High-performance Thin-layer Chromatography Analysis Research Articles

Quantitative determination of lobeglitazone sulfate and glimepiride in combined tablet by robust high‐performance thin layer chromatographic method

AbstractA combination of fixed dose tablet containing 0.5 mg lobeglitazone sulfate (LBZ) and 1 mg glimepiride (GLM) has demonstrated efficacy in enhancing glycemic control in diabetes. The projected work aimed to develop and validate a high‐performance thin‐layer chromatographic (HPTLC) methodology for the precise quantification of both the drugs in tablet formulations. The HPTLC analysis utilized aluminium plates layered with silica gel 60F254, and the solvent system consisted of ethyl acetate, benzene, and hexane (4:3:1 v/v/v) followed by densitometric scanning at 238 nm. The Rf value was found to be 0.68 ± 0.001 for LBZ and 0.48 ± 0.002 for GLM. The methodology exhibited linearity in the range of 100–2000 ng/band for LBZ and 200–4000 ng/band for GLM, with correlation coefficients of 0.9988 and 0.9981, respectively. Exceptional sensitivity was observed, with detection limits of 23.86 ng/band for LBZ and 58.26 ng/band for GLM, along with quantification limits of 72.32 ng/band for LBZ and 176.55 ng/band for GLM. The method demonstrated precision (% relative standard deviation of peak area < 2) and accuracy (recovery between 97% and 102%). The suggested method is suitable for quantifying both the drugs in tablets, making it useful for routine quality control in laboratories.

Determination of Physicochemical Characteristics, Phytochemical Profile and Antioxidant Activity of Various Clover Honeys.

This paper reports on some physicochemical and phytochemical characteristics (i. e. pH, electrical conductivity, colour, moisture content, total phenolic content, sugar profile) and in vitro antioxidant activity of honeys harvested from five legume species, red clover (Trifolium pratense), balansa clover (T. michelianum), Persian clover (T. resupinatum), purple clover (T. purpureum) and sanfoin, also known as holy clover (Onobrychis viciifolia), that were grown in enclosed shade houses to ensure that the honeys' characteristics are reflective of a truly monofloral honey. Glucose and fructose, determined via High-Performance Thin-Layer Chromatography (HPTLC) analysis, were found as the main sugars in all investigated honeys with the ratio of fructose to glucose ranging from 1 : 1.2 to 1 : 1.6. The honeys' pH values ranged from 3.9 to 4.6 which met Codes Alimentarius (CA) requirements. The moisture content was found to be between 17.6 and 22.2 % which in some cases was slightly higher than CA requirements (≤20 %). The honeys' colour values, prior and after filtration, were between 825.5-1149.5 mAU and 532.4-824.8 mAU respectively, illustrating golden yellow to deep yellow hues. The total phenolic content (TPC) of the honeys was determined using a modified Folin-Ciocalteu assay. Their antioxidant activity was captured by the Ferric Reducing-Antioxidant Power (FRAP) assay as well as HPTLC analysis coupled with 2,2-diphenyl-1-picrylhydrazyl (DPPH) derivatisation. The highest total phenolic content was found in red clover honey (45.4 mg GAE/100 g) whereas purple clover honey showed the highest level of activity in the FRAP assay (7.3 mmol Fe2+/kg). HPTLC-DPPH analysis of the honeys' organic extracts demonstrated the presence of various bioactive compounds that contribute to their overall antioxidant activity. This study developed a methodology for producing monofloral clover honeys in a space limited, enclosed production system, which allowed to collate important baseline data for these honeys that can serve as the foundation for their potential future development into commercial honeys, including honeys that can be used for medicinal purposes.

Comparative negation of amphiphile production using nutrition factors: Amyloids versus biosurfactants

Microbial amphiphiles play an important role in environmental activities such as microbial signaling, bioremediation, and biofilm formation. Microorganisms rely on their unique characteristics of interfaces to carry out critical biological functions, which are helped by amphipathic biomolecules known as amphiphiles. Bacillus amyloids aid in cell adhesion and biofilm formation. Pseudomonas sp. are essential in biofilm development and are a vital survival strategy for many bacteria. Furthermore, Pseudomonas and Bacillus are well-known for their ability to produce biosurfactants with a range of applications, including bioremediation and removing biological pollutants from different environments. The study employed 31 different media types and a range of analytical techniques to assess the presence of amyloid proteins and the absence of biosurfactants in Bacillus licheniformis K125 (GQ850525.1) and Pseudomonas fluorescens CHA0. The presence of amyloid proteins was confirmed through Congo red and thioflavin T staining. The carefully constructed medium also efficiently inhibited the synthesis of biosurfactants by these bacteria. Additionally, surface tension measurements, emulsification index, thin-layer chromatography, and high-performance thin-layer chromatography analyses indicated the absence of biosurfactants in the tested media.

