High-performance Thin-layer Chromatography Analysis Research Articles

Combining DNA and HPTLC profiles to differentiate a pain relief herb, Mallotus repandus, from plants sharing the same common name, "Kho-Khlan".

The pain relief formula "Ya Pa Som Kho-Khlan (YPSKK)" or "ยาผสมโคคลาน" in Thai is officially recorded in the Natural List of Essential Medicines (NLEM) of Thailand. The main component is Mallotus repandus (Willd.) Müll. Arg.; however, Anamirta cocculus (L.) Wight & Arn and Croton caudatus Gleiseler share the same common name: "Kho-Khlan". Confused usage of A. cocculus or C. caudatus can have effects via toxicity or unsuccessful treatment. This study aimed to combine a high-performance thin-layer chromatography (HPTLC) technique and DNA barcoding coupled with high-resolution melting (Bar-HRM) to differentiate M. repandus from the other two species. The M. repandus extract exhibited a distinct HPTLC profile that could be used to differentiate it from the others. DNA barcodes of the rbcL, matK, ITS and psbA-trnH intergenic spacer regions of all the plants were established to assist HPTLC analysis. The rbcL region was selected for Bar-HRM analysis. PCR amplification was performed to obtain 102 bp amplicons encompassing nine polymorphic nucleotides. The amplicons were subjected to HRM analysis to obtain melting curve profiles. The melting temperatures (Tm) of authentic A. cocculus (A), C. caudatus (C) and M. repandus (M) were separated at 82.03±0.09°C, 80.93±0.04°C and 80.05±0.07°C, respectively. The protocol was applied to test crude drugs (CD1-6). The HPTLC profiles of CD2-6 showed distinct bands of M. repandus, while CD1 showed unclear band results. The Bar-HRM method was applied to assist the HPTLC and indicated that CD1 was C. caudatus. While ambiguous melting curves from the laboratory-made formulae were obtained, HPTLC analysis helped reveal distinct patterns for the identification of the plant species. The combination of HPTLC and Bar-HRM analysis could be a tool for confirming the identities of plant species sharing the same name, especially for those whose sources are multiple and difficult to identify by either chemical or DNA techniques.

Investigation of Flavonoids in Stereospermum suaveolens DC Leaves (Patala) Using HPTLC Analysis for Inflammatory Pain, Swelling, and Edema Treatment

Background: As per extensive research study, it was observed that Stereospermum suaveolens DC (Patala) is rich sources of flavonoids. These phytoconstituents have played an important role in prevention and maintenance of acute and chronic diseases of pain and inflammation. There is need to explore method for identification and quantification of the presence of flavonoids in S. suaveolens DC leaves (SSL) by qualitatively and quantitatively and to establish its mechanism in pain, swelling and edema Aim: The aim of the study was to identify and assess role of flavonoids in SSL by high-performance thin-layer chromatography (HPTLC) analysis and animal screening models of inflammatory pain, swelling, and edema. Materials and Methods: HPTLC to quantify flavonoid in the ethyl acetate fraction of plant component in comparison with Quarcetin. The inflammatory pain, swelling, and edema method were investigated by acetic acid induced writhing method in mice, histamine-induced edema in Wistar rats, and croton oil-induced ear edema in mice. The test doses 125 mg/kg, 250 mg/kg, and 500 mg/kg oral administration (p.o.) of ethyl acetate fraction of SSL were selected by oral acute toxicity OECD 423. Results: HPTLC analytical method estimated presence of flavonoids and estimated amount was 22.64%. However, inhibition in histamine-induced paw edema and reduction inhibition in ear edema significantly (P < 0.05) observed by dose-dependent effect and antinociceptive activity detected on acetic acid induced writhing response method in mild to moderately using one-way analysis of variance method. Conclusion: It was clear that flavonoids were responsible for direct and indirect release of intermediate inflammatory mediators and promote its role in the treatment of pain and swelling.

Response surface methodology and artificial neural network modelling for optimization of solid-liquid extraction and rapid HPTLC analysis of glycyrrhizin in Glycyrrhiza glabra root

