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.

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.

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.

New Heterocyclic Compounds from Oxazol-5(4H)-one and 1,2,4-Triazin-6(5H)-one Classes: Synthesis, Characterization and Toxicity Evaluation.

This paper describes the synthesis of new heterocycles from oxazol-5(4H)-one and 1,2,4-triazin-6(5H)-one classes containing a phenyl-/4-bromophenylsulfonylphenyl moiety. The oxazol-5(4H)-ones were obtained via condensation of 2-(4-(4-X-phenylsulfonyl)benzamido)acetic acids with benzaldehyde/4-fluorobenzaldehyde in acetic anhydride and in the presence of sodium acetate. The reaction of oxazolones with phenylhydrazine, in acetic acid and sodium acetate, yielded the corresponding 1,2,4-triazin-6(5H)-ones. The structures of the compounds were confirmed using spectral (FT-IR, 1H-NMR, 13C-NMR, MS) and elemental analysis. The toxicity of the compounds was evaluated on Daphnia magna Straus crustaceans and on the budding yeast Saccharomyces cerevisiae. The results indicate that both the heterocyclic nucleus and halogen atoms significantly influenced the toxicity against D. magna, with the oxazolones being less toxic than triazinones. The halogen-free oxazolone had the lowest toxicity, and the fluorine-containing triazinone exhibited the highest toxicity. The compounds showed low toxicity against yeast cells, apparently due to the activity of plasma membrane multidrug transporters Pdr5 and Snq2. The predictive analyses indicated an antiproliferative effect as the most probable biological action. The PASS prediction and CHEMBL similarity studies show evidence that the compounds could inhibit certain relevant oncological protein kinases. These results correlated with toxicity assays suggest that halogen-free oxazolone could be a good candidate for future anticancer investigations.

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.

Interaction of Some Asymmetrical Porphyrins with U937 Cell Membranes-In Vitro and In Silico Studies.

The aim of the present study was to assess the effects exerted in vitro by three asymmetrical porphyrins (5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin, 5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrinatozinc(II), and 5-(2-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrinatocopper(II)) on the transmembrane potential and the membrane anisotropy of U937 cell lines, using bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC4(3)) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), respectively, as fluorescent probes for fluorescence spectrophotometry. The results indicate the hyperpolarizing effect of porphyrins in the concentration range of 0.5, 5, and 50 µM on the membrane of human U937 monocytic cells. Moreover, the tested porphyrins were shown to increase membrane anisotropy. Altogether, the results evidence the interaction of asymmetrical porphyrins with the membrane of U937 cells, with potential consequences on cellular homeostasis. Molecular docking simulations, and Molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) free energy of binding calculations, supported the hypothesis that the investigated porphyrinic compounds could potentially bind to membrane proteins, with a critical role in regulating the transmembrane potential. Thus, both the free base porphyrins and the metalloporphyrins could bind to the SERCA2b (sarco/endoplasmic reticulum ATPase isoform 2b) calcium pump, while the metal complexes may specifically interact and modulate calcium-dependent (large conductance calcium-activated potassium channel, Slo1/KCa1.1), and ATP-sensitive (KATP), potassium channels. Further studies are required to investigate these interactions and their impact on cellular homeostasis and functionality.

Porphyrin Macrocycles: General Properties and Theranostic Potential.

Despite specialists' efforts to find the best solutions for cancer diagnosis and therapy, this pathology remains the biggest health threat in the world. Global statistics concerning deaths associated with cancer are alarming; therefore, it is necessary to intensify interdisciplinary research in order to identify efficient strategies for cancer diagnosis and therapy, by using new molecules with optimal therapeutic potential and minimal adverse effects. This review focuses on studies of porphyrin macrocycles with regard to their structural and spectral profiles relevant to their applicability in efficient cancer diagnosis and therapy. Furthermore, we present a critical overview of the main commercial formulations, followed by short descriptions of some strategies approached in the development of third-generation photosensitizers.

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.

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.

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.

