Quantitation of Usnic acid from three Usnea species, its commercial formulations Using High-Performance Thin-Layer Chromatography-Mass Spectrometry (HPTLC-MS) and their metabolite profiling using UPLC-QTof-MSE.

BACKGROUND: The genus Usnea (Parmeliaceae; lichenized Ascomycetes) is pale grayish-green fruticose lichens which grow as leafless mini-shrubs and comprise about 360 species. Most of the Usnea species are edible and is utilized in preparation of traditional foods as well as in medicines to combat wide range of ailments. OBJECTIVE: The goal of this work was to quantify usnic acid in three Usnea spp. [Usnea ghattensis (UG), Usnea orientalis (UO) and Usnea undulata (UU)] using HPTLC-MS and chemical profiling of acetone extracts using UPLC-QTof-MSE resulted in the identification of sixteen compounds based on their MS/MS fragmentation patterns. METHODS: Hyphenated techniques, HPTLC-MS and UPLC-QTof-MSE have been proposed to quantify usnic acid and analysis of metabolites in the crude extracts qualitatively. This method allowed tentative characterization of metabolites from Usnea spp. RESULTS: The quantification study showed the excellent linearity of the usnic acid at 0.25-1 µg/band with a correlation coefficient r  2>0.99, and LOD, LOQ was found to be 51.7 and 156.6 ng/band, respectively. Further, UPLC-QTof-MSE analysis of crude extract led identification of lichen substances through their exact molecular masses and MS/MS fragmentation studies. CONCLUSIONS: The present study summarizes HPTLC method for quantification of usnic acid in three different Usnea spp. Along with two herbal formulations containing Usnea spp. as the ingredient and developed method was validated as per the ICH guidelines and further UPLC-QTof-MSE analysis provides characterization of the sixteen different secondary metabolites based on their mass fragmentation studies. HIGHLIGHTS: Rapid HPTLC method for quantification of usnic acid in three different Usnea spp. along with two herbal formulations and metabolite profiling using UPLC-QTof-MSE.

HPTLC method to support formulation development and quality control of the tablets prepared from fruit extracts of Piper longum.

Alcoholic extract of fruits of Piper longum is reported to have antihypertensive activity, significantly due to presence of alkaloids like piperine, piperlongumine, piperlonguminine and pipernonaline. A sensitive and specific HPTLC method was developed and validated for simultaneous estimation of piperine, piperlongumine and piperlonguminine in fruit extracts with a purpose to provide the analytical support during the development of formulation as well as to serve as a quality control tool for optimised formulation. Developed method was validated with respect to ICH (International Council for Harmonisation) Q2(R1) and found to be accurate, precise, sensitive, specific and robust for estimation of piperine, piperlongumine and piperlonguminine. Method was successfully applied for assay of dry powdered extract and optimised tablet formulation in terms of content of piperine, piperlongumine and piperlonguminine as well as to study the dissolution profiles of formulation batches during development stage.

Novel eco-friendly HPTLC method using dual-wavelength detection for simultaneous quantification of duloxetine and tadalafil with greenness evaluation and application in human plasma.

A novel, environmentally friendly, and sensitive HPTLC method has been developed and validated for simultaneous quantification of duloxetine (DLX) and tadalafil (TDL) in their pure state, laboratory-prepared mixtures, and spiked human plasma. This method is particularly important for patients dealing with depression and sexual issues, as it allows for the measurement of these co-administered antidepressant and sexual stimulant drugs in biological fluids. The separation process employed a stationary phase of pre-coated silica gel 60 F254 and a mobile phase consisting of ethyl acetate, acetonitrile, and 33% ammonia (8:1:1, v/v). The optimized mobile phase resulted in well-defined bands for DLX and TDL, with Rf values of 0.3 and 0.8, respectively with dual-wavelength detection at 232 nm for DLX and 222 nm for TDL. Polynomial regression analysis revealed exceptional linearity for both drugs, with correlation coefficients of 0.9999 over concentration ranges of 10-900 ng/band for DLX and 10-1200 ng/band for TDL. The quantitation limits were 8.2 ng/band for DLX and 8.6 ng/band for TDL, while the detection limits (LOD) were 2.7 ng/band for DLX and 2.8 ng/band for TDL. The validation of this method followed the guidelines set by the International Council for Harmonization (ICH). Additionally, the suggested method's greenness was assessed by means of four up-to-date ecological tools, namely the Eco-Scale, the National Environmental Method Index (NEMI), the Green Analytical Procedure Index (GAPI), and the Analytical Greenness metric approach (AGREE). The proposed method was also assessed using the Blue Applicability Grade Index (BAGI), a recently developed metric for assessing the practicality (blueness) of procedures.

