[Establishment and Optimization of Quality Standards for the Traditional Tibetan Medicine Preparation of Liuwei Nengxiao Pills]. / è—è¯å ­å‘³èƒ½æ¶ˆä¸¸è´¨é‡æ ‡å‡†çš„å»ºç«‹ä¸Žä¼˜åŒ–.

Objective: To establish quality standards for Liuwei Nengxiao pills, to optimize the quality control method, and to provide references for the quality control of Liuwei Nengxiao pills. Methods: Chebula, dried ginger, and Tibetan liqueur root in Liuwei Nengxiao pills of different batch numbers were analyzed by thin layer chromatography (TLC). Then, the content of chrysophanol in the preparation was determined by high performance liquid chromatography (HPLC). Furthermore, a series of methodological validation, including the investigation of the linear relationship, precision, stability, and reproducibility and sample recovery test, were performed to verify the reliability of the results. Results: The TLC identification method was easy to perform and demonstrated high specificity, clear spots, and good separation effect. In addition, the negative controls showed no interference. The HPLC method showed high accuracy. The results of methodological validation showed that the peak area of chrysophanol had a good linear relationship (r2=1.0) in the range of 0.06-0.80 µg, presenting good precision (with the relative standard deviation being lower than 2.0%), good stability and reproducibility (with the relative standard deviation being lower than 1.0%), and an average recovery rate of 100.8%. Conclusion: TLC and HPLC are easy to perform, showing high accuracy and reproducibility. The quality standards established are scientific, reasonable, stable, and feasible, providing references for the quality control of Liuwei Nengxiao pills.

African Under-Utilized Medicinal Leafy Vegetables Studied by Microtiter Plate Assays and High-Performance Thin-Layer Chromatography-Planar Assays.

Biological activities of six under-utilized medicinal leafy vegetable plants indigenous to Africa, i.e., Basella alba, Crassocephalum rubens, Gnetum africanum, Launaea taraxacifolia, Solanecio biafrae, and Solanum macrocarpon, were investigated via two independent techniques. The total phenolic content (TPC) was determined, and six microtiter plate assays were applied after extraction and fractionation. Three were antioxidant in vitro assays, i.e., ferric reducing antioxidant power (FRAP), cupric reduction antioxidant capacity (CUPRAC), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, and the others were enzyme (acetylcholinesterase, butyrylcholinesterase, and tyrosinase) inhibition assays. The highest TPC and antioxidant activity from all the methods were obtained from polar and medium polar fractions of C. rubens, S. biafrae, and S. macrocarpon. The highest acetyl- and butyrylcholinesterase inhibition was exhibited by polar fractions of S. biafrae, C. rubens, and L. taraxacifolia, the latter comparable to galantamine. The highest tyrosinase inhibition was observed in the n-butanol fraction of C. rubens and ethyl acetate fraction of S. biafrae. In vitro assay results of the different extracts and fractions were mostly in agreement with the bioactivity profiling via high-performance thin-layer chromatography-multi-imaging-effect-directed analysis, exploiting nine different planar assays. Several separated compounds of the plant extracts showed antioxidant, α-glucosidase, α-amylase, acetyl- and butyrylcholinesterase-inhibiting, Gram-positive/-negative antimicrobial, cytotoxic, and genotoxic activities. A prominent apolar bioactive compound zone was tentatively assigned to fatty acids, in particular linolenic acid, via electrospray ionization high-resolution mass spectrometry. The detected antioxidant, antimicrobial, antidiabetic, anticholinesterase, cytotoxic, and genotoxic potentials of these vegetable plants, in particular C. rubens, S. biafrae, and S. macrocarpon, may validate some of their ethnomedicinal uses.

Mycotoxin detection in selected medicinal plants using chromatographic techniques.