Glycyrrhizin, a major bioactive compound of Glycyrrhiza glabra has been used in traditional medicine system to treat several diseases. In the past few decades, its demand has been increased significantly for the active ingredient glycyrrhizin as well as for being a zero calorie sweetener. In the current study, extraction of glycyrrhizin from dry roots of G. glabra was established by reflux method. Quantitative estimation of glycyrrhizin content in the extracted samples was analysed by using high performance thin layer chromatography (HPTLC) system. In this study, response surface methodology (RSM) and artificial neural network (ANN) were used for the first time as a model to optimize the extraction conditions of glycyrrhizin from G. glabra roots in order to compare and establish effective prediction models. The extraction process variables including the time, temperature, solvent composition, particle size, solvent to solute ratio, pH and extraction steps were optimized using a Plackett-Burman design (PBD), Central Composite design (CCD), artificial neural network (ANN) and Multiple Layer Perceptron (MLP) on glycyrrhizin yield. The optimum extraction conditions were 55°C (extraction temperature), 45 min (extraction time), 55% (v/v) (ethanol concentration) and 30 ml/g (solvent to solute ratio) to obtain the maximum glycyrrhizin yield. The contributions of the quadratic model with high determination coefficient (R2=0.9806) were observed and showed good consistency between the experimental value 7.6 mg/g (0.76%) and predicted value 7.51 mg/g (0.751%). Response values were close to the predicted values with prediction accuracy as ANN > RSM, indicating that ANN model has higher prediction accuracy than RSM. The study suggests that RSM and ANN model system can be manipulated for the optimization and production of valuable bioactive compounds.

Sustainable Use of Greek Herbs By-Products, as an Alternative Source of Biologically Active Ingredients for Innovative Products.

The processing of medicinal and aromatic plants (MAPs) results in the production of a significant amount of plant by-products; herbal material of inferior quality and/or unusable plant parts that are not commercially exploitable. An extensive study of Greek native species was performed toward the production of innovative bioactive products using as raw materials the by-products obtained from the processing of cultivated MAPs. Origanum vulgare subsp. hirtum (oregano), Sideritis scardica (Greek mountain tea), Thymus vulgaris (thyme), and Matricaria recutita (chamomile) were selected due to their wide use for the preparation of beverages and culinary purposes. The determination of the percentage of the post-harvest processing by-products was performed for a 3 years period (2018–2020). Results showed that by-products derived from the above-mentioned species' processing constitute 64% (thyme), 54% (oregano), 37% (Greek mountain tea), and 24% (chamomile) of the total processed mass. To value the by-products as a potent source of bioactive ingredients, superior and inferior quality herbal material of the aforementioned plant species were extracted by an ultrasonic assisted extraction method. Hydroalcoholic extracts were chemically investigated using high-performance thin layer chromatography (HPTLC) and liquid chromatography-mass spectrometry (LC-MS) techniques. In addition, their free radical scavenging activity and total phenolic content (TPC) were estimated. Based on the results, herbs by-products revealed similar chemical content to the superior herbal material by the means of HPTLC and LC-MS analysis. In addition, strong free radical scavenging related to a high phenolic content was detected in the case of thyme, oregano, and Greek mountain tea. Moreover, the gas chromatography-mass spectrometry (GC-MS) analyses of the essential oils (EOs) of oregano and thyme by-products revealed the presence of carvacrol, thymol, γ-terpinene, and p-cymene among the major constituents. Finally, the LC-MS analyses of aqueous extracts of Greek mountain tea and chamomile by-products led to the identification of several bioactive compounds, such as flavonoids and phenylpropanoids. Overall, the presence of bioactive constituents in by-products, such as terpenes, phenolic compounds, and flavonoids underly their potent use as food antimicrobial and antioxidant additives, in the preparation of high added-value products, such as enriched aromatic edible oils, and innovative herbal teas, such as instant beverages.

Antidiabetic Potential of Ficus glomerata Roots with a Special Emphasis on Estimation of Bioactive Compounds by a Novel Validated HPTLC Technique

Abstract: Background: The data presented in this article for Ficus glomerata Linn. belonging to family Moraceace which is commonly found all over India. This study aimed towards the development and validation of high-performance thin-layer chromatography (HPTLC) method for simultaneous estimation of lupeol and quercetin from Ficus glomerata and correlate with its antidiabetic potential. Methods: The various fractions of ethanolic extract of Ficus glomerata root were prepared. The HPTLC analysis of quercetin and lupeol which are the important phytoconstituents responsible for various pharmacological actions was carried out at 525 nm. ICH guidelines were followed to validate this method for accuracy, precision and repeatability. Results: The linearity range of quercetin and lupeol were obtained as 400-2400 ng/spot and 1000- 5000 ng/spot respectively. Percent drug content was highest in diethyl ether fraction (quercetin 2531.8 ng and lupeol 1400 ng). The limit of detection value (LOD) obtained for quercetin and lupeol was 3.0793 and 3.1645 ng and the limit of quantification (LOQ) was 9.3314 and 9.5895 ng respectively. This method developed was accurate, precise and simple has shown higher resolution from other phytoconstituents present in the fractions. The method can be very effectively applied for analyzing the quality of herbal material and formulations containing Ficus glomerata. Antidiabetic activity of various fractions of ethanolic extract of Ficus glomerata roots was studied on alloxan-induced diabetic rats. Treatment with fractions was continued for 11 days. The effect of the fractions on glucose was analyzed. Diabetic rats treated with diethyl ether fraction exhibited a significant (p<0.05) decrease in glucose levels, indicating the potential use of Ficus glomerata in diabetes mellitus. Conclusion: As per the ICH guidelines, the HPTLC method used for simultaneous estimation of lupeol and quercetin was accurate, precise and specific. The method used for phytochemical standardization of various fractions of ethanolic extract of the roots of Ficus glomerata and correlated with its antidiabetic activity. Key words: Ficus glomerata, HPTLC, Lupeol, Quercetin, Alloxan, Antidiabetic activity