Chemotaxonomy of Southeast Asian Peperomia (Piperaceae) Using High-Performance Thin-Layer Chromatography Colour Scale Fingerprint Imaging and Gas Chromatography-Mass Spectrometry.

The morphological characters of Southeast Asia's indigenous Peperomia species are very similar, especially in their flower structures. The flowers are simple, hermaphrodite and lack a perianth. Therefore, many species are hard to distinguish using morphological characters alone. Here, we apply chemometric data for species identification and classification, gathered using multiwavelength detection combined with the colour scale High-Performance Thin-Layer Chromatography (HPTLC) fingerprinting procedure and chemical compounds determined by Gas Chromatography-Mass Spectrometry (GC-MS). Fourteen taxa were investigated using hexane, ethyl acetate and ethanol solvent extractions. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were used with the colour scale fingerprints to classify the Peperomia species. The PCA and HCA using the chromatogram profile from hexane divided the taxa into six groups compared to the profile from ethyl acetate and ethanol, which each detected seven groups. The chromatogram from the combined dataset of all three solvents can differentiate all the species. The GC-MS data detected a total of 40 compounds from the hexane extract, and these differed among Peperomia species. This approach based on HPTLC fingerprinting and GC-MS analysis can therefore be used as a tool for authentication and identification studies of Peperomia species.

Fermentation dynamics of bile salt hydrolase production in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012: Addressing ninhydrin assay limitations with a novel HPTLC-MS method.

Bile salt hydrolase (BSH), a pivotal enzyme in cholesterol management, holds significant promise in both human and animal subjects. This study investigated the effect of fermentation dynamics in Heyndrickxia coagulans ATCC 7050 and Lactiplantibacillus plantarum ATCC 10012 to enhance BSH production. Cultivation of cultures in MRS and M17 media revealed that MRS medium enhanced BSH production by 235.98 % in H. coagulans ATCC 7050 and 147.37 % in L. plantarum ATCC 10012, compared to M 17 medium. Additionally, varying oxygen concentration levels indicated that H. coagulans ATCC 7050 exhibited its minimum doubling time of 79.8 ± 0.64 min in anaerobic conditions, whereas L.plantarum ATCC 10012 demonstrated its minimum doubling time of 85.5 ± 1.2 min under microaerophilic conditions. However, their highest BSH activity was observed during the stationary phase under anaerobic conditions, yielding 17.14 ± 0.78 U/mL by H. coagulans ATCC 7050 and 19.04 ± 0.81 U/mL by L.plantarum ATCC 10012. Furthermore, it was observed that both organisms did not retain BSH within their cells. BSH activity was assessed using ninhydrin assay that detected free taurine liberated from sodium taurocholate. However, ninhydrin can yield false-positive results owing to its interaction with other free amino acids. To subjugate this limitation, the study introduced a novel and sensitive HPTLC-MS method capable of accurately detecting taurine. By comprehending fermentation dynamics and selecting appropriate conditions, BSH production increased 2.1-fold in both organisms. These findings illuminate critical insights, offering a pathway for novel strategies to enhance the BSH-producing capabilities of these LAB strains.

Bioassay-Guided Isolation of Guggulsterol I and Evaluation of Its Antioxidant Properties in Ethanol Extracts From Commiphora caudata Leaves.