Mycotoxins are toxic mycological products that when consumed, absorbed or inhaled cause sickness or even the death of humans. Therefore, the present study aimed to evaluate the contamination levels of mycotoxins (aflatoxins, AFB1 , AFB2 , AFG1 , AFG2 , and ochratoxin A, OTA) in selected medicinal herbs and shrubs using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A total of 15 samples of medicinal herbs and shrubs were selected. Among them, four samples were aflatoxin contaminated while two samples were ochratoxin A contaminated. The highest level of aflatoxin was detected in Justicia adhathoda (4,704.94 ppb) through HPLC (153.4 ppb) and through TLC, while the lowest level of aflatoxin was detected in Pegnum harmala (205.1 ppb) through HPLC. Similarly, the highest level of OTA was detected in Dodonia viscosa (0.53 ppb) through HPLC (0.5 ppb) and through TLC, while the lowest level was detected in J. adhathoda (O.11 ppb) through HPLC (0.4 ppb) and through TLC. The OTA concentration was very low, being negligible and below permissible limits. The present study concludes that there is a potential risk for the consumption of herbal decoctions. Therefore, regular monitoring and proper management of mycotoxins, including aflatoxins and OTA, in herbal medicines are needed to ensure the safety of herbal drugs to protect consumers.

Densitometric method for assessment of six specialized metabolites in four Sida sp. and its congener Abutilon indicum: Targeted metabolomics, greenness assessment, and chemometrics analysis.

Sida is one of the most diverse genera, with about 200 species distributed in tropical and subtropical regions of the world. Among 18 species distributed in India, Sida acuta, Sida cordifolia, Sida rhombifolia, and Sida cordata are used in traditional medicines along with its possible adulterant Abutilon indicum for several therapeutic uses. The non-availability of marker-based validated methods for the identification and classification of these species leads to adulteration. Indoloquinoline and quinazoline are the major bioactive alkaloids distributed in Sida spp. First time, a simple, economical and high throughput method was developed and validated for the simultaneous determination of 20-hydroxyecdysone (1), vasicine (2), vasicinone (3), cryptolepine (4), quindolinone (5), and cryptolepinone (6) using HPTLC-UV densitometry. The method was validated to meet globally accepted ICH guidelines. The method was sensitive with LOD and LOQ ranging from 0.38-0.63 and 1.57-2.12 µg/band. The samples were spiked at 3 different concentrations, the recovery values were 93.49-98.88%. In addition, the greenness index of the HPTLC method was estimated using four different greenness assessment techniques. Targeted HPTLC analysis indicated the distribution of specialized metabolites in Sida spp. and A. indicum. However, the occurrence of cryptolepine in A. indicum was not reported in the literature, so this was further confirmed by liquid chromatographic studies of the samples from different locations. The chromatographic data was statistically evaluated by principal component analysis (PCA) and hierarchical clustering (HCA). HPTLC-based targeted metabolite quantitation explains the adulteration/substitution in Sida raw material and derived herbal preparations.

Quality variation of maidong (Ophiopogon japonicus and Liriope spicata) - A HPTLC-based approach.

The tuberous roots of Ophiopogon japonicus and Liriope spicata are used for the same therapeutic purpose in traditional Chinese medicine and are collectively referred to as maidong medicine. Interestingly, it was observed that the price of tuberous roots varies depending on their location on the plant, and fibrous roots are usually discarded post-harvest. Mislabeling might be of concern due to similarities in morphological features between the two species. Moreover, paclobutrazol has been observed to be heavily applied during the production, and therefore might be of health concern. Overall, maidong might suffer from quality inconsistencies while its metabolomic complexity is influenced by growing region and cultivation practices, botanical species, and plant parts. To address these challenges, this study employed High-Performance Thin Layer Chromatography (HPTLC) approach, in which sample preparation and derivatization procedure were optimized to enable to capture more detailed and comprehensive metabolomic fingerprints. By integrating with rTLC algorithm and Multivariate Data Analysis (MVDA), an improved quality assessment was achieved. Samples were collected from four production regions and supplemented with commercial products from markets. The optimized HPTLC analysis recognized species- and region-specific metabolomic patterns of maidong, uncovering a 4% of mislabelled cases. Moreover, findings highlight the underexplored therapeutic potential of fibrous roots, and comparable therapeutic efficacy between different root types. Additionally, complemented by Liquid Chromatography-Mass Spectrometry (LC-MS) for paclobutrazol residue evaluation, 24.66% of the commercial maidong samples surpassed maximum residue limits of paclobutrazol, raising safety concerns. This research represents a significant analytical advancement, offering a robust, cost-effective, and comprehensive method for maidong quality control, and paving the way for more strict residue regulation and updates to herbal pharmacopoeias and monographs.

