High-performance Thin-layer Chromatography Analysis Research Articles

Investigating Flavonoids by HPTLC Analysis Using Aluminium Chloride as Derivatization Reagent.

This is the first study to report on high performance thin layer chromatography (HPTLC) generated spectrophotometric data to systematically capture flavonoid compounds using optimized derivatization with either AlCl3 or NaNO2-AlCl3-NaOH as visualisation reagents. While the traditional AlCl3 colorimetric method using UV-Vis analysis provides valuable insights into the presence of flavonoids and allows derivation of the total flavonoid content (TFC) of a sample, HPTLC fingerprints obtained after spraying with AlCl3 or NaNO2-AlCl3-NaOH enable the visualization of the various flavonoids present in a sample based on their respective absorption shifts, thus complementing the traditional TFC assay. In this study, 40 different flavonoids representing different classes (flavonols, flavanolols, flavan-3-ol, flavones, flavanones, and isoflavonoids) were analysed. Upon derivatization with AlCl3 most of the investigated flavonoids recorded bathochromic shifts, yielding characteristic λmax values between 370 and 420 nm, while spraying with NaNO2-AlCl3-NaOH triggered hyperchromic shifts, and thus an increase in absorbance intensity in flavonoids with particular substitution patterns. A few non-flavonoid components with structural similarities to flavonoids (e.g., rosmarinic acid, gallic acid, aspirin, salicylic acid) served as the negative control in this study to determine whether the derivatization reagents allowed exclusive detection of flavonoids. The method was then applied to the analysis of flavonoid containing supplements as well as red clover honey to demonstrate the method's application in the analysis of natural products.

LC-MS/MS Analysis of crude Flavonoid Compounds from <i>Justicia secunda</i> from Democratic Republic of the Congo and evaluation of their antisickling Activity

<i>Justicia secunda</i> Vahl (Acanthaceae) is a tropical herbaceous plant traditionally used by indigenous peoples of the Democratic Republic of the Congo (DRC) to treat symptoms associated with sickle cell disease (SCD), a genetic condition with life-threatening complications. Given the lack of side-effect-free drugs, alternative herbal therapies that can reduce or reverse red blood cell sickling may serve as safe and effective treatments. The primary objective of this study is to conduct a phytochemical screening of crude flavonoids extracted from <i>J. secunda</i> using LC-ESI/MS/MS. <i>J. secunda</i> extracts were evaluated for their phytoconstituents and anti-sickling properties. The anti-sickling properties were assessed through reverse sickling estimation, polymerization and oxidation inhibition, and osmotic fragility assays. Fractionation of the methanol extract and chemical profiling were performed by flash chromatography and high-performance thin-layer chromatography analysis respectively. Characterization and identification of the crude flavonoids were done using chromatography and spectroscopy methods. Chemical screening of crude flavonoids revealed the presence of 12 flavonoid O-glycosides based on one flavonol (methoxy-kaempferol) and two flavones (luteolin and apigenin). The crude flavonoid extracts demonstrated maximum reversal of sickled red blood cells (91.9 ± 0.8%), significant inhibition of hemoglobin polymerization (77.8 ± 7.6%) and hemoglobin S auto-oxidation (96.3 ± 0.2), and provided maximum protection of red blood cells against hyperhemolysis. The results validate the traditional use of <i>J. secunda</i> and identify potential compounds for the development of novel anti-sickling agents.

HPTLC Phytochemical Profiling and Simultaneous Quantification of Quercetin and Gallic in Prosopis juliflora (Sw.)

Prosopis juliflora (Sw.) commonly known as jangli babool or vilayati babool have several bioactive compounds of therapeutic needs. The presence of various bioactive compounds and markers specifically, quercetin and gallic acid in the methanolic extract of Prosopis juliflora (fruiting aerial part) denotes their valuable pharmacological activities. Therefore, high performance thin layer chromatography (HPTLC) analysis with simultaneous quantification of quercetin and gallic acid was done for their phytochemical profiling.The phytochemical profiling and simultaneous quantification of quercetin and gallic acid of methanolic extract of Prosopis juliflora through CAMAG HPTLC analysis was determined and results were obtained in the form of chromatogram scanned at the wavelength 254nm and 366nm. Phytochemical profiling of the plant was presented in the tables showing the number of peaks, peak area, area percentage and Rf values. 14 compounds were present in sample 1 and 9 compounds were present in sample 2 denoted by the number of peaks as represented in chromatograms of test samples. Peak area and height were used to quantify quercetin and gallic acid. Quercetin was found to be 1.364% in sample 1 and 0.27% in Sample 2 while gallic acid was 0.088% in sample 1 only. Importance of this research is to rationalize the therapeutic properties of Prosopis juliflora in the indian traditional medicinal system and phytochemical profiling and simultaneous quantification of quercetin and gallic acid reveals that Prosopis juliflora have several bioactive compounds and this might be accountable for their future application in the field of pharmacognostic and pharmacological drug discovery.

