High-performance Thin-layer Chromatographic Fingerprint Research Articles

Gas chromatography‐mass spectrophotometric profile of cold‐pressed Celastrus paniculatus seed oil and its high‐performance thin‐layer chromatographic analysis emphasizing squalene content

AbstractCelastrus paniculatus, a medicinal plant with diverse therapeutic activities, is known as ‘malkangani’ in the traditional medicine system of India, Ayurveda. It is well known for its memory‐enhancing potential. The reported work involves the gas chromatography‐mass spectrophotometric analysis of cold‐pressed seed oil of Celastrus paniculatus. It revealed the presence of components such as monosaturated fatty acids, polyunsaturated long‐chain fatty acids, and their derivatives. It also showed the presence of terpenes such as squalene, β amyrone, β amyrin acetate, and long‐chain hydrocarbons. Among all the constituents, squalene was found to be a major component. Hence, considering the potential pharmacological actions of squalene, a high‐performance thin‐layer chromatographic (HPTLC) method has been developed for its quantification and subsequently validated as per the International Conference of Harmonization guidelines. A HPTLC fingerprint of oil was obtained on pre‐coated silica gel 60 F254 aluminum sheets, using n‐hexane‐diethyl ether‐glacial acetic acid (7:3:0.05 v/v/v). A densitometric scan was performed at 470 nm after derivatization with vanillin sulphuric acid to quantify squalene. A well‐resolved squalene peak was obtained at a retardation factor of 0.85. It is a simple approach for the estimation of squalene in Celastrus paniculatus seed oil for quality control.

Extraction solvent selection for Cannabis sativa L. by efficient exploration of cannabinoid selectivity and phytochemical diversity.

Cannabis sativa L. is attracting worldwide attention due to various health-promoting effects. Extraction solvent type is critical for the recovery of bioactive compounds from the plant, especially cannabinoids. However, the choice of solvent is varied and not adequately warranted elsewhere, causing confusion in involved fields. The present work aimed to investigate the effect of extraction solvent on C. sativa (hemp) with regard to cannabinoid recovery and phytochemical profile of the extracts, considering most of the related solvents. The majority of solvents reported for C. sativa (n = 14) were compared using a representative hemp pool. Quantitative results for major and minor cannabinoids were rapidly and reliably obtained using ultrahigh-performance liquid chromatography coupled with photodiode array detection (UPLC-PDA). In parallel, high-performance thin-layer chromatographic (HPTLC) fingerprinting was employed, involving less toxic mobile phase than in relevant reports. Various derivatisation schemes were applied for more comprehensive comparison of extracts. Differential selectivity towards cannabinoids was observed among solvents. MeOH was found particularly efficient for most cannabinoids, in addition to solvent systems such as n-Hex/EtOH 70:30 and ACN/EtOH 80:20, while EtOH was generally inferior. For tetrahydrocannabinol (THC)-type compounds, EtOAc and n-Hex/EtOAc 60:40 outperformed n-Hex, despite its use in the official EU method. Solvents that tend to extract more lipids or more polar compounds were revealed based on HPTLC results. Combining the observations from UPLC quantitation and HPTLC fingerprinting, this work allowed comprehensive evaluation of extraction solvents, in view of robust quality assessment and maximised utilisation of C. sativa.

Chemical Standards and HPTLC Finger Print Profiles of a Siddha Polyherbal Formulation - Kadukkai Legiyam

To study physico-chemical, phytochemical and high performance thin layer chromatography of a Siddha drug “Kadukkai Legiyam” (KL). The prepared Kadukkai Legiyam (KL) was prepared as per the standard operating procedures mentioned in literature. Then the drug was subjected to physicochemical parameters, phytochemical screening, thin layer chromatographic photo documentation (TLC), high performance thin layer chromatographic (HPTLC) finger print profile of hexane, chloroform, ethanol and hydro alcohol (1:1) extracts. Different extracts of the drug showed distinct TLC and HPTLC finger print patterns which will be unique to this drug. This study giving information about physiochemical and phytochemical analysis and HPTLC fingerprint profile of different extracts, the integration spectrum which will useful in standardizing the raw drugs and future comparison studies.

TLC and HPTLC Finger Printing Analysis of Cyperus rotundus (Linn.)