In Vitro Evaluation of Extracts From Ixora Species for a Potential Phytosomal Formulation.

Background Ixora species are perennial shrubs and flowering plants belonging to the family Rubiaceae. The leaf and flower parts of Ixora coccinea (I. coccinea)andIxora alba (I. alba) were aimed at isolating their active fractions. The present study was to determine in vitro antitumor activity against malignant melanoma cell lines for phytosome formulation. Materials and methods Two species, I. coccinea (red flowers and leaves) and I. alba (white flowers and leaves), were selected, and this study focused on determining the active fraction by comparing the in vitro antimicrobial and antioxidant potentials of petroleum ether, chloroform, ethyl acetate, and hydroalcoholic (ethanol:water, 70:30 v/v) extracts. The identified potent extract was subjected to in vitro anticancer activity in malignant melanoma cell lines. Results A phytochemical study revealed phytosterols, flavonoids, proteins, amino acids, alkaloids, carbohydrates, phenols, tannins, and diterpenes. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay was used to evaluate the antioxidant effect of I. coccinea and I. alba leaf and flower extracts. In the DPPH assay, I. coccinea flower hydroalcoholic extract (ICFHA) had an IC50 value of 248.99 µg/mL, and I. coccinea leaf hydroalcoholic extract (ICLHA) had an IC50 value of 268.87 µg/mL. These two extracts had a lower value with a higher antioxidant effect. In the total antioxidant assay, I. coccinea leaf ethyl acetate extract (ICLEA) and I. coccinea leaf chloroform extract (ICLCE) have 77.4 ± 0.05 and 68.9 ± 0.03 mg of ascorbic acid equivalent per gm of extract, respectively. These two extracts exhibited a high antioxidant effect. The antimicrobial potential was evaluated using selected bacterial and fungal strains using the agar-well diffusion method. Petroleum ether and chloroform extracts of I. coccinea and I. alba leaves and flowers did not possess antimicrobial activity with any of the bacterial or fungal strains. An ethyl acetate extract and a hydroalcoholic extract of I. coccinea leaves and flowers showed antimicrobial activity against Enterococcus faecalis, Candida albicans, and Staphylococcus aureus. An ethyl acetate extract of I. coccinea flower and a hydroalcoholic extract of I. alba leaf showed a significant zone of inhibition when compared with standard chloramphenicol for all three selected strains, which may be due to the presence of active phytoconstituents. ICLHA showed a MIC of ≤300 µg/mL for Enterococcus faecalisandStaphylococcus aureusand ≤400 µg/mL for Candida albicans microbial strains. The high total flavonoid content was reported in ICLEA at 771.31 µg/mL and in I. coccinea flower ethyl acetate extract (ICFEA) at 694.69 µg/mL. High-performance thin layer chromatography (HPTLC) analysis showed a high quercetin (QCE) content in the ICLEA extract. To prove the in vitro skin anticancer activity, an MTT assay was performed for the ICLEA extract in a malignant melanoma cell line, and the IC50 value was reported as 7.96 µg/mL. Conclusion I. coccinea leaf ethyl acetate extract revealed a significant total flavonoid content in analysis through the aluminum chloride method, and the presence of a high QCE content was confirmed by HPTLC analysis. The in vitro skin anticancer activity of ICLEA was confirmed by the MTT assay; therefore, it was concluded that the ICLEA extract was a potent fraction and was selected to develop a phytosome.