Physicochemical properties, antioxidant activity and phytochemical profiling of Australian Tetragonula carbonaria cerumen

This study investigated the physicochemical and antioxidant properties of stingless bee (Tetragonula carbonaria) cerumen from Australia, while also identifying some phenolic constituents using High-Performance Thin-Layer Chromatography (HPTLC). The antioxidant activity of the ethanolic T. carbonaria cerumen extracts was determined using a Ferric Reducing Antioxidant Power (FRAP) assay and a HPTLC-DPPH approach was used to identify the constituents that contribute to the antioxidant activity of the cerumen samples. For the determination of the total phenolic content (TPC), a modified Folin-Ciocalteu assay was carried out. The FRAP values ranged from 5.19 ± 0.40 to 16.2 ± 0.70 mmol Fe2+/kg of raw cerumen and the TPC values from 3.77 ± 0.12 to 81.3 ± 2.04 mg GAE/g of raw cerumen. The HPTLC analysis revealed the presence of gallic acid and kaempferol in the ethanolic T. carbonaria cerumen extracts which contribute to the antioxidant activity of the samples. The findings of the study demonstrate the antioxidant activity of Australian T. carbonaria cerumen and highlight the importance of more research into its chemical composition, bioactivity and potential therapeutical use.

Effect-directed analysis of endocrine and neurotoxic effects in stormwater depending discharges

The investigation of pollutant inputs via stormwater runoff and subsequent effects in receiving waters is becoming increasingly urgent in view of climate change with accompanying extreme weather situations such as heavy rainfall events. In this study, two sampling areas, one urban and one rural but dominated by a highway, were investigated using effect-directed analysis to identify endocrine and neurotoxic effects and potentially responsible substances in stormwater structures and receiving waters. For this purpose, a transgenic yeast cell assay for the simultaneous detection of estrogenic, androgenic, and progestogenic effects (YMEES) was performed directly on high-performance thin-layer chromatography (HPTLC) plates. Concomitantly, estrogens were analyzed by GC–MS/MS and other micropollutants typical for wastewater and stormwater by LC-MS/MS. Discharges from the combined sewer overflow (CSO) contribute a large portion of the endocrine load to the studied water body, even surpassing the load from a nearby wastewater treatment plant (WWTP). An effect pattern similar to the CSO sample was shown in the receiving water after the CSO with lower intensities, consisting of an estrogenic, androgenic, and progestogenic effect. In contrast, after the WWTP, only one estrogenic effect with a lower intensity was detected. Concentrations of E1, 17α-E2, 17β-E2, EE2, and E3 in the CSO sample were 2000, 410, 1100, 560, and 2700 pg/L, respectively. HPTLC-YMEES and GC–MS/MS complement each other very well and help to elucidate endocrine stresses. An Acetylcholinesterase (AChE) inhibitory effect could not be assigned to a causative compound by suspect and non-target analysis using LC-HRMS. However, the workflow showed how information from HPTLC separation, effect-based methods, and other meta-information on the sampling area and substance properties can contribute to an identification of effect-responsible substances. Overall, the study demonstrated that effect-based methods in combination with HPTLC and instrumental analysis can be implemented to investigate pollution by stormwater run-off particularly regarding heavy rain events due to climate change.