Derivatization-Based Novel Chromatographic and Spectrophotometric Methods for the Simultaneous Determination of Gymnemic Acid and Resveratrol in Antidiabetic Polyherbal Formulation.

Herbal medicine is widely used for the treatment and prevention of various ailments, highlighting the importance of ensuring its consistency and quality. This research focuses on the simultaneous detection of Gymnemic acid (GYM) and Resveratrol (RES) in an antidiabetic polyherbal formulation as no reported method exists for their simultaneously detection. The objective of this study is to develop and validate novel derivatization-based spectrometric and HPTLC methods for the simultaneous determination of GYM and RES. The spectrophotometric method involved derivatization of GYM with benzoyl chloride, followed by measurement of absorbance at 349 nm an isoabsorptive point. The HPTLC method utilized post derivatization with vanillin-sulfuric acid, and its separation was achieved on pre-coated silica gel 60GF254 using chloroform:methanol:glacial acetic acid (13:4:0.1, v/v/v) as mobile phase and estimated at 575 nm. The developed method exhibits linearity, accuracy, precision, LOD, LOQ, specificity and robustness in accordance with the ICH Q2 (R1) guideline. The percent assay of GYM and RES in the marketed capsule formulation was statistically compared using an unpaired t-test, resulting in a range of 99.51-102.65%. These indicate no significant difference between the proposed method and the marketed formulation. Therefore, both novel methods can be interchangeably used for quality control of GYM and RES in polyherbal formulations.

Species discrimination of multiple botanical origins of Fritillaria species based on infrared spectroscopy, thin layer chromatography-image analysis and untargeted metabolomics.

BACKGROUND: Fritillaria Bulbus (FB), a precious medicinal herb renowned for its heat-clearing, lung-moistening, cough-relieving and phlegm-eliminating effects. In pursuit of profits, unscrupulous merchants have engaged in the substitution or adulteration of valuable varieties with cheaper alternatives. It is, therefore, urgent to develop effective technical approaches to identify FBs from adulterants. METHODS: This paper employed infrared spectroscopy (IR), thin layer chromatography-image analysis (TLC-IA), and untargeted metabolomics techniques to discriminate ten species of FBs. RESULTS: Five species of FBs were successfully differentiated using mid-infrared spectroscopy. Furthermore, the power of TLC-IA technology allowed the differentiation of five species of FBs and two origins of FCBs (Fritillariae Cirrhosae Bulbus). Remarkably, through the application of untargeted metabolomics technique, the precise discrimination of five species of FBs, as well as three origins of FCBs were accomplished. Moreover, a comprehensive identification of 101 markers that reliably distinguished diverse FBs was achieved through the employment of untargeted metabolomics technique. CONCLUSION: The investigation presented powerful means of detection for assuring the quality control of Fritillaria herbs.

Microscopic characterization, TLC fingerprinting and optimization of total lipid content from Euphorbia neriifolia (L.) using response surface methodology.