The secondary metabolites of various parts of Commiphora caudata have shown a range of biological activities under both in vivo and in vitro conditions, particularly anti-inflammatory and antioxidant properties. While E-guggulsterone from this plant has been proven to have anti-inflammatory effects, the antioxidant potential of phytochemicals present in the leaves of C. caudata is less explored. This investigation aimed to isolate an antioxidant phytoconstituent from the ethanolic extract of the dried leaves of C. caudata using a bioassay-guided approach. The dried leaves were successively extracted with organic solvents, including ethanol. The presence of phytochemicals was tested using high-performance thin-layer chromatography (HPTLC), and phytoconstituent from ethanol extract was purified by column chromatography. The antioxidant activity of both the crude extract and the purified compound was evaluated and then compared. The radical scavenging activity was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The ethanolic extract of C. caudata showed 87.6% DPPH radical scavenging activity at a concentration of 250 µg/mL, while standard ascorbic acid showed 94.9% inhibition at the same concentration. The concentrated ethanolic extract of C. caudata was subjected to silica-based column chromatography with an ascending polarity of mobile phase solvents, ethyl acetate, and ethanol (25:75), yielding a single compound. The isolated compound was confirmed as guggulsterol I by ultraviolet (UV), infrared (IR), mass, and nuclear magnetic resonance (NMR) spectroscopy. The radical scavenging activity of the crude ethanolic extract of C. caudata leaves and the isolated compound guggulsterol I was concentration-dependent. The crude ethanolic extract of C. caudata showed significant antioxidant activity in comparison with the standard. However, the isolated guggulsterol I showed less antioxidant activity than the crude ethanolic extract. This study strongly suggests that the crude ethanolic extract of C. caudata leaves had better antioxidant activity due to the synergistic or additive effect of guggulsterol I and other phytoconstituents.

Thevetia thevetioides Cardenolide and Related Cardiac Glycoside Profile in Mature and Immature Seeds by High-Resolution Thin-Layer Chromatography (HPTLC) and Quadrupole Time of Flight-Tandem Mass Spectrometry (Q-TOF MS/MS) Reveals Insights of the Cardenolide Biosynthetic Pathway.

Thevetia thevetioides is a species within the Apocynaceae family known for containing cardenolide-glycosides, commonly referred to as cardiac glycosides, which are characteristic of this genus. The seeds of the Thevetia species are frequently used as a model source for studying cardiac steroids, as these glycosides can be more readily extracted from the oil-rich seeds than from the plant's green tissues. In this work, the cardenolide profile of ripe and immature seeds was determined and compared to establish the main differences. Ripe seeds contain six related cardenolides and triosides, with thevetin B being the predominant component. In contrast, immature seeds exhibit a total of thirteen cardiac glycosides, including monoglycosides such as neriifolin and peruvosides A, B, and C, as well as diglycosides like thevebiosides A, B, and C. Some of these compounds have previously been identified as degradation products of more complex cardiac glycosides; however, their presence in immature seeds, as described in this study, suggests that they may serve as biosynthetic precursors to the triosides observed in mature seeds. The glycoside patterns observed via HPTLC are associated with specific chemical structures characteristic of this genus, typically featuring thevetose or acetyl-thevetose at the first position, followed by glucose or gentibiose in di- or trisaccharides, independent of the trioside aglycones identified: digitoxigenin, cannogenin, or yccotligenin. Ripe seeds predominantly contain triosides, including thevetin B, C, and A, the latter of which has not been previously reported.

Identification of pancreatin inhibitors from Thai medicinal Piper plants for antidiabetic and anti-obesity activities using high-performance thin-layer chromatography-bioautographic assay.

