Phytochemical Characterization and Antioxidant Activity Evaluation for Some Plant Extracts in Conjunction with Pharmacological Mechanism Prediction: Insights into Potential Therapeutic Applications in Dyslipidemia and Obesity.

Lipid metabolism dysregulation can lead to dyslipidemia and obesity, which are major causes of cardiovascular disease and associated mortality worldwide. The purpose of the study was to obtain and characterize six plant extracts (ACE-Allii cepae extractum; RSE-Rosmarini extractum; CHE-Cichorii extractum; CE-Cynarae extractum; AGE-Apii graveolentis extractum; CGE-Crataegi extractum) as promising adjuvant therapies for the prevention and treatment of dyslipidemia and its related metabolic diseases. Phytochemical screening revealed that RSE was the richest extract in total polyphenols (39.62 ± 13.16 g tannic acid/100 g dry extract) and phenolcarboxylic acids (22.05 ± 1.31 g chlorogenic acid/100 g dry extract). Moreover, the spectrophotometric chemical profile highlighted a significant concentration of flavones for CGE (5.32 ± 0.26 g rutoside/100 g dry extract), in contrast to the other extracts. UHPLC-MS quantification detected considerable amounts of phenolic constituents, especially chlorogenic acid in CGE (187.435 ± 1.96 mg/g extract) and rosmarinic acid in RSE (317.100 ± 2.70 mg/g extract). Rosemary and hawthorn extracts showed significantly stronger free radical scavenging activity compared to the other plant extracts (p < 0.05). Pearson correlation analysis and the heatmap correlation matrix indicated significant correlations between phytochemical contents and in vitro antioxidant activities. Computational studies were performed to investigate the potential anti-obesity mechanism of the studied extracts using target prediction, homology modeling, molecular docking, and molecular dynamics approaches. Our study revealed that rosmarinic acid (RA) and chlorogenic acid (CGA) can form stable complexes with the active site of carbonic anhydrase 5A by either interacting with the zinc-bound catalytic water molecule or by directly binding Zn2+. Further studies are warranted to experimentally validate the predicted CA5A inhibitory activities of RA and CGA and to investigate the hypolipidemic and antioxidant activities of the proposed plant extracts in animal models of dyslipidemia and obesity.

In Silico and In Vitro Studies on an Asymmetrical Porphyrin Derivative with Therapeutic Potential in Skin Disorders.

For developing novel photosensitizers with therapeutic potential in non-malignant and malignant cutaneous disorders, the unsymmetrical porphyrin, 5-(2-hydroxy-3-methoxyphenyl)-10, 15, 20-tris-(4-carboxymethylphenyl) porphyrin, was evaluated in silico and in vitro. The cellular uptake of the investigated porphyrin and its ability to perform photodynamic therapy were investigated in terms of the viability, proliferation, and necrosis of human HaCaT keratinocytes and human Hs27 skin fibroblasts, in correlation with the predictions regarding diffusion through cell membranes, ADMET profile (absorption, distribution, metabolism, elimination, toxicity), and potential pharmacological mechanism. Molecular docking and 250 ns molecular dynamics simulations revealed that P5.2 has the potential to form a relatively stable complex with the carbonic anhydrase IX catalytic site, the lowest predicted free energy of binding (MM/PBSA) being -39.097 kcal/mol. The results of the in vitro study showed that P5.2 is incorporated within 24 h in the investigated cells, especially in HaCaT keratinocytes, indicating its photosensitizing ability. Nevertheless, P5.2 does not exert significant cytotoxicity in "dark" conditions. In turn, PDT induced a decrease in the number of metabolically active HaCaT keratinocytes within 24 h, accompanied by a 4-fold increase in lactate dehydrogenase release, indicating its ability to perform PDT in human skin cells. The experimental results suggest that the asymmetrical porphyrin is a promising candidate theranostics agent for skin disorders.

Inhibitory Effects on Staphylococcus aureus Sortase A by Aesculus sp. Extracts and Their Toxicity Evaluation.

A promising strategy for combating bacterial infections involves the development of agents that disarm the virulence factors of pathogenic bacteria, thereby reducing their pathogenicity without inducing direct lethality. Sortase A, a crucial enzyme responsible for anchoring virulence factors to the cell surface of several pathogenic bacteria, has emerged as a possible target for antivirulence strategies. A series of hippocastanum species (Aesculus pavia, A. parviflora, Aesculus x carnea, and A. hippocastanum) were used to prepare ethanol- and water-based extracts for assessing their effect on Staphylococcus aureus sortase A. The extracts were characterized through HPLC analysis, and their polyphenols content was determined using the Folin-Ciocalteu method. The specific toxicity profile was evaluated in Daphnia magna using the median lethal concentration (LC50) and against the fibroblast MRHF cell line. The half maximal inhibitory concentration (IC50) values on sortase A, determined after 30 min of incubation, ranged from 82.70 to 304.31 µg/mL, with the A. pavia water extract exhibiting the highest inhibitory effect. The assessment of the A. pavia water extract on human fibroblasts revealed no significant signs of toxicity, even at a concentration of 500 µg/mL. This reduced toxicity was further validated through the Daphnia assay. These findings highlight the low toxicity and the potential of this extract as a promising source of future development of bacteria antivirulence solutions.