HPTLC estimation and anticancer potential of Aloe perryi petroleum ether extract (APPeE): A mechanistic study on human breast cancer cells (MDA-MB-231)

ObjectivesAloe perryi plant in the genus Aloe (family: Asphodelaceae), have been used in traditional medicine systems. Herein, we aimed to analyze the anticancer effects of A. perryi petroleum ether extract (APPeE) against human breast cancer (MDA-MB-231) and normal (HEK-293) cell lines. Further the biomarker stigmasterol, isolated from A. perryi extract was quantified by a densitometric high-performance thin layer chromatography (HPTLC) method. MethodsThe cytotoxic potential of APPeE was measured by MTT assay, neutral red uptake (NRU) assay, and morphological identification. ResultsAPPeE-induced a concentration dependent strong cytotoxic effects on MDA-MB-231 cells with an IC50 value of 24.5 μg/ml in contrast to HEK-293 (IC50 value of > 100 μg/ml). Similar results were obtained by NRU assay. Further cytotoxic concentrations of APPeE increased reactive oxygen species (ROS) production and activated caspase-3 and −9, leading to apoptosis in MDA-MB-231 cells. Additionally, a significant augment in the expression of p53, Bax, caspase-3 and −9 with a decline in Bcl-2 gene expression confirmed the involvement of apoptosis pathway in MDA-MB-231 cell death. In HPTLC analysis, the solvent system hexane: ethylacetate (8:2 v/v) furnished a sharp peak for stigmasterol (Rf = 0.19 ± 0.001). The low value of % relative standard deviation (RSD) (0.97–1.39) proposed the method is robust. The limit of detection (LOD) and limit of quantification (LOQ) were established as 13.82 and 41.89 ng, respectively. The stigmasterol in APPeE was quantified as 0.238% w/w of dried APPeE. ConclusionsHence, this study concludes the promising anticancer effect of APPeE, which might be endorsed to the existence of known anticancer biomarker stigmasterol.

PHYTOCHEMICAL PROFILING, HPTLC FINGERPRINT AND ANTIBACTERIAL, ANTI-FUNGAL, AND ANTIOXIDANT PROPERTIES OF ESSENTIAL OILS EXTRACTED FROM CUMMINUM CYMINUM, ZINGIBER OFFICINALE, TRACHYSPERMUM AMMI, ALIPNIA GALANGA, CEDRUS DEODARA, AND ELETTARIA CARDAMOMUM

Objective: The objective of the study was to carry out the phytochemical profiling of essential oils (EOs) and evaluation of their anti-microbial activity. Methods: The EOs extracted from Cumminum cyminum, Zingiber officinale, Trachyspermum ammi, Alipnia galanga, Cedrus deodara, and Elettaria cardamomum using clavenger apparatus. Phytochemical analysis and high-performance thin layer chromatography (HPTLC) fingerprinting were carried out for the EO. The antibacterial and antifungal activity were evaluated using agar well-diffusion method against two bacterial strains, Escherichia coli, Staphylococcus aureus and two fungal strains, Candida albicans, and Aspergillus brasiliensis. Positive controls ciprofloxacin-30 mg, azithromycin-15 mg, and nystatin NS-50 mg were used. Antioxidant potential of the EOs was investigated by TLC-bioautography method using 1,1-diphenyl-2-picrylhydrazyl derivatization. Results: The phytochemical analysis reveals presence of various phytochemical such as steroids, terpenoids, and phenylpropanoids. The HPTLC fingerprint is found to be unique for each of the oil. The EO of Z. officinale and T. ammi showed strong antibacterial activity against S. aureus. The EOs of C. cyminum, Tachyspermum ommi and A. galanga displayed prominent antioxidant activity on TLC bioautography. The herbs Cuminum cynimun, T. ammi, C. deodara, and Ellateria cardamomum produce reasonable amount of essentials oil, which can be explored for useful their industrial applications. Conclusions: These EOs can be explored further for their antimicrobial activity. The HPTLC analysis along with derivatization with suitable chromogenic reagents can be a rapid and simple tool for quality control of various EOs.