Exploring the potential of natural products against diabetes and obesity is in demand nowadays. Pancreatic α-amylase and pancreatic lipase are the drug targets to minimize the absorption of glucose from starch and fatty acids from lipids, respectively. In this study, five Piper species, namely P. sarmentosum (Ps), P. wallichii (Pw), P. retrofractum (Pr), P. nigrum (Pn), and P. betle (Pb), which are commonly used as food ingredients and traditional medicines, were evaluated for their inhibitory activities against pancreatin using the microtiter plate method. Additionally, pancreatin inhibitors were identified through a cost-effective high-performance thin-layer chromatography (HPTLC)-bioautography developed using red starch and p-nitrophenyl palmitate, corresponding to anti-amylase and -lipase activities, respectively. Of the 15 samples tested, leaf samples from Pb, which had the highest total phenolic and total flavonoid contents, exhibited remarkable inhibitory activity against pancreatin, with a relative amylase inhibitory capacity (RAIC) ranging between 4.260 × 10-5 and 4.861 × 10-5 and a reciprocal half-maximal inhibitory concentration (1/IC50, PTL) of 0.390-0.510 (mg/mL)-1. Additionally, Ps samples demonstrated the second-ranked anti-pancreatin activity. Principal component analysis indicated that total phenolic content contributed to the anti-pancreatin activities of Pb samples. The anti-pancreatin bands were isolated and identified as caffeic acid, myricetin, genistein, piperine, and eugenol. Myricetin, in the roots of Ps samples, showed notable anti-pancreatin activity, which was consistent with results from the in silico prediction toward pancreatic α-amylase and pancreatic lipase. Caffeic acid and eugenol were present in Pb samples. In conclusion, the developed cost-effective pancreatin HPTLC-bioautography efficiently identified amylase and lipase inhibitors from Piper herbs, which supported the use of these plants for antidiabetes and anti-obesity.

HPTLC-guided flash chromatographic isolation and spectroscopic identification of bioactive compounds from olive flowers.

The goal of preparative chromatography is to isolate suitable amounts of compound(s) at the required purity in the most cost-effective way. This study analyses the power of High-performance thin-layer chromatography (HPTLC) guided preparative flash chromatography to separate and isolate bioactive compounds from an olive flower extract for their further characterisation via spectroscopy. The structure and purity of isolated bioactive compounds were assessed using Fourier-transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy. Flash chromatography of the olive flower extract successfully isolated pure oleanolic and maslinic acids. Moreover, the flash chromatography of the extract allowed isolation and phytochemical analysis of the most lipophilic fraction of the extract, which was found to contain n-eicosane and n-(Z)-eicos-5-ene, that has not been isolated previously with preparative TLC.

Comparative HPTLC analysis of shatavarin IV, sarsasapogenin, caffeic acid, ß-sitosterol and lupeol in roots and cladodes of ten Asparagus L. ssp. from North India.

In the present research, ten species of Asparagus from North India have been analysed for secondary metabolites. The quantitative study of shatavarin IV, sarsasapogenin, caffeic acid, ß-sitosterol, and lupeol in the cladodes and roots of Asparagus was conducted using a validated HPTLC method. The content of sarsasapogenin was found highest in the cladodes of A. racemosus (11.20 ± 0.025 mg/g DW) and roots of A. officinalis (5.95 ± 0.024 mg/g DW). Shatavarin IV was found highest in cladodes of A. densiflorus (6.72 ± 0.02 mg/g DW) and roots of A. adscendens (4.68 ± 0.015 mg/g DW). Caffeic acid was found most abundantly in A. officinalis (65.87 ± 0.021 mg/g DW), while ß-sitosterol (9.36 ± 0.004 mg/g DW) and lupeol (5.91 ± 0.004 mg/g DW) were found highest in A. falcatus among the ten species. Overall findings showed that A. adscendens, A. densiflorus, A. falcatus and A. retrofractus have also rich quantity of examined secondary metabolites as compared to commercially important species (A. officinalis).

Development of a simple and reproducible HPTLC method on protective effect induced by bark of Acacia nilotica on poisoning caused by use of nicotine containing tobacco products.

