medicinal herb over centuries for curing many skin-borne disorders. The present study was designed to validate the tribal knowledge by evaluating antimicrobial potential of purified anthocyanin extracted from in vitro cell suspension culture.Methods: The explants were inoculated on murashige and skoog (MS) medium mixed with various combinations of 2, 4-D a+BAP for callus induction. Green compact callus was initiated within 30 d from the explants on MS medium fortified with benzylaminopurine (BAP) (2.0 mg/l)+2, 4-D (0.5 mg/l). Subsequently, anthocyanin was triggered from the compact callus by subculturing in the medium containing 2, 4-D and Kinetin. Cell suspension culture was also developed. Anthocyanin production was enhanced by elicitation using salicylic acid and others. Three chromatographic methods such as solid phase extraction by Sepharose C18 column, Oasis-MCX and Amberlite XAD 7+Sephadex LH 120 sorbents were used to purify the in vitro synthesized anthocyanin from the cell cultures. HPLC and molar absorptivity assay were carried to check the purity. Antimicrobial analysis was also carried using standard protocols to check minimum inhibitory concentration (MIC) and minimum killing concentration (MKC).Results: The mean purity values obtained by high-performance thin layer chromatography (HPLC) were 90.9%±1.9, 80.60%±2.3 for Oasis MCX, Amberlite XAD-7+Sephadex LH-20 column respectively. However, the purity by molar absorptivity was found to be less. HPLC chromatogram revealed 12 fractions of anthocyanin. Inhibition zone diameter, MIC and MKC values obtained for the purified anthocyanin revealed its antimicrobial potentiality but at different levels among the selected bacteria and fungi. C. albicans, S. aureus, P. aerugenosa showed significant values followed by MRSA, E. coli and A. flavus. The results are comparable with the synthetic antibiotics. However, E. faecalis was more resistance. Mode of action was confirmed from the results of intracellular potassium leakage and bacterial membrane integrity analysis.Conclusion: Thus, the study confirms the efficacy of anthocyanin as natural antimicrobial and suggests the possibility of employing it as drugs for the treatment of infectious diseases caused by the pathogens.

Proof-of-Principle of rTLC, an Open-Source Software Developed for Image Evaluation and Multivariate Analysis of Planar Chromatograms.

High-performance thin-layer chromatography (HPTLC) is an advantageous analytical technique for analysis of complex samples. Combined with multivariate data analysis, it turns out to be a powerful tool for profiling of many samples in parallel. So far, chromatogram analysis has been time-consuming and required the application of at least two software packages to convert HPTLC chromatograms into a numerical data matrix. Hence, this study aimed to develop a powerful, all in one open-source software for user-friendly image processing and multivariate analysis of HPTLC chromatograms. Using the caret package for machine learning, the software was set up in the R programming language with an HTML-user interface created by the shiny package. The newly developed software, called rTLC, is deployed online, and instructions for direct use as a web application and for local installation, if required, are available on GitHub. rTLC was created especially for routine use in planar chromatography. It provides the necessary tools to guide the user in a fast protocol to the statistical data output (e.g., data extraction, preprocessing techniques, variable selection, and data analysis). rTLC offers a standardized procedure and informative visualization tools that allow the user to explore the data in a reproducible and comprehensive way. As proof-of-principle of rTLC, German propolis samples were analyzed using pattern recognition techniques, principal component analysis, hierarchic cluster analysis, and predictive techniques, such as random forest and support vector machines.

Comparative study of different approaches for multivariate image analysis in HPTLC fingerprinting of natural products such as plant resin

Considering the introduction of phytochemical fingerprint analysis, as a method of screening the complex natural products for the presence of most bioactive compounds, use of chemometric classification methods, application of powerful scanning and image capturing and processing devices and algorithms, advancement in development of novel stationary phases as well as various separation modalities, high-performance thin-layer chromatography (HPTLC) fingerprinting is becoming attractive and fruitful field of separation science. Multivariate image analysis is crucial in the light of proper data acquisition. In a current study, different image processing procedures were studied and compared in detail on the example of HPTLC chromatograms of plant resins. In that sense, obtained variables such as gray intensities of pixels along the solvent front, peak area and mean values of peak were used as input data and compared to obtained best classification models. Important steps in image analysis, baseline removal, denoising, target peak alignment and normalization were pointed out. Numerical data set based on mean value of selected bands and intensities of pixels along the solvent front proved to be the most convenient for planar-chromatographic profiling, although required at least the basic knowledge on image processing methodology, and could be proposed for further investigation in HPLTC fingerprinting.