Antioxidant and Cytotoxic Properties of Berberis vulgaris (L.) Stem Bark Dry Extract.

Berberis vulgaris (L.) has remarkable ethnopharmacological properties and is widely used in traditional medicine. The present study investigated B. vulgaris stem bark (Berberidis cortex) by extraction with 50% ethanol. The main secondary metabolites were quantified, resulting in a polyphenols content of 17.6780 ± 3.9320 mg Eq tannic acid/100 g extract, phenolic acids amount of 3.3886 ± 0.3481 mg Eq chlorogenic acid/100 g extract and 78.95 µg/g berberine. The dried hydro-ethanolic extract (BVE) was thoroughly analyzed using Ultra-High-Performance Liquid Chromatography coupled with High-Resolution Mass Spectrometry (UHPLC-HRMS/MS) and HPLC, and 40 bioactive phenolic constituents were identified. Then, the antioxidant potential of BVE was evaluated using three methods. Our results could explain the protective effects of Berberidis cortex EC50FRAP = 0.1398 mg/mL, IC50ABTS = 0.0442 mg/mL, IC50DPPH = 0.2610 mg/mL compared to ascorbic acid (IC50 = 0.0165 mg/mL). Next, the acute toxicity and teratogenicity of BVE and berberine-berberine sulfate hydrate (BS)-investigated on Daphnia sp. revealed significant BS toxicity after 24 h, while BVE revealed considerable toxicity after 48 h and induced embryonic developmental delays. Finally, the anticancer effects of BVE and BS were evaluated in different tumor cell lines after 24 and 48 h of treatments. The MTS assay evidenced dose- and time-dependent antiproliferative activity, which was higher for BS than BVE. The strongest diminution of tumor cell viability was recorded in the breast (MDA-MB-231), colon (LoVo) cancer, and OSCC (PE/CA-PJ49) cell lines after 48 h of exposure (IC50 < 100 µg/mL). However, no cytotoxicity was reported in the normal epithelial cells (HUVEC) and hepatocellular carcinoma (HT-29) cell lines. Extensive data analysis supports our results, showing a significant correlation between the BVE concentration, phenolic compounds content, antioxidant activity, exposure time, and the viability rate of various normal cells and cancer cell lines.

Phytochemical, In Vitro, In Vivo, and In Silico Research on the Extract of Ajuga chamaepitys (L.) Schreb.

The present study focuses on the chemical characterization of a dry extract obtained from the species Ajuga chamaepitys (L.) Schreb, evaluating its antioxidant properties, toxicity, and in silico profile. Quantitative analysis of the dry extract revealed a notable amount of phytochemical compounds: 59.932 ± 21.167 mg rutin equivalents (mg REs)/g dry weight, 45.864 ± 4.434 mg chlorogenic acid equivalents (mg ChAEs)/g dry weight and, respectively, 83.307 ± 3.989 mg tannic acid equivalents (TAEs)/g dry weight. By UHPLC-HRMS/MS, the following were quantified as major compounds: caffeic acid (3253.8 µg/g extract) and kaempherol (3041.5 µg/g extract); more than 11 types of polyphenolic compounds were quantified (genistin 730.2 µg/g extract, naringenin 395 µg/g extract, apigenin 325.7 µg/g extract, galangin 283.3 µg/g extract, ferulic acid 254.3 µg/g extract, p-coumaric acid 198.2 µg/g extract, rutin 110.6 µg/g extract, chrysin 90.22 µg/g extract, syringic acid 84.2 µg/g extract, pinocembrin 32.7 µg/g extract, ellagic acid 18.2 µg/g extract). The antioxidant activity was in accordance with the amount of phytochemical compounds: IC50DPPH = 483.6 ± 41.4 µg/mL, IC50ABTS•+ = 127.4 ± 20.2 µg/mL, and EC50FRAP = 491.6 ± 2 µg/mL. On the larvae of Artemia sp., it was found that the extract has a low cytotoxic action. In silico studies have highlighted the possibility of inhibiting the activity of protein kinases CDK5 and GSK-3b for apigenin, galangin, and kaempferol, with possible utility for treating neurodegenerative pathologies and neuropathic pain. Further studies are warranted to confirm the predicted molecular mechanisms of action and to further investigate the therapeutic potential in animal models of neurological disorders.

Phytochemical Profile, Antioxidant and Cytotoxic Potential of Capsicum annuum (L.) Dry Hydro-Ethanolic Extract.