Euphorbia neriifolia (EN) is a medicinal plant used to treat a variety of ailments in traditional systems. Despite numerous studies on pharmacological activities, no information was available on the microscopic study of this plant. This is the first study that has been attempted to fill this need by performing the light and field emission scanning electron microscopy (FESEM) of leaf, stem, and latex. The powder microscopy of several organs (leaves, stem, and bark) and exudate (latex) of EN was carried out using safranine, fast green, phloroglucinol, and other standard solutions at different magnifications. The chemical fingerprinting of petroleum ether extract was accomplished by using thin layer chromatography. The optimization of total lipid content from the EN leaf under ultrasound-assisted extraction (UAE) and soxhlet extraction (SE) procedure was determined using response surface methodology (RSM). The studied factors that affect the lipid content were: solvent ratio, extraction temperature, and extraction time. Several notable characteristics observed in the leaf of EN are amphistomatic leaves with anticlinical cell walls, anomocytic stomata, spongy mesophyll cells, elongated palisade cells, angular collenchyma, and U-shaped vascular bundle. The plano-convex midrib is covered by polygonal to oval-shaped cuticles and contains anomocytic stomata. The circular petiole has no trichomes and contains laticifers, crystals, and idioblasts. The circular stem was observed with trichomes, hypodermis, collenchyma, parenchymatous cells, central pith, pentagonal stellar region, cambium, and 2-4 times more xylem that of phloem. All of the powdered plant parts and exudate under study contained trichomes, xylem vessels, wood fibers, cork cells, starch grains, calcium oxalate crystals, idioblasts, lignified cork, tannin content, stone cells, and oil globules. The blackish-green colored petroleum ether extract with semi-solid consistency showed the greatest percent (%) yield of 4% in the latex of EN. The thin layer chromatography (TLC) examination of petroleum ether extract of EN leaf produced a maximum 6 spots with Rf values of 0.16, 0.58, 0.62, 0.73, and 0.96 in the mobile phase of petroleum ether-acetone (8:2). In terms of optimization, the dark green colored UAE extract with semi-sticky consistency showed highest % yield of 4.5% whereas the yellowish green colored SE extract of sticky consistency showed the highest % yield of 4.9%. The findings showed that there were not many differences in the total lipid content between UAE (0.16%) and SE (0.11%). However, the best optimum condition for lipid content extraction analysis was obtained as follows: solvent ratio (PE:HE) 50:50, extraction temperature 50°C, extraction time 45 min for UAE, and solvent ratio (PE:HE) 60:40, extraction temperature 45°C, and extraction time of 24 h for SE. Hence, this study signifies the various noteworthy microscopic features along with the presence of different phytocompounds through TLC and best optimized condition for the extraction of lipids from different parts of EN. As no previous study has been reported, the outcomes obtained from the current study prove to be beneficial in the identification of species, quality control, and detection of any adulteration from the laboratory and commercial samples of EN. RESEARCH HIGHLIGHTS: The percent yield was found to be maximum in latex extract (4%). The leaf pet ether extract was separated into 6 bands with different Rf values. The extracted compounds from Euphorbia neriifolia leaves were categorized into non-polar heat tolerant. The highest total lipid yield (0.1119) was obtained at solvent ratios 60:40 of PE:HE (petroleum ether: petroleum hexane).

Bioassay-guided detection, identification and assessment of antibacterial and anti-inflammatory compounds from olive tree flower extracts by high-performance thin-layer chromatography linked to spectroscopy.

Olive trees are one of the most widely cultivated fruit trees in the world. The chemical compositions and biological activities of olive tree fruit and leaves have been extensively researched for their nutritional and health-promoting properties. In contrast, limited data have been reported on olive flowers. The present study aimed to analyse bioactive compounds in olive flower extracts and the effect of fermentation-assisted extraction on phenolic content and antioxidant activity. High-performance thin-layer chromatography (HPTLC) hyphenated with the bioassay-guided detection and spectroscopic identification of bioactive compounds was used for the analysis. Enzymatic and bacterial in situ bioassays were used to detect COX-1 enzyme inhibition and antibacterial activity. Multiple zones of antibacterial activity and one zone of COX-1 inhibition were detected in both, non-fermented and fermented, extracts. A newly developed HPTLC-based experimental protocol was used to measure the high-maximal inhibitory concentrations (IC50) for the assessment of the relative potency of the extracts in inhibiting COX-1 enzyme and antibacterial activity. Strong antibacterial activities detected in zones 4 and 7 were significantly higher in comparison to ampicillin, as confirmed by low IC50 values (IC50 = 57-58 µg in zone 4 and IC50 = 157-167 µg in zone 7) compared to the ampicillin IC50 value (IC50 = 495 µg). The COX-1 inhibition by the extract (IC50 = 76-98 µg) was also strong compared to that of salicylic acid (IC50 = 557 µg). By comparing the locations of the bands to coeluted standards, compounds from detected bioactive bands were tentatively identified. The eluates from bioactive HPTLC zones were further analysed by FTIR NMR, and LC-MS spectroscopy. Multiple zones of antibacterial activity were associated with the presence of triterpenoid acids, while COX-1 inhibition was related to the presence of long-chain fatty acids.