Chemical profiling of endangered Citrus macroptera leaf extracts and evaluation of its cytotoxic activity

The main component of drug production and formulation, with minimal to no adverse effects, is plant-based medicine. Citrus macroptera is an endangered species of North-Eastern India and has a diverse array of pharmaceutical applications owing to the presence of several bioactive compounds. The study aimed to evaluate the presence of bioactive metabolites, antioxidants, and cytotoxic mechanisms of the in vitro and ex-vitro leaf extracts of C.macroptera. Gas chromatography–mass spectrometry (GC-MS) profiling was performed using the ethanolic leaf extracts. Methanolic and aqueous extracts were profiled using High-performance thin-layer chromatography (HPTLC). Moreover, the cytotoxicity of the methanolic leaf extracts was assessed using the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test against SH-SY5Y, human neuroblastoma cell lines. GC-MS analysis revealed the existence of numerous pharmacologically significant components such as esters, hydrocarbons, fatty alcohols, and ester compounds. The presence of flavonoids and stigmasterol was exhibited by the HPTLC profile. The in-situ HPTLC analysis revealed white fluorescence after derivatization under white light that explicitly confirms the antioxidant potential of the extracts and can serve as a lead source of natural antioxidants. The cytotoxic efficacy of the extracts was observed in a concentration-dependent manner. However, the in vitro leaf extract displayed better suppressive effects against the SH-SY5Y cells with a 24-h IC50 of 167.71?g/mL compared to the ex-vitro leaf extract. Further, a comprehensive study is necessary to identify the cytotoxic compounds and their mode of action. The outcome of our investigation provides empirical evidence that phytoconstituents present in the leaf extracts of C. macroptera can be used as an effective pharmaceutical agent to combat several diseases.

Screening of α-amylase/trypsin inhibitor activity in wheat, spelt and einkorn by high-performance thin-layer chromatography.

α-Amylase/trypsin inhibitor proteins (ATI) are discussed as possible triggers for non-celiac gluten sensitivity. The potential of high-performance thin-layer chromatography (HPTLC) was studied for the first time to analyse the inhibitory properties of ATIs from flour of wheat, spelt, and einkorn. Inhibition by each flour of the digestive enzymes trypsin or α-amylase was determined by the reduction of released metabolisation products in comparison to non-digested flour, and positive (acarbose) and negative (water) controls. Firstly, amylolysis was carried out in miniaturized form on the HPTLC surface (HPTLC-nanoGIT) after in-vial pre-incubation of the amylase with the inhibitors from flour. α-Amylase inhibition was evident via the reduction of released saccharides, as analysed by normal phase HPTLC. A strong influence of the flour matrix on the assay results (individual saccharides) was evident, caused by an increased amylolysis of further polysaccharides present, making HPTLC analysis more reliable than currently used spectrophotometric sum value assays. The detection and visualization of such matrix influence helps to understand the problems associated with spectrophotometric assays. Only maltotriose was identified as a reliable marker of the amylolysis. The highest α-amylase inhibition and thus the lowest saccharide response was detected for maltotriose in refined spelt, whereas the lowest α-amylase inhibition and thus the highest saccharide response was detected for maltotriose in refined wheat. A comparison of refined and whole grain flours showed no clear trend in the responses. Secondly, trypsin inhibition and proteolysis were performed in-vial, and any inhibition was evident via the reduction of released peptides, analysed by reversed-phase HPTLC. Based on the product pattern of the proteolysis, einkorn and whole wheat showed the highest trypsin inhibition, whereas refined wheat and refined spelt showed the lowest inhibition. Advantageously, HPTLC analysis provided important information on changes in individual saccharides or peptides, which was more reliable and sustainable than spectrophotometric in-vial assays (only sum value) or liquid column chromatography analysis (targeting only the ATI proteins).

Comprehensive high-performance thin-layer chromatography analysis of Monteverdia ilicifolia leaf and its adulterants

The leaves of Monteverdia ilicifolia (syn. Maytenus ilicifolia) are widely used in traditional South American medicine to treat gastrointestinal problems such as gastritis and ulcers. Several herbal products containing the leaves of M. ilicifolia can be found in the market. However, other species with similar leaf morphology are confounding materials, e.g. Monteverdia aquifolia (Celastraceae), Citronella gongonha (Cardiopteridaceae), Jodina rhombifolia (Santalaceae), Sorocea bonplandii (Moraceae) and Zollernia ilicifolia (Fabaceae). This study aimed to identify M. ilicifolia and distinguish it from its potential adulterants using high-performance thin-layer chromatography (HPTLC) technique. Comprehensive HPTLC analysis revealed specific fingerprints that can be used to assess the minimum content of epicatechin and the quality of commercial espinheira-santa samples. The results of the study demonstrated that the HPTLC method is capable of detecting adulterations and distinguishing M. ilicifolia from all confounding materials in commercial products available on the market, showing that most of the products are of poor quality due to adulterations.