PHYSICO - CHEMICAL AND HPTLC STUDIES OF LIMONIA CRENULATA (ROOT)

Herbal medicines have gained popularity as potential therapeutic agents for the prevention and treatment of many diseases due to their high efficacy and low side effects. With increasing demand for safer drugs, attention has been drawn to the identity, purity and strength of the herbal medicines. Hence, there is a need for standardization and development of reliable quality protocols using modern techniques of analysis for the herbal drugs. In the present paper an attempt was made to lay down the pharmacopoeial standards of Limonia crenulata Roxb. (Root) based on its physico-chemical and HPTLC analysis. Determination of physico-chemical parameters such as total ash, acid insoluble ash, water and alcohol soluble extractive values and High-Performance Thin Layer Chromatography (HPTLC) studies of the chloroform extract of the plant material were carried out. The solvent system, Toluene: Ethyl acetate: Formic acid (5: 2: 0.1) efficiently resolved the components present in the crude extract. The physico-chemical and HPTLC studies on root of Limonia crenulata yielded a set of qualitative and quantitative parameters or standards that can serve as an important source of information to ascertain the identity and to determine the quality and purity of the plant material. These studies enable the identification of the plant material for future investigation and form an important aspect of drug studies.

Qualitative Detection of Triterpenes and Quantification of Betulinic Acid from Hexane Extract of Simarouba glauca Leaves by Gas Chromatography-Mass Spectrometry and High-Performance Thin Layer Chromatography

The present study focuses on the efficiency of high-performance thin layer chromatography and gas chromatography-mass spectrometry analysis in qualitative and quantitative analysis of triterpenoid from the terpene fraction of hexane extract of Simarouba glauca leaves. The total terpenoid content of the hexane extract was assessed which shows that 73.69 % of the extract content is terpenoid. The terpene fraction extracted in petroleum ether from the hexane extract of Simarouba glauca leaves was subjected to gas chromatography-mass spectrometry and high-performance thin layer chromatography analysis. Gas chromatography-mass spectrometry analysis results indicated seventeen compounds out of which only one was triterpene (Squalene an important bioactive triterpenoid). In high-performance thin layer chromatography the separation was achieved by using hexane:ethyl acetate in a 5:5 proportion as mobile phase and anisaldehyde sulphuric acid reagent was used for derivatization which imparted violet color to the triterpenoid containing bands. Detection and quantification of betulinic acid and other triterpenes was done by densitometric scanning at 525 nm. Betulinic acid produced compact spots at Rf 0.66. Linear range of betulinic acid was prepared using concentrations 1-7 μg/spot with a correlation coefficient R2 and standard deviation of 0.96004 %±19.68 % respectively. The results indicated the presence of 10 triterpenoids showing different Rf values including betulinic acid. This study confirmed the presence of betulinic acid in the terpene fraction of the hexane extract of Simarouba glauca leaves.

Phytochemical and Comparative Assessment of Bark and Root Extracts of Ixora barbata Roxb. ex. Sm. and Ixora coccinea L. Using High-Performance Thin Layer Chromatography Assay

The present study deals with the extraction and quantification of phytochemicals, which play a key role in the pharmaceutical world. In this article, we focused on the study of plant secondary metabolites and analyzed ethanolic and methanolic extracts of bark and roots of two species of Ixora using the highperformance thin layer chromatography technique. Alcoholic extracts of bark and root of both the plants revealed the presence of alkaloids, terpenoids, glycosides, phenols, saponins, sterols, tannins, etc., but both extracts were devoid of flavonoids and coumarins. Analysis of alkaloid content revealed the highest amount of total alkaloid content in the methanolic extract of roots of Ixora coccinea (146 mg/ gm) followed by the methanolic extract of bark of Ixora coccinea (138 mg/g). The lowest total alkaloid content was observed in the methanolic extract of bark of Ixora barbata (64 mg/g). Both ethanolic, as well as methanolic extracts of Ixora coccinea, showed higher amounts of alkaloid content when compared to Ixora barbata in similar solvents. As per high-performance thin layer chromatography analysis, the highest amount of camptothecin was recorded in ethanolic extract of root of Ixora coccinea (117 μg/ml) followed by ethanolic extract of bark of Ixora barbata (64.43 μg/ml). The lowest amount of camptothecin was observed in methanolic extract of bark of Ixora coccinea (12.64 μg/ml). The results obtained from the present high-performance thin layer chromatography analysis also revealed that the above-mentioned selected plants are a source of a number of other phytoconstituents. This is the first reported study on the high-performance thin layer chromatography analysis of Ixora barbata. The study includes quantification of camptothecin compound in both methanolic and ethanolic extracts of Ixora barbata for which there are no previously published reports.