Bioassay-Guided Assessment of Antioxidative, Anti-Inflammatory and Antimicrobial Activities of Extracts from Medicinal Plants via High-Performance Thin-Layer Chromatography.

Natural products and their analogues have contributed significantly to treatment options, especially for anti-inflammatory and infectious diseases. Thus, the primary objective of this work was to compare the bioactivity profiles of selected medicinal plants that are historically used in folk medicine to treat inflammation and infections in the body. Chemical HPTLC fingerprinting was used to assess antioxidant, phenolic and flavonoid content, while bioassay-guided HPTLC was used to detect compounds with the highest antibacterial and anti-inflammatory activities. The results of this study showed that green tea leaf, walnut leaf, St. John's wort herb, wild thyme herb, European goldenrod herb, chamomile flower, and immortelle flower extracts were strong radical scavengers. Green tea and nettle extracts were the most active extracts against E. coli, while calendula flower extract showed significant potency against S. aureus. Furthermore, green tea, greater celandine, and fumitory extracts exhibited pronounced potential in suppressing COX-1 activity. The bioactive compounds from the green tea extract, as the most bioactive, were isolated by preparative thin-layer chromatography and characterized with their FTIR spectra. Although earlier studies have related green tea's anti-inflammatory properties to the presence of catechins, particularly epigallocatechin-3-gallate, the FTIR spectrum of the compound from the most intense bioactive zone showed the strongest anti-inflammatory activity can be attributed to amino acids and heterocyclic compounds. As expected, antibacterial activity in extracts was related to fatty acids and monoglycerides.

A high throughput method for detection of cyclooxygenase-2 enzyme inhibitors by effect-directed analysis applying high performance thin layer chromatography-bioassay-mass spectrometry.

A high throughput method was developed to detect bioactive molecules with inhibitory activity over cyclooxygenase (COX-2) enzyme applying effect-directed analysis and planar chromatography hyphenated with bioassay and mass spectrometry. The assay was based on the indirect measurement of arachidonic acid transformation into prostaglandin with the colorimetric co-substrate N,N,N',N'-tetramethyl-p-phenylenediamine. Inhibitory zones were observed as colorless bands over a blue background. Using a central composite design the critical factors like substrate concentration, enzyme: substrate ratio, reaction time, and co-substrate concentration were optimized. Optimal conditions were achieved with 0.03 mg/mL of arachidonic acid, 0.15 U/mL of COX-2, and 8.21 mg/mL of chromogenic reagent. Method usefulness was challenged analyzing fresh Chiloe's giant garlic (Allium ampeloprasum L) ethanol: water (8:2 v/v) extract, finding COX-2 inhibitors that were preliminarily identified as the isomers γ-glutamyl-S-allyl-l-cysteine and γ-glutamyl-S-(trans-1-propenyl)-L- cysteine.

Bioactivity profiles of six baobab fruit pulp powders via planar chromatography hyphenated with effect-directed analysis.