Simultaneous Preconcentration of Fast Green FCF and Rhodamine B Using Deep Eutectic Solvent and Determination via High-Performance Thin Layer Chromatography

The aim of the current investigation is the development of a green, quick, easy, and accurate method for simultaneous preconcentration of fast green FCF (FG) and rhodamine B (Rh B) using a deep eutectic solvent (DES). Then, the high-performance thin layer chromatography (HPTLC) technique was used for the determination of analytes. Decanoic acid and tetrabutylammonium bromide were chosen as the components of DES. HPTLC analysis was performed on an aluminum plate silica gel 60 F254. Methanol-ammonia and ethyl acetate were selected as the mobile phase. Scanning of the plates was accomplished by scanner 3. Effective parameters on the preconcentration process such as concentration of salt, volume of DES, stirrer time, and pH were investigated via central composite design (CCD). Data validation demonstrated good repeatability. The limit of detection for FG and Rh B was obtained as 0.08 and 0.01 µg·mL−1, respectively. The enrichment factor for FG and Rh B was achieved as 7.43 and 10.77, respectively. The linear ranges for FG and Rh B were acquired as 0.10–1.20 and 0.05–1.20 µg·mL−1, respectively. The preconcentration factor for both analytes was 21.66. Finally, the proposed method was successfully used for the quantitation of FG and Rh B in pastille and lipstick.

Quality control of marketed products of Andrographis paniculata by HPLC analysis

Andrographis paniculata is one of the most important medicinal plants having several pharmaceutical properties. Various pharmaceutical products and raw powders of this herb are sold in the market. The high demand for A. paniculata poses the problem of adulteration in their products. Therefore, it becomes necessary to check the quality in terms of their major bioactive compounds. In the present study, several marketed products were analyzed for their quality assessment i.e. for the presence of desired bioactive constituent, andrographolide. Ten tested products of A. paniculata showed the presence of andrographolide. A validated High Performance Thin Layer Chromatography (HPLC) method has been used for the quantitative estimation of andrographolide. andrographolide was detected at a retention time (Rt) of 17.8±0.05 and was quantified using a linear regression equation. The amount of andrographolide in the marketed products was in the range of 0.01 to 7.08 %. The chromatographic fingerprints were developed in all marketed products in the study. Thus, the present study dealing with the detection and quantification of andrographolide through HPLC analysis would have enough significance in the quality control of marketed products of andrographis paniculata.

Influence of in vitro culture age on morphology, antioxidant activities, reserpine production, and genetic fidelity in Indian snakeroot (Rauvolfia serpentina (L.) Benth. ex Kurz.)

The present study was carried out on Rauvolfia serpentina (L.) Benth. ex Kurz., for the first time, to assess the impact of in vitro culture age on morphology, antioxidant activities, and reserpine production. Fresh in vitro multiple shoot culture was established in Murashige and Skoog semisolid medium fortified with 1 mg/l N6-benzyladenine, while another pre-established in vitro multiple shoot culture was maintained in similar medium for two years. Comparisons were made among two-year-old in vitro culture, six-month-old in vitro culture and six-month-old ex vitro plantlet, wherein six-month-old ex vitro plantlet recorded significantly longer shoots than in vitro cultures. The internode lengths of six-month-old ex vitro plantlet, six-month-old in vitro and two-year-old in vitro cultures were significantly different from each other. The leaf area of six-month-old ex vitro plantlet was highest, followed by that of the six-month-old in vitro and two-year-old in vitro cultures, respectively. Reduction of superoxide dismutase activity was recorded in six-month-old in vitro culture than six-month-old ex vitro plantlet, but was found to be comparable to two-year-old in vitro culture. Marginal increase in peroxidase activity was detected in two-year-old in vitro culture when compared to six-month-old in vitro culture. High-performance thin-layer chromatography analysis detected least reserpine accumulation from six-month-old ex vitro plantlet, whereas, a surge in reserpine was recorded from both six-month-old in vitro and two-year-old in vitro cultures. Eventually, inter simple sequence repeats and start codon targeted polymorphism primers revealed a 6.79% of genetic variation in two-year-old in vitro culture from six-month-old ex vitro plantlet and six-month-old in vitro cultures. The study revealed that for morphological traits and antioxidant activities, a small-scale effect was imposed by tissue culture condition for at least up to two years. However, for enhanced (two-fold) accumulation of reserpine, long-term in vitro culture would be of commercial importance but in the cost of certain extent of genetic variation.