Capsicum annuum (L.) is one of the essential spices most frequently used in our daily routine and has remarkable ethnobotanical and pharmacological properties. Its fruits are rich in vitamins, minerals, carotenoids, and numerous other phenolic metabolites with a well-known antioxidant activity. Regular consumption of chili fruits may have a positive influence on human health. Therefore, we investigated a commercially available chili fruit powder in the present study, extracting it with 50% ethanol. The dried hydro-ethanolic extract (CAE) was thoroughly analyzed using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS/MS), and 79 bioactive phenolic constituents were identified. Then, we quantified the main phenolic compounds and found a polyphenol content of 4.725 ± 1.361 mg Eq tannic acid/100 g extract and a flavonoid amount of 1.154 ± 0.044 mg Eq rutin/100 g extract. Phenolic secondary metabolites are known for their dual redox behavior as antioxidants/pro-oxidants, underlying their numerous benefits in health and disease. Thus, the antioxidant potential of CAE was evaluated using three methods; our results could explain the protective effects of chili fruits: IC50DPPH = 1.669 mg/mL, IC50ABTS = 0.200 mg/mL, and EC50FRAP = 0.561 mg/mL. The pro-oxidant potential of phenolic compounds could be a basis for CAE cytotoxicity, investigated in vitro on tumor cell lines and in vivo on Daphnia sp. Results demonstrated the dose- and time-dependent CAE's cytotoxic activity; the highest antiproliferative activity was recorded on colon (LoVo) and breast (MDA-MB-231) cancer cell lines after 48 h of exposure (IC50 values < 200 µg/mL). In vivo testing on Daphnia sp. reported a potent CAE cytotoxicity after 48 h and embryonic developmental delays. Extensive data analyses support our results, showing a significant correlation between the CAE's concentration, phenolic compound content, antioxidant activity, exposure time, and the viability rate of different tested cell lines.

Implications of the Cultivation of Rosemary and Thyme (Lamiaceae) in Plant Communities for the Development of Antioxidant Therapies.

Oxidative stress is the most critical factor in multiple functional disorders' development, and natural antioxidants could protect the human body against it. Our study aims to investigate the polyphenol content of four extracts of two medicinal plants (Rosmarinus officinalis L. and Thymus vulgaris L.) and analyze the correlation with their antioxidant activity. The research was carried out on extracts of rosemary and thyme obtained from species cultivated together in plant communities. Both were compared with extracts from species cultivated in individual crops (control crops). Their polyphenols were determined by spectrophotometric methods (dosage of flavones, phenol carboxylic acids, and total polyphenols) and chromatography (UHPLC-MS and FT-ICR MS). Triterpenic acids were also quantified, having a higher concentration in the thyme extract from the culture. The antioxidant activity of the dry extracts was evaluated in vitro (DPPH, ABTS, and FRAP) and in silico (prediction of interactions with BACH1/BACH2 transcription factors). The concentrations of polyphenols are higher in the extracts obtained from the sources collected from the common crops. These observations were also validated following the chromatographic analysis for some compounds. Statistically significant differences in the increase in the antioxidant effect were observed for the extracts from the common batches compared to those from the individual ones. Following the Pearson analysis, the IC50 values for each plant extract were strongly correlated with the concentration of active phytoconstituents. Molecular docking studies revealed that quercetin could bind to BTB domains of BACH1 and BACH2 transcription factors, likely translating into increased antioxidant enzyme expression. Future studies must validate the in silico findings and further investigate phytosociological cultivation's effects.

Phytochemical Screening and Antioxidant Potential of Selected Extracts from Betula alba var. pendula Roth., Glycyrrhiza glabra L., and Avena sativa L.

The aim of the present study was to obtain, characterize, and evaluate the antioxidant potential of some extracts obtained from the bark of Betula alba var. pendula Roth., the root of Glycyrrhiza glabra L., and the green herb of the Avena sativa. The results revealed that the lowest IC50 value, determined by all three methods, was obtained for Betulae extractum (BE) (73.6 µg/mL-DPPH method, 11.2 µg/mL-ABTS method, and 58.7 µg/mL-FRAP method), followed by Liquiritiae extractum (LE) (805.6 µg/mL, 92.1 µg/mL, and 722 µg/mL) and Avenae extractum (1.13 mg/mL-DPPH method, 99.7 µg/mL-ABTS method, and 135.1 µg/mL-FRAP method). These results correlate with total polyphenols content (expressed in g tannic acid/100 g dry extract), with BE having more polyphenols than LE and AE (47.96 ± 9.7083 for BE, compared with 9.31 ± 0.9913 for LE and 40.55 ± 6.3715 for AE). The total flavonoid content (expressed as g rutoside/100 g dry extract) is similar for BE and LE (3.75 ± 0.3140 and 3.44 ± 0.3037) and smaller for AE (1.95 ± 0.0526). Therefore, Betulae extractum has the strongest antioxidant action, with an IC50 value very close to the standard used as a reference (ascorbic acid-16.5 µg/mL solution). The FT-ICR-MS analysis confirmed the presence of the major compounds in all three extracts. The antioxidant properties of the studied extracts were further supported by molecular docking experiments that revealed the potential of the analyzed phytochemicals to act as both noncovalent and covalent activators of the Nrf2 signaling pathway, with promising benefits in treating various skin disorders.