Phytochemical profiling and HPTLC studies on leaf of Chrozophora rottleri (Geiseler) Spreng

For centuries, plants have been integral to traditional medicine, food preservation, and disease prevention. Chrozophora rottleri (Geiseler) A. Juss. ex Spreng., an annual herb of the Euphorbiaceae family, is widely used across India, Myanmar, Thailand, the Andaman Islands, and Malesia in various ethnomedicinal applications. This study aims to provide a comprehensive phytochemical profile and HPTLC analysis of Chrozophora rottleri leaves to highlight its medicinal potential. The leaves were subjected to hot continuous extraction using a Soxhlet apparatus with solvents of increasing polarity to maximize the yield of bioactive compounds. Phytochemical screening identified the presence of alkaloids, flavonoids, tannins, saponins, glycosides, triterpenoids, phenols, steroids, coumarins, cardiac glycosides, and phytosterols in different extracts. Quantitative analysis revealed that alkaloids were most abundant in chloroform and ethanol extracts (500 mg/g), while flavonoids were prominent in ethyl acetate and ethanol extracts (200 mg/g). Phenols and tannins were concentrated in ethyl acetate and aqueous extracts (200 mg/g). The HPTLC analysis displayed distinct separation of phytochemical bands at varying retention factor (Rf) values, using toluene: ethyl acetate (9:1 v/v) at 254 nm and a combination of ethyl acetate: water: formic acid: glacial acetic acid (100:26:11:11 v/v/v/v) at 366 nm and 540 nm. Chromatograms revealed multiple peaks, signifying a rich diversity of bioactive compounds. These findings support the traditional uses of Chrozophora rottleri and underscore its potential for further pharmacological research. The research concluded that the leaf extract of Chrozophora rottleri is abundant in various phytochemicals, potentially contributing to its therapeutic properties. Further investigation into its individual phytochemical profile is recommended for isolating novel compounds. High-performance thin-layer chromatography (HPTLC) fingerprint analysis of Chrozophora rottleri leaf extract could serve as a reliable method for precise plant identification, acting as both a phytochemical marker and a valuable indicator of genetic variability within plant populations.

Pharmacognostical and Pharmaceutical Analysis of Yavanishadavadi Vati - An Ayurvedic Polyherbal Formulation

Pulmonary Tuberculosis (PTB) is caused by the aerobic acid-fast rod-shaped bacterium Mycobacterium tuberculosis. This tiny bacterium, expelled from a contagious host, can remain suspended in air as droplet nuclei for hours. Multidrug-resistant tuberculosis (MDR-TB) is a form of TB caused by bacteria that do not respond to isoniazid and rifampicin, the 2 most effective first-line TB drugs. In Ayurveda, the term Rajayakshma has been used interchangeably with tuberculosis. Rajayakshma is primarily attributable to Dhatukshaya (tissue emaciation or loss). Aim: To analyze the Pharmacognostical and Pharmaceutical properties of Yavanishadavadi Vati. Materials and Methods: Yavanishadavadi Vati was subjected to microscopic evaluation for pharmacognostical study, physico-chemical analysis like hardness, weight variation, loss on drying, ash value, acid insoluble extract, pH value, water soluble extract, alcohol soluble extract and high performance thin layer chromatography (HPTLC). Result: Pharmacognostical study showed the presence of certain identifying characteristics of all the ingredients of Yavanishadavadi Vati that is Yavani, Tintidaka, Shunthi, Amalvetasa, Dadima, Badara, Dhanyaka, Suvarchal Lavana, Jeeraka, Dalchini, Pippali, Maricha and Sarkara. In pharmaceutical study, preliminary physico-chemical analysis showed that hardness of Yavanishadavadi Vati was hardness 6.6Kg/cm2, ash value 10.59%w/w, loss on drying 8.17%w/w, water soluble extract 28.6%w/w, alcohol soluble extract 20.2%w/w and pH value was 6.5. HPTLC analysis showed eleven spots in 254nm and nine spots in 365nm. Conclusion: Present work was carried out to standardize the polyherbal formulation Yavanishadavadi Vati in terms of its identity, quality and purity. Pharmacognostical and physico-chemical observations revealed the specific characters of all the active constituents in the preparation.