Comparison of the two extraction methods of fruit pulp of Aragvadha (Cassia fistula Linn.) by HPTLC

Background: The existence of significant bioactive compounds in herbs makes them pharmacologically treasured. Aragvadha (Cassia fistula Linn.), generally known as 'golden shower tree' is popular for its medicinal properties in Ayurveda. The fruit pulp of it owns medicinal values. Each plant has certain bioactive important compounds. For the extraction of different vital compounds, there is a need of specific method of extraction, which are known as ‘panchavidha kashaya kalpana’ (five varieties of extraction) in Ayurveda. These are - Svarasa (expressed juice), Kalka (paste), Kvatha (decoction), Hima (cold infusion) and Phanta (hot infusion). From centuries, Ayurvedic physicians are using extraction method, called as 'phanta' (hot infusion) for the fruit pulp of Aragvadha as a conventional technique; Ayurvedic scriptures also recommend the 'kvatha' (decoction) extraction process. To assess the superiority of these two extraction procedures, the present work used high-performance thin layer chromatography (HPTLC) analysis of Aragvadha (Cassia fistula Linn.) fruit pulp methanolic extract for phytochemical profiling. Results: The HPTLC analysis of Aragvadha (Cassia fistula Linn.) fruit pulp methanolic extract was carried out using winCATS Planar Chromatography Manager system, and the outcomes, which were obtained as chromatograms (scanned at the wavelength of 254 nm and 366 nm) representing multiple peaks. The plant's phytochemical profile of both samples were established, and tables indicating the total count of peaks with the height, area, percent area of peaks, and Rf values. Conclusion: The study revealed that the 'kadha' (decoction) of Aragvadha (Cassia fistula Linn.) fruit pulp methanolic extract contains a rich variety of phytochemicals as compare to 'phanta' (hot infusion) which might be accountable for its therapeutic value and thus justifies superiority of the 'kadha' (decoction) extraction method.

Phytochemical, Physicochemical and HPTLC Analysis of Siddha Herbal Formulation Muppirandai Chooranam

Aim: The aim of the study was to evaluate the Phytochemical, Physicochemical, HPTLC analysis of Siddha herbal formulation Muppirandai Chooranam (MRC), indicated for various ailments including menorrhagia.
 Study design
 Place of study: The Phytochemical and Physicochemical analysis were conducted at the Tamilnadu Dr. MGR Medical University, located at no.69, Anna salai, Guindy, Chennai-32. The high-performance thin layer chromatography (HPTLC) was carried out at Noble research solutions, Kolathur, Chennai -99.
 Methodology: Siddha formulation Muppirandai Chooranam was prepared as per good manufacturing practices (GMP) guidelines and the phytochemical analysis, physicochemical analysis were carried out at the Tamilnadu Dr. MGR Medical University, located at no.69, Anna salai, Guindy, Chennai-32. As per PLIM guidelines (Pharmacopeia Laboratory Of Indian Medicine) in accordance with the guidelines established by AYUSH (Ayurveda, Yoga, Unani, Siddha, Homoeopathy), the governing body for traditional health systems in India. The HPTLC analysis was conducted at Noble Research Solutions, Kolathur, Chennai -99.
 Results: The Phytochemical screening of MRC shown the presence of Alkaloids, Carbohydrates, Saponins, Flavanoids, Diterpenes, Gum and Mucilage. The physicochemical analysis of MRC revealed that it had a loss on drying of 0.42% at 105°C, a total ash content of 23.3%, an acid-insoluble ash content of 3.02%, a water-soluble ash content of 2.30%, and a water-soluble extract content of 17.67%, and alcohol soluble extract content of 5.23%.HPTLC finger printing analysis of the sample reveals the presence of five prominent peaks corresponds to the presence of five versatile phytocomponents present within it. Rf value of the peaks ranges from 0.02 to 0.37.
 Conclusion: From the findings, it is concluded that it shown the compendious understanding of presence of Phytochemical components, Physicochemical characteristics, and HPTLC analysis of MRC and it is competent to assess the quality profile of Muppirandai Chooranam as a reference standard for the development of the standardised pharmaceutical product.

Impact of ultrasound-assisted extraction of polyphenols and caffeine from green tea leaves using high-performance thin-layer chromatography.