Outlook on Chronic Venous Disease Treatment: Phytochemical Screening, In Vitro Antioxidant Activity and In Silico Studies for Three Vegetal Extracts.

Chronic venous disease is one of the most common vascular diseases; the signs and symptoms are varied and are often neglected in the early stages. Vascular damage is based on proinflammatory, prothrombotic, prooxidant activity and increased expression of several matrix metalloproteinases (MMPs). The aim of this research is preparation and preliminary characterization of three vegetal extracts (Sophorae flos-SE, Ginkgo bilobae folium-GE and Calendulae flos-CE). The obtained dry extracts were subjected to phytochemical screening (FT-ICR-MS, UHPLC-HRMS/MS) and quantitative analysis (UHPLC-HRMS/MS, spectrophotometric methods). Antioxidant activity was evaluated using three methods: FRAP, DPPH and ABTS. More than 30 compounds were found in each extract. The amount of flavones follows the succession: SE > GE > CE; the amount of phenolcarboxylic acids follows: SE > CE > GE; and the amount of polyphenols follows: SE > GE > CE. Results for FRAP method varied as follows: SE > CE > GE; results for the DPPH method followed: SE > GE > CE; and results for ABTS followed: SE > GE > CE. Strong and very strong correlations (appreciated by Pearson coefficient) have been observed between antioxidant activity and the chemical content of extracts. Molecular docking studies revealed the potential of several identified phytochemicals to inhibit the activity of four MMP isoforms. In conclusion, these three extracts have potential in the treatment of chronic venous disease, based on their phytochemical composition.

Assessment of Some Unsymmetrical Porphyrins as Promising Molecules for Photodynamic Therapy of Cutaneous Disorders.

In order to select for further development novel photosensitizers for photodynamic therapy in cutaneous disorders, three unsymmetrical porphyrins, namely 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.2), 5-(2-hydroxy-5-methoxyphenyl)-10,15,20-tris-(4-carboxymethylphenyl) porphyrin (P3.2), and 5-(2,4-dihydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl) porphyrin (P4.2), along with their fully symmetrical counterparts 5,10,15,20-tetrakis-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.1) and 5,10,15,20-tetrakis-(4-carboxymethylphenyl) porphyrin (P3.1) were comparatively evaluated. The absorption and fluorescence properties, as well as atomic force microscopy measurements were performed to evaluate the photophysical characteristics as well as morphological and textural properties of the mentioned porphyrins. The cellular uptake of compounds and the effect of photodynamic therapy on the viability, proliferation, and necrosis of human HaCaT keratinocytes, human Hs27 skin fibroblasts, human skin SCL II squamous cell carcinoma, and B16F10 melanoma cells were assessed in vitro, in correlation with the structural and photophysical properties of the investigated porphyrins, and with the predictions regarding diffusion through cell membranes and ADMET properties. All samples were found to be isotropic and self-similar, with slightly different degrees of aggregability, had a relatively low predicted toxicity (class V), and a predicted long half-life after systemic administration. The in vitro study performed on non-malignant and malignant skin-relevant cells highlighted that the asymmetric P2.2 porphyrin qualified among the five investigated porphyrins to be a promising photosensitizer candidate for PDT in skin disorders. P2.2 was shown to accumulate well within cells, and induced by PDT a massive decrease in the number of metabolically active skin cells, partly due to cell death by necrosis. P2.2 had in this respect a better behavior than the symmetric P.2.1 compound and the related asymmetric compound P4.2. The strong action of P2.2-mediated PDT on normal skin cells might be an important drawback for further development of this compound. Meanwhile, the P3.1 and P3.2 compounds were not able to accumulate well in skin cells, and did not elicit significant PDT in vitro. Taken together, our experiments suggest that P2.2 can be a promising candidate for the development of novel photosensitizers for PDT in skin disorders.

ROS-Induced DNA-Damage and Autophagy in Oral Squamous Cell Carcinoma by Usnea barbata Oil Extract-An In Vitro Study.

Oxidative stress is associated with aging, cancers, and numerous metabolic and chronic disorders, and phenolic compounds are well known for their health-promoting role due to their free-radical scavenging activity. These phytochemicals could also exhibit pro-oxidant effects. Due to its bioactive phenolic secondary metabolites, Usnea barbata (L.) Weber ex. F.H. Wigg (U. barbata) displays anticancer and antioxidant activities and has been used as a phytomedicine for thousands of years. The present work aims to analyze the properties of U. barbata extract in canola oil (UBO). The UBO cytotoxicity on oral squamous cell carcinoma (OSCC) CLS-354 cell line and blood cell cultures was explored through complex flow cytometry analyses regarding apoptosis, reactive oxygen species (ROS) levels, the enzymatic activity of caspase 3/7, cell cycle, nuclear shrinkage (NS), autophagy (A), and synthesis of deoxyribonucleic acid (DNA). All these studies were concomitantly performed on canola oil (CNO) to evidence the interaction of lichen metabolites with the constituents of this green solvent used for extraction. The obtained data evidenced that UBO inhibited CLS-354 oral cancer cell proliferation through ROS generation (316.67 × 104), determining higher levels of nuclear shrinkage (40.12%), cell cycle arrest in G0/G1 (92.51%; G0 is the differentiation phase, while during G1 phase occurs preparation for cell division), DNA fragmentation (2.97%), and autophagy (62.98%) than in blood cells. At a substantially higher ROS level in blood cells (5250.00 × 104), the processes that lead to cell death-NS (30.05%), cell cycle arrest in G0/G1 (86.30%), DNA fragmentation (0.72%), and autophagy (39.37%)-are considerably lower than in CLS-354 oral cancer cells. Our work reveals the ROS-mediated anticancer potential of UBO through DNA damage and autophagy. Moreover, the present study suggests that UBO pharmacological potential could result from the synergism between lichen secondary metabolites and canola oil phytoconstituents.