Cyperus rotundus (Linn.) is a versatile plant belonging to the family Cyperaceae, used in herbal medicines worldwide to cure various human ailments. The present study attempts to analyze the profiles of flavonoids in different extracts of C. rotundus with the help of thin-layer chromatography (TLC) and high-performance thin-layer chromatographic (HPTLC) fingerprint. Flower and stem extracts of C. rotundus were screens out with the help of TLC, and the Rf values were determined. HPTLC was used to quantify the flavonoid from flower extract of a plant at a 1.0 mg/mL concentration. It revealed the occurrence of flavonoids, especially quercetin in the ethanolic extract of C. rotundus flower, by using mobile phase toluene-ethyl acetate-formic acid (3:4:2.5 v/v), on a pre-coated plate of silica gel and quantified the amount of quercetin by densitometry absorbance mode at 257 nm. The limits of detection and quantification were 30.08 & 91.16 ng/mL, and the relative standard deviation ranged between 1.03 to 1.48 for intra-day and inter-day for HPTLC. The calibration range was 200-700 ng per band (r2 = 0.99321). Quercetin quantity in the ethanolic extract of the flower was found to be 0.011 mg/mL of the extract with an average recovery of 99.01–100.00%. Such fingerprinting is valuable in quality control and checking adulterants of natural drugs. Therefore, it can be helpful for the assessment of different marketable pharmaceuticals preparations.

Densitometric high-performance thin-layer chromatographic fingerprinting method for the determination and quantification of plumbagin in Plumbago zeylanica L. roots

Densitometric high-performance thin-layer chromatographic fingerprinting method for the determination and quantification of plumbagin in Plumbago zeylanica L. roots

Importance of chromatographic and spectrophotometric methods in determining authenticity, classification and bioactivity of honey

Determination of the botanical origin and bioactivity of honey is crucial parameters that may effect health benefits and market value of honey. Melissopalynology is frequently practiced for identifiying its botanical origin, however, this study may sometimes not allow a clear authentication of its origin. Therefore, complementary methods as chromatographic techniques are recommended to accomplish the purpose. In this study, thirty-three Turkish honeys were first authenticated by melissopalynology and classified as honeydew and blossom honeys. Then, phenolic characterization and quantification in honeys were performed by high-performance thin-layer chromatographic (HPTLC) and a validated liquid chromatographic (HPLC) methods, respectively and applicability of these methods in place of melissopalinology was evaluated. Pine, chestnut, and sunflower honeys showed individually characteristic HPTLC fingerprint. Key markers were quantified by HPLC and caffeic acid, one of the marker components, was found to be highest in sunflower honey as 2.63±0.04 mg/g. Honeys were further subjected to principal component (PCA) and hierarchical clustering (HCA) analyses according to the key markers and pine, chestnut, and sunflower honeys were grouped independently. In terms of bioactivity, pine honey showed the highest antioxidant effect among investigated samples. Lastly, some phenolic compounds were found to contribute significantly to the antioxidant effect of honeys by HPTLC-DPPH.

A New Type of Anatolian Propolis: Evaluation of Its Chemical Composition, Activity Profile and Botanical Origin.

This study was undertaken to analyze the phenolic profiles of 19 propolis samples from Turkey by using a high-performance thin-layer chromatographic (HPTLC) method in order to identify their plant origins. Furthermore, their antioxidant and antimicrobial activity profiles were comparatively evaluated. For the appraisal of antioxidant potential, total phenolic (TPC) and total flavonoid contents (TFC) of propolis samples were firstly determined and then their effects on free radicals were evaluated by FRAP, ABTS.+ , CUPRAC, DPPH. and HPTLC-DPPH. methods. Antimicrobial activity of propolis samples against Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 11229) and Candida albicans ATCC 10231 were determined by disc diffusion and broth dilution methods. HPTLC fingerprinting analyses revealed that O-type (botanical origin from Populus nigra L.) was the primarily available propolis type in Turkey. Moreover, 3-O-methylquercetin (3MQ) rich propolis was identified as a new propolis type for the first time. Principal component analysis (PCA) indicated that 3MQ-type propolis differs from the O-type. Antioxidant activity studies showed that O-type of propolis possesses higher antioxidant effect than the other tested propolis types. Quercetin, caffeic acid, caffeic acid phenethyl ester (CAPE) and galangin were determined to contribute significantly to the antioxidant potential of O-type propolis among others. Propolis extracts exerted moderate antimicrobial activity against the tested microorganisms with MIC values between the ranges of 128-512 μg/mL.