Liquisolid Compact Technique for the Enhancement of Solubility and Dissolution Rate of Ambrisentan: Quality by Design Approach

Soluble epoxide hydrolase inhibitors have been reported as antihypertensive, anti-inflammatory, antiulcer and anticancer actions and protect the brain, heart and kidney from damage. Exploring soluble epoxide hydrolase inhibition activity of traditional medicinal plants helps to uncover the new target to treat various complications associated with inflammatory mediators. The present study was planned to explore soluble epoxide hydrolase inhibition activity of fifty medicinal plants commonly used in the traditional medicinal system. The dried plant material of each species was grounded into a coarse powder and separately macerated with absolute methanol and ethyl acetate for 7 d. Then the solvents were evaporated and obtained extracts were solubilized in dimethyl sulfoxide (10 mg/ml) and soluble epoxide hydrolase enzyme inhibitory potencies were evaluated using a fluorescent reporting system. Moreover, preliminary phytochemical screening and high-performance thin layer chromatography analysis were carried out for two extracts that showed potent soluble epoxide hydrolase inhibition activities. The results revealed that 10 methanolic extracts and 20 ethyl acetate extracts were potentially effective in suppressing soluble epoxide hydrolase activity with inhibitory concentration value of less than 10 μg/ml. Among the 30 potentially effective plants, four seed extracts (Celastrus paniculatus, Nigella sativa, Wrightia tinctorial, Vernonia anthhelmintica and Embelia ribes), one leaves extracts (Bergera koengii), one rhizome extracts (Curcuma longa) and one root extracts (Vetiveria zizanioides) were common in both methanolic and ethyl acetate extract. In conclusion, the study report suggests that various natural products used in the traditional medicinal system have many promising soluble epoxide hydrolase inhibitors. Methanolic extracts of seeds of Celastrus paniculatus and Nigella sativa have potent soluble epoxide hydrolase inhibition activities. Further research is warranted to identify a greater number of medicinal plants and active principles responsible for soluble epoxide hydrolase inhibition activities

Evaluation of Soluble Epoxide Hydrolase Inhibition Activity of 50 Traditional Medicinal Plants: Exploring Old Drugs for the New Pharmacological Target

Soluble epoxide hydrolase inhibitors have been reported as antihypertensive, anti-inflammatory, antiulcer and anticancer actions and protect the brain, heart and kidney from damage. Exploring soluble epoxide hydrolase inhibition activity of traditional medicinal plants helps to uncover the new target to treat various complications associated with inflammatory mediators. The present study was planned to explore soluble epoxide hydrolase inhibition activity of fifty medicinal plants commonly used in the traditional medicinal system. The dried plant material of each species was grounded into a coarse powder and separately macerated with absolute methanol and ethyl acetate for 7 d. Then the solvents were evaporated and obtained extracts were solubilized in dimethyl sulfoxide (10 mg/ml) and soluble epoxide hydrolase enzyme inhibitory potencies were evaluated using a fluorescent reporting system. Moreover, preliminary phytochemical screening and high-performance thin layer chromatography analysis were carried out for two extracts that showed potent soluble epoxide hydrolase inhibition activities. The results revealed that 10 methanolic extracts and 20 ethyl acetate extracts were potentially effective in suppressing soluble epoxide hydrolase activity with inhibitory concentration value of less than 10 μg/ml. Among the 30 potentially effective plants, four seed extracts (Celastrus paniculatus, Nigella sativa, Wrightia tinctorial, Vernonia anthhelmintica and Embelia ribes), one leaves extracts (Bergera koengii), one rhizome extracts (Curcuma longa) and one root extracts (Vetiveria zizanioides) were common in both methanolic and ethyl acetate extract. In conclusion, the study report suggests that various natural products used in the traditional medicinal system have many promising soluble epoxide hydrolase inhibitors. Methanolic extracts of seeds of Celastrus paniculatus and Nigella sativa have potent soluble epoxide hydrolase inhibition activities. Further research is warranted to identify a greater number of medicinal plants and active principles responsible for soluble epoxide hydrolase inhibition activities

Qualitative and quantitative estimation of gallic acid in flower galls of Crataeva religiosa through FT-IR and HPTLC techniques

In the present study, FT- IR (FourierTransform Infrared spectroscopy) analysis of galled flower was conducted to qualitatively identify gallic acid while comparative HPTLC (High-Performance ThinLayer Chromatography) studies of normal and galled flower extracts confirmed the gallic acid quantitatively. The quantitative estimation of gallic acid was carried out on silica gel 60 F24HPTLC plates (stationary phase). The plates were developed in a solvent system as prepared with Toluene, Acetone and Glacial acetic acid at a ratio of 3:1:2, in a CAMAG glass twin trough chamber of 20X10 cm, at a temperature of 2 20C. Detection was carried out densitometrically at 220 nm. The FTIR analysis was done with a spectrophotometer system, which was used to identify and analyze the characteristic peaks and their respective functional groups. HPTLC analysis revealed that gallic acid content increased almost two folds in gall tissues as compared to normal tissues. C. religiosa flower galls can be utilized medicinally due to enhanced gallic acid.This is the first report of the occurrence of gallic acid in flower galls of C.religiosa.