In Vitro Anticancer Activity of Mucoadhesive Oral Films Loaded with Usnea barbata (L.) F. H. Wigg Dry Acetone Extract, with Potential Applications in Oral Squamous Cell Carcinoma Complementary Therapy.

Oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy, with a high death rate and an inadequate response to conventional chemotherapeutic drugs. Medical research explores plant extracts' properties to obtain potential nanomaterial-based anticancer drugs. The present study aims to formulate, develop, and characterize mucoadhesive oral films loaded with Usnea barbata (L.) dry acetone extract (F-UBA) and to investigate their anticancer potential for possible use in oral cancer therapy. U. barbata dry acetone extract (UBA) was solubilized in ethanol: isopropanol mixture and loaded in a formulation containing hydroxypropyl methylcellulose (HPMC) K100 and polyethylene glycol 400 (PEG 400). The UBA influence on the F-UBA pharmaceutical characteristics was evidenced compared with the references, i.e., mucoadhesive oral films containing suitable excipients but no active ingredient loaded. Both films were subjected to a complex analysis using standard methods to evaluate their suitability for topical administration on the oral mucosa. Physico-chemical and structural characterization was achieved by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Pharmacotechnical evaluation (consisting of the measurement of specific parameters: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time) proved that F-UBAs are suitable for oral mucosal administration. The brine shrimp lethality (BSL) assay was the F-UBA cytotoxicity prescreen. Cellular oxidative stress, caspase 3/7 activity, nuclear condensation, lysosomal activity, and DNA synthesis induced by F-UBA in blood cell cultures and oral epithelial squamous cell carcinoma (CLS-354) cell line were investigated through complex flow cytometry analyses. Moreover, F-UBA influence on both cell type division and proliferation was determined. Finally, using the resazurin-based 96-well plate microdilution method, the F-UBA antimicrobial potential was explored against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019. The results revealed that each UBA-loaded film contains 175 µg dry extract with a usnic acid (UA) content of 42.32 µg. F-UBAs are very thin (0.060 ± 0.002 mm), report a neutral pH (7.01 ± 0.01), a disintegration time of 146 ± 5.09 s, and an ex vivo mucoadhesion time of 85 ± 2.33 min, and they show a swelling ratio after 6 h of 211 ± 4.31%. They are suitable for topical administration on the oral mucosa. Like UA, they act on CLS-354 tumor cells, considerably increasing cellular oxidative stress, nuclear condensation, and autophagy and inducing cell cycle arrest in G0/G1. The F-UBAs inhibited the bacterial and fungal strains in a dose-dependent manner; they showed similar effects on both Candida sp. and higher inhibitory activity against P. aeruginosa than S. aureus. All these properties lead to considering the UBA-loaded mucoadhesive oral films suitable for potential application as a complementary therapy in OSCC.

Formulation and Development of Bioadhesive Oral Films Containing Usnea barbata (L.) F.H.Wigg Dry Ethanol Extract (F-UBE-HPC) with Antimicrobial and Anticancer Properties for Potential Use in Oral Cancer Complementary Therapy.

Medical research explores plant extracts' properties to obtain potential anticancer drugs. The present study aims to formulate, develop, and characterize the bioadhesive oral films containing Usnea barbata (L.) dry ethanol extract (F-UBE-HPC) and to investigate their anticancer potential for possible use in oral cancer therapy. The physicochemical and morphological properties of the bioadhesive oral films were analyzed through Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), Atomic Force Microscopy (AFM), thermogravimetric analysis (TG), and X-ray diffraction techniques. Pharmacotechnical evaluation (consisting of the measurement of the specific parameters: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time) completed the bioadhesive films' analysis. Next, oxidative stress, caspase 3/7 activity, nuclear condensation, lysosomal activity, and DNA synthesis induced by F-UBE-HPC in normal blood cell cultures and oral epithelial squamous cell carcinoma (CLS-354) cell line and its influence on both cell types' division and proliferation was evaluated. The results reveal that each F-UBE-HPC contains 0.330 mg dry extract with a usnic acid (UA) content of 0.036 mg. The bioadhesive oral films are thin (0.093 ± 0.002 mm), reveal a neutral pH (7.10 ± 0.02), a disintegration time of 118 ± 3.16 s, an ex vivo bioadhesion time of 98 ± 3.58 min, and show a swelling ratio after 6 h of 289 ± 5.82%, being suitable for application on the oral mucosa. They displayed in vitro anticancer activity on CLS-354 tumor cells. By considerably increasing cellular oxidative stress and caspase 3/7 activity, they triggered apoptotic processes in oral cancer cells, inducing high levels of nuclear condensation and lysosomal activity, cell cycle arrest in G0/G1, and blocking DNA synthesis. All these properties lead to considering the UBE-loaded bioadhesive oral films suitable for potential application as a complementary therapy in oral cancer.