Tea is the most popular daily drink consumed globally, with a high concentration of caffeine and polyphenols. In this study, the effects of ultrasonic-assisted extraction and quantification of caffeine and polyphenols from green tea were investigated and optimized using 23 -full factorial design and high-performance thin-layer chromatography. Three parameters were optimized to maximize the concentration of caffeine and polyphenols extracted using ultrasound: crude drug-to-solvent ratio (1:10-1:5), temperature (20-40°C), and ultrasonication time (10-30 min). The optimal conditions achieved from the model for tea extraction were as follows: crude drug-to-solvent ratio, 0.199 g/ml; temperature, 39.9°C; and time, 29.9min; the extractive value was found to be 16.8%. Images from scanning electron microscopy showed that the matrix underwent a physical alteration and cell wall disintegration, which intensified and accelerated the extraction. This process might be simplified using sonication, which results in a higher extractive yield and a significant concentration of caffeine and polyphenols than the traditional approach, with a smaller quantity of solvent and faster analytical times. The result of high-performance thin-layer chromatography analysis proves a significant positive correlation between extractive value and caffeine and polyphenol concentrations.

Validated HPTLC Analysis for Estimation of Quercetin in Seeds of Anethum graveolens

A high-performance thin layer chromatography (HPTLC) method for the quantification of quercetin was developed on methanolic extract of dill (Anethum graveolens) seeds and subsequently validated. A suitable mobile phase was used to establish the HPTLC method, ethyl acetate, toluene, methanol, chloroform, and formic acid (2:3:3:2). A densitometric analysis was done at 366 nm of wavelength. Quercetin has an Rf value of 0.55. In the dilution range of 100 to 800 ng per band, quercetin revealed a linear connection with r2= 0.9938 in the calibration’s linear regression analysis. By conducting replication analysis on 2 separate days and one day, accuracy was verified. The standard addition method was used to conduct recovery studies to validate accuracy. The quercetin recovery rate was 98.60% on average. Five replicates of each of the three standards were used to detect the system suitability parameter. With regard to both the peak area and the Rf value, the %RSD was observed to be under 2%. The mobile phase concentration was altered from Toluene: Ethyl acetate: Chloroform: Methanol (2:3:3:2) to (3:2:2:3) with few drops of formic acid. It was found to have a %RSD of peak area below 10%, the robustness of the method was assessed. The developed HPTLC method was discovered to be easy to use, precise, accurate, suitable, and robust for estimating quercetin from dill seed extract.

Shelf-Life Evaluation and Comparative High-Performance Thin-Layer Chromatography Profile of Amavatari rasa: A Preliminary Study

ABSTRACT Background: In addition to safety and efficacy data, it is important to establish quality parameters for all drugs, including shelf life. Shelf life is the length of time that a product may be stored without becoming unfit for use, consumption, or sale. Every product in this universe has a definite shelf life. In Ayurveda texts, shelf life is known as Saviryata avadhi that is defined as the time period during which the potency (Virya) of any drug remains unaffected. Amavatari Rasa (AR) is an important formulation being used in Ayurvedic therapeutics; however, its shelf life is not reported till date. Aims and Objectives: This study aimed to evaluate shelf life of AR through accelerated stability study. Materials and Methods: Accelerated stability study and comparative high-performance thin-layer chromatography (HPTLC) profile were done to evaluate the shelf life of the AR. The formulation was prepared by using authentic raw material in the departmental laboratory by the following classical guidelines. It was evaluated by analyzing changes in physicochemical profiles of the product after keeping it at specific temperature (40°C ± 2°C) and relative humidity (75% ± 5%). Analysis was carried out at the intervals of 0, 3, and 6 months. Average 10% degradation time was calculated and extrapolated to find the shelf life as per the ICH guidelines. Results and Conclusion: In the present study, shelf life of AR was found to be 48 months; similar Rf values obtained in HPTLC analysis of AR initially and after 6 months showed the minimum deterioration of the product.

Extraction, HPTLC Analysis and Antiobesity Activity of Jatropha tanjorensis and Fraxinus micrantha on High-Fat Diet Model in Rats.