Tobacco (Nicotiana tobacum) and tobacco products are the most critical public health challenges today across the globe. Nicotine is the main chemical composition of tobacco and is associated with withdrawal syndrome. A laboratory animal is commonly employed as a model to investigate nicotine toxicity, drug dependence, reinforcing effects, and the protective effects of samples against nicotine-induced toxicity. The first in-vitro model was developed to prove the protective effect of Babbul (Acacianilotica Linn.) against nicotine poisoning caused by consumption of tobacco products. The HPTLC method for estimating the protective effect against nicotine poisoning was performed by taking the solvent systems dichloromethane, methanol, and liquid ammonia (25 %)(9:1:0.04v/v/v). This in-vitro approach was done by treating the bark of the Acacia nilotica extract with a standard solution of nicotine, which reduced the concentration of nicotine by 39.12 %. The prescribed HPTLC method can be used successfully to assess Acacia nilotica's protective impact against nicotine toxicity caused by intake of nicotine containing tobacco products.

Development of HPTLC method for simultaneous determination of quercetin and kaempferol in leaf extract of Hibiscus mutabilis.

The aim of this study was to develop and validate a densitometric High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous quantification of quercetin (Q) and kaempferol (K) in Hibiscus mutabilis leaf extracts. The analyses were performed on silica gel 60 F254 plates using a mobile phase composed of toluene, formic acid, and ethyl acetate (6:0.4:4, v/v/v). Detection was carried out at a wavelength of 272 nm using a deuterium and tungsten light source. The method exhibited excellent linearity over the concentration range of 100-600 ng/spot for quercetin and 500-3000 ng/spot for kaempferol, with determination coefficients (r2) of 0.9989 and 0.9973, respectively. The method showed no interferences from the plant matrix. The relative standard deviation (RSD) values for intra- and inter-day precision were less than 2% for both flavonoids. Recovery rates ranged from 97.69% to 99.20% for quercetin and from 89.91% to 95.87% for kaempferol. The limits of detection (LOD) were 190.23 ng/spot for quercetin and 187.23 ng/spot for kaempferol, while the limits of quantification (LOQ) were 570.10 ng/spot for quercetin and 566.12 ng/spot for kaempferol. This validated HPTLC method is reliable, precise, and accurate, making it suitable for the quality control of Hibiscus mutabilis leaf extracts. The study's findings can be broadly applied to the quality control of herbal products, pharmacological research, and the development of nutraceuticals. The method's ability to provide rapid and accurate quantification makes it an invaluable tool for researchers across various disciplines.

HPTLC sequentially coupled to UV and SERS: A cost-effective tool for confirmative identification and quantitation of sildenafil in falsified herbal products.

The detection of falsified drugs usually requires multi-disciplinary analysis for confirmative identification. Among hyphenated techniques with high specificity detection, thin-layer chromatography coupled with surface-enhanced Raman spectroscopy (TLC-SERS) is an efficient choice, especially for herbal products with diversified matrix. In this study, HPTLC was coupled to two detection techniques: UV absorption and Raman scattering with silver colloid enhancement for the analysis of sildenafil adulterated in herbal products. With this approach, orthogonal UV and SERS spectral data was collected, so that confirmative results could be obtained within a single TLC analysis. How this approach helped to reduce chances of false positive or false negative results was also discussed. The HPTLC sequentially coupled to UV and SERS (HPTLC-UV-SERS) method was developed and validated parallelly on the UV and SERS signals. To improve the repeatability of the SERS signal, several analytical conditions were optimized, so that direct quantitation with TLC-SERS was feasible without chemometric data extrapolation. The determination was done with UV scanning at 304 nm for HPTLC and with SERS signal at 1580 cm-1 (excitation 633 nm). The TLC-SERS method had a detection limit of 1.65 ng/spot, 95 times lower than HPTLC method (157 ng/spot). The HPTLC-UV-SERS method was applied on 24 real herbal samples collected from the market, among which 3 real samples were positive to sildenafil, and quantitation results by UV and SERS were in consistency. Not only this method was proved feasible for practical applications, but the recommendations for TLC-SERS procedures could also be useful in TLC-SERS method development for other compounds.