The Influence of Phytosociological Cultivation and Fertilization on Polyphenolic Content of Menthae and Melissae folium and Evaluation of Antioxidant Properties through In Vitro and In Silico Methods.

Since medicinal plants are widely used in treating various diseases, phytoconstituents enrichment strategies are of high interest for plant growers. First of all, we investigated the impact of phytosociological cultivation on polyphenolic content (total flavonoids-TFL, and total polyphenols-TPC) of peppermint (Mentha piperita L.) and lemon balm (Melissa officinalis L.) leaves, using spectrophotometric methods. Secondly, the influence of chemical (NPK) and organic (BIO) fertilization on polyphenolic content and plant material quality was also assessed. Dry extracts were obtained from harvested leaves using hydroethanolic extraction solvents for further qualitative and quantitative assessment of phytoconstituents by FT-ICR MS and UHPLC-MS. Furthermore, the antioxidant activity of leaf extracts was determined in vitro using DPPH, ABTS and FRAP methods. Molecular docking simulations were employed to further evaluate the antioxidant potential of obtained extracts, predicting the interactions of identified phytochemicals with sirtuins. The concentration of polyphenols was higher in the plant material harvested from the phytosociological culture. Moreover, the use of BIO fertilizer led to the biosynthesis of a higher content of polyphenols. Higher amounts of phytochemicals, such as caffeic acid, were determined in extracts obtained from phytosociological crops. The antioxidant activity was dependent on polyphenols concentration, more potent inhibition values being observed for the extracts obtained from the phytosociological batches. Molecular docking studies and MM/PBSA calculations revealed that the obtained extracts have the potential to directly activate sirtuins 1, 5 and 6 through several polyphenolic compounds, such as rosmarinic acid, thus complementing the free radical scavenging activity with the potential stimulation of endogenous antioxidant defense mechanisms. In conclusion, growing medicinal plants in phytosociological cultures treated with biofertilizers can have a positive impact on plant material quality, concentration in active constituents and biological activity.

Design, Characterization, and Anticancer and Antimicrobial Activities of Mucoadhesive Oral Patches Loaded with Usnea barbata (L.) F. H. Wigg Ethanol Extract F-UBE-HPMC.

The oral cavity's common pathologies are tooth decay, periodontal disease, and oral cancer; oral squamous cell carcinoma (OSCC) is the most frequent oral malignancy, with a high mortality rate. Our study aims to formulate, develop, characterize, and pharmacologically investigate the oral mucoadhesive patches (F-UBE-HPMC) loaded with Usnea barbata (L.) F.H. Wigg dry ethanol extract (UBE), using HPMC K100 as a film-forming polymer. Each patch contains 312 µg UBE, with a total phenolic content (TPC) of 178.849 µg and 33.924 µg usnic acid. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were performed for their morphological characterization, followed by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). Pharmacotechnical evaluation involved the measurement of the specific parameters for mucoadhesive oral patches as follows: weight uniformity, thickness, folding endurance, tensile strength, elongation, moisture content, pH, disintegration time, swelling rate, and ex vivo mucoadhesion time. Thus, each F-UBE-HPMC has 104 ± 4.31 mg, a pH = 7.05 ± 0.04, a disintegration time of 130 ± 4.14 s, a swelling ratio of 272 ± 6.31% after 6 h, and a mucoadhesion time of 102 ± 3.22 min. Then, F-UBE-HPMCs pharmacological effects were investigated using brine shrimp lethality assay (BSL assay) as a cytotoxicity prescreening test, followed by complex flow cytometry analyses on blood cell cultures and oral epithelial squamous cell carcinoma CLS-354 cell line. The results revealed significant anticancer effects by considerably increasing oxidative stress and blocking DNA synthesis in CLS-354 cancer cells. The antimicrobial potential against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019 was assessed by a Resazurin-based 96-well plate microdilution method. The patches moderately inhibited both bacteria strains growing and displayed a significant antifungal effect, higher on C. albicans than on C. parapsilosis. All these properties lead to considering F-UBE-HPMC suitable for oral disease prevention and therapy.

Evaluation of Usnea barbata (L.) Weber ex F.H. Wigg Extract in Canola Oil Loaded in Bioadhesive Oral Films for Potential Applications in Oral Cavity Infections and Malignancy.