Baobab (Adansonia digitata) fruit pulp has a high nutrient content and has been traditionally used for medicinal purposes (e.g., as an anti-inflammatory and antioxidant agent) that may help protect against chronic diseases. Six different baobab fruit pulp powders were investigated using three different extractants and analyzed by high-performance thin-layer chromatography (HPTLC) hyphenated with antibacterial bioassays and enzyme inhibition assays. The developed non-target effect-directed screening was performed after extraction with pentyl acetate - ethanol 1:1 (V/V) on the HPTLC plate silica gel 60 using toluene - ethyl acetate - methanol 6:3:1 (V/V/V) as mobile phase system and derivatization via the anisaldehyde sulfuric acid reagent for detection. The physico-chemical profiles of the six baobab fruit pulp powder extracts were comparable, although the intensity of some zones was moderately different. The following effect-directed profiling via tyrosinase, α-glucosidase, and acetylcholinesterase inhibition assays as well as antibacterial Aliivibrio fischeri and Bacillus subtilis bioassays revealed one prominent multipotent bioactive compound zone in common, more or less active in all five studied (bio)assays. Via the recording of high-resolution mass spectra, this compound zone was tentatively assigned to coeluting saturated (palmitic acid 16:0 and stearic acid 18:0), monounsaturated (oleic acid 18:1), and polyunsaturated (linoleic acid 18:2 and linolenic acid 18:3) fatty acids. This finding was confirmed by other studies, which already proved individual activities of fatty acids. The first (bio)activity profiling of baobab fruit pulp powders via HPTLC-effect-directed analysis revealed that the baobab fruit could be considered as a functional food, however, further research is needed to study the impact on health and the influences on the bioactivity arising from different climates, years and soils or regions.

Evaluation of bioactive compounds from Ficus carica L. leaf extracts via high-performance thin-layer chromatography combined with effect-directed analysis.

This study compares different solvent systems with the use of spontaneous fermentation on the phytochemical composition of leaf extracts from a locally grown white variety of common fig (Ficus carica Linn.). The aim was to detect and identify bioactive compounds that are responsible for acetylcholinesterase (AChE), α-amylase and cyclooxygenase-1 (COX-1) enzyme inhibition, and compounds that exhibit antimicrobial activity. Bioactive zones in chromatograms were detected by combining High-performance thin-layer chromatography (HPTLC) with enzymatic and biological assays. A new experimental protocol for measuring the relative half-maximum inhibitory concentration (IC50) was designed to evaluate the potency of the extracts compared to the potency of known inhibitors. Although the IC50 of the fig leaf extract for α-amylase and AChE inhibition were significantly higher when compared to IC50 for acarbose and donepezil, the COX-1 inhibition by the extract (IC50 = 627 µg) was comparable to that of salicylic acid (IC50 = 557 µg), and antimicrobial activity of the extract (IC50 = 375-511 µg) was similar to ampicillin (IC50 = 495 µg). Four chromatographic zones exhibited bioactivity. Compounds from detected bioactive bands were provisionally identified by comparing the band positions to coeluted standards, and by Fourier transform infrared (FTIR) spectra from eluted zones. Flash chromatography was used to separate selected extract into fractions and isolate fractions that are rich in bioactive compounds for further characterisation with nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) analysis. The main constituents identified were umbelliferon (zone 1), furocoumarins psoralen and bergapten (zone 2), different fatty acids (zone 3 and 4), and pentacyclic triterpenoids (calotropenyl acetate or lupeol) and stigmasterol (zone 4).

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, 29.9 min; 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.

Single versus Double Coffee-Ring Effect Patterns in Thin-Layer Chromatography Coupled with Surface-Enhanced Raman Spectroscopic Analysis of Anti-Diabetic Drugs Adulterated in Herbal Products.