Synchronized Assessment of Lobeglitazone Sulfate and Metformin Hydrochloride in Tablet by Robust, High-performance Thin-layer Chromatographic Method

Background: A combination of fixed-doses containing 0.5 mg lobeglitazone sulfate and 500 mg metformin hydrochloride has demonstrated efficacy in enhancing glycemic control in diabetes. Aims: The projected work aimed to establish and validate a high-performance thin-layer chromatographic methodology for the quantification of both drugs in tablet formulations. Objectives: The task involves creating and validating a method in accordance with ICH guidelines to quantify two particular drugs in tablet formulations accurately. Methods: The high-performance thin-layer chromatographic analysis utilized aluminum plates layered with silica gel 60F254, and the solvent system consisted of acetonitrile, 1 M ammonium acetate (methanol), toluene, and triethyl amine (1.5:2.5:4:0.2 v/v/v/v), followed by densitometric scanning at 237 nm. Results: The methodology exhibited linearity in the range of 100-1500 ng/band for lobeglitazone sulfate and 1000-15000 ng/band for metformin hydrochloride, with correlation coefficients of 0.9991 and 0.9992, correspondingly. Exceptional sensitivity was observed, with detection limits of 8.17 ng/band for lobeglitazone sulfate and 271.34 ng/band for metformin hydrochloride, along with quantification limits of 24.75 ng/band for lobeglitazone sulfate and 822.24 ng/band for metformin hydrochloride. The method demonstrated precision (% relative standard deviation of peak area <2) and accuracy (recovery between 96 and 103%). Conclusion: The suggested methodology is fit for the concurrent quantification of both drugs in tablet formulations, making it applicable for routine quality control assessments in laboratories.

Cryoethanolic Extraction, Purification of Allicin from Allium sativum and HPTLC analysis

The significance of this research extends beyond the mere extraction and purification of allicin, encompassing its potential applications across diverse industries. Allicin exhibits a plethora of biological activities, including antimicrobial, antifungal, antiviral, and antiprotozoal properties. Its mechanism of action involves the inhibition of thiol-containing enzymes in microorganisms, rendering it effective against a wide array of pathogens. Moreover, allicin has demonstrated promising anticancer properties, eliciting apoptosis and inhibiting cell proliferation in various cancer cell lines. Additionally, its anti-inflammatory and cardioprotective effects underscore its potential therapeutic utility in mitigating cardiovascular diseases and other inflammatory conditions. This study presents a novel method for extracting and purifying allicin from raw garlic cloves. Dry ice ensures the ethanol to reach its cryo temperature (hilled to sub-zero temperatures ranging from -40°C to -80°C). The process involves the use of ethanol, dry ice and ascorbic acid as solvents, vacuum stirring, and subsequent crystallization. The final product, a hygroscopic powder, was analyzed using High-Performance Thin-Layer Chromatography (HPTLC) to determine allicin content. The extraction yielded 5 grams of whitish hygroscopic powder from 21 grams of crude extract, with an allicin content of 5%. Chromatographic conditions included a mobile phase of acetonitrile: water: Formic acid (30:8:2) and derivatization with ninhydrin, with detection under UV light at 366 nm. This method provides an efficient way to isolate and purify allicin for various applications.

Rheum khorasanicum Decreases Migration and Induces Apoptosis in the MDA-MB-231 Breast Cancer Cell Line

aims: N/A background: Rheum khorasanicum belongs to the Polygonaceae family, which is well-known for its anticancer activities. objective: Due to the limited studies on the anticancer activity of R. khorasanicum, the present study aimed to evaluate the apoptotic effect of this medicinal plant on MDA-MB-231 cells as a model for an aggressive triple negative breast cancer that has lower treatment option. method: The high-performance thin layer chromatography (HPTLC) assay was used for determining the main ingredients of the extract. Viability and apoptosis of the cells was evaluated by WST-1 and the annexin-V/PI dual staining assay, respectively. The scratch assay was used to assess the cellular migratory potential, and the western blotting test was employed for determining the expression of proteins involved in the apoptotic pathway. result: The HPTLC analysis indicated that emodin, gallic acid, epicatechin gallate, and rhaponticin are pharmacologically active compounds isolated from the hydro-alcoholic extract of R. khorasanicum. The extract showed a significant toxic effect on MDA-MB-231 cells. R. khorasanicum extracts also inhibited cellular migratory potential at concentrations higher than 60 μg/mL. The plant extract significantly diminished the Bcl-2/Bax ratio and increased cleaved caspase- 3, cleaved caspase-7/ procaspase-7 expression at protein levels compared to the nontreated cells. conclusion: Our findings demonstrated that effect root extract efficiently triggered the death of breast cancer cell lines possibly through the stimulation of the apoptotic signaling pathway. other: N/A

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.