Insight into the Web of Stress Responses Triggered at Gene Expression Level by Porphyrin-PDT in HT29 Human Colon Carcinoma Cells.

Photodynamic therapy (PDT), a highly targeted therapy with acceptable side effects, has emerged as a promising therapeutic option in oncologic pathology. One of the issues that needs to be addressed is related to the complex network of cellular responses developed by tumor cells in response to PDT. In this context, this study aims to characterize in vitro the stressors and the corresponding cellular responses triggered by PDT in the human colon carcinoma HT29 cell line, using a new asymmetric porphyrin derivative (P2.2) as a photosensitizer. Besides investigating the ability of P2.2-PDT to reduce the number of viable tumor cells at various P2.2 concentrations and fluences of the activating light, we assessed, using qRT-PCR, the expression levels of 84 genes critically involved in the stress response of PDT-treated cells. Results showed a fluence-dependent decrease of viable tumor cells at 24 h post-PDT, with few cells that seem to escape from PDT. We highlighted following P2.2-PDT the concomitant activation of particular cellular responses to oxidative stress, hypoxia, DNA damage and unfolded protein responses and inflammation. A web of inter-connected stressors was induced by P2.2-PDT, which underlies cell death but also elicits protective mechanisms that may delay tumor cell death or even defend these cells against the deleterious effects of PDT.

Comparison of the Polyphenolic Profile of Medicago sativa L. and Trifolium pratense L. Sprouts in Different Germination Stages Using the UHPLC-Q Exactive Hybrid Quadrupole Orbitrap High-Resolution Mass Spectrometry.

Identification and quantification of polyphenols in plant material are of great interest since they make a significant contribution to its total bioactivity. In the present study, an UPLC-Orbitrap-MS/MS approach using the variable data acquisition mode (vDIA) was developed and applied for rapid separation, identification, and quantification of the main polyphenolic compounds in Medicago sativa L. and Trifolium pratense L. sprouts in different germination stages. Based on accurate MS data and fragment ions identification strategy, a total of 29 compounds were identified by comparing their accurate masses, fragment ions, retention times, and literatures. Additionally, a number of 30 compounds were quantified by comparing to the reference standards. Data were statistically analysed. For both plant species, the sprouts of the third germination day are valuable sources of bioactive compounds and could be used in phytotherapy and nutrition. Although Trifolium pratense L. (Red Clover) is considered to be a reference for natural remedies in relieving menopause disorders, alfalfa also showed a high level of biological active compounds with estrogenic activity.

Photochemical /Photocytotoxicity Studies of New Tetrapyrrolic Structures as Potential Candidates for Cancer Theranostics.