In thin-layer chromatography coupled with surface-enhanced Raman spectroscopy (TLC-SERS), the coffee ring effect (CRE) describes the formation of a ring-shape spot (blank in the middle and darker on the edge) caused by the aggregation of silver nanoparticles (Ag NPs), alone (single CRE) or with the analytes (double CRE). In this work, the SCRE and DCRE were investigated in two anti-diabetic drugs, hydrophobic glibenclamide (GLB) and more hydrophilic metformin (MET). The SCRE occurred in GLB analysis, as opposed to the DCRE that occurred in MET. It was proven that for optimization of the TLC-SERS analytical procedure, it is necessary to distinguish the CRE patterns of analytes. Additionally, MET and GLB were analyzed with the developed TLC-SERS method and confirmed by another validated method using high-performance liquid chromatography. Four herbal products collected on the market were found to be adulterated with GLB or/and MET; among those, one product was adulterated with both MET and GLB, and two products were adulterated with GLB at a higher concentration than the usual GLB prescription dose. The TLC-SERS method provided a useful tool for the simultaneous detection of adulterated anti-diabetic herbal products, and the comparison of the SCRE and DCRE provided more evidence to predict CRE patterns in TLC-SERS.

Application of effect-directed analysis using TLC-bioautography for rapid isolation and identification of antidiabetic compounds from the leaves of Annona cherimola Mill.

INTRODUCTION: Type 2 diabetes mellitus is a globally prevalent chronic disease characterised by hyperglycaemia and oxidative stress. The search for new natural bioactive compounds that contribute to controlling this condition and the application of analytical methodologies that facilitate rapid detection and identification are important challenges for science. Annona cherimola Mill. is an important source of aporphine alkaloids with many bioactivities. OBJECTIVE: The aim of this study is to isolate and identify antidiabetic compounds from alkaloid extracts with α-glucosidase and α-amylase inhibitory activity from A. cherimola Mill. leaves using an effect-directed analysis by thin-layer chromatography (TLC)-bioautography. METHODOLOGY: Guided fractionation for α-glucosidase and α-amylase inhibitors in leaf extracts was done using TLC-bioassays. The micro-preparative TLC was used to isolate the active compounds, and the identification was performed by mass spectrometry associated with web-based molecular networks. Additionally, in vitro estimation of the inhibitory activity and antioxidant capacity was performed in the isolated compounds. RESULTS: Five alkaloids (liriodenine, dicentrinone, N-methylnuciferine, anonaine, and moupinamide) and two non-alkaloid compounds (3-methoxybenzenepropanoic acid and methylferulate) with inhibitory activity were isolated and identified using a combination of simple methodologies. Anonaine, moupinamide, and methylferulate showed promising results with an outstanding inhibitory activity against both enzymes and antioxidant capacity that could contribute to controlling redox imbalance. CONCLUSIONS: These high-throughput methodologies enabled a rapid isolation and identification of seven compounds with potential antidiabetic activity. To our knowledge, the estimated inhibitory activity of dicentrinone, N-methylnuciferine, and anonaine against α-glucosidase and α-amylase is reported here for the first time.

Assessing radical scavenging capacity of Sempervivum tectorum L. leaf extracts: An integrated high-performance thin-layer chromatography/in silico/chemometrics approach.

High-Performance Thin-Layer Chromatography (HPTLC)-radical scavenging capacity (RSC) assays are standard techniques for the separation and identification of antioxidants from complex mixtures. HPTLC coupled with DPPH· visualization of chromatograms allows for the detection of individual antioxidants. However, other HPTLC-RSC assays that recognize compounds exhibiting different mechanisms of radical-scavenging activity are rarely reported. In this study, we developed an integrated approach that combines five HPTLC-RSC assays, principal component analysis (PCA) and quantum chemical calculations to assess the antioxidant capacity of Sempervivum tectorum L. leaf extracts. Two HPTLC assays - potassium hexacyanoferrate(III) total reducing power assay (TRP) and total antioxidant capacity by phosphomolybdenum method (TAC) - were developed for the first time. The method supports a more in-depth study of the RSC of natural products, as it compares the radical scavenging fingerprints of S. tectorum leaf extracts and recognizes differences in their individual bioactive constituents. Kaempferol, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, caffeic acid, and gallic acid were identified as the compounds that discriminate HPTLC-RSC assays according to their mechanism of action and capture the similarities between 20 S. tectorum samples. Additionally, DFT calculations on M06-2X/6-31+G(d,p) level were applied to map thermodynamic feasibility of hydrogen atom transfer (HAT) and single electron transfer (SET) mechanisms of the identified compounds. Based on experimental and theoretical results, a combination of HPTLC-ABTS and HPTLC-TAC assays were proposed as the optimal method for mapping the antioxidants from S. tectorum. This study represents a step forward in identifying and quantifying individual antioxidants from complex food and natural product matrices in a more rational manner.