Usnea lichens are known for their beneficial pharmacological effects with potential applications in oral medicine. This study aims to investigate the extract of Usnea barbata (L.) Weber ex F.H. Wigg from the Calimani Mountains in canola oil as an oral pharmaceutical formulation. In the present work, bioadhesive oral films (F-UBO) with U. barbata extract in canola oil (UBO) were formulated, characterized, and evaluated, evidencing their pharmacological potential. The UBO-loaded films were analyzed using standard methods regarding physicochemical and pharmacotechnical characteristics to verify their suitability for topical administration on the oral mucosa. F-UBO suitability confirmation allowed for the investigation of antimicrobial and anticancer potential. The antimicrobial properties against Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27353, Candida albicans ATCC 10231, and Candida parapsilosis ATCC 22019 were evaluated by a resazurin-based 96-well plate microdilution method. The brine shrimp lethality assay (BSL assay) was the animal model cytotoxicity prescreen, followed by flow cytometry analyses on normal blood cells and oral epithelial squamous cell carcinoma CLS-354 cell line, determining cellular apoptosis, caspase-3/7 activity, nuclear condensation and lysosomal activity, oxidative stress, cell cycle, and cell proliferation. The results indicate that a UBO-loaded bioadhesive film's weight is 63 ± 1.79 mg. It contains 315 µg UBO, has a pH = 6.97 ± 0.01, a disintegration time of 124 ± 3.67 s, and a bioadhesion time of 86 ± 4.12 min, being suitable for topical administration on the oral mucosa. F-UBO showed moderate dose-dependent inhibitory effects on the growth of both bacterial and fungal strains. Moreover, in CLS-354 tumor cells, F-UBO increased oxidative stress, diminished DNA synthesis, and induced cell cycle arrest in G0/G1. All these properties led to considering UBO-loaded bioadhesive oral films as a suitable phytotherapeutic formulation with potential application in oral infections and neoplasia.

Phenolic Secondary Metabolites and Antiradical and Antibacterial Activities of Different Extracts of Usnea barbata (L.) Weber ex F.H.Wigg from Calimani Mountains, Romania.

Phenolic compounds represent an essential bioactive metabolites group with numerous pharmaceutical applications. Our study aims to identify and quantify phenolic constituents of various liquid and dry extracts of Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) from Calimani Mountains, Romania, and investigate their bioactivities. The extracts in acetone, 96% ethanol, and water with the same dried lichen/solvent ratio (w/v) were obtained through two conventional techniques: maceration (mUBA, mUBE, and mUBW) and Soxhlet extraction (dUBA, dUBE, and dUBW). High-performance liquid chromatography with diode-array detection (HPLC-DAD) was performed for usnic acid (UA) and different polyphenols quantification. Then, the total phenolic content (TPC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity (AA) were determined through spectrophotometric methods. Using the disc diffusion method (DDM), the antibacterial activity was evaluated against Gram-positive and Gram-negative bacteria known for their pathogenicity: Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae (ATCC 49619), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 13883). All extracts contain phenolic compounds expressed as TPC values. Five lichen extracts display various UA contents; this significant metabolite was not detected in dUBW. Six polyphenols from the standards mixture were quantified only in ethanol and water extracts; mUBE has all individual polyphenols, while dUBE shows only two. Three polyphenols were detected in mUBW, but none was found in dUBW. All U. barbata extracts had antiradical activity; however, only ethanol and acetone extracts proved inhibitory activity against P. aeruginosa, S. pneumoniae, and S. aureus. In contrast, K. pneumoniae was strongly resistant (IZD = 0). Data analysis evidenced a high positive correlation between the phenolic constituents and bioactivities of each U. barbata extract. Associating these extracts' properties with both conventional techniques used for their preparation revealed the extraction conditions' significant influence on lichen extracts metabolites profiling, with a powerful impact on their pharmacological potential.

The Polyphenolic Profile and Antioxidant Activity of Five Vegetal Extracts with Hepatoprotective Potential.