BACKGROUND: Detailed photochemical and photocytotoxicity studies of two new porphyrins: 5,10,15,20-meso-tetrakis-(4-acetoxy-3-methoxyphenyl) porphyrin (P2.1) and 5-(4-hydroxy-3- methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (P2.2) are reported, as potential candidates for theranostics. For powdered samples of P2.1 and P2.2 adsorbed onto a powdered biocompatible substrate, polyethylene glycol (PEG), a concentration study was performed, correlating the fluorescence emission intensity with sample absorption to determine the useful concentration range for photodynamic therapy of cancer (PDT) in which aggregation does not occur. Cytotoxicity studies were performed in dark and illuminated conditions. METHODS: The laser induced luminescence set-up is home-made, a N2 laser is used as the excitation source and a time gated charged-coupled device (ICCD) as the detector. Fluorescence lifetime determinations were made using pulsed light sources from the excitation LEDs and measures of the fluorescence intensities at different time delays after the excitation pulse. The singlet oxygen formation quantum yields ΦΔ measurements were obtained by comparing the total area of the emission spectra for the reference compound and also for the samples under study in the same solvent and with the same optical density at the excitation wavelength (405 nm). An integrating sphere for relative and absolute measurements was used in this work as an alternative methodology to obtain the values for the fluorescence emission quantum yields (ΦF) of the adsorbed porphyrin under study. The cytotoxicity evaluation was made in the dark and under irradiation, using four different human tumor cell lines and one non-tumor primary cell culture. RESULTS: In order to establish the useful range of concentrations of the sensitizer for PDT, and due to the use of powdered samples, a special methodology was needed: the variations of the fluorescence lifetimes and fluorescence quantum yields were evaluated as a function of the concentration of the dye, measured by (1-R)*fdye. Both ΦF and τF are constant in the range from 0.002 to about 0.050 µmol g-1, and only after that a concentration quenching effect becomes visible, decreasing both ΦF and τF. This methodology is based in the correlations established between the Remission Function values and ΦF and τF obtained for increasing values of the sensitizer concentrations. CONCLUSIONS: The study of the aggregation effects of P2.1 and P2.2 porphyrins into a PEG matrix allowed us to determine the usable concentration range for photodynamic therapy use, where the aggregation of porphyrins decreases, therefore reducing the PDT action. The use of an integrating sphere for relative and absolute measurements of fluorescence quantum yields and also the lifetime studies as a function of the dye loading confirms the useful range for the use of P2.1 and P2.2 in PEG as powdered samples. The determination of the GI50, the porphyrin concentration which inhibits 50% of the cell growth, evidences that P2.2, the A3B porphyrin overtakes P2.1 (the A4 porphyrin) in terms of PDT efficiency and both porphyrins are much better PDT agents than the unsubstituted porphyrin, TPP. These data clearly show that porphyrins P2.2 and P2.1 exhibit an excellent behaviour in terms of its photocytotoxicity. These results encourage us to pursuit in the study of this family of porphyrins in which a balance of hydrophobic versus hydrophilic substituents in the phenyl group was achieved.

Emerging Therapeutic Targets in Oncologic Photodynamic Therapy.

BACKGROUND: Reactive oxygen species sustain tumorigenesis and cancer progression through deregulated redox signalling which also sensitizes cancer cells to therapy. Photodynamic therapy (PDT) is a promising anti-cancer therapy based on a provoked singlet oxygen burst, exhibiting a better toxicological profile than chemo- and radiotherapy. Important gaps in the knowledge on underlining molecular mechanisms impede on its translation towards clinical applications. AIMS AND METHODS: The main objective of this review is to critically analyse the knowledge lately gained on therapeutic targets related to redox and inflammatory networks underlining PDT and its outcome in terms of cell death and resistance to therapy. Emerging therapeutic targets and pharmaceutical tools will be documented based on the identified molecular background of PDT. RESULTS: Cellular responses and molecular networks in cancer cells exposed to the PDT-triggered singlet oxygen burst and the associated stresses are analysed using a systems medicine approach, addressing both cell death and repair mechanisms. In the context of immunogenic cell death, therapeutic tools for boosting anti-tumor immunity will be outlined. Finally, the transcription factor NRF2, which is a major coordinator of cytoprotective responses, is presented as a promising pharmacologic target for developing co-therapies designed to increase PDT efficacy. CONCLUSION: There is an urgent need to perform in-depth molecular investigations in the field of PDT and to correlate them with clinical data through a systems medicine approach for highlighting the complex biological signature of PDT. This will definitely guide translation of PDT to clinic and the development of new therapeutic strategies aimed at improving PDT.

Studies on the Synthesis, Photophysical and Biological Evaluation of Some Unsymmetrical Meso-Tetrasubstituted Phenyl Porphyrins.