Powder Microscopic, Physicochemical, HPTLC and Antioxidant Studies on Noccik Kudinir Chooranam – A Polyherbal Siddha Formulation

<p style="text-align: justify;"><strong>Objectives:</strong> To study pharmacognostical, physico-chemical, high performance thin layer chromatography and anti-oxidant activity potential of a Siddha drug &ldquo;Noccik Kudinir Chooranam&rdquo; (NKC). <strong>Methods:</strong> The raw drugs were collected, authenticated and the Kudinir Chooranam was prepared. Then the drug was subjected to powder microscopic diagnosis, physicochemical parameters, Thin Layer Chromatographic photo documentation (TLC), High Performance Thin Layer Chromatographic (HPTLC) finger print profile of successive hexane, successive chloroform, successive ethanol, hydro alcohol (1:1) extract, ethyl acetate solubles of water extract, alkaloid fraction and direct ethanol along with ingredients. The methanol and aqueous extracts were evaluated for total phenol and flavonoid, DPPH radical scavenging activity and superoxide scavenging activities.<strong> Results:</strong> Different extracts of the drug showed distinct TLC and HPTLC finger print patterns which will be unique to this drug. The antioxidant activity showed significant results which are comparable to standards. <strong>Conclusion:</strong> The identified powder microscopic characters and the evolved physicochemical parameters would serve as reference tool for quality control and the antioxidant activity would find the much wider therapeutic importance of the drug. <p style="text-align: justify;"><strong>Key words:</strong> Noccik Kudinir, Vitex negundo, Allium sativum, TLC, HPTLC, Antioxidant activity.

High-performance thin-layer chromatographic fingerprinting of sandalwood essential oils

The potential of high-performance thin-layer chromatographic (HPTLC) fingerprinting in identifying differences in sandalwood essential oils from 5 sandalwood species, namely, Santalum album, Santalum spicatum, Santalum austrocaledonicum, Santalum paniculatum, Santalum lanceolatum, and a natural substitute for sandalwood, Osyris lanceolata, was explored. Variation was observed in the profile of bands (RF values and color) and peak intensity profiles displayed by the essential oils across and within the essential oils studied with some bands being unique to the individual species. The potential of HPTLC fingerprinting as a quality control tool in authenticating sandalwood oils in the sandalwood industry was demonstrated in the present study.

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.

Pharmacognostic evaluation and HPTLC fingerprinting of Pirantai Vatakam, a Siddha formulation

The present study is an attempt to evaluate the pharmacognostic parameters and HPTLC fingerprint profiles for a Siddha compound formulation, Pirantai vatakam in which Cissus quadrangularis is the main ingredient. Powder microscopy studies were carried out and different microscopic characters were distinguished. Physico-chemical parameters such as loss on drying at 105⁰C, total ash, acid insoluble ash, water soluble extractive, alcohol soluble extractive AND volatile oil were determined. High performance thin layer chromatographic (HPTLC) study of Pirantai vatakam was performed and the chromatograms were documented. The observations laid down a platform for the standardization of Pirantai vatakam and will help us to determine the genuineness of the drug from the adulterants and substitutes.

High-performance thin-layer chromatography profiling of Jarrah and Manuka honeys

This article presents the findings of an in-depth study on high-performance thin-layer chromatographic (HPTLC) profiling of Jarrah (Eucalyptus marginata) and Manuka (Leptospermum spp.) honeys following a simple one-step solvent extraction process. The study demonstrates that different HPTLC fingerprints are obtained from honeys from varying floral sources and that honeys of the same floral origin present a consistent reproducible HPTLC profile. In addition, the linearity and reproducibility of this technique as well as its ability to detect (accidental or deliberate) contaminations with honeys of different floral sources are demonstrated. The study thus illustrates the usefulness of HPTLC profiling as a potential quality control tool that might complement other analytical techniques used in the authentication of monofloral honeys.

HPTLC Phenolic Profiles as Useful Tools for the Authentication of Honey

The present study reveals the utility of high-performance thin-layer chromatographic (HPTLC) fingerprinting of phenolic constituents for the authentication of monofloral honeys. The obtained data enables a more complete assessment of honey quality and the identification of emerging threats to honey quality. The developed procedure facilitates differentiation of varietal honeys and detection of honey adulterations. We used an HPTLC fingerprint analysis to determine the characteristic patterns of different honey types (willow, buckwheat, heather, pine honeydew, and manuka honey). The HPTLC chromatograms were used to determine the differences in the botanical origin of the honey samples on the basis of the band profiles, which are characteristic for each honey type. Identification of 11 polyphenols was performed by comparison of the color and Rf of the bands with available standards. Additionally, the results were confirmed by an HPLC analysis.