Chromatographic fingerprinting of ipratropium and fenoterol in their novel co-formulated inhaler treating major respiratory disorders; application to delivered dose uniformity testing along with greenness and whiteness assessment.

Ipratropium bromide (IPR) and fenoterol hydrobromide (FEN) have recently been combined in a promising inhaler to treat two prevalent inflammatory illnesses of the airways: bronchial asthma and chronic obstructive pulmonary disease (COPD). The necessity for a single, sensitive, and trustworthy analytical approach to cover the diverse and necessary tests of in-vitro and in-vivo studies is greatly grown with the rising production of new fixed combinations. Two novel, selective and environmentally friendly LC techniques were developed in order to guarantee precise measurement of IPR and FEN in their challenging formulation. The initial technique involved high-performance thin-layer chromatography (HPTLC) in conjunction with densitometric quantification. Chromatographic separation was attained on HPTLC plates utilizing ethyl acetate - ethanol - acetic acid (5.0:5.0:0.1, by volume) as a developing system. Densitometric quantification of the separated bands was carried out at 220.0 nm over concentration ranges of 0.50-15.0 µg/band for IPR and 0.50-12.0 µg/band for FEN. High-performance liquid chromatography (HPLC) paired with diode array detection (DAD) was the core of the second technique. The optimized separation was achieved on a Zorbax SB C18 (150 × 4.6 mm, 5 µm) column with a combination of 10.0 mM potassium dihydrogen orthophosphate, pH 5.0 ± 0.1, adjusted with o-phosphoric acid and methanol (70:30, v/v) as the mobile phase and pumped at flow rate of 1.0 mL/min. The peaks were monitored at 220.0 nm using diode array detection, achieving linearity range of 5.0-200.0 µg/mL for both drugs. The ICH criteria have been verified and both methods have been confirmed to be valid, and successfully applied for assay the cited drugs in the Atrovent® comp HFA metered dose inhaler as well as delivered dose uniformity testing of the final product. Finally, whiteness appraisal and several state-of-the-art green evaluation metrics were applied to evaluate the sustainability of the proposed methods. The suggested approaches produced promising results and are the first simple and sustainable methodologies for the simultaneous quantification of both drugs in different real samples, all of which strongly suggest their application in quality control laboratories.

Physicochemical Properties, Antioxidant Activity, and High-Performance Thin-Layer Chromatography Profiling of Propolis Samples from Western Australia.

This study reports on the physicochemical and antioxidant properties of propolis samples from various regions across Western Australia and identifies some phenolic constituents using high-performance thin-layer chromatography (HPTLC). Total phenolic content (TPC) was determined using a modified Folin-Ciocalteu assay, and antioxidant activity was investigated with the Ferric Reducing Antioxidant Power (FRAP) assay and also visualised and semi-quantified by HPTLC-DPPH analysis. TPC values ranged from 9.26 to 59.3 mg gallic acid equivalent/g of raw propolis and FRAP assay data from 4.34 to 53.8 mmol Fe2+ mmol/kg of raw propolis, although some of these variations might be related to differences in extraction yields obtained with 70% ethanol. The presence of luteolin, taxifolin, naringenin, and 4-hydroxyphenylacetic acid was confirmed based on a comprehensive, validated matching approach against an HPTLC-derived database. The findings of the study highlight the importance of future research on the chemical composition and bioactivity of Western Australian propolis.

Tissue-Specific Natural Synthesis of Galanthaminein Zephyranthes Species and Its Accumulation in Different In Vitro-Grown Organs Following Methyl Jasmonate Treatment.