Abstract: We designed three unsymmetrical meso-tetrasubstituted phenyl porphyrins for further development as theranostic agents for cancer photodynamic therapy (PDT): 5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (P2.2), Zn(II)-5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Zn(II)2.2) and Cu(II)-5-(4-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (Cu(II)2.2). The porphyrinic compounds were synthesized and their structures were confirmed by elemental analysis, FT-IR, UV-Vis, EPR and NMR. The compounds had a good solubility in polar/nonpolar media. P2.2 and, to a lesser extent, Zn(II)2.2 were fluorescent, albeit with low fluoresence quantum yields. P2.2 and Zn(II)2.2 exhibited PDT-acceptable values of singlet oxygen generation. A "dark" cytotoxicity study was performed using cells that are relevant for the tumor niche (HT-29 colon carcinoma cells and L929 fibroblasts) and for blood (peripheral mononuclear cells). Cellular uptake of fluorescent compounds, cell viability/proliferation and death were evaluated. P2.2 was highlighted as a promising theranostic agent for PDT in solid tumors considering that P2.2 generated PDT-acceptable singlet oxygen yields, accumulated into tumor cells and less in blood cells, exhibited good fluorescence within cells for imagistic detection, and had no significant cytotoxicity in vitro against tumor and normal cells. Complexing of P2.2 with Zn(II) or Cu(II) altered several of its PDT-relevant properties. These are consistent arguments for further developing P2.2 in animal models of solid tumors for in vivo PDT.

Synthesis, Spectral Analysis and Preliminary in Vitro Evaluation of Some Tetrapyrrolic Complexes with 3d Metal Ions.

In this paper, two tetrapyrrolic complexes, Zn(II)-5-(3-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin and Cu(II)-5-(3-hydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin were synthesized, and characterized from a spectral and biological point of view. The study provided data concerning the behavior of identical external substituents vs. two different core insertions. Some of the properties of the proposed tetrapyrrolic structures were highlighted, having photodynamic therapy of cancer as a targeted biomedical application. Elemental analysis, NMR, FTIR and UV-Vis data in various solvents were provided. A preliminary in vitro study on normal and cancer cultured cells was carried out for biocompatibility assessment in dark conditions. The preliminary in vitro study performed on human peripheral mononuclear cells exposed to tetrapyrrolic compounds (2 µM) showed that the proposed compounds had a convenient cytotoxic profile on human normal peripheral blood mononuclear cells under dark conditions. Meanwhile, the investigated compounds reduced the number of metabolically active breast tumor MCF-7 cells, with the exception of Zn(II) complex-containing a symmetrical ligand. Accordingly, preliminary in vitro data suggest that the proposed tetrapyrrolic compounds are good candidates for PDT, as they limit tumor expansion even under dark conditions, whilst sparing normal cells.

Synthesis and spectral evaluation of some unsymmetrical mesoporphyrinic complexes.

Synthesis and spectral evaluation of new zinc and copper unsymmetrical mesoporphyrinic complexes are reported. Zn(II)-5-(4-acetoxy-3-methoxyphenyl)-10,15,20- tris-(4-carboxymethylphenyl)porphyrin, Zn(II)-5-[(3,4-methylenedioxy)phenyl]-10,15,20- tris-(4-carboxymethylphenyl)porphyrin, Cu(II)-5-(4-acetoxy-3-methoxyphenyl)-10,15,20- tris-(4-carboxymethylphenyl)porphyrin and Cu(II)-5-[(3,4-methylenedioxy)phenyl]-10,15,20- tris-(4-carboxymethylphenyl)porphyrin were synthesized using microwave-assisted synthesis. The complexes were characterized by elemental analysis, FT-IR, UV-Vis, EPR and NMR spectroscopy, which fully confirmed their structure. The spectral absorption properties of the porphyrinic complexes were studied in solvents with different polarities. Fluorescence emission and singlet oxygen formation quantum yields were evaluated for the compounds under study, revealing high yields for the zinc derivatives. The copper complexes are not emissive and only display residual capacity for singlet oxygen formation.

Microwave synthesis under solvent-free conditions and spectral studies of some mesoporphyrinic complexes.

A series of A3B and A4 type mesoporphyrinic complexes were synthesized with superior yields using microwave irradiation under solvent-free conditions. The structures of the complexes were confirmed using elemental analysis, FT-IR, UV-Vis, EPR and NMR spectral data. The influence of environmental polarity on spectral properties of the mesoporphyrinic complexes was investigated. The obtained results indicate that the shape of absorption and fluorescence spectra does not depend on the solvent polarity under the experimental conditions used. The small shifts of the absorption and emission maximums that occur by increasing of solvent polarity reflects the physical interaction between the porphyrinic substituents and the solvent molecules.