Preparation and standardization of Mathan Tailam: A classical Siddha formulation for diabetic ulcerative wound healing

BackgroundIncreasing popularity of Mathan Tailam (mattan tailam, pachai ennai) for the treatment of diabetic foot ulcer necessitated standardization and quality control of this medicated oil both in large scale production and quality check in marketed drug. ObjectivePresent study aims to develop standard operating procedure for the preparation of Mathan Tailam as per Siddha Formulary of India and its standardization using suitable analytical techniques. Materials and methodsMathan Tailam was prepared as per Siddha Formulary of India. Physicochemical parameters and preliminary phytochemical screening were carried out using standard methods. The in-house prepared sample underwent physico-chemical analysis, qualitative phytochemical analysis, Gas chromatography-mass spectrum (GC-MS) analysis, High performance thin layer chromatographic (HPTLC) fingerprinting profile and inductively coupled plasma-optical emission spectroscopic (ICP-OES) analysis. ResultsPhysico-chemical parameters of the prepared formulation were comparable to that of coconut oil. Aqueous methonolic extract of this drug was found to be positive for alkaloid, saponin, coumarin, steroid, triterpinoid, quinine and furan. The GC-MS values were comparable to that of the base used i.e., the coconut oil. HPTLC fingerprinting profile revealed the presence of phytochemicals in the medicated oil derived from both coconut oil and Datura metel. ICP-OES addressed the mineral portion of the formulation and its safety in heavy metal aspect. ConclusionAll these parameters can be utilized for the overall quality check over its preparation and formulation.

Development and validation of HPTLC fingerprints of three species of Alpinia with biomarker Galangin

BackgroundAlpinia galanga (L.) Willd. commonly called as Rasna, Greater galangal or Kulinjan is a medicinally important rhizome used in Indian traditional system of medicine to cure a number of ailments. A. galanga is the main source of a galangin -a medicinally important flavanol which has a number of pharmacological properties viz. anti-mutagenic, and anti-inflammatory. Due to the high demand for the rhizome of A. galanga traders are now substituting it with rhizomes of A. calcarata and A. officinarum.MethodsThe present study aims to develop high performance thin layer chromatographic (HPTLC) fingerprinting of A. galanga with its adulterants or substitutes and to quantify bioactive galangin present thereof. Methanolic extracts were obtained from rhizomes of the three species of Alpinia used for HPTLC analysis using silica gel 60 F254 plates and hexane: ethyl acetate: acetic acid (6.2: 2.8: 1.0 v/v/v); the densitometric analysis was performed at 272 nm.ResultsBy comparison of Rf values and of the spectra of the bands with those of the standard galangin was identified in all three samples. HPTLC quantitative analysis of the methanolic extracts showed the decline trend in the quantity of the galangin in the three species of Alpinia as A. galanga (7.67 ± 0.36 mg/g) > A. officinarum (5.77 ± 0.71 mg/g) > A. calcarata (4.31 ± 0.44 mg/g). The HPTLC method was validated using International Conference on Harmonization (ICH) guidelines. The HPTLC method showed good linearity, recovery and high precision of biomarker.ConclusionsRapid and reproducible method is useful for routine analysis of galangin and quality control of Alpinia galangal along with its adulterants or substitutes.

Authentication of Turkish propolis through HPTLC fingerprints combined with multivariate analysis and palynological data and their comparative antioxidant activity

Propolis is a honeycomb product having very diverse chemical composition and possessing a broad spectrum of biological activities. This study comprehensively evaluated the phenolic profile of Turkish propolis by using a high performance thin-layer chromatographic (HPTLC) method in combination with image analysis and pattern recognition technique. Also, botanical origin of each propolis sample was determined by comparison of HPTLC fingerprints of propolis samples with that of plant bud extracts and also by palynological analysis. Moreover, HPTLC coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH˙) detection technique was used for screening of antioxidant activity of each separated compounds directly on the plate. Results of the present study have demonstrated that Turkish propolis could be classified under three main types; i.e. orange (O) (originated from Populus nigra L.), blue (B) (originated from Populus tremula L.) and nonphenolic types. Palynological analysis have shown that dominant pollen grains (>%45) in propolis samples were: Fabaceae, Lamiaceae, Rosaceae, Castanea sativa Mill., Lotus corniculatus L., Salix spp. In addition, HPTLC-DPPH˙ results showed that O-type of propolis exerted higher antioxidant activity than the other propolis types. Moreover, quercetin, caffeic acid, caffeic acid phenyl ester, pinobanksin and galangin had significant contribution to the antioxidant activity of propolis.