Identification and quantitative determination of arbutin in the herb of Orthilia secunda

Introduction. Orthilia secunda (L.) House is a perennial herb that grows in Europe, Siberia, Asia Minor and Central Asia. The herb of Orthilia secunda is actively used in folk medicine as a diuretic, wound-healing and anti-inflammatory agent. From literary sources it is known that this medicinal plant raw material (PRM) contains flavonoids, tannins, organic acids, vitamins, as well as simple phenols and their derivatives (arbutin and hydroquinone). The presence of arbutin is responsible for the plant's high antioxidant and anti-inflammatory properties. But the use of Orthilia secunda in official medicine is limited due to the lack of complete information on the chemical composition and criteria for standardization of this type of medicinal product.Aim. Identification and quantification of arbutin by chromatographic methods in Orthilia secunda (L.) House, harvested in various phytocenotic zones.Materials and methods. The investigated medicinal plant material – the herb of Orthilia secunda – was harvested in various phytocenotic zones: in July 2018, harvesting was carried out in the northern part of Kazakhstan (Kokshetau district), in July-August 2019 in the Perm Territory and in the Tyumen Region. Preliminary identification of arbutin and related phenols – gallic acid and hydroquinone – was carried out by high performance thin layer chromatography (HPTLC) on a CAMAG instrument with a UV cabinet (Merck HPTLC silica gel 60 F154 plates, 20 × 10), semi-automatic Linomat 5 applicator (sample application). Elution of the plates was performed in a CAMAG Automatic Developing Chamber (ADC2). Image fixation was performed on a CAMAG Scanner 3 spectrodensitometer. The quantitative determination of arbutin was carried out by the method of highperformance liquid chromatography, which was carried out on a Prominence LC-20 device (Shimadzu, Japan) according to the validated method described in the European Pharmacopoeia 10.0. Diode array detector SPD-M20A, column Intersil C18 column (250–4.6 mm, 5 μm) (Phenomenex, USA). The results were processed using the LabSolution software. The identification and quantification of arbutin was carried out in comparison with a standard solution containing a reference sample (RS) of arbutin (C = 0,025 mg/ml) and RS of hydroquinone (C = 0,0125 mg/ml).Results and discussion. HPTLC analysis made it possible to detect arbutin and gallic acid – the main product of hydrolytic degradation/ precursor of the biosynthesis of tannins of the hydrolysable group – in the herb of Orthilia secunda from different places of growth. HPLC analysis demonstrates a different chromatographic profile of Orthilia herb harvested in different phytocenotic zones. However, in all studied objects, the absence of hydroquinone and the presence of substances that can presumably be attributed to its derivatives were confirmed, which is confirmed by the visual similarity of the spectra of these compounds and the proximity of the extrema. It was found that arbutin does not belong to the marker (majority) compounds of Orthilia. Its content is low and reaches a maximum (about 0,021 %) in the herb of Orthilia secunda growing on the territory of Kazakhstan, while in the herb of Orthilia harvested in the Perm Territory arbutin was not identified. From the data obtained, it follows that the greatest accumulation of arbutin occurs in areas with a warmer and drier climate (northern part of Kazakhstan).Conclusion. HPTLC analysis of the herb Orthilia secunda allowed the identification of arbutin and gallic acid (the main precursor of tannins of the hydrolysable group). The results of HPLC analysis of Orthilia herb harvested in various phytocenotic zones suggest quantitative differences in the content of arbutin depending on the region of growth. From the experimental data, it follows that Orthilia growing in the northern part of Kazakhstan accumulates the maximum (0,021%) amount of arbutin, in comparison with the samples harvested in the Tyumen region and the Perm region. At the same time, Orthilia harvested in the Perm Territory does not accumulate arbutin. The presence of hydroquinone has not been confirmed (by HPTLC and HPLC methods); therefore, it is not justified to talk about the hydrolytic cleavage of arbutin in the process of biosynthesis or drying. However, in all studied objects there are peaks of substances with spectral characteristics like hydroquinone, which makes it possible to assume the presence of its derivatives. Therefore, it is not advisable to position arbutin as a marker compound of Orthilia secunda harvested on the territory of the Russian Federation, and to standardize raw materials for this compound.

Using high performance thin layer chromatography for the detection of pharmacologically active secondary metabolites in Empetrum nigrum L.