Characteristic expression of glycosphingolipid profiles in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60

Changes of glycosphingolipids (GSLs) in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60 cells were investigated by high-performance thin-layer chromatography (HPTLC), with special reference to morphological and functional changes, such as phagocytosis and nitroblue tetrazolium (NBT) reduction. Nine molecular species of neutral GSLs and 13 or more species of sialo-GSLs, ie, gangliosides, were detected on the HPTLC chromatograms for untreated HL-60 cells. The major components were ceramide dihexoside (CDH), GM3, and sialo-paragloboside (SPG). When HL- 60 cells were induced to differentiate into both myeloid mature cells and macrophage-like cells in vitro, no new molecular species of GSLs specific for one of the cell differentiations was induced, but distinctive quantitative changes in the GSL composition were definitely observed between the two cell differentiations. During the myeloid differentiation induced by either dimethylsulfoxide (DMSO) or retinoic acid (RA), CDH, paragloboside (PG), and gangliosides having longer sugar moieties characteristically increased with a concomitant decrease of GSLs with shorter sugar chains, such as ceramide monohexoside (CMH) and GM3, and the GSL composition profile of myeloid differentiation- induced HL-60 cells became more similar to that of normal human granulocytes. However, some marked differences were noted between the induced HL-60 cells and the normal granulocytes, especially in the ganglioside compositions. These differences might reflect either some deficiency in the in vitro myeloid differentiation or some leukemic properties of HL-60 cells. In marked contrast to the change of GSL composition during myeloid differentiation, a remarkable increase of GM3, with a concurrent marked decrease of CDH, was observed in the process of cell differentiation into macrophage-like cells with 12-O- tetradecanoyl-phorbol-13-acetate (TPA), which suggested an increase in the biosynthesis of GM3. These results demonstrate that HL-60 cells express distinct GSL profiles, depending not only on maturation stages but also on differentiation directions.

Characteristic Expression of Glycosphingolipid Profiles in the Bipotential Cell Differentiation of Human Promyelocytic Leukemia Cell Line HL-60

Changes of glycosphingolipids (GSLs) in the bipotential cell differentiation of human promyelocytic leukemia cell line HL-60 cells were investigated by high-performance thin-layer chromatography (HPTLC), with special reference to morphological and functional changes, such as phagocytosis and nitroblue tetrazolium (NBT) reduction. Nine molecular species of neutral GSLs and 13 or more species of sialo-GSLs, ie, gangliosides, were detected on the HPTLC chromatograms for untreated HL-60 cells. The major components were ceramide dihexoside (CDH), GM3, and sialo-paragloboside (SPG). When HL-60 cells were induced to differentiate into both myeloid mature cells and macrophage-like cells in vitro, no new molecular species of GSLs specific for one of the cell differentiations was induced, but distinctive quantitative changes in the GSL composition were definitely observed between the two cell differentiations. During the myeloid differentiation induced by either dimethylsulfoxide (DMSO) or retinoic acid (RA), CDH, paragloboside (PG), and gangliosides having longer sugar moieties characteristically increased with a concomitant decrease of GSLs with shorter sugar chains, such as ceramide monohexoside (CMH) and GM3, and the GSL composition profile of myeloid differentiation-induced HL-60 cells became more similar to that of normal human granulocytes. However, some marked differences were noted between the induced HL-60 cells and the normal granulocytes, especially in the ganglioside compositions. These differences might reflect either some deficiency in the in vitro myeloid differentiation or some leukemic properties of HL-60 cells. In marked contrast to the change of GSL composition during myeloid differentiation, a remarkable increase of GM3, with a concurrent marked decrease of CDH, was observed in the process of cell differentiation into macrophage-like cells with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which suggested an increase in the biosynthesis of GM3. These results demonstrate that HL-60 cells express distinct GSL profiles, depending not only on maturation stages but also on differentiation directions.