Authentication of honeys of different floral origins via high-performance thin-layer chromatographic fingerprinting

This paper explores the high-performance thin-layer chromatographic (HPTLC) fingerprinting of non-sugar constituents for the authentication of honeys using highly antibacterial Jarrah (Eucalyptus marginata) and Marri (Corymbia calophylla) honeys sourced from Western Australia, different Leptospermum-derived Manuka honeys, and a typical table honey from an undisclosed floral source as test samples. As is demonstrated in this study, using HPTLC fingerprinting, it is possible to define differences in botanical origin as the honey fingerprints exhibit a unique profile of bands (i.e., Rf values, color) and peak profiles (i.e., Rf and peak intensity values, peak intensity ratios) that differ distinctly from each other. The identification of patterns of common bands among honeys derived from the same floral source as authentication tool is possible. Further, slight differences among honeys from the same botanical origin might be due to age, processing, or regional factors. The HPTLC analysis of two differently a...

Assessment of antioxidant activity in Victorian marine algal extracts using high performance thin-layer chromatography and multivariate analysis

The aim of this study was to develop and validate a rapid and simple high performance thin layer chromatographic (HPTLC) method to screen for antioxidant activity in algal samples. 16 algal species were collected from local Victorian beaches. Fucoxanthin, one of the most abundant marine carotenoids was quantified directly from the HPTLC plates before derivatization, while derivatization either with 2,2-diphenyl-1-picrylhydrazyl (DPPH) or ferric chloride (FeCl3) was used to analyze antioxidants in marine algae, based on their ability to scavenge non biological stable free radical (DPPH) or to chelate iron ions. Principal component analysis of obtained HPTLC fingerprints has classified algae species into 5 groups according to their chemical/antioxidant profiles. The investigated brown algae samples were found to be rich in non-and moderate-polar compounds and phenolic compounds with antioxidant activity. Most of the phenolic iron chelators also have shown free radical scavenging activity. Strong positive and significant correlations between total phenolic content and DPPH radical scavenging activity showed that, phenolic compounds, including flavonoids are the main contributors of antioxidant activity in these species. The results suggest that certain brown algae possess significantly higher antioxidant potential when compared to red or green algae and could be considered for future applications in medicine, dietary supplements, cosmetics or food industries. Cystophora monilifera extract was found to have the highest antioxidant concentration, followed by Zonaria angustata, Cystophora pectinate, Codium fragile, and Cystophora pectinata. Fucoxanthin was found mainly in the brown algae species. The proposed methods provide an edge in terms of screening for antioxidants and quantification of antioxidant constituents in complex mixtures. The current application also demonstrates flexibility and versatility of a standard HPTLC system in the drug discovery. Proposed methods could be used for the bioassay-guided isolation of unknown natural antioxidants and subsequent identification if combined with spectroscopic identification.

Development and validation of high-performance thin-layer chromatographic–fingerprint analysis ofStemona sessilifolia(Miq.) Miq. with the reference of protostemonine

A high-performance thin-layer chromatographic (HPTLC) method for the analysis of the protostemonine from Stemona sessilifolia (Miq.) Miq. has been developed and validated. According to the HPTLC fingerprint, 12 common peaks were selected to evaluate the similarities among 10 batches of samples with RF values ranging from 0.02 to 0.95. Ten different batches of samples were stable and reliable in quality, all the similarities of which are more than 0.98. The results indicated that this method was sensitive and precise for the quality evaluation of S. sessilifolia samples.

Development and validation of a high-performance thin-layer chromatographic fingerprint method for the evaluation of QiYi capsules with the reference of myotonin

The newly developed high-performance thin-layer chromatography (HPTLC) method allowed for the differentiation of QiYi capsules from 10 batches due to their characteristic fingerprints, proving to be a well-suited method for characterization and assignment of Traditional Chinese Medicine (TCM) preparation. The method was validated for repeatability, intra-precision, and inter-precision. The result was acceptable, with the reference of myotonin, because relative standard deviation (% RSD) was 3.9% in reproducibility test and less than 3.44% in inter- or intra-plate precisions. HPTLC fingerprinting of the extracts showed several peaks with different RF values. Eleven peaks were selected as the common peaks to evaluate the similarities of 10 different samples with RF values ranging from 0.10 to 0.87 according to fingerprint. The similarities of 10 batches of QiYi capsules were more than 0.940; it indicated that the different batches of QiYi capsules were stable and reliable in quality.