The accumulation of body fat due to an imbalance between calorie intake and energy expenditure is called obesity. Metabolic syndrome increases the risk of heart disease, type 2 diabetes, and stroke. The purpose of this study was to determine the effect of Jatropha tanjorensis (J.T.) and Fraxinus micrantha (F.M.) leaf extracts on high-fat diet-induced obesity in rats. Normal control, high-fat diet (HFD) control, orlistat standard, and test groups were created using male Albino Wistar rats (n = 6 per group) weighing 190 ± 15 g. Except for the control group, all regimens were administered orally and continued for 6 weeks while on HFD. Evaluation criteria included body weight, food intake, blood glucose, lipid profile, oxidative stress, and liver histology. High-Performance Thin Layer Chromatography (HPTLC) analysis was performed using a solvent system (7:3 hexane: ethyl acetate for sitosterol solution and Jatropha tanjorensis extracts and 6:4 hexane: ethyl acetate: 1 drop of acetic acid for esculetin and Fraxinus micrantha extracts). There were no deaths during the 14 days before the acute toxicity test, indicating that aqueous and ethanolic extracts of both J.T. and F.M. did not produce acute toxicity at any dose (5, 50, 300, and 2000 mg/kg). The ethanolic and aqueous extracts of J.T. and F.M. leaves at 200 and 400 mg/kg/orally showed a reduction in weight gain, feed intake, and significant decreases in serum glucose and lipid profile. As compared to inducer HFD animals, co-treatment of aqueous and ethanolic extract of both J.T. and F.M. and orlistat increased the levels of antioxidant enzymes and decreased lipid peroxidation. The liver's histological findings showed that the sample had some degree of protection. These results indicate that ethanolic samples of J.T. have antidiabetic potential in diabetic rats fed an HFD. The strong antioxidant potential and restoration of serum lipid levels may be related to this. Co-treatment of samples JTE, JTAQ, FME, FMAQ and orlistat resulted in an increase in antioxidant enzymes and reduction in lipid peroxidation as compared to inducer HFD animals. We report, for the first time, on using these leaves to combat obesity.

In vitro effects of undifferentiated callus extracts from Plantago major L, Rhodiola rosea L and Silybum marianum L in normal and malignant human skin cells

Background and objectivesThe occurrence of non-melanoma and melanoma skin cancers is currently increasing rapidly with one in every three cancers diagnosed as a skin cancer. A useful strategy to control the progression of skin cancer could be the use of plant flavonoids that suppress pro-inflammatory cytokines involved in tumor initiation and progression. In this study, the anti-inflammatory and antioxidant activity of undifferentiated callus extracts from Plantago major L, Silybum marianum L and Rhodiola rosea L was investigated both in normal and malignant skin cells. MethodsAntioxidant activity of the extracts was analyzed by using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. High-Performance Thin-Layer Chromatography (HPTLC) was performed to demonstrate the phytochemical profile, and the total flavonoid content was analyzed with an aluminum chloride colorimetric method. The anti-inflammatory effect was investigated by cell treatments using the plant extracts. Thereafter, the possible suppression of induced IL-6 response was measured from the cultured skin cancer cell lines A2058 and A431, and normal primary keratinocytes with Enzyme-Linked Immunosorbent Assay (ELISA). ResultsThe HPTLC analysis assessed that the extracts contained a complex phytochemical profile that was rich in phenolic and flavonoid compounds. Dose response assays showed that concentrations between 15 and 125 μg/mL of all three plant extracts could be used to investigate an effect on the IL-6 production. The S. marianum extract had the most pronounced anti-inflammatory effect, which significantly inhibited induced IL-6 production in both normal keratinocytes and skin cells derived from epidermal carcinoma. The extract from S. marianum also had the highest flavonoid content and showed the highest antioxidant activity of the three extracts tested. ConclusionAll in all, we have confirmed that undifferentiated callus extracts of S. marianum possess properties such as antioxidant and anti-inflammatory activities in both normal and malignant keratinocytes, and thus could be a promising agent controlling the pro-inflammatory IL-6 production.

HPTLC Analysis and Chemical Composition of Selected Melaleuca Essential Oils.