Oxidative stress is among the major triggers for many important human functional disorders, which often lead to various metabolic or tissue diseases. The aim of the study is to obtain five standardized vegetal extracts (Cynarae extractum-CE, Rosmarini extractum-RE, Taraxaci extractum-TE, Cichorii extractum-CHE, and Agrimoniae extractum-AE) that contain active principles with an essential role in protecting liver cells against free radicals and quantify their antioxidant actions. The compounds of therapeutic interest from the analyzed extracts were identified and quantified using the UHPLC-HRMS/MS technique. Thus, the resulting identified compounds were 28 compounds in CE, 48 compounds in RE, 39 compounds in TE, 43 compounds in CHE, and 31 compounds in AE. These compounds belong to the class of flavonoids, isoflavones, phenolic acids and dicarboxylic acids, depsides, diterpenes, triterpenes, sesquiterpenes, proanthocyanidins, or coumarin derivatives. From the major polyphenolic compounds quantified in all the extracts analyzed by UHPLC-HRMS/MS, considerable amounts have been found for chlorogenic acid (619.8 µg/g extract for TE-2032.4 µg/g extract for AE), rutoside (105.1 µg/g extract for RE-1724.7 µg/g extract for AE), kaempferol (243 µg/g extract for CHE-2028.4 µg/g extract for CE), and for naringenin (383 µg/g extract for CHE-1375.8 µg/g extract for AE). The quantitative chemical analysis showed the highest content of total phenolic acids for AE (24.1528 ± 1.1936 g chlorogenic acid/100 g dry extract), the highest concentration of flavones for RE (6.0847 ± 0.3025 g rutoside/100 g dry extract), and the richest extract in total polyphenols with 31.7017 ± 1.2211 g tannic acid equivalent/100 g dry extract for AE. Several methods (DPPH, ABTS, and FRAP) have been used to determine the in vitro total antioxidant activity of the extracts to evaluate their free radical scavenging ability, influenced by the identified compounds. As a result, the correlation between the content of the polyphenolic compounds and the antioxidant effect of the extracts has been demonstrated. Statistically significant differences were found when comparing the antiradical capacity within the study groups. Although all the analyzed extracts showed good IC50 values, which may explain their antihepatotoxic effects, the highest antioxidant activity was obtained for Agrimoniae extractum (IC50ABTS = 0.0147 mg/mL) and the lowest antioxidant activity was obtained for Cynarae extractum (IC50ABTS = 0.1588 mg/mL). Furthermore, the hepatoprotective potential was evaluated in silico by predicting the interactions between the determined phytochemicals and key molecular targets relevant to liver disease pathophysiology. Finally, the evaluation of the pharmacognostic and phytochemical properties of the studied extracts validates their use as adjuvants in phytotherapy, as they reduce oxidative stress and toxin accumulation and thus exert a hepatoprotective effect at the cellular level.

Antioxidant, Cytotoxic, and Rheological Properties of Canola Oil Extract of Usnea barbata (L.) Weber ex F.H. Wigg from Calimani Mountains, Romania.

Usnea genus (Parmeliaceae, lichenized Ascomycetes) is a potent phytomedicine, due to phenolic secondary metabolites, with various pharmacological effects. Therefore, our study aimed to explore the antioxidant, cytotoxic, and rheological properties of Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) extract in canola oil (UBO) compared to cold-pressed canola seed oil (CNO), as a green solvent used for lichen extraction, which has phytoconstituents. The antiradical activity (AA) of UBO and CNO was investigated using UV-Vis spectrophotometry. Their cytotoxicity was examined in vivo through a brine shrimp lethality (BSL) test after Artemia salina (A. salina) larvae exposure for 6 h to previously emulsified UBO and CNO. The rheological properties of both oil samples (flow behavior, thixotropy, and temperature-dependent viscosity variation) were comparatively analyzed. The obtained results showed that UBO (IC50 = 0.942 ± 0.004 mg/mL) had a higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity than CNO (IC50 = 1.361 ± 0.008 mg/mL). Both UBO and CNO emulsions induced different and progressive morphological changes to A. salina larvae, incompatible with their survival; UBO cytotoxicity was higher than that of CNO. Finally, in the temperature range of 32-37 °C, the UBO and CNO viscosity and viscoelastic behavior indicated a clear weakening of the intermolecular bond when temperature increases, leading to a more liquid state, appropriate for possible pharmaceutical formulations. All quantified parameters were highly intercorrelated. Moreover, their significant correlation with trace/heavy minerals and phenolic compounds can be observed. All data obtained also suggest a possible synergism between lichen secondary metabolites, minerals, and canola oil phytoconstituents.

Antioxidant and Cytotoxic Activities of Usnea barbata (L.) F.H. Wigg. Dry Extracts in Different Solvents.

Lichens represent a significant source of antioxidants due to numerous metabolites that can reduce free radicals. Usnea barbata (L.) F.H. Wigg. has been recognized and used since ancient times for its therapeutic effects, some of which are based on its antioxidant properties. The present study aims to analyze the phytochemical profile and to evaluate the antioxidant and cytotoxic potential of this lichen species. Five dry extracts of U. barbata (UBDE) in different solvents (acetone, ethyl acetate, ethanol, methanol, water) were prepared by refluxing at Soxhlet to achieve these proposed objectives and to identify which solvent is the most effective for the extraction. The usnic acid content (UAC) was quantified by ultra-high performance liquid chromatography (UHPLC). The total polyphenols content (TPC) and tannins content (TC) were evaluated by spectrophotometry, and the total polysaccharides (PSC) were extracted by a gravimetric method. The 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical method was used to assess the antioxidant activity (AA) and the Brine Shrimp Lethality (BSL) assay was the biotest for cytotoxic activity evaluation. The ethyl acetate extract had the highest usnic acid content, and acetone extract had the highest content of total polyphenols and tannins. The most significant antioxidant effect was reported to methanol extract, and all the extracts proved high cytotoxicity. The water extract has the lowest cytotoxicity because usnic acid is slightly soluble in this solvent, and it was not found at UHPLC analysis. All extracts recorded a moderate correlation between the content of usnic acid, polyphenols, tannins, and AA; furthermore, it has been observed that the cytotoxicity varies inversely with the antioxidant effect.