Introduction. The article presents the results of the detection of pharmacologically active secondary metabolites in black crowberry Empetrum nigrum L. using the method of high performance thin layer chromatography (HPTLC).Aim. To show the efficiency of HPTLC for conducting preliminary phytochemical analysis to determine the main groups of metabolites in promising medicinal plant species.Materials and methods. HPTLC analysis was carried out on a CAMAG device (Switzerland), using MERCK HPTLC silica gel 60 F154, 20 × 10 cm plates. For the evaporation of the samples, a Heidolph vacuum rotary evaporator (Germany) was used. The aerial parts (shoots) of Empetrum nigrum were harvested next to St. Petersburg State Chemical and Pharmaceutical University (SPCPU) nursery garden of medicinal plants (Leningrad Region, Vsevolozhsky District, Priozerskoe Highway, 38 km) in August 2019.Results and discussion. In the course of the research, four fractions from the aerial parts of Empetrum nigrum were obtained: hexane, dichloromethane, butanol, and water. Then, these fractions were investigated by HPTLC in two solvent systems – n-butanol : acetic acid : water (BAW) (4 : 1 : 2) and hexane : dichloromethane : methanol (HDM) (1 : 2 : 0.5). After scanning densitometric analysis of the plates eluted in the HDM system, it was revealed, that the hexane and dichloromethane fractions have a similar composition and contain the greatest amount of compounds, compared to the butanol and water fractions, and in the BAW system, it was found, that the butanol fraction contains the greatest variety of metabolites. As a result of UV spectroscopy, it was found, that the main groups of compounds contained in the hexane and dichloromethane fractions are derivatives of chalcones, dihydrochalcones, bibenzyls and 9,10-dihydrophenanthrenes. While in the butanol fraction, the main groups of secondary metabolites were derivatives of flavonoids and tanninsConclusion. The data obtained allow us to note the efficiency, speed and simplicity of HPTLC for conducting preliminary phytochemical analysis to determine the main groups of metabolites of promising medicinal plant species.

High-performance thin-layer chromatographic analysis of three components in Arnebia guttata Bunge

High-performance thin-layer chromatographic analysis of three components in Arnebia guttata Bunge

Standardization of Berberis aristata DC and Nigella sativa L. Using HPTLC and GCMS and Their Antineoplasia Activity in 7,12-Dimethylbenz[a]anthracene-Induced Mouse Models.

Berberis aristata DC and Nigella sativa L. are officially listed in various Indian Pharmacopoeia and AYUSH official documents. Prescribed for different ailments for proven medicinal activities, they thus became part of polyherbal medications. With reverse pharmacology and scientific validation, more than 30 patents are filed on different formulations of B. aristata and granted. Nigella sativa L. has been broadly studied for its therapeutic potential and wide range of activities against cardiovascular, diabetic, cancer, and life style disorders. Thus, this study is aimed at standardizing B. aristata and N. sativa and their antineoplasia activity in 7, 12-dimethylbenz[a]anthracene (DMBA)-induced mouse models. Molecular docking was done using the Schrodinger program Maestro 9.0. Herbal extracts and essential oil (B. aristata and N. sativa) were standardized and quantified using high-performance thin-layer chromatography (HPTLC) (CAMAG) and gas chromatography–mass spectrometry (GCMS) (Agilent 2010GC System) with validated methods. DMBA was administered orally once a week (1mg/200 µL) to each animal except the normal control. Hematology, histopathology, and immunoassays were performed, and data were analyzed and depicted with GraphPad and SPSS. In molecular docking, thymoquinone showed the highest docking score (9.519, 9.211, and 9.042, respectively) in the active site pockets of IL6 (PDB ID: 4CNI and 5FCU), TNF (PDB ID: 2AZ5), and VEGF (PDB ID: 4KZN). Out of all four target sites, thymoquinone and berberine showed good binding affinity with IL6 (PDB ID: 4CNI) compared to α- and β-pinenes. HPTLC analysis of the hydroalcoholic extract showed the presence of berberine both qualitatively and quantitatively (5.4% berberine), and thymoquinone detected 0.17% in the N. sativa extract. GCMS for essential oil showed 26 compounds including ±pinene. Leukocytes and erythrocytes of N. sativa and B. aristata were analyzed, and significant improvements were recorded (P < 0.05) and graphically presented. Mean survival time was calculated by the Kaplan Meier method (119 days). Immunoassay analyses were conducted, namely, TNF-α and VEGF, and interpreted and marked.

Anti-dengue activity of super critical extract and isolated oleanolic acid of Leucas cephalotes using in vitro and in silico approach