Galanthamine is an immensely valuable alkaloid exhibiting anti-cancer and antiviral activity. The cultivation of plant tissues in in vitro conditions is a good source for the synthesis and enrichment of secondary metabolites of commercial interest. In this study, the Amaryllidaceae alkaloid galanthamine was quantified in three Zephyranthes species, such as Zephyranthes candida, Zephyranthes grandiflora, and Zephyranthes citrina, and the impact of the methyl jasmonate (MJ) signaling molecule on galanthamine accumulation was monitored in in vitro-derived plant tissues. This is the first ever study of the MJ-regulated accumulation of galanthamine in in vitro-grown Zephyranthes tissues. Shoot regeneration was obtained in all three Zephyranthes species on Murashige and Skoog (MS) medium containing 2.0 mgL-1 benzylaminopurine (BAP) + 0.5 mgL-1 naphthalene acetic acid (NAA). The regenerated shoots were rooted on a medium containing 2.0 mgL-1 indole butyric acid (IBA). A GC-MS study of Zephyranthes extracts revealed the presence of 34 phyto-compounds of varied levels with therapeutic activities against diseases. The galanthamine content was quantified in plant parts of the three Zephyranthes species using high-performance thin layer chromatography (HPTLC); the maximum was found in Z. candida bulb (2.41 µg g-1 dry wt.), followed by Z. grandiflora (2.13 µg g-1 dry wt.), and then Z. citrina (2.02 µg g-1 dry wt.). The galanthamine content showed bulb > leaf > root source order. The in vitro-generated plantlets were treated with different MJ concentrations, and the galanthamine yield was measured in bulb, leaf, and root tissues. The highest galanthamine content was recorded in bulbs of Z. candida (3.97 µg g-1 dry wt.) treated with 150 µM MJ, showing an increase of 64.73% compared to the control. This accumulation may be attributed to MJ-induced stress, highlighting the potential commercial synthesis of galanthamine in vitro.

Estrogen receptor alpha (ER-α) antagonistic activity of phytoconstituents from Potentilla atrosanguinea and Potentilla fulgens in breast cancer.

The Potentilla genus has long been used traditionally as food and a folklore medicine. In the present study, aerial parts of two Potentilla species, Potentilla fulgens and Potentilla atrosanguinea, of western Himalayan origin, were studied for their anti-breast cancer activity. Ethyl acetate (PAA-EA, PFA-EA), methanolic (PAA-ME, PFA-ME) and hydro-methanolic extract (PAA-HM, PFA-HM) of the plants were tested for their antiproliferative activities against MCF-7 and T-47D breast cancer cell lines. The extracts showed good antiproliferative activity against ER-α dominant breast cancer cell line T-47D, having IC50 values 6.19 ± 0.01 to 33.23 ± 0.04 µg/ml. Eight compounds were isolated, characterized, and quantified from ethyl acetate and methanolic extracts by column chromatography, 1D, 2D-NMR, HRMS and TLC densitometric analysis. Two compounds (4 and 6) have shown better antiproliferative activity than standard bazedoxifene and were further evaluated for their ER-α binding affinity via-fluorescence polarization-based competitive binding assay. The antiestrogenic properties of both compounds were assessed using western blotting. Compounds 4 and 6 were found to have significant affinity for the ER-α and managed to decrease its expression by 38 and 54% respectively. Compounds 4 and 6 also had good stability and reactivity as measured by minimal fluctuations in molecular dynamic simulation analysis, a good dock score in molecular docking, and a respectable HOMO-LUMO energy gap in DFT calculations. Compounds 4 and 6 have shown reliable results and can be used in the development of natural product-based anti-breast cancer agents.

Evaluation of Phytochemistry and Antidiabetic Potential of an Astragalus Species (Astragalus kurdicus Boiss.).