Bioactivity Profiling and Quantification of Gastrodin in Gastrodia elata Cultivated in the Field versus Facility via Hyphenated High-Performance Thin-Layer Chromatography.

Gastrodia elata (Orchidaceae) is native to mountainous areas of Asia and is a plant species used in traditional medicine for more than two thousand years. The species was reported to have many biological activities, such as neuroprotective, antioxidant, and anti-inflammatory activity. After many years of extensive exploitation from the wild, the plant was added to lists of endangered species. Since its desired cultivation is considered difficult, innovative cultivation methods that can reduce the costs of using new soil in each cycle and at the same time avoid contamination with pathogens and chemicals are urgently needed on large scale. In this work, five G. elata samples cultivated in a facility utilizing electron beam-treated soil were compared to two samples grown in the field concerning their chemical composition and bioactivity. Using hyphenated high-performance thin-layer chromatography (HPTLC) and multi-imaging (UV/Vis/FLD, also after derivatization), the chemical marker compound gastrodin was quantified in the seven G. elata rhizome/tuber samples, which showed differences in their contents between facility and field samples and between samples collected during different seasons. Parishin E was also found to be present. Combining HPTLC with on-surface (bio)assays, the antioxidant activity and inhibition of acetylcholinesterase as well as the absence of cytotoxicity against human cells were demonstrated and compared between samples.

Isolation, development and validation of HPTLC method for the estimation of ß-carotene from Gymnosporia senegalensis (Lam.) Loes.

The present study is aimed to isolate terpenoids from Gymnosporia senegalensis through analytical and preparative thin-layer chromatography (TLC) and to determine their antioxidant activity using the 2, 2-diphenyl-1- picrylhydrazyl (DPPH) assay and to find out the presence of ß-carotene through high-performance thin-layer chromatography (HPTLC). The validation included linearity, limit of detection (LOD), limit of quantification (LOQ), specificity, precision, recovery, and robustness. All the isolated compounds from TLC exhibited significant antioxidant activity. Among all, isolated compounds from leaf showed highest IC50 values. The highest total terpenoid content (TTC) was found 51.6 ± 0.06 in stem, then 49.02 ± 0.01 in bark, and 46.27 ± 0.01 in leaf. DPPH results indicated that leaf-isolated compound 1 (LIC1) showed the highest IC50 at 7.55 ± 0.02 and stem-isolated compound 2 (SIC2) showed the lowest IC50 at 0.616 ± 0.01 among all the isolated compounds of G. senegalensis. HPTLC separation was carried out on aluminium plates pre-coated with silica gel 60 F254 as the stationary phase and n-hexane: ethyl acetate (6:4, v/v) as the mobile phase. Quantification was achieved based on a densitometric analysis of ß-carotene in the concentration range of 100-500 ng/band at 254 nm. For the calibration plots, linear regression produced r2 = 0.96450 and Rf = 0.27. The LOD and LOQ were 10.15 and 30.76 ng/mL for HPTLC and relative standard deviation were 137.26 ± 2.03 and 160.43 ± 2.95 (intra-day) and 127.88 ± 2.14 and 157.27 ± 1.90 (inter-day) for 200 and 400 ng/band, respectively. The present study shows the presence of various types of terpenoids through TLC whereas the HPTLC results indicated that the developed methods were accurate and precise. It also shows that the approach is appropriate for its intended use in routine quality control testing of commercially available tablet formulations and drug assay to assist both industries and researchers in making important decisions at a reasonable cost. Moreover, due to the use of a safer and more environmentally friendly mobile phase in comparison to the toxic mobile phases used in recent analytical techniques to estimate ß-carotene, this methodology is also secure and sustainable.

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.