BackgroundsLeucas cephalotes is a common ethnomedicinal plant widely used by traditional healers for the treatment of Malaria and other types of fever. Oleanolic acid and its derivatives have been reported for various types of pharmacological activities, such as anti-inflammatory, antioxidant, anticancer, hepatoprotective, anti-HIV and anti-HCV activity.MethodsL.cephalotes plant extracts were prepared by supercritical fluid extraction (SFE) method and oleanolic acid was isolated by preparatory thin-layer chromatography. The compound was identified and characterize by using ultraviolet-visible spectroscopy (UV-VIS), Fourier transform infra-Red spectroscopy (FT-IR) and high-performance thin-layer chromatography (HPTLC). The structure of the compound was elucidated by proton nuclear magnetic resonance (1HNMR) and carbon nuclear magnetic resonance (1CNMR) and the purity checked by differential scanning calorimetry (DSC). The MTT assay was used to determine the toxicity of plant extract and oleanolic acid using a microplate reader at 595 nm. The anti-dengue activity of plant extract and oleanolic acid was tested in vitro and in silico using real-time RT-PCR.ResultsThe optimum yield of the extract was obtained at 40 °C temperature and 15Mpa pressure. The maximum non-toxic dose (MNTD) of plant extract and oleanolic acid were found as 46.87 μg/ml and 93.75 μg/ml, respectively in C6/36 cell lines. UV spectrophotometer curve of the isolated compound was overlapped with standard oleanolic acid at 232 nm. Superimposed FT-IR structure of the isolated compound was indicated the same spectra at 3433, 2939, 2871, 1690, 1500,1463, 1387, 1250, 1209, 1137 and 656 position as per marker compound. HPTLC analysis showed the retention factor of L. cephalotes extract was 0.19 + 0.06 as similar to the standard oleanolic acid chromatogram. The NMR structure of the isolated compound was identified as similar to the marker oleanolic acid structure. DSC analysis revealed the purity of isolated oleanolic acid was 98.27% with a melting point of 311.16 °C. Real-time RT PCR results revealed that L. cephalotes supercritical extract and isolated oleanolic acid showed 100 and 99.17% inhibition against the dengue − 2 virus when treated with MNTD value of plant extract (46.87 μg/ml) and the test compound (93.75 μg/ml), respectively. The molecular study demonstrated the binding energy of oleanolic acid with NS1and NS5 (non-structural protein) were − 9.42 & -8.32Kcal/mol, respectively.ConclusionsThe SFE extract L. cephalotes and its active compound, oleanolic acid inhibiting the activity of dengue-2 serotype in the in vitro and in silico assays. Thus, the L.cephalotes plant could be an excellent source for drug design for the treatment of dengue infection.

Validated high-performance thin-layer chromatographic analysis of curcumin in the methanolic fraction of Curcuma longa L. rhizomes

BackgroundIn the present study, an HPTLC (high-performance thin-layer chromatography) method was developed for the quantitative determination and validation of the curcumin in the methanolic fraction of Curcuma longa L. For achieving good separation of curcumin, the mobile phase of chloroform:methanol (97:3) was used. The densitometric analysis of curcumin was performed at 420 nm in reflection/absorption mode.ResultsLinearity of the method was obtained in the range of 100‒600 ng per spot. During analysis, the methanolic fraction of the C. longa showed the presence of a quantifiable amount of curcumin. The content of curcumin was found to be 1.5% (per dry weight).ConclusionsThe method is specific, simple, precise, and accurate. The obtained data can have used for the routine analysis of the reported biomarkers in crude drugs and extracts. The quantification and the method validation of curcumin have not yet been reported in C. longa which can be utilized for the proper standardization of the plant.

Scientific-methodological approach for standardization of herbal raw materials and herbal preparations using high-performance thin-layer chromatography procedure

The high-performance thin-layer chromatography (HPTLC) is widely used for quality control of herbal raw materials (HRM).&#x0D; The purpose of this work were to define the problematic issues of quality control of herbal drugs in Ukraine and to develop a systematic scientific and methodological approach for the standardization of HRM and herbal drugs using the modern analytical technique of HPTLC: to optimize the existing and develop new methods of identification and determination of adulterations, and to develop alternative methods of quantitative determination, as well as studying of the stability.&#x0D; As a result of a critical evaluation of existing approaches for the standardization of HRM and herbal drugs of Ukraine’s flora, numerous problematic issues were identified, including the existence of a limited number of national monographs / national parts of monographs on the HRM of the Ukraine’s flora, which were not previously standardized by other pharmacopeias; non-consideration of Ukraine’s flora species (non-pharmacopoeial) in monographs on closely related species that introduced in the State Pharmacopoeia of Ukraine; lack of specificity of combinations of physical and chemical methods used for quality control; lack of specificity and reproducibility, the labor intensity of methods; necessity of usage of toxic solvents and precursors; use of different groups of markers and methods for the HRM and its finished product or HRM and its closely related species, etc. The proposed systematic scientific and methodological approach includes: a preliminary collection of information about the object of study, markers/groups of bioactive substances, approaches to its standardization; theoretical evaluation of existing techniques of quality control in monographs on HRM and experimental approbation using samples of domestic plants, determination of the need for optimization of existed methods by the parallel development of specific, reproducible and documented methods for identification, adulterations’ detection, quantification, and bio-detection (if necessary) by HPTLC method. The approach is illustrated with examples.&#x0D; The proposed approach can be used for comprehensive HPTLC analysis of domestic HRM and herbal drugs for research, prevention of adulteration, standardization of initial HRM and its finished product, pharmaceutical development, and stability studies.