Astragalus kurdicus Boiss. roots are used in folk medicine for antidiabetic purposes. Different Astragalus plant metabolites have a notable potential for antidiabetic activity through varying mechanisms. Herein, this study is designed to assess the antidiabetic activity of Astragalus kurdicus total (AKM: methanol extract, yield: 14.53 %) and sub-extracts (AKB: n-butanol, AKC: chloroform, AKW: water, AKH: hexane extracts), utilizing a range of diabetes-related in vitro methodologies, and to investigate the chemical composition of the plant. The highest astragaloside and saponin content was seen in AKB extract. Among the measured saponins, the abundance of Astragaloside IV (27.41 µg/mg in AKM) was the highest in high-performance thin-layer chromatography (HPTLC) analysis. Furthermore, flavonoid-rich AKC was found to be mostly responsible for the high antioxidant activity. According to the results of the activity tests, AKW was the most active extract in protein tyrosine phosphatase 1 B (PTP1B), dipeptidyl peptidase IV (DPP4), and α-amylase inhibition tests (percent inhibitions are: 87.17 %, 82.4 %, and 91.49 % respectively, at 1 mg/mL). AKM and AKW demonstrated the highest efficacy in stimulating the growth of prebiotic microorganisms and preventing the formation of advanced glycation end products (AGEs). Thus, for the first time, the antidiabetic activity of A. kurdicus was evaluated from various perspectives.

Maclura cochinchinensis (Lour.) Corner Heartwood Extracts Containing Resveratrol and Oxyresveratrol Inhibit Melanogenesis in B16F10 Melanoma Cells.

This study aimed to isolate and purify resveratrol and oxyresveratrol from the heartwoods of Maclura cochinchinensis, and to evaluate their inhibitory effects on melanogenesis in B16F10 murine melanoma cells. A methanol maceration process yielded a crude extract comprising 24.86% of the initial mass, which was subsequently analyzed through HPTLC, HPLC, and LC-MS/MS. These analyses revealed the presence of resveratrol and oxyresveratrol at concentrations of 4.32 mg/g and 33.6 mg/g in the extract, respectively. Initial purification employing food-grade silica gel column chromatography separated the extract into two fractions: FA, exhibiting potent inhibition of both tyrosinase activity and melanogenesis, and FM, showing no such inhibitory activity. Further purification processes led to the isolation of fractions Y11 and Gn12 with enhanced concentrations of resveratrol (94.9 and 110.21 mg/g, respectively) and fractions Gn15 and Gn16 with elevated levels of oxyresveratrol (321.93 and 274.59 mg/g, respectively), all of which significantly reduced melanin synthesis. These outcomes affirm the substantial presence of resveratrol and oxyresveratrol in the heartwood of M. cochinchinensis, indicating their promising role as natural agents for skin lightening.

Exploring the neuropharmacological properties of scopoletin-rich Evolvulus alsinoides extract using in-silico and in-vitro methods.

OBJECTIVES: This study investigates the neuropharmacologic properties of Scopoletin, a bioactive compound in Evolvulus alsinoides (EA) extract, for managing cognitive impairment using in-vitro, in-silico, and zebrafish embryo toxicity assays. METHODS: The study estimates Scopoletin concentration in EA extract using HPTLC, assesses antioxidant properties using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing ability of plasma (FRAP) assays, and uses bioinformatic tools for scopoletin targets. Zebrafish embryo toxicity (ZET) is used to assess its toxicological profile. RESULTS: 0.0076% w/w Scopoletin in the samples was quantified using HPTLC, further studies on the DPPH (0.5 mM) and FRAP gave EC50 at 440.0 µg/ml and 84.29 µg/ml respectively. Twelve common targets associated with cognitive impairment (CI) were identified, along with possible pathways and molecular interactions. Our results indicate significant binding affinities of Scopoletin with ERAP1, SCN3A, and COMT. Molecular dynamics simulations further confirm the stability of these interactions. ZET assessment demonstrated mortality after 450 µg/ml concentration of EA extract. CONCLUSION: The study verifies the presence of Scopoletin in EA, along with their targets playing a crucial role in neurogenesis and neuroplasticity. The ZET demonstrated concentration-dependent effects, emphasizing the importance of dosage considerations in developing new formulations or therapeutics. This comprehensive study contributes valuable insight into the therapeutic potential of Scopoletin from EA for cognitive impairment, paving the way for further research.