Tea tree oil (TTO) is a volatile essential oil obtained by distillation, mainly from the Australian native plant Melaleuca alternifolia (Maiden & Betche) Cheel (Myrtaceae). In this study, a comparative analysis of the chemical constituents of seven tea tree oils (M. alternifolia) and four other Melaleuca spp. oils (M. cajuputi, (MCa), two chemotypes of M. quinquenervia, (MNe and MNi), and M. ericifolia (MRo)) was carried out using gas chromatography-mass spectrometry (GC-MS) and high-performance thin-layer chromatography (HPTLC). Among the seven TTOs, terpinen-4-ol (37.66-44.28%), γ-terpinene (16.42-20.75%), α-terpinene (3.47-12.62%), α-terpineol (3.11-4.66%), and terpinolene (2.75-4.19%) were the most abundant compounds. On the other hand, the most abundant compounds of the other Melaleuca oils varied, such as 1,8-cineole (64.63%) in MCa oil, (E)-nerolidol (48.40%) and linalool (33.30%) in MNe oil, 1,8-cineole (52.20%) in MNi oil, and linalool (38.19%) and 1,8-cineole (27.57%) in MRo oil. HPTLC fingerprinting of Melaleuca oils enabled the discrimination of TTO oils from other Melaleuca spp. oils. Variation was observed in the profile of the Rf values among EOs. The present study shows that HPTLC is one of the best ways to identify and evaluate the quality control in authenticating TTOs, other Melaleuca EOs, or EOs from other species within the Myrtaceae.

Development of a Technique for Chromatographic Analysis of Excipients in a Liposomal Dosage Form of a Photosensitizer Tetra-3-phenylthio-phthalocyanine Hydroxyaluminium

Introduction. The Laboratory of Drug Formulation Development at the N. N. National Medical Research Center of Oncology developed a liposomal dosage form of FS tetra-3-phenylthio-pthalocyanine hydroxyluminide (TPHA) for which a simple, accurate and easily reproducible method of quantitative determination of the main active substance and excipients in the drug product was needed. High-performance thin-layer chromatography (HPTLC) was chosen as such a technique.Aim. The aim of this work was to develop chromatographic methods for the quantitative determination of excipients in a liposomal dosage form of a tetra-3-phenylthiophthalocyanine hydroxyaluminium (TPHA).Materials and methods. Determination of substances was carried out by the method of high-performance thin-layer chromatography (HPTLC) followed by TLC-densitometry. Substance TPHA 98 % (Federal State Unitary Enterprise "SSC "NIOPIK", Russia), placebo lyophilized liposomal form without TFHA in the composition (FSBI "National Medical Research Center of Oncology. N. N. Blokhin" of the Ministry of Health of Russia), egg phosphatidylcholine (96 %, CAS № 97281-44-2, EPC Lipoid, Germany), cholesterol (≥99 %, CAS № C8667, Sigma-Aldrich, Japan), sucrose analytical grade (CHIMMED, Russia) and various organic solvents.Results and discussion. The selected mobile phase for HPTLC analysis: isopropanol: ammonia 25 % aqueous 80 : 20, separation time was 20– 25 min (l = 75mm), t = 20 °С. To visualize the spots of sucrose, phosphatidylcholine, and cholesterol, a 5 % solution of phosphomolybdic acid was chosen. The stability of the standard samples was confirmed on a series of prevalidation studies. Validation of the methodology was carried out.Conclusion. For quantitative determination of sucrose, phosphatidylcholine and cholesterol in a new liposomal dosage form, the method of HPTLC with TLC-densitometry was chosen, which allows identifying all three components in the dosage form simultaneously. The method was validated.

Identification of elite species of Ophiorrhiza utilizing HPTLC analysis and camptothecin as a phytochemical marker: Assessment of extraction effectiveness and organ selection

ObjectiveThe main objective of this experiment is to find the camptothecin (CPT) content in different parts of three Ophiorrhiza spp. MethodsIn this study, three species of Ophiorrhiza viz., O.mungos L., O. hirsutula Wight ex Hook. f. and O.rugosa var prostrata (D.Don) Deb & Mondal were taken. Different parts of plant sample such as inflorescence, leaf, stem, and roots were extracted in methanol (100mg/10 ml w/v) using microwave and screened for camptothecin content by High-performance thin layer chromatography (HPTLC). After the selection of elite species different extraction techniques such as Maceration, Hot water bath extraction, Microwave assisted extraction (MAE), and Ultrasonic assisted extraction (UAE) were performed on the elite species for optimization of extraction technique. ResultsHigh performance thin layer chromatography confirmed the presence of camptothecin in all three species. Highest was observed in O.mungos(0.37%) followed by O.hirsutula(0.11%) and a negligible amount in O.rugosa var prostrata(0.001%). Ultrasonic assisted extraction (UAE) method showed the best results in O.mungos leaves.